U.S. patent application number 11/502008 was filed with the patent office on 2007-11-22 for modulation of telomere length by oligonucleotides having a g-core sequence.
Invention is credited to C. Frank Bennett, David J. Ecker, Timothy Vickers, Jacqueline R. Wyatt.
Application Number | 20070270363 11/502008 |
Document ID | / |
Family ID | 25495059 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070270363 |
Kind Code |
A1 |
Bennett; C. Frank ; et
al. |
November 22, 2007 |
Modulation of telomere length by oligonucleotides having a G-core
sequence
Abstract
Modified oligonucleotides having a GGG motif sequence and a
sufficient number of flanking nucleotides to modulate the telomere
length of a chromosome are provided. Methods of modulating telomere
length of a mammalian chromosome in vitro and in vivo are also
provided, as are methods for inhibiting the division of a malignant
mammalian cell and for modulating the effects of cellular
aging.
Inventors: |
Bennett; C. Frank;
(Carlsbad, CA) ; Ecker; David J.; (Encinitas,
CA) ; Vickers; Timothy; (Oceanside, CA) ;
Wyatt; Jacqueline R.; (Sundance, WY) |
Correspondence
Address: |
ISIS PHARMACEUTICALS INC
1896 RUTHERFORD RD.
CARLSBAD
CA
92008
US
|
Family ID: |
25495059 |
Appl. No.: |
11/502008 |
Filed: |
August 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11436901 |
May 17, 2006 |
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11502008 |
Aug 9, 2006 |
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10038335 |
Jan 2, 2002 |
7067497 |
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11436901 |
May 17, 2006 |
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09299058 |
Apr 23, 1999 |
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10038335 |
Jan 2, 2002 |
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08403888 |
Jun 12, 1995 |
5952490 |
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PCT/US93/09297 |
Sep 29, 1993 |
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09299058 |
Apr 23, 1999 |
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07954185 |
Sep 29, 1992 |
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08403888 |
Jun 12, 1995 |
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Current U.S.
Class: |
514/44R ;
435/375 |
Current CPC
Class: |
C12N 15/1133 20130101;
A61P 31/22 20180101; A61P 31/18 20180101; A61P 35/00 20180101; C12N
2310/151 20130101; C07H 21/00 20130101; C12N 2310/341 20130101;
C12N 15/117 20130101; A61P 31/12 20180101; C12N 15/115 20130101;
A61K 31/70 20130101; A61K 38/00 20130101; C12Y 207/07049 20130101;
C12N 2310/315 20130101; C12Q 1/701 20130101; C12N 15/1137 20130101;
A61P 31/16 20180101; C12Y 301/01004 20130101; C12N 2310/18
20130101; C12N 2310/346 20130101; C12N 2310/321 20130101; C12N
2310/321 20130101; C12N 2310/3521 20130101 |
Class at
Publication: |
514/044 ;
435/375 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1. A method of modulating telomere length of a mammalian chromosome
comprising contacting a mammalian chromosome with a chemically
modified oligonucleotide having no more than 27 nucleic acid base
units, said oligonucleotide having the sequence
(N.sub.XG.sub.3-4).sub.QN.sub.X wherein each X is independently 1
to 8 and Q is 1 to 6, wherein said oligonucleotide modulates
mammalian telomere length.
2. The method of claim 1 which is carried out in vitro.
3. The method of claim 1 which is carried out in vivo.
4. The method of claim 1 wherein the oligonucleotide chemical
modifications are selected from the group consisting of a modified
internucleoside linkage, a modified sugar and a modified base.
5. A method for inhibiting the division of a malignant mammalian
cell comprising contacting said malignant mammalian cell with a
chemically modified oligonucleotide having no more than 27 nucleic
acid base units, said oligonucleotide having the sequence
(N.sub.XG.sub.3-4).sub.QN.sub.X wherein each X is independently 1
to 8 and Q is 1 to 6, wherein said oligonucleotide modulates
mammalian telomere length.
6. The method of claim 5 which is carried out in vitro.
7. The method of claim 5 which is carried out in vivo.
8. The method of claim 5 wherein the oligonucleotide chemical
modifications are selected from the group consisting of a modified
internucleoside linkage, a modified sugar and a modified base.
