Note: Descriptions are shown in the official language in which they were submitted.
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CONJUGATION OF C-MYC ANTISENSE OLIGONUCLEOTIDES WITH
CHOLESTEROL TO SIGNIFICANTLY ENHANCE THEIR INHIBITORY
EFFECT ON NEOINTIMAL HYPERPLASIA
5 RE~ATED APPLICATIONS
This application is based upon provisional applications S.N.
60/005,544 filed October 19, 1995 and S.N. 60/012,037 filed February
21, 1996, both of which concern the conjugation of c-myc antisense
10 oligonucleotides with cholesterol.
BACKGROUND OF THE INVENTION
The ability of antisense oligomers to interact specifically with
15 mRNAs of their corresponding sequences provides a valuable tool for the
control of cellular expression. Antisense oligodeoxynucleotides (ODNs)
have recently been successfully used as "informational drugs" to prevent
smooth muscle cell proliferation in vitro and neointimal hyperplasia after
balloon angioplasty. During development of this strategy, other
20 investigators delivered oligonucleotides through the use of a pluronic gel
surgically applied on the adventitial layer of the artery to permit sustained
and prolonged DNA delivery to the angioplastied vessel. This approach is,
however, inapplicable in human clinical angioplasty procedures. The
importance of continued local presence of the therapeutic molecule at a
25 sufficient concentration at the site of angioplasty must not be
underestimated, since the proliferation window of vascular smooth muscle
cell (SMC) has been shown to last up to 14 days following angioplasty.
Moreover, the inappropriate delivery profile of numerous other molecules
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tried in clinical studies aimed at preventing restenosis may explain in part
the failure seen so far in all these pharmacological trials.
In order to optimize their level of cellular incorporation,
5 oligonucleotides have been covalently linked to a variety of hydrophobic
groups, such as cholesterol. This lipidic conjugation of oligomers have
proven efficient for in vitro inhibition of HIV multiplication. In vitro studieshave also documented enhanced cellular uptake in neutrophils and HeLa
cells. Furthermore, two to three fold improved oligomers nuclease
lo resistance was associated with cholesterol conjugation in hepatoma cells.
Little information, however, is available on the fate of cholesterol-coupled
oligonucleotides in vivo. Association of 5'-cholesterol-bearing
oligonucleotides with low and high density lipoproteins demonstrated a
prolonged plasma half-like in rats, increasing from less than 1 minute to 9-
11 minutes.
SlJMMARY OF THE INVENTION
In order to apply and optimize the efficacy of ODN prevention of
neointimal hyperplasia following balloon angioplasty in a human clinicalsetting, we demonstrate that coupling all oligonucleotide to cholesterol
significantly enhances the ODN efficacy to prevent neointimal hyperplasia
following balloon angioplasty. This enhanced inhibitory effect of
cholesterol conjugation is demonstrated with c-myc mRNA targeting but
may also be applied to any other DNA oligonucleotide targeting mRNA
switched in smooth muscle cell proliferation such as c-myb, PCNA and
others. This invention relies on the demonstration that cholesterol-
conjugation of the oligonucleotide significantly increases the ODN retention
in vascular tissue following in vivo transfection. Accordingly, the purpose
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of this invention evaluates, first, in vitro, the effecl: of conjugation of c-myc
antisense phosphorothioate oligomers with cholesterol on ODNs
pharmacokinetics properties and effect on smooth muscle cell proliferation.
Then, in vivo, following direct arterial transfection, evaluate the long term
5 outcome of transfected cholesterol-antisense oligomers and their effect on
neointimal hyperplasia formation.
DESCRIPTION OF THE DRAWINGS
Figure 1: Sense, antisense and scrambled oligonucleotides
conjugated with cholesterol moieties.
Figure 2: In vitro inhibition percentage of smooth muscle cells
proliferation following incubation with 5 ~M or 10 ,L~M of sense or antisense
15 oligonucleotides either alone or conjugated with cholesterol (n =4 for each
group) .
