WO1994004137A1

WO1994004137A1 - Antisense oligonucleotides to cyclin d1 proto-oncogene

Info

Publication number: WO1994004137A1
Application number: PCT/US1993/007892
Authority: WO
Inventors: Bruno Calabretta
Original assignee: Thomas Jefferson University
Priority date: 1992-08-25
Filing date: 1993-08-23
Publication date: 1994-03-03
Also published as: AU5086393A

Abstract

Oligonucleotides are provided having a nucleotide sequence complementary to at least a portion of the mRNA transcript of the cyclin D1 gene. These 'antisense' oligonucleotides are hybridizable to the cyclin D1 mRNA transcript. Such oligonucleotides are useful in treating neoplastic diseases characterized by amplification of the cyclin D1 gene, or the activation of cyclin D1 gene expression.

Description

ANTISENSE OLIGONUCLEOTIDES TO CYCLIN DI PROTO-ONCOGENE

Field of the Invention The invention relates to antisense oligonucleotides to proto-oncogenes, in particular antisense oligonucleotides to the cyclin DI gene, and the use of such oligonucleotides as antineoplastic agents.

Reference to Government Grant

The invention described hereinwas made in part with government support under grant CA46782 awarded by National Institutes of Health. The government has certain rights in the invention.

Background of the Invention The human gene cyclin DI was independently discovered by several groups of researchers. It is therefore known by several different names: "cyclin DI", "cyclin D"; "PRAD1" and "bcl-1". All refer to the same gene located on chromosome llql3.

Postembryonic cell division is primarily reg¬ ulated during the first gap phase (Gl) of the cell cycle, when cells have completed division and are preparing to replicate their chromosomal DNA. DNA synthesis occurs in the next phase, the S phase. The cyclins, a class of molecules which were originally identified by their cyclic accumulation and destruction at defined points in the cell cycle, bind to inactive p34^cdc2 protein kinase and are essential for kinase activation. The cyclins can be divided into three families, on the basis of their kinetics of oscillation across the cell cycle, their amino acid sequences and, in some cases, genetic experiments in yeast that have determined when their functions are needed. The B-type "mitotic" cyclins drive cells into mitosis. The A-type cyclins may act earlier in the cell cycle. The Gl cyclins are believed to have analogous functions by interacting with cdc2 homologues, driving cells into the S phase. The A, B and Gl cyclins may act as stage-specific regulators of progress across the cell cycle by conferring selective substrate specificity on cdc2 kinase or by selectively targeting cdc2 to different cellular compartments. In a study of CYL1, the murine homologue cyclin DI (Matsushime et al. , Cell. 65, 701-713 (1991)), the protein was shown to be expressed in the Gl phase of the cell cycle in association with a cdc2-related protein.

The human cyclin DI has been isolated, cloned and sequenced as "PRAD1" by Motokura et -al. , Nature 350, 512-14 (1991) ; as "cyclin DI" by Xiong et al. , Cell 65, 691-699 (1991); as "cyclin D" by Lew et al. , Cell 66, 1197-1206 (1991) ; and as "bcl-1" by Withers et al. , Mol. Cell. Biol. 11, 4846-4853 (1991) . The human gene encodes a predicted protein of 295 amino acids. Hereinafter, the human gene will be referred to as "cyclin DI".

Expression of cyclin DI has been reported in a glioblastoma cell line (Xiong et aJL. , supra) ; in certain parathyroid tumors as a result of genomic rearrangement (Rosenberg et al. , Oncogene 6, 449-453 (1991)); and in squamous cell and mammary carcinomas (Lammie et al., Onco¬ gene 6, 439-444 (1991) ; and in primary esophageal tumors (Jiang et al.. Cancer Res.52.2980-2983 (1992)). However, there has been no direct demonstration that cyclin DI can transform cells, or that inhibition of cyclin DI expression will curb cell proliferation. STj-mmarv of the Invention

The invention provides antisense oligonucleotides and pharmaceutical compositions thereof with pharmaceuti¬ cally acceptable carriers. Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human cyclin DI gene. The oligonucleotide is hybridizable to the mRNA transcript. The oligonucleotide is at least an 8-mer oligonucleotide, that is, an oligomer containing at least 8 nucleotide residues, and contains up to 50 nucleotides. In particu¬ lar, 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 statis¬ tically 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. Most preferably, the oligo¬ nucleotide is a 15- to 30-mer oligodeoxynucleotide, more advantageously an 18- to 26-mer.

While in principle oligonucleotides having a sequence complementary to any region of the cyclin DI mRNA find utility in the present invention, oligonucleo¬ tides complementary to a portion of the cyclin DI mRNA transcript including the translation initiation codon are particularly preferred. Also preferred are oligonucleotides complementary to a portion of the cyclin DI mRNA transcript lying within about 50 nucleotides (preferably within about 40 nucleotides) upstream (the 5' direction) , or about 50 (preferably 40) nucleotides downstream (the 3' direction) from the translation initiation codon. The invention provides a method of treating neoplastic disease in vivo or ex vivo comprising adminis¬ tering to an individual or cells harvested from the individual an effective amount of cyclin DI antisense oligonucleotide. The neoplastic diseases treatable include those diseases in which the cyclin DI gene is amplified, or in which expression of the gene is activated.

The invention is also a method for purging bone marrow of metastasized neoplastic cells. Bone marrow aspirated from an inflicted individual is treated with an effective amount of cyclin DI antisense oligonucleotide, and the thus-treated cells are then returned to the body of the afflicted individual.

According to another embodiment, the invention relates to an artificially-constructed gene comprising a transcriptional promotor segment and a segment containing cyclin DI DNA in inverted orientation such that transcrip¬ tion of the artificially-constructed gene produces RNA complementary to at least a portion of the mRNA transcript of the cyclin DI gene. The gene may be introduced into neoplastic cells which are characterized by the amplifica¬ tion of the cyclin DI gene or activation of cyclin DI expression to inhibit the proliferation of those cells. The artificially-constructed gene may be introduced into the neoplastic cells by, for example, transfection, transduction with a viral vector, or microinjection.

