WO1997011172A1 - Antisense oligonucleotide chemotherapy for benign hyperplasia or cancer of the prostate - Google Patents

Abstract

Methods of selectively inhibiting the growth of or killing prostatic cells, using antisense oligonucleotides to prostate specific genes, are disclosed. The oligonucleotides may have natural nucleic acid structures or may be modified oligonucleotides with enhanced stability or tissue specific targeting. The prostate specific genes to which the antisense may be directed include the PSA and the probasin gene. Pharmaceutical compositions including such antisense oligonucleotides are also described for use in the methods. The methods and products are of particular utility in the treatment of benign prostatic hyperplasia or prostate cancer.

Description

ANTISENSE OLIGONUCLEOTIDE CHEMOTHERAPY FOR BENIGN HYPERPLASIA OR CANCER OF THE PROSTATE

Field of the Invention

The present invention relates to the field of chemotherapy for hyperplasias and cancers and, in particular, to chemotherapy for benign hyperplasia or cancer ofthe prostate. In addition, the invention relates to the field of antisense oligonucleotides and their use in human hyperplasia and cancer therapy.

Background of the Invention

Treatment of carcinoma of the prostate was one ofthe first successes of cancer chemotherapy, using the therapeutic program of castration and/or anti-androgen hormonal treatments introduced by Charles Huggins in the 1940s. A remarkable relief of symptoms and objective regression of bony metastases occurs under this endocrine therapeutic program.

Unfortunately, after a "golden period" which lasts roughly 18 months, regrowth ofthe prostate cancer cells occurs and, in the later stages ofthe disease, sensitivity to and repression by anti- androgen hormonal therapy ceases. The conventional regimen of combined chemotherapeutic agents also is typically ineffective after the golden period, and a downhill clinical course follows, terminating in death.

A key problem had been the silent onset of cancer ofthe prostate, with growth beyond its capsule and metastasis to bone too frequently occurring before the first visit to a physician. During the last half dozen years, there has been increasing recognition ofthe importance of early diagnosis and significant improvements in the available tests. As a consequence of early diagnosis, detection of prostatic cancer still contained within its capsule has become more frequent. For this situation, radical prostatectomy has largely supplanted the traditional castration/estrogen therapy. Radiation targeted to the prostate itself and to any proximal capsular infiltration has also become a prominent modality of therapy. When these two therapeutic approaches fail to halt progression ofthe disease, which is all too often (see, e.g., Gittes (1991); and Catalona (1994)), the prospect of benefit from available chemotherapy is gloomy.

Less severe but more common than prostatic cancer is benign prostatic hyperplasia (BPH). This condition may be a precursor to full blown prostatic cancer or may continue for decades without evolving into the deadly carcinoma. Depending upon the degree of hypertrophy and the age ofthe patient, treatment may range from "watchful waiting" to more aggressive approaches employing anti-androgen hormonal therapy, transurethral resection, or radical prostatectomy (see, e.g., Catalona (1994)).

Prostatic specific antigen (PSA) was first described by Wang et al. (1979) as a specific marker for prostate tissue and was subsequently identified by Papsidero et al. (1980) as being present in the sera of prostate cancer patients. Since then, PSA in the sera has become the most prevalent diagnostic marker for cancer ofthe prostate (see, e.g., Gittes (1991); Catalona (1994); Oesterling (1995); and Pienta (1995)). Levels of serum PSA are also elevated in BPH but less so than in progressed prostatic cancer. The complete amino acid sequence of PSA was disclosed by Watt et al. (1986) and the complete gene encoding PSA was disclosed by Lundwall (1989) and Klobeck et al. (1989). PSA is a glycoprotein having a single polypeptide chain and a molecular mass of approximately 34kDa. PSA is produced exclusively by epithelial cells ofthe prostate and is localized to the rough endoplasmic reticulum and associated vesicles until it is secreted into the acini and ducts ofthe prostate (see, e.g., Sinha, et al. (1987)). There PSA functions as a neutral serine protease which serves to liquefy the seminal coagulum by degrading seminal vesicle proteins including fibronectin and semenogelin (see, e.g., Lilja (1985); Warhol and Logtine (1985)). Higher serum PSA levels are correlated with the presence and progression of prostate cancer. Despite variance between patients, PSA levels are useful both in monitoring the progress of individual patients and as an indicator for diagnosing or staging prostate cancer (see, e.g., El-Shirbiny (1994)). Probasin is a prostate specific basic protein first isolated by Matuo et al. (1982) from rat dorsolateral prostate. A cDNA to the rat probasin was disclosed by Spence et al. (1989) and revealed two in-frame translation initiation codons which are believed to account for the secreted and nuclear forms ofthe protein. The physiological role of probasin is unknown but it is a heparin binding protein that co-purifies with heparin binding growth factor- 1 (HBGF-1) and is positively regulated by androgen. Probasin appears to have minor mitogenic activity (0.2-1% of HBGF-1) but this may be an artifact of its co-purification with HBGF-1 (Matuo et al. (1989)).

Summary of the Invention

The present invention provides methods for treating a patient diagnosed as having benign prostatic hyperplasia or a prostatic cancer. The methods include administering to the patient a therapeutically effective amount of a composition comprising an antisense oligonucleotide which selectively hybridizes to a PSA or probasin gene or mRNA sequence ofthe patient, thereby inhibiting the expression ofthe PSA or probasin gene or mRNA sequence. This inhibition of the PSA or probasin genes or mRNAs by antisense oligonucleotides results in a significant inhibition ofthe growth or survival of prostatic cells. As a result, the methods provide a useful new means of treating benign prostatic hyperplasia and prostatic cancer.

The PSA antisense oligonucleotides may comprise at least 10 consecutive bases from SEQ ID NO.: 1, at least 10 consecutive bases from the joined exons of SEQ ID NO.: 1; or oligonucleotides that hybridize to the complements of these sequences under physiological conditions. More preferably, the antisense oligonucleotides comprise at least 15 consecutive bases, and most preferably, 20-30 consecutive bases from the above-described sequences.

The probasin antisense oligonucleotides may comprise at least 10 consecutive bases from SEQ ID NO.: 2, at least 10 consecutive bases from a genomic sequence corresponding to SEQ ID NO.: 2, or oligonucleotides that hybridize to the complements of these sequences under physiological conditions. More preferably, the antisense oligonucleotides comprise at least 15 consecutive bases, and most preferably, 20-30 consecutive bases from the above-described sequences.

Examples of sequences ofthe invention include, but are not limited to, those disclosed as SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, and SEQ ID NO.: 8. In preferred embodiments, all ofthe above-described oligonucleotides are modified oligonucleotides. In one set of embodiments, the modified oligonucleotide includes at least one synthetic internucleoside linkage such as a phosphorothioate, alkylphosphonate, phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, or carboxymethyl ester. In other embodiments with modified oligonucleotides, the modified oligonucleotide has at least one low molecular weight organic group covalently bound to a phosphate group of said oligonucleotide. In another set of embodiments, the modified oligonucleotide has at least one low molecular weight organic group covalently bound to a 2' position of a ribose of said oligonucleotide. Such low molecular weight organic groups include lower alkyl chains or aliphatic groups (e.g., methyl, ethyl, propyl, butyl), substituted alkyl and aliphatic groups (e.g., aminoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), small saccharides or glycosyl groups.

In another set of embodiments the modified oligonucleotide has covalently attached thereto a prostate-targeting compound such as an androgen, androgen derivative, estrogen, estrogen derivative, estramustine, emcyt or estracyt. In preferred embodiments, the antisense oligonucleotides are administered intravenously at a dosage between 1.0 μg and 100 mg per kg body weight ofthe patient.

The present invention also provides for any or all ofthe above-described antisense oligonucleotides, including the various modified oligonucleotides, in a pharmaceutical composition. The antisense oligonucleotides are admixed with a sterile pharmaceutically acceptable carrier in a therapeutically effective amount such that the isolated antisense oligonucleotide selectively hybridizes to the PSA or probasin gene or mFtNA sequence when administered to a patient. A pharmaceutical kit is also provided in which such a pharmaceutical composition is combined with a pharmaceutically acceptable carrier for intravenous administration.

