U.S. patent application number 11/657341 was filed with the patent office on 2007-09-13 for chemically modified oligonucleotides for use in modulating micro rna and uses thereof.
This patent application is currently assigned to The Rockefeller University. Invention is credited to Muthiah Manoharan, Kallanthottathil G. Rajeev, Markus Stoffel.
Application Number | 20070213292 11/657341 |
Document ID | / |
Family ID | 39645514 |
Filed Date | 2007-09-13 |
United States Patent
Application |
20070213292 |
Kind Code |
A1 |
Stoffel; Markus ; et
al. |
September 13, 2007 |
Chemically modified oligonucleotides for use in modulating micro
RNA and uses thereof
Abstract
This invention relates generally to chemically modified
oligonucleotides useful for modulating expression of microRNAs and
pre-microRNAs. More particularly, the invention relates to single
stranded chemically modified oligonucleotides for inhibiting
microRNA and pre-microRNA expression and to methods of making and
using the modified oligonucleotides. Also included in the invention
are compositions and methods for silencing microRNAs in the central
nervous system.
Inventors: |
Stoffel; Markus; (Zurich,
CH) ; Manoharan; Muthiah; (Weston, MA) ;
Rajeev; Kallanthottathil G.; (Cambridge, MA) |
Correspondence
Address: |
DRINKER BIDDLE & REATH;ATTN: INTELLECTUAL PROPERTY GROUP
ONE LOGAN SQUARE
18TH AND CHERRY STREETS
PHILADELPHIA
PA
19103-6996
US
|
Assignee: |
The Rockefeller University
New York
NY
10021
Alnylam Pharmaceuticals, Inc.
Cambridge
MA
02142
|
Family ID: |
39645514 |
Appl. No.: |
11/657341 |
Filed: |
January 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11502158 |
Aug 10, 2006 |
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11657341 |
Jan 24, 2007 |
|
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60706866 |
Aug 10, 2005 |
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60731554 |
Oct 28, 2005 |
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60763201 |
Jan 26, 2006 |
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Current U.S.
Class: |
514/44A ;
514/81 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2310/11 20130101; C12N 2310/345 20130101; C12N 2310/3533
20130101; C12N 2310/3515 20130101; C12N 2310/315 20130101; C12N
2310/346 20130101; C12N 2310/3527 20130101; C12N 2310/3531
20130101; C12N 2310/3521 20130101; C12N 2310/321 20130101 |
Class at
Publication: |
514/044 ;
514/081 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The work described herein was carried out, at least in part,
using funds from the United States government under grant number 1
P01 GM073047-01, awarded by the National Institutes of Health
(NIH). The United States government may therefore have certain
rights in the invention.
Claims
1. A method of reducing the amount of a microRNA (miRNA) in a cell
of the central nervous system (CNS) in a mammal, the method
comprising administering an antagomir to the mammal, wherein the
antagomir comprises a sequence which is substantially complementary
to 12 to 23 contiguous nucleotides of a target sequence, and
wherein the target sequence differs by no more than 1, 2, or 3
nucleotides from a sequence selected from the group consisting of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
2. The method of claim 1, wherein the antagomir comprises a
sequence selected from the group consisting of SEQ ID NOS:
5-39.
3. The method of claim 2, wherein the antagomir further comprises a
phosphorothioate backbone modification.
4. The method of claim 1, wherein the target sequence is SEQ ID NO:
2.
5. The method of claim 2, wherein the antagomir is at least
nineteen nucleotides in length.
6. The method of claim 2, wherein the antagomir is stabilized
against nucleolytic degradation.
7. The method of claim 3, wherein the phosphorothioate modification
is at least at the first two internucleotide linkage at the 5' end
of the nucleotide sequence.
8. The method of claim 3, wherein the phosphorothioate modification
is at least at the first four internucleotide linkage at the 3' end
of the nucleotide sequence.
9. The method of claim 3, wherein the phosphorothioate modification
is at the first two internucleotide linkage at the 5' end of the
nucleotide sequence, and at the first four internucleotide linkage
at the 3' end of the nucleotide sequence.
10. The method of claim 2, wherein the antagomir further comprises
a 2'-modified nucleotide.
11. The method of claim 10, wherein the 2'-modified nucleotide
comprises a modification selected from the group consisting of:
2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl
(2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl
(2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP),
2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and
2'-O--N-methylacetamido (2'-O--NMA).
12. The method of claim 11, wherein the 2'-modified nucleotide
comprises a 2'-O-methyl.
13. The method of claim 2, wherein the antagomir further comprises
a cholesterol molecule attached to the 3' end of the agent.
14. A method of treating a mammal suffering from a disease,
disorder or condition of the central nervous system, the method
comprising administering an antagomir to the mammal, wherein the
antagomir comprises a sequence which is substantially complementary
to 12 to 23 contiguous nucleotides of a target sequence, and
wherein the target sequence differs by no more than 1, 2, or 3
nucleotides from a sequence selected from the group consisting of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, further
wherein the presence of the antagomir in the central nervous system
effects treatment of the disease, disorder or condition.
15. The method of claim 14, wherein the antagomir comprises a
sequence selected from the group consisting of SEQ ID NOS:
5-39.
16. The method of claim 15, wherein the antagomir further comprises
a phosphorothioate backbone modifications.
17. The method of claim 14, wherein the target sequence is SEQ ID
NO: 2.
18. The method of claim 15, wherein the antagomir is at least
nineteen nucleotides in length.
19. The method of claim 15, wherein the antagomir is stabilized
against nucleolytic degradation.
20. The method of claim 16, wherein the phosphorothioate
modification is at least at the first two internucleotide linkage
at the 5' end of the nucleotide sequence.
21. The method of claim 16, wherein the phosphorothioate
modification is at least at the first four internucleotide linkage
at the 3' end of the nucleotide sequence.
22. The method of claim 16, wherein the phosphorothioate
modification is at the first two internucleotide linkage at the 5'
end of the nucleotide sequence, and at the first four
internucleotide linkage at the 3' end of the nucleotide
sequence.
23. The method of claim 15, wherein the antagomir further comprises
a 2'-modified nucleotide.
24. The method of claim 23, wherein the 2'-modified nucleotide
comprises a modification selected from the group consisting of:
2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl
(2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl
(2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP),
2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and
2'-O--N-methylacetamido (2'-O--NMA).
25. The method of claim 24, wherein the 2'-modified nucleotide
comprises a 2'-O-methyl.
26. The method of claim 15, wherein the antagomir further comprises
a cholesterol molecule attached to the 3' end of the agent.
27. The method of claim 14, wherein the mammal is a human.
28. The method of claim 14, wherein said disease, disorder or
condition of the central nervous system is selected from the group
consisting of a genetic disease, a disease associated with
unregulated expression of miR-16.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 11/502,158, filed Aug. 10, 2006, which claims
the benefit of U.S. Provisional Application No. 60/706,866, filed
Aug. 10, 2005; U.S. Provisional Application No. 60/731,554, filed
Oct. 28, 2005, and U.S. Provisional Application No. 60/763,201,
filed Jan. 26, 2006. The contents of each of these priority
applications are incorporated herein by reference in their
entirety.
TECHNICAL FIELD
[0003] This invention relates generally to chemically modified
oligonucleotides (antagomirs) useful for modulating expression of
microRNAs. More particularly, the invention relates to single
stranded, double stranded, partially double stranded and hairpin
structured chemically modified oligonucleotides for inhibiting
microRNA expression and to methods of making and using the modified
oligonucleotides.
BACKGROUND
[0004] A variety of nucleic acid species are capable of modifying
gene expression. These include antisense RNA, siRNA, microRNA, RNA
and DNA aptamers, and decoy RNAs. Each of these nucleic acid
species can inhibit target nucleic acid activity, including gene
expression.
[0005] MicroRNAs (miRNAs) are a class of 18-24 nt non-coding RNAs
(ncRNAs) that exist in a variety of organisms, including mammals,
and are conserved in evolution. miRNAs are processed from hairpin
precursors of 70 nt (pre-miRNA) which are derived from primary
transcripts (pri-miRNA) through sequential cleavage by the RNAse
III enzymes drosha and dicer. Many microRNAs can be encoded in
intergenic regions, hosted within introns of pre-mRNAs or within
ncRNA genes. Many miRNAs also tend to be clustered and transcribed
as polycistrons and often have similar spatial temporal expression
patterns. MiRNAs have been found to have roles in a variety of
biological processes including developmental timing,
differentiation, apoptosis, cell proliferation, organ development,
and metabolism.
[0006] miRNAs are an abundant class of non-coding RNA ranging from
20 to 23 nucleotides of length that are post-transcriptional
regulators of gene expression. miRNAs have been mainly associated
with developmental processes in metazoa such as Caenorhabditis
elegans or Drosophila melanogaster (Ambros, 2004 Nature 431:350-5).
However, evidence also suggests a role for miRNAs in a wide range
of functions in mammals, including insulin secretion, heart,
skeletal muscle and brain development (Kloosterman, et al., 2006
Dev Cell 11:441-50, and Krutzfeldt, et al., 2006 Cell Metab
4:9-12). Furthermore, miRNAs have been implicated in diseases such
as cancer (Esquela-Kerscher, et al., 2006 Nat Rev Cancer 6:259-69)
and hepatitis C (Jopling, et al., 2005 Science 309:1577-81), which
make them attractive new drug targets. In contrast to the widely
used RNAi technology using small interfering RNA (siRNA) duplexes,
strategies to inhibit miRNAs have been less well investigated.
Reverse-complement 2'-O-methyl sugar modified RNA is frequently
being used to block miRNA function in cell-based systems
(Krutzfeldt, et al., 2006 Nat Genet 38:S14-9). The use of miRNA
inhibitors, however, remains challenging. Thus, there is a long
felt need in the art for efficient and directed means of inhibiting
miRNA. The present invention satisfies this need.
SUMMARY
[0007] The present invention is based in part on the discovery that
expression of endogenous microRNAs (miRNAs) or pre-microRNAs
(pre-miRNAs) can be inhibited by an agent herein defined as an
antagomir, e.g., through systemic or local administration of the
antagomir, as well as by parenteral administration of such agents.
However, the invention should not be limited to any particular
route of administration.
[0008] The present invention provides specific compositions and
methods that are useful in reducing miRNA and pre-miRNA levels, in
e.g., a mammal, such as a human. In particular, the present
invention provides specific compositions and methods that are
useful for reducing levels of the miRNAs miR-122, miR-16, miR-192,
and miR-194.
[0009] In one aspect, the invention features antagomirs. Antagomirs
are single stranded, double stranded, partially double stranded and
hairpin structured chemically modified oligonucleotides that target
a microRNA. FIGS. 5-11 provides representative structures of
antagomirs.
[0010] An antagomir consisting essentially of or comprising at
least 12 or more contiguous nucleotides substantially complementary
to an endogenous miRNA and more particularly agents that include 12
or more contiguous nucleotides substantially complementary to a
target sequence of an miRNA or pre-miRNA nucleotide sequence.
Preferably, an antagomir featured in the invention includes a
nucleotide sequence sufficiently complementary to hybridize to a
miRNA target sequence of about 12 to 25 nucleotides, preferably
about 15 to 23 nucleotides. More preferably, the target sequence
differs by no more than 1, 2, or 3 nucleotides from a sequence
shown in Table 1, and in one embodiment, the antagomir is an agent
shown in Table 2a-e, Table 4, and Table 7. In one embodiment, the
antagomir includes a non-nucleotide moiety, e.g., a cholesterol
moiety. The non-nucleotide moiety can be attached, e.g., to the 3'
or 5' end of the oligonucleotide agent. In a preferred embodiment,
a cholesterol moiety is attached to the 3' end of the
oligonucleotide agent.
[0011] Antagomirs are stabilized against nucleolytic degradation
such as by the incorporation of a modification, e.g., a nucleotide
modification. In another embodiment, the antagomir includes a
phosphorothioate at least the first, second, or third
internucleotide linkage at the 5' or 3' end of the nucleotide
sequence. In yet another embodiment, the antagomir includes a
2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro,
2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O--N-methylacetamido (2'-O--NMA). In a particularly preferred
embodiment, the antagomir includes at least one
2'-O-methyl-modified nucleotide, and in some embodiments, all of
the nucleotides of the antagomir include a 2'-O-methyl
modification.
[0012] In one aspect, antagomirs are RNA-like oligonucleotides that
harbor various modifications for RNase protection and pharmacologic
properties such as enhanced tissue and cellular uptake. A preferred
antagomir differs from normal RNA by having complete
2'-O-methylation of sugar, phosphorothioate backbone and a
cholesterol-moiety at 3'-end. Phosphorothioate modifications
provide protection against RNase activity and their lipophilicity
contributes to enhanced tissue uptake. In a preferred embodiment,
the antagomir includes six phosphorothioate backbone modifications;
two phosphorothioates are located at the 5'-end and four at the
3'-end.
[0013] Antagomirs of the present invention can also be modified
with respect to their length or otherwise the number of nucleotides
making up the antagomir. In some instances, it is preferred that
the antagomirs of the present invention are of at least 19
nucleotides in length for optimal function.
[0014] An antagomir that is substantially complementary to a
nucleotide sequence of an miRNA can be delivered to a cell or a
human to inhibit or reduce the activity of an endogenous miRNA,
such as when aberrant or undesired miRNA activity, or insufficient
activity of a target mRNA that hybridizes to the endogenous miRNA,
is linked to a disease or disorder. In one embodiment, an antagomir
featured in the invention has a nucleotide sequence that is
substantially complementary to miR-122 (see Table 1), which
hybridizes to numerous RNAs, including aldolase A mRNA, N-myc
downstram regulated gene (Ndrg3) mRNA, IQ motif containing GTPase
activating protein-1 (Iqgap1) mRNA, HMG-CoA-reductase (Hmgcr) mRNA,
and citrate synthase mRNA and others. In a preferred embodiment,
the antagomir that is substantially complementary to miR-122 is
antagomir-122 (Table 2a-e, Table 4, and Table 7). Aldolase A
deficiencies have been found to be associated with a variety of
disorders, including hemolytic anemia, arthrogryposis complex
congenita, pituitary ectopia, rhabdomyolysis, hyperkalemia. Humans
suffering from aldolase A deficiencies also experience symptoms
that include growth and developmental retardation, midfacial
hypoplasia, hepatomegaly, as well as myopathic symptoms. Thus a
human who has or who is diagnosed as having any of these disorders
or symptoms is a candidate to receive treatment with an antagomir
that hybridizes to miR-122.
[0015] In some embodiments, an antagomir featured in the invention
has a nucleotide sequence that is substantially complementary to
miR-16, miR-192, or miR-194.
[0016] In one aspect, the invention features a method of reducing
the levels of an miRNA or pre-miRNA in a cell of a subject, e.g., a
human subject. In another aspect, the invention includes reducing
the level of an miRNA or pre-miRNA in a cell of the central nervous
system. The method includes the step of administering an antagomir
to the subject, where the antagomir is substantially
single-stranded and includes a sequence that is substantially
complementary to 12 to 23 contiguous nucleotides, and preferably 15
to 23 contiguous nucleotides, of a target sequence of an miRNA or
pre-miRNA nucleotide sequence. Preferably, the target sequence
differs by no more than 1, 2, or 3 nucleotides from a microRNA or
pre-microRNA sequence, such as a microRNA sequence shown in Table
1.
[0017] In one embodiment, the methods featured in the invention are
useful for reducing the level of an endogenous miRNA (e.g.,
miR-122, miR-16, miR-192 or miR-194) or pre-miRNA in a cell, e.g,
in a cell of a subject, such as a human subject. Preferably, the
cell is a cell of the central nervous system. Such methods include
contacting the cell with an antagomir described herein for a time
sufficient to allow uptake of the antagomir into the cell.
[0018] In another aspect, the invention features a pharmaceutical
composition including an antagomir described herein, and a
pharmaceutically acceptable carrier. In a preferred embodiment, the
antagomir included in the pharmaceutical composition hybridizes to
miR-122, miR-16, miR-192, or miR-194.
[0019] In another aspect the invention features a method of
inhibiting miRNA expression (e.g., miR-122, miR-16, miR-192, or
miR-194 expression) or pre-miRNA expression in a cell, e.g., a cell
of a subject. Preferably, the cell is a cell of the central nervous
system. The method includes contacting the cell with an effective
amount of an antagomir described herein, which is substantially
complementary to the nucleotide sequence of the target miRNA or the
target pre-miRNA. Such methods can be performed on a mammalian
subject by administering to a subject one of the oligonucleotide
agents/pharmaceutical compositions described herein.
[0020] In another aspect the invention features a method of
increasing levels of an RNA or protein that are encoded by a gene
whose expression is down-regulated by an miRNA, e.g., an endogenous
miRNA, such as miR-122, miR-16, miR-192 or mir-194. The method
includes contacting the cell with an effective amount of an
antagomir described herein, which is substantially complementary to
the nucleotide sequence of the miRNA that binds to and effectively
inhibits translation of the RNA transcribed from the gene. For
example, the invention features a method of increasing aldolase A
protein levels in a cell. Similarly, the invention features a
method of increasing Ndrg3, Iqgap1, Hmgcr, and/or citrate synthase
protein levels in a cell. The methods include contacting the cell
with an effective amount of an antagomir described herein (e.g.,
antagomir-122, described in Table 2a-e, Table 4, and Table 7),
which is substantially complementary to the nucleotide sequence of
miR-122 (see Table 1).
[0021] In another aspect, the invention provides methods of
increasing expression of a target gene by providing an antagomir to
which a lipophilic moiety is conjugated, e.g., a lipophilic
conjugated antagomir described herein, to a cell. The antagomir
preferably hybridizes to an miRNA (e.g., miR-122, miR-16, miR-192,
or miR-194) or a pre-miRNA. In a preferred embodiment the
conjugated antagomir can be used to increase expression of a target
gene in an organism, e.g., a mammal, e.g., a human, or to increase
expression of a target gene in a cell line or in cells which are
outside an organism. An mRNA transcribed from the target gene
hybridizes to an endogenous miRNA, which consequently results in
downregulation of mRNA expression. An antagomir featured in the
invention hybridizes to the endogenous miRNA and consequently
causes an increase in mRNA expression. In the case of a whole
organism, the method can be used to increase expression of a gene
and treat a condition associated with a low level of expression of
the gene. For example, an antagomir that targets miR-122 (e.g.,
antagomir-122) can be used to increase expression of an aldolase A
gene to treat a subject having, or at risk for developing,
hemolytic anemia, arthrogryposis complex congenita, pituitary
ectopia, rhabdomyolysis, hyperkalemia, or any other disorder
associated with aldolase A deficiency. Administration of an
antagomir that targets miR-122 (e.g., antagomir-122) can be also be
used to increase expression of an Ndrg3, Iqgap1, Hmgcr, or citrate
synthase gene to treat a subject having, or at risk for developing,
a disorder associated with a decreased expression of any one of
these genes.
DESCRIPTION OF DRAWINGS
[0022] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0023] FIG. 1A is a panel of Northern blots of total RNA (15 .mu.g)
isolated from mouse liver 24 h after injection of differently
modified RNAs (240 mg/kg) targeting miR-122. Samples were separated
in 14%-polyacrylamide gels in the absence of formamide, and the
membranes were probed for miR-122. Ethidium bromide staining of
tRNA is shown as a loading control.
[0024] FIG. 1B is a panel of Northern blots of total RNA (15 .mu.g)
isolated from mouse liver 24 h after injection of differently
modified RNAs (240 mg/kg) against miR-122. Samples were separated
in 14%-polyacrylamide gels in the absence of formamide, and the
membranes were probed for miR-122, let7, and miR-22 RNAs. Ethidium
bromide staining of tRNA is shown as a loading control.
[0025] FIG. 1C is a panel of Northern blots of total RNA (15 .mu.g)
isolated from mouse liver 24 h after injection of differently
modified RNAs (240 mg/kg) against miR-122. Samples were separated
in 14%-polyacrylamide gels in the presence of 20% formamide, and
the membranes were probed for miR-122. Ethidium bromide staining of
tRNA is shown as a loading control.
[0026] FIG. 2A is a panel of Northern blots of total RNA (15 .mu.g)
isolated from mouse livers. RNA was isolated 24 h after injection
of 80 mg/kg bodyweight antagomir-122 (n=2) on 1, 2, or 3
consecutive days as indicated. Membranes were probed for both the
endogenous miR-122 and the injected antagomir-122. Ethidium bromide
staining of tRNA is shown as a loading control.
[0027] FIG. 2B is a panel of Northern blots of total RNA (15 .mu.g)
isolated from mouse livers. RNA was isolated 3, 6, 9, 13, and 23
days after injection of antagomir-122. Membranes were probed for
both the endogenous miR-122 and the injected antagomir-122.
Ethidium bromide staining of tRNA is shown as a loading
control.
[0028] FIG. 3A is a panel of Northern blots of total RNA (10-30
.mu.g) isolated from different mouse tissues 24 h after injection
of antagomir-16 (n=3). Membranes were probed for miR-16. The
precursor miR-16 transcript was visible on Northern blots of bone
marrow and expression was similar in all mice. Ethidium bromide
staining of tRNA is shown as a loading control.
[0029] FIG. 3B is a panel of Northern blots of total RNA (10-30
.mu.g) isolated from different mouse tissues 24 h after injection
of antagomir-16 (n=3). Total RNA from 3 mice were pooled for the
detection of miR-16 and the injected antagomir-16. Ethidium bromide
staining of tRNA is shown as a loading control.
[0030] FIG. 4 includes a panel of Northern blots of total RNA
isolated from livers of mice injected with antagomiR-122,
mm-antagomir-122, or PBS. RNA was extracted 24 h after injection by
a bDNA lysis method. Northern blots were probed with miR-122 and
let7 microRNAs. Ethidium bromide staining of tRNA is shown as a
loading control.
[0031] FIG. 5 depicts ligand conjugated oligonucleotide to modulate
expression of miRNA: (a) ligand of interest is conjugated to the
oligonucleotide via a tether and linker; (b) ligand of interest is
conjugated to the oligonucleotide via a linker without a tether or
tether without an additional linker and (c) a ligand of interest is
attached directly to the oligonucleotide.
[0032] FIG. 6 depicts ligand conjugated double stranded
oligonucleotide to modulate expression of miRNA: (a) ligand of
interest is conjugated to the oligonucleotide via a tether and
linker; (b) ligand of interest is conjugated to the oligonucleotide
via a linker without a tether or tether without an additional
linker and (c) a ligand of interest is attached directly to the
oligonucleotide.
[0033] FIG. 7 depicts ligand conjugated antisense strand comprising
partially double stranded oligonucleotides to modulate expression
of miRNA. (a-c) ligand of interest is conjugated to the
oligonucleotide via a tether and linker; (d-f) ligand of interest
is conjugated to the oligonucleotide via a linker without a tether
or tether without an additional linker and (g-i) a ligand of
interest is attached directly to the oligonucleotide.
[0034] FIG. 8 depicts ligand conjugated partial sense strand
comprising partially double stranded oligonucleotides to modulate
expression of miRNA. (a-c) ligand of interest is conjugated to the
oligonucleotide via a tether and linker; (d-f) ligand of interest
is conjugated to the oligonucleotide via a linker without a tether
or tether without an additional linker and (g-i) a ligand of
interest is attached directly to the oligonucleotide.
[0035] FIG. 9 depicts ligand conjugated partial hairpin
oligonucleotides to modulate expression of miRNA. (a-b) ligand of
interest is conjugated to either 3' or 5' end of the hairpin via a
tether and linker; (c-d) ligand of interest is conjugated to the
hairpin via a linker without a tether or tether without an
additional linker and (e-f) a ligand of interest is attached
directly to the oligonucleotide. The hairpin is comprised of
nucleotides or non-nucleotide linkages.
[0036] FIG. 10 depicts ligand conjugated hairpin oligonucleotides
to modulate expression of miRNA. (a) ligand of interest is
conjugated to either 3' or 5' end of the hairpin via a tether and
linker; (b) ligand of interest is conjugated to the hairpin via a
linker without a tether or tether without an additional linker and
(c) a ligand of interest is attached directly to the
oligonucleotide. The hairpin is comprised of nucleotides or
non-nucleotide linkages.
[0037] FIG. 11 depicts cholesterol conjugated oligonucleotides to
modulate expression of miRNA. (a) 5' cholesterol conjugate; (b) 3'
cholesterol conjugate and (c) cholesterol conjugate building blocks
for oligonucleotide synthesis. The oligonucleotide can be miRNA,
anti-miRNA, chemically modified RNA or DNA; DNA or DNA analogues
for antisense application.
[0038] FIG. 12 depicts activity of double-stranded antagomirs (see
Table 2f for the description of agents used).
[0039] FIG. 13 depicts dose response for antagomir-122.
[0040] FIG. 14 depicts mismatch control for antagomir-122.
[0041] FIG. 15 depicts length effect on activity of
antagomir-122.
[0042] FIG. 16 is a schematic representation of various chemical
modifications to an antagomir. The designation of (I), (II), (III),
and (IV) corresponds to miR-122, antagomir-122, antagomir-122 all
P.dbd.S and 5'-Quasar570 labeled antagomir-122, respectively.
[0043] FIG. 17, comprising FIGS. 17A through 17C, is a series of
charts demonstrating the impact of antagomir phosphorothioate
modifications and antagomir length on miR-122 levels. FIG. 17
comprises Northern blots of total RNA isolated from livers of mice
that were treated with different antagomir-122 chemistries at
3.times.20 mg/kg bw. FIG. 17A demonstrates the impact of
mm-antagomir-122, antagomir-122 (no phosphorothioate modification),
and antagomir-122 on the RNA level of miR-122. FIG. 17B
demonstrates the impact of different phosphorothioate modifications
to the antagomir on the RNA level of miR-122. FIG. 17C demonstrates
the impact of different lengths to the antagomir on the RNA level
of miR-122. "P.dbd.S" indicates phosphorothioate modification.
[0044] FIG. 18, comprising FIGS. 18A and 18B, is a series of charts
demonstrating dose- and time-dependency of miR-122 target
regulation by antagomir-122. FIG. 18A depicts a dose-dependent
study. FIG. 18B is a time-course experiment. Also depicted in FIGS.
18A and 18B are the steady-state mRNA levels of miR-122 target
genes in livers of mice treated with the indicated amounts or
duration of antagomir-122. The glyceraldehyde-3-phosphate
dehydrogenase gene (Gapdh) was used as a loading control. The upper
row in each chart shows a Northern blot of liver RNA for
miR-122.
[0045] FIG. 19, comprising FIGS. 19A and 19B, is a series of charts
demonstrating sequence discrimination of antagomir-122. FIG. 19
depicts steady-state mRNA levels of miR-122 target genes in livers
of mice treated with the indicated amounts of antagomir-122 or
antagomir-122 that harbored 4, 2 or 1 nucleotide mismatches (FIG.
19A), or 1 nucleotide mismatch at different positions (FIG.
19B).
[0046] FIG. 20, comprising FIGS. 20A and 20B, is a series of charts
demonstrating the regulation of miR-122 targets by chemically
protected antagomir-122/miR-122-duplexes. FIG. 20A is a schematic
description of two different duplexes used. FIG. 20B depicts the
steady-state mRNA levels of miR-122 target genes in livers of mice
treated with the indicated modified antagomir-122/miR-122-duplexes.
Fold-regulation indicates the ratio of expression levels of the
means of mice treated with antagomir-122/miR-122 duplex compared to
the PBS group. The upper row shows a Northern blot of liver RNA for
miR-122. As controls, duplexes were added to 5 .mu.g total kidney
RNA and loaded on polyacrylamide gels before ("input") or after the
Trizol protocol ("Trizol"). *: p<0.05; **: p<0.01; ***:
p<0.001; student's t-test compared to PBS.
[0047] FIG. 21, comprising FIGS. 21A through 21C, is a series of
charts that demonstrate localization of antagomir-122 and miR-122
in hepatocytes. Liver tissue from mice that were treated with
3.times.80 mg/kg Q570-labeled mm-antagomir-122 was fractionated on
a sucrose gradient following ultracentrifugation. Localization of
Q570-labeled mm-antagomir-122 was analyzed by spectrophotometry
(FIG. 21A). Localization of t-RNA and miR-122 were analyzed using
Northern blotting of total RNA isolated from each fraction (FIG.
21B). For subcellular localization of antagomirs and P-bodies in
mouse liver, mice were treated with Q570-labeled antagomir-122 and
a DNA-plasmid expressing a GFP-GW182 hybrid. P-body and
Q570-antagomir localizations were visualized using laser-scanning
microscopy (FIG. 21C).
[0048] FIG. 22 is a chart depicting Northern blots of miR-16 and
miR-124 from total RNA isolated from mouse cerebral cortex that had
been injected with antagomir-16 or PBS into the right and left
cerebral hemispheres, respectively.
[0049] FIG. 23 is a chart demonstrating that miR-122 regulates mRNA
levels of many targets.
[0050] FIG. 24 is a chart demonstrating that miR-122 regulates the
expression of cholesterol biosynthesis genes.
[0051] FIG. 25 is a chart demonstrating metabolic parameters of
antagomir-122 treated mice.
[0052] The following experiments are designed to study miRNA
function in vivo. Typically, gene expression profiling,
bioinformatics analysis, metabolic profiling, and biochemical
target validation is performed. Using methods discussed elsewhere
herein, miR-122 was observed to regulate levels of many target
genes (FIG. 23). Moreover, miR-122 was observed to regulate the
expression of cholesterol biosynthesis genes (FIG. 24). Based on
the genes observed to be regulated by miR-122, metabolic parameters
of antagomir-122 treated mice were evaluated. The results
demonstrated that mice treated with antagomir-122 exhibited a
decrease in cholesterol as compared with mice treated with
mm-antagomir. The results presented herein characterize the
inhibition of miRNAs with antagomirs in vivo and their therapeutic
use with respect to cholesterol levels.
DETAILED DESCRIPTION
[0053] The present invention is based in part on the discovery that
expression of endogenous microRNAs (miRNAs) or pre-microRNAs
(pre-miRNAs) can be inhibited by an antagomir, e.g., through
systemic administration of an antagomir, as well as by parenteral
administration of such agents. Based on these findings, the present
invention provides specific compositions and methods that are
useful in reducing miRNA and pre-miRNA levels, in e.g., a mammal,
such as a human. In particular, the present invention provides
specific compositions and methods that are useful for reducing
levels of the miRNAs miR-122, miR-16, miR-192, and miR-194, herein
defined as antagomirs.
[0054] In one aspect, the invention features antagomirs. An
antagomir is a single-stranded, double stranded, partially double
stranded or hairpin structured chemically modified oligonucleotide
agents that consisting of, consisting essentially of or comprising
at least 12 or more contiguous nucleotides substantially
complementary to an endogenous miRNA and more particularly agents
that include 12 or more contiguous nucleotides substantially
complementary to a target sequence of an miRNA or pre-miRNA
nucleotide sequence. As used herein partially double stranded
referes to double stranded structures that contain less nucleotides
than the complementary strand. In general, such partial double
stranded agents will have less than 75% double stranded structure,
preferably less than 50%, and more preferably less than 25%, 20% or
15% double stranded structure. FIGS. 5-11 provides representative
structures of antagomirs.
