U.S. patent application number 16/357018 was filed with the patent office on 2020-02-13 for compositions and methods for modulating complement factor b expression.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Susan M. Freier, Tamar R. Grossman, Michael L. McCaleb, Thazha P. Prakash, Punit P. Seth, Eric E. Swayze, Andrew T. Watt.
Application Number | 20200048638 16/357018 |
Document ID | / |
Family ID | 54359510 |
Filed Date | 2020-02-13 |
View All Diagrams
United States Patent
Application |
20200048638 |
Kind Code |
A1 |
Prakash; Thazha P. ; et
al. |
February 13, 2020 |
COMPOSITIONS AND METHODS FOR MODULATING COMPLEMENT FACTOR B
EXPRESSION
Abstract
The present embodiments provide methods, compounds, and
compositions for treating, preventing, or ameliorating a disease
associated with dysregulation of the complement alternative pathway
by administering a Complement Factor B (CFB) specific inhibitor to
a subject.
Inventors: |
Prakash; Thazha P.;
(Carlsbad, CA) ; Seth; Punit P.; (Carlsbad,
CA) ; Swayze; Eric E.; (Encinitas, CA) ;
Grossman; Tamar R.; (La Jolla, CA) ; McCaleb; Michael
L.; (La Jolla, CA) ; Watt; Andrew T.; (San
Diego, CA) ; Freier; Susan M.; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
54359510 |
Appl. No.: |
16/357018 |
Filed: |
March 18, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15307526 |
Oct 28, 2016 |
10280423 |
|
|
PCT/US2015/028916 |
May 1, 2015 |
|
|
|
16357018 |
|
|
|
|
62076273 |
Nov 6, 2014 |
|
|
|
61987471 |
May 1, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/3341 20130101;
C12N 2310/11 20130101; C12N 2310/315 20130101; C12N 2310/346
20130101; A61P 43/00 20180101; C12N 15/113 20130101; C12N 2310/341
20130101; C12N 15/1137 20130101; C12N 2310/351 20130101; A61P 37/02
20180101; A61P 27/02 20180101; C12N 2310/321 20130101; C12Y
304/21047 20130101; A61P 13/12 20180101; C12N 2310/321 20130101;
C12N 2310/3525 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1-263. (canceled)
264. A modified single-stranded oligonucleotide covalently attached
to a conjugate group, wherein the modified single-stranded
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising at least 8 contiguous nucleobases of
any of the nucleobase sequences of SEQ ID NOs: 455, 453, 448, 237,
444, 450, 228, 549, or 198, and wherein the conjugate group
covalently attached to the modified single stranded oligonucleotide
comprises: ##STR00275##
265. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 264, wherein the modified
single-stranded oligonucleotide consists of 20 to 30 linked
nucleosides and has a nucleobase sequence comprising the nucleobase
sequence of any of SEQ ID NOs: 455, 453, 448, 237, 444, 450, 228,
or 198.
266. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 264, wherein the modified
single-stranded oligonucleotide consists of the nucleobase sequence
of any of SEQ ID NOs: 455, 453, 448, 237, 444, 450, 228, 549 or
198.
267. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 264, wherein the modified
single-stranded oligonucleotide has: a gap segment consisting of
linked deoxynucleosides; a 5' wing segment consisting of linked
nucleosides; and a 3' wing segment consisting of linked
nucleosides; wherein the gap segment is positioned between the 5'
wing segment and the 3' wing segment and wherein each nucleoside of
each wing segment comprises a modified sugar.
268. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 264, wherein the modified
single-stranded oligonucleotide consists of 20 linked nucleosides
and has a nucleobase sequence consisting of the nucleobase sequence
of any of SEQ ID NOs: 455, 453, 448, 237, 444, 450, 228, or 198,
and wherein the modified single-stranded oligonucleotide has: a gap
segment consisting of ten linked deoxynucleosides; a 5' wing
segment consisting of five linked nucleosides; and a 3' wing
segment consisting of five linked nucleosides; wherein the gap
segment is positioned between the 5' wing segment and the 3' wing
segment, wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar; wherein each internucleoside linkage of
the modified single-stranded oligonucleotide is a phosphorothioate
linkage, and wherein each cytosine is 5-methylcytosine.
269. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 268, wherein the modified
single-stranded oligonucleotide has a nucleobase sequence
consisting of the nucleobase sequence of SEQ ID NO: 455.
270. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 268, wherein the modified
single-stranded oligonucleotide has a nucleobase sequence
consisting of the nucleobase sequence of SEQ ID NO: 453.
271. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 268, wherein the modified
single-stranded oligonucleotide has a nucleobase sequence
consisting of the nucleobase sequence of SEQ ID NO: 448.
272. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 268, wherein the modified
single-stranded oligonucleotide has a nucleobase sequence
consisting of the nucleobase sequence of SEQ ID NO: 237.
273. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 264, wherein the modified
single-stranded oligonucleotide is at least 85% complementary to
SEQ ID NO: 1.
274. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 264, wherein the modified
single-stranded oligonucleotide comprises at least one modified
internucleoside linkage, at least one modified sugar, or at least
one modified nucleobase.
275. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 274, wherein the modified
single-stranded oligonucleotide comprises at least one modified
internucleoside linkage.
276. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 275, wherein the modified
internucleoside linkage is a phosphorothioate internucleoside
linkage.
277. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 276, wherein the modified
single-stranded oligonucleotide comprises at least 1 phosphodiester
internucleoside linkage.
278. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 277, wherein each
internucleoside linkage of the modified single-stranded
oligonucleotide is selected from a phosphodiester internucleoside
linkage and a phosphorothioate internucleoside linkage.
279. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 274, wherein each
internucleoside linkage of the modified single-stranded
oligonucleotide comprises a phosphorothioate internucleoside
linkage.
280. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 274, wherein the modified
sugar is a bicyclic sugar.
281. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 280, wherein the bicyclic
sugar is selected from the group consisting of: 4'-(CH.sub.2)--O-2'
(LNA); 4'-(CH.sub.2).sub.2--O-2' (ENA); and 4'-CH(CH.sub.3)--O-2'
(cEt).
282. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 274, wherein the modified
sugar is 2'-O-methoxyethyl.
283. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 274, wherein the modified
nucleobase is 5-methylcytosine.
284. A modified double-stranded oligonucleotide comprising the
modified single-stranded oligonucleotide covalently attached to a
conjugate group of claim 264, and a second single stranded
oligonucleotide hybridized to said modified single stranded
oligonucleotide.
285. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 264, wherein the conjugate
group is linked to the modified oligonucleotide at the 5' end of
the modified oligonucleotide.
286. The modified single-stranded oligonucleotide covalently
attached to a conjugate group of claim 264, wherein the conjugate
group is linked to the modified oligonucleotide at the 3' end of
the modified oligonucleotide.
287. A method of treating a disease associated with dysregulation
of the complement alternative pathway in a subject comprising
administering to the subject the modified single-stranded
oligonucleotide covalently attached to a conjugate group of claim
264, or a pharmaceutically acceptable salt thereof, thereby
treating the disease.
288. The method of claim 287, wherein the disease is macular
degeneration, age related macular degeneration (AMD), wet AMD, dry
AMD, or Geographic Atrophy.
289. The method of claim 287, wherein the disease is a kidney
disease.
290. The method of claim 289, wherein the kidney disease is lupus
nephritis, systemic lupus erythematosus (SLE), dense deposit
disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or
atypical hemolytic uremic syndrome (aHUS).
Description
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled BIOL0251USD1SEQ_ST25.txt created Mar. 18, 2019, which
is 204 kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
FIELD
[0002] The present embodiments provide methods, compounds, and
compositions for treating, preventing, or ameliorating a disease
associated with dysregulation of the complement alternative pathway
by administering a Complement Factor B (CFB) specific inhibitor to
a subject.
BACKGROUND
[0003] The complement system is part of the host innate immune
system involved in lysing foreign cells, enhancing phagocytosis of
antigens, clumping antigen-bearing agents, and attracting
macrophages and neutrophils. The complement system is divided into
three initiation pathways--the classical, lectin, and alternative
pathways--that converge at component C3 to generate an enzyme
complex known as C3 convertase, which cleaves C3 into C3a and C3b.
C3b associates with C3 convertase mediated by CFB and results in
generation of C5 convertase, which cleaves C5 into C5a and C5b,
which initiates the membrane attack pathway resulting in the
formation of the membrane attack complex (MAC) comprising
components C5b, C6, C7, C8, and C9. The membrane-attack complex
(MAC) forms transmembrane channels and disrupts the phospholipid
bilayer of target cells, leading to cell lysis.
[0004] In the homeostatic state, the alternative pathway is
continuously activated at a low "tickover" level as a result of
activation of the alternative pathway by spontaneous hydrolysis of
C3 and the production of C3b, which generates C5 convertase.
SUMMARY
[0005] The complement system mediates innate immunity and plays an
important role in normal inflammatory response to injury, but its
dysregulation may cause severe injury. Activation of the
alternative complement pathway beyond its constitutive "tickover"
level can lead to unrestrained hyperactivity and manifest as
diseases of complement dysregulation.
[0006] Certain embodiments provided herein relate to methods of
treating, preventing, or ameliorating a disease associated with
dysregulation of the complement alternative pathway in a subject by
administration of a Complement Factor B (CFB) specific inhibitor.
Several embodiments provided herein are drawn to a method of
inhibiting expression of CFB in a subject having, or at risk of
having, a disease associated with dysregulation of the complement
alternative pathway by administering a CFB specific inhibitor to
the subject. In certain embodiments, a method of reducing or
inhibiting accumulation of C3 deposits in the eye of a subject
having, or at risk of having, a disease associated with
dysregulation of the complement alternative pathway comprises
administering a CFB specific inhibitor to the subject. In several
embodiments, a method of reducing or inhibiting accumulation of C3
deposits in the kidney of a subject having, or at risk of having, a
disease associated with dysregulation of the complement alternative
pathway comprises administering a CFB specific inhibitor to the
subject.
DETAILED DESCRIPTION
[0007] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
[0008] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference for the portions of the document
discussed herein, as well as in their entirety.
[0009] Unless specific definitions are provided, the nomenclature
used in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Standard techniques may be used for
chemical synthesis, and chemical analysis. Certain such techniques
and procedures may be found for example in "Carbohydrate
Modifications in Antisense Research" Edited by Sangvi and Cook,
American Chemical Society, Washington D.C., 1994; "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.,
21.sup.st edition, 2005; and "Antisense Drug Technology,
Principles, Strategies, and Applications" Edited by Stanley T.
Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al.,
"Molecular Cloning, A laboratory Manual," 2.sup.nd Edition, Cold
Spring Harbor Laboratory Press, 1989, which are hereby incorporated
by reference for any purpose. Where permitted, all patents,
applications, published applications and other publications and
other data referred to throughout in the disclosure are
incorporated by reference herein in their entirety.
Unless otherwise indicated, the following terms have the following
meanings:
[0010] "2'-F nucleoside" refers to a nucleoside comprising a sugar
comprising fluorine at the 2' position. Unless otherwise indicated,
the fluorine in a 2'-F nucleoside is in the ribo position
(replacing the OH of a natural ribose).
[0011] "2'-O-methoxyethyl" (also 2'-MOE and
2'-O(CH.sub.2).sub.2--OCH.sub.3) refers to an O-methoxy-ethyl
modification at the 2' position of a furanose ring. A
2'-O-methoxyethyl modified sugar is a modified sugar.
[0012] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside)
means a nucleoside comprising a 2'-MOE modified sugar moiety.
[0013] "2'-substituted nucleoside" means a nucleoside comprising a
substituent at the 2'-position of the furanosyl ring other than H
or OH. In certain embodiments, 2' substituted nucleosides include
nucleosides with bicyclic sugar modifications.
[0014] "3' target site" refers to the nucleotide of a target
nucleic acid which is complementary to the 3'-most nucleotide of a
particular antisense compound.
[0015] "5' target site" refers to the nucleotide of a target
nucleic acid which is complementary to the 5'-most nucleotide of a
particular antisense compound.
[0016] "5-methylcytosine" means a cytosine modified with a methyl
group attached to the 5 position. A 5-methylcytosine is a modified
nucleobase.
[0017] "About" means within .+-.10% of a value. For example, if it
is stated, "the compounds affected at least about 70% inhibition of
CFB", it is implied that CFB levels are inhibited within a range of
60% and 80%.
[0018] "Administration" or "administering" refers to routes of
introducing an antisense compound provided herein to a subject to
perform its intended function. An example of a route of
administration that can be used includes, but is not limited to
parenteral administration, such as subcutaneous, intravenous, or
intramuscular injection or infusion.
[0019] "Alkyl," as used herein, means a saturated straight or
branched hydrocarbon radical containing up to twenty four carbon
atoms. Examples of alkyl groups include without limitation, methyl,
ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and
the like. Alkyl groups typically include from 1 to about 24 carbon
atoms, more typically from 1 to about 12 carbon atoms
(C.sub.1-C.sub.12 alkyl) with from 1 to about 6 carbon atoms being
more preferred.
[0020] As used herein, "alkenyl," means a straight or branched
hydrocarbon chain radical containing up to twenty four carbon atoms
and having at least one carbon-carbon double bond. Examples of
alkenyl groups include without limitation, ethenyl, propenyl,
butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and
the like. Alkenyl groups typically include from 2 to about 24
carbon atoms, more typically from 2 to about 12 carbon atoms with
from 2 to about 6 carbon atoms being more preferred. Alkenyl groups
as used herein may optionally include one or more further
substituent groups.
[0021] As used herein, "alkynyl," means a straight or branched
hydrocarbon radical containing up to twenty four carbon atoms and
having at least one carbon-carbon triple bond. Examples of alkynyl
groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl,
and the like. Alkynyl groups typically include from 2 to about 24
carbon atoms, more typically from 2 to about 12 carbon atoms with
from 2 to about 6 carbon atoms being more preferred. Alkynyl groups
as used herein may optionally include one or more further
substituent groups.
[0022] As used herein, "acyl," means a radical formed by removal of
a hydroxyl group from an organic acid and has the general Formula
--C(O)--X where X is typically aliphatic, alicyclic or aromatic.
Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic
sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic
phosphates, aliphatic phosphates and the like. Acyl groups as used
herein may optionally include further substituent groups.
[0023] As used herein, "alicyclic" means a cyclic ring system
wherein the ring is aliphatic. The ring system can comprise one or
more rings wherein at least one ring is aliphatic. Preferred
alicyclics include rings having from about 5 to about 9 carbon
atoms in the ring. Alicyclic as used herein may optionally include
further substituent groups.
[0024] As used herein, "aliphatic" means a straight or branched
hydrocarbon radical containing up to twenty four carbon atoms
wherein the saturation between any two carbon atoms is a single,
double or triple bond. An aliphatic group preferably contains from
1 to about 24 carbon atoms, more typically from 1 to about 12
carbon atoms with from 1 to about 6 carbon atoms being more
preferred. The straight or branched chain of an aliphatic group may
be interrupted with one or more heteroatoms that include nitrogen,
oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by
heteroatoms include without limitation, polyalkoxys, such as
polyalkylene glycols, polyamines, and polyimines. Aliphatic groups
as used herein may optionally include further substituent
groups.
[0025] As used herein, "alkoxy" means a radical formed between an
alkyl group and an oxygen atom wherein the oxygen atom is used to
attach the alkoxy group to a parent molecule. Examples of alkoxy
groups include without limitation, methoxy, ethoxy, propoxy,
isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy,
neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may
optionally include further substituent groups.
[0026] As used herein, "aminoalkyl" means an amino substituted
C.sub.1-C.sub.12 alkyl radical. The alkyl portion of the radical
forms a covalent bond with a parent molecule. The amino group can
be located at any position and the aminoalkyl group can be
substituted with a further substituent group at the alkyl and/or
amino portions.
[0027] As used herein, "aralkyl" and "arylalkyl" mean an aromatic
group that is covalently linked to a C.sub.1-C.sub.12 alkyl
radical. The alkyl radical portion of the resulting aralkyl (or
arylalkyl) group forms a covalent bond with a parent molecule.
Examples include without limitation, benzyl, phenethyl and the
like. Aralkyl groups as used herein may optionally include further
substituent groups attached to the alkyl, the aryl or both groups
that form the radical group.
[0028] As used herein, "aryl" and "aromatic" mean a mono- or
polycyclic carbocyclic ring system radicals having one or more
aromatic rings. Examples of aryl groups include without limitation,
phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
Preferred aryl ring systems have from about 5 to about 20 carbon
atoms in one or more rings. Aryl groups as used herein may
optionally include further substituent groups.
[0029] "Amelioration" refers to a lessening of at least one
indicator, sign, or symptom of an associated disease, disorder, or
condition. In certain embodiments, amelioration includes a delay or
slowing in the progression of one or more indicators of a condition
or disease. The severity of indicators may be determined by
subjective or objective measures, which are known to those skilled
in the art.
[0030] "Animal" refers to a human or non-human animal, including,
but not limited to, mice, rats, rabbits, dogs, cats, pigs, and
non-human primates, including, but not limited to, monkeys and
chimpanzees.
[0031] "Antisense activity" means any detectable or measurable
activity attributable to the hybridization of an antisense compound
to its target nucleic acid. In certain embodiments, antisense
activity is a decrease in the amount or expression of a target
nucleic acid or protein encoded by such target nucleic acid.
[0032] "Antisense compound" means an oligomeric compound that is is
capable of undergoing hybridization to a target nucleic acid
through hydrogen bonding. Examples of antisense compounds include
single-stranded and double-stranded compounds, such as, antisense
oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based
compounds.
[0033] "Antisense inhibition" means reduction of target nucleic
acid levels in the presence of an antisense compound complementary
to a target nucleic acid compared to target nucleic acid levels in
the absence of the antisense compound.
[0034] "Antisense mechanisms" are all those mechanisms involving
hybridization of a compound with target nucleic acid, wherein the
outcome or effect of the hybridization is either target degradation
or target occupancy with concomitant stalling of the cellular
machinery involving, for example, transcription or splicing.
[0035] "Antisense oligonucleotide" means a single-stranded
oligonucleotide having a nucleobase sequence that permits
hybridization to a corresponding region or segment of a target
nucleic acid.
[0036] "Base complementarity" refers to the capacity for the
precise base pairing of nucleobases of an antisense oligonucleotide
with corresponding nucleobases in a target nucleic acid (i.e.,
hybridization), and is mediated by Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen binding between corresponding
nucleobases.
[0037] "Bicyclic sugar moiety" means a modified sugar moiety
comprising a 4 to 7 membered ring (including but not limited to a
furanosyl) comprising a bridge connecting two atoms of the 4 to 7
membered ring to form a second ring, resulting in a bicyclic
structure. In certain embodiments, the 4 to 7 membered ring is a
sugar ring. In certain embodiments the 4 to 7 membered ring is a
furanosyl. In certain such embodiments, the bridge connects the
2'-carbon and the 4'-carbon of the furanosyl.
[0038] "Bicyclic nucleic acid" or "BNA" or "BNA nucleosides" means
nucleic acid monomers having a bridge connecting two carbon atoms
between the 4' and 2' position of the nucleoside sugar unit,
thereby forming a bicyclic sugar. Examples of such bicyclic sugar
include, but are not limited to A) .alpha.-L-Methyleneoxy
(4'-CH.sub.2--O-2') LNA, (B) .beta.-D-Methyleneoxy
(4'-CH.sub.2--O-2') LNA, (C) Ethyleneoxy
(4'-(CH.sub.2).sub.2--O-2') LNA, (D) Aminooxy
(4'-CH.sub.2--O--N(R)-2') LNA and (E) Oxyamino
(4'-CH.sub.2--N(R)--O-2') LNA, as depicted below.
##STR00001##
[0039] As used herein, LNA compounds include, but are not limited
to, compounds having at least one bridge between the 4' and the 2'
position of the sugar wherein each of the bridges independently
comprises 1 or from 2 to 4 linked groups independently selected
from --[C(R.sub.1)(R.sub.2)].sub.n--,
--C(R.sub.1).dbd.C(R.sub.2)--, --C(R.sub.1).dbd.N--,
--C(.dbd.NR.sub.1)--, --C(.dbd.O)--, --C(.dbd.S)--, --O--,
--Si(R.sub.1).sub.2--, --S(.dbd.O).sub.x and --N(R.sub.1)--;
wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R.sub.1 and
R.sub.2 is, independently, H, a protecting group, hydroxyl,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, a
heterocycle radical, a substituted heterocycle radical, heteroaryl,
substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical,
substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl
(C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.12
alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12
alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12
alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20
aryl, substituted C.sub.5-C.sub.20 aryl, acyl (C(.dbd.O)--H),
substituted acyl, a heterocycle radical, a substituted heterocycle
radical, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12
aminoalkyl or a protecting group.
[0040] Examples of 4'-2' bridging groups encompassed within the
definition of LNA include, but are not limited to one of formulae:
--[C(R.sub.1)(R.sub.2)].sub.n--,
--[C(R.sub.1)(R.sub.2)].sub.n--O--,
--C(R.sub.1R.sub.2)--N(R.sub.1)--O-- or
--C(R.sub.1R.sub.2)--O--N(R.sub.1)--. Furthermore, other bridging
groups encompassed with the definition of LNA are 4'-CH.sub.2-2',
4'--(CH.sub.2).sub.2-2', 4'--(CH.sub.2).sub.3-2',
4'--CH.sub.2--O-2', 4'--(CH.sub.2).sub.2--O-2',
4'--CH.sub.2--O--N(R.sub.1)-2' and
4'-CH.sub.2--N(R.sub.1)--O-2'-bridges, wherein each R.sub.1 and
R.sub.2 is, independently, H, a protecting group or
C.sub.1-C.sub.12 alkyl.
[0041] Also included within the definition of LNA according to the
invention are LNAs in which the 2'-hydroxyl group of the ribosyl
sugar ring is connected to the 4' carbon atom of the sugar ring,
thereby forming a methyleneoxy (4'-CH.sub.2--O-2') bridge to form
the bicyclic sugar moiety. The bridge can also be a methylene
(--CH.sub.2--) group connecting the 2' oxygen atom and the 4'
carbon atom, for which the term methyleneoxy (4'-CH.sub.2--O-2')
LNA is used. Furthermore; in the case of the bicylic sugar moiety
having an ethylene bridging group in this position, the term
ethyleneoxy (4'-CH.sub.2CH.sub.2--O-2') LNA is used.
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2'), an isomer of
methyleneoxy (4'-CH.sub.2--O-2') LNA is also encompassed within the
definition of LNA, as used herein.
[0042] "Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
[0043] "Carbohydrate" means a naturally occurring carbohydrate, a
modified carbohydrate, or a carbohydrate derivative.
[0044] "Carbohydrate cluster" means a compound having one or more
carbohydrate residues attached to a scaffold or linker group. (see,
e.g., Maier et al., "Synthesis of Antisense Oligonucleotides
Conjugated to a Multivalent Carbohydrate Cluster for Cellular
Targeting," Bioconjugate Chemistry, 2003, (14): 18-29, which is
incorporated herein by reference in its entirety, or Rensen et al.,
"Design and Synthesis of Novel N-Acetylgalactosamine-Terminated
Glycolipids for Targeting of Lipoproteins to the Hepatic
Asiaglycoprotein Receptor," J. Med. Chem. 2004, (47): 5798-5808,
for examples of carbohydrate conjugate clusters).
[0045] "Carbohydrate derivative" means any compound which may be
synthesized using a carbohydrate as a starting material or
intermediate.
[0046] "cEt" or "constrained ethyl" means a bicyclic sugar moiety
comprising a bridge connecting the 4'-carbon and the 2'-carbon,
wherein the bridge has the formula: 4'-CH(CH.sub.3)--O-2'.
[0047] "Chemical modification" means a chemical difference in a
compound when compared to a naturally occurring counterpart.
Chemical modifications of oligonucleotides include nucleoside
modifications (including sugar moiety modifications and nucleobase
modifications) and internucleoside linkage modifications. In
reference to an oligonucleotide, chemical modification does not
include differences only in nucleobase sequence.
[0048] "Cleavable bond" means any chemical bond capable of being
split. In certain embodiments, a cleavable bond is selected from
among: an amide, a polyamide, an ester, an ether, one or both
esters of a phosphodiester, a phosphate ester, a carbamate, a
di-sulfide, or a peptide.
[0049] "Cleavable moiety" means a bond or group that is capable of
being split under physiological conditions. In certain embodiments,
a cleavable moiety is cleaved inside a cell or sub-cellular
compartments, such as a lysosome. In certain embodiments, a
cleavable moiety is cleaved by endogenous enzymes, such as
nucleases. In certain embodiments, a cleavable moiety comprises a
group of atoms having one, two, three, four, or more than four
cleavable bonds.
[0050] "Conjugate" or "conjugate group" means an atom or group of
atoms bound to an oligonucleotide or oligomeric compound. In
general, conjugate groups modify one or more properties of the
compound to which they are attached, including, but not limited to
pharmacodynamic, pharmacokinetic, binding, absorption, cellular
distribution, cellular uptake, charge and/or clearance
properties.
[0051] "conjugate linker" or "linker" in the context of a conjugate
group means a portion of a conjugate group comprising any atom or
group of atoms and which covalently link (1) an oligonucleotide to
another portion of the conjugate group or (2) two or more portions
of the conjugate group.
[0052] Conjugate groups are shown herein as radicals, providing a
bond for forming covalent attachment to an oligomeric compound such
as an antisense oligonucleotide. In certain embodiments, the point
of attachment on the oligomeric compound is the 3'-oxygen atom of
the 3'-hydroxyl group of the 3' terminal nucleoside of the
oligomeric compound. In certain embodiments the point of attachment
on the oligomeric compound is the 5'-oxygen atom of the 5'-hydroxyl
group of the 5' terminal nucleoside of the oligomeric compound. In
certain embodiments, the bond for forming attachment to the
oligomeric compound is a cleavable bond. In certain such
embodiments, such cleavable bond constitutes all or part of a
cleavable moiety.
[0053] In certain embodiments, conjugate groups comprise a
cleavable moiety (e.g., a cleavable bond or cleavable nucleoside)
and a carbohydrate cluster portion, such as a GalNAc cluster
portion. Such carbohydrate cluster portion comprises: a targeting
moiety and, optionally, a conjugate linker. In certain embodiments,
the carbohydrate cluster portion is identified by the number and
identity of the ligand. For example, in certain embodiments, the
carbohydrate cluster portion comprises 3 GalNAc groups and is
designated "GalNAc.sub.3". In certain embodiments, the carbohydrate
cluster portion comprises 4 GalNAc groups and is designated
"GalNAc4". Specific carbohydrate cluster portions (having specific
tether, branching and conjugate linker groups) are described herein
and designated by Roman numeral followed by subscript "a".
Accordingly "GalNac3-1a" refers to a specific carbohydrate cluster
portion of a conjugate group having 3 GalNac groups and
specifically identified tether, branching and linking groups. Such
carbohydrate cluster fragment is attached to an oligomeric compound
via a cleavable moiety, such as a cleavable bond or cleavable
nucleoside.
[0054] "Conjugate compound" means any atoms, group of atoms, or
group of linked atoms suitable for use as a conjugate group. In
certain embodiments, conjugate compounds may possess or impart one
or more properties, including, but not limited to pharmacodynamic,
pharmacokinetic, binding, absorption, cellular distribution,
cellular uptake, charge and/or clearance properties.
[0055] "Constrained ethyl nucleoside" (also cEt nucleoside) means a
nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH(CH.sub.3)--O-2' bridge.
[0056] "Complement Factor B (CFB)" means any nucleic acid or
protein of CFB. "CFB nucleic acid" means any nucleic acid encoding
CFB. For example, in certain embodiments, a CFB nucleic acid
includes a DNA sequence encoding CFB, an RNA sequence transcribed
from DNA encoding CFB (including genomic DNA comprising introns and
exons), including a non-protein encoding (i.e. non-coding) RNA
sequence, and an mRNA sequence encoding CFB. "CFB mRNA" means an
mRNA encoding a CFB protein.
[0057] "CFB specific inhibitor" refers to any agent capable of
specifically inhibiting CFB RNA and/or CFB protein expression or
activity at the molecular level. For example, CFB specific
inhibitors include nucleic acids (including antisense compounds),
peptides, antibodies, small molecules, and other agents capable of
inhibiting the expression of CFB RNA and/or CFB protein.
[0058] "Chemically distinct region" refers to a region of an
antisense compound that is in some way chemically different than
another region of the same antisense compound. For example, a
region having 2'-O-methoxyethyl nucleotides is chemically distinct
from a region having nucleotides without 2'-O-methoxyethyl
modifications.
[0059] "Chimeric antisense compounds" means antisense compounds
that have at least 2 chemically distinct regions, each position
having a plurality of subunits.
[0060] "Complementarity" means the capacity for pairing between
nucleobases of a first nucleic acid and a second nucleic acid.
[0061] "Comprise," "comprises" and "comprising" will be understood
to imply the inclusion of a stated step or element or group of
steps or elements but not the exclusion of any other step or
element or group of steps or elements.
[0062] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other.
[0063] "Deoxynucleoside" means a nucleoside comprising 2'-H
furanosyl sugar moiety, as found in naturally occurring
deoxyribonucleosides (DNA). In certain embodiments, a
2'-deoxynucleoside may comprise a modified nucleobase or may
comprise an RNA nucleobase (e.g., uracil).
[0064] "Deoxyribonucleotide" means a nucleotide having a hydrogen
at the 2' position of the sugar portion of the nucleotide.
Deoxyribonucleotides may be modified with any of a variety of
substituents.
[0065] "Designing" or "Designed to" refer to the process of
designing an oligomeric compound that specifically hybridizes with
a selected nucleic acid molecule.
[0066] "Differently modified" mean chemical modifications or
chemical substituents that are different from one another,
including absence of modifications. Thus, for example, a MOE
nucleoside and an unmodified DNA nucleoside are "differently
modified," even though the DNA nucleoside is unmodified. Likewise,
DNA and RNA are "differently modified," even though both are
naturally-occurring unmodified nucleosides. Nucleosides that are
the same but for comprising different nucleobases are not
differently modified. For example, a nucleoside comprising a 2'-OMe
modified sugar and an unmodified adenine nucleobase and a
nucleoside comprising a 2'-OMe modified sugar and an unmodified
thymine nucleobase are not differently modified.
[0067] "Double-stranded" refers to two separate oligomeric
compounds that are hybridized to one another. Such double stranded
compounds may have one or more or non-hybridizing nucleosides at
one or both ends of one or both strands (overhangs) and/or one or
more internal non-hybridizing nucleosides (mismatches) provided
there is sufficient complementarity to maintain hybridization under
physiologically relevant conditions.
[0068] "Effective amount" means the amount of active pharmaceutical
agent sufficient to effectuate a desired physiological outcome in
an individual in need of the agent. The effective amount may vary
among individuals depending on the health and physical condition of
the individual to be treated, the taxonomic group of the
individuals to be treated, the formulation of the composition,
assessment of the individual's medical condition, and other
relevant factors.
[0069] "Efficacy" means the ability to produce a desired
effect.
[0070] "Expression" includes all the functions by which a gene's
coded information is converted into structures present and
operating in a cell. Such structures include, but are not limited
to the products of transcription and translation.
[0071] "Fully complementary" or "100% complementary" means each
nucleobase of a first nucleic acid has a complementary nucleobase
in a second nucleic acid. In certain embodiments, a first nucleic
acid is an antisense compound and a target nucleic acid is a second
nucleic acid.
[0072] "Furanosyl" means a structure comprising a 5-membered ring
comprising four carbon atoms and one oxygen atom.
[0073] "Gapmer" means a chimeric antisense compound in which an
internal region having a plurality of nucleosides that support
RNase H cleavage is positioned between external regions having one
or more nucleosides, wherein the nucleosides comprising the
internal region are chemically distinct from the nucleoside or
nucleosides comprising the external regions. The internal region
may be referred to as the "gap" and the external regions may be
referred to as the "wings."
[0074] "Halo" and "halogen," mean an atom selected from fluorine,
chlorine, bromine and iodine.
[0075] "Heteroaryl," and "heteroaromatic," mean a radical
comprising a mono- or poly-cyclic aromatic ring, ring system or
fused ring system wherein at least one of the rings is aromatic and
includes one or more heteroatoms. Heteroaryl is also meant to
include fused ring systems including systems where one or more of
the fused rings contain no heteroatoms. Heteroaryl groups typically
include one ring atom selected from sulfur, nitrogen or oxygen.
Examples of heteroaryl groups include without limitation,
pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,
thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,
thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl,
benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can
be attached to a parent molecule directly or through a linking
moiety such as an aliphatic group or hetero atom. Heteroaryl groups
as used herein may optionally include further substituent
groups.
[0076] "Hybridization" means the annealing of complementary nucleic
acid molecules. In certain embodiments, complementary nucleic acid
molecules include, but are not limited to, an antisense compound
and a nucleic acid target. In certain embodiments, complementary
nucleic acid molecules include, but are not limited to, an
antisense oligonucleotide and a nucleic acid target.
[0077] "Identifying an animal having, or at risk for having, a
disease, disorder and/or condition" means identifying an animal
having been diagnosed with the disease, disorder and/or condition
or identifying an animal predisposed to develop the disease,
disorder and/or condition. Such identification may be accomplished
by any method including evaluating an individual's medical history
and standard clinical tests or assessments.
[0078] "Immediately adjacent" means there are no intervening
elements between the immediately adjacent elements.
[0079] "Individual" means a human or non-human animal selected for
treatment or therapy.
[0080] "Inhibiting the expression or activity" refers to a
reduction, blockade of the expression or activity and does not
necessarily indicate a total elimination of expression or
activity.
[0081] "Internucleoside linkage" refers to the chemical bond
between nucleosides.
[0082] "Internucleoside neutral linking group" means a neutral
linking group that directly links two nucleosides.
[0083] "Internucleoside phosphorus linking group" means a
phosphorus linking group that directly links two nucleosides.
[0084] "Lengthened" antisense oligonucleotides are those that have
one or more additional nucleosides relative to an antisense
oligonucleotide disclosed herein.
[0085] "Linkage motif" means a pattern of linkage modifications in
an oligonucleotide or region thereof. The nucleosides of such an
oligonucleotide may be modified or unmodified. Unless otherwise
indicated, motifs herein describing only linkages are intended to
be linkage motifs. Thus, in such instances, the nucleosides are not
limited.
[0086] "Linked deoxynucleoside" means a nucleic acid base (A, G, C,
T, U) substituted by deoxyribose linked by a phosphate ester to
form a nucleotide.
[0087] "Linked nucleosides" means adjacent nucleosides linked
together by an internucleoside linkage.
[0088] "Locked nucleic acid nucleoside" or "LNA" means a nucleoside
comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2'
bridge.
[0089] "Mismatch" or "non-complementary nucleobase" refers to the
case when a nucleobase of a first nucleic acid is not capable of
pairing with the corresponding nucleobase of a second or target
nucleic acid.
[0090] "Modified internucleoside linkage" refers to a substitution
or any change from a naturally occurring internucleoside bond (i.e.
a phosphodiester internucleoside bond).
[0091] "Modified nucleobase" means any nucleobase other than
adenine, cytosine, guanine, thymidine, or uracil. An "unmodified
nucleobase" means the purine bases adenine (A) and guanine (G), and
the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
[0092] "Modified nucleoside" means a nucleoside having,
independently, a modified sugar moiety and/or modified
nucleobase.
[0093] "Modified nucleotide" means a nucleotide having,
independently, a modified sugar moiety, modified internucleoside
linkage, or modified nucleobase.
[0094] "Modified oligonucleotide" means an oligonucleotide
comprising at least one modified internucleoside linkage, a
modified sugar, and/or a modified nucleobase.
[0095] "Modified sugar" means substitution and/or any change from a
natural sugar moiety.
[0096] "Modulating" refers to changing or adjusting a feature in a
cell, tissue, organ or organism. For example, modulating CFB mRNA
can mean to increase or decrease the level of CFB mRNA and/or CFB
protein in a cell, tissue, organ or organism. A "modulator" effects
the change in the cell, tissue, organ or organism. For example, a
CFB antisense compound can be a modulator that decreases the amount
of CFB mRNA and/or CFB protein in a cell, tissue, organ or
organism.
[0097] "Monomer" refers to a single unit of an oligomer. Monomers
include, but are not limited to, nucleosides and nucleotides,
whether naturally occuring or modified.
[0098] "Mono or polycyclic ring system" is meant to include all
ring systems selected from single or polycyclic radical ring
systems wherein the rings are fused or linked and is meant to be
inclusive of single and mixed ring systems individually selected
from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl,
heterocyclic, heteroaryl, hetero aromatic and heteroarylalkyl. Such
mono and poly cyclic structures can contain rings that each have
the same level of saturation or each, independently, have varying
degrees of saturation including fully saturated, partially
saturated or fully unsaturated. Each ring can comprise ring atoms
selected from C, N, O and S to give rise to hetero cyclic rings as
well as rings comprising only C ring atoms which can be present in
a mixed motif such as for example benzimidazole wherein one ring
has only carbon ring atoms and the fused ring has two nitrogen
atoms. The mono or polycyclic ring system can be further
substituted with substituent groups such as for example phthalimide
which has two .dbd.O groups attached to one of the rings. Mono or
polycyclic ring systems can be attached to parent molecules using
various strategies such as directly through a ring atom, fused
through multiple ring atoms, through a substituent group or through
a bifunctional linking moiety.
[0099] "Motif" means the pattern of unmodified and modified
nucleosides in an antisense compound.
[0100] "Natural sugar moiety" means a sugar moiety found in DNA
(2'-H) or RNA (2'-OH).
[0101] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0102] "Neutral linking group" means a linking group that is not
charged. Neutral linking groups include without limitation phospho
triesters, methylphosphonates, MMI (--CH2-N(CH3)-O--), amide-3
(--CH2-C(.dbd.O)--N(H)--), amide-4 (--CH2-N(H)--C(.dbd.O)--),
formacetal (--O--CH2-O--), and thioformacetal (--S--CH2-O--).
Further neutral linking groups include nonionic linkages comprising
siloxane (dialkylsiloxane), carboxylate ester, carboxamide,
sulfide, sulfonate ester and amides (See for example: Carbohydrate
Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook
Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65)).
Further neutral linking groups include nonionic linkages comprising
mixed N, O, S and CH2 component parts.
[0103] "Non-complementary nucleobase" refers to a pair of
nucleobases that do not form hydrogen bonds with one another or
otherwise support hybridization.
[0104] "Non-internucleoside neutral linking group" means a neutral
linking group that does not directly link two nucleosides. In
certain embodiments, a non-internucleoside neutral linking group
links a nucleoside to a group other than a nucleoside. In certain
embodiments, a non-internucleoside neutral linking group links two
groups, neither of which is a nucleoside.
[0105] "Non-internucleoside phosphorus linking group" means a
phosphorus linking group that does not directly link two
nucleosides. In certain embodiments, a non-internucleoside
phosphorus linking group links a nucleoside to a group other than a
nucleoside. In certain embodiments, a non-internucleoside
phosphorus linking group links two groups, neither of which is a
nucleoside.
[0106] "Nucleic acid" refers to molecules composed of monomeric
nucleotides. A nucleic acid includes, but is not limited to,
ribonucleic acids (RNA), deoxyribonucleic acids (DNA),
single-stranded nucleic acids, and double-stranded nucleic
acids.
[0107] "Nucleobase" means a heterocyclic moiety capable of pairing
with a base of another nucleic acid.
[0108] "Nucleobase complementarity" refers to a nucleobase that is
capable of base pairing with another nucleobase. For example, in
DNA, adenine (A) is complementary to thymine (T). For example, in
RNA, adenine (A) is complementary to uracil (U). In certain
embodiments, complementary nucleobase refers to a nucleobase of an
antisense compound that is capable of base pairing with a
nucleobase of its target nucleic acid. For example, if a nucleobase
at a certain position of an antisense compound is capable of
hydrogen bonding with a nucleobase at a certain position of a
target nucleic acid, then the position of hydrogen bonding between
the oligonucleotide and the target nucleic acid is considered to be
complementary at that nucleobase pair.
[0109] "Nucleobase modification motif" means a pattern of
modifications to nucleobases along an oligonucleotide. Unless
otherwise indicated, a nucleobase modification motif is independent
of the nucleobase sequence.
[0110] "Nucleobase sequence" means the order of contiguous
nucleobases independent of any sugar, linkage, and/or nucleobase
modification.
[0111] "Nucleoside" means a nucleobase linked to a sugar.
[0112] "Nucleoside mimetic" includes those structures used to
replace the sugar or the sugar and the base and not necessarily the
linkage at one or more positions of an oligomeric compound such as
for example nucleoside mimetics having morpholino, cyclohexenyl,
cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics,
e.g., non furanose sugar units. Nucleotide mimetic includes those
structures used to replace the nucleoside and the linkage at one or
more positions of an oligomeric compound such as for example
peptide nucleic acids or morpholinos (morpholinos linked by
--N(H)--C(.dbd.O)--O-- or other non-phosphodiester linkage). Sugar
surrogate overlaps with the slightly broader term nucleoside
mimetic but is intended to indicate replacement of the sugar unit
(furanose ring) only. The tetrahydropyranyl rings provided herein
are illustrative of an example of a sugar surrogate wherein the
furanose sugar group has been replaced with a tetrahydropyranyl
ring system. "Mimetic" refers to groups that are substituted for a
sugar, a nucleobase, and/or internucleoside linkage. Generally, a
mimetic is used in place of the sugar or sugar-internucleoside
linkage combination, and the nucleobase is maintained for
hybridization to a selected target.
[0113] "Nucleoside motif" means a pattern of nucleoside
modifications in an oligonucleotide or a region thereof. The
linkages of such an oligonucleotide may be modified or unmodified.
Unless otherwise indicated, motifs herein describing only
nucleosides are intended to be nucleoside motifs. Thus, in such
instances, the linkages are not limited.
[0114] "Nucleotide" means a nucleoside having a phosphate group
covalently linked to the sugar portion of the nucleoside.
[0115] "Oligomeric compound" means a polymer of linked monomeric
subunits which is capable of hybridizing to at least a region of a
nucleic acid molecule.
[0116] "Oligonucleoside" means an oligonucleotide in which the
internucleoside linkages do not contain a phosphorus atom.
[0117] "Oligonucleotide" means a polymer of linked nucleosides each
of which can be modified or unmodified, independent one from
another.
[0118] "Parenteral administration" means administration through
injection or infusion. Parenteral administration includes
subcutaneous administration, intravenous administration,
intramuscular administration, intraarterial administration,
intraperitoneal administration, or intracranial administration,
e.g. intrathecal or intracerebroventricular administration.
[0119] "Pharmaceutical composition" means a mixture of substances
suitable for administering to an individual. For example, a
pharmaceutical composition may comprise one or more active
pharmaceutical agents and a sterile aqueous solution.
[0120] "Pharmaceutically acceptable salts" means physiologically
and pharmaceutically acceptable salts of antisense compounds, i.e.,
salts that retain the desired biological activity of the parent
oligonucleotide and do not impart undesired toxicological effects
thereto.
[0121] "Phosphorothioate linkage" means a linkage between
nucleosides where the phosphodiester bond is modified by replacing
one of the non-bridging oxygen atoms with a sulfur atom. A
phosphorothioate linkage is a modified internucleoside linkage.
[0122] "Phosphorus linking group" means a linking group comprising
a phosphorus atom. Phosphorus linking groups include without
limitation groups having the formula:
##STR00002##
wherein:
[0123] R.sub.a and R.sub.d are each, independently, O, S, CH.sub.2,
NH, or NJ.sub.1 wherein J.sub.1 is C.sub.1-C.sub.6 alkyl or
substituted C.sub.1-C.sub.6 alkyl;
[0124] R.sub.b is O or S;
[0125] R.sub.c is OH, SH, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, amino or substituted amino; and
[0126] J.sub.1 is R.sub.b is O or S.
Phosphorus linking groups include without limitation,
phosphodiester, phosphorothioate, phosphorodithioate, phosphonate,
phosphoramidate, phosphorothioamidate, thionoalkylphosphonate,
phosphotriesters, thionoalkylphosphotriester and
boranophosphate.
[0127] "Portion" means a defined number of contiguous (i.e.,
linked) nucleobases of a nucleic acid. In certain embodiments, a
portion is a defined number of contiguous nucleobases of a target
nucleic acid. In certain embodiments, a portion is a defined number
of contiguous nucleobases of an antisense compound
[0128] "Prevent" refers to delaying or forestalling the onset,
development or progression of a disease, disorder, or condition for
a period of time from minutes to indefinitely. Prevent also means
reducing the risk of developing a disease, disorder, or
condition.
[0129] "Prodrug" means an inactive or less active form of a
compound which, when administered to a subject, is metabolized to
form the active, or more active, compound (e.g., drug).
[0130] "Prophylactically effective amount" refers to an amount of a
pharmaceutical agent that provides a prophylactic or preventative
benefit to an animal.
[0131] "Protecting group" means any compound or protecting group
known to those having skill in the art. Non-limiting examples of
protecting groups may be found in "Protective Groups in Organic
Chemistry", T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John
Wiley & Sons, Inc, New York, which is incorporated herein by
reference in its entirety.
[0132] "Region" is defined as a portion of the target nucleic acid
having at least one identifiable structure, function, or
characteristic.
[0133] "Ribonucleotide" means a nucleotide having a hydroxy at the
2' position of the sugar portion of the nucleotide. Ribonucleotides
may be modified with any of a variety of substituents.
[0134] "RISC based antisense compound" means an antisense compound
wherein at least some of the antisense activity of the antisense
compound is attributable to the RNA Induced Silencing Complex
(RISC).
[0135] "RNase H based antisense compound" means an antisense
compound wherein at least some of the antisense activity of the
antisense compound is attributable to hybridization of the
antisense compound to a target nucleic acid and subsequent cleavage
of the target nucleic acid by RNase H.
[0136] "Segments" are defined as smaller or sub-portions of regions
within a target nucleic acid.
[0137] "Separate regions" means portions of an oligonucleotide
wherein the chemical modifications or the motif of chemical
modifications of any neighboring portions include at least one
difference to allow the separate regions to be distinguished from
one another.
[0138] "Sequence motif" means a pattern of nucleobases arranged
along an oligonucleotide or portion thereof. Unless otherwise
indicated, a sequence motif is independent of chemical
modifications and thus may have any combination of chemical
modifications, including no chemical modifications.
[0139] "Side effects" means physiological disease and/or conditions
attributable to a treatment other than the desired effects. In
certain embodiments, side effects include injection site reactions,
liver function test abnormalities, renal function abnormalities,
liver toxicity, renal toxicity, central nervous system
abnormalities, myopathies, and malaise. For example, increased
aminotransferase levels in serum may indicate liver toxicity or
liver function abnormality. For example, increased bilirubin may
indicate liver toxicity or liver function abnormality.
[0140] "Sites," as used herein, are defined as unique nucleobase
positions within a target nucleic acid.
[0141] "Slows progression" means decrease in the development of the
said disease.
[0142] "Specifically hybridizable" refers to an antisense compound
having a sufficient degree of complementarity between an antisense
oligonucleotide and a target nucleic acid to induce a desired
effect, while exhibiting minimal or no effects on non-target
nucleic acids under conditions in which specific binding is
desired, i.e., under physiological conditions in the case of in
vivo assays and therapeutic treatments. "Stringent hybridization
conditions" or "stringent conditions" refer to conditions under
which an oligomeric compound will hybridize to its target sequence,
but to a minimal number of other sequences.
[0143] "Subject" means a human or non-human animal selected for
treatment or therapy.
[0144] "Substituent" and "substituent group," means an atom or
group that replaces the atom or group of a named parent compound.
For example a substituent of a modified nucleoside is any atom or
group that differs from the atom or group found in a naturally
occurring nucleoside (e.g., a modified 2'-substuent is any atom or
group at the 2'-position of a nucleoside other than H or OH).
Substituent groups can be protected or unpro tected. In certain
embodiments, compounds of the present disclosure have substituents
at one or at more than one position of the parent compound.
Substituents may also be further substituted with other substituent
groups and may be attached directly or via a linking group such as
an alkyl or hydro carbyl group to a parent compound.
[0145] Likewise, as used herein, "substituent" in reference to a
chemical functional group means an atom or group of atoms that
differs from the atom or a group of atoms normally present in the
named functional group. In certain embodiments, a substituent
replaces a hydrogen atom of the functional group (e.g., in certain
embodiments, the substituent of a substituted methyl group is an
atom or group other than hydrogen which replaces one of the
hydrogen atoms of an unsubstituted methyl group). Unless otherwise
indicated, groups amenable for use as substituents include without
limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (--C
(O) Raa), carboxyl (--C(O)O-Raa), aliphatic groups, ali-cyclic
groups, alkoxy, substituted oxy (--O--Raa), aryl, aralkyl,
heterocyclic radical, hetero aryl, hetero-arylalkyl, amino (N(Rbb)
(Rcc)), imino(=NRbb), amido (C(O)N (Rbb)(Rcc) or N(Rbb)C(O)Raa),
azido (--N3), nitro (NO2), cyano (--CN), carbamido
(OC(O)N(Rbb)(Rcc) or N(Rbb) C(O) ORaa), ureido (N(Rbb)C(O)
N(Rbb)(Rcc)), thioureido (N(Rbb)C (S)N(Rbb) (Rcc)), guanidinyl
(N(Rbb) C(.dbd.NRbb)-N(Rbb)(Rcc)), amidinyl (C(=NRbb) N(Rbb)(Rcc)
or N(Rbb)C(=NRbb)(Raa)), thiol (--SRbb), sulfinyl (S(O)Rbb),
sulfonyl (--S(O)2Rbb) and sulfonamidyl (--S(O)2N(Rbb)(Rcc) or
N(Rbb) S (O)2Rbb). Wherein each Raa, Rbb and Rcc is, independently,
H, an optionally linked chemical functional group or a further
substituent group with a preferred list including without
limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl,
aralkyl, heteroaryl, alicyclic, heterocyclic and hetero aryl alkyl.
Selected substituents within the compounds described herein are
present to a recursive degree.
[0146] "Substituted sugar moiety" means a furanosyl that is not a
naturally occurring sugar moiety. Substituted sugar moieties
include, but are not limited to furanosyls comprising substituents
at the 2'-position, the 3'-position, the 5'-position and/or the
4'-position. Certain substituted sugar moieties are bicyclic sugar
moieties.
[0147] "Sugar moiety" means a naturally occurring sugar moiety or a
modified sugar moiety of a nucleoside.
[0148] "Sugar motif" means a pattern of sugar modifications in an
oligonucleotide or a region thereof.
[0149] "Sugar surrogate" means a structure that does not comprise a
furanosyl and that is capable of replacing the naturally occurring
sugar moiety of a nucleoside, such that the resulting nucleoside
sub-units are capable of linking together and/or linking to other
nucleosides to form an oligomeric compound which is capable of
hybridizing to a complementary oligomeric compound. Such structures
include rings comprising a different number of atoms than furanosyl
(e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a
furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or
nitrogen); or both a change in the number of atoms and a
replacement of the oxygen. Such structures may also comprise
substitutions corresponding to those described for substituted
sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar
surrogates optionally comprising additional substituents). Sugar
surrogates also include more complex sugar replacements (e.g., the
non-ring systems of peptide nucleic acid). Sugar surrogates include
without limitation morpholinos, cyclohexenyls and
cyclohexitols.
[0150] "Target" refers to a protein, the modulation of which is
desired.
[0151] "Target gene" refers to a gene encoding a target.
[0152] "Targeting" means the process of design and selection of an
antisense compound that will specifically hybridize to a target
nucleic acid and induce a desired effect.
[0153] "Target nucleic acid," "target RNA," "target RNA transcript"
and "nucleic acid target" all mean a nucleic acid capable of being
targeted by antisense compounds.
[0154] "Target region" means a portion of a target nucleic acid to
which one or more antisense compounds is targeted.
[0155] "Target segment" means the sequence of nucleotides of a
target nucleic acid to which an antisense compound is targeted. "5'
target site" refers to the 5'-most nucleotide of a target segment.
"3' target site" refers to the 3'-most nucleotide of a target
segment.
[0156] "Terminal group" means one or more atom attached to either,
or both, the 3' end or the 5' end of an oligonucleotide. In certain
embodiments a terminal group is a conjugate group. In certain
embodiments, a terminal group comprises one or more terminal group
nucleosides.
[0157] "Terminal internucleoside linkage" means the linkage between
the last two nucleosides of an oligonucleotide or defined region
thereof.
[0158] "Therapeutically effective amount" means an amount of a
pharmaceutical agent that provides a therapeutic benefit to an
individual.
[0159] "Treat" refers to administering a pharmaceutical composition
to an animal in order to effect an alteration or improvement of a
disease, disorder, or condition in the animal. In certain
embodiments, one or more pharmaceutical compositions can be
administered to the animal.
[0160] "Unmodified" nucleobases mean the purine bases adenine (A)
and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)
and uracil (U).
[0161] "Unmodified nucleotide" means a nucleotide composed of
naturally occuring nucleobases, sugar moieties, and internucleoside
linkages. In certain embodiments, an unmodified nucleotide is an
RNA nucleotide (i.e. .beta.-D-ribonucleosides) or a DNA nucleotide
(i.e. .beta.-D-deoxyribonucleoside).
Certain Embodiments
[0162] Certain embodiments provide methods, compounds and
compositions for inhibiting Complement Factor B (CFB)
expression.
[0163] Certain embodiments provide antisense compounds targeted to
a CFB nucleic acid. In certain embodiments, the CFB nucleic acid
has the sequence set forth in GENBANK Accession No. NM_001710.5
(incorporated herein as SEQ ID NO: 1), GENBANK Accession No.
NT_007592.15 truncated from nucleotides 31852000 to U.S. Pat. No.
31,861,000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession
No NW_001116486.1 truncated from nucleotides 536000 to 545000
(incorporated herein as SEQ ID NO: 3), GENBANK Accession No.
XM_001113553.2 (incorporated herein as SEQ ID NO: 4), or GENBANK
Accession No. NM_008198.2 (incorporated herein as SEQ ID NO:
5).
[0164] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising at least 8 contiguous nucleobases of
any of the nucleobase sequences of SEQ ID NOs: 6-808.
[0165] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising at least 9 contiguous nucleobases of
any of the nucleobase sequences of SEQ ID NOs: 6-808.
[0166] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising at least 10 contiguous nucleobases
of any of the nucleobase sequences of SEQ ID NOs: 6-808.
[0167] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising at least 11 contiguous nucleobases
of any of the nucleobase sequences of SEQ ID NOs: 6-808.
[0168] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising at least 12 contiguous nucleobases
of any of the nucleobase sequences of SEQ ID NOs: 6-808.
[0169] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising the nucleobase sequence of any one
of SEQ ID NOs: 6-808.
[0170] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of the nucleobase sequence of any one of
SEQ ID NOs: 6-808.
[0171] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides
complementary within nucleobases 30-49, 48-63, 150-169, 151-170,
152-171, 154-169, 154-173, 156-171, 156-175, 157-176, 158-173,
158-177, 480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108,
1135-1154, 1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178,
1162-1181, 1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192,
1175-1190, 1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254,
1298-1317, 1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341,
1328-1347, 1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418,
1405-1424, 1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666,
1689-1708, 1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104,
2166-2185, 2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210,
2195-2214, 2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238,
2223-2242, 2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257,
2241-2260, 2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467,
2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478,
2461-2476, 2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570,
2552-2568, 2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571,
2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572,
2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574,
2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576,
2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576,
2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581,
2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584,
2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583,
2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587,
2570-2589, 2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590,
2572-2591, 2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593,
2575-2590, 2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595,
2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597,
2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597,
2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600,
2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600,
2584-2601, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604,
2586-2601, 2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603,
2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606,
2588-2607, 2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608,
2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609,
2591-2607, 2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608,
2592-2609, 2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610,
2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613,
2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611,
2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612,
2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614,
2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616,
2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618,
2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620,
2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618,
2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620,
2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622,
2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624,
2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626,
2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624,
2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626,
2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628,
2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630,
2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629,
2614-2630, 2614-2631, 2615-2630, 2615-2631, or 2616-2631 of SEQ ID
NO: 1, and wherein said modified oligonucleotide is at least 85%,
at least 90%, at least 95%, or 100% complementary to SEQ ID NO:
1.
[0172] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides having a
nucleobase sequence comprising a portion of at least 8 contiguous
nucleobases complementary to an equal length portion of nucleobases
30-49, 48-63, 150-169, 151-170, 152-171, 154-169, 154-173, 156-171,
156-175, 157-176, 158-173, 158-177, 480-499, 600-619, 638-657,
644-663, 738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166,
1150-1169, 1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186,
1171-1190, 1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196,
1183-1202, 1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329,
1316-1335, 1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374,
1393-1412, 1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640,
1646-1665, 1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782,
1912-1931, 2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208,
2191-2210, 2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212,
2197-2216, 2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245,
2227-2242, 2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380,
2388-2407, 2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472,
2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551,
2550-2569, 2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571,
2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572,
2554-2573, 2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574,
2556-2575, 2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575,
2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577,
2560-2578, 2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580,
2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582,
2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582,
2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586,
2569-2588, 2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588,
2571-2590, 2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592,
2574-2590, 2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592,
2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596,
2578-2594, 2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597,
2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600,
2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600,
2583-2601, 2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603,
2585-2601, 2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604,
2586-2605, 2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603,
2588-2604, 2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605,
2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607,
2590-2608, 2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609,
2591-2610, 2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611,
2593-2608, 2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610,
2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612,
2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614,
2596-2615, 2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615,
2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617,
2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615,
2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617,
2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619,
2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621,
2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623,
2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621,
2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623,
2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625,
2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627,
2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629,
2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627,
2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629,
2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630,
2615-2631, or 2616-2631 of SEQ ID NO:1, and wherein the nucleobase
sequence of the modified oligonucleotide is at least 85%, at least
90%, at least 95%, or 100% complementary to SEQ ID NO: 1.
[0173] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides
complementary within nucleobases 1608-1627, 1685-1704, 1686-1705,
1751-1770, 1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890,
1875-1894, 1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898,
2288-2307, 2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792,
3819-3838, 3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862,
4151-4166, 4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230,
4609-4628, 4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667,
4686-4705, 4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289,
5295-5314, 5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425,
6662-6681, 6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998,
6981-7000, 6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004,
6990-7009, 7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381,
7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709,
7694-7713, 7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801,
7787-7803, 7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806,
7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807,
7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809,
7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811,
7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811,
7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816,
7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819,
7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818,
7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822,
7805-7824, 7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825,
7807-7826, 7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828,
7810-7825, 7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830,
7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832,
7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832,
7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835,
7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835,
7819-7836, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839,
7821-7836, 7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838,
7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840,
7823-7841, 7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841,
7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843,
7825-7844, 7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842,
7827-7843, 7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844,
7828-7845, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847,
7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849,
7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847,
7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849,
7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851,
7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853,
7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855,
7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853,
7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855,
7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857,
7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859,
7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861,
7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859,
7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862,
7846-7861, or 7846-7862 of SEQ ID NO: 2, and wherein said modified
oligonucleotide is at least 85%, at least 90%, at least 95%, or
100% complementary to SEQ ID NO: 2.
[0174] Certain embodiments provide a compound comprising a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides having a
nucleobase sequence comprising a portion of at least 8 contiguous
nucleobases complementary to an equal length portion of nucleobases
1608-1627, 1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890,
1872-1891, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896,
1878-1897, 1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865,
2852-2871, 2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850,
3834-3853, 3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172,
4159-4178, 4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634,
4621-4640, 4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711,
4698-4717, 4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849,
5890-5909, 5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973,
6960-6979, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002,
6984-7003, 6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144,
7151-7170, 7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709,
7692-7707, 7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715,
7767-7786, 7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806,
7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807,
7789-7808, 7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809,
7791-7810, 7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810,
7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812,
7795-7813, 7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815,
7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817,
7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817,
7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821,
7804-7823, 7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823,
7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827,
7809-7825, 7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827,
7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831,
7813-7829, 7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832,
7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835,
7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835,
7818-7836, 7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838,
7820-7836, 7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839,
7821-7840, 7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838,
7823-7839, 7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839,
7824-7840, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840,
7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843,
7826-7844, 7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845,
7827-7846, 7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844,
7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846,
7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848,
7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850,
7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852,
7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850,
7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852,
7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854,
7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856,
7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858,
7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856,
7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858,
7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860,
7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862,
7845-7860, 7845-7861, 7845-7862, 7846-7861, and 7846-7862 of SEQ ID
NO: 2, and wherein the nucleobase sequence of the modified
oligonucleotide is at least 85%, at least 90%, at least 95%, or
100% complementary to SEQ ID NO: 2.
[0175] In certain embodiments, antisense compounds or
oligonucleotides target a region of a CFB nucleic acid. In certain
embodiments, such compounds or oligonucleotides targeted to a
region of a CFB nucleic acid have a contiguous nucleobase portion
that is complementary to an equal length nucleobase portion of the
region. For example, the portion can be at least an 8, 9, 10, 11,
12, 13, 14, 15, or 16 contiguous nucleobase portion complementary
to an equal length portion of a region recited herein. In certain
embodiments, a compound comprises or consists of a conjugate and a
modified oligonucleotide targeting any of the following nucleotide
regions of SEQ ID NO: 1: 30-49, 48-63, 150-169, 151-170, 152-171,
154-169, 154-173, 156-171, 156-175, 157-176, 158-173, 158-177,
480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108, 1135-1154,
1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178, 1162-1181,
1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192, 1175-1190,
1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254, 1298-1317,
1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341, 1328-1347,
1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418, 1405-1424,
1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666, 1689-1708,
1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104, 2166-2185,
2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214,
2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238, 2223-2242,
2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257, 2241-2260,
2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467, 2453-2472,
2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476,
2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570, 2552-2568,
2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572,
2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572, 2555-2574,
2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574, 2557-2575,
2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577,
2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576, 2561-2578,
2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578,
2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583,
2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585,
2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587, 2570-2589,
2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590, 2572-2591,
2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593, 2575-2590,
2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594,
2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597, 2579-2598,
2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598,
2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601,
2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2601,
2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2601,
2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603, 2587-2605,
2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606, 2588-2607,
2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605,
2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609, 2591-2607,
2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608, 2592-2609,
2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610, 2593-2612,
2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610,
2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612,
2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612, 2597-2613,
2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615,
2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617,
2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619,
2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617,
2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619,
2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621,
2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623,
2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625,
2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623,
2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625,
2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627,
2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628, 2611-2629,
2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631,
2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630,
2614-2631, 2615-2630, 2615-2631, and 2616-2631.
[0176] In certain embodiments, antisense compounds or
oligonucleotides target a region of a CFB nucleic acid. In certain
embodiments, such compounds or oligonucleotides targeted to a
region of a CFB nucleic acid have a contiguous nucleobase portion
that is complementary to an equal length nucleobase portion of the
region. For example, the portion can be at least an 8, 9, 10, 11,
12, 13, 14, 15, or 16 contiguous nucleobase portion complementary
to an equal length portion of a region recited herein. In certain
embodiments, a compound comprises or consists of a conjugate and a
modified oligonucleotide targeting the following nucleotide regions
of SEQ ID NO: 2: 1608-1627, 1685-1704, 1686-1705, 1751-1770,
1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890, 1875-1894,
1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898, 2288-2307,
2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792, 3819-3838,
3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862, 4151-4166,
4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230, 4609-4628,
4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667, 4686-4705,
4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289, 5295-5314,
5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425, 6662-6681,
6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998, 6981-7000,
6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004, 6990-7009,
7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381, 7683-7702,
7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7694-7713,
7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801, 7787-7803,
7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807,
7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807, 7790-7809,
7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809, 7792-7810,
7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812,
7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811, 7796-7813,
7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813,
7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818,
7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820,
7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822, 7805-7824,
7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826,
7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828, 7810-7825,
7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829,
7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832, 7814-7833,
7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833,
7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836,
7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7836,
7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7836,
7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838, 7822-7840,
7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840, 7823-7841,
7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841, 7824-7842,
7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844,
7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842, 7827-7843,
7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844, 7828-7845,
7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848,
7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846,
7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848,
7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850,
7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852,
7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854,
7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852,
7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854,
7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856,
7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858,
7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860,
7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858,
7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860,
7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861,
and 7846-7862.
[0177] In certain embodiments, a compound comprises or consists of
a conjugate and a modified oligonucleotide targeting the 3'UTR of a
CFB nucleic acid. In certain aspects, the modified oligonucleotide
targets within nucleotides 2574-2626 of a CFB nucleic acid having
the nucleobase sequence of SEQ ID NO: 1. In certain aspects, the
modified oligonucleotide has at least an 8, 9, 10, 11, 12, 13, 14,
15, or 16 contiguous nucleobase portion complementary to an equal
length portion within nucleotides 2574-2626 of a CFB nucleic acid
having the nucleobase sequence of SEQ ID NO: 1.
