U.S. patent application number 17/398490 was filed with the patent office on 2022-01-27 for conjugated antisense compounds and their use.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Michael Oestergaard, Punit P. Seth, Eric E. Swayze.
Application Number | 20220023429 17/398490 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-27 |
United States Patent
Application |
20220023429 |
Kind Code |
A1 |
Seth; Punit P. ; et
al. |
January 27, 2022 |
CONJUGATED ANTISENSE COMPOUNDS AND THEIR USE
Abstract
The present disclosure provides duplexes comprising a first
oligomeric compound and a second oligomeric compound wherein the
second oligomeric compound comprises a conjugate group. In certain
embodiments, the duplex modulates the amount or activity of a
target nucleic acid in extra hepatic tissues and/or extra hepatic
cells. In certain embodiments, the duplex modulates the amount or
activity of a target nucleic acid in hepatic tissues and/or hepatic
cells.
Inventors: |
Seth; Punit P.; (Carlsbad,
CA) ; Oestergaard; Michael; (Carlsbad, CA) ;
Swayze; Eric E.; (Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Appl. No.: |
17/398490 |
Filed: |
August 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15761786 |
Mar 20, 2018 |
11116843 |
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PCT/US2016/053836 |
Sep 26, 2016 |
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17398490 |
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62233292 |
Sep 25, 2015 |
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International
Class: |
A61K 47/55 20060101
A61K047/55; A61K 47/54 20060101 A61K047/54; A61K 9/00 20060101
A61K009/00; A61K 31/7105 20060101 A61K031/7105; C12N 15/11 20060101
C12N015/11; C12N 15/113 20060101 C12N015/113 |
Claims
1-377. (canceled)
378. A duplex comprising a first oligomeric compound and a second
oligomeric compound wherein: the first oligomeric compound
comprises a first modified oligonucleotide consisting of 10-30
linked nucleosides, having a nucleobase sequence that is at least
80% complementary to the nucleobase sequence of the second
oligomeric compound and that is at least 80% complementary to an
extra-hepatic nucleic acid target; wherein neither the first
oligomeric compound nor the duplex is an RNAi compound; the second
oligomeric compound comprises a conjugate group and a second
modified oligonucleotide consisting of 10-30 linked nucleosides;
wherein the conjugate group comprises a conjugate moiety and a
conjugate linker, wherein the conjugate moiety is cholesterol; and
wherein the conjugate linker comprises at least one cleavable
moiety; and wherein the extra-hepatic nucleic acid target is
expressed in the brain or spinal cord.
379. The duplex of claim 378, wherein the first modified
oligonucleotide comprises at least one modified nucleoside.
380. The duplex of claim 379, wherein the at least one modified
nucleoside of the first modified oligonucleotide comprises a
bicyclic sugar moiety.
381. The duplex of claim 380, wherein at least one bicyclic sugar
moiety comprises a 2'-4' bridge, wherein the 2'-4' bridge is
selected from --O--CH.sub.2--; and --O--CH(CH.sub.3)--.
382. The duplex of claim 379, wherein the at least one modified
nucleoside of the first modified oligonucleotide comprises a
non-bicyclic sugar modification.
383. The duplex of claim 382, wherein at least one non-bicyclic
sugar modification is selected from 2'-MOE and 2'-OMe.
384. The duplex of claim 378, wherein the first modified
oligonucleotide comprises a sugar motif having: a 5'-region
consisting of 1-5 linked 5'-nucleosides; a central region
consisting of 6-10 linked central region nucleosides; and a
3'-region consisting of 1-5 linked 3'-region nucleosides; wherein
the nucleosides of the 5'-region, the 3'-region, and the central
region are contiguous, and the central region nucleosides each
comprise an unmodified DNA sugar moiety.
385. The duplex of claim 384, wherein each of the 5'-region
nucleosides and each of the 3'-region nucleosides comprise a
modified sugar moiety.
386. The duplex of claim 378, wherein the second modified
oligonucleotide comprises at least one modified nucleoside.
387. The duplex of claim 386, wherein the at least one modified
nucleoside of the first modified oligonucleotide comprises a
non-bicyclic sugar modification.
388. The duplex of claim 387, wherein at least one non-bicyclic
sugar modification is selected from 2'-MOE and 2'-OMe.
389. The duplex of claim 378, wherein the second modified
oligonucleotide comprises a sugar motif having: a 5'-region
consisting of 1-5 linked 5'-nucleosides; a central region
consisting of 6-10 linked central region nucleosides; and a
3'-region consisting of 1-5 linked 3'-region nucleosides; wherein
the nucleosides of the 5'-region, the 3'-region, and the central
region are contiguous, and the central region nucleosides each
comprise an unmodified RNA sugar moiety.
390. The duplex of claim 389, wherein each of the 5'-region
nucleosides and each of the 3'-region nucleosides comprise a
modified sugar moiety.
391. The duplex of claim 378, wherein the first modified
oligonucleotide comprises at least one modified internucleoside
linkage.
392. The duplex of claim 391, wherein the at least one modified
internucleoside linkage is a phosphorothioate internucleoside
linkage.
393. The duplex of claim 392, wherein each internucleoside linkage
of the first modified oligonucleotide is a phosphorothioate
internucleoside linkage.
394. The duplex of claim 392, wherein each internucleoside linkage
of the first modified oligonucleotide is either an unmodified
phosphodiester internucleoside linkage or a phosphorothioate
internucleoside linkage.
395. The duplex of claim 378, wherein the second modified
oligonucleotide comprises at least one modified internucleoside
linkage.
396. The duplex of claim 395, wherein the at least one modified
internucleoside linkage is a phosphorothioate internucleoside
linkage.
397. The duplex of claim 396, wherein each internucleoside linkage
of the second modified oligonucleotide is either an unmodified
phosphodiester internucleoside linkage or a phosphorothioate
internucleoside linkage.
398. The duplex of claim 397, wherein 1-3 terminal internucleoside
linkages of the second modified oligonucleotide are
phosphorothioate internucleoside linkages, and each remaining
internucleoside linkage of the second modified oligonucleotide is a
phosphodiester internucleoside linkage.
399. The duplex of claim 378, wherein the first modified
oligonucleotide and the second modified oligonucleotide are the
same length, and each modified oligonucleotide consists of 12-16,
14-16, 14-18, 16-18, 14-20, 16-20, 18-20, 18-22, 14, 16, 18, or 20
linked nucleosides.
400. The duplex of claim 378, wherein the cleavable moiety is a
phosphate group.
401. The duplex of claim 378, wherein the extra-hepatic nucleic
acid target is not expressed in the liver at a significant level.
Description
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled CORE0137USC1SEQ_ST25.txt, created on Aug. 9, 2021,
which is 8 Kb in size. The information in the electronic format of
the sequence listing is incorporated herein by reference in its
entirety.
FIELD
[0002] The present disclosure provides duplexes comprising a first
oligomeric compound and a second oligomeric compound wherein the
second oligomeric compound comprises a conjugate group. In certain
embodiments, the duplex modulates the amount or activity of a
target nucleic acid in extra hepatic tissues and/or extra hepatic
cells. In certain embodiments, the duplex modulates the amount or
activity of a target nucleic acid in hepatic tissues and/or hepatic
cells.
BACKGROUND
[0003] The principle behind antisense technology is that an
antisense compound hybridizes to a target nucleic acid and
modulates the amount, activity, and/or function of the target
nucleic acid. For example in certain instances, antisense compounds
result in altered transcription or translation of a target. Such
modulation of expression can be achieved by, for example, target
mRNA degradation or occupancy-based inhibition. An example of
modulation of RNA target function by degradation is RNase H-based
degradation of the target RNA upon hybridization with a DNA-like
antisense compound. Another example of modulation of gene
expression by target degradation is RNA interference (RNAi). RNAi
refers to antisense-mediated gene silencing through a mechanism
that utilizes the RNA-induced silencing complex (RISC). An
additional example of modulation of RNA target function is by an
occupancy-based mechanism such as is employed naturally by
microRNA. MicroRNAs are small non-coding RNAs that regulate the
expression of protein-coding RNAs. The binding of an antisense
compound to a microRNA prevents that microRNA from binding to its
messenger RNA targets, and thus interferes with the function of the
microRNA. MicroRNA mimics can enhance native microRNA function.
Certain antisense compounds alter splicing of pre-mRNA. Regardless
of the specific mechanism, sequence-specificity makes antisense
compounds attractive as tools for target validation and gene
functionalization, as well as therapeutics to selectively modulate
the expression of genes involved in the pathogenesis of
diseases.
[0004] Antisense technology is an effective means for modulating
the expression of one or more specific gene products and can
therefore prove to be uniquely useful in a number of therapeutic,
diagnostic, and research applications. Chemically modified
nucleosides may be incorporated into antisense compounds to enhance
one or more properties, such as nuclease resistance,
pharmacokinetics or affinity for a target nucleic acid. In 1998,
the antisense compound, Vitravene.RTM. (fomivirsen; developed by
Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first
antisense drug to achieve marketing clearance from the U.S. Food
and Drug Administration (FDA), and is currently a treatment of
cytomegalovirus (CMV)-induced retinitis in AIDS patients. For
another example, an antisense oligonucleotide targeting ApoB,
KYNAMRO.TM., has been approved by the U.S. Food and Drug
Administration (FDA) as an adjunct treatment to lipid-lowering
medications and diet to reduce low density lipoprotein-cholesterol
(LDL-C), ApoB, total cholesterol (TC), and non-high density
lipoprotein-cholesterol (non HDL-C) in patients with homozygous
familial hypercholesterolemia (HoFH).
[0005] New chemical modifications have improved the potency and
efficacy of antisense compounds, uncovering the potential for oral
delivery as well as enhancing subcutaneous administration,
decreasing potential for side effects, and leading to improvements
in patient convenience. Chemical modifications increasing potency
of antisense compounds allow administration of lower doses, which
reduces the potential for toxicity, as well as decreasing overall
cost of therapy. Modifications increasing the resistance to
degradation result in slower clearance from the body, allowing for
less frequent dosing. Different types of chemical modifications can
be combined in one compound to further optimize the compound's
efficacy. Traditionally, antisense compounds, including modified
oligonucleotides, have deomonstrated good functional uptake into
liver tissue. However, there is still a need to facilitate uptake
and distribution of antisense compounds into other cell types.
SUMMARY OF THE INVENTION
[0006] After an oligomeric compound is administered to a subject,
different organs, tissues, and cells receive different amounts of
the oligomeric compound. The distribution of the oligomeric
compound to different organs, tissues, and cells depends on many
factors. For example, the degree to which a given oligomeric
compound binds to plasma proteins may affect the distribution of a
given oligomeric compound to various tissues. In certain
embodiments, the degree to which a given oligomeric compound is
recognized by certain cell-surface receptors may affect the
distribution of a given oligomeric compound to various tissues or
cells.
[0007] Oligomeric compounds typically show good distribution to the
liver after administration to a subject. However, in certain
embodiments a need exists to deliver oligomeric compounds to other
tissues within a subject. For example, a need exists to deliver
oligomeric compounds to one or more extra-hepatic tissues such as
adipose tissue or muscle tissue. In certain embodiments, the
present disclosure provides oligomeric compounds comprising a
modified oligonucleotide and a conjugate group, wherein the
conjugate group enhances delivery of the modified oligonucleotide
to one or more extra-hepatic tissues.
[0008] In certain embodiments, the present disclosure provides a
duplex comprising a first oligomeric compound and a second
oligomeric compound, wherein the second oligomeric compound
comprises a modified oligonucleotide and a conjugate group. In
certain embodiments, the duplex modulates the amount or activity of
a target nucleic acid transcript in an extra-hepatic cell to a
greater extent than a duplex having a second oligomeric compound
that does not comprise a conjugate.
[0009] The present disclosure provides the following non-limiting
numbered embodiments: [0010] Embodiment 1: A duplex comprising a
first oligomeric compound and a second oligomeric compound wherein
[0011] the first oligomeric compound comprises a first modified
oligonucleotide consisting of 10-30 linked nucleosides and has a
nucleobase sequence complementary to the nucleobase sequence of the
second oligomeric compound and to an extra-hepatic nucleic acid
target; and [0012] the second oligomeric compound comprises a
second modified oligonucleotide consisting of 10-30 linked
nucleosides and a conjugate group; [0013] wherein the conjugate
group comprises a conjugate moiety and a conjugate linker, [0014]
wherein the conjugate moiety is selected from among: a lipid,
vitamin, steroid, C.sub.5-C.sub.30 saturated alkyl group,
C.sub.5-C.sub.30 unsaturated alkyl group, fatty acid, and
lipophilic group; [0015] and wherein the conjugate linker comprises
at least one cleavable moiety. [0016] Embodiment 2: A duplex
comprising a first oligomeric compound and a second oligomeric
compound wherein [0017] the first oligomeric compound comprises a
first modified oligonucleotide consisting of 10-30 linked
nucleosides and has a nucleobase sequence complementary to the
nucleobase sequence of the second oligomeric compound and to an
extra-hepatic nucleic acid target or a hepatic nucleic acid target;
and [0018] the second oligomeric compound comprises a second
modified oligonucleotide consisting of 10-30 linked nucleosides and
a conjugate group; [0019] wherein the modified oligonucleotide has
a sugar motif other than: [0020] 1-4 2'-OMethyl-modified
nucleosides at the 5'-end; [0021] 10-15 central nucleosides each
comprising an unmodified RNA sugar moiety; and [0022] 1-4
2'-OMethyl-modified nucleosides at the 3'-end; [0023] wherein the
conjugate group comprises a conjugate moiety and a conjugate
linker, [0024] wherein the conjugate moiety is selected from among:
a lipid, vitamin, steroid, C.sub.5-C.sub.30 saturated alkyl group,
C.sub.5-C.sub.30 unsaturated alkyl group, fatty acid, and
lipophilic group; [0025] and wherein the conjugate linker comprises
at least one cleavable moiety. [0026] Embodiment 3: A duplex
comprising a first oligomeric compound and a second oligomeric
compound wherein [0027] the first oligomeric compound comprises a
first modified oligonucleotide consisting of 10-30 linked
nucleosides and has a nucleobase sequence complementary to the
nucleobase sequence of the second oligomeric compound and to an
extra-hepatic nucleic acid target or a hepatic nucleic acid target;
and [0028] the second oligomeric compound comprises a second
modified oligonucleotide consisting of 10-30 linked nucleosides and
a conjugate group; [0029] wherein the conjugate group comprises a
conjugate moiety and a conjugate linker, [0030] wherein the
conjugate moiety is selected from among: a lipid, steroid,
C.sub.5-C.sub.30 saturated alkyl group, C.sub.5-C.sub.30
unsaturated alkyl group, fatty acid, and a lipophilic group other
than a vitamin; and wherein the conjugate linker comprises at least
one cleavable moiety. [0031] Embodiment 4: The duplex of any of
embodiments 1-3, wherein the extra-hepatic nucleic acid target is
not expressed in the liver at a significant level. [0032]
Embodiment 5: The duplex of any of embodiments 1-3, wherein the
extra-hepatic nucleic acid target is expressed in the liver at a
significant level. [0033] Embodiment 6: The duplex of any of
embodiments 1-5, wherein the extra-hepatic nucleic acid target is
expressed in at least one extra-hepatic cell type selected from
among: white fat cells, brown fat cells, adipocytes, macrophages,
cancer cells, tumor cells, smooth muscle cells, lymphocytes, heart
muscle cells, and pulmonary cells. [0034] Embodiment 7: The duplex
of any of embodiments 1-6, wherein the extra-hepatic nucleic acid
target is expressed in at least two extra-hepatic cell types.
[0035] Embodiment 8: The duplex of any of embodiments 1-7, wherein
the extra-hepatic nucleic acid target is expressed in at least
three extra-hepatic cell types. [0036] Embodiment 9: The duplex of
any of embodiments 1-8, wherein the extra-hepatic nucleic acid
target is expressed in at least four extra-hepatic cell types.
[0037] Embodiment 10: The duplex of any of embodiments 1-9, wherein
the extra-hepatic nucleic acid target is expressed in white fat
cells. [0038] Embodiment 11: The duplex of any of embodiments 1-10,
wherein the extra-hepatic nucleic acid target is expressed in brown
fat cells [0039] Embodiment 12: The duplex of any of embodiments
1-11, wherein the extra-hepatic nucleic acid target is expressed in
adipocytes. [0040] Embodiment 13: The duplex of any of embodiments
1-12, wherein the extra-hepatic nucleic acid target is expressed in
macrophages. [0041] Embodiment 14: The duplex of any of embodiments
1-13, wherein the extra-hepatic nucleic acid target is expressed in
cancer cells. [0042] Embodiment 15: The duplex of any of
embodiments 1-14, wherein the extra-hepatic nucleic acid target is
expressed in tumor cells. [0043] Embodiment 16: The duplex of any
of embodiments 1-15, wherein the extra-hepatic nucleic acid target
is expressed in smooth muscle cells [0044] Embodiment 17: The
duplex of any of embodiments 1-16, wherein the extra-hepatic
nucleic acid target is expressed in heart muscle cells. [0045]
Embodiment 18: The duplex of any of embodiments 1-17, wherein the
extra-hepatic nucleic acid target is expressed in lymphocytes.
[0046] Embodiment 19: The duplex of any of embodiments 1-18,
wherein the extra-hepatic nucleic acid target is expressed in at
least one extra-hepatic tissue selected from among: skeletal
muscle, cardiac muscle, smooth muscle, adipose, white adipose,
brown adipose, spleen, bone, intestine, adrenal, testes, ovary,
pancreas, pituitary, prostate, skin, uterus, bladder, brain,
glomerulus, distal tubular epithelium, breast, lung, heart, kidney,
ganglion, frontal cortex, spinal cord, trigeminal ganglia, sciatic
nerve, dorsal root ganglion, epididymal fat, diaphragm, and colon.
[0047] Embodiment 20: The duplex of any of embodiments 1-19,
wherein the extra-hepatic nucleic acid target is expressed in at
least two extra-hepatic tissues. [0048] Embodiment 21: The duplex
of any of embodiments 1-20, wherein the extra-hepatic nucleic acid
target is expressed in at least three extra-hepatic tissues. [0049]
Embodiment 22: The duplex of any of embodiments 1-21, wherein the
extra-hepatic nucleic acid target is expressed in at least four
extra-hepatic tissues. [0050] Embodiment 23: The duplex of any of
embodiments 1-22, wherein the extra-hepatic nucleic acid target is
expressed in skeletal muscle. [0051] Embodiment 24: The duplex of
any of embodiments 1-23, wherein the extra-hepatic nucleic acid
target is expressed in cardiac muscle. [0052] Embodiment 25: The
duplex of any of embodiments 1-24, wherein the extra-hepatic
nucleic acid target is expressed in smooth muscle. [0053]
Embodiment 26: The duplex of any of embodiments 1-25, wherein the
extra-hepatic nucleic acid target is expressed in epididymal fat.
[0054] Embodiment 27: The duplex of any of embodiments 1-26,
wherein the extra-hepatic nucleic acid target is expressed in white
adipose tissue. [0055] Embodiment 28: The duplex of any of
embodiments 1-27, wherein the extra-hepatic nucleic acid target is
expressed in the spleen. [0056] Embodiment 29: The duplex of any of
embodiments 1-28, wherein the extra-hepatic nucleic acid target is
expressed in bone. [0057] Embodiment 30: The duplex of any of
embodiments 1-29, wherein the extra-hepatic nucleic acid target is
expressed in bone marrow. [0058] Embodiment 31: The duplex of any
of embodiments 1-30, wherein the extra-hepatic nucleic acid target
is expressed in the intestine. [0059] Embodiment 32: The duplex of
any of embodiments 1-31, wherein the extra-hepatic nucleic acid
target is expressed in adrenal tissue. [0060] Embodiment 33: The
duplex of any of embodiments 1-32, wherein the extra-hepatic
nucleic acid target is expressed in the testes. [0061] Embodiment
34: The duplex of any of embodiments 1-33, wherein the
extra-hepatic nucleic acid target is expressed in the ovaries.
