WO1991003260A1

WO1991003260A1 - Antisense oligonucleotides to c-abl proto-oncogene

Info

Publication number: WO1991003260A1
Application number: PCT/US1990/004961
Authority: WO
Inventors: Bruno Calabretta; Alan M. Gewirtz
Original assignee: Temple University Of The Commonwealth System Of Higher Education
Priority date: 1989-09-01
Filing date: 1990-08-29
Publication date: 1991-03-21
Also published as: EP0489846A1; AU646643B2; EP0489846A4; JPH05500217A; CA2065294A1; AU6410790A

Abstract

Oligonucleotides are provided having a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-abl gene. These ''antisense'' oligonucleotides are hybridizable to the c-abl mRNA transcript. Such oligonucleotides are useful in inhibiting proliferation of myeloid cells, particularly in myelo-proliferative disorders such as chronic myelogenous leukemia.

Description

ANTISENSE OLIGONUCLEOTIDES TO C-ABL PROTO-ONCOGENE

Field of the Invention

The invention relates to antisense oligonucleotides to proto-oncogenes, in particular to antisense oligonu- cleotides to the c-abl gene, and the use of such oligonu¬ cleotides to selectively inhibit proliferation of myeloid cells.

Background to the Invention The c-abl proto-oncogene encodes a protein with tyrosine kinase activity. Although the functional signi¬ ficance of the protein is unknown, it is well-established that more than 90% of chronic myelogenous leukemia (CML) patients have c-abl structural alterations in their leu¬ kocyte DNA. Also known as chronic granulocytic leukemia or chronic myeloid leukemia, CML is a clonal cancer aris¬ ing from neoplastic transformation of hematopoietic stem cells.

The structural alterations in leukocyte DNA are caused by the translocation of the c-abl gene from chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22 (t(9; 22)(q34; qll)) , and the resulting formation of a bcr-abl hybrid gene. The translocation results in a truncated chromosome 22, the so-called "Philadelphia chromosome". The fused bcr-abl gene is transcribed into a long primary transcript, which is spliced into a chimeric mRNA. The 8 kilobase (kb) chimeric mRNA is translated into a 210 kd bcr-abl protein unique to CML.

The most characteristic clinical feature of the chronic phase of CML is an increase of mature and imma¬ ture myeloid elements in bone marrow and peripheral blood. Terminal differentiation of cells is maintained, resulting in profoundly elevated counts of circulating mature granulocytes. Kinetic studies indicate that these abnormal cells do not proliferate or mature faster than their normal counterparts. Rather, the basic defect underlying the exuberant granulopoiesis in CML appears to be an expansion of the myeloid progenitor cell pool in bone marrow and peripheral blood. Galbraith et al., Br. J. Haematol. 22, 135 (1972). Although hematopoiesis in the chronic phase of CML is altered, it retains some normal features.

The c-abl proto-oncogene resides on the long arm of chromosome 9 (band q34) . Cloning of the c-abl gene has revealed that it spans at least 230 kb, and contains at least 11 exons. Two alternative first exons exist, name¬ ly exon la and exon lb. Exon la is 19 kb proximal to exon 2. Exon lb is more than 200 kb proximal to exon 2. As a result of this configuration, at least two major c- abl messages are transcribed. Each of exons la and lb are preceded by a transcriptional promotor.

The two distinct c-abl mRNAs differing in their 5' regions have been identified. Shtivelman et al.. Cell 47, 277 (1986); Bernards et al., Mol. Cell. Biol. 7, 3231 (1987) . The 6-kb transcript consists of exons la through 11. The 7-kb transcript begins with exon lb, skips the 200 kb distance to exon 2, omits exon la, and joins to exons 2 through 11. Thus, both c-abl messages share a common set of 3^• exons, starting from the c-abl exon 2. Consequently, the messages code for two proteins that share most of their amino acid sequence, except for the N-termini. Since the coding begins with the first exon, exonic selection will determine the protein product.

While at least two major c-abl messages are trans- cribed, to date only one normal c-abl protein has been identified, a tyrosine protein kinase having a molecular weight of approximately 145 kd.

While antisense RNA probes hybridizable with c- abl mRNA have been used to detect c-abl transcription, Klimfinger et al., Virchos Archiv. B-Cell Phathol. 54, 256-259 (1988), Griel et al.. Lab. Invest. 60, 574-582 (1989) , c-abl antisense has not heretofore been recog¬ nized as being useful for selectively inhibiting myeloid cell proliferation.