Detailed Description of the Invention The present invention provides new methods for the treatment of cancer ofthe prostate and pharmaceutical compositions useful therefor. It is now disclosed that antisense oligonucleotides complementary to genes which are expressed only or predominantly in prostatic cells are effective for inhibiting the growth of and/or killing hypeφlastic or cancerous cells of prostatic origin. In particular, the present invention provides oligonucleotides, including modified oligonucleotides, which have antisense homology to a sufficient portion of either the PSA or probasin gene such that they inhibit the expression of that gene. The expression of both of these genes is believed to be tissue specific to the prostate. Suφrisingly, inhibition of either of these genes, both of which encode secreted proteins with no known function within prostate cells, inhibits the growth of these cells. Because the antisense oligonucleotides ofthe invention can be administered systemically but selectively inhibit prostate cells, the present invention has particular utility in late stage prostate cancer which has metastasized.

Definitions

In order to describe more clearly and concisely the subject matter ofthe present invention, the following definitions are provided for specific terms used in the claims appended hereto:

PSA. As used herein, the abbreviation "PSA" refers to the prostatic specific antigen well known in the art and described in the various references cited herein. Genomic DNA sequences ofthe human PSA gene were disclosed in Lundwall (1989) and Klobeck et al. (1989). The Klobeck et al. (1989) sequence is available on GenBank (Accession number X14810) and is reproduced here as SEQ. ID NO.: 1. The translation initiation codon of this gene is found at base positions 401-403 and the stop codon is at positions 5566-5568 of SEQ ID NO.: 1. The gene consists of five exons which are indicated on SEQ. ID NO.: 1. A TATA signal is found at positions 332-338 and a transcriptional start region appears at positions 355-365. As will be obvious to one of ordinary skill in the art, other alleles ofthe PSA gene, including other human alleles and homologues from other mammalian species, encoding a PSA protein and hybridizing to SEQ ID NO.: 1 under stringent hybridization conditions, will exist in natural populations and are embraced by the term "PSA gene" as used herein. A slightly different sequence for PSA is also available on GenBank (Acession number M27274). The PSA gene ofthe invention is intended to encompass all such sequences.

Probasin. As used herein, the term "probasin" refers to the probasin protein known in the art and described in the various references cited herein. A cDNA to one allele ofthe rat probasin gene was disclosed in Spence, et al. (1989). The Spence et al. (1989) sequence is available on GenBank (Accession number M27156) and is reproduced here as SEQ. ID NO.: 2. This gene has two potential translation initiation start codons which are in frame with each other. The first is at positions 41-43 of SEQ ID NO. 2 and the second is at positions 92-94. The stop codon is at positions 572-574. The bases between the first and second initiation codons encode a hydrophobic sequence consistent with a secretory signal sequence. Thus, it is believed that the initiation of translation from the first start codon leads to production ofthe secreted form of probasin whereas translation from the second results in the nuclear form ofthe protein. As used herein, the term "probasin gene" is specifically intended to include a gene encoding either or both forms ofthe probasin protein. In addition, as will be obvious to one of ordinary skill in the art, other alleles ofthe probasin gene, including other human alleles and homologues from other mammalian species, encoding a probasin protein and hybridizing to SEQ ID NO.: 2 under stringent hybridization conditions, will exist in natural populations and are embraced by the term "probasin gene" as used herein.

Antisense Oligonucleotides. As used herein, the term "antisense oligonucleotide" or "antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA. In particular, by a "PSA-antisense oligonucleotide" and by a "probasin-antisense oligonucleotide" are meant oligonucleotides which hybridize under physiological conditions to the PSA gene/mRNA or probasin gene/mRN A and, thereby, inhibit transcription/translation ofthe PSA and probasin genes/mRNAs, respectively. The antisense molecules are designed so as to interfere with transcription or translation of PSA or probasin upon hybridization with the target. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity will depend upon the specific target selected, including the sequence ofthe target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be selected so as to hybridize selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Stringent hybridization conditions. As used herein, the term "stringent hybridization conditions" means hybridization conditions from 30°C-60°C and from 5x to 0.1 x SSC. Highly stringent hybridization conditions are at 45 °C and O.lx SSC. "Stringent hybridization conditions" is a term of art understood by those of ordinary skill in the art. For any given nucleic acid sequence, stringent hybridization conditions are those conditions of temperature and buffer solution which will permit hybridization of that nucleic acid sequence to its complementary sequence and not to substantially different sequences. The exact conditions which constitute "stringent" conditions, depend upon the length ofthe nucleic acid sequence and the frequency of occurrence of subsets of that sequence within other non-identical sequences. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, one of ordinary skill in the art can, without undue experimentation, determine conditions which will allow a given sequence to hybridize only with identical sequences. Suitable ranges of such stringency conditions are described in Krause, M.H.. and S.A. Aaronson, Methods in Enzymology. 200:546-556 (1991). As used herein with respect to in vivo hybridization conditions, the term "physiological conditions" is considered functionally equivalent to the in vitro stringent hybridization conditions. I. Design of PSA and Probasin Antisense Oligonucleotides The present invention depends, in part, upon the discovery that the selective inhibition of the expression of PSA or probasin by antisense oligonucleotides in prostatic cells effectively inhibits cell growth and/or causes cell death.

Based upon SEQ ID NO.: 1 and SEQ ID NO.: 2, or upon allelic or homologous genomic or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for PSA or probasin inhibition, such antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the PSA or probasin mRNA transcripts. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. Although oligonucleotides may be chosen which are antisense to any region ofthe PSA or probasin genes or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N- terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3'-untranslated regions or telomerase sites may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the PSA or probasin antisense is, preferably, targeted to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al. (1994)) and at which proteins are not expected to bind. With respect to probasin, an N-terminal antisense oligonucleotide may be targeted to either the first or the second initiation codon so as to interfere with translation of both forms or just the secreted form of probasin. Finally, although, SEQ ID NO.: 1 discloses a genomic DNA sequence and SEQ ID NO.: 2 discloses a cDNA sequence, one of ordinary skill in the art may easily derive the cDNA corresponding to the joined exons of SEQ ID NO.: 1 and may easily obtain the genomic DNA sequence corresponding to SEQ ID NO.: 2. Thus, the present invention also provides for antisense oligonucleotides which are complementary to the cDNA corresponding to SEQ ID NO.: 1 and the genomic DNA corresponding to SEQ ID NO.: 2. Similarly, antisense to allelic or homologous cDNAs and genomic DNAs are enabled without undue experimentation. As will be understood by one of ordinary skill in the art, the antisense oligonucleotides of the present invention need not be perfectly complementary to the PSA or probasin genes or mRNA transcripts in order to be effective. Rather, some degree of mismatches will be acceptable if the antisense oligonucleotide is of sufficient length. In all cases, however, the oligonucleotides should have sufficient length and complementarity so as to hybridize to a PSA or probasin transcript under physiological conditions. Preferably, of course, mismatches are absent or minimal. In addition, although it is not recommended, the antisense oligonucleotides may have one or more non-complementary sequences of bases inserted into an otherwise complementary antisense oligonucleotide sequence. Such non-complementary sequences may "loop" out of a duplex formed by a PSA or probasin transcript and the bases flanking the non- complementary region. Therefore, the entire oligonucleotide may retain an inhibitory effect despite an apparently low percentage of complementarity. Of particular importance in this respect is the use of self-stabilized or haiφin oligonucleotides. Such oligonucleotides, or modified oligonucleotides, have a sequence at the 5' and/or 3' end which is capable of folding over and forming a duplex with itself. The duplex region, which is preferably at least 4-6 bases joined by a loop of 3-6 bases, stabilizes the oligonucleotide against degradation. These self- stabilized oligonucleotides are easily designed by adding the inverted complement of a 5' or 3' PSA or probasin sequence to the end ofthe oligonucleotide (see, e.g., Table 1, SEQ ID NO.: 5; Tang, J.-Y., et al. (1993) Nucleic Acids Res. 21 :2729-2735). In one set of embodiments, the PSA and probasin antisense oligonucleotides ofthe invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one nucleotide and the 3' end of another nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer.

