U.S. patent application number 17/398923 was filed with the patent office on 2022-08-18 for reversir tm compounds.
This patent application is currently assigned to ALNYLAM PHARMACEUTICALS, INC.. The applicant listed for this patent is ALNYLAM PHARMACEUTICALS, INC.. Invention is credited to Akin AKINC, Vasant JADHAV, Martin MAIER, Muthiah MANOHARAN, John MARAGANORE, Kallanthottathil G. RAJEEV, Ivan ZLATEV.
Application Number | 20220259589 17/398923 |
Document ID | / |
Family ID | 1000006504985 |
Filed Date | 2022-08-18 |
United States Patent
Application |
20220259589 |
Kind Code |
A9 |
JADHAV; Vasant ; et
al. |
August 18, 2022 |
REVERSIR TM COMPOUNDS
Abstract
The present invention relates, in general to agents that
modulate the pharmacological activity of conjugated siRNAs.
Inventors: |
JADHAV; Vasant; (Cambridge,
MA) ; MARAGANORE; John; (Cambridge, MA) ;
MAIER; Martin; (Cambridge, MA) ; RAJEEV;
Kallanthottathil G.; (Cambridge, MA) ; MANOHARAN;
Muthiah; (Cambridge, MA) ; AKINC; Akin;
(Cambridge, MA) ; ZLATEV; Ivan; (Cambridge,
MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
ALNYLAM PHARMACEUTICALS, INC. |
Cambridge |
MA |
US |
|
|
Assignee: |
ALNYLAM PHARMACEUTICALS,
INC.
Cambridge
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20220127604 A1 |
April 28, 2022 |
|
|
Family ID: |
1000006504985 |
Appl. No.: |
17/398923 |
Filed: |
August 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15537083 |
Jun 16, 2017 |
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PCT/US2015/066465 |
Dec 17, 2015 |
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17398923 |
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62093906 |
Dec 18, 2014 |
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62238467 |
Oct 7, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/3183 20130101;
C12N 2310/113 20130101; C12N 2310/319 20130101; C12N 2310/346
20130101; C12N 2310/315 20130101; C12N 2310/351 20130101; C12N
15/113 20130101; C12N 2310/3341 20130101; C12N 15/111 20130101;
C12N 2310/3231 20130101 |
International
Class: |
C12N 15/11 20060101
C12N015/11; C12N 15/113 20060101 C12N015/113 |
Claims
1.-3. (canceled)
4. A method of inhibiting the activity of a siRNA in a cell
comprising contacting the cell with a REVERSIR compound comprising
a modified oligonucleotide consisting of 6 to 25 linked nucleotides
and having a nucleobase sequence substantially complementary to
antisense strand of the siRNA.
5.-13. (canceled)
14. A method of treating a subject comprising: administering to the
subject a siRNA; monitoring the subject for siRNA activity; if the
siRNA activity becomes higher than desired, administering a
REVERSIR compound to the subject.
15.-27. (canceled)
28. A REVERSIR compound comprising a modified oligonucleotide
consisting of 6 to 20 linked nucleotides and having a nucleobase
sequence substantially complementary to antisense strand of a
siRNA.
29. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide consists of 8-15 linked nucleotides.
30. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide is a single-stranded oligonucleotide having at
least 90% complementary to the antisense strand.
31. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide is substantially complementary to nucleotides 2-16
of the antisense stand.
32. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide is fully complementary to the antisense strand.
33. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide comprises at least one modified internucleotide
linkage.
34. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide comprises at least one modified internucleotide
linkage and at least one unmodified internucleotide linkage.
35. The REVERSIR compound of claim 34, wherein the modified
oligonucleotide comprises an unmodified internucleotide linkage
between the 3'-terminus nucleotide and the penultimate
nucleoside.
36. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide comprises at least one modified nucleobase.
37. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide comprises at least one modified sugar.
38. The REVERSIR compound of claim 37, wherein said at least one
modified sugar is a bicyclic sugar.
39. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide comprises at least one nucleotide wherein 2'
position of furnaosyl is connected to the 4' position by a linker
selected independently from --[C(R1)(R2)].sub.n-,
--[C(R1)(R2)].sub.n-O--, --[C(R1)(R2)].sub.n--N(R1)-,
--[C(R1)(R2)].sub.n--N(R1)-O--, [C(R1R2)].sub.n-O--N(R1)-,
--C(R1)=C(R2)-O--, --C(R1)=N--, --C(R1)=N--O--, C(.dbd.NR1)-,
C(.dbd.NR1)-O--, C(.dbd.O)--, C(.dbd.O)O--, C(.dbd.S)--,
C(.dbd.S)O--, C(.dbd.S)S--, O, Si(R1)2-, S(.dbd.O).sub.x-- and
N(R1)-, wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R1 and
R2 is, independently, H, a protecting group, hydroxyl, C1-C12
alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12
alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl,
substituted C5-C20 aryl, heterocycle radical, substituted
heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7
alicyclic radical, substituted C5-C7 alicyclic radical, halogen,
OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(.dbd.O)--H), substituted acyl,
CN, sulfonyl (S(.dbd.O)2-J1), or sulfoxyl (S(.dbd.O)-J1); and each
J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12
alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl,
substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl,
acyl (C(.dbd.O)--H), substituted acyl, a heterocycle radical, a
substituted heterocycle radical, C1-C12 aminoalkyl, substituted
C1-C12 aminoalkyl or a protecting group.
40. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide is conjugated with a ligand.
41. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide is conjugated with a ligand of structure:
##STR00112##
42. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide is conjugated with a ligand and the ligand is
conjugated to 3'-terminus of the modified oligonucleotide.
43. The REVERSIR compound of claim 28, wherein the modified
oligonucleotide is conjugated with a ligand and the ligand is
conjugated to a nucleoside with a deoxy sugar in the REVERSIR
compound.
44. The REVERSIR compound of claim 43, wherein said deoxy sugar is
a 2'-deoxy ribose.
45. The REVERSIR compound of claim 28, wherein the siRNA is
targeted to an mRNA, a pre-mRNA, a micro-RNA a pre-micro-RNA.
46.-48. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.
119(e) of the U.S. Provisional Application No. 62/093,906, filed
Dec. 18, 2014, and U.S. Provisional Application No. 62/238,467,
filed Oct. 7, 2015, the contents of both which are incorporated
herein by reference in their entireties.
TECHNICAL FIELD
[0002] The present disclosure relates generally to oligomeric
compounds (oligomers), which target siRNAs (e.g. conjugated or
unconjugated siRNAs) in vivo, thereby providing a method of
tailored control of RNAi pharmacology and therefore the therapeutic
activity and/or side effects of siRNA based therapeutics in
vivo.
BACKGROUND
[0003] Conjugated and unconjugated siRNA compounds have been used
to modulate target nucleic acids. Conjugated and unconjugated
siRNAs comprising a variety of modifications and motifs have been
reported. In certain instances, such compounds are useful as
research tools and as therapeutic agents.
SUMMARY OF THE INVENTION
[0004] In certain embodiments, provided herein are REVERSIR
compounds (REVERSIR is a trademark of Alnylam Pharmaceuticals,
Inc.). Such compounds reduce RNAi activity of a siRNA compound, for
example conjugated siRNA or unconjugated siRNA. Generally, the
REVERSIR compounds modulate hybridize or bind siRNA molecule in a
sequence dependent manner and modulate (e.g., inhibit or reverse)
their activity.
[0005] In certain embodiments, the present invention provides
REVERSIR compounds that are complementary to at least one strand of
siRNA compounds (e.g. conjugated or unconjugated siRNA). In some
embodiments, the REVERSIR compounds are complementary to the
antisense strand of siRNA compounds. In some other embodiments, the
REVERSIR compounds are complementary to the sense strand of siRNA
compounds.
[0006] In certain embodiments, the present invention provides
REVERSIR compounds comprising a modified oligonucleotide consisting
of 6 to 30 linked nucleosides and having a nucleobase sequence
substantially complementary to at least one strand of siRNA
compounds (e.g. conjugated or unconjugated siRNA). In some
embodiments, the REVERSIR compounds comprise a modified
oligonucleotide consisting of 6 to 30 linked nucleosides and having
a nucleobase sequence substantially complementary to the antisense
strand of siRNA compounds. In some other embodiments, the REVERSIR
compounds comprise a modified oligonucleotide consisting of 6 to 30
linked nucleosides and having a nucleobase sequence substantially
complementary to the sense strand of siRNA compounds.
[0007] In certain such embodiments, the modified oligonucleotide is
a single-stranded oligonucleotide and/or is at least 90%
complementary to at least one strand of the siRNA. In some
embodiments, the modified oligonucleotide is a single-stranded
oligonucleotide and/or is at least 90% complementary to the
antisense strand of the siRNA. In some other embodiments, the
modified oligonucleotide is a single-stranded oligonucleotide
and/or is at least 90% complementary to the sense strand of the
siRNA.
[0008] In certain embodiments, the REVERSIR compound is fully
complementary to at least one strand of the conjugated or
unconjugated siRNA. In some embodiments, the REVERSIR compound is
fully complementary to the antisense strand of the siRNA. In some
other embodiments, the REVERSIR compound is fully complementary to
the sense strand of the siRNA.
[0009] In certain embodiments, REVERSIR compounds comprise at least
one modified internucleoside or intersugar linkage. In certain such
embodiments, at least one (e.g., one, two, three, four, five, six,
seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,
fifteen or more and upto and including all) internucleoside linkage
is a phosphorothioate internucleoside linkage.
[0010] In certain embodiments, REVERSIR compounds comprise at least
one nucleoside comprising a modified sugar. In certain such
embodiments, the modified sugar is a bicyclic sugar or sugar
comprising a 2'-O-methyl or a 2'-O-methoxyethyl.
[0011] In certain embodiments, REVERSIR compounds comprise one or
more (e.g., one, two, three, four, five, six, seven, eight, nine,
ten, eleven, twelve, thirteen, fourteen, fifteen or more and upto
and including all) locked nucleic acid (LNA) monomers.
[0012] In certain embodiments, REVERSIR compounds comprise at least
one (e.g., one, two, three, four, five, six, seven, eight, nine,
ten or more) nucleotide that does not comprise a 2'-O-methyl group,
i.e., the REVERSIR compound is not fully 2'-O-methyl. In some
embodiments, each nucleoside in the REVERSIR compound is a
2'-O-methyl nucleoside and the REVERSIR compound comprises at least
one (e.g., one, two, three, four, five, six, seven, eight, nine,
ten or more) G-clamp nucleobases.
[0013] In certain embodiments, REVERSIR compounds comprise at least
one nucleoside comprising a modified nucleobase. In certain such
embodiments, the modified nucleobase is a 5-methylcytosine.
[0014] In certain embodiments, REVERSIR compounds comprise at least
one modification. In certain such embodiments, REVERSIR compounds
comprise one or more nucleoside modifications and or one or more
linkage modifications. In certain embodiments, REVERSIR compounds
comprise one or more modifications selected from: sugar
modifications, linkage modifications, nucleobase modifications,
conjugates (e.g., ligands), and any combinations thereof.
[0015] In certain embodiments, REVERSIR compounds comprise a
modified oligonucleotide comprising: a gap segment consisting of
linked deoxynucleosides; a 5' wing segment consisting of linked
nucleosides; a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment
and the 3' wing segment and wherein each nucleoside of each wing
segment comprises a modified sugar.
[0016] In certain embodiments, REVERSIR compounds comprise a
modified oligonucleotide comprising: a gap segment consisting of
ten linked deoxynucleosides; a 5' wing segment consisting of five
linked nucleosides; a 3' wing segment consisting of five linked
nucleosides; wherein the gap segment is positioned between the 5'
wing segment and the 3' wing segment, wherein each nucleoside of
each wing segment comprises a 2'-O-methoxyethyl sugar; and wherein
each internucleoside linkage is a phosphorothioate linkage.
[0017] In certain embodiments, REVERSIR compound comprises a
modified oligonucleotide consisting of 6-17, 7-16 8-15 or 6-25
linked nucleosides. In some embodiments, REVERSIR compound
comprises a modified oligonucleotide consisting of 8, 9, 10, 11,
12, 13, 14, 15 or 20 linked nucleosides.
[0018] In certain embodiments, REVERSIR compound comprises a
modified oligonucleotide wherein each nucleoside is modified.
[0019] In some embodiments, REVERSIR compound comprises or consists
of nine linked nucleosides.
[0020] In some embodiments, REVERSIR compound has low PS content.
By low PS content is meant that the REVERSIR compound has 1, 2, 3,
4 or 5 phosphorothioate linkages per nine nucleosides of the
REVERSIR compound.
[0021] In some embodiments, REVERSIR compound comprises or consists
of nine linked nucleosides and has low PS content.
[0022] In some embodiments, REVERSIR compound consists of nine
linked nucleosides and comprises five phosphorothioate
linkages.
[0023] In some embodiments, REVERSIR compound consists of nine
linked nucleosides, comprises five phosphorothioate linkages and is
linked to a ligand.
[0024] In certain embodiments, REVERSIR compounds are complementary
to the antisense or sense strand of a conjugated or unconjugated
siRNA, wherein the siRNA is targeted to an mRNA. In certain
embodiments, the siRNA is targeted to an mRNA encoding a blood
factor. In certain embodiments, the siRNA is targeted to an mRNA
encoding a protein involved in metabolism. In certain embodiments,
the siRNA is targeted to an mRNA encoding a protein involved in
diabetes. In certain embodiments, the siRNA is targeted to an mRNA
encoding a protein involved in cardiopathology. In certain
embodiments, the siRNA is targeted to an mRNA encoding a protein
expressed in nerve cells. In certain embodiments, the siRNA is
targeted to an mRNA encoding a protein expressed in the central
nervous system. In certain embodiments, the siRNA is targeted to an
mRNA expressed in peripheral nerves.
[0025] In certain embodiments, the conjugated or unconjugated siRNA
is targeted to an mRNA encoding a protein expressed in the liver.
In certain embodiments, the siRNA is targeted to an mRNA encoding a
protein expressed in the kidney.
[0026] In certain embodiments, the conjugated or unconjugated siRNA
is targeted to a pre-mRNA. In certain embodiments, the conjugated
or unconjugated siRNA is targeted to a micro-RNA. In certain
embodiments, the conjugated or unconjugated siRNA activates the
RISC pathway. In some embodiments, the conjugated or unconjugated
siRNA inhibits the expression of a target nucleic acid.
[0027] In certain embodiments, REVERSIR compounds modulate the RISC
pathway. In some embodiments, REVERSIR compounds inhibit the RISC
pathway.
[0028] In certain embodiments, the invention provides a composition
comprising a REVERSIR compound or a pharmaceutically acceptable
salt thereof and a pharmaceutically acceptable carrier or
diluent.
[0029] In certain embodiments, the invention provides methods
comprising administering to a subject (e.g., an animal) a REVERSIR
compound or composition comprising same. In certain embodiments,
the subject is a human. In certain embodiments, the administering
is oral, topical, or parenteral.
[0030] In certain embodiments, the invention provides methods of
inhibiting RNAi activity of a conjugated or unconjugated siRNA in a
cell. The method, generally, comprises contacting the cell with a
REVERSIR compound according the present invention and thereby
inhibiting the RNAi activity in the cell. In certain such
embodiments, the cell is in in vivo. In some embodiments, the cell
is in vitro. In some embodiments the cell is ex vivo. In some
embodiments, the cell is in a subject. In some further embodiments
of this, the cell is an animal. In certain embodiments, the animal
is a human.
[0031] In certain embodiments, the invention provides methods
comprising: contacting a cell with a conjugated or unconjugated
siRNA; detecting RNAi activity; and contacting the cell with a
REVERSIR compound. In certain embodiments, the method the detecting
RANi activity comprises measuring the amount of target mRNA
present, the amount of target protein present, and/or the activity
of a target protein. In certain embodiments, such methods comprise
detecting REVERSIR activity by measuring RNAi activity after
contacting the cell with the REVERSIR compound. In certain such
methods, the cell is in vivo. In some embodiments, the cell is in
an animal. In certain embodiments, the animal is a human.
[0032] In certain embodiments, the invention provides methods of
ameliorating a side-effect of siRNA treatment comprising:
contacting a cell with a conjugated or unconjugated siRNA;
detecting a side-effect; contacting the cell with a REVERSIR
compound; and thereby ameliorating the side effect of the
siRNA.
[0033] In certain embodiments, the invention provides methods of
treating a patient comprising: administering to the patient a
conjugated or unconjugated siRNA; monitoring the patient for siRNA
activity; and if the siRNA activity becomes higher than desired,
administrating a REVERSIR compound. In certain such embodiments,
the monitoring siRNA activity comprises measuring the amount of
target mRNA present, measuring the amount of target protein present
and/or measuring the activity of a target protein. In certain
embodiments, such methods include detecting REVERSIR activity by
measuring siRNA activity after administration of the REVERSIR
compound. In certain embodiments, the patient is a mammal. In some
embodiments, the patient is a human.
[0034] In certain embodiments, the invention provides methods of
treating a patient comprising: administering to the patient a
conjugated or unconjugated siRNA; monitoring the patient for one or
more side effect; and if the one or more side effect reaches an
undesirable level, administrating a REVERSIR compound. In certain
embodiments, the patient is a mammal. In some embodiments, the
patient is a human.
[0035] In certain embodiments, the invention provides a kit
comprising a conjugated or unconjugated siRNA and a REVERSIR
compound; REVERSIR compound and a non-oligomeric REVERSIR; or
conjugated or unconjugated siRNA compound, REVERSIR compound, and a
non-oligomeric REVERSIR. In certain such embodiments, the
non-oligomeric REVERSIR is a target protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 shows in vivo activity of exemplary REVERSIR
compounds targeting antithrombin (AT) siRNAs.
[0037] FIG. 2 shows that reversal of activity of siRNAs by REVERSIR
compounds in vivo is rapid and dose-dependent. Full reversal can be
seen within 4-days of dosing.
[0038] FIG. 3 shows the effect of REVERSIR compound length on the
in vivo activity of exemplary REVERSIR compounds. As seen, shorter
REVERSIR compounds showed better in vivo activity than the longer
REVERSIR compounds.
[0039] FIG. 4 shows the effect of exemplary nucleic acid
modifications on the in vivo activity of REVERSIR compounds.
[0040] FIG. 5 shows the effect of number of phosphorothioate
internucleoside linkages on the in vivo activity of REVERSIR
compounds.
[0041] FIGS. 6 and 7 show that REVERSIR compounds have increased in
vivo potency with decreasing length
[0042] FIGS. 8 and 9 shows effect of number phosphorothioate
linkages on the activity of REVERSIR compounds.
[0043] FIG. 10 shows further improvement in potency for exemplary
REVERSIR compounds.
[0044] FIG. 11 shows in vitro reversal of siRNA activity by free
uptake of exemplary REVERSIR compounds targeting antithrombin siRNA
in primary mouse hepatocytes.
[0045] FIG. 12 shows in vitro reversal of siRNA activity by free
uptake of exemplary REVERSIR compounds targeting antithrombin siRNA
in primary mouse hepatocytes at various concentrations.
[0046] FIGS. 13 and 14 show in vitro reversal of siRNA activity by
free uptake of exemplary REVERSIR compounds targeting Factor IX
siRNAs in primary mouse hepatocytes at various concentrations.
[0047] FIG. 15 shows the effect of high-affinity chemistry on the
in vivo activity of exemplary REVERSIR compounds targeting Factor
IX siRNAs.
[0048] FIGS. 16 and 17 show the effect of REVERSIR compound length
on the in vivo activity of exemplary REVERSIR compounds targeting
Factor IX siRNAs. REVERSIR compounds were administered at 3 mg/kg
(FIG. 16) and 1 mg/kg (FIG. 17)
[0049] FIG. 18 shows the effect of linker, between the REVERSIR
compound and the ligand conjugated with the REVERSIR compound, on
the in vivo activity of exemplary REVERSIR compounds targeting
Factor IX siRNAs.
[0050] FIG. 19 shows the effect of phosphorothioate linkages in the
REVERSIR compound on the in vivo activity of exemplary REVERSIR
compounds targeting Factor IX siRNAs.
[0051] FIG. 20 shows the effect of linker, between the REVERSIR
compound and the ligand conjugated with the REVERSIR compound, on
the in vitro activity of exemplary REVERSIR compounds targeting
Factor IX siRNAs.
[0052] FIG. 21 shows the effect on activity of siRNA by exemplary
REVERSIR compounds matching certain portion of the antisense strand
of the siRNA.
[0053] FIG. 22 shows in vivo dos-dependent effect of exemplary
REVERSIR compounds targeting Factor IX siRNA.
[0054] FIG. 23 shows that REVERSIR compounds are tolerated in
vivo.
[0055] FIGS. 24A and 24B show in vivo reversal of siRNA activity by
some exemplary REVERSIR compounds in non-human primates.
DETAILED DESCRIPTION OF THE INVENTION
[0056] 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 of the invention, as
claimed. 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.
[0057] 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 in their entirety for any purpose.
Definitions
[0058] Unless specific definitions are provided, the nomenclature
utilized 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. Standard techniques may be used for
chemical synthesis, and chemical analysis. Certain such techniques
and procedures may be found for example in "Carbohydrate
Modifications in Antisense Research" Edited by Sangvi and Cook,
American Chemical Society, Washington D.C., 1994; "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th
edition, 1990; and "Antisense Drug Technology, Principles,
Strategies, and Applications" Edited by Stanley T. Crooke, CRC
Press, Boca Raton, Fla.; and Sambrook et al., "Molecular Cloning, A
laboratory Manual," 2.sup.nd Edition, Cold Spring Harbor Laboratory
Press, 1989, which are hereby incorporated by reference for any
purpose. Where permitted, all patents, applications, published
applications and other publications and other data referred to
throughout in the disclosure herein are incorporated by reference
in their entirety.
[0059] Unless otherwise indicated, the following terms have the
following meanings:
[0060] As used herein, the term "nucleoside" means a glycosylamine
comprising a nucleobase and a sugar. Nucleosides includes, but are
not limited to, naturally occurring nucleosides, abasic
nucleosides, modified nucleosides, and nucleosides having mimetic
bases and/or sugar groups.
[0061] As used herein, the term "nucleotide" refers to a
glycosomine comprising a nucleobase and a sugar having a phosphate
group covalently linked to the sugar. Nucleotides may be modified
with any of a variety of substituents.
[0062] As used herein, the term "nucleobase" refers to the base
portion of a nucleoside or nucleotide. A nucleobase may comprise
any atom or group of atoms capable of hydrogen bonding to a base of
another nucleic acid.
[0063] As used herein, the term "heterocyclic base moiety" refers
to a nucleobase comprising a heterocycle.
[0064] As used herein, the term "oligomeric compound" refers to a
polymeric structure comprising two or more sub-structures and
capable of hybridizing to a region of a nucleic acid molecule. In
certain embodiments, oligomeric compounds are oligonucleosides. In
certain embodiments, oligomeric compounds are oligonucleotides. In
certain embodiments, oligomeric compounds are antisense compounds.
In certain embodiments, oligomeric compounds are REVERSIR
compounds. In certain embodiments, oligomeric compounds comprise
conjugate groups.
[0065] As used herein "oligonucleoside" refers to an
oligonucleotide in which the internucleoside linkages do not
contain a phosphorus atom.
[0066] As used herein, the term "oligonucleotide" refers to an
oligomeric compound comprising a plurality of linked nucleosides.
In certain embodiment, one or more nucleotides of an
oligonucleotide is modified. In certain embodiments, an
oligonucleotide comprises ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA). In certain embodiments,
oligonucleotides are composed of naturally- and/or
non-naturally-occurring nucleobases, sugars and covalent
internucleoside linkages, and may further include non-nucleic acid
conjugates.
[0067] As used herein "internucleoside linkage" refers to a
covalent linkage between adjacent nucleosides.
[0068] As used herein "naturally occurring internucleoside linkage"
refers to a 3' to 5' phosphodiester linkage.
[0069] As used herein the term "detecting siRNA activity" or
"measuring siRNA activity" means that a test for detecting or
measuring siRNA activity is performed on a particular sample and
compared to that of a control sample. Such detection and/or
measuring can include values of zero. Thus, if a test for detection
of siRNA activity results in a finding of no siRNA activity (siRNA
activity of zero), the step of "detecting siRNA activity" has
nevertheless been performed.
[0070] As used herein the term "control sample" refers to a sample
that has not been contacted with a reporter oligomeric
compound.
[0071] As used herein, the term "motif" refers to the pattern of
unmodified and modified nucleotides in an oligomeric compound.
[0072] As used herein, the term "REVERSIR compound" refers to an
oligomeric compound that is complementary to and capable of
hybridizing with at least one strand of a conjugated or
unconjugated siRNA. Without limitations, the REVERSIR compound
could not only block unintended target PD effect but also block any
potential off-target activity that could happen with a conjugated
or unconjugated siRNA.
[0073] As used herein, the term "non-oligomeric REVERSIR" refers to
a compound that does not hybridize with a strand of siRNA and that
reduces the amount or duration of a siRNA activity. In certain
embodiments, a non-oligomeric REVERSIR is a target protein.
[0074] As used herein, the term "REVERSIR activity" refers to any
decrease in intensity or duration of any siRNA activity
attributable to hybridization of a REVERSIR compound to one of the
strands of the siRNA.
[0075] As used herein, the term "chimeric oligomer" refers to an
oligomeric compound, having at least one sugar, nucleobase or
internucleoside linkage that is differentially modified as compared
to at least on other sugar, nucleobase or internucleoside linkage
within the same oligomeric compound. The remainder of the sugars,
nucleobases and internucleoside linkages can be independently
modified or unmodified, the same or different.
[0076] As used herein, the term "chimeric oligonucleotide" refers
to an oligonucleotide, having at least one sugar, nucleobase or
internucleoside linkage that is differentially modified as compared
to at least on other sugar, nucleobase or internucleoside linkage
within the same oligonucleotide. The remainder of the sugars,
nucleobases and internucleoside linkages can be independently
modified or unmodified, the same or different.
[0077] As used herein, the term "mixed-backbone oligomeric
compound" refers to an oligomeric compound wherein at least one
internucleoside linkage of the oligomeric compound is different
from at least one other internucleoside linkage of the oligomeric
compound.
[0078] As used herein, the term "target protein" refers to a
protein, the modulation of which is desired.
[0079] As used herein, the term "target gene" refers to a gene
encoding a target protein.
[0080] As used herein, the term "target nucleic acid" refers to any
nucleic acid molecule the expression or activity of which is
capable of being modulated by a conjugated or unconjugated siRNA
compound. Target nucleic acids include, but are not limited to, RNA
(including, but not limited to pre-mRNA and mRNA or portions
thereof) transcribed from DNA encoding a target protein, and also
cDNA derived from such RNA, and miRNA. For example, the target
nucleic acid can be a cellular gene (or mRNA transcribed from the
gene) whose expression is associated with a particular disorder or
disease state, or a nucleic acid molecule from an infectious
agent.
[0081] As used herein, the term "target siRNA" refers to a siRNA
compound that is targeted by a REVERSIR compound.
[0082] As used herein, the term "targeting" or "targeted to" refers
to the association of antisense strand of a siRNA to a particular
target nucleic acid molecule or a particular region of nucleotides
within a target nucleic acid molecule.
[0083] As used herein, the term "nucleobase complementarity" refers
to a nucleobase that is capable of base pairing with another
nucleobase. For example, in DNA, adenine (A) is complementary to
thymine (T). For example, in RNA, adenine (A) is complementary to
uracil (U). In certain embodiments, complementary nucleobase refers
to a nucleobase of an antisense compound that is capable of base
pairing with a nucleobase of its target nucleic acid. For example,
if a nucleobase at a certain position of an antisense compound is
capable of hydrogen bonding with a nucleobase at a certain position
of a target nucleic acid, then the position of hydrogen bonding
between the oligonucleotide and the target nucleic acid is
considered to be complementary at that nucleobase pair.
[0084] As used herein, the term "non-complementary nucleobase"
refers to a pair of nucleobases that do not form hydrogen bonds
with one another or otherwise support hybridization.
[0085] As used herein, the term "complementary" refers to the
capacity of an oligomeric compound to hybridize to another
oligomeric compound or nucleic acid through nucleobase
complementarity. In certain embodiments, an oligomeric compound and
its target are complementary to each other when a sufficient number
of corresponding positions in each molecule are occupied by
nucleobases that can bond with each other to allow stable
association between the antisense compound and the target. One
skilled in the art recognizes that the inclusion of mismatches is
possible without eliminating the ability of the oligomeric
compounds to remain in association. Therefore, described herein are
oligomeric compounds (e.g., REVERSIR compounds, siRNAs, and the
like) that may comprise up to about 20% nucleotides that are
mismatched (i.e., are not nucleobase complementary to the
corresponding nucleotides of the target). Preferably the oligomeric
compounds, such as REVERSIR compounds and siRNAs, contain no more
than about 15%, more preferably not more than about 10%, most
preferably not more than 5% or no mismatches. The remaining
nucleotides are nucleobase complementary or otherwise do not
disrupt hybridization (e.g., universal bases). One of ordinary
skill in the art would recognize the compounds provided herein are
at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% complementary
to a target nucleic acid.
[0086] As used herein, "hybridization" means the pairing of
complementary oligomeric compounds (e.g., an antisense strand of a
siRNA and its target nucleic acid or a REVERSIR to its target
siRNA). While not limited to a particular mechanism, the most
common mechanism of pairing involves hydrogen bonding, which may be
Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,
between complementary nucleoside or nucleotide bases (nucleobases).
For example, the natural base adenine is nucleobase complementary
to the natural nucleobases thymidine and uracil which pair through
the formation of hydrogen bonds. The natural base guanine is
nucleobase complementary to the natural bases cytosine and 5-methyl
cytosine. Hybridization can occur under varying circumstances.
[0087] As used herein, the term "specifically hybridizes" refers to
the ability of an oligomeric compound to hybridize to one nucleic
acid site with greater affinity than it hybridizes to another
nucleic acid site. In certain embodiments, the antisense strand of
an siRNA specifically hybridizes to more than one target site.
[0088] As used herein, "designing" or "designed to" refer to the
process of designing an oligomeric compound that specifically
hybridizes with a selected nucleic acid molecule.
[0089] As used herein, the term "modulation" refers to a
perturbation of function or activity when compared to the level of
the function or activity prior to modulation. For example,
modulation includes the change, either an increase (stimulation or
induction) or a decrease (inhibition or reduction) in gene
expression. As further example, modulation of expression can
include perturbing splice site selection of pre-mRNA
processing.
[0090] As used herein, the term "expression" refers to all the
functions and steps by which a gene's coded information is
converted into structures present and operating in a cell. Such
structures include, but are not limited to the products of
transcription and translation.
[0091] As used herein, "variant" refers to an alternative RNA
transcript that can be produced from the same genomic region of
DNA. Variants include, but are not limited to "pre-mRNA variants"
which are transcripts produced from the same genomic DNA that
differ from other transcripts produced from the same genomic DNA in
either their start or stop position and contain both intronic and
exonic sequence. Variants also include, but are not limited to,
those with alternate splice junctions, or alternate initiation and
termination codons.
[0092] As used herein, "high-affinity modified monomer" refers to a
monomer having at least one modified nucleobase, internucleoside
linkage or sugar moiety, when compared to naturally occurring
monomers, such that the modification increases the affinity of an
antisense compound comprising the high-affinity modified monomer to
its target nucleic acid. High-affinity modifications include, but
are not limited to, monomers (e.g., nucleosides and nucleotides)
comprising 2'-modified sugars.
[0093] As used herein, the term "2'-modified" or "2'-substituted"
means a sugar comprising substituent at the 2' position other than
H or OH. 2'-modified monomers, include, but are not limited to,
BNA's and monomers (e.g., nucleosides and nucleotides) with
2'-substituents, such as allyl, amino, azido, thio, O-allyl,
O--C.sub.1-C.sub.10 alkyl, OCF3, O--(CH.sub.2).sub.2--O--CH.sub.3,
2'-O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(Rm)(Rn),
or O--CH.sub.2--C(.dbd.O) N(Rm)(Rn), where each Rm and Rn is,
independently, H or substituted or unsubstituted C.sub.1-C.sub.10
alkyl. In certain embodiments, oligomeric compounds comprise a 2'
modified monomer that does not have the formula
2'-O(CH.sub.2).sub.nH, wherein n is one to six. In certain
embodiments, oligomeric compounds comprise a 2' modified monomer
that does not have the formula 2'-OCH.sub.3. In certain
embodiments, oligomeric compounds comprise a 2' modified monomer
that does not have the formula or, in the alternative,
2'-O(CH.sub.2).sub.2OCH.sub.3.
[0094] As used herein, the term "locked nucleic acid" or "LNA" or
"locked nucleoside" or "locked nucleotide" refers to a nucleoside
or nucleotide wherein the furanose portion of the nucleoside
includes a bridge connecting two carbon atoms on the furanose ring,
thereby forming a bicyclic ring system. Locked nucleic acids are
also referred to as bicyclic nucleic acids (BNA).
[0095] As used herein, unless otherwise indicated, the term
"methyleneoxy LNA" alone refers to .beta.-D-methyleneoxy LNA.
[0096] As used herein, the term "MOE" refers to a 2'-O-methoxyethyl
substituent.
[0097] As used herein, the term "gapmer" refers to a chimeric
oligomeric compound comprising a central region (a "gap") and a
region on either side of the central region (the "wings"), wherein
the gap comprises at least one modification that is different from
that of each wing. Such modifications include nucleobase, monomeric
linkage, and sugar modifications as well as the absence of
modification (unmodified). Thus, in certain embodiments, the
nucleotide linkages in each of the wings are different than the
nucleotide linkages in the gap. In certain embodiments, each wing
comprises nucleotides with high affinity modifications and the gap
comprises nucleotides that do not comprise that modification. In
certain embodiments the nucleotides in the gap and the nucleotides
in the wings all comprise high affinity modifications, but the high
affinity modifications in the gap are different than the high
affinity modifications in the wings. In certain embodiments, the
modifications in the wings are the same as one another. In certain
embodiments, the modifications in the wings are different from each
other. In certain embodiments, nucleotides in the gap are
unmodified and nucleotides in the wings are modified. In certain
embodiments, the modification(s) in each wing are the same. In
certain embodiments, the modification(s) in one wing are different
from the modification(s) in the other wing. In certain embodiments,
oligomeric compounds are gapmers having 2'-deoxynucleotides in the
gap and nucleotides with high-affinity modifications in the
wing.
[0098] As used herein, the term "prodrug" refers to a therapeutic
agent that is prepared in an inactive form that is converted to an
active form (i.e., drug) within the body or cells thereof by the
action of endogenous enzymes or other chemicals and/or
conditions.
[0099] As used herein, the term "pharmaceutically acceptable salts"
refers to salts of active compounds that retain the desired
biological activity of the active compound and do not impart
undesired toxicological effects thereto.
[0100] As used herein, the term "cap structure" or "terminal cap
moiety" refers to chemical modifications, which have been
incorporated at either terminus of an antisense compound.
[0101] As used herein, the term "prevention" refers to delaying or
forestalling the onset or development of a condition or disease for
a period of time from hours to days, preferably weeks to
months.
[0102] As used herein, the term "amelioration" refers to a
lessening of at least one activity or one indicator of the severity
of a condition or disease. The severity of indicators may be
determined by subjective or objective measures which are known to
those skilled in the art.
[0103] As used herein, the term "treatment" refers to administering
a composition of the invention to effect an alteration or
improvement of the disease or condition. Prevention, amelioration,
and/or treatment may require administration of multiple doses at
regular intervals, or prior to onset of the disease or condition to
alter the course of the disease or condition. Moreover, a single
agent may be used in a single individual for each prevention,
amelioration, and treatment of a condition or disease sequentially,
or concurrently.
[0104] As used herein, the term "pharmaceutical agent" refers to a
substance that provides a therapeutic benefit when administered to
a subject. In certain embodiments, a pharmaceutical agent is an
active pharmaceutical agent. In certain embodiments, a
pharmaceutical agent is a prodrug.
[0105] As used herein, the term "therapeutically effective amount"
refers to an amount of a pharmaceutical agent that provides a
therapeutic benefit to an animal.
[0106] As used herein, "administering" means providing a
pharmaceutical agent to an animal, and includes, but is not limited
to administering by a medical professional and
self-administering.
[0107] As used herein, the term "co-administering" means providing
more than one pharmaceutical agent to an animal. In certain
embodiments, such more than one pharmaceutical agents are
administered together. In certain embodiments, such more than one
pharmaceutical agents are administered separately. In certain
embodiments, such more than one pharmaceutical agents are
administered at the same time. In certain embodiments, such more
than one pharmaceutical agents are administered at different times.
In certain embodiments, such more than one pharmaceutical agents
are administered through the same route of administration. In
certain embodiments, such more than one pharmaceutical agents are
administered through different routes of administration. In certain
embodiments, such more than one pharmaceutical agents are contained
in the same pharmaceutical formulation. In certain embodiments,
such more than one pharmaceutical agents are in separate
formulations.
[0108] As used herein, the term "pharmaceutical composition" refers
to a mixture of substances suitable for administering to an
individual. For example, a pharmaceutical composition may comprise
an antisense oligonucleotide and a sterile aqueous solution. In
certain embodiments, a pharmaceutical composition includes a
pharmaceutical agent and a diluent and/or carrier.
[0109] As used herein, the term "in vitro" refers to events that
occur in an artificial environment, e.g., in a test tube or
reaction vessel, in cell culture, etc., rather than within an
organism (e.g. animal or a plant). As used herein, the term "ex
vivo" refers to cells which are removed from a living organism and
cultured outside the organism (e.g., in a test tube). As used
herein, the term "in vivo" refers to events that occur within an
organism (e.g. animal, plant, and/or microbe).
[0110] As used herein, the term "subject" or "patient" refers to
any organism to which a composition disclosed herein can be
administered, e.g., for experimental, diagnostic, and/or
therapeutic purposes. Typical subjects include animals (e.g.,
mammals such as mice, rats, rabbits, non-human primates, and
humans) and/or plants. Usually the animal is a vertebrate such as a
primate, rodent, domestic animal or game animal. Primates include
chimpanzees, cynomologous monkeys, spider monkeys, and macaques,
e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets,
rabbits and hamsters. Domestic and game animals include cows,
horses, pigs, deer, bison, buffalo, feline species, e.g., domestic
cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,
chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
Patient or subject includes any subset of the foregoing, e.g., all
of the above, but excluding one or more groups or species such as
humans, primates or rodents. In certain embodiments of the aspects
described herein, the subject is a mammal, e.g., a primate, e.g., a
human. The terms, "patient" and "subject" are used interchangeably
herein. A subject can be male or female.
[0111] Preferably, the subject is a mammal. The mammal can be a
human, non-human primate, mouse, rat, dog, cat, horse, or cow, but
are not limited to these examples. Mammals other than humans can be
advantageously used as subjects that represent animal models of
human diseases and disorders. In addition, compounds, compositions
and methods described herein can be used to with domesticated
animals and/or pets.
[0112] In one embodiment, the subject is human. In another
embodiment, the subject is an experimental animal or animal
substitute as a disease model. The term does not denote a
particular age or sex. Thus, adult and newborn subjects, as well as
fetuses, whether male or female, are intended to be covered.
Examples of subjects include humans, dogs, cats, cows, goats, and
mice. The term subject is further intended to include transgenic
species. In some embodiments, the subject can be of European
ancestry. In some embodiments, the subject can be of African
American ancestry. In some embodiments, the subject can be of Asian
ancestry.
[0113] In jurisdictions that forbid the patenting of methods that
are practiced on the human body, the meaning of "administering" of
a composition to a human subject shall be restricted to prescribing
a controlled substance that a human subject will self-administer by
any technique (e.g., orally, inhalation, topical application,
injection, insertion, etc.). The broadest reasonable interpretation
that is consistent with laws or regulations defining patentable
subject matter is intended. In jurisdictions that do not forbid the
patenting of methods that are practiced on the human body, the
"administering" of compositions includes both methods practiced on
the human body and also the foregoing activities.
[0114] As used herein, the term "parenteral administration," refers
to administration through injection or infusion. Parenteral
administration includes, but is not limited to, subcutaneous
administration, intravenous administration, or intramuscular
administration.
[0115] As used herein, the term "subcutaneous administration"
refers to administration just below the skin. "Intravenous
administration" means administration into a vein.
[0116] As used herein, the term "dose" refers to a specified
quantity of a pharmaceutical agent provided in a single
administration. In certain embodiments, a dose may be administered
in two or more boluses, tablets, or injections. For example, in
certain embodiments, where subcutaneous administration is desired,
the desired dose requires a volume not easily accommodated by a
single injection. In such embodiments, two or more injections may
be used to achieve the desired dose. In certain embodiments, a dose
may be administered in two or more injections to minimize injection
site reaction in an individual.
[0117] As used herein, the term "dosage unit" refers to a form in
which a pharmaceutical agent is provided. In certain embodiments, a
dosage unit is a vial comprising lyophilized antisense
oligonucleotide. In certain embodiments, a dosage unit is a vial
comprising reconstituted antisense oligonucleotide.
[0118] As used herein, the term "active pharmaceutical ingredient"
refers to the substance in a pharmaceutical composition that
provides a desired effect.
[0119] As used herein, the term "side effects" refers to
physiological responses attributable to a treatment other than
desired effects. In certain embodiments, side effects include,
without limitation, injection site reactions, liver function test
abnormalities, renal function abnormalities, liver toxicity, renal
toxicity, central nervous system abnormalities, and myopathies. For
example, increased aminotransferase levels in serum may indicate
liver toxicity or liver function abnormality. For example,
increased bilirubin may indicate liver toxicity or liver function
abnormality.
[0120] As used herein, the term "alkyl," as used herein, refers to
a saturated straight or branched hydrocarbon radical containing up
to twenty four carbon atoms. Examples of alkyl groups include, but
are not limited to, methyl, ethyl, propyl, butyl, isopropyl,
n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically
include from 1 to about 24 carbon atoms, more typically from 1 to
about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon
atoms being more preferred. The term "lower alkyl" as used herein
includes from 1 to about 6 carbon atoms. Alkyl groups as used
herein may optionally include one or more further substituent
groups.
[0121] As used herein, the term "alkenyl," as used herein, refers
to a straight or branched hydrocarbon chain radical containing up
to twenty four carbon atoms and having at least one carbon-carbon
double bond. Examples of alkenyl groups include, but are not
limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,
dienes such as 1,3-butadiene and the like. Alkenyl groups typically
include from 2 to about 24 carbon atoms, more typically from 2 to
about 12 carbon atoms with from 2 to about 6 carbon atoms being
more preferred. Alkenyl groups as used herein may optionally
include one or more further substituent groups.
[0122] As used herein, the term "alkynyl," as used herein, refers
to a straight or branched hydrocarbon radical containing up to
twenty four carbon atoms and having at least one carbon-carbon
triple bond. Examples of alkynyl groups include, but are not
limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl
groups typically include from 2 to about 24 carbon atoms, more
typically from 2 to about 12 carbon atoms with from 2 to about 6
carbon atoms being more preferred. Alkynyl groups as used herein
may optionally include one or more further substitutent groups.
[0123] As used herein, the term "aminoalkyl" as used herein, refers
to an amino substituted alkyl radical. This term is meant to
include C1-C12 alkyl groups having an amino substituent at any
position and wherein the alkyl group attaches the aminoalkyl group
to the parent molecule. The alkyl and/or amino portions of the
aminoalkyl group can be further substituted with substituent
groups.
[0124] As used herein, the term "aliphatic," as used herein, refers
to a straight or branched hydrocarbon radical containing up to
twenty four carbon atoms wherein the saturation between any two
carbon atoms is a single, double or triple bond. An aliphatic group
preferably contains from 1 to about 24 carbon atoms, more typically
from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms
being more preferred. The straight or branched chain of an
aliphatic group may be interrupted with one or more heteroatoms
that include nitrogen, oxygen, sulfur and phosphorus. Such
aliphatic groups interrupted by heteroatoms include without
limitation polyalkoxys, such as polyalkylene glycols, polyamines,
and polyimines. Aliphatic groups as used herein may optionally
include further substitutent groups.
