U.S. patent application number 13/125560 was filed with the patent office on 2012-03-08 for methods of using oligomeric compounds comprising 2'-substituted nucleosides.
This patent application is currently assigned to ISIS PHARMACEUTICALS, INC.. Invention is credited to Balkrishen Bhat, Thazha P. Prakash, Andrew M. Siwkowski, Eric E. Swayze.
Application Number | 20120059045 13/125560 |
Document ID | / |
Family ID | 42120004 |
Filed Date | 2012-03-08 |
United States Patent
Application |
20120059045 |
Kind Code |
A1 |
Prakash; Thazha P. ; et
al. |
March 8, 2012 |
METHODS OF USING OLIGOMERIC COMPOUNDS COMPRISING 2'-SUBSTITUTED
NUCLEOSIDES
Abstract
The present disclosure provides oligomeric compounds comprising
at least one 2'-fluoroethoxy modified nucleoside of formula I and
methods of using these oligomeric compounds. The methods provided
herein include contacting a cell or administering to an animal at
least one of the oligomeric compounds. In certain embodiments, the
oligomeric compounds hybridize to a portion of a target RNA
resulting in loss of normal function of the target RNA.
Inventors: |
Prakash; Thazha P.;
(Carlsbad, CA) ; Siwkowski; Andrew M.; (Carlsbad,
CA) ; Swayze; Eric E.; (Encinitas, CA) ; Bhat;
Balkrishen; (Carlsbad, CA) |
Assignee: |
ISIS PHARMACEUTICALS, INC.
Carlsbad
CA
|
Family ID: |
42120004 |
Appl. No.: |
13/125560 |
Filed: |
October 23, 2009 |
PCT Filed: |
October 23, 2009 |
PCT NO: |
PCT/US2009/061916 |
371 Date: |
July 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61108457 |
Oct 24, 2008 |
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61149297 |
Feb 2, 2009 |
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61150492 |
Feb 6, 2009 |
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61163217 |
Mar 25, 2009 |
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61174137 |
Apr 30, 2009 |
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61239672 |
Sep 3, 2009 |
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Current U.S.
Class: |
514/44A ;
435/375; 435/6.11; 435/6.12; 435/6.13 |
Current CPC
Class: |
C07H 21/00 20130101;
A61K 31/00 20130101; C07H 19/10 20130101 |
Class at
Publication: |
514/44.A ;
435/375; 435/6.13; 435/6.12; 435/6.11 |
International
Class: |
A61K 31/712 20060101
A61K031/712; C12Q 1/68 20060101 C12Q001/68; C12N 5/071 20100101
C12N005/071 |
Claims
1. A method comprising contacting a cell with an oligomeric
compound, wherein said oligomeric compound comprises at least one
2'-fluoroethoxy modified nucleoside having formula I: ##STR00007##
wherein independently for each of said 2'-fluoroethoxy modified
nucleosides having formula I: Bx is a heterocyclic base moiety; one
of T.sub.1 and T.sub.2 is an internucleoside linking group
attaching the 2'-fluoroethoxy modified nucleoside of formula I to
the oligomeric compound and the other of T.sub.1 and T.sub.2 is H,
a hydroxyl protecting group, a 5' or 3'-terminal group or an
internucleoside linking group attaching the 2'-fluoroethoxy
modified nucleoside of formula I to the oligomeric compound;
R.sub.1 and R.sub.2 are each independently H or F; and wherein said
oligomeric compound comprises from about 8 to about 40 linked
monomeric subunits and is complementary to at least a portion of a
target RNA.
2. The method of claim 1 wherein R.sub.1 and R.sub.2 are each H for
each of said 2'-fluoroethoxy modified nucleosides of formula I.
3. The method of claim 1 wherein one of R.sub.1 and R.sub.2 is F
for each of said 2'-fluoroethoxy modified nucleosides of formula
I.
4. The method of claim 1 wherein R.sub.1 and R.sub.2 are each F for
each of said 2'-fluoroethoxy modified nucleosides of formula I.
5. The method of claim 1 wherein each Bx is, independently, an
optionally protected uracil, thymine, cytosine, 5-methylcytosine,
adenine or guanine.
6. The method of any claim 1 wherein said oligomeric compound
further comprises a 5' or 3'-terminal group.
7. The method of claim 1 wherein each linked monomeric subunit that
is not a .beta.-D-2'-deoxyribonucleoside or a 2'-fluoroethoxy
modified nucleoside of formula I is a modified nucleoside wherein
each modified nucleoside is, independently, a bicyclic modified
nucleoside, a 2'-modified nucleoside, a 4'-thio modified nucleoside
or a 4'-thio-2'-modified nucleoside.
8-10. (canceled)
11. The method of claim 1 wherein said oligomeric compound
comprises a gapped oligomeric compound comprising two external
regions separated by an internal region wherein each external
region independently comprises from 1 to 5 contiguous
2'-fluoroethoxy modified nucleosides having formula I and the
internal region comprises from 6 to about 23 contiguous monomeric
subunits independently selected from nucleosides and modified
nucleosides.
12. (canceled)
13. The method of claim 11 wherein essentially each monomeric
subunit in the internal region is a
.beta.-D-2'-deoxyribonucleoside.
14. (canceled)
15. The method of claim 11 wherein each external region comprises
from 1 to 3 2'-fluoroethoxy modified nucleosides having formula I
and the internal region comprises from about 10 to about 14
.beta.-D-2'-deoxyribonucleosides.
16-20. (canceled)
21. The method of claim 15 wherein each external region comprises 2
2'-fluoroethoxy modified nucleosides having formula I and the
internal region comprises 10 .beta.-D-2'-deoxyribonucleosides.
22. (canceled)
23. The method of any claim 1 wherein said oligomeric compound
comprises a 5'-terminal 2'-fluoroethoxy modified nucleoside having
formula I wherein T.sub.1 is a phosphate group.
24-25. (canceled)
26. The method of claim 1 wherein each internucleoside linking
group is, independently, a phosphodiester or a
phosphorothioate.
27. (canceled)
28. The method of claim 1 wherein said oligomeric compound
comprises from 8 to about 18 linked monomeric subunits.
29. (canceled)
30. The method of claim 1 wherein said oligomeric compound
comprises from 10 to about 14 linked monomeric subunits.
31. (canceled)
32. The method of claim 1 wherein said oligomeric compound
comprises from 18 to about 21 linked monomeric subunits.
33. The method of claim 1 wherein said oligomeric compound
comprises from 19 to about 20 linked monomeric subunits.
34. The method of claim 1 wherein said cell is in an animal.
35. The method of claim 1 wherein said cell is in a human.
36. The method of claim 1 wherein said target RNA is selected from
mRNA, pre-mRNA and micro RNA.
37. (canceled)
38. The method of claim 36 wherein said target RNA is human
mRNA.
39. The method of claim 1 wherein said target RNA is cleaved
thereby inhibiting its function.
40. The method of claim 1 further comprising evaluating the
antisense activity of said oligomeric compound on said cell.
41. The method of claim 40 wherein said evaluating comprises
detecting the levels of target RNA.
42. The method of claim 40 wherein said evaluating comprises
detecting the levels of a protein.
43. The method of claim 40 wherein said evaluating comprises
detection of one or more phenotypic effects.
44-86. (canceled)
87. The method of claim 7 wherein each linked monomeric subunit
that is not a 2'-fluoroethoxy modified nucleoside of formula I is a
modified nucleoside.
Description
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled CHEM0057WOSEQ.ixt, created on Oct. 23, 2009 which is
8 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present disclosure provides methods of using oligomeric
compounds comprising 2'-fluoroethoxy modified nucleosides. The
present methods include contacting a cell with, or administering to
an animal, at least one of the oligomeric compounds provided
herein. In certain embodiments, the oligomeric compounds hybridize
to a portion of a target RNA resulting in loss of normal function
of the target RNA.
BACKGROUND OF THE INVENTION
[0003] Targeting disease-causing gene sequences was first suggested
more than thirty years ago (Belikova et al., Tet. Lett., 1967, 37,
3557-3562), and antisense activity was demonstrated in cell culture
more than a decade later (Zamecnik et al., Proc. Natl. Acad. Sci.
U.S.A., 1978, 75, 280-284). One advantage of antisense technology
in the treatment of a disease or condition that stems from a
disease-causing gene is that it is a direct genetic approach that
has the ability to modulate (increase or decrease) the expression
of specific disease-causing genes. Another advantage is that
validation of a therapeutic target using antisense compounds
results in direct and immediate discovery of the drug candidate;
the antisense compound is the potential therapeutic agent.
[0004] Generally, the principle behind antisense technology is that
an antisense compound hybridizes to a target nucleic acid and
modulates gene expression activities or function, such as
transcription or translation. The modulation of gene expression can
be achieved by, for example, target degradation or occupancy-based
inhibition. An example of modulation of RNA target function by
degradation is RNase H-based degradation of the target RNA upon
hybridization with an antisense compound. Another example of
modulation of gene expression by target degradation is RNA
interference (RNAi). RNAi generally refers to antisense-mediated
gene silencing involving the introduction of dsRNA leading to the
sequence-specific reduction of targeted endogenous mRNA levels. An
additional example of modulation of RNA target function by an
occupancy-based mechanism is modulation of microRNA function.
MicroRNAs are small non-coding RNAs that regulate the expression of
protein-coding RNAs. The binding of an antisense compound to a
microRNA prevents that microRNA from binding to its messenger RNA
targets, and thus interferes with the function of the microRNA.
Regardless of the specific mechanism, this sequence-specificity
makes antisense compounds extremely attractive as tools for target
validation and gene functionalization, as well as therapeutics to
selectively modulate the expression of genes involved in the
pathogenesis of malignancies and other diseases.
[0005] Antisense technology is an effective means for reducing the
expression of one or more specific gene products and can therefore
prove to be uniquely useful in a number of therapeutic, diagnostic,
and research applications. Chemically modified nucleosides are
routinely used for incorporation into antisense compounds to
enhance one or more properties, such as nuclease resistance,
affinity, specificity or pharmacokinetics for a target RNA. In
1998, the antisense compound, Vitravene.RTM. (fomivirsen; developed
by Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first
antisense drug to achieve marketing clearance from the U.S. Food
and Drug Administration (FDA), and is currently a treatment of
cytomegalovirus (CMV)-induced retinitis in AIDS patients.
[0006] New chemical modifications have improved the potency and
efficacy of antisense compounds, uncovering the potential for oral
delivery as well as enhancing subcutaneous administration,
decreasing potential for side effects, and leading to improvements
in patient convenience. Chemical modifications increasing potency
of antisense compounds allow administration of lower doses, which
reduces the potential for toxicity, as well as decreasing overall
cost of therapy. Modifications increasing the resistance to
degradation result in slower clearance from the body, allowing for
less frequent dosing. Different types of chemical modifications can
be combined in one compound to further optimize the compound's
efficacy. One such group of chemically modified nucleosides
includes 2'-fluoroethoxy modified nucleosides.
[0007] 2'-OCH.sub.2CH.sub.2F modified nucleosides were prepared and
put into 15 and 16mer DNA sequences for affinity measurements
against complementary RNA (U.S. Pat. No. 5,977,332, issued Nov. 2,
1999). Modified nucleosides having 2'-OCH.sub.2R(R.dbd.CH.sub.2F or
CF.sub.3) substituents were also prepared and put into 15 and 16mer
DNA sequences for measurement of affinity against complementary
RNA, measurement of nuclease resistance and analysis of crystal
structure data (Egli et al., Biochemistry, 2005, 44,
9045-9057).
[0008] Consequently, there remains a long-felt need for agents that
specifically regulate gene expression via antisense mechanisms.
Disclosed herein are oligomeric compounds comprising
2'-fluoroethoxy modified nucleosides that are useful for modulating
gene expression pathways, including those relying on mechanisms of
action such as RNaseH, RNAi and dsRNA enzymes, as well as other
antisense mechanisms based on target degradation or target
occupancy. One having skill in the art, once armed with this
disclosure will be able, without undue experimentation, to
identify, prepare and exploit antisense compounds for these
uses.
BRIEF SUMMARY OF THE INVENTION
[0009] Provided herein are methods of using oligomeric compounds
comprising at least one 2'-fluoroethoxy modified nucleoside of
formula I. The present methods include contacting a cell or
administering to an animal at least one of the oligomeric compounds
provided herein. In certain embodiments, the oligomeric compounds
hybridize to a portion of a target RNA resulting in loss of normal
function of the target RNA.
[0010] The variables are defined individually in further detail
herein. It is to be understood that the oligomer compounds
comprising 2'-fluoroethoxy modified nucleosides of formula I and
methods of use thereof provided herein include all combinations of
the embodiments disclosed and variables defined herein.
[0011] In certain embodiments, methods are provided comprising
contacting a cell with an oligomeric compound, wherein said
oligomeric compound comprises at least one 2'-fluoroethoxy modified
nucleoside having formula I:
##STR00001##
wherein independently for each of said 2'-fluoroethoxy modified
nucleosides having formula I:
[0012] Bx is a heterocyclic base moiety;
[0013] one of T.sub.1 and T.sub.2 is an internucleoside linking
group attaching the 2'-fluoroethoxy modified nucleoside of formula
I to the oligomeric compound and the other of T.sub.1 and T.sub.2
is H or a hydroxyl protecting group, a 5' or 3'-terminal group or
an internucleoside linking group attaching the 2'-fluoroethoxy
modified nucleoside of formula I to the oligomeric compound;
[0014] R.sub.1 and R.sub.2 are each independently, H or F; and
[0015] wherein said oligomeric compound comprises from about 8 to
about 40 linked monomeric subunits and is complementary to at least
a portion of a target RNA.
[0016] In certain embodiments, R.sub.1 and R.sub.2 are each H for
each of said 2'-fluoroethoxy modified nucleosides of formula I. In
certain embodiments, one of R.sub.1 and R.sub.2 is F for each of
said 2'-fluoroethoxy modified nucleosides of formula I. In certain
embodiments, R.sub.1 and R.sub.2 are each F for each of said
2'-fluoroethoxy modified nucleosides of formula I.
[0017] In certain embodiments, each Bx is, independently, uracil,
5-methyluracil, thymine, cytosine, 5-methylcytosine,
2,6-diaminopurine, adenine or guanine.
[0018] In certain embodiments, oligomeric compounds are provided
comprising a 5' or 3'-terminal group. In certain embodiments,
oligomeric compounds are provided comprising a 5' and a 3'-terminal
group.
[0019] In certain embodiments, oligomeric compounds are provided
wherein each linked monomeric subunit that is not a
.beta.-D-2'-deoxyribonucleoside or a 2'-fluoroethoxy modified
nucleoside of formula I is a modified nucleoside. In certain
embodiments, each modified nucleoside is, independently, a bicyclic
modified nucleoside, a 2'-modified nucleoside, a 4'-thio modified
nucleoside or a 4'-thio-2'-modified nucleoside.
[0020] In certain embodiments, oligomeric compounds are provided
comprising a blockmer, a 3'-hemimer or 5'-hemimer. In certain
embodiments, each monomeric subunit is, independently, a
.beta.-D-2'-deoxyribonucleoside or a 2'-fluoroethoxy modified
nucleoside of formula I.
[0021] In certain embodiments, gapped oligomeric compounds are
provided comprising two external regions separated by an internal
region wherein each external region independently comprises from 1
to 5 contiguous 2'-fluoroethoxy modified nucleosides having formula
I and the internal region comprises from 6 to about 23 contiguous
monomeric subunits independently selected from nucleosides and
modified nucleosides. In certain embodiments, each monomeric
subunit in the internal region is, independently, a
.beta.-D-2'-deoxyribonucleoside or a modified nucleoside. In
certain embodiments, each monomeric subunit in the internal region
is a .beta.-D-2'-deoxyribonucleoside. In certain embodiments, the
internal region comprises from about 8 to about 12
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, each
external region comprises from 1 to 3 2'-fluoroethoxy modified
nucleoside having formula I. In certain embodiments, the internal
region comprises from about 10 to about 12
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, the
internal region comprises from 11 to about 18
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, each
external region comprises from 1 to 3 2'-fluoroethoxy modified
nucleoside having formula I. In certain embodiments, the internal
region comprises from 12 to about 14
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, each
external region independently comprises from 1 to 3 2'-fluoroethoxy
modified nucleosides having formula I. In certain embodiments, each
external region comprises 2 2'-fluoroethoxy modified nucleosides
having formula I. In certain embodiments, the internal region
comprises 10 .beta.-D-2'-deoxyribonucleosides.
[0022] In certain embodiments, oligomeric compounds are provided
comprising a 5'-terminal 2'-fluoroethoxy modified nucleoside having
formula I wherein T.sub.1 is a phosphate group.
[0023] In certain embodiments, oligomeric compounds are provided
wherein each internucleoside linkage is a phosphodiester. In
certain embodiments, oligomeric compounds are provided wherein each
internucleoside linkage is a phosphorothioate. In certain
embodiments, oligomeric compounds are provided wherein each
internucleoside linking group is, independently, a phosphodiester
or a phosphorothioate. In certain embodiments, oligomeric compounds
are provided wherein each internucleoside linking group is,
independently, a phosphodiester, phosphorothioate, chiral
phosphorothioate, phosphorodithioate, phosphotriester,
aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate,
5'-alkylene phosphonate, chiral phosphonate, phosphinate,
phosphoramidate, 3'-amino phosphoramidate,
aminoalkylphosphoramidate, thionophosphoramidate,
thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate
or boranophosphate.
