U.S. patent application number 10/641521 was filed with the patent office on 2004-06-03 for modified protein kinase a-specifc oligonucleotides and methods of their use.
This patent application is currently assigned to Hybridon, Inc., A Corporation of the State of Delaware. Invention is credited to Agrawal, Sudhir.
Application Number | 20040106570 10/641521 |
Document ID | / |
Family ID | 32398192 |
Filed Date | 2004-06-03 |
United States Patent
Application |
20040106570 |
Kind Code |
A1 |
Agrawal, Sudhir |
June 3, 2004 |
Modified protein kinase A-specifc oligonucleotides and methods of
their use
Abstract
Disclosed are synthetic, modified oligonucleotides complementary
to, and capable of down-regulating the expression of, nucleic acid
encoding protein kinase A subunit RI.sub..alpha.. The modified
oligonucleotides have from about 15 to about 30 nucleotides and are
hybrid, inverted hybrid, or inverted chimeric oligonucleotides.
Also disclosed are therapeutic compositions containing such
oligonucleotides and methods of using the same.
Inventors: |
Agrawal, Sudhir;
(Shrewsbury, MA) |
Correspondence
Address: |
HALE AND DORR, LLP
60 STATE STREET
BOSTON
MA
02109
|
Assignee: |
Hybridon, Inc., A Corporation of
the State of Delaware
Cambridge
MA
|
Family ID: |
32398192 |
Appl. No.: |
10/641521 |
Filed: |
August 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10641521 |
Aug 15, 2003 |
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09022965 |
Feb 12, 1998 |
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6624293 |
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10641521 |
Aug 15, 2003 |
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08532979 |
Sep 22, 1995 |
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5969117 |
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08532979 |
Sep 22, 1995 |
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08516454 |
Aug 17, 1995 |
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5652356 |
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60040740 |
Mar 12, 1997 |
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Current U.S.
Class: |
514/44A ;
536/23.2 |
Current CPC
Class: |
C12N 2310/322 20130101;
C12N 2310/321 20130101; C12N 15/1137 20130101; C07H 21/00 20130101;
A61K 38/00 20130101; C12N 2310/3125 20130101; Y10S 977/898
20130101; Y10S 977/915 20130101; C12N 2310/315 20130101; C12N
2310/345 20130101; C12N 2310/321 20130101; Y10S 977/894 20130101;
C12N 2310/341 20130101; C12N 2310/346 20130101; C12N 2310/3521
20130101 |
Class at
Publication: |
514/044 ;
536/023.2 |
International
Class: |
A61K 048/00; C07H
021/04 |
Claims
What is claimed is:
1. A method of inhibiting the proliferation of cancer cells,
comprising the step of administering to the cells a synthetic,
modified oligonucleotide complementary to, and capable of
down-regulating the expression of, nucleic acid encoding protein
kinase A subunit RI.sub..alpha., the modified oligonucleotide
having from about 15 to about 30 nucleotides and being a hybrid,
inverted hybrid, or inverted chimeric oligonucleotide, the hybrid
oligonucleotide comprising a region of at least two
deoxyribonucleotides, flanked by 3' and 5' flanking ribonucleotide
regions each having at least four ribonucleotides, the inverted
hybrid oligonucleotide comprising a region of at least four
ribonucleotides flanked by 3' and 5' flanking deoxyribonucleotide
regions of at least two deoxyribonucleotides, and the inverted
chimeric oligonucleotide comprising an oligonucleotide nonionic
region of at least four nucleotides flanked by two oligonucleotide
phosphorothioate regions.
2. The method of claim 1, wherein the oligonucleotide is a hybrid
oligonucleotide.
3. The method of claim 2, wherein there is reduced mitogenicity,
reduced activation of complement, or reduced antithrombotic
properties, relative to the side effects caused by an
oligonucleotide which is not hybrid.
4. The method of claim 1, wherein the oligonucleotide is an
inverted hybrid oligonucleotide.
5. The method of claim 4, wherein there is reduced mitogenicity,
reduced activation of complement, or reduced antithrombotic
properties, relative to the side effects caused by an
oligonucleotide which is not inverted hybrid.
6. The method of claim 1, wherein the oligonucleotide is an
inverted chimeric oligonucleotide.
7. The method of claim 6, wherein there is reduced mitogenicity,
reduced activation of complement, or reduced antithrombotic
properties, relative to the side effects caused by an
oligonucleotide which is not inverted chimeric.
8. A method of treating cancer in an afflicted subject, comprising
the step of administering to the subject a therapeutic composition
comprising a synthetic, modified oligonucleotide complementary to,
and capable of down-regulating the expression of, nucleic acid
encoding protein kinase A subunit RI.sub..alpha., the modified
oligonucleotide having from about 15 to about 30 nucleotides and
being a hybrid, inverted hybrid, or inverted chimeric
oligonucleotide, the hybrid oligonucleotide comprising a region of
at least two deoxyribonucleotides, flanked by 3' and 5' flanking
ribonucleotide regions each having at least four ribonucleotides,
the inverted hybrid oligonucleotide comprising a region of at least
four ribonucleotides flanked by 3' and 5' flanking
deoxyribonucleotide regions of at least two deoxyribonucleotides,
and the inverted chimeric oligonucleotide comprising an
oligonucleotide nonionic region of at least four nucleotides
flanked by two oligonucleotide phosphorothioate regions.
9. The method of claim 8, wherein the oligonucleotide is a hybrid
oligonucleotide.
10. The method of claim 9, wherein there is reduced mitogenicity,
reduced activation of complement, or reduced antithrombotic
properties, relative to the side effects caused by an
oligonucleotide which is not hybrid.
11. The method of claim 8, wherein the oligonucleotide is an
inverted hybrid oligonucleotide.
12. The method of claim 11, wherein there is reduced mitogenicity,
reduced activation of complement, or reduced antithrombotic
properties, relative to the side effects caused by an
oligonucleotide which is not inverted hybrid.
13. The method of claim 8, wherein the oligonucleotide is an
inverted chimeric oligonucleotide.
14. The method of claim 13, wherein there is reduced mitogenicity,
reduced activation of complement, or reduced antithrombotic
properties, relative to the side effects caused by an
oligonucleotide which is not inverted chimeric.
Description
[0001] This application is a divisional application of patent
application Ser. No. 09/022,965, filed Feb. 12, 1998, which claims
the benefit of Patent Application No. 60/040,740, filed on Mar. 12,
1997, and is a continuation-in-part application of patent
application Ser. No. 08/532,979, filed Sep. 22, 1995, which issued
as U.S. Pat. No. 5,969,117, which is a continuation-in-part of
patent application Ser. No. 08/516,454, filed Aug. 17, 1995, which
issued as U.S. Pat. No. 5,652,356.
FIELD OF THE INVENTION
[0002] The present invention relates to cancer therapy. More
specifically, the present invention relates to the inhibition of
the proliferation of cancer cells using modified antisense
oligonucleotides complementary to nucleic acid encoding the protein
kinase A RI.sub..alpha. subunit.
BACKGROUND OF THE INVENTION
[0003] The development of effective cancer therapies has been a
major focus of biomedical research. Surgical procedures have been
developed and used to treat patients whose tumors are confined to
particular anatomical sites. However, at present, only about 25% of
patients have tumors that are truly confined and amenable to
surgical treatment alone (Slapak et al. in Harrison's Principles of
Internal Medicine (Isselbacher et al., eds.) McGraw-Hill, Inc., NY
(1994) pp. 1826-1850). Radiation therapy, like surgery, is a local
modality whose usefulness in the treatment of cancer depends to a
large extent on the inherent radiosensitivity of the tumor and its
adjacent normal tissues. However, radiation therapy is associated
with both acute toxicity and long term sequelae. Furthermore,
radiation therapy is known to be mutagenic, carcinogenic, and
teratogenic (Slapak et al., ibid.).
