+

WO1992019252A1 - Antisense oligonucleotides to c-kit proto-oncogene and uses thereof - Google Patents

Antisense oligonucleotides to c-kit proto-oncogene and uses thereof Download PDF

Info

Publication number
WO1992019252A1
WO1992019252A1 PCT/US1992/002854 US9202854W WO9219252A1 WO 1992019252 A1 WO1992019252 A1 WO 1992019252A1 US 9202854 W US9202854 W US 9202854W WO 9219252 A1 WO9219252 A1 WO 9219252A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
mer
oligodeoxynucleotide
oligonucleotide
kit
Prior art date
Application number
PCT/US1992/002854
Other languages
French (fr)
Inventor
Alan M. Gewirtz
Bruno Calabretta
Original Assignee
Temple University Of The Commonwealth System Of Higher Education
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Temple University Of The Commonwealth System Of Higher Education filed Critical Temple University Of The Commonwealth System Of Higher Education
Priority to JP51182692A priority Critical patent/JP2002507186A/en
Publication of WO1992019252A1 publication Critical patent/WO1992019252A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to antisense oligonucleo ⁇ tides to proto-oncogenes, in particular to antisense oligonucleotides to the c-kit gene, and the use of such oligonucleotides to selectively inhibit proliferation of certain cells.
  • the c-kit gene is the normal homologue of v- kit, the HZ4 feline sarcoma virus oncogene. It resides on human chromosome 4.
  • the gene encodes a dimeric trans ⁇ membrane glycoprotein receptor with tyrosine kinase ac ⁇ tivity that appears to be highly related to the receptors for colony stimulating factor-1 and platelet derived growth factor. (Yarden et. al.. , The EMBO Journal. 6, 3341-3351 (1987)). Like these receptors, c-kit also appears to belong to the immunoglobulin gene superfamily.
  • the mouse c-kit gene has been mapped to chromo ⁇ some 5 where it was determined to be allelic with the dominant white spotting locus ( ) (Chabot et al. , Nature 335, 88-89 (1988).
  • C-kit mutations are commonly found in W mice and, in addition to abnormalities affecting coat color and gonadal development, they also have a variety of hematopoietic defects. Macrocytic anemia is one of the most striking and profound of these abnormalities.
  • the W 42 mutation associated with a particularly severe hematologic manifestation, has been shown to be due to a missense mutation leading to replacement of one amino acid and defective tyrosine kinase activity (Tan et al.
  • Such animals are also known to have about one-third of the erythroid burst forming units of healthy wild-type littermates (Goldwather et aJL. , Exp. Heme. 18, 936 (1990)).
  • the ligand for the c-kit receptor has now been identified, molecularly cloned and expressed (Yarden et. al.. The EMBO Journal. 6, 3341-3351 (1987)).
  • the encoded protein known as stem cell factor (SCF) , mast cell growth factor (MGF) , or steel factor (SLF) is the product of a gene which resides at the steel (SI) locus.
  • SCF stem cell factor
  • MMF mast cell growth factor
  • SSF steel factor
  • Mice with SI mutations have phenotypic abnormalities quite similar to those of mice.
  • the mouse lacks, or has defects in, a critical signal transducing receptor encod ⁇ ed by c-kit.
  • the SI mouse has defects in the ligand which stimulates the receptor.
  • mice may have very limited applicability to human systems. Moreover, even if a tissue is shown to express a particular message, the importance of the mes ⁇ sage to expression of a cellular phenotype is not known until the cell is deprived of the encoded protein. Bio ⁇ logical systems are redundant. Lack of a protein can often be compensated by another protein of the same fami- ly. It is therefore not predictable that inhibition of expression of a particular gene will result in altered phenotype.
  • Antisense oligonucleotides and pharmaceutical compositions thereof with pharmaceutical carriers are provided.
  • Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA tran ⁇ script of the human c-kit 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.
  • oligonucleotides smaller than 12-mers may be uti ⁇ lized, 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 desta ⁇ bilize 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 oligo ⁇ deoxynucleotide, more advantageously an 18- to 26-mer. A 15- to 21-mer is most preferred.
  • oligonucleotides having a sequence complementary to any region of the c-kit gene find utility in the present invention
  • oligonucleotides complementary to a portion of the c-kit mRNA transcript including the translation initiation codon are particu ⁇ larly preferred.
  • the invention also provides a method for in ⁇ hibiting proliferation of erythroid cells comprising administering to a host in need of such treatment an effective amount of the c-kit antisense oligonucleotides of the invention.
  • the invention provides a method for treating hematologic neoplasms characterized by c-kit expression comprising administering an effective amount of c-kit antisense oligonucleotide in vivo or ex vivo to a host in need of such treatment, or to cells harvested from the host.
  • oligonu ⁇ cleotide includes both oligomers of ribonucleotide i.e. , oligoribonucleotides, and oligomers of deoxyribonucleo- tide i.e., oligodeoxyribonucleotides (also referred to herein as "oligodeoxynucleotides”) . Oligodeoxynucleo- tides are preferred.
  • oligonucleotide also includes oligomers which may be large enough to be termed “polynucleotides”.
  • polynucleotides oligonucleotide and oligodeoxynuc- leotide.
  • nucleotides of adenine (“A”), deoxyadenine (“dA”) , guanine (“G”) , deoxy- guanine (“dG”) , cytosine (“C”) , deoxycytosine (“dC”) , thymine (“T”) and uracil (“TJ”) but also oligomers and polymers hybridizable to the c-kit mRNA transcript which may contain other nucleotides.
  • oli ⁇ gonucleotide and “oligodeoxynucleotide” include oligo ⁇ mers and polymers wherein one or more purine or pyrimi- dine moieties, sugar moieties or internucleotide linkages is chemically modified.
  • oligonucleotide is thus understood to also include oligomers which may prop- erly be designated as “oligonucleosides" because of modi ⁇ fication of the internucleotide phosphodiester bond.
  • modified oligonucleotides include, for example, the alkylphosphonate oligonucleosides, discussed below.
  • phosphorothioate oligonucleotide means an oligonucleotide wherein one or more of the in ⁇ ternucleotide linkages is a phosphorothioate group,
  • alkylphosphonate oligonucleoside is meant an oligonucleotide wherein one or more of the internuc ⁇ leotide linkages is an alkylphosphonate group
  • R is an alkyl group, preferably methyl or ethyl.
  • downstream when used in reference to a direction along a nucleotide sequence means the 5' ⁇ 3' direction.
  • upstream means the
  • c-kit mRNA transcript means the presently known mRNA transcript of the human c-kit gene, or any further transcripts which may be elucidated. Description of the Figures
  • Figure 2 is a similar autoradiograph indicat ⁇ ing the effect of c-kit oligomer exposure on c-kit mRNA levels in A ' T ' MNC after stimulation by interleukin-3 and erythropoietin in 5% AB serum.
  • lane 3 sense oligomer, 36 hrs
  • lane 4 antisense, 36 hrs
  • lane 5 (scrambled sequence with identical base content, 36 hrs) .
  • Figure 3 shows the effect of c-kit oligode- oxyynucleotides on BFU-E-derived colony formation. Oligomers were added to cultures at time zero, and 50% of the initial dose was again added 18 hours later.
  • the bars on the graph indicate: 1, untreated control cells; 2, antisense treatment of 20 ⁇ g/ml followed by 10 ⁇ g/ml; 3, antisense treatment of 40 ⁇ g/ml followed by 20 ⁇ g/ml; 4, antisense treatment of 100 ⁇ g/ml fol ⁇ lowed by 50 ⁇ g/ml; 5, sense treatment of 100 ⁇ g/ml followed by 50 ⁇ g/ml; 6, scrambled-sequence treatment of 100 ⁇ g/ml followed by 50 ⁇ g/ml.
  • c-kit gene is of predominant importance in human erythropoiesis.
  • the protein which this gene expresses a receptor for tyrosine kinase, transduces a signal which acts in concert with interleukin-3 (IL-3) to optimize cell proliferation, particularly erythroid burst forming units (BFU-E) .
  • IL-3 interleukin-3
  • BFU-E erythroid burst forming units
  • the putative DNA sequence complementary to the mRNA transcript of the human c-kit gene has been reported by Yarden et al. , The EMBO Journal, 6, 3341- 3351 (1987) , the entire disclosure of which is incorpo- rated herein by reference.
  • the nucleotide sequence and predicted amino acid sequence are set forth in Figure 3 thereof.
  • the c-kit polypeptide is synthesized by translation of a single 5-kb mRNA, which contains an open reading frame coding for a 976 amino acid polypep- tide.
  • the antisense oligonucleotides of the inven ⁇ tion may be synthesized by any of the known chemical oligonucleotide synthesis methods. Such methods are generally described, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technology. VCH Verlagsgesellschaft mbH (H. Ibelgaufts trans. 1987).
  • any of the known methods of oligonucleotide synthesis may be utilized in preparing the instant antisense oligonucleotides.
  • the antisense oligonucleotides are most ad ⁇ vantageously prepared by utilizing any of the commer ⁇ cially available, automated nucleic acid synthesizers, for example, the Applied Biosystems 380B DNA Synthe ⁇ sizer, which utilizes /9-cyanoethyl phosphoramidite chemistry.
  • antisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by the oligonucleotide synthesis methods known to those skilled in the art.
  • oligonucleotide may be uti ⁇ lized in the practice of the invention
  • sequences shor ⁇ ter than 12 nucleotides may be less specific in hybrid- izing to the target c-kit mRNA, may be more easily destroyed by enzymatic digestion, and may be destabili ⁇ zed by even a single base pair mismatch.
  • oligo- nucleotides having 12 or more nucleotides . are pre ⁇ ferred.
  • oligomers of 12-40 nucleotides are preferred, more preferably 15-30 nucleotides, most preferably 18-26 nucleotides. Se ⁇ quences of 18-21 nucleotides are particularly pre- ferred.