[0125] As used herein, the term "alicyclic" or "alicyclyl" refers
to a cyclic ring system wherein the ring is aliphatic. The ring
system can comprise one or more rings wherein at least one ring is
aliphatic. Preferred alicyclics include rings having from about 5
to about 9 carbon atoms in the ring. Alicyclic as used herein may
optionally include further substitutent groups. As used herein, the
term "alkoxy," as used herein, refers to a radical formed between
an alkyl group and an oxygen atom wherein the oxygen atom is used
to attach the alkoxy group to a parent molecule. Examples of alkoxy
groups include, but are not limited to, methoxy, ethoxy, propoxy,
isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy,
neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may
optionally include further substitutent groups. As used herein, the
terms "halo" and "halogen," as used herein, refer to an atom
selected from fluorine, chlorine, bromine and iodine.
[0126] As used herein, the terms "aryl" and "aromatic," as used
herein, refer to a mono- or polycyclic carbocyclic ring system
radicals having one or more aromatic rings. Examples of aryl groups
include, but are not limited to, phenyl, naphthyl,
tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl
ring systems have from about 5 to about 20 carbon atoms in one or
more rings. Aryl groups as used herein may optionally include
further substitutent groups.
[0127] As used herein, the terms "aralkyl" and "arylalkyl," as used
herein, refer to a radical formed between an alkyl group and an
aryl group wherein the alkyl group is used to attach the aralkyl
group to a parent molecule. Examples include, but are not limited
to, benzyl, phenethyl and the like. Aralkyl groups as used herein
may optionally include further substitutent groups attached to the
alkyl, the aryl or both groups that form the radical group.
[0128] As used herein, the term "heterocyclic radical" as used
herein, refers to a radical mono-, or poly-cyclic ring system that
includes at least one heteroatom and is unsaturated, partially
saturated or fully saturated, thereby including heteroaryl groups.
Heterocyclic is also meant to include fused ring systems wherein
one or more of the fused rings contain at least one heteroatom and
the other rings can contain one or more heteroatoms or optionally
contain no heteroatoms. A heterocyclic group typically includes at
least one atom selected from sulfur, nitrogen or oxygen. Examples
of heterocyclic groups include, [1,3]dioxolane, pyrrolidinyl,
pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,
piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,
morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,
pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as
used herein may optionally include further substitutent groups. As
used herein, the terms "heteroaryl," and "heteroaromatic," as used
herein, refer to a radical comprising a mono- or poly-cyclic
aromatic ring, ring system or fused ring system wherein at least
one of the rings is aromatic and includes one or more heteroatom.
Heteroaryl is also meant to include fused ring systems including
systems where one or more of the fused rings contain no
heteroatoms. Heteroaryl groups typically include one ring atom
selected from sulfur, nitrogen or oxygen. Examples of heteroaryl
groups include, but are not limited to, pyridinyl, pyrazinyl,
pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,
isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,
quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl,
quinoxalinyl, and the like. Heteroaryl radicals can be attached to
a parent molecule directly or through a linking moiety such as an
aliphatic group or hetero atom. Heteroaryl groups as used herein
may optionally include further substitutent groups.
[0129] As used herein, the term "heteroarylalkyl," as used herein,
refers to a heteroaryl group as previously defined having an alky
radical that can attach the heteroarylalkyl group to a parent
molecule. Examples include, but are not limited to,
pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyl and the
like. Heteroarylalkyl groups as used herein may optionally include
further substitutent groups on one or both of the heteroaryl or
alkyl portions.
[0130] As used herein, the term "mono or poly cyclic structure" as
used in the present invention includes all ring systems that are
single or polycyclic having rings that are fused or linked and is
meant to be inclusive of single and mixed ring systems individually
selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl,
arylalkyl, heterocyclic, heteroaryl, heteroaromatic,
heteroarylalkyl. Such mono and poly cyclic structures can contain
rings that are uniform or have varying degrees of saturation
including fully saturated, partially saturated or fully
unsaturated. Each ring can comprise ring atoms selected from C, N,
O and S to give rise to heterocyclic rings as well as rings
comprising only C ring atoms which can be present in a mixed motif
such as for example benzimidazole wherein one ring has only carbon
ring atoms and the fused ring has two nitrogen atoms. The mono or
poly cyclic structures can be further substituted with substituent
groups such as for example phthalimide which has two .dbd.O groups
attached to one of the rings. In another aspect, mono or poly
cyclic structures can be attached to a parent molecule directly
through a ring atom, through a substituent group or a bifunctional
linking moiety.
[0131] As used herein, the term "acyl," as used herein, refers to a
radical formed by removal of a hydroxyl group from an organic acid
and has the general formula C(O)--X where X is typically aliphatic,
alicyclic or aromatic. Examples include aliphatic carbonyls,
aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls,
aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and
the like. Acyl groups as used herein may optionally include further
substitutent groups.
[0132] As used herein, the term "hydrocarbyl" includes groups
comprising C, O and H. Included are straight, branched and cyclic
groups having any degree of saturation. Such hydrocarbyl groups can
include one or more heteroatoms selected from N, O and S and can be
further mono or poly substituted with one or more substituent
groups.
[0133] As used herein, the terms "substituent" and "substituent
group," as used herein, include groups that are typically added to
other groups or parent compounds to enhance desired properties or
give desired effects. Substituent groups can be protected or
unprotected and can be added to one available site or to many
available sites in a parent compound. Substituent groups may also
be further substituted with other substituent groups and may be
attached directly or via a linking group such as an alkyl or
hydrocarbyl group to a parent compound. Such groups include without
limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl
(--C(O)Raa), carboxyl (--C(O)O--Raa), aliphatic groups, alicyclic
groups, alkoxy, substituted oxo (--O--Raa), aryl, aralkyl,
heterocyclic, heteroaryl, heteroarylalkyl, amino (--NRbbRcc), imino
(.dbd.NRbb), amido (--C(O)N--RbbRcc or N(Rbb)C(O)Raa), azido
(--N3), nitro (--NO2), cyano (--CN), carbamido (--OC(O)NRbbRcc or
N(Rbb)C(O)ORaa), ureido (--N(Rbb)C(O)NRbbRcc), thioureido
(--N(Rbb)C(S)NRbbRcc), guanidinyl (--N(Rbb)C(.dbd.NRbb)NRbbRcc),
amidinyl (--C(.dbd.NRbb)-NRbbRcc or N(Rbb)C(NRbb)Raa), thiol
(--SRbb), sulfinyl (--S(O)Rbb), sulfonyl (--S(O)2Rbb), sulfonamidyl
(--S(O)2NRbbRcc or N(Rbb)S(O)2Rbb) and conjugate groups. Wherein
each Raa, Rbb and Rcc is, independently, H, an optionally linked
chemical functional group or a further substituent group with a
preferred list including without limitation H, alkyl, alkenyl,
alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,
alicyclic, heterocyclic and heteroarylalkyl.
[0134] The REVERSIR compounds disclosed herein are particularly
effective in reducing the activity of siRNAs. For example, the
REVERSIR compounds disclosed herein can reduce the activity of an
siRNA by at least about 50%, or at least about 60%, or at least
about 70%, or at least about 80%, or at least about 90%, or at
least about 95%, or at least about 97%, or at least about 99% or up
to and including a 100% decrease (i.e., absent level as compared to
a reference sample), or any decrease between 50-100% as compared to
a reference level. The reference level can be siRNA activity in
absence of the REVERSIR compound.
[0135] In some embodiments, the REVERSIR compounds describe herein
can reduce the activity of the siRNA by at least 75%, for example
by 80%, 85%, 90%, 95% or more and upto and including completer
reduction or inhibition of siRNA activity, within less than seven
(e.g., within 6 days, five days, four days, three days, two days or
one day) of administering or use of the REVERSIR compound.
[0136] In some embodiments, the REVERSIR compounds can completely
reduce the siRNA activity within four days of administering or use
of the REVERSIR compound. By complete reduction of siRNA activity
is meant a reduction of the siRNA activity by at least 80% relative
to a reference level.
Oligomeric Compounds
[0137] In certain embodiments, the siRNA and/or the REVERSIR
compounds are oligomeric compounds. In certain embodiments, it is
desirable to chemically modify oligomeric compounds, including
siRNAs and/or REVERSIR compounds, compared to naturally occurring
oligomers, such as DNA or RNA. Certain such modifications alter the
activity of the oligomeric compound. Certain such chemical
modifications can alter activity by, for example: increasing
affinity of a siRNA for its target nucleic acid or a REVERSIR for
its target siRNA, increasing its resistance to one or more
nucleases, and/or altering the pharmacokinetics or tissue
distribution of the oligomeric compound. In certain instances, the
use of chemistries that increase the affinity of an oligomeric
compound for its target can allow for the use of shorter oligomeric
compounds.
Monomers
[0138] In certain embodiment, oligomeric compounds comprise one or
more modified monomer. In certain such embodiments, oligomeric
compounds comprise one or more high affinity monomer. In certain
embodiments, such high-affinity monomer is selected from monomers
(e.g., nucleosides and nucleotides) comprising 2'-modified sugars,
including, but not limited to: BNA's and monomers (e.g.,
nucleosides and nucleotides) with 2'-substituents such as allyl,
amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10 alkyl, OCF.sub.3,
O--(CH.sub.2).sub.2--O--CH3, 2'-O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(Rm)(Rn), or
O--CH.sub.2--C(.dbd.O)--N(Rm)(Rn), where each Rm and Rn is,
independently, H or substituted or unsubstituted C.sub.1-C.sub.10
alkyl.
[0139] In certain embodiments, the oligomeric compounds including,
but not limited to REVERSIR compounds and siRNAs of the present
invention, comprise one or more high affinity monomers.
[0140] In certain embodiments, the oligomeric compounds including,
but not limited to REVERSIR compounds and siRNAs of the present
invention, comprise one or more 3-D-Methyleneoxy
(4'-CH.sub.2--O-2') LNA monomers.
[0141] In certain embodiments, the oligomeric compounds including,
including, but not limited to REVERSIR compounds and siRNAs of the
present invention, comprise one or more .alpha.-D-Methyleneoxy
(4'-CH.sub.2--O-2') LNA monomers.
[0142] In certain embodiments, the oligomeric compounds including,
including, but not limited to REVERSIR compounds and siRNAs of the
present invention, comprise one or more (S)-cEt monomers.
[0143] In certain embodiments, the oligomeric compounds including,
but not limited to REVERSIR compounds and siRNAs of the present
invention, comprise one or more high affinity monomers provided
that the oligomeric compound does not comprise a nucleotide
comprising a 2'-O(CH2).sub.nH, wherein n is one to six.
[0144] In certain embodiments, the oligomeric compounds including,
but not limited to REVERSIR compounds and siRNAs, comprise one or
more high affinity monomer provided that the oligomeric compound
does not comprise a nucleotide comprising a 2'-OCH.sub.3 or a
2'-O(CH.sub.2).sub.2OCH.sub.3.
[0145] In certain embodiments, the oligomeric compounds including,
but not limited to REVERSIR compounds and siRNAs, comprise one or
more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or
more) high affinity monomer provided that the oligomeric compound
does not comprise a .alpha.-L-Methyleneoxy (4'-CH.sub.2--O-2')
LNA.
[0146] In certain embodiments, the oligomeric compounds including,
but no limited to REVERSIR compounds and siRNAs, comprise one or
more high affinity monomer provided that the oligomeric compound
does not comprise a .beta.-D-Methyleneoxy (4'-CH.sub.2--O-2')
LNA.
[0147] In certain embodiments, the oligomeric compounds including,
but no limited to REVERSIR compound and siRNAs, comprise one or
more high affinity monomer provided that the oligomeric compound
does not comprise a .alpha.-L-Methyleneoxy (4'-CH.sub.2--O-2') LNA
or .beta.-D-Methyleneoxy (4'-CH.sub.2--O-2') LNA.
Certain Nucleobases
[0148] The naturally occurring base portion of a nucleoside is
typically a heterocyclic base. The two most common classes of such
heterocyclic bases are the purines and the pyrimidines. For those
nucleosides that include a pentofuranosyl sugar, a phosphate group
can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. In
forming oligonucleotides, those phosphate groups covalently link
adjacent nucleosides to one another to form a linear polymeric
compound. Within oligonucleotides, the phosphate groups are
commonly referred to as forming the internucleoside backbone of the
oligonucleotide. The naturally occurring linkage or backbone of RNA
and of DNA is a 3' to 5' phosphodiester linkage.
[0149] In addition to "unmodified" or "natural" nucleobases such as
the purine nucleobases adenine (A) and guanine (G), and the
pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U),
many modified nucleobases or nucleobase mimetics known to those
skilled in the art are amenable with the compounds described
herein. The unmodified or natural nucleobases can be modified or
replaced to provide oligonucleotides having improved properties.
For example, nuclease resistant oligonucleotides can be prepared
with these bases or with synthetic and natural nucleobases (e.g.,
inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or
tubercidine) and any one of the oligomer modifications described
herein. Alternatively, substituted or modified analogs of any of
the above bases and "universal bases" can be employed. When a
natural base is replaced by a non-natural and/or universal base,
the nucleotide is said to comprise a modified nucleobase and/or a
nucleobase modification herein. Modified nucleobase and/or
nucleobase modifications also include natural, non-natural and
universal bases, which comprise conjugated moieties, e.g. a ligand
described herein. Preferred conjugate moieties for conjugation with
nucleobases include cationic amino groups which can be conjugated
to the nucleobase via an appropriate alkyl, alkenyl or a linker
with an amide linkage.
[0150] An oligomeric compound described herein can also include
nucleobase (often referred to in the art simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and
uracil (U). Exemplary modified nucleobases include, but are not
limited to, other synthetic and natural nucleobases such as
inosine, xanthine, hypoxanthine, nubularine, isoguanisine,
tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine,
2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine,
2-(methylthio)-N.sup.6-(isopentenyl)adenine, 6-(alkyl)adenine,
6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine,
8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine,
8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine,
8-(thiol)adenine, N.sup.6-(isopentyl)adenine,
N.sup.6-(methyl)adenine, N.sup.6, N.sup.6-(dimethyl)adenine,
2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine,
6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine,
7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine,
8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine,
8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine,
N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine,
3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine,
5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine,
5-(propynyl)cytosine, 5-(propynyl)cytosine,
5-(trifluoromethyl)cytosine, 6-(azo)cytosine,
N.sup.4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil,
2-(thio)uracil, 5-(methyl)-2-(thio)uracil,
5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil,
5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil,
5-(methyl)-2,4-(dithio)uracil,
5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil,
5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil,
5-(aminoallyl)uracil, 5-(aminoalkyl)uracil,
5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil,
5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil,
5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil,
uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil,
5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil,
5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil,
dihydrouracil, N.sup.3-(methyl)uracil, 5-uracil (i.e.,
pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil,
2,4-(dithio)psuedouracil, 5-(alkyl)pseudouracil,
5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil,
5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil,
5-(methyl)-4-(thio)pseudouracil,
5-(alkyl)-2,4-(dithio)pseudouracil,
5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil,
1-substituted 2(thio)-pseudouracil, 1-substituted
4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil,
1-(aminocarbonylethylenyl)-pseudouracil,
1-(aminocarbonylethylenyl)-2(thio)-pseudouracil,
1-(aminocarbonylethylenyl)-4-(thio)pseudouracil,
1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,
1-(aminoalkylaminocarbonylethylenyl)-pseudouracil,
1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil,
1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil,
1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil,
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine,
hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl,
2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl,
nitrobenzimidazolyl, nitroindazolyl, aminoindolyl,
pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl,
5-(methyl)isocarbostyrilyl,
3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,
6-(methyl)-7-(aza)indolyl, imidizopyridinyl,
9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,
7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,
2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,
phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl,
stilbenyl, tetracenyl, pentacenyl, difluorotolyl,
4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole,
6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,
6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine,
5-substituted pyrimidines, N.sup.2-substituted purines,
N.sup.6-substituted purines, O.sup.6-substituted purines,
substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl,
6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,
pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl,
2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated
derivatives thereof. Alternatively, substituted or modified analogs
of any of the above bases and "universal bases" can be
employed.
[0151] As used herein, a universal nucleobase is any nucleobase
that can base pair with all of the four naturally occurring
nucleobases without substantially affecting the melting behavior,
recognition by intracellular enzymes or activity of the
oligonucleotide duplex. Some exemplary universal nucleobases
include, but are not limited to, 2,4-difluorotoluene,
nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine,
4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3-methyl
isocarbostyrilyl, 5-methyl isocarbostyrilyl, 3-methyl-7-propynyl
isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl,
imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl,
isocarbostyrilyl, 7-propynyl isocarbostyrilyl,
propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl,
4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl,
phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and
structural derivatives thereof (see for example, Loakes, 2001,
Nucleic Acids Research, 29, 2437-2447).
[0152] Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808; those disclosed in International Application No.
PCT/US09/038425, filed Mar. 26, 2009; those disclosed in the
Concise Encyclopedia Of Polymer Science And Engineering, pages
858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990; those
disclosed by English et al., Angewandte Chemie, International
Edition, 1991, 30, 613; those disclosed in Modified Nucleosides in
Biochemistry, Biotechnology and Medicine, Herdewijin, P. Ed.
Wiley-VCH, 2008; and those disclosed by Sanghvi, Y. S., Chapter 15,
dsRNA Research and Applications, pages 289-302, Crooke, S. T. and
Lebleu, B., Eds., CRC Press, 1993. Contents of all of the above are
herein incorporated by reference.
[0153] In certain embodiments, a modified nucleobase is a
nucleobase that is fairly similar in structure to the parent
nucleobase, such as for example a 7-deaza purine, a 5-methyl
cytosine, or a G-clamp. In certain embodiments, nucleobase mimetic
include more complicated structures, such as for example a
tricyclic phenoxazine nucleobase mimetic. Methods for preparation
of the above noted modified nucleobases are well known to those
skilled in the art.
[0154] In some embodiments, the REVERSIR compound comprises at
least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
or more) G-clamp nucleobase selected from the following:
##STR00001## ##STR00002## ##STR00003## ##STR00004##
##STR00005##
where n is 0, 1, 2, 3, 4, 5 or 6.
Certain Sugars
[0155] Oligomeric compounds provided herein can comprise one or
more monomer, including a nucleoside or nucleotide, having a
modified sugar moiety. For example, the furanosyl sugar ring of a
nucleoside can be modified in a number of ways including, but not
limited to, addition of a substituent group, bridging of two
non-geminal ring atoms to form a locked nucleic acid or bicyclic
nucleic acid. In certain embodiments, oligomeric compounds comprise
one or more monomers that are LNA.
[0156] In some embodiments of a locked nucleic acid, the 2'
position of furnaosyl is connected to the 4' position by a linker
selected independently from --[C(R1)(R2)].sub.n-,
--[C(R1)(R2)].sub.n-O--, --[C(R1)(R2)].sub.n-N(R1)-,
--[C(R1)(R2)].sub.n-N(R1)-O--, [C(R1R2)].sub.n-O--N(R1)-,
--C(R1)=C(R2)-O--, --C(R1)=N--, --C(R1)=N--O--, C(.dbd.NR1)-,
C(.dbd.NR1)-O--, C(.dbd.O)--, C(.dbd.O)O--, C(.dbd.S)--,
C(.dbd.S)O--, C(.dbd.S)S--, O, Si(R1)2-, S(.dbd.O).sub.x-- and
N(R1)-;
[0157] wherein:
[0158] x is 0, 1, or 2;
[0159] n is 1, 2, 3, or 4;
[0160] each R1 and R2 is, independently, H, a protecting group,
hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl,
substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12
alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical,
substituted heterocycle radical, heteroaryl, substituted
heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic
radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(.dbd.O) H),
substituted acyl, CN, sulfonyl (S(.dbd.O)2-J1), or sulfoxyl
(S(.dbd.O)-J1); and
[0161] each J1 and J2 is, independently, H, C1-C12 alkyl,
substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12
alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl,
substituted C5-C20 aryl, acyl (C(.dbd.O)--H), substituted acyl, a
heterocycle radical, a substituted heterocycle radical, C1-C12
aminoalkyl, substituted C1-C12 aminoalkyl or a protecting
group.
[0162] In one embodiment, each of the linkers of the LNA compounds
is, independently, [C(R1)(R2)].sub.n-, [C(R1)(R2)].sub.n-O--,
C(R1R2)-N(R1)-O or C(R1R2)-O--N(R1)-. In another embodiment, each
of said linkers is, independently, 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',
4'-(CH.sub.2).sub.2--O-2', 4'-CH.sub.2--O--N(R1)-2' and
4'-CH.sub.2--N(R1)-O-2'- wherein each R1 is, independently, H, a
protecting group or C1-C12 alkyl.
[0163] Certain LNA's have been prepared and disclosed in the patent
literature as well as in scientific literature (Singh et al., Chem.
Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,
3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000,
97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,
2219-2222; WO 94/14226; WO 2005/021570; Singh et al., J. Org.
Chem., 1998, 63, 10035-10039; Examples of issued US patents and
published applications that disclose LNA s include, for example,
U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499;
7,034,133; and 6,525,191; and U.S. Pre-Grant Publication Nos.
2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841;
2004-0143114; and 20030082807.
[0164] Also provided herein are LNAs in which the 2'-hydroxyl group
of the ribosyl sugar ring is linked to the 4' carbon atom of the
sugar ring thereby forming a methyleneoxy (4'-CH.sub.2--O-2')
linkage to form the bicyclic sugar moiety (reviewed in Elayadi et
al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al.,
Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol.
Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and
6,670,461). The linkage can be a methylene (--CH.sub.2--) group
bridging the 2' oxygen atom and the 4' carbon atom, for which the
term methyleneoxy (4'-CH.sub.2--O-2') LNA is used for the bicyclic
moiety; in the case of an ethylene group in this position, the term
ethyleneoxy (4'-CH.sub.2CH.sub.2--O-2') LNA is used (Singh et al.,
Chem. Commun., 1998, 4, 455-456: Morita et al., Bioorganic
Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy
(4'-CH.sub.2--O-2') LNA and other bicyclic sugar analogs display
very high duplex thermal stabilities with complementary DNA and RNA
(Tm=+3 to +10.degree. C.), stability towards 3'-exonucleolytic
degradation and good solubility properties. Potent and nontoxic
antisense oligonucleotides comprising BNAs have been described
(Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97,
5633-5638).
[0165] An isomer of methyleneoxy (4'-CH.sub.2--O-2') LNA that has
also been discussed is alpha-L-methyleneoxy (4'-CH.sub.2--O-2') LNA
which has been shown to have superior stability against a
3'-exonuclease. The alpha-L-methyleneoxy (4'-CH.sub.2--O-2') LNA's
were incorporated into antisense gapmers and chimeras that showed
potent antisense activity (Frieden et al., Nucleic Acids Research,
2003, 21, 6365-6372).
[0166] The synthesis and preparation of the methyleneoxy
(4'-CH.sub.2--O-2') LNA monomers adenine, cytosine, guanine,
5-methyl-cytosine, thymine and uracil, along with their
oligomerization, and nucleic acid recognition properties have been
described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs
and preparation thereof are also described in WO 98/39352 and WO
99/14226.
[0167] Analogs of methyleneoxy (4'-CH.sub.2--O-2') LNA,
phosphorothioate-methyleneoxy (4'-CH.sub.2--O-2') LNA and
2'-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med.
Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside
analogs comprising oligodeoxyribonucleotide duplexes as substrates
for nucleic acid polymerases has also been described (Wengel et
al., WO 99/14226). Furthermore, synthesis of 2'-amino-LNA, a novel
comformationally restricted high-affinity oligonucleotide analog
has been described in the art (Singh et al., J. Org. Chem., 1998,
63, 10035-10039). In addition, 2'-Amino- and 2'-methylamino-LNA's
have been prepared and the thermal stability of their duplexes with
complementary RNA and DNA strands has been previously reported.
[0168] Modified sugar moieties are well known and can be used to
alter, typically increase, the affinity of the antisense compound
for its target and/or increase nuclease resistance. A
representative list of preferred modified sugars includes but is
not limited to bicyclic modified sugars, including methyleneoxy
(4'-CH.sub.2--O-2') LNA and ethyleneoxy (4'-(CH.sub.2).sub.2--O-2'
bridge) ENA; substituted sugars, especially 2'-substituted sugars
having a 2'-F, 2'-OCH.sub.3 or a 2'-O(CH.sub.2).sub.2--OCH.sub.3
substituent group; and 4'-thio modified sugars. Sugars can also be
replaced with sugar mimetic groups among others. Methods for the
preparations of modified sugars are well known to those skilled in
the art. Some representative patents and publications that teach
the preparation of such modified sugars include, but are not
limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920;
6,531,584; and 6,600,032; and WO 2005/121371.
[0169] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR, n=1-50; "locked"
nucleic acids (LNA) in which the furanose portion of the nucleoside
includes a bridge connecting two carbon atoms on the furanose ring,
thereby forming a bicyclic ring system; O-AMINE or
O--(CH.sub.2).sub.nAMINE (n=1-10, AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, ethylene diamine or polyamino); and
O--CH.sub.2CH.sub.2(NCH.sub.2CH.sub.2NMe.sub.2).sub.2.
[0170] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars, which are of particular relevance to the single-strand
overhangs); halo (e.g., fluoro); amino (e.g. NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino); --NHC(O)R (R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar); cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl;
alkenyl and alkynyl, which can be optionally substituted with e.g.,
an amino functionality.
[0171] Other suitable 2'-modifications, e.g., modified MOE, are
described in U.S. Patent Application Publication No. 20130130378,
contents of which are herein incorporated by reference.
[0172] A modification at the 2' position can be present in the
arabinose configuration The term "arabinose configuration" refers
to the placement of a substituent on the C2' of ribose in the same
configuration as the 2'-OH is in the arabinose.
[0173] The sugar can comprise two different modifications at the
same carbon in the sugar, e.g., gem modification. The sugar group
can also contain one or more carbons that possess the opposite
stereochemical configuration than that of the corresponding carbon
in ribose. Thus, an oligomeric compound can include one or more
monomers containing e.g., arabinose, as the sugar. The monomer can
have an alpha linkage at the 1' position on the sugar, e.g.,
alpha-nucleosides. The monomer can also have the opposite
configuration at the 4'-position, e.g., C5' and H4' or substituents
replacing them are interchanged with each other. When the C5' and
H4' or substituents replacing them are interchanged with each
other, the sugar is said to be modified at the 4' position.
[0174] Oligomeric compounds can also include abasic sugars, i.e., a
sugar which lack a nucleobase at C-1' or has other chemical groups
in place of a nucleobase at C1'. See for example U.S. Pat. No.
5,998,203, content of which is herein incorporated in its entirety.
These abasic sugars can also be further containing modifications at
one or more of the constituent sugar atoms. Oligomeric compounds
can also contain one or more sugars that are the L isomer, e.g.
L-nucleosides. Modification to the sugar group can also include
replacement of the 4'-O with a sulfur, optionally substituted
nitrogen or CH2 group. In some embodiments, linkage between C1' and
nucleobase is in a configuration.
[0175] Sugar modifications can also include acyclic nucleotides,
wherein a C--C bonds between ribose carbons (e.g., C1'-C2',
C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and/or at least one
of ribose carbons or oxygen (e.g., C1', C2', C3', C4' or O4') are
independently or in combination absent from the nucleotide. In some
embodiments, acyclic nucleotide is
##STR00006##
wherein B is a modified or unmodified nucleobase, R.sub.1 and
R.sub.2 independently are H, halogen, OR.sub.3, or alkyl; and
R.sub.3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or
sugar).
[0176] In some embodiments, sugar modifications are selected from
the group consisting of 2'-H, 2'-O-Me (2'-O-methyl), 2'-O-MOE
(2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl]
(2'-O-NMA), 2'-S-methyl, 2'-O--CH.sub.2-(4'-C) (LNA),
2'-O--CH.sub.2CH.sub.2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) and gem
2'-OMe/2'F with 2'-O-Me in the arabinose configuration.
[0177] It is to be understood that when a particular nucleotide is
linked through its 2'-position to the next nucleotide, the sugar
modifications described herein can be placed at the 3'-position of
the sugar for that particular nucleotide, e.g., the nucleotide that
is linked through its 2'-position. A modification at the 3'
position can be present in the xylose configuration The term
"xylose configuration" refers to the placement of a substituent on
the C3' of ribose in the same configuration as the 3'-OH is in the
xylose sugar.
[0178] The hydrogen attached to C4' and/or C1' can be replaced by a
straight- or branched-optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, wherein
backbone of the alkyl, alkenyl and alkynyl can contain one or more
of O, S, S(O), SO.sub.2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'),
CH(Z'), phosphorous containing linkage, optionally substituted
aryl, optionally substituted heteroaryl, optionally substituted
heterocyclic or optionally substituted cycloalkyl, where R' is
hydrogen, acyl or optionally substituted aliphatic, Z' is selected
from the group consisting of OR.sub.11, COR.sub.11,
CO.sub.2R.sub.11,
##STR00007##
NR.sub.21R.sub.31, CONR.sub.21R.sub.31, CON(H)NR.sub.21R.sub.31,
ONR.sub.21R.sub.31, CON(H)N.dbd.CR.sub.41R.sub.51,
N(R.sub.21)C(.dbd.NR.sub.31)NR.sub.21R.sub.31,
N(R.sub.21)C(O)NR.sub.21R.sub.31, N(R.sub.21)C(S)NR.sub.21R.sub.31,
OC(O)NR.sub.21R.sub.31, SC(O)NR.sub.21R.sub.31,
N(R.sub.21)C(S)OR.sub.11, N(R.sub.21)C(O)OR.sub.11,
N(R.sub.21)C(O)SR.sub.11, N(R.sub.21)N.dbd.CR.sub.41R.sub.51,
ON.dbd.CR.sub.41R.sub.51, SO.sub.2R.sub.11, SOR.sub.11, SR.sub.11,
and substituted or unsubstituted heterocyclic; R.sub.21 and
R.sub.31 for each occurrence are independently hydrogen, acyl,
unsubstituted or substituted aliphatic, aryl heteroaryl,
heterocyclic, OR.sub.11, COR.sub.11, CO.sub.2R.sub.11, or
NR.sub.11R.sub.11'; or R.sub.21 and R.sub.31, taken together with
the atoms to which they are attached, form a heterocyclic ring;
R.sub.41 and R.sub.51 for each occurrence are independently
hydrogen, acyl, unsubstituted or substituted aliphatic, aryl,
heteroaryl, heterocyclic, OR.sub.11, COR.sub.11, or
CO.sub.2R.sub.11, or NR.sub.11R.sub.11'; and R.sub.11 and R.sub.11'
are independently hydrogen, aliphatic, substituted aliphatic, aryl,
heteroaryl, or heterocyclic. In some embodiments, the hydrogen
attached to the C4' of the 5' terminal nucleotide is replaced.
[0179] In some embodiments, C4' and C5' together form an optionally
substituted heterocyclic, preferably comprising at least one
--PX(Y)--, wherein X is H, OH, OM, SH, optionally substituted
alkyl, optionally substituted alkoxy, optionally substituted
alkylthio, optionally substituted alkylamino or optionally
substituted dialkylamino, where M is independently for each
occurrence an alki metal or transition metal with an overall charge
of +1; and Y is O, S, or NR', where R' is hydrogen, optionally
substituted aliphatic. Preferably this modification is at the 5
terminal of the oligonucleotide.
[0180] In certain embodiments, LNA's include bicyclic nucleoside
having the formula:
##STR00008##
[0181] wherein: [0182] Bx is a heterocyclic base moiety; [0183] T1
is H or a hydroxyl protecting group; [0184] T2 is H, a hydroxyl
protecting group or a reactive phosphorus group; [0185] Z is C1-C6
alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl,
substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl,
substituted acyl, or substituted amide.
[0186] In one embodiment, each of the substituted groups, is,
independently, mono or poly substituted with optionally protected
substituent groups independently selected from halogen, oxo,
hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(.dbd.X)J1, OC(.dbd.X)NJ1J2,
NJ3C(.dbd.X)NJ1J2 and CN, wherein each J1, J2 and J3 is,
independently, H or C1-C6 alkyl, and X is O, S or NJ1.
[0187] In certain such embodiments, each of the substituted groups,
is, independently, mono or poly substituted with substituent groups
independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2,
SJ1, N3, OC(.dbd.X)J1, and NJ3C(.dbd.X)NJ1J2, wherein each J1, J2
and J3 is, independently, H, C1-C6 alkyl, or substituted C1-C6
alkyl and X is O or NJ 1.
[0188] In certain embodiments, the Z group is C1-C6 alkyl
substituted with one or more Xx, wherein each Xx is independently
OJ1, NJ1J2, SJ1, N3, OC(.dbd.X)J1, OC(.dbd.X)NJ1J2,
NJ3C(.dbd.X)NJ1J2 or CN; wherein each J1, J2 and J3 is,
independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another
embodiment, the Z group is C1-C6 alkyl substituted with one or more
Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl,
alkoxy (e.g., CH3O--), substituted alkoxy or azido.
[0189] In certain embodiments, the Z group is --CH2Xx, wherein Xx
is OJ1, NJ1J2, SJ1, N3, OC(.dbd.X)J1, OC(.dbd.X)NJ1J2,
NJ3C(.dbd.X)NJ1J2 or CN; wherein each J1, J2 and J3 is,
independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another
embodiment, the Z group is --CH2Xx, wherein Xx is halo (e.g.,
fluoro), hydroxyl, alkoxy (e.g., CH3O--) or azido.
[0190] In certain such embodiments, the Z group is in the
(R)-configuration:
##STR00009##
[0191] In certain such embodiments, the Z group is in the
(S)-configuration:
##STR00010##
[0192] In certain embodiments, each T1 and T2 is a hydroxyl
protecting group. A preferred list of hydroxyl protecting groups
includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT),
9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl
(MOX). In certain embodiments, T1 is a hydroxyl protecting group
selected from acetyl, benzyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred
hydroxyl protecting group is T1 is 4,4'-dimethoxytrityl.
[0193] In certain embodiments, T2 is a reactive phosphorus group
wherein preferred reactive phosphorus groups include
diisopropylcyanoethoxy phosphoramidite and H-phosphonate. In
certain embodiments T1 is 4,4'-dimethoxytrityl and T2 is
diisopropylcyanoethoxy phosphoramidite.
[0194] In certain embodiments, oligomeric compounds have at least
one monomer of the formula:
##STR00011##
or of the formula:
##STR00012##
or of the formula:
##STR00013##
[0195] wherein [0196] Bx is a heterocyclic base moiety; [0197] T3
is H, a hydroxyl protecting group, a linked conjugate group or an
internucleoside linking group attached to a nucleoside, a
nucleotide, an oligonucleoside, an oligonucleotide, a monomeric
subunit or an oligomeric compound; [0198] T4 is H, a hydroxyl
protecting group, a linked conjugate group or an internucleoside
linking group attached to a nucleoside, a nucleotide, an
oligonucleoside, an oligonucleotide, a monomeric subunit or an
oligomeric compound; [0199] wherein at least one of T3 and T4 is an
internucleoside linking group attached to a nucleoside, a
nucleotide, an oligonucleoside, an oligonucleotide, a monomeric
subunit or an oligomeric compound; and [0200] Z is C1-C6 alkyl,
C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted
C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl,
or substituted amide.
[0201] In one embodiment, each of the substituted groups, is,
independently, mono or poly substituted with optionally protected
substituent groups independently selected from halogen, oxo,
hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(.dbd.X)J1, OC(.dbd.X)NJ1J2,
NJ3C(.dbd.X)NJ1J2 and CN, wherein each J1, J2 and J3 is,
independently, H or C1-C6 alkyl, and X is O, S or NJ1.
[0202] In one embodiment, each of the substituted groups, is,
independently, mono or poly substituted with substituent groups
independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2,
SJ1, N3, OC(.dbd.X)J1, and NJ3C(.dbd.X)NJ1J2, wherein each J1, J2
and J3 is, independently, H or C1-C6 alkyl, and X is O or NJ1.
[0203] In certain such embodiments, at least one Z is C1-C6 alkyl
or substituted C1-C6 alkyl. In certain embodiments, each Z is,
independently, C1-C6 alkyl or substituted C1-C6 alkyl. In certain
embodiments, at least one Z is C1-C6 alkyl. In certain embodiments,
each Z is, independently, C1-C6 alkyl. In certain embodiments, at
least one Z is methyl. In certain embodiments, each Z is methyl. In
certain embodiments, at least one Z is ethyl. In certain
embodiments, each Z is ethyl. In certain embodiments, at least one
Z is substituted C1-C6 alkyl. In certain embodiments, each Z is,
independently, substituted C1-C6 alkyl. In certain embodiments, at
least one Z is substituted methyl. In certain embodiments, each Z
is substituted methyl. In certain embodiments, at least one Z is
substituted ethyl. In certain embodiments, each Z is substituted
ethyl.
[0204] In certain embodiments, at least one substituent group is
C1-C6 alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with
one or more C1-C6 alkoxy). In another embodiment, each substituent
group is, independently, C1-C6 alkoxy (e.g., each Z is,
independently, C1-C6 alkyl substituted with one or more C1-C6
alkoxy).
[0205] In certain embodiments, at least one C1-C6 alkoxy
substituent group is CH3O-- (e.g., at least one Z is
CH.sub.3OCH.sub.2--). In another embodiment, each C1-C6 alkoxy
substituent group is CH.sub.3O-- (e.g., each Z is
CH.sub.3OCH.sub.2--).
[0206] In certain embodiments, at least one substituent group is
halogen (e.g., at least one Z is C1-C6 alkyl substituted with one
or more halogen). In certain embodiments, each substituent group
is, independently, halogen (e.g., each Z is, independently, C1-C6
alkyl substituted with one or more halogen). In certain
embodiments, at least one halogen substituent group is fluoro
(e.g., at least one Z is CH.sub.2FCH.sub.2--, CHF.sub.2CH.sub.2--
or CF.sub.3CH.sub.2--). In certain embodiments, each halo
substituent group is fluoro (e.g., each Z is, independently,
CH.sub.2FCH.sub.2--, CHF.sub.2CH.sub.2-- or
CF.sub.3CH.sub.2--).
[0207] In certain embodiments, at least one substituent group is
hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one
or more hydroxyl). In certain embodiments, each substituent group
is, independently, hydroxyl (e.g., each Z is, independently, C1-C6
alkyl substituted with one or more hydroxyl). In certain
embodiments, at least one Z is HOCH.sub.2--. In another embodiment,
each Z is HOCH.sub.2--.
[0208] In certain embodiments, at least one Z is CH.sub.3--,
CH.sub.3CH.sub.2--, CH.sub.2OCH.sub.3--, CH.sub.2F-- or
HOCH.sub.2--. In certain embodiments, each Z is, independently,
CH.sub.3--, CH.sub.3CH.sub.2--, CH.sub.2OCH.sub.3--, CH.sub.2F-- or
HOCH.sub.2--.
[0209] In certain embodiments, at least one Z group is C1-C6 alkyl
substituted with one or more Xx, wherein each Xx is, independently,
OJ1, NJ1J2, SJ1, N3, OC(.dbd.X)J1, OC(.dbd.X)NJ1J2,
NJ3C(.dbd.X)NJ1J2 or CN; wherein each J1, J2 and J3 is,
independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another
embodiment, at least one Z group is C1-C6 alkyl substituted with
one or more Xx, wherein each Xx is, independently, halo (e.g.,
fluoro), hydroxyl, alkoxy (e.g., CH.sub.3O--) or azido.
[0210] In certain embodiments, each Z group is, independently,
C1-C6 alkyl substituted with one or more Xx, wherein each Xx is
independently OJ1, NJ1J2, SJ1, N3, OC(.dbd.X)J1, OC(.dbd.X)NJ1J2,
NJ3C(.dbd.X)NJ1J2 or CN; wherein each J1, J2 and J3 is,
independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another
embodiment, each Z group is, independently, C1-C6 alkyl substituted
with one or more Xx, wherein each Xx is independently halo (e.g.,
fluoro), hydroxyl, alkoxy (e.g., CH.sub.3O--) or azido.
[0211] In certain embodiments, at least one Z group is
--CH.sub.2Xx, wherein Xx is OJ1, NJ1J2, SJ1, N3, OC(.dbd.X)J1,
OC(.dbd.X)NJ1J2, NJ3C(.dbd.X)NJ1J2 or CN; wherein each J1, J2 and
J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1 In
certain embodiments, at least one Z group is --CH.sub.2Xx, wherein
Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH.sub.3O--) or
azido.
[0212] In certain embodiments, each Z group is, independently,
--CH.sub.2Xx, wherein each Xx is, independently, OJ1, NJ1J2, SJ1,
N3, OC(.dbd.X)J1, OC(.dbd.X)NJ1J2, NJ3C(.dbd.X)NJ1J2 or CN; wherein
each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O,
S or NJ1. In another embodiment, each Z group is, independently,
--CH.sub.2Xx, wherein each Xx is, independently, halo (e.g.,
fluoro), hydroxyl, alkoxy (e.g., CH.sub.3O--) or azido.
[0213] In certain embodiments, at least one Z is CH.sub.3--. In
another embodiment, each Z is, CH.sub.3--.
[0214] In certain embodiments, the Z group of at least one monomer
is in the (R)-configuration represented by the formula:
##STR00014##
or the formula:
##STR00015##
or the formula:
##STR00016##
[0215] IN certain embodiments, the Z group of each monomer of the
formula is in the (R)-configuration.
[0216] In certain embodiments, the Z group of at least one monomer
is in the (S)-configuration represented by the formula:
##STR00017##
or the formula:
##STR00018##
or the formula:
##STR00019##
[0217] In certain embodiments, the Z group of each monomer of the
formula is in the (S)-configuration.
[0218] In certain embodiments, T3 is H or a hydroxyl protecting
group. In certain embodiments, T4 is H or a hydroxyl protecting
group. In a further embodiment T3 is an internucleoside linking
group attached to a nucleoside, a nucleotide or a monomeric
subunit. In certain embodiments, T4 is an internucleoside linking
group attached to a nucleoside, a nucleotide or a monomeric
subunit. In certain embodiments, T3 is an internucleoside linking
group attached to an oligonucleoside or an oligonucleotide. In
certain embodiments, T4 is an internucleoside linking group
attached to an oligonucleoside or an oligonucleotide. In certain
embodiments, T3 is an internucleoside linking group attached to an
oligomeric compound. In certain embodiments, T4 is an
internucleoside linking group attached to an oligomeric compound.
In certain embodiments, at least one of T3 and T4 comprises an
internucleoside linking group selected from phosphodiester or
phosphorothioate.
[0219] In certain embodiments, oligomeric compounds have at least
one region of at least two contiguous monomers of the formula:
##STR00020##
or of the formula:
##STR00021##
or of the formula:
##STR00022##
[0220] In certain such embodiments, LNAs include, but are not
limited to, (A) .alpha.-L-Methyleneoxy (4'-CH2-O-2') LNA, (B)
.beta.-D-Methyleneoxy (4'-CH2-O-2') LNA, (C) Ethyleneoxy
(4'-(CH2)2-O-2') LNA, (D) Aminooxy (4'-CH2-O--N(R)-2') LNA and (E)
Oxyamino (4'-CH2-N(R)--O-2') LNA, as depicted below:
##STR00023##
[0221] In certain embodiments, the oligomeric compound comprises at
least two regions of at least two contiguous monomers of the above
formula. In certain embodiments, the oligomeric compound comprises
a gapped oligomeric compound. In certain embodiments, the
oligomeric compound comprises at least one region of from about 8
to about 14 contiguous .beta.-D-2'-deoxyribofuranosyl nucleosides.
In certain embodiments, the oligomeric compound comprises at least
one region of from about 9 to about 12 contiguous
.beta.-D-2'-deoxyribofuranosyl nucleosides.
[0222] In certain embodiments, the oligomeric compound comprises at
least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
or more)S-cEt monomer of the formula:
##STR00024##
wherein Bx IS heterocyclic base moiety.
[0223] In some embodiments, the oligomeric compound, e.g. REVERSIR
compound, comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15 or more) nucleoside selected from the
following:
##STR00025## ##STR00026## ##STR00027## ##STR00028##
##STR00029##
where B is A-001 to A-026 and n is 0-6 (e.g., 0, 1, 2, 3, 4, 5 or
6).