In preferred embodiments, however, the antisense oligonucleotides ofthe invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting to prostatic cells or which otherwise enhance their therapeutic effectiveness. The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.

Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. Further, one or more of the 5'-3' phosphate group may be covalently joined to a low molecular weight (e.g., 15-500 Da) organic group. Such low molecular weight organic groups include lower alkyl chains or aliphatic groups (e.g., methyl, ethyl, propyl, butyl), substituted alkyl or aliphatic groups (e.g., aminoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), a small saccharides or glycosyl groups. Other low molecular weight organic modifications include additions to the internucleoside phosphate linkages such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose. Oligonucleotides with these linkages or other modifications can be prepared according to known methods (see, e.g., Agrawal and Goodchild (1987); Agrawal et al. (1988); Uhlmann et al. (1990); Agrawal et al. (1992); Agrawal (1993); and U.S. Pat. No. 5,149,798).

The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus modified oligonucleotides may include a 2'-O-alkylated ribose group such as a 2'-O-methylated ribose. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. Alternatively, the modified oligonucleotides may be branched oligonucleotides. Unoxidized or partially oxidized oligonucleotides having a substitution in one or more nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides.

Also considered as modified oligonucleotides are oligonucleotides having prostate- targeting, nuclease resistance-conferring, or other bulky substituents and/or various other structural modifications not found in vivo without human intervention. The androgen receptor and other hormonal receptor sites on prostate cells allow for targeting antisense oligonucleotides specifically or particularly to prostatic cells. Attachment ofthe antisense oligonucleotides by a molecular "tether" (e.g., an alkyl chain) to estramustine, emcyt or estracyt (Sheridan and Tew (1991)), for example, may provide prostatic targeting and the possibility of covalent alkylation of host prostatic DNA. Estramustine targets particularly to the ventral prostate (Forsgren, et al. (1979)). Similarly, one may covalently attach androgen, estrogen, androgen or estrogen derivatives, or other prostate cell ligands to antisense oligonucleotides using tethers and conjugating linkages for prostatic targeting. Finally, one may of course covalently attach other chemotherapeutic agents (e.g., dexamethasone, vinblastine, etoposide) to the antisense oligonucleotides for enhanced effect.

The most preferred modified oligonucleotides are hybrid or chimeric oligonucleotides in which some but not all ofthe phosphodiester linkages, bases or sugars have been modified. Hybrid modified antisense oligonucleotides may be composed, for example, of stretches often 2'-O-alkyl nucleotides or ten phosphorothioate synthetic linkages at the 5' and/or 3' ends, and a segment of seven unmodified oligodeoxynucleotides in the center, or of similar terminal segments of alkyl phosphonates, with central P=S or P=O oligonucleotides (Agrawal, et al. (1990); Metelev, et al. (1994)). The currently most preferred modified oligonucleotides are 2'-O- methylated hybrid oligonucleotides. Since degradation occurs mainly at the 3' end, secondarily at the 5' end, and less in the middle, unmodified oligonucleotides located at this position can activate RNase H. and yet are degraded slowly. Furthermore, the T_m of such a 27-mer is approximately 20 °C higher than that of a 27-mer all phosphorothioate oligodeoxynucleotide. This greater affinity for the targeted genomic area can result in greater inhibiting efficacy.

Obviously, the number of synthetic linkages at the termini need not be ten and synthetic linkages may be combined with other modifications, such as alkylation of a 5' or 3' phosphate, or 2'-O- alkylation. Thus, merely as another example, one may produce a modified oligonucleotide with the following structure, where B represents any base, R is an alkyl, aliphatic or other substituent, the subscript S represents a synthetic (e.g. phosphorothioate) linkage, and each n is an independently chosen integer from 1 to about 20:

OH

⁵(B_s)_nBBBB- ... -BBBB(B_s)_nB— P=O³ I

O— R II. Products and Methods of Treatment for BPH and Prostate Cancer

The methods ofthe present invention represent new and useful additions to the field of benign prostate hypeφlasia or prostate cancer therapy. In particular, the methods ofthe present invention are especially useful for late stage prostate cancer in which metastases have occurred and in which the cells have become resistant to estrogen or anti-androgen therapy. The methods may, however, also be used in benign prostate hypeφlasia or early stage prostate cancer and may provide a substitute for more radical procedures such as transurethral resection, radical prostatectomy, or physical or chemical castration. The products ofthe present invention include the isolated antisense oligonucleotides described above. As used herein, the term "isolated" as applied to an antisense oligonucleotide means not covalently bound to and physically separated from the 5' and 3' sequences which flank the corresponding antisense sequence in nature.

Admimstration ofthe PSA or probasin antisense oligonucleotides may be oral, intravenous, parenteral, cutaneous or subcutaneous. For BPH or when the site of a prostatic tumor is known, the admimstration also may be localized to the prostate or to the region ofthe tumor by injection to or perfusion of the site.

PSA or probasin antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount ofthe antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically acceptable" means a non¬ toxic material that does not interfere with the effectiveness ofthe biological activity ofthe active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics ofthe carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art. The pharmaceutical composition ofthe invention may also contain other active factors and/or agents which inhibit prostate cell growth or increase cell death. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect or to minimize side- effects caused. The pharmaceutical composition ofthe invention may be in the form of a liposome in which the PSA or probasin antisense oligonucleotides are combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which are in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871 ; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323. The pharmaceutical composition ofthe invention may further include compounds such as cyclodextrins and the like which enhance delivery of oligonucleotides into cells. When the composition is not administered systemically but, rather, is injected at the site ofthe target cells, cationic detergents (e.g. Lipofectin) may be added to enhance uptake.

When a therapeutically effective amount of PSA or probasin antisense oligonucleotides is administered orally, the oligonucleotides will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition ofthe invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder may contain from about 5 to 95% ofthe PSA and/or probasin antisense oligonucleotides and preferably from about 25 to 90% ofthe oligonucleotides. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, sesame oil, or synthetic oils may be added. The liquid form ofthe pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition may contain from about 0.5 to 90% by weight of a PSA and/or probasin antisense oligonucleotide and preferably from about 1 to 50% ofthe oligonucleotide.

When a therapeutically effective amount of a PSA or probasin antisense oligonucleotide is administered by intravenous, cutaneous or subcutaneous injection, the oligonucleotides will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the antisense oligonucleotides, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or another vehicle as known in the art. The pharmaceutical composition ofthe present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

In preferred embodiments, when the target cells are readily accessible, administration of the antisense oligonucleotides is localized to the region ofthe targeted cells in order to maximize the delivery ofthe antisense and to minimize the amount of antisense needed per treatment. Thus, in one preferred embodiment, administration is by direct injection at or perfusion ofthe site ofthe targeted cells, such as a tumor. Alternatively, the antisense oligonucleotides may be adhered to small particles (e.g., microscopic gold beads) which are impelled through the membranes ofthe target cells (see, e.g., U.S. Pat. No. 5,149,655).

In another series of embodiments, a recombinant gene is constructed which encodes a PSA or probasin antisense oligonucleotide and this gene is introduced within the targeted cells on a vector. Such a PSA or probasin antisense gene may, for example, consist of the normal PSA or probasin sequence, or a subset ofthe normal sequences, operably joined in reverse orientation to a promoter region. An operable antisense gene may be introduced on an integration vector or may be introduced on an expression vector. In order to be most effective, it is preferred that the antisense sequences be operably joined to a strong eukaryotic promoter which is inducible or constitutively expressed.