  • sequences of 18-21 nucleotides are most particularly preferred, for unmodified oligonucleo ⁇ tides, slightly longer chains of up to about 26 nucleo ⁇ tides, are preferred for modified oligonucleotides such as phosphorothioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides.
  • Oligonucleotides complementary to and hybrid ⁇ izable with any portion of the c-kit 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, oligonuc ⁇ leotides complementary to the 5'-terminal region of the c-kit mRNA transcript are preferred. The oligonucleo- tide is preferably directed to a site at or near the initiation codon for protein synthesis.
  • the following 40-mer oligodeoxynucleotide is complementary to the c- kit mRNA transcript beginning with the initiation codon of the transcript and extending downstream (in the 5' direction) : GAACGCAGAG AAAATCCCAG GCGCCGCGAG
  • oligomers based upon the above se ⁇ quence, in particular oligomers hybridizable to seg ⁇ ments of the c-kit message containing the initian codon, may be utilized. Particularly preferred are the following 15- to 26-mers:
  • Oligonucleotides hybridizable to the c-kit mRNA transcript finding utility according to the pres ⁇ ent invention include not only native oligomers of the biologically significant 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.
  • the oligonucleotides may be any of a number of types, including those having a charged or uncharged backbone.
  • phosphorothioates are stable to nuclease cleavage and soluble in lipid. They may be synthesized by known automatic synthesis methods.
  • the oligonucleotide employed may represent an unmodified oligonucleotide or an oligonucleotide ana ⁇ log.
  • One group of such analogs, the alkyl phosphon ⁇ ates includes but is not limited to the ethyl or meth ⁇ yl phosphonate analogs disclosed by U.S. Patent No. 4,469,863.
  • Non-ionic oligonucleotides 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 insol ⁇ uble polymer supports (Agrawal and Riftina, Nucl. Acids Res. r 6, 3009-3024 (1979); Miller e_ al. , 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.
  • methylphosphonate oligonucleosides The most efficient procedure for preparation of methylphosphonate oligonucleosides involves use of 5'-0-dimethoxytrityldeoxynucleoside-3'-O-diisopropylme- thylphosphoramidite intermediates, which are similar to the methoxy or 0-cyanoethyl phosphoramidite reagents used to prepare oligodeoxyribonucleotides.
  • the methyl ⁇ phosphonate oligomers can be prepared on controlled pore glass polymer supports using anautomated DNA syn ⁇ thesizer (Sarin et al. , Proc. Natl. Acad. Sci. USA r 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 ac- cording 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 phos ⁇ phate analogue. The charge is believed to confer the property of cellular uptake via a receptor (Loke et al.. 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 al. , J. Am. Chem. Soc. 106, 6077 (1984) .
  • the general synthetic method for phosphorothioate oligonucleotides was modified by Stein et al. , Nucl. Acids Res.. 16, 3209-3221 (1988), so that these compounds may readily be synthesized on an auto- matic synthesizer using the phosphoramidite approach.
  • oligoribonucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-0-methylribonucleotides (Inoue et al., Nucleic Acids Res. 15, 6131 (1987) and chimeric oligo ⁇ nucleotides that are composite RNA-DNA analogues (Inoue et al., FEBS Lett. 215, 327 (1987).
  • oligodeoxyribonucleotides are preferred in the practice of the present invention. Oligodeoxyribonucleotides are further preferred because, upon hybridization with c-kit mRNA, the resulting DNA-RNA hybrid duplex is a substrate for RNase H, which specifically attacks the RNA portion of DNA-RNA hybrid.
  • the antisense oligonucleotides of the present invention will have a sequence which is completely complementary to the target portion of the c-kit message. Absolute complementarity is not however required, particularly in larger oligomers.
  • reference herein to a "nucleotide sequence complementa ⁇ ry to at least a portion of the mRNA transcript" of c- kit does not necessarily mean a sequence having 100% complementarity with the transcript.
  • any oligonucleotide having sufficient complementarity to form a stable duplex with c-kit mRNA that is, an oli ⁇ gonucleotide which is "hybridizable" is suitable.
  • Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the de ⁇ gree of complementarity with the target region of the c-kit 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.
  • oligonucleotides capable of stable hybridization with any region of the c-kit message are within the scope of the present invention, oligonucleo ⁇ tides complementary to a region including the initia ⁇ tion codon are believed particularly effective. Par- ticularly preferred are oligonucleotides hybridizable to a region of the c-kit mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direc- tion) of that codon.
  • the antisense oligonucleotides of the inven ⁇ tion inhibit human erythropoiesis, as indicated by inhibition of colony forming unit-erythroid cells (CFU- E) and burst forming unit-erythroyd cells (BFU-E) . However, they do not appear to inhibit proliferation of cells of other lineages, such as colony forming unit- granulocyte-macrophage cells (CFU-GM) and colony form ⁇ ing unit-megakaryocyte cells (CFU-MEG) .
  • CFU-GM cells and CFU-MEG cells are the progenitors of blood granulo- cytes and platelets, respectively.
  • This pharmaceuti ⁇ cally significant differential sensitivity makes the instant oligonucleotides useful in treating disorders characterized by elevated production of red blood cells.
  • the antisense oligonucleotides of the inven ⁇ tion are believed useful in the treatment of any one of a variety of conditions characterized by an elevated hematocrit due to overproduction of erythrocytes.
  • One such disorder, polycythemia may arise from a variety of causes and is classified as either relative, second ⁇ ary or primary polycythemia.
  • Relative polycythemia In relative polycythemia, the red cell mass is normal. Plasma volume is decreased. The increase in erythrocytes is therefore a concentration effect. Relative polycythemia is associated with diabetic aci- dosis, diarrhea, or diabetes insipidus. It is also associated with the intake of diuretics.
  • red cell mass is increased secondarily to elevated erythropoietin (EPO) production.
  • EPO erythropoietin
  • Secondary polycythemia may also occur in patients who have significant pulmonary or cardiac disfunction. Decreased oxygen delivery to tissues simulates anemia which triggers a signal to increase erythrocyte production.
  • Secondary polycythe ⁇ mia may also occur in individuals who have tumors which are capable of synthesizing EPO, as in hypernephroma, cerebellar hemangioma and uterine leiomyoma.
  • Primary polycythemia is characterized by an increase in red cell mass, with either normal or de ⁇ creased EPO levels.
  • Primary polycythemia occurs in the myeloproliferative disorders, in particular polycythe ⁇ mia vera (PV) .
  • PV polycythe ⁇ mia vera
  • Disorders such as PV are true stem cell disorders. Accordingly, the white blood cell count and platelet count may be elevated.
  • control of erythrocyte production is the primary objective in management of PV. Control of PV is usually effected by phlebotomy in secondary causes (if treatment of the primary disease is ineffective) , and by a combination of phlebotomy and chemotherapy.
  • Chemotherapeutic treatment of PV typically utilizes alkylating agents such as busulfan, melphalan, cyclophosphamide, chloram- bucil or radioactive phosphorous in the form of sodium phosphate- 32 P.
  • alkylating agents such as busulfan, melphalan, cyclophosphamide, chloram- bucil or radioactive phosphorous in the form of sodium phosphate- 32 P.
  • the rapid fluid shifts imposed by phlebotomy in the treatment of PV can be dangerous for patients with cardiac/pulmonary disease. Phlebotomy is also associated with a significant risk of fatal thrombosis. (Burk et al. , Semin. Hematol. 23, 132 (1986); Ellis et al. , id. at 144). Control of erythrocyte production by administration of the c-kit antisense oligomers of the present invention is an attractive alternative to phlebotomy and chemotherapy.
  • the antisense oligonucleotides of the inven- tion are further believed to possess utility in the treatment of hematologic malignancies.
  • Hematologic neoplastic cells believed sensitive to the instant c- kit antisense oligonucleotides include, for example, myeloid leukemia cells. The appearance of these cells in the bone marrow and elsewhere in the body is associ ⁇ ated with various disease conditions, such as all of the various French-American-British (FAB) subtypes of acute myeloid leukemia, and chronic myeloid leukemia.
  • FAB French-American-British
  • the c-kit antisense oligonucleotides are believed particularly useful against acute myelogenous leukemia (AML) .
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • CML is characterized by abnormal pro ⁇ liferation of immature granulocytes - neutrophils, eosinophils, and basophils - in the blood, the bone marrow, the spleen, the liver, and sometimes other tissues.
  • the essential feature is accumulation of granulocytic precursors in these tissues.
  • the patient who presents symptoms will characteristically have more than 20,000 white blood cells per ⁇ l, and the count may exceed 400,000.
  • Virtually all CML patients will devel- op "blast crisis", the terminal stage of the disease during which immature blast cells rapidly proliferate, leading to patient death.
  • c-kit function appears to be important for development of melanocytes, i.e., neural crest- derived pigment cells, and germ (gonadal) cells, it is believed that the antisense oligonucleotides of the present invention are useful for the treatment of ma ⁇ lignant melanoma and testicular or ovarian tumors.
  • the antisense oligonucleotides of the inven- tion find utility as bone marrow purging agents. They may be utilized in vitro to cleanse bone marrow contam ⁇ inated by hematologic neoplasms. They are believed useful as purging agents in either allogeneic or autol- ogous bone marrow transplantation. They are believed particularly effective in the treatment of hematologi- cal malignancies or other neoplasias which metastasize in the bone marrow. According to a method for bone marrow purg ⁇ ing, 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.
