CA2065294A1 - Antisense oligonucleotides to c-abl proto-oncogene - Google Patents

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

q ~l 2 ~ PCT/US90/0~961 ., 1 .

ANrI5EN5~ OLI~ON~CLEOTIDES TO C-ABL PROTO-ONCOGENE

Field of the Invention The invention relates to antisense oligonucleotides to proto-oncogenes, in particular to antisense oligonu-cl~otides 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-oncogen~ 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 ~rom neoplastic transformation of he~atopoie~ic stem cells.
The structural alterations in leukocyte DNA are causQd by the translocation o~ the c~abl g~ne from chromosome 9 to the breakpoint cluster region ~bcr) on chromosome 22 (t(9: 22)(q34: qll~, and the resulting ~ormatio~ o~ a ~cr-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 - ..~. ~ .~. -.
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WO91/03260 P~T/USg~/049~1 f~
2~ ~5 2 ~ ~ -2-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 o~ 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. ~aematol. 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 lg kb proximal to exon 2. Exon lb is more than 200 kb proximal to exon 2.
As a result o~ 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-~b transcript begins with exon lb, skips the 200 kb distance to ~xon 2, omits exon la, and joins to exons 2 through 11. Thus, both c-~bl messages share a common set of 3' exons, starting from the c-abl 8xon 2.
Consequently, the messages code for two proteins that ,~". . ' . '-, - ~ :

WO91/03260 ~ PCT/US90/04961 2~ ~L

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 ha~Ying a molecular weight of approximately 145 kd.
While antisense RNA probes hyhridizable with c-abl mRNA have been used to detect c-abl transcription, Klimfinyer et al., Virchos Archiv. B-Cell Phathol. 54, 256-259 (1988), Griel et al., Lab. Inve~t~ 60, 574-582 (1989), c-abl antisense has not heretofore been recog-nized as being useful for selectively inhibiting myeloid cell proliferation.

_ummary 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 ~RNA tran-script of the human c-abl gene. The oligonucleotide is hybridizable to the m~NA 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 frcm the 5' end of the transcript are particularly pre-~erred.
As used in the herein specification and appended claims, unless otherwise indicated, th~ term "oligo-nucleotide1' include both oligomers of ri~onucleotide 3~6~ PCT/US90/04961 h~

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i.e., oligoribonucleotides, and oligomers of deoxyribo-nucleotide i.e., oligodeoxyribonucleotides (also referred to herein as "oligodeoxynucleotides").
As used herein, unless otherwise indicated, the tarm "oligonucleotide" also includes oligomers which may be large enough to be termed "polynucleotides".
The terms "oligonucleotide'l 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 th~ human c-abl gene, or any further transcripts whi~h 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.
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etailed 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 differ~ntly.

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WO9l/03260 PCT/US90/04961 ~5~

Specifically, we have discovered, guite unexpectedly that c-abl antisense oligonucleotides inhibit myeloid, but not erythroid cells. This differenl:ial sensitivity makes possible the use of c-abl antisense to treat dis-orders such as CNL which are characterized by the expan-sion of the myeloid progenitor cell population.
The putative partial DNA sequence complementary to the m~N~ transcript of the human c-abl gene has been reported in Shtivelman et al., Cell ~7, 277-~84 (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 lB 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, ~r~ 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, ~or ex-ample, the Applied Biosystems 380B DNA Synthesizer, which ukilizes ~-cyanoethyl phosphoramidite chemistry.
Since the complete nucleotide synthesis of DNA
complementary to the c-abl ~RNA transcript is known, antisen~e oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by the oligonucle-.
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WO91/~3260 PCT/US90/04961 ~ 6-otide synthesis methods known to those skilled in the art.
While any length oligonucleotide may be utilized in the practice of the invention, sequences ~horter than 15 bases may be less speci~ic in hybridi~ing 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 o~ 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, e~fective for inhibiting translation of the transcript, and capable of inducing the effects herein described. It i5 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 complemen~ary 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 khere are at least two ~RNA 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 o~ the transcript:

-.
:
. . ~ , : -WO91/03260 ~ ~ 2~ ~ PCT/USg~/04~6l 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 mRN~ tran-10script 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' 155'-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'-20terminal regions of either the 6.0 kb or 7.0 kb tr~ns-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 25transcript containing the co~mon exon 2 may be utili2ed.
For example, the following 15- through 21-mer oligodeoxy-nucleotides compliment~ry to a region o~ exon 2 beginning with the second codon thereof is a preferred embodiment of the invention:
305 ? -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' WO 91/032~i0 PCI/IJS9~/04961 i' " ' ' '`

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5'-TAC TGG CCG CTG AAG GGC-3' 5l-AC TGG CCG CTG AAG GGC-3l 5'-C TGG CCG CTG AAG GGC-3' 5'-TGG CCG CTG AAG GGC-3' 5Oligonucleotides hybridi~able to the c-abl mRNA
transcript finding utility according t:o the present invention include not only native oligomers of the bioloyically signi~icant nucleotides, i.e~, A, dA, G, dG~
C, dC, T and U, but also oligonucleotidle species which have been modified for improved stability and/or lipid solubility. For example, it is ~nown 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 b~ synth~sized 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 use~ul in treating myeloproliferative disorders.
Myeloproliferative 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 - ~ - .