As used in the herein specification and appended claims, unless otherwise indicated, the term "oligonucleo¬ tide" includes both oligomers of ribonucleotides, i.e., oligoribonucleotides, and oligomers of deoxyribonucleo- tides, i.e., oligodeoxyribonucleotides (also referred to herein as "oligodeoxynucleotides") . Oligodeoxynucleo- tides are preferred. As used herein, unless otherwise indicated, the term "oligonucleotide" also includes oligomers which may be large enough to be termed "polynucleotides".

The terms "oligonucleotide" and "oligodeoxynuc- leotide" include not only oligomers and polymers of the common biologically significant nucleotides, i.e., the nucleotides adenine ("A") , deoxyadenine ("dA") , guanine ("G") , deoxyguanine ("dG") , cytosine ("C") , deoxycytosine ("dC"), thymine ("T") and uracil ("U") , but also include oligomers and polymers hybridizable to the cyclin DI mRNA transcript which may contain other nucleotides. Likewise, the terms "oligonucleotide" and "oligodeoxynucleotide" includes oligomers and polymers wherein one or more purine orpyrimidine moieties, sugar moieties or internucleotide linkages is chemically modified. The term "oligonucleo¬ tide" 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

-O - P - 0~

as opposed to the phosphodiester group

0

II -0 - P - 0~

I

0^"~

which is characteristic of unmodified oligonucleotides, By "alkylphosphonate oligonucleoside" is meant an oligonucleotide wherein one or more of the internucleo¬ tide linkages is an alkylphosphonate group,

O II

~0 - P - 0-

I

R wherein R is an alkyl group, preferably methyl or ethyl.

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

The term "cyclin DI mRNA transcript" means the presently known mRNA transcript of the human cyclin DI gene and all variations thereof, or any further transcripts which may be elucidated.

Description of the Figures

Fig. 1 shows a 14-day culture of cells transfec- ted with a construct comprising a fragment of cyclin DI cDNA in antisense orientation in a pSV40-polylinker vector.

Fig. 2 shows a 14-day culture of cells transfec- ted with pSV40-polylinker vector without the cyclin DI insert (control) .

Detailed Description of the Invention

The putative DNA sequence complementary to the mRNA transcript of the human cyclin DI gene has been reported by Motokura et al. , Nature 350. 512-14 (1991), Xiong et al. , Cell 65, 691-699 (1991) , Lew et al. , Cell 66, 1197-1206 (1991), and Withers et al. , Mol. Cell. Biol. 11, 4846-4853 (1991) , the entire disclosures of all of which are incorporated herein by reference. These investigators further disclose the predicted 295 amino acid sequence of the putative cyclin DI protein. The initiation codon ATG is preceded by a 5'-untrans- lated region of about 145 nucleotides. The termination codon TGA is followed by a 3'-untranslated region span¬ ning about 3,000 nucleotides, including a consensus polyadenylation signal sequence at the 3' end. The length of the 3'-untranslated region varies depending upon the cell source.

The antisense oligonucleotides of the inven¬ tion, which are complementary to the cyclin DI mRNA, may be synthesized by any of the known chemical oligonucleo¬ tide synthesis methods. Such methods are generally described, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technology. VCH Verlagsges- ellschaft mbH (Ibelgaufts trans. 1987) . The antisense oligonucleotides are most advantageously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers. One such device, the Applied Biosystems 380B DNA Synthesizer, utilizes 3-cyanoethyl phosphoramidite chemistry.

Since the complete nucleotide synthesis of DNA complementary to the cyclin DI mRNA transcript is known, antisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by oligonucleo¬ tide synthesis methods known to those skilled in the art.

While any length oligonucleotide may be uti- lized in the practice of the invention, sequences short¬ er than 12 nucleotides, and in particular sequences shorter than 8 nucleotides, may be less specific in hybridizing to the target cyclin DI mRNA, may be more easily destroyed by enzymatic digestion, and may be destabilized by enzymatic digestion. Hence, oligo- nucleotides having 12 or more nucleotides are preferred. Long sequences, particularly sequences longer than about 50 nucleotides, may be somewhat less effec¬ tive in inhibiting cyclin DI translation because of decreased uptake by the target cell. Thus, oligomers of 12-40 nucleotides are preferred, more preferably 15- 30 nucleotides, most preferably 18-26 nucleotides. While sequences of 18-21 nucleotides are most particu¬ larly preferred for unmodified oligonucleotides, slightly longer chains of up to about 26 nucleotides, are preferred for modified oligonucleotides such as phosphorothioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides.

Oligonucleotides complementary to and hybrid- izable with any portion of the cyclin DI 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 cyclin DI mRNA transcript are preferred.

The antisense oligonucleotide is preferably directed to a site at or near the initiation codon for protein synthesis. Oligonucleotides complementary to the cyclin DI mRNA, including the initiation codon (the first codon at the 5' end of the translated portion of the cyclin DI transcript) are preferred.

While antisense oligomers complementary to the 5'-terminal region of the cyclin DI transcript are pre¬ ferred, 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 of the mRNA transcript, but also includes oligomers complementary to nucleotide sequences contained in, or extending into, the 5'-and 3'-untranslated regions.

The following 50-mer oligodeoxynucleotide is complementary to the cyclin DI mRNA transcript beginning with the initiation codon of the transcript and extend¬ ing downstream thereof (in the 3' direction): SEQ ID NO:l.

Smaller oligomers based upon the above se¬ quence, in particular, oligomers hybridizable to seg- ments of the cyclin DI message containing the initiation codon, may be utilized. Particularly preferred are oligomers containing at least 12 nucleotides, having a nucleotide sequence corresponding to a portion of SEQ ID NO:l. The oligonucleotide employed may represent an unmodified or modified oligonucleotide. Thus, oligo¬ nucleotides hybridizable to the cyclin Dl mRNA tran¬ script finding utility according to the present inven¬ tion include not only oligomers of the biologically sig- nificant native nucleotides, i.e., A, dA, G, dG, C, dC, T and U, but also oligonucleotide species which have been modified for improved stability and/or lipid solu¬ bility. For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion re- suits by substituting an alkyl group or alkoxy group for a phosphate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate oligonucleoside or alkylphosphotriester oligonucleotide. Non-ionic oligonucleotides such as these are characterized by increased resistance to nuclease hydrolysis and/or in¬ creased cellular uptake, while retaining the ability to form stable complexes with complementary nucleic acid sequences. The alkylphosphonates in particular, are stable to nuclease cleavage and soluble in lipid. The preparation of alkylphosphonate oligonucleosides is disclosed in U.S. Patent 4,469,863.

