Note: Descriptions are shown in the official language in which they were submitted.
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WO91/03260 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
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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
WO91/03~6~ 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.
. .
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
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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-
.
.
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:
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WO91/03260 ~ ~ 2~ ~ PCT/USg~/04~6l
-7
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
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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");
.
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
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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
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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).
.
WO91/03260 ~ PCT/VS90/04961
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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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