Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02321189 2000-10-13
FIELD OF THE INVENTION
The present invention pertains to the field of antisense oligonucleotides for
mammalian VEGF
receptor genes and their use as anti-angiogenics and/or anti-inflammatory
agents.
BACKGROUND
Angiogenesis is a process by which new capillary vessels sprout from pre-
existing ones, and can
be summarised as the culmination of i) increased endothelial cell permeability
to plasma
proteins; ii) transmigration of inflammatory cells into extracellular matrix;
iii) synthesis and
release of degrading matrix molecules; iv) release of growth factors; v)
migration and
proliferation of endothelial cells to distant sites; and vi) capillary tube
formation and vascular
wall remodelling. Physiological angiogenesis is a highly co-ordinated process
that exclusively
occurs in healthy individuals under specific conditions, such as during wound
healing, ovulation
and pregnancy. At other times, the vasculature is extremely stable, with very
low rates of new
blood vessels (Fan et al., (1995) Trends Pharmacol. Sci. 16:57-66).
Pathological angiogenesis is present in a number of disease states and
biological conditions,
including tumour growth and metastasis, ocular diseases (diabetic and
perinatal hyperoxic
retinopathies, age-related macular degeneration), arthritis, psoriasis and
atherosclerosis (Folkman
et al., (1987) Science. 235:442-447; Ferrara and Davis-Smyth (1997) Endocrine
Rev. 18:4-25;
Moulton et al., (1999) Circulation 99:1726-1732; Ferrara (1999) J. Mol. Med.
77:527-543;
Folkman (1972) Ann. Surg. 175:409-416; Folkman and Shing (1992) J. Biol. Chem.
267:10931-
10934). Thus, attempts have been made to develop methods of inhibiting
pathological
angiogenesis as potential therapeutic techniques.
Angiogenesis is the coordinated response to several factors including vascular
endothelial growth
3o factor (VEGF), acidic and basic fibroblast growth factors (aFGF, bFGF),
transforming growth
factor-a and -(3 (TGF-a, TGF-(3), hepatocyte growth factor (HGF), tumor-
necrosis factor-a (TNF-
a) angiogenin and others (Ferrara and Davis-Smyth (1997) Endocrine Rev. 18:4-
25; Folkman
and Shing (1992) J. Biol. Chem. 267:10931-10934; Klagsbrun and D'Amore (1991)
Annu. Rev.
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CA 02321189 2000-10-13
Physiol. 53:217-239). Growing evidence suggests that VEGF plays a pivotal role
in the
regulation of normal and pathophysiological angiogenesis (Folkman and Shing
(1992) J. Biol.
Chem. 267:10931-10934; Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-
239;
Breier and Risau (1996) Trends Cell Biol. 6:454-456; Ferrara (1993) Trends
Cardiovasc. Med.
3:244-250). Similar to other growth factors VEGF can induce the proliferation
and migration of
1o endothelial cells, however, VEGF is the only growth factor known, to date,
to have the ability to
augment vascular permeability (Senger et al., (1983) Science. 219:983-985;
Connolly et al.,
(1989) J. Clin. Invest. 84:1470-1478; Favard et al., 1991) Biol. Cell. 73:1-
6).
The actions of VEGF and other family members are mediated by tyrosine kinase
receptors, Flt-1
(VEGFR-1), Flk-1 (VEGFR-2), and Flt-4 (VEGFR-3), which are expressed almost
exclusively
on endothelial cells. VEGF is known to interact with both Flt-1 and Flk-1 in
vivo, but there is no
evidence of its interaction with Flt-4 (Neufeld et al., (1999) FASEB J. 13:9-
22; Petrova et al.,
(1999) Exp. Cell Res. 253:117-130).
2o The importance of VEGF receptors in vascular development has been
illustrated using gene-
targeting approaches. Disruption of Flt-1, Flk-1, and Flt-4 leads to embryonic
lethality (Petrova
et al., (1999) Exp. Cell Res. 253:117-130). Flt-1 and Flk-1 are expressed
predominantly in
endothelial cells, and few other cell types express one or both receptors
(Neufeld et al., (1999)
FASEB J. 13:9-22; Petrova et al., (1999) Exp. Cell Res. 253:117-130; Jussila
et al., (1998)
Cancer Res. 58:1599-1604; de Vries et al., (1992) Science. 255:989-991; Terman
et al., (1992)
Biochem. Biophys. Res. Commun. 34:1578-1586; Shibuya et al., (1990) Oncogene.
8:519-527;
Quinn et al., (1993) Proc. Natl. Acad. Sci. U.S.A. 90:7533-7537). Flt-1 is
expressed on
monocytes, renal mesengial cells, Leydig and Sertoli cells (Barleon et al.,
(1996) Blood.
87:3336-3343; Takahashi et al., (1995) Biochem. Biophys. Res. Commun. 209:218-
226; Ergun et
3o al., (1997) Mol. Cell. Endocrinol. 131:9-20). Flk-1 is also expressed on
Leydig and Sertoli cells
and on hematopoietic stem cells and megakaryocytes (Ergun et al., (1997) Mol.
Cell. Endocrinol.
131:9-20; Katoh et al., (1995 Cancer Res. 55:5687-5692; Yang and Cepko (1996)
J. Neurosci.
16:6089-6099). Further, VEGF exerts its multiple actions by binding to Flt-1
and Flk-1 and not
on Flt-4. Many studies show that Flt-1 and Flk-1 receptors may play a leading
role in VEGF
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induced angiogenesis; however, they seem to be involved in different
biological activities.
Antisense compounds are commonly used as research and diagnostic reagents. For
example,
antisense oligonucleotides, which are able to inhibit gene expression with
exquisite specificity,
are often used by those of ordinary skill in the relevant art the to elucidate
the function of
particular genes. Antisense compounds are also used, for example, to
distinguish between
functions of various members of a biological pathway. Antisense modulation
has, therefore,
been harnessed for research use. The specificity and sensitivity of antisense
is also harnessed by
those of skill in the art for therapeutic uses. Antisense oligonucleotides
have been employed as
therapeutic moieties in the treatment of disease states in animals and man.
Antisense
oligonucleotides have been safely and effectively administered to humans and
numerous clinical
trials are presently underway. It is thus established that oligonucleotides
can be useful
therapeutic modalities that can be configured to be useful in treatment
regimes for treatment of
cells, tissues and animals, especially humans.
2o Antisense technology is emerging as an effective means for blocking or
inhibiting the expression
of specific gene products and, therefore, can be uniquely useful in a number
of therapeutic,
diagnostic, and research applications involving the modulation of VEGF
receptor expression.
The effective regulation of pathological angiogenesis using the antisense
oligonucleotides of the
present invention can be useful in medical treatments for various diseases and
disorders
including, but not limited to, inflammation, tumour growth and metastasis,
ocular diseases,
arthritis, psoriasis and atherosclerosis.
This background information is provided for the purpose of making known
information believed
by the applicant to be of possible relevance to the present invention. No
admission is necessarily
3o intended, nor should be construed, that any of the preceding information
constitutes prior art
against the present invention.
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SUMMARY OF THE INVENTION
An object of the present invention is to provide anti-angiogenic antisense
oligonucleotides
directed toward mammalian VEGF receptors and uses thereof. In accordance with
an aspect of
the present invention, there is provided an antisense oligonucleotide
complementary to a gene
encoding a mammalian vascular endothelial growth factor (VEGF) receptor
selected from the
1o group comprising Flt-1 and Flk-1, wherein said antisense oligonucleotide
comprises about 15 to
about 25 nucleotides complementary to said gene.
In accordance with another aspect of the invention, there is provided a
pharmaceutical
composition comprising a pharmaceutically acceptable diluent and an antisense
oligonucleotide
15 complementary to a gene encoding a mammalian VEGF receptor selected from
the group
comprising Flt-1 and Flk-1, wherein said antisense oligonucleotide comprises
about 15 to about
25 nucleotides complementary to said gene.
In accordance with another aspect of the invention, there is provided a method
of blocking
2o pathological angiogenesis in a mammal in need of such therapy, comprising
the step of
administering to said mammal the antisense oligonucleotide an antisense
oligonucleotide
complementary to a gene encoding a mammalian VEGF receptor selected from the
group
comprising Flt-1 and Flk-1.
25 In accordance with another aspect of the invention, there is provided a
method of blocking
inflammation in a mammal in need of such therapy, comprising the step of
administering to said
mammal the antisense oligonucleotide an antisense oligonucleotide
complementary to a gene
encoding a mammalian VEGF receptor selected from the group comprising Flt-1
and Flk-1.
3o BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Antisense regulation of VEGF receptors expression on bovine aortic
endothelial cells
(BAEC). BAEC were seeded at 1 x 106 cells/100 mm culture plate and grown to
confluence.
Cells were treated either with antisense or scrambled sequences.
Immunoprecipitation was
CA 02321189 2000-10-13
performed on 12 mg of total proteins as described in Example I. The
immunoprecipitated
proteins were subjected to SDS-polyacrylamide gel electrophoresis under
reducing conditions.
Flt-1 and Flk-1 protein expression was revealed by Western blot analysis.
Image densitometry
results are given as relative expression percentage as compared to PBS-treated
cells (control =
Ctrl). A) Flt-1 protein expression of PBS-treated cells (Ctrl), cells treated
with antisense Flt-1
1o oligomers (AS1-Flt and AS2-Flt; 10-'M), or cells treated with the scrambled
Flt-1 oligomer
(SCR-Flt; 10-'M). B) Flk-1 protein expression of PBS-treated cells (Ctrl),
cells treated with
antisense Flk-1 oligomers (AS1-Flk and AS2-Flk; 10-' M), or cells treated with
the scrambled
Flk-1 oligomer (SCR-Flk; 10-' M). C) Flk-1 protein expression of PBS-treated
cells (Ctrl), cells
treated with antisense Flk-1 oligomers (AS1-Flk and AS2-Flk; 5 x 10-' M), or
cells treated with
the scrambled Flk-1 oligomer (SCR-Flk; 5 x 10-' M).
Figure 2: Western blot analysis of antisense cross-reactivity. BAEC were
seeded at 1 x 106
cells/100 mm culture plate and grown to confluence. Cells were treated either
with antisense
AS1-Flk or AS2-Flt. Total proteins were isolated and immunoprecipitated
against the mentioned
receptor. Image densitometry results are given as relative expression (%) as
compared to PBS-
treated cells (Ctrl). A) Flt-1 protein expression of PBS-treated cells (Ctrl),
cells treated with the
more potent antisense Flk-1 oligomer (AS1-Flk; 5 x 10-' M), or cells treated
with the more
potent antisense Flt-1 oligomer (AS2-Flt; 5 x 10-' M). B) Flk-1 protein
expression of PBS-
treated cells (Ctrl), cells treated with the more potent antisense Flk-1
oligomer (AS1-Flk; 5 x 10-'
M), or cells treated with the most potent antisense Flt-1 oligomer (AS2-Flt; 5
x 10-' M).
Figure 3: Antisense regulation of VEGF-induced Flt-1 and Flk-1
phosphorylation. A) Analysis
of Flt-1 phosphorylation of PBS-treated cells (Ctrl), unstimulated (-) or
stimulated (+) with
VEGF, and from cells treated either with the most potent antisense Flk-1
oligomer (AS1-Flk; 5
3o x 10-' M) with VEGF stimulation (+), or cells treated with the most potent
antisense Flt-1
oligomer (AS2-Flt; 5 x 10-' M) with VEGF stimulation (+). B) Analysis of Flk-1
protein
phosphorylation of BAEC treated as described for A.
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Figure 4: Mitogenic effect of VEGF and P1GF on endothelial cell proliferation.
BAEC were
seeded at 1 x 104 cells/well (24-well tissue culture plate) and stimulated for
24 h with DMEM
culture media, 5% FBS. The cells were synchronized in G° by a 48 h
treatment with DMEM,
0.25% FBS. The cells were then stimulated with VEGF (10-", 10-'° and
2.5 x 10-'° M) or P1GF
(10-'°, 2.5 x 10-'°, 10-9 and 10-g M), and cell number was
counted 72 h post-treatment. The values
1o are means of cell count obtained from 6 wells for each treatment. [*, p <
0.05; ***, p < 0.001 as
compared with control (DMEM, 1% FBS) as determined by analysis of variance
followed by an
unpaired Student's t-test.]
Figure 5: Effect of antisense oligomers on VEGF-induced endothelial cell
proliferation. BAEC
were seeded at 1 x 104 cells/well (24-well tissue culture plate) and
stimulated for 24 h with
DMEM culture media and 5% FBS with or without antisense oligomers (10-' M),
the cells were
synchronized by a 48 h treatment with DMEM and 0.25% FBS with or without
antisense
oligomers (10~' M daily). The cells were then stimulated with VEGF (10-9 M)
with or without
antisense oligomers (10-' M daily), and cell number was counted 72 h post-
treatment. The values
2o present are means of cell count obtained from 10 wells for each treatment.
[**, p < 0.01 as
compared with control (DMEM, 1% FBS); tt, p < 0.01 as compared with VEGF (2.5
x 10-'° M)
as determined by analysis of variance followed by an unpaired Student's t-
test.]