9. A method for inhibiting the division of a malignant mammalian
cell comprising contacting said malignant mammalian cell with a
chemically modified oligonucleotide having no more than 27 nucleic
acid base units, said oligonucleotide comprising the sequence
(N.sub.xG.sub.3-4).sub.QN.sub.x wherein X is 1 to 8 and Q is 1 to
6, wherein said oligonucleotide modulates mammalian telomere
length.
10. The method of claim 9 which is carried out in vitro.
11. The method of claim 9 which is carried out in vivo.
12. The method of claim 9 wherein the oligonucleotide chemical
modifications are selected from the group consisting of a modified
internucleoside linkage, a modified sugar and a modified base.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation in part of U.S.
application Ser. No. 11/436,901 filed May 17, 2006, which is a
continuation-in-part of U.S. application Ser. No. 10/038,335, filed
Jan. 2, 2002, which is U.S. Pat. No. 7,067,497 and which is a
continuation-in-part of U.S. application Ser. No. 09/299,058, filed
Apr. 23, 1999, now abandoned, which is a continuation of U.S.
application Ser. No. 08/403,888 filed Jun. 12, 1995, which is U.S.
Pat. No. 5,952,490, the U.S. national phase of PCT Application
Serial No. PCT/US93/09297 filed Sep. 29, 1993, which is a
continuation-in-part of U.S. application Ser. No. 07/954,185 filed
Sep. 29, 1992, now abandoned, each of which is incorporated herein
by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the design and synthesis of
oligonucleotides which can be used to modulate telomere length in
vivo or in vitro. These compounds can be used prophylactically or
therapeutically for diseases associated with abnormal telomere
length, such as aging and hyperproliferative conditions, e.g.,
cancer. Methods for the treatment of cancer and to retard aging are
also contemplated by this invention.
BACKGROUND OF THE INVENTION
Modulation of Telomere Length
[0003] It has been recognized that telomeres, long chains of
repeated nucleotides located at the tip of each chromosome, play a
role in the protection and organization of the chromosome. In human
cells, the sequence TTAGGG is repeated hundreds to thousands of
times at both ends of every chromosome, depending on cell type and
age. Harley, C. B. et al., Nature, 1990, 345, 458-460; Hastie, N.
D. et al., Nature, 1990, 346,866-868. Telomeres also appear to have
a role in cell aging which has broad implications for the study of
aging and cell immortality that is manifested by cancerous
cells.
[0004] Researchers have determined that telomere length is reduced
whenever a cell divides and it has been suggested that telomere
length controls the number of divisions before a cell's innate
lifespan is spent. Harley, C. B. et al., Nature, 1990, 345,
458-460; Hastie, N. D. et al., Nature, 1990, 346,866-868. For
example, normal human cells divide between 70-100 times and appear
to lose about 50 nucleotides of their telomeres with each division.
Some researchers have suggested that there is a strong correlation
between telomere length and the aging of the entire human being.
Greider, C. W., Curr. Opinion Cell Biol., 1991, 3, 444-451. Other
studies have shown that telomeres undergo a dramatic transformation
during the genesis and progression of cancer. Hastie, N. D. et al.,
Nature 1990, 346, 866-868. For example, it has been reported that
when a cell becomes malignant, the telomeres become shortened with
each cell division. Hastie, N. D. et al., Nature 1990, 346,
866-868. Experiments by Greider and Bacchetti and their colleagues
have shown that at a very advanced and aggressive stage of tumor
development, telomere shrinking may cease or even reverse. Counter,
C. M. et al., EMBO J. 1992, 11, 1921-1929. It has been suggested,
therefore, that telomere blockers may be useful for cancer therapy.
In vitro studies have also shown that telomere length can be
altered by electroporation of linearized vector containing human
chromosome fragments into hybrid human-hamster cell lines.
Chromosome fragments consisted of approximately 500 base pairs of
the human telomeric repeat sequence TTAGGG and related variants
such as TTGGGG, along with adjacent GC-rich repetitive sequences.
Farr, C. et al., Proc. Natl. Acad. Sci. USA 1992, 88, 7006-7010.
