U.S. patent application number 15/837785 was filed with the patent office on 2018-08-09 for site-specific delivery of nucleic acids by combining targeting ligands with endosomolytic components.
This patent application is currently assigned to Arbutus Biopharma Corporation. The applicant listed for this patent is Arbutus Biopharma Corporation. Invention is credited to David Butler, Muthusamy Jayaraman, Muthiah Manoharan, Kallanthottathil G. Rajeev.
Application Number | 20180221494 15/837785 |
Document ID | / |
Family ID | 41162667 |
Filed Date | 2018-08-09 |
United States Patent
Application |
20180221494 |
Kind Code |
A1 |
Manoharan; Muthiah ; et
al. |
August 9, 2018 |
SITE-SPECIFIC DELIVERY OF NUCLEIC ACIDS BY COMBINING TARGETING
LIGANDS WITH ENDOSOMOLYTIC COMPONENTS
Abstract
The invention relates to compositions and methods for
site-specific delivery of nucleic acids by combining them with
targeting ligands and endosomolytic components.
Inventors: |
Manoharan; Muthiah;
(Cambridge, MA) ; Rajeev; Kallanthottathil G.;
(Cambridge, MA) ; Butler; David; (Medford, MA)
; Jayaraman; Muthusamy; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Arbutus Biopharma Corporation |
Burnaby |
|
CA |
|
|
Assignee: |
Arbutus Biopharma
Corporation
Burnaby
CA
|
Family ID: |
41162667 |
Appl. No.: |
15/837785 |
Filed: |
December 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15133010 |
Apr 19, 2016 |
9895448 |
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15837785 |
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14043682 |
Oct 1, 2013 |
9345780 |
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15133010 |
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12936961 |
Dec 28, 2010 |
8575123 |
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PCT/US2009/040274 |
Apr 10, 2009 |
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14043682 |
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61044186 |
Apr 11, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1137 20130101;
A61P 31/04 20180101; A61P 25/14 20180101; A61P 31/08 20180101; A61P
31/12 20180101; A61K 47/551 20170801; A61P 29/00 20180101; A61P
37/06 20180101; A61P 19/02 20180101; C12N 2310/3513 20130101; A61P
9/00 20180101; C12N 2320/32 20130101; A61P 11/06 20180101; C12Y
304/21021 20130101; A61P 33/00 20180101; A61P 1/16 20180101; A61P
35/00 20180101; A61P 25/00 20180101; A61P 25/04 20180101; A61P
43/00 20180101; C12N 2310/346 20130101; A61P 19/00 20180101; C12N
2310/14 20130101; A61K 48/0008 20130101; A61P 31/22 20180101; A61P
31/00 20180101; A61P 37/00 20180101; C12N 15/87 20130101; C12N
15/111 20130101; A61P 31/10 20180101; C12N 2310/322 20130101; C12N
2310/351 20130101; A61K 47/554 20170801; A61P 31/18 20180101; A61P
25/16 20180101; C12N 2310/11 20130101; A61K 47/545 20170801; A61P
31/20 20180101; A61P 33/06 20180101; A61P 25/28 20180101; A61P
31/06 20180101; A61P 1/04 20180101; C12N 2310/315 20130101; A61P
3/10 20180101; A61P 35/02 20180101; A61P 9/10 20180101; A61P 31/14
20180101; A61K 47/549 20170801; A61P 17/06 20180101 |
International
Class: |
A61K 47/54 20170101
A61K047/54; A61K 47/55 20170101 A61K047/55; A61K 48/00 20060101
A61K048/00; C12N 15/11 20060101 C12N015/11; C12N 15/113 20100101
C12N015/113; C12N 15/87 20060101 C12N015/87 |
Claims
1. A modular composition, comprising a nucleic acid, at least one
endosomolytic component, and at least one targeting ligand, the
composition having the formula: ##STR00080## wherein ##STR00081##
is the nucleic acid; E is the endosomolytic component; L is the
targeting ligand; x represents independently for each occurrence 0
or 1; y represents independently for each occurrence 1, 2, 3, 4, 5,
or 6; z represents independently for each occurrence 0, 1, 2, 3, 4,
5, or 6; A is a linking moiety selected from the group consisting
of: ##STR00082## wherein Z.sup.1 represents independently for each
occurrence O or S; Z.sup.2 represents independently for each
occurrence --OH, --OM, --Oalkyl, --Oaryl, --Oaralkyl, --SH, --SM,
--Salkyl, --Saryl, -aralkyl, --N(R.sup.3)R.sup.4,
--C(R.sup.11).sub.2).sub.mN(R.sup.11).sub.2,
--N(R.sup.11)(C(R.sup.11).sub.2).sub.mN(R.sup.11).sub.2, or alkyl;
R.sup.3 and R.sup.4 represent independently H or alkyl; or R.sup.3
and R.sup.4 taken together form a 3-, 4-, 5-, 6-, or 7-membered
ring; R.sup.11 represents independently for each occurrence
hydrogen or alkyl; M represents independently for each occurrence
an alkali metal or a transition metal with an overall charge of +1;
R.sup.8 represents independently for each occurrence hydrogen,
alkyl, aryl, aralkyl, acyl, silyl, a bond to the nucleic acid, or,
when combined with B.sup.10, a bond between linking moieties;
B.sup.10 is a bond between A and the nucleic acid or, when combined
with R.sup.8, is a bond between linking moieties; A' is a direct
bond or a tether having the formula:
--[(P-Q-R).sub.q--X--(P'-Q'-R').sub.q'].sub.q''-T-, wherein: P, R,
T, P' and R' are each independently absent, CO, NH, O, S, OC(O),
NHC(O), CH.sub.2, CH.sub.2NH, CH.sub.2O; NHCH(R.sup.a)C(O),
--C(O)--CH(R.sup.a)--NH--, C(O)-(optionally substituted
alkyl)-NH--, CH.dbd.N--O, ##STR00083## cyclyl, heterocyclyl, aryl
or heteroaryl; R.sub.50 and R.sub.51 are independently alkyl,
substituted alkyl, or R.sub.50 and R.sub.51 taken together form a
cyclic ring; Q and Q' are each independently for each occurrence
absent, --(CH.sub.2).sub.n--,
--C(R.sup.40)(R.sup.41)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R.sup.40)(R.sup.41)--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--, aryl, heteroaryl,
cyclyl, or heterocyclyl; X is absent or a cleavable linking group;
R.sup.a is H or an amino acid side chain; R.sup.40 and R.sup.41 are
each independently H, CH.sub.3, OH, SH or N(R.sup.X).sub.2; R.sup.X
is, for each occurrence, H, methyl, ethyl, propyl, isopropyl, butyl
or benzyl; q, q' and q'' are each independently 0-30; n is, for
each occurrence, an integer from 1-20; and m is, for each
occurrence, an integer from 0-50; provided that E and L are each
present at least once.
2. The composition of claim 1, wherein the composition has the
formula: ##STR00084##
3. The composition of claim 1, wherein the composition has the
formula: ##STR00085## wherein each z is independently 1, 2, 3, 4,
5, or 6.
4. The composition of claim 1, wherein the composition has the
formula: ##STR00086## wherein z is 1, 2, 3, 4, 5, or 6 in one
instance, and z is 0, 1, 2, 3, 4, 5, or 6 in the other
instance.
5. The composition of claim 1, wherein the A' tether is an alkyl
diradical, heteroalkyl diradical, alkenyl diradical, alkynyl
diradical, alkylalkynyl diradical, aminoalkyl diradical, thioether,
--C(O)--, --S(O)--, --S(O).sub.2--, --C(R).sub.2--, or has the
formula selected from the group consisting of: ##STR00087## wherein
m represents independently for each occurrence 1, 2, 3, 4, 5, 6, 7,
or 8; m.sup.1 represents independently for each occurrence 0, 1, 2,
3, 4, 5, 6, 7, or 8; p represents independently for each occurrence
1, 2, 3, or 4; and R represents independently for each occurrence
hydrogen or alkyl.
6. The composition of claim 1, wherein the A' tether is selected
from the group consisting of: ##STR00088## ##STR00089##
##STR00090##
7. The composition of claim 1, wherein the endosomolytic component
is selected from the group consisting of imidazoles, poly or
oligoimidazoles, linear or branched polyethyleneimines (PEIs),
linear and branched polyamines, 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, and natural and synthetic
fusogenic lipids.
8. (canceled)
9. (canceled)
10. (canceled)
11. The composition of claim 1, wherein the targeting ligand is
selected from the group consisting of an antibody, a ligand-binding
portion of a receptor, a ligand for a receptor, an aptamer,
D-galactose, N-acetyl-D-galactose (GalNAc), multivalent
N-acetyl-D-galactose, D-mannose, cholesterol, a fatty acid, a
lipoprotein, folate, thyrotropin, melanotropin, surfactant protein
A, mucin, carbohydrate, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose,
multivalent fucose, glycosylated polyaminoacids, transferrin,
bisphosphonate, polyglutamate, polyaspartate, a lipophilic moiety
that enhance plasma protein binding, a steroid, bile acid, vitamin
B.sub.12, biotin, an RGD peptide, an RGD peptide mimetic,
ibuprofen, naproxen, aspirin, folate, and analogs and derivatives
thereof.
12. (canceled)
13. (canceled)
14. The composition of claim 1, wherein the nucleic acid contains
at least one sugar modification.
15. (canceled)
16. (canceled)
17. The composition of claim 1, wherein the nucleic acid contains
at least one backbone modification.
18. (canceled)
19. The composition of claim 1, where in the nucleic acid comprises
at least one nucleobase modification.
20. (canceled)
21. The composition of claim 1, wherein the nucleic acid down
regulates the expression of a target gene.
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. The composition of claim 1, wherein the endosomolytic agent and
the targeting ligand are on the same terminal end of the nucleic
acid.
27. The composition of claim 1, wherein the endosomolytic agent and
the targeting ligands are on the opposite terminal ends of the
nucleic acid.
28. The composition of claim 1, wherein one of the endosomolytic
agent and the targeting ligand is at the terminal end of the
nucleic acid and the other is at an internal, non-terminal position
on the nucleic acid.
29. The composition of claim 1, wherein both the endosomolytic
agent and the targeting ligand are located at internal,
non-terminal positions on the nucleic acid.
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. The composition of claim 1, wherein the tether comprises at
least one redox cleavable linking group.
35. The composition of claim 1, wherein the tether comprises at
least one pH sensitive component.
36. A method of delivering a modular composition to a cell, the
method comprising (a) contacting a cell with a modular composition
of claim 1; and (b) allowing the cell to internalize the modular
composition.
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. A method of inhibiting the expression of one or more genes,
comprising contacting one or more cells with an effective amount of
the modular composition of claim 1, wherein the effective amount is
an amount that suppresses the expression of the one or more genes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation application of U.S.
application Ser. No. 15/133,010, filed Apr. 19, 2016, which is a
Continuation application of U.S. application Ser. No. 14/043,682,
filed Oct. 1, 2013, now U.S. Pat. No. 9,345,780, which is a
Continuation application of U.S. application Ser. No. 12/936,961,
now U.S. Pat. No. 8,575,123, which is a 35 U.S.C. .sctn. 371
application of International Application No. PCT/US2009/040274,
filed Apr. 10, 2009, which claims priority to U.S. Provisional
Application Ser. No. 61/044,186, filed Apr. 11, 2008, all of which
are herein incorporated by reference in their entirety.
BACKGROUND
[0002] RNA interference or "RNAi" is a term initially coined by
Fire and co-workers to describe the observation that certain
double-stranded RNA (dsRNA) can block gene expression when it is
introduced into worms (Fire et al. (1998) Nature 391, 806-811).
Short double-stranded interfering RNA (dsiRNA) directs
gene-specific, post-transcriptional silencing in many organisms,
including vertebrates, and has provided a new tool for studying
gene function. RNAi may involve mRNA degradation.
[0003] Work in this field is typified by comparatively cumbersome
approaches to delivery of dsiRNA to live mammals. E.g., McCaffrey
et al. (Nature 418:38-39, 2002) demonstrated the use of dsiRNA to
inhibit the expression of a luciferase reporter gene in mice. The
dsiRNAs were administered by the method of hydrodynamic tail vein
injections (in addition, inhibition appeared to depend on the
injection of greater than 2 mg/kg dsiRNA). The inventors have
discovered, inter alia, that the unwieldy methods typical of some
reported work are not needed to provide effective amounts of dsiRNA
to mammals and in particular not needed to provide therapeutic
amounts of dsiRNA to human subjects. The advantages of the current
invention include practical, uncomplicated methods of
administration and therapeutic applications.
SUMMARY
[0004] The invention relates to compositions and methods for
delivery of an iRNA agent, (e.g., an iRNA agent or siRNA agent) or
other nucleic acid. In some embodiments, the nucleic acids which
may be used in the modular composition and methods of the invention
include iRNAs, siRNAs, single-stranded iRNAs, antagomirs, aptamers,
antisense nucleic acids, decoy oligonucleotides, microRNAs
(miRNAs), miRNA mimics, antimir, activating RNAs (RNAa), ribozymes,
supermirs, U1 adaptor and the like. Derivatives of these nucleic
acids may also be used.
[0005] Accordingly, in one aspect, the invention features a modular
composition, comprising a nucleic acid (e.g., an iRNA agent or
siRNA agent), an endosomolytic component, and a targeting
ligand.
[0006] The endosomolytic component may be a polyanionic peptide or
peptidomimetic which shows pH-dependent membrane activity and
fusogenicity. In certain embodiments, the endosomolytic component
assumes its active conformation at endosomal pH. The "active"
conformation is that conformation in which the endosomolytic
component promotes lysis of the endosome and/or transport of the
modular composition of the invention, or its components, from the
endosome to the cytoplasm of the cell.
[0007] Also provided are methods for screening libraries of
compounds for their differential membrane activity at endosomal pH
(e.g., pH 5-6) versus neutral pH. In certain embodiments, these
method may utilize a hemolysis assay. Promising candidates isolated
by this method may be useful as components of the modular
compositions of the invention.
[0008] The targeting ligand may be any moiety that, for example,
alters the pharmacokinetics, biodistribution, or cellular uptake of
the modular composition of the invention. Exemplary targeting
ligands include, for example, those which influence binding to
proteins in the blood and those that target specific organs,
tissues, or cell types. Targeting may be active or passive. Active
targeting may be achieved, for example, by using an antibody or a
binding partner for a cell-surface antigen, as the targeting
ligand. Passive targeting may be achieved, for example, by altering
the physicochemical properties (e.g., molecular weight) of the
modular composition of the invention to influence biodistribution.
One example of passive targeting is the enhanced permeability and
retention (EPR) effect.
[0009] The endosomolytic component and the targeting ligand of the
present invention may be linked to the nucleic acid in a number of
different manners, which will be more fully described in the
Detailed Description. In certain embodiments, a linker may be used
covalently couple the endosomolytic component or targeting ligand
to another component of the modular composition of the invention.
The nature of this linker (e.g., cleavable or non-cleavable) will
depend on the application.
[0010] In a certain embodiments, the modular composition may be
used to deliver an iRNA agent, or other nucleic acid, to a subject
in need thereof, e.g., to deliver an iRNA agent to a subject having
a disorder, e.g., a disorder described herein.
[0011] In certain embodiments, the modular composition may be kept,
maintained or stored at a pH greater than that typically found
inside an endosome (e.g., greater than pH 6.5, greater than pH 6.8,
greater than pH 7.0, greater than pH 7.5, greater than pH 8.0, or
higher).
[0012] In another aspect, the invention includes a method of
delivering an iRNA agent, or other nucleic acid, to a cell. The
method includes (a) providing or obtaining a modular composition of
the invention; (b) contacting a cell with the modular composition;
and (c) allowing the cell to internalize the modular
composition.
[0013] The method can be performed in vitro, ex vivo or in vivo,
e.g., to treat a subject identified as being in need of an iRNA
agent or other nucleic acid. A subject in need of a said agent is a
subject, e.g., a human, in need of having the expression of a gene
or genes, e.g., a gene related to a disorder, downregulated or
silenced.
[0014] In a certain embodiments, the modular composition may be
internalized by the cell into an endosome, e.g., by endocytosis. In
such embodiments, the modular composition can disrupt the endosome,
e.g., resulting in the release of the modular composition, or any
of its components (e.g., the iRNA agent), into the cytoplasm of the
cell.
[0015] In one aspect, the invention provides a method for
inhibiting the expression of one or more genes. The method
comprising contacting one or more cells with an effective amount of
an oligonucleotide of the invention, wherein the effective amount
is an amount that suppresses the expression of the one or more
genes. The method can be performed in vitro, ex vivo or in
vivo.
[0016] In another aspect, the invention features a kit. The kit may
include (a) an endosomolytic component; (b) a targeting ligand; and
(c) instructions for attaching an iRNA agent or other nucleic acid
to the endosomolytic component and targeting ligand. In some
embodiments, the kit may include an iRNA agent or other nucleic
acid. In some other embodiments, the kit may include the modular
composition of the invention, with instructions for in vitro, ex
vivo, or in vivo administration.
[0017] The methods and compositions of the invention, e.g., the
modular composition described herein, can be used with any of the
iRNA agents or nucleic acids described herein. The methods and
compositions of the invention may also be used with any of the iRNA
agents or nucleic acids known in the art. In addition, the methods
and compositions of the invention can be used for the treatment of
any disease or disorder described herein, and for the treatment of
any subject, e.g., any animal, any mammal, such as any human. One
of ordinary skill in the art will also recognize that the methods
and compositions of the invention may be used for the treatment of
any disease that would benefit from downregulating or silencing a
gene or genes.
[0018] The methods and compositions of the invention, e.g., the
modular composition described herein, may be used with any dosage
and/or formulation described herein, or any dosage or formulation
known in the art. In addition to the routes of administration
described herein, an ordinarily skilled artisan will also
appreciate that other routes of administration may be used to
administer the modular composition of the invention.
[0019] An "RNA agent" as used herein, is an unmodified RNA,
modified RNA, or nucleoside surrogate, all of which are defined
herein, see the section herein entitled RNA Agents. While numerous
modified RNAs and nucleoside surrogates are described herein,
examples include those which have greater resistance to nuclease
degradation than do unmodified RNAs. Further examples include those
which have a 2' sugar modification, a modification in a single
strand overhang, for example a 3' single strand overhang, or,
particularly if single stranded, a 5' modification which includes
one or more phosphate groups or one or more analogs of a phosphate
group.
[0020] An "iRNA agent", as used herein, is an RNA agent which can,
or which can be cleaved into an RNA agent which can, downregulate
the expression of a target gene, for example an endogenous or
pathogen target RNA. While not wishing to be bound by theory, an
iRNA agent may act by one or more of a number of mechanisms,
including post-transcriptional cleavage of a target mRNA sometimes
referred to in the art as RNAi, or pre-transcriptional or
pre-translational mechanisms. An iRNA agent can include a single
strand or can include more than one strands, e.g., it can be a
double stranded iRNA agent. If the iRNA agent is a single strand it
may include a 5' modification which includes one or more phosphate
groups or one or more analogs of a phosphate group.
[0021] In one aspect, the modular composition of the invention can
modulate the expression of a target gene. By "modulate" is meant
that the expression of the gene, or level of RNA molecule or
equivalent of RNA molecules encoding one or more proteins or
protein subunits, or activity od 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 use of the word "modulate" is not limited to
this definition.
[0022] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from this description, and from the claims. A person of
ordinary skill in the art will readily recognize that additional
embodiments of the invention exist. This application incorporates
all cited references, patents, and patent applications by reference
in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A-1D. Schematic representation of some exemplary
modular compositions comprising a nucleic acid (e.g., iRNA), a
targeting ligand, and endosomolytic component: FIG. 1A) conjugation
of the targeting ligand and endosomolytic component to the same
terminal side on the nucleic acid (same strand and terminus); FIG.
1B) conjugation of the targeting ligand and endosomolytic component
to the same strand but opposite termini; FIG. 1C) conjugation of
the targeting ligand and endosomolytic component to opposite
strands but same termini; FIG. 1D) conjugation of targeting ligand
and endosomolytic component to opposite strands and opposite
termini. In this embodiment, cholesterol is used as a targeting
ligand. The targeting ligand may also be conjugated to the
endosomolytic component as shown schematically in FIG. 2.
[0024] FIG. 2. Nucleic acid (e.g., iRNA) conjugate in which the
targeting ligand is covalently attached to the endosomolytic
component (nucleic acid-endosomolytic component-targeting ligand).
The arrangement nucleic acid-targeting ligand-endosomolytic
component is also contemplated.
[0025] FIG. 3. Some exemplary strategies for conjugating targeting
ligands and endosomolytic agents to oligonucleotides (e.g., SEQ ID
NO:189) with pH sensitive linkers.
[0026] FIG. 4. Schematic representation of targeting ligand
(folate) and endosomolytic agent (spermine) conjugated to an
oligonucleotide.
[0027] FIG. 5. Some exemplary synthetic monomers for conjugating
endosomolytic agents and targeting ligands with
oligonucleotides.
[0028] FIG. 6. Exemplary modular compositions having different
endosomolytic agents using GalNAc2 as the targeting ligand.
[0029] FIG. 7. Depicts the chemical structures of GalNAc2 and
GalNAc3.
[0030] FIG. 8. Depicts biotinylated GalNAc2 and GalNAc3.
DETAILED DESCRIPTION
[0031] In one aspect, the invention features a modular composition,
comprising a nucleic acid, at least one endosomolytic component,
and at least one targeting ligand. The endosomolytic component is
also referred to as endosomolytic ligand and endosomolytic agent
herein.
[0032] The modular composition may have the formula:
##STR00001##
wherein
##STR00002##
is the nucleic acid; E is the endosomolytic component; L is the
targeting ligand; x represents independently for each occurrence 0
or 1; y represents independently for each occurrence 1, 2, 3, 4, 5,
or 6; z represents independently for each occurrence 0, 1, 2, 3, 4,
5, or 6. In the modular composition, E and L are each present at
least once.
[0033] Preferred modular compositions include a modular composition
having the formula:
##STR00003##
a modular composition having the formula:
##STR00004##
wherein each z is independently 1, 2, 3, 4, 5, or 6; and a modular
composition having the formula:
##STR00005##
wherein z is 1, 2, 3, 4, 5, or 6 in one instance, and z is 0, 1, 2,
3, 4, 5, or 6 in the other instance.
[0034] The A moiety is a linking moiety. Any moiety known in the
art may be used to attach the endosomolytic component and/or the
targeting ligand to the nucleic acid. Preferably, A is a linking
moiety selected from the group consisting of:
##STR00006##
wherein
[0035] Z.sup.1 represents independently for each occurrence O or
S;
[0036] Z.sup.2 represents independently for each occurrence --OH,
--OM, --Oalkyl, --Oaryl, --Oaralkyl, --SH, --SM, --Salkyl, --Saryl,
-aralkyl, --N(R.sup.3)R.sup.4,
--C(R.sup.11).sub.2).sub.mN(R.sup.11).sub.2,
--N(R.sup.11)(C(R.sup.11).sub.2).sub.mN(R.sup.11).sub.2, or
alkyl;
[0037] R.sup.3 and R.sup.4 represent independently H or alkyl; or
R.sup.3 and R.sup.4 taken together form a 3-, 4-, 5-, 6-, or
7-membered ring;
[0038] R.sup.11 represents independently for each occurrence
hydrogen or alkyl;
[0039] M represents independently for each occurrence an alkali
metal or a transition metal with an overall charge of +1;
[0040] R.sup.8 represents independently for each occurrence
hydrogen, alkyl, aryl, aralkyl, acyl, silyl, a bond to the nucleic
acid, or, when combined with B.sup.10, a bond between linking
moieties;
[0041] B.sup.10 represents a bond between A and the nucleic acid
or, when combined with R.sup.8, is a bond between linking
moieties.
[0042] Because A can be more than one, the linking moieties may be
bonded to each other, and then bonded to an endosomolytic component
and/or targeting ligand. In a preferred embodiment, at least two A
linking moieties are bonded together and then bonded to the nucleic
acid. The endosomolytic component and targeting ligand may both be
bonded together through the same linking moiety, or the
endosomolytic component may be bonded to one linking moiety and the
targeting ligand to another linking moiety.
[0043] The linking may moiety may contain a tether (A') between the
linking moiety and the endosomolytic component and/or targeting
ligand. Thus, A' is a direct bond or a tether having the formula:
--[(P-Q-R).sub.q--X--(P'-Q'-R').sub.q'].sub.q''-T-, wherein:
[0044] P, R, T, P' and R' are each independently absent, CO, NH, O,
S, OC(O), NHC(O), CH.sub.2, CH.sub.2NH, CH.sub.2O;
NHCH(R.sup.a)C(O), --C(O)--CH(R.sup.a)--NH--, C(O)-(optionally
substituted alkyl)-NH--, CH.dbd.N--O,
##STR00007##
cyclyl, heterocyclyl, aryl or heteroaryl; where R.sub.50 and
R.sub.51 are independently alkyl, substituted alkyl, or R.sub.50
and R.sub.51 taken together form a cyclic ring;
[0045] Q and Q' are each independently for each occurrence absent,
--(CH.sub.2).sub.n--, --C(R.sup.40)(R.sup.41)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R.sup.40)(R.sup.41)--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--, aryl, heteroaryl,
cyclyl, or heterocyclyl;
[0046] X is absent or a cleavable linking group;
[0047] R.sup.a is H or an amino acid side chain;
[0048] R.sup.40 and R.sup.41 are each independently H, CH.sub.3,
OH, SH or N(R.sup.X).sub.2;
[0049] R.sup.X is, for each occurrence, H, methyl, ethyl, propyl,
isopropyl, butyl or benzyl;
[0050] q, q' and q'' are each independently 0-30;
[0051] n is, for each occurrence, an integer from 1-20; and
[0052] m is, for each occurrence, an integer from 0-50.
[0053] In preferred embodiments, the tether contains a redox
cleavable linking group, a pH sensitive component, or a combination
thereof. Preferred tethers include an alkyl diradical, heteroalkyl
diradical, alkenyl diradical, alkynyl diradical, alkylalkynyl
diradical, aminoalkyl diradical, thioether, --C(O)--, --S(O)--,
--S(O).sub.2--, --C(R).sub.2--, or a tether having the formula:
##STR00008##
wherein m represents independently for each occurrence 1, 2, 3, 4,
5, 6, 7, or 8; m.sup.1 represents independently for each occurrence
0, 1, 2, 3, 4, 5, 6, 7, or 8; p represents independently for each
occurrence 1, 2, 3, or 4; and R represents independently for each
occurrence hydrogen or alkyl.
[0054] More preferred tethers include
##STR00009## ##STR00010##
[0055] The endosomolytic component of this invention is a cellular
compartmental release component, and may be any compound capable of
releasing from any of the cellular compartments known in the art,
such as the endosome, lysosome, endoplasmic reticulum (ER), golgi
apparatus, microtubule, peroxisome, or other vesicular bodies with
the cell.
[0056] The endosomolytic ligand and the targeting ligand can be
located anywhere in the nucleic acid. Exemplary locations are the
5'-terminal end, the 3'-terminal end and/or internal non-terminal
positions. The term "terminal end" includes the first 5 nucleotides
from the end of the oligonucleotide. Thus the first 5 nucleotides
from the end are considered to be in the terminal end.
[0057] For instance, the 5'-terminal end may contain both the
endosomolytic ligand and targeting ligand on one or both strands of
the nucleic acid; the 3'-terminal end may contain both the
endosomolytic ligand and the targeting ligand on one or both
strands of the nucleic acid; the endosomolytic ligand and the
targeting ligand may both be present on an internal non-terminus
position on the nucleic acid; or any combination of the above. The
endosomolytic ligand and the targeting ligand may be attached
through one another and/or through linking moieties. While the
modular composition contains at least one endosomolytic component
and targeting ligand, it can contain six or more endosomolytic
components and six or more targeting ligands. A different number of
endosomolytic components and targeting ligands may be present on
the modular composition depending on the desired functionality of
the composition.
[0058] In some embodiments, both the endosomolytic ligand and the
targeting ligand are on the same terminal end of the
oligonucleotide.
[0059] In some embodiments, both the endosomolytic ligand and the
targeting ligand are on the 5'-terminal end of the
oligonucleotide.
[0060] In some embodiments, both the endosomolytic ligand and the
targeting ligand are on the 3'-terminal end of the
oligonucleotide.
[0061] When both the endosomolytic ligand and the targeting ligand
are on the same terminal end, one of them may be linked
[0062] In some embodiments, the endosomolytic ligand and the
targeting ligand are on opposite terminal ends of the
oligonucleotide.
[0063] In some embodiments, the endosomolytic ligand is on the
5'-terminal end of the oligonucleotide and the targeting ligand is
on the 3'-terminal end of the oligonucleotide.
[0064] In some embodiments, the endosomolytic ligand is on the
3'-terminal end of the oligonucleotide and the targeting ligand is
on the 5'-terminal end of the oligonucleotide.
[0065] In some embodiments, one of the endosomolytic ligand and the
targeting ligand is on the terminal while the other is located at
an internal non-terminal part of the oligonucleotide.
[0066] In some embodiments, the endosomolytic ligand is on the
terminal end and the targeting ligand is located at an internal
position.
[0067] In some embodiments, the endosomolytic ligand is on the
5'-terminal end and the targeting ligand is located at an internal
position.
[0068] In some embodiments, the endosomolytic ligand is on the
3'-terminal end and the targeting ligand is located at an internal
position.
[0069] In some embodiments, the targeting ligand is on the terminal
end and the endosomolytic ligand is located at an internal
position.
[0070] In some embodiments, the targeting ligand is on the
5'-terminal end and the endosomolytic ligand is located at an
internal position.
[0071] In some embodiments, the targeting ligand is on the
3'-terminal end and the endosomolytic ligand is located at an
internal position.
[0072] In some embodiments, both the endosomolytic ligand and the
targeting ligand are located at internal non-terminal
positions.
[0073] When the oligonucleotide is a double-stranded
oligonucleotide the endosomolytic ligand and the targeting ligand
may both be located on the same strand or on different strands.
[0074] In some embodiments, the endosomolytic ligand and the
targeting ligand are both on the same strand.
[0075] In some embodiments, the endosomolytic ligand and the
targeting ligand are both on the sense strand.
[0076] In some embodiments, the endosomolytic ligand and the
targeting ligand are both on the antisense strand.
[0077] In some embodiments, the endosomolytic ligand and the
targeting ligand are located on different strands.
[0078] In some embodiments, the endosomolytic ligand is on the
sense strand while the targeting ligand is on the antisense
strand.
[0079] In some embodiments, the endosomolytic ligand is on the
sense strand while the targeting ligand is on the antisense
strand.
[0080] In some embodiments, the endosomolytic ligand and the
targeting ligand are on different strands but on the same terminal
end of the double-stranded oligonucleotide.
[0081] In some embodiments, the endosomolytic ligand and the
targeting ligand are on different strands and on the opposite
terminal ends of the double-stranded oligonucleotide.
[0082] In some embodiments, the endosomolytic ligand and the
targeting ligand are on different strands and one of them is on the
terminal end and the other is located at an internal non-terminal
position of the double stranded oligonucleotide.
[0083] In some embodiments, the endosomolytic ligand and/or the
targeting ligand is attached to the oligonucleotides via an
intervening tether/linker. In some embodiments, the endosomolytic
ligand and/or the targeting ligand is linked to a monomer before
conjugation to the oligonucleotide. The endosomolytic ligand and/or
the targeting ligand may be linked directly or through a linker to
the monomer. The endosomolytic ligand and/or the targeting ligand
may be present on a monomer when said monomer is incorporated into
the growing strand. In some embodiments, the ligand may 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.,
X--(CH.sub.2).sub.nNH.sub.2 may be incorporated into a growing
oligonucleotide 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.
[0084] In another example, a monomer having a chemical group
suitable for taking part in Click Chemistry reaction may 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.
[0085] In some embodiments, the endosomolytic agents and/or
targeting ligands can be conjugated to nucleobases, sugar moieties,
or internucleosidic linkages of nucleic acid molecules, either
directly or through a linker. 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. 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', 4' 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, phosphorodithioate, 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.
[0086] 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.
[0087] 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.
[0088] In some embodiments, one endosomolytic ligand and two or
more targeting ligands are present in the oligonucleotide.
[0089] In some embodiments, one targeting ligand and two or more
targeting ligands are present in the oligonucleotide.
[0090] In some embodiments, two or more endosomolytic ligands and
two or more targeting ligands are present.
[0091] When the oligonucleotide is a double-stranded
oligonucleotide and multiple endosomolytic ligands and/or targeting
ligands are present, such multiple endosomolytic ligands and/or
targeting ligands may all be present in one strand or both strands
of the double stranded oligonucleotide.
[0092] When multiple endosomolytic ligands and/or targeting ligands
are present, they may all be the same or different.
[0093] In some embodiments, the ligands, e.g. endosomolytic ligands
and/or targeting ligands, are linked to a monomer which is then
incorporated into the growing oligonucleotide strand. Such monomers
are also referred to as carrier monomers herein.
[0094] In some embodiments, oligonucleotide comprises at least one
carrier monomer of formula (I)
##STR00011## [0095] wherein: [0096] A and B are independently for
each occurrence hydrogen, protecting group, optionally substituted
aliphatic, optionally substituted aryl, optionally substituted
heteroaryl, polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)--, or --P(Z.sup.1)(Z.sup.2)--; [0097] Z.sup.1
and Z.sup.2 are each independently for each occurrence O, S,
N(alkyl) or optionally substituted alkyl; [0098] J.sup.1 and
J.sup.2 are independently O, S, NR.sup.N, optionally substituted
alkyl, OC(O)NH, NHC(O)O, C(O)NH, NHC(O), OC(O), C(O)O, OC(O)O,
NHC(O)NH, NHC(S)NH, OC(S)NH, OP(N(R.sup.P).sub.2)O, or
OP(N(R.sup.P).sub.2);
##STR00012##
[0098] is cyclic group or acyclic group; [0099] L.sup.1 is -L.sup.2
or -L.sup.2-linker-L.sup.3; [0100] L.sup.2 and L.sup.3 are each
independently an endosomolytic agent or a targeting ligand; and
[0101] p is 1-3.
[0102] In some embodiments, p is 1.
[0103] In some embodiments, carrier monomer is cyclic and
##STR00013##
is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,
imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,
[1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,
thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,
tetrahydrofuryl and decalin.
[0104] In some embodiments, carrier monomer is acyclic and
##STR00014##
is selected from serinol backbone or diethanolamine backbone.
[0105] In some embodiment, L.sup.1 is -L.sup.2.
[0106] In some embodiments, the carrier monomer is based on the
pyrroline ring system as shown in formula (II)
##STR00015##
[0107] wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH,
SO, SO.sub.2, or SO.sub.2NH;
[0108] R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16,
R.sup.17, and R.sup.18 are each independently for each occurrence
H, --CH.sub.2OR.sup.a, or OR.sup.b,
[0109] R.sup.a and R.sup.b are each independently for each
occurrence hydrogen, hydroxyl protecting group, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted alkenyl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)-- or --P(Z.sup.1)(O-linker-R.sup.L)--;
[0110] R.sup.30 is independently for each occurrence
-linker-R.sup.L;
[0111] R.sup.L is hydrogen, -L.sup.2 or
-L.sup.2-linker-L.sup.3;
[0112] L.sup.2 and L.sup.3 are each independently an endosomolytic
agent or a targeting ligand; and
[0113] Z.sup.1 and Z.sup.2 are each independently for each
occurrence O, S, N(alkyl) or optionally substituted alkyl;
[0114] provided that R.sup.L is -L.sup.2 or -L.sup.2-linker-L.sup.3
at least once.
[0115] In some embodiments, the oligonucleotide comprises at least
one monomer of formula (II).
[0116] In some embodiment, R.sup.L is -L.sup.2.
[0117] For the pyrroline-based monomers, R.sup.11 is
--CH.sub.2OR.sup.a and R1.sup.3 is OR.sup.b; or R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.9 is OR.sup.b; or R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.17 is OR.sup.b; or R.sup.13 is
--CH.sub.2OR.sup.a and R.sup.11 is OR.sup.b; or R.sup.13 is
--CH.sub.2OR.sup.a and R.sup.15 is OR.sup.b; or R.sup.13 is
--CH.sub.2OR.sup.a and R.sup.17 is OR.sup.b. In certain
embodiments, CH.sub.2OR.sup.a and OR.sup.b may be geminally
substituted. For the 4-hydroxyproline-based monomers, R.sup.11 is
--CH.sub.2OR.sup.a and R.sup.17 is OR.sup.b. The pyrroline- and
4-hydroxyproline-based compounds may therefore contain linkages
(e.g., carbon-carbon bonds) wherein bond rotation is restricted
about that particular linkage, e.g. restriction resulting from the
presence of a ring. Thus, CH.sub.2OR.sup.a and OR.sup.b may be cis
or trans with respect to one another in any of the pairings
delineated above Accordingly, all cis/trans isomers are expressly
included. The compounds may also contain one or more asymmetric
centers and thus occur as racemates and racemic mixtures, single
enantiomers, individual diastereomers and diastereomeric mixtures.
All such isomeric forms of the compounds are expressly included
(e.g., the centers bearing CH.sub.2OR.sup.a and OR.sup.b can both
have the R configuration; or both have the S configuration; or one
center can have the R configuration and the other center can have
the S configuration and vice versa).
[0118] In some embodiments, R.sup.11 is CH.sub.2OR.sup.a and
R.sup.15 is OR.sup.b.
[0119] In some embodiments, R.sup.b is a solid support.
[0120] In one embodiment, carrier of formula (II) is a
phosphoramidite, i.e., one of R.sup.a or R.sup.b is
--P(O-alkyl)N(alkyl).sub.2, e.g.,
--P(OCH.sub.2CH.sub.2CN)N(i-propyl).sub.2. In one embodiment,
R.sup.b is --P(O-alkyl)N(alkyl).sub.2.
[0121] In some embodiments, the carrier monomer is based on the
ribose ring system as shown in formula (III).
##STR00016##
[0122] wherein:
[0123] X is O, S, NR.sup.N or CR.sup.P.sub.2;
[0124] B is independently for each occurrence hydrogen, optionally
substituted natural or non-natural nucleobase, optionally
substituted natural nucleobase conjugated with -linker-R.sup.L or
optionally substituted non-natural nucleobase conjugated with
-linker-R.sup.L;
[0125] R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are each
independently for each occurrence H, OR.sup.6, F, N(R.sup.N).sub.2,
or -J-linker-R.sup.L;
[0126] J is absent, O, S, NR.sup.N, OC(O)NH, NHC(O)O, C(O)NH,
NHC(O), NHSO, NHSO.sub.2, NHSO.sub.2NH, OC(O), C(O)O, OC(O)O,
NHC(O)NH, NHC(S)NH, OC(S)NH, OP(N(R.sup.P).sub.2)O, or
OP(N(R.sup.P).sub.2);
[0127] R.sup.6 is independently for each occurrence hydrogen,
hydroxyl protecting group, optionally substituted alkyl, optionally
substituted aryl, optionally substituted cycloalkyl, optionally
substituted aralkyl, optionally substituted alkenyl, optionally
substituted heteroaryl, polyethyleneglycol (PEG), a phosphate, a
diphosphate, a triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)-- or --P(Z.sup.1)(O-linker-R.sup.L)--;
[0128] R.sup.N is independently for each occurrence H, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted heteroaryl or an amino protecting group;
[0129] R.sup.P is independently for each occurrence H, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted cycloalkyl or optionally substituted heteroaryl;
[0130] R.sup.L is hydrogen, -L.sup.2 or
-L.sup.2-linker-L.sup.3;
[0131] L.sup.2 and L.sup.3 are each independently an endosomolytic
agent or a targeting ligand; and
[0132] Z.sup.1 and Z.sup.2 are each independently for each
occurrence O, S N(alkyl) or optionally substituted alkyl;
[0133] provided that R.sup.L is present at least once and further
provided that R.sup.L is -L.sup.2 or -L.sup.2-linker-L.sup.3 at
least once.
[0134] In some embodiments, the oligonucleotide comprises at least
one monomer of formula (III).
[0135] In some embodiment, R.sup.L is -L.sup.2.
[0136] In some embodiments, the carrier monomer is based on an
acyclic group and is termed an "acyclic carrier". Preferred acyclic
carriers can have the structure shown in formula (IV) or formula
(V) below.
[0137] In some embodiments, the acyclic carrier has the structure
shown in formula (IV).
##STR00017##
[0138] wherein:
[0139] W is absent, O, S and N(R.sup.N), where R.sup.N is
independently for each occurrence H, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aryl, optionally substituted cycloalkyl,
optionally substituted aralkyl, optionally substituted heteroaryl
or an amino protecting group;
[0140] E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO,
SO.sub.2, or SO.sub.2NH;
[0141] R.sup.a and R.sup.b are each independently for each
occurrence hydrogen, hydroxyl protecting group, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted alkenyl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)-- or --P(Z.sup.1)(O-linker-R.sup.L)--;
[0142] R.sup.30 is independently for each occurrence
-linker-R.sup.L;
[0143] R.sup.L is hydrogen, -L.sup.2 or
-L.sup.2-linker-L.sup.3;
[0144] L.sup.2 and L.sup.3 are each independently an endosomolytic
agent or a targeting ligand;
[0145] Z.sup.1 and Z.sup.2 are each independently for each
occurrence O, S, N(alkyl) or optionally substituted alkyl; and
[0146] r, s and t are each independently for each occurrence 0, 1,
2 or 3;
[0147] provided that R.sup.L is -L.sup.2 or -L.sup.2-linker-L.sup.3
at least once.
[0148] When r and s are different, then the tertiary carbon can be
either the R or S configuration. In preferred embodiments, x and y
are one and z is zero (e.g. carrier is based on serinol). The
acyclic carriers can optionally be substituted, e.g. with hydroxy,
alkoxy, perhaloalkyl.
[0149] In one embodiment, the oligonucleotide comprises at least
one monomer of formula (IV).
[0150] In some embodiment, R.sup.L is -L.sup.2.
[0151] In some embodiments, the acyclic carrier has the structure
shown in formula (V)
##STR00018##
[0152] wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH,
SO, SO.sub.2, or SO.sub.2NH;
[0153] R.sup.a and R.sup.b are each independently for each
occurrence hydrogen, hydroxyl protecting group, optionally
substituted alkyl, optionally substituted aryl, optionally
substituted cycloalkyl, optionally substituted aralkyl, optionally
substituted alkenyl, optionally substituted heteroaryl,
polyethyleneglycol (PEG), a phosphate, a diphosphate, a
triphosphate, a phosphonate, a phosphonothioate, a
phosphonodithioate, a phosphorothioate, a phosphorothiolate, a
phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a
phosphotriester, an activated phosphate group, an activated
phosphite group, a phosphoramidite, a solid support,
--P(Z.sup.1)(Z.sup.2)-- or --P(Z.sup.1)(O-linker-R.sup.L)--;
[0154] R.sup.30 is independently for each occurrence
-linker-R.sup.L;
[0155] R.sup.L is hydrogen, -L.sup.2 or
-L.sup.2-linker-L.sup.3;
[0156] L.sup.2 and L.sup.3 are each independently an endosomolytic
agent or a targeting ligand;
[0157] Z.sup.1 and Z.sup.2 are each independently for each
occurrence O, S, N(alkyl) or optionally substituted alkyl; and
[0158] r and s are each independently for each occurrence 0, 1, 2
or 3;
[0159] provided that R.sup.L is -L.sup.2 or -L.sup.2-linker-L.sup.3
at least once.
[0160] In some embodiments, the oligonucleotide comprises at least
one monomer of formula (V).
[0161] In some embodiment, R.sup.L is -L.sup.2 In some embodiments,
the oligonucleotide comprises at least two different monomers
selected from formula (I)-formula (V).
Interfering RNA
[0162] Double-stranded interfering RNA (dsiRNA) directs the
sequence-specific silencing of mRNA through a process known as RNA
interference (RNAi). The process occurs in a wide variety of
organisms, including mammals and other vertebrates.
[0163] It has been demonstrated that 21-23 nt fragments of dsiRNA
are sequence-specific mediators of RNA silencing, e.g., by causing
RNA degradation. While not wishing to be bound by theory, it may be
that a molecular signal, which may be merely the specific length of
the fragments, present in these 21-23 nt fragments recruits
cellular factors that mediate RNAi. Described herein are methods
for preparing and administering these 21-23 nt fragments, and other
iRNAs agents, and their use for specifically inactivating gene
function. The use of iRNA agents (or recombinantly produced or
chemically synthesized oligonucleotides of the same or similar
nature) enables the targeting of specific mRNAs for silencing in
mammalian cells. In addition, longer dsiRNA agent fragments can
also be used, e.g., as described below.
[0164] Although, in mammalian cells, long dsiRNAs can induce the
interferon response which is frequently deleterious, siRNAs do not
trigger the interferon response, at least not to an extent that is
deleterious to the cell and host. In particular, the length of the
iRNA agent strands in an siRNA agent can be less than 31, 30, 28,
25, or 23 nt, e.g., sufficiently short to avoid inducing a
deleterious interferon response. Thus, the administration of a
composition of siRNA agent (e.g., formulated as described herein)
to a mammalian cell can be used to silence expression of a target
gene while circumventing the interferon response. Further, use of a
discrete species of iRNA agent can be used to selectively target
one allele of a target gene, e.g., in a subject heterozygous for
the allele.
[0165] Moreover, in one embodiment, a mammalian cell is treated
with an iRNA agent that disrupts a component of the interferon
response, e.g., dsiRNA-activated protein kinase PKR. Such a cell
can be treated with a second iRNA agent that includes a sequence
complementary to a target RNA and that has a length that might
otherwise trigger the interferon response.
[0166] In a typical embodiment, the subject is a mammal such as a
cow, horse, mouse, rat, dog, pig, goat, or a primate. The subject
can be a dairy mammal (e.g., a cow, or goat) or other farmed animal
(e.g., a chicken, turkey, sheep, pig, fish, shrimp). In certain
embodiments, the subject is a human, e.g., a normal individual or
an individual that has, is diagnosed with, or is predicted to have
a disease or disorder.
[0167] Further, because iRNA agent mediated silencing persists for
several days after administering the iRNA agent composition, in
many instances, it is possible to administer the composition with a
frequency of less than once per day, or, for some instances, only
once for the entire therapeutic regimen. For example, treatment of
some cancer cells may be mediated by a single bolus administration,
whereas a chronic viral infection may require regular
administration, e.g., once per week or once per month.
[0168] A number of exemplary routes of delivery are described that
can be used to administer an iRNA agent to a subject. In addition,
the iRNA agent can be formulated according to any exemplary method
described herein.
[0169] The present invention relates to modular compositions and
methods for delivery of a nucleic acid (e.g., an iRNA or siRNA).
One aspect of the invention relates to a modular composition
comprising an endosomolytic component, a targeting ligand, and a
nucleic acid (e.g., an iRNA agent or siRNA agent).
[0170] For ease of exposition, certain formulations, compositions
and methods in this application are discussed largely with regard
to unmodified nucleic acids or iRNAs. It may be understood,
however, that these formulations, compositions and methods can be
practiced with modified nucleic acids, and that such practices are
within the scope of the invention.
Endosomolytic Components
[0171] For macromolecular drugs and hydrophilic drug molecules,
which cannot easily cross bilayer membranes, entrapment in
endosomal/lysosomal compartments of the cell is thought to be the
biggest hurdle for effective delivery to their site of action. In
recent years, a number of approaches and strategies have been
devised to address this problem. For liposomal formulations, the
use of fusogenic lipids in the formulation has been the most common
approach (Singh, R. S., Goncalves, C. et al. (2004). On the Gene
Delivery Efficacies of pH-Sensitive Cationic Lipids via Endosomal
Protonation. A Chemical Biology Investigation. Chem. Biol. 11,
713-723.). Other components, which exhibit pH-sensitive
endosomolytic activity through protonation and/or pH-induced
conformational changes, include charged polymers and peptides.
Examples may be found in Hoffman, A. S., Stayton, P. S. et al.
(2002). Design of "smart" polymers that can direct intracellular
drug delivery. Polymers Adv. Technol. 13, 992-999; Kakudo, Chaki,
T., S. et al. (2004). Transferrin-Modified Liposomes Equipped with
a pH-Sensitive Fusogenic Peptide: An Artificial Viral-like Delivery
System. Biochemistry 436, 5618-5628; Yessine, M. A. and Leroux, J.
C. (2004). Membrane-destabilizing polyanions: interaction with
lipid bilayers and endosomal escape of biomacromolecules. Adv. Drug
Deliv. Rev. 56, 999-1021; Oliveira, S., van Rooy, I. et al. (2007).
Fusogenic peptides enhance endosomal escape improving siRNA-induced
silencing of oncogenes. Int. J Pharm. 331, 211-4. They have
generally been used in the context of drug delivery systems, such
as liposomes or lipoplexes. For folate receptor-mediated delivery
using liposomal formulations, for instance, a pH-sensitive
fusogenic peptide has been incorporated into the liposomes and
shown to enhance the activity through improving the unloading of
drug during the uptake process (Turk, M. J., Reddy, J. A. et al.
(2002). Characterization of a novel pH-sensitive peptide that
enhances drug release from folate-targeted liposomes at endosomal
pHs. Biochim. Biophys. Acta 1559, 56-68).
[0172] In certain embodiments, the endosomolytic components of the
present invention may be polyanionic peptides or peptidomimetics
which show pH-dependent membrane activity and/or fusogenicity. A
peptidomimetic may be a small protein-like chain designed to mimic
a peptide. A peptidomimetic may arise from modification of an
existing peptide in order to alter the molecule's properties, or
the synthesis of a peptide-like molecule using unnatural amino
acids or their analogs. In certain embodiments, they have improved
stability and/or biological activity when compared to a peptide. In
certain embodiments, the endosomolytic component assumes its active
conformation at endosomal pH (e.g., pH 5-6). The "active"
conformation is that conformation in which the endosomolytic
component promotes lysis of the endosome and/or transport of the
modular composition of the invention, or its any of its components
(e.g., a nucleic acid), from the endosome to the cytoplasm of the
cell.
[0173] Libraries of compounds may be screened for their
differential membrane activity at endosomal pH versus neutral pH
using a hemolysis assay. Promising candidates isolated by this
method may be used as components of the modular compositions of the
invention. A method for identifying an endosomolytic component for
use in the compositions and methods of the present invention may
comprise: providing a library of compounds; contacting blood cells
with the members of the library, wherein the pH of the medium in
which the contact occurs is controlled; determining whether the
compounds induce differential lysis of blood cells at a low pH
(e.g., about pH 5-6) versus neutral pH (e.g., about pH 7-8).
[0174] Exemplary endosomolytic components include the GALA peptide
(Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA
peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586),
and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002,
1559: 56-68). In certain embodiments, the endosomolytic component
may contain a chemical group (e.g., an amino acid) which will
undergo a change in charge or protonation in response to a change
in pH. The endosomolytic component may be linear or branched.
Exemplary primary sequences of endosomolytic components include
H.sub.2N-(AALEALAEALEALAEALEALAEAAAAGGC)-CO.sub.2H (SEQ ID NO:1);
H.sub.2N-(AALAEALAEALAEALAEALAEALAAAAGGC)-CO.sub.2H (SEQ ID NO:2);
and H.sub.2N-(ALEALAEALEALAEA)-CONH.sub.2 (SEQ ID NO:3).
[0175] Further examples of endosomolytic components include those
in Table 1:
TABLE-US-00001 TABLE 1 Exemplary Endosomolytic Components Name
Sequence (N to C) SEQ ID NO. GALA AALEALAEALEALAEALEALAEAAAAGGC 1
EALA AALAEALAEALAEALAEALAEALAAAAGGC 2 ALEALAEALEALAEA 3 INF-7
GLFEMEGFIENGWEGMIWDYG 4 Inf HA-2 GLFGAIAGFIENGWEGMIDGWYG 5 diINF-7
GLF EAI EGFI ENGW EGMI DGWYGC 6 GLF EAI EGFI ENGW EGMI DGWYGC
diINF3 GLF EAI EGFI ENGW EGMI DGGC 7 GLF EAI EGFI ENGW EGMI DGGC
GLF GLFGALAEALAEALAEHLAEALAEALEALAAGGSC 8 GALA-INF3
GLFEMEGFIENGWEGLAEALAEALEALAAGGSC 9 INF-5 GLF EAI EGFI ENGW EGnI DG
K 10 GLF EAI EGFI ENGW EGnI DG JTS-1 GLFEALLELLESLWELLLEA 11 ppTG1
GLFKALLKLLKSLWKLLLKA 12 ppTG20 GLFRALLRLLRSLWRLLLRA 13 KALA
WEAKLAKALAKALAKHLAKALAKALKACEA 14 HA GLFFEAIAEFIEGGWEGLIEGC 15
Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 16 Histidine CHK.sub.6HC 17
rich n, norleucine
[0176] In some embodiments, endosomolytic ligands can include
imidazoles, poly or oligoimidazoles, linear or branched
polyethyleneimines (PEIs), linear and branched 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.
[0177] Preferred synthetic polymers with endosomolytic activity,
are described in the following United States Patent Application
Publications Nos. 2009/0048410; 20090023890; 2008/0287630;
20080287628; 2008/0281044; 2008/0281041; 2008/0269450;
2007/0105804; 20070036865; and 2004/0198687, contents of which are
hereby incorporated by reference in their entirety.
[0178] In some embodiments, the membrane active functionality of
the endosomolytic agent is masked when said endosomolytic agent is
conjugated with the oligonucleotide. When the oligonucleotide
reaches the endosome, the membrane active functionality is unmasked
and the agent becomes active. The unmasking may be carried out more
readily under the conditions found in the endosome than outside the
endosome. For example, the membrane active functionality can be
masked with a molecule through a cleavable linker that under goes
cleavage in the endosome. Without wishing to be bound by theory, it
is envisioned that upon entry into the endosome, such a linkage
will be cleaved and the masking agent released from the
endosomolytic agent.
[0179] In some embodiments, the masking agent has a cleavable
linker that upon cleavage release a functional group that can
cleave the linkage between the masking agent and the active
functional group of the endosomolytic agent. One example is a
masking agent linked to the endosomolytic agent through a amide
type linkage, and having a S--S bond. Upon entry into the endosome,
the S--S bond can be cleaved releasing free thiols that can then
cleave the amide linkage between the masking agent and the
endosomolytic agents either inter or intra molecularly. United
States Patent Application Publication No. 2008/0281041 describes
some masked endosomolytic polymers that are amenable to the present
invention.
[0180] Lipids having membrane activity are also amenable to the
present invention as endosomolytic agents. Such lipids are also
described as fusogenic lipids. These fusogenic lipids are thought
to fuse with and consequently destabilize a membrane. Fusogenic
lipids usually have small head groups and unsaturated acyl chains.
Exemplary fusogenic lipids include
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 (XTC).
[0181] The histidine-rich peptide H5WYG (SEQ ID NO:18) is a
derivative of the N-terminal sequence of the HA-2 subunit of the
influenza virus hemagglutinin in which 5 of the amino acids have
been replaced with histidine residues. H5WYG is able to selectively
destabilize membranes at a slightly acidic pH as the histidine
residues are protonated.
[0182] In some embodiments, the endosomolytic ligand is a
cell-permeation agent, preferably a helical cell-permeation agent.
Preferably, the agent is amphipathic. The helical agent is
preferably an alpha-helical agent, which preferably has a
lipophilic and a lipophobic phase. A cell-permeation agent can be,
for example, a cell permeation peptide, cationic peptide,
amphipathic peptide or hydrophobic peptide, e.g. consisting
primarily of Tyr, Trp and Phe, dendrimer peptide, constrained
peptide or crosslinked peptide. In some embodiments, the cell
permeation peptide can include a hydrophobic membrane translocation
sequence (MTS). An exemplary hydrophobic MTS-containing peptide is
RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID
NO:19). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP
(SEQ ID NO:20)) containing a hydrophobic MTS can also be a
targeting ligand. The cell permeation peptide can be a "delivery"
peptide, which can carry large polar molecules including peptides,
oligonucleotides, and protein across cell membranes. Some exemplary
cell-permeation peptides are shown in Table 2.