[0004] Systemic chemotherapy alone or in combination with surgery
and/or radiation therapy is currently the primary treatment
available for disseminated malignancies. However, conventional
chemotherapeutic agents which either block enzymatic pathways or
randomly interact with DNA irrespective of the cell phenotype, lack
specificity for killing neoplastic cells. Thus, systemic toxicity
often results from standard cytotoxic chemotherapy. More recently,
the development of agents that block replication, transcription, or
translation in transformed cells, and at the same time defeat the
ability of cells to become resistant, has been the goal of many
approaches to chemotherapy.
[0005] One strategy is to down regulate the expression of a gene
associated with the neoplastic phenotype in a cell. A technique for
turning off a single activated gene is the use of antisense
oligodeoxynucleotides and their analogues for inhibition of gene
expression (Zamecnik et al. (1978) Proc. Natl. Acad. Sci. (USA)
75:280-284). An antisense oligonucleotide targeted at a gene
involved in the neoplastic cell growth should specifically
interfere only with the expression of that gene, resulting in
arrest of cancer cell growth. The ability to specifically block or
down-regulate expression of such genes provides a powerful tool to
explore the molecular basis of normal growth regulation, as well as
the opportunity for therapeutic intervention (see, e.g., Cho-Chung
(1993) Curr. Opin. Thera. Patents 3:1737-1750). The identification
of genes that confer a growth advantage to neoplastic cells as well
as other genes causally related to cancer and the understanding of
the genetic mechanism(s) responsible for their activation makes the
antisense approach to cancer treatment possible.
[0006] One such gene encodes the RI.sub..alpha. subunit of cyclic
AMP (cAMP)-dependent protein kinase A (PKA) (Krebs (1972) Curr.
Topics Cell. Regul. 5:99-133). Protein kinase is bound by cAMP,
which is thought to have a role in the control of cell
proliferation and differentiation (see, e.g., Cho-Chung (1980) J.
Cyclic Nucleotide Res. 6:163-167). There are two types of PKA, type
I (PKA-I) and type II (PKA-II), both of which share a common C
subunit but each containing distinct R subunits, RI and RII,
respectively (Beebe et al. in The Enzymes: Control by
Phosphorylation, 17(A):43-1 11 (Academic, New York, 1986). The R
subunit isoforms differ in tissue distribution (.O slashed.yen et
al. (1988) FEBS Lett. 229:391-394; Clegg et al. (1988) Proc. Natl.
Acad. Sci. (USA) 85:3703-3707) and in biochemical properties (Beebe
et al. in The Enzymes: Control by Phosphorylation, 17(A):43-1 11
(Academic Press, NY, 1986); Cadd et al. (1990) J. Biol. Chem.
265:19502-19506). The two general isoforms of the R subunit also
differ in their subcellular localization: RI is found throughout
the cytoplasm; whereas RII localizes to nuclei, nucleoli, Golgi
apparatus and the microtubule-organizing center (see, e.g., Lohmann
in Advances in Cyclic Nucleotide and Protein Phosphorylation
Research, 18:63-117 (Raven, New York, 1984; and Nigg et al. (1985)
Cell 41:1039-1051).
[0007] An increase in the level of RI.sub..alpha. expression has
been demonstrated in human cancer cell lines and in primary tumors,
as compared with normal counterparts, in cells after transformation
with the Ki-ras oncogene or transforming growth factor-.alpha., and
upon stimulation of cell growth with granulocyte-macrophage
colony-stimulating factor (GM-CSF) or phorbol esters (Lohmann in
Advances in Cyclic Nucleotide and Protein Phosphorylation Research,
18:63-117 (Raven, New York, 1984); and Cho-Chung (1990) Cancer Res.
50:7093-7100). Conversely, a decrease in the expression of
RI.sub..alpha. has been correlated with growth inhibition induced
by site-selective cAMP analogs in a broad spectrum of human cancer
cell lines (Cho-Chung (1990) Cancer Res. 50:7093-7100). It has also
been determined that the expression of RI/PKA-I and RII/PKA-II has
an inverse relationship during ontogenic development and cell
differentiation (Lohmann in Advances in Cyclic Nucleotide and
Protein Phosphorylation Research, Vol. 18, 63-117 (Raven, New York,
1984); Cho-Chung (1990) Cancer Res. 50:7093-7100). The
RI.sub..alpha. subunit of PKA has thus been hypothesized to be an
ontogenic growth-inducing protein whose constitutive expression
disrupts normal ontogenic processes, resulting in a pathogenic
outgrowth, such as malignancy (Nesterova et al. (1995) Nature
Medicine 1:528-533).
[0008] Antisense oligonucleotides directed to the RI.sub..alpha.
gene have been prepared. U.S. Pat. No. 5,271,941 describes
phosphodiester-linked antisense oligonucleotides complementary to a
region of the first 100 N-terminal amino acids of RI.sub..alpha.
which inhibit the expression of RI.sub..alpha. in leukemia cells in
vitro. In addition, antisense phosphorothioate
oligodeoxynucleotides corresponding to the N-terminal 8-13 codons
of the RI.sub..alpha. gene was found to reduce in vivo tumor growth
in nude mice (Nesterova et al. (1995) Nature Med. 1:528-533).
[0009] Unfortunately, problems have been encountered with the use
of phosphodiester-linked (PO) oligonucleotides and some
phosphorothioate-linked (PS) oligonucleotides. It is known that
nucleases in the serum readily degrade PO oligonucleotides.
Replacement of the phosphodiester internucleotide linkages with
phosphorothioate internucleotide linkages has been shown to
stabilize oligonucleotides in cells, cell extracts, serum, and
other nuclease-containing solutions (see, e.g., Bacon et al. (1990)
Biochem. Biophys. Meth. 20:259) as well as in vivo (Iversen (1993)
Antisense Research and Application (Crooke, ed) CRC Press, 461).
However, some PS oligonucleotides have been found to exhibit an
immunostimulatory response, which in certain cases may be
undesirable. For example, Galbraith et al. (Antisense Res. &
Dev. (1994) 4:201-206) disclose complement activation by some PS
oligonucleotides. Henry et al. (Pharm. Res. (1994) 11: PPDM8082)
disclose that some PS oligonucleotides may potentially interfere
with blood clotting.
[0010] There is, therefore, a need for modified oligonucleotides
directed to cancer-related genes that retain gene expression
inhibition properties while producing fewer side effects than
conventional oligonucleotides.
SUMMARY OF THE INVENTION
[0011] The present invention relates to modified oligonucleotides
useful for studies of gene expression and for the antisense
therapeutic approach. The invention provides modified
oligonucleotides that down-regulate the expression of the
RI.sub..alpha. gene while producing fewer side effects than
conventional oligonucleotides. In particular, the invention
provides modified oligonucleotides that demonstrate reduced
mitogenicity, reduced activation of complement and reduced
antithrombotic properties, relative to conventional
oligonucleotides.
[0012] It is also known that some PS oligonucleotides cause an
immunostimulatory response in subjects to whom they have been
administered, which may be undesirable in some cases.
[0013] It is known that exclusively phosphodiester- or exclusively
phosphorothioate-linked oligonucleotides directed to the first 100
nucleotides of the RI.sub..alpha. nucleic acid inhibit cell
proliferation.
[0014] It has now been discovered that modified oligonucleotides
complementary to the protein kinase A RI.sub..alpha. subunit gene
inhibit the growth of tumors in vivo. With at least the activity of
a comparable PO- or PS-linked oligonucleotide with fewer side
effects.
[0015] This finding has been exploited to produce the present
invention, which in a first aspect, includes synthetic hybrid,
inverted hybrid, and inverted chimeric oligonucleotides and
compositions of matter for specifically down-regulating protein
kinase A subunit RI.sub..alpha. gene expression with reduced side
effects. Such inhibition of gene expression is useful as an
alternative to mutant analysis for determining the biological
function and role of protein kinase A-related genes in cell
proliferation and tumor growth. Such inhibition of RI.sub..alpha.
gene expression can also be used to therapeutically treat diseases
and disorders that are caused by the over-expression or
inappropriate expression of the gene.