  • This suspension of leukocytes is treated with c-kit antisense oligonuc ⁇ leotides 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.
  • One or more hematopoietic growth factors may be added to the aspirated marrow or buffy coat prepara ⁇ tion to stimulate growth of hematopoietic neoplasms, and thereby increase their sensitivity to the toxicity of the c-kit antisense oligonucleotides.
  • hemato ⁇ poietic growth factors include, for example, IL-3 and granulocyte macrophage colony stimulating factor (GM- CSF) .
  • GM- CSF granulocyte macrophage colony stimulating factor
  • the cells to be transferred are washed with autologous plasma or buffer to remove unincorporated oligomer. The washed cells are then infused back into the patient.
  • the antisense oligonucleo- tides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • 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 commer ⁇ cially available. Illustrative thereof are distilled water, physiological saline, aqueous solution of dex ⁇ trose, and the like.
  • the c-kit mRNA antisense oligonucleotides are preferably administered parenterally, most preferably intravenously. The vehi- cle is designed accordingly.
  • lympho ⁇ cytes from peripheral blood, treating them with the antisense oligonucleotides, then returning the treated lymphocytes to the peripheral blood of the donor.
  • Ex vivo techniques have been utilized in treatment of cancer patients with interleukin-2 activated lympho ⁇ cytes, and are well-known to those skilled in the art.
  • the antisense oligonucleotides may be administered by a variety of specialized oligonucleo ⁇ tide delivery techniques.
  • oligonucleo ⁇ tides may be encapsulated in liposomes 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 diacetylphos- phate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. Oligonucleotides have been successfully encapsulated in unilameller liposomes.
  • RNA and DNA have been successfully used to deliver RNA and DNA to cells. Arad et al., Biochem. Biophy. Acta. 859, 88-94 (1986).
  • the oligonucleotides may be conjugated to poly(L-lysine) to increase cell penetration. Such conjugates are described by Lemaitre et aj.. , Proc. Natl. Acad. Sci. USA. 84, 648-652 (1987).
  • the proce ⁇ dure 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 c-kit antisense oligonucleotides may be administered in amounts effective to kill neoplastic cells while main- taining the viability of normal hematologic cells. Such amounts may vary depending on the nature and ex ⁇ tent of the neoplasm, the particular oligonucleotide utilized, the relative sensitivity of the neoplasm to the oligonucleotide, and other factors. Concentrations from about 10 to 200 ⁇ g/ml per 10 5 cells may be employ ⁇ ed, 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.
  • the c-kit antisense oligonu ⁇ cleotides may be administered in an amount sufficient to result in extracellular concentrations approximating the above stated in vitro concentrations.
  • the intracellular concentration is in the range of from about 10 to about 100 ⁇ g/ml.
  • the actual dosage admin ⁇ istered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health and sex of the patient, the route of administra ⁇ tion, and other factors. Those skilled in the art should be readily able to derive suitable dosages and schedules of administration to suit the specific cir ⁇ cumstance.
  • the daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 1,000 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 circumstances and needs of the patient.
  • c-kit antisense oligonucleotide on hematopoietic progenitor cell cloning efficiency and development was systematically investigated by assess ⁇ ing CFU-E, burst-forming unit-erythroid (BFU-E) , CFU- GM, and CFU-MEG growth after oligomer exposure.
  • BMC Human bone marrow cells
  • adherent, T lymphocyte-de ⁇ pleted population was further enriched by positively selecting CD34 + cells with immunomagnetic beads (Dynal A.S., Oslo, Norway).
  • the A ' T ' MNC cells were suspended in supplemented alpha medium and incu- bated with mouse anti-HPCA-I antibody in 1:20 dilution, 45 minutes, at 4 ⁇ C with gentle inverting of tubes. Cells were washed x 3 in Supplemented alpha medium, and then incubated with beads coated with the Fc fragment of goat anti-mouse IgG 1 (75 ⁇ l of immunobeads/10 7 A ' T " MNC) . After 45 minutes of incubation (4 ⁇ C), cells ad ⁇ herent to the beads were positively selected using a magnetic particle concentrator as directed by the manu ⁇ facturer.
  • Oligodeoxynucleotides Unmodified, 18-nucle- otide oligodeoxynucleotides (oligomers) were synthe ⁇ sized as previously reported (Gewirtz et al. , Science 242, 1303-1306 (1988)). In brief, oligomers were syn ⁇ thesized on an Applied Biosystems 380B DNA synthesizer by means of a 0-cyanoethyl phosphoramidite chemistry. Oligomers were purified by ethanol precipitation and multiple washes in 70% ethanol. They were then lyophi- lized to dryness and redissolved in culture medium prior to use at a concentration of l ⁇ g/ ⁇ l (0.175 ⁇ M) .
  • Oligomer sequences employed corresponding to codons 1- 6 of the published human c-kit cDNA sequence (Yarden, et al., The EMBO Journal 6, 3341-3351 (1987)), were as follows: ATGAGAGGCG CTCGCGGC (SEQ ID NO:14), sense oligomer; GCCGCGAGCG CCTCTCAT (SEQ ID NO:10), antisense oligomer; and GCACCGTCTG CCAGTCGC (SEQ ID NO:15) , scrambled sequence oligomer.
  • Oligomer Treatment of Cells Cells were exposed to oligomers as previously described (Gewirtz et al.. Science 242, 1303-1306 (1988)). 2 x 10 5 A ' T ' MNC or CD34 + MNC were incubated in 5 ml polypropylene tubes (Fisher Scientific, Pittsburgh, PA) in a total volume of 0.4 ml of Iscove's modified Dulbecco's medium (IMDM) containing 2% human AB serum and 10 mM Hepes buffer. Oligomers were added at time zero (2.5-100 ⁇ g/ml), and 50% of the initial dose was added again 18 hours later (final total concentration -0.6-26 ⁇ M) .
  • IMDM Iscove's modified Dulbecco's medium
  • cells (10 5 A " T ' MNC or 5 x 10 3 CD34 + MNC) were resuspended in IMDM sup ⁇ plemented with 30% human AB serum, 1% BSA, 10 "4 M mer- captoethanol, and 10% citrated bovine plasma (Hyclone Laboratories, Denver, CO) . Addition of the appropriate recombinant human growth factors allowed for stimula ⁇ tion of the following cell types:
  • BFU-E 20 U/ml IL-3 and 5 U/ml EPO, or
  • CFU-GM 20 U/ml IL-3 and 5 ng/ml granulocyte- macrophage colony stimulating factor
  • CFU-MEG 20 U/ml IL-3 and 100 ng/ml IL-6.
  • CFU-E colo ⁇ nies were scored at day 7, BFU-E colonies at day 14, CFU-MEG at day 12, and CFU-GM at day 11 of incubation.
  • RT-PCR Reverse Transcription-Polvmerase Chain Reaction
  • RNA pel ⁇ let was resuspended in -400 ⁇ l of water, precipitated with 0.3 M potassium acetate, washed twice in 75% etha ⁇ nol, and then stored at -70 ⁇ C until used.
  • RNA was reverse-transcribed with 500 U of Molo- ney murine leukemia virus reverse transcriptase (MoMLV- RT) and 50 pmol of a 22-nucleotide oligodeoxynucleotide 3' primer complementary to nucleotides 1201-1179 (CTAGG- AATGT GTAAGTGCCT CC, SEQ ID NO:16) of the published c- kit cDNA sequence.
  • MoMLV- RT Molo- ney murine leukemia virus reverse transcriptase
  • the resulting cDNA fragment was am- plified using 5 U of Thermus aquaticus (Tag) polymerase and a 22-nucleotide oligodeoxynucleotide 5' primer spe ⁇ cific for c-kit nucleotides 842-864 (GGTTGACTAT CAGTTCA- GCG AG, SEQ ID NO:17). Twenty-five ⁇ l of amplified pro ⁇ duct was electrophoresed on 4% agarose gel and subse- quently transferred to a nylon filter. Filters were pre-hybridized, and then probed with a 32 P end-labeled oligonucleotide probe (Caracciolo et aj..
  • a ' T ' MNC cells were kept for 24 hours at 4°C in IMDM containing 2% human AB serum, then shifted to 37°C and stimulated with IL-3 (20 U/ml) and EPO (5 U/ml) in
  • Antisense-treated cells had no detect ⁇ able c-kit mRNA, while sense (lane 3) and scrambled se- quence (lane 5) treated cells had levels which were sim ⁇ ilar to those observed in untreated control cells at the same time point (lane 2) .
  • Example 2
  • c-kit antisense oligomers are believed particularly useful as CML bone marrow pur ⁇ ging agents.
  • c-kit oligomers are useful in controlling the mark ⁇ edly elevated hemoglobin/hematocrit found in patients with PV, another myeloproliferative disorder.
  • CD34 + MNC (2 x 10 4 ) were cloned in the presence of 5 units of EPO and 100 ng of SCF per ml alone or with sense, antisense, or scram- bled-sequence c-kit oligomers (final concentration, 150 ⁇ g/ml (-26 ⁇ M) ) .
  • 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 trans ⁇ ferred 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 pro-halted 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 pla ⁇ telets) .
  • 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 car ⁇ ried 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 ad ⁇ justed to a cell concentration of about 2 x 10 7 /ml in TC- 199 containing about 20% autologous plasma.
  • C-kit anti ⁇ sense oligodeoxynucleotide for example, in a concentra- tion of about 8 mg/ml, is added to the transfer packs containing the cell suspension.
  • Recombinant human hema ⁇ topoietic growth factors e.g., rH IL-3 or rH GM-CSF
  • rH IL-3 or rH GM-CSF may be added to the suspension to stimulate growth of hematopoietic neoplasms and thereby increase their sen- sitivity c-kit 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% autol- ogous 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Oligonucleotides are provided having a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-kit gene. These 'antisense' oligonucleotides are hybridizable to the c-kit mRNA transcript. Such oligonucleotides are useful in selectively inhibiting proliferation of erythroid cells, particularly in disorders characterized by an elevated hematocrit due to overproduction of erythrocytes. The antisense oligomers also have activity agent hematologic neoplastic cells and are therefore suitable as bone marrow purging agents.