Figure 6: Chemotactic effect of VEGF and P1GF on endothelial cell migration.
BAEC were
trypsinized and resuspended in DMEM, 1% FBS, and antibiotics; and 5 x 104
cells were added in
the higher chamber of the modified Boyden chamber apparatus, and the lower
chamber was filled
with DMEM, 1% FBS and antibiotics with or without VEGF or P1GF. Five hours (5
h) post-
incubation at 37 °C, the migrated cells were stained and counted by
using a microscope adapted
to a digitized videocamera. The values are means of migrating cells/mm2 from 6
chambers for
3o each treatment. [**, p < 0.01; ***, p < 0.001 as compared with control
buffer (PBS) as
determined by analysis of variance followed by an unpaired Student's t-test.]
Figure 7: Antisense oligomer effects on VEGF-induced endothelial cell
migration. BAEC were
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trypsinized and seeded at 2.5 x 106 cells/well of 6-well tissue culture plate,
stimulated for 24 h in
DMEM, 5% FBS, and antibiotics with or without antisense oligomers (10-' M),
starved for 48 h
in DMEM, 0.25% FBS, and antibiotics with or without antisense oligomers (10-'
M daily). Cells
were harvested by trypsinization, resuspended in DMEM, 1% FBS, and
antibiotics. Cells, 5 x
104, with or without antisense oligomers (10-' M) were added in the higher
chamber of the
modified Boyden chamber apparatus, and the lower chamber was filled with DMEM,
1% FBS,
and antibiotics plus VEGF. Five (5) h post-incubation at 37 °C, the
migrated cells were stained
and counted by using a microscope adapted to a digitized videocamera. The
values are means of
migrating cells/mmz from 6 chambers for each treatment. [*, p < 0.05; **, p <
0.01 as compared
with control-PBS. t1', p < 0.01; 1't1', p < 0.001 as compared with control-
VEGF (10-9 M) as
determined by analysis of variance followed by an unpaired Student's t-test.]
Figure 8: VEGF and placental growth factor (P1GF) effect on endothelial cell
platelet activating
factor (PAF) synthesis. Confluent BAEC (6-well tissue culture plate) were
incubated with 3H-
acetate and were stimulated with either VEGF or P1GF for 15 min. The
radioactive polar lipids
samples were extracted by the Bligh and Dyer procedure (Bligh and Dyer (1959)
Can. J.
Biochem. Physiol. 37, 911). The samples were injected into a 4.6 x 250 mm
Varian Si-5 column
and eluted with a mobile phase (HzO:CHCI3:MeOH; 5:40:55; 0.5 ml/min).
Fractions were
collected every minute after injection, and radioactivity was determined with
a (3-counter. The
values are means of at least eight experiments. [*, p < 0.05; ***, p < 0.001
as compared with
control buffer (PBS) as determined by analysis of variance followed by an
unpaired Student's t-
test.]
Figure 9: Effect of antisense oligomers on VEGF-induced PAF synthesis to
assess the role of
VEGF receptors on PAF synthesis. BAEC were seeded at 2.5 x 105 cells/well of 6
well tissue
3o culture plate, stimulated for 24 h in DMEM, 5% FBS, and antibiotics with or
without antisense
oligomers (10-' - 5 x 10-' M) and starved for 48 h in DMEM, 0.25% FBS, and
antibiotics with or
without antisense oligomers (10-' - 5 x 10-' M daily) for Go synchronization.
The cells were then
grown to confluence for 24 h in DMEM, 1% FBS, and antibiotics with or without
antisense
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oligomers (10-' - 5 x 10-' M) and starved for 8 h in DMEM, 0.25% FBS, and
antibiotics with or
without antisense oligomers (10-' - 5 x 10-' M) to induce an upregulation of
VEGF receptor
expression. Then the cells were incubated with 3H-acetate, and treated with
VEGF (10-9 M). The
values are means of at least eight experiments. [*, p < 0.05; **, p < 0.01;
and ***, p < 0.001, as
compared with control buffer (PBS). t'f't, p < 0.001 as compared with VEGF (10-
9 M) as
l0 determined by analysis of variance followed by an unpaired Student's t-
test.]
Figure 10: Assessment of the correlation between antisense Flk-1 oligomer
regulation of Flk-1
expression and VEGF-induced PAF synthesis. Shown is the expression of Flk-1
protein
expression of BAEC untreated or treated with antisense Flk-1 oligomers (10-' -
5 x 10-' M)
versus PAF synthesis elicited by a treatment with VEGF (10-9 M).
Figure 11: Effect of Flt-1 and Flk-1 antisense oligomers on VEGF-induced
angiogenesis. A
new model of angiogenesis was developed and used to measure the ability of
VEGF to induce
angiogenesis in mouse testis and the ability of Flt-1 and Flk-1 antisense
oligomers to abolish
2o VEGF-induced angiogenesis. The angiogenesis mouse model is described in
Examples II. The
values shown are means of at least 5-8 experiments. [A) **, p < 0.05 as
compared with control
buffer (PBS); B) **, p < 0.05 as compared with VEGF (2.5 ~g/200 ~1) as
determined by analysis
of variance followed by an unpaired Student's t-test.]
Figure 12: Photomicrograph depicting the effect of Flt-1 and Flk-1 antisense
oligomers on
VEGF-induced angiogenesis in a mouse model. Experimental C57BL/6 mice were
treated with
PBS (control), VEGF (2.5 ~g/200 pl), VEGF (2.5 pg/100 pl) + ASFIk-1 (200
pg/100 pl) or
VEGF (2.5 pg/100 ~1) + ASFIt-1 (200 pg/100 pl) for a period of 14 days as
detailed in the
Example II. Photomicrographs of the level of angiogenesis under each treatment
were taken at
day 0 (before treatment) and day 14 (following treatment) using standard
histological techniques.
All images were analysed with NIH image 1.6 software. The number, diameter and
the length of
newly formed and pre-existing blood vessels were measured and compared
following each
treatment. Both Flk-1 and Flt-1 antisense oligomers dramatically decreased the
VEGF-induced
increase in angiogenesis in the mouse testis model.
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Figure 13: Efficiency and selectivity of the Flk-1 antisense oligomer on Flk-1
protein
expression in mouse testis. The immunohistochemical analysis demonstrated that
neither PBS,
VEGF, AS-Flt-1 nor AS-SCR, affected the level of Flk-1 protein expression in
mouse testis,
panels A, B, D and E, respectively. The specificity of the Flk-1 antisense
oligomer depicted in
1o panel C is evident by the lack of positive staining following incubation
with Flk-1 antibody.
Figure 14: Efficiency and selectivity of the Flt-1 antisense oligomer on Flt-1
protein expression
in mouse testis. The immunohistochemical analysis demonstrated that neither
PBS, VEGF, AS-
Flk-1 nor AS-SCR, affected the level of Flt-1 protein expression in mouse
testis, panels A, B, C
and E, respectively. The specificity of the Flt-1 antisense oligomer depicted
in panel D is evident
by the lack of positive staining following incubation with Flt-1 antibody.
Figure 15: Effect of Flk-1 and Flt-1 antisense oligomers on endothelial cell
nitric oxide
synthase (ecNOS) protein expression in rat testis. The immunohistochemical
analysis
2o demonstrated that neither PBS, VEGF, AS-Flk-1, AS-Flt-1 nor AS-SCR,
affected the level of
ecNOS protein expression in mouse testis, panels A - E, respectively.
DETAILED DESCRIPTION OF THE INVENTION
The present invention employs antisense oligonucleotides for use in modulating
the function of
nucleic acid molecules encoding vascular endothelial growth factor (VEGF)
receptors Flt-1 and
Flk-l, ultimately modulating the amount of VEGF receptor protein produced.
This is
accomplished by providing antisense compounds which specifically hybridise
with one or more
nucleic acids encoding vascular endothelial growth factor (VEGF) receptors Flt-
1 and Flk-1.
The specific hybridisation of an oligonucleotide with its target nucleic acid
interferes with the
3o normal function of the nucleic acid. This modulation of function of a
target nucleic acid by
compounds which specifically hybridise to it is generally referred to as
"antisense". The
functions of DNA to be interfered with include replication and transcription.
The functions of
RNA to be interfered with include all vital functions such as, for example,
translocation of the
CA 02321189 2000-10-13
RNA to the site of protein translation, translation of protein from the RNA,
splicing of the RNA
to yield one or more mRNA species, and catalytic activity which may be engaged
in or facilitated
by the RNA. The overall effect of such interference with target nucleic acid
function is
modulation of the expression of vascular endothelial growth factor (VEGF)
receptors, Flt-1
and/or Flk-1.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as
commonly understood by one of ordinary skill in the art to which this
invention belongs.
"Antisense oligonucleotide", as used herein, refers to any oligonucleotide
that is complementary
to the target gene. The antisense oligonucleotide may be in the form of DNA,
RNA or any
combination thereof.
"Corresponds to" refers to a polynucleotide sequence is homologous (i.e., is
identical, not strictly
evolutionarily related) to all or a portion of a reference polynucleotide
sequence, or that a
2o polypeptide sequence is identical to a reference polypeptide sequence.
"Naturally-occurring", as used herein, as applied to an object, refers to the
fact that an object can
be found in nature. For example, a polypeptide or polynucleotide sequence that
is present in an
organism (including viruses) that can be isolated from a source in nature and
which has not been
intentionally modified in the laboratory is naturally-occurring.
"Nucleic acid" refers to DNA and RNA and can be either double stranded or
single stranded.
The invention also includes nucleic acid sequences which are complementary to
the claimed
nucleic acid sequences.
"Oligonucleotide", as used herein, refers to an oligomer or polymer of
ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA) or mimetics thereof. This term includes
oligonucleotides
composed of naturally-occurring nucleobases, sugars and covalent
internucleoside (backbone)
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linkages as well as oligonucleotides having non-naturally-occurring portions
which function
similarly. Such modified or substituted oligonucleotides are often preferred
over native forms
because of desirable properties such as, for example, enhanced cellular
uptake, enhanced affinity
for nucleic acid target and increased stability in the presence of nucleases.
"Polynucleotide" refers to a polymeric form of nucleotides of at least 10
bases in length, either
ribonucleotides or deoxynucleotides or a modified form of either type of
nucleotide. The term
includes single and double stranded forms of DNA or RNA.
"Protein", as used herein, refers to a whole protein, or fragment thereof,
such as a protein domain
or a binding site for a second messenger, co-factor, ion, etc. It can be a
peptide or an amino acid
sequence that functions as a signal for another protein in the system, such as
a proteolytic
cleavage site.
Other biochemistry and chemistry terms herein are used according to
conventional usage in the
2o art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (ed.
Parker, S., 1985),
McGraw-Hill, San Francisco).
In one embodiment of the present invention antisense oligonucleotides are
designed that are
complementary to specific regions of mammalian Flt-1 and Flk-1 genes. In a
specific
embodiment of the present invention antisense oligonucleotides are designed
that are
complementary to specific regions of the human Flt-1 and Flk-1 genes.
Exemplary antisense oligonucleotide sequences of the present invention are
listed below. It
should be apparent to one skilled in the art that other antisense
oligonucleotide sequences that are
3o complementary to specific regions of mammalian Flt-1 and Flk-1 genes are
within the scope of
the present invention.
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BOVINE FLT-1
AS1-bFlt-1: 5'-CAA AGA TGG ACT CGG GAG-3' (SEQ ID NO:1)
AS2-bFlt-1: 5'-GTC GCT CTT GGT GCT ATA-3' (SEQ ID N0:2)
BOVINE FLK-1
ASl-bFlk-1: 5'-GCT GCT CTG ATT GTT GGG-3' (SEQ ID N0:3)
1o AS2-bFlk-1: 5'-CCT CCA CTC TTT TCT CAG-3' (SEQ ID N0:4)
MURINE FLT-1
AS1-mFlt-1: 5'-AAG CAG ACA CCC GAG CAG-3' (SEQ ID N0:5)
AS2-mFlt-1: 5'-CCC TGA GCC ATA TCC TGT-3~ (SEQ ID N0:6)
MURINE FLK-1
AS1-mFlk-1: 5'-AGA ACC ACA GAG CGA CAG-3' (SEQ ID N0:7)
AS2-mFlk-1: 5'-AGT ATG TCT TTC TGT GTG-3' (SEQ ID N0:8)
HUMAN FLT-1
ASl-hFlt-1: 5'-CTG TTT CCT TCT TCT (SEQ ID N0:9)
TTG-3'
AS2-hFlt-1: 5~-TCC TTA CTC ACC ATT (SEQ ID NO:10)
TCA -3'
2o AS3-hFlt-1:5'-TGT TTC CTT CTT CTT (SEQ ID NO:11)
TGA-3'
AS4-hFlt-1: 5'-TAC TCA CCA TTT CAG (SEQ ID N0:12)
GCA-3'
AS5-hFlt-l: 5'-ACT CAC CAT TTC AGG
CAA -3' (SEQ ID N0:13)
HUMAN FLK-1/KDR
AS1-hFlk-1: 5'-AGT ATG TCT TTT TGT ATG-3'(SEQ ID N0:14)
AS2-hFlk-1:5'-TGA AGA GTT GTA TTA GCC-3'(SEQ ID N0:15)
AS3-hFlk-l: 5'-ACT GCC ACT CTG ATT ATT-3'(SEQ ID N0:16)
AS4-hFlk-1: 5'-TTT GCT CAC TGC CAC TCT-3'(SEQ ID N0:17)
ASS-hFlk-1: 5'-GTC TTT TTG TAT GCT GAG-3'(SEQ ID N0:18)
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Design and Preparation of Antisense Oligonucleotides
"Targeting" an antisense compound to a particular nucleic acid, in the context
of this invention,
is a multistep process. The process usually begins with the identification of
a nucleic acid
sequence whose function is to be modulated. This may be, for example, a
cellular gene (or
mRNA transcribed from the gene) whose expression is associated with a
particular disorder or
disease state, or a nucleic acid molecule from an infectious agent. In the
present invention, the
target is a nucleic acid molecule encoding a mammalian VEGF receptor that is
Flt-1 or Flk-1. As
used herein the "gene encoding a VEGF receptor" refers to any gene which
encodes a protein
that is capable of acting as a VEGF receptor. Gene sequences are often
available on electronic
databases, for example, GenBank. In the present invention the bovine antisense
oligonucleotides
were designed from the sequence in GenBank Accession Nos. X94263 and X94298,
the murine
antisense oligonucleotides were designed from the sequence in GenBank
Accession Nos.
