WO1994007538A1

WO1994007538A1 - ANTISENSE OLIGONUCLEOTIDES TO B-m^_y^_b^_ PROTO-ONCOGENE

Info

Publication number: WO1994007538A1
Application number: PCT/US1993/009013
Authority: WO
Inventors: Bruno Calabretta
Original assignee: Thomas Jefferson University
Priority date: 1992-10-01
Filing date: 1993-09-23
Publication date: 1994-04-14
Also published as: AU5136693A

Abstract

Oligonucleotides are provided having a nucleotide sequence complementary to at least a portion of the mRNA transcript of the B-m^_y^_b^_ gene. These 'antisense' oligonucleotides are hybridizable to the B-m^_y^_b^_ mRNA transcript. Such oligonucleotides are useful in treating neoplastic diseases characterized by activation of B-m^_y^_b^_ gene expression.

Description

Φ

ANTISENSE OLIGONUCLEOTIDES TO B-mvb PROTO-ONCOGENE

Field of the invention

The invention relates to antisense 5 oligonucleotides to proto-oncogenes, in particular antisense oligonucleotides to the B-myb gene, and the use of such oligonucleotides as antineoplastic agents.

Reference to Government Grant

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

15 Background of the Invention

Two genes, A-myb and B-myb. have recently been isolated that are related to the proto-oncogene c-myb, the cellular homolog of the transforming gene of the avian myeloblastosis virus (AMV) and the avian leukemia

20 virus E26 which causes myeloblastic leukemia in chickens and transform myelomonocytic hematopoietic cells in culture.

Recent evidence suggests that c-mvb plays an important role in the regulation of normal and leukemic

25 hematopoiesis and T-lymphocyte proliferation. See, for example, Gewirtz and Calabretta. Science 242, 1303 (1988) . Inhibition of c-myb expression prevents G^/S transition in normal T-lymphocytes and is associated with selective down-regulation of DNA polymerase-α expression

30 (Venturelli et al. , Proc. Natl. Acad. Sci. USA 87, 5963 (1989)), suggesting direct involvement of c-myb in an essential biochemical pathway leading to DNA synthesis. Moreover, constitutive expression of the human c-myb gene in murine fibroblasts abrogates the requirement for the cell cycle progression factor IGF-1 (Travali et al. , Mol. Cell. Biol.11, 731 (1990) , further supporting the notion that c-myb plays a general role in cellular prolifera¬ tion. The inhibition of normal and leukemic cells with antisense oligonucleotides which hybridize to the c-mvb mRNA is described in U.S. Patent 5,098,890, the entire disclosure of which is incorporated herein by reference. The c-myb gene is necessary for proliferation of both normal and leukemic hematopoietic cells.

The product of the c-myb gene is a nuclear binding protein. B-myb is homologous to c-mvb in the DNA binding domain and its pattern of expression does not appear to be restricted to hematopoietic cells. The cDNA nucleotide sequence of the human B-mvb gene, and the deduced 700-amino acid sequence, are set forth in Nomura et al. , Nucleic Acids Res. 16.11705-11089 (1988), the entire disclosure of which is incorporated herein by reference. Like c-mvb, B-myb acts as a trans- activating factor. In transient expression assays, a constitutively expressed B-myb cDNA transactivates a reporter gene linked to the SV40 early promoter and enhancer through interaction of the encoded protein with B-myb binding sites in the SV40 early promoter (Mizuguchi et al., J. Biol. Chem. 265. 9280 (1990)).

The expression of the B-myb in normal he ato- poietic cells has been shown to correlate with prolifera¬ tion and c-myb expression in these cells (Golay et ai. , Blood 77, 149 (1991)). B-myb is also expressed in Balb/c3T3 fibroblasts at the G^/S boundary, and appears to be growth-regulated at that boundary. However, despite the recent advances in the study of the B-myb gene, its role in maintaining proliferation of trans¬ formed cells has not been demonstrated.

Summary of the Invention The invention provides antisense oligonucleotides andpharmaceutical compositions thereof with pharmaceutically acceptable carriers. Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human B-myb gene. The oligonucleotide is hybridizable to the mRNA transcript, but does not hybridize to the c-myb gene. The oligonucleotide is at least an 8-mer oligo¬ nucleotide, that is, an oligomer containing at least 8 - nucleotide residues, and contains up to 50 nucleotides. 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. Most preferably, the oligonucleotide is a 15- to 30-mer oligodeoxynucleotide, more advantageously an 18- to 26- er.

