JPH07501525A - Treatment of melanoma with antisense oligonucleotides against the c-myb proto-oncogene - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] by antisense oligonucleotide against the c-myb proto-oncogene Treatment of Melanoma Technical Field of the Invention The present invention provides antisense oligonucleotides against proto-oncogenes, particularly Antisense oligonucleotides against the c-myb gene and tumorigenic prevention The present invention relates to the use of these oligonucleotides as inhibitors.

Explanation regarding government approval The invention disclosed herein has been approved by the National Institute of Health Partially supported by the government under No. 4678. The government has certain rights in this invention. Ru.

Background of the invention Proto-oncogene c-myb is an avian myeloid leukemia virus transforming gene. It is the normal cellular homologue of the child v-myb. c-myb is primarily expressed in hematopoietic cells. encodes the expressed nuclear protein. This is a proto-oncogene, i.e. a positive coats proteins required for normal, non-tumor cell survival. Genes change appropriately This then has the potential to become an oncogene. Oncogenes are This is a gene that plays a role in the transformation of normal cells into tumor cells. Ru.

The human c-myb gene has been isolated, cloned and sequenced [Majuro et al., Proceedings National Academy of Sciences, USA, No. 83 Vol., pp. 9636-9640 (1986)]. Human c-m-y nucleus The oligonucleotide having the code sequence was published by the applicant on October 27, 1989. filed in U.S. Patent Application No. 427,659 and corresponding International Patent Application No. WO 90 No. 105,445, the entire disclosure of which is hereby incorporated by reference. So Here, C-myb antisense oligonucleotides have been shown to be useful in the treatment of hematological tumors and It is disclosed that it is useful for immunosuppression and immunosuppression.

Melanoma, also known as “malignant melanoma” or “cutaneous melanoma”, is an aggressive and It is a melanocytic tumor with metastatic potential. Melanospheres are melanosome-containing cells. It is involved in the biosynthesis and transport of melamine pigment. Black spheres are found in the basal layer of the epidermis. Present in the skin. Under various stimuli, they produce melamine pigments. Mela Min synthesis occurs in melanosomes, distinct intracellular organelles within the melanosome. Cheating.

Once thought to be rare, the incidence of melanoma is increasing, excluding bronchial cancer. It is bigger than any other cancer. The incidence of melanoma is highest in Caucasian peoples. shadows are most commonly caused by exposure to ultraviolet light, as well as geographic and occupational factors. ing. Nitrosoureas are affected by DTIC. The incidence of melanoma is increasing in the United States and other countries. It is increasing rapidly everywhere, apparently doubling every 10 to 17 years. the current , melanoma accounts for approximately 1% of cancer cases in the United States, and approximately the same proportion of cancer deaths occur. Ru. This only accounts for about 3% of skin tumors, and melanoma is the killer of all skin cancers. They account for two-thirds of the total population.

In most cases, melanoma initially progresses through a radial growth phase at the site of the primary lesion. Ru. This initial period is characterized by little or no capacity for metastasis.

Melanoma during this period can generally be treated by surgery. deeper During the vertical growth phase, which is characterized by invasion of deep skin tissue, melanomas are unable to metastasize. acquire the ability to Once metastases occur, surgery alone is not effective in treating melanoma. Not.

Chemotherapy alone or in combination with surgery has been used to treat melanoma. Jime Tiltriazenoimidazole (DTIC) is the most popular treatment for metastatic melanoma. is the only active drug, but has an overall objective response rate of only 21%. stomach. DTIC can lead to impairment of liver function. This is a slight hematological toxicity. has.

Slightly less effective in treating malignant melanoma are synthetic nitrosoureas; Of these, 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1 -(2-Chlorethyl)-3-cyclohequinru-1-ditrourea (CCNU) , methyl-CCNU and chlorozotoxyisotopy. preferably shows significant hematological toxicity with slightly higher oligonucleotide levels. The reaction ratio is 10-1 The range is 8%.

Unfortunately, no single drug or There are no combination prescriptions. There is an urgent need to develop more effective drugs .

Key points of the invention The present invention provides methods for treating melanoma. For individuals who require this type of treatment, an effective amount complementary to at least a portion of one or its progeny mRNA transcripts; It has a unique nucleotide sequence. This oligonucleotide binds to the mRNA transcript. Can be bridized. Preferably, the oligonucleotide contains at least one 2m oligonucleotide, i.e. at least 12 nucleotide residues. It is an oligomer having a group. In particular, the oligomer is preferably a 12- to 4-mer oligomer. Preferably, it is an oligodeoxynucleotide. Oligonucleus smaller than monomer Reotides may also be used; their statistical tendency to hybridize to non-target sequences is for this reason, the specificity is low. In addition, a single mismatch is a hybrid destabilizes. Oligonucleotides larger than a four-part structure may also be used; absorption is more difficult. Furthermore, partial alignment of long sequences can lead to non-specific hybridization. The oligodeoxynucleotides and nonspecific oligodeoxynucleotides are 15-trimeric oligodeoxynucleotides, and are more abundant. It is preferably a 18 to 2 parter.

In principle, an oligonucleotide having a sequence complementary to any region of the c-myb gene c-myb mRNA transcripts containing the translation initiation codon can be used in the present invention. Particularly preferred are oligodeoxynucleotides that are complementary to a portion of. Additionally, translation Approximately 40 nucleotides upstream (5' direction) or approximately 40 nucleotides from the start codon Complementary to part of the c-myb mRNA transcript located downstream (3' direction) Also suitable are oligonucleotides.

Additionally, the present invention provides a method for purifying bone marrow of metastatic melanocytes. Melanoma Bone marrow aspirated from an individual is treated with an effective amount of c-myb antisense oligonucleotide. treatment and then returning the treated cells to the body of the affected individual. Bone marrow purification technology is a high It can also be used for autologous bone marrow repair (transplant) in combination with Yokuma's chemotherapy process. .

Furthermore, the present invention provides pharmaceutically acceptable carriers and c-myb antisense oligos. The present invention also relates to a composition for treating melanoma comprising a nucleotide.

As used herein, unless otherwise specified, the term "oligonucleotide J" Oligomers of ribonucleotides, i.e. oligoribonucleotides and oligoribonucleotides Oligomers of xyribonucleotides, i.e. oligodeoxyribonucleotides (herein also referred to as "oligodeoxynucleotides"). O Rigodeoxynucleotides are preferred.

As used herein, unless otherwise specified, the term "oligonucleotide" It also includes oligomers that are large enough to be called "polynucleotides". do.

The terms "oligonucleotide" and "oligodeoxynucleotide" refer to A common biologically important nucleotide, namely the nucleotide adenine (rA J’), deoxyadenine (rdΔ”), guanine (rGJ), deoxyg anine (“dG”), cytosine (“C”), deoxycytosine (rdcJ), chi Only oligomers and polymers of min (rTJ) and uracil (rUJ) hybridizes to the c-myb mRNA transcript, which may contain other nucleotides. It also includes oligomers and polymers that can be mixed. Similarly, “oligonucleotide The terms "deoxynucleotide" and "oligodeoxynucleotide" refer to one or more The purine or pyrimidine moiety, sugar moiety or internucleotide bond on Also included are modified oligomers and polymers. "oligonucleotide" Therefore, the term ``optional It is understood that oligomers, which may also be referred to as "ligonucleosides", are also included.