[0016] As used herein, the term "synthetic oligonucleotide"
includes chemically synthesized polymers of three up to 50,
preferably from about 15 to about 30, and most preferably, 18
ribonucleotide and/or deoxyribonucleotide monomers connected
together or linked by at least one, and preferably more than one,
5' to 3' internucleotide linkage.
[0017] For purposes of the invention, the term "oligonucleotide
sequence that is complementary to a genomic region or an RNA
molecule transcribed therefrom" is intended to mean an
oligonucleotide that binds to the nucleic acid sequence under
physiological conditions, e.g., by Watson-Crick base pairing
(interaction between oligonucleotide and single-stranded nucleic
acid) or by Hoogsteen base pairing (interaction between
oligonucleotide and double-stranded nucleic acid) or by any other
means including in the case of a oligonucleotide binding to RNA,
causing pseudoknot formation. Binding by Watson-Crick or Hoogsteen
base pairing under physiological conditions is measured as a
practical matter by observing interference with the function of the
nucleic acid sequence.
[0018] In one preferred embodiment according to this aspect of the
invention, the oligonucleotide is a core region hybrid
oligonucleotide comprising a region of at least two
deoxyribonucleotides, flanked by 5' and 3' ribonucleotide regions,
each having at least four ribonucleotides. A hybrid oligonucleotide
having the sequence set forth in the Sequence Listing as SEQ ID
NO:4 is one particular embodiment. In some embodiments, each of the
3' and 5' flanking ribonucleotide regions of an oligonucleotide of
the invention comprises at least four contiguous, 2'-O-substituted
ribonucleotides.
[0019] For purposes of the invention, the term "2'-O-substituted"
means substitution of the 2' position of the pentose moiety with an
--O-lower alkyl group containing 1-6 saturated or unsaturated
carbon atoms, or with an --O-aryl or allyl group having 2-6 carbon
atoms, wherein such alkyl, aryl or allyl group may be unsubstituted
or may be substituted, e.g., with halo, hydroxy, trifluoromethyl,
cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or
amino groups; or with a hydroxy, an amino or a halo group, but not
with a 2'-H group.
[0020] In some embodiments, each of the 3' and 5' flanking
ribonucleotide regions of an oligonucleotide of the invention
comprises at least one 2'-O-alkyl substituted ribonucleotide. In
one preferred embodiment, the 2'-O-alkyl-substituted nucleotide is
a 2'-O-methyl ribonucleotide. In other preferred embodiments, the
3' and 5' flanking ribonucleotide regions of an oligonucleotide of
the invention comprises at least four 2'-O-methyl ribonucleotides.
In preferred embodiments, the ribonucleotides and
deoxyribonucleotides of the hybrid oligonucleotide are linked by
phosphorothioate internucleotide linkages. In particular
embodiments, this phosphorothioate region or regions have from
about four to about 18 nucleosides joined to each other by 5' to 3'
phosphorothioate linkages, and preferably from about 5 to about 18
such phosphorothioate-linked nucleosides. The phosphorothioate
linkages may be mixed R.sub.p and S.sub.p enantiomers, or they may
be stereoregular or substantially stereoregular in either R.sub.p
or S.sub.p form (see Iyer et al. (1995) Tetrahedron Asymmetry
6:1051-1054).
[0021] In another preferred embodiment according to this aspect of
the invention, the oligonucleotide is an inverted hybrid
oligonucleotide comprising a region of at least four
ribonucleotides flanked by 3' and 5' deoxyribonucleotide regions of
at least two deoxyribonucleotides. The structure of this
oligonucleotide is "inverted" relative to traditional hybrid
oligonucleotides. In some embodiments, the 2'-O-substituted RNA
region has from about four to about ten 2'-O-substituted
nucleosides joined to each other by 5' to 3' internucleoside
linkages, and most preferably from about four to about six such
2'-O-substituted nucleosides. In some embodiments, the
oligonucleotides of the invention have a ribonucleotide region that
comprises at least five contiguous ribonucleotides. In one
particularly preferred embodiment, the overall size of the inverted
hybrid oligonucleotide is 18. In preferred embodiments, the
2'-O-substituted ribonucleosides are linked to each other through a
5' to 3' phosphorothioate, phosphorodithioate, phosphotriester, or
phosphodiester linkages. The phosphorothioate 3' or 5' flanking
region (or regions) has from about four to about 18 nucleosides
joined to each other by 5' to 3' phosphorothioate linkages, and
preferably from about 5 to about 18 such phosphorothioate-linked
nucleosides. In preferred embodiments, the phosphorothioate regions
will have at least 5 phosphorothioate-linked nucleosides. One
specific embodiment is an oligonucleotide having substantially the
nucleotide sequence set forth in the Sequence Listing as SEQ ID
NO:6. In preferred embodiments of this aspect of the invention, the
ribonucleotide region comprises 2'-O-substituted ribonucleotides,
such as 2'-O-alkyl substituted ribonucleotides. One particularly
preferred embodiment is an inverted hybrid oligonucleotide whose
ribonucleotide region comprises at least one 2'-O-methyl
ribonucleotide.
[0022] In some embodiments, all of the nucleotides in the inverted
hybrid oligonucleotide are linked by phosphorothioate
internucleotide linkages. In particular embodiments, the
deoxyribonucleotide flanking region or regions has from about four
to about 18 nucleosides joined to each other by 5' to 3'
phosphorothioate linkages, and preferably from about 5 to about 18
such phosphorothioate-linked nucleosides. In some embodiments, the
deoxyribonucleotide 3' and 5' flanking regions of the inverted
hybrid oligonucleotides of the invention have about 5
phosphorothioate-linked nucleosides. The phosphorothioate linkages
may be mixed R.sub.p and S.sub.p enantiomers, or they may be
stereoregular or substantially stereoregular in either R.sub.p or
S.sub.p form (see Iyer et al. (1995) Tetrahedron Asymmetry
6:1051-1054).
[0023] Another embodiment is a composition of matter for inhibiting
the expression of protein kinase A subunit RI.sub..alpha. with
reduced side effects, the composition comprising an inverted hybrid
oligonucleotide according to the invention.
[0024] Yet another preferred embodiment according to this aspect of
the invention is an inverted chimeric oligonucleotide comprising an
oligonucleotide nonionic region of at least four nucleotides
flanked by one or more, and preferably two oligonucleotide
phosphorothioate regions. Such a chimeric oligonucleotide has a
structure that is "inverted" relative to traditional chimeric
oligonucleotides. In one particular embodiment, an inverted
chimeric oligonucleotide of the invention has substantially the
nucleotide sequence set forth in the Sequence Listing as SEQ ID
NO:1. In preferred embodiments, the oligonucleotide nonionic region
comprises about four to about 12 nucleotides joined to each other
by 5' to 3' nonionic linkages. In some embodiments, the nonionic
region contains alkylphosphonate and/or phosphoramidate and/or
phosphotriester internucleoside linkages. In one particular
embodiment, the oligonucleotide nonionic region comprises six
nucleotides. In some preferred embodiments, the oligonucleotide has
a nonionic region having from about six to about eight
methylphosphonate-linked nucleosides, flanked on either side by
phosphorothioate regions, each having from about six to about ten
phosphorothioate-linked nucleosides. In preferred embodiments, the
flanking region or regions are phosphorothioate nucleotides. In
some embodiments, the flanking region or regions have from about
four to about 24 nucleosides joined to each other by 5' to 3'
phosphorothioate linkages, and preferably from about six to about
16 such phosphorothioate-linked nucleosides. In preferred
embodiments, the phosphorothioate regions have from about five to
about 15 phosphorothioate-linked nucleosides. The phosphorothioate
linkages may be mixed R.sub.p and S.sub.p enantiomers, or they may
be stereoregular or substantially stereoregular in either R.sub.p
or S.sub.p form (see Iyer et al. (1995) Tetrahedron Asymmetry
6:1051-1054).