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W091/03260 PCT/US9~/04961 f ~
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granulocytic precursors in the blood, bone marrow, and spleen. The patient who presents symptoms will charac-teristically have more than 20,000 white bloo~ cells per ~1, and the count may exceed 400,000. Some 60 to 80 percent o~ C~L 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 ~-abl proto-oncogene are use-ful for controlling or arresting such myeloproliferative disorders, in particular in arresting the abnormal myelo-poiesis which characterizes CML. We have found that substantial, speci~ic reduction in myeloid cell prolifer-ation rasults from treatment o~ normal and abnormal cells, with little or no effect on erythroid cells. The sparing o~ erythroid lineage cells is not without sig-nificance, since individuals afflicted with myelopro-liferative 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 myeloproliferative disorders with conventional chemical agents. The anemia is typically treated by transfusion th2rapy, 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 iD 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 ~onventional and commercially available.
Illustrative thereof are distilled water, physiological saline, aqueous solution of dextrose, and the like. The WO91/03260 PCT/~S90/04961 lo- !

c-abl mRNA antisense oligonucleotides are preferably administered intravenously.
While i~hibition of c-abl mRNA translation is pos~
sible utilizing either antisense oligoribonucleotides or oligodeoxyribonucleotides, oligoribonucleotides are more susceptible to enzymatic attack by ribonuclaases than deoxyribonucleotides. Hence, oligodeoxyribonucleotides are preferred in the practice of the present invention.
In addition to administration with conventional 19 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 liposomesO
Reconstituted Sendai virus envelopes have been success-fully used to deliver RNA and DNA to cells. Arad et al., Biochem. Biophy. Acta. 359, 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 suf~icient oligonucleotide to result in substantial reduction of the myeloid cell population without significantly affecting erythroid cell numbers. The actual dosage administared 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 Ool mg to 1 y oligo~uc-leotide per day, preferably ~rom about lO to about l,OO0 mg per day. Greater or les~er amounts of oligonucleotide may be administered, as required. Based upo~ the experi-ments hereinafter described, a dosage sufficient to provide a plasma antisense oligonucleotide concentration o~ about 14 ~M may be utilized. Other dosages will be WV91/03260 ~ PCT/US90/Q4961 ~ '' ' . .
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apparent to those skilled in the axt by routine experi mentatio~. .
The prPsent invention is described in greater detail in the followin~ non-limiting examples.

E L~ 1 Effect of c-abl_antisense oliqomer on bone marrow call~. The following experiment was per~ormed to es-tablish the lineage-specific inhibitory ef~ect o~ c-abl antisense oligonucleotide on cells. Adhexent- and T-cell depleted low density bone marrow cells were exposed to the following oligomer preparations, final concentration 14 ~M, for 15-18 hoursO
(i) the c-abl antisense 18-mer, 5'-TTT TCC
AGG TGC CTG CCC-3', which is complement~ry to the 7.0 kb c-abl mRNA transcript beginning with the second codon (hereinafter "c-abl 2");
(ii) the c-abl antisen~e 18-mer, 5'-CTT CAG
GCA GAT CTC CAA-3l, 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 lB nucleo-tides of the second exon of c abl (codons 2 through 7), which is common to both c abl mRNAs (hereinafter "c-abl 2~ 6");
(iv) the 18-mer sense oligomer corresponding to c-abl 2, having the sequ~nce 5' GG~ 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 3l7);
(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");

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WO~/03260 9 PCT/US90/0496a ~v~k~ -12~

~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 I .
script (hereinafter "bcr 2"); and (viii) the 18-mer sense oligomer corresponding to bcr 2, having the se~uence 5'-GTG GAC CIC~ GTC GGG TTC-3' (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 ~
~-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 t"CFU-E"): 3 U/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-CSFIl), and 20 U/ml interleukin 3 ("IL-3");
(c~ Colony forming unit-granulocyte-macro-phage ("CFU-GMI'): 0.3% agar in the presence of 10 ng/ml GM-CSF and 20 U/ml IL-3;
(d) Colony forming unit-granulocytes ("CFU-G'l): 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 obtain~d 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 lQ%.
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.