Modified oligonucleotides of this type include, for example, the methylphosphonate oligonucleotides described below. Includes nucleosides.

The term "phosphorothioate oligonucleotide" refers to one or more of the expression % expression % refers to an oligonucleotide with a phosphorothioate group, which refers to an unmodified oligonucleotide. Formula% which is a characteristic of gonucleotides is different from the phosphodiester group of

The term "methylphosphorate oligonucleoside" refers to the internucleotide bond one or more of the methylphosphonate groups ~o-po-o~ H3 means an oligonucleotide that is

The term "downstream" as used with respect to the direction along a nucleotide sequence is 5'-3' means direction. Similarly, the term "upstream" refers to the 3'-5' direction.

means any other transcript that is specified.

Brief description of the drawing Figure 1 shows c-myb activation of human melanoma cells (CIIP) in hydrotherapy. is a graph showing the effect of antisense and antisense oligonucleotides on cells. (i) no oligomer treatment (“comparison”), (ii) c-myb mRNA transcript Indication of one-day body oligodeoxynucleotides complementary to codon cost 2-7 of translation position. ``antisense'') or (ii) the corresponding sense 1 Treated in the body for days ("Sense").

Figure 2 is similar to Figure 1, but oligonucleotide treatment is extended to 2 days. did. Oligonucleotide concentrations are integrated over the 2-day treatment period.

Figure 3 is similar to Figure 2, except that the treatment was extended to 5 consecutive days. origone Creotide concentration is cumulative.

Figure 4 is similar to Figure 1, except that other human melanoma cell lines (SKME L-37) was used instead of CHP cells.

Figure 5 shows human black N (SKMEL-) transplanted into mice with severe combined immunodeficiency. 37) Effect of luc-myb sense and antisense oligomers on cell growth This is a plot showing the results. For 7 days, mice (i) c-myb mRNA A 24-mer phosphorothioate oligonucleotide complementary to codons 2-9 of the translated portion of the A transcript. Godeoxynucleotide (“antisense”; 2 mice), (ii) corresponding serum sense 24-mer phosphorothioate oligodeoxynucleotide (“sense”; (1 mouse) or (ii) without oligomer/“comparison”; 1 mouse ) was taken daily at 100 μg.

It was determined that it is important for reproduction. This proto-oncology in cell proliferation The role of these cells is not limited to cells of hematopoietic origin, as previously thought. epithelial tumor form Adult cells are larger than previously thought.

The deduced DNA sequence complementary to the mRNA transcript of the human c-myb gene is B et al., Proceedings National Academy of Sciences, USA, No. 8 3, pp. 9636-9640 (1986).

Furthermore, Majuro et al. Disclosed. The initiation codon ATG is present at position 114, preceded by a 5'-untranslated There is a translation area. Approximately 1200 nucleotides following the stop codon TGA at position 2034 There is a 3'-untranslated region spaced between cleotides followed by approximately 140 nucleotides. followed by a poly(A) terminus.

The antisense oligonucleotides of the present invention may be any known chemical oligonucleotide. It can be synthesized by the tide synthesis method. This kind of method is generally used for example Nnatuka-1 “From genes to clones: Introduction to genetic engineering”, ■CH Fuera Eibergarach Translation, 1. 987). Antisense oligonucleotides are most advantageously Created using any commercially available automated nucleic acid synthesizer. One device of this kind, The Applied Biosystems 380B DNA synthesizer is β-cyanoe. Using tylphosphoramidite chemistry.

The complete nucleotide synthesis of NA complementary to the c-myb mRNA transcript is not publicly available. Since it is known that antibodies that can hybridize to any part of the mRNA transcript Sense oligonucleotides are produced by oligonucleotide synthesis methods known to those skilled in the art. can be achieved.

Although oligonucleotides of any length can be used in the practice of the invention, oligonucleotides shorter than 12 bases A dark sequence has low specificity for hybridizing to the target c-myb mRNA, and the enzyme It is easily destroyed by cleavage and can be further destabilized by enzymatic cleavage. did Therefore, oligonucleotides with 12 or more nucleotides are preferred. suitable.

Long sequences, especially sequences longer than about 40 nucleotides, have low uptake by target cells. It is slightly less effective in suppressing c-myb translation. Therefore 12-4 Oligomers of 0 nucleotides are preferred, more preferably 15-30 nucleotides. nucleotides, particularly preferably 18 to 26 nucleotides. Unmodified oligonucleotide Sequences of 18 to 21 nucleotides are most preferred for oligonucleotides, but For cleotides (e.g. phosphorothioate oligonucleotides) about 2 Slightly longer chains of up to 6 nucleotides are preferred and are less likely than unmodified oligonucleotides. It also hybridizes to mRNA with low intensity.

Complementary and hybridizable to any part of the c-myb mRNA transcript In principle, these oligonucleotides are effective in suppressing the translation of transcripts, and The described effects can be induced. Translation begins at or near the start codon. It is thought that the most effective suppression is achieved by blocking the mRNA at the desired site.

Therefore, an origin complementary to the 5'-terminal region of the c-myb mRNA transcript Gonucleotides are preferred. Secondary or tertiary that impedes hybridization Structure appears to be minimal in this region. Furthermore, in the 3′ direction from the starting site Sequences that are too far apart may cause hybridization of mRNA transcripts due to the “read-through” phenomenon. It has also been suggested that ribosomes are less effective in Unraveling the sense/sense duplex to enable message translation [For example, Shakin, Journal Biochemistry, Vol. 261] , p. 16018 (1986)].

Preferably the antisense oligonucleotide targets the initiation codon for protein synthesis. Directed at or near the area of Start codon (nucleotide 11 of complete transcript) Oligonucleotides complementary to 4~ are preferred.

complementary to the 5'-terminal region of the c-myb transcript, particularly the region containing the initiation codon. Although antisense oligomers are preferred, useful antisense oligomers include mR Complementary to the sequence present in the translated portion of NA (nucleotides 114-2031) including, but not limited to, the 5′- and 3′-untranslated regions or extending thereto. Also included are oligomers that are complementary to the extending nucleotide sequence. Seems to be compatible Ru.

Preferred oligonucleotides complementary to the 5'-untranslated region of the transcript include cap C containing nucleotides (i.e. the nucleotides at the 5'-most end of the transcript) - a molecule with a nucleotide sequence complementary to a portion of the myb mRNA transcript; include. Molecular Cloning, 2nd edition [edited by Samprutsuk et al. (1989)] , pp. 7゜66-7.70, cited here for reference], 31 nucleases Determine the position of the c-m-y-b-cap region by applying substantial force to the analysis method, and mRNA isolated from high levels of c-myEM was used. longest clear view The band that appears is located 90 base pairs upstream of the published c-myb cDNA [map Juro et al., Proceedings of the National Academy of Sciences, USA. , Vol. 83, pp. 9536-9640 (1986)], Principles of Estimation of Cap Site shows. The location of this site was determined by c-m cloned from CCRF-CEM cells. yb completely matches the length of cDNA [Clarke et al., Molecular Cellular Biology, Vol. 8, pp. 884-892 (1988)]. In addition, S1 protection The analysis also showed a thin band in addition to the main band corresponding to the cap site. these Other bands indicate rare or unstable c-myb mRNA transcripts. transcription opening The first multiple sites are found in genes such as c-myb, which lacks a complete TATAA frame. Not rare in children. 5'-end of mRNA transcript starting with cap nucleotide The nucleotide sequence of Antisense oligonucleotides can be created that can hybridize.

