CA2239976A1 - 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 AR and the .alpha.FGF 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

W O 97/11170 PCT~US96/15081 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 of the 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 of the first successes of cancer chemotherapy, using the therapeutic program of castration and/or anti-androgen hormonal tre~tment~ introduced by Charles Huggins in the 1 940s. A r~m~rk~hle relief of symptoms and objective regression of bony metastases occurs under this endocrine therapeutic program.
U~ ately, after a "golden period" which lasts roughly 18 months, regrowth of the prostate cancer cells occurs and, in the later stages of the 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, t~rmin~ting in death.
A key problem had been the silent onset of cancer of the 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 of the 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 of the 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 ~l~ç~(les without evolving into the deadly carcinoma. Depending upon the degree of hypertrophy WO 97/11170 PCT~US96/15081 and the age of the 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)).
The androgen receptor (AR) binds the male hormone testosterone and, acting at the 5 transcriptional level, regulates the growth of normal prostatic cells. A cDNA for the human AR
was disclosed by T nh~hn et al. (1988). As noted above, anti-androgen or estrogen hormonal therapy, including physical or chemical castration, may be effective against early stage prostate cancer but, after a period of roughly 18 months, the patient becomes refractory to the hormonal therapy. The relapse is believed to be the result of the development or clonal selection of 10 androgen-independent tumor cells in which the AR has mutated or been lost (see, e.g., Taplin, et al. (1995); Klocker, et al. (1994). Interestingly, in murine androgen-independent prostatic cancer cells, transfection ~ith an AR cDNA has been shown to inhibit growth in the presence of testosterone (Suzuki, et al. (1994)).
The acidic fibroblast growth factor (ocFGF), also known as the heparin binding growth 15 factor type one (HBGF- 1), is an androgen-regulated mitogen produced by prostatic cells. An mRNA sequence for a human allele of aFGF was disclosed in Harris, et al. (1991). Mansson, et al. (1989) found that o~FGF was expressed in normal imm~tnre rat prostate but not in normal mature rat prostate. In cancerous rat prostatic cell lines, they found ccFGF expression similar to that in imm~tllre rat prostate.

Summar~ 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 ~nnini~t~ring to the patient a therapeutically effective amount of a composition comprising an antisense oligonucleotide which 25 selectively hybridizes to an AR or c~FGF gene or mRNA sequence of the patient, thereby inhibiting the expression of the AR or ocFGF gene or mRNA sequence. This inhibition of the AR
or ocFGF genes or mRNAs by antisense oligonucleotides results in a significant inhibition of the 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 methods are particularly useful in 30 treating prostate cancer patients who have become refractory to anti-androgen hormonal therapy.
The AR antisense oligonucleotides may comprise at least 10 consecutive bases from SEQ

W O 97/11170 PCT~US96/15081 ID NO.: 1, at least 10 consecutive bases from a genomic sequence corresponding to 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 S sequences.
The aFGF antisense oligonucleotides may comprise at least 10 consecutive bases from any one of SEQ ID NO.: 2, SEQ ID NO.: 3 or SEQ ID NO.: 4, at least 10 consecutive bases from the joined exons of SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4, or oligonucleotides that hybridize to the complements of these sequences under physiological conditions. More 10 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 of the invention include, but are not limited to, those disclosed as SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, and SEQ ID NO.: 8.
In ~lt;r~ ed embo-liment.~, all of the above-described oligonucleotides are modified 15 oligonucleotides. In one set of embo-limentc, the modified oligonucleotide includes at least one synthetic intermlcleoside 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 20 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., 25 aminoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), small saccharides or glycosyl groups.
In another set of embo-1iment~ the modified oligonucleotide has covalently attached thereto a prostate-targeting compound such as an androgen, androgen derivative, estrogen, estrogen derivative, estr~mll~tine, emcyt or estracyt.
In pl~r~lled embodiments, the ~nti~en~e oligonucleotides are :~mini~tered intravenously at a dosage between 1.0 ~Lg and 100 mg per kg body weight of the patient.

W O 97/11170 PCT~US96/15081 The present invention also provides for any or all of the above-described antisense oligonucleotides, including the various modified oligonucleotides, in a ph~rmslreutical composition. The antisense oligonucleotides are admixed with a sterile ph~nn:~relltically acceptable carrier in a therapeutically effective amount such that the isolated ~nti~t-n~e 5 oligonucleotide selectively hybridizes to the AR or ~FGF gene or mRNA sequence when ~rlmini.stered to a patient. A pharmaceutical kit is also provided in which such a ph~rm~eeutical composition is combined with a ph~rrn~reutically acceptable carrier for intravenous s~1mini~tration.
The methods and products of the present invention further include ~nti~çn~e 10 oligonucleotides, as described above, directed at a PSA gene, a probasin gene, an estrogen receptor gene, a telomerase gene, a prohibitin gene, a src gene, a ras gene, a myc gene, a blc-2 gene, a protein kinase-A gene, a plasminogen activator urokinase gene and a methyl transferase gene.

