EP0851919A1 - Chimiotherapie par oligonucleotides antisens de l'hypertrophie ou du cancer de la prostate - Google Patents

Chimiotherapie par oligonucleotides antisens de l'hypertrophie ou du cancer de la prostate

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Publication number
EP0851919A1
EP0851919A1 EP96935879A EP96935879A EP0851919A1 EP 0851919 A1 EP0851919 A1 EP 0851919A1 EP 96935879 A EP96935879 A EP 96935879A EP 96935879 A EP96935879 A EP 96935879A EP 0851919 A1 EP0851919 A1 EP 0851919A1
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Prior art keywords
seq
oligonucleotides
oligonucleotide
gene
group
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EP96935879A
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German (de)
English (en)
Inventor
Paul A. Zamecnik
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University of Massachusetts UMass
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University of Massachusetts UMass
Worcester Foundation for Biomedical Research
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Publication of EP0851919A1 publication Critical patent/EP0851919A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • 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.
  • the invention relates to the field of antisense oligonucleotides and their use in human hyperplasia and cancer therapy.
  • BPH Bacillus subtilis
  • This condition may be a precursor to full blown prostatic cancer or may continue for decades without evolving into the deadly carcinoma.
  • 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 transcriptional level, regulates the growth of normal prostatic cells.
  • a cDNA for the human AR was disclosed by Lubahn et al. (1988).
  • 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 ofthe development or clonal selection of androgen-independent tumor cells in which the AR has mutated or been lost (see, e.g., Taplin, et al. (1995); Klocker, et al. (1994).
  • transfection with an AR cDNA has been shown to inhibit growth in the presence of testosterone (Suzuki, et al. (1994)).
  • the acidic fibroblast growth factor also known as the heparin binding growth factor type one (HBGF-1)
  • HBGF-1 heparin binding growth factor type one
  • An mRNA sequence for a human allele of ⁇ FGF was disclosed in Harris, et al. (1991). Mansson. et al. (1989) found that ⁇ FGF was expressed in normal immature rat prostate but not in normal mature rat prostate. In cancerous rat prostatic cell lines, they found ⁇ FGF expression similar to that in immature rat prostate.
  • the present invention provides methods for treating a patient diagnosed as having benign prostatic hype ⁇ lasia 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 an AR or ⁇ FGF gene or mRNA sequence ofthe patient, thereby inhibiting the expression ofthe AR or ⁇ FGF gene or mRNA sequence.
  • This inhibition of the AR or ⁇ FGF genes or mRNAs by antisense oligonucleotides results in a significant inhibition ofthe growth or survival of prostatic cells.
  • the methods provide a useful new means of treating benign prostatic hype ⁇ lasia and prostatic cancer.
  • the methods are particularly useful in 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 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 sequences.
  • the ⁇ FGF 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 preferably, the antisense oligonucleotides comprise at least 15 consecutive bases, and most preferably, 20-30 consecutive bases from the above-described sequences.
  • sequences ofthe 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.
  • all ofthe above-described oligonucleotides are modified oligonucleotides.
  • 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.
  • 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.
  • the modified oligonucleotide has covalently attached thereto a prostate-targeting compound such as an androgen, androgen derivative, estrogen, estrogen derivative, estramustine, emcyt or estracyt.
  • a prostate-targeting compound such as an androgen, androgen derivative, estrogen, estrogen derivative, estramustine, emcyt or estracyt.
  • the antisense oligonucleotides are administered intravenously at a dosage between 1.0 ⁇ g and 100 mg per kg body weight of the 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 AR or ⁇ FGF gene or mRNA 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.
  • the methods and products ofthe present invention further include antisense 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.
  • 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 predominantly or strongly in prostatic cells are effective for inhibiting the growth of and/or killing hype ⁇ lastic or cancerous cells of prostatic origin.
  • the present invention provides oligonucleotides, including modified oligonucleotides. which have antisense homology to a sufficient portion of either the AR or ⁇ FGF gene such that they inhibit the expression of that gene. Su ⁇ risingly, inhibition of either of these genes, even in androgen-resistant prostatic cancer 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.
  • AR refers to the androgen receptor well known in the art and described in the various references cited herein.
  • a cDNA sequence ofthe human AR gene was disclosed in Lubahn et al. (1988). The Lubahn et al. (1988)sequence is available on GenBank (Accession number 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 at positions 3120-3122 of SEQ ID NO. : 1. As will be obvious to one of ordinary skill in the art.