Figure 3: Sucrose gradient distribution of fractions derived from in
vitro transfected SMCs with 10 ~M of either cholesterol-conjugated or
20 unconjugated antisense oligonucleotides. Each experiment was repeated
in triplicates producing similar results. Nucleotidase activity of each
fraction is presented as a ratio of the highest value obtained within the
sucrose gradient. For the comparison between 35S-labeled cholesterol-
conjugated and unconjugated ODNs association with gradient fractions,
25 35S radioactivity quantified was standardized for a same amount of
evaluated 5'-nucleotidase activity.
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Figure 4: Intra-arterial distribution of 35S-labeled unconjugated and
conjugated ODNs (80 ~M) transfected into isolated rabbit carotid arteries
(1 cm, 20 mg of tissue) for 30 minutes. n=3 for each group.
Figure 5: Photomicrograph of histological section derived from rabbit
carotid artery treated with 80,uM of fluoresceine-conjugated cholesterol
antisense ODNs for 30 minutes in an isolated arterial segment (1 cm, 20
mg of tissue). "IEL" indicates internal elastic lamina and "M" indicates
medial layer. Original magnification x 1000.
Figure 6: 35S-oDNs retention into normal carotid arteries was
evaluated at 3, 6, 12, 24, 48, 72 and 168 hours following transfection
with c-myc antisense ODNs (80 ~M, 30 min), either alone or conjugated
with cholesterol (n = 3 for each group) . The retained 35-S labeled
oligomers were evaluated by radioactivity quantification. Post-transfection
at O hours, corresponding to 30 minutes of arterial exposure to
oligonucleotides, was considered as "100% incorporation".
Figure 7: 35S-labeled oligonucleotides extraction from normal
arterial segments exposed to oligomers (80 ~M, 30 minutes). Each
experiment was repeated in triplicata, producing similar results.
Figure 8-1 1: Fig. 8-unconjugated sense ODNs, Fig. 9 = unconjugated
antisense ODNs, Fig. 1 0 = cholesterol-conjugated sense ODNs, Fig. 11 -
cholesterol-conjugated antisense ODNs. Photomicrographs of histological
sections derived from rabbit doubled injured carotid arteries treated with
80 IIM of oligonucleotides. "IEL" indicates internal elastic lamina and "N"
indicates neointimal hyperplasia. Original magnification x 1000.
(Hematoxylin and Eosin stain).
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Figure 12: Ratio of neointima/media areas of double injured arterial
segments treated with control (100 ~l of NaCI 0.9%), 80 ,L/M of sense
ODNs, antisense ODNs, cholesterol-conjugated sense ODNs, cholesterol-
conjugated antisense ODNs or cholesterol-conjugated scrambled ODNs.
5 Intimal/medial areas were evaluated by computer analysis on histological
section derived from transfected arteries two weeks following the second
injury and compound transfection (n = 6 for each group~.
lO DETAILED DESCRIPTION OF THE INVENTION
Material and Methods
Cholesterol conjugation of oligonucleotides
Phosphorothioate antisense ODNs, 1 5 mer in length, complementary
to c-myc (5'-CAC GTT GAG GGG CAT-3'), the corresponding sense
sequence (5'-ATG CCC CAC AAC GTG-3') and scrambled sequence (5'-
CAC TGT TAG GGG AAG-3') were synthesized on a 392 DNA/RNA
20 synthesizer following standard procedure (Applied Biosystems) . This target
was selected to inhibit smooth muscle cell proliferation in vitro.
Conjugation of oligomers with cholesterol was achieved with 3'-
cholesterol-VN CPG (Clontech), a virtual nucleotide (VN) glass reagent that
introduces a cholesterol label to the 3' terminus of an oligonucleotide via
25 solid-phase synthesis. When incorporated into an oligonucleotide, 3'-
cholesterol-VN CPG introduces a deoxyribose sugar moiety that mimics a
~ natural nucleotide base unit. As a result, the identical sugar-phosphate
DNA backbone is maintained: stereochemical definition, enantiomeric
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purity, conformational rigidity, and internucleotide phosphate distance are
all conserved (Figure 1 ) .