Summary of the Invention

Antisense oligonucleotides and pharmaceutical com¬ positions thereof with pharmaceutical carriers are pro¬ vided. Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA tran- script of the human c-abl gene. The oligonucleotide is hybridizable to the mRNA transcript. Preferably, the oligonucleotide is at least a 15-mer oligodeoxynucleo- tide, that is, an oligomer containing at least 15 deoxy- nucleotide residues. Most preferably, the oligodeoxy- nucleotide is a 15- to 21-mer. While in principle oligo¬ nucleotides having a sequence complementary to any region of the c-abl gene find utility in the present invention, oligodeoxynucleotides complementary to a portion of the c-abl mRNA transcript beginning with the second codon from the 5* end of the transcript are particularly pre¬ ferred.

As used in the herein specification and appended claims, unless otherwise indicated, the term "oligo¬ nucleotide" includes both oligomers of ribonucleotide i.e., oligoribonucleotides, and oligomers of deoxyribo- nucleotide i.e., oligodeoxyribonucleotides (also referred to herein as "oligodeoxynucleotides") .

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 "oligodeoxynucleo- tide" include not only oligomers and polymers of the biologically significant nucleotides, i.e. nucleotides of adenine ("A"), deoxyadenine ("dA") , guanine ("G") , deoxy- guanine ("dG") , cytosine ("C") , deoxycytosine ("dC") , thymine ("T") and uracil ("U") , but also oligomers and polymers hybridizable to the c-abl mRNA transcript which may contain other nucleotides. Likewise, the terms "oligonucleotide" and "oligodeoxynucleotide" include oligomers and polymers wherein one or more purine or pyrimidine moieties, sugar moieties or internucleotide linkages is chemically modified.

The term "c-abl mRNA transcript" means either or both of the presently known mRNA transcripts of the human c-abl gene, or any further transcripts which may be elucidated.

The invention provides a method for inhibiting proliferation of myeloid cells comprising administering to an individual or cells harvested from the individual, c-abl antisense oligonucleotide.

Detailed Description of the Invention We have discovered that the c-abl gene plays a critical role in regulating normal human hematopoiesis, and that its function is lineage-specific. We have found that exposure to c-abl antisense oligonucleotides, that is, oligonucleotides complementary to and hybridizable with the mRNA transcript of the human c-abl gene, effects two major populations of hematopoietic cells differently. Specifically, we have discovered, quite unexpectedly, that c-abl antisense oligonucleotides inhibit myeloid, but not erythroid cells. This differential sensitivity makes possible the use of c-abl antisense to treat dis- orders such as CML which are characterized by the expan¬ sion of the myeloid progenitor cell population.

The putative partial DNA sequence complementary to the mRNA transcript of the human c-abl gene has been reported in Shtivelman et al.. Cell 47, 277-284 (1986), the entire disclosure of which is incorporated herein by reference. The nucleotide sequence and predicted amino acid sequence of the open reading from the initiation codon are set forth in Figure IB of Shtivelman et al. The open reading frame spans the region between nucleo- tides 148 and 3537 of the cDNA and codes for a protein of 1130 amino acids.

The antisense oligonucleotides of the invention 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 Verlags- gesellschaft mbH (H. Ibelgaufts trans. 1987) .

Any of the known methods of oligonucleotide syn¬ thesis may be utilized in preparing the instant antisense oligonucleotides.

The antisense oligonucleotides are most advantag¬ eously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers, for ex¬ ample, the Applied Biosystems 380B DNA Synthesizer, which utilizes y9-cyanoethyl phosphoramidite chemistry.

Since the complete nucleotide synthesis of DNA complementary to the c-abl mRNA transcript is known, antisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by the oligonucle- otide synthesis methods known to those skilled in the art.