D28498 and X70842, and the human antisense oligonucleotides were designed from
the
sequence in GenBank Accession Nos AF063658 and X51602.
The targeting process also includes determination of a site or sites within
this gene for the
antisense interaction to occur such that the desired effect, e.g., detection
or modulation of
expression of the protein, will result. Within the context of the present
invention, a possible
intragenic site is the region encompassing the translation initiation or
termination codon of the
open reading frame (ORF) of the gene. It is known in the art that eukaryotic
and prokaryotic
genes may have two or more alternative start codons, any one of which may be
utilized for
translation initiation in a particular cell type or tissue, or under a
particular set of conditions. In
the context of the invention, "start codon" and "translation initiation codon"
refer to the codon or
codons that are used in vivo to initiate translation of an mRNA molecule
transcribed from a gene
encoding a mammalian VEGF receptor that is Flt-1 or Flk-1, regardless of the
sequences) of
3o such codons.
The open reading frame (ORF) or "coding region," which is known in the art to
refer to the
region between the translation initiation codon and the translation
termination codon, is also a
region which may be targeted effectively. Other target regions include the 5'
untranslated region
14
CA 02321189 2000-10-13
(5'UTR), known in the art to refer to the portion of an mRNA in the 5'
direction from the
translation initiation codon, and thus including nucleotides between the 5'
cap site and the
translation initiation codon of an mRNA or corresponding nucleotides on the
gene, and the 3'
untranslated region (3'UTR), known in the art to refer to the portion of an
mRNA in the 3'
direction from the translation termination codon, and thus including
nucleotides between the
1o translation termination codon and 3' end of an mRNA or corresponding
nucleotides on the gene.
The 5' cap of an mRNA comprises an N'-methylated guanosine residue joined to
the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an
mRNA is
considered to include the 5' cap structure itself as well as the first 50
nucleotides adjacent to the
cap. The 5' cap region may also be a preferred target region.
Although some eukaryotic mRNA transcripts are directly translated, many
contain one or more
regions, known as "introns," which are excised from a transcript before it is
translated. The
remaining (and therefore translated) regions are known as "exons" and are
spliced together to
form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon
junctions, may also be
2o preferred target regions, and are particularly useful in situations where
aberrant splicing is
implicated in disease, or where an overproduction of a particular mRNA splice
product is
implicated in disease. Aberrant fusion junctions due to rearrangements or
deletions are also
potential targets. It has also been found that introns can be effective target
regions for antisense
compounds targeted, for example, to DNA or pre-mRNA.
Once one or more target sites have been identified, oligonucleotides are
chosen which are
sufficiently complementary to the target, i.e., hybridise sufficiently well
and with sufficient
specificity, to give the desired effect.
3o In the context of this invention, "hybridisation" means hydrogen bonding,
which may be
Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between
complementary
nucleoside or nucleotide bases. For example, adenine and thymine are
complementary
nucleobases which pair through the formation of hydrogen bonds.
"Complementary," as used
herein, refers to the capacity for precise pairing between two nucleotides.
For example, if a
CA 02321189 2000-10-13
nucleotide at a certain position of an oligonucleotide is capable of hydrogen
bonding with a
nucleotide at the same position of a DNA or RNA molecule, then the
oligonucleotide and the
DNA or RNA are considered to be complementary to each other at that position.
The
oligonucleotide and the DNA or RNA are complementary to each other when a
sufficient number
of corresponding positions in each molecule are occupied by nucleotides which
can hydrogen
1o bond with each other. Thus, "specifically hybridisable" and "complementary"
are terms which
are used to indicate a sufficient degree of complementarity or precise pairing
such that stable and
specific binding occurs between the oligonucleotide and the DNA or RNA target.
It is
understood in the art that the sequence of an antisense compound need not be
100%
complementary to that of its target nucleic acid to be specifically
hybridisable. An antisense
compound is specifically hybridisable when binding of the compound to the
target DNA or RNA
molecule interferes with the normal function of the target DNA or RNA to cause
a loss of utility,
and there is a sufficient degree of complementarity to avoid non-specific
binding of the antisense
compound to non-target sequences under conditions in which specific binding is
desired, i.e.,
under physiological conditions in the case of in vivo assays or therapeutic
treatment, and in the
2o case of in vitro assays, under conditions in which the assays are
performed.
In one embodiment of the present invention the antisense oligonucleotides are
selected to have
the following characteristics:
i) no more than three, or preferably less, consecutive guanosines;
ii) incapacity to form hairpin structures;
iii) minimal capacity to form homodimers; and
iv) contain between about 15 and about 25 nucleotides that are complementary
to the
target gene.
The antisense oligonucleotides can be selected, based on the above
characteristics, using
3o commercially available computer software, for example OLIGO~ Primer
Analysis.
While antisense oligonucleotides are one form of antisense compounds, the
present invention
contemplates other oligomeric antisense compounds, including but not limited
to oligonucleotide
mimetics such as are described below. As is known in the art, a nucleoside is
a base-sugar
16
CA 02321189 2000-10-13
combination. The base portion of the nucleoside is normally a heterocyclic
base. The two most
common classes of such heterocyclic bases are the purines and the pyrimidines.
Nucleotides are
nucleosides that further include a phosphate group covalently linked to the
sugar portion of the
nucleoside. For those nucleosides that include a pentofuranosyl sugar, the
phosphate group can
be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming
oligonucleotides, the
phosphate groups covalently link adjacent nucleosides to one another to form a
linear polymeric
compound. In turn the respective ends of this linear polymeric structure can
be further joined to
form a circular structure, however, open linear structures are generally
preferred. Within the
oligonucleotide structure, the phosphate groups are commonly referred to as
forming the
internucleoside backbone of the oligonucleotide. The normal linkage or
backbone of RNA and
DNA is a 3' to S' phosphodiester linkage.
Specific examples of antisense compounds useful in this invention include
oligonucleotides
containing modified backbones or non-natural internucleoside linkages. As
defined in this
specification, oligonucleotides having modified backbones include those that
retain a phosphorus
2o atom in the backbone and those that do not have a phosphorus atom in the
backbone. For the
purposes of this specification, and as sometimes referenced in the art,
modified oligonucleotides
that do not have a phosphorus atom in their internucleoside backbone can also
be considered to
be oligonucleosides.
2s Alternative modified oligonucleotide backbones include, for example,
phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl
and other alkyl phosphonates including 3'-alkylene phosphonates and chiral
phosphonates,
phosphinates, phosphoramidates including 3'amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,
30 thionoalkylphosphotriesters, and boranophosphates having normal 3'-5'
linkages, 2'-5' linked
analogs of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside
units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts
and free acid forms are
also included.
17
CA 02321189 2000-10-13
Alternative modified oligonucleotide backbones that do not include a
phosphorus atom therein
have backbones that are formed by short chain alkyl or cycloalkyl
internucleoside linkages,
mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or
more short chain
heteroatomic or heterocyclic internucleoside linkages. These include those
having morpholino
linkages (formed in part from the sugar portion of a nucleoside); siloxane
backbones; sulfide,
1o sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones;
methylene
formacetyl and thioformacetyl backbones; alkene containing backbones;
sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide
backbones;
amide backbones; and others having mixed N, O, S and CHZ component parts.
In alternative oligonucleotide mimetics, both the sugar and the
internucleoside linkage, i.e., the
backbone, of the nucleotide units are replaced with novel groups. The base
units are maintained
for hybridization with an appropriate nucleic acid target compound. One such
oligomeric
compound, an oligonucleotide mimetic that has been shown to have excellent
hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds,
the sugar-
2o backbone of an oligonucleotide is replaced with an amide containing
backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are bound
directly or indirectly
to aza nitrogen atoms of the amide portion of the backbone. Representative
United States patents
that teach the preparation of PNA compounds include, but are not limited to,
U.S. Pat Nos.:
5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be
found in
Nielsen et al (1991) Science, 254, 1497-1500.
Modified oligonucleotides may also contain one or more substituted sugar
moieties. For
example, oligonucleotides may comprise one of the following at the 2'
position: OH; F; O-, S-,
or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl,
wherein the alkyl,
3o alkenyl and alkynyl may be substituted or unsubstituted C, to C,o alkyl or
CZ to C,o alkenyl and
alkynyl. Particularly preferred are O[(CHZ)~ O]", CH3, O(CHZ)~ OCH3, O(CHZ)~
NH2, O(CHZ)"
CH3, O(CHZ)~ ONHZ, and O(CHZ)~ON[(CHZ)" CH3)]2, where n and m are from 1 to
about 10.
Other preferred oligonucleotides comprise one of the following at the 2'
position: C, to C,o lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,
SCH3, OCN, Cl, Br,
18
CA 02321189 2000-10-13
CN, CF3, OCF3, SOCH3, SOZ CH3, ONOZ, NOZ, N3, NHZ, heterocycloalkyl,
heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an
intercalator, a group for improving the pharmacokinetic properties of an
oligonucleotide, or a
group for improving the pharmacodynamic properties of an oligonucleotide, and
other
substituents having similar properties.
Other modifications include 2'-methoxy (2'-O--CH3), 2'-aminopropoxy (2'-OCHZ
CHZ CHZ NHZ)
and 2'-fluoro (2'-F). Similar modifications may also be made at other
positions on the
oligonucleotide, particularly the 3' position of the sugar on the 3' terminal
nucleotide or in 2'-5'
linked oligonucleotides and the 5' position of 5' terminal nucleotide.
Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl
sugar.
Oligonucleotides may also include nucleobase (often referred to in the art
simply as "base")
modifications or substitutions. As used herein, "unmodified" or "natural"
nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine
(T), cytosine (C)
2o and uracil (U). Modified nucleobases include other synthetic and natural
nucleobases such as 5-
methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-
aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and
other alkyl derivatives
of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-
halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine,
5-uracil
(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-
hydroxyl and other 8-
substituted adenines and guanines, 5-halo particularly 5-bromo, 5-
trifluoromethyl and other 5-
substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-
azaguanine and 8-
azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-
deazaadenine.
Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those
disclosed in The
Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859,
Kroschwitz, J. L,
ed. John Wiley & Sons, 1990, those disclosed by Englisch et al (1991)
Angewandte Chemie,
International Edition, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter
15, Antisense
Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed.,
CRC Press, 1993.
Certain of these nucleobases are particularly useful for increasing the
binding affinity of the
19
CA 02321189 2000-10-13
oligomeric compounds of the invention. These include 5-substituted
pyrimidines, 6-
azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-
aminopropyladenine, 5-
propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have
been shown to
increase nucleic acid duplex stability by 0.6-1.2'-'C. (Sanghvi, Y. S.,
Crooke, S. T. and Lebleu, B.,
eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278), even
1o more particularly when combined with 2'-O-methoxyethyl sugar modifications.
Another modification of the oligonucleotides of the invention involves
chemically linking to the
oligonucleotide one or more moieties or conjugates which enhance the activity,
cellular
distribution or cellular uptake of the oligonucleotide. Such moieties include
but are not limited to
lipid moieties such as a cholesterol moiety (Letsinger et al (1989) Proc.
Natl. Acid. Sci. USA,
86, 6553-6556), cholic acid (Manoharan et al (1994) Bioorg. Med. Chem. Lett.,
4, 1053-1060), a
thioether, e.g., hexyl-S-tritylthiol (Manoharan et al (1992) Ann. N. Y. Acid.