Detailed De~el ;I lion of the Invention The present invention provides new methods for the tre~tment of cancer of the prostate and pharmaceutical compositions useful therefor. It is now disclosed that antisense oligonucleotides complementary to genes which are expressed predomin~ntly or strongly in prostatic cells are effective for inhibiting the growth of and/or killing hyperplastic or cancerous 20 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 AR or c~FGF gene such that they inhibit the t;x~lession of that gene. Surprisingly, inhibition of either of these genes, even in androgen-resistant prostatic cancer cells, inhibits the growth of these cells. Because the ~nti~n~e oligonucleotides ofthe invention can be ~lmini~tered 25 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 of the present invention, the following definitions are provided for specific terms used in the claims appended 30 hereto:
AR. As used herein, the abbreviation "AR" refers to the androgen receptor well known W O 97tlll70 PCTfUS96/15081 s in the art and described in the various references cited herein. A cDNA sequence of the hurnan AR gene was disclosed in Lubahn et al. (1988). The Lubahn et al. (1988)sequence is available on GenBank (Accession nurnber J03180) and is reproduced here as SEQ. ID NO.: 1. The translation initiation codon of this gene is found at base positions 363-365 and the stop codon is 5 at positions 3120-3122 of SEQ ID NO.: 1. As will be obvious to one of ordinary skill in the art, other alleles of the AR gene, including other human alleles and homologues from other m~mm~ n species, encoding an AR protein and hybridizing to SEQ ID NO.: 1 under stringent hybridization conditions, will exist in natural populations and are embraced by the term "AR
gene" as used herein.
~aFGF. As used herein, the term "aFGF" refers to the aFGF protein known in the art and described in the various references cited herein. The genomic DNA of one allele of the human aFGF gene has been partially sequenced and was disclosed in Wang et al. (1989). The Wang et al.(l 989) sequences cover the three exons of the aFGF gene as well as some 5', 3' and intron sequences. These sequences are available on GenBank (Accession numbers M23017, M23086 15 and M23087) and are reproduced here as SEQ. ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4.
A partial cDNA sequence for a human aFGF gene also may be found in Harris et al. (1991). The locations of the exons are located in the sequence listings. The translation initiation codon is found at positions 602-604 of SEQ ID NO.: 2 and the stop codon is found at positions 496-498.
In addition, as will be obvious to one of ordinary skill in the art, other alleles of the c~FGF gene, 20 including other human alleles and homologues from other m~mm~ n species, encoding an aFGF protein and hybridizing to one or more of SEQ ID NO.: 2, SEQ ID NO.: 3 or SEQ ID
NO.: 4 under stringent hybridization conditions, will exist in natural populations and are embraced by the terrn "aFGF gene" as used herein.
.~nti~e~e Oli~onucleotides. As used herein, the term "antisense oligonucleotide" or 25 "~nti~n~e" 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 an "AR-antisense oligonucleotide" and bv an ~ 30 "aFGF-antisense oligonucleotide" are meant oligonucleotides which hybridize under physiological conditions to the AR gene/mRNA or aFGF gene/mRNA and, thereby. inhibit transcription/translation of the AR and aFGF genes/mRNAs, respectively. The antisense molecules are designed so as to h~ r~L~; with transcription or translation of AR or aFGF 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 S selected, including the sequence of the 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.
Strin~e~t hvbridization conditions. As used herein, the term "stringent hybridization 10 conditions" means hybridization conditions from 30~C-60~C and from 5x to O.lx 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 15 sequence and not to substantially dirr~lcllL sequences. The exact conditions which constitute "stringent" conditions, depend upon the length of the 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 20 e~ hllentation, 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 Enz,vmolo~y. 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.
25 I Des;~n of AR and aFGF Anti~ n~e Oli~onucleotides The present invention depends, in part, upon the discovery that the selective inhibition of the expression of AR or aFGF by antisense oligonucleotides in prostatic cells effectively inhibits cell growth and/or causes cell death.
Based upon SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4, or 30 upon allelic or homologous genomic or cDNA sequences, one of skill in the art can easily choose and synth~ci7~ any of a number of al)plopl;ate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for AR or aFGF inhibition, such ~3ntieçnee oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the AR or aFGF mRNA transcripts. Most preferably, the ~ntieçnee oligonucleotides comprise a complementary sequence of 20-30 bases.
5 Although oligonucleotides may be chosen which are ~ntieçnee to any region of the AR or aFGF
genes or mRNA transcripts, in preferred embo(1imente the ~ntiei nee oligonucleotides correspond to N-tçrminsll or 5' upstream sites such as kanslation initiation, transcription initiation or promoter sites. In addition, 3'-untr:~nel~t~-l 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 10 alternative mRNA splicing occurs. In addition, the AR or aFGF ~ntie~nee 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. Finally, although, SEQ ID NO.: 1 discloses a cDNA
sequence and SEQ ID NO.: 2, SEQ ID NO.:3 and SEQ ID NO.: 4 disclose genomic DNA
sequences, one of ordinary skill in the art may easily derive the genomic DNA corresponding to 15 the cDNA of SEQ ID NO.: 1 and may easily obtain the cDNA sequence corresponding to SEQ
ID NO.: 2, SEQ ID NO.:3 and SEQ ID NO.: 4. Thus, the present invention also provides for ~ntiecnee oligonucleotides which are complementary to the genomic DNA corresponding to SEQ
ID NO.: 1 and the cDNA corresponding to SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4.
Similarly, ~ntieçnee to allelic or homologous cDNAs and genomic DNAs are enabled without 20 undue ~ hnentation.
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 AR or aFGF genes or mRNA
transcripts in order to be effective. Rather, some degree of miem~tches will be acceptable if the slntieenee oligonucleotide is of sufficient length. In all cases, however, the oligonucleotides 25 should have sufficient length and complementarity so as to hybridize to an AR or ocFGF
transcript under physiological conditions. Preferably, of course, mi~m~trhes are absent or minim~l 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 30 duplex formed by an AR or aFGF transcript and the bases fl~nkin~ 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 hairpin 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 5 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' AR or o~FGF
sequence to the end of the oligonucleotide (see, e.g., Table 1, SEQ ID NO.: 6 and SEQ ID NO.:
7, Tang, J.-Y., et al. (1993) Nucleic Acids Res. 21:2729-2735).
In one set of embodiments, the AR and ~FGF ~nti~en.~e oligonucleotides of the invention 10 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 ca~ied out manually or by an automated synthesizer.
In preferred embo~liment~, however, the antisense oligonucleotides of the 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 20 in which (1) at least two of its nucleotides are covalently linked via a synthetic intf?n~llcleoside 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 int~ rmlcleoside linkages are phosphorothioates, aLkylphosphonates, 25 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 and aliphatic groups (e.g., 30 arninoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), small saccharides or glycosyl groups. Other low molecular weight organic modifications include additions to the WO97/11170 PCT~US96/15081 int~rmlrleoside phosphate linkages such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and t~rmin~l 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 S 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 10 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. ~ltern~tively, 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 15 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 hurnan intervention. The androgen receptor and other hormonal receptor sites on prostate cells allow for targeting antisense oligonucleotides 20 specifically or particularly to prostatic cells. Attachment of the antisense oligonucleotides by a molecular "tether" (e.g., an alkyl chain) to estrz~n~ tine, 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 25 der*atives, or other prostate cell ligands to ~nti~ence 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 plc~r~lled modified oligonucleotides are hybrid or chimeric oligonucleotides in 30 which some but not all of the phosphodiester linkages, bases or sugars have been modified.
Hybrid modified ~nti~cn~e oligonucleotides may be composed, for example, of stretches of ten W O 97/11170 PCT~US96/15081 2'-O-alkyl nucleotides or ten phosphorothioate synthetic linkages at the S' and/or 3' ends, and a segment of seven unmodified oligodeoxynucleotides in the center, or of similar terrninal segment~ 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-5 methylated hybrid oligonucleotides. Since degradation occurs mainly at the 3' end, secondarilyat the 5' end, and less in the middle, unmodified oligonucleotides located at this position can acJiv~te Rl~a~e H, ~nd yet arç degraded slowly. F~hermorç, the T~n of suçh 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.
10 Obviously, the nurnber 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 15 independently chosen integer from 1 to about 20:
OH