  • ⁇ FGF refers to the ⁇ FGF protein known in the art and described in the various references cited herein.
  • the genomic DNA of one allele ofthe human ⁇ FGF gene has been partially sequenced and was disclosed in Wang et al. (1989). The Wang et al.(1989) sequences cover the three exons ofthe ⁇ FGF gene as well as some 5', 3' and intron sequences.
  • GenBank accesion numbers M23017, M23086 and M23087
  • SEQ. ID NO.: 2 SEQ ID NO.: 3
  • SEQ ID NO.: 4 A partial cDNA sequence for a human ⁇ FGF gene also may be found in Harris et al. (1991). The locations ofthe 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.
  • ⁇ FGF gene including other human alleles and homologues from other mammalian species, encoding an ⁇ FGF 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 term " ⁇ FGF gene" as used herein.
  • 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.
  • an "AR-antisense oligonucleotide” and by an “ ⁇ FGF-antisense oligonucleotide” are meant oligonucleotides which hybridize under physiological conditions to the AR gene/mRNA or ⁇ FGF gene/mRNA and, thereby, inhibit transcription/translation ofthe AR and ⁇ FGF genes/mRNAs, respectively.
  • the antisense molecules are designed so as to interfere with transcription or translation of AR or ⁇ FGF upon hybridization with the target.
  • 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 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.
  • 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.
  • the present invention depends, in part, upon the discovery that the selective inhibition of the expression of AR or ⁇ FGF by antisense oligonucleotides in prostatic cells effectively inhibits cell growth and/or causes cell death.
  • antisense oligonucleotides Based upon SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4, 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.
  • such antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the AR or ⁇ FGF mRNA transcripts. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region of the AR or ⁇ FGF 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.
  • the AR or ⁇ FGF antisense is, preferably, targeted to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al.
  • SEQ ID NO.: 1 discloses a cDNA sequence
  • SEQ ID NO.: 2 disclose genomic DNA sequences
  • one of ordinary skill in the art may easily derive the genomic DNA corresponding to 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.
  • the present invention also provides for antisense 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.
  • antisense to allelic or homologous cDNAs and genomic DNAs are enabled without undue experimentation.
  • the antisense oligonucleotides of the present invention need not be perfectly complementary to the AR or ⁇ FGF 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 an AR or ⁇ FGF transcript under physiological conditions. Preferably, of course, mismatches are absent or minimal.
  • 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 an AR or ⁇ FGF 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.
  • self-stabilized or hai ⁇ in oligonucleotides are examples of self-stabilized or hai ⁇ in 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' AR or ⁇ 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).
  • the AR and ⁇ FGF 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 intemucleoside 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.
  • modified oligonucleotide describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic intemucleoside 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.
  • a synthetic intemucleoside linkage i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide
  • Preferred synthetic intemucleoside 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.
  • a low molecular weight e.g. 15-500 Da
  • 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.
  • Other low molecular weight organic modifications include additions to the intemucleoside 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).
  • modified oligonucleotide also encompasses oligonucleotides with a covalently modified base and/or sugar.
  • 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.
  • modified oligonucleotides may include a 2'-O-alkylated ribose group such as a 2'-O-methylated ribose.
  • modified oligonucleotides may include sugars such as arabinose instead of ribose.
  • 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.
  • modified oligonucleotides are oligonucleotides having prostate- targeting, nuclease resistance-conferring, or other bulky substituents and/or various other stmctural 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
  • Estramustine targets particularly to the ventral prostate (Forsgren, et al. (1979)).
  • chemotherapeutic agents e.g., dexamethasone, vinblastine, etoposide
  • modified oligonucleotides are hybrid or chimeric oligonucleotides in which some but not all ofthe phosphodiester linkages, bases or sugars have been modified.
  • 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.
  • 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.
  • synthetic linkages may be combined with other modifications, such as alkylation of a 5' or 3' phosphate, or 2'-O- alkylation.
  • one may produce a modified oligonucleotide with the following stmcture, 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:
  • the methods ofthe present invention represent new and useful additions to the field of benign prostate hype ⁇ lasia or prostate cancer therapy.
  • 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 hype ⁇ lasia 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 of the present invention include the isolated antisense oligonucleotides described above.
  • 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.
  • Administration ofthe AR or ⁇ FGF antisense oligonucleotides may be oral, intravenous,
  • parenteral cutaneous or subcutaneous.
  • the administration also may be localized to the prostate or to the region ofthe tumor by injection to or perfusion ofthe site.