The following 40-mer oligodeoxynucleotides begin at the start codon of the transcript. is complementary to the c-myb mRNA transcript extending downstream (in the 3' direction). SEQ ID NO:I.

Small oligomers based on the above sequences (especially c-myb oligomers with start codons) oligomers that can hybridize to the segment of the page. Ru.

The following 26 to 15 as a whole are particularly suitable SEQ ID NO: 2, SEQ ID NO: 3, SEQ TD 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: 1O1 SEQ ID NO:]I, SEQ ID NO:]2 and SEQ ID No. 13゜ The second codon of the translated portion of the transcript (nucleotides 117-119 of the complete transcript) Complementary oligodeoxynucleotides to the starting c-myb mRNA transcript are other preferred oligomer groups. Examples of this type of oligomer are the following 26-15 1 including the body: SEQ ID NO: i4, SEQ ID No. 15, SEQ ID No. 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:2’01 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID No. 23, SEQ ID No. 24 and SEQ ID NO: 25゜ The oligonucleotides used may be unmodified or modified. Can show the punch line.

That is, hybridization for c-myb mRNA transcripts that have uses according to the present invention. Redizable oligonucleotides are biologically important natural nucleotides, i.e. AldA, G, dG, C, dCST and U oligomers as well as improved oligonucleotide types modified for stability and/or lipid solubility. Also includes.

For example, improved lipid solubility and/or resistance to nuclease cleavage , a substitute for the phosphate oxygen in the internucleotide phosphodiester bond. Alkylphosphonate oligonucleosylation using alkyl or alkoxy groups In order to generate oligonucleotides or alkyl phosphotriester oligonucleotides, It is known that this occurs more frequently. For example, these nonionic oligonucleotides improved resistance to nuclease hydrolysis and/or increased cellular resistance to nuclease hydrolysis. characterized by absorption and retains the ability to form stable complexes with complementary nucleic acid sequences. hold In particular, alkylphosphonates are stable against nuclease cleavage and Soluble in lipids. About the production of alkylphosphonate oligonucleosides is disclosed in US Pat. No. 4,469,863.

Methylphosphonate oligomers can be produced both in solution and on an insoluble polymer support. can be created by various methods [Agrawal and Liftina, Kraitsk Acids Research, Vol. 6, pp. 3009-3024 (1979) ); Miller et al., Biochemistry, Vol. 18, pp. 5134-5142 (1, 979), Miller et al., Journal Biological Chemistry, Vol. 255 , pp. 9659-9665 (1980); Miller et al. Research, Vol. 11, pp. 5189-5204 (1983), Miller et al., Nucle. Itsuk Assos Research, Vol. 11, pp. 6225-6242 (1,983 ), Miller et al., Biochemistry, Vol. 25, pp. 5092-5097 (19 86); Engels and Jaeger, Angewante hemi-sublument, no. 912 pages (1982); Shin 71 et al., Tetrahedron Letters, Vol. 24, No. pp. 877-880 (1983); Tolman et al., Tetrahedron, Vol. 40, No. pp. 95-1.02 (1984): Schagger and Engels, Tetrahedron. Letters, Vol. 25, pp. 1437-1440 (1984): Nople et al., Nuku. Reitzk Acids Research, Vol. 12, pp. 3387-3404 (1984) ); Callahan et al., Proceedings National Academy of Sciences; USA, Vol. 83, pp. 1617-1621 (1986); Tsu et al., Chemi-Riki Scribta, Vol. 26, pp. 251-260 (1986) Near Growal and Gu Sotchild, Tetrahedron Letters, Volume 38, pp. 3539-3542 (1,987), Lesnikowski et al., Tetrahedron ・Letters, Vol. 28, pp. 5535-5538 (1987); Sarin et al., Pro. Seeding National Academy of Sciences, USA.

Vol. 85, pp. 7448-7451 (1988)].

The most efficient method for producing methylphosphonate oligonucleosides is Metquin or β-cyano used to make xyliphonucleotides 5'-　-　-dimethoxytritylthi, which is similar to the ethylphosphoramidite reagent. Oquine nucleoside-3'-　-　-diisopropylmethylphosphoramidite Including the use of interbody. Methylphosphonate oligomers can be used in automated DNA synthesis equipment. can be made from glass polymer supports with controlled porosity [Sarin, etc.] Rossing National Academy of Sciences, USA, Volume 85, No. 7448-7451 (1988)].

Furthermore, resistance to nuclease cleavage is also present at both the 5' and 3' ends. Internucleotide bonds were determined by the method of Dagle et al. 18, pp. 4751-4757 (1990)]. can be achieved by changing.

Phosphorothioate oligonucleotides contain internucleotide phosphodiester bonds. In some cases, there is a substitution of sulfur and oxygen. Phosphorothioate oligonucleotides are Effective hybridization properties and substantial nuclei for duplex formation It has both anti-seizure resistance and water solubility of charged phosphate congeners.

Electrostatic charges appear to confer receptor-mediated cellular absorption properties [Roaf et al. Rossing National Academy of Sciences, USA, Volume 86, No. 3474-3478 (1989)].

Phosphorothioate oligodeoxynucleotides are manufactured by Laplancier et al. Qu Acids Research, Vol. 14, p. 9081 (1986) and Steck. et al., Journal of the American Chemical Society, Volume 106, No. 6077. (1984).

A general method for the synthesis of phosphorothioate oligonucleotides is described by Stein et al. Tsuku Acids Research, Vol. 16, pp. 3209-3221 (1988) These compounds were synthesized by automated synthesis equipment using the phosphoroamitite method. made it easy to synthesize.

Furthermore, recent advances in the production of oligoribonucleotide analogues have enabled the above-mentioned purposes. and other reagents, such as 2'-0-methylribonucleotides [Inolab et al. Ituku Acids Research, Vol. 15, p. 6131 (1987)] and Chimeric oligonucleotides that are synthetic RNA-DNA homologs [Inolab et al., FEB S Letters, Vol. 215, p. 327 (1987)] is also used to mean scales. do.

c-Machi Riyom RN A translation may be suppressed by antisense oligoribonucleotides. Although it is possible to use either oligodeoxyribonucleotides or oligodeoxyribonucleotides, it is possible to Rigoribonucleotides are more ribonucleans than oligodeoxyribonucleotides. It is highly susceptible to enzymatic attack by enzymes. Therefore, the practice of the present invention requires Godeoxyribonucleotides are preferred. Oligodeoxyribonucleotides are , DNA-RNA hybridization obtained during hybridization with c-myb mRNA Hybrid duplex is RNase H (this is a DNA-RNA hybrid) It is more suitable because it is a substrate for (specifically attacks the RNA portion of). Deupe Degradation of the Rex mRNA strand results in hives with other c-myb messages. Releases antisense oligodeoxynucleotide strands for lidding .