[0025] Another embodiment of this aspect of the invention is a
composition of matter for inhibiting the expression of protein
kinase A subunit RI.sub..alpha. with reduced side effects, the
composition comprising an inverted chimeric oligonucleotide
according to the invention.
[0026] Another aspect of the invention is a method of inhibiting
the proliferation of cancer cells in vitro. In this method, an
oligonucleotide of the invention is administered to the cells.
[0027] Yet another aspect is a therapeutic composition comprising
an oligonucleotide of the invention in a pharmaceutically
acceptable carrier.
[0028] A method of treating cancer in an afflicted subject with
reduced side effects is another aspect of the invention. This
method comprises administering a therapeutic composition of the
invention to the subject in which the protein kinase A subunit
RI.sub..alpha. gene is being over-expressed.
[0029] Those skilled in the art will recognize that the elements of
these preferred embodiments can be combined and the inventor does
contemplate such combination. For example, 2'-O-substituted
ribonucleotide regions may well include from one to all nonionic
internucleoside linkages. Alternatively, nonionic regions may have
from one to all 2'-O-substituted ribonucleotides. Moreover,
oligonucleotides according to the invention may contain
combinations of one or more 2'-O-substituted ribonucleotide region
and one or more nonionic region, either or both being flanked by
phosphorothioate regions. (See Nucleosides & Nucleotides
14:1031-1035 (1995) for relevant synthetic techniques.)
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The foregoing and other objects of the present invention,
the various features thereof, as well as the invention itself may
be more fully understood from the following description, when read
together with the accompanying drawings in which:
[0031] FIG. 1 is a graphic representation showing the effect of
modified oligonucleotides of the invention on tumor size in a mouse
relative to various controls.
DETAILED DESCRIPTION
[0032] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. The issued U.S. patents, allowed applications, published
foreign applications, and references cited herein are hereby
incorporated by reference.
[0033] Synthetic oligonucleotides of the hybrid, inverted hybrid,
and inverted chimeric oligonucleotides as described above.
[0034] Such synthetic hybrid, inverted hybrid, and inverted
chimeric oligonucleotides of the invention have a nucleotide
sequence complementary to a genomic region or an RNA molecule
transcribed therefrom encoding the RI.sub..alpha. subunit of PKA.
These oligonucleotides are about 15 to about 30 nucleotides in
length, preferably about 15 to 25 nucleotides in length, but most
preferably, are about 18 nucleotides long. The sequence of this
gene is known. Thus, an oligonucleotide of the invention can have
any nucleotide sequence complementary to any region of the gene.
Three non-limiting examples of an 18mer of the invention has the
sequence set forth below in TABLE 1 as SEQ ID NOS:1, 4, and 6.
1TABLE 1 Oligo SEQ ID # Sequence (5'.fwdarw.3') Type NO: 164 GCG
TGC CTC CTC ACT GGC Antisense 1 167 GCG CGC CTC CTC GCT GGC
Mismatched 2 Control 188 GCA TGC TTC CAC ACA GGC Mismatched 3
Control *** * * *** 165 GCG UGC CTC CTC ACU GGC Hybrid 4 *** * *
*** Mismatched 168 GCG CGC CTC CTC GCU GGC Hybrid 5 (Control) ***
** 166 GCG TGC CUC CUC ACT GGC Inverted 6 Hybrid *** ** Mismatched
169 GCG CGC CUC CUC GCT GGC Inverted 7 Hybrid (Control) *** **
Mismatched 189 GCA TGC AUC CGC ACA GGC Inverted 8 Hybrid (Control)
.cndot..cndot..cndot. .cndot..cndot..cndot. 190 GCG TGC CTC CTC ACT
GGC Inverted 1 Chimeric .cndot..cndot..cndot. .cndot..cndot..cndot.
Mismatched Inverted 191 GCG CGC CTC CTC GCT GGC Chimeric 2
(Control) X = mismatched bases * ribonucleotide .cndot.
methylphosphonate nucleotide
[0035] Oligonucleotides having greater than 18 oligonucleotides are
also contemplated by the invention. These oligonucleotides have up
to 25 additional nucleotides extending from the 3', or 5' terminus,
or from both the 3' and 5' termini of, for example, the 18mer with
SEQ ID NOS:1, 4, or 6, without diminishing the ability of these
oligonucleotides to down regulate RI.sub..alpha. gene expression.
Alternatively, other oligonucleotides of the invention may have
fewer nucleotides than, for example, oligonucleotides having SEQ ID
NOS:1, 4, or 6. Such shortened oligonucleotides maintain at least
the antisense activity of the parent oligonucleotide to
down-regulate the expression of the RI.sub..alpha. gene, or have
greater activity.
[0036] The oligonucleotides of the invention can be prepared by art
recognized methods. Oligonucleotides with phosphorothioate linkages
can be prepared manually or by an automated synthesizer and then
processed using methods well known in the field such as
phosphoramidite (reviewed in Agrawal et al. (1992) Trends
Biotechnol. 10:152-158, see, e.g., Agrawal et al. (1988) Proc.
Natl. Acad. Sci. (USA) 85:7079-7083) or H-phosphonate (see, e.g.,
Froehler (1986) Tetrahedron Lett. 27:5575-5578) chemistry. The
synthetic methods described in Bergot et al. (J. Chromatog. (1992)
559:35-42) can also be used. Examples of other chemical groups
include alkylphosphonates, phosphorodithioates,
alkylphosphonothioates, phosphoramidates, 2'-O-methyls, carbamates,
acetamidate, carboxymethyl esters, carbonates, and phosphate
triesters. Oligonucleotides with these linkages can be prepared
according to known methods (see, e.g., Goodchild (1990)
Bioconjugate Chem. 2:165-187; Agrawal et al. (Proc. Natl. Acad.
Sci. (USA) (1988) 85:7079-7083); Uhlmann et al. (Chem. Rev. (1990)
90:534-583; and Agrawal et al. (Trends Biotechnol. (1992)
10:152-158)).
[0037] Preferred hybrid, inverted hybrid, and inverted chimeric
oligonucleotides of the invention may have other modifications
which do not substantially affect their ability to specifically
down-regulate RI.sub..alpha. gene expression. These modifications
include those which are internal or are at the end(s) of the
oligonucleotide molecule and include additions to the molecule at
the internucleoside phosphate linkages, such as cholesteryl or
diamine compounds with varying numbers of carbon residues between
the two amino groups, and terminal ribose, deoxyribose and
phosphate modifications which cleave, or crosslink to the opposite
chains or to associated enzymes or other proteins which bind to the
RI.sub..alpha. nucleic acid. Examples of such oligonucleotides
include those with a modified base and/or sugar such as arabinose
instead of ribose, or a 3', 5'-substituted oligonucleotide having a
sugar which, at one or both its 3' and 5' positions is attached to
a chemical group other than a hydroxyl or phosphate group (at its
3' or 5' position). Other modified oligonucleotides are capped with
a nuclease resistance-conferring bulky substituent at their 3'
and/or 5' end(s), or have a substitution in one or both nonbridging
oxygens per nucleotide. Such modifications can be at some or all of
the internucleoside linkages, as well as at either or both ends of
the oligonucleotide and/or in the interior of the molecule
(reviewed in Agrawal et al. (1992) Trends Biotechnol.
10:152-158).
[0038] The invention also provides therapeutic compositions
suitable for treating undesirable, uncontrolled cell proliferation
or cancer comprising at least one oligonucleotide in accordance
with the invention, capable of specifically down-regulating
expression of the RI.sub..alpha. gene, and a pharmaceutically
acceptable carrier or diluent. It is preferred that an
oligonucleotide used in the therapeutic composition of the
invention be complementary to at least a portion of the
RI.sub..alpha. genomic region, gene, or RNA transcript thereof.