In certain embodiments, a compound comprises or consists of a
conjugate and a modified oligonucleotide targeting a region of a
CFB nucleic acid having the nucleobase sequence of SEQ ID NO: 1
within nucleobases 2457-2631, 2457-2472, 2457-2474, 2457-2476,
2457-2566, 2457-2570, 2457-2571, 2457-2572, 2457-2573, 2457-2574,
2457-2575, 2457-2576, 2457-2577, 2457-2578, 2457-2579, 2457-2580,
2457-2581, 2457-2582, 2457-2583, 2457-2584, 2457-2585, 2457-2586,
2457-2587, 2457-2588, 2457-2589, 2457-2590, 2457-2591, 2457-2592,
2457-2593, 2457-2594, 2457-2595, 2457-2596, 2457-2597, 2457-2598,
2457-2599, 2457-2600, 2457-2601, 2457-2602, 2457-2603, 2457-2604,
2457-2605, 2457-2606, 2457-2607, 2457-2608, 2457-2609, 2457-2610,
2457-2611, 2457-2612, 2457-2613, 2457-2614, 2457-2615, 2457-2616,
2457-2617, 2457-2618, 2457-2619, 2457-2620, 2457-2621, 2457-2622,
2457-2623, 2457-2624, 2457-2625, 2457-2626, 2457-2627, 2457-2628,
2457-2629, 2457-2630, 2457-2631, 2459-2474, 2459-2476, 2459-2566,
2459-2570, 2459-2571, 2459-2572, 2459-2573, 2459-2574, 2459-2575,
2459-2576, 2459-2577, 2459-2578, 2459-2579, 2459-2580, 2459-2581,
2459-2582, 2459-2583, 2459-2584, 2459-2585, 2459-2586, 2459-2587,
2459-2588, 2459-2589, 2459-2590, 2459-2591, 2459-2592, 2459-2593,
2459-2594, 2459-2595, 2459-2596, 2459-2597, 2459-2598, 2459-2599,
2459-2600, 2459-2601, 2459-2602, 2459-2603, 2459-2604, 2459-2605,
2459-2606, 2459-2607, 2459-2608, 2459-2609, 2459-2610, 2459-2611,
2459-2612, 2459-2613, 2459-2614, 2459-2615, 2459-2616, 2459-2617,
2459-2618, 2459-2619, 2459-2620, 2459-2621, 2459-2622, 2459-2623,
2459-2624, 2459-2625, 2459-2626, 2459-2627, 2459-2628, 2459-2629,
2459-2630, 2459-2631, 2461-2476, 2461-2566, 2461-2570, 2461-2571,
2461-2572, 2461-2573, 2461-2574, 2461-2575, 2461-2576, 2461-2577,
2461-2578, 2461-2579, 2461-2580, 2461-2581, 2461-2582, 2461-2583,
2461-2584, 2461-2585, 2461-2586, 2461-2587, 2461-2588, 2461-2589,
2461-2590, 2461-2591, 2461-2592, 2461-2593, 2461-2594, 2461-2595,
2461-2596, 2461-2597, 2461-2598, 2461-2599, 2461-2600, 2461-2601,
2461-2602, 2461-2603, 2461-2604, 2461-2605, 2461-2606, 2461-2607,
2461-2608, 2461-2609, 2461-2610, 2461-2611, 2461-2612, 2461-2613,
2461-2614, 2461-2615, 2461-2616, 2461-2617, 2461-2618, 2461-2619,
2461-2620, 2461-2621, 2461-2622, 2461-2623, 2461-2624, 2461-2625,
2461-2626, 2461-2627, 2461-2628, 2461-2629, 2461-2630, 2461-2631,
2551-2566, 2551-2570, 2551-2571, 2551-2572, 2551-2573, 2551-2574,
2551-2575, 2551-2576, 2551-2577, 2551-2578, 2551-2579, 2551-2580,
2551-2581, 2551-2582, 2551-2583, 2551-2584, 2551-2585, 2551-2586,
2551-2587, 2551-2588, 2551-2589, 2551-2590, 2551-2591, 2551-2592,
2551-2593, 2551-2594, 2551-2595, 2551-2596, 2551-2597, 2551-2598,
2551-2599, 2551-2600, 2551-2601, 2551-2602, 2551-2603, 2551-2604,
2551-2605, 2551-2606, 2551-2607, 2551-2608, 2551-2609, 2551-2610,
2551-2611, 2551-2612, 2551-2613, 2551-2614, 2551-2615, 2551-2616,
2551-2617, 2551-2618, 2551-2619, 2551-2620, 2551-2621, 2551-2622,
2551-2623, 2551-2624, 2551-2625, 2551-2626, 2551-2627, 2551-2628,
2551-2629, 2551-2630, 2551-2631, 2553-2570, 2553-2571, 2553-2572,
2553-2573, 2553-2574, 2553-2575, 2553-2576, 2553-2577, 2553-2578,
2553-2579, 2553-2580, 2553-2581, 2553-2582, 2553-2583, 2553-2584,
2553-2585, 2553-2586, 2553-2587, 2553-2588, 2553-2589, 2553-2590,
2553-2591, 2553-2592, 2553-2593, 2553-2594, 2553-2595, 2553-2596,
2553-2597, 2553-2598, 2553-2599, 2553-2600, 2553-2601, 2553-2602,
2553-2603, 2553-2604, 2553-2605, 2553-2606, 2553-2607, 2553-2608,
2553-2609, 2553-2610, 2553-2611, 2553-2612, 2553-2613, 2553-2614,
2553-2615, 2553-2616, 2553-2617, 2553-2618, 2553-2619, 2553-2620,
2553-2621, 2553-2622, 2553-2623, 2553-2624, 2553-2625, 2553-2626,
2553-2627, 2553-2628, 2553-2629, 2553-2630, 2553-2631, 2554-2573,
2554-2574, 2554-2575, 2554-2576, 2554-2577, 2554-2578, 2554-2579,
2554-2580, 2554-2581, 2554-2582, 2554-2583, 2554-2584, 2554-2585,
2554-2586, 2554-2587, 2554-2588, 2554-2589, 2554-2590, 2554-2591,
2554-2592, 2554-2593, 2554-2594, 2554-2595, 2554-2596, 2554-2597,
2554-2598, 2554-2599, 2554-2600, 2554-2601, 2554-2602, 2554-2603,
2554-2604, 2554-2605, 2554-2606, 2554-2607, 2554-2608, 2554-2609,
2554-2610, 2554-2611, 2554-2612, 2554-2613, 2554-2614, 2554-2615,
2554-2616, 2554-2617, 2554-2618, 2554-2619, 2554-2620, 2554-2621,
2554-2622, 2554-2623, 2554-2624, 2554-2625, 2554-2626, 2554-2627,
2554-2628, 2554-2629, 2554-2630, 2554-2631, 2555-2572, 2555-2573,
2555-2574, 2555-2575, 2555-2576, 2555-2577, 2555-2578, 2555-2579,
2555-2580, 2555-2581, 2555-2582, 2555-2583, 2555-2584, 2555-2585,
2555-2586, 2555-2587, 2555-2588, 2555-2589, 2555-2590, 2555-2591,
2555-2592, 2555-2593, 2555-2594, 2555-2595, 2555-2596, 2555-2597,
2555-2598, 2555-2599, 2555-2600, 2555-2601, 2555-2602, 2555-2603,
2555-2604, 2555-2605, 2555-2606, 2555-2607, 2555-2608, 2555-2609,
2555-2610, 2555-2611, 2555-2612, 2555-2613, 2555-2614, 2555-2615,
2555-2616, 2555-2617, 2555-2618, 2555-2619, 2555-2620, 2555-2621,
2555-2622, 2555-2623, 2555-2624, 2555-2625, 2555-2626, 2555-2627,
2555-2628, 2555-2629, 2555-2630, 2555-2631, 2556-2573, 2556-2574,
2556-2575, 2556-2576, 2556-2577, 2556-2578, 2556-2579, 2556-2580,
2556-2581, 2556-2582, 2556-2583, 2556-2584, 2556-2585, 2556-2586,
2556-2587, 2556-2588, 2556-2589, 2556-2590, 2556-2591, 2556-2592,
2556-2593, 2556-2594, 2556-2595, 2556-2596, 2556-2597, 2556-2598,
2556-2599, 2556-2600, 2556-2601, 2556-2602, 2556-2603, 2556-2604,
2556-2605, 2556-2606, 2556-2607, 2556-2608, 2556-2609, 2556-2610,
2556-2611, 2556-2612, 2556-2613, 2556-2614, 2556-2615, 2556-2616,
2556-2617, 2556-2618, 2556-2619, 2556-2620, 2556-2621, 2556-2622,
2556-2623, 2556-2624, 2556-2625, 2556-2626, 2556-2627, 2556-2628,
2556-2629, 2556-2630, 2556-2631, 2557-2574, 2557-2575, 2557-2576,
2557-2577, 2557-2578, 2557-2579, 2557-2580, 2557-2581, 2557-2582,
2557-2583, 2557-2584, 2557-2585, 2557-2586, 2557-2587, 2557-2588,
2557-2589, 2557-2590, 2557-2591, 2557-2592, 2557-2593, 2557-2594,
2557-2595, 2557-2596, 2557-2597, 2557-2598, 2557-2599, 2557-2600,
2557-2601, 2557-2602, 2557-2603, 2557-2604, 2557-2605, 2557-2606,
2557-2607, 2557-2608, 2557-2609, 2557-2610, 2557-2611, 2557-2612,
2557-2613, 2557-2614, 2557-2615, 2557-2616, 2557-2617, 2557-2618,
2557-2619, 2557-2620, 2557-2621, 2557-2622, 2557-2623, 2557-2624,
2557-2625, 2557-2626, 2557-2627, 2557-2628, 2557-2629, 2557-2630,
2557-2631, 2558-2575, 2558-2576, 2558-2577, 2558-2578, 2558-2579,
2558-2580, 2558-2581, 2558-2582, 2558-2583, 2558-2584, 2558-2585,
2558-2586, 2558-2587, 2558-2588, 2558-2589, 2558-2590, 2558-2591,
2558-2592, 2558-2593, 2558-2594, 2558-2595, 2558-2596, 2558-2597,
2558-2598, 2558-2599, 2558-2600, 2558-2601, 2558-2602, 2558-2603,
2558-2604, 2558-2605, 2558-2606, 2558-2607, 2558-2608, 2558-2609,
2558-2610, 2558-2611, 2558-2612, 2558-2613, 2558-2614, 2558-2615,
2558-2616, 2558-2617, 2558-2618, 2558-2619, 2558-2620, 2558-2621,
2558-2622, 2558-2623, 2558-2624, 2558-2625, 2558-2626, 2558-2627,
2558-2628, 2558-2629, 2558-2630, 2558-2631, 2559-2576, 2559-2577,
2559-2578, 2559-2579, 2559-2580, 2559-2581, 2559-2582, 2559-2583,
2559-2584, 2559-2585, 2559-2586, 2559-2587, 2559-2588, 2559-2589,
2559-2590, 2559-2591, 2559-2592, 2559-2593, 2559-2594, 2559-2595,
2559-2596, 2559-2597, 2559-2598, 2559-2599, 2559-2600, 2559-2601,
2559-2602, 2559-2603, 2559-2604, 2559-2605, 2559-2606, 2559-2607,
2559-2608, 2559-2609, 2559-2610, 2559-2611, 2559-2612, 2559-2613,
2559-2614, 2559-2615, 2559-2616, 2559-2617, 2559-2618, 2559-2619,
2559-2620, 2559-2621, 2559-2622, 2559-2623, 2559-2624, 2559-2625,
2559-2626, 2559-2627, 2559-2628, 2559-2629, 2559-2630, 2559-2631,
2560-2577, 2560-2578, 2560-2579, 2560-2580, 2560-2581, 2560-2582,
2560-2583, 2560-2584, 2560-2585, 2560-2586, 2560-2587, 2560-2588,
2560-2589, 2560-2590, 2560-2591, 2560-2592, 2560-2593, 2560-2594,
2560-2595, 2560-2596, 2560-2597, 2560-2598, 2560-2599, 2560-2600,
2560-2601, 2560-2602, 2560-2603, 2560-2604, 2560-2605, 2560-2606,
2560-2607, 2560-2608, 2560-2609, 2560-2610, 2560-2611, 2560-2612,
2560-2613, 2560-2614, 2560-2615, 2560-2616, 2560-2617, 2560-2618,
2560-2619, 2560-2620, 2560-2621, 2560-2622, 2560-2623, 2560-2624,
2560-2625, 2560-2626, 2560-2627, 2560-2628, 2560-2629, 2560-2630,
2560-2631, 2561-2578, 2561-2579, 2561-2580, 2561-2581, 2561-2582,
2561-2583, 2561-2584, 2561-2585, 2561-2586, 2561-2587, 2561-2588,
2561-2589, 2561-2590, 2561-2591, 2561-2592, 2561-2593, 2561-2594,
2561-2595, 2561-2596, 2561-2597, 2561-2598, 2561-2599, 2561-2600,
2561-2601, 2561-2602, 2561-2603, 2561-2604, 2561-2605, 2561-2606,
2561-2607, 2561-2608, 2561-2609, 2561-2610, 2561-2611, 2561-2612,
2561-2613, 2561-2614, 2561-2615, 2561-2616, 2561-2617, 2561-2618,
2561-2619, 2561-2620, 2561-2621, 2561-2622, 2561-2623, 2561-2624,
2561-2625, 2561-2626, 2561-2627, 2561-2628, 2561-2629, 2561-2630,
2561-2631, 2562-2577, 2562-2578, 2562-2579, 2562-2580, 2562-2581,
2562-2582, 2562-2583, 2562-2584, 2562-2585, 2562-2586, 2562-2587,
2562-2588, 2562-2589, 2562-2590, 2562-2591, 2562-2592, 2562-2593,
2562-2594, 2562-2595, 2562-2596, 2562-2597, 2562-2598, 2562-2599,
2562-2600, 2562-2601, 2562-2602, 2562-2603, 2562-2604, 2562-2605,
2562-2606, 2562-2607, 2562-2608, 2562-2609, 2562-2610, 2562-2611,
2562-2612, 2562-2613, 2562-2614, 2562-2615, 2562-2616, 2562-2617,
2562-2618, 2562-2619, 2562-2620, 2562-2621, 2562-2622, 2562-2623,
2562-2624, 2562-2625, 2562-2626, 2562-2627, 2562-2628, 2562-2629,
2562-2630, 2562-2631, 2563-2580, 2563-2581, 2563-2582, 2563-2583,
2563-2584, 2563-2585, 2563-2586, 2563-2587, 2563-2588, 2563-2589,
2563-2590, 2563-2591, 2563-2592, 2563-2593, 2563-2594, 2563-2595,
2563-2596, 2563-2597, 2563-2598, 2563-2599, 2563-2600, 2563-2601,
2563-2602, 2563-2603, 2563-2604, 2563-2605, 2563-2606, 2563-2607,
2563-2608, 2563-2609, 2563-2610, 2563-2611, 2563-2612, 2563-2613,
2563-2614, 2563-2615, 2563-2616, 2563-2617, 2563-2618, 2563-2619,
2563-2620, 2563-2621, 2563-2622, 2563-2623, 2563-2624, 2563-2625,
2563-2626, 2563-2627, 2563-2628, 2563-2629, 2563-2630, 2563-2631,
2564-2581, 2564-2582, 2564-2583, 2564-2584, 2564-2585, 2564-2586,
2564-2587, 2564-2588, 2564-2589, 2564-2590, 2564-2591, 2564-2592,
2564-2593, 2564-2594, 2564-2595, 2564-2596, 2564-2597, 2564-2598,
2564-2599, 2564-2600, 2564-2601, 2564-2602, 2564-2603, 2564-2604,
2564-2605, 2564-2606, 2564-2607, 2564-2608, 2564-2609, 2564-2610,
2564-2611, 2564-2612, 2564-2613, 2564-2614, 2564-2615, 2564-2616,
2564-2617, 2564-2618, 2564-2619, 2564-2620, 2564-2621, 2564-2622,
2564-2623, 2564-2624, 2564-2625, 2564-2626, 2564-2627, 2564-2628,
2564-2629, 2564-2630, 2564-2631, 2565-2584, 2565-2585, 2565-2586,
2565-2587, 2565-2588, 2565-2589, 2565-2590, 2565-2591, 2565-2592,
2565-2593, 2565-2594, 2565-2595, 2565-2596, 2565-2597, 2565-2598,
2565-2599, 2565-2600, 2565-2601, 2565-2602, 2565-2603, 2565-2604,
2565-2605, 2565-2606, 2565-2607, 2565-2608, 2565-2609, 2565-2610,
2565-2611, 2565-2612, 2565-2613, 2565-2614, 2565-2615, 2565-2616,
2565-2617, 2565-2618, 2565-2619, 2565-2620, 2565-2621, 2565-2622,
2565-2623, 2565-2624, 2565-2625, 2565-2626, 2565-2627, 2565-2628,
2565-2629, 2565-2630, 2565-2631, 2566-2583, 2566-2584, 2566-2585,
2566-2586, 2566-2587, 2566-2588, 2566-2589, 2566-2590, 2566-2591,
2566-2592, 2566-2593, 2566-2594, 2566-2595, 2566-2596, 2566-2597,
2566-2598, 2566-2599, 2566-2600, 2566-2601, 2566-2602, 2566-2603,
2566-2604, 2566-2605, 2566-2606, 2566-2607, 2566-2608, 2566-2609,
2566-2610, 2566-2611, 2566-2612, 2566-2613, 2566-2614, 2566-2615,
2566-2616, 2566-2617, 2566-2618, 2566-2619, 2566-2620, 2566-2621,
2566-2622, 2566-2623, 2566-2624, 2566-2625, 2566-2626, 2566-2627,
2566-2628, 2566-2629, 2566-2630, 2566-2631, 2567-2584, 2567-2585,
2567-2586, 2567-2587, 2567-2588, 2567-2589, 2567-2590, 2567-2591,
2567-2592, 2567-2593, 2567-2594, 2567-2595, 2567-2596, 2567-2597,
2567-2598, 2567-2599, 2567-2600, 2567-2601, 2567-2602, 2567-2603,
2567-2604, 2567-2605, 2567-2606, 2567-2607, 2567-2608, 2567-2609,
2567-2610, 2567-2611, 2567-2612, 2567-2613, 2567-2614, 2567-2615,
2567-2616, 2567-2617, 2567-2618, 2567-2619, 2567-2620, 2567-2621,
2567-2622, 2567-2623, 2567-2624, 2567-2625, 2567-2626, 2567-2627,
2567-2628, 2567-2629, 2567-2630, 2567-2631, 2568-2585, 2568-2586,
2568-2587, 2568-2588, 2568-2589, 2568-2590, 2568-2591, 2568-2592,
2568-2593, 2568-2594, 2568-2595, 2568-2596, 2568-2597, 2568-2598,
2568-2599, 2568-2600, 2568-2601, 2568-2602, 2568-2603, 2568-2604,
2568-2605, 2568-2606, 2568-2607, 2568-2608, 2568-2609, 2568-2610,
2568-2611, 2568-2612, 2568-2613, 2568-2614, 2568-2615, 2568-2616,
2568-2617, 2568-2618, 2568-2619, 2568-2620, 2568-2621, 2568-2622,
2568-2623, 2568-2624, 2568-2625, 2568-2626, 2568-2627, 2568-2628,
2568-2629, 2568-2630, 2568-2631, 2569-2586, 2569-2587, 2569-2588,
2569-2589, 2569-2590, 2569-2591, 2569-2592, 2569-2593, 2569-2594,
2569-2595, 2569-2596, 2569-2597, 2569-2598, 2569-2599, 2569-2600,
2569-2601, 2569-2602, 2569-2603, 2569-2604, 2569-2605, 2569-2606,
2569-2607, 2569-2608, 2569-2609, 2569-2610, 2569-2611, 2569-2612,
2569-2613, 2569-2614, 2569-2615, 2569-2616, 2569-2617, 2569-2618,
2569-2619, 2569-2620, 2569-2621, 2569-2622, 2569-2623, 2569-2624,
2569-2625, 2569-2626, 2569-2627, 2569-2628, 2569-2629, 2569-2630,
2569-2631, 2569-2586, 2569-2587, 2569-2588, 2569-2589, 2569-2590,
2569-2591, 2569-2592, 2569-2593, 2569-2594, 2569-2595, 2569-2596,
2569-2597, 2569-2598, 2569-2599, 2569-2600, 2569-2601, 2569-2602,
2569-2603, 2569-2604, 2569-2605, 2569-2606, 2569-2607, 2569-2608,
2569-2609, 2569-2610, 2569-2611, 2569-2612, 2569-2613, 2569-2614,
2569-2615, 2569-2616, 2569-2617, 2569-2618, 2569-2619, 2569-2620,
2569-2621, 2569-2622, 2569-2623, 2569-2624, 2569-2625, 2569-2626,
2569-2627, 2569-2628, 2569-2629, 2569-2630, 2569-2631, 2571-2588,
2571-2589, 2571-2590, 2571-2591, 2571-2592, 2571-2593, 2571-2594,
2571-2595, 2571-2596, 2571-2597, 2571-2598, 2571-2599, 2571-2600,
2571-2601, 2571-2602, 2571-2603, 2571-2604, 2571-2605, 2571-2606,
2571-2607, 2571-2608, 2571-2609, 2571-2610, 2571-2611, 2571-2612,
2571-2613, 2571-2614, 2571-2615, 2571-2616, 2571-2617, 2571-2618,
2571-2619, 2571-2620, 2571-2621, 2571-2622, 2571-2623, 2571-2624,
2571-2625, 2571-2626, 2571-2627, 2571-2628, 2571-2629, 2571-2630,
2571-2631, 2572-2589, 2572-2590, 2572-2591, 2572-2592, 2572-2593,
2572-2594, 2572-2595, 2572-2596, 2572-2597, 2572-2598, 2572-2599,
2572-2600, 2572-2601, 2572-2602, 2572-2603, 2572-2604, 2572-2605,
2572-2606, 2572-2607, 2572-2608, 2572-2609, 2572-2610, 2572-2611,
2572-2612, 2572-2613, 2572-2614, 2572-2615, 2572-2616, 2572-2617,
2572-2618, 2572-2619, 2572-2620, 2572-2621, 2572-2622, 2572-2623,
2572-2624, 2572-2625, 2572-2626, 2572-2627, 2572-2628, 2572-2629,
2572-2630, 2572-2631, 2573-2590, 2573-2591, 2573-2592, 2573-2593,
2573-2594, 2573-2595, 2573-2596, 2573-2597, 2573-2598, 2573-2599,
2573-2600, 2573-2601, 2573-2602, 2573-2603, 2573-2604, 2573-2605,
2573-2606, 2573-2607, 2573-2608, 2573-2609, 2573-2610, 2573-2611,
2573-2612, 2573-2613, 2573-2614, 2573-2615, 2573-2616, 2573-2617,
2573-2618, 2573-2619, 2573-2620, 2573-2621, 2573-2622, 2573-2623,
2573-2624, 2573-2625, 2573-2626, 2573-2627, 2573-2628, 2573-2629,
2573-2630, 2573-2631, 2574-2591, 2574-2592, 2574-2593, 2574-2594,
2574-2595, 2574-2596, 2574-2597, 2574-2598, 2574-2599, 2574-2600,
2574-2601, 2574-2602, 2574-2603, 2574-2604, 2574-2605, 2574-2606,
2574-2607, 2574-2608, 2574-2609, 2574-2610, 2574-2611, 2574-2612,
2574-2613, 2574-2614, 2574-2615, 2574-2616, 2574-2617, 2574-2618,
2574-2619, 2574-2620, 2574-2621, 2574-2622, 2574-2623, 2574-2624,
2574-2625, 2574-2626, 2574-2627, 2574-2628, 2574-2629, 2574-2630,
2574-2631, 2575-2592, 2575-2593, 2575-2594, 2575-2595, 2575-2596,
2575-2597, 2575-2598, 2575-2599, 2575-2600, 2575-2601, 2575-2602,
2575-2603, 2575-2604, 2575-2605, 2575-2606, 2575-2607, 2575-2608,
2575-2609, 2575-2610, 2575-2611, 2575-2612, 2575-2613, 2575-2614,
2575-2615, 2575-2616, 2575-2617, 2575-2618, 2575-2619, 2575-2620,
2575-2621, 2575-2622, 2575-2623, 2575-2624, 2575-2625, 2575-2626,
2575-2627, 2575-2628, 2575-2629, 2575-2630, 2575-2631, 2576-2593,
2576-2594, 2576-2595, 2576-2596, 2576-2597, 2576-2598, 2576-2599,
2576-2600, 2576-2601, 2576-2602, 2576-2603, 2576-2604, 2576-2605,
2576-2606, 2576-2607, 2576-2608, 2576-2609, 2576-2610, 2576-2611,
2576-2612, 2576-2613, 2576-2614, 2576-2615, 2576-2616, 2576-2617,
2576-2618, 2576-2619, 2576-2620, 2576-2621, 2576-2622, 2576-2623,
2576-2624, 2576-2625, 2576-2626, 2576-2627, 2576-2628, 2576-2629,
2576-2630, 2576-2631, 2577-2594, 2577-2595, 2577-2596, 2577-2597,
2577-2598, 2577-2599, 2577-2600, 2577-2601, 2577-2602, 2577-2603,
2577-2604, 2577-2605, 2577-2606, 2577-2607, 2577-2608, 2577-2609,
2577-2610, 2577-2611,
2577-2612, 2577-2613, 2577-2614, 2577-2615, 2577-2616, 2577-2617,
2577-2618, 2577-2619, 2577-2620, 2577-2621, 2577-2622, 2577-2623,
2577-2624, 2577-2625, 2577-2626, 2577-2627, 2577-2628, 2577-2629,
2577-2630, 2577-2631, 2578-2597, 2578-2598, 2578-2599, 2578-2600,
2578-2601, 2578-2602, 2578-2603, 2578-2604, 2578-2605, 2578-2606,
2578-2607, 2578-2608, 2578-2609, 2578-2610, 2578-2611, 2578-2612,
2578-2613, 2578-2614, 2578-2615, 2578-2616, 2578-2617, 2578-2618,
2578-2619, 2578-2620, 2578-2621, 2578-2622, 2578-2623, 2578-2624,
2578-2625, 2578-2626, 2578-2627, 2578-2628, 2578-2629, 2578-2630,
2578-2631, 2579-2598, 2579-2599, 2579-2600, 2579-2601, 2579-2602,
2579-2603, 2579-2604, 2579-2605, 2579-2606, 2579-2607, 2579-2608,
2579-2609, 2579-2610, 2579-2611, 2579-2612, 2579-2613, 2579-2614,
2579-2615, 2579-2616, 2579-2617, 2579-2618, 2579-2619, 2579-2620,
2579-2621, 2579-2622, 2579-2623, 2579-2624, 2579-2625, 2579-2626,
2579-2627, 2579-2628, 2579-2629, 2579-2630, 2579-2631, 2580-2598,
2580-2599, 2580-2600, 2580-2601, 2580-2602, 2580-2603, 2580-2604,
2580-2605, 2580-2606, 2580-2607, 2580-2608, 2580-2609, 2580-2610,
2580-2611, 2580-2612, 2580-2613, 2580-2614, 2580-2615, 2580-2616,
2580-2617, 2580-2618, 2580-2619, 2580-2620, 2580-2621, 2580-2622,
2580-2623, 2580-2624, 2580-2625, 2580-2626, 2580-2627, 2580-2628,
2580-2629, 2580-2630, 2580-2631, 2581-2597, 2581-2598, 2581-2599,
2581-2600, 2581-2601, 2581-2602, 2581-2603, 2581-2604, 2581-2605,
2581-2606, 2581-2607, 2581-2608, 2581-2609, 2581-2610, 2581-2611,
2581-2612, 2581-2613, 2581-2614, 2581-2615, 2581-2616, 2581-2617,
2581-2618, 2581-2619, 2581-2620, 2581-2621, 2581-2622, 2581-2623,
2581-2624, 2581-2625, 2581-2626, 2581-2627, 2581-2628, 2581-2629,
2581-2630, 2581-2631, 2582-2600, 2582-2601, 2582-2602, 2582-2603,
2582-2604, 2582-2605, 2582-2606, 2582-2607, 2582-2608, 2582-2609,
2582-2610, 2582-2611, 2582-2612, 2582-2613, 2582-2614, 2582-2615,
2582-2616, 2582-2617, 2582-2618, 2582-2619, 2582-2620, 2582-2621,
2582-2622, 2582-2623, 2582-2624, 2582-2625, 2582-2626, 2582-2627,
2582-2628, 2582-2629, 2582-2630, 2582-2631, 2583-2601, 2583-2602,
2583-2603, 2583-2604, 2583-2605, 2583-2606, 2583-2607, 2583-2608,
2583-2609, 2583-2610, 2583-2611, 2583-2612, 2583-2613, 2583-2614,
2583-2615, 2583-2616, 2583-2617, 2583-2618, 2583-2619, 2583-2620,
2583-2621, 2583-2622, 2583-2623, 2583-2624, 2583-2625, 2583-2626,
2583-2627, 2583-2628, 2583-2629, 2583-2630, 2583-2631, 2585-2603,
2585-2604, 2585-2605, 2585-2606, 2585-2607, 2585-2608, 2585-2609,
2585-2610, 2585-2611, 2585-2612, 2585-2613, 2585-2614, 2585-2615,
2585-2616, 2585-2617, 2585-2618, 2585-2619, 2585-2620, 2585-2621,
2585-2622, 2585-2623, 2585-2624, 2585-2625, 2585-2626, 2585-2627,
2585-2628, 2585-2629, 2585-2630, 2585-2631, 2586-2604, 2586-2605,
2586-2606, 2586-2607, 2586-2608, 2586-2609, 2586-2610, 2586-2611,
2586-2612, 2586-2613, 2586-2614, 2586-2615, 2586-2616, 2586-2617,
2586-2618, 2586-2619, 2586-2620, 2586-2621, 2586-2622, 2586-2623,
2586-2624, 2586-2625, 2586-2626, 2586-2627, 2586-2628, 2586-2629,
2586-2630, 2586-2631, 2587-2605, 2587-2606, 2587-2607, 2587-2608,
2587-2609, 2587-2610, 2587-2611, 2587-2612, 2587-2613, 2587-2614,
2587-2615, 2587-2616, 2587-2617, 2587-2618, 2587-2619, 2587-2620,
2587-2621, 2587-2622, 2587-2623, 2587-2624, 2587-2625, 2587-2626,
2587-2627, 2587-2628, 2587-2629, 2587-2630, 2587-2631, 2588-2606,
2588-2607, 2588-2608, 2588-2609, 2588-2610, 2588-2611, 2588-2612,
2588-2613, 2588-2614, 2588-2615, 2588-2616, 2588-2617, 2588-2618,
2588-2619, 2588-2620, 2588-2621, 2588-2622, 2588-2623, 2588-2624,
2588-2625, 2588-2626, 2588-2627, 2588-2628, 2588-2629, 2588-2630,
2588-2631, 2589-2607, 2589-2608, 2589-2609, 2589-2610, 2589-2611,
2589-2612, 2589-2613, 2589-2614, 2589-2615, 2589-2616, 2589-2617,
2589-2618, 2589-2619, 2589-2620, 2589-2621, 2589-2622, 2589-2623,
2589-2624, 2589-2625, 2589-2626, 2589-2627, 2589-2628, 2589-2629,
2589-2630, 2589-2631, 2590-2606, 2590-2607, 2590-2608, 2590-2609,
2590-2610, 2590-2611, 2590-2612, 2590-2613, 2590-2614, 2590-2615,
2590-2616, 2590-2617, 2590-2618, 2590-2619, 2590-2620, 2590-2621,
2590-2622, 2590-2623, 2590-2624, 2590-2625, 2590-2626, 2590-2627,
2590-2628, 2590-2629, 2590-2630, 2590-2631, 2591-2610, 2591-2611,
2591-2612, 2591-2613, 2591-2614, 2591-2615, 2591-2616, 2591-2617,
2591-2618, 2591-2619, 2591-2620, 2591-2621, 2591-2622, 2591-2623,
2591-2624, 2591-2625, 2591-2626, 2591-2627, 2591-2628, 2591-2629,
2591-2630, 2591-2631, 2592-2611, 2592-2612, 2592-2613, 2592-2614,
2592-2615, 2592-2616, 2592-2617, 2592-2618, 2592-2619, 2592-2620,
2592-2621, 2592-2622, 2592-2623, 2592-2624, 2592-2625, 2592-2626,
2592-2627, 2592-2628, 2592-2629, 2592-2630, 2592-2631, 2593-2608,
2593-2612, 2593-2613, 2593-2614, 2593-2615, 2593-2616, 2593-2617,
2593-2618, 2593-2619, 2593-2620, 2593-2621, 2593-2622, 2593-2623,
2593-2624, 2593-2625, 2593-2626, 2593-2627, 2593-2628, 2593-2629,
2593-2630, 2593-2631, 2594-2612, 2594-2613, 2594-2614, 2594-2615,
2594-2616, 2594-2617, 2594-2618, 2594-2619, 2594-2620, 2594-2621,
2594-2622, 2594-2623, 2594-2624, 2594-2625, 2594-2626, 2594-2627,
2594-2628, 2594-2629, 2594-2630, 2594-2631, 2595-2611, 2595-2612,
2595-2613, 2595-2614, 2595-2615, 2595-2616, 2595-2617, 2595-2618,
2595-2619, 2595-2620, 2595-2621, 2595-2622, 2595-2623, 2595-2624,
2595-2625, 2595-2626, 2595-2627, 2595-2628, 2595-2629, 2595-2630,
2595-2631, 2596-2614, 2596-2615, 2596-2616, 2596-2617, 2596-2618,
2596-2619, 2596-2620, 2596-2621, 2596-2622, 2596-2623, 2596-2624,
2596-2625, 2596-2626, 2596-2627, 2596-2628, 2596-2629, 2596-2630,
2596-2631, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616,
2597-2617, 2597-2618, 2597-2619, 2597-2620, 2597-2621, 2597-2622,
2597-2623, 2597-2624, 2597-2625, 2597-2626, 2597-2627, 2597-2628,
2597-2629, 2597-2630, 2597-2631, 2598-2613, 2598-2614, 2598-2615,
2598-2616, 2598-2617, 2598-2618, 2598-2619, 2598-2620, 2598-2621,
2598-2622, 2598-2623, 2598-2624, 2598-2625, 2598-2626, 2598-2627,
2598-2628, 2598-2629, 2598-2630, 2598-2631, 2599-2614, 2599-2615,
2599-2616, 2599-2617, 2599-2618, 2599-2619, 2599-2620, 2599-2621,
2599-2622, 2599-2623, 2599-2624, 2599-2625, 2599-2626, 2599-2627,
2599-2628, 2599-2629, 2599-2630, 2599-2631, 2600-2615, 2600-2616,
2600-2617, 2600-2618, 2600-2619, 2600-2620, 2600-2621, 2600-2622,
2600-2623, 2600-2624, 2600-2625, 2600-2626, 2600-2627, 2600-2628,
2600-2629, 2600-2630, 2600-2631, 2601-2616, 2601-2617, 2601-2618,
2601-2619, 2601-2620, 2601-2621, 2601-2622, 2601-2623, 2601-2624,
2601-2625, 2601-2626, 2601-2627, 2601-2628, 2601-2629, 2601-2630,
2601-2631, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2602-2622,
2602-2623, 2602-2624, 2602-2625, 2602-2626, 2602-2627, 2602-2628,
2602-2629, 2602-2630, 2602-2631, 2603-2620, 2603-2621, 2603-2622,
2603-2623, 2603-2624, 2603-2625, 2603-2626, 2603-2627, 2603-2628,
2603-2629, 2603-2630, 2603-2631, 2604-2619, 2604-2620, 2604-2621,
2604-2622, 2604-2623, 2604-2624, 2604-2625, 2604-2626, 2604-2627,
2604-2628, 2604-2629, 2604-2630, 2604-2631, 2605-2620, 2605-2621,
2605-2622, 2605-2623, 2605-2624, 2605-2625, 2605-2626, 2605-2627,
2605-2628, 2605-2629, 2605-2630, 2605-2631, 2606-2621, 2606-2622,
2606-2623, 2606-2624, 2606-2625, 2606-2626, 2606-2627, 2606-2628,
2606-2629, 2606-2630, 2606-2631, 2607-2622, 2607-2623, 2607-2624,
2607-2625, 2607-2626, 2607-2627, 2607-2628, 2607-2629, 2607-2630,
2607-2631, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627,
2608-2628, 2608-2629, 2608-2630, 2608-2631, 2609-2624, 2609-2625,
2609-2626, 2609-2627, 2609-2628, 2609-2629, 2609-2630, 2609-2631,
2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2610-2630,
2610-2631, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630,
2611-2631, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631,
2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630,
2614-2631, 2615-2630, 2615-2631, or 2616-2631. In certain aspects,
antisense compounds or oligonucleotides target at least an 8, 9,
10, 11, 12, 13, 14, 15, or 16 contiguous nucleobases within the
aforementioned nucleobase regions.
[0179] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, display at least 50% inhibition: 30-49, 48-63,
150-169, 151-170, 152-171, 154-169, 154-173, 156-171, 156-175,
157-176, 158-173, 158-177, 480-499, 600-619, 638-657, 644-663,
738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166, 1150-1169,
1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186, 1171-1190,
1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196, 1183-1202,
1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329, 1316-1335,
1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374, 1393-1412,
1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640, 1646-1665,
1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782, 1912-1931,
2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208, 2191-2210,
2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212, 2197-2216,
2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245, 2227-2242,
2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380, 2388-2407,
2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476,
2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551, 2550-2569,
2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571, 2553-2568,
2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573,
2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575,
2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576,
2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578,
2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580, 2562-2577,
2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581,
2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584,
2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588,
2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588, 2571-2590,
2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2590,
2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592, 2575-2594,
2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594,
2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598,
2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598,
2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601,
2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603, 2585-2601,
2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604, 2586-2605,
2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604,
2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605, 2589-2606,
2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608,
2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609, 2591-2610,
2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611, 2593-2608,
2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610, 2594-2611,
2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613,
2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615,
2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616,
2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614,
2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616,
2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618,
2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620,
2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622,
2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620,
2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622,
2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624,
2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626,
2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628,
2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626,
2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628,
2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630,
2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631,
and 2616-2631.
[0180] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 2, when targeted by antisense compounds or
oligonucleotides, display at least 50% inhibition: 1608-1627,
1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890, 1872-1891,
1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1878-1897,
1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865, 2852-2871,
2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850, 3834-3853,
3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172, 4159-4178,
4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634, 4621-4640,
4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711, 4698-4717,
4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849, 5890-5909,
5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973, 6960-6979,
6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6984-7003,
6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144, 7151-7170,
7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709, 7692-7707,
7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715, 7767-7786,
7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806, 7788-7803,
7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808,
7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810,
7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811,
7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813,
7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815, 7797-7812,
7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816,
7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819,
7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823,
7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823, 7806-7825,
7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7825,
7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827, 7810-7829,
7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829,
7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833,
7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833,
7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836,
7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838, 7820-7836,
7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839, 7821-7840,
7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839,
7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839, 7824-7840,
7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841,
7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843, 7826-7844,
7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845, 7827-7846,
7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844, 7829-7845,
7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847,
7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849,
7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851,
7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849,
7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851,
7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853,
7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855,
7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857,
7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855,
7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857,
7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859,
7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861,
7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860,
7845-7861, 7845-7862, 7846-7861, and 7846-7862.
[0181] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, display at least 60% inhibition: 48-63, 150-169,
152-171, 154-169, 154-173, 156-171, 156-175, 158-173, 158-177,
600-619, 1135-1154, 1141-1160, 1147-1166, 1153-1172, 1171-1186,
1173-1188, 1175-1190, 1749-1768, 1763-1782, 1763-1782, 1912-1931,
2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2197-2212,
2197-2216, 2223-2238, 2225-2240, 2227-2242, 2238-2257, 2448-2467,
2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478,
2461-2476, 2461-2480, 2550-2569, 2551-2566, 2552-2571, 2553-2568,
2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573,
2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2574,
2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576,
2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2578,
2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578,
2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583,
2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585,
2568-2587, 2569-2586, 2569-2588, 2570-2587, 2570-2589, 2571-2588,
2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2591, 2574-2593,
2575-2590, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594,
2577-2595, 2577-2596, 2578-2594, 2578-2597, 2579-2598, 2580-2596,
2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599,
2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599,
2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2602, 2584-2603,
2585-2601, 2585-2603, 2585-2604, 2586-2602, 2586-2604, 2586-2605,
2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2606,
2588-2607, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605,
2590-2606, 2590-2607, 2590-2608, 2590-2609, 2591-2607, 2591-2609,
2591-2610, 2592-2608, 2592-2609, 2592-2611, 2593-2608, 2593-2609,
2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613,
2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611,
2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2613,
2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615,
2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617,
2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619,
2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617,
2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619,
2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621,
2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623,
2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625,
2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623,
2608-2624, 2608-2625, 2608-2625, 2608-2626, 2608-2627, 2609-2624,
2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626,
2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2626, 2611-2627,
2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629,
2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631,
2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2630, 2615-2631,
2615-2631, and 2616-2631.
[0182] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 2, when targeted by antisense compounds or
oligonucleotides, display at least 60% inhibition: 1685-1704,
1686-1705, 1769-1784, 1871-1890, 1873-1892, 1875-1890, 1875-1894,
1877-1892, 1877-1896, 1879-1894, 1879-1898, 2808-2827, 3819-3838,
3825-3844, 3831-3850, 3837-3856, 4151-4166, 5890-5909, 5904-5923,
5904-5923, 6406-6425, 6977-6996, 6979-6998, 6981-7000, 6983-6998,
6983-7002, 6985-7000, 6985-7004, 7122-7141, 7683-7702, 7688-7707,
7690-7709, 7692-7707, 7692-7711, 7694-7709, 7696-7711, 7696-7715,
7786-7801, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807,
7789-7806, 7789-7807, 7789-7808, 7790-7807, 7790-7809, 7791-7808,
7791-7809, 7791-7810, 7792-7809, 7792-7810, 7792-7811, 7793-7810,
7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812,
7795-7813, 7795-7814, 7796-7813, 7796-7814, 7796-7815, 7797-7812,
7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816,
7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819,
7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823,
7805-7822, 7805-7824, 7806-7823, 7806-7825, 7807-7824, 7807-7825,
7807-7826, 7808-7825, 7808-7827, 7809-7826, 7809-7828, 7810-7825,
7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830,
7812-7831, 7813-7829, 7813-7832, 7814-7833, 7815-7831, 7815-7832,
7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835,
7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835,
7818-7836, 7818-7837, 7819-7835, 7819-7837, 7819-7838, 7820-7836,
7820-7838, 7820-7839, 7821-7837, 7821-7839, 7821-7840, 7822-7838,
7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7841, 7823-7842,
7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841,
7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7844, 7826-7845,
7827-7843, 7827-7844, 7827-7846, 7828-7843, 7828-7844, 7828-7847,
7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845,
7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847,
7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849,
7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851,
7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853,
7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851,
7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853,
7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855,
7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857,
7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859,
7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857,
7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859,
7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861,
7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, 7846-7862,
and 7847-7862.
[0183] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, display at least 70% inhibition: 48-63, 150-169,
152-171, 154-169, 154-173, 156-171, 156-175, 158-173, 158-177,
1135-1154, 1141-1160, 1147-1166, 1171-1186, 1173-1188, 1175-1190,
1749-1768, 1763-1782, 1912-1931, 2193-2212, 2195-2210, 2195-2214,
2197-2212, 2197-2216, 2223-2238, 2225-2240, 2227-2242, 2453-2472,
2455-2474, 2457-2472, 2457-2476, 2459-2474, 2461-2476, 2461-2480,
2550-2569, 2551-2566, 2552-2571, 2553-2570, 2553-2571, 2553-2572,
2554-2571, 2554-2572, 2554-2573, 2554-2573, 2555-2572, 2555-2574,
2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2574, 2557-2576,
2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578,
2560-2577, 2560-2578, 2560-2579, 2561-2578, 2561-2579, 2561-2580,
2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582,
2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582,
2567-2584, 2567-2586, 2568-2585, 2568-2587, 2569-2586, 2569-2588,
2570-2587, 2570-2589, 2571-2588, 2571-2590, 2572-2589, 2572-2591,
2573-2590, 2573-2592, 2574-2591, 2574-2593, 2575-2592, 2575-2594,
2576-2593, 2576-2595, 2577-2594, 2577-2596, 2578-2597, 2579-2598,
2580-2596, 2580-2598, 2580-2599, 2581-2597, 2581-2600, 2582-2598,
2582-2600, 2582-2601, 2583-2599, 2583-2601, 2583-2602, 2584-2600,
2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2605,
2587-2606, 2588-2604, 2588-2606, 2588-2607, 2589-2605, 2589-2606,
2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2609,
2591-2607, 2591-2610, 2592-2611, 2593-2608, 2593-2612, 2594-2609,
2594-2610, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612,
2595-2613, 2595-2614, 2596-2611, 2596-2614, 2596-2615, 2597-2612,
2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614,
2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616,
2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618,
2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620,
2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2619,
2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621,
2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623,
2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625,
2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623,
2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625,
2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627,
2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2629, 2611-2630,
2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628,
2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631,
2615-2630, 2615-2630, 2615-2631, and 2616-2631.
[0184] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 2, when targeted by antisense compounds or
oligonucleotides, display at least 70% inhibition: 1685-1704,
1686-1705, 1769-1784, 1871-1890, 1873-1892, 1875-1890, 1875-1894,
1877-1892, 1877-1896, 1879-1894, 1879-1898, 3819-3838, 3825-3844,
3831-3850, 4151-4166, 5890-5909, 5904-5923, 5904-5923, 6406-6425,
6983-6998, 6983-7002, 6985-7000, 6985-7004, 7688-7707, 7690-7709,
7692-7707, 7692-7711, 7694-7709, 7696-7711, 7696-7715, 7786-7801,
7787-7806, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807,
7789-7808, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810,
7792-7809, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811,
7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7813,
7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813,
7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818,
7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7820, 7803-7822,
7804-7821, 7804-7823, 7805-7822, 7805-7824, 7806-7823, 7806-7825,
7807-7824, 7807-7826, 7808-7825, 7808-7827, 7809-7826, 7809-7828,
7810-7827, 7811-7828, 7811-7830, 7812-7829, 7812-7831, 7813-7832,
7814-7833, 7815-7831, 7815-7833, 7815-7834, 7816-7832, 7816-7835,
7817-7833, 7817-7835, 7817-7836, 7818-7834, 7818-7836, 7818-7837,
7819-7835, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839,
7821-7840, 7822-7841, 7823-7839, 7823-7841, 7823-7842, 7824-7840,
7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842,
7825-7844, 7826-7842, 7826-7845, 7827-7846, 7828-7843, 7828-7847,
7829-7844, 7829-7845, 7829-7847, 7829-7848, 7830-7845, 7830-7846,
7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7849, 7831-7850,
7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848,
7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850,
7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852,
7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854,
7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856,
7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855,
7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857,
7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859,
7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861,
7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859,
7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862,
7846-7861, 7846-7862, and 7847-7862.
[0185] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, display at least 80% inhibition: 152-171,
154-169, 156-171, 158-173, 1135-1154, 1171-1186, 1173-1188,
1175-1190, 1763-1782, 1912-1931, 2197-2212, 2223-2238, 2225-2240,
2227-2242, 2457-2472, 2459-2474, 2461-2476, 2551-2566, 2553-2570,
2553-2571, 2553-2572, 2554-2573, 2555-2572, 2555-2574, 2556-2573,
2556-2574, 2556-2575, 2557-2574, 2557-2576, 2558-2575, 2558-2576,
2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2578,
2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2580,
2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2567-2584,
2567-2586, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2587,
2571-2588, 2571-2590, 2572-2589, 2572-2591, 2573-2590, 2573-2592,
2574-2591, 2574-2593, 2575-2592, 2576-2593, 2576-2595, 2577-2594,
2577-2596, 2578-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2600,
2582-2601, 2583-2602, 2584-2603, 2585-2604, 2586-2605, 2587-2606,
2588-2607, 2589-2608, 2590-2606, 2590-2607, 2590-2609, 2591-2610,
2592-2611, 2593-2608, 2593-2612, 2594-2613, 2595-2611, 2595-2614,
2596-2615, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616,
2598-2613, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617,
2599-2614, 2599-2617, 2599-2618, 2600-2615, 2600-2617, 2600-2618,
2600-2619, 2601-2616, 2601-2617, 2601-2619, 2601-2620, 2602-2618,
2602-2621, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620,
2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622,
2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624,
2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626,
2608-2623, 2608-2624, 2608-2625, 2608-2627, 2609-2624, 2609-2626,
2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2628, 2610-2629,
2611-2626, 2611-2627, 2611-2629, 2611-2630, 2612-2627, 2612-2628,
2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2631, 2614-2629,
2614-2630, 2614-2631, 2615-2630, and 2616-2631.
[0186] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 2, when targeted by antisense compounds or
oligonucleotides, display at least 80% inhibition: 1685-1704,
1686-1705, 1873-1892, 1875-1890, 1877-1892, 1879-1894, 3819-3838,
4151-4166, 5904-5923, 6406-6425, 6985-7000, 7692-7707, 7694-7709,
7696-7711, 7786-7801, 7788-7805, 7788-7806, 7788-7807, 7789-7808,
7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7809,
7792-7811, 7793-7810, 7793-7811, 7794-7812, 7794-7813, 7795-7812,
7795-7813, 7795-7814, 7796-7813, 7796-7814, 7796-7815, 7797-7812,
7797-7814, 7797-7816, 7798-7815, 7798-7817, 7799-7816, 7799-7818,
7800-7819, 7801-7818, 7802-7819, 7802-7821, 7803-7820, 7803-7822,
7804-7821, 7804-7823, 7805-7822, 7806-7823, 7806-7825, 7807-7824,
7807-7826, 7808-7825, 7808-7827, 7809-7826, 7809-7828, 7810-7827,
7811-7828, 7812-7829, 7812-7831, 7813-7832, 7814-7833, 7815-7834,
7816-7832, 7816-7835, 7817-7836, 7818-7837, 7819-7838, 7820-7839,
7821-7840, 7822-7841, 7823-7842, 7824-7843, 7825-7841, 7825-7842,
7825-7844, 7826-7845, 7827-7846, 7828-7843, 7828-7847, 7829-7848,
7830-7846, 7830-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849,
7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851,
7833-7852, 7834-7849, 7834-7852, 7834-7853, 7835-7850, 7835-7852,
7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7854, 7836-7855,
7837-7853, 7837-7856, 7838-7855, 7838-7856, 7838-7857, 7839-7854,
7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856,
7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858,
7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860,
7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7862, 7844-7859,
7844-7861, 7844-7862, 7845-7860, 7845-7861, 7846-7862, and
7847-7862.
[0187] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, display at least 90% inhibition: 154-169,
156-171, 158-173, 1135-1154, 1171-1186, 1173-1188, 1763-1782,
1912-1931, 2223-2238, 2227-2242, 2459-2474, 2461-2476, 2554-2573,
2555-2574, 2560-2577, 2561-2578, 2561-2579, 2562-2581, 2563-2580,
2563-2582, 2564-2581, 2566-2583, 2567-2584, 2568-2585, 2568-2587,
2569-2586, 2570-2587, 2576-2593, 2577-2594, 2577-2596, 2578-2597,
2580-2599, 2581-2600, 2582-2601, 2583-2602, 2584-2603, 2586-2605,
2587-2605, 2587-2606, 2588-2607, 2589-2608, 2590-2607, 2590-2609,
2592-2611, 2595-2614, 2596-2615, 2597-2612, 2597-2613, 2597-2615,
2597-2616, 2598-2613, 2598-2613, 2598-2617, 2599-2614, 2599-2618,
2600-2615, 2600-2619, 2601-2617, 2601-2620, 2602-2621, 2603-2622,
2604-2623, 2605-2621, 2605-2622, 2605-2624, 2606-2625, 2607-2626,
2608-2623, 2608-2625, 2609-2628, 2611-2627, 2611-2630, 2612-2628,
2612-2631, 2613-2629, 2614-2629, 2615-2630, and 2616-2631.
[0188] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 2, when targeted by antisense compounds or
oligonucleotides, display at least 90% inhibition: 1685-1704,
1686-1705, 1875-1890, 1877-1892, 1879-1894, 3819-3838, 5904-5923,
6406-6425, 7694-7709, 7696-7711, 7789-7808, 7790-7809, 7795-7812,
7795-7813, 7796-7813, 7796-7814, 7797-7814, 7797-7816, 7798-7815,
7798-7817, 7799-7816, 7801-7818, 7802-7819, 7803-7820, 7803-7822,
7804-7821, 7805-7822, 7811-7828, 7812-7829, 7812-7831, 7813-7832,
7815-7834, 7818-7837, 7819-7838, 7821-7840, 7822-7840, 7822-7841,
7825-7842, 7832-7847, 7832-7848, 7832-7850, 7833-7848, 7833-7852,
7834-7849, 7834-7853, 7835-7850, 7836-7852, 7836-7855, 7837-7856,
7838-7856, 7839-7857, 7839-7858, 7840-7856, 7840-7857, 7840-7859,
7843-7858, 7843-7860, and 7846-7862.
[0189] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least a 50% inhibition of a CFB mRNA, ISIS NOs: 516350, 532614,
532632, 532635, 532638, 532639, 532686, 532687, 532688, 532689,
532690, 532691, 532692, 532692, 532693, 532694, 532695, 532696,
532697, 532698, 532699, 532700, 532701, 532702, 532703, 532704,
532705, 532706, 532707, 532770, 532775, 532778, 532780, 532791,
532800, 532809, 532810, 532811, 532917, 532952, 588509, 588510,
588511, 588512, 588513, 588514, 588515, 588516, 588517, 588518,
588519, 588520, 588522, 588523, 588524, 588525, 588527, 588528,
588529, 588530, 588531, 588532, 588533, 588534, 588535, 588536,
588537, 588538, 588539, 588540, 588541, 588542, 588543, 588544,
588545, 588546, 588547, 588548, 588549, 588550, 588551, 588552,
588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560,
588561, 588562, 588563, 588564, 588565, 588566, 588567, 588568,
588569, 588570, 588571, 588572, 588573, 588574, 588575, 588576,
588577, 588580, 588581, 588585, 588586, 588589, 588590, 588599,
588603, 588606, 588608, 588610, 588614, 588616, 588628, 588631,
588632, 588634, 588636, 588638, 588640, 588645, 588646, 588654,
588656, 588658, 588660, 588662, 588664, 588670, 588672, 588676,
588682, 588688, 588696, 588698, 588807, 588808, 588809, 588813,
588814, 588815, 588819, 588820, 588822, 588823, 588838, 588839,
588840, 588841, 588842, 588846, 588847, 588848, 588849, 588850,
588851, 588852, 588853, 588854, 588855, 588856, 588857, 588858,
588859, 588860, 588861, 588862, 588863, 588864, 588865, 588866,
588867, 588868, 588870, 588871, 588872, 588873, 588874, 588875,
588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883,
588884, 598999, 599000, 599001, 599002, 599003, 599004, 599005,
599006, 599007, 599008, 599009, 599010, 599011, 599012, 599013,
599014, 599015, 599018, 599019, 599023, 599024, 599025, 599026,
599027, 599028, 599029, 599030, 599031, 599032, 599033, 599034,
599035, 599058, 599062, 599063, 599064, 599065, 599070, 599071,
599072, 599073, 599074, 599076, 599077, 599078, 599079, 599080,
599081, 599082, 599083, 599084, 599085, 599086, 599087, 599088,
599089, 599090, 599091, 599092, 599093, 599094, 599095, 599096,
599097, 599098, 599102, 599119, 599123, 599124, 599125, 599126,
599127, 599128, 599132, 599133, 599134, 599135, 599136, 599137,
599138, 599139, 599140, 599141, 599142, 599143, 599144, 599145,
599147, 599148, 599149, 599150, 599151, 599152, 599153, 599154,
599155, 599156, 599157, 599158, 599159, 599178, 599179, 599180,
599181, 599182, 599186, 599187, 599188, 599189, 599190, 599191,
599192, 599193, 599194, 599195, 599196, 599197, 599198, 599199,
599200, 599201, 599202, 599203, 599204, 599205, 599206, 599207,
599208, 599209, 599210, 599211, 599212, 599213, 599214, 599215,
599216, 599217, 599218, 599219, 599220, 599221, 599221, 599222,
599223, 599224, 599225, 599226, 599227, 599228, 599229, 599230,
599231, 599232, 599233, 599234, 599235, 599236, 599241, 599247,
599248, 599249, 599255, 599256, 599257, 599258, 599260, 599261,
599262, 599263, 599264, 599265, 599266, 599267, 599268, 599269,
599270, 599271, 599272, 599273, 599274, 599275, 599276, 599277,
599278, 599279, 599280, 599297, 599299, 599306, 599307, 599308,
599309, 599311, 599312, 599313, 599314, 599315, 599316, 599317,
599318, 599319, 599320, 599321, 599322, 599323, 599324, 599325,
599326, 599327, 599328, 599329, 599330, 599338, 599349, 599353,
599354, 599355, 599356, 599357, 599358, 599359, 599360, 599361,
599362, 599363, 599364, 599369, 599371, 599372, 599373, 599376,
599378, 599379, 599382, 599383, 599384, 599385, 599386, 599387,
599388, 599389, 599390, 599391, 599392, 599393, 599394, 599395,
599396, 599397, 599398, 599399, 599400, 599401, 599402, 599403,
599404, 599405, 599406, 599407, 599408, 599409, 599410, 599412,
599413, 599414, 599415, 599416, 599417, 599418, 599419, 599420,
599421, 599422, 599423, 599424, 599425, 599426, 599433, 599434,
599435, 599436, 599437, 599438, 599439, 599440, 599441, 599442,
599443, 599444, 599445, 599446, 599447, 599448, 599450, 599454,
599455, 599456, 599467, 599468, 599469, 599471, 599472, 599473,
599474, 599475, 599476, 599477, 599478, 599479, 599480, 599481,
599482, 599483, 599484, 599485, 599486, 599487, 599488, 599489,
599490, 599491, 599492, 599493, 599494, 599495, 599496, 599497,
599498, 599499, 599500, 599501, 599502, 599503, 599504, 599505,
599506, 599507, 599508, 599509, 599512, 599515, 599518, 599531,
599541, 599541, 599546, 599547, 599548, 599549, 599550, 599552,
599553, 599554, 599555, 599557, 599558, 599561, 599562, 599563,
599564, 599565, 599566, 599567, 599568, 599569, 599570, 599577,
599578, 599579, 599580, 599581, 599581, 599582, 599584, 599585,
599586, 599587, 599588, 599589, 599590, 599591, 599592, 599593,
599594, 599595, 601321, 601322, 601323, 601325, 601327, 601328,
601329, 601330, 601332, 601333, 601334, 601335, 601336, 601337,
601338, 601339, 601341, 601342, 601343, 601344, 601345, 601346,
601347, 601348, 601349, 601362, 601367, 601368, 601369, 601371,
601372, 601373, 601374, 601375, 601377, 601378, 601380, 601381,
601382, 601383, 601384, 601385, 601386, 601387, and 601388.
[0190] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least a 50% inhibition of a CFB mRNA, SEQ ID NOs: 12, 30, 33,
36, 37, 84, 85, 86, 87, 88, 89, 90, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 198, 203, 206, 208, 219, 228,
237, 238, 239, 317, 395, 396, 397, 398, 399, 400, 401, 402, 403,
404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,
417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,
430, 431, 432, 433, 434, 434, 435, 436, 437, 438, 439, 440, 441,
442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,
455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 468, 472,
473, 475, 478, 479, 488, 492, 494, 495, 498, 499, 500, 502, 503,
509, 510, 511, 512, 513, 514, 515, 517, 518, 522, 523, 524, 525,
529, 530, 531, 534, 535, 537, 540, 541, 542, 543, 544, 545, 546,
547, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 563,
564, 565, 569, 570, 572, 573, 577, 588, 589, 590, 591, 592, 594,
595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607,
608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 623,
640, 641, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654,
655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667,
668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,
681, 682, 683, 684, 685, 686, 687, 688, 689, 700, 704, 705, 706,
707, 708, 709, 711, 712, 713, 714, 715, 716, 717, 718, 720, 721,
722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734,
735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 745, 746,
747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 758, 759, 760,
761, 762, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776,
777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789,
790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 813, 833, 834,
841, 846, 849, 850, 867, and 873.
[0191] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least a 60% inhibition of a CFB mRNA, ISIS NOs: 516350, 532614,
532635, 532686, 532687, 532688, 532689, 532770, 532800, 532809,
532810, 532811, 532917, 532952, 588512, 588513, 588514, 588515,
588516, 588517, 588518, 588519, 588522, 588523, 588524, 588525,
588527, 588528, 588529, 588530, 588531, 588532, 588533, 588534,
588535, 588536, 588537, 588538, 588539, 588540, 588541, 588542,
588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550,
588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558,
588559, 588560, 588561, 588562, 588563, 588564, 588565, 588566,
588567, 588568, 588569, 588570, 588571, 588572, 588573, 588574,
588575, 588576, 588577, 588636, 588638, 588640, 588664, 588676,
588696, 588698, 588807, 588808, 588814, 588815, 588819, 588820,
588840, 588842, 588846, 588847, 588848, 588849, 588850, 588851,
588852, 588853, 588854, 588855, 588856, 588857, 588858, 588859,
588860, 588861, 588862, 588863, 588864, 588866, 588867, 588868,
588870, 588871, 588872, 588873, 588874, 588875, 588876, 588877,
588878, 588879, 588880, 588881, 588882, 588883, 588884, 598999,
599000, 599001, 599002, 599003, 599004, 599005, 599006, 599007,
599008, 599009, 599010, 599011, 599012, 599013, 599014, 599015,
599019, 599024, 599025, 599026, 599027, 599028, 599029, 599030,
599031, 599032, 599033, 599034, 599035, 599064, 599065, 599071,
599072, 599077, 599078, 599079, 599080, 599083, 599084, 599085,
599086, 599087, 599088, 599089, 599090, 599091, 599092, 599093,
599094, 599095, 599096, 599097, 599125, 599126, 599127, 599133,
599134, 599135, 599136, 599138, 599139, 599140, 599141, 599142,
599148, 599149, 599150, 599151, 599152, 599154, 599155, 599156,
599157, 599158, 599159, 599178, 599179, 599180, 599181, 599187,
599188, 599190, 599192, 599193, 599194, 599195, 599196, 599197,
599198, 599199, 599200, 599201, 599202, 599203, 599204, 599205,
599206, 599207, 599208, 599209, 599210, 599211, 599212, 599213,
599214, 599215, 599216, 599217, 599218, 599219, 599220, 599221,
599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229,
599230, 599231, 599232, 599233, 599234, 599235, 599236, 599247,
599255, 599256, 599257, 599263, 599264, 599265, 599266, 599270,
599271, 599272, 599273, 599274, 599275, 599276, 599277, 599278,
599279, 599280, 599306, 599307, 599308, 599311, 599312, 599313,
599314, 599315, 599316, 599317, 599318, 599319, 599320, 599321,
599322, 599323, 599324, 599325, 599327, 599328, 599329, 599330,
599349, 599353, 599355, 599356, 599357, 599358, 599359, 599360,
599361, 599362, 599363, 599364, 599369, 599371, 599372, 599373,
599376, 599378, 599379, 599382, 599384, 599386, 599387, 599388,
599389, 599390, 599391, 599392, 599393, 599394, 599395, 599396,
599397, 599398, 599399, 599400, 599401, 599402, 599403, 599404,
599405, 599406, 599407, 599408, 599409, 599410, 599412, 599413,
599414, 599415, 599416, 599417, 599418, 599419, 599420, 599421,
599422, 599423, 599424, 599425, 599433, 599434, 599435, 599436,
599437, 599438, 599439, 599440, 599441, 599442, 599443, 599444,
599445, 599446, 599447, 599448, 599456, 599467, 599468, 599471,
599472, 599473, 599474, 599475, 599476, 599477, 599478, 599479,
599480, 599481, 599482, 599483, 599484, 599485, 599486, 599487,
599488, 599489, 599490, 599491, 599492, 599493, 599494, 599495,
599496, 599497, 599498, 599499, 599500, 599501, 599502, 599503,
599504, 599505, 599506, 599507, 599508, 599512, 599531, 599547,
599548, 599549, 599552, 599553, 599554, 599555, 599557, 599558,
599562, 599563, 599564, 599565, 599566, 599567, 599568, 599569,
599570, 599577, 599578, 599579, 599580, 599581, 599582, 599584,
599585, 599586, 599587, 599588, 599589, 599590, 599591, 599592,
599593, 599594, 599595, 601323, 601327, 601329, 601332, 601333,
601333, 601334, 601335, 601336, 601338, 601339, 601341, 601342,
601343, 601344, 601345, 601346, 601347, 601348, 601349, 601368,
601369, 601371, 601372, 601374, 601375, 601377, 601378, 601380,
601381, 601382, 601383, 601384, 601385, 601386, 601387, and
601388.
[0192] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least a 60% inhibition of a CFB mRNA, SEQ ID NOs: 12, 33, 84,
85, 86, 87, 198, 228, 237, 238, 239, 317, 395, 396, 397, 398, 399,
400, 401, 402, 403, 404, 405, 406, 407, 408, 410, 411, 412, 413,
414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,
427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439,
440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,
453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,
472, 473, 513, 514, 515, 531, 537, 541, 542, 543, 544, 545, 546,
547, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 564, 565,
569, 570, 577, 590, 592, 595, 596, 597, 598, 599, 600, 601, 602,
603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615,
616, 617, 618, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653,
654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666,
667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679,
680, 682, 683, 684, 685, 686, 687, 688, 689, 700, 704, 706, 707,
708, 709, 711, 712, 713, 714, 715, 716, 717, 720, 721, 722, 723,
724, 725, 726, 727, 727, 728, 729, 730, 731, 732, 733, 734, 736,
737, 738, 739, 740, 741, 742, 743, 744, 745, 745, 746, 747, 748,
749, 750, 751, 752, 753, 754, 755, 756, 758, 759, 760, 761, 767,
768, 770, 772, 773, 774, 775, 775, 776, 776, 777, 777, 778, 779,
780, 781, 782, 783, 783, 784, 784, 785, 786, 787, 788, 789, 790,
791, 792, 793, 794, 795, 796, 797, 798, 799, 813, 833, 834, 841,
846, 849, and 850.
[0193] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least a 70% inhibition of a CFB mRNA, ISIS NOs: 516350, 532614,
532686, 532687, 532688, 532770, 532800, 532809, 532810, 532811,
532917, 532952, 588512, 588513, 588514, 588515, 588516, 588517,
588518, 588524, 588529, 588530, 588531, 588532, 588533, 588534,
588535, 588536, 588537, 588538, 588539, 588540, 588541, 588542,
588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550,
588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558,
588559, 588560, 588561, 588562, 588563, 588564, 588565, 588568,
588569, 588570, 588571, 588572, 588573, 588574, 588575, 588577,
588636, 588638, 588640, 588696, 588698, 588807, 588814, 588815,
588819, 588842, 588847, 588848, 588849, 588850, 588851, 588852,
588853, 588856, 588857, 588858, 588859, 588860, 588861, 588862,
588863, 588866, 588867, 588870, 588871, 588872, 588873, 588874,
588875, 588876, 588877, 588878, 588879, 588880, 588881, 588882,
588883, 588884, 599000, 599001, 599003, 599004, 599005, 599008,
599009, 599010, 599011, 599014, 599015, 599024, 599025, 599027,
599028, 599029, 599030, 599031, 599032, 599033, 599034, 599072,
599077, 599080, 599085, 599086, 599087, 599088, 599089, 599090,
599091, 599093, 599094, 599095, 599096, 599097, 599125, 599126,
599134, 599138, 599139, 599148, 599149, 599150, 599151, 599152,
599154, 599155, 599156, 599157, 599158, 599187, 599188, 599193,
599195, 599196, 599197, 599198, 599199, 599200, 599201, 599202,
599203, 599204, 599205, 599206, 599207, 599208, 599210, 599211,
599212, 599213, 599214, 599215, 599216, 599217, 599218, 599219,
599220, 599221, 599222, 599223, 599224, 599225, 599226, 599227,
599228, 599229, 599230, 599231, 599232, 599233, 599234, 599235,
599236, 599266, 599272, 599272, 599273, 599274, 599275, 599277,
599278, 599279, 599280, 599280, 599306, 599311, 599312, 599313,
599314, 599315, 599316, 599317, 599318, 599319, 599320, 599321,
599322, 599323, 599325, 599327, 599328, 599329, 599330, 599355,
599357, 599358, 599359, 599360, 599361, 599362, 599363, 599364,
599369, 599371, 599372, 599373, 599378, 599379, 599382, 599384,
599386, 599387, 599388, 599389, 599390, 599391, 599392, 599393,
599394, 599395, 599396, 599397, 599398, 599399, 599400, 599401,
599402, 599403, 599404, 599405, 599406, 599407, 599408, 599409,
599410, 599413, 599414, 599415, 599416, 599417, 599418, 599419,
599420, 599421, 599422, 599423, 599424, 599433, 599434, 599435,
599436, 599437, 599438, 599439, 599440, 599441, 599442, 599443,
599445, 599446, 599447, 599448, 599472, 599473, 599474, 599475,
599476, 599477, 599478, 599479, 599480, 599481, 599482, 599483,
599484, 599485, 599486, 599487, 599488, 599489, 599490, 599491,
599492, 599493, 599494, 599495, 599496, 599497, 599498, 599499,
599500, 599501, 599502, 599503, 599504, 599505, 599506, 599507,
599508, 599512, 599547, 599548, 599552, 599553, 599554, 599555,
599558, 599562, 599563, 599564, 599566, 599567, 599568, 599569,
599570, 599577, 599578, 599579, 599580, 599581, 599582, 599585,
599586, 599587, 599588, 599589, 599590, 599591, 599592, 599593,
599594, 599595, 601332, 601335, 601341, 601343, 601344, 601345,
601346, 601347, 601348, 601349, 601371, 601372, 601380, 601382,
601383, 601384, 601385, 601386, and 601387.