Generally, the antisense oligonucleotide used in the method of the present invention is a c-m sequence. It has a sequence that is completely complementary to the target portion of the jaw message. However, especially Absolute complementarity is not required for large oligomers. Therefore, c-myb( 7) a nucleotide sequence complementary to at least a portion of the rmRNA transcript. The expression does not necessarily imply a sequence with 100% complementarity to the transcript.

generally sufficient to form a stable duplex with c-myb mRNA. Oligonucleotides with high complementarity are suitable. stable duplex Formation depends on the sequence and length of the hybridizing oligonucleotide and the c-m It depends on the degree of complementarity of the yb message to the target region. In general, Note Eve The larger the oligomer that undergoes re-soto, the larger the misma-sochi is tolerated. Can be done. Two or more mismatches probably result in an antisense of less than about 21 nucleotides. It is not allowed for oligomers. Those skilled in the art will recognize any given antisense origin. The degree of mismatch that can be tolerated between the target c-myb message sequence and the target c-myb message sequence Based on the melting point (and therefore the stability of the resulting duplex) can be determined. Melting point of Deubre Socs with a given base pair composition standard literature [e.g. molecular cloning, laboratory manuals] (2nd ed., 1989), edited by Sampurczuk et al.]. Within the scope of the invention are oligonucleotides that can Oligonucleotides that are complementary to this appear to be particularly effective. start codon Up to 40 nucleotides upstream (5' direction) or 40 nucleotides below the start codon Oxygen that can hybridize to the region of c-myb mRNA up to the stream (3' direction) Particularly preferred are oligonucleotides.

For therapeutic applications, antisense oligonucleotides may be packaged, e.g., in a suitable liquid vehicle. or in combination with pharmaceutical carriers such as excipients and appropriate auxiliary additives. can. Liquid vehicles and excipients are conventional and commercially available. So Examples are distilled water, saline, and dextrose solutions. c-myb m RNA antisense oligonucleotides are preferably parenteral [1, particularly preferably is administered intravenously. The vehicle is designed accordingly. Or Origonu Creotide can also be administered subcutaneously via modified release dosage forms.

Oligonucleotides are conjugated to poly(L-lysine) to increase cell permeability can be done. This type of conjugate is described in Lematowa et al. Null Academy Science, USA, Vol. 84, pp. 648-652 (19 87). This method uses a 3′-terminal nucleotide It needs to be a punch line. The resulting aldehyde group is then converted into poly(L-lysine) ) Random bonding to the ε-amino group in the Norizine residue by Schiff base generation and then reduced with sodium cyanoborohydride. This method uses the 3'-terminal The ribose ring is converted into an antisense oligomer with a morpholine structure.

In addition to administration through conventional carriers, antisense oligonucleotides can be Administration can also be by special oligonucleotide delivery techniques. For example, Ligonucleotides can also be encapsulated for therapeutic delivery. Oligonuku Reotide can be present in both the aqueous layer and the lipid layer depending on its solubility. Alternatively, they may be present in what is commonly referred to as a liposome suspension. hydrophobic The layer generally contains, but is not limited to, lecithin and sphingomyelin. Phospholipids: for example cholesterol Steroids: for example diacetyl phosphatide Ionic surfaces such as sphate, stearylamine or phosphatidic acid active agents; and/or other substances with hydrophobic properties. oligonucleotide has been successfully encapsulated in unilamellar liposomes.

RNA and DNA using reassembled Sendai virus envelopes have been successfully supplied to cells [Arad et al. 859, pp. 88-94 (1986)] as the main therapy. can be given. Alternatively, oligonucleotides may be used to treat patients who do not have the disease but have a high recurrence rate. It may be given as an adjunctive measure after surgical removal of melanoma in patients at high risk. It comes with me.

A preferred method of administering oligonucleotides to treat melanoma is localized administration as needed. or systemic perfusion. According to local perfusion method, disease Separate the import and export pipes that supply the area with the nest, and connected to a low flow perfusion pump in series with the water heater and heat exchanger. by iliac vessels Lower (III) Possible to perfuse the f+ page area・Banshi axillary blood IBH for upper lesions t = Irrigation is performed at a high position in the armpit. adding oligonucleotides to the perfusion circuit, tract; Perfusion is continued for a suitable period of time (eg, 1 hour). 100-150m1/ A perfusion rate of min is used for the lower area lesions, while half that rate is used for the upper areas It can be used on lesions. Systemic heparinization is used during perfusion, Reverse after completion.

This isolated perfusion technique tolerates injection into the arterial or venous systemic circulation. allows the administration of higher doses of chemotherapeutic agents.

For systemic injection, the oligonucleotide is preferably injected into a suitable continuous infusion device. Supplied via a central venous catheter connected to the The catheter placed inside the body , allows for long-term administration into the intravenous circulation for frequent drug administration over long periods of time. Make it. Generally, the catheter is inserted into the lateral cephalic vein under general or local anesthesia. The tube is surgically inserted into the medial artery and vein. Other common sites for catheter insertion are the clavicular region. It is the pulse. The infusion pump can be external or e.g. -Inflation available from Corporation, Norwood, Massachusetts. User boat system and Pharmacia Laboratories, Bisryotaue B. Like the PORT-A-CATH system obtained from New Chassis. It may also form part of a completely implantable central venous system. these The device is implanted into a subcutaneous pocket under local anesthesia. connected to the pump pouring boat A catheter is attached to the superior vena cava via the clavicular vein. The graft is placed in an oligomer in the reservoir. The cleotide supply section includes a self-sealing container in the reservoir, which can also contain the drug if necessary. It can be refilled by further injections through the eaphragm and through the hypodermic needle. complete transfer Implantable injection devices are preferred. This is because these devices are easy to use and easy to maintain. because it is better accepted by patients because of its ease and cosmetic benefits. Ru.

High-dose chemotherapy combined with autologous bone marrow recovery (transplant) in attempts to treat melanoma I'm being forced to. Although high-dose chemotherapy has given a significantly higher therapeutic response, The survival rate for patients is generally not long-term [Jones et al. ) 7-Macology, Vol. 26, pp. 155-6 (1,990) (Carboplatin , cyclophosphamide and BCNU); Null Cancer, Vol. 61, pp. 330-4 (1990) (Melf 7 runs) Kepler et al., Oncology, Vol. 12, pp. 277-9 (1989) (Mel. Hualan and BCNU); Sarachar et al., Cancer, Vol. 63, No. 1296 ~302 pages (1989) (DTIC); Tsuruf et al., Journal Clinical Oncology, Vol. 7, pp. 245-9 (1989) (thiotepa): Shea et al., A. Architecture Dermatology, Volume 124, Pages 878-84 (1988) (cyclophosphamide, cisplatin and carmustine); Chekmezian et al., Journal Clinical Oncology, Volume 4, pp. 1811-8 (19 86) (BCNU, Melphalan or both); Thomas et al., Oncology , Vol. 43, pp. 273-7 (198t3) (BCNU and Melphalan).

Melanoma, especially in advanced stages, is essentially metastatic. Therefore melanoma One possibility is that high-dose chemotherapy and autologous bone marrow cleansing have a low success rate when treating The reason for this is that the recovered bone marrow of malignant cells that have metastasized to the bone marrow cannot be properly purified. Ru. According to the present invention, c-myb antisense oligonucleotides can be used in melanoma cells. High doses of conventional chemicals as bone marrow purifiers for in vitro purification of bone marrow in Can be used in combination with therapy. Normal hematopoietic cells are c-myb antisene These are commonly filed in co-pending U.S. patent application No. 427. No. 659 and the corresponding international patent application WO 90/malignant cells. child Differences in susceptibility to c-myb antisense oligos in clearing the bone marrow of tumor cells Allows the use of nucleotides.