[0039] As used herein, a "pharmaceutically or physiologically
acceptable carrier" includes any and all solvents (including but
not limited to lactose), dispersion media, coatings, antibacterial
and antifungal agents, isotonic and absorption delaying agents and
the like. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions of the
invention is contemplated. Supplementary active ingredients can
also be incorporated into the compositions.
[0040] Several preferred therapeutic compositions of the invention
suitable for inhibiting cell proliferation in vitro or in vivo or
for treating cancer in humans in accordance with the methods of the
invention comprise about 25 to 75 mg of a lyophilized
oligonucleotide(s) having SEQ ID NOS: 1, 4, and/or 6 and 20-75 mg
lactose, USP, which is reconstituted with sterile normal saline to
the therapeutically effective dosages described herein.
[0041] The invention also provides methods for treating humans
suffering from disorders or diseases wherein the RI.sub..alpha.
gene is incorrectly or over-expressed. Such a disorder or disease
that could be treated using this method includes tumor-forming
cancers such as, but not limited to, human colon carcinoma, breast
carcinoma, gastric carcinoma, and neuroblastoma. In the method of
the invention, a therapeutically effective amount of a composition
of the invention is administered to the human. Such methods of
treatment according to the invention, may be administered in
conjunction with other therapeutic agents.
[0042] As used herein, the term "therapeutically effective amount"
means the total amount of each active component of the
pharmaceutical formulation or method that is sufficient to show a
meaningful subject or patient benefit, i.e., a reduction in tumor
growth or in the expression of proteins which cause or characterize
the cancer. When applied to an individual active ingredient,
administered alone, the term refers to that ingredient alone. When
applied to a combination, the term refers to combined amounts of
the active ingredients that result in the therapeutic effect,
whether administered in combination, serially or
simultaneously.
[0043] A "therapeutically effective manner" refers to a route,
duration, and frequency of administration of the pharmaceutical
formulation which ultimately results in meaningful patient benefit,
as described above. In some embodiments of the invention, the
pharmaceutical formulation is administered via injection,
sublingually, rectally, intradermally, orally, or enterally in
bolus, continuous, intermittent, or continuous, followed by
intermittent, regimens.
[0044] The therapeutically effective amount of synthetic
oligonucleotide in the pharmaceutical composition of the present
invention will depend upon the nature and severity of the condition
being treated, and on the nature of prior treatments which the
patient has undergone. Ultimately, the attending physician will
decide the amount of synthetic oligonucleotide with which to treat
each individual patient. Initially, the attending physician will
administer low doses of the synthetic oligonucleotide and observe
the patient's response. Larger doses of synthetic oligonucleotide
may be administered until the optimal therapeutic effect is
obtained for the patient, and at that point the dosage is not
increased further. It is contemplated that the dosages of the
pharmaceutical compositions administered in the method of the
present invention should contain about 0.1 to 5.0 mg/kg body weight
per day, and preferably 0.1 to 2.0 mg/kg body weight per day. When
administered systemically, the therapeutic composition is
preferably administered at a sufficient dosage to attain a blood
level of oligonucleotide from about 0.01 .mu.M to about 10 .mu.M.
Preferably, the concentration of oligonucleotide at the site of
aberrant gene expression should be from about 0.01 .mu.M to about
10 .mu.M, and most preferably from about 0.05 .mu.M to about 5
.mu.M. However, for localized administration, much lower
concentrations than this may be effective, and much higher
concentrations may be tolerated. It may be desirable to administer
simultaneously or sequentially a therapeutically effective amount
of one or more of the therapeutic compositions of the invention to
an individual as a single treatment episode.
[0045] Administration of pharmaceutical compositions in accordance
with the invention or to practice the method of the present
invention can be carried out in a variety of conventional ways,
such as by oral ingestion, enteral, rectal, or transdermal
administration, inhalation, sublingual administration, or
cutaneous, subcutaneous, intramuscular, intraocular,
intraperitoneal, or intravenous injection, or any other route of
administration known in the art for administrating therapeutic
agents.
[0046] When the composition is to be administered orally,
sublingually, or by any non-injectable route, the therapeutic
formulation will preferably include a physiologically acceptable
carrier, such as an inert diluent or an assimilable edible carrier
with which the composition is administered. Suitable formulations
that include pharmaceutically acceptable excipients for introducing
compounds to the bloodstream by other than injection routes can be
found in Remington's Pharmaceutical Sciences (18th ed.) (Genarro,
ed. (1990) Mack Publishing Co., Easton, Pa.). The oligonucleotide
and other ingredients may be enclosed in a hard or soft shell
gelatin capsule, compressed into tablets, or incorporated directly
into the individual's diet. The therapeutic compositions may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. When the therapeutic composition is
administered orally, it may be mixed with other food forms and
pharmaceutically acceptable flavor enhancers. When the therapeutic
composition is administered enterally, they may be introduced in a
solid, semi-solid, suspension, or emulsion form and may be
compounded with any number of well-known, pharmaceutically
acceptable additives. Sustained release oral delivery systems
and/or enteric coatings for orally administered dosage forms are
also contemplated such as those described in U.S. Pat. Nos.
4,704,295, 4,556,552, 4,309,404, and 4,309,406.
[0047] When a therapeutically effective amount of composition of
the invention is administered by injection, the synthetic
oligonucleotide will preferably be in the form of a pyrogen-free,
parenterally-acceptable- , aqueous solution. The preparation of
such parenterally-acceptable solutions, having due regard to ph,
isotonicity, stability, and the like, is within the skill in the
art. A preferred pharmaceutical composition for injection should
contain, in addition to the synthetic oligonucleotide, an isotonic
vehicle such as Sodium Chloride Injection, Ringer's Injection,
Dextrose Injection, Dextrose and Sodium Chloride Injection,
Lactated Ringer's Injection, or other vehicle as known in the art.
The pharmaceutical composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of skill in the art.
[0048] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile. It must be
stable under the conditions of manufacture and storage and may be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents.
Prolonged absorption of the injectable therapeutic agents can be
brought about by the use of compositions of agents delaying
absorption. Sterile injectable solutions are prepared by
incorporating the oligonucleotide in the required amount in the
appropriate solvent, followed by filtered sterilization.
[0049] The pharmaceutical formulation can be administered in bolus,
continuous, or intermittent dosages, or in a combination of
continuous and intermittent dosages, as determined by the physician
and the degree and/or stage of illness of the patient. The duration
of therapy using the pharmaceutical composition of the present
invention will vary, depending on the unique characteristics of the
oligonucleotide and the particular therapeutic effect to be
achieved, the limitations inherent in the art of preparing such a
therapeutic formulation for the treatment of humans, the severity
of the disease being treated and the condition and potential
idiosyncratic response of each individual patient. Ultimately the
attending physician will decide on the appropriate duration of
intravenous therapy using the pharmaceutical composition of the
present invention.
[0050] Compositions of the invention are useful for inhibiting or
reducing the proliferation of cancer or tumor cells in vitro. A
synthetic oligonucleotide of the invention is administered to the
cells in an amount sufficient to enable the binding of the
oligonucleotide to a complementary genomic region or RNA molecule
transcribed therefrom encoding the RI.sub..alpha. subunit. In this
way, expression of PKA is decreased, thus inhibiting or reducing
cell proliferation.
[0051] Compositions of the invention are also useful for treating
cancer or uncontrolled cell proliferation in humans. In this
method, a therapeutic formulation including an antisense
oligonucleotide of the invention is provided in a physiologically
acceptable carrier. The individual is then treated with the
therapeutic formulation in an amount sufficient to enable the
binding of the oligonucleotide to the PKA RI.sub..alpha. genomic
region or RNA molecule transcribed therefrom in the infected cells.
In this way, the binding of the oligonucleotide inhibits or
down-regulates RI.sub..alpha. expression and hence the activity of
PKA.