[0062] Embodiment 35: The duplex of any of embodiments 1-34,
wherein the extra-hepatic nucleic acid target is expressed in the
pancreas. [0063] Embodiment 36: The duplex of any of embodiments
1-35, wherein the extra-hepatic nucleic acid target is expressed in
the pituitary. [0064] Embodiment 37: The duplex of any of
embodiments 1-36, wherein the extra-hepatic nucleic acid target is
expressed in the prostate. [0065] Embodiment 38: The duplex of any
of embodiments 1-37, wherein the extra-hepatic nucleic acid target
is expressed in the skin. [0066] Embodiment 39: The duplex of any
of embodiments 1-38, wherein the extra-hepatic nucleic acid target
is expressed in the uterus. [0067] Embodiment 40: The duplex of any
of embodiments 1-39, wherein the extra-hepatic nucleic acid target
is expressed in the bladder. [0068] Embodiment 41: The duplex of
any of embodiments 1-40, wherein the extra-hepatic nucleic acid
target is expressed in the brain. [0069] Embodiment 42: The duplex
of any of embodiments 1-41, wherein the extra-hepatic nucleic acid
target is expressed in the glomerulus. [0070] Embodiment 43: The
duplex of any of embodiments 1-42, wherein the extra-hepatic
nucleic acid target is expressed in the distal tubular epithelium.
[0071] Embodiment 44: The duplex of any of embodiments 1-43,
wherein the extra-hepatic nucleic acid target is expressed in the
breast. [0072] Embodiment 45: The duplex of any of embodiments
1-44, wherein the extra-hepatic nucleic acid target is expressed in
the lung. [0073] Embodiment 46: The duplex of any of embodiments
1-45, wherein the extra-hepatic nucleic acid target is expressed in
the heart. [0074] Embodiment 47: The duplex of any of embodiments
1-46, wherein the extra-hepatic nucleic acid target is expressed in
the kidney. [0075] Embodiment 48: The duplex of any of embodiments
1-47, wherein the extra-hepatic nucleic acid target is expressed in
the colon. [0076] Embodiment 49: The duplex of any of embodiments
1-48, wherein the extra-hepatic nucleic acid target is expressed in
the ganglion. [0077] Embodiment 50: The duplex of any of
embodiments 1-49, wherein the extra-hepatic nucleic acid target is
expressed in the frontal cortex. [0078] Embodiment 51: The duplex
of any of embodiments 1-50, wherein the extra-hepatic nucleic acid
target is expressed in the spinal cord. [0079] Embodiment 52: The
duplex of any of embodiments 1-51, wherein the extra-hepatic
nucleic acid target is expressed in the trigeminal ganglia. [0080]
Embodiment 53: The duplex of any of embodiments 1-52, wherein the
extra-hepatic nucleic acid target is expressed in the sciatic
nerve. [0081] Embodiment 54: The duplex of any of embodiments 1-53,
wherein the extra-hepatic nucleic acid target is expressed in the
dorsal root ganglion. [0082] Embodiment 55: The duplex of any of
embodiments 1-54, wherein the extra-hepatic nucleic acid target is
an endogenous RNA transcript. [0083] Embodiment 56: The duplex of
embodiment 55, wherein the RNA transcript is a pre-mRNA. [0084]
Embodiment 57: The duplex of embodiment 55, wherein the RNA
transcript is an mRNA. [0085] Embodiment 58: The duplex of
embodiment 55, wherein the RNA transcript is a toxic RNA. [0086]
Embodiment 59: The duplex of embodiment 55, wherein the RNA
transcript is a non-coding RNA. [0087] Embodiment 60: The duplex of
embodiment 55, wherein the RNA transcript is a microRNA. [0088]
Embodiment 61: The duplex of any of embodiments 1-54, wherein the
extra-hepatic nucleic acid target is viral nucleic acid. [0089]
Embodiment 62: The duplex of any of embodiments 1-58, wherein the
extra-hepatic nucleic acid target is selected from among: ATGL,
CD40, TNF-.alpha., CD36, DMPK, DNM2, DMD, DUX4, LMNA, ZFN9, SGLT2,
and GCCR. [0090] Embodiment 63: The duplex of any of embodiments
1-58, wherein the extra-hepatic nucleic acid target is selected
from among: Androgen Receptor (AR), ANGPTL3, DGAT2, eIF4E, Factor
XI, FGFR4, GCCR, GCGR, GHR, PTP1B, SMRT, STAT3, Them1, TRPV4, FTO,
MC4R, TMEM18, KCTD15, GNPDA2, SH2B1, MTCH2, NEGR1, BDNF, ETV5,
Leptin, leptin receptor, FAIM2, KCNMA1, MAF, NRXN3, TFAP2B, MSRA,
AGPAT2, BSCL2, AKT2, PPAR.gamma., LMNA, ZMPSTE24, DGAT1,
TNF.alpha., IL-6, Resistin, PAI-1, TBC1D1, METAP2, VEGF, AIF-1,
JNK1, CB1, RIP140, TIF2, ANGPT1, ANGPT2, EIF4EBP2, CDK5, SLC13A5,
Perilipin 1, Perilipin 2, Perilipin 3, Perilipin 4, HGF, GDF3,
TNKs, KATNA1, ChREBP, ATF4, BASP-1, NNMT. [0091] Embodiment 64: The
duplex of any of embodiments 1-61, wherein the extra-hepatic
nucleic acid target is other than any of: Androgen Receptor (AR),
ANGPTL3, DGAT2, eIF4E, Factor XI, FGFR4, GCCR, GCGR, GHR, PTP1B,
SMRT, STAT3, Them1, TRPV4, FTO, MC4R, TMEM18, KCTD15, GNPDA2,
SH2B1, MTCH2, NEGR1, BDNF, ETV5, Leptin, leptin receptor, FAIM2,
KCNMA1, MAF, NRXN3, TFAP2B, MSRA, AGPAT2, BSCL2, AKT2, PPAR.gamma.,
LMNA, ZMPSTE24, DGAT1, TNF.alpha., IL-6, Resistin, PAI-1, TBC1D1,
METAP2, VEGF, AIF-1, JNK1, CB1, RIP140, TIF2, ANGPT1, ANGPT2,
EIF4EBP2, CDK5, SLC13A5, Perilipin 1, Perilipin 2, Perilipin 3,
Perilipin 4, HGF, GDF3, TNKs, KATNA1, ChREBP, ATF4, BASP-1, NNMT.
[0092] Embodiment 65: The duplex of any of embodiments 1-64,
wherein the first modified oligonucleotide has a nucleobase
sequence that is at least 80% complementary to the nucleobase
sequence of the extra-hepatic nucleic acid target, when measured
across the entire nucleobase sequence of the modified
oligonucleotide. [0093] Embodiment 66: The duplex of embodiment 65,
wherein the first modified oligonucleotide has a nucleobase
sequence that is at least 90% complementary to the nucleobase
sequence of the extra-hepatic nucleic acid target, when measured
across the entire nucleobase sequence of the modified
oligonucleotide. [0094] Embodiment 67: The duplex of embodiment 65,
wherein the first modified oligonucleotide has a nucleobase
sequence that is 100% complementary to the nucleobase sequence of
the extra-hepatic nucleic acid target, when measured across the
entire nucleobase sequence of the modified oligonucleotide. [0095]
Embodiment 68: The duplex of any of embodiments 1-55, wherein the
first modified oligonucleotide has at least 8 contiguous
nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, or 23. [0096] Embodiment 69: The duplex of any of
embodiments 1-55, wherein the first modified oligonucleotide has at
least 9 contiguous nucleobases of any of the nucleobase sequences
of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, or 23. [0097] Embodiment 70: The duplex
of any of embodiments 1-55, wherein the first modified
oligonucleotide has at least 10 contiguous nucleobases of any of
the nucleobase sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
[0098] Embodiment 71: The duplex of any of embodiments 1-55,
wherein the first modified oligonucleotide consists of the
nucleobase sequence of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
[0099] Embodiment 72: The duplex of any of embodiments 1-55,
wherein the first modified oligonucleotide has at least 12
contiguous nucleobases of any of the nucleobase sequences of SEQ ID
NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, or 23. [0100] Embodiment 73: The duplex of any of
embodiments 1-72, wherein the first modified oligonucleotide does
not have any 2'-deoxynucleosides. [0101] Embodiment 74: The duplex
of any of embodiments 1-72, wherein the first modified
oligonucleotide comprises at least one modified nucleoside. [0102]
Embodiment 75: The duplex of embodiment 74, wherein the first
modified oligonucleotide comprises a least one modified nucleoside
comprising a modified sugar moiety. [0103] Embodiment 76: The
duplex of embodiment 75, wherein the first modified oligonucleotide
comprises at least one modified nucleoside comprising a bicyclic
sugar moiety. [0104] Embodiment 77: The duplex of embodiment 76,
wherein the first modified oligonucleotide comprises at least one
modified nucleoside comprising a bicyclic sugar moiety having a
2'-4' bridge, wherein the 2'-4' bridge is selected from
--O--CH.sub.2--; and --O--CH(CH.sub.3)--. [0105] Embodiment 78: The
duplex of any of embodiments 73-77, wherein the first modified
oligonucleotide comprises at least one modified nucleoside
comprising a modified non-bicyclic sugar moiety. [0106] Embodiment
79: The duplex of embodiment 78, wherein the first modified
oligonucleotide comprises at least one modified nucleoside
comprising a non-bicyclic sugar moiety comprising a 2'-MOE or
2'-O-Methyl modified sugar moiety. [0107] Embodiment 80: The duplex
of any of embodiments 73-79, wherein the first modified
oligonucleotide comprises at least one modified nucleoside
comprising a sugar surrogate. [0108] Embodiment 81: The duplex of
embodiment 80, wherein the first modified oligonucleotide comprises
at least one modified nucleoside comprising a sugar surrogate
selected from a morpholino, a PNA, a F-HNA, a THP, or a modified
THP. [0109] Embodiment 82: The duplex of any of embodiments 1-72 or
74-81, wherein the first modified oligonucleotide comprises a sugar
motif having: [0110] a 5'-region consisting of 1-5 linked
5'-nucleosides; [0111] a central region consisting of 6-10 linked
central region nucleosides; and [0112] a 3'-region consisting of
1-5 linked 3'-region nucleosides; wherein [0113] the nucleosides of
the 5'-region, the 3'-region, and the central region are
contiguous, and the central region nucleosides each comprise an
unmodified DNA sugar moiety. [0114] Embodiment 83: The duplex of
embodiment 82, wherein each of the 5'-region nucleosides and each
of the 3'-region nucleosides comprise a modified sugar moiety.
[0115] Embodiment 84: The duplex of any of embodiments 1-83,
wherein the first modified oligonucleotide comprises at least one
modified internucleoside linkage. [0116] Embodiment 85: The duplex
of embodiment 84, wherein each internucleoside linkage of the first
modified oligonucleotide is a modified internucleoside linkage.
[0117] Embodiment 86: The duplex of embodiment 84 or 85 wherein at
least one internucleoside linkage is a phosphorothioate
internucleoside linkage. [0118] Embodiment 87: The duplex of
embodiment 84 or 86 wherein the first modified oligonucleotide
comprises at least one unmodified phosphodiester internucleoside
linkage. [0119] Embodiment 88: The duplex of embodiment 87, wherein
each internucleoside linkage is either an unmodified phosphodiester
internucleoside linkage or a phosphorothioate internucleoside
linkage. [0120] Embodiment 89: The duplex of embodiment 85, wherein
each internucleoside linkage is a phosphorothioate internucleoside
linkage. [0121] Embodiment 90: The duplex of any of embodiments
1-89, wherein the first modified oligonucleotide comprises at least
one modified nucleobase. [0122] Embodiment 91: The duplex of
embodiment 90, wherein the first modified nucleobase is a 5-Methyl
cytosine. [0123] Embodiment 92: The duplex of any of embodiments
1-91 wherein each nucleobase of each nucleoside of the first
modified oligonucleotide is either an unmodified nucleobase or is
5-Methyl cytosine. [0124] Embodiment 93: The duplex of any of
embodiments 1-92, wherein the first modified oligonucleotide
consists of 12-22 linked nucleosides. [0125] Embodiment 94: The
duplex of any of embodiments 1-92, wherein the first modified
oligonucleotide consists of 12-20 linked nucleosides. [0126]
Embodiment 95: The duplex of any of embodiments 1-92, wherein the
first modified oligonucleotide consists of 14-20 linked
nucleosides. [0127] Embodiment 96: The duplex of any of embodiments
1-92, wherein the first modified oligonucleotide consists of 16-20
linked nucleosides. [0128] Embodiment 97: The duplex of any of
embodiments 1-92, wherein the first modified oligonucleotide
consists of 18-20 linked nucleosides. [0129] Embodiment 98: The
duplex of any of embodiments 1-92, wherein the first modified
oligonucleotide consists of 20 linked nucleosides. [0130]
Embodiment 99: The duplex of any of embodiments 1-92, wherein the
first modified oligonucleotide consists of 19 linked nucleosides.
[0131] Embodiment 100: The duplex of any of embodiments 1-92,
wherein the first modified oligonucleotide consists of 18 linked
nucleosides. [0132] Embodiment 101: The duplex of any of
embodiments 1-92, wherein the first modified oligonucleotide
consists of 17 linked nucleosides. [0133] Embodiment 102: The
duplex of any of embodiments 1-92, wherein the first modified
oligonucleotide consists of 16 linked nucleosides. [0134]
Embodiment 103: The duplex of any of embodiments 1-102, wherein the
second modified oligonucleotide does not have any
2'deoxynucleosides. [0135] Embodiment 104: The duplex of any of
embodiments 1-103, wherein the second modified oligonucleotide
comprises at least one modified nucleoside. [0136] Embodiment 105:
The duplex of embodiment 104, wherein the second modified
oligonucleotide comprises a least one modified nucleoside
comprising a modified sugar moiety. [0137] Embodiment 106: The
duplex of embodiment 105, wherein the second modified
oligonucleotide comprises at least one modified nucleoside
comprising a bicyclic sugar moiety. [0138] Embodiment 107: The
duplex of embodiment 106, wherein the second modified
oligonucleotide comprises at least one modified nucleoside
comprising a bicyclic sugar moiety having a 2'-4' bridge, wherein
the 2'-4' bridge is selected from --O--CH.sub.2--; and
--O--CH(CH.sub.3)--. [0139] Embodiment 108: The duplex of any of
embodiments 103-107, wherein the second modified oligonucleotide
comprises at least one modified nucleoside comprising a modified
non-bicyclic sugar moiety. [0140] Embodiment 109: The duplex of
embodiment 108, wherein the first modified oligonucleotide
comprises at least one modified nucleoside comprising a
non-bicyclic sugar moiety comprising a 2'-MOE or 2'-O-Methyl
modification. [0141] Embodiment 110: The duplex of any of
embodiments 103-109 wherein the second modified oligonucleotide
does not comprise any nucleosides comprising 2'-OMe. [0142]
Embodiment 111: The duplex of any of embodiments 103-110, wherein
the second modified oligonucleotide comprises at least one modified
nucleoside comprising a sugar surrogate. [0143] Embodiment 112: The
duplex of embodiment 111, wherein the second modified
oligonucleotide comprises at least one modified nucleoside
comprising a sugar surrogate selected from a morpholino, a PNA, a
F-HNA, a THP, or a modified THP. [0144] Embodiment 113: The duplex
of any of embodiments 104-112, wherein the second modified
oligonucleotide has a sugar motif comprising: [0145] a 5'-region
consisting of 1-5 linked 5'-nucleosides; [0146] a central region
consisting of 6-10 linked central region nucleosides; and [0147] a
3'-region consisting of 1-5 linked 3'-region nucleosides; wherein
[0148] the nucleosides of the 5'-region, the 3'-region, and the
central region are contiguous; and the central region nucleosides
each comprise an unmodified 2'-deoxy sugar moiety. [0149]
Embodiment 114: The duplex of embodiment 113 wherein each of the
5'-region nucleosides and each of the 3'-region nucleosides
comprise a modified sugar moiety. [0150] Embodiment 115: The duplex
of embodiment 114, wherein the 5'-region nucleosides and the
3'-region nucleosides of the second modified oligonucleotide are
selected from: F-RNA modified nucleosides, 2'-MOE modified
nucleosides, LNA nucleosides, and cEt nucleosides. [0151]
Embodiment 116: The duplex of any of embodiments 104-112 wherein
the second modified oligonucleotide has sugar motif comprising
alternating 2'-deoxynucleosides and 2'-MOE modified nucleosides.
[0152] Embodiment 117: The duplex of any of embodiments 103-116,
wherein the second modified oligonucleotide comprises at least one
modified internucleoside linkage. [0153] Embodiment 118: The duplex
of embodiment 117, wherein each internucleoside linkage of the
second modified oligonucleotide is a modified internucleoside
linkage. [0154] Embodiment 119: The duplex of embodiment 117 or 118
wherein at least one internucleoside linkage of the second
oligonucleotide is a phosphorothioate internucleoside linkage.
[0155] Embodiment 120: The duplex of embodiment 117 or 119 wherein
the second modified oligonucleotide comprises at least one
unmodified phosphodiester internucleoside linkage. [0156]
Embodiment 121: The duplex of embodiment 120, wherein each
internucleoside linkage of the second oligonucleotide is either an
unmodified phosphodiester internucleoside linkage or a
phosphorothioate internucleoside linkage. [0157] Embodiment 122:
The duplex of embodiment 118, wherein each internucleoside linkage
of the second oligonucleotide is a phosphorothioate internucleoside
linkage. [0158] Embodiment 123: The duplex of any of embodiments
117 or 119-121 wherein the second oligonucleotide has a center
region comprising unmodified phosphodiester internucleoside
linkages. [0159] Embodiment 124: The duplex of embodiment 123,
wherein the second oligonucleotide has 1 or 2 terminal
phosphorothioate linkages on one end or on both ends. [0160]
Embodiment 125: The duplex of any of embodiments 103-124, wherein
the second modified oligonucleotide comprises at least one modified
nucleobase. [0161] Embodiment 126: The duplex of embodiment 125,
wherein the second modified nucleobase is a 5-Methyl cytosine.
[0162] Embodiment 127: The duplex of any of embodiments 103-126
wherein each nucleobase of each nucleoside of the second modified
oligonucleotide is either an unmodified nucleobase or is 5-Methyl
cytosine. [0163] Embodiment 128: The duplex of any of embodiments
103-127, wherein the second modified oligonucleotide consists of
12-22 linked nucleosides. [0164] Embodiment 129: The duplex of any
of embodiments 103-127, wherein the second modified oligonucleotide
consists of 12-20 linked nucleosides. [0165] Embodiment 130: The
duplex of any of embodiments 103-127, wherein the second modified
oligonucleotide consists of 14-20 linked nucleosides. [0166]
Embodiment 131: The duplex of any of embodiments 103-127, wherein
the second modified oligonucleotide consists of 16-20 linked
nucleosides. [0167] Embodiment 132: The duplex of any of
embodiments 103-127, wherein the second modified oligonucleotide
consists of 18-20 linked nucleosides. [0168] Embodiment 133: The
duplex of any of embodiments 103-127, wherein the second modified
oligonucleotide consists of 20 linked nucleosides. [0169]
Embodiment 134: The duplex of any of embodiments 103-127, wherein
the second modified oligonucleotide consists of 19 linked
nucleosides. [0170] Embodiment 135: The duplex of any of
embodiments 103-127, wherein the second modified oligonucleotide
consists of 18 linked nucleosides. [0171] Embodiment 136: The
duplex of any of embodiments 103-127, wherein the second modified
oligonucleotide consists of 17 linked nucleosides. [0172]
Embodiment 137: The duplex of any of embodiments 103-127, wherein
the second modified oligonucleotide consists of 16 linked
nucleosides. [0173] Embodiment 138: The duplex of any of
embodiments 1-137, wherein the conjugate linker comprises 1-5
linker-nucleosides. [0174] Embodiment 139: The duplex of embodiment
138, wherein the conjugate linker comprises 3 linker-nucleosides.