While this research suggests that telomere length affects cell
division, no effective method for control of the aging process or
cancer has been discovered. Therefore, there is an unmet need to
identify effective modulators of telomere length.
[0005] Guanosine nucleotides, both as mononucleotides and in
oligonucleotides or polynucleotides, are able to form arrays known
as guanine quartets or G-quartets. For review, see Williamson, J.
R., (1993) Curr. Opin. Struct. Biol. 3:357-362. G-quartets have
been known for years, although interest has increased in the past
several years because of their possible role in telomere structure
and function.
[0006] In addition to their natural role (in telomeres, for
example, though there may be others), oligonucleotides which have a
GGGG motif or one or more GGG motifs are useful for inhibiting
viral gene expression and viral growth and for inhibiting PLA.sub.2
enzyme activity and have long been believed to be useful as
modulators of telomere length. Chemical modification of the
oligonucleotides for such use is desirable and, in some cases,
necessary for maximum activity.
[0007] It has now been clearly demonstrated both in vitro and in
vivo that oligonucleotides containing a GGG motif are capable of
modulating telomere length on mammalian chromosomes. Herbert et
al., 1999, Proceedings Natl. Acad. Sci., USA, 96, 14276-14281.
SUMMARY OF THE INVENTION
[0008] It has been discovered that oligonucleotides containing at
least one GGG motif are effective inhibitors of telomere length on
chromosomes.
[0009] The formula for an active sequence is generally
(N.sub.XG.sub.3-4).sub.QN.sub.X wherein each X is independently 1-8
and Q is 1-6. The sequence (N.sub.XG.sub.4N.sub.Y).sub.Q or
(G.sub.3-4N.sub.XG.sub.3-4).sub.Q wherein X and Y are 1-8, and Q is
1-4 is also believed to be useful in some embodiments of the
invention. Compositions and methods for modulating, preferably
shortening, telomere length are provided. Preferably the telomeres
are mammalian telomeres, i.e., found on mammalian chromosomes, and
more preferably are human telomeres. Methods of modulating
mammalian telomere length are also provided, as are methods for
inhibiting the division of a malignant mammalian cell and for
modulating cellular aging.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0010] It has been discovered that oligonucleotides containing one
or more GGG motifs, or G-cores, wherein G is a guanine-containing
nucleotide or analog, are effective inhibitors of telomere length
on chromosomes. Although the GGG core sequence(s) or G
pharmacophore is necessary, sequences flanking the GGG sequence
have been found to play an important role in inhibitory activity
because it has been found that activity can be modulated by
substituting or deleting the surrounding sequences. In the context
of this invention, the term "modulate" means to increase or
decrease.
[0011] In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA) or mimetics thereof. This term includes
oligonucleotides composed of naturally-occurring nucleobases,
sugars and covalent internucleoside (backbone) linkages as well as
oligonucleotides having non-naturally-occurring portions which
function similarly. Such modified or substituted oligonucleotides
are often preferred over native forms because of desirable
properties such as, for example, enhanced cellular uptake, enhanced
affinity for nucleic acid target and increased stability in the
presence of nucleases.
[0012] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base. The two most common classes of such heterocyclic
bases are the purines and the pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to the 2', 3' or 5' hydroxyl moiety of the
sugar. In forming oligonucleotides, the phosphate groups covalently
link adjacent nucleosides to one another to form a linear polymeric
compound. In turn the respective ends of this linear polymeric
structure can be further joined to form a circular structure,
however, open linear structures are generally preferred. Within the
oligonucleotide structure, the phosphate groups are commonly
referred to as forming the internucleoside backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage.
[0013] Specific examples of preferred compounds useful in this
invention include oligonucleotides containing modified backbones or
non-natural internucleoside linkages. As defined in this
specification, oligonucleotides having modified backbones include
those that retain a phosphorus atom in the backbone and those that
do not have a phosphorus atom in the backbone. For the purposes of
this specification, and as sometimes referenced in the art,
modified oligonucleotides that do not have a phosphorus atom in
their internucleoside backbone can also be considered to be
oligonucleosides.
