WO1992020348A1

WO1992020348A1 - Treatment of colorectal carcinoma with antisense oligonucleotides to c-myb proto-oncogene

Info

Publication number: WO1992020348A1
Application number: PCT/US1992/004318
Authority: WO
Inventors: Bruno Calabretta; Alan M. Gewirtz
Original assignee: Temple University - Of The Commonwealth System Of Higher Education
Priority date: 1991-05-23
Filing date: 1992-05-22
Publication date: 1992-11-26
Also published as: EP0585399A4; CA2109829A1; JPH06508130A; EP0585399A1

Abstract

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

Description

TREATMENT OF COLORECTAL CARCINOMA WITH ANTISENSE OLIGONUCLEOTIDES TO C-MYB PROTO-ONCOGENE

Field of the Invention

The invention relates to antisense oligonucleotides to proto-oncogenes, in particular antisense oligonucleotides to the c-myb gene, and the use of such oligonucleotides as antineoplastic agents.

Reference to Government Grant The invention described herein was supported in part by National Institutes of Health grant CA4678.

Background of the Invention

The proto-oncogene c-myb is the normal cellular homologue of the avian myeloblastosis virus-transforming gene v-myb. The c-myjo gene codes for a nuclear protein expressed primarily in hematopoietic cells. It is a proto-oncogene, that is, it codes for a protein which is required for the survival of normal, non-tumor cells.

When the gene is altered in the appropriate manner, it has the potential to become an oncogene. Oncogenes are genes whose expression within a cell provides some function in the transformation from normal to tumor cell.

The human c-myb gene has been isolated, cloned, and seguenced. Maj llo et al. , Proc. Natl. Acad. Sci.

U.S.A. 83, 9636-9640 (1986). Antisense oligonucleotides to human c-myb mRNA that is, oligonucleotides having a nucleotide sequence complementary to the mRNA transcript of the c-myb gene, are disclosed in our commonly assigned, U.S. Patent No. 5,098,890, the entire disclosure of which is incorporated herein by reference. C-myb antisense oligonucleotides are disclosed therein as being useful for the treatment of hematologic neoplasms, and for immunosuppression. C-myb antisense oligonucleotides have not heretofore been known to have antiproliferative activity against neoplastic diseases other than those of a hematologic nature. Colorectal cancer is the second most common malignancy of the adult population in the United States (excluding skin cancer) . Its occurrence is exceeded only by lung cancer in males and breast cancer among females. A similar incidence has been reported in many other countries. A large majority of rectal and colonic malignancies are adenocarcinomas, i.e., they originate in glandular epithelium. Colorectal carcinoma generally progresses from adenoma, to in situ adenocarcinoma , and then to invasive adenocarcinoma. Colorectal cancers are classified based on the "Duke's classification" into four different stages: A, B, C and D. At stage A, the cancer is limited to the mucosa and submucosa, with a 90% five- year survival. At stage B, the cancer extends into muscularis or serosa, with a 60-75% five-year survival. Stage C cancer involves regional lymph nodes, with a 30- 40% five-year survival. At stage D, metastases in the liver, bone and lungs are commonly found. Stage D patients typically have only a 5% five-year survival rate. The current approach to colorectal cancer treatment is primarily surgical. The overall five-year survival rate for all patients undergoing resection for colonic malignancy is approximately 50%. Surgical cure is possible only when the tumor is confined to the bowel wall. There has been disappointingly little progress in the development of effective chemotherapeutic agents for colorectal carcinoma. 5-fluorouracil (5-FU) remains the most widely used agent. Recently, 5-FU has been used in combination with levamisole to treat stage C colorectal cancer, with some success (Martell et al. , N. Enql. J. Med. 322, 352-358 (1990)). 5-FU is administered to patients with metastases to the liver, but temporary improvement is obtained only in 25% or less of cases, and overall survival is not significantly affected. 5-FU treatment does not spare normal dividing cells, and is associated with the usual toxicity of chemotherapy, i.e. , anemia, infections and haemorrhagy diarrhea, vomitus and hair loss. The rather slow cellular doubling time of colorectal cancers has been offered as an explanation for the general unresponsiveness to chemotherapeutic agents. Clearly what it is needed is a more effective alternative to presently available treatments for colorectal cancer.

Elevated c-myb expression has been associated with colon carcinoma (Trainer et a_L. , Int. J. Cancer. 41,