Description

ANTISENSE OLIGONUCLEOTIDES TO C-KIT PROTO-ONCOGENE AND USES THEREOF
Field of the Invention
The invention relates to antisense oligonucleo¬ tides to proto-oncogenes, in particular to antisense oligonucleotides to the c-kit gene, and the use of such oligonucleotides to selectively inhibit proliferation of certain cells.
Reference to Government Grant
The invention described herein was supported in part by National Institutes of Health grants CA36896 and CA01324. The United States government has certain rights in the invention.
Background of the Invention
The c-kit gene is the normal homologue of v- kit, the HZ4 feline sarcoma virus oncogene. It resides on human chromosome 4. The gene encodes a dimeric trans¬ membrane glycoprotein receptor with tyrosine kinase ac¬ tivity that appears to be highly related to the receptors for colony stimulating factor-1 and platelet derived growth factor. (Yarden et. al.. , The EMBO Journal. 6, 3341-3351 (1987)). Like these receptors, c-kit also appears to belong to the immunoglobulin gene superfamily. The mouse c-kit gene has been mapped to chromo¬ some 5 where it was determined to be allelic with the dominant white spotting locus ( ) (Chabot et al. , Nature 335, 88-89 (1988). C-kit mutations are commonly found in W mice and, in addition to abnormalities affecting coat color and gonadal development, they also have a variety of hematopoietic defects. Macrocytic anemia is one of the most striking and profound of these abnormalities. The W42 mutation, associated with a particularly severe hematologic manifestation, has been shown to be due to a missense mutation leading to replacement of one amino acid and defective tyrosine kinase activity (Tan et al. , Science 247, 209 (1990)) . Such animals are also known to have about one-third of the erythroid burst forming units of healthy wild-type littermates (Goldwather et aJL. , Exp. Heme. 18, 936 (1990)).
The ligand for the c-kit receptor has now been identified, molecularly cloned and expressed (Yarden et. al.. The EMBO Journal. 6, 3341-3351 (1987)). The encoded protein, known as stem cell factor (SCF) , mast cell growth factor (MGF) , or steel factor (SLF) is the product of a gene which resides at the steel (SI) locus. Mice with SI mutations have phenotypic abnormalities quite similar to those of mice. The mouse lacks, or has defects in, a critical signal transducing receptor encod¬ ed by c-kit. The SI mouse has defects in the ligand which stimulates the receptor.
The importance of the c-kit ligand-receptor system in human hematopoiesis is unclear. No human uta- tions at the corresponding locii have been described.
Studies in mice may have very limited applicability to human systems. Moreover, even if a tissue is shown to express a particular message, the importance of the mes¬ sage to expression of a cellular phenotype is not known until the cell is deprived of the encoded protein. Bio¬ logical systems are redundant. Lack of a protein can often be compensated by another protein of the same fami- ly. It is therefore not predictable that inhibition of expression of a particular gene will result in altered phenotype.
Summary of the invention
Antisense oligonucleotides and pharmaceutical compositions thereof with pharmaceutical carriers are provided. Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA tran¬ script of the human c-kit 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 uti¬ lized, 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 desta¬ bilize 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 oligo¬ deoxynucleotide, more advantageously an 18- to 26-mer. A 15- to 21-mer is most preferred.
While in principle oligonucleotides having a sequence complementary to any region of the c-kit gene find utility in the present invention, oligonucleotides complementary to a portion of the c-kit mRNA transcript including the translation initiation codon are particu¬ larly preferred. Also preferred are oligonucleotides complementary to a portion of the c-kit mRNA transcript lying within about 40 nucleotides upstream (the 5' direc¬ tion) or about 40 nucleotides downstream (the 3' direc¬ tion) from the translation initiation codon. The invention also provides a method for in¬ hibiting proliferation of erythroid cells comprising administering to a host in need of such treatment an effective amount of the c-kit antisense oligonucleotides of the invention.
The invention provides a method for treating hematologic neoplasms characterized by c-kit expression comprising administering an effective amount of c-kit antisense oligonucleotide in vivo or ex vivo to a host in need of such treatment, or to cells harvested from the host.
Administration of the c-kit oligonucleotides is also useful in treatment of malignant melanoma, and tes- ticular or ovarian tumors. As used in the herein specification and append¬ ed claims, unless otherwise indicated, the term "oligonu¬ cleotide" includes both oligomers of ribonucleotide i.e. , oligoribonucleotides, and oligomers of deoxyribonucleo- tide i.e., oligodeoxyribonucleotides (also referred to herein as "oligodeoxynucleotides") . Oligodeoxynucleo- tides are preferred.
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 biologically significant nucleotides, i.e. nucleotides of adenine ("A"), deoxyadenine ("dA") , guanine ("G") , deoxy- guanine ("dG") , cytosine ("C") , deoxycytosine ("dC") , thymine ("T") and uracil ("TJ") , but also oligomers and polymers hybridizable to the c-kit mRNA transcript which may contain other nucleotides. Likewise, the terms "oli¬ gonucleotide" and "oligodeoxynucleotide" include oligo¬ mers and polymers wherein one or more purine or pyrimi- dine moieties, sugar moieties or internucleotide linkages is chemically modified. The term "oligonucleotide" is thus understood to also include oligomers which may prop- erly be designated as "oligonucleosides" because of modi¬ fication of the internucleotide phosphodiester bond. Such modified oligonucleotides include, for example, the alkylphosphonate oligonucleosides, discussed below. The term "phosphorothioate oligonucleotide" means an oligonucleotide wherein one or more of the in¬ ternucleotide linkages is a phosphorothioate group,
Figure imgf000007_0001
as opposed to the phosphodiester group
0
» -0 - P - 0~
I o-
which is characteristic of unmodified oligonucleotides. By "alkylphosphonate oligonucleoside" is meant an oligonucleotide wherein one or more of the internuc¬ leotide linkages is an alkylphosphonate group,
Figure imgf000007_0002
wherein R is an alkyl group, preferably methyl or ethyl. 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-kit mRNA transcript" means the presently known mRNA transcript of the human c-kit gene, or any further transcripts which may be elucidated. Description of the Figures
Figure 1 is an autoradiograph of a reverse transcription-polymerase chain recaction gel indicating the increase in c-kit mRNA at intervals following stim- ulation of adherent, T lymphocyte-depleted human bone marrow cells (A'T'MNC) with 20 U/ml interleukin-3 and 5 U/ml erythropoietin: Lane 1 (t = 0) ; lane 2 (2 hrs) ; lane 3 (8 hrs) ; lane 4 (12 hrs) ; lane 5 (24 hrs) ; lane 6 (36 hrs) ; lane 7 (48 hrs) ; lane 8 (H20 control) . Figure 2 is a similar autoradiograph indicat¬ ing the effect of c-kit oligomer exposure on c-kit mRNA levels in A'T'MNC after stimulation by interleukin-3 and erythropoietin in 5% AB serum. Lane 1 (no oligo¬ mer, t = 0) ; lane 2 (no oligomer, t = 36 hrs) ; lane 3 (sense oligomer, 36 hrs) ; lane 4 (antisense, 36 hrs) ; lane 5 (scrambled sequence with identical base content, 36 hrs) .
Figure 3 shows the effect of c-kit oligode- oxyynucleotides on BFU-E-derived colony formation. Oligomers were added to cultures at time zero, and 50% of the initial dose was again added 18 hours later. The bars on the graph indicate: 1, untreated control cells; 2, antisense treatment of 20 μg/ml followed by 10 μg/ml; 3, antisense treatment of 40 μg/ml followed by 20 μg/ml; 4, antisense treatment of 100 μg/ml fol¬ lowed by 50 μg/ml; 5, sense treatment of 100 μg/ml followed by 50 μg/ml; 6, scrambled-sequence treatment of 100 μg/ml followed by 50 μg/ml.
Detailed Description of the Invention
We have discovered that the c-kit gene is of predominant importance in human erythropoiesis. We have found that the protein which this gene expresses, a receptor for tyrosine kinase, transduces a signal which acts in concert with interleukin-3 (IL-3) to optimize cell proliferation, particularly erythroid burst forming units (BFU-E) . The putative DNA sequence complementary to the mRNA transcript of the human c-kit gene has been reported by Yarden et al. , The EMBO Journal, 6, 3341- 3351 (1987) , the entire disclosure of which is incorpo- rated herein by reference. The nucleotide sequence and predicted amino acid sequence are set forth in Figure 3 thereof. The c-kit polypeptide is synthesized by translation of a single 5-kb mRNA, which contains an open reading frame coding for a 976 amino acid polypep- tide.
The antisense oligonucleotides of the inven¬ tion may be synthesized by any of the known chemical oligonucleotide synthesis methods. Such methods are generally described, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technology. VCH Verlagsgesellschaft mbH (H. Ibelgaufts trans. 1987).
Any of the known methods of oligonucleotide synthesis may be utilized in preparing the instant antisense oligonucleotides. The antisense oligonucleotides are most ad¬ vantageously prepared by utilizing any of the commer¬ cially available, automated nucleic acid synthesizers, for example, the Applied Biosystems 380B DNA Synthe¬ sizer, which utilizes /9-cyanoethyl phosphoramidite chemistry.
Since the complete nucleotide synthesis of DNA complementary to the c-kit mRNA transcript is known, antisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by the oligonucleotide synthesis methods known to those skilled in the art.
While any length oligonucleotide may be uti¬ lized in the practice of the invention, sequences shor¬ ter than 12 nucleotides may be less specific in hybrid- izing to the target c-kit mRNA, may be more easily destroyed by enzymatic digestion, and may be destabili¬ zed by even a single base pair mismatch. Hence, oligo- nucleotides having 12 or more nucleotides . are pre¬ ferred.
Long sequences, particularly sequences longer than about 40 nucleotides, may be somewhat less effec- tive in inhibiting c-kit translation because of de¬ creased uptake by the target cell. Thus, oligomers of 12-40 nucleotides are preferred, more preferably 15-30 nucleotides, most preferably 18-26 nucleotides. Se¬ quences of 18-21 nucleotides are particularly pre- ferred. While sequences of 18-21 nucleotides are most particularly preferred, for unmodified oligonucleo¬ tides, slightly longer chains of up to about 26 nucleo¬ tides, are preferred for modified oligonucleotides such as phosphorothioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides.