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

Oligonucleotides complementary to and hybridizable with any portion of the c-abl mRNA transcript are, in principle, effective for inhibiting translation of the transcript, and capable of inducing the effects herein described. It is believed that translation is most effectively inhibited by blocking the mRNA at a site at or near the initiation codon. Thus, oligonucleotides complementary to the 5'-terminal region of the c-abl mRNA transcript are preferred. The oligonucleotide is prefer¬ ably directed to a site at or near the initiation codon for protein synthesis. Oligonucleotides complementary to the c-abl mRNA, beginning with the codon adjacent to the initiation codon (the second codon from the 5' end of the transcript) , may be thus advantageously employed. Since there are at least two mRNA c-abl transcripts, a 6.0 kb transcript containing exon la, and a 7.0 kb transcript containing alternative exon lb, two sets of preferred 15- 21 nucleotide oligomers are possible.

The following 15- through 21-mer oligodeoxynucleo- tides are complementary to the 6.0 c-abl mRNA transcript beginning with the second codon of the transcript: 5'-CAG CTT CAG GCA GAT CTC CAA-3•

5'-AG CTT CAG GCA GAT CTC CAA-3'

5'-G CTT CAG GCA GAT CTC CAA-3'

5'-CTT CAG GCA GAT CTC CAA-3' 5'-TT CAG GCA GAT CTC CAA-3'

5'-T CAG GCA GAT CTC CAA-3'

5'-CAG GCA GAT CTC CAA-3'

The following 15- through 21-mer oligodeoxynucleo- tides are complimentary to the 7.0 kb c-abl mRNA tran- script beginning with the second codon of the transcript:

5'-TAC TTT TCC AGG CTG CTG CCC-3•

5'-AC TTT TCC AGG CTG CTG CCC-3'

5'-C TTT TCC AGG CTG CTG CCC-3'

5'-TTT TCC AGG CTG CTG CCC-3' 5'-TT TCC AGG CTG CTG CCC-3'

5'-T TCC AGG CTG CTG CCC-3' 5'-TCC AGG CTG CTG CCC-3' In addition to blocking translation of the c-abl transcript with oligonucleotides complimentary to the 5'- terminal regions of either the 6.0 kb or 7.0 kb trans¬ cripts, translation may be effectively blocked by oligo¬ nucleotides complimentary to common sequences shared by both transcripts. In particular, oligonucleotides complimentary to and hybridizable with any portion of the transcript containing the common exon 2 may be utilized. For example, the following 15- through 21-mer oligodeoxy- nucleotides complimentary to a region of exon 2 beginning with the second codon thereof is a preferred embodiment of the invention: 5'-TGC TAC TGG CCG CTG AAG GGC-3'

5'-GC TAC TGG CCG CTG AAG GGC-3' 5'-C TAC TGG CCG CTG AAG GGC-3' 5'-TAC TGG CCG CTG AAG GGC-3' 5'-AC TGG CCG CTG AAG GGC-3' 5'-C TGG CCG CTG AAG GGC-3' 5'-TGG CCG CTG AAG GGC-3' Oligonucleotides hybridizable to the c-abl mRNA transcript finding utility according to the present invention include not only native oligomers of the biologically significant nucleotides, i.e.. A, dA, G, dG, C, dC, T and U, but also oligonucleotide species which have been modified for improved stability and/or lipid solubility. For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting a methyl group or sulfur atom for a phosphate oxygen in the internucleotide phosphodiester linkage. The phosphorothioates, in particular, are stable to nuclease cleavage and soluble in lipid. They may be synthesized by known automatic synthesis methods.

The antisense oligonucleotides of the invention inhibit human myelopoiesis. However, they do not affect erythropoiesis. This pharmaceutically significant dif¬ ferential sensitivity makes the instant oligonucleotides very useful in treating myeloproliterative disorders.

Myeloproliterative disorders refer to certain diseases in which the marrow and sometimes hematopoietic stem cells in extramedullary sites proliferate more or less en masse. The proliferation is self-perpetuating, resembling neoplastic disease. Such disorders -include for example, CML, polycythemia vera, myelofibrosis with myeloid metaplasia, and essential (idiopathic) throm- bocythemia.