s'(BS)nBBBB- - ~BBBB(Bs)nB - P=O3' O--R

II. Products and Methods of Tr~tment for BPH and Prostate Cancer The methods of the present invention represent new and useful additions to the field of benign prostate hyperplasia or prostate cancer therapy. In particular, the methods of the present 25 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 hyperplasia or early stage prostate cancer and may provide a snkstit~lte for more radical procedures such as transurethral resection, radical prostatectomy, or physical or chemical castration. The products of the present invention include 30 the isolated anti~n~e oligonucleotides described above. As used herein, the term "isolated" as applied to an anti~n~e oligonucleotide means not covalently bound to and physically separated from the S' and 3' sequences which flank the corresponding antisense sequence in nature.
Atlmini~tration of the AR or o~FGF ~nti~çn~e oligonucleotides may be oral, intravenous, 77454.1 W O 97/11170 PCT~US96/15081 11 parenteral, cutaneous or subcutaneous. For BPH or when the site of a prostatic tumor is known, the ~mini.~tration also may be localized to the prostate or to the region of the tumor by injection to or perfusion of the site.
AR or aFGF antisense oligonucleotides may be ~(lmini~tered as part of a pharrnaceutical 5 composition. Such a ph~rm~reutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or ph~rm~reutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the ~nti.~f n~e oligonucleotides in a unit of weight or volume suitable for ~1mini~tration to a patient. The term "ph~rm~relltically acceptable" means a non-toxic material 10 that does not interfere with the effectiveness of the biological activity of the active ingredients.
The term "physiologically acceptable" refers to a non-toxic m~t~ri~l that is compatible with a biological system such as a cell, cell culture, tissue, or org~ni~m. The characteristics of the carrier will depend on the route of ~-1mini~tration. Physiologically and ph~rm~ceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other 15 m~t~ri~l~ which are well known in the art. The pharmaceutical composition of the 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 phzlrm~ eutical composition to produce a synergistic effect or to minimi7f side-effects caused.
The pharrnaceutical composition of the invention may be in the forrn of a liposome in 20 which the AR or aFGF ~nti~çn~e oligonucleotides are combined, in addition to other ph~rm~eutically 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 forrnulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the 25 like. P~ udlion 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 ph~rmzlreutical composition of the invention may further include compounds such as cyclodextrins and the like which enhance delivery of oligonucleotides into cells. When the 30 composition is not al1mini~tered systemically but, rather, is injected at the site of the target cells, cationic detergents (e.g. Lipofectin) may be added to enhance uptake.