Methylphosphonate oligomers can be prepared by a variety of methods, both in solution and on insolu- ble polymer supports (Agrawal and Riftina, Nucl. Acids Res. , 6, 3009-3024 (1979); Miller et al. , Biochemistry, 18, 5134-5142 (1979), Miller et aJL. , J. Biol. Chem.. 255, 9659-9665 (1980); Miller et al. , Nucl. Acids Res.. 11, 5189-5204 (1983), Miller et al. , Nucl. Acids Res.. 11, 6225-6242 (1983), Miller et al. , Biochemistry. 25, 5092-5097 (1986) ; Engels and Jager, Angew. Chem. Suppl. 912 (1982); Sinha et al. , Tetrahedron Lett. 24. 877-880 (1983); Dorman et al, Tetrahedron. 40, 95-102 (1984); Jager and Engels, Tetrahedron Lett.. 25, 1437-1440 (1984); Noble et al., Nucl. Acids Res.. 12, 3387-3404 (1984); Callahan et al. , Proc. Natl. Acad. Sci. USA. 83, 1617-1621 (1986); Koziolkiewicz et aJL. , Chemica Scripta. 26, 251-260 (1986) ; Agrawal and Goodchild, Tetrahedron Lett.. 38, 3539-3542 (1987); Lesnikowski et al. , Tetra- hedron Lett.. 28, 5535-5538 (1987); Sarin et al. , Proc. Natl. Acad. Sci. USA. 85, 7448-7451 (1988)).

The most efficient procedure for preparation of methylphosphonate oligonucleosides involves use of 5'-0-dimethoxytrityldeoxynucleoside-3'-O-diisopropyl- methylphosphoramidite intermediates, which are similar to the methoxy or 3-cyanoethyl phosphoramidite reagents used to prepare oligodeoxyribonucleotides. The methylphosphonate oligomers can be prepared on con¬ trolled 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 ac- cording to the procedure of Dagle et al., Nucl. Acids Res. 18, 4751-4757 (1990).

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

Phosphorothioate oligodeoxynucleotide are described by LaPlanche, et aJL. , Nucleic Acids Research 14, 9081 (1986) and by Stec et a_l. , J. Am. Chem. Soc. 106, 6077 (1984). The general synthetic method for phosphorothioate oligonucleotides was modified by Stein et al., Nucl. Acids Res. , 16, 3209-3221 (1988), so that these compounds may readily be synthesized on an auto¬ matic synthesizer using the phosphoramidite approach. Furthermore, recent advances in the production of oligoribonucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-0-methylribonucleotides (Inove et al., Nucleic Acids Res. 15, 6131 (1987) and chimeric oligonucleotides that are composite RNA-DNA analogues (Inove et aJL. , FEBS Lett. 215. 327 (1987).

While inhibition of cyclin DI mRNA translation is possible utilizing either antisense oligoribonucleo- tides or oligodeoxyribonucleotides, free oligoribo- nucleotides are more susceptible to enzymatic attack by ribonucleases than oligodeoxyribonucleotides. Hence, oligodeoxyribonucleotides are preferred in the practice of the present invention. Oligodeoxyribonucleotides are further preferred because, upon hybridization with cyclin DI mRNA, the resulting DNA-RNA hybrid duplex is a substrate for RNase H, which specifically attacks the RNA portion of DNA-RNA hybrid. Degradation of the mRNA strand of the duplex releases the antisense oligodeoxy- nucleotide strand for hybridization with additional cyclin DI messages.

In general, the antisense oligonucleotides used in the method of the present invention will have a sequence which is completely complementary to the target portion of the cyclin DI message. Absolute com¬ plementarity is not however required, particularly in larger oligomers. Thus, reference herein to a "nucleo¬ tide sequence complementary to at least a portion of the mRNA transcript" of cyclin DI does not necessarily mean a sequence having 100% complementarity with the tran¬ script. In general, any oligonucleotide having suffi¬ cient complementarity to form a stable duplex with cy¬ clin DI mRNA, that is, an oligonucleotide which is "hy¬ bridizable", is suitable. Stable duplex formation de- pends on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity with the target region of the cyclin DI 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 read¬ ily determine the degree of mismatching which may be tolerated between any given antisense oligomer and the target cyclin DI message sequence, based upon the melt- ing point, and therefore the stability, of the resulting duplex. Melting points of duplexes of a given base pair composition can be readily determined from standard texts, such as Molecular Cloning: A Laboratory Manual. (2nd edition, 1989), J. Sambrook et aJL. , eds. While oligonucleotides capable of stable hy¬ bridization with any region of the cyclin DI message are within the scope of the present invention, oligonucleotides complementary to a region including the initiation codon are believed particularly effective. Particularlypreferred are oligonucleotides hybridizable to a region of the cyclin DI mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direction) of that codon.

For therapeutic use, the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liquid vehicles and excipients are conventional and commercially available. Illustrative thereof are dis¬ tilled water, physiological saline, aqueous solution of dextrose, and the like. The cyclin DI mRNA antisense oligonucleotides are preferably administered parenter- ally, most preferably intravenously. The vehicle is designed accordingly. Alternatively, oligonucleotide may be administered subcutaneously via controlled re¬ lease dosage forms.

In addition to administration with convention- al carriers, the antisense oligonucleotides may be ad¬ ministered 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 sus¬ pension. The hydrophobic layer, generally but not ex¬ clusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydro- phobic nature. Oligonucleotides have been successfully encapsulated in unilamellar liposomes.

Reconstituted Sendai virus envelopes have been successfully used to deliver RNA and DNA to cells. Arad et ajL. , Biochem. Biophv. Acta. 859, 88-94 (1986).