TABLE-US-00002 TABLE 2 Exemplary Cell Permeation Peptides. Cell
Permeation Peptide Amino acid Sequence Reference Penetratin
RQIKIWFQNRRMKWKK (SEQ ID NO: 21) Derossi et al., J. Biol. Chem.
269:10444, 1994 Tat fragment GRKKRRQRRRPPQC (SEQ ID NO: 22) Vives
et al., J. Biol. (48-60) Chem., 272 :16010, 1997 Signal
Sequence-based GALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin et al., Biochem.
peptide (SEQ ID NO: 23) Biophys. Res. Commun., 243 :601, 1998 PVEC
LLIILRRRIRKQAHAHSK (SEQ ID NO: 24) Elmquist et al., Exp. Cell Res.,
269 :237, 2001 Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga et al.,
FASEB J., (SEQ ID NO: 25) 12:67, 1998 Amphiphilic model peptide
KLALKLALKALKAALKLA (SEQ ID Oehlke et al., Mol. Ther., NO: 26)
2:339, 2000 Arg.sub.9 RRRRRRRRR (SEQ ID NO: 27) Mitchell et al., J.
Pept. Res., 56:318, 2000 Bacterial cell wall KFFKFFKFFK (SEQ ID NO:
28) permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN LVPRTES (SEQ ID
NO: 29) Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQGGP R (SEQ ID NO:
30) .alpha.-defensin ACYCRIPACIAGERRYGTCIYQGRLWAFC C (SEQ ID NO:
31) b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTCYR GKAKCCK (SEQ ID NO:
32) Bactenecin RKCRIVVIRVCR (SEQ ID NO: 33) PR-3
RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPP RFPPRFPGKR-NH2 (SEQ ID NO: 34)
Indolicidin ILPWKWPWWPWRR-NH2 (SEQ ID NO: 35)
[0183] Cell-permeation peptides can be linear or cyclic, and
include D-amino acids, non-peptide or pseudo-peptide linkages,
peptidyl mimics. In addition the peptide and peptide mimics can be
modified, e.g. glycosylated or methylated. Synthetic mimics of
targeting peptides are also included.
[0184] In certain embodiments, more than one endosomolytic
component may be incorporated in the modular composition of the
invention. In some embodiments, this will entail incorporating more
than one of the same endosomolytic component into the modular
composition. In other embodiments, this will entail incorporating
two or more different endosomolytic components into the modular
composition.
[0185] These endosomolytic components may mediate endosomal escape
by, for example, changing conformation at endosomal pH. In certain
embodiments, the endosomolytic components may exist in a random
coil conformation at neutral pH and rearrange to an amphipathic
helix at endosomal pH. As a consequence of this conformational
transition, these peptides may insert into the lipid membrane of
the endosome, causing leakage of the endosomal contents into the
cytoplasm. Because the conformational transition is pH-dependent,
the endosomolytic components can display little or no fusogenic
activity while circulating in the blood (pH .about.7.4). Fusogenic
activity is defined as that activity which results in disruption of
a lipid membrane by the endosomolytic component. One example of
fusogenic activity is the disruption of the endosomal membrane by
the endosomolytic component, leading to endosomal lysis or leakage
and transport of one or more components of the modular composition
of the invention (e.g., the nucleic acid) from the endosome into
the cytoplasm.
[0186] In addition to the hemolysis assay described herein,
suitable endosomolytic components can be tested and identified by a
skilled artisan using other methods. For example, the ability of a
compound to respond to, e.g., change charge depending on, the pH
environment can be tested by routine methods, e.g., in a cellular
assay. In certain embodiments, a test compound is combined with or
contacted with a cell, and the cell is allowed to internalize the
test compound, e.g., by endocytosis. An endosome preparation can
then be made from the contacted cells and the endosome preparation
compared to an endosome preparation from control cells. A change,
e.g., a decrease, in the endosome fraction from the contacted cell
vs. the control cell indicates that the test compound can function
as a fusogenic agent. Alternatively, the contacted cell and control
cell can be evaluated, e.g., by microscopy, e.g., by light or
electron microscopy, to determine a difference in the endosome
population in the cells. The test compound and/or the endosomes can
labeled, e.g., to quantify endosomal leakage.
[0187] In another type of assay, a modular composition described
herein is constructed using one or more test or putative fusogenic
agents. The modular composition can be constructed using a labeled
nucleic acid. The ability of the endosomolytic component to promote
endosomal escape, once the modular composition is taken up by the
cell, can be evaluated, e.g., by preparation of an endosome
preparation, or by microscopy techniques, which enable
visualization of the labeled nucleic acid in the cytoplasm of the
cell. In certain other embodiments, the inhibition of gene
expression, or any other physiological parameter, may be used as a
surrogate marker for endosomal escape.
[0188] In other embodiments, circular dichroism spectroscopy can be
used to identify compounds that exhibit a pH-dependent structural
transition.
[0189] A two-step assay can also be performed, wherein a first
assay evaluates the ability of a test compound alone to respond to
changes in pH, and a second assay evaluates the ability of a
modular composition that includes the test compound to respond to
changes in pH.
Targeting Ligands
[0190] The modular compositions of the present invention comprise a
targeting ligand. In some embodiments, this targeting ligand may
direct the modular composition to a particular cell. For example,
the targeting ligand may specifically or non-specifically bind with
a molecule on the surface of a target cell. The targeting moiety
can be a molecule with a specific affinity for a target cell.
Targeting moieties can include antibodies directed against a
protein found on the surface of a target cell, or the ligand or a
receptor-binding portion of a ligand for a molecule found on the
surface of a target cell. For example, the targeting moiety can
recognize a cancer-specific antigen (e.g., CA15-3, CA19-9, CEA, or
HER2/neu) or a viral antigen, thus delivering the iRNA to a cancer
cell or a virus-infected cell. Exemplary targeting moieties include
antibodies (such as IgM, IgG, IgA, IgD, and the like, or a
functional portions thereof), ligands for cell surface receptors
(e.g., ectodomains thereof).
[0191] Table 3 provides examples of a number of antigens which can
be used to target selected cells.
TABLE-US-00003 TABLE 3 Exemplary antigens for targeting specific
cells ANTIGEN Exemplary tumor tissue CEA (carcinoembryonic antigen)
colon, breast, lung PSA (prostate specific antigen) prostate cancer
CA-125 ovarian cancer CA 15-3 breast cancer CA 19-9 breast cancer
HER2/neu breast cancer .alpha.-feto protein testicular cancer,
hepatic cancer .beta.-HCG (human chorionic testicular cancer,
choriocarcinoma gonadotropin) MUC-1 breast cancer Estrogen receptor
breast cancer, uterine cancer Progesterone receptor breast cancer,
uterine cancer EGFr (epidermal growth factor bladder cancer
receptor)
[0192] Ligand-mediated targeting to specific tissues through
binding to their respective receptors on the cell surface offers an
attractive approach to improve the tissue-specific delivery of
drugs. Specific targeting to disease-relevant cell types and
tissues may help to lower the effective dose, reduce side effects
and consequently maximize the therapeutic index. Carbohydrates and
carbohydrate clusters with multiple carbohydrate motifs represent
an important class of targeting ligands, which allow the targeting
of drugs to a wide variety of tissues and cell types. For examples,
see Hashida, M., Nishikawa, M. et al. (2001) Cell-specific delivery
of genes with glycosylated carriers. Adv. Drug Deliv. Rev. 52,
187-9; Monsigny, M., Roche, A.-C. et al. (1994). Glycoconjugates as
carriers for specific delivery of therapeutic drugs and genes. Adv.
Drug Deliv. Rev. 14, 1-24; Gabius, S., Kayser, K. et al. (1996).
Endogenous lectins and neoglycoconjugates. A sweet approach to
tumor diagnosis and targeted drug delivery. Eur. J. Pharm. and
Biopharm. 42, 250-261; Wadhwa, M. S., and Rice, K. G. (1995)
Receptor mediated glycotargeting. J. Drug Target. 3, 111-127.
[0193] One of the best characterized receptor-ligand pairs is the
asialoglycoprotein receptor (ASGP-R), which is highly expressed on
hepatocytes and which has a high affinity for D-galactose as well
as N-acetyl-D-galactose (GalNAc). Those carbohydrate ligands have
been successfully used to target a wide variety of drugs and even
liposomes or polymeric carrier systems to the liver parenchyma. For
examples, see Wu, G. Y., and Wu, C. H. (1987) Receptor-mediated in
vitro gene transformation by a soluble DNA carrier system. J. Biol.
Chem. 262, 4429-4432; Biessen, E. A. L., Vietsch, H., Rump, E. T.,
Flutter, K., Bijsterbosch, M. K., and Van Berkel, T. J. C. (2000)
Targeted delivery of antisense oligonucleotides to parenchymal
liver cells in vivo. Methods Enzymol. 313, 324-342; Zanta, M.-A.,
Boussif, O., Adib, A., and Behr, J.-P. (1997) In Vitro Gene
Delivery to Hepatocytes with Galactosylated Polyethylenimine.
Bioconjugate Chem. 8, 839-844; Managit, C., Kawakami, S. et al.
(2003). Targeted and sustained drug delivery using PEGylated
galactosylated liposomes. Int. J. Pharm. 266, 77-84; Sato, A.,
Takagi, M. et al. (2007). Small interfering RNA delivery to the
liver by intravenous administration of galactosylated cationic
liposomes in mice. Biomaterials 28; 1434-42.
[0194] The Mannose receptor, with its high affinity to D-mannose
represents another important carbohydrate-based ligand-receptor
pair. The mannose receptor is highly expressed on specific cell
types such as macrophages and possibly dendritic cells Mannose
conjugates as well as mannosylated drug carriers have been
successfully used to target drug molecules to those cells. For
examples, see Biessen, E. A. L., Noorman, F. et al. (1996).
Lysine-based cluster mannosides that inhibit ligand binding to the
human mannose receptor at nanomolar concentration. J. Biol. Chem.
271, 28024-28030; Kinzel, O., Fattori, D. et al. (2003). Synthesis
of a functionalized high affinity mannose receptor ligand and its
application in the construction of peptide-, polyamide- and
PNA-conjugates. J Peptide Sci. 9, 375-385; Barratt, G., Tenu, J. P.
et al. (1986). Preparation and characterization of liposomes
containing mannosylated phospholipids capable of targeting drugs to
macrophages. Biochim. Biophys. Acta 862, 153-64; Diebold, S. S.,
Plank, C. et al. (2002). Mannose Receptor-Mediated Gene Delivery
into Antigen Presenting Dendritic Cells. Somat. Cell Mol. Genetics
27, 65-74.
[0195] 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-glucosamine, multivalent fucose,
glycosylated polyaminoacids and lectins. The term multivalent
indicates that more than one monosaccharide unit is present. Such
monosaccharide subunits may be linked to each other through
glycosidic linkages or linked to a scaffold molecule.
[0196] Lipophilic moieties, such as cholesterol or fatty acids,
when attached to highly hydrophilic molecules such as nucleic acids
can substantially enhance plasma protein binding and consequently
circulation half life. In addition, binding to certain plasma
proteins, such as lipoproteins, has been shown to increase uptake
in specific tissues expressing the corresponding lipoprotein
receptors (e.g., LDL-receptor or the scavenger receptor SR-B1). For
examples, see Bijsterbosch, M. K., Rump, E. T. et al. (2000).
Modulation of plasma protein binding and in vivo liver cell uptake
of phosphorothioate oligodeoxynucleotides by cholesterol
conjugation. Nucleic Acids Res. 28, 2717-25; Wolfrum, C., Shi, S.
et al. (2007). Mechanisms and optimization of in vivo delivery of
lipophilic siRNAs. Nat. Biotechnol. 25, 1149-57. Lipophilic
conjugates can therefore also be considered as a targeted delivery
approach and their intracellular trafficking could potentially be
further improved by the combination with endosomolytic agents.
[0197] Exemplary lipophilic moieties that enhance plasma protein
binding include, but are not limited to, sterols, cholesterol,
fatty acids, cholic acid, lithocholic acid, dialkylglycerides,
diacylglyceride, phospholipids, sphingolipids, 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, phenoxazine, aspirin,
naproxen, ibuprofen, vitamin E and biotin etc.
[0198] Folates represent another class of ligands which has been
widely used for targeted drug delivery via the folate receptor.
This receptor is highly expressed on a wide variety of tumor cells,
as well as other cells types, such as activated macrophages. For
examples, see Matherly, L. H. and Goldman, I. D. (2003). Membrane
transport of folates. Vitamins Hormones 66, 403-456; Sudimack, J.
and Lee, R. J. (2000). Targeted drug delivery via the folate
receptor. Adv. Drug Delivery Rev. 41, 147-162. Similar to
carbohydrate-based ligands, folates have been shown to be capable
of delivering a wide variety of drugs, including nucleic acids and
even liposomal carriers. For examples, see Reddy, J. A., Dean, D.
et al. (1999). Optimization of Folate-Conjugated Liposomal Vectors
for Folate Receptor-Mediated Gene Therapy. J. Pharm. Sci. 88,
1112-1118; Lu, Y. and Low P. S. (2002). Folate-mediated delivery of
macromolecular anticancer therapeutic agents. Adv. Drug Delivery
Rev. 54, 675-693; Zhao, X. B. and Lee, R. J. (2004).
Tumor-selective targeted delivery of genes and antisense
oligodeoxyribonucleotides via the folate receptor; Leamon, C. P.,
Cooper, S. R. et al. (2003). Folate-Liposome-Mediated Antisense
Oligodeoxynucleotide Targeting to Cancer Cells: Evaluation in Vitro
and in Vivo. Bioconj. Chem. 14, 738-747.
[0199] U.S. patent application Ser. No. 12/328,537, filed Dec. 4,
2008 and Ser. No. 12/328,528, filed Dec. 4, 2008 describe a number
of folate and carbohydrate targeting ligands that are amenable to
the modular compositions of the present invention. Contents of
these patent applications are herein incorporated by reference in
their entirety.
[0200] The targeting ligands also include proteins, peptides and
peptidomimetics that can target cell markers, e.g. markers enriched
in proliferating cells. 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 moiety 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 Such peptides include, but are not limited to, RGD
containing peptides and peptidomimetics that can target cancer
cells, in particular cells that exhibit .alpha..sub.v.beta..sub.3
(alpha.v.beta.3) integrin. Targeting peptides can be linear or
cyclic, and include D-amino acids, non-peptide or pseudo-peptide
linkages, peptidyl mimics. In addition the peptide and peptide
mimics can be modified, e.g. glycosylated or methylated. Synthetic
mimics of targeting peptides are also included.
[0201] The targeting ligands can also include other receptor
binding ligands such as hormones and hormone receptor binding
ligands. A targeting ligand can be a thyrotropin, melanotropin,
lectin, glycoprotein, surfactant protein A, mucin, glycosylated
polyaminoacids, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, folate, vitamin B12, biotin, or an aptamer.
Table 4 shows some examples of targeting ligands and their
associated receptors.
TABLE-US-00004 TABLE 4 Liver Targeting Ligands and their associated
receptors Liver Cells Ligand Receptor 1) Parenchymal Cell (PC)
Galactose ASGP-R (Hepatocytes) (Asiologlycoprotein receptor) Gal
NAc ASPG-R (n-acetyl-galactosamine) Gal NAc Receptor Lactose
Asialofetuin ASPG-r 2) Sinusoidal Endothelial Hyaluronan Hyaluronan
receptor Cell (SEC) Procollagen Procollagen receptor Negatively
charged molecules Scavenger receptors Mannose Mannose receptors
N-acetyl Glucosamine Scavenger receptors Immunoglobulins Fc
Receptor LPS CD14 Receptor Insulin Receptor mediated transcytosis
Transferrin Receptor mediated transcytosis Albumins Non-specific
Sugar-Albumin conjugates Mannose-6-phosphate Mannose-6-phosphate
receptor 3) Kupffer Cell (KC) Mannose Mannose receptors Fucose
Fucose receptors Albumins Non-specific Mannose-albumin
conjugates
[0202] When two or more targeting ligands are present, such
targeting ligands may all be the same or different targeting
ligands that target the same cell/tissue/organ.
[0203] In addition to the endosomolytic ligand and the targeting
ligand, the modular composition may comprise one or more other
moieties/ligands that may enhance circulation half life and/or
cellular uptake. These can include naturally occurring substances,
such as a protein (e.g., human serum albumin (HSA), low-density
lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); or
a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,
inulin, cyclodextrin or hyaluronic acid). These moieties may also
be a recombinant or synthetic molecule, such as a synthetic polymer
or synthetic polyamino acids. Examples include polylysine (PLL),
poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid
anhydride copolymer, poly(L-lactide-co-glycolide) copolymer,
divinyl ether-maleic anhydride copolymer,
N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene
glycol (PEG, e.g., PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K,
PEG-40K), methyl-PEG (mPEG), [mPEG].sub.2, polyvinyl alcohol (PVA),
polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide
polymers, or polyphosphazine. Example of polyamines include:
polyethylenimine, polylysine (PLL), spermine, spermidine,
polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,
dendrimer polyamine, arginine, amidine, protamine, cationic lipid,
cationic porphyrin, quaternary salt of a polyamine, or an alpha
helical peptide.
[0204] Oligonucleotides and oligomeric compounds that comprise a
number of phosphorothioate linkages are known in the art to bind to
serum protein, thus short oligonucleotides, e.g. oligonucleotides
of about 5 bases, 10 bases, 15 bases or 20 bases, and
non-nucleosidic oligomeric compounds comprising multiple
phosphorothioate linkages can be used to enhance the circulation
half life of the modular composition of the invention. In addition,
oligonucleotides, e.g. aptamers, that bind serum ligands (e.g.
serum proteins) can also be used to enhance the circulation half
life of the modular composition of the invention. These
oligonucleotides and aptamers may comprise any nucleic acid
modification, e.g. sugar modification, backbone modification or
nucleobase modification, described in this application.
[0205] Ligands that increase the cellular uptake of the modular
composition, may also be present in addition to the endosomolytic
ligand and the targeting ligand. Exemplary ligands that enhance
cellular uptake include vitamins. These are particularly useful for
targeting cells/tissues/organs characterized by unwanted cell
proliferation, e.g., of the malignant or non-malignant type, e.g.,
cancer cells. Exemplary vitamins include vitamin A, E, and K. Other
exemplary vitamins include B vitamin, e.g., folic acid, B12,
riboflavin, biotin, pyridoxal or other vitamins or nutrients taken
up by cancer cells.
[0206] The ligand can be a substance, e.g, a drug, which can
increase the uptake of the modular composition into the cell, for
example, by disrupting the cell's cytoskeleton, e.g., by disrupting
the cell's microtubules, microfilaments, and/or intermediate
filaments. The drug can be, for example, taxon, vincristine,
vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin
A, phalloidin, swinholide A, indanocine, or myoservin.
[0207] The ligand can increase the uptake of the modular
composition into the cell by activating an inflammatory response,
for example. Exemplary ligands that would have such an effect
include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta,
or gamma interferon.
[0208] In some embodiments, such a ligand is a cell-permeation
agent, preferably a helical cell-permeation agent. Preferably, the
agent is amphipathic. The helical agent is preferably an
alpha-helical agent, which preferably has a lipophilic and a
lipophobic phase.
[0209] Other ligands that can be present in the modular composition
of the invention include, dyes and reporter groups for monitoring
distribution, intercalating agents (e.g. acridines), cross-linkers
(e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin,
Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine,
dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating
agents, phosphate, mercapto, amino, polyamino, alkyl, substituted
alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),
synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine,
imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes
of tetraazamacrocycles, dinitrophenyl, HRP and AP.
[0210] In some embodiments, a single ligand may have more than one
property, e.g. ligand has both endosomolytic and targeting
properties.
Enhanced Permeability and Retention
[0211] In certain embodiments, the modular composition of the
invention may be targeted to a site via the enhanced permeability
and retention (EPR) effect. The EPR effect is the property by which
certain sizes of molecules, typically macromolecules, tend to
accumulate in, for example, tumor tissue to a greater extent than
in normal tissue. Without being bound by theory, the general
explanation for this phenomenon is that the blood vessels supplying
a tumor are typically abnormal in their architecture, containing
wide fenestrations which permit the diffusion of macromolecules
from the blood. Moreover, tumors typically lack effective lymphatic
drainage, leading to the accumulation of molecules that diffuse
from the blood. A person of ordinary skill in the art will
recognize that such methods of targeting may also be useful for
other conditions in which abnormal vasculature enable access to a
specific site, with or without compromised lymphatic drainage.
[0212] Representative United States patents that teach the
preparation of oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,149,782; 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; each of which is herein
incorporated by reference.
Linkers
[0213] In certain embodiments, the covalent linkages between any of
the three components of the modular composition of the invention
may be mediated by a linker. This linker may be cleavable or
non-cleavable, depending on the application. In certain
embodiments, a cleavable linker may be used to release the nucleic
acid after transport from the endosome to the cytoplasm. The
intended nature of the conjugation or coupling interaction, or the
desired biological effect, will determine the choice of linker
group.
[0214] Linker groups may be connected to the oligonucleotide
strand(s) at a linker group attachment point (LAP) and may include
any C.sub.1-C.sub.100 carbon-containing moiety, (e.g.,
C.sub.1-C.sub.75, C.sub.1-C.sub.50, C.sub.1-C.sub.20,
C.sub.1-C.sub.10; C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5,
C.sub.6, C.sub.7, C.sub.8, C.sub.9, or C.sub.10), in some
embodiments having at least one oxygen atom, at least one
phosphorous atom, and/or at least one nitrogen atom. In some
embodiments, the phosphorous atom forms part of a terminal
phosphate, or phosphorothioate, group on the linker group, which
may serve as a connection point for the nucleic acid strand. In
certain embodiments, the nitrogen atom forms part of a terminal
ether, ester, amino or amido (NHC(O)--) group on the linker group,
which may serve as a connection point for the endosomolytic
component or targeting ligand. Preferred linker groups (underlined)
include LAP-X--(CH.sub.2).sub.nNH--;
LAP-X--C(O)(CH.sub.2).sub.nNH--; LAP-X--NR''''(CH.sub.2).sub.nNH--,
LAP-X--C(O)--(CH.sub.2).sub.n--C(O)--;
LAP-X--C(O)--(CH.sub.2).sub.n--C(O)O--; LAP-X--C(O)--O--;
LAP-X--C(O)--(CH.sub.2).sub.n--NH--C(O)--;
LAP-X--C(O)--(CH.sub.2).sub.n--; LAP-X--C(O)--NH--; LAP-X--C(O)--;
LAP-X--(CH.sub.2).sub.n--C(O)--; LAP-X--(CH.sub.2).sub.n--C(O)O--;
LAP-X--(CH.sub.2).sub.n--; or LAP-X--(CH.sub.2).sub.n--NH--C(O)--;
in which --X is (--O--(R''''O)P(O)--O).sub.m,
(--O--(R''''O)P(S)--O--).sub.m, (--O--(R''''S)P(O)--O).sub.m,
(--O--(R''''S)P(S)--O).sub.m, (--O--(R''''O)P(O)--S).sub.m,
(--S--(R''''O)P(O)--O).sub.m, or nothing, n is 1-20 (e.g., 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), m
is 1 to 3, and R'''' is H or C.sub.1-C.sub.6 alkyl. Preferably, n
is 5, 6, or 11. In other embodiments, the nitrogen may form part of
a terminal oxyamino group, e.g., --ONH.sub.2, or hydrazino group,
--NHNH.sub.2. The linker group may optionally be substituted, e.g.,
with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with
one or more additional heteroatoms, e.g., N, O, or S. Certain
linker groups may include, e.g., LAP-X--(CH.sub.2).sub.nNH--;
LAP-X--C(O)(CH.sub.2).sub.nNH--; LAP-X--NR''''(CH.sub.2).sub.nNH--;
LAP-X--(CH.sub.2).sub.nONH--; LAP-X--C(O)(CH.sub.2).sub.nONH--;
LAP-X--NR''''(CH.sub.2).sub.nONH--;
LAP-X--(CH.sub.2).sub.nNHNH.sub.2--,
LAP-X--C(O)(CH.sub.2).sub.nNHNH.sub.2--;
LAP-X--NR''''(CH.sub.2).sub.nNHNH.sub.2--;
LAP-X--C(O)--(CH.sub.2).sub.n--C(O)--;
LAP-X--C(O)--(CH.sub.2).sub.n--C(O)O--; LAP-X--C(O)--O--;
LAP-X--C(O)--(CH.sub.2).sub.n--NH--C(O)--;
LAP-X--C(O)--(CH.sub.2).sub.n--; LAP-X--C(O)--NH--; LAP-X--C(O)--;
LAP-X--(CH.sub.2).sub.n--C(O)--; LAP-X--(CH.sub.2).sub.n--C(O)O--;
LAP-X--(CH.sub.2).sub.n--; or LAP-X--(CH.sub.2).sub.n--NH--C(O)--.
In some embodiments, amino terminated linker groups (e.g.,
NH.sub.2, ONH.sub.2, NH.sub.2NH.sub.2) can form an imino bond
(i.e., C.dbd.N) with the ligand. In some embodiments, amino
terminated linker groups (e.g., NH.sub.2, ONH.sub.2,
NH.sub.2NH.sub.2) can be acylated, e.g., with C(O)CF.sub.3.
[0215] In some embodiments, the linker group can terminate with a
mercapto group (i.e., SH) or an olefin (e.g., CH.dbd.CH.sub.2). For
example, the linker group can be LAP-X--(CH.sub.2).sub.n--SH,
LAP-X--C(O)(CH.sub.2).sub.nSH,
LAP-X--(CH.sub.2).sub.n--(CH.dbd.CH.sub.2), or
LAP-X--C(O)(CH.sub.2).sub.n(CH.dbd.CH.sub.2), in which X and n can
be as described for the linker groups above. In certain
embodiments, the olefin can be a Diels-Alder diene or dienophile.
The linker group may optionally be substituted, e.g., with hydroxy,
alkoxy, perhaloalkyl, and/or optionally inserted with one or more
additional heteroatoms, e.g., N, O, or S. The double bond can be
cis or trans or E or Z.
[0216] In other embodiments the linker group may include an
electrophilic moiety, preferably at the terminal position of the
linker group. Certain electrophilic moieties include, e.g., an
aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate,
or an activated carboxylic acid ester, e.g., an NHS ester, or a
pentafluorophenyl ester. Other linker groups (underlined) include
LAP-X--(CH.sub.2).sub.nCHO; LAP-X--C(O)(CH.sub.2).sub.nCHO; or
LAP-X--NR''''(CH.sub.2).sub.nCHO, in which n is 1-6 and R'''' is
C.sub.1-C.sub.6 alkyl; or LAP-X--(CH.sub.2).sub.nC(O)ONHS:
LAP-X--C(O)(CH.sub.2).sub.nC(O)ONHS; or
LAP-X--NR''''(CH.sub.2).sub.nC(O)ONHS, in which n is 1-6 and R''''
is C.sub.1-C.sub.6 alkyl;
LAP-X--(CH.sub.2).sub.nC(O)OC.sub.6F.sub.5;
LAP-X--C(O)(CH.sub.2).sub.nC(O)OC.sub.6F.sub.5; or
LAP-X--NR''''(CH.sub.2).sub.nC(O)OC.sub.6F.sub.5, in which n is
1-11 and R'''' is C.sub.1-C.sub.6 alkyl; or
--(CH.sub.2).sub.nCH.sub.2LG;
LAP-X--C(O)(CH.sub.2).sub.nCH.sub.2LG; or
LAP-X--NR''''(CH.sub.2).sub.nCH.sub.2LG, in which X, R'''' and n
can be as described for the linker groups above (LG can be a
leaving group, e.g., halide, mesylate, tosylate, nosylate,
brosylate). In some embodiments, coupling the -linker group to the
endosomolytic component or targeting ligand can be carried out by
coupling a nucleophilic group of the endosomolytic component or
targeting ligand with an electrophilic group on the linker
group.
[0217] In other embodiments, other protected amino groups can be at
the terminal position of the linker group, e.g., alloc, monomethoxy
trityl (MMT), trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the
aryl portion can be ortho-nitrophenyl or ortho,
para-dinitrophenyl).
[0218] In any of the above linker groups, in addition, one, more
than one, or all, of the n --CH.sub.2-- groups may be replaced by
one or a combination of, e.g., X, as defined above,
--Y--(CH.sub.2).sub.m--, --Y--(C(CH.sub.3)H).sub.m--,
--Y--C((CH.sub.2).sub.pCH.sub.3)H).sub.m--,
--Y--(CH.sub.2--C(CH.sub.3)H).sub.m--,
--Y--(CH.sub.2--C((CH.sub.2).sub.pCH.sub.3)H).sub.m--,
--CH.dbd.CH--, or --C.ident.C--, wherein Y is O, S, Se, S--S, S(O),
S(O).sub.2, m is 1-4 and p is 0-4.
[0219] Where more than one endosomolytic component or targeting
ligand is present on the same modular composition, the more than
one endosomolytic component or targeting ligand may be linked to
the oligonucleotide strand or an endosomolytic component or
targeting ligand in a linear fashion, or by a branched linker
group.
[0220] In some embodiments, the linker group is a branched linker
group, and more in ceratin cases a symmetric branched linker group.
The branch point may be an at least trivalent, but may be a
tetravalent, pentavalent, or hexavalent atom, or a group presenting
such multiple valencies. In some embodiments, the branch point is a
glycerol, or glycerol triphosphate, group.
[0221] 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 other
embodiments, the branchpoint is a glycerol derivative.
[0222] In one embodiment, the linker is
--[(P-Q-R).sub.q--X--(P'-Q'-R').sub.q'].sub.q''-T-, wherein:
[0223] P, R, T, P' and R' 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(R.sup.a)C(O),
--C(O)--CH(R.sup.a)--NH--, C(O)-(optionally substituted
alkyl)-NH--, CH.dbd.N--O,
##STR00019##
cyclyl, heterocycyclyl, aryl or heteroaryl;
[0224] Q and Q' are each independently for each occurrence absent,
--(CH.sub.2).sub.n--, --C(R.sup.100)(R.sup.200)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R.sup.100)(R.sup.200)--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--, aryl, heteroaryl,
cyclyl, or heterocyclyl;
[0225] X is absent or a cleavable linking group;
[0226] R.sup.a is H or an amino acid side chain;
[0227] R.sup.100 and R.sup.200 are each independently for each
occurrence H, CH.sub.3, OH, SH or N(R.sup.X).sub.2;
[0228] R.sup.X is independently for each occurrence H, methyl,
ethyl, propyl, isopropyl, butyl or benzyl;
[0229] q, q' and q'' are each independently for each occurrence
0-30 and wherein the repeating unit can be the same or
different;
[0230] n is independently for each occurrence 1-20; and
[0231] m is independently for each occurrence 0-50.
[0232] In some embodiments, a carrier monomer is also considered a
linker. In those instances the term linker comprises the carrier
monomer and the linker between the monomer and the ligand, e.g.
endosomolytic ligand and targeting ligand.
[0233] In some embodiments, the linker comprises at least one
cleavable linking group.
Cleavable Linking Groups
[0234] 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 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).
[0235] 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; 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
phosphatases.
[0236] A cleavable linkage group, such as a disulfide bond can be
susceptible to pH. The pH of human serum is 7.4, while the average
intracellular pH is slightly lower, ranging from about 7.1-7.3.
Endosomes have a more acidic pH, in the range of 5.5-6.0, and
lysosomes have an even more acidic pH at around 5.0. Some linkers
will have a cleavable linking group that is cleaved at a preferred
pH, thereby releasing the cationic lipid from the ligand inside the
cell, or into the desired compartment of the cell.
[0237] 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.
[0238] Linkers that contain peptide bonds can be used when
targeting cell types rich in peptidases, such as liver cells and
synoviocytes.
[0239] In general, the suitability of a candidate cleavable linking
group can be evaluated by testing the ability of a degradative
agent (or condition) to cleave the candidate linking group. It will
also be desirable to also test the candidate cleavable linking
group for the ability to resist cleavage in the blood or when in
contact with other non-target tissue. Thus one can determine the
relative susceptibility to cleavage between a first and a second
condition, where the first is selected to be indicative of cleavage
in a target cell and the second is selected to be indicative of
cleavage in other tissues or biological fluids, e.g., blood or
serum. The evaluations can be carried out in cell free systems, in
cells, in cell culture, in organ or tissue culture, or in whole
animals. It may be useful to make initial evaluations in cell-free
or culture conditions and to confirm by further evaluations in
whole animals. In preferred embodiments, useful candidate compounds
are cleaved at least 2, 4, 10 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).
Redox Cleavable Linking Groups
[0240] One class of cleavable linking groups are redox cleavable
linking groups that are cleaved upon reduction or oxidation. An
example of reductively cleavable linking group is a disulphide
linking group (--S--S--). To determine if a candidate cleavable
linking group is a suitable "reductively cleavable linking group,"
or for example is suitable for use with a particular iRNA moiety
and particular targeting agent one can look to methods described
herein. For example, a candidate can be evaluated by incubation
with dithiothreitol (DTT), or other reducing agent using reagents
know in the art, which mimic the rate of cleavage which would be
observed in a cell, e.g., a target cell. The candidates can also be
evaluated under conditions which are selected to mimic blood or
serum conditions. In a preferred embodiment, candidate compounds
are cleaved by at most 10% in the blood. In preferred embodiments,
useful candidate compounds are degraded at least 2, 4, 10 or 100
times faster in the cell (or under in vitro conditions selected to
mimic intracellular conditions) as compared to blood (or under in
vitro conditions selected to mimic extracellular conditions). The
rate of cleavage of candidate compounds can be determined using
standard enzyme kinetics assays under conditions chosen to mimic
intracellular media and compared to conditions chosen to mimic
extracellular media.
Phosphate-Based Cleavable Linking Groups
[0241] Phosphate-based cleavable linking groups are cleaved by
agents that degrade or hydrolyze the phosphate group. An example of
an agent that cleaves phosphate groups in cells are enzymes such as
phosphatases in cells. Examples of phosphate-based linking groups
are --O--P(O)(ORk)-O--, --O--P(S)(ORk)-O--, --O--P(S)(SRk)-O--,
--S--P(O)(ORk)-O--, --O--P(O)(ORk)-S--, --S--P(O)(ORk)-S--,
--O--P(S)(ORk)-S--, --S--P(S)(ORk)-O--, --O--P(O)(Rk)-O--,
--O--P(S)(Rk)-O--, --S--P(O)(Rk)-O--, --S--P(S)(Rk)-O--,
--S--P(O)(Rk)-S--, --O--P(S)(Rk)-S--. Preferred embodiments are
--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--, --O--P(S)(H)--S--. A preferred embodiment is
--O--P(O)(OH)--O--. These candidates can be evaluated using methods
analogous to those described above.
Acid Cleavable Linking Groups
[0242] Acid cleavable linking groups are linking groups that are
cleaved under acidic conditions. In preferred embodiments acid
cleavable linking groups are cleaved in an acidic environment with
a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower),
or by agents such as enzymes that can act as a general acid. In a
cell, specific low pH organelles, such as endosomes and lysosomes
can provide a cleaving environment for acid cleavable linking
groups. Examples of acid cleavable linking groups include but are
not limited to hydrazones, esters, and esters of amino acids. Acid
cleavable groups can have the general formula --C.dbd.NN--, C(O)O,
or --OC(O). A preferred embodiment is when the carbon attached to
the oxygen of the ester (the alkoxy group) is an aryl group,
substituted alkyl group, or tertiary alkyl group such as dimethyl
pentyl or t-butyl. These candidates can be evaluated using methods
analogous to those described above.
Ester-Based Cleavable Linking Groups
[0243] Ester-based cleavable linking groups are cleaved by enzymes
such as esterases and amidases in cells. Examples of ester-based
cleavable linking groups include but are not limited to esters of
alkylene, alkenylene and alkynylene groups. Ester cleavable linking
groups have the general formula --C(O)O--, or --OC(O)--. These
candidates can be evaluated using methods analogous to those
described above.
Peptide-Based Cleaving Linking Groups
[0244] Peptide-based cleavable linking groups are cleaved by
enzymes such as peptidases and proteases in cells. Peptide-based
cleavable linking groups are peptide bonds formed between amino
acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)
and polypeptides. A peptide bond is a special type of amide bond
formed between amino acids to yield peptides and proteins. The
peptide based cleavage group is generally limited to the peptide
bond (i.e., the amide bond) formed between amino acids yielding
peptides and proteins and does not include the entire amide
functional group. Peptide-based cleavable linking groups have the
general formula --NHCHR.sup.AC(O)NHCHR.sup.BC(O)--, where R.sup.A
and R.sup.B are the R groups of the two adjacent amino acids. These
candidates can be evaluated using methods analogous to those
described above.
[0245] Where more than one endosomolytic ligand or targeting ligand
is present on the same modular composition, the more than one
endosomolytic ligand or targeting ligand may be linked to the
oligonucleotide strand or an endosomolytic ligand or targeting
ligand in a linear fashion, or by a branched linker group.
iRNA Agents
[0246] The iRNA agent should include a region of sufficient
homology to the target gene, and be of sufficient length in terms
of nucleotides, such that the iRNA agent, or a fragment thereof,
can mediate downregulation of the target gene. (For ease of
exposition the term nucleotide or ribonucleotide is sometimes used
herein in reference to one or more monomeric subunits of an RNA
agent. It will be understood herein that the usage of the term
"ribonucleotide" or "nucleotide", herein can, in the case of a
modified RNA or nucleotide surrogate, also refer to a modified
nucleotide, or surrogate replacement moiety at one or more
positions.) Thus, the iRNA agent is or includes a region which is
at least partially, and in some embodiments fully, complementary to
the target RNA. It is not necessary that there be perfect
complementarity between the iRNA agent and the target, but the
correspondence must be sufficient to enable the iRNA agent, or a
cleavage product thereof, to direct sequence specific silencing,
e.g., by RNAi cleavage of the target RNA, e.g., mRNA.
Complementarity, or degree of homology with the target strand, is
most critical in the antisense strand. While perfect
complementarity, particularly in the antisense strand, is often
desired some embodiments can include, particularly in the antisense
strand, one or more, or for example, 6, 5, 4, 3, 2, or fewer
mismatches (with respect to the target RNA). The mismatches,
particularly in the antisense strand, are most tolerated in the
terminal regions and if present may be in a terminal region or
regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5' and/or 3'
termini. The sense strand need only be sufficiently complementary
with the antisense strand to maintain the over all double stranded
character of the molecule.
[0247] As discussed elsewhere herein, and in the material
incorporated by reference in its entirety, an iRNA agent will often
be modified or include nucleoside surrogates. Single stranded
regions of an iRNA agent will often be modified or include
nucleoside surrogates, e.g., the unpaired region or regions of a
hairpin structure, e.g., a region which links two complementary
regions, can have modifications or nucleoside surrogates.
Modification to stabilize one or more 3'- or 5'-termini of an iRNA
agent, e.g., against exonucleases, or to favor the antisense siRNA
agent to enter into RISC are also envisioned. Modifications can
include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl
linkers, non-nucleotide spacers (C3, C6, C9, C12, abasic,
triethylene glycol, hexaethylene glycol), special biotin or
fluorescein reagents that come as phosphoramidites and that have
another DMT-protected hydroxyl group, allowing multiple couplings
during RNA synthesis.
[0248] iRNA agents include: molecules that are long enough to
trigger the interferon response (which can be cleaved by Dicer
(Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC
(RNAi-induced silencing complex)); and, molecules which are
sufficiently short that they do not trigger the interferon response
(which molecules can also be cleaved by Dicer and/or enter a RISC),
e.g., molecules which are of a size which allows entry into a RISC,
e.g., molecules which resemble Dicer-cleavage products. Molecules
that are short enough that they do not trigger an interferon
response are termed siRNA agents or shorter iRNA agents herein.
"siRNA agent or shorter iRNA agent" as used herein, refers to an
iRNA agent, e.g., a double stranded RNA agent or single strand
agent, that is sufficiently short that it does not induce a
deleterious interferon response in a human cell, e.g., it has a
duplexed region of less than 60, 50, 40, or 30 nucleotide pairs.
The siRNA agent, or a cleavage product thereof, can down regulate a
target gene, e.g., by inducing RNAi with respect to a target RNA,
wherein the target may comprise an endogenous or pathogen target
RNA.
[0249] Each strand of an siRNA agent can be equal to or less than
30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand may
be at least 19 nucleotides in length. For example, each strand can
be between 21 and 25 nucleotides in length. siRNA agents may have a
duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide
pairs, and one or more overhangs, or one or two 3' overhangs, of
2-3 nucleotides.
[0250] In addition to homology to target RNA and the ability to
down regulate a target gene, an iRNA agent may have one or more of
the following properties: [0251] (1) it may be of the Formula VI
set out in the RNA Agent section below; [0252] (2) if single
stranded it may have a 5' modification which includes one or more
phosphate groups or one or more analogs of a phosphate group;
[0253] (3) it may, despite modifications, even to a very large
number, or all of the nucleosides, have an antisense strand that
can present bases (or modified bases) in the proper three
dimensional framework so as to be able to form correct base pairing
and form a duplex structure with a homologous target RNA which is
sufficient to allow down regulation of the target, e.g., by
cleavage of the target RNA; [0254] (4) it may, despite
modifications, even to a very large number, or all of the
nucleosides, still have "RNA-like" properties, i.e., it may possess
the overall structural, chemical and physical properties of an RNA
molecule, even though not exclusively, or even partly, of
ribonucleotide-based content. For example, an iRNA agent can
contain, e.g., a sense and/or an antisense strand in which all of
the nucleotide sugars contain e.g., 2' fluoro in place of 2'
hydroxyl. This deoxyribonucleotide-containing agent can still be
expected to exhibit RNA-like properties. While not wishing to be
bound by theory, the electronegative fluorine prefers an axial
orientation when attached to the C2' position of ribose. This
spatial preference of fluorine can, in turn, force the sugars to
adopt a C.sub.3'-endo pucker. This is the same puckering mode as
observed in RNA molecules and gives rise to the RNA-characteristic
A-family-type helix. Further, since fluorine is a good hydrogen
bond acceptor, it can participate in the same hydrogen bonding
interactions with water molecules that are known to stabilize RNA
structures. A modified moiety at the 2' sugar position may be able
to enter into H bonding which is more characteristic of the OH
moiety of a ribonucleotide than the H moiety of a
deoxyribonucleotide. Certain iRNA agents will: exhibit a
C.sub.3'-endo pucker in all, or at least 50, 75, 80, 85, 90, or 95%
of its sugars; exhibit a C.sub.3'-endo pucker in a sufficient
amount of its sugars that it can give rise to a the
RNA-characteristic A-family-type helix; will have no more than 20,
10, 5, 4, 3, 2, or 1 sugar which is not a C.sub.3'-endo pucker
structure. Regardless of the nature of the modification, and even
though the RNA agent can contain deoxynucleotides or modified
deoxynucleotides, particularly in overhang or other single strand
regions, it is certain DNA molecules, or any molecule in which more
than 50, 60, or 70% of the nucleotides in the molecule, or more
than 50, 60, or 70% of the nucleotides in a duplexed region are
deoxyribonucleotides, or modified deoxyribonucleotides which are
deoxy at the 2' position, are excluded from the definition of RNA
agent.
[0255] A "single strand iRNA agent" as used herein, is an iRNA
agent which is made up of a single molecule. It may include a
duplexed region, formed by intra-strand pairing, e.g., it may be,
or include, a hairpin or pan-handle structure. Single strand iRNA
agents may be antisense with regard to the target molecule. In
certain embodiments single strand iRNA agents 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)2(O)P--S-5'); any additional combination
of oxygen/sulfur replaced monophosphate, diphosphate and
triphosphates (e.g., 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO)2(O)P--NH-5', (HO)(NH2)(O)P--O-5'), 5'-alkylphosphonates
(R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.,
RP(OH)(O)--O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates
(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.,
RP(OH)(O)--O-5'-). (These modifications can also be used with the
antisense strand of a double stranded iRNA.)
[0256] A single strand iRNA agent may be sufficiently long that it
can enter the RISC and participate in RISC mediated cleavage of a
target mRNA. A single strand iRNA agent is at least 14, and in
other embodiments at least 15, 20, 25, 29, 35, 40, or 50
nucleotides in length. In certain embodiments, it is less than 200,
100, or 60 nucleotides in length.
[0257] Hairpin iRNA agents will have a duplex region equal to or at
least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The
duplex region will may be equal to or less than 200, 100, or 50, in
length. In certain embodiments, ranges for the duplex region are
15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in
length. The hairpin may have a single strand overhang or terminal
unpaired region, in some embodiments at the 3', and in certain
embodiments on the antisense side of the hairpin. In some
embodiments, the overhangs are 2-3 nucleotides in length.
[0258] A "double stranded (ds) iRNA agent" as used herein, is an
iRNA agent which includes more than one, and in some cases two,
strands in which interchain hybridization can form a region of
duplex structure.
[0259] The antisense strand of a double stranded iRNA agent may be
equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60
nucleotides in length. It may be equal to or less than 200, 100, or
50, nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19
to 21 nucleotides in length.
[0260] The sense strand of a double stranded iRNA agent may be
equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60
nucleotides in length. It may be equal to or less than 200, 100, or
50, nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19
to 21 nucleotides in length.
[0261] The double strand portion of a double stranded iRNA agent
may be equal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23,
24, 25, 29, 40, or 60 nucleotide pairs in length. It may be equal
to or less than 200, 100, or 50, nucleotides pairs in length.
Ranges may be 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides
pairs in length.
[0262] In many embodiments, the ds iRNA agent is sufficiently large
that it can be cleaved by an endogenous molecule, e.g., by Dicer,
to produce smaller ds iRNA agents, e.g., siRNAs agents.
[0263] The present invention further includes iRNA agents that
target within the sequence targeted by one of the iRNA agents of
the present invention. As used herein a second iRNA agent is said
to target within the sequence of a first iRNA agent if the second
iRNA agent cleaves the message anywhere within the mRNA that is
complementary to the antisense strand of the first iRNA agent. Such
a second agent will generally consist of at least 15 contiguous
nucleotides coupled to additional nucleotide sequences taken from
the region contiguous to the selected sequence in the target
gene.
[0264] The dsiRNAs of the invention can contain one or more
mismatches to the target sequence. In a preferred embodiment, the
dsiRNA of the invention contains no more than 3 mismatches. If the
antisense strand of the dsiRNA contains mismatches to a target
sequence, it is preferable that the area of mismatch not be located
in the center of the region of complementarity. If the antisense
strand of the dsiRNA contains mismatches to the target sequence, it
is preferable that the mismatch be restricted to 5 nucleotides from
either end, for example 5, 4, 3, 2, or 1 nucleotide from either the
5' or 3' end of the region of complementarity. For example, for a
23 nucleotide dsiRNA strand which is complementary to a region of
the target gene, the dsRNA generally does not contain any mismatch
within the central 13 nucleotides. The methods described within the
invention can be used to determine whether a dsiRNA containing a
mismatch to a target sequence is effective in inhibiting the
expression of the target gene. Consideration of the efficacy of
dsiRNAs with mismatches in inhibiting expression of the target gene
may be important, especially if the particular region of
complementarity in the target gene is known to have polymorphic
sequence variation within the population.
[0265] In some embodiments, the sense-strand comprises a mismatch
to the antisense strand. In some embodiments, the mismatch is at
the 5 nucleotides from the 3'-end, for example 5, 4, 3, 2, or 1
nucleotide from the end of the region of complementarity. In some
embodiments, the mismatch is located in the target cleavage site
region. In one embodiment, the sense strand comprises no more than
1, 2, 3, 4 or 5 mismatches to the antisense strand. In preferred
embodiments, the sense strand comprises no more than 3 mismatches
to the antisense strand.
[0266] In certain embodiments, the sense strand comprises a
nucleobase modification, e.g. an optionally substituted natural or
non-natural nucleobase, a universal nucleobase, in the target
cleavage site region.
[0267] The "target cleavage site" herein means the backbone linkage
in the target gene, e.g. target mRNA, or the sense strand that is
cleaved by the RISC mechanism by utilizing the iRNA agent. And the
"target cleavage site region" comprises at least one or at least
two nucleotides on both side of the cleavage site. For the sense
strand, the target cleavage site is the backbone linkage in the
sense strand that would get cleaved if the sense strand itself was
the target to be cleaved by the RNAi mechanism. The target cleavage
site can be determined using methods known in the art, for example
the 5'-RACE assay as detailed in Soutschek et al., Nature (2004)
432, 173-178. As is well understood in the art, the cleavage site
region for a conical double stranded RNAi agent comprising two
21-nucleotides long strands (wherein the strands form a double
stranded region of 19 consecutive basepairs having 2-nucleotide
single stranded overhangs at the 3'-ends), the cleavage site region
corresponds to positions 9-12 from the 5'-end of the sense
strand.
[0268] The present invention also includes nucleic acids which are
chimeric compounds. "Chimeric" nucleic acid compounds or
"chimeras," in the context of this invention, are nucleic acid
compounds, which contain two or more chemically distinct regions,
each made up of at least one monomer unit, i.e., a nucleotide in
the case of a nucleic acid compound. These nucleic acids typically
contain at least one region wherein the nucleic acid is modified so
as to confer upon the it increased resistance to nuclease
degradation, increased cellular uptake, and/or increased binding
affinity for the target nucleic acid. An additional region of the
nucleic acid may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an
RNA:DNAduplex. Activation of RNase H, therefore, results in
cleavage of the RNA target, thereby greatly enhancing the
efficiency of dsRNA inhibition of gene expression.
[0269] The present invention also includes ds iRNAs wherein the two
strands are linked together. The two strands can be linked together
by a polynucleotide linker such as (dT).sub.n; wherein n is 4-10,
and thus forming a hairpin. 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 polynucleotide linker.
[0270] The double stranded oligonucleotides can be optimized for
RNA interference by increasing the propensity of the duplex to
disassociate or melt (decreasing the free energy of duplex
association), in the region of the 5' end of the antisense strand
This can be accomplished, e.g., by the inclusion of modifications
or modified nucleosides which increase the propensity of the duplex
to disassociate or melt in the region of the 5' end of the
antisense strand. It can also be accomplished by inclusion of
modifications or modified nucleosides or attachment of a ligand
that increases the propensity of the duplex to disassociate of melt
in the region of the 5'end of the antisense strand. While not
wishing to be bound by theory, the effect may be due to promoting
the effect of an enzyme such as helicase, for example, promoting
the effect of the enzyme in the proximity of the 5' end of the
antisense strand.
[0271] Modifications which increase the tendency of the 5' end of
the antisense strand in the duplex to dissociate can be used alone
or in combination with other modifications described herein, e.g.,
with modifications which decrease the tendency of the 3' end of the
antisense in the duplex to dissociate. Likewise, modifications
which decrease the tendency of the 3' end of the antisense in the
duplex to dissociate can be used alone or in combination with other
modifications described herein, e.g., with modifications which
increase the tendency of the 5' end of the antisense in the duplex
to dissociate.
[0272] Nucleic acid base pairs can be ranked on the basis of their
propensity to promote dissociation or melting (e.g., on the free
energy of association or dissociation of a particular pairing, the
simplest approach is to examine the pairs on an individual pair
basis, though next neighbor or similar analysis can also be used).
In terms of promoting dissociation: A:U is preferred over G:C; G:U
is preferred over G:C; I:C is preferred over G:C (I=inosine);
mismatches, e.g., non-canonical or other than canonical pairings
are preferred over canonical (A:T, A:U, G:C) pairings; pairings
which include a universal base are preferred over canonical
pairings.
[0273] It is preferred that pairings which decrease the propensity
to form a duplex are used at 1 or more of the positions in the
duplex at the 5' end of the antisense strand. The terminal pair
(the most 5' pair in terms of the antisense strand), and the
subsequent 4 base pairing positions (going in the 3' direction in
terms of the antisense strand) in the duplex are preferred for
placement of modifications to decrease the propensity to form a
duplex. More preferred are placements in the terminal most pair and
the subsequent 3, 2, or 1 base pairings. It is preferred that at
least 1, and more preferably 2, 3, 4, or 5 of the base pairs from
the 5'-end of antisense strand in the duplex be chosen
independently from the group of: A:U, G:U, I:C, mismatched pairs,
e.g., non-canonical or other than canonical pairings or pairings
which include a universal base. In a preferred embodiment at least
one, at least 2, or at least 3 base-pairs include a universal
base.
[0274] Modifications or changes which promote dissociation are
preferably made in the sense strand, though in some embodiments,
such modifications/changes will be made in the antisense
strand.
[0275] Nucleic acid base pairs can also be ranked on the basis of
their propensity to promote stability and inhibit dissociation or
melting (e.g., on the free energy of association or dissociation of
a particular pairing, the simplest approach is to examine the pairs
on an individual pair basis, though next neighbor or similar
analysis can also be used). In terms of promoting duplex stability:
G:C is preferred over A:U, Watson-Crick matches (A:T, A:U, G:C) are
preferred over non-canonical or other than canonical pairings,
analogs that increase stability are preferred over Watson-Crick
matches (A:T, A:U, G:C), e.g. 2-amino-A:U is preferred over A:U,
2-thio U or 5 Me-thio-U:A, are preferred over U:A, G-clamp (an
analog of C having 4 hydrogen bonds):G is preferred over C:G,
guanadinium-G-clamp:G is preferred over C:G, psuedo uridine:A, is
preferred over U:A, sugar modifications, e.g., 2' modifications,
e.g., 2'F, ENA, or LNA, which enhance binding are preferred over
non-modified moieties and can be present on one or both strands to
enhance stability of the duplex.
[0276] It is preferred that pairings which increase the propensity
to form a duplex are used at 1 or more of the positions in the
duplex at the 3' end of the antisense strand. The terminal pair
(the most 3' pair in terms of the antisense strand), and the
subsequent 4 base pairing positions (going in the 5' direction in
terms of the antisense strand) in the duplex are preferred for
placement of modifications to decrease the propensity to form a
duplex. More preferred are placements in the terminal most pair and
the subsequent 3, 2, or 1 base pairings. It is preferred that at
least 1, and more preferably 2, 3, 4, or 5 of the pairs of the
recited regions be chosen independently from the group of: G:C, a
pair having an analog that increases stability over Watson-Crick
matches (A:T, A:U, G:C), 2-amino-A:U, 2-thio U or 5 Me-thio-U:A,
G-clamp (an analog of C having 4 hydrogen bonds):G,
guanadinium-G-clamp:G, psuedo uridine:A, a pair in which one or
both subunits has a sugar modification, e.g., a 2' modification,
e.g., 2'F, ENA, or LNA, which enhance binding. In some embodiments,
at least one, at least, at least 2, or at least 3, of the base
pairs promote duplex stability.
[0277] In a preferred embodiment at least one, at least 2, or at
least 3, of the base pairs are a pair in which one or both subunits
has a sugar modification, e.g., a 2' modification, e.g.,
2'-O-methyl, 2'-O-Me (2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl),
2'-F, 2'-O--CH.sub.2-(4'-C) (LNA) and 2'-O--CH.sub.2CH.sub.2-(4'-C)
(ENA), which enhances binding.