W O 97/11170 PCTrUS96/15081 When a therapeutically effective amount of AR or o~FGF antisense oligonucleotides is ~lmini~tered orally, the oligonucleotides will be in the form of a tablet, capsule, powder, solution or elixir. When ~fimini~tered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and 5 powder may contain from about 5 to 95% of the AR and/or aFGF antisense oligonucleotides and preferably from about 25 to 90% of the oligonucleotides. When ~lmini~tered in liquid form, a liquid carrier such as water, pekoleum, 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 of the I)h~rmzlreutical composition may further contain physiological saline solution, dexkose or other 10 saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
When ~lmini~trred in liquid form, the ph~rm~reutical composition may contain from about 0.5 to 90% by weight of an AR and/or aFGF antisense oligonucleotide and preferably from about 1 to 50% ofthe oligonucleotide.
When a therapeutically effective amount of an AR or o~FGF antisense oligonucleotide is 15 ~r1mini~trred by inkavenous, cutaneous or subcutaneous injection, the oligonucleotides will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The p~ ~dlion of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharrnaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the ~nti~en~e oligonucleotides, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dexkose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or another vehicle as known in the art.
l~he ph~rrn~reutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
In ~ler~ d embodiments, when the target cells are readily accessible, ~-1mini~tration of the ~nti~çn~e oligonucleotides is localized to the region of the targeted cells in order to m~lcimi71 the delivery of the ~nti~en~e and to minimi7P the amount of antisense needed per treatment.
Thus, in one preferred embodiment, ~flmini~kation is by direct injection at or perfusion of the site of the targeted cells, such as a tumor. Alternatively, the ~nti~.on~e oligonucleotides may be adhered to small particles (e.g., microscopic gold beads) which are impelled through the 30 membranes of the target cells (see, e.g., U.S. Pat. No. 5,149,655).
In another series of embodiments, a recombinant gene is constructed which encodes an CA 02239976 l998-03-l7 W O 97/11170 PCTAUS96/lS081 AR or aFGF antisense oligonucleotide and this gene is introduced within the targeted cells on a vector. Such an AR or aFGF antisense gene may, for example, consist of the normal AR or aFGF sequence, or a subset of the normal sequences, operably joined in reverse orientation to a promoter region. An operable antisense gene may be introduced on an integration vector or may 5 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 con~liLuliv~ly ~ essed.
In all of the above-described methods of treatment, the AR and/or aFGF antisenseoligonucleotides are ~mini~t~red in therapeutically effective amounts. As used herein, the term lO "therapeutically effective amount" means that amount of antisense which, under the conditions of a~lmini~tration, including mode of ~lmini~tration and presence of other active components, is sufficient to result in a me~ningful patient benefit, i.e., the killing or inhibition of the gro~,-vth of target cells.
The amount of AR and/or aFGF antisense oligonucleotides in the ph~rm~(~eutical 15 composition of the present invention will depend not only upon the potency of the :~nti~çn~e but also upon the nature and severity of the condition being treated, and on the nature of prior tre~tments which the patient has undergone. Ultimately, the attending physician will decide the amount of ~nti~çn~e with which to treat each individual patient. Initially, the ~tt~-ntiing physician will ~t1mini~tcr low doses ofthe inhibitor and observe the patient's response. Larger doses of 20 antisense may be ~lmini~tered 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 rh~rm~t~e~ltical compositions used to practice the method of the present invention should contain about 1.0 llg to about 100 mg of oligonucleotide per kg body weight.
The duration of intravenous therapy using the ph~rm~eutical compositions of the present 25 invention will vary, depending on the severity of the 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 ~1mini~tration is preferred. Thus, intravenous ;~mini~tration of therapeutically effective amounts over a 12-24 30 hour period are contemplated. Ultimately the attending physician will decide on the ap~ liate duration of intravenous therapy using the pharmaceutical composition of the present invention.

77454.1 W O97/11170 PCT~US96/15081 The following examples of the use of AR and ocFGF antisense are presented merely to illustrate some of the oligonucleotides, including modified oligonucleotides, that may be employed according to the present invention. The particular oligonucleotides used, therefore, should not be conskued as limiting of the invention but, rather, as indicative of the wide range of 5 oligonucleotides which may be employed. As will be obvious to one of ordinary skill in the art in light of the present disclosure, a great many equivalents to the presently disclosed ~nti~n~e oligonucleotides and disclosed methods are now available. In particular, other ~nti~en~e oligonucleotides subst~nti~lly complementary to subsets of SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3 or SEQ ID NO.: 4 and chemical modifications of the same which do not prevent 10 hybridization under physiological conditions, are contemplated as equivalents of the 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. In particular, the use of antisense oligonucleotides to the eskogen receptor, PSA, probasin, telomerase, prohibitin, src, ras, myc, blc-2, protein kinase-A, plasminogenctivator urokinase and 15 methyl kansferase genes is contemplated for the tre~ttnent of benign prostatic hyperplasia or prostatic cancer.