The oligonucleotides may be conjugated to poly(L-lysine) to increase cell penetration. Such con¬ jugates are described by Lemaitre et al. , Proc. Natl. Acad. Sci. USA. 84, 648-652 (1987). The procedure re¬ quires that the 3'-terminal nucleotide be a ribonucleo- tide. The resulting aldehyde groups are then randomly coupled to the epsilon-amino groups of lysine residues of poly(L-lysine) by Schiff base formation, and then reduced with sodium cyanoborohydride. This procedure converts the 3'-terminal ribose ring into a morpholine structure antisense oligomers.

The oligonucleotides may be conjugated for therapeutic administration to ligand-binding molecules which recognize cell-surface molecules, such as accord¬ ing to International Patent Application WO 91/04753. In particular, transferrin-polylysine-oligonucleotide complexes may be prepared for uptake by cells expressing high levels of transferrin receptor. The preparation of such complexes as carriers of oligonucleotide uptake into cells is described by Wagner et al- , Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). Inhibition of leukemia cell proliferation by transferrin receptor- mediated uptake of c-myb antisense oligonucleotides conjugated to transferrin was demonstrated by Citro et al. , Proc. Natl. Acad. Sci. USA 89, 7031-7035 (1992).

The disorders treatable with the antisense oligonucleotides of the invention include neoplastic diseases wherein the cyclin DI gene is amplified, that is, the copy number of the gene is enhanced above normal levels. The disorders treatable with oligonucleotide therapy also include those disorders characterized by the activation of cyclin DI expression, signalled by the appearance of cyclin DI mRNA transcripts and/or the 295 amino acid protein product. Cyclin DI gene amplifica¬ tion expression may be assayed by conventional probing techniques, such as described by Lammie etal., Oncogene 6, 439-444 (1991). Briefly, tumor DNA is digested with a restriction enzyme, e.g. PstI, and fractionated by electrophoresis on a 0.8% agarose gel for Southern blot analysis. After transfer to the appropriate membrane, the tumor DNA is hybridized to a radiolabelled DNA frag¬ ment of cyclin DI. As a control, DNA of contiguous normal tissue is also analyzed. Amplification is as- sessed relative to the copy number for the same gene in normal cells from the contiguous normal tissue. In a similar fashion, the level of cyclin DI expression is determined by probing total cellular RNA from tumor cells with a complementary probe for cyclin DI mRNA. Total RNA from the tumor cells is fractionated in a glyoxal/agarose gel, transferred to nylon and hybridized to an appropriately labelled nucleic acid probe for cyclin DI mRNA. See Lammie et aj.. , supra. The number of cyclin DI mRNA transcripts found in the tumor cells is compared to that found in normal cells from the same tissue.

At least a 10-fold amplification of cyclin DI in patient neoplastic cells over normal cells from the same tissue would be indicative that the patient's di- sease would be susceptible to treatment by cyclin DI antisense. Similarly, an at least 10-fold increase in cyclin DI expression in neoplastic cells over expression in normal cells from the same tissue would indicate that the disease would respond to antisense treatment. These thresholds are based upon correlations between the level of gene amplification/expression and the extent of the disease state for various other oncogenes. See, for example, Slamon et al. , Science 235. 177-182 (1988) and Science. 244, 707-712 (1989). (Correlation between erb- b2 amplification/expression and breast or ovarian can¬ cer) ; Alitalo et al. , Advances in Cancer Research. 47, 235-282 (1986) .

Disease conditions characterized by cyclin DI gene amplification and/or activated expression include, for example, malignant melanoma, neuroectodermal cancers such as neuroblastoma and neuroepithelioma, esophageal cancer, breast cancer, squamous cell cancers, parathy¬ roid adenomas, and leukemia and lymphoma, particularly leukemias and lymphomas characterized by the t(ll;14) (ql3;q32) chromosomal translocation. Cyclin DI expression is deregulated in leukemias with this trans- location, which has been associated with human B-lympho- cytic malignancy. Similarly, cyclin DI expression is deregulated in parathyroid tumors as a result of a chro- mosomal rearrangement that juxtaposes it to the enhancer of the parathyroid hormone gene. See Motokura et al.. Nature 350, 512-515 (1991).

A preferred method of administration of ol¬ igonucleotide comprises either regional or systemic per- fusion, as is appropriate. According to a method of regional perfusion, the afferent and efferent vessels supplying the extremity containing the lesion are iso¬ lated and connected to a low-flow perfusion pump in continuity with an oxygenator and a heat exchanger. The iliac vessels may be used for perfusion of the lower extremity. The axillary vessels are cannulated high in the axilla for upper extremity lesions. Oligonucleotide is added to the perfusion circuit, and the perfusion is continued for an appropriate time period, e.g., one hour. Perfusion rates of from 100 to 150 ml/minute may be employed for lower extremity lesions, while half that rate should be employed for upper extremity lesions. Systemic heparinization may be used throughout the per¬ fusion, and reversed after the perfusion is complete. This isolation perfusion technique permits administra¬ tion of higher doses of chemotherapeutic agent than would otherwise be tolerated upon infusion into the arterial or venous systemic circulation.

For systemic infusion, the oligonucleotides are preferably delivered via a central venous catheter, which is connected to an appropriate continuous infusion device. Indwelling catheters provide long term access to the intravenous circulation for frequent administra¬ tion of drugs over extended time periods. They are generally surgically inserted into the external cephalic or internal jugular vein under general or local anesthe¬ sia. The subclavian vein is another common site of catheterization. The infuser pump may be external, or may form part of an entirely implantable central venous system such as the INFUSAPORT system available from Infusaid Corp. , Norwood, MA and the PORT-A-CATH system available from Pharmacia Laboratories, Piscataway, NJ. These devices are implanted into a subcutaneous pocket under local anesthesia. A catheter, connected to the pump injection port, is threaded through the subclavian vein to the superior vena cava. The implant contains a supply of oligonucleotide in a reservoir which may be replenished as needed by injection of additional drug from a hypodermic needle through a self-sealing dia- phragm in the reservoir. Completely implantable infus¬ ers are preferred, as they are generally well accepted by patients because of the convenience, ease of mainten¬ ance and cosmetic advantage of such devices.