[0175] Embodiment 140: The duplex of embodiment 139, wherein the 3
linker-nucleosides have a TCA motif. [0176] Embodiment 141: The
duplex of embodiment 138, wherein 1-5 linker-nucleosides do not
comprise a TCA motif. [0177] Embodiment 142: The duplex of any of
embodiments 1-137, wherein the conjugate group does not comprise
linker-nucleosides. [0178] Embodiment 143: The duplex of any of
embodiments 1-142, wherein the conjugate linker comprises a
hexylamino group. [0179] Embodiment 144: The duplex of any of
embodiments 1-143, wherein the conjugate linker comprises a
polyethylene glycol group. [0180] Embodiment 145: The duplex of any
of embodiments 1-144, wherein the conjugate linker comprises a
triethylene glycol group. [0181] Embodiment 146: The duplex of any
of embodiments 1-145, wherein the conjugate linker comprises a
phosphate group. [0182] Embodiment 147: The duplex of any of
embodiments 1-146, wherein the conjugate linker comprises:
[0182] ##STR00001## [0183] X directly or indirectly attaches to the
conjugate moiety; and [0184] Y directly or indirectly attaches to
the second modified oligonucleotide. [0185] Embodiment 148: The
duplex of embodiment 147, wherein X comprises 0. [0186] Embodiment
149: The duplex of embodiment 147 or 148, wherein Y comprises a
phosphate group. [0187] Embodiment 150: The duplex of any of
embodiments 1-146, wherein the conjugate linker comprises:
[0187] ##STR00002## [0188] X directly or indirectly attaches to the
conjugate moiety; and [0189] T.sub.1 comprises a linking group,
nucleoside, or a modified oligonucleotide. [0190] Embodiment 151:
The duplex of any of embodiments 1-146, wherein the conjugate
linker comprises:
[0190] ##STR00003## [0191] X directly or indirectly attaches to the
conjugate moiety; and [0192] wherein T.sub.1 comprises a nucleotide
or a modified oligonucleotide; and B.sub.x is a modified or
unmodified nucleobase. [0193] Embodiment 152: The duplex of any of
embodiments 1-151, wherein the conjugate moiety comprises
lipophilic group. [0194] Embodiment 153: The duplex of embodiment
152, wherein the lipophilic group is selected from among:
cholesterol, C.sub.10-C.sub.26 saturated fatty acid,
C.sub.10-C.sub.26 unsaturated fatty acid, C.sub.10-C.sub.26 alkyl,
triglyceride, tocopherol, or cholic acid. [0195] Embodiment 154:
The duplex of embodiment 153, wherein the conjugate moiety is a
saturated fatty acid or an unsaturated fatty acid. [0196]
Embodiment 155: The duplex of embodiment 153, wherein the conjugate
moiety is C.sub.16 lipid. [0197] Embodiment 156: The duplex of
embodiment 153, wherein the conjugate moiety is C.sub.18 lipid.
[0198] Embodiment 157: The duplex of embodiment 153, wherein the
conjugate moiety is C.sub.16 alkyl. [0199] Embodiment 158: The
duplex of embodiment 153, wherein the conjugate moiety is C.sub.18
alkyl. [0200] Embodiment 159: The duplex of embodiment 153, wherein
the conjugate moiety is cholesterol. [0201] Embodiment 160: The
duplex of embodiment 153, wherein the conjugate moiety is
tocopherol. [0202] Embodiment 161: The duplex of any of embodiments
1-160, wherein the conjugate group is attached to the second
modified oligonucleotide at the 5'-end of the second modified
oligonucleotide. [0203] Embodiment 162: The duplex of any of
embodiments 1-160, wherein the conjugate group is attached to the
second modified oligonucleotide at the 3'-end of the second
modified oligonucleotide. [0204] Embodiment 163: The duplex of any
of embodiments 1-162 comprising a terminal group. [0205] Embodiment
164: An antisense compound consisting of the duplex of any of
embodiments 1-163. [0206] Embodiment 165: An antisense compound
comprising the duplex of any of embodiments 1-163. [0207]
Embodiment 166: The antisense compound of embodiment 164 or 165
that is an RNase H antisense compound. [0208] Embodiment 167: The
antisense compound of embodiment 164 or 165 that is an RNAi
antisense compound. [0209] Embodiment 168: The antisense compound
of any of embodiments 164-167 that is capable of reducing the
amount or activity of the extra-hepatic nucleic acid target by at
least 20% when tested at a concentration of 1.0 nM in a standard
cell assay. [0210] Embodiment 169: The antisense compound of
embodiment 168 that is capable of reducing the amount or activity
of the extra-hepatic nucleic acid target by at least 40% in the
standard cell assay. [0211] Embodiment 170: The antisense compound
of embodiment 168 that is capable of reducing the amount or
activity of the extra-hepatic nucleic acid target by at least 80%
in the standard cell assay. [0212] Embodiment 171: The antisense
compound of any of embodiments 164-170 that is capable of reducing
the amount or activity of the extra-hepatic nucleic acid target in
an extra-hepatic tissue by at least 20% when provided at a dose of
100 mg/kg in a standard animal experiment. [0213] Embodiment 172:
The antisense compound of embodiment 171 that is capable of
reducing the amount or activity of the extra-hepatic nucleic acid
target in the extra-hepatic tissue by at least 40%. [0214]
Embodiment 173: The antisense compound of embodiment 171 that is
capable of reducing the amount or activity of the extra-hepatic
nucleic acid target in the extra-hepatic tissue by at least 80%.
[0215] Embodiment 174: The antisense compound of embodiment 164 or
165 that alters the RNA processing of the extra-hepatic nucleic
acid target. [0216] Embodiment 175: A method comprising contacting
a cell with the duplex of any of embodiments 1-163. [0217]
Embodiment 176: A method comprising contacting a cell with the
antisense compound of any of embodiments 164-174. [0218] Embodiment
177: A method of modulating the amount or activity of an
extra-hepatic nucleic acid target in a cell comprising contacting
the cell with the duplex or antisense compound of any of
embodiments 1-174 and thereby modulating the amount or activity of
the extra-hepatic nucleic acid target in the cell. [0219]
Embodiment 178: The method of embodiment 177, wherein the amount or
activity of the extra-hepatic nucleic acid target is reduced.
[0220] Embodiment 179: The method of any of embodiments 175-178,
wherein the cell is in vitro. [0221] Embodiment 180: The method of
any of embodiments 175-178, wherein the cell is in an animal.
[0222] Embodiment 181: The method of embodiment 180, wherein the
animal is a human. [0223] Embodiment 182: A method of modulating
the amount or activity of a hepatic nucleic acid target in a cell
comprising contacting the cell with the duplex or antisense
compound of any of embodiments 1-174 and thereby modulating the
amount or activity of the hepatic nucleic acid target in the cell.
[0224] Embodiment 183: The method of embodiment 182, wherein the
amount or activity of the extra-hepatic nucleic acid target is
reduced. [0225] Embodiment 184: The method of embodiment 182 or
183, wherein the cell is in vitro. [0226] Embodiment 185: The
method of embodiment 182 or 183, wherein the cell is in an animal.
[0227] Embodiment 186: The method of embodiment 185, wherein the
animal is a human. [0228] Embodiment 187: A pharmaceutical
composition comprising a duplex of any embodiments 1-163 and a
pharmaceutically acceptable carrier or diluent. [0229] Embodiment
188: A pharmaceutical composition comprising an antisense compound
of any of embodiments 164-174 and a pharmaceutically acceptable
carrier or diluent. [0230] Embodiment 189: A method comprising
administering to an animal a pharmaceutical composition of
embodiment 187 or 188. [0231] Embodiment 190: A method of treating
a disease associated with an extra-hepatic nucleic acid target
comprising administering to an individual having or at risk for
developing a disease associated with the extra-hepatic nucleic acid
target a therapeutically effective amount of a pharmaceutical
composition according to embodiment 187 or 188; and thereby
treating the disease associated with the extra-hepatic nucleic acid
target. [0232] Embodiment 191: The method of embodiment 190,
wherein the extra-hepatic nucleic acid target is selected from
among: ATGL, CD40, CD36, DMPK, DNM2, DMD, DUX4, LMNA, ZFN9, SGLT2,
or GCCR. [0233] Embodiment 192: The method of embodiment 190,
wherein the extra-hepatic nucleic acid target transcript is
selected from among: Androgen Receptor (AR), ANGPTL3, DGAT2, eIF4E,
Factor XI, FGFR4, GCCR, GCGR, GHR, PTP1B, SMRT, STAT3, Them1,
TRPV4, FTO, MC4R, TMEM18, KCTD15, GNPDA2, SH2B1, MTCH2, NEGR1,
BDNF, ETV5, Leptin, leptin receptor, FAIM2, KCNMA1, MAF, NRXN3,
TFAP2B, MSRA, AGPAT2, BSCL2, AKT2, PPAR.gamma., LMNA, ZMPSTE24,
DGAT1, TNF.alpha., IL-6, Resistin, PAI-1, TBC1D1, METAP2, VEGF,
AIF-1, JNK1, CB1, RIP140, TIF2, ANGPT1, ANGPT2, EIF4EBP2, CDK5,
SLC13A5, Perilipin 1, Perilipin 2, Perilipin 3, Perilipin 4, HGF,
GDF3, TNKs, KATNA1, ChREBP, ATF4, BASP-1, NNMT. [0234] Embodiment
193: The method of embodiment 190, wherein the extra-hepatic
nucleic acid target transcript is not selected from among: Androgen
Receptor (AR), ANGPTL3, DGAT2, eIF4E, Factor XI, FGFR4, GCCR, GCGR,
GHR, PTP1B, SMRT, STAT3, Them1, TRPV4, FTO, MC4R, TMEM18, KCTD15,
GNPDA2, SH2B1, MTCH2, NEGR1, BDNF, ETV5, Leptin, leptin receptor,
FAIM2, KCNMA1, MAF, NRXN3, TFAP2B, MSRA, AGPAT2, BSCL2, AKT2,
PPAR.gamma., LMNA, ZMPSTE24, DGAT1, TNF.alpha., IL-6, Resistin,
PAI-1, TBC1D1, METAP2, VEGF, AIF-1, JNK1, CB1, RIP140, TIF2,
ANGPT1, ANGPT2, EIF4EBP2, CDK5, SLC13A5, Perilipin 1, Perilipin 2,
Perilipin 3, Perilipin 4, HGF, GDF3, TNKs, KATNA1, ChREBP, ATF4,
BASP-1, NNMT. [0235] Embodiment 194: The method of any of
embodiments 190-193, wherein at least one symptom of a disease
associated with an extra-hepatic nucleic acid target is
ameliorated. [0236] Embodiment 195: The method of any of
embodiments 190-194, wherein the disease is selected from among:
diabetes, metabolic syndrome, cardiac disease, muscular dystrophy,
myotonic dystrophy, Becker muscular dystrophy, congenital muscular
dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy,
Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular
dystrophy, limb-girdle muscular dystrophy, or oculopharyngeal
muscular dystrophy. [0237] Embodiment 196: The method of any of
embodiments 189-195 wherein the amount or activity of the
extra-hepatic nucleic acid target is modulated in at least one
tissue type other than liver. [0238] Embodiment 197: The method of
embodiment 196, wherein the amount of activity of the extra-hepatic
nucleic acid target is modulated in at least two tissue types.
[0239] Embodiment 198: The method of embodiment 197, wherein at
least one of the at least two tissue types is selected from among:
liver, skeletal muscle, cardiac muscle, smooth muscle, adipose,
white adipose, spleen, bone, intestine, adrenal, testes, ovary,
pancreas, pituitary, prostate, skin, uterus, bladder, brain,
glomerulus, distal tubular epithelium, breast, lung, heart, kidney,
ganglion, frontal cortex, spinal cord, trigeminal ganglia, sciatic
nerve, dorsal root ganglion, epididymal fat, diaphragm, and colon.
[0240] Embodiment 199: The method of embodiment 198, wherein at
least two tissue types are selected from among: liver, skeletal
muscle, cardiac muscle, smooth muscle, adipose, white adipose,
spleen, bone, intestine, adrenal, testes, ovary, pancreas,
pituitary, prostate, skin, uterus, bladder, brain, glomerulus,
distal tubular epithelium, breast, lung, heart, kidney, ganglion,
frontal cortex, spinal cord, trigeminal ganglia, sciatic nerve,
dorsal root ganglion, epididymal fat, diaphragm, and colon. [0241]
Embodiment 200: A method of treating a multi-tissue disease or
condition, comprising administering a therapeutically effective
amount of the pharmaceutical composition of embodiment 187 or 188
to a subject, and thereby modulating the amount or activity of a
target nucleic acid in two or more tissues. [0242] Embodiment 201:
A method of treating a disease or condition, comprising
administering a therapeutically effective amount of the
pharmaceutical composition of embodiment 187 or 188 to a subject,
and thereby modulating the amount or activity of a target nucleic
acid in two or more cell types. [0243] Embodiment 202: A method of
treating a multi-tissue disease or condition, comprising
administering a therapeutically effective amount of the
pharmaceutical composition of embodiment 187 or 188 to a subject,
and thereby modulating the amount or activity of a target nucleic
acid in two or more cell types. [0244] Embodiment 203: The method
of embodiment 201 or 202, wherein the two or more cell types are
selected from among: hepatocytes, white fat cells, brown fat cells,
adipocytes, macrophages, cancer cells, tumor cells, smooth muscle
cells, lymphocytes, and heart muscle cells. [0245] Embodiment 204:
The method of any of embodiments 189-203, wherein the
pharmaceutical composition is administered subcutaneously. [0246]
Embodiment 205: The method of any of embodiments 189-203, wherein
the pharmaceutical composition is administered intravenously.
[0247] Embodiment 206: The method of any of embodiments 189-203,
wherein the pharmaceutical composition is administered by
parenteral administration. [0248] Embodiment 207: The method of any
of embodiments 189-203, wherein the pharmaceutical composition is
administered by intraperitoneal administration.
DETAILED DESCRIPTION OF THE INVENTION
[0249] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive. Herein, the use of
the singular includes the plural unless specifically stated
otherwise. As used herein, the use of "or" means "and/or" unless
stated otherwise. Furthermore, the use of the term "including" as
well as other forms, such as "includes" and "included", is not
limiting. Also, terms such as "element" or "component" encompass
both elements and components comprising one unit and elements and
components that comprise more than one subunit, unless specifically
stated otherwise.
[0250] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated-by-reference for the portions of the document
discussed herein, as well as in their entirety.
Definitions
[0251] Unless specific definitions are provided, the nomenclature
used in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Where permitted, all patents,
applications, published applications and other publications and
other data referred to throughout in the disclosure are
incorporated by reference herein in their entirety.
[0252] Unless otherwise indicated, the following terms have the
following meanings:
[0253] "2'-deoxynucleoside" means a nucleoside comprising 2'-H(H)
furanosyl sugar moiety, as found in naturally occurring
deoxyribonucleic acids (DNA). In certain embodiments, a
2'-deoxynucleoside may comprise a modified nucleobase or may
comprise an RNA nucleobase (uracil).
[0254] "2'-substituted nucleoside" or "2-modified nucleoside" means
a nucleoside comprising a 2'-substituted or 2'-modified sugar
moiety. As used herein, "2'-substituted" or "2-modified" in
reference to a sugar moiety means a sugar moiety comprising at
least one 2'-substituent group other than H or OH.
[0255] "Antisense activity" means any detectable and/or measurable
change attributable to the hybridization of an antisense compound
to its target nucleic acid. In certain embodiments, antisense
activity is a decrease in the amount or expression of a target
nucleic acid or protein encoded by such target nucleic acid
compared to target nucleic acid levels or target protein levels in
the absence of the antisense compound. In certain embodiments,
antisense activity is a change in splicing of a pre-mRNA nucleic
acid target. In certain embodiments, antisense activity is an
increase in the amount or expression of a target nucleic acid or
protein encoded by such target nucleic acid compared to target
nucleic acid levels or target protein levels in the absence of the
antisense compound.
[0256] "Antisense compound" means a compound comprising an
antisense oligonucleotide and optionally one or more additional
features, such as a conjugate group or terminal group.
[0257] "Antisense oligonucleotide" means an oligonucleotide that
(1) has a nucleobase sequence that is at least partially
complementary to a target nucleic acid and that (2) is capable of
producing an antisense activity in a cell or animal.
[0258] "Ameliorate" in reference to a treatment means improvement
in at least one symptom relative to the same symptom in the absence
of the treatment. In certain embodiments, amelioration is the
reduction in the severity or frequency of a symptom or the delayed
onset or slowing of progression in the severity or frequency of a
symptom.
[0259] "Bicyclic nucleoside" or "BNA" means a nucleoside comprising
a bicyclic sugar moiety. As used herein, "bicyclic sugar" or
"bicyclic sugar moiety" means a modified sugar moiety comprising
two rings, wherein the second ring is formed via a bridge
connecting two of the atoms in the first ring thereby forming a
bicyclic structure. In certain embodiments, the first ring of the
bicyclic sugar moiety is a furanosyl moiety. In certain
embodiments, the bicyclic sugar moiety does not comprise a
furanosyl moiety.
[0260] "Branching group" means a group of atoms having at least 3
positions that are capable of forming covalent linkages to at least
3 groups. In certain embodiments, a branching group provides a
plurality of reactive sites for connecting tethered ligands to an
oligonucleotide via a conjugate linker and/or a cleavable
moiety.
[0261] "Cell-targeting moiety" means a conjugate group or portion
of a conjugate group that is capable of binding to a particular
cell type or particular cell types.
[0262] "Cleavable moiety" means a bond or group of atoms that is
cleaved under physiological conditions, for example, inside a cell,
an animal, or a human.
[0263] "Complementary" in reference to an oligonucleotide means
that at least 70% of the nucleobases of such oligonucleotide or one
or more regions thereof and the nucleobases of another nucleic acid
or one or more regions thereof are capable of hydrogen bonding with
one another when the nucleobase sequence of the oligonucleotide and
the other nucleic acid are aligned in opposing directions.
Complementary nucleobases means nucleobases that are capable of
forming hydrogen bonds with one another. Complementary nucleobase
pairs include, but unless otherwise specific are not limited to,
adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine
(C) and guanine (G), 5-methyl cytosine (mC) and guanine (G).
Complementary oligonucleotides and/or nucleic acids need not have
nucleobase complementarity at each nucleoside. Rather, some
mismatches are tolerated. As used herein, "fully complementary" or
"100% complementary" in reference to oligonucleotides means that
such oligonucleotides are complementary to another oligonucleotide
or nucleic acid at each nucleoside of the oligonucleotide.
[0264] "Conjugate group" means a group of atoms that is directly or
indirectly attached to an oligonucleotide. Conjugate groups include
a conjugate moiety and a conjugate linker that attaches the
conjugate moiety to the oligonucleotide.
[0265] "Conjugate linker" means a group of atoms comprising at
least one bond that connects a conjugate moiety to an
oligonucleotide.
[0266] "Conjugate moiety" means a group of atoms that is attached
to an oligonucleotide via a conjugate linker.
[0267] "Contiguous" in the context of an oligonucleotide refers to
nucleosides, nucleobases, sugar moieties, or internucleoside
linkages that are immediately adjacent to each other. For example,
"contiguous nucleobases" means nucleobases that are immediately
adjacent to each other in a sequence.
[0268] "Duplex" means two oligomeric compounds that are paired. In
certain embodiments, the two oligomeric compounds are paired via
hybridization of complementary nucleobases.