While in principle oligonucleotides having a sequence complementary to any region of the B-mvb mRNA find utility in the present invention, oligonucleotides complementary to a portion of the B-mvb mRNA transcript including the translation initiation codon are par¬ ticularlypreferred. Also preferred are oligonucleotides complementary to a portion of the B-myb 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 administering to an individual or cells harvested from the individual an effective amount of B-mvb antisense oligonucleotide. The neoplastic diseases treatable include those diseases in which the B-myb gene is activated.

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

According to another embodiment, the invention relates to an artificially-constructed gene comprising a transcriptional promotor segment and a segment containing B-myb DNA in inverted orientation such that transcription of the artificially-constructed gene produces RNA complementary to at least a portion of the mRNA transcript of the B-mvb gene. The gene may be introduced into cells which are characterized by the activation of B-myb expression to inhibit the prolifer¬ ation of those cells. The artificially-constructed gene may be introduced into the neoplastic cells by, for example, transfection, transduction with a viral vector, or icroinjection. As used in the herein specification and appended claims, unless otherwise indicated, the term "oligonucleotide" includes both oligomers of ribonucleo- tides, i.e., oligoribonucleotides, and oligomers of deoxyribonucleotides, i.e., oligodeoxyribonucleotides (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 "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"), thy ine ("T") and uracil ("U") , but also include oligomers and polymers hybridizable to the B-myb mRNA transcript which may contain other nucleotides. Likewise, the terms "oligonucleotide" and "oligodeoxy- nucleotide" includes 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 "oligo- nucleosides" because of modification of the inter¬ nucleotide phosphodiester bond. Such modified oligonucleotides include, for example, the alkylphos- phonate oligonucleosides, discussed below.

The term "phosphorothioate oligonucleotide" means an oligonucleotide wherein one or more of the internucleotide linkages is a phosphorothioate group,

O

-O - P - 0~

I s-

s opposed to the phosphodiester group O

II -o - p - o- I

0^~

which is characteristic of unmodified oligonucleotides. By "alkylphosphonate oligonucleoside" ismeant an oligonucleotide wherein one or more of the inter¬ nucleotide linkages is an alkylphosphonate group,

0

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 "B-myb mRNA transcript" means the presently known mRNA transcript of the human B-mvb gene and all variations thereof, or any further transcripts which may be elucidated.

Description of the Fiσures

Fig. 1A shows a Balb/c3T3 cell culture transfected with pSV40 polylinker (control) .

Fig. IB shows a Balb/c3T3 cell culture transfected with a pSV/B-mvb construct.

Fig. 1C shows a Balb/c3T3 cell culture transfected with a pSV/anti-B-myb construct (antisense orientation) . Fig. 2A is similar to Fig. IB and shows a LAN-5 neuroblastoma cell culture transfectedwith the pSV/B-myb construct.

Fig. 2B is similar to Fig. 1C and shows a LAN-5 cell culture transfected with the pSV/anti-B-myb construct.

Fig. 3 is a cell count of LAN-5 cells treated with B-mvb antisense ("AS") or sense ("S") oligomer, or of untreated LAN-5 cells ("C") .

Detailed Description of the Invention

The putative DNA sequence complementary to the mRNA transcript of the human B-myb gene has been re¬ ported by Nomura et aJL. , Nucleic Acids Res. 16, 11705- 11089 (1988) , the entire disclosure of which is incor¬ porated herein by reference. These investigators further disclose the predicted 700 amino acid sequence of the putative B-myb protein. The initiation codon ATG is preceded by a 5'-untranslated region of about 127 nucle- otides. The termination codon TGA is followed by a 3'- untranslated region, which includes a putative poly- adenylation signal (AATAAA) located seventy nucleotides downstream of the termination codon.

The antisense oligonucleotides of the inven- tion, which are complementary to the B-myb mRNA, may be synthesized by any of the known chemical oligonucleotide synthesis methods. Such methods are generally de¬ scribed, 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 ?-cyanoethyl phosphoramidite chemistry. Since the complete nucleotide synthesis of DNA complementary to the B-myb mRNA transcript is known, an¬ tisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by oligonucleo- tide synthesis methods known to those skilled in the art.