[0055] Preferably, an antagomir featured in the invention includes
a nucleotide sequence sufficiently complementary to hybridize to an
miRNA target sequence of about 12 to 25 nucleotides, preferably
about 15 to 23 nucleotides. More preferably, the target sequence
differs by no more than 1, 2, or 3 nucleotides from a sequence
shown in Table 1, and in one embodiment, the antagomir is an agent
shown in Table 2a-e, Table 4 and Table 7. In one embodiment, the
antagomir includes a non-nucleotide moiety, e.g., a cholesterol
moiety. The non-nucleotide moiety can be attached, e.g., to the 3'
or 5' end of the oligonucleotide agent. In a preferred embodiment,
a cholesterol moiety is attached to the 3' end of the
oligonucleotide agent.
[0056] In another aspect, the length of the antagimor can
contribute to the biochemical function of the antagimor with
respect to the ability to decrease expression levels of a desired
miRNA. An miRNA-type antagomir can be, for example, from about 12
to 30 nucleotides in length, preferably about 15 to 28 nucleotides
in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27
nucleotides in length). In some instances, antagomirs may require
at least 19 nucleotides in length for optimal function.
[0057] The antagomir is further stabilized against nucleolytic
degradation such as by the incorporation of a modification, e.g., a
nucleotide modification. The antagomir includes a phosphorothioate
at least the first, second, or third internucleotide linkage at the
5' or 3' end of the nucleotide sequence. In one embodiment, the
antagomir includes a 2'-modified nucleotide, e.g., a 2'-deoxy,
2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O--N-methylacetamido (2'-O--NMA). In a particularly preferred
embodiment, the antagomir includes at least one
2'-O-methyl-modified nucleotide, and in some embodiments, all of
the nucleotides of the antagomir include a 2'-O-methyl
modification. In yet another preferred embodiment, the antagomir
includes six phosphorothioate backbone modifications; two
phosphorothioates are located at the 5'-end and four at the 3'-end.
In a preferred embodiment, the antagimor comprises 19 nucleotides
and six phosphorothioate backbone modifications.
[0058] The antagomir is further modified so as to be attached to a
ligand that is selected to improve stability, distribution or
cellular uptake of the agent, e.g., cholesterol. In a preferred
embodiment, the antagimor comprises 19 nucleotides, six
phosphorothioate backbone modifications and a ligand to improve
stability, distribution or cellular uptake of the antagomir. The
oligonucleotide antagomir can further be in isolated form or can be
part of a pharmaceutical composition used for the methods described
herein, particularly as a pharmaceutical composition formulated for
parental administration. The pharmaceutical compositions can
contain one or more oligonucleotide agents, and in some
embodiments, will contain two or more oligonucleotide agents, each
one directed to a different miRNA.
[0059] An antagomir that is substantially complementary to a
nucleotide sequence of an miRNA can be delivered to a cell or a
human to inhibit or reduce the activity of an endogenous miRNA,
such as when aberrant or undesired miRNA activity, or insufficient
activity of a target mRNA that hybridizes to the endogenous miRNA,
is linked to a disease or disorder. In one embodiment, an antagomir
featured in the invention has a nucleotide sequence that is
substantially complementary to miR-122 (see Table 1), which
hybridizes to numerous RNAs, including aldolase A mRNA, N-myc
downstram regulated gene (Ndrg3) mRNA, IQ motif containing GTPase
activating protein-1 (Iqgap1) mRNA, HMG-CoA-reductase (Hmgcr) mRNA,
and citrate synthase mRNA and others. In a preferred embodiment,
the antagomir that is substantially complementary to miR-122 is
antagomir-122 (Table 2a-e, Table 4 and Table 7). Aldolase A
deficiencies have been found to be associated with a variety of
disorders, including hemolytic anemia, arthrogryposis complex
congenita, pituitary ectopia, rhabdomyolysis, hyperkalemia. Humans
suffering from aldolase A deficiencies also experience symptoms
that include growth and developmental retardation, midfacial
hypoplasia, hepatomegaly, as well as myopathic symptoms. Thus a
human who has or who is diagnosed as having any of these disorders
or symptoms is a candidate to receive treatment with an antagomir,
such as a single-stranded oligonucleotide agent, that hybridizes to
miR-122.
[0060] In some embodiments, an antagomir featured in the invention
has a nucleotide sequence that is substantially complementary to
miR-16, miR-192, or miR-194 (see Table 1).
[0061] In one embodiment, the antagomiris selected from those shown
in Table 2a-e, Table 4 and Table 7. The single-stranded
oligonucleotide agents of Table 2a-e, Table 4 and Table 7 are
complementary to and hybridize to the corresponding miRNAs of Table
1. TABLE-US-00001 TABLE 1 Exemplary miRNAs identified in mus
musculus miRNA Sequence SEQ ID NO: miR-122
5'-UGGAGUGUGACAAUGGUGUUUGU-3' 1 miR-16 5'-UAGCAGCACGUAAAUAUUGGCG-3'
2 miR-192 5'-CUGACCUAUGAAUUGACAGCC-3' 3 miR-194
5'-UGUAACAGCAACUCCAUGUGGA-3' 4
[0062] TABLE-US-00002 TABLE 2a Oligonucleotide agents targeting mus
musculus miRNAs RNA Sequence SEQ ID NO: antagomir-122
5'-a.sub.sc.sub.saaacaccauugucacacu.sub.sc.sub.sc.sub.sa.sub.s-Chol-3'
5 antagomir-165
5'-c.sub.sg.sub.sccaau.sub.suuuacgugcug.sub.sc.sub.su.sub.sa.sub.s-Chol-3-
' 6 antagomir-192
5'-g.sub.sg.sub.scugucaauucauaggu.sub.sc.sub.sa.sub.sg.sub.s-Chol-3'
7 antagomir-194
5'-u.sub.sc.sub.scacauggaguugcuguu.sub.sa.sub.sc.sub.sa.sub.s-Chol-3'
lower case letters represent 2'-O-methyl modified nucleotides;
subscript `s` represents a phosphorothioate linkage; "Chol"
indicates cholesterol conjugate
[0063] TABLE-US-00003 TABLE 2b Double stranded oligonucleotides to
modulate microRNAs Duplex ID Sequence ID and sequence AL-DP-3018:
AL-SQ-3035: UGGAGUGUGACAAUGGUGUUUGU (SEQ ID NO:1) AL-SQ-3037:
oAsoCsoAsoAsoAsoCsoAsoCsoCsoAsoUsoUso
GsoUsoCsoAsoCsoAsoCsoUsoCsoCsoAs-Chol (SEQ ID NO:10) AL-DP-3019
AL-SQ-3035: UGGAGUGUGACAAUGGUGUUUGU (SEQ ID NO:1) AL-SQ-3038:
oAsoCsoAoAoAoCoAoCoCoAoUoUoGoUoCoAoCoAo CoUsoCsoCsoAs-Chol (SEQ ID
NO:11) AL-DP-3020 AL-SQ-3036: (mismatch) UGGAAUGUGACAGUGUUGUGUGU
(SEQ ID NQ:12) AL-SQ-3039: oAsoCsoAsoCsoAsoCsoAsoAsoCsoAsoCsoUso
GsoUsoCsoAsoCsoAsoUsoUsoCsoCsoAs-Chol (SEQ ID NO:13) AL-DP-3021
AL-SQ-3036: (mismatch) UGGAAUGUGACAGUGUUGUGUGU (SEQ ID NO:12)
AL-SQ-3040: oAsoCsoAoCoAoCoAoAoCoAoCoUoGoUoCoAoCo
AoUoUsoCsoCsoAs-Chol (SEQ ID NO:14) Note: oN represents 2'-O-Me
ribo sugar modification, dN represents deoxyribo sugar modification
and `s` stands for phosphorothioate linkage
[0064] TABLE-US-00004 TABLE 2c Partial double stranded and hairpin
structured oligonucleotides to modulate microRNA-122 Sequence ID
Sequence AL-SQ-3384 oAoCoAoAoAoCoAoCoCoAoUoUoGoUoCoAoCoAoCo
UoCoCoAdTdTdTdToUoGoGoAs-Chol (SEQ ID NO:15) AL-SQ-3385
oAoCoAoAoAoCoAoCoCoAoUoUoGoUoCoAoCoAoCo
UoCoCoAsdTsdTsdTsdTsoUoGoGoAs-Chol (SEQ ID NO:16) Note: oN
represents 2'-O-Me ribo sugar modification, dN represents deoxyribo
sugar modification and `s` stands for phosphorothioate linkage
[0065] TABLE-US-00005 TABLE 2d Partial double stranded
oligonucleotides to modulate microRNA-122 SEQ ID Duplex ID Sequence
ID and sequence NO: AL-DP-3043 AL-SQ-3038: 11
oAsoCsoAoAoAoCoAoCoCoAoUoUoGoUoCoAoCoAo CoUsoCsoCsoAs-Chol
AL-SQ-3400: 17 oUoGoGoAoGoUoG (7-mer at the 3'-end) AL-DP-3044
AL-SQ-3038: 11 oAsoCsoAoAoAoCoAoCoCoAoUoUoGoUoCoAo
CoAoCoUsoCsoCsoAs-Chol AL-SQ-3401: 18 oGoAoCoAoAoUoG (7-mer at nts
9-15) AL-DP-3045 AL-SQ-3040: 14 oAsoCsoAoCoAoCoAoAoCoAoCoUoGoUoCoAo
CoAoUoUsoCsoCsoAs-Chol AL-SQ-3402: 19 oUoGoGoAoAoUoG (7-mer at the
3'-end) AL-DP-3046 AL-SQ-3040: 14
oAsoCsoAoCoAoCoAoAoCoAoCoUoGoUoCoAo CoAoUoUsoCsoCsoAs-Chol
AL-SQ-3403: 20 oGoAoCoAoGoUoG (7-mer at nts 9-15) Note: oN
represents 2'-O-Me ribo sugar modification, dN represents deoxyribo
sugar modification and `s` stands for phosphorothioate linkage
[0066] TABLE-US-00006 TABLE 2e Single stranded oligonucleotides to
modulate microRNAs SEQ ID Sequence ID Sequence NO: AL-SQ-3035
UGGAGUGUGACAAUGGUGUUUGU 1 AL-SQ-3036 UGGAAUGUGACAGUGUUGUGUGU 12
AL-SQ-3037 oAsoCsoAsoAsoAsoCsoAsoCsoCsoAsoUso 10
UsoGsoUsoCsoAsoCsoAsoCsoUsoCsoCsoAs- Chol AL-SQ-3038
oAsoCsoAoAoAoCoAoCoCoAoUoUoGoUoCoAo 11 CoAoCoUsoCsoCsoAs-chol
AL-SQ-3039 oAsoCsoAsoCsoAsoCsoAsoAsoCsoAsoCso 13
UsoGsoUsoCsoAsoCsoAsoUsoUsoCsoCsoAs- Chol AL-SQ-3040
oAsoCsoAoCoAoCoAoAoCoAoCoUoGoUoCoAo 14 CoAoUoUsoCsoCsoAs-chol
AL-SQ-3223 oUsoGsoGoAoGoUoGoUoGoAoCoAoAoUoGoGo 21
UoGoUoUsoUsoGsoUs-chol AL-SQ-3224
oUsoGsoGoAoAoUoGoUoGoAoCoAoGoUoGoUo 22 UoGoUoGsoUsoGsoUs-chol
AL-SQ-3225 oAsoCsoAsoAsoAsoCsoAsoCsoCsoAsoUso 23
UsoGsoUsoCsoAsoCsoAsoCsoUsoCsoCsoA AL-SQ-3226
oAsoCsoAoAoAoCoAoCoCoAoUoUoGoUoCo 24 AoCoAOCoUoC*oC*Oa AL-SQ-3227
oCsoGsoCoCoAoAoUoAoUoUoUoAoCoGoUo 25 GoCoUoGoC*oU*oA*-chol
AL-SQ-3228 oGsoGsoCoUoGoUoCoAoAoUoUoCoAoUoAo 26
GoGoU*oC*oA*oG*-chol AL-SQ-3229 oUsoCsoCoAoCoAoUoGoGoAoGoUoUoGoCo
27 UoGoUoUoNoCoA-chol AL-SQ-3230 oUsoCsoAoCoGoCoGoAoGoCoCoGoAoAoCo
28 GoAoAoCsoAsoAsQAs-chol AL-SQ-3344 UGGIGUGUGICIIUGGUGUUUGU 29
AL-SQ-3350 oAoCoAoAoAoCoAoCoCoAoUoUoGoUoCoAo 30 CoAoCoUoCoCoA-Chol
AL-SQ-3351 oCsoAsoCoAoAoAoCoAoCoCoAoUoUoGoUo 31
CoAoCoAoCoUoCsoCsoAsoCs-Chol AL-SQ-3352
oCsoAsoAoAoCoAoCoCoAoUoUoGoUoCoAo 32 CoAoCsoUsoCsoCs-Chol
AL-SQ-3353 oAsoAsoAoCoAoCoCoAoUoUoGoUoCoAoCo 33 AsoCsoUsoCs-Chol
AL-SQ-3354 oAsoAsoCoAoCoCoAoUoUoGoUoCoAoCso 34 AsoCsoUs-Chol
AL-SQ-3355 oAsoCsoAoAoAoCoAoAoCoAoCoUoGoUoCo 35
AoCoAoUoUsoCsoCsoAs-Chol AL-SQ-3356
oAsoCsoAoAoAoCoAoCoCoAoCoUoGoUoCo 36 AoCoAoUoUsoCsoCsoAs-Chol
AL-SQ-3357 oAsoCsoAoAoAoCoAoCoCoAoUoUoGoUoCo 37
AoCoAoUoUsoCsoCsoAs-Chol AL-SQ-3358
Cy-5-soAsoCoAoAoAoCoAoCoCoAoUoUo 38 GoUoCoAoCoAoCoUsoCsoCsoAs-Chol
AL-SQ-3359 Cy-3-soAsoCoAoCoAoCoAoAoCoAoCoUo 39
GoUoCoAoCoAoUoUsoCsoCsoAs-Chol Cy-5 and Cy-3 are dyes used for
localization studies.
[0067] TABLE-US-00007 TABLE 2f Description of sequences listed in
Table 2b-2e Sequence # Description AL-SQ-3035 complementary to
antagomir-122 AL-SQ-3036 complementary to mm-antagomir-122
AL-SQ-3037 antagomir-122-fullyPS AL-SQ-3038 antagomir-122
AL-SQ-3039 mm-antagomir-122-fullyPS AL-SQ-3040 mm-antagomir-122
AL-SQ-3223 complementary to antagomir-122 AL-SQ-3224 complementary
to mm-antagomir-122 AL-SQ-3225 anti-122fs AL-SQ-3226 anti-122ps
AL-SQ-3227 antagomir-16 AL-SQ-3228 antagomir-192 AL-SQ-3229
antagomir-194 AL-SQ-3230 antagomir-375 AL-SQ-3344 complementary to
antagomir-122 with A -> I modification AL-SQ-3350
antagomir-122-noPS AL-SQ-3351 antagomir-122-25mer AL-SQ-3352
antagomir-122-21mer AL-SQ-3353 antagomir-122-19mer AL-SQ-3354
antagomir-122-17mer AL-SQ-3355 mismatch -antagomir-122-3mm
AL-SQ-3356 mismatch-antagomir-122-2mm AL-SQ-3357
mismatch-antagomir-122-1mm AL-SQ-3358 antagomir-122-5'-Cy5
AL-SQ-3359 antagomir-122-5'-Cy3 AL-SQ-3400 7-mer complementary to
3'-end of antagomir-122 AL-SQ-3401 7-mer complementary to
nucleotides 9-15 of antagomir-122 AL-SQ-3402 7-mer complementary to
3-end of mismatch -antagomir-122 AL-SQ-3403 7-mer complementary to
nucleotides 9-15 of mismatch -antagomir-122
[0068] In one aspect, the invention features an antagomir, such as
a single-stranded oligonucleotide agent, that includes a nucleotide
sequence that is substantially identical to a nucleotide sequence
of an miRNA, such as an endogenous miRNA listed in Table 1. An
oligonucleotide sequence that is substantially identical to an
endogenous miRNA sequence is 70%, 80%, 90%, or more identical to
the endogenous miRNA sequence. Preferably, the agent is identical
in sequence with an endogenous miRNA. An antagomir that is
substantially identical to a nucleotide sequence of an miRNA can be
delivered to a cell or a human to replace or supplement the
activity of an endogenous miRNA, such as when an miRNA deficiency
is linked to a disease or disorder, or aberrant or unwanted
expression of the mRNA that is the target of the endogenous miRNA
is linked to a disease or disorder. In one embodiment, an antagomir
agent featured in the invention can have a nucleotide sequence that
is substantially identical to miR-122 (see Table 1). An miR-122
binds to numerous RNAs including aldolase A mRNA, which has been
shown to be overexpressed in different cancers, including lung
cancer and breast cancer, and is overexpressed in adenocarcinomas
of various different tissues origins. Thus a single stranded
antagomir that is substantially identical to miR-122 can be
administered as a therapeutic composition to a subject having or at
risk for developing lung cancer or breast cancer, for example.
[0069] An miR-122 binds other mRNAs, including N-myc downstram
regulated gene (Ndrg3) mRNA, IQ motif containing GTPase activating
protein-1 (Iqgap1) mRNA, HMG-CoA-reductase (Hmgcr) mRNA, and
citrate synthase mRNA. Iqgap1 overexpression is associated with
gastric cancer and colorectal cancer. Thus a single stranded
antagomir that is substantially identical to miR-122 can be useful
for downregulating Iqgap1 expression, and can be administered as a
therapeutic composition to a subject having or at risk for
developing gastric cancer and colorectal cancer. Hmgcr inhibitors
are useful to treat hyperglycemia and to reduce the risk of stroke
and bone fractures. Thus a single stranded antagomir that is
substantially identical to miR-122 can be useful for downregulating
Hmgcr expression, and can be administered as a therapeutic
composition to a subject having or at risk for developing
hyperglycemia, stroke, or a bone fracture. A single stranded
antagomir that is substantially identical to miR-122 can be
administered as a therapeutic composition to a subject having or at
risk for developing a disorder characterized by the aberrant or
unwanted expression of any of these genes, or any other gene
downregulated by miR-122.
[0070] In one embodiment, an antagomir, such as a single-stranded
oligonucleotide agent, can have a nucleotide sequence that is
substantially identical to miR-16, miR-192, or miR-194.
Single-stranded oligonucleotide agents that are substantially
identical to at least a portion of an miRNA, such as those
described above, can be administered to a subject to treat the
disease or disorder associated with the downregulation of an
endogenous miRNA, or the aberrant or unwanted expression of an mRNA
target of the endogenous miRNA.
[0071] In one aspect, the invention features a method of reducing
the levels of an miRNA or pre-miRNA in a cell of a subject, e.g., a
human subject. The method includes the step of administering an
antagomir to the subject, where the antagomir is substantially
single-stranded and includes a sequence that is substantially
complementary to 12 to 23 contiguous nucleotides, and preferably 15
to 23 contiguous nucleotides, of a target sequence of an miRNA or
pre-miRNA nucleotide sequence. Preferably, the target sequence
differs by no more than 1, 2, or 3 nucleotides from a microRNA or
pre-microRNA sequence, such as a microRNA sequence shown in Table
1.
[0072] The antagomir may be administered into a recipient in a wide
variety of ways. Preferred modes of administration are parenteral,
intraperitoneal, intravenous, intradermal, epidural, intraspinal,
intrasternal, intra-articular, intra-synovial, intrathecal,
intra-arterial, intracardiac, intramuscular, intranasal,
subcutaneous, intraorbital, intracapsular, topical, transdermal
patch, via rectal, vaginal or urethral administration including via
suppository, percutaneous, nasal spray, surgical implant, internal
surgical paint, infusion pump, or via catheter.
[0073] In one embodiment, the methods featured in the invention are
useful for reducing the level of an endogenous miRNA (e.g.,
miR-122, miR-16, miR-192 or miR-194) or pre-miRNA in a cell, e.g,
in a cell of a subject, such as a human subject. Such methods
include contacting the cell with an antagomir, such as a
single-stranded oligonucleotide agent, described herein for a time
sufficient to allow uptake of the antagomir into the cell.
[0074] In another aspect, the invention features a method of making
an antagomir, such as a single-stranded oligonucleotide agent,
described herein. In one embodiment, the method includes
synthesizing an oligonucleotide agent, including incorporating a
nucleotide modification that stabilizes the antagomir against
nucleolytic degradation.
[0075] In another aspect, the invention features a pharmaceutical
composition including an antagomir, such as a single-stranded
oligonucleotide agent, described herein, and a pharmaceutically
acceptable carrier. In a preferred embodiment, the antagomir, such
as a single-stranded oligonucleotide agent, included in the
pharmaceutical composition hybridizes to miR-122, miR-16, miR-192,
or miR-194.
[0076] In another aspect the invention features a method of
inhibiting miRNA expression (e.g., miR-122, miR-16, miR-192, or
miR-194 expression) or pre-miRNA expression in a cell, e.g., a cell
of a subject. The method includes contacting the cell with an
effective amount of an antagomir, such as a single-stranded
oligonucleotide agent, described herein, which is substantially
complementary to the nucleotide sequence of the target miRNA or the
target pre-miRNA. Such methods can be performed on a mammalian
subject by administering to a subject one of the oligonucleotide
agents/pharmaceutical compositions described herein.
[0077] In another aspect the invention features a method of
increasing levels of an RNA or protein that are encoded by a gene
whose expression is down-regulated by an miRNA, e.g., an endogenous
miRNA, such as miR-122, miR-16, miR-192 or mir-194. The method
includes contacting the cell with an effective amount of an
antagomir, such as a single-stranded oligonucleotide agent,
described herein, which is substantially complementary to the
nucleotide sequence of the miRNA that binds to and effectively
inhibits translation of the RNA transcribed from the gene. For
example, the invention features a method of increasing aldolase A
protein levels in a cell. Similarly, the invention features a
method of increasing Ndrg3, Iqgap1, Hmgcr, and/or citrate synthase
protein levels in a cell. The methods include contacting the cell
with an effective amount of an antagomir described herein (e.g.,
antagomir-122, described in Table 2a-e, Table 4 and Table 7), which
is substantially complementary to the nucleotide sequence of
miR-122 (see Table 1).
[0078] Preferably, an antagomir, such as a single-stranded
oligonucleotide agent, (a term which is defined below) will include
a ligand that is selected to improve stability, distribution or
cellular uptake of the agent. Compositions featured in the
invention can include conjugated single-stranded oligonucleotide
agents as well as conjugated monomers that are the components of or
can be used to make the conjugated oligonucleotide agents. The
conjugated oligonucleotide agents can modify gene expression by
targeting and binding to a nucleic acid, such as an miRNA (e.g.,
miR-122, miR-16, miR-192, or miR-194) or pre-miRNA.
[0079] In a preferred embodiment, the ligand is a lipophilic
moiety, e.g., cholesterol, which enhances entry of the antagomir,
such as a single-stranded oligonucleotide agent, into a cell, such
as a hepatocyte, synoviocyte, myocyte, keratinocyte, leukocyte,
endothelial cell (e.g., a kidney cell), B-cell, T-cell, epithelial
cell, mesodermal cell, myeloid cell, neural cell, neoplastic cell,
mast cell, or fibroblast cell. In some embodiments, a myocyte is a
smooth muscle cell or a cardiac myocyte. A fibroblast cell can be a
dermal fibroblast, and a leukocyte can be a monocyte. In another
embodiment, the cell is from an adherent tumor cell line derived
from a tissue, such as bladder, lung, breast, cervix, colon,
pancreas, prostate, kidney, liver, skin, or nervous system (e.g.,
central nervous system).
[0080] In another aspect, the invention provides methods of
increasing expression of a target gene by providing an antagomir to
which a lipophilic moiety is conjugated, e.g., a lipophilic
conjugated antagomir described herein, to a cell. The antagomir
preferably hybridizes to an miRNA (e.g., miR-122, miR-16, miR-192,
or miR-194) or a pre-miRNA. In a preferred embodiment the
conjugated antagomir can be used to increase expression of a target
gene in an organism, e.g., a mammal, e.g., a human, or to increase
expression of a target gene in a cell line or in cells which are
outside an organism. An mRNA transcribed from the target gene
hybridizes to an endogenous miRNA, which consequently results in
downregulation of mRNA expression. An antagomir, such as a
single-stranded oligonucleotide agent, featured in the invention
hybridizes to the endogenous miRNA and consequently causes an
increase in mRNA expression. In the case of a whole organism, the
method can be used to increase expression of a gene and treat a
condition associated with a low level of expression of the gene.
For example, an antagomir, such as a single-stranded
oligonucleotide agent, that targets miR-122 (e.g., antagomir-122)
can be used to increase expression of an aldolase A gene to treat a
subject having, or at risk for developing, hemolytic anemia,
arthrogryposis complex congenita, pituitary ectopia,
rhabdomyolysis, hyperkalemia, or any other disorder associated with
aldolase A deficiency. Administration of an antagomir, such as a
single-stranded oligonucleotide agent, that targets miR-122 (e.g.,
antagomir-122) can be also be used to increase expression of an
Ndrg3, Iqgap1, Hmgcr, or citrate synthase gene to treat a subject
having, or at risk for developing, a disorder associated with a
decreased expression of any one of these genes.
[0081] In another aspect, the invention provides compositions and
methods for treating a disease, disorder or condition of the
central nervous system. One such disease, disorder or condition of
the central nervous system is associated with abnormal expression
of a target gene or otherwise an abnormal decreased expression of a
target gene when compared with the normal expression of the
otherwise identical gene. Such an abnormal decreased expression of
a target gene may be the result of a genetic mutation in the gene.
Regardless, the term "disease, disorder or condition of the central
nervous system" should also be construed to encompass other
pathologies in the central nervous system which are not the result
of a genetic defect per se in cells of the central nervous system,
but rather are the result of infiltration of the central nervous
system by cells which do not originate in the central nervous
system, for example, metastatic tumor formation in the central
nervous system. The term should also be construed to include stroke
or trauma to the central nervous system induced by direct injury to
the tissues of the central nervous system.
[0082] Diseases, disorders or conditions of the CNS also
encompasses pathologies including neurodegenerative diseases,
spinal cord injury, head trauma or surgery, viral infections that
result in tissue, organ, or gland disfunction, and the like. Such
neurodegenerative diseases include but are not limited to, AIDS
dementia complex; demyelinating diseases, such as multiple
sclerosis and acute transferase myelitis; extrapyramidal and
cerebellar disorders, such as lesions of the ecorticospinal system;
disorders of the basal ganglia or cerebellar disorders;
hyperkinetic movement disorders, such as Huntington's Chorea and
senile chorea; drug-induced movement disorders, such as those
induced by drugs that block CNS dopamine receptors; hypokinetic
movement disorders, such as Parkinson's disease; progressive
supra-nucleo palsy; structural lesions of the cerebellum;
spinocerebellar degenerations, such as spinal ataxia, Friedreich's
ataxia, cerebellar cortical degenerations, multiple systems
degenerations (Mencel, Dejerine Thomas, Shi-Drager, and
Machado-Joseph), systermioc disorders, such as Rufsum's disease,
abetalipoprotemia, ataxia, telangiectasia; and mitochondrial
multi-system disorder; demyelinating core disorders, such as
multiple sclerosis, acute transverse myelitis; and disorders of the
motor unit, such as neurogenic muscular atrophies (anterior horn
cell degeneration, such as amyotrophic lateral sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy
body disease; Senile Demetia of Lewy body type; Wernicke-Korsakoff
syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute
sclerosing panencephalitis hallerrorden-Spatz disease; and Dementia
pugilistica.
[0083] The invention includes compositions and methods for
decreasing miRNA levels in the CNS, preferably the brain of a
mammal. By way of a non-limiting example, miR-16 levels can
effectively be decreased by local injection of an antagomir
directed to miR-16 to a mouse brain. The decrease expression of
miR-16 levels in turn can increase expression of a target gene
where expression therefrom is inhibited by miR-16. Therefore, an
antagimor can effectively increase expression levels of a desired
target gene in the CNS of a mammal. In another aspect, the
antagimor can effectively increase expression levels of a desired
target gene in a cell of the CNS.
[0084] In one embodiment, the antagomir, such as a single-stranded
oligonucleotide agent, to which a lipophilic moiety is conjugated
is used to increase expression of a gene in a cell that is not part
of a whole organism, such as when the cell is part of a primary
cell line, secondary cell line, tumor cell line, or transformed or
immortalized cell line. Cells that are not part of a whole organism
can be used in an initial screen to determine if an antagomir, such
as a single-stranded oligonucleotide agent, is effective in
increasing target gene expression levels, or decreasing levels of a
target miRNA or pre-miRNA. A test in cells that are not part of a
whole organism can be followed by test of the antagomir in a whole
animal. In some embodiments, the antagomir that is conjugated to a
lipophilic moiety is administered to an organism, or contacted with
a cell that is not part of an organism, in the absence of (or in a
reduced amount of) other reagents that facilitate or enhance
delivery, e.g., a compound which enhances transmit through the cell
membrane. (A reduced amount can be an amount of such reagent which
is reduced in comparison to what would be needed to get an equal
amount of nonconjugated antagomir into the target cell). For
example, the antagomir that is conjugated to a lipophilic moiety is
administered to an organism, or contacted with a cell that is not
part of an organism, in the absence (or reduced amount) of (i) an
additional lipophilic moiety; (ii) a transfection agent (e.g., an
ion or other substance which substantially alters cell permeability
to an oligonucleotide agent); or (iii) a commercial transfecting
agent such as Lipofectamine.TM. (Invitrogen, Carlsbad, Calif.),
Lipofectamine 2000.TM., TransIT-TKO.TM. (Mirus, Madison, Wis.),
FuGENE 6 (Roche, Indianapolis, Ind.), polyethylenimine, X-tremeGENE
Q2 (Roche, Indianapolis, Ind.), DOTAP, DOSPER, Metafectene.TM.
(Biontex, Munich, Germany), and the like.
[0085] Cationic lipid particles have been used to encapsulate
oligonucleotide reagents. For e.g. Cationic lipid saturation
influences intracellular delivery of encapsulated nucleic acids.
Heyes, James; Palmer, Lorne; Bremner, Kaz; MacLachlan, Ian.,
Journal of Controlled Release (2005), 107(2), 276-287.
[0086] An analogous series of cationic lipids
(1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),
1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA),
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA) and
1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA))
possessing 0, 1, 2 or 3 double bonds per alkyl chain resp., was
synthesized to det. the correlation between lipid satn.,
fusogenicity and efficiency of intracellular nucleic acid delivery.
31P-NMR anal. suggests that as satn. increases, from 2 to 0 double
bonds, lamellar (L.alpha.) to reversed hexagonal (HII) phase
transition temp. increases, indicating decreasing fusogenicity.
This trend is largely reflected by the efficiency of gene silencing
observed in vitro when the lipids are formulated as Stable Nucleic
Acid Lipid Particles (SNALPs) encapsulating small inhibitory RNA
(siRNA). Uptake expts. suggest that despite their lower gene
silencing efficiency, the less fusogenic particles are more readily
internalized by cells. Microscopic visualization of fluorescently
labeled siRNA uptake was supported by quant. data acquired using
radiolabeled prepns. Since electrostatic binding is a precursor to
uptake, the pKa of each cationic lipid was detd. The results
support a transfection model in which endosomal release, mediated
by fusion with the endosomal membrane, results in cytoplasmic
translocation of the nucleic acid payload.
[0087] In a preferred embodiment, the antagomir is suitable for
delivery to a cell in vivo, e.g., to a cell in an organism. In
another embodiment, the antagomir is suitable for delivery to a
cell in vitro, e.g., to a cell in a cell line.
