WO1993009789A1 - Treatment of melanoma with antisense oligonucleotides to c-myb proto-oncogene - Google Patents

Treatment of melanoma with antisense oligonucleotides to c-myb proto-oncogene Download PDF

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WO1993009789A1
WO1993009789A1 PCT/US1992/009656 US9209656W WO9309789A1 WO 1993009789 A1 WO1993009789 A1 WO 1993009789A1 US 9209656 W US9209656 W US 9209656W WO 9309789 A1 WO9309789 A1 WO 9309789A1
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seq
oligonucleotide
mer
oligodeoxynucleotide
myb
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PCT/US1992/009656
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French (fr)
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Alan M. Gewirtz
Bruno Calabretta
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Temple University - Of The Commonwealth System Of Higher Education
The University Of Pennsylvania
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Priority to JP5509357A priority Critical patent/JPH07501525A/en
Priority to CA002123611A priority patent/CA2123611A1/en
Priority to EP92924379A priority patent/EP0667778A4/en
Publication of WO1993009789A1 publication Critical patent/WO1993009789A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N15/1135—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 oncogenes or tumor suppressor genes
    • 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
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the invention relates to antisense oligonucleotides to proto-oncogenes, in particular antisense oligonucleotides to the c-myb gene, and the use 10 of such oligonucleotides as antineoplastic agents.
  • the proto-oncogene c-myb is the normal cellular 20 homologue of the avian myeloblastosis virus-transforming gene v-myb.
  • the c-myb gene codes for a nuclear protein expressed primarily in hematopoietic cells. It is a proto-oncogene, that is, it codes for a protein which is required for the survival of normal, non-tumor cells. 25 When the gene is altered in the appropriate manner, it has the potential to become an oncogene.
  • Oncogenes are genes whose expression within a cell provides some function in the transformation from normal to tumor cell.
  • the human c-myb gene has been isolated, cloned, 30 and sequenced. Majello et al. , Proc. Natl.
  • Antisense oligonucleotides to human c-myb mRNA that is, oligonucleotides having a nucleotide sequence complementary to the mRNA transcript of the c-myb gene, are disclosed in our allowed, co- pending application Serial No. 427,659, filed October 27, 1989, and corresponding international patent application WO90/05445, the entire disclosures of which are incorporated herein by reference.
  • C-myb antisense oligonucleotides are disclosed therein as being useful for the treatment of hematologic neoplasms, and for immunosuppression.
  • Melanoma also known as "malignant melanoma” or “cutaneous melanoma” is a neoplasm of melanocytes that has the potential for invasion and metastasis.
  • Melanocytes are elanosome-containing cells that specialize in the biosynthesis and transport of melanin pigment. Melanocytes reside in the skin at the basal layer of the epidermis. Under a variety of stimuli, they elaborate melanin pigment. Melanin synthesis occurs on the melanosome, a well-defined intracellular organelle within the melanosome.
  • the rate of increase in the incidence of melanoma is greater than for any other cancer, with the exception of bronchogenic carcinoma.
  • the incidence of melanoma is greatest among Caucasians, and is influenced by ultraviolet light exposure, and by geographical and occupational factors.
  • the incidence of melanoma is increasing rapidly in the United states and elsewhere, with an apparent doubling every ten to seventeen years.
  • melanoma accounts for roughly one percent of cancers in the United States, and about the same proportion of cancer deaths. While it represents only about three percent of cutaneous neoplasms, melanoma accounts for two thirds of all skin cancer fatalities. For the most part, melanoma first progresses through a radial growth phase at the site of the primary lesion.
  • This initial phase is characterized by little or no competence to metastasize.
  • Melanomas in this phase are generally treatable by surgical procedures.
  • a primary melanoma acquires competence to metastasize.
  • Surgery alone is ineffective in treating the melanoma, once metastasis has occurred.
  • Chemotherapy either alone or in combination with surgery, has been utilized in the treatment of melanoma.
  • Dimethyltriazeno-imidazole (DTIC) is the most active single agent for the treatment of metastatic melanoma, with an overall objective response rate of only 21%. DTIC can cause disturbances in liver function. It possesses modest hematologic toxicity.
  • nitrosoureas Somewhat less effective in treating malignant melanoma are the synthetic nitrosoureas, of which 1,3- bis(2-chloroethyl) -1-nitrosourea (BCNU) , l-(2- chloroethyl)-3-cyclohexyl-l-nitrourea (CCNU) , methyl-CCNU and chlorozotocin are best known.
  • BCNU 1,3- bis(2-chloroethyl) -1-nitrosourea
  • CCNU l-(2- chloroethyl)-3-cyclohexyl-l-nitrourea
  • chlorozotocin chlorozotocin
  • the invention provides a method for treating melanoma.
  • An effective amount of one or more c-myb antisense oligonucleotides is administered to an individual in need of such treatment.
  • Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene.
  • the oligonucleotide is hybridizable to the mRNA transcript.
  • the oligonucleotide is at least a 12-mer oligonucleotide, that is, an oligomer containing at least 12 nucleotide residues.
  • the oligomer is advantageously a 12-mer to a 40-mer, preferably an oligodeoxynucleotide. While oligonucleotides smaller than 12-mers may be utilized, they are statistically more likely to hybridize with non- targeted sequences, and for this reason may be less specific. In addition, a single mismatch may destabilize the hybrid. While oligonucleotides larger than 40-mers may be utilized, uptake may be more difficult. Moreover, partial matching of long sequences may lead to non ⁇ specific hybridization, and non-specific effects.
  • the oligonucleotide is a 15- to 30-mer oligodeoxynucleotide, more advantageously an 18- to 26- mer.
  • oligonucleotides having a sequence complementary to any region of the c-myb gene find utility in the present invention
  • oligodeoxynucleo ⁇ tides complementary to a portion of the c-myb mRNA trans ⁇ cript including the translation initiation codon are particularly preferred.
  • the invention is also a method for purging bone marrow of metastasized melanoma cells.
  • Bone marrow aspirated from amelanoma-inflicted individual is treated with an effective amount of c-myb antisense oligonucleotide, and the thus-treated cells are then returned to the body of the afflicted individual.
  • the bone marrow purging technique may be utilized for an autologous bone marrow rescue (transplantation) , in connection with a course of high dose chemotherapy.
  • the invention is also a composition for the treatment of melanoma comprising a pharmaceutically acceptable carrier and c-myb antisense oligonucleotide.
  • oligonucleotide includes both oligomers of ribonucleotides, i.e., oligoribonucleotides, and oligomers of deoxyribonucleotides, i.e., oligo- deoxyribonucleotides (also referred to herein as "oligo- deoxynucleotides”) . Oligodeoxynucleotides arepreferred. As used herein, unless otherwise indicated, the term “oligonucleotide” also includes oligomers which may be large enough to be termed "polynucleotides”.
  • oligonucleotide and “oligodeoxynuc- leotide” include not only oligomers and polymers of the common biologically significant nucleotides, i.e., the nucleotides adenine ("A") , deoxyadenine ("dA”) , guanine (“G”) , deoxyguanine (“dG”) , cytosine (“C”) , deoxycytosine (“dC”), thymine (“T”) and uracil (“U”) , but also include oligomers and polymers hybridizable to the c-myb mRNA transcript which may contain other nucleotides.
  • A adenine
  • dA deoxyadenine
  • G guanine
  • dG deoxyguanine
  • C cytosine
  • C deoxycytosine
  • T thymine
  • U uracil
  • oligonucleotide and “oligodeoxynucleotide” includes oligomers and polymers wherein one or more purine or pyrimidine moieties, sugar moieties or internucleotide linkages is chemically modified.
  • oligonucleotide is thus understood to also include oligomers which may properly be designated as “oligonucleosides" because of modification of the internucleotide phosphodiester bond.
  • modified oligonucleotides include, for example, the methylphosphonate oligonucleosides, discussed below.
  • phosphorothioate oligonucleotide means an oligonucleotide wherein one or more of the internucleotide linkages is a phosphorothioate group, 0
  • methylphosphorateoligonucleoside ismeant an oligonucleotide wherein one or more of the internucleotide linkages is a methylphosphonate group
  • downstream when used in reference to a direction along a nucleotide sequence means the 5' ⁇ 3' direction.
  • upstream means the 3' ⁇ 5' direction.
  • c-myb mRNA transcript means the presently known mRNA transcript of the human c-myb gene, or any further transcripts which may be elucidated.
  • Fig. 1 is a graph of the effect of c-myb sense and antisense oligonucleotide on human melanoma cells
  • Control (ii) the indicated concentrations of an 18- er oligodeoxynucleotide complementary to codons 2-7 of the translated position of the c-myb mRNA transcript ("Antisense”) , or (iii) the corresponding sense 18-mer (“Sense”) .
  • Fig. 2 is similar to Fig. 1, except that the oligonucleotide treatment was extended for two days.
  • the oligonucleotide concentrations are cumulative of the two day treatment period.
  • Fig. 3 is similar to Fig. 2, except that the treatment was extended to five consecutive days. The oligonucleotide concentrations are cumulative.
  • Fig. 4 is similar to Fig. 1, except that another human melanoma cell line (SK MEL-37) was substituted for the CHP cells.
  • SK MEL-37 human melanoma cell line
  • Fig. 5 is a plot of the effect of c-myb sense and antisense oligomers on the growth of human melanoma
  • mice received 100 ⁇ g per day for 7 days of (i) a 24-mer phosphorothioate oligodeoxynucleotide complementary to codons 2-9 of the translated portion of the c-myb mRNA transcript
  • Antisense 2 mice
  • Sense 24- mer phosphorothioate oligodeoxynucleotide
  • Control no oligomer
  • the putative DNA sequence complementary to the mRNA transcript of the human c-myb gene has been reported in Majello et al. , Proc. Natl. Acad. Sci. U.S.A. 83, 9636-9640 (1986) .
  • Majello et aJL. further disclose the predicted 640 amino acid sequence of the putative c-myb protein.
  • the initiation codon ATG appears at position 114, preceded by a 5'-untranslated region.
  • the termination codon TGA at position 2034 is followed by a 3'-untranslated region spanning about 1200 nucleotides, which is followed by a poly(A) tail of about 140 nucleotides .
  • the antisense oligonucleotides of the invention may be synthesized by any of the known chemical oligonu- cleotide synthesis methods. Such methods are generally described, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technology. VCH Verlagsges- ellschaft mbH (Ibelgaufts trans. 1987).
  • the antisense oligonucleotides are most advantageously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers.
  • One such device, the Applied Biosyste s 380B DNA Synthesizer utilizes ?-cyanoethyl phosphoramidite chemistry.