When ODN synthesis was completed, radiolabeled oligomers were
5 removed from the column with 30% NH40H (1 hour at room temperature),
and then deprotected for 8 hours at 60~C. Oligomers were purified and
detritylated with oligonucleotide purification cartridges (Applied
Biosystems), and then Iyophilized with a centrifugal evaporator (Savant
SpeedVac). Prior to transfection, oligomer concentration was assessed by
spectrophotometry at 260 nm. Following ethidium bromide staining (0.3
/~9l~l), the comparison of oligomer migration with standard DNA, under UV
light, showed only 15 mer ODNs in the final preparation to be transfected.
35S labellinq of oligomers
In order to permit detection and quantification of transfected
oligonucleotides, these oligomers were internally radiolabeled by replacing
the standard sulfurizing step of base 3, 6, and 9 with a radiolabelling 35S
solution (200 ,ul of TEDT/Acetonitrile + 5,L~1 of 35S [0.373 mCi]).
ODN purification was performed as described above, with
oligonucleotide purification cartridges. 35S radioactivity quantification was
evaluated with a Beckman LS 8100 scintillation counter, and 35S-oDN
integrity was verified, as described above, by electrophoresis on a 20%
25 urea-polyacrylamid gel. Furthermore, radioactivity emission of 35S-oDN
was trapped on a film (Kodak) for several days (in darkness at -80~C), and
oli,aomer location was indicated by a dark band. Only 15 mer oligomers
were detected on these radiographic films.
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Assessment of smooth muscle cell Droliferation index in vitro
Primary cultures of rabbit vascular smooth muscle cells were
obtained from explants of New-Zealand rabbit thoracic aorta which were
surgically removed, cleaned and kept at 37~C under 5% C02 in 10 ml of
D-MEM medium supplemented with 10% fetal bovine serum ~FBS), 2 mM
glutamine, penicillin (100 U/ml) and streptomycine (100 U/ml). Cells from
passage 3 to 5 were used in this study.
lo Inhibitory effect of oligonucleotides on smooth muscle cell
proliferation was assessed by thymidine incorporation index of untreated
cells and also cells treated with 5 ,uM or 10 ~M c-myc antisense
(cholesterol-conjugated or not), and c-myc sense (cholesterol-conjugated
or not). Cells were plated at a density of 5000 cells/cm2, and allowed to
recover 24 hours in D-MEM (Gibco-BRL) supplemented with 10% FBS.
Cells were synchronized to a same state of proliferation by supplementing
the medium with 0.5% FBS for 48 h. Increase of serum content to 10%
FBS activated cell proliferation and oligonucleotides were added to the
medium. After 12 hours of incubation, 3H thymidine was added (5 uCi/ml)
and 12 hours later the medium was removed. Cells were washed with
cold phosphate buffered saline, fixed for 10 minutes in ethanol:acetic acid
(3:1), washed with distilled water and incubated for 15 minutes in ice-cold
0.5N perchloric acid. Following a rapid wash in 0.5N perchloric acid, cells
were incubated for 20 minutes at 80~C in 0.5N perchloric acid, and
evaluated in a scintillation counter for 3H radioactivity emission. All
experiments were performed at least in triplicates.
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Sucrose aradient of transfected SMCs
To assess potential intracellular distribution sites of cholesterol-
conjugated ODNs we evaluated the association of 35S-labeled oligomers
with cellular membranes. Smooth muscle cells were synchronized to a
quiescent state by supplementing the D-MEM medium with 0.5% FBS for
48 h. Cells were stimulated to proliferate by changing the medium to 10%
FBS D-MEM and unconjugated or cholesterol-conjugated antisense
oligonucleotides (10 ~M) were then added to the medium. After 24 hours
of incubation, cells were removed from culture plates with 4 ml of trypsine
and neutralized with 10 ml of cold PBS. Cells were then centrifugated at
1900 rpm for 7 minutes (4~C), and the resulting pellet was resuspended
in a solution of 200 mM sucrose:5 mM NaP04 (pH=7.5). Smooth muscle
cells plasmic membranes were permanently destabilized by applying a high
power ultrasound (2x15 seconds) resulting in intracellular components
release. By centrifugating this solution at 800 9 x 5 minutes, the few cells
maintaining their integrity were pulled apart in the pellet. The supernatant
containing the intracellular components and plasmic membranes from
destabilized cells was then deposed on a 20-54% sucrose gradient
prepared with 5mM of NaP04 (pH = 7.5). An ultracentrifugation at 38000
rpm for 16 hours (3~C) allowed cell particles to sedimentate through the
gradient in separate zones characterized by their sedimentation rate.