The B-myb and c-myb cDNA nucleotide sequences are characterized by varying degrees of homology in three distinct domains, corresponding to the following amino acid residues (I) B-myb 33-193, c-myb 42-202; (II) B-mvb 468-545, c-mvb 414-498; and (III) B-mvb 663-681, c-myb 608-626. The homology is primarily in the DNA- binding domain. The strongest homology lies from about B-myb amino acid 63 to about 193. Preferably, the antisense oligonucleotides of the invention are selected so as to be specific for hybridizing to B-mvb, and not c-myb mRNA. The use of such B-mvb specific antisense oligonucleotide would minimize the side effects which could result from inhi- bition of c-mvb expression in normal cells. The com¬ bined effect of B-mvb and c-myb expression inhibition might have an.adverse effect particularly on hematopoi¬ etic cells, both of which are characterized by both c- vb and B-myb expression. The combination effect is avoided by selecting the portion of the B-myb mRNA tar¬ geted for hybridization from the regions which have a lesser degree of homology with the corresponding c-mvb sequence. The selection of the particular sequence for the antisense oligonucleotide is therefore of necessity made not only on the basis of the B-myb mRNA nucleotide sequence, but also the c-myb sequence. The complete cDNA nucleotide sequence of the human c-mvb gene is disclosed by Majello et al.. , Proc. Natl. Acad. Sci. U.S.A. 38, 9636-9640 (1986), and U.S. Patent 5,098,890, the entire disclosures of both of which are incorporated herein by reference.

The areas of the B-mvb mRNA which are most preferred for targeting for antisense hybridization are those sequences lying completely outside the regions of homology with c-mvb. that is, the preferred areas for targeting include nucleotides 1-226, 708-1531, 1764-2116 and 2172-2630 (nucleotide 128 being the translation initiation codon) . 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 mRNA, may be more easily de- stroyed 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 50 nucleotides, may be somewhat less effec- tive in inhibiting B-myb translation because of de¬ creased uptake by the target cell. Thus, oligomers of 12-40 nucleotides are preferred, more preferably 15-30 nucleotides, most preferably 18-26 nucleotides. While sequences of 18-21 nucleotides are most particularly preferred for unmodified oligonucleotides, slightly longer chains of up to about 26 nucleotides, are pre¬ ferred for modified oligonucleotides such as phosphor¬ othioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides. It may be appreciated that oligonucleotides appreciably larger than 50 nucleotides may be freely utilized where oligo¬ nucleotide delivery is via transfection of targeted cells with a vector coding for B-mvb antisense oligo¬ nucleotide. Oligonucleotides complementary to and hybrid¬ izable with any portion of the B-mvb 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. This region shares essentially no homology with c-mvb. Thus, oligonucleotides complementary to the 5'-terminal region of the B-mvb mRNA transcript are preferred. The anti¬ sense oligonucleotide is preferably directed to a site at or near the initiation codon for protein synthesis. Oligonucleotides complementary to the B-myb mRNA, in¬ cluding the initiation codon (the first codon at the 5' end of the translated portion of the B-myb transcript) are preferred.

While antisense oligomers complementary to the 5'-terminal region of the B-myb 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 B-mvb mRNA transcript beginning with the initiation codon of the transcript and extend¬ ing downstream thereof (in the 3' direction): SEQ ID NO:l.

Particularly preferred smaller oligonucleo¬ tides hybridizable to segments of the B-myb message containing the initiation codon are the oligomers having a nucleotide sequence selected from the nucleotide se- quence of the following 15- to 30-mers: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,

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

SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and

SEQ ID NO: 17. Smaller oligomers based upon the above se¬ quence, in particular, oligomers hybridizable to seg¬ ments of the B-mvb 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 B-myb mRNA transcript finding utility according to the present invention in¬ clude not only oligomers of the biologically significant native nucleotides, i.e.. A, dA, G, dG, C, dC, T and U, but also oligonucleotide species which have been modi¬ fied for improved stability and/or lipid solubility. For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by sub¬ stituting an alkyl group or alkoxy group for a phosphate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate oligonucleoside or alkylphos- photriesteroligonucleotide. 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 clea¬ vage and soluble in lipid. The preparation of alkyl¬ phosphonate 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 al. , J. Biol. Chem.. 255, 9659-9665 (1980); Miller et al. , Nucl. Acids Res.. 11, 5189-5204 (1983), Miller et al. , Nucl. Acids Res.. 11, 6225-6242 (1983), Miller et al. , Biochemistry. 25, 5092-5097 (1986) ; Engels and Jager, Ancrew. 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 . , Che ica 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 e_t al. , Proc. Natl. Acad. Sci. U.S.A. 86. 3474-3478 (1989)).

Phosphorothioate oligodeoxynucleotide are described by LaPlanche, et al. , Nucleic Acids Research 14, 9081 (1986) and by Stec et al. , J. Am. Chem. Soc. 106, 6077 (1984). The general synthetic method for phosphorothioate oligonucleotides was modified by Stein et al., Nucl. Acids Res.. 16, 3209-3221 (1988), so that these compounds may readily be synthesized on an auto¬ matic synthesizer using the phosphoramidite approach.