  • an ⁇ tisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by oligonucleotide synthesis methods known to those skilled in the art.
  • oligonucleotide While any length oligonucleotide may be utilized in the practice of the invention, sequences shorter than 12 bases may be less specific in hybridizing to the target c-myb mRNA, may be more easily destroyed by enzymatic digestion, and may be destabilized by enzymatic digestion. Hence, oligonucleotides having 12 or more nucleotides are preferred.
  • oligomers of 12-40 nucleotides are preferred, more preferably 15-30 nucleotides, most preferably 18-26 nucleotides. While sequences of 18-21 nucleotides are most particularly preferred, for unmodified oligonucleotides, slightly longer chains of up to about 26 nucleotides, are preferred for modified oligonucleotides such as phosphorothioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides.
  • Oligonucleotides complementary to and hybridizable with any portion of the c-myb mRNA transcript are, in principle, effective for inhibiting translation of the transcript, and capable of inducing the effects herein described. It is believed that translation is most effectively inhibited by blocking the mRNA at a site at or near the initiation codon. Thus, oligonucleotides complementary to the 5'-terminal region of the c-myb mRNA transcript are preferred. It is believed that secondary or tertiary structure which might interfere with hybridization is minimal in this region.
  • sequences that are too distant in the 3'-direction from the initiation site may be less effective in hybridizing the mRNA transcripts because of a "read-through” phenomenon whereby the ribosome is postulated to unravel the antisense/sense duplex to permit translation of the message. See, e.g., Shakin, J. Biochemistry 261, 16018 (1986).
  • the antisense oligonucleotide is preferably directed to a site at or near the initiation codon for protein synthesis. Oligonucleotides complementary to the c-myb mRNA, including the initiation codon (the first codon at the 5' end of the translated portion of the c- myb transcript, comprising nucleotides 114-116 of the complete transcript) are preferred.
  • antisense oligomers complementary to the 5'-terminal region of the c-myb transcript are preferred, particularly the region including the initiation codon, it should be appreciated that useful antisense oligomers are not limited to those complementary to the sequences found in the translated portion (nucleotides 114 to 2031) of the mRNA transcript, but also includes oligomers complementary to nucleotide sequences contained in, or extending into, the 5'-and 3'-untranslated regions. Oligomers whose complementarity extends into the 5'- untranslated region of the c-myb transcript are believed particularly effective in inhibiting c-myb translation.
  • Preferredoligonucleotidescomplementarytothe 5 '-untranslated region of the transcript include molecules having a nucleotide sequence complementary to a portion of the c-myb mRNA transcript including the cap nucleotide, that is, the nucleotide at the extreme 5'-end of the transcript.
  • nucleotide sequence of the mRNA transcript 5'-terminus beginning with the cap nucleotide may be readily established, and antisense oligonuc ⁇ leotides complementary and hybridizable thereto may be prepared.
  • the following 40-mer oligodeoxynucleotide is complementary to the c-myb mRNA transcript beginning with the initiation codon of the transcript and extending downstream thereof (in the 3' direction): SEQ ID NO:l.
  • oligomers based upon the above sequence, in particular, oligomers hybridizable to segments of the c-myb message containing the initiation codon, may be utilized. Particularly preferred are the following 26- to 15-mers: SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
  • Oligodeoxynucleotides complementary to the c- myb mRNA transcript beginning with the second codon of the translated portion of the transcript are another group of preferred oligomers.
  • Such oligomers include, for example, the following 26- to 15-mers: SEQ ID NO: 14,
  • SEQ ID NO:20 SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and
  • oligonucleotide employed may represent an un ⁇ modified or modified oligonucleotide.
  • oligo- nucleotides hybridizable to the c-myb mRNA transcript finding utility according to the present invention include not only oligomers of the biologically sig- nificant native nucleotides, i.e.. A, dA, G, dG, C, dC, T and U, but also oligonucleotide species which have been modified for improved stability and/or lipid solubility.
  • alkylphosphonate oligonucleoside or allylphosphotriester oligonucleotide For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting an alkyl group or alkoxy group for a phos ⁇ phate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate oligonucleoside or allylphosphotriester oligonucleotide.
  • Non-ionic oligonucleotides such as these are characterized by increased resistance to nuclease hydrolysis and/or increased cellular uptake, while retaining the ability to form stable complexes with complementary nucleic acid sequences.
  • the alkylphosphonates in particular, are stable to nuclease cleavage and soluble in lipid.
  • the preparation of alkylphosphonate oligonucleosides is disclosed in U.S. Patent 4,469,863.
  • Methylphosphonate oligomers can be prepared by a variety of methods, both in solution and on insoluble polymer supports (Agrawal and Riftina, Nucl. Acids Res. , 6, 3009-3024 (1979); Miller et ______ , Biochemistry. 18,
  • methylphosphonate oligonucleosides The most efficient procedure for preparation of methylphosphonate oligonucleosides involves use of 5'-0- dimethoxytrityldeoxynucleoside-3 ' - 0_- diisopropylmethylphosphoramidite intermediates, which are similar to the methoxy or ⁇ -cyanoethyl phosphoramidite reagents used to prepare oligodeoxyribonucleotides.
  • the methylphosphonate oligomers can be prepared on controlled pore glass polymer supports using anautomated DNA synthesizer (Sarin et al . , Proc. Natl. Acad. Sci. USA, 85, 7448-7451 (1988)).
  • Resistance to nuclease digestion may also be achieved by modifying the internucleotide linkage at both the 5' and 3' termini with phosphoroamidites according to the procedure of Dagle et al., Nucl. Acids Res. 18, 4751-4757 (1990) .
  • Phosphorothioate oligonucleotides contain a sulfur- for-oxygen substitution in the internucleotide phosphodiester bond. Phosphorothioate oligonucleotides combine the properties of effective hybridization for duplex formation with substantial nuclease resistance, while retaining the water solubility of a charged phosphate analogue. The charge is believed to confer the property of cellular uptake via a receptor (Loke et aJL. , Proc. Natl. Acad. Sci. U.S.A. 86, 3474-3478 (1989)). Phosphorothioate oligodeoxynucleotide are described by LaPlanche, et al.
  • oligoribonucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-0- methylribonucleotides (Inove et al. , Nucleic Acids Res. 15, 6131 (1987) and chimeric oligonucleotides that are composite RNA-DNA analogues (Inove et aJL. , FEBS Lett. 215, 327 (1987) .
  • oligodeoxyribonucleotides are preferred in the practice of the present invention. Oligodeoxyribonucleotides are further preferred because, upon hybridization with c-myb mRNA, the resulting DNA-RNA hybrid duplex is a substrate for RNase H, which specifically attacks the RNA portion of DNA-RNA hybrid. Degradation of the mRNA strand of the duplexreleases the antisense oligodeoxynucleotide strand for hybridization with additional c-myb messages.
  • the antisense oligonucleotides used in the method of the present invention will have a sequence which is completely complementary to the target portion of the c-myb message. Absolute complementarity is not however required, particularly in larger oligomers.
  • reference herein to a "nucleotide sequence complementary to at least a portion of the mRNA transcript" of c-myb does not necessarily mean a sequence having 100% complementarity with the transcript. In gene- ral, any oligonucleotide having sufficient com ⁇ plementarity to form a stable duplex with c-myb mRNA is suitable.
  • Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity with the target region of the c-myb message. Generally, the larger the hybridizing oligomer, the more mismatches may be tolerated. More than one mismatch probably will not be tolerated for antisense oligomers of less than about 21 nucleotides.
  • One skilled in the art may readily determine the degree of mismatching which may be tolerated between any given antisense oligomer and the target c-myb message sequence, based upon the melting point, and therefore the stability, of the resulting duplex. Melting points of duplexes of a given base pair composition can be readily determined from standard texts, such as Molecular Cloning: A Laboratory Manual. (2nd edition, 1989), J. Sambrook et al. , eds.
  • oligonucleotides capable of stable hybridiza ⁇ tion with any region of the c-myb message are within the scope of the present invention, oligonucleotides complementary to a region including the initiation codon are believed particularly effective. Particularly preferred are oligonucleotides hybridizable to a region of the c-myb mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direction) of that codon.
  • the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • a suitable liquid vehicle or excipient such as distilled water, physiological saline, aqueous solution of dextrose, and the like.
  • the c-myb mRNA antisense oligonucleotides are preferably administeredparenterally, mostpreferably intravenously.
  • the vehicle is designed accordingly.
  • oligonucleotide may be administered subcutaneously via controlled release dosage forms.
  • the oligonucleotides may be conjugated to poly(L- lysime) to increase cell penetration.
  • Such conjugates are described by Lemaitre et al. , Proc. Natl. Acad. Sci. USA, 84, 648-652 (1987).
  • the procedure requires that the 3'-terminal nucleotide be a ribonucleotide.
  • the resulting aldehyde groups are then randomly coupled to the epsilon-amino groups of lysine residues of poly(L- lysine) by Schiff base formation, and then reduced with sodium cyanoborohydride. This procedure converts the 3'- terminal ribose ring into a morpholine structure antisense oligomers.
  • the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques.
  • oligonucleotides may be encapsulated in for therapeutic delivery.
  • the oligonucleotide depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension.
  • the hydrophobic layer generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • Oligonucleotides have been successfully encapsulated in unilameller liposomes. Reconstituted Sendaivirus envelopes havebeen suc ⁇ cessfully used to deliver RNA and DNA to cells. Arad et al., Biochem. Biophy. Acta. 859, 88-94 (1986).
  • the c-myb antisense oligonucleotides may be administered de novo as the primary therapy.
  • the oligonucleotides may be administered as an adjuvant following surgical removal of a melanoma to patients who may be disease-free but at high risk of recurrence.
  • a preferred method of administration of oligonucleotide for treatment of melanoma comprises either regional or systemic perfusion, as is appropriate.
  • the afferent and efferent vessels supplying the extremity containing the lesion are isolated and connected to a low-flow perfusion pump in continuity with an oxygenator and a heat exchanger.
  • the iliac vessels may be used for perfusion of the lower extremity.
  • the axillary vessels are cannulated high in the axilla for upper extremity lesions.
  • Oligonucleotide is added to the perfusion circuit, and the perfusion is continued for an appropriate time period, e.g. , one hour.
  • Perfusion rates of from 100 to 150 ml/minute may be employed for lower extremity lesions, while half that rate should be employed for upper extremity lesions.
  • Systemic heparinization may be used throughout the perfusion, and reversed after the perfusion is complete. This isolation perfusion technique permits administration of higher doses of chemotherapeutic agent than would otherwise be tolerated upon infusion into the arterial or venous sys ⁇ temic circulation.
