Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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OLIGONUCLEOTIDES TARGETED TO ANGIOTENSINOGEN mRNA
Backqround Of The Invention
Technical Field
This invention relates to compositions and methods
which are useful for reducing hypertension in humans.
More particularly, it relates to oligonucleotide
compounds capable of binding to angiotensinogen mRNA to
inhibit expression of angiotensinogen, and thereby,
essential hypertension.
Back~round Art
Angiotensinogen (AGT), which is produced largely by
the liver, is the substrate for the protein renin,
produced by the kidneys. The action of renin on
angiotensinogen results in the formation of the
decapeptide angiotensin I. Angiotensin I is further
converted to the octapeptide angiotensin II, through the
action of angiotensin converting enzyme (ACE) as it
circulates through the vasculature. Angiotensin II is a
potent vasoconstrictor and regulator of blood pressure
and volume homeostasis. An overactive renin angiotensin
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system (RAS) has been implicated in the development and
maintenance of essential hypertension and related forms
of hypertension in humans which affects approximately 40
million adults in the United States. Through the
inhibition of angiotensinogen the production of
angiotensin II can be inhibited, subsequently decreasing
hypertensive blood pressures.
Genetic variants of AGT have been associated with
elevated plasma AGT levels in hypertensive patients and a
predisposition to preeclampsia or atherosclerosis in
specific populations (Jeunmatre, X., et al., Cell
71(1):169-80, 1992; Hixon, J. E. and Powers, P. K., Human
Genet. 96:110-112, 1995). The RAS can be inhibited by
several mechanisms, which include ACE inhibitors, renin
inhibitors and angiotensin antagonists but as yet there
has been no specific inhibitor of AGT. The production of
AGT is controlled mainly by hormones that act upon gene
transcription and affect the concentration of the mRNA in
tissues.
The most important role played by hormones in the
regulation of AGT may be to maintain its synthesis and
constitutive secretion during its rapid consumption by
high levels of renin. Hepatic AGT is regulated mainly at
the transcriptional level in hepatocytes, by hormones
acting at the genomic level such as steroids, thyroid
hormones, glucocorticoids and inflammatory cytokines via
activation of DNA binding proteins that interact with the
appropriate response elements and multi-hormone response
sites like the hormone inducible enhancer unit ~HIEU) at
nucleotides -615 tc -440 upstream of the major
transcription start site (Peters, J., su~ra, 1995;
Brashier, A. R., et al., ~idney International~ 46:1564-
1566, 1994).
A large number of antihypertensive agents are
commercially available, however most have severe side
effects which generally require the action of a second
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group of agents. This, in combination with the nature of
the disease, tends to result in poor patient compliance.
A number of renin angiotensin system inhibitors presently
under use include: renin inhibitors, angiotensin
converting enzyme inhibitors, and angiotensin receptor
- blockers. These agents also lack specificity, have
unwanted side effects, require frequent dosing regimens,
and do not completely prevent the formation of
angiotensin II.
An alternative approach has been proposed to reduce
hypertension. A number of researchers have developed
antisense sequences for the inhibition of renin-
angiotensin system components.
Antisense oligodeoxynucleotides have been used to
successfully inhibit protein synthesis in a number of
biological systems (Yakubov, L., et al., Proc. Natl.
Acad. Sci. USA 86:6454-6458, 1989; Wahlstedt, D., et al.,
Science 259:528-531, 1993i Wahlstedt, D., et al., Nature
363:260-263, 1993; Wielbo, D., et al., Hypertension
25:314-319, 1995). This paradigm of gene regulation has
many potential therapeutic applications and is currently
being developed for application as anticancer,
antianxlety, antiviral and antiparasitic agents.
Antisense regulation or attenuation of protein synthesis
can be applied to any candidate gene with known molecular
sequence.
Antisense molecules are short strands of DNA or RNA,
usually 12-18 bases in length which are synthesized to
complement a target region of a candidate gene. The
antisense molecule binds to its complementary region and
via a number of mechanisms inhibits or attenuates gene
expression (Helene, C. C. and Toulme, J. J., Biochemica
et Biophysica Acta 1049:99-125, 1990).
Advances in understanding the function, metabolism
and structure of these molecules has lead to the
development of antisense molecules with enhanced nuclease
.. ..
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resistance and increased specificity, selectivity and
potency (Crooke, S. T., FASEB J. 7:533-539, 1993).
Phosphorothioated ASODN are modified phosphodiester
oligonucleotides where one of the non-bridged oxygen
atoms of the internucleotide linkage has been replaced
with a sulfur. Fully thioated antisense molecules are
more resistant to nucleases but also exhibit several non
sequence-specific effects in doses generally greater than
1 ~m (Wagner, R. W., Nature 372:333-335, 1994).
Phosphorothioation also stimulates the activity of
RNase H. This enzyme recognizes the DNA-mRNA duplex as a
substrate and cleaves the mRNA portion of the duplex,
freeing the DNA antisense molecule to bind to other mRNA
strands promoting a type of catalytic effect (Helene, C.
C. and Toulme, J. J., supra, 1990; Boiziau, C., et al.,
Biochemical Society Transactions 20:764-767, 1992).
We and others have utilized the concept of antisense
technology as a physiological tool to provide information
on cardiovascular function and hypertension. Sakai and
Meng demonstrated that angiotensin II type 1 (AT1)
receptor antisense oligonucleotides inhibit dipsogenic
responses to AngII (Sakai, R. R., et al., J. Neurochem.
62:2053-2056, 1994; Meng, H., et al., Regulatory Peptides
54:543-551, 1994) and Morishita and coworkers
successfully used antisense targeted to proliferating
cell nuclear antigens to inhibit neointima formation
after balloon catheter angioplasty (Morishita, R., et
al., Proc. Natl. Acad. Sci. USA 90 (18):8474-8, 1993).
Gyurko was able to show decreases in blood pressure
after central administration of ASO~N targeted to the AT1
receptor (Gyurko, R., et al., Regulatory Peptides 49:167-
174, 1993) and similar effects were observed when Wielbo
targeted central AGT (Wielbo, D., et al., supra, 1995).
Recently, Tomita was able to decrease blood pressure in
the SHR using three contiguous antisense oligonucleotide
sequences to target peripheral AGT and also showed
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decreases in AGT mRN-A (Tomita, N., et al., Hypertension
24(3):65, 397, 1994; Tomita, N., et al., Hypertension
26:131-136, 1995). Wielbo, et al. also decreased
hypertensive blood pressures in a rat model using a
liposome encapsulated 18-mer ODN sequence but no changes
in mRNA were observed (Wielbo, D., et al., Hypertension
28:147-151, 1996).