Furthermore, recent advances in the 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 B-mvb mRNA translation is possibleutilizingeitherantisense oligoribonucleotides or oligodeoxyribonucleotides, free oligoribonucleotides are more susceptible to enzymatic attack by ribonu- cleases than oligodeoxyribonucleotides. Hence, oligo¬ deoxyribonucleotides are preferred in the practice of the present invention. Oligodeoxyribonucleotides are further preferred because, upon hybridization with B-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 B-myb 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 B-myb message. Absolute comple¬ mentarity is not however required, particularly in larg¬ er oligomers. Thus, reference herein to a "nucleotide sequence complementary to at least a portion of the mRNA transcript" of B-myb does not necessarily mean a se- quence having 100% complementarity with the transcript. In general, any oligonucleotide having sufficient com¬ plementarity to form a stable duplex with B-myb mRNA, that is, an oligonucleotide which is "hybridizable", is suitable. Stable duplex formation depends on the se- quence and length of the hybridizing oligonucleotide and the degree of complementarity with the target region of the B-mvb message. Generally, the larger the hybrid¬ izing oligomer, the more mismatches may be tolerated. More than one mismatch probably will not be tolerated for antisense oligomers of less than about 21 nucleo¬ tides. One skilled in the art may readily determine the degree of mismatching which may be tolerated between any given antisense oligomer and the target B-myb mes¬ sage sequence, based upon the melting point, and there- fore 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 Mo¬ lecular Cloning: A Laboratory Manual. (2nd edition, 1989), J. Sambrook et al. , eds. While oligonucleotides capable of stable hy¬ bridization with any region of the B-myb message are within the scope of the present invention, oligonucleotides complementary to a region including the translation initiation codon are believed particularly effective. Particularly preferred are oligonucleotides hybridizable to a region of the B-myb mRNA up to 40 nucleotides upstream (in the 5' direction) of the ini¬ tiation 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 B-mvb mRNA antisense oligonucleotides arepreferably administeredparenteral¬ ly, most preferably intravenously. The vehicle is de- signed accordingly. Alternatively, oligonucleotide may be administered subcutaneously via controlled release dosage forms.

In addition to administration with conven¬ tional 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 sus- pension. The hydrophobic layer, generally but not ex¬ clusively, comprises phospholipids such as lecithin and sphingo yelin, steroids such as cholesterol, ionic surfactants such as diacetylphosphate, stearyla ine, 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 al., Biochem. Biophy. 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 ribonu- cleotide. The resulting aldehyde groups are then ran¬ domly coupled to the epsilon-amino groups of lysine residues of poly(L-lysine) by Schiff base formation, and then reduced with sodium cyanoborohydride. This proce- dure converts the 3'-terminal ribose ring into a morpho- line 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-mvb 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 characterized by the activation of B-myb ex¬ pression, signalled by the appearance of B-myb mRNA transcripts and/or the 700-amino acid protein product. The level of B-myb expression may be determined, for example, by probing total cellular RNA from tumor cells with a complementary probe for B-myb mRNA, according to Nomura et al. , Nucleic Acids Res. 16 (28), 11075-11089 (1988) . Total cytoplasmic RNA from the tumor cells is passed over oligo (dT)-cellulose. The glyoxylated poly(A)⁺ is fractionated on a 0.7% agarose gel, trans¬ ferred to a filter and hybridized to an appropriately labelled nucleic acid probe for B-myb mRNA. The number of B-myb mRNA transcripts found in the tumor cells is compared to that found in normal cells from the same tissue. An at least 10-fold increase in B-myb expres¬ sion in neoplastic cells over expression in normal cells from the same tissue indicates activation of B-myb ex- pression, and would indicate that the disease would respond to B-myb antisense treatment. This threshold is based upon correlations between the level of gene ex¬ pression and the extent of the disease state for various other oncogenes. See, for example, Slamon et al. , Sci- ence 235, 177-182 (1988) and Science. 244, 707-712

(1989) (correlationbetweenerb-b2 amplification/expres¬ sion and breast or ovarian cancer) ; Alitalo et al. , Advances in Cancer Research. 47, 235-282 (1986) .

Disease conditions characterized by B-myb activated expression include, for example, neuroectoder- al cancers such as neuroblastoma and neuroepithelioma, malignant melanoma, breast cancer, prostate carcinoma, colon cancer, renal carcinoma and leukemia and lymphoma.