[0024] In certain embodiments, oligomeric compounds are provided
comprising from 8 to about 18 linked monomeric subunits. In certain
embodiments, oligomeric compounds are provided comprising from 10
to about 16 linked monomeric subunits. In certain embodiments,
oligomeric compounds are provided comprising from 10 to about 14
linked monomeric subunits. In certain embodiments, oligomeric
compounds are provided comprising from 17 to about 26 linked
monomeric subunits. In certain embodiments, oligomeric compounds
are provided comprising from 18 to about 21 linked monomeric
subunits. In certain embodiments, oligomeric compounds are provided
comprising from 19 to about 20 linked monomeric subunits.
[0025] In certain embodiments, methods are provided comprising
contacting a cell with an oligomeric compound as provided herein,
wherein the cell is in an animal. In certain embodiments, the cell
is in a human.
[0026] In certain embodiments, methods are provided comprising
contacting a cell with an oligomeric compound that is complementary
to at least a portion of a target RNA wherein the target RNA is
selected from mRNA, pre-mRNA and micro RNA. In certain embodiments,
the target RNA is mRNA. In certain embodiments, the target RNA is
human mRNA.
[0027] In certain embodiments, methods are provided comprising
contacting a cell with an oligomeric compound that is complementary
to at least a portion of a target RNA wherein the target RNA is
cleaved thereby inhibiting its function.
[0028] In certain embodiments, methods are provided comprising
contacting a cell with an oligomeric compound and evaluating the
antisense activity of the oligomeric compound on the cell. In
certain embodiments, the evaluating comprises detecting the levels
of target RNA. In certain embodiments, the evaluating comprises
detecting the levels of a protein. In certain embodiments, the
evaluating comprises detection of one or more phenotypic
effects.
[0029] In certain embodiments, methods are provided comprising
administering an oligomeric compound to an animal, wherein said
oligomeric compound comprises at least one 2'-fluoroethoxy modified
nucleoside having formula I:
##STR00002##
wherein independently for each of said 2'-fluoroethoxy modified
nucleosides having formula I:
[0030] Bx is a heterocyclic base moiety;
[0031] one of T.sub.1 and T.sub.2 is an internucleoside linking
group attaching the 2'-fluoroethoxy modified nucleoside of formula
I to the oligomeric compound and the other of T.sub.1 and T.sub.2
is H or a hydroxyl protecting group, a 5' or 3'-terminal group or
an internucleoside linking group attaching the 2'-fluoroethoxy
modified nucleoside of formula I to the oligomeric compound;
[0032] R.sub.1 and R.sub.2 are each independently, H or F; and
[0033] wherein said oligomeric compound comprises from about 8 to
about 40 linked monomeric subunits and is complementary to at least
a portion of a target RNA.
[0034] In certain embodiments, R.sub.1 and R.sub.2 are each H for
each of said 2'-fluoroethoxy modified nucleosides of formula I. In
certain embodiments, R.sub.1 and R.sub.2 is F for each of said
2'-fluoroethoxy modified nucleosides of formula I. In certain
embodiments, R.sub.1 and R.sub.2 are each F for each of said
2'-fluoroethoxy modified nucleosides of formula I.
[0035] In certain embodiments, each Bx is, independently, uracil,
5-methyluracil, thymine, cytosine, 5-methylcytosine,
2,6-diaminopurine, adenine or guanine.
[0036] In certain embodiments, oligomeric compounds are provided
comprising a 5' or 3'-terminal group. In certain embodiments,
oligomeric compounds are provided comprising a 5' and a 3'-terminal
group.
49. The method of any one of claims 44 to 48 wherein said
oligomeric compound further comprises a 5' or 3'-terminal
group.
[0037] In certain embodiments, oligomeric compounds are provided
wherein each linked monomeric subunit that is not a
.beta.-D-2'-deoxyribonucleoside or a 2'-fluoroethoxy modified
nucleoside of formula I is a modified nucleoside. In certain
embodiments, each modified nucleoside is, independently, a bicyclic
modified nucleoside, a 2'-modified nucleoside, a 4'-thio modified
nucleoside or a 4'-thio-2'-modified nucleoside.
[0038] In certain embodiments, oligomeric compounds are provided
comprising a blockmer, a 3'-hemimer or 5'-hemimer. In certain
embodiments, each monomeric subunit is, independently, a
.beta.-D-2'-deoxyribonucleoside or a 2'-fluoroethoxy modified
nucleoside of formula I.
[0039] In certain embodiments, gapped oligomeric compounds are
provided comprising two external regions separated by an internal
region wherein each external region independently comprises from 1
to 5 contiguous 2'-fluoroethoxy modified nucleosides having formula
I and the internal region comprises from 6 to about 23 contiguous
monomeric subunits independently selected from nucleosides and
modified nucleosides. In certain embodiments, each monomeric
subunit in the internal region is, independently, a
.beta.-D-2'-deoxyribonucleoside or a modified nucleoside. In
certain embodiments, each monomeric subunit in the internal region
is a .beta.-D-2'-deoxyribonucleoside. In certain embodiments, the
internal region comprises from about 8 to about 12
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, each
external region comprises from 1 to 3 2'-fluoroethoxy modified
nucleoside having formula I. In certain embodiments, the internal
region comprises from about 10 to about 12
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, the
internal region comprises from 11 to about 18
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, each
external region comprises from 1 to 3 2'-fluoroethoxy modified
nucleoside having formula I. In certain embodiments, the internal
region comprises from 12 to about 14
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, each
external region independently comprises from 1 to 3 2'-fluoroethoxy
modified nucleosides having formula I. In certain embodiments, each
external region comprises 2 2'-fluoroethoxy modified nucleosides
having formula I. In certain embodiments, the internal region
comprises 10 .beta.-D-2'-deoxyribonucleosides.
[0040] In certain embodiments, oligomeric compounds are provided
comprising a 5'-terminal 2'-fluoroethoxy modified nucleoside having
formula I wherein T.sub.1 is a phosphate group.
[0041] In certain embodiments, oligomeric compounds are provided
wherein each internucleoside linkage is a phosphodiester. In
certain embodiments, each internucleoside linkage is a
phosphorothioate. In certain embodiments, each internucleoside
linking group is, independently, a phosphodiester or a
phosphorothioate. In certain embodiments, each internucleoside
linking group is, independently, a phosphodiester,
phosphorothioate, chiral phosphorothioate, phosphorodithioate,
phosphotriester, aminoalkylphosphotriester, methyl phosphonate,
alkyl phosphonate, 5'-alkylene phosphonate, chiral phosphonate,
phosphinate, phosphoramidate, 3'-amino phosphoramidate,
aminoalkylphosphoramidate, thionophosphoramidate,
thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate
or boranophosphate.
[0042] In certain embodiments, oligomeric compounds are provided
comprising from 8 to about 18 linked monomeric subunits. In certain
embodiments, oligomeric compounds are provided comprising from 10
to about 16 linked monomeric subunits. In certain embodiments,
oligomeric compounds are provided comprising from 10 to about 14
linked monomeric subunits. In certain embodiments, oligomeric
compounds are provided comprising from 17 to about 26 linked
monomeric subunits. In certain embodiments, oligomeric compounds
are provided comprising from 18 to about 21 linked monomeric
subunits. In certain embodiments, oligomeric compounds are provided
comprising from 19 to about 20 linked monomeric subunits.
[0043] In certain embodiments, methods are provided comprising
administering an oligomeric compound to an animal, wherein the
animal is a mammal. In certain embodiments, methods are provided
comprising administering an oligomeric compound to an animal,
wherein the animal is a human.
[0044] In certain embodiments, methods are provided comprising
contacting a cell with an oligomeric compound that is complementary
to at least a portion of a target RNA wherein the target RNA is
selected from mRNA, pre-mRNA and micro RNA. In certain embodiments,
the target RNA is mRNA. In certain embodiments, the target RNA is
human mRNA.
[0045] In certain embodiments, methods are provided comprising
contacting a cell with an oligomeric compound that is complementary
to at least a portion of a target RNA wherein the target RNA is
cleaved thereby inhibiting its function.
[0046] In certain embodiments, methods are provided comprising
contacting a cell with an oligomeric compound and evaluating the
antisense activity of the oligomeric compound on the cell. In
certain embodiments, the evaluating comprises detecting the levels
of target RNA. In certain embodiments, the evaluating comprises
detecting the levels of a protein. In certain embodiments, the
evaluating comprises detection of one or more phenotypic
effects.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present disclosure provides methods of using oligomeric
compounds wherein each oligomeric compound comprises at least one
2'-fluoroethoxy modified nucleosides of formula I. The present
methods include contacting a cell with at least one of the
oligomeric compounds. The present methods also include
administering at least one of the oligomeric compounds to an
animal. In certain embodiments, the oligomeric compounds hybridize
to a portion of a target RNA resulting in loss of normal function
of the target RNA. The oligomeric compounds are also expected to be
useful as primers and probes in diagnostic applications.
[0048] In certain embodiments, methods are provided comprising
contacting a cell with an oligomeric compound, wherein said
oligomeric compound comprises at least one 2'-fluoroethoxy modified
nucleoside having formula I:
##STR00003##
wherein independently for each of said 2'-fluoroethoxy modified
nucleosides having formula I:
[0049] Bx is a heterocyclic base moiety;
[0050] one of T.sub.1 and T.sub.2 is an internucleoside linking
group attaching the 2'-fluoroethoxy modified nucleoside of formula
I to the oligomeric compound and the other of T.sub.1 and T.sub.2
is H or a hydroxyl protecting group, a 5' or 3'-terminal group or
an internucleoside linking group attaching the 2'-fluoroethoxy
modified nucleoside of formula I to the oligomeric compound;
[0051] R.sub.1 and R.sub.2 are each independently, H or F; and
[0052] wherein said oligomeric compound comprises from about 8 to
about 40 linked monomeric subunits and is complementary to at least
a portion of a target RNA.
[0053] In certain embodiments, the contacting step is performed on
a cell or group of cells. In certain embodiments, the cells are in
an animal. In a preferred embodiment, the cells are in a human.
[0054] In certain embodiments, methods are provided comprising
administering an oligomeric compound to an animal, wherein said
oligomeric compound comprises at least one 2'-fluoroethoxy modified
nucleoside having formula I:
##STR00004##
wherein independently for each of said 2'-fluoroethoxy modified
nucleosides having formula I:
[0055] Bx is a heterocyclic base moiety;
[0056] one of T.sub.1 and T.sub.2 is an internucleoside linking
group attaching the 2'-fluoroethoxy modified nucleoside of formula
I to the oligomeric compound and the other of T.sub.1 and T.sub.2
is H or a hydroxyl protecting group, a 5' or 3'-terminal group or
an internucleoside linking group attaching the 2'-fluoroethoxy
modified nucleoside of formula I to the oligomeric compound;
[0057] R.sub.1 and R.sub.2 are each independently, H or F; and
[0058] wherein said oligomeric compound comprises from about 8 to
about 40 linked monomeric subunits and is complementary to at least
a portion of a target RNA.
[0059] In one embodiment, the target RNA is selected from mRNA,
pre-mRNA and micro RNA. A preferred target RNA is mRNA with human
mRNA being more preferred.
[0060] The present methods can be evaluated using methods that are
known in the art. One particular method of evaluating the methods
is to evaluate the antisense activity of the oligomeric compounds
on a cell, a group of cells or in an animal. Preferred animals for
evaluating the antisense activity of the oligomeric compounds are
mammals with humans being more preferred.
[0061] Another method of evaluating the activity of the oligomeric
compounds is to monitor the levels of target RNA or one or more
proteins affected by a change in target RNA concentration.
Phenotypic effects can also be monitored.
[0062] In certain embodiments, the 2'-fluoroethoxy modified
nucleosides of formula I provided herein are useful for modifying
oligomeric compounds at one or more positions. Such modified
oligomeric compounds can be described as having a particular motif.
Motifs include without limitation, gapped motifs, hemimer motifs,
blockmer motifs, uniformly fully modified motifs, positionally
modified motifs and alternating motifs. In conjunction with these
motifs a wide variety of internucleoside linkages can also be used
including but not limited to phosphodiester and phosphorothioate
internucleoside linkages which can be incorporated uniformly or in
various combinations. The oligomeric compounds can further include
at least one 5' or 3' terminal group such as for example a
conjugate or reporter group. The positioning of the 2'-fluoroethoxy
modified nucleosides of formula I provided herein, the use of
linkage strategies and 5' or 3' terminal groups can be easily
optimized to enhance a desired activity for a selected target.
[0063] As used herein the term "motif" refers to the pattern
created by the relative positioning of monomer subunits within an
oligomeric compound wherein the pattern is determined by comparing
the sugar groups. The only determinant for the motif of an
oligomeric compound is the differences or lack of differences
between the sugar groups. As used herein the term "sugar group" as
it applies to motifs includes naturally occurring sugars having a
furanose ring, sugars having a modified furanose ring and sugar
surrogates wherein the furanose ring has been replaced with another
ring system such as for example a morpholino or hexitol ring
system. When each sugar group is the same (DNA, RNA, modified or
surrogate) the motif is termed uniformly fully modified. When two
or more types of sugar groups are present the motif is defined by
the pattern created from the positioning of monomer subunits having
one type of sugar group relative to the positioning of monomer
subunits having different types of sugar groups within an
oligomeric compound.
[0064] Illustrative examples of some different types of sugar
groups useful in the preparation of oligomeric compounds having
motifs include without limitation, .beta.-D-ribose,
.beta.-D-2'-deoxyribose, substituted sugars (such as 2', 5' and bis
substituted sugars), 4'-S-sugars (such as 4'-S-ribose,
4'-S-2'-deoxyribose and 4'-S-2'-substituted ribose), bicyclic
modified sugars (such as the 2'-O--CH.sub.2-4' or
2'-O--(CH.sub.2).sub.2-4' bridged ribose derived bicyclic sugars)
and sugar surrogates (such as when the ribose ring has been
replaced with a morpholino or a hexitol ring system). The type of
heterocyclic base and internucleoside linkage used at each position
is variable and is not a factor in determining the motif. The
presence of one or more other groups including but not limited to
capping groups, conjugate groups and other 5' or 3'-terminal groups
is also not a factor in determining the motif.
[0065] Representative U.S. patents that teach the preparation of
motifs include without limitation, 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.
Motifs are also disclosed in International Applications
PCT/US2005/019219, filed Jun. 2, 2005 and published as WO
2005/121371 on Dec. 22, 2005 and PCT/US2005/019220, filed Jun. 2,
2005 and published as WO 2005/121372 on Dec. 22, 2005; each of
which is incorporated by reference herein in its entirety.
[0066] As used herein the term "alternating motif" refers to a an
oligomeric compound comprising a contiguous sequence of linked
monomer subunits wherein the monomer subunits have two different
types of sugar groups that alternate for essentially the entire
sequence of the oligomeric compound. Oligomeric compounds having an
alternating motif can be described by the formula:
5'-A(-L-B-L-A).sub.n(-L-B).sub.nn-3' where A and B are monomer
subunits that have different sugar groups, each L is,
independently, an internucleoside linking group, n is from about 4
to about 12 and nn is 0 or 1. The heterocyclic base and
internucleoside linkage is independently variable at each position.
The motif further optionally includes the use of one or more other
groups including but not limited to capping groups, conjugate
groups and other 5' or 3'-terminal groups. This permits alternating
oligomeric compounds from about 9 to about 26 monomer subunits in
length. This length range is not meant to be limiting as longer and
shorter oligomeric compounds are also amenable to oligomeric
compounds provided herein. In certain embodiments, each A or each B
comprises 2'-fluoroethoxy modified nucleosides of formula I.
[0067] As used herein the term "uniformly fully modified motif"
refers to an oligomeric compound comprising a contiguous sequence
of linked monomer subunits that each have the same type of sugar
group. The heterocyclic base and internucleoside linkage is
independently variable at each position. The motif further
optionally includes the use of one or more other groups including
but not limited to capping groups, conjugate groups and other 5' or
3'-terminal groups. In certain embodiments, the uniformly fully
modified motif includes a contiguous sequence of 2'-fluoroethoxy
modified nucleosides of formula I. In certain embodiments, one or
both of the 5' and 3'-ends of the contiguous sequence of
2'-fluoroethoxy modified nucleosides of formula I, comprise 5' or
3'-terminal groups such as one or more unmodified nucleosides.
[0068] As used herein the term "hemimer motif" refers to an
oligomeric compound comprising a contiguous sequence of monomer
subunits that each have the same type of sugar group with a further
short contiguous sequence of monomer subunits located at the 5' or
the 3' end that have a different type of sugar group. The
heterocyclic base and internucleoside linkage is independently
variable at each position. The motif further optionally includes
the use of one or more other groups including but not limited to
capping groups, conjugate groups and other 5' or 3'-terminal
groups. In general, a hemimer is an oligomeric compound of uniform
sugar groups further comprising a short region (1, 2, 3, 4 or about
5 monomer subunits) having uniform but different sugar groups
located on either the 3' or the 5' end of the oligomeric
compound.