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WO9l/03260 PCT/US90/04961 -13- 20~2~ !

As set forth in Table 1, exposure of bone marrow mononuclear cells to c-abl antisense ol:igomers did not effect erythroid colony formation deriving from BFU-E and CFU-G progenitors, but markedly inhibited (10% to 20~ o~~.
residual growth in comparison to controls) myeloid colony formation deriving from CFU-G and CFU-GM progenitors. In ~ddition, the residual myeloid colonies we.re 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: 1 Colonies or clusters found Oliqodeo~ynucleotide BFU-E CFU-E CFU G~ CFU-G
CONTROL
(no oligomer added) 54.21 298~20 120~5 327~38 c-abl 1 (sense) 51~8 276~31 98~10 298~12 c abl 2 (antisense) 48~6 258~18 24+5 75+10 CONTROL 60~8 280116 113~4 300~10 c-abl 3 (sense) 55~10 274~12 120~10 285~8 c-abl 4 (antisense) 52~8 268~10 16~8 60+10 CO~TROL 75+5 294+30 93~5 280_38 c-~bl 5 (sense) 56~8 255_28 74~24 270~25 c-abl 6 (antisense) 45~4 246~27 8~458~10 CONTROL 65~10 290~18 138~10 280~10 bcr 1 (sense) 68~4 320~12 120~8 270+20 bcr 2 (anti~ense) 66~8 340~20 135~12 320~20 1 Values represent mean ~ standard de.viation of quadruplicat2 control cultures (no oligodeoxynucleotide added~ and duplicate experimental cultures from three separate experiments for each colony type.

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W091/03260 ~, PCT/U~0/04~6~
~ Q - 14-EXAMPLE ?
Effect of c-abl oliqomer on CD34~ cells. We analyzed the effect of c-abl antisense oligomers on the growth of marrow proyanitors selected on the basis of their expression of the MylO antigen (CD34~ cells) (Civan et al., J. Immunol. 133, 157 (1984)). This population is rich in primitive BFU-E and CFU-GM (Br,andt et al., JO
Clin. Invest. 82, 1017 (19~8)), but does not contain CFU~
E or CFU-G progenitors. Based on the model that hemato-poiesis is a developmental continuum, MylO+ progenitors correspond to a more homogeneous, less mature population of colony-forming units than the population assayed from partially purified bone marrow cells in Example 1.

4 X 103 MylO+ cells were isolated by immunoro~
setting as described by Civin et al., J. Immunol. 133, 157 (1985) and plated in culture dishes. The experimen-tal conditions were as described in Example 1. GFU-GM
colonies were obtained after fourteen days from cultures stimulated by GM-CSF (10 ng/ml) and Ih-3 (100 U/ml)O
BFU-E colonies were obtained after fourteen days from cultures stimulated by GM-CSF (10 ng/ml, I~-3 (100 U/ml) and rh Epo (3 U/ml). As set forth in Table 2, it was observed that c-abl antisense oligomers inhibited the formation of ~yeloid tCFU-GM) growth, but did not effect primitive erythroid colony (BFU-E) growth. A ~cr anti-sense oligomer did not have any ef~ect on colony number or colony size.