[0088] An antagomir to which a lipophilic moiety is attached can
target any miRNA (e.g., miR-122, miR-16, miR-192, or miR-194) or
pre-miRNA described herein and can be delivered to any cell type
described herein, e.g., a cell type in an organism, tissue, or cell
line. Delivery of the antagomir can be in vivo, e.g., to a cell in
an organism, or in vitro, e.g., to a cell in a cell line.
[0089] In another aspect, the invention provides compositions
including single-stranded oligonucleotide agents described herein,
and in particular, compositions including an antagomir to which a
lipophilic moiety is conjugated, e.g., a lipophilic conjugated
antagomir that hybridizes to miR-122, miR-16, miR-192, or miR-194.
In a preferred embodiment the composition is a pharmaceutically
acceptable composition.
[0090] In one embodiment the composition is suitable for delivery
to a cell in vivo, e.g., to a cell in an organism. In another
aspect, the antagomir is suitable for delivery to a cell in vitro,
e.g., to a cell in a cell line.
[0091] An "antagomir" or "oligonucleotide agent" of the present
invention referes to a single stranded, double stranded or
partially double stranded oligomer or polymer of ribonucleic acid
(RNA) or deoxyribonucleic acid (DNA) or both or modifications
thereof, which is antisense with respect to its target. This term
includes oligonucleotides composed of naturally-occurring
nucleobases, sugars and covalent internucleoside (backbone)
linkages and which contain at least one non-naturally-occurring
portions which function similarly. Such modified or substituted
oligonucleotides are preferred over native forms because of
desirable properties such as, for example, enhanced cellular
uptake, enhanced affinity for nucleic acid target and increased
stability in the presence of nucleases. In a preferred embodiment,
the antagomir does not include a sense strand, and in another
preferred embodiment, the antagomir does not self-hybridize to a
significant extent. An antagomir featured in the invention can have
secondary structure, but it is substantially single-stranded under
physiological conditions. An antagomir that is substantially
single-stranded is single-stranded to the extent that less than
about 50% (e.g., less than about 40%, 30%, 20%, 10%, or 5%) of the
antagomir is duplexed with itself. FIGS. 5-11 provides
representative structures of antagomirs.
[0092] "Substantially complementary" means that two sequences are
substantially complementary that a duplex can be formed between
them. The duplex may have one or more mismatches but the region of
duplex formation is sufficient to down-regulate expression of the
target nucleic acid. The region of substantial complementarity can
be perfectly paired. In other embodiments, there will be nucleotide
mismatches in the region of substantial complementarity. In a
preferred embodiment, the region of substantial complementarity
will have no more than 1, 2, 3, 4, or 5 mismatches.
[0093] The antagomirs featured in the invention include oligomers
or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid
(DNA) or both or modifications thereof. This term includes
oligonucleotides composed of naturally occurring nucleobases,
sugars, and covalent internucleoside (backbone) linkages as well as
oligonucleotides having non-naturally-occurring portions that
function similarly. Such modified or substituted oligonucleotides
are often preferred over native forms because of desirable
properties such as, for example, enhanced cellular uptake, enhanced
affinity for nucleic acid target, and/or increased stability in the
presence of nucleases. The oligonucleotide agents can be about 12
to about 30 nucleotides long, e.g., about 15 to about 25, or about
18 to about 25 nucleotides long (e.g., about 19, 20, 21, 22, 23, 24
nucleotides long).
[0094] The antagomirs featured in the invention can target RNA,
e.g., an endogenous pre-miRNA or miRNA of the subject or an
endogenous pre-miRNA or miRNA of a pathogen of the subject. For
example, the oligonucleotide agents can target an miRNA of the
subject, such as miR-122, miR-16, miR-192, or miR-194. Such
single-stranded oligonucleotide can be useful for the treatment of
diseases involving biological processes that are regulated by
miRNAs, including developmental timing, differentiation, apoptosis,
cell proliferation, organ development, and metabolism.
MicroRNA-Type Oligonucleotide Agents
[0095] The antagomir featured in the invention include
microRNA-type (miRNA-type) oligonucleotide agents, e.g., the
miRNA-type oligonucleotide agents listed in Table 2a-f. MicroRNAs
are small noncoding RNA molecules that are capable of causing
post-transcriptional silencing of specific genes in cells such as
by the inhibition of translation or through degradation of the
targeted mRNA. An miRNA can be completely complementary or can have
a region of noncomplementarity with a target nucleic acid,
consequently resulting in a "bulge" at the region of
non-complementarity. The region of noncomplementarity (the bulge)
can be flanked by regions of sufficient complementarity, preferably
complete complementarity to allow duplex formation. Preferably, the
regions of complementarity are at least 8, 9, or 10 nucleotides
long. An miRNA can inhibit gene expression by repressing
translation, such as when the microRNA is not completely
complementary to the target nucleic acid, or by causing target RNA
degradation, which is believed to occur only when the miRNA binds
its target with perfect complementarity. The invention also can
include double-stranded precursors of miRNAs that may or may not
form a bulge when bound to their targets.
[0096] An miRNA or pre-miRNA can be 18-100 nucleotides in length,
and more preferably from 18-80 nucleotides in length. Mature miRNAs
can have a length of 19-30 nucleotides, preferably 21-25
nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides.
MicroRNA precursors typically have a length of about 70-100
nucleotides and have a hairpin conformation. MicroRNAs are
generated in vivo from pre-miRNAs by the enzymes Dicer and Drosha,
which specifically process long pre-miRNA into functional miRNA.
The miRNA-type oligonucleotide agents, or pre-miRNA-type
oligonucleotide agents featured in the invention can be synthesized
in vivo by a cell-based system or in vitro by chemical synthesis.
MicroRNA-type oligonucleotide agents can be synthesized to include
a modification that imparts a desired characteristic. For example,
the modification can improve stability, hybridization
thermodynamics with a target nucleic acid, targeting to a
particular tissue or cell-type, or cell permeability, e.g., by an
endocytosis-dependent or -independent mechanism. Modifications can
also increase sequence specificity, and consequently decrease
off-site targeting. Methods of synthesis and chemical modifications
are described in greater detail below.
[0097] Given a sense strand sequence (e.g., the sequence of a sense
strand of a cDNA molecule), an miRNA-type antagomir can be designed
according to the rules of Watson and Crick base pairing. The
miRNA-type antagomir can be complementary to a portion of an RNA,
e.g., an miRNA, pre-miRNA, or mRNA. For example, the miRNA-type
antagomir can be complementary to an miRNA endogenous to a cell,
such as miR-122, miR-16, miR-192, or miR-194. An miRNA-type
antagomir can be, for example, from about 12 to 30 nucleotides in
length, preferably about 15 to 28 nucleotides in length (e.g., 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in
length).
[0098] Based on the present disclosure, the length of the antagimor
can contribute to the biochemical function of the antagimor with
respect to its ability to decrease the expression levels of a
desired miRNA. In some instances, antagomirs may require at least
19 nucleotides in length for optimal function.
[0099] In particular, an miRNA-type antagomir featured in the
invention can have a chemical modification on a nucleotide in an
internal (i.e., non-terminal) region having noncomplementarity with
the target nucleic acid. For example, a modified nucleotide can be
incorporated into the region of an miRNA that forms a bulge. The
modification can include a ligand attached to the miRNA, e.g., by a
linker. The modification can, for example, improve pharmacokinetics
or stability of a therapeutic miRNA-type oligonucleotide agent, or
improve hybridization properties (e.g., hybridization
thermodynamics) of the miRNA-type antagomir to a target nucleic
acid. In some embodiments, it is preferred that the orientation of
a modification or ligand incorporated into or tethered to the bulge
region of an miRNA-type antagomir is oriented to occupy the space
in the bulge region. For example, the modification can include a
modified base or sugar on the nucleic acid strand or a ligand that
functions as an intercalator. These are preferably located in the
bulge. The intercalator can be an aromatic, e.g., a polycyclic
aromatic or heterocyclic aromatic compound. A polycyclic
intercalator can have stacking capabilities, and can include
systems with 2, 3, or 4 fused rings. The universal bases described
below can be incorporated into the miRNA-type oligonucleotide
agents. In some embodiments, it is preferred that the orientation
of a modification or ligand incorporated into or tethered to the
bulge region of an miRNA-type antagomir is oriented to occupy the
space in the bulge region. This orientation facilitates the
improved hybridization properties or an otherwise desired
characteristic of the miRNA-type oligonucleotide agent.
[0100] In one embodiment, an miRNA-type antagomir or a pre-miRNA
can include an aminoglycoside ligand, which can cause the
miRNA-type antagomir to have improved hybridization properties or
improved sequence specificity. Exemplary aminoglycosides include
glycosylated polylysine; galactosylated polylysine; neomycin B;
tobramycin; kanamycin A; and acridine conjugates of
aminoglycosides, such as Neo-N-acridine, Neo-S-acridine,
Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an
acridine analog can increase sequence specificity. For example,
neomycin B has a high affinity for RNA as compared to DNA, but low
sequence-specificity. In some embodiments the guanidine analog (the
guanidinoglycoside) of an aminoglycoside ligand is tethered to an
oligonucleotide agent. In a guanidinoglycoside, the amine group on
the amino acid is exchanged for a guanidine group. Attachment of a
guanidine analog can enhance cell permeability of an
oligonucleotide agent.
[0101] In one embodiment, the ligand can include a cleaving group
that contributes to target gene inhibition by cleavage of the
target nucleic acid. Preferably, the cleaving group is tethered to
the miRNA-type antagomir in a manner such that it is positioned in
the bulge region, where it can access and cleave the target RNA.
The cleaving group can be, for example, a bleomycin (e.g.,
bleomycin-A.sub.5, bleomycin-A.sub.2, or bleomycin-B.sub.2),
pyrene, phenanthroline (e.g., O-phenanthroline), a polyamine, a
tripeptide (e.g., lys-tyr-lys tripeptide), or metal ion chelating
group. The metal ion chelating group can include, e.g., an Lu(III)
or EU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthroline
derivative, a Cu(II) terpyridine, or acridine, which can promote
the selective cleavage of target RNA at the site of the bulge by
free metal ions, such as Lu(III). In some embodiments, a peptide
ligand can be tethered to an miRNA or a pre-miRNA to promote
cleavage of the target RNA, e.g., at the bulge region. For example,
1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be
conjugated to a peptide (e.g., by an amino acid derivative) to
promote target RNA cleavage. The methods and compositions featured
in the invention include miRNA-type oligonucleotide agents that
inhibit target gene expression by a cleavage or non-cleavage
dependent mechanism.
[0102] An miRNA-type antagomir or pre-miRNA-type antagomir can be
designed and synthesized to include a region of noncomplementarity
(e.g., a region that is 3, 4, 5, or 6 nucleotides long) flanked by
regions of sufficient complementarity to form a duplex (e.g.,
regions that are 7, 8, 9, 10, or 11 nucleotides long) with a target
RNA, e.g., an miRNA, such as miR-122, miR-16, miR-192, or
miR-194.
[0103] For increased nuclease resistance and/or binding affinity to
the target, the single-stranded oligonucleotide agents featured in
the invention can include 2'-O-methyl, 2'-fluorine,
2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and/or
phosphorothioate linkages. Inclusion of locked nucleic acids (LNA),
ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene-bridged nucleic
acids, and certain nucleobase modifications such as 2-amino-A,
2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase
binding affinity to the target. The inclusion of pyranose sugars in
the oligonucleotide backbone can also decrease endonucleolytic
cleavage. An antagomir can be further modified by including a 3'
cationic group, or by inverting the nucleoside at the 3'-terminus
with a 3'-3' linkage. In another alternative, the 3'-terminus can
be blocked with an aminoalkyl group, e.g., a 3' C5-aminoalkyl dT.
Other 3' conjugates can inhibit 3'-5' exonucleolytic cleavage.
While not being bound by theory, a 3' conjugate, such as naproxen
or ibuprofen, may inhibit exonucleolytic cleavage by sterically
blocking the exonuclease from binding to the 3' end of the
oligonucleotide. Even small alkyl chains, aryl groups, or
heterocyclic conjugates or modified sugars (D-ribose, deoxyribose,
glucose etc.) can block 3'-5'-exonucleases.
[0104] With respect to phosphorothioate linkages that serve to
increase protection against RNase activity, the antagomir can
include a phosphorothioate at least the first, second, or third
internucleotide linkage at the 5' or 3' end of the nucleotide
sequence. In one embodiment, the antagomir includes a 2'-modified
nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl,
2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O--N-methylacetamido (2'-O--NMA). In a preferred embodiment, the
antagomir includes at least one 2'-O-methyl-modified nucleotide,
and in some embodiments, all of the nucleotides of the antagomir
include a 2'-O-methyl modification. In yet another the preferred
embodiment, the antagomir includes six phosphorothioate backbone
modifications; two phosphorothioates are located at the 5'-end and
four at the 3'-end.
[0105] The 5'-terminus can be blocked with an aminoalkyl group,
e.g., a 5'-O-alkylamino substitutent. Other 5' conjugates can
inhibit 5'-3' exonucleolytic cleavage. While not being bound by
theory, a 5' conjugate, such as naproxen or ibuprofen, may inhibit
exonucleolytic cleavage by sterically blocking the exonuclease from
binding to the 5' end of the oligonucleotide. Even small alkyl
chains, aryl groups, or heterocyclic conjugates or modified sugars
(D-ribose, deoxyribose, glucose etc.) can block
3'-5'-exonucleases.
[0106] In one embodiment, an antagomir, such as a single-stranded
oligonucleotide agent, includes a modification that improves
targeting, e.g. a targeting modification described herein. Examples
of modifications that target single-stranded oligonucleotide agents
to particular cell types include carbohydrate sugars such as
galactose, N-acetylgalactosamine, mannose; vitamins such as
folates; other ligands such as RGDs and RGD mimics; and small
molecules including naproxen, ibuprofen or other known
protein-binding molecules.
[0107] An antagomir, such as a single-stranded oligonucleotide
agent, featured in the invention can be constructed using chemical
synthesis and/or enzymatic ligation reactions using procedures
known in the art. For example, an antagomir can be chemically
synthesized using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological stability
of the molecules or to increase the physical stability of the
duplex formed between the antagomir and target nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides
can be used. Other appropriate nucleic acid modifications are
described herein. Alternatively, the antagomir can be produced
biologically using an expression vector into which a nucleic acid
has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense
orientation to a target nucleic acid of interest (e.g., an miRNA or
pre-miRNA)).
[0108] Chemical Definitions
[0109] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine.
[0110] The term "alkyl" refers to a hydrocarbon chain that may be a
straight chain or branched chain, containing the indicated number
of carbon atoms. For example, C.sub.1-C.sub.12 alkyl indicates that
the group may have from 1 to 12 (inclusive) carbon atoms in it. The
term "haloalkyl" refers to an alkyl in which one or more hydrogen
atoms are replaced by halo, and includes alkyl moieties in which
all hydrogens have been replaced by halo (e.g., perfluoroalkyl).
Alkyl and haloalkyl groups may be optionally inserted with O, N, or
S. The terms "aralkyl" refers to an alkyl moiety in which an alkyl
hydrogen atom is replaced by an aryl group. Aralkyl includes groups
in which more than one hydrogen atom has been replaced by an aryl
group. Examples of "aralkyl" include benzyl, 9-fluorenyl,
benzhydryl, and trityl groups.
[0111] The term "alkenyl" refers to a straight or branched
hydrocarbon chain containing 2-8 carbon atoms and characterized in
having one or more double bonds. Examples of a typical alkenyl
include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl
and 3-octenyl groups. The term "alkynyl" refers to a straight or
branched hydrocarbon chain containing 2-8 carbon atoms and
characterized in having one or more triple bonds. Some examples of
a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and
propargyl. The sp.sup.2 and sp.sup.3 carbons may optionally serve
as the point of attachment of the alkenyl and alkynyl groups,
respectively.
[0112] The terms "alkylamino" and "dialkylamino" refer to
--NH(alkyl) and --NH(alkyl).sub.2 radicals respectively. The term
"aralkylamino" refers to a --NH(aralkyl) radical. The term "alkoxy"
refers to an --O-alkyl radical, and the terms "cycloalkoxy" and
"aralkoxy" refer to an --O-cycloalkyl and O-aralkyl radicals
respectively. The term "siloxy" refers to a R.sub.3SiO-radical. The
term "mercapto" refers to an SH radical. The term "thioalkoxy"
refers to an --S-alkyl radical.
[0113] The term "alkylene" refers to a divalent alkyl (i.e.,
--R--), e.g., --CH.sub.2--, --CH.sub.2CH.sub.2--, and
--CH.sub.2CH.sub.2CH.sub.2--. The term "alkylenedioxo" refers to a
divalent species of the structure --O--R--O--, in which R
represents an alkylene.
[0114] The term "aryl" refers to an aromatic monocyclic, bicyclic,
or tricyclic hydrocarbon ring system, wherein any ring atom can be
substituted. Examples of aryl moieties include, but are not limited
to, phenyl, naphthyl, anthracenyl, and pyrenyl.
[0115] The term "cycloalkyl" as employed herein includes saturated
cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups
having 3 to 12 carbons, wherein any ring atom can be substituted.
The cycloalkyl groups herein described may also contain fused
rings. Fused rings are rings that share a common carbon-carbon bond
or a common carbon atom (e.g., spiro-fused rings). Examples of
cycloalkyl moieties include, but are not limited to, cyclohexyl,
adamantyl, and norbornyl, and decalin.
[0116] The term "heterocyclyl" refers to a nonaromatic 3-10
membered monocyclic, 8-12 membered bicyclic, or 11-14 membered
tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6
heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said
heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3,
1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or
tricyclic, respectively), wherein any ring atom can be substituted.
The heterocyclyl groups herein described may also contain fused
rings. Fused rings are rings that share a common carbon-carbon bond
or a common carbon atom (e.g., spiro-fused rings). Examples of
heterocyclyl include, but are not limited to tetrahydrofuranyl,
tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and
pyrrolidinyl.
[0117] The term "cycloalkenyl" as employed herein includes
partially unsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or
polycyclic hydrocarbon groups having 5 to 12 carbons, preferably 5
to 8 carbons, wherein any ring atom can be substituted. The
cycloalkenyl groups herein described may also contain fused rings.
Fused rings are rings that share a common carbon-carbon bond or a
common carbon atom (e.g., spiro-fused rings). Examples of
cycloalkenyl moieties include, but are not limited to cyclohexenyl,
cyclohexadienyl, or norbornenyl.
[0118] The term "heterocycloalkenyl" refers to a partially
saturated, nonaromatic 5-10 membered monocyclic, 8-12 membered
bicyclic, or 11-14 membered tricyclic ring system having 1-3
heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9
heteroatoms if tricyclic, said heteroatoms selected from O, N, or S
(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S
if monocyclic, bicyclic, or tricyclic, respectively), wherein any
ring atom can be substituted. The heterocycloalkenyl groups herein
described may also contain fused rings. Fused rings are rings that
share a common carbon-carbon bond or a common carbon atom (e.g.,
spiro-fused rings). Examples of heterocycloalkenyl include but are
not limited to tetrahydropyridyl and dihydropyran.
[0119] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein any ring atom can be substituted. The
heteroaryl groups herein described may also contain fused rings
that share a common carbon-carbon bond.
[0120] The term "oxo" refers to an oxygen atom, which forms a
carbonyl when attached to carbon, an N-oxide when attached to
nitrogen, and a sulfoxide or sulfone when attached to sulfur.
[0121] The term "acyl" refers to an alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or
heteroarylcarbonyl substitutent, any of which may be further
substituted by substitutents.
[0122] The term "substitutents" refers to a group "substituted" on
an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl,
heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any
atom of that group. Suitable substitutents include, without
limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano,
nitro, amino, SO.sub.3H, sulfate, phosphate, perfluoroalkyl,
perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo,
thioxo, imino (alkyl, aryl, aralkyl), S(O).sub.nalkyl (where n is
0-2), S(O).sub.naryl (where n is 0-2), S(O).sub.nheteroaryl (where
n is 0-2), S(O).sub.nheterocyclyl (where n is 0-2), amine (mono-,
di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations
thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-,
alkyl, aralkyl, heteroaralkyl, and combinations thereof),
sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and
combinations thereof), unsubstituted aryl, unsubstituted
heteroaryl, unsubstituted heterocyclyl, and unsubstituted
cycloalkyl. In one aspect, the substitutents on a group are
independently any one single, or any subset of the aforementioned
substitutents.
[0123] The terms "adeninyl, cytosinyl, guaninyl, thyminyl, and
uracilyl" and the like refer to radicals of adenine, cytosine,
guanine, thymine, and uracil.
[0124] A "protected" moiety refers to a reactive functional group,
e.g., a hydroxyl group or an amino group, or a class of molecules,
e.g., sugars, having one or more functional groups, in which the
reactivity of the functional group is temporarily blocked by the
presence of an attached protecting group. Protecting groups useful
for the monomers and methods described herein can be found, e.g.,
in Greene, T. W., Protective Groups in Organic Synthesis (John
Wiley and Sons: New York), 1981, which is hereby incorporated by
reference.
[0125] Antagomir Structure
[0126] An antagomir, such as a single-stranded oligonucleotide
agent, featured in the invention includes a region sufficient
complementarity to the target nucleic acid (e.g., target miRNA,
pre-miRNA or mRNA), and is of sufficient length in terms of
nucleotides, such that the antagomir forms a duplex with the target
nucleic acid. The antagomir can modulate the function of the
targeted molecule. For example, when the targeted molecule is an
miRNA, such as miR-122, miR-16, miR-192, or miR-194, the antagomir
can inhibit the gene silencing activity of the target miRNA, which
action will up-regulate expression of the mRNA targeted by the
target miRNA. When the target is an mRNA, the antagomir can replace
or supplement the gene silencing activity of an endogenous
miRNA.
[0127] For ease of exposition the term nucleotide or ribonucleotide
is sometimes used herein in reference to one or more monomeric
subunits of an oligonucleotide agent. It will be understood herein
that the usage of the term "ribonucleotide" or "nucleotide" herein
can, in the case of a modified RNA or nucleotide surrogate, also
refer to a modified nucleotide, or surrogate replacement moiety at
one or more positions.
[0128] An antagomir featured in the invention is, or includes, a
region that is at least partially, and in some embodiments fully,
complementary to the target RNA. It is not necessary that there be
perfect complementarity between the antagomir and the target, but
the correspondence must be sufficient to enable the oligonucleotide
agent, or a cleavage product thereof, to modulate (e.g., inhibit)
target gene expression.
[0129] An antagomir will preferably have one or more of the
following properties: [0130] (1) it will be of the Formula 1, 2, 3,
or 4 described below; [0131] (2) it will have a 5' modification
that includes one or more phosphate groups or one or more analogs
of a phosphate group; [0132] (3) it will, despite modifications,
even to a very large number of bases specifically base pair and
form a duplex structure with a homologous target RNA of sufficient
thermodynamic stability to allow modulation of the activity of the
targeted RNA; [0133] (4) it will, despite modifications, even to a
very large number, or all of the nucleosides, still have "RNA-like"
properties, i.e., it will possess the overall structural, chemical
and physical properties of an RNA molecule, even though not
exclusively, or even partly, of ribonucleotide-based content. For
example, all of the nucleotide sugars can contain e.g., 2'OMe, 2'
fluoro in place of 2' hydroxyl. This deoxyribonucleotide-containing
agent can still be expected to exhibit RNA-like properties. While
not wishing to be bound by theory, an electronegative fluorine
prefers an axial orientation when attached to the C2' position of
ribose. This spatial preference of fluorine can, in turn, force the
sugars to adopt a C.sub.3'-endo pucker. This is the same puckering
mode as observed in RNA molecules and gives rise to the
RNA-characteristic A-family-type helix. Further, since fluorine is
a good hydrogen bond acceptor, it can participate in the same
hydrogen bonding interactions with water molecules that are known
to stabilize RNA structures. (Generally, it is preferred that a
modified moiety at the 2' sugar position will be able to enter into
hydrogen-bonding which is more characteristic of the 2'-OH moiety
of a ribonucleotide than the 2'-H moiety of a deoxyribonucleotide.
A preferred antagomir will: exhibit a C.sub.3'-endo pucker in all,
or at least 50, 75, 80, 85, 90, or 95% of its sugars; exhibit a
C.sub.3'-endo pucker in a sufficient amount of its sugars that it
can give rise to the RNA-characteristic A-family-type helix; will
have no more than 20, 10, 5, 4, 3, 2, or 1 sugar which is not a
C.sub.3'-endo pucker structure.
[0134] Preferred 2'-modifications with C3'-endo sugar pucker
include:
[0135] 2'-OH, 2'-O-Me, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-F,
2'-O--CH.sub.2--CO--NHMe,
2'-O--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--N(Me)2, and
LNA.
[0136] Preferred 2'-modifications with a C2'-endo sugar pucker
include:
[0137] 2'-H, 2'-Me, 2'-S-Me, 2'-Ethynyl, 2'-ara-F.
[0138] Sugar modifications can also include L-sugars and
2'-5'-linked sugars.
[0139] As used herein, "specifically hybridizable" and
"complementary" are terms that are used to indicate a sufficient
degree of complementarity such that stable and specific binding
occurs between an antagomir of the invention and a target RNA
molecule, e.g., an miRNA or a pre-miRNA. Specific binding requires
a sufficient lack of complementarity to non-target sequences under
conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or
therapeutic treatment, or in the case of in vitro assays, under
conditions in which the assays are performed. It has been shown
that a single mismatch between targeted and non-targeted sequences
are sufficient to provide discrimination for siRNA targeting of an
mRNA (Brummelkamp et al., Cancer Cell, 2002, 2:243).
[0140] In one embodiment, an antagomir is "sufficiently
complementary" to a target RNA, such that the antagomir inhibits
production of protein encoded by the target mRNA. The target RNA
can be, e.g., a pre-mRNA, mRNA, or miRNA endogenous to the subject.
In another embodiment, the antagomir is "exactly complementary"
(excluding the SRMS containing subunit(s)) to a target RNA, e.g.,
the target RNA and the antagomir can anneal to form a hybrid made
exclusively of Watson-Crick base pairs in the region of exact
complementarity. A "sufficiently complementary" target RNA can
include a region (e.g., of at least 7 nucleotides) that is exactly
complementary to a target RNA. Moreover, in some embodiments, the
antagomir specifically discriminates a single-nucleotide
difference. In this case, the antagomir only down-regulates gene
expression if exact complementarity is found in the region of the
single-nucleotide difference.
[0141] Oligonucleotide agents discussed herein include otherwise
unmodified RNA and DNA as well as RNA and DNA that have been
modified, e.g., to improve efficacy, and polymers of nucleoside
surrogates. Unmodified RNA refers to a molecule in which the
components of the nucleic acid, namely sugars, bases, and phosphate
moieties, are the same or essentially the same as that which occur
in nature, preferably as occur naturally in the human body. The art
has referred to rare or unusual, but naturally occurring, RNAs as
modified RNAs, see, e.g., Limbach et al. (Nucleic Acids Res., 1994,
22:2183-2196). Such rare or unusual RNAs, often termed modified
RNAs, are typically the result of a post-transcriptional
modification and are within the term unmodified RNA as used herein.
Modified RNA, as used herein, refers to a molecule in which one or
more of the components of the nucleic acid, namely sugars, bases,
and phosphate moieties, are different from that which occur in
nature, preferably different from that which occurs in the human
body. While they are referred to as "modified RNAs" they will of
course, because of the modification, include molecules that are
not, strictly speaking, RNAs. Nucleoside surrogates are molecules
in which the ribophosphate backbone is replaced with a
non-ribophosphate construct that allows the bases to be presented
in the correct spatial relationship such that hybridization is
substantially similar to what is seen with a ribophosphate
backbone, e.g., non-charged mimics of the ribophosphate backbone.
Examples of all of the above are discussed herein.
[0142] As nucleic acids are polymers of subunits or monomers, many
of the modifications described below occur at a position which is
repeated within a nucleic acid, e.g., a modification of a base, or
a phosphate moiety, or a non-linking O of a phosphate moiety. In
some cases the modification will occur at all of the subject
positions in the nucleic acid but in many, and in fact in most
cases it will not. By way of example, a modification may only occur
at a 3' or 5' terminal position, in a terminal region, e.g., at a
position on a terminal nucleotide, or in the last 2, 3, 4, 5, or 10
nucleotides of a strand. The ligand can be attached at the 3' end,
the 5' end, or at an internal position, or at a combination of
these positions. For example, the ligand can be at the 3' end and
the 5' end; at the 3' end and at one or more internal positions; at
the 5' end and at one or more internal positions; or at the 3' end,
the 5' end, and at one or more internal positions. For example, a
phosphorothioate modification at a non-linking O position may only
occur at one or both termini, or may only occur in a terminal
region, e.g., at a position on a terminal nucleotide or in the last
2, 3, 4, 5, or 10 nucleotides of the oligonucleotide. The 5' end
can be phosphorylated.
[0143] Modifications and nucleotide surrogates are discussed below.
##STR1##
[0144] The scaffold presented above in Formula 1 represents a
portion of a ribonucleic acid. The basic components are the ribose
sugar, the base, the terminal phosphates, and phosphate
internucleotide linkers. Where the bases are naturally occurring
bases, e.g., adenine, uracil, guanine or cytosine, the sugars are
the unmodified 2' hydroxyl ribose sugar (as depicted) and W, X, Y,
and Z are all O. Formula 1 represents a naturally occurring
unmodified oligoribonucleotide.
[0145] Unmodified oligoribonucleotides may be less than optimal in
some applications, e.g., unmodified oligoribonucleotides can be
prone to degradation by e.g., cellular nucleases. Nucleases can
hydrolyze nucleic acid phosphodiester bonds. However, chemical
modifications to one or more of the above RNA components can confer
improved properties, and, for example, can render
oligoribonucleotides more stable to nucleases. Unmodified
oligoribonucleotides may also be less than optimal in terms of
offering tethering points for attaching ligands or other moieties
to an oligonucleotide agent.
[0146] Modified nucleic acids and nucleotide surrogates can include
one or more of:
[0147] (i) alteration, e.g., replacement, of one or both of the
non-linking (X and Y) phosphate oxygens and/or of one or more of
the linking (W and Z) phosphate oxygens (When the phosphate is in
the terminal position, one of the positions W or Z will not link
the phosphate to an additional element in a naturally occurring
ribonucleic acid. However, for simplicity of terminology, except
where otherwise noted, the W position at the 5' end of a nucleic
acid and the terminal Z position at the 3' end of a nucleic acid,
are within the term "linking phosphate oxygens" as used
herein.);
[0148] (ii) alteration, e.g., replacement, of a constituent of the
ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar, or
wholesale replacement of the ribose sugar with a structure other
than ribose, e.g., as described herein;
[0149] (iii) wholesale replacement of the phosphate moiety (bracket
I) with "dephospho" linkers;
[0150] (iv) modification or replacement of a naturally occurring
base;
[0151] (v) replacement or modification of the ribose-phosphate
backbone (bracket II);
[0152] (vi) modification of the 3' end or 5' end of the RNA, e.g.,
removal, modification or replacement of a terminal phosphate group
or conjugation of a moiety, such as a fluorescently labeled moiety,
to either the 3' or 5' end of RNA.
[0153] The terms replacement, modification, alteration, and the
like, as used in this context, do not imply any process limitation,
e.g., modification does not mean that one must start with a
reference or naturally occurring ribonucleic acid and modify it to
produce a modified ribonucleic acid but rather modified simply
indicates a difference from a naturally occurring molecule.