N. Tomita, et al., 26 HYpertension 131-136 (1995)
demonstrated that the administration of an antisense
molecule targeted to the exon/intron junctions of nascent
mRNA transiently decreases blood pressure in a rat model
of hypertension. These antisense molecules were
administered directly into the hepatic portal vein via a
liposomal delivery mechanism incorporating a viral
antigen to facilitate hepatic uptake. This research
group demonstrated a decrease in blood pressures, with
subsequent decreases in peripheral angiotensinogen and
angiotensinogen mRNA. Note that no informaiton was given
regarding dosage rates or number of injections. However,
dose response studies were carried out using up to
15~Mol/L to get significant decreases in AGT. This data
was represented as ratios probably due to non-specific
effects observed by control oligos at such high doses.
One drawback of the Tomita, et al. method is that an
invasive surgical procedure is required to administer the
liposomes via the hepatic portal vein. Another drawback
to the injection of antisense molecules directly into the
liver (at the volumes used by Tomita) is the risk of
damaging liver tissue. Also, viral antigen modification
must be done to facilitate hepatic uptake.
Unfortunately, the art has not yet developed an
approach which avoids the forgoing problems. Thus, the
need exists for effective ASODN hypertension treatment
compositions which avoid toxic side effects and are
efficacious in small dosages, and a method which does not
require invasive surgical administration techniques.
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Di~closure Of The In~ention
The invention resides in the discovery that AGT
expression can be inhibited in humans by administration
of oligonucleotide compounds of Formula I:
5'
ROCH2yOyB
X OtCH2yOyB
~ p/~ Y
X O~CH2yOyB
~ 3
RO Y
S in which:
each X independently is O, S, or Clg alkyl; each B
independently is adenine, guanine, cytosine, or thymine
selected such that the oligonucleotide is capable of
binding to the sense mRNA strand coding for human
angiotensinogen to thereby lnhibit the translation
thereof; each R independently is H or Clg alkyl or
P(0)(0)-substituted acridine; each Y independently is H
or OH; and n is 8 to 23. The oligonucleotide compound of
Formula I may also be 2 pharmaceutically acceptable salt
lS or hydrate thereof. Preferably, B is selected such that
the oligonucleotide is antisense to the sense mRNA strand
for human angiotensinogen. More preferably, B is
selected such that the oligonucleotide is capable of
binding to the mRNA base region encompassing the AUG
initiation codon.
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Another aspect-of the invention provides a
pharmaceutical composition useful for inhibiting
expression of angiotensinogen comprising a pharmaceutical
carrier and oligonucleotides of the above kind. The
pharmaceutical carrier may be a liposome, viral vector,
or protein conjugate formulation.
Another aspect of the invention provides a method
for treating hypertension in a human comprising
administering to a subject an effective amount of
oligonucleotides or compositions of the above kind.
The objects of the present invention therefore
include providing compounds, compositions and methods of
the above kind that:
(a~ avoid toxic side effects;
(b) avoid invasive surgical procedures;
(c) are effective in small dosages; and
(d) specifically target angiotensinogen mRNA.
These and still other objects and advantages of the
present invention will be apparent from the description
below.
Brief Description Of The Drawinqs
Fig. l is a bar graph depicting the percent
expresslon of angiotensinogen using one antisense
oligonucleotide of the present invention;
Figs. 2a and 2b are micrographs of rat liver tissue
one hour after injection of unencapsulated antisense
oligonucleotide and liposome encapsulated
oligonucleotide, respectively;
Fig. 3 is a bar graph depicting the change in mean
arterial pressure after treating spontaneously
hypertensive rats with liposome encapsulated antisense
oligonucleotide and control compositions;
Fig. 4 is a bar graph depicting the level of plasma
angiotensin II of spontaneously hypertensive rats after
.
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treating them with liposome encapsulated antlsense
oligonucleotide and control compositions;
Fig. 5 is a bar graph depicting the level of plasma
angiotensinogen of spontaneously hypertensive rats after
treating them with liposome encapsulated antisense
oligonucleotide and control compositions;
Fig. 6 is a graph showing comparative data using a
viral vector delivery system;
Fig. 7a shows a Northern blot hybridization for AGT
and the control gene cathepsin D;
Fig. 7b is a bar graph showing the relative
intensity of AGT mRNA expression after treatment compared
to angiotensinogen expression in untreated control cells;
Fig. 8 is a bar graph showing the effect of cationic
liposome con~ugated ASODN on angiotensinogen production;
Fig. 9a shows a Northern blot hybridization for AGT
and the control gene cathepsin D;
Fig. 9b is a bar graph showing the relative
expression of AGT mRNA compared to control treated
samples; and
Fig. l0 is a bar graph showing the dose dependent
decreases in angiotensinogen protein after cationic
liposomal delivery of ASODN.
Best ModeQ For CarrYinq Out The Invention
The native DNA segment coding for angiotensinogen
(AGT), as all such mammalian DNA strands, has two
strands: a sense strand and an antisense strand held
together by hydrogen bonding. The messenger RNA coding
for AGT has the same nucleotide sequence as the sense DNA
strand except that the DNA thymidine is replaced by
uridine. Thus, synthetic antisense nucleotide sequences
should bind with the DNA and RNA coding for AGT.
The oligonucleotide compounds of the invention bind
to the messenger RNA coding for human AGT thereby
inhibiting expression of this protein. Preferred
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_g_
compounds of the inv-ention are antisense to the sense DNA
sequence coding for human AGT as shown in Fig. 2 of
Fukamizu, et al. 265 J. Biol. Chem. 7576-7582 (1990).
Especially preferred oligonucleotide compounds are those
in which B of Formula I is selected such that the base
sequence of the oligonucleotide is 5'-CTCGCTTCCGCATACCCT-3'
(SEQ ID NO:1).
In the specification and claims, the letters, A, G,
C, T, and U respectively indicate nucleotides in which
the nucleoside is Adenosine (Ade), Guanosine (Gua),
Cytidine (Cyt), Thymidine (Thy), and Uridine (Ura). As
used in the specification and claims, compounds that are
antisense to the AGT DNA or mRNA sense strand are
compounds which have a nucleoside sequence complementary
to the sense strand. Table 1 shows the four possible
sense strand nucleosides and their complements present in
an antisense compound.
TABLE 1
¦ Sense ¦ Antisense
Ade Thy
Gua Cyt
Cyt Gua
Thy Ade
Ura Ade
It will be understood by those skilled in the art
that the present invention broadly includes
oligonucleotide compounds which are capable of binding to
the sense mRNA strand coding for angiotensinogen. Thus,
the invention includes compounds which are not strictly
antisense: the compounds may have some non-complementary
- bases provided such compounds have sufficient binding
affinity for angiotensinogen mRNA to inhibit expression.