287-286 (1988); Alitalo et al. , Proc. Natl. Acad. Sci. USA, 81, 4534-4538 (1984); Torelli et al. , Cancer Research. 47, 5266-5269 (1987); Untawali et al. , Anticancer Research. 8, 1-8 (1988)). Notwithstanding these reports, recent efforts to identify the genetic cause of colorectal cancer have focused not on c-myb but on other genes. For example, suppression of human colorectal cell proliferation has been achieved through transfection with the wild-type human p53 gene, suggesting that the p53 gene product may function as a suppressor of neoplastic growth (Baker et al. , Science. 249, 912-915 (1990)). Mutation in the ras gene has been proposed as the initiating event in at least a subset of colorectal carcinomas (Feron et al. , Cell. 61, 759-767 (1990)). Thus, genetic events other than c-myb expres¬ sion have been suggested as the major events in color- ectal carcinoma. Summary of the Invention The invention provides a method for treating human colorectal carcinomas characterized by c-myb gene expression. An effective amount of one or more c-myb antisense oligonucleotides is administered to an individual in need of such treatment. Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA trans¬ cript of the human c-myb gene. The oligonucleotide is hybridizable to the mRNA transcript. Preferably, the oligonucleotide is at least a 12-mer oligonucleotide, that is, an oligomer containing at least 12 nucleotide residues. In particular, the oligomer is advantageously a 12-mer to a 40-mer, preferably an oligodeoxynucleotide. While oligonucleotides smaller than 12-mers may be utilized, they are statistically more likely to hybridize with non-targeted sequences, and for this reason may be less specific. In addition, a single mismatch may destabilize the hybrid. While oligonucleotides larger than 40-mers may be utilized, uptake may be more difficult. Moreover, partial matching of long sequences may lead to non-specific hybridization, and non-specific effects. Preferably, the oligonucleotide is a 15- to 30-mer oligodeoxynucleotide, more advantageously an 18- to 26-mer.

Most preferably, the oligonucleotide is a 15- to 21-mer oligodeoxynucleotide.

While in principle oligonucleotides having a sequence complementary to any region of the c-myb gene find utility in the present invention, oligodeoxynucleotides complementary to a portion of the c-myb mRNA transcript including the translation initiation codon are particularly preferred. Also preferred are oligonucleotides complementary to a portion of the c-myb mRNA transcript lying within about 40 nucleotides upstream (the 5' direction) or about 40 nucleotides downstream (the 3' direction) from the translation initiation codon.

As used in the herein specification and appended claims, unless otherwise indicated, the term "oligonucleotide" includes both oligomers of ribonucleotide, i.e., oligoribonucleotides, and oligomers of deoxyribonucleotide, i.e., oligo- deoxyribonucleotides (also referred to herein as "oligodeoxynucleotides") . Oligodeoxynucleotides are preferred.

As used herein, unless otherwise indicated, the term "oligonucleotide" also includes oligomers which may be large enough to be termed "polynucleo- tides".

The terms "oligonucleotide" and "oligodeoxynucleotide" include not only oligomers and polymers of the common biologically significant nucleotides, i.e., the nucleotides adenine ("A"), deoxyadenine ("dA") , guanine ("G") , deoxyguanine ("dG") , cytosine ("C") , deoxycytosine ("dC") , thymine ("T") and uracil ("U") , but also include oligomers and polymers hybridizable to the c-myb mRNA transcript which may contain other nucleotides. Likewise, the terms "oligonucleotide" and "oligodeoxynucleotide" may include oligomers and polymers wherein one or more purine or pyrimidine moieties, sugar moieties or internucleotide linkages is chemically modified. The term "oligonucleotide" is thus understood to also include oligomers which may properly be designated as "oligonucleosides" because of modification of the internucleotide phosphodiester bond. Such modified oligonucleotides include, for example, the alkylphosphonate oligonucleosides, discussed below. The term "phosphorothioate oligonucleotide" means an oligonucleotide wherein one or more of the internucleotide linkages is a phosphorothioate group,

0 II

-O - P - 0~

I

S-

as opposed to the phosphodiester group

which is characteristic of unmodified oligonucleotides.

By "alkylphosphonate oligonucleoside" is meant an oligonucleotide wherein one or more of the internucleotide linkages is an alkylphosphonate group,

where R is an alkyl group preferably methyl or ethyl.

The term "downstream" when used in reference to a direction along a nucleotide sequence means the 5'→3' direction. Similarly, the term "upstream" means the 3'→5' direction.

The term "c-myb mRNA transcript" means the presently known MRNA transcript of the human c-myb gene, or any further transcripts which may be elucidated. Brief Description of the Figures

Figures 1A and IB comprise graphs recording the (A) proliferation and (B) ³H-thymidine incorporation, of colon carcinoma cell lines LoVo/Dx, LoVo, Colo 205 and HT 29, cultured in the presence of culture medium alone ("MEDIUM") , or medium to which has been added c-myb sense ("SENSE") or antisense ("ANTISENSE") oligodeoxynucleotide.

Figures 2A and 2B record the dose-depeident inhibition of (A) ³H-thymidine uptake and (B) ' cell proliferation, of carcinoma cell lines Colo 205, LoVo/Dx and LoVo treated with c-myb sense ("SENSE") or varying amounts of antisense ("AS") oligodeoxynucleotide.