[0224] In certain embodiments, monomers include sugar mimetics. In
certain such embodiments, a mimetic is used in place of the sugar
or sugar-internucleoside linkage combination, and the nucleobase is
maintained for hybridization to a selected target. Representative
examples of a sugar mimetics include, but are not limited to,
cyclohexenyl or morpholino. Representative examples of a mimetic
for a sugar-internucleoside linkage combination include, but are
not limited to, peptide nucleic acids (PNA) and morpholino groups
linked by uncharged achiral linkages. In some instances a mimetic
is used in place of the nucleobase. Representative nucleobase
mimetics are well known in the art and include, but are not limited
to, tricyclic phenoxazine analogs and universal bases (Berger et
al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by
reference). Methods of synthesis of sugar, nucleoside and
nucleobase mimetics are well known to those skilled in the art.
[0225] In certain embodiments, the REVERSIR compound comprises at
least one monomer that is LNA and at least one G-clamp nucleobase.
For example, the REVERSIR compound can comprise 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15 or more monomers that are LNA 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more G-clamp
nucleobases.
[0226] In some embodiments, the REVERSIR compound comprises at
least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
or more) peptide nucleic acid monomer. In certain embodiments, the
REVERSIR compound comprises at least one monomer that is LNA and at
least one monomer that is PNA. For example, the REVERSIR compound
can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or
more monomers that are LNA 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15 or more monomers that are PNA.
[0227] In certain embodiments, the REVERSIR compound comprises at
least one PNA monomer and at least one G-clamp nucleobase. For
example, the REVERSIR compound can comprise 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15 or more PNA monomers and 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more G-clamp nucleobases.
[0228] In certain embodiments, the REVERSIR compound comprises at
least one LNA monomer, at least one PNA monomer and at least one
G-clamp nucleobase. For example, the REVERSIR compound can comprise
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more LNA
monomers; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more
PNA monomers and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
or more G-clamp nucleobases.
Monomeric Linkages
[0229] Described herein are linking groups that link monomers
(including, but not limited to, modified and unmodified nucleosides
and nucleotides) together, thereby forming an oligomeric compound.
Such linking groups are also referred to as intersugar linkage. The
two main classes of linking groups are defined by the presence or
absence of a phosphorus atom. Representative phosphorus containing
linkages include, but are not limited to, phosphodiesters
(P.dbd.O), phosphotriesters, methylphosphonates, phosphoramidate,
and phosphorothioates (P.dbd.S). Representative non-phosphorus
containing linking groups include, but are not limited to,
methylenemethylimino (--CH2-N(CH3)-O--CH2-), thiodiester
(--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane
(--O--Si(H)2-O--); and N,N'-dimethylhydrazine
(--CH2-N(CH3)-N(CH3)-). Oligomeric compounds having non-phosphorus
linking groups are referred to as oligonucleosides. Modified
linkages, compared to natural phosphodiester linkages, can be used
to alter, typically increase, nuclease resistance of the oligomeric
compound. In certain embodiments, linkages having a chiral atom can
be prepared a racemic mixtures, as separate enantomers.
Representative chiral linkages include, but are not limited to,
alkylphosphonates and phosphorothioates. Methods of preparation of
phosphorous-containing and non-phosphorous-containing linkages are
well known to those skilled in the art.
[0230] The phosphate group in the linking group can be modified by
replacing one of the oxygens with a different substituent. One
result of this modification can be increased resistance of the
oligonucleotide to nucleolytic breakdown. Examples of modified
phosphate groups include phosphorothioate, phosphoroselenates,
borano phosphates, borano phosphate esters, hydrogen phosphonates,
phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
In some embodiments, one of the non-bridging phosphate oxygen atoms
in the linkage can be replaced by any of the following: S, Se,
BR.sub.3 (R is hydrogen, alkyl, aryl), C (i.e. an alkyl group, an
aryl group, etc. . . . ), H, NR.sub.2 (R is hydrogen, optionally
substituted alkyl, aryl), or OR (R is optionally substituted alkyl
or aryl). The phosphorous atom in an unmodified phosphate group is
achiral. However, replacement of one of the non-bridging oxygens
with one of the above atoms or groups of atoms renders the
phosphorous atom chiral; in other words a phosphorous atom in a
phosphate group modified in this way is a stereogenic center. The
stereogenic phosphorous atom can possess either the "R"
configuration (herein Rp) or the "S" configuration (herein Sp).
[0231] Phosphorodithioates have both non-bridging oxygens replaced
by sulfur. The phosphorus center in the phosphorodithioates is
achiral which precludes the formation of oligonucleotides
diastereomers. Thus, while not wishing to be bound by theory,
modifications to both non-bridging oxygens, which eliminate the
chiral center, e.g. phosphorodithioate formation, can be desirable
in that they cannot produce diastereomer mixtures. Thus, the
non-bridging oxygens can be independently any one of O, S, Se, B,
C, H, N, or OR (R is alkyl or aryl).
[0232] The phosphate linker can also be modified by replacement of
bridging oxygen, (i.e. oxygen that links the phosphate to the sugar
of the monomer), with nitrogen (bridged phosphoroamidates), sulfur
(bridged phosphorothioates) and carbon (bridged
methylenephosphonates). The replacement can occur at the either one
of the linking oxygens or at both linking oxygens. When the
bridging oxygen is the 3'-oxygen of a nucleoside, replacement with
carbon is preferred. When the bridging oxygen is the 5'-oxygen of a
nucleoside, replacement with nitrogen is preferred.
[0233] Modified phosphate linkages where at least one of the oxygen
linked to the phosphate has been replaced or the phosphate group
has been replaced by a non-phosphorous group, are also referred to
as "non-phosphodiester intersugar linkage" or "non-phosphodiester
linker."
[0234] In certain embodiments, the phosphate group can be replaced
by non-phosphorus containing connectors, e.g. dephospho linkers.
Dephospho linkers are also referred to as non-phosphodiester
linkers herein. While not wishing to be bound by theory, it is
believed that since the charged phosphodiester group is the
reaction center in nucleolytic degradation, its replacement with
neutral structural mimics should impart enhanced nuclease
stability. Again, while not wishing to be bound by theory, it can
be desirable, in some embodiment, to introduce alterations in which
the charged phosphate group is replaced by a neutral moiety.
[0235] Examples of moieties which can replace the phosphate group
include, but are not limited to, amides (for example amide-3
(3'-CH.sub.2--C(.dbd.O)--N(H)-5') and amide-4
(3'-CH.sub.2--N(H)--C(.dbd.O)-5')), hydroxylamino, siloxane
(dialkylsiloxxane), carboxamide, carbonate, carboxymethyl,
carbamate, carboxylate ester, thioether, ethylene oxide linker,
sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal
(3'-S--CH.sub.2--O-5'), formacetal (3'-O--CH.sub.2--O-5'), oxime,
methyleneimino, methykenecarbonylamino, methylenemethylimino (MMI,
3'-CH.sub.2--N(CH.sub.3)--O-5'), methylenehydrazo,
methylenedimethylhydrazo, methyleneoxymethylimino, ethers
(C3'-O--C5'), thioethers (C3'-S--C5'), thioacetamido
(C3'-N(H)--C(.dbd.O)--CH.sub.2--S--C5', C3'-O--P(O)--O--SS--C5',
C3'-CH.sub.2--NH--NH--C5', 3'-NHP(O)(OCH.sub.3)--O-5' and
3'-NHP(O)(OCH.sub.3)--O-5' and nonionic linkages containing mixed
N, O, S and CH.sub.2 component parts. See for example, Carbohydrate
Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook
Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65).
Preferred embodiments include methylenemethylimino (MMI),
methylenecarbonylamino, amides, carbamate and ethylene oxide
linker.
[0236] One skilled in the art is well aware that in certain
instances replacement of a non-bridging oxygen can lead to enhanced
cleavage of the intersugar linkage by the neighboring 2'-OH, thus
in many instances, a modification of a non-bridging oxygen can
necessitate modification of 2'-OH, e.g., a modification that does
not participate in cleavage of the neighboring intersugar linkage,
e.g., arabinose sugar, 2'-O-alkyl, 2'-F, LNA and ENA.
[0237] Preferred non-phosphodiester intersugar linkages include
phosphorothioates, phosphorothioates with an at least 1%, 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more enantiomeric
excess of Sp isomer, phosphorothioates with an at least 1%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more
enantiomeric excess of Rp isomer, phosphorodithioates,
phsophotriesters, aminoalkylphosphotrioesters, alkyl-phosphonaters
(e.g., methyl-phosphonate), selenophosphates, phosphoramidates
(e.g., N-alkylphosphoramidate), and boranophosphonates.
[0238] In some embodiments, the oligomeric compound, e.g., REVERSIR
compound or siRNA, comprises at least one (e.g., 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15 or more and upto including all)
modified or nonphosphodiester linkages. In one embodiment, the
oligomeric compound, e.g., REVERSIR compound or siRNA, comprises at
least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
or more and upto including all) phosphorothioate linkages.
[0239] In some embodiments, all internucleoside linkages in the
reverser compounds are phosphorothioate (PS) internucleoside
linkages. In certain embodiments, the REVERSIR compounds comprise
at least one phosphorothioate (PS) internucleoside linkage, but not
all internucleoside linkages in said REVERSIR compound are a
phosphorothioate linkage. In other words, in some embodiments, less
than 100% (e.g., 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%,
45%, 40% or fewer) of the internucleoside linkages are
phosphorothioate linkages.
[0240] In some embodiments, the REVERSIR compounds comprise at
least one phosphorothioate internucleoside linkage and at least one
internucleoside linkage that is not a phosphorothioate. For
example, the REVERSIR compounds comprise at least one
phosphorothioate internucleoside linkage and at least one
phosphodiester internucleoside linkage. In some embodiments, the
non-phosphorothioate internucleoside linkage is between the
terminus and the penultimate nucleosides.
[0241] In some embodiments, the internucleoside linkage between the
nucleobase at the 3'-terminus of the REVERSIR compound and the rest
of the REVERSIR compound is a phosphodiester linkage. In some
embodiments, all internucleoside linkages in the reverser compounds
are phosphorothioate except for the internucleoside linkage between
the nucleoside at the 3'-terminus of the REVERSIR compound and the
rest of the REVERSIR compound.
[0242] Oligomeric compounds can also be constructed wherein the
phosphate linker and the sugar are replaced by nuclease resistant
nucleoside or nucleotide surrogates. While not wishing to be bound
by theory, it is believed that the absence of a repetitively
charged backbone diminishes binding to proteins that recognize
polyanions (e.g. nucleases). Again, while not wishing to be bound
by theory, it can be desirable in some embodiment, to introduce
alterations in which the bases are tethered by a neutral surrogate
backbone. Examples include the morpholino, cyclobutyl, pyrrolidine,
peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA) and
backnone-extended pyrrolidine PNA (bepPNA) nucleoside surrogates. A
preferred surrogate is a PNA surrogate.
[0243] The oligomeric compounds described herein contain one or
more asymmetric centers and thus give rise to enantiomers,
diastereomers, and other stereoisomeric configurations that may be
defined, in terms of absolute stereochemistry, as (R) or (S), such
as for sugar anomers, or as (D) or (L) such as for amino acids et
al. Included in the antisense compounds provided herein are all
such possible isomers, as well as their racemic and optically pure
forms.
Terminal Modifications
[0244] Ends of the oligomeric compound can be modified. Such
modifications can be at one end or both ends. For example, the 3'
and/or 5' ends of an oligonucleotide can be conjugated to other
functional molecular entities such as labeling moieties, e.g.,
fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or
protecting groups (based e.g., on sulfur, silicon, boron or ester).
The functional molecular entities can be attached to the sugar
through a phosphate group and/or a linker. The terminal atom of the
linker can connect to or replace the linking atom of the phosphate
group or the C-3' or C-5' O, N, S or C group of the sugar.
Alternatively, the linker can connect to or replace the terminal
atom of a nucleotide surrogate (e.g., PNAs).
[0245] When a linker/phosphate-functional molecular
entity-linker/phosphate array is interposed between two strands of
a double stranded oligomeric compound, this array can substitute
for a hairpin loop in a hairpin-type oligomeric compound.
[0246] Terminal modifications useful for modulating activity
include modification of the 5' end of oligomeric compound with
phosphate or phosphate analogs. In certain embodiments, the 5'end
of oligomeric compound is phosphorylated or includes a phosphoryl
analog. Exemplary 5'-phosphate modifications include those which
are compatible with RISC mediated gene silencing. Modifications at
the 5'-terminal end can also be useful in stimulating or inhibiting
the immune system of a subject. In some embodiments, the 5'-end of
the oligomeric compound comprises the modification
##STR00030##
wherein W, X and Y are each independently selected from the group
consisting of 0, OR (R is hydrogen, alkyl, aryl), S, Se, BR.sub.3
(R is hydrogen, alkyl, aryl), BH.sub.3.sup.-, C (i.e. an alkyl
group, an aryl group, etc. . . . ), H, NR.sub.2 (R is hydrogen,
alkyl, aryl), or OR (R is hydrogen, alkyl or aryl); A and Z are
each independently for each occurrence absent, O, S, CH.sub.2, NR
(R is hydrogen, alkyl, aryl), or optionally substituted alkylene,
wherein backbone of the alkylene can comprise one or more of O, S,
SS and NR (R is hydrogen, alkyl, aryl) internally and/or at the
end; and n is 0-2. In some embodiments, n is 1 or 2. It is
understood that A is replacing the oxygen linked to 5' carbon of
sugar. When n is 0, W and Y together with the P to which they are
attached can form an optionally substituted 5-8 membered
heterocyclic, wherein W an Y are each independently O, S, NR' or
alkylene. Preferably the heterocyclic is substituted with an aryl
or heteroaryl. In some embodiments, one or both hydrogen on C5' of
the 5'-terminal nucleotides are replaced with a halogen, e.g.,
F.
[0247] Exemplary 5'-modifications include, but are not limited to,
5'-monophosphate ((HO).sub.2(O)P--O-5'); 5'-diphosphate
((HO).sub.2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate
((HO).sub.2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5');
5'-monothiophosphate (phosphorothioate; (HO).sub.2(S)P--O-5');
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO)2(O)P--S-5'); 5'-alpha-thiotriphosphate;
5'-beta-thiotriphosphate; 5'-gamma-thiotriphosphate;
5'-phosphoramidates ((HO).sub.2(O)P--NH-5',
(HO)(NH.sub.2)(O)P--O-5'). Other 5'-modification include
5'-alkylphosphonates (R(OH)(O)P--O-5', R=alkyl, e.g., methyl,
ethyl, isopropyl, propyl, etc. . . . ), 5'-alkyletherphosphonates
(R(OH)(O)P--O-5', R=alkylether, e.g., methoxymethyl (CH2OMe),
ethoxymethyl, etc. . . . ). Other exemplary 5'-modifications
include where Z is optionally substituted alkyl at least once,
e.g.,
((HO).sub.2(X)P--O[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
((HO).sub.2(X)P--O[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5',
((HO).sub.2(X)P--[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5';
dialkyl terminal phosphates and phosphate mimics:
HO[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
H.sub.2N[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
H[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
Me.sub.2N[--(CH.sub.2).sub.a--O--P(X)(OH)--O].sub.b-5',
HO[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5',
H.sub.2N[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5',
H[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5',
Me.sub.2N[--(CH.sub.2).sub.a--P(X)(OH)--O].sub.b-5', wherein a and
b are each independently 1-10. Other embodiments, include
replacement of oxygen and/or sulfur with BH.sub.3, BH.sub.3.sup.-
and/or Se.
[0248] Terminal modifications can also be useful for monitoring
distribution, and in such cases the preferred groups to be added
include fluorophores, e.g., fluorescein or an Alexa dye, e.g.,
Alexa 488. Terminal modifications can also be useful for enhancing
uptake, useful modifications for this include targeting ligands.
Terminal modifications can also be useful for cross-linking an
oligonucleotide to another moiety; modifications useful for this
include mitomycin C, psoralen, and derivatives thereof.
Oligomeric Compounds
[0249] In certain embodiments, provided herein are oligomeric
compounds having reactive phosphorus groups useful for forming
linkages including for example phosphodiester and phosphorothioate
internucleoside linkages. Methods of preparation and/or
purification of precursors or oligomeric compounds are not a
limitation of the compositions or methods provided herein. Methods
for synthesis and purification of oligomeric compounds including
DNA, RNA, oligonucleotides, oligonucleosides, and antisense
compounds are well known to those skilled in the art.
[0250] Generally, oligomeric compounds comprise a plurality of
monomeric subunits linked together by linking groups. Non-limiting
examples of oligomeric compounds include primers, probes, antisense
compounds, antisense oligonucleotides, external guide sequence
(EGS) oligonucleotides, alternate splicers, and siRNAs. As such,
these compounds can be introduced in the form of single-stranded,
double-stranded, circular, branched or hairpins and can contain
structural elements such as internal or terminal bulges or loops.
Oligomeric double-stranded compounds can be two strands hybridized
to form double-stranded compounds or a single strand with
sufficient self-complementarity to allow for hybridization and
formation of a fully or partially double-stranded compound.
[0251] In certain embodiments, the present invention provides
chimeric oligomeric compounds. In certain such embodiments,
chimeric oligomeric compounds are chimeric oligonucleotides. In
certain such embodiments, the chimeric oligonucleotides comprise
differently modified nucleotides. In certain embodiments, chimeric
oligonucleotides are mixed-backbone antisense oligonucleotides.
[0252] In general a chimeric oligomeric compound will have modified
nucleosides that can be in isolated positions or grouped together
in regions that will define a particular motif. Any combination of
modifications and/or mimetic groups can comprise a chimeric
oligomeric compound as described herein.
[0253] In certain embodiments, chimeric oligomeric compounds
typically comprise at least one region modified so as to confer
increased resistance to nuclease degradation, increased cellular
uptake, and/or increased binding affinity for the target nucleic
acid. In certain embodiments, an additional region of the
oligomeric compound may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids.
[0254] In certain embodiments, chimeric oligomeric compounds are
gapmers. In certain such embodiments, a mixed-backbone oligomeric
compound has one type of internucleotide linkages in one or both
wings and a different type of internucleoside linkages in the gap.
In certain such embodiments, the mixed-backbone oligonucleotide has
phosphodiester linkages in the wings and phosphorothioate linkages
in the gap. In certain embodiments in which the internucleoside
linkages in a wing is different from the internucleoside linkages
in the gap, the internucleoside linkage bridging that wing and the
gap is the same as the internucleoside linkage in the wing. In
certain embodiments in which the internucleoside linkages in a wing
is different from the internucleoside linkages in the gap, the
internucleoside linkage bridging that wing and the gap is the same
as the internucleoside linkage in the gap.
[0255] In certain embodiments, the present invention provides
oligomeric compounds, including siRNAs and REVERSIR compounds of
any of a variety of ranges of lengths. In certain embodiments, the
invention provides oligomeric compounds consisting of X--Y linked
oligonucleosides, where X and Y are each independently selected
from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X<Y.
For example, in certain embodiments, the invention provides
oligomeric compounds comprising: 8-9, 8-10, 8-11, 8-12, 8-13, 8-14,
8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25,
8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15,
9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26,
9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16,
10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25,
10-26, 10-27, 10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15,
11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24,
11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 12-13, 12-14, 12-15,
12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24,
12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16,
13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25,
13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18,
14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27,
14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21,
15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30,
16-17, 16-18, 16-19, 16-25, 16-21, 16-22, 16-23, 16-24, 16-25,
16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17-19, 17-20, 17-21,
17-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30,
18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27,
18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19-23, 19-24, 19-25,
19-26, 19-29, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24,
20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24,
21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25,
22-26, 22-27, 22-28, 22-29, 22-30, 23-24, 23-25, 23-26, 23-27,
23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28, 24-29, 24-30,
25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30,
27-28, 27-29, 27-30, 28-29, 28-30, or 29-30 linked nucleosides.
[0256] As noted-above, REVERSIR compounds can be of any length. For
example, in some embodiments, the REVERSIR compound is a modified
oligonucleotide consisting of 6-30 nucleotides. For example, the
REVERSIR compound can consist of 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
linked nucleobases. In some embodiments, the REVERSIR compound
consists of 6-17, 7-16 or 8-15 linked nucleobases.
[0257] The inventors have discovered inter alia that REVERSIR
compounds, i.e., modified oligonucleotides, consisting of 15 or
fewer nucleosides are particularly effective in reversing the siRNA
activity. Accordingly, in some embodiments, the REVERSIR compound
is a modified oligonucleotide consisting of 8-15 (e.g., 8, 9, 10,
11, 12, 13, 14 or 15) linked nucleosides. In some embodiments, the
REVERSIR compound is a modified oligonucleotide consisting of 6-12,
7-11 or 8-10 linked nucleobases. In some embodiments, the REVERSIR
compound consists of 8-9 linked nucleobases.
[0258] As discussed herein, REVERSIR compounds are modified
oligonucleotides that are substantially complementary to at least
one strand of an siRNA. Now without wishing to be bound by a
theory, REVERSIR compounds that are substantially complementary to
the seed region of the antisense strand of the siRNA (i.e., at
positions 2-8 of the 5'-end of the antisense strand) are
particularly effective in reducing siRNA activity. Thus, in many
embodiments, the REVERSIR compound is substantially complementary
to nucleosides 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15 or 2-16
of the antisense strand of the siRNA. By substantially
complementary in this context is meant a complementarity of at
least 90%, preferably at least 95%, and more preferably complete
complementarity.
Ligands
[0259] In certain embodiments, oligomeric compounds are modified by
covalent attachment of one or more conjugate groups. In general,
conjugate groups modify one or more properties of the attached
oligomeric compound including but not limited to pharmacodynamic,
pharmacokinetic, binding, absorption, cellular distribution,
cellular uptake, charge and clearance. Conjugate groups are
routinely used in the chemical arts and are linked directly or via
an optional linking moiety or linking group to a parent compound
such as an oligomeric compound. A preferred list of conjugate
groups includes without limitation, intercalators, reporter
molecules, polyamines, polyamides, polyethylene glycols,
thioethers, polyethers, cholesterols, thiocholesterols, cholic acid
moieties, folate, lipids, phospholipids, biotin, phenazine,
phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,
rhodamines, coumarins and dyes.
[0260] Preferred conjugate groups amenable to the present invention
include lipid moieties such as a cholesterol moiety (Letsinger et
al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid
(Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a
thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem.
Let., 1993, 3, 2765); a thiocholesterol (Oberhauser et al., Nucl.
Acids Res., 1992, 20, 533); an aliphatic chain, e.g., dodecandiol
or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al.,
Biochimie, 1993, 75, 49); a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or
triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al.,
Nucl. Acids Res., 1990, 18, 3777); a polyamine or a polyethylene
glycol chain (Manoharan et al., Nucleosides & Nucleotides,
1995, 14, 969); adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651); a palmityl moiety (Mishra et
al., Biochim. Biophys. Acta, 1995, 1264, 229); or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923).
[0261] Generally, a wide variety of entities, e.g., ligands, can be
coupled to the oligomeric compounds described herein. Ligands can
include naturally occurring molecules, or recombinant or synthetic
molecules. Exemplary ligands include, but are not limited to,
polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,
styrene-maleic acid anhydride copolymer,
poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic
anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer
(HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K,
PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl
alcohol (PVA), polyurethane, poly(2-ethylacryllic acid),
N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine,
cationic groups, spermine, spermidine, polyamine,
pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer
polyamine, arginine, amidine, protamine, cationic lipid, cationic
porphyrin, quaternary salt of a polyamine, thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, mucin,
glycosylated polyaminoacids, transferrin, bisphosphonate,
polyglutamate, polyaspartate, aptamer, asialofetuin, hyaluronan,
procollagen, immunoglobulins (e.g., antibodies), insulin,
transferrin, albumin, sugar-albumin conjugates, intercalating
agents (e.g., acridines), cross-linkers (e.g. psoralen, mitomycin
C), porphyrins (e.g., TPPC4, texaphyrin, Sapphyrin), polycyclic
aromatic hydrocarbons (e.g., phenazine, dihydrophenazine),
artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g,
steroids, bile acids, cholesterol, cholic acid, adamantane acetic
acid, 1-pyrene butyric acid, dihydrotestosterone,
1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl
group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,
O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine),
peptides (e.g., an alpha helical peptide, amphipathic peptide, RGD
peptide, cell permeation peptide, endosomolytic/fusogenic peptide),
alkylating agents, phosphate, amino, mercapto, polyamino, alkyl,
substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.
biotin), transport/absorption facilitators (e.g., naproxen,
aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g.,
imidazole, bisimidazole, histamine, imidazole clusters,
acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones
and hormone receptors, lectins, carbohydrates, multivalent
carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K,
vitamin B, e.g., folic acid, B12, riboflavin, biotin and
pyridoxal), vitamin cofactors, lipopolysaccharide, an activator of
p38 MAP kinase, an activator of NF-.kappa.B, taxon, vincristine,
vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin
A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis
factor alpha (TNFalpha), interleukin-1 beta, gamma interferon,
natural or recombinant low density lipoprotein (LDL), natural or
recombinant high-density lipoprotein (HDL), and a cell-permeation
agent (e.g., a. helical cell-permeation agent).
[0262] Peptide and peptidomimetic ligands include those having
naturally occurring or modified peptides, e.g., D or L peptides;
.alpha., .beta., or .gamma. peptides; N-methyl peptides;
azapeptides; peptides having one or more amide, i.e., peptide,
linkages replaced with one or more urea, thiourea, carbamate, or
sulfonyl urea linkages; or cyclic peptides. A peptidomimetic (also
referred to herein as an oligopeptidomimetic) is a molecule capable
of folding into a defined three-dimensional structure similar to a
natural peptide. The peptide or peptidomimetic ligand can be about
5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40,
45, or 50 amino acids long.
[0263] Exemplary amphipathic peptides include, but are not limited
to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like
peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides,
hagfish intestinal antimicrobial peptides (HFIAPs), magainines,
brevinins-2, dermaseptins, melittins, pleurocidin, H2A peptides,
Xenopus peptides, esculentinis-1, and caerins.
[0264] As used herein, the term "endosomolytic ligand" refers to
molecules having endosomolytic properties. Endosomolytic ligands
promote the lysis of and/or transport of the composition of the
invention, or its components, from the cellular compartments such
as the endosome, lysosome, endoplasmic reticulum (ER), golgi
apparatus, microtubule, peroxisome, or other vesicular bodies
within the cell, to the cytoplasm of the cell. Some exemplary
endosomolytic ligands include, but are not limited to, imidazoles,
poly or oligoimidazoles, linear or branched polyethyleneimines
(PEIs), linear and brached polyamines, e.g. spermine, cationic
linear and branched polyamines, polycarboxylates, polycations,
masked oligo or poly cations or anions, acetals, polyacetals,
ketals/polyketals, orthoesters, linear or branched polymers with
masked or unmasked cationic or anionic charges, dendrimers with
masked or unmasked cationic or anionic charges, polyanionic
peptides, polyanionic peptidomimetics, pH-sensitive peptides,
natural and synthetic fusogenic lipids, natural and synthetic
cationic lipids.
[0265] Exemplary endosomolytic/fusogenic peptides include, but are
not limited to, AALEALAEALEALAEALEALAEAAAAGGC (GALA);
AALAEALAEALAEALAEALAEALAAAAGGC (EALA); ALEALAEALEALAEA;
GLFEAIEGFIENGWEGMIWDYG (INF-7); GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2);
GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7);
GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3);
GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF);
GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3); GLF EAI EGFI ENGW
EGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, n is norleucine);
LFEALLELLESLWELLLEA (JTS-1); GLFKALLKLLKSLWKLLLKA (ppTG1);
GLFRALLRLLRSLWRLLLRA (ppTG20); WEAKLAKALAKALAKHLAKALAKALKACEA
(KALA); GLFFEAIAEFIEGGWEGLIEGC (HA); GIGAVLKVLTTGLPALISWIKRKRQQ
(Melittin); H.sub.5WYG; and CHK.sub.6HC.
[0266] Without wishing to be bound by theory, fusogenic lipids fuse
with and consequently destabilize a membrane. Fusogenic lipids
usually have small head groups and unsaturated acyl chains.
Exemplary fusogenic lipids include, but are not limited to,
1,2-dileoyl-sn-3-phosphoethanolamine (DOPE),
phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine
(POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol
(Di-Lin),
N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanam-
ine (DLin-k-DMA) and
N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethan-
amine (also refered to as XTC herein).
[0267] Synthetic polymers with endosomolytic activity amenable to
the present invention are described in U.S. Pat. App. Pub. Nos.
2009/0048410; 2009/0023890; 2008/0287630; 2008/0287628;
2008/0281044; 2008/0281041; 2008/0269450; 2007/0105804;
20070036865; and 2004/0198687, contents of which are hereby
incorporated by reference in their entirety.
[0268] Exemplary cell permeation peptides include, but are not
limited to, RQIKIWFQNRRMKWKK (penetratin); GRKKRRQRRRPPQC (Tat
fragment 48-60); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence based
peptide); LLIILRRRIRKQAHAHSK (PVEC); GWTLNSAGYLLKINLKALAALAKKIL
(transportan); KLALKLALKALKAALKLA (amphiphilic model peptide);
RRRRRRRRR (Arg9); KFFKFFKFFK (Bacterial cell wall permeating
peptide); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37);
SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1);
ACYCRIPACIAGERRYGTCIYQGRLWAFCC (.alpha.-defensin);
DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (.beta.-defensin);
RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39);
ILPWKWPWWPWRR-NH2 (indolicidin); AAVALLPAVLLALLAP (RFGF);
AALLPVLLAAP (RFGF analogue); and RKCRIVVIRVCR (bactenecin).
[0269] Exemplary cationic groups include, but are not limited to,
protonated amino groups, derived from e.g., O-AMINE
(AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene
diamine, polyamino); aminoalkoxy, e.g., O(CH.sub.2).sub.nAMINE,
(e.g., AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino); amino (e.g. NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, or amino acid); and
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino).
[0270] As used herein the term "targeting ligand" refers to any
molecule that provides an enhanced affinity for a selected target,
e.g., a cell, cell type, tissue, organ, region of the body, or a
compartment, e.g., a cellular, tissue or organ compartment. Some
exemplary targeting ligands include, but are not limited to,
antibodies, antigens, folates, receptor ligands, carbohydrates,
aptamers, integrin receptor ligands, chemokine receptor ligands,
transferrin, biotin, serotonin receptor ligands, PSMA, endothelin,
GCPII, somatostatin, LDL and HDL ligands.
[0271] Carbohydrate based targeting ligands include, but are not
limited to, D-galactose, multivalent galactose,
N-acetyl-D-galactose (GalNAc), multivalent GalNAc, e.g. GalNAc2 and
GalNAc3; D-mannose, multivalent mannose, multivalent lactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine, multivalent fucose,
glycosylated polyaminoacids and lectins. The term multivalent
indicates that more than one monosaccharide unit is present. Such
monosaccharide subunits can be linked to each other through
glycosidic linkages or linked to a scaffold molecule.
[0272] A number of folate and folate analogs amenable to the
present invention as ligands are described in U.S. Pat. Nos.
2,816,110; 51,410,104; 5,552,545; 6,335,434 and 7,128,893, contents
of which are herein incorporated in their entireties by
reference.
[0273] As used herein, the terms "PK modulating ligand" and "PK
modulator" refers to molecules which can modulate the
pharmacokinetics of the composition of the invention. Some
exemplary PK modulator include, but are not limited to, lipophilic
molecules, bile acids, sterols, phospholipid analogues, peptides,
protein binding agents, vitamins, fatty acids, phenoxazine,
aspirin, naproxen, ibuprofen, suprofen, ketoprofen,
(S)-(+)-pranoprofen, carprofen, PEGs, biotin, and
transthyretia-binding ligands (e.g., tetraiidothyroacetic acid, 2,
4, 6-triiodophenol and flufenamic acid). Oligomeric compounds that
comprise a number of phosphorothioate intersugar linkages are also
known to bind to serum protein, thus short oligomeric compounds,
e.g. oligonucleotides of comprising from about 5 to 30 nucleotides
(e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g.,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
nucleotides), and that comprise a plurality of phosphorothioate
linkages in the backbone are also amenable to the present invention
as ligands (e.g. as PK modulating ligands). The PK modulating
oligonucleotide can comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15 or more phosphorothioate and/or phosphorodithioate
linkages. In some embodiments, all internucleotide linkages in PK
modulating oligonucleotide are phosphorothioate and/or
phosphorodithioates linkages. In addition, aptamers that bind serum
components (e.g. serum proteins) are also amenable to the present
invention as PK modulating ligands. Binding to serum components
(e.g. serum proteins) can be predicted from albumin binding assays,
such as those described in Oravcova, et al., Journal of
Chromatography B (1996), 677: 1-27.
[0274] When two or more ligands are present, the ligands can all
have same properties, all have different properties or some ligands
have the same properties while others have different properties.
For example, a ligand can have targeting properties, have
endosomolytic activity or have PK modulating properties. In a
preferred embodiment, all the ligands have different
properties.
[0275] In some embodiments, ligand on one strand of a
double-stranded oligomeric compound has affinity for a ligand on
the second strand. In some embodiments, a ligand is covalently
linked to both strands of a double-stranded oligomeric compound. As
used herein, when a ligand is linked to more than oligomeric
strand, point of attachment for an oligomeric compound can be an
atom of the ligand self or an atom on a carrier molecule to which
the ligand itself is attached.
[0276] Ligands can be coupled to the oligomeric compounds at
various places, for example, 3'-end, 5'-end, and/or at an internal
position. When two or more ligands are present, the ligand can be
on opposite ends of an oligomeric compound. In preferred
embodiments, the ligand is attached to the oligomeric compound via
an intervening tether/linker. The ligand or tethered ligand can be
present on a monomer when said monomer is incorporated into the
growing strand. In some embodiments, the ligand can be incorporated
via coupling to a "precursor" monomer after said "precursor"
monomer has been incorporated into the growing strand. For example,
a monomer having, e.g., an amino-terminated tether (i.e., having no
associated ligand), e.g., monomer-linker-NH.sub.2 can be
incorporated into a growing oligomeric compound strand. In a
subsequent operation, i.e., after incorporation of the precursor
monomer into the strand, a ligand having an electrophilic group,
e.g., a pentafluorophenyl ester or aldehyde group, can subsequently
be attached to the precursor monomer by coupling the electrophilic
group of the ligand with the terminal nucleophilic group of the
precursor monomer's tether.
[0277] In another example, a monomer having a chemical group
suitable for taking part in Click Chemistry reaction can be
incorporated e.g., an azide or alkyne terminated tether/linker. In
a subsequent operation, i.e., after incorporation of the precursor
monomer into the strand, a ligand having complementary chemical
group, e.g. an alkyne or azide can be attached to the precursor
monomer by coupling the alkyne and the azide together.
[0278] For double-stranded oligomeric compounds, ligands can be
attached to one or both strands. In some embodiments, an siRNA
comprises a ligand conjugated to the sense strand. In other
embodiments, an siRNA comprises a ligand conjugated to the
antisense strand.
[0279] In some embodiments, ligand can be conjugated to
nucleobases, sugar moieties, or internucleosidic linkages of
oligomeric compound. Conjugation to purine nucleobases or
derivatives thereof can occur at any position including, endocyclic
and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or
8-positions of a purine nucleobase are attached to a conjugate
moiety. Conjugation to pyrimidine nucleobases or derivatives
thereof can also occur at any position. In some embodiments, the
2-, 5-, and 6-positions of a pyrimidine nucleobase can be
substituted with a conjugate moiety. When a ligand is conjugated to
a nucleobase, the preferred position is one that does not interfere
with hybridization, i.e., does not interfere with the hydrogen
bonding interactions needed for base pairing.
[0280] Conjugation to sugar moieties of nucleosides can occur at
any carbon atom. Example carbon atoms of a sugar moiety that can be
attached to a conjugate moiety include the 2', 3', and 5' carbon
atoms. The 1' position can also be attached to a conjugate moiety,
such as in an abasic residue. Internucleosidic linkages can also
bear conjugate moieties. For phosphorus-containing linkages (e.g.,
phosphodiester, phosphorothioate, phosphorodithiotate,
phosphoroamidate, and the like), the conjugate moiety can be
attached directly to the phosphorus atom or to an O, N, or S atom
bound to the phosphorus atom. For amine- or amide-containing
internucleosidic linkages (e.g., PNA), the conjugate moiety can be
attached to the nitrogen atom of the amine or amide or to an
adjacent carbon atom.
[0281] Inventors have discovered inter alia that REVERSIR compounds
conjugated with a ligand are particularly effective in reducing
activity of siRNAs. Without wishing to be bound by a theory, a
ligand can increase or enhance the ability of a REVERSIR compound
by delivering the REVERSIR compound to the desired location of
action. Accordingly, in some embodiments, the REVERSIR compound is
conjugated with a ligand.
[0282] While useful in delivery of the REVERSIR compound to a
desired location of action, the ligand conjugated with the REVERSIR
compound can negatively affect the ability of the REVERSIR compound
to reduce siRNA activity. Therefore, in some embodiments, the
linkage between the ligand and the REVERSIR compound can be
designed to undergo cleavage after the REVERSIR compound reaches a
desired location of action. This can be accomplished in a number of
ways. For example, the linker connecting the REVERSIR compound to
the ligand can be a cleavable linker.
[0283] The inventors have also discovered that the nucleoside in
the REVERSIR compound that is connected with the ligand can have an
effect on the ability of the REVERSIR compound to reduce activity
of the siRNA. Inventors have discovered that ligand conjugated
nucleosides comprising deoxy sugars (e.g., 2'-deoxy ribose) are
particularly effective in enhancing the ability of REVERSIR
compounds to reduce siRNA activity. Accordingly, in some
embodiments, the nucleoside conjugated with the ligand comprises a
deoxy sugar, for example, a 2'-deoxy sugar.
[0284] In some embodiments of the various aspects disclosed herein,
the ligand is attached to the nucleoside at the 3'-terminus of the
REVERSIR compound. The inventors have discovered inter alia that
internucleotide linkage between the ligand conjugated nucleotide
and the rest of the REVERSIR compound can also have an effect on
the ability of the REVERSIR compound to reduce siRNA activity.
Without wishing to be bound by a theory, readily cleavable
internucleotide linkages were found to be particularly effective in
enhancing the ability of REVERSIR compounds to reduce siRNA
activity. Accordingly, in some embodiments, the ligand conjugated
nucleotide is attached to the rest of the REVERSIR compound via a
cleavable internucleotide linage. In some embodiment, the cleavable
internucleotide linkage is a phosphodiester internucleotide
linkage.
[0285] In some embodiments, the ligand conjugated nucleotide
comprises a deoxy sugar and is linked to rest of the REVERSIR
compound via a cleavable internucleotide linkage. In some further
embodiments, of this the cleavable internucleotide linkage is a
phosphodiester linkage.
[0286] In some embodiments, the ligand conjugated nucleotide
comprises a deoxy sugar and is linked to rest of the REVERSIR
compound via an internucleotide linkage that is not a
phosphodiester linkage.
[0287] In some embodiments, the ligand is conjugated to the
nucleotide at the 3'-terminus of the REVERSIR compound.
[0288] In some embodiments, the ligand is conjugated at the
5'-terminus of the REVERSIR compound. In some embodiments, a first
ligand is conjugated at the 5'-terminus of the REVERSIR compound
and a second ligand conjugated to the first ligand.
[0289] There are numerous methods for preparing conjugates of
oligomeric compounds. Generally, an oligomeric compound is attached
to a conjugate moiety by contacting a reactive group (e.g., OH, SH,
amine, carboxyl, aldehyde, and the like) on the oligomeric compound
with a reactive group on the conjugate moiety. In some embodiments,
one reactive group is electrophilic and the other is
nucleophilic.
[0290] For example, an electrophilic group can be a
carbonyl-containing functionality and a nucleophilic group can be
an amine or thiol. Methods for conjugation of nucleic acids and
related oligomeric compounds with and without linking groups are
well described in the literature such as, for example, in Manoharan
in Antisense Research and Applications, Crooke and LeBleu, eds.,
CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is
incorporated herein by reference in its entirety.
[0291] Representative U.S. patents that teach the preparation of
conjugates of oligomeric compounds, e.g., oligonucleotides,
include, but are not limited to, U.S. Pat. Nos. 4,828,979;
4,948,882; 5,218, 105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;
5,578, 717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118, 802;
5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578, 718; 5,608,046;
4,587,044; 4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941;
4,835,263; 4,876,335; 4,904, 582; 4,958,013; 5,082,830; 5,112,963;
5,214,136; 5,082, 830; 5,112,963; 5,149,782; 5,214,136; 5,245,022;
5,254, 469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;
5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510, 475; 5,512,667;
5,514,785; 5,565,552; 5,567,810; 5,574, 142; 5,585,481; 5,587,371;
5,595,726; 5,597,696; 5,599, 923; 5,599,928; 5,672,662; 5,688,941;
5,714,166; 6,153, 737; 6,172,208; 6,300,319; 6,335,434; 6,335,437;
6,395, 437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; 6,559, 279;
contents of which are herein incorporated in their entireties by
reference.
[0292] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand having a structure shown below:
##STR00031##
wherein: [0293] L.sup.G is independently for each occurrence a
ligand, e.g., carbohydrate, e.g. monosaccharide, disaccharide,
trisaccharide, tetrasaccharide, polysaccharide; and [0294] Z', Z'',
Z''' and Z'''' are each independently for each occurrence O or
S.
[0295] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of Formula (II), (III), (IV) or (V):
##STR00032##
[0296] wherein:
[0297] q.sup.2A, q.sup.2B, q.sup.3A, q.sup.3B, q4.sup.A, q.sup.4B,
q.sup.5A, q.sup.5B and q.sup.5C represent independently for each
occurrence 0-20 and wherein the repeating unit can be the same or
different;
Q and Q' are independently for each occurrence is absent,
--(P.sup.7-Q.sup.7-R.sup.7).sub.p-T.sup.7- or
-T.sup.7-Q.sup.7-T.sup.7'-B-T.sup.8'-Q.sup.8-T.sup.8; P.sup.2A,
P.sup.2B, P.sup.3A, P.sup.3B, P.sup.4A, P.sup.4B, P.sup.5A,
P.sup.5B, P.sup.5C, P.sup.7, T.sup.2A, T.sup.2B, T.sup.3A,
T.sup.3B, T.sup.4A, T.sup.4B, T.sup.4A, T.sup.5B, T.sup.5C,
T.sup.7, T.sup.7', T.sup.8 and T.sup.8' are each independently for
each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2,
CH.sub.2NH or CH.sub.2O; B is --CH.sub.2--N(B.sup.L)--CH.sub.2--;
B.sup.L is -T.sup.B-Q.sup.B-T.sup.B'-R.sup.x;
[0298] Q.sup.2A, Q.sup.2B, Q.sup.3A, Q.sup.3B, Q.sup.4A, Q.sup.4B,
Q.sup.5A, Q.sup.5B, Q.sup.5C, Q.sup.7, Q.sup.8 and Q.sup.B are
independently for each occurrence absent, alkylene, substituted
alkylene and wherein one or more methylenes can be interrupted or
terminated by one or more of O, S, S(O), SO.sub.2, N(R.sup.N),
C(R').dbd.C(R'), C.ident.C or C(O);
[0299] T.sup.B and T.sup.B' are each independently for each
occurrence absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH,
NHC(O)O, CH.sub.2, CH.sub.2NH or CH.sub.2O;
[0300] R.sup.x is a lipophile (e.g., cholesterol, cholic acid,
adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,
1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl
group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,
O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a
vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a
peptide, a carbohydrate (e.g., monosaccharide, disaccharide,
trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide),
an endosomolytic component, a steroid (e.g., uvaol, hecigenin,
diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin,
Friedelin, epifriedelanol derivatized lithocholic acid), or a
cationic lipid;
[0301] R.sup.1, R.sup.2, R.sup.2A, R.sup.2B, R.sup.3A, R.sup.3B,
R.sup.4A, R.sup.4B, R.sup.5A, R.sup.5B, R.sup.5C, R.sup.7 are each
independently for each occurrence absent, NH, O, S, CH.sub.2,
C(O)O, C(O)NH, NHCH(R.sup.a)C(O), --C(O)--CH(R.sup.a)--NH--, CO,
CH.dbd.N--O,
##STR00033##
or heterocyclyl;
[0302] L.sup.1, L.sup.2A, L.sup.2B, L.sup.3A, L.sup.3B, L.sup.4A,
L.sup.4B, L.sup.5A, L.sup.5B and L.sup.5C are each independently
for each occurrence a carbohydrate, e.g., monosaccharide,
disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and
polysaccharide;
[0303] R' and R'' are each independently H, C.sup.1-C.sub.6 alkyl,
OH, SH, or N(R.sup.N).sub.2;
[0304] R.sup.N is independently for each occurrence H, methyl,
ethyl, propyl, isopropyl, butyl or benzyl;
[0305] R.sup.a is H or amino acid side chain;
[0306] Z', Z'', Z''' and Z'''' are each independently for each
occurrence O or S; p represent independently for each occurrence
0-20.