Experimental Exa~nples The PC3- 1435 permanent cell line of human prostatic cancer, obtained from the 20 American Type Culture Collection, was grown in monolayer culture: The PC3-1435 cells are from an osseous metastasis and are androgen-insensitive. Cells were grown in Dulbecco's medium supplemented with 10 percent fetal calf serum, gl~lt~m:~te, pyruvate, penicillin and skeptomycin, in 25-150 cm flasks, incubated at 37~C in 6 percent CO2-air.
A number of AR and o~FGF antisense oligonucleotides were tested for their inhibitory 25 effect on prostatic cells. The base sequences of these oligonucleotides are disclosed as SEQ ID
NO.: 5 through SEQ ID NO.: 8. SEQ ID NO.: 5 is antisense to positions 927-953 of the AR
gene (SEQ ID NO.: 1). SEQ ID NO.: 6 is a self-stabilized or hairpin oligonucleotide. The first 21 bases are complementary to positions 916-936 of the AR gene. The rem~ining eight are identical to positions 920-927 of the gene, allowing formation of a 3' hairpin. SEQ ID NO.: 7 is 30 another self-stabilized antisense oligonucleotide. The first 21 bases of this oligonucleotide are complementary to positions 927-947 of the AR gene. The rem~inin~ eight are identical to WO 97/11170 PCT~US96/15081 positions 931 -938 of the gene, allowing for formation of a 3' hairpin. Finally, SEQ ID NO.: 8 is an ~ntic~n~e sequence corresponding to positions 611-635 of the oLFGF gene.
Table 1 shows some ofthe ~nti~t?n~e oligonucleotides tested. The numbers at the left of each sequence correspond to the sequence numbers in the sequence listing. ~nti~en~e oligonucleotides with unmodified or natural intern~lcleoside linkages (P=O) and oligonucleotides with all phosphorothioate synthetic linkages (P=S) were tested. In addition, modified oligonucleotides were tested in which just the termin~l 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 organic chemical groups (e.g., 2-hydroxy-3-amino-10 propyl, propylamine) were added to the 3' termin~l phosphate or the penllltim~te 3' phosphate.
Growth of the PC3-1435 cell line in tissue culture monolayers was con~i~tently inhibited by addition of phosphorothioate-modified oligodeoxynucleotides t~rgeted against the AR or o~FGF genes and incubation for 24-48 hours thereafter. As the concentration of modified oligonucleotides is decreased from the 10-20 ~lM level, most effective inhibition occurs with 15 specific ~nti~n~e oligodeoxynucleotides at the 2-5 ~LM level, as contrasted with mi~m~tched 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 of the inhibition events using AR antisense oligonucleotides against PC3-1435 cells. The first evidence of antisense inhibition is rupture of 20 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 e~mined on successive days. There is an early loss of adhesiveness to the floor of the plastic wells. These changes are more severe (see Table 4) than those measured by 3H-thymidine incorporation into DNA, in other words more drastic than the i",pai""ent of DNA synthesis.
Each of the above-mentioned references and patents are incorporated by reference.

Antisense Oligonucleotides Sequence Target #5 5'CTG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CAT3 Androgen receptor, P=S
#5 5'CTG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CAT3 Androgen 10 receptor, P=O
#5 5 ' CSTsG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CsAsT3' Androgen receptor, P=S termini lS #5 5 ' CTG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CAT3 Androgen receptor, ¦ modi~ied with O organic group +
H3N-CH2cHcH2O-pl=o OH OH

O Androgen 25 receptor, ll modi~ied with #5 5'CTG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CA-o-P-o-T3 organic group #6 5 ' GGA-GTT-GCA-TGG-TGC-TGG-CCT-CAG-CAC-CA3' Androgen receptor 3' hairpin, P=S
#7 5 ' CTG-TTG-CTG-AAG-GAG-TTG-CAT-AAC-TCC-TT3' Androgen receptor 3' hairpin, P=S
#8 5 GGG-CTG-TGA-AGG-TGG-TGA-TTT-CCC-C3' . ~FGF, P=S

W O 97/11170 PCT~US96/15081 #85 ' GGG-CTG-TGA-AGG-TGG-TGA-TTT-CCC- C3 ' o~FGF, P=O

3H-thymidine incorporation into DNA PC3-1435 human prostate cancer tissue culture 10 Genes Tar~eted Concentration (,uM) CPM~ % inhibition Control (no oligo) -- 38,000 0 Androgen receptor, (P = S) 2015,000 60 20,000 48 Androgen receptor, (P = S)~ 2010,200 68 24,000 25 l~i~mzlt~h (P = S) 2020,000 47 s 27,000 30 Averages of 3 separate wells * 3' phosphate modified with -CH2CHOHCHNH3+

Degree of inhibition of DNA synthesis in PC3-1435 prostate cancer tissue cultures Genes targeted Concentration (,uM) CPM ~ % inhibition Control (no oligo) -- 14,700 0 ccFGF (P=S) 20 2,485 83 4,500 69 Mismatch 20 6,990 51 s 10,750 27 35 ~ Averages of 3 separate wells.