  • the oligonucleotides are preferably delivered via a central venous catheter, which is connected to an appropriate continuous infusion device.
  • Indwelling catheters provide long term access to the intravenous circulation for frequent administration of drugs over extended time periods. They are generally surgically inserted into the external cephalic or internal jugular vein under general or local anesthesia.
  • the subclavian vein is another common site of catheterization.
  • the infuser pump may be external, or may form part of an entirely implantable central venous system such as the INFUSAPORT system available from Infusaid Corp., Norwood, MA and the PORT-A-CATH system available from Pharmacia Laboratories, Piscataway, NJ. These devices are implanted into a subcutaneous pocket under local anesthesia.
  • the implant contains a supply of oligonucleotide in a reservoir which may be replenished as needed by injection of additional drug from a hypodermic needle through a self-sealing diaphragm in the reservoir.
  • Completely implantable infusers are preferred, as they are generally well accepted by patients because of the convenience, ease of maintenance and cosmetic advantage of such devices.
  • High dose chemotherapy has been coupled with autologous bone marrow rescue (transplantation) in an attempt to treat melanoma. While high dose chemotherapy has resulted in significantly higher therapeutic responses, patient survival is not generally prolonged: Jones et al . . , Cancer Chemother. Pharmacol.
  • c-myb antisense oligonucleotides may be used as bone marrow purging agents for the in vitro cleansing of bone marrow of melanoma cells in conjunction with high dose conventional chemotherapy. While normal hematopoietic cells are sensitive to c-myb antisense, they are less sensitive than malignant cells expressing c-myb, as taught in our copending patent application Serial No.
  • bone marrow is harvested from a donor by standard operating room procedures from the iliac bones of the donor.
  • Methods of aspirating bone marrow from donors are well- known in the art. Examples of apparatus and processes for aspirating bone marrow from donors are disclosed in U.S. Patents 4,481,946 and 4,486,188, incorporated herein by reference.
  • Sufficient marrow is withdrawn so that the recipient, who is either the donor (autologous transplant) or another individual (allogeneic transplant) , may receive from about 4 x 10 8 to about 8 x 10 8 processed marrow cells per kg of bodyweight. This generally requires aspiration of about 750 to about 1000 ml of marrow. The aspirated marrow is filtered until a single cell suspension, known to those skilled in the art as a "buffy coat" preparation, is obtained. This suspension of leukocytes is treated with c-myb antisense oligonucleotides in a suitable carrier, advantageously in a concentration of about 8 mg/ml.
  • the leucocyte suspension may be stored in liquid nitrogen using standard procedures known to those skilled in the art until purging is carried out.
  • the purged marrow can be stored frozen in liquid nitrogen until ready for use. Methods of freezing bone marrow and biological substances are disclosed, for example, in U.S. Patents 4,107,937 and 4,117,881.
  • bone marrow for treat- ment with c-myb antisense may be utilized, which methods may result in even more purified preparations of hemato ⁇ poietic cells than the aforesaid buffy coat preparation.
  • One or more growth factors may be added to the aspirated marrow or buffy coat preparation to stimulate growth of neoplasms, and thereby increase their sensi ⁇ tivity to the toxicity of the c-myb antisense oligonucleotides.
  • the cells to be transferred are washed with auto- logous plasma or buffer to remove unincorporated oligomer.
  • the washed cells are then infused back into the patient.
  • the c-myb antisense oligonucleotides may be administered in a dosage effective for inhibiting the proliferation of melanoma cells in the afflicted individual, while maintaining the viability of normal cells. Such amounts may vary depending on the nature and extent of the neoplasm, the particular oligonucleotide utilized, and other factors.
  • the actual dosage admin ⁇ istered may take into account the size and weight of the patient, whether the nature of the treatment is prophy- lactic or therapeutic in nature, the age, health and sex of the patient, the route of administration, whether the treatment is regional or systemic, and other factors. Inhibition of melanoma cell proliferation has been observed at antisense concentrations of as low as 10 ⁇ g/ml. At 20 ⁇ g/ml, inhibition was profound. Concen ⁇ trations of from about 1 to about 100 ⁇ g/ml may be employed, preferably from about 10 ⁇ g/ml to about 100 ⁇ g/ml, most preferably from about 20 ⁇ g/ml to about 60 ⁇ g/ml.
  • the patient should receive a sufficient daily dosage of antisense oligonucleotide to achieve these intercellular concentrations of drug.
  • the daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 700 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required.
  • a course of treatment may advantageously comprise infusion of the recommended daily dose of oligonucleotide for a period of from about 3 to about 28 days, more preferably from about 7 to about 10 days.
  • a daily dose of about 350 mg oligonucleotide is believed sufficient, to achieve an effective intercellular concentration of 20 ⁇ g.
  • the c-myb antisense oligonucleotides may be administered in amounts effective to kill neoplastic cells while maintaining the viability of normal hematologic cells. Such amounts may vary depending on the extent to which melanoma cells may have metastasized to the bone marrow, the particular oligonucleotide utilized, the relative sensitivity of the neoplastic cells to the oligo ⁇ nucleotide, and other factors. Concentrations from about 10 to 200 ⁇ g/ml per 10 5 cells may be employed, preferably from about 40 to 150 ⁇ g/ml per 10 5 cells.
  • Supplemental dosing of the same or lesser amounts of oligonucleotide are advantageous to optimize the treatment.
  • dosages of from about 2 to 40 mg antisense per ml of marrow may be effectively utilized, preferably from about 8 to 24 mg/ml.
  • Greater or lesser amounts of oligonucleotide may be employed.
  • CHP melanoma cells (Children's Hospital of Philadelphia, Philadelphia, PA) were grown in RPMI culture medium containing 2% oxalophosphate and 5% bovine calf serum at 37°C in 5% C0 2 . Cells (5,000/ml) were seeded in 200 ⁇ l volumes into individual wells of a 96 well Costar plate at day -3, and allowed to grow for three days.
  • the unmodified antisense 18-mer oligodeoxynucleotide (SEQ ID NO:22) complementary to codons 2-7 of the translated portion of the c-myb mRNA, or the corresponding sense 18-mer (SEQ ID NO:26) were added to the cultures at concentrations of 0, 10, 20, 50 and 100 ⁇ g/ml for 1, 2 or 5 consecutive days. On day 7, 100 ⁇ l of fresh medium was added to the cultures. Cell viability/proliferation was determined on day 8 utilizing a commercially available kit (CELLTITER 96TM Promega, Madison, WI) .
  • the assay is based on the ability of viable cells to convert a tetrazolium salt into a blue formazan product which can be quantified by measuring the absorbance at 570 nm with a conventional microplate reader.
  • the extent of absorbance at 570 nm is proportional to the amount of formazan produced, and thus the number of viable cells remaining. Accordingly, 10 ⁇ l of 3-(4,5-dimethylthiazol-2-yl) -2,5 diphenyltetrazolium bromide solution (5 mg/ml) was added to 110 ⁇ l of cell suspension and incubated for 4 hours at 37 ⁇ C.
  • the results of the viability assay are set forth in Figures 1 (one-day treatment) , 2 (two-day treatment) and 3 (five-day treatment) .
  • the oligonucleotide concentrations indicated in Figures 2 and 3 are cumulative dosages for the two- and five-day treatments, respectively. It may be appreciated from the figures that treatment of the melanoma cells with antisense oligomer resulted in sequence-specific killing. The effect was most pronounced when the melanoma cells were treated for one day with an antisense oligomer concen ⁇ tration of at least 50 ⁇ g/ml, or when the cells were treated on consecutive days with as little as 20 ⁇ g/ml oligomer.
  • SK MEL-37 250,000 melanoma cells
  • C.B-17/LCRTAC-SCID DF from Fox Chase Cancer Institute, Philadelphia PA
  • the tumors were allowed to grow to a size of approximately 5 mm in diameter.
  • ALZET constant infusion pumps Alza Corporation, Palo Alto, CA
  • delivering a total dosage of 700 ⁇ g oligomer over 7 days were surgically implanted into the mice.
  • the tumor area was then monitored daily.
  • Another mouse received a 24-mer "sense" phosphorothioate oligonucleotide (corresponding to codons 2-9 of the translated c-myb mRNA) .
  • Two mice received a 24-mer anti- sense phosphorothioate oligonucleotide having the nucleotide sequence of SEQ ID NO: 16 (TATGCTGTGC CGGGGGT- CTTC GGGC) , complementary to c-myb mRNA codons 2-9.
  • the results are shown in Figure 5. Each curve represents one animal. In the two antisense-treated animals, the tumor size stayed the same or regressed. In contrast, the tumors continued to grow in the sense-treated and control animals. The animals were sacrificed on day 19.
  • the tumors were removed and weighed.
  • the weights of the tumors in the control and sense-treated animals were 9.9 and 9.6 grams, respectively.
  • the tumor weights in the two anti- sense treated animals were only 0.1 and 0.2 grams.
  • the following non-limiting example illustrates one methodology for purging bone marrow of metastatic melanoma.
  • Bone marrow is harvested from the iliac bones of a donor under general anesthesia in an operating room using standard techniques. Multiple aspirations are taken into heparinized syringes. Sufficient marrow is withdrawn so that the marrow recipient will be able to receive about 4 x 10 8 to about 8 x 10 8 processed marrow cells per kg of body weight. Thus, about 750 to 1000 ml of marrow is withdrawn. The aspirated marrow is transferred immediately into a transport medium (TC-199, Gibco, Grand Island, New York) containing 10,000 units of preservative-free heparin per 100 ml of medium.
  • TC-199 Gibco, Grand Island, New York
  • the aspirated marrow is filtered through three progressively finer meshes until a single cell suspension results, i.e., a suspension devoid of cellular aggregates, debris and bone particles.
  • the filtered marrow is then processed further into an automated cell separator (e.g.,
  • Cobe 2991 Cell Processor which prepares a "buffy coat" product, (i.e., leukocytes devoid of red cells and platelets) .
  • the buffy coat preparation is then placed in a transfer pack for further processing and storage.
  • the purging procedure may be carried out as follows.
  • Cells in the buffy coat preparation are adjusted to a cell concentration of about 2 x 10 7 / ⁇ LL in TC-199 containing about 20% autologous plasma.
  • C-myb antisense oligodeoxynucleotide for example, in a concentration of about 8 mg/ml, is added to the transfer packs containing the cell suspension.
  • Recombinant human hematopoietic growth factors e.g. , rH IL-3 or rH GM-CSF, may be added to the suspension to stimulate growth of neoplasms and thereby increase their sensitivity c-myb antisense oligonucleotide toxicity.