CML, in particular, is characterized by abnormal proliferation of immature granulocytes - neutrophils, eosinophils, and basophils - in the blood, the bone marrow, the spleen, the liver, and sometimes other tissues. The essential feature is accumulation of granulocytic precursors in the blood, bone marrow, and spleen. The patient who presents symptoms will charac¬ teristically have more than 20,000 white blood cells per μl, and the count may exceed 400,000. Some 60 to 80 percent of CML patients will develop "blast crisis", the terminal stage of the disease during which immature blast cells rapidly proliferate, leading to patient death. Antisense oligomers to the c-abl proto-oncogene are use¬ ful for controlling or arresting such myeloproliterative disorders, in particular in arresting the abnormal myelo- poiesis which characterizes CML. We have found that substantial, specific reduction in myeloid cell prolifer¬ ation results from treatment of normal and abnormal cells, with little or no effect on erythroid cells. The sparing of erythroid lineage cells is not without sig¬ nificance, since individuals afflicted with myelopro- 1iterative disorders in many cases suffer from anemia of varying degree, due to the crowding out of erythroid cells in response to myeloid expansion. Moreover, anemia results from prolonged chemotherapeutic treatment of myeloproliterative disorders with conventional chemical agents. The anemia is typically treated by transfusion therapy, which is expensive and not without possible short and long term side effects. Treatment with c-abl antisense oligonucleotide permits the substantial reduc¬ tion of myeloid cell numbers, without sacrificing erthy- roid cells and aggravating the anemic condition.

For in vivo use, a myeloid cell proliferation inhibiting-amount of the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liquid vehicles and excipients are conventional and commercially available. Illustrative thereof are distilled water, physiological saline, aqueous solution of dextrose, and the like. The c-abl mRNA antisense oligonucleotides are preferably administered intravenously.

While inhibition of c-abl mRNA translation is pos¬ sible utilizing either antisense oligoribonucleotides or oligodeoxyribonucleotides, oligoribonucleotides are more susceptible to enzymatic attack by ribonucleases than deoxyribonucleotides. Hence, oligodeoxyribonucleotides are preferred in the practice of the present invention.

In addition to administration with conventional carriers, the antisense oligonucleotides may be adminis¬ tered by a variety of specialized oligonucleotide deliv¬ ery techniques. For example, oligonucleotides have been successfully encapsulated in unilameller liposomes. Reconstituted Sendai virus envelopes have been success- fully used to deliver RNA and DNA to cells. Arad et al., Biochem. Biophy. Acta. 859, 88-94 (1986).

The c-abl antisense oligonucleotides may be admin¬ istered to an individual suffering from a myeloprolifera- tive disorder in an amount sufficient to inhibit prolif- eration of myeloid lineage cells. Generally, it will be desirable to administer sufficient oligonucleotide to result in substantial reduction of the myeloid cell population without significantly affecting erythroid cell numbers. The actual dosage administered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health and sex of the patient, the route of administration, and other factors. The daily dosage may range from about 0.1 mg to 1 g oligonuc- leotide per day, preferably from about 10 to about 1,000 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required. Based upon the experi¬ ments hereinafter described, a dosage sufficient to provide a plasma antisense oligonucleotide concentration of about 14 μl may be utilized. Other dosages will be apparent to those skilled in the art by routine experi¬ mentation.

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

EXAMPLE 1

Effect of c-abl antisense oliσomer on bone marrow cells. The following experiment was performed to es¬ tablish the lineage-specific inhibitory effect of c-abl antisense oligonucleotide on cells. Adherent- and T-cell depleted low density bone marrow cells were exposed to the following oligomer preparations, final concentration 14 μM, for 15-18 hours:

(i) the c-abl antisense 18-mer, 5'-TTT TCC AGG TGC CTG CCC-3• , which is complementary to the 7.0 kb c-abl mRNA transcript beginning with the second codon (hereinafter "c-abl 2") ;

(ii) the c-abl antisense 18-mer, 5'-CTT CAG GCA GAT CTC CAA-3' , which is complementary to the 6.0 kb c-abl mRNA transcript beginning with the second codon (hereinafter "c-abl 4") ;

(iii) the c-abl antisense 18-mer, 5'-TAC TGG CCG CTG AAG GGC-3' , which is complementary to 18 nucleo¬ tides of the second exon of c-abl (codons 2 through 7) , which is common to both c-abl mRNAs (hereinafter "c-abl 6") ;

(iv) the 18-mer sense oligomer corresponding to σ-abl 2, having the sequence 5'-GGG CAG CAG CCT GGA AAA-3' (hereinafter "c-abl 1") ;

(v) the 18-mer sense oligomer corresponding to c-abl 4, having the sequence 5'-TTG GAG ATC TGC CTG AAG-3' (hereinafter "c-abl 3") ;

(vi) the 18-mer sense oligomer corresponding to c-abl 6, having the sequence 5*-GCC CTT CAG CGG CCA GTA-3' (hereinafter "c-abl 5"); (vii) the bcr antisense 18-mer, 5'-GAA GCC CAC CGG GTC CAC-3* , which is complementary to a region from the second to the seventh codon of the bcr mRNA tran¬ script (hereinafter "bcr 2") ; and (viii) the 18-mer sense oligomer corresponding to bcr 2, having the sequence 5*-GTG GAC CCG GTC GGG TIC¬ S' (hereafter "bcr 1").