According to bone marrow purification, bone marrow is collected from the donor's ilium using standard operating room procedures. Melt. Methods for aspirating bone marrow from donors are well known in the art. bone from donor Examples of devices and methods for aspirating marrow are U.S. Pat. ．． No. 486,188 (incorporated herein by reference). donor( Recipe ends that are either autografts) or other individuals (allografts) are approximately 4 Sufficient bone to receive 10 to about 8 x 10 treated bone marrow cells per kg body weight Remove the pith. This generally requires aspiration of about 750 to about 10,100 O of bone marrow. . The aspirated bone marrow is combined into a single Filter until a cell suspension is obtained. This suspension of leukocytes was mixed with c-myb antibody. Advantageously about 8 mg/g in a carrier more suitable for oligonucleotides. Treat at a concentration of mI. Alternatively, leukocyte suspensions can be prepared using standard methods known to those skilled in the art. It can be stored in liquid nitrogen until used and purified. Purified bone marrow is used It can be stored frozen in liquid nitrogen until used. bone marrow and biological materials Freezing methods are described, for example, in U.S. Pat. It is disclosed in the publication No. 17.881.

A method of making the buffy coat may also be used, which is much more effective than the buffy coat preparation described above. A preparation of purified hematopoietic cells can be provided.

Adding one or more growth factors to the aspirated bone marrow or buffy coat preparation and stimulate tumor growth, thereby inhibiting c-myb antisense oligonucleotides. can increase susceptibility to toxicity.

After treatment with antisense oligonucleotides, cells to be transferred are transferred to autologous plasma or is washed with buffer to remove unincorporated oligomers. Then washed The cells are then injected back into the patient.

c-myb antisense oligonucleotides can be used to treat melanoma tumors in affected individuals. Administered at a dose effective to suppress cell proliferation and maintain normal cell survival. can do. This amount depends on the nature and extent of the tumor and the particular oligonucleotide used. Can vary depending on location and other factors. Actual dose administered the size and weight of the patient, whether the treatment is preventive or therapeutic in nature; The patient's age, health status, and gender; whether the procedure is local or systemic; However, the route of administration, and other factors can be taken into account. Suppression of black cell proliferation It has been observed at antisense concentrations as low as 10 μg/ml. 20 μg / The suppression was remarkable in ml. Concentrations of about 1 to about 100 μg/ml, preferred Preferably about 10 to about 100 μg/ml, particularly preferably about 20 to about 60 μg/ml Concentrations of μg/ml can be used. The patient obtains drug concentrations between these cells. A dose of antisense oligonucleotide II sufficient to No.

10 doses per dose [1 about 0.1 to 1.000'mg of oligonucleotide, preferably The dosage may range from about 10 to about 700 mg daily. more or Less warm oligonucleotides can also be administered if desired.

Those skilled in the art will be able to determine dosages and dosing schedules appropriate to a patient's particular circumstances and needs. can be easily obtained. Based on the in vivo studies described herein, treatment is advantageous. The aging process is for about 3 to about 28 days, more preferably about 7 to about 10 days. It can consist of an infusion of the recommended daily dose of cleotide. Those skilled in the art should The optimum dosage can be easily determined in each case.

Approximately 350 mg of oligonucleotide per adult is required to obtain an effective intercellular concentration of 20 μg. It seems that the 111 temperature of cleotide is sufficient.

For ex vivo tumor prevention applications, e.g. in bone marrow purification, c-myb Thisense oligonucleotide kills tumor cells and kills normal hematopoietic cells. This can be administered in an amount effective to maintain survival. This amount is the amount that black cells The degree of metastasis to the spinal cord, the specific oligonucleotide used, and the oligonucleotide can vary depending on the relative susceptibility of tumor cells to, as well as other factors . Approximately 10 to 200 μg/'ml per 105 cells, preferably approximately Concentrations of 40-150 μg/ml can be used. To optimize treatment, Additional administration of the same or smaller amount of oligonucleotide is advantageous. did Therefore, for example, bone marrow 1. Purify bone marrow containing 2X10' cells per mI 2 to 40 mg of antisense per ml of bone marrow, preferably about 8 to 24 mg of antisense per ml of bone marrow. Doses of g/ml can be effectively used. more or less than that Oligonucleotides can also be used.

Hereinafter, the present invention will be explained in more detail by way of non-limiting examples.

CHP melanoma cells (Children's Hospital of Philadelphia, Philadelphia, Pencil) RP containing 2% oxalophosphate and 5% calf serum The cells were grown in MI medium at 37°C and 5% CO2. Cells (5゜000 cells/　m　 1) in a volume of 200 μl into individual wells of a 96-well Coscuk plate 3 days in advance. Seed was then allowed to grow for 3 days. At this point (day 0), c-mybmR An unmodified antisense 18-mer oligodeline complementary to codons 2-7 of the translated portion of NA. Oxynucleotide (SEQ ID NO: 22) or corresponding sense 18-mer (SEQ ID NO: 26) at 0110.20.50 and 100μg/ It was added to the culture continuously for 1, 2 or 5 days at a concentration of ml.

On day 7, 1°0 μm of fresh medium was added to the culture. cell viability/proliferation, Commercially available kits (CellTiter 96 (registered trademark) Promega, Madison, Wisconsin The measurement was carried out on the 8th day using the following method (Sin State). Analysis of tetrazolium salts as blue formosan Based on the ability of living cells to convert the formozan product into a Quantification can be performed by measuring absorbance at 570 nm with a small plate meter. 57 The degree of absorbance at 0 nm is a measure of the amount of formosan produced and therefore remaining. is proportional to the number of viable cells. Therefore, 10 μl of 3-(4,5-dibromide) Methylthiazol-2-yl)-2,5-diphenyltetrazolium solution (5 m g/ml) was added to a 110 μm cell suspension and cultured at 37° C. for 4 hours. Next 150 μm acidified isopropanol (25 ml isopropanol) +0.1 ml of 12N HCI) was added and the solution was mixed. Optical density 570 Measured in nm. Standard measurement of pack ground from cell debris at 630 nm The value was subtracted and removed.

The results of survival rate analysis are shown in Figure 1 (1-day treatment), Figure 2 (2-day treatment), and Figure 3 (5-day treatment). processing). The oligonucleotide concentrations shown in Figures 2 and 3 are These are the cumulative doses for the 2-day and 5-day treatments. As you can see from the drawing, Treatment of melanoma cells with antisense oligomers resulted in sequence-specific killing.

This effect is effective against melanoma cells with at least 50 μg/ml of antisense oligonucleotide. When treated with sesame concentration for 1 day, or cells were treated with 20 μg/m of 1 species. It was most pronounced when treated with oligomers on consecutive days.

Other human melanoma lines SK MEL-37 (Sloan Kettering Inst. c-myb antisense receptors against The effect of goscleotide was measured. Cells were grown on day 0 according to the procedure of Example 1. were treated with sense and antisense oligomers at the same concentration. Cell viability was analyzed on the 50th day. The results are shown in Figure 4.

Again, black with a single 50 μg/ml dose of antisense oligomer. Treatment of chromoma cells results in substantial cell death compared to sense-treated or untreated cells. It was enough to induce extinction.