  • the transfer packs are then placed in a 37°C waterbath and incubated for 18 - 24 hours with gentle shaking.
  • the cells may then either be frozen in liquid nitrogen or washed once at 4°C in TC-199 containing about 20% autologous plasma to remove unincorporated oligomer. Washed cells are then infused into the recipient. Care must be taken to work under sterile conditions wherever possible and to maintain scrupulous aseptic techniques at all times.

Abstract

Melanoma is treated by administering oligonucleotides having a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene. These 'antisense' oligonucleotides are hybridizable to the c-myb mRNA transcript.

Description

I f-
TREAT ENT OF MELANOMA WITH ANTISENSE OLIGONUCLEOTIDES TO C-MYB PROTO-ONCOGENE
5 Field of the Invention
The invention relates to antisense oligonucleotides to proto-oncogenes, in particular antisense oligonucleotides to the c-myb gene, and the use 10 of such oligonucleotides as antineoplastic agents.
Reference to Government Grant
The invention described herein was made in part with government support under grant CA 54384 awarded by 15 National Institutes of Health. The government has certain rights in the invention.
Background of the Invention
The proto-oncogene c-myb is the normal cellular 20 homologue of the avian myeloblastosis virus-transforming gene v-myb. The c-myb gene codes for a nuclear protein expressed primarily in hematopoietic cells. It is a proto-oncogene, that is, it codes for a protein which is required for the survival of normal, non-tumor cells. 25 When the gene is altered in the appropriate manner, it has the potential to become an oncogene. Oncogenes are genes whose expression within a cell provides some function in the transformation from normal to tumor cell. The human c-myb gene has been isolated, cloned, 30 and sequenced. Majello et al. , Proc. Natl. Acad. Sci. U.S.A. 83, 9636-9640 (1986) . Antisense oligonucleotides to human c-myb mRNA that is, oligonucleotides having a nucleotide sequence complementary to the mRNA transcript of the c-myb gene, are disclosed in our allowed, co- pending application Serial No. 427,659, filed October 27, 1989, and corresponding international patent application WO90/05445, the entire disclosures of which are incorporated herein by reference. C-myb antisense oligonucleotides are disclosed therein as being useful for the treatment of hematologic neoplasms, and for immunosuppression.
Melanoma, also known as "malignant melanoma" or "cutaneous melanoma", is a neoplasm of melanocytes that has the potential for invasion and metastasis. Melanocytes are elanosome-containing cells that specialize in the biosynthesis and transport of melanin pigment. Melanocytes reside in the skin at the basal layer of the epidermis. Under a variety of stimuli, they elaborate melanin pigment. Melanin synthesis occurs on the melanosome, a well-defined intracellular organelle within the melanosome.
At one time considered rare, the rate of increase in the incidence of melanoma is greater than for any other cancer, with the exception of bronchogenic carcinoma. The incidence of melanoma is greatest among Caucasians, and is influenced by ultraviolet light exposure, and by geographical and occupational factors. The incidence of melanoma is increasing rapidly in the United states and elsewhere, with an apparent doubling every ten to seventeen years. Presently, melanoma accounts for roughly one percent of cancers in the United States, and about the same proportion of cancer deaths. While it represents only about three percent of cutaneous neoplasms, melanoma accounts for two thirds of all skin cancer fatalities. For the most part, melanoma first progresses through a radial growth phase at the site of the primary lesion. This initial phase is characterized by little or no competence to metastasize. Melanomas in this phase are generally treatable by surgical procedures. In the vertical growth phase, characterized by penetration into deeper cutaneous tissues, a primary melanoma acquires competence to metastasize. Surgery alone is ineffective in treating the melanoma, once metastasis has occurred. Chemotherapy, either alone or in combination with surgery, has been utilized in the treatment of melanoma. Dimethyltriazeno-imidazole (DTIC) is the most active single agent for the treatment of metastatic melanoma, with an overall objective response rate of only 21%. DTIC can cause disturbances in liver function. It possesses modest hematologic toxicity.
Somewhat less effective in treating malignant melanoma are the synthetic nitrosoureas, of which 1,3- bis(2-chloroethyl) -1-nitrosourea (BCNU) , l-(2- chloroethyl)-3-cyclohexyl-l-nitrourea (CCNU) , methyl-CCNU and chlorozotocin are best known. The nitrosoureas display a somewhat more severe hematologic toxicity than DTIC. Response rates range from 10% to 18%.
Unfortunately, there are no single agents or combination regimens that induce a substantial number of complete remissions in melanoma patients. There is an urgent need for the development of more effective agents.
Summary of the Invention The invention provides a method for treating melanoma. An effective amount of one or more c-myb antisense oligonucleotides is administered to an individual in need of such treatment. Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene. The oligonucleotide is hybridizable to the mRNA transcript. Preferably, the oligonucleotide is at least a 12-mer oligonucleotide, that is, an oligomer containing at least 12 nucleotide residues. In particular, the oligomer is advantageously a 12-mer to a 40-mer, preferably an oligodeoxynucleotide. While oligonucleotides smaller than 12-mers may be utilized, they are statistically more likely to hybridize with non- targeted sequences, and for this reason may be less specific. In addition, a single mismatch may destabilize the hybrid. While oligonucleotides larger than 40-mers may be utilized, uptake may be more difficult. Moreover, partial matching of long sequences may lead to non¬ specific hybridization, and non-specific effects. Preferably, the oligonucleotide is a 15- to 30-mer oligodeoxynucleotide, more advantageously an 18- to 26- mer.
While in principle oligonucleotides having a sequence complementary to any region of the c-myb gene find utility in the present invention, oligodeoxynucleo¬ tides complementary to a portion of the c-myb mRNA trans¬ cript including the translation initiation codon are particularly preferred. Also preferred are oligonucleotides complementary to a portion of the c-myb mRNA transcript lying within about 40 nucleotides upstream (the 5' direction) or about 40 nucleotides down¬ stream (the 3' direction) from the translation initiation codon.
The invention is also a method for purging bone marrow of metastasized melanoma cells. Bone marrow aspirated from amelanoma-inflicted individual is treated with an effective amount of c-myb antisense oligonucleotide, and the thus-treated cells are then returned to the body of the afflicted individual. The bone marrow purging technique may be utilized for an autologous bone marrow rescue (transplantation) , in connection with a course of high dose chemotherapy.
The invention is also a composition for the treatment of melanoma comprising a pharmaceutically acceptable carrier and c-myb antisense oligonucleotide.
As used in the herein specification and appended claims, unless otherwise indicated, the term "oligonucleotide" includes both oligomers of ribonucleotides, i.e., oligoribonucleotides, and oligomers of deoxyribonucleotides, i.e., oligo- deoxyribonucleotides (also referred to herein as "oligo- deoxynucleotides") . Oligodeoxynucleotides arepreferred. As used herein, unless otherwise indicated, the term "oligonucleotide" also includes oligomers which may be large enough to be termed "polynucleotides".
Theterms "oligonucleotide" and "oligodeoxynuc- leotide" include not only oligomers and polymers of the common biologically significant nucleotides, i.e., the nucleotides adenine ("A") , deoxyadenine ("dA") , guanine ("G") , deoxyguanine ("dG") , cytosine ("C") , deoxycytosine ("dC"), thymine ("T") and uracil ("U") , but also include oligomers and polymers hybridizable to the c-myb mRNA transcript which may contain other nucleotides. Likewise, the terms "oligonucleotide" and "oligodeoxynucleotide" includes oligomers and polymers wherein one or more purine or pyrimidine moieties, sugar moieties or internucleotide linkages is chemically modified. The term "oligonucleotide" is thus understood to also include oligomers which may properly be designated as "oligonucleosides" because of modification of the internucleotide phosphodiester bond. Such modified oligonucleotides include, for example, the methylphosphonate oligonucleosides, discussed below.
The term "phosphorothioate oligonucleotide" means an oligonucleotide wherein one or more of the internucleotide linkages is a phosphorothioate group, 0
II ~o - P - o~ as opposed to the phosphodiester group
Figure imgf000008_0001
which is characteristic of unmodified oligonucleotides. By "methylphosphorateoligonucleoside" ismeant an oligonucleotide wherein one or more of the internucleotide linkages is a methylphosphonate group,
0
~0 - P - 0~
CH„ The term "downstream" when used in reference to a direction along a nucleotide sequence means the 5'→3' direction. Similarly, the term "upstream" means the 3'→5' direction.
The term "c-myb mRNA transcript" means the presently known mRNA transcript of the human c-myb gene, or any further transcripts which may be elucidated.
Brief Description of the Figures
Fig. 1 is a graph of the effect of c-myb sense and antisense oligonucleotide on human melanoma cells
(CHP) in vitro. Cells were treated with (i) no oligomer
("Control") , (ii) the indicated concentrations of an 18- er oligodeoxynucleotide complementary to codons 2-7 of the translated position of the c-myb mRNA transcript ("Antisense") , or (iii) the corresponding sense 18-mer ("Sense") .
Fig. 2 is similar to Fig. 1, except that the oligonucleotide treatment was extended for two days. The oligonucleotide concentrations are cumulative of the two day treatment period. Fig. 3 is similar to Fig. 2, except that the treatment was extended to five consecutive days. The oligonucleotide concentrations are cumulative.
Fig. 4 is similar to Fig. 1, except that another human melanoma cell line (SK MEL-37) was substituted for the CHP cells.
Fig. 5 is a plot of the effect of c-myb sense and antisense oligomers on the growth of human melanoma
(SK MEL-37) cells transplanted into severe combined immunodeficient mice. The mice received 100 μg per day for 7 days of (i) a 24-mer phosphorothioate oligodeoxynucleotide complementary to codons 2-9 of the translated portion of the c-myb mRNA transcript
("Antisense"; 2 mice), (ii) the corresponding sense 24- mer phosphorothioate oligodeoxynucleotide ("Sense"; 1 mouse) or (iii) no oligomer ("Control"; 1 mouse).
Detailed Description of the invention
It has now been discovered that the expression of the human c-myb gene is important for the proliferation of malignant melanoma. The role of this proto-oncogene in cell proliferation is not restricted to cells of hematopoietic origin, as previously thought. The proliferation of malignant melanocytes, which are neoplastic cells of epidermal and not hematologic origin, is maintained by c-myb expression. Thus, the role of c- myb is more general than previously thought.