[0307] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00034##
[0308] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00035##
[0309] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00036##
[0310] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00037##
[0311] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00038##
[0312] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00039##
[0313] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00040##
[0314] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00041##
[0315] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00042##
[0316] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00043##
[0317] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00044##
[0318] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00045##
[0319] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00046##
[0320] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00047##
[0321] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00048##
[0322] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00049##
[0323] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00050##
[0324] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00051##
[0325] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00052##
[0326] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00053##
[0327] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00054##
[0328] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00055##
[0329] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00056##
[0330] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00057##
[0331] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00058##
[0332] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00059##
[0333] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00060##
[0334] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00061##
[0335] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00062##
[0336] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00063##
[0337] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00064##
[0338] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00065##
[0339] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00066##
[0340] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00067##
[0341] In some embodiments both L.sup.2A and L.sup.2B are
different.
[0342] In some preferred embodiments both L.sup.3A and L.sup.3B are
the same.
[0343] In some embodiments both L.sup.3A and L.sup.3B are
different.
[0344] In some preferred embodiments both L.sup.4A and L.sup.4B are
the same.
[0345] In some embodiments both L.sup.4A and L.sup.4B are
different.
[0346] In some preferred embodiments all of L.sup.5A, L.sup.5B and
L.sup.5C are the same.
[0347] In some embodiments two of L.sup.5A, L.sup.5B and L.sup.5C
are the same
[0348] In some embodiments L.sup.5A and L.sup.5B are the same.
[0349] In some embodiments L.sup.5A and L.sup.5C are the same.
[0350] In some embodiments L.sup.5B and L.sup.5C are the same.
[0351] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00068##
[0352] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00069##
[0353] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00070##
[0354] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00071##
wherein Y is O or S and n is 3-6.
[0355] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00072##
wherein Y is O or S and n is 3-6.
[0356] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00073##
[0357] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00074##
wherein X is O or S.
[0358] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer selected from the group consisting of:
##STR00075##
[0359] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00076##
wherein R is OH or NHCOOH.
[0360] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00077##
wherein R is OH or NHCOOH.
[0361] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00078##
wherein R is O or S.
[0362] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00079##
wherein R is OH or NHCOOH.
[0363] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00080##
[0364] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00081##
where in R is OH or NHCOOH.
[0365] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00082##
wherein R is OH or NHCOOH.
[0366] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00083##
wherein R is OH or NHCOOH.
[0367] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00084##
wherein R is OH or.
[0368] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a monomer of structure:
##STR00085##
[0369] In the above described monomers, X and Y are each
independently for each occurrence H, a protecting group, a
phosphate group, a phosphodiester group, an activated phosphate
group, an activated phosphite group, a phosphoramidite, a solid
support, --P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a
lipid, a PEG, a steroid, a polymer, a nucleotide, a nucleoside, or
an oligonucleotide; and Z' and Z'' are each independently for each
occurrence O or S.
[0370] In certain embodiments, the REVERSIR compound is conjugated
with a ligand of structure:
##STR00086##
[0371] In certain embodiments, the conjugated siRNA comprises a
ligand of structure:
##STR00087##
[0372] In certain embodiments, the REVERSIR compound comprises a
monomer of structure:
##STR00088##
[0373] Synthesis of above described ligands and monomers is
described, for example, in U.S. Pat. No. 8,106,022, content of
which is incorporated herein by reference in its entirety.
[0374] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand of structure:
##STR00089##
[0375] In certain embodiments, the oligomeric compound described
herein, including but not limited to REVERSIR compounds and siRNAs,
comprises a ligand from those described in U.S. Pat. No. 9,181,549
to Prakash et al., the content of which is incorporated herein by
reference in its entirety.
[0376] Linking groups or bifunctional linking moieties such as
those known in the art are amenable to the compounds provided
herein. Linking groups are useful for attachment of chemical
functional groups, conjugate groups, reporter groups and other
groups to selective sites in a parent compound such as for example
an oligomeric compound. In general a bifunctional linking moiety
comprises a hydrocarbyl moiety having two functional groups. One of
the functional groups is selected to bind to a parent molecule or
compound of interest and the other is selected to bind essentially
any selected group such as chemical functional group or a conjugate
group. In some embodiments, the linker comprises a chain structure
or an oligomer of repeating units such as ethylene glycol or amino
acid units. Examples of functional groups that are routinely 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 some
embodiments, bifunctional linking moieties include amino, hydroxyl,
carboxylic acid, thiol, unsaturations (e.g., double or triple
bonds), and the like. Some nonlimiting examples of bifunctional
linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO),
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)
and 6-aminohexanoic acid (AHEX or AHA). Other linking groups
include, but are not limited to, substituted C1-C10 alkyl,
substituted or unsubstituted C2-C10 alkenyl or substituted or
unsubstituted C2-C10 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.
[0377] In certain embodiments, the ligand is conjugated with the
oligomeric compound via a linker.
[0378] As used herein, the term "linker" means an organic moiety
that connects two parts of a compound. Linkers typically comprise a
direct bond or an atom such as oxygen or sulfur, a unit such as
NR.sup.1, C(O), C(O)NH, SO, SO.sub.2, SO.sub.2NH or a chain of
atoms, such as substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl,
heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl,
heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl,
heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,
alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,
alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,
alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,
alkylheteroarylalkenyl, alkylheteroarylalkynyl,
alkenylheteroarylalkyl, alkenylheteroarylalkenyl,
alkenylheteroarylalkynyl, alkynylheteroarylalkyl,
alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
alkylheterocyclylalkyl, alkylheterocyclylalkenyl,
alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,
alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,
alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,
alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or
more methylenes can be interrupted or terminated by O, S, S(O),
SO.sub.2, N(R.sup.1).sub.2, C(O), cleavable linking group,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocyclic; where
R.sup.1 is hydrogen, acyl, aliphatic or substituted aliphatic.
[0379] In one embodiment, the linker is
--[(P-Q''-R).sub.q--X--(P'--Q'''--R').sub.q'].sub.q''-T-, wherein:
P, R, T, P', R' and T are each independently for each occurrence
absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2, CH.sub.2NH,
CH.sub.2O; NHCH(Ra)C(O), --C(O)--CH(Ra)--NH--, CH.dbd.N--O,
##STR00090##
or heterocyclyl; Q'' and Q''' are each independently for each
occurrence absent, --(CH.sub.2).sub.n--,
--C(R.sup.1)(R.sup.2)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R.sup.1)(R.sup.2)--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--; X is absent or a
cleavable linking group; R.sup.a is H or an amino acid side chain;
R.sup.1 and R.sup.2 are each independently for each occurrence H,
CH.sub.3, OH, SH or N(R.sup.N).sub.2; R.sup.N is independently for
each occurrence H, methyl, ethyl, propyl, isopropyl, butyl or
benzyl; q, q' and q'' are each independently for each occurrence
0-20 and wherein the repeating unit can be the same or different; n
is independently for each occurrence 1-20; and m is independently
for each occurrence 0-50.
[0380] In some embodiments, the linker comprises at least one
cleavable linking group.
[0381] In some embodiments, the linker is a branched linker. The
branchpoint of the branched linker may be at least trivalent, but
can be a tetravalent, pentavalent or hexavalent atom, or a group
presenting such multiple valencies. In some embodiments, the
branchpoint is, --N, --N(Q)-C, --O--C, --S--C, --SS--C,
--C(O)N(Q)-C, --OC(O)N(Q)-C, --N(Q)C(O)--C, or --N(Q)C(O)O--C;
wherein Q is independently for each occurrence H or optionally
substituted alkyl. In some embodiments, the branchpoint is glycerol
or derivative thereof.
[0382] A cleavable linking group is one which is sufficiently
stable outside the cell, but which upon entry into a target cell is
cleaved to release the two parts the linker is holding together. In
a preferred embodiment, the cleavable linking group is cleaved at
least 10 times or more, preferably at least 100 times faster in the
target cell or under a first reference condition (which can, e.g.,
be selected to mimic or represent intracellular conditions) than in
the blood or serum of a subject, or under a second reference
condition (which can, e.g., be selected to mimic or represent
conditions found in the blood or serum).
[0383] Cleavable linking groups are susceptible to cleavage agents,
e.g., pH, redox potential or the presence of degradative molecules.
Generally, cleavage agents are more prevalent or found at higher
levels or activities inside cells than in serum or blood. Examples
of such degradative agents include: redox agents which are selected
for particular substrates or which have no substrate specificity,
including, e.g., oxidative or reductive enzymes or reductive agents
such as mercaptans, present in cells, that can degrade a redox
cleavable linking group by reduction; esterases; amidases;
endosomes or agents that can create an acidic environment, e.g.,
those that result in a pH of five or lower; enzymes that can
hydrolyze or degrade an acid cleavable linking group by acting as a
general acid, peptidases (which can be substrate specific) and
proteases, and phosphatases.
[0384] A linker can include a cleavable linking group that is
cleavable by a particular enzyme. The type of cleavable linking
group incorporated into a linker can depend on the cell to be
targeted. For example, liver targeting ligands can be linked to the
cationic lipids through a linker that includes an ester group.
Liver cells are rich in esterases, and therefore the linker will be
cleaved more efficiently in liver cells than in cell types that are
not esterase-rich. Other cell-types rich in esterases include cells
of the lung, renal cortex, and testis.
[0385] Linkers that contain peptide bonds can be used when
targeting cell types rich in peptidases, such as liver cells and
synoviocytes.
[0386] In some embodiments, cleavable linking group is cleaved at
least 1.25, 1.5, 1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster
in the cell (or under in vitro conditions selected to mimic
intracellular conditions) as compared to blood or serum (or under
in vitro conditions selected to mimic extracellular conditions). In
some embodiments, the cleavable linking group is cleaved by less
than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% in the
blood (or in vitro conditions selected to mimic extracellular
conditions) as compared to in the cell (or under in vitro
conditions selected to mimic intracellular conditions).
[0387] Exemplary cleavable linking groups include, but are not
limited to, redox cleavable linking groups (e.g., --S--S-- and
--C(R).sub.2--S--S--, wherein R is H or C1-C6 alkyl and at least
one R is C.sub.1-C.sub.6 alkyl such as CH.sub.3 or
CH.sub.2CH.sub.3); phosphate-based cleavable linking groups (e.g.,
--O--P(O)(OR)--O--, --O--P(S)(OR)--O--, --O--P(S)(SR)--O--,
--S--P(O)(OR)--O--, --O--P(O)(OR)--S--, --S--P(O)(OR)--S--,
--O--P(S)(ORk)-S--, --S--P(S)(OR)--O--, --O--P(O)(R)--O--,
--O--P(S)(R)--O--, --S--P(O)(R)--O--, --S--P(S)(R)--O--,
--S--P(O)(R)--S--, --O--P(S)(R)--S--, --O--P(O)(OH)--O--,
--O--P(S)(OH)--O--, --O--P(S)(SH)--O--, --S--P(O)(OH)--O--,
--O--P(O)(OH)--S--, --S--P(O)(OH)--S--, --O--P(S)(OH)--S--,
--S--P(S)(OH)--O--, --O--P(O)(H)--O--, --O--P(S)(H)--O--,
--S--P(O)(H)--O--, --S--P(S)(H)--O--, --S--P(O)(H)--S--, and
--O--P(S)(H)--S--, wherein R is optionally substituted linear or
branched C.sub.1-C.sub.10 alkyl); acid celavable linking groups
(e.g., hydrazones, esters, and esters of amino acids, --C.dbd.NN--
and --OC(O)--); ester-based cleavable linking groups (e.g.,
--C(O)O--); peptide-based cleavable linking groups, (e.g., linking
groups that are cleaved by enzymes such as peptidases and proteases
in cells, e.g., --NHCHR.sup.AC(O)NHCHR.sup.BC(O)--, where R.sup.A
and RB are the R groups of the two adjacent amino acids). A peptide
based cleavable linking group comprises two or more amino acids. In
some embodiments, the peptide-based cleavage linkage comprises the
amino acid sequence that is the substrate for a peptidase or a
protease found in cells.
[0388] In some embodiments, an acid cleavable linking group is
cleaveable in an acidic environment with a pH od about 6.5 or lower
(e.g., about 6-, 5.5, 5.0, or lower), or by agents such as enzymes
that can act as a general acid.
[0389] In some embodiments, the linker is an oligonucleotide linker
including, but not limited to, (N).sub.n; wherein N is
independently a modified or unmodified nucleotide and n is 1-23. In
some embodiments, n is 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10. In some embodiments, the oligonucleotide linker is selected
from the group consisting of GNRA, (G).sub.4, (U).sub.4, and
(dT).sub.4, wherein N is a modified or unmodified nucleotide and R
is a modified or unmodified purine nucleotide. Some of the
nucleotides in the linker can be involved in base-pair interactions
with other nucleotides in the linker. It will be appreciated by one
of skill in the art that any oligonucleotide chemical modifications
or variations describe herein can be used in the oligonucleotide
linker. In certain embodiments, the linker is dA.
Motifs
[0390] The present invention also includes oligomeric compounds
which are chimeric oligomeric compounds. "Chimeric" oligomeric
compounds or "chimeras," in the context of this invention, are
oligomeric compounds which contain two or more chemically distinct
regions, each made up of at least one monomer unit, i.e., a
modified or unmodified nucleotide in the case of an
oligonucleotide. Chimeric oligomeric compounds can be described as
having a particular motif. In some embodiments, the motifs include,
but are not limited to, an alternating motif, a gapped motif, a
hemimer motif, a uniformly fully modified motif and a positionally
modified motif. As used herein, the phrase "chemically distinct
region" refers to an oligomeric region which is different from
other regions by having a modification that is not present
elsewhere in the oligomeric compound or by not having a
modification that is present elsewhere in the oligomeric compound.
An oligomeric compound can comprise two or more chemically distinct
regions. As used herein, a region that comprises no modifications
is also considered chemically distinct.
[0391] A chemically distinct region can be repeated within an
oligomeric compound. Thus, a pattern of chemically distinct regions
in an oligomeric compound can be realized such that a first
chemically distinct region is followed by one or more second
chemically distinct regions. This sequence of chemically distinct
regions can be repeated one or more times. Preferably, the sequence
is repeated more than one time. Both strands of a double-stranded
oligomeric compound can comprise these sequences. Each chemically
distinct region can actually comprise as little as a single
monomers, e.g., nucleotides. In some embodiments, each chemically
distinct region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17 or 18 monomers, e.g., nucleotides.
[0392] In some embodiments, alternating nucleotides comprise the
same modification, e.g. all the odd number nucleotides in a strand
have the same modification and/or all the even number nucleotides
in a strand have the similar modification to the first strand. In
some embodiments, all the odd number nucleotides in an oligomeric
compound have the same modification and all the even numbered
nucleotides have a modification that is not present in the odd
number nucleotides and vice versa.
[0393] When both strands of a double-stranded oligomeric compound
comprise the alternating modification patterns, nucleotides of one
strand can be complementary in position to nucleotides of the
second strand which are similarly modified. In an alternative
embodiment, there is a phase shift between the patterns of
modifications of the first strand, respectively, relative to the
pattern of similar modifications of the second strand. Preferably,
the shift is such that the similarly modified nucleotides of the
first strand and second strand are not in complementary position to
each other.
[0394] In some embodiments, the first strand has an alternating
modification pattern wherein alternating nucleotides comprise a
2'-modification, e.g., 2'-O-Methyl modification. In some
embodiments, the first strand comprises an alternating 2'-O-Methyl
modification and the second strand comprises an alternating
2'-fluoro modification. In other embodiments, both strands of a
double-stranded oligonucleotide comprise alternating 2'-O-methyl
modifications.
[0395] When both strands of a double-stranded oligonucleotide
comprise alternating 2'-O-methyl modifications, such 2'-modified
nucleotides can be in complementary position in the duplex region.
Alternatively, such 2'-modified nucleotides may not be in
complementary positions in the duplex region.
[0396] In some embodiments, the oligonucleotide comprises two
chemically distinct regions, wherein each region is 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10 nucleotides in length.
[0397] In other embodiments, the oligomeric compound comprises
three chemically distinct region. The middle region is about 5-15,
(e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) nucleotide in
length and each flanking or wing region is independently 1-10
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) nucleotides in length. All
three regions can have different modifications or the wing regions
can be similarly modified to each other. In some embodiments, the
wing regions are of equal length, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or
10 nucleotides long.
[0398] As used herein the term "alternating motif" refers to an
oligomeric compound comprising a contiguous sequence of linked
monomer subunits wherein the monomer subunits have two different
types of sugar groups that alternate for essentially the entire
sequence of the oligomeric compound. Oligomeric compounds having an
alternating motif can be described by the formula:
5'-A(-L-B-L-A)n(-L-B)nn-3' where A and B are monomelic subunits
that have different sugar groups, each L is an internucleoside
linking group, n is from about 4 to about 12 and nn is 0 or 1. This
permits alternating oligomeric compounds from about 9 to about 26
monomer subunits in length. This length range is not meant to be
limiting as longer and shorter oligomeric compounds are also
amenable to the present invention. In one embodiment, one of A and
B is a 2'-modified nucleoside as provided herein.
[0399] As used herein, "type of modification" in reference to a
nucleoside or a nucleoside of a "type" refers to the modification
of a nucleoside and includes modified and unmodified nucleosides.
Accordingly, unless otherwise indicated, a "nucleoside having a
modification of a first type" may be an unmodified nucleoside.
[0400] As used herein, "type region" refers to a portion of an
oligomeric compound wherein the nucleosides and internucleoside
linkages within the region all comprise the same type of
modifications; and the nucleosides and/or the internucleoside
linkages of any neighboring portions include at least one different
type of modification. As used herein the term "uniformly fully
modified motif" refers to an oligonucleotide comprising a
contiguous sequence of linked monomer subunits that each have the
same type of sugar group. In one embodiment, the uniformly fully
modified motif includes a contiguous sequence of nucleosides of the
invention. In one embodiment, one or both of the 3' and 5'-ends of
the contiguous sequence of the nucleosides provided herein,
comprise terminal groups such as one or more unmodified
nucleosides.
[0401] As used herein the term "hemimer motif" refers to an
oligomeric compound having a short contiguous sequence of monomer
subunits having one type of sugar group located at the 5' or the 3'
end wherein the remainder of the monomer subunits have a different
type of sugar group. In general, a hemimer is an oligomeric
compound of uniform sugar groups further comprising a short region
(1, 2, 3, 4 or about 5 monomelic subunits) having uniform but
different sugar groups and located on either the 3' or the 5' end
of the oligomeric compound. In one embodiment, the hemimer motif
comprises a contiguous sequence of from about 10 to about 28
monomer subunits of one type with from 1 to 5 or from 2 to about 5
monomer subunits of a second type located at one of the termini. In
one embodiment, a hemimer is a contiguous sequence of from about 8
to about 20 .beta.-D-2'-deoxyribonucleosides having from 1-12
contiguous nucleosides of the invention located at one of the
termini. In one embodiment, a hemimer is a contiguous sequence of
from about 8 to about 20 .beta.-D-2'-deoxyribonucleosides having
from 1-5 contiguous nucleosides of the invention located at one of
the termini. In one embodiment, a hemimer is a contiguous sequence
of from about 12 to about 18 .beta.-D-2'-deoxyribo-nucleosides
having from 1-3 contiguous nucleosides of the invention located at
one of the termini. In one embodiment, a hemimer is a contiguous
sequence of from about 10 to about 14
.beta.-D-2'-deoxyribonucleosides having from 1-3 contiguous
nucleosides of the invention located at one of the termini.
[0402] As used herein the term "blockmer motif" refers to an
oligonucleotide comprising an otherwise contiguous sequence of
monomer subunits wherein the sugar groups of each monomer subunit
is the same except for an interrupting internal block of contiguous
monomer subunits having a different type of sugar group. A blockmer
overlaps somewhat with a gapmer in the definition but typically
only the monomer subunits in the block have non-naturally occurring
sugar groups in a blockmer and only the monomer subunits in the
external regions have non-naturally occurring sugar groups in a
gapmer with the remainder of monomer subunits in the blockmer or
gapmer being .beta.-D-2'-deoxyribonucleosides or
.beta.-D-ribonucleosides. In one embodiment, blockmer
oligonucleotides are provided herein wherein all of the monomer
subunits comprise non-naturally occurring sugar groups.
[0403] As used herein the term "positionally modified motif" is
meant to include an otherwise contiguous sequence of monomer
subunits having one type of sugar group that is interrupted with
two or more regions of from 1 to about 5 contiguous monomer
subunits having another type of sugar group. Each of the two or
more regions of from 1 to about 5 contiguous monomer subunits are
independently uniformly modified with respect to the type of sugar
group. In one embodiment, each of the two or more regions have the
same type of sugar group. In one embodiment, each of the two or
more regions have a different type of sugar group. In one
embodiment, positionally modified oligonucleotides are provided
comprising a sequence of from 8 to 20
.beta.-D-2'-deoxyribonucleosides that further includes two or three
regions of from 2 to about 5 contiguous nucleosides of the
invention. Positionally modified oligonucleotides are distinguished
from gapped motifs, hemimer motifs, blockmer motifs and alternating
motifs because the pattern of regional substitution defined by any
positional motif does not fit into the definition provided herein
for one of these other motifs. The term positionally modified
oligomeric compound includes many different specific substitution
patterns.
[0404] As used herein the term "gapmer" or "gapped oligomeric
compound" refers to an oligomeric compound having two external
regions or wings and an internal region or gap. The three regions
form a contiguous sequence of monomer subunits with the sugar
groups of the external regions being different than the sugar
groups of the internal region and wherein the sugar group of each
monomer subunit within a particular region is the same. When the
sugar groups of the external regions are the same the gapmer is a
symmetric gapmer and when the sugar group used in the 5'-external
region is different from the sugar group used in the 3'-external
region, the gapmer is an asymmetric gapmer. In one embodiment, the
external regions are small (each independently 1, 2, 3, 4 or about
5 monomer subunits) and the monomer subunits comprise non-naturally
occurring sugar groups with the internal region comprising
.beta.-D-2'-deoxyribonucleosides. In one embodiment, the external
regions each, independently, comprise from 1 to about 5 monomer
subunits having non-naturally occurring sugar groups and the
internal region comprises from 6 to 18 unmodified nucleosides. The
internal region or the gap generally comprises
.beta.-D-2'-deoxyribo-nucleosides but can comprise non-naturally
occurring sugar groups.
[0405] In one embodiment, the gapped oligomeric compounds comprise
an internal region of .beta.-D-2'-deoxyribonucleosides with one of
the external regions comprising nucleosides of the invention. In
one embodiment, the gapped oligonucleotide comprise an internal
region of .beta.-D-2'-deoxyribonucleosides with both of the
external regions comprising nucleosides of the invention. In one
embodiment, the gapped oligonucleotide comprise an internal region
of .beta.-D-2'-deoxyribonucleosides with both of the external
regions comprising nucleosides of the invention. In one embodiment,
gapped oligonucleotides are provided herein wherein all of the
monomer subunits comprise non-naturally occurring sugar groups. In
one embodiment, gapped oliogonucleotides are provided comprising
one or two nucleosides of the invention at the 5'-end, two or three
nucleosides of the invention at the 3'-end and an internal region
of from 10 to 16 .beta.-D-2'-deoxyribonucleosides. In one
embodiment, gapped oligonucleotides are provided comprising one
nucleoside of the invention at the 5'-end, two nucleosides of the
invention at the 3'-end and an internal region of from 10 to 16
.beta.-D-2'-deoxyribonucleosides. In one embodiment, gapped
oligonucleotides are provided comprising two nucleosides of the
invention at the 5'-end, two nucleosides of the invention at the
3'-end and an internal region of from 10 to 14
.beta.-D-2'-deoxyribonucleosides. In one embodiment, gapped
oligonucleotides are provided that are from about 10 to about 21
monomer subunits in length. In one embodiment, gapped
oligonucleotides are provided that are from about 12 to about 16
monomer subunits in length. In one embodiment, gapped
oligonucleotides are provided that are from about 12 to about 14
monomer subunits in length.
[0406] In certain embodiments, the 5'-terminal monomer of an
oligomeric compound of the invention comprises a phosphorous moiety
at the 5'-end. In certain embodiments the 5'-terminal monomer
comprises a 2'-modification. In certain such embodiments, the
2'-modification of the 5'-terminal monomer is a cationic
modification. In certain embodiments, the 5'-terminal monomer
comprises a 5'-modification. In certain embodiments, the
5'-terminal monomer comprises a 2'-modification and a
5'-modification. In certain embodiments, the 5'-terminal monomer is
a 5'-stabilizing nucleoside. In certain embodiments, the
modifications of the 5'-terminal monomer stabilize the
5'-phosphate. In certain embodiments, oligomeric compounds
comprising modifications of the 5'-terminal monomer are resistant
to exonucleases. In certain embodiments, oligomeric compounds
comprising modifications of the 5'-terminal monomer have improved
REVERSIR properties. In certain such embodiments, oligomeric
compound comprising modifications of the 5'-terminal monomer have
improved association with a strand of the siRNA.
[0407] In certain embodiments, the 5'terminal monomer is attached
to rest of the oligomeric compound a modified linkage. In certain
such embodiments, the 5'terminal monomer is attached to rest of the
oligomeric compound by a phosphorothioate linkage.
[0408] In certain embodiments, oligomeric compounds of the present
invention comprise one or more regions of alternating
modifications. In certain embodiments, oligomeric compounds
comprise one or more regions of alternating nucleoside
modifications. In certain embodiments, oligomeric compounds
comprise one or more regions of alternating linkage modifications.
In certain embodiments, oligomeric compounds comprise one or more
regions of alternating nucleoside and linkage modifications.
[0409] In certain embodiments, oligomeric compounds of the present
invention comprise one or more regions of alternating 2'-F modified
nucleosides and 2'-OMe modified nucleosides. In certain such
embodiments, such regions of alternating 2'F modified and 2'OMe
modified nucleosides also comprise alternating linkages. In certain
such embodiments, the linkages at the 3' end of the 2'-F modified
nucleosides are phosphorothioate linkages. In certain such
embodiments, the linkages at the 3'end of the 2'OMe nucleosides are
phosphodiester linkages.
[0410] In certain embodiments, such alternating regions are:
(2'-F)--(PS)-(2'-OMe)-(PO)
[0411] In certain embodiments, oligomeric compounds comprise 2, 3,
4, 5, 6, 7, 8, 9, 10, or 11 such alternating regions. Such regions
may be contiguous or may be interrupted by differently modified
nucleosides or linkages.
[0412] In certain embodiments, one or more alternating regions in
an alternating motif include more than a single nucleoside of a
type. For example, oligomeric compounds of the present invention
may include one or more regions of any of the following nucleoside
motifs:
[0413] ABA;
[0414] ABBA;
[0415] AABA;
[0416] AABBAA;
[0417] ABBABB;
[0418] AABAAB;
[0419] ABBABAABB;
[0420] ABABAA;
[0421] AABABAB;
[0422] ABABAA;
[0423] ABBAABBABABAA;
[0424] BABBAABBABABAA; or
[0425] ABABBAABBABABAA;
wherein A is a nucleoside of a first type and B is a nucleoside of
a second type. In certain embodiments, A and B are each selected
from 2'-F, 2'-OMe, LNA, DNA and MOE.
[0426] In certain embodiments, A is DNA. In certain embodiments B
is DNA. In some embodiments, A is 4'-CH.sub.2O-2'-LNA. In certain
embodiments, B is 4'-CH.sub.2O-2'-LNA. In certain embodiments, A is
DNA and B is 4'-CH.sub.2O-2'-LNA. In certain embodiments A is
4'-CH.sub.2O-2'-LNA and B is DNA.
[0427] In certain embodiments, A is 2'-OMe. In certain embodiments
B is 2'-OMe. In certain embodiments, A is 2'-OMe and B is
4'-CH.sub.2O-2'-LNA. In certain embodiments A is
4'-CH.sub.2O-2'-LNA and B is 2'-OMe. In certain embodiments, A is
2'-OMe and B is DNA. In certain embodiments A is DNA and B is
2'-OMe.
[0428] In certain embodiments, A is (S)-cEt. In some embodiments, B
is (S)-cEt. In certain embodiments, A is 2'-OMe and B is (S)-cEt.
In certain embodiments A is (S)-cEt and B is 2'-OMe. In certain
embodiments, A is DNA and B is (S)-cEt. In certain embodiments A is
(S)-cEt and B is DNA.
[0429] In certain embodiments, A is 2'-F. In certain embodiments B
is 2'-F. In certain embodiments, A is 2'-F and B is
4'-CH.sub.2O-2'-LNA. In certain embodiments A is
4'-CH.sub.2O-2'-LNA and B is 2'-F. In certain embodiments, A is
2'-F and B is (S)-cEt. In certain embodiments A is (S)-cEt and B is
2'-F. In certain embodiments, A is 2'-F and B is DNA. In certain
embodiments A is DNA and B is 2'-F. In certain embodiments, A is
2'-OMe and B is 2'-F. In certain embodiments, A is DNA and B is
2'-OMe. In certain embodiments, A is 2'-OMe and B is DNA.
[0430] In certain embodiments, oligomeric compounds having such an
alternating motif also comprise a 5' terminal nucleoside comprising
a phosphate stabilizing modification. In certain embodiments,
oligomeric compounds having such an alternating motif also comprise
a 5' terminal nucleoside comprising a 2'-cationic modification. In
certain embodiments, oligomeric compounds having such an
alternating motif also comprise a 5' terminal modification.
Two-Two-Three Motifs
[0431] In certain embodiments, oligomeric compounds of the present
invention comprise a region having a 2-2-3 motif. Such regions
comprises the following motif:
5'-(E).sub.w-(A).sub.2-(B).sub.x-(A).sub.2-(C).sub.y-(A).sub.3-(D).sub.z
[0432] wherein: A is a first type of modified nucleoside;
[0433] B, C, D, and E are nucleosides that are differently modified
than A, however, B, C, D, and E may have the same or different
modifications as one another;
[0434] w and z are from 0 to 15;
[0435] x and y are from 1 to 15.
[0436] In certain embodiments, A is a 2'-OMe modified nucleoside.
In certain embodiments, B, C, D, and E are all 2'-F modified
nucleosides. In certain embodiments, A is a 2'-OMe modified
nucleoside and B, C, D, and E are all 2'-F modified
nucleosides.
[0437] In certain embodiments, the linkages of a 2-2-3 motif are
all modified linkages. In certain embodiments, the linkages are all
phosphorothioate linkages. In certain embodiments, the linkages at
the 3'-end of each modification of the first type are
phosphodiester.
[0438] In certain embodiments, Z is 0. In such embodiments, the
region of three nucleosides of the first type are at the 3'-end of
the oligonucleotide. In certain embodiments, such region is at the
3'-end of the oligomeric compound, with no additional groups
attached to the 3' end of the region of three nucleosides of the
first type. In certain embodiments, an oligomeric compound
comprising an oligonucleotide where Z is 0, may comprise a terminal
group attached to the 3'-terminal nucleoside. Such terminal groups
may include additional nucleosides. Such additional nucleosides are
typically non-hybridizing nucleosides.
[0439] In certain embodiments, Z is 1-3. In certain embodiments, Z
is 2. In certain embodiments, the nucleosides of Z are 2'-MOE
nucleosides. In certain embodiments, Z represents non-hybridizing
nucleosides. To avoid confusion, it is noted that such
non-hybridizing nucleosides might also be described as a
3'-terminal group with Z.dbd.O.
Combination Motifs
[0440] It is to be understood, that certain of the above described
motifs and modifications can be combined. Since a motif may
comprises only a few nucleosides, a particular oligomeric compound
can comprise two or more motifs. By way of non-limiting example, in
certain embodiments, oligomeric compounds can have two or more
nucleoside motifs selected from LNAs, phosphorthioate linkages,
2'-OMe, conjugated ligand(s).
[0441] Oligomeric compounds having any of the various nucleoside
motifs described herein, can have also have any linkage motif. For
example, in the oligomeric compounds first 1, 2, 3, 4 or 5 at the
5'-end be modified intrersugar linkages and first 4, 5, 6, 7 or 8
intersugar linkages at the 3'-end can be modified intersugar
linkages. The central region of such modified oligomeric compound
can have intersugar linkages based on the any of the other motifs
described herein, for example, uniform, alternating, hemimer,
gapmer, and the like. In some embodiments, the oligomeric compound
comprise a phosphorothioate linkage between the first and second
monomer at the 5'-terminus, alternating
phosphorothioate/phosphodiester linkages in the central region and
6, 7, or 8 phosphorothioate linkages at the 3'-terminus.
[0442] It is to be noted that the lengths of the regions defined by
a nucleoside motif and that of a linkage motif need not be the
same.
[0443] In some embodiments, single-stranded oligomeric compounds or
at least one strand of a double-stranded oligomeric compound,
includes at least one of the following motifs: [0444] (a)
5'-phosphorothioate or 5'-phosphorodithioate; [0445] (b) a cationic
modification of nucleotides 1 and 2 on the 5' terminal, wherein the
cationic modification is at C5 position of pyrimidines and C2, C6,
C8, exocyclic N2 or exocyclic N6 of purines; [0446] (c) at least
one G-clamp nucleotide in the first two terminal nucleotides at the
5' end and the other nucleotide having a cationic modification,
wherein the cationic modification is at C5 position of pyrimidines
or C2, C6, C8, exocyclic N2 or exocyclic N6 position of purines;
[0447] (d) at least one 2'-F modified nucleotide comprising a
nucleobase base modification; [0448] (e) at least one
gem-2'-O-methyl/2'-F modified nucleotide comprising a nucleobase
modification, preferably the methyl substituent is in the up
configuration, e.g. in the arabinose configuration; [0449] (f) a
5'-PuPu-3' dinucleotide at the 3' terminal wherein both nucleotides
comprise a modified MOE at 2'-position as described in U.S. Patent
Application Publication No. 20130130378, content of which is
incorporated herein by reference in its entirety., [0450] (g) a
5'-PuPu-3' dinucleotide at the 5' terminal wherein both nucleotides
comprise a modified MOE at 2'-position as described in U.S. Patent
Application Publication No. 20130130378; [0451] (h) nucleotide at
the 5' terminal having a modified MOE at 2'-position as described
in U.S. Patent Application Publication No. 20130130378; [0452] (i)
nucleotide at the 5' terminal having a 3'-F modification; [0453]
(j) 5' terminal nucleotide comprising a 4'-substituent; [0454] (k)
5' terminal nucleotide comprising a 04' modification; [0455] (l) 3'
terminal nucleotide comprising a 4'-substituent; and [0456] (m)
combinations thereof.
[0457] In some embodiments, both strands of a double-stranded
oligomeric compound independently comprise at least one of the
above described motifs. In some other embodiments, both strands of
a double-stranded oligomeric compound comprise at least one at
least one of the above described motifs, which motifs can be same
or different or some combination of same and different.
[0458] The above examples are provided solely to illustrate how the
described motifs may be used in combination and are not intended to
limit the invention to the particular combinations or the
particular modifications used in illustrating the combinations.
Further, specific examples herein, including, but not limited to
those in the above table are intended to encompass more generic
embodiments. For example, column A in the above table exemplifies a
region of alternating 2'-OMe and 2'-F nucleosides. Thus, that same
disclosure also exemplifies a region of alternating different
2'-modifications. It also exemplifies a region of alternating
2'-O-alkyl and 2'-halogen nucleosides. It also exemplifies a region
of alternating differently modified nucleosides. All of the
examples throughout this specification contemplate such generic
interpretation.
[0459] It is also noted that the lengths of oligomeric compounds,
such as those exemplified in the above tables, can be easily
manipulated by lengthening or shortening one or more of the
described regions, without disrupting the motif.
[0460] In some embodiments, oligomeric compound comprises two or
more chemically distinct regions and has a structure as described
in International Application No. PCT/US09/038433, filed Mar. 26,
2009, contents of which are herein incorporated in their
entirety.
Synthesis, Purification and Analysis
[0461] Oligomerization of modified and unmodified nucleosides and
nucleotides can be routinely performed according to literature
procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed.
Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001),
23, 206-217. Gait et al., Applications of Chemically synthesized
RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et
al., Tetrahedron (2001), 57, 5707-5713).
[0462] Oligomeric compounds provided herein can be conveniently and
routinely made through the well-known technique of solid phase
synthesis. Equipment for such synthesis is sold by several vendors
including, for example, Applied Biosystems (Foster City, Calif.).
Any other means for such synthesis known in the art may
additionally or alternatively be employed. It is well known to use
similar techniques to prepare oligonucleotides such as the
phosphorothioates and alkylated derivatives. The invention is not
limited by the method of antisense compound synthesis.
[0463] Methods of purification and analysis of oligomeric compounds
are known to those skilled in the art. Analysis methods include
capillary electrophoresis (CE) and electrospray-mass spectroscopy.
Such synthesis and analysis methods can be performed in multi-well
plates. The method of the invention is not limited by the method of
oligomer purification.
[0464] The oligomeric compounds of the invention can be prepared
using solution-phase or solid-phase organic synthesis, or
enzymatically by methods known in the art. Organic synthesis offers
the advantage that the oligomeric strands comprising non-natural or
modified nucleotides can be easily prepared. Any other means for
such synthesis known in the art can additionally or alternatively
be employed. It is also known to use similar techniques to prepare
other oligomeric compounds, such as those comprising
phosphorothioates, phosphorodithioates and alkylated derivatives of
intersugar linkages. The double-stranded oligomeric compounds of
the invention can be prepared using a two-step procedure. First,
the individual strands of the double-stranded molecule are prepared
separately. Then, the component strands are annealed.
[0465] Regardless of the method of synthesis, the oligomeric
compounds can be prepared in a solution (e.g., an aqueous and/or
organic solution) that is appropriate for formulation. For example,
the oligonmeric preparation can be precipitated and redissolved in
pure double-distilled water, and lyophilized. The dried oligomeric
compound can then be resuspended in a solution appropriate for the
intended formulation process.
[0466] Teachings regarding the synthesis of particular modified
oligomeric compounds can be found in the following U.S. patents or
pending patent applications: U.S. Pat. Nos. 5,138,045 and
5,218,105, drawn to polyamine conjugated oligonucleotides; U.S.
Pat. No. 5,212,295, drawn to monomers for the preparation of
oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos.
5,378,825 and 5,541,307, drawn to oligonucleotides having modified
backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified
oligonucleotides and the preparation thereof through reductive
coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases
based on the 3-deazapurine ring system and methods of synthesis
thereof, U.S. Pat. No. 5,459,255, drawn to modified nucleobases
based on N--2 substituted purines; U.S. Pat. No. 5,521,302, drawn
to processes for preparing oligonucleotides having chiral
phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide
nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides
having beta-lactam backbones; U.S. Pat. No. 5,571,902, drawn to
methods and materials for the synthesis of oligonucleotides; U.S.
Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups,
wherein such groups can be used as linkers to other moieties
attached at any of a variety of positions of the nucleoside; U.S.
Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having
phosphorothioate linkages of high chiral purity; U.S. Pat. No.
5,506,351, drawn to processes for the preparation of 2'-O-alkyl
guanosine and related compounds, including 2,6-diaminopurine
compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides
having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to
oligonucleotides having 3-deazapurines; U.S. Pat. Nos. 5,223,168,
and 5,608,046, both drawn to conjugated 4'-desmethyl nucleoside
analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to
backbone-modified oligonucleotide analogs; and U.S. Pat. Nos.
6,262,241, and 5,459,255, drawn to, inter alia, methods of
synthesizing 2'-fluoro-oligonucleotides.
Compositions and Methods for Formulating Pharmaceutical
Compositions
[0467] Oligomeric compounds can 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 are
dependent upon a number of criteria, including, but not limited to,
route of administration, extent of disease, or dose to be
administered.
[0468] Oligomeric compounds, including siRNAs and/or REVERSIR
compounds, can be utilized in pharmaceutical compositions by
combining such oligomeric compounds with a suitable
pharmaceutically acceptable diluent or carrier. A pharmaceutically
acceptable diluent includes phosphate-buffered saline (PBS). PBS is
a diluent suitable for use in compositions to be delivered
parenterally. Accordingly, in one embodiment, employed in the
methods described herein is a pharmaceutical composition comprising
an antisense compound and/or antidote compound and a
pharmaceutically acceptable diluent. In certain embodiments, the
pharmaceutically acceptable diluent is PBS.
[0469] Pharmaceutical compositions comprising oligomeric compounds
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters. In certain embodiments, pharmaceutical compositions
comprising oligomeric compounds comprise one or more
oligonucleotide which, upon administration to an animal, including
a human, is 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.
[0470] A prodrug can include the incorporation of additional
nucleosides at one or both ends of an oligomeric compound which are
cleaved by endogenous nucleases within the body, to form the active
oligomeric compound.
[0471] The pharmaceutical compositions of the present invention may
be administered in a number of ways depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Administration may be topical (e.g., by a transdermal patch),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal, oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; subdermal,
e.g., via an implanted device; or intracranial, e.g., by
intraparenchymal, intrathecal or intraventricular,
administration.
[0472] The oligomeric compounds can be delivered in a manner to
target a particular tissue, such as the liver (e.g., the
hepatocytes of the liver).
[0473] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful. Suitable
topical formulations include those in which the iRNAs featured in
the invention are in admixture with a topical delivery agent such
as lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Suitable lipids and liposomes
include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl
choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and
cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, iRNAs may be complexed to lipids, in particular to
cationic lipids. Suitable fatty acids and esters include but are
not limited to arachidonic acid, oleic acid, eicosanoic acid,
lauric acid, caprylic acid, capric acid, myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a C.sub.1-20 alkyl ester (e.g., isopropylmyristate IPM),
monoglyceride, diglyceride or pharmaceutically acceptable salt
thereof. Topical formulations are described in detail in U.S. Pat.
No. 6,747,014, which is incorporated herein by reference.
[0474] There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles, such as liposomes, have attracted great
interest because of their specificity and the duration of action
they offer from the standpoint of drug delivery. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0475] Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous
interior. The aqueous portion contains the composition to be
delivered. Cationic liposomes possess the advantage of being able
to fuse to the cell wall. Non-cationic liposomes, although not able
to fuse as efficiently with the cell wall, are taken up by
macrophages in vivo.
[0476] Further advantages of liposomes include; liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
[0477] Liposomes are useful for the transfer and delivery of active
ingredients to the site of action. Because the liposomal membrane
is structurally similar to biological membranes, when liposomes are
applied to a tissue, the liposomes start to merge with the cellular
membranes and as the merging of the liposome and cell progresses,
the liposomal contents are emptied into the cell where the active
agent may act.
[0478] Liposomal formulations have been the focus of extensive
investigation as the mode of delivery for many drugs. There is
growing evidence that for topical administration, liposomes present
several advantages over other formulations. Such advantages include
reduced side-effects related to high systemic absorption of the
administered drug, increased accumulation of the administered drug
at the desired target, and the ability to administer a wide variety
of drugs, both hydrophilic and hydrophobic, into the skin.
[0479] Several reports have detailed the ability of liposomes to
deliver agents including high-molecular weight DNA into the skin.
Compounds including analgesics, antibodies, hormones and
high-molecular weight DNAs have been administered to the skin. The
majority of applications resulted in the targeting of the upper
epidermis
[0480] Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. The positively
charged DNA/liposome complex binds to the negatively charged cell
surface and is internalized in an endosome. Due to the acidic pH
within the endosome, the liposomes are ruptured, releasing their
contents into the cell cytoplasm (Wang et al., Biochem. Biophys.