The antisense oligonucleotides may also be administered locally, as contrasted to regional or sys- temic administration. Local administration of poly- nucleotides have been carried out by direct injection into muscle. Local administration of oligonucleotides may be particularly useful in treating neuroectodermal tumors, esophageal tumors and melanoma. For treatment of esophageal tumors, a pharmaceutical preparation of antisense oligonucleotide may be delivered locally to the tumor site by means of a catheter. Such catheters have been used to deliver drugs for local cardiovascular treatment and can be adapted for use in delivering drug directly to esophageal lesions. For treatment of mela¬ noma, the oligonucleotides may be delivered by skin infiltration. Methods for delivering therapeutic oligo¬ nucleotide and polynucleotides by local infiltration are known to those skilled in the art.

As an alternative to treatment with exogenous oligonucleotide, antisense oligonucleotide synthesismay be induced jLn situ by local treatment of the targeted neoplastic cell with a vector containing an artificial- ly-constructed gene comprising a transcriptional promo- tor and cyclin DI DNA in inverted orientation. The cyclin DI for insertion into the artificial gene in inverted orientation comprises cDNA which may be pre¬ pared, for example, by reverse transcriptase polymerase chain reaction from RNA using primers derived from the published cDNA sequence of cyclin DI. Upon transcrip¬ tion, the inverted cyclin DI gene segment, which is complementary to at least a portion of the cyclin DI mRNA, is produced jLn situ in the targeted cell. The endogenously produced RNA hybridizes to cyclin DI mRNA, resulting in interference with cyclin DI function and inhibition of the proliferation of the targeted cell.

The promotor segment of the artificially-con¬ structed gene serves as a signal conferring expression of the inverted cyclin DI sequence which lies downstream thereof. It will include all of the signals necessary for initiating transcription of the sequence. The pro- motor may be of any origin as long as it specifies a rate of transcription which will produce sufficient antisense mRNA to inhibit the expression of the cyclin DI gene, and therefore the proliferation of the tumor cells. Preferably, a highly efficient promotor such as a viral promotor is employed. Other sources of potent promotors include cellular genes that are expressed at high levels. The promotor segment may comprise a con¬ stitutive or a regulatable promotor. Described in the hereinafter Example 1 is a typical construct which util¬ izes the SV40 promotor.

The artificial gene may be introduced by any of the methods described in U.S. Patent 4,740,463, in¬ corporated herein by reference. One technique is transfection, which can be done by several different methods. One method of transfection involves the addi¬ tion of DEAE-dextran to increase the uptake of the naked DNA molecules by a recipient cell. See McCutchin, J.H. and Pagano, J.S., J. Natl. Cancer Inst. 41, 351-7 (1968) . Another method of transfection is the calcium phosphate precipitation technique which depends upon the addition of Ca⁺⁺ to a phosphate-containing DNA solution. The resulting precipitate apparently includes DNA in association with calcium phosphate crystals. These crystals settle onto a cell monolayer; the resulting apposition of crystals and cell surface appears to lead to uptake of the DNA. A small proportion of the DNA taken up becomes expressed in a transfectant, as well as in its clonal descendants. See Graham, F.L. and van der Eb, A.J., Virology 52, 456-467 (1973) and Virology 54, 536-539 (1973) .

Transfection may also be carried out by cat- ionic phospholipid-mediated delivery. In particular, polycationic liposomes can be formed from N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) . See Feigner et al., Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987) (DNA-transfection) ; Malone et al. , Proc. Natl. Acad. Sci. USA. 86, 6077-6081 (1989) (RNA- transfection) .

Alternatively, the artificially-constructed gene can be introduced in to cells, in vitro or in vivo, via a transducing viral vector. See Tabin et a_l. , Mol. Cel. Biol. 2, 426-436 (1982). Use of a retrovirus, for example, will infect a variety of cells and cause the artificial gene to be inserted into the genome of in¬ fected cells. Such infection could either be done with the aid of a helper retrovirus, which would allow the virus to spread through the organism, or the antisense retrovirus could be produced in a helper-free system, such as ψ2-like cells (See Mann et ai. , Cell 33, 153- 160, 1983) that package amphotropic viruses. A helper- free virus might be employed to minimize spread through- out the organism. Viral vectors in addition to retro¬ viruses can also be employed, such as paporaviruses, SV40-like viruses, or papillo a viruses. The use of retroviruses for gene transfer has been reviewed by Eglitis and Anderson, BioTechniques 6, 608-614 (1988) . Vesicle fusion could also be employed to de¬ liver the artificial gene. Vesicle fusion may be physi¬ cally targeted to the tumor tissue if the vesicle were approximately designed to be taken up by the cells con¬ taining cyclin DI. Such a delivery system would be expected to have a lower efficiency of integration and expression of the artificial gene delivered, but would have a higher specificity than a retroviral vector. A combination strategy of targeted vesicles containing papilloma virus or retrovirus DNA molecules might pro- vide a method for increasing the efficiency of expres¬ sion of targeted molecules.

Still another alternative is to introduce the artificial gene via micro-injection. See for example, Laski et al. , Cell. 1982.

Particulate systems and polymers for in vitro and in vivo delivery of polynucleotides was extensively reviewed by Feigner in Advanced Drug Delivery Reviews 5, 163-187 (1990) . Techniques for direct delivery of purified genes in vivo, without the use of retroviruses, has been reviewed by Feigner in Nature 349, 351-352 (1991) . Such methods of direct delivery of polynucleo¬ tides may be utilized for local delivery of either ex¬ ogenous cyclin DI antisense oligonucleotide or arti- ficially-constructed genes producing DI antisense oligo¬ nucleotide in situ.

Recently, Wolf et ai. demonstrated that direct injection of non-replicating gene sequences in a non- viral vehicle is possible. See Science. 247, 1465-1468 (1990) . DNA injected directly into mouse muscle did not integrate into the host genome, and plasmid essentially identical to the starting material was recovered from the muscle months after injection. Interestingly, no special delivery system is required. Simple saline or sucrose solutions are sufficient to delivery DNA and RNA.