[0278] G-clamps and guanidinium G-clamps are discussed in the
following references: Holmes and Gait, "The Synthesis of
2'-O-Methyl G-Clamp Containing Oligonucleotides and Their
Inhibition of the HIV-1 Tat-TAR Interaction," Nucleosides,
Nucleotides & Nucleic Acids, 22:1259-1262, 2003; Holmes et al.,
"Steric inhibition of human immunodeficiency virus type-1
Tat-dependent trans-activation in vitro and in cells by
oligonucleotides containing 2'-O-methyl G-clamp ribonucleoside
analogues," Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et
al., "Structural basis for recognition of guanosine by a synthetic
tricyclic cytosine analogue: Guanidinium G-clamp," Helvetica
Chimica Acta, 86:966-978, 2003; Rajeev, et al., "High-Affinity
Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine
Analogues," Organic Letters, 4:4395-4398, 2002; Ausin, et al.,
"Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers," Organic
Letters, 4:4073-4075, 2002; Maier et al., "Nuclease resistance of
oligonucleotides containing the tricyclic cytosine analogues
phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") and
origins of their nuclease resistance properties," Biochemistry,
41:1323-7, 2002; Flanagan, et al., "A cytosine analog that confers
enhanced potency to antisense oligonucleotides," Proceedings Of The
National Academy Of Sciences Of The United States Of America,
96:3513-8, 1999.
[0279] As is discussed above, ds iRNA can be modified to both
decrease the stability of the antisense 5'end of the duplex and
increase the stability of the antisense 3' end of the duplex. This
can be effected by combining one or more of the stability
decreasing modifications in the antisense 5' end of the duplex with
one or more of the stability increasing modifications in the
antisense 3' end of the duplex.
[0280] It may be desirable to modify one or both of the antisense
and sense strands of a double strand iRNA agent. In some cases they
will have the same modification or the same class of modification
but in other cases the sense and antisense strand will have
different modifications, e.g., in some cases it is desirable to
modify only the sense strand. It may be desirable to modify only
the sense strand, e.g., to inactivate it, e.g., the sense strand
can be modified in order to inactivate the sense strand and prevent
formation of an active siRNA/protein or RISC. This can be
accomplished by a modification which prevents 5'-phosphorylation of
the sense strand, e.g., by modification with a 5'-O-methyl
ribonucleotide (see Nykanen et al., (2001) ATP requirements and
small interfering RNA structure in the RNA interference pathway.
Cell 107, 309-321.) Other modifications which prevent
phosphorylation can also be used, e.g., simply substituting the
5'-OH by H rather than O-Me. Alternatively, a large bulky group may
be added to the 5'-phosphate turning it into a phosphodiester
linkage, though this may be less desirable as phosphodiesterases
can cleave such a linkage and release a functional siRNA 5'-end.
Antisense strand modifications include 5' phosphorylation as well
as any of the other 5' modifications discussed herein, particularly
the 5' modifications discussed above in the section on single
stranded iRNA molecules.
[0281] The sense and antisense strands may be chosen such that the
ds iRNA agent includes a single strand or unpaired region at one or
both ends of the molecule. Thus, a ds iRNA agent may contain sense
and antisense strands, paired to contain an overhang, e.g., one or
two 5' or 3' overhangs, or a 3' overhang of 2-3 nucleotides. Many
embodiments will have a 3' overhang. Certain siRNA agents will have
single-stranded overhangs, in some embodiments 3' overhangs, of 1
or 2 or 3 nucleotides in length at each end. The overhangs can be
the result of one strand being longer than the other, or the result
of two strands of the same length being staggered. 5' ends may be
phosphorylated.
[0282] In one embodiment, the single-stranded overhang has the
sequence 5'-GCNN-3', wherein N is independently for each
occurrence, A, G, C, U, dT, dU or absent. Double-stranded iRNA
having only one overhang has proven particularly stable and
effective in vivo, as well as in a variety of cells, cell culture
mediums, blood, and serum. The dsRNA may also have a blunt end,
generally located at the 5'-end of the antisense strand.
[0283] In one embodiment, the antisense strand of the ds iRNA has
1-10 nucleotides overhangs each at the 3' end and the 5' end over
the sense strand. In one embodiment, the sense strand of the ds
iRNA has 1-10 nucleotides overhangs each at the 3' end and the 5'
end over the antisense strand.
[0284] In some embodiments, the length for the duplexed region is
between 15 and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in
length, e.g., in the siRNA agent range discussed above. siRNA
agents can resemble in length and structure the natural Dicer
processed products from long dsiRNAs. Embodiments in which the two
strands of the siRNA agent are linked, e.g., covalently linked are
also included. Hairpin, or other single strand structures which
provide the required double stranded region, and a 3' overhang are
also within the invention.
[0285] In some embodiments, the length for the duplexed region is
between 10-15, e.g. 10, 11, 12, 13, 14 and 15 nucleotides in length
and the antisense strand has 1-10 nucleotides single-strand
overhangs each at the 3' end and the 5' end over the sense
strand.
[0286] The isolated iRNA agents described herein, including ds iRNA
agents and siRNA agents can mediate silencing of a target RNA,
e.g., mRNA, e.g., a transcript of a gene that encodes a protein.
For convenience, such mRNA is also referred to herein as mRNA to be
silenced. Such a gene is also referred to as a target gene. In
general, the RNA to be silenced is an endogenous gene or a pathogen
gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral
RNAs, can also be targeted.
[0287] As used herein, the phrase "mediates RNAi" refers to the
ability to silence, in a sequence specific manner, a target RNA.
While not wishing to be bound by theory, it is believed that
silencing uses the RNAi machinery or process and a guide RNA, e.g.,
an siRNA agent of 21 to 23 nucleotides.
[0288] As used herein, "specifically hybridizable" and
"complementary" are terms which are used to indicate a sufficient
degree of complementarity such that stable and specific binding
occurs between a compound of the invention and a target RNA
molecule. 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.
[0289] In one embodiment, an iRNA agent is "sufficiently
complementary" to a target RNA, e.g., a target mRNA, such that the
iRNA agent silences production of protein encoded by the target
mRNA. In another embodiment, the iRNA agent is "exactly
complementary" to a target RNA, e.g., the target RNA and the iRNA
agent anneal, for example to form a hybrid made exclusively of
Watson-Crick base pairs in the region of exact complementarity. A
"sufficiently complementary" target RNA can include an internal
region (e.g., of at least 10 nucleotides) that is exactly
complementary to a target RNA. Moreover, in some embodiments, the
iRNA agent specifically discriminates a single-nucleotide
difference. In this case, the iRNA agent only mediates RNAi if
exact complementary is found in the region (e.g., within 7
nucleotides of) the single-nucleotide difference.
[0290] As used herein, the term "oligonucleotide" refers to a
nucleic acid molecule (RNA or DNA) for example of length less than
100, 200, 300, or 400 nucleotides.
[0291] RNA agents discussed herein include unmodified RNA as well
as RNA which have been modified, e.g., to improve efficacy, and
polymers of nucleoside surrogates. Unmodified RNA refers to a
molecule in which the components of the nucleic acid, namely
sugars, bases, and phosphate moieties, are the same or essentially
the same as that which occur in nature, for example as occur
naturally in the human body. The art has often referred to rare or
unusual, but naturally occurring, RNAs as modified RNAs, see, e.g.,
Limbach et al., (1994) Summary: the modified nucleosides of RNA,
Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often
termed modified RNAs (apparently because the are typically the
result of a post transcriptionally modification) are within the
term unmodified RNA, as used herein. Modified RNA refers to a
molecule in which one or more of the components of the nucleic
acid, namely sugars, bases, and phosphate moieties, are different
from that which occur in nature, for example, different from that
which occurs in the human body. While they are referred to as
modified "RNAs," they will of course, because of the modification,
include molecules which are not RNAs. Nucleoside surrogates are
molecules in which the ribophosphate backbone is replaced with a
non-ribophosphate construct that allows the bases to the presented
in the correct spatial relationship such that hybridization is
substantially similar to what is seen with a ribophosphate
backbone, e.g., non-charged mimics of the ribophosphate backbone.
Examples of all of the above are discussed herein.
[0292] Much of the discussion below refers to single strand
molecules. In many embodiments of the invention a double stranded
iRNA agent, e.g., a partially double stranded iRNA agent, is
envisioned. Thus, it is understood that that double stranded
structures (e.g., where two separate molecules are contacted to
form the double stranded region or where the double stranded region
is formed by intramolecular pairing (e.g., a hairpin structure))
made of the single stranded structures described below are within
the invention. Lengths are described elsewhere herein.
[0293] As nucleic acids are polymers of subunits, many of the
modifications described below occur at a position which is repeated
within a nucleic acid, e.g., a modification of a base, or a
phosphate moiety, or the a non-linking O of a phosphate moiety. In
some cases the modification will occur at all of the subject
positions in the nucleic acid but in many cases it will not. By way
of example, a modification may only occur at a 3' or 5' terminal
position, may only occur in a terminal regions, e.g., at a position
on a terminal nucleotide or in the last 2, 3, 4, 5, or 10
nucleotides of a strand. A modification may occur in a double
strand region, a single strand region, or in both. A modification
may occur only in the double strand region of an RNA or may only
occur in a single strand region of an RNA. E.g., a phosphorothioate
modification at a non-linking O position may only occur at one or
both termini, may only occur in a terminal regions, e.g., at a
position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10
nucleotides of a strand, or may occur in double strand and single
strand regions, particularly at termini. The 5' end or ends can be
phosphorylated.
[0294] A modification described herein may be the sole
modification, or the sole type of modification included on multiple
nucleotides, or a modification can be combined with one or more
other modifications described herein. The modifications described
herein can also be combined onto an oligonucleotide, e.g. different
nucleotides of an oligonucleotide have different modifications
described herein.
[0295] In some embodiments it is possible, e.g., to enhance
stability, to include particular bases in overhangs, or to include
modified nucleotides or nucleotide surrogates, in single strand
overhangs, e.g., in a 5' or 3' overhang, or in both. E.g., it can
be desirable to include purine nucleotides in overhangs. In some
embodiments all or some of the bases in a 3' or 5' overhang will be
modified, e.g., with a modification described herein. Modifications
can include, e.g., the use of modifications at the 2' OH group of
the ribose sugar, e.g., the use of deoxyribonucleotides, e.g.,
deoxythymidine, instead of ribonucleotides, and modifications in
the phosphate group, e.g., phosphothioate modifications. Overhangs
need not be homologous with the target sequence.
[0296] Modifications and nucleotide surrogates are discussed
below.
##STR00020##
Formula VI
[0297] The scaffold presented above in Formula VI represents a
portion of a ribonucleic acid. The basic components are the ribose
sugar, the base, the terminal phosphates, and phosphate
internucleotide linkers. Where the bases are naturally occurring
bases, e.g., adenine, uracil, guanine or cytosine, the sugars are
the unmodified 2' hydroxyl ribose sugar (as depicted) and W, X, Y,
and Z are all O, Formula VI represents a naturally occurring
unmodified oligoribonucleotide.
[0298] Unmodified oligoribonucleotides may be less than optimal in
some applications, e.g., unmodified oligoribonucleotides can be
prone to degradation by e.g., cellular nucleases. Nucleases can
hydrolyze nucleic acid phosphodiester bonds. However, chemical
modifications to one or more of the above RNA components can confer
improved properties, and, e.g., can render oligoribonucleotides
more stable to nucleases.
[0299] Modified nucleic acids and nucleotide surrogates can include
one or more of:
[0300] (i) alteration, e.g., replacement, of one or both of the
non-linking (X and Y) phosphate oxygens and/or of one or more of
the linking (W and Z) phosphate oxygens (When the phosphate is in
the terminal position, one of the positions W or Z will not link
the phosphate to an additional element in a naturally occurring
ribonucleic acid. However, for simplicity of terminology, except
where otherwise noted, the W position at the 5' end of a nucleic
acid and the terminal Z position at the 3' end of a nucleic acid,
are within the term "linking phosphate oxygens" as used
herein);
[0301] (ii) alteration, e.g., replacement, of a constituent of the
ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;
[0302] (iii) wholesale replacement of the phosphate moiety (bracket
I) with "dephospho" linkers;
[0303] (iv) modification or replacement of a naturally occurring
base;
[0304] (v) replacement or modification of the ribose-phosphate
backbone (bracket II);
[0305] (vi) modification of the 3' end or 5' end of the RNA, e.g.,
removal, modification or replacement of a terminal phosphate group
or conjugation of a moiety, e.g., a fluorescently labeled moiety,
to either the 3' or 5' end of RNA.
[0306] The terms replacement, modification, alteration, and the
like, as used in this context, do not imply any process limitation,
e.g., modification does not mean that one must start with a
reference or naturally occurring ribonucleic acid and modify it to
produce a modified ribonucleic acid bur rather modified simply
indicates a difference from a naturally occurring molecule.
[0307] It is understood that the actual electronic structure of
some chemical entities cannot be adequately represented by only one
canonical form (i.e., Lewis structure). While not wishing to be
bound by theory, the actual structure can instead be some hybrid or
weighted average of two or more canonical forms, known collectively
as resonance forms or structures. Resonance structures are not
discrete chemical entities and exist only on paper. They differ
from one another only in the placement or "localization" of the
bonding and nonbonding electrons for a particular chemical entity.
It can be possible for one resonance structure to contribute to a
greater extent to the hybrid than the others. Thus, the written and
graphical descriptions of the embodiments of the present invention
are made in terms of what the art recognizes as the predominant
resonance form for a particular species. For example, any
phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)
would be represented by X.dbd.O and Y.dbd.N in the above
figure.
[0308] Specific modifications are discussed in more detail
below.
The Phosphate Group
[0309] The phosphate group is a negatively charged species. The
charge is distributed equally over the two non-linking oxygen atoms
(i.e., X and Y in Formula VI above). However, the phosphate group
can be modified by replacing one of the oxygens with a different
substituent. One result of this modification to RNA phosphate
backbones can be increased resistance of the oligoribonucleotide to
nucleolytic breakdown. Thus while not wishing to be bound by
theory, it can be desirable in some embodiments to introduce
alterations which result in either an uncharged linker or a charged
linker with unsymmetrical charge distribution.
[0310] Examples of modified phosphate groups include
phosphorothioate, phosphoroselenates, borano phosphates, borano
phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl
or aryl phosphonates and phosphotriesters. Phosphorodithioates have
both non-linking oxygens replaced by sulfur. Unlike the situation
where only one of X or Y is altered, the phosphorus center in the
phosphorodithioates is achiral which precludes the formation of
oligoribonucleotides diastereomers. Diastereomer formation can
result in a preparation in which the individual diastereomers
exhibit varying resistance to nucleases. Further, the hybridization
affinity of RNA containing chiral phosphate groups can be lower
relative to the corresponding unmodified RNA species. Thus, while
not wishing to be bound by theory, modifications to both X and Y
which eliminate the chiral center, e.g., phosphorodithioate
formation, may be desirable in that they cannot produce
diastereomer mixtures. Thus, X can be any one of S, Se, B, 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, alkyl, aryl,
etc.), or OR (R is alkyl or aryl). Thus Y can be any one of S, Se,
B, 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, alkyl,
aryl, etc. . . . ), or OR (R is alkyl or aryl). Replacement of X
and/or Y with sulfur is possible.
[0311] When the modification of the phosphate leads to phosphorous
atom becoming stereogenic, such chiral phosphate can posses either
the "R" configuration (herein Rp) or the "S" configuration (herein
Sp).
[0312] The phosphate linker can also be modified by replacement of
a linking oxygen (i.e., W or Z in Formula VI) with nitrogen
(bridged phosphoroamidates), sulfur (bridged phosphorothioates) and
carbon (bridged methylenephosphonates). The replacement can occur
at a terminal oxygen (position W (3') or position Z (5').
Replacement of W with carbon or Z with nitrogen is possible. When
the bridging oxygen is 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.
[0313] Candidate agents can be evaluated for suitability as
described below.
The Sugar Group
[0314] A modified RNA can include modification of all or some of
the sugar groups of the ribonucleic acid. E.g., the 2' hydroxyl
group (OH) can be modified or replaced with a number of different
"oxy" or "deoxy" substituents. While not being bound by theory,
enhanced stability is expected since the hydroxyl can no longer be
deprotonated to form a 2' alkoxide ion. The 2' alkoxide can
catalyze degradation by intramolecular nucleophilic attack on the
linker phosphorus atom. Again, while not wishing to be bound by
theory, it can be desirable to some embodiments to introduce
alterations in which alkoxide formation at the 2' position is not
possible.
[0315] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar; ENA in
which the 2' hydroxyl is connected by a ethylene bridge, to the 4'
carbon of the same ribose sugar; O-AMINE (AMINE=NH.sub.2,
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, ethylene diamine, polyamino
and aminoalkoxy), O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2,
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, ethylene diamine, polyamino
and aminoalkoxy). It is noteworthy that oligonucleotides containing
only the methoxyethyl group (MOE), (OCH.sub.2CH.sub.2OCH.sub.3, a
PEG derivative), exhibit nuclease stabilities comparable to those
modified with the robust phosphorothioate modification.
[0316] "Deoxy" modifications include hydrogen (i.e., deoxyribose
sugars, which are of particular relevance to the overhang portions
of partially ds RNA); halo (e.g., fluoro); amino (e.g., NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino), --NHC(O)R (R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl
and alkynyl, which may be optionally substituted with halo,
hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl,
alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino,
acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl,
alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl,
alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,
alkylcarbonyl, acyloxy, cyano, or ureido. Other substitutents of
certain embodiments include 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl,
2'-C-allyl, and 2'-fluoro.
[0317] Other preferred substitutents are
2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), 2'-NH.sub.2,
2'-O-amine, 2'-SH, 2'-S-alkyl, 2'-S-allyl, 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) and
2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE).
[0318] In some embodiments, the 2'- and the 4'-carbons of the same
ribose sugar may be linked together by a linker described
herein.
[0319] The sugar group can also contain one or more carbons that
possess the opposite stereochemical configuration than that of the
corresponding carbon in ribose. Thus, a modified RNA can include
nucleotides containing e.g., arabinose, as the sugar.
[0320] The sugar group can also have an alpha linkage at the 1'
position on the sugar, e.g., alpha-nucleosides.
[0321] The sugar group can also be a L-sugar, e.g.
L-nucleosides.
[0322] Modified RNA's can also include "abasic" sugars, which lack
a nucleobase at C-1'. These abasic sugars can also be further
contain modifications at one or more of the constituent sugar
atoms.
[0323] To maximize nuclease resistance, the 2' modifications can be
used in combination with one or more phosphate linker modifications
(e.g., phosphorothioate). The so-called "chimeric" oligonucleotides
are those that contain two or more different modifications.
[0324] Candidate modifications can be evaluated as described
below.
Replacement of the Phosphate Group
[0325] The phosphate group can be replaced by non-phosphorus
containing connectors (cf Bracket I in Formula VI above). While not
wishing to be bound by theory, it is believed that since the
charged phosphodiester group is the reaction center in nucleolytic
degradation, its replacement with neutral structural mimics should
impart enhanced nuclease stability. Again, while not wishing to be
bound by theory, it can be desirable, in some embodiment, to
introduce alterations in which the charged phosphate group is
replaced by a neutral moiety.
[0326] Examples of moieties which can replace the phosphate group
include siloxane, carbonate, carboxymethyl, carbamate, amide,
thioether, ethylene oxide linker, sulfonate, sulfonamide,
thioformacetal, formacetal, oxime, methyleneimino,
methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo
and methyleneoxymethylimino. In certain embodiments, replacements
may include the methylenecarbonylamino and methylenemethylimino
groups.
[0327] Candidate modifications can be evaluated as described
below.
Replacement of Ribophosphate Backbone
[0328] Oligonucleotide-mimicking scaffolds can also be constructed
wherein the phosphate linker and ribose sugar are replaced by
nuclease resistant nucleoside or nucleotide surrogates (see Bracket
II of Formula 1 above). While not wishing to be bound by theory, it
is believed that the absence of a repetitively charged backbone
diminishes binding to proteins that recognize polyanions (e.g.,
nucleases). Again, while not wishing to be bound by theory, it can
be desirable in some embodiment, to introduce alterations in which
the bases are tethered by a neutral surrogate backbone.
[0329] Examples include the mophilino, cyclobutyl, pyrrolidine and
peptide nucleic acid (PNA) nucleoside surrogates. In certain
embodiments, PNA surrogates may be used.
[0330] Modified phosphate linkages where at least one of the
oxygens 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 backbone linkage."
[0331] Preferred backbone modifications are phosphorothioate,
phosphorodithioate, phosphoramidate, phosphonate, alkylphosphonate,
siloxane, carbonate, carboxymethyl, carbamate, amide, thioether,
ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,
formacetal, oxime, methyleneimino, methyleneaminocarbonyl,
methylenemethylimino (MMI), methylenehydrazo,
methylenedimethylhydrazo (MDH) and methyleneoxymethylimino.
[0332] Candidate modifications can be evaluated as described
below.
Types of Backbone Linkages
[0333] The canonical 3'-5' backbone linkage can also be replaced
with linkage between other positions on the nucleosides. In some
embodiments, the oligonucleotide comprises at least one of 5'-5',
3'-3', 3'-2', 2'-3', 2'-3' or 2'-5' backbone linkage.
[0334] In some embodiments, the last nucleotide on the end of the
oligonucleotide is linked via a 5'-5', 3'-3', 3'-2', 2'-3' or 2'-3'
backbone linkage to the rest of the oligonucleotide.
Terminal Modifications
[0335] The 3' and 5' ends of an oligonucleotide can be modified.
Such modifications can be at the 3' end, 5' end or both ends of the
molecule. They can include modification or replacement of an entire
terminal phosphate or of one or more of the atoms of the phosphate
group. E.g., the 3' and 5' ends of an oligonucleotide can be
conjugated to other functional molecular entities such as labeling
moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3
or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon,
boron or ester). The functional molecular entities can be attached
to the sugar through a phosphate group and/or a spacer. The
terminal atom of the spacer can connect to or replace the linking
atom of the phosphate group or the C-3' or C-5' O, N, S or C group
of the sugar. Alternatively, the spacer can connect to or replace
the terminal atom of a nucleotide surrogate (e.g., PNAs). These
spacers or linkers can include e.g., --(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nN--, --(CH.sub.2).sub.nO--,
--(CH.sub.2).sub.nS--, O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OH
(e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine,
oxyimine, thioether, disulfide, thiourea, sulfonamide, or
morpholino, or biotin and fluorescein reagents. When a
spacer/phosphate-functional molecular entity-spacer/phosphate array
is interposed between two strands of iRNA agents, this array can
substitute for a hairpin RNA loop in a hairpin-type RNA agent. The
3' end can be an --OH group. While not wishing to be bound by
theory, it is believed that conjugation of certain moieties can
improve transport, hybridization, and specificity properties.
Again, while not wishing to be bound by theory, it may be desirable
to introduce terminal alterations that improve nuclease resistance.
Other examples of terminal modifications include dyes,
intercalating agents (e.g., acridines), cross-linkers (e.g.,
psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin),
polycyclic aromatic hydrocarbons (e.g., phenazine,
dihydrophenazine), artificial endonucleases (e.g., EDTA),
lipophilic carriers (e.g., cholesterol, cholic acid, adamantane
acetic acid, 1-pyrene butyric acid, dihydrotestosterone,
1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl
group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,
O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and
peptide conjugates (e.g., antennapedia peptide, Tat peptide),
alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K),
MPEG, [MPEG].sub.2, polyamino, alkyl, substituted alkyl,
radiolabeled markers, enzymes, haptens (e.g., biotin),
transport/absorption facilitators (e.g., aspirin, vitamin E, folic
acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,
histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+
complexes of tetraazamacrocycles).
[0336] Terminal modifications can be added for a number of reasons,
including as discussed elsewhere herein to modulate activity or to
modulate resistance to degradation. Terminal modifications useful
for modulating activity include modification of the 5' end with
phosphate or phosphate analogs. E.g., in certain embodiments iRNA
agents, especially antisense strands, 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)2(O)P--S-5'); any additional combination
of oxygen/sulfur replaced monophosphate, diphosphate and
triphosphates (e.g., 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO)2(O)P--NH-5', (HO)(NH2)(O)P--O-5'), 5'-alkylphosphonates
(R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.,
RP(OH)(O)--O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates
(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.,
RP(OH)(O)--O-5'-).
[0337] Terminal modifications can also be useful for monitoring
distribution, and in such cases the groups to be added may include
fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488.
Terminal modifications can also be useful for enhancing uptake,
useful modifications for this include cholesterol. Terminal
modifications can also be useful for cross-linking an RNA agent to
another moiety; modifications useful for this include mitomycin
C.
[0338] Candidate modifications can be evaluated as described
below.
The Bases
[0339] Adenine, guanine, cytosine and uracil are the most common
bases found in RNA. These bases can be modified or replaced to
provide RNA's having improved properties. E.g., nuclease resistant
oligoribonucleotides can be prepared with these bases or with
synthetic and natural nucleobases (e.g., inosine, thymine,
xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine)
and any one of the above modifications. Alternatively, substituted
or modified analogs of any of the above bases and "universal bases"
can be employed. Examples include 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine
and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,
5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino,
thiol, thioalkyl, hydroxyl and other 8-substituted adenines and
guanines, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine, 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine,
5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles,
2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil,
uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,
5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil, 3-(3-amino-3
carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,
N.sup.4-acetyl cytosine, 2-thiocytosine, N6-methyladenine,
N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine,
N-methylguanines, or O-alkylated bases. Further purines and
pyrimidines include those disclosed in U.S. Pat. No. 3,687,808,
those disclosed in the Concise Encyclopedia Of Polymer Science And
Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley &
Sons, 1990, and those disclosed by Englisch et al., Angewandte
Chemie, International Edition, 1991, 30, 613.
[0340] Generally, base changes are not used for promoting
stability, but they can be useful for other reasons, e.g., some,
e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent.
Modified bases can reduce target specificity. This may be taken
into consideration in the design of iRNA agents.
[0341] In some embodiments, nucleobase is chosen from a group
consisting of inosine, thymine, 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-(alk-
yl)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, and any O-alkylated or N-alkylated
derivatives thereof.
[0342] Candidate modifications can be evaluated as described
below.
Cationic Groups
[0343] Modifications to oligonucleotides can also include
attachment of one or more cationic groups to the sugar, base,
and/or the phosphorus atom of a phosphate or modified phosphate
backbone moiety. A cationic group can be attached to any atom
capable of substitution on a natural, unusual or universal base. A
preferred position is one that does not interfere with
hybridization, i.e., does not interfere with the hydrogen bonding
interactions needed for base pairing. A cationic group can be
attached e.g., through the C2' position of a sugar or analogous
position in a cyclic or acyclic sugar surrogate. Cationic groups
can include e.g., 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);
or 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).
Placement of Modifications within an Oligonucleotide
[0344] Some modifications may preferably be included on an
oligonucleotide at a particular location, e.g., at an internal
position of a strand, or on the 5' or 3' end of an oligonucleotide.
A preferred location of a modification on an oligonucleotide, may
confer preferred properties on the agent. For example, preferred
locations of particular modifications may confer optimum gene
silencing properties, or increased resistance to endonuclease or
exonuclease activity.
[0345] One or more nucleotides of an oligonucleotide may have a
2'-5' linkage. One or more nucleotides of an oligonucleotide may
have inverted linkages, e.g. 3'-3', 3'-2', 5'-5', 2'-2' or 2'-3'
linkages.
[0346] An oligonucleotide may comprise at least one
5'-pyrimidine-purine-3' (5'-PyPu-3') dinucleotide wherein the
pyrimidine is modified with a modification chosen independently
from a group consisting of 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) and
2'-O--CH.sub.2CH.sub.2-(4'-C) (ENA).
[0347] In one embodiment, the 5'-most pyrimidines in all
occurrences of sequence motif 5'-pyrimidine-purine-3' (5'-PyPu-3')
dinucleotide in the oligonucleotide are modified with a
modification chosen from a group consisting of 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) and 2'-O--CH.sub.2CH.sub.2-(4'-C)
(ENA).
[0348] A double-stranded oligonucleotide may include at least one
5'-uridine-adenine-3' (5'-UA-3') dinucleotide wherein the uridine
is a 2'-modified nucleotide, or a 5'-uridine-guanine-3' (5'-UG-3')
dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide,
or a terminal 5'-cytidine-adenine-3' (5'-CA-3') dinucleotide,
wherein the 5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-uridine-uridine-3' (5'-UU-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide, or a terminal
5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-cytidine-uridine-3' (5'-CU-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-uridine-cytidine-3' (5'-UC-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide. Double-stranded
oligonucleotides including these modifications are particularly
stabilized against endonuclease activity.
Evaluation of Candidate RNAs
[0349] One can evaluate a candidate RNA agent, e.g., a modified
RNA, for a selected property by exposing the agent or modified
molecule and a control molecule to the appropriate conditions and
evaluating for the presence of the selected property. For example,
resistance to a degradant can be evaluated as follows. A candidate
modified RNA (and a control molecule, usually the unmodified form)
can be exposed to degradative conditions, e.g., exposed to a
milieu, which includes a degradative agent, e.g., a nuclease. E.g.,
one can use a biological sample, e.g., one that is similar to a
milieu, which might be encountered, in therapeutic use, e.g., blood
or a cellular fraction, e.g., a cell-free homogenate or disrupted
cells. The candidate and control could then be evaluated for
resistance to degradation by any of a number of approaches. For
example, the candidate and control could be labeled prior to
exposure, with, e.g., a radioactive or enzymatic label, or a
fluorescent label, such as Cy3 or Cy5. Control and modified RNA's
can be incubated with the degradative agent, and optionally a
control, e.g., an inactivated, e.g., heat inactivated, degradative
agent. A physical parameter, e.g., size, of the modified and
control molecules are then determined. They can be determined by a
physical method, e.g., by polyacrylamide gel electrophoresis or a
sizing column, to assess whether the molecule has maintained its
original length, or assessed functionally. Alternatively, Northern
blot analysis can be used to assay the length of an unlabeled
modified molecule.
[0350] A functional assay can also be used to evaluate the
candidate agent. A functional assay can be applied initially or
after an earlier non-functional assay, (e.g., assay for resistance
to degradation) to determine if the modification alters the ability
of the molecule to silence gene expression. For example, a cell,
e.g., a mammalian cell, such as a mouse or human cell, can be
co-transfected with a plasmid expressing a fluorescent protein,
e.g., GFP, and a candidate RNA agent homologous to the transcript
encoding the fluorescent protein (see, e.g., WO 00/44914). For
example, a modified dsiRNA homologous to the GFP mRNA can be
assayed for the ability to inhibit GFP expression by monitoring for
a decrease in cell fluorescence, as compared to a control cell, in
which the transfection did not include the candidate dsiRNA, e.g.,
controls with no agent added and/or controls with a non-modified
RNA added. Efficacy of the candidate agent on gene expression can
be assessed by comparing cell fluorescence in the presence of the
modified and unmodified dsiRNA agents.
[0351] In an alternative functional assay, a candidate dsiRNA agent
homologous to an endogenous mouse gene, for example, a maternally
expressed gene, such as c-mos, can be injected into an immature
mouse oocyte to assess the ability of the agent to inhibit gene
expression in vivo (see, e.g., WO 01/36646). A phenotype of the
oocyte, e.g., the ability to maintain arrest in metaphase II, can
be monitored as an indicator that the agent is inhibiting
expression. For example, cleavage of c-mos mRNA by a dsiRNA agent
would cause the oocyte to exit metaphase arrest and initiate
parthenogenetic development (Colledge et al. Nature 370: 65-68,
1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the
modified agent on target RNA levels can be verified by Northern
blot to assay for a decrease in the level of target mRNA, or by
Western blot to assay for a decrease in the level of target
protein, as compared to a negative control. Controls can include
cells in which with no agent is added and/or cells in which a
non-modified RNA is added.
RNA Structure References
[0352] The disclosure of all publications, patents, and published
patent applications listed herein are hereby incorporated by
reference.
General References
[0353] The oligoribonucleotides and oligoribonucleosides used in
accordance with this invention may be with solid phase synthesis,
see for example "Oligonucleotide synthesis, a practical approach",
Ed. M. J. Gait, IRL Press, 1984; "Oligonucleotides and Analogues, A
Practical Approach", Ed. F. Eckstein, IRL Press, 1991 (especially
Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide
synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3,
2'-O-Methyloligoribonucleotide-s: synthesis and applications,
Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis
of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of
oligo-2'-deoxyribonucleoside methylphosphonates, and. Chapter 7,
Oligodeoxynucleotides containing modified bases. Other particularly
useful synthetic procedures, reagents, blocking groups and reaction
conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,
486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,
2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993,
49, 6123-6194, or references referred to therein. Modification
described in WO 00/44895, WO01/75164, or WO02/44321 can be used
herein.
Phosphate Group References
[0354] The preparation of phosphinate oligoribonucleotides is
described in U.S. Pat. No. 5,508,270. The preparation of alkyl
phosphonate oligoribonucleotides is described in U.S. Pat. No.
4,469,863. The preparation of phosphoramidite oligoribonucleotides
is described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
The preparation of phosphotriester oligoribonucleotides is
described in U.S. Pat. No. 5,023,243. The preparation of borano
phosphate oligoribonucleotide is described in U.S. Pat. Nos.
5,130,302 and 5,177,198. The preparation of 3'-Deoxy-3'-amino
phosphoramidate oligoribonucleotides is described in U.S. Pat. No.
5,476,925. 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is
described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.
Preparation of sulfur bridged nucleotides is described in Sproat et
al. Nucleosides Nucleotides 1988, 7, 651 and Crosstick et al.
Tetrahedron Lett. 1989, 30, 4693.
Sugar Group References
[0355] Modifications to the 2' modifications can be found in Verma,
S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references
therein. Specific modifications to the ribose can be found in the
following references: 2'-fluoro (Kawasaki et. al., J. Med. Chem.,
1993, 36, 831-841), 2'-MOE (Martin, P. Helv. Chim. Acta 1996, 79,
1930-1938), "LNA" (Wengel, J. Acc. Chem. Res. 1999, 32,
301-310).
Replacement of the Phosphate Group References
[0356] Methylenemethylimino linked oligoribonucleosides, also
identified herein as MMI linked oligoribonucleosides,
methylenedimethylhydrazo linked oligoribonucleosides, also
identified herein as MDH linked oligoribonucleosides, and
methylenecarbonylamino linked oligonucleosides, also identified
herein as amide-3 linked oligoribonucleosides, and
methyleneaminocarbonyl linked oligonucleosides, also identified
herein as amide-4 linked oligoribonucleosides as well as mixed
backbone compounds having, as for instance, alternating MMI and PO
or PS linkages can be prepared as is described in U.S. Pat. Nos.
5,378,825, 5,386,023, 5,489,677 and in published PCT applications
PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and
92/20823, respectively). Formacetal and thioformacetal linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
Nos. 5,264,562 and 5,264,564. Ethylene oxide linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
No. 5,223,618. Siloxane replacements are described in Cormier, J.
F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements
are described in Tittensor, J. R. J. Chem. Soc. C 1971, 1933.
Carboxymethyl replacements are described in Edge, M. D. et al. J.
Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are
described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.
Replacement of the Phosphate-Ribose Backbone References
[0357] Cyclobutyl sugar surrogate compounds can be prepared as is
described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate
can be prepared as is described in U.S. Pat. No. 5,519,134.
Morpholino sugar surrogates can be prepared as is described in U.S.
Pat. Nos. 5,142,047 and 5,235,033, and other related patent
disclosures. Peptide Nucleic Acids (PNAs) are known per se and can
be prepared in accordance with any of the various procedures
referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties
and Potential Applications, Bioorganic & Medicinal Chemistry,
1996, 4, 5-23. They may also be prepared in accordance with U.S.
Pat. No. 5,539,083.
Terminal Modification References
[0358] Terminal modifications are described in Manoharan, M. et al.
Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and
references therein.
Base References
[0359] N-2 substituted purine nucleoside amidites can be prepared
as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine
nucleoside amidites can be prepared as is described in U.S. Pat.
No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can
be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl
pyrimidine nucleoside amidites can be prepared as is described in
U.S. Pat. No. 5,484,908. Additional references can be disclosed in
the above section on base modifications.
Terminal Modification References
[0360] Terminal modifications are described in Manoharan, M. et al.
Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and
references therein.
Definitions
[0361] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine. The term "alkyl" refers to saturated and
unsaturated non-aromatic hydrocarbon chains that may be a straight
chain or branched chain, containing the indicated number of carbon
atoms (these include without limitation propyl, allyl, or
propargyl), which may be optionally inserted with N, O, or S. For
example, C.sub.1-C.sub.10 indicates that the group may have from 1
to 10 (inclusive) carbon atoms in it. The term "alkoxy" refers to
an --O-alkyl radical. The term "alkylene" refers to a divalent
alkyl (i.e., --R--). The term "alkylenedioxo" refers to a divalent
species of the structure --O--R--O--, in which R represents an
alkylene. The term "aminoalkyl" refers to an alkyl substituted with
an amino. The term "mercapto" refers to an --SH radical. The term
"thioalkoxy" refers to an --S-- alkyl radical.
[0362] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon
bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of
each ring may be substituted by a substituent. Examples of aryl
groups include phenyl, naphthyl and the like. The term "arylalkyl"
or the term "aralkyl" refers to alkyl substituted with an aryl. The
term "arylalkoxy" refers to an alkoxy substituted with aryl.
[0363] The term "cycloalkyl" as employed herein includes saturated
and partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, for example, 3 to 8 carbons, and, for example, 3 to 6
carbons, wherein the cycloalkyl group additionally may be
optionally substituted. Cycloalkyl groups include, without
limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,
cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
[0364] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be
substituted by a substituent. Examples of heteroaryl groups include
pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl,
thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the
like. The term "heteroarylalkyl" or the term "heteroaralkyl" refers
to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0365] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring may be
substituted by a substituent. Examples of heterocyclyl groups
include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl,
tetrahydrofuranyl, and the like.
[0366] The term "oxo" refers to an oxygen atom, which forms a
carbonyl when attached to carbon, an N-oxide when attached to
nitrogen, and a sulfoxide or sulfone when attached to sulfur.
[0367] The term "acyl" refers to an alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or
heteroarylcarbonyl substituent, any of which may be further
substituted by substituents.
[0368] The term "substituents" refers to a group "substituted" on
an alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at
any atom of that group. Suitable substituents include, without
limitation, halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl,
aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl,
arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,
alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,
aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido
groups.
Palindromes
[0369] The iRNA agents of the invention can target more than one
RNA region. For example, an iRNA agent can include a first and
second sequence that are sufficiently complementary to each other
to hybridize. The first sequence can be complementary to a first
target RNA region and the second sequence can be complementary to a
second target RNA region. The first and second sequences of the
iRNA agent can be on different RNA strands, and the mismatch
between the first and second sequences can be less than 50%, 40%,
30%, 20%, 10%, 5%, or 1%. The first and second sequences of the
iRNA agent are on the same RNA strand, and in a related embodiment
more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNA agent can
be in bimolecular form. The first and second sequences of the iRNA
agent can be fully complementary to each other.
[0370] The first target RNA region can be encoded by a first gene
and the second target RNA region can encoded by a second gene, or
the first and second target RNA regions can be different regions of
an RNA from a single gene. The first and second sequences can
differ by at least 1 nucleotide.
[0371] The first and second target RNA regions can be on
transcripts encoded by first and second sequence variants, e.g.,
first and second alleles, of a gene. The sequence variants can be
mutations, or polymorphisms, for example. The first target RNA
region can include a nucleotide substitution, insertion, or
deletion relative to the second target RNA region, or the second
target RNA region can a mutant or variant of the first target
region.
[0372] The first and second target RNA regions can comprise viral
or human RNA regions. The first and second target RNA regions can
also be on variant transcripts of an oncogene or include different
mutations of a tumor suppressor gene transcript. In addition, the
first and second target RNA regions can correspond to hot-spots for
genetic variation.
[0373] The compositions of the invention can include mixtures of
iRNA agent molecules. For example, one iRNA agent can contain a
first sequence and a second sequence sufficiently complementary to
each other to hybridize, and in addition the first sequence is
complementary to a first target RNA region and the second sequence
is complementary to a second target RNA region. The mixture can
also include at least one additional iRNA agent variety that
includes a third sequence and a fourth sequence sufficiently
complementary to each other to hybridize, and where the third
sequence is complementary to a third target RNA region and the
fourth sequence is complementary to a fourth target RNA region. In
addition, the first or second sequence can be sufficiently
complementary to the third or fourth sequence to be capable of
hybridizing to each other. The first and second sequences can be on
the same or different RNA strands, and the third and fourth
sequences can be on the same or different RNA strands.
[0374] The target RNA regions can be variant sequences of a viral
or human RNA, and in certain embodiments, at least two of the
target RNA regions can be on variant transcripts of an oncogene or
tumor suppressor gene. The target RNA regions can correspond to
genetic hot-spots.
[0375] Methods of making an iRNA agent composition can include
obtaining or providing information about a region of an RNA of a
target gene (e.g., a viral or human gene, or an oncogene or tumor
suppressor, e.g., p53), where the region has high variability or
mutational frequency (e.g., in humans). In addition, information
about a plurality of RNA targets within the region can be obtained
or provided, where each RNA target corresponds to a different
variant or mutant of the gene (e.g., a region including the codon
encoding p53 248Q and/or p53 249S). The iRNA agent can be
constructed such that a first sequence is complementary to a first
of the plurality of variant RNA targets (e.g., encoding 249Q) and a
second sequence is complementary to a second of the plurality of
variant RNA targets (e.g., encoding 249S), and the first and second
sequences can be sufficiently complementary to hybridize.
[0376] Sequence analysis, e.g., to identify common mutants in the
target gene, can be used to identify a region of the target gene
that has high variability or mutational frequency. A region of the
target gene having high variability or mutational frequency can be
identified by obtaining or providing genotype information about the
target gene from a population.
[0377] Expression of a target gene can be modulated, e.g.,
downregulated or silenced, by providing an iRNA agent that has a
first sequence and a second sequence sufficiently complementary to
each other to hybridize. In addition, the first sequence can be
complementary to a first target RNA region and the second sequence
can be complementary to a second target RNA region.
[0378] An iRNA agent can include a first sequence complementary to
a first variant RNA target region and a second sequence
complementary to a second variant RNA target region. The first and
second variant RNA target regions can correspond to first and
second variants or mutants of a target gene, e.g., viral gene,
tumor suppressor or oncogene. The first and second variant target
RNA regions can include allelic variants, mutations (e.g., point
mutations), or polymorphisms of the target gene. The first and
second variant RNA target regions can correspond to genetic
hot-spots.
[0379] A plurality of iRNA agents (e.g., a panel or bank) can be
provided.
Other Embodiments
[0380] In yet another embodiment, iRNAs agents are produced in a
cell in vivo, e.g., from exogenous DNA templates that are delivered
into the cell. For example, the DNA templates can be inserted into
vectors and used as gene therapy vectors. Gene therapy vectors can
be delivered to a subject by, for example, intravenous injection,
local administration (U.S. Pat. No. 5,328,470), or by stereotactic
injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA
91:3054-3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. The DNA templates, for example, can
include two transcription units, one that produces a transcript
that includes the top strand of a iRNA agent and one that produces
a transcript that includes the bottom strand of a iRNA agent. When
the templates are transcribed, the iRNA agent is produced, and
processed into siRNA agent fragments that mediate gene
silencing.
[0381] In certain embodiments, the nucleic acid may be modified
with a 2'-OMe modification (Kraynack & Baker, RNA, 12: 163-176,
2006). In other embodiments, the nucleic acid may comprise an
ethylene-bridged nucleic acid (ENA) (Morita et al., Nucleic Acids
Res., 2001, Suppl. 1, 241-242; Surono A et al., Human Gene Therapy,
15: 749-757, 2004).
MicroRNAs
[0382] MicroRNAs (miRNAs or mirs) are a highly conserved class of
small RNA molecules that are transcribed from DNA in the genomes of
plants and animals, but are not translated into protein.
Pre-microRNAs are processed into miRNAs. Processed microRNAs are
single stranded .about.17-25 nucleotide (nt) RNA molecules that
become incorporated into the RNA-induced silencing complex (RISC)
and have been identified as key regulators of development, cell
proliferation, apoptosis and differentiation. They are believed to
play a role in regulation of gene expression by binding to the
3'-untranslated region of specific mRNAs. RISC mediates
down-regulation of gene expression through translational
inhibition, transcript cleavage, or both. RISC is also implicated
in transcriptional silencing in the nucleus of a wide range of
eukaryotes.
[0383] The number of miRNA sequences identified to date is large
and growing, illustrative examples of which can be found, for
example, in: "miRBase: microRNA sequences, targets and gene
nomenclature" Griffiths-Jones S, Grocock R J, van Dongen S, Bateman
A, Enright A J. NAR, 2006, 34, Database Issue, D140-D144; "The
microRNA Registry" Griffiths-Jones S. NAR, 2004, 32, Database
Issue, D109-D111.
[0384] Single-stranded oligonucleotides, including those described
and/or identified as microRNAs or mirs which may be used as targets
or may serve as a template for the design of oligonucleotides of
the invention are taught in, for example, Esau, et al. US
Publication No. 20050261218 (U.S. Ser. No. 10/909,125) entitled
"Oligomeric compounds and compositions for use in modulation small
non-coding RNAs" the entire contents of which is incorporated
herein by reference. It will be appreciated by one of skill in the
art that any oligonucleotide chemical modifications or variations
describe herein also apply to single stranded oligonucleotides.
miRNA Mimics
[0385] miRNA mimics represent a class of molecules that can be used
to imitate the gene silencing ability of one or more miRNAs. Thus,
the term "microRNA mimic" refers to synthetic non-coding RNAs (i.e.
the miRNA is not obtained by purification from a source of the
endogenous miRNA) that are capable of entering the RNAi pathway and
regulating gene expression. miRNA mimics can be designed as mature
molecules (e.g. single stranded) or mimic precursors (e.g., pri- or
pre-miRNAs). miRNA mimics can be comprised of nucleic acid
(modified or modified nucleic acids) including oligonucleotides
comprising, without limitation, RNA, modified RNA, DNA, modified
DNA, locked nucleic acids, or 2'-O,4'-C-ethylene-bridged nucleic
acids (ENA), or any combination of the above (including DNA-RNA
hybrids). In addition, miRNA mimics can comprise conjugates that
can affect delivery, intracellular compartmentalization, stability,
specificity, functionality, strand usage, and/or potency. In one
design, miRNA mimics are double stranded molecules (e.g., with a
duplex region of between about 16 and about 31 nucleotides in
length) and contain one or more sequences that have identity with
the mature strand of a given miRNA. Modifications can comprise 2'
modifications (including 2'-O methyl modifications and 2' F
modifications) on one or both strands of the molecule and
internucleotide modifications (e.g. phosphorthioate modifications)
that enhance nucleic acid stability and/or specificity. In
addition, miRNA mimics can include overhangs. The overhangs can
consist of 1-6 nucleotides on either the 3' or 5' end of either
strand and can be modified to enhance stability or functionality.
In one embodiment, a miRNA mimic comprises a duplex region of
between 16 and 31 nucleotides and one or more of the following
chemical modification patterns: the sense strand contains
2'-O-methyl modifications of nucleotides 1 and 2 (counting from the
5' end of the sense oligonucleotide), and all of the Cs and Us; the
antisense strand modifications can comprise 2' F modification of
all of the Cs and Us, phosphorylation of the 5' end of the
oligonucleotide, and stabilized internucleotide linkages associated
with a 2 nucleotide 3' overhang.
Supermirs
[0386] A supermir refers to a single stranded, double stranded or
partially double stranded oligomer or polymer of ribonucleic acid
(RNA) or deoxyribonucleic acid (DNA) or both or modifications
thereof, which has a nucleotide sequence that is substantially
identical to an miRNA and that is antisense with respect to its
target. This term includes oligonucleotides composed of
naturally-occurring nucleobases, sugars and covalent
internucleoside (backbone) linkages and which contain at least one
non-naturally-occurring portion which functions similarly. Such
modified or substituted oligonucleotides are preferred over native
forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced affinity for nucleic acid target
and increased stability in the presence of nucleases. In a
preferred embodiment, the supermir does not include a sense strand,
and in another preferred embodiment, the supermir does not
self-hybridize to a significant extent. An supermir featured in the
invention can have secondary structure, but it is substantially
single-stranded under physiological conditions. An supermir that is
substantially single-stranded is single-stranded to the extent that
less than about 50% (e.g., less than about 40%, 30%, 20%, 10%, or
5%) of the supermir is duplexed with itself. The supermir can
include a hairpin segment, e.g., sequence, preferably at the 3' end
can self hybridize and form a duplex region, e.g., a duplex region
of at least 1, 2, 3, or 4 and preferably less than 8, 7, 6, or n
nucleotides, e.g., 5 nucleotides. The duplexed region can be
connected by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or
6 dTs, e.g., modified dTs. In another embodiment the supermir is
duplexed with a shorter oligo, e.g., of 5, 6, 7, 8, 9, or 10
nucleotides in length, e.g., at one or both of the 3' and 5' end or
at one end and in the non-terminal or middle of the supermir.
Antimir or miRNA Inhibitor
[0387] The terms "antimir" "microRNA inhibitor", "miR inhibitor",
or "inhibitor" are synonymous and refer to oligonucleotides or
modified oligonucleotides that interfere with the ability of
specific miRNAs. In general, the inhibitors are nucleic acid or
modified nucleic acids in nature including oligonucleotides
comprising RNA, modified RNA, DNA, modified DNA, locked nucleic
acids (LNAs), or any combination of the above. Modifications
include 2' modifications (including 2'-0 alkyl modifications and 2'
F modifications) and internucleotide modifications (e.g.
phosphorothioate modifications) that can affect delivery,
stability, specificity, intracellular compartmentalization, or
potency. In addition, miRNA inhibitors can comprise conjugates that
can affect delivery, intracellular compartmentalization, stability,
and/or potency. Inhibitors can adopt a variety of configurations
including single stranded, double stranded (RNA/RNA or RNA/DNA
duplexes), and hairpin designs, in general, microRNA inhibitors
comprise contain one or more sequences or portions of sequences
that are complementary or partially complementary with the mature
strand (or strands) of the miRNA to be targeted, in addition, the
miRNA inhibitor may also comprise additional sequences located 5'
and 3' to the sequence that is the reverse complement of the mature
miRNA. The additional sequences may be the reverse complements of
the sequences that are adjacent to the mature miRNA in the
pri-miRNA from which the mature miRNA is derived, or the additional
sequences may be arbitrary sequences (having a mixture of A, G, C,
or U). In some embodiments, one or both of the additional sequences
are arbitrary sequences capable of forming hairpins. Thus, in some
embodiments, the sequence that is the reverse complement of the
miRNA is flanked on the 5' side and on the 3' side by hairpin
structures. Micro-RNA inhibitors, when double stranded, may include
mismatches between nucleotides on opposite strands. Furthermore,
micro-RNA inhibitors may be linked to conjugate moieties in order
to facilitate uptake of the inhibitor into a cell. For example, a
micro-RNA inhibitor may be linked to cholesteryl
5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3 hydroxypentylcarbamate)
which allows passive uptake of a micro-RNA inhibitor into a cell.
Micro-RNA inhibitors, including hairpin miRNA inhibitors, are
described in detail in Vermeulen et al., "Double-Stranded Regions
Are Essential Design Components Of Potent Inhibitors of RISC
Function," RNA 13: 723-730 (2007) and in WO2007/095387 and WO
2008/036825 each of which is incorporated herein by reference in
its entirety. A person of ordinary skill in the art can select a
sequence from the database for a desired miRNA and design an
inhibitor useful for the methods disclosed herein.
U1 Adaptors
[0388] U1 adaptors inhibit polyA sites and are bifunctional
oligonucleotides with a target domain complementary to a site in
the target gene's terminal exon and a `U1 domain` that binds to the
U1 smaller nuclear RNA component of the U1 snRNP (Goraczniak, et
al., 2008, Nature Biotechnology, 27(3), 257-263, which is expressly
incorporated by reference herein, in its entirety). U1 snRNP is a
ribonucleoprotein complex that functions primarily to direct early
steps in spliceosome formation by binding to the pre-mRNA
exon-intron boundary (Brown and Simpson, 1998, Annu Rev Plant
Physiol Plant Mol Biol 49:77-95). Nucleotides 2-11 of the 5'end of
U1 snRNA base pair bind with the 5'ss of the pre mRNA. In one
embodiment, oligonucleotides of the invention are U1 adaptors. In
one embodiment, the U1 adaptor can be administered in combination
with at least one other iRNA agent.
Antagomirs
[0389] Antagomirs are RNA-like oligonucleotides that harbor various
modifications for RNAse protection and pharmacologic properties,
such as enhanced tissue and cellular uptake. They differ from
normal RNA by, for example, complete 2'-O-methylation of sugar,
phosphorothioate backbone and, for example, a cholesterol-moiety at
3'-end. Antagomirs may be used to efficiently silence endogenous
miRNAs by forming duplexes comprising the antagomir and endogenous
miRNA, thereby preventing miRNA-induced gene silencing. An example
of antagomir-mediated miRNA silencing is the silencing of miR-122,
described in Krutzfeldt et al, Nature, 2005, 438: 685-689, which is
expressly incorporated by reference herein, in its entirety.
Antagomir RNAs may be synthesized using standard solid phase
oligonucleotide synthesis protocols. See U.S. patent application
Ser. Nos. 11/502,158 and 11/657,341 (the disclosure of each of
which are incorporated herein by reference). An antagomir can
include ligand-conjugated monomer subunits and monomers for
oligonucleotide synthesis. Exemplary monomers are described in U.S.
application Ser. No. 10/916,185, filed on Aug. 10, 2004. An
antagomir can have a ZXY structure, such as is described in PCT
Application No. PCT/US2004/07070 filed on Mar. 8, 2004. An
antagomir can be complexed with an amphipathic moiety. Exemplary
amphipathic moieties for use with oligonucleotide agents are
described in PCT Application No. PCT/US2004/07070, filed on Mar. 8,
2004.
[0390] Antagomirs may be single stranded, double stranded,
partially double stranded or hairpin-structured, chemically
modified oligonucleotides that target a microRNA. An antagomir may
consist essentially of or comprise about 12 or more contiguous
nucleotides substantially complementary to an endogenous miRNA, and
more particularly, agents that include about 12 or more contiguous
nucleotides substantially complementary to a target sequence of an
miRNA or pre-miRNA nucleotide sequence. In certain embodiments, an
antagomir featured in the invention includes a nucleotide sequence
sufficiently complementary to hybridize to a miRNA target sequence
of about 12 to 25 nucleotides, in some instances about 15 to 23
nucleotides.
Decoy Oligonucleotides
[0391] Because transcription factors can recognize their relatively
short binding sequences, even in the absence of surrounding genomic
DNA, short oligonucleotides bearing the consensus binding sequence
of a specific transcription factor can be used as tools for
manipulating gene expression in living cells. This strategy
involves the intracellular delivery of such "decoy
oligonucleotides", which are then recognized and bound by the
target factor. Occupation of the transcription factor's DNA-binding
site by the decoy renders the transcription factor incapable of
subsequently binding to the promoter regions of target genes.
Decoys can be used as therapeutic agents, either to inhibit the
expression of genes that are activated by a transcription factor,
or to upregulate genes that are suppressed by the binding of a
transcription factor. Examples of the utilization of decoy
oligonucleotides may be found in Mann et al., J. Clin. Invest.,
2000, 106: 1071-1075, which is expressly incorporated by reference
herein, in its entirety.
[0392] An oligonucleotide agent featured in the invention can also
be a decoy nucleic acid, e.g., a decoy RNA. A decoy nucleic acid
resembles a natural nucleic acid, but may be modified in such a way
as to inhibit or interrupt the activity of the natural nucleic
acid. For example, a decoy RNA can mimic the natural binding domain
for a ligand. The decoy RNA, therefore, competes with natural
binding domain for the binding of a specific ligand. The natural
binding target can be an endogenous nucleic acid, e.g., a
pre-miRNA, miRNA, pre-mRNA, mRNA or DNA. For example, it has been
shown that over-expression of HIV trans-activation response (TAR)
RNA can act as a "decoy" and efficiently bind HIV tat protein,
thereby preventing it from binding to TAR sequences encoded in the
HIV RNA. In certain embodiments, a decoy RNA may include a
modification that improves targeting, e.g., a targeting
modification described herein.