Oligonucleotides complementary to and hybrid¬ izable with any portion of the c-kit 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, oligonuc¬ leotides complementary to the 5'-terminal region of the c-kit mRNA transcript are preferred. The oligonucleo- tide is preferably directed to a site at or near the initiation codon for protein synthesis. The following 40-mer oligodeoxynucleotide is complementary to the c- kit mRNA transcript beginning with the initiation codon of the transcript and extending downstream (in the 5' direction) : GAACGCAGAG AAAATCCCAG GCGCCGCGAG
CGCCTCTCAT (SEQ ID N0:1).
Smaller oligomers based upon the above se¬ quence, in particular oligomers hybridizable to seg¬ ments of the c-kit message containing the initian codon, may be utilized. Particularly preferred are the following 15- to 26-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)
(SEQ ID NO:13) Oligonucleotides hybridizable to the c-kit mRNA transcript finding utility according to the pres¬ ent invention include not only native oligomers of the biologically significant 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. The oligonucleotides may be any of a number of types, including those having a charged or uncharged backbone. For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting an alkyl group or sulfur atom for a phosphate oxygen in the internucleo- tide phosphodiester linkage to form alkylphosphonate oligonucleotide or phosphorothioate oligonucleotides. The phosphorothioates, in particular, are stable to nuclease cleavage and soluble in lipid. They may be synthesized by known automatic synthesis methods.
The oligonucleotide employed may represent an unmodified oligonucleotide or an oligonucleotide ana¬ log. One group of such analogs, the alkyl phosphon¬ ates, includes but is not limited to the ethyl or meth¬ yl phosphonate analogs disclosed by U.S. Patent No. 4,469,863. Non-ionic oligonucleotides 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 insol¬ uble polymer supports (Agrawal and Riftina, Nucl. Acids Res. r 6, 3009-3024 (1979); Miller e_ al. , 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) ; Dor an 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'-O-diisopropylme- thylphosphoramidite intermediates, which are similar to the methoxy or 0-cyanoethyl phosphoramidite reagents used to prepare oligodeoxyribonucleotides. The methyl¬ phosphonate oligomers can be prepared on controlled pore glass polymer supports using anautomated DNA syn¬ thesizer (Sarin et al. , Proc. Natl. Acad. Sci. USAr 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 ac- cording 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 phos¬ phate analogue. The charge is believed to confer the property of cellular uptake via a receptor (Loke et al.. 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 al. , J. Am. Chem. Soc. 106, 6077 (1984) . The general synthetic method for phosphorothioate oligonucleotides was modified by Stein et al. , Nucl. Acids Res.. 16, 3209-3221 (1988), so that these compounds may readily be synthesized on an auto- matic synthesizer using the phosphoramidite approach.
Furthermore, recent advances in the produc¬ tion of oligoribonucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-0-methylribonucleotides (Inoue et al., Nucleic Acids Res. 15, 6131 (1987) and chimeric oligo¬ nucleotides that are composite RNA-DNA analogues (Inoue et al., FEBS Lett. 215, 327 (1987).
While inhibition of c-kit mRNA translation is possible utilizing either antisense oligoribonucleo- tides or oligodeoxyribonucleotides, free oligoribonuc- leotides 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-kit 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 duplex releases the antisense oligodeoxy- nucleotide for hybridization with additional c-kit messages. In general, the antisense oligonucleotides of the present invention will have a sequence which is completely complementary to the target portion of the c-kit message. Absolute complementarity is not however required, particularly in larger oligomers. Thus, reference herein to a "nucleotide sequence complementa¬ ry to at least a portion of the mRNA transcript" of c- kit does not necessarily mean a sequence having 100% complementarity with the transcript. In general, any oligonucleotide having sufficient complementarity to form a stable duplex with c-kit mRNA, that is, an oli¬ gonucleotide which is "hybridizable", is suitable. Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the de¬ gree of complementarity with the target region of the c-kit 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 de- gree of mismatching which may be tolerated between any given antisense oligomer and the target c-kit 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 determined from standard texts, such as Molecular Clon¬ ing: A Laboratory Manual. (2nd edition, 1989) , J. Sam- brook et al. , eds.
While oligonucleotides capable of stable hybridization with any region of the c-kit message are within the scope of the present invention, oligonucleo¬ tides complementary to a region including the initia¬ tion codon are believed particularly effective. Par- ticularly preferred are oligonucleotides hybridizable to a region of the c-kit mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direc- tion) of that codon.
The antisense oligonucleotides of the inven¬ tion inhibit human erythropoiesis, as indicated by inhibition of colony forming unit-erythroid cells (CFU- E) and burst forming unit-erythroyd cells (BFU-E) . However, they do not appear to inhibit proliferation of cells of other lineages, such as colony forming unit- granulocyte-macrophage cells (CFU-GM) and colony form¬ ing unit-megakaryocyte cells (CFU-MEG) . CFU-GM cells and CFU-MEG cells are the progenitors of blood granulo- cytes and platelets, respectively. This pharmaceuti¬ cally significant differential sensitivity makes the instant oligonucleotides useful in treating disorders characterized by elevated production of red blood cells. The antisense oligonucleotides of the inven¬ tion are believed useful in the treatment of any one of a variety of conditions characterized by an elevated hematocrit due to overproduction of erythrocytes. One such disorder, polycythemia, may arise from a variety of causes and is classified as either relative, second¬ ary or primary polycythemia.
In relative polycythemia, the red cell mass is normal. Plasma volume is decreased. The increase in erythrocytes is therefore a concentration effect. Relative polycythemia is associated with diabetic aci- dosis, diarrhea, or diabetes insipidus. It is also associated with the intake of diuretics.
In secondary polycythemia, red cell mass is increased secondarily to elevated erythropoietin (EPO) production. This occurs in individuals who have locat¬ ed to higher altitudes, since decreased oxygen simu¬ lates anemia, which is a triggering signal for increase of erythrocyte production. Secondary polycythemia may also occur in patients who have significant pulmonary or cardiac disfunction. Decreased oxygen delivery to tissues simulates anemia which triggers a signal to increase erythrocyte production. Secondary polycythe¬ mia may also occur in individuals who have tumors which are capable of synthesizing EPO, as in hypernephroma, cerebellar hemangioma and uterine leiomyoma.
Primary polycythemia is characterized by an increase in red cell mass, with either normal or de¬ creased EPO levels. Primary polycythemia occurs in the myeloproliferative disorders, in particular polycythe¬ mia vera (PV) . Disorders such as PV are true stem cell disorders. Accordingly, the white blood cell count and platelet count may be elevated. However, control of erythrocyte production is the primary objective in management of PV. Control of PV is usually effected by phlebotomy in secondary causes (if treatment of the primary disease is ineffective) , and by a combination of phlebotomy and chemotherapy. Chemotherapeutic treatment of PV typically utilizes alkylating agents such as busulfan, melphalan, cyclophosphamide, chloram- bucil or radioactive phosphorous in the form of sodium phosphate-32P. The rapid fluid shifts imposed by phlebotomy in the treatment of PV can be dangerous for patients with cardiac/pulmonary disease. Phlebotomy is also associated with a significant risk of fatal thrombosis. (Burk et al. , Semin. Hematol. 23, 132 (1986); Ellis et al. , id. at 144). Control of erythrocyte production by administration of the c-kit antisense oligomers of the present invention is an attractive alternative to phlebotomy and chemotherapy.
The antisense oligonucleotides of the inven- tion are further believed to possess utility in the treatment of hematologic malignancies. Hematologic neoplastic cells believed sensitive to the instant c- kit antisense oligonucleotides include, for example, myeloid leukemia cells. The appearance of these cells in the bone marrow and elsewhere in the body is associ¬ ated with various disease conditions, such as all of the various French-American-British (FAB) subtypes of acute myeloid leukemia, and chronic myeloid leukemia.
The c-kit antisense oligonucleotides are believed particularly useful against acute myelogenous leukemia (AML) . Significant activity against chronic myelogenous leukemia (CML) has also been demonstrated. CML, in particular, is characterized by abnormal pro¬ liferation of immature granulocytes - neutrophils, eosinophils, and basophils - in the blood, the bone marrow, the spleen, the liver, and sometimes other tissues. The essential feature is accumulation of granulocytic precursors in these tissues. The patient who presents symptoms will characteristically have more than 20,000 white blood cells per μl, and the count may exceed 400,000. Virtually all CML patients will devel- op "blast crisis", the terminal stage of the disease during which immature blast cells rapidly proliferate, leading to patient death.
Since c-kit function appears to be important for development of melanocytes, i.e., neural crest- derived pigment cells, and germ (gonadal) cells, it is believed that the antisense oligonucleotides of the present invention are useful for the treatment of ma¬ lignant melanoma and testicular or ovarian tumors.