[0269] "Extra-hepatic cell type" means a cell type that is not a
hepatocyte.
[0270] "Extra-hepatic nucleic acid target" means a target nucleic
acid that is expressed in tissues other than liver. In certain
embodiments, extra-hepatic nucleic acid targets are not expressed
in the liver or not expressed in the liver at a significant level.
In certain embodiments, extra-hepatic nucleic acid targets are
expressed outside the liver and also in the liver.
[0271] "Extra-hepatic tissue" means a tissue other than liver.
[0272] "Fully modified" in reference to a modified oligonucleotide
means a modified oligonucleotide in which each sugar moiety is
modified. "Uniformly modified" in reference to a modified
oligonucleotide means a fully modified oligonucleotide in which
each sugar moiety is the same. For example, the nucleosides of a
uniformly modified oligonucleotide can each have a 2'-MOE
modification but different nucleobase modifications, and the
internucleoside linkages may be different.
[0273] "Gapmer" means an antisense oligonucleotide comprising an
internal region having a plurality of nucleosides that support
RNase H cleavage positioned between external regions having one or
more nucleosides, wherein the nucleosides comprising the internal
region are chemically distinct from the nucleoside or nucleosides
comprising the external regions. The internal region may be
referred to as the "gap" and the external regions may be referred
to as the "wings."
[0274] "Hybridization" means the pairing or annealing of
complementary oligonucleotides and/or nucleic acids. While not
limited to a particular mechanism, the most common mechanism of
hybridization involves hydrogen bonding, which may be Watson-Crick,
Hoogsteen or reversed Hoogsteen hydrogen bonding, between
complementary nucleobases.
[0275] "Inhibiting the expression or activity" refers to a
reduction or blockade of the expression or activity relative to the
expression of activity in an untreated or control sample and does
not necessarily indicate a total elimination of expression or
activity.
[0276] "Internucleoside linkage" means a group or bond that forms a
covalent linkage between adjacent nucleosides in an
oligonucleotide. As used herein "modified internucleoside linkage"
means any internucleoside linkage other than a naturally occurring,
phosphate internucleoside linkage. Non-phosphate linkages are
referred to herein as modified internucleoside linkages.
"Phosphorothioate linkage" means a modified phosphate linkage in
which one of the non-bridging oxygen atoms is replaced with a
sulfur atom. A phosphorothioate internucleoside linkage is a
modified internucleoside linkage.
[0277] "Linker-nucleoside" means a nucleoside that links, either
directly or indirectly, an oligonucleotide to a conjugate moiety.
Linker-nucleosides are located within the conjugate linker of an
oligomeric compound. Linker-nucleosides are not considered part of
the oligonucleotide portion of an oligomeric compound even if they
are contiguous with the oligonucleotide.
[0278] "Lipophilic group" or "lipophilic" in reference to a
chemical group means a group of atoms that is more soluble in
lipids or organic solvents than in water and/or has a higher
affinity for lipids than for water. In certain embodiments,
lipophilic groups comprise a lipid. As used herein "lipid" means a
molecule that is not soluble in water or is less soluble in water
than in organic solvents. In certain embodiments, compounds of the
present invention comprise lipids selected from saturated or
unsaturated fatty acids, steroids, fat soluble vitamins,
phospholipids, sphingolipids, hydrocarbons, mono-, di-, and
tri-glycerides, and synthetic derivatives thereof.
[0279] "Non-bicyclic modified sugar" or "non-bicyclic modified
sugar moiety" means a modified sugar moiety that comprises a
modification, such as a substitutent, that does not form a bridge
between two atoms of the sugar to form a second ring.
[0280] "Linked nucleosides" are nucleosides that are connected in a
continuous sequence (i.e. no additional nucleosides are present
between those that are linked).
[0281] "Mismatch" or "non-complementary" means a nucleobase of a
first oligonucleotide that is not complementary with the
corresponding nucleobase of a second oligonucleotide or target
nucleic acid when the first and second oligomeric compound are
aligned.
[0282] "MOE" means methoxyethyl. "2'-MOE" means a
--OCH.sub.2CH.sub.2OCH.sub.3 group at the 2' position of a
furanosyl ring.
[0283] "Motif" means the pattern of unmodified and/or modified
sugar moieties, nucleobases, and/or internucleoside linkages, in an
oligonucleotide.
[0284] "Multi-tissue disease or condition" means a disease or
condition affects or is effected by more than one tissue. In
treating a multi-tissue disease or condition, it is desirable to
affect more than one tissue type. In certain embodiments, treatment
of disease or condition may be enhanced by treating the disease or
condition in multiple tissues. For example, in certain embodiments,
a disease or condition may manifest itself in the liver tissue and
the muscle tissue. In certain embodiments, treating the disease or
condition in the liver tissue and the muscle tissue will be more
effective than treating the disease in either the liver tissue or
the muscle tissue.
[0285] "Naturally occurring" means found in nature.
[0286] "Nucleobase" means an unmodifiednucleobase or a modified
nucleobase. As used herein a "an "unmodified nucleobase" is adenine
(A), thymine (T), cytosine (C), uracil (U), and guanine (G). As
used herein, a "modified nucleobase" is a group of atoms other than
unmodified A, T, C, U, or G capable of pairing with at least one
unmodified nucleobase. A universal base is a modified nucleobase
that can pair with any one of the five unmodified nucleobases. As
used herein, "nucleobase sequence" means the order of contiguous
nucleobases in a nucleic acid or oligonucleotide independent of any
sugar or internucleoside linkage modification.
[0287] "Nucleoside" means a compound comprising a nucleobase and a
sugar moiety. The nucleobase and sugar moiety are each,
independently, unmodified or modified. As used herein, "modified
nucleoside" means a nucleoside comprising a modified nucleobase
and/or a modified sugar moiety. Modified nucleosides include abasic
nucleosides, which lack a nucleobase.
[0288] "Oligomeric compound" means a compound consisting of an
oligonucleotide and optionally one or more additional features,
such as a conjugate group or terminal group.
[0289] "Oligonucleotide" means a strand of linked nucleosides
connected via internucleoside linkages, wherein each nucleoside and
internucleoside linkage may be modified or unmodified. Unless
otherwise indicated, oligonucleotides consist of 8-50 linked
nucleosides. As used herein, "modified oligonucleotide" means an
oligonucleotide, wherein at least one nucleoside or internucleoside
linkage is modified. As used herein, "unmodified oligonucleotide"
means an oligonucleotide that does not comprise any nucleoside
modifications or internucleoside modifications.
[0290] "Pharmaceutically acceptable carrier or diluent" means any
substance suitable for use in administering to an animal. Certain
such carriers enable pharmaceutical compositions to be formulated
as, for example, tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspension and lozenges for the oral ingestion by
a subject. In certain embodiments, a pharmaceutically acceptable
carrier or diluent is sterile water; sterile saline; or sterile
buffer solution.
[0291] "Pharmaceutically acceptable salts" means physiologically
and pharmaceutically acceptable salts of compounds, such as
oligomeric compounds, i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto.
[0292] "Pharmaceutical composition" means a mixture of substances
suitable for administering to a subject. For example, a
pharmaceutical composition may comprise an antisense compound and a
sterile aqueous solution. In certain embodiments, a pharmaceutical
composition shows activity in free uptake assay in certain cell
lines.
[0293] "Phosphorus moiety" means a group of atoms comprising a
phosphorus atom. In certain embodiments, a phosphorus moiety
comprises a mono-, di-, or tri-phosphate, or phosphorothioate.
[0294] "Prodrug" means a therapeutic agent in a form outside the
body that is converted to a different form within the body or cells
thereof. Typically conversion of a prodrug within the body is
facilitated by the action of an enzymes (e.g., endogenous or viral
enzyme) or chemicals present in cells or tissues and/or by
physiologic conditions.
[0295] "RNAi compound" means an antisense compound that acts, at
least in part, through RISC or Ago2 to modulate a target nucleic
acid and/or protein encoded by a target nucleic acid. RNAi
compounds include, but are not limited to double-stranded siRNA,
single-stranded RNA (ssRNA), and microRNA, including microRNA
mimics. In certain embodiments, an RNAi compound modulates the
amount, activity, and/or splicing of a target nucleic acid. The
term RNAi compound excludes antisense oligonucleotides that act
through RNase H.
[0296] "Single-stranded" in reference to an oligomeric compound
means such a compound that is not paired with a second oligomeric
compound to form a duplex. "Self-complementary" in reference to an
oligonucleotide means an oligonucleotide that at least partially
hybridizes to itself. A compound consisting of one oligomeric
compound, wherein the oligonucleotide of the oligomeric compound is
self-complementary, is a single-stranded compound. A
single-stranded antisense or oligomeric compound may be capable of
binding to a complementary oligomeric compound to form a duplex, in
which case it would no longer be single-stranded.
[0297] "Standard cell assay" means the assay described in Example 1
and reasonable variations thereof
[0298] "Standard in vivo experiment" means the procedure described
in Example 5 and reasonable variations thereof.
[0299] "Sugar moiety" means an unmodified sugar moiety or a
modified sugar moiety. As used herein, "unmodified sugar moiety"
means a 2'-OH(H) furanosyl moiety, as found in RNA (an "unmodified
RNA sugar moiety"), or a 2'-H(H) moiety, as found in DNA (an
"unmodified DNA sugar moiety"). Unmodified sugar moieties have one
hydrogen at each of the 1', 3', and 4' positions, an oxygen at the
3' position, and two hydrogens at the 5' position. As used herein,
"modified sugar moiety" or "modified sugar" means a modified
furanosyl sugar moiety or a sugar surrogate. As used herein,
modified furanosyl sugar moiety means a furanosyl sugar comprising
a non-hydrogen substituent in place of at least one hydrogen of an
unmodified sugar moiety. In certain embodiments, a modified
furanosyl sugar moiety is a 2'-substituted sugar moiety. Such
modified furanosyl sugar moieties include bicyclic sugars and
non-bicyclic sugars. As used herein, "sugar surrogate" means a
modified sugar moiety having other than a furanosyl moiety that can
link a nucleobase to another group, such as an internucleoside
linkage, conjugate group, or terminal group in an oligonucleotide.
Modified nucleosides comprising sugar surrogates can be
incorporated into one or more positions within an oligonucleotide
and such oligonucleotides are capable of hybridizing to
complementary oligomeric compounds or nucleic acids.
[0300] "Target nucleic acid" means a naturally occurring,
identified nucleic acid. In certain embodiments, target nucleic
acids are endogenous cellular nucleic acids, including, but not
limited to RNA transcripts, pre-mRNA, mRNA, microRNA. In certain
embodiments, target nucleic acids are viral nucleic acids. In
certain embodiments, target nucleic acids are nucleic acids that an
antisense compound is designed to affect.
[0301] "Target region" means a portion of a target nucleic acid to
which an antisense compound is designed to hybridize.
[0302] "TCA motif" means three nucleosides having the nucleobase
sequence TCA (5'-3'). Such nucleosides may have modified sugar
moieties and/or modified internucleosides linkages. Unless
otherwise indicated, the nucleosides of TCA motifs comprise
unmodified 2'-deoxy sugar moieties and unmodified phosphodiester
internucleoside linkages.
[0303] "Terminal group" means a chemical group or group of atoms
that is covalently linked to a terminus of an oligonucleotide.
[0304] "CNS" means central nervous system. The CNS includes, the
spine and the brain and the cerebrospinal fluid.
[0305] "Cerebrospinal fluid" or "CSF" means the fluid filling the
space around the brain and spinal cord.
[0306] "Nervous system" means the network of nerve cells and fibers
that transmits nerve impulses between parts of the body. The
nervous system includes glial cells and neurons. The nervous system
includes the central nervous system and the peripheral nervous
system.
[0307] I. Certain Oligonucleotides
[0308] In certain embodiments, the invention provides a duplex
comprising a first oligomeric compound and a second oligomeric
compound. In certain embodiments an oligomeric compound comprises
an oligonucleotide, which consists of linked nucleosides.
Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or
may be modified oligonucleotides. Modified oligonucleotides, for
example the first modified oligonucleotide or the second modified
oligonucleotide, comprise at least one modification relative to
unmodified RNA or DNA (i.e., comprise at least one modified
nucleoside (comprising a modified sugar moiety and/or a modified
nucleobase) and/or at least one modified internucleoside
linkage).
[0309] A. Certain Modified Nucleosides
[0310] Modified nucleosides comprise a modified sugar moiety or a
modified nucleobase or both a modified sugar moiety and a modified
nucleobase.
[0311] 1. Certain Sugar Moieties
[0312] In certain embodiments, modified sugar moieties are
non-bicyclic modified sugar moieties. In certain embodiments,
modified sugar moieties are bicyclic or tricyclic sugar moieties.
In certain embodiments, modified sugar moieties are sugar
surrogates. Such sugar surrogates may comprise one or more
substitutions corresponding to those of other types of modified
sugar moieties.
[0313] In certain embodiments, modified sugar moieties are
non-bicyclic modified sugar moieties comprising a furanosyl ring
with one or more acyclic substituent, including but not limited to
substituents at the 2', 4', and/or 5' positions. In certain
embodiments one or more acyclic substituent of non-bicyclic
modified sugar moieties is branched. Examples of 2'-substituent
groups suitable for non-bicyclic modified sugar moieties include
but are not limited to: 2'-F, 2'-OCH.sub.3 ("OMe" or "O-methyl"),
and 2'-O(CH.sub.2).sub.2OCH.sub.3 ("MOE"). In certain embodiments,
2'-substituent groups are selected from among: halo, allyl, amino,
azido, SH, CN, OCN, CF.sub.3, OCF.sub.3, O--C.sub.1-C.sub.10
alkoxy, O--C.sub.1-C.sub.10 substituted alkoxy, O--C.sub.1-C.sub.10
alkyl, O--C.sub.1-C.sub.10 substituted alkyl, S-alkyl,
N(R.sub.m)-alkyl, O-alkenyl, S-alkenyl, N(R.sub.m)-alkenyl,
O-alkynyl, S-alkynyl, N(R.sub.m)-alkynyl, O-alkylenyl-O-alkyl,
alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ON(R.sub.m)(R.sub.n)
or OCH.sub.2C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H, an amino protecting group, or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl, and the
2'-substituent groups described in Cook et al., U.S. Pat. No.
6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al.,
U.S. Pat. No. 6,005,087. Certain embodiments of these
2'-substituent groups can be further substituted with one or more
substituent groups independently selected from among: hydroxyl,
amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol,
thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.
Examples of 4'-substituent groups suitable for non-bicyclic
modified sugar moieties include but are not limited to alkoxy
(e.g., methoxy), alkyl, and those described in Manoharan et al., WO
2015/106128. Examples of 5'-substituent groups suitable for
non-bicyclic modified sugar moieties include but are not limited
to: 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain
embodiments, non-bicyclic modified sugars comprise more than one
non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar
moieties and the modified sugar moieties and modified nucleosides
described in Migawa et al., WO 2008/101157 and Rajeev et al.,
US2013/0203836.).
[0314] In certain embodiments, a 2'-substituted nucleoside or
2'-non-bicyclic modified nucleoside comprises a sugar moiety
comprising a non-bridging 2'-substituent group selected from: F,
NH.sub.2, N.sub.3, OCF.sub.3, OCH.sub.3, O(CH.sub.2).sub.3NH.sub.2,
CH.sub.2CH.dbd.CH.sub.2, OCH.sub.2CH.dbd.CH.sub.2,
OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2ON(R.sub.m)(R.sub.n),
O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
N-substituted acetamide (OCH.sub.2C(.dbd.O)--N(R.sub.m)(R.sub.n)),
where each R.sub.m and R.sub.n is, independently, H, an amino
protecting group, or substituted or unsubstituted C.sub.1-C.sub.10
alkyl.
[0315] In certain embodiments, a 2'-substituted nucleoside or
2'-non-bicyclic modified nucleoside comprises a sugar moiety
comprising a non-bridging 2'-substituent group selected from: F,
OCF.sub.3, OCH.sub.3, OCH.sub.2CH.sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ON(CH.sub.3).sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
OCH.sub.2C(.dbd.O)--N(H)CH.sub.3 ("NMA").
[0316] In certain embodiments, a 2'-substituted nucleoside or
2'-non-bicyclic modified nucleoside comprises a sugar moiety
comprising a non-bridging 2'-substituent group selected from: F,
OCH.sub.3, and OCH.sub.2CH.sub.2OCH.sub.3.
[0317] Nucleosides comprising modified sugar moieties, such as
non-bicyclic modified sugar moieties, may be referred to by the
position(s) of the substitution(s) on the sugar moiety of the
nucleoside. For example, nucleosides comprising 2'-substituted or
2-modified sugar moieties are referred to as 2'-substituted
nucleosides or 2-modified nucleosides.
[0318] Certain modified sugar moieties comprise a bridging sugar
substituent that forms a second ring resulting in a bicyclic sugar
moiety. In certain such embodiments, the bicyclic sugar moiety
comprises a bridge between the 4' and the 2' furanose ring atoms.
Examples of such 4' to 2' bridging sugar substituents include but
are not limited to: 4'-CH.sub.2-2', 4'-(CH.sub.2).sub.2-2',
4'-(CH.sub.2).sub.3-2', 4'-CH.sub.2--O-2' ("LNA"),
4'-CH.sub.2--S-2', 4'-(CH.sub.2).sub.2--O-2' ("ENA"),
4'-CH(CH.sub.3)--O-2' (referred to as "constrained ethyl" or "cEt"
when in the S configuration), 4'-CH.sub.2--O--CH.sub.2-2',
4'-CH.sub.2--N(R)-2', 4'-CH(CH.sub.2OCH.sub.3)--O-2' ("constrained
MOE" or "cMOE") and analogs thereof (see, e.g., Seth et al., U.S.
Pat. No. 7,399,845, Bhat et al., U.S. Pat. No. 7,569,686, Swayze et
al., U.S. Pat. No. 7,741,457, and Swayze et al., U.S. Pat. No.
8,022,193), 4'-C(CH.sub.3)(CH.sub.3)--O-2' and analogs thereof
(see, e.g., Seth et al., U.S. Pat. No. 8,278,283),
4'-CH.sub.2--N(OCH.sub.3)-2' and analogs thereof (see, e.g.,
Prakash et al., U.S. Pat. No. 8,278,425),
4'-CH.sub.2--O--N(CH.sub.3)-2' (see, e.g., Allerson et al., U.S.
Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745),
4'-CH.sub.2--C(H)(CH.sub.3)-2' (see, e.g., Zhou, et al., J. Org.
Chem., 2009, 74, 118-134), 4'-CH.sub.2--C(.dbd.CH.sub.2)-2' and
analogs thereof (see e.g., Seth et al., U.S. Pat. No. 8,278,426),
4'-C(R.sub.aR.sub.b)--N(R)--O-2', 4'-C(R.sub.aR.sub.b)--O--N(R)-2',
4'-CH.sub.2--O--N(R)-2', and 4'-CH.sub.2--N(R)--O-2', wherein each
R, R.sub.a, and R.sub.b is, independently, H, a protecting group,
or C.sub.1-C.sub.12 alkyl (see, e.g. Imanishi et al., U.S. Pat. No.
7,427,672).
[0319] In certain embodiments, such 4' to 2' bridges independently
comprise from 1 to 4 linked groups independently selected from:
--[C(R.sub.a)(R.sub.b)].sub.n--,
--[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.a).dbd.C(R.sub.b)--,
--C(R.sub.a).dbd.N--, --C(.dbd.NR.sub.a)--, --C(.dbd.O)--,
--C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--,
and --N(R.sub.a)--;
[0320] wherein:
[0321] x is 0, 1, or 2;
[0322] n is 1, 2, 3, or 4;
[0323] each R.sub.a and R.sub.b is, independently, H, a protecting
group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted
C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle
radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7
alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical,
halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1,
acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.12
alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12
alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12
alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20
aryl, substituted C.sub.5-C.sub.20 aryl, acyl (C(.dbd.O)--H),
substituted acyl, a heterocycle radical, a substituted heterocycle
radical, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12
aminoalkyl, or a protecting group.