The compounds of Formula I also differ from native
DNA in that some or all of the phosphates in the
nucleotides are replaced by phosphorothioates (X=S) or
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methylphosphonates (~=CH3) or other Cl9 alkylphosphonates.
The compounds of Formula I optionally may be further
differentiated from native DNA by replacing one or both
of the free hydroxy groups of the sense molecule with Cl4
alkoxy groups (R=Cl4 alkoxy). As used herein, Cl4 alkyl
means a branched or unbranched hydrocarbon having 1 to 4
carbon atoms.
Formula I compounds also may be substituted at the
3~ and/or 5' ends by a substituted acridine derivative.
As used herein, "substituted acridine" means any acridine
derivative capable of intercalating nucleotide strands
such as DNA. Preferred substituted acridines are 2-
methoxy-6-chloro-9-pentylaminoacridine, N-(6-chloro-2-
methoxyacridinyl)-O-methoxydiisopropylaminophosphinyl-3-
aminopropanol, and N-(6-chloro-2-methoxyacridinyl)-O-
methoxydiisopropylaminophosphinyl-5-aminopentanol. Other
sultable acridine derivatives are readily apparent to
persons skilled in the art. Additionally, as used herein
"P(0)(0)-substituted acridine" means a phosphate
covalently linked to a substitute acridine.
Formula I compounds also may include ribozyme
sequences inserted into their nucleotide sequence. The
ribozyme sequences are inserted into Formula I compounds
such that they are immediately preceded by AUC, WC, GUA,
GW, GUC, or, preferably, CUC. The ribozyme sequence is
any sequence which can be inserted and causes self-
cleavage of messenger RNA. The sequence CUG AUG AGU CCG
UGA CGA A is preferred. Other such sequences can be
prepared as described by Haseloff and Gerlach, 334 Nature
585-591 (1988).
The compounds of Formula I have about 10 to 25
nucleotides. As used herein, the term "nucleotides"
includes nucleotides in which the phosphate moiety is
replaced by phosphorothioate or alkylphosphonate and the
nucleotides may be substituted by substituted acridines.
Preferred Formula I compounds have 13 to 22 nucleotides.
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More preferred are compounds having 16 to 20 nucleotides.
Most preferred are compounds having 18 nucleotides.
Compounds having fewer than 10 nucleotides are less
desirable because they generally have less specificity
and compounds having greater than 25 nucleotides are less
desirable because their physical size and charge will
attenuate the crossing of the lipophilic cell membrane.
Thus, they are less likely to enter cells.
Although Formula I compounds that are antisense to
human AGT mRNA are preferred, Formula I includes
nucleotide compounds which lack a complement for each
nucleotide in a segment of the mRNA sense strand provided
such compounds have sufficient binding affinity for human
AGT mRNA to inhibit expression. The procedures of
Examples 1 and 5 are useful for screening whether
specific oligonucleotides of the present invention are
effective in inhibiting angiotensinogen expression.
Formula I compounds in which R is H are preferred.
R, however, can be Cl 4 alkyl provided the resulting
compounds retains sufficient binding affinity for the AGT
mRNA sense strand to inhibit expression of AGT.
Formula I compounds in which at least one X is S are
prepared by the following published procedures: W.J.
Stec, et al., 106 J. Am. Chem. Soc. 6077-6079 (1984);
S.P. Adams, et al., 105 ~. Am. Chem. Soc. 661 (1983);
M.H. Caruthers, et al., 4 Genetic Enqineerinq 1, Settlow,
J. Hollander. A. Eds; Plenum Press: New York (1982); M.S.
Broido, et al., 119 Biochem. Biophys. Res. Commun. 663
(1984). The reaction scheme described in these published
procedures is conducted on a solid support.
The reaction scheme involves lH-tetrazole-catalyzed
coupling of phosphoramidites to give phosphate
intermediates which are reacted with sulfur in 2,6-
lutidine to give phosphate compounds. Oligonucleotide
~ 35 compounds are prepared by treating the phosphate
compounds with thiophenoxide (1:2:2
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thiophenol/triethylamine/tetra-hydrofuran, room
temperature, 1 hour). The reaction sequence is repeated
until an oligonucleotide compound of the desired length
has been prepared ~Formula 1). The Formula I compounds
are cleaved from the support by treating with ammonium
hydroxide at room temperature for 1 hour and then are
further deprotected by heating at about 50~C overnight to
yield Formula I compounds. Formula I compounds in which
at least one X is oxygen are prepared by substituting I2-
HzO for the sulfur in 2,6-lutidine.
Formula I compounds in which at least X is CH3 or
other Cl4 alkyl are prepared by the following published
procedure: K.L. Aqarwal and F. Riftina, 6 Nucl. Acids
Res. 3009-3023 ~1979). The reaction sequence is
conducted on a solid support. The reaction procedure
involves phosphorylation of the 3'-hydroxyl group of a
5'-protected nucleoside using methylphosphonoditriazolide
as the phosphorylating reagent followed by benzene
sulfonyl-catalyzed coupling of the methylphosphonates to
yield the methyl phosphonate oligonucleotide.
Methylphosphonoditriazolide is prepared in situ from
equimolar quantities of methylphosphono-dichloridate,
triethylamine, and triazole. Benzene sulfonyl tetrazole
also was prepared in situ from pyridine, benezene
sulfonic acid and triethylamine. Repeating this reaction
sequence followed by cleavage from the support and
deprotection yield Formula I compounds.
Formula I compounds in which R is Cl4 alkyl are
prepared by replacing the DMT-protected compounds with
Cl4 alkylethers.
Formula I compounds in which R is P(0)(0)-
substituted acridine also are prepared by the following
published procedures: U. Asseline and N.T. Thuong,
30(19) Tet. Letters 2521-2524 (lg89) C.A. Stein, et al.,
72 G_ 333-341 (1988). These published procedures
include synthesis of a nucleoside phosphoramidite-bearing
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acridine derivative which then is reacted with 2, 2'-
dithiodiethanol attached to a support. The elongation
chain then is carried out on an automatic solid-phase DNA
synthesized as described above. These published
procedures also include synthesis of nucleoside
phosphoramidite-bearing acridine derivatives by reacting
substituted 9-(3-hydroxypropyl) amino acridines with N-
ethyldiisopropylamine followed by N,N-
diisopropylmethylphosphonamidic chloride. Using an
automated DNA synthesizer, Formula I compounds in which R
is P(0)(0)-substituted acridine are prepared by an extra
round of synthesis using the acridinyl phosphoramidites
in acetonitrile.