250,000 melanoma cells (SK MEL-37) in 4 animals with significant combined immunity deficiency mouse (C, B-17/LCRTAC-3CID DF, Fox Chi Ace Cancer Institute, Philadelphia, Pennsylvania ) was injected subcutaneously 21[1 days ago. Growth of tumor to approximately 5mm in diameter I let it happen. Deliver a total dose of 700 μg of oligomer in one day over 7 days. ALZET constant infusion pump (Alza Corporation needle, Palo Aldo, CA) Lihornia) were surgically transplanted into mice. The tumor area was then monitored every 11 days. 1 One mouse received no oligomer. Other mice have 24-mer “Sense” Phosphorothioate oligonucleotide (translated c-myb mRNA codon 2 ~9) was given. Two mice had a c-myb mRNA codon 2-9. Complementary SEQ II) NO+16CTATGCTGTGCCGGGGGGTC A 24-mer antisense phosphorotinase having the nucleotide sequence of oate oligonucleotide was given.

The results are shown in Figure 5. Each curve represents one animal. Two animals were treated with antisense. In animals treated, tumor size remained at the same level or regressed. On the other hand, Tumors continued to grow in sense-treated and comparison animals.

The animals were sacrificed for 199 days, the tumors removed and weighed. Compare and Sen Tumor weights in the S-treated animals were 9,9 g and 9.6 g, respectively.

Tumor weights in two antisense-treated animals were only 0,1 g and 0. ．． It was 2g The following non-limiting example describes one method for purifying the bone marrow of metastatic melanoma. exemplify.

Bone marrow was collected by standard techniques in the operating room from the donor's ilium under general anesthesia. . Multiple aspirates were placed into heparinized syringes. Bone marrow recipe end is weight Sufficient bone to receive about 4 x 108 to about 8 x 108 treated bone marrow cells per kg The pith was removed. Therefore, approximately 750-10100 O of bone marrow was extracted. sucked Immediately add 10,000 units of preservative-free heparin to 100ml of medium. Transport medium (TC-199, Gibco, Grand Island, moved to New York). The aspirated bone marrow is passed through three sequentially fine meshes. until a single cell suspension results, i.e., cell aggregates, debris and bone particles. Once filtered until no suspension occurs, the filtered bone marrow is further passed through an automated cell separator ( For example, Kozo 2991 cell processing equipment) to produce a “Packcoat” product. (i.e., white blood cells without red blood cells and platelets). Then Buffy The coat preparation was placed in transport packages for further processing and storage. purify this It can be stored by standard methods in liquid nitrogen until it reaches its maximum capacity. Or clean it immediately. The purified bone marrow is then stored frozen in liquid nitrogen until ready for transplantation. You can also do that.

The purification method can be carried out as follows. Cells in buffy coat preparation Approximately 2X107 cells/ml in TC-199 containing approximately 20% autologous plasma Adjust to concentration. For example, C-myb at a concentration of about 8 mg/ml Transport packaging containing antisense oligodeoxynucleotides and cell suspensions add to something Recombinant human hematopoietic growth factors (e.g. rHIL-3 or rII) GM-C3F) was added to the suspension to stimulate tumor growth, which b Can increase susceptibility to antisense oligonucleotide toxicity Ru. The transport package was then placed in a 37°C water bath and heated for 18 to 2 hours with gentle shaking. Incubate for 4 hours. Cells are then frozen in liquid nitrogen or frozen for about 20 minutes. Wash once at 4°C with TC-199 containing % autologous plasma to remove any unincorporated orifices. Sesame can be removed. Then, inject the washed cells into the recipe end do. Work under aseptic conditions when possible and maintain careful aseptic technique at all times You have to be careful.

The invention may be embodied in other specific forms without departing from its spirit or substantial characteristics. Therefore, in order to indicate the scope of the invention, it is necessary to refer not only to the above description but also to the claims. You should also refer to range.

All publications cited herein with respect to synthetic, preparative and analytical procedures are incorporated herein by reference. I quote it for consideration.

Arino [λri-riage] (1) General information: (i) Applicant: Temple University of the Commonwealth TEM OF HIRE EDUCATION (a) Inventors: Gewirtz, Alain Mkaraprecta, Bruno (if) Title of the invention: antisense ori against proto-oncogene on c-1L Treatment of melanoma with gonucleotides (i　i　i) Number of arrays: 26 (iv) Communication destination; (A) Address: Temple University-of-the-Commonwealth Sith TEM OF HIRE EDUCATION (B) Street name = 406 University Service Building (C) Book title: Philadelphia (D) State name: Pennsylvania (E) Country name: United States of America (F)ZIP: 19122 (v) Computer-deciphered form: (A) Media type: Diskette, 3.50 inches, 720Kb (B) Computer: IBM PS/2 (C) Operating system: MS-DO3 (D) Software: Word Perfect 5. 1(vi) Application data: (A) Application number/ (B) Application date (C) Classification/ (vii) Prior application data: (A) Application number: U.S. Patent Application No. 792,999 (B) Filing date: January 1991 January 15th (viii) Agent information.

(A) Name: Monaco, Daniel A (B) Registration number: 30.480 (C) Reference number: 6056-159 pcT 1 (ix) Telegraphic information (A) Telephone・(215)568-8383(B) Telefax・ (2]5)568-5549 (C) Telex None 2) SEQ ID No. 1 information (i) Sequence characteristics.

(A) Length: 40 nucleotides (B) Type: Nucleic acid (C) Chain wheel/single chain (D) Topology/linearity (xi) Sequence description SEQ ID NO: ICGTCACTGCT ATATA TGCTG　TGCCGGGGGTCTTCGGGCCAT　40(2)SEQ　 Information on ID NO:2.

(i) Sequence characteristics: (A) Length: 26 nucleotides (B) Type: Nucleic acid (C) Chain wheel single strand (D) Topology: Linear (xi) Sequence description: SEQ ID NO: 2: ATGCTGTGCCGGGGT C’ITCG GGCCAT 26(2) SEQ ID NO:3 information (i ) Array characteristics: (A) Length/25 nucleotides (B) Type/nucleic acid (C) Chain wheel single strand (D) Topology: Linear (xi) Sequence explanation SEQ ID NO:3:TGCTGTGCCG GGGT CTTCGG GCCAT 25 (2) SEQ ID NO: 4 information.

(i) Sequence characteristics・ (A) Length: 24 nucleotides (B) Type: Nucleic acid (C) Chain wheel single strand (D) Topology Linear (x i) Sequence description: SEQ ID NO: 4: GCTGTGCCGG GGT CTTCGGG CCAT 24(2) SEQ ID NO:5 information (i) Array characteristics (A) Length, 23 nucleotides (B) Type: Nucleic acid (C) Armor type, single chain (D) Topology Linear (xi) Sequence description: SEQ ID NO: 5: CTGTGCCGGG GTCT TCGGGCCAT 23 (2) SEQ ID NO:6 ftl information (l array special sex (A) Length: 22 nucleotides (B) Type/nucleic acid (C) Mirror-shaped double strand (D) Topology Linear (xi) Sequence description: SEQ ID NO+6: TGTGCCGGGGG TCTT CGGGCCAT 22(2) SEQ ID NO Near information: (i) Sequence characteristics .