Gradient fractions of 250 ~I were collected, each fraction being examined
for associated 35S labeled antisense oligonucleotides and 5'-Nucleotidase
activity indicating the presence of membranes. 35S labeled ODNs (1OO~
of sample) were quantified on a Beckman LS 8100 scintillation counter.
For 5'-nucleotidase activity quantification, fraction samples of 100
~I were mixed with 650 ~l of reactional solution (50 mM of glycine, pH = 9;
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g
0.4 mM of MgCI2; 0.16 mM of 5'AMP) and incubated for 30 minutes at
- 37~C. The reaction was stopped with successive addition of 39.5 ~l of
ZnS04 750 mM and 474 ,LII of Ba(OH)2 100 mM (final concentration of
37.5 mM) at 4~C. Fotlowing a centrifugation at 14,000 rpm for 5
5 minutes, 5' nucleotidase activity was quantified on a spectrophotometer
at 260 nm.
In vivo arterial ODN transfection: oharmacokinetic studies
New Zealand rabbits male or female (2 Kg) were intramusculary
sedated with xylazine (2 mg/Kg) and anesthetized with ketamine (100
mg/Kg) prior to surgical exposure of left carotid artery. Additional doses
of anesthesia were given intramusculary throughout the experiment, as
needed. Segments (10 mm, corresponding approximately to 20 mg of
15 tissue) of carotids were transiently isolated by temporary ligatures and
rinsed with 0.9% sodium chloride via a cannula until there was no more
visible evidence of blood components. Carotid arteries (n = 57) were
transfected with 80 ,uM of c-myc 35S labeled antisense ODNs in a 1 cm
portion either alone (n=27) or conjugated to cholesterol (n=30) for a
period of 30 minutes. The volume infused was 100 ~I, and no visible loss
of volume was noted throughout the incubation period. Following
transfection, the treated segments were rinsed with 0.9% sodium chloride
(3 x 100 111) and upon cannula removal, the arteriotomy site was repaired
with microsutures (100 microns nylon monofilament, Pike Surgical Inc.
Calgary, Canada), restoring normal blood flow. The neck wound was then
closed and no adverse neurological or vascular effects were observed in
- any animal undergoing this procedure. Following surgery, the rabbits were
allowed to recuperate, housed in approved facilities with free access to
rabbit chow and water, and exposed to a 1 2-hour light dark cycle.
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To assess arterial retention of transfected oligomers, rabbits were
sacrificed at 3, 6, 12, 24, 48, 72 and 168 hours post-transfection. Both
carotid segments, from the same animal, were removed as the right
common carotid artery was considered as an internal control for 35S
5 contamination. Incorporation and retention levels of 35-S ODNs were
evaluated by scintillation counting following exposure of treated segments
to liquid nitrogen, thus releasing the incorporated oligomers.
Phvsical integritY of oligos
Oligonucleotide physical integrity was assessed following oligomers
extraction from the arterial segments at 72 hours and 168 hours post-
transfection. The transfected segments were removed surgically and
crushed into fine powder following exposure to liquid nitrogen. 35S-
15 labeled ODN were then extracted from the vascular cells and wereseparated from cellular DNA by further extraction with an equal volume of
phenol (pH = 6.8). Oligos recovered in the aqueous phase were then
reextracted with a 50:50 mixture of phenol and equilibrated
chloroform:isoamyl t24: 1), followed by an equal volume of
20 chloroform:isoamyl. The recovered 35S-labeled ODN were desalted with
oligonucleotide purification cartridges (Applied Biosystems), the cartridges
being rinsed with 5 ml of acetonitrile (99%) of 5 ml of TEAA 2M prior to
oligo application. Oligomers retained by the cartridges were rinsed with 10
ml of H20 and were eluted with 20% of acetonitrile, llyophilized and then
25 solubilized in deionized water. Recovery of 35S-labeled oligos applied on
the cartridge varied between 80% and 90%. Oligomers physical integrity
was verified by electrophoresis on a 20% urea-polyacrylamid gel, oligos
migraton being compared to standard DNA following ethidium bromide
coloration. Furthermore, visualization by autoradiography indicated that
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the totality of 35S radioactivity recovered from the transfected arteries
~ was coupled to the 15 mer oligos.