The compounds of Formula I can be incorporated into
convenient pharmaceutical dosage forms such as capsules,
tablets, or injectable preparations. Solid or liquid
pharmaceutical carriers can be employed. Solid carriers
include starch, lacrose, calcium sulfate dehydrate, terra
alba, sucrose, talc, gelatin, agar, pectin, acacia,
magnesium stearate, and stearic acid. Liquid carriers
include syrup, peanut oil, olive oil, saline and water.
Liposomal, viral vector, and protein conjugate
preparations can also be used as carriers. Similarly,
the carrier or diluent may include any prolonged release
material, such as glyceryl monosteararate of glyceryl
disteararate, alone or with a wax. The amount of solid
carrier varies widely but, preferably, will be from about
25 mg to about l g per dosage unit. When a liquid
carrier is used, the preparation will be in the form of a
syrup, elixir, emulsion, soft gelatin capsule, sterile
injectable liquid such as an ampoule, or an aqueous or
nonaqueous liquid suspension. When a liquid carrier is
used it will most often be a saline solution or phosphate
buffered saline solution.
The pharmaceutical preparations are made following
conventional techniques of a pharmaceutical chemist
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involving mixing, granulating and compressing, when
necessary, for tablet forms, or mixing, filling, and
dissolving the ingredients, as appropriate, to give the
desired oral or parenteral products.
Doses of the present Formula I compounds (in a
pharmaceutical dosage unit as described above) will be an
efficacious, nontoxic quantity selected from the range of
1 ng/kg to 500 mg/kg of active compound, preferably less
than 1 mg/kg. The selected dose is administered to a
human patient in need of inhibition of AGT expression
from 1-6 or more times daily, orally, rectally, by
injection, or continuously by infusion. Oral
formulations would generally require somewhat larger
dosages to overcome the effects of gastric decomposition.
Intravenous or intraarterial administration would
generally require minimum doses since the drug is placed
directly into the systemic circulation. Therefore, the
dose will depend on the actual route of administration.
By peripheral administration we mean by any other
route of delivery apart from oral or central (into the
brain). Peripherally administering the oligonucleotide
compounds of~the present invention via an artery
(carotid), vein (tail vein in rats or arm vein in
humans), or intraperitoneally (in mammals) allows
delivery to the liver without having to surgically open
the abdominal cavity for injection into the hepatic vein
or artery as ln Tomita, et al., suPra.
The following Examples are illustrative of Formula
(I) compounds and their preparation. The Examples are
not intended to limit the scope of the invention as
defined above and claimed below.
This invention relates to an oligonucleotide
compound which binds to a region of angiotensinogen mRNA
preventing the production of angiotensinogen protein
which is involved in the development and maintenance of
hypertensive blood pressure. By encapsulating the
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antisense molecules in pharmaceutical carriers, such as
liposomes, it is possible to obtain compositions which
can be used in a method to target the delivery of the
antisense molecules directly to the liver, the major site
of angiotensinogen production in the body. Liver
targeting can also be accomplished by packaging the
antisense molecules with viral vectors and protein
conjugates.
The compounds and compositions of the present
invention are unique because there is no commercially
available agent which specifically inhibits the action of
angiotensinogen. Additionally, due to the chemical
nature of antisense molecules the action is highly
specific, allowing the administration of small doses,
with potentially fewer side effects than conventional
antihypertensive agents.
The liposome encapsulated oligonucleotide compounds
of the present invention were demonstrated using an
antisense oligodeoxynucleotide for inhibiting the
angiotensinogen mRNA of spontaneously hypertensive rats.
The sequence for rat mRNA is disclosed in Fig. 3 of
Ohkubo, et al., 80 PNAS USA 2196-2200 (1983). One
version of the invention is an 18 base oligomer,
synthesized to complement the -5 to +13 base region of
rat angiotensinogen mRNA, which encompasses the AUG
translation initiation codon, the oligomer being composed
of the following base sequence: 5'-CCGTGGGAGT
CATCACGG-3' (SEQ ID NO:2). A phosphorothioated backbone
modification can be included on every base to confer
nuclease resistance.
The compositions of the present invention comprise
novel DNA sequences capable of binding to angiotensinogen
mRNA, preferably encapsulated in a pharmaceutical
delivery system, which results in the ASODN being
delivered directly to the liver, the major site of
angiotensinogen production. No viral antigen
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modification of the antisense oligonucleotide is
necessary.
Liposomes (80% phosphatidylcholine, 20g6 cholesterol)
are prepared using a rotary evaporator apparatus for
drying and rehydration of the lipid film. The liposomes
are subjected to multiple freeze thaw cycles which
enhances the entrapment of the antisense molecules and
are then passed through an ~xtruder (0.1 ~lm filter) in
order to reduce their size. Size can then be determined
by dynamic light scattering. For further details on
various methods of making liposome-encapsulated
biologically active compounds see U.S. patents 4,311,712;
4,370,349; 4,963,362; 5,264,221; 5,417,978; and
5,422,120.
As well as facilitating peripheral delivery,
liposomal encapsulation facilitates cellular uptake of
the antisense molecules resulting in an increased
efficiency of delivery. This has allowed us to
administer the antisense composition peripherally into
the blood stream and obtain physiological responses with
doses which produced no physiological response when
previously tested (see Gyurko, et al., and Wielbo, et
al., supra). Once the antisense molecule enters the
cells it binds to the targeted region of angiotensinogen
mRNA forming an mRNA/DNA duplex. This duplex formation
serves to prevent the assembly of ribosomal sub units and
the subsequent reading of the protein message, thereby
inhibiting angiotensinogen production. The
phosphorothioated form of the antisense molecule has
enhanced nuclease resistance and additionally stimulates
the action of RNase H, an enzyme which cleaves the mRNA
portion of the duplex, subsequently freeing the antisense
molecule to bind to another target mRNA.
We have shown that hypertensive blood pressure in
animal models of hypertension can be decreased with a
single, intra-arterial dose of a composition comprising a
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liposome encapsulated antisense DNA fragment targeted to
angiotensinogen. As well as being able to elicit a
physiological response we also have been able to produce
biochemical data which shows a subsequent decrease in
angiotensin II in the periphery at the time of blood
pressure decreases. Our previous studies have also shown
that a single dose of the antisense molecule into the
brain of hypertensive animals will also decrease blood
pressures for up to seven days.