The antisense oligonucleotides of the inven- tion find utility as bone marrow purging agents. They may be utilized in vitro to cleanse bone marrow contam¬ inated by hematologic neoplasms. They are believed useful as purging agents in either allogeneic or autol- ogous bone marrow transplantation. They are believed particularly effective in the treatment of hematologi- cal malignancies or other neoplasias which metastasize in the bone marrow. According to a method for bone marrow purg¬ ing, 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. 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-kit antisense oligonuc¬ leotides 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 treatment with c-kit antisense may be utilized, which methods may result in even more purified preparations of hematopoietic cells than the aforesaid buffy coat preparation.
One or more hematopoietic growth factors may be added to the aspirated marrow or buffy coat prepara¬ tion to stimulate growth of hematopoietic neoplasms, and thereby increase their sensitivity to the toxicity of the c-kit antisense oligonucleotides. Such hemato¬ poietic growth factors include, for example, IL-3 and granulocyte macrophage colony stimulating factor (GM- CSF) . The recombinant human versions of such growth factors are advantageously employed.
After treatment with the antisense oligonuc- leotides, the cells to be transferred are washed with autologous plasma or buffer to remove unincorporated oligomer. The washed cells are then infused back into the patient.
For in vivo use, the antisense oligonucleo- tides 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 commer¬ cially available. Illustrative thereof are distilled water, physiological saline, aqueous solution of dex¬ trose, and the like. For in vivo antineoplastic use and in vivo erythroid cell reduction, the c-kit mRNA antisense oligonucleotides are preferably administered parenterally, most preferably intravenously. The vehi- cle is designed accordingly. It is also possible to administer such compounds gx vivo by isolating lympho¬ cytes from peripheral blood, treating them with the antisense oligonucleotides, then returning the treated lymphocytes to the peripheral blood of the donor. Ex vivo techniques have been utilized in treatment of cancer patients with interleukin-2 activated lympho¬ cytes, and are well-known to those skilled in the art.
In addition to administration with conven¬ tional carriers, the antisense oligonucleotides may be administered by a variety of specialized oligonucleo¬ tide delivery techniques. For example, oligonucleo¬ tides may be encapsulated in liposomes 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 diacetylphos- phate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. Oligonucleotides have been successfully encapsulated in unilameller liposomes.
Reconstituted Sendai virus envelopes have been successfully used to deliver RNA and DNA to cells. Arad et al., Biochem. Biophy. Acta. 859, 88-94 (1986). The oligonucleotides may be conjugated to poly(L-lysine) to increase cell penetration. Such conjugates are described by Lemaitre et aj.. , Proc. Natl. Acad. Sci. USA. 84, 648-652 (1987). The proce¬ dure 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.
For ex vivo antineoplastic application, such as, for example, in bone marrow purging, the c-kit antisense oligonucleotides may be administered in amounts effective to kill neoplastic cells while main- taining the viability of normal hematologic cells. Such amounts may vary depending on the nature and ex¬ tent of the neoplasm, the particular oligonucleotide utilized, the relative sensitivity of the neoplasm to the oligonucleotide, and other factors. Concentrations from about 10 to 200 μg/ml per 105 cells may be employ¬ ed, 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 contain- ing 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.
For in vivo use, the c-kit antisense oligonu¬ cleotides may be administered in an amount sufficient to result in extracellular concentrations approximating the above stated in vitro concentrations. Preferably, the intracellular concentration is in the range of from about 10 to about 100 μg/ml. The actual dosage admin¬ istered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health and sex of the patient, the route of administra¬ tion, and other factors. Those skilled in the art should be readily able to derive suitable dosages and schedules of administration to suit the specific cir¬ cumstance. The daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 1,000 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 circumstances and needs of the patient.
The present invention is described in greater detail in the following non-limiting examples.
Example 1
Effect of c-kit Antisense Oligomer Exposure on Normal Hematopoietic Progenitor Cell Growth.
The effect of c-kit antisense oligonucleotide on hematopoietic progenitor cell cloning efficiency and development was systematically investigated by assess¬ ing CFU-E, burst-forming unit-erythroid (BFU-E) , CFU- GM, and CFU-MEG growth after oligomer exposure.
Cells: Human bone marrow cells (BMC) were obtained from normal, healthy volunteers by Ficoll- Hypaque density gradient centrifugation, and were par¬ tially enriched for hematopoietic progenitors by remov¬ al of adherent, phagoc tic elements and T lymphocytes (Gewirtz et al. , J. Immunol. 139, 2915-2925 (1987)). For some experiments, the adherent, T lymphocyte-de¬ pleted population (A"T"MNC) was further enriched by positively selecting CD34+ cells with immunomagnetic beads (Dynal A.S., Oslo, Norway). The A'T'MNC cells were suspended in supplemented alpha medium and incu- bated with mouse anti-HPCA-I antibody in 1:20 dilution, 45 minutes, at 4βC with gentle inverting of tubes. Cells were washed x 3 in Supplemented alpha medium, and then incubated with beads coated with the Fc fragment of goat anti-mouse IgG1 (75 μl of immunobeads/107 A'T" MNC) . After 45 minutes of incubation (4βC), cells ad¬ herent to the beads were positively selected using a magnetic particle concentrator as directed by the manu¬ facturer.
Oligodeoxynucleotides: Unmodified, 18-nucle- otide oligodeoxynucleotides (oligomers) were synthe¬ sized as previously reported (Gewirtz et al. , Science 242, 1303-1306 (1988)). In brief, oligomers were syn¬ thesized on an Applied Biosystems 380B DNA synthesizer by means of a 0-cyanoethyl phosphoramidite chemistry. Oligomers were purified by ethanol precipitation and multiple washes in 70% ethanol. They were then lyophi- lized to dryness and redissolved in culture medium prior to use at a concentration of lμg/μl (0.175 μM) . Oligomer sequences employed, corresponding to codons 1- 6 of the published human c-kit cDNA sequence (Yarden, et al., The EMBO Journal 6, 3341-3351 (1987)), were as follows: ATGAGAGGCG CTCGCGGC (SEQ ID NO:14), sense oligomer; GCCGCGAGCG CCTCTCAT (SEQ ID NO:10), antisense oligomer; and GCACCGTCTG CCAGTCGC (SEQ ID NO:15) , scrambled sequence oligomer.
Oligomer Treatment of Cells: Cells were exposed to oligomers as previously described (Gewirtz et al.. Science 242, 1303-1306 (1988)). 2 x 105 A'T'MNC or CD34+MNC were incubated in 5 ml polypropylene tubes (Fisher Scientific, Pittsburgh, PA) in a total volume of 0.4 ml of Iscove's modified Dulbecco's medium (IMDM) containing 2% human AB serum and 10 mM Hepes buffer. Oligomers were added at time zero (2.5-100 μg/ml), and 50% of the initial dose was added again 18 hours later (final total concentration -0.6-26 μM) . Twenty-four hours after the first addition of oligomers, cells were prepared for plating in plasma clot or methylcellulose cultures. Cells (1 x 105 A"T*MNC or 1 x 104 CD34+MNC per dish) were not washed before plating. Control cultures were manipulated in an identical manner but were not treated with oligomers. Colony Assays: Assays for hematopoietic progenitor cells of varying lineages were carried out essentially as reported (Id.). In brief, cells (105 A" T'MNC or 5 x 103 CD34+MNC) were resuspended in IMDM sup¬ plemented with 30% human AB serum, 1% BSA, 10"4 M mer- captoethanol, and 10% citrated bovine plasma (Hyclone Laboratories, Denver, CO) . Addition of the appropriate recombinant human growth factors allowed for stimula¬ tion of the following cell types:
CFU-E: 5 U/ml EPO;
BFU-E: 20 U/ml IL-3 and 5 U/ml EPO, or
100 ng/ml SCF and 5 U/ml EPO; CFU-GM: 20 U/ml IL-3 and 5 ng/ml granulocyte- macrophage colony stimulating factor; CFU-MEG: 20 U/ml IL-3 and 100 ng/ml IL-6.
One ml volumes were cultured in 35 mm petri dishes at 37°C, 5% C02, and 95% humidity. CFU-E colo¬ nies were scored at day 7, BFU-E colonies at day 14, CFU-MEG at day 12, and CFU-GM at day 11 of incubation.
Colony identification was carried out as previously described (Id.) . Statistics: Statistical significance of' differences between means of test groups was assessed by Mann-Whitney non-parametric analysis using a statis¬ tical software package (Statview 512+, Brainpower, Inc., Calabasas, CA) . The results appear in Tables 1 and 2. Values presented are actual colonies counted, pooled from two or three individual studies.
£ 0
Table 1
Effect of c-kit on A'T" cell derived colony formation
Progenitor Control Sense Scrambled Antisense Cell Type Sequence
CFU-E 182, 209 153, 142 119, 128 33, 59 1943, 543 1635, 1135 627, 649 243, 213
148, 100 129, 176 149, 206 97, 107 Mean (±SE)= Mean (±SE)= Mean (±SE)= Mean (±SE)=
522±291 562±268 3131103 125±34
BFU-E 133, 152 117, 106 94, 64 60, 149
534, 392 601, 249 273, 246 126, 113
206, 172 215, 258 162, 246 59, 51
Mean (±SE)= Mean (±SE)= Mean (1SE)= Mean (+SE)=
265±66 258174 181+36 76+14
CHMΞM 212, 189 231, 179 282, 193 195, 220 412, 408 395, 421 457, 384 407, 471 217, 241 230, 237 201, 199 293, 187 209, 246 Mean (1SE)= Mean (1SE)= Mean (1SE)= Mean (±SE)= 282+41 286+46 296+49 280+42
CFU-MEG 114, 107 133, 117 154, 113 127, 112 93, 100 58, 52 53, 40 47, 54 Mean (1SE)= Mean (1SE)= Mean (1SE)= Mean (1SE)= 10415 90120 90+27 85120 Table 2
Effect of c-kit oligomers on CD3 + dell derived colony formation
As shown in Table 1, when A*T"MNC were employed as indicator cells, c-kit antisense oligomers inhibited growth of CFU-E ~75%, and BFU-E ~71%, when employed at the highest doses. Inhibition was sequence-specific since neither sense, nor scrambled sequence oligomers, significantly affected colony growth in comparison to untreated controls. In contrast to these results, CFU- GM and CFU-MEG derived colony formation was unaffected by exposure to any of the oligomers, at any of the doses employed (Table 1) .