[0194] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least a 70% inhibition of a CFB mRNA, SEQ ID NOs: 12, 84, 85,
86, 198, 228, 237, 238, 239, 317, 395, 396, 397, 398, 399, 402,
403, 404, 405, 407, 408, 410, 411, 412, 412, 413, 414, 415, 416,
417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,
430, 431, 432, 433, 433, 434, 435, 436, 437, 438, 439, 440, 441,
442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,
455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 464, 465, 472,
473, 513, 514, 515, 541, 542, 543, 544, 545, 546, 547, 549, 550,
551, 552, 553, 554, 555, 556, 557, 564, 565, 569, 592, 595, 596,
597, 598, 599, 600, 601, 602, 603, 604, 606, 607, 608, 609, 610,
611, 612, 613, 614, 615, 616, 617, 618, 645, 646, 647, 648, 649,
650, 653, 654, 655, 656, 659, 660, 662, 663, 664, 665, 666, 668,
669, 670, 671, 672, 673, 674, 675, 676, 677, 677, 678, 679, 680,
682, 683, 684, 686, 687, 688, 689, 706, 708, 709, 711, 712, 713,
714, 715, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730,
731, 732, 733, 734, 736, 737, 738, 739, 740, 741, 742, 743, 744,
745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 767,
768, 773, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785,
786, 787, 788, 789, 790, 791, 792, 793, 793, 794, 795, 797, 798,
799, 813, 833, 834, 841, 846, 849, 867, and 873.
[0195] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least an 80% inhibition of a CFB mRNA, ISIS NOs: 532686, 532809,
532810, 532811, 532917, 532952, 588512, 588517, 588518, 588533,
588534, 588535, 588536, 588537, 588538, 588539, 588540, 588542,
588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550,
588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558,
588559, 588560, 588561, 588562, 588563, 588564, 588565, 588571,
588638, 588640, 588696, 588698, 588807, 588814, 588849, 588850,
588851, 588853, 588857, 588858, 588859, 588860, 588861, 588862,
588863, 588866, 588867, 588871, 588872, 588873, 588874, 588875,
588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883,
599001, 599024, 599025, 599033, 599086, 599087, 599088, 599089,
599093, 599094, 599095, 599096, 599134, 599139, 599148, 599149,
599151, 599154, 599155, 599156, 599158, 599188, 599195, 599196,
599198, 599201, 599202, 599203, 599204, 599205, 599206, 599207,
599212, 599213, 599215, 599216, 599217, 599218, 599219, 599220,
599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228,
599229, 599230, 599231, 599232, 599233, 599234, 599235, 599236,
599272, 599273, 599275, 599277, 599278, 599279, 599280, 599311,
599313, 599314, 599316, 599317, 599318, 599320, 599321, 599322,
599323, 599327, 599328, 599329, 599330, 599355, 599357, 599358,
599359, 599360, 599361, 599362, 599363, 599364, 599371, 599372,
599373, 599378, 599379, 599382, 599384, 599386, 599387, 599388,
599389, 599390, 599391, 599392, 599393, 599397, 599398, 599399,
599400, 599401, 599403, 599404, 599405, 599407, 599408, 599409,
599410, 599413, 599414, 599415, 599416, 599417, 599418, 599419,
599420, 599421, 599422, 599423, 599424, 599433, 599434, 599435,
599436, 599437, 599438, 599439, 599440, 599441, 599445, 599446,
599447, 599448, 599474, 599476, 599477, 599479, 599481, 599482,
599483, 599485, 599486, 599487, 599488, 599489, 599490, 599491,
599492, 599494, 599495, 599496, 599497, 599498, 599499, 599500,
599502, 599503, 599504, 599505, 599506, 599507, 599508, 599547,
599552, 599553, 599554, 599558, 599563, 599567, 599568, 599569,
599570, 599577, 599578, 599581, 599582, 599585, 599587, 599588,
599590, 599591, 599592, 599593, 599594, 601332, 601344, 601345,
601382, 601383, and 601385.
[0196] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least a 80% inhibition of a CFB mRNA, SEQ ID NOs: 84, 237, 238,
239, 317, 395, 397, 411, 412, 413, 414, 415, 417, 418, 419, 420,
421, 422, 423, 425, 426, 427, 429, 430, 431, 433, 434, 435, 436,
437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,
450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,
463, 464, 465, 472, 473, 514, 515, 542, 543, 544, 545, 546, 547,
550, 551, 552, 553, 554, 555, 556, 557, 564, 595, 599, 600, 601,
602, 603, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616,
617, 618, 646, 655, 660, 662, 663, 666, 669, 670, 671, 672, 673,
675, 676, 677, 678, 679, 682, 684, 686, 687, 688, 689, 706, 708,
709, 711, 712, 713, 714, 715, 720, 722, 723, 724, 725, 726, 727,
729, 730, 731, 732, 733, 736, 737, 738, 739, 740, 741, 742, 743,
744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756,
768, 775, 776, 778, 781, 782, 783, 784, 785, 787, 788, 789, 790,
791, 792, 793, 794, 799, 813, 833, 834, 841, 849, 867, and 873.
[0197] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least a 90% inhibition of a CFB mRNA, ISIS NOs: 532686, 532811,
532917, 588536, 588537, 588538, 588539, 588544, 588545, 588546,
588548, 588551, 588552, 588553, 588554, 588555, 588556, 588557,
588558, 588559, 588560, 588561, 588562, 588564, 588638, 588640,
588696, 588698, 588849, 588850, 588851, 588860, 588866, 588867,
588872, 588873, 588874, 588876, 588877, 588878, 588879, 588881,
588883, 599149, 599188, 599203, 599206, 599220, 599221, 599222,
599223, 599224, 599225, 599226, 599227, 599228, 599229, 599235,
599236, 599279, 599280, 599314, 599321, 599362, 599378, 599390,
599391, 599398, 599399, 599404, 599413, 599414, 599416, 599419,
599420, 599422, 599435, 599437, 599438, 599441, 599483, 599494,
599508, 599552, 599553, 599554, 599568, 599570, 599577, 599581,
599591, 599592, and 599593.
[0198] In certain embodiments, the following antisense compounds or
oligonucleotides target a region of a CFB nucleic acid and effect
at least a 90% inhibition of a CFB mRNA, SEQ ID NOs: 84, 238, 239,
317, 412, 413, 420, 421, 426, 434, 436, 437, 438, 439, 440, 442,
443, 444, 445, 446, 448, 451, 452, 453, 454, 455, 456, 457, 458,
459, 460, 461, 462, 464, 465, 472, 473, 514, 515, 542, 543, 544,
545, 546, 551, 553, 555, 556, 599, 600, 601, 602, 610, 616, 617,
618, 662, 666, 670, 676, 677, 678, 688, 689, 713, 723, 729, 730,
740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 755, 756, 768,
783, 793, 833, and 867.
[0199] In certain embodiments, a compound can comprise or consist
of any oligonucleotide targeted to CFB described herein and a
conjugate group.
[0200] In certain embodiments, a compound comprises a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides
complementary within nucleotides 2193-2212, 2195-2210, 2457-2476,
2571-2590, 2584-2603, 2588-2607, 2592-2611, 2594-2613, 2597-2616,
2600-2619, or 2596-2611 of SEQ ID NO: 1.
[0201] In certain embodiments, a compound comprises a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides having a
nucleobase sequence comprising any one of SEQ ID NOs: 198, 228,
237, 440, 444, 448, 450, 453, 455, 549, and 598.
[0202] In certain embodiments, a compound comprises a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide has a nucleobase sequence consisting of any one of
SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and
598.
[0203] In certain embodiments, any of the foregoing compounds or
oligonucleotides can comprise at least one modified internucleoside
linkage, at least one modified sugar, and/or at least one modified
nucleobase.
[0204] In certain aspects, any of the foregoing compounds or
oligonucleotides can comprise at least one modified sugar. In
certain aspects, at least one modified sugar comprises a
2'-O-methoxyethyl group. In certain aspects, at least one modified
sugar is a bicyclic sugar, such as a 4'-CH(CH.sub.3)--O-2' group, a
4'-CH.sub.2--O-2' group, or a 4'-(CH.sub.2).sub.2--O-2' group.
[0205] In certain aspects, the modified oligonucleotide comprises
at least one modified internucleoside linkage, such as a
phosphorothioate internucleoside linkage.
[0206] In certain embodiments, the modified oligonucleotide
comprises at least 1, 2, 3, 4, 5, 6, or 7 phosphodiester
internucleoside linkages.
[0207] In certain embodiments, each internucleoside linkage of the
modified oligonucleotide is selected from a phosphodiester
internucleoside linkage and a phosphorothioate internucleoside
linkage.
[0208] In certain embodiments, each internucleoside linkage of the
modified oligonucleotide is a phosphorothioate linkage.
[0209] In certain embodiments, any of the foregoing compounds or
oligonucleotides comprises at least one modified nucleobase, such
as 5-methylcytosine.
[0210] In certain embodiments, a compound comprises a conjugate
group and a modified oligonucleotide comprising: [0211] a gap
segment consisting of linked deoxynucleosides; [0212] a 5' wing
segment consisting of linked nucleosides; and [0213] a 3' wing
segment consisting of linked nucleosides;
[0214] wherein the gap segment is positioned between the 5' wing
segment and the 3' wing segment and wherein each nucleoside of each
wing segment comprises a modified sugar. In certain embodiments,
the oligonucleotide consists of 10 to 30 linked nucleosides having
a nucleobase sequence comprising the sequence recited in SEQ ID NO:
198, 228, 237, 440, 444, 448, 450, 453, 455, 549, or 598.
[0215] In certain embodiments, the modified oligonucleotide has a
nucleobase sequence comprising or consisting of the sequence
recited in SEQ ID NO: 198, 228, 237, 440, 444, 448, 450, 453, or
455, wherein the modified oligonucleotide comprises:
[0216] a gap segment consisting of ten linked deoxynucleosides;
[0217] a 5' wing segment consisting of five linked nucleosides;
and
[0218] a 3' wing segment consisting of five linked nucleosides;
[0219] wherein the gap segment is positioned between the 5' wing
segment and the 3' wing segment, wherein each nucleoside of each
wing segment comprises a 2'-O-methoxyethyl sugar; wherein each
internucleoside linkage is a phosphorothioate linkage and wherein
each cytosine is a 5-methylcytosine.
[0220] In certain embodiments, a compound comprises or consists of
a single-stranded modified oligonucleotide and a conjugate group,
wherein the modified oligonucleotide consists of 20 linked
nucleosides having a nucleobase sequence consisting of the sequence
recited in SEQ ID NO: 198, 228, 237, 440, 444, 448, 450, 453, or
455, wherein the oligonucleotide comprises:
[0221] a gap segment consisting of ten linked deoxynucleosides;
[0222] a 5' wing segment consisting of five linked nucleosides;
and
[0223] a 3' wing segment consisting of five linked nucleosides;
[0224] wherein the gap segment is positioned between the 5' wing
segment and the 3' wing segment, wherein each nucleoside of each
wing segment comprises a 2'-O-methoxyethyl sugar; wherein each
internucleoside linkage is a phosphorothioate linkage; and wherein
each cytosine is a 5-methylcytosine.
[0225] In certain embodiments, a compound comprises or consists of
ISIS 588540 and a conjugate group. In certain embodiments, ISIS
588540 has the following chemical structure:
##STR00003##
[0226] In certain embodiments, the modified oligonucleotide has a
nucleobase sequence comprising or consisting of the sequence
recited in SEQ ID NO: 549, wherein the modified oligonucleotide
comprises
[0227] a gap segment consisting of ten linked deoxynucleosides;
[0228] a 5' wing segment consisting of three linked nucleosides;
and
[0229] a 3' wing segment consisting of three linked
nucleosides;
[0230] wherein the gap segment is positioned between the 5' wing
segment and the 3' wing segment; wherein each nucleoside of each
wing segment comprises a cEt sugar; wherein each internucleoside
linkage is a phosphorothioate linkage; and wherein each cytosine is
a 5-methylcytosine.
[0231] In certain aspects, the modified oligonucleotide has a
nucleobase sequence comprising or consisting of the sequence
recited in SEQ ID NO: 598, wherein the modified oligonucleotide
comprises
[0232] a gap segment consisting of ten linked deoxynucleosides;
[0233] a 5' wing segment consisting of three linked nucleosides;
and
[0234] a 3' wing segment consisting of three linked
nucleosides;
[0235] wherein the gap segment is positioned between the 5' wing
segment and the 3' wing segment; wherein the 5' wing segment
comprises a 2'-O-methoxyethyl sugar, 2'-O-methoxyethyl sugar, and
cEt sugar in the 5' to 3' direction; wherein the 3' wing segment
comprises a cEt sugar, cEt sugar, and 2'-O-methoxyethyl sugar in
the 5' to 3' direction; wherein each internucleoside linkage is a
phosphorothioate linkage; and wherein each cytosine is a
5-methylcytosine.
[0236] In any of the foregoing embodiments, the compound or
oligonucleotide can be at least 85%, at least 90%, at least 95%, at
least 98%, at least 99%, or 100% complementary to a nucleic acid
encoding CFB.
[0237] In any of the foregoing embodiments, the compound or
oligonucleotide can be single-stranded.
[0238] In certain embodiments, the conjugate group is linked to the
modified oligonucleotide at the 5' end of the modified
oligonucleotide. In certain embodiments, the conjugate group is
linked to the modified oligonucleotide at the 3' end of the
modified oligonucleotide. In certain embodiments, the conjugate
group comprises at least one N-Acetylgalactosamine (GalNAc), at
least two N-Acetylgalactosamines (GalNAcs), or at least three
N-Acetylgalactosamines (GalNAcs).
[0239] In certain embodiments, a compound having the following
chemical structure comprises or consists of ISIS 588540 with a
5'-X, wherein X is a conjugate group comprising GalNAc as described
herein:
##STR00004##
[0240] In certain embodiments, a compound comprises or consists of
SEQ ID NO: 440, 5'-GalNAc, and chemical modifications as
represented by the following chemical structure:
##STR00005##
wherein either R.sup.1 is --OCH.sub.2CH.sub.2OCH.sub.3 (MOE) and
R.sup.2 is H; or R.sup.1 and R.sup.2 together form a bridge,
wherein R.sup.1 is --O-- and R.sup.2 is --CH.sub.2--,
--CH(CH.sub.3)--, or --CH.sub.2CH.sub.2--, and R.sup.1 and R.sup.2
are directly connected such that the resulting bridge is selected
from: --O--CH.sub.2--, --O--CH(CH.sub.3)--, and
--O--CH.sub.2CH.sub.2--; And for each pair of R.sup.3 and R.sup.4
on the same ring, independently for each ring: either R.sup.3 is
selected from H and --OCH.sub.2CH.sub.2OCH.sub.3 and R.sup.4 is H;
or R.sup.3 and R.sup.4 together form a bridge, wherein R.sup.3 is
--O--, and R.sup.4 is --CH.sub.2--, --CH(CH.sub.3)--, or
--CH.sub.2CH.sub.2-- and R.sup.3 and R.sup.4 are directly connected
such that the resulting bridge is selected from: --O--CH.sub.2--,
--O--CH(CH.sub.3)--, and --O--CH.sub.2CH.sub.2--; And R.sup.5 is
selected from H and --CH.sub.3; And Z is selected from S.sup.- and
O.sup.-.
[0241] In certain embodiments, a compound comprises ISIS 696844. In
certain embodiments, a compound consists of ISIS 696844. In certain
embodiments, ISIS 696844 has the following chemical structure:
##STR00006##
[0242] In certain embodiments, a compound comprises ISIS 696845. In
certain embodiments, a compound consists of ISIS 696845. In certain
embodiments, ISIS 696845 has the following chemical structure:
##STR00007##
[0243] In certain embodiments, a compound comprises ISIS 698969. In
certain embodiments, a compound consists of ISIS 698969. In certain
embodiments, ISIS 698969 has the following chemical structure:
##STR00008##
[0244] In certain embodiments, a compound comprises ISIS 698970. In
certain embodiments, a compound consists of ISIS 698970. In certain
embodiments, ISIS 698970 has the following chemical structure:
##STR00009##
[0245] Certain embodiments provide compositions comprising any of
the compounds comprising or consisting of a modified
oligonucleotide targeted to CFB or salt thereof and a conjugate
group, and at least one of a pharmaceutically acceptable carrier or
diluent.
[0246] In certain embodiments, the compounds or compositions as
described herein are efficacious by virtue of having at least one
of an in vitro IC.sub.50 of less than 250 nM, less than 200 nM,
less than 150 nM, less than 100 nM, less than 90 nM, less than 80
nM, less than 70 nM, less than 65 nM, less than 60 nM, less than 55
nM, less than 50 nM, less than 45 nM, less than 40 nM, less than 35
nM, less than 30 nM, less than 25 nM, or less than 20 nM.
[0247] In certain embodiments, the compounds or compositions as
described herein are highly tolerable as demonstrated by having at
least one of an increase an ALT or AST value of no more than 4
fold, 3 fold, or 2 fold over saline treated animals or an increase
in liver, spleen, or kidney weight of no more than 30%, 20%, 15%,
12%, 10%, 5%, or 2%. In certain embodiments, the compounds or
compositions as described herein are highly tolerable as
demonstrated by having no increase of ALT or AST over saline
treated animals. In certain embodiments, the compounds or
compositions as described herein are highly tolerable as
demonstrated by having no increase in liver, spleen, or kidney
weight over saline treated animals.
[0248] Certain embodiments provide a composition comprising the
compound of any of the aforementioned embodiments or salt thereof
and at least one of a pharmaceutically acceptable carrier or
diluent. In certain aspects, the composition has a viscosity less
than about 40 centipoise (cP), less than about 30 centipose (cP),
less than about 20 centipose (cP), less than about 15 centipose
(cP), or less than about 10 centipose (cP). In certain aspects, the
composition having any of the aforementioned viscosities comprises
a compound provided herein at a concentration of about 100 mg/mL,
about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL,
about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, or about 300
mg/mL. In certain aspects, the composition having any of the
aforementioned viscosities and/or compound concentrations has a
temperature of room temperature or about 20.degree. C., about
21.degree. C., about 22.degree. C., about 23.degree. C., about
24.degree. C., about 25.degree. C., about 26.degree. C., about
27.degree. C., about 28.degree. C., about 29.degree. C., or about
30.degree. C.
[0249] In certain embodiments, a method of treating, preventing, or
ameliorating a disease associated with dysregulation of the
complement alternative pathway in a subject comprises administering
to the subject a compound or composition described herein, thereby
treating, preventing, or ameliorating the disease. In certain
aspects, the complement alternative pathway is activated greater
than normal. In certain embodiments, a method of treating,
preventing, or ameliorating a disease associated with dysregulation
of the complement alternative pathway in a subject comprises
administering to the subject a compound comprising or consisting of
a modified oligonucleotide and a conjugate group, wherein the
modified oligonucleotide consists of 10 to 30 linked nucleosides
and has a nucleobase sequence comprising the nucleobase sequence of
any one of SEQ ID NOs: 6-808. In certain embodiments, a method of
treating, preventing, or ameliorating a disease associated with
dysregulation of the complement alternative pathway in a subject
comprises administering to the subject a compound comprising or
consisting of a modified oligonucleotide and a conjugate group,
wherein the modified oligonucleotide consists of 10 to 30 linked
nucleosides having a nucleobase sequence comprising any one of SEQ
ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598.
In certain embodiments, a method of treating, preventing, or
ameliorating a disease associated with dysregulation of the
complement alternative pathway in a subject comprises administering
to the subject a compound comprising or consisting of ISIS 696844,
ISIS 696845, ISIS 698969, or ISIS 698970.
[0250] In certain embodiments, a method of treating, preventing, or
ameliorating macular degeneration, such as age-related macular
degeneration (AMD) in a subject comprises administering to the
subject a compound or composition described herein, thereby
treating, preventing, or ameliorating AMD. In certain aspects, the
complement alternative pathway is activated greater than normal. In
certain aspects, the AMD is wet AMD. In certain aspects, the AMD is
dry AMD, such as Geographic Atrophy. In certain embodiments, a
method of treating, preventing, or ameliorating macular
degeneration in a subject, such as age-related macular degeneration
(AMD), wet AMD, dry AMD, or Geographic Atrophy comprises
administering to the subject a a compound comprising or consisting
of a modified oligonucleotide and a conjugate group, wherein the
modified oligonucleotide consists of 10 to 30 linked nucleosides
and has a nucleobase sequence comprising the nucleobase sequence of
any one of SEQ ID NOs: 6-808. In certain embodiments, a method of
treating, preventing, or ameliorating macular degeneration, such as
age-related macular degeneration (AMD), wet AMD, dry AMD, or
Geographic Atrophy in a subject comprises administering to the
subject a comprises administering to the subject a compound
comprising or consisting of a modified oligonucleotide and a
conjugate group, wherein the modified oligonucleotide consists of
10 to 30 linked nucleosides having a nucleobase sequence comprising
any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455,
549, and 598. In certain embodiments, a method of treating,
preventing, or ameliorating macular degeneration, such as
age-related macular degeneration (AMD), wet AMD, dry AMD, or
Geographic Atrophy in a subject comprises administering to the
subject a compound comprising or consisting of ISIS 696844, ISIS
696845, ISIS 698969, or ISIS 698970. In certain aspects, the
compound or composition is administered to the subject
parenterally.
[0251] In certain embodiments, a method of treating, preventing, or
ameliorating a kidney disease associated with dysregulation of the
complement alternative pathway in a subject comprises administering
to the subject a compound or composition described herein, thereby
treating, preventing, or ameliorating the kidney disease. In
certain embodiments, a method of treating, preventing, or
ameliorating a kidney disease associated with dysregulation of the
complement alternative pathway in a subject comprises administering
to the subject a compound comprising or consisting of a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising the nucleobase sequence of any one
of SEQ ID NOs: 6-808. In certain embodiments, a method of treating,
preventing, or ameliorating a kidney disease associated with
dysregulation of the complement alternative pathway in a subject
comprises administering to the subject a compound comprising or
consisting of a modified oligonucleotide and a conjugate group,
wherein the modified oligonucleotide consists of 10 to 30 linked
nucleosides having a nucleobase sequence comprising any one of SEQ
ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598.
In certain embodiments, a method of treating, preventing, or
ameliorating a kidney disease associated with dysregulation of the
complement alternative pathway in a subject comprises administering
to the subject a compound comprising or consisting of ISIS 696844,
ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, the
complement alternative pathway is activated greater than normal. In
certain aspects, the kidney disease is lupus nephritis, systemic
lupus erythematosus (SLE), dense deposit disease (DDD), C3
glomerulonephritis (C3GN), CFHR5 nephropathy, or atypical hemolytic
uremic syndrome (aHUS), or any combination thereof. In certain
aspects, the kidney disease is associated with C3 deposits, such as
C3 deposits in the glomerulus. In certain aspects, the kidney
disease is associated with lower than normal circulating C3 levels,
such as serum or plasma C3 levels. In certain aspects,
administering the compound or composition reduces or inhibits
accumulation of ocular C3 levels, such as C3 protein levels. In
certain aspects, administering the compound or composition reduces
the level of ocular C3 deposits or inhibits accumulation of ocular
C3 deposits. In certain aspects, the compound or composition is
administered to the subject parenterally. In certain aspects,
administering the compound or composition reduces or inhibits
accumulation of C3 levels in the kidney, such as C3 protein levels.
In certain aspects, administering the compound or composition
reduces the level of kidney C3 deposits or inhibits accumulation of
kidney C3 deposits, such as C3 levels in the glomerulus. In certain
aspects, the subject is identified as having or at risk of having a
disease associated with dysregulation of the complement alternative
pathway, for example by detecting complement levels or
membrane-attack complex levels in the subject's blood and/or
performing a genetic test for gene mutations of complement factors
associated with the disease.
[0252] In certain embodiments, a method of inhibiting expression of
Complement Factor B (CFB) in a subject having, or at risk of
having, a disease associated with dysregulation of the complement
alternative pathway comprises administering a compound or
composition described herein to the subject, thereby inhibiting
expression of CFB in the subject. In certain embodiments, a method
of inhibiting expression of Complement Factor B (CFB) in a subject
having, or at risk of having, a disease associated with
dysregulation of the complement alternative pathway comprises
administering to the subject a compound comprising or consisting of
a modified oligonucleotide and a conjugate group, wherein the
modified oligonucleotide consists of 10 to 30 linked nucleosides
and has a nucleobase sequence comprising the nucleobase sequence of
any one of SEQ ID NOs: 6-808. In certain embodiments, a method of
inhibiting expression of Complement Factor B (CFB) in a subject
having, or at risk of having, a disease associated with
dysregulation of the complement alternative pathway comprises
administering to the subject a compound comprising or consisting of
a modified oligonucleotide and a conjugate group, wherein the
modified oligonucleotide consists of 10 to 30 linked nucleosides
having a nucleobase sequence comprising any one of SEQ ID NOs: 198,
228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain
embodiments, a method of inhibiting expression of Complement Factor
B (CFB) in a subject having, or at risk of having, a disease
associated with dysregulation of the complement alternative pathway
comprises administering to the subject a compound comprising or
consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS
698970. In certain aspects, administering the compound or
composition inhibits expression of CFB in the eye. In certain
aspects, the subject has, or is at risk of having, age related
macular degeneration (AMD), such as wet AMD and dry AMD. In certain
aspects, dry AMD can be Geographic Atrophy. Geographic Atrophy is
considered an advanced form of dry AMD involving degeneration of
the retina. In certain aspects, administering the compound or
composition inhibits expression of CFB in the kidney, such as in
the glomerulus. In certain aspects, the subject has, or is at risk
of having, lupus nephritis, systemic lupus erythematosus (SLE),
dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5
nephropathy, or atypical hemolytic uremic syndrome (aHUS), or any
combination thereof.
[0253] In certain embodiments, a method of reducing or inhibiting
accumulation of C3 deposits in the eye of a subject having, or at
risk of having, a disease associated with dysregulation of the
complement alternative pathway comprises administering a compound
or composition described herein to the subject, thereby reducing or
inhibiting accumulation of C3 deposits in the eye of the subject.
In certain embodiments, a method of reducing or inhibiting
accumulation of C3 deposits in the eye of a subject having, or at
risk of having, a disease associated with dysregulation of the
complement alternative pathway comprises administering to the
subject a compound comprising or consisting of a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising the nucleobase sequence of any one
of SEQ ID NOs: 6-808. In certain embodiments, a method of reducing
or inhibiting accumulation of C3 deposits in the eye of a subject
having, or at risk of having, a disease associated with
dysregulation of the complement alternative pathway comprises
administering to the subject a compound comprising or consisting of
a modified oligonucleotide and a conjugate group, wherein the
modified oligonucleotide consists of 10 to 30 linked nucleosides
having a nucleobase sequence comprising any one of SEQ ID NOs: 198,
228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain
embodiments, a method of reducing or inhibiting accumulation of C3
deposits in the eye of a subject having, or at risk of having, a
disease associated with dysregulation of the complement alternative
pathway comprises administering to the subject a compound
comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969,
or ISIS 698970. In certain aspects, the subject has, or is at risk
of having, age related macular degeneration (AMD), such as wet AMD
and dry AMD. In certain aspects, dry AMD can be Geographic Atrophy.
In certain aspects, the compound or composition is administered to
the subject parenterally.
[0254] In certain embodiments, a method of reducing or inhibiting
accumulation of C3 deposits in the kidney of a subject having, or
at risk of having, a disease associated with dysregulation of the
complement alternative pathway comprises administering a compound
or composition described herein to the subject, thereby reducing or
inhibiting accumulation of C3 deposits in the kidney of the
subject. In certain embodiments, a method of reducing or inhibiting
accumulation of C3 deposits in the kidney of a subject having, or
at risk of having, a disease associated with dysregulation of the
complement alternative pathway comprises administering to the
subject a compound comprising or consisting of a modified
oligonucleotide and a conjugate group, wherein the modified
oligonucleotide consists of 10 to 30 linked nucleosides and has a
nucleobase sequence comprising the nucleobase sequence of any one
of SEQ ID NOs: 6-808. In certain embodiments, a method of reducing
or inhibiting accumulation of C3 deposits in the kidney of a
subject having, or at risk of having, a disease associated with
dysregulation of the complement alternative pathway comprises
administering to the subject a compound comprising or consisting of
a modified oligonucleotide and a conjugate group, wherein the
modified oligonucleotide consists of 10 to 30 linked nucleosides
having a nucleobase sequence comprising any one of SEQ ID NOs: 198,
228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain
embodiments, a method of reducing or inhibiting accumulation of C3
deposits in the kidney of a subject having, or at risk of having, a
disease associated with dysregulation of the complement alternative
pathway comprises administering to the subject a compound
comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969,
or ISIS 698970. In certain aspects, the subject has, or is at risk
of having, lupus nephritis, systemic lupus erythematosus (SLE),
dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5
nephropathy, or atypical hemolytic uremic syndrome (aHUS), or any
combination thereof. In certain aspects, the compound or
composition is administered to the subject parenterally.
[0255] Certain embodiments are drawn to use of a compound or
composition described herein for treating a disease associated with
dysregulation of the complement alternative pathway. Certain
embodiments are drawn to use of a compound comprising or consisting
of a modified oligonucleotide and a conjugate group, wherein the
modified oligonucleotide consists of 10 to 30 linked nucleosides
and has a nucleobase sequence comprising the nucleobase sequence of
any one of SEQ ID NOs: 6-808, for treating a disease associated
with dysregulation of the complement alternative pathway. Certain
embodiments are drawn to use of a compound comprising or consisting
of a modified oligonucleotide and a conjugate group, wherein the
modified oligonucleotide consists of 10 to 30 linked nucleosides
having a nucleobase sequence comprising any one of SEQ ID NOs: 198,
228, 237, 440, 444, 448, 450, 453, 455, 549, and 598, for treating
a disease associated with dysregulation of the complement
alternative pathway. Certain embodiments are drawn to use of a
compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS
698969, or ISIS 698970 for treating a disease associated with
dysregulation of the complement alternative pathway. In certain
aspects, the complement alternative pathway is activated greater
than normal. In certain aspects, the disease is macular
degeneration, such as age related macular degeneration (AMD), which
can be wet AMD or dry AMD. In certain aspects, dry AMD can be
Geographic Atrophy. In certain aspects, the disease is a kidney
disease such as lupus nephritis, systemic lupus erythematosus
(SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN),
CFHR5 nephropathy, or atypical hemolytic uremic syndrome (aHUS), or
any combination thereof. In certain aspects, the compound or
composition is administered to the subject parenterally.
[0256] In certain embodiments, a compound or composition described
herein is administered parenterally. For example, in certain
embodiments the compound or composition can be administered through
injection or infusion. Parenteral administration includes
subcutaneous administration, intravenous administration,
intramuscular administration, intraarterial administration,
intraperitoneal administration, or intracranial administration,
e.g. intrathecal or intracerebroventricular administration.
Antisense Compounds
[0257] Oligomeric compounds include, but are not limited to,
oligonucleotides, oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics, antisense compounds, antisense
oligonucleotides, and siRNAs. An oligomeric compound may be
"antisense" to a target nucleic acid, meaning that is is capable of
undergoing hybridization to a target nucleic acid through hydrogen
bonding.
[0258] In certain embodiments, an antisense compound has a
nucleobase sequence that, when written in the 5' to 3' direction,
comprises the reverse complement of the target segment of a target
nucleic acid to which it is targeted.
[0259] In certain embodiments, an antisense compound is 10 to 30
subunits in length. In certain embodiments, an antisense compound
is 12 to 30 subunits in length. In certain embodiments, an
antisense compound is 12 to 22 subunits in length. In certain
embodiments, an antisense compound is 14 to 30 subunits in length.
In certain embodiments, an antisense compound is 14 to 20 subunits
in length. In certain embodiments, an antisense compound is 15 to
30 subunits in length. In certain embodiments, an antisense
compound is 15 to 20 subunits in length. In certain embodiments, an
antisense compound is 16 to 30 subunits in length. In certain
embodiments, an antisense compound is 16 to 20 subunits in length.
In certain embodiments, an antisense compound is 17 to 30 subunits
in length. In certain embodiments, an antisense compound is 17 to
20 subunits in length. In certain embodiments, an antisense
compound is 18 to 30 subunits in length. In certain embodiments, an
antisense compound is 18 to 21 subunits in length. In certain
embodiments, an antisense compound is 18 to 20 subunits in length.
In certain embodiments, an antisense compound is 20 to 30 subunits
in length. In other words, such antisense compounds are from 12 to
30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15
to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20
subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits,
18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to
22 linked subunits, respectively. In certain embodiments, an
antisense compound is 14 subunits in length. In certain
embodiments, an antisense compound is 16 subunits in length. In
certain embodiments, an antisense compound is 17 subunits in
length. In certain embodiments, an antisense compound is 18
subunits in length. In certain embodiments, an antisense compound
is 19 subunits in length. In certain embodiments, an antisense
compound is 20 subunits in length. In other embodiments, the
antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to
30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17
to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30,
19 to 50, or 20 to 30 linked subunits. In certain such embodiments,
the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits
in length, or a range defined by any two of the above values. In
some embodiments the antisense compound is an antisense
oligonucleotide, and the linked subunits are nucleotides.
[0260] In certain embodiments antisense oligonucleotides may be
shortened or truncated. For example, a single subunit may be
deleted from the 5' end (5' truncation), or alternatively from the
3' end (3' truncation). A shortened or truncated antisense compound
targeted to an CFB nucleic acid may have two subunits deleted from
the 5' end, or alternatively may have two subunits deleted from the
3' end, of the antisense compound. Alternatively, the deleted
nucleosides may be dispersed throughout the antisense compound, for
example, in an antisense compound having one nucleoside deleted
from the 5' end and one nucleoside deleted from the 3' end.
[0261] When a single additional subunit is present in a lengthened
antisense compound, the additional subunit may be located at the 5'
or 3' end of the antisense compound. When two or more additional
subunits are present, the added subunits may be adjacent to each
other, for example, in an antisense compound having two subunits
added to the 5' end (5' addition), or alternatively to the 3' end
(3' addition), of the antisense compound. Alternatively, the added
subunits may be dispersed throughout the antisense compound, for
example, in an antisense compound having one subunit added to the
5' end and one subunit added to the 3' end.
[0262] It is possible to increase or decrease the length of an
antisense compound, such as an antisense oligonucleotide, and/or
introduce mismatch bases without eliminating activity. For example,
in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a
series of antisense oligonucleotides 13-25 nucleobases in length
were tested for their ability to induce cleavage of a target RNA in
an oocyte injection model. Antisense oligonucleotides 25
nucleobases in length with 8 or 11 mismatch bases near the ends of
the antisense oligonucleotides were able to direct specific
cleavage of the target mRNA, albeit to a lesser extent than the
antisense oligonucleotides that contained no mismatches. Similarly,
target specific cleavage was achieved using 13 nucleobase antisense
oligonucleotides, including those with 1 or 3 mismatches.
[0263] Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March
2001) demonstrated the ability of an oligonucleotide having 100%
complementarity to the bcl-2 mRNA and having 3 mismatches to the
bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in
vitro and in vivo. Furthermore, this oligonucleotide demonstrated
potent anti-tumor activity in vivo.
[0264] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988)
tested a series of tandem 14 nucleobase antisense oligonucleotides,
and a 28 and 42 nucleobase antisense oligonucleotides comprised of
the sequence of two or three of the tandem antisense
oligonucleotides, respectively, for their ability to arrest
translation of human DHFR in a rabbit reticulocyte assay. Each of
the three 14 nucleobase antisense oligonucleotides alone was able
to inhibit translation, albeit at a more modest level than the 28
or 42 nucleobase antisense oligonucleotides.
Certain Antisense Compound Motifs and Mechanisms
[0265] In certain embodiments, antisense compounds have chemically
modified subunits arranged in patterns, or motifs, to confer to the
antisense compounds properties such as enhanced inhibitory
activity, increased binding affinity for a target nucleic acid, or
resistance to degradation by in vivo nucleases.
[0266] Chimeric antisense compounds typically contain at least one
region modified so as to confer increased resistance to nuclease
degradation, increased cellular uptake, increased binding affinity
for the target nucleic acid, and/or increased inhibitory activity.
A second region of a chimeric antisense compound may confer another
desired property e.g., serve as a substrate for the cellular
endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA
duplex.
[0267] Antisense activity may result from any mechanism involving
the hybridization of the antisense compound (e.g., oligonucleotide)
with a target nucleic acid, wherein the hybridization ultimately
results in a biological effect. In certain embodiments, the amount
and/or activity of the target nucleic acid is modulated. In certain
embodiments, the amount and/or activity of the target nucleic acid
is reduced. In certain embodiments, hybridization of the antisense
compound to the target nucleic acid ultimately results in target
nucleic acid degradation. In certain embodiments, hybridization of
the antisense compound to the target nucleic acid does not result
in target nucleic acid degradation. In certain such embodiments,
the presence of the antisense compound hybridized with the target
nucleic acid (occupancy) results in a modulation of antisense
activity. In certain embodiments, antisense compounds having a
particular chemical motif or pattern of chemical modifications are
particularly suited to exploit one or more mechanisms. In certain
embodiments, antisense compounds function through more than one
mechanism and/or through mechanisms that have not been elucidated.
Accordingly, the antisense compounds described herein are not
limited by particular mechanism.
[0268] Antisense mechanisms include, without limitation, RNase H
mediated antisense; RNAi mechanisms, which utilize the RISC pathway
and include, without limitation, siRNA, ssRNA and microRNA
mechanisms; and occupancy based mechanisms. Certain antisense
compounds may act through more than one such mechanism and/or
through additional mechanisms.
[0269] RNase H-Mediated Antisense
[0270] In certain embodiments, antisense activity results at least
in part from degradation of target RNA by RNase H. RNase H is a
cellular endonuclease that cleaves the RNA strand of an RNA:DNA
duplex. It is known in the art that single-stranded antisense
compounds which are "DNA-like" elicit RNase H activity in mammalian
cells. Accordingly, antisense compounds comprising at least a
portion of DNA or DNA-like nucleosides may activate RNase H,
resulting in cleavage of the target nucleic acid. In certain
embodiments, antisense compounds that utilize RNase H comprise one
or more modified nucleosides. In certain embodiments, such
antisense compounds comprise at least one block of 1-8 modified
nucleosides. In certain such embodiments, the modified nucleosides
do not support RNase H activity. In certain embodiments, such
antisense compounds are gapmers, as described herein. In certain
such embodiments, the gap of the gapmer comprises DNA nucleosides.
In certain such embodiments, the gap of the gapmer comprises
DNA-like nucleosides. In certain such embodiments, the gap of the
gapmer comprises DNA nucleosides and DNA-like nucleosides.
[0271] Certain antisense compounds having a gapmer motif are
considered chimeric antisense compounds. In a gapmer an internal
region having a plurality of nucleotides that supports RNaseH
cleavage is positioned between external regions having a plurality
of nucleotides that are chemically distinct from the nucleosides of
the internal region. In the case of an antisense oligonucleotide
having a gapmer motif, the gap segment generally serves as the
substrate for endonuclease cleavage, while the wing segments
comprise modified nucleosides. In certain embodiments, the regions
of a gapmer are differentiated by the types of sugar moieties
comprising each distinct region. The types of sugar moieties that
are used to differentiate the regions of a gapmer may in some
embodiments include .beta.-D-ribonucleosides,
.beta.-D-deoxyribonucleosides, 2'-modified nucleosides (such
2'-modified nucleosides may include 2'-MOE and 2'-O--CH.sub.3,
among others), and bicyclic sugar modified nucleosides (such
bicyclic sugar modified nucleosides may include those having a
constrained ethyl). In certain embodiments, nucleosides in the
wings may include several modified sugar moieties, including, for
example 2'-MOE and bicyclic sugar moieties such as constrained
ethyl or LNA. In certain embodiments, wings may include several
modified and unmodified sugar moieties. In certain embodiments,
wings may include various combinations of 2'-MOE nucleosides,
bicyclic sugar moieties such as constrained ethyl nucleosides or
LNA nucleosides, and 2'-deoxynucleosides.
[0272] Each distinct region may comprise uniform sugar moieties,
variant, or alternating sugar moieties. The wing-gap-wing motif is
frequently described as "X--Y--Z", where "X" represents the length
of the 5'-wing, "Y" represents the length of the gap, and "Z"
represents the length of the 3'-wing. "X" and "Z" may comprise
uniform, variant, or alternating sugar moieties. In certain
embodiments, "X" and "Y" may include one or more
2'-deoxynucleosides. "Y" may comprise 2'-deoxynucleosides. As used
herein, a gapmer described as "X--Y--Z" has a configuration such
that the gap is positioned immediately adjacent to each of the
5'-wing and the 3' wing. Thus, no intervening nucleotides exist
between the 5'-wing and gap, or the gap and the 3'-wing. Any of the
antisense compounds described herein can have a gapmer motif. In
certain embodiments, "X" and "Z" are the same; in other embodiments
they are different. In certain embodiments, "Y" is between 8 and 15
nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more
nucleosides.
[0273] In certain embodiments, the antisense compound targeted to a
CFB nucleic acid has a gapmer motif in which the gap consists of 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.
[0274] In certain embodiments, the antisense oligonucleotide has a
sugar motif described by Formula A as follows:
(J).sub.m-(B).sub.n-(J).sub.p-(B).sub.r-(A).sub.t-(D).sub.g-(A).sub.v-(B)-
.sub.w-(J).sub.x-(B).sub.y-(J).sub.z
[0275] wherein:
[0276] each A is independently a 2'-substituted nucleoside;
[0277] each B is independently a bicyclic nucleoside;
[0278] each J is independently either a 2'-substituted nucleoside
or a 2'-deoxynucleoside;
[0279] each D is a 2'-deoxynucleoside;
[0280] m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2;
w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;
[0281] provided that:
[0282] at least one of m, n, and r is other than 0;
[0283] at least one of w and y is other than 0;
[0284] the sum of m, n, p, r, and t is from 2 to 5; and
[0285] the sum of v, w, x, y, and z is from 2 to 5.
[0286] RNAi Compounds
[0287] In certain embodiments, antisense compounds are interfering
RNA compounds (RNAi), which include double-stranded RNA compounds
(also referred to as short-interfering RNA or siRNA) and
single-stranded RNAi compounds (or ssRNA). Such compounds work at
least in part through the RISC pathway to degrade and/or sequester
a target nucleic acid (thus, include microRNA/microRNA-mimic
compounds). In certain embodiments, antisense compounds comprise
modifications that make them particularly suited for such
mechanisms.
[0288] i. ssRNA Compounds
[0289] In certain embodiments, antisense compounds including those
particularly suited for use as single-stranded RNAi compounds
(ssRNA) comprise a modified 5'-terminal end. In certain such
embodiments, the 5'-terminal end comprises a modified phosphate
moiety. In certain embodiments, such modified phosphate is
stabilized (e.g., resistant to degradation/cleavage compared to
unmodified 5'-phosphate). In certain embodiments, such 5'-terminal
nucleosides stabilize the 5'-phosphorous moiety. Certain modified
5'-terminal nucleosides may be found in the art, for example in
WO/2011/139702.
[0290] In certain embodiments, the 5'-nucleoside of an ssRNA
compound has Formula IIc:
##STR00010##
wherein:
[0291] T.sub.1 is an optionally protected phosphorus moiety;
[0292] T.sub.2 is an internucleoside linking group linking the
compound of Formula IIc to the oligomeric compound;
[0293] A has one of the formulas:
##STR00011##
[0294] Q.sub.1 and Q.sub.2 are each, independently, H, halogen,
C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy,
C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6 alkynyl or
N(R.sub.3)(R.sub.4);
[0295] Q.sub.3 is O, S, N(R.sub.5) or C(R.sub.6)(R.sub.7);
[0296] each R.sub.3, R.sub.4 R.sub.5, R.sub.6 and R.sub.7 is,
independently, H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy;
[0297] M.sub.3 is O, S, NR.sub.14, C(R.sub.15)(R.sub.16),
C(R.sub.15)(R.sub.16)C(R.sub.17)(R.sub.18),
C(R.sub.15).dbd.C(R.sub.17), OC(R.sub.15)(R.sub.16) or
OC(R.sub.15)(Bx.sub.2);
[0298] R.sub.14 is H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl;
[0299] R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are each,
independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl;
[0300] Bx.sub.1 is a heterocyclic base moiety;
[0301] or if Bx.sub.2 is present then Bx.sub.2 is a heterocyclic
base moiety and Bx.sub.1 is H, halogen, C.sub.1-C.sub.6 alkyl,
substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
substituted C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl,
substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or
substituted C.sub.2-C.sub.6 alkynyl;
[0302] J.sub.4, J.sub.5, J.sub.6 and J.sub.7 are each,
independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl;
[0303] or J.sub.4 forms a bridge with one of J.sub.5 or J.sub.7
wherein said bridge comprises from 1 to 3 linked biradical groups
selected from O, S, NR.sub.19, C(R.sub.20)(R.sub.21),
C(R.sub.20).dbd.C(R.sub.21), C[.dbd.C(R.sub.20)(R.sub.21)] and
C(.dbd.O) and the other two of J.sub.5, J.sub.6 and J.sub.7 are
each, independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl; [0304] each R.sub.19, R.sub.20 and
R.sub.21 is, independently, H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl;
[0305] G is H, OH, halogen or
O-[C(R.sub.8)(R.sub.9)].sub.n--[(C.dbd.O).sub.m--X.sub.1].sub.j--Z;
[0306] each R.sub.8 and R.sub.9 is, independently, H, halogen,
C.sub.1-C.sub.6 alkyl or substituted C.sub.1-C.sub.6 alkyl;
[0307] X.sub.1 is O, S or N(E.sub.1);
[0308] Z is H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted
C.sub.2-C.sub.6 alkynyl or N(E.sub.2)(E.sub.3);
[0309] E.sub.1, E.sub.2 and E.sub.3 are each, independently, H,
C.sub.1-C.sub.6 alkyl or substituted C.sub.1-C.sub.6 alkyl;
[0310] n is from 1 to about 6;
[0311] m is 0 or 1;
[0312] j is 0 or 1;
[0313] each substituted group comprises one or more optionally
protected substituent groups independently selected from halogen,
OJ.sub.1, N(J.sub.1)(J.sub.2), =NJ.sub.1, SJ.sub.1, N.sub.3, CN,
OC(.dbd.X.sub.2)J.sub.1, OC(.dbd.X.sub.2)N(J.sub.1)(J.sub.2) and
C(.dbd.X.sub.2)N(J.sub.1)(J.sub.2);
[0314] X.sub.2 is O, S or NJ.sub.3;
[0315] each J.sub.1, J.sub.2 and J.sub.3 is, independently, H or
C.sub.1-C.sub.6 alkyl;
[0316] when j is 1 then Z is other than halogen or
N(E.sub.2)(E.sub.3); and
[0317] wherein said oligomeric compound comprises from 8 to 40
monomeric subunits and is hybridizable to at least a portion of a
target nucleic acid.
[0318] In certain embodiments, M.sub.3 is O, CH.dbd.CH, OCH.sub.2
or OC(H)(Bx.sub.2). In certain embodiments, M.sub.3 is O.
[0319] In certain embodiments, J.sub.4, J.sub.5, J.sub.6 and
J.sub.7 are each H. In certain embodiments, J.sub.4 forms a bridge
with one of J.sub.5 or J.sub.7.
[0320] In certain embodiments, A has one of the formulas:
##STR00012##
wherein:
[0321] Q.sub.1 and Q.sub.2 are each, independently, H, halogen,
C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy or substituted C.sub.1-C.sub.6 alkoxy. In
certain embodiments, Q.sub.1 and Q.sub.2 are each H. In certain
embodiments, Q.sub.1 and Q.sub.2 are each, independently, H or
halogen. In certain embodiments, Q.sub.1 and Q.sub.2 is H and the
other of Q.sub.1 and Q.sub.2 is F, CH.sub.3 or OCH.sub.3.
[0322] In certain embodiments, T.sub.1 has the formula:
##STR00013##
wherein:
[0323] R.sub.a and R.sub.c are each, independently, protected
hydroxyl, protected thiol, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, protected amino or substituted amino;
and
[0324] R.sub.b is O or S. In certain embodiments, R.sub.1, is O and
R. and R, are each, independently, OCH.sub.3, OCH.sub.2CH.sub.3 or
CH(CH.sub.3).sub.2.
[0325] In certain embodiments, G is halogen, OCH.sub.3, OCH.sub.2F,
OCHF.sub.2, OCF.sub.3, OCH.sub.2CH.sub.3, O(CH.sub.2).sub.2F,
OCH.sub.2CHF.sub.2, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2,
O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--SCH.sub.3,
O(CH.sub.2).sub.2--OCF.sub.3,
O(CH.sub.2).sub.3--N(R.sub.10)(R.sub.11),
O(CH.sub.2).sub.2--ON(R.sub.10)(R.sub.11),
O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(R.sub.10)(R.sub.11),
OCH.sub.2C(.dbd.O)--N(R.sub.10)(R.sub.11),
OCH.sub.2C(.dbd.O)--N(R.sub.12)--(CH.sub.2).sub.2--N(R.sub.10)(R.sub.11)
or
O(CH.sub.2).sub.2--N(R.sub.12)--C(.dbd.NR.sub.13)[N(R.sub.10)(R.sub.11-
)] wherein R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are each,
independently, H or C.sub.1-C.sub.6 alkyl. In certain embodiments,
G is halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3,
OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2,
O(CH.sub.2).sub.2--OCH.sub.3,
O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2,
OCH.sub.2C(.dbd.O)--N(H)CH.sub.3,
OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2 or
OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2. In certain embodiments, G is
F, OCH.sub.3 or O(CH.sub.2).sub.2--OCH.sub.3. In certain
embodiments, G is O(CH.sub.2).sub.2--OCH.sub.3.
[0326] In certain embodiments, the 5'-terminal nucleoside has
Formula IIe:
##STR00014##
[0327] In certain embodiments, antisense compounds, including those
particularly suitable for ssRNA comprise one or more type of
modified sugar moieties and/or naturally occurring sugar moieties
arranged along an oligonucleotide or region thereof in a defined
pattern or sugar modification motif. Such motifs may include any of
the sugar modifications discussed herein and/or other known sugar
modifications.
[0328] In certain embodiments, the oligonucleotides comprise or
consist of a region having uniform sugar modifications. In certain
such embodiments, each nucleoside of the region comprises the same
RNA-like sugar modification. In certain embodiments, each
nucleoside of the region is a 2'-F nucleoside. In certain
embodiments, each nucleoside of the region is a 2'-OMe nucleoside.
In certain embodiments, each nucleoside of the region is a 2'-MOE
nucleoside. In certain embodiments, each nucleoside of the region
is a cEt nucleoside. In certain embodiments, each nucleoside of the
region is an LNA nucleoside. In certain embodiments, the uniform
region constitutes all or essentially all of the oligonucleotide.
In certain embodiments, the region constitutes the entire
oligonucleotide except for 1-4 terminal nucleosides.
[0329] In certain embodiments, oligonucleotides comprise one or
more regions of alternating sugar modifications, wherein the
nucleosides alternate between nucleotides having a sugar
modification of a first type and nucleotides having a sugar
modification of a second type. In certain embodiments, nucleosides
of both types are RNA-like nucleosides. In certain embodiments the
alternating nucleosides are selected from: 2'-OMe, 2'-F, 2'-MOE,
LNA, and cEt. In certain embodiments, the alternating modifications
are 2'-F and 2'-OMe. Such regions may be contiguous or may be
interrupted by differently modified nucleosides or conjugated
nucleosides.
[0330] In certain embodiments, the alternating region of
alternating modifications each consist of a single nucleoside
(i.e., the pattern is (AB).sub.xA.sub.y wherein A is a nucleoside
having a sugar modification of a first type and B is a nucleoside
having a sugar modification of a second type; x is 1-20 and y is 0
or 1). In certain embodiments, one or more alternating regions in
an alternating motif includes more than a single nucleoside of a
type. For example, oligonucleotides may include one or more regions
of any of the following nucleoside motifs:
TABLE-US-00001 AABBAA; ABBABB; AABAAB; ABBABAABB; ABABAA; AABABAB;
ABABAA; ABBAABBABABAA; BABBAABBABABAA; or ABABBAABBABABAA;
[0331] wherein A is a nucleoside of a first type and B is a
nucleoside of a second type. In certain embodiments, A and B are
each selected from 2'-F, 2'-OMe, BNA, and MOE.
[0332] In certain embodiments, oligonucleotides having such an
alternating motif also comprise a modified 5' terminal nucleoside,
such as those of formula IIc or IIe.
[0333] In certain embodiments, oligonucleotides comprise a region
having a 2-2-3 motif. Such regions comprises the following
motif:
-(A).sub.2-(B).sub.x-(A).sub.2-(C).sub.y-(A).sub.3-
[0334] wherein: A is a first type of modified nucleoside;
[0335] B and C, are nucleosides that are differently modified than
A, however, B and C may have the same or different modifications as
one another;
[0336] x and y are from 1 to 15.
[0337] In certain embodiments, A is a 2'-OMe modified nucleoside.
In certain embodiments, B and C are both 2'-F modified nucleosides.
In certain embodiments, A is a 2'-OMe modified nucleoside and B and
C are both 2'-F modified nucleosides.
[0338] In certain embodiments, oligonucleosides have the following
sugar motif:
5'-(Q)-(AB).sub.xA.sub.y-(D).sub.z
wherein:
[0339] Q is a nucleoside comprising a stabilized phosphate moiety.
In certain embodiments, Q is a nucleoside having Formula IIc or
IIe;
[0340] A is a first type of modified nucleoside;
[0341] B is a second type of modified nucleoside;
[0342] D is a modified nucleoside comprising a modification
different from the nucleoside adjacent to it. Thus, if y is 0, then
D must be differently modified than B and if y is 1, then D must be
differently modified than A. In certain embodiments, D differs from
both A and B.
[0343] X is 5-15;
[0344] Y is 0 or 1;
[0345] Z is 0-4.
[0346] In certain embodiments, oligonucleosides have the following
sugar motif:
5'-(Q)-(A).sub.x-(D).sub.z
wherein:
[0347] Q is a nucleoside comprising a stabilized phosphate moiety.
In certain embodiments, Q is a nucleoside having Formula IIc or
IIe;
[0348] A is a first type of modified nucleoside;
[0349] D is a modified nucleoside comprising a modification
different from A.
[0350] X is 11-30;
[0351] Z is 0-4.
[0352] In certain embodiments A, B, C, and D in the above motifs
are selected from: 2'-OMe, 2'-F, 2'-MOE, LNA, and cEt. In certain
embodiments, D represents terminal nucleosides. In certain
embodiments, such terminal nucleosides are not designed to
hybridize to the target nucleic acid (though one or more might
hybridize by chance). In certain embodiments, the nucleobase of
each D nucleoside is adenine, regardless of the identity of the
nucleobase at the corresponding position of the target nucleic
acid. In certain embodiments the nucleobase of each D nucleoside is
thymine.
[0353] In certain embodiments, antisense compounds, including those
particularly suited for use as ssRNA comprise modified
internucleoside linkages arranged along the oligonucleotide or
region thereof in a defined pattern or modified internucleoside
linkage motif. In certain embodiments, oligonucleotides comprise a
region having an alternating internucleoside linkage motif. In
certain embodiments, oligonucleotides comprise a region of
uniformly modified internucleoside linkages. In certain such
embodiments, the oligonucleotide comprises a region that is
uniformly linked by phosphorothioate internucleoside linkages. In
certain embodiments, the oligonucleotide is uniformly linked by
phosphorothioate internucleoside linkages. In certain embodiments,
each internucleoside linkage of the oligonucleotide is selected
from phosphodiester and phosphorothioate. In certain embodiments,
each internucleoside linkage of the oligonucleotide is selected
from phosphodiester and phosphorothioate and at least one
internucleoside linkage is phosphorothioate.
[0354] In certain embodiments, the oligonucleotide comprises at
least 6 phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least 8
phosphorothioate internucleoside linkages. In certain embodiments,
the oligonucleotide comprises at least 10 phosphorothioate
internucleoside linkages. In certain embodiments, the
oligonucleotide comprises at least one block of at least 6
consecutive phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least one block of at
least 8 consecutive phosphorothioate internucleoside linkages. In
certain embodiments, the oligonucleotide comprises at least one
block of at least 10 consecutive phosphorothioate internucleoside
linkages. In certain embodiments, the oligonucleotide comprises at
least one block of at least one 12 consecutive phosphorothioate
internucleoside linkages. In certain such embodiments, at least one
such block is located at the 3' end of the oligonucleotide. In
certain such embodiments, at least one such block is located within
3 nucleosides of the 3' end of the oligonucleotide.
[0355] Oligonucleotides having any of the various sugar motifs
described herein, may have any linkage motif. For example, the
oligonucleotides, including but not limited to those described
above, may have a linkage motif selected from non-limiting the
table below:
TABLE-US-00002 5' most linkage Central region 3'-region PS
Alternating PO/PS 6 PS PS Alternating PO/PS 7 PS PS Alternating
PO/PS 8 PS
[0356] ii. siRNA Compounds
[0357] In certain embodiments, antisense compounds are
double-stranded RNAi compounds (siRNA). In such embodiments, one or
both strands may comprise any modification motif described above
for ssRNA. In certain embodiments, ssRNA compounds may be
unmodified RNA. In certain embodiments, siRNA compounds may
comprise unmodified RNA nucleosides, but modified internucleoside
linkages.
[0358] Several embodiments relate to double-stranded compositions
wherein each strand comprises a motif defined by the location of
one or more modified or unmodified nucleosides. In certain
embodiments, compositions are provided comprising a first and a
second oligomeric compound that are fully or at least partially
hybridized to form a duplex region and further comprising a region
that is complementary to and hybridizes to a nucleic acid target.
It is suitable that such a composition comprise a first oligomeric
compound that is an antisense strand having full or partial
complementarity to a nucleic acid target and a second oligomeric
compound that is a sense strand having one or more regions of
complementarity to and forming at least one duplex region with the
first oligomeric compound.
[0359] The compositions of several embodiments modulate gene
expression by hybridizing to a nucleic acid target resulting in
loss of its normal function. In some embodiments, the target
nucleic acid is CFB. In certain embodiment, the degradation of the
targeted CFB is facilitated by an activated RISC complex that is
formed with compositions of the invention.
[0360] Several embodiments are directed to double-stranded
compositions wherein one of the strands is useful in, for example,
influencing the preferential loading of the opposite strand into
the RISC (or cleavage) complex. The compositions are useful for
targeting selected nucleic acid molecules and modulating the
expression of one or more genes. In some embodiments, the
compositions of the present invention hybridize to a portion of a
target RNA resulting in loss of normal function of the target
RNA.
[0361] Certain embodiments are drawn to double-stranded
compositions wherein both the strands comprises a hemimer motif, a
fully modified motif, a positionally modified motif or an
alternating motif. Each strand of the compositions of the present
invention can be modified to fulfil a particular role in for
example the siRNA pathway. Using a different motif in each strand
or the same motif with different chemical modifications in each
strand permits targeting the antisense strand for the RISC complex
while inhibiting the incorporation of the sense strand. Within this
model, each strand can be independently modified such that it is
enhanced for its particular role. The antisense strand can be
modified at the 5'-end to enhance its role in one region of the
RISC while the 3'-end can be modified differentially to enhance its
role in a different region of the RISC.
[0362] The double-stranded oligonucleotide molecules can be a
double-stranded polynucleotide molecule comprising
self-complementary sense and antisense regions, wherein the
antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The double-stranded oligonucleotide
molecules can be assembled from two separate oligonucleotides,
where one strand is the sense strand and the other is the antisense
strand, wherein the antisense and sense strands are
self-complementary (i.e. each strand comprises nucleotide sequence
that is complementary to nucleotide sequence in the other strand;
such as where the antisense strand and sense strand form a duplex
or double-stranded structure, for example wherein the
double-stranded region is about 15 to about 30, e.g., about 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base
pairs; the antisense strand comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense strand comprises
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof (e.g., about 15 to about 25 or more
nucleotides of the double-stranded oligonucleotide molecule are
complementary to the target nucleic acid or a portion thereof).
Alternatively, the double-stranded oligonucleotide is assembled
from a single oligonucleotide, where the self-complementary sense
and antisense regions of the siRNA are linked by means of a nucleic
acid based or non-nucleic acid-based linker(s).
[0363] The double-stranded oligonucleotide can be a polynucleotide
with a duplex, asymmetric duplex, hairpin or asymmetric hairpin
secondary structure, having self-complementary sense and antisense
regions, wherein the antisense region comprises nucleotide sequence
that is complementary to nucleotide sequence in a separate target
nucleic acid molecule or a portion thereof and the sense region
having nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The double-stranded oligonucleotide
can be a circular single-stranded polynucleotide having two or more
loop structures and a stem comprising self-complementary sense and
antisense regions, wherein the antisense region comprises
nucleotide sequence that is complementary to nucleotide sequence in
a target nucleic acid molecule or a portion thereof and the sense
region having nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof, and wherein the
circular polynucleotide can be processed either in vivo or in vitro
to generate an active siRNA molecule capable of mediating RNAi.
[0364] In certain embodiments, the double-stranded oligonucleotide
comprises separate sense and antisense sequences or regions,
wherein the sense and antisense regions are covalently linked by
nucleotide or non-nucleotide linkers molecules as is known in the
art, or are alternately non-covalently linked by ionic
interactions, hydrogen bonding, van der waals interactions,
hydrophobic interactions, and/or stacking interactions. In certain
embodiments, the double-stranded oligonucleotide comprises
nucleotide sequence that is complementary to nucleotide sequence of
a target gene. In another embodiment, the double-stranded
oligonucleotide interacts with nucleotide sequence of a target gene
in a manner that causes inhibition of expression of the target
gene.
[0365] As used herein, double-stranded oligonucleotides need not be
limited to those molecules containing only RNA, but further
encompasses chemically modified nucleotides and non-nucleotides. In
certain embodiments, the short interfering nucleic acid molecules
lack 2'-hydroxy (2'-OH) containing nucleotides. In certain
embodiments short interfering nucleic acids optionally do not
include any ribonucleotides (e.g., nucleotides having a 2'--OH
group). Such double-stranded oligonucleotides that do not require
the presence of ribonucleotides within the molecule to support RNAi
can however have an attached linker or linkers or other attached or
associated groups, moieties, or chains containing one or more
nucleotides with 2'--OH groups. Optionally, double-stranded
oligonucleotides can comprise ribonucleotides at about 5, 10, 20,
30, 40, or 50% of the nucleotide positions. As used herein, the
term siRNA is meant to be equivalent to other terms used to
describe nucleic acid molecules that are capable of mediating
sequence specific RNAi, for example short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA
(shRNA), short interfering oligonucleotide, short interfering
nucleic acid, short interfering modified oligonucleotide,
chemically modified siRNA, post-transcriptional gene silencing RNA
(ptgsRNA), and others. In addition, as used herein, the term RNAi
is meant to be equivalent to other terms used to describe sequence
specific RNA interference, such as post transcriptional gene
silencing, translational inhibition, or epigenetics. For example,
double-stranded oligonucleotides can be used to epigenetically
silence genes at both the post-transcriptional level and the
pre-transcriptional level. In a non-limiting example, epigenetic
regulation of gene expression by siRNA molecules of the invention
can result from siRNA mediated modification of chromatin structure
or methylation pattern to alter gene expression (see, for example,
Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al.,
2004, Science, 303, 669-672; Allshire, 2002, Science, 297,
1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,
2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,
2232-2237).
[0366] It is contemplated that compounds and compositions of
several embodiments provided herein can target CFB by a
dsRNA-mediated gene silencing or RNAi mechanism, including, e.g.,
"hairpin" or stem-loop double-stranded RNA effector molecules in
which a single RNA strand with self-complementary sequences is
capable of assuming a double-stranded conformation, or duplex dsRNA
effector molecules comprising two separate strands of RNA. In
various embodiments, the dsRNA consists entirely of ribonucleotides
or consists of a mixture of ribonucleotides and deoxynucleotides,
such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364,
filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21,
1999. The dsRNA or dsRNA effector molecule may be a single molecule
with a region of self-complementarity such that nucleotides in one
segment of the molecule base pair with nucleotides in another
segment of the molecule. In various embodiments, a dsRNA that
consists of a single molecule consists entirely of ribonucleotides
or includes a region of ribonucleotides that is complementary to a
region of deoxyribonucleotides. Alternatively, the dsRNA may
include two different strands that have a region of complementarity
to each other.
[0367] In various embodiments, both strands consist entirely of
ribonucleotides, one strand consists entirely of ribonucleotides
and one strand consists entirely of deoxyribonucleotides, or one or
both strands contain a mixture of ribonucleotides and
deoxyribonucleotides. In certain embodiments, the regions of
complementarity are at least 70, 80, 90, 95, 98, or 100%
complementary to each other and to a target nucleic acid sequence.
In certain embodiments, the region of the dsRNA that is present in
a double-stranded conformation includes at least 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000
or 5000 nucleotides or includes all of the nucleotides in a cDNA or
other target nucleic acid sequence being represented in the dsRNA.
In some embodiments, the dsRNA does not contain any single stranded
regions, such as single stranded ends, or the dsRNA is a hairpin.
In other embodiments, the dsRNA has one or more single stranded
regions or overhangs. In certain embodiments, RNA/DNA hybrids
include a DNA strand or region that is an antisense strand or
region (e.g, has at least 70, 80, 90, 95, 98, or 100%
complementarity to a target nucleic acid) and an RNA strand or
region that is a sense strand or region (e.g, has at least 70, 80,
90, 95, 98, or 100% identity to a target nucleic acid), and vice
versa.
[0368] In various embodiments, the RNA/DNA hybrid is made in vitro
using enzymatic or chemical synthetic methods such as those
described herein or those described in WO 00/63364, filed Apr. 19,
2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other
embodiments, a DNA strand synthesized in vitro is complexed with an
RNA strand made in vivo or in vitro before, after, or concurrent
with the transformation of the DNA strand into the cell. In yet
other embodiments, the dsRNA is a single circular nucleic acid
containing a sense and an antisense region, or the dsRNA includes a
circular nucleic acid and either a second circular nucleic acid or
a linear nucleic acid (see, for example, WO 00/63364, filed Apr.
19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.)
Exemplary circular nucleic acids include lariat structures in which
the free 5' phosphoryl group of a nucleotide becomes linked to the
2' hydroxyl group of another nucleotide in a loop back fashion.
[0369] In other embodiments, the dsRNA includes one or more
modified nucleotides in which the 2' position in the sugar contains
a halogen (such as fluorine group) or contains an alkoxy group
(such as a methoxy group) which increases the half-life of the
dsRNA in vitro or in vivo compared to the corresponding dsRNA in
which the corresponding 2' position contains a hydrogen or an
hydroxyl group. In yet other embodiments, the dsRNA includes one or
more linkages between adjacent nucleotides other than a
naturally-occurring phosphodiester linkage. Examples of such
linkages include phosphoramide, phosphorothioate, and
phosphorodithioate linkages. The dsRNAs may also be chemically
modified nucleic acid molecules as taught in U.S. Pat. No.
6,673,661. In other embodiments, the dsRNA contains one or two
capped strands, as disclosed, for example, by WO 00/63364, filed
Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21,
1999.