We have previously shown that a single 50 ~g dose of
antisense oligonucleotide targeted to angiotensinogen
mRNA, administered directly into the brains of
spontaneously hypertensive rats resulted in a significant
and profound decrease in hypertensive blood pressures for
extended periods of time. The following figures
demonstrate the physiological effects of the liposome
encapsulated antisense molecules targeted to
angiotensinogen in the liver of hypertensive rats.
An alternative delivery system that can be used is a
viral vector. Fig. 6 shows the effect of a viral vector
antisense delivery system on angiotensin II type l
receptor expression in a neuroma-glioma brain tumor cell
line. In this study, antisense (the entire length of the
protein sequence) was targeted to the mRNA of the
angiotensin II type l receptor. Cells were either
treated with transfectam--an agent available to enhance
the uptake of the viral vector; an empty, mock plasmid
viral vector; paAT, the adeno-associated virus (AAV)
vector containing the antisense sequence; and paAT + Ad,
the adeno-associated viral vector containing the
antisense and a helper virus for adeno-associated virus
which enhances the AAV's p40 promoter which drives the
expression of the antisense mRNA. Substantially less
receptor binding was observed in the paAT + Ad treated
cells. This indicates that mRNA expression is attenuated
with the paAT + Ad treatment.
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Another delivery system which can be used in the
present invention is a protein conjugate oligonucleotide
composition. This technique is based on the construction
of DNA-protein complexes that are recognized by the liver
specific asialoglycoprotein receptor. Binding of poly(l-
lysine)-asialoorosomucoid (ASoR) protein conjugates with
phosphorothioated antisense can facilitate cellular
uptake in the liver. The conjugates use receptor
mediated endocytotic mechanisms for delivering genes into
cells. By covalently linking a specific receptor ligand,
such as asialoorosomucoid or transferrin to the
polylysine to form DNA protein complexes, it has been
possible to target cell specific surface receptors and
transfix antisense DNA into cells. See Bunnell, et al.,
18 Somatic Cell Molec. Gen. 559-569 (1992).
~xample 1
Fig. 1 demonstrates the specificity of the target
antisense molecule (SEQ ID NO:2) to the chosen target,
the rat protein angiotensinogen. In these experiments,
transcription and translation of the target protein was
- carried out in vitro, utilizing complimentary DNA (cDNA)
to angiotensinogen. The in vi tro reactions were carried
out in the presence and absence of antisense molecules
and scrambled or sense control oligonucleotide molecules.
These in vi tro reactions can also be employed to evaluate
the ability of the oligonucleotides of the present
invention to inhibit expression of human angiotensinogen.
Data is presented as percent expression of
angiotensinogen compared to the control reaction. Dose
response studies were carried out encompassing a range of
30 ~M oligonucleotide to 0.3 ~M oligonucleotide. At
higher doses of antisense, 3-30 ~M, the expression of
angiotensinogen was decreased to approximately 5-10% of
the control. However, non-specific attenuation of
angiotensinogen expression was observed with the
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scrambled and sense oligonucleotides, a phenomenon
recently described in the literature. In this regard, in
Tomita, su~ra, the degree of response seen with doses of
15~Mol/L is most likely due to this phenomenon. At lower
doses of oligonucleotides, 0.3-1 ~M, reactions carried
out in the presence of antisense show profound
attenuation of angiotensinogen production (45~), with no
observed non-specific attenuation with the scrambled
oligonucleotide. This suggests that, at low doses, the
antisense oligodeoxynucleotide profoundly and
specifically attenuates the translation of
angiotensinogen in vitro.
Example 2
Figs. 2a and 2b show two confocal micrographs of rat
liver tissue, one hour after injection of 50 ~g of (a)
unencapsulated, fluorescently labeled antisense (SEQ ID
NO:2), or (b) liposome encapsulated fluorescently labeled
antisense (SEQ ID NO:2) directly into the carotid artery.
Micrograph (a) shows little or no distribution of
fluorescent signal within the liver tissue. Micrograph
(b) shows an intense and evenly distributed fluorescent
signal throughout the tissue, indicating that liposome
encapsulation facilitates the delivery of the antisense
to the target organ.
To demonstrate the physiological responses to
intraarterially, peripherally administered compositions
comprising liposome encapsulated mRNA, antisense
oligonucleotide targeted to liver angiotensinogen mRNA,
groups of male, spontaneously hypertensive rats (250-
275 g) were catheterized via the carotid artery and
allowed 24 hours to recover from surgery. Baseline mean
arterial pressure (MAP) was then measured via direct
blood pressure transducer, attached to the indwelling
arterial catheter. A single 50 ~g dose of either a
composition comprising liposome encapsulated (25 mg
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lipid) antisense oligonucleotide (AS/L), or liposome
encapsulated scrambled oligonucleotide (Scr/L), or empty
liposomes (25 mg lipid) (Lipo), or 50 ~g unencapsulated
antisense oligonucleotide (ASODN) was administered via
the carotid artery. 24 hours later, mean arterial
pressure was measured to determine blood pressure
changes.
Example 3
This example demonstrates the effect of liposome
encapsulated ASODN (SEQ ID NO:2) on mean arterial
pressure (MAP) in spontaneously hypertensive rats.
Baseline MAP was established in groups of rats. Then 50
~g of liposome encapsulated (25 mg lipid) ASODN (AS/L);
liposome encapsulated ScrODN (Scr/L); empty liposomes (25
mg) (Lipo); or 50 ~g unencapsulated ASODN was
administered intra-arterially. Fig. 3 shows the blood
pressure changes observed 24 hours after each treatment.
Mean arterial pressure was significantly decreased in the
group treated with liposome encapsulated antisense
composition, (-24.66 mmHg + 2.43). However, no
significant blood pressure changes were observed in the
Scr/L (1.34 mmHg + 3.98); Lipo (-5.34 mmHg + 3.71) or
ASODN (-6.02 mmHg + 8.68) treatment groups. Results are
expressed as Mean + SEM, P c 0.013, n=6 per group. This
data supports the concept that peripherally administered
compositions comprising the liposome encapsulated
antisense (AS/L) will decrease hypertensive blood
pressures in the SHR model of hypertension (which is
predictive of what will occur in humans).
To determine biochemical changes, groups of animals
(n=3/group) were sacrificed 24 hours after treatment and
plasma angiotensin II levels were measured by
radioimmunoassay. Fig. 4 shows the effect of liposome
encapsulated antisense (AS/L) treatment on plasma
angiotensin II levels 24 hours after administration.
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Example 4
This example demonstrates the effect of liposome
encapsulated ASODN (SEQ ID NO:2) on angiotensin II.
Animals were sacriflced 24 hours after treatment. Plasma
angiotensin II levels were measured by radioimmunoassay.