Detailed Description of the Invention It has now been discovered that the expression of the human c-myb gene is important for the proliferation of certain colorectal carcinomas. The role of this proto-oncogene in cell proliferation is not restricted to cells of hematopoietic origin, as previously thought. The proliferation of colorectal carcinoma cells, which are neoplastic cells of epithelial and not hematologic origin, is maintained by high levels of c-myb expression. Thus, the role of c-myb is more general than previously thought. Surprisingly, however, c-myb expression is not an absolute requirement for the proliferation of neoplastic epithelial cells as shown by the complete independence of proliferation of some colorectal car¬ cinoma cell lines from the synthesis of c-myb protein. Whether a particular patient's carcinoma expresses c- myb. and is therefore potentially treatable with antisense oligonucleotides, can be readily determined by hybridization studies utilizing appropriate oligonucleotide probes for detecting c-myb mRNA. A representative screening technique is described hereinafter in Example 2, it being understood that other methods for determining the level of a gene's expression are well-known to those skilled in the art. Such other methods include, for example, reverse transcriptase polymerase chain reaction (RT-PCR) analysis.

The putative DNA sequence complementary to the mRNA transcript of the human c-myb gene has been reported in Majello et a_L. , Proc. Natl. Acad. Sci. U.S.A. 83, 9636-9640 (1986) and in U.S. Patent No. 5,098,890. Majello et al. further disclose the predicted 640 amino acid sequence of the putative c- mvb protein. The initiation codon ATG appears at position 114, preceded by a 5'-untranslated region. The termination codon TGA at position 2034 is followed by a 3'-untranslated region spanning about 1200 nucleotides, which is followed by a poly(A) tail of about 140 nucleotides. The antisense oligonucleotides of the invention may be synthesized by any of the known chemical oligonucleotide synthesis methods. Such methods are generally described, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technolog . 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 advantageously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers. One such device, the Applied Biosystems 380B DNA Synthesizer, utilizes 3-cyanoethyl phosphoramidite chemistry. Since the complete nucleotide synthesis of DNA complementary to the c-mvb mRNA transcript is known, antisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by oligonucleotide synthesis methods known to those skilled in the art.

While any length oligonucleotide may be utilized in the practice of the invention, sequences shorter than 12 bases may be less specific in hybridizing to the target c-myb mRNA, may be more easily destroyed by enzymatic digestion, and may be destabilized by enzymatic digestion. Hence, oligonucleotides having 12 or more nucleotides are preferred. Long sequences, particularly sequences longer than about 40 nucleotides, may be somewhat less effective in inhibiting c-myb translation because of decreased uptake by the target cell. Thus, oligomers of 12-40 nucleotides are preferred, more preferably 15-30 nucleotides, most preferably 18-26 nucleotides. Sequences of 18-21 nucleotides are most particularly preferred.

Oligonucleotides complementary to and hybridizable with any portion of the c-myb mRNA transcript are, in principle, effective for inhibiting translation of the transcript, and capable of inducing the effects herein described. It is believed that translation is most effectively inhibited by blocking the mRNA at a site at or near the initiation codon. Thus, oligonucleotides complementary to the 5'- terminal region of the c-myb mRNA transcript are preferred. It is believed that secondary or tertiary structure which might interfere with hybridization is minimal in this region. Moreover, it has been suggested that sequences that are too distant in the 3'-direction from the initiation site may be less effective in hybridizing the mRNA transcripts because of a "read-through" phenomenon whereby the ribosome is postulated to unravel the antisense/sense duplex to permit translation of the message. See, e.g., Shakin, J. Biochemistry 261. 16018 (1986).

The antisense oligonucleotide is preferably directed to a site at or near the initiation codon for protein synthesis. Oligonucleotides complementary to the c-myb mRNA, including the . initiation codon (the first codon at the 5' end of the translated port: ai of the c-myb transcript, comprising nucleotides 114-116 of the complete transcript) are preferred.

While antisense oligomers complementary to the 5'-terminal region of the c-myb transcript are preferred, particularly the region including the initiation codon, it should be appreciated that useful antisense oligomers are not limited to those complementary to the sequences found in the translated portion (nucleotides 114 to 2031) of the mRNA transcript, but also includes oligomers complementary to nucleotide sequences contained in, or extending into, the 5'-and 3'-untranslated regions. Oligomers whose complementarity extends into the 5'-untranslated region of the c-myb transcript are believed particularly effective in inhibiting c-mvb translation.

Preferred oligonucleotides complementary to the 5'-untranslated region of the transcript include molecules having a nucleotide sequence complementary to a portion of the c-myb mRNA transcript including the cap nucleotide, that is, the nucleotide at the extreme 5'-end of the transcript. The SI nuclease assay procedure of Molecular Cloning. 2nd edition (Sambrook et a_L. , Eds. 1989), pages 7.66-7.70 (incor- porated herein by reference) was essentially followed to map the location of c-myb cap sites using mRNA isolated from the leukemic cell line CCRF-CEM, which expresses high levels of c-mvb mRNA. The longest clearly visible band was located 90 base pairs upstream of the published c-myb cDNA (Majello et al. , Proc. Natl. Acad. Sci. U.S.A.. 83, 9536-9640 (1986)), indicating the putative principle cap site. The position of this site is in perfect agreement with the length of the c-myb cDNA cloned from CCRF-CEM cells (Clarke et al. , Mol. Cell. Biol.. 8, 884-892 (1988)). SI protection assays also revealed faint bands in addition to the main band corresponding to the cap site. These other bands may represent rare or unstable c-myb mRNA transcripts. Multiple sites of transcription initiation are not uncommon in genes such as c-myb which lack a perfect TATAA box. The nucleotide sequence of the mRNA transcript 5'-terminus beginning with the cap nucleotide may be readily established, and antisense oligonucleotides complementary and hybridizable thereto may be prepared.