Many neoplasms, such as neuroblastoma, melano¬ ma and breast cancer, may be substantially metastatic, particularly in advanced stages. In particular, malig- nant cells may metastasize to the bone marrow. Patients with disseminated disease may have bone marrow etas- tases. It is therefore necessary to develop an effec¬ tive method to purge bone marrow of all remaining neo¬ plastic cells if autologous bone marrow transplantation is used in conjunction with aggressive chemotherapy. According to the present invention, cyclin DI antisense oligonucleotides may be used as bone marrow purging agents for the in vitro cleansing of bone marrow of malignant cells. According to a method for bone marrow purging, bone marrow is harvested from a donor by standard oper¬ ating room procedures from the iliac bones of the donor. Methods of aspirating bone marrow from donors are well- known in the art. Examples of apparatus and processes for aspirating bone marrow from donors are disclosed in U.S. Patents 4,481,946 and 4,486,188, incorporated here¬ in by reference. Sufficient marrow is withdrawn so that the recipient, who is either the donor (autologous transplant) or another individual (allogeneic trans- plant) , may receive from about 4 x 10⁸ to about 8 x 10⁸ processed marrow cells per kg of bodyweight. This gen¬ erally requires aspiration of about 750 to about 1000 ml of marrow. The aspirated marrow is filtered until a single cell suspension, known to those skilled in the art as a "buffy coat" preparation, is obtained. This suspension of leukocytes is treated with cyclin DI antisense oligonucleotides in a suitable carrier, advan¬ tageously in a concentration of about 50-100 μg/ml. Alternatively, the leucocyte suspension may be stored in liquid nitrogen using standard procedures known to those skilled in the art until purging is carried out. The purged marrow can be stored frozen in liquid nitro¬ gen until ready for use. Methods of freezing bone mar¬ row and biological substances are disclosed, for example, in U.S. Patents 4,107,937 and 4,117,881.

Other methods of preparing bone marrow for treatment with cyclin DI antisense may be utilized, which methods may result in even more purified prepara¬ tions of hematopoietic cells than the aforesaid buffy coat preparation. After treatment with the antisense oligonucleotides, the cells to be transferred are washed with autologous plasma or buffer to remove unincor¬ porated oligomer. The washed cells are then infused back into the patient.

The amount of antisense oligonucleotide may vary depending on the nature and extent of the neoplasm, the particular oligonucleotide utilized, and other fac¬ tors. The actual dosage administered may take into ac- count 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, whether the treatment is re¬ gional or systemic, and other factors. Concentrations of from about 1 to about 100 μg/ml may be employed, preferably from about 10 μg/ml to about 100 μg/ml, most preferably from about 20 μg/ml to about 60 μg/ml. The patient should receive a sufficient daily dosage of antisense oligonucleotide to achieve these intercellular concentrations of drug. The daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferab¬ ly from about 10 to about 700 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient.

It is believed that a course of treatment may advantageously comprise infusion of the recommended daily dose of oligonucleotide for a period of from about 3 to about 28 days, more preferably from about 7 to about 10 days. Those skilled in the art should readily be able to determine the optimal dosage in each case. For modified oligonucleotides, such as phosphorothioate oligonucleotides, which have a half life of from 24 to 48 hours, the treatment regimen may comprise dosing on alternate days.

For an about 70 kg adult human being, a daily dose of about 350 mg oligonucleotide is believed suffi- cient, to achieve an effective extracellular concentra¬ tion of 2-20 μM. For children, the daily dosage is reduced proportionately according to the weight of the patient.

For ex vivo antineoplastic application, such as, for example, in bone marrow purging, the cyclin DI antisense oligonucleotides may be administered in amounts effective to kill neoplastic cells. Such amounts may vary depending on the extent to which malig¬ nant cells may have metastasized to the bone marrow, the particular oligonucleotide utilized, the relative sensi¬ tivity of the neoplastic cells to the oligonucleotide, and other factors. Concentrations from about 10 to 200 μg/ml per 10⁵ cells may be employed, preferably from about 40 to 150 μg/ml per 10⁵ cells. Supplemental dosing of the same or lesser amounts of oligonucleotide are advantageous to optimize the treatment. Thus, for purg¬ ing bone marrow containing 2 x 10⁷ cell per ml of marrow volume, dosages of from about 2 to 40 mg antisense per ml of marrow may be effectively utilized, preferably from about 8 to 24 mg/ml. Greater or lesser amounts of oligonucleotide may be employed.

The effectiveness of the treatment may be assessed by routine methods which are used for determin¬ ing whether or not remission has occurred. Such methods generally depend upon some combination of morphological, cytochemical, cytogenetic, i munologic and molecular analyses. In addition, remission can be assessed gene¬ tically by probing the level of expression of the cyclin DI oncogene. The reverse transcriptase polymerase chain reaction methodology can be used to detect even very low numbers of mRNA transcript.

Typically, therapeutic success is assessed by the decrease in the extent of the primary and any met- astatic diseases lesions. For solid tumors, decreasing tumor size is the primary indicia of successful treat¬ ment. Neighboring tissues should be biopsied to deter¬ mine the extent to which metastasis has occurred. Tis¬ sue biopsy methods are known to those skilled in the art. For non-solid tumors, i.e. the leukemias, treat¬ ment is monitored primarily by histological examination of the bone marrow for surviving leukemic cells. How¬ ever, a significant number of leukemic cells may still exist when marrow examination provides normal results. For this reason, more recent methods for detecting leu¬ kemic cells have focused on detecting the presence of the gene for the relevant oncogene, or its corresponding mRNA, in cells of the bone marrow as a more sensitive test. See for example the following U.S. Patents: 4,681,840, 4,857,466 and 4,874,853. The presence of even a few copies of the target oncogene can be effec¬ tively detected by amplification using reverse tran- scriptase polymerase chain reaction technology. For a detailed discussion of such methods, see for example, Cancer: Principles & Practice of Oncology, edited by V. T. DeVita, S. Hellman and S.A. Rosenberg, J.B. Lip- pincott Company, Philadelphia, PA (3rd ed. , 1989), in¬ corporated herein by reference. Methods for diagnosing and monitoring the progress of neoplastic disorders vary depending upon the nature of the particular disease.