[0370] In other embodiments, the dsRNA can be any of the at least
partially dsRNA molecules disclosed in WO 00/63364, as well as any
of the dsRNA molecules described in U.S. Provisional Application
60/399,998; and U.S. Provisional Application 60/419,532, and
PCT/US2003/033466, the teaching of which is hereby incorporated by
reference. Any of the dsRNAs may be expressed in vitro or in vivo
using the methods described herein or standard methods, such as
those described in WO 00/63364.
[0371] Occupancy
[0372] In certain embodiments, antisense compounds are not expected
to result in cleavage or the target nucleic acid via RNase H or to
result in cleavage or sequestration through the RISC pathway. In
certain such embodiments, antisense activity may result from
occupancy, wherein the presence of the hybridized antisense
compound disrupts the activity of the target nucleic acid. In
certain such embodiments, the antisense compound may be uniformly
modified or may comprise a mix of modifications and/or modified and
unmodified nucleosides.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
[0373] Nucleotide sequences that encode Complement Factor B (CFB)
include, without limitation, the following: GENBANK Accession No.
NM_001710.5 (incorporated herein as SEQ ID NO: 1), GENBANK
Accession No. NT_007592.15 truncated from nucleotides 31852000 to
U.S. Pat. No. 31,861,000 (incorporated herein as SEQ ID NO: 2),
GENBANK Accession No NW_001116486.1 truncated from nucleotides
536000 to 545000 (incorporated herein as SEQ ID NO: 3), GENBANK
Accession No. XM_001113553.2 (incorporated herein as SEQ ID NO: 4),
or GENBANK Accession No. NM_008198.2 (incorporated herein as SEQ ID
NO: 5).
Hybridization
[0374] In some embodiments, hybridization occurs between an
antisense compound disclosed herein and a CFB nucleic acid. The
most common mechanism of hybridization involves hydrogen bonding
(e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen
bonding) between complementary nucleobases of the nucleic acid
molecules.
[0375] Hybridization can occur under varying conditions. Stringent
conditions are sequence-dependent and are determined by the nature
and composition of the nucleic acid molecules to be hybridized.
[0376] Methods of determining whether a sequence is specifically
hybridizable to a target nucleic acid are well known in the art. In
certain embodiments, the antisense compounds provided herein are
specifically hybridizable with a CFB nucleic acid.
Complementarity
[0377] An antisense compound and a target nucleic acid are
complementary to each other when a sufficient number of nucleobases
of the antisense compound can hydrogen bond with the corresponding
nucleobases of the target nucleic acid, such that a desired effect
will occur (e.g., antisense inhibition of a target nucleic acid,
such as a CFB nucleic acid).
[0378] Non-complementary nucleobases between an antisense compound
and a CFB nucleic acid may be tolerated provided that the antisense
compound remains able to specifically hybridize to a target nucleic
acid. Moreover, an antisense compound may hybridize over one or
more segments of a CFB nucleic acid such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure, mismatch or hairpin structure).
[0379] In certain embodiments, the antisense compounds provided
herein, or a specified portion thereof, are, or are at least, 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% complementary to a CFB nucleic acid, a
target region, target segment, or specified portion thereof.
Percent complementarity of an antisense compound with a target
nucleic acid can be determined using routine methods.
[0380] For example, an antisense compound in which 18 of 20
nucleobases of the antisense compound are complementary to a target
region, and would therefore specifically hybridize, would represent
90 percent complementarity. In this example, the remaining
noncomplementary nucleobases may be clustered or interspersed with
complementary nucleobases and need not be contiguous to each other
or to complementary nucleobases. As such, an antisense compound
which is 18 nucleobases in length having four noncomplementary
nucleobases which are flanked by two regions of complete
complementarity with the target nucleic acid would have 77.8%
overall complementarity with the target nucleic acid and would thus
fall within the scope of the present invention. Percent
complementarity of an antisense compound with a region of a target
nucleic acid can be determined routinely using BLAST programs
(basic local alignment search tools) and PowerBLAST programs known
in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410;
Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology,
sequence identity or complementarity, can be determined by, for
example, the Gap program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, Madison Wis.), using default settings, which uses the
algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482
489).
[0381] In certain embodiments, the antisense compounds provided
herein, or specified portions thereof, are fully complementary
(i.e. 100% complementary) to a target nucleic acid, or specified
portion thereof. For example, an antisense compound may be fully
complementary to a CFB nucleic acid, or a target region, or a
target segment or target sequence thereof. As used herein, "fully
complementary" means each nucleobase of an antisense compound is
capable of precise base pairing with the corresponding nucleobases
of a target nucleic acid. For example, a 20 nucleobase antisense
compound is fully complementary to a target sequence that is 400
nucleobases long, so long as there is a corresponding 20 nucleobase
portion of the target nucleic acid that is fully complementary to
the antisense compound. Fully complementary can also be used in
reference to a specified portion of the first and/or the second
nucleic acid. For example, a 20 nucleobase portion of a 30
nucleobase antisense compound can be "fully complementary" to a
target sequence that is 400 nucleobases long. The 20 nucleobase
portion of the 30 nucleobase oligonucleotide is fully complementary
to the target sequence if the target sequence has a corresponding
20 nucleobase portion wherein each nucleobase is complementary to
the 20 nucleobase portion of the antisense compound. At the same
time, the entire 30 nucleobase antisense compound may or may not be
fully complementary to the target sequence, depending on whether
the remaining 10 nucleobases of the antisense compound are also
complementary to the target sequence.
[0382] The location of a non-complementary nucleobase may be at the
5' end or 3' end of the antisense compound. Alternatively, the
non-complementary nucleobase or nucleobases may be at an internal
position of the antisense compound. When two or more
non-complementary nucleobases are present, they may be contiguous
(i.e. linked) or non-contiguous. In one embodiment, a
non-complementary nucleobase is located in the wing segment of a
gapmer antisense oligonucleotide.
[0383] In certain embodiments, antisense compounds that are, or are
up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in
length comprise no more than 4, no more than 3, no more than 2, or
no more than 1 non-complementary nucleobase(s) relative to a target
nucleic acid, such as a CFB nucleic acid, or specified portion
thereof.
[0384] In certain embodiments, antisense compounds that are, or are
up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30 nucleobases in length comprise no more than
6, no more than 5, no more than 4, no more than 3, no more than 2,
or no more than 1 non-complementary nucleobase(s) relative to a
target nucleic acid, such as a CFB nucleic acid, or specified
portion thereof.
[0385] The antisense compounds provided also include those which
are complementary to a portion of a target nucleic acid. As used
herein, "portion" refers to a defined number of contiguous (i.e.
linked) nucleobases within a region or segment of a target nucleic
acid. A "portion" can also refer to a defined number of contiguous
nucleobases of an antisense compound. In certain embodiments, the
antisense compounds, are complementary to at least an 8 nucleobase
portion of a target segment. In certain embodiments, the antisense
compounds are complementary to at least a 9 nucleobase portion of a
target segment. In certain embodiments, the antisense compounds are
complementary to at least a 10 nucleobase portion of a target
segment. In certain embodiments, the antisense compounds are
complementary to at least an 11 nucleobase portion of a target
segment. In certain embodiments, the antisense compounds are
complementary to at least a 12 nucleobase portion of a target
segment. In certain embodiments, the antisense compounds are
complementary to at least a 13 nucleobase portion of a target
segment. In certain embodiments, the antisense compounds are
complementary to at least a 14 nucleobase portion of a target
segment. In certain embodiments, the antisense compounds are
complementary to at least a 15 nucleobase portion of a target
segment. Also contemplated are antisense compounds that are
complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, or more nucleobase portion of a target segment, or a range
defined by any two of these values.
Identity
[0386] The antisense compounds provided herein may also have a
defined percent identity to a particular nucleotide sequence, SEQ
ID NO, or compound represented by a specific Isis number, or
portion thereof. As used herein, an antisense compound is identical
to the sequence disclosed herein if it has the same nucleobase
pairing ability. For example, a RNA which contains uracil in place
of thymidine in a disclosed DNA sequence would be considered
identical to the DNA sequence since both uracil and thymidine pair
with adenine. Shortened and lengthened versions of the antisense
compounds described herein as well as compounds having
non-identical bases relative to the antisense compounds provided
herein also are contemplated. The non-identical bases may be
adjacent to each other or dispersed throughout the antisense
compound. Percent identity of an antisense compound is calculated
according to the number of bases that have identical base pairing
relative to the sequence to which it is being compared.
[0387] In certain embodiments, the antisense compounds, or portions
thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to one or more of the antisense compounds or
SEQ ID NOs, or a portion thereof, disclosed herein.
[0388] In certain embodiments, a portion of the antisense compound
is compared to an equal length portion of the target nucleic acid.
In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to
an equal length portion of the target nucleic acid.
[0389] In certain embodiments, a portion of the antisense
oligonucleotide is compared to an equal length portion of the
target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase
portion is compared to an equal length portion of the target
nucleic acid.
Modifications
[0390] A nucleoside is a base-sugar combination. The nucleobase
(also known as base) portion of the nucleoside is normally a
heterocyclic base moiety. Nucleotides are nucleosides that further
include a phosphate group covalently linked to the sugar portion of
the nucleoside. For those nucleosides that include a pentofuranosyl
sugar, the phosphate group can be linked to the 2', 3' or 5'
hydroxyl moiety of the sugar. Oligonucleotides are formed through
the covalent linkage of adjacent nucleosides to one another, to
form a linear polymeric oligonucleotide. Within the oligonucleotide
structure, the phosphate groups are commonly referred to as forming
the internucleoside linkages of the oligonucleotide.
[0391] Modifications to antisense compounds encompass substitutions
or changes to internucleoside linkages, sugar moieties, or
nucleobases. Modified antisense compounds are often preferred over
native forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced affinity for nucleic acid
target, increased stability in the presence of nucleases, or
increased inhibitory activity. Chemically modified nucleosides may
also be employed to increase the binding affinity of a shortened or
truncated antisense oligonucleotide for its target nucleic acid.
Consequently, comparable results can often be obtained with shorter
antisense compounds that have such chemically modified
nucleosides.
Modified Internucleoside Linkages
[0392] The naturally occurring internucleoside linkage of RNA and
DNA is a 3' to 5' phosphodiester linkage. Antisense compounds
having one or more modified, i.e. non-naturally occurring,
internucleoside linkages are often selected over antisense
compounds having naturally occurring internucleoside linkages
because of desirable properties such as, for example, enhanced
cellular uptake, enhanced affinity for target nucleic acids, and
increased stability in the presence of nucleases.
[0393] Oligonucleotides having modified internucleoside linkages
include internucleoside linkages that retain a phosphorus atom as
well as internucleoside linkages that do not have a phosphorus
atom. Representative phosphorus containing internucleoside linkages
include, but are not limited to, phosphodiesters, phosphotriesters,
methylphosphonates, phosphoramidate, and phosphorothioates. Methods
of preparation of phosphorous-containing and
non-phosphorous-containing linkages are well known.
[0394] In certain embodiments, antisense compounds targeted to a
CFB nucleic acid comprise one or more modified internucleoside
linkages. In certain embodiments, the modified internucleoside
linkages are phosphorothioate linkages. In certain embodiments,
each internucleoside linkage of an antisense compound is a
phosphorothioate internucleoside linkage.
[0395] In certain embodiments, oligonucleotides comprise modified
internucleoside linkages arranged along the oligonucleotide or
region thereof in a defined pattern or modified internucleoside
linkage motif. In certain embodiments, internucleoside linkages are
arranged in a gapped motif. In such embodiments, the
internucleoside linkages in each of two wing regions are different
from the internucleoside linkages in the gap region. In certain
embodiments the internucleoside linkages in the wings are
phosphodiester and the internucleoside linkages in the gap are
phosphorothioate. The nucleoside motif is independently selected,
so such oligonucleotides having a gapped internucleoside linkage
motif may or may not have a gapped nucleoside motif and if it does
have a gapped nucleoside motif, the wing and gap lengths may or may
not be the same.
[0396] In certain embodiments, oligonucleotides comprise a region
having an alternating internucleoside linkage motif. In certain
embodiments, oligonucleotides of the present invention comprise a
region of uniformly modified internucleoside linkages. In certain
such embodiments, the oligonucleotide comprises a region that is
uniformly linked by phosphorothioate internucleoside linkages. In
certain embodiments, the oligonucleotide is uniformly linked by
phosphorothioate. In certain embodiments, each internucleoside
linkage of the oligonucleotide is selected from phosphodiester and
phosphorothioate. In certain embodiments, each internucleoside
linkage of the oligonucleotide is selected from phosphodiester and
phosphorothioate and at least one internucleoside linkage is
phosphorothioate.
[0397] In certain embodiments, the oligonucleotide comprises at
least 6 phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least 8
phosphorothioate internucleoside linkages. In certain embodiments,
the oligonucleotide comprises at least 10 phosphorothioate
internucleoside linkages. In certain embodiments, the
oligonucleotide comprises at least one block of at least 6
consecutive phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least one block of at
least 8 consecutive phosphorothioate internucleoside linkages. In
certain embodiments, the oligonucleotide comprises at least one
block of at least 10 consecutive phosphorothioate internucleoside
linkages. In certain embodiments, the oligonucleotide comprises at
least block of at least one 12 consecutive phosphorothioate
internucleoside linkages. In certain such embodiments, at least one
such block is located at the 3' end of the oligonucleotide. In
certain such embodiments, at least one such block is located within
3 nucleosides of the 3' end of the oligonucleotide.
[0398] In certain embodiments, oligonucleotides comprise one or
more methylphosponate linkages. In certain embodiments,
oligonucleotides having a gapmer nucleoside motif comprise a
linkage motif comprising all phosphorothioate linkages except for
one or two methylphosponate linkages. In certain embodiments, one
methylphosponate linkage is in the central gap of an
oligonucleotide having a gapmer nucleoside motif.
[0399] In certain embodiments, it is desirable to arrange the
number of phosphorothioate internucleoside linkages and
phosphodiester internucleoside linkages to maintain nuclease
resistance. In certain embodiments, it is desirable to arrange the
number and position of phosphorothioate internucleoside linkages
and the number and position of phosphodiester internucleoside
linkages to maintain nuclease resistance. In certain embodiments,
the number of phosphorothioate internucleoside linkages may be
decreased and the number of phosphodiester internucleoside linkages
may be increased. In certain embodiments, the number of
phosphorothioate internucleoside linkages may be decreased and the
number of phosphodiester internucleoside linkages may be increased
while still maintaining nuclease resistance. In certain embodiments
it is desirable to decrease the number of phosphorothioate
internucleoside linkages while retaining nuclease resistance. In
certain embodiments it is desirable to increase the number of
phosphodiester internucleoside linkages while retaining nuclease
resistance.
Modified Sugar Moieties
[0400] Antisense compounds can optionally contain one or more
nucleosides wherein the sugar group has been modified. Such sugar
modified nucleosides may impart enhanced nuclease stability,
increased binding affinity, or some other beneficial biological
property to the antisense compounds. In certain embodiments,
nucleosides comprise chemically modified ribofuranose ring
moieties. Examples of chemically modified ribofuranose rings
include without limitation, addition of substitutent groups
(including 5' and 2' substituent groups, bridging of non-geminal
ring atoms to form bicyclic nucleic acids (BNA), replacement of the
ribosyl ring oxygen atom with S, N(R), or C(R.sub.1)(R.sub.2) (R,
R.sub.1 and R.sub.2 are each independently H, C.sub.1-C.sub.12
alkyl or a protecting group) and combinations thereof. Examples of
chemically modified sugars include 2'-F-5'-methyl substituted
nucleoside (see PCT International Application WO 2008/101157
Published on Aug. 21, 2008 for other disclosed 5',2'-bis
substituted nucleosides) or replacement of the ribosyl ring oxygen
atom with S with further substitution at the 2'-position (see
published U.S. Patent Application US2005-0130923, published on Jun.
16, 2005) or alternatively 5'-substitution of a BNA (see PCT
International Application WO 2007/134181 Published on Nov. 22, 2007
wherein LNA is substituted with for example a 5'-methyl or a
5'-vinyl group).
[0401] Examples of nucleosides having modified sugar moieties
include without limitation nucleosides comprising 5'-vinyl,
5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH.sub.3,
2'--OCH.sub.2CH.sub.3, 2'-OCH.sub.2CH.sub.2F and
2'-O(CH.sub.2).sub.2OCH.sub.3 substituent groups. The substituent
at the 2' position can also be selected from allyl, amino, azido,
thio, O-allyl, O--C.sub.1-C.sub.10 alkyl, OCF.sub.3, OCH.sub.2F,
O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n),
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), and
O--CH.sub.2--C(.dbd.O)--N(R.sub.1)--(CH.sub.2).sub.2--N(R.sub.m)(R.sub.n)-
, where each R.sub.l, R.sub.m and R.sub.n is, independently, H or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl.
[0402] As used herein, "bicyclic nucleosides" refer to modified
nucleosides comprising a bicyclic sugar moiety. Examples of
bicyclic nucleosides include without limitation nucleosides
comprising a bridge between the 4' and the 2' ribosyl ring atoms.
In certain embodiments, antisense compounds provided herein include
one or more bicyclic nucleosides comprising a 4' to 2' bridge.
Examples of such 4' to 2' bridged bicyclic nucleosides, include but
are not limited to one of the formulae: 4'-(CH.sub.2)--O-2' (LNA);
4'-(CH.sub.2)--S-2; 4'--(CH.sub.2).sub.2--O-2' (ENA);
4'-CH(CH.sub.3)--O-2' (also referred to as constrained ethyl or
cEt) and 4'-CH(CH.sub.2OCH.sub.3)--O-2' (and analogs thereof see
U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008);
4'-C(CH.sub.3)(CH.sub.3)--O-2' (and analogs thereof see published
International Application WO 2009/006478, published Jan. 8, 2009);
4'-CH.sub.2--N(OCH.sub.3)-2' (and analogs thereof see published
International Application WO/2008/150729, published Dec. 11, 2008);
4'-CH.sub.2--O--N(CH.sub.3)-2' (see published U.S. Patent
Application US2004-0171570, published Sep. 2, 2004);
4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or
a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23,
2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see Zhou et al., J. Org.
Chem., 2009, 74, 118-134); and 4'-CH.sub.2--C(.dbd.CH.sub.2)-2'
(and analogs thereof see published International Application WO
2008/154401, published on Dec. 8, 2008).
[0403] Further reports related to bicyclic nucleosides can also be
found in published literature (see for example: Singh et al., Chem.
Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,
3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U S. A., 2000,
97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,
2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;
Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379;
Elayadi et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561;
Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr.
Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,268,490;
6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207;
7,399,845; 7,547,684; 8,530,640; and 7,696,345; U.S. Patent
Publication No. US2008-0039618; US2009-0012281; U.S. Patent Ser.
Nos. 61/026,995 and 61/097,787; Published PCT International
applications; WO 2009/067647; WO 2011/017521; WO 2010/036698 WO
1999/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO
2008/150729; WO 2008/154401; and WO 2009/006478. Each of the
foregoing bicyclic nucleosides can be prepared having one or more
stereochemical sugar configurations including for example
.alpha.-L-ribofuranose and .beta.-D-ribofuranose (see PCT
international application PCT/DK98/00393, published on Mar. 25,
1999 as WO 99/14226).
[0404] In certain embodiments, bicyclic sugar moieties of BNA
nucleosides include, but are not limited to, compounds having at
least one bridge between the 4' and the 2' position of the
pentofuranosyl sugar moiety wherein such bridges independently
comprises 1 or from 2 to 4 linked groups independently selected
from --[C(R.sub.a)(R.sub.b)].sub.n--,
--C(R.sub.a).dbd.C(R.sub.b)--, --C(R.sub.a).dbd.N--, --C(.dbd.O)--,
--C(.dbd.NR.sub.a)--, --C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--,
--S(.dbd.O).sub.x--, and --N(R.sub.a)--;
[0405] wherein:
[0406] x is 0, 1, or 2;
[0407] n is 1, 2, 3, or 4;
[0408] each R.sub.a and R.sub.b is, independently, H, a protecting
group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted
C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle
radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7
alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical,
halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1,
acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
[0409] each J.sub.1 and J.sub.2 is, independently, H,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl
(C(.dbd.O)--H), substituted acyl, a heterocycle radical, a
substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl,
substituted C.sub.1-C.sub.12 aminoalkyl or a protecting group.
[0410] In certain embodiments, the bridge of a bicyclic sugar
moiety is --[C(R.sub.a)(R.sub.b)].sub.n--,
--[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)--N(R)--O--
or --C(R.sub.aR.sub.b)--O--N(R)--. In certain embodiments, the
bridge is 4'-CH.sub.2-2', 4'--(CH.sub.2).sub.2-2',
4'--(CH.sub.2).sub.3-2', 4'--CH.sub.2--O-2',
4'--(CH.sub.2).sub.2--O-2', 4'--CH.sub.2--O--N(R)-2' and
4'-CH.sub.2--N(R)--O-2'- wherein each R is, independently, H, a
protecting group or C.sub.1-C.sub.12 alkyl.
[0411] In certain embodiments, bicyclic nucleosides are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-2' methylene-oxy bridge, may be in the .alpha.-L
configuration or in the .beta.-D configuration. Previously,
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') BNA's have been
incorporated into antisense oligonucleotides that showed antisense
activity (Frieden et al., Nucleic Acids Research, 2003, 21,
6365-6372).
[0412] In certain embodiments, bicyclic nucleosides include, but
are not limited to, (A) .alpha.-L-methyleneoxy (4'-CH.sub.2--O-2')
BNA, (B) .beta.-D-methyleneoxy (4'-CH.sub.2--O-2') BNA, (C)
ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') BNA, (D) aminooxy
(4'-CH.sub.2--O--N(R)-2') BNA, (E) oxyamino
(4'-CH.sub.2--N(R)--O-2') BNA, and (F) methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') BNA, (G) methylene thio (4'-CH.sub.2--S-2')
BNA, (H) methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl
carbocyclic (4'-CH.sub.2--CH(CH.sub.3)-2') BNA, (J) propylene
carbocyclic (4'-(CH.sub.2).sub.3-2') BNA and (K) vinyl BNA as
depicted below:
##STR00015## ##STR00016##
[0413] wherein Bx is the base moiety and R is independently H, a
protecting group, C.sub.1-C.sub.12 alkyl or C.sub.1-C.sub.12
alkoxy.
[0414] In certain embodiments, bicyclic nucleosides are provided
having Formula I:
##STR00017##
wherein:
[0415] Bx is a heterocyclic base moiety;
[0416] -Q.sub.a-Q.sub.b-Q.sub.c- is
--CH.sub.2--N(R.sub.c)--CH.sub.2--,
--C(.dbd.O)--N(R.sub.c)--CH.sub.2--, --CH.sub.2--O--N(R.sub.c)--,
--CH.sub.2--N(R.sub.c)--O-- or --N(R.sub.c)--O--CH.sub.2;
[0417] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting
group; and
[0418] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium.
[0419] In certain embodiments, bicyclic nucleosides are provided
having Formula II:
##STR00018##
wherein:
[0420] Bx is a heterocyclic base moiety;
[0421] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0422] Z.sub.a is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkynyl, acyl, substituted acyl, substituted amide, thiol or
substituted thio.
[0423] In one embodiment, each of the substituted groups is,
independently, mono or poly substituted with substituent groups
independently selected from halogen, oxo, hydroxyl, OJ.sub.c,
NJ.sub.cJ.sub.d, SJ.sub.c, N.sub.3, OC(.dbd.X)J.sub.c, and
NJ.sub.eC(.dbd.X)NJ.sub.cJ.sub.d, wherein each J.sub.c, J.sub.d and
J.sub.e is, independently, H, C.sub.1-C.sub.6 alkyl, or substituted
C.sub.1-C.sub.6 alkyl and X is O or NJ.sub.c.
[0424] In certain embodiments, bicyclic nucleosides are provided
having Formula III:
##STR00019##
wherein:
[0425] Bx is a heterocyclic base moiety;
[0426] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0427] Z.sub.b is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkynyl or substituted acyl (C(.dbd.O)--).
[0428] In certain embodiments, bicyclic nucleosides are provided
having Formula IV:
##STR00020##
wherein:
[0429] Bx is a heterocyclic base moiety;
[0430] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0431] R.sub.d is C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl;
[0432] each q.sub.a, q.sub.b, q.sub.c and q.sub.d is,
independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, substituted
C.sub.1-C.sub.6 alkoxyl, acyl, substituted acyl, C.sub.1-C.sub.6
aminoalkyl or substituted C.sub.1-C.sub.6 aminoalkyl;
[0433] In certain embodiments, bicyclic nucleosides are provided
having Formula V:
##STR00021##
wherein:
[0434] Bx is a heterocyclic base moiety;
[0435] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0436] q.sub.a, q.sub.b, q.sub.e and q.sub.f are each,
independently, hydrogen, halogen, C.sub.1-C.sub.12 alkyl,
substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl,
substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl,
substituted C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxy,
substituted C.sub.1-C.sub.12 alkoxy, OJ.sub.j, SJ.sub.j, SOJ.sub.j,
SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j,
C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j,
O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k,
N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k;
[0437] or q.sub.e and q.sub.f together are
.dbd.C(q.sub.g)(q.sub.h);
[0438] q.sub.g and q.sub.h are each, independently, H, halogen,
C.sub.1-C.sub.12 alkyl or substituted C.sub.1-C.sub.12 alkyl.
[0439] The synthesis and preparation of the methyleneoxy
(4'-CH.sub.2--O-2') BNA monomers adenine, cytosine, guanine,
5-methyl-cytosine, thymine and uracil, along with their
oligomerization, and nucleic acid recognition properties have been
described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs
and preparation thereof are also described in WO 98/39352 and WO
99/14226.
[0440] Analogs of methyleneoxy (4'-CH.sub.2--O-2') BNA and
2'-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med.
Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside
analogs comprising oligodeoxyribonucleotide duplexes as substrates
for nucleic acid polymerases has also been described (Wengel et
al., WO 99/14226). Furthermore, synthesis of 2'-amino-BNA, a novel
comformationally restricted high-affinity oligonucleotide analog
has been described in the art (Singh et al., J. Org. Chem., 1998,
63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA's
have been prepared and the thermal stability of their duplexes with
complementary RNA and DNA strands has been previously reported.
[0441] In certain embodiments, bicyclic nucleosides are provided
having Formula VI:
##STR00022##
wherein:
[0442] Bx is a heterocyclic base moiety;
[0443] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0444] each q.sub.i, q.sub.j, q.sub.k and q.sub.l is,
independently, H, halogen, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxyl, substituted
C.sub.1-C.sub.12 alkoxyl, OJ.sub.j, SJ.sub.j, SOJ.sub.j,
SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j,
C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j,
--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k,
N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k;
and
[0445] q.sub.i and q.sub.j or q.sub.l and q.sub.k together are
.dbd.C(q.sub.g)(q.sub.h), wherein q.sub.g and q.sub.h are each,
independently, H, halogen, C.sub.1-C.sub.12 alkyl or substituted
C.sub.1-C.sub.12 alkyl.
[0446] One carbocyclic bicyclic nucleoside having a
4'-(CH.sub.2).sub.3-2' bridge and the alkenyl analog bridge
4'-CH.dbd.CH--CH.sub.2-2' have been described (Freier et al.,
Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,
J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation
of carbocyclic bicyclic nucleosides along with their
oligomerization and biochemical studies have also been described
(Srivastava et al., J. Am. Chem. Soc., 2007, 129(26),
8362-8379).
[0447] As used herein, "4'-2' bicyclic nucleoside" or "4' to 2'
bicyclic nucleoside" refers to a bicyclic nucleoside comprising a
furanose ring comprising a bridge connecting two carbon atoms of
the furanose ring connects the 2' carbon atom and the 4' carbon
atom of the sugar ring.
[0448] As used herein, "monocylic nucleosides" refer to nucleosides
comprising modified sugar moieties that are not bicyclic sugar
moieties. In certain embodiments, the sugar moiety, or sugar moiety
analogue, of a nucleoside may be modified or substituted at any
position.
[0449] As used herein, "2'-modified sugar" means a furanosyl sugar
modified at the 2' position. In certain embodiments, such
modifications include substituents selected from: a halide,
including, but not limited to substituted and unsubstituted alkoxy,
substituted and unsubstituted thioalkyl, substituted and
unsubstituted amino alkyl, substituted and unsubstituted alkyl,
substituted and unsubstituted allyl, and substituted and
unsubstituted alkynyl. In certain embodiments, 2' modifications are
selected from substituents including, but not limited to:
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nF,
O(CH.sub.2).sub.nONH.sub.2, OCH.sub.2C(.dbd.O)N(H)CH.sub.3, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m
are from 1 to about 10. Other 2'-substituent groups can also be
selected from: C.sub.1-C.sub.12 alkyl, substituted alkyl, alkenyl,
alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3,
OCN, Cl, Br, CN, F, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving
pharmacokinetic properties, or a group for improving the
pharmacodynamic properties of an antisense compound, and other
substituents having similar properties. In certain embodiments,
modified nucleosides comprise a 2'-MOE side chain (Baker et al., J.
Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have
been described as having improved binding affinity compared to
unmodified nucleosides and to other modified nucleosides, such as
2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having
the 2'-MOE substituent also have been shown to be antisense
inhibitors of gene expression with promising features for in vivo
use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al.,
Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans.,
1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,
1997, 16, 917-926).
[0450] As used herein, a "modified tetrahydropyran nucleoside" or
"modified THP nucleoside" means a nucleoside having a six-membered
tetrahydropyran "sugar" substituted in for the pentofuranosyl
residue in normal nucleosides (a sugar surrogate). Modified THP
nucleosides include, but are not limited to, what is referred to in
the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA),
manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002,
10, 841-854) or fluoro HNA (F-HNA) having a tetrahydropyran ring
system as illustrated below:
##STR00023##
[0451] In certain embodiments, sugar surrogates are selected having
Formula VII:
##STR00024##
wherein independently for each of said at least one tetrahydropyran
nucleoside analog of Formula VII:
[0452] Bx is a heterocyclic base moiety;
[0453] T.sub.a and T.sub.b are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the antisense compound or one of T.sub.a and
T.sub.b is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound and the
other of T.sub.a and T.sub.b is H, a hydroxyl protecting group, a
linked conjugate group or a 5' or 3'-terminal group;
[0454] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and
q.sub.7 are each independently, H, C.sub.1-C.sub.6 alkyl,
substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or
substituted C.sub.2-C.sub.6 alkynyl; and each of R.sub.1 and
R.sub.2 is selected from hydrogen, hydroxyl, halogen, substituted
or unsubstituted alkoxy, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3,
OC(.dbd.X)J.sub.1, OC(.dbd.X)NJ.sub.1J.sub.2,
NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2 and CN, wherein X is O, S or
NJ.sub.1 and each J.sub.1, J.sub.2 and J.sub.3 is, independently, H
or C.sub.1-C.sub.6 alkyl.
[0455] In certain embodiments, the modified THP nucleosides of
Formula VII are provided wherein q.sub.1, q.sub.2, q.sub.3,
q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, THP
nucleosides of Formula VII are provided wherein one of R.sub.1 and
R.sub.2 is fluoro. In certain embodiments, R.sub.1 is fluoro and
R.sub.2 is H; R.sub.1 is methoxy and R.sub.2 is H, and R.sub.1 is
methoxyethoxy and R.sub.2 is H.
[0456] In certain embodiments, sugar surrogates comprise rings
having more than 5 atoms and more than one heteroatom. For example
nucleosides comprising morpholino sugar moieties and their use in
oligomeric compounds has been reported (see for example: Braasch et
al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos.
5,698,685; 5,166,315; 5,185,444; and 5,034,506).sub.n As used here,
the term "morpholino" means a sugar surrogate having the following
formula:
##STR00025##
In certain embodiments, morpholinos may be modified, for example by
adding or altering various substituent groups from the above
morpholino structure. Such sugar surrogates are referred to herein
as "modified morpholinos."
[0457] Combinations of modifications are also provided without
limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT
International Application WO 2008/101157 published on Aug. 21, 2008
for other disclosed 5', 2'-bis substituted nucleosides) and
replacement of the ribosyl ring oxygen atom with S and further
substitution at the 2'-position (see published U.S. Patent
Application US2005-0130923, published on Jun. 16, 2005) or
alternatively 5'-substitution of a bicyclic nucleic acid (see PCT
International Application WO 2007/134181, published on Nov. 22,
2007 wherein a 4'-CH.sub.2--O-2' bicyclic nucleoside is further
substituted at the 5' position with a 5'-methyl or a 5'-vinyl
group). The synthesis and preparation of carbocyclic bicyclic
nucleosides along with their oligomerization and biochemical
studies have also been described (see, e.g., Srivastava et al., J.
Am. Chem. Soc. 2007, 129(26), 8362-8379).
[0458] In certain embodiments, antisense compounds comprise one or
more modified cyclohexenyl nucleosides, which is a nucleoside
having a six-membered cyclohexenyl in place of the pentofuranosyl
residue in naturally occurring nucleosides. Modified cyclohexenyl
nucleosides include, but are not limited to those described in the
art (see for example commonly owned, published PCT Application WO
2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am.
Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron
Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem.
Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides
& Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al.,
Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al.,
Acta Crystallographica, Section F: Structural Biology and
Crystallization Communications, 2005, F61(6), 585-586; Gu et al.,
Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides,
2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68,
4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24),
4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et
al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),
785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published
PCT application, WO 06/047842; and Published PCT Application WO
01/049687; the text of each is incorporated by reference herein, in
their entirety). Certain modified cyclohexenyl nucleosides have
Formula X.
##STR00026##
[0459] wherein independently for each of said at least one
cyclohexenyl nucleoside analog of Formula X:
[0460] Bx is a heterocyclic base moiety;
[0461] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the cyclohexenyl nucleoside
analog to an antisense compound or one of T.sub.3 and T.sub.4 is an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to an antisense compound and the other of T.sub.3
and T.sub.4 is H, a hydroxyl protecting group, a linked conjugate
group, or a 5'-or 3'-terminal group; and
[0462] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6,
q.sub.7, q.sub.8 and q.sub.9 are each, independently, H,
C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6 alkynyl or
other sugar substituent group.
[0463] As used herein, "2'-modified" or "2'-substituted" refers to
a nucleoside comprising a sugar comprising a substituent at the 2'
position other than H or OH. 2'-modified nucleosides, include, but
are not limited to, bicyclic nucleosides wherein the bridge
connecting two carbon atoms of the sugar ring connects the 2'
carbon and another carbon of the sugar ring; and nucleosides with
non-bridging 2' substituents, such as allyl, amino, azido, thio,
O-allyl, O--C.sub.1-C.sub.10 alkyl, --OCF.sub.3,
O--(CH.sub.2).sub.2--O--CH.sub.3, 2'-O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl. 2'-modified nucleosides may further
comprise other modifications, for example at other positions of the
sugar and/or at the nucleobase.
[0464] As used herein, "2'-F" refers to a nucleoside comprising a
sugar comprising a fluoro group at the 2' position of the sugar
ring.
[0465] As used herein, "2'-OMe" or "2'-OCH.sub.3" or "2'-O-methyl"
each refers to a nucleoside comprising a sugar comprising an
--OCH.sub.3 group at the 2' position of the sugar ring.
[0466] As used herein, "MOE" or "2'-MOE" or
"2'-OCH.sub.2CH.sub.2OCH.sub.3" or "2'-O-methoxyethyl" each refers
to a nucleoside comprising a sugar comprising a
--OCH.sub.2CH.sub.2OCH.sub.3 group at the 2' position of the sugar
ring.
[0467] As used herein, "oligonucleotide" refers to a compound
comprising a plurality of linked nucleosides. In certain
embodiments, one or more of the plurality of nucleosides is
modified. In certain embodiments, an oligonucleotide comprises one
or more ribonucleosides (RNA) and/or deoxyribonucleosides
(DNA).
[0468] Many other bicyclo and tricyclo sugar surrogate ring systems
are also known in the art that can be used to modify nucleosides
for incorporation into antisense compounds (see for example review
article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring
systems can undergo various additional substitutions to enhance
activity.
[0469] Methods for the preparations of modified sugars are well
known to those skilled in the art. Some representative U.S. patents
that teach the preparation of such modified sugars include without
limitation, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032
and International Application PCT/US2005/019219, filed Jun. 2, 2005
and published as WO 2005/121371 on Dec. 22, 2005, and each of which
is herein incorporated by reference in its entirety.
[0470] In nucleotides having modified sugar moieties, the
nucleobase moieties (natural, modified or a combination thereof)
are maintained for hybridization with an appropriate nucleic acid
target.
[0471] In certain embodiments, antisense compounds comprise one or
more nucleosides having modified sugar moieties. In certain
embodiments, the modified sugar moiety is 2'-MOE. In certain
embodiments, the 2'-MOE modified nucleosides are arranged in a
gapmer motif. In certain embodiments, the modified sugar moiety is
a bicyclic nucleoside having a (4'-CH(CH.sub.3)--O-2') bridging
group. In certain embodiments, the (4'-CH(CH.sub.3)--O-2') modified
nucleosides are arranged throughout the wings of a gapmer
motif.
Modified Nucleobases
[0472] Nucleobase (or base) modifications or substitutions are
structurally distinguishable from, yet functionally interchangeable
with, naturally occurring or synthetic unmodified nucleobases. Both
natural and modified nucleobases are capable of participating in
hydrogen bonding. Such nucleobase modifications can impart nuclease
stability, binding affinity or some other beneficial biological
property to antisense compounds. Modified nucleobases include
synthetic and natural nucleobases such as, for example,
5-methylcytosine (5-me-C). Certain nucleobase substitutions,
including 5-methylcytosine substitutions, are particularly useful
for increasing the binding affinity of an antisense compound for a
target nucleic acid. For example, 5-methylcytosine substitutions
have been shown to increase nucleic acid duplex stability by
0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B.,
eds., Antisense Research and Applications, CRC Press, Boca Raton,
1993, pp. 276-278).
[0473] Additional modified nucleobases include 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine,
5-propynyl (--C.ident.C--CH3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine.
[0474] Heterocyclic base moieties can also include those in which
the purine or pyrimidine base is replaced with other heterocycles,
for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and
2-pyridone. Nucleobases that are particularly useful for increasing
the binding affinity of antisense compounds include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted
purines, including 2 aminopropyladenine, 5-propynyluracil and
5-propynylcytosine.
[0475] In certain embodiments, antisense compounds targeted to a
CFB nucleic acid comprise one or more modified nucleobases. In
certain embodiments, shortened or gap-widened antisense
oligonucleotides targeted to a CFB nucleic acid comprise one or
more modified nucleobases. In certain embodiments, the modified
nucleobase is 5-methylcytosine. In certain embodiments, each
cytosine is a 5-methylcytosine.
Conjugated Antisense compounds
[0476] In certain embodiments, the present disclosure provides
conjugated antisense compounds. In certain embodiments, the present
disclosure provides conjugated antisense compounds comprising an
antisense oligonucleotide complementary to a nucleic acid
transcript. In certain embodiments, the present disclosure provides
methods comprising contacting a cell with a conjugated antisense
compound comprising an antisense oligonucleotide complementary to a
nucleic acid transcript. In certain embodiments, the present
disclosure provides methods comprising contacting a cell with a
conjugated antisense compound comprising an antisense
oligonucleotide and reducing the amount or activity of a nucleic
acid transcript in a cell.
[0477] The asialoglycoprotein receptor (ASGP-R) has been described
previously. See e.g., Park et al., PNAS vol. 102, No. 47, pp
17125-17129 (2005). Such receptors are expressed on liver cells,
particularly hepatocytes. Further, it has been shown that compounds
comprising clusters of three N-acetylgalactosamine (GalNAc) ligands
are capable of binding to the ASGP-R, resulting in uptake of the
compound into the cell. See e.g., Khorev et al., Bioorganic and
Medicinal Chemistry, 16, 9, pp 5216-5231 (May 2008). Accordingly,
conjugates comprising such GalNAc clusters have been used to
facilitate uptake of certain compounds into liver cells,
specifically hepatocytes. For example it has been shown that
certain GalNAc-containing conjugates increase activity of duplex
siRNA compounds in liver cells in vivo. In such instances, the
GalNAc-containing conjugate is typically attached to the sense
strand of the siRNA duplex. Since the sense strand is discarded
before the antisense strand ultimately hybridizes with the target
nucleic acid, there is little concern that the conjugate will
interfere with activity. Typically, the conjugate is attached to
the 3' end of the sense strand of the siRNA. See e.g., U.S. Pat.
No. 8,106,022. Certain conjugate groups described herein are more
active and/or easier to synthesize than conjugate groups previously
described.
[0478] In certain embodiments of the present invention, conjugates
are attached to single-stranded antisense compounds, including, but
not limited to RNase H based antisense compounds and antisense
compounds that alter splicing of a pre-mRNA target nucleic acid. In
such embodiments, the conjugate should remain attached to the
antisense compound long enough to provide benefit (improved uptake
into cells) but then should either be cleaved, or otherwise not
interfere with the subsequent steps necessary for activity, such as
hybridization to a target nucleic acid and interaction with RNase H
or enzymes associated with splicing or splice modulation. This
balance of properties is more important in the setting of
single-stranded antisense compounds than in siRNA compounds, where
the conjugate may simply be attached to the sense strand. Disclosed
herein are conjugated single-stranded antisense compounds having
improved potency in liver cells in vivo compared with the same
antisense compound lacking the conjugate. Given the required
balance of properties for these compounds such improved potency is
surprising.
[0479] In certain embodiments, conjugate groups herein comprise a
cleavable moiety. As noted, without wishing to be bound by
mechanism, it is logical that the conjugate should remain on the
compound long enough to provide enhancement in uptake, but after
that, it is desirable for some portion or, ideally, all of the
conjugate to be cleaved, releasing the parent compound (e.g.,
antisense compound) in its most active form. In certain
embodiments, the cleavable moiety is a cleavable nucleoside. Such
embodiments take advantage of endogenous nucleases in the cell by
attaching the rest of the conjugate (the cluster) to the antisense
oligonucleotide through a nucleoside via one or more cleavable
bonds, such as those of a phosphodiester linkage. In certain
embodiments, the cluster is bound to the cleavable nucleoside
through a phosphodiester linkage. In certain embodiments, the
cleavable nucleoside is attached to the antisense oligonucleotide
(antisense compound) by a phosphodiester linkage. In certain
embodiments, the conjugate group may comprise two or three
cleavable nucleosides. In such embodiments, such cleavable
nucleosides are linked to one another, to the antisense compound
and/or to the cluster via cleavable bonds (such as those of a
phosphodiester linkage). Certain conjugates herein do not comprise
a cleavable nucleoside and instead comprise a cleavable bond. It is
shown that that sufficient cleavage of the conjugate from the
oligonucleotide is provided by at least one bond that is vulnerable
to cleavage in the cell (a cleavable bond).
[0480] In certain embodiments, conjugated antisense compounds are
prodrugs. Such prodrugs are administered to an animal and are
ultimately metabolized to a more active form. For example,
conjugated antisense compounds are cleaved to remove all or part of
the conjugate resulting in the active (or more active) form of the
antisense compound lacking all or some of the conjugate.
[0481] In certain embodiments, conjugates are attached at the 5'
end of an oligonucleotide. Certain such 5'-conjugates are cleaved
more efficiently than counterparts having a similar conjugate group
attached at the 3' end. In certain embodiments, improved activity
may correlate with improved cleavage. In certain embodiments,
oligonucleotides comprising a conjugate at the 5' end have greater
efficacy than oligonucleotides comprising a conjugate at the 3' end
(see, for example, Examples 56, 81, 83, and 84). Further,
5'-attachment allows simpler oligonucleotide synthesis. Typically,
oligonucleotides are synthesized on a solid support in the 3' to 5'
direction. To make a 3'-conjugated oligonucleotide, typically one
attaches a pre-conjugated 3' nucleoside to the solid support and
then builds the oligonucleotide as usual. However, attaching that
conjugated nucleoside to the solid support adds complication to the
synthesis. Further, using that approach, the conjugate is then
present throughout the synthesis of the oligonucleotide and can
become degraded during subsequent steps or may limit the sorts of
reactions and reagents that can be used. Using the structures and
techniques described herein for 5'-conjugated oligonucleotides, one
can synthesize the oligonucleotide using standard automated
techniques and introduce the conjugate with the final (5'-most)
nucleoside or after the oligonucleotide has been cleaved from the
solid support.
[0482] In view of the art and the present disclosure, one of
ordinary skill can easily make any of the conjugates and conjugated
oligonucleotides herein. Moreover, synthesis of certain such
conjugates and conjugated oligonucleotides disclosed herein is
easier and/or requires few steps, and is therefore less expensive
than that of conjugates previously disclosed, providing advantages
in manufacturing. For example, the synthesis of certain conjugate
groups consists of fewer synthetic steps, resulting in increased
yield, relative to conjugate groups previously described. Conjugate
groups such as GalNAc.sub.3-10 in Example 46 and GalNAc.sub.3-7 in
Example 48 are much simpler than previously described conjugates
such as those described in U.S. Pat. Nos. 8,106,022 or 7,262,177
that require assembly of more chemical intermediates. Accordingly,
these and other conjugates described herein have advantages over
previously described compounds for use with any oligonucleotide,
including single-stranded oligonucleotides and either strand of
double-stranded oligonucleotides (e.g., siRNA).
[0483] Similarly, disclosed herein are conjugate groups having only
one or two GalNAc ligands. As shown, such conjugates groups improve
activity of antisense compounds. Such compounds are much easier to
prepare than conjugates comprising three GalNAc ligands. Conjugate
groups comprising one or two GalNAc ligands may be attached to any
antisense compounds, including single-stranded oligonucleotides and
either strand of double-stranded oligonucleotides (e.g.,
siRNA).
[0484] In certain embodiments, the conjugates herein do not
substantially alter certain measures of tolerability. For example,
it is shown herein that conjugated antisense compounds are not more
immunogenic than unconjugated parent compounds. Since potency is
improved, embodiments in which tolerability remains the same (or
indeed even if tolerability worsens only slightly compared to the
gains in potency) have improved properties for therapy.
[0485] In certain embodiments, conjugation allows one to alter
antisense compounds in ways that have less attractive consequences
in the absence of conjugation. For example, in certain embodiments,
replacing one or more phosphorothioate linkages of a fully
phosphorothioate antisense compound with phosphodiester linkages
results in improvement in some measures of tolerability. For
example, in certain instances, such antisense compounds having one
or more phosphodiester are less immunogenic than the same compound
in which each linkage is a phosphorothioate. However, in certain
instances, as shown in Example 26, that same replacement of one or
more phosphorothioate linkages with phosphodiester linkages also
results in reduced cellular uptake and/or loss in potency. In
certain embodiments, conjugated antisense compounds described
herein tolerate such change in linkages with little or no loss in
uptake and potency when compared to the conjugated
full-phosphorothioate counterpart. In fact, in certain embodiments,
for example, in Examples 44, 57, 59, and 86, oligonucleotides
comprising a conjugate and at least one phosphodiester
internucleoside linkage actually exhibit increased potency in vivo
even relative to a full phosphorothioate counterpart also
comprising the same conjugate. Moreover, since conjugation results
in substantial increases in uptake/potency a small loss in that
substantial gain may be acceptable to achieve improved
tolerability. Accordingly, in certain embodiments, conjugated
antisense compounds comprise at least one phosphodiester
linkage.
[0486] In certain embodiments, conjugation of antisense compounds
herein results in increased delivery, uptake and activity in
hepatocytes. Thus, more compound is delivered to liver tissue.
However, in certain embodiments, that increased delivery alone does
not explain the entire increase in activity. In certain such
embodiments, more compound enters hepatocytes. In certain
embodiments, even that increased hepatocyte uptake does not explain
the entire increase in activity. In such embodiments, productive
uptake of the conjugated compound is increased. For example, as
shown in Example 102, certain embodiments of GalNAc-containing
conjugates increase enrichment of antisense oligonucleotides in
hepatocytes versus non-parenchymal cells. This enrichment is
beneficial for oligonucleotides that target genes that are
expressed in hepatocytes.
[0487] In certain embodiments, conjugated antisense compounds
herein result in reduced kidney exposure. For example, as shown in
Example 20, the concentrations of antisense oligonucleotides
comprising certain embodiments of GalNAc-containing conjugates are
lower in the kidney than that of antisense oligonucleotides lacking
a GalNAc-containing conjugate. This has several beneficial
therapeutic implications. For therapeutic indications where
activity in the kidney is not sought, exposure to kidney risks
kidney toxicity without corresponding benefit. Moreover, high
concentration in kidney typically results in loss of compound to
the urine resulting in faster clearance. Accordingly for non-kidney
targets, kidney accumulation is undesired.
[0488] In certain embodiments, the present disclosure provides
conjugated antisense compounds represented by the formula:
A-B-C-D E-F).sub.q
[0489] wherein
[0490] A is the antisense oligonucleotide;
[0491] B is the cleavable moiety
[0492] C is the conjugate linker
[0493] D is the branching group
[0494] each E is a tether;
[0495] each F is a ligand; and
[0496] q is an integer between 1 and 5.
[0497] In the above diagram and in similar diagrams herein, the
branching group "D" branches as many times as is necessary to
accommodate the number of (E-F) groups as indicated by "q". Thus,
where q=1, the formula is:
A-B-C-D-E-F
[0498] where q=2, the formula is:
##STR00027##
[0499] where q=3, the formula is:
##STR00028##
[0500] where q=4, the formula is:
##STR00029##
[0501] where q=5, the formula is:
##STR00030##
[0502] In certain embodiments, conjugated antisense compounds are
provided having the structure:
##STR00031##
[0503] In certain embodiments, conjugated antisense compounds are
provided having the structure:
##STR00032##
[0504] In certain embodiments, conjugated antisense compounds are
provided having the structure:
##STR00033##
[0505] In certain embodiments, conjugated antisense compounds are
provided having the structure:
##STR00034##
[0506] The present disclosure provides the following non-limiting
numbered embodiments:
Embodiment 1
[0507] The conjugated antisense compound of any of embodiments 1179
to 1182, wherein the tether has a structure selected from
among:
##STR00035##
wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.
Embodiment 2
[0508] The conjugated antisense compound of any of embodiments 1179
to 1182, wherein the tether has the structure:
##STR00036##
Embodiment 3
[0509] The conjugated antisense compound of any of embodiments 1179
to 1182 or 1688 to 1689, wherein the linker has a structure
selected from among:
##STR00037##
Embodiment 4
[0510] The conjugated antisense compound of any of embodiments 1179
to 1182 or 1688 to 1689, wherein the linker has a structure
selected from among:
##STR00038##
[0511] wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or
7.
Embodiment 5
[0512] The conjugated antisense compound of any of embodiments 1179
to 1182 or 1688 to 1689, wherein the linker has the structure:
##STR00039##
[0513] In embodiments having more than one of a particular variable
(e.g., more than one "m" or "n"), unless otherwise indicated, each
such particular variable is selected independently. Thus, for a
structure having more than one n, each n is selected independently,
so they may or may not be the same as one another.
[0514] i. Certain Cleavable Moieties
[0515] In certain embodiments, a cleavable moiety is a cleavable
bond. In certain embodiments, a cleavable moiety comprises a
cleavable bond. In certain embodiments, the conjugate group
comprises a cleavable moiety. In certain such embodiments, the
cleavable moiety attaches to the antisense oligonucleotide. In
certain such embodiments, the cleavable moiety attaches directly to
the cell-targeting moiety. In certain such embodiments, the
cleavable moiety attaches to the conjugate linker. In certain
embodiments, the cleavable moiety comprises a phosphate or
phosphodiester. In certain embodiments, the cleavable moiety is a
cleavable nucleoside or nucleoside analog. In certain embodiments,
the nucleoside or nucleoside analog comprises an optionally
protected heterocyclic base selected from a purine, substituted
purine, pyrimidine or substituted pyrimidine. In certain
embodiments, the cleavable moiety is a nucleoside comprising an
optionally protected heterocyclic base selected from uracil,
thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine,
4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine
and 2-N-isobutyrylguanine. In certain embodiments, the cleavable
moiety is 2'-deoxy nucleoside that is attached to the 3' position
of the antisense oligonucleotide by a phosphodiester linkage and is
attached to the linker by a phosphodiester or phosphorothioate
linkage. In certain embodiments, the cleavable moiety is 2'-deoxy
adenosine that is attached to the 3' position of the antisense
oligonucleotide by a phosphodiester linkage and is attached to the
linker by a phosphodiester or phosphorothioate linkage. In certain
embodiments, the cleavable moiety is 2'-deoxy adenosine that is
attached to the 3' position of the antisense oligonucleotide by a
phosphodiester linkage and is attached to the linker by a
phosphodiester linkage.
[0516] In certain embodiments, the cleavable moiety is attached to
the 3' position of the antisense oligonucleotide. In certain
embodiments, the cleavable moiety is attached to the 5' position of
the antisense oligonucleotide. In certain embodiments, the
cleavable moiety is attached to a 2' position of the antisense
oligonucleotide. In certain embodiments, the cleavable moiety is
attached to the antisense oligonucleotide by a phosphodiester
linkage. In certain embodiments, the cleavable moiety is attached
to the linker by either a phosphodiester or a phosphorothioate
linkage. In certain embodiments, the cleavable moiety is attached
to the linker by a phosphodiester linkage. In certain embodiments,
the conjugate group does not include a cleavable moiety.
[0517] In certain embodiments, the cleavable moiety is cleaved
after the complex has been administered to an animal only after
being internalized by a targeted cell. Inside the cell the
cleavable moiety is cleaved thereby releasing the active antisense
oligonucleotide. While not wanting to be bound by theory it is
believed that the cleavable moiety is cleaved by one or more
nucleases within the cell. In certain embodiments, the one or more
nucleases cleave the phosphodiester linkage between the cleavable
moiety and the linker. In certain embodiments, the cleavable moiety
has a structure selected from among the following:
##STR00040##
wherein each of Bx, Bx.sub.1, Bx.sub.2, and Bx.sub.3 is
independently a heterocyclic base moiety. In certain embodiments,
the cleavable moiety has a structure selected from among the
following:
##STR00041##
[0518] ii. Certain Linkers
[0519] In certain embodiments, the conjugate groups comprise a
linker. In certain such embodiments, the linker is covalently bound
to the cleavable moiety. In certain such embodiments, the linker is
covalently bound to the antisense oligonucleotide. In certain
embodiments, the linker is covalently bound to a cell-targeting
moiety. In certain embodiments, the linker further comprises a
covalent attachment to a solid support. In certain embodiments, the
linker further comprises a covalent attachment to a protein binding
moiety. In certain embodiments, the linker further comprises a
covalent attachment to a solid support and further comprises a
covalent attachment to a protein binding moiety. In certain
embodiments, the linker includes multiple positions for attachment
of tethered ligands. In certain embodiments, the linker includes
multiple positions for attachment of tethered ligands and is not
attached to a branching group. In certain embodiments, the linker
further comprises one or more cleavable bond. In certain
embodiments, the conjugate group does not include a linker.
[0520] In certain embodiments, the linker includes at least a
linear group comprising groups selected from alkyl, amide,
disulfide, polyethylene glycol, ether, thioether (--S--) and
hydroxylamino (--O--N(H)--) groups. In certain embodiments, the
linear group comprises groups selected from alkyl, amide and ether
groups. In certain embodiments, the linear group comprises groups
selected from alkyl and ether groups. In certain embodiments, the
linear group comprises at least one phosphorus linking group. In
certain embodiments, the linear group comprises at least one
phosphodiester group. In certain embodiments, the linear group
includes at least one neutral linking group. In certain
embodiments, the linear group is covalently attached to the
cell-targeting moiety and the cleavable moiety. In certain
embodiments, the linear group is covalently attached to the
cell-targeting moiety and the antisense oligonucleotide. In certain
embodiments, the linear group is covalently attached to the
cell-targeting moiety, the cleavable moiety and a solid support. In
certain embodiments, the linear group is covalently attached to the
cell-targeting moiety, the cleavable moiety, a solid support and a
protein binding moiety. In certain embodiments, the linear group
includes one or more cleavable bond.
[0521] In certain embodiments, the linker includes the linear group
covalently attached to a scaffold group. In certain embodiments,
the scaffold includes a branched aliphatic group comprising groups
selected from alkyl, amide, disulfide, polyethylene glycol, ether,
thioether and hydroxylamino groups. In certain embodiments, the
scaffold includes a branched aliphatic group comprising groups
selected from alkyl, amide and ether groups. In certain
embodiments, the scaffold includes at least one mono or polycyclic
ring system. In certain embodiments, the scaffold includes at least
two mono or polycyclic ring systems. In certain embodiments, the
linear group is covalently attached to the scaffold group and the
scaffold group is covalently attached to the cleavable moiety and
the linker. In certain embodiments, the linear group is covalently
attached to the scaffold group and the scaffold group is covalently
attached to the cleavable moiety, the linker and a solid support.
In certain embodiments, the linear group is covalently attached to
the scaffold group and the scaffold group is covalently attached to
the cleavable moiety, the linker and a protein binding moiety. In
certain embodiments, the linear group is covalently attached to the
scaffold group and the scaffold group is covalently attached to the
cleavable moiety, the linker, a protein binding moiety and a solid
support. In certain embodiments, the scaffold group includes one or
more cleavable bond.
[0522] In certain embodiments, the linker includes a protein
binding moiety. In certain embodiments, the protein binding moiety
is a lipid such as for example including but not limited to
cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric
acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol,
geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,
1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,
O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin
A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g.,
monosaccharide, disaccharide, trisaccharide, tetrasaccharide,
oligosaccharide, polysaccharide), an endosomolytic component, a
steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g.,
triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol
derivatized lithocholic acid), or a cationic lipid. In certain
embodiments, the protein binding moiety is a C16 to C22 long chain
saturated or unsaturated fatty acid, cholesterol, cholic acid,
vitamin E, adamantane or 1-pentafluoropropyl.
[0523] In certain embodiments, a linker has a structure selected
from among:
##STR00042## ##STR00043##
[0524] wherein each n is, independently, from 1 to 20; and p is
from 1 to 6.
[0525] In certain embodiments, a linker has a structure selected
from among:
##STR00044## ##STR00045##
[0526] wherein each n is, independently, from 1 to 20.
[0527] In certain embodiments, a linker has a structure selected
from among:
##STR00046## ##STR00047##
[0528] wherein n is from 1 to 20.
[0529] In certain embodiments, a linker has a structure selected
from among:
##STR00048## ##STR00049##
[0530] wherein each L is, independently, a phosphorus linking group
or a neutral linking group; and
[0531] each n is, independently, from 1 to 20.
[0532] In certain embodiments, a linker has a structure selected
from among:
##STR00050## ##STR00051## ##STR00052##
[0533] In certain embodiments, a linker has a structure selected
from among:
##STR00053## ##STR00054##
[0534] In certain embodiments, a linker has a structure selected
from among:
##STR00055## ##STR00056##
[0535] In certain embodiments, a linker has a structure selected
from among:
##STR00057##
[0536] wherein n is from 1 to 20.
[0537] In certain embodiments, a linker has a structure selected
from among:
##STR00058##
[0538] In certain embodiments, a linker has a structure selected
from among:
##STR00059##
[0539] In certain embodiments, a linker has a structure selected
from among:
##STR00060##
[0540] In certain embodiments, the conjugate linker has the
structure:
##STR00061##
[0541] In certain embodiments, the conjugate linker has the
structure:
##STR00062##
[0542] In certain embodiments, a linker has a structure selected
from among:
##STR00063##
[0543] In certain embodiments, a linker has a structure selected
from among:
##STR00064##
[0544] wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or
7.
[0545] iii. Certain Cell-Targeting Moieties
[0546] In certain embodiments, conjugate groups comprise
cell-targeting moieties. Certain such cell-targeting moieties
increase cellular uptake of antisense compounds. In certain
embodiments, cell-targeting moieties comprise a branching group,
one or more tether, and one or more ligand. In certain embodiments,
cell-targeting moieties comprise a branching group, one or more
tether, one or more ligand and one or more cleavable bond.
[0547] 1. Certain Branching Groups
[0548] In certain embodiments, the conjugate groups comprise a
targeting moiety comprising a branching group and at least two
tethered ligands. In certain embodiments, the branching group
attaches the conjugate linker. In certain embodiments, the
branching group attaches the cleavable moiety. In certain
embodiments, the branching group attaches the antisense
oligonucleotide. In certain embodiments, the branching group is
covalently attached to the linker and each of the tethered ligands.
In certain embodiments, the branching group comprises a branched
aliphatic group comprising groups selected from alkyl, amide,
disulfide, polyethylene glycol, ether, thioether and hydroxylamino
groups. In certain embodiments, the branching group comprises
groups selected from alkyl, amide and ether groups. In certain
embodiments, the branching group comprises groups selected from
alkyl and ether groups. In certain embodiments, the branching group
comprises a mono or polycyclic ring system. In certain embodiments,
the branching group comprises one or more cleavable bond. In
certain embodiments, the conjugate group does not include a
branching group.
[0549] In certain embodiments, a branching group has a structure
selected from among:
##STR00065## ##STR00066## ##STR00067##
[0550] wherein each n is, independently, from 1 to 20;
[0551] j is from 1 to 3; and
[0552] m is from 2 to 6.
[0553] In certain embodiments, a branching group has a structure
selected from among:
##STR00068## ##STR00069##
[0554] wherein each n is, independently, from 1 to 20; and
[0555] m is from 2 to 6.
[0556] In certain embodiments, a branching group has a structure
selected from among:
##STR00070## ##STR00071## ##STR00072##
[0557] In certain embodiments, a branching group has a structure
selected from among:
##STR00073## [0558] wherein each A.sub.1 is independently, O, S,
C.dbd.O or NH; and [0559] each n is, independently, from 1 to
20.
[0560] In certain embodiments, a branching group has a structure
selected from among:
##STR00074## [0561] wherein each A.sub.1 is independently, O, S,
C.dbd.O or NH; and [0562] each n is, independently, from 1 to
20.
[0563] In certain embodiments, a branching group has a structure
selected from among:
##STR00075## [0564] wherein A.sub.1 is O, S, C.dbd.O or NH; and
[0565] each n is, independently, from 1 to 20.
[0566] In certain embodiments, a branching group has a structure
selected from among:
##STR00076##
[0567] In certain embodiments, a branching group has a structure
selected from among:
##STR00077##
[0568] In certain embodiments, a branching group has a structure
selected from among:
##STR00078##
[0569] 2. Certain Tethers
[0570] In certain embodiments, conjugate groups comprise one or
more tethers covalently attached to the branching group. In certain
embodiments, conjugate groups comprise one or more tethers
covalently attached to the linking group. In certain embodiments,
each tether is a linear aliphatic group comprising one or more
groups selected from alkyl, ether, thioether, disulfide, amide and
polyethylene glycol groups in any combination. In certain
embodiments, each tether is a linear aliphatic group comprising one
or more groups selected from alkyl, substituted alkyl, ether,
thioether, disulfide, amide, phosphodiester and polyethylene glycol
groups in any combination. In certain embodiments, each tether is a
linear aliphatic group comprising one or more groups selected from
alkyl, ether and amide groups in any combination. In certain
embodiments, each tether is a linear aliphatic group comprising one
or more groups selected from alkyl, substituted alkyl,
phosphodiester, ether and amide groups in any combination. In
certain embodiments, each tether is a linear aliphatic group
comprising one or more groups selected from alkyl and
phosphodiester in any combination. In certain embodiments, each
tether comprises at least one phosphorus linking group or neutral
linking group.
[0571] In certain embodiments, the tether includes one or more
cleavable bond. In certain embodiments, the tether is attached to
the branching group through either an amide or an ether group. In
certain embodiments, the tether is attached to the branching group
through a phosphodiester group. In certain embodiments, the tether
is attached to the branching group through a phosphorus linking
group or neutral linking group. In certain embodiments, the tether
is attached to the branching group through an ether group. In
certain embodiments, the tether is attached to the ligand through
either an amide or an ether group. In certain embodiments, the
tether is attached to the ligand through an ether group. In certain
embodiments, the tether is attached to the ligand through either an
amide or an ether group. In certain embodiments, the tether is
attached to the ligand through an ether group.
[0572] In certain embodiments, each tether comprises from about 8
to about 20 atoms in chain length between the ligand and the
branching group. In certain embodiments, each tether group
comprises from about 10 to about 18 atoms in chain length between
the ligand and the branching group. In certain embodiments, each
tether group comprises about 13 atoms in chain length.
[0573] In certain embodiments, a tether has a structure selected
from among:
##STR00079##
[0574] wherein each n is, independently, from 1 to 20; and
[0575] each p is from 1 to about 6.
[0576] In certain embodiments, a tether has a structure selected
from among:
##STR00080##
[0577] In certain embodiments, a tether has a structure selected
from among:
##STR00081## [0578] wherein each n is, independently, from 1 to
20.
[0579] In certain embodiments, a tether has a structure selected
from among:
##STR00082## [0580] wherein L is either a phosphorus linking group
or a neutral linking group; [0581] Z.sub.1 is C(.dbd.O)O--R.sub.2;
[0582] Z.sub.2 is H, C.sub.1-C.sub.6 alkyl or substituted
C.sub.1-C.sub.6 alky; [0583] R.sub.2 is H, C.sub.1-C.sub.6 alkyl or
substituted C.sub.1-C.sub.6 alky; and [0584] each m.sub.1 is,
independently, from 0 to 20 wherein at least one m.sub.1 is greater
than O for each tether.
[0585] In certain embodiments, a tether has a structure selected
from among:
##STR00083##
[0586] In certain embodiments, a tether has a structure selected
from among:
##STR00084## [0587] wherein Z.sub.2 is H or CH.sub.3; and [0588]
each m.sub.1 is, independently, from 0 to 20 wherein at least one
m.sub.1 is greater than 0 for each tether.
[0589] In certain embodiments, a tether has a structure selected
from among:
##STR00085##
wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7. [0590]
In certain embodiments, a tether comprises a phosphorus linking
group. In certain embodiments, a tether does not comprise any amide
bonds. In certain embodiments, a tether comprises a phosphorus
linking group and does not comprise any amide bonds.
[0591] 3. Certain Ligands
[0592] In certain embodiments, the present disclosure provides
ligands wherein each ligand is covalently attached to a tether. In
certain embodiments, each ligand is selected to have an affinity
for at least one type of receptor on a target cell. In certain
embodiments, ligands are selected that have an affinity for at
least one type of receptor on the surface of a mammalian liver
cell. In certain embodiments, ligands are selected that have an
affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In
certain embodiments, each ligand is a carbohydrate. In certain
embodiments, each ligand is, independently selected from galactose,
N-acetyl galactoseamine, mannose, glucose, glucosamone and fucose.
In certain embodiments, each ligand is N-acetyl galactoseamine
(GalNAc). In certain embodiments, the targeting moiety comprises 2
to 6 ligands. In certain embodiments, the targeting moiety
comprises 3 ligands. In certain embodiments, the targeting moiety
comprises 3 N-acetyl galactoseamine ligands.
[0593] In certain embodiments, the ligand is a carbohydrate,
carbohydrate derivative, modified carbohydrate, multivalent
carbohydrate cluster, polysaccharide, modified polysaccharide, or
polysaccharide derivative. In certain embodiments, the ligand is an
amino sugar or a thio sugar. For example, amino sugars may be
selected from any number of compounds known in the art, for example
glucosamine, sialic acid, .alpha.-D-galactosamine,
N-Acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose
(GalNAc),
2-Amino-3-O--[(R)-1-carboxyethyl]-2-deoxy-.beta.-D-glucopyranose
(.beta.-muramic acid), 2-Deoxy-2-methylamino-L-glucopyranose,
4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,
2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and
N-Glycoloyl-.alpha.-neuraminic acid. For example, thio sugars may
be selected from the group consisting of
5-Thio-.beta.-D-glucopyranose, Methyl
2,3,4-tri-O-acetyl-1-thio-6-O-trityl-.alpha.-D-glucopyranoside,
4-Thio-.beta.-D-galactopyranose, and ethyl
3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-.alpha.-D-gluco-heptopyranoside-
.