[0014] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters,
selenophosphates and boranophosphates having normal 3'-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3', 5' to
5' or 2' to 2' linkage. Preferred oligonucleotides having inverted
polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e., a single inverted nucleoside residue
which may be a basic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0015] Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;
5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;
5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555;
5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are
commonly owned with this application, and each of which is herein
incorporated by reference in its entirety.
[0016] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
[0017] Representative United States patents that teach the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos.: 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608;
5,646,269 and 5,677,439, certain of which are commonly owned with
this application, and each of which is herein incorporated by
reference in its entirety.
[0018] In other preferred oligonucleotide mimetics, both the sugar
and the internucleoside linkage, i.e., the backbone, of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative United States patents that
teach the preparation of PNA compounds include, but are not limited
to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of
which is herein incorporated by reference in its entirety. Further
teaching of PNA compounds can be found in Nielsen et al., Science,
1991, 254, 1497-1500.
[0019] Most preferred embodiments of the invention are
oligonucleotides with phosphorothioate backbones and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- (known as a methylene
(methylimino) or MMI backbone),
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- (wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--) of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0020] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH; F; O--, S--, or N-alkyl;
O--, S--, or N-alkenyl; O--, S-- or N-alkynyl; or O-alkyl-O-alkyl,
wherein the alkyl, alkenyl and alkynyl may be substituted or
unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10
alkenyl and alkynyl. Particularly preferred are
O((CH.sub.2).sub.nO).sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON((CH.sub.2).sub.nCH.sub.3)).sub.2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples herein below, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples hereinbelow.
[0021] A further preferred modification includes Locked Nucleic
Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or
4' carbon atom of the sugar ring thereby forming a bicyclic sugar
moiety. The linkage is preferably a methylene (--CH.sub.2--).sub.n
group bridging the 2' oxygen atom and the 4' carbon atom wherein n
is 1 or 2. LNAs and preparation thereof are described in WO
98/39352 and WO 99/14226.
[0022] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747;
and 5,700,920, certain of which are commonly owned with the instant
application, and each of which is herein incorporated by reference
in its entirety.
[0023] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl
(--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further modified nucleobases include tricyclic
pyrimidines such as phenoxazine
cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole
cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. Certain of these nucleobases are particularly useful
for increasing the binding affinity of the oligomeric compounds of
the invention. These include 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C.
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense
Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions, even more
particularly when combined with 2'-O-methoxyethyl sugar
modifications.
[0024] Representative United States patents that teach the
preparation of certain of the above noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.:
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653;
5,763,588; 6,005,096; and 5,681,941, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference in its entirety, and U.S. Pat. No.
5,750,692, which is commonly owned with the instant application and
also herein incorporated by reference in its entirety.
[0025] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. The
compounds of the invention can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include intercalators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugate groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve oligomer
uptake, enhance oligomer resistance to degradation, and/or
strengthen sequence-specific hybridization with RNA. Groups that
enhance the pharmacokinetic properties, in the context of this
invention, include groups that improve oligomer uptake,
distribution, metabolism or excretion. Representative conjugate
groups are disclosed in International Patent Application
PCT/US92/09196, filed Oct. 23, 1992, the entire disclosure of which
is incorporated herein by reference in its entirety. Conjugate
moieties include but are not limited to lipid moieties such as a
cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.
Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
1111-1118; Kadbanov et al., FEBS Lett., 1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the
invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) which is incorporated herein by reference in its
entirety.
[0026] Representative United States patents that teach the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941, certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference in its entirety.
[0027] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes oligonucleotides which are
chimeric compounds. "Chimeric" oligonucleotides or "chimeras," in
the context of this invention, are oligonucleotides which contain
two or more chemically distinct regions, each made up of at least
one monomer unit, i.e., a nucleotide in the case of an
oligonucleotide compound. These oligonucleotides typically contain
at least one region wherein the oligonucleotide is modified so as
to confer upon the oligonucleotide increased resistance to nuclease
degradation, increased cellular uptake, and/or increased binding
affinity for the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of
oligonucleotide inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0028] Chimeric oligonucleotides of the invention may be formed as
composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Representative United States patents
that teach the preparation of such hybrid structures include, but
are not limited to, U.S. Pat. Nos.: 5,013,830; 5,149,797;
5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350;
5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which
are commonly owned with the instant application, and each of which
is herein incorporated by reference in its entirety.