The practice of the invention is illustrated by the non-limiting examples, below. The effect of cyclin DI antisense oligonucleotide in inhibiting cell proliferation was demonstrated on a murine fibroblast cell line (Balb/B-myb) engineered to express high levels of exogenous human B-myb mRNA. The B-myb gene is homol¬ ogous to c-myb and is expressed in a wide variety of tissues. The Balb/B-myb cell line has at least two characteristics of a tumorigenic cell. First, the cells proliferate in low serum conditions, that is, they are growth factor-independent. Second, they grow in soft agar. Their growth is not contact inhibited.

Example 1

Inhibition of Cloning Efficiency of Balb/B-myb Cells Transfected with a pSV/anti-Cvclin DI Construct

A. Establishment of Balb/B-mvb Cell line A cell line (Balb/B-mvb) expressing high lev¬ els of exogenous human B-myb was established by trans- fecting Balb/c3T3 cells with a plasmid containing full- length B-mvb cDNA. A human lymphoma cDNA library cloned in a γgtll vector was screened with a 1.4 kilobase ra- diolabelled B-myb fragment (Nomura et al. , Nucleic Acids Res. 16, 11075 (1988)). A 1,469-bp fragment was sub- cloned into an SK-plasmid vector (Stragene, La Jolla, CA) . The remaining 5' portion of the cDNA was cloned by polymerase chain reaction amplification of reverse- transcribed B-myb mRNA from HL-60 cells. Full-length B-myb cDNA was subsequently eluted from the SK vector, digested with Clal and Xbal and subcloned into the pSV40 polylinker vector, which contains a polycloning site located in between the pSV40 early promoter and the SV40 polyadenylation signals.

B. pSV/anti-Cvclin DI Construct

A construct containing a 600 bp cyclin DI cDNA fragment cloned in the antisense orientation with re- spect to the SV40 promoter was obtained as follows. A

606 bp cyclin DI cDNA fragment (from nucleotide 188 to nucleotide 743) was synthesized by reverse transcriptase polymerase chain reaction from Balb/c3T3 RNA with a 5' primer (5'-ATGGAACACC AGCTCCTG-3' , SEQ ID NO:2) and a 3' primer (5'-CATGGAGGGT GGCTGGAAA, SEQ ID NO:3) derived from the published cDNA sequence of the murine homolog of cyclin DI (Matsushime et al., Cell 65, 701-713 (1991)), and cloned into the vector PCR 1000 (Invitro- gen, LaJolla, CA) . Sequence analysis revealed that the cloned fragment was identical to the published murine cyclin DI sequence. The cyclin DI fragment was subse¬ quently eluted from the vector PCR 1000 by digestion with Spel and EcoRl restriction enzymes and cloned in antisense orientation into the pSV40-polylinker vector digested with EcoRl and Xbal.

C. Transfection of Balb/B-mvb Cells

Plasmids of the pSV/anti-cyclin DI construct (10 μg/10⁶ cells) were transfected into Balb/B-myb cells by calcium phosphate precipitation in the presence of 1 μg of pRSV-Neo which carries the neomycin resistance gene. Colonies were scored after 14 days of selection in medium containing G418 (800 μg/ml) , and stained by crystal violet. The results are shown in Figure 1 and 2: 1, pSV/anti-cyclin Dl-transfected cells; 2, pSV40 polylinker-transfected cells (control) . A drastically reduced number of G418 resistant colonies (-80% inhibi¬ tion) formed upon transfection with the antisense con¬ struct as compared to the Balb/B-myb cells transfected with the vector only.

The following example describes a method of bone marrow purging using cyclin DI antisense oligo¬ nucleotide to purge marrow of malignant cells. Example 2

Bone Marrow Purging with Cyclin DI Antisense Oligonucleotide

Bone marrow is harvested from the iliac bones of a donor under general anesthesia in an operating room using standard techniques. Multiple aspirations are taken into heparinized syringes. Sufficient marrow is withdrawn so that the marrow recipient will be able to receive about 4 x 10⁸ to about 8 x 10⁸ processed marrow cells per kg of body weight. Thus, about 750 to 1000 ml of marrow is withdrawn. The aspirated marrow is transferred immediately into a transport medium (TC-199, Gibco, Grand Island, New York) containing 10,000 units of preservative-free heparin per 100 ml of medium. The aspirated marrow is filtered through three progressively finer meshes until a single cell suspension results, i.e., a suspension devoid of cellular aggregates, debris and bone particles. The filtered marrow is then pro- cessed further into an automated cell separator (e.g. , Cobe 2991 Cell Processor) which prepares a "buffy coat" product, (i.e., leukocytes devoid of red cells and platelets) . The buffy coat preparation is then placed in a transfer pack for further processing and storage. It may be stored until purging in liquid nitrogen using standard procedures. Alternatively, purging can be carried out immediately, then the purged marrow may be stored frozen in liquid nitrogen until it is ready for transplantation. The purging procedure may be carried out as follows. Cells in the buffy coat preparation are ad¬ justed to a cell concentration of about 2 x 10⁷/ml in TC- 199 containing about 20% autologous plasma. Cyclin DI antisense oligodeoxynucleotide, for example, in a con- centration of about 50-100 μg/ml, is added to the trans- fer packs containing the cell suspension. The transfer packs are then placed in a 37°C waterbath and incubated for 18 - 24 hours with gentle shaking. The cells may then either be frozen in liquid nitrogen or washed once at 4°C in TC-199 containing about 20% autologous plasma to remove unincorporated oligomer. Washed cells are then infused into the recipient. Care must be taken to work under sterile conditions wherever possible and to maintain scrupulous aseptic techniques at all times.

The present invention may be embodied in other 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.

All references cited herein with respect to synthetic, preparative and analytical procedures are incorporated by reference.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Thomas Jefferson University (A) INVENTOR: Calabretta, Bruno

(ii) TITLE OF INVENTION: Antisense Oligonu¬ cleotides to Cyclin DI Proto-oncogene. (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEES: Thomas Jefferson University

10th and Locust Streets Philadelphia Pennsylvania U.S.A.