Arterial distribution of ODNs
Oligos biodistribution in the vessel wall was evaluated by dot
counting on histological sections derived from transfected arteries.
Representative sections of carotid arteries were fixed in 4~/0 paraformalin
and embedded in paraffin, and 5-~m sections were cut. 35S radioactivity
10 emission from the vascular cells was trapped by photographic emulsion (in
darkness at 4~C, Kodak autoradiography emulsion). Following
development (2.5 minutes in Kodak developer) and fixation (5 minutes in
Kodak fixer), 35S-labeled oligomers were visualized as black dots.
Histological sections were stained with hematoxylin/eosin (for cells
5 identification) and examined under light microscopy for oligomers
transfection into the arterial wall. The average dots counted in the
different layers of the carotid artery were extrapolated for the segment
treated. Oligomers distribution percentage in a specific layer was then
compared to the entire segment. A high correlation factor (r2=0.96) was
20 found between dot and scintillation counting.
Thereafter, in vivo intracellular distribution of oligomers was
visualized by conjugation with a fluoreseine-isothyocyanate molecule
(FITC), introduced at the 5' end of c-myc cholesterol-antisense ODNs via
25 standard automated synthesis ~Applied Biosystems). Oligomer purification
and detritylation was performed, as described above, with ODN purification
cartridges. Oligomer integrity was verified by electrophoresis on a 20%
urea-polyacrylamid gel, and oligomer migration was compared to standard
~ DNA under UV light. The totality of fluoresceine molecules detected were
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coupled to 15 mer oligomers. Fluorescine-conjugated antisense ODNs (80
~M) were locally transfected into rabbit carotid arteries for 30 minutes and
histological sections were derived from these carotid arteries.
Representative sections were fixed in 4% paraformalin, embedded in
5 paraffin.
Neointimal hvoer~lasia inhibition
A total of 36 New Zealand white rabbit carotid arteries were inured
with a 2.5 mm balloon catheter serially inflated for 1 minute to 4, 6, 8 and
10 atm with gentle traction allowing 45 seconds between inflations. Two
weeks later, a second injury was imposed at the same arterial site which
was then transfected in a 1 cm portion with 80 IlM (100 JJL of volume
injected) of either c-myc antisense alone or conjugated with cholesterol,
15 with c-myc sense alone or conjugated with cholesterol, with cholesterol-
conjugated scrambled, or with 100 ~L of NaCI 0.9% as control.
Intimal/medial areas were evaluated by computer analysis on histological
sections derived from transfected arteries two weeks following the second
injury and transfection procedure.
~itatistical analysis
All values are expressed as mean + SEM. Analysis of Student's t
test was used to determine significant differences between groups. A
25 value of P<0.05 was considered significant.
-
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In vitro studies
In order to determine the potential usefulness of conjugating c-myc
antisense oligomers with cholesterol in the prevention of neointimal
5 hyperplasia formation, we first studied the effect of each type of oligomer,
sense and antisense, conjugated or not with cholesterol, on primary
cultures of rabbit vascular smooth muscle cells. Consistent with previous
studies, unconjugated antisense oligomers manifested a dose-response
effect on SMCs growth inhibition, reaching a miximal inhibition potential
of 72.8 + 2.9 % at 10 ~M (P=0.02 vs 58.0 + 4.4 % at 5 ~M) (Fig. 2).
Interestingly, at a dose of 10 ~M conjugated-antisense oligonucleotides
manifested significant advantage over unconjugated oligomers as their
antiproliferative potential increased to 91.3+2.1% of control (P=0.005
vs. unconjugated oligomers). Although cholesterol-conjugated sense
15 sequences presented a discrete inhibitory effect at both 5~M and 10 ~M
concentrations, this effect did not reach 20 % inhibition (P=0.0001 vs.
corresponding antisense). Importantly, all treated cells preserved their
normal morphology in presence of conjugated as well as unconjugated
oligomers. Also, cholesterol alone, added to our cells cultures, did not
20 affect SMC appearance or proliferation rate (data not shown).