[0069] In certain embodiments, the hemimer motif comprises a
contiguous sequence of from about 10 to about 28 monomer subunits
having one type of sugar group with from 1 to 5 or from 2 to about
5 monomer subunits having a second type of sugar group located at
one of the termini. In certain embodiments, the hemimer is a
contiguous sequence of from about 8 to about 20
.beta.-D-2'-deoxyribonucleosides having from 1-12 contiguous
2'-fluoroethoxy modified nucleosides of formula I located at one of
the termini. In certain embodiments, the hemimer is a contiguous
sequence of from about 8 to about 20
.beta.-D-2'-deoxyribonucleosides having from 1-5 contiguous
2'-fluoroethoxy modified nucleosides of formula I located at one of
the termini. In certain embodiments, the hemimer is a contiguous
sequence of from about 12 to about 18
.beta.-D-2'-deoxyribonucleosides having from 1-3 contiguous
2'-fluoroethoxy modified nucleosides of formula I located at one of
the termini. In certain embodiments, the hemimer is a contiguous
sequence of from about 10 to about 14
.beta.-D-2'-deoxyribonucleosides having from 1-3 contiguous
2'-fluoroethoxy modified nucleosides of formula I located at one of
the termini.
[0070] As used herein the term "blockmer motif" refers to an
oligomeric compound comprising an otherwise contiguous sequence of
monomer subunits wherein the sugar groups of each monomer subunit
is the same except for an interrupting internal block of contiguous
monomer subunits having a different type of sugar group. The
heterocyclic base and internucleoside linkage is independently
variable at each position of a blocker oligomeric compound. The
motif further optionally includes the use of one or more other
groups including but not limited to capping groups, conjugate
groups and other 5' or 3'-terminal groups. A blockmer overlaps
somewhat with a gapmer in the definition but typically only the
monomer subunits in the block have non-naturally occurring sugar
groups in a blockmer and only the monomer subunits in the external
regions have non-naturally occurring sugar groups in a gapmer with
the remainder of monomer subunits in the blockmer or gapmer being
.beta.-D-2'-deoxyribonucleosides or .beta.-D-ribonucleosides. In
certain embodiments, blockmer oligomeric compounds are provided
herein wherein all of the monomer subunits comprise non-naturally
occurring sugar groups.
[0071] As used herein the term "positionally modified motif" is
meant to include an otherwise contiguous sequence of monomer
subunits having one type of sugar group that is interrupted with
two or more regions of from 1 to about 5 contiguous monomer
subunits having another type of sugar group. Each of the two or
more regions of from 1 to about 5 contiguous monomer subunits are
independently uniformly modified with respect to the type of sugar
group. In certain embodiments, each of the two or more regions have
the same type of sugar group. In certain embodiments, each of the
two or more regions have a different type of sugar group. In
certain embodiments, each of the two or more regions,
independently, have the same or a different type of sugar group.
The heterocyclic base and internucleoside linkage is independently
variable at each position of a positionally modified oligomeric
compound. The motif further optionally includes the use of one or
more other groups including but not limited to capping groups,
conjugate groups and other 5' or 3'-terminal groups. In certain
embodiments, positionally modified oligomeric compounds are
provided comprising a sequence of from 8 to 20
.beta.-D-2'-deoxyribonucleosides that further includes two or three
regions of from 2 to about 5 contiguous 2'-fluoroethoxy modified
nucleosides of formula I each. Positionally modified oligomeric
compounds are distinguished from gapped motifs, hemimer motifs,
blockmer motifs and alternating motifs because the pattern of
regional substitution defined by any positional motif does not fit
into the definition provided herein for one of these other motifs.
The term positionally modified oligomeric compound includes many
different specific substitution patterns.
[0072] As used herein the term "gapmer" or "gapped oligomeric
compound" refers to an oligomeric compound having two external
regions or wings and an internal region or gap. The three regions
form a contiguous sequence of monomer subunits with the sugar
groups of the external regions being different than the sugar
groups of the internal region and wherein the sugar group of each
monomer subunit within a particular region is essentially the same.
In certain embodiments, each monomer subunit within a particular
region has the same sugar group. When the sugar groups of the
external regions are the same the gapmer is a symmetric gapmer and
when the sugar group used in the 5'-external region is different
from the sugar group used in the 3'-external region, the gapmer is
an asymmetric gapmer. In certain embodiments, the external regions
are small (each independently 1, 2, 3, 4 or about 5 monomer
subunits) and the monomer subunits comprise non-naturally occurring
sugar groups with the internal region comprising
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, the
external regions each, independently, comprise from 1 to about 5
monomer subunits having non-naturally occurring sugar groups and
the internal region comprises from 6 to 18 unmodified nucleosides.
The internal region or the gap generally comprises
.beta.-D-2'-deoxyribonucleosides but can comprise non-naturally
occurring sugar groups. The heterocyclic base and internucleoside
linkage is independently variable at each position of a gapped
oligomeric compound. The motif further optionally includes the use
of one or more other groups including but not limited to capping
groups, conjugate groups and other 5' or 3'-terminal groups.
[0073] In certain embodiments, the gapped oligomeric compounds
comprise an internal region of .beta.-D-2'-deoxyribonucleosides
with one of the external regions comprising 2'-fluoroethoxy
modified nucleosides of formula I as provided herein. In certain
embodiments, the gapped oligomeric compounds comprise an internal
region of .beta.-D-2'-deoxyribonucleosides with both of the
external regions comprising 2'-fluoroethoxy modified nucleosides of
formula I as provided herein. In certain embodiments, gapped
oligomeric compounds are provided herein wherein all of the monomer
subunits comprise non-naturally occurring sugar groups.
[0074] In certain embodiments, gapped oligomeric compounds are
provided comprising one or two 2'-fluoroethoxy modified nucleosides
of formula I at the 5'-end, two or three 2'-fluoroethoxy modified
nucleosides of formula I at the 3'-end and an internal region of
from 10 to 16 .beta.-D-2'-deoxyribonucleosides. In certain
embodiments, gapped oligomeric compounds are provided comprising
one 2'-fluoroethoxy modified nucleosides of formula I at the
5'-end, two 2'-fluoroethoxy modified nucleosides of formula I at
the 3'-end and an internal region of from 10 to 16
.beta.-D-2'-deoxyribonucleosides. In certain embodiments, gapped
oligomeric compounds are provided comprising one 2'-fluoroethoxy
modified nucleosides of formula I at the 5'-end, two
2'-fluoroethoxy modified nucleosides of formula I at the 3'-end and
an internal region of from 10 to 14
.beta.-D-2'-deoxyribonucleosides.
[0075] In certain embodiments, gapped oligomeric compounds are
provided that are from about 10 to about 21 monomer subunits in
length. In certain embodiments, gapped oligomeric compounds are
provided that are from about 12 to about 16 monomer subunits in
length. In certain embodiments, gapped oligomeric compounds are
provided that are from about 12 to about 14 monomer subunits in
length.
[0076] The terms "substituent" and "substituent group," as used
herein, are meant to include groups that are typically added to
other groups or parent compounds to enhance desired properties or
provide other desired effects. Substituent groups can be protected
or unprotected and can be added to one available site or to many
available sites in a parent compound. Substituent groups may also
be further substituted with other substituent groups and may be
attached directly or via a linking group such as an alkyl or
hydrocarbyl group to a parent compound.
[0077] Substituent groups amenable herein include without
limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl
(--C(O)R.sub.aa), carboxyl (--C(O)O--R.sub.aa), aliphatic groups,
alicyclic groups, alkoxy, substituted oxy (--O--R.sub.aa), aryl,
aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino
(--N(R.sub.bb)(R.sub.cc)), imino(.dbd.NR.sub.bb), amido
(--C(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)R.sub.aa), azido
(--N.sub.3), nitro (--NO.sub.2), cyano (--CN), carbamido
(--OC(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)OR.sub.aa),
ureido (--N(R.sub.bb)C(O)N(R.sub.bb)(R.sub.cc)), thioureido
(--N(R.sub.bb)C(S)N(R.sub.bb)(R.sub.cc)), guanidinyl
(--N(R.sub.bb)C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc)), amidinyl
(--C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc) or
N(R.sub.bb)C(.dbd.NR.sub.bb)(R.sub.aa)), thiol (--SR.sub.bb),
sulfinyl (--S(O)R.sub.bb), sulfonyl (--S(O).sub.2R.sub.bb) and
sulfonamidyl (--S(O).sub.2N(R.sub.bb)(R.sub.cc) or
--N(R.sub.bb)S--(O).sub.2R.sub.bb). Wherein each R.sub.aa, R.sub.bb
and R.sub.cc is, independently, H, an optionally linked chemical
functional group or a further substituent group with a preferred
list including without limitation, H, alkyl, alkenyl, alkynyl,
aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,
heterocyclic and heteroarylalkyl. Selected substituents within the
compounds described herein are present to a recursive degree.
[0078] In this context, "recursive substituent" means that a
substituent may recite another instance of itself. Because of the
recursive nature of such substituents, theoretically, a large
number may be present in any given claim. One of ordinary skill in
the art of medicinal chemistry and organic chemistry understands
that the total number of such substituents is reasonably limited by
the desired properties of the compound intended. Such properties
include, by way of example and not limitation, physical properties
such as molecular weight, solubility or log P, application
properties such as activity against the intended target and
practical properties such as ease of synthesis.
[0079] Recursive substituents are an intended aspect of the
invention. One of ordinary skill in the art of medicinal and
organic chemistry understands the versatility of such substituents.
To the degree that recursive substituents are present in a claim of
the invention, the total number will be determined as set forth
above.
[0080] The terms "stable compound" and "stable structure" as used
herein are meant to indicate a compound that is sufficiently robust
to survive isolation to a useful degree of purity from a reaction
mixture, and formulation into an efficacious therapeutic agent.
Only stable compounds are contemplated herein.
[0081] The term "alkyl," as used herein, refers to a saturated
straight or branched hydrocarbon radical containing up to twenty
four carbon atoms. Examples of alkyl groups include without
limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl,
octyl, decyl, dodecyl and the like. Alkyl groups typically include
from 1 to about 24 carbon atoms, more typically from 1 to about 12
carbon atoms (C.sub.1-C.sub.12 alkyl) with from 1 to about 6 carbon
atoms being more preferred. The term "lower alkyl" as used herein
includes from 1 to about 6 carbon atoms. Alkyl groups as used
herein may optionally include one or more further substitutent
groups.
[0082] The term "alkenyl," as used herein, refers to a straight or
branched hydrocarbon chain radical containing up to twenty four
carbon atoms and having at least one carbon-carbon double bond.
Examples of alkenyl groups include without limitation, ethenyl,
propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as
1,3-butadiene and the like. Alkenyl groups typically include from 2
to about 24 carbon atoms, more typically from 2 to about 12 carbon
atoms with from 2 to about 6 carbon atoms being more preferred.
Alkenyl groups as used herein may optionally include one or more
further substitutent groups.
[0083] The term "alkynyl," as used herein, refers to a straight or
branched hydrocarbon radical containing up to twenty four carbon
atoms and having at least one carbon-carbon triple bond. Examples
of alkynyl groups include, without limitation, ethynyl, 1-propynyl,
1-butynyl, and the like. Alkynyl groups typically include from 2 to
about 24 carbon atoms, more typically from 2 to about 12 carbon
atoms with from 2 to about 6 carbon atoms being more preferred.
Alkynyl groups as used herein may optionally include one or more
further substitutent groups.
[0084] The term "acyl," as used herein, refers to a radical formed
by removal of a hydroxyl group from an organic acid and has the
general Formula --C(O)--X where X is typically aliphatic, alicyclic
or aromatic. Examples include aliphatic carbonyls, aromatic
carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic
sulfinyls, aromatic phosphates, aliphatic phosphates and the like.
Acyl groups as used herein may optionally include further
substitutent groups.
[0085] The term "alicyclic" refers to a cyclic ring system wherein
the ring is aliphatic. The ring system can comprise one or more
rings wherein at least one ring is aliphatic. Preferred alicyclics
include rings having from about 5 to about 9 carbon atoms in the
ring. Alicyclic as used herein may optionally include further
substitutent groups.
[0086] The term "aliphatic," as used herein, refers to a straight
or branched hydrocarbon radical containing up to twenty four carbon
atoms wherein the saturation between any two carbon atoms is a
single, double or triple bond. An aliphatic group preferably
contains from 1 to about 24 carbon atoms, more typically from 1 to
about 12 carbon atoms with from 1 to about 6 carbon atoms being
more preferred. The straight or branched chain of an aliphatic
group may be interrupted with one or more heteroatoms that include
nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups
interrupted by heteroatoms include without limitation, polyalkoxys,
such as polyalkylene glycols, polyamines, and polyimines. Aliphatic
groups as used herein may optionally include further substitutent
groups.
[0087] The term "alkoxy," as used herein, refers to a radical
formed between an alkyl group and an oxygen atom wherein the oxygen
atom is used to attach the alkoxy group to a parent molecule.
Examples of alkoxy groups include without limitation, methoxy,
ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy,
n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used
herein may optionally include further substitutent groups.
[0088] The term "aminoalkyl" as used herein, refers to an amino
substituted C.sub.1-C.sub.12 alkyl radical. The alkyl portion of
the radical forms a covalent bond with a parent molecule. The amino
group can be located at any position and the aminoalkyl group can
be substituted with a further substituent group at the alkyl and/or
amino portions.
[0089] The terms "aralkyl" and "arylalkyl," as used herein, refer
to an aromatic group that is covalently linked to a
C.sub.1-C.sub.12 alkyl radical. The alkyl radical portion of the
resulting aralkyl (or arylalkyl) group forms a covalent bond with a
parent molecule. Examples include without limitation, benzyl,
phenethyl and the like. Aralkyl groups as used herein may
optionally include further substitutent groups attached to the
alkyl, the aryl or both groups that form the radical group.
[0090] The terms "aryl" and "aromatic," as used herein, refer to a
mono- or polycyclic carbocyclic ring system radicals having one or
more aromatic rings. Examples of aryl groups include without
limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl
and the like. Preferred aryl ring systems have from about 5 to
about 20 carbon atoms in one or more rings. Aryl groups as used
herein may optionally include further substitutent groups.
[0091] The terms "halo" and "halogen," as used herein, refer to an
atom selected from fluorine, chlorine, bromine and iodine.
[0092] The terms "heteroaryl," and "heteroaromatic," as used
herein, refer to a radical comprising a mono- or poly-cyclic
aromatic ring, ring system or fused ring system wherein at least
one of the rings is aromatic and includes one or more heteroatoms.
Heteroaryl is also meant to include fused ring systems including
systems where one or more of the fused rings contain no
heteroatoms. Heteroaryl groups typically include one ring atom
selected from sulfur, nitrogen or oxygen. Examples of heteroaryl
groups include without limitation, pyridinyl, pyrazinyl,
pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,
isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,
quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl,
quinoxalinyl and the like. Heteroaryl radicals can be attached to a
parent molecule directly or through a linking moiety such as an
aliphatic group or hetero atom. Heteroaryl groups as used herein
may optionally include further substitutent groups.
[0093] The term "heteroarylalkyl," as used herein, refers to a
heteroaryl group as previously defined that further includes a
covalently attached C.sub.1-C.sub.12 alkyl radical. The alkyl
radical portion of the resulting heteroarylalkyl group is capable
of forming a covalent bond with a parent molecule. Examples include
without limitation, pyridinylmethyl, pyrimidinylethyl,
napthyridinylpropyl and the like. Heteroarylalkyl groups as used
herein may optionally include further substitutent groups on one or
both of the heteroaryl or alkyl portions.
[0094] The term "heterocyclic radical" as used herein, refers to a
radical mono-, or poly-cyclic ring system that includes at least
one heteroatom and is unsaturated, partially saturated or fully
saturated, thereby including heteroaryl groups. Heterocyclic is
also meant to include fused ring systems wherein one or more of the
fused rings contain at least one heteroatom and the other rings can
contain one or more heteroatoms or optionally contain no
heteroatoms. A heterocyclic radical typically includes at least one
atom selected from sulfur, nitrogen or oxygen. Examples of
heterocyclic radicals include, [1,3]dioxolanyl, pyrrolidinyl,
pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,
piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,
morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,
pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as
used herein may optionally include further substitutent groups.
[0095] The term "hydrocarbyl" includes radical groups that comprise
C, O and H. Included are straight, branched and cyclic groups
having any degree of saturation. Such hydrocarbyl groups can
include one or more heteroatoms selected from N, O and S and can be
further mono or poly substituted with one or more substituent
groups.