The following 40-mer oligodeoxynucleotide is complementary to the c-myb mRNA transcript beginning with the initiation codon of the transcript and extending downstream thereof (in the 5' direction): SEQ ID NO:l.

Smaller oligomers based upon the above sequence, in particular, oligomers hybridizable to segments of the c-myb message containing the initial codon, may be utilized. Particularly preferred are the following 26- to 15-mers:

SEQ ID NO:2,

SEQ ID NO:3,

SEQ ID NO:4, SEQ ID NO:5,

SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,

SEQ ID NO:12 and SEQ ID NO:13.

Oligodeoxynucleotides . complementary to the c-myb mRNA transcript beginning with the second codon of the translated portion of the transcript (nucleotides 117-119 of the complete transcript) are another group of preferred oligomers. Such oligomers include, for example, the following 21- to 15-mers:

SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,

SEQ ID NO:19 and SEQ ID NO:20.

The oligonucleotide employed may represent an unmodified oligonucleotide or an oligonucleotide analog. Thus, oligonucleotides hybridizable to the c- myb mRNA transcript finding utility according to the present invention include not only oligomers of the biologically significant native nucleotides, i.e., A, dA, G, dG, C, dC, T and ϋ, but also oligonucleotide species which have been modified for improved stability and/or lipid solubility. For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting an alkyl or alkoxy group for a phosphate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate o 1 i gonuc 1 e o s i de or alkylphosphotriester oligonucleotide. The phosphoro- thioates, in particular, are stable to nuclease cleavage and soluble in lipid. They may be synthesized by known automatic synthesis methods. Non-ionic oligonucleotides such as these are characterized by increased resistance to nuclease hydrolysis and/or increased cellular uptake, while retaining the ability to form . stable complexes with complementary nucleic acid sequences. The alkyl- phosphonates 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. The methylphosphonates, in particular, are preferred.

Methylphosphonate oligomers can be prepared by a variety of methods, both in solution and on insoluble polymer supports (Agrawal and Riftina, Nucl. Acids Res.. 6, 3009-3024 (1979); Miller et a__. , Biochemistry. 18, 5134-5142 (1979), Miller et al. , J. Biol. Chem.. 255, 9659-9665 (1980); Miller et a_L. , Nucl. Acids Res.. 11, 5189-5204 (1983), Miller et al.. Nucl. Acids Res.. 11, 6225-6242 (1983), Miller et al. , Biochemistry, 25, 5092-5097 (1986) ; Engels and Jager, Angew. Chem. Suppl. 912 (1982); Sinha et al.. Tetrahedron Lett. 24, 877-880 (1983); Dorman et al, Tetrahedron, 40, 95-102 (1984) ; Jager and Engels, Tetrahedron Lett.. 25, 1437-1440 (1984); Noble et al. , Nucl. Acids Res.. 12, 3387-3404 (1984); Callahan et al. , Proc. Natl. Acad. Sci. USA. 83, 1617-1621 (1986); Koziolkiewicz et al. , Chemica Scripta. 26, 251-260 (1986) ; Agrawal and Goodchild, Tetrahedron Lett.. 38, 3539-3542 (1987); Lesnikowski et al. , Tetrahedron Lett.. 28, 5535-5538 (1987); Sarin et al. , Proc. Natl. Acad. Sci. USA. 85, 7448-7451 (1988)). The most efficient procedure for preparation of methylphosphonate oligonucleosides involves use of 5 ' -0_-dimethoxytrityldeoxynucleoside-3 ' -0_- diisopropylmethylphosphoramidite intermediates, which are similar to the methoxy or S-cyanoethyl phosphoramidite reagents used to prepare oligodeoxy- ribonucleotides. The methylphosphonate oligomers can be prepared on controlled pore glass polymer supports using an automated DNA synthesizer (Sarin et al., Proc. Natl. Acad. Sci. USA. 85, 7448-7451 (1988)).

Resistance to nuclease digestion may also be achieved by modifying the internucleotide linkage at both the 5' and 3' termini with phosphoroamidites according to the procedure of Dagle et al., Nucl. Acids Res. 18. 4751-4757 (1990).

Suitable nucleotide analogs for preparation of the antisense oligonucleotides described herein include but are not limited to the ethyl or methyl phosphonate analogs disclosed in U.S. Patent No. 4,469,863.