The cells (2.5 x 10* cells) were plated in 1 ml of IMDM supplemented with 30% fetal bovine serum, 5 x 10^""ε jS-2-mercaptoethanol and 0.9% methylcellulose and cultured in the presence of optimum concentration of the growth factors listed below. Each growth factor is specific for the indicated cell subset:

(a) Colony forming unit-erythroid cells ("CFU-E") : 3 ϋ/ml recombinant erythropoietin ("rh Epo") ;

(b) Burst-forming unit-erythroid cells ("BFU- E"): 3 U/ml rh Epo, 5 ng/ml granulocyte-macrophage colony stimulating factor ("GM-CSF") , and 20 ϋ/ml interleukin 3 ("IL-3") ; (c) Colony forming unit-granulocyte-macro- phage ("CFU-GM") : 0.3% agar in the presence of 10 ng/ml GM-CSF and 20 ϋ/ml IL-3;

(d) Colony forming unit-granulocytes ("CFU- G") : 10% conditioned medium of Chinese hamster ovary cells producing granulocyte-colony stimulating factor ("G-CSF") (Tweardy et al., Oncogene Res. 1, 209 (1987)). The conditioned medium was obtained by introducing by transfection a human G-CSF cDNA into Chinese hamster ovary cells. Twenty-four hours following transfection, the supernatant containing secreted G-CSF was collected and used as a source of G-CSF at 10%.

CFU-E and CFU-G colonies were scored after seven and nine days of growth, respectively. CFU-GM and BFU-E colonies were scored after fourteen days of culture. As set forth in Table 1, exposure of bone marrow mononuσlear cells to c-abl antisense oligomers did not effect erythroid colony formation deriving from BFU-E and CFU-G progenitors, but markedly inhibited (10% to 20% of residual growth in comparison to controls) myeloid colony formation deriving from CFU-G and CFU-GM progenitors. In addition, the residual myeloid colonies were much smaller than those formed in the presence of c-abl sense oligo¬ mers. A bcr antisens oligomer did not have any effect on colony number or colony size.

TABLE

01igodeoxynucleotide

CONTROL

(no oligomer added) c-abl 1 (sense) c-abl 2 (antisense)

CONTROL c-abl 3 (sense) c-abl 4 (antisense)

CONTROL c-abl 5 (sense) σ-abl 6 (antisense)

CONTROL

bcr 1 (sense) bcr 2 (antisense)

¹ Values represent mean ± standard deviation of quadruplicate control σultures (no oligodeoxynucleotide added) and duplicate experimental σultures from three separate experiments for eaσh σolony type. EXAMPLE 2 Effect of σ-abl oligomer on CD34⁺ σells. We analyzed the effeσt of σ-abl antisense oligomers on the growth of marrow progenitors seleσted on the basis of their expression of the MylO antigen (CD34⁺ σells) (Civan et al., J. Immunol. 133, 157 (1984)). This population is riσh in primitive BFU-E and CFU-GM (Brandt et al., J. Clin. Invest. 82, 1017 (1988)), but does not σontain CFU- E or CFU-G progenitors. Based on the model that hemato- poiesis is a developmental σontinuu , MylO⁺ progenitors σorrespond to a more homogeneous, less mature population of σolony-forming units than the population assayed from partially purified bone marrow σells in Example 1.