[0096] The term "mono or poly cyclic structure" as used herein
includes all ring systems selected from single or polycyclic
radical ring systems wherein the rings are fused or linked and is
meant to be inclusive of single and mixed ring systems individually
selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl,
arylalkyl, heterocyclic, heteroaryl, heteroaromatic and
heteroarylalkyl. Such mono and poly cyclic structures can contain
rings that each have the same level of saturation or each,
independently, have varying degrees of saturation including fully
saturated, partially saturated or fully unsaturated. Each ring can
comprise ring atoms selected from C, N, O and S to give rise to
heterocyclic rings as well as rings comprising only C ring atoms
which can be present in a mixed motif such as for example
benzimidazole wherein one ring has only carbon ring atoms and the
fused ring has two nitrogen atoms. The mono or poly cyclic
structures can be further substituted with substituent groups such
as for example phthalimide which has two .dbd.O groups attached to
one of the rings. Mono or poly cyclic structures can be attached to
parent molecules using various strategies such as directly through
a ring atom, through a substituent group or through a bifunctional
linking moiety.
[0097] The term "oxo" refers to the group (.dbd.O).
[0098] Linking groups or bifunctional linking moieties such as
those known in the art are useful for attachment of chemical
functional groups, conjugate groups, reporter groups and other
groups to selective sites in a parent compound such as for example
an oligomeric compound. In general, a bifunctional linking moiety
comprises a hydrocarbyl moiety having two functional groups. One of
the functional groups is selected to bind to a parent molecule or
compound of interest and the other is selected to bind to
essentially any selected group such as a chemical functional group
or a conjugate group. In some embodiments, the linker comprises a
chain structure or a polymer of repeating units such as ethylene
glycols or amino acid units. Examples of functional groups that are
routinely used in bifunctional linking moieties include without
limitation, electrophiles for reacting with nucleophilic groups and
nucleophiles for reacting with electrophilic groups. In some
embodiments, bifunctional linking moieties include amino, hydroxyl,
carboxylic acid, thiol, unsaturations (e.g., double or triple
bonds), and the like. Some nonlimiting examples of bifunctional
linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO),
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)
and 6-aminohexanoic acid (AHEX or AHA). Other linking groups
include without limitation, substituted C.sub.1-C.sub.10 alkyl,
substituted or unsubstituted C.sub.2-C.sub.10 alkenyl or
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein a
nonlimiting list of preferred substituent groups includes hydroxyl,
amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy,
halogen, alkyl, aryl, alkenyl and alkynyl.
[0099] In certain embodiments, the oligomeric compounds as provided
herein can be modified by covalent attachment of one or more
conjugate groups. In general, conjugate groups modify one or more
properties of the oligomeric compounds they are attached to. Such
oligonucleotide properties include without limitation,
pharmacodynamics, pharmacokinetics, binding, absorption, cellular
distribution, cellular uptake, charge and clearance. Conjugate
groups are routinely used in the chemical arts and are linked
directly or via an optional linking moiety or linking group to a
parent compound such as an oligomeric compound. A preferred list of
conjugate groups includes without limitation, intercalators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
thioethers, polyethers, cholesterols, thiocholesterols, cholic acid
moieties, folate, lipids, phospholipids, biotin, phenazine,
phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,
rhodamines, coumarins and dyes.
[0100] In certain embodiments, the oligomeric compounds as provided
herein can be modified by covalent attachment of one or more 5' or
3'-terminal groups. The terms "5' or 3'-terminal groups",
"5-terminal group" and "3'-terminal group" as used herein are meant
to include useful groups known to the art skilled that can be
placed on one or both of the 5' and 3'-ends of an oligomeric
compound respectively, for various purposes such as enabling the
tracking of the oligomeric compound (a fluorescent label or other
reporter group), improving the pharmacokinetics or pharmacodynamics
of the oligomeric compound (a group for enhancing uptake and/or
delivery) or enhancing one or more other desirable properties of
the oligomeric compound (a group for improving nuclease stability
or binding affinity). In certain embodiments, 5' and 3'-terminal
groups include without limitation, modified or unmodified
nucleosides; two or more linked nucleosides that are independently,
modified or unmodified; conjugate groups; capping groups; phosphate
moieties; and protecting groups.
[0101] The term "phosphate moiety" as used herein, refers to a
terminal phosphate group that includes phosphates as well as
modified phosphates. The phosphate moiety can be located at either
terminus but is preferred at the 5'-terminal nucleoside. In certain
embodiments, the terminal phosphate is unmodified having the
formula --O--P(.dbd.O)(OH)OH. In certain embodiments, the terminal
phosphate is modified such that one or more of the O and OH groups
are replaced with H, O, S, N(R) or alkyl where R is H, an amino
protecting group or unsubstituted or substituted alkyl. In certain
embodiments, the terminal phosphate is modified to have the
formula:
##STR00005##
wherein:
[0102] R.sub.a and R.sub.c are each, independently, OH, SH,
C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy, amino
or substituted amino; and
[0103] R.sub.b and R.sub.d are each, independently, O or S.
[0104] The term "protecting group," as used herein, refers to a
labile chemical moiety which is known in the art to protect
reactive groups including without limitation, hydroxyl, amino and
thiol groups, against undesired reactions during synthetic
procedures. Protecting groups are typically used selectively and/or
orthogonally to protect sites during reactions at other reactive
sites and can then be removed to leave the unprotected group as is
or available for further reactions. Protecting groups as known in
the art are described generally in Greene's Protective Groups in
Organic Synthesis, 4th edition, John Wiley & Sons, New York,
2007.
[0105] Groups can be selectively incorporated into oligomeric
compounds as provided herein as precursors. For example an amino
group can be placed into a compound as provided herein as an azido
group that can be chemically converted to the amino group at a
desired point in the synthesis. Generally, groups are protected or
present as precursors that will be inert to reactions that modify
other areas of the parent molecule for conversion into their final
groups at an appropriate time. Further representative protecting or
precursor groups are discussed in Agrawal et al., Protocols for
Oligonucleotide Conjugates, Humana Press; New Jersey, 1994, 26,
1-72.
[0106] The term "orthogonally protected" refers to functional
groups which are protected with different classes of protecting
groups, wherein each class of protecting group can be removed in
any order and in the presence of all other classes (see, Barany et
al., J. Am. Chem. Soc., 1977, 99, 7363-7365; Barany et al., J. Am.
Chem. Soc., 1980, 102, 3084-3095). Orthogonal protection is widely
used in for example automated oligonucleotide synthesis. A
functional group is deblocked in the presence of one or more other
protected functional groups which is not affected by the deblocking
procedure. This deblocked functional group is reacted in some
manner and at some point a further orthogonal protecting group is
removed under a different set of reaction conditions. This allows
for selective chemistry to arrive at a desired compound or
oligomeric compound.
[0107] Examples of hydroxyl protecting groups include without
limitation, acetyl, t-butyl, t-butoxymethyl, methoxymethyl,
tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl,
p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,
diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl
(ACE),2-trimethylsilylethyl, trimethylsilyl, triethylsilyl,
t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,
[(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl,
trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl,
p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate,
triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMT),
trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP),
9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl
(MOX). Wherein more commonly used hydroxyl protecting groups
include without limitation, benzyl, 2,6-dichlorobenzyl,
t-butyldimethylsilyl, t-butyl-diphenylsilyl, benzoyl, mesylate,
tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and
9-(p-methoxyphenyl)xanthine-9-yl (MOX).
[0108] Examples of amino protecting groups include without
limitation, carbamate-protecting groups, such as
2-trimethylsilylethoxycarbonyl (Teoc),
1-methyl-1-(4-biphenyl)-ethoxycarbonyl (Bpoc), t-butoxycarbonyl
(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl
(Fmoc), and benzyloxycarbonyl (Cbz); amide-protecting groups, such
as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;
sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and
imine- and cyclic imide-protecting groups, such as phthalimido and
dithiasuccinoyl.
[0109] Examples of thiol protecting groups include without
limitation, triphenylmethyl (trityl), benzyl (Bn), and the
like.
[0110] In certain embodiments, oligomeric compounds as provided
herein can be prepared having one or more optionally protected
phosphorus containing internucleoside linkages. Representative
protecting groups for phosphorus containing internucleoside
linkages such as phosphodiester and phosphorothioate linkages
include .beta.-cyanoethyl, diphenylsilylethyl,
.delta.-cyanobutenyl, cyano p-xylyl (CPX),
N-methyl-N-trifluoroacetyl ethyl (META), acetoxy phenoxy ethyl
(APE) and butene-4-yl groups. See for example U.S. Pat. Nos.
4,725,677 and Re. 34,069 (.beta.-cyanoethyl); Beaucage et al.,
Tetrahedron, 1993, 49(10), 1925-1963; Beaucage et al., Tetrahedron,
1993, 49(46), 10441-10488; Beaucage et al., Tetrahedron, 1992,
48(12), 2223-2311.
[0111] In certain embodiments, compounds having reactive phosphorus
groups are provided that are useful for forming internucleoside
linkages including for example phosphodiester and phosphorothioate
internucleoside linkages. Such reactive phosphorus groups are known
in the art and contain phosphorus atoms in P.sup.III or P.sup.V
valence state including, but not limited to, phosphoramidite,
H-phosphonate, phosphate triesters and phosphorus containing chiral
auxiliaries. In certain embodiments, reactive phosphorus groups are
selected from diisopropylcyanoethoxy phosphoramidite
(--O*--P[N[(CH(CH.sub.3).sub.2].sub.2]O(CH.sub.2).sub.2CN) and
H-phosphonate (--O*--P(.dbd.O)(H)OH), wherein the O* is provided
from the Markush group for the monomer. A preferred synthetic solid
phase synthesis utilizes phosphoramidites (P.sup.III chemistry) as
reactive phosphites. The intermediate phosphite compounds are
subsequently oxidized to the phosphate or thiophosphate (P.sup.V
chemistry) using known methods to yield, phosphodiester or
phosphorothioate internucleoside linkages. Additional reactive
phosphates and phosphites are disclosed in Tetrahedron Report
Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48,
2223-2311).
[0112] As used herein the term "internucleoside linkage" or
"internucleoside linking group" is meant to include all manner of
internucleoside linking groups known in the art including but not
limited to, phosphorus containing internucleoside linking groups
such as phosphodiester and phosphorothioate, and non-phosphorus
containing internucleoside linking groups such as formacetyl and
methyleneimino. Internucleoside linkages also includes neutral
non-ionic internucleoside linkages such as amide-3
(3'-CH.sub.2--C(.dbd.O)--N(H)-5'), amide-4
(3'-CH.sub.2--N(H)--C(.dbd.O)-5') and methylphosphonate wherein a
phosphorus atom is not always present.
[0113] In certain embodiments, oligomeric compounds as provided
herein can be prepared having one or more internucleoside linkages
containing modified e.g. non-naturally occurring internucleoside
linkages. The two main classes of internucleoside linkages are
defined by the presence or absence of a phosphorus atom. Modified
internucleoside linkages having a phosphorus atom include without
limitation, 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. Oligonucleotides having inverted polarity
can comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0114] Representative U.S. patents that teach the preparation of
the above phosphorus containing linkages include without
limitation, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,194,599; 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; 5,527,899; 5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,565,555; 5,571,799; 5,587,361;
5,625,050; 5,672,697 and 5,721,218, certain of which are commonly
owned with this application, and each of which is herein
incorporated by reference.
[0115] In certain embodiments, oligomeric compounds as provided
herein can be prepared having one or more non-phosphorus containing
internucleoside linkages. Such oligomeric compounds include without
limitation, those 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 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.
[0116] Representative U.S. patents that teach the preparation of
the above oligonucleosides include without limitation, 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;
5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;
5,677,437; 5,677,439; 5,646,269 and 5,792,608, certain of which are
commonly owned with this application, and each of which is herein
incorporated by reference.
[0117] As used herein the phrase "neutral internucleoside linkage"
is intended to include internucleoside linkages that are non-ionic.
Neutral internucleoside linkages include without limitation,
phosphotriesters, methylphosphonates, MMI
(3'-CH.sub.2--N(CH.sub.3)--O-5'), amide-3
(3'-CH.sub.2--C(.dbd.O)--N(H)-5'), amide-4
(3'-CH.sub.2--N(H)--C(.dbd.O)-5'), formacetal
(3'-O--CH.sub.2--O-5'), and thioformacetal (3'-S--CH.sub.2--O-5').
Further neutral internucleoside linkages include nonionic linkages
comprising siloxane (dialkylsiloxane), carboxylate ester,
carboxamide, sulfide, sulfonate ester and amides (See for example:
Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and
P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4,
40-65). Further neutral internucleoside linkages include nonionic
linkages comprising mixed N, O, S and CH.sub.2 component parts.
[0118] The 2'-fluoroethoxy modified nucleosides of formula I
provided herein can be prepared by any of the applicable techniques
of organic synthesis, as, for example, illustrated in the examples
below. Many such techniques are well known in the art. However,
many of the known techniques are elaborated in Compendium of
Organic Synthetic Methods, John Wiley & Sons, New York: Vol. 1,
Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison
and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade,
1977; Vol. 4, Leroy G. Wade Jr., 1980; Vol. 5, Leroy G. Wade Jr.,
1984; and Vol. 6, Michael B. Smith; as well as March, J., Advanced
Organic Chemistry, 3rd Edition, John Wiley & Sons, New York,
1985; Comprehensive Organic Synthesis. Selectivity, Strategy &
Efficiency in Modern Organic Chemistry, in 9 Volumes, Barry M.
Trost, Editor-in-Chief, Pergamon Press, New York, 1993; Advanced
Organic Chemistry, Part B: Reactions and Synthesis, 4th Edition;
Carey and Sundberg, Kluwer Academic/Plenum Publishers, New York,
2001; Advanced Organic Chemistry, Reactions, Mechanisms, and
Structure, 2nd Edition, March, McGraw Hill, 1977; Greene, T. W.,
and Wutz, P. G. M., Protecting Groups in Organic Synthesis, 4th
Edition, John Wiley & Sons, New York, 1991; and Larock, R. C.,
Comprehensive Organic Transformations, 2nd Edition, John Wiley
& Sons, New York, 1999.
[0119] The compounds described herein contain one or more
asymmetric centers and thus give rise to enantiomers,
diastereomers, and other stereoisomeric forms that may be defined,
in terms of absolute stereochemistry, as (R)- or (S)-, .alpha. or
.beta., or as (D)- or (L)- such as for amino acids. Included herein
are all such possible isomers, as well as their racemic and
optically pure forms. Optical isomers may be prepared from their
respective optically active precursors by the procedures described
above, or by resolving the racemic mixtures. The resolution can be
carried out in the presence of a resolving agent, by chromatography
or by repeated crystallization or by some combination of these
techniques which are known to those skilled in the art. Further
details regarding resolutions can be found in Jacques, et al.,
Enantiomers, Racemates, and Resolutions, John Wiley & Sons,
1981. When the compounds described herein contain olefinic double
bonds, other unsaturation, or other centers of geometric asymmetry,
and unless specified otherwise, it is intended that the compounds
include both E and Z geometric isomers or cis- and trans-isomers.
Likewise, all tautomeric forms are also intended to be included.
The configuration of any carbon-carbon double bond appearing herein
is selected for convenience only and is not intended to limit a
particular configuration unless the text so states.
[0120] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base moiety. The two most common classes of such
heterocyclic bases are purines and pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. The respective ends of this linear
polymeric structure can be joined to form a circular structure by
hybridization or by formation of a covalent bond. However, open
linear structures are generally desired. Within the oligonucleotide
structure, the phosphate groups are commonly referred to as forming
the internucleoside linkages of the oligonucleotide. The normal
internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester
linkage.
[0121] The term "nucleotide mimetic" as used herein is meant to
include monomers that incorporate into oligomeric compounds with
sugar and linkage surrogate groups, such as for example peptide
nucleic acids (PNA) or morpholinos (linked by
--N(H)--C(.dbd.O)--O--). In general, the heterocyclic base at each
position is maintained for hybridization to a nucleic acid target
but the sugar and linkage is replaced with surrogate groups that
are expected to function similar to native groups but have one or
more enhanced properties.
[0122] As used herein the term "nucleoside mimetic" is intended to
include those structures used to replace the sugar and the base at
one or more positions of an oligomeric compound. Examples of
nucleoside mimetics include without limitation replacement of the
heterocyclic base moiety with a mimetic thereof such as a
phenoxazine moiety (for example the
9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one group, also referred
to as a G-clamp which forms four hydrogen bonds when hybridized
with a guanosine base) and further replacement of the sugar group
with a group such as for example a morpholino, a cyclohexenyl or a
bicyclo[3.1.0]hexyl.
[0123] As used herein the term "modified nucleoside" is meant to
include all manner of modified nucleosides that can be incorporated
into an oligomeric compound using oligomer synthesis. The term is
intended to include modifications made to a nucleoside such as
modified stereochemical configurations, one or more substitutions,
and deletion of groups as opposed to the use of surrogate groups
which are described elsewhere herein. The term includes nucleosides
having a furanose sugar (or 4'-S analog) portion and can include a
heterocyclic base but abasic modified nucleosides are also
envisioned. One group of representative modified nucleosides
includes without limitation, substituted nucleosides (such as 2',
5', and/or 4' substituted nucleosides) 4'-S-modified nucleosides,
(such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and
4'-S-2'-substituted ribonucleosides), bicyclic modified nucleosides
(such as for example, bicyclic nucleosides wherein the sugar group
has a 2'-O--CHR.sub.a-4' bridging group, wherein R.sub.a is H,
alkyl or substituted alkyl) and base modified nucleosides. The
sugar can be modified with more than one of these modifications
listed such as for example a bicyclic modified nucleoside further
including a 5'-substitution or a 5' or 4' substituted nucleoside
further including a 2' substitutent. The term modified nucleoside
also includes combinations of these modifications such as a base
and sugar modified nucleosides. These modifications are meant to be
illustrative and not exhaustive as other modifications are known in
the art and are also envisioned as possible modifications for the
modified nucleosides described herein.