A preferred method of administration of oli- gonucleotide 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 main¬ tenance 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 and melanoma. 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 neuroplastic lesions. For treatment of melanoma, 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 synthesis may be induced in situ by local treatment of the targeted neoplastic cell with a vector containing an artificial- ly-constructed gene comprising a transcriptional promo- tor and B-myb DNA in inverted orientation. The B-mvb for insertion into the artificial gene in inverted ori¬ entation comprises cDNA which may be prepared, for exam¬ ple, by reverse transcriptase polymerase chain reaction from RNA using primers derived from the published cDNA sequence of B-myb. Upon transcription, the inverted B- myb gene segment, which is complementary to at least a portion of the B-myb mRNA, is produced jln situ in the targeted cell. The endogenously produced RNA hybridizes to B-myb mRNA, resulting in interference with B-myb 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 B-myb 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 B-mvb 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 uti¬ lizes 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 onolayer; 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 jLn vivo, via a transducing viral vector. See Tabin et al. , 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 al. , 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 papilloma viruses. The use of retroviruses for gene transfer has been reviewed by Eglitis and Anderson, BioTechnigues 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 B-myb. 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 high¬ er specificity than a retroviral vector. A combination strategy of targeted vesicles containing papillo a virus or retrovirus DNA molecules might provide a method for increasing the efficiency of expression 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 polynucle¬ otides may be utilized for local delivery of either exogenous B-myb antisense oligonucleotide or artificial- ly-constructed genes producing B-mvb antisense oligonu¬ cleotide in situ.

Recently, Wolf et al. 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.

The B-myb antisense oligonucleotides may be administered to the patient in the form of an appropri- ate pharmaceutical composition. Alternatively, the antisense oligonucleotides may be administered ex vivo, to cells harvested from the patient. Thus, according to a preferred embodiment of the invention, the B-myb antisense oligonucleotides are utilized as bone marrow purging agents for in vitro cleansing of the patient's bone marrow contaminated by leukemic cells. The antisense oligonucleotides are believed useful as purg¬ ing agents in either allogeneic or autologous bone mar¬ row transplantation. 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 metas- 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, B-myb antisense oligonucleotides may be used as bone marrow purging agents for the in vitro cleansing of bone marrow of malignant cells which have metastasized to the bone marrow from other tissues.

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 tran¬ splant) or another individual (allogeneic transplant) , may receive from about 4 x 10⁸ to about 8 x 10⁸ processed marrow cells per kg of bodyweight. This generally re¬ quires aspiration of about 750 to about 1000 ml of mar¬ row. 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 suspen- sion of leukocytes is treated with B-myb antisense oligonucleotides in a suitable carrier, advantageously in a concentration of about 50-100 μg/ml. Alter¬ natively, the leucocyte suspension may be stored in liquid nitrogen using standard procedures known to those skilled in the art until purging is carried out. The purged marrow can be stored frozen in liquid nitrogen until ready for use. Methods of freezing bone marrow and biological substances are disclosed, for example, in U.S. Patents 4,107,937 and 4,117,881. Other methods of preparing bone marrow for treatment with B-myb antisense may be utilized, which methods may result in even more purified preparations 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 unincorpor¬ ated 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 concen- tration 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 B-myb 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 deter¬ mining whether or not remission has occurred. Such methods generally depend upon some combination of mor- phological, cytochemical, cytogenetic, immunologic and molecular analyses. In addition, remission can be as¬ sessed genetically by probing the level of expression of the B-mvb oncogene. The reverse transcriptase poly¬ merase 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 meta- static 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.

According to one embodiment, B-myb antisense inhibition of cell proliferation is demonstrated below using Balb/c3T3 fibroblasts. These cells have a well- defined growth-factor requirement, which allows deter¬ mination of the stage in G₀/transition that requires B- myb function. B-myb is expressed in Balb/c3T3 fibro- blasts at the G.,/S boundary with a kinetics similar to that of c-myb in phytohemagglutinin-stimulated normal T-lymphocytes; B-myb is believed to be growth-regulated at the G,/S boundary (Golay et al. , Blood 77, 149 (1991) ; Reiss et al. , J. Cell. Phvsiol. 148, 338 (1991)). In connection with demonstrating B-myb antisense inhibition of Balb/c3T3 fibroblasts, the onco- genic potential of B-myb was determined by examining the effects of B-myb constitutive expression on the growth of Balb/c3T3. This was achieved by engineering a cell line (Balb/B-myb) which expresses high levels of exo- genous human B-myb. The cell line was established by transfecting Balb/c3T3 cells with a plasmid containing full-length B-myb cDNA.