4 x 10^s Myl0⁺ σells were isolated by immunoro- setting as desσribed by Civin et al., J. Immunol. 133, 157 (1985) and plated in σulture dishes. The experimen¬ tal σonditions were as desσribed in Example 1. GFU-GM σolonies were obtained after fourteen days from σultures stimulated by GM-CSF (10 ng/ml) and IL-3 (100 U/ml) . BFU-E σolonies were obtained after fourteen days from σultures stimulated by GM-CSF (10 ng/ml, IL-3 (100 U/ml) and rh Epo (3 ϋ/ml). As set forth in Table 2, it was observed that σ-abl antisense oligomers inhibited the formation of myeloid (CFU-GM) growth, but did not effeσt primitive erythroid σolony (BFU-E) growth. A bcr anti- sense oligomer did not have any effeσt on σolony number or σolony size. TABLE 2

Colonies or clusters found

CFU-GM

75±5

70±4

20±2

70±3

62±2

10±3

51±3

46±3

10±2

68±5

88±8

86+10

Effect of σ-abl oligomer on normal peripheral blood progenitors.

Peripheral blood progenitors are antigeniσally distinct from, and less differentiated than, progenitors found in the bone marrow. Ferrero et al., Proc. Natl. Sci. USA 80, 4114 (1983) . CFU-GM σolonies were grown in the presenσe of reσombinant GM-CSF and IL-3 from adherent- and T-σell depleted peripheral blood mononuσlear σells after sixteen days of σulture. CFU-GM σolonies formed from peripheral blood progenitors in the presenσe of the σ-abl antisense oligomer were indistinguishable from those derived from similarly treated bone marrow progenitors, and were muσh smaller than progenitors arising in the presenσe of σ-abl sense oligomer. In addition, the number of σolonies formed was inhibited essentially to the same degree (75% to 85%) as that observed for bone marrow progenitors. Growth of erythroid progenitors was inhibited slightly more (20% to 25%) than we had observed for bone marrow erythroid progenitors (10% to 20%) .

The above experiments indiσate that the effeσt of c-abl antisense on progenitor cells is lineage-specifiσ. The σ-abl funσtion is required for the formation of myeloid σolonies, but is apparently unneσessary for the formation of erythroid σolonies. In addition, the above experiments indiσate that σ-abl's funσtional requirements are indepen¬ dent of proliferative aσtivity and differentiation stage of myeloid progenitor σells.

The effeσt of σ-abl antisense oligomers on myeloid σolony formation was observed with either abl 2 or abl 4, whiσh are σomplimentary to the first respeσtive exons of the 6.0 and 7.0 σ-abl mRNAs. Sinσe the two known speσies of σ-abl mRNA differ only in the region σorresponding to the two distinσt first exons (la and lb) of the σ-abl gene, our results suggest that expression of both the 6.0 and 7.0 kb transσripts are required for myelopoiesis. Inhibition of either transσript inhibits myeloid prolifera- tion.

C-abl antisense oligomer σomplementary to the common seσond σ-abl exon was also found to inhibit the expression of the hybrid bcr-abl product in K562 cells, a CML cell line σontaining multiple σopies of the hybrid bσr- abl gene. The K562 line has been isolated from a Philadel¬ phia σhromosome positive patient with CML in blast σrisis. In these leukemiσ σells, the σ-abl seσond exon is spliσed to bσr exons "2" and "3", Shtivelman el al.. Cell 47, 277 (1986) . While bcr-abl protein levels were unaffeσted by exposure to a σ-abl sense oligomer (abl 5) by an immuno- fluoresσenσe assay (Gewirtz et al.. Science 242, 1303 (1988) , protein levels were significantly reduσed in the presenσe of σ-abl antisense oligomer (abl 6) . The present invention may be embodied in other speσifiσ forms without departing from the spirit or essential attributes thereof and, aσσordingly, referenσe should be made to the appended σlaims, rather than to the foregoing speσifiσation, as indiσating the scope of the invention.

Claims

CIAIMS

1. A pharmaσeutiσal σomposition σomprising a pharmaσeutiσal σarrier and an oligonuσleotide whiσh has a nuσleotide sequenσe σomplementary to at least a portion of the mRNA transσript of the human σ-abl gene, said oligo¬ nuσleotide being hybridizable to said mRNA transσript.

2. A σomposition aσσording to σlaim 1 wherein the oligonuσleotide is an at least 15-mer oligodeoxynuσleotide.

3. A σomposition aσσording to σlaim 1 or 2 wherein the oligodeoxynuσleotide has a deoxynuσleotide sequenσe σomplementary to a portion of the σ-abl mRNA transσript beginning with the seσond σodon from the 5* end of said transσript.