Res. Commun., 1987, 147, 980-985).
[0481] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. Nevertheless, some DNA is entrapped within the
aqueous interior of these liposomes. pH-sensitive liposomes have
been used to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture. Expression of the exogenous gene was
detected in the target cells (Zhou et al., Journal of Controlled
Release, 1992, 19, 269-274).
[0482] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0483] Several studies have assessed the topical delivery of
liposomal drug formulations to the skin. Application of liposomes
containing interferon to guinea pig skin resulted in a reduction of
skin herpes sores while delivery of interferon via other means
(e.g., as a solution or as an emulsion) were ineffective (Weiner et
al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
[0484] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome.TM. I
(glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether)
and Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).
[0485] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.M1, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765).
[0486] Various liposomes comprising one or more glycolipids are
known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci.,
1987, 507, 64) reported the ability of monosialoganglioside
G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to
improve blood half-lives of liposomes. These findings were
expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A.,
1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to
Allen et al., disclose liposomes comprising (1) sphingomyelin and
(2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester.
U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes
comprising sphingomyelin. Liposomes comprising
1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499
(Lim et al).
[0487] Many liposomes comprising lipids derivatized with one or
more hydrophilic polymers, and methods of preparation thereof, are
known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53,
2778) described liposomes comprising a nonionic detergent,
2C.sub.1215G, that contains a PEG moiety. Illum et al. (FEBS Lett.,
1984, 167, 79) noted that hydrophilic coating of polystyrene
particles with polymeric glycols results in significantly enhanced
blood half-lives. Synthetic phospholipids modified by the
attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)
are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899).
Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments
demonstrating that liposomes comprising phosphatidylethanolamine
(PE) derivatized with PEG or PEG stearate have significant
increases in blood circulation half-lives. Blume et al. (Biochimica
et Biophysica Acta, 1990, 1029, 91) extended such observations to
other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from
the combination of distearoylphosphatidylethanolamine (DSPE) and
PEG. Liposomes having covalently bound PEG moieties on their
external surface are described in European Patent No. EP 0 445 131
B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20
mole percent of PE derivatized with PEG, and methods of use
thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556
and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and
European Patent No. EP 0 496 813 B1). Liposomes comprising a number
of other lipid-polymer conjugates are disclosed in WO 91/05545 and
U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073
(Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids
are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935
(Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.)
describe PEG-containing liposomes that can be further derivatized
with functional moieties on their surfaces.
[0488] A number of liposomes comprising nucleic acids are known in
the art. WO 96/40062 to Thierry et al. discloses methods for
encapsulating high molecular weight nucleic acids in liposomes.
U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded
liposomes and asserts that the contents of such liposomes may
include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes
certain methods of encapsulating oligodeoxynucleotides in
liposomes. WO 97/04787 to Love et al. discloses liposomes
comprising dsRNAs targeted to the raf gene.
[0489] Transfersomes are yet another type of liposomes, and are
highly deformable lipid aggregates which are attractive candidates
for drug delivery vehicles. Transfersomes may be described as lipid
droplets which are so highly deformable that they are easily able
to penetrate through pores which are smaller than the droplet.
Transfersomes are adaptable to the environment in which they are
used, e.g., they are self-optimizing (adaptive to the shape of
pores in the skin), self-repairing, frequently reach their targets
without fragmenting, and often self-loading. To make transfersomes
it is possible to add surface edge-activators, usually surfactants,
to a standard liposomal composition. Transfersomes have been used
to deliver serum albumin to the skin. The transfersome-mediated
delivery of serum albumin has been shown to be as effective as
subcutaneous injection of a solution containing serum albumin.
[0490] Liposome compositions can be prepared by a variety of
methods that are known in the art. See e.g., U.S. Pat. Nos.
4,235,871; 4,737,323; 4,897,355 and 5,171,678; published
International Applications WO 96/14057 and WO 96/37194; Felgner, P.
L. et al., Proc. Natl. Acad. Sci., USA (1987) 8:7413-7417, Bangham,
et al. M. Mol. Biol. (1965) 23:238, Olson, et al. Biochim. Biophys.
Acta (1979) 557:9, Szoka, et al. Proc. Natl. Acad. Sci. (1978) 75:
4194, Mayhew, et al. Biochim. Biophys. Acta (1984) 775:169, Kim, et
al. Biochim. Biophys. Acta (1983) 728:339, and Fukunaga, et al.
Endocrinol. (1984) 115:757.
[0491] Surfactants find wide application in formulations such as
emulsions (including microemulsions) and liposomes. The most common
way of classifying and ranking the properties of the many different
types of surfactants, both natural and synthetic, is by the use of
the hydrophile/lipophile balance (HLB). The nature of the
hydrophilic group (also known as the "head") provides the most
useful means for categorizing the different surfactants used in
formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0492] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical and cosmetic products and are usable
over a wide range of pH values. In general their HLB values range
from 2 to about 18 depending on their structure. Nonionic
surfactants include nonionic esters such as ethylene glycol esters,
propylene glycol esters, glyceryl esters, polyglyceryl esters,
sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
alkanolamides and ethers such as fatty alcohol ethoxylates,
propoxylated alcohols, and ethoxylated/propoxylated block polymers
are also included in this class. The polyoxyethylene surfactants
are the most popular members of the nonionic surfactant class.
[0493] If the surfactant molecule carries a negative charge when it
is dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
[0494] If the surfactant molecule carries a positive charge when it
is dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
[0495] If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0496] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Lipid Particles
[0497] In some embodiments, the REVERSIR can be fully encapsulated
in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid
particle. The REVERSIR encapsulated in the lipid formulation can be
unconjugated or conjugated with a ligand (i.e., a conjugated
REVERSIR).
[0498] As used herein, the term "LNP" refers to a stable nucleic
acid-lipid particle. LNPs contain a cationic lipid, a non-cationic
lipid, and a lipid that prevents aggregation of the particle (e.g.,
a PEG-lipid conjugate). LNPs are extremely useful for systemic
applications, as they exhibit extended circulation lifetimes
following intravenous (i.v.) injection and accumulate at distal
sites (e.g., sites physically separated from the administration
site). LNPs include "pSPLP," which include an encapsulated
condensing agent-nucleic acid complex as set forth in PCT
Publication No. WO 00/03683. The particles of the present invention
typically have a mean diameter of about 50 nm to about 150 nm, more
typically about 60 nm to about 130 nm, more typically about 70 nm
to about 110 nm, most typically about 70 nm to about 90 nm, and are
substantially nontoxic. In addition, the nucleic acids when present
in the nucleic acid-lipid particles of the present invention are
resistant in aqueous solution to degradation with a nuclease.
Nucleic acid-lipid particles and their method of preparation are
disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;
6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT
Publication No. WO 96/40964.
[0499] In some embodiments, the lipid to drug ratio (mass/mass
ratio) (e.g., lipid to REVERSIR ratio) will be in the range of from
about 1:1 to about 50:1, from about 1:1 to about 25:1, from about
3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to
about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the
above recited ranges are also contemplated to be part of the
invention.
[0500] The cationic lipid can be, for example,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N--(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N--(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),
1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),
1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane
(DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA) or analogs thereof,
(3aR,5s,6aS)--N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydr-
o-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100),
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (MC3),
1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)ami-
no)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol, or a mixture
thereof. The cationic lipid can comprise from about 20 mol % to
about 50 mol % or about 40 mol % of the total lipid present in the
particle.
[0501] In some embodiments, the compound
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to
prepare lipid-REVERSIR nanoparticles. Synthesis of
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in
International application no. PCT/US2009/061897, published as
WO/2010/048536, which is herein incorporated by reference.
[0502] In some embodiments, the lipid-REVERSIR particle includes
40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC:
40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size
of 63.0.+-.20 nm and a 0.027 REVERSIR/Lipid Ratio.
[0503] The ionizable/non-cationic lipid can be an anionic lipid or
a neutral lipid including, but not limited to,
distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or
a mixture thereof. The non-cationic lipid can be from about 5 mol %
to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol
is included, of the total lipid present in the particle.
[0504] The conjugated lipid that inhibits aggregation of particles
can be, for example, a polyethyleneglycol (PEG)-lipid including,
without limitation, a PEG-diacylglycerol (DAG), a
PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide
(Cer), or a mixture thereof. The PEG-DAA conjugate can be, for
example, a PEG-dilauryloxypropyl (C.sub.12), a
PEG-dimyristyloxypropyl (C.sub.14), a PEG-dipalmityloxypropyl
(C.sub.16), or a PEG-distearyloxypropyl (C.sub.18). The conjugated
lipid that prevents aggregation of particles can be from 0 mol % to
about 20 mol % or about 2 mol % of the total lipid present in the
particle.
[0505] In some embodiments, the nucleic acid-lipid particle further
includes cholesterol at, e.g., about 10 mol % to about 60 mol % or
about 48 mol % of the total lipid present in the particle.
[0506] Additional exemplary lipid-REVERSIR formulations are
described in Table 1 below.
TABLE-US-00001 TABLE 1 Exemplary lipid REVERSIR formulations
cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate
Formulation Ionizable/Cationic Lipid Lipid:REVERSIR ratio
LNP_DLinDMA 1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA)
(57.1/7.1/34.4/1.4) lipid:REVERSIR~7:1 2-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4
lipid:REVERSIR~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5
lipid:REVERSIR~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5
lipid:REVERSIR~11:1 LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5,
lipid:REVERSIR~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5,
lipid:REVERSIR~11:1 LNP09
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5
Lipid:REVERSIR 10:1 LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-
ALN100/DSPC/Cholesterol/PEG-DMG
octadeca-9,12-dienyl)tetrahydro-3aH- 50/10/38.5/1.5
cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:REVERSIR 10:1 LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-
MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl
4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3) Lipid:REVERSIR 10:1
LNP12 1,1'-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG-DMG
hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5
hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:REVERSIR 10:1
yl)ethylazanediyl)didodecan-2-ol (C12-200> LNP13 XTC
XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:REVERSIR: 33:1 LNP14 MC3
MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:REVERSIR: 11:1 LNP15 MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG- DSG 50/10/35/4.5/0.5
Lipid:REVERSIR: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5
Lipid:REVERSIR: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5
Lipid:REVERSIR: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5
Lipid:REVERSIR: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5
Lipid:REVERSIR: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5
Lipid:REVERSIR: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG
50/10/38.5/1.5 Lipid:REVERSIR: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG
50/10/38.5/1.5 Lipid:REVERSIR: 10:1 LNPX
(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-
(13Z,16Z)-N,N-dimethyl-3-nonyldocosa- 13,16-dien-1-amine
13,16-dien-1-amine/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:REVERSIR:
10:1 LNPY Biodegradable lipid Biodegradable lipid/DSPC/Chol/PEG-DSG
50/10/38.5/1.5 Lipid:REVERSIR: 10:1 *The REVERSIR can be an
unconjugated or conjugated with a ligand (i.e., conjugated
REVERSIR).
[0507] Abbreviations in Table 1 include the following: DSPC:
distearoylphosphatidylcholine; DPPC:
dipalmitoylphosphatidylcholine; PEG-DMG: PEG-didimyristoyl glycerol
(C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000); PEG-DSG:
PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of
2000); PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG
with avg mol wt of 2000).
[0508] DLinDMA (1,2-Dilinolenyloxy-N,N-dimethylaminopropane)
comprising formulations are described in International Publication
No. WO2009/127060, filed Apr. 15, 2009, which is hereby
incorporated by reference.
[0509] XTC comprising formulations are described, e.g., in U.S.
Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S.
Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S.
Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Ser.
No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No.
61/239,686, filed Sep. 3, 2009, and International Application No.
PCT/US2010/022614, filed Jan. 29, 2010, which are hereby
incorporated by reference.
[0510] MC3 comprising formulations are described, e.g., in U.S.
Publication No. 2010/0324120, filed Jun. 10, 2010, the entire
contents of which are hereby incorporated by reference.
[0511] Biodegradable lipid comprising formulations are described,
e.g., PCT Publications No. WO2011/153493, filed Jun. 3, 2011 and
WO/2013/086354, filed Dec. 7, 2012, the entire contents of which
are hereby incorporated by reference.
[0512] (13Z,16Z)--N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine
comprising formulations are described, e.g., in PCT Publications
No. WO/2012/040184, filed Sep. 20, 2011, the entire contents of
which are hereby incorporated by reference.
[0513] The oligomeric compounds of the invention can be prepared
and formulated as micelles. As used herein, "micelles" are a
particular type of molecular assembly in which amphipathic
molecules are arranged in a spherical structure such that all
hydrophobic portions on the molecules are directed inward, leaving
the hydrophilic portions in contact with the surrounding aqueous
phase. The converse arrangement exists if the environment is
hydrophobic.
[0514] In some embodiments, the formulations comprises micelles
formed from an oligonucleotide of the invention and at least one
amphiphilic carrier, in which the micelles have an average diameter
of less than about 100 nm, preferably. More preferred embodiments
provide micelles having an average diameter less than about 50 nm,
and even more preferred embodiments provide micelles having an
average diameter less than about 30 nm, or even less than about 20
nm.
[0515] Micelle formulations can be prepared by mixing an aqueous
solution of the oligonucleotide composition, an alkali metal
C.sub.8 to C.sub.22 alkyl sulphate, and an amphiphilic carrier. The
amphiphilic carrier can be added at the same time or after addition
of the alkali metal alkyl sulphate. Micelles will form with
substantially any kind of mixing of the ingredients but vigorous
mixing in order to provide smaller size micelles.
[0516] The oligomeric compounds of the present invention can be
prepared and formulated as emulsions. As used herein, "emulsion" is
a heterogenous system of one liquid dispersed in another in the
form of droplets.
[0517] Emulsions are often biphasic systems comprising two
immiscible liquid phases intimately mixed and dispersed with each
other. In general, emulsions may be of either the water-in-oil
(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is
finely divided into and dispersed as minute droplets into a bulk
oily phase, the resulting composition is called a water-in-oil
(w/o) emulsion. Alternatively, when an oily phase is finely divided
into and dispersed as minute droplets into a bulk aqueous phase,
the resulting composition is called an oil-in-water (o/w) emulsion.
Emulsions may contain additional components in addition to the
dispersed phases, and the active drug which may be present as a
solution in either the aqueous phase, oily phase or itself as a
separate phase. Pharmaceutical excipients such as emulsifiers,
stabilizers, dyes, and anti-oxidants may also be present in
emulsions as needed. Pharmaceutical emulsions may also be multiple
emulsions that are comprised of more than two phases such as, for
example, in the case of oil-in-water-in-oil (o/w/o) and
water-in-oil-in-water (w/o/w) emulsions. Such complex formulations
often provide certain advantages that simple binary emulsions do
not. Multiple emulsions in which individual oil droplets of an o/w
emulsion enclose small water droplets constitute a w/o/w emulsion.
Likewise a system of oil droplets enclosed in globules of water
stabilized in an oily continuous phase provides an o/w/o
emulsion.
[0518] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0519] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (Rieger, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
Surfactants are typically amphiphilic and comprise a hydrophilic
and a hydrophobic portion. The ratio of the hydrophilic to the
hydrophobic nature of the surfactant has been termed the
hydrophile/lipophile balance (HLB) and is a valuable tool in
categorizing and selecting surfactants in the preparation of
formulations. Surfactants may be classified into different classes
based on the nature of the hydrophilic group: nonionic, anionic,
cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0520] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0521] A large variety of non-emulsifying materials is also
included in emulsion formulations and contributes to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0522] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0523] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that may
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0524] In some embodiments, the compositions are formulated as
microemulsions. As used herein, "microemulsion" refers to a system
of water, oil and amphiphile which is a single optically isotropic
and thermodynamically stable liquid solution. Microemuslions also
include thermodynamically stable, isotropically clear dispersions
of two immiscible liquids that are stabilized by interfacial films
of surface-active molecules.
[0525] A microemulsion may be defined as a system of water, oil and
amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution (Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically
microemulsions are systems that are prepared by first dispersing an
oil in an aqueous surfactant solution and then adding a sufficient
amount of a fourth component, generally an intermediate
chain-length alcohol to form a transparent system. Therefore,
microemulsions have also been described as thermodynamically
stable, isotropically clear dispersions of two immiscible liquids
that are stabilized by interfacial films of surface-active
molecules (Leung and Shah, in: Controlled Release of Drugs:
Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
[0526] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0527] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750),
decaglycerol decaoleate (DAO750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized C8-C10
glycerides, vegetable oils and silicone oil.
[0528] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13,
205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form
spontaneously when their components are brought together at ambient
temperature. This may be particularly advantageous when formulating
thermolabile drugs, peptides or dsRNAs. Microemulsions have also
been effective in the transdermal delivery of active components in
both cosmetic and pharmaceutical applications. It is expected that
the microemulsion compositions and formulations of the present
invention will facilitate the increased systemic absorption of
dsRNAs and nucleic acids from the gastrointestinal tract, as well
as improve the local cellular uptake of dsRNAs and nucleic
acids.
[0529] Microemulsions of the present invention may also contain
additional components and additives such as sorbitan monostearate
(Grill 3), Labrasol, and penetration enhancers to improve the
properties of the formulation and to enhance the absorption of the
dsRNAs and nucleic acids of the present invention. Penetration
enhancers used in the microemulsions of the present invention may
be classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
[0530] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature, for example see Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199;
Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; and Block, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., volume 1, p. 335, contents of which are herein incorporated
by reference in their entirety.
[0531] The oligomeric compounds of the present invention can be
prepared and formulated as lipid particles, e.g., formulated lipid
particles (FLiPs) comprising (a) an oligonucleotide of the
invention, where said oligonucleotide has been conjugated to a
lipophile and (b) at least one lipid component, for example an
emulsion, liposome, isolated lipoprotein, reconstituted lipoprotein
or phospholipid, to which the conjugated oligonucleotide has been
aggregated, admixed or associated. The stoichiometry of
oligonucleotide to the lipid component can be 1:1. Alternatively
the stoichiometry can be 1:many, many:1 or many:many, where many is
two or more.
[0532] The FLiP can comprise triacylglycerols, phospholipids,
glycerol and one or several lipid-binding proteins aggregated,
admixed or associated via a lipophilic linker molecule with an
oligonucleotide. Surprisingly, it has been found that due to said
one or several lipid-binding proteins in combination with the above
mentioned lipids, the FLiPs show affinity to liver, gut, kidney,
steroidogenic organs, heart, lung and/or muscle tissue. These FLiPs
can therefore serve as carrier for oligonucleotides to these
tissues. For example, lipid-conjugated oligonucleotides, e.g.,
cholesterol-conjugated oligonucleotides, bind to HDL and LDL
lipoprotein particles which mediate cellular uptake upon binding to
their respective receptors thus directing oligonucleotide delivery
into liver, gut, kidney and steroidogenic organs, see Wolfrum et
al. Nature Biotech. (2007), 25:1145-1157.
[0533] The FLiP can be a lipid particle comprising 15-25%
triacylglycerol, about 0.5-2% phospholipids and 1-3% glycerol, and
one or several lipid-binding proteins. FLiPs can be a lipid
particle having about 15-25% triacylglycerol, about 1-2%
phospholipids, about 2-3% glycerol, and one or several
lipid-binding proteins. In some embodiments, the lipid particle
comprises about 20% triacylglycerol, about 1.2% phospholipids and
about 2.25% glycerol, and one or several lipid-binding
proteins.
[0534] Another suitable lipid component for FLiPs is lipoproteins,
for example isolated lipoproteins or more preferably reconstituted
lipoproteins. Exemplary lipoproteins include chylomicrons, VLDL
(Very Low Density Lipoproteins), IDL (Intermediate Density
Lipoproteins), LDL (Low Density Lipoproteins) and HDL (High Density
Lipoproteins). Methods of producing reconstituted lipoproteins are
known in the art, for example see A. Jones, Experimental Lung Res.
6, 255-270 (1984), U.S. Pat. Nos. 4,643,988 and 5,128,318, PCT
publication WO87/02062, Canadian Pat. No. 2,138,925. Other methods
of producing reconstituted lipoproteins, especially for
apolipoproteins A-I, A-II, A-IV, apoC and apoE have been described
in A. Jonas, Methods in Enzymology 128, 553-582 (1986) and G.
Franceschini et al. J. Biol. Chem., 260(30), 16321-25 (1985).
[0535] One preferred lipid component for FLiP is Intralipid.
Intralipid.RTM. is a brand name for the first safe fat emulsion for
human use. Intralipid.RTM. 20% (a 20% intravenous fat emulsion) is
made up of 20% soybean oil, 1.2% egg yolk phospholipids, 2.25%
glycerin, and water for injection. It is further within the present
invention that other suitable oils, such as saflower oil, can serve
to produce the lipid component of the FLiP.
[0536] FLiP can range in size from about 20-50 nm or about 30-50
nm, e.g., about 35 nm or about 40 nm. In some embodiments, the FLiP
has a particle size of at least about 100 nm. FLiPs can
alternatively be between about 100-150 nm, e.g., about 110 nm,
about 120 nm, about 130 nm, or about 140 nm, whether characterized
as liposome- or emulsion-based. Multiple FLiPs can also be
aggregated and delivered together, therefore the size can be larger
than 100 nm.
[0537] The process for making the lipid particles comprises the
steps of: (a) mixing a lipid components with one or several
lipophile (e.g. cholesterol) conjugated oligonucleotides that can
be chemically modified; and (b) fractionating this mixture. In some
embodiments, the process comprises the additional step of selecting
the fraction with particle size of 30-50 nm, preferably of about 40
nm in size.
[0538] Some exemplary lipid particle formulations amenable to the
invention are described in U.S. patent application Ser. No.
12/412,206, filed Mar. 26, 2009, content of which is herein
incorporated by reference in its entirety.
[0539] In some embodiments, the oligomeric compounds can be
formulated in yeast cell wall particles ("YCWP"). A yeast cell wall
particle comprises an extracted yeast cell wall exterior and a
core, the core comprising a payload (e.g., oligonucleotides).
Exterior of the particle comprises yeast glucans (e.g. beta
glucans, beta-1,3-glucans, beta-1,6-glucans), yeast mannans, or
combinations thereof. Yeast cell wall particles are typically
spherical particles about 1-4 .mu.m in diameter.
[0540] Preparation of yeast cell wall particles is known in the
art, and is described, for example in U.S. Pat. Nos. 4,992,540;
5,082,936; 5,028,703; 5,032,401; 5,322,841; 5,401,727; 5,504,079;
5,607,677; 5,741,495; 5,830,463; 5,968,811; 6,444,448; and
6,476,003, U.S. Pat. App. Pub. Nos. 2003/0216346 and 2004/0014715,
and Int. App. Pub. No. WO 2002/12348, contents of which are herein
incorporated by reference in their entirety. Applications of yeast
cell like particles for drug delivery are described, for example in
U.S. Pat. Nos. 5,032,401; 5,607,677; 5,741,495; and 5,830,463, and
U.S. Pat. Pub Nos. 2005/0281781 and 2008/0044438, contents of which
are herein incorporated by reference in their entirety. U.S. Pat.
App. Pub. No. 2009/0226528, contents of which are herein
incorporated by reference, describes formulation of nucleic acids
with yeast cell wall particles for delivery of oligonucleotide to
cells.
[0541] Exemplary formulations for oligomeric compounds are
described in U.S. Pat. Nos. 4,897,355; 4,394,448; 4,235,871;
4,231,877; 4,224,179; 4,753,788; 4,673,567; 4,247,411; 4,814,270;
5,567,434; 5,552,157; 5,565,213; 5,738,868; 5,795,587; 5,922,859;
6,077,663; 7,906,484; and 8,642,076; PCT Publication No.
WO2009/132131 and U.S. Pat. Pub. Nos. 2006/0240093, 2007/0135372,
2011/0117125, 2009/0291131, 2012/0316220, 2009/0163705 and
2013/0129785, contents of all of which is herein incorporated by
reference in its entirety. Behr (1994) Bioconjugate Chem.
5:382-389, and Lewis et al. (1996) PNAS 93:3176-3181), also
describe formulations for oligonucleotides that are amenable to the
invention, contents of which are herein incorporated by reference
in their entirety.
siRNA
[0542] As used herein, the term "siRNA" refers to an agent that
mediates the targeted cleavage of an RNA transcript. These agents
associate with a cytoplasmic multi-protein complex known as
RNAi-induced silencing complex (RISC). Agents that are effective in
inducing RNA interference are also referred to as siRNA, RNAi
agent, or iRNA agent, herein. As used herein, the term siRNA
includes microRNAs and pre-microRNAs.
[0543] As used herein, the term "siRNA" refers to an agent that
mediates the targeted cleavage of an RNA transcript. These agents
associate with a cytoplasmic multi-protein complex known as
RNAi-induced silencing complex (RISC). Agents that are effective in
inducing RNA interference are also referred to as siRNA, dsRNA,
RNAi agent, or iRNA agent herein.
[0544] As used herein, the terms "siRNA activity" and "RNAi
activity" refer to gene silencing by an siRNA.
[0545] As used herein, "gene silencing" by a RNA interference
molecule refers to a decrease in the mRNA level in a cell for a
target gene by at least about 5%, at least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 99% up to and
including 100%, and any integer in between of the mRNA level found
in the cell without the presence of the miRNA or RNA interference
molecule. In one preferred embodiment, the mRNA levels are
decreased by at least about 70%, at least about 80%, at least about
90%, at least about 95%, at least about 99%, up to and including
100% and any integer in between 5% and 100%."
[0546] As used herein the term "modulate gene expression" means
that expression of the gene, or level of RNA molecule or equivalent
RNA molecules encoding one or more proteins or protein subunits is
up regulated or down regulated, such that expression, level, or
activity is greater than or less than that observed in the absence
of the modulator. For example, the term "modulate" can mean
"inhibit," but the use of the word "modulate" is not limited to
this definition.
[0547] As used herein, gene expression modulation happens when the
expression of the gene, or level of RNA molecule or equivalent RNA
molecules encoding one or more proteins or protein subunits is at
least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
2-fold, 3-fold, 4-fold, 5-fold or more different from that observed
in the absence of the siRNA, e.g., RNAi agent. The % and/or fold
difference can be calculated relative to the control or the
non-control, for example,
% .times. .times. difference = [ expression .times. .times. with
.times. .times. siRNA - expression .times. .times. without .times.
.times. siRNA ] expression .times. .times. without .times. .times.
siRNA ##EQU00001## .times. or ##EQU00001.2## % .times. .times.
difference = [ expression .times. .times. with .times. .times.
siRNA - expression .times. .times. without .times. .times. siRNA ]
expression .times. .times. without .times. .times. siRNA
##EQU00001.3##
[0548] As used herein, the term "inhibit", "down-regulate", or
"reduce" in relation to gene expression, means that the expression
of the gene, or level of RNA molecules or equivalent RNA molecules
encoding one or more proteins or protein subunits, or activity of
one or more proteins or protein subunits, is reduced below that
observed in the absence of modulator. The gene expression is
down-regulated when expression of the gene, or level of RNA
molecules or equivalent RNA molecules encoding one or more proteins
or protein subunits, or activity of one or more proteins or protein
subunits, is reduced at least 10% lower relative to a corresponding
non-modulated control, and preferably at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or most preferably, 100%
(i.e., no gene expression).
[0549] As used herein, the term "increase" or "up-regulate" in
relation to gene expression, means that the expression of the gene,
or level of RNA molecules or equivalent RNA molecules encoding one
or more proteins or protein subunits, or activity of one or more
proteins or protein subunits, is increased above that observed in
the absence of modulator. The gene expression is up-regulated when
expression of the gene, or level of RNA molecules or equivalent RNA
molecules encoding one or more proteins or protein subunits, or
activity of one or more proteins or protein subunits, is increased
at least 10% relative to a corresponding non-modulated control, and
preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold,
3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more.
[0550] The term "increased" or "increase" as used herein generally
means an increase by a statically significant amount; for the
avoidance of any doubt, "increased" means an increase of at least
10% as compared to a reference level, for example an increase of at
least about 20%, or at least about 30%, or at least about 40%, or
at least about 50%, or at least about 60%, or at least about 70%,
or at least about 80%, or at least about 90% or up to and including
a 100% increase or any increase between 10-100% as compared to a
reference level, or at least about a 2-fold, or at least about a
3-fold, or at least about a 4-fold, or at least about a 5-fold or
at least about a 10-fold increase, or any increase between 2-fold
and 10-fold or greater as compared to a reference level.
[0551] The term "reduced" or "reduce" as used herein generally
means a decrease by a statistically significant amount. However,
for avoidance of doubt, "reduced" means a decrease by at least 10%
as compared to a reference level, for example a decrease by at
least about 20%, or at least about 30%, or at least about 40%, or
at least about 50%, or at least about 60%, or at least about 70%,
or at least about 80%, or at least about 90% or up to and including
a 100% decrease (i.e. absent level as compared to a reference
sample), or any decrease between 10-100% as compared to a reference
level.
[0552] The skilled person is well aware that double-stranded
oligonucleotides comprising a duplex structure of between 20 and
23, but specifically 21, base pairs have been hailed as
particularly effective in inducing RNA interference (Elbashir et
al., EMBO 2001, 20:6877-6888). However, others have found that
shorter or longer double-stranded oligonucleotides can be effective
as well.
[0553] The double-stranded oligonucleotides comprise two
oligonucleotide strands that are sufficiently complementary to
hybridize to form a duplex structure. Generally, the duplex
structure is between 15 and 30, more generally between 18 and 25,
yet more generally between 19 and 24, and most generally between 19
and 21 base pairs in length. In some embodiments, longer
double-stranded oligonucleotides of between 25 and 30 base pairs in
length are preferred. In some embodiments, shorter double-stranded
oligonucleotides of between 10 and 15 base pairs in length are
preferred. In another embodiment, the double-stranded
oligonucleotide is at least 21 nucleotides long.
[0554] In some embodiments, the double-stranded oligonucleotide
comprises a sense strand and an antisense strand, wherein the
antisense RNA strand has a region of complementarity which is
complementary to at least a part of a target sequence, and the
duplex region is 14-30 nucleotides in length. Similarly, the region
of complementarity to the target sequence is between 14 and 30,
more generally between 18 and 25, yet more generally between 19 and
24, and most generally between 19 and 21 nucleotides in length.
[0555] The phrase "antisense strand" as used herein, refers to an
oligomeric compound that is substantially or 100% complementary to
a target sequence of interest. The phrase "antisense strand"
includes the antisense region of both oligomeric compounds that are
formed from two separate strands, as well as unimolecular
oligomeric compounds that are capable of forming hairpin or
dumbbell type structures. The terms "antisense strand" and "guide
strand" are used interchangeably herein.
[0556] The phrase "sense strand" refers to an oligomeric compound
that has the same nucleoside sequence, in whole or in part, as a
target sequence such as a messenger RNA or a sequence of DNA. The
terms "sense strand" and "passenger strand" are used
interchangeably herein.
[0557] By "specifically hybridizable" and "complementary" is meant
that a nucleic acid can form hydrogen bond(s) with another nucleic
acid sequence by either traditional Watson-Crick or other
non-traditional types. In reference to the nucleic molecules of the
present invention, the binding free energy for a nucleic acid
molecule with its complementary sequence is sufficient to allow the
relevant function of the nucleic acid to proceed, e.g., RNAi
activity. Determination of binding free energies for nucleic acid
molecules is well known in the art (see, e.g., Turner et al, 1987,
CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc.
Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem.
Soc. 109:3783-3785). A percent complementarity indicates the
percentage of contiguous residues in a nucleic acid molecule that
can form hydrogen bonds (e.g., Watson-Crick base pairing) with a
second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10
being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly
complementary" or 100% complementarity means that all the
contiguous residues of a nucleic acid sequence will hydrogen bond
with the same number of contiguous residues in a second nucleic
acid sequence. Less than perfect complementarity refers to the
situation in which some, but not all, nucleoside units of two
strands can hydrogen bond with each other. "Substantial
complementarity" refers to polynucleotide strands exhibiting 90% or
greater complementarity, excluding regions of the polynucleotide
strands, such as overhangs, that are selected so as to be
noncomplementary. Specific binding requires a sufficient degree of
complementarity to avoid non-specific binding of the oligomeric
compound to non-target sequences under conditions in which specific
binding is desired, i.e., under physiological conditions in the
case of in vivo assays or therapeutic treatment, or in the case of
in vitro assays, under conditions in which the assays are
performed. The non-target sequences typically differ by at least 5
nucleotides.
[0558] The term "off-target" and the phrase "off-target effects"
refer to any instance in which an siRNA against a given target
causes an unintended affect by interacting either directly or
indirectly with another mRNA sequence, a DNA sequence or a cellular
protein or other moiety. For example, an "off-target effect" may
occur when there is a simultaneous degradation of other transcripts
due to partial homology or complementarity between that other
transcript and the sense and/or antisense strand of an siRNA.
[0559] In some embodiments, the double-stranded region of a
double-stranded oligomeric compound is equal to or at least, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26,
27, 28, 29, or 30 nucleotide pairs in length.
[0560] In some embodiments, the antisense strand of a
double-stranded oligomeric compound is equal to or at least 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides in length.
[0561] In some embodiments, the sense strand of a double-stranded
oligomeric compound is equal to or at least 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides in length.
[0562] In some embodiments, one strand has at least one stretch of
1-5 single-stranded nucleotides in the double-stranded region. By
"stretch of single-stranded nucleotides in the double-stranded
region" is meant that there is present at least one nucleotide base
pair at both ends of the single-stranded stretch. In some
embodiments, both strands have at least one stretch of 1-5 (e.g.,
1, 2, 3, 4, or 5) single-stranded nucleotides in the double
stranded region. When both strands have a stretch of 1-5 (e.g., 1,
2, 3, 4, or 5) single-stranded nucleotides in the double stranded
region, such single-stranded nucleotides can be opposite to each
other (e.g., a stretch of mismatches) or they can be located such
that the second strand has no single-stranded nucleotides opposite
to the single-stranded oligonucleotides of the first strand and
vice versa (e.g., a single-stranded loop). In some embodiments, the
single-stranded nucleotides are present within 8 nucleotides from
either end, for example 8, 7, 6, 5, 4, 3, or 2 nucleotide from
either the 5' or 3' end of the region of complementarity between
the two strands.
[0563] In some embodiments, each strand of the double-stranded
oligonucleotide has a ZXY structure, such as is described in PCT
Publication No. 2004080406, content of which is hereby incorporated
in its entireties.
[0564] In certain embodiment, the two strands of double-stranded
oligomeric compound can be linked together. The two strands can be
linked to each other at both ends, or at one end only. By linking
at one end is meant that 5'-end of first strand is linked to the
3'-end of the second strand or 3'-end of first strand is linked to
5'-end of the second strand. When the two strands are linked to
each other at both ends, 5'-end of first strand is linked to 3'-end
of second strand and 3'-end of first strand is linked to 5'-end of
second strand. The two strands can be linked together by an
oligonucleotide linker including, but not limited to, (N).sub.n;
wherein N is independently a modified or unmodified nucleotide and
n is 3-23. In some embodiments, n is 3-10, e.g., 3, 4, 5, 6, 7, 8,
9, or 10. In some embodiments, the oligonucleotide linker is
selected from the group consisting of GNRA, (G).sub.4, (U).sub.4,
and (dT).sub.4, wherein N is a modified or unmodified nucleotide
and R is a modified or unmodified purine nucleotide. Some of the
nucleotides in the linker can be involved in base-pair interactions
with other nucleotides in the linker. The two strands can also be
linked together by a non-nucleosidic linker, e.g. a linker
described herein. It will be appreciated by one of skill in the art
that any oligonucleotide chemical modifications or variations
describe herein can be used in the oligonucleotide linker.
[0565] Hairpin and dumbbell type oligomeric compounds will have a
duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29,
21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be
equal to or less than 200, 100, or 50, in length. In some
embodiments, ranges for the duplex region are 15-30, 17 to 23, 19
to 23, and 19 to 21 nucleotides pairs in length.
[0566] The hairpin oligomeric compounds can have a single strand
overhang or terminal unpaired region, in some embodiments at the
3', and in some embodiments on the antisense side of the hairpin.
In some embodiments, the overhangs are 1-4, more generally 2-3
nucleotides in length. The hairpin oligomeric compounds that can
induce RNA interference are also referred to as "shRNA" herein.
[0567] In certain embodiments, two oligomeric strands specifically
hybridize when there is a sufficient degree of complementarity to
avoid non-specific binding of the antisense compound to non-target
nucleic acid sequences under conditions in which specific binding
is desired, i.e., under physiological conditions in the case of in
vivo assays or therapeutic treatment, and under conditions in which
assays are performed in the case of in vitro assays.
[0568] As used herein, "stringent hybridization conditions" or
"stringent conditions" refers to conditions under which an
antisense compound will hybridize to its target sequence, but to a
minimal number of other sequences. Stringent conditions are
sequence-dependent and will be different in different
circumstances, and "stringent conditions" under which antisense
compounds hybridize to a target sequence are determined by the
nature and composition of the antisense compounds and the assays in
which they are being investigated.
[0569] It is understood in the art that incorporation of nucleotide
affinity modifications may allow for a greater number of mismatches
compared to an unmodified compound. Similarly, certain
oligonucleotide sequences may be more tolerant to mismatches than
other oligonucleotide sequences. One of ordinary skill in the art
is capable of determining an appropriate number of mismatches
between oligonucleotides, or between an oligonucleotide and a
target nucleic acid, such as by determining melting temperature
(Tm). Tm or .DELTA.Tm can be calculated by techniques that are
familiar to one of ordinary skill in the art. For example,
techniques described in Freier et al. (Nucleic Acids Research,
1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to
evaluate nucleotide modifications for their ability to increase the
melting temperature of an RNA:DNA duplex.
Modulation of Target Expression
[0570] In certain embodiments, a target nucleic acid is a mRNA. In
certain such embodiments, siRNAs are designed to modulate that
target mRNA or its expression. In certain embodiments, designing an
antisense compound to a target nucleic acid molecule can be a
multistep process. Typically the process begins with the
identification of a target protein, the activity of which is to be
modulated, and then identifying the nucleic acid the expression of
which yields the target protein. In certain embodiments, designing
of an antisense compound results in an antisense compound that is
hybridizable to the targeted nucleic acid molecule. In certain
embodiments, the antisense compound is an antisense oligonucleotide
or antisense oligonucleoside. In certain embodiments, an antisense
compound and a target nucleic acid are complementary to one
another. In certain such embodiments, an antisense compound is
perfectly complementary to a target nucleic acid. In certain
embodiments, an antisense compound includes one mismatch. In
certain embodiments, an antisense compound includes two mismatches.
In certain embodiments, an antisense compound includes three or
more mismatches.
[0571] Modulation of expression of a target nucleic acid can be
achieved through alteration of any number of nucleic acid
functions. In certain embodiments, the functions of RNA to be
modulated include, but are not limited to, translocation functions,
which include, but are not limited to, translocation of the RNA to
a site of protein translation, translocation of the RNA to sites
within the cell which are distant from the site of RNA synthesis,
and translation of protein from the RNA. RNA processing functions
that can be modulated include, but are not limited to, splicing of
the RNA to yield one or more RNA species, capping of the RNA, 3'
maturation of the RNA and catalytic activity or complex formation
involving the RNA which may be engaged in or facilitated by the
RNA. Modulation of expression can result in the increased level of
one or more nucleic acid species or the decreased level of one or
more nucleic acid species, either temporally or by net steady state
level. Thus, in one embodiment modulation of expression can mean
increase or decrease in target RNA or protein levels. In another
embodiment modulation of expression can mean an increase or
decrease of one or more RNA splice products, or a change in the
ratio of two or more splice products.
[0572] In certain embodiments, the siRNA is a conjugated siRNA. As
used herein, the term "conjugated siRNA" refers to an RNAi agent
that is conjugated with a ligand. For Example, an RNAi agent
conjugated with a ligand described herein.
[0573] In some other embodiments, the siRNA is an unconjugated
siRNA. As used herein, the term "unconjugated siRNA" refers to an
RNAi agent that is not conjugated with a ligand, e.g., a ligand
described herein.
[0574] In one aspect, the invention relates to a double-stranded
RNA (dsRNA) agent, i.e., siRNA, for inhibiting the expression of a
target gene. The dsRNA agent comprises a sense strand and an
antisense strand, each strand having 14 to 40 nucleotides. The
dsRNA agent is represented by formula (I):
##STR00091##
[0575] In formula (I), B1, B2, B3, B1', B2', B3', and B4' each are
independently a nucleotide containing a modification selected from
the group consisting of 2'-O-alkyl, 2'-substituted alkoxy,
2'-substituted alkyl, 2'-halo, ENA, and BNA/LNA. In one embodiment,
B1, B2, B3, B1', B2', B3', and B4' each contain 2'-OMe
modifications.
[0576] C1 is a thermally destabilizing nucleotide placed at a site
opposite to the seed region of the antisense strand (i.e., at
positions 2-8 of the 5'-end of the antisense strand). For example,
C1 is at a position of the sense strand that pairs with a
nucleotide at positions 2-8 of the 5'-end of the antisense strand.
C1 nucleotide bears the thermally destabilizing modification which
can include abasic modification; mismatch with the opposing
nucleotide in the duplex; and sugar modification such as 2'-deoxy
modification or acyclic nucleotide e.g., unlocked nucleic acids
(UNA) or glycerol nuceltic acid (GNA). In one embodiment, C1 has
thermally destabilizing modification selected from the group
consisting of: i) mismatch with the opposing nucleotide in the
antisense strand; ii) abasic modification selected from the group
consisting of:
##STR00092##
and iii) sugar modification selected from the group consisting
of:
##STR00093##
wherein B is a modified or unmodified nucleobase, R.sup.1 and
R.sup.2 independently are H, halogen, OR.sub.3, or alkyl; and
R.sub.3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or
sugar. In one embodiment, the thermally destabilizing modification
in C1 is a mismatch selected from the group consisting of G:G, G:A,
G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and
optionally, at least one nucleobase in the mismatch pair is a
2'-deoxy nucleobase. In one example, the thermally destabilizing
modification in C1 is GNA or
##STR00094##
[0577] T1, T1', T2', and T3' each independently represent a
nucleotide comprising a modification providing the nucleotide a
steric bulk that is less or equal to the steric bulk of a 2'-OMe
modification. The modification can be at the 2' position of a
ribose sugar of the nucleotide, or a modification to a non-ribose
nucleotide, acyclic nucleotide, or the backbone of the nucleotide
that is similar or equivalent to the 2' position of the ribose
sugar, and provides the nucleotide a steric bulk that is less than
or equal to the steric bulk of a 2'-OMe modification. For example,
T1, T1', T2', and T3' are each independently selected from DNA,
RNA, LNA, 2'-F, and 2'-F-5'-methyl. In one embodiment, T1 is DNA.
In one embodiment, T1' is DNA, RNA or LNA. In one embodiment, T2'
is DNA or RNA. In one embodiment, T3' is DNA or RNA.
[0578] n.sup.1, n.sup.3, and q.sup.1 are independently 4 to 15
nucleotides in length.
[0579] n.sup.5, q.sup.3, and q.sup.7 are independently 1-6
nucleotide(s) in length.
[0580] n.sup.4, q.sup.2, and q.sup.6 are independently 1-3
nucleotide(s) in length.
[0581] q.sup.5 is independently 0-10 nucleotide(s) in length.
[0582] n.sup.2 and q.sup.4 are independently 0-3 nucleotide(s) in
length.
[0583] Alternatively, n.sup.4 is 0-3 nucleotide(s) in length.
[0584] In one embodiment, n.sup.4 can be 0. In one example, n.sup.4
is 0, and q.sup.2 and q.sup.6 are 1. In another example, n.sup.4 is
0, and q.sup.2 and q.sup.6 are 1, with two phosphorothioate
internucleotide linkage modifications within position 1-5 of the
sense strand (counting from the 5'-end of the sense strand), and
two phosphorothioate internucleotide linkage modifications at
positions 1 and 2 and two phosphorothioate internucleotide linkage
modifications within positions 18-23 of the antisense strand
(counting from the 5'-end of the antisense strand).
[0585] In one embodiment, n.sup.4, q.sup.2, and q.sup.6 are each
1.