[0052] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of at least one or
more therapeutic compositions of the invention is administered to a
subject afflicted with a cancer. An anticancer response showing a
decrease in tumor growth or size or a decrease in RI.sub..alpha.
expression is considered to be a positive indication of the ability
of the method and pharmaceutical formulation to inhibit or reduce
cell growth and thus, to treat cancer in humans.
[0053] At least one therapeutic composition of the invention may be
administered in accordance with the method of the invention either
alone or in combination with other known therapies for cancer such
as cisplatin, carboplatin, paclitaxel, tamoxifen, taxol, interferon
.alpha. and doxorubicin. When co-administered with one or more
other therapies, the compositions of the invention may be
administered either simultaneously with the other treatment(s), or
sequentially. If administered sequentially, the attending physician
will decide on the appropriate sequence of administering the
compositions of the invention in combination with the other
therapy.
[0054] The following examples illustrate the preferred modes of
making and practicing the present invention, but are not meant to
limit the scope of the invention since alternative methods may be
utilized to obtain similar results.
EXAMPLE 1
Synthesis, Deprotection, and Purification of Oligonucleotides
[0055] Oligonucleotides were synthesized using standard
phosphoramidite chemistry (Beaucage (1993) Meth. Mol. Biol.
20:33-61) on an automated DNA synthesizer (Model 8700, Biosearch,
Bedford, Mass.) using a beta-cyanoethyl phosphoramidate
approach.
[0056] Oligonucleotide phosphorothioates were synthesized using an
automated DNA synthesizer (Model 8700, Biosearch, Bedford, Mass.)
using a beta-cyanoethyl phosphoramidate approach on a 10 micromole
scale. To generate the phosphorothioate linkages, the intermediate
phosphite linkage obtained after each coupling was oxidized using
3H, 1,2-benzodithiole-3H-one-1,1-dioxide (see Beaucage, in
Protocols for Oligonucleotides and Analogs: Synthesis and
Properties, Agrawal (ed.), (1993) Humana Press, Totowa, N.J., pp.
33-62). Similar synthesis was carried out to generate
phosphodiester linkages, except that a standard oxidation was
carried out using standard iodine reagent. Synthesis of inverted
chimeric oligonucleotide was carried out in the same manner, except
that methylphosphonate linkages were assembled using nucleoside
methylphosphonamidite (Glen Research, Sterling, Va.), followed by
oxidation with 0.1 M iodine in tetrahydrofuran/2,6-lutidine/water
(75:25:0.25) (see Agrawal & Goodchild (1987) Tet. Lett.
28:3539-3542). Hybrids and inverted hybrid oligonucleotides were
synthesized similarly, except that the segment containing
2'-O-methylribonucleotides was assembled using
2'-O-methylribonucleoside phosphorarnidite, followed by oxidation
to a phosphorothioate or phosphodiester linkage as described above.
Deprotection and purification of oligonucleotides was carried out
according to standard procedures, (see Padmapriya et al. (1994)
Antisense Res. & Dev. 4:185-199), except for oligonucleotides
containing methylphosphonate-containing regions. For those
oligonucleotides, the CPG-bound oligonucleotide was treated with
concentrated ammonium hydroxide for 1 hour at room temperature, and
the supernatant was removed and evaporated to obtain a pale yellow
residue, which was then treated with a mixture of
ethylenediamine/ethanol (1:1 v/v) for 6 hours at room temperature
and dried again under reduced pressure.
EXAMPLE 2
In Vitro Complement Activation Studies
[0057] To determine the relative effect of inverted hybrid or
inverted chimeric structure on oligonucleotide-mediated depletion
of complement, the following experiments were performed. Venous
blood was collected from healthy adult human volunteers. Serum was
prepared for hemolytic complement assay by collecting blood into
vacutainers (Becton Dickinson #6430 Franklin Lakes, N.J.) without
commercial additives. Blood was allowed to clot at room temperature
for 30 minutes, chilled on ice for 15 minutes, then centrifuged at
4.degree. C. to separate serum. Harvested serum was kept on ice for
same day assay or, alternatively, stored at -70.degree. C. Buffer,
or an oligonucleotide sample was then incubated with the serum. The
oligonucleotides tested were 25mer oligonucleotide phosphodiesters
or phosphorothioates, 25mer hybrid oligonucleotides, 25mer inverted
hybrid oligonucleotides, 25mer chimeric oligonucleotides, and 25mer
inverted chimeric oligonucleotides. Representative hybrid
oligonucleotides were composed of seven to 13 2'-O-methyl
ribonucleotides flanked by two regions of six to nine
deoxyribonucleotides each. Representative 25mer inverted hybrid
oligonucleotides were composed of 17 deoxyribonucleotides flanked
by two regions of four ribonucleotides each. Representative 25mer
chimeric oligonucleotides were composed of six methylphosphonate
deoxyribonucleotides and 19 phosphorothioate deoxyribonucleotides.
Representative inverted chimeric oligonucleotides were composed of
from 16 to 17 phosphorothioate deoxyribonucleotides flanked by
regions of from two to seven methylphosphonate
deoxyribonucleotides, or from six to eight methylphosphonate
deoxyribonucleotides flanked by nine to ten phosphorothioate
deoxyribonucleotides, or two phosphorothioate regions ranging from
two to 12 oligonucleotides, flanked by three phosphorothioate
regions ranging in size from two to six nucleotides in length. A
standard CH50 assay (See Kabat and Mayer (eds), Experimental
ImmunoChemistry, 2d Ed., Springfield, Ill., CC Thomas, p. 125) for
complement-mediated lysis of sheep red blood cells (Colorado Serum
Co.) sensitized with anti-sheep red blood cell antibody (hemolysin,
Diamedix, Miami, Fla.) was performed, using duplicate
determinations of at least five dilutions of each test serum, then
hemoglobin release into cell-free supernates was measured
spectrophotometrically at 541 nm.
EXAMPLE 3
In Vitro Mitogenicity Studies
[0058] To determine the relative effect of inverted hybrid or
inverted chimeric structure on oligonucleotide-mediated
mitogenicity, the following experiments were performed. Spleen was
taken from a male CD1 mouse (4-5 weeks, 20-22 g; Charles River,
Wilmington, Mass.). Single cell suspensions were prepared by gently
mincing with frosted edges of glass slides. Cells were then
cultured in RPMI complete media (RPMI media supplemented with 10%
fetal bovine serum (FBS), 50 micromolar 2-mercaptoethanol (2-ME),
100 U/ml penicillin, 100 micrograms/ml streptomycin, 2 mM
L-glutamine). To minimize oligonucleotide degradation, FBS was
first heated for 30 minutes at 65.degree. C.
(phosphodiester-containing oligonucleotides) or 56.degree. C. (all
other oligonucleotides). Cells were plated in 96 well dishes at
100,000 cells per well (volume of 100 microliters/well). One type
of each oligonucleotide described in Example 2 above in 10
microliters TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) was added
to each well. After 44 hours of culturing at 37.degree. C., one
microcurie tritiated thymidine (Amersham, Arlington Heights, Ill.)
was added in 20 microliters RPMI media for a 4 hour pulse
labelling. The cells were then harvested in an automatic cell
harvester (Skatron, Sterling, Va.) and the filters were assessed
using a scintillation counter. In control experiments for
mitogenicity, cells were treated identically, except that either
media (negative control) or concanavalin A (positive control) was
added to the cells in place of the oligonucleotides.
[0059] All of the inverted hybrid oligonucleotides proved to be
less immunogenic than phosphorothioate oligonucleotides. Inverted
hybrid oligonucleotides having phosphodiester linkages in the
2'-O-methyl region appeared to be slightly less immunogenic than
those containing phosphorothioate linkages in that region. No
significant difference in mitogenicity was observed when the
2'-O-methyl ribonucleotide region was pared down from 13 to 11 or
to 9 nucleotides. Inverted chimeric oligonucleotides were also
generally less mitogenic than phosphorothioate oligonucleotides. In
addition, these oligonucleotides appeared to be less mitogenic than
traditional chimeric oligonucleotides, at least in cases in which
the traditional chimeric oligonucleotides had significant numbers
of methylphosphonate linkages near the 3' end. Increasing the
number of methylphosphonate linkers in the middle of the
oligonucleotide from 5 to 6 or 7 did not appear to have a
significant effect on mitogenicity. These results indicate that
incorporation of inverted hybrid or inverted chimeric structure
into an oligonucleotide can reduce its mitogenicity.