  • AR or ⁇ FGF antisense oligonucleotides may be administered as part of a pharmaceutical composition.
  • 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.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness ofthe biological activity ofthe active ingredients.
  • 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 AR or ⁇ FGF 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.
  • cationic detergents e.g. Lipofectin
  • the oligonucleotides will be in the form of a tablet, capsule, powder, solution or elixir.
  • 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 AR and/or ⁇ FGF antisense oligonucleotides and preferably from about 25 to 90% ofthe oligonucleotides.
  • 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.
  • the pharmaceutical composition may contain from about 0.5 to 90% by weight of an AR and or ⁇ FGF antisense oligonucleotide and preferably from about 1 to 50% ofthe oligonucleotide.
  • the oligonucleotides When a therapeutically effective amount of an AR or ⁇ FGF 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.
  • 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.
  • 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.
  • 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.
  • administration is by direct injection at or perfusion ofthe site ofthe targeted cells, such as a tumor.
  • the antisense oligonucleotides may be adhered to small particles (e.g., microscopic gold beads) which are impelled through the membranes of the target cells (see, e.g., U.S. Pat. No. 5,149,655).
  • a recombinant gene is constmcted which encodes an AR or ⁇ FGF antisense oligonucleotide and this gene is introduced within the targeted cells on a vector.
  • an AR or ⁇ FGF antisense gene may, for example, consist ofthe normal AR or ⁇ FGF 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.
  • the AR and/or ⁇ FGF antisense oligonucleotides are administered in therapeutically effective amounts.
  • 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 AR and/or ⁇ FGF 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 of the present invention should contain about 1.0 ⁇ g 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 ofthe present invention.
  • antisense oligonucleotides substantially complementary to subsets of SEQ ID NO.: 1, SEQ ID NO.: 2.
  • SEQ ID NO.: 3 or SEQ ID NO.: 4 and chemical modifications ofthe same which do not prevent hybridization under physiological conditions are contemplated as equivalents ofthe examples presented below.
  • the use of prostate specific antisense oligonucleotides is contemplated as a method of selectively inhibiting the growth of or killing prostatic cells.
  • antisense oligonucleotides to the estrogen receptor, PSA, probasin, telomerase, prohibitin, src, ras, myc, blc-2, protein kinase- A, pla ' sminogenctivator urokinase and methyl transferase genes is contemplated for the treatment of benign prostatic hype ⁇ lasia or prostatic cancer.
  • the PC3-1435 permanent cell line of human prostatic cancer obtained from the 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 semm, glutamate, pymvate, penicillin and streptomycin, in 25-150 cm flasks, incubated at 37°C in 6 percent CO 2 -air.
  • SEQ ID NO.: 5 is antisense to positions 927-953 ofthe AR gene (SEQ ID NO.: 1).
  • SEQ ID NO.: 6 is a self-stabilized or hai ⁇ in oligonucleotide. The first 21 bases are complementary to positions 916-936 ofthe AR gene. The remaining eight are identical to positions 920-927 ofthe gene, allowing formation of a 3' hai ⁇ in.
  • SEQ ID NO.: 7 is another self-stabilized antisense oligonucleotide.
  • SEQ ID NO.: 8 is an antisense sequence corresponding to positions 611-635 of the ⁇ FGF gene.
  • 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 organic chemical groups (e.g., 2-hydroxy-3-amino- propyl, propylamine) were added to the 3' terminal phosphate or the penultimate 3' phosphate.
  • small organic chemical groups e.g., 2-hydroxy-3-amino- propyl, propylamine
  • ADDRESSEE WOLF, GREENFIELD & SACKS, P.C.