Sci. , 660, 306-309;
Manoharan et al (1993) Bioorg. Med. Chem. Lett., 3, 2765-2770), a
thiocholesterol (Oberhauser
et al (1992) Nucl. Acids Res., 20, 533-538), an aliphatic chain, e.g.,
dodecandiol or undecyl
2o residues (Saison-Behmoaras et al (1991) EMBO J., 10, 1111-1118), a
phospholipid, e.g., di-
hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-
phosphonate
(Manoharan et al (1995) Tetrahedron Lett., 36, 3651-3654; Shea et al (1990)
Nucl. Acids Res.,
18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al
(1995)
Nucleosides & Nucleotides, 14, 969-973), or adamantine acetic acid (Manoharan
et al (1995)
Tetrahedron Lett., 36, 3651-3654), a palmityl moiety (Mishra et al (1995)
Biochim. Biophys.
Acta, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-
oxycholesterol moiety
(Crooke et al (1996) J. Pharmacol. Exp. Ther. , 277, 923-937.
It is not necessary for all positions in a given compound to be uniformly
modified, and in fact
more than one of the aforementioned modifications may be incorporated in a
single compound or
even at a single nucleoside within an oligonucleotide. The present invention
also includes
antisense compounds which are chimeric compounds. "Chimeric" antisense
compounds or
"chimeras," in the context of this invention, are antisense compounds,
particularly
oligonucleotides, which contain two or more chemically distinct regions, each
made up of at least
CA 02321189 2000-10-13
one monomer unit, i.e., a nucleotide in the case of an oligonucleotide
compound. These
oligonucleotides may contain at least one region wherein the oligonucleotide
is modified so as to
confer upon the oligonucleotide increased resistance to nuclease degradation,
increased cellular
uptake, and/or increased binding affinity for the target nucleic acid. An
additional region of the
oligonucleotide may serve as a substrate for enzymes capable of cleaving
RNA:DNA or
1o RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease
which cleaves the
RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage of
the RNA target, thereby greatly enhancing the efficiency of oligonucleotide
inhibition of gene
expression. Consequently, comparable results can often be obtained with
shorter oligonucleotides
when chimeric oligonucleotides are used, compared to phosphorothioate
deoxyoligonucleotides
hybridizing to the same target region. Cleavage of the RNA target can be
routinely detected by
gel electrophoresis and, if necessary, associated nucleic acid hybridization
techniques known in
the art.
Chimeric antisense compounds of the invention may be formed as composite
structures of two or
2o more oligonucleotides, modified oligonucleotides, oligonucleosides and/or
oligonucleotide
mimetics as described above. Such compounds have also been referred to in the
art as hybrids or
gapmers.
The antisense compounds used in accordance with this invention may be
conveniently and
routinely made through the well-known technique of solid phase synthesis.
Equipment for such
synthesis is sold by several vendors including, for example, Applied
Biosystems (Foster City,
Calif.). Any other means for such synthesis known in the art may additionally
or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the
phosphorothioates and alkylated derivatives.
The compounds of the invention may also be admixed, encapsulated, conjugated
or otherwise
associated with other molecules, molecule structures or mixtures of compounds,
as for example,
liposomes, receptor targeted molecules, oral, rectal, topical or other
formulations, for assisting in
uptake, distribution and/or absorption.
21
CA 02321189 2000-10-13
The antisense compounds of the invention encompass any pharmaceutically
acceptable salts,
esters, or salts of such esters, or any other compound which, upon
administration to an animal
including a human, is capable of providing (directly or indirectly) the
biologically active
metabolite or residue thereof. Accordingly, for example, the disclosure is
also drawn to prodrugs
l0 and pharmaceutically acceptable salts of the compounds of the invention,
pharmaceutically
acceptable salts of such prodrugs, and other bioequivalents.
The term "prodrug" indicates a therapeutic agent that is prepared in an
inactive form that is
converted to an active form (i.e., drug) within the body or cells thereof by
the action of
15 endogenous enzymes or other chemicals and/or conditions. In particular,
prodrug versions of the
oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-
thioethyl)phosphate]
derivatives according to the methods disclosed in WO 93/24510 to Gosselin et
al., published
Dec. 9, 1993 or in WO 94/26764 to Imbach et al.
2o The term "pharmaceutically acceptable salts" refers to physiologically and
pharmaceutically
acceptable salts of the compounds of the invention: i.e., salts that retain
the desired biological
activity of the parent compound and do not impart undesired toxicological
effects thereto.
For oligonucleotides, examples of pharmaceutically acceptable salts include
but are not limited
25 to (a) salts formed with cations such as sodium, potassium, ammonium,
magnesium, calcium,
polyamines such as spermine and spermidine, etc.; (b) acid addition salts
formed with inorganic
acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid,
phosphoric acid, nitric acid
and the like; (c) salts formed with organic acids such as, for example, acetic
acid, oxalic acid,
tartaric acid, succinic acid, malefic acid, fumaric acid, gluconic acid,
citric acid, malic acid,
3o ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid,
polyglutamic acid,
naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic
acid, polygalacturonic acid, and the like; and (d) salts formed from elemental
anions such as
chlorine, bromine, and iodine.
22
CA 02321189 2000-10-13
An expression vector comprising the antisense oligonucleotide sequence may be
constructed
having regard to the sequence of the oligonucleotide and using procedures
known in the art.
Vectors can be constructed by those skilled in the art to contain all the
expression elements
required to achieve the desired transcription of the antisense oligonucleotide
sequences.
to Therefore, the invention provides vectors comprising a transcription
control sequence operatively
linked to a sequence which encodes an antisense oligonucleotide. Suitable
transcription and
translation elements may be derived from a variety of sources, including
bacterial, fungal, viral,
mammalian or insect genes. Selection of appropriate elements is dependent on
the host cell
chosen.
Testing Activity of Antisense Oligonucleotidcs
One embodiment of the present invention provides methods for testing the
activity of the
antisense oligonucleotides. Commonly the antisense oligonucleotides are first
tested in vitro to
determine modulation of VEGF receptor expression and the subsequent effect of
this modulation.
2o The oligonucleotides can then be tested using in vivo techniques, using
animal models, prior to
their testing and subsequent use in humans.
In Vitro Assays
The in vitro assays can be performed using cultures of any cell line that
expresses the Flt-1
and/or the Flk-1 VEGF receptors. For example, bovine aortic endothelial cells
(BAEC) can be
used to test bovine antisense oligonucleotides of the present invention and
human umbilical vein
endothelial cells (HUVEC) can be used to test human antisense oligonucleotides
of the present
invention.
Various assays can be performed using cell cultures including those used to
determine protein
3o expression from the target Flt-1 and/or Flk-1 genes and the downstream
effects of decreased
protein expression.
Western blot and/or immunohistochemical analysis of Flt-1 and Flk-1 protein
expression can be
23
CA 02321189 2000-10-13
carried out using standard techniques and antibodies specific for Flt-1 or Flk-
1. A decrease in
protein expression, following treatment of cells in culture with the candidate
antisense
oligonucleotide, in comparison to untreated cells, is indicative of an
effective antisense
oligonucleotide. This is demonstrated in Example 1. Western blot and/or
immunohistochemical
analysis can also be used to determine the degree of VEGF-induced Flt-1 and/or
Flk-1
1o phosphorylation. An effective antisense oligonucleotide will cause a
decrease in
phosphorylation, as demonstrated in Example I.
Mitogenic assays can be performed to monitor endothelial cell proliferation in
the presence and
absence of a candidate antisense oligonucleotide. Effective antisense
oligonucleotides of the
present invention (i.e. those that are capable of down-regulating Flt-1 and/or
Flk-1 protein
expression) can block or inhibit the mitogenic effect of VEGF and thereby
reduce endothelial
cell proliferation. These assays can be performed using standard techniques
well known to those
skilled in the art. One example of a mitogenic assay using BAEC cultures is
provided in
Example I. As indicated above, this assay can be adapted for use with any Flt-
1 and/or Flk-1
2o expressing cell lines.
Chemotactic assays can be performed to evaluate the effect of candidate
antisense
oligonucleotides on VEGF-mediated cell migration. Effective antisense
oligonucleotides of the
present invention (i.e. those that are capable of down-regulating Flt-1 and/or
Flk-1 protein
expression) can block or inhibit the chemotactic effect of VEGF. These assays
can be performed
using standard techniques well known to those skilled in the art. One example
of a chemotactic
assay using BAEC cultures is provided in Example I. As indicated above, this
assay can be
adapted for use with any Flt-1 and/or Flk-1 expressing cell lines.
3o VEGF, as a result of interaction with VEGF receptors, has been shown to
enhance vascular
permeability through platelet activating factor (PAF) synthesis. A reduction
in PAF synthesis,
therefore, can be indicative of a successful antisense effect. By monitoring
the amount of PAF
produced in response to VEGF, in the presence and absence of a candidate
antisense
oligonucleotide of the present invention, it is possible to identify a
reduction in VEGF activity.
24
CA 02321189 2000-10-13
Methods of monitoring PAF production are well known to those skilled in the
art. One example
of a PAF production assay using BAEC cultures is provided in Example I. As
indicated above,
this assay can be adapted for use with any Flt-1 and/or Flk-1 expressing cell
lines.
In Vivo
Once a candidate antisense oligonucleotide is demonstrated to have an
effective in vitro effect, it
1o can then be tested in vivo. These assays are generally performed using
animal models, for
example the mouse testes model presented in Example II. In general, in vivo
assays involve the
administration or introduction of a candidate antisense oligonucleotide to a
subject and
monitoring its effect on Flt-1 and Flk-1 protein production and
phosphorylation and angiogensis.
Protein production and phosphorylation can be assayed by standard techniques,
including
15 Western blot and/or immunohistochemical analysis of tissue extracts.
Successful candidate
antisense oligonucleotides will demonstrate reduced Flt-1 and Flk-1 protein
production and
phosphorylation and a reduction in VEGF-mediated angiogenesis and/or
inflammation.
Histological and microscopic analysis can also be used, according to standard
techniques known
2o in the art, to view formation of blood vessels as an indication of
angiogensis. Successful
candidate antisense oligonucleotides will demonstrate reduced angiogenesis
and, therefore, a
reduction in the formation of blood vessels in comparison to tissue from
untreated subjects.
Use of Antisense Oligonucleotides
25 Antisense compounds are commonly used as research reagents and diagnostics.
For example,
antisense oligonucleotides, which are able to inhibit gene expression with
exquisite specificity,
are often used by those of ordinary skill to elucidate the function of
particular genes. Antisense
compounds are also used, for example, to distinguish between functions of
various members of a
biological pathway. Antisense modulation has, therefore, been harnessed for
research use.
In one embodiment of the present invention the antisense oligonucleotides are
used to block
VEGF-mediated effects in a mammal suffering from pathological angiogensis.
Pathological
angiogenesis is present in tumour growth and metastasis, ocular diseases
(diabetic and perinatal
CA 02321189 2000-10-13
hyperoxic retinopathies, age-related macular degeneration), arthritis,
psoriasis and
atherosclerosis. In a related embodiment of the present invention the
antisense oligonucleotides
are used to inhibit pathological angiogenesis in a mammal in need of such
therapy.
In an alternative embodiment of the present invention the antisense
oligonucleotides are used to
to reduce PAF synthesis and inflammation in a mammal in need of such therapy.
The antisense compounds of the present invention are also useful for research
and diagnostics,
because these compounds hybridize to nucleic acids encoding a mammalian VEGF
receptor that
is Flt-1 or Flk-1, enabling sandwich and other assays to easily be constructed
to exploit this fact.
15 Hybridization of the antisense oligonucleotides of the invention with a
nucleic acid encoding a
mammalian VEGF receptor that is Flt-1 or Flk-1 can be detected by means known
in the art.
Such means may include linkage of a fluorophore to the oligonucleotide,
attachment of a reporter
gene to the oligonucleotide, conjugation of an enzyme to the oligonucleotide,
radiolabelling of
the oligonucleotide or any other suitable detection means. Kits using such
detection means for
20 detecting the level of a mammalian VEGF receptor that is Flt-1 or Flk-1 in
a sample may also be
prepared.
Antisense Oligonucleotide Administration
When employed as pharmaceuticals, the antisense oligonucleotides are usually
administered in
25 the form of pharmaceutical compositions. The pharmaceutical compositions
are prepared by
adding an effective amount of an antisense oligonucleotide to a suitable
pharmaceutically
acceptable diluent or carrier. As such, one embodiment of the present
invention provides
pharmaceutical compositions and formulations which include the antisense
oligonucleotides of
the invention.
The pharmaceutical compositions of the present invention may be administered
in a number of
ways depending upon whether local or systemic treatment is desired and upon
the area to be
treated. Administration may be topical (including ophthalmic and to mucous
membranes
26
CA 02321189 2000-10-13
including vaginal and rectal delivery), pulmonary, e.g., by inhalation or
insufflation of powders
or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and
transdermal), oral or
parenteral. Parenteral administration includes intravenous, intraarterial,
subcutaneous,
intraperitoneal or intramuscular injection or infusion; or intracranial, e.g.,
intrathecal or
intraventricular, administration. Oligonucleotides with at least one 2'-O-
methoxyethyl
to modification are believed to be particularly useful for oral
administration.