[0594] In certain embodiments, "GalNac" or "Gal-NAc" refers to
2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in
the literature as N-acetyl galactosamine. In certain embodiments,
"N-acetyl galactosamine" refers to
2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments,
"GalNac" or "Gal-NAc" refers to
2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments,
"GalNac" or "Gal-NAc" refers to
2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both the
.beta.-form: 2-(Acetylamino)-2-deoxy-.beta.-D-galactopyranose and
.alpha.-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain
embodiments, both the .beta.-form:
2-(Acetylamino)-2-deoxy-.beta.-D-galactopyranose and .alpha.-form:
2-(Acetylamino)-2-deoxy-D-galactopyranose may be used
interchangeably. Accordingly, in structures in which one form is
depicted, these structures are intended to include the other form
as well. For example, where the structure for an .alpha.-form:
2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure
is intended to include the other form as well. In certain
embodiments, In certain preferred embodiments, the .beta.-form
2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred
embodiment.
##STR00086##
[0595] In certain embodiments one or more ligand has a structure
selected from among:
##STR00087##
[0596] wherein each R.sub.1 is selected from OH and NHCOOH.
[0597] In certain embodiments one or more ligand has a structure
selected from among:
##STR00088##
[0598] In certain embodiments one or more ligand has a structure
selected from among:
##STR00089##
[0599] In certain embodiments one or more ligand has a structure
selected from among:
##STR00090##
[0600] i. Certain Conjugates
[0601] In certain embodiments, conjugate groups comprise the
structural features above. In certain such embodiments, conjugate
groups have the following structure:
##STR00091##
[0602] wherein each n is, independently, from 1 to 20.
[0603] In certain such embodiments, conjugate groups have the
following structure:
##STR00092##
[0604] In certain such embodiments, conjugate groups have the
following structure:
##STR00093##
[0605] wherein each n is, independently, from 1 to 20;
[0606] Z is H or a linked solid support;
[0607] Q is an antisense compound;
[0608] X is O or S; and
[0609] Bx is a heterocyclic base moiety.
[0610] In certain such embodiments, conjugate groups have the
following structure:
##STR00094##
[0611] In certain such embodiments, conjugate groups have the
following structure:
##STR00095##
[0612] In certain such embodiments, conjugate groups have the
following structure:
##STR00096##
[0613] In certain such embodiments, conjugate groups have the
following structure:
##STR00097##
[0614] In certain such embodiments, conjugate groups have the
following structure:
##STR00098##
[0615] In certain such embodiments, conjugate groups have the
following structure:
##STR00099##
[0616] In certain such embodiments, conjugate groups have the
following structure:
##STR00100##
[0617] In certain such embodiments, conjugate groups have the
following structure:
##STR00101##
[0618] In certain embodiments, conjugates do not comprise a
pyrrolidine.
[0619] In certain such embodiments, conjugate groups have the
following structure:
##STR00102##
[0620] In certain such embodiments, conjugate groups have the
following structure:
##STR00103##
[0621] In certain such embodiments, conjugate groups have the
following structure:
##STR00104##
[0622] In certain such embodiments, conjugate groups have the
following structure:
##STR00105##
[0623] In certain such embodiments, conjugate groups have the
following structure:
##STR00106##
[0624] In certain such embodiments, conjugate groups have the
following structure:
##STR00107##
[0625] In certain such embodiments, conjugate groups have the
following structure:
##STR00108##
[0626] In certain such embodiments, conjugate groups have the
following structure:
##STR00109##
[0627] In certain such embodiments, conjugate groups have the
following structure:
##STR00110##
[0628] In certain such embodiments, conjugate groups have the
following structure:
##STR00111##
[0629] In certain such embodiments, conjugate groups have the
following structure:
##STR00112##
In certain embodiments, the cell-targeting moiety of the conjugate
group has the following structure:
##STR00113##
wherein X is a substituted or unsubstituted tether of six to eleven
consecutively bonded atoms. In certain embodiments, the
cell-targeting moiety of the conjugate group has the following
structure:
##STR00114##
wherein X is a substituted or unsubstituted tether of ten
consecutively bonded atoms. In certain embodiments, the
cell-targeting moiety of the conjugate group has the following
structure:
##STR00115##
wherein X is a substituted or unsubstituted tether of four to
eleven consecutively bonded atoms and wherein the tether comprises
exactly one amide bond. In certain embodiments, the cell-targeting
moiety of the conjugate group has the following structure:
##STR00116##
wherein Y and Z are independently selected from a C.sub.1-C.sub.12
substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a
group comprising an ether, a ketone, an amide, an ester, a
carbamate, an amine, a piperidine, a phosphate, a phosphodiester, a
phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a
thioether. In certain such embodiments, the cell-targeting moiety
of the conjugate group has the following structure:
##STR00117##
wherein Y and Z are independently selected from a C.sub.1-C.sub.12
substituted or unsubstituted alkyl group, or a group comprising
exactly one ether or exactly two ethers, an amide, an amine, a
piperidine, a phosphate, a phosphodiester, or a phosphorothioate.
In certain such embodiments, the cell-targeting moiety of the
conjugate group has the following structure:
##STR00118##
wherein Y and Z are independently selected from a C.sub.1-C.sub.12
substituted or unsubstituted alkyl group. In certain such
embodiments, the cell-targeting moiety of the conjugate group has
the following structure:
##STR00119##
wherein m and n are independently selected from 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, and 12. In certain such embodiments, the
cell-targeting moiety of the conjugate group has the following
structure:
##STR00120##
wherein m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4. In certain
embodiments, the cell-targeting moiety of the conjugate group has
the following structure:
##STR00121##
wherein X is a substituted or unsubstituted tether of four to
thirteen consecutively bonded atoms, and wherein X does not
comprise an ether group. In certain embodiments, the cell-targeting
moiety of the conjugate group has the following structure:
##STR00122##
wherein X is a substituted or unsubstituted tether of eight
consecutively bonded atoms, and wherein X does not comprise an
ether group. In certain embodiments, the cell-targeting moiety of
the conjugate group has the following structure:
##STR00123##
wherein X is a substituted or unsubstituted tether of four to
thirteen consecutively bonded atoms, and wherein the tether
comprises exactly one amide bond, and wherein X does not comprise
an ether group. In certain embodiments, the cell-targeting moiety
of the conjugate group has the following structure:
##STR00124##
wherein X is a substituted or unsubstituted tether of four to
thirteen consecutively bonded atoms and wherein the tether consists
of an amide bond and a substituted or unsubstituted
C.sub.2-C.sub.11 alkyl group. In certain embodiments, the
cell-targeting moiety of the conjugate group has the following
structure:
##STR00125##
wherein Y is selected from a C.sub.1-C.sub.12 substituted or
unsubstituted alkyl, alkenyl, or alkynyl group, or a group
comprising an ether, a ketone, an amide, an ester, a carbamate, an
amine, a piperidine, a phosphate, a phosphodiester, a
phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a
thioether. In certain such embodiments, the cell-targeting moiety
of the conjugate group has the following structure:
##STR00126##
wherein Y is selected from a C.sub.1-C.sub.12 substituted or
unsubstituted alkyl group, or a group comprising an ether, an
amine, a piperidine, a phosphate, a phosphodiester, or a
phosphorothioate. In certain such embodiments, the cell-targeting
moiety of the conjugate group has the following structure:
##STR00127##
wherein Y is selected from a C.sub.1-C.sub.12 substituted or
unsubstituted alkyl group. In certain such embodiments, the
cell-targeting moiety of the conjugate group has the following
structure:
##STR00128##
Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
[0630] In certain such embodiments, the cell-targeting moiety of
the conjugate group has the following structure:
##STR00129##
wherein n is 4, 5, 6, 7, or 8.
[0631] In certain embodiments, conjugates do not comprise a
pyrrolidine.
[0632] a Certain Conjugated Antisense Compounds
[0633] In certain embodiments, the conjugates are bound to a
nucleoside of the antisense oligonucleotide at the 2', 3', of 5'
position of the nucleoside. In certain embodiments, a conjugated
antisense compound has the following structure:
A-B-C-D E-F).sub.q
[0634] wherein
[0635] A is the antisense oligonucleotide;
[0636] B is the cleavable moiety
[0637] C is the conjugate linker
[0638] D is the branching group
[0639] each E is a tether;
[0640] each F is a ligand; and
[0641] q is an integer between 1 and 5.
In certain embodiments, a conjugated antisense compound has the
following structure:
A-C-D E-F).sub.q
[0642] wherein
[0643] A is the antisense oligonucleotide;
[0644] C is the conjugate linker
[0645] D is the branching group
[0646] each E is a tether;
[0647] each F is a ligand; and
[0648] q is an integer between 1 and 5.
[0649] In certain such embodiments, the conjugate linker comprises
at least one cleavable bond.
[0650] In certain such embodiments, the branching group comprises
at least one cleavable bond.
[0651] In certain embodiments each tether comprises at least one
cleavable bond.
In certain embodiments, the conjugates are bound to a nucleoside of
the antisense oligonucleotide at the 2', 3', of 5' position of the
nucleoside.
[0652] In certain embodiments, a conjugated antisense compound has
the following structure:
A-B-C E-F).sub.q
[0653] wherein
[0654] A is the antisense oligonucleotide;
[0655] B is the cleavable moiety
[0656] C is the conjugate linker
[0657] each E is a tether;
[0658] each F is a ligand; and
[0659] q is an integer between 1 and 5.
In certain embodiments, the conjugates are bound to a nucleoside of
the antisense oligonucleotide at the 2', 3', of 5' position of the
nucleoside. In certain embodiments, a conjugated antisense compound
has the following structure:
A-C E-F).sub.q
[0660] wherein
[0661] A is the antisense oligonucleotide;
[0662] C is the conjugate linker
[0663] each E is a tether;
[0664] each F is a ligand; and
[0665] q is an integer between 1 and 5.
In certain embodiments, a conjugated antisense compound has the
following structure:
A-B-D E-F).sub.q
[0666] wherein
[0667] A is the antisense oligonucleotide;
[0668] B is the cleavable moiety
[0669] D is the branching group
[0670] each E is a tether;
[0671] each F is a ligand; and
[0672] q is an integer between 1 and 5.
In certain embodiments, a conjugated antisense compound has the
following structure:
A-D E-F).sub.q
[0673] wherein
[0674] A is the antisense oligonucleotide;
[0675] D is the branching group
[0676] each E is a tether;
[0677] each F is a ligand; and
[0678] q is an integer between 1 and 5.
[0679] In certain such embodiments, the conjugate linker comprises
at least one cleavable bond.
[0680] In certain embodiments each tether comprises at least one
cleavable bond.
[0681] In certain embodiments, a conjugated antisense compound has
a structure selected from among the following:
##STR00130##
[0682] In certain embodiments, a conjugated antisense compound has
a structure selected from among the following:
##STR00131##
[0683] In certain embodiments, a conjugated antisense compound has
a structure selected from among the following:
##STR00132##
[0684] Representative United States patents, United States patent
application publications, and international patent application
publications that teach the preparation of certain of the above
noted conjugates, conjugated antisense compounds, tethers, linkers,
branching groups, ligands, cleavable moieties as well as other
modifications include without limitation, U.S. Pat. Nos. 5,994,517,
6,300,319, 6,660,720, 6,906,182, 7,262,177, 7,491,805, 8,106,022,
7,723,509, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO
2012/037254, each of which is incorporated by reference herein in
its entirety.
[0685] Representative publications that teach the preparation of
certain of the above noted conjugates, conjugated antisense
compounds, tethers, linkers, branching groups, ligands, cleavable
moieties as well as other modifications include without limitation,
BIESSEN et al., "The Cholesterol Derivative of a Triantennary
Galactoside with High Affinity for the Hepatic Asialoglycoprotein
Receptor: a Potent Cholesterol Lowering Agent" J. Med. Chem. (1995)
38:1846-1852, BIESSEN et al., "Synthesis of Cluster Galactosides
with High Affinity for the Hepatic Asialoglycoprotein Receptor" J.
Med. Chem. (1995) 38:1538-1546, LEE et al., "New and more efficient
multivalent glyco-ligands for asialoglycoprotein receptor of
mammalian hepatocytes" Bioorganic & Medicinal Chemistry (2011)
19:2494-2500, RENSEN et al., "Determination of the Upper Size Limit
for Uptake and Processing of Ligands by the Asialoglycoprotein
Receptor on Hepatocytes in Vitro and in Vivo" J. Biol. Chem. (2001)
276(40):37577-37584, RENSEN et al., "Design and Synthesis of Novel
N-Acetylgalactosamine-Terminated Glycolipids for Targeting of
Lipoproteins to the Hepatic Asialoglycoprotein Receptor" J. Med.
Chem. (2004) 47:5798-5808, SLIEDREGT et al., "Design and Synthesis
of Novel Amphiphilic Dendritic Galactosides for Selective Targeting
of Liposomes to the Hepatic Asialoglycoprotein Receptor" J. Med.
Chem. (1999) 42:609-618, and Valentijn et al., "Solid-phase
synthesis of lysine-based cluster galactosides with high affinity
for the Asialoglycoprotein Receptor" Tetrahedron, 1997, 53(2),
759-770, each of which is incorporated by reference herein in its
entirety.
[0686] In certain embodiments, conjugated antisense compounds
comprise an RNase H based oligonucleotide (such as a gapmer) or a
splice modulating oligonucleotide (such as a fully modified
oligonucleotide) and any conjugate group comprising at least one,
two, or three GalNAc groups. In certain embodiments a conjugated
antisense compound comprises any conjugate group found in any of
the following references: Lee, Carbohydr Res, 1978, 67, 509-514;
Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int
J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984,
23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328;
Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et
al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al.,
Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997,
38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato
et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem,
2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362,
38-43; Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et
al., Bioorg Med Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et
al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg
Med Chem, 2008, 16, 5216-5231; Lee et al., Bioorg Med Chem, 2011,
19, 2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46;
Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 7445-7448;
Biessen et al., J Med Chem, 1995, 38, 1846-1852; Sliedregt et al.,
J Med Chem, 1999, 42, 609-618; Rensen et al., J Med Chem, 2004, 47,
5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26,
169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato
et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J Org
Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14,
1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et
al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug
Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12,
5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12,
103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et
al., Bioorg Med Chem, 2013, 21, 5275-5281; International
applications WO1998/013381; WO2011/038356; WO1997/046098;
WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053;
WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230;
WO2013/075035; WO2012/083185; WO2012/083046; WO2009/082607;
WO2009/134487; WO2010/144740; WO2010/148013; WO1997/020563;
WO2010/088537; WO2002/043771; WO2010/129709; WO2012/068187;
WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352;
WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat. Nos.
4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319;
8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;
6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772;
8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182;
6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent
Application Publications US2011/0097264; US2011/0097265;
US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044;
US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869;
US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042;
US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115;
US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509;
US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512;
US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and
US2009/0203132; each of which is incorporated by reference in its
entirety.
In Vitro Testing of Antisense Oligonucleotides
[0687] Described herein are methods for treatment of cells with
antisense oligonucleotides, which can be modified appropriately for
treatment with other antisense compounds.
[0688] Cells may be treated with antisense oligonucleotides when
the cells reach approximately 60-80% confluency in culture.
[0689] One reagent commonly used to introduce antisense
oligonucleotides into cultured cells includes the cationic lipid
transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.).
Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM
1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final
concentration of antisense oligonucleotide and a LIPOFECTIN
concentration that may range from 2 to 12 ug/mL per 100 nM
antisense oligonucleotide.
[0690] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in
OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to
achieve the desired concentration of antisense oligonucleotide and
a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per
100 nM antisense oligonucleotide.
[0691] Another technique used to introduce antisense
oligonucleotides into cultured cells includes electroporation.
[0692] Yet another technique used to introduce antisense
oligonucleotides into cultured cells includes free uptake of the
oligonucleotides by the cells.
[0693] Cells are treated with antisense oligonucleotides by routine
methods. Cells may be harvested 16-24 hours after antisense
oligonucleotide treatment, at which time RNA or protein levels of
target nucleic acids are measured by methods known in the art and
described herein. In general, when treatments are performed in
multiple replicates, the data are presented as the average of the
replicate treatments. The concentration of antisense
oligonucleotide used varies from cell line to cell line. Methods to
determine the optimal antisense oligonucleotide concentration for a
particular cell line are well known in the art. Antisense
oligonucleotides are typically used at concentrations ranging from
1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense
oligonucleotides are used at higher concentrations ranging from 625
to 20,000 nM when transfected using electroporation.
RNA Isolation
[0694] RNA analysis can be performed on total cellular RNA or
poly(A)+ mRNA. Methods of RNA isolation are well known in the art.
RNA is prepared using methods well known in the art, for example,
using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according
to the manufacturer's recommended protocols.
Certain Indications
[0695] Certain embodiments provided herein relate to methods of
treating, preventing, or ameliorating a disease associated with
dysregulation of the complement alternative pathway in a subject by
administration of a CFB specific inhibitor, such as an antisense
compound targeted to CFB.
[0696] Examples of renal diseases associated with dysregulation of
the complement alternative pathway treatable, preventable, and/or
ameliorable with the methods provided herein include C3
glomerulopathy, atypical hemolytic uremic syndrome (aHUS), dense
deposit disease (DDD; also known as MPGN Type II or C3Neph), and
CFHR5 nephropathy.
[0697] Additional renal diseases associated with dysregulation of
the complement alternative pathway treatable, preventable, and/or
ameliorable with the methods provided herein include IgA
nephropathy;
[0698] mesangiocapillary (membranoproliferative) glomerulonephritis
(MPGN); autoimmune disorders including lupus nephritis and systemic
lupus erythematosus (SLE); infection-induced glomerulonephritis
(also known as Postinfectious glomerulonephritis); and renal
ischemia-reperfusion injury, for example post-transplant renal
ischemia-reperfusion injury.
[0699] Examples of non-renal disorders associated with
dysregulation of the complement alternative pathway treatable
and/or preventable with the methods provided herein include ocular
diseases such as macular degeneration, for example age-related
macular degeneration (AMD), including wet AMD and dry AMD, such as
Geographic Atrophy; neuromyelitis optica; corneal disease, such as
corneal inflammation; autoimmune uveitis; and diabetic retinopathy.
It has been reported that complement system is involved in ocular
diseases. Jha P, et al., Mol Immunol (2007) 44(16): 3901-3908.
Additional examples of non-renal disorders associated with
dysregulation of the complement alternative pathway treatable
and/or preventable with the methods provided herein include
ANCA-assocaited vasculitis, antiphospholipid syndrome (also known
as antiphospholipid antibody syndrome (APS)), asthma, rheumatoid
arthritis, Myasthenia Gravis, and multiple sclerosis.
[0700] Certain embodiments provided herein relate to methods of
treating, preventing, or ameliorating a renal disease associated
with dysregulation of the complement alternative pathway in a
subject by administration of a CFB specific inhibitor, such as an
antisense compound targeted to CFB. In certain aspects, the renal
disease is lupus nephritis, systemic lupus erythematosus (SLE),
dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5
nephropathy, or atypical hemolytic uremic syndrome (aHUS), or any
combination thereof.
[0701] Certain embodiments provided herein relate to methods of
treating, preventing, or ameliorating macular degeneration, such as
age-related macular degeneration (AMD), in a subject by
administration of a CFB specific inhibitor, such as an antisense
compound targeted to CFB. In certain aspects, the AMD is wet AMD or
dry AMD. In certain aspects, dry AMD can be Geographic Atrophy.
Studies have demonstrated the association of complement alternative
pathway dysregulation and AMD. Complement components are common
constituents of ocular drusen, the extracellular material that
accumulates in the macula of AMD patients. Furthermore, it has been
reported that CFH and CFB variants account for nearly 75% of AMD
cases in northern Europe and North America. It has also been found
that a specific CFB polymorphism confers protection against AMD.
Patel, N. et al., Eye (2008) 22(6):768-76. Additionally, CFB
homozygous null mice have lower complement pathway activity,
exhibit smaller ocular lesions, and choroidal neovascularization
(CNV) after laser photocoagulation. Rohrer, B. et al., Invest
Ophthalmol Vis Sci. (2009) 50(7):3056-64. Furthermore, CFB siRNA
treatment protects mice from laser induced CNV. Bora, N S et al., J
Immunol. (2006) 177(3):1872-8. Studies have also shown that the
kidney and eye share developmental pathways and structural features
including basement membrane collagen IV protomer composition and
vascularity. Savige et al., J Am Soc Nephrol. (2011) 22(8):1403-15.
There is evidence that the complement pathway is involved in renal
and ocular diseases. For instance, inherited complement regulatory
protein deficiency causes predisposition to atypical hemolytic
uremic syndrome and AMD. Richards A et al., Adv Immunol. (2007)
96:141-77. Additionally, chronic kidney disease has been associated
with AMD. Nitsch, D. et al., Ophthalmic Epidemiol. (2009)
16(3):181-6; Choi, J. et al, Ophthalmic Epidemiol. (2011)
18(6):259-63. Dense deposit disease (DDD), a kidney disease
associated with dysregulated complement alternative pathway, is
characterized by acute nephritic syndrome and ocular drusen. Cruz
and Smith, GeneReviews (2007) July 20. Moreover, mice harboring
genetic deletion of a component of the complement alternative
pathway have coexisting renal and ocular disease phenotypes. It has
been reported that CFH homozygous null mice develop DDD and present
retinal abnormalities and visual dysfunction. Pickering et al., Nat
Genet. (2002) 31(4):424-8. Mouse models of renal diseases
associated with dysregulation of the complement alternative pathway
are also accepted as models of AMD. Pennesi M E et al., Mol Aspects
Med (2012) 33:487-509. CFH null mice, for example, are an accepted
model for renal diseases, such as DDD, and AMD. Furthermore, it has
been reported that AMD is associated with the systemic source of
complement factors, which accumulate locally in the eye to drive
alternative pathway complement activation. Loyet et al., Invest
Ophthalmol Vis Sci. (2012) 53(10):6628-37.
EXAMPLES
[0702] The following examples illustrate certain embodiments of the
present disclosure and are not limiting. Moreover, where specific
embodiments are provided, the inventors have contemplated generic
application of those specific embodiments. For example, disclosure
of an oligonucleotide having a particular motif provides reasonable
support for additional oligonucleotides having the same or similar
motif. And, for example, where a particular high-affinity
modification appears at a particular position, other high-affinity
modifications at the same position are considered suitable, unless
otherwise indicated.
Example 1: General Method for the Preparation of Phosphoramidites,
Compounds 1, 1a and 2
##STR00133##
[0704] Bx is a heterocyclic base;
[0705] Compounds 1, 1a and 2 were prepared as per the procedures
well known in the art as described in the specification herein (see
Seth et al., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org.
Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series,
2008, 52(1), 553-554); and also see published PCT International
Applications (WO 2011/115818, WO 2010/077578, WO2010/036698,
WO2009/143369, WO 2009/006478, and WO 2007/090071), and U.S. Pat.
No. 7,569,686).
Example 2: Preparation of Compound 7
##STR00134##
[0707] Compounds 3
(2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-.beta.-Dgalactopyranose
or galactosamine pentaacetate) is commercially available. Compound
5 was prepared according to published procedures (Weber et al., J.
Med. Chem., 1991, 34, 2692).
Example 3: Preparation of Compound 11
##STR00135##
[0709] Compounds 8 and 9 are commercially available.
Example 4: Preparation of Compound 18
##STR00136## ##STR00137##
[0711] Compound 11 was prepared as per the procedures illustrated
in Example 3. Compound 14 is commercially available. Compound 17
was prepared using similar procedures reported by Rensen et al., J.
Med. Chem., 2004, 47, 5798-5808.
Example 5: Preparation of Compound 23
##STR00138##
[0713] Compounds 19 and 21 are commercially available.
Example 6: Preparation of Compound 24
##STR00139##
[0715] Compounds 18 and 23 were prepared as per the procedures
illustrated in Examples 4 and 5.
Example 7: Preparation of Compound 25
##STR00140##
[0717] Compound 24 was prepared as per the procedures illustrated
in Example 6.
Example 8: Preparation of Compound 26
##STR00141##
[0719] Compound 24 is prepared as per the procedures illustrated in
Example 6.
Example 9: General Preparation of Conjugated ASOs Comprising
GalNAc.sub.3-1 at the 3' Terminus, Compound 29
##STR00142## ##STR00143##
[0721] Wherein the protected GalNAc.sub.3-1 has the structure:
##STR00144##
[0722] The GalNAc.sub.3 cluster portion of the conjugate group
GalNAc.sub.3-1 (GalNAc.sub.3-1.sub.a) can be combined with any
cleavable moiety to provide a variety of conjugate groups. Wherein
GalNAc.sub.3-1.sub.a has the formula:
##STR00145##
[0723] The solid support bound protected GalNAc.sub.3-1, Compound
25, was prepared as per the procedures illustrated in Example 7.
Oligomeric Compound 29 comprising GalNAc.sub.3-1 at the 3' terminus
was prepared using standard procedures in automated DNA/RNA
synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45,
3623-3627). Phosphoramidite building blocks, Compounds 1 and 1a
were prepared as per the procedures illustrated in Example 1. The
phosphoramidites illustrated are meant to be representative and not
intended to be limiting as other phosphoramidite building blocks
can be used to prepare oligomeric compounds having a predetermined
sequence and composition. The order and quantity of
phosphoramidites added to the solid support can be adjusted to
prepare gapped oligomeric compounds as described herein. Such
gapped oligomeric compounds can have predetermined composition and
base sequence as dictated by any given target.
Example 10: General Preparation Conjugated ASOs Comprising
GalNAc.sub.3-1 at the 5' Terminus, Compound 34
##STR00146## ##STR00147##
[0725] The Unylinker.TM. 30 is commercially available. Oligomeric
Compound 34 comprising a GalNAc.sub.3-1 cluster at the 5' terminus
is prepared using standard procedures in automated DNA/RNA
synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45,
3623-3627). Phosphoramidite building blocks, Compounds 1 and 1a
were prepared as per the procedures illustrated in Example 1. The
phosphoramidites illustrated are meant to be representative and not
intended to be limiting as other phosphoramidite building blocks
can be used to prepare an oligomeric compound having a
predetermined sequence and composition. The order and quantity of
phosphoramidites added to the solid support can be adjusted to
prepare gapped oligomeric compounds as described herein. Such
gapped oligomeric compounds can have predetermined composition and
base sequence as dictated by any given target.
Example 11: Preparation of Compound 39
##STR00148## ##STR00149##
[0727] Compounds 4, 13 and 23 were prepared as per the procedures
illustrated in Examples 2, 4, and 5. Compound 35 is prepared using
similar procedures published in Rouchaud et al., Eur. J. Org.
Chem., 2011, 12, 2346-2353.
Example 12: Preparation of Compound 40
##STR00150##
[0729] Compound 38 is prepared as per the procedures illustrated in
Example 11.
Example 13: Preparation of Compound 44
##STR00151## ##STR00152##
[0731] Compounds 23 and 36 are prepared as per the procedures
illustrated in Examples 5 and 11. Compound 41 is prepared using
similar procedures published in WO 2009082607.
Example 14: Preparation of Compound 45
##STR00153##
[0733] Compound 43 is prepared as per the procedures illustrated in
Example 13.
Example 15: Preparation of Compound 47
##STR00154##
[0735] Compound 46 is commercially available.
Example 16: Preparation of Compound 53
##STR00155## ##STR00156##
[0737] Compounds 48 and 49 are commercially available. Compounds 17
and 47 are prepared as per the procedures illustrated in Examples 4
and 15.
Example 17: Preparation of Compound 54
##STR00157##
[0739] Compound 53 is prepared as per the procedures illustrated in
Example 16.
Example 18: Preparation of Compound 55
##STR00158##
[0741] Compound 53 is prepared as per the procedures illustrated in
Example 16.
Example 19: General Method for the Preparation of Conjugated ASOs
Comprising GalNAc.sub.3-1 at the 3' Position Via Solid Phase
Techniques (Preparation of ISIS 647535, 647536 and 651900)
[0742] Unless otherwise stated, all reagents and solutions used for
the synthesis of oligomeric compounds are purchased from commercial
sources. Standard phosphoramidite building blocks and solid support
are used for incorporation nucleoside residues which include for
example T, A, G, and .sup.mC residues. A 0.1 M solution of
phosphoramidite in anhydrous acetonitrile was used for
.beta.-D-2'-deoxyribonucleoside and 2'-MOE.
[0743] The ASO syntheses were performed on ABI 394 synthesizer (1-2
.mu.mol scale) or on GE Healthcare Bioscience AKTA oligopilot
synthesizer (40-200 .mu.mol scale) by the phosphoramidite coupling
method on an GalNAc.sub.3-1 loaded VIMAD solid support (110
.mu.mol/g, Guzaev et al., 2003) packed in the column. For the
coupling step, the phosphoramidites were delivered 4 fold excess
over the loading on the solid support and phosphoramidite
condensation was carried out for 10 min. All other steps followed
standard protocols supplied by the manufacturer. A solution of 6%
dichloroacetic acid in toluene was used for removing
dimethoxytrityl (DMT) group from 5'-hydroxyl group of the
nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH.sub.3CN
was used as activator during coupling step. Phosphorothioate
linkages were introduced by sulfurization with 0.1 M solution of
xanthane hydride in 1:1 pyridine/CH.sub.3CN for a contact time of 3
minutes. A solution of 20% tert-butylhydroperoxide in CH.sub.3CN
containing 6% water was used as an oxidizing agent to provide
phosphodiester internucleoside linkages with a contact time of 12
minutes.
[0744] After the desired sequence was assembled, the cyanoethyl
phosphate protecting groups were deprotected using a 1:1 (v/v)
mixture of triethylamine and acetonitrile with a contact time of 45
minutes. The solid-support bound ASOs were suspended in aqueous
ammonia (28-30 wt %) and heated at 55.degree. C. for 6 h.
[0745] The unbound ASOs were then filtered and the ammonia was
boiled off. The residue was purified by high pressure liquid
chromatography on a strong anion exchange column (GE Healthcare
Bioscience, Source 30Q, 30 .mu.m, 2.54.times.8 cm, A=100 mM
ammonium acetate in 30% aqueous CH.sub.3CN, B=1.5 M NaBr in A,
0-40% of B in 60 min, flow 14 mL min-1, .lamda.=260 nm). The
residue was desalted by HPLC on a reverse phase column to yield the
desired ASOs in an isolated yield of 15-30% based on the initial
loading on the solid support. The ASOs were characterized by
ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.
[0746] Antisense oligonucleotides not comprising a conjugate were
synthesized using standard oligonucleotide synthesis procedures
well known in the art.
[0747] Using these methods, three separate antisense compounds
targeting ApoC III were prepared. As summarized in Table 17, below,
each of the three antisense compounds targeting ApoC III had the
same nucleobase sequence; ISIS 304801 is a 5-10-5 MOE gapmer having
all phosphorothioate linkages; ISIS 647535 is the same as ISIS
304801, except that it had a GalNAc.sub.3-1 conjugated at its 3'
end; and ISIS 647536 is the same as ISIS 647535 except that certain
internucleoside linkages of that compound are phosphodiester
linkages. As further summarized in Table 17, two separate antisense
compounds targeting SRB-1 were synthesized. ISIS 440762 was a
2-10-2 cEt gapmer with all phosphorothioate internucleoside
linkages; ISIS 651900 is the same as ISIS 440762, except that it
included a GalNAc.sub.3-1 at its 3'-end.
TABLE-US-00003 TABLE 17 Modified ASO targeting ApoC III and SRB-1
SEQ CalCd Observed ID ASO Sequence (5' to 3') Target Mass Mass No.
ISIS
A.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.dsT.sub.dsT-
.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC-
.sub.dsT.sub.esT.sub.esT.sub.esA.sub.esT.sub.e ApoC 7165.4 7164.4
821 304801 III ISIS
A.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.dsT.sub.dsT-
.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC-
.sub.dsT.sub.esT.sub.esT.sub.esA.sub.es ApoC 9239.5 9237.8 822
647535 III ISIS
A.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.dsT.sub.dsT-
.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC-
.sub.dsT.sub.eoT.sub.eoT.sub.esA.sub.es ApoC 9142.9 9140.8 822
647536 III ISIS
T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT-
.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub.k
SRB- 4647.0 4646.4 823 440762 1 ISIS
T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT-
.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.m SRB-
6721.1 6719.4 824 651900 1
Subscripts: "e" indicates 2'-MOE modified nucleoside; "d" indicates
.beta.-D-2'-deoxyribonucleoside; "k" indicates 6'-(S)--CH.sub.3
bicyclic nucleoside (e.g. cEt); "s" indicates phosphorothioate
internucleoside linkages (PS); "o" indicates phosphodiester
internucleoside linkages (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Superscript "m" indicates 5-methylcytosines.
"GalNAc.sub.3-1" indicates a conjugate group having the structure
shown previously in Example 9. Note that GalNAc.sub.3-1 comprises a
cleavable adenosine which links the ASO to remainder of the
conjugate, which is designated "GalNAc.sub.3-1a." This nomenclature
is used in the above table to show the full nucleobase sequence,
including the adenosine, which is part of the conjugate. Thus, in
the above table, the sequences could also be listed as ending with
"GalNAc.sub.3-1" with the "Ado" omitted. This convention of using
the subscript "a" to indicate the portion of a conjugate group
lacking a cleavable nucleoside or cleavable moiety is used
throughout these Examples. This portion of a conjugate group
lacking the cleavable moiety is referred to herein as a "cluster"
or "conjugate cluster" or "GalNAc.sub.3 cluster." In certain
instances it is convenient to describe a conjugate group by
separately providing its cluster and its cleavable moiety.
Example 20: Dose-Dependent Antisense Inhibition of Human ApoC III
in huApoC III Transgenic Mice
[0748] ISIS 304801 and ISIS 647535, each targeting human ApoC III
and described above, were separately tested and evaluated in a
dose-dependent study for their ability to inhibit human ApoC III in
human ApoC III transgenic mice.
Treatment
[0749] Human ApoCIII transgenic mice were maintained on a 12-hour
light/dark cycle and fed ad libitum Teklad lab chow. Animals were
acclimated for at least 7 days in the research facility before
initiation of the experiment. ASOs were prepared in PBS and
sterilized by filtering through a 0.2 micron filter. ASOs were
dissolved in 0.9% PBS for injection.
[0750] Human ApoC III transgenic mice were injected
intraperitoneally once a week for two weeks with ISIS 304801 or
647535 at 0.08, 0.25. 0.75, 2.25 or 6.75 .mu.mol/kg or with PBS as
a control. Each treatment group consisted of 4 animals. Forty-eight
hours after the administration of the last dose, blood was drawn
from each mouse and the mice were sacrificed and tissues were
collected.
ApoC III mRNA Analysis
[0751] ApoC III mRNA levels in the mice's livers were determined
using real-time PCR and RIBOGREEN.RTM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.) according to standard
protocols. ApoC III mRNA levels were determined relative to total
RNA (using Ribogreen), prior to normalization to PBS-treated
control. The results below are presented as the average percent of
ApoC III mRNA levels for each treatment group, normalized to
PBS-treated control and are denoted as "% PBS". The half maximal
effective dosage (ED.sub.50) of each ASO is also presented in Table
18, below.
[0752] As illustrated, both antisense compounds reduced ApoC III
RNA relative to the PBS control. Further, the antisense compound
conjugated to GalNAc.sub.3-1 (ISIS 647535) was substantially more
potent than the antisense compound lacking the GalNAc.sub.3-1
conjugate (ISIS 304801).sub.n
TABLE-US-00004 TABLE 18 Effect of ASO treatment on ApoC III mRNA
levels in human ApoC III transgenic mice Dose % ED.sub.50
Internucleoside SEQ ASO (.mu.mol/kg) PBS (.mu.mol/kg) 3' Conjugate
linkage/Length ID No. PBS 0 100 -- -- -- ISIS 0.08 95 0.77 None
PS/20 821 304801 0.75 42 2.25 32 6.75 19 ISIS 0.08 50 0.074
GalNAc.sub.3-1 PS/20 822 647535 0.75 15 2.25 17 6.75 8
ApoC III Protein Analysis (Turbidometric Assay)
[0753] Plasma ApoC III protein analysis was determined using
procedures reported by Graham et al, Circulation Research,
published online before print Mar. 29, 2013.
[0754] Approximately 100 .mu.l of plasma isolated from mice was
analyzed without dilution using an Olympus Clinical Analyzer and a
commercially available turbidometric ApoC III assay (Kamiya, Cat #
KAI-006, Kamiya Biomedical, Seattle, Wash.). The assay protocol was
performed as described by the vendor.
[0755] As shown in the Table 19 below, both antisense compounds
reduced ApoC III protein relative to the PBS control. Further, the
antisense compound conjugated to GalNAc.sub.3-1 (ISIS 647535) was
substantially more potent than the antisense compound lacking the
GalNAc.sub.3-1 conjugate (ISIS 304801).
TABLE-US-00005 TABLE 19 Effect of ASO treatment on ApoC III plasma
protein levels in human ApoC III transgenic mice Dose % ED.sub.50
Internucleoside SEQ ASO (.mu.mol/kg) PBS (.mu.mol/kg) 3' Conjugate
Linkage/Length ID No. PBS 0 100 -- -- -- ISIS 0.08 86 0.73 None
PS/20 821 304801 0.75 51 2.25 23 6.75 13 ISIS 0.08 72 0.19
GalNAc.sub.3-1 PS/20 822 647535 0.75 14 2.25 12 6.75 11
[0756] Plasma triglycerides and cholesterol were extracted by the
method of Bligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J.
Biochem. Physiol. 37: 911-917, 1959) (Bligh, E and Dyer, W, Can J
Biochem Physiol, 37, 911-917, 1959) (Bligh, E and Dyer, W, Can J
Biochem Physiol, 37, 911-917, 1959) and measured by using a
Beckmann Coulter clinical analyzer and commercially available
reagents.
[0757] The triglyceride levels were measured relative to PBS
injected mice and are denoted as "% PBS". Results are presented in
Table 20. As illustrated, both antisense compounds lowered
triglyceride levels. Further, the antisense compound conjugated to
GalNAc.sub.3-1 (ISIS 647535) was substantially more potent than the
antisense compound lacking the GalNAc.sub.3-1 conjugate (ISIS
304801).
TABLE-US-00006 TABLE 20 Effect of ASO treatment on triglyceride
levels in transgenic mice Dose % ED.sub.50 Internucleoside SEQ ASO
(.mu.mol/kg) PBS (.mu.mol/kg) 3' Conjugate Linkage/Length ID No.
PBS 0 100 -- -- -- ISIS 0.08 87 0.63 None PS/20 821 304801 0.75 46
2.25 21 6.75 12 ISIS 0.08 65 0.13 GalNAc.sub.3-1 PS/20 822 647535
0.75 9 2.25 8 6.75 9
[0758] Plasma samples were analyzed by HPLC to determine the amount
of total cholesterol and of different fractions of cholesterol (HDL
and LDL). Results are presented in Tables 21 and 22. As
illustrated, both antisense compounds lowered total cholesterol
levels; both lowered LDL; and both raised HDL. Further, the
antisense compound conjugated to GalNAc.sub.3-1 (ISIS 647535) was
substantially more potent than the antisense compound lacking the
GalNAc.sub.3-1 conjugate (ISIS 304801). An increase in HDL and a
decrease in LDL levels is a cardiovascular beneficial effect of
antisense inhibition of ApoC III.
TABLE-US-00007 TABLE 21 Effect of ASO treatment on total
cholesterol levels in transgenic mice Inter- Total nucleoside Dose
Cholesterol Linkage/ SEQ ASO (.mu.mol/kg) (mg/dL) 3' Conjugate
Length ID No. PBS 0 257 -- -- ISIS 0.08 226 None PS/20 821 304801
0.75 164 2.25 110 6.75 82 ISIS 0.08 230 GalNAc.sub.3-1 PS/20 822
647535 0.75 82 2.25 86 6.75 99
TABLE-US-00008 TABLE 22 Effect of ASO treatment on HDL and LDL
cholesterol levels in transgenic mice Dose HDL LDL Internucleoside
SEQ ASO (.mu.mol/kg) (mg/dL) (mg/dL) 3' Conjugate Linkage/Length ID
No. PBS 0 17 28 -- -- ISIS 0.08 17 23 None PS/20 821 304801 0.75 27
12 2.25 50 4 6.75 45 2 ISIS 0.08 21 21 GalNAc.sub.3-1 PS/20 822
647535 0.75 44 2 2.25 50 2 6.75 58 2
Pharmacokinetics Analysis (PK)
[0759] The PK of the ASOs was also evaluated. Liver and kidney
samples were minced and extracted using standard protocols. Samples
were analyzed on MSD1 utilizing IP-HPLC-MS. The tissue level
(.mu.g/g) of full-length ISIS 304801 and 647535 was measured and
the results are provided in Table 23. As illustrated, liver
concentrations of total full-length antisense compounds were
similar for the two antisense compounds. Thus, even though the
GalNAc.sub.3-1-conjugated antisense compound is more active in the
liver (as demonstrated by the RNA and protein data above), it is
not present at substantially higher concentration in the liver.
Indeed, the calculated EC.sub.50 (provided in Table 23) confirms
that the observed increase in potency of the conjugated compound
cannot be entirely attributed to increased accumulation. This
result suggests that the conjugate improved potency by a mechanism
other than liver accumulation alone, possibly by improving the
productive uptake of the antisense compound into cells.
[0760] The results also show that the concentration of
GalNAc.sub.3-1 conjugated antisense compound in the kidney is lower
than that of antisense compound lacking the GalNAc conjugate. This
has several beneficial therapeutic implications. For therapeutic
indications where activity in the kidney is not sought, exposure to
kidney risks kidney toxicity without corresponding benefit.
Moreover, high concentration in kidney typically results in loss of
compound to the urine resulting in faster clearance. Accordingly,
for non-kidney targets, kidney accumulation is undesired. These
data suggest that GalNAc.sub.3-1 conjugation reduces kidney
accumulation.
TABLE-US-00009 TABLE 23 PK analysis of ASO treatment in transgenic
mice Dose Liver Kidney Liver EC.sub.50 Internucleoside SEQ ASO
(.mu.mol/kg) (.mu.g/g) (.mu.g/g) (.mu.g/g) 3' Conjugate
Linkage/Length ID No. ISIS 0.1 5.2 2.1 53 None PS/20 821 304801 0.8
62.8 119.6 2.3 142.3 191.5 6.8 202.3 337.7 ISIS 0.1 3.8 0.7 3.8
GalNAc.sub.3-1 PS/20 822 647535 0.8 72.7 34.3 2.3 106.8 111.4 6.8
237.2 179.3
[0761] Metabolites of ISIS 647535 were also identified and their
masses were confirmed by high resolution mass spectrometry
analysis. The cleavage sites and structures of the observed
metabolites are shown below. The relative % of full length ASO was
calculated using standard procedures and the results are presented
in Table 23a. The major metabolite of ISIS 647535 was full-length
ASO lacking the entire conjugate (i.e. ISIS 304801), which results
from cleavage at cleavage site A, shown below. Further, additional
metabolites resulting from other cleavage sites were also observed.
These results suggest that introducing other cleabable bonds such
as esters, peptides, disulfides, phosphoramidates or
acyl-hydrazones between the GalNAc.sub.3-1 sugar and the ASO, which
can be cleaved by enzymes inside the cell, or which may cleave in
the reductive environment of the cytosol, or which are labile to
the acidic pH inside endosomes and lyzosomes, can also be
useful.
TABLE-US-00010 TABLE 23a Observed full length metabolites of ISIS
647535 Cleavage Relative Metabolite ASO site % 1 ISIS 304801 A 36.1
2 ISIS 304801 + dA B 10.5 3 ISIS 647535 minus [3 GalNAc] C 16.1 4
ISIS 647535 minus D 17.6 [3 GalNAc + 1 5-hydroxy-pentanoic acid
tether] 5 ISIS 647535 minus D 9.9 [2 GalNAc + 2 5-hydroxy-pentanoic
acid tether] 6 ISIS 647535 minus D 9.8 [3 GalNAc + 3
5-hydroxy-pentanoic acid tether]
##STR00159## ##STR00160##
Example 21: Antisense Inhibition of Human ApoC III in Human ApoC
III Transgenic Mice in Single Administration Study
[0762] ISIS 304801, 647535 and 647536 each targeting human ApoC III
and described in Table 17, were further evaluated in a single
administration study for their ability to inhibit human ApoC III in
human ApoC III transgenic mice.
Treatment
[0763] Human ApoCIII transgenic mice were maintained on a 12-hour
light/dark cycle and fed ad libitum Teklad lab chow. Animals were
acclimated for at least 7 days in the research facility before
initiation of the experiment. ASOs were prepared in PBS and
sterilized by filtering through a 0.2 micron filter. ASOs were
dissolved in 0.9% PBS for injection.
[0764] Human ApoC III transgenic mice were injected
intraperitoneally once at the dosage shown below with ISIS 304801,
647535 or 647536 (described above) or with PBS treated control. The
treatment group consisted of 3 animals and the control group
consisted of 4 animals. Prior to the treatment as well as after the
last dose, blood was drawn from each mouse and plasma samples were
analyzed. The mice were sacrificed 72 hours following the last
administration.
[0765] Samples were collected and analyzed to determine the ApoC
III mRNA and protein levels in the liver; plasma triglycerides; and
cholesterol, including HDL and LDL fractions were assessed as
described above (Example 20). Data from those analyses are
presented in Tables 24-28, below. Liver transaminase levels,
alanine aminotransferase (ALT) and aspartate aminotransferase
(AST), in serum were measured relative to saline injected mice
using standard protocols. The ALT and AST levels showed that the
antisense compounds were well tolerated at all administered
doses.
[0766] These results show improvement in potency for antisense
compounds comprising a GalNAc.sub.3-1 conjugate at the 3' terminus
(ISIS 647535 and 647536) compared to the antisense compound lacking
a GalNAc.sub.3-1 conjugate (ISIS 304801). Further, ISIS 647536,
which comprises a GalNAc.sub.3-1 conjugate and some phosphodiester
linkages was as potent as ISIS 647535, which comprises the same
conjugate and all internucleoside linkages within the ASO are
phosphorothioate.
TABLE-US-00011 TABLE 24 Effect of ASO treatment on ApoC III mRNA
levels in human ApoC III transgenic mice Dose % ED.sub.50
Internucleoside SEQ ASO (mg/kg) PBS (mg/kg) 3' Conjugate
linkage/Length ID No. PBS 0 99 -- -- -- ISIS 1 104 13.2 None PS/20
821 304801 3 92 10 71 30 40 ISIS 0.3 98 1.9 GalNAc.sub.3-1 PS/20
822 647535 1 70 3 33 10 20 ISIS 0.3 103 1.7 GalNAc.sub.3-1 PS/PO/20
822 647536 1 60 3 31 10 21
TABLE-US-00012 TABLE 25 Effect of ASO treatment on ApoC III plasma
protein levels in human ApoC III transgenic mice Dose % ED.sub.50
Internucleoside SEQ ASO (mg/kg) PBS (mg/kg) 3' Conjugate
Linkage/Length ID No. PBS 0 99 -- -- -- ISIS 1 104 23.2 None PS/20
821 304801 3 92 10 71 30 40 ISIS 0.3 98 2.1 GalNAc.sub.3-1 PS/20
822 647535 1 70 3 33 10 20 ISIS 0.3 103 1.8 GalNAc.sub.3-1 PS/PO/20
822 647536 1 60 3 31 10 21
TABLE-US-00013 TABLE 26 Effect of ASO treatment on triglyceride
levels in transgenic mice Dose % ED.sub.50 3' Internucleoside SEQ
ASO (mg/kg) PBS (mg/kg) Conjugate Linkage/Length ID No. PBS 0 98 --
-- -- ISIS 1 80 29.1 None PS/20 304801 3 92 821 10 70 30 47 ISIS
0.3 100 2.2 GalNAc.sub.3-1 PS/20 822 647535 1 70 3 34 10 23 ISIS
0.3 95 1.9 GalNAc.sub.3-1 PS/PO/20 822 647536 1 66 3 31 10 23
TABLE-US-00014 TABLE 27 Effect of ASO treatment on total
cholesterol levels in transgenic mice Dose % Internucleoside SEQ
ASO (mg/kg) PBS 3' Conjugate Linkage/Length ID No. PBS 0 96 -- --
ISIS 1 104 None PS/20 821 304801 3 96 10 86 30 72 ISIS 0.3 93
GalNAc.sub.3-1 PS/20 822 647535 1 85 3 61 10 53 ISIS 0.3 115
GalNAc.sub.3-1 PS/PO/20 822 647536 1 79 3 51 10 54
TABLE-US-00015 TABLE 28 Effect of ASO treatment on HDL and LDL
cholesterol levels in transgenic mice Dose HDL LDL Internucleoside
SEQ ASO (mg/kg) % PBS % PBS 3' Conjugate Linkage/Length ID No. PBS
0 131 90 -- -- ISIS 1 130 72 None PS/20 821 304801 3 186 79 10 226
63 30 240 46 ISIS 0.3 98 86 GalNAc.sub.3-1 PS/20 822 647535 1 214
67 3 212 39 10 218 35 ISIS 0.3 143 89 GalNAc.sub.3-1 PS/PO/20 822
647536 1 187 56 3 213 33 10 221 34
[0767] These results confirm that the GalNAc.sub.3-1 conjugate
improves potency of an antisense compound. The results also show
equal potency of a GalNAc.sub.3-1 conjugated antisense compounds
where the antisense oligonucleotides have mixed linkages (ISIS
647536 which has six phosphodiester linkages) and a full
phosphorothioate version of the same antisense compound (ISIS
647535).
[0768] Phosphorothioate linkages provide several properties to
antisense compounds. For example, they resist nuclease digestion
and they bind proteins resulting in accumulation of compound in the
liver, rather than in the kidney/urine. These are desirable
properties, particularly when treating an indication in the liver.
However, phosphorothioate linkages have also been associated with
an inflammatory response. Accordingly, reducing the number of
phosphorothioate linkages in a compound is expected to reduce the
risk of inflammation, but also lower concentration of the compound
in liver, increase concentration in the kidney and urine, decrease
stability in the presence of nucleases, and lower overall potency.
The present results show that a GalNAc.sub.3-1 conjugated antisense
compound where certain phosphorothioate linkages have been replaced
with phosphodiester linkages is as potent against a target in the
liver as a counterpart having full phosphorothioate linkages. Such
compounds are expected to be less proinflammatory (See Example 24
describing an experiment showing reduction of PS results in reduced
inflammatory effect).
Example 22: Effect of GalNAc.sub.3-1 Conjugated Modified ASO
Targeting SRB-1 In Vivo
[0769] ISIS 440762 and 651900, each targeting SRB-1 and described
in Table 17, were evaluated in a dose-dependent study for their
ability to inhibit SRB-1 in Balb/c mice.
Treatment
[0770] Six week old male Balb/c mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously once at the dosage shown
below with ISIS 440762, 651900 or with PBS treated control. Each
treatment group consisted of 4 animals. The mice were sacrificed 48
hours following the final administration to determine the SRB-1
mRNA levels in liver using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. SRB-1 mRNA levels were determined
relative to total RNA (using Ribogreen), prior to normalization to
PBS-treated control. The results below are presented as the average
percent of SRB-1 mRNA levels for each treatment group, normalized
to PBS-treated control and is denoted as "% PBS".
[0771] As illustrated in Table 29, both antisense compounds lowered
SRB-1 mRNA levels. Further, the antisense compound comprising the
GalNAc.sub.3-1 conjugate (ISIS 651900) was substantially more
potent than the antisense compound lacking the GalNAc.sub.3-1
conjugate (ISIS 440762). These results demonstrate that the potency
benefit of GalNAc.sub.3-1 conjugates are observed using antisense
oligonucleotides complementary to a different target and having
different chemically modified nucleosides, in this instance
modified nucleosides comprise constrained ethyl sugar moieties (a
bicyclic sugar moiety).
TABLE-US-00016 TABLE 29 Effect of ASO treatment on SRB-1 mRNA
levels in Balb/c mice Dose Liver ED.sub.50 Internucleoside SEQ ASO
(mg/kg) % PBS (mg/kg) 3' Conjugate linkage/Length ID No. PBS 0 100
-- -- ISIS 0.7 85 2.2 None PS/14 823 440762 2 55 7 12 20 3 ISIS
0.07 98 0.3 GalNAc.sub.3-1 PS/14 824 651900 0.2 63 0.7 20 2 6 7
5
Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) Assay
Protocol
[0772] The hPBMC assay was performed using BD Vautainer CPT tube
method. A sample of whole blood from volunteered donors with
informed consent at US HealthWorks clinic (Faraday & El Camino
Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer
CPT 8 ml tubes (VWR Cat. # BD362753).sub.n The approximate starting
total whole blood volume in the CPT tubes for each donor was
recorded using the PBMC assay data sheet.
[0773] The blood sample was remixed immediately prior to
centrifugation by gently inverting tubes 8-10 times. CPT tubes were
centrifuged at rt (18-25.degree. C.) in a horizontal (swing-out)
rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman
Allegra 6R). The cells were retrieved from the buffy coat interface
(between Ficoll and polymer gel layers); transferred to a sterile
50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical
tube/donor. The cells were then washed twice with PBS (Ca.sup.++,
Mg.sup.++ free; GIBCO). The tubes were topped up to 50 ml and mixed
by inverting several times. The sample was then centrifuged at
330.times.g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R)
and aspirated as much supernatant as possible without disturbing
pellet. The cell pellet was dislodged by gently swirling tube and
resuspended cells in RPMI+10% FBS+pen/strep (.about.1 ml/10 ml
starting whole blood volume). A 60 .mu.l sample was pipette into a
sample vial (Beckman Coulter) with 600 .mu.l VersaLyse reagent
(Beckman Coulter Cat # A09777) and was gently vortexed for 10-15
sec. The sample was allowed to incubate for 10 min. at rt and being
mixed again before counting. The cell suspension was counted on
Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell
type (dilution factor of 1:11 was stored with other parameters).
The live cell/ml and viability were recorded. The cell suspension
was diluted to 1.times.10.sup.7 live PBMC/ml in RPMI+10%
FBS+pen/strep.
[0774] The cells were plated at 5.times.10.sup.5 in 50 .mu.l/well
of 96-well tissue culture plate (Falcon Microtest). 50 .mu.l/well
of 2.times. concentration oligos/controls diluted in RPMI+10%
FBS+pen/strep. was added according to experiment template
(100.sub.11.1/well total). Plates were placed on the shaker and
allowed to mix for approx. 1 min. After being incubated for 24 hrs
at 37.degree. C.; 5% CO.sub.2, the plates were centrifuged at
400.times.g for 10 minutes before removing the supernatant for MSD
cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-1).
Example 24: Evaluation of Proinflammatory Effects in hPBMC Assay
for GalNAc.sub.3-1 Conjugated ASOs
[0775] The antisense oligonucleotides (ASOs) listed in Table 30
were evaluated for proinflammatory effect in hPBMC assay using the
protocol described in Example 23. ISIS 353512 is an internal
standard known to be a high responder for IL-6 release in the
assay. The hPBMCs were isolated from fresh, volunteered donors and
were treated with ASOs at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and
200 .mu.M concentrations. After a 24 hr treatment, the cytokine
levels were measured.
[0776] The levels of IL-6 were used as the primary readout. The
EC.sub.50 and E.sub.max was calculated using standard procedures.
Results are expressed as the average ratio of E.sub.max/EC.sub.50
from two donors and is denoted as "E.sub.max/EC.sub.50." The lower
ratio indicates a relative decrease in the proinflammatory response
and the higher ratio indicates a relative increase in the
proinflammatory response.
[0777] With regard to the test compounds, the least proinflammatory
compound was the PS/PO linked ASO (ISIS 616468).sub.n The
GalNAc.sub.3-1 conjugated ASO, ISIS 647535 was slightly less
proinflammatory than its non-conjugated counterpart ISIS 304801.
These results indicate that incorporation of some PO linkages
reduces proinflammatory reaction and addition of a GalNAc.sub.3-1
conjugate does not make a compound more proinflammatory and may
reduce proinflammatory response. Accordingly, one would expect that
an antisense compound comprising both mixed PS/PO linkages and a
GalNAc.sub.3-1 conjugate would produce lower proinflammatory
responses relative to full PS linked antisense compound with or
without a GalNAc.sub.3-1 conjugate. These results show that
GalNAc.sub.3-1 conjugated antisense compounds, particularly those
having reduced PS content are less proinflammatory.
[0778] Together, these results suggest that a GalNAc.sub.3-1
conjugated compound, particularly one with reduced PS content, can
be administered at a higher dose than a counterpart full PS
antisense compound lacking a GalNAc.sub.3-1 conjugate. Since
half-life is not expected to be substantially different for these
compounds, such higher administration would result in less frequent
dosing. Indeed such administration could be even less frequent,
because the GalNAc.sub.3-1 conjugated compounds are more potent
(See Examples 20-22) and re-dosing is necessary once the
concentration of a compound has dropped below a desired level,
where such desired level is based on potency.
TABLE-US-00017 TABLE 30 Modified ASOs SEQ ID ASO Sequence (5' to
3') Target No. ISIS
G.sub.es.sup.mC.sub.esT.sub.esG.sub.esA.sub.esT.sub.dsT.sub.dsA.sub.d-
sG.sub.dsA.sub.dsG.sub.ds TNF.alpha. 825 104838
A.sub.dsG.sub.dsA.sub.dsG.sub.dsG.sub.esT.sub.es.sup.mC.sub.es.sup.-
mC.sub.es.sup.mC.sub.e ISIS
T.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.dsA.sub.dsT.sub.dsT.s-
ub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds CRP 826 353512
G.sub.dsA.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsT.sub.e-
sG.sub.esG.sub.e ISIS
A.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.dsT.sub.dsT-
.sub.dsG.sub.dsT.sub.ds ApoC 821 304801
.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.esT-
.sub.esT.sub.esA.sub.esT.sub.e III ISIS
A.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.dsT.sub.dsT-
.sub.dsG.sub.dsT.sub.ds 647535
.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.esT-
.sub.esT.sub.esA.sub.es ApoC 822 III ISIS
A.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.dsT.sub.dsT-
.sub.dsG.sub.dsT.sub.ds ApoC 821 616468
.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.eoT-
.sub.eoT.sub.esA.sub.esT.sub.e III
[0779] Subscripts: "e" indicates 2'-MOE modified nucleoside; "d"
indicates .beta.-D-2'-deoxyribonucleoside; "k" indicates
6'-(S)--CH3 bicyclic nucleoside (e.g. cEt); "s" indicates
phosphorothioate internucleoside linkages (PS); "o" indicates
phosphodiester internucleoside linkages (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Superscript "m" indicates 5-methylcytosines.
"A.sub.do'-GalNAc.sub.3-1a" indicates a conjugate having the
structure GalNAc.sub.3-1 shown in Example 9 attached to the 3'-end
of the antisense oligonucleotide, as indicated.
TABLE-US-00018 TABLE 31 Proinflammatory Effect of ASOs targeting
ApoC III in hPBMC assay EC.sub.50 E.sub.max Internucleoside SEQ ASO
(.mu.M) (.mu.M) E.sub.max/EC.sub.50 3' Conjugate Linkage/Length ID
No. ISIS 353512 0.01 265.9 26,590 None PS/20 826 (high responder)
ISIS 304801 0.07 106.55 1,522 None PS/20 821 ISIS 647535 0.12 138
1,150 GalNAc.sub.3-1 PS/20 822 ISIS 616468 0.32 71.52 224 None
PS/PO/20 821
Example 25: Effect of GalNAc.sub.3-1 Conjugated Modified ASO
Targeting Human ApoC III In Vitro
[0780] ISIS 304801 and 647535 described above were tested in vitro.
Primary hepatocyte cells from transgenic mice at a density of
25,000 cells per well were treated with 0.03, 0.08, 0.24, 0.74,
2.22, 6.67 and 20 .mu.M concentrations of modified
oligonucleotides. After a treatment period of approximately 16
hours, RNA was isolated from the cells and mRNA levels were
measured by quantitative real-time PCR and the hApoC III mRNA
levels were adjusted according to total RNA content, as measured by
RIBOGREEN.
[0781] The IC.sub.50 was calculated using the standard methods and
the results are presented in Table 32. As illustrated, comparable
potency was observed in cells treated with ISIS 647535 as compared
to the control, ISIS 304801.
TABLE-US-00019 TABLE 32 Modified ASO targeting human ApoC III in
primary hepatocytes IC.sub.50 Internucleoside SEQ ASO (.mu.M) 3'
Conjugate linkage/Length ID No. ISIS 0.44 None PS/20 821 304801
ISIS 0.31 GalNAc.sub.3-1 PS/20 822 647535
[0782] In this experiment, the large potency benefits of
GalNAc.sub.3-1 conjugation that are observed in vivo were not
observed in vitro. Subsequent free uptake experiments in primary
hepatocytes in vitro did show increased potency of oligonucleotides
comprising various GalNAc conjugates relative to oligonucleotides
that lacking the GalNAc conjugate. (see Examples 60, 82, and
92)
Example 26: Effect of PO/PS Linkages on ApoC III ASO Activity
[0783] Human ApoC III transgenic mice were injected
intraperitoneally once at 25 mg/kg of ISIS 304801, or ISIS 616468
(both described above) or with PBS treated control once per week
for two weeks. The treatment group consisted of 3 animals and the
control group consisted of 4 animals. Prior to the treatment as
well as after the last dose, blood was drawn from each mouse and
plasma samples were analyzed. The mice were sacrificed 72 hours
following the last administration.
[0784] Samples were collected and analyzed to determine the ApoC
III protein levels in the liver as described above (Example 20).
Data from those analyses are presented in Table 33, below.
[0785] These results show reduction in potency for antisense
compounds with PO/PS (ISIS 616468) in the wings relative to full PS
(ISIS 304801).
TABLE-US-00020 TABLE 33 Effect of ASO treatment on ApoC III protein
levels in human ApoC III transgenic mice Dose % Internucleoside SEQ
ASO (mg/kg) PBS 3' Conjugate linkage/Length ID No. PBS 0 99 -- --
ISIS 25 24 None Full PS 821 304801 mg/kg/wk for 2 wks ISIS 25 40
None 14 PS/6 PO 821 616468 mg/kg/wk for 2 wks
Example 27: Compound 56
##STR00161##
[0787] Compound 56 is commercially available from Glen Research or
may be prepared according to published procedures reported by
Shchepinov et al., Nucleic Acids Research, 1997, 25(22),
4447-4454.
Example 28: Preparation of Compound 60
##STR00162##
[0789] Compound 4 was prepared as per the procedures illustrated in
Example 2. Compound 57 is commercially available. Compound 60 was
confirmed by structural analysis.
[0790] Compound 57 is meant to be representative and not intended
to be limiting as other mono-protected substituted or unsubstituted
alkyl diols including but not limited to those presented in the
specification herein can be used to prepare phosphoramidites having
a predetermined composition.
Example 29: Preparation of Compound 63
##STR00163##
[0792] Compounds 61 and 62 are prepared using procedures similar to
those reported by Tober et al., Eur. J. Org. Chem., 2013, 3,
566-577; and Jiang et al., Tetrahedron, 2007, 63(19),
3982-3988.
[0793] Alternatively, Compound 63 is prepared using procedures
similar to those reported in scientific and patent literature by
Kim et al., Synlett, 2003, 12, 1838-1840; and Kim et al., published
PCT International Application, WO 2004063208.Example 30:
Preparation of Compound 63b
##STR00164##
[0794] Compound 63a is prepared using procedures similar to those
reported by Hanessian et al., Canadian Journal of Chemistry, 1996,
74(9), 1731-1737.
Example 31: Preparation of Compound 63d
##STR00165##
[0796] Compound 63c is prepared using procedures similar to those
reported by Chen et al., Chinese Chemical Letters, 1998, 9(5),
451-453.
Example 32: Preparation of Compound 67
##STR00166##
[0798] Compound 64 was prepared as per the procedures illustrated
in Example 2. Compound 65 is prepared using procedures similar to
those reported by Or et al., published PCT International
Application, WO 2009003009. The protecting groups used for Compound
65 are meant to be representative and not intended to be limiting
as other protecting groups including but not limited to those
presented in the specification herein can be used.
Example 33: Preparation of Compound 70
##STR00167##
[0800] Compound 64 was prepared as per the procedures illustrated
in Example 2. Compound 68 is commercially available. The protecting
group used for Compound 68 is meant to be representative and not
intended to be limiting as other protecting groups including but
not limited to those presented in the specification herein can be
used.
Example 34: Preparation of Compound 75a
##STR00168##
[0802] Compound 75 is prepared according to published procedures
reported by Shchepinov et al., Nucleic Acids Research, 1997,
25(22), 4447-4454.
Example 35: Preparation of Compound 79
##STR00169##
[0804] Compound 76 was prepared according to published procedures
reported by Shchepinov et al., Nucleic Acids Research, 1997,
25(22), 4447-4454.
Example 36: Preparation of Compound 79a
##STR00170##
[0806] Compound 77 is prepared as per the procedures illustrated in
Example 35.
Example 37: General Method for the Preparation of Conjugated
Oligomeric Compound 82 Comprising a Phosphodiester Linked
GalNAc.sub.3-2 Conjugate at 5' Terminus Via Solid Support (Method
I)
##STR00171## ##STR00172##
[0807] wherein GalNAc.sub.3-2 has the structure:
##STR00173##
[0808] The GalNAc.sub.3 cluster portion of the conjugate group
GalNAc.sub.3-2 (GalNAc.sub.3-2.sub.a) can be combined with any
cleavable moiety to provide a variety of conjugate groups. Wherein
GalNAc.sub.3-2.sub.a has the formula:
##STR00174##
[0809] The VIMAD-bound oligomeric compound 79b was prepared using
standard procedures for automated DNA/RNA synthesis (see Dupouy et
al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627).sub.n The
phosphoramidite Compounds 56 and 60 were prepared as per the
procedures illustrated in Examples 27 and 28, respectively. The
phosphoramidites illustrated are meant to be representative and not
intended to be limiting as other phosphoramidite building blocks
including but not limited those presented in the specification
herein can be used to prepare an oligomeric compound having a
phosphodiester linked conjugate group at the 5' terminus. The order
and quantity of phosphoramidites added to the solid support can be
adjusted to prepare the oligomeric compounds as described herein
having any predetermined sequence and composition.
Example 38: Alternative Method for the Preparation of Oligomeric
Compound 82 Comprising a Phosphodiester Linked GalNAc.sub.3-2
Conjugate at 5' Terminus (Method II)
##STR00175##
[0811] The VIMAD-bound oligomeric compound 79b was prepared using
standard procedures for automated DNA/RNA synthesis (see Dupouy et
al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627).sub.n The
GalNAc.sub.3-2 cluster phosphoramidite, Compound 79 was prepared as
per the procedures illustrated in Example 35. This alternative
method allows a one-step installation of the phosphodiester linked
GalNAc.sub.3-2 conjugate to the oligomeric compound at the final
step of the synthesis. The phosphoramidites illustrated are meant
to be representative and not intended to be limiting, as other
phosphoramidite building blocks including but not limited to those
presented in the specification herein can be used to prepare
oligomeric compounds having a phosphodiester conjugate at the 5'
terminus. The order and quantity of phosphoramidites added to the
solid support can be adjusted to prepare the oligomeric compounds
as described herein having any predetermined sequence and
composition.