[0154] It is understood that the actual electronic structure of
some chemical entities cannot be adequately represented by only one
canonical form (i.e. Lewis structure). While not wishing to be
bound by theory, the actual structure can instead be some hybrid or
weighted average of two or more canonical forms, known collectively
as resonance forms or structures. Resonance structures are not
discrete chemical entities and exist only on paper. They differ
from one another only in the placement or "localization" of the
bonding and nonbonding electrons for a particular chemical entity.
It can be possible for one resonance structure to contribute to a
greater extent to the hybrid than the others. Thus, the written and
graphical descriptions of the embodiments of the present invention
are made in terms of what the art recognizes as the predominant
resonance form for a particular species. For example, any
phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)
would be represented by X.dbd.O and Y.dbd.N in the above
figure.
[0155] Specific modifications are discussed in more detail
below.
[0156] The Phosphate Group
[0157] The phosphate group is a negatively charged species. The
charge is distributed equally over the two non-linking oxygen atoms
(i.e., X and Y in Formula 1 above). However, the phosphate group
can be modified by replacing one of the oxygens with a different
substitutent. One result of this modification to RNA phosphate
backbones can be increased resistance of the oligoribonucleotide to
nucleolytic breakdown. Thus while not wishing to be bound by
theory, it can be desirable in some embodiments to introduce
alterations which result in either an uncharged linker or a charged
linker with unsymmetrical charge distribution.
[0158] Examples of modified phosphate groups include
phosphorothioate, phosphoroselenates, borano phosphates, borano
phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl
or aryl phosphonates and phosphotriesters. Phosphorodithioates have
both non-linking oxygens replaced by sulfur. Unlike the situation
where only one of X or Y is altered, the phosphorus center in the
phosphorodithioates is achiral which precludes the formation of
oligoribonucleotides diastereomers. Diastereomer formation can
result in a preparation in which the individual diastereomers
exhibit varying resistance to nucleases. Further, the hybridization
affinity of RNA containing chiral phosphate groups can be lower
relative to the corresponding unmodified RNA species. Thus, while
not wishing to be bound by theory, modifications to both X and Y
which eliminate the chiral center, e.g., phosphorodithioate
formation, may be desirable in that they cannot produce
diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N,
or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C,
H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with
sulfur is preferred.
[0159] The phosphate linker can also be modified by replacement of
a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged
phosphoroamidates), sulfur (bridged phosphorothioates) and carbon
(bridged methylenephosphonates). The replacement can occur at a
terminal oxygen (position W (3') or position Z (5')). Replacement
of W with carbon or Z with nitrogen is preferred.
[0160] Candidate agents can be evaluated for suitability as
described below.
[0161] The Sugar Group
[0162] A modified RNA can include modification of all or some of
the sugar groups of the ribonucleic acid. For example, the 2'
hydroxyl group (OH) can be modified or replaced with a number of
different "oxy" or "deoxy" substitutents. While not being bound by
theory, enhanced stability is expected since the hydroxyl can no
longer be deprotonated to form a 2' alkoxide ion. The 2' alkoxide
can catalyze degradation by intramolecular nucleophilic attack on
the linker phosphorus atom. Again, while not wishing to be bound by
theory, it can be desirable to some embodiments to introduce
alterations in which alkoxide formation at the 2' position is not
possible.
[0163] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge or ethylene bridge (e.g., 2'-4'-ethylene bridged
nucleic acid (ENA)), to the 4' carbon of the same ribose sugar;
amino, O-AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino,
heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or
diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy,
O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, or diheteroaryl amino, ethylene diamine, polyamino). It is
noteworthy that oligonucleotides containing only the methoxyethyl
group (MOE), (OCH.sub.2CH.sub.2OCH.sub.3, a PEG derivative),
exhibit nuclease stabilities comparable to those modified with the
robust phosphorothioate modification.
[0164] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars); halo (e.g., fluoro); amino (e.g. NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino), --NHC(O)R(R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl
and alkynyl, which may be optionally substituted with e.g., an
amino functionality. Preferred substitutents are 2'-methoxyethyl,
2'-OCH3, 2'-O-allyl, 2'-C-- allyl, and 2'-fluoro.
[0165] The sugar group can also contain one or more carbons that
possess the opposite stereochemical configuration than that of the
corresponding carbon in ribose. Thus, a modified RNA can include
nucleotides containing e.g., arabinose, as the sugar.
[0166] Modified RNAs can also include "abasic" sugars, which lack a
nucleobase at C-1'. These abasic sugars can also be further contain
modifications at one or more of the constituent sugar atoms.
[0167] To maximize nuclease resistance, the 2' modifications can be
used in combination with one or more phosphate linker modifications
(e.g., phosphorothioate). The so-called "chimeric" oligonucleotides
are those that contain two or more different modifications.
[0168] The modification can also entail the wholesale replacement
of a ribose structure with another entity (an SRMS) at one or more
sites in the oligonucleotide agent.
[0169] Candidate modifications can be evaluated as described
below.
[0170] Replacement of the Phosphate Group
[0171] The phosphate group can be replaced by non-phosphorus
containing connectors (cf. Bracket I in Formula 1 above). While not
wishing to be bound by theory, it is believed that since the
charged phosphodiester group is the reaction center in nucleolytic
degradation, its replacement with neutral structural mimics should
impart enhanced nuclease stability. Again, while not wishing to be
bound by theory, it can be desirable, in some embodiment, to
introduce alterations in which the charged phosphate group is
replaced by a neutral moiety.
[0172] Examples of moieties which can replace the phosphate group
include siloxane, carbonate, carboxymethyl, carbamate, amide,
thioether, ethylene oxide linker, sulfonate, sulfonamide,
thioformacetal, formacetal, oxime, methyleneimino,
methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo
and methyleneoxymethylimino. Preferred replacements include the
methylenecarbonylamino and methylenemethylimino groups.
[0173] Candidate modifications can be evaluated as described
below.
[0174] Replacement of Ribophosphate Backbone
[0175] Oligonucleotide-mimicking scaffolds can also be constructed
wherein the phosphate linker and ribose sugar are replaced by
nuclease resistant nucleoside or nucleotide surrogates (see Bracket
II of Formula 1 above). While not wishing to be bound by theory, it
is believed that the absence of a repetitively charged backbone
diminishes binding to proteins that recognize polyanions (e.g.
nucleases). Again, while not wishing to be bound by theory, it can
be desirable in some embodiment, to introduce alterations in which
the bases are tethered by a neutral surrogate backbone.
[0176] Examples include the mophilino, cyclobutyl, pyrrolidine and
peptide nucleic acid (PNA) nucleoside surrogates. A preferred
surrogate is a PNA surrogate.
[0177] Candidate modifications can be evaluated as described
below.
[0178] Terminal Modifications
[0179] The 3' and 5' ends of an oligonucleotide can be modified.
Such modifications can be at the 3' end, 5' end or both ends of the
molecule. They can include modification or replacement of an entire
terminal phosphate or of one or more of the atoms of the phosphate
group. E.g., the 3' and 5' ends of an oligonucleotide can be
conjugated to other functional molecular entities such as labeling
moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3
or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon,
boron or ester). The functional molecular entities can be attached
to the sugar through a phosphate group and/or a spacer. The
terminal atom of the spacer can connect to or replace the linking
atom of the phosphate group or the C-3' or C-5' O, N, S or C group
of the sugar. Alternatively, the spacer can connect to or replace
the terminal atom of a nucleotide surrogate (e.g., PNAs). These
spacers or linkers can include e.g., --(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nN--, --(CH.sub.2).sub.nO--,
--(CH.sub.2).sub.nS--, O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OH
(e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine,
oxyimine, thioether, disulfide, thiourea, sulfonamide, or
morpholino, or biotin and fluorescein reagents. While not wishing
to be bound by theory, it is believed that conjugation of certain
moieties can improve transport, hybridization, and specificity
properties. Again, while not wishing to be bound by theory, it may
be desirable to introduce terminal alterations that improve
nuclease resistance. Other examples of terminal modifications
include dyes, intercalating agents (e.g. acridines), cross-linkers
(e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin,
Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine,
dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic
carriers (e.g., cholesterol, cholic acid, adamantane acetic acid,
1-pyrene butyric acid, dihydrotestosterone,
1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl
group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,
O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and
peptide conjugates (e.g., antennapedia peptide, Tat peptide),
alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K),
MPEG, [MPEG].sub.2, polyamino, alkyl, substituted alkyl,
radiolabeled markers, enzymes, haptens (e.g. biotin),
transport/absorption facilitators (e.g., aspirin, vitamin E, folic
acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,
histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+
complexes of tetraazamacrocycles).
[0180] Terminal modifications can be added for a number of reasons,
including as discussed elsewhere herein to modulate activity or to
modulate resistance to degradation. Preferred modifications include
the addition of a methylphosphonate at the 3'-most terminal
linkage; a 3' C5-aminoalkyl-dT; 3' cationic group; or another 3'
conjugate to inhibit 3'-5' exonucleolytic degradation.
[0181] Terminal modifications useful for modulating activity
include modification of the 5' end with phosphate or phosphate
analogs. E.g., in preferred embodiments oligonucleotide agents are
5' phosphorylated or include a phosphoryl analog at the 5'
terminus. 5'-phosphate modifications include those which are
compatible with RISC mediated gene silencing. Suitable
modifications include: 5'-monophosphate ((HO)2(O)P--O-5');
5'-diphosphate ((HO)2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate
((HO)2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap structure
(N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5');
5'-monothiophosphate (phosphorothioate; (HO)2(S)P--O-5');
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO)2(O)P--S-5'); any additional combination
of oxygen/sulfur replaced monophosphate, diphosphate and
triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO)2(O)P--NH-5', (HO)(NH2)(O)P--O-5'), 5'-alkylphosphonates
(R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.
RP(OH)(O)--O-5'-, (OH).sub.2(O)P-5'-CH.sub.2--),
5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2--),
ethoxymethyl, etc., e.g. RP(OH)(O)--O-5'-).
[0182] Terminal modifications can also be useful for monitoring
distribution, and in such cases the preferred groups to be added
include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa
488. Terminal modifications can also be useful for enhancing
uptake, useful modifications for this include cholesterol. Terminal
modifications can also be useful for cross-linking anantagomir to
another moiety; modifications useful for this include mitomycin
C.
[0183] Candidate modifications can be evaluated as described
below.
[0184] The Bases
[0185] Adenine, guanine, cytosine and uracil are the most common
bases found in RNA. These bases can be modified or replaced to
provide RNA's having improved properties. E.g., nuclease resistant
oligoribonucleotides can be prepared with these bases or with
synthetic and natural nucleobases (e.g., inosine, thymine,
xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine)
and any one of the above modifications. Alternatively, substituted
or modified analogs of any of the above bases, e.g., "unusual
bases" and "universal bases" described herein, can be employed.
Examples include without limitation 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine
and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,
5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino,
thiol, thioalkyl, hydroxyl and other 8-substituted adenines and
guanines, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine, 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine,
5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles,
2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil,
uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,
5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil,
3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine,
5-methylcytosine, N.sup.4-acetyl cytosine, 2-thiocytosine,
N6-methyladenine, N6-isopentyladenine,
2-methylthio-N6-isopentenyladenine, N-methylguanines, or
O-alkylated bases. Further purines and pyrimidines include those
disclosed in U.S. Pat. No. 3,687,808, those disclosed in the
Concise Encyclopedia Of Polymer Science And Engineering, pages
858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and
those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613.
[0186] Candidate modifications can be evaluated as described
below.
[0187] Evaluation of Candidate Oligonucleotide Agents
[0188] One can evaluate a candidate single-stranded oligonucleotide
agent, e.g., a modified candidate single-stranded oligonucleotide
agent, for a selected property by exposing the agent or modified
molecule and a control molecule to the appropriate conditions and
evaluating for the presence of the selected property. For example,
resistance to a degradent can be evaluated as follows. A candidate
modified antagomir (and preferably a control single-stranded
oligonucleotide agent, usually the unmodified form) can be exposed
to degradative conditions, e.g., exposed to a milieu, which
includes a degradative agent, e.g., a nuclease. For example, one
can use a biological sample, e.g., one that is similar to a milieu,
which might be encountered, in therapeutic use, e.g., blood or a
cellular fraction, e.g., a cell-free homogenate or disrupted cells.
The candidate and control can then be evaluated for resistance to
degradation by any of a number of approaches. For example, the
candidate and control could be labeled, preferably prior to
exposure, with, e.g., a radioactive or enzymatic label, or a
fluorescent label, such as Cy3 or Cy5. Control and modified
oligonucleotide agents can be incubated with the degradative agent,
and optionally a control, e.g., an inactivated, e.g., heat
inactivated, degradative agent. A physical parameter, e.g., size,
of the modified and control molecules are then determined. They can
be determined by a physical method, e.g., by polyacrylamide gel
electrophoresis or a sizing column, to assess whether the molecule
has maintained its original length, or assessed functionally.
Alternatively, Northern blot analysis can be used to assay the
length of an unlabeled modified molecule.
[0189] A functional assay can also be used to evaluate the
candidate agent. A functional assay can be applied initially or
after an earlier non-functional assay, (e.g., assay for resistance
to degradation) to determine if the modification alters the ability
of the molecule to inhibit gene expression. For example, a cell,
e.g., a mammalian cell, such as a mouse or human cell, can be
co-transfected with a plasmid expressing a fluorescent protein,
e.g., GFP, and a candidate antagomir homologous to the transcript
encoding the fluorescent protein (see, e.g., WO 00/44914). For
example, a modified antagomir homologous to the GFP mRNA can be
assayed for the ability to inhibit GFP expression by monitoring for
a decrease in cell fluorescence, as compared to a control cell, in
which the transfection did not include the candidate
oligonucleotide agent, e.g., controls with no agent added and/or
controls with a non-modified RNA added. Efficacy of the candidate
agent on gene expression can be assessed by comparing cell
fluorescence in the presence of the modified and unmodified
oligonucleotide agent.
[0190] In an alternative functional assay, a candidate antagomir
homologous to an endogenous mouse gene, preferably a maternally
expressed gene, such as c-mos, can be injected into an immature
mouse oocyte to assess the ability of the agent to inhibit gene
expression in vivo (see, e.g., WO 01/36646). A phenotype of the
oocyte, e.g., the ability to maintain arrest in metaphase II, can
be monitored as an indicator that the agent is inhibiting
expression. For example, cleavage of c-mos mRNA by an antagomir
would cause the oocyte to exit metaphase arrest and initiate
parthenogenetic development (Colledge et al. Nature 370: 65-68,
1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the
modified agent on target RNA levels can be verified by Northern
blot to assay for a decrease in the level of target RNA, or by
Western blot to assay for a decrease in the level of target
protein, as compared to a negative control. Controls can include
cells in which with no agent is added and/or cells in which a
non-modified RNA is added.
[0191] Preferred Oligonucleotide Agents
[0192] Preferred single-stranded oligonucleotide agents have the
following structure (see Formula 2 below): ##STR2##
[0193] Referring to Formula 2 above, R.sup.1, R.sup.2, and R.sup.3
are each, independently, H, (i.e. abasic nucleotides), adenine,
guanine, cytosine and uracil, inosine, thymine, xanthine,
hypoxanthine, nubularine, tubercidine, isoguanisine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,
6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl
uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other
8-substituted adenines and guanines, 5-trifluoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine, 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted
purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine,
2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil,
substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole,
3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,
5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,
5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,
3-methylcytosine, 5-methylcytosine, N.sup.4-acetyl cytosine,
2-thiocytosine, N6-methyladenine, N6-isopentyladenine,
2-methylthio-N6-isopentenyladenine, N-methylguanines, or
O-alkylated bases.
[0194] R.sup.4, R.sup.5, and R.sup.6 are each, independently,
OR.sup.8, O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2OR.sup.8;
O(CH.sub.2).sub.nR.sup.9; O(CH.sub.2).sub.nOR.sup.9, H; halo;
NH.sub.2; NHR.sup.8; N(R.sup.8).sub.2;
NH(CH.sub.2CH.sub.2NH).sub.mCH.sub.2CH.sub.2NHR.sup.9; NH
C(O)R.sup.8; cyano; mercapto, SR.sup.8; alkyl-thio-alkyl; alkyl,
aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of
which may be optionally substituted with halo, hydroxy, oxo, nitro,
haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino,
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,
arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,
alkanesulfonyl, alkanesulfonamido, arenesulfonamido,
aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or
R.sup.4, R.sup.5, or R.sup.6 together combine with R.sup.7 to form
an [--O--CH.sub.2--] covalently bound bridge between the sugar 2'
and 4' carbons.
[0195] A.sup.1 is: ##STR3##
[0196] ; H; OH; OCH.sub.3; W.sup.1; an abasic nucleotide; or
absent;
[0197] (a preferred A1, especially with regard to anti-sense
strands, is chosen from 5'-monophosphate ((HO).sub.2(O)P--O-5'),
5'-diphosphate ((HO).sub.2(O)P--O--P(HO)(O)--O-5'), 5'-triphosphate
((HO).sub.2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'), 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'), 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap structure
(N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'),
5'-monothiophosphate (phosphorothioate; (HO).sub.2(S)P--O-5'),
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO).sub.2(O)P--S-5'); any additional
combination of oxygen/sulfur replaced monophosphate, diphosphate
and triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO).sub.2(O)P--NH-5', (HO)(NH.sub.2)(O)P--O-5'),
5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl,
etc., e.g. RP(OH)(O)--O-5'-, (OH).sub.2(O)P-5'-CH.sub.2--),
5'-alkyletherphosphonates (R=alkylether=ethoxymethyl
(MeOCH.sub.2--), ethoxymethyl, etc., e.g. RP(OH)(O)--O-5'-)).
[0198] A.sup.2 is: ##STR4##
[0199] A.sup.3 is: ##STR5##
[0200] ; and
[0201] A.sup.4 is: ##STR6##
[0202] ; H; Z.sup.4; an inverted nucleotide; an abasic nucleotide;
or absent.
[0203] W.sup.1 is OH, (CH.sub.2).sub.nR.sup.10,
(CH.sub.2).sub.nNHR.sup.10, (CH.sub.2).sub.nOR.sup.10,
(CH.sub.2).sub.nSR.sup.10; O(CH.sub.2).sub.nR.sup.10;
O(CH.sub.2).sub.nOR.sup.10, O(CH.sub.2).sub.nNR.sup.10,
O(CH.sub.2).sub.nSR.sup.10;
O(CH.sub.2).sub.nSS(CH.sub.2).sub.nOR.sup.10,
O(CH.sub.2).sub.nC(O)OR.sup.10, NH(CH.sub.2).sub.nR.sup.10;
NH(CH.sub.2).sub.nNR.sup.10; NH(CH.sub.2).sub.nOR.sup.10,
NH(CH.sub.2).sub.nSR.sup.10; S(CH.sub.2).sub.nR.sup.10,
S(CH.sub.2).sub.nNR.sup.10, S(CH.sub.2).sub.nOR.sup.10,
S(CH.sub.2).sub.nSR.sup.10O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2OR.su-
p.10; O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NHR.sup.10,
NH(CH.sub.2CH.sub.2NH).sub.mCH.sub.2CH.sub.2NHR.sup.10; Q-R',
O-Q-R.sup.10N-Q-R.sup.10, S-Q-R.sup.10 or --O--. W.sup.4 is O,
CH.sub.2, NH, or S.
[0204] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are each,
independently, O or S.
[0205] Y.sup.1, Y.sup.2, Y.sup.3, and Y.sup.4 are each,
independently, OH, O.sup.-, OR.sup.8, S, Se, BH.sub.3.sup.-, H,
NHR.sup.9, N(R.sup.9).sub.2 alkyl, cycloalkyl, aralkyl, aryl, or
heteroaryl, each of which may be optionally substituted.
[0206] Z.sup.1, Z.sup.2, and Z.sup.3 are each independently O,
CH.sub.2, NH, or S. Z.sup.4 is OH, (CH.sub.2).sub.nR.sup.10,
(CH.sub.2).sub.nNHR.sup.10, (CH.sub.2).sub.nOR.sup.10,
(CH.sub.2).sub.nSR.sup.10; O(CH.sub.2).sub.nR.sup.10;
O(CH.sub.2).sub.nOR.sup.10,
O(CH.sub.2).sub.nNR.sup.10O(CH.sub.2).sub.nSR.sup.10,
O(CH.sub.2).sub.nSS(CH.sub.2).sub.nOR.sup.10,
O(CH.sub.2).sub.nC(O)OR.sup.10; NH(CH.sub.2).sub.nR.sup.10;
NH(CH.sub.2).sub.nNR.sup.10; NH(CH.sub.2).sub.nOR.sup.10,
NH(CH.sub.2).sub.nSR.sup.10; S(CH.sub.2).sub.nR.sup.10,
S(CH.sub.2).sub.nNR.sup.10, S(CH.sub.2).sub.nOR.sup.10,
S(CH.sub.2).sub.nSR.sup.10O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2OR.su-
p.10, O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NHR.sup.10,
NH(CH.sub.2CH.sub.2NH).sub.mCH.sub.2CH.sub.2NHR.sup.10; Q-R.sup.10,
O-Q-R.sup.10N-Q-R.sup.10, S-Q-R.sup.10.
[0207] X is 5-100, chosen to comply with a length for an antagomir
described herein.
[0208] R.sup.7 is H; or is together combined with R.sup.4, R.sup.5,
or R.sup.6 to form an [--O--CH.sub.2--] covalently bound bridge
between the sugar 2' and 4' carbons.
[0209] R.sup.8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,
heteroaryl, amino acid, or sugar; R.sup.9 is NH.sub.2, alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, or amino acid; and R.sup.10 is H;
fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur,
silicon, boron or ester protecting group; intercalating agents
(e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),
porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic
hydrocarbons (e.g., phenazine, dihydrophenazine), artificial
endonucleases (e.g. EDTA), lipophilic carriers (cholesterol, cholic
acid, adamantane acetic acid, 1-pyrene butyric acid,
dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl
group, hexadecylglycerol, borneol, menthol, 1,3-propanediol,
heptadecyl group, palmitic acid, myristic acid,
O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,
antennapedia peptide, Tat peptide), alkylating agents, phosphate,
amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG].sub.2,
polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl;
radiolabelled markers, enzymes, haptens (e.g. biotin),
transport/absorption facilitators (e.g., aspirin, vitamin E, folic
acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,
histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+
complexes of tetraazamacrocycles); or an oligonucleotide agent. M
is 0-1,000,000, and n is 0-20. Q is a spacer selected from the
group consisting of abasic sugar, amide, carboxy, oxyamine,
oxyimine, thioether, disulfide, thiourea, sulfonamide, or
morpholino, biotin or fluorescein reagents.
[0210] Preferred oligonucleotide agents in which the entire
phosphate group has been replaced have the following structure (see
Formula 3 below): ##STR7##
[0211] Referring to Formula 3, A.sup.10-A.sup.40 is L-G-L; A.sup.10
and/or A.sup.40 may be absent, in which L is a linker, wherein one
or both L may be present or absent and is selected from the group
consisting of CH.sub.2(CH.sub.2).sub.g; N(CH.sub.2).sub.g;
O(CH.sub.2).sub.g; S(CH.sub.2).sub.g. G is a functional group
selected from the group consisting of siloxane, carbonate,
carboxymethyl, carbamate, amide, thioether, ethylene oxide linker,
sulfonate, sulfonamide, thioformacetal, formacetal, oxime,
methyleneimino, methylenemethylimino, methylenehydrazo,
methylenedimethylhydrazo and methyleneoxymethylimino.
[0212] R.sup.10, R.sup.20, and R.sup.30 are each, independently, H,
(i.e. abasic nucleotides), adenine, guanine, cytosine and uracil,
inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine,
isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil
and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 5-halouracil,
5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino,
thiol, thioalkyl, hydroxyl and other 8-substituted adenines and
guanines, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine, 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine,
2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil
substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole,
3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,
5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,
5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,
3-methylcytosine, 5-methylcytosine, N.sup.4-acetyl cytosine,
2-thiocytosine, N6-methyladenine, N6-isopentyladenine,
2-methylthio-N6-isopentenyladenine, N-methylguanines, or
O-alkylated bases.
[0213] R.sup.40, R.sup.50, and R.sup.60 are each, independently,
OR.sup.8, O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2OR.sup.8;
O(CH.sub.2).sub.nR.sup.9; O(CH.sub.2).sub.nOR.sup.9, H; halo;
NH.sub.2; NHR.sup.8; N(R.sup.8).sub.2;
NH(CH.sub.2CH.sub.2NH).sub.mCH.sub.2CH.sub.2R.sup.9; NHC(O)R.sup.8;
cyano; mercapto, SR.sup.7; alkyl-thio-alkyl; alkyl, aralkyl,
cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may
be optionally substituted with halo, hydroxy, oxo, nitro,
haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino,
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,
arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,
alkanesulfonyl, alkanesulfonamido, arenesulfonamido,
aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido
groups; or R.sup.40, R.sup.50, or R.sup.60 together combine with
R.sup.70 to form an [--O--CH.sub.2--] covalently bound bridge
between the sugar 2' and 4' carbons.
[0214] X is 5-100 or chosen to comply with a length for an
antagomir described herein.
[0215] R.sup.70 is H; or is together combined with R.sup.40,
R.sup.50, or R.sup.60 to form an [--O--CH.sub.2--] covalently bound
bridge between the sugar 2' and 4' carbons.
[0216] R.sup.8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,
heteroaryl, amino acid, or sugar; and R.sup.9 is NH.sub.2,
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid. M is
0-1,000,000, n is 0-20, and g is 0-2.
[0217] Preferred nucleoside surrogates have the following structure
(see Formula 4 below): SLR-(M-SLR.sup.200).sub.x-M-SLR.sup.300
FORMULA 4
[0218] S is a nucleoside surrogate selected from the group
consisting of mophilino, cyclobutyl, pyrrolidine and peptide
nucleic acid. L is a linker and is selected from the group
consisting of CH.sub.2(CH.sub.2).sub.g; N(CH.sub.2).sub.g;
O(CH.sub.2).sub.g; S(CH.sub.2).sub.g; --C(O)(CH.sub.2).sub.n-- or
may be absent. M is an amide bond; sulfonamide; sulfinate;
phosphate group; modified phosphate group as described herein; or
may be absent.
[0219] R.sup.100, R.sup.200, and R.sup.300 are each, independently,
H (i.e., abasic nucleotides), adenine, guanine, cytosine and
uracil, inosine, thymine, xanthine, hypoxanthine, nubularine,
tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl
derivatives of adenine and guanine, 2-propyl and other alkyl
derivatives of adenine and guanine, 5-halouracil and cytosine,
5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine,
5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,
5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino,
thiol, thioalkyl, hydroxyl and other 8-substituted adenines and
guanines, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine, 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine,
2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil
substituted 1,2,4,-triazoles, 2-pyridinones, 5-nitroindole,
3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,
5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,
5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,
3-methylcytosine, 5-methylcytosine, N.sup.4-acetyl cytosine,
2-thiocytosine, N6-methyladenine, N6-isopentyladenine,
2-methylthio-N6-isopentenyladenine, N-methylguanines, or
O-alkylated bases.
[0220] X is 5-100, or chosen to comply with a length for an
antagomir described herein; and g is 0-2.
[0221] An antagomir can include an internucleotide linkage (e.g.,
the chiral phosphorothioate linkage) useful for increasing nuclease
resistance. In addition, or in the alternative, an antagomir can
include a ribose mimic for increased nuclease resistance. Exemplary
internucleotide linkages and ribose mimics for increased nuclease
resistance are described in co-owned PCT Application No.
PCT/US2004/07070 filed on Mar. 8, 2004.
[0222] An antagomir can include ligand-conjugated monomer subunits
and monomers for oligonucleotide synthesis. Exemplary monomers are
described in co-owned U.S. application Ser. No. 10/916,185, filed
on Aug. 10, 2004.
[0223] An antagomir can have a ZXY structure, such as is described
in co-owned PCT Application No. PCT/US2004/07070 filed on Mar. 8,
2004.
[0224] An antagomir can be complexed with an amphipathic moiety.
Exemplary amphipathic moieties for use with oligonucleotide agents
are described in co-owned PCT Application No. PCT/US2004/07070
filed on Mar. 8, 2004.
[0225] In another embodiment, the antagomir can be complexed to a
delivery agent that features a modular complex. The complex can
include a carrier agent linked to one or more of (preferably two or
more, more preferably all three of): (a) a condensing agent (e.g.,
an agent capable of attracting, e.g., binding, a nucleic acid,
e.g., through ionic or electrostatic interactions); (b) a fusogenic
agent (e.g., an agent capable of fusing and/or being transported
through a cell membrane); and (c) a targeting group, e.g., a cell
or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or
protein, e.g., an antibody, that binds to a specified cell type.
oligonucleotide agents complexed to a delivery agent are described
in co-owned PCT Application No. PCT/US2004/07070 filed on Mar. 8,
2004.
[0226] Enhanced Nuclease Resistance
[0227] An antagomir, such as a single-stranded oligonucleotide
agent, featured in the invention can have enhanced resistance to
nucleases.
[0228] For increased nuclease resistance and/or binding affinity to
the target, an oligonucleotide agent, e.g., the oligonucleotide
agent, can include, for example, 2'-modified ribose units and/or
phosphorothioate linkages. E.g., the 2' hydroxyl group (OH) can be
modified or replaced with a number of different "oxy" or "deoxy"
substitutents.
[0229] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar; amine,
O-AMINE and aminoalkoxy, O(CH.sub.2).sub.nAMINE, (e.g.,
AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl amino,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino). It is noteworthy that
oligonucleotides containing only the methoxyethyl group (MOE),
(OCH.sub.2CH.sub.2OCH.sub.3, a PEG derivative), exhibit nuclease
stabilities comparable to those modified with the robust
phosphorothioate modification.
[0230] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars); halo (e.g., fluoro); amino (e.g. NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl
amino, heteroaryl amino, or diheteroaryl amino), --NHC(O)R(R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl
and alkynyl, which may be optionally substituted with e.g., an
amino functionality.
[0231] Preferred substitutents are 2'-methoxyethyl, 2'-OCH3,
2'-O-allyl, 2'-C-- allyl, and 2'-fluoro.
[0232] One way to increase resistance is to identify cleavage sites
and modify such sites to inhibit cleavage, as described in co-owned
U.S. Application No. 60/559,917, filed on May 4, 2004. For example,
the dinucleotides 5'-UA-3',5'-UG-3',5'-CA-3',5'-UU-3', or 5'-CC-3'
can serve as cleavage sites. Enhanced nuclease resistance can
therefore be achieved by modifying the 5' nucleotide, resulting,
for example, in at least one 5'-uridine-adenine-3' (5'-UA-3')
dinucleotide wherein the uridine is a 2'-modified nucleotide; at
least one 5'-uridine-guanine-3' (5'-UG-3') dinucleotide, wherein
the 5'-uridine is a 2'-modified nucleotide; at least one
5'-cytidine-adenine-3' (5'-CA-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide; at least one
5'-uridine-uridine-3' (5'-UU-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide; or at least one
5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide. The antagomir can include
at least 2, at least 3, at least 4 or at least 5 of such
dinucleotides. In certain embodiments, all the pyrimidines of an
antagomir carry a 2'-modification, and the antagomir therefore has
enhanced resistance to endonucleases.