To demonstrate that B-myb expression alters the growth characteristics of the transfected cells, Balb/B-myb and control Balb/c3T3 cells transfected with the pSV40 vector lacking the B-mvb cDNA in medium con¬ taining 1% serum which induces entry into a quiescent state. After 9 days of culture, the number of Balb/B- myb cells was significantly higher than that of the control cells (about 10⁴ cells/well in plates for Balb/B-myb as compared to about x 10⁴ cells of Balb/c3T3 transfected with the vector alone) , indicating that Balb/B-myb cells can still proliferate in low serum conditions, although at a reduced rate (doubling time of 48 hours in 1% serum as compared to 24 hours in 10% serum) . The reduced serum requirements of randomly picked single clones from different transfections cor¬ related well with mRNA levels of exogenous B-myb. as indicated by the capacity of clones with high B-myb expression to grow in 1% serum, whereas Balb/c3T3 cells require platelet-derived growth and insulin or insulin¬ like growth factor for survival in serum-free medium. Balb/B-myb cells not only survived but also grew in serum-free medium, although with a very slow (about 96 hours) doubling time. To determine whether the growth- factor independence of Balb/B-myb cells was an indica¬ tion of progression toward a transformed phenotype, we plated in soft agar both Balb/B-myb and the parental Balb/c3T3 cell line. After 10 days of culture, Balb/B- myb cells formed colonies in agar with high efficiency (about 50%) , while Balb/c3T3 cells formed small colo¬ nies, with an efficiency of only about 1-5%.

These findings indicate that cells consti- tutively expressing B-myb have at least two charac- teristics of a tumorigenic cell. First, the cells pro¬ liferate in low serum conditions, that is, they are growth factor-independent. Second, they grow in soft agar. Their growth is not contact inhibited. The effect on cell proliferation of inhibiting

B-mvb expression with antisense oligonucleotide is dem¬ onstrated by Examples 1, 2 and 3. According to Example 1, the blockage of B-mvb mRNA function in Balb/c3T3 cells transfected with a human B-mvb antisense construct inhibited the proliferation of the Balb/c3T3 cells. According to Example 2, the proliferation of neuro- blastoma cells is likewise inhibited by transfection with the antisense construct. In Example 3, the pro¬ liferation of neuroblastoma cells was inhibited by exo- genous B-myb antisense oligonucleotide.

Example 1

Inhibition of Cloning Efficiency of Balb/c3T3 Cells Transfected with a pSV/anti-B-myb Construct

A. pSV/B-mvb Construct

A human lymphoma cDNA library cloned in a γgtll vector was screened with a 1.4 kilobase radio- labelled B-mvb 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 (pSKB-myb) , digested with Clal and Xbal and subcloned into the pSV40 polylinker vector, which contains a poly- cloning site located in between the pSV40 early promoter and the SV40 polyadenylation signals. The resulting construct was designated pSV/B-myb.

B. pSV/anti-B-myb Construct The SKB-myb plasmid was linearized by Xhol digestion. The restriction site was filled by Klenow treatment before digesting with Spel. A 2 kb B-mvb cDNA fragment (Xhol blunted-Spel) was cloned in the antisense orientation with respect to the SV40 promoter into the pSV40-polylinker vector linearized with Xbal, Klenow- filled and subsequently digested with Spel. The result¬ ing construct was designated pSV/anti-B-myb.

C. Transfection of Balb/c3T3 Cells

Plasmids of the pSV/B-myb and pSV/anti-B-myb constructs (10 μg/10⁶ cells) were transfected into Balb/- c3T3 cells by calcium phosphate precipitation in the presence of 1 μg of pLHL4 which carries the hygromycin resistance gene (Furukawa et al. , Science 250, 805

(1990); Lee et al. , Nature 336, 738 (1988); Draetta et al. , Oncogene 2, 55 (1988)). Hygromycin resistant clones were selected in hygromycin-containing medium (250 μg/ml) , scored 12 days after transfection and stained with crystal violet. The results are shown in Figures 1A-1C: 1A, pSV40 polylinker-transfected cells; IB, pSV/B-m b-transfected cells; 1C, pSV/anti-B-myb- transfected cells. A drastically reduced number of hygromycin-resistant colonies (about 90% inhibition) is observed for cells treated with the antisense construct (Fig. 1C) , as compared to Balb/c3T3 cells transfected with the vector only (Fig. 1A) or sense construct (Fig. IB) . Example 2