Methods of delivery of foreign nucleic acids, such as antisense
oligonucleotides, are known in
the art, such as containing the nucleic acid in a liposome and infusing the
preparation into an
artery (LeClerc G. et al., (1992) J Clin Invest. 90: 936-44), transthoracic
injection (Gal, D. et al.,
(1993) Lab Invest. 68: 18-25.). Other methods of delivery may include coating
a balloon catheter
with polymers impregnated with the foreign DNA and inflating the balloon in
the region of
arteriosclerosis, thus combining balloon angioplasty and gene therapy (Nabel,
E.G. et al., (1994)
Hum Gene Ther. 5: 1089-94.)
2o Another method of delivery involves "shotgun" delivery of the naked
antisense oligonucleotides
across the dermal layer. The delivery of "naked" antisense oligonucleotides is
well known in the
art. See, for example, Felgner et al.,U.S. Patent No. 5,580,859. It is
contemplated that the
antisense oligonucleotides may be packaged in a lipid vesicle before "shotgun"
delivery of the
antisense oligonucleotide.
Another method of delivery involves the use of electroporation to facilitate
entry of the nucleic
acid into the cells of the mammal. This method can be useful for targeting the
antisense
oligonucleotides to the cells to be treated, for example, a tumour, since the
electroporation would
be performed at selected treatment areas.
In one embodiment of the present invention the antisense oligonucleotides or
the
pharmaceutical compositions comprising the antisense oligonucleotides may be
packaged into
convenient kits providing the necessary materials packaged into suitable
containers.
27
CA 02321189 2000-10-13
To gain a better understanding of the invention described herein, the
following examples are set
forth. It should be understood that these examples are for illustrative
purposes only. Therefore,
they should not limit the scope of this invention in any way.
EXAMPLES
1o EXAMPLE l: VEGFEFFECT ON ENDOTHELIAL CELL PROLIFERATION, MIGRATION
AND PAF SYNTHESIS
To discriminate the contribution of Flt-1 and Flk-1 receptors upon endothelial
cell (EC)
stimulation by VEGF, selective antisense deoxyribophosphorothioate oligomers,
which
hybridized specifically with a complementary mRNA sequence and prevented the
translation of
the targeted mRNA into its protein (Crooke R., (1991) Anticancer Drug Des. 6,
609-646; Loke,
S. et al (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3474-3478; Yakubov, L.A. et
al (1989) Proc.
Natl. Acad. Sci. U.S.A 86, 6454-6458), were used. This antisense gene
expression knockdown
approach resulted in downregulation of the protein expression of Flt-1 or Flk-
1 in a highly
selective fashion and thus to evaluate their contribution to the biological
activities mediated by
VEGF.
The mitogenic, chemotactic and PAF synthesis activities of VEGF on BAEC were
studied.
Furthermore, the ability of antisense oligonucleotide sequences complementary
to Flt-1 or Flk-1
mRNA to modulate VEGF-mediated effects is demonstrated. The activation of Flk-
1 was found
to be sufficient to mediate the VEGF actions on EC in vitro.
Materials and Methods
Cell Culture: BAEC expressing both VEGF receptors (Barleon, B. et al (1994) J.
Cell. Biochem.
54, 56-66) were isolated from freshly harvested aorta, cultured in Dulbecco's
modified eagle
medium (DMEM; Life Technologies, Burlington, ON) containing 5% fetal bovine
serum
(Hyclone Lab., Logan UT), and antibiotics (Sigma Chem., St-Louis, MO). BAEC
were
characterized by their cobblestone monolayer morphology and Factor VIII
immunohistochemistry, and were not passaged for more than 9 cycles.
28
CA 02321189 2000-10-13
S
Antisense Oligonucleotide Therapy: To discriminate the contribution of Flt-1
and Flk-1 upon
stimulation of EC by VEGF, BAEC were treated with antisense oligonucleotide
sequences
complementary to bovine Flt-1 or Flk-1 mRNA (GenBank Accession Numbers X94263
and
94298). A total of four different antisense oligonucleotide phosphorothioate
backbone sequences
1o were used, two targeting bovine Flt-1 mRNA (antisense 1, AS1-bFlt: 5'-CAA
AGA TGG ACT
CGG GAG-3' (SEQ ID NO:1); antisense 2, AS2-bFlt: S'-GTC GCT CTT GGT GCT ATA-3'
(SEQ ID N0:2)), and two targeting bovine Flk-1 mRNA (antisense l, AS1-bFlk: 5'-
GCT GCT
CTG ATT GTT GGG-3' (SEQ ID N0:3); antisense 2, AS2-bFlk: 5'-CCT CCA CTC TTT
TCT
CAG-3' (SEQ ID N0:4)). Two scrambled phosphorothioate sequences (scrambled
Flt, SCR-Flt:
15 5'-AGC TAG GCA CGA GAG TGA-3' (SEQ ID N0:19); scrambled Flk, SCR-Flk: 5'-
TGC
TGG CAT GTG CGT TGT-3' (SEQ ID N0:20)) were also used as negative controls.
These
sequences were designed with no more than three consecutive guanosines and by
minimizing
their capacity to form hairpins and homodimers. All sequences were synthesized
at the Armand
Frappier Institute (Laval, Canada). After synthesis, the oligonucleotides were
dried, resuspended
2o in sterile water and quantified by spectrophotometry. The antisense
oligomer solutions were by-
products-free, as confirmed using denaturing polyacrylamide gel
electrophoresis (20%; 7 M
urea), based on the known length of the oligonucleotide.
Western blot analysis of Flt-1 and Flk-1 protein expression: The efficiency
and specificity of the
25 antisense sequences to block the targeted protein expression were evaluated
by Western blot
analysis. Confluent BAEC (100 mm tissue culture plate) were washed with DMEM
and
trypsinized (trypsin-EDTA; Life Technologies). Cells were resuspended in DMEM
containing
5% of fetal bovine serum and antibiotics, and a cell count was obtained with a
Coulter counter
Zl (Coulter Electronics, Luton, UK). Cells were seeded at 1 x 106 cells / 100
mm tissue culture
30 plate (Becton-Dickinson, Rutherford, NJ), stimulated for 24 h in DMEM / 5%
FBS / antibiotics ~
antisense oligonucleotides (10-' - 5 x 10-' M) and starved for 48 h in DMEM /
0.25% FBS /
antibiotics ~ antisense oligonucleotides (10-' M daily) for Go
synchronization. The cells were
then grown to confluence for 16 h in DMEM / 1% FBS / antibiotics ~ antisense
oligonucleotides
29
CA 02321189 2000-10-13
(10-' - 5 x 10-' M) and starved for 8 h in DMEM / 0.25% FBS / antibiotics ~
antisense
oligonucleotides (10-' - 5 x 10-' M) to induce an upregulation of the VEGF
receptors expression.
The culture medium was removed and cells were rinsed twice with ice-cold DMEM.
Total
proteins were prepared by the addition of 500 pl of lysis buffer containing
phenylmethylsulfonyl
fluoride 1 mM (Sigma), leupeptin 10 ~g/ml (Sigma), aprotinin 30 pg/ml (Sigma)
and NaV03 1
1o mM (Sigma). Plates were incubated at 4 °-C for 30 min, scraped and
the protein concentration
was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).
Immunoprecipitation
was performed on 12 mg of total proteins for each sample by incubation with
rabbit anti-mouse
Flk-1 IgG or rabbit anti-human Flt-1 IgG polyclonal antibodies (Santa Cruz
Biotech., Santa
Cruz, CA) bound to protein A-Sepharose beads at 4 °-C for 1 h. Both
antibodies were specific for
their targeted protein and do not cross react with each other. After washing 3
times with lysis
buffer, the immunoprecipitates were dissolved in Laemmli's buffer, boiled for
5 min in reducing
conditions, separated by a 10%-20% gradient SDS-PAGE (Protean II kit; Bio-Rad)
and
transblotted onto a 0.45-pm polyvinylidene difluoride membranes (Milipore
Corp., Bedford,
MA). The membranes were blocked in 5% Blotto-TTBS (5% nonfat dry milk, Bio-
Rad; Tween-
20 0.05%, 0.15M NaCI, 25mM Tris-HC1 pH 7.5) for 2 h at room temperature with
gentle
agitation and incubated for 45 min in 1% Blotto-TTBS containing the desired
antisera (anti-Flt-1
or anti-Flk-1; dilution 1:100). Membranes were washed 3 times with TTBS,
reblocked for 10
min in 1% Blotto-TTBS and incubated with a horseradish peroxidase goat anti-
rabbit IgG
antibodies (dilution 1:7500, Santa Cruz) in 5% Blotto-TTBS for 30 min.
Membranes were
washed with TTBS, and horseradish peroxidase bound to secondary antibody was
revealed by
chemiluminescence (Renaissance kit, New England Nuclear, Boston, MA).
Kaleidoscope
molecular weight and SDS-PAGE broad range marker proteins (Bio-Rad) were used
as standards
for SDS-PAGE. Digital image densitometry (PDI Bioscience, NY) was performed on
X-ray
films to determine relative percentages of Flt-1 or Flk-1 protein expression.
Western blot analysis of Flt-1 and Flk-1 protein phosphorylation: BAEC were
pretreated with
the antisense sequences as described above for Western blot analysis. Cells
were then rinsed
with DMEM, incubated on ice in DMEM + 1 mg/ml BSA + VEGF (10-9 M) for 30 min,
CA 02321189 2000-10-13
incubated at 37 °C for 7 min and then brought back on ice. Cells were
rinsed with DMEM +
NaV03 (1mM), and total proteins were prepared as described.
Immunoprecipitation was
performed on 500 pg of total proteins with rabbit anti-mouse Flk-1 IgG or
rabbit anti-human Flt-
1 IgG polyclonal antibodies (Santa Cruz Biotech.) bound to protein G-Sepharose
4 Fast Flow
(Amersham, Uppsala, Sweden) at 4 °C for 1 h. After 3 washes with lysis
buffer, the
1o immunoprecipitates were dissolved in Laemmli's buffer, boiled for 5 min in
reducing conditions,
separated by a 6% SDS-PAGE (Mini-Protean II kit; Bio-Rad) and transblotted
onto a 0.45 ~m
PVDF membrane. The membranes were blocked in 3%-BSA-PBST (Tween 0.1%) for 1 h
at
room temperature and incubated overnight with the primary antisera (mouse anti-
phosphotyrosine clone 4610; dilution 1:3000, Upstate Biotechnology Inc, Lake
Placid, NY).
Membranes were washed with PBST, incubated with an anti-mouse IgG (dilution
1:4000, Santa
Cruz), washed with PBST and chemiluminescence protocol was followed as
described above.
Mitogenic assays: Confluent BAEC were washed with DMEM, and trypsinized. Cells
were
resuspended in 9 ml of DMEM / 5% FBS / antibiotics, and a cell count was
obtained. BAEC
2o were seeded at 1x104 cells / well of 24-well tissue culture plates,
stimulated for 24 h in DMEM /
5% FBS / antibiotics ~ antisenses (10-' M) and starved for 48 h in DMEM /
0.25% FBS /
antibiotics ~ antisenses (10-' M daily) for G~ synchronization. The cells were
stimulated for 72 h
in DMEM / 1% FBS / antibiotics ~ antisenses (10-' M daily) with different
concentrations of
VEGF or P1GF (human recombinant vascular growth factor, VEGF,bs; PeproTech
Inc., Rocky
Hill, NJ., and human placenta growth factor, PIGF,sz; R & D Systems,
Minneapolis, MN.). The
cells were then trypsinized and cell number was determined by using a Coulter
counter.
Chemotaxis assays: Cell migration was evaluated using a modified Boyden 48-
well
microchamber kit (NeuroProbe, Cabin John, MD). Near confluent BAEC (100 mm
tissue culture
plate) were washed with DMEM, and trypsinized. Cells were resuspended in DMEM
/ 5% FBS /
antibiotics, and a cell count was obtained. BAEC were seeded at 2.5 x lOs
cells / well of 6-well
tissue culture plates, stimulated for 24 h in DMEM / 5% FBS / antibiotics ~
antisense
oligonucleoitdes (10-' M), starved for 48 h in DMEM / 0.25% FBS / antibiotics
~ antisense
oligonucleotides (10-' M daily). Cells were harvested by trypsinisation,
resuspended in DMEM /
31
CA 02321189 2000-10-13
1% FBS / antibiotics at a concentration of 1 x 106 cells / ml. Fifty
microliters of this solution t
antisense oligonucleotides (10-' M) was added in the higher chamber of the
modified Boyden
chamber apparatus, and the lower chamber was filled with DMEM / 1% FBS /
antibiotics plus
the proper concentration of agonist (VEGF or P1GF). The two sections of the
system were
separated by a porous polycarbonate filter (5 pm pores) pretreated with a
gelatin solution (1.5 mg
/ ml), and assembled. Five hours post-incubation at 37 °C, the non-
migrated cells were scraped
with a plastic policeman, the migrated cells were stained using Quick-Diff
solutions. The filter
was then mounted on a glass slide and migrated cells were counted using a
microscope adapted
to a video camera to obtain a computer-digitized image.
Measurement of PAF synthesis: PAF production by BAEC was measured by
incorporation of 3H-
acetate into lyso-PAF (Sirois, M.G., and Edelman, E.R. (1997) Am. J. Physiol.