19107

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.50 inch, 720 Kb

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS (D) SOFTWARE: WordPerfect 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 935,075

(B) FILING DATE: 25 August 1992 (viϋ) ATTORNEY/AGENT INFORMATION:

(A) NAME: Monaco, Daniel A. (B) REGISTRATION NUMBER: 30,480

(C) REFERENCE/DOCKET NUMBER: 8321-2 (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: 50 Nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ACCTTGTGGT CGAGGACACG ACGCTTCACC TTTGGTAGGC GGCGCGCATG 50

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

(A) LENGTH: 18 Nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATGGAACACC AGCTCCTG 18

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

(A) LENGTH: 19 Nucleotides

(B) TYPE: nucleic acid

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CATGGAGGGT GGCTGGAAA 19

Claims

1. A method for the treatment of a neo- plastic disease characterized by the amplification of the cyclin DI gene or activation of cyclin DI expression comprising 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 cyclin DI gene, said oligonucleotide being hybridizable to said mRNA transcript.

2. A method according to claim 1 wherein the oligonucleotide is an at least 8-mer.

3. A method according to claim 2 'wherein the oligonucleotide is an alkylphosphonate oligonucleoside or phosphorothioate oligonucleotide.

4. A method according to claim 2 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the cyclin DI mRNA lying within about 50 nucleotides of the translation initiation codon.

5. A method according to claim 4 wherein the oligonucleotide is a phosphorothioate oligodeoxy¬ nucleotide or methylphosphonate oligodeoxynucleoside.

6. A method according to claim 2 wherein the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the cyclin DI mRNA transcript including the translation initiation codon of said transcript.

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

8. A method according to claim 7 wherein the oligonucleotide is an alkylphosphonate oligonucleoside or a phosphorothioate oligonucleotide.

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

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

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

12. A method according to claim 9 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence of SEQ ID NO:l, or at least 12-mer portion thereof.

13. A method according to claim 1 wherein the neoplastic disease is selected from the group consisting of malignant melanoma, neuroectodermal cancers, esopha¬ geal cancer, breast cancer, squamous cell cancer, para¬ thyroid adenoma, leukemia and lymphoma.

14. A method according to claim 1 wherein the antisense oligonucleotide is administered locally.

15. A method for purging bone marrow of metas- tasized cells comprising treating bone marrow cells aspirated from an individual afflicted with a neoplastic disease characterized by the amplification of the cyclin DI gene or activation of cyclin DI expression with an effective amount of an oligo¬ nucleotide which has a nucleotide sequence com- plementary to at least a portion of the mRNA transcript of the cyclin DI gene, said oligonu¬ cleotide being hybridizable to said mRNA trans¬ cript, and returning the thus-treated cells to the body of the afflicted individual.

16. A method according to claim 15 wherein the oligonucleotide is an at least 8-mer.

17. A method according to claim 16 wherein the oligonucleotide is a alkylphosphonate oligonucleoside or phosphorothioate oligonucleotide.

18. A method according to claim 16 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the cyclin DI mRNA lying within about 50 nucleotides of the translation initiation codon.

19. A method according to claim 16 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.

20. An oligonucleotide comprising from 8 to 50 nucleotides which has a nucleotide sequence complement- ary to at least a portion of the mRNA transcript of the cyclin DI gene, said oligonucleotide being hybridizable to said mRNA transcript.

21. An oligonucleotide according to claim 20 wherein the oligonucleotide is an alkylphosphonate oli¬ gonucleoside or phosphorothioate oligonucleotide.

22. An oligonucleotide according to claim 20 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the cyclin DI transcript lying within about 50 nucleotides of the translation initiation codon.

23. An oligonucleotide according to claim 20 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the cyclin DI mRNA trans¬ cript including the translation initiation codon of said transcript.

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

25. An oligonucleotide according to claim 24 wherein the oligonucleotide is from a 15-mer to a 30- mer.

26. An oligonucleotide according to claim 25 wherein the oligonucleotide is from a 18-mer to a 26- mer.

27. An oligonucleotide according to claim 26 wherein the oligonucleotide is from a 18-mer to a 21- mer.

28. An oligonucleotide according to claim 20 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence corresponding to SEQ ID NO:l, or at least 12-mer position thereof.

29. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one oligonucleotide which has a nucleotide sequence comple¬ mentary to at least a portion of the mRNA transcript of the cyclin DI gene, said oligonucleotide being hybridiz¬ able to said mRNA transcript.

30. A composition according to claim 29 wherein the oligonucleotide is an alkylphosphonate oligo¬ nucleoside or phosphorothioate oligonucleotide.

31. A composition according to claim 29 wherein the oligonucleotide has a nucleotide sequence comple¬ mentary to a portion of the cyclin DI mRNA transcript lying within about 50 nucleotides of the translation initiation codon.

32. A composition according to claim 29 wherein the oligonucleotide has a nucleotide sequence comple¬ mentary to a portion of the cyclin DI mRNA transcript including the translation initiation codon of said transcript.

33. A composition according to claim 29 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.

34. A composition according to claim 33 wherein the oligonucleotide is from a 15-mer to a 30-mer.

35. A composition according to claim 34 wherein the oligonucleotide is from a 18-mer to 26-mer.

36. A composition according to claim 35 wherein the oligonucleotide is from a 18-mer to 21-mer.

37. A composition according to claim 29 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence corresponding to SEQ ID NO:l, or at least a 12-mer portion thereof.

38. An artificially-constructed gene comprising a transcriptional promotor segment and a segment con¬ taining a cyclin DI DNA in inverted orientation such that transcription of said artificially-constructed gene produces RNA complementary to at least a portion of the mRNA transcript of the cyclin DI gene.

39. A method according to claim 38 wherein the promotor segment comprises SV40 promotor.

40. A method for inhibiting the proliferation of neoplastic cells characterized by the amplification of the cyclin DI gene or activation of cyclin DI expres¬ sion comprising introducing into such cells an artifi¬ cially-constructed gene which, upon transcription in said cells, produces RNA complementary to the mRNA transcript of the cyclin DI gene.

41. A method according to claim 40 wherein the artificially-constructed gene is introduced into said cells by transfection, by a transducing viral vector or by microinjection.