Similar results were obtained after exposure of CD34+ cells to c-kit oligomers. As shown in Table 2, the mean number of CFU-E colonies decreased by ~66% after exposure to c-kit antisense oligomers while the number of BFU-E colonies decreased -71%. Neither sense nor scrambled sequence oligomers inhibited colony formation. As was also noted with the less purified cell prepara¬ tion, neither antisense nor control oligomers inhibited CFU-GM colony formation. The failure of c-kit antisense to inhibit CFU-GM and CFU-MEG is surprising since W/Wv mice have been reported to have defective granulopoiesis and megakaryocytopoiesis (Chervenick et al. , Proc. Soc. EXP. Biol. Med. 152. 398-402 (1976)). Erythroid colony formation was inhibited in a dose-dependent fashion. See Fig. 3, showing the effect of various concentrations of c-kit antisense oligomer on BFU-E-derived colony formation. Moreover, residual col¬ onies were much smaller for the antisense-treated cells versus untreated controls (data not shown) .
Reverse Transcription-Polvmerase Chain Reaction (RT-PCR) : As additional proof that the antisense effect was due to a specific decrement in c-kit mRNA levels, the kinetics of c-kit message expression in marrow ono- nuclear cells were examined, and the effect of oligomer exposure on c-kit mRNA levels was then assessed by the following RT-PCR procedure. Total RNA was extracted from cells using the quanidine isothiocyante method of Chirgwin et al. , Bio¬ chemistry. 18, 5294 (1979). Cells (2-5 x 106) were lysed in 250 μl of guanidine thiocyanate buffer (4M guanidine thiocyanate; 50 mM sodium acetate pH 5.0; 1 mM EDTA; 1M S-mercaptoethanol; 0.5% sarσosyl) and then layered over 250 μl of a cesium chloride (5.7 M) cushion in Beckman open-top ultra clear centrifuge tubes (0.8 ml). Tubes were centrifuged (Beckman TL-100 Ultracentrifuge; 100,000 RMP; 1.5 hours; 20°C) and the resulting RNA pel¬ let was resuspended in -400 μl of water, precipitated with 0.3 M potassium acetate, washed twice in 75% etha¬ nol, and then stored at -70βC until used.
RNA was reverse-transcribed with 500 U of Molo- ney murine leukemia virus reverse transcriptase (MoMLV- RT) and 50 pmol of a 22-nucleotide oligodeoxynucleotide 3' primer complementary to nucleotides 1201-1179 (CTAGG- AATGT GTAAGTGCCT CC, SEQ ID NO:16) of the published c- kit cDNA sequence. The resulting cDNA fragment was am- plified using 5 U of Thermus aquaticus (Tag) polymerase and a 22-nucleotide oligodeoxynucleotide 5' primer spe¬ cific for c-kit nucleotides 842-864 (GGTTGACTAT CAGTTCA- GCG AG, SEQ ID NO:17). Twenty-five μl of amplified pro¬ duct was electrophoresed on 4% agarose gel and subse- quently transferred to a nylon filter. Filters were pre-hybridized, and then probed with a 32P end-labeled oligonucleotide probe (Caracciolo et aj.. , Science 245, 1107-1109 (1989)) corresponding to the 21-nucleotide c- kit sequence (GATCCACTGC TGGTGTTCAG G, SEQ ID NO:18) contained within the amplified region (nucleotides 1068- 1047) . Autoradiography was performed by exposing fil¬ ters on Kodak X-ray film at -70"C using intensifying screens.
A'T'MNC cells were kept for 24 hours at 4°C in IMDM containing 2% human AB serum, then shifted to 37°C and stimulated with IL-3 (20 U/ml) and EPO (5 U/ml) in
5% human AB serum. C-kit expression was determined at intervals according to RT-PCR. The results are indicat¬ ed in Figure 1. The Figure 1 lanes indicate relative c- kit transcript amounts determined by autoradiography of RT-PCR hybridization gels at the following intervals after stimulation:
Fig. 1 Lane Time to RT-PCR Assay (hrs)
1 0 2 2
3 8
4 12
5 24
6 36 7 48
9 H20
Lane 8 contained H20 as a control. Expression appeared to peak at -36 hours. A"T"MNC cells stimulated (t=0) in the same manner were exposed to c-kit sense, antisense or mismatch oli¬ gomers. c-kit expression was assayed by RT-PCR (t=36 hrs) as above. The results appear in Figure 2. The lanes are identified as follows:
Fig.2 Lane Treatment
1 control cells (t=0)
2 control cells (t=36 hrs)
3 sense (t=36 hrs) 4 antisense (t=36 hrs)
5 mismatch (t=36 hrs)
Antisense-treated cells (lane 4) had no detect¬ able c-kit mRNA, while sense (lane 3) and scrambled se- quence (lane 5) treated cells had levels which were sim¬ ilar to those observed in untreated control cells at the same time point (lane 2) . Example 2
Effect of c-kit Antisense Oligomer Exposure on Malignant Hematopoietic Progenitor Cell Growth
To explore the importance of the c-kit receptor in regulating malignant hematopoietic cell growth, we employed a strategy which we have successfully employed in the past (Calabretta et al. , Proc. Nat. Acad. Sci. USA. 88, 2351 (1991)), and which is essentially identi- cal to that described above. A'T'MNC were obtained from patients with a variety of hematologic malignancies and exposed to the c-kit oligomers utilized in the preceding normal cell studies. Effects on the ability of malig¬ nant CFU-GM to form colonies, an index of the effect on malignant cell survival and proliferative activity, was then assessed. A total of twenty-two patients were studied, three with acute lymphocytic leukemia, four with acute myelogenous leukemia, ten with chronic myelo¬ genous leukemia, and one with a myelodysplastic (pre- leukemic) syndrome and four with polycythemia vera. Overall response rates, and response rate by disease type, is given in Table 3.
Table 3
Effect of c-kit oligomers on growth of malignant hematopoietic colony forming cells
Disease Type No. Pts Responders % Decrease Non- Colonies Respon- ders
Acute 68% lymphocytic leukemia
Acute myelogenous leukemia 63%
Chronic 10 84% myelogenous 33% leukemia 40% 90% 78% Mean=65%+26%
Figure imgf000031_0001
While the number of patients in categories other than CML is small, the data nonetheless suggest that pa¬ tients with CML are particularly likely to respond to c- kit antisense. Accordingly, c-kit antisense oligomers are believed particularly useful as CML bone marrow pur¬ ging agents. In addition, because of their marked inhi¬ bition of erythroid progenitor cells, it is believed that c-kit oligomers are useful in controlling the mark¬ edly elevated hemoglobin/hematocrit found in patients with PV, another myeloproliferative disorder. Example 3
Effect of c-kit Antisense Oligomer Exposure on BFU-E Responsiveness to Stem Cell Factor
To provide proof that c-kit antisense mediated inhibition of erythropoiesis was due to the absense of KIT receptor function, we sought to demonstrate that BFU-E responsiveness to stem cell factor (SCF) could be abolished in a sequence-specified manner after exposure to c-kit oligomers. Accordingly, CD34 + MNC (2 x 104) were cloned in the presence of 5 units of EPO and 100 ng of SCF per ml alone or with sense, antisense, or scram- bled-sequence c-kit oligomers (final concentration, 150 μg/ml (-26 μM) ) . In four experiments, 191 + 19 BFU-E (mean + SD) were grown in the presence of the growth factors alone. These numbers were not statistically different from those cloned with sense (183 + 29; P = 0.654) or scrambled-sequence oligomers (180 + 20; P = 0.758) . In the presence of the c-kit antisense oligo- mers, BFU-E-derived colony formation was completely abo¬ lished (0.4 + 0.7; P<0.0001), suggesting that KIT recep¬ tor was no longer present to interact with its ligand.
The following non-limiting example illustrates one methodology for bone marrow purging according to the present invention.
Figure imgf000032_0001
Bone Marrow Purging with c-kit 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 trans¬ ferred 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 pro- cessed 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 pla¬ telets) . 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 car¬ ried 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 ad¬ justed to a cell concentration of about 2 x 107/ml in TC- 199 containing about 20% autologous plasma. C-kit anti¬ sense oligodeoxynucleotide, for example, in a concentra- tion of about 8 mg/ml, is added to the transfer packs containing the cell suspension. Recombinant human hema¬ topoietic growth factors, e.g., rH IL-3 or rH GM-CSF, may be added to the suspension to stimulate growth of hematopoietic neoplasms and thereby increase their sen- sitivity c-kit 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% autol- ogous 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 specific forms without departing from the spirit or es- sential 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 syn- thetic, preparative and analytical procedures are incor¬ porated herein by reference.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Gewirtz, Alan M. Calabretta, Bruno
(ii) TITLE OF INVENTION: Antisense Oligonucleo¬ tides to c-kit Proto-Oncogene and Uses Thereof (iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Temple University - Of The Com¬ monwealth 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:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 07/682,812
(B) FILING DATE: April 9, 1991 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Monaco, Daniel A.
(B) REGISTRATION NUMBER: 30,480
(C) REFERENCE/DOCKET NUMBER: 6056-129 PC (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: GAACGCAGAG AAAATCCCAG GCGCCGCGAG CGCCTCTCAT 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: TCCCAGGCGC CGCGAGCGCC TCTCAT 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:
CCCAGGCGCC GCGAGCGCCT CTCAT 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: CCAGGCGCCG CGAGCGCCTC TCAT 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: CAGGCGCCGC GAGCGCCTCT 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:
AGGCGCCGCG AGCGCCTCTC 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
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGCGCCGCGA GCGCCTCTCA 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
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GCGCCGCGAG CGCCTCTCAT 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 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CGCCGCGAGC GCCTCTCAT 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: GCCGCGAGCG CCTCTCAT 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: CCGCGAGCGC CTCTCAT 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: CGCGAGCGCC TCTCAT 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCGAGCGCCT CTCAT 15
(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 Nucleotides (B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATGAGAGGCG CTCGCGGC 18
(2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15*. GCACCGTCTG CCAGTCGC 18
(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 Nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTAGGAATGT GTAAGTGCCT CC 22
(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GGTTGACTAT CAGTTCAGCG AG 22
(2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 Nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GATCCACTGC TGGTGTTCAG G 21