[0586] In one embodiment, n.sup.2, n.sup.4, q.sup.2, q.sup.4, and
q.sup.6 are each 1.
[0587] In one embodiment, C1 is at position 14-17 of the 5'-end of
the sense strand, when the sense strand is 19-22 nucleotides in
length, and n.sup.4 is 1.
[0588] In one embodiment, T3' starts at position 2 from the 5' end
of the antisense strand. In one example, T3' is at position 2 from
the 5' end of the antisense strand and q.sup.6 is equal to 1.
[0589] In one embodiment, T1' starts at position 14 from the 5' end
of the antisense strand. In one example, T1' is at position 14 from
the 5' end of the antisense strand and q.sup.2 is equal to 1.
[0590] In one embodiment, T1' and T3' are separated by 11
nucleotides in length (i.e. not counting the T1' and T3'
nucleotides.
[0591] In one embodiment, T1' is at position 14 from the 5' end of
the antisense strand. In one example, T1' is at position 14 from
the 5' end of the antisense strand and q.sup.2 is equal to 1, and
the modification at the 2' position or positions in a non-ribose,
acyclic or backbone that provide less steric bulk than a 2'-OMe
ribose.
[0592] In one embodiment, T3' is at position 2 from the 5' end of
the antisense strand. In one example, T3' is at position 2 from the
5' end of the antisense strand and q.sup.6 is equal to 1, and the
modification at the 2' position or positions in a non-ribose,
acyclic or backbone that provide less than or equal to steric bulk
than a 2'-OMe ribose.
[0593] In one embodiment, T1 is at cleavage site of the sense
strand. In one example, T1 is at position 11 from the 5' end of the
sense strand, when the sense strand is 19-22 nucleotides in length,
and n.sup.2 is 1.
[0594] In one embodiment, T2' starts at position 6 from the 5' end
of the antisense strand. In one example, T2' is at positions 6-10
from the 5' end of the antisense strand, and q.sup.4 is 1.
[0595] In one embodiment, B1 is 2'-OMe or 2'-F, n.sup.1 is 8, T1 is
2'F, n.sup.2 is 3, B2 is 2'-OMe, n.sup.3 is 7, n.sup.4 is 0, B3 is
2'OMe, n.sup.5 is 3, B1' is 2'-OMe or 2'-F, q.sup.1 is 9, T1' is
2'-F, q.sup.2 is 1, B2' is 2'-OMe or 2'-F, q.sup.3 is 4, T2' is
2'-F, q.sup.4 is 2, B3' is 2'-OMe or 2'-F, q.sup.5 is 5, T3' is
2'-F, q.sup.6 is 1, B4' is 2'-OMe, and q.sup.7 is 1.
[0596] In one embodiment, B1 is 2'-OMe or 2'-F, n.sup.1 is 8, T1 is
2'F, n.sup.2 is 3, B2 is 2'-OMe, n.sup.3 is 7, n.sup.4 is 0, B3 is
2'-OMe, n.sup.5 is 3, B1' is 2'-OMe or 2'-F, q.sup.1 is 9, T1' is
2'-F, q.sup.2 is 1, B2' is 2'-OMe or 2'-F, q.sup.3 is 4, T2' is
2'-F, q.sup.4 is 2, B3' is 2'-OMe or 2'-F, q.sup.5 is 5, T3' is
2'-F, q.sup.6 is 1, B4' is 2'-OMe, and q.sup.7 is 1; with two
phosphorothioate internucleotide linkage modifications within
position 1-5 of the sense strand (counting from the 5'-end of the
sense strand), and two phosphorothioate internucleotide linkage
modifications at positions 1 and 2 and two phosphorothioate
internucleotide linkage modifications within positions 18-23 of the
antisense strand (counting from the 5'-end of the antisense
strand).
[0597] In one embodiment, B1 is 2'-OMe or 2'-F, n.sup.1 is 8, T1 is
2'F, n.sup.2 is 3, B2 is 2'-OMe, n.sup.3 is 7, n.sup.4 is 0, B3 is
2'-OMe, n.sup.5 is 3, B1' is 2'-OMe or 2'-F, q.sup.1 is 9, T1' is
2'-F, q.sup.2 is 1, B2' is 2'-OMe or 2'-F, q.sup.3 is 4, q.sup.4 is
0, B3' is 2'-OMe or 2'-F, q.sup.5 is 7, T3' is 2'-F, q.sup.6 is 1,
B4' is 2'-OMe, and q.sup.7 is 1.
[0598] In one embodiment, B1 is 2'-OMe or 2'-F, n.sup.1 is 8, T1 is
2'F, n.sup.2 is 3, B2 is 2'-OMe, n.sup.3 is 7, n.sup.4 is 0, B3 is
2'-OMe, n.sup.5 is 3, B1' is 2'-OMe or 2'-F, q.sup.1 is 9, T1' is
2'-F, q.sup.2 is 1, B2' is 2'-OMe or 2'-F, q.sup.3 is 4, q.sup.4 is
0, B3' is 2'-OMe or 2'-F, q.sup.5 is 7, T3' is 2'-F, q.sup.6 is 1,
B4' is 2'-OMe, and q.sup.7 is 1; with two phosphorothioate
internucleotide linkage modifications within position 1-5 of the
sense strand (counting from the 5'-end), and two phosphorothioate
internucleotide linkage modifications at positions 1 and 2 and two
phosphorothioate internucleotide linkage modifications within
positions 18-23 of the antisense strand (counting from the
5'-end).
[0599] In one embodiment, B1 is 2'-OMe or 2'-F, n.sup.1 is 8, T1 is
2'F, n.sup.2 is 3, B2 is 2'-OMe, n.sup.3 is 7, n.sup.4 is 0, B3 is
2'OMe, n.sup.5 is 3, B1' is 2'-OMe or 2'-F, q.sup.1 is 9, T1' is
2'-F, q.sup.2 is 1, B2' is 2'-OMe or 2'-F, q.sup.3 is 4, T2' is
2'-F, q.sup.4 is 2, B3' is 2'-OMe or 2'-F, q.sup.5 is 5, T3' is
2'-F, q.sup.6 is 1, B4' is 2'-F, and q.sup.7 is 1.
[0600] In one embodiment, B1 is 2'-OMe or 2'-F, n.sup.1 is 8, T1 is
2'F, n.sup.2 is 3, B2 is 2'-OMe, n.sup.3 is 7, n.sup.4 is 0, B3 is
2'-OMe, n.sup.5 is 3, B1' is 2'-OMe or 2'-F, q.sup.1 is 9, T1' is
2'-F, q.sup.2 is 1, B2' is 2'-OMe or 2'-F, q.sup.3 is 4, T2' is
2'-F, q.sup.4 is 2, B3' is 2'-OMe or 2'-F, q.sup.5 is 5, T3' is
2'-F, q.sup.6 is 1, B4' is 2'-F, and q.sup.7 is 1; with two
phosphorothioate internucleotide linkage modifications within
position 1-5 of the sense strand (counting from the 5'-end of the
sense strand), and two phosphorothioate internucleotide linkage
modifications at positions 1 and 2 and two phosphorothioate
internucleotide linkage modifications within positions 18-23 of the
antisense strand (counting from the 5'-end of the antisense
strand).
[0601] In one embodiment, B1 is 2'-OMe or 2'-F, n.sup.1 is 8, T1 is
2'F, n.sup.2 is 3, B2 is 2'-OMe, n.sup.3 is 7, n.sup.4 is 0, B3 is
2'-OMe, n.sup.5 is 3, B1' is 2'-OMe or 2'-F, q.sup.1 is 9, T1' is
2'-F, q.sup.2 is 1, B2' is 2'-OMe or 2'-F, q.sup.3 is 4, q.sup.4 is
0, B3' is 2'-OMe or 2'-F, q.sup.5 is 7, T3' is 2'-F, q.sup.6 is 1,
B4' is 2'-F, and q.sup.7 is 1.
[0602] In one embodiment, B1 is 2'-OMe or 2'-F, n.sup.1 is 8, T1 is
2'F, n.sup.2 is 3, B2 is 2'-OMe, n.sup.3 is 7, n.sup.4 is 0, B3 is
2'-OMe, n.sup.5 is 3, B1' is 2'-OMe or 2'-F, q.sup.1 is 9, T1' is
2'-F, q.sup.2 is 1, B2' is 2'-OMe or 2'-F, q.sup.3 is 4, q.sup.4 is
0, B3' is 2'-OMe or 2'-F, q.sup.5 is 7, T3' is 2'-F, q.sup.6 is 1,
B4' is 2'-F, and q.sup.7 is 1; with two phosphorothioate
internucleotide linkage modifications within position 1-5 of the
sense strand (counting from the 5'-end of the sense strand), and
two phosphorothioate internucleotide linkage modifications at
positions 1 and 2 and two phosphorothioate internucleotide linkage
modifications within positions 18-23 of the antisense strand
(counting from the 5'-end of the antisense strand).
[0603] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%,
60%, 55%, 50%, 45%, 40%, 35% or 30% of the dsRNA agent of the
invention is modified.
[0604] In one embodiment, each of the sense and antisense strands
of the dsRNA agent is independently modified with acyclic
nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl,
2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro,
2'-O--N-methylacetamido (2'-O-NMA), a 2'-O-dimethylaminoethoxyethyl
(2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.
[0605] In one embodiment, each of the sense and antisense strands
of the dsRNA agent contains at least two different
modifications.
[0606] In one embodiment, the dsRNA agent of Formula (I) further
comprises 3' and/or 5' overhang(s) of 1-10 nucleotides in length.
In one example, dsRNA agent of formula (I) comprises a 3' overhang
at the 3'-end of the antisense strand and a blunt end at the 5'-end
of the antisense strand. In another example, the dsRNA agent has a
5' overhang at the 5'-end of the sense strand.
[0607] In one embodiment, the dsRNA agent of the invention does not
contain any 2'-F modification.
[0608] In one embodiment, the sense strand and/or antisense strand
of the dsRNA agent comprises one or more blocks of phosphorothioate
or methylphosphonate internucleotide linkages. In one example, the
sense strand comprises one block of two phosphorothioate or
methylphosphonate internucleotide linkages. In one example, the
antisense strand comprises two blocks of two phosphorothioate or
methylphosphonate internucleotide linkages. For example, the two
blocks of phosphorothioate or methylphosphonate internucleotide
linkages are separated by 16-18 phosphate internucleotide
linkages.
[0609] In one embodiment, each of the sense and antisense strands
of the dsRNA agent has 15-30 nucleotides. In one example, the sense
strand has 19-22 nucleotides, and the antisense strand has 19-25
nucleotides. In another example, the sense strand has 21
nucleotides, and the antisense strand has 23 nucleotides.
[0610] In one embodiment, the nucleotide at position 1 of the
5'-end of the antisense strand in the duplex is selected from the
group consisting of A, dA, dU, U, and dT. In one embodiment, at
least one of the first, second, and third base pair from the 5'-end
of the antisense strand is an AU base pair.
[0611] In one embodiment, the antisense strand of the dsRNA agent
of the invention is 100% complementary to a target RNA to hybridize
thereto and inhibits its expression through RNA interference. In
another embodiment, the antisense strand of the dsRNA agent of the
invention is at least 95%, at least 90%, at least 85%, at least
80%, at least 75%, at least 70%, at least 65%, at least 60%, at
least 55%, or at least 50% complementary to a target RNA.
[0612] In one aspect, the invention relates to a dsRNA agent
capable of inhibiting the expression of a target gene. The dsRNA
agent comprises a sense strand and an antisense strand, each strand
having 14 to 40 nucleotides. The sense strand contains at least one
thermally destabilizing nucleotide, wherein at at least one said
thermally destabilizing nucleotide occurs at or near the site that
is opposite to the seed region of the antisense strand (i.e. at
position 2-8 of the 5'-end of the antisense strand), For example,
the thermally destabilizing nucleotide occurs between positions
14-17 of the 5'-end of the sense strand when the sense strand is 21
nucleotides in length. The antisense strand contains at least two
modified nucleic acids that are smaller than a sterically demanding
2'-OMe modification. Preferably, the two modified nucleic acids
that is smaller than a sterically demanding 2'-OMe are separated by
11 nucleotides in length. For example, the two modified nucleic
acids are at positions 2 and 14 of the 5'end of the antisense
strand.
[0613] In one embodiment, the sense strand sequence of the dsRNA
agent is represented by formula (Is):
##STR00095##
[0614] wherein: [0615] B1, B2, and B3 each independently represent
a nucleotide containing a modification selected from the group
consisting of 2'-Oalkyl, 2'-substituted alkoxy, 2'-substituted
alkyl, 2'-halo, ENA, and BNA/LNA; [0616] C1 is a thermally
destabilizing nucleotide (e.g., acyclic nucleotide such as UNA or
GNA, mismatch, abasic, or DNA) placed at the opposite of the
antisense seed region (i.e., positions 2-8 of the 5'-end of the
antisense strand); [0617] T1 represents a nucleotide comprising a
chemical modification at the 2' position or equivalent position in
a non-ribose, acyclic or backbone that provide the nucleotide a
less steric bulk than a 2'-OMe modification; for example, T1 is
selected from the group consisting of DNA, RNA, LNA, 2'-F, and
2'-F-5'-methyl; [0618] n.sup.1 or n.sup.3 is independently 4 to 15
nucleotides in length; [0619] n.sup.5 is 1-6 nucleotide(s) in
length; [0620] n.sup.4 is 1-3 nucleotide(s) in length; and [0621]
n.sup.2 is 0-3 nucleotide(s) in length.
[0622] In one embodiment, the sense strand sequence having 19, 20,
21, or 22 nucleotides in length of the dsRNA agent is represented
by formula (Is):
##STR00096##
[0623] wherein: [0624] B1, B2, and B3 each independently represent
a nucleotide containing a modification selected from the group
consisting of 2'-Oalkyl, 2'-substituted alkoxy, 2'-substituted
alkyl, 2'-halo, ENA, and BNA/LNA; [0625] C1 is a thermally
destabilizing nucleotide (e.g., acyclic nucleotide such as UNA or
GNA, mismatch, abasic, or DNA) placed at the opposite of the
antisense seed region (i.e., positions 2-8 of the 5'-end of the
antisense strand); [0626] T1 represents a nucleotide comprising a
chemical modification selected from the group consisting of DNA,
RNA, LNA, 2'-F, and 2'-F-5'-methyl; [0627] n.sup.1 or n.sup.3 is
independently 4 to 15 nucleotides in length; [0628] n.sup.5 is 1-6
nucleotide(s) in length; [0629] n.sup.4 is 1-3 nucleotide(s) in
length; and [0630] n.sup.2 is 0-3 nucleotide(s) in length.
[0631] In one embodiment, the dsRNA agent of formula (Is) further
comprises 3' and/or 5' overhang(s) of 1-10 nucleotides in length.
In one example, the dsRNA agent of formula (Is) comprises a 5'
overhang.
[0632] In one embodiment, C1 comprises one thermally destabilizing
nucleotide at position 14, 15, 16 or 17 from the 5'-end of the
sense strand. For example, C1 is an acyclic nucleotide (e.g., UNA
or GNA), mismatch, abasic, or DNA. In one specific example, C1 is a
GNA.
[0633] In one embodiment, T1 comprises a DNA, RNA, LNA, 2'-F, or
2'-F-5'-methyl at position 11 from the 5'-end of the sense
strand.
[0634] In one embodiment, the dsRNA agent of the invention
comprises a sense strand (Is), wherein C1 is an acyclic nucleotide
(e.g., UNA or GNA), mismatch, abasic, or DNA; and T1 comprises a
DNA, RNA, LNA, 2'-F, or 2'-F-5'-methyl at position 11 from the
5'-end of the sense strand.
[0635] In one embodiment, the antisense strand sequence of the
dsRNA agent is represented by formula (Ia):
##STR00097##
[0636] wherein: [0637] B1', B2', B3', and B4' each independently
represent a nucleotide containing a modification selected from the
group consisting of 2'-Oalkyl, 2'-substituted alkoxy,
2'-substituted alkyl, 2'-halo, ENA, and BNA/LNA; [0638] T1', T2',
and T3' each independently represent a nucleotide comprising a
chemical modification at the 2' position or equivalent position in
a non-ribose, acyclic or backbone that provide the nucleotide a
less steric bulk than a 2'-OMe modification; [0639] for example,
T1', T2', and T3' each are independently selected from the group
consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; [0640] q is
independently 4 to 15 nucleotides in length; [0641] q.sup.3 or
q.sup.7 is independently 1-6 nucleotide(s) in length; [0642]
q.sup.2 or q.sup.6 is independently 1-3 nucleotide(s) in length;
[0643] q.sup.4 is independently 0-3 nucleotide(s) in length; and
[0644] q.sup.5 is independently 0-10 nucleotide(s) in length.
[0645] In one embodiment, the antisense strand sequence having 19,
20, 21, 22, 23, 24, or 25 nucleotides in length of the dsRNA agent
is represented by formula (Ia):
##STR00098##
[0646] wherein: [0647] B1', B2', B3', and B4' each independently
represent a nucleotide containing a modification selected from the
group consisting of 2'-Oalkyl, 2'-substituted alkoxy,
2'-substituted alkyl, 2'-halo, ENA, and BNA/LNA; [0648] T1', T2',
and T3' each independently represent a nucleotide comprising a
chemical modification selected from the group consisting of DNA,
RNA, LNA, 2'-F, and 2'-F-5'-methyl; [0649] q is independently 4 to
15 nucleotides in length; [0650] q.sup.3 or q.sup.7 is
independently 1-6 nucleotide(s) in length; [0651] q.sup.2 or
q.sup.6 is independently 1-3 nucleotide(s) in length; [0652]
q.sup.4 is independently 0-3 nucleotide(s) in length; and [0653]
q.sup.5 is independently 0-10 nucleotide(s) in length.
[0654] In one embodiment, dsRNA of formula (Ia) further comprises
3' and/or 5' overhang(s) of 1-10 nucleotides in length. In one
example, dsRNA of formula (Ia) comprises a 3' overhang.
[0655] In one embodiment, the invention relates to a
double-stranded RNA (dsRNA) agent for inhibiting the expression of
a target gene. The dsRNA agent comprises a sense strand and an
antisense strand, each strand having 14 to 40 nucleotides:
##STR00099##
[0656] wherein: [0657] B1, B2, B3, B1', B2', B3', and B4' each
independently represent a nucleotide containing a modification
selected from the group consisting of 2'-Oalkyl, 2'-substituted
alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA/LNA; C1 is an
acyclic nucleotide (e.g., UNA or GNA); [0658] T1, T1', T2', and T3'
each independently represent a nucleotide comprising a chemical
modification selected from the group consisting of DNA, RNA, LNA,
2'-F, and 2'-F-5'-methyl; [0659] n.sup.1, n.sup.3, or q.sup.1 is
independently 4 to 15 nucleotides in length; [0660] n.sup.5,
q.sup.3 or q.sup.7 is independently 1-6 nucleotide(s) in length;
[0661] n.sup.4, q.sup.2 or q.sup.6 is independently 1-3
nucleotide(s) in length; [0662] n.sup.2 or q.sup.4 is independently
0-3 nucleotide(s) in length; [0663] q.sup.5 is independently 0-10
nucleotide(s) in length; and [0664] wherein the dsRNA agent has 3'
and/or 5' overhang(s) of 1-10 nucleotides in length of the
antisense and/or sense strand(s).
[0665] In one embodiment, the invention relates to a
double-stranded RNA (dsRNA) agent for inhibiting the expression of
a target gene. The dsRNA agent comprises a sense strand and an
antisense strand, each strand having 14 to 40 nucleotides:
##STR00100##
[0666] wherein: [0667] B1, B2, B3, B1', B2', B3', and B4' each
independently represent a nucleotide containing a modification
selected from the group consisting of 2'-Oalkyl, 2'-substituted
alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA/LNA; C1 is an
acyclic nucleotide (e.g., UNA or GNA); [0668] T1, T1', T2', and T3'
each independently represent a nucleotide comprising a chemical
modification selected from the group consisting of DNA, RNA, LNA,
2'-F, and 2'-F-5'-methyl; [0669] n.sup.1, n.sup.3, or q.sup.1 is
independently 4 to 15 nucleotides in length; [0670] n.sup.5,
q.sup.3 or q.sup.7 is independently 1-6 nucleotide(s) in length;
[0671] n.sup.4, q.sup.2 or q.sup.6 is independently 1-3
nucleotide(s) in length; [0672] n.sup.2 or q.sup.4 is independently
0-3 nucleotide(s) in length; [0673] q.sup.5 is independently 0-10
nucleotide(s) in length; and [0674] wherein the dsRNA agent has a
3' overhang of 2 nucleotides in length at the 3'-end of the
antisense.
[0675] In one embodiment, the invention relates to a
double-stranded RNA (dsRNA) agent for inhibiting the expression of
a target gene. The dsRNA agent comprises a sense strand and an
antisense strand, each strand having 15-30 nucleotides:
##STR00101##
[0676] wherein: [0677] B1, B2, B3, B1', B2', B3', and B4' each
independently represent a nucleotide containing a modification
2'-OMe; [0678] C1 is an acyclic nucleotide GNA; [0679] T1, T1',
T2', and T3' each are independently DNA or RNA; [0680] n.sup.1,
n.sup.3, or q.sup.1 is independently 4 to 15 nucleotides in length;
[0681] n.sup.5, q.sup.3 or q.sup.7 is independently 1-6
nucleotide(s) in length; [0682] n.sup.4, q.sup.2 or q.sup.6 is
independently 1-3 nucleotide(s) in length; [0683] n.sup.2 or
q.sup.4 is independently 0-3 nucleotide(s) in length; [0684]
q.sup.5 is independently 0-10 nucleotide(s) in length; and [0685]
wherein the dsRNA agent has a 3' overhang of 1-6 nucleotides in
length at the 3'-end of the antisense.
[0686] In one embodiment, the invention relates to a
double-stranded RNA (dsRNA) agent for inhibiting the expression of
a target gene. The dsRNA agent comprises a sense strand and an
antisense strand, each strand having 19-23 nucleotides:
##STR00102##
[0687] wherein: [0688] B1, B2, B3, B1', B2', B3', and B4' each
independently represent a nucleotide containing a 2'-OMe
modification; [0689] C1 is an acyclic nucleotide GNA; [0690] T1,
T1', T2', and T3' are independently DNA or RNA; [0691] n.sup.1,
n.sup.3, q.sup.1, or q.sup.3 is independently 4 to 15 nucleotides
in length; [0692] n.sup.5, q.sup.3 or q.sup.7 is independently 1-6
nucleotide(s) in length; [0693] n.sup.4, q.sup.2 or q.sup.6 is
independently 1-3 nucleotide(s) in length; [0694] n.sup.2, q.sup.4
or q.sup.5 is independently 0-3 nucleotide(s) in length; [0695]
q.sup.5 is independently 0-10 nucleotide(s) in length; and [0696]
wherein the dsRNA agent has a 3' overhang of 2 nucleotides in
length at the 3'-end of the antisense.
[0697] In one embodiment, the invention relates to a
double-stranded RNA (dsRNA) agent for inhibiting the expression of
a target gene. The dsRNA agent comprises a sense strand and an
antisense strand, each strand having 14 to 40 nucleotides:
##STR00103## [0698] wherein: [0699] B1, B2, B3, B1', B2', B3', and
B4' each independently represent a nucleotide containing a
modification selected from the group consisting of 2'-Oalkyl,
2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and
BNA/LNA; [0700] C1 is an acyclic nucleotide (e.g., UNA or GNA);
[0701] T1, T1', T2', and T3' each independently represent a
nucleotide comprising a chemical modification selected from the
group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; [0702]
n.sup.1, n.sup.3, or q.sup.1 is independently 4 to 15 nucleotides
in length; [0703] n.sup.5, q.sup.3 or q.sup.7 is independently 1-6
nucleotide(s) in length; [0704] n.sup.4, q.sup.2 or q.sup.6 is
independently 1-3 nucleotide(s) in length; [0705] n.sup.2 or
q.sup.4 is independently 0-3 nucleotide(s) in length; [0706]
q.sup.5 is independently 0-10 nucleotide(s) in length; and [0707]
wherein the dsRNA agent has a 5' overhang of 1-10 nucleotides in
length at the 5'-end of the sense.
[0708] In one embodiment, the invention relates to a
double-stranded RNA (dsRNA) agent for inhibiting the expression of
a target gene. The dsRNA agent comprises a sense strand and an
antisense strand, each strand having 14 to 40 nucleotides:
##STR00104## [0709] wherein: [0710] B1, B2, B3, B1', B2', B3', and
B4' each independently represent a nucleotide containing a
modification selected from the group consisting of 2'-Oalkyl,
2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and
BNA/LNA; [0711] C1 is an acyclic nucleotide (e.g., UNA or GNA);
[0712] T1, T1', T2', and T3' each independently represent a
nucleotide comprising a chemical modification selected from the
group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; [0713]
n.sup.1, n.sup.3, or q.sup.1 is independently 4 to 15 nucleotides
in length; [0714] n.sup.5, q.sup.3 or q.sup.7 is independently 1-6
nucleotide(s) in length; [0715] n.sup.4, q.sup.2 or q.sup.6 is
independently 1-3 nucleotide(s) in length; [0716] n.sup.2 or
q.sup.4 is independently 0-3 nucleotide(s) in length; [0717]
q.sup.5 is independently 0-10 nucleotide(s) in length; and [0718]
wherein the dsRNA agent has a 5' overhang of 1-6 nucleotides in
length at the 5'-end of the sense.
[0719] In one embodiment, the invention relates to a
double-stranded RNA (dsRNA) agent for inhibiting the expression of
a target gene. The dsRNA agent comprises a sense strand and an
antisense strand, each strand having 14 to 40 nucleotides:
##STR00105## [0720] wherein: [0721] B1, B2, B3, B1', B2', B3', and
B4' each independently represent a nucleotide containing a
modification selected from the group consisting of 2'-Oalkyl,
2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and
BNA/LNA; [0722] C1 is an acyclic nucleotide (e.g., UNA or GNA);
[0723] T1, T1', T2', and T3' each independently represent a
nucleotide comprising a chemical modification selected from the
group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; [0724]
n.sup.1, n.sup.3, or q.sup.1 is independently 4 to 15 nucleotides
in length; [0725] n.sup.5, q.sup.3 or q.sup.7 is independently 1-6
nucleotide(s) in length; [0726] n.sup.4, q.sup.2 or q.sup.6 is
independently 1-3 nucleotide(s) in length; [0727] n.sup.2 or
q.sup.4 is independently 0-3 nucleotide(s) in length; [0728]
q.sup.5 is independently 0-10 nucleotide(s) in length; and [0729]
wherein the dsRNA agent has a 5' overhang of 1-10 nucleotides in
length at the 5'-end of the sense and a 3' overhang of 1-10
nucleotides in length at the 5'-end of the antisense strand.
Thermally Destabilizing Modifications
[0730] The dsRNA agent can be optimized for RNA interference by
increasing the propensity of the dsRNA duplex to disassociate or
melt (decreasing the free energy of duplex association) by
introducing a thermally destabilizing modification in the sense
strand at a site opposite to the seed region of the antisense
strand (i.e., at positions 2-8 of the 5'-end of the antisense
strand). This modification can increase the propensity of the
duplex to disassociate or melt in the seed region of the antisense
strand.
[0731] The thermally destabilizing modifications can include abasic
modification; mismatch with the opposing nucleotide in the opposing
strand; and sugar modification such as 2'-deoxy modification or
acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycerol
nuceltic acid (GNA).
[0732] Exemplified abasic modifications are:
##STR00106##
[0733] Exemplified sugar modifications are:
##STR00107##
[0734] The term "acyclic nucleotide" refers to any nucleotide
having an acyclic ribose sugar, for example, where any of bonds
between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4',
C4'-O4', or C1'-O4') is absent and/or at least one of ribose
carbons or oxygen (e.g., C1', C2', C3', C4' or O4') are
independently or in combination absent from the nucleotide. In some
embodiments, acyclic nucleotide is
##STR00108##
wherein B is a modified or unmodified nucleobase, R.sup.1 and
R.sup.2 independently are H, halogen, OR.sub.3, or alkyl; and
R.sub.3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or
sugar). The term "UNA" refers to unlocked acyclic nucleic acid,
wherein any of the bonds of the sugar has been removed, forming an
unlocked "sugar" residue. In one example, UNA also encompasses
monomers with bonds between C1'-C4' being removed (i.e. the
covalent carbon-oxygen-carbon bond between the C1' and C4'
carbons). In another example, the C2'-C3' bond (i.e. the covalent
carbon-carbon bond between the C2' and C3' carbons) of the sugar is
removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059
(1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which
are hereby incorporated by reference in their entirety). The
acyclic derivative provides greater backbone flexibility without
affecting the Watson-Crick pairings. The acyclic nucleotide can be
linked via 2'-5' or 3'-5' linkage.
[0735] The term `GNA` refers to glycol nucleic acid which is a
polymer similar to DNA or RNA but differing in the composition of
its "backbone" in that is composed of repeating glycerol units
linked by phosphodiester bonds:
##STR00109##
[0736] The thermally destabilizing modification can be mismatches
(i.e., noncomplementary base pairs) between the thermally
destabilizing nucleotide and the opposing nucleotide in the
opposite strand within the dsRNA duplex. Exemplary mismatch
basepairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U,
T:T, U:T, or a combination thereof. Other mismatch base pairings
known in the art are also amenable to the present invention. A
mismatch can occur between nucleotides that are either naturally
occurring nucleotides or modified nucleotides, i.e., the mismatch
base pairing can occur between the nucleobases from respective
nucleotides independent of the modifications on the ribose sugars
of the nucleotides. In certain embodiments, the dsRNA agent
contains at least one nucleobase in the mismatch pairing that is a
2'-deoxy nucleobase; e.g., the 2'-deoxy nucleobase is in the sense
strand.
[0737] More examples of abasic nucleotide, acyclic nucleotide
modifications (including UNA and GNA), and mismatch modifications
have been described in detail in WO 2011/133876, which is herein
incorporated by reference in its entirety.
[0738] The thermally destabilizing modifications may also include
universal base with reduced or abolished capability to form
hydrogen bonds with the opposing bases, and phosphate
modifications.
[0739] Nucleobase modifications with impaired or completely
abolished capability to form hydrogen bonds with bases in the
opposite strand have been evaluated for destabilization of the
central region of the dsRNA duplex as described in WO 2010/0011895,
which is herein incorporated by reference in its entirety.
Exemplary nucleobase modifications are:
##STR00110##
[0740] Exemplary phosphate modifications known to decrease the
thermal stability of dsRNA duplexes compared to natural
phosphodiester linkages are:
##STR00111##
[0741] In one embodiment, the dsRNA agent of the invention can
comprise 2'-5' linkages (with 2'-H, 2'-OH and 2'-OMe and with
P.dbd.O or P.dbd.S). For example, the 2'-5' linkages modifications
can be used to promote nuclease resistance or to inhibit binding of
the sense to the antisense strand, or can be used at the 5' end of
the sense strand to avoid sense strand activation by RISC.
[0742] In another embodiment, the dsRNA agent of the invention can
comprise L sugars (e.g., L ribose, L-arabinose with 2'-H, 2'-OH and
2'-OMe). For example, these L sugars modifications can be used to
promote nuclease resistance or to inhibit binding of the sense to
the antisense strand, or can be used at the 5' end of the sense
strand to avoid sense strand activation by RISC.
[0743] In one embodiment, the dsRNA agent is a multimer containing
at least two duplexes represented by formula (I), wherein said
duplexes are connected by a linker. The linker can be cleavable or
non-cleavable. Optionally, said multimer further comprise a ligand.
Each of the dsRNA agent can target the same gene or two different
genes; or each of the dsRNA agent can target same gene at two
different target sites.
[0744] In one embodiment, the dsRNA agent is a multimer containing
three, four, five, six or more duplexes represented by formula (I),
wherein said duplexes are connected by a linker. The linker can be
cleavable or non-cleavable. Optionally, said multimer further
comprises a ligand. Each of the dsRNA agent can target the same
gene or two different genes; or each of the dsRNA agent can target
same gene at two different target sites.
[0745] In one embodiment, two dsRNA agent represented by formula
(I) are linked to each other at the 5' end, and one or both of the
3' ends of the are optionally conjugated to a ligand. Each of the
dsRNA can target the same gene or two different genes; or each of
the dsRNA can target same gene at two different target sites.
[0746] Various publications described multimeric siRNA and can all
be used with the dsRNA of the invention. Such publications include
WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511,
WO2007/117686, WO2009/014887 and WO2011/031520 which are hereby
incorporated by their entirely.
[0747] The dsRNA agent that contains conjugations of one or more
carbohydrate moieties to a dsRNA agent can optimize one or more
properties of the dsRNA agent. In many cases, the carbohydrate
moiety will be attached to a modified subunit of the dsRNA agent.
E.g., the ribose sugar of one or more ribonucleotide subunits of a
dsRNA agent can be replaced with another moiety, e.g., a
non-carbohydrate (preferably cyclic) carrier to which is attached a
carbohydrate ligand. A ribonucleotide subunit in which the ribose
sugar of the subunit has been so replaced is referred to herein as
a ribose replacement modification subunit (RRMS). A cyclic carrier
may be a carbocyclic ring system, i.e., all ring atoms are carbon
atoms, or a heterocyclic ring system, i.e., one or more ring atoms
may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic
carrier may be a monocyclic ring system, or may contain two or more
rings, e.g. fused rings. The cyclic carrier may be a fully
saturated ring system, or it may contain one or more double
bonds.
[0748] The ligand may be attached to the polynucleotide via a
carrier. The carriers include (i) at least one "backbone attachment
point," preferably two "backbone attachment points" and (ii) at
least one "tethering attachment point." A "backbone attachment
point" as used herein refers to a functional group, e.g. a hydroxyl
group, or generally, a bond available for, and that is suitable for
incorporation of the carrier into the backbone, e.g., the
phosphate, or modified phosphate, e.g., sulfur containing,
backbone, of a ribonucleic acid. A "tethering attachment point"
(TAP) in some embodiments refers to a constituent ring atom of the
cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from
an atom which provides a backbone attachment point), that connects
a selected moiety. The moiety can be, e.g., a carbohydrate, e.g.
monosaccharide, disaccharide, trisaccharide, tetrasaccharide,
oligosaccharide and polysaccharide. Optionally, the selected moiety
is connected by an intervening tether to the cyclic carrier. Thus,
the cyclic carrier will often include a functional group, e.g., an
amino group, or generally, provide a bond, that is suitable for
incorporation or tethering of another chemical entity, e.g., a
ligand to the constituent ring.
[0749] In one embodiment the dsRNA agent of the invention is
conjugated to a ligand via a carrier, wherein the carrier can be
cyclic group or acyclic group; preferably, the cyclic group is
selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,
imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,
[1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,
thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,
tetrahydrofuryl and decalin; preferably, the acyclic group is
selected from serinol backbone or diethanolamine backbone.
[0750] The double-stranded RNA (dsRNA) agent of the invention may
optionally be conjugated to one or more ligands. The ligand can be
attached to the sense strand, antisense strand or both strands, at
the 3'-end, 5'-end or both ends. For instance, the ligand may be
conjugated to the sense strand, in particular, the 3'-end of the
sense strand.
[0751] In one embodiment dsRNA agents of the invention are 5'
phosphorylated or include a phosphoryl analog at the 5' prime
terminus. 5'-phosphate modifications include those which are
compatible with RISC mediated gene silencing. Suitable
modifications include: 5'-monophosphate ((HO)2(O)P--O-5');
5'-diphosphate ((HO)2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate
((HO)2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap structure
(N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5');
5'-monothiophosphate (phosphorothioate; (HO)2(S)P--O-5');
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO).sub.2(O)P--S--5'); any additional
combination of oxygen/sulfur replaced monophosphate, diphosphate
and triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO).sub.2(O)P--NH-5', (HO)(NH.sub.2)(O)P--O-5'),
5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl,
etc., e.g. RP(OH)(O)--O-5'-, 5'-alkenylphosphonates (i.e. vinyl,
substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates
(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.
RP(OH)(O)--O-5'-). In one example, the modification can in placed
in the antisense strand of a dsRNA agent.
REVERSIR Compounds
[0752] In certain instances it is desirable to inhibit siRNA
activity. For example, in certain embodiments where the siRNA
target is an mRNA, it is may be desirable to inhibit siRNA activity
and thereby restore expression of a target protein. For example,
certain siRNAs have been used therapeutically. In certain such
uses, siRNAs are long-acting. In certain instances, such long
acting siRNAs are desirable, for their convenience. In such
instances, though, it can also be desirable to have a means to
reverse the activity of an siRNA. For example, a patient may
respond poorly to treatment or receive too high a dose. In such an
instance, a reverser compound can be administered to at least
partially reduce the RNAi activity of the siRNA. In certain
embodiments, the long-lasting effect of siRNA makes waiting for
that effect to slowly diminish through natural clearance an
unattractive option.
[0753] By way of example, and without limiting the present
invention, certain siRNAs are useful for inhibiting blood clotting
factors (e.g., Factor II (prothrombin), Factor VII, Factor IX,
etc.). Such siRNAs have therapeutic potential as anticoagulants.
Long half-lives make such siRNAs particularly attractive, however,
if a patient receives too high a dose, has surgery (where
anti-coagulation is undesirable) or otherwise desires a decrease in
the anti-coagulant effect, a reverser compound to the
anti-coagulant siRNA can be administered. Such REVERSIR compound
will restore coagulation function more quickly than simply waiting
for natural clearance of the siRNA. This example is provided for
illustrative purposes. Many siRNAs have been designed to a vast
number of targets, including without limitation, a vast number of
messenger RNA (mRNA) targets and pre-mRNA targets, as well as a
vast number of non-coding RNA targets. REVERSIR compounds provided
herein are suitable for any siRNA, regardless of the target or
mechanism of the siRNA compound.
[0754] In certain embodiments, the invention provides REVERSIR
compounds to an siRNA targeted to an mRNA. In certain such
embodiments, the target mRNA encodes a protein involved in
metabolism. In certain such embodiments, the target mRNA encodes a
protein involved in cardiac function. In certain embodiments, the
target mRNA encodes a protein involved in blood-clotting. Exemplary
siRNA compounds targeting any of a variety of target proteins are
known in the art. Further, methods for preparing siRNA against a
target gene are well known in the art and readily available to one
of skill in the art.
[0755] Without limitations, target genes for siRNAs include, but
are not limited to genes promoting unwanted cell proliferation,
growth factor gene, growth factor receptor gene, genes expressing
kinases, an adaptor protein gene, a gene encoding a G protein super
family molecule, a gene encoding a transcription factor, a gene
which mediates angiogenesis, a viral gene, a gene required for
viral replication, a cellular gene which mediates viral function, a
gene of a bacterial pathogen, a gene of an amoebic pathogen, a gene
of a parasitic pathogen, a gene of a fungal pathogen, a gene which
mediates an unwanted immune response, a gene which mediates the
processing of pain, a gene which mediates a neurological disease,
an allene gene found in cells characterized by loss of
heterozygosity, or one allege gene of a polymorphic gene.
[0756] Specific exemplary target genes for the siRNAs include, but
are not limited to, AT3, AGT, ALAS1, TMPR, HAO1, AGT, C5, CCR-5,
PDGF beta gene; Erb-B gene, Src gene; CRK gene; GRB2 gene; RAS
gene; MEKK gene; JNK gene; RAF gene; Erk1/2 gene; PCNA(p21) gene;
MYB gene; c-MYC gene; JUN gene; FOS gene; BCL-2 gene; Cyclin D
gene; VEGF gene; EGFR gene; Cyclin A gene; Cyclin E gene; WNT-1
gene; beta-catenin gene; c-MET gene; PKC gene; NFKB gene; STAT3
gene; survivin gene; Her2/Neu gene; topoisomerase I gene;
topoisomerase II alpha gene; p73 gene; p21(WAF1/CIP1) gene,
p27(KIP1) gene; PPM1D gene; caveolin I gene; MIB I gene; MTAI gene;
M68 gene; tumor suppressor genes; p53 gene; DN-p63 gene; pRb tumor
suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor
gene; PTEN tumor suppressor gene; MLL fusion genes, e.g., MLL-AF9,
BCR/ABL fusion gene; TEL/AML1 fusion gene; EWS/FLI1 fusion gene;
TLS/FUS1 fusion gene; PAX3/FKHR fusion gene; AML1/ETO fusion gene;
alpha v-integrin gene; Flt-1 receptor gene; tubulin gene; Human
Papilloma Virus gene, a gene required for Human Papilloma Virus
replication, Human Immunodeficiency Virus gene, a gene required for
Human Immunodeficiency Virus replication, Hepatitis A Virus gene, a
gene required for Hepatitis A Virus replication, Hepatitis B Virus
gene, a gene required for Hepatitis B Virus replication, Hepatitis
C Virus gene, a gene required for Hepatitis C Virus replication,
Hepatitis D Virus gene, a gene required for Hepatitis D Virus
replication, Hepatitis E Virus gene, a gene required for Hepatitis
E Virus replication, Hepatitis F Virus gene, a gene required for
Hepatitis F Virus replication, Hepatitis G Virus gene, a gene
required for Hepatitis G Virus replication, Hepatitis H Virus gene,
a gene required for Hepatitis H Virus replication, Respiratory
Syncytial Virus gene, a gene that is required for Respiratory
Syncytial Virus replication, Herpes Simplex Virus gene, a gene that
is required for Herpes Simplex Virus replication, herpes
Cytomegalovirus gene, a gene that is required for herpes
Cytomegalovirus replication, herpes Epstein Barr Virus gene, a gene
that is required for herpes Epstein Barr Virus replication,
Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is
required for Kaposi's Sarcoma-associated Herpes Virus replication,
JC Virus gene, human gene that is required for JC Virus
replication, myxovirus gene, a gene that is required for myxovirus
gene replication, rhinovirus gene, a gene that is required for
rhinovirus replication, coronavirus gene, a gene that is required
for coronavirus replication, West Nile Virus gene, a gene that is
required for West Nile Virus replication, St. Louis Encephalitis
gene, a gene that is required for St. Louis Encephalitis
replication, Tick-borne encephalitis virus gene, a gene that is
required for Tick-borne encephalitis virus replication, Murray
Valley encephalitis virus gene, a gene that is required for Murray
Valley encephalitis virus replication, dengue virus gene, a gene
that is required for dengue virus gene replication, Simian Virus 40
gene, a gene that is required for Simian Virus 40 replication,
Human T Cell Lymphotropic Virus gene, a gene that is required for
Human T Cell Lymphotropic Virus replication, Moloney-Murine
Leukemia Virus gene, a gene that is required for Moloney-Murine
Leukemia Virus replication, encephalomyocarditis virus gene, a gene
that is required for encephalomyocarditis virus replication,
measles virus gene, a gene that is required for measles virus
replication, Vericella zoster virus gene, a gene that is required
for Vericella zoster virus replication, adenovirus gene, a gene
that is required for adenovirus replication, yellow fever virus
gene, a gene that is required for yellow fever virus replication,
poliovirus gene, a gene that is required for poliovirus
replication, poxvirus gene, a gene that is required for poxvirus
replication, plasmodium gene, a gene that is required for
plasmodium gene replication, Mycobacterium ulcerans gene, a gene
that is required for Mycobacterium ulcerans replication,
Mycobacterium tuberculosis gene, a gene that is required for
Mycobacterium tuberculosis replication, Mycobacterium leprae gene,
a gene that is required for Mycobacterium leprae replication,
Staphylococcus aureus gene, a gene that is required for
Staphylococcus aureus replication, Streptococcus pneumoniae gene, a
gene that is required for Streptococcus pneumoniae replication,
Streptococcus pyogenes gene, a gene that is required for
Streptococcus pyogenes replication, Chlamydia pneumoniae gene, a
gene that is required for Chlamydia pneumoniae replication,
Mycoplasma pneumoniae gene, a gene that is required for Mycoplasma
pneumoniae replication, an integrin gene, a selectin gene,
complement system gene, chemokine gene, chemokine receptor gene,
GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIG gene,
Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTES
gene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,
CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component
of an ion channel, a gene to a neurotransmitter receptor, a gene to
a neurotransmitter ligand, amyloid-family gene, presenilin gene, HD
gene, DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene,
SCA7 gene, SCA8 gene, allele gene found in loss of heterozygosity
(LOH) cells, one allele gene of a polymorphic gene and combinations
thereof.