[0233] To maximize nuclease resistance, the 2' modifications can be
used in combination with one or more phosphate linker modifications
(e.g., phosphorothioate). The so-called "chimeric" oligonucleotides
are those that contain two or more different modifications.
[0234] The inclusion of furanose sugars in the oligonucleotide
backbone can also decrease endonucleolytic cleavage. An antagomir
can be further modified by including a 3' cationic group, or by
inverting the nucleoside at the 3'-terminus with a 3'-3' linkage.
In another alternative, the 3'-terminus can be blocked with an
aminoalkyl group, e.g., a 3' C5-aminoalkyl dT. Other 3' conjugates
can inhibit 3'-5' exonucleolytic cleavage. While not being bound by
theory, a 3' conjugate, such as naproxen or ibuprofen, may inhibit
exonucleolytic cleavage by sterically blocking the exonuclease from
binding to the 3'-end of oligonucleotide. Even small alkyl chains,
aryl groups, or heterocyclic conjugates or modified sugars
(D-ribose, deoxyribose, glucose etc.) can block
3'-5'-exonucleases.
[0235] Similarly, 5' conjugates can inhibit 5'-3' exonucleolytic
cleavage. While not being bound by theory, a 5' conjugate, such as
naproxen or ibuprofen, may inhibit exonucleolytic cleavage by
sterically blocking the exonuclease from binding to the 5'-end of
oligonucleotide. Even small alkyl chains, aryl groups, or
heterocyclic conjugates or modified sugars (D-ribose, deoxyribose,
glucose etc.) can block 3'-5'-exonucleases.
[0236] Thus, an antagomir can include modifications so as to
inhibit degradation, e.g., by nucleases, e.g., endonucleases or
exonucleases, found in the body of a subject. These monomers are
referred to herein as NRMs, or Nuclease Resistance promoting
Monomers, the corresponding modifications as NRM modifications. In
many cases these modifications will modulate other properties of
the antagomir as well, e.g., the ability to interact with a
protein, e.g., a transport protein, e.g., serum albumin, or a
member of the RISC, or the ability of the antagomir to form a
duplex with another sequence, e.g., a target molecule, such as an
miRNA or pre-miRNA.
[0237] One or more different NRM modifications can be introduced
into an antagomir or into a sequence of an oligonucleotide agent.
An NRM modification can be used more than once in a sequence or in
an oligonucleotide agent.
[0238] NRM modifications include some which can be placed only at
the terminus and others which can go at any position. Some NRM
modifications that can inhibit hybridization are preferably used
only in terminal regions, and more preferably not at the cleavage
site or in the cleavage region of the oligonucleotide agent.
[0239] Modifications which interfere with or inhibit endonuclease
cleavage should not be inserted in the region which is subject to
RISC mediated cleavage, e.g., the cleavage site or the cleavage
region (As described in Elbashir et al., Genes and Dev. 15: 188,
2001, hereby incorporated by reference). Cleavage of the target
occurs about in the middle of a 20 or 21 nt oligonucleotide agent,
or about 10 or 11 nucleotides upstream of the first nucleotide on
the target mRNA which is complementary to the oligonucleotide
agent. As used herein, cleavage site refers to the nucleotides on
either side of the site of cleavage, on the target mRNA or on the
antagomir which hybridizes to it. Cleavage region means the
nucleotides within 1, 2, or 3 nucleotides of the cleavage site, in
either direction.
[0240] Such modifications can be introduced into the terminal
regions, e.g., at the terminal position or with 2, 3, 4, or 5
positions of the terminus, of a sequence which targets or a
sequence which does not target a sequence in the subject.
Delivery of Single-Stranded Oligonucleotide Agents to Tissues and
Cells
[0241] Formulation
[0242] The single-stranded oligonucleotide agents described herein
can be formulated for administration to a subject.
[0243] For ease of exposition, the formulations, compositions, and
methods in this section are discussed largely with regard to
unmodified oligonucleotide agents. It should be understood,
however, that these formulations, compositions, and methods can be
practiced with other oligonucleotide agents, e.g., modified
oligonucleotide agents, and such practice is within the
invention.
[0244] A formulated antagomir composition can assume a variety of
states. In some examples, the composition is at least partially
crystalline, uniformly crystalline, and/or anhydrous (e.g., less
than 80, 50, 30, 20, or 10% water). In another example, the
antagomir is in an aqueous phase, e.g., in a solution that includes
water, this form being the preferred form for administration via
inhalation.
[0245] The aqueous phase or the crystalline compositions can be
incorporated into a delivery vehicle, e.g., a liposome
(particularly for the aqueous phase), or a particle (e.g., a
microparticle as can be appropriate for a crystalline composition).
Generally, the antagomir composition is formulated in a manner that
is compatible with the intended method of administration.
[0246] An antagomir preparation can be formulated in combination
with another agent, e.g., another therapeutic agent or an agent
that stabilizes an oligonucleotide agent, e.g., a protein that
complexes with the oligonucleotide agent. Still other agents
include chelators, e.g., EDTA (e.g., to remove divalent cations
such as Mg.sup.2+), salts, RNAse inhibitors (e.g., a broad
specificity RNAse inhibitor such as RNAsin) and so forth.
[0247] In one embodiment, the antagomir preparation includes
another antagomir, e.g., a second antagomir that can down-regulate
expression of a second gene. Still other preparations can include
at least three, five, ten, twenty, fifty, or a hundred or more
different oligonucleotide species. In some embodiments, the agents
are directed to the same target nucleic acid but different target
sequences. In another embodiment, each antagomir is directed to a
different target. In one embodiment the antagomir preparation
includes a double stranded RNA that targets an RNA (e.g., an mRNA)
for donwregulation by an RNAi silencing mechanism.
[0248] Treatment Methods and Routes of Delivery
[0249] A composition that includes an antagomir featured in the
invention, e.g., an antagomir that targets an miRNA or pre-miRNA
(e.g., miR-122, miR-16, miR-192, or miR-194) can be delivered to a
subject by a variety of routes. Exemplary routes include
inhalation, intrathecal, parenchymal, intravenous, nasal, oral, and
ocular delivery.
[0250] An antagomir can be incorporated into pharmaceutical
compositions suitable for administration. For example, compositions
can include one or more oligonucleotide agents and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0251] The pharmaceutical compositions featured in the invention
may be administered in a number of ways depending upon whether
local or systemic treatment is desired and upon the area to be
treated. Administration may be topical (including ophthalmic,
intranasal, transdermal, intrapulmonary), oral or parenteral.
Parenteral administration includes intravenous drip, subcutaneous,
intraperitoneal or intramuscular injection, or intrathecal or
intraventricular administration.
[0252] In general, delivery of an antagomir featured in the
invention directs the agent to the site of infection in a subject.
The preferred means of delivery is through local administration
directly to the site of infection, or by systemic administration,
e.g. parental administration.
[0253] Formulations for direct injection and parenteral
administration are well known in the art. Such formulations may
include sterile aqueous solutions which may also contain buffers,
diluents and other suitable additives. For intravenous use, the
total concentration of solutes should be controlled to render the
preparation isotonic.
[0254] Administration of Oligonucleotide Agents
[0255] A patient who has been diagnosed with a disorder
characterized by unwanted miRNA expression (e.g., unwanted
expression of miR-122, miR-16, miR-192, or miR-194) can be treated
by administration of an antagomir described herein to block the
negative effects of the miRNA, thereby alleviating the symptoms
associated with the unwanted miRNA expression. Similarly, a human
who has or is at risk for developing a disorder characterized by
underexpression of a gene that is regulated by an miRNA can be
treated by the administration of an antagomir that targets the
miRNA. For example, a human diagnosed with hemolytic anemia, and
who carries a mutation in the aldolase A gene, expresses a
compromised form of the enzyme. The patient can be administered an
antagomir that targets endogenous miR-122, which binds aldolase A
RNA in vivo, presumably to downregulate translation of the aldolase
A mRNA and consequently downregulate aldolase A protein levels.
Administration of an antagomir that targets the endogenous miR-122
in a patient having hemolytic anemia will decrease miR-122
activity, which will result in the upregulation of aldolase A
expression and an increase in aldolase A protein levels. Although
the enzyme activity of the mutant aldolase A is suboptimal, an
increase in protein levels may be sufficient to relieve the disease
symptoms. A human who has or who is at risk for developing
arthrogryposis multiplex congenital, pituitary ectopia,
rhabdomyolysis, or hyperkalemia, or who suffers from a myopathic
symptom, is also a suitable candidate for treatment with an
antagomir that targets miR-122. A human who carries a mutation in
the aldolase A gene can be a candidate for treatment with an
antagomir that targets miR-122. A human who carries a mutation in
the aldolase A gene can have a symptom characterizing aldolase A
deficiency including growth and developmental retardation,
midfacial hypoplasia, and hepatomegaly.
[0256] In another example, a human who has or who is at risk for
developing a disorder associated with overexpression of a gene
regulated by an miRNA or by an miRNA deficiency, e.g., an miR-122,
miR-16, miR-192, or miR-194 deficiency, can be treated by the
administration of an antagomir, such as a single-stranded
oligonucleotide agent, that is substantially identical to the
deficient miRNA.
[0257] The single-stranded oligonucleotide agents featured in the
invention can be administered systemically, e.g., orally or by
intramuscular injection or by intravenous injection, in admixture
with a pharmaceutically acceptable carrier adapted for the route of
administration. An antagomir can include a delivery vehicle, such
as liposomes, for administration to a subject, carriers and
diluents and their salts, and/or can be present in pharmaceutically
acceptable formulations. Methods for the delivery of nucleic acid
molecules are described in Akhtar et al., Trends in Cell Bio.
2:139, 1992; Delivery Strategies for Antisense Oligonucleotide
Therapeutics, ed. Akhtar, 1995; Maurer et al., Mol. Membr. Biol.,
16:129, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137:165,
1999; and Lee et al., ACS Symp. Ser. 752:184, 2000, all of which
are incorporated herein by reference. Beigelman et al., U.S. Pat.
No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe
the general methods for delivery of nucleic acid molecules. Nucleic
acid molecules can be administered to cells by a variety of methods
known to those of skill in the art, including, but not restricted
to, encapsulation in liposomes, by ionophoresis, or by
incorporation into other vehicles, such as hydrogels, cyclodextrins
(see for example Gonzalez et al., Bioconjugate Chem. 10:1068,
1999), biodegradable nanocapsules, and bioadhesive microspheres, or
by proteinaceous vectors (O'Hare and Normand, International PCT
Publication No. WO 00/53722).
[0258] In the present methods, the antagomir can be administered to
the subject either as a naked oligonucleotide agent, in conjunction
with a delivery reagent, or as a recombinant plasmid or viral
vector which expresses the oligonucleotide agent. Preferably, the
antagomir is administered as a naked oligonucleotide agent.
[0259] An antagomir featured in the invention can be administered
to the subject by any means suitable for delivering the agent to
the cells of the tissue at or near the area of unwanted target
nucleic acid expression (e.g., target miRNA or pre-miRNA
expression). For example, an antagomir that targets miR-122 can be
delivered directly to the liver, or can be conjugated to a molecule
that targets the liver. Exemplary delivery methods include
administration by gene gun, electroporation, or other suitable
parenteral administration route.
[0260] Suitable enteral administration routes include oral
delivery.
[0261] Suitable parenteral administration routes include
intravascular administration (e.g., intravenous bolus injection,
intravenous infusion, intra-arterial bolus injection,
intra-arterial infusion and catheter instillation into the
vasculature); peri- and intra-tissue injection (e.g., intraocular
injection, intra-retinal injection, or sub-retinal injection);
subcutaneous injection or deposition including subcutaneous
infusion (such as by osmotic pumps); direct application to the area
at or near the site of neovascularization, for example by a
catheter or other placement device (e.g., a retinal pellet or an
implant comprising a porous, non-porous, or gelatinous
material).
[0262] An antagomir featured in the invention can be delivered
using an intraocular implant. Such implants can be biodegradable
and/or biocompatible implants, or may be non-biodegradable
implants. The implants may be permeable or impermeable to the
active agent, and may be inserted into a chamber of the eye, such
as the anterior or posterior chambers, or may be implanted in the
sclera, transchoroidal space, or an avascularized region exterior
to the vitreous. In a preferred embodiment, the implant may be
positioned over an avascular region, such as on the sclera, so as
to allow for transscleral diffusion of the drug to the desired site
of treatment, e.g., the intraocular space and macula of the eye.
Furthermore, the site of transscleral diffusion is preferably in
proximity to the macula.
[0263] An antagomir featured in the invention can also be
administered topically, for example, by patch or by direct
application to the eye, or by iontophoresis. Ointments, sprays, or
droppable liquids can be delivered by ocular delivery systems known
in the art such as applicators or eyedroppers. The compositions can
be administered directly to the surface of the eye or to the
interior of the eyelid. Such compositions can include mucomimetics
such as hyaluronic acid, chondroitin sulfate, hydroxypropyl
methylcellulose or poly(vinyl alcohol), preservatives such as
sorbic acid, EDTA or benzylchronium chloride, and the usual
quantities of diluents and/or carriers.
[0264] An antagomir featured in the invention may be provided in
sustained release compositions, such as those described in, for
example, U.S. Pat. Nos. 5,672,659 and 5,595,760. The use of
immediate or sustained release compositions depends on the nature
of the condition being treated. If the condition consists of an
acute or over-acute disorder, treatment with an immediate release
form will be preferred over a prolonged release composition.
Alternatively, for certain preventative or long-term treatments, a
sustained release composition may be appropriate.
[0265] An antagomir can be injected into the interior of the eye,
such as with a needle or other delivery device.
[0266] An antagomir featured in the invention can be administered
in a single dose or in multiple doses. Where the administration of
the antagomir is by infusion, the infusion can be a single
sustained dose or can be delivered by multiple infusions. Injection
of the agent can be directly into the tissue at or near the site of
aberrant or unwanted target gene expression (e.g., aberrant or
unwanted miRNA or pre-miRNA expression). Multiple injections of the
agent can be made into the tissue at or near the site.
[0267] Dosage levels on the order of about 1 .mu.g/kg to 100 mg/kg
of body weight per administration are useful in the treatment of a
disease. One skilled in the art can also readily determine an
appropriate dosage regimen for administering the antagomir of the
invention to a given subject. For example, the antagomir can be
administered to the subject once, e.g., as a single injection or
deposition at or near the site on unwanted target nucleic acid
expression. Alternatively, the antagomir can be administered once
or twice daily to a subject for a period of from about three to
about twenty-eight days, more preferably from about seven to about
ten days. In a preferred dosage regimen, the antagomir is injected
at or near a site of unwanted target nucleic acid expression once a
day for seven days. Where a dosage regimen comprises multiple
administrations, it is understood that the effective amount of
antagomir administered to the subject can include the total amount
of antagomir administered over the entire dosage regimen. One
skilled in the art will appreciate that the exact individual
dosages may be adjusted somewhat depending on a variety of factors,
including the specific antagomir being administered, the time of
administration, the route of administration, the nature of the
formulation, the rate of excretion, the particular disorder being
treated, the severity of the disorder, the pharmacodynamics of the
oligonucleotide agent, and the age, sex, weight, and general health
of the patient. Wide variations in the necessary dosage level are
to be expected in view of the differing efficiencies of the various
routes of administration. For instance, oral administration
generally would be expected to require higher dosage levels than
administration by intravenous or intravitreal injection. Variations
in these dosage levels can be adjusted using standard empirical
routines of optimization, which are well-known in the art. The
precise therapeutically effective dosage levels and patterns are
preferably determined by the attending physician in consideration
of the above-identified factors.
[0268] In addition to treating pre-existing diseases or disorders,
oligonucleotide agents featured in the invention (e.g.,
single-stranded oligonucleotide agents targeting miR-122, miR-16,
miR-192, or miR-194) can be administered prophylactically in order
to prevent or slow the onset of a particular disease or disorder.
In prophylactic applications, an antagomir is administered to a
patient susceptible to or otherwise at risk of a particular
disorder, such as disorder associated with aberrant or unwanted
expression of an miRNA or pre-miRNA.
[0269] The oligonucleotide agents featured by the invention are
preferably formulated as pharmaceutical compositions prior to
administering to a subject, according to techniques known in the
art. Pharmaceutical compositions featured in the present invention
are characterized as being at least sterile and pyrogen-free. As
used herein, "pharmaceutical formulations" include formulations for
human and veterinary use. Methods for preparing pharmaceutical
compositions are within the skill in the art, for example as
described in Remington's Pharmaceutical Science, 18th ed., Mack
Publishing Company, Easton, Pa. (1990), and The Science and
Practice of Pharmacy, 2003, Gennaro et al., the entire disclosures
of which are herein incorporated by reference.
[0270] The present pharmaceutical formulations include an antagomir
featured in the invention (e.g., 0.1 to 90% by weight), or a
physiologically acceptable salt thereof, mixed with a
physiologically acceptable carrier medium. Preferred
physiologically acceptable carrier media are water, buffered water,
normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the
like.
[0271] Pharmaceutical compositions featured in the invention can
also include conventional pharmaceutical excipients and/or
additives. Suitable pharmaceutical excipients include stabilizers,
antioxidants, osmolality adjusting agents, buffers, and pH
adjusting agents. Suitable additives include physiologically
biocompatible buffers (e.g., tromethamine hydrochloride), additions
of chelants (such as, for example, DTPA or DTPA-bisamide) or
calcium chelate complexes (as for example calcium DTPA,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium
salts (for example, calcium chloride, calcium ascorbate, calcium
gluconate or calcium lactate). Pharmaceutical compositions can be
packaged for use in liquid form, or can be lyophilized.
[0272] For solid compositions, conventional non-toxic solid
carriers can be used; for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like.
[0273] For example, a solid pharmaceutical composition for oral
administration can include any of the carriers and excipients
listed above and 10-95%, preferably 25%-75%, of one or more
single-stranded oligonucleotide agents featured in the
invention.
[0274] By "pharmaceutically acceptable formulation" is meant a
composition or formulation that allows for the effective
distribution of the nucleic acid molecules of the instant invention
in the physical location most suitable for their desired activity.
Non-limiting examples of agents suitable for formulation with the
nucleic acid molecules of the instant invention include:
P-glycoprotein inhibitors (such as PluronicP85), which can enhance
entry of drugs into the CNS (Jolliet-Riant and Tillement, Fundam.
Clin. Pharmacol. 13:16, 1999); biodegradable polymers, such as
poly(DL-lactide-coglycolide) microspheres for sustained release
delivery. Other non-limiting examples of delivery strategies for
the nucleic acid molecules featured in the instant invention
include material described in Boado et al., J. Pharm. Sci. 87:1308,
1998; Tyler et al., FEBS Lett. 421:280, 1999; Pardridge et al.,
PNAS USA. 92:5592, 1995; Boado, Adv. Drug Delivery Rev. 15:73,
1995; Aldrian-Herrada et al., Nucleic Acids Res. 26:4910, 1998; and
Tyler et al., PNAS USA 96:7053, 1999.
[0275] The invention also features the use of a composition that
includes surface-modified liposomes containing poly(ethylene
glycol) lipids (PEG-modified, or long-circulating liposomes or
stealth liposomes). These formulations offer a method for
increasing the accumulation of drugs in target tissues. This class
of drug carriers resists opsonization and elimination by the
mononuclear phagocytic system (MPS or RES), thereby enabling longer
blood circulation times and enhanced tissue exposure for the
encapsulated drug (Lasic et al., Chem. Rev. 95:2601, 1995; Ishiwata
et al., Chem. Phare. Bull. 43:1005, 1995).
[0276] Such liposomes have been shown to accumulate selectively in
tumors, presumably by extravasation and capture in the
neovascularized target tissues (Lasic et al., Science 267:1275,
1995; Oku et al., Biochim. Biophys. Acta 1238:86, 1995). The
long-circulating liposomes enhance the pharmacokinetics and
pharmacodynamics of DNA and RNA, particularly compared to
conventional cationic liposomes which are known to accumulate in
tissues of the MPS (Liu et al., J. Biol. Chem. 42:24864, 1995; Choi
et al., International PCT Publication No. WO 96/10391; Ansell et
al., International PCT Publication No. WO 96/10390; Holland et al.,
International PCT Publication No. WO 96/10392). Long-circulating
liposomes are also likely to protect drugs from nuclease
degradation to a greater extent compared to cationic liposomes,
based on their ability to avoid accumulation in metabolically
aggressive MPS tissues such as the liver and spleen.
[0277] The present invention also features compositions prepared
for storage or administration that include a pharmaceutically
effective amount of the desired oligonucleotides in a
pharmaceutically acceptable carrier or diluent. Acceptable carriers
or diluents for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.
1985), hereby incorporated by reference herein. For example,
preservatives, stabilizers, dyes and flavoring agents can be
provided. These include sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid. In addition, antioxidants and suspending
agents can be used.
[0278] The nucleic acid molecules of the present invention can also
be administered to a subject in combination with other therapeutic
compounds to increase the overall therapeutic effect. The use of
multiple compounds to treat an indication can increase the
beneficial effects while reducing the presence of side effects.
[0279] Alternatively, certain single-stranded oligonucleotide
agents featured in the instant invention can be expressed within
cells from eukaryotic promoters (e.g., Izant and Weintraub, Science
229:345, 1985; McGarry and Lindquist, Proc. Natl. Acad. Sci. USA
83:399, 1986; Scanlon et al., Proc. Natl. Acad. Sci. USA 88:10591,
1991; Kashani-Sabet et al., Antisense Res. Dev. 2:3, 1992; propulic
et al., J. Virol. 66:1432, 1992; Weerasinghe et al., J. Virol.
65:5531, 1991; Ojwang et al., Proc. Natl. Acad. Sci. USA 89:10802,
1992; Chen et al., Nucleic Acids Res. 20:4581, 1992; Sarver et al.,
Science 247:1222, 1990; Thompson et al., Nucleic Acids Res.
23:2259, 1995; Good et al., Gene Therapy 4:45, 1997). Those skilled
in the art realize that any nucleic acid can be expressed in
eukaryotic cells from the appropriate DNA/RNA vector. The activity
of such nucleic acids can be augmented by their release from the
primary transcript by a enzymatic nucleic acid (Draper et al., PCT
WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al.,
Nucleic Acids Symp. Ser. 27:156, 1992; Taira et al., Nucleic Acids
Res. 19:5125, 1991; Ventura et al., Nucleic Acids Res. 21:3249,
1993; Chowrira et al., J. Biol. Chem. 269:25856, 1994).
[0280] In another aspect of the invention, RNA molecules of the
present invention can be expressed from transcription units (see
for example Couture et al., Trends in Genetics 12:510, 1996)
inserted into DNA or RNA vectors. The recombinant vectors can be
DNA plasmids or viral vectors. Oligonucleotide agent-expressing
viral vectors can be constructed based on, but not limited to,
adeno-associated virus, retrovirus, adenovirus, or alphavirus. In
another embodiment, pol III based constructs are used to express
nucleic acid molecules of the invention (see for example Thompson,
U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors
capable of expressing the oligonucleotide agents can be delivered
as described above, and can persist in target cells. Alternatively,
viral vectors can be used that provide for transient expression of
nucleic acid molecules. Such vectors can be repeatedly administered
as necessary. Once expressed, the antagomir interacts with the
target RNA (e.g., miRNA or pre-miRNA) and inhibits miRNA activity.
In a preferred embodiment, the antagomir forms a duplex with the
target miRNA, which prevents the miRNA from binding to its target
mRNA, which results in increased translation of the target mRNA.
Delivery of oligonucleotide agent-expressing vectors can be
systemic, such as by intravenous or intra-muscular administration,
by administration to target cells ex-planted from a subject
followed by reintroduction into the subject, or by any other means
that would allow for introduction into the desired target cell (for
a review see Couture et al., Trends in Genetics 12:510, 1996).
[0281] The term "therapeutically effective amount" is the amount
present in the composition that is needed to provide the desired
level of drug in the subject to be treated to give the anticipated
physiological response.
[0282] The term "physiologically effective amount" is that amount
delivered to a subject to give the desired palliative or curative
effect.
[0283] The term "pharmaceutically acceptable carrier" means that
the carrier can be taken into the subject with no significant
adverse toxicological effects on the subject.
[0284] The term "co-administration" refers to administering to a
subject two or more single-stranded oligonucleotide agents. The
agents can be contained in a single pharmaceutical composition and
be administered at the same time, or the agents can be contained in
separate formulation and administered serially to a subject. So
long as the two agents can be detected in the subject at the same
time, the two agents are said to be co-administered.
[0285] The types of pharmaceutical excipients that are useful as
carrier include stabilizers such as human serum albumin (HSA),
bulking agents such as carbohydrates, amino acids and polypeptides;
pH adjusters or buffers; salts such as sodium chloride; and the
like. These carriers may be in a crystalline or amorphous form or
may be a mixture of the two.
[0286] Bulking agents that are particularly valuable include
compatible carbohydrates, polypeptides, amino acids or combinations
thereof. Suitable carbohydrates include monosaccharides such as
galactose, D-mannose, sorbose, and the like; disaccharides, such as
lactose, trehalose, and the like; cyclodextrins, such as
2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as
raffinose, maltodextrins, dextrans, and the like; alditols, such as
mannitol, xylitol, and the like. A preferred group of carbohydrates
includes lactose, threhalose, raffinose maltodextrins, and
mannitol. Suitable polypeptides include aspartame. Amino acids
include alanine and glycine, with glycine being preferred.
[0287] Suitable pH adjusters or buffers include organic salts
prepared from organic acids and bases, such as sodium citrate,
sodium ascorbate, and the like; sodium citrate is preferred.
[0288] Dosage
[0289] An antagomir can be administered at a unit dose less than
about 75 mg per kg of bodyweight, or less than about 70, 60, 50,
40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or
0.0005 mg per kg of bodyweight, and less than 200 nmol of antagomir
(e.g., about 4.4.times.10.sup.16 copies) per kg of bodyweight, or
less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075,
0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmol of antagomir per kg of
bodyweight. The unit dose, for example, can be administered by
injection (e.g., intravenous or intramuscular, intrathecally, or
directly into an organ), inhalation, or a topical application.
[0290] Delivery of an antagomir directly to an organ (e.g.,
directly to the liver) can be at a dosage on the order of about
0.00001 mg to about 3 mg per organ, or preferably about
0.0001-0.001 mg per organ, about 0.03-3.0 mg per organ, about
0.1-3.0 mg per organ or about 0.3-3.0 mg per organ.
[0291] The dosage can be an amount effective to treat or prevent a
disease or disorder.
[0292] In one embodiment, the unit dose is administered less
frequently than once a day, e.g., less than every 2, 4, 8 or 30
days. In another embodiment, the unit dose is not administered with
a frequency (e.g., not a regular frequency). For example, the unit
dose may be administered a single time. Because oligonucleotide
agent-mediated silencing can persist for several days after
administering the antagomir composition, in many instances, it is
possible to administer the composition with a frequency of less
than once per day, or, for some instances, only once for the entire
therapeutic regimen.
[0293] In one embodiment, a subject is administered an initial
dose, and one or more maintenance doses of an antagomir. The
maintenance dose or doses are generally lower than the initial
dose, e.g., one-half less of the initial dose. A maintenance
regimen can include treating the subject with a dose or doses
ranging from 0.01 .mu.g to 75 mg/kg of body weight per day, e.g.,
70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,
0.001, or 0.0005 mg per kg of bodyweight per day. The maintenance
doses are preferably administered no more than once every 5, 10, or
30 days. Further, the treatment regimen may last for a period of
time which will vary depending upon the nature of the particular
disease, its severity and the overall condition of the patient. In
preferred embodiments the dosage may be delivered no more than once
per day, e.g., no more than once per 24, 36, 48, or more hours,
e.g., no more than once every 5 or 8 days. Following treatment, the
patient can be monitored for changes in his condition and for
alleviation of the symptoms of the disease state. The dosage of the
compound may either be increased in the event the patient does not
respond significantly to current dosage levels, or the dose may be
decreased if an alleviation of the symptoms of the disease state is
observed, if the disease state has been ablated, or if undesired
side-effects are observed.
[0294] The effective dose can be administered in a single dose or
in two or more doses, as desired or considered appropriate under
the specific circumstances. If desired to facilitate repeated or
frequent infusions, implantation of a delivery device, e.g., a
pump, semi-permanent stent (e.g., intravenous, intraperitoneal,
intracisternal or intracapsular), or reservoir may be
advisable.
[0295] Following successful treatment, it may be desirable to have
the patient undergo maintenance therapy to prevent the recurrence
of the disease state, wherein the compound of the invention is
administered in maintenance doses, ranging from 0.01 .mu.g to 100 g
per kg of body weight (see U.S. Pat. No. 6,107,094).
[0296] The concentration of the antagomir composition is an amount
sufficient to be effective in treating or preventing a disorder or
to regulate a physiological condition in humans. The concentration
or amount of antagomir administered will depend on the parameters
determined for the agent and the method of administration, e.g.
direct administration to the eye. For example, eye formulations
tend to require much lower concentrations of some ingredients in
order to avoid irritation or burning of the ocular tissues. It is
sometimes desirable to dilute an oral formulation up to 10-100
times in order to provide a suitable ocular formulation.
[0297] Certain factors may influence the dosage required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. It will also be appreciated that the effective dosage of
the antagomir used for treatment may increase or decrease over the
course of a particular treatment. Changes in dosage may result and
become apparent from the results of diagnostic assays. For example,
the subject can be monitored after administering an antagomir
composition. Based on information from the monitoring, an
additional amount of the antagomir composition can be
administered.
[0298] Dosing is dependent on severity and responsiveness of the
disease condition to be treated, with the course of treatment
lasting from several days to several months, or until a cure is
effected or a diminution of disease state is achieved. Optimal
dosing schedules can be calculated from measurements of drug
accumulation in the body of the patient. Persons of ordinary skill
can easily determine optimum dosages, dosing methodologies and
repetition rates. Optimum dosages may vary depending on the
relative potency of individual compounds, and can generally be
estimated based on EC.sub.50s found to be effective in in vitro and
in vivo animal models.
Therapeutic and Applications for Treating CNS Disorders
[0299] The present invention provides a method for inhibiting
expression levels of an miRNA in a mammalian CNS tissue, preferably
the brain of a mammal. In addition, the invention encompasses
methods for administering a desirable antagomir to a mammal
suffering from a disease, disorder or condition of the CNS. A
mammal can be a rodent, rabbit, primate, human, etc. The antagomir
can be transplanted directly into the defective region of the brain
or into the penumbral tissue, which is a tissue adjacent to a
lesion or defective region. The tissue adjacent to the lesion
provides a receptive environment, similar to that of a developing
brain.
[0300] The compositions of the present invention can be
administered into including, but not limited to ischemic brain,
injured brain, injured spinal cord, and into brain that exhibits
symptoms of degeneration. Administration of the composition into
the mammal can also be performed in combination with growth factors
including, but not limited to brain derived neurotrophic factor
(BDNF), nerve growth factor (NGF), and the like.
[0301] The present invention is based on the discovery that an
antagomir directed to miR-16 efficiently decreased miR-16 levels in
mouse brain when injected locally. For example, local injection of
a small amount of antagomir-16 efficiently reduced expression of
miR-16 in the cortex. This inhibition was specific since the
expression of other miRNAs was not affected and no alteration in
miR-16 levels were measured in the contra-lateral hemisphere that
was injected with PBS. Based on the present disclosure, a skilled
artisan would appreciate that any antagomir presented herein can
decrease expression levels of the corresponding miRNA. The ability
to regulate expression of a desired miRNA in vivo provides a
strategy to regulate target genes that are regulated by a
particular miRNA. As such, the invention encompasses inhibiting an
miRNA in order to increase expression of a target gene that is
regulated by the miRNA. The increase expression of a target gene
can in turn increase the protein levels corresponding to the target
gene.