Claims

1. A pharmaceutical composition comprising a pharmaceutical carrier and an oligonucleotide which has a nucleotide sequence complementary to at least a por¬ tion of the mRNA transcript of the human c-kit gene, said oligonucleotide being hybridizable to said mRNA transcript.
2. A composition according to claim 1 wherein the oligonucleotide comprises an at least 12-mer.
3. A composition according to claim 2 wherein the oligonucleotide has a nucleotide sequence complemen¬ tary to a portion of the c-kit mRNA lying within about 40 nucleotides of the translation initiation codon.
4. A composition according to claim 2 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-kit mRNA transcript including the translation ini¬ tiation codon of said transcript.
5. A composition according to claim 2 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
6. A composition according to claim 5 wherein the oligodeoxynucleotide is an alkylphosphonate oligode¬ oxynucleotide or a phosphorotioate oligodeoxynucleotide.
7. A composition according to claim 5 wherein the oligodeoxynucleotide comprises from a 15-mer to a 30-mer.
8. A composition according to claim 7 wherein the oligodeoxynucleotide comprises from an 18-mer to a 26-mer.
9. A composition according to claim 8 wherein the oligodeoxynucleotide comprises from an 18-mer to a 21-mer.
10. A composition according to claim 7 wherein the oligodeoxynucleotide has a sequence selected from the group of sequences 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.
11. A composition according to claim 10 wherein the oligodeoxynucleotide has a nucleotide sequence of SEQ ID NO:10.
12. An oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-kit gene, said oligonucleotide being hybridizable to said mRNA transcript.
13. An oligonucleotide according to claim 12 which comprises an at least 12-mer.
14. An oligonucleotide according to claim 13 hav¬ ing a nucleotide sequence complementary to a portion of the c-kit mRNA lying within about 40 nucleotides of the translation initiation codon.
15. An oligodeoxynucleotide according to claim 13 which is an oligodeoxynucleotide having a deoxynucleo¬ tide sequence complementary to a portion of the c-kit mRNA transcript including the translation initiation codon of said transcript.
16. An oligodeoxynucleotide according to claim 13 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.
17. An oligodeoxynucleotide according to claim 16 which is an alkylphosphonate oligodeoxynucleoside or a phosphorothioate oligodeoxynucleotide.
18. An oligodeoxynucleotide according to claim 16 which comprises from a 15-mer to a 30-mer.
19. An oligdeoxyonucleotide according to claim 18 which comprises from a 18-mer to a 26-mer.
20. An oligodeoxynucleotide according to claim 19 which comprises from a 18-mer to a 21-mer.
21. An oligodeoxynucleotide according to claim 16 selected from the group of oligodeoxynucleotides having sequences 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 .
22. An oligodeoxynucleotide according to claim 21 having the nucleotide sequence SEQ ID NO:10.
23. A method for in vivo or ex vivo treatment of hematologic neoplasms characterized by c-kit expression comprising administering to a host in need of such treatment, or to cells harvested from such host, an ef¬ fective amount of an oligonucleotide which has a nucleo¬ tide sequence complementary to at least a portion of the mRNA transcript of the human c-kit gene, said oligonuc¬ leotide being hybridizable to said mRNA transcript.
24. The method according to claim 23 wherein the oligonucleotide is an at least 12-mer.
25. A method according to claim 24 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-kit mRNA lying within about 40 nucleotides of the translation initiation codon.
26. A method according to claim 24 wherein the oligodeoxynucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-kit mRNA transcript including the translation ini¬ tiation codon of said transcript.
27. A method according to claim 24 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oli¬ godeoxynucleotide.
28. A method according to claim 27 wherein the oligodeoxynucleotide is an alkylphosphonate oligodeo¬ xynucleoside or a phosphorothioate oligodeoxynucleotide.
29. A method according to claim 27 wherein the oligodeoxynucleotide is selected from the group of oli¬ godeoxynucleotides having sequences 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.
30. A method according to any of claims 23, 24, 25, 26, 27, 28 or 29 comprising treating aspirated bone marrow cells and returning the aspirated cells to the host following treatment.
31. A method according to claim 23 wherein the hematologic neoplasm comprises chronic myelogenous leu¬ kemia.
32. A method according to claim 23 wherein the hematologic neoplasm comprises acute myelogenous leuke¬ mia.
33. A method for inhibiting proliferation of ery¬ throid cells comprising administering to a host an ef¬ fective amount of an oligonucleotide which has a nucleo¬ tide sequence complementary to at least a portion of the mRNA transcript of the human c-kit gene, said oligonu¬ cleotide being hybridizable to said mRNA transcript.
34. A method according to claim 33 wherein the oligonucleotide is an at least 12-mer.
35. A method according to claim 34 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-kit mRNA lying within about 40 nucleotides of the translation initiation codon.
36. A method according to claim 34 wherein the oligonucleotide is an oligodeoxynucleotide having a deo¬ xynucleotide sequence complementary to a portion of the c-kit mRNA transcript including the translation initia¬ tion codon of said transcript.
37. A method according to claim 34 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oli¬ godeoxynucleotide.
38. A method according to claim 37 wherein the oligodeoxynucleotide is an alkylphosphonate oligodeo¬ xynucleoside or a phosphorothioate oligodeoxynucleotide.
39. A method according to claim 37 wherein the oligodeoxynucleotide is from a 15-mer to a 30-mer.
40. A method according to claim 39 wherein the oligodeoxynucleotide is from an 18-mer to a 26-mer.
41. A method according to claim 40 wherein the oligodeoxynucleotide is from an 18-mer to a 21-mer.
42. A method according to claim 41 wherein the oligodeoxynucleotide has a nucleotide sequence selected from the group of sequences 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.
43. A method for treating malignant melanoma com¬ prising administering to a host in need thereof an ef¬ fective amount of an oligonucleotide which has a nucleo¬ tide sequence complementary to at least a portion of the mRNA transcript of the human c-kit gene, said oligonu¬ cleotide being hybridizable to said mRNA transcript.
44. A method according to claim 43 wherein the oligonucleotide comprises an at least 12-mer.
45. A method according to claim 44 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oli¬ godeoxynucleotide.
46. A method according to claim 45 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-kit mRNA lying within about 40 nucleotides of the translation initiation codon.
47. A method according to claim 45 wherein the oligonucleotide is an alkylphosphonate oligodeoxynucleo¬ side or a phosphorothioate oligodeoxynucleotide.
48. A method for treating testicular or ovarian tumors comprising administering to a host in need there¬ of 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-kit gene, said oligonucleotide being hybridizable to said mRNA trans¬ cript.
49. A method according to claim 48 wherein the oligonucleotide comprises an at least 12-mer.
50. A method according to claim 49 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oli¬ godeoxynucleotide.
51. A method according to claim 50 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-kit mRNA lying within about 40 nucleotides of the translation initiation codon.
52. A method according to claim 50 wherein the oligonucleotide is an alkylphosphonate oligodeoxynucleo¬ side or a phosphorothioate oligodeoxynucleotide.
PCT/US1992/002854 1991-04-09 1992-04-08 Antisense oligonucleotides to c-kit proto-oncogene and uses thereof WO1992019252A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP51182692A JP2002507186A (en) 1991-05-09 1992-04-08 Antisense oligonucleotides to c-kit proto-oncogene and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US68281291A 1991-05-09 1991-05-09
US682,812 1991-05-09

Publications (1)

Publication Number Publication Date
WO1992019252A1 true WO1992019252A1 (en) 1992-11-12

Family

ID=24741242

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/002854 WO1992019252A1 (en) 1991-04-09 1992-04-08 Antisense oligonucleotides to c-kit proto-oncogene and uses thereof

Country Status (5)

Country Link
EP (1) EP0580795A4 (en)
JP (1) JP2002507186A (en)
AU (1) AU2335192A (en)
CA (1) CA2107915A1 (en)
WO (1) WO1992019252A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5681600A (en) * 1995-12-18 1997-10-28 Abbott Laboratories Stabilization of liquid nutritional products and method of making
US6756482B1 (en) 1998-05-25 2004-06-29 Ajinomoto Co., Inc. Purified human activin and process for producing the same
GB2425311A (en) * 2005-04-15 2006-10-25 Ist Superiore Sanita Micro RNA against kit protein
CN112771162A (en) * 2018-08-27 2021-05-07 北卡罗来纳州立大学 Targeting KIT with splice switching oligonucleotides to induce mast cell apoptosis

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Cancer Research, Volume 51, issued 01 April 1991, T. STROHMEYER et al., "Expression of the hst-1 and c-kit Protooncogenes in Human Testicular Germ Cell Tumors", pages 1811-1816, especially "Results" pages 1812-1815, entire document. *
EMBO Journal, Volume 7, Number 4, issued 1988, F. QIU et al., "Primary Structure of c-kit: Relationship with the CSF-1/PDGF Receptor Kinase Family - Oncogenic Activation of v-kit involves deletion of Extracellular Domain and C Terminus", pages 1003-1011, especially figure 2. *
Journal of Immunology, Volume 140, Number 7, issued 01 April 1988, D. HAREL-BELLAN et al., "Specific Inhibition of c-myc Protein Biosynthesis using an Antisense Synthetic Deoxy-Oligonucleotide in Human T Lymphocytes", pages 2431-2435, entire document. *
See also references of EP0580795A4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5681600A (en) * 1995-12-18 1997-10-28 Abbott Laboratories Stabilization of liquid nutritional products and method of making
US6756482B1 (en) 1998-05-25 2004-06-29 Ajinomoto Co., Inc. Purified human activin and process for producing the same
GB2425311A (en) * 2005-04-15 2006-10-25 Ist Superiore Sanita Micro RNA against kit protein
CN112771162A (en) * 2018-08-27 2021-05-07 北卡罗来纳州立大学 Targeting KIT with splice switching oligonucleotides to induce mast cell apoptosis
US20210363531A1 (en) * 2018-08-27 2021-11-25 North Carolina State University Targeting kit with splice switching oligonucleotides to induce apoptosis of mast cells
EP3844278A4 (en) * 2018-08-27 2022-05-25 North Carolina State University TARGETING KIT BY SPLICING SWITCHING OLIGONUCLEOTIDES TO INDUCE MASTOCYTE APOPTOSIS

Also Published As

Publication number Publication date
EP0580795A4 (en) 1995-11-15
JP2002507186A (en) 2002-03-05
AU2335192A (en) 1992-12-21
CA2107915A1 (en) 1992-11-12
EP0580795A1 (en) 1994-02-02

Similar Documents

Publication Publication Date Title
AU641636B2 (en) Antisense oligonucleotides to C-MYB proto-oncogene and uses thereof
US5618709A (en) Antisense oligonucleotides specific for STK-1 and method for inhibiting expression of the STK-1 protein
US5652222A (en) Selective inhibition of leukemic cell proliferation by bcr-abl antisense oligonucleotides
US5744460A (en) Combination for treatment of proliferative diseases
KR101015913B1 (en) Methods of treating genetic variation of hematopoietic stem cells and uses of the mutant cells
EP0841068A1 (en) Leukemic cell growth inhibitor containing antisense oligonucleotide derivative against wilms&#39; tumor gene (wt1)
HUT69922A (en) Oligonucleotide modification of cell adhesion
EP0668782B1 (en) Combination of antineoplastic agent and antisense oligonucleotides for treatment of cancer
US20040072259A1 (en) Methods and products for manipulating hematopoietic stem cells
US5612212A (en) Selective inhibition of cell proliferation by vav antisense oligonucleotides
US5989849A (en) Antisense of oligonucleotides to c-kit proto-oncogene and in vitro methods
JP2003511393A (en) Antisense compositions and methods of treating cancer
WO1992019252A1 (en) Antisense oligonucleotides to c-kit proto-oncogene and uses thereof
AU646643B2 (en) Antisense oligonucleotides to C-ABL proto-oncogene
JPH07501525A (en) Treatment of melanoma with antisense oligonucleotides against the c-myb proto-oncogene
EP0585399A1 (en) Treatment of colorectal carcinoma with antisense oligonucleotides to c-myb proto-oncogene
US20080057579A1 (en) Methods and products for manipulating hematopoietic stem cells
JP2003527096A (en) C-MYC antisense-treated hematopoietic stem cell composition and method
WO2025077790A1 (en) Engineered immune cells and uses thereof
WO1994004137A1 (en) Antisense oligonucleotides to cyclin d1 proto-oncogene
CA2004364C (en) Antisense oligonucleotides to c-myb oncogene and uses thereof
WO1994007538A1 (en) ANTISENSE OLIGONUCLEOTIDES TO B-m^_y^_b^_ PROTO-ONCOGENE

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH CS DE DK ES FI GB HU JP KP KR LK LU MG MN MW NL NO PL RO RU SD SE US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CI CM DE DK ES FR GA GB GN GR IT LU MC ML MR NL SE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 1992915785

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2107915

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 1993 129123

Country of ref document: US

Date of ref document: 19931007

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 1992915785

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1992915785

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载