EXAMPLE 4
In Vitro Studies
[0060] To determine the relative effect of inverted hybrid or
inverted chimeric structure on oligonucleotide-induced
mitogenicity, the following experiments were performed. Venous
blood was collected from healthy adult human volunteers. Plasma for
clotting time assay was prepared by collecting blood into
siliconized vacutainers with sodium citrate (Becton Dickinson
#367705), followed by two centrifugations at 4.degree. C. to
prepare platelet-poor plasma. Plasma aliquots were kept on ice,
spiked with various test oligonucleotides described in Example 2
above, and either tested immediately or quickly frozen on dry ice
for subsequent storage at -20.degree. C. prior to coagulation
assay. Activated partial thromboplastin time (aPTT) was performed
in duplicate on an Electra 1000C (Medical Laboratory Automation,
Mount Vernon, N.Y.) according to the manufacturer's recommended
procedures, using Actin FSL (Baxter Dade, Miami, Fla.) and calcium
to initiate clot formation, which was measured photometrically.
Prolongation of aPTT was taken as an indication of clotting
inhibition side effect produced by the oligonucleotide.
[0061] Traditional phosphorothioate oligonucleotides produced the
greatest prolongation of aPTT, of all of the oligonucleotides
tested. Traditional hybrid oligonucleotides produced somewhat
reduced prolongation of aPTT. In comparison with traditional
phosphorothioate or traditional hybrid oligonucleotides, all of the
inverted hybrid oligonucleotides tested produced significantly
reduced prolongation of aPTT. Inverted hybrid oligonucleotides
having phosphodiester linkages in the 2'-O-substituted
ribonucleotide region had the greatest reduction in this side
effect, with one such oligonucleotide having a 2'-O-methyl RNA
phosphodiester region of 13 nucleotides showing very little
prolongation of aPTT, even at oligonucleotide concentrations as
high as 100 micrograms/ml. Traditional chimeric oligonucleotides
produce much less prolongation of aPTT than do traditional
phosphorothioate oligonucleotides. Generally, inverted chimeric
oligonucleotides retain this characteristic. At least one inverted
chimeric oligonucleotide, having a methylphosphonate region of
seven nucleotides flanked by phosphorothioate regions of nine
nucleotides, gave better results in this assay than the traditional
chimeric oligonucleotides at all but the highest oligonucleotide
concentrations tested. These results indicate that inverted hybrid
and inverted chimeric oligonucleotides may provide advantages in
reducing the side effect of clotting inhibition when they are
administered to modulate gene expression in vivo.
EXAMPLE 5
In Vivo Complement Activation Studies
[0062] Rhesus monkeys (4-9 kg body weight) are acclimatized to
laboratory conditions for at least 7 days prior to the study. On
the day of the study, each animal is lightly sedated with
ketamine-HCl (10 mg/kg) and diazepam (0.5 mg/kg). Surgical level
anesthesia is induced and maintained by continuous ketamine
intravenous drip throughout the procedure. The oligonucleotides
described in Example 2 above are dissolved in normal saline and
infused intravenously via a cephalic vein catheter, using a
programmable infusion pump at a delivery rate of 0.42 mg/minute.
For each oligonucleotide, doses of 0, 0.5, 1, 2, 5 and 10 mg/kg are
administered to two animals each over a 10 minute infusion period.
Arterial blood samples are collected 10 minutes prior to
oligonucleotide administration and 2, 5, 10, 20, 40 and 60 minutes
after the start of the infusion, as well as 24 hours later. Serum
is used for determining complement CH50, using the conventional
complement-dependent lysis of sheep erythrocyte procedure (see
Kabat and Mayer, 1961, supra). At the highest dose,
phosphorothioate oligonucleotide causes a decrease in serum
complement CH50 beginning within 5 minutes of the start of
infusion. Inverted hybrid and chimeric oligonucleotides are
expected to show a much reduced or undetectable decrease in serum
complement CH50 under these conditions.
EXAMPLE 6
In Vivo Mitogenicity Studies
[0063] CD1 mice are injected intraperitoneally with a dose of 50
mg/kg body weight of oligonucleotide described in Example 2 above.
Forty-eight hours later, the animals are euthanized and the spleens
are removed and weighed. Animals treated with inverted hybrid or
inverted hybrid oligonucleotides are expected to show no
significant increase in spleen weight, while those treated with
oligonucleotide phosphorothioates are expected to show modest
increases in spleen weight.
EXAMPLE 7
In Vivo Clotting Studies
[0064] Rhesus monkeys are treated as in Example 5. From the whole
blood samples taken, plasma for clotting assay is prepared, and the
assay performed, as described in Example 4. It is expected that
prolongation of aPTT will be substantially reduced for both
inverted hybrid oligonucleotides and for inverted chimeric
oligonucleotide, relative to traditional oligonucleotide
phosphorothioates.
EXAMPLE 8
RNase H Activity Studies
[0065] To determine the ability of inverted hybrid oligonucleotides
and inverted chimeric oligonucleotides to activate RNase H when
bound to a complementary RNA molecule, the following experiments
were performed. Each type of oligonucleotide described in Example 2
above was incubated together with a molar equivalent quantity of
complimentary oligoribonucleotide (0.266 micromolar concentration
of each), in a cuvette containing a final volume of 1 ml RNase H
buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl.sub.2, 0.1 M KCl, 2%
glycerol, 0.1 mM DTT). The samples were heated to 95.degree. C.,
then cooled gradually to room temperature to allow annealing to
form duplexes. Annealed duplexes were incubated for 10 minutes at
37.degree. C., then 5 units RNase H was added and data collection
commenced over a three hour period. Data was collected using a
spectrophotometer (GBC 920, GBC Scientific Equipment, Victoria,
Australia) at 259 nm. RNase H degradation was determined by
hyperchromic shift.
[0066] As expected, phosphodiester oligonucleotides behaved as very
good co-substrates for RNase H-mediated degradation of RNA, with a
degradative half-life of 8.8 seconds. Phosphorothioate
oligonucleotides produced an increased half-life of 22.4 seconds.
Introduction of a 2'-O-methyl ribonucleotide segment at either end
of the oligonucleotide further worsened RNase H activity
(half-life=32.7 seconds). In contrast, introducing a 2'-O-methyl
segment into the middle of the oligonucleotide (inverted hybrid
structure) always resulted in improved RNase H-mediated
degradation. When a region of 13 2'-O-methylribonucleoside
phosphodiesters was flanked on both sides by phosphorothioate DNA,
the best RNase H activity was observed, with a half-life of 7.9
seconds. Introduction of large blocks of methylphosphonate-linked
nucleosides at the 3' end of the oligonucleotide either had no
effect or caused further deterioration of RNase H activity even
when in a chimeric configuration. Introduction of methylphosphonate
linked nucleosides at the 5' end, however, improved RNase H
activity, particularly in a chimeric configuration with a single
methylphosphonate linker at the 3' end (best half-life=8.1
seconds). All inverted chimeric oligonucleotides with
methylphosphonate core regions flanked by phosphorothioate regions
gave good RNase results, with a half-life range of 9.3 to 14.4
seconds. These results indicate that the introduction of inverted
hybrid or inverted chimeric structure into
phosphorothioate-containing oligonucleotides can restore some or
all of the ability of the oligonucleotide to act as a co-substrate
for RNase H, a potentially important attribute for an effective
antisense agent.