(A) Length: 21 nucleotides (B) Type: Nucleic acid (C) Armor type/single chain (D) Topology, linear (xi) Sequence description: SEQ ID NO Near: GTGCCGGGGT CTTC GGGCCA T 2/1 (2) SEQ ID NO:8 information/(i) Array characteristics sex: (A) Length/20 nucleotides (B) Type: Nucleic acid (C) Mirror-shaped double strand (D) Topology/linearity (xi) Sequence description: SEQ ID NO: 8゜’rcccccccTc TTC GGGCCAT 20 (2) SEQ If) Information on NO:9 (i) Sequence characteristics (A) Length, 19 nucleotides (B) Type, nucleic acid (C) Mirror-shaped double strand (D) Topology bilinear (xl) Sequence description: SEQ ID NO: 9GCCGGGGTCT TCGGG CCAT 19(2) SEQ ID NO:10 information (i) Sequence characteristics: (A) Length/18 nucleotides (B) Type/nucleic acid (C) Armor type single strand (D) Topology: Linear (x i) Sequence description: SEQ ID NO: 10: CCGGGGTCTT CG GGCCAT 1B (2) SEQ ID NO: 11 information (i) Sequence characteristics・ (A) Length: 17 nucleotides (B) Type: Nucleic acid (C) Armor type, - wooden chain (D) Topology/linearity (xi) Sequence description SEQ ID NO: 1ICGGGGTCTTCGGGCC AT 17 (2) SEQ ID No. 12 information (summer) sequence characteristics (A) Length °16 nucleotides (B) Type: Nucleic acid (C) Armor type single strand (D) Topolon-linear (xi) Sequence explanation SEQ ID NO: 12:GGGGTCTTCG GGC CAT 16(2) SEQ [D NO:13 information (i) Sequence characteristics (A) Length/15 nucleotides (B) Type: Nucleic acid (C) Armor type/single chain (D) Topology/linearity (xi) Sequence description: SEQ’ID NO: 13:GGGTCTTCGG GCC AT 15 (2) SEQ ID NO: 14 information.

(i) Sequence characteristics: (A) Length, 26 nucleotides (B) Type: Nucleic acid (C) Mirror-shaped double strand (D) Topology/linearity (xi) Sequence explanation SEQ ID NO: 14: TATATGCTGT GCC GGGGTCT TCGGGC26 (2) SEQ ID NO+15 information: ( 1) Array characteristics・ (A) Length, 25 nucleotides (B) Type: Nucleic acid (C) Armor type/single chain (D) Topology Linear (Xl) Sequence explanation/SEQ ID NO: 15:ATATGCTGTG CCG GGGTCTr CGGGC25 (2) SEQ ID NO:16 information: (i ) Array characteristics: (A) Length: 24 nucleotides (B) Type/nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology: Linear (x i) Sequence description: SEQ ID NO: 16: TATGCTGTGCCGG GGTC’I’rCGGGC24(2) SEQ ID NO:17 information・(i ) array characteristics (A) Length: 23 nucleotides (B) Type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology Linear (xi) Sequence description: SEQ ID NO: 17ATGCTGTGCCGGGGT CTTCG GGC23(2) SEQ 10 NO:18 information (i) Sequence characteristics ・ (A) Length: 22 nucleotides (B) Type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology Linear (xi) Sequence explanation SEQ ID NO: 18・TGCTGTGCCG GGG TC? TCGG GC22 (2) SEQ ID NO:19 information: (i) Array Characteristic: (A) Length: 21 nucleotides (B) Type, nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology bilinear (xi) Sequence description: SEQ rD NO: 19: GCTGTGCCGG GGT CTTCGGG C21 (2) SEQ ID NO:20 information: (i) Array special sex: (A) Length = 20 nucleotides (B) Type: Nucleic acid (C) Mirror type - heavy chain (D) Topology/linearity (xi) Sequence description: SEQ ID NO: 20: CTGTGCCGGG GTC TTCGGGC20(2) SEQ ID NO:21 information (i) Sequence characteristics: (A) Length: 19 nucleotides (B) Type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology, linear (xi) Sequence explanation: SEQ ID NO: 21: (2) SEQ ID NO: 2 Information in 2: (i) Sequence characteristics: (A) Length: 18 nucleotides (B) Type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology: Linear (xi) Sequence description: SEQ ID NO: 22: GTGCCGGGGT CTT CGGGC:L8(2) SEQ ID NO:23 information.

(i) Sequence characteristics・ (A) Length = 17 nucleotides (B) Type: Nucleic acid (C) Mirror type; - heavy chain (D) Topology/linearity (xi) Sequence description: SEQ ID NO: 23TGCCGGGGTCTrCGG GC17(2) SEQ ID NO:24 information: (i) Sequence characteristics: (A) Length: 16 nucleotides (B) Type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology, linear (xi) Sequence description: SEQ ID NO: 24: GCCGGGGTCT TCG GGC16(2) SEQ ID NO:25 information: (i) Sequence characteristics: (A) Length = 15 nucleotides (B) Type; Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology Linear (xi) Sequence explanation SEQ ID NO: 25: CCGGGGTCTT CGG GC15(2) SEQ ID No. 26 information: (i) Sequence characteristics: (A) Length = 18 nucleotides (B) Type: Nucleic acid (C) Mirror type - heavy chain (D) Topology bilinear (xi) Sequence description: SEQ ID NO: 26:GCCCGAAGACCCCG GCAC18FIG, l concentration (pg/m1) FIG, 2a degree (p g/m1) FIG, 3 1 degree (Jug/m1) FIG, 4 1 degree (loJJg/ml) FIG, 5 days Continuation of front page (72) Inventor: Gewirtz, Alan M. Pennsylvania, United States of America 19130 Philadelphia North Touninty Fourth Street 83 7 (72) Inventor Karaprecta, Bruno United States Pennsylvania 1 9103 Philadelphia Pine Street

Claims (59)