Antisense Oligonucleotides
[0393] Antisense oligonucleotides are single strands of DNA or RNA
that are at least partially complementary to a chosen sequence. In
the case of antisense RNA, they prevent translation of
complementary RNA strands by binding to it. Antisense DNA can also
be used to target a specific, complementary (coding or non-coding)
RNA. If binding takes place, the DNA/RNA hybrid can be degraded by
the enzyme RNase H. Examples of the utilization of antisense
oligonucleotides may be found in Dias et al., Mol. Cancer Ther.,
2002, 1: 347-355, which is expressly incorporated by reference
herein, in its entirety.
[0394] The single-stranded oligonucleotide agents featured in the
invention include antisense nucleic acids. An "antisense" nucleic
acid includes a nucleotide sequence that is complementary to a
"sense" nucleic acid encoding a gene expression product, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an RNA sequence, e.g., a pre-mRNA,
mRNA, miRNA, or pre-miRNA. Accordingly, an antisense nucleic acid
may form hydrogen bonds with a sense nucleic acid target.
[0395] Given a coding strand sequence (e.g., the sequence of a
sense strand of a cDNA molecule), antisense nucleic acids can be
designed according to the rules of Watson and Crick base pairing.
The antisense nucleic acid molecule can be complementary to a
portion of the coding or noncoding region of an RNA, e.g., a
pre-mRNA or mRNA. For example, the antisense oligonucleotide can be
complementary to the region surrounding the translation start site
of a pre-mRNA or mRNA, e.g., the 5' UTR. An antisense
oligonucleotide can be, for example, about 10 to 25 nucleotides in
length (e.g., about 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23,
or 24 nucleotides in length). An antisense oligonucleotide can also
be complementary to a miRNA or pre-miRNA.
[0396] In certain embodiments, an antisense nucleic acid can be
constructed using chemical synthesis and/or enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and target nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. Other appropriate nucleic acid
modifications are described herein. Alternatively, the antisense
nucleic acid can be produced biologically using an expression
vector into which a nucleic acid has been subcloned in an antisense
orientation (i.e., RNA transcribed from the inserted nucleic acid
will be of an antisense orientation to a target nucleic acid of
interest).
[0397] An antisense agent can include ribonucleotides only,
deoxyribonucleotides only (e.g., oligodeoxynucleotides), or both
deoxyribonucleotides and ribonucleotides. For example, an antisense
agent consisting only of ribonucleotides can hybridize to a
complementary RNA, and prevent access of the translation machinery
to the target RNA transcript, thereby preventing protein synthesis.
An antisense molecule including only deoxyribonucleotides, or
deoxyribonucleotides and ribonucleotides, e.g., DNA sequence
flanked by RNA sequence at the 5' and 3' ends of the antisense
agent, can hybridize to a complementary RNA, and the RNA target can
be subsequently cleaved by an enzyme, e.g., RNAse H. Degradation of
the target RNA prevents translation. The flanking RNA sequences can
include 2'-O-methylated nucleotides, and phosphorothioate linkages,
and the internal DNA sequence can include phosphorothioate
internucleotide linkages. In some embodiments, the internal DNA
sequence may be at least five nucleotides in length when targeting
by RNAseH activity is desired.
[0398] For increased nuclease resistance, an antisense agent can be
further modified by inverting the nucleoside at the 3'-terminus
with a 3'-3' linkage. In another alternative, the 3'-terminus can
be blocked with an aminoalkyl group.
[0399] In other embodiments, an antisense oligonucleotide agent may
include a modification that improves targeting, e.g., a targeting
modification described herein.
Aptamers
[0400] Aptamers are nucleic acid molecules that bind a specific
target molecule or molecules. Aptamers may be RNA or DNA based, and
may include a riboswitch. A riboswitch is a part of an mRNA
molecule that can directly bind a small target molecule, and whose
binding of the target affects the gene's activity. Thus, an mRNA
that contains a riboswitch is directly involved in regulating its
own activity, depending on the presence or absence of its target
molecule.
[0401] An oligonucleotide agent featured in the invention can be an
aptamer. An aptamer binds to a non-nucleic acid ligand, such as a
small organic molecule or protein, e.g., a transcription or
translation factor, and subsequently modifies (e.g., inhibits)
activity. An aptamer can fold into a specific structure that
directs the recognition of the targeted binding site on the
non-nucleic acid ligand. An aptamer can contain any of the
modifications described herein.
[0402] Ribozymes are oligonucleotides having specific catalytic
domains that possess endonuclease activity (Kim and Cech, Proc Natl
Acad Sci USA. 1987 December; 84(24):8788-92; Forster and Symons,
Cell. 1987 Apr. 24; 49(2):211-20). At least six basic varieties of
naturally-occurring enzymatic RNAs are known presently. In general,
enzymatic nucleic acids act by first binding to a target RNA. Such
binding occurs through the target binding portion of an enzymatic
nucleic acid which is held in close proximity to an enzymatic
portion of the molecule that acts to cleave the target RNA. Thus,
the enzymatic nucleic acid first recognizes and then binds a target
RNA through complementary base-pairing, and once bound to the
correct site, acts enzymatically to cut the target RNA. Strategic
cleavage of such a target RNA will destroy its ability to direct
synthesis of an encoded protein. After an enzymatic nucleic acid
has bound and cleaved its RNA target, it is released from that RNA
to search for another target and can repeatedly bind and cleave new
targets.
[0403] Methods of producing a ribozyme targeted to any target
sequence are known in the art. Ribozymes may be designed as
described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat.
Appl. Publ. No. WO 94/02595, each specifically incorporated herein
by reference, and synthesized to be tested in vitro and in vivo, as
described therein.
Physiological Effects
[0404] The iRNA agents described herein can be designed such that
determining therapeutic toxicity is made easier by the
complementarity of the iRNA agent with both a human and a non-human
animal sequence. By these methods, an iRNA agent can consist of a
sequence that is fully complementary to a nucleic acid sequence
from a human and a nucleic acid sequence from at least one
non-human animal, e.g., a non-human mammal, such as a rodent,
ruminant or primate. For example, the non-human mammal can be a
mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus, Pan
troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence of
the iRNA agent could be complementary to sequences within
homologous genes, e.g., oncogenes or tumor suppressor genes, of the
non-human mammal and the human. By determining the toxicity of the
iRNA agent in the non-human mammal, one can extrapolate the
toxicity of the iRNA agent in a human. For a more strenuous
toxicity test, the iRNA agent can be complementary to a human and
more than one, e.g., two or three or more, non-human animals.
[0405] The methods described herein can be used to correlate any
physiological effect of an iRNA agent on a human, e.g., any
unwanted effect, such as a toxic effect, or any positive, or
desired effect.
Increasing Cellular Uptake of dsiRNAs
[0406] A method of the invention that includes administering an
iRNA agent and a drug that affects the uptake of the iRNA agent
into the cell. The drug can be administered before, after, or at
the same time that the iRNA agent is administered. The drug can be
covalently linked to the iRNA agent. The drug can be, for example,
a lipopolysaccharide, an activator of p38 MAP kinase, or an
activator of NF-.kappa.B. The drug can have a transient effect on
the cell.
[0407] The drug can increase the uptake of the iRNA agent into the
cell, for example, by disrupting the cell's cytoskeleton, e.g., by
disrupting the cell's microtubules, microfilaments, and/or
intermediate filaments. The drug can be, for example, taxon,
vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide,
latrunculin A, phalloidin, swinholide A, indanocine, or
myoservin.
[0408] The drug can also increase the uptake of the iRNA agent into
the cell by activating an inflammatory response, for example.
Exemplary drug's that would have such an effect include tumor
necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma
interferon.
iRNA Conjugates
[0409] An iRNA agent can be coupled, e.g., covalently coupled, to a
second agent. For example, an iRNA agent used to treat a particular
disorder can be coupled to a second therapeutic agent, e.g., an
agent other than the iRNA agent. The second therapeutic agent can
be one which is directed to the treatment of the same disorder. For
example, in the case of an iRNA used to treat a disorder
characterized by unwanted cell proliferation, e.g., cancer, the
iRNA agent can be coupled to a second agent which has an
anti-cancer effect. For example, it can be coupled to an agent
which stimulates the immune system, e.g., a CpG motif, or more
generally an agent that activates a toll-like receptor and/or
increases the production of gamma interferon.
iRNA Production
[0410] An iRNA can be produced, e.g., in bulk, by a variety of
methods. Exemplary methods include: organic synthesis and RNA
cleavage, e.g., in vitro cleavage. Organic synthesis offers the
advantage that the oligonucleotide strands comprising non-natural
or modified nucleotides can be easily prepared. Any other means for
such synthesis known in the art may additionally or alternatively
be employed. It is also known to use similar techniques to prepare
other oligonucleotides, such as the phosphorothioates,
phosphorodithioates and alkylated derivatives.
[0411] Regardless of the method of synthesis, the oligonucleotide
can be prepared in a solution (e.g., an aqueous and/or organic
solution) that is appropriate for formulation. For example, the
oligonucleotide preparation can be precipitated and redissolved in
pure double-distilled water, and lyophilized. The dried
oligonucleotide can then be resuspended in a solution appropriate
for the intended formulation process.
[0412] Teachings regarding the synthesis of particular modified
oligonucleotides may 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 may 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. No. 5,223,168,
and U.S. Pat. No. 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.
Organic Synthesis
[0413] An iRNA can be made by separately synthesizing each
respective strand of a double-stranded RNA molecule. The component
strands can then be annealed.
[0414] A large bioreactor, e.g., the OligoPilot II from Pharmacia
Biotec AB (Uppsala Sweden), can be used to produce a large amount
of a particular RNA strand for a given iRNA. The OligoPilotII
reactor can efficiently couple a nucleotide using only a 1.5 molar
excess of a phosphoramidite nucleotide. To make an RNA strand,
ribonucleotides amidites are used. Standard cycles of monomer
addition can be used to synthesize the 21 to 23 nucleotide strand
for the iRNA. Typically, the two complementary strands are produced
separately and then annealed, e.g., after release from the solid
support and deprotection.
[0415] Organic synthesis can be used to produce a discrete iRNA
species. The complementary of the species to a particular target
gene can be precisely specified. For example, the species may be
complementary to a region that includes a polymorphism, e.g., a
single nucleotide polymorphism. Further the location of the
polymorphism can be precisely defined. In some embodiments, the
polymorphism is located in an internal region, e.g., at least 4, 5,
7, or 9 nucleotides from one or both of the termini.
dsiRNA Cleavage
[0416] iRNAs can also be made by cleaving a larger ds iRNA. The
cleavage can be mediated in vitro or in vivo. For example, to
produce iRNAs by cleavage in vitro, the following method can be
used:
[0417] In vitro transcription. dsiRNA is produced by transcribing a
nucleic acid (DNA) segment in both directions. For example, the
HiScribe.TM. RNAi transcription kit (New England Biolabs) provides
a vector and a method for producing a dsiRNA for a nucleic acid
segment that is cloned into the vector at a position flanked on
either side by a T7 promoter. Separate templates are generated for
T7 transcription of the two complementary strands for the dsiRNA.
The templates are transcribed in vitro by addition of T7 RNA
polymerase and dsiRNA is produced. Similar methods using PCR and/or
other RNA polymerases (e.g., T3 or SP6 polymerase) can also be
used. In one embodiment, RNA generated by this method is carefully
purified to remove endotoxins that may contaminate preparations of
the recombinant enzymes.
[0418] In vitro cleavage. dsiRNA is cleaved in vitro into iRNAs,
for example, using a Dicer or comparable RNAse III-based activity.
For example, the dsiRNA can be incubated in an in vitro extract
from Drosophila or using purified components, e.g., a purified
RNAse or RISC complex (RNA-induced silencing complex). See, e.g.,
Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9. and Hammond
Science 2001 Aug. 10; 293(5532):1146-50.
[0419] dsiRNA cleavage generally produces a plurality of iRNA
species, each being a particular 21 to 23 nt fragment of a source
dsiRNA molecule. For example, iRNAs that include sequences
complementary to overlapping regions and adjacent regions of a
source dsiRNA molecule may be present.
[0420] Regardless of the method of synthesis, the iRNA preparation
can be prepared in a solution (e.g., an aqueous and/or organic
solution) that is appropriate for formulation. For example, the
iRNA preparation can be precipitated and redissolved in pure
double-distilled water, and lyophilized. The dried iRNA can then be
resuspended in a solution appropriate for the intended formulation
process.
Formulation
[0421] The iRNA agents described herein can be formulated for
administration to a subject
[0422] For ease of exposition the formulations, compositions and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It may be understood, however, that these
formulations, compositions and methods can be practiced with other
iRNA agents, e.g., modified iRNA agents, and such practice is
within the invention.
[0423] A formulated iRNA composition can assume a variety of
states. In some examples, the composition is at least partially
crystalline, uniformly crystalline, and/or anhydrous (e.g., less
than 80, 50, 30, 20, or 10% water). In another example, the iRNA is
in an aqueous phase, e.g., in a solution that includes water.
[0424] The aqueous phase or the crystalline compositions can, e.g.,
be incorporated into a delivery vehicle, e.g., a liposome
(particularly for the aqueous phase) or a particle (e.g., a
microparticle as can be appropriate for a crystalline composition).
Generally, the iRNA composition is formulated in a manner that is
compatible with the intended method of administration (see,
below).
[0425] In particular embodiments, the composition is prepared by at
least one of the following methods: spray drying, lyophilization,
vacuum drying, evaporation, fluid bed drying, or a combination of
these techniques; or sonication with a lipid, freeze-drying,
condensation and other self-assembly.
[0426] A iRNA preparation can be formulated in combination with
another agent, e.g., another therapeutic agent or an agent that
stabilizes a iRNA, e.g., a protein that complexes with iRNA to form
an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to
remove divalent cations such as Mg.sup.2+), salts, RNAse inhibitors
(e.g., a broad specificity RNAse inhibitor such as RNAsin) and so
forth.
[0427] In one embodiment, the iRNA preparation includes another
iRNA agent, e.g., a second iRNA that can mediated RNAi with respect
to a second gene, or with respect to the same gene. Still other
preparation can include at least 3, 5, ten, twenty, fifty, or a
hundred or more different iRNA species. Such iRNAs can mediated
RNAi with respect to a similar number of different genes.
[0428] In one embodiment, the iRNA preparation includes at least a
second therapeutic agent (e.g., an agent other than an RNA or a
DNA). For example, a iRNA composition for the treatment of a viral
disease, e.g., HIV, might include a known antiviral agent (e.g., a
protease inhibitor or reverse transcriptase inhibitor). In another
example, a iRNA composition for the treatment of a cancer might
further comprise a chemotherapeutic agent.
[0429] Exemplary formulations are discussed below:
Liposomes
[0430] For ease of exposition the formulations, compositions and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It may be understood, however, that these
formulations, compositions and methods can be practiced with other
iRNA agents, e.g., modified iRNA s agents, and such practice is
within the invention. An iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof) preparation can be formulated for
delivery in a membranous molecular assembly, e.g., a liposome or a
micelle. As used herein, the term "liposome" refers to a vesicle
composed of amphiphilic lipids arranged in at least one bilayer,
e.g., one bilayer or a plurality of bilayers. Liposomes include
unilamellar and multilamellar vesicles that have a membrane formed
from a lipophilic material and an aqueous interior. The aqueous
portion contains the iRNA composition. The lipophilic material
isolates the aqueous interior from an aqueous exterior, which
typically does not include the iRNA composition, although in some
examples, it may. 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 liposomal bilayer fuses
with bilayer of the cellular membranes. As the merging of the
liposome and cell progresses, the internal aqueous contents that
include the iRNA are delivered into the cell where the iRNA can
specifically bind to a target RNA and can mediate RNAi. In some
cases the liposomes are also specifically targeted, e.g., to direct
the iRNA to particular cell types.
[0431] A liposome containing a iRNA can be prepared by a variety of
methods.
[0432] In one example, the lipid component of a liposome is
dissolved in a detergent so that micelles are formed with the lipid
component. For example, the lipid component can be an amphipathic
cationic lipid or lipid conjugate. The detergent can have a high
critical micelle concentration and may be nonionic. Exemplary
detergents include cholate, CHAPS, octylglucoside, deoxycholate,
and lauroyl sarcosine. The iRNA preparation is then added to the
micelles that include the lipid component. The cationic groups on
the lipid interact with the iRNA and condense around the iRNA to
form a liposome. After condensation, the detergent is removed,
e.g., by dialysis, to yield a liposomal preparation of iRNA.
[0433] If necessary a carrier compound that assists in condensation
can be added during the condensation reaction, e.g., by controlled
addition. For example, the carrier compound can be a polymer other
than a nucleic acid (e.g., spermine or spermidine). pH can also
adjusted to favor condensation.
[0434] Further description of methods for producing stable
polynucleotide delivery vehicles, which incorporate a
polynucleotide/cationic lipid complex as structural components of
the delivery vehicle, are described in, e.g., WO 96/37194. Liposome
formation can also include one or more aspects of exemplary methods
described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA
8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No.
5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et
al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl.
Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta
775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983;
and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used
techniques for preparing lipid aggregates of appropriate size for
use as delivery vehicles include sonication and freeze-thaw plus
extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161,
1986). Microfluidization can be used when consistently small (50 to
200 nm) and relatively uniform aggregates are desired (Mayhew, et
al. Biochim. Biophys. Acta 775:169, 1984). These methods are
readily adapted to packaging iRNA preparations into liposomes.
[0435] Liposomes that are pH-sensitive or negatively-charged,
entrap nucleic acid molecules rather than complex with them. Since
both the nucleic acid molecules and the lipid are similarly
charged, repulsion rather than complex formation occurs.
Nevertheless, some nucleic acid molecules are 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, 19, (1992) 269-274).
[0436] 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.
[0437] Examples of other methods to introduce liposomes into cells
in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No.
5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol.
Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993;
Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143,
1993; and Strauss EMBO J. 11:417, 1992.
[0438] In one embodiment, cationic liposomes are used. Cationic
liposomes possess the advantage of being able to fuse to the cell
membrane. Non-cationic liposomes, although not able to fuse as
efficiently with the plasma membrane, are taken up by macrophages
in vivo and can be used to deliver iRNAs to macrophages.
[0439] 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 iRNAs in their internal
compartments from metabolism and degradation (Rosoff, in
"Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.),
1988, 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.
[0440] A positively charged synthetic cationic lipid,
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA) can be used to form small liposomes that interact
spontaneously with nucleic acid to form lipid-nucleic acid
complexes which are capable of fusing with the negatively charged
lipids of the cell membranes of tissue culture cells, resulting in
delivery of iRNA (see, e.g., Felgner, P. L. et al., Proc. Natl.
Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a
description of DOTMA and its use with DNA).
[0441] A DOTMA analogue,
1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used
in combination with a phospholipid to form DNA-complexing vesicles.
Lipofectin.TM. Bethesda Research Laboratories, Gaithersburg, Md.)
is an effective agent for the delivery of highly anionic nucleic
acids into living tissue culture cells that comprise positively
charged DOTMA liposomes which interact spontaneously with
negatively charged polynucleotides to form complexes. When enough
positively charged liposomes are used, the net charge on the
resulting complexes is also positive. Positively charged complexes
prepared in this way spontaneously attach to negatively charged
cell surfaces, fuse with the plasma membrane, and efficiently
deliver functional nucleic acids into, for example, tissue culture
cells. Another commercially available cationic lipid,
1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP")
(Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in
that the oleoyl moieties are linked by ester, rather than ether
linkages.
[0442] Other reported cationic lipid compounds include those that
have been conjugated to a variety of moieties including, for
example, carboxyspermine which has been conjugated to one of two
types of lipids and includes compounds such as
5-carboxyspermylglycine dioctaoleoylamide ("DOGS")
(Transfectam.TM., Promega, Madison, Wis.) and
dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide
("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).
[0443] Another cationic lipid conjugate includes derivatization of
the lipid with cholesterol ("DC-Chol") which has been formulated
into liposomes in combination with DOPE (See, Gao, X. and Huang,
L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine,
made by conjugating polylysine to DOPE, has been reported to be
effective for transfection in the presence of serum (Zhou, X. et
al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines,
these liposomes containing conjugated cationic lipids, are said to
exhibit lower toxicity and provide more efficient transfection than
the DOTMA-containing compositions. Other commercially available
cationic lipid products include DMRIE and DMRIE-HP (Vical, La
Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc.,
Gaithersburg, Md.). Other cationic lipids suitable for the delivery
of oligonucleotides are described in WO 98/39359 and WO 96/37194.
Other cationic lipids suitable for liposome formation are described
in U.S. Provisional applications No. 61/018,616 (filed Jan. 2,
2008), No. 61/039,748 (filed Mar. 26, 2008), No. 61/047,087 (filed
Apr. 22, 2008) and No. 61/051,528 (filed May 21-2008), all of which
are incorporated by reference in their entireties for all
purposes.
[0444] Liposomal formulations are particularly suited 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 iRNA, into the skin. In some
implementations, liposomes are used for delivering iRNA to
epidermal cells and also to enhance the penetration of iRNA into
dermal tissues, e.g., into skin. For example, the liposomes can be
applied topically. Topical delivery of drugs formulated as
liposomes to the skin has been documented (see, e.g., Weiner et
al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du
Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R.
J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T.
et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz.
149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.
Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl.
Acad. Sci. USA 84:7851-7855, 1987).
[0445] 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 I (glyceryl
dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and
Novasome II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver a drug into the dermis of mouse skin. Such formulations
with iRNA are useful for treating a dermatological disorder.
[0446] Liposomes that include iRNA can be made highly deformable.
Such deformability can enable the liposomes to penetrate through
pore that are smaller than the average radius of the liposome. For
example, transfersomes are a type of deformable liposomes.
Transferosomes can be made by adding surface edge activators,
usually surfactants, to a standard liposomal composition.
Transfersomes that include iRNA can be delivered, for example,
subcutaneously by infection in order to deliver iRNA to
keratinocytes in the skin. In order to cross intact mammalian skin,
lipid vesicles must pass through a series of fine pores, each with
a diameter less than 50 nm, under the influence of a suitable
transdermal gradient. In addition, due to the lipid properties,
these transferosomes can be self-optimizing (adaptive to the shape
of pores, e.g., in the skin), self-repairing, and can frequently
reach their targets without fragmenting, and often
self-loading.
Surfactants
[0447] For ease of exposition the formulations, compositions and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It may be understood, however, that these
formulations, compositions and methods can be practiced with other
iRNA agents, e.g., modified iRNA agents, and such practice is
within the invention. Surfactants find wide application in
formulations such as emulsions (including microemulsions) and
liposomes (see above). iRNA (or a precursor, e.g., a larger dsiRNA
which can be processed into a iRNA, or a DNA which encodes a iRNA
or precursor) compositions can include a surfactant. In one
embodiment, the iRNA is formulated as an emulsion that includes a
surfactant. 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 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).
[0448] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical 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.
[0449] 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.
[0450] 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.
[0451] 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.
[0452] 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).
Micelles and Other Membranous Formulations
[0453] For ease of exposition the micelles and other formulations,
compositions and methods in this section are discussed largely with
regard to unmodified iRNA agents. It may be understood, however,
that these micelles and other formulations, compositions and
methods can be practiced with other iRNA agents, e.g., modified
iRNA agents, and such practice is within the invention. The iRNA
agent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a
precursor, e.g., a larger iRNA agent which can be processed into a
siRNA agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof))
composition can be provided as a micellar formulation. "Micelles"
are defined herein as a particular type of molecular assembly in
which amphipathic molecules are arranged in a spherical structure
such that all the hydrophobic portions of 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.
[0454] A mixed micellar formulation suitable for delivery through
transdermal membranes may be prepared by mixing an aqueous solution
of the iRNA composition, an alkali metal C.sub.8 to C.sub.22 alkyl
sulphate, and a micelle forming compounds. Exemplary micelle
forming compounds include lecithin, hyaluronic acid,
pharmaceutically acceptable salts of hyaluronic acid, glycolic
acid, lactic acid, chamomile extract, cucumber extract, oleic acid,
linoleic acid, linoleic acid, monoolein, monooleates, monolaurates,
borage oil, evening of primrose oil, menthol, trihydroxy oxo
cholanyl glycine and pharmaceutically acceptable salts thereof,
glycerin, polyglycerin, lysine, polylysine, triolein,
polyoxyethylene ethers and analogues thereof, polidocanol alkyl
ethers and analogues thereof, chenodeoxycholate, deoxycholate, and
mixtures thereof. The micelle forming compounds may be added at the
same time or after addition of the alkali metal alkyl sulphate.
Mixed micelles will form with substantially any kind of mixing of
the ingredients but vigorous mixing in order to provide smaller
size micelles.
[0455] In one method a first micellar composition is prepared which
contains the iRNA composition and at least the alkali metal alkyl
sulphate. The first micellar composition is then mixed with at
least three micelle forming compounds to form a mixed micellar
composition. In another method, the micellar composition is
prepared by mixing the iRNA composition, the alkali metal alkyl
sulphate and at least one of the micelle forming compounds,
followed by addition of the remaining micelle forming compounds,
with vigorous mixing.
[0456] Phenol and/or m-cresol may be added to the mixed micellar
composition to stabilize the formulation and protect against
bacterial growth. Alternatively, phenol and/or m-cresol may be
added with the micelle forming ingredients. An isotonic agent such
as glycerin may also be added after formation of the mixed micellar
composition.
[0457] For delivery of the micellar formulation as a spray, the
formulation can be put into an aerosol dispenser and the dispenser
is charged with a propellant. The propellant, which is under
pressure, is in liquid form in the dispenser. The ratios of the
ingredients are adjusted so that the aqueous and propellant phases
become one, i.e., there is one phase. If there are two phases, it
is necessary to shake the dispenser prior to dispensing a portion
of the contents, e.g., through a metered valve. The dispensed dose
of pharmaceutical agent is propelled from the metered valve in a
fine spray.
[0458] Propellants may include hydrogen-containing
chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl
ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2
tetrafluoroethane) may be used.
[0459] The specific concentrations of the essential ingredients can
be determined by relatively straightforward experimentation. For
absorption through the oral cavities, it is often desirable to
increase, e.g., at least double or triple, the dosage for through
injection or administration through the gastrointestinal tract.
Particles
[0460] For ease of exposition the particles, formulations,
compositions and methods in this section are discussed largely with
regard to unmodified iRNA agents. It may be understood, however,
that these particles, formulations, compositions and methods can be
practiced with other iRNA agents, e.g., modified iRNA agents, and
such practice is within the invention. In another embodiment, an
iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent,
(e.g., a precursor, e.g., a larger iRNA agent which can be
processed into a siRNA agent, or a DNA which encodes an iRNA agent,
e.g., a double-stranded iRNA agent, or siRNA agent, or precursor
thereof) preparations may be incorporated into a particle, e.g., a
microparticle. Microparticles can be produced by spray-drying, but
may also be produced by other methods including lyophilization,
evaporation, fluid bed drying, vacuum drying, or a combination of
these techniques. See below for further description.
[0461] Sustained-Release Formulations. An iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, (e.g., a precursor,
e.g., a larger iRNA agent which can be processed into a siRNA
agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof)
described herein can be formulated for controlled, e.g., slow
release. Controlled release can be achieved by disposing the iRNA
within a structure or substance which impedes its release. E.g.,
iRNA can be disposed within a porous matrix or in an erodable
matrix, either of which allow release of the iRNA over a period of
time.
[0462] Polymeric particles, e.g., polymeric in microparticles can
be used as a sustained-release reservoir of iRNA that is taken up
by cells only released from the microparticle through
biodegradation. The polymeric particles in this embodiment should
therefore be large enough to preclude phagocytosis (e.g., larger
than 10 .mu.m or larger than 20 .mu.m). Such particles can be
produced by the same methods to make smaller particles, but with
less vigorous mixing of the first and second emulsions. That is to
say, a lower homogenization speed, vortex mixing speed, or
sonication setting can be used to obtain particles having a
diameter around 100 .mu.m rather than 10 .mu.m. The time of mixing
also can be altered.
[0463] Larger microparticles can be formulated as a suspension, a
powder, or an implantable solid, to be delivered by intramuscular,
subcutaneous, intradermal, intravenous, or intraperitoneal
injection; via inhalation (intranasal or intrapulmonary); orally;
or by implantation. These particles are useful for delivery of any
iRNA when slow release over a relatively long term is desired. The
rate of degradation, and consequently of release, varies with the
polymeric formulation.
[0464] Microparticles may include pores, voids, hollows, defects or
other interstitial spaces that allow the fluid suspension medium to
freely permeate or perfuse the particulate boundary. For example,
the perforated microstructures can be used to form hollow, porous
spray dried microspheres.
[0465] Polymeric particles containing iRNA (e.g., a siRNA) can be
made using a double emulsion technique, for instance. First, the
polymer is dissolved in an organic solvent. A polymer may be
polylactic-co-glycolic acid (PLGA), with a lactic/glycolic acid
weight ratio of 65:35, 50:50, or 75:25. Next, a sample of nucleic
acid suspended in aqueous solution is added to the polymer solution
and the two solutions are mixed to form a first emulsion. The
solutions can be mixed by vortexing or shaking, and in the mixture
can be sonicated. Any method by which the nucleic acid receives the
least amount of damage in the form of nicking, shearing, or
degradation, while still allowing the formation of an appropriate
emulsion is possible. For example, acceptable results can be
obtained with a Vibra-cell model VC-250 sonicator with a 1/8''
microtip probe, at setting #3.
Spray Drying
[0466] An iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, (e.g., a precursor, e.g., a larger iRNA agent which can be
processed into a siRNA agent, or a DNA which encodes an iRNA agent,
e.g., a double-stranded iRNA agent, or siRNA agent, or precursor
thereof)) can be prepared by spray drying. Spray dried iRNA can be
administered to a subject or be subjected to further formulation. A
pharmaceutical composition of iRNA can be prepared by spray drying
a homogeneous aqueous mixture that includes a iRNA under conditions
sufficient to provide a dispersible powdered composition, e.g., a
pharmaceutical composition. The material for spray drying can also
include one or more of: a pharmaceutically acceptable excipient, or
a dispersibility-enhancing amount of a physiologically acceptable,
water-soluble protein. The spray-dried product can be a dispersible
powder that includes the iRNA.
[0467] Spray drying is a process that converts a liquid or slurry
material to a dried particulate form. Spray drying can be used to
provide powdered material for various administrative routes
including inhalation. See, for example, M. Sacchetti and M. M. Van
Oort in: Inhalation Aerosols: Physical and Biological Basis for
Therapy, A. J. Hickey, ed. Marcel Dekkar, New York, 1996.
[0468] Spray drying can include atomizing a solution, emulsion, or
suspension to form a fine mist of droplets and drying the droplets.
The mist can be projected into a drying chamber (e.g., a vessel,
tank, tubing, or coil) where it contacts a drying gas. The mist can
include solid or liquid pore forming agents. The solvent and pore
forming agents evaporate from the droplets into the drying gas to
solidify the droplets, simultaneously forming pores throughout the
solid. The solid (typically in a powder, particulate form) then is
separated from the drying gas and collected.
[0469] Spray drying includes bringing together a highly dispersed
liquid, and a sufficient volume of air (e.g., hot air) to produce
evaporation and drying of the liquid droplets. The preparation to
be spray dried can be any solution, course suspension, slurry,
colloidal dispersion, or paste that may be atomized using the
selected spray drying apparatus. Typically, the feed is sprayed
into a current of warm filtered air that evaporates the solvent and
conveys the dried product to a collector. The spent air is then
exhausted with the solvent. Several different types of apparatus
may be used to provide the desired product. For example, commercial
spray dryers manufactured by Buchi Ltd. or Niro Corp. can
effectively produce particles of desired size.
[0470] Spray-dried powdered particles can be approximately
spherical in shape, nearly uniform in size and frequently hollow.
There may be some degree of irregularity in shape depending upon
the incorporated medicament and the spray drying conditions. In
many instances the dispersion stability of spray-dried microspheres
appears to be more effective if an inflating agent (or blowing
agent) is used in their production. Certain embodiments may
comprise an emulsion with an inflating agent as the disperse or
continuous phase (the other phase being aqueous in nature). An
inflating agent may be dispersed with a surfactant solution, using,
for instance, a commercially available microfluidizer at a pressure
of about 5000 to 15,000 psi. This process forms an emulsion, which
may be stabilized by an incorporated surfactant, typically
comprising submicron droplets of water immiscible blowing agent
dispersed in an aqueous continuous phase. The formation of such
dispersions using this and other techniques are common and well
known to those in the art. The blowing agent may be a fluorinated
compound (e.g., perfluorohexane, perfluorooctyl bromide,
perfluorodecalin, perfluorobutyl ethane) which vaporizes during the
spray-drying process, leaving behind generally hollow, porous
aerodynamically light microspheres. As will be discussed in more
detail below, other suitable blowing agents include chloroform,
freons, and hydrocarbons. Nitrogen gas and carbon dioxide are also
contemplated as a suitable blowing agent.
[0471] Although the perforated microstructures may be formed using
a blowing agent as described above, it will be appreciated that, in
some instances, no blowing agent is required and an aqueous
dispersion of the medicament and surfactant(s) are spray dried
directly. In such cases, the formulation may be amenable to process
conditions (e.g., elevated temperatures) that generally lead to the
formation of hollow, relatively porous microparticles. Moreover,
the medicament may possess special physicochemical properties
(e.g., high crystallinity, elevated melting temperature, surface
activity, etc.) that make it particularly suitable for use in such
techniques.
[0472] The perforated microstructures may optionally be associated
with, or comprise, one or more surfactants. Moreover, miscible
surfactants may optionally be combined with the suspension medium
liquid phase. It will be appreciated by those skilled in the art
that the use of surfactants may further increase dispersion
stability, simplify formulation procedures or increase
bioavailability upon administration. Of course combinations of
surfactants, including the use of one or more in the liquid phase
and one or more associated with the perforated microstructures are
contemplated as being within the scope of the invention. By
"associated with or comprise" it is meant that the structural
matrix or perforated microstructure may incorporate, adsorb,
absorb, be coated with or be formed by the surfactant.
[0473] Surfactants suitable for use include any compound or
composition that aids in the formation and maintenance of the
stabilized respiratory dispersions by forming a layer at the
interface between the structural matrix and the suspension medium.
The surfactant may comprise a single compound or any combination of
compounds, such as in the case of co-surfactants. Particularly
certain surfactants are substantially insoluble in the propellant,
nonfluorinated, and selected from the group consisting of saturated
and unsaturated lipids, nonionic detergents, nonionic block
copolymers, ionic surfactants, and combinations of such agents. It
may be emphasized that, in addition to the aforementioned
surfactants, suitable (i.e., biocompatible) fluorinated surfactants
are compatible with the teachings herein and may be used to provide
the desired stabilized preparations.
[0474] Lipids, including phospholipids, from both natural and
synthetic sources may be used in varying concentrations to form a
structural matrix. Generally, compatible lipids comprise those that
have a gel to liquid crystal phase transition greater than about
40.degree. C. In certain embodiments, the incorporated lipids are
relatively long chain (i.e., C.sub.6-C.sub.22) saturated lipids and
may comprise phospholipids. Exemplary phospholipids useful in the
disclosed stabilized preparations comprise egg phosphatidylcholine,
dilauroylphosphatidylcholine, dioleylphosphatidylcholine,
dipalmitoylphosphatidyl-choline, disteroylphosphatidylcholine,
short-chain phosphatidylcholines, phosphatidylethanolamine,
dioleylphosphatidylethanolamine, phosphatidylserine,
phosphatidylglycerol, phosphatidylinositol, glycolipids,
ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin;
lipids bearing polymer chains such as, polyethylene glycol, chitin,
hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated
mono-, di-, and polysaccharides; fatty acids such as palmitic acid,
stearic acid, and oleic acid; cholesterol, cholesterol esters, and
cholesterol hemisuccinate. Due to their excellent biocompatibility
characteristics, phospholipids and combinations of phospholipids
and poloxamers are particularly suitable for use in the stabilized
dispersions disclosed herein.
[0475] Compatible nonionic detergents comprise: sorbitan esters
including sorbitan trioleate (Spans.TM. 85), sorbitan sesquioleate,
sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20)
sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate,
oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether,
lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose
esters. Other suitable nonionic detergents can be easily identified
using McCutcheon's Emulsifiers and Detergents (McPublishing Co.,
Glen Rock, N.J.). Certain block copolymers include diblock and
triblock copolymers of polyoxyethylene and polyoxypropylene,
including poloxamer 188 (Pluronic.RTM. F68), poloxamer 407
(Pluronic.RTM. F-127), and poloxamer 338. Ionic surfactants such as
sodium sulfosuccinate, and fatty acid soaps may also be utilized.
In certain embodiments, the microstructures may comprise oleic acid
or its alkali salt.
[0476] In addition to the aforementioned surfactants, cationic
surfactants or lipids may be used, especially in the case of
delivery of an iRNA agent, e.g., a double-stranded iRNA agent, or
siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which
can be processed into a siRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or siRNA agent, or
precursor thereof). Examples of suitable cationic lipids include:
DOTMA,
N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride;
DOTAP, 1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB,
1,2-dioleyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol.
Polycationic amino acids such as polylysine, and polyarginine are
also contemplated.
[0477] For the spraying process, such spraying methods as rotary
atomization, pressure atomization and two-fluid atomization can be
used. Examples of the devices used in these processes include
"Parubisu [phonetic rendering] Mini-Spray GA-32" and "Parubisu
Spray Drier DL-41", manufactured by Yamato Chemical Co., or "Spray
Drier CL-8," "Spray Drier L-8," "Spray Drier FL-12," "Spray Drier
FL-16" or "Spray Drier FL-20," manufactured by Okawara Kakoki Co.,
can be used for the method of spraying using rotary-disk
atomizer.
[0478] While no particular restrictions are placed on the gas used
to dry the sprayed material, it is recommended to use air, nitrogen
gas or an inert gas. The temperature of the inlet of the gas used
to dry the sprayed materials such that it does not cause heat
deactivation of the sprayed material. The range of temperatures may
vary between about 50.degree. C. to about 200.degree. C., for
example, between about 50.degree. C. and 100.degree. C. The
temperature of the outlet gas used to dry the sprayed material, may
vary between about 0.degree. C. and about 150.degree. C., for
example, between 0.degree. C. and 90.degree. C., and for example
between 0.degree. C. and 60.degree. C.
[0479] The spray drying is done under conditions that result in
substantially amorphous powder of homogeneous constitution having a
particle size that is respirable, a low moisture content and flow
characteristics that allow for ready aerosolization. In some cases,
the particle size of the resulting powder is such that more than
about 98% of the mass is in particles having a diameter of about 10
.mu.m or less with about 90% of the mass being in particles having
a diameter less than 5 .mu.m. Alternatively, about 95% of the mass
will have particles with a diameter of less than 10 .mu.m with
about 80% of the mass of the particles having a diameter of less
than 5 .mu.m.
[0480] The dispersible pharmaceutical-based dry powders that
include the iRNA preparation may optionally be combined with
pharmaceutical carriers or excipients which are suitable for
respiratory and pulmonary administration. Such carriers may serve
simply as bulking agents when it is desired to reduce the iRNA
concentration in the powder which is being delivered to a patient,
but may also serve to enhance the stability of the iRNA
compositions and to improve the dispersibility of the powder within
a powder dispersion device in order to provide more efficient and
reproducible delivery of the iRNA and to improve handling
characteristics of the iRNA such as flowability and consistency to
facilitate manufacturing and powder filling.
[0481] Such carrier materials may be combined with the drug prior
to spray drying, i.e., by adding the carrier material to the
purified bulk solution. In that way, the carrier particles will be
formed simultaneously with the drug particles to produce a
homogeneous powder. Alternatively, the carriers may be separately
prepared in a dry powder form and combined with the dry powder drug
by blending. The powder carriers will usually be crystalline (to
avoid water absorption), but might in some cases be amorphous or
mixtures of crystalline and amorphous. The size of the carrier
particles may be selected to improve the flowability of the drug
powder, typically being in the range from 25 .mu.m to 100 .mu.m. A
carrier material may be crystalline lactose having a size in the
above-stated range.
[0482] Powders prepared by any of the above methods will be
collected from the spray dryer in a conventional manner for
subsequent use. For use as pharmaceuticals and other purposes, it
will frequently be desirable to disrupt any agglomerates which may
have formed by screening or other conventional techniques. For
pharmaceutical uses, the dry powder formulations will usually be
measured into a single dose, and the single dose sealed into a
package. Such packages are particularly useful for dispersion in
dry powder inhalers, as described in detail below. Alternatively,
the powders may be packaged in multiple-dose containers.
[0483] Methods for spray drying hydrophobic and other drugs and
components are described in U.S. Pat. Nos. 5,000,888; 5,026,550;
4,670,419, 4,540,602; and 4,486,435. Bloch and Speison (1983)
Pharm. Acta Helv 58:14-22 teaches spray drying of
hydrochlorothiazide and chlorthalidone (lipophilic drugs) and a
hydrophilic adjuvant (pentaerythritol) in azeotropic solvents of
dioxane-water and 2-ethoxyethanol-water. A number of Japanese
Patent application Abstracts relate to spray drying of
hydrophilic-hydrophobic product combinations, including JP 806766;
JP 7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP
4036233. Other foreign patent publications relevant to spray drying
hydrophilic-hydrophobic product combinations include FR 2594693; DE
2209477; and WO 88/07870.
Lyophilization
[0484] An iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, (e.g., a precursor, e.g., a larger iRNA agent which can be
processed into a siRNA agent, or a DNA which encodes an iRNA agent,
e.g., a double-stranded iRNA agent, or siRNA agent, or precursor
thereof) preparation can be made by lyophilization. Lyophilization
is a freeze-drying process in which water is sublimed from the
composition after it is frozen. The particular advantage associated
with the lyophilization process is that biologicals and
pharmaceuticals that are relatively unstable in an aqueous solution
can be dried without elevated temperatures (thereby eliminating the
adverse thermal effects), and then stored in a dry state where
there are few stability problems. With respect to the instant
invention such techniques are particularly compatible with the
incorporation of nucleic acids in perforated microstructures
without compromising physiological activity. Methods for providing
lyophilized particulates are known to those of skill in the art and
it would clearly not require undue experimentation to provide
dispersion compatible microstructures in accordance with the
teachings herein. Accordingly, to the extent that lyophilization
processes may be used to provide microstructures having the desired
porosity and size, they are conformance with the teachings herein
and are expressly contemplated as being within the scope of the
instant invention.
Genes
[0485] In one aspect, the invention features, a method of treating
a subject at risk for or afflicted with a disease that may benefit
from the administration of the modular composition of the
invention. The method comprises administering the modular
composition of the invention to a subject in need thereof, thereby
treating the subject. The nucleic acid that is administered will
depend on the disease being treated.
[0486] In certain embodiments, the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences a growth factor
or growth factor receptor gene, a kinase, e.g., a protein tyrosine,
serine or threonine kinase gene, an adaptor protein gene, a gene
encoding a G protein superfamily molecule, or a gene encoding a
transcription factor.
[0487] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the PDGF beta
gene, and thus can be used to treat a subject having or at risk for
a disorder characterized by unwanted PDGF beta expression, e.g.,
testicular and lung cancers.
[0488] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the Erb-B gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted Erb-B expression, e.g., breast
cancer.
[0489] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the Src gene, and
thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted Src expression, e.g., colon
cancers.
[0490] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the CRK gene, and
thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted CRK expression, e.g., colon and
lung cancers.
[0491] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the GRB2 gene, and
thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted GRB2 expression, e.g., squamous
cell carcinoma.
[0492] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the RAS gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted RAS expression, e.g.,
pancreatic, colon and lung cancers, and chronic leukemia.
[0493] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the MEKK gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted MEKK expression, e.g., squamous
cell carcinoma, melanoma or leukemia.
[0494] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the JNK gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted JNK expression, e.g., pancreatic
or breast cancers.
[0495] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the RAF gene, and
thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted RAF expression, e.g., lung
cancer or leukemia.
[0496] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the Erk1/2 gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted Erk1/2 expression, e.g., lung
cancer.
[0497] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the PCNA(p21)
gene, and thus can be used to treat a subject having or at risk for
a disorder characterized by unwanted PCNA expression, e.g., lung
cancer.
[0498] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the MYB gene, and
thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted MYB expression, e.g., colon
cancer or chronic myelogenous leukemia.
[0499] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the c-MYC gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted c-MYC expression, e.g.,
Burkitt's lymphoma or neuroblastoma.
[0500] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the JUN gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted JUN expression, e.g., ovarian,
prostate or breast cancers.
[0501] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the FOS gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted FOS expression, e.g., skin or
prostate cancers.
[0502] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the BCL-2 gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted BCL-2 expression, e.g., lung or
prostate cancers or Non-Hodgkin lymphoma.
[0503] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the Cyclin D gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted Cyclin D expression, e.g.,
esophageal and colon cancers.
[0504] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the VEGF gene, and
thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted VEGF expression, e.g.,
esophageal and colon cancers.
[0505] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the EGFR gene, and
thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted EGFR expression, e.g., breast
cancer.
[0506] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the Cyclin A
gene, and thus can be used to treat a subject having or at risk for
a disorder characterized by unwanted Cyclin A expression, e.g.,
lung and cervical cancers.
[0507] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the Cyclin E
gene, and thus can be used to treat a subject having or at risk for
a disorder characterized by unwanted Cyclin E expression, e.g.,
lung and breast cancers.
[0508] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the WNT-1 gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted WNT-1 expression, e.g., basal
cell carcinoma.
[0509] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the
beta-catenin gene, and thus can be used to treat a subject having
or at risk for a disorder characterized by unwanted beta-catenin
expression, e.g., adenocarcinoma or hepatocellular carcinoma.
[0510] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the c-MET gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted c-MET expression, e.g.,
hepatocellular carcinoma.
[0511] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the PKC gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted PKC expression, e.g., breast
cancer.
[0512] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the NFKB gene, and
thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted NFKB expression, e.g., breast
cancer.
[0513] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the STAT3 gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted STAT3 expression, e.g., prostate
cancer.
[0514] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the survivin
gene, and thus can be used to treat a subject having or at risk for
a disorder characterized by unwanted survivin expression, e.g.,
cervical or pancreatic cancers.
[0515] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the Her2/Neu
gene, and thus can be used to treat a subject having or at risk for
a disorder characterized by unwanted Her2/Neu expression, e.g.,
breast cancer.
[0516] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the
topoisomerase I gene, and thus can be used to treat a subject
having or at risk for a disorder characterized by unwanted
topoisomerase I expression, e.g., ovarian and colon cancers.
[0517] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the topoisomerase
II alpha gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted topoisomerase II
expression, e.g., breast and colon cancers.
[0518] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
p73 gene, and thus can be used to treat a subject having or at risk
for a disorder characterized by unwanted p73 expression, e.g.,
colorectal adenocarcinoma.
[0519] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
p21(WAF1/CIP1) gene, and thus can be used to treat a subject having
or at risk for a disorder characterized by unwanted p21(WAF1/CIP1)
expression, e.g., liver cancer.
[0520] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
p27(KIP1) gene, and thus can be used to treat a subject having or
at risk for a disorder characterized by unwanted p27(KIP1)
expression, e.g., liver cancer.
[0521] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
PPM1D gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted PPM1D expression,
e.g., breast cancer.
[0522] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
RAS gene, and thus can be used to treat a subject having or at risk
for a disorder characterized by unwanted RAS expression, e.g.,
breast cancer.
[0523] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences mutations in
the caveolin I gene, and thus can be used to treat a subject having
or at risk for a disorder characterized by unwanted caveolin I
expression, e.g., esophageal squamous cell carcinoma.
[0524] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences mutations in
the MIB I gene, and thus can be used to treat a subject having or
at risk for a disorder characterized by unwanted MIB I expression,
e.g., male breast carcinoma (MBC).
[0525] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences mutations in
the MTAI gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted MTAI expression,
e.g., ovarian carcinoma.
[0526] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences mutations in
the M68 gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted M68 expression, e.g.,
human adenocarcinomas of the esophagus, stomach, colon, and
rectum.
[0527] In certain embodiments the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences mutations in
tumor suppressor genes, and thus can be used as a method to promote
apoptotic activity in combination with chemotherapeutics.
[0528] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
p53 tumor suppressor gene, and thus can be used to treat a subject
having or at risk for a disorder characterized by unwanted p53
expression, e.g., gall bladder, pancreatic and lung cancers.
[0529] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
p53 family member DN-p63, and thus can be used to treat a subject
having or at risk for a disorder characterized by unwanted DN-p63
expression, e.g., squamous cell carcinoma
[0530] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
pRb tumor suppressor gene, and thus can be used to treat a subject
having or at risk for a disorder characterized by unwanted pRb
expression, e.g., oral squamous cell carcinoma
[0531] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
APC1 tumor suppressor gene, and thus can be used to treat a subject
having or at risk for a disorder characterized by unwanted APC1
expression, e.g., colon cancer.
[0532] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
BRCA1 tumor suppressor gene, and thus can be used to treat a
subject having or at risk for a disorder characterized by unwanted
BRCA1 expression, e.g., breast cancer.
[0533] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences mutations in the
PTEN tumor suppressor gene, and thus can be used to treat a subject
having or at risk for a disorder characterized by unwanted PTEN
expression, e.g., hamartomas, gliomas, and prostate and endometrial
cancers.
[0534] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences MLL fusion genes,
e.g., MLL-AF9, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted MLL fusion gene
expression, e.g., acute leukemias.
[0535] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the BCR/ABL
fusion gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted BCR/ABL fusion gene
expression, e.g., acute and chronic leukemias.
[0536] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the TEL/AML1
fusion gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted TEL/AML1 fusion gene
expression, e.g., childhood acute leukemia.
[0537] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the EWS/FLI1
fusion gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted EWS/FLI1 fusion gene
expression, e.g., Ewing Sarcoma.
[0538] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the TLS/FUS1
fusion gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted TLS/FUS1 fusion gene
expression, e.g., Myxoid liposarcoma.
[0539] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the PAX3/FKHR
fusion gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted PAX3/FKHR fusion gene
expression, e.g., Myxoid liposarcoma.
[0540] In another embodiment the iRNA agent (e.g., the iRNA agent
in a modular composition described herein) silences the AML1/ETO
fusion gene, and thus can be used to treat a subject having or at
risk for a disorder characterized by unwanted AML1/ETO fusion gene
expression, e.g., acute leukemia.
Diseases
Angiogenesis
[0541] In another aspect, the invention features, a method of
treating a subject, e.g., a human, at risk for or afflicted with a
disease or disorder that may benefit by angiogenesis inhibition,
e.g., cancer. The method comprises administering the modular
composition of the invention to a subject in need thereof, thereby
treating the subject. The nucleic acid that is administered will
depend on the type of angiogenesis-related gene being treated.
[0542] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the alpha
v-integrin gene, and thus can be used to treat a subject having or
at risk for a disorder characterized by unwanted alpha V integrin,
e.g., brain tumors or tumors of epithelial origin.
[0543] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the Flt-1 receptor
gene, and thus can be used to treat a subject having or at risk for
a disorder characterized by unwanted Flt-1 receptors, eg. cancer
and rheumatoid arthritis.
[0544] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the tubulin gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted tubulin, eg. cancer and retinal
neovascularization.
[0545] In some embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences the tubulin gene,
and thus can be used to treat a subject having or at risk for a
disorder characterized by unwanted tubulin, eg. cancer and retinal
neovascularization.
Viral Diseases
[0546] In yet another aspect, the invention features a method of
treating a subject infected with a virus or at risk for or
afflicted with a disorder or disease associated with a viral
infection. The method comprises administering the modular
composition of the invention to a subject in need thereof, thereby
treating the subject. The nucleic acid that is administered will
depend on the type of viral disease being treated. In some
embodiments, the nucleic acid may target a viral gene. In other
embodiments, the nucleic acid may target a host gene.
[0547] Thus, the invention provides for a method of treating
patients infected by the Human Papilloma Virus (HPV) or at risk for
or afflicted with a disorder mediated by HPV, e.g, cervical cancer.
HPV is linked to 95% of cervical carcinomas and thus an antiviral
therapy is an attractive method to treat these cancers and other
symptoms of viral infection. In some embodiments, the expression of
a HPV gene is reduced. In another embodiment, the HPV gene is one
of the group of E2, E6, or E7. In some embodiments the expression
of a human gene that is required for HPV replication is
reduced.
[0548] The invention also includes a method of treating patients
infected by the Human Immunodeficiency Virus (HIV) or at risk for
or afflicted with a disorder mediated by HIV, e.g., Acquired Immune
Deficiency Syndrome (AIDS). In some embodiments, the expression of
a HIV gene is reduced. In another embodiment, the HIV gene is CCR5,
Gag, or Rev. In some embodiments the expression of a human gene
that is required for HIV replication is reduced. In another
embodiment, the gene is CD4 or Tsg101.
[0549] The invention also includes a method for treating patients
infected by the Hepatitis B Virus (HBV) or at risk for or afflicted
with a disorder mediated by HBV, e.g., cirrhosis and hepatocellular
carcinoma. In some embodiments, the expression of a HBV gene is
reduced. In another embodiment, the targeted HBV gene encodes one
of the group of the tail region of the HBV core protein, the
pre-cregious (pre-c) region, or the cregious (c) region. In another
embodiment, a targeted HBV-RNA sequence is comprised of the poly(A)
tail. In certain embodiment the expression of a human gene that is
required for HBV replication is reduced.
[0550] The invention also provides for a method of treating
patients infected by the Hepatitis A Virus (HAV), or at risk for or
afflicted with a disorder mediated by HAV. In some embodiments the
expression of a human gene that is required for HAV replication is
reduced.
[0551] The present invention provides for a method of treating
patients infected by the Hepatitis C Virus (HCV), or at risk for or
afflicted with a disorder mediated by HCV, e.g., cirrhosis. In some
embodiments, the expression of a HCV gene is reduced. In another
embodiment the expression of a human gene that is required for HCV
replication is reduced.
[0552] The present invention also provides for a method of treating
patients infected by the any of the group of Hepatitis Viral
strains comprising hepatitis D, E, F, G, or H, or patients at risk
for or afflicted with a disorder mediated by any of these strains
of hepatitis. In some embodiments, the expression of a Hepatitis,
D, E, F, G, or H gene is reduced. In another embodiment the
expression of a human gene that is required for hepatitis D, E, F,
G or H replication is reduced.
[0553] Methods of the invention also provide for treating patients
infected by the Respiratory Syncytial Virus (RSV) or at risk for or
afflicted with a disorder mediated by RSV, e.g, lower respiratory
tract infection in infants and childhood asthma, pneumonia and
other complications, e.g., in the elderly. In some embodiments, the
expression of a RSV gene is reduced. In another embodiment, the
targeted HBV gene encodes one of the group of genes N, L, or P. In
some embodiments the expression of a human gene that is required
for RSV replication is reduced.
[0554] Methods of the invention provide for treating patients
infected by the Herpes Simplex Virus (HSV) or at risk for or
afflicted with a disorder mediated by HSV, e.g, genital herpes and
cold sores as well as life-threatening or sight-impairing disease
mainly in immunocompromised patients. In some embodiments, the
expression of a HSV gene is reduced. In another embodiment, the
targeted HSV gene encodes DNA polymerase or the helicase-primase.
In some embodiments the expression of a human gene that is required
for HSV replication is reduced.
[0555] The invention also provides a method for treating patients
infected by the herpes Cytomegalovirus (CMV) or at risk for or
afflicted with a disorder mediated by CMV, e.g., congenital virus
infections and morbidity in immunocompromised patients. In some
embodiments, the expression of a CMV gene is reduced. In some
embodiments the expression of a human gene that is required for CMV
replication is reduced.