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WO91/n3260 PCT/US90/0~961 (~ . q~
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TABLE ~2 Colonies or clusters ~fot~d BFU-E CFU-GM
CONTROL (no oligomer added) 56~3 75+5 c-abl 1 ~sense) 58~4 70+4 c-~bl 2 (antisense) 4842 20+2 CONTROL 58~6 70~3 c-abl 3 (sense) 60~8 62~2 c-abl ~ tantisense) 52~2 10'3 CONTROL 84~7 51~3 c-abl S tsense) 68~3 46~3 c-abl 6 (antisense) 56~4 10~2 CONTROL 64~6 68~5 bcr 1 (sense~ 84~6 88~8 bcr 2 ~antisense) 76~6 86~10 EXAMPL~ 3 Effect of c-abl oliaomer on normal peripheral blood proaenitors.
Peripheral blood progenitors are antigenically distinct from, and less differentiat~d than, progenitors found in the bone marrow. Ferrero et al., Proc. Natl. Sci.
~SA 80, 4114 (1983). CFU-GM colonies were grown in the presence of recombinant GM-CSF and IL-3 from adherent- and T-cell depleted peripheral blood mononuclear cells after sixteen days of culture. CFU-GM colonies formed from peripheral blood progenitors in the presence of the c-abl antisenss oligomer were indistinguishable from those derived from similarly treated bone marrow progenitors, and were much smaller than progenitors arising in the presence of c abl sense oligomer. In addition, the number of colonies formed was inhibited essentially to the same degree (75~ to 85%) as that observed for bone marrow progenitors. Growth of erythroid progenitors was inhibited . -, ~ , : ~
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WO91/03260 ~ PCT/US90/04961 ~q -16-slightly more (20% to 25%) than we had observed for bone marrow erythroid progenitors (10~ to 20%).
The above experiments indicate that: the effect oP
c-abl antisense on progenitor cells is lineage-specificO
The c-abl function is required for the formation of myeloid colonies, but is apparently unnecessary for the formation of erythroid colonies. In addition, the above experiments indicate that c-abl's functional requirements are indepen~
dent of proliferative activity and di~ferentiation stage of myeloid progenitor cells.
The effect of c-abl antisense oligomers on myeloid colony formation was observed with either abl 2 or abl 4, which are complimentary to the first respective exons of the 6.0 and 7.0 c~abl ~RNAs. Since the two known species of c-abl mRNA differ only in the region corresponding to the two distinct first exons (la and lb) of the c-abl gene, our results suggest that expression of both the 6.0 and 7.0 kb transcripts are required for myelopoiesis.
Inhibition of either transcript inhibits myeloid prolifera-tion.
C-abl antisense oligomer complementary to the common second c-abl exon was also found to inhibit the expression of the hybrid bcr-abl product in K562 cells, a CML cell line containing multiple copies of the hybrid bcr-abl gene. The K562 line has been isolated from a Philadel-phia chromosome positive patient with CML in blast crisis.
In these leukemic cells, the c-abl second exon is spliced to bcr exons "2" and "3", Shtivelman el al., Cell 47, 277 (1986). While bcr-abl protein levels were unaffected by exposure to a c-abl sense oligomer (abl 5) by an immuno-fluorescence assay (Gewirtz et al., Sci~nce ~42, 1303 (1988), protein levels were significantly reduced in the presence of c-abl antisense oligomer (abl 6).

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WO91/03260 ~ PCT/VS90/04961 The present invention may be embodied in other specific forms without departing from the spirit or essential attributas thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

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Claims (17)

1. A pharmaceutical composition comprising pharmaceutical carrier and an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-abl gene, said oligo-nucleotide being hybridizable to said mRNA transcript.

2. A composition according to claim 1 wherein the oligonucleotide is an at least 15-mer oligocleoxynucleotide.

3. A composition according to claim 1 or 2 wherein the oligodeoxynucleotide has a deoxynucleotide sequence complementary to a portion of the c-abl mRNA
transcript beginning with the second codon from the 5' end of said transcript.

4. A composition according to claim 2 or 3 wherein the oligodeoxynucleotide is from a 15-mer to a 21-mer.

5. A composition according to claim 3 wherein the deoxyoligonucleotide is hybridizable to the c-abl about 6.0 kb mRNA transcript and is selected from the group of deoxyoligonucleotides consisting 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 composition according to claim 5 wherein the oligodeoxynucleotide is 5'-CTT CAG GCA GAT CTC CAA-3'.

7. A composition according to claim 3 wherein the deoxyoligonucleotide is hybridizable to the c-abl about 7.0 kb mRNA transcript and is selected from the group of deoxyoligonucleotides consisting 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 according to claim 7 wherein the oligodeoxynucleotide is 5'-TTT TCC AGG CGG CTG CCC-3'.

9. A method for inhibiting proliferation of ryeloid cells comprising administering to an individual an oligonucleotide which has a nucleotide sequence complemen-tary to at laast a portion of the mRNA transcript of the human c-abl gene, said oligonucleotide being hybridizable to said mRNA transcript.

10. A method according to claim 9 wherein the oligonucleotide is an at least 15-mer oligodeoxynucleotide.

11. A method according to claim 9 or 10 wherein the oligodeoxynucleotide has a deoxynucleotide sequence complementary to a portion of the c-abl mRNA transcript beginning with the second codon from the 5' end of said transcript.

12. A method according to claim 10 or 11 wherein the oligodeoxynucleotide is from a 15-mer to a 21-mer.

13. A method according to claim 11 wherein the deoxyoligonucleotide is selected from the group of deoxyoligonucleotides consisting 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 according to claim 13 wherein the oligodeoxynucleotide is 5'-CTT CAG GCA GAT TCT CAA-3'.

15. A method according to claim 11 wherein the deoxyoligonucleotide is selected from the group of deoxy-oligonucleotides consisting 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 according to claim 15 wherein the oligodeoxynucleotide is 5'-TTT TCC AGG CGG CTG CCC-3'.

17. A method according to claim 9 for inhibiting myeloid cell proliferation in an individual afflicted with chronic myelogenous leukemia.