[0124] As used herein the term "monomer subunit" is meant to
include all manner of monomer units that are amenable to oligomer
synthesis with one preferred list including monomer subunits such
as .beta.-D-ribonucleosides, .beta.-D-2'-deoxyribonucleosides,
substituted nucleosides (such as 2',5' and bis substituted
nucleosides), 4'-S-modified nucleosides, (such as
4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and
4'-S-2'-substituted ribonucleosides), bicyclic modified nucleosides
(such as bicyclic nucleosides wherein the sugar group has a
2'-O--CHR.sub.a-4' bridging group, wherein R.sub.a is H, alkyl or
substituted alkyl), other modified nucleosides, nucleoside mimetics
and nucleosides having sugar surrogates.
[0125] The term "oligonucleotide" refers to an oligomer or polymer
of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). This term
includes oligonucleotides composed of naturally-occurring
nucleobases, sugars and covalent internucleoside linkages. The term
"oligonucleotide analog" refers to oligonucleotides that have one
or more non-naturally occurring portions. Such non-naturally
occurring oligonucleotides are often desired over naturally
occurring forms because of desirable properties such as, for
example, enhanced cellular uptake, enhanced affinity for nucleic
acid target and/or increased stability in the presence of
nucleases.
[0126] The term "oligonucleoside" refers to a sequence of
nucleosides that are joined by internucleoside linkages that do not
have phosphorus atoms. Internucleoside linkages of this type
include short chain alkyl, cycloalkyl, mixed heteroatom alkyl,
mixed heteroatom cycloalkyl, one or more short chain heteroatomic
and one or more short chain heterocyclic. These internucleoside
linkages include without limitation, siloxane, sulfide, sulfoxide,
sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl,
thioformacetyl, alkeneyl, sulfamate, methyleneimino,
methylenehydrazino, sulfonate, sulfonamide, amide and others having
mixed N, O, S and CH.sub.2 component parts.
[0127] The terms "heterocyclic base moiety" and "nucleobase" as
used herein, include unmodified or naturally occurring nucleobases,
modified or non-naturally occurring nucleobases as well as
synthetic mimetics thereof (such as for example phenoxazines). In
general, a heterocyclic base moiety is heterocyclic system that
contains one or more atoms or groups of atoms capable of hydrogen
bonding to a base of a nucleic acid.
[0128] As used herein the terms, "unmodified nucleobase" and
"naturally occurring nucleobase" 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,
3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic
bases, promiscuous bases, size-expanded bases, and fluorinated
bases as defined herein. 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',':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-deaza-adenine, 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, Kroschwitz, J. I., Ed., John Wiley &
Sons, 1990, 858-859; 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,
Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
[0129] The heterocyclic base moiety of each of the 2'-fluoroethoxy
modified nucleosides of formula I can be modified with one or more
substituent groups to enhance one or more properties such as
affinity for a target strand or affect some other property in an
advantageous manner. Modified nucleobases include without
limitation, universal bases, hydrophobic bases, promiscuous bases,
size-expanded bases, and fluorinated bases as defined herein.
Certain of these nucleobases are particularly useful for increasing
the binding affinity of the oligomeric compounds as provided
herein. 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. (Antisense Research and
Applications, Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds.,
CRC Press, Boca Raton, 1993, 276-278).
[0130] Representative United States patents that teach the
preparation of certain of the above noted modified nucleobases as
well as other modified nucleobases include without limitation, U.S.
Pat. Nos. 3,687,808; 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; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and
6,005,096, certain of which are commonly owned with the instant
application, and each of which is herein incorporated by reference
in its entirety.
[0131] In general, the term "oligomeric compound" refers to a
contiguous sequence of linked monomer subunits. In general, each
linked monomer subunit is directly or indirectly attached to a
heterocyclic base moiety but abasic sites are also possible. At
least some and generally most if not essentially all of the
heterocyclic bases in an oligomeric compound are capable of
hybridizing to a nucleic acid molecule, normally a preselected RNA
target. The term "oligomeric compound" therefore includes
oligonucleotides, oligonucleotide analogs and oligonucleosides. It
also includes polymers having a plurality of non-naturally
occurring nucleoside mimetics and or nucleosides having sugar
surrogate groups. In certain embodiments, oligomeric compounds
comprise a plurality of monomer subunits independently selected
from naturally occurring nucleosides, non-naturally occurring
nucleosides, modified nucleosides, nucleoside mimetics, and
nucleosides having sugar surrogate groups.
[0132] When preparing oligomeric compounds having specific motifs
as disclosed herein it can be advantageous to mix non-naturally
occurring monomer subunits such as the 2'-fluoroethoxy modified
nucleosides of formula I as provided herein with other
non-naturally occurring monomer subunits, naturally occurring
monomer subunits (nucleosides) or mixtures thereof. In certain
embodiments, oligomeric compounds are provided herein comprising a
contiguous sequence of linked monomer subunits wherein at least one
monomer subunit is a 2'-fluoroethoxy modified nucleoside of formula
I as provided herein. In certain embodiments, oligomeric compounds
are provided comprising a plurality of 2'-fluoroethoxy modified
nucleosides of formula I as provided herein.
[0133] Oligomeric compounds are routinely prepared linearly but can
also be joined or otherwise prepared to be circular and/or can be
prepared to include branching. Oligomeric compounds can form double
stranded constructs such as for example two strands hybridized to
form a double stranded composition. Double stranded compositions
can be linked or separate and can include various other groups such
as conjugates and/or overhangs on the ends.
[0134] Oligomeric compounds provided herein can optionally contain
one or more nucleosides wherein the sugar group has been modified.
Such sugar modified nucleosides may impart enhanced nuclease
stability, increased binding affinity or some other beneficial
biological property to the oligomeric compounds. As used herein the
term "modified sugar" refers to modifications that can be made to
the furanose sugar portion of otherwise unmodified or modified
nucleosides useful herein. Such modified sugars include without
limitation substitution with one or more substituent groups,
bridging of two non-geminal ring carbon atoms to form a bicyclic
nucleoside or substitution of the 4'-O atom with a disubstituted
methylene group [C(R).sub.2] or a heteroatom or substituted
heteroatom (NR). Modified sugar moieties can also comprise mixtures
of these modifications such as for example putting a 5'-substituent
group on a bicyclic nucleoside.
[0135] Examples of substituent groups useful for modifying sugar
moieties of nucleosides include without limitation 2'-F, 2'-allyl,
2'-amino, 2'-azido, 2'-thio, 2'-O-allyl, 2'-OCF.sub.3,
2'-O--C.sub.1-C.sub.10 alkyl, 2'-O--CH.sub.3, OCF.sub.3,
2'-O--CH.sub.2CH.sub.3, 2'-O--(CH.sub.2).sub.2CH.sub.3,
2'-O--(CH.sub.2).sub.2--O--CH.sub.3, 2'-O(CH.sub.2).sub.2SCH.sub.3,
2'-O--CH.sub.2--CH.dbd.CH.sub.2 (MOE),
2'-O--(CH.sub.2).sub.3--N(R.sub.m)(R.sub.n),
2'-O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n),
2'-O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--N(R.sub.m)(R.sub.n),
2'-O--CH.sub.2C(.dbd.O)--N(R.sub.m)(R.sub.n),
2'-O--CH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(R.sub.m)(R.sub.n)
and 2'-O--CH.sub.2--N(H)--C(.dbd.NR.sub.m)[N(R.sub.m)(R.sub.n)],
5'-vinyl, 5'-methyl (R or S) and 4'-S wherein each R.sub.m and
R.sub.n is, independently, H, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl or a protecting group. Further examples of
modified sugar moieties include without limitation bicyclic sugars
(e.g. bicyclic nucleic acids or bicyclic nucleosides discussed
below).
[0136] Combinations of these modifications are also provided for
herein without limitation, such as 2'-F-5'-methyl substituted
nucleosides (see PCT International Application WO 2008/101157
Published on Aug. 21, 2008 for other disclosed 5',2'-bis
substituted nucleosides) and replacement of the ribosyl ring oxygen
atom with S and further substitution at the 2'-position (see
published U.S. Patent Application US2005-0130923, published on Jun.
16, 2005) or alternatively 5'-substitution of a bicyclic nucleic
acid (see PCT International Application WO 2007/134181, published
on Nov. 22, 2007 wherein a 4'-CH.sub.2--O-2' bicyclic nucleoside is
further substituted at the 5' position with a 5'-methyl or a
5'-vinyl group).
[0137] As used herein the terms "bicyclic nucleic acid" and
"bicyclic nucleoside" refer to nucleosides wherein the sugar
portion of the nucleoside is bicyclic (e.g. bicyclic sugar). In
certain embodiments, a bicyclic nucleic acid comprises a nucleoside
wherein the furanose ring comprises a bridge between two
non-geminal ring carbon atoms. Examples of bicyclic nucleosides
include without limitation nucleosides comprising a bridge between
the 4' and the 2' ribosyl ring atoms. In certain embodiments,
oligomeric compounds provided herein include one or more bicyclic
nucleosides wherein the bridge comprises one of the formulae:
4'-(CH.sub.2)--O-2' (LNA); 4'-(CH.sub.2)--S-2';
4'-(CH.sub.2).sub.2--O-2' (ENA); 4'-CH(CH.sub.3)--O-2' and
4'-CH(CH.sub.2OCH.sub.3)--O-2' (and analogs thereof see U.S. Pat.
No. 7,399,845, issued on Jul. 15, 2008);
4'-C(CH.sub.3)(CH.sub.3)--O-2' (and analogs thereof see published
International Application WO/2009/006478, published Jan. 8, 2009);
4'-CH.sub.2--N(OCH.sub.3)-2' (and analogs thereof see published
International Application WO/2008/150729, published Dec. 11, 2008);
4'-CH.sub.2--O--N(CH.sub.3)-2' (see published U.S. Patent
Application US2004-0171570, published Sep. 2, 2004);
4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or
a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23,
2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see Chattopadhyaya, et al.,
J. Org. Chem., 2009, 74, 118-134); and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2' (and analogs thereof see published
International Application WO 2008/154401, published on Dec. 8,
2008). Each of the foregoing bicyclic nucleosides can be prepared
having one or more stereochemical sugar configurations including
for example .alpha.-L-ribofuranose and .beta.-D-ribofuranose (see
PCT international application PCT/DK98/00393, published on Mar. 25,
1999 as WO 99/14226).
[0138] As used herein the term "sugar surrogate" refers to
replacement of the nucleoside furanose ring with a non-furanose (or
4'-substituted furanose) group with another structure such as
another ring system or open system. Such structures can be as
simple as a six membered ring as opposed to the five membered
furanose ring or can be more complicated as is the case with the
non-ring system used in peptide nucleic acid. The term is meant to
include replacement of the sugar group with all manner of sugar
surrogates know in the art and includes without limitation sugar
surrogate groups such as morpholinos, cyclohexenyls and
cyclohexitols. In most monomer subunits having a sugar surrogate
group the heterocyclic base moiety is generally maintained to
permit hybridization.
[0139] In certain embodiments, nucleosides having sugar surrogate
groups include without limitation, replacement of the ribosyl ring
with a surrogate ring system such as a tetrahydropyranyl ring
system (also referred to as hexitol) as illustrated below:
##STR00006##
[0140] Many other monocyclic, bicyclic and tricyclic ring systems
are known in the art and are suitable as sugar surrogates that can
be used to modify nucleosides for incorporation into oligomeric
compounds as provided herein (see for example review article:
Leumann, Christian J.). Such ring systems can undergo various
additional substitutions to further enhance their activity.
[0141] Some representative U.S. patents that teach the preparation
of such modified sugars include without limitation, 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; 5,670,633;
5,700,920; 5,792,847 and 6,600,032 and International Application
PCT/US2005/019219, filed Jun. 2, 2005 and published as WO
2005/121371 on Dec. 22, 2005 certain of which are commonly owned
with the instant application, and each of which is herein
incorporated by reference in its entirety.
[0142] Those skilled in the art, having possession of the present
disclosure will be able to prepare oligomeric compounds, comprising
a contiguous sequence of linked monomer subunits, of essentially
any viable length to practice the methods disclosed herein. Such
oligomeric compounds will include at least one and preferably a
plurality of the 2'-fluoroethoxy modified nucleosides of formula I
provided herein and may also include other monomer subunits
including but not limited to nucleosides, modified nucleosides,
nucleosides comprising sugar surrogate groups and nucleoside
mimetics.
[0143] In certain embodiments, oligomeric compounds provided herein
comprise from about 8 to about 80 monomer subunits in length. One
having ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 monomer subunits in
length, or any range therewithin.
[0144] In certain embodiments, oligomeric compounds provided herein
comprise from about 8 to 40 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39 or 40 monomer subunits in length, or any range
therewithin.
[0145] In certain embodiments, oligomeric compounds provided herein
comprise from about 8 to 20 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19 or 20 monomer subunits in length, or any range therewithin.
[0146] In certain embodiments, oligomeric compounds provided herein
comprise from about 8 to 16 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomer
subunits in length, or any range therewithin.
[0147] In certain embodiments, oligomeric compounds provided herein
comprise from about 10 to 14 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 10, 11, 12, 13 or 14 monomer subunits in
length, or any range therewithin.
[0148] In certain embodiments, oligomeric compounds provided herein
comprise from about 10 to 18 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 10, 11, 12, 13, 14, 15, 16, 17 or 18
monomer subunits in length, or any range therewithin.
[0149] In certain embodiments, oligomeric compounds provided herein
comprise from about 10 to 21 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
or 21 monomer subunits in length, or any range therewithin.
[0150] In certain embodiments, oligomeric compounds provided herein
comprise from about 12 to 14 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 12, 13 or 14 monomer subunits in length, or
any range therewithin.
[0151] In certain embodiments, oligomeric compounds provided herein
comprise from about 12 to 18 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 12, 13, 14, 15, 16, 17 or 18 monomer
subunits in length, or any range therewithin.
[0152] In certain embodiments, oligomeric compounds provided herein
comprise from about 12 to 21 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21
monomer subunits in length, or any range therewithin.
[0153] In certain embodiments, oligomeric compounds provided herein
comprise from about 14 to 18 monomer subunits in length. One having
ordinary skill in the art will appreciate that this embodies
oligomeric compounds of 14, 15, 16, 17 or 18 monomer subunits in
length, or any range therewithin.
[0154] In certain embodiments, oligomeric compounds of any of a
variety of ranges of lengths of linked monomer subunits are
provided. In certain embodiments, oligomeric compounds are provided
consisting of X-Y linked monomer subunits, where X and Y are each
independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50;
provided that X<Y. For example, in certain embodiments, this
provides oligomeric compounds comprising: 8-9, 8-10, 8-11, 8-12,
8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23,
8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13,
9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24,
9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14,
10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23,
10-24, 10-25, 10-26, 10-27, 10-28, 10-29, 10-30, 11-12, 11-13,
11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22,
11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 12-13,
12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22,
12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14,
13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23,
13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16,
14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25,
14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19,
15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28,
15-29, 15-30, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16-23,
16-24, 16-25, 16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17-19,
17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28,
17-29, 17-30, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25,
18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19-23,
19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 20-21, 20-22,
20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22,
21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23,
22-24, 22-25, 22-26, 22-27, 22-28, 22-29, 22-30, 23-24, 23-25,
23-26, 23-27, 23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28,
24-29, 24-30, 25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28,
26-29, 26-30, 27-28, 27-29, 27-30, 28-29, 28-30, or 29-30 linked
monomer subunits.
[0155] In certain embodiments, the ranges for the oligomeric
compounds listed herein are meant to limit the number of monomer
subunits in the oligomeric compounds, however such oligomeric
compounds may further include 5' and/or 3'-terminal groups
including but not limited to protecting groups such as hydroxyl
protecting groups, optionally linked conjugate groups and/or other
substituent groups.
[0156] In certain embodiments, the preparation of oligomeric
compounds as disclosed herein is performed according to literature
procedures for DNA: Protocols for Oligonucleotides and Analogs,
Agrawal, Ed., Humana Press, 1993, and/or RNA: Scaringe, Methods,
2001, 23, 206-217; Gait et al., Applications of Chemically
synthesized RNA in RNA: Protein Interactions, Smith, Ed., 1998,
1-36; Gallo et al., Tetrahedron , 2001, 57, 5707-5713. Additional
methods for solid-phase synthesis may be found in Caruthers U.S.
Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679;
and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re.
34,069.
[0157] Oligomeric compounds are routinely prepared using solid
support methods as opposed to solution phase methods. Commercially
available equipment commonly used for the preparation of oligomeric
compounds that utilize the solid support method is sold by several
vendors including, for example, Applied Biosystems (Foster City,
Calif.). Any other means for such synthesis known in the art may
additionally or alternatively be employed. Suitable solid phase
techniques, including automated synthesis techniques, are described
in Oligonucleotides and Analogues, a Practical Approach, F.