[0302] Based on the present disclosure, the administered antagomir
decreases a desired miRNA in a cell of a mammal and hence the
increase expression level of a desired target gene. However, the
invention should also encompass a secondary effect as a result of
the targeted decreased expression level of the desired miRNA. For
example, if a target gene is a factor that is secreted from a cell,
than the increased expression of the target gene (e.g. secreted
factor) results in the increased amount of the factor being
secreted. Non-limiting factors include, but are not limited to,
leukemia inhibitory factor (LIF), brain-derived neurotrophic factor
(BDNF), epidermal growth factor receptor (EGF), basic fibroblast
growth factor (bFGF), FGF-6, glial-derived neurotrophic factor
(GDNF), granulocyte colony-stimulating factor (GCSF), hepatocyte
growth factor (HGF), IFN-.gamma., insulin-like growth factor
binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte
chemotacetic protein (MCP-1), mononuclear phagocyte
colony-stimulating factor (M-CSF), neurotrophic factors (NT3),
tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor
necrosis factor (TNF-.beta.), vascular endothelial growth factor
(VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR),
bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stem cell
factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet
derived growth factor-BB (PDGFBB), transforming growth factors beta
TGF.beta.-1 and TGF.beta.-3.
[0303] As such, the present invention also includes a method for
regulating the secretion of a factor from a cell, whereby the
secreted factor can have a beneficial effect on neighboring and/or
distal cells. For example, in neurodegenerative disorders, the
secreted factors can activate endogenous cells to proliferate and
differentiate into cells of the CNS. In another aspect, the factors
secreted by the cell targeted by the antagomir can serve to
activate endogenous stem cells and/or epedymal cells in the brain
and/or spinal cord to proliferate and differentiate into
parenchymal cells, including, but not limited to neurons. Thus, the
present invention includes a method of using an antigomir to
directly and/or indirectly promote repair and plasticity of a CNS
tissue in a mammal including, but not limited to brain and spinal
cord diseases.
[0304] The mode of administration of the compositions of the
invention to the CNS of the mammal may vary depending on several
factors including the type of disease or disorder being treated,
the age of the mammal, whether the compositions have been modified,
or the like. For example, an antagomir can be introduced into the
brain of a mammal by intracerebral administration. The compositions
may be introduced to the desired site by direct injection, or by
any other means used in the art for the introduction of compounds
into the CNS.
[0305] Administration of the compositions of the present invention
can be accomplished using techniques well known in the art as well
as those described herein or as developed in the future.
Exemplified herein are methods for administering compositions of
the invention into a brain of a mammal, but the present invention
is not limited to such anatomical sites. Rather, the composition
can be injected into a number of sites, including the
intraventricular region, the parenchyma (either as a blind
injection or to a specific site by stereotaxic injections), and the
subarachnoid or subpial spaces. Specific sites of injection can be
portions of the cortical gray matter, white matter, basal ganglia,
and spinal cord. Without wishing to be bound to any particular
theory, any mammal affected by a CNS disorder, as described
elsewhere herein, can be so treated by one or more of the
methodologies described herein.
[0306] According to the present invention, administration of the
compositions into selected regions of a mammal's brain may be made
by drilling a hole and piercing the dura to permit the needle of a
microsyringe to be inserted. Alternatively, the compositions can be
injected intrathecally into a spinal cord region.
[0307] The types of diseases which are treatable using the
compositions of the present invention are limitless. For example,
among neonates and children, the compositions may be used for
treatment of a number of genetic diseases of the CNS, including,
but not limited to, Tay-Sachs disease and the related Sandhoff's
disease, Hurler's syndrome and related mucopolysaccharidoses and
Krabbe's disease. To varying extents, these diseases also produce
lesions in the spinal cord and peripheral nerves. In addition, in
neonates and children, treatment of head trauma during birth or
following birth is treatable by introducing the compositions into
the CNS of the individual. CNS tumor formation in children is also
treatable using the methods of the present invention.
[0308] With respect to adult diseases of the CNS, the cells of the
present invention are useful for treatment of Parkinson's disease,
Alzheimer's disease, spinal cord injury, stroke, trauma, tumors,
degenerative diseases of the spinal cord such as amyotropic lateral
sclerosis, Huntington's disease, epilepsy and the like. Treatment
of multiple sclerosis is also comtemplated.
[0309] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
Example 1
Single Stranded Oligonucleotide Agents Inhibited miRNA Activity
[0310] Chemically-stabilized, cholesterol-conjugated
single-stranded RNAs complementary to miRNAs were designed and
synthesized. These single-stranded modified RNAs are referred to
herein as "antagomirs" (see below). To explore the potential of
these synthetic RNAs to silence endogenous miRNAs, antagomir-122
was designed to target miR-122, an miRNA expressed in the liver.
The sequence of antagomir-122 is shown in Table 3. Antagomir-122
was administered to mice by intravenous injection in a small volume
(0.2 ml, 80 mg/kg, 3 consecutive days) and normal pressure.
Administration of antagomir-122 resulted in a striking reduction of
endogenous miR-122 levels as detected by Northern blot analysis
(FIG. 1A). Administration of unmodified single-stranded RNA
(anti-122) had no effect on hepatic miR-122 expression levels (FIG.
1A), while injection of unconjugated, but chemically-stabilized
single-stranded RNAs with partial (pS) or complete (fS)
phosphorothioate backbone and 2'-O-methyl sugar modifications
(anti-122fS, anti-122pS, see Table 3) led to an incomplete effect
(FIG. 1A). The effects of antagomir-122 were found to be specific
as animals injected with a control antagomir-122 derivative that
harbored four mismatch mutations (mm-antagomir-122) had no effect
on miR-122 expression in the liver. Furthermore, expression levels
of miR-let7 and miR-22 were unaffected in antagomir-122 and
mm-antagomir-122 treated mice, suggesting that silencing was
miRNA-specific (FIG. 1B). The structure of the single stranded RNAs
injected into mice is described in Table 3. TABLE-US-00008 TABLE 3
Antagomirs RNA Sequence AL-SQ NO: SEQ ID NO: Anti-122
5'-ACAAACACCAUUGUCACACUCCA-3' 3033 40 Anti-122pS
5'-a.sub.sc.sub.saaacaccauugucacac.sub.su.sub.sc.sub.sc.sub.sa-- 3'
3226 24 Anti-122fS
5'-a.sub.sc.sub.sa.sub.sa.sub.sc.sub.sa.sub.sc.sub.sc.sub.sa.su-
b.su.sub.su.sub.sg.sub.su.sub.sc.sub.sa.sub.sc.sub.sa.sub.sc.sub.su.sub.sc-
.sub.sc.sub.sa-3' 3037 10 antagomir-122
5'-a.sub.sc.sub.saaacaccauugucacacu.sub.sc.sub.sc.sub.sa.sub.s-Chol-3'
3038 5 mm-antagomir-122
5'-a.sub.sc.sub.scacaacacugucacauu.sub.sc.sub.sc.sub.sa.sub.s-Chol-3'
3040 14 antagomir-122(I)
5'-u.sub.sg.sub.sgagugugacaaugguguu.sub.su.sub.sg.sub.su.sub.s-Chol-3'
3223 21 antagomir-122(11)
5'-u.sub.sg.sub.sgaaggugacaguguuguu.sub.su.sub.sg.sub.su.sub.s-Chol-3'
3224 22 antagomir-122(111)
5'-u.sub.sc.sub.sacgcgagccgaacgaac.sub.sa.sub.sa.sub.sa.sub.s-Chol-3'
3230 28 antagomir-16
5'-c.sub.sg.sub.sccaauauuuacgugcug.sub.sc.sub.su.sub.sa.sub.s-Chol-3'
3227 6 antagomir-192 5'-g.sub.spl
g.sub.scugucaauucauaggu.sub.sc.sub.sa.sub.sg.sub.s-Chol-3' 3228 7
antagomir-194
5'-u.sub.sc.sub.scacauggaguugcuguu.sub.sa.sub.sc.sub.sa.sub.s-Chol-3'
3229 8 lower case letters represent 2'-O-methyl modified
nucleotides; subscript `s` represent phosphorothioate linkage;
"Chol" indicates cholesterol conjugate
[0311] MiR-122 is expressed at high levels in hepatocytes with over
50,000 copies per cell (Chang J. et al., RNA Biology 1:2, 106-113,
2004). To determine whether the silencing of miR-122 following
antagomir treatment was caused by stoichiometric duplex formation
between miR-122 and antagomir-122 or by catalytic degradation of
miR-122, total RNA from livers of mice treated with unconjugated
single-stranded anti-miR-122 RNAs (anti-122fS, anti-122pS) or
antagomir-122 were examined under stringent, formamide-containing
denaturing conditions (FIG. 1C). No difference in miR-122 levels
could be detected between PBS and unconjugated anti-miR-122
RNA-treated livers, showing that the decrease in miR-122 levels
observed under non-stringent conditions was not caused by
degradation, but instead by the formation of miR-122/RNA duplexes.
In contrast, miR-122 remained undetectable in livers of mice
treated with antagomir-122. These data suggest that the silencing
of miRNA-122 in livers of mice treated with antagomir-122 was due
to degradation of the miRNA, and the ability of antagomir-122, but
not unconjugated anti-122 RNAs, to result in miR-122 degradation
may be due to efficient delivery of antagomirs to hepatocytes.
[0312] To determine the dose of antagomir-122 that can completely
silence miR-122, mice were injected with 80, 160 or 240 mg/kg
bodyweight antagomir-122 and miR-122 expression levels were
measured. The highest dose (240 mg/kg bodyweight) resulted in a
complete loss of miR-122 signal and was subsequently used for all
other experiments (FIG. 2A).
[0313] The duration of silencing with antagomir-122 was also
measured. Levels of miR-122 were undetectable for as long as 23
days post-injection (FIG. 2B), indicating that silencing of miRNAs
using antagomirs is long lasting. The injected antagomirs were well
tolerated even during the course of the prolonged treatment; no
alterations in bodyweight or serum markers of liver toxicity
(alanine aminotransferase) were detected. To test the
bioavailability of antagomirs in vivo and their ability to silence
miRNA expression in different tissues, mice were injected with
antagomir-16 directed to miR-16, which is abundantly expressed in
all tissues (miR-16 is predicted to target one or both of Activin
type II receptor gene, which is involved in TGFbeta signaling, and
Hox-A51 (John et al., PLoS Biology 2:1862-1878, 2004; correction in
PLoS Biology 3:1328, 2005)). Tissues were harvested one day after
the final injection, and miRNA expression levels were compared to
PBS-injected mice. Northern blot analysis revealed that expression
of miR-16 was efficiently silenced in all tissues tested except
brain (FIG. 3A). Antagomir-16 did not affect the expression of the
89 nt precursor of miR-16 as detected in bone marrow. The
bioavailability of antagomir-16 was also assessed by Northern
blotting in the above mentioned tissue samples. In concordance with
the ability to silence miR-16 levels, significant levels of
antagomir-16 were detected in all tissues except brain (FIG. 3B).
Together, these data demonstrate that antagomirs achieve broad
biodistribution and can efficiently silence miRNAs in most tissues
in vivo.
[0314] Many miRNA genes have been found to be located in close
proximity and to be coordinately transcribed. These polycistronic
miRNA genes are transcribed to generate long primary transcripts
(pri-miRNAs), which are processed by multiple enzymes in the
nucleus and cytoplasm to generate the mature miRNA. To investigate
if antagomirs targeting polycistronic miRNAs retain their target
specificity with no effect on the expression of neighboring miRNAs,
mice were injected with antagomirs targeting either miR-192 or
miR-194 of the bicistronic cluster miR-192/194. Administration of
antagomir-192 into mice resulted in silencing of miR-192 in liver
and kidney, with no effect on the expression levels of miR-194.
Conversely, injection of antagomir-194 into mice abolished miR-194
expression but had no demonstrable effect on the miR-192 levels
compared to PBS-injected mice. These data demonstrate that
antagomirs have the ability to differentially silence specific
miRNAs that derive from the same primary transcript.
[0315] To test whether silencing of an miRNA can cause a
corresponding increase in target protein and possibly mRNA levels,
the expression of aldolase A, a gene that is repressed in
hepatocytes and predicted to be a target of miR-122, was examined.
The aldolase-A mRNA has a conserved nucleotide sequence with
perfect sequence complementarity to miR-122 between nucleotides 29
and 36 downstream of the open reading frame. Aldolase-A expression
was increased 4-5 fold in livers of mice injected with
antagomir-122 compared to a scrambled control (mm-antagomir-122).
This regulation was observed in multiple experiments and different
time points after injection. The target was also independently
confirmed by cloning the 3'UTR Aldolase-A downstream of the
luciferase open reading frame and cotransfecting this vector with
control miRNAs (miR-124 (5'-UAAGGCACGCGGUGAAUGCCA-3 SEQ ID NO:41);
see Krek et al., Nature Genetics 37:495-500, 2005, and Lim et al.,
Nature 433:769-773, 2005) and miR-192) and miR-122 into HEK293
cells, which lack miR-122 expression. Cotransfection of miR-122
resulted in a significant reduction in luciferase activity compared
to miR-124 and miR-192 transfected cells. Together, these data
indicate that aldolase-A is a physiological target of miR-122.
[0316] The upregulation of aldolase-A in mice treated with
antagomir-122 demonstrates functional silencing of this miRNA.
Aldolase-A is a housekeeping gene expressed in all cells. This gene
is produced in large amounts in muscle where it can be as much as
5% of total cellular protein. In adult liver, aldolase-A expression
is repressed and aldolase-B is produced. Conversely,
dedifferentiated hepatocytes and transformed liver cells have
increased aldolase-A expression levels and can even replace
aldolase-B. Expression of miR-122 shows an inverse relationship
with aldolase-A expression, with highest levels in differentiated
adult hepatocytes and complete absence in undifferentiated cells
such as HepG2. In contrast, the mRNA levels of aldolase-B, which
lacks miR-122 target sites, were unaffected by antagomir-122. These
findings provide non-genetic, pharmacologic evidence in mammals
that microRNAs define tissue-specific gene expression.
[0317] To identify other genes regulated by miR-122, we carried out
gene expression analysis using Affymetrix.TM. arrays (Affymetrix,
Inc., Santa Clara, Calif.) in livers from mm-antagomir-122 and
antagomir-122 treated mice. We identified 493 gene transcripts that
were up-regulated (.gtoreq.1,4-fold) and 365 transcripts
(.ltoreq.1.4-fold) that were down-regulated in antagomir-122
treated mice compared to controls. The regulation of genes that
were up-regulated in antagomir-122-treated livers was confirmed by
RT-PCR. Strikingly, these included those members of gene families
that are usually repressed in hepatocytes, including alodolase A
(aldo-A), N-Myc downstream regulated gene (Ndrg3), IQ motif
containing GTPase activating protein-1 (Iqgap1). MiR-122 could
therefore contribute to the maintenance of the adult liver
phenotype, as previously suggested for two other specific miRNAs
(Lim et al., Nature 433:769-773, 2005). To assess further the motif
contents of significantly up- and down-regulated genes, we analyzed
the 3'UTR sequences of 9554 mRNAs. Of these, 142 mRNAs were
significantly up-regulated and had a fold-change of at least 1.4.
We observed a highly significant 2,6-fold increase in the
probability to have at least one miR-122 nucleus in the 3'UTR of
up-regulated genes compared to genes with no change in mRNA levels.
Interestingly, the majority of the miR-122 nuclei in up-regulated
genes had not been detected by previous prediction methods (see,
e.g., Lewis et al., Cell 120:15-20, 2005), indicating that the
number of direct miRNA targets is significantly larger then
previously estimated.
[0318] To experimentally validate the link between repression and
the presence of miR-122 nuclei matches within the 3'UTR, we cloned
the 3' UTR of four genes repressed by antagomir-122 and containing
a miR-122 nucleus into a luciferase reporter system. When
co-transfected with miR-122, all reporters exhibited significant
repression relative to co-transfections with control miRNA
(si-124), suggesting that miR-122 binding to its nucleus
contributes directly to mRNA repression. Surprisingly, we also
observed that the probability for down-regulated genes to harbor a
miR-122 nucleus was reduced by almost the same factor of 2,7-fold.
To further analyze if over-representation and under-representation
of miR-122 nuclei is specific, we analyzed the abundance of all
4096 possible 6-mer motifs across down-, up-, and "no change"
transcripts. When comparing up-regulated versus no change genes,
the miR-122 nucleus (CACTCC) was the most significantly
over-represented 6-mer. Similarly, the miR-122 nucleus was within
the top 0.5% of under-represented motifs for down-regulated
transcripts, indicating an evolutionary tendency of down-regulated
genes to lack binding sites for miR-122. These results indicate
that up-regulated mRNAs are directly targeted and repressed by
miR-122, but also that a significant number of down-regulated genes
are likely to be either directly or indirectly activated by
miR-122.
[0319] To assess the functional significance of altered gene
regulation by miR-122 we analyzed the functional annotation of
regulated genes for enrichment in Gene Ontology categories (see
Methods). The top ranking functional category was "cholesterol
biosynthesis" with a p-value of 1.6.times.10.sup.-11 and was found
for gene transcripts down-regulated by antagomir-122. The
expression of at least 13 genes involved in cholesterol
biosynthesis was decreased between 1.4 to 2,3-fold in antagomir-122
treated mice; some of these were confirmed by RT-PCR.
Interestingly, mice injected with an adenovirus expressing miR-122
(Ad-122) increased expression of some of these genes. One of these
gene transcripts down-regulated by antagomir-122 treatment was
HMG-CoA-reductase (Hmgcr), a rate-limiting enzyme of endogenous
cholesterol biosynthesis and the target for statin-based drugs. We
measured the enzymatic activity of Hmgcr in liver extracts and
found a .about.45% reduction in Hmgcr activity in antagomir-122 vs.
mm-antagomir-122 treated mice (9.7.+-.1.0 versus 17.2.+-.2.3
pmol/mg microsomal protein/min, respectively; n=5, P=0.02).
Consistent with this effect on Hmgcr activity, plasma cholesterol
levels were decreased >40% in antagomir-122 treated animals
while there was no detectable effect on plasma free fatty acids
(FFA), triglyceride, bile acid and glucose levels. No decrease in
plasma cholesterol was observed with antagomir-192, -194 and -16,
showing that together with the absence of effects by
mm-antagomir-122, the effects of antagomir-122 are sequence
specific and unrelated to the use of a cholesterol-conjugated
oligonucleotide per se. Reduced plasma cholesterol levels in
antagomir-122 treated mice lasted for at least 2 weeks. Together,
these data demonstrate that miR-122 is a regulator of the
cholesterol biosynthetic pathway.
[0320] To test whether double stranded antagomirs were effective,
six-week old female C57BL/6 mice were injected via the tail vein
with 80 mg/kg/day on three consecutive days with either PBS, or
compounds AP-3018, -3019, -3020 in a total volume of 0.2 ml. The
liver was harvested 24 hrs after the last injection and total RNA
was isolated using Trizol (Invitrogen). 10 .mu.g of total RNA was
run on formamide containing polyacrylamide gels, blotted and probed
for miR-122 using a 32P-labeled antisense oligo. Two different
autoradiography exposures are shown (see FIG. 12). Each lane
represents an individual animal. Double stranded antagomirs were
able to reduce mircoRNA levels.
[0321] Our data demonstrate that antagomirs are effective
inhibitors of miRNAs in vivo. Silencing of miR-122 by antagomirs
allowed us to study gene regulation by a tissue-specific miRNA in
vivo. Of the genes that were up-regulated, only 12 genes (including
AldoA, citrate synthase and Iqgap1) had previously been predicted
using bioinformatic approaches (Krek et al., Nat. Genet.
37:495-500, 2005). Since 52% of all up-regulated genes have at
least one miR-122 nucleus in their 3'UTR sequence this indicates
that they are likely direct targets. Notably, silencing of miR-122
also led to a reduction of a significant number of genes. We found
that these genes have a drastically reduced probability to contain
a miR-122 nucleus in their 3'UTR.
[0322] Methods
[0323] Synthesis of antagomirs RNAs were synthesized using
commercially available
5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-(2-cyanoethyl-N,N--
diisopropyl) RNA phosphoramidite monomers of 6-N-benzoyladenosine
(ABz), 4-N-benzoylcytidine (CBz), 2-N-isobutyrylguanosine (GiBu),
and uridine (U), according to standard solid phase oligonucleotide
synthesis protocols (Damha and Ogilvie, Methods Mol. Biol.
20:81-114, 1993). For antagomirs, i.e., cholesterol conjugated
RNAs, the synthesis started from a controlled-pore glass solid
support carrying a cholesterol-hydroxyprolinol linker (Manoharan et
al., U.S. Pat. Appl. Publ. 20050107325). Antagomirs with
phosphorothioate backbone at a given position were achieved by
oxidation of phosphite with phenylacetyl disulfide (PADS) during
oligonucleotide synthesis (Cheruvallath et al., Nucleosides
Nucleotides 18:485-492, 1999). After cleavage and de-protection,
antagomirs were purified by reverse-phase high-performance liquid
chromatography, while the unconjugated RNA oligonucleotides were
purified by anion-exchange high-performance liquid chromatography.
Purified oligonucleotides were characterized by ES mass
spectrometry and capillary gel electrophoresis.
[0324] Animals. All animal models were maintained in C57B1/6J
background on a 12 hours light/dark cycle in a pathogen-free animal
facility at Rockefeller University. Six week old mice received, on
one to three consecutive days, tail vein injections of saline or
different RNAs. RNAs were administered at doses of 80 mg/kg body
weight in 0.2 ml per injection. Measurements of miRNA levels in
tissues were performed 24 h after the last injection unless
indicated otherwise. Tissues were harvested, snap frozen and stored
at -80.degree. C.
[0325] Generation of recombinant adenovirus. The recombinant
adenovirus used to express miR-122 (Ad-122) was generated by PCR,
amplifying a 344 bp miRNA precursor sequence with primers
5'-AGTCAGATGTACAGTTATAAGCACAAGAGGACCAG-3' (SEQ ID NO:42) and
5'-TTATTCAAGATCCCGGGGCTCTTCC-3' (SEQ ID NO:43). The fragment was
cloned into vector Ad5CMV-KnpA. Ad-EGFP (ViraQuest, North Liberty,
Iowa) was used as a control. Mice were infected with
1.times.10.sup.9 pfu/mouse by tail vein injection.
[0326] Gene expression analysis. Total RNA of mice treated with
antagomirs or recombinant adenovirus was isolated three days after
treatment. RNA was pooled from four animals for each group. The
integrity of the RNA sample was assessed by denaturing formamide
gel analysis. First strand cDNA synthesis was completed with total
RNA (30 .mu.g) cleaned with RNAeasy columns (Qiagen, Valencia,
Calif.) and the Superscript Choice cDNA synthesis protocol
(Invitrogen, Carlsbad, Calif.), except and HPLC purified
T7-promoter-dT30 primer (Proligo LLC, Boulder, Colo.) was used to
initiate the first strand reaction. Biotin labeled cRNA was
synthesized from T7 cDNA using the RNA transcript labeling kit
(Enzo Biochem, Farmingdale, N.Y.), supplemented with biotin 11-CTP
and biotin-UTP (Enzo Biochem, Farmingdale, N.Y.) as specified by
the Affymetrix protocol. The sample was cleaned with an RNAeasy
column (Qiagen, Valencia, Calif.) to remove free nucleotides and
then quantitated spectrophotometrically. Biotin-labeled cRNA was
fragmented and hybridized to Mouse et 430 arrays (Affymetrix, Inc.,
Santa Clara, Calif.) according to the manufacturer's manual with a
final concentration of fragmented cRNA of 0.05 .mu.g/.mu.l. The
arrays were scanned using a Hewlett Packard confocal laser scanner
and analyzed using ArrayAssist Lite and Affymetrix.RTM. Microarray
Suite v.5 (MAS5) software.
[0327] Northern blotting analysis. Total RNA was isolated using the
Trizol.RTM. reagent (Invitrogen, Carlsbad, Calif.) and ethanol
precipitation. RNA was separated at 45 mA on 14%-polyacrylamide
gels that contained 8 M urea and 20% fommamide. Antisense probes
were designed according to the "microRNA registry"
(Griffiths-Jones, NAR 32:D109-D111, 2004).
[0328] RT-PCR. Extraction of total RNA, synthesis of cDNA, and PCR
were carried out as described in Shih et al. (Proc. Natl. Acad.
Sci. U.S.A. 99:3818-3823, 2002).
[0329] Assay of luciferase activity. The mouse full length
adolase-A 3'UTR was PCR-amplified using the following primers: 5'
d-(CCAGAGCTGAACTAAGGCTGCTCCA)-3' (SEQ ID NO:44) and 5'
d-(CCCCTTAAATAGTTGTTTAT TGGCA)-3' (SEQ ID NO:9) and cloned
downstream of the stop codon in pRL-TK (Promega, Madison, Wis.).
HEK293 cells were cultured in 24-well plates and each transfected
with 50 ng of pRL-TK (Rr-luc), 50 ng of pGL3 control vector
(Pp-luc) (Promega, Madison, Wis.) and 200 ng of double-stranded
siRNA (Dharmacon, Lafayette, Colo.). Cells were harvested and
assayed 24-30 h post-transfection.
[0330] 3' UTR sequences and mapping of array probes to transcripts.
We extracted mouse 3' UTRs using the Refseq data set (Pruitt et
al., Nucleic Acids Res. 33:D501-D504, 2005). 17264 3' UTR sequences
of at least 30 nucleotides in length were obtained. Affymetrix
probe identifiers were assigned to the Refseq transcripts by using
a mapping provided by Ensembl software (Hubbard et al., Nucleic
Acids Res. 33 Database issue:D447-D453, 2005). When only one probe
identifier mapped to a transcript, the significance call for a fold
change and the fold change itself, as provided by the Affymetrix
software, was taken at face value. When more than one probe
identifier mapped to a transcript, we insisted that the
significance call was consistent for all probes. Transcripts were
discarded otherwise. The fold change assigned to a transcript was
the average of all probes that mapped to the transcript. Finally, a
cut-off of 0.5 in the logarithm (base 2) of fold changes was
applied.
[0331] Gene Ontology analysis. Refseq identifiers were mapped to
MGI identifiers using a map provided by Ensembl software (Hubbard
et al., Nucleic Acids Res. 33 Database issue:D447-D453, 2005). We
then used the program FuncAssociate (Castillo-Davis and Hartl,
Bioinformatics 19:891-892, 2003) with default settings to search
for overrepresented Gene Ontology terms. Results were sorted by LOD
scores. Independently, we obtained very similar results with the
program GeneMerge v.1.2 and by applying a conservative Bonferroni
correction for multiple testing.
[0332] HMG-CoA reductase (HMGR) activity assay. Hepatic microsomal
HMGR activity was assayed by a method modified from a previously
published procedure (Nguyen, et al., J. Clin. Invest. 86:923-931,
1990). Briefly, hepatic microsomal protein extracts were
preincubated with an NADPH-generating system (3.4 mM NADP+/30 mM
glucose 6-phosphate/0.3 units of glucose-6-phosphate dehydrogenase)
in buffer (50 mM K.sub.2PHO4/70 mM KCl/10 mM DTT/30 mM EDTA, pH
7.4). The reaction was started with the addition of 15 .mu.l
.sup.14C-labeled substrate ([.sup.14C]HMG CoA, (Amersham,
Piscataway, N.J.)). The mixture was incubated for 15 min. and
stopped with 15 .mu.l 6 M HCl. [.sup.3H]mevalonolactone and
unlabeled mevalonolactone were added for recovery standard and
product marker, respectively. After lactonization the products were
extracted with ether and separated by TLC on Silica Gel 60 plates
(VWR Scientific, West Chester, Pa.) with benzene/acetone (1:1,
vol/vol) as the solvent system. The immediate product
(.sup.14C-labeled mevalonolactone) was quantitated by scintillation
spectrometry.
[0333] Statistical analysis. Results are given as mean.+-.s.d.
Statistical analyses were performed by using Student's t-test, and
the null hypothesis was rejected at the 0.05 level.
Example 2
Trizol.RTM. Reagent and Ethanol can be Used to Precipitate a
miR-122/Antagomir-122 Duplex
[0334] A Trizol.RTM. (Invitrogen, Carlsbad, Calif.) protocol was
modified for precipitation of a miR-122/antagomir 122 duplex. A
duplex containing a synthetic miR-122 (22 nt, Dharmacon, Lafayette,
Colo.) and antagomir-122 molecule was formed by incubating equal
amounts of miR-122 and antagomir-122 in water for 1 min. at
95.degree. C. followed by an incubation at 37.degree. C. for 1 hr.
The duplex was then added to the aqueous phase of a
Trizol.RTM./liver extract and aliquots were subjected to different
precipitation methods (10 min. at room temperature with 50% or 80%
isopropanol followed by 10 min. centrifugation at 13,200 rpm at
4.degree. C.; or 30 min. at -80.degree. C. with 70% ethanol and 0.5
M ammonium acetate or 0.08 M sodium acetate followed by 20 min.
centrifugation at 13,200 rpm at 4.degree. C.). The precipitates
were washed once with 85% ethanol, dissolved in water, separated on
a 14% sequencing gel and visualized using ethidium bromide. The
respective input of the duplex was loaded in comparison (1.4
mg/lane).
[0335] The duplex did precipitate in (i) 80% isopropanol, (ii) in
70% ethanol/0.5M NH.sub.4-Acetate, and (iii) in 70% ethanol/0.08M
Na-acetate. The duplex would not precipitate in 50% isopropanol,
which follows the conventional Trizol.RTM. protocol.
Example 3
Trizol.RTM. Did not Precipitate a miR/Antagomir Duplex in the
Absence of Ethanol or Isopropanol
[0336] Liver tissue was homogenized in Trizol.RTM., aliquoted into
eppendorf tubes (1 ml volume each) and preformed duplex
miR-122/antagomir-122 or miR-16/antagomir-16 was added. Samples
were vortexed and left at room temperature for 10 minutes. 200 ml
of chloroform were added and samples vortexed for 2 min at room
temperature, followed by centrifugation at 13,200 rpm at room
temperature for 15 minutes. 400 ml of the supernatant were added to
1 ml 100% ethanol and 40 ml 3 M sodium acetate, pH 5.2. After 30
min. at -80.degree. C. samples were centrifuged for 20 min. at
4.degree. C. Precipitates were washed with 85% ethanol, dissolved
in water, separated on a 14% sequencing gel and visualized using
ethidium bromide. The input was loaded for comparison (5 mg per
lane). The miR/antagomir duplex was not precipitated by this
Trizol.RTM. protocol, which lacked ethanol or isopropanol, although
a faint signal indicated some recovery of single-stranded miR.
[0337] In a similar assay, liver/Trizol.RTM. homogenates were
processed exactly as described, but 4 mg each of (i) miR-122, (ii)
antagomir-122, (iii) anti122pS, or (iv) miR-122 (4 mg) and
antagomir-122 (4 mg) together were added to the homogenates. After
the incubation and precipitation steps, only miR-122 was isolated.
This was the result whether miR-122 alone was added to the
homogenate, or whether miR-122 was added in combination with
antagomir-122.