[0324] Additional bicyclic sugar moieties are known in the art,
see, for example: Freier et al., Nucleic Acids Research, 1997,
25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71,
7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin
et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg.
Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem.,
1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 20017,
129, 8362-8379; Wengel et a., U.S. Pat. No. 7,053,207; Imanishi et
al., U.S. Pat. No. 6,268,490; Imanishi et al. U.S. Pat. No.
6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. Pat.
No. 6,794,499; Wengel et al., U.S. Pat. No. 6,670,461; Wengel et
al., U.S. Pat. No. 7,034,133; Wengel et al., U.S. Pat. No.
8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al.,
U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582;
and Ramasamy et al., U.S. Pat. No. 6,525,191; Torsten et al., WO
2004/106356; Wengel et al., WO 1999/014226; Seth et al., WO
2007/134181; Seth et al., U.S. Pat. No. 7,547,684; Seth et al.,
U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth
et al., U.S. Pat. No. 7,750,131; Seth et al., U.S. Pat. No.
8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al., U.S.
Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa et
al., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805;
and U.S. Patent Publication Nos. Allerson et al., US2008/0039618
and Migawa et al., US2015/0191727.
[0325] In certain embodiments, bicyclic sugar moieties and
nucleosides incorporating such bicyclic sugar moieties are further
defined by isomeric configuration. For example, an LNA nucleoside
(described herein) may be in the .alpha.-L configuration or in the
.beta.-D configuration.
##STR00004##
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') or .alpha.-L-LNA
bicyclic nucleosides have been incorporated into antisense
oligonucleotides that showed antisense activity (Frieden et al.,
Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general
descriptions of bicyclic nucleosides include both isomeric
configurations. When the positions of specific bicyclic nucleosides
(e.g., LNA or cEt) are identified in exemplified embodiments
herein, they are in the .beta.-D configuration, unless otherwise
specified.
[0326] In certain embodiments, modified sugar moieties comprise one
or more non-bridging sugar substituent and one or more bridging
sugar substituent (e.g., 5'-substituted and 4'-2' bridged
sugars).
[0327] In certain embodiments, modified sugar moieties are sugar
surrogates. In certain such embodiments, the oxygen atom of the
sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen
atom. In certain such embodiments, such modified sugar moieties
also comprise bridging and/or non-bridging substituents as
described herein. For example, certain sugar surrogates comprise a
4'-sulfur atom and a substitution at the 2'-position (see, e.g.,
Bhat et al., U.S. Pat. No. 7,875,733 and Bhat et al., U.S. Pat. No.
7,939,677) and/or the 5' position.
[0328] In certain embodiments, sugar surrogates comprise rings
having other than 5 atoms. For example, in certain embodiments, a
sugar surrogate comprises a six-membered tetrahydropyran ("THP").
Such tetrahydropyrans may be further modified or substituted.
Nucleosides comprising such modified tetrahydropyrans include but
are not limited to hexitol nucleic acid ("HNA"), anitol nucleic
acid ("ANA"), manitol nucleic acid ("MNA") (see, e.g., Leumann, C
J. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA:
##STR00005##
("F-HNA", see e.g. Swayze et al., U.S. Pat. No. 8,088,904; Swayze
et al., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. No.
8,796,437; and Swayze et al., U.S. Pat. No. 9,005,906; F-HNA can
also be referred to as a F-THP or 3'-fluoro tetrahydropyran), and
nucleosides comprising additional modified THP compounds having the
formula:
##STR00006##
wherein, independently, for each of said modified THP
nucleoside:
[0329] Bx is a nucleobase moiety;
[0330] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the modified THP nucleoside
to the remainder of an oligonucleotide or one of T.sub.3 and
T.sub.4 is an internucleoside linking group linking the modified
THP nucleoside to the remainder of an oligonucleotide and the other
of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a linked
conjugate group, or a 5' or 3'-terminal group; q.sub.1, q.sub.2,
q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each,
independently, H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted
C.sub.2-C.sub.6 alkynyl; and
[0331] each of R1 and R2 is independently selected from among:
hydrogen, halogen, substituted or unsubstituted alkoxy,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1,
OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2, and
CN, wherein X is O, S or NJ', and each J.sub.1, J.sub.2, and
J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl.
[0332] In certain embodiments, modified THP nucleosides are
provided wherein q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5,
q.sub.6 and are each H. In certain embodiments, at least one of
q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 is
other than H. In certain embodiments, at least one of q.sub.1,
q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 is methyl.
In certain embodiments, modified THP nucleosides are provided
wherein one of R.sub.1 and R.sub.2 is F. In certain embodiments,
R.sub.1 is F and R.sub.2 is H, in certain embodiments, R.sub.1 is
methoxy and R.sub.2 is H, and in certain embodiments, R.sub.1 is
methoxyethoxy and R.sub.2 is H.
[0333] In certain embodiments, sugar surrogates comprise rings
having more than 5 atoms and more than one heteroatom. For example,
nucleosides comprising morpholino sugar moieties and their use in
oligonucleotides have been reported (see, e.g., Braasch et al.,
Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Pat.
No. 5,698,685; Summerton et al., U.S. Pat. No. 5,166,315; Summerton
et al., U.S. Pat. No. 5,185,444; and Summerton et al., U.S. Pat.
No. 5,034,506). As used here, the term "morpholino" means a sugar
surrogate having the following structure:
##STR00007##
In certain embodiments, morpholinos may be modified, for example by
adding or altering various substituent groups from the above
morpholino structure. Such sugar surrogates are referred to herein
as "modified morpholinos."
[0334] In certain embodiments, sugar surrogates comprise acyclic
moieties. Examples of nucleosides and oligonucleotides comprising
such acyclic sugar surrogates include but are not limited to:
peptide nucleic acid ("PNA"), acyclic butyl nucleic acid (see,
e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and
nucleosides and oligonucleotides described in Manoharan et al.,
WO2011/133876.
Many other bicyclic and tricyclic sugar and sugar surrogate ring
systems are known in the art that can be used in modified
nucleosides).
[0335] 1. Certain Modified Nucleobases
[0336] In certain embodiments, the first modified oligonucleotide
comprises one or more nucleoside comprising an unmodified
nucleobase. In certain embodiments, the second modified
oligonucleotide comprises one or more nucleoside comprising an
unmodified nucleobase. In certain embodiments, modified
oligonucleotides, for example the first modified oligonucleotide or
the second modified oligonucleotide, comprise one or more
nucleoside comprising a modified nucleobase. In certain
embodiments, modified oligonucleotides, for example the first
modified oligonucleotide or the second modified oligonucleotide,
comprise one or more nucleoside that does not comprise a
nucleobase, referred to as an abasic nucleoside.
[0337] In certain embodiments, modified nucleobases are selected
from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl
substituted pyrimidines, alkyl substituted purines, and N-2, N-6
and 0-6 substituted purines. In certain embodiments, modified
nucleobases are selected from: 2-aminopropyladenine,
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-N-methylguanine, 6-N-methyladenine, 2-propyladenine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl
(--C.ident.C--CH.sub.3) uracil, 5-propynylcytosine, 6-azouracil,
6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl,
8-aza and other 8-substituted purines, 5-halo, particularly
5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine,
7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine,
7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine,
6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine,
4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl
4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous
bases, size-expanded bases, and fluorinated bases. Further modified
nucleobases include tricyclic pyrimidines, such as
1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and
9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified
nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in Merigan et al., U.S.
Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of
Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley
& Sons, 1990, 858-859; Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15,
Antisense Research and Applications, Crooke, S. T. and Lebleu, B.,
Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6
and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press,
2008, 163-166 and 442-443.
[0338] Publications that teach the preparation of certain of the
above noted modified nucleobases as well as other modified
nucleobases include without limitation, Manohara et al.,
US2003/0158403; Manoharan et al., US2003/0175906; Dinh et al., U.S.
Pat. No. 4,845,205; Spielvogel et al., U.S. Pat. No. 5,130,302;
Rogers et al., U.S. Pat. No. 5,134,066; Bischofberger et al., U.S.
Pat. No. 5,175,273; Urdea et al., U.S. Pat. No. 5,367,066; Benner
et al., U.S. Pat. No. 5,432,272; Matteucci et al., U.S. Pat. No.
5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cook et al.,
U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No. 5,484,908;
Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al., U.S.
Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540;
Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat.
No. 5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et
al., U.S. Pat. No. 5,614,617; Froehler et al., U.S. Pat. No.
5,645,985; Cook et al., U.S. Pat. No. 5,681,941; Cook et al., U.S.
Pat. No. 5,811,534; Cook et al., U.S. Pat. No. 5,750,692; Cook et
al., U.S. Pat. No. 5,948,903; Cook et al., U.S. Pat. No. 5,587,470;
Cook et al., U.S. Pat. No. 5,457,191; Matteucci et al., U.S. Pat.
No. 5,763,588; Froehler et al., U.S. Pat. No. 5,830,653; Cook et
al., U.S. Pat. No. 5,808,027; Cook et al., U.S. Pat. No. 6,166,199;
and Matteucci et al., U.S. Pat. No. 6,005,096.
[0339] B. Certain Modified Internucleoside Linkages
[0340] In certain embodiments, nucleosides of modified
oligonucleotides may be linked together using any internucleoside
linkage. In certain embodiments, nucleosides of the first modified
oligonucleotide may be linked together using any internucleoside
linkage. In certain embodiments, nucleosides of the second modified
oligonucleotide may be linked together using any internucleoside
linkage. The two main classes of internucleoside linking groups are
defined by the presence or absence of a phosphorus atom.
Representative phosphorus-containing internucleoside linkages
include but are not limited to phosphates, which contain a
phosphodiester bond ("P.dbd.O") (also referred to as unmodified or
naturally occurring linkages), phosphotriesters,
methylphosphonates, phosphoramidates, and phosphorothioates
("P=5"), and phosphorodithioates ("HS--P.dbd.S"). Representative
non-phosphorus containing internucleoside linking groups include
but are not limited to methylenemethylimino
(--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--), thiodiester,
thionocarbamate (--O--C(.dbd.O)(NH)--S--); siloxane
(--O--SiH.sub.2--O--); and N,N'-dimethylhydrazine
(--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--). Modified internucleoside
linkages, compared to naturally occurring phosphate linkages, can
be used to alter, typically increase, nuclease resistance of the
oligonucleotide. In certain embodiments, internucleoside linkages
having a chiral atom can be prepared as a racemic mixture, or as
separate enantiomers. Representative chiral internucleoside
linkages include but are not limited to alkylphosphonates and
phosphorothioates. Methods of preparation of phosphorous-containing
and non-phosphorous-containing internucleoside linkages are well
known to those skilled in the art.
[0341] Neutral internucleoside linkages include, without
limitation, phosphotriesters, methylphosphonates, MMI
(3'-CH.sub.2--N(CH.sub.3)--O-5'), amide-3
(3'-CH.sub.2--C(.dbd.O)--N(H)-5'), amide-4
(3'-CH.sub.2--N(H)--C(.dbd.O)-5'), formacetal
(3'-O--CH.sub.2--O-5'), methoxypropyl, and thioformacetal
(3'-S--CH.sub.2--O-5'). Further neutral internucleoside linkages
include nonionic linkages comprising siloxane (dialkylsiloxane),
carboxylate ester, carboxamide, sulfide, sulfonate ester and amides
(See for example: Carbohydrate Modifications in Antisense Research;
Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580;
Chapters 3 and 4, 40-65). Further neutral internucleoside linkages
include nonionic linkages comprising mixed N, O, S and CH.sub.2
component parts.
[0342] C. Certain Motifs
[0343] In certain embodiments, the first modified oligonucleotide
comprises one or more modified nucleoside comprising a modified
sugar. In certain embodiments, the first modified oligonucleotide
comprises one or more modified nucleosides comprising a modified
nucleobase. In certain embodiments, the first modified
oligonucleotide comprises one or more modified internucleoside
linkage. In such embodiments, the modified, unmodified, and
differently modified sugar moieties, nucleobases, and/or
internucleoside linkages of the first modified oligonucleotide
define a pattern or motif. In certain embodiments, the patterns of
sugar moieties, nucleobases, and internucleoside linkages are each
independent of one another. Thus, the first modified
oligonucleotide may be described by its sugar motif, nucleobase
motif and/or internucleoside linkage motif (as used herein,
nucleobase motif describes the modifications to the nucleobases
independent of the sequence of nucleobases).
[0344] In certain embodiments, the second modified oligonucleotide
comprises one or more modified nucleoside comprising a modified
sugar. In certain embodiments, the second modified oligonucleotide
comprises one or more modified nucleosides comprising a modified
nucleobase. In certain embodiments, the second modified
oligonucleotide comprises one or more modified internucleoside
linkage. In such embodiments, the modified, unmodified, and
differently modified sugar moieties, nucleobases, and/or
internucleoside linkages of the second modified oligonucleotide
define a pattern or motif. In certain embodiments, the patterns of
sugar moieties, nucleobases, and internucleoside linkages are each
independent of one another. Thus, the second modified
oligonucleotide may be described by its sugar motif, nucleobase
motif and/or internucleoside linkage motif (as used herein,
nucleobase motif describes the modifications to the nucleobases
independent of the sequence of nucleobases).
[0345] In certain embodiments, modified oligonucleotides comprise
one or more modified nucleoside comprising a modified sugar. In
certain embodiments, modified oligonucleotides comprise one or more
modified nucleosides comprising a modified nucleobase. In certain
embodiments, modified oligonucleotides comprise one or more
modified internucleoside linkage. In such embodiments, the
modified, unmodified, and differently modified sugar moieties,
nucleobases, and/or internucleoside linkages of a modified
oligonucleotide define a pattern or motif. In certain embodiments,
the patterns of sugar moieties, nucleobases, and internucleoside
linkages are each independent of one another. Thus, a modified
oligonucleotide may be described by its sugar motif, nucleobase
motif and/or internucleoside linkage motif (as used herein,
nucleobase motif describes the modifications to the nucleobases
independent of the sequence of nucleobases).
[0346] 1. Certain Sugar Motifs
[0347] In certain embodiments, oligonucleotides comprise one or
more type of modified sugar and/or unmodified sugar moiety arranged
along the oligonucleotide or region thereof in a defined pattern or
sugar motif. In certain instances, such sugar motifs include but
are not limited to any of the sugar modifications discussed
herein.
[0348] In certain embodiments, modified oligonucleotides, for
example the first modified oligonucleotide, comprise or consist of
a region having a gapmer motif, which comprises two external
regions or "wings" and a central or internal region or "gap." The
three regions of a gapmer motif (the 5'-wing, the gap, and the
3'-wing) form a contiguous sequence of nucleosides wherein at least
some of the sugar moieties of the nucleosides of each of the wings
differ from at least some of the sugar moieties of the nucleosides
of the gap. Specifically, at least the sugar moieties of the
nucleosides of each wing that are closest to the gap (the 3'-most
nucleoside of the 5'-wing and the 5'-most nucleoside of the
3'-wing) differ from the sugar moiety of the neighboring gap
nucleosides, thus defining the boundary between the wings and the
gap (i.e., the wing/gap junction). In certain embodiments, the
sugar moieties within the gap are the same as one another. In
certain embodiments, the gap includes one or more nucleoside having
a sugar moiety that differs from the sugar moiety of one or more
other nucleosides of the gap. In certain embodiments, the sugar
motifs of the two wings are the same as one another (symmetric
gapmer). In certain embodiments, the sugar motif of the 5'-wing
differs from the sugar motif of the 3'-wing (asymmetric
gapmer).
[0349] In certain embodiments, the wings of a gapmer comprise 1-5
nucleosides. In certain embodiments, the wings of a gapmer comprise
2-5 nucleosides. In certain embodiments, the wings of a gapmer
comprise 3-5 nucleosides. In certain embodiments, the nucleosides
of a gapmer are all modified nucleosides.
[0350] In certain embodiments, the gap of a gapmer comprises 7-12
nucleosides. In certain embodiments, the gap of a gapmer comprises
7-10 nucleosides. In certain embodiments, the gap of a gapmer
comprises 8-10 nucleosides. In certain embodiments, the gap of a
gapmer comprises 10 nucleosides. In certain embodiment, each
nucleoside of the gap of a gapmer is an unmodified 2'-deoxy
nucleoside.
[0351] In certain embodiments, the gapmer is a deoxy gapmer. In
such embodiments, the nucleosides on the gap side of each wing/gap
junction are unmodified 2'-deoxy nucleosides and the nucleosides on
the wing sides of each wing/gap junction are modified nucleosides.
In certain such embodiments, each nucleoside of the gap is an
unmodified 2'-deoxy nucleoside. In certain such embodiments, each
nucleoside of each wing is a modified nucleoside.
[0352] In certain embodiments, the first modified oligonucleotide
and/or the second modified oligonucleotide comprise or consist of a
region having a fully modified sugar motif. In such embodiments,
each nucleoside of the fully modified region of the modified
oligonucleotide comprises a modified sugar moiety. In certain such
embodiments, each nucleoside to the entire modified oligonucleotide
(either the first modified oligonucleotide and/or the second
modified oligonucleotide) comprises a modified sugar moiety. In
certain embodiments, modified oligonucleotides (either the first
modified oligonucleotide and/or the second modified
oligonucleotide) comprise or consist of a region having a fully
modified sugar motif, wherein each nucleoside within the fully
modified region comprises the same modified sugar moiety, referred
to herein as a uniformly modified sugar motif. In certain
embodiments, a fully modified oligonucleotide is a uniformly
modified oligonucleotide. In certain embodiments, each nucleoside
of a uniformly modified comprises the same 2'-modification.
[0353] 2. Certain Nucleobase Motifs
[0354] In certain embodiments, oligonucleotides (including the
first modified oligonucleotide and/or the second modified
oligonucleotide) comprise modified and/or unmodified nucleobases
arranged along the oligonucleotide or region thereof in a defined
pattern or motif. In certain embodiments, each nucleobase is
modified. In certain embodiments, none of the nucleobases are
modified. In certain embodiments, each purine or each pyrimidine is
modified. In certain embodiments, each adenine is modified. In
certain embodiments, each guanine is modified. In certain
embodiments, each thymine is modified. In certain embodiments, each
uracil is modified. In certain embodiments, each cytosine is
modified. In certain embodiments, some or all of the cytosine
nucleobases in a modified oligonucleotide are
5-methylcytosines.
[0355] In certain embodiments, modified oligonucleotides (for
example the first modified oligonucleotide or the second modified
oligonucleotide) comprise a block of modified nucleobases. In
certain such embodiments, the block is at the 3'-end of the
oligonucleotide. In certain embodiments the block is within 3
nucleosides of the 3'-end of the oligonucleotide. In certain
embodiments, the block is at the 5'-end of the oligonucleotide. In
certain embodiments the block is within 3 nucleosides of the 5'-end
of the oligonucleotide.
[0356] In certain embodiments, oligonucleotides having a gapmer
motif comprise a nucleoside comprising a modified nucleobase. In
certain such embodiments, one nucleoside comprising a modified
nucleobase is in the central gap of an oligonucleotide having a
gapmer motif. In certain such embodiments, the sugar moiety of said
nucleoside is a 2'-deoxyribosyl moiety. In certain embodiments, the
modified nucleobase is selected from: a 2-thiopyrimidine and a
5-propynepyrimidine. In certain embodiments, the first modified
oligonucleotide has a gapmer motif. In certain embodiments, the
first modified oligonucleotide has a gapmer motif and the second
modified oligonucleotide does not have a gapmer motif. In certain
embodiments, the first modified oligonucleotide has a gapmer motif
and the second modified oligonucleotide has a fully modified
motif
[0357] 3. Certain Internucleoside Linkage Motifs
[0358] In certain embodiments, oligonucleotides, for example the
first modified oligonucleotide and/or the second modified
oligonucleotide, comprise modified and/or unmodified
internucleoside linkages arranged along the oligonucleotide or
region thereof in a defined pattern or motif. In certain
embodiments, essentially each internucleoside linking group is a
phosphate internucleoside linkage (P.dbd.O). In certain
embodiments, each internucleoside linking group of a modified
oligonucleotide is a phosphorothioate (P.dbd.S). In certain
embodiments, essentially each internucleoside linking group of the
first modified oligonucleotide is a phosphate internucleoside
linkage (P.dbd.O). In certain embodiments, each internucleoside
linking group of the first modified oligonucleotide is a
phosphorothioate (P.dbd.S). In certain embodiments, essentially
each internucleoside linking group of the second modified
oligonucleotide is a phosphate internucleoside linkage (P.dbd.O).