[0029] The oligonucleotides in accordance with this invention
preferably comprise from about 6 to about 27 nucleic acid base
units. It is preferred that such oligonucleotides have from about 6
to 24 nucleic acid base units. As will be appreciated, a nucleic
acid base unit is a base-sugar combination suitably bound to
adjacent nucleic acid base unit through phosphodiester or other
bonds.
[0030] The oligonucleotides used in accordance with this invention
may be conveniently and routinely made through the well-known
technique of solid phase synthesis. Equipment for such synthesis is
sold by several vendors including Applied Biosystems. Any other
means for such synthesis may also be employed, however the actual
synthesis of the oligonucleotides are well within the talents of
the routineer. It is also well known to use similar techniques to
prepare other oligonucleotides such as the phosphorothioates and
alkylated derivatives.
[0031] The essential feature of the invention is a conserved
G.sub.3 or G.sub.4 core sequence and a sufficient number of
additional flanking bases to significantly inhibit activity. It has
also been discovered that chemical modifications are tolerated in
the oligonucleotide. For example, phosphorothioate and 2'-O-methyl
modifications have been incorporated.
[0032] The formula for an active sequence is:
(N.sub.XG.sub.3-4).sub.QN.sub.X wherein each X is independently 1-8
and Q is 1-6.
[0033] In some embodiments of the present invention, the sequence
(N.sub.x G.sub.4 N.sub.y).sub.Q or (G.sub.4 N.sub.x G.sub.4).sub.Q
where G=a guanine-containing nucleotide or analog,
[0034] N=any nucleotide,
[0035] X=1-8,
[0036] Y=1-8,
[0037] and Q=1-4 is believed to be active.
Modulation of Telomere Length
[0038] Oligonucleotides capable of modulating telomere length are
contemplated by this invention. In human cells, the sequence TTAGGG
is repeated from hundreds to thousands of times at both ends of
every chromosome, depending on cell type and age. It is believed
that oligonucleotides having a sequence
(N.sub.XG.sub.3-4).sub.QN.sub.x wherein each X is independently 1-8
and Q is 1-6 would be useful for modulating telomere length.
[0039] Since telomeres appear to have a role in cell aging, i.e.,
telomere length decreases with each cell division, it is believed
that such oligonucleotides would be useful for modulating the
cell's aging process. Altered telomeres are also found in cancerous
cells; it is therefore also believed that such oligonucleotides
would be useful for controlling malignant cell growth. Therefore,
modulation of telomere length using oligonucleotides of the present
invention could prove useful for the treatment of cancer or in
controlling the aging process.
[0040] It has now been demonstrated that oligonucleotides having a
sequence (N.sub.XG.sub.3-4).sub.QN.sub.X wherein each X is
independently 1-8 and Q is 1-6 are able to modulate telomere
length. Herbert et al. (Herbert et al., Proc. Natl. Acad. Sci. USA,
1999, 96, 14276-14281) have demonstrated telomere shortening in
human mammary epithelial (HME) cells and prostate tumor-derived
DU145 cells treated with a 2'-O-methyl chimeric oligonucleotide
having the sequence CAGUUAGGGUUAG (SEQ ID NO: 1). Telomere length
was reduced from 2000 to 1700 base pairs in HME cells (60 day
treatment) and from 3600 base pairs to 2200 base pairs in DU145
cells (76 day treatment). Treatment of DU145 cells with a peptide
nucleic acid of the sequence Gly-CAGTTAGGGTTAG-Lys (SEQ ID NO:2
with a glycine residue covalently attached to the N-terminus and a
lysine residue covalently attached to the C-terminus) caused
similar telomere shortening to that caused by the 2'-O-methyl
oligonucleotide.
[0041] Telomere shortening has also been demonstrated in mice
treated with an 2'-O-methyl oligonucleotide, ISIS 24691, having the
sequence CAGTTAGGGTTAG (SEQ ID NO:2) and a 2'O-methyl sugar
modification at every position and a phosphorothioate backbone
throughout.
[0042] The following examples are provided for illustrative
purposes only and are not intended to limit the invention.
