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WO1992019252A1 - Oligonucleotides non codants du proto-ontogene c-kit et leurs utilisations - Google Patents

Oligonucleotides non codants du proto-ontogene c-kit et leurs utilisations Download PDF

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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
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seq
mer
oligodeoxynucleotide
oligonucleotide
kit
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Alan M. Gewirtz
Bruno Calabretta
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Temple Univ School of Medicine
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Temple Univ School of Medicine
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    • 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.

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Abstract

L'invention concerne des oligonucléotides ayant une séquence de nucléotides d'au moins une partie du transcript d'ARN messager du gène humain c-kit. Ces oligonucléotides 'non codants' sont hybridables avec le transcript d'ARN messager. Lesdits oligonucléotides sont utiles pour inhiber sélectivement la prolifération de cellules érythrocytes, notamment dans les troubles caractérisés par un hématocyte élevé dû à la surproduction d'érythrocytes. Les oligomères non codants présentent également des celllules néoplastiques hématologiques constituant des agents actifs et sont par conséquent adaptés à une utilisation comme agents purgatifs de la moëlle osseuse.
PCT/US1992/002854 1991-04-09 1992-04-08 Oligonucleotides non codants du proto-ontogene c-kit et leurs utilisations Ceased WO1992019252A1 (fr)

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JP51182692A JP2002507186A (ja) 1991-05-09 1992-04-08 ｃ−ｋｉｔプロト−オンコ遺伝子に対するアンチセンスオリゴヌクレオチドおよびその使用

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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 (zh) * 2018-08-27 2021-05-07 北卡罗来纳州立大学 用剪接转换寡核苷酸靶向kit诱导肥大细胞凋亡

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 (zh) * 2018-08-27 2021-05-07 北卡罗来纳州立大学 用剪接转换寡核苷酸靶向kit诱导肥大细胞凋亡
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 (fr) * 2018-08-27 2022-05-25 North Carolina State University Ciblage de kit par des oligonucléotides de commutation d'épissage pour induire l'apoptose de mastocytes

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EP0580795A1 (fr) 1994-02-02
EP0580795A4 (en) 1995-11-15
CA2107915A1 (fr) 1992-11-12
AU2335192A (en) 1992-12-21

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