272, H2746-
H2756). Confluent BAEC (100 mm tissue culture plate) were washed with DMEM and
trypsinized. Cells were resuspended in DMEM / 5% FBS / antibiotics, and a cell
count was
obtained. Cells were seeded at 5 x 105 cells / well of 6 well tissue culture
plates, stimulated for
24 h in DMEM / 5% FBS / antibiotics ~ antisense oligonucleotides (10-' M - 5 x
10-' M) and
starved for 48 h in DMEM / 0.25% FBS / antibiotics ~ antisense
oligonucleotides (10-' M - 5 x
10-' M daily) for Go synchronization. The cells were then grown to confluence
for 24 h in
DMEM / 1% FBS / antibiotics ~ antisense oligonucleotides (10-' M - 5 x 10-' M)
and starved for
8 h in DMEM / 0.25% FBS / antibiotics ~ antisense oligonucleotides (10-' M - 5
x 10-' M) to
induce an upregulation of VEGF receptor expression. Culture medium was removed
and cells
were rinsed twice with HBSS (Hank's balanced salt solution) / HEPES (10 mM; pH
7.4). Cells
were then stimulated for 15 min in 1 ml of HBSS-HEPES (10 mM, pH 7.4) + CaCl2
(10 mM) +
3H-acetate (25 pCi) plus the appropriate concentration of agonist (VEGF or
P1GF). The reaction
was stopped by addition of acidified methanol (50 mM acetic acid), the wells
were scraped and
added to chloroform (2.5 ml) and 0.1 M sodium acetate (1 ml) mixture. Culture
plates were
washed twice with 1 ml of methanol, added to the chloroform mixture and
centrifuged for 2 min
at 1 700 rpm. The upper phase was discarded and the chloroform phase was
washed twice with 2
ml of the organic phase of a HBSS-HEPES (10 mM)nmethanol-chloroform-sodium
acetate
(O.1M) solution (1:2.5:3.75:1). Isolated lipids were evaporated under a stream
of NZ gas,
32
CA 02321189 2000-10-13
resuspended in 175 pl of mobile phase solvent (water-chloroform-methanol
5:40:55) and purified
by HPLC. Samples were injected into a silica-based normal-phase HPLC column
(4.5 x 250
mm, 5 p.m silica particle size; Varian, Harbour City, CA) and eluted with the
mobile phase
solvent at a 0.5 ml / min flow rate. Fractions were collected every min and
the amount of 3H-
PAF synthesised was quantified by counting radioactivity with a (3-counter.
The authenticity of
synthesized 3H-PAF was confirmed by an HPLC elution pattern similar to
standard 3H-PAF
(New England Nuclear), and by its ability to induce platelet aggregation
similar to standard PAF
(Avanti Polar Lipids, Alabaster, AL) (Sirois, M.G., and Edelman, E.R. (1997)
Am. J. Physiol.
272, H2746-H2756).
Statistical Analysis: Data are mean ~ SEM. Statistical comparisons were made
by analysis of
variance followed by an unpaired Student's t-test. Data were considered
significantly different if
values of P < 0.05 were observed.
Results
Modulation of Flt-1 or Flk-1 protein expression by antisense oligonucleotides:
In order to
determine the potency of antisense oligonucleotides to inhibit the targeted
protein expression,
BAEC were pretreated with either the antisense or the scrambled
oligonucleotide sequences.
Total proteins were extracted, quantified by bioassay, immunoprecipitated with
an anti-Flt-1 or
an anti-Flk-1 antibody, and the expression of each receptors was determined by
Western blot
analysis. Digital image densitometry was performed and results were expressed
as relative
expression percentages when compared with control PBS-treated cells. The basal
protein
expression of Flt-1 (Ctrl) was inhibited when the BAEC were pretreated with
the two antisense
complementary to Flt-1 mRNA (10~' M); the first antisense sequence (ASl-Flt)
suppressed Flt-1
protein expression by 91%, while the second antisense sequence (AS2-Flt)
showed a 94%
3o inhibition effect (Figure lA). Similar treatment with the two antisense
sequences (ASl-bFlk-1
and AS2-bFlk-1; 10-' M) complementary to Flk-1 mRNA suppressed basal Flk-1
protein
expression by 80% and 78%, respectively (Figure 1B). Two scrambled sequences
(SCR-Flt and
SCR-Flk; 10-' M) had no inhibitory effect on the studied receptor expression
as compared to
33
CA 02321189 2000-10-13
control cells (Figure lA and B). To achieve a greater inhibition of Flk-1
protein expression,
BAEC were pretreated with a higher concentration of antisense (AS1-bFlk and
AS2-bFlk; 5 x
10-' M), resulting in a 99% and 94% suppression of Flk-1 protein expression
respectively (Figure
1C). The scrambled sequence (5 x 10~' M) showed a slight reduction by 16% of
Flk-1 protein
expression (Figure 1C).
To ensure that the antisenses designed to downregulate the expression of Flk-1
would not affect
Flt-1 receptor expression and vice versa, a Western blot analysis was
performed to evaluate the
specificity of our most potent antisenses. A pretreatment with the more potent
antisense for the
downregulation of Flk-1 expression (AS1-bFlk; 5 x10-' M) did not significantly
affect Flt-1 basal
expression (Figure 2A) while the more potent antisense designed for the
blockade of Flt-1
receptor expression (AS2-bFlt; 5 x 10-' M) almost completely blocked Flt-1
receptor expression
(Figure 2A). A pretreatment with AS1-bFlk (5 x 10-' M) severely impaired Flk-1
protein
expression as compared to non-treated cells, while AS2-bFlt (5 x 10-' M) was
without significant
effect (Figure 2B).
Inhibition of VEGF-induced Flt-1 or Flk-I phosphorylation by antisense
oligomers: Since the
herein described antisense sequences were found to be specific at blocking the
targeted receptor
expression, it was then necessary to determine their potency to modulate Flt-1
and Flk-1 protein
phosphorylation upon stimulation with VEGF. First, the stimulation of BAEC
with VEGF (10-9
M) induced an increase of Flt-1 and Flk-1 phosphorylation by up to 1.5 and
13.2-fold
respectively, over PBS-treated cells (Figure 3A and B). Pretreatment with the
more potent
antisense directed against Flt-1 mRNA (AS2-bFlt; 5 x 10-' M) reduced by 50%
the VEGF-
induced phosphorylation of Flt-1 protein, while the more potent antisense
directed against Flk-1
mRNA increased its phosphorylation by 13% (Figure 3A). A similar pretreatment
with the AS1-
bFlk (5 x 10-' M) inhibited by as much as 87% the phosphorylation of Flk-1
receptor (Figure
3B), while a pretreatment with AS2-bFlt (5 x 10-' M) slightly decreased Flk-1
phosphorylation in
response to VEGF (10-9 M) by 18% (Figure 3B).
VEGF and PIGF mitogenic activity on BAEC: The VEGF and P1GF mitogenic effects
were
34
CA 02321189 2000-10-13
examined in order to discriminate the involvement of the two VEGF receptors on
BAEC
proliferation. Stimulation of quiescent BAEC with DMEM / 1% FBS raised the
cell count from
080 ~ 520 to 19 180 ~ 600 cells within 72 h. The addition of VEGF (10-", 10-
'° and 2.5 x 10-
'o M) increased endothelial cell proliferation dose-dependently with maximal
induction of 62%,
183% and 219% respectively as compared to DMEM / 1% FBS (Figure 4). In
contrast, P1GF
10 (10-", 10-'°, 10-9 and 10-8 M) did not show any mitogenic activity
on BAEC as compared with
DMEM / 1% FBS (Figure 4).
Effects of antisense oligonucleotides complementary to Flk-1 and Flt-1 mRNA on
VEGF
mitogenic activity: By downregulating the protein expression of Flk-1 and Flt-
1 by antisense
gene targeting, it was possible to determine the contribution of each receptor
type to VEGF's
mitogenic effect on BAEC. FBS (1%) increased BAEC count from 9 860 ~ 640 to 37
260 ~ 2
260 cells. The addition of VEGF (2.5 x 10-'° M) increased BAEC
proliferation by an additional
105% (P < 0.01) (Figure 5). Treatment of BAEC with the two antisense sequences
directed
against the Flk-1 mRNA completely blocked VEGF's mitogenic activity. The
scrambled
oligonucleotide sequences also failed to block VEGF-induced proliferation of
BAEC.
VEGF and PIGF chemotactic activity on BAEC: Using a modified Boyden chamber
assay, the
chemotactic response of BAEC to VEGF and P1GF was studied. VEGF (10-'°,
2.5 x 10-'° and 10-
9 M) induced a dose-dependent increase (46%, 83%, and 130% respectively) of
BAEC migration
as compared to PBS-stimulated cells, raising the migrated cell count from 120
~ 4 (PBS) to 276
~ 8 cells / mmz (VEGF 10-9 M; P < 0.001) 5 hours post-treatment (Figure 6).
Checkerboard
analysis revealed that the response of BAEC to VEGF was a result of chemotaxis
and not
chemokinesis. Treatment with P1GF (10-'°, 10-9 and 10-g M) had no
significant effect on the basal
migration of BAEC as compared to PBS-stimulated cells (Figure 6).
Effects of Flk-1 and Flt-1 mRNA antisense oligonucleotides on VEGF chemotactic
activity: Non-
stimulated BAEC (PBS) showed a basal migration count of 105 ~ 7 cells / mm2
(Fig. 7).
Stimulation with VEGF (109 M) increased the migrated cell count to 205 ~ 5
cells / mm2.
CA 02321189 2000-10-13
Pretreatment of BAEC with any of the four antisense sequences (AS1 or AS2-
bFlk, AS1 or
AS2-bFlt; 10-' M) or scrambled sequences (SCR-Flt or SCR-Flk; 10-' M) did not
significantly
affect basal migration in the absence of VEGF. In contrast, the antisense
oligonucleotide
sequences complementary to Flk-1 mRNA, AS1-bFlk and AS2-bFlk (10-' M),
decreased by 91%
and 80% respectively the migration elicited by VEGF. The use of the antisense
sequences to Flt-
1 mRNA (10-' M) did not alter VEGF-induced chemoattraction of BAEC. The
scrambled
oligonucleotide sequences did not significantly affect the chemotactic
properties of VEGF
(Figure 7).
VEGF and PIGF effects on endothelial cell PAF synthesis: To determine whether
VEGF and
P1GF stimulated PAF synthesis in EC, confluent BAEC were incubated with growth
factors and
PAF synthesis was determined by metabolic incorporation of 3H-acetate into
lyso-PAF, the
precursor of PAF synthesis. VEGF (10-'°, 10-y and 10-8 M) dose-
dependently elicited the
synthesis of PAF, with increases of 7.2-, 20.4- and 35.9-fold respectively as
compared to PBS-
treated cells (Figure 8). Treatment with P1GF (10-'°, 10-9 M) did not
significantly affect the basal
2o PAF synthesis of BAEC. However, at 108 M, P1GF induced a slight but
significant increase in
PAF synthesis (67%) as compared to PBS-treated cells (Figure 8).
Effects of Flk-1 and Flt-1 mRNA antisercse oligonucleotides on VEGF-induced
PAF synthesis: In
order to determine the basal and maximal PAF synthesis by BAEC, a group of
cells were left
untreated and others were treated with VEGF (10-9 M) for 15 minutes. The
synthesis of 3H-
labelled PAF increased from 781 ~ 86 to 8 254 ~ 292 DPM (Figure 9). Treatment
of BAEC with
the antisense oligonucleotide sequences complementary to Flk-1 mRNA, AS1-Flk
and AS2-Flk,
(10-' M) reduced by 77% and 75% respectively the synthesis of PAF elicited by
a VEGF
treatment (Figure 9). In contrast, pretreatment with the antisense
oligonucleotide sequences
complementary to Flt-1 mRNA (10-' M) failed to inhibit VEGF's inflammatory
activity on
BAEC. The scrambled oligonucleotide sequences (SCR; 10-' M) also failed to
affect VEGF-
induced PAF synthesis (Figure 9). Since both antisense oligonucleotide
sequences
complementary to Flk-1 mRNA (10'' M) failed to fully inhibit PAF synthesis
induced by VEGF
(10-9 M), the concentration of antisense directed against Flk-1 mRNA was
increased to 5 x 10-'
36
CA 02321189 2000-10-13
M during BAEC treatment. The application of AS1-Flk and AS2-Flk (5 x 10-' M)
caused a near
complete inhibition of Flk-1 protein expression (Figure 1 C) and a reduction
of PAF synthesis by
85% and 82% respectively in response to VEGF (10-9 M), while the two antisense
sequences
complementary to Flt-1 mRNA (5 x 10-' M) did not inhibit VEGF-induced PAF
synthesis
(Figure 9). The absence of nonspecific inhibitory effects was furthermore
confirmed by
1o pretreating BAEC with the scrambled sequences (5 x 10-' M) which did not
affect PAF synthesis.