In addition, to further characterize the enhanced antiproliferative
potential of cholesterol-conjugated antisense ODNs, we sought to examine
intracellular distribution sites of transfected radiolabelled oligomers.
25 Following SMCs particle sedimentation on sucrose gradient we observed
a well defined peak for both gradients performed on conjugated and
- unconjugated-ODN transfected cells (Fig. 3). Radioactive intensity of this
peak was evaluated at 2484 cpm for cholesterol-conjugated ODNs and at
863 cpm for unconjugated oligomers. This represented a 2.9-times
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increase in cholesterol-conjugated ODN uptake and retention level when
compared to unconjugated ODNs (P=0.001). The significant increase
observed supports the concept that cholesterol conjugation of oligomers
may favorably influence the pharmacokinetic properties of these
therapeutic molecules. We sought to further examine the nature of cellular
fractions corresponding with oligomers presence, as we evaluated
membranal fractions by dosage of 5'-nucleotidase activity. Measurement
of this enzyme activity revealed high levels of 5'-nucleotidase in fractions
where oligomers were the most concentrated (Fig. 3), suggesting that
oligomers preferentially locate in membrane components of SMCs.
In vivo studies
In order to efficiently prevent in vivo neointimal hyperplasia
formation following balloon angioplasty, the active molecule must be
efficaciously delivered to the vessel wall and preferably impose its presence
to the injured area as long as possible during the proliferation window that
occurs following the trauma. Accordingly, we examined in this series of
experiments the level of ODNs successfully transfected in the target vessel
wall, its tissue distribution, and vascular retention.
At O hours post-transfection, cholesterol-conjugated antisense ODNs
were more efficiently transfected into injured carotid arteries when
compared to unconjugated ODNs and reached, respectively, 40.3 + 0.4 %
and 28.2 + 0.4 % (P = 0.03) of available oligomer exposed to the vessel
wall, while incorporation in normal arteries was of similar extent for both
antisense formulations (29.2 + 1.3 % for unconjugated antisense,
30.1 + 1.7% for cholesterol-conjugated antisense, P =NS). With respect
to oligomer distribution ~Fig. 4), we observed that both conjugated and
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unconjugated oligomers were mainly located in the medial layer of
- transfected arterial segments (unconjugated O D N:83.0 + 0.9 % of total
ODN incorporated, cholesterol-conjugated ODN: 85.1 +0.4% of total ODN
incorporated, P - NS). In order to further characterize oligomer distribution
and to assess their cellular incorporation, we conjugated short DNA
sequences with fluoresceine-cholesterol molecules. As seen on Figure 5,
transfected cholesterol oligomers were mainly located in the medial layer
with preferential nuclear accumulation. Also, a high level of transfected
cells were present in this layer, being suggested by the finding that over
lo 85% of cells showed fluoresceine activity.
With respect to vascular retention (Fig. 6), we observed that
cholesterol conjugation of DNA conferred a significant advantage over
unconjugated DNA, in normal as in injured arteries, increasing retention
levels at 7 days by 2.7-fold, with halftime retention enhanced from 2.2
hours for unconjugated ODNs to 5.9 for cholesterol-conjugated ODNs. At
one week, 24.2 + 1.3% of the transfected conjugated ODN was still
present in the vessel wall, compared to 6.2+1.1% (P=0.001) of the
unconjugated ODN. Furthermore, DNA extraction from transfected carotid
arteries revealed that oligomers remained intact, respectively for
unconjugated and cholesterol-conjugated ODNs, for at least 72 hours and
168 hours post-transfection (Fig. 7).