Example 39: General Method for the Preparation of Oligomeric
Compound 83h Comprising a GalNAc.sub.3-3 Conjugate at the 5'
Terminus (GalNAc.sub.3-1 Modified for 5' End Attachment) Via Solid
Support
##STR00176## ##STR00177##
[0813] Compound 18 was prepared as per the procedures illustrated
in Example 4. Compounds 83a and 83b are commercially available.
Oligomeric Compound 83e comprising a phosphodiester linked
hexylamine was prepared using standard oligonucleotide synthesis
procedures. Treatment of the protected oligomeric compound with
aqueous ammonia provided the 5'-GalNAc.sub.3-3 conjugated
oligomeric compound (83h).
[0814] Wherein GalNAc.sub.3-3 has the structure:
##STR00178##
[0815] The GalNAc.sub.3 cluster portion of the conjugate group
GalNAc.sub.3-3 (GalNAc.sub.3-3.sub.a) can be combined with any
cleavable moiety to provide a variety of conjugate groups. Wherein
GalNAc.sub.3-3.sub.a has the formula:
##STR00179##
Example 40: General Method for the Preparation of Oligomeric
Compound 89 Comprising a Phosphodiester Linked GalNAc.sub.3-4
Conjugate at the 3' Terminus Via Solid Support
##STR00180## ##STR00181##
[0816] Wherein GalNAc.sub.3-4 has the structure:
##STR00182##
[0817] Wherein CM is a cleavable moiety. In certain embodiments,
cleavable moiety is:
##STR00183##
[0818] The GalNAc.sub.3 cluster portion of the conjugate group
GalNAc.sub.3-4 (GalNAc.sub.3-4.sub.a) can be combined with any
cleavable moiety to provide a variety of conjugate groups. Wherein
GalNAc.sub.3-4.sub.a has the formula:
##STR00184##
[0819] The protected Unylinker functionalized solid support
Compound 30 is commercially available. Compound 84 is prepared
using procedures similar to those reported in the literature (see
Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454;
Shchepinov et al., Nucleic Acids Research, 1999, 27, 3035-3041; and
Hornet et al., Nucleic Acids Research, 1997, 25, 4842-4849).
[0820] The phosphoramidite building blocks, Compounds 60 and 79a
are prepared as per the procedures illustrated in Examples 28 and
36. The phosphoramidites illustrated are meant to be representative
and not intended to be limiting as other phosphoramidite building
blocks can be used to prepare an oligomeric compound having a
phosphodiester linked conjugate at the 3' terminus with a
predetermined sequence and composition. The order and quantity of
phosphoramidites added to the solid support can be adjusted to
prepare the oligomeric compounds as described herein having any
predetermined sequence and composition.
Example 41: General Method for the Preparation of ASOs Comprising a
Phosphodiester Linked GalNAc.sub.3-2 (See Example 37, Bx is
Adenine) Conjugate at the 5' Position Via Solid Phase Techniques
(Preparation of ISIS 661134)
[0821] Unless otherwise stated, all reagents and solutions used for
the synthesis of oligomeric compounds are purchased from commercial
sources. Standard phosphoramidite building blocks and solid support
are used for incorporation nucleoside residues which include for
example T, A, G, and .sup.mC residues. Phosphoramidite compounds 56
and 60 were used to synthesize the phosphodiester linked
GalNAc.sub.3-2 conjugate at the 5' terminus. A 0.1 M solution of
phosphoramidite in anhydrous acetonitrile was used for
.beta.-D-2'-deoxyribonucleoside and 2'-MOE.
[0822] The ASO syntheses were performed on ABI 394 synthesizer (1-2
.mu.mol scale) or on GE Healthcare Bioscience AKTA oligopilot
synthesizer (40-200 .mu.mol scale) by the phosphoramidite coupling
method on VIMAD solid support (110 .mu.mol/g, Guzaev et al., 2003)
packed in the column. For the coupling step, the phosphoramidites
were delivered at a 4 fold excess over the initial loading of the
solid support and phosphoramidite coupling was carried out for 10
min. All other steps followed standard protocols supplied by the
manufacturer. A solution of 6% dichloroacetic acid in toluene was
used for removing the dimethoxytrityl (DMT) groups from 5'-hydroxyl
groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous
CH.sub.3CN was used as activator during the coupling step.
Phosphorothioate linkages were introduced by sulfurization with 0.1
M solution of xanthane hydride in 1:1 pyridine/CH.sub.3CN for a
contact time of 3 minutes. A solution of 20%
tert-butylhydroperoxide in CH.sub.3CN containing 6% water was used
as an oxidizing agent to provide phosphodiester internucleoside
linkages with a contact time of 12 minutes.
[0823] After the desired sequence was assembled, the cyanoethyl
phosphate protecting groups were deprotected using a 20%
diethylamine in toluene (v/v) with a contact time of 45 minutes.
The solid-support bound ASOs were suspended in aqueous ammonia
(28-30 wt %) and heated at 55.degree. C. for 6 h. The unbound ASOs
were then filtered and the ammonia was boiled off. The residue was
purified by high pressure liquid chromatography on a strong anion
exchange column (GE Healthcare Bioscience, Source 30Q, 30 .mu.m,
2.54.times.8 cm, A=100 mM ammonium acetate in 30% aqueous
CH.sub.3CN, B=1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL
min-1, =260 nm). The residue was desalted by HPLC on a reverse
phase column to yield the desired ASOs in an isolated yield of
15-30% based on the initial loading on the solid support. The ASOs
were characterized by ion-pair-HPLC coupled MS analysis with
Agilent 1100 MSD system.
TABLE-US-00021 TABLE 34 ASO comprising a phosphodiester linked
GalNAc.sub.3-2 conjugate at the 5' position targeting SRB-1 SEQ
ISIS CalCd Observed ID No. Sequence (5' to 3') Mass Mass No. 661134
6482.2 6481.6 827
T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.s-
ub.dsG.sub.ds
A.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub.k
[0824] Subscripts: "e" indicates 2'-MOE modified nucleoside; "d"
indicates .beta.-D-2'-deoxyribonucleoside; "k" indicates
6'-(S)--CH3 bicyclic nucleoside (e.g. cEt); "s" indicates
phosphorothioate internucleoside linkages (PS); "o" indicates
phosphodiester internucleoside linkages (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Superscript "m" indicates 5-methylcytosines.
The structure of GalNAc.sub.3-2.sub.a is shown in Example 37.
Example 42: General Method for the Preparation of ASOs Comprising a
GalNAc.sub.3-3 Conjugate at the 5' Position Via Solid Phase
Techniques (Preparation of ISIS 661166)
[0825] The synthesis for ISIS 661166 was performed using similar
procedures as illustrated in Examples 39 and 41.
[0826] ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5' position
comprises a GalNAc.sub.3-3 conjugate. The ASO was characterized by
ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.
TABLE-US-00022 TABLE 34a ASO comprising a GalNAc.sub.3-3 conjugate
at the 5' position via a hexylamino phosphodiester linkage
targeting Malat-1 SEQ ISIS Calcd Observed ID No. Sequence (5' to
3') Conjugate Mass Mass No. 661166 5'-
'.sup.mC.sub.esG.sub.esG.sub.esT.sub.esG.sub.es 8992.16 8990.51 828
.sup.mC.sub.dsA.sub.dsA.sub.dsG.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.dsA.sub.dsG.sub.ds G.sub.esA.sub.esA.sub.esT.sub.esT.sub.e
[0827] Subscripts: "e" indicates 2'-MOE modified nucleoside; "d"
indicates .beta.-D-2'-deoxyribonucleoside; "s" indicates
phosphorothioate internucleoside linkages (PS); "o" indicates
phosphodiester internucleoside linkages (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Superscript "m" indicates 5-methylcytosines.
The structure of "5'-GalNAc.sub.3-3a" is shown in Example 39.
Example 43: Dose-Dependent Study of Phosphodiester Linked
GalNAc.sub.3-2 (See Examples 37 and 41, Bx is Adenine) at the 5'
Terminus Targeting SRB-1 In Vivo
[0828] ISIS 661134 (see Example 41) comprising a phosphodiester
linked GalNAc.sub.3-2 conjugate at the 5' terminus was tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
Unconjugated ISIS 440762 and 651900 (GalNAc.sub.3-1 conjugate at 3'
terminus, see Example 9) were included in the study for comparison
and are described previously in Table 17.
Treatment
[0829] Six week old male Balb/c mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously once at the dosage shown
below with ISIS 440762, 651900, 661134 or with PBS treated control.
Each treatment group consisted of 4 animals. The mice were
sacrificed 72 hours following the final administration to determine
the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN.RTM.
RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. SRB-1 mRNA levels were determined
relative to total RNA (using Ribogreen), prior to normalization to
PBS-treated control. The results below are presented as the average
percent of SRB-1 mRNA levels for each treatment group, normalized
to PBS-treated control and is denoted as "% PBS". The ED.sub.50s
were measured using similar methods as described previously and are
presented below.
[0830] As illustrated in Table 35, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner. Indeed, the antisense oligonucleotides comprising the
phosphodiester linked GalNAc.sub.3-2 conjugate at the 5' terminus
(ISIS 661134) or the GalNAc.sub.3-1 conjugate linked at the 3'
terminus (ISIS 651900) showed substantial improvement in potency
compared to the unconjugated antisense oligonucleotide (ISIS
440762).sub.n Further, ISIS 661134, which comprises the
phosphodiester linked GalNAc.sub.3-2 conjugate at the 5' terminus
was equipotent compared to ISIS 651900, which comprises the
GalNAc.sub.3-1 conjugate at the 3' terminus.
TABLE-US-00023 TABLE 35 ASOs containing GalNAc.sub.3-1 or
GalNAc.sub.3-2 targeting SRB-1 SRB-1 ISIS Dosage mRNA levels
ED.sub.50 SEQ No. (mg/kg) (% PBS) (mg/kg) Conjugate ID No. PBS 0
100 -- -- 440762 0.2 116 2.58 No conjugate 823 0.7 91 2 69 7 22 20
5 651900 0.07 95 0.26 3' GalNAc.sub.3-1 824 0.2 77 0.7 28 2 11 7 8
661134 0.07 107 0.25 5' GalNAc.sub.3-2 827 0.2 86 0.7 28 2 10 7
6
[0831] Structures for 3' GalNAc.sub.3-1 and 5' GalNAc.sub.3-2 were
described previously in Examples 9 and 37.
Pharmacokinetics Analysis (PK)
[0832] The PK of the ASOs from the high dose group (7 mg/kg) was
examined and evaluated in the same manner as illustrated in Example
20. Liver sample was minced and extracted using standard protocols.
The full length metabolites of 661134 (5' GalNAc.sub.3-2) and ISIS
651900 (3' GalNAc.sub.3-1) were identified and their masses were
confirmed by high resolution mass spectrometry analysis. The
results showed that the major metabolite detected for the ASO
comprising a phosphodiester linked GalNAc.sub.3-2 conjugate at the
5' terminus (ISIS 661134) was ISIS 440762 (data not shown). No
additional metabolites, at a detectable level, were observed.
Unlike its counterpart, additional metabolites similar to those
reported previously in Table 23a were observed for the ASO having
the GalNAc.sub.3-1 conjugate at the 3' terminus (ISIS 651900).sub.n
These results suggest that having the phosphodiester linked
GalNAc.sub.3-1 or GalNAc.sub.3-2 conjugate may improve the PK
profile of ASOs without compromising their potency.
Example 44: Effect of PO/PS Linkages on Antisense Inhibition of
ASOs Comprising GalNAc.sub.3-1 Conjugate (See Example 9) at the 3'
Terminus Targeting SRB-1
[0833] ISIS 655861 and 655862 comprising a GalNAc.sub.3-1 conjugate
at the 3' terminus each targeting SRB-1 were tested in a single
administration study for their ability to inhibit SRB-1 in mice.
The parent unconjugated compound, ISIS 353382 was included in the
study for comparison.
[0834] The ASOs are 5-10-5 MOE gapmers, wherein the gap region
comprises ten 2'-deoxyribonucleosides and each wing region
comprises five 2'-MOE modified nucleosides. The ASOs were prepared
using similar methods as illustrated previously in Example 19 and
are described Table 36, below.
TABLE-US-00024 TABLE 36 Modified ASOs comprising GalNAc.sub.3-1
conjugate at the 3' terminus targeting SRB-1 SEQ ISIS ID No.
Sequence (5' to 3') Chemistry No. 353382
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ds Full PS no 829 (parent)
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.-
mC.sub.esT.sub.esT.sub.e conjugate 655861
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ds Full PS 830
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.es with conjugate 655862
G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ds Mixed
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.e-
sT.sub.es PS/PO 830 with conjugate
[0835] Subscripts: "e" indicates 2'-MOE modified nucleoside; "d"
indicates .beta.-D-2'-deoxyribonucleoside; "s" indicates
phosphorothioate internucleoside linkages (PS); "o" indicates
phosphodiester internucleoside linkages (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Superscript "m" indicates 5-methylcytosines.
The structure of "GalNAc.sub.3-1" is shown in Example 9.
Treatment
[0836] Six week old male Balb/c mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously once at the dosage shown
below with ISIS 353382, 655861, 655862 or with PBS treated control.
Each treatment group consisted of 4 animals. Prior to the treatment
as well as after the last dose, blood was drawn from each mouse and
plasma samples were analyzed. The mice were sacrificed 72 hours
following the final administration to determine the liver SRB-1
mRNA levels using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. SRB-1 mRNA levels were determined
relative to total RNA (using Ribogreen), prior to normalization to
PBS-treated control. The results below are presented as the average
percent of SRB-1 mRNA levels for each treatment group, normalized
to PBS-treated control and is denoted as "% PBS". The ED.sub.50s
were measured using similar methods as described previously and are
reported below.
[0837] As illustrated in Table 37, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner compared to PBS treated control. Indeed, the antisense
oligonucleotides comprising the GalNAc.sub.3-1 conjugate at the 3'
terminus (ISIS 655861 and 655862) showed substantial improvement in
potency comparing to the unconjugated antisense oligonucleotide
(ISIS 353382). Further, ISIS 655862 with mixed PS/PO linkages
showed an improvement in potency relative to full PS (ISIS
655861).
TABLE-US-00025 TABLE 37 Effect of PO/PS linkages on antisense
inhibition of ASOs comprising GalNAc.sub.3-1 conjugate at 3'
terminus targeting SRB-1 SRB-1 ISIS Dosage mRNA levels ED.sub.50
SEQ No. (mg/kg) (% PBS) (mg/kg) Chemistry ID No. PBS 0 100 -- --
353382 3 76.65 10.4 Full PS without 829 (parent) 10 52.40 conjugate
30 24.95 655861 0.5 81.22 2.2 Full PS with 830 1.5 63.51
GalNAc.sub.3-1 5 24.61 conjugate 15 14.80 655862 0.5 69.57 1.3
Mixed PS/PO 830 1.5 45.78 with GalNAc.sub.3-1 5 19.70 conjugate 15
12.90
[0838] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were measured
relative to saline injected mice using standard protocols. Organ
weights were also evaluated. The results demonstrated that no
elevation in transaminase levels (Table 38) or organ weights (data
not shown) were observed in mice treated with ASOs compared to PBS
control. Further, the ASO with mixed PS/PO linkages (ISIS 655862)
showed similar transaminase levels compared to full PS (ISIS
655861).
TABLE-US-00026 TABLE 38 Effect of PO/PS linkages on transaminase
levels of ASOs comprising GalNAc.sub.3-1 conjugate at 3' terminus
targeting SRB-1 ISIS Dosage ALT AST SEQ No. (mg/kg) (U/L) (U/L)
Chemistry ID No. PBS 0 28.5 65 -- 353382 3 50.25 89 Full PS without
829 (parent) 10 27.5 79.3 conjugate 30 27.3 97 655861 0.5 28 55.7
Full PS with 830 1.5 30 78 GalNAc.sub.3-1 5 29 63.5 15 28.8 67.8
655862 0.5 50 75.5 Mixed PS/PO 830 1.5 21.7 58.5 with 5 29.3 69
GalNAc.sub.3-1 15 22 61
Example 45: Preparation of PFP Ester, Compound 110a
##STR00185## ##STR00186##
[0840] Compound 4 (9.5 g, 28.8 mmoles) was treated with compound
103a or 103b (38 mmoles), individually, and TMSOTf (0.5 eq.) and
molecular sieves in dichloromethane (200 mL), and stirred for 16
hours at room temperature. At that time, the organic layer was
filtered thru celite, then washed with sodium bicarbonate, water
and brine. The organic layer was then separated and dried over
sodium sulfate, filtered and reduced under reduced pressure. The
resultant oil was purified by silica gel chromatography (2%->10%
methanol/dichloromethane) to give compounds 104a and 104b in
>80% yield. LCMS and proton NMR was consistent with the
structure.
[0841] Compounds 104a and 104b were treated to the same conditions
as for compounds 100a-d (Example 47), to give compounds 105a and
105b in >90% yield. LCMS and proton NMR was consistent with the
structure.
[0842] Compounds 105a and 105b were treated, individually, with
compound 90 under the same conditions as for compounds 901a-d, to
give compounds 106a (80%) and 106b (20%). LCMS and proton NMR was
consistent with the structure.
[0843] Compounds 106a and 106b were treated to the same conditions
as for compounds 96a-d (Example 47), to give 107a (60%) and 107b
(20%). LCMS and proton NMR was consistent with the structure.
[0844] Compounds 107a and 107b were treated to the same conditions
as for compounds 97a-d (Example 47), to give compounds 108a and
108b in 40-60% yield. LCMS and proton NMR was consistent with the
structure.
[0845] Compounds 108a (60%) and 108b (40%) were treated to the same
conditions as for compounds 100a-d (Example 47), to give compounds
109a and 109b in >80% yields. LCMS and proton NMR was consistent
with the structure.
[0846] Compound 109a was treated to the same conditions as for
compounds 101a-d (Example 47), to give Compound 110a in 30-60%
yield. LCMS and proton NMR was consistent with the structure.
Alternatively, Compound 110b can be prepared in a similar manner
starting with Compound 109b.
Example 46: General Procedure for Conjugation with PFP Esters
(Oligonucleotide 111); Preparation of ISIS 666881
(GalNAc.sub.3-10)
[0847] A 5'-hexylamino modified oligonucleotide was synthesized and
purified using standard solid-phase oligonucleotide procedures. The
5'-hexylamino modified oligonucleotide was dissolved in 0.1 M
sodium tetraborate, pH 8.5 (200 .mu.L) and 3 equivalents of a
selected PFP esterified GalNAc.sub.3 cluster dissolved in DMSO (50
.mu.L) was added. If the PFP ester precipitated upon addition to
the ASO solution DMSO was added until all PFP ester was in
solution. The reaction was complete after about 16 h of mixing at
room temperature. The resulting solution was diluted with water to
12 mL and then spun down at 3000 rpm in a spin filter with a mass
cut off of 3000 Da. This process was repeated twice to remove small
molecule impurities. The solution was then lyophilized to dryness
and redissolved in concentrated aqueous ammonia and mixed at room
temperature for 2.5 h followed by concentration in vacuo to remove
most of the ammonia. The conjugated oligonucleotide was purified
and desalted by RP-HPLC and lyophilized to provide the GalNAc.sub.3
conjugated oligonucleotide.
##STR00187##
[0848] Oligonucleotide 111 is conjugated with GalNAc.sub.3-10. The
GalNAc.sub.3 cluster portion of the conjugate group GalNAc.sub.3-10
(GalNAc.sub.3-10.sub.a) can be combined with any cleavable moiety
to provide a variety of conjugate groups. In certain embodiments,
the cleavable moiety is --P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)-- as
shown in the oligonucleotide (ISIS 666881) synthesized with
GalNAc.sub.3-10 below. The structure of GalNAc.sub.3-10
(GalNAc.sub.3-10.sub.a-CM-) is shown below:
##STR00188##
[0849] Following this general procedure ISIS 666881 was prepared.
5'-hexylamino modified oligonucleotide, ISIS 660254, was
synthesized and purified using standard solid-phase oligonucleotide
procedures. ISIS 660254 (40 mg, 5.2 .mu.mol) was dissolved in 0.1 M
sodium tetraborate, pH 8.5 (200 .mu.L) and 3 equivalents PFP ester
(Compound 110a) dissolved in DMSO (50 .mu.L) was added. The PFP
ester precipitated upon addition to the ASO solution requiring
additional DMSO (600 .mu.L) to fully dissolve the PFP ester. The
reaction was complete after 16 h of mixing at room temperature. The
solution was diluted with water to 12 mL total volume and spun down
at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This
process was repeated twice to remove small molecule impurities. The
solution was lyophilized to dryness and redissolved in concentrated
aqueous ammonia with mixing at room temperature for 2.5 h followed
by concentration in vacuo to remove most of the ammonia. The
conjugated oligonucleotide was purified and desalted by RP-HPLC and
lyophilized to give ISIS 666881 in 90% yield by weight (42 mg, 4.7
.mu.mol).
TABLE-US-00027 GalNAc.sub.3-10 conjugated oligonucleotide SEQ 5' ID
ASO Sequence (5' to 3') group No. ISIS NH.sub.2(CH.sub.2).sub.6-
Hexylamine 831 660254
.sub.oA.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA-
.sub.dsG.sub.dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS 831 666881
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
[0850] Capital letters indicate the nucleobase for each nucleoside
and .sup.mC indicates a 5-methyl cytosine. Subscripts: "e"
indicates a 2'-MOE modified nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside; "s" indicates a phosphorothioate
internucleoside linkage (PS); "o" indicates a phosphodiester
internucleoside linkage (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Conjugate groups are in bold.
Example 47: Preparation of Oligonucleotide 102 Comprising
GalNAc.sub.3-8
##STR00189## ##STR00190## ##STR00191##
[0852] The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120
mL) and N,N-Diisopropylethylamine (12.35 mL, 72 mmoles).
Pentafluorophenyl trifluoroacetate (8.9 mL, 52 mmoles) was added
dropwise, under argon, and the reaction was allowed to stir at room
temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was
added, along with N,N-Diisopropylethylamine (12.35 mL, 72 mmoles),
and the reaction was allowed to stir at room temperature for 16
hours. At that time, the DMF was reduced by >75% under reduced
pressure, and then the mixture was dissolved in dichloromethane.
The organic layer was washed with sodium bicarbonate, water and
brine. The organic layer was then separated and dried over sodium
sulfate, filtered and reduced to an oil under reduced pressure. The
resultant oil was purified by silica gel chromatography (2%->10%
methanol/dichloromethane) to give compounds 92a and 92b in an
approximate 80% yield. LCMS and proton NMR were consistent with the
structure.
[0853] Compound 92a or 92b (6.7 mmoles) was treated with 20 mL of
dichloromethane and 20 mL of trifluoroacetic acid at room
temperature for 16 hours. The resultant solution was evaporated and
then dissolved in methanol and treated with DOWEX-OH resin for 30
minutes. The resultant solution was filtered and reduced to an oil
under reduced pressure to give 85-90% yield of compounds 93a and
93b.
[0854] Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7
g, 9.6 mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL)
for 15 minutes. To this was added either compounds 93a or 93b (3
mmoles), and allowed to stir at room temperature for 16 hours. At
that time, the DMF was reduced by >75% under reduced pressure,
and then the mixture was dissolved in dichloromethane. The organic
layer was washed with sodium bicarbonate, water and brine. The
organic layer was then separated and dried over sodium sulfate,
filtered and reduced to an oil under reduced pressure. The
resultant oil was purified by silica gel chromatography (5%->20%
methanol/dichloromethane) to give compounds 96a-d in 20-40% yield.
LCMS and proton NMR was consistent with the structure.
[0855] Compounds 96a-d (0.75 mmoles), individually, were
hydrogenated over Raney Nickel for 3 hours in Ethanol (75 mL). At
that time, the catalyst was removed by filtration thru celite, and
the ethanol removed under reduced pressure to give compounds 97a-d
in 80-90% yield. LCMS and proton NMR were consistent with the
structure.
[0856] Compound 23 (0.32 g, 0.53 mmoles) was treated with HBTU (0.2
g, 0.53 mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14
mmoles) in DMF (30 mL) for 15 minutes. To this was added compounds
97a-d (0.38 mmoles), individually, and allowed to stir at room
temperature for 16 hours. At that time, the DMF was reduced by
>75% under reduced pressure, and then the mixture was dissolved
in dichloromethane. The organic layer was washed with sodium
bicarbonate, water and brine. The organic layer was then separated
and dried over sodium sulfate, filtered and reduced to an oil under
reduced pressure. The resultant oil was purified by silica gel
chromatography (2%->20% methanol/dichloromethane) to give
compounds 98a-d in 30-40% yield. LCMS and proton NMR was consistent
with the structure.
[0857] Compound 99 (0.17 g, 0.76 mmoles) was treated with HBTU
(0.29 g, 0.76 mmoles) and N,N-Diisopropylethylamine (0.35 mL, 2.0
mmoles) in DMF (50 mL) for 15 minutes. To this was added compounds
97a-d (0.51 mmoles), individually, and allowed to stir at room
temperature for 16 hours. At that time, the DMF was reduced by
>75% under reduced pressure, and then the mixture was dissolved
in dichloromethane. The organic layer was washed with sodium
bicarbonate, water and brine. The organic layer was then separated
and dried over sodium sulfate, filtered and reduced to an oil under
reduced pressure. The resultant oil was purified by silica gel
chromatography (5%->20% methanol/dichloromethane) to give
compounds 100a-d in 40-60% yield. LCMS and proton NMR was
consistent with the structure.
[0858] Compounds 100a-d (0.16 mmoles), individually, were
hydrogenated over 10% Pd(OH).sub.2/C for 3 hours in methanol/ethyl
acetate (1:1, 50 mL). At that time, the catalyst was removed by
filtration thru celite, and the organics removed under reduced
pressure to give compounds 101a-d in 80-90% yield. LCMS and proton
NMR was consistent with the structure.
[0859] Compounds 101a-d (0.15 mmoles), individually, were dissolved
in DMF (15 mL) and pyridine (0.016 mL, 0.2 mmoles).
Pentafluorophenyl trifluoroacetate (0.034 mL, 0.2 mmoles) was added
dropwise, under argon, and the reaction was allowed to stir at room
temperature for 30 minutes. At that time, the DMF was reduced by
>75% under reduced pressure, and then the mixture was dissolved
in dichloromethane. The organic layer was washed with sodium
bicarbonate, water and brine. The organic layer was then separated
and dried over sodium sulfate, filtered and reduced to an oil under
reduced pressure. The resultant oil was purified by silica gel
chromatography (2%->5% methanol/dichloromethane) to give
compounds 102a-d in an approximate 80% yield. LCMS and proton NMR
were consistent with the structure.
##STR00192##
[0860] Oligomeric Compound 102, comprising a GalNAc.sub.3-8
conjugate group, was prepared using the general procedures
illustrated in Example 46. The GalNAc.sub.3 cluster portion of the
conjugate group GalNAc.sub.3-8 (GalNAc.sub.3-8.sub.a) can be
combined with any cleavable moiety to provide a variety of
conjugate groups. In a preferred embodiment, the cleavable moiety
is --P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--.
[0861] The structure of GalNAc.sub.3-8 (GalNAc.sub.3-8.sub.a-CM-)
is shown below:
##STR00193##
Example 48: Preparation of Oligonucleotide 119 Comprising
GalNAc.sub.3-7
##STR00194## ##STR00195##
[0863] Compound 112 was synthesized following the procedure
described in the literature (J. Med. Chem. 2004, 47,
5798-5808).
[0864] Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1
methanol/ethyl acetate (22 mL/22 mL). Palladium hydroxide on carbon
(0.5 g) was added. The reaction mixture was stirred at room
temperature under hydrogen for 12 h. The reaction mixture was
filtered through a pad of celite and washed the pad with 1:1
methanol/ethyl acetate. The filtrate and the washings were combined
and concentrated to dryness to yield Compound 105a (quantitative).
The structure was confirmed by LCMS.
[0865] Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and
DIEA (2.8 mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL)
and the reaction mixture was stirred at room temperature for 5 min.
To this a solution of Compound 105a (3.77 g, 8.4 mmol) in anhydrous
DMF (20 mL) was added. The reaction was stirred at room temperature
for 6 h. Solvent was removed under reduced pressure to get an oil.
The residue was dissolved in CH.sub.2Cl.sub.2 (100 mL) and washed
with aqueous saturated NaHCO.sub.3 solution (100 mL) and brine (100
mL). The organic phase was separated, dried (Na.sub.2SO.sub.4),
filtered and evaporated. The residue was purified by silica gel
column chromatography and eluted with 10 to 20% MeOH in
dichloromethane to yield Compound 114 (1.45 g, 30%). The structure
was confirmed by LCMS and .sup.1H NMR analysis.
[0866] Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1
methanol/ethyl acetate (4 mL/4 mL). Palladium on carbon (wet, 0.14
g) was added. The reaction mixture was flushed with hydrogen and
stirred at room temperature under hydrogen for 12 h. The reaction
mixture was filtered through a pad of celite. The celite pad was
washed with methanol/ethyl acetate (1:1). The filtrate and the
washings were combined together and evaporated under reduced
pressure to yield Compound 115 (quantitative). The structure was
confirmed by LCMS and .sup.1H NMR analysis.
[0867] Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol)
and DIEA (0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL)
and the reaction mixture was stirred at room temperature for 5 min.
To this a solution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous
DMF was added and the reaction was stirred at room temperature for
6 h. The solvent was removed under reduced pressure and the residue
was dissolved in CH.sub.2Cl.sub.2. The organic layer was washed
aqueous saturated NaHCO.sub.3 solution and brine and dried over
anhydrous Na.sub.2SO.sub.4 and filtered. The organic layer was
concentrated to dryness and the residue obtained was purified by
silica gel column chromatography and eluted with 3 to 15% MeOH in
dichloromethane to yield Compound 116 (0.84 g, 61%). The structure
was confirmed by LC MS and .sup.1H NMR analysis.
##STR00196##
[0868] Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1
methanol/ethyl acetate (5 mL/5 mL). Palladium on carbon (wet, 0.074
g) was added. The reaction mixture was flushed with hydrogen and
stirred at room temperature under hydrogen for 12 h. The reaction
mixture was filtered through a pad of celite. The celite pad was
washed with methanol/ethyl acetate (1:1). The filtrate and the
washings were combined together and evaporated under reduced
pressure to yield compound 117 (0.73 g, 98%). The structure was
confirmed by LCMS and .sup.1H NMR analysis.
[0869] Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous
DMF (3 mL). To this solution N,N-Diisopropylethylamine (70 .mu.L,
0.4 mmol) and pentafluorophenyl trifluoroacetate (72 .mu.L, 0.42
mmol) were added. The reaction mixture was stirred at room
temperature for 12 h and poured into a aqueous saturated
NaHCO.sub.3 solution. The mixture was extracted with
dichloromethane, washed with brine and dried over anhydrous
Na.sub.2SO.sub.4. The dichloromethane solution was concentrated to
dryness and purified with silica gel column chromatography and
eluted with 5 to 10% MeOH in dichloromethane to yield compound 118
(0.51 g, 79%). The structure was confirmed by LCMS and .sup.1H and
.sup.1H and .sup.19F NMR.
##STR00197##
[0870] Oligomeric Compound 119, comprising a GalNAc.sub.3-7
conjugate group, was prepared using the general procedures
illustrated in Example 46. The GalNAc.sub.3 cluster portion of the
conjugate group GalNAc.sub.3-7 (GalNAc.sub.3-7.sub.a) can be
combined with any cleavable moiety to provide a variety of
conjugate groups. In certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--.
[0871] The structure of GalNAc.sub.3-7 (GalNAc.sub.3-7.sub.a-CM-)
is shown below:
##STR00198##
Example 49: Preparation of Oligonucleotide 132 Comprising
GalNAc.sub.3-5
##STR00199##
[0873] Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol)
were dissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL,
80.35 mmol) was added and stirred for 5 min. The reaction mixture
was cooled in an ice bath and a solution of compound 121 (10 g,
mmol) in anhydrous DMF (20 mL) was added. Additional triethylamine
(4.5 mL, 32.28 mmol) was added and the reaction mixture was stirred
for 18 h under an argon atmosphere. The reaction was monitored by
TLC (ethyl acetate:hexane; 1:1; Rf=0.47).sub.n The solvent was
removed under reduced pressure. The residue was taken up in EtOAc
(300 mL) and washed with 1M NaHSO.sub.4 (3.times.150 mL), aqueous
saturated NaHCO.sub.3 solution (3.times.150 mL) and brine
(2.times.100 mL). Organic layer was dried with Na.sub.2SO.sub.4.
Drying agent was removed by filtration and organic layer was
concentrated by rotary evaporation. Crude mixture was purified by
silica gel column chromatography and eluted by using 35-50% EtOAc
in hexane to yield a compound 122 (15.50 g, 78.13%). The structure
was confirmed by LCMS and .sup.1H NMR analysis. Mass m/z 589.3
[M+H].sup.+.
[0874] A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10
mL) was added to a cooled solution of Compound 122 (7.75 g, 13.16
mmol) dissolved in methanol (15 mL). The reaction mixture was
stirred at room temperature for 45 min. and monitored by TLC
(EtOAc:hexane; 1:1). The reaction mixture was concentrated to half
the volume under reduced pressure. The remaining solution was
cooled an ice bath and neutralized by adding concentrated HCl. The
reaction mixture was diluted, extracted with EtOAc (120 mL) and
washed with brine (100 mL). An emulsion formed and cleared upon
standing overnight. The organic layer was separated dried
(Na.sub.2SO.sub.4), filtered and evaporated to yield Compound 123
(8.42 g). Residual salt is the likely cause of excess mass. LCMS is
consistent with structure. Product was used without any further
purification. M.W.cal:574.36; M.W.fd:575.3 [M+H].sup.+.
##STR00200##
[0875] Compound 126 was synthesized following the procedure
described in the literature (J. Am. Chem. Soc. 2011, 133,
958-963).
##STR00201## ##STR00202##
[0876] Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82
mmol) and compound 126 (6.33 g, 16.14 mmol) were dissolved in and
DMF (40 mL) and the resulting reaction mixture was cooled in an ice
bath. To this N,N-Diisopropylethylamine (4.42 mL, 25.82 mmol),
PyBop (8.7 g, 16.7 mmol) followed by Bop coupling reagent (1.17 g,
2.66 mmol) were added under an argon atmosphere. The ice bath was
removed and the solution was allowed to warm to room temperature.
The reaction was completed after 1 h as determined by TLC
(DCM:MeOH:AA; 89:10:1). The reaction mixture was concentrated under
reduced pressure. The residue was dissolved in EtOAc (200 mL) and
washed with 1 M NaHSO.sub.4 (3.times.100 mL), aqueous saturated
NaHCO.sub.3 (3.times.100 mL) and brine (2.times.100 mL). The
organic phase separated dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by silica gel column
chromatography with a gradient of 50% hexanes/EtOAC to 100% EtOAc
to yield Compound 127 (9.4 g) as a white foam. LCMS and .sup.1H NMR
were consistent with structure. Mass m/z 778.4 [M+H].sup.+.
[0877] Trifluoroacetic acid (12 mL) was added to a solution of
compound 127 (1.57 g, 2.02 mmol) in dichloromethane (12 mL) and
stirred at room temperature for 1 h. The reaction mixture was
co-evaporated with toluene (30 mL) under reduced pressure to
dryness. The residue obtained was co-evaporated twice with
acetonitrile (30 mL) and toluene (40 mL) to yield Compound 128
(1.67 g) as trifluoro acetate salt and used for next step without
further purification. LCMS and .sup.1H NMR were consistent with
structure. Mass m/z 478.2 [M+H].sup.+.
[0878] Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol),
and HOAt (0.035 g, 0.26 mmol) were combined together and dried for
4 h over P.sub.2O.sub.5 under reduced pressure in a round bottom
flask and then dissolved in anhydrous DMF (1 mL) and stirred for 5
min. To this a solution of compound 128 (0.20 g, 0.26 mmol) in
anhydrous DMF (0.2 mL) and N,N-Diisopropylethylamine (0.2 mL) was
added. The reaction mixture was stirred at room temperature under
an argon atmosphere. The reaction was complete after 30 min as
determined by LCMS and TLC (7% MeOH/DCM). The reaction mixture was
concentrated under reduced pressure. The residue was dissolved in
DCM (30 mL) and washed with 1 M NaHSO.sub.4 (3.times.20 mL),
aqueous saturated NaHCO.sub.3 (3.times.20 mL) and brine (3.times.20
mL). The organic phase was separated, dried over Na.sub.2SO.sub.4,
filtered and concentrated. The residue was purified by silica gel
column chromatography using 5-15% MeOH in dichloromethane to yield
Compound 129 (96.6 mg). LC MS and .sup.1H NMR are consistent with
structure. Mass m/z 883.4 [M+2H].sup.+.
[0879] Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol
(5 mL) in 20 mL scintillation vial. To this was added a small
amount of 10% Pd/C (0.015 mg) and the reaction vessel was flushed
with H.sub.2 gas. The reaction mixture was stirred at room
temperature under H.sub.2 atmosphere for 18 h. The reaction mixture
was filtered through a pad of Celite and the Celite pad was washed
with methanol. The filtrate washings were pooled together and
concentrated under reduced pressure to yield Compound 130 (0.08 g).
LCMS and .sup.1H NMR were consistent with structure. The product
was used without further purification. Mass m/z 838.3
[M+2H].sup.+.
[0880] To a 10 mL pointed round bottom flask were added compound
130 (75.8 mg, 0.046 mmol), 0.37 M pyridine/DMF (200 .mu.L) and a
stir bar. To this solution was added 0.7 M pentafluorophenyl
trifluoroacetate/DMF (100 .mu.L) drop wise with stirring. The
reaction was completed after 1 h as determined by LC MS. The
solvent was removed under reduced pressure and the residue was
dissolved in CHCl.sub.3 (.about.10 mL). The organic layer was
partitioned against NaHSO.sub.4 (1 M, 10 mL), aqueous saturated
NaHCO.sub.3 (10 mL) and brine (10 mL) three times each. The organic
phase separated and dried over Na.sub.2SO.sub.4, filtered and
concentrated to yield Compound 131 (77.7 mg). LCMS is consistent
with structure. Used without further purification. Mass m/z 921.3
[M+2H].sup.+.
##STR00203##
[0881] Oligomeric Compound 132, comprising a GalNAc.sub.3-5
conjugate group, was prepared using the general procedures
illustrated in Example 46. The GalNAc.sub.3 cluster portion of the
conjugate group GalNAc.sub.3-5 (GalNAc.sub.3-5.sub.a) can be
combined with any cleavable moiety to provide a variety of
conjugate groups. In certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--.
[0882] The structure of GalNAc.sub.3-5 (GalNAc.sub.3-5.sub.a-CM-)
is shown below:
##STR00204##
Example 50: Preparation of Oligonucleotide 144 Comprising
GalNAc.sub.4-11
##STR00205## ##STR00206##
[0884] Synthesis of Compound 134. To a Merrifield flask was added
aminomethyl VIMAD resin (2.5 g, 450 .mu.mol/g) that was washed with
acetonitrile, dimethylformamide, dichloromethane and acetonitrile.
The resin was swelled in acetonitrile (4 mL). Compound 133 was
pre-activated in a 100 mL round bottom flask by adding 20 (1.0
mmol, 0.747 g), TBTU (1.0 mmol, 0.321 g), acetonitrile (5 mL) and
DIEA (3.0 mmol, 0.5 mL). This solution was allowed to stir for 5
min and was then added to the Merrifield flask with shaking. The
suspension was allowed to shake for 3 h. The reaction mixture was
drained and the resin was washed with acetonitrile, DMF and DCM.
New resin loading was quantitated by measuring the absorbance of
the DMT cation at 500 nm (extinction coefficient=76000) in DCM and
determined to be 238 .mu.mol/g. The resin was capped by suspending
in an acetic anhydride solution for ten minutes three times.
[0885] The solid support bound compound 141 was synthesized using
iterative Fmoc-based solid phase peptide synthesis methods. A small
amount of solid support was withdrawn and suspended in aqueous
ammonia (28-30 wt %) for 6 h. The cleaved compound was analyzed by
LC-MS and the observed mass was consistent with structure. Mass m/z
1063.8 [M+2H].sup.+.
[0886] The solid support bound compound 142 was synthesized using
solid phase peptide synthesis methods.
##STR00207##
[0887] The solid support bound compound 143 was synthesized using
standard solid phase synthesis on a DNA synthesizer.
[0888] The solid support bound compound 143 was suspended in
aqueous ammonia (28-30 wt %) and heated at 55.degree. C. for 16 h.
The solution was cooled and the solid support was filtered. The
filtrate was concentrated and the residue dissolved in water and
purified by HPLC on a strong anion exchange column. The fractions
containing full length compound 144 were pooled together and
desalted. The resulting GalNAc.sub.4-11 conjugated oligomeric
compound was analyzed by LC-MS and the observed mass was consistent
with structure.
[0889] The GalNAc.sub.4 cluster portion of the conjugate group
GalNAc.sub.4-11 (GalNAc.sub.4-11.sub.a) can be combined with any
cleavable moiety to provide a variety of conjugate groups. In
certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--.
[0890] The structure of GalNAc.sub.4-11 (GalNAc.sub.4-11.sub.a-CM)
is shown below:
##STR00208##
Example 51: Preparation of Oligonucleotide 155 Comprising
GalNAc.sub.3-6
##STR00209##
[0892] Compound 146 was synthesized as described in the literature
(Analytical Biochemistry 1995, 229, 54-60).
##STR00210##
[0893] Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams,
57 mmol) were dissolved in CH.sub.2Cl.sub.2 (200 ml). Activated
molecular sieves (4 .ANG.. 2 g, powdered) were added, and the
reaction was allowed to stir for 30 minutes under nitrogen
atmosphere. TMS-OTf was added (4.1 ml, 22.77 mmol) and the reaction
was allowed to stir at room temp overnight. Upon completion, the
reaction was quenched by pouring into solution of saturated aqueous
NaHCO.sub.3 (500 ml) and crushed ice (.about.150 g). The organic
layer was separated, washed with brine, dried over MgSO.sub.4,
filtered, and was concentrated to an orange oil under reduced
pressure. The crude material was purified by silica gel column
chromatography and eluted with 2-10% MeOH in CH.sub.2Cl.sub.2 to
yield Compound 112 (16.53 g, 63%). LCMS and .sup.1H NMR were
consistent with the expected compound.
[0894] Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1
MeOH/EtOAc (40 ml). The reaction mixture was purged by bubbling a
stream of argon through the solution for 15 minutes. Pearlman's
catalyst (palladium hydroxide on carbon, 400 mg) was added, and
hydrogen gas was bubbled through the solution for 30 minutes. Upon
completion (TLC 10% MeOH in CH.sub.2Cl.sub.2, and LCMS), the
catalyst was removed by filtration through a pad of celite. The
filtrate was concentrated by rotary evaporation, and was dried
briefly under high vacuum to yield Compound 105a (3.28 g). LCMS and
1H NMR were consistent with desired product.
[0895] Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous
DMF (100 mL). N,N-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was
added, followed by HBTU (4 g, 10.5 mmol). The reaction mixture was
allowed to stir for .about.15 minutes under nitrogen. To this a
solution of compound 105a (3.3 g, 7.4 mmol) in dry DMF was added
and stirred for 2 h under nitrogen atmosphere. The reaction was
diluted with EtOAc and washed with saturated aqueous NaHCO.sub.3
and brine. The organics phase was separated, dried (MgSO.sub.4),
filtered, and concentrated to an orange syrup. The crude material
was purified by column chromatography 2-5% MeOH in CH.sub.2Cl.sub.2
to yield Compound 148 (3.44 g, 73%). LCMS and .sup.1H NMR were
consistent with the expected product.
[0896] Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1
MeOH/EtOAc (75 ml). The reaction mixture was purged by bubbling a
stream of argon through the solution for 15 minutes. Pearlman's
catalyst (palladium hydroxide on carbon) was added (350 mg).
Hydrogen gas was bubbled through the solution for 30 minutes. Upon
completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was
removed by filtration through a pad of celite. The filtrate was
concentrated by rotary evaporation, and was dried briefly under
high vacuum to yield Compound 149 (2.6 g). LCMS was consistent with
desired product. The residue was dissolved in dry DMF (10 ml) was
used immediately in the next step.
##STR00211##
[0897] Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF
(20 ml). To this DIEA (450 .mu.L, 2.6 mmol, 1.5 eq.) and HBTU (1.96
g, 0.5.2 mmol) were added. The reaction mixture was allowed to stir
for 15 minutes at room temperature under nitrogen. A solution of
compound 149 (2.6 g) in anhydrous DMF (10 mL) was added. The pH of
the reaction was adjusted to pH=9-10 by addition of DIEA (if
necessary). The reaction was allowed to stir at room temperature
under nitrogen for 2 h. Upon completion the reaction was diluted
with EtOAc (100 mL), and washed with aqueous saturated aqueous
NaHCO.sub.3, followed by brine. The organic phase was separated,
dried over MgSO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography and eluted with 2-10%
MeOH in CH.sub.2Cl.sub.2 to yield Compound 150 (0.62 g, 20%). LCMS
and .sup.1H NMR were consistent with the desired product.
[0898] Compound 150 (0.62 g) was dissolved in 1:1 MeOH/EtOAc (5 L).
The reaction mixture was purged by bubbling a stream of argon
through the solution for 15 minutes. Pearlman' s catalyst
(palladium hydroxide on carbon) was added (60 mg). Hydrogen gas was
bubbled through the solution for 30 minutes. Upon completion (TLC
10% MeOH in DCM, and LCMS), the catalyst was removed by filtration
(syringe-tip Teflon filter, 0.45 .mu.m). The filtrate was
concentrated by rotary evaporation, and was dried briefly under
high vacuum to yield Compound 151 (0.57 g). The LCMS was consistent
with the desired product. The product was dissolved in 4 mL dry DMF
and was used immediately in the next step.
##STR00212##
[0899] Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous
DMF (5 mL) and NN-Diisopropylethylamine (75 .mu.L, 1 mmol) and
PFP-TFA (90 .mu.L, 0.76 mmol) were added. The reaction mixture
turned magenta upon contact, and gradually turned orange over the
next 30 minutes. Progress of reaction was monitored by TLC and
LCMS. Upon completion (formation of the PFP ester), a solution of
compound 151 (0.57 g, 0.33 mmol) in DMF was added. The pH of the
reaction was adjusted to pH=9-10 by addition of
N,N-Diisopropylethylamine (if necessary). The reaction mixture was
stirred under nitrogen for .about.30 min. Upon completion, the
majority of the solvent was removed under reduced pressure. The
residue was diluted with CH.sub.2Cl.sub.2 and washed with aqueous
saturated NaHCO.sub.3, followed by brine. The organic phase
separated, dried over MgSO.sub.4, filtered, and concentrated to an
orange syrup. The residue was purified by silica gel column
chromatography (2-10% MeOH in CH.sub.2Cl.sub.2) to yield Compound
152 (0.35 g, 55%). LCMS and .sup.1H NMR were consistent with the
desired product.
[0900] Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1
MeOH/EtOAc (10 mL). The reaction mixture was purged by bubbling a
stream of argon thru the solution for 15 minutes. Pearlman's
catalyst (palladium hydroxide on carbon) was added (35 mg).
Hydrogen gas was bubbled thru the solution for 30 minutes. Upon
completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was
removed by filtration (syringe-tip Teflon filter, 0.45 .mu.m). The
filtrate was concentrated by rotary evaporation, and was dried
briefly under high vacuum to yield Compound 153 (0.33 g,
quantitative). The LCMS was consistent with desired product.
[0901] Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous
DMF (5 mL) with stirring under nitrogen. To this
N,N-Diisopropylethylamine (65 .mu.L, 0.37 mmol) and PFP-TFA (35
.mu.L, 0.28 mmol) were added. The reaction mixture was stirred
under nitrogen for .about.30 min. The reaction mixture turned
magenta upon contact, and gradually turned orange. The pH of the
reaction mixture was maintained at pH=9-10 by adding more
N,-Diisopropylethylamine. The progress of the reaction was
monitored by TLC and LCMS. Upon completion, the majority of the
solvent was removed under reduced pressure. The residue was diluted
with CH.sub.2Cl.sub.2 (50 mL), and washed with saturated aqueous
NaHCO.sub.3, followed by brine. The organic layer was dried over
MgSO.sub.4, filtered, and concentrated to an orange syrup. The
residue was purified by column chromatography and eluted with 2-10%
MeOH in CH.sub.2Cl.sub.2 to yield Compound 154 (0.29 g, 79%). LCMS
and .sup.1H NMR were consistent with the desired product.
##STR00213##
[0902] Oligomeric Compound 155, comprising a GalNAc.sub.3-6
conjugate group, was prepared using the general procedures
illustrated in Example 46. The GalNAc.sub.3 cluster portion of the
conjugate group GalNAc.sub.3-6 (GalNAc.sub.3-6.sub.a) can be
combined with any cleavable moiety to provide a variety of
conjugate groups. In certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--.
[0903] The structure of GalNAc.sub.3-6 (GalNAc.sub.3-6.sub.a-CM-)
is shown below:
##STR00214##
Example 52: Preparation of Oligonucleotide 160 Comprising
GalNAc.sub.3-9
##STR00215##
[0905] Compound 156 was synthesized following the procedure
described in the literature (J. Med. Chem. 2004, 47,
5798-5808).
[0906] Compound 156, (18.60 g, 29.28 mmol) was dissolved in
methanol (200 mL). Palladium on carbon (6.15 g, 10 wt %, loading
(dry basis), matrix carbon powder, wet) was added. The reaction
mixture was stirred at room temperature under hydrogen for 18 h.
The reaction mixture was filtered through a pad of celite and the
celite pad was washed thoroughly with methanol. The combined
filtrate was washed and concentrated to dryness. The residue was
purified by silica gel column chromatography and eluted with 5-10%
methanol in dichloromethane to yield Compound 157 (14.26 g, 89%).
Mass m/z 544.1 [M-H].sup.-.
[0907] Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF
(30 mL). HBTU (3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine
(13.73 mL, 78.81 mmol) were added and the reaction mixture was
stirred at room temperature for 5 minutes. To this a solution of
compound 47 (2.96 g, 7.04 mmol) was added. The reaction was stirred
at room temperature for 8 h. The reaction mixture was poured into a
saturated NaHCO.sub.3 aqueous solution. The mixture was extracted
with ethyl acetate and the organic layer was washed with brine and
dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue
obtained was purified by silica gel column chromatography and
eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25
g, 73.3%). The structure was confirmed by MS and .sup.1H NMR
analysis.
[0908] Compound 158 (7.2 g, 7.61 mmol) was dried over
P.sub.2O.sub.5 under reduced pressure. The dried compound was
dissolved in anhydrous DMF (50 mL). To this 1H-tetrazole (0.43 g,
6.09 mmol) and N-methylimidazole (0.3 mL, 3.81 mmol) and
2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite (3.65 mL,
11.50 mmol) were added. The reaction mixture was stirred t under an
argon atmosphere for 4 h. The reaction mixture was diluted with
ethyl acetate (200 mL). The reaction mixture was washed with
saturated NaHCO.sub.3 and brine. The organic phase was separated,
dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was
purified by silica gel column chromatography and eluted with 50-90%
ethyl acetate in hexane to yield Compound 159 (7.82 g, 80.5%). The
structure was confirmed by LCMS and .sup.31P NMR analysis.
##STR00216##
[0909] Oligomeric Compound 160, comprising a GalNAc.sub.3-9
conjugate group, was prepared using standard oligonucleotide
synthesis procedures. Three units of compound 159 were coupled to
the solid support, followed by nucleotide phosphoramidites.
Treatment of the protected oligomeric compound with aqueous ammonia
yielded compound 160. The GalNAc.sub.3 cluster portion of the
conjugate group GalNAc.sub.3-9 (GalNAc.sub.3-9.sub.a) can be
combined with any cleavable moiety to provide a variety of
conjugate groups. In certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--. The structure of
GalNAc.sub.3-9 (GalNAc.sub.3-9.sub.a-CM) is shown below:
##STR00217##
Example 53: Alternate Procedure for Preparation of Compound 18
(GalNAc.sub.3-1a and GalNAc.sub.3-3a)
##STR00218##
[0911] Lactone 161 was reacted with diamino propane (3-5 eq) or
Mono-Boc protected diamino propane (1 eq) to provide alcohol 162a
or 162b. When unprotected propanediamine was used for the above
reaction, the excess diamine was removed by evaporation under high
vacuum and the free amino group in 162a was protected using CbzCl
to provide 162b as a white solid after purification by column
chromatography. Alcohol 162b was further reacted with compound 4 in
the presence of TMSOTf to provide 163a which was converted to 163b
by removal of the Cbz group using catalytic hydrogenation. The
pentafluorophenyl (PFP) ester 164 was prepared by reacting triacid
113 (see Example 48) with PFPTFA (3.5 eq) and pyridine (3.5 eq) in
DMF (0.1 to 0.5 M). The triester 164 was directly reacted with the
amine 163b (3-4 eq) and DIPEA (3-4 eq) to provide Compound 18. The
above method greatly facilitates purification of intermediates and
minimizes the formation of byproducts which are formed using the
procedure described in Example 4.
Example 54: Alternate Procedure for Preparation of Compound 18
(GalNAc.sub.3-1a and GalNAc.sub.3-3a)
##STR00219##
[0913] The triPFP ester 164 was prepared from acid 113 using the
procedure outlined in example 53 above and reacted with mono-Boc
protected diamine to provide 165 in essentially quantitative yield.
The Boc groups were removed with hydrochloric acid or
trifluoroacetic acid to provide the triamine which was reacted with
the PFP activated acid 166 in the presence of a suitable base such
as DIPEA to provide Compound 18.
[0914] The PFP protected Gal-NAc acid 166 was prepared from the
corresponding acid by treatment with PFPTFA (1-1.2 eq) and pyridine
(1-1.2 eq) in DMF. The precursor acid in turn was prepared from the
corresponding alcohol by oxidation using TEMPO (0.2 eq) and BAIB in
acetonitrile and water. The precursor alcohol was prepared from
sugar intermediate 4 by reaction with 1,6-hexanediol (or
1,5-pentanediol or other diol for other n values) (2-4 eq) and
TMSOTf using conditions described previously in example 47.
Example 55: Dose-Dependent Study of Oligonucleotides Comprising
Either a 3' or 5'-Conjugate Group (Comparison of GalNAc.sub.3-1, 3,
8 and 9) Targeting SRB-1 In Vivo
[0915] The oligonucleotides listed below were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
Unconjugated ISIS 353382 was included as a standard. Each of the
various GalNAc.sub.3 conjugate groups was attached at either the 3'
or 5' terminus of the respective oligonucleotide by a
phosphodiester linked 2'-deoxyadenosine nucleoside (cleavable
moiety).
TABLE-US-00028 TABLE 39 Modified ASO targeting SRB-1 SEQ ASO
Sequence (5' to 3') Motif Conjugate ID No. ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 none
829 353382
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT-
.sub.e (parent) ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 830
655861
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.es
ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 830
664078
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.es
ISIS 831 661161
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
ISIS 665001
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 831
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
[0916] Capital letters indicate the nucleobase for each nucleoside
and .sup.mC indicates a 5-methyl cytosine. Subscripts: "e"
indicates a 2'-MOE modified nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside; "s" indicates a phosphorothioate
internucleoside linkage (PS); "o" indicates a phosphodiester
internucleoside linkage (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Conjugate groups are in bold.
[0917] The structure of GalNAc.sub.3-1.sub.a was shown previously
in Example 9. The structure of GalNAc.sub.3-9 was shown previously
in Example 52. The structure of GalNAc.sub.3-3 was shown previously
in Example 39. The structure of GalNAc.sub.3-8 was shown previously
in Example 47.
Treatment
[0918] Six week old male Balb/c mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously once at the dosage shown
below with ISIS 353382, 655861, 664078, 661161, 665001 or with
saline. Each treatment group consisted of 4 animals. The mice were
sacrificed 72 hours following the final administration to determine
the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN.RTM.
RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. The results below are presented as
the average percent of SRB-1 mRNA levels for each treatment group,
normalized to the saline control.
[0919] As illustrated in Table 40, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner. Indeed, the antisense oligonucleotides comprising the
phosphodiester linked GalNAc.sub.3-1 and GalNAc.sub.3-9 conjugates
at the 3' terminus (ISIS 655861 and ISIS 664078) and the
GalNAc.sub.3-3 and GalNAc.sub.3-8 conjugates linked at the 5'
terminus (ISIS 661161 and ISIS 665001) showed substantial
improvement in potency compared to the unconjugated antisense
oligonucleotide (ISIS 353382).sub.n Furthermore, ISIS 664078,
comprising a GalNAc.sub.3-9 conjugate at the 3' terminus was
essentially equipotent compared to ISIS 655861, which comprises a
GalNAc.sub.3-1 conjugate at the 3' terminus. The 5' conjugated
antisense oligonucleotides, ISIS 661161 and ISIS 665001, comprising
a GalNAc.sub.3-3 or GalNAc.sub.3-9, respectively, had increased
potency compared to the 3' conjugated antisense oligonucleotides
(ISIS 655861 and ISIS 664078).sub.n
TABLE-US-00029 TABLE 40 ASOs containing GalNAc.sub.3-1, 3, 8 or 9
targeting SRB-1 ISIS Dosage SRB-1 mRNA No. (mg/kg) (% Saline)
Conjugate Saline n/a 100 353382 3 88 none 10 68 30 36 655861 0.5 98
GalNac.sub.3-1 (3') 1.5 76 5 31 15 20 664078 0.5 88 GalNac.sub.3-9
(3') 1.5 85 5 46 15 20 661161 0.5 92 GalNac.sub.3-3 (5') 1.5 59 5
19 15 11 665001 0.5 100 GalNac.sub.3-8 (5') 1.5 73 5 29 15 13
[0920] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were measured
relative to saline injected mice using standard protocols. Total
bilirubin and BUN were also evaluated. The change in body weights
was evaluated with no significant change from the saline group.
ALTs, ASTs, total bilirubin and BUN values are shown in the table
below.
TABLE-US-00030 TABLE 41 ISIS Dosage Total No. mg/kg ALT AST
Bilirubin BUN Conjugate Saline 24 59 0.1 37.52 353382 3 21 66 0.2
34.65 none 10 22 54 0.2 34.2 30 22 49 0.2 33.72 655861 0.5 25 62
0.2 30.65 GalNac.sub.3-1 (3') 1.5 23 48 0.2 30.97 5 28 49 0.1 32.92
15 40 97 0.1 31.62 664078 0.5 40 74 0.1 35.3 GalNac.sub.3-9 (3')
1.5 47 104 0.1 32.75 5 20 43 0.1 30.62 15 38 92 0.1 26.2 661161 0.5
101 162 0.1 34.17 GalNac.sub.3-3 (5') 1.5 g 42 100 0.1 33.37 5 g 23
99 0.1 34.97 15 53 83 0.1 34.8 665001 0.5 28 54 0.1 31.32
GalNac.sub.3-8 (5') 1.5 42 75 0.1 32.32 5 24 42 0.1 31.85 15 32 67
0.1 31.
Example 56: Dose-Dependent Study of Oligonucleotides Comprising
Either a 3' or 5'-Conjugate Group (Comparison of GalNAc.sub.3-1, 2,
3, 5, 6, 7 and 10) Targeting SRB-1 In Vivo
[0921] The oligonucleotides listed below were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
Unconjugated ISIS 353382 was included as a standard. Each of the
various GalNAc.sub.3 conjugate groups was attached at the 5'
terminus of the respective oligonucleotide by a phosphodiester
linked 2'-deoxyadenosine nucleoside (cleavable moiety) except for
ISIS 655861 which had the GalNAc.sub.3 conjugate group attached at
the 3' terminus.
TABLE-US-00031 TABLE 42 Modified ASO targeting SRB-1 SEQ ASO
Sequence (5' to 3') Motif Conjugate ID No. ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 no
conjugate 829 353382
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT-
.sub.e (parent) ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 830
655861
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.es
ISIS 5/10/5 831 664507
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS 661161
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 831
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.m.sub.Ces.sup.mC.sub.esT.sub.esT.sub.e
ISIS 5/10/5 831 666224
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS 5/10/5 831
666961
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS 5/10/5 831
666981
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS 5/10/5 831
666881
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
[0922] Capital letters indicate the nucleobase for each nucleoside
and .sup.mC indicates a 5-methyl cytosine. Subscripts: "e"
indicates a 2'-MOE modified nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside; "s" indicates a phosphorothioate
internucleoside linkage (PS); "o" indicates a phosphodiester
internucleoside linkage (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Conjugate groups are in bold.
[0923] The structure of GalNAc.sub.3-1.sub.a was shown previously
in Example 9. The structure of GalNAc.sub.3-2.sub.a was shown
previously in Example 37. The structure of GalNAc.sub.3-3.sub.a was
shown previously in Example 39. The structure of
GalNAc.sub.3-5.sub.a was shown previously in Example 49. The
structure of GalNAc.sub.3-6.sub.a was shown previously in Example
51. The structure of GalNAc.sub.3-7.sub.a was shown previously in
Example 48. The structure of GalNAc.sub.3-10.sub.a was shown
previously in Example 46.
Treatment
[0924] Six week old male Balb/c mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously once at the dosage shown
below with ISIS 353382, 655861, 664507, 661161, 666224, 666961,
666981, 666881 or with saline. Each treatment group consisted of 4
animals. The mice were sacrificed 72 hours following the final
administration to determine the liver SRB-1 mRNA levels using
real-time PCR and RIBOGREEN.RTM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.) according to standard
protocols. The results below are presented as the average percent
of SRB-1 mRNA levels for each treatment group, normalized to the
saline control.
[0925] As illustrated in Table 43, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner. Indeed, the conjugated antisense oligonucleotides showed
substantial improvement in potency compared to the unconjugated
antisense oligonucleotide (ISIS 353382). The 5' conjugated
antisense oligonucleotides showed a slight increase in potency
compared to the 3' conjugated antisense oligonucleotide.
TABLE-US-00032 TABLE 43 ISIS Dosage SRB-1 mRNA No. (mg/kg) (%
Saline) Conjugate Saline n/a 100.0 353382 3 96.0 none 10 73.1 30
36.1 655861 0.5 99.4 GalNac.sub.3-1 (3') 1.5 81.2 5 33.9 15 15.2
664507 0.5 102.0 GalNac.sub.3-2 (5') 1.5 73.2 5 31.3 15 10.8 661161
0.5 90.7 GalNac.sub.3-3 (5') 1.5 67.6 5 24.3 15 11.5 666224 0.5
96.1 GalNac.sub.3-5 (5') 1.5 61.6 5 25.6 15 11.7 666961 0.5 85.5
GalNAc.sub.3-6 (5') 1.5 56.3 5 34.2 15 13.1 666981 0.5 84.7
GalNAc.sub.3-7 (5') 1.5 59.9 5 24.9 15 8.5 666881 0.5 100.0
GalNAc.sub.3-10 (5') 1.5 65.8 5 26.0 15 13.0
[0926] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were measured
relative to saline injected mice using standard protocols. Total
bilirubin and BUN were also evaluated. The change in body weights
was evaluated with no significant change from the saline group.
ALTs, ASTs, total bilirubin and BUN values are shown in Table 44
below.
TABLE-US-00033 TABLE 44 ISIS Dosage Total No. mg/kg ALT AST
Bilirubin BUN Conjugate Saline 26 57 0.2 27 353382 3 25 92 0.2 27
none 10 23 40 0.2 25 30 29 54 0.1 28 655861 0.5 25 71 0.2 34
GalNac.sub.3-1 (3') 1.5 28 60 0.2 26 5 26 63 0.2 28 15 25 61 0.2 28
664507 0.5 25 62 0.2 25 GalNac.sub.3-2 (5') 1.5 24 49 0.2 26 5 21
50 0.2 26 15 59 84 0.1 22 661161 0.5 20 42 0.2 29 GalNac.sub.3-3
(5') 1.5 g 37 74 0.2 25 5 g 28 61 0.2 29 15 21 41 0.2 25 666224 0.5
34 48 0.2 21 GalNac.sub.3-5 (5') 1.5 23 46 0.2 26 5 24 47 0.2 23 15
32 49 0.1 26 666961 0.5 17 63 0.2 26 GalNAc.sub.3-6 (5') 1.5 23 68
0.2 26 5 25 66 0.2 26 15 29 107 0.2 28 666981 0.5 24 48 0.2 26
GalNAc.sub.3-7 (5') 1.5 30 55 0.2 24 5 46 74 0.1 24 15 29 58 0.1 26
666881 0.5 20 65 0.2 27 GalNAc.sub.3-10 (5') 1.5 23 59 0.2 24 5 45
70 0.2 26 15 21 57 0.2 24
Example 57: Duration of Action Study of Oligonucleotides Comprising
a 3'-Conjugate Group Targeting ApoC III In Vivo
[0927] Mice were injected once with the doses indicated below and
monitored over the course of 42 days for ApoC-III and plasma
triglycerides (Plasma TG) levels. The study was performed using 3
transgenic mice that express human APOC-III in each group.
TABLE-US-00034 TABLE 45 Modified ASO targeting ApoC III SEQ Link-
ID ASO Sequence (5' to 3') ages No. ISIS
A.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.dsT.sub.dsT-
.sub.dsG.sub.dsT.sub.ds PS 821 304801
.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.esT-
.sub.esT.sub.esA.sub.esT.sub.e ISIS
A.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.dsT.sub.dsT-
.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds PS 822 647535
A.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.esT.sub.esT.sub.esA.sub.es ISIS
A.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.dsT.sub.dsT-
.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds PO/PS 822
647536
A.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.eoT.sub.eoT.sub.esA.sub.es
[0928] Capital letters indicate the nucleobase for each nucleoside
and .sup.mC indicates a 5-methyl cytosine. Subscripts: "e"
indicates a 2'-MOE modified nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside; "s" indicates a phosphorothioate
internucleoside linkage (PS); "o" indicates a phosphodiester
internucleoside linkage (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Conjugate groups are in bold.
[0929] The structure of GalNAc.sub.3-1.sub.a was shown previously
in Example 9.
TABLE-US-00035 TABLE 46 ApoC III mRNA (% Saline on Day 1) and
Plasma TG Levels (% Saline on Day 1) ASO Dose Target Day 3 Day 7
Day 14 Day 35 Day 42 Saline 0 mg/kg ApoC-III 98 100 100 95 116 ISIS
304801 30 mg/kg ApoC-III 28 30 41 65 74 ISIS 647535 10 mg/kg
ApoC-III 16 19 25 74 94 ISIS 647536 10 mg/kg ApoC-III 18 16 17 35
51 Saline 0 mg/kg Plasma TG 121 130 123 105 109 ISIS 304801 30
mg/kg Plasma TG 34 37 50 69 69 ISIS 647535 10 mg/kg Plasma TG 18 14
24 18 71 ISIS 647536 10 mg/kg Plasma TG 21 19 15 32 35
[0930] As can be seen in the table above the duration of action
increased with addition of the 3'-conjugate group compared to the
unconjugated oligonucleotide. There was a further increase in the
duration of action for the conjugated mixed PO/PS oligonucleotide
647536 as compared to the conjugated full PS oligonucleotide
647535.