In all ofthe above-described methods of treatment, the PSA and/or probasin antisense oligonucleotides are administered in therapeutically effective amounts. As used herein, the term "therapeutically effective amount" means that amount of antisense which, under the conditions of administration, including mode of administration and presence of other active components, is sufficient to result in a meaningful patient benefit, i.e., the killing or inhibition ofthe growth of target cells.

The amount of PSA and/or probasin antisense oligonucleotides in the pharmaceutical composition ofthe present invention will depend not only upon the potency ofthe antisense but also upon the nature and severity ofthe condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of antisense with which to treat each individual patient. Initially, the attending physician will administer low doses ofthe inhibitor and observe the patient's response. Larger doses of antisense may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. In preferred embodiments, it is contemplated that the various pharmaceutical compositions used to practice the method ofthe present invention should contain about 1.0 μg to about 100 mg of oligonucleotide per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositions ofthe present invention will vary, depending on the severity ofthe disease being treated and the condition and potential idiosyncratic response of each individual patient. Because a bolus of oligonucleotides, particularly highly negatively-charged phosphorothioate modified oligonucleotides, may have adverse side effects (e.g., rapid lowering of blood pressure), slow intravenous administration is preferred. Thus, intravenous administration of therapeutically effective amounts over a 12-24 hour period are contemplated. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention. The following examples ofthe use of PSA and probasin antisense are presented merely to illustrate some ofthe oligonucleotides, including modified oligonucleotides, that may be employed according to the present invention. The particular oligonucleotides used, therefore, should not be construed as limiting ofthe invention but, rather, as indicative ofthe wide range of oligonucleotides which may be employed. As will be obvious to one of ordinary skill in the art in light ofthe present disclosure, a great many equivalents to the presently disclosed antisense oligonucleotides and disclosed methods are now available. In particular, other antisense oligonucleotides substantially complementary to subsets of SEQ ID NO.: 1 or SEQ ID NO.: 2, and chemical modifications ofthe same which do not prevent hybridization under physiological conditions, are contemplated as equivalents ofthe examples presented below. In general, the use of prostate specific antisense oligonucleotides is contemplated as a method of selectively inhibiting the growth of or killing prostatic cells.

Experimental Examples

Three permanent cell lines of human prostatic cancer were grown in monolayer culture:

LNCaP, PC3-1435, and DU145, all obtained from the American Type Culture Collection. The LNCaP cells grow as stellate cells in a monolayer, retain hormone sensitivity and, of particular importance, secrete PSA into the tissue culture medium (Oesterling (1995)). Cells were grown in Dulbecco's medium supplemented with 10 percent fetal calf serum, glutamate, pyruvate, penicillin and streptomycin, in 25-150 cm flasks, incubated at 37 °C in 6 percent CO -air.

A number of PSA and probasin antisense oligonucleotides were tested for their inhibitory effect on prostatic cells. The base sequences of these oligonucleotides are disclosed as SEQ ID NO. : 3 through SEQ ID NO. : 9. SEQ ID NO. : 3 is antisense to positions 92- 118 of the probasin gene (SEQ ID NO.: 1). SEQ ID NO.: 4 is antisense to a region upstream ofthe probasin gene at positions 76-99. SEQ ID NO.: 5 is a self-stabilized or haiφin oligonucleotide. The first 21 bases are complementary to positions 80-100 ofthe probasin gene. The remaining eight are identical to positions 84-91 ofthe gene, allowing formation of a 3' haiφin. SEQ ID NO.: 6 is another self-stabilized antisense oligonucleotide. The first 21 bases of this oligonucleotide are complementary to positions 92-112 ofthe probasin gene. The remaining eight are identical to positions 96-103 ofthe gene, allowing for formation of a 3' haiφin. SEQ ID NO.: 7 and SEQ ID NO.: 8 are antisense sequences corresponding to positions 401-427 and 384-410 ofthe PSA gene. Table 1 shows some ofthe antisense oligonucleotides tested. The numbers at the left of each sequence correspond to the sequence numbers in the sequence listing. Antisense oligonucleotides with unmodified or natural internucleoside linkages (P=O) and oligonucleotides with all phosphorothioate synthetic linkages (P=S) were tested. In addition, modified oligonucleotides were tested in which just the terminal two phosphodiester linkages at each end had been replaced by phosphorothioate synthetic linkages (shown as a subscript S between nucleotides in Table 1) and/or in which small aliphatic chemical groups (e.g., 2-hydroxy-3- amino-propyl) were added to the 3' terminal phosphate.

Growth ofthe PC3-1435 cell line in tissue culture monolayers was consistently inhibited by addition of phosphorothioate-modified oligodeoxynucleotides targeted against the PSA or probasin genes and incubation for 24-48 hours thereafter. As the concentration of modified oligonucleotides is decreased from the 10-20 μM level, most effective inhibition occurs with specific antisense oligodeoxynucleotides at the 2-5 μM level, as contrasted with mismatched oligodeoxynucleotides (see Tables 2 and 3).

While the effects on cell growth (i.e. cell numbers) are readily manifest, visual substage microscopy of wells revealed additional features ofthe inhibition events using PSA antisense oligonucleotides against PC3-1435 cells. The first evidence of antisense inhibition is rupture of the monolayer fabric. The stellate cells in a confluent culture lose contact with their neighbors, round up individually or in clumps, become pyknotic, and cease growing, as examined on successive days. There is an early loss of adhesiveness to the floor ofthe plastic wells. These changes are more severe (see Table 4) than those measured by ³H-thymidine incoφoration into DNA, in other words more drastic than the impairment of DNA synthesis.

PSA protein was measured in the tissue culture medium in which LNCaP cells were grown in multi-well plates. This provided a quantitative assay, using a ^l25I-anti-PSA labeled antibody sandwich assay. We measured PSA levels in tissue cultures in as little as 2.5 μl of incubation medium by means ofthe iodinated antibody ¹²⁵I-technique. The labeled antibody is available from Hybritech (San Diego, CA). Table 5 shows the results from two negative controls (no treatment), two positive controls employing an arbitrary oligonucleotide which is antisense to a portion ofthe HIV genome, and the SEQ ID NO.: 8 modified oligonucleotide of Table 1. Using this sandwich assay, the PSA output into the tissue culture medium of LNCaP cells was reduced 51 percent at the 5 μM level, and 45 percent at the 2.5 μM level at a 48 hour time period when the PSA gene was targeted by antisense oligonucleotides.

Each ofthe above-mentioned references and patents is hereby incoφorated by reference.

TABLE 1 Antisense Oligonucleotides

Sequence Target

#3 ^s ' CTT -TTT -GAG -ATT - CTT -GTC -TGT - CAT - CAT³ ' Probasin, P=S #3 ⁵ ' CTT - TTT - GAG -ATT - CTT -GTC -TGT - CAT- CAT³ ' Probasin, P=0 #4 ⁵ 'GTC-ATC-ATA-CTG-GAG-ACA- CCT-AGC³ ' Probasin- upstream, P=S

#5 ⁵'TGT-CAT-CAT-ACT-GGA-GAC-ACC-TCT-CCA-GT³ Probasin 3 'end hairpin, P=S

#6 ⁵ 'GAG-ATT- CTT-GTC-TGT-CAT- CAT-TGA- CAG-AC³ ' Probasin 3 ' end hairpin, P=S #7 ⁵ ' GGT -GAG -GAA -GAC -AAC - CGG -GAC - CCA - CAT³ ' PSA, P=S #8 ⁵'GGA- CCC-ACA-TGG-TGA- CAC-AGC-TCT- CCG³ ' PSA, P=S

77418.1 TABLE 2

³H-thymidine incorporation into DNA PC3-1435 human prostate cancer tissue culture

Gene? Targeted Concentration (μM. CPMt % inhibition

Control (no oligo) — 38,000 0 Probasin (P = S) 20 13,700 64

5 18,000 52

Mismatch (P = S) 20 20,000 47

5 27,000 30 t Averages of 3 separate wells

TABLE 3

Comparisons of degree of inhibition of DNA synthesis in human PC3-1435 prostate cancer tissue cultures for one target and a mix of targets