W O97/11170 PCT~US96/15081 Morphological Comparison of Treated and Control Cells Concentration IlM
Gene Tar~et 20 10 5 2 o~FGF ~P=S) 4+ 4+ 1-1/2+ 1+
Androgen receptor (P=S) 3+ 3+ 1+ 1+
Mismatch (P=S) 1-1/2+ 1/2+ 0 0 Observation 24 hours after oligonucleotide addition. Damage: 4+ dev~.~t~1;ng; 3+ severe, 2+
serious; l+ visible; 1/2+ slight; 0 none W O 97/11170 PCTnUS96/15081 References Agrawal (ed.) Meth. Mol. Biol.~ HumanaPress, Totowa, NJ (1993) Vol. 20.
Agrawal and Goodchild (1987) Tetrahedron Lett. 28:3539-3542.
S Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083.
Agrawal et al.(l990) Proc. Natl. Acad. Sci. (~JSA) 87: 1401-1405.
Agrawal et al. (1992) Trends Biotechnol.10:152-158.
Catalona(1994)N.E. J. Med. 331:996-1004.
Forsgren et al. (1979) CaIlcer Res. 39:5155-5164.
10 Gittes (1991) N,F. J. Med. 324: 236-245.
Harris et al. (1991) in Mol. and Cell. Biol. of Prostate Cancer~ Karr et al (eds.) Plenum Press, NY, pp. 315-330.
Klocker, et al. (1994) The Prostate 25:266-273.
15 Lubahn et al. (1988) Mol. F,ntlocr;nl l. 2(12):1265-1275.
Mansson, et al. (1989) Cancer Res. 49:2485-2494.
Metelev et al. (1994) Bioorg,. Medicinal Chem. T~ett~ 4: 2929-2934.
Sainio et al. (1994) Cell. Mol. Neurobiol 14(5):439-457.
Sheridan and Tew (1991) C~ncer Surveys 11 :239-254.
20 Suzuki, et al. (1994) The Prostate 25:310-319.
Taplin et al. (1995) N.E.J. Med. 332(21):1393-1398.
Uhlmann et al. (1990) Chem. Rev. 90:534-583 Wang et al. (1979) Invest. Urol. 17:159-163.
Wang et al. (1989) Mol. Cell. Biol. 9(6):2387-2395.

WO97/11170 PCTrUS96/15081 SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: WORCESTER FOUNDATION FOR BIOMEDICAL 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, GR~N~lhLD & 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 #l.0, Version #l.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:

CA 02239976 l998-03-l7 WO97/11170 PCT~S96/15081 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: TWOMEY, MICHAEL ~
(B) REGISTRATION NUMBER: 38,349 (C) REFERENCE/DOCKET NUMBER: W0461/7035 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617-720-3500 (B) TELEFAX: 617-720-2441 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3569 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: l 1n~

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

(iv) ANTI-SENSE: NO

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

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 363.. 3122 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