[0757] The loss of heterozygosity (LOH) can result in hemizygosity
for sequence, e.g., genes, in the area of LOH. This can result in a
significant genetic difference between normal and disease-state
cells, e.g., cancer cells, and provides a useful difference between
normal and disease-state cells, e.g., cancer cells. This difference
can arise because a gene or other sequence is heterozygous in
duploid cells but is hemizygous in cells having LOH. The regions of
LOH will often include a gene, the loss of which promotes unwanted
proliferation, e.g., a tumor suppressor gene, and other sequences
including, e.g., other genes, in some cases a gene which is
essential for normal function, e.g., growth. Methods of the
invention rely, in part, on the specific modulation of one allele
of an essential gene with a composition of the invention.
[0758] In certain embodiments, the invention provides REVERSIR
compound to an siRNA that modulates a micro-RNA.
[0759] REVERSIR compounds are oligomeric compounds. Accordingly, in
certain embodiments, REVERSIR compounds comprise, for example and
without limitation, any of the modifications and motifs described
in the discussion herein for oligomeric compounds.
[0760] In certain embodiments, motifs are designed with
consideration given to both the siRNA and the REVERSIR compound. In
certain embodiments, a REVERSIR compound could comprise 4 or more
contiguous DNA-like monomers. In certain embodiments, the resulting
RNA/DNA duplex could activate RNase H, resulting in cleavage of the
RNA-like antisense compound. In certain embodiments, REVERSIR
activity does not depend on enzymatic activity. In certain such
embodiments, compounds designed without regard for enzymatic
compatibility may incorporate modifications to improve other
attributes. For example, certain motifs yield oligomeric compounds
with high affinity for a target nucleic acid, but that are unable
to elicit enzymatic cleavage of that target. Such motifs may be
useful for REVERSIR compounds in embodiments where cleavage of the
siRNA is not necessary.
[0761] In certain embodiments, one strand of the siRNA, e.g., the
strand complementary to REVERSIR compound, and corresponding
REVERSIR compound are the same length. In some embodiments, one
strand of the siRNA, e.g., the strand complementary to REVERSIR
compound, and corresponding REVERSIR compound are different
lengths. In some embodiments, the REVERSIR compound is shorter than
the corresponding complementary strand from the siRNA. In some
embodiments, the REVERSIR compound is shorter by 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or more nucleotides than the corresponding
complementary strand from the siRNA.
[0762] In certain embodiments, antisense strand of the siRNA and
corresponding REVERSIR compound are the same length. In some
embodiments, antisense strand of the siRNA and corresponding
REVERSIR compound are different lengths. In some embodiments, the
REVERSIR compound is shorter than the corresponding complementary
antisense strand from the siRNA. In some embodiments, the REVERSIR
compound is shorter by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
nucleotides than the corresponding complementary antisense strand
from the siRNA.
[0763] In certain embodiments, sense strand of the siRNA and
corresponding REVERSIR compound are the same length. In some
embodiments, sense strand of the siRNA and corresponding REVERSIR
compound are different lengths. In some embodiments, the REVERSIR
compound is shorter than the corresponding complementary sense
strand from the siRNA. In some embodiments, the REVERSIR compound
is shorter by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides
than the corresponding complementary sense strand from the
siRNA.
[0764] In certain embodiments, an siRNA and a REVERSIR compound are
administered to a patient. In certain such embodiments,
pharmaceutical compositions comprising an siRNA and those
comprising a REVERSIR compound comprise the same formulation. In
certain embodiments, pharmaceutical compositions comprising an
siRNA and those comprising a REVERSIR compound comprise different
formulations. In certain embodiments an siRNA and a REVERSIR
compound are administered by the same route. In certain embodiments
an siRNA and a REVERSIR compound are administered by different
routes. For example, in certain embodiments, an siRNA is
administered orally and a REVERSIR compound is administered by
injection. In certain embodiments, the dosages of the siRNA and the
REVERSIR compound are the same. In certain embodiments, the dosages
of the siRNA and the REVERSIR compound are different.
[0765] In certain embodiments, the toxicity profiles of the siRNA
and the REVERSIR compound are similar. In certain embodiments, such
toxicity profiles are different. For example, in certain
embodiments, an siRNA can be intended for chronic administration
and the REVERSIR compound is only intended for acute use as needed.
In such embodiments, the tolerance for toxic side-effects of the
REVERSIR compound can be higher. Accordingly, modifications and
motifs that may be too toxic for use in an siRNA can be acceptable
in a REVERSIR compound. For example, in certain embodiments,
oligomeric compounds comprising one or more LNA nucleoside have
been shown to have high affinity for a target nucleic acid, but in
certain embodiments have been shown to cause toxicity at relatively
low concentrations. For certain siRNAs, where chronic
administration is intended, certain such compounds comprising LNA
may not be suitable. However, in embodiments where a REVERSIR
compound is not intended for chronic administration, but rather for
acute administration when siRNA activity is problematic, such LNA
modifications in an antidote compound are acceptable. The increased
affinity of LNA can improve the REVERSIR effect and since the
REVERSIR compound is only administered for a short period of time,
and possibly when the patient is in distress, the increased
toxicity of LNA may be justified. Other high affinity, but
potentially toxic modifications are also known.
[0766] In certain embodiments, activity of siRNA is counteracted by
a non-oligomeric REVERSIR. For example, in certain embodiments,
when the target nucleic acid is a target mRNA encoding a protein it
is desirable to reduce the activity of siRNA and to increase in the
amount of the target protein (e.g., target protein amount has gone
too low, or circumstances have changed resulting in the desire to
restore target protein amount). In such embodiments, one can simply
administer the target protein itself. Such administration will
immediately reverse the siRNA activity of target protein reduction.
However, it can also be desirable to administer an oligomeric
REVERSIR compound according to the present invention. For example,
the target protein may have a short half-life in the animal.
Accordingly, to maintain the restored target protein concentration
would require repeated administration of target protein until the
siRNA has cleared and normal protein expression is restored. In
certain such embodiments, it is still desirable to administer an
REVERSIR compound to shorten the duration of the siRNA activity. In
certain embodiments an oligomeric REVERSIR compound is
co-administered with a non-oligomeric REVERSIR. In certain such
embodiments, the non-oligomeric REVERSIR is a target protein. In
certain embodiments, the non-oligomeric REVERSIR compound is a
protein having similar physiological effect as a target protein or
that stimulates expression of the target protein.
Research Tools
[0767] In certain instances, siRNAs have been used as research
tools. For example, researchers investigating the function of a
particular gene product can design siRNAs to reduce the amount of
that gene product present in a cell or an animal and observe
phenotypic changes in the cell or animal. In certain embodiments,
the present invention provides methods for reducing the amount of a
gene product in a cell or animal through RNAi and then reducing
that RNAi activity, thereby restoring the inhibited gene product.
In certain embodiments, investigators can use such techniques to
characterize proteins or untranslated nucleic acids. In certain
embodiments, investigators can vary the amount of time between
siRNA and REVERSIR compounds administration. In certain
embodiments, such experiments are used to investigate kinetics
and/or turnover of gene products and/or certain cellular
functions.
[0768] As described herein, the invention provides methods
comprising administering to a subject a siRNA followed by
administering a REVERSIR compound or composition comprising same.
Without limitation, the siRNA and the REVERSIR compound can be
conjugated or unconjugated. Further, the siRNA and the REVERSIR
compound can be independently encapsulated in a lipid formulation,
e.g., a LNP, or other nucleic acid-lipid particle. Moreover, the
siRNA and the REVERSIR compound can be administered, independently,
via any appropriate route or mode of administration. For example,
the siRNA and the REVERSIR compound can be independently
administered via intravenous administration (IV) or via
subcutaneous administration (SC).
[0769] Accordingly, in some embodiments, the invention provides
methods comprising administering to a subject an unconjugated siRNA
followed by administering a conjugated REVERSIR compound, wherein
the REVERSIR compound is encapsulated in a lipid formulation, e.g.,
a LNP, or other nucleic acid-lipid particle, and wherein the
REVERSIR compound is administered via intravenous
administration.
[0770] In some other embodiments, the invention provides methods
comprising administering to a subject an unconjugated siRNA
followed by administering a conjugated REVERSIR compound, wherein
the REVERSIR compound is encapsulated in a lipid formulation and
the REVERSIR compound is administered via subcutaneous
administration.
[0771] In yet some other embodiments, the invention provides
methods comprising administering to a subject a conjugated siRNA
followed by administering a conjugated REVERSIR compound, wherein
the REVERSIR compound is encapsulated in a lipid formulation and
the REVERSIR compound is administered via intravenous
administration.
[0772] In still some other embodiments, the invention provides
methods comprising administering to a subject a conjugated siRNA
followed by administering a conjugated REVERSIR compound, wherein
the REVERSIR compound is encapsulated in a lipid formulation and
the REVERSIR compound is administered via subcutaneous
administration.
[0773] While the above described embodiments specify conjugated
REVERSIR compounds, unconjugated REVERSIR compounds can also be
used. Thus, in some embodiments, the invention provides methods
comprising administering to a subject an unconjugated siRNA
followed by administering an unconjugated REVERSIR compound,
wherein the REVERSIR compound is encapsulated in a lipid
formulation and the REVERSIR compound is administered via
intravenous administration. In some other embodiments, the
invention provides methods comprising administering to a subject an
unconjugated siRNA followed by administering an unconjugated
REVERSIR compound, wherein the REVERSIR compound is encapsulated in
a lipid formulation and the REVERSIR compound is administered via
subcutaneous administration. In yet some other embodiments, the
invention provides methods comprising administering to a subject a
conjugated siRNA followed by administering an unconjugated REVERSIR
compound, wherein the REVERSIR compound is encapsulated in a lipid
formulation and the REVERSIR compound is administered via
intravenous administration.
In still some other embodiments, the invention provides methods
comprising administering to a subject a conjugated siRNA followed
by administering an unconjugated REVERSIR compound, wherein the
REVERSIR compound is encapsulated in a lipid formulation and the
REVERSIR compound is administered via subcutaneous
administration.
Kits
[0774] In certain embodiments, the present invention provides kits
comprising one or more siRNAs and one or more corresponding
REVERSIR compound. In certain embodiments, such kits are intended
for therapeutic application. In certain embodiments, such kits are
intended for research use.
[0775] 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.
[0776] The nucleoside sequences set forth in the sequence listing
and Examples, are independent of any modification to a sugar
moiety, a monomeric linkage, or a nucleobase. As such, oligomeric
compounds defined by a SEQ ID NO can comprise, independently, one
or more modifications to a sugar moiety, an internucleoside
linkage, or a nucleobase.
EXAMPLES
Example 1: Reversal of Antithrombin (AT) Knockdown in Wild-Type
Mice
[0777] Sixty wild-type mice (C57BL/6, female) were bled on Day -1
to obtain pre-dose blood samples. All animals were subsequently
injected subcutaneously with a single dose of ALN-57213 at 3 mg/kg
on Day 0. On Day 3, 3 mice per group received a single subcutaneous
injection of one of 19 different reversal agents (Table 2) at a
dose of 10 mg/kg. Three animals did not receive an injection on Day
3 and served as an untreated control. All animals were bled on Days
7, 11, and 15 to obtain serum samples. Serum samples were then
analyzed for AT antigen level by AT ELISA and were normalized to
the pre-dose AT level for each animal. FIG. 1 displays normalized
group mean (.+-.S.D.) AT levels. As indicated in FIG. 1, multiple
reversal agents reduced the level of AT knockdown mediated by a
single subcutaneous dose of ALN-57213.
TABLE-US-00002 TABLE 2 Design and Synthesis of 19 exemplary
REVERSIR compounds (primarily varying length and LNAs) SEQ Design
ID length feature REVERSIR sequence Alnylam # NO (nt) #LNAs
Description Basic cugguuaacaccauuuacuucadAL96 A-132289 1 22 0 Full
length 1-22; all design OMe; No PS linkages
csusgguuaacaccauuuacuuscsadAL96 A-132290 2 22 0 Full length 1-22;
all OMe; two PS linkages each at both ends Length
csusgsgsususasascsascscsasususu A-132291 3 22 0 Full length 1-22;
all reduction sascsususcsadAL96 OMe gsususasascsascscsasusususascsu
A-132292 4 18 0 Fragment 4-22; all suscsadAL96 OMe
ascsascscsasusususascsususcsadAL96 A-132293 5 15 0 Fragment 8-22;
all OMe LNAs in cs(Tlns)gsgsususasascsascscsasu A-132294 6 22 2
Full length 1-22; all full sususascsususcs(Alns)adAL96 OMe except
LNA length; @ n2, 22 22-mer cs(Tlns)gs(Glns)ususasascsascscsa
A-132295 7 22 4 Full length 1-22; all
susususascsus(Tlns)cs(Alns)adAL96 OMe except LNA @ n2, 4, 20, 22
cs(Tlns)gs(Glns)ususasascsascscsa A-132296 8 22 5 Full length 1-22;
all susususas(m5clns)us(Tlns)cs(Alns) OMe except LNA adAL96 @ n2,
4, 18, 20, 22 cs(Tlns)gs(Glns)ususasascs(Alns) A-132297 9 22 6 Full
length 1-22; all cscsasusususas(m5clns)us(Tlns)cs OMe except LNA @
n2, (Aln)dAL96 4, 10, 18, 20, 22 LNAs in
(Glns)ususasascsascscsasusususas A-132298 10 18 2 Fragment 4-22;
all 4-22 csususcs(Aln)dAL96 OMe except LNA fragment; @ n4, 22
18-mer (Glns)ususasascsascscsasusususas A-132299 11 18 3 Fragment
4-22; all csus(Tlns)cs(Aln)dAL96 OMe except LNA @ n4, 20, 22
(Glns)ususasascsascscsasusususas A-132300 12 18 4 Fragment 4-22;
all (m5clns)us(Tlns)cs(Aln)dAL96 OMe except LNA @ n4, 18, 20, 22
(Glns)ususasascs(Alns)cscsasusu A-132301 13 18 5 Fragment 4-22; all
susas(m5clns)us(Tlns)cs(Aln)dAL96 OMe except LNA @ n4, 10, 18, 20,
22 LNAs in (Alns)csascscsasusususascsususcs A-132302 14 15 2
Fragment 8-22; all 8-22 (Aln)dAL96 OMe except LNA fragment; @ n8,
22 15-mer (Alns)(m5clns)ascscsasusususascsus A-132303 15 15 4
Fragment 8-22; all (Tlns)cs(Aln)dAL96 OMe except LNA @ n8, 9, 20,
22 (Alns)cs(Alns)cscsasusususas A-132304 16 15 5 Fragment 8-22; all
(m5Clns)us(Tlns)cs(Aln)dAL96 OMe except LNA @ 8, 10, 18, 20, 22 DNA
nts cs(Tlns)dGs(Glns)ususasascs(Alns)dCscsa A-132305 17 22 6 Full
length; LNA in LNA susususas(m5clns)dTs(Tlns)dCs(Aln)dAL96 @ n2, 4,
10, 18, 20, region; 22; DNA @ 3, 11, varying 19, 21 length
(Glns)ususasascs(Alns)dCscsasusususas A-132306 18 22 5 Fragment
4-22; all (m5clns) dTs(Tlns)dCs(Aln)dAL96 OMe except LNA @ n4, 10,
18, 20, 22; DNA @ 11, 19, 21
(Alns)cs(Alns)dCscsasusususas(m5clns)dTs A-132307 19 22 5 Fragment
8-22; all (Tlns)dCs(Aln)dAL96 OMe except LNA @ 8, 10, 18, 20, 22;
DNA @ 11, 19, 21 If, not indicated, all OMe with full PS
linkages
TABLE-US-00003 TABLE 3 Inclusion of DNA residues may reduce
activity Serum AT levels SEQ length # Day Day Day ID REVERSIR
sequence (nt) LNAs 7 11 15 Alnylam # NO
cs(Tlns)gs(Glns)ususasascs(Alns)cscsasusususas 22 6 0.92 1.06 1.03
A-132297 9 (m5clns)us(Tlns)cs(Aln)dAL96
cs(Tlns)dGs(Glns)ususasascs(Alns)dCscsasusususas 22 6 0.86 1.00
0.97 A-132305 17 (m5clns)dTs(Tlns)dCs(Aln)dAL96
(Glns)ususasascs(Alns)cscsasusususas(m5clns)us 18 5 0.85 0.98 1.01
A-132301 13 (Tlns)cs(Aln)dAL96
(Glns)ususasascs(Alns)dCscsasusususas(m5clns) 18 5 0.61 0.66 0.68
A-132306 18 dTs(Tlns)dCs(Aln)dAL96
(Alns)cs(Alns)cscsasusususas(m5Clns)us(Tlns)cs 15 5 1.00 0.80 0.91
A-132304 16 (Aln)dAL96
(Alns)cs(Alns)dCscsasusususas(m5clns)dTs(Tlns) 15 5 0.88 0.77 0.72
A-132307 19 dCs(Aln)dAL96
TABLE-US-00004 TABLE 4 In vitro Transfection of REVERSIR compounds
targeting AD-57213 In vitro dual-luc-2pt siRNA then REVERSIR dose
response REVERSIR then siRNA 1 10 transfection transfection
REVERSIR nM nM IC.sub.50 IC.sub.50 ID avg SD avg SD (nM) (nM)
A-132289 21.1 1.4 40.5 5.6 0.786 0.650 A-132290 41.5 5.5 77.0 7.2
0.569 0.955 A-132291 24.4 1.6 46.5 6.1 0.965 0.749 A-132292 24.6
1.8 47.2 7.9 0.498 0.341 A-132293 16.3 0.9 17.5 2.0 0.119 N/A
A-132294 30.6 6.0 61.3 3.2 0.322 0.440 A-132295 45.3 5.2 82.3 6.0
0.408 0.132 A-132296 50.4 6.0 79.3 5.3 0.381 0.053 A-132297 54.8
16.6 83.4 4.6 0.355 0.061 A-132298 24.1 3.3 56.0 4.0 A-132299 26.7
4.4 56.6 4.0 A-132300 40.3 5.9 70.1 1.6 A-132301 69.7 6.9 88.4 8.7
0.383 0.075 A-132302 18.0 2.7 31.7 2.7 A-132303 17.8 1.0 45.4 8.8
A-132304 25.1 3.3 57.1 7.3 1.060 0.382 A-132305 48.6 3.8 75.8 10.0
A-132306 53.4 4.0 72.2 7.4 A-132307 18.2 1.6 46.1 9.4
TABLE-US-00005 TABLE 5 4-dose free-uptake in vitro of REVERSIR
compounds targeting AD-57213 SEQ 100 10 1 0.1 ID nM nM nM nM
REVERSIR NO 5'-Sequence-3' avg SD avg SD avg SD avg SD A-132301 13
(Glns)ususasascs(Alns)cscsasusususas 76.9 5.9 61.5 5.8 19.6 16.7
3.8 1.3 (m5Clns)us(Tlns)cs(Aln)dAL96 A-135676 20
(Glns)ususasascs(Alns)csgsususususas 2.9 0.7 7.4 4.2 2.8 2.0 3.8
1.1 (Glns)as(Tlns)cs(Aln)dAL96 A-135677 21
(Alns)cscsususas(Tlns)asusascsgsasus 3.5 2.1 6.4 1.9 2.5 1.2 4.7
1.0 (Tlns)as(m5Clns)us(m5Cln)dAL96 A-135678 22
gs(Tces)usasascsas(m5Cces)csasusususas 80.4 16.9 104.6 23.4 35.0
27.5 13.4 3.4 (m5Cces)us(Tces)(m5Cces)adAL96 A-135679 23
gs(Tlns)usasascsas(m5Clns)csasusususas 104.9 19.1 97.4 32.9 37.9
16.5 20.5 5.5 (m5Clns)us(Tlns)(m5Clns)adAL96 A-135680 24
gs(Tces)usasascsas(m5Cces)csausususas 85.9 16.1 128.3 18.2 27.8
23.2 10.5 4.6 (m5Cces)us(Tces)(m5Cces)adAL96 A-135681 25
gs(Tces)usasascsas(m5Cces)csdAusususas 88.0 29.1 137.6 30.3 36.3
21.1 13.8 6.0 (m5Cces)us(Tces)(m5Cces)adAL96 A-135682 26
gsususasascsascscsas(Tces)us(Tces)as 82.8 17.0 129.2 47.8 31.6 23.0
11.6 4.7 (m5Cces)us(Tces)(m5Cces)adAL96 A-135683 27
gsususasascsascscsas(Tlns)us(Tlns)as 97.5 22.4 100.5 25.6 41.2 19.2
14.6 4.4 (m5Clns)us(Tlns)(m5Clns)adAL96 A-135684 28
gs(Tces)(Tces)asas(m5Cces)as(m5Cces) 72.4 23.5 95.8 40.8 7.6 0.9
6.4 0.9 (m5Cces)asusususascsususcsadAL96 A-135685 29
gs(Tlns)(Tlns)asas(m5Clns)as(m5Clns) 67.7 3.4 84.2 7.8 14.2 5.8 5.0
3.0 (m5Clns)asusususascsususcsadAL96 A-135686 30
gsususasascsas(m5Cces)(m5Cces)as(Tces) 64.8 13.6 115.7 34.8 30.3
25.8 4.3 1.8 (Tces)(Tces)ascsususcsadAL96 A-135687 31
gsususasascsas(m5Clns)(m5Clns)as(Tlns) 69.1 12.9 42.4 15.9 14.0 4.5
2.9 1.1 (Tlns)(Tlns)ascsususcsadAL96 A-135688 32
gs(Tces)usasas(m5Cces)ascscsas(Tces) 87.6 16.5 44.4 13.7 22.5 2.9
7.1 3.7 ususas(m5Cces)usus(m5Cces)adAL96 A-135689 33
gs(Tlns)usasas(m5Clns)ascscsas(Tlns) 95.8 20.5 102.5 20.0 53.3 22.3
16.7 7.9 ususas(m5Clns)usus(m5Clns)adAL96 A-135690 34
gsususasas(m5Cces)as(m5Cces)(m5Cces) 96.7 16.9 66.7 25.9 29.1 3.8
13.2 1.0 asusususas(m5Cces)usus(m5Cces)adAL96 A-135691 35
gsususasas(m5Clns)as(m5Clns)(m5Clns) 125.8 27.0 93.7 52.2 36.7 21.4
15.8 11.2 asusususas(m5Clns)usus(m5Clns)adAL96 A-135692 36
gsususasas(m5Cces)ascs(m5Cces)asusus 110.9 16.0 106.8 20.1 15.6 6.0
11.0 4.0 usas(m5Cces)usus(m5Cces)adAL96 A-135693 37
gsususasas(m5Clns)ascs(m5Clns)asusus 118.1 15.3 93.2 40.3 62.6 33.9
19.4 7.2 usas(m5Clns)usus(m5Clns)adAL96 A-135694 38
gsususasas(m5Cces)ascs(m5Cces)as(Tces) 85.4 9.1 69.7 15.1 22.1 13.4
11.1 3.0 ususascsusus(m5Cces)adAL96 A-135695 39
gsususasascsascs(m5Cces)as(Tces)ususas 66.3 11.3 56.3 19.4 14.9 9.3
8.0 2.1 csus(Tces)(m5Cces)adAL96 A-135696 40
gsususasascsascscsas(Tces)us(Tces)ascs 80.5 15.7 96.0 23.2 18.6 3.3
8.5 2.7 (Tces)us(m5Cces)adAL96 A-135697 41
gsususasascsascscsas(Tces)ususascs 32.6 6.8 47.5 10.0 8.4 4.4 3.4
1.5 (Tces)(Tces)(m5Cces)adAL96 A-135698 42
gs(Tces)usasas(m5Cces)ascs(m5Cces)as 83.2 36.7 37.4 10.0 14.5 5.7
4.3 2.4 (Tces)ususascsususcsadAL96 A-135699 43
gs(Tces)(Tces)asas(m5Cces)ascscsas 56.3 10.4 40.3 21.7 9.1 4.7 2.6
1.1 (Tces)ususascsususcsadAL96 A-135700 44
gsususasascsascscsas(Tces)ususascs 56.3 15.9 46.5 24.4 9.4 5.8 5.8
1.0 (Tces)us(m5Cces)adAL96 A-135701 45
gs(Tces)usasas(m5Cces)ascscsas(Tces) 90.1 8.3 68.3 12.3 14.3 11.9
12.5 4.9 ususascsususcsadAL96 A-135702 46
gs(Tces)(Tces)asascsascscsas(Tces) 68.9 11.9 43.7 24.4 9.5 3.9 13.1
3.3 ususascsususcsadAL96 A-135703 47 gs(Tces)(Tces)asas(m5Cces)
57.7 7.7 38.9 16.9 11.1 5.3 14.8 1.0 ascscsasusususascsususcsadAL96
A-135704 48 gs(Tces)usaaca(m5Cce)casusuusa 108.7 17.2 80.6 20.4
52.4 31.7 23.2 5.6 (m5Cce)us(Tce)(m5Cces)adAL96 A-135705 49
gs(Tce)usaaca(m5Cce)casuuua(m5Cce) 94.4 19.0 89.6 7.5 49.1 21.6
21.8 3.5 us(Tce)(m5Cce)adAL96 A-135706 50
gs(Tces)uaaca(m5Cce)cauuua(m5Cce) 69.5 7.7 52.9 13.6 20.2 6.2 11.9
3.8 u(Tces)(m5Cces)adAL96 A-135707 51
gs(Tce)uaaca(m5Cce)cauuua(m5Cce) 71.1 19.5 58.7 10.0 28.3 23.8 14.2
4.6 u(Tce)(m5Cces)adAL96 A-135708 52
gs(Tces)uaacaccauuuacuuscsadAL96 44.5 7.6 47.2 27.9 11.4 4.4 8.6
4.9 A-135709 53 gsus(Tce)aacaccauuuacuuscsadAL96 33.2 6.1 43.8 17.3
7.4 3.6 6.1 1.6 A-135710 54 gsusuaa(m5Cce)accauuuacuuscsadAL96 34.8
15.8 67.0 9.7 16.4 8.3 5.7 1.8 A-135711 55
gsusuaaca(m5Cce)cauuuacuuscsadAL96 66.8 15.6 29.9 18.5 4.2 1.1 3.6
1.5 A-135712 56 gsusuaacac(m5Cce)auuuacuuscsadAL96 43.0 11.0 35.6
11.4 9.6 5.1 6.6 2.8 A-135713 57 gsusuaacacca(Tce)uuacuuscsadAL96
48.8 7.2 39.1 6.3 14.7 6.2 8.4 2.8 A-135714 58
gsusuaacaccau(Tce)uacuuscsadAL96 44.1 17.1 32.0 13.4 20.7 12.1 9.5
2.2 A-135715 59 gsusuaacaccauu(Tce)acuuscsadAL96 51.7 13.8 42.6
20.5 19.7 13.9 8.7 4.5 A-135716 60
gsusuaacaccauuua(m5Cce)uuscsadAL96 84.0 8.2 73.9 36.3 27.0 21.5
14.9 7.8 A-135717 61 gsusuaacaccauuuac(Tce)uscsadAL96 61.5 20.7
41.9 5.5 13.9 8.1 7.7 2.8 A-135718 62
gsusuaacaccauuuacu(Tces)csadAL96 51.8 13.2 33.1 13.9 7.4 1.5 7.4
2.6 A-135719 63 gsusuaacaccauuuacuus(m5Cces)adAL96 61.0 12.9 38.1
4.4 13.6 7.8 9.9 4.8 A-135720 64 gs(Tlns)uaacaccauuuacuuscsadAL96
61.3 10.7 43.5 14.8 16.2 12.4 5.6 2.4 A-135721 65
gsus(Tln)aacaccauuuacuuscsadAL96 35.4 7.5 40.8 17.9 9.1 4.2 5.2 2.0
A-135722 66 gsusuaa(m5Cln)accauuuacuuscsadAL96 24.6 13.4 38.8 11.2
7.9 1.7 3.6 1.6 A-135723 67 gsusuaaca(m5Cln)cauuuacuuscsadAL96 47.8
15.5 17.6 4.3 4.4 1.0 2.0 1.0 A-135724 68
gsusuaacac(m5Cln)auuuacuuscsadAL96 56.4 4.0 18.6 7.0 8.6 5.9 4.6
3.1 A-135725 69 gsusuaacacca(Tln)uuacuuscsadAL96 55.1 13.9 22.8 7.1
14.2 3.5 8.3 3.4 A-135726 70 gsusuaacaccau(Tln)uacuuscsadAL96 39.8
4.3 23.1 9.4 6.3 2.9 10.2 4.4 A-135727 71
gsusuaacaccauu(Tln)acuuscsadAL96 54.4 15.0 25.3 5.6 12.0 4.8 8.8
3.3 A-135728 72 gsusuaacaccauuua(m5Cln)uuscsadAL96 89.1 32.8 45.7
18.9 27.5 4.5 18.1 4.8 A-135729 73 gsusuaacaccauuuac(Tln)uscsadAL96
57.7 21.0 32.3 18.0 11.4 5.6 9.0 2.3 A-135730 74
gsusuaacaccauuuacu(Tlns)csadAL96 54.9 10.3 25.2 8.9 20.6 16.3 11.1
3.8 A-135731 75 gsusuaacaccauuuacuus(m5Clns)adAL96 58.2 8.4 28.8
8.1 10.1 7.0 9.8 1.7 A-135732 76
gsususasasY5sascscsasusususascsusus 72.6 4.5 39.3 17.7 7.5 1.9 6.6
2.7 Y5sadAL96 A-135733 77 gsususasasY5sascscsasusususasY5susu 54.3
10.2 30.2 20.7 3.4 2.2 5.6 4.0 scsadAL96 A-135734 78
gsususasascsasY5scsasusususascsusus 55.4 31.8 43.9 25.4 10.8 4.8
3.0 0.4 Y5sadAL96 A-135735 79 gsususasascsascsY5sasusususasY5susu
65.1 10.4 27.0 14.7 5.1 1.7 2.5 1.4 scsadAL96 A-135736 80
gsususasasY5sasY5scsasusususascsusu 64.8 31.1 17.1 12.4 4.5 2.7 3.7
1.1 scsadAL96 A-135737 81 gsususasasY5sascsY5sasusususascsusu 64.3
20.4 26.9 15.7 4.7 2.3 4.6 2.0 scsadAL96 A-135738 82
gsususasascsascscsasusususasY5susus 60.5 20.5 25.2 12.3 8.9 3.8 7.2
4.0 Y5sadAL96 A-135739 83 gsususasasY5sascscsasusususascsusus 76.9
12.2 30.3 15.6 11.5 5.7 7.4 2.4 csadAL96 A-135740 84
gsususasascsasY5scsasusususascsusus 99.1 38.2 43.2 20.0 9.4 6.0 8.4
4.4 csadAL96 A-135741 85 gsususasascsascsY5sasusususascsusus 86.5
12.9 55.1 33.0 7.2 1.3 5.1 3.0 csadAL96
A-135742 86 gsususasascsascscsasusususasY5susus 81.2 10.4 33.4 17.8
13.4 3.8 9.6 2.4 csadAL96 A-135743 87
gsususasascsascscsasusususascsususY 69.0 13.7 36.6 20.8 15.3 16.7
7.5 3.5 5sadAL96 A-135744 88 gsususasasY24sascsY24sasusususascsu
30.1 6.3 19.7 6.2 7.8 3.1 5.0 1.8 susY24sadAL96 A-135745 89
gsususasasY24sascscsasusususasY24su 9.6 2.8 8.7 5.6 5.8 3.1 3.0 0.8
susY24sadAL96 A-135746 90 gsususasasY24sascscsasusususascsusu 6.7
2.9 11.9 7.8 2.8 1.2 2.5 1.2 sY24sadAL96 A-135747 91
gsususasasY24sascscsasusususasY24su 22.8 11.8 5.1 1.7 2.0 0.7 2.0
0.6 suscsadAL96 A-135748 92 gsususasascsascsY24sasusususasY24su
28.3 1.9 7.0 3.2 2.9 2.3 4.2 1.5 suscsadAL96 A-135749 93
gsususasasY24sasY24scsasusususascsu 24.1 6.6 13.6 4.4 4.9 2.3 3.5
2.0 suscsadAL96 A-135750 94 gsususasascsascscsasusususasY24susu
16.7 5.1 12.7 10.0 9.9 10.1 6.7 4.3 sY24sadAL96 A-135751 95
gsususasasY27sascscsasusususasY27 60.8 23.5 16.6 7.7 8.6 5.9 4.8
2.6 sususY27sadAL96 A-135752 96 gsusuaacaccauuuacuuscsadAL96 58.1
29.6 32.3 17.0 14.7 12.5 7.6 3.4 A-135753 97
gsUfsusAfsasCfsasCfscsAfsusUf 48.0 10.1 23.0 12.4 8.0 4.4 3.6 2.8
susAfscsUfsusCfsadAL96 A-135754 98 GfsusUfsasAfscsAfscscsasUfsus
84.3 18.9 29.3 14.2 12.7 9.2 5.8 2.6 UfsasCfsusUfscsAfdAL96
A-135755 99 gsUfsusAfsasCfsasCfscsasusUfsus 32.9 6.8 15.4 4.0 7.5
5.4 5.3 1.9 AfscsUfsusCfsadAL96 A-135756 100
gsUfsuAfsaCfsaCfscasuUfsuAfscUfsus 42.4 13.7 29.1 13.4 4.9 3.7 6.3
3.7 CfsadAL96 A-135757 101 gs(Tces)usAfsas(m5Cces)asCfscsasus 44.3
12.7 20.6 5.1 4.2 3.7 2.9 1.7 UfsusAfscsUfsusCfsadAL96 A-135758 102
gsUfsusAfsasCfsasCfscsasusUfsusAfscs 27.4 9.7 20.4 11.6 12.1 8.0
2.8 3.5 (Tces)us(m5Cces)adAL96 A-135759 103
gsUfsusAfsasCfsasCfs(m5Cces)as 68.3 15.7 26.9 11.7 4.1 1.1 2.4 0.3
(Tces)UfsusAfscsUfsusCfsadAL96 A-135760 104
gs(Tces)usAfsas(m5Cces)asCfscsasus 61.4 14.2 22.3 1.4 2.5 1.0 2.1
0.4 (Tces)usAfscsUfsusCfsadAL96 A-135761 105
gsgs(Tces)usasascsas(m5Cces)csasusus 71.2 18.9 82.9 11.2 12.8 6.7
6.0 3.3 usas(m5Cces)us(Tces)(m5Cce)dAL96 A-135762 106
(Tces)gsgs(Tces)usasascsas(m5Cces) 19.3 7.6 17.3 4.1 5.9 5.4 3.8
1.9 csasusususas(m5Cces)us(Tce)dCL96 A-135763 107
cs(Tces)gsgs(Tces)usasascsas(m5Cces) 6.1 3.5 7.9 4.5 3.7 3.1 5.3
3.0 csasusus(Tces)as(m5Cces)udTL96 A-135764 108
gs(Tces)UfsAfsAfsCfsAfs(m5Cces)CfsAfsUfs 82.6 8.4 47.2 32.8 6.5 3.3
5.6 2.7 UfsUfsAfs(m5Cces)Ufs(Tces)(m5Cces)adAL96 A-135765 109
gsUfsUfsAfsAfsCfsAfsCfsCfsAfsUfsUfsUfsA 88.7 12.8 30.2 7.5 7.0 2.5
3.7 2.1 fsCfsUfsUfsCfsadAL96 A-135766 110
gsUfsUfAfAfCfAfCfCfAfUfUfUfAfCfUfUfs 6.1 3.4 5.5 3.1 2.5 1.6 4.0
1.0 CfsadAL96
TABLE-US-00006 TABLE 6 IC.sub.50 free-uptake in vitro of REVERSIR
compounds targeting AD-57213 REVERSIR SEQ ID ID NO 5'-Sequence-3'
IC.sub.50 (nM) A-138952 111
csusgsgsususasascsascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96
>100 A-135683 112
gsususasascsascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96
9.6 A-138953 113
ascsascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 2.5
A-138954 114
csascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 0.49
A-138955 115 ascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96
0.71 A-138956 116
cscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 0.29 A-138957
117 csas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 0.27
A-138958 118 as(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 0.22
A-138959 119 (Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96
<0.015
TABLE-US-00007 TABLE 7 REVERSIR compounds targeting AD-57213 used
in the in vivo experiments SEQ ID siRNA NO 5'-Sequence-3' AD-57213
120 GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 121
usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg REVERSIR ID 5'-Sequence-3'
A-132293 5 ascsascscsasusususascsususcsadAL96 A-132302 14
(Alns)csascscsasusususascsususcs(Aln)dAL96 A-132303 15
(Alns)(m5Clns)ascscsasusususascsus(Tlns)cs(Aln)dAL96 A-132304 16
(Alns)cs(Alns)cscsasusususas(m5Clns)us(Tlns)cs(Aln)dAL96 A-132296 8
cs(Tlns)gs(Glns)ususasascsascscsasusususas(m5Clns)us(Tlns)cs(Al-
ns)adAL96 A-132301 13
(Glns)ususasascs(Alns)cscsasusususas(m5Clns)us(Tlns)cs(Aln)dAL96
A-135678 22
gs(Tces)usasascsas(m5Cces)csasusususas(m5Cces)us(Tces)(m5Cces)adAL96
A-135679 23
gs(Tlns)usasascsas(m5Clns)csasusususas(m5Clns)us(Tlns)(m5Clns)adAL96
A-135704 48
gs(Tces)usaaca(m5Cce)casusuusa(m5Cce)us(Tce)(m5Cces)adAL96 A-138953
113 ascsascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96
A-140335 122 (Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 A-138955 115
ascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 A-140336 123
(Tlns)as(m5Clns)(Tlns)(Tlns)(m5Clns)adAL96 A-138957 117
csas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 A-138959 119
(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 A-140339 124
csas(Tln)us(Tln)as(m5Cln)us(Tln)(m5Clns)adAL96 A-140340 125
(Tlns)us(Tln)as(m5Cln)us(Tln)(m5Clns)adAL96 A-140337 126
ascsascscsas(Tln)us(Tln)as(m5Cln)us(Tln)(m5Clns)adAL96 A-140338 127
ascscsas(Tln)us(Tln)as(m5Cln)us(Tln)(m5Clns)adAL96
TABLE-US-00008 TABLE 8 4-dose free-uptake in vitro of REVERSIR
compounds targeting AD-66568 SEQ 100 10 1 0.1 ID nM nM nM nM
REVERSIR NO 5'-Sequence-3' avg SD avg SD avg SD avg SD A-138962.1
128 ususcsasgsusascscsususasgsasgsususcscsas 47.6 12.3 50.2 6.3
23.1 4.8 15.7 3.6 csusdAL96 A-138963.1 129
ususcsasgsusascscsususasgsasgsususcscsas 48.9 13.8 57.6 7.7 33.4
3.2 22.8 5.4 cs(Tln)dAL96 A-138964.1 130
ususcsasgsusascscsususasgsasgsususcscsas 67.4 17.0 67.1 10.2 23.6
6.8 19.8 4.8 (m5Clns)(Tln)dAL96 A-138965.1 131
ususcsasgsusascscsususasgsasgsususcs(m5Clns) 69.7 11.1 65.1 11.9
33.8 11.3 21.3 3.3 as(m5Clns)(Tln)dAL96 A-138966.1 132
ususcsasgsusascscsususasgsasgsus(Tlns)cs 63.8 13.5 63.9 6.4 22.2
3.6 15.9 3.8 (m5Clns)as(m5Clns)(Tln)dAL96 A-138967.1 133
ususcsasgsusascscsususasgsasgs(Tlns)(Tlns) 53.1 3.2 60.9 6.4 21.4
3.7 16.1 1.6 cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138968.1 134
ususcsasgsusascscsus(Tlns)asgsasgs(Tlns) 57.9 8.6 59.4 14.8 20.8
4.1 17.3 0.7 (Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138969.1 135
ususcsasgsusas(m5Clns)csus(Tlns)asgsasgs 71.0 12.6 63.4 8.6 24.0
1.8 16.0 3.5 (Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138970.1
136 uscsasgsusascscsususasgsasgs(Tlns)(Tlns) 55.5 7.0 43.3 5.8 20.3
3.2 18.1 2.7 cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138971.1 137
csasgsusascscsususasgsasgs(Tlns)(Tlns) 44.3 20.0 65.0 13.8 20.3 3.3
21.5 3.7 cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138972.1 138
asgsusascscsususasgsasgs(Tlns)(Tlns)cs(m5Clns) 56.8 17.3 59.3 10.2
26.2 9.5 20.2 4.4 as(m5Clns)(Tln)dAL96 A-138973.1 139
gsusascscsususasgsasgs(Tlns)(Tlns)cs(m5Clns) 42.3 16.0 53.7 7.7
29.1 7.0 18.6 4.2 as(m5Clns)(Tln)dAL96 A-138974.1 140
usascscsususasgsasgs(Tlns)(Tlns)cs(m5Clns) 59.7 18.3 55.8 6.9 27.9
3.8 20.6 5.8 as(m5Clns)(Tln)dAL96 A-138975.1 141
ascscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as 50.0 5.0 51.2 5.4 38. 6
12.3 22.4 2.0 (m5Clns)(Tln)dAL96 A-138976.1 142
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 82.6 17.8 65.4
13.9 31.9 3.5 20.4 3.7 (Tln)dAL96 A-138977.1 143
csususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 74.4 7.8 60.4 9.4
27.8 6.9 17.8 4.1 (Tln)dAL96 A-138978.1 144
ususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 77.0 6.5 62.5 10.7
22.6 2.4 19.8 5.0 (Tln)dAL96 A-138979.1 145
usasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 73.7 15.1 57.6 8.8 31.4
8.2 17.6 2.4 (Tln)dAL96 A-138980.1 146
asgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln) 72.2 12.7 61.0 4.9
32.5 7.4 16.8 2.7 dAL96 A-138981.1 147
gsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 81.6 9.0 66.4 7.7
33.3 6.2 20.2 1.5 A-138982.1 148
asgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 104.3 8.9 61.1 12.0
41.2 8.2 24.5 2.6 A-138983.1 149
gs(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 84.6 22.8 83.1 12.6
52.0 14.6 24.4 2.6 A-138984.1 150
(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 54.2 14.0 55.9 3.5 22.7
4.0 19.4 3.1 A-138985.1 151
csususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 52.1 14.5 53.9 4.1
19.2 7.9 18.4 1.3 (Tlns)adAL96 A-138976.2 152
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 68.6 26.2 48.6 4.1
29.0 6.2 24.1 3.4 (Tln)dAL96 A-138986.1 153
ascscsus(Tlns)asgsasgs(Tlns)(Tlns)cs(m5Clns)as 84.0 20.6 54.6 22.8
37. 5 11.8 28.0 9.9 (m5Cln)dAL96 A-138987.1 154
usascs(m5Clns)us(Tlns)asgsasgs(Tlns)(Tlns)cs 75.5 4.8 57.4 10.6
29.5 4.2 19.6 2.5 (m5Clns)adAL96 A-138988.1 155
gsusascs(m5Clns)us(Tlns)asgsasgs(Tlns)(Tlns) 63.1 6.9 58.2 15.3
24.0 5.1 21.6 5.6 cs(m5Cln)dAL96 A-138989.1 156
asgs(Tlns)ascs(m5Clns)us(Tlns)asgsasgs(Tlns) 43.8 3.5 38.1 6.3 18.3
1.5 18.2 4.3 (Tlns)cdAL96 A-138990.1 157
csasgs(Tlns)ascs(m5Clns)us(Tlns)asgsasgs(Tlns) 29.7 12.3 26.2 1.8
18.1 3.4 15.5 3.1 (Tln)dAL96 A-138991.1 158
us(m5Clns)asgs(Tlns)ascs(m5Clns)us(Tlns)asgsasgs 34.0 7.2 31.1 7.8
18.2 3.4 18.2 1.7 (Tln)dAL96 A-138992.1 159
(Tlns)us(m5Clns)asgs(Tlns)ascs(m5Clns)us(Tlns)as 37.9 7.7 31.1 6.1
20.0 2.3 18.4 0.7 gsasgdAL96 A-138976.3 160
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 69.2 12.9 65.4 8.8
23.0 3.8 21.2 3.9 (Tln)dAL96 A-138993.1 161
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 59.3 14.8 65.1 6.8
25.0 5.0 20.8 3.4 (Tlns)dAL96 A-138994.1 162
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 46.2 10.7 59.1
14.9 21.9 2.8 22.1 2.4 (Tlns)aL96 A-138995.1 163
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 52.7 5.8 53.4 6.9
20.9 4.9 20.1 2.6 (Tlns)asL96 A-138996.1 164
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 65.6 8.3 50.6 1.9
34.9 13.2 28.0 5.5 (Tln)dTL96 A-138997.1 165
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 79.9 6.4 56.8 6.5
41.2 12.8 29.3 10.0 (Tln)dGL96 A-138998.1 166
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 50.7 5.9 42.9 8.0
32.5 3.2 23.6 1.8 (Tln)dCL96 A-138976.4 167
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 83.3 24.1 69.5 6.1
28.0 3.5 26.1 6.0 (Tln)dAL96 A-138999.1 168
(m5Clns)(m5Clns)us(Tlns)asgsasgs(Tlns)(Tlns)cscs 113.8 15.2 78.2
12.6 31.6 8.1 18.7 4.1 ascsudAL96 A-139000.1 169
cscs(Tlns)(Tlns)asgsasgs(Tlns)(Tlns)cs(m5Clns) 98.7 14.8 73.6 2.5
20.9 7.2 19.4 1.5 ascsudAL96 A-139001.1 170
(m5Clns)csus(Tlns)asgsasgs(Tlns)uscs(m5Clns) 82.2 25.7 77.3 5.9
32.0 5.1 21.3 8.9 ascs(Tln)dAL96 A-139002.1 171
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 29.6 11.0 44.1
14.7 16.3 1.7 17.6 4.8 (Tln) A-139003.1 172
cscsuuagag(Tln)(Tln)c(m5Cln)a(m5Clns)(Tln) 20.8 4.5 29.7 5.6 11.8
3.9 17.3 2.7 A-139004.1 173
cscsususasgsasgs(Tln)(Tln)cs(m5Cln)as(m5Clns) 29.1 8.8 51.6 7.2
18.2 4.8 20.8 4.2 (Tln) A-139005.1 174
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 27.1 3.9 36.0 13.1
14.5 4.0 20.9 3.3 (Tln)dAL10 A-139006.1 175
cscsuuagag(Tln)(Tln)c(m5Cln)a(m5Clns)(Tln)dAL10 44.0 14.0 40.9 4.4
18.7 3.9 22.0 4.7 A-139007.1 176
cscsususasgsasgs(Tln)(Tln)cs(m5Cln)as(m5Clns) 28.1 12.2 29.1 3.6
24.2 6.6 32.8 3.2 (Tln)dAL10 A-139008.1 177
Q173Q173cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns) 81.3 7.7 65.0 5.5
30.4 7.5 21.3 3.4 as(m5Clns)(Tln) A-139009.1 178
cscsuuagag(Tln)(Tln)c(m5Cln)a(m5Clns)(Tln)dAL96 96.7 12.8 65.9 8.6
55.9 8.6 26.8 6.1 A-139010.1 179
cscsususasgsasgs(Tln)(Tln)cs(m5Cln)as(m5Clns) 105.5 15.8 73.1 9.4
41.5 14.9 28.2 8.6 (Tln)dAL96 A-139011.1 180
cscsususasgsasgs(Tln)(Tln)cs(m5Cln)as(m5Cln) 103.1 20.5 76.2 13.9
30.7 6.3 22.4 5.4 (Tln)dAL96 A-139012.1 181
cscsususasgsasgsususY5scsascsudAL96 67.3 9.4 46.7 5.6 21.4 4.3 19.4
1.7 A-139013.1 182 cscsususasgsasgsususcsY5sascsusdAL96 67.6 12.9
46.5 5.3 26.8 5.7 18.0 3.0 A-139014.1 183
cscsususasgsasgsususcscsasY5sudAL96 37.0 13.2 34.9 12.8 23.6 6.2
18.6 2.2 A-139015.1 184 csY5sususasgsasgsususcscsascsudAL96 68.4
13.9 61.3 9.9 20.8 5.0 19.9 2.8 A-139016.1 185
(m5Clns)csususasgsasgsususcscsascsudAL96 44.2 8.7 51.2 8.7 18.6 3.2
20.7 1.7 A-139017.1 186 cs(m5Clns)ususasgsasgsususcscsascsudAL96
64.5 4.1 55.7 7.6 24.0 3.6 23.3 4.5 A-139018.1 187
cscs(Tlns)usasgsasgsususcscsascsudAL96 54.1 15.2 41.4 3.8 20.9 8.1
24.6 2.5 A-139019.1 188 cscsus(Tlns)asgsasgsususcscsascsudAL96 59.2
13.0 52.1 4.5 26.4 6.6 29.3 6.7 A-139020.1 189
cscsusus(Alns)gsasgsususcscsascsudAL96 54.4 25.0 45.6 7.4 23.1 7.9
19.8 2.1 A-139021.1 190 cscsususas(Glns)asgsususcscsascsudAL96 69.0
6.4 60.3 8.4 33.2 8.9 19.3 3.6 A-139022.1 191
cscsususasgs(Alns)gsususcscsascsudAL96 57.2 6.5 42.7 13.2 36.7 12.0
22.5 5.7 A-139023.1 192 cscsususasgsas(Glns)ususcscsascsudAL96 58.9
4.9 43.2 10.4 22.6 4.5 21.9 6.1 A-139024.1 193
cscsususasgsasgs(Tlns)uscscsascsudAL96 70.6 12.4 48.2 3.3 24.1 2.9
19.1 3.6
A-139025.1 194 cscsususasgsasgsus(Tlns)cscsascsudAL96 64.7 24.2
49.5 8.1 25.5 6.7 16.2 2.5 A-139026.1 195
cscsususasgsasgsusus(m5Clns)csascsudAL96 99.4 10.2 71.4 10.6 35.6
5.2 20.0 3.1 A-139027.1 196
cscsususasgsasgsususcs(m5Clns)ascsudAL96 59.2 8.7 51.8 8.5 22.6 6.8
16.6 3.1 A-139028.1 197 cscsususasgsasgsususcscs(Alns)csudAL96 45.6
8.7 46.8 15.6 22.9 4.6 20.2 2.3 A-139029.1 198
cscsususasgsasgsususcscsas(m5Clns)usdAL96 42.6 4.2 39.5 12.5 20.0
4.7 20.5 4.5 A-139030.1 199 cscsususasgsasgsususcscsascs(Tln)dAL96
36.9 5.1 30.7 3.4 21.2 5.0 25.2 3.7 A-139031.1 200
cscsususasgsasgs(Tlns)(m5Clns)gs(m5Clns) 28.3 1.6 19.2 3.4 19.1 1.9
31.3 7.3 us(m5Clns)(m5Cln)dAL96 A-139032.1 201
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)us(m5Clns) 46.1 10.7 46.2
13.8 29.6 6.6 20.0 2.3 (m5Cln)dAL96 A-139033.1 202
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 46.9 19.5 48.5 3.8
26.9 6.5 23.1 6.1 (m5Cln)dAL96 A-139034.1 203
cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)us(m5Clns) 30.8 11.4 40.7
12.4 23.6 3.5 21.6 5.5 (Tln)dAL96 A-139035.1 204
gsasgsasususcscs(m5Clns)(m5Clns)us(m5Clns)cs 23.3 3.3 18.1 3.7 20.9
1.7 18.2 2.2 (Tlns)(m5Cln)dAL96 A-139036.1 205
(m5Clns)(Tlns)usasgsasgsususcscsas(m5Clns)(Tln) 80.6 3.9 45.7 20.1
32.8 12.7 20.6 2.3 dAL96 A-139037.1 206
(m5Clns)(Tlns)dTsdAsdGsdAsdGsdTsdTsdCsdCsd 27.5 1.9 23.3 0.7 21.3
3.5 18.0 1.3 As(m5Clns)(Tln)dAL96 A-139038.1 207
(m5Clns)asgs(Tlns)(Alns)cscsususasgsasgsususcs 82.9 23.8 76.1 12.8
34.5 6.3 22.1 1.6 (m5Clns)as(m5Clns)(Tln)dAL96 A-139039.1 208
(m5Clns)asgs(Tlns)(Alns)dCsdCsdTsdTsdAsdGsdA 73.9 12.3 64.8 9.1
23.9 5.7 20.7 5.0 sdGsdTsdTscs(m5Clns)as(m5Clns)(Tln)dAL96 AD-66568
209 CfsasGfuAfcCfuUfAfGfaGfuticCfaCfuAfL96 101.9 21.1 100.1 5.7
100.2 7.3 100.6 11.6 210 usAfsgUfgGfaAfalfcuaAfgGfuAfcUfgsasa
Example 2: In Vitro Reversal of Antithrombin Knockdown by Free
Uptake in Primary Mouse Hepatocytes
[0778] In vitro assay: siRNA Transfection followed by REVERSIR Free
Uptake: Mouse primary hepatocytes were transfected with 1 nM siRNA
by adding 4.9 .mu.L of Opti-MEM plus 0.1 L of Lipofectamine RNAiMax
per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 .mu.L
siRNA per well into a 384-well plate and incubated at room
temperature for 15 minutes. 40 .mu.L of William's media containing
.about.5.times.10.sup.3 cells were then added to the siRNA mixture,
yielding a final siRNA concentration of 1 nM. Cells were incubated
at 37.degree. C. After 4 h, hepatocytes were washed and REVERSIR
compounds were added by free uptake in 50 .mu.L media for 48 h at
37.degree. C.