Genes targeted Concentration f μM) CPM t % inhibition

Control (ho oligo) -- 14,700 0

Mismatch 20 6,990 51

5 10,750 27

Mix* 20 4,930 66

5 6,054 59 * Mix: Probasin, PSA. PSA upstream, PSA farther upstream. 5 μM each at 20 μM total; 1.25 μM each at 5 μM total. t Averages of 3 separate wells. TABLE 4 Morphological Comparison of Treated and Control Cells

Concentration μM

Gene Target 20 10 5 2

PSA gene (P=S) 4+ 3+ 2-1/2+ 1 +

Mismatch (P=S) 1-1/2+ 1/2+ 0 0

Observation 24 hours after oligonucleotide addition. Damage: 4+ devastating; 3+ severe; 2+ serious; 1+ visible; 1/2+ slight; 0 none

TABLE 5

PSA Levels in Media of Cultured LNCaP Cells 24 hours after

Oligodeoxynucleotide Phosphorothioate Treatment

Sample No. Description CPM PSA (ng/ml)

1 No treatment 1 1,940 33

2 No treatment 1 1,389 31

3 + Control 5 μM 8,31 1 23

4 + Control 1 μM 8,892 25

5 PSA antisense 5 μM 5,765 16

6 PSA antisense 1 μM 6,375 17

References

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Agrawal and Goodchild (1987) Tetrahedron Lett. 28:3539-3542.

Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA . 85:7079-7083. Agrawal et al.(1990) Proc. Natl. Acad. Sci. (USA1 87: 1401-1405.

Agrawal et al. (1992 , Trends Biotechnol. 10:152-158.

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Lundwall (19891 Biochem. Biophvs. Res. Commun. 161 (31:1 151-1 159.

Matuo et al. (1982) Biochem. Biophvs. Res. Commun. 109:334-340. Matuo et al. (1989) In Vitro Cell. Devel. Biol. 25(6):581-584.

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Oesterling (1995) Michigan One, J, 6:8-12.

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Pienta (1995) Michigan One, J, 6:4-7. Sainio et al. (1994) Cell. Mol. Neurobiol. 14(5):439-457.

Sheridan and Tew (1991 ) Cancer Surveys 11 :239-254.

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Spence et al. (1989) Proc. Natl. Acad. Sci. (USA1 86:7843-7847.

Uhlmann et al. (1990) Chem. Rev. 90:534-583. Wang et al. (19791 Invest. Urol. 17:159-163.

Warhol and Logtine (1985) J. Urol. 134:607. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: WORCESTER FOUNDATION FOR BIOLOGICAL RESEARCH INC.

(ii) TITLE OF INVENTION: ANTISENSE OLIGONUCLEOTIDE CHEMOTHERAPY FOR BENIGN HYPERPLASIA OR CANCER OF THE PROSTATE

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: WOLF, GREENFIELD & SACKS, P.C.

(B) STREET: 600 ATLANTIC AVENUE (C) CITY: BOSTON

(D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 02210

(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: TWOMEY, MICHAEL J.

(B) REGISTRATION NUMBER: 38,349

(C) REFE___NCE/DOCKET NUMBER: W0461/7029

(ix) ____ECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-720-3500

(B) TELEFAX: 617-720-2441

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5873 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: HOMO SAPIENS

(G) CELT, TYPE: LYMPHOID (H) CELL LINE: GM 607

(ix) FEATURE: (A) NAME/KEY: TATA_signal

(B) LOCATION: 332.-338 ( ix) FEATURE :

(A) NAME/KEY: misc_signal

(B) LOCATION: 355..365

(D) OTHER INFORMATION: /note= "TRANSCRIPTIONAL START REGION"

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 401..446

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 1688..1847

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 3477..3763

(ix) FEATURE: (A) NAME/KEY: exon

(B) LOCATION: 3907..4043

(ix) FEATURE:

(A) NAME/KEY: exon (B) LOCATION: 5413..5568

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(401..446, 1688..1847, 3477..3763, 3907..4043, 5413..5568) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GG-GTCTTAG GCA(_ACTGGT CITGGAGTGC AAAGGATCTA GGCACGTGAG GCTTTGTATG 60

AAGAATCGGG GATCGTACCC ACCCCCTGTT TCTGTITCAT CCTGGGCATG TCTCCTCTGC 120

CTTTGTCCCC TAGATGAAGT CTCCATGAGC TACAAGGGCC TGGTGCATCC AGGGTGATCT 180

AGTAATTGCA GAACAGCAAG TGCTAG TCT CCCTCCCCTT CCACAGCTCT GGGTGTGGGA 240

GGGGGTTGTC CAGCCTCCAG CAGCATGGGG AGGGCCTTGG TCAGCCTCTG GGTGCCAGCA 300

GGGCAGGGGC GGAGTCCTGG GGAATGAAGG TTTTATAGGG CTCCTGGGGG AGGCTCCCCA 360

GCCCCAAGCT TACCACCTGC ACCCGGAGAG CTGTGTCACC ATG TGG GTC CCG GTT 415

Met Trp Val Pro Val 1 5

GTC TTC CTC ACC CTG TCC GTG ACG TGG ATT G GTGAGAGGGG CCATGGTTGG 466 Val Phe Leu Thr Leu Ser Val Thr Trp He

10 15

GGGGATGCAG GAGAGGGAGC CAGCCCTGAC TGTCAAGCTG AGGCTCTTTC CCCCCCAACC 526

CAGCACCCCA GCCCAGACAG GGAGCTGGGC TC TTTCTGT CTCTCCCAGC CCCACTTCAA 586

GCCCATACCC CCAGTCCCCT CCATATTGCA ACAGTCCTCA CTCCCACACC AGGTCCCCGC 646

TCCCTCCCAC TTACCCCAGA ACTTTCTTCC CATTTGCCCA GCCAGCTCCC TGCTCCCAGC 706

TGCTTTACTA AAGGGGAAGT TCCTGGGCAT CTCCXπ ITT CTCTTTGTGG GGCTCAAAAC 766 CTCCAAGGAC CTCTCTCAAT GCCATTGGTT CCTTGGACCG TATCACTGGT CCATCTCCTG 826

AGCCCCTCAA TCCTATCACA GTCTACTGAC TTTTCCCATT CAGCTGTGAG TGTCCAACCC 886

TATCCCAGAG ACCTTGATGC TTGGCCTCCC AATCTTGCCC TAGGATACCC AGATGCCAAC 946

CAGACACCTC CTTCTTTCCT AGCCAGGCTA TCTGGCCTGA GACAACAAAT GGGTCCCTCA 1006

GTCTGGCAAT GGGACTCTGA GAACTCCTCA TTCCCTGACT CTTAGCCCCA GACTCTTCAT 1066

TCAGTGGCCC ACATTTTCCT TAGGAAAAAC ATGAGCATCC CCAGCCACAA CTGCCAGCTC 1126

TCTGAGTCCC CAAATCTGCA TCCTTTTCAA AACCTAAAAA CAAAAAGAAA AACAAATAAA 1186

ACAAAACCAA CTCAGACCAG AACTGTTTTC TCAACCTGGG ACTTCCTAAA CTTTCCAAAA 1246

CCTTCCTCTT CCAGCAACTG AACCTCGCCA TAAGGCACTT ATCCCTGGTT CCTAGCACCC 1306

CTTATCCCCT CAGAATCCAC AACTTGTACC AAGTTTCCCT TCTCCCAGTC CAAGACCCCA 1366

AATCACCACA AAGGACCCAA TCCCCAGACT CAAGATATGG TCTGGGCGCT GTCTTGTGTC 1426

TCCTACCCTG ATCCCTGGGT TCAACTCTGC TCCCAGAGCA TGAAGCCTCT CCACCAGCAC 1486

CAGCCACCAA CCTGCAAACC TAGGGAAGAT TGACAGAATT CCCAGCCTTT CCCAGCTCCC 1546

CCTGCCCATG TCCCAGGACT CCCAGCCTTG GTTCTCTGCC CCCGTGTCTT TTCAAACCCA 1606

CATCCTAAAT CCATCTCCTA TCCGAGTCCC CCAGTTCCCC CTGTCAACCC TGATTCCCCT 1666 GATCTAGCAC CCCCTCTGCA G GC GCT GCG CCC CTC ATC CTG TCT CGG ATT 1716