As shown in Fig. 4, angiotensinogen II was significantly
lower in liposome encapsulated ASODN (AS/L) treated rats
(30.3 + 11.4 pg/mL, n=5) compared to controls: liposome
encapsulated ScrODN (Scr/L) (103.1 + 33.2 pgtmL, n=3);
empty liposomes (Lipo) (233.5 + 71.1 pg/mL, n=3); and
unencapsulated ASODN (201.4 + 88.5 pg/mL, n=3), Pc0.05.
This data suggest that the antisense effects are mediated
via the proposed mechanism of action, attenuating
angiotensinogen production with a subsequent decrease in
plasma angiotensin II.
ExamPle 5
This example demonstrates the effect of liposome
encapsulated ASODN (SEQ ID NO:2) on plasma AGT. Animals
were sacrificed 24 hours after treatment and plasma AGT
levels were measured by radioimmunoassay. As shown in
Fig. 5, AGT was significantly lower in liposome
encapsulated ASODN (AS/L) treated rats (58.5 + 3.71
pg/mL, n=5) compared to controls: liposome encapsulated
ScrODN (Scr/L) (79.0 + 8.72 pg/mL, n=3); empty liposomes
(Lipo) (85 + 5.72 pg/mL, n=3); and unencapsulated ASODN
(77.8 + 3.25 pg/mL, n=3), P<0.05.
Liposomes are drug vesicles in which an aqueous
phase is enclosed in a membrane of phospholipid
molecules, which form spontaneously when the lipids are
dispersed in an aqueous medium. These vesicles range in
size from nanometers to microns, and can be constructed
to entrap quantities of materials in both the aqueous
compartment and within the membrane. Advances in
targeted drug delivery now enable liposomal encapsulation
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of drug molecules to-improve protection, sustained
release and more efficient cellular uptake.
Phosphatidylcholine liposomes have been shown to be
efficient carriers of oligonucleotides, increasing
efficiency of cellular uptake and also increasing the
stability of the oligonucleotides in culture medium.
Cationic liposomes have been shown to be especially
efficient in cellular uptake, due to complexing of the
negatively charged oligonucleotides on the liposome
surface via electrostatic interactions (Ahktar, S. and
Juliano, R. L., Trends in Cell Biology 2 :139-144, 1992).
In this study we use an AGT-secreting Hepatoma cell line
to test the hypothesis that a single 18 mer AGT antisense
oligonucleotide conjugated to cationic liposomes will
enhance its delivery and cellular uptake and thereby
inhibit angiotensinogen production at lower doses and in
a dose dependant manner. In previous studies, although
we successfully decreased target protein production in
vitro and in vivo at high doses of naked ASODN, we did
2 0 not observe decreases in target mRNA, thus leaving doubt
as to the mechanism and specificity of protein
inhibition.
Material~ and Method~ (Examples 6-9)
Antisense Synthesis:
Using the AGT sequence established by Ohkubo
(Ohkubo, H., et al. Proc. Natl. Acad. sci. USA 80:2196-
200, 1983), an 18 mer ASODN strand was synthesized to the
-5 to +13 base sequence of the angiotensinogen mRNA, 5-
CCGTGGGAGTCATCACGG-3' (SEQ ID NO: 2 ) . This region covers
the AUG translation initiation codon. Control scrambled
ODN (ScrODN) had the same base composition as the ASODN
strand but in random order. These oligonucleotides are
modified by phosphorotioation. ODNs were synthesized in
the DNA Synthesis Laboratory, University of Florida. All
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oligonucleotide sequences were surveyed via the National
Center for Biotechnology Information BLAST network server
and Genebank data program for homology with related
proteins.
Liposome Synthesi~:
Cationic liposomes composed of
dimethyldiactadecylamonium bromide (DDAB) and
dioleoylphosphatidylethanolamine (DPOE) (2:5, w/w)
(Avanti Polar Lipids Inc., Alabaster, Alabama) were
dissolved in 30 mL chloroform and the solvent evaporated
by heating at 55~C under partial vacuum. Liposomes were
prepared by resuspending the lipids in 1 mL sterile
deionized water and sonication on ice until the solution
was almost clear. ASODN or ScrODN were then added to
give a concentration of l uM. The -/+ charge ratios of
ODS/cationic lipids was 0.18, giving a net positive
charge to the ODN-liposome conjugates to allow fusion
between the cell membranes and ODN-liposome conjugates.
For dose response studies five different oligonucleotide
concentrations were prepared with different
concentrations of DDA}3 to make ODN/cationic lipid
complexes with the same-/+ charge ratios.
Cell Culture:
H-4-II E, Hepatoma, Reuber H35, rat cells were
purchased from ATCC (Rockville, Maryland). Cells were
grown in monolayer culture in 12 mL of Eagles Minimum
Essential Medium (EMEM) supplemented with 10% fetal
bovine serum, 10% calf serum and incubated in 95%
air - 5% CO2 at 37~C. Cultures were fed everyday and
passaged when confluent at a ratio of 1:6 using 1.0 mL of
0.25% trypsin-EDTA. Cells were grown on 10 cm petri
dishes until experimentation. Cultures were then washed
with EMEM to remove any serum supplemented media and then
treated with varying concentrations of oligonucleotides
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or liposome complexe~ oligonucleotides and control
treatments.
Angiotensinogen Assay:
500 ~l aliquots of culture medium were evaporated to
dryness. The dried samples were assayed for
angiotensinogen by the direct radioimmunoassay method of
Sernia (Sernia, C., et al., Neuroendocrinology 55: 308-
316, 1992). AGT sample content was measured from a
standard curve of pure rat AGT diluted in the same cell
culture medium as the medium as the sample. The assay
sensitivity was 0.3 ng/tube, and an inter-assay and
intra-assay variability of 14% and 9% respectively.
Northern blot:
Northern blot analysis was carried by the method of
Chomczynski and Sacchi (Chomczynski, P. and Sacchi, N.,
Anal . Biochem. 162:156-9, 1987) and quantified by
densitometric methods. Cells were lysed using
mercaptoethanol then RNA was extracted by treatment with
guanidium thiocyanate followed by phenol-chloroform
extraction. RNA was precipitated by isopropanol at -
20~C. After centrifugation the amount of RNA was
calculated by spectrophotometry. 20 ~g aliquots of RNA
were electrophoresed on an agarose formaldehyde gel at 25
V for 16 hours. Adequate separation of mRNA was observed
using an RNA ladder and ethidium bromide staining. RNA
was transferred to a nylon membrane by Northern blot,
after prehybridization for 4 hours at 56~C with 1 x
Denhardts SSPE solution, 5 x SSPE, 0.1~ SDS and 50
formamide. The solution also contained 250 mg/mL
denatured salmon sperm DNA. Hybridization was carried
out under the same conditions with a labeled riboprobe to
the specific mRNA sequences. After wash steps the
membrane was exposed to x-ray film then developed.