Phosphorothioate oligonucleotides contain a sulfur-for-oxygen substitution in the internucleotide phosphodiester bond. Phosphorothioate oligonucleotides combine the properties of effective hybridization for duplex formation with substantial nuclease resistance, while retaining the water solubility of a charged phosphate analogue. The charge is believed to confer the property of cellular uptake via a receptor (Loke et al. , Proc. Natl. Acad. Sci. U.S.A. 86. 3474-3478 (1989)).

Phosphorothioate modified oligodeoxynu¬ cleotide 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 automatic synthesizer using the phosphoramidite approach.

Furthermore, recent advances in the production of oligoribonucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-0-methylribonucleotides (Inove et al. , Nucleic Acids Res. 15, 6131 (1987) and chimeric oligonucleotides that .are composite RNA-DNA analogues (Inove et al. , FEBS Lett. 215, 327 (1987).

While inhibition of c-myb mRNA translation is possible utilizing either antisense oligoribonucleotides or oligodeoxyribonucleotides, free oligoribonucleotides are more susceptible to enzymatic attack by ribonucleases than oligodeoxyribonucleotides . Hence, oligodeoxyribonucleotides are preferred in the practice of the present invention. Oligo¬ deoxyribonucleotides are further preferred because, upon hybridization with c-myb mRNA, the resulting DNA- RNA hybrid duplex is a substrate for RNase H, which specifically attacks the RNA portion of DNA-RNA hybrid. Degradation of the mRNA strand of the duplex releases the antisense oligodeoxynucleotide strand for hybridization with additional c-mvb messages.

In general, the antisense oligonucleotides of the present invention will have a sequence which is completely complementary to the target portion of the c-myb message. Absolute complementarity is not however required, particularly in larger oligomers. Thus, reference herein to a "nucleotide sequence complementary to at least a portion of the mRNA transcript" of c-myb does not necessarily mean a sequence having 100% complementarity with the transcript. In general, any oligonucleotide having sufficient complementarity to form a stable duplex with c-mvb mRNA is suitable. Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity with the target region of the c-myb message. Generally, the larger the hybridizing oligomer, the more mismatches may be tolerated. More than one mismatch probably will not be tolerated for antisense oligomers of less than about 21 nucleotides. One skilled in the art may readily determine the- degree of mismatching which may be tolerated between any given antisense oligomer and the target c-mvb message sequence, based upon the melting point, and therefore the stability, of the resulting duplex. Melting points of duplexes of a given base pair composition can be readily deter- mined from standard texts, such as Molecular Cloning: A Laboratory Manual. (2nd edition, 1989) , J. Sambrook et al. , eds.

While oligonucleotides capable of stable hybridization with any region of the c-myb message are within the scope of the present invention, oligonucleotides complementary to a region including the initiation codon are believed particularly effective. Particularly preferred are oligonu¬ cleotides hybridizable to a region of the c-myb mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direction) of that codon.

The antisense oligonucleotides of the invention have been observed to inhibit normal human hematopoiesis. However, they inhibit the growth of c- myb colorectal carcinoma cells at a significantly lower concentration than such normal cells. As hereinafter established, normal colon cells do not express detectable levels of c-myb transcript. Their growth would not therefore be inhibited by c-myb antisense at selected dosages fatal to colon carcinoma cells. This pharmaceutically significant differential sensitivity makes the instant oligonucleotides useful in treating colorectal carcinoma.

For therapeutic use, the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liquid vehicles and excipients are conventional and commercially available. Illustrative thereof are distilled water, physiological s^: ...ine, aqueous solution of dextrose, and the like. The c-myb mRNA antisense oligonucleotides are preferably administered parenterally, most preferably intra¬ venously. The vehicle is designed accordingly. For treatment of liver metastases arising from colorectal carcinoma, direct intra-arterial administration of oligonucleotides may be utilized. Other possible routes of oligomer administration include rectal and oral. In addition to administration with conventional carriers, the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides 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 sphin- gomyelin, steroids such as cholesterol, ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. Oligonucleotides have been successfully encapsulated in unilameller liposomes. Reconstituted Sendai virus envelopes have been successfully used to deliver RNA and DNA to cells. Arad et al. , Biochem. Biophy. Acta. 859, 88-94 (1986) . Antisense oligomers have also been delivered in the form of poly(L-lysine) conjugates. Such conjugates are described by Lemaitre et al. , Proc. Natl. Acad. Sci. USA. 84, 648-652 (1987).

The c-mvb antisense oligonucleotides may be administered in a dosage effective for inhibiting the proliferation of colorectal carcinoma cells in the afflicted individual, while maintaining the viability of normal cells. Such amounts may vary depending on the nature and extent of the neoplasm, the particular oligonucleotide utilized, and other factors. The actual dosage administered 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, health and sex of the patient, the route of administration, and other factors. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient. Significant inhibition of colorectal carcinoma cell proliferation has been observed at antisense concentrations of as low as 1 μg/ml for some cell lines. At 40 μg/ml, inhibition was most profound. Concentrations of from about 1 to about 100 μg/ml may be employed, preferably from about 10 μg/ml to abut 100 μg/ml, most preferably from about 40 μg/ml to about 60 μg/ml. The patient should receive a sufficient daily dosage of antisense oligonucleotide to achieve these intercellular concentrations of drug. The daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 1,000 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required. Those skilled in the art should readily be able to determine the optimal dosage in each case.