EXAMPLES
Example 1
Oligonucleotide Synthesis
[0043] Oligonucleotides may be purchased commercially or
synthesized as follows. DNA synthesizer reagents, controlled-pore
glass (CPG)-bound and B-cyanoethyldiisopropylphosphoramidites were
purchased from Applied Biosystems (Foster City, Calif.).
2'-O-Methyl B-cyanoethyldiisopropylphosphoramidites were purchased
from Chemgenes (Needham, Mass.). Phenoxyacetyl-protected
phosphoramadites for RNA synthesis were purchased from BioGenex
(Hayward, Calif.).
[0044] Oligonucleotides were synthesized on an automated DNA
synthesizer (Applied Biosystems model 380B). 2'-O-Methyl
oligonucleotides were synthesized using the standard cycle for
unmodified oligonucleotides, except the wait step after pulse
delivery of tetrazole and base was increased to 360 seconds. The 3'
base bound to the CPG used to start the synthesis was a
2'-deoxyribonucleotide. After cleavage from the CPG column and
deblocking in concentrated ammonium hydroxide at 55.degree. C. (18
hours), the oligonucleotides were purified by precipitation two
times out of 0.5 M NaCl solution with 2.5 volumes ethanol.
Analytical gel electrophoresis was accomplished in 20% acrylamide,
8 M urea, 45 mM Tris-borate buffer, pH=7.0. Oligonucleotides were
judged from polyacrylamide gel electrophoresis to be greater than
85% full length material.
Example 2
Modulation of Telomere Length by G.sub.4 Phosphorothioate
Oligonucleotides
[0045] The amount and length of telomeric DNA in human fibroblasts
has been shown to decrease during aging as a function of serial
passage in vitro. To examine the effect of G.sub.4 phosphorothioate
oligonucleotides on this process, human skin biopsy fibroblasts are
grown as described in Harley, C. B., Meth. Molec. Biol. 1990, 5,
25-32. Cells are treated with the oligonucleotides shown in Table
1, by adding the oligonucleotide to the medium to give a final
concentration of 1 .mu.M, 3 .mu.M or 10 .mu.M; control cells
receive no oligonucleotide. Population doublings are counted and
DNA is isolated at regular intervals. Telomere length is determined
by Southern blot analysis and plotted against number of population
doublings as described in Harley, C. B. et al., Nature 1990, 345,
458-460. The slope of the resulting linear regression lines
indicates a loss of approximately 50 bp of telomere DNA per mean
population doubling in untreated fibroblasts. Harley, C. B. et al.,
Nature 1990, 345, 458-460. Treatment with oligonucleotides of Table
1 is expected to result in modulation of telomere length.
TABLE-US-00001 TABLE 1 Effect of G.sub.4 Phosphorothioate
Oligonucleotides on Telomere Length in Aging Fibroblasts ISIS NO.
SEQUENCE SEQ ID NO: TT AGGG 5739 TT GGGG 5756 TT AGGG TT 5320 TT
GGGG TT 5675 TT GGGG TT GGGG TT 3 5651 TT GGGG TT GGGG TT GGGG TT 4
GGGG TTTT GGGG TTTA GGGG 5673 GGGG
Example 3
Chimeric 2'-O-methyl G.sub.4 Oligonucleotides with Deoxy Gaps
[0046] A series of phosphorothioate oligonucleotides were
synthesized having a 2'-O-methyl substitution on the sugar of each
nucleotide in the flanking regions, and 2'-deoxynucleotides in the
center portion of the oligonucleotide (referred to as the "deoxy
gap"). Deoxy gaps varied from zero to seven nucleotides in length.
Additional chimeric oligonucleotides were synthesized having the
sequences GTTGGAGACCGGGGTTGGGG (SEQ ID NO:5) and
CACGGGGTCGCCGATGAACC (SEQ ID NO:6). These oligonucleotides were
2'-O-methyl oligonucleotides with deoxy gaps as described above,
but instead of a uniform phosphorothioate backbone, these compounds
had phosphorothioate internucleotide linkages in the deoxy gap
region and phosphodiester linkages in the flanking region.