As the inhibition of Flk-1 expression had a direct effect on PAF synthesis, a
correlation analysis
was performed. The synthesis of PAF by BAEC treated with VEGF (10-9 M) showed
a linear
correlation increment with Flk-1 protein expression [PAF synthesis % = m x Flk-
1 expression
+ b] , where m is the slope and b is the linearity constant. Our data showed a
slope m of 0.89
and a linear constant b of 9.81 (r = 0.984; Figure 10) .
Discussion
Angiogenesis is a tightly regulated process, integral to normal and
pathological conditions.
Crucial steps in the angiogenic process support an early increase in vascular
permeability
(Dvorak, H.F., et al (1995) Am. J. Pathol. 146, 1029-1039), closely followed
by migration and
2o proliferation of EC. Much evidence implicates VEGF and its two tyrosine
kinase receptors Flt-1
and Flk-1 as major regulators of these events (Waltenberger, J., et al (1994)
J. Biol. Chem. 269,
26988-26995; Brown, L.F., et al (1995) Human Pathol. 26, 86-91; Ravindrath,
N., et al (1992)
Endocrinology 94, 1192-1199; and Breier, G., et al (1992) Development 114, 521-
532). VEGF,
unlike any other growth factors studied to date, is capable of inducing
protein extravasation and
it is likely that its angiogenic properties are mediated in large part through
the induction of
plasma protein leakage (Dvorak, H.F., et al (1995) Am. J. Pathol. 146, 1029-
1039). It was
recently shown that VEGF's effect on vascular permeability was mediated
through the synthesis
of PAF by EC (Sirois, M.G., and Edelman, E.R. (1997) Am. J. Physiol. 272,
H2746-H2756).
The present invention demonstrates that the proliferation, migration and PAF
synthesis elicited
by VEGF in cultured BAEC are dose-dependent (Figures 4, 6 and 8) and above
all, these effects
were completely (proliferation) or almost completely (migration and PAF
synthesis) inhibited by
treating the cells with specific antisense oligonucleotide sequences
complementary to Flk-1
37
CA 02321189 2000-10-13
receptor mRNA.
Antisense oligomers specifically inhibit Flt-1 or Flk-1 receptor expression.
Both Flt-1 and Flk-1 are cell surface-associated receptors deemed to play a
role in VEGF-
induced EC activation. Recent studies have investigated their signal
transduction properties
using porcine aortic endothelial cells or NIH 3T3 cells transfected with a
plasmid coding either
for Flk-1 or Flt-1 (Waltenberger, J., et al (1994) J. Biol. Chem. 269, 26988-
26995; Seetharam,
L., et al (1995) Oncogene 10, 135-147). Recently, many novel VEGF-related
molecules (P1GF,
VEGF-C, VEGF-C-ONOC 1565 mutant) which vary in their potency to activate one
of the two
VEGF receptors preferentially were isolated and characterized (Park, J.E., et
al (1994) J. Biol.
Chem. 269, 25646-25654; Joukov, V., et al (1998) J. Biol. Chem. 273, 6599-
6602; and Clauss,
M., et al (1996) J. Biol. Chem. 271, 17629-17634). Although the use of these
analogs suggested
that both receptors could mediate biological actions, they do not assess the
possible formation of
heterodimers, which has been proposed to occur between VEGF receptors when
signaling
(Waltenberger, J., et al (1994) J. Biol. Chem. 269, 26988-26995).
2o In the present invention antisense gene therapy was used to suppress
specifically the Flt-1 and
Flk-1 gene products. This approach allowed the use of fresh non-transfected
endothelial cells
which endogenously express the two VEGF receptors and the intracellular
pathways found in
native EC. In addition, since it was possible to inhibit separately the Flt-1
and Flk-1 protein
expression, the present system provided the possibility to evaluate if Flt-1
and Flk-1
heterodimerization was required to observe the VEGF biological activity.
This example made use of two selective antisense oligonucleotide sequences for
the Flt-1
receptor mRNA, and two others for the Flk-1 receptor mRNA. These sequences did
not contain
more than three consecutive guanosines to avoid a possible interference with
serum proteins
3o including growth factors like VEGF (Stein, C.A. (1995) Nature Med. 1, 1119-
1121). Having the
assurance that BAEC express both VEGF receptors (Pepper, M.S., et al (1998)
.l. Cell. Physiol.
177, 439-452), the ability of antisense oligomers to specifically inhibit the
expression and
phosphorylation patterns of Flt-1 and Flk-1 was determined. As shown by
Western blot analysis,
38
CA 02321189 2000-10-13
BAEC expressed Flt-1 and Flk-1 proteins (Figure lA, B and C) which were both
phosphorylated
by a VEGF treatment (Figure 3A and B). Treatment of BAEC with the antisense
Flt-1 oligomers
(up to 5 x 10-' M) for a 4 day period decreased the protein expression of Flt-
1 receptor by as
much as 94% (AS2-bFlt; Fig. lA) and inhibited its phosphorylation by up to 50%
in response to
a VEGF stimulation (10-9 M; Figure 3A). Treatment with the antisense Flk-1
oligomers (10-' M)
to was also effective at modulating Flk-1 receptor expression, with a maximum
inhibition of 80%
(AS1-bFlk). The difference in the inhibitory percentage is in accordance with
previous reports
which showed that the biological effects of antisense oligomers are dictated
in part by the
kinetics of antisense target gene expression (Edelman, E.R., et al (1995)
Circ. Res. 76, 176-182).
The difference between Flk-1 and Flt-1 oligomers was overcome by increasing
the antisense Flk-
1 oligomer concentration to 5 x 10-' M, resulting in a greater reduction in
the residual Flk-1
expression when compared with the 10-' M treatment, from a 80% to a 99%
inhibition of Flk-1
protein expression. This latter pretreatment also prevented VEGF-induced Flk-1
protein
phosphorylation by as much as 87% (Figure 3B).
2o Previous reports have raised concerns that the inhibitory activities of
antisense oligonucleotides
may arise from non-specific rather than hybridization-dependent mechanisms
(Burgess, T.L., et
al (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4051-4055; and Guvakova, M.A., et
al (1995) J. Biol.
Chem. 270, 2620-2627). To address this issue more definitely, two groups of
BAEC were
pretreated with two different scrambled oligomers at similar concentrations
(10-' - 5 x 10-' M).
In contrast to the VEGF receptor antisense oligomers, the scrambled oligomers
(10-' M) failed to
modulate the normal pattern of VEGF receptors protein expression by BAEC,
although it
showed a slight reduction at a higher concentration (5 x 10-' M). In addition,
no cross-reactivity
was observed between the Flk-1-directed antisense sequences and Flt-1
expression and vice versa
(Figure 2A and B). It is to be noted also that the scrambled oligomers (10-' -
5 x 10-' M) did not
inhibit VEGF effect on EC proliferation, migration and PAF synthesis.
Antisense oligomer-directed modulation of VEGF activities
Since the antisense sequences used in this example specifically prevented both
the protein
39
CA 02321189 2000-10-13
expression and phosphorylation of Flt-1 or Flk-1 genes, they were tested for
their ability to
modulate VEGF properties on EC. A treatment with ASl-Flk (10-' M) was
sufficient to provide
a complete inhibition of VEGF mitogenic effect (Figure 5), and abolished
almost completely
(91% inhibition) the cellular migration induced by VEGF (Figure 7). However,
this approach
inhibited by 75% the synthesis of PAF (Figure 9). A higher concentration of
AS1-Flk (5 x 10-'
1o M) induced not only a higher inhibition of Flk-1 protein expression (Figure
1B), but also blocked
the PAF synthesis elicited by VEGF by as much as 85% (Figure 9). This
demonstration suggests
that Flk-1 plays a role in mediating VEGF effects on BAEC. The correlation
between the
synthesis of PAF from BAEC stimulated with VEGF (10-9M) and the expressed Flk-
1 receptors
on these EC was also demonstrated. A linear correlation was established and
suggested that a
complete inhibition of Flk-1 protein expression by antisense oligomers against
Flk-1 mRNA
would still permit VEGF to induce a 9.8% residual PAF synthesis by treated
BAEC (Figure 10).
Such a minor effect can possibly be explained either by Western blot analysis
limitation to fully
detect residual Flk-1 protein expression or by a partial contribution of Flt-1
stimulation. Though
a 99% inhibition of Flk-1 protein expression was observed, it is possible that
more than 1% of
2o Flk-1 receptors were still present on BAEC surface, which could not be
detect by either the
immunoprecipitation process or the protein revelation by chemiluminescence
after a Western
blot study. The other possibility to explain the residual PAF synthesis may
involve a partial
effect through the activation of Flt-1 receptors. This latter hypothesis is
supported by the data
from P1GF treatment of BAEC.
P1GF is a secreted growth factor expressed by umbilical vein EC and placenta
(Maglione, D., et
al (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9267-9271; and Hauser, S., and
Weich, H. (1993)
Growth Factors 9, 259-268). According to its amino acid sequence, P1GF shows a
partial
homology to VEGF (53% homology), which might explain its ability to bind
uniquely to Flt-1
(Park, J.E., et al (1994) J. Biol. Chem. 269, 25646-25654; and Clauss, M., et
al (1996) J. Biol.
Chem. 271, 17629-17634). Therefore, P1GF can be used to study the effect of
Flt-1 activation on
EC. Although various concentrations of P1GF (10-'° - 10-8 M) failed to
elicit EC proliferation
and migration, P1GF at 10-~ M induced a slight but significant increment of
PAF synthesis over
control levels, suggesting that Flt-1 may indeed participate in mediating P1GF
and VEGF action
CA 02321189 2000-10-13
on EC. This is in agreement with previous reports which have shown that Flt-1
stimulation
either by P1GF or VEGF can induce Flt-1 phosphorylation (Waltenberger, J., et
al (1994) J. Biol.
Chem. 269, 26988-26995; Cunningham, S.A., et al (1997) Biochem. Biophys. Res.
Commun.
240, 635-639; and Sawano, A., et al (1997) Biochem. Biophys. Res. Commun. 238,
487-491).
However, the biological activities mediated by either VEGF or P1GF upon Flt-1
l0 activation/phosphorylation on intracellular Ca2+ elevation, cellular
proliferation, migration and
procoagulant tissue factor production observed were either absent or weak
(Waltenberger, J., et
al (1994) J. Biol. Chem. 269, 26988-26995; Clauss, Met al (1996) J. Biol.
Chem. 271, 17629-
17634; Hauser, S., and Weich, H. (1993) Growth Factors 9, 259-268; Cunningham,
S.A., et al
(1999) Am. .l. Physiol. 276, C176-C181) as compared to Flk-1
activation/phosphorylation.
Consequently, the residual PAF synthesis (10%) that was observed following an
antisense Flk-1
oligomer treatment as estimated by the linear correlation may in fact be due
to: 1) an incomplete
suppression of the Flk-1 protein expression and/or 2) from intracellular
signaling through Flt-1
receptor activation. In addition, VEGF may interact with Flt-1 differently
than P1GF and induce
a greater PAF synthesis. Therefore, these data support the hypothesis that Flt-
1 stimulation is
2o capable of mediating biological response, but to a lower extent than Flk-1
stimulation.
VEGF, Flt-1 and Flk-1
Many studies suggest that VEGF and its two receptors may take part in the
angiogenesis
phenomenon. For instance, homozygous disruption of the Flk-1 gene leads to
embryonic death
due to failure of vasculogenesis whereas homozygous Flt-1 disruption allows
normal vascular
endothelial differentiation and development but leads to a failure to assemble
normal vascular
channels and death (Fong, G.H., et al (1995) Nature 376, 66-70; and Sharma,
H.S., et al (1992)
Exper. Suppl. 61, 255-260). In this example, the inhibition of Flk-1 protein
expression severely
impaired VEGF effects on EC, which supports the importance of this receptor
for VEGF activity.
In summary, this example demonstrates that antisense oligomer-directed
inhibition of Flk-1
receptor expression severely impaired VEGF-induced EC proliferation, migration
and PAF
synthesis.
41
CA 02321189 2000-10-13
EXAMPLE IL~ VEGF-MEDIATED ANGIOGENESIS - ROLE OF FLK-1 AND FLT 1
RECEPTORS
A new model of angiogenesis was developed using mice testes. Briefly, the
model was created
using the following steps:
1o (i) the inguinal canal was cut open to isolate the right testis;
(ii) a PE-10 catheter was inserted through the tunicae vaginalis and
positioned in the
right testis;
(iii) the catheter was secured with a microsuture (8.0 silk) outside the
testis;
(iv) the abdominal rectus aponevrosis was sutured to recreate the inguinal
canal; and
(v) the other extremity of the PE-10 catheter was adapted to an Alzet pump
loaded
with a candidate antisense oligonucleotide or combination of antisense
oligonucleotides and placed subcutaneously on the abdominal-lateral side.
Alzet pumps 2002 were implanted in C57BL/6 wild type mice to deliver either
control buffer
2o (PBS; 200 pl) or VEGF (1, 2.5 and 5 pg/200 p,l). Under anesthesia, pictures
of the superficial
testis vascular network were taken at day 0 (before treatment) and at day 14
(before sacrifice).