The present invention is described in greater detail in the following non-limiting examples.

Example 1

Inhibition of Colon Carcinoma Cells with c-myb Antisense Oligonucleotide. LoVo, Colo 205 and HT 29 cell lines, all of which are derived from human colon adenocarcinomas (Trainer et al. , Int. J. Cancer 41, 287-296 (1988)) were utilized in the following experiments. Doxorubicin resistance was induced in a portion of LoVo cells to form the LoVo/Dx subline by prolonged culture in medium containing doxorubicin according to the procedure of Grandi et al. , Br. J. Cancer 54, 515- 518 (1986).

Cells were cultured in Ham's F 12 medium (Gibco) supplemented with 10% fetal calf serum (Biological Industries) , pretreated for 15 min. at 65°C with 2mM glutamine and antibiotics (MA Bioprod- ucts) . 3 x 10⁴ cells were seeded in 24 well plates (Costar) and c-mvb sense(S) (SEQ ID NO:21) or antisense (AS) oligodeoxynucleotides (SEQ ID NO:17) were added to the culture medium for 3 days at 40μg/ml, 30μg/ml and 20μg/ml, respectively. The S and AS oligonucleotides correspond to codons 2-7 of the translated c-myb message. Cells were exposed to three different decreasing doses of AS to obtain concentrations which were 1/3, 1/10 and 1/40 of the initial dosage described above (40 μg/ml) . Sense oligomers were only given at the highest dose. Control cells were cultured in the same conditions without oligodeoxynucleotides. After 3 days, cells were recovered by mechanical detachment, counted and analyzed for viability by trypan blue dye exclusion. To determine the cell proliferation rate, 3 x 10⁴ S- and AS-treated and untreated cells were washed after treatment with oligodeoxynucleotides, pulsed with ⁸H- TdR (1 μCi/well) and incubated for 6 hours at 37^*C. Cells were then washed and fixed on glass fiber dishes by using a cell-harvester apparatus (Titertek Cell Harvester 550, Flow Laboratories) . Radioactivity was measured using a beta counter. Non-specific radioactivity was measured in the culture medium and eliminated from the counts.

As shown in Figure 1A, the number of proliferating LoVo, LoVo/Dx and Colo 205 cells exposed to the highest dose, of c-myb AS oligodeoxynucleotides for 3 days was reduced by 55%, 56% and 73% as compared to cells grown in culture medium alone. ³H-TdR incorporation was reduced by 84% in LoVo, 83% in LoVo/Dx and 68% in Colo 205 cells after exposure to AS oligodeoxynucleotides. (Fig. IB) . No differences in proliferative activity were detected in untreated or c-myb sense- treated cells. Proliferation of HT 29 cells, in which the expression of c-myb mRNA was not detected, did not appear to be significantly affected by c-myb AS treatment. According to Figure 2, LoVo/Dx and Colo 205 cells were not inhibited by the lowest dose of AS oligodeoxynucleotide which, instead, was still effective on LoVo cells. Thus, lowering the AS dosage may result in dose-dependent differential inhibition. The inhibition of cell proliferation in the various cell lines correlated with c-myb mRNA levels, as described in Example 2. Example 2 Measurement of c-mvb Expression Steady-state c-myb mRNA levels were measured by Northern blot analysis of LoVo, LoVo/Dx, Colo 205 and HT 29 human carcinoma cell lines as follows. Simultaneous isolation of RNA and DNA from the above- identified colon carcinoma cell lines, and from fresh normal colonic mucosa, was performed by the guanidine isothiocyanate/cesium chloride method. (Davis et al. , Basic Method in Molecular Biology (Elsevier, New York, 1986) . Cells were dissolved in 4M guanidine isothiocyanate/25mM sodium citrate/0.5% Sarkosyl/ 0.7% /3-mercaptoethanol by vortexing. Cell lysate was loaded on a cesium chloride cushion (4.95 M CsCl/0.1 M sodium acetate/5mM EDTA) and centrifuged 16 hours at 35000 rpm at 25°C. Total RNA was recovered from the pellet at the bottom of the tube upon ultracentri- fugation. Total RNA was extracted twice with chloroform/isobutanol and ethanol- precipitated. Total RNA (20μg) was denatured, separated on 1% agarose/formaldehyde gel and transferred to nylon filters (Hybond N, A ersham) . Nucleic acids were bound to filters at 80°C for 2 hrs. Prehybridization and Northern blot hybridization were performed as described by Colombo et al. , Immunogenetics. 26, 99- 104 (1987). Filters were washed twice in 2X SSC/0.1% SDS at room temperature for 15 min. and then two or three times in 0.2X SSC/1.0% SDS at 52°C for 30 min. To quantitate c-myb expression, densitometric scanning of the Northern blots was performed on a LKB Ultroscan XL.