Gly Ala Ala Pro Leu He Leu Ser Arg He

20 25

GTG GGA GGC TGG GAG TGC GAG AAG CAT TCC CAA CCC TGG CAG GTG CTT 1764 Val Gly Gly Trp Glu Cys Glu Lys His Ser Gin Pro Trp Gin Val Leu

30 35 40

GTG GCC TCT CGT GGC AGG GCA GTC TGC GGC GGT GTT CTG GTG CAC CCC 1812 Val Ala Ser Arg Gly Arg Ala Val Cys Gly Gly Val Leu Val His Pro 45 50 55

CAG TGG GTC CTC ACA GCT GCC CAC TGC ATC AGG AA GTGAGTAGGG 1857 Gin Trp Val Leu Thr Ala Ala His Cys He Arg Asn 60 65

GCCTGGGGTC TGGGGAGCAG GTGTCTGTGT CCCAGAGGAA TAACAGCTGG GCATTTTCCC 1917

CAGGATAACC TCTAAGGCCA GCCTTGGGAC TGGGGGAGAG AGGGAAAGTT CTGGTTCAGG 1977

TCACATGGGG AGGCAGGGTT GGGGCTGGAC CACCCTCCCC ATGGCTGCCT GGGTCTCCAT 2037

CTGTGTCCCT CTATGTCTCT TTGTGTCGCT TTCATTATGT CTCTTGGTAA CTGGCTTCGG 2097

TTGTGTCTCT CCGTGTGACT ATTTTGTTCT CTCTCTCCCT CTCTTCTCTG TCTTCAGTCT 2157

CCATATCTCC CCCTCTCTCT GTCCTTCTCT GGTCCCTCTC TAGCCAGTGT GTCTCACCCT 2217

GTATCTCTCT GCCAGGCTCT GTCTCTCGGT CTCTGTCTCA CCTGTGCCTT CTCCCTACTG 2277

AACACACGCA CGGGATGGGC CTGGGGGACC CTGAGAAAAG GAAGGGCTTT GGCTGGGCGC 2337 GGTGGCTCAC ACCTGTAATC CCAGCACTTT GGGAGGCCAA GGCAGGTAGA TCACCTGAGG 2397

TCAGGAGTTC GAGACCAGCC TGGCCAACTG GTGAAACCCC ATCTCTACTA AAAATACAAA 2457

AAATTAGCCA GGCGTGGTGG CGCATGCCTG TAGTCCCAGC TACTCAGGAG CTGAGGGAGG 2517

AGAATTGCAT TGAACCTGGA GGTTGAGGTT GCAGTGAGCC GAGACCGTGC CACTGCACTC 2577

CAGCCTGGGT GACAGAGTGA GACTCCGCCT CAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2637

AGAAAAGAAA AGAAAAGAAA AGGAAGTGTT TTATCCCTGA TGTGTGTGGG TATGAGGGTA 2697

TGAGAGGGCC CCTCTCACTC CATTCCTTCT CCAGGACATC CCTCCACTCT TGGGAGACAC 2757

AGAGAAGGGC TGGTTCCAGC TGGAGCTGGG AGGGGCAATT GAGGGAGGAG GAAGGAGAAG 2817

GGGGAAGGAA AACAGGGTAT GGGGGAAAGG ACCCTGGGGA GCGAAGTGGA GGATACAACC 2877

TTGGGCCTGC AGGCAGGCTA CCTACCCACT TGGAAACCCA CGCCAAAGCC GCAT TACAG 2937

CTGAGCCACT CTGAGGCCTC CCCTCCCCGG CGGTCCCCAC TCAGCTCCAA AGTCTCTCTC 2997

CCTTTTCTCT CCCΑCACTTT ATCATCCCCC GGATTCCTCT CTACTTGGTT CTCATTCTTC 3057

CTTTGACTTC CTGCTTCCCT TTCTCATTCA TCTGTTTCTC ACTTTCTGCC TGGTTTTGTT 3117

CTTCTCTCTC TCTTTCTCTG GCCCATGTCT GTTTCTCTAT GTTTCTGTCT TTTCTTTCTC 3177

ATCCTGTGTA TTTTCGGCTC ACCITGTTTG TCACTGTTCT CCCCTCTGCC CTTTCATTCT 3237

CTCTGCCCTT TTACCCTCTT CCTTTTCCCT TGGTTCTCTC AGTTCTGTAT CTGCCCTTCA 3297 CCCTCTCACA CTGCTGTTTC CCAACTCGTT CTCTGTATTT TGGCCTGAAC TGTGTCTTCC 3357

CAACCCTGTG TTTTCTCACT GTTTCTTTTT CTCTTTTGGA GCCTCCTCCT TGCTCCTCTG 3417

TCCCTTCTCT CTTTCCTTAT CATCCTCGCT CCTCATTCCT GO__CTGCTT CCTCCCCAGC 3477

AAA AGC GTG ATC TTG CTG GGT CGG CAC AGC CTG TTT CAT CCT GAA GAC 3525 Lys Ser Val He Leu Leu Gly Arg His Ser Leu Phe His Pro Glu Asp 70 75 80 85

ACA GGC CAG GTA TTT CAG GTC AGC CAC AGC TTC CCA CAC CCG CTC TAC 3573 Thr Gly Gin Val Phe Gin Val Ser His Ser Phe Pro His Pro Leu Tyr

90 95 100

GAT ATG AGC CTC CTG AAG AAT CGA TTC CTC AGG CCA GGT GAT GAC TCC 3621 Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser 105 110 115

AGC CAC GAC CTC ATG CTG CTC CGC CTG TCA GAG CCT GCC GAG CTC ACG 3669 Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu Thr 120 125 130

GAT GCT GTG AAG GTC ATG GAC CTG CCC ACC CAG GAG CCA GCA CTG GGG 3717 Asp Ala Val Lys Val Met Asp Leu Pro Thr Gin Glu Pro Ala Leu Gly 135 140 145

ACC ACC TGC TAC GCC TCA GGC TGG GGC AGC ATT GAA CCA GAG GAG T 3763 Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser He Glu Pro Glu Glu 150 155 160

GTACGCCTGG GCCAGATGGT GCAGCCGGGA GCCCAGATGC CTGGGTCTGA GGGAGGAGGG 3823 GACAGGACTC CTGGGTCTGA GGGAGGAGGG CCAAGGAACC AGGTGGGGTC CAGCCCACAA 3883

CAGTG'1____' GCCTGGCCCG TAG TC TTG ACC CCA AAG AAA CTT CAG TGT 3932

Phe Leu Thr Pro Lys Lys Leu Gin Cys 165 170

GTG GAC CTC CAT GTT ATT TCC AAT GAC GTG TGT GCG CAA GTT CAC CCT 3980

Val Asp Leu His Val He Ser Asn Asp Val Cys Ala Gin Val His Pro 175 180 185

CAG AAG GTG ACC AAG TTC ATG CTG TGT GCT GGA CGC TGG ACA GGG GGC 4028

Gin Lys Val Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly 190 195 200 205