EXAMPLE 9
Melting Temperature Studies
[0067] To determine the effect of inverted hybrid or inverted
chimeric structure on stability of the duplex formed between an
antisense oligonucleotide and a target molecule, the following
experiments were performed. Thermal melting (Tm) data were
collected using a spectrophotometer (GBC 920, GBC Scientific
Equipment, Victoria, Australia), which has six 10 mm cuvettes
mounted in a dual carousel. In the Tm experiments, the temperature
was directed and controlled through a peltier effect temperature
controller by a computer, using software provided by GBC, according
to the manufacturer's directions. Tm data were analyzed by both the
first derivative method and the mid-point method, as performed by
the software. Tm experiments were performed in a buffer containing
10 mM PIPES, pH 7.0, 1 mM EDTA, 1 M NaCl. A refrigerated bath (VWR
1166, VWR, Boston, Mass.) was connected to the peltier-effect
temperature controller to absorb the heat. Oligonucleotide strand
concentration was determined using absorbance values at 260 nm,
taking into account extinction coefficients.
EXAMPLE 10
Tumor Growth and Antisense Treatment
[0068] LS-174T human colon carcinoma cells (1.times.10.sup.6 cells)
were inoculated subcutaneously (s.c.) into the left flank of
athymic mice. A single dose of RI.sub..alpha. antisense hybrid
(Oligo 165, SEQ ID NO:4), inverted hybrid (Oligo 166, SEQ ID NO:6),
or antisense (Oligo 164, SEQ ID NO:1) oligonucleotides or control
oligonucleotide (Oligo 169, SEQ ID NO:7); Oligo 168 (SEQ ID NO:5);
Oligo 188, (SEQ ID NO:3) as shown in Table 1 (1 mg per 0.1 ml
saline per mouse), or saline (0.1 ml per mouse), was injected s.c.
into the right flank of mice when tumor size reached 80 to 100 mg,
about 1 week after cell inoculation. Tumor volumes were obtained
from daily measurement of the longest and shortest diameters and
calculation by the formula, 4/3.pi.r.sup.3 where
r=(length+width)/4. At each indicated time, two animals from the
control and antisense-treated groups were killed, and tumors were
removed and weighed. The results are shown in FIG. 1. These results
show that the size of the tumor in the animal treated with the
inverted hybrid oligonucleotide 166 having SEQ ID NO:6 was
surprisingly smaller from three days after injection onward than
the phosphorothioate oligonucleotide 164 having SEQ ID NO:1. That
this effect was sequence-specific is also demonstrated in FIG. 1:
control oligonucleotide 168 (SEQ ID NO:5) has little ability to
keep tumor size at a minimum relative to the hybrid and inverted
hybrid oligonucleotides.
EXAMPLE 11
Photoaffinity Labelling and Immunoprecipitation of RI.sub..alpha.
Subunits
[0069] The tumors are homogenized with a Teflon/glass homogenizer
in ice-cold buffer 10 (Tris-HCl, pH 7.4, 20 mM; NaCl, 100 mM;
NP-40, 1%; sodium deoxycholate, 0.5%; MgCl.sub.2, 5 mM; pepstatin,
0.1 mM; antipain, 0.1 mM; chymostatin, 0.1 mM; leupeptin, 0.2 mM;
aprotinin, 0.4 mg/ml; and soybean trypsin inhibitor, 0.5 mg/ml;
filtered through a 0.45-.mu.m pore size membrane), and centrifuged
for 5 min in an Eppendorf microfuge at 4.degree. C. The
supernatants are used as tumor extracts.
[0070] The amount of PKA RI.sub..alpha. subunits in tumors is
determined by photoaffinity labelling with 8-N.sub.3-[.sup.32P]cAMP
followed by immunoprecipitation with RI.sub..alpha. antibodies as
described by Tortora et al. (Proc. Natl. Acad. Sci. (USA) (1990)
87:705-708). The photoactivated incorporation of
8-N.sub.3-[.sup.32P]cAMP (60.0 Ci/m-mol), and the
immunoprecipitation using the anti-RI.sub..alpha. or
anti-RII.sub..beta. antiserum and protein A Sepharose and SDS-PAGE
of solubilized antigen-antibody complex follows the method
previously described (Tortora et al. (1990) Proc. Natl. Acad. Sci.
(USA) 87:705-708; Ekanger et al. (1985) J. Biol. Chem.
260:3393-3401). It is expected that the amount of RI.sub..alpha. in
tumors treated with hybrid, inverted hybrid, and inverted chimeric
oligonucleotides of the invention will be reduced compared with the
amount in tumors treated with mismatch, straight phosphorothioate,
or straight phosphodiester oligonucleotide controls, saline, or
other controls.
EXAMPLE 12
cAMP-Dependent Protein Kinase Assays
[0071] Extracts (10 mg protein) of tumors from antisense-, control
antisense-, or saline-treated animals are loaded onto DEAE
cellulose columns (1.times.10 cm) and fractionated with a linear
salt gradient (Rohlff et al. (1993) J. Biol. Chem. 268:5774-5782).
PKA activity is determined in the absence or presence of 5 .mu.M
cAMP as described below (Rohlff et al. (1993) J. Biol. Chem.
268:5774-5782). cAMP-binding activity is measured by the method
described previously and expressed as the specific binding
(Tagliaferri et al. (1988) J. Biol. Chem. 263:409-416).
[0072] After two washes with Dulbecco's phosphate-buffered saline,
cell pellets (2.times.10.sup.6 cells) are lysed in 0.5 ml of 20 mM
Tris (pH 7.5), 0.1 mM sodium EDTA, 1 mM dithiothreitol, 0.1 mM
pepstatin, 0.1 mM antipain, 0.1 mM chymostatin, 0.2 mM leupeptin,
0.4 mg/ml aprotinin, and 0.5 mg/ml soybean trypsin inhibitor, using
100 strokes of a Dounce homogenizer. After centrifugation
(Eppendorf 5412) for 5 min, the supernatants are adjusted to 0.7 mg
protein/ml and assayed (Uhler et al. (1987) J. Biol. Chem.
262:15202-15207) immediately. Assays (40 .mu.L total volume) are
performed for 10 min at 300.degree. C. and contained 200 .mu.M ATP,
2.7.times.10.sup.6 cpm .gamma.[.sup.32P]ATP, 20 mM MgCl.sub.2, 100
.mu.M Kemptide (Sigma K-1127) (Kemp et al. (1977) J. Biol. Chem.
252:4888-4894), 40 mM Tris (pH 7.5), .+-.100 .mu.M protein kinase
inhibitor (Sigma P-3294) (Cheng et al. (1985) Biochem. J.
231:655-661), .+-.8 .mu.M cAMP and 7 .mu.g of cell extract. The
phosphorylation of Kemptide is determined by spotting 20 .mu.l of
incubation mixture on phosphocellulose filters (Whatman, P81) and
washing in phosphoric acid as described (Roskoski (1983) Methods
Enzymol. 99:3-6). Radioactivity is measured by liquid scintillation
using Econofluor-2 (NEN Research Products NEF-969). It is expected
that PKA and cAMP binding activity will be reduced in extracts of
tumors treated with the hybrid, inverted hybrid, and inverted
chimeric oligonucleotides of the invention.
EQUIVALENTS
[0073] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific substances and procedures described
herein. Such equivalents are considered to be within the scope of
this invention, and are covered by the following claims.
Sequence CWU 1
1
8 1 18 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 1 gcgtgcctcc tcactggc 18 2 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 2 gcgcgcctcc tcgctggc 18 3 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 3 gcatgcttcc acacaggc 18 4 18 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 4 gcgugcctcc tcacuggc 18 5 18 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 5 gcgcgcctcc tcgcuggc 18 6 18 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 6 gcgtgccucc ucactggc 18 7 18 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 7 gcgcgccucc ucgctggc 18 8 18 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 8 gcatgcaucc gcacaggc 18
* * * * *