The above Northern blot hybridization revealed that c-mvb was expressed in both LoVo and LoVo/Dx cell lines, and overexpressed in Colo 205

(probably due to gene amplification) , while HT 29 as well as normal colonic mucosa did not show detectable level of transcript at an exposure time which was never longer than six days. Densitometric scanning of the Northern blot revealed that c-myb expression was 15 times higher in LoVo/Dx than in LoVo. The greater abundance of c-myb mRNA in LoVo/Dx than in LoVo cells, appeared to correlate with the higher proliferative activity of this cell line, as demonstrated by the above ΘH-TdR incorporation assay.

Example 3 Antisense Inhibition of c-mvb Expression Detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Inhibition of c-myb expression in AS-treated

LoVo/Dx cells was demonstrated by RT-PCR and Southern blotting of the PCR products.

From LoVo/Dx total RNA, a 234 nucleotide segment corresponding to the 3'-untranslated region of c-myb mRNA from nucleotides 2254-2487 was generated as follows by means of reverse transcription and PCR amplification with synthetic human c-myb specific primers (Caraciolo et al. , J. Clin. Invest.. 41, 287- 296 (1988)). Total RNA was extracted from 3-8 x 10⁵ LoVo/Dx cells treated with c-myb oligodeoxynucleotide as above. RNA was precipitated with 20 μg of E. coli tRNA and resuspended in lx reverse transcriptase buffer (BRL) ; 100 ng of 3' c-myb primer (synthetically prepared) was annealed at 55°C for 90 min. and the first strand of cDNA was synthesized using 400 U of Moloney murine leukemia virus reverse transcriptase (BRL) at 37°C for 1 hr. The reaction was stopped and the mixture diluted in lx Thermus aquaticus (Taq) polymerase buffer. 5' and 3' primers were added to a final concentration of 2 ng/ 1 each, together with 2.5 U of Taq polymerase (Cetus) for amplification of the cDNA fragments. Sixty cycles of PCR were performed in a Perkin Elmer Thermal Cycler by annealing at 55°C, synthesizing at 72^βC and denaturing at 95°C. Positive and negative controls were used to exclude amplification of contaminating sequences. The PCR products were separated on a 2% Nusieve agarose gel and transferred to a nylon filter (Hybond N, Amersham) by alkaline blotting. The resulting blot was hybridized at 50°C with a 50-mer c-myb synthetic oligonucleotide probe (made by a DNA synthesizer) complementary to c-myb cDNA from nucleotides 2,351 to 2,400. The probe was 5'end-labelled with T4 polynucleotide kinase (Promega Biotech, Madison, WI) and γ-³²P-ATP. After hybridization, the filters were washed in 2X SSC/0.1% SDS at room temperature for 10 min. and once at 50"C for 30 min. before overnight exposure to X-ray films at 80^βC.

Analysis of c-myb mRNA levels in the oligodeoxynucleotide-treated and untreated LoVo/DX cells revealed the presence of c-myb transcripts in untreated or c-myb sense-treated cells but not in LoVo/Dx cells exposed to c-myb antisense oligodeoxynucleotide. 2-Microglobulin mRNA levels were constant in all the samples. This demonstrates the specific inhibition of gene expression by anti¬ sense oligodeoxynucleotides. In LoVo/Dx cells exposed to c-myb antisense oligonucleotides, ΘH-thymidine incorporation was more sharply inhibited than cell proliferation, suggesting that inhibition of c-myb expression prevented DNA synthesis in most colon carcinoma cells.

The data presented herein indicate that c- myb gene function is required for proliferation of colon carcinoma cells expressing c-myb. The role of c-myb is thus not limited to cells of hematopoietic origin. In fact, the proliferation of colon carcinoma cells expressing c-myb can be effectively suppressed with antisense oligonucleotides. The dose of antisense exerting an inhibitory effect is related to the level of c-myb mRNA expression, proliferation of colon carcinoma cells, and therefore the attendant tiαmor growth and disease progression, is maintained by high levels of c-myb expression.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: Temple University - Of The Commonwealth System of Higher Education

(ii) TITLE OF INVENTION: Treatment of Colon Carcinoma with Antisense Oligonucleotides to c-myb Proto-oncogene.

(iϋ) NUMBER OF SEQUENCES: 21 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Temple University - Of The Commonwealth System of Higher Education

(B) STREET: 406 University Services Building

(C) CITY: Philadelphia (D) STATE: Pennsylvania

(E) COUNTRY: U.S.A.

(F) ZIP: 19122

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.50 inch, 720 Kb (B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WordPerfect 5.1 (Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Unknown (B) FILING DATE: Concurrently herewith (C) CLASSIFICATION: Unknown (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/704,862

(B) FILING DATE: May 23, 1991 (Viϋ) ATTORNEY/AGENT INFORMATION:

(A) NAME: Monaco, Daniel A.