[0556] Methods of the invention also provide for a method of
treating patients infected by the herpes Epstein Barr Virus (EBV)
or at risk for or afflicted with a disorder mediated by EBV, e.g.,
NK/T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease. In
some embodiments, the expression of a EBV gene is reduced. In some
embodiments the expression of a human gene that is required for EBV
replication is reduced.
[0557] Methods of the invention also provide for treating patients
infected by Kaposi's Sarcoma-associated Herpes Virus (KSHV), also
called human herpesvirus 8, or patients at risk for or afflicted
with a disorder mediated by KSHV, e.g., Kaposi's sarcoma,
multicentric Castleman's disease and AIDS-associated primary
effusion lymphoma. In some embodiments, the expression of a KSHV
gene is reduced. In some embodiments the expression of a human gene
that is required for KSHV replication is reduced.
[0558] The invention also includes a method for treating patients
infected by the JC Virus (JCV) or a disease or disorder associated
with this virus, e.g., progressive multifocal leukoencephalopathy
(PML). In some embodiments, the expression of a JCV gene is
reduced. In certain embodiments the expression of a human gene that
is required for JCV replication is reduced.
[0559] Methods of the invention also provide for treating patients
infected by the myxovirus or at risk for or afflicted with a
disorder mediated by myxovirus, e.g., influenza. In some
embodiments, the expression of a myxovirus gene is reduced. In some
embodiments the expression of a human gene that is required for
myxovirus replication is reduced.
[0560] Methods of the invention also provide for treating patients
infected by the rhinovirus or at risk for of afflicted with a
disorder mediated by rhinovirus, e.g., the common cold. In some
embodiments, the expression of a rhinovirus gene is reduced. In
certain embodiments the expression of a human gene that is required
for rhinovirus replication is reduced.
[0561] Methods of the invention also provide for treating patients
infected by the coronavirus or at risk for of afflicted with a
disorder mediated by coronavirus, e.g., the common cold. In some
embodiments, the expression of a coronavirus gene is reduced. In
certain embodiments the expression of a human gene that is required
for coronavirus replication is reduced.
[0562] Methods of the invention also provide for treating patients
infected by the flavivirus West Nile or at risk for or afflicted
with a disorder mediated by West Nile Virus. In some embodiments,
the expression of a West Nile Virus gene is reduced. In another
embodiment, the West Nile Virus gene is one of the group comprising
E, NS3, or NS5. In some embodiments the expression of a human gene
that is required for West Nile Virus replication is reduced.
[0563] Methods of the invention also provide for treating patients
infected by the St. Louis Encephalitis flavivirus, or at risk for
or afflicted with a disease or disorder associated with this virus,
e.g., viral haemorrhagic fever or neurological disease. In some
embodiments, the expression of a St. Louis Encephalitis gene is
reduced. In some embodiments the expression of a human gene that is
required for St. Louis Encephalitis virus replication is
reduced.
[0564] Methods of the invention also provide for treating patients
infected by the Tick-borne encephalitis flavivirus, or at risk for
or afflicted with a disorder mediated by Tick-borne encephalitis
virus, e.g., viral haemorrhagic fever and neurological disease. In
some embodiments, the expression of a Tick-borne encephalitis virus
gene is reduced. In some embodiments the expression of a human gene
that is required for Tick-borne encephalitis virus replication is
reduced.
[0565] Methods of the invention also provide for methods of
treating patients infected by the Murray Valley encephalitis
flavivirus, which commonly results in viral haemorrhagic fever and
neurological disease. In some embodiments, the expression of a
Murray Valley encephalitis virus gene is reduced. In some
embodiments the expression of a human gene that is required for
Murray Valley encephalitis virus replication is reduced.
[0566] The invention also includes methods for treating patients
infected by the dengue flavivirus, or a disease or disorder
associated with this virus, e.g., dengue haemorrhagic fever. In
some embodiments, the expression of a dengue virus gene is reduced.
In some embodiments the expression of a human gene that is required
for dengue virus replication is reduced.
[0567] Methods of the invention also provide for treating patients
infected by the Simian Virus 40 (SV40) or at risk for or afflicted
with a disorder mediated by SV40, e.g., tumorigenesis. In some
embodiments, the expression of a SV40 gene is reduced. In some
embodiments the expression of a human gene that is required for
SV40 replication is reduced.
[0568] The invention also includes methods for treating patients
infected by the Human T Cell Lymphotropic Virus (HTLV), or a
disease or disorder associated with this virus, e.g., leukemia and
myelopathy. In some embodiments, the expression of a HTLV gene is
reduced. In another embodiment the HTLV1 gene is the Tax
transcriptional activator. In some embodiments the expression of a
human gene that is required for HTLV replication is reduced.
[0569] Methods of the invention also provide for treating patients
infected by the Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk
for or afflicted with a disorder mediated by Mo-MuLV, e.g., T-cell
leukemia. In some embodiments, the expression of a Mo-MuLV gene is
reduced. In some embodiments the expression of a human gene that is
required for Mo-MuLV replication is reduced.
[0570] Methods of the invention also provide for treating patients
infected by the encephalomyocarditis virus (EMCV) or at risk for or
afflicted with a disorder mediated by EMCV, e.g., myocarditis. EMCV
leads to myocarditis in mice and pigs and is capable of infecting
human myocardial cells. This virus is therefore a concern for
patients undergoing xenotransplantation. In some embodiments, the
expression of a EMCV gene is reduced. In some embodiments the
expression of a human gene that is required for EMCV replication is
reduced.
[0571] The invention also includes a method for treating patients
infected by the measles virus (MV) or at risk for or afflicted with
a disorder mediated by MV, e.g., measles. In some embodiments, the
expression of a MV gene is reduced. In some embodiments the
expression of a human gene that is required for MV replication is
reduced.
[0572] The invention also includes a method for treating patients
infected by the Varicella zoster virus (VZV) or at risk for or
afflicted with a disorder mediated by VZV, e.g., chicken pox or
shingles (also called zoster). In some embodiments, the expression
of a VZV gene is reduced. In some embodiments the expression of a
human gene that is required for VZV replication is reduced.
[0573] The invention also includes a method for treating patients
infected by an adenovirus or at risk for or afflicted with a
disorder mediated by an adenovirus, e.g., respiratory tract
infection. In some embodiments, the expression of an adenovirus
gene is reduced. In some embodiments the expression of a human gene
that is required for adenovirus replication is reduced.
[0574] The invention includes a method for treating patients
infected by a yellow fever virus (YFV) or at risk for or afflicted
with a disorder mediated by a YFV, e.g., respiratory tract
infection. In some embodiments, the expression of a YFV gene is
reduced. In another embodiment, the gene may be one of a group that
includes the E, NS2A, or NS3 genes. In some embodiments the
expression of a human gene that is required for YFV replication is
reduced.
[0575] Methods of the invention also provide for treating patients
infected by the poliovirus or at risk for or afflicted with a
disorder mediated by poliovirus, e.g., polio. In some embodiments,
the expression of a poliovirus gene is reduced. In some embodiments
the expression of a human gene that is required for poliovirus
replication is reduced.
[0576] Methods of the invention also provide for treating patients
infected by a poxvirus or at risk for or afflicted with a disorder
mediated by a poxvirus, e.g., smallpox. In some embodiments, the
expression of a poxvirus gene is reduced. In some embodiments the
expression of a human gene that is required for poxvirus
replication is reduced.
Other Pathogens
[0577] In another, aspect the invention features methods of
treating a subject infected with a pathogen, e.g., a bacterial,
amoebic, parasitic, or fungal pathogen. The method comprises
administering the modular composition of the invention to a subject
in need thereof, thereby treating the subject. The nucleic acid
that is administered will depend on the type of pathogen being
treated. In some embodiments, the nucleic acid may target a
pathogen gene. In other embodiments, the nucleic acid may target a
host gene.
[0578] The target gene can be one involved in growth, cell wall
synthesis, protein synthesis, transcription, energy metabolism,
e.g., the Krebs cycle, or toxin production.
[0579] Thus, the present invention provides for a method of
treating patients infected by a plasmodium that causes malaria. In
some embodiments, the expression of a plasmodium gene is reduced.
In another embodiment, the gene is apical membrane antigen 1
(AMA1). In some embodiments the expression of a human gene that is
required for plasmodium replication is reduced.
[0580] The invention also includes methods for treating patients
infected by the Mycobacterium ulcerans, or a disease or disorder
associated with this pathogen, e.g., Buruli ulcers. In some
embodiments, the expression of a Mycobacterium ulcerans gene is
reduced. In some embodiments the expression of a human gene that is
required for Mycobacterium ulcerans replication is reduced.
[0581] The invention also includes methods for treating patients
infected by the Mycobacterium tuberculosis, or a disease or
disorder associated with this pathogen, e.g., tuberculosis. In some
embodiments, the expression of a Mycobacterium tuberculosis gene is
reduced. In some embodiments the expression of a human gene that is
required for Mycobacterium tuberculosis replication is reduced.
[0582] The invention also includes methods for treating patients
infected by the Mycobacterium leprae, or a disease or disorder
associated with this pathogen, e.g., leprosy. In some embodiments,
the expression of a Mycobacterium leprae gene is reduced. In some
embodiments the expression of a human gene that is required for
Mycobacterium leprae replication is reduced.
[0583] The invention also includes methods for treating patients
infected by the bacteria Staphylococcus aureus, or a disease or
disorder associated with this pathogen, e.g., infections of the
skin and muscous membranes. In some embodiments, the expression of
a Staphylococcus aureus gene is reduced. In some embodiments the
expression of a human gene that is required for Staphylococcus
aureus replication is reduced.
[0584] The invention also includes methods for treating patients
infected by the bacteria Streptococcus pneumoniae, or a disease or
disorder associated with this pathogen, e.g., pneumonia or
childhood lower respiratory tract infection. In some embodiments,
the expression of a Streptococcus pneumoniae gene is reduced. In
some embodiments the expression of a human gene that is required
for Streptococcus pneumoniae replication is reduced.
[0585] The invention also includes methods for treating patients
infected by the bacteria Streptococcus pyogenes, or a disease or
disorder associated with this pathogen, e.g., Strep throat or
Scarlet fever. In some embodiments, the expression of a
Streptococcus pyogenes gene is reduced. In some embodiments the
expression of a human gene that is required for Streptococcus
pyogenes replication is reduced.
[0586] The invention also includes methods for treating patients
infected by the bacteria Chlamydia pneumoniae, or a disease or
disorder associated with this pathogen, e.g., pneumonia or
childhood lower respiratory tract infection. In some embodiments,
the expression of a Chlamydia pneumoniae gene is reduced. In some
embodiments the expression of a human gene that is required for
Chlamydia pneumoniae replication is reduced.
[0587] The invention also includes methods for treating patients
infected by the bacteria Mycoplasma pneumoniae, or a disease or
disorder associated with this pathogen, e.g., pneumonia or
childhood lower respiratory tract infection. In some embodiments,
the expression of a Mycoplasma pneumoniae gene is reduced. In some
embodiments the expression of a human gene that is required for
Mycoplasma pneumoniae replication is reduced.
Immune Disorders
[0588] In one aspect, the invention features, a method of treating
a subject, e.g., a human, at risk for or afflicted with a disease
or disorder characterized by an unwanted immune response, e.g., an
inflammatory disease or disorder, or an autoimmune disease or
disorder. The method comprises administering the modular
composition of the invention to a subject in need thereof, thereby
treating the subject. The nucleic acid that is administered will
depend on the type of immune disorder being treated.
[0589] In some embodiments the disease or disorder is an ischemia
or reperfusion injury, e.g., ischemia or reperfusion injury
associated with acute myocardial infarction, unstable angina,
cardiopulmonary bypass, surgical intervention e.g., angioplasty,
e.g., percutaneous transluminal coronary angioplasty, the response
to a transplanted organ or tissue, e.g., transplanted cardiac or
vascular tissue; or thrombolysis.
[0590] In some embodiments the disease or disorder is restenosis,
e.g., restenosis associated with surgical intervention e.g.,
angioplasty, e.g., percutaneous transluminal coronary
angioplasty.
[0591] In certain embodiments the disease or disorder is
Inflammatory Bowel Disease, e.g., Crohn's Disease or Ulcerative
Colitis.
[0592] In certain embodiments the disease or disorder is
inflammation associated with an infection or injury.
[0593] In certain embodiments the disease or disorder is asthma,
lupus, multiple sclerosis, diabetes, e.g., type II diabetes,
arthritis, e.g., rheumatoid or psoriatic.
[0594] In certain other embodiments the iRNA agent (e.g., the iRNA
agent in a modular composition described herein) silences an
integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.
[0595] In certain other embodiments the iRNA agent (e.g., the iRNA
agent in a modular composition described herein) silences a
selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM),
I-selectin, P-selectin glycoprotein-1 (PSGL-1).
[0596] In certain other embodiments the iRNA agent (e.g., the iRNA
agent in a modular composition described herein) silences a
component of the complement system, e.g., C3, C5, C3aR, C5aR, C3
convertase, C5 convertase.
[0597] In certain other embodiments the iRNA agent (e.g., the iRNA
agent in a modular composition described herein) silences a
chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J,
IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE,
SCYA11, CCR3.
[0598] In other embodiments the iRNA agent (e.g., the iRNA agent in
a modular composition described herein) silences GCSF, Gro1, Gro2,
Gro3, PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J,
RANTES, MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1,
I-309.
Pain
[0599] In one aspect, the invention features, a method of treating
a subject, e.g., a human, at risk for or afflicted with acute pain
or chronic pain. The method comprises administering the modular
composition of the invention to a subject in need thereof, thereby
treating the subject. The nucleic acid that is administered will
depend on the type of pain being treated.
[0600] In certain other embodiments the iRNA agent (e.g., the iRNA
agent in a modular composition described herein) silences a
component of an ion channel.
[0601] In certain other embodiments the iRNA agent (e.g., the iRNA
agent in a modular composition described herein) silences a
neurotransmitter receptor or ligand.
[0602] In one aspect, the invention features, a method of treating
a subject, e.g., a human, at risk for or afflicted with a
neurological disease or disorder. The method includes:
[0603] providing an iRNA agent (e.g., the iRNA agent in a modular
composition described herein) which iRNA is homologous to and can
silence, e.g., by cleavage, a gene which mediates a neurological
disease or disorder;
[0604] administering the to a subject,
[0605] thereby treating the subject.
Neurological Disorders
[0606] In certain embodiments the disease or disorder is a
neurological disorder, including Alzheimer's Disease or Parkinson
Disease. The method comprises administering the modular composition
of the invention to a subject in need thereof, thereby treating the
subject. The nucleic acid that is administered will depend on the
type of neurological disorder being treated.
[0607] In certain other embodiments the iRNA agent (e.g., the iRNA
agent in a modular composition described herein) silences an
amyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 and
PSEN2, or I-synuclein.
[0608] In some embodiments the disease or disorder is a
neurodegenerative trinucleotide repeat disorder, e.g., Huntington
disease, dentatorubral pallidoluysian atrophy or a spinocerebellar
ataxia, e.g., SCA1, SCA2, SCA3 (Machado-Joseph disease), SCA7 or
SCA8.
[0609] In certain other embodiments the iRNA agent (e.g., the iRNA
agent in a modular composition described herein) silences HD,
DRPLA, SCA1, SCA2, MJD1, CACNL1A4, SCA7, SCA8.
Loss of Heterozygosity
[0610] 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
euploid 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 cleavage or silencing of
one allele of an essential gene with an iRNA agent (e.g., the iRNA
agent in a modular composition described herein) of the invention.
The iRNA agent (e.g., the iRNA agent in a modular composition
described herein) is selected such that it targets the single
allele of the essential gene found in the cells having LOH but does
not silence the other allele, which is present in cells which do
not show LOH. In essence, it discriminates between the two alleles,
preferentially silencing the selected allele. In essence
polymorphisms, e.g., SNPs of essential genes that are affected by
LOH, are used as a target for a disorder characterized by cells
having LOH, e.g., cancer cells having LOH.
[0611] One of ordinary skill in the art can identify essential
genes which are in proximity to tumor suppressor genes, and which
are within a LOH region which includes the tumor suppressor gene.
The gene encoding the large subunit of human RNA polymerase II,
POLR2A, a gene located in close proximity to the tumor suppressor
gene p53, is such a gene. It frequently occurs within a region of
LOH in cancer cells. Other genes that occur within LOH regions and
are lost in many cancer cell types include the group comprising
replication protein A 70-kDa subunit, replication protein A 32-kD,
ribonucleotide reductase, thymidilate synthase, TATA associated
factor 2H, ribosomal protein S14, eukaryotic initiation factor 5 A,
alanyl tRNA synthetase, cysteinyl tRNA synthetase, NaK ATPase,
alpha-1 subunit, and transferrin receptor.
[0612] Accordingly, the invention features, a method of treating a
disorder characterized by LOH, e.g., cancer. The method comprises
optionally, determining the genotype of the allele of a gene in the
region of LOH and determining the genotype of both alleles of the
gene in a normal cell; providing an iRNA agent (e.g., the iRNA
agent in a modular composition described herein) which
preferentially cleaves or silences the allele found in the LOH
cells; and administering the iRNA to the subject, thereby treating
the disorder.
[0613] The invention also includes a iRNA agent (e.g., the iRNA
agent in a modular composition described herein) disclosed herein,
e.g, an iRNA agent (e.g., the iRNA agent in a modular composition
described herein) which can preferentially silence, e.g., cleave,
one allele of a polymorphic gene.
[0614] In another aspect, the invention provides a method of
cleaving or silencing more than one gene with an iRNA agent (e.g.,
the iRNA agent in a modular composition described herein). In these
embodiments the iRNA agent (e.g., the iRNA agent in a modular
composition described herein) is selected so that it has sufficient
homology to a sequence found in more than one gene. For example,
the sequence AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO:36) is conserved
between mouse lamin B1, lamin B2, keratin complex 2-gene 1 and
lamin A/C. Thus an iRNA agent (e.g., the iRNA agent in a modular
composition described herein) targeted to this sequence would
effectively silence the entire collection of genes.
[0615] The invention also includes an iRNA agent (e.g., the iRNA
agent in a modular composition described herein) disclosed herein,
which can silence more than one gene.
Routes of Delivery
[0616] For ease of exposition the formulations, compositions and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It may be understood, however, that these
formulations, compositions and methods can be practiced with other
iRNA agents, e.g., modified iRNA agents, and such practice is
within the invention. A composition that includes a iRNA can be
delivered to a subject by a variety of routes. Exemplary routes
include: intravenous, topical, rectal, anal, vaginal, nasal,
pulmonary, ocular.
[0617] The iRNA molecules of the invention can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically include one or more species of iRNA and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0618] 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 (including ophthalmic, vaginal,
rectal, intranasal, transdermal), oral or parenteral. Parenteral
administration includes intravenous drip, subcutaneous,
intraperitoneal or intramuscular injection, or intrathecal or
intraventricular administration.
[0619] The route and site of administration may be chosen to
enhance targeting. For example, to target muscle cells,
intramuscular injection into the muscles of interest would be a
logical choice. Lung cells might be targeted by administering the
iRNA in aerosol form. The vascular endothelial cells could be
targeted by coating a balloon catheter with the iRNA and
mechanically introducing the DNA.
[0620] 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.
[0621] Compositions for oral administration include powders or
granules, suspensions or solutions in water, syrups, elixirs or
non-aqueous media, tablets, capsules, lozenges, or troches. In the
case of tablets, carriers that can be used include lactose, sodium
citrate and salts of phosphoric acid. Various disintegrants such as
starch, and lubricating agents such as magnesium stearate, sodium
lauryl sulfate and talc, are commonly used in tablets. For oral
administration in capsule form, useful diluents are lactose and
high molecular weight polyethylene glycols. When aqueous
suspensions are required for oral use, the nucleic acid
compositions can be combined with emulsifying and suspending
agents. If desired, certain sweetening and/or flavoring agents can
be added.
[0622] Compositions for intrathecal or intraventricular
administration may include sterile aqueous solutions which may also
contain buffers, diluents and other suitable additives.
[0623] Formulations for parenteral administration may include
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives. Intraventricular injection may be
facilitated by an intraventricular catheter, for example, attached
to a reservoir. For intravenous use, the total concentration of
solutes may be controlled to render the preparation isotonic.
[0624] For ocular administration, ointments or droppable liquids
may be delivered by ocular delivery systems known to the art such
as applicators or eye droppers. Such compositions can include
mucomimetics such as hyaluronic acid, chondroitin sulfate,
hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives
such as sorbic acid, EDTA or benzylchronium chloride, and the usual
quantities of diluents and/or carriers.
Topical Delivery
[0625] For ease of exposition the formulations, compositions and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It may be understood, however, that these
formulations, compositions and methods can be practiced with other
iRNA agents, e.g., modified iRNA agents, and such practice is
within the invention. In some embodiments, an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, (e.g., a precursor,
e.g., a larger iRNA agent which can be processed into a siRNA
agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof)
is delivered to a subject via topical administration. "Topical
administration" refers to the delivery to a subject by contacting
the formulation directly to a surface of the subject. The most
common form of topical delivery is to the skin, but a composition
disclosed herein can also be directly applied to other surfaces of
the body, e.g., to the eye, a mucous membrane, to surfaces of a
body cavity or to an internal surface. As mentioned above, the most
common topical delivery is to the skin. The term encompasses
several routes of administration including, but not limited to,
topical and transdermal. These modes of administration typically
include penetration of the skin's permeability barrier and
efficient delivery to the target tissue or stratum. Topical
administration can be used as a means to penetrate the epidermis
and dermis and ultimately achieve systemic delivery of the
composition. Topical administration can also be used as a means to
selectively deliver oligonucleotides to the epidermis or dermis of
a subject, or to specific strata thereof, or to an underlying
tissue.
[0626] The term "skin," as used herein, refers to the epidermis
and/or dermis of an animal. Mammalian skin consists of two major,
distinct layers. The outer layer of the skin is called the
epidermis. The epidermis is comprised of the stratum corneum, the
stratum granulosum, the stratum spinosum, and the stratum basale,
with the stratum corneum being at the surface of the skin and the
stratum basale being the deepest portion of the epidermis. The
epidermis is between 50 .mu.m and 0.2 mm thick, depending on its
location on the body.
[0627] Beneath the epidermis is the dermis, which is significantly
thicker than the epidermis. The dermis is primarily composed of
collagen in the form of fibrous bundles. The collagenous bundles
provide support for, inter alia, blood vessels, lymph capillaries,
glands, nerve endings and immunologically active cells.
[0628] One of the major functions of the skin as an organ is to
regulate the entry of substances into the body. The principal
permeability barrier of the skin is provided by the stratum
corneum, which is formed from many layers of cells in various
states of differentiation. The spaces between cells in the stratum
corneum is filled with different lipids arranged in lattice-like
formations that provide seals to further enhance the skins
permeability barrier.
[0629] The permeability barrier provided by the skin is such that
it is largely impermeable to molecules having molecular weight
greater than about 750 Da. For larger molecules to cross the skin's
permeability barrier, mechanisms other than normal osmosis must be
used.
[0630] Several factors determine the permeability of the skin to
administered agents. These factors include the characteristics of
the treated skin, the characteristics of the delivery agent,
interactions between both the drug and delivery agent and the drug
and skin, the dosage of the drug applied, the form of treatment,
and the post treatment regimen. To selectively target the epidermis
and dermis, it is sometimes possible to formulate a composition
that comprises one or more penetration enhancers that will enable
penetration of the drug to a preselected stratum.
[0631] Transdermal delivery is a valuable route for the
administration of lipid soluble therapeutics. The dermis is more
permeable than the epidermis and therefore absorption is much more
rapid through abraded, burned or denuded skin. Inflammation and
other physiologic conditions that increase blood flow to the skin
also enhance transdermal adsorption. Absorption via this route may
be enhanced by the use of an oily vehicle (inunction) or through
the use of one or more penetration enhancers. Other effective ways
to deliver a composition disclosed herein via the transdermal route
include hydration of the skin and the use of controlled release
topical patches. The transdermal route provides a potentially
effective means to deliver a composition disclosed herein for
systemic and/or local therapy.
[0632] In addition, iontophoresis (transfer of ionic solutes
through biological membranes under the influence of an electric
field) (Lee et al., Critical Reviews in Therapeutic Drug Carrier
Systems, 1991, p. 163), phonophoresis or sonophoresis (use of
ultrasound to enhance the absorption of various therapeutic agents
across biological membranes, notably the skin and the cornea) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
p. 166), and optimization of vehicle characteristics relative to
dose position and retention at the site of administration (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.
168) may be useful methods for enhancing the transport of topically
applied compositions across skin and mucosal sites.
[0633] The compositions and methods provided may also be used to
examine the function of various proteins and genes in vitro in
cultured or preserved dermal tissues and in animals. The invention
can be thus applied to examine the function of any gene. The
methods of the invention can also be used therapeutically or
prophylactically. For example, for the treatment of animals that
are known or suspected to suffer from diseases such as psoriasis,
lichen planus, toxic epidermal necrolysis, erythema multiforme,
basal cell carcinoma, squamous cell carcinoma, malignant melanoma,
Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease
and viral, fungal and bacterial infections of the skin.
Pulmonary Delivery
[0634] For ease of exposition the formulations, compositions and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It may be understood, however, that these
formulations, compositions and methods can be practiced with other
iRNA agents, e.g., modified iRNA agents, and such practice is
within the invention. A composition that includes an iRNA agent,
e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a
precursor, e.g., a larger iRNA agent which can be processed into a
siRNA agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof)
can be administered to a subject by pulmonary delivery. Pulmonary
delivery compositions can be delivered by inhalation by the patient
of a dispersion so that the composition, for example, iRNA, within
the dispersion can reach the lung where it can be readily absorbed
through the alveolar region directly into blood circulation.
Pulmonary delivery can be effective both for systemic delivery and
for localized delivery to treat diseases of the lungs.
[0635] Pulmonary delivery can be achieved by different approaches,
including the use of nebulized, aerosolized, micellar and dry
powder-based formulations. Delivery can be achieved with liquid
nebulizers, aerosol-based inhalers, and dry powder dispersion
devices. Metered-dose devices are may be used. One of the benefits
of using an atomizer or inhaler is that the potential for
contamination is minimized because the devices are self contained.
Dry powder dispersion devices, for example, deliver drugs that may
be readily formulated as dry powders. A iRNA composition may be
stably stored as lyophilized or spray-dried powders by itself or in
combination with suitable powder carriers. The delivery of a
composition for inhalation can be mediated by a dosing timing
element which can include a timer, a dose counter, time measuring
device, or a time indicator which when incorporated into the device
enables dose tracking, compliance monitoring, and/or dose
triggering to a patient during administration of the aerosol
medicament.
[0636] The term "powder" means a composition that consists of
finely dispersed solid particles that are free flowing and capable
of being readily dispersed in an inhalation device and subsequently
inhaled by a subject so that the particles reach the lungs to
permit penetration into the alveoli. Thus, the powder is said to be
"respirable." For example, the average particle size is less than
about 10 .mu.m in diameter with a relatively uniform spheroidal
shape distribution. In some embodiments, the diameter is less than
about 7.5 .mu.m and in some embodiments less than about 5.0 .mu.m.
Usually the particle size distribution is between about 0.1 .mu.m
and about 5 .mu.m in diameter, sometimes about 0.3 .mu.m to about 5
.mu.m.
[0637] The term "dry" means that the composition has a moisture
content below about 10% by weight (% w) water, usually below about
5% w and in some cases less it than about 3% w. A dry composition
can be such that the particles are readily dispersible in an
inhalation device to form an aerosol.
[0638] The term "therapeutically effective amount" is the amount
present in the composition that is needed to provide the desired
level of drug in the subject to be treated to give the anticipated
physiological response.
[0639] The term "physiologically effective amount" is that amount
delivered to a subject to give the desired palliative or curative
effect.
[0640] The term "pharmaceutically acceptable carrier" means that
the carrier can be taken into the lungs with no significant adverse
toxicological effects on the lungs.
[0641] The types of pharmaceutical excipients that are useful as
carrier include stabilizers such as human serum albumin (HSA),
bulking agents such as carbohydrates, amino acids and polypeptides;
pH adjusters or buffers; salts such as sodium chloride; and the
like. These carriers may be in a crystalline or amorphous form or
may be a mixture of the two.
[0642] Bulking agents that are particularly valuable include
compatible carbohydrates, polypeptides, amino acids or combinations
thereof. Suitable carbohydrates include monosaccharides such as
galactose, D-mannose, sorbose, and the like; disaccharides, such as
lactose, trehalose, and the like; cyclodextrins, such as
2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as
raffinose, maltodextrins, dextrans, and the like; alditols, such as
mannitol, xylitol, and the like. A group of carbohydrates may
includes lactose, trehalose, raffinose maltodextrins, and mannitol.
Suitable polypeptides include aspartame. Amino acids include
alanine and glycine, with glycine being used in some
embodiments.
[0643] Additives, which are minor components of the composition of
this invention, may be included for conformational stability during
spray drying and for improving dispersibility of the powder. These
additives include hydrophobic amino acids such as tryptophan,
tyrosine, leucine, phenylalanine, and the like.
[0644] Suitable pH adjusters or buffers include organic salts
prepared from organic acids and bases, such as sodium citrate,
sodium ascorbate, and the like; sodium citrate may be used in some
embodiments.
[0645] Pulmonary administration of a micellar iRNA formulation may
be achieved through metered dose spray devices with propellants
such as tetrafluoroethane, heptafluoroethane,
dimethylfluoropropane, tetrafluoropropane, butane, isobutane,
dimethyl ether and other non-CFC and CFC propellants.
Oral or Nasal Delivery
[0646] For ease of exposition the formulations, compositions and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It may be understood, however, that these
formulations, compositions and methods can be practiced with other
iRNA agents, e.g., modified iRNA agents, and such practice is
within the invention. Both the oral and nasal membranes offer
advantages over other routes of administration. For example, drugs
administered through these membranes have a rapid onset of action,
provide therapeutic plasma levels, avoid first pass effect of
hepatic metabolism, and avoid exposure of the drug to the hostile
gastrointestinal (GI) environment. Additional advantages include
easy access to the membrane sites so that the drug can be applied,
localized and removed easily.
[0647] In oral delivery, compositions can be targeted to a surface
of the oral cavity, e.g., to sublingual mucosa which includes the
membrane of ventral surface of the tongue and the floor of the
mouth or the buccal mucosa which constitutes the lining of the
cheek. The sublingual mucosa is relatively permeable thus giving
rapid absorption and acceptable bioavailability of many drugs.
Further, the sublingual mucosa is convenient, acceptable and easily
accessible.
[0648] The ability of molecules to permeate through the oral mucosa
appears to be related to molecular size, lipid solubility and
peptide protein ionization. Small molecules, less than 1000 daltons
appear to cross mucosa rapidly. As molecular size increases, the
permeability decreases rapidly. Lipid soluble compounds are more
permeable than non-lipid soluble molecules. Maximum absorption
occurs when molecules are un-ionized or neutral in electrical
charges. Therefore charged molecules present the biggest challenges
to absorption through the oral mucosae.
[0649] A pharmaceutical composition of iRNA may also be
administered to the buccal cavity of a human being by spraying into
the cavity, without inhalation, from a metered dose spray
dispenser, a mixed micellar pharmaceutical formulation as described
above and a propellant. In one embodiment, the dispenser is first
shaken prior to spraying the pharmaceutical formulation and
propellant into the buccal cavity.
Devices
[0650] For ease of exposition the devices, formulations,
compositions and methods in this section are discussed largely with
regard to unmodified iRNA agents. It may be understood, however,
that these devices, formulations, compositions and methods can be
practiced with other iRNA agents, e.g., modified iRNA agents, and
such practice is within the invention. An iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, (e.g., a precursor,
e.g., a larger iRNA agent which can be processed into a siRNA
agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof)
can be disposed on or in a device, e.g., a device which implanted
or otherwise placed in a subject. Exemplary devices include devices
which are introduced into the vasculature, e.g., devices inserted
into the lumen of a vascular tissue, or which devices themselves
form a part of the vasculature, including stents, catheters, heart
valves, and other vascular devices. These devices, e.g., catheters
or stents, can be placed in the vasculature of the lung, heart, or
leg.
[0651] Other devices include non-vascular devices, e.g., devices
implanted in the peritoneum, or in organ or glandular tissue, e.g.,
artificial organs. The device can release a therapeutic substance
in addition to a iRNA, e.g., a device can release insulin.
[0652] Other devices include artificial joints, e.g., hip joints,
and other orthopedic implants.
[0653] In one embodiment, unit doses or measured doses of a
composition that includes iRNA are dispensed by an implanted
device. The device can include a sensor that monitors a parameter
within a subject. For example, the device can include pump, e.g.,
and, optionally, associated electronics.
[0654] Tissue, e.g., cells or organs can be treated with An iRNA
agent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a
precursor, e.g., a larger iRNA agent which can be processed into a
siRNA agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof)
ex vivo and then administered or implanted in a subject.
[0655] The tissue can be autologous, allogeneic, or xenogeneic
tissue. E.g., tissue can be treated to reduce graft v. host
disease. In other embodiments, the tissue is allogeneic and the
tissue is treated to treat a disorder characterized by unwanted
gene expression in that tissue. E.g., tissue, e.g., hematopoietic
cells, e.g., bone marrow hematopoietic cells, can be treated to
inhibit unwanted cell proliferation.
[0656] Introduction of treated tissue, whether autologous or
transplant, can be combined with other therapies.
[0657] In some implementations, the iRNA treated cells are
insulated from other cells, e.g., by a semi-permeable porous
barrier that prevents the cells from leaving the implant, but
enables molecules from the body to reach the cells and molecules
produced by the cells to enter the body. In one embodiment, the
porous barrier is formed from alginate.
[0658] In one embodiment, a contraceptive device is coated with or
contains an iRNA agent, e.g., a double-stranded iRNA agent, or
siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which
can be processed into a siRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or siRNA agent, or
precursor thereof). Exemplary devices include condoms, diaphragms,
IUD (implantable uterine devices, sponges, vaginal sheaths, and
birth control devices. In one embodiment, the iRNA is chosen to
inactive sperm or egg. In another embodiment, the iRNA is chosen to
be complementary to a viral or pathogen RNA, e.g., an RNA of an
STD. In some instances, the iRNA composition can include a
spermicide.
Dosage
[0659] In one aspect, the invention features a method of
administering an iRNA agent, e.g., a double-stranded iRNA agent, or
siRNA agent, to a subject (e.g., a human subject). The method
includes administering a unit dose of the iRNA agent, e.g., a siRNA
agent, e.g., double stranded siRNA agent that (a) the
double-stranded part is 19-25 nucleotides (nt) long, for example,
21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous
or pathogen target RNA), and, optionally, (c) includes at least one
3' overhang 1-5 nucleotide long. In one embodiment, the unit dose
is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1,
0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or
0.00001 mg per kg of bodyweight, and less than 200 nanomole of RNA
agent (e.g., about 4.4.times.10.sup.16 copies) per kg of
bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5,
0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nanomole
of RNA agent per kg of bodyweight.
[0660] The defined amount can be an amount effective to treat or
prevent a disease or disorder, e.g., a disease or disorder
associated with the target RNA. The unit dose, for example, can be
administered by injection (e.g., intravenous or intramuscular), an
inhaled dose, or a topical application. In some embodiments dosages
may be less than 2, 1, or 0.1 mg/kg of body weight.
[0661] In some embodiments, the unit dose is administered less
frequently than once a day, e.g., less than every 2, 4, 8 or 30
days. In another embodiment, the unit dose is not administered with
a frequency (e.g., not a regular frequency). For example, the unit
dose may be administered a single time.
[0662] In one embodiment, the effective dose is administered with
other traditional therapeutic modalities. In one embodiment, the
subject has a viral infection and the modality is an antiviral
agent other than an iRNA agent, e.g., other than a double-stranded
iRNA agent, or siRNA agent. In another embodiment, the subject has
atherosclerosis and the effective dose of an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, is administered in
combination with, e.g., after surgical intervention, e.g.,
angioplasty.
[0663] In one embodiment, a subject is administered an initial dose
and one or more maintenance doses of an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, (e.g., a precursor,
e.g., a larger iRNA agent which can be processed into a siRNA
agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof).
The maintenance dose or doses are generally lower than the initial
dose, e.g., one-half less of the initial dose. A maintenance
regimen can include treating the subject with a dose or doses
ranging from 0.01 Lg to 1.4 mg/kg of body weight per day, e.g., 10,
1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day.
The maintenance doses are, for example, administered no more than
once every 5, 10, or 30 days. Further, the treatment regimen may
last for a period of time which will vary depending upon the nature
of the particular disease, its severity and the overall condition
of the patient. In certain embodiments the dosage may be delivered
no more than once per day, e.g., no more than once per 24, 36, 48,
or more hours, e.g., no more than once for every 5 or 8 days.
Following treatment, the patient can be monitored for changes in
his condition and for alleviation of the symptoms of the disease
state. The dosage of the compound may either be increased in the
event the patient does not respond significantly to current dosage
levels, or the dose may be decreased if an alleviation of the
symptoms of the disease state is observed, if the disease state has
been ablated, or if undesired side-effects are observed.
[0664] The effective dose can be administered in a single dose or
in two or more doses, as desired or considered appropriate under
the specific circumstances. If desired to facilitate repeated or
frequent infusions, implantation of a delivery device, e.g., a
pump, semi-permanent stent (e.g., intravenous, intraperitoneal,
intracisternal or intracapsular), or reservoir may be
advisable.
[0665] In one embodiment, the iRNA agent pharmaceutical composition
includes a plurality of iRNA agent species. In another embodiment,
the iRNA agent species has sequences that are non-overlapping and
non-adjacent to another species with respect to a naturally
occurring target sequence. In another embodiment, the plurality of
iRNA agent species is specific for different naturally occurring
target genes. In another embodiment, the iRNA agent is allele
specific.
[0666] In some cases, a patient is treated with a iRNA agent in
conjunction with other therapeutic modalities. For example, a
patient being treated for a viral disease, e.g., an HIV associated
disease (e.g., AIDS), may be administered a iRNA agent specific for
a target gene essential to the virus in conjunction with a known
antiviral agent (e.g., a protease inhibitor or reverse
transcriptase inhibitor). In another example, a patient being
treated for cancer may be administered a iRNA agent specific for a
target essential for tumor cell proliferation in conjunction with a
chemotherapy.
[0667] Following successful treatment, it may be desirable to have
the patient undergo maintenance therapy to prevent the recurrence
of the disease state, wherein the compound of the invention is
administered in maintenance doses, ranging from 0.01 .mu.g to 100 g
per kg of body weight (see U.S. Pat. No. 6,107,094).
[0668] The concentration of the iRNA agent composition is an amount
sufficient to be effective in treating or preventing a disorder or
to regulate a physiological condition in humans. The concentration
or amount of iRNA agent administered will depend on the parameters
determined for the agent and the method of administration, e.g.,
nasal, buccal, pulmonary. For example, nasal formulations tend to
require much lower concentrations of some ingredients in order to
avoid irritation or burning of the nasal passages. It is sometimes
desirable to dilute an oral formulation up to 10-100 times in order
to provide a suitable nasal formulation.
[0669] Certain factors may influence the dosage required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of an iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof) can include a single treatment or, for
example, can include a series of treatments. It will also be
appreciated that the effective dosage of a iRNA agent such as a
siRNA agent used for treatment may increase or decrease over the
course of a particular treatment. Changes in dosage may result and
become apparent from the results of diagnostic assays as described
herein. For example, the subject can be monitored after
administering a iRNA agent composition. Based on information from
the monitoring, an additional amount of the iRNA agent composition
can be administered.
[0670] Dosing is dependent on severity and responsiveness of the
disease condition to be treated, with the course of treatment
lasting from several days to several months, or until a cure is
effected or a diminution of disease state is achieved. Optimal
dosing schedules can be calculated from measurements of drug
accumulation in the body of the patient. Persons of ordinary skill
can easily determine optimum dosages, dosing methodologies and
repetition rates. Optimum dosages may vary depending on the
relative potency of individual compounds, and can generally be
estimated based on EC50s found to be effective in in vitro and in
vivo animal models. In some embodiments, the animal models include
transgenic animals that express a human gene, e.g., a gene that
produces a target RNA. The transgenic animal can be deficient for
the corresponding endogenous RNA. In another embodiment, the
composition for testing includes a iRNA agent that is
complementary, at least in an internal region, to a sequence that
is conserved between the target RNA in the animal model and the
target RNA in a human.
[0671] The inventors have discovered that iRNA agents described
herein can be administered to mammals, particularly large mammals
such as nonhuman primates or humans in a number of ways.
[0672] In one embodiment, the administration of the iRNA agent,
e.g., a double-stranded iRNA agent, or siRNA agent, composition is
parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible
infusion), intradermal, intraperitoneal, intramuscular,
intrathecal, intraventricular, intracranial, subcutaneous,
transmucosal, buccal, sublingual, endoscopic, rectal, oral,
vaginal, topical, pulmonary, intranasal, urethral or ocular.
Administration can be provided by the subject or by another person,
e.g., a health care provider. The medication can be provided in
measured doses or in a dispenser which delivers a metered dose.
Selected modes of delivery are discussed in more detail below.
[0673] The invention provides methods, compositions, and kits, for
rectal administration or delivery of iRNA agents described
herein.
[0674] Accordingly, an iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or
siRNA agent, or precursor thereof) described herein, e.g., a
therapeutically effective amount of a iRNA agent described herein,
e.g., a iRNA agent having a double stranded region of less than 40,
and, for example, less than 30 nucleotides and having one or two
1-3 nucleotide single strand 3' overhangs can be administered
rectally, e.g., introduced through the rectum into the lower or
upper colon. This approach is particularly useful in the treatment
of, inflammatory disorders, disorders characterized by unwanted
cell proliferation, e.g., polyps, or colon cancer.
[0675] The medication can be delivered to a site in the colon by
introducing a dispensing device, e.g., a flexible, camera-guided
device similar to that used for inspection of the colon or removal
of polyps, which includes means for delivery of the medication.
[0676] The rectal administration of the iRNA agent is by means of
an enema. The iRNA agent of the enema can be dissolved in a saline
or buffered solution. The rectal administration can also by means
of a suppository, which can include other ingredients, e.g., an
excipient, e.g., cocoa butter or hydroxypropylmethylcellulose.
[0677] Any of the iRNA agents described herein can be administered
orally, e.g., in the form of tablets, capsules, gel capsules,
lozenges, troches or liquid syrups. Further, the composition can be
applied topically to a surface of the oral cavity.
[0678] Any of the iRNA agents described herein can be administered
buccally. For example, the medication can be sprayed into the
buccal cavity or applied directly, e.g., in a liquid, solid, or gel
form to a surface in the buccal cavity. This administration is
particularly desirable for the treatment of inflammations of the
buccal cavity, e.g., the gums or tongue, e.g., in one embodiment,
the buccal administration is by spraying into the cavity, e.g.,
without inhalation, from a dispenser, e.g., a metered dose spray
dispenser that dispenses the pharmaceutical composition and a
propellant.
[0679] Any of the iRNA agents described herein can be administered
to ocular tissue. For example, the medications can be applied to
the surface of the eye or nearby tissue, e.g., the inside of the
eyelid. They can be applied topically, e.g., by spraying, in drops,
as an eyewash, or an ointment. Administration can be provided by
the subject or by another person, e.g., a health care provider. The
medication can be provided in measured doses or in a dispenser
which delivers a metered dose. The medication can also be
administered to the interior of the eye, and can be introduced by a
needle or other delivery device which can introduce it to a
selected area or structure. Ocular treatment is particularly
desirable for treating inflammation of the eye or nearby
tissue.
[0680] Any of the iRNA agents described herein can be administered
directly to the skin. For example, the medication can be applied
topically or delivered in a layer of the skin, e.g., by the use of
a microneedle or a battery of microneedles which penetrate into the
skin, but, for example, not into the underlying muscle tissue.
Administration of the iRNA agent composition can be topical.
Topical applications can, for example, deliver the composition to
the dermis or epidermis of a subject. Topical administration can be
in the form of transdermal patches, ointments, lotions, creams,
gels, drops, suppositories, sprays, liquids or powders. A
composition for topical administration can be formulated as a
liposome, micelle, emulsion, or other lipophilic molecular
assembly. The transdermal administration can be applied with at
least one penetration enhancer, such as iontophoresis,
phonophoresis, and sonophoresis.
[0681] Any of the iRNA agents described herein can be administered
to the pulmonary system. Pulmonary administration can be achieved
by inhalation or by the introduction of a delivery device into the
pulmonary system, e.g., by introducing a delivery device which can
dispense the medication. Certain embodiments may use a method of
pulmonary delivery by inhalation. The medication can be provided in
a dispenser which delivers the medication, e.g., wet or dry, in a
form sufficiently small such that it can be inhaled. The device can
deliver a metered dose of medication. The subject, or another
person, can administer the medication.
[0682] Pulmonary delivery is effective not only for disorders which
directly affect pulmonary tissue, but also for disorders which
affect other tissue.
[0683] iRNA agents can be formulated as a liquid or nonliquid,
e.g., a powder, crystal, or aerosol for pulmonary delivery.
[0684] Any of the iRNA agents described herein can be administered
nasally. Nasal administration can be achieved by introduction of a
delivery device into the nose, e.g., by introducing a delivery
device which can dispense the medication. Methods of nasal delivery
include spray, aerosol, liquid, e.g., by drops, or by topical
administration to a surface of the nasal cavity. The medication can
be provided in a dispenser with delivery of the medication, e.g.,
wet or dry, in a form sufficiently small such that it can be
inhaled. The device can deliver a metered dose of medication. The
subject, or another person, can administer the medication.
[0685] Nasal delivery is effective not only for disorders which
directly affect nasal tissue, but also for disorders which affect
other tissue
[0686] iRNA agents can be formulated as a liquid or nonliquid,
e.g., a powder, crystal, or for nasal delivery.
[0687] An iRNA agent can be packaged in a viral natural capsid or
in a chemically or enzymatically produced artificial capsid or
structure derived therefrom.
[0688] The dosage of a pharmaceutical composition including a iRNA
agent can be administered in order to alleviate the symptoms of a
disease state, e.g., cancer or a cardiovascular disease. A subject
can be treated with the pharmaceutical composition by any of the
methods mentioned above.
[0689] Gene expression in a subject can be modulated by
administering a pharmaceutical composition including an iRNA
agent.
[0690] A subject can be treated by administering a defined amount
of an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, (e.g., a precursor, e.g., a larger iRNA agent which can be
processed into a siRNA agent) composition that is in a powdered
form, e.g., a collection of microparticles, such as crystalline
particles. The composition can include a plurality of iRNA agents,
e.g., specific for one or more different endogenous target RNAs.
The method can include other features described herein.
[0691] A subject can be treated by administering a defined amount
of an iRNA agent composition that is prepared by a method that
includes spray-drying, i.e., atomizing a liquid solution, emulsion,
or suspension, immediately exposing the droplets to a drying gas,
and collecting the resulting porous powder particles. The
composition can include a plurality of iRNA agents, e.g., specific
for one or more different endogenous target RNAs. The method can
include other features described herein.
[0692] The iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, (e.g., a precursor, e.g., a larger iRNA agent which can be
processed into a siRNA agent, or a DNA which encodes an iRNA agent,
e.g., a double-stranded iRNA agent, or siRNA agent, or precursor
thereof), can be provided in a powdered, crystallized or other
finely divided form, with or without a carrier, e.g., a micro- or
nano-particle suitable for inhalation or other pulmonary delivery.
This can include providing an aerosol preparation, e.g., an
aerosolized spray-dried composition. The aerosol composition can be
provided in and/or dispensed by a metered dose delivery device.
[0693] The subject can be treated for a condition treatable by
inhalation, e.g., by aerosolizing a spray-dried iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, (e.g., a precursor,
e.g., a larger iRNA agent which can be processed into a siRNA
agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof)
composition and inhaling the aerosolized composition. The iRNA
agent can be an siRNA. The composition can include a plurality of
iRNA agents, e.g., specific for one or more different endogenous
target RNAs. The method can include other features described
herein.
[0694] A subject can be treated by, for example, administering a
composition including an effective/defined amount of an iRNA agent,
e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a
precursor, e.g., a larger iRNA agent which can be processed into a
siRNA agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof),
wherein the composition is prepared by a method that includes
spray-drying, lyophilization, vacuum drying, evaporation, fluid bed
drying, or a combination of these techniques.
[0695] In another aspect, the invention features a method that
includes: evaluating a parameter related to the abundance of a
transcript in a cell of a subject; comparing the evaluated
parameter to a reference value; and if the evaluated parameter has
a preselected relationship to the reference value (e.g., it is
greater), administering a iRNA agent (or a precursor, e.g., a
larger iRNA agent which can be processed into a siRNA agent, or a
DNA which encodes a iRNA agent or precursor thereof) to the
subject. In one embodiment, the iRNA agent includes a sequence that
is complementary to the evaluated transcript. For example, the
parameter can be a direct measure of transcript levels, a measure
of a protein level, a disease or disorder symptom or
characterization (e.g., rate of cell proliferation and/or tumor
mass, viral load).
[0696] In another aspect, the invention features a method that
includes: administering a first amount of a composition that
comprises an iRNA agent, e.g., a double-stranded iRNA agent, or
siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which
can be processed into a siRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or siRNA agent, or
precursor thereof) to a subject, wherein the iRNA agent includes a
strand substantially complementary to a target nucleic acid;
evaluating an activity associated with a protein encoded by the
target nucleic acid; wherein the evaluation is used to determine if
a second amount may be administered. In some embodiments the method
includes administering a second amount of the composition, wherein
the timing of administration or dosage of the second amount is a
function of the evaluating. The method can include other features
described herein.
[0697] In another aspect, the invention features a method of
administering a source of a double-stranded iRNA agent (ds iRNA
agent) to a subject. The method includes administering or
implanting a source of a ds iRNA agent, e.g., a siRNA agent, that
(a) includes a double-stranded region that is 19-25 nucleotides
long, for example, 21-23 nucleotides, (b) is complementary to a
target RNA (e.g., an endogenous RNA or a pathogen RNA), and,
optionally, (c) includes at least one 3' overhang 1-5 nt long. In
one embodiment, the source releases ds iRNA agent over time, e.g.,
the source is a controlled or a slow release source, e.g., a
microparticle that gradually releases the ds iRNA agent. In another
embodiment, the source is a pump, e.g., a pump that includes a
sensor or a pump that can release one or more unit doses.
[0698] In one aspect, the invention features a pharmaceutical
composition that includes an iRNA agent, e.g., a double-stranded
iRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof) including a nucleotide sequence
complementary to a target RNA, e.g., substantially and/or exactly
complementary. The target RNA can be a transcript of an endogenous
human gene. In one embodiment, the iRNA agent (a) is 19-25
nucleotides long, for example, 21-23 nucleotides, (b) is
complementary to an endogenous target RNA, and, optionally, (c)
includes at least one 3' overhang 1-5 nt long. In one embodiment,
the pharmaceutical composition can be an emulsion, microemulsion,
cream, jelly, or liposome.
[0699] In one example the pharmaceutical composition includes an
iRNA agent mixed with a topical delivery agent. The topical
delivery agent can be a plurality of microscopic vesicles. The
microscopic vesicles can be liposomes. In some embodiments the
liposomes are cationic liposomes.
[0700] In another aspect, the pharmaceutical composition includes
an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent
(e.g., a precursor, e.g., a larger iRNA agent which can be
processed into a siRNA agent, or a DNA which encodes an iRNA agent,
e.g., a double-stranded iRNA agent, or siRNA agent, or precursor
thereof) admixed with a topical penetration enhancer. In one
embodiment, the topical penetration enhancer is a fatty acid. The
fatty acid can be arachidonic acid, oleic acid, lauric acid,
caprylic acid, capric acid, myristic acid, palmitic acid, stearic
acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
monolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a C.sub.1-10 alkyl ester, monoglyceride, diglyceride or
pharmaceutically acceptable salt thereof.
[0701] In another embodiment, the topical penetration enhancer is a
bile salt. The bile salt can be cholic acid, dehydrocholic acid,
deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic
acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic
acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate,
sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a
pharmaceutically acceptable salt thereof.
[0702] In another embodiment, the penetration enhancer is a
chelating agent. The chelating agent can be EDTA, citric acid, a
salicyclate, a N-acyl derivative of collagen, laureth-9, an N-amino
acyl derivative of a beta-diketone or a mixture thereof.
[0703] In another embodiment, the penetration enhancer is a
surfactant, e.g., an ionic or nonionic surfactant. The surfactant
can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether,
polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or
mixture thereof.
[0704] In another embodiment, the penetration enhancer can be
selected from a group consisting of unsaturated cyclic ureas,
1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal
anti-inflammatory agents and mixtures thereof. In yet another
embodiment the penetration enhancer can be a glycol, a pyrrol, an
azone, or a terpenes.
[0705] In one aspect, the invention features a pharmaceutical
composition including an iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof) in a form suitable for oral delivery.
In one embodiment, oral delivery can be used to deliver an iRNA
agent composition to a cell or a region of the gastro-intestinal
tract, e.g., small intestine, colon (e.g., to treat a colon
cancer), and so forth. The oral delivery form can be tablets,
capsules or gel capsules. In one embodiment, the iRNA agent of the
pharmaceutical composition modulates expression of a cellular
adhesion protein, modulates a rate of cellular proliferation, or
has biological activity against eukaryotic pathogens or
retroviruses. In another embodiment, the pharmaceutical composition
includes an enteric material that substantially prevents
dissolution of the tablets, capsules or gel capsules in a mammalian
stomach. In some embodiments the enteric material is a coating. The
coating can be acetate phthalate, propylene glycol, sorbitan
monoleate, cellulose acetate trimellitate, hydroxy propyl
methylcellulose phthalate or cellulose acetate phthalate.
[0706] In another embodiment, the oral dosage form of the
pharmaceutical composition includes a penetration enhancer. The
penetration enhancer can be a bile salt or a fatty acid. The bile
salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts
thereof. The fatty acid can be capric acid, lauric acid, and salts
thereof.
[0707] In another embodiment, the oral dosage form of the
pharmaceutical composition includes an excipient. In one example
the excipient is polyethyleneglycol. In another example the
excipient is precirol.
[0708] In another embodiment, the oral dosage form of the
pharmaceutical composition includes a plasticizer. The plasticizer
can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl
phthalate or triethyl citrate.
[0709] In one aspect, the invention features a pharmaceutical
composition including an iRNA agent and a delivery vehicle. In one
embodiment, the iRNA agent is (a) is 19-25 nucleotides long, for
example, 21-23 nucleotides, (b) is complementary to an endogenous
target RNA, and, optionally, (c) includes at least one 3' overhang
1-5 nucleotides long.
[0710] In one embodiment, the delivery vehicle can deliver an iRNA
agent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a
precursor, e.g., a larger iRNA agent which can be processed into a
siRNA agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof)
to a cell by a topical route of administration. The delivery
vehicle can be microscopic vesicles. In one example the microscopic
vesicles are liposomes. In some embodiments the liposomes are
cationic liposomes. In another example the microscopic vesicles are
micelles. In one aspect, the invention features a pharmaceutical
composition including an iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof) in an injectable dosage form. In one
embodiment, the injectable dosage form of the pharmaceutical
composition includes sterile aqueous solutions or dispersions and
sterile powders. In some embodiments the sterile solution can
include a diluent such as water; saline solution; fixed oils,
polyethylene glycols, glycerin, or propylene glycol.
[0711] In one aspect, the invention features a pharmaceutical
composition including an iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof) in oral dosage form. In one
embodiment, the oral dosage form is selected from the group
consisting of tablets, capsules and gel capsules. In another
embodiment, the pharmaceutical composition includes an enteric
material that substantially prevents dissolution of the tablets,
capsules or gel capsules in a mammalian stomach. In some
embodiments the enteric material is a coating. The coating can be
acetate phthalate, propylene glycol, sorbitan monoleate, cellulose
acetate trimellitate, hydroxy propyl methyl cellulose phthalate or
cellulose acetate phthalate. In one embodiment, the oral dosage
form of the pharmaceutical composition includes a penetration
enhancer, e.g., a penetration enhancer described herein.