4. A σomposition aσσording to σlaim 2 or 3 wherein the oligodeoxynuσleotide is from a 15-mer to a 21- mer.

5. A σomposition aσσording to σlaim 3 wherein the deoxyoligonuσleotide is hybridizable to the σ-abl about 6.0 kb mRNA transσript and is seleσted from the group of deoxyoligonuσleotides σonsisting of:

5'-CAG CTT CAG GCA GAT CTC CAA-3' ,

5'-AG CTT CAG GCA GAT CTC CAA-3',

5'-G CTT CAG GCA GAT CTC CAA-3' ,

5'-CTT CAG GCA GAT CTC CAA-3' ,

5*-TT CAG GCA GAT CTC CAA-3',

5'-T CAG GCA GAT CTC CAA-3' and

5'-CAG GCA GAT CTC CAA 3'.

6. A σomposition aσσording to σlaim 5 wherein the oligodeoxynuσleotide is 5'-CTT CAG GCA GAT CTC CAA-3* .

7. A σomposition aσσording to σlaim 3 wherein the deoxyoligonuσleotide is hybridizable to the σ-abl about 7.0 kb mRNA transσript and is seleσted from the group of deoxyoligonuσleotides σonsisting of: 5'-TAC TTT TCC AGG CTG CTG CCC-3' ,

5•-AC TTT TCC AGG CTG CTG CCC-3' ,

5'-C TTT TCC AGG CTG CTG CCC-3',

5'-TTT TCC AGG CTG CTG CCC-3',

5'-TT TCC AGG CTG CTG CCC-3^» ,

5'-T TCC AGG CTG CTG CCC-3' and

5'-TCC AGG CTG CTG CCC-3' .

8. A composition acσording to σlaim 7 wherein the oligodeoxynuσleotide is 5'-TTT TCC AGG CGG CTG CCC-3*.

9. A method for inhibiting proliferation of p.yeloid σells σomprising administering to an individual an oligonuσleotide whiσh has a nuσleotide sequenσe σomplemen¬ tary to at least a portion of the mRNA transσript of the human σ-abl gene, said oligonuσleotide being hybridizable to said mRNA transσript.

10. A method aσσording to σlaim 9 wherein the oligonuσleotide is an at least 15-mer oligodeoxynuσleotide.

11. A method aσσording to σlaim 9 or 10 wherein the oligodeoxynuσleotide has a deoxynuσleotide sequenσe σomplementary to a portion of the σ-abl mRNA transσript beginning with the seσond σodon from the 5' end of said transσript.

12. A method aσσording to σlaim 10 or 11 wherein the oligodeoxynuσleotide is from a 15-mer to a 21-mer.

13. A method aσσording to σlaim 11 wherein the deoxyoligonuσleotide is seleσted from the group of deoxyoligonuσleotides σonsisting of:

5'-CAG CTT CAG GCA GAT CTC CAA-3',

5'-AG CTT CAG GCA GAT CTC CAA-3',

5'-G CTT CAG GCA GAT CTC CAA-3• ,

5'-CTT CAG GCA GAT CTC CAA-3',

5'-TT CAG GCA GAT CTC CAA-3• ,

5'-T CAG GCA GAT CTC CAA-3' and

5'-CAG GCA GAT CTC CAA 3' .

14. A method aσσording to σlaim 13 wherein the oligodeoxynuσleotide is 5'-CTT CAG GCA GAT TCT CAA-3*.

15. A method aσσording to σlaim 11 wherein the deoxyoligonuσleotide is seleσted from the group of deoxy¬ oligonuσleotides σonsisting of:

5'-TAC TTT TCC AGG CTG CTG CCC-3' ,

5'-AC TTT TCC AGG CTG CTG CCC-3',

5 --C TTT TCC AGG CTG CTG CCC-3' ,

5'-TTT TCC AGG CTG CTG CCC-3',

5'-TT TCC AGG CTG CTG CCC-3' ,

5'-T TCC AGG CTG CTG CCC-3' and

5'-TCC AGG CTG CTG CCC-3' .

16. A method aσσording to σlaim 15 wherein the oligodeoxynuσleotide is 5'-TTT TCC AGG CGG CTG CCC-3• .

17. A method aσσording to σlaim 9 for inhibiting myeloid σell proliferation in an individual affliσted with σhroniσ myelogenous leukemia.