The putative DNA sequence complementary to the mRNA transcript of the human c-myb gene has been reported in Majello et al. , Proc. Natl. Acad. Sci. U.S.A. 83, 9636-9640 (1986) . Majello et aJL. further disclose the predicted 640 amino acid sequence of the putative c-myb protein. The initiation codon ATG appears at position 114, preceded by a 5'-untranslated region. The termination codon TGA at position 2034 is followed by a 3'-untranslated region spanning about 1200 nucleotides, which is followed by a poly(A) tail of about 140 nucleotides .
The antisense oligonucleotides of the invention may be synthesized by any of the known chemical oligonu- cleotide synthesis methods. Such methods are generally described, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technology. VCH Verlagsges- ellschaft mbH (Ibelgaufts trans. 1987). The antisense oligonucleotides are most advantageously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers. One such device, the Applied Biosyste s 380B DNA Synthesizer, utilizes ?-cyanoethyl phosphoramidite chemistry.
Since the complete nucleotide synthesis of DNA complementary to the c-myb mRNA transcript is known, an¬ tisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by oligonucleotide synthesis methods known to those skilled in the art.
While any length oligonucleotide may be utilized in the practice of the invention, sequences shorter than 12 bases may be less specific in hybridizing to the target c-myb mRNA, may be more easily destroyed by enzymatic digestion, and may be destabilized by enzymatic digestion. Hence, oligonucleotides having 12 or more nucleotides are preferred.
Long sequences, particularly sequences longer than about 40 nucleotides, may be somewhat less effective in inhibiting c-myb translation because of decreased uptake by the target cell. Thus, oligomers of 12-40 nucleotides are preferred, more preferably 15-30 nucleotides, most preferably 18-26 nucleotides. While sequences of 18-21 nucleotides are most particularly preferred, for unmodified oligonucleotides, slightly longer chains of up to about 26 nucleotides, are preferred for modified oligonucleotides such as phosphorothioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides. Oligonucleotides complementary to and hybridizable with any portion of the c-myb mRNA transcript are, in principle, effective for inhibiting translation of the transcript, and capable of inducing the effects herein described. It is believed that translation is most effectively inhibited by blocking the mRNA at a site at or near the initiation codon. Thus, oligonucleotides complementary to the 5'-terminal region of the c-myb mRNA transcript are preferred. It is believed that secondary or tertiary structure which might interfere with hybridization is minimal in this region. Moreover, it has been suggested that sequences that are too distant in the 3'-direction from the initiation site may be less effective in hybridizing the mRNA transcripts because of a "read-through" phenomenon whereby the ribosome is postulated to unravel the antisense/sense duplex to permit translation of the message. See, e.g., Shakin, J. Biochemistry 261, 16018 (1986).
The antisense oligonucleotide is preferably directed to a site at or near the initiation codon for protein synthesis. Oligonucleotides complementary to the c-myb mRNA, including the initiation codon (the first codon at the 5' end of the translated portion of the c- myb transcript, comprising nucleotides 114-116 of the complete transcript) are preferred.
While antisense oligomers complementary to the 5'-terminal region of the c-myb transcript are preferred, particularly the region including the initiation codon, it should be appreciated that useful antisense oligomers are not limited to those complementary to the sequences found in the translated portion (nucleotides 114 to 2031) of the mRNA transcript, but also includes oligomers complementary to nucleotide sequences contained in, or extending into, the 5'-and 3'-untranslated regions. Oligomers whose complementarity extends into the 5'- untranslated region of the c-myb transcript are believed particularly effective in inhibiting c-myb translation. Preferredoligonucleotidescomplementarytothe 5 '-untranslated region of the transcript include molecules having a nucleotide sequence complementary to a portion of the c-myb mRNA transcript including the cap nucleotide, that is, the nucleotide at the extreme 5'-end of the transcript. The SI nuclease assay procedure of Molecular Cloning, 2nd edition (Sa brook et al. , Eds. 1989) , pages 7.66-7.70 (incorporated herein by reference) was essentially followed to map the location of c-myb cap sites using mRNA isolated from the leukemic cell line CCRF-CEM, which expresses high levels of c-myb mRNA. The longest clearly visible band was located 90 base pairs upstream of the published c-myb cDNA (Majello et al. , Proc. Natl. Acad. Sci. U.S.A., 83, 9536-9640 (1986)), indicating the putative principle cap site. The position of this site is in perfect agreement with the length of the c-myb cDNA cloned from CCRF-CEM cells (Clarke et al. , Mol. Cell. Biol.. 8, 884-892 (1988)). SI protection assays also revealed faint bands in addition to the main band corresponding to the cap site. These other bands may -represent rare or unstable c-myb mRNA transcripts. Multiple sites of transcription initiation are not uncommon in genes such as c-myb which lack a perfect TATAA box. The nucleotide sequence of the mRNA transcript 5'-terminus beginning with the cap nucleotide may be readily established, and antisense oligonuc¬ leotides complementary and hybridizable thereto may be prepared.
The following 40-mer oligodeoxynucleotide is complementary to the c-myb mRNA transcript beginning with the initiation codon of the transcript and extending downstream thereof (in the 3' direction): SEQ ID NO:l.
Smaller oligomers based upon the above sequence, in particular, oligomers hybridizable to segments of the c-myb message containing the initiation codon, may be utilized. Particularly preferred are the following 26- to 15-mers: SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11,
SEQ ID NO: 12 and SEQ ID NO:13.
Oligodeoxynucleotides complementary to the c- myb mRNA transcript beginning with the second codon of the translated portion of the transcript (nucleotides 117-119 of the complete transcript) are another group of preferred oligomers. Such oligomers include, for example, the following 26- to 15-mers: SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and
SEQ ID NO:25.
The oligonucleotide employed may represent an un¬ modified or modified oligonucleotide. Thus, oligo- nucleotides hybridizable to the c-myb mRNA transcript finding utility according to the present invention include not only oligomers of the biologically sig- nificant native nucleotides, i.e.. A, dA, G, dG, C, dC, T and U, but also oligonucleotide species which have been modified for improved stability and/or lipid solubility. For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting an alkyl group or alkoxy group for a phos¬ phate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate oligonucleoside or allylphosphotriester oligonucleotide. Non-ionic oligonucleotides such as these are characterized by increased resistance to nuclease hydrolysis and/or increased cellular uptake, while retaining the ability to form stable complexes with complementary nucleic acid sequences. The alkylphosphonates in particular, are stable to nuclease cleavage and soluble in lipid. The preparation of alkylphosphonate oligonucleosides is disclosed in U.S. Patent 4,469,863.
Methylphosphonate oligomers can be prepared by a variety of methods, both in solution and on insoluble polymer supports (Agrawal and Riftina, Nucl. Acids Res. , 6, 3009-3024 (1979); Miller et ______ , Biochemistry. 18,
5134-5142 (1979), Miller et al., J. Biol. Chem.. 255, 9659-9665 (1980); Miller et al. , Nucl. Acids Res.. 11, 5189-5204 (1983), Miller et al.. Nucl. Acids Res.. 11, 6225-6242 (1983), Miller et al., Biochemistry. 25, 5092- 5097 (1986) ; Engels and Jager, Angew. Chem. Suppl. 912 (1982); Sinha et al. , Tetrahedron Lett. 24, 877-880 (1983); Dorman et al. Tetrahedron, 40, 95-102 (1984); Jager and Engels, Tetrahedron Lett. , 25, 1437-1440 (1984); Noble et al., Nucl. Acids Res.. 12, 3387-3404 (1984) ; Callahan et al. , Proc. Natl. Acad. Sci. USA, 83, 1617-1621 (1986) ; Koziolkiewicz et al. , Chemica Scripta. 26, 251-260 (1986) ; Agrawal and Goodchild, Tetrahedron Lett.. 38, 3539-3542 (1987); Lesnikowski et al.. Tetrahedron Lett.. 28, 5535-5538 (1987); Sarin et al.. Proc. Natl. Acad. Sci. USA. 85, 7448-7451 (1988)). The most efficient procedure for preparation of methylphosphonate oligonucleosides involves use of 5'-0- dimethoxytrityldeoxynucleoside-3 ' - 0_- diisopropylmethylphosphoramidite intermediates, which are similar to the methoxy or β-cyanoethyl phosphoramidite reagents used to prepare oligodeoxyribonucleotides. The methylphosphonate oligomers can be prepared on controlled pore glass polymer supports using anautomated DNA synthesizer (Sarin et al . , Proc. Natl. Acad. Sci. USA, 85, 7448-7451 (1988)).
Resistance to nuclease digestion may also be achieved by modifying the internucleotide linkage at both the 5' and 3' termini with phosphoroamidites according to the procedure of Dagle et al., Nucl. Acids Res. 18, 4751-4757 (1990) .
Phosphorothioate oligonucleotides contain a sulfur- for-oxygen substitution in the internucleotide phosphodiester bond. Phosphorothioate oligonucleotides combine the properties of effective hybridization for duplex formation with substantial nuclease resistance, while retaining the water solubility of a charged phosphate analogue. The charge is believed to confer the property of cellular uptake via a receptor (Loke et aJL. , Proc. Natl. Acad. Sci. U.S.A. 86, 3474-3478 (1989)). Phosphorothioate oligodeoxynucleotide are described by LaPlanche, et al. , Nucleic Acids Research 14, 9081 (1986) and by Stec et __1. , J. Am. Chem. Soc. 106, 6077 (1984) . The general synthetic method for phosphorothio¬ ate oligonucleotides was modified by Stein et a]L. , Nucl. Acids Res.. 16, 3209-3221 (1988) , so that these compounds may readily be synthesized on an automatic synthesizer using the phosphoramidite approach.
Furthermore, recent advances in the production of oligoribonucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-0- methylribonucleotides (Inove et al. , Nucleic Acids Res. 15, 6131 (1987) and chimeric oligonucleotides that are composite RNA-DNA analogues (Inove et aJL. , FEBS Lett. 215, 327 (1987) .
While inhibition of c-myb mRNA translation is possible utilizing either antisense oligoribonucleotides or oligodeoxyribonucleotides, free oligoribonucleotides are more susceptible to enzymatic attack by ribonucleases than oligodeoxyribonucleotides. Hence, oligodeoxyribonucleotides are preferred in the practice of the present invention. Oligodeoxyribonucleotides are further preferred because, upon hybridization with c-myb mRNA, the resulting DNA-RNA hybrid duplex is a substrate for RNase H, which specifically attacks the RNA portion of DNA-RNA hybrid. Degradation of the mRNA strand of the duplexreleases the antisense oligodeoxynucleotide strand for hybridization with additional c-myb messages.