Inhibition of Cloning Efficiency of Neuroblastoma Cells Transfected v/ith a pSV/anti-B-myb Construct

Neuroblastoma cell line LAN-5 (Seeger et al.. J. Immunol. 128, 983-989 (1982)) was grown in RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO.) supple¬ mented with fetal bovine serum (FEBS) (Sigma) . The procedure of Example 1 was repeated substituting LAN-5 cells for Balb/c3T3. Again, a drastically reduced num¬ ber of hygromycin-resistant colonies was observed for cells treated with the antisense construct (Fig. 2B) , as compared to cells transfected with the sense con¬ struct (Fig. 2A) .

Example 3

B-mvb Antisense Oligonucleotide Inhibition of Neuroblastoma Cell Proliferation

In a typical experiment, 1 x 10⁴ LAN-5 cells were seeded in the presence of antisense (SEQ ID NO: 5) or sense (SEQ ID NO: 18) phosphorothioate oligodeoxy¬ nucleotides (80 μg/ml at zero hours, 40 μg/ml after 18 hours, and 40 μg/ml after 36 hours) . Cells were counted after 7 or 9 days. The results are set forth in Fig. 2: C, no oligomer: S, sense oligomer; and AS, antisense oligomer. Cell proliferation was inhibited by the antisense oligomer.

Example 4

Bone Marrow Purging with B-myb 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 with¬ drawn. 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 parti¬ cles. The filtered marrow is then processed 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 proce¬ dures. Alternatively, purging can be carried out imme¬ diately, 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. B-myb 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 B-myb Proto-oncogene. (iii) NUMBER OF SEQUENCES: 18 (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: 07/956,698

(B) FILING DATE: 01 October 1992 (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Monaco, Daniel A.

(B) REGISTRATION NUMBER: 30,480

(C) REFERENCE/DOCKET NUMBER: 8321-3 (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:

TCCTGGTAGT GCAGCTCATC CAGATCCTCG 30 CAGCGCGTCC GCCGAGACAT 50

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 Nucleotides

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

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

CAGATCCTCG CAGCGCGTCC GCCGAGACAT 30

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 Nucleotides

(B) TYPE: nucleic acid

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AGATCCTCGC AGCGCGTCCG CCGAGACAT 29 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 Nucleotides

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

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GATCCTCGCA GCGCGTCCGC CGAGACAT 28

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 Nucleotides

(B) TYPE: nucleic acid

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ATCCTCGCAG CGCGTCCGCC GAGACAT 27

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

(A) LENGTH: 26 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

TCCTCGCAGC GCGTCCGCCG AGACAT 26

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCTCGCAGCG CGTCCGCCGA GACAT 25 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 Nucleotides

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

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CTCGCAGCGC GTCCGCCGAG ACAT 24

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 Nucleotides

(B) TYPE: nucleic acid

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TCGCAGCGCG TCCGCCGAGA CAT 23

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

(A) LENGTH: 22 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

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

CGCAGCGCGT CCGCCGAGAC AT 22

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GCAGCGCGTC CGCCGAGACA T 21 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 Nucleotides

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

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CAGCGCGTCC GCCGAGACAT 20

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 Nucleotides

(B) TYPE: nucleic acid

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AGCGCGTCCG CCGAGACAT 19

(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

GCGCGTCCGC CGAGACAT 18

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CGCGTCCGCC GAGACAT 17 (2) INFORMATION FOR SEQ ID NO:16

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 Nucleotides

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

(D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCGTCCGCCG AGACAT 16

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 Nucleotides

(B) TYPE: nucleic acid

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CGTCCGCCGA GACAT 15

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

(A) LENGTH: 25 Nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

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

ATGTCGGC GGACGCGCTG CGAGGAT 25

Claims

1. A-method for the treatment of a neoplastic disease characterized by the activation of B-myb expres¬ sion comprising administering to an individual in need of such treatment an effective amount of an oligo¬ nucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the B- myb gene, said oligonucleotide being hybridizable to said mRNA transcript.

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

3. A method according to claim 2 where¬ in the oligonucleotide does not hybridize to c-myb mRNA.

4. A method according to claim 2 where- in the oligonucleotide is an alkylphosphonate oligo- nucleoside or phosphorothioate oligonucleotide.

5. A method according to claim 2 where¬ in the oligonucleotide has a nucleotide sequence comple- mentary to a portion of the B-myb mRNA lying within about 50 nucleotides of the translation initiation codon.