In certain embodiments, each internucleoside linking group of the
second modified oligonucleotide is a phosphorothioate (P.dbd.S). In
certain embodiments, each internucleoside linking group of the
first modified oligonucleotide is independently selected from a
phosphorothioate and phosphate internucleoside linkage. In certain
embodiments, each internucleoside linking group of the second
modified oligonucleotide is independently selected from a
phosphorothioate and phosphate internucleoside linkage. In certain
embodiments, the sugar motif of the first modified oligonucleotide
is a gapmer and the internucleoside linkages within the gap are all
modified. In certain such embodiments, some or all of the
internucleoside linkages in the wings are unmodified phosphate
linkages. In certain embodiments, the terminal internucleoside
linkages are modified.
[0359] D. Certain Lengths
[0360] In certain embodiments, oligonucleotides (including modified
oligonucleotides) can have any of a variety of ranges of lengths.
In certain embodiments, the first modified oligonucleotide can have
any of a variety of ranges of lengths. In certain embodiments, the
second modified oligonucleotide can have any of a variety of ranges
of lengths. In certain embodiments, oligonucleotides consist of X
to Y linked nucleosides, where X represents the fewest number of
nucleosides in the range and Y represents the largest number
nucleosides in the range. In certain such embodiments, X and Y are
each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and
50; provided that X.ltoreq.Y. For example, in certain embodiments,
oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16,
12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to
23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12
to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19,
13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to
26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14
to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23,
14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to
30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15
to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28,
15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to
21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16
to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21,
17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to
28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18
to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29,
18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to
25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20
to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28,
20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to
26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22
to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24,
23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to
25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25
to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29,
26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29
to 30 linked nucleosides
[0361] E. Certain Modified Oligonucleotides
[0362] In certain embodiments, the above modifications (sugar,
nucleobase, internucleoside linkage) are incorporated into a
modified oligonucleotide. In certain embodiments, modified
oligonucleotides are characterized by their modification motifs and
overall lengths. In certain embodiments, such parameters are each
independent of one another. Thus, unless otherwise indicated, each
internucleoside linkage of an oligonucleotide having a gapmer sugar
motif may be modified or unmodified and may or may not follow the
gapmer modification pattern of the sugar modifications. For
example, the internucleoside linkages within the wing regions of a
sugar gapmer may be the same or different from one another and may
be the same or different from the internucleoside linkages of the
gap region of the sugar motif. Likewise, such sugar gapmer
oligonucleotides may comprise one or more modified nucleobase
independent of the gapmer pattern of the sugar modifications.
Furthermore, in certain instances, an oligonucleotide is described
by an overall length or range and by lengths or length ranges of
two or more regions (e.g., a regions of nucleosides having
specified sugar modifications), in such circumstances it may be
possible to select numbers for each range that result in an
oligonucleotide having an overall length falling outside the
specified range. In such circumstances, both elements must be
satisfied. For example, in certain embodiments, a modified
oligonucleotide consists if of 15-20 linked nucleosides and has a
sugar motif consisting of three regions, A, B, and C, wherein
region A consists of 2-6 linked nucleosides having a specified
sugar motif, region B consists of 6-10 linked nucleosides having a
specified sugar motif, and region C consists of 2-6 linked
nucleosides having a specified sugar motif. Such embodiments do not
include modified oligonucleotides where A and C each consist of 6
linked nucleosides and B consists of 10 linked nucleosides (even
though those numbers of nucleosides are permitted within the
requirements for A, B, and C) because the overall length of such
oligonucleotide is 22, which exceeds the upper limit of the overall
length of the modified oligonucleotide (20). Herein, if a
description of an oligonucleotide is silent with respect to one or
more parameter, such parameter is not limited. Thus, a modified
oligonucleotide described only as having a gapmer sugar motif
without further description may have any length, internucleoside
linkage motif, and nucleobase motif. Unless otherwise indicated,
all modifications are independent of nucleobase sequence.
[0363] F. Nucleobase Sequence
[0364] In certain embodiments, oligonucleotides (unmodified or
modified oligonucleotides) are further described by their
nucleobase sequence. In certain embodiments oligonucleotides have a
nucleobase sequence that is complementary to a second
oligonucleotide or an identified reference nucleic acid, such as a
target nucleic acid. In certain such embodiments, a region of an
oligonucleotide has a nucleobase sequence that is complementary to
a second oligonucleotide or an identified reference nucleic acid,
such as a target nucleic acid. In certain embodiments, the
nucleobase sequence of a region or entire length of an
oligonucleotide is at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, or 100% complementary to the
second oligonucleotide or nucleic acid, such as a target nucleic
acid.
[0365] II. Certain Oligomeric Compounds
[0366] In certain embodiments, the invention provides oligomeric
compounds, which consist of an oligonucleotide (modified or
unmodified or a first modified oligonucleotide or a second modified
oligonucleotide) and optionally one or more conjugate groups and/or
terminal groups. Conjugate groups consist of one or more conjugate
moiety and a conjugate linker which links the conjugate moiety to
the oligonucleotide. Conjugate groups may be attached to either or
both ends of an oligonucleotide and/or at any internal position. In
certain embodiments, conjugate groups are attached to the
2'-position of a nucleoside of a modified oligonucleotide. In
certain embodiments, conjugate groups that are attached to either
or both ends of an oligonucleotide are terminal groups. In certain
such embodiments, conjugate groups or terminal groups are attached
at the 3' and/or 5'-end of oligonucleotides. In certain such
embodiments, conjugate groups (or terminal groups) are attached at
the 3'-end of oligonucleotides. In certain embodiments, conjugate
groups are attached near the 3'-end of oligonucleotides. In certain
embodiments, conjugate groups (or terminal groups) are attached at
the 5'-end of oligonucleotides. In certain embodiments, conjugate
groups are attached near the 5'-end of oligonucleotides.
[0367] Examples of terminal groups include but are not limited to
conjugate groups, capping groups, phosphate moieties, protecting
groups, modified or unmodified nucleosides, and two or more
nucleosides that are independently modified or unmodified.
[0368] A. Certain Conjugate Groups
[0369] In certain embodiments, oligonucleotides, including a first
modified oligonucleotide or a second modified oligonucleotide, are
covalently attached to one or more conjugate groups. In certain
embodiments, a second modified oligonucleotide is covalently
attached to one or more conjugate groups. In certain embodiments, a
second modified oligonucleotide is covalently attached to one or
more conjugate groups and the first modified oligonucleotide is not
attached to a conjugate group. In certain embodiments, conjugate
groups modify one or more properties of the attached
oligonucleotide, including but not limited to pharmacodynamics,
pharmacokinetics, stability, binding, absorption, tissue
distribution, cellular distribution, cellular uptake, charge and
clearance. In certain embodiments, conjugate groups impart a new
property on the attached oligonucleotide, e.g., fluorophores or
reporter groups that enable detection of the oligonucleotide.
Certain conjugate groups and conjugate moieties have been described
previously, for example: cholesterol moiety (Letsinger et al.,
Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid
(Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a
thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.
Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et
al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain,
e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al.,
EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990,
259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid a palmityl moiety (Mishra et
al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an
octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke
et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), a tocopherol
group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4,
e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or
a GalNAc cluster (e.g., WO2014/179620).
[0370] 1. Conjugate Moieties
[0371] Conjugate moieties include, without limitation,
intercalators, reporter molecules, polyamines, polyamides,
peptides, carbohydrates, vitamin moieties, polyethylene glycols,
thioethers, polyethers, cholesterols, thiocholesterols, cholic acid
moieties, folate, lipids, phospholipids, biotin, phenazine,
phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,
rhodamines, coumarins, fluorophores, and dyes.
[0372] In certain embodiments, a conjugate moiety comprises an
active drug substance, for example, aspirin, warfarin,
phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen,
(S)-(+)-pranoprofen, carprofen, dansylsarcosine,
2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic
acid, a benzothiadiazide, chlorothiazide, a diazepine,
indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
[0373] 2. Conjugate Linkers
[0374] Conjugate moieties are attached to oligonucleotides through
conjugate linkers. In certain oligomeric compounds, the conjugate
linker is a single chemical bond (i.e., the conjugate moiety is
attached directly to an oligonucleotide through a single bond). In
certain embodiments, the conjugate linker comprises a chain
structure, such as a hydrocarbyl chain, or an oligomer of repeating
units such as ethylene glycol, nucleosides, or amino acid
units.
[0375] In certain embodiments, a conjugate linker comprises one or
more groups selected from alkyl, amino, oxo, amide, disulfide,
polyethylene glycol, ether, thioether, and hydroxylamino. In
certain such embodiments, the conjugate linker comprises groups
selected from alkyl, amino, oxo, amide and ether groups. In certain
embodiments, the conjugate linker comprises groups selected from
alkyl and amide groups. In certain embodiments, the conjugate
linker comprises groups selected from alkyl and ether groups. In
certain embodiments, the conjugate linker comprises at least one
phosphorus moiety. In certain embodiments, the conjugate linker
comprises at least one phosphate group. In certain embodiments, the
conjugate linker includes at least one neutral linking group.
[0376] In certain embodiments, conjugate linkers, including the
conjugate linkers described above, are bifunctional linking
moieties, e.g., those known in the art to be useful for attaching
conjugate groups to parent compounds, such as the oligonucleotides
provided herein. In general, a bifunctional linking moiety
comprises at least two functional groups. One of the functional
groups is selected to bind to a particular site on a parent
compound and the other is selected to bind to a conjugate group.
Examples of functional groups used in a bifunctional linking moiety
include but are not limited to electrophiles for reacting with
nucleophilic groups and nucleophiles for reacting with
electrophilic groups. In certain embodiments, bifunctional linking
moieties comprise one or more groups selected from amino, hydroxyl,
carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.
[0377] Examples of conjugate linkers include but are not limited to
pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and
6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include
but are not limited to substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl or substituted or unsubstituted
C.sub.2-C.sub.10 alkynyl, wherein a nonlimiting list of preferred
substituent groups includes hydroxyl, amino, alkoxy, carboxy,
benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,
alkenyl and alkynyl.
[0378] In certain embodiments, conjugate linkers comprise 1-10
linker-nucleosides. In certain embodiments, conjugate linkers
comprise 2-5 linker-nucleosides. In certain embodiments, conjugate
linkers comprise exactly 3 linker-nucleosides. In certain
embodiments, conjugate linkers comprise the TCA motif. In certain
embodiments, such linker-nucleosides are modified nucleosides. In
certain embodiments such linker-nucleosides comprise a modified
sugar moiety. In certain embodiments, linker-nucleosides are
unmodified. In certain embodiments, linker-nucleosides comprise an
optionally protected heterocyclic base selected from a purine,
substituted purine, pyrimidine or substituted pyrimidine. In
certain embodiments, a cleavable moiety is a nucleoside selected
from uracil, thymine, cytosine, 4-N-benzoylcytosine,
5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,
6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is
typically desirable for linker-nucleosides to be cleaved from the
oligomeric compound after it reaches a target tissue. Accordingly,
linker-nucleosides are typically linked to one another and to the
remainder of the oligomeric compound through cleavable bonds. In
certain embodiments, such cleavable bonds are phosphodiester
bonds.
[0379] Herein, linker-nucleosides are not considered to be part of
the oligonucleotide. Accordingly, in embodiments in which an
oligomeric compound comprises an oligonucleotide consisting of a
specified number or range of linked nucleosides and/or a specified
percent complementarity to a reference nucleic acid and the
oligomeric compound also comprises a conjugate group comprising a
conjugate linker comprising linker-nucleosides, those
linker-nucleosides are not counted toward the length of the
oligonucleotide and are not used in determining the percent
complementarity of the oligonucleotide for the reference nucleic
acid. For example, an oligomeric compound may comprise (1) a
modified oligonucleotide consisting of 8-30 nucleosides and (2) a
conjugate group comprising 1-10 linker-nucleosides that are
contiguous with the nucleosides of the modified oligonucleotide.
The total number of contiguous linked nucleosides in such an
oligomeric compound is more than 30. Alternatively, an oligomeric
compound may comprise a modified oligonucleotide consisting of 8-30
nucleosides and no conjugate group. The total number of contiguous
linked nucleosides in such an oligomeric compound is no more than
30. Unless otherwise indicated conjugate linkers comprise no more
than 10 linker-nucleosides. In certain embodiments, conjugate
linkers comprise no more than 5 linker-nucleosides. In certain
embodiments, conjugate linkers comprise no more than 3
linker-nucleosides. In certain embodiments, conjugate linkers
comprise no more than 2 linker-nucleosides. In certain embodiments,
conjugate linkers comprise no more than 1 linker-nucleoside.
[0380] In certain embodiments, it is desirable for a conjugate
group to be cleaved from the oligonucleotide. For example, in
certain circumstances oligomeric compounds comprising a particular
conjugate moiety are better taken up by a particular cell type, but
once the oligomeric compound has been taken up, it is desirable
that the conjugate group be cleaved to release the unconjugated or
parent oligonucleotide. Thus, certain conjugate linkers may
comprise one or more cleavable moieties. In certain embodiments, a
cleavable moiety is a cleavable bond. In certain embodiments, a
cleavable moiety is a group of atoms comprising at least one
cleavable bond. In certain embodiments, a cleavable moiety
comprises a group of atoms having one, two, three, four, or more
than four cleavable bonds. In certain embodiments, a cleavable
moiety is selectively cleaved inside a cell or subcellular
compartment, such as a lysosome. In certain embodiments, a
cleavable moiety is selectively cleaved by endogenous enzymes, such
as nucleases.
[0381] In certain embodiments, a cleavable bond is selected from
among: an amide, an ester, an ether, one or both esters of a
phosphodiester, a phosphate ester, a carbamate, or a disulfide. In
certain embodiments, a cleavable bond is one or both of the esters
of a phosphodiester. In certain embodiments, a cleavable moiety
comprises a phosphate or phosphodiester. In certain embodiments,
the cleavable moiety is a phosphate linkage between an
oligonucleotide and a conjugate moiety or conjugate group.
[0382] In certain embodiments, a cleavable moiety comprises or
consists of one or more linker-nucleosides. In certain such
embodiments, the one or more linker-nucleosides are linked to one
another and/or to the remainder of the oligomeric compound through
cleavable bonds. In certain embodiments, such cleavable bonds are
unmodified phosphodiester bonds. In certain embodiments, a
cleavable moiety is 2'-deoxy nucleoside that is attached to either
the 3' or 5'-terminal nucleoside of an oligonucleotide by a
phosphate internucleoside linkage and covalently attached to the
remainder of the conjugate linker or conjugate moiety by a
phosphate or phosphorothioate linkage. In certain such embodiments,
the cleavable moiety is 2'-deoxyadenosine.
[0383] III. Certain Antisense Compounds
[0384] In certain embodiments, the present invention provides
antisense compounds, which comprise or consist of an oligomeric
compound comprising an antisense oliognucleotide, having a
nucleobase sequences complementary to that of a target nucleic
acid. In certain embodiments, antisense compounds are
single-stranded. Such single-stranded antisense compounds typically
comprise or consist of an oligomeric compound that comprises or
consists of a modified oligonucleotide and optionally a conjugate
group. In certain embodiments, antisense compounds are
double-stranded. Such double-stranded antisense compounds comprise
a first oligomeric compound having a region complementary to a
target nucleic acid and a second oligomeric compound having a
region complementary to the first oligomeric compound. The first
oligomeric compound of such double stranded antisense compounds
typically comprises or consists of a modified oligonucleotide and
optionally a conjugate group. The oligonucleotide of the second
oligomeric compound of such double-stranded antisense compound may
be modified or unmodified. Either or both oligomeric compounds of a
double-stranded antisense compound may comprise a conjugate group.
The oligomeric compounds of double-stranded antisense compounds may
include non-complementary overhanging nucleosides.
[0385] In certain embodiments, oligomeric compounds of antisense
compounds are capable of hybridizing to a target nucleic acid,
resulting in at least one antisense activity. In certain
embodiments, antisense compounds selectively affect one or more
target nucleic acid. Such selective antisense compounds comprises a
nucleobase sequence that hybridizes to one or more target nucleic
acid, resulting in one or more desired antisense activity and does
not hybridize to one or more non-target nucleic acid or does not
hybridize to one or more non-target nucleic acid in such a way that
results in significant undesired antisense activity.
[0386] In certain antisense activities, hybridization of an
antisense compound to a target nucleic acid results in recruitment
of a protein that cleaves the target nucleic acid. For example,
certain antisense compounds result in RNase H mediated cleavage of
the target nucleic acid. RNase H is a cellular endonuclease that
cleaves the RNA strand of an RNA:DNA duplex. The DNA in such an
RNA:DNA duplex need not be unmodified DNA. In certain embodiments,
the invention provides antisense compounds that are sufficiently
"DNA-like" to elicit RNase H activity. Further, in certain
embodiments, one or more non-DNA-like nucleoside in the gap of a
gapmer is tolerated.
[0387] In certain antisense activities, an antisense compound or a
portion of an antisense compound is loaded into an RNA-induced
silencing complex (RISC), ultimately resulting in cleavage of the
target nucleic acid. For example, certain antisense compounds
result in cleavage of the target nucleic acid by Argonaute.
Antisense compounds that are loaded into RISC are RNAi compounds.
RNAi compounds may be double-stranded (siRNA) or single-stranded
(ssRNA).
[0388] In certain embodiments, hybridization of an antisense
compound to a target nucleic acid does not result in recruitment of
a protein that cleaves that target nucleic acid. In certain such
embodiments, hybridization of the antisense compound to the target
nucleic acid results in alteration of splicing of the target
nucleic acid. In certain embodiments, hybridization of an antisense
compound to a target nucleic acid results in inhibition of a
binding interaction between the target nucleic acid and a protein
or other nucleic acid. In certain such embodiments, hybridization
of an antisense compound to a target nucleic acid results in
alteration of translation of the target nucleic acid.
[0389] Antisense activities may be observed directly or indirectly.
In certain embodiments, observation or detection of an antisense
activity involves observation or detection of a change in an amount
of a target nucleic acid or protein encoded by such target nucleic
acid, a change in the ratio of splice variants of a nucleic acid or
protein, and/or a phenotypic change in a cell or animal.
[0390] IV. Certain Duplexes
[0391] In certain embodiments, the present invention provides
duplexes comprising a first oligomeric compound comprising a first
modified oligonucleotide and a second oligomeric compound
comprising a second oligonucleotide, wherein the first and second
modified oligonucleotides comprise regions of complementarity
sufficient to form a duplex. In certain such embodiments, the first
modified oligonucleotide is complementary to a target nucleic acid.
In certain such embodiments, the first modified oligonucleotide is
a gapmer as described above. Thus, in such embodiments, the first
oligomeric compound is capable of hybridizing to a target nucleic
acid and eliciting cleavage of the target nucleic acid by RNase H.