Eckstein, Ed., Oxford University Press, New York, 1991.
[0158] The synthesis of RNA and related analogs relative to the
synthesis of DNA and related analogs has been increasing as efforts
in RNA interference and micro RNA increase. The primary RNA
synthesis strategies that are presently being used commercially
include 5'-O-DMT-2'-O-t-butyldimethylsilyl (TBDMS),
5'-O-DMT-2'-O[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP),
2'-O-[(triisopropylsilyl)oxy]methyl
(2'-O--CH.sub.2--O--Si(iPr).sub.3 (TOM) and the 5'-O-silyl
ether-2'-ACE (5'-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether
(DOD)-2'-O-bis(2-acetoxyethoxy)-methyl (ACE). A current list of
some of the major companies currently offering RNA products include
Pierce Nucleic Acid Technologies, Dharmacon Research Inc., Ameri
Biotechnologies Inc., and Integrated DNA Technologies, Inc. One
company, Princeton Separations, is marketing an RNA synthesis
activator advertised to reduce coupling times especially with TOM
and TBDMS chemistries. The primary groups being used for commercial
RNA synthesis are: TBDMS: 5'-O-DMT-2'-O-t-butyldimethylsilyl; TOM:
2'-O-[(triisopropylsilyl)oxy]methyl; DOD/ACE:
(5'-.beta.-bis(trimethylsiloxy)cyclododecyloxysilyl
ether-2'-O-bis(2-acetoxyethoxy)methyl; and FPMP:
5'-.beta.-DMT-2'-O-[1(2-fluorophenyl)-4-ethoxypiperidin-4-yl]. In
certain embodiments, each of the aforementioned RNA synthesis
strategies can be used herein. In certain embodiments, the
aforementioned RNA synthesis strategies can be performed together
in a hybrid fashion e.g. using a 5'-protecting group from one
strategy with a 2'-O-protecting from another strategy.
[0159] As used herein the term "hybridization" includes the pairing
of complementary strands of oligomeric compounds such as including
the binding of an oligomeric compound as provided herein to a
target nucleic acid. In certain embodiments, the mechanism of
pairing involves hydrogen bonding, which may be Watson-Crick,
Hoogsteen or reversed Hoogsteen hydrogen bonding, between
complementary heterocyclic base moieties of nucleosides (or monomer
subunits) that are in close enough proximity to hydrogen bond. For
example, adenine and thymine are complementary nucleobases which
pair through the formation of hydrogen bonds. Hybridization can
occur under varying circumstances.
[0160] An oligomeric compound is specifically hybridizable when
binding of the compound to the target nucleic acid interferes with
the normal function of the target nucleic acid resulting in a loss
of activity. To be specifically hybridizable also requires a
sufficient degree of complementarity to avoid non-specific binding
of the oligomeric compound to non-target nucleic acid sequences
under the conditions in which specific binding is desired, i.e.,
under physiological conditions (for in vivo assays or therapeutic
treatment) or other diagnostic conditions (for performing in vitro
assays).
[0161] As used herein the term "complementary," refers to the
capacity for precise pairing of two nucleobases regardless of where
the two nucleobases are located. For example, if a nucleobase at a
certain position of an oligomeric compound is capable of hydrogen
bonding with a nucleobase at a certain position of a target nucleic
acid, the target nucleic acid being a DNA, RNA, or oligonucleotide
molecule, then the position of hydrogen bonding between the
oligonucleotide and the target nucleic acid is considered to be a
complementary position. The oligomeric compound and the further
DNA, RNA, or oligonucleotide molecule are complementary to each
other when a sufficient number of complementary positions in each
molecule are occupied by nucleobases which can hydrogen bond with
each other. Thus, "specifically hybridizable" and "complementary"
are terms which are used to indicate a sufficient degree of precise
pairing or complementarity over a sufficient number of nucleobases
such that stable and specific binding occurs between an oligomeric
compound and its target nucleic acid.
[0162] It is understood in the art that the sequence of an
oligomeric compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. Moreover, an
oligomeric compound may hybridize over one or more segments such
that intervening or adjacent segments are not involved in the
hybridization event (e.g., a loop structure or hairpin structure).
In certain embodiments, oligomeric compounds can comprise at least
about 70%, at least about 80%, at least about 90%, at least about
95%, or at least about 99% sequence complementarity to a target
region within the target nucleic acid sequence to which they are
targeted. For example, an oligomeric compound in which 18 of 20
nucleobases of the oligomeric compound are complementary to a
target region, and would therefore specifically hybridize, would
represent 90 percent complementarity. In this example, the
remaining noncomplementary nucleobases may be clustered or
interspersed with complementary nucleobases and need not be
contiguous to each other or to complementary nucleobases. As such,
an oligomeric compound which is 18 nucleobases in length having 4
(four) noncomplementary nucleobases which are flanked by two
regions of complete complementarity with the target nucleic acid
would have 77.8% overall complementarity with the target nucleic
acid and would thus fall within this scope. Percent complementarity
of an oligomeric compound with a region of a target nucleic acid
can be determined routinely using BLAST programs (basic local
alignment search tools) and PowerBLAST programs known in the art
(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and
Madden, Genome Res., 1997, 7, 649-656).
[0163] Further included herein are oligomeric compounds such as
antisense oligomeric compounds, antisense oligonucleotides,
ribozymes, external guide sequence (EGS) oligonucleotides,
alternate splicers, primers, probes, and other oligomeric compounds
which hybridize to at least a portion of the target nucleic acid.
As such, these oligomeric compounds may be introduced in the form
of single-stranded, double-stranded, circular or hairpin oligomeric
compounds and may contain structural elements such as internal or
terminal bulges or loops. Once introduced to a system, the
oligomeric compounds provided herein may elicit the action of one
or more enzymes or structural proteins to effect modification of
the target nucleic acid.
[0164] One non-limiting example of such an enzyme is RNAse H, a
cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. It is known in the art that single-stranded oligomeric
compounds which are "DNA-like" elicit RNAse H. Activation of RNase
H, therefore, results in cleavage of the RNA target, thereby
greatly enhancing the efficiency of oligonucleotide-mediated
inhibition of gene expression. Similar roles have been postulated
for other ribonucleases such as those in the RNase III and
ribonuclease L family of enzymes.
[0165] While one form of oligomeric compound is a single-stranded
antisense oligonucleotide, in many species the introduction of
double-stranded structures, such as double-stranded RNA (dsRNA)
molecules, has been shown to induce potent and specific
antisense-mediated reduction of the function of a gene or its
associated gene products. This phenomenon occurs in both plants and
animals and is believed to have an evolutionary connection to viral
defense and transposon silencing.
[0166] In some embodiments, "suitable target segments" may be
employed in a screen for additional oligomeric compounds that
modulate the expression of a selected protein. "Modulators" are
those oligomeric compounds that decrease or increase the expression
of a nucleic acid molecule encoding a protein and which comprise at
least an 8-nucleobase portion which is complementary to a suitable
target segment. The screening method comprises the steps of
contacting a suitable target segment of a nucleic acid molecule
encoding a protein with one or more candidate modulators, and
selecting for one or more candidate modulators which decrease or
increase the expression of a nucleic acid molecule encoding a
protein. Once it is shown that the candidate modulator or
modulators are capable of modulating (e.g. either decreasing or
increasing) the expression of a nucleic acid molecule encoding a
peptide, the modulator may then be employed herein in further
investigative studies of the function of the peptide, or for use as
a research, diagnostic, or therapeutic agent.
[0167] Suitable target segments may also be combined with their
respective complementary oligomeric compounds provided herein to
form stabilized double-stranded (duplexed) oligonucleotides. Such
double stranded oligonucleotide moieties have been shown in the art
to modulate target expression and regulate translation as well as
RNA processing via an antisense mechanism. Moreover, the
double-stranded moieties may be subject to chemical modifications
(Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature,
1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et
al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl.
Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev.,
1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498;
Elbashir et al., Genes Dev., 2001, 15, 188-200). For example, such
double-stranded moieties have been shown to inhibit the target by
the classical hybridization of antisense strand of the duplex to
the target, thereby triggering enzymatic degradation of the target
(Tijsterman et al., Science, 2002, 295, 694-697).
[0168] The oligomeric compounds provided herein can also be applied
in the areas of drug discovery and target validation. In certain
embodiments, provided herein is the use of the oligomeric compounds
and targets identified herein in drug discovery efforts to
elucidate relationships that exist between proteins and a disease
state, phenotype, or condition. These methods include detecting or
modulating a target peptide comprising contacting a sample, tissue,
cell, or organism with one or more oligomeric compounds provided
herein, measuring the nucleic acid or protein level of the target
and/or a related phenotypic or chemical endpoint at some time after
treatment, and optionally comparing the measured value to a
non-treated sample or sample treated with a further oligomeric
compound as provided herein. These methods can also be performed in
parallel or in combination with other experiments to determine the
function of unknown genes for the process of target validation or
to determine the validity of a particular gene product as a target
for treatment or prevention of a particular disease, condition, or
phenotype. In certain embodiments, oligomeric compounds are
provided for use in therapy. In certain embodiments, the therapy is
reducing target messenger RNA.
[0169] As used herein, the term "dose" refers to a specified
quantity of a pharmaceutical agent provided in a single
administration. In certain embodiments, a dose may be administered
in two or more boluses, tablets, or injections. For example, in
certain embodiments, where subcutaneous administration is desired,
the desired dose requires a volume not easily accommodated by a
single injection. In such embodiments, two or more injections may
be used to achieve the desired dose. In certain embodiments, a dose
may be administered in two or more injections to minimize injection
site reaction in an individual.
[0170] In certain embodiments, chemically-modified oligomeric
compounds are provided herein that may have a higher affinity for
target RNAs than does non-modified DNA. In certain such
embodiments, higher affinity in turn provides increased potency
allowing for the administration of lower doses of such compounds,
reduced potential for toxicity, improvement in therapeutic index
and decreased overall cost of therapy.
[0171] Effect of nucleoside modifications on RNAi activity is
evaluated according to existing literature (Elbashir et al.,
Nature, 2001, 411, 494-498; Nishikura et al., Cell, 2001, 107,
415-416; and Bass et al., Cell, 2000, 101, 235-238.)
[0172] In certain embodiments, oligomeric compounds provided herein
can be utilized for diagnostics, therapeutics, prophylaxis and as
research reagents and kits. Furthermore, antisense
oligonucleotides, which are able to inhibit gene expression with
exquisite specificity, are often used by those of ordinary skill to
elucidate the function of particular genes or to distinguish
between functions of various members of a biological pathway. In
certain embodiments, oligomeric compounds provided herein can be
utilized either alone or in combination with other oligomeric
compounds or other therapeutics as tools in differential and/or
combinatorial analyses to elucidate expression patterns of a
portion or the entire complement of genes expressed within cells
and tissues. Oligomeric compounds can also be effectively used as
primers and probes under conditions favoring gene amplification or
detection, respectively. These primers and probes are useful in
methods requiring the specific detection of nucleic acid molecules
encoding proteins and in the amplification of the nucleic acid
molecules for detection or for use in further studies.
Hybridization of oligomeric compounds as provided herein,
particularly the primers and probes, with a nucleic acid can be
detected by means known in the art. Such means may include
conjugation of an enzyme to the oligonucleotide, radiolabelling of
the oligonucleotide or any other suitable detection means. Kits
using such detection means for detecting the level of selected
proteins in a sample may also be prepared.
[0173] As one nonlimiting example, expression patterns within cells
or tissues treated with one or more of the oligomeric compounds
provided herein are compared to control cells or tissues not
treated with oligomeric compounds and the patterns produced are
analyzed for differential levels of gene expression as they
pertain, for example, to disease association, signaling pathway,
cellular localization, expression level, size, structure or
function of the genes examined. These analyses can be performed on
stimulated or unstimulated cells and in the presence or absence of
other compounds and or oligomeric compounds which affect expression
patterns.
[0174] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,
2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression) (Madden, et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999,
303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et
al., Proc. Natl. Acad. Sci. USA, 2000, 97, 1976-81), protein arrays
and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed
sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000,
480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57),
subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.
Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,
203-208), subtractive cloning, differential display (DD) (Jurecic
and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative
genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl.,
1998, 31, 286-96), FISH (fluorescent in situ hybridization)
techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35,
1895-904) and mass spectrometry methods (To, Comb. Chem. High
Throughput Screen, 2000, 3, 235-41).
[0175] While in certain embodiments, oligomeric compounds provided
herein can be utilized as described, the following examples serve
only to illustrate and are not intended to be limiting.
EXAMPLES
General
[0176] .sup.1H and .sup.13C NMR spectra were recorded on a 300 MHz
and 75 MHz Bruker spectrometer, respectively.
Example 1
Synthesis of Nucleoside Phosphoramidites
[0177] The preparation of nucleoside phosphoramidites is performed
following procedures that are illustrated herein and in the art
such as but not limited to U.S. Pat. No. 6,426,220 and published
PCT WO 02/36743.
Example 2
Synthesis of Oligomeric Compounds
[0178] The oligomeric compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as alkylated derivatives and those having
phosphorothioate linkages.
[0179] Oligomeric compounds: Unsubstituted and substituted
phosphodiester (P.dbd.O) oligomeric compounds, including without
limitation, oligonucleotides can be synthesized on an automated DNA
synthesizer (Applied Biosystems model 394) using standard
phosphoramidite chemistry with oxidation by iodine.
[0180] In certain embodiments, phosphorothioate internucleoside
linkages (P.dbd.S) are synthesized similar to phosphodiester
internucleoside linkages with the following exceptions: thiation is
effected by utilizing a 10% w/v solution of
3-H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time is increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligomeric compounds are recovered by precipitating with greater
than 3 volumes of ethanol from a 1 M NH.sub.4OAc solution.
Phosphinate internucleoside linkages can be prepared as described
in U.S. Pat. No. 5,508,270.
[0181] Alkyl phosphonate internucleoside linkages can be prepared
as described in U.S. Pat. No. 4,469,863.
[0182] 3'-Deoxy-3'-methylene phosphonate internucleoside linkages
can be prepared as described in U.S. Pat. No. 5,610,289 or
5,625,050.
[0183] Phosphoramidite internucleoside linkages can be prepared as
described in U.S. Pat. No., 5,256,775 or U.S. Pat. No.
5,366,878.
[0184] Alkylphosphonothioate internucleoside linkages can be
prepared as described in published PCT applications PCT/US94/00902
and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively).
[0185] 3'-Deoxy-3'-amino phosphoramidate internucleoside linkages
can be prepared as described in U.S. Pat. No. 5,476,925.
[0186] Phosphotriester internucleoside linkages can be prepared as
described in U.S. Pat. No. 5,023,243.
[0187] Borano phosphate internucleoside linkages can be prepared as
described in U.S. Pat. Nos. 5,130,302 and 5,177,198.
[0188] Oligomeric compounds having one or more non-phosphorus
containing internucleoside linkages including without limitation
methylenemethylimino linked oligonucleosides, also identified as
MMI linked oligonucleosides, methylenedimethylhydrazo linked
oligonucleosides, also identified as MDH linked oligonucleosides,
methylenecarbonylamino linked oligonucleosides, also identified as
amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone oligomeric compounds
having, for instance, alternating MMI and P.dbd.O or P.dbd.S
linkages can be prepared as described in U.S. Pat. Nos. 5,378,825,
5,386,023, 5,489,677, 5,602,240 and 5,610,289.
[0189] Formacetal and thioformacetal internucleoside linkages can
be prepared as described in U.S. Pat. Nos. 5,264,562 and
5,264,564.
[0190] Ethylene oxide internucleoside linkages can be prepared as
described in U.S. Pat. No. 5,223,618.
Example 3
Isolation and Purification of Oligomeric Compounds
[0191] After cleavage from the controlled pore glass solid support
or other support medium and deblocking in concentrated ammonium
hydroxide at 55.degree. C. for 12-16 hours, the oligomeric
compounds, including without limitation oligonucleotides and
oligonucleosides, are recovered by precipitation out of 1 M
NH.sub.4OAc with >3 volumes of ethanol. Synthesized oligomeric
compounds are analyzed by electrospray mass spectroscopy (molecular
weight determination) and by capillary gel electrophoresis. The
relative amounts of phosphorothioate and phosphodiester linkages
obtained in the synthesis is determined by the ratio of correct
molecular weight relative to the -16 amu product (+/-32 +/-48). For
some studies oligomeric compounds are purified by HPLC, as
described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171.
Results obtained with HPLC-purified material are generally similar
to those obtained with non-HPLC purified material.
Example 4
Synthesis of Oligomeric Compounds Using the 96 Well Plate
Format
[0192] Oligomeric compounds, including without limitation
oligonucleotides, can be synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a 96-well format.
Phosphodiester internucleoside linkages are afforded by oxidation
with aqueous iodine. Phosphorothioate internucleoside linkages are
generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard
base-protected beta-cyanoethyl-diiso-propyl phosphoramidites can be
purchased from commercial vendors (e.g. PE-Applied Biosystems,
Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard
nucleosides are synthesized as per standard or patented methods and
can be functionalized as base protected beta-cyanoethyldiisopropyl
phosphoramidites.