AAA AGC ACC TGC TCG GTGAGTCATC CCTACTCCCA AGATCTTGAG GGAAAGGTGA 4083 Lys Ser Thr Cys Ser 210

GTGGGACCTT AATTCTGGGC TGGGGTCTAG AAGCCAACAA GGCGTCTGCC TCCCCTGCTC 4143

CCCAGCTGTA GCCATGCCAC CTCCCCGTGT CTCATCTCAT TCCCTCCTTC CCTCTTCTTT 4203

GACTCCCTCA AGGCAATAGG TTATTCTTAC AGCACAACTC ATCTGTTCCT GCGTTCAGCA 4263

CACGGTTACT AGGCACCTGC TATGCACCCA GCACTGCCCT AGAGCCTGGG ACATAGCAGT 4323

GAACAGACAG AGAGCAGCCC CTCCCTTCTG TAGCCCCCAA GCCAGTGAGG GGCACAGGCA 4383

GGAACAGGGA CCACAACACA GAAAAGCTGG AGGGTGTCAG GAGGTGATCA GGCTCTCGGG 4443

GAGGGAGAAG GGGTGGGGAG TGTGACTGGG AGGAGACATC CTGCAGAAGG TGGGAGTGAG 4503 CAAACACCTG CX3CAGGGGAG GGGAGGGCCT GCGGCACCTG GGGGAGCAGA GGGAACAGCA 4563

TCTGGCCAGG CCTGGGAGGA GGGGCCTAGA GGGCGTCAGG AGCAGAGAGG AGGTTGCCTG 4623

GCTGGAGTGA AGGATCGGGG CAGGGTGCGA GAGGGAACAA AGGACCCCTC CTGCAGGGCC 4683

TCACCTGGGC CACAGGAGGA CACTGCITIT CCTCTGAGGA GTCAGGAACT GTGGATGGTG 4743

CTGGACAGAA GCAGGACAGG GCCTGGCTCA GGTGTCCAGA GGCTGCGCTG GCCTCCTATG 4803

GGATCAGACT GCAGGGAGGG AGGGCAGCAG GGATGTGGAG GGAGTGATGA TGGGGCTGAC 4863

CTGGGGGTGG CTCCAGGCAT TGTCCCCACC TGGGCCCTTA CCCAGCCTCC CTCACAGGCT 4923

CCTGGCCCTC AGTCTCTCCC CTCCACTCCA TTCTCCACCT ACCCACAGTG GGTCATTCTG 4983

ATCACCGAAC TGACCATGCC AGCCCTGCCG ATGGTCCTCC ATGGCTCCCT AGTGCCCTGG 5043

AGAGGAGGTG TCTAGTCAGA GAGTAGTCCT GGAAGGTGGC CTCTGTGAGG AGCCACGGGG 5103

ACAGCATCCT GCAGATGGTC CTGGCCCTTG TCCCACCGAC CTGTCTACAA GGACTGTCCT 5163

CGTGGACCCT CCCCTCTGCA CAGGAGCTGG ACCCTGAAGT CCCTTCCTAC CGGCCAGGAC 5223

TGGAGCCCCT ACCCCTCTGT TGGAATCCCT GCCCACCTTC TTCTGGAAGT CGGCTCTGGA 5283

GACATTTCTC TCTTCTTCCA AAGCTGGGAA CTGCTATCTG TTATCTGCCT GTCCAGGTCT 5343

GAAAGATAGG ATTGCCCAGG CAGAAACTGG GACTGACCTA TCTCACTCTC TCCCTGCTTT 5403

TACCCTTAG GGT GAT TCT GGG GGC CCA CTT GTC TGT AAT GGT GTG CTT 5451 Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu 215 220

CAA GGT ATC ACG TCA TGG GGC AGT GAA CCA TGT GCC CTG CCC GAA AGG 5499 Gin Gly He Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg 225 230 235

CCT TCC CTG TAC ACC AAG GTG GTG CAT TAC CGG AAG TGG ATC AAG GAC 5547 Pro Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp He Lys Asp 240 245 250 255

ACC ATC GTG GCC AAC CCC TGAGCACCCC TATCAAGTCC CTATTGTAGT 5595

Thr He Val Ala Asn Pro 260

AAACTTGGAA CCTTGGAAAT GACCAGGCCA AGACTCAAGC CTCCCCAGTT CTACTGACCT 5655

TTGTCCTTAG GTGTGAGGTC (_AGGGTTGCT AGGAAAAGAA ATCAGCAGAC ACAGGTGTAG 5715

ACCAGAGTGT TTCTTAAATG GTGTAATTTT GTCCTCTCTG TCTCCTGGGG AATACTGGCC 5775

ATGCCTGGAG ACATATCACT CAATTTCTCT GAGGACACAG TTAGGATGGG GTGTCTGTGT 5835

TATTTGTGGG ATACAGAGAT GAAAGAGGGG TGGGATCC 5873

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 776 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: mRNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM : RATTUS NORVEGICUS

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 41..574

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 41..91

(ix) FEATURE:

(A) NAME/KEY: tτιat_peptide

(B) LOCATION: 92..571

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 :

CTTGTCAGTG AGGTCCAGAT ACCTACAGAG CTCACACACG ATG AGG GTC ATC CTC 55

Met Arg Val He Leu -17 -15 CTC CTG CTC ACA CTG GAT GTG CTA GGT GTC TCC AGT ATG ATG ACA GAC 103 Leu Leu Leu Thr Leu Asp Val Leu Gly Val Ser Ser Met Met Thr Asp -10 -5 1

AAG AAT CTC AAA AAG AAG ATT GAA GGG AAT TGG AGA ACC GTT TAC TTA 151 Lys Asn Leu Lys Lys Lys He Glu Gly Asn Trp Arg Thr Val Tyr Leu 5 10 15 20

GCT GCC AGT AGC GTG GAG AAG ATA AAT GAA GGC TCA CCA TTG AGG ACC 199 Ala Ala Ser Ser Val Glu Lys He Asn Glu Gly Ser Pro Leu Arg Thr

25 30 35

TAC TTC CGT CGC ATT GAG TGT GGG AAG AGA TGC AAC CGA ATC AAT CTC 247 Tyr Phe Arg Arg He Glu Cys Gly Lys Arg Cys Asn Arg He Asn Leu 40 45 50

TAC TTT TAT ATT AAG AAA GGG GCC AAG TGC CAA CAG TTT AAA ATC GTG 295 Tyr Phe Tyr He Lys Lys Gly Ala Lys Cys Gin Gin Phe Lys He Val 55 60 65

GGA AGG AGA TCC CAA GAC GTT TAC TAT GCA AAG TAT GAA GGG AGC ACG 343 Gly Arg Arg Ser Gin Asp Val Tyr Tyr Ala Lys Tyr Glu Gly Ser Thr 70 75 80

GCA TTC ATG TTA AAG ACA GTG AAT GAG AAG ATA TTG CTG TTT GAT TAT 391 Ala Phe Met Leu Lys Thr Val Asn Glu Lys He Leu Leu Phe Asp Tyr 85 90 95 100

TTT AAC AGA AAC AGA AGA AAT GAC GTT ACA CGA GTG GCT GGA GTT TTG 439 Phe Asn Arg Asn Arg Arg Asn Asp Val Thr Arg Val Ala Gly Val Leu

105 110 115 GCG AAA GGC AGG CAA CTG ACT AAG GAT GAG ATG ACA GAA TAC ATG AAC 487 Ala Lys Gly Arg Gin Leu Thr Lys Asp Glu Met Thr Glu Tyr Met Asn 120 125 130

TTC GTG GAA GAA ATG GGC ATT GAG GAT GAG AAT GTA CAA CGT GTC ATG 535 Phe Val Glu Glu Met Gly He Glu Asp Glu Asn Val Gin Arg Val Met 135 140 145

GAC ACA GAC ACC TGT CCA AAC AAG ATC AGA ATT AGA TGACATCAGG 581 Asp Thr Asp Thr Cys Pro Asn Lys He Arg He Arg 150 155 160

AATTTTCCAC TATATTCTTC CTGGAACCTG AAACATCAAT ATGAAGATGA AGCAATCTTT 641

TCTTTCAGAT CATATCTTCC TATTTGCTGC AAATTACAAT TCTTGTCTCC ATACTTTCTC 701

TTTCATTCAT ACTTTCCCAT GTTCTAATTG GTATTAGTAC ATCTTTGAAT GTTTAAATAA 761

ATCTATTTCA CTTGC 776

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: SYNTHETIC OLIGONUCLEOTIDE

(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION: 1..27

(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS 92-118 OF SEQ ID NO.: 2."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CTTTTTGAGA TTCTTGTCTG TCATCAT 27