[0338] When preformed duplex of miR-122/antagomir-122 (4 mg of
each) were added to the liver/Trizol.RTM. homogenates, miR-122, but
not antagomir-122, was isolated from the preformed duplex.
[0339] To further test the Trizol.RTM. precipitation protocol,
total liver RNA was isolated (using the protocol), and then the RNA
was dissolved in water. 40 mg of the isolated RNA were incubated in
a total volume of 50 ml water with the increasing amounts of
antagomir-122 for 5 min. at 65.degree. C. followed by 2 h at
37.degree. C. and 30 min. at room temperature. The amounts of
antagomir-122 tested were 6.4 pg, 320 pg, 16 ng, 0.8 .mu.g, 40
.mu.g, and 120 .mu.g. 1 ml of Trizol.RTM. was added to the samples
and Trizol.RTM. extraction was performed again as described above.
The precipitates were dissolved in 60 ml water, and then 30 ml of
each sample was separated on 14% polyacrylamide gels with or
without 20% formamide. miR-122 was then detected using Northern
blotting.
[0340] The miR-122 was precipitated and detected by gel
electrophoresis in the presence of formamide regardless of the
amount of antagomir-122 added to the precipitation mix. When 40
.mu.g or 120 .mu.g were used in the precipitation mix, a duplex
between miR-122 and antagomir was visible when analyzed on a
sequencing gel. Minor amounts of antagomir were always retrieved by
the Trizol.RTM. protocol.
[0341] To test the precipitation protocol with smaller amounts of
RNA, total RNA was isolated from liver or kidney using the
Trizol.RTM. protocol and then the RNA was dissolved in water. 10 mg
of RNA were incubated for 3 h at 37.degree. C. in a total volume of
50 ml water together with 20 mg antagomir-122. The kidney RNA,
which does not contain endogenous miR-122, was spiked with 20
fmoles of synthetic miR-122. 1 ml of Trizol.RTM. was added and then
Trizol.RTM. extraction was performed again as described above.
Precipitates were dissolved in 30 ml water and separated in a 14%
polyacrylamide gel containing 20% formamide. First, miR-122 was
detected using Northern blotting, then the membrane was re-probed
against antagomir-122, which detected the presence of the
antagomir-122. Incubation with antagomir-122 did not alter the
miR-122 signal detected on the gel.
[0342] Mice were administered 80 mg/kg/day antagomir-122 or a
scrambled control (mm-antagmir122) via tail-vein on three separate
days. Livers were harvested at days 3, 6, 9, 13, and 23
post-injection, and subjected to Trizol.RTM. isolation as described
above. Approximately 50 mg liver were homogenized in 1 ml
Trizol.RTM.. Analysis by Northern blot indicated that antagomir-122
could be detected for at least 23 days post-injection.
Example 4
A bDNA Lysis Protocol Allows Quantitative Isolation of
miR-122/Antagomir-122 Duplexes and miR-122 Single-Stranded
Molecules
[0343] 100 mg liver were sonicated in 2 ml T+C (Epicentre.RTM.,
Madison, Wis.) in the presence of 350 mg proteinase K. 100 ml of
the homogenate was supplemented with either miR-122/antagomir-122
duplexes (8 mg per lane), miR-122 (4 mg per lane) or antagomir-122
(4 mg per lane). 200 ml 1.times.STE-buffer was added, then 200 ml
of phenol, pH 4 or pH 8. Samples were vortexed for 30 sec and left
on ice for 2 min. After centrifugation for 10 min at 13,200 rpm
(4.degree. C.), 280 ml of the supernatant was added to 900 ml 100%
ethanol and incubated for 90 min at 90.degree. C. RNA was
precipitated for 10 min at 13,200 rpm, washed once with 85%
ethanol, dissolved in water and separated on a sequencing gel.
miR-122/antagomir-122 duplexes and miR-122 (but not antagomir-122)
were detected by ethidium bromide staining. The duplexes and
miR-122 were successfully isolated with phenol at pH4 and at pH
8.
Example 5
Antagomir-122 Caused a Decrease in miR-122 Levels In Vivo
[0344] To test the effect of antagomir-122 on miRNA levels in vivo,
mice were administered PBS, mm-antagomir-122 or antagomir-122 (80
mg/kg/day) via tail-vein injection for 3 subsequent days. Livers
were harvested 24 hrs after the last injection and .about.20 mg of
tissue was sonicated in 1 ml T+C (Epicentre.RTM., Madison, Wis.)
and proteinase K and processed as described in example 4.
Precipitated RNA was dissolved in water and analyzed on 14%
polyacrylamide gels that contained 20% formamide. Northern blotting
was performed for miR-122 and let7 miRNAs. Mice injected with
antagomir-122 revealed a striking decrease in the amount of miR-122
isolated from liver, as compared to mice injected with PBS or
mm-antagomir-122 (FIG. 4). The level of let7 miRNA isolated from
liver was similar in the three test groups.
[0345] To test the long-term effect of antagomir-122 on miRNA
levels in vivo, mice were administered PBS, mm-antagomir-122 or
antagomir-122 (80 mg/kg/day) via tail-vein injection for 3
subsequent days. Livers were then harvested 72 h, 10 days or 27
days after the last injection. Tissues were processed as described
in example 4, and Northern blot analysis revealed a marked
reduction of miR-122 in the antagomir-treated mice, even 27 days
post-injection. As a further note, the Northern blot analysis
revealed an RNA of higher molecular weight specifically detected by
the miR-122 probe. This molecule is most likely an miR-122
precursor.
Example 6
Dose Response for Antagomir-122
[0346] Three groups of mice n=3, C57/B16, female, 6-8 weeks old)
were injected with antagomir-122 at a dose of 3.times.20,
3.times.40 or 3.times.80 mg/kg body weight in a total volume of 0.2
ml on three consecutive days. Phosphate buffered saline (PBS)
injected mice (3.times.0.2 ml) served as controls. Mice were
sacrificed on day 4 and total RNA was extracted from livers for
Northern blotting. Mir-122 expression was analyzed following
Northern blotting using a .sup.32P-labeled oligonucleotide with
complementary sequence to miR-122. Expression levels of aldolase A,
a validated target gene of miR-122, were analyzed by RT-PCR. Gapdh
served as a loading control, Gapdh-RT indicates the absence of
reverse transcriptase as a control for DNA contamination.
[0347] Results demonstrate that reduction of miR-122 can be
achieved at a dose of 3.times.20 mg/kg bodyweight. A >90%
reduction in miR-122 levels is required to detect a significant
increase in aldolaseA expression (FIG. 13).
Example 7
Mismatch Control of Antagomir-122 Activity
[0348] Five groups of mice n=3, C57/B16, female, 6-8 weeks old)
were injected with antagomir-122 harboring 4, 3, 2, 1 or no
nucleotide exchange in the sequence of antagomir-122 (4 mm, 3 mm, 2
mm, 1 mm, antagomir-122, respectively). Mice were injected at a
dose of 3.times.80 mg/kg body weight in a total volume of 0.2 ml on
three consecutive days. Mice were sacrificed on day 4 and total RNA
was extracted from livers for Northern blotting. Mir-122 expression
was analyzed by Northern blotting using a .sup.32P labeled
oligonucleotide with complementary sequence to miR-122.
[0349] Results demonstrate that 4, 3 (data not shown), or 2
mismatches in antagomir-122 had no effect on miR-122 expression
levels. A single mismatch resulted in a 20-30% reduction in miR-122
expression. These data demonstrate that antagomirs have exquisite
target specificity (FIG. 14). TABLE-US-00009 4 mm:
oA*oC*oAoCoAoCoAoAoCoAoCoUoGoUoCoAo (SEQ ID NO:1)
CoAoUoU*oC*oC*oA*-CHOL 3 mm: oA*oC*oAoAoAoCoAoAoCoAoCoUoGoUoCoAo
(SEQ ID NO:35) CoAoUoU*oC*oC*oA*-CHOL 2 mm:
oA*oC*oAoAoAoCoAoCoCoAoCoUoGoUoCoAo (SEQ ID NO:36)
CoAoUoU*oC*oC*oA*-CHOL 1 mm: oA*oC*oAoAoAoCoAoCoCoAoUoUoGoUoCoAo
(SEQ ID NO:37) CoAoUoU*oC*oC*oA*-CHOL Antagomir-122
oA*oC*oAoAoAoCoAoCoCoAoUoUoGoUoCoAo (SEQ OD NO:11)
CoAoCoU*oC*oC*oA*-CHOL
Example 8
Length Effect on Antagomir-122 Activity
[0350] Six groups of mice (n=3, C57/B16, female, 6-8 weeks old;
only n=2 shown) were injected with antagomir-122, which differed in
length between 25, 23 (antagomir-122), 21, 19 and 17 bp. Mice were
injected at a dose of 3.times.80 mg/kg body weight in a total
volume of 0.2 ml on three consecutive days. Mice were sacrificed on
day 4 and total RNA was extracted from livers for Northern
blotting. Mir-122 expression was analyzed by Northern blotting
using a .sup.32P-labeled oligonucleotide with complementary
sequence to miR-122.
[0351] Results demonstrate that an additional nucleotide at both 3'
and 5' ends of antagomir-122 (25-mer), or a deletion at either end
(21-mer) has no effect of the ability of antagomirs to silence
miR-122. Further shortening of antagomirs (19- and 17-mers) result
in a loss of antagomir activity (FIG. 15). TABLE-US-00010
Antagomir-122 (23-mer) oA*oC*oAoAoAoCoAoCoCoAoUoUoGoUoCoAo (SEQ ID
NO:5) CoAoCoU*oC*oC*oA*-CHOL 25-mer:
oC*oA*oCoAoAoAoCoAoCoCoAoUoUoGoUoCo (SEQ ID NO:31)
AoCoAoCoUoC*oC*oA*oC*-CHOL 21-mer:
oC*oA*oAoAoCoAoCoCoAoUoUoGoUoCoAoCo (SEQ ID NO:32)
AoC*oU*oC*oC*-CHOL 19-mer: oA*oA*oAoCoAoCoCoAoUoUoGoUoCoAoCo (SEQ
ID NO:33) A*oC*oU*oC*-CHOL 17-mer:
oA*oA*oCoAoCoCoAoUoUoGoUoCoAoC*oA*o (SEQ ID NO:34) C*oU*-CHOL
Example 9
Synthesis of Antagomirs
[0352] Step 1. Oligonucleotide Synthesis
[0353] All oligonucleotides were synthesized on an AKTAoligopilot
synthesizer or on an ABI 394 DNA/RNA synthesizer. Commercially
available controlled pore glass solid supports (rU-CPG, 2'-O-methly
modified rA-CPG and 2'-O-methyl modified rG-CPG from Prime
Synthesis) or the in-house synthesized solid support
hydroxyprolinol-cholesterol-CPG were used for the synthesis. RNA
phosphoramidites and 2'-O-methyl modified RNA phosphoramidites with
standard protecting groups
(5'-O-dimethoxytrityl-N-6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O--
-N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-O--N,N-
'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethylsilyl-guanosine-3'-O--
-N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O--N,N'-diisoprop-
yl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N-6-benzoyl-2'-O-methyl-adenosine-3'-O--N,N'-diisopr-
opyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-O-methyl-cytidine-3'-O--N,N'-diisopropy-
l-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N2-isobutryl-2'-O-methyl-guanosine-3'-O--N,N'-diisop-
ropyl-2-cyanoethylphosphoramidite, and
5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O--N,N'-diisopropyl-2-cyanoet-
hylphosphoramidite) were obtained from Pierce Nucleic Acids
Technologies and ChemGenes Research. The Quasar 570 phosphoramidite
was obtained from Biosearch Technologies. The
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-inosine-3'-O--N,N'-diisoprop-
yl-2-cyanoethylphosphoramidite was obtained from ChemGenes
Research. For the syntheses on AKTAoligopilot synthesizer, all
phosphoramidites were used at a concentration of 0.2 M in
CH.sub.3CN except for guanosine and 2'-O-methyl-uridine, which were
used at 0.2 M concentration in 10% THF/CH.sub.3CN (v/v).
Coupling/recycling time of 16 minutes was used for all
phosphoramidite couplings. The activator was 5-ethyl-thio-tetrazole
(0.75 M, American International Chemicals). For the PO-oxidation,
50 mM iodine in water/pyridine (10:90 v/v) was used and for the
PS-oxidation 2% PADS (GL Synthesis) in 2,6-lutidine/CH.sub.3CN (1:1
v/v) was used. For the syntheses on ABI 394 DNA/RNA synthesizer,
all phosphoramidites were used at a concentration of 0.15 M in
CH.sub.3CN except for 2'-O-methyl-uridine, which was used at 0.15 M
concentration in 10% THF/CH.sub.3CN (v/v). Coupling time of 10
minutes was used for all phosphoramidite couplings. The activator
was 5-ethyl-thio-tetrazole (0.25 M, Glen Research). For the
PO-oxidation, 20 mM iodine in water/pyridine (Glen Research) was
used and for the PS-oxidation 0.1M DDTT (AM Chemicals) in pyridine
was used. Coupling of the Quasar 570 phosphoramidite was carried
out on the ABI DNA/RNA synthesizer. The Quasar 570 phosphoramidite
was used at a concentration of 0.1M in CH.sub.3CN with a coupling
time of 10 mins. The activator was 5-ethyl-thio-tetrazole (0.25 M,
Glen Research) and 0.1M DDTT (AM Chemicals) in pyridine was used
for PS oxidation.
[0354] Step 2. Deprotection of Oligonucleotides
[0355] A. Sequences Synthesized on the AKTAoligopilot
Synthesizer
[0356] After completion of synthesis, the support was transferred
to a 100 mL glass bottle (VWR). The oligonucleotide was cleaved
from the support with simultaneous deprotection of base and
phosphate groups with 40 mL of a 40% aq. methyl amine (Aldrich) 90
mins at 45.degree. C. The bottle was cooled briefly on ice and then
the methylamine was filtered into a new 500 mL bottle. The CPG
washed three times with 40 mL portions of DMSO. The mixture was
then cooled on dry ice.
[0357] In order to remove the tert-butyldimethylsilyl (TBDMS)
groups at the 2' position, 60 mL triethylamine trihydrofluoride
(Et3N--HF) was added to the above mixture. The mixture was heated
at 40.degree. C. for 60 minutes. The reaction was then quenched
with 220 mL of 50 mM sodium acetate (pH 5.5) and stored in the
freezer until purification.
[0358] B. Sequences Synthesized on the ABI DAN/RNA Synthesizer
[0359] After completion of synthesis, the support was transferred
to a 15 mL tube (VWR). The oligonucleotide was cleaved from the
support with simultaneous deprotection of base and phosphate groups
with 7 mL of a 40% aq. methyl amine (Aldrich) 15 mins at 65.degree.
C. The bottle was cooled briefly on ice and then the methylamine
was filtered into a 100 mL bottle (VWR). The CPG washed three times
with 7 mL portions of DMSO. The mixture was then cooled on dry
ice.
[0360] In order to remove the tert-butyldimethylsilyl (TBDMS)
groups at the 2' position, 10.5 mL triethylamine trihydrofluoride
(Et3N--HF) was added to the above mixture. The mixture was heated
at 60.degree. C. for 15 minutes. The reaction was then quenched
with 38.5 mL of 50 mM sodium acetate (pH 5.5) and stored in the
freezer until purification.
[0361] Step 3. Quantitation of Crude Oligonucleotides
[0362] For all samples, a 10 .mu.L aliquot was diluted with 990
.mu.L of deionised nuclease free water (1.0 mL) and the absorbance
reading at 260 nm was obtained.
[0363] Step 4. Purification of Oligonucleotides
[0364] (a) Unconjugated Oligonucleotides
[0365] The unconjugated crude oligonucleotides were first analyzed
by HPLC (Dionex PA 100). The buffers were 20 mM phosphate, pH 11
(buffer A); and 20 mM phosphate, 1.8 M NaBr, pH 11 (buffer B). The
flow rate 1.0 mL/min and monitored wavelength was 260-280 nm.
Injections of 5-15 .mu.L were done for each sample.
[0366] The unconjugated samples were purified by HPLC on a TSK-Gel
SuperQ-5PW (20) column packed in house (17.3.times.5 cm) or on a
commercially available TSK-Gel SuperQ-5PW column (15.times.0.215
cm) available from TOSOH Bioscience. The buffers were 20 mM
phosphate in 10% CH.sub.3CN, pH 8.5 (buffer A) and 20 mM phosphate,
1.0 M NaBr in 10% CH.sub.3CN, pH 8.5 (buffer B). The flow rate was
50.0 mL/min for the in house packed column and 10.0 ml/min for the
commercially obtained column. Wavelengths of 260 and 294 nm were
monitored. The fractions containing the full-length
oligonucleotides were pooled together, evaporated, and
reconstituted to .about.100 mL with deionised water.
[0367] (b) Cholesterol-Conjugated Oligonucleotides
[0368] The cholesterol-conjugated crude oligonucleotides were first
analyzed by LC/MS to determine purity. The cholesterol conjugated
sequences were HPLC purified on RPC-Source15 reverse-phase columns
packed in house (17.3.times.5 cm or 15.times.2 cm). The buffers
were 20 mM NaOAc in 10% CH.sub.3CN (buffer A) and 20 mM NaOAc in
70% CH.sub.3CN (buffer B). The flow rate was 50.0 mL/min for the
17.3.times.5 cm column and 12.0 ml/min for the 15.times.2 cm
column. Wavelengths of 260 and 284 nm were monitored. The fractions
containing the full-length oligonucleotides were pooled,
evaporated, and reconstituted to 100 mL with deionised water.
[0369] Step 5. Desalting of Purified Oligonucleotides
[0370] The purified oligonucleotides were desalted on either an
AKTA Explorer or an AKTA Prime system (Amersham Biosciences) using
a Sephadex G-25 column packed in house. First, the column washed
with water at a flow rate of 40 mL/min for 20-30 min. The sample
was then applied in 40-60 mL fractions. The eluted salt-free
fractions were combined, dried, and reconstituted in .about.50 mL
of RNase free water.
[0371] Step 6. Purity Analysis by Capillary Gel Electrophoresis
(CGE), Ion-Exchange HPLC (IEX), and Electrospray LC/Ms
[0372] Approximately 0.3 OD of each of the desalted
oligonucleotides were diluted in water to 300 .mu.L and were
analyzed by CGE, ion exchange HPLC, and LC/MS.
[0373] Step 7. Duplex Formation
[0374] For the fully double stranded duplexes, equal amounts, by
weight, of two RNA strands were mixed together. The mixtures were
frozen at -80.degree. C. and dried under vacuum on a speed vac.
Dried samples were then dissolved in 1.times.PBS to a final
concentration of 40 mg/ml. The dissolved samples were heated to
95.degree. C. for 5 min and slowly cooled to room temperature.
TABLE-US-00011 TABLE 4 Oligonucleotides synthesized to modulate
microRNAs (SEQ ID NOs 1, 12, 10, 11, 13, 14, 21-37, 39, 30 and 10
respectively) AL-SQ Calc Found Purity # Sequence Target Mass Mass
(%) 3035 UGG AGU GUG ACA AUG GUG UUU GU miR-122A 7422.44 7422.20
94.1* 3036 UGG AAU GUG ACA GUG UUG UGU GU miR-122A 7422.42 7422.24
95.3* 3037
A.sub.OMesC.sub.OMesA.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMe-
sC.sub.OMesA.sub.OMesU.sub.OMesU.sub.OMesG.sub.OMes miR-122A
8613.43 8614.53 82.7
U.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesU.sub.OMesC.s-
ub.OMesC.sub.OMesA.sub.OMes-Chol 3038
A.sub.OMesC.sub.OMesA.sub.OMesA.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMe-
sC.sub.OMesC.sub.OMesA.sub.OMesU.sub.OMesU.sub.OMes miR-122A
8340.09 8341.23 99.2
G.sub.OMesU.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesU.s-
ub.OMesV.sub.OMesV.sub.OMes-Chol 3039
A.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMe-
sA.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesU.sub.OMes miR-122A
8613.43 8614.75 86.6
G.sub.OMesU.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMesU.sub.OMesU.s-
ub.OMesC.sub.OMesC.sub.OMesA.sub.OMes-Chol 3040
A.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMe-
sA.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesU.sub.OMes miR-122A
8340.09 8341.15 85.2
G.sub.OMesU.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMesU.sub.OMesU.s-
ub.OMesC.sub.OMesA.sub.OMes-Chol 3223
U.sub.OMesG.sub.OMesG.sub.OMesA.sub.OMesG.sub.OMesU.sub.OMesG.sub.OMe-
sU.sub.OMesG.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMes miR-122A
8545.13 8546.19 95.8
A.sub.OMesU.sub.OMesG.sub.OMesG.sub.OMesU.sub.OMesG.sub.OMesU.sub.OMesU.s-
ub.OMesU.sub.OMesG.sub.OMesU.sub.OMes-Chol 3224
U.sub.OMesG.sub.OMesG.sub.OMesA.sub.OMesA.sub.OMesU.sub.OMesG.sub.OMe-
sU.sub.OMesG.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMes miR-122A
8545.13 8546.28 92.3
G.sub.OMesU.sub.OMesG.sub.OMesU.sub.OMesU.sub.OMesG.sub.OMesU.sub.OMesG.s-
ub.OMesU.sub.OMesG.sub.OMesU.sub.OMes-Chol 3225
A.sub.OMesC.sub.OMesA.sub.OMesA.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMe-
sC.sub.OMesC.sub.OMesA.sub.OMesU.sub.OMesU.sub.OMes miR-122A
7892.09 7892.92 84.0
G.sub.OMesU.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesA.sub.OMesC.sub.OMesU.s-
ub.OMesC.sub.OMesC.sub.OMesA.sub.OMe 3226
A.sub.OMesC.sub.OMeA.sub.OMeA.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.sub-
.OMeC.sub.OMeA.sub.OMeU.sub.OMeUOMe miR-122A 7604.09 7604.04 81.5
G.sub.OMeU.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeU.sub.OMeC-
.sub.OMeC.sub.OMeAOMe 3227
COMeG.sub.OMeC.sub.OMeC.sub.OMeA.sub.OMeA.sub.OMeU.sub.OMeA.sub.OMeU.-
sub.OMeU.sub.OMeU.sub.OMeAOMe miR-16 8047.82 8048.88 94.0*
COMeG.sub.OMeUOMeG.sub.OMeC.sub.OMeUOMeG.sub.OMesoC.sub.OMesoU.sub.OMesoA-
.sub.OMes-Chol 3228
G.sub.OMesG.sub.OMesC.sub.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMeA.su-
b.OMeA.sub.OMeU.sub.OMeU.sub.OMeC.sub.OMe miR-192 7807.68 7808.49
97.1*
A.sub.OMeU.sub.OMeA.sub.OMeG.sub.OMeG.sub.OMeU.sub.OMesC.sub.OMesA.sub.OM-
esG.sub.OMes-Chol 3229
U.sub.OMesC.sub.OMesC.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeU.sub.OMeG.su-
b.OMeG.sub.OMeA.sub.OMeG.sub.OMeU.sub.OMeU.sub.OMeG.sub.OMeC.sub.OMe
miR-194 8088.84 8089.69 92.7*
U.sub.OMeG.sub.OMeU.sub.OMeU.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMes-Chol
3230
U.sub.OMesC.sub.OMesA.sub.OMeC.sub.OMeG.sub.OMeC.sub.OMeG.sub.OMeA.su-
b.OMeG.sub.OMeC.sub.OMeC.sub.OMeG.sub.OMeA.sub.OMeA.sub.OMeC.sub.OMe
miR-375 8178.03 8178.77 100*
G.sub.OMeA.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMesA.sub.OMesA.sub.OMes-Chol
3344 UGG IGU GUG ICI IUG GUG UUU GU miR-122A 7120.19 7119.36 83.0*
3350
A.sub.OMeC.sub.OMeA.sub.OMeA.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.sub.-
OMeC.sub.OMeA.sub.OMeU.sub.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMeA.sub.OM-
e miR-122A 8244.09 8244.13 8.0*
C.sub.OMeA.sub.OMeC.sub.OMeU.sub.OMeC.sub.OMeC.sub.OMeA.sub.OMe-Chol
3351
C.sub.OMesA.sub.OMes.sub.OMeC.sub.OMeA.sub.OMeA.sub.OMeA.sub.OMeC.sub-
.OMeA.sub.OMeC.sub.OMeC.sub.OMeA.sub.OMeU.sub.OMeU.sub.OMeG.sub.OMe
miR-122A 8978.51 8979.07 97.1*
U.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeU.sub.OMeC.sub.OMes-
C.sub.OMesA.sub.OMesC.sub.OMes-Chol 3352
C.sub.OMesA.sub.OMesA.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeC.su-
b.OMeA.sub.OMeU.sub.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMeA.sub.OMe
miR-122A 7653.61 7653.92 89.0*
C.sub.OMeA.sub.OMeC.sub.OMesU.sub.OMesC.sub.OMesC.sub.OMes-Chol
3353
A.sub.OMesA.sub.OMesA.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeC.sub.OMeA.su-
b.OMeU.sub.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMe
miR-122A 7015.19 7015.7 97.9*
A.sub.OMesC.sub.OMesU.sub.OMesC.sub.OMes-Chol 3354
A.sub.OMesA.sub.OMesC.sub.OMeA.sub.OMeC.sub.OMeC.sub.OMeA.sub.OMeU.su-
b.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMes
miR-122A 6352.74 6353.29 97.9* A.sub.OMesC.sub.OMesU.sub.OMes-Chol
3355
A.sub.OMesC.sub.OMesA.sub.OMeA.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeA.su-
b.OMeC.sub.OMeA.sub.OMeC.sub.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMe
miR-122A 8364.13 8364.45 90.2*
A.sub.OMeC.sub.OMeA.sub.OMeU.sub.OMeU.sub.OMeC.sub.OMesC.sub.OMesA.sub.OM-
es-Chol 3356
A.sub.OMesC.sub.OMesA.sub.OMeA.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.su-
b.OMeC.sub.OMeA.sub.OMeC.sub.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMe
miR-122A 8340.09 8340.46 86.0*
A.sub.OMeC.sub.OMeA.sub.OMeU.sub.OMeU.sub.OMeC.sub.OMesC.sub.OMesA.sub.OM-
es-Chol 3357
A.sub.OMesC.sub.OMesA.sub.OMeA.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.su-
b.OMeC.sub.OMeA.sub.OMeU.sub.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMe
miR-122A 8341.08 8341.43 79.0*
A.sub.OMeC.sub.OMeA.sub.OMeU.sub.OMeU.sub.OMesC.sub.OMesA.sub.OMes-Chol
3359
Quasar5sA.sub.OMesC.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeA.sub.-
OMeA.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeU.sub.OMeG.sub.OMe miR-122A
8960.91 8960.78 93.0*
U.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeU.sub.OMeU.sub.OMesC.sub.OMe-
sC.sub.OMesA.sub.OMes-Chol 3383
A.sub.OMeC.sub.OMeA.sub.OMeA.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.sub.-
OMeC.sub.OMeA.sub.OMeU.sub.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMeA.sub.OM-
e miR-122A 8260.09 8260.13 86.0*
C.sub.OMeA.sub.OMeC.sub.OMeU.sub.OMeC.sub.OMeC.sub.OMeAs-Chol 3474
Quasar5sA.sub.OMesC.sub.OMeA.sub.OMeA.sub.OMeA.sub.OMeC.sub.OMeA.sub.-
OMeC.sub.OMeC.sub.OMeA.sub.OMeU.sub.OMeU.sub.OMeG.sub.OMe miR-122A
8960.91 8960.65 98.9*
U.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeA.sub.OMeC.sub.OMeU.sub.OMesC.sub.OMe-
sC.sub.OMesA.sub.OMes-Chol
[0375] The strands are shown written 5' to 3'. Lower case "s"
indicates a phosphorothioate linkage. "Chol-" indicates a
hydroxyprolinol cholesterol conjugate. Subscript "OMe" indicates a
2'-O-methyl sugar. "I" is ribo-Inosine nucleoside. Purity was
determined by CGE except for the where indicated by (*), in these
cases purity was determined by anion-exchange HPLC. TABLE-US-00012
TABLE 5 Double stranded oligonucleotides to modulate microRNAs
AL-DP # Strand 1 Strand 2 Target 3018 AL-SQ-3035 AL-SQ-3037
miR-122A 3019 AL-SQ-3035 AL-SQ-3038 miR-122A 3020 AL-SQ-3036
AL-SQ-3039 miR-122A 3021 AL-SQ-3036 AL-SQ-3040 miR-122A
[0376] TABLE-US-00013 TABLE 6 Description of oligonucleotides
synthesized to modulate microRNAs Sequence # Description 3035
complementary to antagomir-122 3036 complementary to
mm-antagomir-122 3037 antagomir-122-fullyPS 3038 antagomir-122 3039
mm-antagomir-122-fullyPS 3040 mm-antagomir-122 3223 complementary
to antagomir-122 3224 complementary to mm-antagomir-122 3225
anti-122-fully-PS 3226 anti-122-partial_PS 3227 antagomir-16 3228
antagomir-192 3229 antagomir-194 3230 antagomir-375 3344
complementary to antagomir-122 with Adenosine -> Inosine
modification 3350 antagomir-122-noPS 3351 antagomir-122-25mer 3352
antagomir-122-21mer 3353 antagomir-122-19mer 3354
antagomir-122-17mer 3355 mm-antagomir-122-3 mm 3356
mm-antagomir-122-2 mm 3357 mm-antagomir-122-1 mm 3359
mm-antagomir-122-5'-Quasar570 3383 Same as AL-3350 with P.dbd.S
between 3'-end and Cholesterol 3474 antagomir-122-5'-Quasar570
Example 10
Characterization of Antagomirs
[0377] The following experiments were designed to further
characterize the properties and function of antagomirs in mice. The
results presented herein demonstrate that antagomirs that have
optimized phosphorothioate modification and are preferably greater
than 19 nucleotides in length exhibit the highest biological
efficiency, and can discriminate between single nucleotide
mismatches of the targeted miRNA.
[0378] The observation that degradation of different chemically
protected miRNA/antagomir duplexes in mouse livers and localization
of antagomirs in a cytosolic compartment that is distinct from
processing (P)-bodies indicates a degradation mechanism independent
of the RNA interference (RNAi) pathway.
[0379] It was also observed that although antagomirs are incapable
of silencing miRNAs in the central nervous system (CNS) when
injected systemically, the antagomirs efficiently targeted miRNAs
in the CNS when injected locally into the mouse cortex. The data
presented herein further validate the effectiveness of antagomirs
in vivo, particularly in clinically relevant settings.
[0380] The materials and methods employed in the experiments
disclosed herein are now described.
Synthesis of Antagomirs
[0381] Single-stranded RNAs and modified RNA analogs (antagomirs)
were synthesized as previously elsewhere herein. The
oligonucleotides used in this study are listed in Table 7 and a
schematic representation of the chemical modifications is shown in
FIG. 16. Quasar-570 (Q570) phosphoramidite from Biosearch
Technologies was coupled to the 5'-terminal under standard solid
phase phosphoramidite synthesis conditions to obtain fluorophore
tagged antagomir 122 and mm-antagomir-122. Extended 15 minute
coupling of quasar-570 phosphoramidite was carried out at a
concentration of 0.1M in CH.sub.3CN in the presence of
5-(ethylthio)-1H-tetrazole activator followed by standard capping,
oxidation and deprotection afforded labeled oligonucleotides. The
Q570 conjugated sequences were HPLC purified on an in-house packed
RPC-Source15 reverse-phase column. The buffers were 20 mM NaOAc in
10% CH.sub.3CN (buffer A) and 20 mM NaOAc in 70% CH.sub.3CN (buffer
B). Fractions containing full-length oligonucleotides were pooled
and desalted. Analytical HPLC, CGE and ES LC-MS established the
integrity of the compounds. For duplex generation, equal molar
mounts of miR-122 and antagomir were heated in 1.times.PBS at
95.degree. C. for 5 minutes and slowly cooled to room temperature.