In general, the antisense oligonucleotides used in the method of the present invention will have a sequence which is completely complementary to the target portion of the c-myb message. Absolute complementarity is not however required, particularly in larger oligomers. Thus, reference herein to a "nucleotide sequence complementary to at least a portion of the mRNA transcript" of c-myb does not necessarily mean a sequence having 100% complementarity with the transcript. In gene- ral, any oligonucleotide having sufficient com¬ plementarity to form a stable duplex with c-myb mRNA is suitable. Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity with the target region of the c-myb message. Generally, the larger the hybridizing oligomer, the more mismatches may be tolerated. More than one mismatch probably will not be tolerated for antisense oligomers of less than about 21 nucleotides. One skilled in the art may readily determine the degree of mismatching which may be tolerated between any given antisense oligomer and the target c-myb message sequence, based upon the melting point, and therefore the stability, of the resulting duplex. Melting points of duplexes of a given base pair composition can be readily determined from standard texts, such as Molecular Cloning: A Laboratory Manual. (2nd edition, 1989), J. Sambrook et al. , eds.
While oligonucleotides capable of stable hybridiza¬ tion with any region of the c-myb message are within the scope of the present invention, oligonucleotides complementary to a region including the initiation codon are believed particularly effective. Particularly preferred are oligonucleotides hybridizable to a region of the c-myb mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direction) of that codon.
For therapeutic use, the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liquid vehicles and excipients are conventional and commercially available. Illustrative thereof are distilled water, physiological saline, aqueous solution of dextrose, and the like. The c-myb mRNA antisense oligonucleotides are preferably administeredparenterally, mostpreferably intravenously. The vehicle is designed accordingly. Alternatively, oligonucleotide may be administered subcutaneously via controlled release dosage forms.
The oligonucleotides may be conjugated to poly(L- lysime) to increase cell penetration. Such conjugates are described by Lemaitre et al. , Proc. Natl. Acad. Sci. USA, 84, 648-652 (1987). The procedure requires that the 3'-terminal nucleotide be a ribonucleotide. The resulting aldehyde groups are then randomly coupled to the epsilon-amino groups of lysine residues of poly(L- lysine) by Schiff base formation, and then reduced with sodium cyanoborohydride. This procedure converts the 3'- terminal ribose ring into a morpholine structure antisense oligomers.
In addition to administration with conventional carriers, the antisense oligonucleotidesmay be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides may be encapsulated in for therapeutic delivery. The oligonucleotide, depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. Oligonucleotides have been successfully encapsulated in unilameller liposomes. Reconstituted Sendaivirus envelopes havebeen suc¬ cessfully used to deliver RNA and DNA to cells. Arad et al., Biochem. Biophy. Acta. 859, 88-94 (1986).
The c-myb antisense oligonucleotides may be administered de novo as the primary therapy. Alternatively, the oligonucleotides may be administered as an adjuvant following surgical removal of a melanoma to patients who may be disease-free but at high risk of recurrence.
A preferred method of administration of oligonucleotide for treatment of melanoma comprises either regional or systemic perfusion, as is appropriate. According to a method of regional perfusion, the afferent and efferent vessels supplying the extremity containing the lesion are isolated and connected to a low-flow perfusion pump in continuity with an oxygenator and a heat exchanger. The iliac vessels may be used for perfusion of the lower extremity. The axillary vessels are cannulated high in the axilla for upper extremity lesions. Oligonucleotide is added to the perfusion circuit, and the perfusion is continued for an appropriate time period, e.g. , one hour. Perfusion rates of from 100 to 150 ml/minute may be employed for lower extremity lesions, while half that rate should be employed for upper extremity lesions. Systemic heparinization may be used throughout the perfusion, and reversed after the perfusion is complete. This isolation perfusion technique permits administration of higher doses of chemotherapeutic agent than would otherwise be tolerated upon infusion into the arterial or venous sys¬ temic circulation.
For systemic infusion, the oligonucleotides are preferably delivered via a central venous catheter, which is connected to an appropriate continuous infusion device. Indwelling catheters provide long term access to the intravenous circulation for frequent administration of drugs over extended time periods. They are generally surgically inserted into the external cephalic or internal jugular vein under general or local anesthesia. The subclavian vein is another common site of catheterization. The infuser pump may be external, or may form part of an entirely implantable central venous system such as the INFUSAPORT system available from Infusaid Corp., Norwood, MA and the PORT-A-CATH system available from Pharmacia Laboratories, Piscataway, NJ. These devices are implanted into a subcutaneous pocket under local anesthesia. A catheter, connected to the pump injection port, is threaded through the subclavian vein to the superior vena cava. The implant contains a supply of oligonucleotide in a reservoir which may be replenished as needed by injection of additional drug from a hypodermic needle through a self-sealing diaphragm in the reservoir. Completely implantable infusers are preferred, as they are generally well accepted by patients because of the convenience, ease of maintenance and cosmetic advantage of such devices. High dose chemotherapy has been coupled with autologous bone marrow rescue (transplantation) in an attempt to treat melanoma. While high dose chemotherapy has resulted in significantly higher therapeutic responses, patient survival is not generally prolonged: Jones et al.. , Cancer Chemother. Pharmacol. 26, 155-6 (1990) (carboplatin, cyclophosphamide and BCNU) ; Lakhani et al., Br. J. Cancer 61, 330-4 (1990) (melphalan) ; Koeppler et al. , Onkologie 12, 277-9 (1989) (melphalan and BCNU); Thatcher et al., Cancer 63, 1296-302 (1989) (DTIC); Wolff et al., J. Clin. Oncol. 7, 245-9 (1989) (thiotepa) ; Shea et al. , Arch. Dermatol. 124, 878-84 (1988) (cyclophosphamide, cisplatin and carmustine) ; Tchekmedian et al. , J. Clin. Oncol. 4, 1811-8 (1986) (BCNU, melphalan, or both); Thomas et al. , Oncology 43, 273-7 (1986) (BCNU and melphalan) .
Melanoma, particularly in advanced stages, may be substantially metastatic. Thus, one possible reason for the lack of success of high dose chemotherapy and autologous bone marrow purging in treating melanoma may be a failure to properly purge the harvested marrow of malignant cells which have metastasized to the bone marrow. According to the present invention, c-myb antisense oligonucleotides may be used as bone marrow purging agents for the in vitro cleansing of bone marrow of melanoma cells in conjunction with high dose conventional chemotherapy. While normal hematopoietic cells are sensitive to c-myb antisense, they are less sensitive than malignant cells expressing c-myb, as taught in our copending patent application Serial No. 427,659 and corresponding international application WO90/05445. This differential sensitivity makes possible the use of c-myb antisense oligonucleotides in purging bone marrow of neoplastic cells. According to a method for bone marrow purging, bone marrow is harvested from a donor by standard operating room procedures from the iliac bones of the donor. Methods of aspirating bone marrow from donors are well- known in the art. Examples of apparatus and processes for aspirating bone marrow from donors are disclosed in U.S. Patents 4,481,946 and 4,486,188, incorporated herein by reference. Sufficient marrow is withdrawn so that the recipient, who is either the donor (autologous transplant) or another individual (allogeneic transplant) , may receive from about 4 x 108 to about 8 x 108 processed marrow cells per kg of bodyweight. This generally requires aspiration of about 750 to about 1000 ml of marrow. The aspirated marrow is filtered until a single cell suspension, known to those skilled in the art as a "buffy coat" preparation, is obtained. This suspension of leukocytes is treated with c-myb antisense oligonucleotides in a suitable carrier, advantageously in a concentration of about 8 mg/ml. Alternatively, the leucocyte suspension may be stored in liquid nitrogen using standard procedures known to those skilled in the art until purging is carried out. The purged marrow can be stored frozen in liquid nitrogen until ready for use. Methods of freezing bone marrow and biological substances are disclosed, for example, in U.S. Patents 4,107,937 and 4,117,881.
Other methods of preparing bone marrow for treat- ment with c-myb antisense may be utilized, which methods may result in even more purified preparations of hemato¬ poietic cells than the aforesaid buffy coat preparation. One or more growth factors may be added to the aspirated marrow or buffy coat preparation to stimulate growth of neoplasms, and thereby increase their sensi¬ tivity to the toxicity of the c-myb antisense oligonucleotides.
After treatment with the antisense oligonucleo¬ tides, the cells to be transferred are washed with auto- logous plasma or buffer to remove unincorporated oligomer. The washed cells are then infused back into the patient. The c-myb antisense oligonucleotides may be administered in a dosage effective for inhibiting the proliferation of melanoma cells in the afflicted individual, while maintaining the viability of normal cells. Such amounts may vary depending on the nature and extent of the neoplasm, the particular oligonucleotide utilized, and other factors. The actual dosage admin¬ istered may take into account the size and weight of the patient, whether the nature of the treatment is prophy- lactic or therapeutic in nature, the age, health and sex of the patient, the route of administration, whether the treatment is regional or systemic, and other factors. Inhibition of melanoma cell proliferation has been observed at antisense concentrations of as low as 10 μg/ml. At 20 μg/ml, inhibition was profound. Concen¬ trations of from about 1 to about 100 μg/ml may be employed, preferably from about 10 μg/ml to about 100 μg/ml, most preferably from about 20 μg/ml to about 60 μg/ml. The patient should receive a sufficient daily dosage of antisense oligonucleotide to achieve these intercellular concentrations of drug. The daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 700 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient. Based upon the in vivo study described herein, it is believed that a course of treatment may advantageously comprise infusion of the recommended daily dose of oligonucleotide for a period of from about 3 to about 28 days, more preferably from about 7 to about 10 days. Those skilled in the art should readily be able to determine the optimal dosage in each case. For an adult human being, a daily dose of about 350 mg oligonucleotide is believed sufficient, to achieve an effective intercellular concentration of 20 μg.
For ex vivo antineoplastic application, such as, for example, in bone marrow purging, the c-myb antisense oligonucleotides may be administered in amounts effective to kill neoplastic cells while maintaining the viability of normal hematologic cells. Such amounts may vary depending on the extent to which melanoma cells may have metastasized to the bone marrow, the particular oligonucleotide utilized, the relative sensitivity of the neoplastic cells to the oligo¬ nucleotide, and other factors. Concentrations from about 10 to 200 μg/ml per 105 cells may be employed, preferably from about 40 to 150 μg/ml per 105 cells. Supplemental dosing of the same or lesser amounts of oligonucleotide are advantageous to optimize the treatment. Thus, for purging bone marrow containing 2 x 107 cell per ml of marrow volume, dosages of from about 2 to 40 mg antisense per ml of marrow may be effectively utilized, preferably from about 8 to 24 mg/ml. Greater or lesser amounts of oligonucleotide may be employed.