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Membranes were standrardized by reprobing for Cathepsin D
mRNA.
Stati~tical Analysis:
Statistical analysis was performed by ANOVA for
treatment effect and Duncan multiple range test was used
for individual comparisons. Radioimmunoassay data for
individual time points was analyzed by students
independent T-test. P~0.05 is considered statistically
slgnificant.
Determination of in vitro effects of ASODN on
angiotensinogen production and angiotensinogen mRNA
expression in Hepatoma H4 cell culture.
H4 Hepatoma cell cultures were grown to confluence
then treated with cationic liposome conjugated ASODN and
ScrODN (l ~M) or l ~M naked ASODN or cationic lipid
control solutions. Cultures were incubated for 24 hours.
Media was decanted and after l00 ~l aliquots were
collected for AGT assay, cells were recovered from the
petri dishes. Combing two treatment plates to ensure
sufficient cells, mRNA was extracted and analyzed by
Northern blot to determine AGT mRNA levels. Northern
blots were quantified by densitometry. Media aliquots
were lyophilized and assayed for AGT levels by RIA. Six,
l00 mm plates were combined for each sample assay.
Do~e response relationships of cationic lipoQome
conjugated ASODN in Hepatoma H4 cell culture.
H4 Hepatoma cell cultures were grown to confluence
then treated with cationic liposome complexed ASODN at
the following concentrations: 0, 0.0l, 0.05, 0.l, 0.5
and l.0 ~M. Control groups consisted of increasing
amounts of cationic lipid in amounts necessary to
maintain an ASODN lipid ratio of -/+. ODN/cationic
lipid, 0.18. Cultures were incubated for 24 hours.
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Media was decanted and 100 ~l aliquots were lyophilized
and assayed for AGT levels by RIA. Cells were recovered
from petri dishes after collection of the media,
combining two treatment plates to ensure sufficient
cells, mRNA was extracted and analyzed by Northern blot
analysis to determlne ASODN dose effects on AGT mRNA.
Northern blots were quantified by densitometric methods.
Example 6
To determine the effect of cationic liposome
complexed ASODN and control treatments on mRNA expression
cell cultures were treated with 1 ~M of cationic liposome
complexed ASODN, complexed ScrODN, cationic liposomes and
1 ~M naked ASODN. Fig. 7a shows the result of Northern
hybridization for AGT (1.9 Kb) and the control gene
cathepsin (2.1 Kb). Samples 1-5 correspond to the
following treatments: 1. Naked antisense (ASODN); 2.
Empty cationic liposome control (CL); 3. Cationic
liposome complexed ScrODN control (Scr/CL); 4. Cationic
liposome complexed ASODN (AS/CL) and 5. Non-treated
control (CTRL). Fig. 7b corresponds to the relative
intensity of AGT mRNA expression after treatment compared
to AGT expression in untreated control cells. Expression
is presented as percent control. Cells treated with
naked ASODN (ASODN) appeared to have AGT expression
attenuated to 70~. Control treatments of uncomplexed
liposomes (CL) and liposome complexed ScrODN (Scr/CL)
resulted in little change from baseline levels with
approximately 90~ expression. However, cells treated
with liposome complexed ASODN (AS/CL) only show 22
expression.
ExamPle 7
To determine the effect of ASODN on AGT production
in Hepatoma H4 cell culture, cell cultures were treated
as before and culture media was analyzed for AGT protein
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levels using RIA. Fig. 8 shows the amount of AGT
produced by Hepatoma cell culture after control and ASODN
treatments. This graphs shows the effect of cationic
liposome complexed ASODN on AGT production. Cells were
incubated with 1 ~M of ASODN in the presence or absence
of cationic liposomes and appropriate controls. In
untreated controls (CTRL) the baseline AGT level was
52.0+2.46 ng/mL. There was no significant decrease in
AGT production from baseline levels in the empty liposome
(CL) and liposome complexed ScrODN (Scr/CL) control
groups (45.53+5.9; 47.03+6.9 ng/mL; nc3, respectively).
Cells treated with naked ASODN and cationic liposome
complexed ASODN (AS/CL) had AGT levels significants
decreased from baseline levels (30.2+3.0 ng/mL, n=3,
*=Ps0.05 and 5.61+.95 ng/mL; n=3, **=p~0.01,
respectively).
ExamPle 8
Dose response relationships of cationic liposome
complexed ASODN in Hepatoma H4 cell culture were then
conducted. Fig. 9a and 9b show a dose dependant
attenuation of AGT mRNA using cationic liposomes as
delivery mechanisms. Fig. 9a shows the result of
Northern hybridization for AGT and the control gene
cathepsin D. Samples 1-5 shows representative blots of
cells treated with only cationic liposomes. Each
oligonucleotide concentration was accompanied by a
separate control of uncomplexed liposomes as the
concentration of liposome per treatment increased
proportionally with each increase in oligonucleotide
concentration. Samples 6-10 correspond to cells treated
with 0.01, 0.05, 0.1, 0.5 and 1.0 ~M cationic liposome
complexed ASODN. Sample 11 shows AGT mRNA expression in
untreated control cells, which is used as the baseline
expression of AGT for the comparison of attenuation after
test ASODN treatments. Fig. 9b graphs the relative
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expression of the mRNA compared to the control treated
samples. The average expression for cells treated with
control cationic liposomes was 92%. Cells treated with
0.01 and 0.05 ~M ASODN had approximately 92% expression
which was similar to the control cationic liposome
treatments. At higher doses of ASODN, at 0.1, 0.5 and 1
~M the expression was decreased in a dose dependant
manner to 60%, 56% and 24% of control expression,
respectively. No changes were observed in the expression
of the Cathepsin D mRNA after each treatment.
ExEmple 9
Fig. 10 shows dose dependant decreases in AGT
protein after cationic liposomal delivery of ASODN.
Again each oligonucleotide concentration was accompanied
by a separate control of uncomplexed liposomes as the
concentration of liposome per treatment increased
proportionally with each increase in oligonucleotide
concentration. The baseline level of AGT protein was
51.77+3.7 ng/mL in the untreated controls (n=3). No
changes in baseline AGT production were observed using
empty cationic liposomes, mean AGT levels were 48. 5 ng/mL
for each increasing dose. Consistent with decreases in
mRNA levels, no significant decreases in AGT were
observed with lower doses of ASODN (0.01 ~M or 0.05 ~M)
AGT protein levels were 47.6+7.4 and 40.29+4.0 ng/mL,
respectively, although a decreasing trend is apparent.