In certain such embodiments, the second oligomeric compound
improves a property of the first oligomeric compound compared to
the property in the absence of the second oligomeric compound. In
certain embodiments, that improved property is one or more of:
distribution to a target tissue, uptake into a target cell,
potency, and efficacy. In certain embodiments, the target tissue is
liver (hepatic). In certain embodiments, the target tissue is other
than liver (extra-hepatic). In certain embodiments, it is desirable
to reduce target in more than one tissue. In certain such
embodiments, it is desirable to reduce target in the liver and one
or more other tissues. In certain embodiments, it is desirable to
reduce target in more than one extra-hepatic tissue.
[0392] In certain embodiments, the first oligonucleotide of a
duplex is a gapmer. In certain such embodiments, the wings of the
gapmer comprise 2'-MOE modified nucleosides. In certain
embodiments, the wings of the gapmer comprise cEt nucleosides. In
certain embodiments the wings of the gapmer comprise LNA
nucleosides. In certain embodiments, the wings of a gapmer comprise
at least one 2'-MOE modified nucleoside and at least one bicyclic
nucleoside. In certain such embodiments, each such bicyclic
nucleoside is selected from among an LNA nucleoside and a cEt
nucleoside. In certain embodiments, the gap constitutes 7-10
2'-deoxynucleosides.
[0393] In certain embodiments, the second oligonucleotide comprises
at least one 2'-MOE nucleoside. In certain embodiments, the second
oligonucleotide comprises 2'-MOE and 2'-deoxynucleosides. In
certain embodiments, the second oligonucleotide has sugar motif of
alternating modification types (including no modification). In
certain such embodiments, the sugar motif of the second
oligonucleotide alternates between 2'-MOE nucleosides and
2'-deoxynucleosides. In certain embodiments, the second
oligonucleotide has a sugar motif similar to a gapmer (as described
above) except that it may not elicit cleavage of a target nucleic
acid. Such gapmer-like motifs have a central region and flanking
wing regions. In certain such embodiment, the central region is
comprises of 2'-deoxynucleosides and the wing regions are 2'-MOE
modified nucleosides. The internucleoside linkages of the second
oligonucleotide may be modified or phosphodiester. In certain
embodiments, the internucleoside linkages of the second
oligonucleotide follow a gapmer-like motif--phosphorothioate wings
and phosphdiester in the center. Such internucleoside linkage motif
may or may not track the sugar motif. Though duplexes comprising
second oligomeric compounds with second oligonucleotides having
central regions comprising RNA are shown herein to have enhanced
activity (compared to the first oligomeric compound alone and not
in a duplex) it is noted that such oligonucleotides comprising RNA
are expensive to manufacture and relatively unstable when compared
to oligonucleotides that comprise modified nucleosides or DNA
nucleosides.
[0394] In certain embodiments, at least one of the first and second
oligomeric compounds comprises a conjugate group (as described
above). Typically, the second oligomeric compound comprises a
conjugate group. The conjugate group may be attached at either end
of the oligomeric compound. In certain embodiments, a conjugate
group is attached to both ends.
[0395] V. Certain Target Nucleic Acids
[0396] In certain embodiments, antisense compounds comprise or
consist of an oligonucleotide comprising a region that is
complementary to a target nucleic acid. In certain embodiments, the
target nucleic acid is an endogenous RNA molecule. In certain
embodiments, the target nucleic acid encodes a protein. In certain
such embodiments, the target nucleic acid is selected from: an mRNA
and a pre-mRNA, including intronic, exonic and untranslated
regions. In certain embodiments, the target RNA is an mRNA. In
certain embodiments, the target nucleic acid is a pre-mRNA. In
certain such embodiments, the target region is entirely within an
intron. In certain embodiments, the target region spans an
intron/exon junction. In certain embodiments, the target region is
at least 50% within an intron.
[0397] In certain embodiments, the target nucleic acid is a
non-coding RNA. In certain such embodiments, the target non-coding
RNA is selected from: a long-non-coding RNA, a short non-coding
RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA
(including pre-microRNA and mature microRNA), a ribosomal RNA, and
promoter directed RNA. In certain embodiments, the target nucleic
acid is a nucleic acid other than a mature mRNA. In certain
embodiments, the target nucleic acid is a nucleic acid other than a
mature mRNA or a microRNA. In certain embodiments, the target
nucleic acid is a non-coding RNA other than a microRNA. In certain
embodiments, the target nucleic acid is a non-coding RNA other than
a microRNA or an intronic region of a pre-mRNA. In certain
embodiments, the target nucleic acid is a long non-coding RNA. In
certain embodiments, the target nucleic acid is a non-coding RNA
associated with splicing of other pre-mRNAs. In certain
embodiments, the target nucleic acid is a nuclear-retained
non-coding RNA.
[0398] In certain embodiments, antisense compounds described herein
are complementary to a target nucleic acid comprising a
single-nucleotide polymorphism (SNP). In certain such embodiments,
the antisense compound is capable of modulating expression of one
allele of the SNP-containing target nucleic acid to a greater or
lesser extent than it modulates another allele. In certain
embodiments, an antisense compound hybridizes to a (SNP)-containing
target nucleic acid at the single-nucleotide polymorphism site.
[0399] In certain embodiments, antisense compounds are at least
partially complementary to more than one target nucleic acid. For
example, antisense compounds of the present invention may mimic
microRNAs, which typically bind to multiple targets.
[0400] A. Complementarity/Mismatches to the Target Nucleic Acid
[0401] In certain embodiments, antisense compounds comprise
antisense oligonucleotides that are complementary to the target
nucleic acid over the entire length of the oligonucleotide. In
certain embodiments, such oligonucleotides are 99% complementary to
the target nucleic acid. In certain embodiments, such
oligonucleotides are 95% complementary to the target nucleic acid.
In certain embodiments, such oligonucleotides are 90% complementary
to the target nucleic acid. In certain embodiments, such
oligonucleotides are 85% complementary to the target nucleic acid.
In certain embodiments, such oligonucleotides are 80% complementary
to the target nucleic acid. In certain embodiments, antisense
oligonucleotides are at least 80% complementary to the target
nucleic acid over the entire length of the oligonucleotide and
comprise a region that is 100% or fully complementary to a target
nucleic acid. In certain such embodiments, the region of full
complementarity is from 6 to 20 nucleobases in length. In certain
such embodiments, the region of full complementarity is from 10 to
18 nucleobases in length. In certain such embodiments, the region
of full complementarity is from 18 to 20 nucleobases in length.
[0402] In certain embodiments, the oligomeric compounds of
antisense compounds comprise one or more mismatched nucleobases
relative to the target nucleic acid. In certain such embodiments,
antisense activity against the target is reduced by such mismatch,
but activity against a non-target is reduced by a greater amount.
Thus, in certain such embodiments selectivity of the antisense
compound is improved. In certain embodiments, the mismatch is
specifically positioned within an oligonucleotide having a gapmer
motif. In certain such embodiments, the mismatch is at position 1,
2, 3, 4, 5, 6, 7, or 8 from the 5'-end of the gap region. In
certain such embodiments, the mismatch is at position 9, 8, 7, 6,
5, 4, 3, 2, 1 from the 3'-end of the gap region. In certain such
embodiments, the mismatch is at position 1, 2, 3, or 4 from the
5'-end of the wing region. In certain such embodiments, the
mismatch is at position 4, 3, 2, or 1 from the 3'-end of the wing
region.
[0403] B. Certain Target Nucleic Acids in Certain Tissues
[0404] In certain embodiments, antisense compounds comprise or
consist of an oligonucleotide comprising a region that is
complementary to a target nucleic acid, wherein the target nucleic
acid is expressed in an extra-hepatic tissue. Extra-hepatic tissues
include, but are not limited to: skeletal muscle, cardiac muscle,
smooth muscle, adipose, white adipose, spleen, bone, intestine,
adrenal, testes, ovary, pancreas, pituitary, prostate, skin,
uterus, bladder, brain, glomerulus, distal tubular epithelium,
breast, lung, heart, kidney, ganglion, frontal cortex, spinal cord,
trigeminal ganglia, sciatic nerve, dorsal root ganglion, epididymal
fat, diaphragm, pancreas, and colon.
[0405] A. Certain First Modified Oligonucleotides
[0406] In certain embodiments, disclosed here in are first modified
oligonucleotides designed to target certain nucleic acid targets.
Tables A and B below describe certain modified oligonucleotides
targeted to certain nucleic acid transcripts. In Tables A and B
below, subscript "s" represents a phosphorothioate internucleoside
linkage, subscript "o" represents a phosphate internucleoside
linkage, subscript "d" represents a 2'-deoxynucleoside, subscript
"e" represents a 2'-MOE modified nucleoside, and subscript "k"
represents a cEt modified nucleoside. In tables A and B below,
superscript "m" before a C represents a 5-methylcysteine.
TABLE-US-00001 TABLE A Certain First Modified Oligonucleotides SEQ
Isis ID Target No. Sequence (5'-3') Motif NO: CRP 329993
AGCATAGTTAACGAGCTCCC 5-10-5 MOE 14 PTPB1B 404173
AATGGTTTATTCCATGGCCA 5-10-5 MOE 15 GCCR 426115 GCAGCCATGGTGATCAGGAG
5-10-5 MOE 16 GCGR 449884 GGTTCCCGAGGTGCCCA 3-10-4 MOE 17 FGFR4
463588 GCACACTCAGCAGGACCCCC 5-10-5 MOE 18 GHr 532401
CCACCTTTGGGTGAATAGCA 5-10-5 MOE 19 DGAT2 484137
TGCCATTTAATGAGCTTCAC 5-10-5 MOE 20 DMPK 598769 TCCCGAATGTCCGACA
Mixed wing 21 CFB 696844 ATCCCACGCCCCTGTCCAGC 5-10-5 MOE 22
GalNAc
TABLE-US-00002 TABLE B Certain First Modified Oligonucleotides
Target Isis No. Motif (5'-3') CRP 329993
A.sub.esG.sub.es.sup.mC.sub.esA.sub.esT.sub.esA.sub.dsG.sub.dsT-
.sub.dsT.sub.dsA.sub.dsA.sub.ds.sup.mC.sub.dsG.sub.dsA.sub.dsG.sub.ds.sup.-
mC.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e PTPB1B
404173
A.sub.esA.sub.esT.sub.esG.sub.esG.sub.esT.sub.dsT.sub.dsT.sub.dsA.sub.dsT-
.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.esG.sub.e-
s.sup.mC.sub.es.sup.mC.sub.esA.sub.e GCCR 426115
G.sub.es.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.es.sup.mC.sub.dsA.sub.d-
sT.sub.dsG.sub.dsG.sub.dsT.sub.dsG.sub.asA.sub.dsT.sub.ds.sup.mC.sub.dsA.s-
ub.esG.sub.esG.sub.esA.sub.esG.sub.e GCGR 449884
G.sub.esG.sub.esT.sub.esT.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.d-
sG.sub.dsA.sub.dsG.sub.dsG.sub.dsT.sub.dsG.sub.ds.sup.mC.sub.es.sup.mC.sub-
.es.sup.mC.sub.esA.sub.e FGFR4 463588
G.sub.es.sup.mC.sub.esA.sub.es.sup.mC.sub.esA.sub.es.sup.mC.sub.dsT.sub.d-
s.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.dsG.sub.dsA.sub-
.ds.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e
GHr 532401
.sup.mC.sub.es.sup.mC.sub.esA.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.dsG.sub.dsT.sub.dsG.sub.dsA.sub.dsA-
.sub.dsT.sub.esA.sub.esG.sub.es.sup.mC.sub.esA.sub.e DGAT2 484137
T.sub.esG.sub.es.sup.mC.sub.es.sup.mC.sub.esA.sub.esT.sub.dsT.sub.dsT.sub-
.dsA.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.esT-
.sub.es.sup.mC.sub.esA.sub.es.sup.mC.sub.e DMPK 598769
T.sub.es.sup.mC.sub.es.sup.mC.sub.ks.sup.mC.sub.ksG.sub.dsA.sub.dsA.sub.d-
sT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsG.sub.ksA.sub.ks.sup.-
mC.sub.esA.sub.e CFB 696844
A.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.esA.sub-
.ds.sup.mC.sub.dsG.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC-
.sub.dsT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.es.sup.mC.sub.esA.sub.esG.sub.e-
s.sup.mC.sub.e
The first modified oligonucleotides provided above can be paired
with the second modified oligonucleotide of a second oligomeric
compound to form a duplex.
[0407] I. Certain Pharmaceutical Compositions
[0408] In certain embodiments, the present invention provides
pharmaceutical compositions comprising one or more antisense
compound or a salt thereof. In certain such embodiments, the
pharmaceutical composition comprises a suitable pharmaceutically
acceptable diluent or carrier. In certain embodiments, a
pharmaceutical composition comprises a sterile saline solution and
one or more antisense compound. In certain embodiments, such
pharmaceutical composition consists of a sterile saline solution
and one or more antisense compound. In certain embodiments, the
sterile saline is pharmaceutical grade saline. In certain
embodiments, a pharmaceutical composition comprises one or more
antisense compound and sterile water. In certain embodiments, a
pharmaceutical composition consists of one antisense compound and
sterile water. In certain embodiments, the sterile water is
pharmaceutical grade water. In certain embodiments, a
pharmaceutical composition comprises one or more antisense compound
and phosphate-buffered saline (PBS). In certain embodiments, a
pharmaceutical composition consists of one or more antisense
compound and sterile PBS. In certain embodiments, the sterile PBS
is pharmaceutical grade PBS.
[0409] In certain embodiments, pharmaceutical compositions comprise
one or more or antisense compound and one or more excipients. In
certain such embodiments, excipients are selected from water, salt
solutions, alcohol, polyethylene glycols, gelatin, lactose,
amylase, magnesium stearate, talc, silicic acid, viscous paraffin,
hydroxymethylcellulose and polyvinylpyrrolidone.
[0410] In certain embodiments, antisense compounds may be admixed
with pharmaceutically acceptable active and/or inert substances for
the preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical
compositions depend on a number of criteria, including, but not
limited to, route of administration, extent of disease, or dose to
be administered.
[0411] In certain embodiments, pharmaceutical compositions
comprising an antisense compound encompass any pharmaceutically
acceptable salts of the antisense compound, esters of the antisense
compound, or salts of such esters. In certain embodiments,
pharmaceutical compositions comprising antisense compounds
comprising one or more antisense oligonucleotide, upon
administration to an animal, including a human, are capable of
providing (directly or indirectly) the biologically active
metabolite or residue thereof. Accordingly, for example, the
disclosure is also drawn to pharmaceutically acceptable salts of
antisense compounds, prodrugs, pharmaceutically acceptable salts of
such prodrugs, and other bioequivalents. Suitable pharmaceutically
acceptable salts include, but are not limited to, sodium and
potassium salts. In certain embodiments, prodrugs comprise one or
more conjugate group attached to an oligonucleotide, wherein the
conjugate group is cleaved by endogenous nucleases within the
body.
[0412] Lipid moieties have been used in nucleic acid therapies in a
variety of methods. In certain such methods, the nucleic acid, such
as an antisense compound, is introduced into preformed liposomes or
lipoplexes made of mixtures of cationic lipids and neutral lipids.
In certain methods, DNA complexes with mono- or poly-cationic
lipids are formed without the presence of a neutral lipid. In
certain embodiments, a lipid moiety is selected to increase
distribution of a pharmaceutical agent to a particular cell or
tissue. In certain embodiments, a lipid moiety is selected to
increase distribution of a pharmaceutical agent to fat tissue. In
certain embodiments, a lipid moiety is selected to increase
distribution of a pharmaceutical agent to muscle tissue.
[0413] In certain embodiments, pharmaceutical compositions comprise
a delivery system. Examples of delivery systems include, but are
not limited to, liposomes and emulsions. Certain delivery systems
are useful for preparing certain pharmaceutical compositions
including those comprising hydrophobic compounds. In certain
embodiments, certain organic solvents such as dimethylsulfoxide are
used.
[0414] In certain embodiments, pharmaceutical compositions comprise
one or more tissue-specific delivery molecules designed to deliver
the one or more pharmaceutical agents of the present invention to
specific tissues or cell types. For example, in certain
embodiments, pharmaceutical compositions include liposomes coated
with a tissue-specific antibody.
[0415] In certain embodiments, pharmaceutical compositions comprise
a co-solvent system. Certain of such co-solvent systems comprise,
for example, benzyl alcohol, a nonpolar surfactant, a
water-miscible organic polymer, and an aqueous phase. In certain
embodiments, such co-solvent systems are used for hydrophobic
compounds. A non-limiting example of such a co-solvent system is
the VPD co-solvent system, which is a solution of absolute ethanol
comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant
Polysorbate 80.TM. and 65% w/v polyethylene glycol 300. The
proportions of such co-solvent systems may be varied considerably
without significantly altering their solubility and toxicity
characteristics. Furthermore, the identity of co-solvent components
may be varied: for example, other surfactants may be used instead
of Polysorbate 80.TM.; the fraction size of polyethylene glycol may
be varied; other biocompatible polymers may replace polyethylene
glycol, e.g., polyvinyl pyrrolidone; and other sugars or
polysaccharides may substitute for dextrose.
[0416] In certain embodiments, pharmaceutical compositions are
prepared for oral administration. In certain embodiments,
pharmaceutical compositions are prepared for buccal administration.
In certain embodiments, a pharmaceutical composition is prepared
for administration by injection (e.g., intravenous, subcutaneous,
intramuscular, etc.). In certain of such embodiments, a
pharmaceutical composition comprises a carrier and is formulated in
aqueous solution, such as water or physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. In certain embodiments, other
ingredients are included (e.g., ingredients that aid in solubility
or serve as preservatives). In certain embodiments, injectable
suspensions are prepared using appropriate liquid carriers,
suspending agents and the like. Certain pharmaceutical compositions
for injection are presented in unit dosage form, e.g., in ampoules
or in multi-dose containers. Certain pharmaceutical compositions
for injection are suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Certain solvents
suitable for use in pharmaceutical compositions for injection
include, but are not limited to, lipophilic solvents and fatty
oils, such as sesame oil, synthetic fatty acid esters, such as
ethyl oleate or triglycerides, and liposomes. Aqueous injection
suspensions may contain.
Nonlimiting Disclosure and Incorporation by Reference
[0417] Each of the literature and patent publications listed herein
is incorporated by reference in its entirety. While certain
compounds, compositions and methods described herein have been
described with specificity in accordance with certain embodiments,
the following examples serve only to illustrate the compounds
described herein and are not intended to limit the same. Each of
the references, GenBank accession numbers, and the like recited in
the present application is incorporated herein by reference in its
entirety.
[0418] Although the sequence listing accompanying this filing
identifies each sequence as either "RNA" or "DNA" as required, in
reality, those sequences may be modified with any combination of
chemical modifications. One of skill in the art will readily
appreciate that such designation as "RNA" or "DNA" to describe
modified oligonucleotides is, in certain instances, arbitrary. For
example, an oligonucleotide comprising a nucleoside comprising a
2'-OH sugar moiety and a thymine base could be described as a DNA
having a modified sugar (2'-OH in place of one 2'-H of DNA) or as
an RNA having a modified base (thymine (methylated uracil) in place
of a uracil of RNA). Accordingly, nucleic acid sequences provided
herein, including, but not limited to those in the sequence
listing, are intended to encompass nucleic acids containing any
combination of natural or modified RNA and/or DNA, including, but
not limited to such nucleic acids having modified nucleobases. By
way of further example and without limitation, an oligomeric
compound having the nucleobase sequence "ATCGATCG" encompasses any
oligomeric compounds having such nucleobase sequence, whether
modified or unmodified, including, but not limited to, such
compounds comprising RNA bases, such as those having sequence
"AUCGAUCG" and those having some DNA bases and some RNA bases such
as "AUCGATCG" and oligomeric compounds having other modified
nucleobases, such as "ATmCGAUCG," wherein .sup.mC indicates a
cytosine base comprising a methyl group at the 5-position.