Example 58: Dose-Dependent Study of Oligonucleotides Comprising a
3'-Conjugate Group (Comparison of GalNAc.sub.3-1 and GalNAc4-11)
Targeting SRB-1 In Vivo
[0931] The oligonucleotides listed below were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
Unconjugated ISIS 440762 was included as an unconjugated standard.
Each of the conjugate groups were attached at the 3' terminus of
the respective oligonucleotide by a phosphodiester linked
2'-deoxyadenosine nucleoside cleavable moiety.
[0932] The structure of GalNAc.sub.3-1.sub.a was shown previously
in Example 9. The structure of GalNAc.sub.3-11.sub.a was shown
previously in Example 50.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously once at the dosage shown
below with ISIS 440762, 651900, 663748 or with saline. Each
treatment group consisted of 4 animals. The mice were sacrificed 72
hours following the final administration to determine the liver
SRB-1 mRNA levels using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. The results below are presented as
the average percent of SRB-1 mRNA levels for each treatment group,
normalized to the saline control.
[0933] As illustrated in Table 47, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner. The antisense oligonucleotides comprising the
phosphodiester linked GalNAc.sub.3-1 and GalNAc4-11 conjugates at
the 3' terminus (ISIS 651900 and ISIS 663748) showed substantial
improvement in potency compared to the unconjugated antisense
oligonucleotide (ISIS 440762). The two conjugated oligonucleotides,
GalNAc.sub.3-1 and GalNAc4-11, were equipotent.
TABLE-US-00036 TABLE 47 Modified ASO targeting SRB-1 Dose % Saline
SEQ ID ASO Sequence (5' to 3') mg/kg control No. Saline 100 ISIS
T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT-
.sub.dsG.sub.dsA.sub.ds 0.6 73.45 823 440762
.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub.k 2 59.66 6 23.50 ISIS
T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT-
.sub.dsG.sub.dsA.sub.ds 0.2 62.75 824 651900
.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.m 0.6 29.14 2 8.61 6 5.62 ISIS
T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT-
.sub.dsG.sub.dsA.sub.ds 0.2 63.99 824 663748
.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.m 0.6 33.53 2 7.58 6 5.52
[0934] Capital letters indicate the nucleobase for each nucleoside
and .sup.mC indicates a 5-methyl cytosine. Subscripts: "e"
indicates a 2'-MOE modified nucleoside; "k" indicates
6'-(S)--CH.sub.3 bicyclic nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside; "s" indicates a phosphorothioate
internucleoside linkage (PS); "o" indicates a phosphodiester
internucleoside linkage (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Conjugate groups are in bold.
[0935] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were measured
relative to saline injected mice using standard protocols. Total
bilirubin and BUN were also evaluated. The change in body weights
was evaluated with no significant change from the saline group.
ALTs, ASTs, total bilirubin and BUN values are shown in Table 48
below.
TABLE-US-00037 TABLE 48 ISIS Dosage Total No. mg/kg ALT AST
Bilirubin BUN Conjugate Saline 30 76 0.2 40 440762 0.60 32 70 0.1
35 none 2 26 57 0.1 35 6 31 48 0.1 39 651900 0.2 32 115 0.2 39
GalNac.sub.3-1 (3') 0.6 33 61 0.1 35 2 30 50 0.1 37 6 34 52 0.1 36
663748 0.2 28 56 0.2 36 GalNac.sub.4-11 (3') 0.6 34 60 0.1 35 2 44
62 0.1 36 6 38 71 0.1 33
Example 59: Effects of GalNAc.sub.3-1 Conjugated ASOs Targeting FXI
In Vivo
[0936] The oligonucleotides listed below were tested in a multiple
dose study for antisense inhibition of FXI in mice. ISIS 404071 was
included as an unconjugated standard. Each of the conjugate groups
was attached at the 3' terminus of the respective oligonucleotide
by a phosphodiester linked 2'-deoxyadenosine nucleoside cleavable
moiety.
TABLE-US-00038 TABLE 49 Modified ASOs targeting FXI SEQ Link- ID
ASO Sequence (5' to 3') ages No. ISIS
T.sub.esG.sub.esG.sub.esT.sub.esA.sub.esA.sub.dsT.sub.ds.sup.mC.sub.d-
s.sup.mC.sub.dsA.sub.ds.sup.mC.sub.ds PS 832 404071
T.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.esG.sub.esA.sub.esG.sub-
.esG.sub.e ISIS
T.sub.esG.sub.esG.sub.esT.sub.esA.sub.esA.sub.dsT.sub.ds.sup.mC.sub.d-
s.sup.mC.sub.dsA.sub.ds.sup.mC.sub.ds PS 833 656172
T.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.esG.sub.esA.sub.esG.sub-
.es ISIS
T.sub.esG.sub.eoG.sub.eoT.sub.eoA.sub.eoA.sub.dsT.sub.ds.sup.mC.sub.d-
s.sup.mC.sub.dsA.sub.ds.sup.mC.sub.ds PO/PS 833 656173
T.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.eoG.sub.eoA.sub.esG.sub-
.es
[0937] Capital letters indicate the nucleobase for each nucleoside
and .sup.mC indicates a 5-methyl cytosine. Subscripts: "e"
indicates a 2'-MOE modified nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside; "s" indicates a phosphorothioate
internucleoside linkage (PS); "o" indicates a phosphodiester
internucleoside linkage (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Conjugate groups are in bold.
[0938] The structure of GalNAc.sub.3-1.sub.a was shown previously
in Example 9.
Treatment
[0939] Six week old male Balb/c mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously twice a week for 3 weeks
at the dosage shown below with ISIS 404071, 656172, 656173 or with
PBS treated control. Each treatment group consisted of 4 animals.
The mice were sacrificed 72 hours following the final
administration to determine the liver FXI mRNA levels using
real-time PCR and RIBOGREEN.RTM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.) according to standard
protocols. Plasma FXI protein levels were also measured using
ELISA. FXI mRNA levels were determined relative to total RNA (using
RIBOGREEN.RTM.), prior to normalization to PBS-treated control. The
results below are presented as the average percent of FXI mRNA
levels for each treatment group. The data was normalized to
PBS-treated control and is denoted as "% PBS". The ED.sub.50s were
measured using similar methods as described previously and are
presented below.
TABLE-US-00039 TABLE 50 Factor XI mRNA (% Saline) Dose % ASO mg/kg
Control Conjugate Linkages Saline 100 none ISIS 3 92 none PS 404071
10 40 30 15 ISIS 0.7 74 GalNAc.sub.3-1 PS 656172 2 33 6 9 ISIS 0.7
49 GalNAc.sub.3-1 PO/PS 656173 2 22 6 1
[0940] As illustrated in Table 50, treatment with antisense
oligonucleotides lowered FXI mRNA levels in a dose-dependent
manner. The oligonucleotides comprising a 3'-GalNAc.sub.3-1
conjugate group showed substantial improvement in potency compared
to the unconjugated antisense oligonucleotide (ISIS 404071).
Between the two conjugated oligonucleotides an improvement in
potency was further provided by substituting some of the PS
linkages with PO (ISIS 656173).
[0941] As illustrated in Table 50a, treatment with antisense
oligonucleotides lowered FXI protein levels in a dose-dependent
manner. The oligonucleotides comprising a 3'-GalNAc.sub.3-1
conjugate group showed substantial improvement in potency compared
to the unconjugated antisense oligonucleotide (ISIS 404071).
Between the two conjugated oligonucleotides an improvement in
potency was further provided by substituting some of the PS
linkages with PO (ISIS 656173).
TABLE-US-00040 TABLE 50a Factor XI protein (% Saline) Dose Protein
ASO mg/kg (% Control) Conjugate Linkages Saline 100 none ISIS 3 127
none PS 404071 10 32 30 3 ISIS 0.7 70 GalNAc.sub.3-1 PS 656172 2 23
6 1 ISIS 0.7 45 GalNAc.sub.3-1 PO/PS 656173 2 6 6 0
[0942] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were measured
relative to saline injected mice using standard protocols. Total
bilirubin, total albumin, CRE and BUN were also evaluated. The
change in body weights was evaluated with no significant change
from the saline group. ALTs, ASTs, total bilirubin and BUN values
are shown in the table below.
TABLE-US-00041 TABLE 51 ISIS Dosage Total Total No. mg/kg ALT AST
Albumin Bilirubin CRE BUN Conjugate Saline 71.8 84.0 3.1 0.2 0.2
22.9 404071 3 152.8 176.0 3.1 0.3 0.2 23.0 none 10 73.3 121.5 3.0
0.2 0.2 21.4 30 82.5 92.3 3.0 0.2 0.2 23.0 656172 0.7 62.5 111.5
3.1 0.2 0.2 23.8 GalNac.sub.3-1 (3') 2 33.0 51.8 2.9 0.2 0.2 22.0 6
65.0 71.5 3.2 0.2 0.2 23.9 656173 0.7 54.8 90.5 3.0 0.2 0.2 24.9
GalNac.sub.3-1 (3') 2 85.8 71.5 3.2 0.2 0.2 21.0 6 114.0 101.8 3.3
0.2 0.2 22.7
Example 60: Effects of Conjugated ASOs Targeting SRB-1 In Vitro
[0943] The oligonucleotides listed below were tested in a multiple
dose study for antisense inhibition of SRB-1 in primary mouse
hepatocytes. ISIS 353382 was included as an unconjugated standard.
Each of the conjugate groups were attached at the 3' or 5' terminus
of the respective oligonucleotide by a phosphodiester linked
2'-deoxyadenosine nucleoside cleavable moiety.
TABLE-US-00042 TABLE 52 Modified ASO targeting SRB-1 SEQ ID ASO
Sequence (5' to 3') Motif Conjugate No. ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 none
829 353382
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT-
.sub.e ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 830
655861
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.es
ISIS
G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 830
655862
.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.es
ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.ds
5/10/5 831 661161
T.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.d-
sT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.ds
5/10/5 831 665001
T.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.d-
sT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT-
.sub.dsmC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds 5/10/5 830 664078
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.es
ISIS
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.ds
5/10/5 831 666961
T.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.d-
sT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS
5/10/5 831 664507
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS 5/10/5 831
666881
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS 5/10/5 831
666224
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e ISIS 5/10/5 831
666981
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
[0944] Capital letters indicate the nucleobase for each nucleoside
and .sup.mC indicates a 5-methyl cytosine. Subscripts: "e"
indicates a 2'-MOE modified nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside; "s" indicates a phosphorothioate
internucleoside linkage (PS); "o" indicates a phosphodiester
internucleoside linkage (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Conjugate groups are in bold.
[0945] The structure of GalNAc.sub.3-1.sub.a was shown previously
in Example 9. The structure of GalNAc.sub.3-3a was shown previously
in Example 39. The structure of GalNAc.sub.3-8a was shown
previously in Example 47. The structure of GalNAc.sub.3-9a was
shown previously in Example 52. The structure of GalNAc.sub.3-6a
was shown previously in Example 51. The structure of
GalNAc.sub.3-2a was shown previously in Example 37. The structure
of GalNAc.sub.3-10a was shown previously in Example 46. The
structure of GalNAc.sub.3-5a was shown previously in Example 49.
The structure of GalNAc.sub.3-7a was shown previously in Example
48.
Treatment
[0946] The oligonucleotides listed above were tested in vitro in
primary mouse hepatocyte cells plated at a density of 25,000 cells
per well and treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20
nM modified oligonucleotide. After a treatment period of
approximately 16 hours, RNA was isolated from the cells and mRNA
levels were measured by quantitative real-time PCR and the SRB-1
mRNA levels were adjusted according to total RNA content, as
measured by RIBOGREEN.RTM..
[0947] The IC.sub.50 was calculated using standard methods and the
results are presented in Table 53. The results show that, under
free uptake conditions in which no reagents or electroporation
techniques are used to artificially promote entry of the
oligonucleotides into cells, the oligonucleotides comprising a
GalNAc conjugate were significantly more potent in hepatocytes than
the parent oligonucleotide (ISIS 353382) that does not comprise a
GalNAc conjugate.
TABLE-US-00043 TABLE 53 IC.sub.50 Internucleoside SEQ ASO (nM)
linkages Conjugate ID No. ISIS 353382 190.sup.a PS none 829 ISIS
655861 .sup. 11.sup.a PS GalNAc.sub.3-1 830 ISIS 655862 3 PO/PS
GalNAc.sub.3-1 830 ISIS 661161 .sup. 15.sup.a PS GalNAc.sub.3-3 831
ISIS 665001 20 PS GalNAc.sub.3-8 831 ISIS 664078 55 PS
GalNAc.sub.3-9 830 ISIS 666961 .sup. 22.sup.a PS GalNAc.sub.3-6 831
ISIS 664507 30 PS GalNAc.sub.3-2 831 ISIS 666881 30 PS
GalNAc.sub.3-10 831 ISIS 666224 .sup. 30.sup.a PS GalNAc.sub.3-5
831 ISIS 666981 40 PS GalNAc.sub.3-7 831 .sup.aAverage of multiple
runs.
Example 61: Preparation of Oligomeric Compound 175 Comprising
GalNAc.sub.3-12
##STR00220## ##STR00221## ##STR00222##
[0948] Compound 169 is commercially available. Compound 172 was
prepared by addition of benzyl (perfluorophenyl) glutarate to
compound 171. The benzyl (perfluorophenyl) glutarate was prepared
by adding PFP-TFA and DIEA to 5-(benzyloxy)-5-oxopentanoic acid in
DMF. Oligomeric compound 175, comprising a GalNAc.sub.3-12
conjugate group, was prepared from compound 174 using the general
procedures illustrated in Example 46. The GalNAc.sub.3 cluster
portion of the conjugate group GalNAc.sub.3-12
(GalNAc.sub.3-12.sub.a) can be combined with any cleavable moiety
to provide a variety of conjugate groups. In a certain embodiments,
the cleavable moiety is --P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--.
The structure of GalNAc.sub.3-12 (GalNAc.sub.3-12.sub.a-CM-) is
shown below:
##STR00223##
Example 62: Preparation of Oligomeric Compound 180 Comprising
GalNAc.sub.3-13
##STR00224## ##STR00225##
[0949] Compound 176 was prepared using the general procedure shown
in Example 2. Oligomeric compound 180, comprising a GalNAc.sub.3-13
conjugate group, was prepared from compound 177 using the general
procedures illustrated in Example 49. The GalNAc.sub.3 cluster
portion of the conjugate group GalNAc.sub.3-13
(GalNAc.sub.3-13.sub.a) can be combined with any cleavable moiety
to provide a variety of conjugate groups. In a certain embodiments,
the cleavable moiety is --P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--.
The structure of GalNAc.sub.3-13 (GalNAc.sub.3-13.sub.a-CM-) is
shown below:
##STR00226##
Example 63: Preparation of Oligomeric Compound 188 Comprising
GalNAc.sub.3-14
##STR00227## ##STR00228## ##STR00229##
[0950] Compounds 181 and 185 are commercially available. Oligomeric
compound 188, comprising a GalNAc.sub.3-14 conjugate group, was
prepared from compound 187 using the general procedures illustrated
in Example 46. The GalNAc.sub.3 cluster portion of the conjugate
group GalNAc.sub.3-14 (GalNAc.sub.3-14.sub.a) can be combined with
any cleavable moiety to provide a variety of conjugate groups. In
certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--. The structure of
GalNAc.sub.3-14 (GalNAc.sub.3-14.sub.a-CM-) is shown below:
##STR00230##
Example 64: Preparation of Oligomeric Compound 197 Comprising
GalNAc.sub.3-15
##STR00231## ##STR00232##
[0951] Compound 189 is commercially available. Compound 195 was
prepared using the general procedure shown in Example 31.
Oligomeric compound 197, comprising a GalNAc.sub.3-15 conjugate
group, was prepared from compounds 194 and 195 using standard
oligonucleotide synthesis procedures. The GalNAc.sub.3 cluster
portion of the conjugate group GalNAc.sub.3-15
(GalNAc.sub.3-15.sub.a) can be combined with any cleavable moiety
to provide a variety of conjugate groups. In certain embodiments,
the cleavable moiety is --P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--.
The structure of GalNAc.sub.3-15 (GalNAc.sub.3-15.sub.a-CM-) is
shown below:
##STR00233##
Example 65: Dose-Dependent Study of Oligonucleotides Comprising a
5'-Conjugate Group (Comparison of GalNAc.sub.3-3, 12, 13, 14, and
15) Targeting SRB-1 In Vivo
[0952] The oligonucleotides listed below were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
Unconjugated ISIS 353382 was included as a standard. Each of the
GalNAc.sub.3 conjugate groups was attached at the 5' terminus of
the respective oligonucleotide by a phosphodiester linked
2'-deoxyadenosine nucleoside (cleavable moiety).
TABLE-US-00044 TABLE 54 Modified ASOs targeting SRB-1 ISIS SEQ No.
Sequence (5' to 3') Conjugate ID No. 353382
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub-
.dsT.sub.es none 829 .sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
661161 GalNAc.sub.3-3 831
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.su-
b.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.ds
T.sub.es.sup.m.sub.Ces.sup.mC.sub.esT.sub.esT.sub.e 671144
GalNAc.sub.3-12 831
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.su-
b.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.ds
T.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 670061
GalNAc.sub.3-13 831
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.su-
b.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.ds
T.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 671261
GalNAc.sub.3-14 831
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.su-
b.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.ds
T.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 671262
GalNAc.sub.3-15 831
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.su-
b.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.ds
T.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
Capital letters indicate the nucleobase for each nucleoside and
.sup.mC indicates a 5-methyl cytosine. Subscripts: "e" indicates a
2'-MOE modified nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside; "s" indicates a phosphorothioate
internucleoside linkage (PS); "o" indicates a phosphodiester
internucleoside linkage (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Conjugate groups are in bold.
[0953] The structure of GalNAc.sub.3-3.sub.a was shown previously
in Example 39. The structure of GalNAc.sub.3-12a was shown
previously in Example 61. The structure of GalNAc.sub.3-13a was
shown previously in Example 62. The structure of GalNAc.sub.3-14a
was shown previously in Example 63. The structure of
GalNAc.sub.3-15a was shown previously in Example 64.
Treatment
[0954] Six to eight week old C.sub.57bl6 mice (Jackson Laboratory,
Bar Harbor, Me.) were injected subcutaneously once or twice at the
dosage shown below with ISIS 353382, 661161, 671144, 670061,
671261, 671262, or with saline. Mice that were dosed twice received
the second dose three days after the first dose. Each treatment
group consisted of 4 animals. The mice were sacrificed 72 hours
following the final administration to determine the liver SRB-1
mRNA levels using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. The results below are presented as
the average percent of SRB-1 mRNA levels for each treatment group,
normalized to the saline control.
[0955] As illustrated in Table 55, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner. No significant differences in target knockdown were
observed between animals that received a single dose and animals
that received two doses (see ISIS 353382 dosages 30 and 2.times.15
mg/kg; and ISIS 661161 dosages 5 and 2.times.2.5 mg/kg). The
antisense oligonucleotides comprising the phosphodiester linked
GalNAc.sub.3-3, 12, 13, 14, and 15 conjugates showed substantial
improvement in potency compared to the unconjugated antisense
oligonucleotide (ISIS 335382).
TABLE-US-00045 TABLE 55 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1
mRNA ED.sub.50 No. (mg/kg) (% Saline) (mg/kg) Conjugate Saline n/a
100.0 n/a n/a 353382 3 85.0 22.4 none 10 69.2 30 34.2 2 .times. 15
36.0 661161 0.5 87.4 2.2 GalNAc.sub.3-3 1.5 59.0 5 25.6 2 .times.
2.5 27.5 15 17.4 671144 0.5 101.2 3.4 GalNAc.sub.3-12 1.5 76.1 5
32.0 15 17.6 670061 0.5 94.8 2.1 GalNAc.sub.3-13 1.5 57.8 5 20.7 15
13.3 671261 0.5 110.7 4.1 GalNAc.sub.3-14 1.5 81.9 5 39.8 15 14.1
671262 0.5 109.4 9.8 GalNAc.sub.3-15 1.5 99.5 5 69.2 15 36.1
[0956] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were measured
relative to saline injected mice using standard protocols. Total
bilirubin and BUN were also evaluated. The changes in body weights
were evaluated with no significant differences from the saline
group (data not shown). ALTs, ASTs, total bilirubin and BUN values
are shown in Table 56 below.
TABLE-US-00046 TABLE 56 Total ISIS Dosage ALT AST Bilirubin BUN No.
(mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Conjugate Saline n/a 28 60 0.1
39 n/a 353382 3 30 77 0.2 36 none 10 25 78 0.2 36 30 28 62 0.2 35 2
.times. 15 22 59 0.2 33 661161 0.5 39 72 0.2 34 GalNAc.sub.3-3 1.5
26 50 0.2 33 5 41 80 0.2 32 2 .times. 2.5 24 72 0.2 28 15 32 69 0.2
36 671144 0.5 25 39 0.2 34 GalNAc.sub.3-12 1.5 26 55 0.2 28 5 48 82
0.2 34 15 23 46 0.2 32 670061 0.5 27 53 0.2 33 GalNAc.sub.3-13 1.5
24 45 0.2 35 5 23 58 0.1 34 15 24 72 0.1 31 671261 0.5 69 99 0.1 33
GalNAc.sub.3-14 1.5 34 62 0.1 33 5 43 73 0.1 32 15 32 53 0.2 30
671262 0.5 24 51 0.2 29 GalNAc.sub.3-15 1.5 32 62 0.1 31 5 30 76
0.2 32 15 31 64 0.1 32
Example 66: Effect of Various Cleavable Moieties on Antisense
Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a
5'-GalNAc.sub.3 Cluster
[0957] The oligonucleotides listed below were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
Each of the GalNAc.sub.3 conjugate groups was attached at the 5'
terminus of the respective oligonucleotide by a phosphodiester
linked nucleoside (cleavable moiety (CM)).
TABLE-US-00047 TABLE 57 Modified ASOs targeting SRB-1 ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 661161
GalNAc.sub.3-3.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT-
.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.d-
s GalNAc.sub.3-3a A.sub.d 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.esT.sub.e 670699
GalNAc.sub.3-3.sub.a-.sub.o'T.sub.doG.sub.es.sup.mC.sub.eoT.sub.eoT-
.sub.eo.sup.mC.sub.eoA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.d-
s GalNAc.sub.3-3a T.sub.d 834
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.e-
sT.sub.esT.sub.e 670700
GalNAc.sub.3-3.sub.a-.sub.o'A.sub.eoG.sub.es.sup.mC.sub.eoT.sub.eoT-
.sub.eo.sup.mC.sub.eoA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.d-
s GalNAc.sub.3-3a A.sub.e 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.e-
sT.sub.esT.sub.e 670701
GalNAc.sub.3-3.sub.a-o'T.sub.eoG.sub.es.sup.mC.sub.eoT.sub.eoT.sub.-
eo.sup.mC.sub.eoA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ds
GalNAc.sub.3-3a T.sub.e 834
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.e-
sT.sub.esT.sub.e 671165
GalNAc.sub.3-13.sub.a-o'A.sub.doG.sub.es.sup.mC.sub.eoT.sub.eoT.sub-
.eo.sup.mC.sub.eoA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ds
GalNAc.sub.3-13a A.sub.d 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.e-
sT.sub.esT.sub.e
Capital letters indicate the nucleobase for each nucleoside and
.sup.mC indicates a 5-methyl cytosine. Subscripts: "e" indicates a
2'-MOE modified nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside;
[0958] "s" indicates a phosphorothioate internucleoside linkage
(PS); "o" indicates a phosphodiester internucleoside linkage (PO);
and "o'" indicates --O--P(.dbd.O)(OH)--. Conjugate groups are in
bold.
[0959] The structure of GalNAc.sub.3-3.sub.a was shown previously
in Example 39. The structure of GalNAc.sub.3-13a was shown
previously in Example 62.
Treatment
[0960] Six to eight week old C.sub.57bl6 mice (Jackson Laboratory,
Bar Harbor, Me.) were injected subcutaneously once at the dosage
shown below with ISIS 661161, 670699, 670700, 670701, 671165, or
with saline. Each treatment group consisted of 4 animals. The mice
were sacrificed 72 hours following the final administration to
determine the liver SRB-1 mRNA levels using real-time PCR and
RIBOGREEN.RTM. RNA quantification reagent (Molecular Probes, Inc.
Eugene, Oreg.) according to standard protocols. The results below
are presented as the average percent of SRB-1 mRNA levels for each
treatment group, normalized to the saline control.
[0961] As illustrated in Table 58, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner. The antisense oligonucleotides comprising various cleavable
moieties all showed similar potencies.
TABLE-US-00048 TABLE 58 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1
mRNA GalNAc.sub.3 No. (mg/kg) (% Saline) Cluster CM Saline n/a
100.0 n/a n/a 661161 0.5 87.8 GalNAc.sub.3-3a A.sub.d 1.5 61.3 5
33.8 15 14.0 670699 0.5 89.4 GalNAc.sub.3-3a T.sub.d 1.5 59.4 5
31.3 15 17.1 670700 0.5 79.0 GalNAc.sub.3-3a A.sub.e 1.5 63.3 5
32.8 15 17.9 670701 0.5 79.1 GalNAc.sub.3-3a T.sub.e 1.5 59.2 5
35.8 15 17.7 671165 0.5 76.4 GalNAc.sub.3-13a A.sub.d 1.5 43.2 5
22.6 15 10.0
[0962] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were measured
relative to saline injected mice using standard protocols. Total
bilirubin and BUN were also evaluated. The changes in body weights
were evaluated with no significant differences from the saline
group (data not shown). ALTs, ASTs, total bilirubin and BUN values
are shown in Table 56 below.
TABLE-US-00049 TABLE 59 Total ISIS Dosage ALT AST Bilirubin BUN
GalNAc.sub.3 No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Cluster CM
Saline n/a 24 64 0.2 31 n/a n/a 661161 0.5 25 64 0.2 31
GalNAc.sub.3-3a A.sub.d 1.5 24 50 0.2 32 5 26 55 0.2 28 15 27 52
0.2 31 670699 0.5 42 83 0.2 31 GalNAc.sub.3-3a T.sub.d 1.5 33 58
0.2 32 5 26 70 0.2 29 15 25 67 0.2 29 670700 0.5 40 74 0.2 27
GalNAc.sub.3-3a A.sub.e 1.5 23 62 0.2 27 5 24 49 0.2 29 15 25 87
0.1 25 670701 0.5 30 77 0.2 27 GalNAc.sub.3-3a T.sub.e 1.5 22 55
0.2 30 5 81 101 0.2 25 15 31 82 0.2 24 671165 0.5 44 84 0.2 26
GalNAc.sub.3-13a A.sub.d 1.5 47 71 0.1 24 5 33 91 0.2 26 15 33 56
0.2 29
Example 67: Preparation of Oligomeric Compound 199 Comprising
GalNAc.sub.3-16
##STR00234##
[0963] Oligomeric compound 199, comprising a GalNAc.sub.3-16
conjugate group, is prepared using the general procedures
illustrated in Examples 7 and 9. The GalNAc.sub.3 cluster portion
of the conjugate group GalNAc.sub.3-16 (GalNAc.sub.3-16.sub.a) can
be combined with any cleavable moiety to provide a variety of
conjugate groups. In certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--. The structure of
GalNAc.sub.3-16 (GalNAc.sub.3-16.sub.a-CM-) is shown below:
##STR00235##
Example 68: Preparation of Oligomeric Compound 200 Comprising
GalNAc.sub.3-17
##STR00236##
[0964] Oligomeric compound 200, comprising a GalNAc.sub.3-17
conjugate group, was prepared using the general procedures
illustrated in Example 46. The GalNAc.sub.3 cluster portion of the
conjugate group GalNAc.sub.3-17 (GalNAc.sub.3-17.sub.a) can be
combined with any cleavable moiety to provide a variety of
conjugate groups. In certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--. The structure of
GalNAc.sub.3-17 (GalNAc.sub.3-17.sub.a-CM-) is shown below:
##STR00237##
Example 69: Preparation of Oligomeric Compound 201 Comprising
GalNAc.sub.3-18
##STR00238##
[0965] Oligomeric compound 201, comprising a GalNAc.sub.3-18
conjugate group, was prepared using the general procedures
illustrated in Example 46. The GalNAc.sub.3 cluster portion of the
conjugate group GalNAc.sub.3-18 (GalNAc.sub.3-18.sub.a) can be
combined with any cleavable moiety to provide a variety of
conjugate groups. In certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--. The structure of
GalNAc.sub.3-18 (GalNAc.sub.3-18.sub.a-CM-) is shown below:
##STR00239##
Example 70: Preparation of Oligomeric Compound 204 Comprising
GalNAc.sub.3-19
##STR00240##
[0966] Oligomeric compound 204, comprising a GalNAc.sub.3-19
conjugate group, was prepared from compound 64 using the general
procedures illustrated in Example 52. The GalNAc.sub.3 cluster
portion of the conjugate group GalNAc.sub.3-19
(GalNAc.sub.3-19.sub.a) can be combined with any cleavable moiety
to provide a variety of conjugate groups. In certain embodiments,
the cleavable moiety is --P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--.
The structure of GalNAc.sub.3-19 (GalNAc.sub.3-19.sub.a-CM-) is
shown below:
##STR00241##
Example 71: Preparation of Oligomeric Compound 210 Comprising
GalNAc.sub.3-20
##STR00242## ##STR00243##
[0967] Compound 205 was prepared by adding PFP-TFA and DIEA to
6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which
was prepared by adding triflic anhydride to 6-aminohexanoic acid.
The reaction mixture was heated to 80.degree. C., then lowered to
rt. Oligomeric compound 210, comprising a GalNAc.sub.3-20 conjugate
group, was prepared from compound 208 using the general procedures
illustrated in Example 52. The GalNAc.sub.3 cluster portion of the
conjugate group GalNAc.sub.3-20 (GalNAc.sub.3-20.sub.a) can be
combined with any cleavable moiety to provide a variety of
conjugate groups. In certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--. The structure of
GalNAc.sub.3-20 (GalNAc.sub.3-20.sub.a-CM-) is shown below:
##STR00244##
Example 72: Preparation of Oligomeric Compound 215 Comprising
GalNAc.sub.3-21
##STR00245## ##STR00246##
[0968] Compound 211 is commercially available. Oligomeric compound
215, comprising a GalNAc.sub.3-21 conjugate group, was prepared
from compound 213 using the general procedures illustrated in
Example 52. The GalNAc.sub.3 cluster portion of the conjugate group
GalNAc.sub.3-21 (GalNAc.sub.3-21.sub.a) can be combined with any
cleavable moiety to provide a variety of conjugate groups. In
certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--. The structure of
GalNAc.sub.3-21 (GalNAc.sub.3-21.sub.a-CM-) is shown below:
##STR00247##
Example 73: Preparation of Oligomeric Compound 221 Comprising
GalNAc.sub.3-22
##STR00248## ##STR00249##
[0969] Compound 220 was prepared from compound 219 using
diisopropylammonium tetrazolide. Oligomeric compound 221,
comprising a GalNAc.sub.3-21 conjugate group, is prepared from
compound 220 using the general procedure illustrated in Example 52.
The GalNAc.sub.3 cluster portion of the conjugate group
GalNAc.sub.3-22 (GalNAc.sub.3-22.sub.a) can be combined with any
cleavable moiety to provide a variety of conjugate groups. In
certain embodiments, the cleavable moiety is
--P(.dbd.O)(OH)-A.sub.d-P(.dbd.O)(OH)--. The structure of
GalNAc.sub.3-22 (GalNAc.sub.3-22.sub.a-CM-) is shown below:
##STR00250##
Example 74: Effect of Various Cleavable Moieties on Antisense
Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a
5'-GalNAc.sub.3 Conjugate
[0970] The oligonucleotides listed below were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
Each of the GalNAc.sub.3 conjugate groups was attached at the 5'
terminus of the respective oligonucleotide.
TABLE-US-00050 TABLE 60 Modified ASOs targeting SRB-1 ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 353382
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub-
.dsT.sub.es n/a n/a 829 .sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
661161
GalNAc.sub.3-3.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT-
.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.d-
s GalNAc.sub.3-3a A.sub.d 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.esT.sub.e 666904
GalNAc.sub.3-3.sub.a-.sub.o'G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.-
sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ds
GalNAc.sub.3-3a PO 829
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.esT.sub.e 675441
GalNAc.sub.3-17.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.es-
T.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.-
ds GalNAc.sub.3-17a A.sub.d 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.esT.sub.e 675442
GalNAc.sub.3-18.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.es-
T.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.-
ds GalNAc.sub.3-18a A.sub.d 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.esT.sub.e
In all tables, capital letters indicate the nucleobase for each
nucleoside and .sup.mC indicates a 5-methyl cytosine. Subscripts:
"e" indicates a 2'-MOE modified nucleoside; "d" indicates a
.beta.-D-2'-deoxyribonucleoside; "s" indicates a phosphorothioate
internucleoside linkage (PS); "o" indicates a phosphodiester
internucleoside linkage (PO); and "o'" indicates
--O--P(.dbd.O)(OH)--. Conjugate groups are in bold.
[0971] The structure of GalNAc.sub.3-3.sub.a was shown previously
in Example 39. The structure of GalNAc.sub.3-17a was shown
previously in Example 68, and the structure of GalNAc.sub.3-18a was
shown in Example 69.
Treatment
[0972] Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously once at the dosage shown
below with an oligonucleotide listed in Table 60 or with saline.
Each treatment group consisted of 4 animals. The mice were
sacrificed 72 hours following the final administration to determine
the SRB-1 mRNA levels using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. The results below are presented as
the average percent of SRB-1 mRNA levels for each treatment group,
normalized to the saline control.
[0973] As illustrated in Table 61, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner. The antisense oligonucleotides comprising a GalNAc
conjugate showed similar potencies and were significantly more
potent than the parent oligonucleotide lacking a GalNAc
conjugate.
TABLE-US-00051 TABLE 61 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1
mRNA GalNAc.sub.3 No. (mg/kg) (% Saline) Cluster CM Saline n/a
100.0 n/a n/a 353382 3 79.38 n/a n/a 10 68.67 30 40.70 661161 0.5
79.18 GalNAc.sub.3-3a A.sub.d 1.5 75.96 5 30.53 15 12.52 666904 0.5
91.30 GalNAc.sub.3-3a PO 1.5 57.88 5 21.22 15 16.49 675441 0.5
76.71 GalNAc.sub.3-17a A.sub.d 1.5 63.63 5 29.57 15 13.49 675442
0.5 95.03 GalNAc.sub.3-18a A.sub.d 1.5 60.06 5 31.04 15 19.40
[0974] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were measured
relative to saline injected mice using standard protocols. Total
bilirubin and BUN were also evaluated. The change in body weights
was evaluated with no significant change from the saline group
(data not shown). ALTs, ASTs, total bilirubin and BUN values are
shown in Table 62 below.
TABLE-US-00052 TABLE 62 Total ISIS Dosage ALT AST Bilirubin BUN
GalNAc.sub.3 No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Cluster CM
Saline n/a 26 59 0.16 42 n/a n/a 353382 3 23 58 0.18 39 n/a n/a 10
28 58 0.16 43 30 20 48 0.12 34 661161 0.5 30 47 0.13 35
GalNAc.sub.3-3a A.sub.d 1.5 23 53 0.14 37 5 26 48 0.15 39 15 32 57
0.15 42 666904 0.5 24 73 0.13 36 GalNAc.sub.3-3a PO 1.5 21 48 0.12
32 5 19 49 0.14 33 15 20 52 0.15 26 675441 0.5 42 148 0.21 36
GalNAc.sub.3-17a A.sub.d 1.5 60 95 0.16 34 5 27 75 0.14 37 15 24 61
0.14 36 675442 0.5 26 65 0.15 37 GalNAc.sub.3-18a A.sub.d 1.5 25 64
0.15 43 5 27 69 0.15 37 15 30 84 0.14 37
Example 75: Pharmacokinetic Analysis of Oligonucleotides Comprising
a 5'-Conjugate Group
[0975] The PK of the ASOs in Tables 54, 57 and 60 above was
evaluated using liver samples that were obtained following the
treatment procedures described in Examples 65, 66, and 74. The
liver samples were minced and extracted using standard protocols
and analyzed by IP-HPLC-MS alongside an internal standard. The
combined tissue level (.mu.g/g) of all metabolites was measured by
integrating the appropriate UV peaks, and the tissue level of the
full-length ASO missing the conjugate ("parent," which is Isis No.
353382 in this case) was measured using the appropriate extracted
ion chromatograms (EIC).
TABLE-US-00053 TABLE 63 PK Analysis in Liver Total Parent ASO
Tissue Level Tissue Level ISIS Dosage by UV by EIC GalNAc.sub.3 No.
(mg/kg) (.mu.g/g) (.mu.g/g) Cluster CM 353382 3 8.9 8.6 n/a n/a 10
22.4 21.0 30 54.2 44.2 661161 5 32.4 20.7 GalNAc.sub.3-3a A.sub.d
15 63.2 44.1 671144 5 20.5 19.2 GalNAc.sub.3-12a A.sub.d 15 48.6
41.5 670061 5 31.6 28.0 GalNAc.sub.3-13a A.sub.d 15 67.6 55.5
671261 5 19.8 16.8 GalNAc.sub.3-14a A.sub.d 15 64.7 49.1 671262 5
18.5 7.4 GalNAc.sub.3-15a A.sub.d 15 52.3 24.2 670699 5 16.4 10.4
GalNAc.sub.3-3a T.sub.d 15 31.5 22.5 670700 5 19.3 10.9
GalNAc.sub.3-3a A.sub.e 15 38.1 20.0 670701 5 21.8 8.8
GalNAc.sub.3-3a T.sub.e 15 35.2 16.1 671165 5 27.1 26.5
GalNAc.sub.3-13a A.sub.d 15 48.3 44.3 666904 5 30.8 24.0
GalNAc.sub.3-3a PO 15 52.6 37.6 675441 5 25.4 19.0 GalNAc.sub.3-17a
A.sub.d 15 54.2 42.1 675442 5 22.2 20.7 GalNAc.sub.3-18a A.sub.d 15
39.6 29.0
[0976] The results in Table 63 above show that there were greater
liver tissue levels of the oligonucleotides comprising a
GalNAc.sub.3 conjugate group than of the parent oligonucleotide
that does not comprise a GalNAc.sub.3 conjugate group (ISIS 353382)
72 hours following oligonucleotide administration, particularly
when taking into consideration the differences in dosing between
the oligonucleotides with and without a GalNAc.sub.3 conjugate
group. Furthermore, by 72 hours, 40-98% of each oligonucleotide
comprising a GalNAc.sub.3 conjugate group was metabolized to the
parent compound, indicating that the GalNAc.sub.3 conjugate groups
were cleaved from the oligonucleotides.
Example 76: Preparation of Oligomeric Compound 230 Comprising
GalNAc.sub.3-23
##STR00251## ##STR00252##
[0978] Compound 222 is commercially available. 44.48 ml (0.33 mol)
of compound 222 was treated with tosyl chloride (25.39 g, 0.13 mol)
in pyridine (500 mL) for 16 hours. The reaction was then evaporated
to an oil, dissolved in EtOAc and washed with water, sat.
NaHCO.sub.3, brine, and dried over Na.sub.2SO.sub.4. The ethyl
acetate was concentrated to dryness and purified by column
chromatography, eluted with EtOAc/hexanes (1:1) followed by 10%
methanol in CH.sub.2Cl.sub.2 to give compound 223 as a colorless
oil. LCMS and NMR were consistent with the structure. 10 g (32.86
mmol) of 1-Tosyltriethylene glycol (compound 223) was treated with
sodium azide (10.68 g, 164.28 mmol) in DMSO (100 mL) at room
temperature for 17 hours. The reaction mixture was then poured onto
water, and extracted with EtOAc. The organic layer was washed with
water three times and dried over Na.sub.2SO.sub.4. The organic
layer was concentrated to dryness to give 5.3 g of compound 224
(92%). LCMS and NMR were consistent with the structure.
1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol) and
compound 4 (6 g, 18.22 mmol) were treated with 4 A molecular sieves
(5 g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100 mL)
under an inert atmosphere. After 14 hours, the reaction was
filtered to remove the sieves, and the organic layer was washed
with sat. NaHCO.sub.3, water, brine, and dried over
Na.sub.2SO.sub.4. The organic layer was concentrated to dryness and
purified by column chromatography, eluted with a gradient of 2 to
4% methanol in dichloromethane to give compound 225. LCMS and NMR
were consistent with the structure. Compound 225 (11.9 g, 23.59
mmol) was hydrogenated in EtOAc/Methanol (4:1, 250 mL) over
Pearlman's catalyst. After 8 hours, the catalyst was removed by
filtration and the solvents removed to dryness to give compound
226. LCMS and NMR were consistent with the structure.
[0979] In order to generate compound 227, a solution of
nitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig's
base (10.3 ml, 60.17 mmol) in DMF (100 mL) were treated dropwise
with pentaflourotrifluoro acetate (9.05 ml, 52.65 mmol). After 30
minutes, the reaction was poured onto ice water and extracted with
EtOAc. The organic layer was washed with water, brine, and dried
over Na.sub.2SO.sub.4. The organic layer was concentrated to
dryness and then recrystallized from heptane to give compound 227
as a white solid. LCMS and NMR were consistent with the structure.
Compound 227 (1.5 g, 1.93 mmol) and compound 226 (3.7 g, 7.74 mmol)
were stirred at room temperature in acetonitrile (15 mL) for 2
hours. The reaction was then evaporated to dryness and purified by
column chromatography, eluting with a gradient of 2 to 10% methanol
in dichloromethane to give compound 228. LCMS and NMR were
consistent with the structure. Compound 228 (1.7 g, 1.02 mmol) was
treated with Raney Nickel (about 2 g wet) in ethanol (100 mL) in an
atmosphere of hydrogen. After 12 hours, the catalyst was removed by
filtration and the organic layer was evaporated to a solid that was
used directly in the next step. LCMS and NMR were consistent with
the structure. This solid (0.87 g, 0.53 mmol) was treated with
benzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and
DIEA (273.7 .mu.l, 1.6 mmol) in DMF (5 mL). After 16 hours, the DMF
was removed under reduced pressure at 65.degree. C. to an oil, and
the oil was dissolved in dichloromethane. The organic layer was
washed with sat. NaHCO.sub.3, brine, and dried over
Na.sub.2SO.sub.4. After evaporation of the organic layer, the
compound was purified by column chromatography and eluted with a
gradient of 2 to 20% methanol in dichloromethane to give the
coupled product. LCMS and NMR were consistent with the structure.
The benzyl ester was deprotected with Pearlman's catalyst under a
hydrogen atmosphere for 1 hour. The catalyst was them removed by
filtration and the solvents removed to dryness to give the acid.
LCMS and NMR were consistent with the structure. The acid (486 mg,
0.27 mmol) was dissolved in dry DMF (3 mL). Pyridine (53.61 .mu.l,
0.66 mmol) was added and the reaction was purged with argon.
Pentaflourotriflouro acetate (46.39 .mu.l, 0.4 mmol) was slowly
added to the reaction mixture. The color of the reaction changed
from pale yellow to burgundy, and gave off a light smoke which was
blown away with a stream of argon. The reaction was allowed to stir
at room temperature for one hour (completion of reaction was
confirmed by LCMS). The solvent was removed under reduced pressure
(rotovap) at 70.degree. C. The residue was diluted with DCM and
washed with 1N NaHSO.sub.4, brine, saturated sodium bicarbonate and
brine again. The organics were dried over Na.sub.2SO.sub.4,
filtered, and were concentrated to dryness to give 225 mg of
compound 229 as a brittle yellow foam. LCMS and NMR were consistent
with the structure.
[0980] Oligomeric compound 230, comprising a GalNAc.sub.3-23
conjugate group, was prepared from compound 229 using the general
procedure illustrated in Example 46. The GalNAc.sub.3 cluster
portion of the GalNAc.sub.3-23 conjugate group
(GalNAc.sub.3-23.sub.a) can be combined with any cleavable moiety
to provide a variety of conjugate groups. The structure of
GalNAc.sub.3-23 (GalNAc.sub.3-23.sub.a-CM) is shown below:
##STR00253##
Example 77: Antisense Inhibition In Vivo by Oligonucleotides
Targeting SRB-1 Comprising a GalNAc.sub.3 Conjugate
[0981] The oligonucleotides listed below were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
TABLE-US-00054 TABLE 64 Modified ASOs targeting SRB-1 ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 661161
GalNAc.sub.3-3.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT-
.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.d-
s GalNAc.sub.3-3a A.sub.d 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.esT.sub.e 666904
GalNAc.sub.3-3.sub.a-.sub.o'G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.-
sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ds
GalNAc.sub.3-3a PO 829
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.esT.sub.e 673502
GalNAc.sub.3-10.sub.a-.sub.o'AdoG.sub.es.sup.mC.sub.eoT.sub.eoT.sub-
.eo.sup.mC.sub.eoA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsAdT.sub.ds
GalNAc.sub.3-10a A.sub.d 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.e-
sT.sub.esT.sub.e 677844
GalNAc.sub.3-9.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT-
.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.d-
s GalNAc.sub.3-9a A.sub.d 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.esT.sub.e 677843
GalNAc.sub.3-23.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.es-
T.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.-
ds GalNAc.sub.3-23a A.sub.d 831
G.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e-
sT.sub.esT.sub.e 655861
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub-
.dsT.sub.es.sup.mC.sub.es GalNAc.sub.3-1a A.sub.d 830
.sup.mC.sub.esT.sub.esT.sub.eoA.sub.do'-GalNAc.sub.3-1.sub.a 677841
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub-
.dsT.sub.es.sup.mC.sub.es GalNAc.sub.3-19a A.sub.d 830
.sup.mC.sub.esT.sub.esT.sub.eoA.sub.do'-GalNAc.sub.3-19.sub.a
677842
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub-
.dsT.sub.es.sup.mC.sub.es GalNAc.sub.3-20a A.sub.d 830
.sup.mC.sub.esT.sub.esT.sub.eoA.sub.do'-GalNAc.sub.3-20.sub.a
[0982] The structure of GalNAc.sub.3-1.sub.a was shown previously
in Example 9, GalNAc.sub.3-3.sub.a was shown in Example 39,
GalNAc.sub.3-9a was shown in Example 52, GalNAc.sub.3-10a was shown
in Example 46, GalNAc.sub.3-19.sub.a was shown in Example 70,
GalNAc.sub.3-20.sub.a was shown in Example 71, and
GalNAc.sub.3-23.sub.a was shown in Example 76.
Treatment
[0983] Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar
Harbor, Me.) were each injected subcutaneously once at a dosage
shown below with an oligonucleotide listed in Table 64 or with
saline. Each treatment group consisted of 4 animals. The mice were
sacrificed 72 hours following the final administration to determine
the SRB-1 mRNA levels using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. The results below are presented as
the average percent of SRB-1 mRNA levels for each treatment group,
normalized to the saline control.
[0984] As illustrated in Table 65, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner.
TABLE-US-00055 TABLE 65 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1
mRNA GalNAc.sub.3 No. (mg/kg) (% Saline) Cluster CM Saline n/a
100.0 n/a n/a 661161 0.5 89.18 GalNAc.sub.3-3a A.sub.d 1.5 77.02 5
29.10 15 12.64 666904 0.5 93.11 GalNAc.sub.3-3a PO 1.5 55.85 5
21.29 15 13.43 673502 0.5 77.75 GalNAc.sub.3-10a A.sub.d 1.5 41.05
5 19.27 15 14.41 677844 0.5 87.65 GalNAc.sub.3-9a A.sub.d 1.5 93.04
5 40.77 15 16.95 677843 0.5 102.28 GalNAc.sub.3-23a A.sub.d 1.5
70.51 5 30.68 15 13.26 655861 0.5 79.72 GalNAc.sub.3-1a A.sub.d 1.5
55.48 5 26.99 15 17.58 677841 0.5 67.43 GalNAc.sub.3-19a A.sub.d
1.5 45.13 5 27.02 15 12.41 677842 0.5 64.13 GalNAc.sub.3-20a
A.sub.d 1.5 53.56 5 20.47 15 10.23
[0985] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were also measured
using standard protocols. Total bilirubin and BUN were also
evaluated. Changes in body weights were evaluated, with no
significant change from the saline group (data not shown). ALTs,
ASTs, total bilirubin and BUN values are shown in Table 66
below.
TABLE-US-00056 TABLE 66 Total ISIS Dosage ALT AST Bilirubin BUN
GalNAc.sub.3 No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Cluster CM
Saline n/a 21 45 0.13 34 n/a n/a 661161 0.5 28 51 0.14 39
GalNAc.sub.3-3a A.sub.d 1.5 23 42 0.13 39 5 22 59 0.13 37 15 21 56
0.15 35 666904 0.5 24 56 0.14 37 GalNAc.sub.3-3a PO 1.5 26 68 0.15
35 5 23 77 0.14 34 15 24 60 0.13 35 673502 0.5 24 59 0.16 34
GalNAc.sub.3-10a A.sub.d 1.5 20 46 0.17 32 5 24 45 0.12 31 15 24 47
0.13 34 677844 0.5 25 61 0.14 37 GalNAc.sub.3-9a A.sub.d 1.5 23 64
0.17 33 5 25 58 0.13 35 15 22 65 0.14 34 677843 0.5 53 53 0.13 35
GalNAc.sub.3-23a A.sub.d 1.5 25 54 0.13 34 5 21 60 0.15 34 15 22 43
0.12 38 655861 0.5 21 48 0.15 33 GalNAc.sub.3-1a A.sub.d 1.5 28 54
0.12 35 5 22 60 0.13 36 15 21 55 0.17 30 677841 0.5 32 54 0.13 34
GalNAc.sub.3-19a A.sub.d 1.5 24 56 0.14 34 5 23 92 0.18 31 15 24 58
0.15 31 677842 0.5 23 61 0.15 35 GalNAc.sub.3-20a A.sub.d 1.5 24 57
0.14 34 5 41 62 0.15 35 15 24 37 0.14 32
Example 78: Antisense Inhibition In Vivo by Oligonucleotides
Targeting Angiotensinogen Comprising a GalNAc.sub.3 Conjugate
[0986] The oligonucleotides listed below were tested in a
dose-dependent study for antisense inhibition of Angiotensinogen
(AGT) in normotensive Sprague Dawley rats.
TABLE-US-00057 TABLE 67 Modified ASOs targeting AGT ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 552668
.sup.mC.sub.esA.sub.es.sup.mC.sub.esT.sub.esG.sub.esA.sub.dsT.sub.d-
sT.sub.dsT.sub.dsT.sub.dsT.sub.dsG.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.-
mC.sub.dsA.sub.esG.sub.es n/a n/a 835 G.sub.esA.sub.esT.sub.e
669509
.sup.mC.sub.esA.sub.es.sup.mC.sub.esT.sub.esG.sub.esA.sub.dsT.sub.d-
sT.sub.dsT.sub.dsT.sub.dsT.sub.dsG.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.-
mC.sub.dsA.sub.esG.sub.es GalNAc.sub.3-1.sub.a A.sub.d 836
G.sub.esA.sub.esT.sub.eoA.sub.do'-GalNAC.sub.3-1.sub.a
The structure of GalNAc.sub.3-1.sub.a was shown previously in
Example 9.
Treatment
[0987] Six week old, male Sprague Dawley rats were each injected
subcutaneously once per week at a dosage shown below, for a total
of three doses, with an oligonucleotide listed in Table 67 or with
PBS. Each treatment group consisted of 4 animals. The rats were
sacrificed 72 hours following the final dose. AGT liver mRNA levels
were measured using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. AGT plasma protein levels were
measured using the Total Angiotensinogen ELISA (Catalog # JP27412,
IBL International, Toronto, ON) with plasma diluted 1:20,000. The
results below are presented as the average percent of AGT mRNA
levels in liver or AGT protein levels in plasma for each treatment
group, normalized to the PBS control.
[0988] As illustrated in Table 68, treatment with antisense
oligonucleotides lowered AGT liver mRNA and plasma protein levels
in a dose-dependent manner, and the oligonucleotide comprising a
GalNAc conjugate was significantly more potent than the parent
oligonucleotide lacking a GalNAc conjugate.
TABLE-US-00058 TABLE 68 AGT liver mRNA and plasma protein levels
AGT liver AGT plasma ISIS Dosage mRNA protein GalNAc.sub.3 No.
(mg/kg) (% PBS) (% PBS) Cluster CM PBS n/a 100 100 n/a n/a 552668 3
95 122 n/a n/a 10 85 97 30 46 79 90 8 11 669509 0.3 95 70
GalNAc.sub.3-1a A.sub.d 1 95 129 3 62 97 10 9 23
[0989] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in plasma and body weights
were also measured at time of sacrifice using standard protocols.
The results are shown in Table 69 below.
TABLE-US-00059 TABLE 69 Liver transaminase levels and rat body
weights Body Weight ISIS Dosage ALT AST (% of GalNAc.sub.3 No.
(mg/kg) (U/L) (U/L) baseline) Cluster CM PBS n/a 51 81 186 n/a n/a
552668 3 54 93 183 n/a n/a 10 51 93 194 30 59 99 182 90 56 78 170
669509 0.3 53 90 190 GalNAc.sub.3-1a A.sub.d 1 51 93 192 3 48 85
189 10 56 95 189
Example 79: Duration of Action In Vivo of Oligonucleotides
Targeting APOC-III Comprising a GalNAc.sub.3 Conjugate
[0990] The oligonucleotides listed in Table 70 below were tested in
a single dose study for duration of action in mice.
TABLE-US-00060 TABLE 70 Modified ASOs targeting APOC-III ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 304801
A.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.dsT.sub.d-
sT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.-
mC.sub.dsT.sub.esT.sub.es n/a n/a 821 T.sub.esA.sub.esT.sub.e
647535
A.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.dsT.sub.d-
sT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.-
mC.sub.dsT.sub.esT.sub.es GalNAc.sub.3-1a A.sub.d 822
T.sub.esA.sub.esT.sub.eoA.sub.do'-GalNAc.sub.3-1.sub.a 663083
GalNAc3-3.sub.a-.sub.o'A.sub.doA.sub.esG.sub.es.sup.mC.sub.esT.sub.-
esT.sub.es.sup.mC.sub.dsT.sub.dsT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds
GalNAc.sub.3-3a A.sub.d 837
.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.esT.sub.esT.sub.esA.sub-
.esT.sub.e 674449
GalNAC.sub.3-7.sub.a-.sub.o'A.sub.doA.sub.esG.sub.es.sup.mC.sub.esT-
.sub.esT.sub.es.sup.mC.sub.dsT.sub.dsT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.d-
s GalNAc.sub.3-7a A.sub.d 837
.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.esT.sub.esT.sub.esA.sub-
.esT.sub.e 674450
GalNAc.sub.3-10.sub.a-.sub.o'A.sub.doA.sub.esG.sub.es.sup.mC.sub.es-
T.sub.esT.sub.es.sup.mC.sub.dsT.sub.dsT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.-
ds GalNAc.sub.3-10a A.sub.d 837
.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.esT.sub.esT.sub.esA.sub-
.esT.sub.e 674451
GalNAc.sub.3-13.sub.a-.sub.o'A.sub.doA.sub.esG.sub.es.sup.mC.sub.es-
T.sub.esT.sub.es.sup.mC.sub.dsT.sub.dsT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.-
ds GalNAc.sub.3-13a A.sub.d 837
.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.esT.sub.esT.sub.esA.sub-
.esT.sub.e
The structure of GalNAc.sub.3-1.sub.a was shown previously in
Example 9, GalNAc.sub.3-3.sub.a was shown in Example 39,
GalNAc.sub.3-7.sub.a was shown in Example 48, GalNAc.sub.3-10.sub.a
was shown in Example 46, and GalNAc.sub.3-13.sub.a was shown in
Example 62.
Treatment
[0991] Six to eight week old transgenic mice that express human
APOC-III were each injected subcutaneously once with an
oligonucleotide listed in Table 70 or with PBS. Each treatment
group consisted of 3 animals. Blood was drawn before dosing to
determine baseline and at 72 hours, 1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, and 6 weeks following the dose. Plasma triglyceride
and APOC-III protein levels were measured as described in Example
20. The results below are presented as the average percent of
plasma triglyceride and APOC-III levels for each treatment group,
normalized to baseline levels, showing that the oligonucleotides
comprising a GalNAc conjugate group exhibited a longer duration of
action than the parent oligonucleotide without a conjugate group
(ISIS 304801) even though the dosage of the parent was three times
the dosage of the oligonucleotides comprising a GalNAc conjugate
group.
TABLE-US-00061 TABLE 71 Plasma triglyceride and APOC-III protein
levels in transgenic mice Time point ISIS Dosage (days post-
Triglycerides APOC-III protein GalNAc.sub.3 No. (mg/kg) dose) (%
baseline) (% baseline) Cluster CM PBS n/a 3 97 102 n/a n/a 7 101 98
14 108 98 21 107 107 28 94 91 35 88 90 42 91 105 304801 30 3 40 34
n/a n/a 7 41 37 14 50 57 21 50 50 28 57 73 35 68 70 42 75 93 647535
10 3 36 37 GalNAc.sub.3-1a A.sub.d 7 39 47 14 40 45 21 41 41 28 42
62 35 69 69 42 85 102 663083 10 3 24 18 GalNAc.sub.3-3a A.sub.d 7
28 23 14 25 27 21 28 28 28 37 44 35 55 57 42 60 78 674449 10 3 29
26 GalNAc.sub.3-7a A.sub.d 7 32 31 14 38 41 21 44 44 28 53 63 35 69
77 42 78 99 674450 10 3 33 30 GalNAc.sub.3-10a A.sub.d 7 35 34 14
31 34 21 44 44 28 56 61 35 68 70 42 83 95 674451 10 3 35 33
GalNAc.sub.3-13a A.sub.d 7 24 32 14 40 34 21 48 48 28 54 67 35 65
75 42 74 97
Example 80: Antisense Inhibition In Vivo by Oligonucleotides
Targeting Alpha-1 Antitrypsin (A1AT) Comprising a GalNAc.sub.3
Conjugate
[0992] The oligonucleotides listed in Table 72 below were tested in
a study for dose-dependent inhibition of A1AT in mice.
TABLE-US-00062 TABLE 72 Modified ASOs targeting A1AT ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 476366
A.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.esA.sub.esA.sub.dsT-
.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.dsA.sub.dsA.sub.dsG.sub.dsG.sub-
.dsA.sub.esA.sub.es n/a n/a 838 G.sub.esG.sub.esA.sub.e 656326
A.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.esA.sub.esA.sub.dsT-
.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.dsA.sub.dsA.sub.dsG.sub.dsG.sub-
.dsA.sub.esA.sub.es GalNAc.sub.3-1a A.sub.d 839
G.sub.esG.sub.esA.sub.eoA.sub.do'-GalNAc.sub.3-1.sub.a 678381
GalNAc.sub.3-3.sub.a-.sub.o'A.sub.doA.sub.es.sup.mC.sub.es.sup.mC.s-
ub.es.sup.mC.sub.esA.sub.esA.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsG-
.sub.dsA.sub.ds GalNAc.sub.3-3a A.sub.d 840
A.sub.dsG.sub.dsG.sub.dsA.sub.esA.sub.esG.sub.esG.sub.esA.sub.e
678382
GalNAC.sub.3-7.sub.a-.sub.o'A.sub.doA.sub.es.sup.mC.sub.es.sup.mC.s-
ub.es.sup.mC.sub.esA.sub.esA.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsG-
.sub.dsA.sub.ds GalNAc.sub.3-7a A.sub.d 840
A.sub.dsG.sub.dsG.sub.dsA.sub.esA.sub.esG.sub.esG.sub.esA.sub.e
678383
GalNAc.sub.3-10.sub.a-.sub.o'A.sub.doA.sub.es.sup.mC.sub.es.sup.mC.-
sub.es.sup.mC.sub.esA.sub.esA.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds-
G.sub.ds GalNAc.sub.3-10a A.sub.d 840
A.sub.dsA.sub.dsG.sub.dsG.sub.dsA.sub.esA.sub.esG.sub.esG.sub.esA.sub.e
678384
GalNAc.sub.3-13.sub.a-.sub.o'A.sub.doA.sub.es.sup.mC.sub.es.sup.mC.-
sub.es.sup.mC.sub.esA.sub.esA.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds-
G.sub.ds GalNAc.sub.3-13a A.sub.d 840
A.sub.dsA.sub.dsG.sub.dsG.sub.dsA.sub.esA.sub.esG.sub.esG.sub.esA.sub.e
The structure of GalNAc.sub.3-1.sub.a was shown previously in
Example 9, GalNAc.sub.3-3.sub.a was shown in Example 39,
GalNAc.sub.3-7.sub.a was shown in Example 48, GalNAc.sub.3-10.sub.a
was shown in Example 46, and GalNAc.sub.3-13.sub.a was shown in
Example 62.
Treatment
[0993] Six week old, male C57BL/6 mice (Jackson Laboratory, Bar
Harbor, Me.) were each injected subcutaneously once per week at a
dosage shown below, for a total of three doses, with an
oligonucleotide listed in Table 72 or with PBS. Each treatment
group consisted of 4 animals. The mice were sacrificed 72 hours
following the final administration. A1AT liver mRNA levels were
determined using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. A1AT plasma protein levels were
determined using the Mouse Alpha 1-Antitrypsin ELISA (catalog
#41-A1AMS-E01, Alpco, Salem, N.H.). The results below are presented
as the average percent of A1AT liver mRNA and plasma protein levels
for each treatment group, normalized to the PBS control.
[0994] As illustrated in Table 73, treatment with antisense
oligonucleotides lowered A1AT liver mRNA and A1AT plasma protein
levels in a dose-dependent manner. The oligonucleotides comprising
a GalNAc conjugate were significantly more potent than the parent
(ISIS 476366).sub.n
TABLE-US-00063 TABLE 73 A1AT liver mRNA and plasma protein levels
A1AT liver A1AT plasma ISIS Dosage mRNA protein GalNAc.sub.3 No.
(mg/kg) (% PBS) (% PBS) Cluster CM PBS n/a 100 100 n/a n/a 476366 5
86 78 n/a n/a 15 73 61 45 30 38 656326 0.6 99 90 GalNAc.sub.3-1a
A.sub.d 2 61 70 6 15 30 18 6 10 678381 0.6 105 90 GalNAc.sub.3-3a
A.sub.d 2 53 60 6 16 20 18 7 13 678382 0.6 90 79 GalNAc.sub.3-7a
A.sub.d 2 49 57 6 21 27 18 8 11 678383 0.6 94 84 GalNAc.sub.3-10a
A.sub.d 2 44 53 6 13 24 18 6 10 678384 0.6 106 91 GalNAc.sub.3-13a
A.sub.d 2 65 59 6 26 31 18 11 15
[0995] Liver transaminase and BUN levels in plasma were measured at
time of sacrifice using standard protocols. Body weights and organ
weights were also measured. The results are shown in Table 74
below. Body weight is shown as % relative to baseline. Organ
weights are shown as % of body weight relative to the PBS control
group.
TABLE-US-00064 TABLE 74 ISIS Dosage ALT AST BUN Body weight Liver
weight Kidney weight Spleen weight No. (mg/kg) (U/L) (U/L) (mg/dL)
(% baseline) (Rel % BW) (Rel % BW) (Rel % BW) PBS n/a 25 51 37 119
100 100 100 476366 5 34 68 35 116 91 98 106 15 37 74 30 122 92 101
128 45 30 47 31 118 99 108 123 656326 0.6 29 57 40 123 100 103 119
2 36 75 39 114 98 111 106 6 32 67 39 125 99 97 122 18 46 77 36 116
102 109 101 678381 0.6 26 57 32 117 93 109 110 2 26 52 33 121 96
106 125 6 40 78 32 124 92 106 126 18 31 54 28 118 94 103 120 678382
0.6 26 42 35 114 100 103 103 2 25 50 31 117 91 104 117 6 30 79 29
117 89 102 107 18 65 112 31 120 89 104 113 678383 0.6 30 67 38 121
91 100 123 2 33 53 33 118 98 102 121 6 32 63 32 117 97 105 105 18
36 68 31 118 99 103 108 678384 0.6 36 63 31 118 98 103 98 2 32 61
32 119 93 102 114 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101
104
Example 81: Duration of Action In Vivo of Oligonucleotides
Targeting A1AT Comprising a GalNAc.sub.3 Cluster
[0996] The oligonucleotides listed in Table 72 were tested in a
single dose study for duration of action in mice.
Treatment
[0997] Six week old, male C57BL/6 mice were each injected
subcutaneously once with an oligonucleotide listed in Table 72 or
with PBS. Each treatment group consisted of 4 animals. Blood was
drawn the day before dosing to determine baseline and at 5, 12, 19,
and 25 days following the dose. Plasma A1AT protein levels were
measured via ELISA (see Example 80). The results below are
presented as the average percent of plasma A1AT protein levels for
each treatment group, normalized to baseline levels. The results
show that the oligonucleotides comprising a GalNAc conjugate were
more potent and had longer duration of action than the parent
lacking a GalNAc conjugate (ISIS 476366). Furthermore, the
oligonucleotides comprising a 5'-GalNAc conjugate (ISIS 678381,
678382, 678383, and 678384) were generally even more potent with
even longer duration of action than the oligonucleotide comprising
a 3'-GalNAc conjugate (ISIS 656326).
TABLE-US-00065 TABLE 75 Plasma A1AT protein levels in mice Time
point A1AT ISIS Dosage (days post- (% base- GalNAc.sub.3 No.
(mg/kg) dose) line) Cluster CM PBS n/a 5 93 n/a n/a 12 93 19 90 25
97 476366 100 5 38 n/a n/a 12 46 19 62 25 77 656326 18 5 33
GalNAc.sub.3-1a A.sub.d 12 36 19 51 25 72 678381 18 5 21
GalNAc.sub.3-3a A.sub.d 12 21 19 35 25 48 678382 18 5 21
GalNAc.sub.3-7a A.sub.d 12 21 19 39 25 60 678383 18 5 24
GalNAc.sub.3-10a A.sub.d 12 21 19 45 25 73 678384 18 5 29
GalNAc.sub.3-13a A.sub.d 12 34 19 57 25 76
Example 82: Antisense Inhibition In Vitro by Oligonucleotides
Targeting SRB-1 Comprising a GalNAc.sub.3 Conjugate
[0998] Primary mouse liver hepatocytes were seeded in 96 well
plates at 15,000 cells/well 2 hours prior to treatment. The
oligonucleotides listed in Table 76 were added at 2, 10, 50, or 250
nM in Williams E medium and cells were incubated overnight at
37.degree. C. in 5% CO.sub.2. Cells were lysed 16 hours following
oligonucleotide addition, and total RNA was purified using RNease
3000 BioRobot (Qiagen). SRB-1 mRNA levels were determined using
real-time PCR and RIBOGREEN.RTM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.) according to standard
protocols. IC.sub.50 values were determined using Prism 4 software
(GraphPad). The results show that oligonucleotides comprising a
variety of different GalNAc conjugate groups and a variety of
different cleavable moieties are significantly more potent in an in
vitro free uptake experiment than the parent oligonucleotides
lacking a GalNAc conjugate group (ISIS 353382 and 666841).