[0712] In another embodiment, the oral dosage form of the
pharmaceutical composition includes an excipient. In one example
the excipient is polyethyleneglycol. In another example the
excipient is precirol.
[0713] In another embodiment, the oral dosage form of the
pharmaceutical composition includes a plasticizer. The plasticizer
can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl
phthalate or triethyl citrate.
[0714] In one aspect, the invention features a pharmaceutical
composition including an iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof) in a rectal dosage form. In one
embodiment, the rectal dosage form is an enema. In another
embodiment, the rectal dosage form is a suppository.
[0715] In one aspect, the invention features a pharmaceutical
composition including an iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof) in a vaginal dosage form. In one
embodiment, the vaginal dosage form is a suppository. In another
embodiment, the vaginal dosage form is a foam, cream, or gel.
[0716] In one aspect, the invention features a pharmaceutical
composition including an iRNA agent, e.g., a double-stranded iRNA
agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof) in a pulmonary or nasal dosage form.
In one embodiment, the iRNA agent is incorporated into a particle,
e.g., a macroparticle, e.g., a microsphere. The particle can be
produced by spray drying, lyophilization, evaporation, fluid bed
drying, vacuum drying, or a combination thereof. The microsphere
can be formulated as a suspension, a powder, or an implantable
solid.
[0717] In one aspect, the invention features a spray-dried iRNA
agent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a
precursor, e.g., a larger iRNA agent which can be processed into a
siRNA agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof)
composition suitable for inhalation by a subject, including: (a) a
therapeutically effective amount of a iRNA agent suitable for
treating a condition in the subject by inhalation; (b) a
pharmaceutically acceptable excipient selected from the group
consisting of carbohydrates and amino acids; and (c) optionally, a
dispersibility-enhancing amount of a physiologically-acceptable,
water-soluble polypeptide.
[0718] In one embodiment, the excipient is a carbohydrate. The
carbohydrate can be selected from the group consisting of
monosaccharides, disaccharides, trisaccharides, and
polysaccharides. In some embodiments the carbohydrate is a
monosaccharide selected from the group consisting of dextrose,
galactose, mannitol, D-mannose, sorbitol, and sorbose. In another
embodiment the carbohydrate is a disaccharide selected from the
group consisting of lactose, maltose, sucrose, and trehalose.
[0719] In another embodiment, the excipient is an amino acid. In
one embodiment, the amino acid is a hydrophobic amino acid. In some
embodiments the hydrophobic amino acid is selected from the group
consisting of alanine, isoleucine, leucine, methionine,
phenylalanine, proline, tryptophan, and valine. In yet another
embodiment the amino acid is a polar amino acid. In some
embodiments the amino acid is selected from the group consisting of
arginine, histidine, lysine, cysteine, glycine, glutamine, serine,
threonine, tyrosine, aspartic acid and glutamic acid.
[0720] In one embodiment, the dispersibility-enhancing polypeptide
is selected from the group consisting of human serum albumin,
.alpha.-lactalbumin, trypsinogen, and polyalanine.
[0721] In one embodiment, the spray-dried iRNA agent composition
includes particles having a mass median diameter (MMD) of less than
10 microns. In another embodiment, the spray-dried iRNA agent
composition includes particles having a mass median diameter of
less than 5 microns. In yet another embodiment the spray-dried iRNA
agent composition includes particles having a mass median
aerodynamic diameter (MMAD) of less than 5 microns.
[0722] In certain other aspects, the invention provides kits that
include a suitable container containing a pharmaceutical
formulation of an iRNA agent, e.g., a double-stranded iRNA agent,
or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which
can be processed into a siRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or siRNA agent, or
precursor thereof). In certain embodiments the individual
components of the pharmaceutical formulation may be provided in one
container. Alternatively, it may be desirable to provide the
components of the pharmaceutical formulation separately in two or
more containers, e.g., one container for an iRNA agent preparation,
and at least another for a carrier compound. The kit may be
packaged in a number of different configurations such as one or
more containers in a single box. The different components can be
combined, e.g., according to instructions provided with the kit.
The components can be combined according to a method described
herein, e.g., to prepare and administer a pharmaceutical
composition. The kit can also include a delivery device.
[0723] In another aspect, the invention features a device, e.g., an
implantable device, wherein the device can dispense or administer a
composition that includes an iRNA agent, e.g., a double-stranded
iRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a siRNA agent, or a DNA which
encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA
agent, or precursor thereof), e.g., a iRNA agent that silences an
endogenous transcript. In one embodiment, the device is coated with
the composition. In another embodiment the iRNA agent is disposed
within the device. In another embodiment, the device includes a
mechanism to dispense a unit dose of the composition. In other
embodiments the device releases the composition continuously, e.g.,
by diffusion. Exemplary devices include stents, catheters, pumps,
artificial organs or organ components (e.g., artificial heart, a
heart valve, etc.), and sutures.
[0724] As used herein, the term "crystalline" describes a solid
having the structure or characteristics of a crystal, i.e.,
particles of three-dimensional structure in which the plane faces
intersect at definite angles and in which there is a regular
internal structure. The compositions of the invention may have
different crystalline forms. Crystalline forms can be prepared by a
variety of methods, including, for example, spray drying.
[0725] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference.
EXAMPLES
Example 1: Synthesis of a Modular Composition Comprising an iRNA,
an Endosomolytic Component, and a Targeting Ligand
[0726] An iRNA, or other nucleic acid (e.g., antagomir, aptamer,
antisense oligonucleotide, decoy oligonucleotide) is provided. The
endosomolytic component and the targeting ligand may be conjugated
to the selected iRNA or nucleic acid in a variety of formats (FIGS.
1 and 2). In certain embodiments, a linker may be used between the
nucleic acid and the endosomolytic component, the nucleic acid and
the targeting ligand, and/or the targeting ligand and the
endosomolytic component. This linker may be cleavable or
non-cleavable, depending on the application.
Example 2: Oligonucleotide Synthesis and Purification
Step 1. Oligonucleotide Synthesis
[0727] All oligonucleotides were synthesized on an AKTAoligopilot
synthesizer or on an ABI 394 DNA/RNA synthesizer. Commercially
available controlled pore glass solid supports (rU-CPG, 2'-O-methyl
modified rA-CPG and 2'-O-methyl modified rG-CPG from Prime
Synthesis) or the in-house synthesized solid support
hydroxyprolinol-cholesterol-CPG were used for the synthesis. RNA
phosphoramidites and 2'-O-methyl modified RNA phosphoramidites with
standard protecting groups
(5'-O-dimethoxytrityl-N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O---
N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethyl
silyl-cytidine-3'-O--N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethyl
silyl-guanosine-3'-O--N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O--N,N'-diisoprop-
yl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N6-benzoyl-2'-O-methyl-adenosine-3'-O--N,N'-diisopro-
pyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-O-methyl-cytidine-3'-O--N,N'-diisopropy-
l-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N2-isobutryl-2'-O-methyl-guanosine-3'-O--N,N'-diisop-
ropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O--N,N'-diisopropyl-2-cyanoet-
hylphosphoramidite and
5'-O-dimethoxytrityl-2'-deoxy-thymidine-3'-O--N,N'-diisopropyl-2-cyanoeth-
ylphosphoramidite) were obtained from Pierce Nucleic Acids
Technologies and ChemGenes Research. The Quasar 570 phosphoramidite
was obtained from Biosearch Technologies. The
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-inosine-3'-O--N,N'-diisoprop-
yl-2-cyanoethylphosphoramidite was obtained from ChemGenes
Research. The
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-(2,4)-diflurotolyl-3'-O--N,N-
'-diisopropyl-2-cyanoethylphosphoramidite (DFT-phosphoramidite) and
the
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-9-(2-aminoethoxy)-phenoxazin-
e-3'-O--N,N'-diisopropyl-2-cyanoethylphosphoramidite (G-clamp
phosphoramidite) were synthesized in house.
[0728] For the syntheses on AKTAoligopilot synthesizer, all
phosphoramidites were used at a concentration of 0.2 M in
CH.sub.3CN except for guanosine and 2'-O-methyl-uridine, which were
used at 0.2 M concentration in 10% THF/CH.sub.3CN (v/v). A
coupling/recycling time of 16 minutes was used for all
phosphoramidite couplings. The activator was 5-ethyl-thio-tetrazole
(0.75 M, American International Chemicals). For the PO-oxidation,
50 mM iodine in water/pyridine (10:90 v/v) was used and for the
PS-oxidation 2% PADS (GL Synthesis) in 2,6-lutidine/CH.sub.3CN (1:1
v/v) was used. For the syntheses on ABI 394 DNA/RNA synthesizer,
all phosphoramidites, including DFT and G-clamp phosphoramidites
were used at a concentration of 0.15 M in CH.sub.3CN except for
2'-O-methyl-uridine, which was used at 0.15 M concentration in 10%
THF/CH.sub.3CN (v/v). A coupling time of 10 minutes was used for
all phosphoramidite couplings. The activator was
5-ethyl-thio-tetrazole (0.25 M, Glen Research). For the
PO-oxidation, 20 mM iodine in water/pyridine (Glen Research) was
used and for the PS-oxidation 0.1M DDTT (AM Chemicals) in pyridine
was used. Coupling of the Quasar 570 phosphoramidite was carried
out on the ABI DNA/RNA synthesizer. The Quasar 570 phosphoramidite
was used at a concentration of 0.1M in CH.sub.3CN with a coupling
time of 10 mins. The activator was 5-ethyl-thio-tetrazole (0.25 M,
Glen Research) and 0.1M DDTT (AM Chemicals) in pyridine was used
for PS oxidation.
Step 2. Deprotection of Oligonucleotides
[0729] A. Sequences Synthesized on the AKTAoligopilot
Synthesizer
[0730] After completion of synthesis, the support was transferred
to a 100 mL glass bottle (VWR). The oligonucleotide was cleaved
from the support with simultaneous deprotection of base and
phosphate groups with 40 mL of a 40% aq. methyl amine (Aldrich) 90
mins at 45.degree. C. The bottle was cooled briefly on ice and then
the methylamine was filtered into a new 500 mL bottle. The CPG was
washed three times with 40 mL portions of DMSO. The mixture was
then cooled on dry ice.
[0731] In order to remove the tert-butyldimethylsilyl (TBDMS)
groups at the 2' position, 60 mL triethylamine trihydrofluoride
(Et3N--HF) was added to the above mixture. The mixture was heated
at 40.degree. C. for 60 minutes. The reaction was then quenched
with 220 mL of 50 mM sodium acetate (pH 5.5) and stored in the
freezer until purification.
[0732] B. Sequences Synthesized on the ABI DAN/RNA Synthesizer
[0733] After completion of synthesis, the support was transferred
to a 15 mL tube (VWR). The oligonucleotide was cleaved from the
support with simultaneous deprotection of base and phosphate groups
with 7 mL of a 40% aq. methyl amine (Aldrich) 15 mins at 65.degree.
C. The bottle was cooled briefly on ice and then the methylamine
was filtered into a 100 mL bottle (VWR). The CPG was washed three
times with 7 mL portions of DMSO. The mixture was then cooled on
dry ice.
[0734] In order to remove the tert-butyldimethylsilyl (TBDMS)
groups at the 2' position, 10.5 mL triethylamine trihydrofluoride
(Et3N--HF) was added to the above mixture. The mixture was heated
at 60.degree. C. for 15 minutes. The reaction was then quenched
with 38.5 mL of 50 mM sodium acetate (pH 5.5) and stored in the
freezer until purification.
Step 3. Quantitation of Crude Oligonucleotides
[0735] For all samples, a 10 .mu.L aliquot was diluted with 990
.mu.L of deionised nuclease free water (1.0 mL) and the absorbance
reading at 260 nm was obtained.
Step 4. Purification of Oligonucleotides
[0736] (a) Unconjugated Oligonucleotides
[0737] The unconjugated crude oligonucleotides were first analyzed
by HPLC (Dionex PA 100). The buffers were 20 mM phosphate, pH 11
(buffer A); and 20 mM phosphate, 1.8 M NaBr, pH 11 (buffer B). The
flow rate 1.0 mL/min and monitored wavelength was 260-280 nm.
Injections of 5-15 .mu.L were done for each sample.
[0738] The unconjugated samples were purified by HPLC on a TSK-Gel
SuperQ-5PW (20) column packed in house (17.3.times.5 cm) or on a
commercially available TSK-Gel SuperQ-5PW column (15.times.0.215
cm) available from TOSOH Bioscience. The buffers were 20 mM
phosphate in 10% CH.sub.3CN, pH 8.5 (buffer A) and 20 mM phosphate,
1.0 M NaBr in 10% CH.sub.3CN, pH 8.5 (buffer B). The flow rate was
50.0 mL/min for the in house packed column and 10.0 ml/min for the
commercially obtained column. Wavelengths of 260 and 294 nm were
monitored. The fractions containing the full-length
oligonucleotides were pooled together, evaporated, and
reconstituted to .about.100 mL with deionised water.
[0739] (b) Cholesterol-Conjugated Oligonucleotides
[0740] The cholesterol-conjugated crude oligonucleotides were first
analyzed by LC/MS to determine purity. The cholesterol conjugated
sequences were HPLC purified on RPC-Source15 reverse-phase columns
packed in house (17.3.times.5 cm or 15.times.2 cm). The buffers
were 20 mM NaOAc in 10% CH.sub.3CN (buffer A) and 20 mM NaOAc in
70% CH.sub.3CN (buffer B). The flow rate was 50.0 mL/min for the
17.3.times.5 cm column and 12.0 mL/min for the 15.times.2 cm
column. Wavelengths of 260 and 284 nm were monitored. The fractions
containing the full-length oligonucleotides were pooled,
evaporated, and reconstituted to 100 mL with deionised water.
Step 5. Desalting of Purified Oligonucleotides
[0741] The purified oligonucleotides were desalted on either an
AKTA Explorer or an AKTA Prime system (Amersham Biosciences) using
a Sephadex G-25 column packed in house. First, the column was
washed with water at a flow rate of 40 mL/min for 20-30 min. The
sample was then applied in 40-60 mL fractions. The eluted salt-free
fractions were combined, dried, and reconstituted in .about.50 mL
of RNase free water.
Step 6. Purity Analysis by Capillary Gel Electrophoresis (CGE),
Ion-exchange HPLC (IEX), and Electrospray LC/Ms
[0742] Approximately 0.3 OD of each of the desalted
oligonucleotides were diluted in water to 300 .mu.L and were
analyzed by CGE, ion exchange HPLC, and LC/MS.
Step 7. Duplex Formation
[0743] For the fully double stranded duplexes, equimolar amounts of
two strands were mixed together. The mixtures were frozen at
-80.degree. C. and dried under vacuum on a speed vac. Dried samples
were then dissolved in 1.times.PBS to a final concentration of 40
mg/mL. The dissolved samples were heated to 95.degree. C. for 5 min
and slowly cooled to room temperature.
Step 8. Tm Determination
[0744] For the partial double stranded duplexes and hairpins
melting temperatures were determined. For the duplexes, equimolar
amounts of the two single stranded RNAs were mixed together. The
mixtures were frozen at -80.degree. C. and dried under vacuum on a
speed vac. Dried samples were then dissolved in 1.times.PBS to a
final concentration of 2.5 .mu.M. The dissolved samples were heated
to 95.degree. C. for 5 min and slowly cooled to room temperature.
Denaturation curves were acquired between 10-90.degree. C. at 260
nm with temperature ramp of 0.5.degree. C./min using a Beckman
spectrophotometer fitted with a 6-sample thermostated cell block.
The Tm was then determined using the 1st derivative method of the
manufacturer's supplied program.
Example 3: 2'-F Oligonucleotide Synthesis
1. Oligonucleotide Synthesis.
[0745] All oligonucleotides were synthesized on an AKTAoligopilot
synthesizer. Commercially available controlled pore glass solid
support (dT-CPG, 500 .ANG., Prime Synthesis) and RNA
phosphoramidites with standard protecting groups,
5'-O-dimethoxytrityl N6-benzoyl-2'-t-butyldimethyl
silyl-adenosine-3'-O--N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-O--N,N-
'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethylsilyl-guanosine-3'-O--
-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and
5'-O-dimethoxytrityl-2'-t-butyldimethyl
silyl-uridine-3'-O--N,N'-diisopropyl-2-cyanoethylphosphoramidite
(Pierce Nucleic Acids Technologies) were used for the
oligonucleotide synthesis. The 2'-F phosphoramidites,
5'-O-dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3'-O--N,N'-diisopropyl-2-
-cyanoethyl-phosphoramidite and
5'-O-dimethoxytrityl-2'-fluro-uridine-3'-O--N,N'-diisopropyl-2-cyanoethyl-
-phosphoramidite were purchased from (Promega). All
phosphoramidites were used at a concentration of 0.2M in
acetonitrile (CH.sub.3CN) except for guanosine which was used at
0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of
16 minutes was used. The activator was 5-ethyl thiotetrazole
(0.75M, American International Chemicals), for the PO-oxidation
Iodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in
2,6-lutidine/ACN (1:1 v/v) was used. The cholesterol
phosphoramidite was synthesized in house, and used at a
concentration of 0.1 M in dichloromethane. Coupling time for the
cholesterol phosphoramidite was 16 minutes.
2. Deprotection-I (Nucleobase Deprotection)
[0746] After completion of synthesis, the support was transferred
to a 100 mL glass bottle (VWR). The oligonucleotide was cleaved
from the support with simultaneous deprotection of base and
phosphate groups with 80 mL of a mixture of ethanolic ammonia
[ammonia:ethanol (3:1)] for 6.5 h at 55.degree. C. The bottle was
cooled briefly on ice and then the ethanolic ammonia mixture was
filtered into a new 250 mL bottle. The CPG was washed with
2.times.40 mL portions of ethanol/water (1:1 v/v). The volume of
the mixture was then reduced to 30 mL by roto-vap. The mixture was
then frozen on dyince and dried under vacuum on a speed vac.
3. Deprotection-II (Removal of 2' TBDMS Group)
[0747] The dried residue was resuspended in 26 mL of triethylamine,
triethylamine trihydrofluoride (TEA.3HF) and DMSO (3:4:6) and
heated at 60.degree. C. for 90 minutes to remove the
tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The
reaction was then quenched with 50 mL of 20 mM sodium acetate and
pH adjusted to 6.5, and stored in freezer until purification.
4. Quantitation of Crude Oligomer or Raw Analysis
[0748] For all samples, a 10 .mu.l aliquot was diluted with 990
.mu.l of deionised nuclease free water (1.0 mL) and absorbance
reading obtained at 260 nm.
5. Purification of Oligomers
[0749] (a) HPLC Purification
[0750] The crude oligomers were first analyzed by HPLC (Dionex PA
100). The buffer system was: A=20 mM phosphate pH 11, B=20 mM
phosphate, 1.8 M NaBr, pH 11, flow rate 1.0 mL/min, and wavelength
260-280 nm. Inject 5-15 .mu.l of the each sample. The unconjugated
samples were purified by HPLC on a TSK-Gel SuperQ-5PW (20) column
packed in house (17.3.times.5 cm). The buffer system was: A=20 mM
phosphate in 10% ACN, pH 8.5 and B=20 mM phosphate, 1.0 M NaBr in
10% ACN, pH 8.5, with a flow rate of 50.0 mL/min, and wavelength
260 and 294. The 5'-cholesterol conjugated sequences were HPLC
purified using a reverse-phase column. The buffer system was: A=20
mM TEAA in 10% ACN and B=20 mM TEAA in 70% ACN. The fractions
containing the full length oligonucleotides were then pooled
together, evaporated and reconstituted to 100 mL with deionised
water.
6. Desalting of Purified Oligomer
[0751] The purified oligonucleotides were desalted using AKTA
Explorer (Amersham Biosciences) using Sephadex G-25 column. First
column was washed with water at a flow rate of 25 mL/min for 20-30
min. The sample was then applied in 25 mL fractions. The eluted
salt-free fractions were combined together, dried down and
reconstituted in 50 mL of RNase free water.
7. Capillary Gel Electrophoresis (CGE) and Electrospray LC/MS
[0752] Approximately 0.15 OD of desalted oligonucleotides were
diluted in water to 150 .mu.l and then pipetted into vials for CGE
and LC/MS analysis.
Table 5 lists some of the oligonucleotides synthesized.
TABLE-US-00005 TABLE 5 Factor VII siRNA and the corresponding
scrambled control modified with 2'-F and with or without
cholesterol conjugation at the 5'-end of sense sequences. Sequence
Cal Found CGE ID Sequence* Mass Mass (%) AL-2918
Q11GGAfUfCAfUfCfUfCAAGfUfCfUfUAfCdT*dT 7332.93 7333.61 99.91 (SEQ
ID NO: 37) AL-2919 Q11GGAfCfUAfCfUfCfUAAGfUfUfCfUAfCdT*dT 7332.93
7333.62 99.59 (SEQ ID NO: 38) AL-2920
GGAfCfUAfCfUfCfUAAGfUfUfCfUAfCdT*dT (SEQ 6628.93 6628.45 99.61 ID
NO: 39) AL-2921 GfUAGAAfCfUfUAGAGfUAGfUfCfCdT*dT (SEQ 6726.04
6725.78 96.01 ID NO: 40) AL-4723
GGAfUfCAfUfCfUfCAAGfUfCfUfUAfCdT*dT (SEQ 6628.93 6628.47 98.94 ID
NO: 41) AL-4724 GfUAAGAfCfUfUGAGAfUGAfUfCfCdT*dT (SEQ 6726.04
6725.56 96.29 ID NO: 42) Q11 = cholesterol, fN = 2'-fluro and * =
thioate linkage
TABLE-US-00006 TABLE 6 Listing of some Factor VII siRNA
sequencesconjugated with endosomolytic agents and targeting
ligands. Strand Target Seq. ID (S/AS) Sequence (5' to 3') FVII 43 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsL8 FVII 44 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsL99 FVII 45 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8L99 FVII 46 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96L8 FVII 47 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8L99 FVII 48 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96L8 FVII 49 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11L8 FVII 50 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11L99 FVII 51 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8L10 FVII 52 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96L10 FVII 53 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8L50 FVII 54 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96L8 FVII 55 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsL50 FVII 56 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8Q11L50 FVII 57 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96Q11L8 FVII 58 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8L110 FVII 59 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96L110 FVII 60 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8Q11L110 FVII 61 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96Q8L110 FVII 62 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQl1Q8L110 FVII 63 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQl1Q96L110 FVII 64 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8L80 FVII 65 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96L80 FVII 66 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8Q11L80 FVII 67 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96Q8L80 FVII 68 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQl1Q8L80 FVII 69 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11Q96L80 FVII 70 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8L90 FVII 71 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96L90 FVII 72 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ8Q11L90 FVII 73 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96Q8L90 FVII 74 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11Q8L90 FVII 75 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11Q96L90 FVII 76 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)L50, where n = 1 to 10
FVII 77 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)L135,
where n = 0 to 10 FVII 78 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q11L50, where n = 1 to 10 FVII 79 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11(n x Q94)L50; where n = 1 to
10 FVII 80 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q96L10;
where n = 1 to 10 FVII 81 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)L10; where n = 0 to
10 FVII 82 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96Q11(n x
Q94)L135; where n = 1 to 10 FVII 83 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11(n x Q94)L50; where n = 0 to
10 FVII 84 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x
Q94)L50, where n = 1 to 10 FVII 85 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x Q94)L50; where
n = 1 to 10 FVII 86 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q96(n x Q94)L10; where n = 1 to 10 FVII 87 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q96(n x Q94)L10; where
n = 0 to 10 FVII 88 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x
Q94)Q11(n x Q94)L135; where n = 1 to 10 FVII 89 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x Q94)L50; where
n = 0 to 10 FVII 90 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)L96L110, where n = 1 to 10 FVII 91 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)L110, where n = 0 to
10 FVII 92 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11L110,
where n = 1 to 10 FVII 93 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11(n x Q94)L110; where n = 1 to
10 FVII 94 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q96L110;
where n = 1 to 10 FVII 95 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q96Q11L110; where n = 1 to 10 FVII 96 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11Q96L110; where n = 1
to 10 FVII 97 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q11Q96(n x Q94)L110; where n = 1 to 10 FVII 98 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11 (n x Q94) Q96L110;
where n = 1 to 10 FVII 99 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)L110; where n = 0 to
10 FVII 100 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96Q11(n x
Q94)L110; where n = 1 to 10 FVII 101 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11(n x Q94)L110; where n = 0 to
10 FVII 102 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x
Q94)Q96L110, where n = 1 to 10 FVII 103 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x Q94)L110; where
n = 1 to 10 FVII 104 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q11(n x Q94)Q11L110; where n = 1 to 10 FVII 105 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q96(n x Q94)Q11L110;
where n = 1 to 10 FVII 106 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q96(n x Q94)Q11L110; where n = 0 to 10 FVII 107 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)Q11(n x Q94)L110;
where n = 1 to 10 FVII 108 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q11(n x Q94)Q96L110; where n = 0 to 10 FVII 109 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)L96L80, where n = 1 to
10 FVII 110 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)L80,
where n = 0 to 10 FVII 111 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q11L80, where n = 1 to 10 FVII 112 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11(n x Q94)L80; where n = 1 to
10 FVII 113 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q96L80;
where n = 1 to 10 FVII 114 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q96Q11L80; where n = 1 to 10 FVII 115 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11Q96L80; where n = 1
to 10 FVII 116 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q11Q96(n x Q94)L80; where n = 1 to 10 FVII 117 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11 (n x Q94) Q96L80;
where n = 1 to 10 FVII 118 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)L80; where n = 0 to
10 FVII 119 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96Q11(n x
Q94)L80; where n = 1 to 10 FVII 120 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11(n x Q94)L80; where n = 0 to
10 FVII 121 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x
Q94)Q96L80, where n = 1 to 10 FVII 122 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x Q94)L80; where
n = 1 to 10 FVII 123 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q11(n x Q94)Q11L80; where n = 1 to 10 FVII 124 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q96(n x Q94)Q11L80;
where n = 1 to 10 FVII 125 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q96(n x Q94)Q11L80; where n = 0 to 10 FVII 126 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)Q11(n x Q94)L80;
where n = 1 to 10 FVII 127 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q11(n x Q94)Q96L80; where n = 0 to 10 FVII 128 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)L96L90, where n = 1 to
10 FVII 129 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)L90,
where n = 0 to 10 FVII 130 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q11L90, where n = 1 to 10 FVII 131 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11(n x Q94)L 90; where n = 1 to
10 FVII 132 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q96L90;
where n = 1 to 10 FVII 133 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q96Q11L90; where n = 1 to 10 FVII 134 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11Q96L90; where n = 1
to 10
FVII 135 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11Q96(n x
Q94)L90; where n = 1 to 10 FVII 136 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11 (n x Q94) Q96L90;
where n = 1 to 10 FVII 137 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)L90; where n = 0 to
10 FVII 138 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96Q11(n x
Q94)L90; where n = 1 to 10 FVII 139 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ11(n x Q94)L90; where n = 0 to
10 FVII 140 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x
Q94)Q96L90, where n = 1 to 10 FVII 141 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x Q94)L90; where
n = 1 to 10 FVII 142 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q11(n x Q94)Q11L90; where n = 1 to 10 FVII 143 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q96(n x Q94)Q11L90;
where n = 1 to 10 FVII 144 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q96(n x Q94)Q11L90; where n = 0 to 10 FVII 145 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ96(n x Q94)Q11(n x Q94)L90;
where n = 1 to 10 FVII 146 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q11(n x Q94)Q96L90; where n = 0 to 10 FVII 147 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsL132 FVII 148 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsL133 FVII 149 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsL134 FVII 150 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsL136 FVII 151 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95L99 FVII 152 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95L50 FVII 153 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95L8 FVII 154 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95Q96L110 FVII 155 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95Q8L110 FVII 156 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95L110 FVII 157 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95Q96L80 FVII 158 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95Q8L80 FVII 159 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95L80 FVII 160 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95Q96L90 FVII 161 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95Q8L90 FVII 162 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ95L90 FVII 163 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)L133, where n = 1 to 10
FVII 164 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)L133, where
n = 0 to 10 FVII 165 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q7L110, where n = 1 to 10 FVII 166 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ7(n x Q94)L110; where n = 1 to
10 FVII 167 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q7L110;
where n = 1 to 10 FVII 168 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ7(n x Q94)L110; where n = 0 to
10 FVII 169 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ7Q11(n x
Q94)L110; where n = 1 to 10 FVII 170 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q7(n x Q94)L110, where
n = 1 to 10 FVII 171 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x
Q94)Q11(n x Q94)Q7L110; where n = 0 to 10 FVII 172 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q7L80, where n = 1 to
10 FVII 173 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ7(n x Q94)L80;
where n = 1 to 10 FVII 174 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q7L80; where n = 1 to 10 FVII 175 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ7(n x Q94)L80; where n = 0 to
10 FVII 176 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ7Q11(n x Q94)L80;
where n = 1 to 10 FVII 177 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q7(n x Q94)L80, where n = 1 to 10 FVII 178 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x Q94)Q7L80;
where n = 0 to 10 FVII 179 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q7L90, where n = 1 to 10 FVII 180 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ7(n x Q94)L90; where n = 1 to
10 FVII 181 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q7L90;
where n = 1 to 10 FVII 182 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ7(n x Q94)L80; where n = 0 to
10 FVII 183 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTsQ7Q11(n x Q94)L90;
where n = 1 to 10 FVII 184 S GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n
x Q94)Q7(n x Q94)L90, where n = 1 to 10 FVII 185 S
GGAufcfAufcfufcfAAGufcfufufAcfdTdTs(n x Q94)Q11(n x Q94)Q7L90;
where n = 0 to 10 FVII 186 AS GufAAGAcfufufGAGAufGAufcfcfdTsdT
Note: nf is 2'-fluoro modified nucleotide, s is phsophorothioate
linkage, other symbols are as shown below.
##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025##
[0753] While this invention has been particularly shown and
described with reference to certain embodiments thereof, it will be
understood by those skilled in the art that various changes in form
and details may be made therein without departing from the scope of
the invention encompassed by the appended claims. For example, the
compositions and methods described herein may be useful when used
in combination with any number of oligonucleotide modifications,
targeting ligands, or membrane active agents, and methods related
thereto, as well as other compositions and methods known in the
art.
[0754] In particular, non-limiting examples of such compositions
and methods, which may be used with the instant invention, may be
found in the following co-owned applications, all of which are
expressly incorporated by reference in their entirety: U.S. Ser.
No. 11/115,989, filed Apr. 27, 2005; U.S. Ser. No. 11/119,533,
filed Apr. 29, 2005; U.S. Ser. No. 11/186,915, filed Jul. 21, 2005;
U.S. Ser. No. 11/834,140, filed Aug. 26, 2007; U.S. Ser. No.
11/197,753, filed Aug. 4, 2005; U.S. Ser. No. 11/099,430, filed
Apr. 5, 2005; U.S. Ser. No. 11/170,798, filed Jun. 29, 2005; and
U.S. Ser. No. 11/560,336, filed Jun. 14, 2004.
##STR00026##
[0755] Synthesis of Compound 901:
[0756] Hydrazone monohydrate (0.80 g, 16 mmol) was added to a
solution of commercially available compound 900 (2.00 g, 6.40 mmol)
in THF (30 mL) and the mixture was stirred for 1 h at room
temperature. Filtration to remove NHS, removal of solvent then
column chromatography gave pure compound 901, 0.89 g, 61%. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 9.06 (s, 1H), 8.43 (d, J=4.1,
1H), 7.81 (td, J=7.9, 1.8, 1H), 7.73 (d, J=8.1, 1H), 7.26-7.18 (m,
1H), 2.99 (t, J=7.1, 2H), 2.51-2.45 (m, 2H), 2.42 (t, J=7.1, 2H).
.sup.13C NMR (101 MHz, DMSO-d.sub.6) .delta. 170.16, 160.01,
150.56, 138.80, 122.15, 120.11, 34.85, 33.70. Electrospray/APCI
dual mode MS (+ve): Molecular weight for
C.sub.8H.sub.12N.sub.3O.sub.8S.sub.2 (MH).sup.+ Calc. 230.0, Found
229.9.
[0757] Synthesis of Compound 903:
[0758] Compound 902 was prepared as previously described
(Bioconjugate Chem. 2002, 13, 47-58). An ethanolic solution (40 mL)
of compound 902 (0.85 g, 3.84 mmol), compound 901 (0.88 g, 3.84
mmol) and AcOH (2 mL) was heated for 6 h at 45.degree. C. On
cooling to room temperature, the resulting colorless precipitate
was collected by filtration and dried under suction. Yield of pure
compound 903 was 1.49 g, 90%. .sup.1H NMR (400 MHz, DMSO run at
80.degree. C.) .delta. 11.82 (s, 1H), 10.18 (s, 1H), 8.48 (d,
J=4.1, 1H), 7.81 (d, J=4.0, 2H), 7.69 (s, 2H), 7.24 (d, J=3.9, 1H),
6.96 (d, J=8.6, 2H), 4.10 (t, J=6.5, 2H), 3.20 (t, J=6.8, 2H), 3.07
(s, 2H), 2.43 (t, J=7.3, 2H), 2.24 (s, 3H), 2.02 (p, J=6.9, 2H).
.sup.13C NMR (101 MHz, DMSO-d.sub.6, run at 80.degree. C.) .delta.
174.16, 160.00, 159.90, 149.95, 137.96, 131.38, 127.97, 121.57,
120.16, 114.94, 67.57, 34.67, 30.74, 24.89. Electrospray/APCI dual
mode MS (+ve): Molecular weight for
C.sub.20H.sub.24N.sub.3O.sub.4S.sub.2 (MH).sup.+ Calc. 434.1, Found
434.1.
[0759] Synthesis of Compound 904:
[0760] A DMF solution (4 mL) of DCC (181 mg, 0.878 mmol) was added
to a DMF solution (1 mL) of NaSO.sub.3NHS (173 mg, 0.798 mmol),
DMAP (5 mg, 0.04 mmol) and compound 903 (346 mg, 0.798 mmol) and
stirred at room temperature for 18 h. The mixture was cooled on an
ice bath for 2 h, the dicyclohexylurea was removed by filtration
and the product was precipitated by addition of EtOAc (100 mL) to
the filtrate with stirring. The pale cream solid, compound 904 (426
mg, 84%) was collected by filtration and dried in vacuo. The
material was found to contain 10 mol % of NaSO.sub.3NHS and 30 mol
% water by elemental analysis. .sup.1H NMR shows an E/Z mixture of
isomers in 3:1 ratio which resolves to a single compound on
heating. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.41
(2.times.s, 1H), 8.43 (2.times.d, J=4.6, 1H), 7.85-7.75 (m, 2H),
7.65 (2.times.d, J=8.8, 2H), 7.32-7.12 (m, 1H), 6.94 (2.times.d,
J=8.9, 2H), 4.07 (t, J=6.2, 2H), 3.94 (s, 1H), 3.15-3.01 (m, 4H),
2.91-2.78 (m, 3H), 2.74 (2.times.d, 5.4, 1H), 2.18 (2.times.s, 3H),
2.13-2.00 (m, 2H). Elemental Analysis: Calculated for
(C.sub.24H.sub.25N.sub.4NaO.sub.9S.sub.3)
(C.sub.4H.sub.4NNaO.sub.6S).sub.0.1(H.sub.2O).sub.0.3: C: 44.42, H:
3.98, N: 8.71, S: 15.04, Found: C: 44.32, H: 4.01, N: 8.68, S:
15.26.
[0761] Synthesis of Compound 906:
[0762] Compound 905 was prepared as previously described
(Paramonov, Sergey E.; Bachelder, Eric M.; Beaudette, Tristan T.;
Standley, Stephany M.; Lee, Cameron C.; Dashe, Jesse; Frechet, Jean
M. J. Bioconjugate Chemistry (2008), 19(4), 911-919)
[0763] A DCM solution (10 mL) of compound 900 (1.76 g, 5.6 mmol)
was added to a cooled (-78.degree. C.) methanolic solution (30 mL)
of compound 905 (1.00 g, 6.16 mmol) and NEt.sub.3 (1.4 g, 14 mmol).
The mixture was warmed to room temperature over 2 h. The solvent
was removed and the residue was subjected to column chromatography
(0-10% of 7N methanolic NH.sub.3 in DCM) to give compound 906 (1.38
g, 69%) as an unstable colorless oil which was used without delay
in the next step of the reaction. Electrospray/APCI dual mode MS
(+ve): Molecular weight for C.sub.15H.sub.26N.sub.3O.sub.3S.sub.2
(MH).sup.+ Calc. 360.1, Found 360.1.
[0764] Synthesis of Compound 907:
[0765] Succinic anhydride (578 mg, 5.77 mmol) was added to a DCM
solution (50 mL) of freshly prepared compound 906 (1.38 g, 3.84
mmol) and NEt.sub.3 (1.6 g, 15.4 mmol) and stirred at room
temperature for 1 h. The mixture was subjected to column
chromatographic purification to give compound 907 (1.83 g, 95%) as
a partial triethylammonium salt. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 11.99 (s, 1H), 8.44 (dd, J=4.8, 0.8, 1H),
8.04 (d, J=5.4, 1H), 7.92 (d, J=5.5, 1H), 7.86-7.79 (m, 1H), 7.75
(d, J=8.1, 1H), 7.23 (ddd, J=7.3, 4.8, 1.0, 1H), 3.32 (q, J=5.9,
6H), 3.14 (dq, J=11.8, 5.9, 4H), 2.99 (t, J=7.1, 2H), 2.37 (t,
J=6.6, 2H), 2.28 (t, J=6.6, 2H), 1.24 (s, 6H). Electrospray/APCI
dual mode MS (+ve): Molecular weight for
C.sub.19H.sub.28N.sub.3O.sub.6S.sub.2 (M-H).sup.+ Calc. 458.2,
Found 458.0.
[0766] Synthesis of Compound 909:
[0767] A DCM solution (8 mL) of compound 908 (385 mg, 0.54 mmol)
was added all at once to a DMF solution (8 mL) of compound 907 (248
mg, 0.54 mmol), .sup.iPr.sub.2NEt (0.17 g, 1.35 mmol) and HBTU (205
mg, 0.54 mmol) and the mixture was stirred at room temperature for
1 h. Aqueous workup then column chromatography gave pure 909 (488
mg, 78%) as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.58-8.41 (m, 1H), 7.77-7.59 (m, 2H), 7.12 (ddd, J=6.7,
4.9, 1.8, 1H), 7.05 (s, 1H), 6.31 (s, 1H), 6.20 (s, 1H), 5.45-5.21
(m, 8H), 4.27-4.15 (m, 1H), 4.13-4.03 (m, 1H), 3.60-3.34 (m, 9H),
3.22 (dd, J=13.1, 6.9, 2H), 3.09 (t, J=6.8, 2H), 2.78 (t, J=6.5,
4H), 2.66 (t, J=6.8, 2H), 2.59-2.47 (m, 5H), 2.47-2.31 (m, 3H),
2.27 (s, 3H), 2.13-1.98 (m, 8H), 1.68-1.54 (m, 8H), 1.54-1.42 (m,
4H), 1.42-1.19 (m, 42H), 0.90 (t, J=6.9, 6H). Electrospray/APCI
dual mode MS (+ve): Molecular weight for:
C.sub.66H.sub.116N.sub.5O.sub.7S.sub.2 (MH).sup.+ Calc. 1154.8,
Found 1154.8.
Example 4
##STR00027##
[0769] Synthesis of Compound 112:
[0770] To the suspension of compound 111 (1.0 g, 1.629 mmol, 1.0
eq) and NaBH(OAc).sub.3 (0.46 g, 2.1 mmol, 1.3 eq) in DCM (12.0 ml)
at 0.degree. C. was added a solution of compound 110 (0.28 g, 1.79
mmol, 1.1 eq) in DCM (3.0 ml) at 0.degree. C. under argon and the
reaction was continued at room temperature for 1 hour. After
completion of the starting material the reaction was quenched with
1N MaHCO.sub.3, diluted with DCM, separated the two layers and the
combined organics were washed with brine, dried over MgSO.sub.4,
concentrated and purified by column chromatography using DCM:MeOH
(5%):triethylamine (0.5%) as a gradients to get pure 0.87 g (70%)
of the ester compound 112. MS: Calc. mass for
C.sub.49H.sub.89N.sub.2O.sub.4: 756.68, found: 757.7
(MH).sup.+.
[0771] Synthesis of Compound 114:
[0772] To a solution of compound 113 (3.0 g, 4.12 mmol, 1.0 eq) in
dichloromethane (40.0 ml) was added solid N-hydroxysuccinimide
(NHS) (0.52 g, 4.53 mmol, 1.1 eq) followed by DCC (0.93 g, 4.53
mmol, 1.1 eq), DMAP (0.05 g, 0.45 mmol, 0.1 eq) and the reaction
was continued at room temperature under argon atmosphere. A white
precipitate of urea derivative appears slowly in the reaction,
continued the reaction for overnight, after complete consumption of
the staring material, dichloromoethane was evaporated. Dissolved
the reaction mixture in the ethylacetate (25.0 ml), cooled to ice
bath temperature to complete precipitate out the DCC urea
derivative. Filtered the solid and washed the solid with cold
ethylacetate (2.times.6.0 ml), evaporated the solvent and dried on
vacuum to give 3.4 g of the pure NHS ester of the compound 114 in
quantitative yields, this was directly used in the next step
without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 5.50-5.16 (m, 8H), 4.52 (brs, 1H), 4.25-4.05 (m, 1H), 3.52
(t, J=7.9, 1H), 2.95-2.52 (m, 16H), 2.11-1.97 (m, 8H), 1.82-1.76
(m, 3H), 1.64-1.42 (m, 6H), 1.40-1.11 (m, 38H), 0.88 (t, J=6.8,
6H); MS: Calc. mass for C.sub.51H.sub.88N.sub.2O.sub.6: 824.6,
found: 825.5 (MH).sup.+.
Example 5
##STR00028## ##STR00029##
[0774] Synthesis of Compound 116:
[0775] A suspension of 6-bromo-1-hexanol 115 (55.2 mmol, 10 g),
sodium azide (132 mmol, 8.6 g) in DMF (200 mL) was heated to
100.degree. C. in an autoclave for 15 hours (TLC). The reaction
mixture was then cooled to room temperature, diluted with diethyl
ether (500 ml), washed with water (4.times.100 ml) followed by
brine (1.times.100 ml), dried over sodium sulfate and evaporated at
reduced pressure to obtain the desired product. The crude product
was taken to the next stage without further purification. Yield
(7.5 g, 94.5% as pale yellow oil). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 1.41 (t, 4H, J=3.6 Hz), 1.61 (m, 4H), 3.28 (t,
2H, J=6.8 Hz), 3.66 (t, 2H, J=6.8 Hz).
[0776] Synthesis of Compound 117:
[0777] PCC (17 g, 78.6 mol) adsorbed on silica gel (17 g) was added
portion wise over a period of 5 minutes to a solution of 116 (7.5
g, 52.4 mmol) product in dichloromethane (75 mL) at RT. After
addition, the reaction mixture was stirred for 40 minutes (TLC).
Reaction mass was filtered through celite bed, the filtrate was
dried over sodium sulfate and evaporated at reduced pressure to
obtain the required product as a pale yellow oil. The crude product
was taken as such to the next stage without further purification
(Yield, 6.60 g, 90%) .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
1.42 (m, 2H), 1.65 (m, 4H), 2.47 (t, 2H, J=7.2 Hz), 3.29 (t, 2H,
J=6.8 Hz), 9.78 (s, 1H).
[0778] Synthesis of Compound 118:
[0779] Sodium triacetoxyborohydride (5.36 g, 24.36) was added
portion-wise during 5 minutes to a solution of 111 (12 g, 19.50
mmol) in DCM (180 ml) at 0.degree. C. To this suspension, was added
117 (3.03 g, 21.48 mmol) product drop-wise at 0.degree. C. After
complete addition, the reaction mixture was warmed to room
temperature and stirred for another half an hour (TLC). Reaction
mixture quenched with 1N NaHCO.sub.3 (1.times.50 ml and, diluted
with DCM (200 ml). Organic layer was separated, washed with water
(2.times.100 ml), brine (1.times.100 ml), dried over sodium
sulfate, filtered and evaporated at reduced pressure to obtained
crude product, which was purified by flash silica gel
chromatography. The required product was eluted with 7%
EtOAc/Hexane (12.20 g, 84% as pale green oil). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 0.89 (s, 6H), 1.27 (s, 40H), 1.47 (t, 2H,
J=6.8 Hz), 1.58 (t, 6H, J=4.4 Hz), 2.05 (d, 8H, J=6 Hz), 2.27 (s,
3H), 2.43 (m, 3H), 2.56 (m, 1H), 2.77 (t, 4H, J=5.6 Hz), 3.26 (t,
2H, J=6.8 Hz), 3.53 (t, 1H, J=7.6 Hz), 4.08 (t, 1H, J=7.6 Hz), 4.2
(t, 1H, J=5.6 Hz), 5.33 m, 8H). .sup.13C NMR (100 MHz, CDCl.sub.3):
.delta. 14.1, 22.6, 23.7, 25.6, 27.0, 27.1, 27.2, 28.8, 29.2, 29.3,
29.32, 29.5, 29.6, 29.7, 29.9, 31.5, 37.3, 37.7, 43.1, 51.4, 58.3,
60.1, 69.1, 74.4, 76.7, 77.0, 77.7. MS: Calc. mass for
C.sub.47H.sub.86N.sub.4O.sub.2: 738.68, Found; 739.65
(MH).sup.+.
[0780] Synthesis of Compound 119:
[0781] To a solution of azido compound 118 (1.0 g, 1.35 mmol, 1.0
eq) in anhydrous THF (12.0 ml) was added 1M solution of LAH (2.03
ml, 2.03 mmol, 1.5 eq) in THF drop wise at 0.degree. C. under
argon. Reaction temperature was slowly increased and continued the
reaction at room temperature for 2 hours. After complete
consumption of the staring material, reaction was quenched with
drop wise addition of saturated Na.sub.2SO.sub.4 solution at
0.degree. C. Separated the solution form the solid and extracted
into ethylacetate (25.0 ml), aqueous was extracted with
ethylacetate until there is no product appears. Dried the combined
organics over Na.sub.2SO.sub.4, filtered, evaporated the solvent
and further dried under vacuum gave 0.82 g (86%) of the neat
product 119, which was directly used in the next step without
further purification. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
5.46-5.22 (m, 8H), 4.24-4.14 (m, 1H), 4.08-4.05 (m, 1H), 3.52 (t,
J=7.7, 1H), 2.77 (t, J=6.4, 4H), 2.67 (t, J=6.9, 2H), 2.54 (dd,
J=12.8, 6.2, 1H), 2.46-2.30 (m, 3H), 2.26 (s, 3H), 2.10-1.96 (m,
8H), 1.57-1.52 (m, 4H), 1.45-1.40 (m, 4H), 1.39-1.16 (m, 42H), 0.88
(t, J=6.8, 6H); Calc. mass for C.sub.47H.sub.88N.sub.2O.sub.2:
712.6, abs. mass: 713.7 (MH).sup.+.
[0782] Synthesis of Compound 120:
[0783] Compound 903 (0.26 g, 0.6 mmol, 1.0 eq) was dissolved in 2.5
ml of DMF and added HBTU (0.27 g, 0.72 mmol, 1.2 eq), followed by
DIPEA (0.33 ml, 1.8 mmol, 3.0 eq) at room temperature and stirred
for 10 minutes under argon atmosphere. A solution of compound 119
(0.42 g, 0.6 mmol, 1.0 eq) in DMF:DCM (1:2, 3.0 ml) was added
slowly to the above mixture at room temperature and continued the
reaction until there is no starting material. After 2 hours,
reaction mixture was poured onto ice and extracted with the DCM,
the organic layer was washed with excess amount of water to remove
all DMF present in the organic solution, further washed with
NaHCO.sub.3, dried on MgOS.sub.4, concentrated and purified by
column chromatography using DCM:MeOH (5%):triethylamine (0.5%) as a
gradients to get 0.57 g (85%) of the pure compound 120. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.63 (s, 1H), 8.43 (dd, J=4.8, 0.8,
1H), 7.76 (d, J=8.1, 1H), 7.64-7.59 (m, 2H), 7.07-7.00 (m, 1H),
6.87 (d, J=8.9, 2H), 5.52 (s, 1H), 5.43-5.19 (m, 8H), 4.27-4.12 (m,
1H), 4.05 (dd, J=13.2, 7.0, 3H), 3.52 (t, J=7.7, 1H), 3.30-3.11 (m,
6H), 2.76 (t, J=6.4, 4H), 2.53 (dd, J=12.7, 6.2, 1H), 2.45-2.31 (m,
5H), 2.25 (s, 3H), 2.19-2.10 (m, 5H), 2.04 (q, J=6.7, 8H),
1.64-1.40 (m, 9H), 1.37-1.18 (m, 40H), 0.88 (t, J=6.8, 6H); Calc.
mass for C.sub.67H.sub.109N.sub.5O.sub.5S.sub.2: 1127.7, abs. mass:
1128.5 (MH).sup.+.
Example 6
##STR00030##
[0785] Synthesis of Compound 6b:
[0786] To a flame dried 500 mL RB flask, freshly activated Mg
turnings (2.4 g, 100 mmol) were added and the flask was equipped
with a magnetic stir bar, an addition funnel and a reflux
condenser. This set-up was degassed and flushed with argon and 10
mL of anhydrous ether was added to the flask via syringe. The
bromide 3 (26.5 g, 80.47 mmol) was dissolved in anhydrous ether (50
mL) and added to the addition funnel. About 5 mL of this ether
solution was added to the Mg turnings while stirring vigorously. An
exothermic reaction was noticed (to confirm/accelerate the Grignard
reagent formation, 5 mg of iodine was added and immediate
decolorization was observed confirming the formation of the
Grignard reagent) and the ether started refluxing. The rest of the
solution of the bromide was added dropwise while keeping the
reaction under gentle reflux by cooling the flask in water. After
the completion of the addition the reaction mixture was kept at
35.degree. C. for 1 h and then cooled in ice bath. Ethyl formate
(2.68 g, 36.2 mmol) was dissolved in anhydrous ether (40 mL) and
transferred to the addition funnel and added dropwise to the
reaction mixture with stirring. An exothermic reaction was observed
and the reaction mixture started refluxing. After the initiation of
the reaction the rest of the ethereal solution of formate was
quickly added as a stream and the reaction mixture was stirred for
a further period of 1 h at ambient temperature. The reaction was
quenched by adding 10 mL of acetone dropwise followed by ice cold
water (60 mL). The reaction mixture was treated with aq.
H.sub.2SO.sub.4 (10% by volume, 300 mL) until the solution became
homogeneous and the layers were separated. The aq. phase was
extracted with ether (2.times.100 mL). The combined ether layers
were dried (Na.sub.2SO.sub.4) and concentrated to get the crude
product which was purified by column (silica gel, 0-10% ether in
hexanes) chromatography. The slightly less polar fractions were
concentrated to get the formate 6a (1.9 g) and the pure product
fractions were evaporated to provide the pure product 6b as a
colorless oil (14.6 g, 78%).
[0787] Synthesis of Compound 7:
[0788] To a solution of the alcohol 6b (3 g, 5.68 mmol) in
CH.sub.2Cl.sub.2 (60 mL), freshly activated 4 A molecular sieves
(50 g) were added and to this solution powdered PCC (4.9 g, 22.7
mmol) was added portion wise over a period of 20 minutes and the
mixture was further stirred for 1 hour (Note: careful monitoring of
the reaction is necessary in order to get good yields since
prolonged reaction times leads to lower yields) and the TLC of the
reaction mixture was followed every 10 minutes (5% ether in
hexanes) After completion of the reaction, the reaction mixture was
filtered through a pad of silica gel and the residue was washed
with CH.sub.2Cl.sub.2 (400 mL). The filtrate was concentrated and
the thus obtained crude product was further purified by column
chromatography (silica gel, 1% Et.sub.2O in hexanes) to isolate the
pure product 7 (2.9 g, 97%) as a colorless oil. .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta.=5.33-5.21 (m, 8H), 2.69 (t, 4H), 2.30
(t, 4H), 2.05-1.95 (m, 8H), 1.55-1.45 (m, 2H), 1.35-1.15 (m, 18H),
0.82 (t, 3H). .sup.13C NMR (CDCl.sub.3) .delta.=211.90, 130.63,
130.54, 128.47, 128.41, 43.27, 33.04, 32.01, 30.93, 29.89, 29.86,
29.75, 29.74, 27.69, 26.11, 24.35, 23.06, 14.05. MS. Molecular
weight calculated for C.sub.37H.sub.66O, Cal. 526.92, Found 528.02
(MH).sup.+.
##STR00031##
[0789] Synthesis of Compound 9:
[0790] A mixture of compound 8 (10.6 g, 100 mmol), compound 7
(10.54 g, 20 mmol) and PTSA (0.1 eq) was heated under toluene
reflux with Soxhlet extractor containing activated 4 .ANG.
molecular sieves for 3 h. Removal of solvent then column
purification (silica gel, 0-30% EtOAc in hexanes) gave compound 9
(11 g, 90%) as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 5.45-5.24 (m, 8H), 4.30-4.17 (m, 1H), 4.08 (dd, J=7.8, 6.1,
1H), 3.80 (dd, J=10.6, 5.0, 3H), 3.53 (t, J=8.0, 1H), 2.77 (t,
J=6.4, 5H), 2.29-2.18 (m, 1H), 2.05 (q, J=6.7, 9H), 1.86-1.74 (m,
2H), 1.59 (dd, J=18.3, 9.7, 5H), 1.42-1.18 (m, 43H), 0.89 (t,
J=6.8, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 130.39,
130.36, 130.35, 128.14, 112.80, 77.54, 77.22, 76.90, 75.74, 70.14,
61.08, 37.97, 37.50, 35.56, 31.74, 30.14, 30.13, 29.88, 29.80,
29.73, 29.57, 29.53, 27.45, 27.41, 25.84, 24.20, 24.00, 22.79,
14.30.
[0791] Synthesis of Compound 10:
[0792] To an ice-cold solution of compound 9 (10.5 g, 17 mmol) and
NEt.sub.3 (5 mL) in DCM (100 mL) a solution of MsCl (2.96 g, 20.5
mmol) in DCM (20 mL) was added dropwise with stirring. After 1 h at
r.t., aqueous workup gave a pale yellow oil of 10 which was column
purified (silica gel, 0-30% EtOAc in hexanes) to provide the pure
mesylate (11.1 g, 94%) as a colorless oil. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 5.44-5.26 (m, 8H), 4.37 (m, 2H), 4.26-4.13 (m,
1H), 4.10 (m, 1H), 3.53 (m, 1H), 3.02 (s, 3H), 2.76 (d, J=6.4, 4H),
2.05 (d, J=6.9, 10H), 1.55 (s, 4H), 1.29 (d, J=9.8, 34H), 0.88 (t,
J=6.9, 6H). Electrospray MS (+ve): Molecular weight for C42H76O5S
(MH).sup.+ Calc. 693.5, Found 693.4.
Example 7
##STR00032##
[0794] Synthesis of Compound 11:
[0795] To a solution of methanesulfonyl derivative of compound 10
(12.0 g, 17.31 mmol, 1.0 eq) in ethanol (20.0 ml) was added excess
amount of 33% methylamine in ethanol (200 ml) in the Parr reactor
and continued the reaction at 70.degree. C. for 4 h hours, after
complete consumption of the starting material, cooled the reaction,
transferred into a round bottom flask, concentrated. Dissolved the
reaction product in the ethylacetate and washed with water, dried
on Na.sub.2SO.sub.4, evaporated and further dried on vacuum gave
10.3 g (95%) of the pure product 11, this was used for the next
step without further purification. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 5.47-5.21 (m, 8H), 4.14-4.04 (m, 2H), 3.49 (t,
J=7.6, 1H), 3.00-2.83 (m, 1H), 2.80-2.74 (m, 5H), 2.60 (s, 3H),
2.04 (q, J=6.8, 8H), 1.94-1.81 (m, 2H), 1.60-1.52 (m, 4H),
1.41-1.15 (m, 37H), 0.88 (t, J=6.8, 6H); Calc. mass for
C.sub.42H.sub.77NO.sub.2: 627.6, abs. mass: 628.5 (MH).sup.+.