[0779] Total RNA isolation using DYNABEADS mRNA Isolation Kit: RNA
was isolated from hepatocytes by using an automated protocol on a
BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012).
Briefly, 50 .mu.L of Lysis/Binding Buffer and 25 .mu.L of lysis
buffer containing 3 .mu.L of magnetic beads were added to the plate
with cells. Plates were incubated on an electromagnetic shaker for
10 minutes at room temperature and then magnetic beads were
captured and the supernatant was removed. Bead-bound RNA was then
washed 2 times with 150 .mu.L Wash Buffer A and once with Wash
Buffer B. Beads were then washed with 150 .mu.L Elution Buffer,
re-captured and supernatant removed.
[0780] cDNA synthesis using ABI High capacity cDNA reverse
transcription kit (Applied Biosystems, Foster City, Calif., Cat
#4368813): 10 .mu.L of a master mix containing 1 .mu.L 10.times.
Buffer, 0.4 .mu.L 25.times.dNTPs, 1 .mu.L 10.times. Random primers,
0.5 .mu.L Reverse Transcriptase, 0.5 .mu.L RNase inhibitor and 6.6
.mu.L of water per reaction was added to RNA isolated above. Plates
were sealed, mixed, and incubated on an electromagnetic shaker for
10 minutes at room temperature, followed by 2 h 37.degree. C.
[0781] Real time PCR: 2 .mu.L of cDNA were added to a master mix
containing 0.5 .mu.L of GAPDH TaqMan Probe, 0.5 .mu.L RVR probe
(Mm01302526_m1) and 5 .mu.L Lightcycler 480 probe master mix (Roche
Cat #04887301001) per well in a 384 well plates (Roche cat
#04887301001). Real time PCR was done in a LightCycler480 Real Time
PCR system (Roche) using the .DELTA..DELTA.Ct(RQ) assay. Each
REVERSIR was tested in four independent transfections.
[0782] To calculate relative fold change, real time data were
analyzed using the .DELTA..DELTA.Ct method and normalized to assays
performed with cells transfected with 10 nM AD-1955, or mock
transfected cells. Results are shown in FIGS. 3-14.
Example 3: In Vitro Reversal of Factor IX Knockdown
[0783] Results are shown in FIGS. 15-21.
Example 4: REVERSIR Compounds are Well Tolerated In Vivo
[0784] As shown in FIG. 23, the various REVERSIR compounds tested
for in vivo toxicity showed little or no change in body weight
gain. Further, no liver enzyme elevation was observed across
dosses, e.g., 20 and 100 mg/kg. Moreover, no liver enzyme elevation
was observed across time points, e.g., day 4 and day 8. Thus, the
REVERSIR compounds of the invention have good in vivo tolerability
and safety profile.
Example 5: Reversal of Antithrombin Knockdown by REVERSIR Compounds
in Non-Human Primates
[0785] A single dose exploratory pharmacology study of REVERSIR
compounds was carried out in male Cynomolgus monkeys.
Experimental Design
[0786] Animals judged to be suitable for testing were arbitrarily
assigned to the study and arranged in seven groups as shown in
Table 9. One day 0 (the first day of dosing), all animals received
a single subcutaneous dose of antithrombin siRNA (ALN-AT3SC). The
pharmacology and toxicology of ALN-AT3SC has previously been
evaluated in rodents, dog, rabbit and non-human primate (cynomolgus
monkey). Thus, single administration of 7.5 mg/kg was expected to
be well-tolerated with no adverse physiological or histological
effects.
TABLE-US-00009 TABLE 9 ALN-AT3SC Dose Number Group Dose Level
Concentration Volume of Number (mg/kg) (mg/mL) (mL/kg) Males 1 7.5
3.75 2 3 2 7.5 3.75 2 3 3 7.5 3.75 2 3 4 7.5 3.75 2 3 5 7.5 3.75 2
3 6 7.5 3.75 2 3 7 7.5 3.75 2 3
[0787] One day 14, animals received a single subcutaneous dose of
REVERSIR compounds (A-138959, A-140340 or A-140337) or saline as
shown in Table 10. The dose levels of the REVERSIR compounds
replicated levels previously evaluated in subcutaneous
pharmacokinetic/pharmacodynamic studies in mice.
TABLE-US-00010 TABLE 10 Test Article Dose Dose Group Level
Concentration Volume Number of Number Test Article (mg/kg (mg/mL)
(mL/kg) Males 1 0.9% Saline 0 0 2 3 2 A-138959 0.25 0.125 2 3 3
A-138959 2.5 1.25 2 3 4 A-140340 0.25 0.125 2 3 5 A-140340 2.5 1.25
2 3 6 A-140337 0.25 0.125 2 3 7 A-140337 2.5 1.25 2 3
[0788] All subcutaneous doses were performed with the appropriately
sized syringe per standard operating procedure. Following
administration on day 14, animals were maintained on study for a
6-week non-dosing period.
Pharmacokinetics
[0789] Animals receiving the REVERSIR compounds were bled 2 and 8
hours post-dose on day 14 and once on days 15, 16, 17, 18 and 21.
About 1.0 ml of blood (per time point) was collected from the
femoral or other suitable vein. K.sub.2EDTA was used as the
anticoagulant. Samples were kept chilled (wet ice, as appropriate)
during collection and during processing. The samples were
centrifuged 2400-2700 rpm at approximately 4.degree. C. for
approximately 10 minutes. The maximum amount of plasma recovered
was divided into two approximately equal volume aliquots into
appropriately labeled tubes. Aliquoted sample were designated as
"Aliquot 1" or "Aliquot 2". Both sets of samples were maintained
frozen (-65.degree. C. to -85.degree. C.) until bioanalytical
evaluation.
Pharmacodynamics (Plasma AT)
[0790] All animals were bled twice during pretest (Days -5 and -2)
and on days 0, 7, 14, 15, 16, 17, 18, 21, 28, 35, 42, 49, and 56.
About 1.0 ml of blood (per time point) was collected from the
femoral or other suitable vein. K.sub.2EDTA was used as the
anticoagulant. Samples were kept chilled (wet ice, as appropriate)
during collection and during processing. The samples were
centrifuged 2400-2700 rpm at approximately 4.degree. C. for
approximately 10 minutes. The maximum amount of plasma recovered
was divided into two approximately equal volume aliquots into
appropriately labeled tubes. Aliquoted sample were designated as
"Aliquot 1" or "Aliquot 2". Both sets of samples were maintained
frozen (-65.degree. C. to -85.degree. C.) until evaluation of
plasma antithrombin via ELISA.
Immunostimulation (Plasma Cytokines)
[0791] All animals receiving the REVERSIR compounds on day 14 prior
to dosing and at 4 and 24 hours post-dosing. About 0.6 ml of blood
(per time point) was collected from the femoral or other suitable
vein. K.sub.2EDTA was used as the anticoagulant. Samples were kept
chilled (wet ice, as appropriate) during collection and during
processing. The samples were centrifuged 2400-2700 rpm at
approximately 4.degree. C. for approximately 10 minutes. The
maximum amount of plasma recovered was divided into two
approximately equal volume aliquots into appropriately labeled
tubes. Aliquoted sample were designated as "Aliquot 1" or "Aliquot
2". Both sets of samples were maintained frozen (-65.degree. C. to
-85.degree. C.) until evaluation of plasma cytokines/chemokines by
multiplex assay.
[0792] Clinical pathology: During pretest and on days 16 and 56,
various hematology and serum chemistry parameters were evaluated
from all animals (Groups 1-7) once during the pretest period and on
days 16 and 56 as a clinical pathology screen for the purpose of
animal selection/confirmation of health. Blood samples were
collected from fasted animals via a femoral vein (or other suitable
vein). The anticoagulant used was K.sub.2EDTA for the hematology
samples and sodium citrate for the coagulation samples. Samples for
serum chemistry were collected without anticoagulant.
[0793] Hematology parameters included differential leukocyte count,
erythrocyte count, hemoglobin, hemoglobin distribution width
hematocrit, mean corpuscular hemoglobin, mean corpuscular volume,
mean corpuscular hemoglobin concentration, platelet count, red cell
distribution width, reticulocyte count and total leukocyte
count.
[0794] Serum chemistry parameters included alanine
aminotransferase, albumin, alkaline phosphatase, albumin/globulin
ratio (calculated), aspartate aminotransferase, calcium chloride,
creatinine, gamma glutamyltransferase, globulin (calculated),
glucose, phosphorus, potassium, sodium, sorbitol dehydrogenase,
total bilirubin, total cholesterol, total protein, triglycerides,
urea nitrogen, and appearance.
[0795] As shown in FIGS. 24A and 24B, the tested exemplary REVERSIR
compounds reversed the activity of the antithrombin siRNA in
non-human primates. In FIG. 24A, REVERSIR compounds were
administered at a dose of 2.5 mg/kg (0.75 molar eq. of the siRNA,
ALN-AT3). In FIG. 24B, REVERSIR compounds were administered at a
dose of 0.25 mg/kg (0.075 molar eq. of the siRNA, ALN-AT3).
Surprisingly, REVERSIR A-140340 (a 9-mer with low phosphorothioate
content, 5 PS) showed complete reversal of ALN-AT3 activity within
4 days of dosing and was active at 30-fold lower dose than the
conjugate (13 molar eq.).
[0796] Abbreviations used in describing the sequences, e.g.,
sequences described in Tables 2-8 are collected and described in
Table 11 for convenience.
TABLE-US-00011 TABLE 11 Abbreviations of nucleotide monomers used
in nucleic acid sequence representation. Abbreviation Nucleotide(s)
A Adenosine-3'-phosphate Ab beta-L-adenosine-3'-phosphate Af
2'-fluoroadenosine-3'-phosphate Afs
2'-fluoroadenosine-3'-phosphorothioate As
adenosine-3'-phosphorothioate C cytidine-3'-phosphate Cb
beta-L-cytidine-3'-phosphate Cf 2'-fluorocytidine-3'-phosphate Cfs
2'-fluorocytidine-3'-phosphorothioate Cs
cytidine-3'-phosphorothioate G guanosine-3'-phosphate Gb
beta-L-guanosine-3'-phosphate Gbs
beta-L-guanosine-3'-phosphorothioate Gf
2'-fluoroguanosine-3'-phosphate Gfs
2'-fluoroguanosine-3'-phosphorothioate Gs
guanosine-3'-phosphorothioate T 5'-methyluridine-3'-phosphate Tf
2'-fluoro-5-methyluridine-3'-phosphate Tfs
2'-fluoro-5-methyluridine-3'-phosphorothioate Ts
5-methyluridine-3'-phosphorothioate U Uridine-3'-phosphate Uf
2'-fluorouridine-3'-phosphate Ufs
2'-fluorouridine-3'-phosphorothioate Us uridine-3'-phosphorothioate
N any nucleotide (G, A, C, T or U) a
2'-O-methyladenosine-3'-phosphate as
2'-O-methyladenosine-3'-phosphorothioate c
2'-O-methylcytidine-3'-phosphate cs
2'-O-methylcytidine-3'-phosphorothioate g
2'-O-methylguanosine-3'-phosphate gs
2'-O-methylguanosine-3'-phosphorothioate t
2'-O-methyl-5-methyluridine-3'-phosphate ts
2'-O-methyl-5-methyluridine-3'-phosphorothioate u
2'-O-methyluridine-3'-phosphate us
2'-O-methyluridine-3'-phosphorothioate dT 2'-deoxythymidine dTs
2'-deoxythymidine-3'-phosphorothioate dU 2'-deoxyuridine s
phosphorothioate linkage L96
N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol
Hyp-(GalNAc-alkyl)3 (Aeo) 2'-O-methoxyethyladenosine-3'-phosphate
(Aeos) 2'-O-methoxyethyladenosine-3'-phosphorothioate (Geo)
2'-O-methoxyethylguanosine-3'-phosphate (Geos)
2'-O-methoxyethylguanosine-3'-phosphorothioate (Teo)
2'-O-methoxyethyl-5-methyluridine-3'-phosphate (Teos)
2'-O-methoxyethyl-5-methyluridine-3'-phosphorothioate (m5Ceo)
2'-O-methoxyethyl-5-methylcytidine-3'-phosphate (m5Ceos)
2'-O-methoxyethyl-5-methylcytidine-3'-phosphorothioate (A3m)
3'-O-methyladenosine-2'-phosphate (A3mx)
3'-O-methyl-xylofuranosyladenosine-2'-phosphate (G3m)
3'-O-methylguanosine-2'-phosphate (G3mx)
3'-O-methyl-xylofuranosylguanosine-2'-phosphate (C3m)
3'-O-methylcytidine-2'-phosphate (C3mx)
3'-O-methyl-xylofuranosylcytidine-2'-phosphate (U3m)
3'-O-methyluridine-2'-phosphate (U3mx)
3'-O-methylxylouridine-2'-phosphate (Chd)
2'-O-hexadecyl-cytidine-3'-phosphate (pshe)
Hydroxyethylphosphorothioate (Uhd)
2'-O-hexadecyl-uridine-3'-phosphate (Tgn) Thymidine-glycol nucleic
acid (GNA) S-Isomer (Cgn) Cytidine-glycol nucleic acid (GNA) (Chd)
2'-O-hexadecyl-cytidine-3'-phosphate (Ggn)
2'-O-hexadecyl-cytidine-3'-phosphate (Agn) Adenosine-glycol nucleic
acid (GNA) P 5'-phosphate (m5Cam)
2'-O-(N-methylacetamide)-5-methylcytidine-3'-phosphate (m5Cams)
2'-O-(N-methylacetamide)-5-methylcytidine-3'-phosphorothioate (Tam)
2'-O-(N-methylacetamide)thymidine-3'-phosphate (Tams)
2'-O-(N-methylacetamide)thymidine-3'-phosphorothioate (Aam)
2'-O-(N-methylacetamide)adenosine-3'-phosphate (Aams)
2'-O-(N-methylacetamide)adenosine-3'-phosphorothioate (Gam)
2'-O-(N-methylacetamide)guanosine-3'-phosphate (Gams)
2'-O-(N-methylacetamide)guanosine-3'-phosphorothioate Y44
2-hydroxymethyl-tetrahydrofurane-5-phosphate Q173
N-((GalNAc)-amidopentanoyl)-prolinol-4-phosphate
(Hyp-C5-(GalNAc))
[0797] All patents and other publications identified in the
specification and examples are expressly incorporated herein by
reference for all purposes. These publications are provided solely
for their disclosure prior to the filing date of the present
application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention or for any other reason.
All statements as to the date or representation as to the contents
of these documents is based on the information available to the
applicants and does not constitute any admission as to the
correctness of the dates or contents of these documents.
[0798] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow. Further, to the extent not already indicated, it will be
understood by those of ordinary skill in the art that any one of
the various embodiments herein described and illustrated can be
further modified to incorporate features shown in any of the other
embodiments disclosed herein.
Sequence CWU 1
1
245123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 1cugguuaaca ccauuuacuu caa
23223DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 2cugguuaaca ccauuuacuu caa
23323DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 3cugguuaaca ccauuuacuu caa
23420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 4guuaacacca uuuacuucaa 20516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 5acaccauuua cuucaa 16624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 6ctgguuaaca ccauuuacuu caaa 24724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 7ctgguuaaca ccauuuacut caaa 24824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 8ctgguuaaca ccauuuacut caaa 24923DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 9ctgguuaaca ccauuuacut caa 231020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 10guuaacacca uuuacuucaa 201120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 11guuaacacca uuuacutcaa 201220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 12guuaacacca uuuacutcaa 201320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 13guuaacacca uuuacutcaa 201416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 14acaccauuua cuucaa 161516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 15acaccauuua cutcaa 161616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 16acaccauuua cutcaa 161723DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 17ctgguuaaca ccauuuactt caa 231820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 18guuaacacca uuuacttcaa 201916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 19acaccauuua cttcaa 162020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 20guuaacacgu uuuagatcaa 202120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 21accuuataua cgautacuca 202220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 22gtuaacacca uuuacutcaa 202320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 23gtuaacacca uuuacutcaa 202420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 24gtuaacacca uuuacutcaa 202520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 25gtuaacacca uuuacutcaa 202620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 26guuaacacca tutacutcaa 202720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 27guuaacacca tutacutcaa 202820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 28gttaacacca uuuacuucaa 202920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 29gttaacacca uuuacuucaa 203020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 30guuaacacca tttacuucaa 203120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 31guuaacacca tttacuucaa 203220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 32gtuaacacca tuuacuucaa 203320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 33gtuaacacca tuuacuucaa 203420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 34guuaacacca uuuacuucaa 203520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 35guuaacacca uuuacuucaa 203620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 36guuaacacca uuuacuucaa 203720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 37guuaacacca uuuacuucaa 203820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 38guuaacacca tuuacuucaa 203920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 39guuaacacca tuuacutcaa 204020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 40guuaacacca tutactucaa 204120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 41guuaacacca tuuacttcaa 204220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 42gtuaacacca tuuacuucaa 204320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 43gttaacacca tuuacuucaa 204420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 44guuaacacca tuuactucaa 204520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 45gtuaacacca tuuacuucaa 204620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 46gttaacacca tuuacuucaa 204720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 47gttaacacca uuuacuucaa 204820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 48gtuaacacca uuuacutcaa 204920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 49gtuaacacca uuuacutcaa 205020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 50gtuaacacca uuuacutcaa 205120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 51gtuaacacca uuuacutcaa 205220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 52gtuaacacca uuuacuucaa 205320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 53gutaacacca uuuacuucaa 205420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 54guuaacacca uuuacuucaa 205520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 55guuaacacca uuuacuucaa 205620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 56guuaacacca uuuacuucaa 205720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 57guuaacacca tuuacuucaa 205820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 58guuaacacca utuacuucaa 205920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 59guuaacacca uutacuucaa 206020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 60guuaacacca uuuacuucaa 206120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 61guuaacacca uuuactucaa 206220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 62guuaacacca uuuacutcaa 206320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 63guuaacacca uuuacuucaa 206420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 64gtuaacacca uuuacuucaa 206520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 65gutaacacca uuuacuucaa 206620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 66guuaacacca uuuacuucaa 206720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 67guuaacacca uuuacuucaa 206820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 68guuaacacca uuuacuucaa 206920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 69guuaacacca tuuacuucaa 207020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 70guuaacacca utuacuucaa 207120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 71guuaacacca uutacuucaa 207220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 72guuaacacca uuuacuucaa
207320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 73guuaacacca uuuactucaa
207420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 74guuaacacca uuuacutcaa
207520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 75guuaacacca uuuacuucaa
207620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-(9-aminoethoxy)phenoxazin-
e
ribonucleotide-3'-phosphatemodified_base(18)..(18)2'-O-methyl-(9-am-
inoethoxy)phenoxazine ribonucleotide-3'-phosphate 76guuaanacca
uuuacuunaa 207720DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA
Molecule Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-(9-aminoethoxy)-
phenoxazine
ribonucleotide-3'-phosphatemodified_base(15)..(15)2'-O-methyl-(9-aminoeth-
oxy)phenoxazine ribonucleotide-3'-phosphate 77guuaanacca uuuanuucaa
207820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(8)..(8)2'-O-methyl-(9-aminoethoxy)phenoxazin-
e
ribonucleotide-3'-phosphatemodified_base(18)..(18)2'-O-methyl-(9-am-
inoethoxy)phenoxazine ribonucleotide-3'-phosphate 78guuaacanca
uuuacuunaa 207920DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA
Molecule Synthetic
oligonucleotidemodified_base(9)..(9)2'-O-methyl-(9-aminoethoxy)-
phenoxazine
ribonucleotide-3'-phosphatemodified_base(15)..(15)2'-O-methyl-(9-aminoeth-
oxy)phenoxazine ribonucleotide-3'-phosphate 79guuaacacna uuuanuucaa
208020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-(9-aminoethoxy)phenoxazin-
e
ribonucleotide-3'-phosphatemodified_base(8)..(8)2'-O-methyl-(9-amin-
oethoxy)phenoxazine ribonucleotide-3'-phosphate 80guuaananca
uuuacuucaa 208120DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA
Molecule Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-(9-aminoethoxy)-
phenoxazine
ribonucleotide-3'-phosphatemodified_base(9)..(9)2'-O-methyl-(9-aminoethox-
y)phenoxazine ribonucleotide-3'-phosphate 81guuaanacna uuuacuucaa
208220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(15)..(15)2'-O-methyl-(9-aminoethoxy)phenoxaz-
ine
ribonucleotide-3'-phosphatemodified_base(18)..(18)2'-O-methyl-(9--
aminoethoxy)phenoxazine ribonucleotide-3'-phosphate 82guuaacacca
uuuanuunaa 208320DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA
Molecule Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-(9-aminoethoxy)-
phenoxazine ribonucleotide-3'-phosphate 83guuaanacca uuuacuucaa
208420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(8)..(8)2'-O-methyl-(9-aminoethoxy)phenoxazin-
e ribonucleotide-3'-phosphate 84guuaacanca uuuacuucaa
208520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(9)..(9)2'-O-methyl-(9-aminoethoxy)phenoxazin-
e ribonucleotide-3'-phosphate 85guuaacacna uuuacuucaa
208620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(15)..(15)2'-O-methyl-(9-aminoethoxy)phenoxaz-
ine ribonucleotide-3'-phosphate 86guuaacacca uuuanuucaa
208720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(18)..(18)2'-O-methyl-(9-aminoethoxy)phenoxaz-
ine ribonucleotide-3'-phosphate 87guuaacacca uuuacuunaa
208820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-phenoxazine
ribonucleotide-3'-
phosphatemodified_base(9)..(9)2'-O-methyl-phenoxazine
ribonucleotide-3'-
phosphatemodified_base(18)..(18)2'-O-methyl-phenoxazine
ribonucleotide-3'- phosphate 88guuaanacna uuuacuunaa
208920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-phenoxazine
ribonucleotide-3'-
phosphatemodified_base(15)..(15)2'-O-methyl-phenoxazine
ribonucleotide-3'-
phosphatemodified_base(18)..(18)2'-O-methyl-phenoxazine
ribonucleotide-3'- phosphate 89guuaanacca uuuanuunaa
209020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-phenoxazine
ribonucleotide-3'-
phosphatemodified_base(18)..(18)2'-O-methyl-phenoxazine
ribonucleotide-3'- phosphate 90guuaanacca uuuacuunaa
209120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-phenoxazine
ribonucleotide-3'-
phosphatemodified_base(15)..(15)2'-O-methyl-phenoxazine
ribonucleotide-3'- phosphate 91guuaanacca uuuanuucaa
209220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(9)..(9)2'-O-methyl-phenoxazine
ribonucleotide-3'-
phosphatemodified_base(15)..(15)2'-O-methyl-phenoxazine
ribonucleotide-3'- phosphate 92guuaacacna uuuanuucaa
209320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(6)..(6)2'-O-methyl-phenoxazine
ribonucleotide-3'-
phosphatemodified_base(8)..(8)2'-O-methyl-phenoxazine
ribonucleotide-3'- phosphate 93guuaananca uuuacuucaa
209420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(15)..(15)2'-O-methyl-phenoxazine
ribonucleotide-3'-
phosphatemodified_base(18)..(18)2'-O-methyl-phenoxazine
ribonucleotide-3'- phosphate 94guuaacacca uuuanuunaa
209520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(6)..(6)2'-deoxy-2'-fluoro-phenoxazine
ribonucleotide-
3'-phosphatemodified_base(15)..(15)2'-deoxy-2'-fluoro-phenoxazine
ribonucleotide-
3'-phosphatemodified_base(18)..(18)2'-deoxy-2'-fluoro-phenoxazine
ribonucleotide- 3'-phosphate 95guuaanacca uuuanuunaa
209620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 96guuaacacca uuuacuucaa
209720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 97guuaacacca uuuacuucaa
209820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 98guuaacacca uuuacuucaa
209920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 99guuaacacca uuuacuucaa
2010020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 100guuaacacca uuuacuucaa
2010120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 101gtuaacacca uuuacuucaa
2010220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 102guuaacacca uuuactucaa
2010320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 103guuaacacca tuuacuucaa
2010420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 104gtuaacacca utuacuucaa
2010520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 105ggtuaacacc auuuacutca
2010620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 106tggtuaacac cauuuacutc
2010720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 107ctggtuaaca ccauutacut
2010820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 108gtuaacacca uuuacutcaa
2010920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 109guuaacacca uuuacuucaa
2011020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 110guuaacacca uuuacuucaa
2011123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 111cugguuaaca ccatutacut caa
2311220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 112guuaacacca tutacutcaa
2011316DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 113acaccatuta cutcaa 1611415DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 114caccatutac utcaa 1511514DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 115accatutacu tcaa 1411613DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 116ccatutacut caa 1311712DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 117catutacutc aa 1211811DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 118atutacutca a 1111910DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 119tutacutcaa 1012021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 120gguuaacacc auuuacuuca a 2112123RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 121uugaaguaaa ugguguuaac cag 231228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 122tacutcaa 81238DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic oligonucleotide 123tacttcaa
812412DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 124catutacutc aa 1212510DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 125tutacutcaa 1012616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 126acaccatuta cutcaa 1612714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 127accatutacu tcaa
1412823DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 128uucaguaccu uagaguucca cua
2312923DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 129uucaguaccu uagaguucca cta
2313023DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 130uucaguaccu uagaguucca cta
2313123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 131uucaguaccu uagaguucca cta
2313223DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 132uucaguaccu uagagutcca cta
2313323DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 133uucaguaccu uagagttcca cta
2313423DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 134uucaguaccu tagagttcca cta
2313523DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 135uucaguaccu tagagttcca cta
2313622DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 136ucaguaccuu agagttccac ta
2213721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 137caguaccuua gagttccact a
2113820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 138aguaccuuag agttccacta
2013919DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 139guaccuuaga gttccacta
1914018DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 140uaccuuagag ttccacta
1814117DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 141accuuagagt tccacta
1714216DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 142ccuuagagtt ccacta 1614315DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 143cuuagagttc cacta 1514414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 144uuagagttcc acta 1414513DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 145uagagttcca cta 1314612DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 146agagttccac ta 1214711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 147gagttccact a 1114810DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 148agttccacta 101499DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 149gttccacta 91508DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic oligonucleotide 150ttccacta
815116DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 151cuuagagttc cactaa 1615216DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 152ccuuagagtt ccacta 1615316DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 153accutagagt tccaca 1615416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 154uaccutagag ttccaa 1615516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 155guaccutaga gttcca 1615616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 156agtaccutag agttca 1615716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 157cagtaccuta gagtta 1615816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 158ucagtaccut agagta 1615916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 159tucagtaccu tagaga 1616016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 160ccuuagagtt ccacta 1616116DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 161ccuuagagtt ccacta 1616216DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 162ccuuagagtt ccacta 1616316DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 163ccuuagagtt ccacta 1616416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 164ccuuagagtt ccactt 1616516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 165ccuuagagtt ccactg 1616616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 166ccuuagagtt ccactc 1616716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 167ccuuagagtt ccacta 1616816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 168ccutagagtt ccacua 1616916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 169ccttagagtt ccacua 1617016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 170ccutagagtu ccacta 1617115DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 171ccuuagagtt ccact 1517215DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 172ccuuagagtt ccact 1517315DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 173ccuuagagtt ccact 1517416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 174ccuuagagtt ccacta 1617516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 175ccuuagagtt ccacta 1617616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 176ccuuagagtt ccacta 1617715DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 177ccuuagagtt ccact 1517816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 178ccuuagagtt ccacta 1617916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 179ccuuagagtt ccacta 1618016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 180ccuuagagtt ccacta 1618116DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(11)..(11)2'-O-methyl-(9-aminoethoxy)phenoxaz-
ine ribonucleotide-3'-phosphate 181ccuuagaguu ncacua
1618216DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(12)..(12)2'-O-methyl-(9-aminoethoxy)phenoxaz-
ine ribonucleotide-3'-phosphate 182ccuuagaguu cnacua
1618316DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(14)..(14)2'-O-methyl-(9-aminoethoxy)phenoxaz-
ine ribonucleotide-3'-phosphate 183ccuuagaguu ccanua
1618416DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotidemodified_base(2)..(2)2'-O-methyl-(9-aminoethoxy)phenoxazin-
e ribonucleotide-3'-phosphate 184cnuuagaguu ccacua
1618516DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 185ccuuagaguu ccacua 1618616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 186ccuuagaguu ccacua 1618716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 187cctuagaguu ccacua 1618816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 188ccutagaguu ccacua 1618916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 189ccuuagaguu ccacua 1619016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 190ccuuagaguu ccacua 1619116DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 191ccuuagaguu ccacua 1619216DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 192ccuuagaguu ccacua 1619316DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 193ccuuagagtu ccacua 1619416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 194ccuuagagut ccacua 1619516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 195ccuuagaguu ccacua 1619616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 196ccuuagaguu ccacua 1619716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 197ccuuagaguu ccacua
1619816DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 198ccuuagaguu ccacua 1619916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 199ccuuagaguu ccacta 1620016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 200ccuuagagtc gcucca 1620116DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 201ccuuagagtt ccucca 1620216DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 202ccuuagagtt ccacca 1620316DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 203ccuuagagtt ccucta 1620416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 204gagauucccc ucctca 1620515DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 205ctuagaguuc cacta 1520615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 206cttagagttc cacta 1520721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 207cagtaccuua gaguuccact a 2120821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 208cagtacctta gagttccact a 2120921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 209caguaccuua gaguuccacu a 2121023RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 210uaguggaacu cuaagguacu gaa 2321129PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 211Ala
Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala1 5 10
15Leu Glu Ala Leu Ala Glu Ala Ala Ala Ala Gly Gly Cys 20
2521230PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 212Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala
Leu Ala Glu Ala Leu Ala1 5 10 15Glu Ala Leu Ala Glu Ala Leu Ala Ala
Ala Ala Gly Gly Cys 20 25 3021315PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 213Ala Leu Glu Ala Leu Ala
Glu Ala Leu Glu Ala Leu Ala Glu Ala1 5 10 1521422PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 214Gly
Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10
15Met Ile Trp Asp Tyr Gly 2021523PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 215Gly Leu Phe Gly Ala Ile
Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile Asp Gly Trp
Tyr Gly 2021648PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 216Gly Leu Phe Glu Ala Ile Glu Gly
Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile Asp Gly Trp Tyr Gly
Cys Gly Leu Phe Glu Ala Ile Glu Gly 20 25 30Phe Ile Glu Asn Gly Trp
Glu Gly Met Ile Asp Gly Trp Tyr Gly Cys 35 40 4521744PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
217Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly1
5 10 15Met Ile Asp Gly Gly Cys Gly Leu Phe Glu Ala Ile Glu Gly Phe
Ile 20 25 30Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Gly Cys 35
4021835PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 218Gly Leu Phe Gly Ala Leu Ala Glu Ala Leu
Ala Glu Ala Leu Ala Glu1 5 10 15His Leu Ala Glu Ala Leu Ala Glu Ala
Leu Glu Ala Leu Ala Ala Gly 20 25 30Gly Ser Cys
3521934PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 219Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile
Glu Asn Gly Trp Glu Gly1 5 10 15Leu Ala Glu Ala Leu Ala Glu Ala Leu
Glu Ala Leu Ala Ala Gly Gly 20 25 30Ser Cys22041PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMOD_RES(17)..(17)NorleucineMOD_RES(38)..(38)Norleucine
220Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly1
5 10 15Xaa Ile Asp Gly Lys Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile
Glu 20 25 30Asn Gly Trp Glu Gly Xaa Ile Asp Gly 35
4022119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 221Leu Phe Glu Ala Leu Leu Glu Leu Leu Glu Ser
Leu Trp Glu Leu Leu1 5 10 15Leu Glu Ala22220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 222Gly
Leu Phe Lys Ala Leu Leu Lys Leu Leu Lys Ser Leu Trp Lys Leu1 5 10
15Leu Leu Lys Ala 2022320PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 223Gly Leu Phe Arg Ala Leu
Leu Arg Leu Leu Arg Ser Leu Trp Arg Leu1 5 10 15Leu Leu Arg Ala
2022430PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 224Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala
Lys Ala Leu Ala Lys His1 5 10 15Leu Ala Lys Ala Leu Ala Lys Ala Leu
Lys Ala Cys Glu Ala 20 25 3022522PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 225Gly Leu Phe Phe Glu Ala
Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu1 5 10 15Gly Leu Ile Glu Gly
Cys 2022626PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 226Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr
Gly Leu Pro Ala Leu1 5 10 15Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln
20 252278PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 227His His His His His Trp Tyr Gly1
522810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 228Cys His Lys Lys Lys Lys Lys Lys His Cys1 5
1022916PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 229Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg
Met Lys Trp Lys Lys1 5 10 1523014PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 230Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg Pro Pro Gln Cys1 5 1023127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 231Gly
Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly1 5 10
15Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20
2523218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 232Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys
Gln Ala His Ala His1 5 10 15Ser Lys23326PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 233Gly
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Ile Asn Leu Lys1 5 10
15Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 2523418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 234Lys
Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys1 5 10
15Leu Ala2359PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 235Arg Arg Arg Arg Arg Arg Arg Arg Arg1
523610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 236Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys1 5
1023737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 237Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys
Glu Lys Ile Gly Lys Glu1 5 10 15Phe Lys Arg Ile Val Gln Arg Ile Lys
Asp Phe Leu Arg Asn Leu Val 20 25 30Pro Arg Thr Glu Ser
3523831PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 238Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu
Glu Asn Ser Ala Lys Lys1 5 10 15Arg Ile Ser Glu Gly Ile Ala Ile Ala
Ile Gln Gly Gly Pro Arg 20 25 3023930PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
239Ala Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr1
5 10 15Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 20
25 3024036PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 240Asp His Tyr Asn Cys Val Ser Ser Gly Gly
Gln Cys Leu Tyr Ser Ala1 5 10 15Cys Pro Ile Phe Thr Lys Ile Gln Gly
Thr Cys Tyr Arg Gly Lys Ala 20 25 30Lys Cys Cys Lys
3524142PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 241Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro
Arg Pro Arg Pro Pro Pro1 5 10 15Phe Phe Pro Pro Arg Leu Pro Pro Arg
Ile Pro Pro Gly Phe Pro Pro 20 25 30Arg Phe Pro Pro Arg Phe Pro Gly
Lys Arg 35 4024213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 242Ile Leu Pro Trp Lys Trp Pro Trp Trp
Pro Trp Arg Arg1 5 1024316PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 243Ala Ala Val Ala Leu Leu
Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 1524411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 244Ala
Ala Leu Leu Pro Val Leu Leu Ala Ala Pro1 5 1024512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 245Arg
Lys Cys Arg Ile Val Val Ile Arg Val Cys Arg1 5 10
* * * * *