The present invention is described in greater detail in the following non-limiting examples.
Example 1
Inhibition of CHP Melanoma Growth By c-mγb Antisense Oligonucleotide
CHP melanoma cells (Children's Hospital of Philadelphia, Philadelphia, PA) were grown in RPMI culture medium containing 2% oxalophosphate and 5% bovine calf serum at 37°C in 5% C02. Cells (5,000/ml) were seeded in 200 μl volumes into individual wells of a 96 well Costar plate at day -3, and allowed to grow for three days. At this time (day 0) , the unmodified antisense 18-mer oligodeoxynucleotide (SEQ ID NO:22) complementary to codons 2-7 of the translated portion of the c-myb mRNA, or the corresponding sense 18-mer (SEQ ID NO:26) , were added to the cultures at concentrations of 0, 10, 20, 50 and 100 μg/ml for 1, 2 or 5 consecutive days. On day 7, 100 μl of fresh medium was added to the cultures. Cell viability/proliferation was determined on day 8 utilizing a commercially available kit (CELLTITER 96™ Promega, Madison, WI) . The assay is based on the ability of viable cells to convert a tetrazolium salt into a blue formazan product which can be quantified by measuring the absorbance at 570 nm with a conventional microplate reader. The extent of absorbance at 570 nm is proportional to the amount of formazan produced, and thus the number of viable cells remaining. Accordingly, 10 μl of 3-(4,5-dimethylthiazol-2-yl) -2,5 diphenyltetrazolium bromide solution (5 mg/ml) was added to 110 μl of cell suspension and incubated for 4 hours at 37βC. 150 μl of acidified isopropanol (25 ml of isopropanol plus 0.1 ml of 12 N HC1) was then added and the solution mixed. The optical density was measured at 570 nm. Background from cell debris was eliminated by subtracting a reference measurement at 630 nm.
The results of the viability assay are set forth in Figures 1 (one-day treatment) , 2 (two-day treatment) and 3 (five-day treatment) . The oligonucleotide concentrations indicated in Figures 2 and 3 are cumulative dosages for the two- and five-day treatments, respectively. It may be appreciated from the figures that treatment of the melanoma cells with antisense oligomer resulted in sequence-specific killing. The effect was most pronounced when the melanoma cells were treated for one day with an antisense oligomer concen¬ tration of at least 50 μg/ml, or when the cells were treated on consecutive days with as little as 20 μg/ml oligomer. Example 2
Inhibition of SK MEL-37 Melanoma By c-myb Antisense Oligonucleotide The effect of c-myb antisense oligonucleotide on another human melanoma line, SK MEL-37 (Sloan Kettering Institute, New York, NY) was determined. Cells were treated according to the procedure of Example 1 on day 0 with the same sense and antisense oligomers, in the same concentrations. Cell viability was assayed on day 5. The results are shown in Figure 4.
Again, treatment of melanoma cells with a single 50 μg/ml dose of antisense oligomer was sufficient to induce substantial cell killing, in comparison to sense- treated or untreated cells.
Example 3
Inhibition of SK MEL-37 Melanoma Growth in vivo By c-myjb Antisense Oligonucleotide
The effect of c-myb antisense oligonucleotide on the growth of melanoma cells in vivo was investigated.
250,000 melanoma cells (SK MEL-37) were injected subcutaneously into each of four severe combined immunodeficient mice (C.B-17/LCRTAC-SCID DF from Fox Chase Cancer Institute, Philadelphia PA) at day -21. The tumors were allowed to grow to a size of approximately 5 mm in diameter. On day 1, ALZET constant infusion pumps (Alza Corporation, Palo Alto, CA) delivering a total dosage of 700 μg oligomer over 7 days were surgically implanted into the mice. The tumor area was then monitored daily. One mouse received no oligomer. Another mouse received a 24-mer "sense" phosphorothioate oligonucleotide (corresponding to codons 2-9 of the translated c-myb mRNA) . Two mice received a 24-mer anti- sense phosphorothioate oligonucleotide having the nucleotide sequence of SEQ ID NO: 16 (TATGCTGTGC CGGGGGT- CTTC GGGC) , complementary to c-myb mRNA codons 2-9. The results are shown in Figure 5. Each curve represents one animal. In the two antisense-treated animals, the tumor size stayed the same or regressed. In contrast, the tumors continued to grow in the sense-treated and control animals. The animals were sacrificed on day 19. The tumors were removed and weighed. The weights of the tumors in the control and sense-treated animals were 9.9 and 9.6 grams, respectively. The tumor weights in the two anti- sense treated animals were only 0.1 and 0.2 grams. The following non-limiting example illustrates one methodology for purging bone marrow of metastatic melanoma.
Example 4
Bone Marrow Purging with c-myb Antisense Oligonucleotide
Bone marrow is harvested from the iliac bones of a donor under general anesthesia in an operating room using standard techniques. Multiple aspirations are taken into heparinized syringes. Sufficient marrow is withdrawn so that the marrow recipient will be able to receive about 4 x 108 to about 8 x 108 processed marrow cells per kg of body weight. Thus, about 750 to 1000 ml of marrow is withdrawn. The aspirated marrow is transferred immediately into a transport medium (TC-199, Gibco, Grand Island, New York) containing 10,000 units of preservative-free heparin per 100 ml of medium. The aspirated marrow is filtered through three progressively finer meshes until a single cell suspension results, i.e., a suspension devoid of cellular aggregates, debris and bone particles. The filtered marrow is then processed further into an automated cell separator (e.g.,
Cobe 2991 Cell Processor) which prepares a "buffy coat" product, (i.e., leukocytes devoid of red cells and platelets) . The buffy coat preparation is then placed in a transfer pack for further processing and storage.
It may be stored until purging in liquid nitrogen using standard procedures. Alternatively, purging can be carried out immediately, then the purged marrow may be stored frozen in liquid nitrogen until it is ready for transplantation.
The purging procedure may be carried out as follows. Cells in the buffy coat preparation are adjusted to a cell concentration of about 2 x 107/ΠLL in TC-199 containing about 20% autologous plasma. C-myb antisense oligodeoxynucleotide, for example, in a concentration of about 8 mg/ml, is added to the transfer packs containing the cell suspension. Recombinant human hematopoietic growth factors, e.g. , rH IL-3 or rH GM-CSF, may be added to the suspension to stimulate growth of neoplasms and thereby increase their sensitivity c-myb antisense oligonucleotide toxicity. The transfer packs are then placed in a 37°C waterbath and incubated for 18 - 24 hours with gentle shaking. The cells may then either be frozen in liquid nitrogen or washed once at 4°C in TC-199 containing about 20% autologous plasma to remove unincorporated oligomer. Washed cells are then infused into the recipient. Care must be taken to work under sterile conditions wherever possible and to maintain scrupulous aseptic techniques at all times.
The present invention may be embodied in other spe¬ cific forms without departing from the spirit or essen- tial attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.
All references cited herein with respect to synthetic, preparative and analytical procedures are incorporated by reference. SEQUENCE LISTING
(1) GENERAL INFORMATION: (i) APPLICANT: TEMPLE UNIVERSITY - OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION
(a) INVENTORS: Gewirtz, Alan M.
Calabretta, Bruno (ii) TITLE OF INVENTION: Treatment of Melanoma with Antisense Oligonucleotides to c-myb Proto-oncogene. (iϋ) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Temple University - of the Common¬ wealth System of Higher Education
(B) STREET: 406 University Services Building (C) CITY: Philadelphia
(D) STATE: Pennsylvania
(E) COUNTRY: U.S.A.
(F) ZIP: 19122
(V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.50 inch, 720 Kb
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WordPerfect 5.1 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(VU) PRIORITY APPLICATION DATA:
(A) APPLICATIONNUMBER: U.S. Application Serial No. 792,999
(B) FILING DATE: 15 November 1991 (Viϋ) ATTORNEY/AGENT INFORMATION:
(A) NAME: Monaco, Daniel A.
(B) REGISTRATION NUMBER: 30,480 (C) REFERENCE/DOCKET NUMBER: 6056-159 PCT 1 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (215) 568-8383 (B) TELEFAX: (215) 568-5549
(C) TELEX: None
(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CGTCACTGCT ATATATGCTG TGCCGGGGTC TTCGGGCCAT 40
(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 Nucleotides (B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATGCTGTGCC GGGGTCTTCG GGCCAT 26
(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGCTGTGCCG GGGTCTTCGG GCCAT 25
(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCTGTGCCGG GGTCTTCGGG CCAT 24 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTGTGCCGGG GTCTTCGGGC CAT 23
(2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:6: TGTGCCGGGG TCTTCGGGCC AT 22
(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:7: GTGCCGGGGT CTTCGGGCCA T 21
(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:8: TGCCGGGGTC TTCGGGCCAT 20 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCCGGGGTCT TCGGGCCAT 19
(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CCGGGGTCTT CGGGCCAT 18
(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CGGGGTCTTC GGGCCAT 17
(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGGTCTTCG GGCCAT 16
(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGGTCTTCGG GCCAT 15
(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:14: TATATGCTGT GCCGGGGTCT TCGGGC 26
(2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:15: ATATGCTGTG CCGGGGTCTT CGGGC 25
(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 Nucleotides (B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:16: TATGCTGTGC CGGGGTCTTC GGGC 24
(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATGCTGTGCC GGGGTCTTCG GGC 23
(2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TGCTGTGCCG GGGTCTTCGG GC 22
(2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 Nucleotides (B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GCTGTGCCGG GGTCTTCGGG C 21
(2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CTGTGCCGGG GTCTTCGGGC 20
(2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 Nucleotides (B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:21: TGTGCCGGGG TCTTCGGGC 19
(2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 Nucleotides (B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GTGCCGGGGT CTTCGGGC 18
(2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear ( i) SEQUENCE .DESCRIPTION: SEQ ID NO:23: TGCCGGGGTC TTCGGGC 17
(2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:24: GCCGGGGTCT TCGGGC 16
(2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: CCGGGGTCTT CGGGC 15
(2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GCCCGAAGAC CCCGGCAC 18

Claims

1. A method for the treatment of melanoma com¬ prising administering to an individual in need of such treatment an effective amount of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene, said oligonucleotide being hybridizable to said mRNA transcript.
2. A method according to claim 1 wherein the oligonucleotide is an at least 12-mer.
3. A method according to claim 2 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.
4. A method according to claim 2 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.
5. A method according to claim 2 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-myb mRNA transcript including the translation initiation codon of said transcript and/or the codon immediately downstream from the initiation codon.