Neointimal hv~erDlasia inhibition
In order to assess the potential advantage of DNA conjugation with
cholesterol on in vivo neointimal hyperplasia inhibition, we examined the
efficiency of the five different oligomer formulations to prevent neointimal
hyperplasia (Fig. 8-11). Following arterial double bailoon injury, maximal
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ratio of intimal/medial area was observed in the control group
(0.79+0.04), unconjugated sense ODNs group (0.72+0.05, P=NS vs.
control) (Fig. 8) and cholesterol-conjugated sense ODNs group
(0.72 + 0.04, P--NS vs. control) (Fig. 10) . Although unconjugated
5 antisense ODNs and cholesterol-conjugated scrambled ODNs reduced
intimallmedial ratio area respectively to 0.47+0.02 (P=0.001 vs.
unconjugated sense ODNs) (Fig. 9) and 0.54+0.02 (P=0.002 vs.
cholesterol-conjugated sense ODNs), cholesterol-conjugated antisense
ODNs were far more effective, the intimal/medial area ratio being estimated
at 0.1 1+0.01 (P=0.0001 vs. cholesterol-conjugated sense ODNs,
cholesterol-conjugated scrambled ODN and control) (Fig. 11). Thus,
arterial neointimal hyperplasia was specifically reduced by 34.7% following
transfection of unconjugated antisense c-myc ODNs, and by 84.7%
following transfection of cholesterol-conjugated antisense ODNs (Fig. 12).
15 Not only was the antiproliferative potential of antisense ODNs increased
2.4 times following oligomers conjugation with cholesterol, but this effect
was homogeneously found over the entire neointimal areas.
Antisense oligonucleotide transfection has recently emerged as a
2 o potential therapeutic pathway to prevent neointimal proliferation
component of restenosis. Several studies have demonstrated that
antisense directed against c-myc mRNA not only inhibits smooth muscle
cell proliferation but also smooth muscle cell migration, which constitutes
another critical component of smooth muscle cell response to vascular
25 injury and, presumably, therefore of the restenosis process. However, the
development of antisense oligonucleotide therapy has not been as simple
as first believed, and many critical issues have been highlighted. These
include concerns about cellular uptake, sequence-specific and non-
sequence-specific biological effects, pharmacokinetics and
,
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pharmacodynamics. Key elements of antisense strategy's success may
- rely on high transfection efficiency but also, importantiy, on sufficient
retention of this therapeutic molecule during the proliferation window of
SMCs following angioplasty, which occurs mostly during the first week.
This is the first invention to report the perceived advantage of
conju~ating cholesterol to a therapeutic molecuie in arterial application.
This approach permitted, potentially through advantageous
pharmacokinetic properties of the conjugate, to significantly improve the
0 efficiency to inhibit in vitro smooth muscle cells proliferation and in vivo
neointimal hyperplasia formation.
In vitro, conjugation of antisense oligonucleotides with cholesterol
moieties was extremely beneficial for oligomers ef~ect, since their inhibitory
potential reached 91.3% at a dose of 10 ,uM. This enhanced
antiproliferative effect is correlated with increased intracellular levels of
these therapeutic molecules, our study demonstrating that cholesterol-
conjugated c-myc antisense ODNs are associated at a level with SMCs
membrane components at a level 2.9 times higher than that seen with
20 unconjugated oligomers.
Although the precise mechanisms responsible for inhibition of
protein expression in eukariotic cells are not precisely known, intracellular
trafficking and distribution of oligonucleotides determine the extent to
25 which an "internalized" oligonucleotide is available to interact directly with
its biological targets.
.
Once internalized, 35S-labeled phosphorothioates oligonucleotides
were shown to accumulate in vesicular structures and in the nucleus but
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18
significantly less in the cytoplasm. Consistent with this invention,
conjugated as well as unconjugated oligonucleotides entering in vivo SMCs
migrate rapidly into the nucleus. It appears that cholesterol-conjugated
ODNs are anchored to cellular membranes via cholesterol moieties,
5 rendering oligomers available in the cytoplasmic and/or the nucleoplasm
compartment. This is also consistent with our in vivo findings where
cholesterol-conjugated oligomers incorporation potential was superior to
unconjugated oligomers in injured arteries, suggesting a different
intracellular compartmentalization and/or hybridization to corresponding
10 mRNA, the latest being generally available 30 minutes following cell
stimulation. Moreover, cholesteryl group attachment at the 3'-terminus of
DNA is known to confer an enhance resistance to cellular nucleases and
may increase the stability of mRNA/antisense oligomer complexe, thus
enhancing antisense ODNs availability within the cell which in vivo could
translate as increased vascular retention and enhanced antiproliferative
effect on neointimal hyperplasia.