[0796] Compound 13:
[0797] To the suspension of compound 11 (1.0 g, 1.59 mmol, 1.0 eq)
and NaBH(OAc).sub.3 (0.44 g, 2.0 mmol, 1.3 eq) in DCM (12.0 ml) at
0.degree. C. was added a solution of compound 12 (0.25 g, 1.75
mmol, 1.1 eq) in DCM (3.0 ml) at 0.degree. C. under argon and the
reaction was continued at room temperature for 1 hour. After
completion of the starting material the reaction was quenched with
1N MaHCO.sub.3, diluted with DCM, separated the two layers and the
combined organics were washed with brine, dried over MgSO.sub.4,
concentrated and purified by column chromatography using DCM:MeOH
(5%):triethylamine (0.5%) as a gradients to get pure 0.86 g (72%)
of the ester compound 13. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
5.47-5.24 (m, 8H), 4.09-4.02 (m, 2H), 3.66 (s, 3H), 3.47 (t, J=7.0,
1H), 2.77 (t, J=6.4, 4H), 2.44-2.41 (m, 1H), 2.31 (t, J=7.5, 4H),
2.19 (s, 3H), 2.04 (q, J=6.7, 8H), 1.88-1.74 (m, 1H), 1.71-1.52 (m,
7H), 1.49-1.45 (m, 2H), 1.40-1.16 (m, 39H), 0.89 (t, J=6.8, 6H);
Calc. mass for C.sub.49H.sub.89NO.sub.4: 755.6, abs. mass: 756.5
(MH).sup.+.
[0798] Synthesis of Compound 14:
[0799] To a solution of compound 13 (0.86 g, 1.12 mmol, 1.0 eq) in
THF (4.0 ml) was added a aqueous solution of LiOH.H.sub.2O (0.14 g,
3.36 mmol, 3.0 eq) in water (2.0 ml) drop wise at 0.degree. C. and
continued the reaction at room temperature until there is no
starting material. After complete consumption of the staring
material, THF was evaporated and the aqueous reaction mixture was
acidified to pH 6.0 by drop wise addition of acetic acid. Extracted
the reaction product into ethyl acetate, organic layer was washed
with water, brine, dried over Na.sub.2SO.sub.4, evaporated the
solvent and further dried on vacuum to get 0.73 g (89%) of the
clean product 14, this was used directly without further
purification. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.46-5.24
(m, 8H), 4.08 (dd, J=8.2, 4.3, 2H), 3.53 (s, 1H), 3.23-2.93 (m,
4H), 2.77-2.71 (m, 7H), 2.37-2.14 (m, 3H), 2.04 (q, J=6.8, 8H),
1.97-1.77 (m, 3H), 1.70-1.47 (m, 6H), 1.44-1.14 (m, 38H), 0.88 (t,
J=6.8, 6H); Calc. mass for C.sub.48H.sub.87NO.sub.4: 741.6, abs.
mass: 742.5 (MH).sup.+.
[0800] Synthesis of Compound 15:
[0801] To a solution of compound 14 (0.73 g, 0.98 mmol, 1.0 eq) in
dichloromethane (8.0 ml) was added solid N-hydroxysuccinimide (NHS)
(0.12 g, 1.08 mmol, 1.1 eq) followed by DCC (0.22 g, 1.08 mmol, 1.1
eq), DMAP (0.012 g, 0.098 mmol, 0.1 eq) and the reaction was
continued at room temperature under argon atmosphere. A white
precipitate of urea derivative appears slowly in the reaction,
continued the reaction for overnight. After complete consumption of
the staring material, dichloromoethane was evaporated, dissolved
the reaction mixture in the ethylacetate (6.0 ml), cooled to ice
bath temperature to complete precipitate out the DCC urea
derivative. Filtered the solid and washed the solid with cold
ethylacetate (2.times.2.0 ml), evaporated the solvent and dried on
vacuum to give 0.82 g of the pure NHS ester of the compound 15 in
quantitative yields, this was directly used in the next step
without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 5.45-5.22 (m, 8H), 4.13-3.99 (m, 2H), 3.56-3.44 (m, 1H),
2.95-2.79 (m, 6H), 2.76 (t, J=6.4, 4H), 2.62 (t, J=7.1, 2H), 2.57
(s, 2H), 2.03 (q, J=6.8, 8H), 1.95-1.86 (m, 2H), 1.83-1.63 (m, 5H),
1.61-1.40 (m, 6H), 1.39-1.15 (m, 38H), 0.87 (t, J=6.8, 6H); Calc.
mass for C.sub.52H.sub.90N.sub.2O.sub.6: 838.6, abs. Mass: 839.5
(MH).sup.+.
Example 8
##STR00033##
[0803] Synthesis of Compound 17:
[0804] To the suspension of compound 11 (2.0 g, 3.18 mmol, 1.0 eq)
and NaBH(OAc).sub.3 (0.87 g, 4.13 mmol, 1.3 eq) in DCM (15.0 ml) at
0.degree. C. was added a solution of compound 16 (0.49 g, 3.5 mmol,
1.1 eq) in DCM (5.0 ml) at 0.degree. C. under argon and the
reaction was continued at room temperature for 1 hour. After
completion of the reaction was quenched with 1N MaHCO.sub.3,
diluted with DCM, separated the two layers and the combined
organics were washed with brine, dried over MgSO.sub.4,
concentrated and purified by column chromatography using DCM:MeOH
(5%):triethylamine (0.5%) as a gradients to get 1.85 g (77%) of the
pure ester compound 17. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
5.44-5.25 (m, 8H), 4.14-3.95 (m, 2H), 3.47 (t, J=7.1, 1H), 3.26 (t,
J=6.9, 2H), 2.77 (t, J=6.4, 3H), 2.49-2.25 (m, 3H), 2.20 (s, 3H),
2.05 (q, J=6.7, 8H), 1.86-1.76 (m, 1H), 1.71-1.51 (m, 7H),
1.49-1.13 (m, 44H), 0.89 (t, J=6.8, 6H); Calc. mass for
C.sub.48H.sub.88N.sub.4O.sub.2: 752.7, abs. mass: 753.5
(MH).sup.+.
[0805] Synthesis of Compound 18:
[0806] To a solution of azido derivative 17 (1.5 g, 2.0 mmol, 1.0
eq) in anhydrous THF (12.0 ml) was added 1M solution of LAH (3.0
ml, 3.0 mmol, 1.5 eq) in THF drop wise at 0.degree. C. under argon.
Reaction temperature was slowly increased and continued the
reaction at room temperature for 2 hours. After complete
consumption of the staring material, reaction was quenched with
drop wise addition of saturated Na.sub.2SO.sub.4 solution at
0.degree. C. Separated the solution form the solid and extracted
into ethylacetate (25.0 ml), aqueous was extracted with
ethylacetate until there is no product appears. Dried the combined
organics over Na.sub.2SO.sub.4, filtered, evaporated the solvent
and further dried under vacuum gave 1.31 g (90%) of the neat
product 18, which was directly used in the step without further
purification. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.45-5.24
(m, 8H), 4.13-3.96 (m, 2H), 3.47 (t, J=7.1, 1H), 2.77 (t, J=6.4,
4H), 2.68 (t, J=6.9, 2H), 2.49-2.26 (m, 4H), 2.20 (s, 3H), 2.04 (q,
J=6.7, 8H), 1.88-1.73 (m, 1H), 1.70-1.51 (m, 5H), 1.50-1.40 (m,
4H), 1.39-1.19 (m, 40H), 0.89 (t, J=6.8, 6H); Calc. mass for
C.sub.48H.sub.90N.sub.2O.sub.2: 726.7, abs. mass: 727.8
(MH).sup.+.
[0807] Synthesis of Compound 19:
[0808] Compound 903 (0.6 g, 0.82 mmol, 1.0 eq) was dissolved in 3.0
ml of DMF and added HBTU (0.37 g, 0.99 mmol, 1.2 eq), followed by
DIPEA (0.45 ml, 2.47 mmol, 3.0 eq) at room temperature and stirred
for 10 minutes under argon atmosphere. A solution of compound 18
(0.35 g, 0.82 mmol, 1.0 eq) in DMF:DCM (1:2, 3.0 ml) was added
slowly to the above mixture at room temperature and continued the
reaction until there is no starting material. After 2 hours,
reaction mixture was poured onto ice and extracted with the DCM,
the organic layer was washed with excess amount of water to remove
all DMF present in the organic layer, further washed with
NaHCO.sub.3, dried on MgSO.sub.4, concentrated and purified by
column chromatography using DCM:MeOH (5%):triethylamine (0.5%) as a
gradients to get 0.81 g (86%) of the pure compound 19. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.63 (s, 1H), 8.47-8.36 (m, 1H), 7.76
(d, J=8.1, 1H), 7.61 (d, J=8.8, 2H), 7.07-6.99 (m, 1H), 6.87 (d,
J=8.9, 2H), 5.52 (s, 1H), 5.43-5.25 (m, 8H), 4.13-3.95 (m, 4H),
3.46 (t, J=6.9, 1H), 3.28-3.13 (m, 6H), 2.80-2.75 (m, 4H),
2.42-2.28 (m, 6H), 2.16 (dd, J=19.3, 9.6, 8H), 2.04 (q, J=6.7, 8H),
1.86-1.74 (m, 1H), 1.70-1.40 (m, 8H), 1.39-1.18 (m, 42H), 0.88 (t,
J=6.8, 6H); Calc. mass for C.sub.68H.sub.111N.sub.5O.sub.5S.sub.2:
1141.8, abs. mass: 1142.7 (MH).sup.+.
Example 9
##STR00034##
[0810] Synthesis of Compound 301:
[0811] Cholesterol acid 300 (4.10 g, 7.53 mmol), N-hydroxy
succinimide (1.29 g, 11.29 mmol) and EDAC (2.16 g, 11.26 mol) were
taken in a mixture of DCM/DMF. To that DMAP (0.183 g) was added to
that and stirred the mixture overnight at ambient temperature.
Solvents were removed and the residue dissolved in DCM and washed
with water couple of times. TLC showed complete conversion of the
starting material. This was used for the next reaction without
further purification (white solid, 4.40 g, 92%). MS Cal. for
C.sub.38H.sub.60N.sub.2O.sub.6 640.45; Found. 641.40
(MH).sup.+.
[0812] Synthesis of Compound 302:
[0813] Compound 301 (2.00 g, 3.12 mmol) was dissolved in THF to
that hydrazine hydrate (0.200 mL, 1.2 eq) and TEA (1 mL, excess)
were added and stirred overnight. TLC checked and removed the
solvent. The residue washed with a mixture of EtOAc/Hexane and
dried overnight to get the compound as a white solid (2.10 g, 95%).
MS Cal. for C.sub.34H.sub.59N.sub.3O.sub.3 557.46; Found. 558.44
(MH.sup.+).
[0814] Synthesis of Compound 303:
[0815] Compound 302 (1.81 g, 3.24 mmol) and ketone (0.722 g, 3.24
mmol) were taken in ethanol (20 mL). To that AcOH (2 mL) was added
and stirred the mixture at 50 C for 6 h. Cooled the reaction
mixture and kept in the freezer overnight. Filtered and washed with
EtOAc and anhydrous ether. Dried under vacuum to get the compound
as a white solid (1.68 g, 71%). This compound used for the next
reaction without further purification. MS Cal. for
C.sub.46H.sub.71N.sub.3O.sub.6 761.53; Found: 760.52 (M-H). .sup.1H
NMR (400 MHz, DMSO) .delta. 11.95 (bs, 1H), 9.73 (m, 1H), 7.93-7.48
(m, 5H), 7.09-6.80 (m, 8H), 5.29 (s, 2H), 4.26 (s, 2H), 4.09-3.87
(m, 6H), 3.48-2.02 (m, 20), 2.02-0.55 (m, 20H).
[0816] Synthesis of Compound 304:
[0817] Compound 303 (0.500 g, 0.656 mmol), N-hydroxy succinimide
(0.113 g, 1.5 eq.) and EDAC (0.188 g, 1.5 eq.) were taken in DCM.
To that DMAP (20 mg) was added to that and stirred the mixture
overnight at ambient temperature. The reaction mixture diluted with
DCM, washed with water couple of times and dried. TLC showed
complete conversion of the starting material. Solvents were removed
and the residue dried overnight to get a white solid (480 mg, 92%).
MS Cal. For C.sub.50H.sub.74N.sub.4O.sub.8 858.55; Found: 859.53
(MH).sup.+.
Example 10
##STR00035## ##STR00036## ##STR00037##
[0819] Synthesis of 502:
[0820] The pteroic acid precursor 500 (1.88 g, 3 mmol) was
dissolved in anhydrous DMF (20 mL), HBTU (1.14 g, 1 eq.) followed
by DIEA (1.3 g, 3 eq.) were added and stirred for 20 minutes. To
this reaction mixture the amine 501 (0.961 g, 3 mmol) was added as
a solution in DMF (20 mL). Reaction was monitored by TLC (10%
MeOH/DCM, PMA stain). TLC of the reaction mixture showed completion
of the reaction after 1 h. The reaction mixture was slowly poured
in ice with vigorous stirring. The mixture was extracted with ethyl
acetate and the combined organic layers were dried
(Na.sub.2SO.sub.4) and concentrated to give the crude product. The
thus obtained crude product was further purified by column
chromatography (silica gel, 0-10% MeOH in DCM) to obtain 502 as a
white foam (Yield=2.4 g, 87%). MS. Molecular weight calculated for
C.sub.41H.sub.56F.sub.3N.sub.9O.sub.12, Cal. 923.40, Found 924.5
(MH).sup.+.
[0821] Synthesis of 503:
[0822] The Boc protected pteroic acid 502 (2.3 g) was treated with
2M HCl in ether (100 mL) and the solution was stirred overnight
until all the starting material had disappeared. The reaction
mixture was concentrated and dried to isolate the amine
hydrochloride salt as a waxy solid (1.9 g 100%). MS. Molecular
weight calculated for C.sub.36H.sub.48F.sub.3N.sub.9O.sub.10, Cal.
823.35, Found 824.35 (MH).sup.+.
[0823] Synthesis of 504:
[0824] The amine hydrochloride salt 503 (1.8 g) was dissolved in
100 mL THF/MeOH (1:1) and to it an aqueous solution of LiOH (0.4 g
in 10 mL of water) was added and the mixture was stirred at room
temperature for 2 h. The MS of the reaction mixture showed the
complete disappearance of the starting material and only product
peak was seen. The reaction mixture was concentrated to remove the
organic solvents and the remaining aqueous solution was neutralized
to pH 6.8 by adding acetic acid. The thus obtained aqueous solution
was freeze dried and triturated with ethyl acetate to get 504 (2.1
g along with inorganic impurities) as an orange crystalline solid.
The LC trace showed that the amine is 91% pure and the MS showed
the correct mass. This was used as such in the next step. MS.
Molecular weight calculated for C.sub.29H.sub.41N.sub.9O.sub.8,
Cal. 643.31, Found 624.35 (MH).sup.+.
[0825] Synthesis of 505:
[0826] To a solution of the amine 504 (64 mg, 0.1 mmol) in
anhydrous DMF (20 mL) Hunig's base (0.3 mL) was added followed by
the activated ester 904 (63 mg, 0.1 mmol). The reaction mixture was
stirred at room temperature for 2 h after which the MS showed the
complete disappearance of the starting materials. The reaction
mixture was concentrated and the residue was triturated with ethyl
acetate to obtain the product as yellow solid (67 mg, 52%). MS.
Molecular weight calculated for
C.sub.49H.sub.62N.sub.12O.sub.11S.sub.2, Cal. 1058.41, Found 1057.4
(M-H.sup.-).
Example 11
##STR00038##
[0828] Synthesis of Compound 52:
[0829] To a solution of compound 511 (3.78 g, 7.52 mmol) in
benzylalcohol (24 mL), benzyl acrylate (1.22 g, 7.52 mmol) and
saturated boric acid aq. (0.5 mL) were dropwisely added. The
reaction mixture was stirred for 5 h at room temperature. Then,
di-tert-butyl dicarbonate (2.46 g, 11.3 mmol) was added to the
mixture. Stirring was continued overnight and the reaction mixture
was directly purified by silica gel column chromatography
(hexane:ethyl acetate=4:1 to 2:1) to give 52 (4.45 g, 5.82 mmol,
77%, R.sub.f=0.26 developed with hexane:ethyl acetate=2:1). .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 7.34-7.36 (m, 5H), 6.76 (brs,
1H), 5.08 (s, 2H), 3.39 (t, J=7.0 Hz, 2H), 3.09 (brs, 10H), 2.88
(dd, J=12.4 Hz, 6.0 Hz, 2H), 2.54-2.61 (m, 2H), 1.37-1.62 (m, 44H).
.sup.13C NMR (DMSO-d.sub.6, 100 MHz) .delta. 171.1, 155.4, 154.5,
154.4, 154.3, 135.9, 128.3, 127.9, 127.8, 78.6, 78.2, 78.1, 77.3,
65.5, 44.3, 44.2, 42.7, 37.5, 28.1, 27.9. Molecular weight for
C.sub.40H.sub.68N.sub.4NaO.sub.10 (M+Na).sup.+ Calc. 787.48, Found
787.4. [0830] (1) (a) Geall, A. J., Blagbrough, I. S. Tetrahedron
Letters (1998), 39, 443-446. (b) Geall, A. J., Taylor, R. J.,
Earll, M. E., Eaton, M. A. W., Blagbrough, I. S. Bioconjugate Chem.
(2000), 11, 314-326.
[0831] Synthesis of Compound 53:
[0832] To a solution of 52 (4.20 g, 5.49 mmol) in CH.sub.2Cl.sub.2
(120 mL), trifluoroacetic acid (30 mL) was slowly added with
cooling in ice bath. The reaction mixture was kept stirring for 15
min, then for 2 h at room temperature. The solvent was removed,
coevaporated with toluene, and dried in vacuo to give a crude
material. The crude was resuspended in CH.sub.2Cl.sub.2 with
cooling in ice bath, then trifluoroacetic anhydride (6.87 mL, 49.4
mmol) and triethylamine (9.18 mL, 65.9 mmol) were slowly added. The
reaction mixture was stirred for 15 min in ice bath and for 16 h at
room temperature. The reaction mixture was extracted with
CH.sub.2Cl.sub.2 (300 mL) and saturated NaHCO.sub.3 aq. (150 mL),
dried over anhydrous Na.sub.2SO.sub.4, and purified by silica gel
column chromatography (hexane:ethyl acetate=4:1 to 1:1) to give 53
(3.61 g, 4.82 mmol, 88%, R.sub.f=0.36 developed with hexane:ethyl
acetate=1:1). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.45-9.52
(m, 1H), 7.33-7.37 (m, 5H), 5.11 (d, J=7.6 Hz, 2H), 3.61-3.69 (m,
2H), 3.34-3.39 (m, 10H), 3.21-3.22 (m, 2H), 2.79 (t, J=7.4 Hz, 1H),
2.71 (t, J=7.2 Hz, 1H), 1.75-1.91 (m, 4H), 1.51-1.54 (m, 4H).
.sup.19F NMR (376 MHz, DMSO-d.sub.6) .delta. -68.15, -68.19,
-68.21, -68.26, -68.29, -68.31, -68.33, -68.38, -68.40, -68.41,
-68.48, -68.49, -74.47, -74.49, -74.50, -74.61. .sup.13C NMR
(DMSO-d.sub.6, 100 MHz) .delta..quadrature. 170.7, 170.3, 156.8,
156.5, 156.4, 156.1, 156.0, 155.9, 155.8, 155.7, 155.6, 155.5,
155.3, 155.2, 154.9, 154.8, 135.8, 135.7, 129.2, 128.8, 128.4,
128.1, 127.6, 127.3, 120.6, 120.4, 120.1, 117.7, 117.6, 117.5,
117.3, 114.8, 114.7, 114.6, 112.0, 111.8, 111.6, 66.6, 66.4, 65.8,
65.7, 64.9, 64.8, 64.2, 61.2, 61.1, 59.8, 59.7, 58.3, 54.9, 46.8,
46.7, 46.6, 46.0, 45.9, 45.8, 45.7, 45.5, 45.4, 45.3, 45.2, 45.0,
44.9, 44.7, 44.6, 44.5, 44.4, 44.2, 44.1, 44.0, 43.9, 43.7, 43.6,
42.8, 42.7, 42.6, 40.5, 40.3, 40.2, 36.8, 36.4, 32.7, 32.6, 31.6,
31.5, 31.2, 31.1, 31.0, 30.0, 29.6, 27.7, 27.4, 26.5, 26.4, 26.2,
25.8, 25.4, 25.3, 25.1, 25.0, 24.6, 23.9, 23.5, 23.4, 23.3, 20.2,
19.8, 18.9, 14.6, 14.0, 13.4, 12.2. Molecular weight for
C.sub.28H.sub.32F.sub.12N.sub.4NaO.sub.6 (M+Na).sup.+ Calc. 771.20,
Found 771.0.
[0833] Synthesis of Compound 54:
[0834] To a solution of 53 (3.57 g, 4.77 mmol) in EtOAc (50 mL),
Palladium on carbon (10 wt. %, Degussa type E101 NE/W: 600 mg) was
added. The reaction mixture was stirred under H.sub.2 atmosphere
for 16 h. After filtration through Celite, the filtrate was removed
in vacuo to give 54 (3.04 g, 4.62 mmol, 97%). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 9.45-9.55 (m, 1H), 3.54-3.63 (m, 2H),
3.36-3.39 (m, 10H), 3.19-3.24 (m, 2H), 2.62 (t, J=7.2 Hz, 1H), 2.56
(t, J=9.0 Hz, 1H), 1.75-1.91 (m, 4H), 1.52-1.55 (m, 4H). .sup.19F
NMR (376 MHz, DMSO-d.sub.6) .delta. -68.19, -68.22, -68.23, -68.24,
-68.33, -68.35, -68.38, -68.41, -68.44, -68.48, -68.50, -68.51,
-74.50, -74.52, -74.53, -74.64. .sup.13C NMR (DMSO-d.sub.6, 100
MHz) .delta. 172.4, 172.0, 156.5, 156.4, 156.2, 156.1, 155.9,
155.8, 155.6, 155.5, 155.4, 155.3, 155.2, 154.9, 120.6, 120.5,
120.2, 117.7, 117.6, 117.3, 114.9, 114.8, 114.5, 112.0, 111.6,
46.7, 46.1, 46.0, 45.9, 45.8, 45.0, 44.6, 44.2, 44.0, 43.7, 42.9,
40.1, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 36.9, 36.4, 33.1, 31.2,
27.7, 27.5, 25.8, 25.4, 25.1, 24.0, 23.6, 23.3. Molecular weight
for C.sub.21H.sub.25F.sub.12N.sub.4O.sub.6 (M-H).sup.- Calc.
657.16, Found 657.0.
[0835] Synthesis of Compound 56:
[0836] A solution of compound 54 (3.0 g, 4.57 mmol) and HBTU (1.91
g, 5.03 mmol) in DMF (50 mL) was treated successively with
iPr.sub.2NEt (3.98 mL, 22.9 mmol) and compound 55 (2.68 g, 5.03
mmol) then allowed to stir for 14 h at room temperature. Extraction
with Et.sub.2O and sat. NaHCO.sub.3 aq. and then column
chromatography (0-5% in CH.sub.2Cl.sub.2) gave compound 56 (3.70 g,
3.15 mmol, 69%, R.sub.f=0.59 developed with 10% MeOH in
CH.sub.2Cl.sub.2). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
9.44-9.52 (m, 1H), 7.92-7.98 (m, 1H), 7.16-7.32 (m, 9H), 6.86-6.89
(m, 4H), 4.88-4.98 (m, 1H), 4.28-4.40 (m, 1H), 4.09-4.15 (m, 1H),
3.72 (s, 6H), 3.54-3.62 (m, 3H), 3.29-3.36 (m, 10H), 3.13-3.23 (m,
3H), 2.99-3.02 (m, 3H), 2.36-2.44 (m, 2H), 1.74-2.21 (m, 9H),
1.23-1.53 (m, 10H). .sup.19F NMR (376 MHz, DMSO-d.sub.6) .delta.
-68.17, -68.19, -68.22, -68.23, -68.26, -68.29, -68.32, -68.34,
-68.40, -68.42, -68.49, -68.51, -74.49, -74.51, -74.52, -74.63.
.sup.13C NMR (DMSO-d.sub.6, 100 MHz) .delta. 170.9, 170.8, 169.4,
169.0, 158.1, 158.0, 156.5, 156.2, 155.6, 155.3, 145.1, 144.7,
135.9, 135.7, 135.5, 135.4, 129.6, 129.5, 127.9, 127.8, 127.6,
126.7, 126.6, 117.7, 117.3, 114.9, 114.5, 113.2, 113.1, 85.8, 85.1,
68.6, 67.4, 65.2, 63.3, 55.9, 53.4, 46.7, 46.0, 45.9, 45.8, 45.1,
44.7, 44.2, 44.1, 43.9, 43.7, 43.6, 38.4, 38.0, 36.9, 36.4, 36.3,
34.5, 34.1, 32.6, 32.5, 28.9, 27.7, 27.5, 26.2, 25.9, 25.8, 25.4,
25.1, 24.5, 24.1, 23.9, 23.6, 23.3. Molecular weight for
C.sub.53H.sub.64F.sub.12N.sub.6NaO.sub.10 (M+Na).sup.+ Calc.
1195.44, Found 1195.2.
[0837] Synthesis of Compound 57:
[0838] To a solution of compound 56 (1.00 g, 0.852 mmol) in
CH.sub.2Cl.sub.2 (14 mL), diisopropylethylamine (0.742 mL, 4.26
mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.248
mL, 1.11 mmol) were added at 0.degree. C. The reaction mixture was
stirred for 30 min at 0.degree. C. then for 30 min at room
temperature under argon atmosphere. The reaction mixture was
diluted with CH.sub.2Cl.sub.2 (100 mL) and washed with saturated
NaHCO.sub.3 aq. (50 mL). The organic layer was separated and dried
over anhydrous Na.sub.2SO.sub.4. The filtrate was concentrated and
the resulting crude material was purified by silica gel column
chromatography (hexane:EtOAc=1:4 to 100% EtOAc) to give 57 (892 mg,
0.650 mmol, 76%, R.sub.f=0.18 developed with EtOAc). .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 9.34-9.52 (m, 1H), 7.92-7.95 (m,
1H), 7.16-7.31 (m, 9H), 6.83-6.87 (m, 4H), 4.62-4.64 (m, 1H),
4.11-4.17 (m, 1H), 3.65-3.72 (m, 9H), 3.49-3.56 (m, 5H), 3.30-3.42
(m, 9H), 3.15-3.21 (m, 3H), 2.95-3.02 (m, 3H), 2.72-2.76 (m, 2H),
2.37-2.40 (m, 2H), 2.10-2.20 (4H), 1.75-1.87 (m, 5H), 1.07-1.54 (m,
22H). .sup.31P NMR (DMSO-d.sub.6, 162 MHz) .delta. 146.93, 146.71,
146.51, 146.20. Molecular weight for
C.sub.62H.sub.81F.sub.12N.sub.8NaO.sub.11P (M+Na).sup.+ Calc.
1395.55, Found 1395.2.
[0839] Synthesis of Compound 58:
[0840] Compound 56 (1.01 g, 0.861 mmol) in CH.sub.2Cl.sub.2 (30 mL)
was treated with DMAP (314 mg, 2.57 mmol) and succinic anhydride
(171 mg, 1.71 mmol) then stirred for 16 h at room temperature.
Column chromatography (4% MeOH/4% Et.sub.3N in CH.sub.2Cl.sub.2) of
the crude mixture without aqueous work-up gave the compound 58 as
the corresponding triethylammonium salt (1.18 g, 99.8%,
R.sub.f=0.21 developed with 4% MeOH/4% Et.sub.3N in
CH.sub.2Cl.sub.2). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
9.51-9.58 (m, 1H), 8.01-8.10 (m, 1H), 7.17-7.32 (m, 9H), 6.84-6.88
(m, 4H), 5.22-5.33 (m, 1H), 4.18-4.19 (m, 1H), 3.72 (s, 6H),
3.54-3.62 (m, 3H), 3.35-3.37 (m, 10H), 3.18-3.22 (m, 3H), 2.99-3.06
(m, 3H), 2.34-2.44 (m, 6H), 1.74-2.22 (m, 9H), 1.10-1.53 (m, 10H).
.sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. 173.9, 172.3, 170.7,
170.6, 169.3, 168.9, 158.0, 157.9, 156.5, 156.1, 156.0, 155.9,
155.8, 155.5, 155.4, 155.3, 155.2, 155.1, 144.9, 144.6, 135.7,
135.5, 135.3, 129.6, 129.5, 129.4, 127.8, 127.7, 127.5, 126.7,
126.5, 120.5, 117.7, 117.6, 117.3, 114.8, 114.7, 114.4, 113.1,
113.0, 111.9, 85.9, 85.2, 72.6, 63.2, 54.9, 54.8, 52.1, 51.9, 46.7,
46.6, 46.5, 45.4, 43.6, 38.3, 36.8, 36.4, 34.4, 33.9, 33.0, 32.5,
32.4, 29.8, 29.7, 29.4, 28.9, 28.8, 27.7, 26.0, 25.9, 25.8, 25.7,
25.3, 25.1, 25.0, 24.4, 24.0, 23.5, 23.3, 7.11. Molecular weight
for C.sub.57H.sub.67F.sub.12N.sub.6O.sub.13 (M-H).sup.- Calc.
1271.46, Found 1271.2.
[0841] Synthesis of Compound 59:
[0842] Compound 58 (1.15 g, 0.837 mmol) was dissolved in DMF (80
mL). HBTU (350 mg, 0.921 mmol) then iPr.sub.2NEt (0.730 mL, 4.19
mmol) and finally CPG-NH.sub.2 (Prime Synthesis CPG-500, NH.sub.2
loading=147 .mu.mol/g) (6.27 g, 0.921 mmol) were added in
succession. The mixture was shaken for 2.5 h at room temperature,
then the solid was collected by filtration, washed with
CH.sub.2Cl.sub.2 (100 mL), then 50% MeOH/CH.sub.2Cl.sub.2 (300 mL)
and dried in vacuo. The residual amino groups were capped by
shaking for 1 h with Ac.sub.2O/Pyridine/Et.sub.3N (25 mL/75 mL/5
mL). Filtration and washing with CH.sub.2Cl.sub.2 (100 mL), then
50% MeOH/CH.sub.2Cl.sub.2 (300 mL) then drying overnight in vacuo
gave compound 59 (6.56 g, 53 .mu.mol/g).
Example 12. Spermine Azide Derivatives for Post-Synthetic
Conjugation
##STR00039##
[0844] Synthesis of Compound 63:
[0845] Spermine 60 (11.25 g, 55.6 mmol) was dissolved in
acetonitrile (50 mL). Then, copper sulfate pentahydrate (139 mg,
0.556 mmol) and triethylamine (15.5 mL, 111.2 mmol) were added to
the solution while stirring. The mixture was cooled in an ice bath,
then, a solution of triflic azide 61 in acetonitrile (0.55 M, 100
mL) prepared by the method previously reported.sup.2 was added to
the mixture slowly. The reaction mixture was allowed to warm to
room temperature and run overnight. Di-tert-butyl dicarbonate (60.7
g, 278 mmol) was slowly added to the solution and the reaction
mixture was stirred for 2 hours. After evaporation, the crude was
extracted with EtOAc and H.sub.2O, dried over anhydrous
Na.sub.2SO.sub.4, and purified by silica gel column chromatography
(Hexane:EtOAc=2:1, R.sub.f=0.46) to give 63 (13.12 g, 24.8 mmol,
45%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.77 (brs, 1H),
3.30-3.34 (m, 2H), 3.11-3.19 (m, 8H), 2.86-2.90 (m, 2H), 1.71-1.72
(m, 2H), 1.54-1.56 (m, 2H), 1.37-1.39 (m, 29H). Molecular weight
for C.sub.25H.sub.48N.sub.6NaO.sub.6 (M+Na).sup.+ Calc. 551.35,
Found 551.2. [0846] (2) Yan, R. B., Yang, F., Wu, Y., Zhang, L. H.,
Ye, X. S. Tetrahedron Letters (2005), 46, 8993-8995.
[0847] Synthesis of Compound 64:
[0848] To a solution of compound 63 (1.37 g, 2.59 mmol) in
CH.sub.2Cl.sub.2 (36 mL), TFA (4 mL) was slowly added with cooling
in ice bath. The mixture was stirred for 5 hour at room
temperature. The reaction mixture was evaporated and then
coevaporated with toluene. The residue was precipitated with ether
and the white solid (TFA salt of 64) collected. Yield: 950 mg, 1.67
mmol, 64%. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.48 (t,
J=6.4 Hz, 2H), 2.93-2.96 (m, 10H), 1.62-1.93 (m, 8H). Molecular
weight for C.sub.10H.sub.25N.sub.6 (MH).sup.+ Calc. 229.21, Found
229.3.
[0849] Synthesis of Compound 65:
[0850] To a solution of compound 64 (324 mg, 0.568 mmol) in
pyridine (5 mL), trifluoroacetic anhydride (0.361 mL, 2.27 mmol)
was slowly added with cooling in ice bath. The mixture was stirred
for 16 h at room temperature. The reaction mixture was extracted
with CH.sub.2Cl.sub.2 and sat. NaHCO.sub.3 aq., then dried over
anhydrous Na.sub.2SO.sub.4. The filtrate was concentrated and the
resulting crude material was purified by silica gel column
chromatography (hexane:EtOAc=4:1 to 1:1) to give 65 (214 mg, 0.414
mmol, 73%, R.sub.f=0.33 developed with hexane:EtOAc=1:1). .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 9.48-9.53 (m, 1H), 3.32-3.43
(m, 10H), 3.18-3.23 (m, 2H), 1.75-1.84 (m, 4H), 1.51-1.54 (m, 4H).
.sup.19F NMR (376 MHz, DMSO-d.sub.6) .delta. -70.92, -70.95,
-70.98, -71.02, -71.05, -71.15, -71.18, -77.23, -77.26, -77.38,
-77.38. Molecular weight for C.sub.10H.sub.25N.sub.6 (MH).sup.+
Calc. 229.21, Found 229.3.
Example 13
##STR00040##
[0852] Synthesis of Compound 68:
[0853] Compound 68 was prepared from compound 66.sup.3 as described
for compound 63. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.73
(brs, 1H), 3.05-3.33 (m, 18H), 2.86-2.88 (m, 2H), 1.36-1.70 (m,
57H). Molecular weight for C.sub.41H.sub.78N.sub.8NaO.sub.10
(M+Na).sup.+ Calc. 865.57, Found 865.5. [0854] (3) Miller, K. A.,
Suresh Kumar, E. V. K., Wood, S. J., Cromer, J. R., Datta, A.,
David, S. A. Journal of Medicinal Chemistry (2005), 48,
2589-2599.
[0855] Synthesis of Compound 69:
[0856] Compound 69 was prepared from compound 68 (275 mg, 0.326
mmol) as described for compound 64. Yield: 235 mg (0.257 mmol,
79%). .sup.1H NMR (400 MHz, D.sub.2O) .delta. 3.50 (t, J=6.0 Hz,
2H), 3.09-3.15 (m, 18H), 2.06-2.10 (m, 6H), 1.96 (t, J=6.0 Hz, 2H),
1.75-1.77 (m, 4H). Molecular weight for C.sub.16H.sub.39N.sub.8
(MH).sup.+ Calc. 343.33, Found 343.2.
Example 14
##STR00041## ##STR00042##
[0858] Synthesis of Compound 70:
[0859] For alkylation at N1 position of pseudouridine, a reported
method by Seela et al. for the 2'-deoxypseudouridine analog was
used..sup.4 To a solution of pseudouridine (20 g, 81.9 mmol) in 1M
triethylammoniumbicarbonate buffer (pH 8.5, 780 mL) and EtOH (940
mL), methyl acrylate was dropwisely added. The reaction mixture was
stirred overnight. After 16 hours, TLC showed a complete reaction.
The solvent was removed and dried in vacuo to give a white foam.
The crude material was purified by silica gel column chromatography
(10% MeOH in CH.sub.2Cl.sub.2, R.sub.f=0.23) to give 70 (26.6 g,
80.5 mmol, 98%). .sup.1H NMR (400 MHz, MeOH-d.sub.4) .delta. 7.77
(d, J=0.8 Hz, 1H), 4.58 (d, J=4.8 Hz, 1H), 4.15 (t, J=5.2 Hz, 1H),
4.05 (t, J=5.0 Hz, 1H), 3.98-4.02 (m, 2H), 3.91-3.94 (m, 1H), 3.80
(dd, J=12.0 Hz, 3.3 Hz, 1H), 3.67 (s, 3H), 3.66 (dd, J=12.0 Hz, 3.3
Hz, 1H), 2.73-2.77 (m, 2H). .sup.13C NMR (100 MHz, MeOH-d.sub.4)
.delta. 173.1, 165.4, 152.5, 145.8, 112.9, 85.6, 81.5, 75.6, 72.6,
63.3, 52.5, 46.2, 33.7. Molecular weight for
C.sub.13H.sub.19N.sub.2O.sub.8 (MH).sup.+ Calc. 330.11, Found
331.0. [0860] (4) Ramzaeva, N.; Rosemeyer, H.; Leonard, P.;
Muhlegger, K.; Bergmann, F.; Von der Eltz, H.; Seela, F. Helvetica
Chimica Acta 2000, 83, 1108-1126.
[0861] Synthesis of Compound 71:
[0862] To a solution of compound 70 (5.00 g, 15.1 mmol) in THF (100
mL) and H.sub.2O (20 mL), lithium hydroxide monohydrate (1.03 g,
25.5 mmol) was added. The reaction mixture was stirred overnight.
Additional lithium hydroxide monohydrate (500 mg, 11.9 mmol) was
added. After 2 hours, the reaction mixture was treated with
Amberlite IR-120 (plus) ion exchange resin. The resin was filtered
off and washed with THF/H.sub.2O. The filtrate was evaporated to
give compound 71 as a white solid (4.78 g, quantitatively). .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 11.34 (s, 1H), 7.75 (s, 1H),
4.92-4.93 (m, 1H), 4.70-4.72 (m, 1H), 4.45 (d, J=4.0 Hz, 1H),
3.80-3.93 (m, 4H), 3.68-3.72 (m, 1H), 3.61 (dd, J=12.0 Hz, 3.2 Hz,
1H), 3.47 (dd, J=12.0 Hz, 4.0 Hz, 1H), 3.17 (d, J=3.2 Hz, 1H), 2.59
(t, J=7.0 Hz, 2H). .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta.
172.1, 163.0, 150.4, 143.6, 111.4, 83.2, 79.0, 73.7, 70.4, 61.3,
44.1, 32.7. Molecular weight for C.sub.12H.sub.15N.sub.2O.sub.8
(M-H).sup.- Calc. 315.08, Found 315.1.
[0863] Synthesis of Compound 72:
[0864] To a solution of compound 71 (4.78 g, 15.1 mmol) in DMF (150
mL), HBTU (6.89 g, 18.17 mmol) and diisopropylethylamine (13.19 mL,
7.57 mL) were added. After stirring for 10 minute, a solution of
compound 51 in CH.sub.2Cl.sub.2 (100 mL) was added. The reaction
mixture was stirred for 16 h. Aqueous work-up and silica gel column
chromatography (0-10% MeOH in CH.sub.2Cl.sub.2) gave compound 72
(7.51 g, 9.38 mmol, 62%, R.sub.f=0.30 developed with 10% MeOH in
CH.sub.2Cl.sub.2). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
11.34 (s, 1H), 7.94 (brs, 1H), 7.67 (s, 1H), 6.76 (brs, 1H), 4.92
(d, J=2.6 Hz, 1H), 4.72-4.75 (m, 2H), 4.43 (d, J=2.2 Hz, 1H),
3.81-3.94 (m, 4H), 3.68-3.72 (m, 1H), 3.57-3.62 (m, 1H), 3.44-3.49
(m, 1H), 3.09 (s, 8H), 2.99 (dd, J=6.0 Hz, 12.4 Hz, 2H), 2.88 (dd,
J=6.4 Hz, 12.4 Hz, 2H), 2.43 (t, J=6.8 Hz, 2H), 1.37-1.56 (m, 35H).
.sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. 169.5, 163.2, 155.8,
154.9, 150.5, 144.0, 111.4, 83.5, 79.1, 78.6, 77.8, 73.8, 70.7,
61.5, 54.9, 46.7, 46.6, 46.2, 45.0, 44.6, 44.5, 37.6, 36.4, 34.3,
28.4, 28.2, 25.8, 25.7, 25.4, 25.3, 25.2, 22.4. Molecular weight
for C.sub.37H.sub.64N.sub.6NaO.sub.13 (M+Na).sup.+ Calc. 823.44,
Found 823.2.
[0865] Synthesis of Compound 74:
[0866] To a solution of 72 (2.48 g, 3.10 mmol) in CH.sub.2Cl.sub.2
(40 mL), trifluoroacetic acid (10 mL) was slowly added with cooling
in ice bath. The reaction mixture was kept stirring for 10 min,
then for 1 h at room temperature. The solvent was removed,
coevaporated with toluene, and dried in vacuo to give a crude
material 73 (Molecular weight for C.sub.22H.sub.41N.sub.6O.sub.7
(M+H).sup.+ Calc. 501.30, Found 501.2.) The crude was dissolved in
pyridine (30 mL), then trifluoroacetic anhydride (2.15 mL, 15.5
mmol) was slowly added with cooling in ice bath. The reaction
mixture was stirred for 16 h then quenched by addition of H.sub.2O
(15 mL). After stirring for 1 h, the mixture was evaporated and
dried in vacuo. Silica gel column chromatography (0-20% MeOH in
CH.sub.2Cl.sub.2) gave compound 74 (1.34 g, 1.70 mmol, 55%,
R.sub.f=0.56 developed with 20% MeOH in CH.sub.2Cl.sub.2). .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 11.35 (s, 1H), 9.45-9.53 (m,
1H), 8.00-8.08 (m, 1H), 7.66 (s, 1H), 4.92 (brs, 1H), 4.74 (brs,
1H), 4.42 (d, J=4.4 Hz, 1H), 3.83-4.10 (m, 6H), 3.58-3.71 (m, 2H),
3.34-3.48 (m, 10H), 3.19-3.24 (m, 2H), 2.44 (t, J=7.0 Hz, 2H),
1.52-1.82 (m, 8H). .sup.19F NMR (376 MHz, DMSO-d.sub.6) .delta.
-68.18, -68.19, -68.24, -68.27, -68.31, -68.38, -68.41, -74.47,
-74.50, -74.63. .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. 169.4,
169.3, 163.0, 150.4, 146.0, 143.6, 140.9, 125.4, 111.3, 83.3, 78.9,
73.7, 70.5, 61.4, 46.8, 46.7, 46.6, 46.0, 45.8, 45.6, 45.0, 44.7,
44.6, 44.5, 44.2, 36.8, 36.4, 36.1, 35.7, 34.2, 34.1, 28.5, 27.7,
26.5, 25.8, 25.4, 25.1, 23.5, 23.3. Molecular weight for
C.sub.28H.sub.37F.sub.9N.sub.6NaO.sub.10 (M+Na).sup.+ Calc. 811.23,
Found 811.2.
[0867] Synthesis of Compound 75:
[0868] To a solution of compound 74 (450 mg, 0.571 mmol) in
pyridine (4 mL), DMTrCl (193 mg, 0.571 mmol) was added. The
reaction mixture was stirred for 2 h at room temperature and then
evaporated all. The crude was extracted with CH.sub.2Cl.sub.2 and
H.sub.2O, dried over anhydrous Na.sub.2SO.sub.4, and purified by
silica gel column chromatography (0-10% MeOH in CH.sub.2Cl.sub.2)
to give 75 (300 mg, 0.275 mmol, 48%, R.sub.f=0.44 developed with
10% MeOH in CH.sub.2Cl.sub.2). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 11.33 (s, 1H), 9.42-9.53 (m, 1H), 7.91-8.00 (m, 1H),
7.18-7.43 (m, 10H), 6.87-6.89 (m, 4H), 5.02 (dd, J=3.0 Hz, 4.6 Hz,
1H), 4.76 (t, J=6.0 Hz, 1H), 4.50-4.51 (m, 1H), 3.79-3.92 (m, 3H),
3.73 (s, 6H), 3.53-3.71 (m, 2H), 2.98-3.37 (m, 14H), 2.36 (t, J=6.4
Hz, 2H), 1.48-1.82 (m, 8H). .sup.19F NMR (376 MHz, DMSO-d.sub.6)
.delta. -68.17, -68.18, -68.19, -68.24, -68.28, -68.31, -68.34,
-68.37, -68.40, -74.45, -74.48, -74.60, -74.61. Molecular weight
for C.sub.49H.sub.55F.sub.9N.sub.6NaO.sub.12 (M+Na).sup.+ Calc.
1113.36, Found 1114.2; C.sub.49H.sub.54F.sub.9N.sub.6O.sub.12
(M-H).sup.- Calc. 1089.37, Found 1089.3.
Example 15
##STR00043##
[0870] Synthesis of Compound 307:
[0871] Dilinoleyl alcohol 6b (5.60 g, 10.50 mmol) and DSC (4.00 g,
15.87 mmol) were taken in anhydrous DCM (100 mL) and cooled in
ice-water mixture. TEA (4.27 ml, 3 eq.) was added and stirred the
mixture for two days. Reaction was monitored by TLC. Reaction
mixture diluted with DCM and washed with water (two times) dried
over sodium sulfate. Solvent was removed and the residue dried
overnight. This material was used as such for the next reaction
without any purification. To a solution of compound 305 (10.50
mmol) from the above reaction and hydroxyl proline derivative 55
(5.59 g, 10.50 mmol) in DCM (100 mL) pyridine (10 mL) was added and
stirred the mixture overnight at ambient temperature. Reaction was
monitored by TLC and removed solvent under reduced pressure.
Dissolved the residue in DCM and washed with water, sodium
bicarbonate solution and brine. Crude product was purified by
chromatography 30-80% EtOAc/Hexane) to get the required product
(6.64 g, 54%). MS: Molecular weight cal. for
C.sub.70H.sub.106N.sub.2O.sub.7 1086.80, Found: 1109.79 (M+Na).
[0872] Synthesis of Compound 308:
[0873] To a solution of compound 307 (1.42 g, 1.30 mmol) in DCM was
added DMAP (0.475 g, 3.90 mmol) followed by succinic anhydride
(0.201 g, 2 eq.) and the reaction was continued for overnight at
room temperature under argon. After completion of the reaction was
cooled under ice cold temperature, cooled reaction mixture was
washed with 10% cold citric acid solution followed by cold water.
Combined organics were washed with brine, dried over
Na.sub.2SO.sub.4, evaporated the solvent and purified the mixture
through small bed of silica gel using DCM:MeOH (10%):triethylamine
(0.5%) as gradients to get pure 308 (1.50 g quantitative yields).
MS: Molecular weight cal. for C.sub.74H.sub.110N.sub.2O.sub.10
1186.82, Found: 1185.78 (M-1).
[0874] Synthesis of CPG Derivative 309:
[0875] To a solution of compound 308 (1.50 g, 1.30 mmol) in DMF was
added DIEA (0.700 ml, 3.0 eq), HBTU (0.740 g, 1.5 eq) followed by
long alkylamino CPG (10 g, loading: 124 .mu.mol/g) and continued on
shaker for 4 hours. Filtered the resin, washed with DCM (2.times.),
10% MeOH in DCM (2.times.), DCM (2.times.), finally with ether
(2.times.) and dried on vacuum. Capping: Dried resin was capped in
a solution of pyridine:acetic anhydride (20%) mixture (100 ml) and
continued on shaker for 45 minutes. After completion, the resin was
filtered, washed with DCM (2.times.), 10% MeOH in DCM (2.times.),
DCM (2.times.), finally with ether (2.times.) and dried on vacuum
(10.35 g, loading: 73 .mu.mol/g).
Example 16
##STR00044##
[0877] Synthesis of Compound 310:
[0878] To a solution of compound 307 (3.12 g, 2.86 mmol) in DCM (50
mL). was added DIEA (1.00 mL, 2 eq) followed by choloroamidite
reagent (0.850 ml, 1.30 eq). and stirred the mixture under argon.
Reaction was monitored by TLC and after 15 minutes the mixture
diluted with DCM, washed with sodium bicarbonate solution, water
and dried. Crude product was purified by chromatography (40-80%
EtOAc/Hexane with 1% TEA) to get the required compound (2.86 g,
78%), .sup.31P NMR (400 MHz, DMSO-d6) .delta. 151.87, 151.69,
151.52, 151.20.
Example 17
##STR00045##
[0880] Synthesis of Compound 313:
[0881] To a solution of compound 311 (6.28 g, 15.0 mmol, 1.0 eq)
and DIPEA (8.3 ml, 45.0 mmol, 3.0 eq) in DCM was added a solution
of activated ester of compound 312 (6.33 g, 15.0 mmol, 1.0 eq) in
DCM at room temperature and the reaction was continued for 4 hours,
after completion of the reaction, solvent was evaporated and
purified directly over silica gel using
hexane:ethylacetate:triethylamine (0.5%) as gradients to get 9.47 g
(87%) of the pure product 313. Ms: Calc. mass for
C.sub.40H.sub.42N.sub.2O.sub.7S.sub.2 is 726.2, found: 749.2
(M+Na).
[0882] Synthesis of Compound 314:
[0883] To a solution of compound 313 (9.46 g, 13.03 mmol, 1.0 eq)
in DCM was added DMAP (4.76 g, 39.09 mmol, 3.0 eq) followed by
succinic anhydride (2.6 g, 26.06 g, 2.0 eq) and the reaction was
continued for overnight at room temperature under argon. After
completion of the reaction was cooled under ice cold temperature,
cooled reaction mixture was washed with 10% citric acid solution
followed by cold water. Combined organics were washed with brine,
dried over Na.sub.2SO.sub.4, evaporated the solvent and purified
the mixture through small bed of silica gel using DCM:MeOH
(10%):triethylamine (0.5%) as gradients to get pure 10.7 g
(quantitative yields) of the compound 314. MS: Calc. mass for
C.sub.44H.sub.46N.sub.2O.sub.10S.sub.2 is 826.2, found 826.2.
[0884] Synthesis of Compound 315 (Loading on CPG-NH.sub.2):
[0885] To a solution of compound 314 (10.65 g, 12.9 mmol, 1.0 eq)
in DMF was added DIPEA (6.98 ml, 37.92 mmol, 3.0 eq), HBTU (7.19 g,
18.96 mmol, 1.5 eq) followed by CPG-NH.sub.2 (105 g, 13.02 mmol,
1.01 eq, load: 124 mol/g) and continued on shaker for 4 hours.
Filtered the resin, washed with DCM (2.times.), 10% MeOH in DCM
(2.times.), DCM (2.times.), finally with ether (2.times.) and dried
on vacuum. Capping: Dried resin was capped in a solution of
pyridine:acetic anhydride (20%) mixture (400 ml) and continued on
shaker for 45 minutes. After completion, the resin was filtered,
washed with DCM (2.times.), 10% MeOH in DCM (2.times.), DCM
(2.times.), finally with ether (2.times.) and dried on vacuum to
the resin 315 (110 g. Loading: 66 .mu.mol/g).
Example 18
##STR00046##
[0887] Synthesis of Compound 316:
[0888] To a solution of compound 313 (5.36, 7.37 mmol) in DCM (50
mL) was added DIEA (3.30 mL, 2.5 eq) followed by choloroamidite
reagent (2.14 ml, 1.30 eq). and stirred the mixture under argon.
Reaction was monitored by TLC and after 15 minutes the mixture
diluted with DCM, washed with sodium bicarbonate solution, water
and dried. Crude product was purified by chromatography (40-100%
EtOAc/Hexane with 1% TEA) to get the required compound (5.63, 82%),
.sup.31P NMR (400 MHz, DMSO-d6) .delta. 151.92, 151.74, 151.53,
151.28.
Example 19: Synthesis of Spermine and Cholesterol Oligonucleotide
Conjugate
##STR00047##
[0890] The oligonucleotide (A-40867: AAUCuuAuAuuuGAUCcAAsQ94L10
(SEQ ID NO:187) (target ApoB) was synthesized on ABI 392 DNA/RNA
Synthesizer at 40 umol scale using standard phosphoramidite
chemistry as outlined in Example 2. The solid support was then
treated with 0.5M piperidine in acetonitrile for 5 to 10 mins
followed 3 times acetonitrile wash. The cleavage from the solid
support and nucleobase deprotection was done with 3:1 mixture of
Ammonia/ethanol at 55.degree. C. for 5 hours. Reaction mixture was
cooled and filtered, the support was washed the 3.times. volume of
DMSO. Added 1.5.times. volume TEA3HF into the reaction mixture and
heated at 40 C for 1.5 hours. The crude product was purified with
reverse phase HPLC and desalted with size exclusion column.
Calculated MW: 8010.8, observed MW: 8009.1
Example 20: Conjugation of of DLink-DMA with Hydrazone Linkage to
Oligonucleotide
##STR00048##
[0892] Oligonucleotide (A30861) (SEQ ID NO:188) was synthesized and
purified using standard protocols outlined in example 2. Purified
A30861 was dissolved in 50 mM TEAA buffer (pH=7) and added 2-3
equivalents of TCEP (0.1M in water). Incubated at 40.degree. C. for
30 mins or room temp for overnight. Removed excess TCEP on C18
column and collected the RNA in 50 mM TEAA buffer with 25%
acetonitrile. Dissolved 2-3 equivalents DLinK-DMA pyridyl disulfide
in 1:1 mixture of DMSO and acetonitrile and added into the RNA
solution. Coupling reaction was performed at room temperature for
30 minutes. Calculated MW: 8056.1, Observed MW: 8053.8 (major
impurity: 8038.6).
Example 21. Post Synthesis Conjugation of Activated DLin-k-DMA with
Thiol Modified Oligonucleotides
##STR00049##
[0894] Oligonucleotide (40377.1: AcAuGAAGcAGcACGACuUdTsdTQ8L99 (SEQ
ID NO:189), Q8, Hyp-Amino, L99=Hyp-S--S-amino) was synthesized and
purified using standard protocols outlined in example 2. DLin-k-DMA
was then conjugated with the oligonucleotide as follow:
Reduction of Disulfide Bond:
[0895] The oligonucleotide (10 mg) was dissolved in 1.0 ml of 0.5 M
Ammonium acetate. 0.2 M TCEP solution (280 ul) was added and put at
heating block at 45.degree. C. for 90 min. The excess of TCEP was
removed by using Sephadex G-25 Nap-25 column. The identity of the
compound was confirmed by Ion exchange analysis as well as LC-MS
analysis.
Conjugation of Thiol Modified Oligonucleotides with
Acid-Cleavable-DLin-k-DMA.
[0896] The Thiol containing sample was dissolved in 450 .mu.l of
0.2M Sodium bicarbonate buffer (pH 8.5). To the dissolved
oligonucleotide activated acid-cleavable-DLin-k-DMA (10 equiv.) in
50 .mu.l of DMF was added and the reaction mixture was shaken over
night at room temperature. Progress of reaction was analyzed by
HPLC. LC-MS analysis of conjugated oligonucleotides is in
progress.