6. A method according to claim 2 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
7. A method according to claim 6 wherein the oligonucleotide is a methylphosphonate oligonucleoside or a phosphorothioate oligonucleotide.
8. A method according to claim 6 wherein the oligonucleotide is from a 15-mer to 30-mer.
9. A method according to claim 8 wherein the oligonucleotide is from a 18-mer to 26-mer.
10. A method according to claim 9 wherein the oligonucleotide is from a 18-mer to 21-mer.
11. A method according to claim 8 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of:
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
12. A method according to claim 8 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of
SEQ ID NO:14,
SEQ ID NO:15,
SEQ ID NO:16,
SEQ ID NO: 17,
SEQ ID NO:18,
SEQ ID NO:19,
SEQ ID NO:20,
SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25.
13. A method according to claim 12 wherein the oligonucleotide comprises SEQ ID NO: 22.
14. A method according to claim 8 wherein the oligonucleotide is a phosphorothioate oligodeoxyncucleotide or methylphosphonate oligodeoxynucleoside having a nucleotide/nucleoside sequence corresponding to any of the following:
SEQ ID NO: 2
Figure imgf000038_0001
15. A method according to claim 14 wherein the oligonucleotide comprises a phosphorothioate oligodeoxynucleotide.
16. A method according to claim 15 wherein the phosphorothioate oligodeoxynucleotide has a nucleotide sequence corresponding to SEQ ID NO:16.
17. A method for purging bone marrow of metastasized melanoma cells comprising treating bone marrow cells aspirated from a melanoma-inflicted individual with an effective amount of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene, said oligonucleotide being hybridizable to said m-RNA transcript, and returning the thus-treated cells to the body of the afflicted individual.
18. A method according to claim 17 wherein the oligonucleotide is an at least 12-mer.
19. A method according to claim 18 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.
20. A method according to claim 18 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.
21. A method according to claim 18 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
22. A method according to claim 21 wherein the oligonucleotide is from a 15-mer to 30-mer.
23. A method according to claim 22 wherein the oligonucleotide is from a 18-mer to 26-mer.
24. A method according to claim 23 wherein the oligonucleotide is from a 18-mer to 21-mer.
25. A method according to claim 22 wherein the oligonucleotide is an unmodified oligodeoxynucleotide, phosphorothioate oligodeoxynucleotide or methylphosphonate oligodeoxynucleoside having a nucleotide/nucleoside sequence selected from the group consisting of:
SEQ ID NO:2,
SEQ ID NO:3,
SEQ ID NO:4,
SEQ ID NO:5,
SEQ ID NO:6,
SEQ ID NO:7,
SEQ ID NO:8,
SEQ ID NO:9,
SEQ ID NO:10,
SEQ ID NO:11,
SEQ ID NO:12,
SEQ ID NO:13,
SEQ ID NO:14,
SEQ ID NO:15,
SEQ ID NO:16,
SEQ ID NO:17,
SEQ ID NO:18,
SEQ ID NO:19,
SEQ ID NO:20,
SEQ ID NO:21,
SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.
26. A method according to claim 25 wherein the oligonucleotide has the nucleotide sequence SEQ ID NO:22.
27. A method according to claim 25 wherein the oligonucleotide has the nucleotide sequence SEQ ID NO:16.
28. Use of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene, said oligonucleotide being hybridizable to said mRNA transcript, for the manufacture of a medicament for treatment of melanoma.
29. Use according to claim 28 wherein the oligonucleotide is an at least 12-mer.
30. Use according to claim 29 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.
31. Use according to claim 29 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.
32. Use according to claim 29 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-myb mRNA transcript including the translation initiation codon of said transcript and/or the codon immediately downstream from the initiation codon.
33. Use according to claim 29 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
34. Use according to claim 33 wherein the oligonucleotide is a methylphosphonate oligonucleoside or a phosphorothioate oligonucleotide.
35. Use according to claim 33 wherein the oligonucleotide is from a 15-mer to 30-mer.
36. Use according to claim 35 wherein the oligonucleotide is from a 18-mer to 26-mer.
37. Use according to claim 36 wherein the oligonucleotide is from a 18-mer to 21-mer.
38. Use according to claim 35 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of:
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
39. Use according to claim 35 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of
SEQ ID NO:14, SEQ ID NO: 15,
SEQ ID NO: 16,
SEQ ID NO: 17,
SEQ ID NO: 18,
SEQ ID NO: 19,
SEQ ID NO: 20,
SEQ ID NO: 21,
SEQ ID NO: 22,
SEQ ID NO: 23,
SEQ ID NO: 24 and
SEQ ID NO: 25.
40. Use according to claim 39 wherein the oligonucleotide comprises SEQ ID NO: 22.
41. Use according to claim 35 wherein the oligonucleotide is a phosphorothioate oligodeoxynucleotide or methylphosphonate oligodeoxynucleoside having a nucleotide/nucleoside sequence corresponding to any of the following:
Figure imgf000043_0001
SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 or SEQ ID NO:25.
42. Use according to claim 41 wherein the oligonucleotide comprises a phosphorothioate oligodeoxynucleotide.
43. Use according to claim 42 wherein the phosphorothioate oligodeoxynucleotide has a nucleotide sequence corresponding to SEQ ID NO:16.
44. A composition for the treatment of melanoma comprising a pharmaceutically acceptable carrier and an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene, said oligonucleotide being hybridizable to said mRNA transcript.
45. A composition accordingto claim 44 wherein the oligonucleotide is an at least 12-mer.
46. A composition according to claim 45 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.
47. Acomposition according to claim 45 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.
48. A composition according to claim 45 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-myb mRNA transcript including the translation initiation codon of said transcript and/or the codon immediately downstream from the initiation codon.
49. A composition according to claim 45 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
50. A composition according to claim 49 wherein the oligonucleotide is a methylphosphonate oligonucleoside or a phosphorothioate oligonucleotide.
51. A composition according to claim 49 wherein the oligonucleotide is from a 15-mer to 30-mer.
52. A composition according to claim 51 wherein the oligonucleotide is from a 18-mer to 26-mer.
53. A composition according to claim 52 wherein the oligonucleotide is from a 18-mer to 21-mer.
54. A composition according to claim 51 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of:
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12 and SEQ ID NO: 13.
55. A composition according to claim 51 wherein the oligonucleotide is an oligodeoxynucleotide selected from the group consisting of
SEQ ID NO:14,
SEQ ID NO:15,
SEQ ID NO:16,
SEQ ID NO:17,
SEQ ID NO:18,
SEQ ID NO:19,
SEQ ID NO:20,
SEQ ID NO:21,
SEQ ID NO:22,
SEQ ID NO:23,
SEQ ID NO:24 and
SEQ ID NO:25.
56. A composition according to claim 55 wherein the oligonucleotide comprises SEQ ID NO:22.
57. A composition according to claim 51 wherein the oligonucleotide is a phosphorothioate oligodeoxynucleotide or methylphosphonate oligodeoxynucleoside having a nucleotide/nucleoside sequence corresponding to any of the following:
SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 or SEQ ID NO:25.
58. A composition according to claim 57 wherein the oligonucleotide comprises a phosphorothioate oligodeoxynucleotide.
59. A composition according to claim 58 wherein the phosphorothioate oligodeoxynucleotide has a nucleotide sequence corresponding to SEQ ID NO:16.
PCT/US1992/009656 1991-11-15 1992-11-12 Treatment of melanoma with antisense oligonucleotides to c-myb proto-oncogene WO1993009789A1 (en)

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JP5509357A JPH07501525A (en) 1991-11-15 1992-11-12 Treatment of melanoma with antisense oligonucleotides against the c-myb proto-oncogene
CA002123611A CA2123611A1 (en) 1991-11-15 1992-11-12 Treatment of melanoma with antisense oligonucleotides to c-myb proto-oncogene
EP92924379A EP0667778A4 (en) 1991-11-15 1992-11-12 Treatment of melanoma with antisense oligonucleotides to c-myb proto-oncogene.

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US5618709A (en) * 1994-01-14 1997-04-08 University Of Pennsylvania Antisense oligonucleotides specific for STK-1 and method for inhibiting expression of the STK-1 protein
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US5658780A (en) * 1992-12-07 1997-08-19 Ribozyme Pharmaceuticals, Inc. Rel a targeted ribozymes
US5749847A (en) * 1988-01-21 1998-05-12 Massachusetts Institute Of Technology Delivery of nucleotides into organisms by electroporation
US5989849A (en) * 1991-05-09 1999-11-23 Temple University Of The Commonwealth System Of Higher Education Antisense of oligonucleotides to c-kit proto-oncogene and in vitro methods
US5994320A (en) * 1995-02-06 1999-11-30 Regents Of The University Of Minnesota Antisense oligonucleotides and methods for treating central nervous system tumors
US7888327B2 (en) 1994-07-15 2011-02-15 University Of Iowa Research Foundation Methods of using immunostimulatory nucleic acid molecules to treat allergic conditions

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5749847A (en) * 1988-01-21 1998-05-12 Massachusetts Institute Of Technology Delivery of nucleotides into organisms by electroporation
US5989849A (en) * 1991-05-09 1999-11-23 Temple University Of The Commonwealth System Of Higher Education Antisense of oligonucleotides to c-kit proto-oncogene and in vitro methods
US5646042A (en) * 1992-08-26 1997-07-08 Ribozyme Pharmaceuticals, Inc. C-myb targeted ribozymes
US5817796A (en) * 1992-08-26 1998-10-06 Stinchcomb; Dan T. C-myb ribozymes having 2'-5'-linked adenylate residues
US5658780A (en) * 1992-12-07 1997-08-19 Ribozyme Pharmaceuticals, Inc. Rel a targeted ribozymes
US6410224B1 (en) 1992-12-07 2002-06-25 Ribozyme Pharmaceuticals, Inc. Ribozyme treatment of diseases or conditions related to levels of NF-κB
WO1995011301A1 (en) * 1993-10-19 1995-04-27 The Regents Of The University Of Michigan P53-mediated apoptosis
US5618709A (en) * 1994-01-14 1997-04-08 University Of Pennsylvania Antisense oligonucleotides specific for STK-1 and method for inhibiting expression of the STK-1 protein
US7888327B2 (en) 1994-07-15 2011-02-15 University Of Iowa Research Foundation Methods of using immunostimulatory nucleic acid molecules to treat allergic conditions
US5994320A (en) * 1995-02-06 1999-11-30 Regents Of The University Of Minnesota Antisense oligonucleotides and methods for treating central nervous system tumors

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AU3070992A (en) 1993-06-15
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EP0667778A4 (en) 1997-04-16

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