Interestingly, we notice that in vivo cholesterol-conjugated antisense
oligomers were retained in vascular cells at a level 2.7 times superior in
comparison to unconjugated oligomers Moreover, we observed a
sustained retention of the conjugate's physical integrity, even at 1 week,
supporting the hypothesis that the conjugate may still be active, with
possibly a higher resistance to nucleases. Thus, an increase in ODN's in
vivo stability may provide longer duration of action of these molecules,
leading to a decrease in the frequency of administration, ensuring a higher
vessel to appropriately targeted antisense oligonucleotides may
permanently inhibit the restenosis process.
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-- 19
Single transcatheter and adventitial administration of unconjugated
- ODNs in vivo have been reported to reduce restenosis by others. Thisinvention suggests, however, that conjugation of ODNs with cholesterol
will lead to a significantly superior inhibition of neointimal formation
(84.7% with conjugated ODNs vs 36.7% with unconjugated ODNs).
Although the presence of the four contiguous guanines has been shown to
manifest unusual non-sequence-specific effects, here, the 2.~fold
enhancement of hyperplasia inhibition by cholesterol-conjugated antisenses
is highly specific and is not dependant upon the presence of 4-G sequence,
as shown by the low inhibition percentage (24.2%) of cholesterol-
conjugated scrambled 4-G sequence. Also, it seems unlikely that
differences in inhibitory potential of antisense and sense sequences may
be due to variations in cellular uptake efficacy, since it has been
established by other studies that pharmacokinetic and pharmacodynamic
properties of phosphorothioate-oligonucleotides are largely independent of
base composition.
For many in vivo applications, oligonucieotides must be administered
repeatedly in order to obtain the desired biological effects, partly because
oligonucleotides are rapidly eliminated, and also because the target gene
products have long half-lives. It appears reasonable to infer that if
antisense therapy is to be effective in inhibiting the SMCs proliferation
process, the antisense molecules should be actively available in the injured
region for at least the duration of the proliferative window period. Thus,
by improving oligomer phamacokinetics following 3'-end conjugation with
cholesterol, the need for repeated oligomer delivery may be eliminated.
~ Local intravascular delivery of cholesterol-conjugated antisense
oligonucleotides into double injured rabbit carotid arteries appears to have
an increased potential therapeutic effect over non conjugated-
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oligonucleotides. Although protection of oligonucleotides from nucleases
digestion, and maintaining intracellular high levels of antisense
oligonucleotides, represent potential explanation for this effect, it is not
excluded that cholesterol-conjugation may enhance binding affinities of
ODNs.
Although protection of oligonucleotides from nucleases digestion,
and maintaining intracellular high levels of antisense oligonucleotides,
represent potential explanation for this effect, it is not excluded that
10 cholesterol-conjugation may enhance binding affinities of ODNs.
Our invention demonstrates, that conjugation of c-myc antisense
oligodeoxynucleotides with cholesterol moieties increases in vitro ODN
uptake/retention levels in vascular smooth muscle cells, alters intracellular
15 ODN distribution, and consequently increases ODN efficiency to inhibit
SMC growth. In vivo, following transfection to the vessel wall, this
conjugate is retained at levels significantly higher at one week when
compared to unconjugated ODNs, with a preserved physical integrity.
Importantly, conjugation of c-myc antisense oligodeoxynucleotides with
cholesterol group confers a significant advantage over unconjugated ODNs
in specifically inhibiting neointimal hyperplasia formation in vivo. These
findings raise the possibility of multiple therapeutic opportunities and also
the development of cholesterol-conjugated antisense olionucleotides into
therapeutically viable drugs for the treatment of restenosis. This finding
iS important in view of the multiple failed attempts in past clinical trial of
restenosis prevention. Such trials may benefit from the optimization of the
molecule pharmacokinetic properties prior to entering the clinical arena.
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. 21 -
Naturally, as with all inventions, this invention is not iimited to the
present specification, but rather by the claims and their equivalents.