~L'l"l"l'~'l"l"l"L'l' TTCTTTTAAG ATCTGGGCAT ~'~ "l'~AATC TACCCTTCAA GTATTAAGAG 120 ACAGACTGTG AGCCTAGCAG GGCAGATCTT GTCCACCGTG TGTCTTCTTC TGCACGAGAC l80 Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Ary Pro Pro l 5 lO 15 Ser Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ala GCA CCT CCC GGC GCC AGT TTG CTG CTG CTG CAG CAG CAG CAG CAG CAG 55l Ala Pro Pro Gly Ala Ser Leu Leu Leu Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Thr Ser Pro Arg Gln Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser Pro 9o 95 0 Gln Ala His Arg Arg Gly Pro Thr Gly Tyr Leu Val Leu Asp Glu Glu Gln Gln Pro Ser Gln Pro Gln Ser Ala Leu Glu Cys His Pro Glu Arg Gly Cys Val Pro Glu Pro Gly Ala Ala Val Ala Ala Ser Lys Gly Leu Pro Gln Gln Leu Pro Ala Pro Pro Asp Glu Asp Asp Ser Ala Ala Pro Ser Thr Leu Ser Leu l~eu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys Ser Ala Asp Leu Lys Asp Ile Leu Ser Glu Ala Ser Thr Met Gln Leu Leu Gln Gln Gln Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser Gly CA 02239976 l998-03-l7 WO97/11170 PCT~US96/lS081 Arg Ala Arg Glu Ala Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn Tyr Leu Gly Gly Thr Ser Thr Ile Ser Asp Asn Ala Lys Glu Leu Cys Lys 0 Ala Val Ser Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu His Leu Ser Pro Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Pro Leu Leu Gly Val Pro Pro Ala Val Arg Pro Thr Pro Cys Ala Pro Leu Ala Glu TGC A~A GGT TCT CTG CTA GAC GAC AGC GCA GGC A~G AGC ACT GAA GAT 1271 Cys Lys Gly Ser Leu Leu Asp Asp Ser Ala Gly Lys Ser Thr Glu Asp 25 .ACT GCT GAG TAT TCC CCT TTC AAG GGA GGT TAC ACC A~A GGG CTA GAA 1319 Thr Ala Glu Tyr Ser Pro Phe Lys Gly Gly Tyr Thr Lys Gly Leu Glu Gly Glu Ser Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly Thr Leu Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly Ala Leu CA 02239976 l998-03-l7 WO97/11170 PCT~S96/15081 Asp Glu Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro Leu Ala Leu Ala Gly Pro Pro Pro Pro Pro Pro Pro Pro His Pro His Ala CGC ATC AAG CTG GAG A~C CCG CTG GAC TAC GGC AGC GCC TGG GCG GCT 1559 0 Arg Ile Lys Leu Glu Asn Pro ~eu Asp Tyr Gly Ser Ala Trp Ala Ala Ala Ala Ala Gln Cys Arg Tyr Gly Asp Leu Ala Ser Leu His Gly Ala Gly Ala Ala Gly Pro Gly Ser Gly Ser Pro Ser Ala Ala Ala Ser Ser Ser Trp His Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro Cys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Ala Gly Ala Val Ala Pro Tyr Gly Tyr Thr Arg Pro Pro Gln Gly ~eu Ala Gly Gln Glu Ser Asp W O 97/11170 PCTrUS96/15081 Phe Thr Ala Pro Asp Val Trp Tyr Pro Gly Gly Met Val Ser Arg Val 5 CCC TAT CCC AGT CCC ACT TGT GTC A~A AGC GAA ATG GGC CCC TGG ATG 1943 Pro Tyr Pro Ser Pro Thr Cys Val Lys Ser Glu Met Gly Pro Trp Met 0 Asp Ser Tyr Ser Gly Pro Tyr Gly Asp Met Arg Leu Glu Thr Ala Arg Asp His Val Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr Cys Leu Ile Cys Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Ala Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala Ala Glu Gly Lys Gln Lys Tyr Leu Cys Ala Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg 30 Arg Lys Asn Cys Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu Gly Ala Arg Lys Leu Lys Lys Leu Gly Asn Leu Lys Leu CA 02239976 l998-03-l7 WO97/11170 PCT~US96/lS081 Gln Glu Glu Gly Glu Ala Ser Ser Thr Thr Ser Pro Thr Glu Glu Thr Thr Gln Lys Leu Thr Val Ser His Ile Glu Gly Tyr Glu Cys Gln Pro 0 Ile Phe Leu Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val Cys Ala Gly His Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser Leu Asn Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp Ala Lys Ala Leu Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln Met Ala Val Ile Gln Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met Gly Trp Arg Ser Phe Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp Leu Val Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln -CA 02239976 l998-03-l7 WO97/11170 PCT~S96/15081 Cys Val Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln Ile ACC CCC CAG GAA TTC CTG TGC ATG A~A GCA CTG CTA CTC TTC AGC ATT 2807 Thr Pro Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile ATT CCA GTG GAT GGG CTG AAA AAT CAA A~A TTC TTT GAT GAA CTT CGA 2855 0 Ile Pro Val Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg ATG AAC TAC ATC AAG GAA CTC GAT CGT ATC ATT GCA TGC A~A AGA AAA 2903 Met Asn Tyr Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn Pro Thr Ser Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser Val Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met Met GCA GAG ATC ATC TCT GTG ~AA GTG CCC AAG ATC CTT TCT GGG AAA GTC 3095 Ala Glu Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val goo 905 910 AAG CCC ATC TAT TTC CAC ACC CAG TGAAGCATTG GA~ACCCTAT TTCCCCACCC 3149 Lys Pro Ile Tyr Phe His Thr Gln WO97/11170 PCT~US96/lS081 CAGCTCATGC CCCCTTTCAG ATGTCTTCTG C~l~lLATAA CTCTGCACTA CTCCTCTGCA 3209 ATTTGCTGGG ~'l"l"L'l"l"l"l"l"l' CTCTTTCTCT CCTTTCTTTT TCTTCTTCCC TCCCTATCTA 3329 ACCCTCCCAT GGCACCTTCA GACTTTGCTT CCCATTGTGG CTCCTATCTG ~l~llllGAAT 3389 TTTAGAGAGC TAAGATTATC TGGGGAAATC A~AACAAAAA ACAAGCAAAC AAA~U~AA~ 3569 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1082 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: dou~le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

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

(ix) FEATURE:
(A) NAME/KEY: exon 35 (B~ LOCATION: 602.. 770 WO 97/11170 PCT~US96/15081 (D) OTHER INFOR~L~TION: /note= "SEGMENT 1 OF 3.1' WO97/11170 PCT~US96/15081 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TGTGGACCAG AAGGAGGGAC CAAAACATGA TTCTTTTCCC CATGGTCAGA TGATTA~ATT 300 TGAAGTTCTA AAAAATGCAG TTTGGTCCAA AGCTGTGTCC AATTGGGAAG AGAGA~AAAT 360 TGCCCTGGAG CTGTCCTTCC TCTCTTCA~A GTGCATTTTG TGCGTTTGCT GGAAGAACCG 540 ACTACAGGTT TGTTCAATTT CTTACAGTCT TGA~AGCGCC ACAAGCAGCA GCTGCTGAGC 600 CATGGCTGAA GGGGA~ATCA CCACCTTCAC AGCCCTGACC GAGAAGTTTA ATCTGCCTCC 660 TGGAGTCACA TAAATGCGTA A~ATGTCTGG GA~ATGAA~A TAGGGACTTG TGGGGGCCAC 960 CA 02239976 l998-03-l7 WO97/1l170 PCTAJS96/15081 ACGGGCTGCC GGTGCTCAAT TGCTGTATGT TTTCCCAGGT TTCTGTAACT AGTGA~AGAT 1080 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 427 base pairs (B) TYPE: nucleic acid lC) STRANDEDNESS: double (D) TOPOLOGY: l;n~

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYP~~ CAL: NO

(iv) ANTI-SENSE: NO

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

(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION: 186..289 (D) OTHER INFORMATION: /note= "SEGMENT 2 OF 3. UNKNOWN
NUMBER OF BP AFTER SEGMENT 1."