At 0.1, 0.5 and 0.1 ~M ASODN the protein levels were
significantly decreased from baseline levels in a dose
dependant manner. AGT protein levels were 26.86+5.3;
23.27+6.1 and 5.67+0.3 ng/mL, n=3 per group; *=psO.05 and
**=P50 . 01 .
Discussion of ExamPleq 6-9
Although successful target protein attenuation has
been achieved using antisense technology in a wide range
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of biological systems, the development of antisense
therapy has not been as smooth as once anticipated.
Problems have been encountered in the delivery of the
molecules to the target site. Cellular uptake of ASODN
molecules is thought to be via specific receptor
interaction or via receptor mediated endocytosis
(Morishita, R., et al ., suPra~ 1993; Loke, S. L., et al.,
Proc. Natl. Acad. Sci. USA 86:3474-3478, 1989; Temsamani,
J., et al., Antisense Research and Development 4:35-42,
1994; Wickstrom, E., Trends-Biotechnol., 10(8):281-7,
1992), but in general uptake efficiency is poor with as
little as 2% of ASODN entering the cells and as few as 1%
of cells being transfected (Ahktar, S. and Juliano, R.
L., supra, 1992). The amount of naked oligonucleotides
taken up by viable cells ranges from 1-10~ with the rate
of transfection being variable between cell types.
Liposomal delivery systems have been shown to facilitate
and increase the efficiency of cellular uptake of ASODN,
by protecting the antisense from degradation and by
bringing the antisense molecules into closer proximity to
the cells thereby facilitating the uptake process. In
particular, cationic liposomes have been shown to
increase uptake efficiency by 30% (Lappalainen, K., et
al., Biochimica et Biophysica Acta 1196:201-208, 1994).
Consequently due to the expected enhanced delivery and
cellular transfection of cationic liposomes we expected
that AGT protein and mRNA levels would be decreased
accordingly.
The above data suggests that cationic liposomes are
efficient delivery systems for an antisense molecule
targeted to angiotensinogen mRNA. Our data shows that at
1 ~M doses, liposome complexed ASODN specifically
decreases target protein and mRNA more profoundly than
naked ASODN alone. These observations can be attributed
to the specific inhibition of target mRNA as no
significant decreases in target protein were observed
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with liposome complexed ScrODN or control cationic
lipids. Furthermore, the same dose of liposome complexed
ASODN also resulted in significant decreases in target
mRNA from baseline levels. This suggests that the mode
of oligonucleotide action may be through the stimulation
of RNase H. This enzyme recognizes the mRNA-DNA double-
strand as a substrate and subsequently degrades the mRNA
portion of the duplex. Hence the observed decrease in
angiotensinogen mRNA although this particular
oligonucleotide was designed to inhibit translational
processes and ribosome binding at the AUG initiation
codon.
Interestingly, although mRNA expression was less
than baseline levels (but not significantly) after naked
ASODN treatment, we observed that naked ASODN did
significantly decrease AGT protein levels, although not
as profoundly as the cationic liposome complexed ASODN.
This observation is probably due to the cationic
liposomes resulting in an increase in oligonucleotide
delivery due to the negatively charged oligonucleotide
and the cationic charge of the lipids resulting in a
substantial amount of liposome/oligonucleotide surface
binding.
We conclude that efficient cellular delivery of
antisense oligonucleotides is an important consideration
in obtaining efficacy of protein attenuation and that
such delivery may be facilitated through the use of
liposomal delivery systems. Cationic liposomes may be
utilized to substantially increase the delivery
efficiency of oligonucleotides in cell culture to the
extent that intracellular ASODN levels elicit both
attenuation of target protein and target mRNA. This
delivery system offers a simple approach for the
determination of basic ASODN properties, enabling
sufficient intracellular levels of ASODN for activity
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without the need for-viral vector delivery or targeted
cellular delivery.
Example l0
In this study, baby SH rats which are normotensive
until about 8 weeks of age, were injected with a single
dose (50~g oligo) of liposome encapsulated
oligonucleotide (the base sequence of the oligonucleotide
being in accordance with SEQ ID NO: 2) via the tail vein,
once a week for four weeks. This was done after weaning
from age 4 weeks to 8 weeks.
At 8, 12, and 18 weeks their blood pressure was
measured indirectly, using the tail cuff method. At 8
weeks of age the liposome antisense treated rats had
blood pressures at 126.2 mm Hg + 7.49. This was
significantly lower than the control treatment groups
(P~0.05). This lowering of blood pressure maintained
pressures in the normotensive range whereas the control
groups had blood pressures in the hyper~ensive range. At
12 weeks of age, 8 weeks after the final ASODN-liposome
treatment, animals still had lower blood pressures
compared to the control treatment group. Although this
lowered blood pressure was not significantly lower than
the control groups and was still in the hypertensive
range, a trend was evident and significance could
probably be achieved by increasing the sample size. At
18 weeks of age all groups of rats had blood pressures in
the hypertensive range.
Thus, administration of liposome encapsulated
antisense during the critical growth phase of baby rats
can transiently, but significantly attenuate the
development of hypertension. The duration of this
attenuation could possibly be extended by increasing
dosing frequency or amount of ASODN. That attenuation
can be achieved by simple venous administration without
the need for invasive surgery.
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Although the present invention has been described
with reference to certain preferred embodiments, other
variants are possible. For example, the present
invention includes oligonucleotide compounds which lack a
complement for each nucleotide in a particular segment of
the angiotensinogen mRNA, provided such compounds have
sufficient binding affinity for the AGT mRNA to inhibit
expression thereof. In this regard, the materials and
methods of Examples 6-9, su~ra, are useful in determining
whether specific oligonucleotides are effective in
inhibiting angiotensinogen expression. Obviously, the
above methods would be modified when evaluating specific
compounds for the inhibition of human angiotensinogen.
For example, human cells such as human embryonic cell
lines or human hepotoma cell lines could be used for the
cell culture. Also, the present invention is intended to
include mixtures of oligonucleotide compounds, each
compound being capable of binding to angiotensinogen
mRNA.
Therefore, the scope of the claims is not limited to
the specific examples of the preferred versions herein.
Rather, the claims should be looked to in order to ~udge
the full scope of the invention.
Industrial APPlicabilitY
The oligonucleotides disclosed herein are useful for
inhibiting the expression of angiotensinogen to thereby
control angiotensinogen induced hypertension.
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- SEQUENCE LISTING
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CA 02248932 1998-09-15
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CCGTGGGAGT CATCACGG 18