[0193] Oligomeric compounds can be cleaved from support and
deprotected with concentrated NH.sub.4OH at elevated temperature
(55-60.degree. C.) for 12-16 hours and the released product then
dried in vacuo. The dried product is then re-suspended in sterile
water to afford a master plate from which all analytical and test
plate samples are then diluted utilizing robotic pipettors.
Example 5
Analysis of Oligomeric Compounds Using the 96-Well Plate Format
[0194] The concentration of oligomeric compounds in each well can
be assessed by dilution of samples and UV absorption spectroscopy.
The full-length integrity of the individual products can be
evaluated by capillary electrophoresis (CE) in either the 96-well
format (Beckman P/ACE.TM. MDQ) or, for individually prepared
samples, on a commercial CE apparatus (e.g., Beckman P/ACE.TM.
5000, ABI 270). Base and backbone composition is confirmed by mass
analysis of the oligomeric compounds utilizing electrospray-mass
spectroscopy. All assay test plates are diluted from the master
plate using single and multi-channel robotic pipettors. Plates are
judged to be acceptable if at least 85% of the oligomeric compounds
on the plate are at least 85% full length.
Example 6
In Vitro Treatment of Cells with Oligomeric Compounds
[0195] The effect of oligomeric compounds on target nucleic acid
expression is tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. This
can be routinely determined using, for example, PCR or Northern
blot analysis. Cell lines derived from multiple tissues and species
can be obtained from American Type Culture Collection (ATCC,
Manassas, Va.).
[0196] The following cell type is provided for illustrative
purposes, but other cell types can be routinely used, provided that
the target is expressed in the cell type chosen. This can be
readily determined by methods routine in the art, for example
Northern blot analysis, ribonuclease protection assays or
RT-PCR.
[0197] b.END cells: The mouse brain endothelial cell line b.END was
obtained from Dr. Werner Risau at the Max Plank Institute (Bad
Nauheim, Germany). b.END cells are routinely cultured in DMEM, high
glucose (Invitrogen Life Technologies, Carlsbad, Calif.)
supplemented with 10% fetal bovine serum (Invitrogen Life
Technologies, Carlsbad, Calif.). Cells are routinely passaged by
trypsinization and dilution when they reached approximately 90%
confluence. Cells are seeded into 96-well plates (Falcon-Primaria
#353872, BD Biosciences, Bedford, Mass.) at a density of
approximately 3000 cells/well for uses including but not limited to
oligomeric compound transfection experiments.
[0198] Experiments involving treatment of cells with oligomeric
compounds:
[0199] When cells reach appropriate confluency, they are treated
with oligomeric compounds using a transfection method as
described.
[0200] LIPOFECTIN.TM.
[0201] When cells reached 65-75% confluency, they are treated with
one or more oligomeric compounds. The oligomeric compound is mixed
with LIPOFECTIN.TM. Invitrogen Life Technologies, Carlsbad, Calif.)
in Opti-MEM.TM.-1 reduced serum medium (Invitrogen Life
Technologies, Carlsbad, Calif.) to achieve the desired
concentration of the oligomeric compound(s) and a LIPOFECTIN.TM.
concentration of 2.5 or 3 .mu.g/mL per 100 nM oligomeric
compound(s). This transfection mixture is incubated at room
temperature for approximately 0.5 hours. For cells grown in 96-well
plates, wells are washed once with 100 .mu.L OPTI-MEM.TM.-1 and
then treated with 130 .mu.L of the transfection mixture. Cells
grown in 24-well plates or other standard tissue culture plates are
treated similarly, using appropriate volumes of medium and
oligomeric compound(s). Cells are treated and data are obtained in
duplicate or triplicate. After approximately 4-7 hours of treatment
at 37.degree. C., the medium containing the transfection mixture is
replaced with fresh culture medium. Cells are harvested 16-24 hours
after treatment with oligomeric compound(s).
[0202] Other suitable transfection reagents known in the art
include, but are not limited to, CYTOFECTIN.TM., LIPOFECTAMINE.TM.,
OLIGOFECTAMINE.TM., and FUGENE.TM.. Other suitable transfection
methods known in the art include, but are not limited to,
electroporation.
Example 7
Real-time Quantitative PCR Analysis of Target mRNA Levels
[0203] Quantitation of target mRNA levels is accomplished by
real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. This is a
closed-tube, non-gel-based, fluorescence detection system which
allows high-throughput quantitation of polymerase chain reaction
(PCR) products in real-time. As opposed to standard PCR in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied
Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda,
Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is
attached to the 5' end of the probe and a quencher dye (e.g.,
TAMRA, obtained from either PE-Applied Biosystems, Foster City,
Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 3' end of
the probe. When the probe and dyes are intact, reporter dye
emission is quenched by the proximity of the 3' quencher dye.
During amplification, annealing of the probe to the target sequence
creates a substrate that can be cleaved by the 5'-exonuclease
activity of Taq polymerase. During the extension phase of the PCR
amplification cycle, cleavage of the probe by Taq polymerase
releases the reporter dye from the remainder of the probe (and
hence from the quencher moiety) and a sequence-specific fluorescent
signal is generated. With each cycle, additional reporter dye
molecules are cleaved from their respective probes, and the
fluorescence intensity is monitored at regular intervals by laser
optics built into the ABI PRISM.TM. Sequence Detection System. In
each assay, a series of parallel reactions containing serial
dilutions of mRNA from untreated control samples generates a
standard curve that is used to quantitate the percent inhibition
after antisense oligonucleotide treatment of test samples.
[0204] Prior to quantitative PCR analysis, primer-probe sets
specific to the target gene being measured are evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction. In
multiplexing, both the target gene and the internal standard gene
GAPDH are amplified concurrently in a single sample. In this
analysis, mRNA isolated from untreated cells is serially diluted.
Each dilution is amplified in the presence of primer-probe sets
specific for GAPDH only, target gene only ("single-plexing"), or
both (multiplexing). Following PCR amplification, standard curves
of GAPDH and target mRNA signal as a function of dilution are
generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of
their corresponding values generated from the single-plexed
samples, the primer-probe set specific for that target is deemed
multiplexable. Other methods of PCR are also known in the art.
[0205] RT and PCR reagents are obtained from Invitrogen Life
Technologies (Carlsbad, Calif.). RT, real-time PCR is carried out
by adding 20 .mu.l PCR cocktail (2.5.times.PCR buffer minus
MgCl.sub.2, 6.6 mM MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP
and dGTP, 375 nM each of forward primer and reverse primer, 125 nM
of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5
Units MuLV reverse transcriptase, and 2.5.times.ROX dye) to 96-well
plates containing 30 .mu.L, total RNA solution (20-200 ng). The RT
reaction is carried out by incubation for 30 minutes at 48.degree.
C. Following a 10 minute incubation at 95.degree. C. to activate
the PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol are
carried out: 95.degree. C. for 15 seconds (denaturation) followed
by 60.degree. C. for 1.5 minutes (annealing/extension).
[0206] Gene target quantities obtained by RT, real-time PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RIBOGREEN.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RIBOGREEN.TM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374).
[0207] In this assay, 170 .mu.L of RIBOGREEN.TM. working reagent
(RIBOGREEN.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L
purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE
Applied Biosystems) with excitation at 485 nm and emission at 530
nm.
Example 8
Analysis of Inhibition of Target Expression
[0208] Antisense modulation of a target expression can be assayed
in a variety of ways known in the art. For example, a target mRNA
levels can be quantitated by, e.g., Northern blot analysis,
competitive polymerase chain reaction (PCR), or real-time PCR.
Real-time quantitative PCR is presently desired. RNA analysis can
be performed on total cellular RNA or poly(A)+ mRNA. One method of
RNA analysis of the present disclosure is the use of total cellular
RNA as described in other examples herein. Methods of RNA isolation
are well known in the art. Northern blot analysis is also routine
in the art. Real-time quantitative (PCR) can be conveniently
accomplished using the commercially available ABI PRISM.TM. 7600,
7700, or 7900 Sequence Detection System, available from PE-Applied
Biosystems, Foster City, Calif. and used according to
manufacturer's instructions.
[0209] Protein levels of a target can be quantitated in a variety
of ways well known in the art, such as immunoprecipitation, Western
blot analysis (immunoblotting), enzyme-linked immunosorbent assay
(ELISA) or fluorescence-activated cell sorting (FACS). Antibodies
directed to a target can be identified and obtained from a variety
of sources, such as the MSRS catalog of antibodies (Aerie
Corporation, Birmingham, Mich.), or can be prepared via
conventional monoclonal or polyclonal antibody generation methods
well known in the art. Methods for preparation of polyclonal
antisera are taught in, for example, Ausubel, F. M. et al., Current
Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John
Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies
is taught in, for example, Ausubel, F. M. et al., Current Protocols
in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley
& Sons, Inc., 1997.
[0210] Immunoprecipitation methods are standard in the art and can
be found at, for example, Ausubel, F. M. et al., Current Protocols
in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley
& Sons, Inc., 1998. Western blot (immunoblot) analysis is
standard in the art and can be found at, for example, Ausubel, F.
M. et al., Current Protocols in Molecular Biology, Volume 2, pp.
10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked
immunosorbent assays (ELISA) are standard in the art and can be
found at, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley &
Sons, Inc., 1991.
Example 9
Design of Phenotypic Assays and In Vivo Studies for the Use of
Target Inhibitors
Phenotypic Assays
[0211] Once target inhibitors have been identified by the methods
disclosed herein, the oligomeric compounds are further investigated
in one or more phenotypic assays, each having measurable endpoints
predictive of efficacy in the treatment of a particular disease
state or condition.
[0212] Phenotypic assays, kits and reagents for their use are well
known to those skilled in the art and are herein used to
investigate the role and/or association of a target in health and
disease. Representative phenotypic assays, which can be purchased
from any one of several commercial vendors, include those for
determining cell viability, cytotoxicity, proliferation or cell
survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston,
Mass.), protein-based assays including enzymatic assays (Panvera,
LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene
Research Products, San Diego, Calif.), cell regulation, signal
transduction, inflammation, oxidative processes and apoptosis
(Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation
(Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube
formation assays, cytokine and hormone assays and metabolic assays
(Chemicon International Inc., Temecula, Calif.; Amersham
Biosciences, Piscataway, N.J.).
[0213] In one non-limiting example, cells determined to be
appropriate for a particular phenotypic assay (i.e., MCF-7 cells
selected for breast cancer studies; adipocytes for obesity studies)
are treated with a target inhibitors identified from the in vitro
studies as well as control compounds at optimal concentrations
which are determined by the methods described above. At the end of
the treatment period, treated and untreated cells are analyzed by
one or more methods specific for the assay to determine phenotypic
outcomes and endpoints.
[0214] Phenotypic endpoints include changes in cell morphology over
time or treatment dose as well as changes in levels of cellular
components such as proteins, lipids, nucleic acids, hormones,
saccharides or metals. Measurements of cellular status which
include pH, stage of the cell cycle, intake or excretion of
biological indicators by the cell, are also endpoints of
interest.
[0215] Measurement of the expression of one or more of the genes of
the cell after treatment is also used as an indicator of the
efficacy or potency of the target inhibitors. Hallmark genes, or
those genes suspected to be associated with a specific disease
state, condition, or phenotype, are measured in both treated and
untreated cells.
In Vivo Studies
[0216] The individual subjects of the in vivo studies described
herein are warm-blooded vertebrate animals, which includes
humans.
Example 10
RNA Isolation
[0217] Poly(A)+ mRNA Isolation
[0218] Poly(A)+ mRNA is isolated according to Miura et al., (Clin.
Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA
isolation are routine in the art. Briefly, for cells grown on
96-well plates, growth medium is removed from the cells and each
well is washed with 200 .mu.L cold PBS. 60 .mu.L, lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) is added to each well, the plate is
gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate is transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates are incubated for
60 minutes at room temperature, washed 3 times with 200 .mu.L of
wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After
the final wash, the plate is blotted on paper towels to remove
excess wash buffer and then air-dried for 5 minutes. 60 .mu.L of
elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70.degree. C.,
is added to each well, the plate is incubated on a 90.degree. C.
hot plate for 5 minutes, and the eluate is then transferred to a
fresh 96-well plate.
[0219] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Total RNA Isolation
[0220] Total RNA is isolated using an RNEASY 96.TM. kit and buffers
purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium is removed from the cells and each
well is washed with 200 .mu.l cold PBS. 150 .mu.L Buffer RLT is
added to each well and the plate vigorously agitated for 20
seconds. 150 .mu.L of 70% ethanol is then added to each well and
the contents mixed by pipetting three times up and down. The
samples are then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum is applied for 1
minute. 500 .mu.L of Buffer RW1 is added to each well of the RNEASY
96.TM. plate and incubated for 15 minutes and the vacuum is again
applied for 1 minute. An additional 500 .mu.L of Buffer RW1 is
added to each well of the RNEASY 96.TM. plate and the vacuum is
applied for 2 minutes. 1 mL of Buffer RPE is then added to each
well of the RNEASY 96.TM. plate and the vacuum applied for a period
of 90 seconds. The Buffer RPE wash is then repeated and the vacuum
is applied for an additional 3 minutes. The plate is then removed
from the QIAVAC.TM. manifold and blotted dry on paper towels. The
plate is then re-attached to the QIAVAC.TM. manifold fitted with a
collection tube rack containing 1.2 mL collection tubes. RNA is
then eluted by pipetting 140 .mu.L of RNAse free water into each
well, incubating 1 minute, and then applying the vacuum for 3
minutes.
[0221] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 11
Target-Specific Primers and Probes
[0222] Probes and primers may be designed to hybridize to a target
sequence, using published sequence information.
[0223] For example, for human PTEN, the following primer-probe set
was designed using published sequence information (GENBANK.TM.
accession number U92436.1, SEQ ID NO: 1).
TABLE-US-00001 (SEQ ID NO: 2) Forward primer:
AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 3) Reverse primer:
TGCACATATCATTACACCAGTTCGT
And the PCR probe:
[0224] FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4),
where FAM is the fluorescent dye and TAMRA is the quencher dye.
Example 12
Western Blot Analysis of Target Protein Levels
[0225] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 .mu.l/well), boiled for 5 minutes and loaded on
a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to a target is used, with a radiolabeled or
fluorescently labeled secondary antibody directed against the
primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. (Molecular Dynamics, Sunnyvale Calif.).
Example 13
PTEN Single Administration In Vivo Study with 2-10-2 and 2-14-2
Gapmers
[0226] Six week old Balb/c mice (Jackson Laboratory, Bar Harbor,
Me.) were injected once with the gapped oligomeric compounds
targeted to PTEN at a dose of 3.2, 10, 32 or 100 mg/kg. The mice
were sacrificed 72 hours following last administration. Liver
tissues were homogenized and mRNA levels were quantitated using
real-time PCR as described herein for comparison to untreated
control levels (% UTC). Estimated ED.sub.50 concentrations for each
oligomeric compound were calculated using Graphpad Prism and are
shown below. Tms were determined in 100 mM phosphate buffer, 0.1 mM
EDTA, pH 7, at 260 nm using 4 .mu.M of the modified oligomers
listed below and 4 .mu.M of complementary RNA.
TABLE-US-00002 SEQ ID NO./ Composition ED.sub.50 ISIS NO. (5' to
3') Motif Tm (mg/kg) 05/397770
.sup.meC.sub.eT.sub.eTAGCACTGGC.sup.meC.sub.eT.sub.e (2/10/2) 51.3
61 05/413058 .sup.meC.sub.xT.sub.xTAGCACTGGC.sup.meC.sub.xT.sub.x
(2/10/2) 51.0 19 06/394420
.sup.meC.sub.eT.sub.eGCTAGCCTCTGGATT.sub.eT.sub.e (2/14/2) 55.6 28
06/413059 .sup.meC.sub.xT.sub.xGCTAGCCTCTGGATT.sub.xT.sub.x
(2/14/2) 55.9 24 07/116847
.sup.meC.sub.eT.sub.eG.sub.e.sup.meC.sub.eT.sub.eAG.sup.meC.sup.-
meCT.sup.meC- (5/10/5) 67.9 32
TGGAT.sub.eT.sub.eT.sub.eG.sub.eA.sub.e
[0227] Each internucleoside linking group is a phosphorothioate,
nucleosides not followed by a subscript are
.beta.-D-2'-deoxyribonucleosides, superscript Me indicates that the
following C is a 5-methyl C, subscript e indicates that the
preceding nucleoside is a 2'-O(CH.sub.2).sub.2--O--CH.sub.3 (MOE)
substituted nucleoside and subscript x indicates that the preceding
nucleoside is a 2'-OCH.sub.2CH.sub.2F (FEt) substituted
nucleoside.
TABLE-US-00003 % UTC at dosage SEQ ID NO./ 3.2 10 32 100 ISIS NO.
mg/kg mg/kg mg/kg mg/kg Motif 05/397770 86.6 92.0 71.0 38.2 (2/10/2
MOE) 05/413058 88.4 76.1 37.8 6.6 (2/10/2 FEt) 06/394420 1.03 83.1
51.0 10.8 (2/14/2 MOE) 06/413059 93.2 83.9 40.7 16.3 (2/14/2 FEt)
07/116847 98.3 85.0 52.3 21.4 (5/10/5 MOE) Saline % UTC = 100
[0228] Liver transaminase levels, alanine aminotranferease (ALT)
and aspartate aminotransferase (AST), in serum were also measured
relative to saline injected mice. The approximate liver
transaminase levels are listed in the table below.