  • B STREET: 600 ATLANTIC AVENUE
  • TAATAACTCA GTTCTTATTT GCACCTACTT CAGTGGACAC TGAATTTGGA AGGTGGAGGA 60 TTTTGTTTTT TTCTTTTAAG ATCTGGGCAT CTTTTGAATC TACCCTTCAA GTATTAAGAG 120
  • AAG CCC ATC TAT TTC CAC ACC CAG TGAAGCATTG GAAACCCTAT TTCCCCACCC 3149 Lys Pro He Tyr Phe His Thr Gin 915 920 CAGCTCATGC CCCCTTTCAG ATGTCTTCTG CCTGTTATAA CTCTGCACTA CTCCTCTGCA 3209
  • MOLECULE TYPE DNA (genomic)
  • CTCTATGGCA CCCCCCTTCC CTTTCTGACA TCTTCTGTAG TCAAGGTGGG AGGAAGGTGC 840
  • MOLECULE TYPE DNA (genomic)
  • CTGCCGGGTC CTATCGGCAA AAGCGTAGTG TTTATTTACT TTTGCTCGTG TTATTTTTAT 180 TCCAGTTCAG CTGCAGCTCA GTGCGGAAAG CGTGGGGGAG GTGTATATAA AGAGTACCGA 240
  • GCTGACATGC TTCCAGACGT TGGCCAAGGT TTGAGGTTTC CAGAAATCTT GTTACATGGA 360
  • MOLECULE TYPE DNA (genomic)
  • TAGCAGACAC CAAATGAGGA ATGTTTGTTC CTGGAAAGGC TGGAGGAGAA CCATTACAAC 360

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Abstract

L'invention porte sur un procédé inhibant sélectivement la croissance des cellules prostatiques ou les tuant à l'aide d'oligonucléotides antisens de gènes spécifiques de la prostate. Lesdits oligonucléotides peuvent présenter des structures d'acides nucléiques naturels ou peuvent être des oligonucléotides modifiés à stabilité renforcée ou ciblant des tissus spécifiques. Les gènes spécifiques de la prostate vers lesquels les antisens peuvent être dirigés comportent les gènes AR et αFGF. L'invention porte également sur des préparations pharmaceutiques comprenant lesdits oligonucléotides antisens utilisées dans ces procédés. Lesdits procédés et produits s'avèrent notamment utiles pour le traitement de l'hypertrophie et du cancer de la prostate.
EP96935879A 1995-09-20 1996-09-20 Chimiotherapie par oligonucleotides antisens de l'hypertrophie ou du cancer de la prostate Withdrawn EP0851919A1 (fr)

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US401895P 1995-09-20 1995-09-20
PCT/US1996/015081 WO1997011170A1 (fr) 1995-09-20 1996-09-20 Chimiotherapie par oligonucleotides antisens de l'hypertrophie ou du cancer de la prostate
US4018 2001-10-18

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US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
AUPP249298A0 (en) 1998-03-20 1998-04-23 Ag-Gene Australia Limited Synthetic genes and genetic constructs comprising same I
JP4187413B2 (ja) 1998-03-20 2008-11-26 コモンウェルス サイエンティフィック アンドインダストリアル リサーチ オーガナイゼーション 遺伝子発現の制御
CN1213057C (zh) * 1998-11-09 2005-08-03 中国人民解放军军事医学科学院放射医学研究所 抑制端粒酶活性反义寡核苷酸结构及用途
US6423885B1 (en) 1999-08-13 2002-07-23 Commonwealth Scientific And Industrial Research Organization (Csiro) Methods for obtaining modified phenotypes in plant cells
DK1309726T4 (en) 2000-03-30 2019-01-28 Whitehead Inst Biomedical Res RNA Sequence-Specific Mediators of RNA Interference
CZ308053B6 (cs) 2000-12-01 2019-11-27 Max Planck Gesellschaft Izolovaná molekula dvouřetězcové RNA, způsob její výroby a její použití
EP1229134A3 (fr) 2001-01-31 2004-01-28 Nucleonics, Inc Utilisation de l'inhibition post-transcriptionnelle pour l'identification des séquences d'acides nucléiques qui modifient la fonction d'une cellule
US8729036B2 (en) 2002-08-07 2014-05-20 University Of Massachusetts Compositions for RNA interference and methods of use thereof
AR041407A1 (es) * 2003-09-26 2005-05-18 Nestor Alberto Kerner Oligonucleotidos antiandrogenos utilizables en el tratamiento de patologias dermatologicas relacionadas con el metabolismo de los androgenos, sus composiciones farmaceuticas y sus usos y metodos de tratamiento
US20100286229A1 (en) * 2005-09-30 2010-11-11 Katerina Gurova Modulation of Androgen Receptor for Treatment of Prostate Cancer
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UA100253C2 (uk) 2007-11-26 2012-12-10 Сантаріс Фарма А/С Lna-антагоністи андрогенного рецептора
AU2011326034B2 (en) 2010-11-12 2016-02-04 Roche Innovation Center Copenhagen A/S Compositions and methods for treating androgen receptor dependent disorders including cancers
AR092982A1 (es) 2012-10-11 2015-05-13 Isis Pharmaceuticals Inc Modulacion de la expresion de receptores androgenicos
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