Images were analysed with NIH image 1.6. The number, the diameter and the
length of newly
formed and pre-existing blood vessels were measured and compared. After
sacrifice of the
animals, testes were isolated, fixed in 10% formalin PBS-buffered solution and
processed for
standard histological procedures. As shown in Figures 11 and 12, a sustained
infusion of PBS
had no angiogenic effect (Figures 11A and 12), whereas VEGF (1 - 5 pg) induced
a marked
angiogenic response as demonstrated by the increased number of patent vessels
displaying red
blood cell transit (Figures 11A and 12). The vessel density increased by 250%>
compared to PBS
(Figure 11A). The neovessels had a diameter of 8 to 10 pm and a length of 300
pm. VEGF-
3o induced angiogenesis was abolished by simultaneous infusion of antisense
directed against either
Flt-1 or Flk-1 receptor mRNA (Figures 11B and 12). A scrambled oligomer failed
to prevent
VEGF-induced angiogenesis (Figure 11B). The efficiency and selectivity of the
antisense
oligomers was validated using standard immunohistochemistry techniques.
42
CA 02321189 2000-10-13
Figure 11A depicts the results from an experiment wherein animals were treated
with PBS, or
VEGF at 1, 2.5 and 5 pg/200 pl (0.125, 0.312 and 0.625 pM) for a period of 14
days. After
sacrifice of the animals, testes were isolated, fixed in 10% formalin PBS-
buffered solution and
processed using standard histological procedures (Sirois, Simons, Edelman
(1997) Circulation.
95, 669-676; Noiseux, Boucher, Cartier, Sirois (2000) Circulation. 102, 1330-
1336). In the
1o presence of PBS no angiogenic effect was observed, whereas, VEGF (1-5 pg)
induced a marked
angiogenic response as demonstrated by the 2.5-fold increase in the number of
newly formed
blood vessels when compared to PBS controls.
Figure 11B depicts the results from an experiment wherein animals were treated
with either PBS,
VEGF (2.5 pg/200 pl) alone, or VEGF (2.5 pg/100 pl) combined with Flt-1, Flk-1
or scrambled
antisense oligomers (200 pg/100 pl) for 14 days. In another set of experiments
Flt-1, Flk-1 or
scrambled antisense oligomers alone (in absence of VEGF) (200 pg/200 pl) were
delivered for
14 days. The number of newly formed blood vessels were measured as above and
the infusion of
either Flt-1 or Flk-1, but not scrambled, antisense oligomers, significantly
reduced VEGF-
induced angiogenesis. The use of the Flt-1 or Flk-1 antisense or scrambled
oligomers alone (i.e.
in the absence of VEGF), did not alter the basal level of blood vessels (data
not shown).
The application of antisense oligonucleotides directed against Flk-1 mRNA
sequences abrogated
Flk-1 protein expression with no effect on Flt-1 protein expression.
Similarly, the application of
antisense oligonucleotides directed against Flt-1 mRNA sequences abrogated Flt-
1 protein
expression with no effect on Flk-1 protein expression. Scrambled oligomers had
no effect on Flt-
1 and Flk-1 protein expression (Figures 13 and 14). In addition, the
application of antisense or
scrambled oligomers did not alter the expression of endothelial cell nitric
oxide synthase
(ecNOS) (Figure 15). Primary antibodies were tested, to confirm their
specificity and non-
crossreactivity, using standard techniques.
Figure 13 depicts the results from an experiment designed to demonstrate the
efficiency and
selectivity of the Flk-1 antisense oligonucleotides on Flk-1 protein
expression in mouse testis.
Immunohistochemical analyses on mouse treated testis, as detailed above.
Briefly, treated mouse
43
CA 02321189 2000-10-13
testis were fixed in 10% formalin PBS-buffered solution. The testis were
embedded in paraffin
and sections of 6 hum were obtained by microtome along the length of the
specimen. The testes
were deparaffinized in xylene and ethanol baths, endogenous peroxidase
activity was quenched
in a solution of methanol (200 ml) plus hydrogen peroxide (3%, 50 ml), and non-
specific binding
antibody binding prevented by preincubating the tissues with serum (1:10) from
species other
to than those used to raise the primary antibody. Arterial sections were then
exposed to the primary
antibody, mouse monoclonal anti-human Flk-1 IgG (which recognises mouse Flk-1
protein; and
without cross-reactivity with Flt-1 protein) (Santa Cruz Biotech) diluted
(1:500; 2 hrs), or rinsed
with PBS, and incubated with a biotinylated goat anti-mouse IgG (1:400; 1 hr)
(Vector Labs
Inc.). Peroxidase labelling was achieved with an incubation using
avidin/peroxidase complex
is (Vector Labs Inc.), and antibody visualisation established after a 5 min
exposure to 0.05% 3,3'-
diaminobenzidine (Sigma Chem.) in 0.05 M Tris-HCl at pH 7.6 with 0.003%
hydrogen peroxide.
The testes were counterstained in Gill's hematoxylin #3 solution, and rinsed
in tap and distilled
water. Neither PBS, VEGF, AS-Flt-1 nor AS-SCR, affected the level of Flk-1
protein expression
in mouse testis, panels A, B, D and E, respectively. The specificity of the
Flk-1 antisense
20 oligomer depicted in panel C is evident by the lack of positive staining
following incubation with
Flk-1 antibody.
Figure 14 depicts the results from an experiment designed to study the
efficiency and selectivity
of the Flt-1 antisense oligomer on Flt-1 protein expression in mouse testis.
Immunohistochemical
25 analyses on mouse treated testis were performed as detailed above. Briefly,
treated mouse testis
were fixed in 10% formalin PBS-buffered solution. The testis were embedded in
paraffin and
sections of 6 hum were obtained by microtome along the length of the specimen.
The testes were
deparaffinized in xylene and ethanol baths, endogenous peroxidase activity was
quenched in a
solution of methanol (200 ml) plus hydrogen peroxide (3%, 50 ml), and
nonspecific binding
30 antibody binding prevented by preincubating the tissues with serum (1:10)
from species other
than those used to raise the primary antibody. Arterial sections were then
exposed to the primary
antibody, rabbit polyclonal anti-human Flt-1 IgG (which reco;nises mouse Flt-1
protein; and
without cross-reactivity with Flk-1 protein) (Santa Cruz Biotech) diluted
(1:250; 2 hrs), or rinsed
with PBS, and incubated with a biotinylated goat anti-mouse IgG (1:400; 1 hr)
(Vector Labs
44
CA 02321189 2000-10-13
Inc.). Peroxidase labelling was achieved with an incubation using
avidin/peroxidase complex
(Vector Labs Inc.), and antibody visualisation established after a 5 min
exposure to 0.05% 3,3'-
diaminobenzidine (Sigma Chem.) in 0.05 M Tris-HCl at pH 7.6 with 0.003%
hydrogen peroxide.
The testes were counterstained in Gill's hematoxylin #3 solution, and rinsed
in tap and distilled
water. Neither PBS, VEGF, AS-Flk-1 nor AS-SCR, affected the level of Flt-1
protein expression
l0 in mouse testis, see Figure 14, panels A, B, C and E, respectively. The
specificity of the Flt-1
antisense oligomer depicted in Figure 15, panel D is evident by the lack of
positive staining
following incubation with Flt-1 antibody.
Figure 15 depicts the results from experiments designed to demonstrate the
effect of Flk-1 and
Flt-1 antisense oligomers on endothelial cell nitric oxide synthase (ecNOS)
protein expression in
rat testis. Immunohistochemical analyses on mouse treated testis as detailed
in Figure 12 were
performed as described previously. Briefly, treated mouse testis were fixed in
10% formalin
PBS-buffered solution. The testis were embedded in paraffin and sections of 6
~m were obtained
by microtome along the length of the specimen. The testes were deparaffinized
in xylene and
ethanol baths, endogenous peroxidase activity was quenched in a solution of
methanol (200 ml)
plus hydrogen peroxide (3%, 50 ml), and nonspecific binding antibody binding
prevented by
preincubating the tissues with serum (1:10) from species other than those used
to raise the
primary antibody. Arterial sections were then exposed to the primary antibody,
mouse
monoclonal anti-human ecNOS IgG (which recognises mouse ecNOS protein)
(Transduction
Laboratories) diluted (1:250; 1 hr), or rinsed with PBS, and incubated with a
biotinylated goat
anti-mouse IgG (1:400; 1 hr) (Vector Labs Inc.). Peroxidase labelling was
achieved with an
incubation using avidin/peroxidase complex (Vector Labs Inc.), and antibody
visualisation
established after a 5 min exposure to 0.05% 3,3'-diaminobenzidine (Sigma
Chem.) in 0.05 M
Tris-HCl at pH 7.6 with 0.003% hydrogen peroxide. The testes were
counterstained in Gill's
hematoxylin #3 solution, and rinsed in tap and distilled water. Neither PBS,
VEGF, AS-Flk-1,
AS-Flt-1 nor AS-SCR, affected the level of ecNOS protein expression in mouse
testis, see Figure
15, panels A - E, respectively.
To exclude the possibility that VEGF recruited pre-existing capillary vessels
through
CA 02321189 2000-10-13
vasodilation, the effect of transient vascular occlusion of the testicular
supply in anaesthetised
mice was examined. Following a two minute complete vascular occlusion,
vascular density did
not significantly increase upon release of the vascular clip. Adequacy of
occlusion and
reperfusion were demonstrated by red blood cell stasis during occlusion and
sustained red blood
cell transit upon reperfusion. It was further established that at 14 days the
angiogenic process is
near completion because of non-significant increase in PCNA staining in VEGF-
treated
compared to PBS-treated testes, which is agreement with previous report in a
different VEGF-
mediated angiogenic model (Palacios I, et al (1999) J. Rheumatol. 26: 1080-
1086).
Antisense sequences
The antisense oligonucleotides were selected and designed in function of
specific characteristics
such as no more than three consecutive guanosines, the incapacity to form
hairpins and a
minimal capacity to dimerize together, and the length of the antisense
oligonucleotides is
generally between 15 to 25 bases. The murine Flt-1 and Flk-1 cDNA were
obtained from
GENBANK (GenBank Accession Numbers D28498 and X70842) respectively. A total of
four
different antisense oligonucleotide phosphorothioate backbone sequences were
selected, two
targeting mice Flt-1 mRNA (ASl-mFlt: 5'-AAG CAG ACA CCC GAG CAG-3' (SEQ ID
N0:5); AS2-mFlt: 5'-CCC TGA GCC ATA TCC TGT-3' (Sf~:(~ ID N0:6)), and two
targeting
mice Flk-1 mRNA (AS1-mFlk: 5'-AGA ACC ACA GAG CCiA CAG-3' (SEQ ID N0:7); AS2-
mFlk: 5'-AGT ATG TCT TTC TGT GTG-3' (SEQ ID N0:8).
Two scrambled phosphorothioate sequences (scrambled Flt, SCR2-Flt: 5'-ACT GTC
CAC TCG
CAG TTC-3' (SEQ ID N0:21 ); scrambled Flk, SCR2-Flk: 5'-TTT CTG GTA TGC ATT
GTG-3'
(SEQ ID N0:22)) were also selected as negative controls. All sequences were
synthesised at the
Armand Frappier Institute (Laval, Canada). After synthesis, the
oligonucleotides were dried,
resuspended in sterile PBS, filtered (0.2 trm pore size) and quantified by
spectrophotometry. The
assurance that the antisense oligomer solutions were by-products-free will be
confirmed by
denaturing polyacrylamide gel electrophoresis (20%; 7M urea), based on the
known length of the
oligonucleotide.
46
CA 02321189 2000-10-13
Statistical analysis
All the statistical analysis are expressed as mean ~ SE. Statistical
comparisons were made by
analysis of variance followed by an unpaired (or paired; when applicable)
Student's t-test. Data
were considered significantly different if values of P<0.05 were observed.
1o
The invention being thus described, it will be obvious that the same may be
varied in many ways.
Such variations are not to be regarded as a departure from the spirit and
scope of the invention,
and all such modifications as would be obvious to one skilled in the art are
intended to be
included within the scope of the following claims.
47
CA 02321189 2000-10-13
SEQUENCE LISTING
<110> Sirois, Martin G.
Institut de Cardiologie de Montreal
<120> Antisense Oligonucleotides Directed Toward Mammalian
VEGF Receptor Genes and Uses Thereof
<130> 712-103
<140>
<141>
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caaagatgga
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gctgctctga
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48
CA 02321189 2000-10-13
<220>
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agtatgtctt tctgtgtg 18
49
CA 02321189 2000-10-13
<210> 9
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tgtttccttc ttctttga 18
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tactcaccat ttcaggca lg
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50
CA 02321189 2000-10-13
<400> 13
actcaccatt tcaggcaa 18
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agtatgtctt tttgtatg 18
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tgaagagttg tattagcc 18
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actgccactc tgattatt 18
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tttgctcact gccactct 18
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51
CA 02321189 2000-10-13
<220>
<223> Description of Artificial Sequence:antisense
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gtctttttgt atgctgag 18
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tgctggcatg tgcgttgt 18
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actgtccact cgcagttc 18
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<223> Description of Artificial Sequence:antisense
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tttctggtat gcattgtg 18
52