TABLE-US-00014 TABLE 7* Antagomirs of mir-122 and mir-16 S. No
Sequence Description 1 5'-UGGAGUGUGACAAUGGUGUUUGU-3' Mir-122 5
##STR8## Antagomir-122 (23nt, 6xP = S) 31
5'-c.sub.sa.sub.scaaacaccauugucacacuc.sub.sc.sub.sa.sub.sc.sub.s-Chol-3-
' Antagomir-122 (25nt, 6xP = S) 32
5'-c.sub.sa.sub.saacaccauugucacac.sub.su.sub.sc.sub.sc.sub.s-Chol-3'
Antagomir-122 (21nt, 6xP = S) 33
5'-a.sub.sa.sub.sacaccauugucaca.sub.sc.sub.su.sub.sc.sub.s-Chol-3'
Antagomir-122 (19nt, 6xP = S) 34
5'-a.sub.sa.sub.scaccauugucac.sub.sa.sub.sc.sub.su.sub.s-Chol-3'
Antagomir-122 (17nt, 6xP = S) 30 5'-acaaacaccauugucacacucca-Chol-3'
Antagomir-122 (23nt, no P = S) 45
5'-acaaacaccauugucacacucca.sub.s-Chol-3' Antagomir-122, (23nt, 1xP
= S) 9
5'-a.sub.sc.sub.sa.sub.sa.sub.sa.sub.sc.sub.sa.sub.sc.sub.sc.sub.sa.sub.-
su.sub.su.sub.sg.sub.su.sub.sc.sub.sa.sub.sc.sub.sa.sub.sc.sub.su.sub.sc.s-
ub.sc.sub.sa.sub.s-Chol-3' Antagomir-122, (23nt, 23xP = S) 14
5'-a.sub.sc.sub.sacacaacacugucacauu.sub.sc.sub.sc.sub.sa.sub.s-Chol-3'
mm-antagomir-122 (23nt, 6xP = S, 4mm) 36 ##STR9## mm-antagomir-122
(23nt, 6xP = S, 2mm) 37 ##STR10## mm-antagomir-122 (23nt, 6xP = S,
1mm at nt19) 46 ##STR11## mm-antagomir-122 (23nt, 6xP = S, 1mm at
nt1) 47 ##STR12## mm-antagomir-122 (23nt, 6xP = S, 1mm at nt11) 48
5'-UGGAGUGUGACAauGGUGUUUGU-3' miR-122 (2'-O-Me at 10 & 11) 49
5'-U.sub.sG.sub.sGAGUGUGACAAUGGUGUUU.sub.sG.sub.sU-3' miR-122 (2xP
= S at each end) 50
5'-Q570.sub.s-a.sub.scaaacaccauugucacacu.sub.sc.sub.sc.sub.sa.sub.s-Cho-
l-3' Antagomir-122 (5'-Quasar570) 51
5'-Q570.sub.s-a.sub.scacacaacacugucacauu.sub.sc.sub.sc.sub.sa.sub.s-Cho-
l-3' mm-antagomir-122 (4mm, 5'-Quasar570) 6
5'-c.sub.sg.sub.sccaauauuuacgugcug.sub.sc.sub.su.sub.sa.sub.s-Chol-3'
Antagomir-16 *Lower case letters indicate 2'-O-methyl-modified
nucleotides; subscript `s` indicates a phosphorothioate linkage and
`chol` represents cholesterol linked through a hydroxyprolinol
linkage.
Animals
[0382] All animals were maintained in a C57B1/6J background on a 12
hour light/dark cycle in a pathogen-free animal facility at
Rockefeller University. Six-week-old mice received on three
consecutive days tail vein injections of saline or different RNAs
in 0.2 ml per injection at normal pressure. Liver tissue was
harvested 24 hours after the last injection or as otherwise
indicated.
[0383] For cerebral injections, mice were anaesthetized and
antagomir-16 was injected into the left frontal cortex (.about.800
ng). Injections of equal volume PBS into the contralateral area of
the right hemisphere served as controls. After 72 hours, mice were
sacrificed, blood was removed through systemic perfusion of the
left ventricle with PBS and a .about.0.4 cm.sup.3 area surrounding
the injection site was excised from the cortex for analysis.
Northern Blotting Analysis
[0384] Total RNA was isolated using the Trizol protocol with
ethanol precipitation and Northern blot analysis was performed
using formamide-containing PAGE as described elsewhere herein.
RT-PCR
[0385] Extraction of total RNA, synthesis of cDNA, and PCR were
performed as described elsewhere herein.
Sucrose Density Gradient Fractionation of Liver Homogenates
[0386] Mice were perfused with ice-cold PBS through the left
ventricle and .about.100 mg liver tissue excised. Cells were
fractionated on continuous sucrose density gradients from 0.4-2M.
Fractions were separated on 14% PAGE containing 8M urea and 20%
formamide. Concentration of 5'-Q570-labeled antagomir in liver
fractions was measured using an fmax spectrophotometer from
Molecular devices.
Immunofluorescence
[0387] For immunofluorescence of P-bodies and antagomirs, 1 mg of
Q570-labeled antagomirs were injected in 0.2 ml and normal pressure
on day 1, followed by injection of 50 .mu.g of a
Gfp-GW182-expressing DNA-plasmid (10) in 2 ml PBS and high pressure
on day 2. On day 3, mice were anesthetized and perfused through the
left ventricle with 2% paraformaldehyde. Livers were incubated
overnight at 4.degree. C. in 4% paraformaldehyde, followed by a 16
hour incubation period in 30% sucrose/PBS (v/v). Frozen sections (7
.mu.m) were mounted on glass slides and analyzed using a
laser-scanning microscope.
Statistical Analysis
[0388] Results are given as means.+-.standard errors. Statistical
analysis was performed with Student's t-test, and the null
hypothesis was rejected at the 0.05 level. Results are typically
presented as means.+-.standard errors.
[0389] The results of the experiments presented in this Example are
now described.
Phosphorothioate Modifications and Length of Antagomir-122
[0390] The antagomir-122 chemistry includes 6 phosphorothioate
backbone modifications. Two phosphorothioates are located at the
5'-end and four at the 3'-end. The following experiment was
designed to test whether the number of phosphorothioates is
critical for the ability of antagomir-122 to silence miR-122. Four
different antagomir-122 molecules that only differ in the number of
phosphorothioate modification (P.dbd.S) were compared. Injection of
antagomir-122 at 3.times.20 mg/kg bw with no P.dbd.S into mice did
not influence miR-122 levels in the liver (FIG. 17A). The addition
of a single P.dbd.S did not significantly alter miR-122 levels as
compared to the antagomir-122 with six P.dbd.S (FIG. 17B). Complete
P.dbd.S modification of the antagomir-122 did not further increase
the effect on miR-122 levels (FIG. 17B). These results demonstrate
that phosphorothioates are important for antagomir-122 function.
However, it was observed that complete replacement of P.dbd.O by
P.dbd.S decreases its efficiency. Without wishing to be bound by
any particular theory, the result can be explained by the enhanced
stability provided by the terminal P.dbd.S linked antagomir and the
reduced thermodynamic stability of the fully modified P.dbd.S
antagomir duplex with targeted miRNA.
[0391] Experiments were also designed to determine the optimal
nucleotide length of antagomirs for silencing endogenous miR-122
levels in vivo. It was observed that the addition of two
nucleotides or shortening of antagomir-122 by two nucleotides did
not significantly alter its efficiency (FIG. 17C). However,
silencing of miR-122 was abolished at 3.times.20 mg/kg bw when the
length of antagomir-122 was reduced to 19 nucleotides. Together,
these results demonstrate a preferred optimal number of
phosphorothioate modifications and minimum length of antagomirs for
their biological function in vivo. Without wishing to be bound by
any particular theory, it is believed that the tendency for
improved activity of 25mer antagomir can be explained on the basis
of improved thermodynamic binding affinity of the 25mer, which
should also have higher biostability from exonucleases for the core
23mer.
Dose- and Time Dependency of Antagomir-122
[0392] To investigate the optimal dose- and time-dependency of
antagomir-122, miR-122 levels as well as mRNA levels of endogenous
miR-122 targets were analyzed. Northern blots show that optimal
reduction of miR-122 levels is achieved at antagomir concentrations
between 3.times.40 and 3.times.80 mg/kg bw (FIG. 18A). The effect
of 3.times.40 mg/kg on miR-122 levels is stable for at least 8 days
(=5 days after the last injection) (FIG. 18B). The expression of
miR-122 targets correlated with the reduction of miR-122 levels in
Northern blots and showed highest upregulation at antagomir
concentrations between 3.times.40 and 3.times.80 mg/kg bw (FIG.
18A). All targets analyzed showed stable upregulation for at least
5 days after the last injection (FIG. 18A). The results presented
herein demonstrate that robust and lasting upregulation of miR-122
targets is achieved at antagomir concentration between 3.times.40
and 3.times.80 mg/kg bw as early as 24 hours after the last
injection.
Mismatch Discrimination of Antagomirs
[0393] The following set of experiments were conducted to test the
impact of different mismatch numbers in the antagomir sequence on
miR-122 levels and miR-122 targets. Four mismatches, two mismatches
or a single mismatch at position 19 was sufficient to prevent
downregulation of miR-122 and upregulation of three different
miR-122 targets (AldoA, Tmed3 and Hfe2) as measured by RT-PCR (FIG.
19A). However, it was observed that single nucleotide mismatches at
two different positions (nt1 or nt11) did not prevent
downregulation of miR-122 levels or target regulation (FIG. 4B).
Without wishing to be bound by any particular theory, these data
demonstrate that antagomirs can exhibit high sequence specificity.
However, discrimination at the single nucleotide level is
position-dependent and testing for each microRNA sequence that is
being targeted may be necessary. However, once armed with the
present invention, such testing is well within the skill of the
artisan trained in the field.
Regulation of miR-122 Targets by Stabilized Duplexes of
miR-122/Antagomir-122
[0394] The next series of experiments were designed to analyze the
ability of antagomirs to induce degradation of preformed duplexes
in order to address whether antagomir-mediated miRNA silencing
involves degradation of the miRNA. Duplexes of antagomir-122 and a
synthetic miR-122 that harbors modifications to protect against
different RNAse activities were synthesized. MiR-122 was either
protected at the outside ("out") with a phosphorothioate
modification to protect against exonucleases or at two consecutive
internal positions (nt13 and nt14 of miR-122; "in") using
2'-O-methyl sugar modification (FIG. 20A) to protect against
endonuclease activity. Injection of both types of duplexes led to
the appearance of degradation products of the synthetic miR-122
(FIG. 20B). These degradation products did not appear when the
duplexes were directly analyzed on the polyacrylamide gels or after
they had been subjected to the RNA isolation protocol (FIG. 20B,
"spiked control"). Furthermore, the spiked control data demonstrate
that the synthetic miRNA was not lost during the isolation
procedure. These data establish that both types of stabilized
miR-122 that were bound to the antagomir-122 had been degraded.
Accordingly, both types of protected duplexes upregulated three
different miR-122 targets (FIG. 20B). Without wishing to be bound
by any particular theory, it is believed that
antagomir-mediated-silencing of miRNAs involves target degradation.
However, this process does not depend on exonuclease activity and
differs from the RNAi pathway.
Cellular Localization of Antagomirs
[0395] To localize antagomirs and miRNA in subcellular
compartments, 5'-Q570-labeled antagomir-122 or Q570-labeled
mm-antagomir-122 was used. It was observed that Q570-labeling did
not impair antagomir-122 function, although silencing efficiency
was slightly decreased. Liver homogenates from mice that had been
treated with Q570-mm-antagomir-122 were fractionated by
ultracentrifugation on sucrose gradients. Northern blot analysis of
various fractions showed a single peak of tRNA in fraction 2 (FIG.
21A). In contrast, miR-122 and mm-antagomir-122 localized both to
two peaks, fraction 2/3 and fraction 7/8 (FIG. 21).
[0396] The next set of experiments were designed to investigate
whether co-localization of antagomirs and miRNAs involves the
P-body compartment. In order to visualize P-bodies in mouse liver
in vivo, a GFP-expressing construct (GFP-GW182) that has previously
been demonstrated to act as a marker for the P-body compartment was
used (10). GFP-GW182 was overexpressed in liver using high-pressure
high-volume tail vein injections. GFP- and Q570-fluorescence was
analyzed using laser-scanning microscopy. It was observed that
Q570-labeled antagomirs were exclusively localized in the cytosol
and were distinct from P-bodies (FIG. 21C). There was no observable
overlap between these two compartments. Without wishing to be bound
by any particular theory, it is believed that antagomirs and miRNA
interact in a cytoplasmic compartment upstream of P-bodies.
Intracerebral Application of Antagomirs
[0397] It has been disclosed elsewhere herein that systemic
injections of antagomir-16 into tail veins of mice did not
influence the steady-state levels of miR-16 in the brain even
though miR-16 is ubiquitously expressed in neurons. The following
experiments were designed to determine whether antagomir-16 can
decrease miRNA levels in the brain when injected directly into the
cortex of anesthetized mice. PBS-injections into the contra-lateral
side of the same animal served as a control. A single injection of
about 0.8 .mu.g of antagomir led to an observable decrease in
miR-16 expression at 3 days after the injection (FIG. 22). These
results demonstrate that direct application of antagomirs can
efficiently target miRNAs in tissues that cannot be reached through
tail vein injections.
Example 11
Specificity, Duplex Degradation and Subcellular Localization of
Antagomirs
[0398] The results presented herein characterize the inhibition of
miRNAs with antagomirs in vivo and their therapeutic use. Our study
provides a unique platform since its major read-out is based on the
dose- and time-dependent regulation of several endogenous and
validated targets of miR-122.
[0399] Specificity of drug-like oligonucleotides is important to
minimize off-target effects and to discriminate between related
miRNAs that sometimes differ by only a single nucleotide. In line
with this, antagomir chemistry enables discrimination of a single
nucleotide. This effect depends on the position of the mismatch
within the antagomir sequence. It has been observed that nucleotide
exchanges at the very 5'-end of the antagomir or in the center did
not prevent downregulation of miR-122 levels in Northern blots and
upregulation of miR-122 targets. Without wishing to be bound by any
particular theory, it appears that asymmetry of a single nucleotide
mismatch may therefore be more detrimental for targeting miRNAs
than symmetric changes. These data are important for the design of
antagomirs that target specific members of miRNA families or when
off-target effects are being considered.
[0400] The experiments presented herein were designed to address
whether antagomir-mediated silencing of miRNA involves a
RNA-induced silencing complex (RISC)-dependent cleavage mechanism.
In the RNAi pathway, the siRNA duplex of passenger strand and guide
strand is integrated into the RISC complex and the argonaute-2
(Ago2) protein subsequently cleaves the passenger strand across
from the guide strand's phosphate bond between position 10 and 11
(Rand, et al., 2005 Cell, 123:621-9, and Matranga, et al., 2005
Cell 123:607-20). This cleavage was inhibited by a single
2'-O-methylation of the passenger strand corresponding to
nucleotide 11 of the guide strand (Rand, et al., 2005 Cell,
123:621-9). It is believed that, antagomirs could cleave miRNAs
within RISC with the antagomir acting as the guide strand.
[0401] miRNA/antagomir-duplexes were injected into mice that
harbored a 2'-O-methyl endonuclease protection of the microRNA
corresponding to nucleotide 10 and 11 of the antagomir. However,
endonuclease protection between nucleotides 10 and 11 did not
prevent the degradation of the miRNA as demonstrated by abundant
miRNA fragments in Northern analysis, nor did it prevent the
upregulation of miR-122 targets. Thus Ago2-mediated cleavage is
unlikely to mediate this process. Similar results were obtained
when the miRNA was protected at the outside positions using
phosphorothioates, indicating that the miRNA targeting does not
dependent on exonuclease activity either. However, the fact that
miRNA/antagomir-duplexes regulated miRNA targets suggests antagomir
recycling. The appearance of miRNA fragments of decreased length
also suggests that degradation is involved in this recycling
process.
[0402] To address the subcellular compartment where interaction of
miRNA and antagomir occurs, fluorophore labeled antagomirs were
engineered. Fluorophore labeling of siRNA has previously been used
to evaluate cellular uptake of siRNA (Grunweller, et al., 2003
Oligonucleotides, 13:345-52, and Lingor, et al., 2005 Brain
128:550-8). Q570-labeled antagomirs were cleared from the plasma at
a t.sub.1/2 of approximately 30 minutes, which is considerably
faster than the plasma-clearance of cholesterol-conjugated siRNA of
about 90 minutes (Soutschek, et al., 2004 Nature 432:173-8). A
striking overlap of the subcellular localization profiles of
antagomirs and miRNAs by sucrose gradient ultracentrifugation
analysis of liver homogenates indicates that they may share
subcellular compartments. It was observed that antagomir
localization within hepatocytes was strictly limited to the
cytosol. Without wishing to be bound by any particular theory, it
is believed that antagmir localization to the cytosol explains why
antagomirs did not influence steady-state levels of the nuclear
precursors of miRNAs (Krutzfeldt, et al., 2005 Nature
438:685-9).
[0403] Experiments were also designed to address whether antagomirs
could localize to P-bodies, since this compartment has been linked
to the miRNA pathway. P-bodies are enriched in Ago2 as well as mRNA
that is targeted by miRNAs. There was no observation of any
co-localization of antagomirs with P-bodies. Therefore, it is
believed that the interaction of antagomirs with miRNAs occurs
upstream of this compartment.
[0404] Different types of chemical modifications on antagomirs were
also assessed. Phosphorothioate modifications provide protection
against RNase activity and their lipophilicity contributes to
enhanced tissue uptake. Phosphorothioates also decrease the melting
temperature of RNA duplexes (Davis, et al., 2006 Nucleic Acids Res
34:2294-304) and have been shown to be general inhibitors of
cellular RNAse activity (Crooke, et al., 2000 J Pharmacol Exp Ther
292:140-9). The results presented herein indicate a critical
balance of the number of phosphorothioates within the antagomir
chemistry. While a significant number of phosphorothioates
increases efficiency, complete phosphorothioate modification
decreased efficiency. For example, it was demonstrated that
antagomirs require >19 nucleotides length for optimal
function.
[0405] Results presented herein also demonstrate that antagomirs
can efficiently decrease miR-16 levels in mouse brain when injected
locally. Systemic infusions of antagomir-16 do not result in an
observable change in the brain levels of miR-16. This is because it
is believed that antagomir-16 does not have the ability to cross
the blood-brain barrier. Local injections of small amounts of
antagomir-16 efficiently reduced expression of this miR-16 in the
cortex. This inhibition was specific since the expression of other
miRNAs was not affected and no alteration in miR-16 levels were
measured in the contra-lateral hemisphere that was injected with
PBS. These results suggest that miRNA-inhibitors could facilitate
the elucidation of miRNA function in the CNS.
[0406] For further characterization of antagomirs, expression
levels of endogenous miR-122 targets were used as a read-out. The
results presented herein demonstrate that antagomirs can be used in
a time and dose-dependent fashion to study miRNA targets.
Furthermore, the characterization of the antagomirs with regard to
specificity, functional minimal length requirements and
effectiveness in the CNS following direct application further
support the use of miRNA inhibitors in a clinical setting as a
therapeutic composition.
Example 12
Strategy to Study miRNA Function In Vivo
[0407] The following experiments are designed to study miRNA
function in vivo. Typically, gene expression profiling,
bioinformatics analysis, metabolic profiling, and biochemical
target validation is performed. Using methods discussed elsewhere
herein, miR-122 was observed to regulate levels of many target
genes (FIG. 23). Moreover, miR-122 was observed to regulate the
expression of cholesterol biosynthesis genes (FIG. 24). Based on
the genes observed to be regulated by miR-122, metabolic parameters
of antagomir-122 treated mice were evaluated. The results
demonstrated that mice treated with antagomir-122 exhibited a
decreased levels of at least cholesterol as compared with mice
treated with mm-antagomir (FIG. 25). The results presented herein
characterize the inhibition of miRNAs with antagomirs in vivo and
their therapeutic use with respect to cholesterol levels.
Other Embodiments
[0408] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
[0409] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
Sequence CWU 1
1
51 1 23 RNA Mus musculus 1 uggaguguga caaugguguu ugu 23 2 22 RNA
Mus musculus 2 uagcagcacg uaaauauugg cg 22 3 21 RNA Mus musculus 3
cugaccuaug aauugacagc c 21 4 22 RNA Mus musculus 4 uguaacagca
acuccaugug ga 22 5 23 RNA Artificial Sequence Synthetically
generated oligonucleotide modified_base 1, 2, 20, 21, 22, 23
/2'-O-methyl modification phosphorothioate linkage corresponding
base modified_base (3)...(19) 2'-O-methyl modified nucleotides
corresponding base 5 acaaacacca uugucacacu cca 23 6 22 RNA
Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 2, 19, 20, 21, 22 ' 2'-O-methyl modification
phosphorothioate linkage corresponding base modified_base
(3)...(18) 2'-O-methyl modified nucleotides corresponding base 6
cgccaauauu uacgugcugc ua 22 7 21 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base 1, 2, 18, 19,
20, 21 2'-O-methyl modification phosphorothioate linkage
coresponding base modified_base (3)...(17) 2'-O-methyl modified
nucleotides corresponding base 7 ggcugucaau ucauagguca g 21 8 22
RNA Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 2, 19, 20, 21, 22 2'-O-methyl modification
phosphorothioate linkage corresponding base modified_base
(3)...(18) 2'-O-methyl modified nucleotides corresponding base 8
uccacaugga guugcuguua ca 22 9 25 DNA Artificial Sequence Primer 9
ccccttaaat agttgtttat tggca 25 10 23 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base (1)...(23)
2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base 10 acaaacacca uugucacacu cca 23 11 23 RNA
Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 2, 20, 21, 22, 23 2'-O-Me ribo sugar modification
phosphorothioate linkage corresponding base modified_base
(3)...(19) 2'-O-Me ribo sugar modification corresponding base 11
acaaacacca uugucacacu cca 23 12 23 RNA Artificial Sequence
Synthetically generated oligonucleotide 12 uggaauguga caguguugug
ugu 23 13 23 RNA Artificial Sequence Synthetically generated
oligonucleotide modified_base (1)...(23) 2'-O-Me ribo sugar
modification phosphorothioate linkage corresponding base 13
acacacaaca cugucacauu cca 23 14 23 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base 1, 2, 20, 21,
22, 23 2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base modified_base (3)...(19) 2'-O-Me ribo sugar
modification corresponding base 14 acacacaaca cugucacauu cca 23 15
31 DNA Artificial Sequence Synthetically generated oligonucleotide
modified_base (1)...(23) 2'-O-Me ribo sugar modification
corresponding base modified_base 24, 25, 26, 27 deoxyribo sugar
modification modified_base 28, 29, 30 2'-O-Me ribo sugar
modification corresponding base modified_base 31 2'-O-Me ribo sugar
modification phosphorothioate linkage 15 acaaacacca uugucacacu
ccattttugg a 31 16 31 DNA Artificial Sequence Synthetically
generated oligonucleotide modified_base (1)...(22) 2'-O-Me ribo
sugar modification corresponding base modified_base 23, 31 2'-O-Me
ribo sugar modification phosphorothioate linkage corresponding base
modified_base 24, 25, 26, 27 deoxyribo sugar modification
phosphorothioate linkage modified_base 28, 29, 30 2'-O-Me ribo
sugar modification corresponding base 16 acaaacacca uugucacacu
ccattttugg a 31 17 7 RNA Artificial Sequence Synthetically
generated oligonucleotide modified_base (1)...(7) 2'-O-Me ribo
sugar modification corresponding base 17 uggagug 7 18 7 RNA
Artificial Sequence Synthetically generated oligonucleotide
modified_base (1)...(7) 2'-O-Me ribo sugar modification
corresponding base 18 gacaaug 7 19 7 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base (1)...(7)
2'-O-Me ribo sugar modification corresponding base 19 uggaaug 7 20
7 RNA Artificial Sequence Synthetically generated oligonucleotide
modified_base (1)...(7) 2'-O-Me ribo sugar modification
corresponding base 20 gacagug 7 21 23 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base 1, 2, 19, 20,
21, 22 2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base modified_base (3)...(18) 2'-O-Me ribo sugar
modification corresponding base modified_base 23 2'-O-Me ribo sugar
modification corresponding base 21 uggaguguga caaugguguu ugu 23 22
23 RNA Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 2, 19, 20, 21, 22 2'-O-Me ribo sugar modification
phosphorothioate linkage corresponding base modified_base
(3)...(18) 2'-O-Me ribo sugar modification corresponding base
modified_base 23 2'-O-Me ribo sugar modification corresponding base
22 uggaauguga caguguugug ugu 23 23 23 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base (1)..(22)
2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base modified_base 23 2'-O-Me ribo sugar modification
corresponding base 23 acaaacacca uugucacacu cca 23 24 23 RNA
Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 2, 19, 20, 21, 22 2'-O-Me ribo sugar modification
phosphorothioate linkage corresponding base modified_base
(3)...(18) 2'-O-Me ribo sugar modification corresponding base
modified_base 23 2'-O-Me ribo sugar modification corresponding base
24 acaaacacca uugucacacu cca 23 25 22 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base 1, 2, 19, 20,
21, 22 2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base modified_base (3)...(18) 2'-O-Me ribo sugar
modification corresponding base 25 cgccaauauu uacgugcugc ua 22 26
21 RNA Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 2, 18, 20, 21 2'-O-Me ribo sugar modification
phosphorothioate linkage corresponding base modified_base (3)...(17
2'-O-Me ribo sugar modification corresponding base 26 ggcugucaau
ucauagguca g 21 27 22 RNA Artificial Sequence Synthetically
generated oligonucleotide modified_base 1, 2, 19, 20, 21, 22
2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base modified_base (3)...(18) 2'-O-Me ribo sugar
modification corresponding base 27 uccacaugga guugcuguua ca 22 28
22 RNA Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 2, 19, 20, 21, 22 2'-O-Me ribo sugar modification
phosphorothioate linkage corresponding base modified_base
(3)...(18) 2'-O-Me ribo sugar modification corresponding base 28
ucacgcgagc cgaacgaaca aa 22 29 23 RNA Artificial Sequence
Synthetically generated oligonucleotide misc_feature 4, 10, 12, 13
n = inosine 29 uggngugugn cnnugguguu ugu 23 30 23 RNA Artificial
Sequence Synthetically generated oligonucleotide modified_base
(1)..(23) 2'-O-Me ribo sugar modification corresponding base 30
acaaacacca uugucacacu cca 23 31 25 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base 1, 2, 22, 23,
24, 25 2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base modified_base (3)...(21) 2'-O-Me ribo sugar
modification corresponding base 31 cacaaacacc auugucacac uccac 25
32 21 RNA Artificial Sequence Synthetically generated
oligonucleotide modified_base 1, 2, 18, 19, 20, 21 2'-O-Me ribo
sugar modification phosphorothioate linkage corresponding base
modified_base (3)...(17) 2'-O-Me ribo sugar modification
corresponding base 32 caaacaccau ugucacacuc c 21 33 19 RNA
Artificial Sequence Synthetically generated oligonucleotide
modified_base (1)..(15) 2'-O-Me ribo sugar modification
corresponding base modified_base 16, 17, 18, 19 2'-O-Me ribo sugar
modification phosphorothioate linkage corresponding base 33
aaacaccauu gucacacuc 19 34 17 RNA Artificial Sequence Synthetically
generated oligonucleotide modified_base (3)..(13) 2'-O-Me ribo
sugar modification corresponding base modified_base 1, 2, 14,15,
16, 17 2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base 34 aacaccauug ucacacu 17 35 23 RNA Artificial
Sequence Synthetically generated oligonucleotide modified_base 1,
2, 20, 21, 22, 23 2'-O-Me ribo sugar modification phosphorothioate
linkage corresponding base modified_base (3)...(19) 2'-O-Me ribo
sugar modification corresponding base 35 acaaacaaca cugucacauu cca
23 36 23 RNA Artificial Sequence Synthetically generated
oligonucleotide 36 acaaacacca cugucacauu cca 23 37 23 RNA
Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 2, 20, 21, 22, 23 2'-O-Me ribo sugar modification
phosphorothioate linkage corresponding base modified_base
(3)...(19) 2'-O-Me ribo sugar modification corresponding base 37
acaaacacca uugucacauu cca 23 38 23 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base 1, 20, 21,
22, 23 2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base modified_base (2)...(19) 2'-O-Me ribo sugar
modification corresponding base 38 acaaacacca uugucacacu cca 23 39
23 RNA Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 20, 21, 22, 23 2'-O-Me ribo sugar modification
phosphorothioate linkage corresponding base modified_base
(2)...(19) 2'-O-Me ribo sugar modification corresponding base 39
acacacaaca cugucacauu cca 23 40 23 RNA Artificial Sequence
Synthetically generated oligonucleotide 40 acaaacacca uugucacacu
cca 23 41 21 RNA Artificial Sequence Synthetically generated
oligonucleotide 41 uaaggcacgc ggugaaugcc a 21 42 35 DNA Artificial
Sequence Primer 42 agtcagatgt acagttataa gcacaagagg accag 35 43 25
DNA Artificial Sequence Primer 43 ttattcaaga tcccggggct cttcc 25 44
25 DNA Artificial Sequence Primer 44 ccagagctga actaaggctg ctcca 25
45 23 RNA Artificial Sequence Synthetically generated
oligonucleotide modified_base 23 2'-O-methyl modification
phosphorothioate linkage corresponding base modified_base
(1)...(22) 2'-O-methyl modified nucleotides corresponding base 45
acaaacacca uugucacacu cca 23 46 23 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base 1, 2, 20, 21,
22, 23 2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base modified_base (3)...(19) 2'-O-Me ribo sugar
modification corresponding base 46 ccaaacacca uugucacacu cca 23 47
23 RNA Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 2, 20, 21, 22, 23 2'-O-Me ribo sugar modification
phosphorothioate linkage corresponding base modified_base
(3)...(19) 2'-O-Me ribo sugar modification corresponding base 47
acaaacacca cugucacacu cca 23 48 23 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base 13, 14
2'-O-Me ribo sugar modification corresponding base 48 uggaguguga
caaugguguu ugu 23 49 23 RNA Artificial Sequence Synthetically
generated oligonucleotide modified_base 1, 2, 20, 21, 22 2'-O-Me
ribo sugar modification phosphorothioate linkage corresponding base
49 uggaguguga caaugguguu ugu 23 50 23 RNA Artificial Sequence
Synthetically generated oligonucleotide modified_base 1, 20, 21,
22, 23 2'-O-Me ribo sugar modification phosphorothioate linkage
corresponding base 50 acaaacacca uugucacacu cca 23 51 23 RNA
Artificial Sequence Synthetically generated oligonucleotide
modified_base 1, 20, 21, 22, 23 2'-O-Me ribo sugar modification
phosphorothioate linkage corresponding base 51 acacacaaca
cugucacauu cca 23
* * * * *