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

CAGCTTTCTT TGGAAGGCAA AGA~AAAGGG ACTGTATTTC TATGTTTTGA TTAATCTGAG 60 GCTCATCCTG AGGGCTCCGT GA~ATGAATG AGCAGAATTT TCCATGGCCA ACTGTCCTGG 120 GCTGACATGC TTCCAGACGT TGGCCAAGGT TTGAGGTTTC CAGA~ATCTT GTTACATGGA 360 GTGAGGCA~A CTATA~AGCA ACAATTAGTC TCTGTTTGTT A~ CCA GAAGGATTCC 420 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 664 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

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

(ix) FEATURE:
(A) NAME/KEY: exon 30 (B) LOCATION: 304.. 498 (D) OTHER INFORMATION: /note= "SEGMENT 3 OF 3. UNKNOWN
NUMBER OF BP AFTER SEGMENT 2."

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

WO97/11170 PCT~US96/15081 TGAGGACTCT TAGAAGTGCT CTTATCAGTA GCATCTTAAT TACTTTACA~ TGGATTTTAA 60 ATGGA~AGGA AGTTTACAAT AATAGCA~AT GCATATTGAC AGCTCTTTAG TGCCCGGTGC 120 TGTTCTAAGT CCTTATGACT ACCCTGTGAA ATAAGTTCCA CCATGACCCC AATTTTCCTG l80 AAAAGGAGAC TGAGGCATGG AGAGCTTTAG TATTTTGCCC AATGTCACAC AGCTAGTA~A 240 TAGCAGACAC CAAATGAGGA ATGTTTGTTC CTGGA~AGGC TGGAGGAGAA CCATTACAAC 360 AGCTGCA~AC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTG 480 (2) INFORMATION FOR SEQ ID ~0:5:

(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPO~OGY: linear (ii) MoLEcu-rlE TYPE: cDNA

35 (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:
(A) ORGANISM: ~YNl~llC OLIGONUCLEOTIDE

(ix) FEATURE:
(A) NAME/KEY: misc_~eature (B) LOCATION: l..27 (D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS
927-953 OF SEQ ID NO.: l."

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

(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: ~YN'l'~'llC OLIGONUCLEOTIDE

(ix) FEATURE:
(A) NAME/KEY: misc_~eature CA 02239976 l998-03-l7 WO97/11170 PCT~S96/15081 (B) LOCATION: 1 .21 (D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS
916-936 OF SEQ ID NO.: 1."

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

10 ~(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l; n (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:
(A) ORGANISM: ~YN'l'~'l'lC OLIGONUCLEOTIDE

(ix) FEATURE:
(A) NAME/KEY: misc_~eature (B) LOCATION: 1..21 (D) OTHER INFORMATION: /note= "AMTISENSE TO POSITIONS
927-947 OF SEQ ID NO.: 1.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

~lGll~CTGA AGGAGTTGCA TAACTCCTT 29 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 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: ~YN'l'~'l'lC OLIGONUCLEOTIDE

(ix) FEATURE:
(A) NAME/KEY: misc_~eature (B) LOCATION: l..25 (D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS
611-635 OF SEQ ID NO.: 2."

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

Claims (29)

38We claim.

1. A method for treating a patient diagnosed as having benign prostatic hyperplasia 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 an AR and an .alpha.FGF 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 a genomic sequence corresponding to SEQ ID NO.: 1, and (c) oligonucleotides that hybridize to the complements of the 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 10 consecutive bases from a genomic sequence corresponding to SEQ ID NO.: 1, and (c) oligonucleotides that hybridize to the complements of the 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 the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4;
(b) oligonucleotides comprising at least 10 consecutive bases from the joined exons of SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4; 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 the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4;
(b) oligonucleotides comprising at least 20 consecutive bases from the joined exons of SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4; and (c) oligonucleotides that hybridize to the complements of the 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.: 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 method as in claim 1 wherein said patient has a prostatic cancer which is refractory to anti-androgen or estrogen hormonal therapy.

15. 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 an AR and an .alpha.FGF gene or mRNA sequence.

16. A composition as in claim 15 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.: 1; and (c) oligonucleotides that hybridize to the complements of the oligonucleotides of (a) or (b) under physiological conditions.

17. A composition as in claim 15 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.: 1, and (c) oligonucleotides that hybridize to the complements of the oligonucleotides of (a) or (b) under physiological conditions.

18. A composition as in claim 15 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.

19. A composition as in claim 15 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 of the oligonucleotides of (a) or (b) under physiological conditions.

20. A composition as in claim 15 wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, and SEQ ID NO.: 9.

21. A composition as in claim 15 wherein said oligonucleotide is a modified oligonucleotide.

22. A composition as in claim 15 wherein said oligonucleotide is a modified oligonucleotide including at least one synthetic internucleoside linkage.

23. A composition as in claim 22 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.

24. A composition as in claim 21 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.

25. A composition as in claim 21 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.

26. A composition as in claim 21 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.

27. A pharmaceutical kit comprising the pharmaceutical composition of claim 15 in a pharmaceutically acceptable carrier for intravenous administration.

28. A method for treating a patient diagnosed as having benign prostatic hyperplasia 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 antisense inhibits or represses prostatic cell growth.

29. A method as in claim 28 wherein said gene is selected from the group consisting of a PSA gene, a probasin gene, an .alpha.FGF gene, an androgen receptor gene, an estrogen receptor gene, a telomerase gene, a prohibitin gene, a src gene, a ras gene, a myc gene, a blc-2 gene, a protein kinase-A gene, a plasminogen activator urokinase gene and a methyl transferase gene.