(B) REGISTRATION NUMBER: 30,480

(C) REFERENCE/DOCKET NUMBER: 6056-141 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (215) 568-8383 (B) TELEFAX: (215) 568-5549 (C) TELEX: None

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CGTCACTGCT ATATATGCTG TGCCGGGGTC TTCGGGCCAT 40

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 Nucleotides (B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATGCTGTGCC GGGGTCTTCG GGCCAT 26

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 Nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGCTGTGCCG GGGTCTTCGG GCCAT 25 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 Nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCTGTGCCGG GGTCTTCGGG CCAT 24

(2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTGTGCCGGG GTCTTCGGGC CAT 23

(2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TGTGCCGGGG TCTTCGGGCC AT 22

(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GTGCCGGGGT CTTCGGGCCA T 21 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 Nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TGCCGGGGTC TTCGGGCCAT 20

(2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCCGGGGTCT TCGGGCCAT 19

(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CCGGGGTCTT CGGGCCAT 18

(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 Nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:11: CGGGGTCTTC GGGCCAT 17

(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGGTCTTCG GGCCAT 16

(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGGTCTTCGG GCCAT 15

(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCTGTGCCGG GGTCTTCGGG C 21 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 Nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:15: CTGTGCCGGG GTCTTCGGGC 20

(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TGTGCCGGGG TCTTCGGGC 19

(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GTGCCGGGGT CTTCGGGC 18

(2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 Nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:18: TGCCGGGGTC TTCGGGC 17

(2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GCCGGGGTCT TCGGGC 16

(2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CCGGGGTCTT CGGGC 15

(2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCCCGAAGAC CCCGGCAC 18

Claims

1. A method for the treatment of a colorectal carcinoma which expressesthe c-myb gene, characterized by administering to an individual in need of such treatment an effective amount of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene, said oligonucleot- ide being hybridizable to said mRNA transcript.

2. A method according to claim 1 wherein the oligonucleotide is at least a 12-mer.

3. A method according to claim 2 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.

4. A method according to claim 2 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.

5. A method according to claim 2 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-mvb mRNA transcript including the translation initiation codon of said transcript and/or the codon immediately downstream from the initiation codon.

6. A method according to claim 2 wherein the oligonucleotide comprises from a 12-mer to a 40- mer oligodeoxynucleotide.

7. A method according to claim 6 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.

8. A method according to claim 6 wherein the oligonucleotide is from a 15-mer to 30-mer.

9. A method according to claim 6 wherein the oligonucleotide is from a 18-mer to 26-mer.

10. A method according to claim 8 wherein the oligomer is from a 18-mer to 21-mer.

11. A method according to claim 8 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence selected from the group consisting of:

SEQ ID NO:2,

SEQ ID NO:3,

SEQ ID NO:4,. SEQ ID NO:5,

SEQ ID NO:6,

SEQ ID NO:7,

SEQ ID NO:8,

SEQ ID NO:9, SEQ ID NO:10,

SEQ ID NO:11,

SEQ ID NO:12 and

SEQ ID NO:13.

12. A method according to claim 9 wherein the oligodeoxynucleotide has a nucleotide sequence corresponding to SEQ ID NO:10.

13. A method according to claim 8 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence selected from the group consisting of

SEQ ID NO:14,

SEQ ID NO:15,

SEQ ID NO:16, SEQ ID NO:17,

SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.

14. A method according to claim 13 wherein the oligonucleotide has a nucleotide sequence corresponding to SEQ ID NO:17.

15. The use of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene, said oligonucleotide being hybridizable to said mRNA transcript, for the manufacture of a medicament for treatment of a colorectal carcinoma which expresses the c-myb gene.

16. Use according to claim 15 wherein the oligonucleotide is at least a 12-mer.

17. Use according to claim 16 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.

18. Use according to claim 16 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the c-myb mRNA lying within about 40 nucleotides of the translation initiation codon.

19. Use according to claim 16 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the c-myb mRNA transcript including the translation initiation codon of said transcript and/or the codon immediately downstream from the initiation codon.

20. Use according to claim 16 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.

21. Use according to claim 20 wherein the oligonucleotide is a methylphosphonate oligonucleoside or phosphorothioate oligonucleotide.

22. Use according to claim 20 wherein the oligonucleotide is from a 15-mer to 30-mer.

23. Use according to claim 2 wherein the oligonucleotide is from a 18-mer to 26-mer.

24. Use according to claim 3 wherein the oligomer is from a 18-mer to 21-mer.

25. Use according to claim 2 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence selected from the group consisting of:

SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

26. Use according to claim 5 wherein the oligodeoxynucleotide has a nucleotide sequence corresponding to SEQ ID NO:10.

27. Use according to claim 2 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence selected from the group consisting of

SEQ ID NO:14,

SEQ ID NO:15,

SEQ ID NO:16, SEQ ID NO:17,

SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.

28. Use according to claim 27 wherein the oligonucleotide has a nucleotide sequence corresponding to SEQ ID NO: 17.