Example 22
##STR00050## ##STR00051## ##STR00052##
[0898] Amino RNA 350 is treated with compound 15 under basic pH to
get compound 351. It is purified and anneal with corresponding
complementary sequence to get the duplex 352. This compound is used
for all biological experiments.
Example 23
##STR00053## ##STR00054## ##STR00055##
[0900] Thiol RNA 353 is treated with compound 19 under basic pH to
get compound 354. It is purified and anneal with corresponding
complementary sequence to get the duplex 355. This compound is used
for all biological experiments.
Example 24
##STR00056## ##STR00057## ##STR00058##
[0902] Bifunctional RNA 357 is annealed with corresponding
complementary strand to get the duplex 358. This compound is
treated with compound 506 under basic pH initially to get the
intermediate. The intermediate is then treat with the NHS ester 114
to get the final compound 359. It is purified and is used for all
biological experiments.
Example 25
##STR00059## ##STR00060##
[0904] The ketal 366 was synthesized using a reported procedure
(Paramonov, S. E.; Bachelder, E. M.; Beaudette, T. T.; Standley, S.
M.; Lee, C. C.; Dashe, J.; Frechet, Jean M. J. Fully
Acid-Degradable Biocompatible Polyacetal Microparticles for Drug
Delivery. Bioconjugate Chemistry (2008), 19 (4), 911-919). The
transient protection of the ketal was carried out in two steps in
one pot first by treating the diamine with one equivalent of
ethyltrifluoroacetate followed by one equivalent of Cbz-OSu to
provide the di protected derivative 367 in 80% yield after column
purification. The protected amine 367 on treatment with aqueous
LiOH provided the maine 368 in quantitative yield. Coupling of this
amine 368 (0.5 g) with the protected folic acid 113 (1 g) provided
the coupled product 369 (1.1 g) which on hydrogenation provided the
amine 370 in quantitative yield. Coupling of amine 370 was carried
out with the maleimidopropionic acid 371 to give the coupled
product 372 in good yields. The final deprotection of all the
protecting group in 372 is carried out using ice-cold aqueous LiOH
in THF to afford the precursor 373 as an orange solid.
Example 26
##STR00061## ##STR00062##
[0906] In another embodiment, the dimethyl acetal group was
replaced by a cyclopentenyl group. The ketal 374 is converted to
the monoprotected amine 376 via the TFA derivative 375 as shown in
the following Scheme. The amine 376 on treatment with the acid 500
provided the coupled product 377 which is hydrogenated to give the
amine 378 which is coupled with the maleimide 379 and deprotected
to give the derivative 381.
Example 27
##STR00063## ##STR00064##
[0908] In another embodiment, the dimethyl acetal group was
replaced by a cyclohexyl group. The ketal 382 is converted to the
monoprotected amine 384 via the TFA derivative 383 as shown in the
following Scheme. The amine 384 on treatment with the acid 500
provided the coupled product 385 which is hydrogenated to give the
amine 386 which is coupled with the maleimide 387 and deprotected
to give the derivative 389.
Example 28
##STR00065## ##STR00066##
[0910] In another embodiment, the dimethyl acetal group was
replaced by a methyl t-butyl group. The ketal 390 is converted to
the monoprotected amine 392 via the TFA derivative 391 as shown in
the following Scheme. The amine 392 on treatment with the acid 500
provided the coupled product 393 which is hydrogenated to give the
amine 394 which is coupled with the maleimide 395 and deprotected
to give the derivative 397.
Example 29
##STR00067## ##STR00068## ##STR00069##
[0912] Compound 403 is prepared as outlined above and then
conjugated to nucleic acid.
Example 30
##STR00070## ##STR00071## ##STR00072##
[0914] Compound 407 is prepared as outlined above and then
conjugated to nucleic acid.
Example 31
##STR00073## ##STR00074## ##STR00075##
[0916] Compound 411 is prepared as outlined above and then
conjugated to nucleic acid.
Example 32
##STR00076## ##STR00077## ##STR00078##
[0918] Compound 415 is prepared as outlined above and then
conjugated to nucleic acid.
Example 33
##STR00079##
[0920] Compound 416, 417, 418, 419 are prepared as outlined above
and then conjugated to nucleic acid.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 189 <210> SEQ ID NO 1 <211> LENGTH: 29 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Endosomolytic Component:
GALA <400> SEQUENCE: 1 Ala Ala Leu Glu Ala Leu Ala Glu Ala
Leu Glu Ala Leu Ala Glu Ala 1 5 10 15 Leu Glu Ala Leu Ala Glu Ala
Ala Ala Ala Gly Gly Cys 20 25 <210> SEQ ID NO 2 <211>
LENGTH: 30 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Endosomolytic Component: EALA <400> SEQUENCE: 2 Ala Ala Leu
Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala 1 5 10 15 Glu
Ala Leu Ala Glu Ala Leu Ala Ala Ala Ala Gly Gly Cys 20 25 30
<210> SEQ ID NO 3 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component. <400>
SEQUENCE: 3 Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu
Ala 1 5 10 15 <210> SEQ ID NO 4 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endosomolytic
Component: INF-7 <400> SEQUENCE: 4 Gly Leu Phe Glu Ala Ile
Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Trp Asp
Tyr Gly 20 <210> SEQ ID NO 5 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endosomolytic
Component: INF HA-2 <400> SEQUENCE: 5 Gly Leu Phe Gly Ala Ile
Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly
Trp Tyr Gly 20 <210> SEQ ID NO 6 <211> LENGTH: 48
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endosomolytic
Component: diINF-7 <400> SEQUENCE: 6 Gly Leu Phe Glu Ala Ile
Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly
Trp Tyr Gly Cys Gly Leu Phe Glu Ala Ile Glu Gly 20 25 30 Phe Ile
Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Cys 35 40 45
<210> SEQ ID NO 7 <211> LENGTH: 44 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: diINF3
<400> SEQUENCE: 7 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu
Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly Gly Cys Gly Leu Phe
Glu Ala Ile Glu Gly Phe Ile 20 25 30 Glu Asn Gly Trp Glu Gly Met
Ile Asp Gly Gly Cys 35 40 <210> SEQ ID NO 8 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Endosomolytic Component: GLF <400> SEQUENCE: 8 Gly Leu Phe
Gly Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu 1 5 10 15 His
Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly 20 25
30 Gly Ser Cys 35 <210> SEQ ID NO 9 <211> LENGTH: 34
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endosomolytic
Component: GALA-INF3 <400> SEQUENCE: 9 Gly Leu Phe Glu Ala
Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Leu Ala Glu
Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly 20 25 30 Ser
Cys <210> SEQ ID NO 10 <211> LENGTH: 41 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Endosomolytic Component:
INF-5 <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (17)..(17) <223> OTHER INFORMATION: Nle
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (38)..(38) <223> OTHER INFORMATION: Nle <400>
SEQUENCE: 10 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly
Trp Glu Gly 1 5 10 15 Leu Ile Asp Gly Lys Gly Leu Phe Glu Ala Ile
Glu Gly Phe Ile Glu 20 25 30 Asn Gly Trp Glu Gly Leu Ile Asp Gly 35
40 <210> SEQ ID NO 11 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Endosomolytic Component:
JTS-1 <400> SEQUENCE: 11 Gly Leu Phe Glu Ala Leu Leu Glu Leu
Leu Glu Ser Leu Trp Glu Leu 1 5 10 15 Leu Leu Glu Ala 20
<210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: ppTG1
<400> SEQUENCE: 12 Gly Leu Phe Lys Ala Leu Leu Lys Leu Leu
Lys Ser Leu Trp Lys Leu 1 5 10 15 Leu Leu Lys Ala 20 <210>
SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: ppTG20
<400> SEQUENCE: 13 Gly Leu Phe Arg Ala Leu Leu Arg Leu Leu
Arg Ser Leu Trp Arg Leu 1 5 10 15 Leu Leu Arg Ala 20 <210>
SEQ ID NO 14 <211> LENGTH: 30 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: KALA
<400> SEQUENCE: 14 Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala
Lys Ala Leu Ala Lys His 1 5 10 15 Leu Ala Lys Ala Leu Ala Lys Ala
Leu Lys Ala Cys Glu Ala 20 25 30 <210> SEQ ID NO 15
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Endosomolytic Component: HA <400> SEQUENCE: 15
Gly Leu Phe Phe Glu Ala Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu 1 5
10 15 Gly Leu Ile Glu Gly Cys 20 <210> SEQ ID NO 16
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Endosomolytic Component: Melittin <400>
SEQUENCE: 16 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu
Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25
<210> SEQ ID NO 17 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: Histidine
rich <400> SEQUENCE: 17 Cys His Lys Lys Lys Lys Lys Lys His
Cys 1 5 10 <210> SEQ ID NO 18 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Derivative of
the N-terminal sequence of the HA-2 subunit of the influenza virus
hemagglutinin <400> SEQUENCE: 18 His His His His His Trp Tyr
Gly 1 5 <210> SEQ ID NO 19 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Hydrophobic MTS-containing
peptide: RFGF <400> SEQUENCE: 19 Ala Ala Val Ala Leu Leu Pro
Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 <210> SEQ ID NO
20 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: RFGF analogue <400> SEQUENCE: 20 Ala Ala
Leu Leu Pro Val Leu Leu Ala Ala Pro 1 5 10 <210> SEQ ID NO 21
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Cell Permeation Peptide: Penetratin <400>
SEQUENCE: 21 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys
Trp Lys Lys 1 5 10 15 <210> SEQ ID NO 22 <211> LENGTH:
14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Cell Permeation
Peptide: Tat fragment (48-60) <400> SEQUENCE: 22 Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Cys 1 5 10 <210> SEQ
ID NO 23 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Cell Permeation Peptide: Signal Sequence-based
peptide <400> SEQUENCE: 23 Gly Ala Leu Phe Leu Gly Trp Leu
Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys
Lys Lys Arg Lys Val 20 25 <210> SEQ ID NO 24 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Cell
Permeation Peptide: PVEC <400> SEQUENCE: 24 Leu Leu Ile Ile
Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys
<210> SEQ ID NO 25 <211> LENGTH: 26 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide: Transportan
<400> SEQUENCE: 25 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu
Leu Lys Ile Asn Leu Lys 1 5 10 15 Ala Leu Ala Ala Leu Ala Lys Lys
Ile Leu 20 25 <210> SEQ ID NO 26 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Cell Permeation
Peptide: Amphiphilic model peptide <400> SEQUENCE: 26 Lys Leu
Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15
Leu Ala <210> SEQ ID NO 27 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Cell Permeation Peptide:
Arg9 <400> SEQUENCE: 27 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1
5 <210> SEQ ID NO 28 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide: Bacterial
cell wall permeating <400> SEQUENCE: 28 Lys Phe Phe Lys Phe
Phe Lys Phe Phe Lys 1 5 10 <210> SEQ ID NO 29 <211>
LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Cell
Permeation Peptide: LL-37 <400> SEQUENCE: 29 Leu Leu Gly Asp
Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 Phe Lys
Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25 30
Pro Arg Thr Glu Ser 35 <210> SEQ ID NO 30 <211> LENGTH:
31 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Cell Permeation
Peptide: Cecropin P1 <400> SEQUENCE: 30 Ser Trp Leu Ser Lys
Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys 1 5 10 15 Arg Ile Ser
Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg 20 25 30
<210> SEQ ID NO 31 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide:
Alpha-defensin <400> SEQUENCE: 31 Ala Cys Tyr Cys Arg Ile Pro
Ala Cys Ile Ala Gly Glu Arg Arg Tyr 1 5 10 15 Gly Thr Cys Ile Tyr
Gln Gly Arg Leu Trp Ala Phe Cys Cys 20 25 30 <210> SEQ ID NO
32 <211> LENGTH: 36 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Cell Permeation Peptide: b-defensin <400>
SEQUENCE: 32 Asp His Tyr Asn Cys Val Ser Ser Gly Gly Gln Cys Leu
Tyr Ser Ala 1 5 10 15 Cys Pro Ile Phe Thr Lys Ile Gln Gly Thr Cys
Tyr Arg Gly Lys Ala 20 25 30 Lys Cys Cys Lys 35 <210> SEQ ID
NO 33 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Cell Permeation Peptide: Bactenecin <400>
SEQUENCE: 33 Arg Lys Cys Arg Ile Val Val Ile Arg Val Cys Arg 1 5 10
<210> SEQ ID NO 34 <211> LENGTH: 42 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide: PR-3
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (42)..(42) <223> OTHER INFORMATION: NH2
modification of C-terminus <400> SEQUENCE: 34 Arg Arg Arg Pro
Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro 1 5 10 15 Phe Phe
Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro 20 25 30
Arg Phe Pro Pro Arg Phe Pro Gly Lys Arg 35 40 <210> SEQ ID NO
35 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Cell Permeation Peptide: Indolicidin <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: NH2 modification to
C-terminus <400> SEQUENCE: 35 Ile Leu Pro Trp Lys Trp Pro Trp
Trp Pro Trp Arg Arg 1 5 10 <210> SEQ ID NO 36 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Conserved sequence between mouse lamin B1, lamin B2, keratin
complex 2-gene 1 and lamin A/C <400> SEQUENCE: 36 aagctggccc
tggacatgga gat 23 <210> SEQ ID NO 37 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Conjugation of Cholesterol to 5' end
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(20)..(21) <223> OTHER INFORMATION: Thioate linkage
<400> SEQUENCE: 37 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 38 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Conjugation of Cholesterol to 5' end <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (20)..(21)
<223> OTHER INFORMATION: Thioate linkage <400>
SEQUENCE: 38 ggacuacucu aaguucuact t 21 <210> SEQ ID NO 39
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(20)..(21) <223> OTHER INFORMATION: Thioate linkage
<400> SEQUENCE: 39 ggacuacucu aaguucuact t 21 <210> SEQ
ID NO 40 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(9) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(14)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(17)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (20)..(21) <223> OTHER INFORMATION: Thioate linkage
<400> SEQUENCE: 40 guagaacuua gaguagucct t 21 <210> SEQ
ID NO 41 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (20)..(21) <223> OTHER INFORMATION: Thioate linkage
<400> SEQUENCE: 41 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 42 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(9) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(14)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(17)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (20)..(21) <223> OTHER INFORMATION: Thioate linkage
<400> SEQUENCE: 42 guaagacuug agaugaucct t 21 <210> SEQ
ID NO 43 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to L8 through phosphorothioate linkage <400> SEQUENCE:
43 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 44 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to L99 through
phosphorothioate linkage <400> SEQUENCE: 44 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 45 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q8 through
phosphorothioate linkage, and then link to L99 <400>
SEQUENCE: 45 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 46
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q96 through phosphorothioate linkage, and then link to L8
<400> SEQUENCE: 46 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 47 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to L99
<400> SEQUENCE: 47 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 48 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to L8
<400> SEQUENCE: 48 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 49 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to L8
<400> SEQUENCE: 49 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 50 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to
L99 <400> SEQUENCE: 50 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 51 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and link to L10
<400> SEQUENCE: 51 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 52 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
L10 <400> SEQUENCE: 52 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 53 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to L50
<400> SEQUENCE: 53 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 54 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to L8
<400> SEQUENCE: 54 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 55 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to L50 through phosphorothioate linkage <400>
SEQUENCE: 55 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 56
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q8 through phosphorothioate linkage, and then link to Q11 and L50
<400> SEQUENCE: 56 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 57 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
Q11 and L8 <400> SEQUENCE: 57 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 58 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to
L110 <400> SEQUENCE: 58 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 59 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
L110 <400> SEQUENCE: 59 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 60 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to Q11
and L110 <400> SEQUENCE: 60 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 61 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to Q8
and L110 <400> SEQUENCE: 61 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 62 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to Q8
and L110 <400> SEQUENCE: 62 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 63 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to
Q96 and L110 <400> SEQUENCE: 63 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 64 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to L80
<400> SEQUENCE: 64 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 65 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
L80 <400> SEQUENCE: 65 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 66 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to Q11
and L80 <400> SEQUENCE: 66 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 67 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to Q8
and L80 <400> SEQUENCE: 67 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 68 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to Q8
and L80 <400> SEQUENCE: 68 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 69 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to
Q96 and L80 <400> SEQUENCE: 69 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 70 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to L90
<400> SEQUENCE: 70 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 71 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
L90 <400> SEQUENCE: 71 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 72 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to Q11
and L90 <400> SEQUENCE: 72 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 73 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to Q8
and L90 <400> SEQUENCE: 73 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 74 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to Q8
and L90 <400> SEQUENCE: 74 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 75 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to
Q96 and L90 <400> SEQUENCE: 75 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 76 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94) and L50,
where n = 1 to 10 <400> SEQUENCE: 76 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 77 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q96 through phosphorothioate
linkage, and then link to (n x Q94) and L135, where n = 0 to 10
<400> SEQUENCE: 77 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 78 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11 and
L50, where n = 1 to 10 <400> SEQUENCE: 78 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 79 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q11 through
phosphorothioate linkage, and then link to (n x Q94) and L50, where
n = 1 to 10 <400> SEQUENCE: 79 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 80 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96 and
L10, where n = 1 to 10 <400> SEQUENCE: 80 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 81 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q96 through
phosphorothioate linkage, and then link to (n x Q94) and L10, where
n = 0 to 10 <400> SEQUENCE: 81 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 82 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
Q11, (n x Q94) and L135, where n = 1 to 10 <400> SEQUENCE: 82
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 83 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q11 through
phosphorothioate linkage, and then link to (n x Q94) and L50, where
n = 0 to 10 <400> SEQUENCE: 83 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 84 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) and L50, where n = 1 to 10 <400> SEQUENCE: 84 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 85 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11, (n x Q94) and L50, where n = 1 to 10
<400> SEQUENCE: 85 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 86 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96, (n x
Q94) and L10, where n = 1 to 10 <400> SEQUENCE: 86 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 87 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q96, (n x Q94) and L10, where n = 0 to 10
<400> SEQUENCE: 87 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 88 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to (n
x Q94), Q11, (n x Q94) and L135, where n = 1 to 10 <400>
SEQUENCE: 88 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 89
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) and L50, where n = 0 to 10 <400> SEQUENCE: 89 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 90 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), L96 and L110, where n = 1 to 10 <400>
SEQUENCE: 90 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 91
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q96 through phosphorothioate linkage, and then link to (n x Q94)
and L110, where n = 0 to 10 <400> SEQUENCE: 91 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 92 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11 and L110, where n = 1 to 10 <400>
SEQUENCE: 92 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 93
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q11 through phosphorothioate linkage, and then link to (n x Q94)
and L110, where n = 1 to 10 <400> SEQUENCE: 93 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 94 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q96, and L110, where n = 1 to 10
<400> SEQUENCE: 94 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 95 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96, Q11
and L110, where n = 1 to 10 <400> SEQUENCE: 95 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 96 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11, Q96 and L110, where n = 1 to 10
<400> SEQUENCE: 96 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 97 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, Q96,
(n x Q94) and L110, where n = 1 to 10 <400> SEQUENCE: 97
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 98 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q96 and L110,
where n = 1 to 10 <400> SEQUENCE: 98 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 99 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q96 through phosphorothioate
linkage, and then link to (n x Q94) and L110, where n = 0 to 10
<400> SEQUENCE: 99 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 100 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
Q11, (n x Q94) and L110, where n = 1 to 10 <400> SEQUENCE:
100 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 101
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q11 through phosphorothioate linkage, and then link to (n x Q94)
and L110, where n = 0 to 10 <400> SEQUENCE: 101 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 102 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11, (n x Q94), Q96 and L110, where n = 1
to 10 <400> SEQUENCE: 102 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 103 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) and L110, where n = 1 to 10 <400> SEQUENCE: 103
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 104 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q11 and L110,
where n = 1 to 10 <400> SEQUENCE: 104 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 105 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q96, (n x Q94), Q11 and L110, where n = 1 to 10
<400> SEQUENCE: 105 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 106 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96, (n x
Q94), Q11 and L110, where n = 0 to 10 <400> SEQUENCE: 106
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 107 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q96 through
phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) and L110, where n = 1 to 10 <400> SEQUENCE: 107
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 108 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q96 and L110,
where n =0 to 10 <400> SEQUENCE: 108 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 109 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), L96 and L80, where n = 1 to 10 <400>
SEQUENCE: 109 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 110
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q96 through phosphorothioate linkage, and then link to (n x Q94)
and L80, where n = 0 to 10 <400> SEQUENCE: 110 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 111 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11 and L80, where n = 1 to 10 <400>
SEQUENCE: 111 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 112
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q11 through phosphorothioate linkage, and then link to (n x Q94)
and L80, where n = 1 to 10 <400> SEQUENCE: 112 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 113 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q96 and L80, where n = 1 to 10 <400>
SEQUENCE: 113 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 114
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94), Q96, Q11 and
L80, where n = 1 to 10 <400> SEQUENCE: 114 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 115 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11, Q96 and L80, where n = 1 to 10
<400> SEQUENCE: 115 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 116 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, Q96,
(n x Q94) and L80, where n = 1 to 10 <400> SEQUENCE: 116
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 117 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q96 and L80,
where n = 1 to 10 <400> SEQUENCE: 117 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 118 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q96 through phosphorothioate
linkage, and then link to (n x Q94) and L80, where n = 0 to 10
<400> SEQUENCE: 118 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 119 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
Q11, (n x Q94) and L80, where n = 1 to 10 <400> SEQUENCE: 119
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 120 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q11 through
phosphorothioate linkage, and then link to (n x Q94) and L80, where
n = 0 to 10 <400> SEQUENCE: 120 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 121 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q96 and L80, where n = 1 to 10 <400> SEQUENCE: 121
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 122 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94) and L80, where
n = 1 to 10 <400> SEQUENCE: 122 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 123 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q11 and L80, where n = 1 to 10 <400> SEQUENCE: 123
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 124 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q96, (n x Q94), Q11 and L80,
where n = 1 to 10 <400> SEQUENCE: 124 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 125 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q96, (n x Q94), Q11 and L80, where n = 0 to 10
<400> SEQUENCE: 125 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 126 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to (n
x Q94), Q11, (n x Q94) and L80, where n = 1 to 10 <400>
SEQUENCE: 126 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 127
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q96 and L80, where n = 0 to 10 <400> SEQUENCE: 127
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 128 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), L96 and L90, where n = 1 to 10
<400> SEQUENCE: 128 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 129 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to (n
x Q94) and L90, where n = 0 to 10 <400> SEQUENCE: 129
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 130 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11 and L90, where n = 1 to 10
<400> SEQUENCE: 130 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 131 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to (n
x Q94) and L90, where n = 1 to 10 <400> SEQUENCE: 131
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 132 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q96 and L90, where n = 1 to 10
<400> SEQUENCE: 132 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 133 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96, Q11
and L90, where n = 1 to 10 <400> SEQUENCE: 133 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 134 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11, Q96 and L90, where n = 1 to 10
<400> SEQUENCE: 134 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 135 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, Q96,
(n x Q94) and L90, where n = 1 to 10 <400> SEQUENCE: 135
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 136 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q96 and L90,
where n = 1 to 10 <400> SEQUENCE: 136 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 137 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q96 through phosphorothioate
linkage, and then link to (n x Q94) and L90, where n = 0 to 10
<400> SEQUENCE: 137 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 138 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
Q11, (n x Q94) and L90, where n = 1 to 10 <400> SEQUENCE: 138
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 139 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q11 through
phosphorothioate linkage, and then link to (n x Q94) and L90, where
n = 0 to 10 <400> SEQUENCE: 139 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 140 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q96 and L90, where n = 1 to 10 <400> SEQUENCE: 140
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 141 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94) and L90, where
n = 1 to 10 <400> SEQUENCE: 141 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 142 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(1) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q11 and L90, where n = 1 to 10 <400> SEQUENCE: 142
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 143 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q96, (n x Q94) Q11 and L90,
where n = 1 to 10 <400> SEQUENCE: 143 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 144 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q96, (n x Q94), Q11 and L90, where n = 0 to 10
<400> SEQUENCE: 144 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 145 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to (n
x Q94), Q11, (n x Q94) and L90, where n = 1 to 10 <400>
SEQUENCE: 145 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 146
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) Q96 and L90, where n = 0 to 10 <400> SEQUENCE: 146
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 147 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to L132 through
phosphorothioate linkage <400> SEQUENCE: 147 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 148 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to L133 through
phosphorothioate linkage <400> SEQUENCE: 148 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 149 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to L134 through
phosphorothioate linkage <400> SEQUENCE: 149 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 150 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to L136 through
phosphorothioate linkage <400> SEQUENCE: 150 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 151 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q95 through
phosphorothioate linkage, and then link to L99 <400>
SEQUENCE: 151 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 152
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q95 through phosphorothioate linkage, and then link to L50
<400> SEQUENCE: 152 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 153 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to L8
<400> SEQUENCE: 153 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 154 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
Q96 and L110 <400> SEQUENCE: 154 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 155 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to Q8
and L110 <400> SEQUENCE: 155 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 156 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
L110 <400> SEQUENCE: 156 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 157 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
Q96 and L80 <400> SEQUENCE: 157 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 158 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to Q8
and L80 <400> SEQUENCE: 158 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 159 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
L80 <400> SEQUENCE: 159 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 160 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
Q96 and L90 <400> SEQUENCE: 160 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 161 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to Q8
and L90 <400> SEQUENCE: 161 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 162 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
L90 <400> SEQUENCE: 162 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 163 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94) and L133,
where n = 1 to 10 <400> SEQUENCE: 163 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 164 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94) and L133, where n = 0 to 10 <400> SEQUENCE:
164 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 165
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94), Q7 and L110,
where n = 1 to 10 <400> SEQUENCE: 165 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 166 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q7 through phosphorothioate
linkage, and then link to (n x Q94) and L110, where n = 1 to 10
<400> SEQUENCE: 166 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 167 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q7 and
L110, where n = 1 to 10 <400> SEQUENCE: 167 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 168 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q7 through
phosphorothioate linkage, and then link to (n x Q94) and L110,
where n = 0 to 10 <400> SEQUENCE: 168 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 169 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q7 through phosphorothioate
linkage, and then link to Q11, (n x Q94) and L110, where n = 1 to
10 <400> SEQUENCE: 169 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 170 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q7, (n x
Q94) and L110, where n = 1 to 10 <400> SEQUENCE: 170
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 171 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q7 and L110,
where n = 0 to 10 <400> SEQUENCE: 171 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 172 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q7 and L80, where n = 1 to 10 <400>
SEQUENCE: 172 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 173
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q7 through phosphorothioate linkage, and then link to (n x Q94) and
L80, where n = 1 to 10 <400> SEQUENCE: 173 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 174 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q7 and L80, where n = 1 to 10 <400>
SEQUENCE: 174 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 175
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q7 through phosphorothioate linkage, and then link to (n x Q94) and
L80, where n = 0 to 10 <400> SEQUENCE: 175 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 176 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q7 through
phosphorothioate linkage, and then link to Q11, (n x Q94) and L80,
where n = 1 to 10 <400> SEQUENCE: 176 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 177 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q7, (n x Q94) and L80, where n = 1 to 10
<400> SEQUENCE: 177 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 178 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q7 and L80, where n = 0 to 10 <400> SEQUENCE: 178
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 179 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q7 and L90, where n = 1 to 10
<400> SEQUENCE: 179 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 180 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q7 through phosphorothioate linkage, and then link to (n
x Q94) and L90, where n = 1 to 10 <400> SEQUENCE: 180
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 181 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q7 and L90, where n = 1 to 10
<400> SEQUENCE: 181 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 182 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q7 through phosphorothioate linkage, and then link to (n
x Q94) and L80, where n = 0 to 10 <400> SEQUENCE: 182
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 183 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q7 through
phosphorothioate linkage, and then link to Q11, (n x Q94) and L90,
where n = 1 to 10 <400> SEQUENCE: 183 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 184 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q7, (n x Q94) and L90, where n = 1 to 10
<400> SEQUENCE: 184 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 185 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q7 and L90, where n = 0 to 10 <400> SEQUENCE: 185
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 186 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(9) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (17)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (20)..(21)
<223> OTHER INFORMATION: Phosphorothioate linkage <400>
SEQUENCE: 186 guaagacuug agaugaucct t 21 <210> SEQ ID NO 187
<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide:
A-40867 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Link the 3' end to Q94 through phosphorothioate linkage, and then
link to L10 <400> SEQUENCE: 187 aaucuuauau uugauccaa 19
<210> SEQ ID NO 188 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide: A30861 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to DLink-DMA with hydrazone linkage
<400> SEQUENCE: 188 ggaaucuuau auuugaucca a 21 <210>
SEQ ID NO 189 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (20)..(21) <223> OTHER
INFORMATION: Phosphorothioate linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to Q8 and L99, and
then conjugate to DLin-k-DMA <400> SEQUENCE: 189 acaugaagca
gcacgacuut t 21
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 189
<210> SEQ ID NO 1 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: GALA
<400> SEQUENCE: 1 Ala Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu
Ala Leu Ala Glu Ala 1 5 10 15 Leu Glu Ala Leu Ala Glu Ala Ala Ala
Ala Gly Gly Cys 20 25 <210> SEQ ID NO 2 <211> LENGTH:
30 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endosomolytic
Component: EALA <400> SEQUENCE: 2 Ala Ala Leu Ala Glu Ala Leu
Ala Glu Ala Leu Ala Glu Ala Leu Ala 1 5 10 15 Glu Ala Leu Ala Glu
Ala Leu Ala Ala Ala Ala Gly Gly Cys 20 25 30 <210> SEQ ID NO
3 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Endosomolytic Component. <400> SEQUENCE: 3
Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala 1 5 10
15 <210> SEQ ID NO 4 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: INF-7
<400> SEQUENCE: 4 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu
Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Trp Asp Tyr Gly 20
<210> SEQ ID NO 5 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: INF HA-2
<400> SEQUENCE: 5 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu
Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly Trp Tyr Gly 20
<210> SEQ ID NO 6 <211> LENGTH: 48 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: diINF-7
<400> SEQUENCE: 6 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu
Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly Trp Tyr Gly Cys Gly
Leu Phe Glu Ala Ile Glu Gly 20 25 30 Phe Ile Glu Asn Gly Trp Glu
Gly Met Ile Asp Gly Trp Tyr Gly Cys 35 40 45 <210> SEQ ID NO
7 <211> LENGTH: 44 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Endosomolytic Component: diINF3 <400>
SEQUENCE: 7 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp
Glu Gly 1 5 10 15 Met Ile Asp Gly Gly Cys Gly Leu Phe Glu Ala Ile
Glu Gly Phe Ile 20 25 30 Glu Asn Gly Trp Glu Gly Met Ile Asp Gly
Gly Cys 35 40 <210> SEQ ID NO 8 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endosomolytic
Component: GLF <400> SEQUENCE: 8 Gly Leu Phe Gly Ala Leu Ala
Glu Ala Leu Ala Glu Ala Leu Ala Glu 1 5 10 15 His Leu Ala Glu Ala
Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly 20 25 30 Gly Ser Cys 35
<210> SEQ ID NO 9 <211> LENGTH: 34 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: GALA-INF3
<400> SEQUENCE: 9 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu
Asn Gly Trp Glu Gly 1 5 10 15 Leu Ala Glu Ala Leu Ala Glu Ala Leu
Glu Ala Leu Ala Ala Gly Gly 20 25 30 Ser Cys <210> SEQ ID NO
10 <211> LENGTH: 41 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Endosomolytic Component: INF-5 <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(17)..(17) <223> OTHER INFORMATION: Nle <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (38)..(38)
<223> OTHER INFORMATION: Nle <400> SEQUENCE: 10 Gly Leu
Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15
Leu Ile Asp Gly Lys Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu 20
25 30 Asn Gly Trp Glu Gly Leu Ile Asp Gly 35 40 <210> SEQ ID
NO 11 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Endosomolytic Component: JTS-1 <400>
SEQUENCE: 11 Gly Leu Phe Glu Ala Leu Leu Glu Leu Leu Glu Ser Leu
Trp Glu Leu 1 5 10 15 Leu Leu Glu Ala 20 <210> SEQ ID NO 12
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Endosomolytic Component: ppTG1 <400> SEQUENCE:
12 Gly Leu Phe Lys Ala Leu Leu Lys Leu Leu Lys Ser Leu Trp Lys Leu
1 5 10 15 Leu Leu Lys Ala 20 <210> SEQ ID NO 13 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Endosomolytic Component: ppTG20 <400> SEQUENCE: 13 Gly Leu
Phe Arg Ala Leu Leu Arg Leu Leu Arg Ser Leu Trp Arg Leu 1 5 10 15
Leu Leu Arg Ala 20 <210> SEQ ID NO 14 <211> LENGTH: 30
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endosomolytic
Component: KALA <400> SEQUENCE: 14 Trp Glu Ala Lys Leu Ala
Lys Ala Leu Ala Lys Ala Leu Ala Lys His 1 5 10 15 Leu Ala Lys Ala
Leu Ala Lys Ala Leu Lys Ala Cys Glu Ala 20 25 30 <210> SEQ ID
NO 15
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Endosomolytic Component: HA <400> SEQUENCE: 15
Gly Leu Phe Phe Glu Ala Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu 1 5
10 15 Gly Leu Ile Glu Gly Cys 20 <210> SEQ ID NO 16
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Endosomolytic Component: Melittin <400>
SEQUENCE: 16 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu
Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25
<210> SEQ ID NO 17 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endosomolytic Component: Histidine
rich <400> SEQUENCE: 17 Cys His Lys Lys Lys Lys Lys Lys His
Cys 1 5 10 <210> SEQ ID NO 18 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Derivative of
the N-terminal sequence of the HA-2 subunit of the influenza virus
hemagglutinin <400> SEQUENCE: 18 His His His His His Trp Tyr
Gly 1 5 <210> SEQ ID NO 19 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Hydrophobic MTS-containing
peptide: RFGF <400> SEQUENCE: 19 Ala Ala Val Ala Leu Leu Pro
Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 <210> SEQ ID NO
20 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: RFGF analogue <400> SEQUENCE: 20 Ala Ala
Leu Leu Pro Val Leu Leu Ala Ala Pro 1 5 10 <210> SEQ ID NO 21
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Cell Permeation Peptide: Penetratin <400>
SEQUENCE: 21 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys
Trp Lys Lys 1 5 10 15 <210> SEQ ID NO 22 <211> LENGTH:
14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Cell Permeation
Peptide: Tat fragment (48-60) <400> SEQUENCE: 22 Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Cys 1 5 10 <210> SEQ
ID NO 23 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Cell Permeation Peptide: Signal Sequence-based
peptide <400> SEQUENCE: 23 Gly Ala Leu Phe Leu Gly Trp Leu
Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys
Lys Lys Arg Lys Val 20 25 <210> SEQ ID NO 24 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Cell
Permeation Peptide: PVEC <400> SEQUENCE: 24 Leu Leu Ile Ile
Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys
<210> SEQ ID NO 25 <211> LENGTH: 26 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide: Transportan
<400> SEQUENCE: 25 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu
Leu Lys Ile Asn Leu Lys 1 5 10 15 Ala Leu Ala Ala Leu Ala Lys Lys
Ile Leu 20 25 <210> SEQ ID NO 26 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Cell Permeation
Peptide: Amphiphilic model peptide <400> SEQUENCE: 26 Lys Leu
Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15
Leu Ala <210> SEQ ID NO 27 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Cell Permeation Peptide:
Arg9 <400> SEQUENCE: 27 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1
5 <210> SEQ ID NO 28 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide: Bacterial
cell wall permeating <400> SEQUENCE: 28 Lys Phe Phe Lys Phe
Phe Lys Phe Phe Lys 1 5 10 <210> SEQ ID NO 29 <211>
LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Cell
Permeation Peptide: LL-37 <400> SEQUENCE: 29 Leu Leu Gly Asp
Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 Phe Lys
Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25 30
Pro Arg Thr Glu Ser 35 <210> SEQ ID NO 30 <211> LENGTH:
31 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Cell Permeation
Peptide: Cecropin P1 <400> SEQUENCE: 30 Ser Trp Leu Ser Lys
Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys 1 5 10 15 Arg Ile Ser
Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg 20 25 30
<210> SEQ ID NO 31 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide:
Alpha-defensin <400> SEQUENCE: 31 Ala Cys Tyr Cys Arg Ile Pro
Ala Cys Ile Ala Gly Glu Arg Arg Tyr 1 5 10 15
Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 20 25 30
<210> SEQ ID NO 32 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide: b-defensin
<400> SEQUENCE: 32 Asp His Tyr Asn Cys Val Ser Ser Gly Gly
Gln Cys Leu Tyr Ser Ala 1 5 10 15 Cys Pro Ile Phe Thr Lys Ile Gln
Gly Thr Cys Tyr Arg Gly Lys Ala 20 25 30 Lys Cys Cys Lys 35
<210> SEQ ID NO 33 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide: Bactenecin
<400> SEQUENCE: 33 Arg Lys Cys Arg Ile Val Val Ile Arg Val
Cys Arg 1 5 10 <210> SEQ ID NO 34 <211> LENGTH: 42
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Cell Permeation
Peptide: PR-3 <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (42)..(42) <223> OTHER
INFORMATION: NH2 modification of C-terminus <400> SEQUENCE:
34 Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro
1 5 10 15 Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe
Pro Pro 20 25 30 Arg Phe Pro Pro Arg Phe Pro Gly Lys Arg 35 40
<210> SEQ ID NO 35 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Cell Permeation Peptide: Indolicidin
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: NH2
modification to C-terminus <400> SEQUENCE: 35 Ile Leu Pro Trp
Lys Trp Pro Trp Trp Pro Trp Arg Arg 1 5 10 <210> SEQ ID NO 36
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Conserved sequence between mouse lamin B1, lamin B2,
keratin complex 2-gene 1 and lamin A/C <400> SEQUENCE: 36
aagctggccc tggacatgga gat 23 <210> SEQ ID NO 37 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Conjugation of Cholesterol
to 5' end <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (4)..(5) <223> OTHER INFORMATION:
2'-fluro modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(20)..(21) <223> OTHER INFORMATION: Thioate linkage
<400> SEQUENCE: 37 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 38 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Conjugation of Cholesterol to 5' end <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (20)..(21)
<223> OTHER INFORMATION: Thioate linkage <400>
SEQUENCE: 38 ggacuacucu aaguucuact t 21 <210> SEQ ID NO 39
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(20)..(21) <223> OTHER INFORMATION: Thioate linkage
<400> SEQUENCE: 39 ggacuacucu aaguucuact t 21 <210> SEQ
ID NO 40 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(9) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(14)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(17)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (20)..(21) <223> OTHER INFORMATION: Thioate linkage
<400> SEQUENCE: 40 guagaacuua gaguagucct t 21 <210> SEQ
ID NO 41 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (19)..(19) <223> OTHER INFORMATION:
2'-fluro modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (20)..(21) <223> OTHER
INFORMATION: Thioate linkage <400> SEQUENCE: 41 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 42 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(2)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(9) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (17)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (20)..(21) <223>
OTHER INFORMATION: Thioate linkage <400> SEQUENCE: 42
guaagacuug agaugaucct t 21 <210> SEQ ID NO 43 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to L8 through
phosphorothioate linkage <400> SEQUENCE: 43 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 44 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to L99 through
phosphorothioate linkage <400> SEQUENCE: 44 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 45 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q8 through
phosphorothioate linkage, and then link to L99 <400>
SEQUENCE: 45 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 46
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q96 through phosphorothioate linkage, and then link to L8
<400> SEQUENCE: 46 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 47 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to L99
<400> SEQUENCE: 47 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 48 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to L8
<400> SEQUENCE: 48 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 49 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q11 through
phosphorothioate linkage, and then link to L8 <400> SEQUENCE:
49 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 50 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q11 through
phosphorothioate linkage, and then link to L99 <400>
SEQUENCE: 50 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 51
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q8 through phosphorothioate linkage, and link to L10 <400>
SEQUENCE: 51 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 52
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q96 through phosphorothioate linkage, and then link to L10
<400> SEQUENCE: 52 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 53 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to L50
<400> SEQUENCE: 53 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 54 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to L8
<400> SEQUENCE: 54 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 55 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to L50 through phosphorothioate linkage <400>
SEQUENCE: 55 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 56
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to Q11
and L50 <400> SEQUENCE: 56 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 57 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
Q11 and L8 <400> SEQUENCE: 57 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 58 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to
L110 <400> SEQUENCE: 58 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 59 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
L110 <400> SEQUENCE: 59 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 60 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to Q11
and L110 <400> SEQUENCE: 60 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 61 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to Q8
and L110 <400> SEQUENCE: 61 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 62 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to Q8
and L110 <400> SEQUENCE: 62 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 63 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to
Q96 and L110
<400> SEQUENCE: 63 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 64 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to L80
<400> SEQUENCE: 64 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 65 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
L80 <400> SEQUENCE: 65 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 66 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to Q11
and L80 <400> SEQUENCE: 66 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 67 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to Q8
and L80 <400> SEQUENCE: 67 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 68 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to Q8
and L80 <400> SEQUENCE: 68 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 69 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to
Q96 and L80 <400> SEQUENCE: 69 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 70 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q8 through phosphorothioate linkage, and then link to L90
<400> SEQUENCE: 70 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 71 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q96 through
phosphorothioate linkage, and then link to L90 <400>
SEQUENCE: 71 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 72
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q8 through phosphorothioate linkage, and then link to Q11 and L90
<400> SEQUENCE: 72 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 73 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to Q8
and L90 <400> SEQUENCE: 73 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 74 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to Q8
and L90 <400> SEQUENCE: 74 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 75 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to
Q96 and L90 <400> SEQUENCE: 75 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 76 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94) and L50,
where n = 1 to 10 <400> SEQUENCE: 76 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 77 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q96 through phosphorothioate
linkage, and then link to (n x Q94) and L135, where n = 0 to 10
<400> SEQUENCE: 77 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 78 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q11 and L50, where n = 1 to 10 <400>
SEQUENCE: 78 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 79
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q11 through phosphorothioate linkage, and then link to (n x Q94)
and L50, where n = 1 to 10 <400> SEQUENCE: 79 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 80 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q96 and L10, where n = 1 to 10 <400>
SEQUENCE: 80 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 81
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q96 through phosphorothioate linkage, and then link to (n x Q94)
and L10, where n = 0 to 10 <400> SEQUENCE: 81 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 82 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q96 through
phosphorothioate linkage, and then link to Q11, (n x Q94) and L135,
where n = 1 to 10 <400> SEQUENCE: 82 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 83 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q11 through phosphorothioate
linkage, and then link to (n x Q94) and L50, where n = 0 to 10
<400> SEQUENCE: 83 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 84 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) and L50, where n = 1 to 10 <400> SEQUENCE: 84 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 85 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94) and L50, where
n = 1 to 10 <400> SEQUENCE: 85 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 86 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96, (n x
Q94) and L10, where n = 1 to 10 <400> SEQUENCE: 86 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 87 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q96, (n x Q94) and L10, where n = 0 to 10
<400> SEQUENCE: 87 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 88 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to (n
x Q94), Q11, (n x Q94) and L135, where n = 1 to 10 <400>
SEQUENCE: 88 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 89
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) and L50, where n = 0 to 10 <400> SEQUENCE: 89 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 90 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), L96 and L110, where n = 1 to 10 <400>
SEQUENCE: 90 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 91
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q96 through phosphorothioate linkage, and then link to (n x Q94)
and L110, where n = 0 to 10 <400> SEQUENCE: 91 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 92 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11 and L110, where n = 1 to
10 <400> SEQUENCE: 92 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 93 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q11 through phosphorothioate linkage, and then link to (n
x Q94) and L110, where n = 1 to 10 <400> SEQUENCE: 93
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 94 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q96, and L110, where n = 1 to
10 <400> SEQUENCE: 94 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 95 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96, Q11
and L110, where n = 1 to 10 <400> SEQUENCE: 95 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 96 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11, Q96 and L110, where n = 1 to 10
<400> SEQUENCE: 96 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 97 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, Q96,
(n x Q94) and L110, where n = 1 to 10 <400> SEQUENCE: 97
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 98 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q96 and L110,
where n = 1 to 10 <400> SEQUENCE: 98 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 99 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q96 through phosphorothioate
linkage, and then link to (n x Q94) and L110, where n = 0 to 10
<400> SEQUENCE: 99 ggaucaucuc aagucuuact t 21 <210> SEQ
ID NO 100 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
Q11, (n x Q94) and L110, where n = 1 to 10 <400> SEQUENCE:
100 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 101
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q11 through phosphorothioate linkage, and then link to (n x Q94)
and L110, where n = 0 to 10 <400> SEQUENCE: 101 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 102 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11, (n x Q94), Q96 and L110, where n = 1
to 10 <400> SEQUENCE: 102 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 103 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) and L110, where n = 1 to 10 <400> SEQUENCE: 103
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 104 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q11 and L110,
where n = 1 to 10 <400> SEQUENCE: 104 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 105 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q96, (n x Q94), Q11 and L110, where n = 1 to 10
<400> SEQUENCE: 105 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 106 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96, (n x
Q94), Q11 and L110, where n = 0 to 10 <400> SEQUENCE: 106
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 107 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q96 through
phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) and L110, where n = 1 to 10 <400> SEQUENCE: 107
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 108 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q96 and L110,
where n =0 to 10 <400> SEQUENCE: 108 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 109 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), L96 and L80, where n = 1 to 10 <400>
SEQUENCE: 109 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 110
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q96 through phosphorothioate linkage, and then link to (n x Q94)
and L80, where n = 0 to 10 <400> SEQUENCE: 110 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 111 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11 and L80, where n = 1 to 10 <400>
SEQUENCE: 111 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 112
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q11 through phosphorothioate linkage, and then link to (n x Q94)
and L80, where n = 1 to 10 <400> SEQUENCE: 112 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 113 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q96 and L80, where n = 1 to 10 <400>
SEQUENCE: 113 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO
114
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94), Q96, Q11 and
L80, where n = 1 to 10 <400> SEQUENCE: 114 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 115 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q11, Q96 and L80, where n = 1 to 10
<400> SEQUENCE: 115 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 116 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, Q96,
(n x Q94) and L80, where n = 1 to 10 <400> SEQUENCE: 116
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 117 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q96 and L80,
where n = 1 to 10 <400> SEQUENCE: 117 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 118 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q96 through phosphorothioate
linkage, and then link to (n x Q94) and L80, where n = 0 to 10
<400> SEQUENCE: 118 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 119 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to
Q11, (n x Q94) and L80, where n = 1 to 10 <400> SEQUENCE: 119
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 120 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q11 through
phosphorothioate linkage, and then link to (n x Q94) and L80, where
n = 0 to 10 <400> SEQUENCE: 120 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 121 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q96 and L80, where n = 1 to 10 <400> SEQUENCE: 121
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 122 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94) and L80, where
n = 1 to 10 <400> SEQUENCE: 122 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 123 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q11 and L80, where n = 1 to 10 <400> SEQUENCE: 123
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 124 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q96, (n x Q94), Q11 and L80,
where n = 1 to 10 <400> SEQUENCE: 124 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 125 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q96, (n x Q94), Q11 and L80, where n = 0 to 10
<400> SEQUENCE: 125 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 126 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to (n
x Q94), Q11, (n x Q94) and L80, where n = 1 to 10 <400>
SEQUENCE: 126 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 127
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q96 and L80, where n = 0 to 10 <400> SEQUENCE: 127
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 128 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), L96 and
L90, where n = 1 to 10 <400> SEQUENCE: 128 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 129 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q96 through
phosphorothioate linkage, and then link to (n x Q94) and L90, where
n = 0 to 10 <400> SEQUENCE: 129 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 130 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11 and
L90, where n = 1 to 10 <400> SEQUENCE: 130 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 131 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q11 through
phosphorothioate linkage, and then link to (n x Q94) and L90, where
n = 1 to 10 <400> SEQUENCE: 131 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 132 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96 and
L90, where n = 1 to 10 <400> SEQUENCE: 132 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 133 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q96, Q11 and L90, where n = 1 to 10
<400> SEQUENCE: 133 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 134 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, Q96
and L90, where n = 1 to 10 <400> SEQUENCE: 134 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 135 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, Q96, (n x Q94) and L90,
where n = 1 to 10 <400> SEQUENCE: 135 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 136 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q11, (n x Q94), Q96 and L90, where n = 1 to 10
<400> SEQUENCE: 136 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 137 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q96 through phosphorothioate linkage, and then link to (n
x Q94) and L90, where n = 0 to 10 <400> SEQUENCE: 137
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 138 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q96 through
phosphorothioate linkage, and then link to Q11, (n x Q94) and L90,
where n = 1 to 10 <400> SEQUENCE: 138 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 139 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to Q11 through phosphorothioate
linkage, and then link to (n x Q94) and L90, where n = 0 to 10
<400> SEQUENCE: 139 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 140 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q96 and L90, where n = 1 to 10 <400> SEQUENCE: 140
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 141 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94) and L90, where
n = 1 to 10 <400> SEQUENCE: 141 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 142 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature
<222> LOCATION: (7)..(10) <223> OTHER INFORMATION:
2'-fluro modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(1)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94), Q11 and L90,
where n = 1 to 10 <400> SEQUENCE: 142 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 143 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q96, (n x Q94) Q11 and L90, where n = 1 to 10
<400> SEQUENCE: 143 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 144 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q96, (n x
Q94), Q11 and L90, where n = 0 to 10 <400> SEQUENCE: 144
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 145 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q96 through
phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94) and L90, where n = 1 to 10 <400> SEQUENCE: 145
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 146 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q11, (n x Q94) Q96 and L90,
where n = 0 to 10 <400> SEQUENCE: 146 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 147 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Conjugate the 3' end to L132 through phosphorothioate
linkage <400> SEQUENCE: 147 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 148 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to L133 through phosphorothioate linkage <400>
SEQUENCE: 148 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 149
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature
<222> LOCATION: (14)..(17) <223> OTHER INFORMATION:
2'-fluro modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to L134 through
phosphorothioate linkage <400> SEQUENCE: 149 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 150 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to L136 through
phosphorothioate linkage <400> SEQUENCE: 150 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 151 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q95 through
phosphorothioate linkage, and then link to L99 <400>
SEQUENCE: 151 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 152
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q95 through phosphorothioate linkage, and then link to L50
<400> SEQUENCE: 152 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 153 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to L8
<400> SEQUENCE: 153 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 154 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
Q96 and L110 <400> SEQUENCE: 154 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 155 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to Q8
and L110 <400> SEQUENCE: 155 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 156 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
L110 <400> SEQUENCE: 156
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 157 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q95 through
phosphorothioate linkage, and then link to Q96 and L80 <400>
SEQUENCE: 157 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 158
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q95 through phosphorothioate linkage, and then link to Q8 and L80
<400> SEQUENCE: 158 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 159 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
L80 <400> SEQUENCE: 159 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 160 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
Q96 and L90 <400> SEQUENCE: 160 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 161 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to Q8
and L90 <400> SEQUENCE: 161 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 162 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q95 through phosphorothioate linkage, and then link to
L90 <400> SEQUENCE: 162 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 163 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94) and L133,
where n = 1 to 10 <400> SEQUENCE: 163 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 164 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94) and L133,
where n = 0 to 10 <400> SEQUENCE: 164 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 165 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q7 and L110, where n = 1 to 10 <400>
SEQUENCE: 165 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 166
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q7 through phosphorothioate linkage, and then link to (n x Q94) and
L110, where n = 1 to 10 <400> SEQUENCE: 166 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 167 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Link the 3' end to phosphorothioate linkage, and
then link to (n x Q94), Q7 and L110, where n = 1 to 10 <400>
SEQUENCE: 167 ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 168
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(5) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(10) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Conjugate the 3' end to
Q7 through phosphorothioate linkage, and then link to (n x Q94) and
L110, where n = 0 to 10 <400> SEQUENCE: 168 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 169 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q7 through
phosphorothioate linkage, and then link to Q11, (n x Q94) and L110,
where n = 1 to 10 <400> SEQUENCE: 169 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 170 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q7, (n x Q94) and L110, where n = 1 to 10
<400> SEQUENCE: 170 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 171 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q11, (n x Q94), Q7 and L110, where n = 0 to 10
<400> SEQUENCE: 171 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 172 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q7 and
L80, where n = 1 to 10 <400> SEQUENCE: 172 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 173 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q7 through
phosphorothioate linkage, and then link to (n x Q94) and L80, where
n = 1 to 10 <400> SEQUENCE: 173 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 174 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q7 and
L80, where n = 1 to 10 <400> SEQUENCE: 174 ggaucaucuc
aagucuuact t 21 <210> SEQ ID NO 175 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Combined
DNA/RNA Molecule, Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(7)..(10) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (14)..(17) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Conjugate the 3' end to Q7 through
phosphorothioate linkage, and then link to (n x Q94) and L80, where
n = 0 to 10 <400> SEQUENCE: 175 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 176 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q7 through phosphorothioate linkage, and then link to
Q11, (n x Q94) and L80, where n = 1 to 10 <400> SEQUENCE: 176
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 177 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q7, (n x Q94) and L80, where n
= 1 to 10 <400> SEQUENCE: 177 ggaucaucuc aagucuuact t 21
<210> SEQ ID NO 178 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Link the 3' end
to phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q7 and L80, where n = 0 to 10 <400> SEQUENCE: 178
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 179 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q7 and L90, where n = 1 to 10
<400> SEQUENCE: 179 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 180 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q7 through phosphorothioate linkage, and then link to (n
x Q94) and L90, where n = 1 to 10 <400> SEQUENCE: 180
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 181 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to phosphorothioate
linkage, and then link to (n x Q94), Q7 and L90, where n = 1 to 10
<400> SEQUENCE: 181 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 182 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(19)..(19) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Conjugate the
3' end to Q7 through phosphorothioate linkage, and then link to (n
x Q94) and L80, where n = 0 to 10 <400> SEQUENCE: 182
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 183 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(10) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(17) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Conjugate the 3' end to Q7 through
phosphorothioate linkage, and then link to Q11, (n x Q94) and L90,
where n = 1 to 10 <400> SEQUENCE: 183 ggaucaucuc aagucuuact t
21 <210> SEQ ID NO 184 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Combined DNA/RNA Molecule,
Synthetic Oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(5) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(14)..(17) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (19)..(19) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to phosphorothioate linkage, and then
link to (n x Q94), Q7, (n x Q94) and L90, where n = 1 to 10
<400> SEQUENCE: 184 ggaucaucuc aagucuuact t 21 <210>
SEQ ID NO 185 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(17)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)
<223> OTHER INFORMATION: 2'-fluro modification <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Link the 3' end to
phosphorothioate linkage, and then link to (n x Q94), Q11, (n x
Q94), Q7 and L90, where n = 0 to 10 <400> SEQUENCE: 185
ggaucaucuc aagucuuact t 21 <210> SEQ ID NO 186 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Combined DNA/RNA Molecule, Synthetic Oligonucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: 2'-fluro modification
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(9) <223> OTHER INFORMATION: 2'-fluro
modification <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: 2'-fluro modification <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (17)..(19) <223>
OTHER INFORMATION: 2'-fluro modification <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (20)..(21)
<223> OTHER INFORMATION: Phosphorothioate linkage <400>
SEQUENCE: 186 guaagacuug agaugaucct t 21 <210> SEQ ID NO 187
<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Combined DNA/RNA Molecule, Synthetic Oligonucleotide:
A-40867 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Link the 3' end to Q94 through phosphorothioate linkage, and then
link to L10 <400> SEQUENCE: 187 aaucuuauau uugauccaa 19
<210> SEQ ID NO 188 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide: A30861 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Link the 3' end to DLink-DMA with hydrazone linkage
<400> SEQUENCE: 188 ggaaucuuau auuugaucca a 21 <210>
SEQ ID NO 189 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Combined DNA/RNA Molecule, Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (20)..(21) <223> OTHER
INFORMATION: Phosphorothioate linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Link the 3' end to Q8 and L99, and
then conjugate to DLin-k-DMA <400> SEQUENCE: 189 acaugaagca
gcacgacuut t 21
* * * * *