[0047] Additional oligonucleotides were synthesized with
2'-O-propyl modifications. 2'-O-propyl oligonucleotides were
prepared from 2'-deoxy-2'-O-propyl ribosides of nucleic acid bases
A, G, U(T), and C which were prepared by modifications of
literature procedures described by B. S. Sproat, et al., Nucleic
Acids Research 18:41-49 (1990) and H. Inoue, et al., Nucleic Acids
Research 15:6131-6148 (1987). ISIS 7114 is a phosphorothioate which
has SEQ ID NO:6, and has a 2'-O-propyl modification on each sugar.
ISIS 7171 is a phosphorothioate gapped 2'-O-propyl oligonucleotide
with the same sequence, and 2'-O-propyl modifications at positions
1-7 and 14-20 (6-deoxy gap).
Example 4
Cell Lines
[0048] Cell lines were produced and grown as described in Herbert
et al., 1999, Proc. Natl. Acad. Sci. USA, 96, 14276-14281. Briefly,
spontaneously immortalized human mammary epithelial (HME) cells
were grown in supplemented serum-free medium and used between
population doublings 100 and 150. Prostate tumor-derived DU145
cells were maintained in DMEM containing fetal calf serum and
antibiotics.
Example 5
Oligonucleotides
[0049] The oligonucleotides are fully described in Herbert et al.,
1999, Proc. Natl. Acad. Sci. USA, 96, 14276-14281. 2'-O-methyl
oligonucleotides were purchased from Oligos Etc. and Oligo
Therapeutics (Wilsonville Oreg.). The "match" phosphorothioate
modified 2'-O-methyl RNA oligonucleotide has the sequence
CAGUUAGGGUUAG (SEQ ID NO:1) wherein the bold nucleotides have
phosphorothioate linkages. The mismatch 2'-O-methyl oligonucleotide
is CAGUUAGAAUUAG (SEQ ID NO:7). Match and mismatch PNAs were
synthesized automatically with a PerSeptive Biosystems (Framingham
Mass.) Expedite 8909 Synthesizer using Fmoc protocols and reagents
obtained from PE Biosystems. PNAs were purified by HPLC and
characterized by matrix-assisted laser desorption time-of-flight
mass spectrometry. The match PNA has the sequence
Gly-CAGTTAGGGTTAG-Lys (SEQ ID NO:2 with a glycine residue
covalently attached to the N-terminus and a lysine residue
covalently attached to the C-terminus); the mismatch is
Gly-CAGTTAGAATTAG-Lys (SEQ ID NO:8 with a glycine residue
covalently attached to the N-terminus and a lysine residue
covalently attached to the C-terminus). DNA oligonucleotides used
for transfection of PNA/DNA complexes were obtained from Life
Technologies (Gaithersburg, Md.). The DNA oligonucleotide complexed
to the match PNA has the sequence TCTAACCCTAA (SEQ ID NO:9); the
DNA oligonucleotide complexed to the mismatch PNA has the sequence
TCTAATTCTAA (SEQ ID NO: 10).
Example 6
Transfection of Cells with Oligonucleotides
[0050] Cells were transfected as described in Herbert et al., 1999,
Proc. Natl. Acad. Sci. USA, 96, 14276-14281. Briefly, HME cells
were transfected with 2'-O-methyl RNA and mismatch control
oligonucleotides using the FuGENE6 Transfection Reagent protocol
(Roche Molecular Biochemicals). DU145 cells were plated at 25,000
cells per well in a 24 well plate. For DNA/PNA transfections, 100
.mu.M PNA was hybridized with 109 .mu.M of the appropriate DNA
oligonucleotide in 0.5.times.PBS. Cells were allowed to adhere and
transfected with 2.0 .mu.l (7 .mu.g/ml) of Lipofectamine (Life
Technologies) and 0.5 .mu.M 2'-O-methyl RNA oligonucleotide or 1
.mu.M PNA/DNA complex in 200 .mu.l total Opti-MEM (Life
Technologies) according to the manufacturer's instructions. Cells
were transfected with oligonucleotides at 3 to 4 day intervals for
120 days.