[Claims] 1. Low levels of human c-myb gene mRNA transcripts for individuals in need of treatment. An effective amount of an oligonucleotide having a nucleotide sequence complementary to at least one part of the the oligonucleotide hybridizes to the mRNA transcript. A method for treating melanoma, which is characterized in that it can be redized. 2. Claim 1, wherein the oligonucleotide is at least a dodecamer. Method. 3. If the oligonucleotide is a methylphosphonate oligonucleoside or 3. The method according to claim 2, wherein the oligonucleotide is a holotioate oligonucleotide. 4. The oligonucleotide is within approximately 40 nucleotides of the translation initiation codon Claim 2 having a nucleotide sequence complementary to a part of c-myb mRNA The method described in section. 5. The oligonucleotide is located at the translation initiation codon of said transcript and/or its opening codon. A fragment complementary to the part of c-myb mRNA that includes the codon immediately downstream from the start codon. Claim No. 1, which is an oligodeoxynucleotide having a xynucleotide sequence. The method described in Section 2. 6. The oligonucleotide consists of 12- to 40-mer oligodeoxynucleotides The method according to claim 2. 7. If the oligonucleotide is a methylphosphonate oligonucleoside or 7. The method according to claim 6, wherein the oligonucleotide is a holotioate oligonucleotide. 8. The method according to claim 6, wherein the oligonucleotide is a 15-30 mer. . 9. The method according to claim 8, wherein the oligonucleotide is a 18-26 mer. . 10. The method according to claim 9, wherein the oligonucleotide is an 18-21 mer. Law. 11. The oligonucleotide: 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 Claim 8 which is an oligodeoxynucleotide selected from the group consisting of The method described in. 12. The oligonucleotide: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 Claim 8 which is an oligodeoxynucleotide selected from the group consisting of The method described in. 13. Claim 12: The oligonucleotide consists of SEQ ID NO: 22. The method described in. 14. The oligonucleotide: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 Phosphorothioe with a nucleotide/nucleoside sequence corresponding to either oligodeoxynucleotide or methylphosphonate oligodeoxynucleotide 9. The method according to claim 8, wherein the method is a creoside. 15. Is the oligonucleotide a phosphorothioate oligodeoxynucleotide? 15. The method according to claim 14, comprising: 16. Phosphorothioate oligodeoxynucleotide is SEQ ID NO: 16 16. The method according to claim 15, having a nucleotide sequence corresponding to. 17. A method of purifying the bone marrow of metastatic melanoma cells, which is aspirated from individuals affected by melanoma. bone marrow cells that are homologous to at least a portion of the human c-myb gene mRNA transcript. has a complementary nucleotide sequence and is capable of hybridizing to said mRNA transcript. treated with an effective amount of oligonucleotide that A method of purification of bone marrow characterized by returning the treated cells to the body of an affected individual. 18. Claim 17, wherein the oligonucleotide is at least a dodecamer. How to put it on. 19. If the oligonucleotide is a methylphosphonate oligonucleoside or 19. The method according to claim 18, which is a sulforthioate oligonucleotide. 20. The oligonucleotide is within approximately 40 nucleotides of the translation initiation codon. Claim 1, which has a nucleotide sequence complementary to a part of c-myb mRNA. The method described in item 18. 21. The oligonucleotide is composed of 12- to 40-mer oligodeoxynucleotides. 19. The method according to claim 18. 22. Claim 21, wherein the oligonucleotide is a 15-30 mer. Method. 23. Claim 22, wherein the oligonucleotide is a 18-26 mer. Method. 24. Claim 23, wherein the oligonucleotide is an 18-21 mer. Method. 25. The oligonucleotide: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 an unmodified oligonucleotide having a nucleotide/nucleoside sequence selected from the group consisting of Godeoxynucleotides, phosphorothioate oligodeoxynucleotides Claim 22 is a methylphosphonate oligodeoxynucleoside. The method described in. 26. The oligonucleotide has the nucleotide sequence SEQ ID NO: 22. Scope of Claim 25. The method according to item 25. 27. The oligonucleotide has the nucleotide sequence SEQ ID NO: 16. Scope of Claim 25. The method according to item 25. 28. A nucleic acid complementary to at least a portion of the mRNA transcript of the human c-myb gene an oligonucleotide having a leotide sequence and capable of hybridizing to the mRNA transcript; Use of cleotide to manufacture a drug to treat melanoma. 29. Claim 28, wherein the oligonucleotide is at least a dodecamer. Use of listed. 30. If the oligonucleotide is a methylphosphonate oligonucleoside or The use according to claim 29, which is a sulforthioate oligonucleotide. 31. The oligonucleotide is within approximately 40 nucleotides of the translation initiation codon. Claim 1, which has a nucleotide sequence complementary to a part of c-myb mRNA. Uses as described in Section 29. 32. The oligonucleotide is located at the translation start codon of the transcript and/or at this start codon. A fragment complementary to the portion of the c-myb mRNA transcript containing the codon immediately downstream of the codon. The claim is an oligodeoxynucleotide having an oxynucleotide sequence. Uses as described in Section 29. 33. The oligonucleotide is composed of 12- to 40-mer oligodeoxynucleotides. The use according to claim 29. 34. If the oligonucleotide is a methylphosphonate oligonucleoside or 34. The use according to claim 33, which is a sulforothioate oligonucleotide. 35. Claim 33, wherein the oligonucleotide is a 15-30 mer. use. 36. Claim 35, wherein the oligonucleotide is a 18-26 mer. use. 37. Claim 36, wherein the oligonucleotide is an 18-21 mer. use. 38. The oligonucleotide: 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 Claim 35 is an oligodeoxynucleotide selected from the group consisting of Uses as described in Section. 39. The oligonucleotide: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 Claim 35 is an oligodeoxynucleotide selected from the group consisting of Uses as described in Section. 40. Claim 39: The oligonucleotide consists of SEQ ID NO: 22. Uses as described in. 41. The oligonucleotide: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 Phosphorothioe with a nucleotide/nucleoside sequence corresponding to either oligodeoxynucleotide or methylphosphonate oligodeoxynucleotide The use according to claim 35, which is a creoside. 42. Is the oligonucleotide a phosphorothioate oligodeoxynucleotide? The use according to claim 41 consisting of: 43. Phosphorothioate oligodeoxynucleotide is SEQ ID NO: 16 43. The use according to claim 42, having a nucleotide sequence corresponding to. 44. a pharmaceutically acceptable carrier and an mRNA transcript of the human c-myb gene. has a nucleotide sequence complementary to at least a portion thereof and has a nucleotide sequence complementary to the mRNA transcript; melanoma, which is characterized by being composed of hybridizable oligonucleotides. Composition for treatment. 45. Claim 44, wherein the oligonucleotide is at least a dodecamer. composition. 46. If the oligonucleotide is a methylphosphonate oligonucleoside or The composition according to claim 45, which is a sulforothioate oligonucleotide. . 47. The oligonucleotide is within approximately 40 nucleotides of the translation initiation codon. Claim 1, which has a nucleotide sequence complementary to a part of c-myb mRNA. Composition according to item 45. 48. The oligonucleotide is located at the translation start codon of the transcript and/or at this start codon. An oligonucleotide complementary to the portion of the c-myb mRNA transcript containing the codon immediately downstream of the codon 46. The composition of claim 45, which is a oligodeoxynucleotide. 49. The oligonucleotide is composed of 12- to 40-mer oligodeoxynucleotides. 46. The composition according to claim 45. 50. If the oligonucleotide is a methylphosphonate oligonucleoside or The composition according to claim 49, which is a sulforothioate oligonucleotide. . 51. Claim 49, wherein the oligonucleotide is a 15-30 mer. Composition. 52. Claim 51, wherein the oligonucleotide is an 18-26 mer. Composition. 53. Claim 52, wherein the oligonucleotide is an 18-21 mer. Composition. 54. The oligonucleotide: 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 Claim 51, which is an oligodeoxynucleotide selected from the group consisting of The composition described in Section. 55. The oligonucleotide: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 Claim 51, which is an oligodeoxynucleotide selected from the group consisting of Composition as described in Section. 56. Claim 55: The oligonucleotide consists of SEQ ID NO: 22. The composition described in . 57. The oligonucleotide: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 Phosphorothioe with the corresponding nucleotide/nucleoside sequence of either oligodeoxynucleotide or methylphosphonate oligodeoxynucleotide 52. The composition of claim 51, which is a creoside. 58. Is the oligonucleotide a phosphorothioate oligodeoxynucleotide? 58. The composition of claim 57, comprising: 59. Phosphorothioate oligodeoxynucleotide is SEQ ID NO: 16 59. A composition according to claim 58, having a nucleotide sequence corresponding to.