[0419] Certain compounds described herein (e.g., modified
oligonucleotides) have one or more asymmetric center and thus give
rise to enantiomers, diastereomers, and other stereoisomeric
configurations that may be defined, in terms of absolute
stereochemistry, as (R) or (S), as a or 13 such as for sugar
anomers, or as (D) or (L), such as for amino acids, etc. Included
in the compounds provided herein are all such possible isomers,
including their racemic and optically pure forms, unless specified
otherwise. Likewise, all cis- and trans-isomers and tautomeric
forms are also included unless otherwise indicated. Unless
otherwise indicated, compounds described herein are intended to
include corresponding salt forms.
EXAMPLES
[0420] The following examples illustrate certain embodiments of the
present disclosure and are not limiting. Moreover, where specific
embodiments are provided, the inventors have contemplated generic
application of those specific embodiments. For example, disclosure
of an oligonucleotide having a particular motif provides reasonable
support for additional oligonucleotides having the same or similar
motif. And, for example, where a particular high-affinity
modification appears at a particular position, other high-affinity
modifications at the same position are considered suitable, unless
otherwise indicated.
Example 1: Effects of Duplexes Comprising a Lipophilic Conjugate
Group In Vivo
[0421] Duplexes, each consisting of two oligomeric compounds, are
described in the table below. One oligomeric compound of each
duplex comprises an antisense oligonucleotide (Isis No. 626112)
that is complementary to both human and mouse Metastasis Associated
Lung Adenocarcinoma Transcript 1 (MALAT-1) transcripts. The other
oligomeric compound of each duplex comprises an oligonucleotide and
a lipophilic conjugate group. The effects of the duplexes on
MALAT-1 expression were tested in vivo. Wild type C57bl/6 mice each
received an intravenous injection, via the tail vein, of a duplex
listed in the table below, Isis No. 626112 alone as a control, or
saline vehicle alone. Each injection contained 100 mg/kg of the
antisense oligonucleotide (Isis No. 626112). Each treatment group
consisted of four mice. Eight days after the injection, the animals
were sacrificed. MALAT-1 RNA expression was analyzed in liver,
kidney, lung, trigeminal ganglia, frontal cortex, and spinal cord
by RT-qPCR and normalized to total RNA using RiboGreen (Thermo
Fisher Scientific, Carlsbad, Calif.). The average results for each
group are shown below as the percent normalized MALAT-1 RNA levels
relative to average results for the vehicle treated animals.
TABLE-US-00003 TABLE 1 MALAT-1 expression in vivo MALAT-1 RNA level
(% Vehicle) SEQ Fr. Sp. ID Duplex Isis No. Sequence (5' to 3')
Liver Kidney Lung Ganglia Cor. Cord NO. n/a 626112 G.sub.es
.sup.mC.sub.eo .sup.mC.sub.eo A.sub.eo G.sub.eo G.sub.ds 26 67 62
65 101 104 1 .sup.mC.sub.ds T.sub.ds G.sub.ds G.sub.ds T.sub.ds
T.sub.ds A.sub.ds T.sub.ds G.sub.ds A.sub.eo .sup.mC.sub.eo
T.sub.es .sup.mC.sub.es A.sub.e 1 626112 G.sub.es .sup.mC.sub.eo
.sup.mC.sub.eo A.sub.eo G.sub.eo G.sub.ds 59 59 89 55 119 110 1
.sup.mC.sub.ds T.sub.ds G.sub.ds G.sub.ds T.sub.ds T.sub.ds
A.sub.ds T.sub.ds G.sub.ds A.sub.eo .sup.mC.sub.eo T.sub.es
.sup.mC.sub.es A.sub.e 719228 Toco-TEG-U.sub.ms G.sub.ms A.sub.ro 2
G.sub.ro U.sub.ro C.sub.ro A.sub.ro U.sub.ro A.sub.ro A.sub.ro
C.sub.ro C.sub.ro A.sub.ro G.sub.ro C.sub.ro C.sub.ro U.sub.ro
G.sub.rs G.sub.ms C.sub.m 2 626112 G.sub.es .sup.mC.sub.eo
.sup.mC.sub.eo A.sub.eo G.sub.eo G.sub.ds 38 39 76 47 107 88 1
.sup.mC.sub.ds T.sub.ds G.sub.ds G.sub.ds T.sub.ds T.sub.ds
A.sub.ds T.sub.ds G.sub.ds A.sub.eo .sup.mC.sub.eo T.sub.es
.sup.mC.sub.es A.sub.e 719232 C10-TEG-U.sub.ms G.sub.ms A.sub.ro
G.sub.ro 2 U.sub.ro C.sub.ro A.sub.ro U.sub.ro A.sub.ro A.sub.ro
C.sub.ro C.sub.ro A.sub.ro G.sub.ro C.sub.ro C.sub.ro U.sub.ro
G.sub.rs G.sub.ms C.sub.m 3 626112 G.sub.es .sup.mC.sub.eo
.sup.mC.sub.eo A.sub.eo G.sub.eo G.sub.ds 27 63 86 38 120 75 1
.sup.mC.sub.ds T.sub.ds G.sub.ds G.sub.ds T.sub.ds T.sub.ds
A.sub.ds T.sub.ds G.sub.ds A.sub.eo .sup.mC.sub.eo T.sub.es
.sup.mC.sub.es A.sub.e 719233 C16-TEG-U.sub.ms G.sub.ms A.sub.ro
G.sub.ro 2 U.sub.ro C.sub.ro A.sub.ro U.sub.ro A.sub.ro A.sub.ro
C.sub.ro C.sub.ro A.sub.ro G.sub.ro C.sub.ro C.sub.ro U.sub.ro
G.sub.rs G.sub.ms C.sub.m 4 626112 G.sub.es .sup.mC.sub.eo
.sup.mC.sub.eo A.sub.eo G.sub.eo G.sub.ds 18 29 22 21 92 51 1
.sup.mC.sub.ds T.sub.ds G.sub.ds G.sub.ds T.sub.ds T.sub.ds
A.sub.ds T.sub.ds G.sub.ds A.sub.eo .sup.mC.sub.eo T.sub.es
.sup.mC.sub.es A.sub.e 719234 Chol-TEG-U.sub.ms G.sub.ms A.sub.ro 2
G.sub.ro U.sub.ro C.sub.ro A.sub.ro U.sub.ro A.sub.ro A.sub.ro
C.sub.ro C.sub.ro A.sub.ro G.sub.ro C.sub.ro C.sub.ro U.sub.ro
G.sub.rs G.sub.ms C.sub.m
Subscripts in the table above: "s" represents a phosphorothioate
internucleoside linkage, "o" represents a phosphate internucleoside
linkage, "d" represents a 2'-deoxynucleoside, "e" represents a
2'-MOE modified nucleoside, "r" represents a 2'-ribonucleoside
(2'-hydroxy), "m" represents 2'-O-methyl modified nucleoside.
Superscripts: "m" before a C represents a 5-methylcysteine. The
structures of "Chol-TEG-", "Toco-TEG-", "C10-TEG-", and "C16-TEG-"
are shown below.
##STR00008##
[0422] wherein n is 1 in "C10-TEG-", and n is 7 in "C16-TEG-".
Example 2: Effects of Duplexes Comprising a Lipophilic Conjugate
Group In Vivo
[0423] Duplexes, each consisting of two oligomeric compounds, are
described in the table below. One oligomeric compound of each
duplex comprises an antisense oligonucleotide (Isis No. 626112 or
Isis No. 556089) that is complementary to both human and mouse
MALAT-1 transcripts. The other oligomeric compound of each duplex
comprises an oligonucleotide and a lipophilic conjugate group. The
effects of the duplexes on MALAT-1 expression were tested in vivo.
Wild type C57bl/6 mice each received an intravenous injection, via
the tail vein, of a duplex listed in the table below, Isis No.
626112 alone, Isis No. 556089 alone, or saline. The dosages listed
in the table below indicate the amount of Isis No. 626112 or Isis
No. 556089 that was administered in each injection. Each treatment
group consisted of three or four mice. Three days after the
injection, the animals were sacrificed. MALAT-1 RNA expression was
analyzed in heart, macrophages (Macs), trigeminal ganglia (TG),
sciatic nerve (SN), and dorsal root ganglion (DRG) by RT-qPCR and
normalized to total RNA using RiboGreen (Thermo Fisher Scientific,
Carlsbad, Calif.). The average results for each group are shown
below as the percent normalized MALAT-1 RNA levels relative to
average results for the vehicle treated animals.
TABLE-US-00004 TABLE 2 MALAT-1 expression in vivo MALAT-1 RNA level
SEQ Dosage (% Vehicle) ID Duplex Isis No. Sequence (5' to 3')
(.mu.mol/kg) Heart Macs TG SN DRG NO. n/a 626112 G.sub.es
.sup.mC.sub.eo .sup.mC.sub.eo A.sub.eo G.sub.eo G.sub.ds
.sup.mC.sub.ds T.sub.ds 14 48 69 45 67 79 1 G.sub.ds G.sub.ds
T.sub.ds T.sub.ds A.sub.ds T.sub.ds G.sub.ds A.sub.eo
.sup.mC.sub.eo T.sub.es .sup.mC.sub.es A.sub.e 4 626112 See above
14 17 16 32 31 44 1 719234 Chol-TEG-U.sub.ms G.sub.ms A.sub.ro
G.sub.ro U.sub.ro C.sub.ro A.sub.ro U.sub.ro A.sub.ro A.sub.ro
C.sub.ro C.sub.ro A.sub.ro G.sub.ro 2 C.sub.ro C.sub.ro U.sub.ro
G.sub.rs G.sub.ms C.sub.m n/a 556089 G.sub.ks .sup.mC.sub.ks
A.sub.ks T.sub.ds T.sub.ds .sup.mC.sub.ds T.sub.ds A.sub.ds 4.5 67
48 79 77 88 3 A.sub.ds T.sub.ds A.sub.ds G.sub.ds .sup.mC.sub.ds
A.sub.ks G.sub.ks .sup.mC.sub.k 5 556089 See above 4.5 67 40 51 64
59 3 827936 Toco-TEG-G.sub.es .sup.mC.sub.es T.sub.eo G.sub.do
.sup.mC.sub.do 4 T.sub.do A.sub.do T.sub.do T.sub.do A.sub.do
G.sub.do A.sub.do A.sub.do T.sub.es G.sub.es .sup.mC.sub.e 6 556089
See above 4.5 56 53 64 81 41 3 827937 Toco-G.sub.ms C.sub.ms
U.sub.ro G.sub.ro C.sub.ro U.sub.ro A.sub.ro 5 U.sub.ro U.sub.ro
A.sub.ro G.sub.ro A.sub.ro A.sub.ro U.sub.rs G.sub.ms C.sub.m
See legend for Table 1 for subscripts and superscript key.
Subscript "k" represents a cEt modified bicyclic sugar moiety. The
structures of "Chol-TEG-" and "Toco-TEG-", are shown in Example 1.
The structure of "Toco-" is:
##STR00009##
Example 3: Effects of Duplexes Comprising a Lipophilic Conjugate
Group In Vivo
[0424] A duplex, consisting of two oligomeric compounds, is
described in the table below. One oligomeric compound of the duplex
comprises an antisense oligonucleotide (Isis No. 486178) that is
complementary to both human and mouse dystrophia myotonica-protein
kinase (DMPK) transcripts. The other oligomeric compound of the
duplex comprises an oligonucleotide and a lipophilic conjugate
group. The effects of the duplex on DMPK expression were tested in
vivo. Wild type Balb-C mice each received an intravenous injection
of the duplex listed in the table below, Isis No. 486178 alone, or
saline once per week for four weeks. The dosages listed in the
table below indicate the amount of Isis No. 486178 that was
administered in each injection. Each treatment group consisted of
three or four mice. Seven days after the final injection, the
animals were sacrificed. DMPK mRNA expression was analyzed in
liver, diaphragm (Dia), quadriceps (Quad), tibialis anterior (TA),
heart, and gastrocnemius (Gast) using RT-qPCR and normalized to
total RNA using RiboGreen (Thermo Fisher Scientific, Carlsbad,
Calif.). The average results for each group are shown below as the
percent normalized DMPK RNA levels relative to average results for
the vehicle treated animals.
TABLE-US-00005 TABLE 3 DMPK expression in vivo SEQ Dosage DMPK mRNA
level (% Vehicle) ID Isis No. Sequence (5' to 3') Duplex (mg/kg)
Liver Dia Quad TA Heart Gast NO. 486178 A.sub.ks .sup.mC.sub.ks
A.sub.ks A.sub.ds T.sub.ds A.sub.ds A.sub.ds n/a 25 8 41 32 26 55
32 6 A.sub.ds T.sub.ds A.sub.ds .sup.mC.sub.ds .sup.mC.sub.ds
G.sub.ds 50 6 30 13 21 21 18 A.sub.ks G.sub.ks G.sub.k 486178 See
above 7 25 5 27 8 15 22 18 6 694790 Toco-C.sub.ms C.sub.ms U.sub.ro
C.sub.ro G.sub.ro 50 5 14 8 16 10 13 7 G.sub.ro U.sub.ro A.sub.ro
U.sub.ro U.sub.ro U.sub.ro A.sub.ro U.sub.ro U.sub.rs G.sub.ms
U.sub.m
See legends of Table 1 and 2 for subscripts and superscripts key.
The structure of "Toco-" is shown in Example 2.
Example 4: Effects of Duplexes Comprising a Lipophilic Conjugate
Group In Vivo
[0425] Duplexes, consisting of two oligomeric compounds, are
described in the table below. One oligomeric compound of each
duplex comprises an antisense oligonucleotide (Isis No. 440762)
that is complementary to mouse SCARB1 scavenger receptor class B,
member 1 (SRB-1) transcript. The other oligomeric compound of the
duplex comprises an oligonucleotide and a lipophilic conjugate
group. The effects of the duplex on SRB-1 expression were tested in
vivo. Wild type mice each received an intravenous injection of a
duplex listed in the table below, Isis No. 440762 alone, or saline
once per. The dosages listed in the table below indicate the amount
of Isis No. 440762 that was administered in each injection. Each
treatment group consisted of three or four mice. After the final
injection, the animals were sacrificed. SRB-1 mRNA expression was
analyzed in liver using RT-qPCR and normalized to total RNA using
RiboGreen (Thermo Fisher Scientific, Carlsbad, Calif.). The average
results for each group are shown below as the percent normalized
SRB-1 RNA levels relative to average results for the vehicle
treated animals.
TABLE-US-00006 TABLE 4 SRB-1 expression in vivo SRB-1 mRNA SEQ
Dosage level (% ID Isis No. Sequence (5' to 3') Duplex (mg/kg)
Vehicle) NO. 440762 T.sub.ks .sup.mC.sub.ks A.sub.ds G.sub.ds
T.sub.ds .sup.mC.sub.ds A.sub.ds T.sub.ds G.sub.ds n/a 3 51 8
A.sub.ds .sup.mC.sub.ds T.sub.ds T.sub.ks .sup.mC.sub.k 440762 See
above 8 1 33 8 655462 Toco-G.sub.ms A.sub.ms A.sub.ro G.sub.ro
U.sub.ro C.sub.ro A.sub.ro 9 U.sub.ro G.sub.ro A.sub.ro C.sub.ro
U.sub.rs G.sub.ms A.sub.m 440762 See above 9 1 50 8 663429
Toco-G.sub.eo A.sub.eo A.sub.do G.sub.do T.sub.eo .sup.mC.sub.do
A.sub.eo 10 T.sub.do G.sub.eo A.sub.do .sup.mC.sub.eo T.sub.do
G.sub.eo A.sub.e 440762 See above 10 1 51 8 663430 Toco-G.sub.es
A.sub.es A.sub.do G.sub.do T.sub.eo .sup.mC.sub.do A.sub.eo 10
T.sub.do G.sub.eo A.sub.do .sup.mC.sub.eo T.sub.ds G.sub.es A.sub.e
440762 See above 11 1 97 8 663752 TOCO-G.sub.fs A.sub.fs A.sub.fo
G.sub.fo U.sub.fo C.sub.fo A.sub.fo U.sub.fo 9 G.sub.fo A.sub.fo
C.sub.fo U.sub.fs Gfs A.sub.f 440762 See above 12 1 34 8 671663
Toco-G.sub.ms A.sub.ms A.sub.ro G.sub.fo U.sub.ro C.sub.ro A.sub.fo
9 U.sub.ro G.sub.ro A.sub.fo C.sub.ro U.sub.rs G.sub.ms A.sub.m
440762 See above 13 1 30 8 671221 Toco-G.sub.ks A.sub.ks A.sub.ro
G.sub.ro U.sub.ro C.sub.ro A.sub.ro 9 U.sub.ro G.sub.ro A.sub.ro
C.sub.ro U.sub.rs G.sub.ks A.sub.k 440762 See above 14 1 44 8
674021 Toco-G.sub.ms A.sub.ms A.sub.do G.sub.do T.sub.do C.sub.do
A.sub.do 10 T.sub.do G.sub.do A.sub.do C.sub.do T.sub.ds G.sub.ms
A.sub.m 440762 See above 15 1 36 8 675421 Toco-G.sub.es A.sub.es
A.sub.ro G.sub.ro U.sub.ro C.sub.ro A.sub.ro U.sub.ro 9 G.sub.ro
A.sub.ro C.sub.ro U.sub.rs G.sub.es A.sub.e
See legends of Table 1 and 2 for subscripts and superscripts key.
Subscript "f" indicates a 2'-fluoro modification. The structure of
"Toco-" is shown in Example 2.
Sequence CWU 1
1
22120DNAArtificial sequenceSynthetic oligonucleotide 1gccaggctgg
ttatgactca 20223DNAArtificial sequenceSynthetic oligonucleotide
2tcagccaggc tggttatgac tca 23316DNAArtificial sequenceSynthetic
oligonucleotide 3gcattctaat agcagc 16419DNAArtificial
sequenceSynthetic oligonucleotide 4tcagcattct aatagcagc
19516DNAArtificial sequenceSynthetic oligonucleotide 5aggatatgga
accaaa 16619DNAArtificial sequenceSynthetic oligonucleotide
6tcaaggatat ggaaccaaa 19716DNAArtificial sequenceSynthetic
oligonucleotide 7gacaacttgg agcttg 16819DNAArtificial
sequenceSynthetic oligonucleotide 8tcagacaact tggagcttg
19916DNAArtificial sequenceSynthetic oligonucleotide 9ctggtatgag
gcctga 161019DNAArtificial sequenceSynthetic oligonucleotide
10tcactggtat gaggcctga 191116DNAArtificial sequneceSynthetic
oligonucleotide 11acaataaata ccgagg 161219DNAArtificial
sequenceSynthetic oligonucleotide 12tcaacaataa ataccgagg
191314DNAArtificial sequenceSynthetic
oligonucleotidemisc_feature(1)..(2)bases at these positions are
RNAmisc_feature(4)..(8)bases at these positions are
RNAmisc_feature(10)..(14)bases at these positions are RNA
13cutagcactg gccu 141420DNAArtificial sequenceSynthetic
oligonucleotide 14agcatagtta acgagctccc 201520DNAArtificial
sequenceSynthetic oligonucleotide 15aatggtttat tccatggcca
201620DNAArtificial sequenceSynthetic oligonucleotide 16gcagccatgg
tgatcaggag 201717DNAArtificial sequenceSynthetic oligonucleotide
17ggttcccgag gtgccca 171820DNAArtificial sequenceSynthetic
oligonucleotide 18gcacactcag caggaccccc 201920DNAArtificial
sequenceSynthetic oligonucleotide 19ccacctttgg gtgaatagca
202020DNAArtificial sequenceSynthetic oligonucleotide 20tgccatttaa
tgagcttcac 202116DNAArtificial sequenceSynthetic oligonucleotide
21tcccgaatgt ccgaca 162220DNAArtificial sequenceSynthetic
oligonucleotide 22atcccacgcc cctgtccagc 20
* * * * *