6. A method according to claim 5 where- in the oligonucleotide is a phosphorothioate oligodeoxy¬ nucleotide or methylphosphonate oligodeoxynucleoside.

7. A method according to claim 2 where¬ in the oligonucleotide is an oligodeoxynucleotide having a deoxynucleotide sequence complementary to a portion of the B-mvb mRNA transcript including the translation initiation codon of said transcript.

8. A method according to claim 2 where- in the oligonucleotide comprises from a 12-mer to a 40- mer oligodeoxynucleotide.

9. A method according to claim 8 where¬ in the oligonucleotide is an alkylphosphonate oligo- nucleoside or a phosphorothioate oligonucleotide.

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

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

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

13. A method according to claim 5 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence of SEQ ID NO:l, or an at least 12- mer portion thereof.

14. A method according to claim 1 wherein the neoplastic disease is selected from the group consisting of malignant melanoma, neuroectodermal cancers, breast cancer, prostate carcinoma, colon cancer, renal car- cinoma, leukemia and lymphoma.

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

16. A method for purging bone marrow of neo¬ plastic cells comprising treating bone marrow cells aspirated from an individual afflicted with a neoplastic dis- ease characterized by the activation of B-mvb 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 B-myb gene, said oligonucleo- tide being hybridizable to said mRNA transcript, and returning the thus-treated cells to the body of the afflicted individual.

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

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

19. A method according to claim 17 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the B-mvb mRNA lying within about 50 nucleotides of the translation initiation codon.

20. A method according to claim 17 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.

21. An oligonucleotide comprising from 8 to 50 nucleotides which has a nucleotide sequence comple¬ mentary to at least a portion of the mRNA transcript of the B-myb gene, said oligonucleotide being hybridizable to said mRNA transcript.

22. An oligonucleotide according to claim 21 which does not hybridize to c-mvb mRNA.

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

24. An oligonucleotide according to claim 21 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the B-myb transcript lying within about 50 nucleotides of the translation initia¬ tion codon.

25. An oligonucleotide according to claim 21 wherein the oligonucleotide has a nucleotide sequence complementary to a portion of the B-mvb mRNA transcript including the translation initiation codon of said tran¬ script.

26. An oligonucleotide according to claim 21 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.

27. An oligonucleotide according to claim 26 wherein the oligonucleotide is from a 15-mer to a 30- mer.

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

29. An oligonucleotide according to claim 28 wherein the oligonucleotide is from a 18-mer to a 21- mer.

30. An oligonucleotide according to claim 21 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence corresponding to SEQ ID NO:l, or at least 12-mer portion thereof.

31. 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 B-myb gene, said oligonucleotide being hybridizable to said mRNA transcript.

32. A composition according to claim 31 wherein the oligonucleotide does not hybridize to c-mvb mRNA.

33. A composition according to claim 31 wherein the oligonucleotide is an alkylphosphonate oligonucleo- side or phosphorothioate oligonucleotide.

34. A composition according to claim 31 wherein the oligonucleotide has a nucleotide sequence complemen¬ tary to a portion of the B-myb mRNA transcript lying within about 50 nucleotides of the translation initia¬ tion codon.

35. A composition according to claim 31 wherein the oligonucleotide has a nucleotide sequence complemen¬ tary to a portion of the B-mvb mRNA transcript including the translation initiation codon of said transcript.

36. A composition according to claim 31 wherein the oligonucleotide comprises from a 12-mer to a 40-mer oligodeoxynucleotide.

37. A composition according to claim 36 wherein the oligonucleotide is from a 15-mer to a 30-mer.

38. A composition according to claim 37 wherein the oligonucleotide is from a 18-mer to 26-mer.

39. A composition according to claim 38 wherein the oligonucleotide is from a 18-mer to 21-mer.

40. A composition according to claim 31 wherein the oligonucleotide is an oligodeoxynucleotide having a nucleotide sequence corresponding to SEQ ID NO:l, or at least a 12-mer portion thereof.

41. An artificially-constructed gene comprising a transcriptional promotor segment and a segment con¬ taining a B-myb 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 B-myb gene.

42. A method according to claim 41 wherein the promotor segment comprises SV40 promotor.

43. A method for inhibiting the proliferation of neoplastic cells characterized by the activation of B-myb expression comprising introducing into such cells an artificially-constructed gene which, upon transcrip¬ tion in said cells, produces RNA complementary to the mRNA transcript of the B-mvb gene.

44. A method according to claim 43 wherein the artificially-constructed gene is introduced into said cells by transfection, by a transducing viral vector or by microinjection.