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(A) ORGANISM: SYNTHETIC OLIGONUCLEOTIDE

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..24

(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS 76-99 OF SEQ ID NO. : 2. "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GTCATCATAC TGGAGACACC TAGC 24

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: SYNTHETIC OLIGONUCLEOTIDE (ix) FEATURE :

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..21

(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS 80-100 OF SEQ ID NO. : 2."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

TGTCATCATA CTGGAGACAC CTCTCCAGT 29

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: SYNTHETIC OLIGONUCLEOTIDE

(ix) FEATURE: (A) NAME/KEY: misc_feature

(B) LOCATION: 1..21 (D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS 92-112 OF SEQ ID NO. : 2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GAGATTCTTG TCTGTCATCA TTGACAGAC 29

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE: (A) ORGANISM: SYNTHETIC OLIGONUCLEOTIDE

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..27 (D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS

401-427 OF SEQ ID NO. : 1." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

GGTGAGGAAG ACAACCGGGA CCCACAT 27

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: SYNTHETIC OLIGONUCLEOTIDE

(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION: 1..27

(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS 384-410 OF SEQ ID NO. : 1."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GGACCCACAT GGTGACACAG CTCTCCG 27

Claims

CLAIMSWe claim:

1. A method for treating a patient diagnosed as having benign prostatic hypeφlasia or a prostatic cancer comprising administering to said patient a therapeutically effective amount of a composition comprising an antisense oligonucleotide which selectively hybridizes to a gene or mRNA sequence of said patient; wherein said antisense inhibits expression of said gene or mRNA sequence; and wherein said gene or mRNA sequence is selected from the group consisting of a PSA and a probasin gene or mRNA sequence.

2. A method as in claim 1 wherein said oligonucleotide is selected from the group consisting of

(a) oligonucleotides comprising at least 10 consecutive bases from SEQ ID NO.: 1 ;

(b) oligonucleotides comprising at least 10 consecutive bases from the joined exons of SEQ ID NO.: l; and

(c) oligonucleotides that hybridize to the complements ofthe oligonucleotides of (a) or (b) under physiological conditions.

3. A method as in claim 1 wherein said oligonucleotide is selected from the group consisting of

(a) oligonucleotides comprising at least 20 consecutive bases from SEQ ID NO.: 1 ; (b) oligonucleotides comprising at least 20 consecutive bases from the joined exons of

SEQ ID NO.: l; and

(c) oligonucleotides that hybridize to the complements ofthe oligonucleotides of (a) or (b) under physiological conditions.

4. A method as in claim 1 wherein said oligonucleotide is selected from the group consisting of (a) oligonucleotides comprising at least 10 consecutive bases from SEQ ID NO.: 2;

(b) oligonucleotides comprising at least 10 consecutive bases from a genomic sequence corresponding to SEQ ID NO.: 2; and

(c) oligonucleotides that hybridize to the complements of the oligonucleotides of (a) or (b) under physiological conditions.

5. A method as in claim 1 wherein said oligonucleotide is selected from the group consisting of

(a) oligonucleotides comprising at least 20 consecutive bases from SEQ ID NO.: 2; (b) oligonucleotides comprising at least 20 consecutive bases from a genomic sequence corresponding to SEQ ID NO.: 2; and

(c) oligonucleotides that hybridize to the complements ofthe oligonucleotides of (a) or (b) under physiological conditions.

6. A method as in claim 1 wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, and SEQ ID NO.: 8.

7. A method as in claim 1 wherein said oligonucleotide is a modified oligonucleotide.

8. A method as in claim 7 wherein said oligonucleotide is a modified oligonucleotide including at least one synthetic internucleoside linkage.

9. A method as in claim 8 wherein said synthetic internucleoside linkage is selected from the group consisting of phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters. acetamidates, and carboxymethyl esters.

10. A method as in claim 7 wherein said oligonucleotide is a modified oligonucleotide having at least one low molecular weight organic group covalently bound to a phosphate group of said oligonucleotide.

11. A method as in claim 7 wherein said oligonucleotide is a modified oligonucleotide having at least one low molecular weight organic group covalently bound to a 2' position of a ribose of said oligonucleotide.

12. A method as in claim 7 wherein said oligonucleotide is a modified oligonucleotide having covalently attached thereto a compound selected from the group consisting of androgen, androgen derivatives, estrogen, estrogen derivatives, estramustine, emcyt and estracyt.

13. A method as in claim 1 wherein said oligonucleotide is administered intravenously at a dosage between 1.0 μg and 100 mg per kg body weight of said patient.

14. A pharmaceutical composition comprising a sterile pharmaceutically acceptable carrier; and a therapeutically effective amount of an isolated antisense oligonucleotide which selectively hybridizes to a gene or mRNA sequence of a patient; wherein said antisense inhibits expression of said gene or mRNA sequence; and wherein said gene or mRNA sequence is selected from the group consisting of a PSA and a probasin gene or mRNA sequence.

15. A composition as in claim 14 wherein said oligonucleotide is selected from the group consisting of

(a) oligonucleotides comprising at least 10 consecutive bases from SEQ ID NO.: 1 ;

(b) oligonucleotides comprising at least 10 consecutive bases from the joined exons of SEQ ID NO.: l; and (c) oligonucleotides that hybridize to the complements ofthe oligonucleotides of (a) or

(b) under physiological conditions.

16. A composition as in claim 14 wherein said oligonucleotide is selected from the group consisting of (a) oligonucleotides comprising at least 20 consecutive bases from SEQ ID NO.: 1 ;

(b) oligonucleotides comprising at least 20 consecutive bases from the joined exons of SEQ ID NO.: l; and

(c) oligonucleotides that hybridize to the complements ofthe oligonucleotides of (a) or (b) under physiological conditions.

17. A composition as in claim 14 wherein said oligonucleotide is selected from the group consisting of

(a) oligonucleotides comprising at least 10 consecutive bases from SEQ ID NO.: 2;

(b) oligonucleotides comprising at least 10 consecutive bases from a genomic sequence corresponding to SEQ ID NO.: 2; and (c) oligonucleotides that hybridize to the complements of the oligonucleotides of (a) or

(b) under physiological conditions.

18. A composition as in claim 14 wherein said oligonucleotide is selected from the group consisting of (a) oligonucleotides comprising at least 20 consecutive bases from SEQ ID NO.: 2;

(b) oligonucleotides comprising at least 20 consecutive bases from a genomic sequence corresponding to SEQ ID NO.: 2; and

(c) oligonucleotides that hybridize to the complements ofthe oligonucleotides of (a) or (b) under physiological conditions.

19. A composition as in claim 14 wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, and SEQ ID NO.: 9.

20. A composition as in claim 14 wherein said oligonucleotide is a modified oligonucleotide.

21. A composition as in claim 14 wherein said oligonucleotide is a modified oligonucleotide including at least one synthetic internucleoside linkage.

22. A composition as in claim 21 wherein said synthetic internucleoside linkage is selected from the group consisting of phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, and carboxymethyl esters.

23. A composition as in claim 20 wherein said oligonucleotide is a modified oligonucleotide having at least one low molecular weight organic group covalently bound to a phosphate group of said oligonucleotide.

24. A composition as in claim 20 wherein said oligonucleotide is a modified oligonucleotide having at least one low molecular weight organic group covalently bound to a 2' position of a ribose of said oligonucleotide.

25. A composition as in claim 20 wherein said oligonucleotide is a modified oligonucleotide having covalently attached thereto a compound selected from the group consisting of androgen, androgen derivatives, estrogen, estrogen derivatives, estramustine, emcyt and estracyt.

26. A pharmaceutical kit comprising the pharmaceutical composition of claim 14 in a pharmaceutically acceptable carrier for intravenous administration.