Example 7
Measurement of Reduction of Telomere Length in Cell Culture by
Oligonucleotide Treatment
[0051] Telomere length was measured as described by Herbert et al.,
1999, Proc. Natl. Acad. Sci. USA, 96, 14276-14281. Briefly, mean
telomere length was evaluated by using telomere restriction
fragment analysis, a variation of standard Southern analysis, and
was quantitated as described by Shay et al., 1994, Methods Mol.
Genet., 5, 263-268. Digested samples were resolved on a 0.7%
agarose gel and hybridized to a telomeric probe
((.sup.32P)(TTAGGG).sub.4 oligonucleotide).
[0052] Within 60 days of treatment, the mean telomere length of
HME-50 cells treated with the 2'-O-methyl RNA G-core
oligonucleotide of sequence CAGUUAGGGUUAG (SEQ ID NO:1) was reduced
from 2000 to 1700 base pairs. This decrease in measured telomere
restriction fragment length may be an underestimate of the total
loss of telomere length because little telomeric DNA remained to
hybridize with the labeled probe. The telomere restriction fragment
length of cells treated with the mismatch 2'-O-methyl RNA
oligonucleotide lacking the GGG sequence was unchanged at 2000 base
pairs.
[0053] DU145 cells have longer telomeres than HME-50 cells. In
DU145 cells treated with the 2'-O-methyl G-core oligonucleotide of
sequence CAGUUAGGGUUAG (SEQ ID NO:1) for 76 days, the mean telomere
restriction fragment length decreased from 3600 base pairs to 2200
base pairs. Again the signal was greatly reduced due to reduction
in telomere repeat number.
[0054] A PNA oligonucleotide of sequence Gly-CAGTTAGGGTTAG-Lys (SEQ
ID NO:2 with an amino acid residue tethered to each end) caused
telomere shortening in DU145 cells similar to that caused by the
2'-O-methyl oligonucleotide.
[0055] The above-described results are shown in FIG. 4 of Herbert
et al., 1999, Proc. Natl. Acad. Sci. USA. 96, 14276-14281.
Example 8
Measurement of Reduction of Telomere Length in vivo by
Oligonucleotide Treatment
[0056] Du145 xenografts were implanted in seven nude mice. Mice
were injected on bilateral flanks with 3 million DU145 cells. Once
tumors reached 100-500 mm.sup.3, mice were injected
intraperitoneally with either PBS (control) or ISIS 24691 antisense
oligonucleotide. Mice 1-3 were controls, mice 4-7 were treated
long-term with 2'-O-methyl oligonucleotide ISIS 24691
(CAGTTAGGGTTAG; SEQ ID NO:2). This oligonucleotide has a
2'-O-methyl sugar modification at every position and a
phosphorothioate backbone throughout. The C residue is a 5-methyl C
residue.
[0057] The results are shown in Table 2. TABLE-US-00002 TABLE 2
Approximate Telomere Mouse length (bp) Treatment 1 4400 Control 2
3900 Control 3 3900 Control 4 2200 ISIS 24691 5 2800 ISIS 24691 6
3000 ISIS 24691 7 3800 ISIS 24691
[0058] As shown in the table, the average telomere length was
approximately 4067 for control mice and 2950 for oligonucleotide
treated mice, a reduction in telomere length of 27.5% after
oligonucleotide treatment.
Sequence CWU 1
1
10 1 13 RNA Artificial Sequence Oligomeric compound 1 caguuagggu
uag 13 2 13 DNA Artificial Sequence Oligomeric compound 2
cagttagggt tag 13 3 14 DNA Artificial Sequence Oligomeric compound
3 ttggggttgg ggtt 14 4 24 DNA Artificial Sequence Oligomeric
compound 4 ttggggttgg ggttggggtt gggg 24 5 20 DNA Artificial
Sequence Oligomeric compound 5 gttggagacc ggggttgggg 20 6 20 DNA
Artificial Sequence Oligomeric compound 6 cacggggtcg ccgatgaacc 20
7 13 RNA Artificial Sequence Oligomeric compound 7 caguuagaau uag
13 8 13 DNA Artificial Sequence Oligomeric compound 8 cagttagaat
tag 13 9 11 DNA Artificial Sequence Oligomeric compound 9
tctaacccta a 11 10 11 DNA Artificial Sequence Oligomeric compound
10 tctaattcta a 11
* * * * *