TABLE-US-00004 ALT (IU/L) at dosage SEQ ID NO./ 3.2 10 32 100 ISIS
NO. mg/kg mg/kg mg/kg mg/kg Motif 05/397770 27.6 56.8 33.0 22.1
(2/10/2 MOE) 05/413058 22.9 23.5 24.7 21.0 (2/10/2 FEt) 06/394420
43.8 24.9 29.0 34.4 (2/14/2 MOE) 06/413059 20.7 28.5 30.1 39.2
(2/14/2 FEt) 07/116847 28.7 24.3 30.4 34.0 (5/10/5 MOE) AST (IU/L)
at dosage SEQ ID NO./ 3.2 10 32 100 ISIS NO. mg/kg mg/kg mg/kg
mg/kg Motif 05/397770 57.7 248.1 108.9 60.4 (2/10/2 MOE) 05/413058
62.0 94.7 92.8 39.0 (2/10/2 FEt) 06/394420 158.4 143.2 167.8 96.9
(2/14/2 MOE) 06/413059 40.7 118.2 132.4 129.6 (2/14/2 FEt)
07/116847 61.3 63.1 130.8 98.4 (5/10/5 MOE).
Example 14
PTEN Multiple Administration In Vivo Study with 2-10-2 Gapmers
[0229] Six week old Balb/c mice (Jackson Laboratory, Bar Harbor,
Me.) were injected twice per week for three weeks with gapped
oligomeric compounds targeted to PTEN at a dose of 0.5, 1.5, 5.0 or
15 mg/kg. The mice were sacrificed 48 hours following last
administration. Liver tissues were homogenized and mRNA levels were
quantitated using real-time PCR as described herein for comparison
to untreated control levels (% UTC). Plasma chemistry analysis was
completed. Tms were determined in 100 mM phosphate buffer, 0.1 mM
EDTA, pH 7, at 260 nm using 4 .mu.M of the modified oligomers
listed below and 4 .mu.M of the complementary RNA.
TABLE-US-00005 SEQ ID NO./ Composition ED.sub.50 ISIS NO. (5' to
3') Tm (mg/kg) 05/413058
.sup.meC.sub.xT.sub.xTAGCACTGGC.sup.meC.sub.xT.sub.x 51.0 13.0
[0230] Each internucleoside linking group is a phosphorothioate,
nucleosides not followed by a subscript are
.beta.-D-2'-deoxyribonucleosides, superscript Me indicates that the
following C is a 5-methyl C and subscript x indicates that the
preceding nucleoside is a 2'-OCH.sub.2CH.sub.2F (FEt) substituted
nucleoside.
TABLE-US-00006 SEQ ID NO./ % UTC at dosage ISIS NO. 0.5 mg/kg 1.5
mg/kg 5.0 mg/kg 15 mg/kg 05/413058 93.0 76.7 78.4 43.2 Saline % UTC
= 100
[0231] Liver transaminase levels, alanine aminotranferease (ALT)
and aspartate aminotransferase (AST), in serum were also measured
relative to saline injected mice. The approximate liver
transaminase levels are listed in the table below.
TABLE-US-00007 SEQ ID NO./ ALT (IU/L) at dosage ISIS NO. 0.5 mg/kg
1.5 mg/kg 5.0 mg/kg 15 mg/kg 05/413058 20.0 26.2 18.25 18.0 SEQ ID
NO./ AST (IU/L) at dosage ISIS NO. 0.5 mg/kg 1.5 mg/kg 5.0 mg/kg 15
mg/kg 05/413058 47.7 74.5 52.0 44.0.
Example 15
Oligomeric Compounds Targeted to PTEN
In Vitro Study
[0232] In accordance with the present disclosure, oligomeric
compounds were synthesized and tested for their ability to reduce
PTEN expression over a range of doses. Human HeLa cells were
treated with either ISIS 404320, 418030, 418031, or ISIS 418032 at
concentrations of 1.56, 3.13, 6.25, 12.5, 25 and 50 nM. Expression
levels of PTEN were determined using real-time PCR and normalized
to RIBOGREEN.TM. using methods described herein. The percent
inhibition of PTEN mRNA was determined and the resulting
dose-response curves were used to determine the EC.sub.50 as listed
below.
TABLE-US-00008 SEQ ID NO./ Composition EC.sub.50 ISIS NO. (5' to
3') (nM) 08/404320
U.sub.fU.sub.fG.sub.fU.sub.fC.sub.fU.sub.fC.sub.fU.sub.fG.sub.fG-
.sub.fU.sub.fC.sub.fC.sub.fU.sub.fU.sub.fA.sub.fC.sub.fU.sub.fU.sub.fA.sub-
.eA.sub.e 6.4 08/418030
U.sub.eU.sub.fG.sub.fU.sub.fC.sub.fU.sub.fC.sub.fU.sub.fG.sub.fG-
.sub.fU.sub.fC.sub.fC.sub.fU.sub.fU.sub.fA.sub.fC.sub.fU.sub.fU.sub.fA.sub-
.eA.sub.e 8.5 09/418031
T.sub.eU.sub.fG.sub.fU.sub.fC.sub.fU.sub.fC.sub.fU.sub.fG.sub.fG-
.sub.fU.sub.fC.sub.fC.sub.fU.sub.fU.sub.fA.sub.fC.sub.fU.sub.fU.sub.fA.sub-
.eA.sub.e 3.9 09/418032
T.sub.xU.sub.fG.sub.fU.sub.fC.sub.fU.sub.fC.sub.fU.sub.fG.sub.fG-
.sub.fU.sub.fC.sub.fC.sub.fU.sub.fU.sub.fA.sub.fC.sub.fU.sub.fU.sub.fA.sub-
.eA.sub.e 2.9
[0233] Underlined nucleosides are connected to the following
nucleoside by a phosphorothioate internucleoside linkage and all
other internucleoside linkages are phosphodiester (going 5' to 3').
Nucleosides followed by a subscript e indicates the preceding
nucleoside is a 2'-O(CH.sub.2).sub.2--O--CH.sub.3 (MOE) substituted
nucleoside, subscript f indicates the preceding nucleoside is a
2'-fluoro substituted nucleoside, and subscript x indicates the
preceding nucleoside is a 2'-OCH.sub.2CH.sub.2F (FEt) substituted
nucleoside.
TABLE-US-00009 SEQ ID NO./ISIS NO. % UTC Dose (nM) 08/404320
08/418030 09/418031 09/418032 1.56 72.8 83.6 66.0 65.2 3.13 63.3
63.0 55.9 47.5 6.25 46.2 52.6 44.3 31.6 12.5 37.4 38.9 31.3 24.4 25
29.3 33.6 25.5 21.0 50 26.1 27.7 28.6 20.3 Saline % UTC = 100.
Example 16
Derepression of ALDOA mRNA in Anti-miR-122 Treated Mouse
Hepatocytes
[0234] Primary hepatocytes were isolated from Balb/c mice (see for
example: Neufeld, D. S. Methods Mol. Biol. 1997, 75, 145-151).
Cells were seeded into 96-well plates in culture medium (Williams'
Medium E supplemented with 10% fetal bovine serum, 10 nM HEPES, and
penicillin/streptomycin) and cultured overnight before
transfection. Transfection of oligonucleotide in Cytofectin
(Invitrogen) was performed in triplicate, according to the
manufacturer's instructions. Cells were lysed 24 hrs after
transfection, and total RNA was harvested using Qiagen RNeasy 96
columns on a Bio Robot 3000 (Qiagen). Total RNA was used for
real-time quantitative RT-PCR analysis with a Prism 7700 Sequence
Detector (Applied Biosystems). mRNA levels were normalized to total
RNA for each sample, as measured by Ribogreen. An increase in mRNA
level over untreated control is indicative of activity in this
derepression assay. All reagents were from Invitrogen.
TABLE-US-00010 SEQ ID NO./ ISIS NO. Composition (5' to 3')
10/327895
A.sub.e.sup.meC.sub.eA.sub.eA.sub.eA.sub.e.sup.meC.sub.eA.sub.e.-
sup.meC.sub.e.sup.meC.sub.eA.sub.eT.sub.eT.sub.eG.sub.eT.sub.e.sup.meC.sub-
.eA.sub.e.sup.meC.sub.eA.sub.e.sup.meC.sub.eT.sub.e.sup.meC.sub.e.sup.meC.-
sub.eA.sub.e 10/410146
A.sub.e.sup.meC.sub.xA.sub.eA.sub.eA.sub.e.sup.meC.sub.xA.sub.e.-
sup.meC.sub.x.sup.meC.sub.xA.sub.eT.sub.xT.sub.xG.sub.eT.sub.x.sup.meC.sub-
.xA.sub.e.sup.meC.sub.xA.sub.e.sup.meC.sub.xT.sub.x.sup.meC.sub.x.sup.meC.-
sub.xA.sub.e 11/407588
A.sub.e.sup.meC.sub.eA.sub.eA.sub.e.sup.meC.sub.e.sup.meC.sub.eA-
.sub.e.sup.meC.sub.eT.sub.eA.sub.eT.sub.e.sup.meC.sub.eG.sub.e.sup.meC.sub-
.e.sup.meC.sub.eA.sub.eT.sub.eA.sub.eA.sub.eT.sub.e.sup.meC.sub.e.sup.meC.-
sub.eA.sub.e
[0235] Each internucleoside linking group is a phosphorothioate.
Nucleosides followed by a superscript Me indicates the following C
is a 5-methyl C, subscript e indicates the preceding nucleoside is
a 2'-O(CH.sub.2).sub.2--O--CH.sub.3 (MOE) substituted nucleoside
and subscript x indicates the preceding nucleoside is a
2'-OCH.sub.2CH.sub.2F (FEt) substituted nucleoside.
TABLE-US-00011 SEQ ID NO./ISIS NO. % UTC Dose (nM) 10/327895
010/410146 11/407588 12.5 105.4 102.2 130.4 25 112.7 115.8 129.5 50
145.3 157.5 132.8 100 137.6 181.0 116.4 200 151.4 214.2 147.9 400
103.0 185.5 119.6 Saline % UTC = 100
Sequence CWU 1
1
1113160DNAH. sapiens 1cctcccctcg cccggcgcgg tcccgtccgc ctctcgctcg
cctcccgcct cccctcggtc 60ttccgaggcg cccgggctcc cggcgcggcg gcggaggggg
cgggcaggcc ggcgggcggt 120gatgtggcag gactctttat gcgctgcggc
aggatacgcg ctcggcgctg ggacgcgact 180gcgctcagtt ctctcctctc
ggaagctgca gccatgatgg aagtttgaga gttgagccgc 240tgtgaggcga
ggccgggctc aggcgaggga gatgagagac ggcggcggcc gcggcccgga
300gcccctctca gcgcctgtga gcagccgcgg gggcagcgcc ctcggggagc
cggccggcct 360gcggcggcgg cagcggcggc gtttctcgcc tcctcttcgt
cttttctaac cgtgcagcct 420cttcctcggc ttctcctgaa agggaaggtg
gaagccgtgg gctcgggcgg gagccggctg 480aggcgcggcg gcggcggcgg
cggcacctcc cgctcctgga gcggggggga gaagcggcgg 540cggcggcggc
cgcggcggct gcagctccag ggagggggtc tgagtcgcct gtcaccattt
600ccagggctgg gaacgccgga gagttggtct ctccccttct actgcctcca
acacggcggc 660ggcggcggcg gcacatccag ggacccgggc cggttttaaa
cctcccgtcc gccgccgccg 720caccccccgt ggcccgggct ccggaggccg
ccggcggagg cagccgttcg gaggattatt 780cgtcttctcc ccattccgct
gccgccgctg ccaggcctct ggctgctgag gagaagcagg 840cccagtcgct
gcaaccatcc agcagccgcc gcagcagcca ttacccggct gcggtccaga
900gccaagcggc ggcagagcga ggggcatcag ctaccgccaa gtccagagcc
atttccatcc 960tgcagaagaa gccccgccac cagcagcttc tgccatctct
ctcctccttt ttcttcagcc 1020acaggctccc agacatgaca gccatcatca
aagagatcgt tagcagaaac aaaaggagat 1080atcaagagga tggattcgac
ttagacttga cctatattta tccaaacatt attgctatgg 1140gatttcctgc
agaaagactt gaaggcgtat acaggaacaa tattgatgat gtagtaaggt
1200ttttggattc aaagcataaa aaccattaca agatatacaa tctttgtgct
gaaagacatt 1260atgacaccgc caaatttaat tgcagagttg cacaatatcc
ttttgaagac cataacccac 1320cacagctaga acttatcaaa cccttttgtg
aagatcttga ccaatggcta agtgaagatg 1380acaatcatgt tgcagcaatt
cactgtaaag ctggaaaggg acgaactggt gtaatgatat 1440gtgcatattt
attacatcgg ggcaaatttt taaaggcaca agaggcccta gatttctatg
1500gggaagtaag gaccagagac aaaaagggag taactattcc cagtcagagg
cgctatgtgt 1560attattatag ctacctgtta aagaatcatc tggattatag
accagtggca ctgttgtttc 1620acaagatgat gtttgaaact attccaatgt
tcagtggcgg aacttgcaat cctcagtttg 1680tggtctgcca gctaaaggtg
aagatatatt cctccaattc aggacccaca cgacgggaag 1740acaagttcat
gtactttgag ttccctcagc cgttacctgt gtgtggtgat atcaaagtag
1800agttcttcca caaacagaac aagatgctaa aaaaggacaa aatgtttcac
ttttgggtaa 1860atacattctt cataccagga ccagaggaaa cctcagaaaa
agtagaaaat ggaagtctat 1920gtgatcaaga aatcgatagc atttgcagta
tagagcgtgc agataatgac aaggaatatc 1980tagtacttac tttaacaaaa
aatgatcttg acaaagcaaa taaagacaaa gccaaccgat 2040acttttctcc
aaattttaag gtgaagctgt acttcacaaa aacagtagag gagccgtcaa
2100atccagaggc tagcagttca acttctgtaa caccagatgt tagtgacaat
gaacctgatc 2160attatagata ttctgacacc actgactctg atccagagaa
tgaacctttt gatgaagatc 2220agcatacaca aattacaaaa gtctgaattt
ttttttatca agagggataa aacaccatga 2280aaataaactt gaataaactg
aaaatggacc tttttttttt taatggcaat aggacattgt 2340gtcagattac
cagttatagg aacaattctc ttttcctgac caatcttgtt ttaccctata
2400catccacagg gttttgacac ttgttgtcca gttgaaaaaa ggttgtgtag
ctgtgtcatg 2460tatatacctt tttgtgtcaa aaggacattt aaaattcaat
taggattaat aaagatggca 2520ctttcccgtt ttattccagt tttataaaaa
gtggagacag actgatgtgt atacgtagga 2580attttttcct tttgtgttct
gtcaccaact gaagtggcta aagagctttg tgatatactg 2640gttcacatcc
tacccctttg cacttgtggc aacagataag tttgcagttg gctaagagag
2700gtttccgaaa ggttttgcta ccattctaat gcatgtattc gggttagggc
aatggagggg 2760aatgctcaga aaggaaataa ttttatgctg gactctggac
catataccat ctccagctat 2820ttacacacac ctttctttag catgctacag
ttattaatct ggacattcga ggaattggcc 2880gctgtcactg cttgttgttt
gcgcattttt ttttaaagca tattggtgct agaaaaggca 2940gctaaaggaa
gtgaatctgt attggggtac aggaatgaac cttctgcaac atcttaagat
3000ccacaaatga agggatataa aaataatgtc ataggtaaga aacacagcaa
caatgactta 3060accatataaa tgtggaggct atcaacaaag aatgggcttg
aaacattata aaaattgaca 3120atgatttatt aaatatgttt tctcaattgt
aaaaaaaaaa 3160226DNAArtificial SequencePrimer 2aatggctaag
tgaagatgac aatcat 26325DNAArtificial SequencePrimer 3tgcacatatc
attacaccag ttcgt 25430DNAArtificial SequenceProbe 4ttgcagcaat
tcactgtaaa gctggaaagg 30514DNAArtificial SequenceSynthetic
Oligonucleotide 5cttagcactg gcct 14618DNAArtificial
SequenceSynthetic Oligonucleotide 6ctgctagcct ctggattt
18720DNAArtificial SequenceSynthetic Oligonucleotide 7ctgctagcct
ctggatttga 20821RNAArtificial SequenceSynthetic Oligonucleotide
8uugucucugg uccuuacuua a 21921DNAArtificial SequenceSynthetic
Oligonucleotide 9tugucucugg uccuuacuua a 211023DNAArtificial
SequenceSynthetic Oligonucleotide 10acaaacacca ttgtcacact cca
231123DNAArtificial SequenceSynthetic Oligonucleotide 11acaaccacta
tcgccataat cca 23
* * * * *