TABLE-US-00066 TABLE 76 Inhibition of SRB-1 expression in vitro
ISIS GalNAc IC.sub.50 SEQ No. Sequences (5' to 3') Linkages Cluster
CM (nM) ID No. 353382
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.ds PS n/a n/a 250 829
A.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.e-
sT.sub.e 655861
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.ds PS GalNAc.sub.3-
A.sub.d 40 830
A.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.e-
sT.sub.eoA.sub.do'- 1.sub.a GalNAc.sub.3-1.sub.a 661161
GalNAc.sub.3-3.sub.a- PS GalNAc.sub.3- A.sub.d 40 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 3.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 661162
GalNAc.sub.3-3.sub.a- PO/PS GalNAc.sub.3- A.sub.d 8 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mT.sub.eoA.sub.-
dsG.sub.dsT.sub.ds 3.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT.sub.e 664078
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.ds PS GalNAc.sub.3-
A.sub.d 20 830
A.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.e-
sT.sub.eoA.sub.do'- 9.sub.a GalNAc.sub.3-9.sub.a 665001
GalNAc.sub.3-8.sub.a- PS GalNAc.sub.3- A.sub.d 70 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds.sup.mC.sub.ds- 8.sub.a
A.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub-
.es.sup.mC.sub.esT.sub.esT.sub.e 666224 GalNAc.sub.3-5.sub.a- PS
GalNAc.sub.3- A.sub.d 80 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 5.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mc.sub.es.sup.mc.sub.esT.sub.esT.sub.e 666841
G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.ds PO/PS n/a n/a
>250 829
A.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.e-
sT.sub.e 666881 GalNAc.sub.3-10.sub.a- PS GalNAc.sub.3- A.sub.d 30
831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 10.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 666904
GalNAc.sub.3-3.sub.a- PS GalNAc.sub.3- PO 9 829
.sub.o'G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.-
dsT.sub.ds.sup.mC.sub.ds 3.sub.a
A.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub-
.es.sup.mC.sub.esT.sub.esT.sub.e 666924 GalNAc.sub.3-3.sub.a- PS
GalNAc.sub.3- T.sub.d 15 834
.sub.o'T.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 3.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 666961
GalNAc.sub.3-6.sub.a- PS GalNAc.sub.3- A.sub.d 150 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 6.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 666981
GalNAc.sub.3-7.sub.a- PS GalNAc.sub.3- A.sub.d 20 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 7.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 670061
GalNAc.sub.3-13.sub.a- PS GalNAc.sub.3- A.sub.d 30 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 13.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 670699
GalNAc.sub.3-3.sub.a- PO/PS GalNAc.sub.3- T.sub.d 15 834
.sub.o'T.sub.doG.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mT.sub.eoA.sub.-
dsG.sub.dsT.sub.ds 3.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT.sub.e 670700
GalNAc3-3.sub.a- PO/PS GalNAc.sub.3- A.sub.e 30 831
.sub.o'A.sub.eoG.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.-
dsG.sub.dsT.sub.ds 3.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT 670701
GalNAc.sub.3-3.sub.a- PO/PS GalNAc.sub.3- T.sub.e 25 834
.sub.o'T.sub.eoG.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.-
dsG.sub.dsT.sub.ds 3.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT.sub.e 671144
GalNAc.sub.3-12.sub.a- PS GalNAc.sub.3- A.sub.d 40 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 12.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 671165
GalNAc.sub.3-13.sub.a- PO/PS GalNAc.sub.3- A.sub.d 8 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mT.sub.eoA.sub.-
dsG.sub.dsT.sub.ds 13.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT 671261
GalNAc.sub.3-14.sub.a- PS GalNAc.sub.3- A.sub.d >250 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 14.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 671262
GalNAc.sub.3-15.sub.a- PS GalNAc.sub.3- A.sub.d >250 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 15.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 673501
GalNAc.sub.3-7.sub.a- PO/PS GalNAc.sub.3- A.sub.d 30 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mT.sub.eoA.sub.-
dsG.sub.dsT.sub.ds 7.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT.sub.e 673502
GalNAc.sub.3-10.sub.a- PO/PS GalNAc.sub.3- A.sub.d 8 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.-
dsG.sub.dsT.sub.ds 10.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT.sub.e 675441
GalNAc.sub.3-17.sub.a- PS GalNAc.sub.3- A.sub.d 30 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 17.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 675442
GalNAc.sub.3-18.sub.a- PS GalNAc.sub.3- A.sub.d 20 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 18.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 677841
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.ds PS GalNAc.sub.3-
A.sub.d 40 830
A.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.e-
sT.sub.eoA.sub.do'- 19.sub.a GalNAc.sub.3-19.sub.a 677842
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.ds PS GalNAc.sub.3-
A.sub.d 30 830 A.sub.ds
.sup.mCasTasT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.eo-
A.sub.do'- 20.sub.a GalNAc.sub.3-20.sub.a 677843
GalNAc.sub.3-23.sub.a- PS GalNAc.sub.3- A.sub.d 40 831
.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
dsG.sub.dsT.sub.ds 23.sub.a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
The structure of GalNAc.sub.3-1.sub.a was shown previously in
Example 9, GalNAc.sub.3-3.sub.a was shown in Example 39,
GalNAc.sub.3-5.sub.a was shown in Example 49, GalNAc.sub.3-6.sub.a
was shown in Example 51, GalNAc.sub.3-7.sub.a was shown in Example
48, GalNAc.sub.3-8.sub.a was shown in Example 47,
GalNAc.sub.3-9.sub.a was shown in Example 52, GalNAc.sub.3-10.sub.a
was shown in Example 46, GalNAc.sub.3-12.sub.a was shown in Example
61, GalNAc.sub.3-13.sub.a was shown in Example 62,
GalNAc.sub.3-14.sub.a was shown in Example 63,
GalNAc.sub.3-15.sub.a was shown in Example 64,
GalNAc.sub.3-17.sub.a was shown in Example 68,
GalNAc.sub.3-18.sub.a was shown in Example 69,
GalNAc.sub.3-19.sub.a was shown in Example 70,
GalNAc.sub.3-20.sub.a was shown in Example 71, and
GalNAc.sub.3-23.sub.a was shown in Example 76.
Example 83: Antisense Inhibition In Vivo by Oligonucleotides
Targeting Factor XI Comprising a GalNAc.sub.3 Cluster
[0999] The oligonucleotides listed in Table 77 below were tested in
a study for dose-dependent inhibition of Factor XI in mice.
TABLE-US-00067 TABLE 77 Modified oligonucleotides targeting Factor
XI ISIS GalNAc SEQ No. Sequences (5' to 3') Cluster CM ID No.
404071
T.sub.esG.sub.esG.sub.esT.sub.esA.sub.esA.sub.dsT.sub.ds.sup.mC.sub-
.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.dsT.sub.ds.sup.mC.sub-
.dsA.sub.es n/a n/a 832 G.sub.esA.sub.esG.sub.esGe 656173
T.sub.esG.sub.esG.sub.eoT.sub.eoA.sub.eoA.sub.dsT.sub.ds.sup.mC.sub-
.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.dsT.sub.ds.sup.mC.sub-
.dsA.sub.eo GalNAc.sub.3- A.sub.d 833
G.sub.eoA.sub.esG.sub.esG.sub.eoA.sub.do'-GalNAc.sub.3-1.sub.a
1.sub.a 663086 GalNAc.sub.3-3.sub.a- GalNAc.sub.3- A.sub.d 841
.sub.o'A.sub.doT.sub.esG.sub.eoG.sub.eoT.sub.eoA.sub.eoA.sub.dsT.sub.ds.s-
up.mC.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.ds 3.sub.a
T.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.eoG.sub.eoA.sub.esG.sub.esG.sub.e
678347 GalNAc.sub.3-7.sub.a- GalNAc.sub.3- A.sub.d 841
.sub.o'A.sub.doT.sub.esG.sub.eoG.sub.eoT.sub.eoA.sub.eoA.sub.dsT.sub.ds.s-
up.mC.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.ds 7.sub.a
T.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.eoG.sub.eoA.sub.esG.sub.esG.sub.e
678348 GalNAc.sub.3-10.sub.a- GalNAc.sub.3- A.sub.d 841
.sub.o'A.sub.doT.sub.esG.sub.eoG.sub.eoT.sub.eoA.sub.eoA.sub.dsT.sub.ds.s-
up.mC.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.ds 10.sub.a
T.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.eoG.sub.eoA.sub.esG.sub.esG.s-
ub.e 678349 GalNAc.sub.3-13.sub.a- GalNAc.sub.3- A.sub.d 841
.sub.o'A.sub.doT.sub.esG.sub.eoG.sub.eoT.sub.eoA.sub.eoA.sub.dsT.sub.ds.s-
up.mC.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.ds 13.sub.a
T.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.eoG.sub.eoA.sub.esG.sub.esG.s-
ub.e
The structure of GalNAc.sub.3-1.sub.a was shown previously in
Example 9, GalNAc.sub.3-3.sub.a was shown in Example 39,
GalNAc.sub.3-7.sub.a was shown in Example 48, GalNAc.sub.3-10.sub.a
was shown in Example 46, and GalNAc.sub.3-13.sub.a was shown in
Example 62.
Treatment
[1000] Six to eight week old mice were each injected subcutaneously
once per week at a dosage shown below, for a total of three doses,
with an oligonucleotide listed below or with PBS. Each treatment
group consisted of 4 animals. The mice were sacrificed 72 hours
following the final dose. Factor XI liver mRNA levels were measured
using real-time PCR and normalized to cyclophilin according to
standard protocols. Liver transaminases, BUN, and bilirubin were
also measured. The results below are presented as the average
percent for each treatment group, normalized to the PBS
control.
[1001] As illustrated in Table 78, treatment with antisense
oligonucleotides lowered Factor XI liver mRNA in a dose-dependent
manner. The results show that the oligonucleotides comprising a
GalNAc conjugate were more potent than the parent lacking a GalNAc
conjugate (ISIS 404071). Furthermore, the oligonucleotides
comprising a 5'-GalNAc conjugate (ISIS 663086, 678347, 678348, and
678349) were even more potent than the oligonucleotide comprising a
3'-GalNAc conjugate (ISIS 656173).
TABLE-US-00068 TABLE 78 Factor XI liver mRNA, liver transaminase,
BUN, and bilirubin levels Factor XI ISIS Dosage mRNA ALT AST BUN
Bilirubin GalNAc.sub.3 SEQ No. (mg/kg) (% PBS) (U/L) (U/L) (mg/dL)
(mg/dL) Cluster ID No. PBS n/a 100 63 70 21 0.18 n/a n/a 404071 3
65 41 58 21 0.15 n/a 832 10 33 49 53 23 0.15 30 17 43 57 22 0.14
656173 0.7 43 90 89 21 0.16 GalNAc.sub.3-1a 833 2 9 36 58 26 0.17 6
3 50 63 25 0.15 663086 0.7 33 91 169 25 0.16 GalNAc.sub.3-3a 841 2
7 38 55 21 0.16 6 1 34 40 23 0.14 678347 0.7 35 28 49 20 0.14
GalNAc.sub.3-7a 841 2 10 180 149 21 0.18 6 1 44 76 19 0.15 678348
0.7 39 43 54 21 0.16 GalNAc.sub.3-10a 841 2 5 38 55 22 0.17 6 2 25
38 20 0.14 678349 0.7 34 39 46 20 0.16 GalNAc.sub.3-13a 841 2 8 43
63 21 0.14 6 2 28 41 20 0.14
Example 84: Duration of Action In Vivo of Oligonucleotides
Targeting Factor XI Comprising a GalNAc.sub.3 Conjugate
[1002] The oligonucleotides listed in Table 77 were tested in a
single dose study for duration of action in mice.
Treatment
[1003] Six to eight week old mice were each injected subcutaneously
once with an oligonucleotide listed in Table 77 or with PBS. Each
treatment group consisted of 4 animals. Blood was drawn by tail
bleeds the day before dosing to determine baseline and at 3, 10,
and 17 days following the dose. Plasma Factor XI protein levels
were measured by ELISA using Factor XI capture and biotinylated
detection antibodies from R & D Systems, Minneapolis, Minn.
(catalog # AF2460 and # BAF2460, respectively) and the OptEIA
Reagent Set B (Catalog #550534, BD Biosciences, San Jose, Calif.).
The results below are presented as the average percent of plasma
Factor XI protein levels for each treatment group, normalized to
baseline levels. The results show that the oligonucleotides
comprising a GalNAc conjugate were more potent with longer duration
of action than the parent lacking a GalNAc conjugate (ISIS 404071).
Furthermore, the oligonucleotides comprising a 5'-GalNAc conjugate
(ISIS 663086, 678347, 678348, and 678349) were even more potent
with an even longer duration of action than the oligonucleotide
comprising a 3'-GalNAc conjugate (ISIS 656173).
TABLE-US-00069 TABLE 79 Plasma Factor XI protein levels in mice
Time point ISIS Dosage (days post- Factor XI GalNAc.sub.3 SEQ No.
(mg/kg) dose) (% baseline) Cluster CM ID No. PBS n/a 3 123 n/a n/a
n/a 10 56 17 100 404071 30 3 11 n/a n/a 832 10 47 17 52 656173 6 3
1 GalNAc.sub.3-1a A.sub.d 833 10 3 17 21 663086 6 3 1
GalNAc.sub.3-3a A.sub.d 841 10 2 17 9 678347 6 3 1 GalNAc.sub.3-7a
A.sub.d 841 10 1 17 8 678348 6 3 1 GalNAc.sub.3-10a A.sub.d 841 10
1 17 6 678349 6 3 1 GalNAc.sub.3-13a A.sub.d 841 10 1 17 5
Example 85: Antisense Inhibition In Vivo by Oligonucleotides
Targeting SRB-1 Comprising a GalNAc.sub.3 Conjugate
[1004] Oligonucleotides listed in Table 76 were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
Treatment
[1005] Six to eight week old C57BL/6 mice were each injected
subcutaneously once per week at a dosage shown below, for a total
of three doses, with an oligonucleotide listed in Table 76 or with
saline. Each treatment group consisted of 4 animals. The mice were
sacrificed 48 hours following the final administration to determine
the SRB-1 mRNA levels using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. The results below are presented as
the average percent of liver SRB-1 mRNA levels for each treatment
group, normalized to the saline control.
[1006] As illustrated in Tables 80 and 81, treatment with antisense
oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent
manner.
TABLE-US-00070 TABLE 80 SRB-1 mRNA in liver ISIS Dosage SRB-1 mRNA
GalNAc.sub.3 No. (mg/kg) (% Saline) Cluster CM Saline n/a 100 n/a
n/a 655861 0.1 94 GalNAc.sub.3-1a A.sub.d 0.3 119 1 68 3 32 661161
0.1 120 GalNAc.sub.3-3a A.sub.d 0.3 107 1 68 3 26 666881 0.1 107
GalNAc.sub.3-10a A.sub.d 0.3 107 1 69 3 27 666981 0.1 120
GalNAc.sub.3-7a A.sub.d 0.3 103 1 54 3 21 670061 0.1 118
GalNAc.sub.3-13a A.sub.d 0.3 89 1 52 3 18 677842 0.1 119
GalNAc.sub.3-20a A.sub.d 0.3 96 1 65 3 23
TABLE-US-00071 TABLE 81 SRB-1 mRNA in liver ISIS Dosage SRB-1 mRNA
GalNAc.sub.3 No. (mg/kg) (% Saline) Cluster CM 661161 0.1 107
GalNAc.sub.3-3a A.sub.d 0.3 95 1 53 3 18 677841 0.1 110
GalNAc.sub.3-19a A.sub.d 0.3 88 1 52 3 25
[1007] Liver transaminase levels, total bilirubin, BUN, and body
weights were also measured using standard protocols. Average values
for each treatment group are shown in Table 82 below.
TABLE-US-00072 TABLE 82 ISIS Dosage ALT AST Bilirubin BUN Body
Weight GalNAc.sub.3 No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (%
baseline) Cluster CM Saline n/a 19 39 0.17 26 118 n/a n/a 655861
0.1 25 47 0.17 27 114 GalNAc.sub.3-1a A.sub.d 0.3 29 56 0.15 27 118
1 20 32 0.14 24 112 3 27 54 0.14 24 115 661161 0.1 35 83 0.13 24
113 GalNAc.sub.3-3a A.sub.d 0.3 42 61 0.15 23 117 1 34 60 0.18 22
116 3 29 52 0.13 25 117 666881 0.1 30 51 0.15 23 118
GalNAc.sub.3-10a A.sub.d 0.3 49 82 0.16 25 119 1 23 45 0.14 24 117
3 20 38 0.15 21 112 666981 0.1 21 41 0.14 22 113 GalNAc.sub.3-7a
A.sub.d 0.3 29 49 0.16 24 112 1 19 34 0.15 22 111 3 77 78 0.18 25
115 670061 0.1 20 63 0.18 24 111 GalNAc.sub.3-13a A.sub.d 0.3 20 57
0.15 21 115 1 20 35 0.14 20 115 3 27 42 0.12 20 116 677842 0.1 20
38 0.17 24 114 GalNAc.sub.3-20a A.sub.d 0.3 31 46 0.17 21 117 1 22
34 0.15 21 119 3 41 57 0.14 23 118
Example 86: Antisense Inhibition In Vivo by Oligonucleotides
Targeting TTR Comprising a GalNAc.sub.3 Cluster
[1008] Oligonucleotides listed in Table 83 below were tested in a
dose-dependent study for antisense inhibition of human
transthyretin (TTR) in transgenic mice that express the human TTR
gene.
Treatment
[1009] Eight week old TTR transgenic mice were each injected
subcutaneously once per week for three weeks, for a total of three
doses, with an oligonucleotide and dosage listed in the tables
below or with PBS. Each treatment group consisted of 4 animals. The
mice were sacrificed 72 hours following the final administration.
Tail bleeds were performed at various time points throughout the
experiment, and plasma TTR protein, ALT, and AST levels were
measured and reported in Tables 85-87. After the animals were
sacrificed, plasma ALT, AST, and human TTR levels were measured, as
were body weights, organ weights, and liver human TTR mRNA levels.
TTR protein levels were measured using a clinical analyzer (AU480,
Beckman Coulter, Calif.). Real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were
used according to standard protocols to determine liver human TTR
mRNA levels. The results presented in Tables 84-87 are the average
values for each treatment group. The mRNA levels are the average
values relative to the average for the PBS group. Plasma protein
levels are the average values relative to the average value for the
PBS group at baseline. Body weights are the average percent weight
change from baseline until sacrifice for each individual treatment
group. Organ weights shown are normalized to the animal's body
weight, and the average normalized organ weight for each treatment
group is then presented relative to the average normalized organ
weight for the PBS group.
[1010] In Tables 84-87, "BL" indicates baseline, measurements that
were taken just prior to the first dose. As illustrated in Tables
84 and 85, treatment with antisense oligonucleotides lowered TTR
expression levels in a dose-dependent manner. The oligonucleotides
comprising a GalNAc conjugate were more potent than the parent
lacking a GalNAc conjugate (ISIS 420915). Furthermore, the
oligonucleotides comprising a GalNAc conjugate and mixed PS/PO
internucleoside linkages were even more potent than the
oligonucleotide comprising a GalNAc conjugate and full PS
linkages.
TABLE-US-00073 TABLE 83 Oligonucleotides targeting human TTR ISIS
GalNAc SEQ No. Sequences (5' to 3') Linkages Cluster CM ID No.
420915
T.sub.es.sup.mC.sub.esT.sub.esT.sub.esG.sub.esG.sub.dsT.sub.dsT.sub-
.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds PS n/a
n/a 842
A.sub.dsA.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e
660261
T.sub.es.sup.mC.sub.esT.sub.esT.sub.esG.sub.esG.sub.dsT.sub.dsT.sub-
.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds PS
GalNAc.sub.3-1a A.sub.d 843
A.sub.dsA.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.eoA.sub.d-
o'-GalNAC.sub.3-1.sub.a 682883 GalNAc.sub.3-3.sub.a- PS/PO
GalNAc.sub.3-3a PO 842
.sub.o'T.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.sub.dsT.su-
b.dsA.sub.ds.sup.mC.sub.dsA.sub.ds
T.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.eoT.sub.eo.sup.mC.sub.es.sup.mC.sub-
.es.sup.mC.sub.e 682884 GalNAc.sub.3-7.sub.a- PS/PO GalNAc.sub.3-7a
PO 842
.sub.o'T.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.sub.dsT.su-
b.dsA.sub.ds.sup.mC.sub.dsA.sub.ds
T.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.eoT.sub.eo.sup.mC.sub.es.sup.mC.sub-
.es.sup.mC.sub.e 682885 GalNAc.sub.3-10.sub.a- PS/PO GalNAc.sub.3-
PO 842
.sub.o'T.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.sub.dsT.su-
b.dsA.sub.ds.sup.mC.sub.ds 10a
A.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.eoT.sub.eo.sup.mC.sub.es.su-
p.mC.sub.es.sup.mC.sub.e 682886 GalNAc.sub.3-13.sub.a- PS/PO
GalNAc.sub.3- PO 842
.sub.o'T.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.sub.dsT.su-
b.dsA.sub.ds.sup.mC.sub.ds 13a
A.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.eoT.sub.eo.sup.mC.sub.es.su-
p.mC.sub.es.sup.mC.sub.e 684057
T.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.sub.dsT.sub-
.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds PS/PO
GalNAc.sub.3-19a A.sub.d 843
A.sub.dsA.sub.eoT.sub.eo.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.eoA.sub.d-
o'-GalNAc.sub.3-19.sub.a
The legend for Table 85 can be found in Example 74. The structure
of GalNAc.sub.3-1 was shown in Example 9. The structure of
GalNAc.sub.3-3.sub.a was shown in Example 39. The structure of
GalNAc.sub.3-7.sub.a was shown in Example 48. The structure of
GalNAc.sub.3-10.sub.a was shown in Example 46. The structure of
GalNAc.sub.3-13.sub.a was shown in Example 62. The structure of
GalNAc.sub.3-19.sub.a was shown in Example 70.
TABLE-US-00074 TABLE 84 Antisense inhibition of human TTR in vivo
Plasma TTR Isis Dosage TTR mRNA protein GalNAc SEQ No. (mg/kg) (%
PBS) (% PBS) cluster CM ID No. PBS n/a 100 100 n/a n/a 420915 6 99
95 n/a n/a 842 20 48 65 60 18 28 660261 0.6 113 87 GalNAc.sub.3-1a
A.sub.d 843 2 40 56 6 20 27 20 9 11
TABLE-US-00075 TABLE 85 Antisense inhibition of human TTR in vivo
TTR Plasma TTR protein (% PBS at BL) Isis Dosage mRNA Day 17 GalNAc
SEQ No. (mg/kg) (% PBS) BL Day 3 Day 10 (After sac) cluster CM ID
No. PBS n/a 100 100 96 90 114 n/a n/a 420915 6 74 106 86 76 83 n/a
n/a 842 20 43 102 66 61 58 60 24 92 43 29 32 682883 0.6 60 88 73 63
68 GalNAc.sub.3-3a PO 842 2 18 75 38 23 23 6 10 80 35 11 9 682884
0.6 56 88 78 63 67 GalNAc.sub.3-7a PO 842 2 19 76 44 25 23 6 15 82
35 21 24 682885 0.6 60 92 77 68 76 GalNAc.sub.3-10a PO 842 2 22 93
58 32 32 6 17 85 37 25 20 682886 0.6 57 91 70 64 69
GalNAc.sub.3-13a PO 842 2 21 89 50 31 30 6 18 102 41 24 27 684057
0.6 53 80 69 56 62 GalNAc.sub.3-19a A.sub.d 843 2 21 92 55 34 30 6
11 82 50 18 13
TABLE-US-00076 TABLE 86 Transaminase levels, body weight changes,
and relative organ weights ALT (U/L) AST (U/L) Isis Dosage Day Day
Day Day Day Day Body Liver Spleen Kidney SEQ No. (mg/kg) BL 3 10 17
BL 3 10 17 (% BL) (% PBS) (% PBS) (% PBS) ID No. PBS n/a 33 34 33
24 58 62 67 52 105 100 100 100 n/a 420915 6 34 33 27 21 64 59 73 47
115 99 89 91 842 20 34 30 28 19 64 54 56 42 111 97 83 89 60 34 35
31 24 61 58 71 58 113 102 98 95 660261 0.6 33 38 28 26 70 71 63 59
111 96 99 92 843 2 29 32 31 34 61 60 68 61 118 100 92 90 6 29 29 28
34 58 59 70 90 114 99 97 95 20 33 32 28 33 64 54 68 95 114 101 106
92
TABLE-US-00077 TABLE 87 Transaminase levels, body weight changes,
and relative organ weights ALT (U/L) AST (U/L) Isis Dosage Day Day
Day Day Day Day Body Liver Spleen Kidney SEQ No. (mg/kg) BL 3 10 17
BL 3 10 17 (% BL) (% PBS) (% PBS) (% PBS) ID No. PBS n/a 32 34 37
41 62 78 76 77 104 100 100 100 n/a 420915 6 32 30 34 34 61 71 72 66
102 103 102 105 842 20 41 34 37 33 80 76 63 54 106 107 135 101 60
36 30 32 34 58 81 57 60 106 105 104 99 682883 0.6 32 35 38 40 53 81
74 76 104 101 112 95 842 2 38 39 42 43 71 84 70 77 107 98 116 99 6
35 35 41 38 62 79 103 65 105 103 143 97 682884 0.6 33 32 35 34 70
74 75 67 101 100 130 99 842 2 31 32 38 38 63 77 66 55 104 103 122
100 6 38 32 36 34 65 85 80 62 99 105 129 95 682885 0.6 39 26 37 35
63 63 77 59 100 109 109 112 842 2 30 26 38 40 54 56 71 72 102 98
111 102 6 27 27 34 35 46 52 56 64 102 98 113 96 682886 0.6 30 40 34
36 58 87 54 61 104 99 120 101 842 2 27 26 34 36 51 55 55 69 103 91
105 92 6 40 28 34 37 107 54 61 69 109 100 102 99 684057 0.6 35 26
33 39 56 51 51 69 104 99 110 102 843 2 33 32 31 40 54 57 56 87 103
100 112 97 6 39 33 35 40 67 52 55 92 98 104 121 108
Example 87: Duration of Action In Vivo by Single Doses of
Oligonucleotides Targeting TTR Comprising a GalNAc.sub.3
Cluster
[1011] ISIS numbers 420915 and 660261 (see Table 83) were tested in
a single dose study for duration of action in mice. ISIS numbers
420915, 682883, and 682885 (see Table 83) were also tested in a
single dose study for duration of action in mice.
Treatment
[1012] Eight week old, male transgenic mice that express human TTR
were each injected subcutaneously once with 100 mg/kg ISIS No.
420915 or 13.5 mg/kg ISIS No. 660261. Each treatment group
consisted of 4 animals. Tail bleeds were performed before dosing to
determine baseline and at days 3, 7, 10, 17, 24, and 39 following
the dose. Plasma TTR protein levels were measured as described in
Example 86. The results below are presented as the average percent
of plasma TTR levels for each treatment group, normalized to
baseline levels.
TABLE-US-00078 TABLE 88 Plasma TTR protein levels Time point ISIS
Dosage (days post- TTR GalNAc.sub.3 SEQ No. (mg/kg) dose) (%
baseline) Cluster CM ID No. 420915 100 3 30 n/a n/a 842 7 23 10 35
17 53 24 75 39 100 660261 13.5 3 27 GalNAc.sub.3-1a A.sub.d 843 7
21 10 22 17 36 24 48 39 69
Treatment
[1013] Female transgenic mice that express human TTR were each
injected subcutaneously once with 100 mg/kg ISIS No. 420915, 10.0
mg/kg ISIS No. 682883, or 10.0 mg/kg 682885. Each treatment group
consisted of 4 animals. Tail bleeds were performed before dosing to
determine baseline and at days 3, 7, 10, 17, 24, and 39 following
the dose. Plasma TTR protein levels were measured as described in
Example 86. The results below are presented as the average percent
of plasma TTR levels for each treatment group, normalized to
baseline levels.
TABLE-US-00079 TABLE 89 Plasma TTR protein levels Time point ISIS
Dosage (days post- TTR GalNAc.sub.3 SEQ No. (mg/kg) dose) (%
baseline) Cluster CM ID No. 420915 100 3 48 n/a n/a 842 7 48 10 48
17 66 31 80 682883 10.0 3 45 GalNAc.sub.3-3a PO 842 7 37 10 38 17
42 31 65 682885 10.0 3 40 PO 842 7 33 GalNAc.sub.3-10a 10 34 17 40
31 64
The results in Tables 88 and 89 show that the oligonucleotides
comprising a GalNAc conjugate are more potent with a longer
duration of action than the parent oligonucleotide lacking a
conjugate (ISIS 420915).
Example 88: Splicing Modulation In Vivo by Oligonucleotides
Targeting SMN Comprising a GalNAc.sub.3 Conjugate
[1014] The oligonucleotides listed in Table 90 were tested for
splicing modulation of human survival of motor neuron (SMN) in
mice.
TABLE-US-00080 TABLE 90 Modified ASOs targeting SMN ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 387954
A.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.es.sup.mC.sub.esT.sub.e-
sT.sub.esT.sub.es.sup.mC.sub.esA.sub.esT.sub.esA.sub.esA.sub.esT.sub.esG.s-
ub.es.sup.m n/a n/a 844 C.sub.esT.sub.esG.sub.esG.sub.e 699819
GalNAc.sub.3-7.sub.a-o'A.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.-
es.sup.mC.sub.esT.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.esT.sub.esA.su-
b.esA.sub.es GalNAc.sub.3- PO 844
T.sub.esG.sub.es.sup.mC.sub.esT.sub.esG.sub.esG.sub.e 7a 699821
GalNAc.sub.3-7.sub.a-.sub.o'A.sub.esT.sub.eoT.sub.eo.sup.mC.sub.eoA-
.sub.eo.sup.mC.sub.eoT.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.eoT.sub.e-
oA.sub.eo GalNAc.sub.3- PO 844
A.sub.eoT.sub.eoG.sub.eo.sup.mC.sub.eoT.sub.esG.sub.esG.sub.e 7a
700000
A.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.es.sup.mC.sub.esT.sub.e-
sT.sub.esT.sub.es.sup.mC.sub.esA.sub.esT.sub.esA.sub.esA.sub.esT.sub.esG.s-
ub.es.sup.mC.sub.esT.sub.es GalNAc.sub.3- A.sub.d 845
G.sub.esG.sub.eoA.sub.do'-GalNAc.sub.3-1.sub.a 1a 703421
X-ATT.sup.mCA.sup.mCTTT.sup.mCATAATG.sup.mCTGG n/a n/a 844 703422
GalNAc.sub.3-7.sub.b-X-ATT.sup.mCA.sup.mCTTT.sup.mCATAATG.sup.mCTGG
GalNAc.sub.3- n/a 844 7b
The structure of GalNAc.sub.3-7.sub.a was shown previously in
Example 48. "X" indicates a 5' primary amine generated by Gene
Tools (Philomath, Oreg.), and GalNAc.sub.3-7.sub.b indicates the
structure of GalNAc.sub.3-7.sub.a lacking the --NH--C.sub.6--O
portion of the linker as shown below:
##STR00254##
ISIS numbers 703421 and 703422 are morphlino oligonucleotides,
wherein each nucleotide of the two oligonucleotides is a morpholino
nucleotide.
Treatment
[1015] Six week old transgenic mice that express human SMN were
injected subcutaneously once with an oligonucleotide listed in
Table 91 or with saline. Each treatment group consisted of 2 males
and 2 females. The mice were sacrificed 3 days following the dose
to determine the liver human SMN mRNA levels both with and without
exon 7 using real-time PCR according to standard protocols. Total
RNA was measured using Ribogreen reagent. The SMN mRNA levels were
normalized to total mRNA, and further normalized to the averages
for the saline treatment group. The resulting average ratios of SMN
mRNA including exon 7 to SMN mRNA missing exon 7 are shown in Table
91. The results show that fully modified oligonucleotides that
modulate splicing and comprise a GalNAc conjugate are significantly
more potent in altering splicing in the liver than the parent
oligonucleotides lacking a GlaNAc conjugate. Furthermore, this
trend is maintained for multiple modification chemistries,
including 2'-MOE and morpholino modified oligonucleotides.
TABLE-US-00081 TABLE 91 Effect of oligonucleotides targeting human
SMN in vivo ISIS Dose GalNAc.sub.3 SEQ No. (mg/kg) +Exon 7/-Exon 7
Cluster CM ID No. Saline n/a 1.00 n/a n/a n/a 387954 32 1.65 n/a
n/a 844 387954 288 5.00 n/a n/a 844 699819 32 7.84 GalNAc.sub.3-7a
PO 844 699821 32 7.22 GalNAc.sub.3-7a PO 844 700000 32 6.91
GalNAc.sub.3-1a A.sub.d 845 703421 32 1.27 n/a n/a 844 703422 32
4.12 GalNAc.sub.3-7b n/a 844
Example 89: Antisense Inhibition In Vivo by Oligonucleotides
Targeting Apolipoprotein A (Apo(a)) Comprising a GalNAc.sub.3
Conjugate
[1016] The oligonucleotides listed in Table 92 below were tested in
a study for dose-dependent inhibition of Apo(a) in transgenic
mice.
TABLE-US-00082 TABLE 92 Modified ASOs targeting Apo(a) ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 494372
T.sub.esG.sub.es.sup.mC.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.dsG-
.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.dsT.sub.dsG.sub.ds.sup.mC.sub.ds
n/a n/a 847 T.sub.dsT.sub.esG.sub.esT.sub.esT.sub.es.sup.mC.sub.e
681257
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eo.-
sup.mC.sub.eo.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.ds
GalNAc.sub.3-7a PO 847
T.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eoG.sub.eoT.sub.esT.sub.es.su-
p.mC.sub.e
The structure of GalNAc.sub.3-7.sub.a was shown in Example 48.
Treatment
[1017] Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar
Harbor, Me.) were each injected subcutaneously once per week at a
dosage shown below, for a total of six doses, with an
oligonucleotide listed in Table 92 or with PBS. Each treatment
group consisted of 3-4 animals. Tail bleeds were performed the day
before the first dose and weekly following each dose to determine
plasma Apo(a) protein levels. The mice were sacrificed two days
following the final administration. Apo(a) liver mRNA levels were
determined using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)
according to standard protocols. Apo(a) plasma protein levels were
determined using ELISA, and liver transaminase levels were
determined. The mRNA and plasma protein results in Table 93 are
presented as the treatment group average percent relative to the
PBS treated group. Plasma protein levels were further normalized to
the baseline (BL) value for the PBS group. Average absolute
transaminase levels and body weights (% relative to baseline
averages) are reported in Table 94.
[1018] As illustrated in Table 93, treatment with the
oligonucleotides lowered Apo(a) liver mRNA and plasma protein
levels in a dose-dependent manner. Furthermore, the oligonucleotide
comprising the GalNAc conjugate was significantly more potent with
a longer duration of action than the parent oligonucleotide lacking
a GalNAc conjugate. As illustrated in Table 94, transaminase levels
and body weights were unaffected by the oligonucleotides,
indicating that the oligonucleotides were well tolerated.
TABLE-US-00083 TABLE 93 Apo(a) liver mRNA and plasma protein levels
Apo(a) Apo(a) plasma protein (% PBS) ISIS Dosage mRNA Week Week
Week Week Week Week No. (mg/kg) (% PBS) BL 1 2 3 4 5 6 PBS n/a 100
100 120 119 113 88 121 97 494372 3 80 84 89 91 98 87 87 79 10 30 87
72 76 71 57 59 46 30 5 92 54 28 10 7 9 7 681257 0.3 75 79 76 89 98
71 94 78 1 19 79 88 66 60 54 32 24 3 2 82 52 17 7 4 6 5 10 2 79 17
6 3 2 4 5
TABLE-US-00084 TABLE 94 ISIS Dosage ALT AST Body weight No. (mg/kg)
(U/L) (U/L) (% baseline) PBS n/a 37 54 103 494372 3 28 68 106 10 22
55 102 30 19 48 103 681257 0.3 30 80 104 1 26 47 105 3 29 62 102 10
21 52 107
Example 90: Antisense Inhibition In Vivo by Oligonucleotides
Targeting TTR Comprising a GalNAc.sub.3 Cluster
[1019] Oligonucleotides listed in Table 95 below were tested in a
dose-dependent study for antisense inhibition of human
transthyretin (TTR) in transgenic mice that express the human TTR
gene.
Treatment
[1020] TTR transgenic mice were each injected subcutaneously once
per week for three weeks, for a total of three doses, with an
oligonucleotide and dosage listed in Table 96 or with PBS. Each
treatment group consisted of 4 animals. Prior to the first dose, a
tail bleed was performed to determine plasma TTR protein levels at
baseline (BL). The mice were sacrificed 72 hours following the
final administration. TTR protein levels were measured using a
clinical analyzer (AU480, Beckman Coulter, Calif.). Real-time PCR
and RIBOGREEN.RTM. RNA quantification reagent (Molecular Probes,
Inc. Eugene, Oreg.) were used according to standard protocols to
determine liver human TTR mRNA levels. The results presented in
Table 96 are the average values for each treatment group. The mRNA
levels are the average values relative to the average for the PBS
group. Plasma protein levels are the average values relative to the
average value for the PBS group at baseline. "BL" indicates
baseline, measurements that were taken just prior to the first
dose. As illustrated in Table 96, treatment with antisense
oligonucleotides lowered TTR expression levels in a dose-dependent
manner. The oligonucleotides comprising a GalNAc conjugate were
more potent than the parent lacking a GalNAc conjugate (ISIS
420915), and oligonucleotides comprising a phosphodiester or
deoxyadenosine cleavable moiety showed significant improvements in
potency compared to the parent lacking a conjugate (see ISIS
numbers 682883 and 666943 vs 420915 and see Examples 86 and
87).
TABLE-US-00085 TABLE 95 Oligonucleotides targeting human TTR ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Linkages Cluster CM ID
No. 420915
T.sub.es.sup.mC.sub.esT.sub.esT.sub.esG.sub.esG.sub.dsT.sub.dsT.sub-
.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds PS n/a
n/a 842
A.sub.dsA.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e
682883 GalNAc.sub.3-3.sub.a- PS/PO GalNAc.sub.3- PO 842
.sub.o'T.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.sub.dsT.su-
b.dsA.sub.ds.sup.mC.sub.dsA.sub.ds 3a
T.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.eoT.sub.eo.sup.mC.sub.es.sup.mC.sub-
.es.sup.mC.sub.e 666943 GalNAc.sub.3-3.sub.a- PS/PO GalNAc.sub.3-
A.sub.d 846
.sub.o'A.sub.doT.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.su-
b.dsT.sub.dsA.sub.ds 3a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.eoT.sub.eo.su-
p.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e 682887 GalNAc.sub.3-7.sub.a-
PS/PO GalNAc.sub.3- A.sub.d 846
.sub.o'A.sub.doT.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.su-
b.dsT.sub.dsA.sub.ds 7a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.eoT.sub.eo.su-
p.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e 682888
GalNAc.sub.3-10.sub.a- PS/PO GalNAc.sub.3- A.sub.d 846
.sub.o'A.sub.doT.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.su-
b.dsT.sub.dsA.sub.ds 10a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.eoT.sub.eo.su-
p.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e 682889
GalNAc.sub.3-13.sub.a- PS/PO GalNAc.sub.3- A.sub.d 846
.sub.o'A.sub.doT.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.su-
b.dsT.sub.dsA.sub.ds 13a
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.eoT.sub.eo.su-
p.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e
The legend for Table 95 can be found in Example 74. The structure
of GalNAc.sub.3-3.sub.a was shown in Example 39. The structure of
GalNAc.sub.3-7.sub.a was shown in Example 48. The structure of
GalNAc.sub.3-10.sub.a was shown in Example 46. The structure of
GalNAc.sub.3-13.sub.a was shown in Example 62.
TABLE-US-00086 TABLE 96 Antisense inhibition of human TTR in vivo
Isis Dosage TTR mRNA TTR protein GalNAc No. (mg/kg) (% PBS) (% BL)
cluster CM PBS n/a 100 124 n/a n/a 420915 6 69 114 n/a n/a 20 71 86
60 21 36 682883 0.6 61 73 GalNAc.sub.3-3a PO 2 23 36 6 18 23 666943
0.6 74 93 GalNAc.sub.3-3a A.sub.d 2 33 57 6 17 22 682887 0.6 60 97
GalNAc.sub.3-7a A.sub.d 2 36 49 6 12 19 682888 0.6 65 92
GalNAc.sub.3-10a A.sub.d 2 32 46 6 17 22 682889 0.6 72 74
GalNAc.sub.3-13a A.sub.d 2 38 45 6 16 18
Example 91: Antisense Inhibition In Vivo by Oligonucleotides
Targeting Factor VII Comprising a GalNAc.sub.3 Conjugate in
Non-Human Primates
[1021] Oligonucleotides listed in Table 97 below were tested in a
non-terminal, dose escalation study for antisense inhibition of
Factor VII in monkeys.
Treatment
[1022] Non-naive monkeys were each injected subcutaneously on days
0, 15, and 29 with escalating doses of an oligonucleotide listed in
Table 97 or with PBS. Each treatment group consisted of 4 males and
1 female. Prior to the first dose and at various time points
thereafter, blood draws were performed to determine plasma Factor
VII protein levels. Factor VII protein levels were measured by
ELISA. The results presented in Table 98 are the average values for
each treatment group relative to the average value for the PBS
group at baseline (BL), the measurements taken just prior to the
first dose. As illustrated in Table 98, treatment with antisense
oligonucleotides lowered Factor VII expression levels in a
dose-dependent manner, and the oligonucleotide comprising the
GalNAc conjugate was significantly more potent in monkeys compared
to the oligonucleotide lacking a GalNAc conjugate.
TABLE-US-00087 TABLE 97 Oligonucleotides targeting Factor VII ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Linkages Cluster CM ID
No. 407935
A.sub.esT.sub.esG.sub.es.sup.mC.sub.esA.sub.esT.sub.dsG.sub.dsG.sub-
.dsT.sub.dsG.sub.dsA.sub.dsT.sub.dsG.sub.ds.sup.m PS n/a n/a 848
C.sub.dsT.sub.dsT.sub.es.sup.mC.sub.esT.sub.esG.sub.esA.sub.e
686892 GalNAc.sub.3-10.sub.a- PS GalNAc.sub.3- PO 848
o'A.sub.esT.sub.esG.sub.es.sup.mC.sub.esA.sub.esT.sub.dsG.sub.dsG.sub.dsT-
.sub.dsG.sub.ds 10a
A.sub.dsT.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.esT.sub-
.esG.sub.esA.sub.e
The legend for Table 97 can be found in Example 74. The structure
of GalNAc.sub.3-10, was shown in Example 46.
TABLE-US-00088 TABLE 98 Factor VII plasma protein levels ISIS Dose
Factor VII No. Day (mg/kg) (% BL) 407935 0 n/a 100 15 10 87 22 n/a
92 29 30 77 36 n/a 46 43 n/a 43 686892 0 3 100 15 10 56 22 n/a 29
29 30 19 36 n/a 15 43 n/a 11
Example 92: Antisense Inhibition in Primary Hepatocytes by
Antisense Oligonucleotides Targeting Apo-CIII Comprising a
GalNAc.sub.3 Conjugate
[1023] Primary mouse hepatocytes were seeded in 96-well plates at
15,000 cells per well, and the oligonucleotides listed in Table 99,
targeting mouse ApoC-III, were added at 0.46, 1.37, 4.12, or 12.35,
37.04, 111.11, or 333.33 nM or 1.00 .mu.M. After incubation with
the oligonucleotides for 24 hours, the cells were lysed and total
RNA was purified using RNeasy (Qiagen). ApoC-III mRNA levels were
determined using real-time PCR and RIBOGREEN.RTM. RNA
quantification reagent (Molecular Probes, Inc.) according to
standard protocols. IC.sub.50 values were determined using Prism 4
software (GraphPad). The results show that regardless of whether
the cleavable moiety was a phosphodiester or a
phosphodiester-linked deoxyadensoine, the oligonucleotides
comprising a GalNAc conjugate were significantly more potent than
the parent oligonucleotide lacking a conjugate.
TABLE-US-00089 TABLE 99 Inhibition of mouse APOC-III expression in
mouse primary hepatocytes ISIS IC.sub.50 SEQ No. Sequences (5' to
3') CM (nM) ID No. 440670
.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.d-
sA.sub.dsT.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.s-
ub.esA.sub.esG.sub.es.sup.m n/a 13.20 849 C.sub.esA.sub.e 661180
.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.d-
sA.sub.dsT.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.s-
ub.es A.sub.d 1.40 850
A.sub.esG.sub.es.sup.mC.sub.esA.sub.eoA.sub.do'-GalNAC.sub.3-1.sub.a
680771 GalNAc.sub.3-3.sub.a- PO 0.70 849
o'.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.dsA.s-
ub.dsT.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.e-
s A.sub.esG.sub.es.sup.mC.sub.esA.sub.e 680772
GalNAc.sub.3-7.sub.a- PO 1.70 849
o'.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.dsA.s-
ub.dsT.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.e-
s A.sub.esG.sub.es.sup.mC.sub.esA.sub.e 680773
GalNAc.sub.3-10.sub.a- PO 2.00 849
o'.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.dsA.s-
ub.dsT.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.e-
s A.sub.esG.sub.es.sup.mC.sub.esA.sub.e 680774
GalNAc.sub.3-13.sub.a- PO 1.50 849
o'.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.dsA.s-
ub.dsT.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.e-
s A.sub.esG.sub.es.sup.mC.sub.esA.sub.e 681272
GalNAc.sub.3-3.sub.a- PO <0.46 849
o'.sup.mC.sub.esA.sub.eoG.sub.eo.sup.mC.sub.eoT.sub.eoT.sub.dsT.sub.dsA.s-
ub.dsT.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.e-
o A.sub.eoG.sub.es.sup.mC.sub.esA.sub.e 681273
GalNAc.sub.3-3.sub.a- A.sub.d 1.10 851
o'Ado.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.ds-
A.sub.dsT.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds
.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esA.sub.e 683733
.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.d-
sA.sub.dsT.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.s-
ub.es A.sub.d 2.50 850
A.sub.esG.sub.es.sup.mC.sub.esA.sub.eoA.sub.do'-GalNAc.sub.3-19.sub.a
The structure of GalNAc.sub.3-1.sub.a was shown previously in
Example 9, GalNAc.sub.3-3.sub.a was shown in Example 39,
GalNAc.sub.3-7.sub.a was shown in Example 48, GalNAc.sub.3-10.sub.a
was shown in Example 46, GalNAc.sub.3-13.sub.a was shown in Example
62, and GalNAc.sub.3-19.sub.a was shown in Example 70.
Example 93: Antisense Inhibition In Vivo by Oligonucleotides
Targeting SRB-1 Comprising Mixed Wings and a 5'-GalNAc.sub.3
Conjugate
[1024] The oligonucleotides listed in Table 100 were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
TABLE-US-00090 TABLE 100 Modified ASOs targeting SRB-1 ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 449093
T.sub.ksT.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.d-
sA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub-
.ks.sup.mC.sub.k n/a n/a 852 699806
GalNAc.sub.3-3.sub.a-.sub.o'T.sub.ksT.sub.ks.sup.mC.sub.ksA.sub.dsG-
.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.d-
s GalNAc.sub.3- PO 852 T.sub.dsT.sub.ks.sup.mC.sub.ks.sup.mC.sub.k
3a 699807
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.ksT.sub.ks.sup.mC.sub.ksA.sub.dsG-
.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.d-
s GalNAc.sub.3- PO 852 T.sub.dsT.sub.ks.sup.mC.sub.ks.sup.mC.sub.k
7a 699809
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.ksT.sub.ks.sup.mC.sub.ksA.sub.dsG-
.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.d-
s GalNAc.sub.3- PO 852 T.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.e
7a 699811
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG-
.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.d-
s GalNAc.sub.3- PO 852 T.sub.dsT.sub.ks.sup.mC.sub.ks.sup.mC.sub.k
7a 699813
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.ksT.sub.ds.sup.mC.sub.ksA.sub.dsG-
.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.d-
s GalNAc.sub.3- PO 852 T.sub.dsT.sub.ks.sup.mC.sub.ds.sup.mC.sub.k
7a 699815
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.esT.sub.ks.sup.mC.sub.ksA.sub.dsG-
.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.d-
s GalNAc.sub.3- PO 852 T.sub.dsT.sub.ks.sup.mC.sub.ks.sup.mC.sub.e
7a
The structure of GalNAc.sub.3-3.sub.a was shown previously in
Example 39, and the structure of GalNAc.sub.3-7a was shown
previously in Example 48. Subscripts: "e" indicates 2'-MOE modified
nucleoside; "d" indicates .beta.-D-2'-deoxyribonucleoside; "k"
indicates 6'-(S)--CH.sub.3 bicyclic nucleoside (cEt); "s" indicates
phosphorothioate internucleoside linkages (PS); "o" indicates
phosphodiester internucleoside linkages (PO). Supersript "m"
indicates 5-methylcytosines.
Treatment
[1025] Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously once at the dosage shown
below with an oligonucleotide listed in Table 100 or with saline.
Each treatment group consisted of 4 animals. The mice were
sacrificed 72 hours following the final administration. Liver SRB-1
mRNA levels were measured using real-time PCR. SRB-1 mRNA levels
were normalized to cyclophilin mRNA levels according to standard
protocols. The results are presented as the average percent of
SRB-1 mRNA levels for each treatment group relative to the saline
control group. As illustrated in Table 101, treatment with
antisense oligonucleotides lowered SRB-1 mRNA levels in a
dose-dependent manner, and the gapmer oligonucleotides comprising a
GalNAc conjugate and having wings that were either full cEt or
mixed sugar modifications were significantly more potent than the
parent oligonucleotide lacking a conjugate and comprising full cEt
modified wings.
[1026] Body weights, liver transaminases, total bilirubin, and BUN
were also measured, and the average values for each treatment group
are shown in Table 101. Body weight is shown as the average percent
body weight relative to the baseline body weight (% BL) measured
just prior to the oligonucleotide dose.
TABLE-US-00091 TABLE 101 SRB-1 mRNA, ALT, AST, BUN, and total
bilirubin levels and body weights SRB-1 ISIS Dosage mRNA ALT AST
Body weight No. (mg/kg) (% PBS) (U/L) (U/L) Bil BUN (% BL) PBS n/a
100 31 84 0.15 28 102 449093 1 111 18 48 0.17 31 104 3 94 20 43
0.15 26 103 10 36 19 50 0.12 29 104 699806 0.1 114 23 58 0.13 26
107 0.3 59 21 45 0.12 27 108 1 25 30 61 0.12 30 104 699807 0.1 121
19 41 0.14 25 100 0.3 73 23 56 0.13 26 105 1 24 22 69 0.14 25 102
699809 0.1 125 23 57 0.14 26 104 0.3 70 20 49 0.10 25 105 1 33 34
62 0.17 25 107 699811 0.1 123 48 77 0.14 24 106 0.3 94 20 45 0.13
25 101 1 66 57 104 0.14 24 107 699813 0.1 95 20 58 0.13 28 104 0.3
98 22 61 0.17 28 105 1 49 19 47 0.11 27 106 699815 0.1 93 30 79
0.17 25 105 0.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106
Example 94: Antisense Inhibition In Vivo by Oligonucleotides
Targeting SRB-1 Comprising 2'-Sugar Modifications and a
5'-GalNAc.sub.3 Conjugate
[1027] The oligonucleotides listed in Table 102 were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
TABLE-US-00092 TABLE 102 Modified ASOs targeting SRB-1 ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 353382
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub-
.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.es n/a n/a 829 T.sub.esT.sub.e
700989
G.sub.msC.sub.msU.sub.msU.sub.msC.sub.msA.sub.dsG.sub.dsT.sub.ds.su-
p.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsU.sub.msC-
.sub.msC.sub.ms n/a n/a 853 U.sub.msU.sub.m 666904
GalNAc.sub.3-3.sub.a-.sub.o'G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.-
sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.d-
sA.sub.ds GalNAc.sub.3- PO 829
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e
3a 700991
GalNAc.sub.3-7.sub.a-.sub.o'G.sub.msC.sub.msU.sub.msU.sub.msC.sub.m-
sA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.ds
Ga1NAc.sub.3- PO 853
A.sub.ds.sup.mC.sub.dsT.sub.dsU.sub.msC.sub.msC.sub.msU.sub.msU.sub.m
7a
Subscript "m" indicates a 2'-O-methyl modified nucleoside. See
Example 74 for complete table legend. The structure of
GalNAc.sub.3-3.sub.a was shown previously in Example 39, and the
structure of GalNAc.sub.3-7a was shown previously in Example
48.
Treatment
[1028] The study was completed using the protocol described in
Example 93. Results are shown in Table 103 below and show that both
the 2'-MOE and 2'-OMe modified oligonucleotides comprising a GalNAc
conjugate were significantly more potent than the respective parent
oligonucleotides lacking a conjugate. The results of the body
weights, liver transaminases, total bilirubin, and BUN measurements
indicated that the compounds were all well tolerated.
TABLE-US-00093 TABLE 103 SRB-1 mRNA ISIS No. Dosage (mg/kg) SRB-1
mRNA (% PBS) PBS n/a 100 353382 5 116 15 58 45 27 700989 5 120 15
92 45 46 666904 1 98 3 45 10 17 700991 1 118 3 63 10 14
Example 95: Antisense Inhibition In Vivo by Oligonucleotides
Targeting SRB-1 Comprising Bicyclic Nucleosides and a
5'-GalNAc.sub.3 Conjugate
[1029] The oligonucleotides listed in Table 104 were tested in a
dose-dependent study for antisense inhibition of SRB-1 in mice.
TABLE-US-00094 TABLE 104 Modified ASOs targeting SRB-1 ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No 440762
T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.d-
sT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub.k
n/a n/a 823 666905
GalNAc.sub.3-3.sub.a-.sub.o'T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.d-
sT.sub.ks.sup.mC.sub.k GalNAc.sub.3-3.sub.a PO 823 699782
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.d-
sT.sub.ks.sup.mC.sub.k GalNAc.sub.3-7.sub.a PO 823 699783
GalNAc.sub.3-3.sub.a-.sub.o'T.sub.ls.sup.mC.sub.lsA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.d-
sT.sub.ls.sup.mC.sub.l GalNAc.sub.3-3.sub.a PO 823 653621
T.sub.ls.sup.mC.sub.lsA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.d-
sT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.lsmC.sub.loA.sub.do'--
GalNAC.sub.3-1.sub.a GalNAc.sub.3-1.sub.a A.sub.d 824 439879
T.sub.gs.sup.mC.sub.gsA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.d-
sT.sub.dG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.gs.sup.mC.sub.g
n/a n/a 823 699789
GalNAc.sub.3-3.sub.a-.sub.o'T.sub.gs.sup.mC.sub.gsA.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.ds-
T.sub.gs.sup.mC.sub.g GalNAc.sub.3-3.sub.a PO 823
Subscript "g" indicates a fluoro-HNA nucleoside, subscript "1"
indicates a locked nucleoside comprising a 2'-O--CH.sub.2-4'
bridge. See the Example 74 table legend for other abbreviations.
The structure of GalNAc.sub.3-1.sub.a was shown previously in
Example 9, the structure of GalNAc.sub.3-3.sub.a was shown
previously in Example 39, and the structure of GalNAc.sub.3-7a was
shown previously in Example 48.
Treatment
[1030] The study was completed using the protocol described in
Example 93. Results are shown in Table 105 below and show that
oligonucleotides comprising a GalNAc conjugate and various bicyclic
nucleoside modifications were significantly more potent than the
parent oligonucleotide lacking a conjugate and comprising bicyclic
nucleoside modifications. Furthermore, the oligonucleotide
comprising a GalNAc conjugate and fluoro-HNA modifications was
significantly more potent than the parent lacking a conjugate and
comprising fluoro-HNA modifications. The results of the body
weights, liver transaminases, total bilirubin, and BUN measurements
indicated that the compounds were all well tolerated.
TABLE-US-00095 TABLE 105 SRB-1 mRNA, ALT, AST, BUN, and total
bilirubin levels and body weights ISIS No. Dosage (mg/kg) SRB-1
mRNA (% PBS) PBS n/a 100 440762 1 104 3 65 10 35 666905 0.1 105 0.3
56 1 18 699782 0.1 93 0.3 63 1 15 699783 0.1 105 0.3 53 1 12 653621
0.1 109 0.3 82 1 27 439879 1 96 3 77 10 37 699789 0.1 82 0.3 69 1
26
Example 96: Plasma Protein Binding of Antisense Oligonucleotides
Comprising a GalNAc.sub.3 Conjugate Group
[1031] Oligonucleotides listed in Table 70 targeting ApoC-III and
oligonucleotides in Table 106 targeting Apo(a) were tested in an
ultra-filtration assay in order to assess plasma protein
binding.
TABLE-US-00096 TABLE 106 Modified oligonucleotides targeting Apo(a)
ISIS GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No
494372
T.sub.esG.sub.es.sup.mC.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.dsG-
.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.dsT.sub.dsG.sub.ds.sup.mC.sub.dsT.sub-
.dsT.sub.esG.sub.esT.sub.es n/a n/a 847 T.sub.es.sup.mC.sub.e
693401
T.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eo.sup.mC.sub.eo.sup.mC.sub.dsG-
.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.dsT.sub.dsG.sub.ds.sup.mC.sub.dsT.sub-
.dsT.sub.eoG.sub.eoT.sub.es n/a n/a 847 T.sub.es.sup.mC.sub.e
681251
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.esG.sub.es.sup.mC.sub.esT.sub.es.-
sup.mC.sub.es.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.dsT.sub.d-
sG.sub.ds.sup.mC.sub.ds GalNAc.sub.3- PO 847
T.sub.dsT.sub.esG.sub.esT.sub.esT.sub.es.sup.mC.sub.e 7.sub.a
681257
GalNAC.sub.3-7.sub.a-.sub.o'T.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eo.-
sup.mC.sub.eo.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.dsT.sub.d-
sG.sub.ds.sup.mC.sub.ds GalNAc.sub.3- PO 847
T.sub.dsT.sub.eoG.sub.eoT.sub.esT.sub.es.sup.mC.sub.e 7.sub.a
See the Example 74 for table legend. The structure of
GalNAc.sub.3-7a was shown previously in Example 48.
[1032] Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding
regenerated cellulose membrane, Millipore, Bedford, Mass.) were
pre-conditioned with 300 .mu.L of 0.5% Tween 80 and centrifuged at
2000 g for 10 minutes, then with 3004, of a 300 .mu.g/mL solution
of a control oligonucleotide in H.sub.2O and centrifuged at 2000 g
for 16 minutes. In order to assess non-specific binding to the
filters of each test oligonucleotide from Tables 70 and 106 to be
used in the studies, 300 .mu.L of a 250 ng/mL solution of
oligonucleotide in H.sub.2O at pH 7.4 was placed in the
pre-conditioned filters and centrifuged at 2000 g for 16 minutes.
The unfiltered and filtered samples were analyzed by an ELISA assay
to determine the oligonucleotide concentrations. Three replicates
were used to obtain an average concentration for each sample. The
average concentration of the filtered sample relative to the
unfiltered sample is used to determine the percent of
oligonucleotide that is recovered through the filter in the absence
of plasma (% recovery).
[1033] Frozen whole plasma samples collected in K3-EDTA from
normal, drug-free human volunteers, cynomolgus monkeys, and CD-1
mice, were purchased from Bioreclamation LLC (Westbury, N.Y.). The
test oligonucleotides were added to 1.2 mL aliquots of plasma at
two concentrations (5 and 150 .mu.g/mL). An aliquot (300 .mu.L) of
each spiked plasma sample was placed in a pre-conditioned filter
unit and incubated at 37.degree. C. for 30 minutes, immediately
followed by centrifugation at 2000 g for 16 minutes. Aliquots of
filtered and unfiltered spiked plasma samples were analyzed by an
ELISA to determine the oligonucleotide concentration in each
sample. Three replicates per concentration were used to determine
the average percentage of bound and unbound oligonucleotide in each
sample. The average concentration of the filtered sample relative
to the concentration of the unfiltered sample is used to determine
the percent of oligonucleotide in the plasma that is not bound to
plasma proteins (% unbound). The final unbound oligonucleotide
values are corrected for non-specific binding by dividing the %
unbound by the % recovery for each oligonucleotide. The final %
bound oligonucleotide values are determined by subtracting the
final % unbound values from 100. The results are shown in Table 107
for the two concentrations of oligonucleotide tested (5 and 150
.mu.g/mL) in each species of plasma. The results show that GalNAc
conjugate groups do not have a significant impact on plasma protein
binding. Furthermore, oligonucleotides with full PS internucleoside
linkages and mixed PO/PS linkages both bind plasma proteins, and
those with full PS linkages bind plasma proteins to a somewhat
greater extent than those with mixed PO/PS linkages.
TABLE-US-00097 TABLE 107 Percent of modified oligonucleotide bound
to plasma proteins Human plasma Monkey plasma Mouse plasma ISIS 5
150 5 150 5 150 No. .mu.g/mL .mu.g/mL .mu.g/mL .mu.g/mL .mu.g/mL
.mu.g/mL 304801 99.2 98.0 99.8 99.5 98.1 97.2 663083 97.8 90.9 99.3
99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.6 89.3 494372 94.1
89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.0 94.6 90.2
681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.6 93.7
95.6 92.7
Example 97: Modified Oligonucleotides Targeting TTR Comprising a
GalNAc.sub.3 Conjugate Group
[1034] The oligonucleotides shown in Table 108 comprising a GalNAc
conjugate were designed to target TTR.
TABLE-US-00098 TABLE 108 Modified oligonucleotides targeting TTR
ISIS GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No
666941
GalNAc.sub.3-3.sub.a-.sub.o'A.sub.doT.sub.es.sup.mC.sub.esT.sub.esT-
.sub.esG.sub.esG.sub.dsT.sub.dsT.sub.dsA.sub.ds GalNAc.sub.3-3
A.sub.d 846
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.esT.sub.es.su-
p.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e 666942
T.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eoG.sub.eoG.sub.dsT.sub.dsT.sub-
.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.ds GalNAc.sub.3-1
A.sub.d 843
A.sub.dsA.sub.dsA.sub.eoT.sub.eo.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e-
oA.sub.do'-GalNAc.sub.3-3.sub.a 682876
GalNAc.sub.3-3.sub.a-.sub.o'T.sub.es.sup.mC.sub.esT.sub.esT.sub.esG-
.sub.esG.sub.dsT.sub.dsT.sub.dsA.sub.ds.sup.mC.sub.ds
GalNAc.sub.3-3 PO 842
A.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.esT.sub.es.sup.mC.sub.es.su-
p.mC.sub.es.sup.mC.sub.e 682877
GalNAC.sub.3-7.sub.a-.sub.o'T.sub.es.sup.mC.sub.esT.sub.esT.sub.esG-
.sub.esG.sub.dsT.sub.dsT.sub.dsA.sub.ds.sup.mC.sub.ds
GalNAc.sub.3-7 PO 842
A.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.esT.sub.es.sup.mC.sub.es.su-
p.mC.sub.es.sup.mC.sub.e 682878
GalNAC.sub.3-10.sub.a-.sub.o'T.sub.es.sup.mC.sub.esT.sub.esT.sub.es-
G.sub.esG.sub.dsT.sub.dsT.sub.dsA.sub.ds.sup.mC.sub.ds
GalNAc.sub.3-10 PO 842
A.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.esT.sub.es.sup.mC.sub.es.su-
p.mC.sub.es.sup.mC.sub.e 682879
GalNAC.sub.3-13.sub.a-.sub.o'T.sub.es.sup.mC.sub.esT.sub.esT.sub.es-
G.sub.esG.sub.dsT.sub.dsT.sub.dsA.sub.ds.sup.mC.sub.ds
GalNAc.sub.3-13 PO 842
A.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.esT.sub.es.sup.mC.sub.es.su-
p.mC.sub.es.sup.mC.sub.e 682880
GalNAC.sub.3-7.sub.a-.sub.o'A.sub.doT.sub.es.sup.mC.sub.esT.sub.esT-
.sub.esG.sub.esG.sub.dsT.sub.dsT.sub.dsA.sub.ds GalNAc.sub.3-7
A.sub.d 846
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.esT.sub.es.su-
p.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e 682881
GalNAC.sub.3-10.sub.a-.sub.o'A.sub.doT.sub.es.sup.mC.sub.esT.sub.es-
T.sub.esG.sub.esG.sub.dsT.sub.dsT.sub.dsA.sub.ds GalNAc.sub.3-10
A.sub.d 846
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.esT.sub.es.su-
p.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e 682882
GalNAC.sub.3-13.sub.a-.sub.o'A.sub.doT.sub.es.sup.mC.sub.esT.sub.es-
T.sub.esG.sub.esG.sub.dsT.sub.dsT.sub.dsA.sub.ds GalNAc.sub.3-13
A.sub.d 846
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.dsA.sub.dsA.sub.esT.sub.es.su-
p.mC.sub.es.sup.mC.sub.es.sup.mC.sub.e 684056
T.sub.es.sup.mC.sub.esT.sub.esT.sub.esG.sub.esG.sub.dsT.sub.dsT.sub-
.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.3-19 A.sub.d 846
A.sub.dsA.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.eoA.sub.d-
o'-GalNAc.sub.3-19.sub.a
The legend for Table 108 can be found in Example 74. The structure
of GalNAc.sub.3-1 was shown in Example 9. The structure of
GalNAc.sub.3-3.sub.a was shown in Example 39. The structure of
GalNAc.sub.3-7.sub.a was shown in Example 48. The structure of
GalNAc.sub.3-10.sub.a was shown in Example 46. The structure of
GalNAc.sub.3-13.sub.a was shown in Example 62. The structure of
GalNAc.sub.3-19.sub.a was shown in Example 70.
Example 98: Evaluation of Pro-Inflammatory Effects of
Oligonucleotides Comprising a GalNAc Conjugate in hPMBC Assay
[1035] The oligonucleotides listed in Table 109 and were tested for
pro-inflammatory effects in an hPMBC assay as described in Examples
23 and 24. (See Tables 30, 83, 95, and 108 for descriptions of the
oligonucleotides.) ISIS 353512 is a high responder used as a
positive control, and the other oligonucleotides are described in
Tables 83, 95, and 108. The results shown in Table 109 were
obtained using blood from one volunteer donor. The results show
that the oligonucleotides comprising mixed PO/PS internucleoside
linkages produced significantly lower pro-inflammatory responses
compared to the same oligonucleotides having full PS linkages.
Furthermore, the GalNAc conjugate group did not have a significant
effect in this assay.
TABLE-US-00099 TABLE 109 ISIS No. E.sub.max/EC.sub.50 GalNAc.sub.3
cluster Linkages CM 353512 3630 n/a PS n/a 420915 802 n/a PS n/a
682881 1311 GalNAc.sub.3-10 PS A.sub.d 682888 0.26 GalNAc.sub.3-10
PO/PS A.sub.d 684057 1.03 GalNAc.sub.3-19 PO/PS A.sub.d
Example 99: Binding Affinities of Oligonucleotides Comprising a
GalNAc Conjugate for the Asialoglycoprotein Receptor
[1036] The binding affinities of the oligonucleotides listed in
Table 110 (see Table 76 for descriptions of the oligonucleotides)
for the asialoglycoprotein receptor were tested in a competitive
receptor binding assay. The competitor ligand, .alpha.1-acid
glycoprotein (AGP), was incubated in 50 mM sodium acetate buffer
(pH 5) with 1 U neuraminidase-agarose for 16 hours at 3TC, and
>90% desialylation was confirmed by either sialic acid assay or
size exclusion chromatography (SEC). Iodine monochloride was used
to iodinate the AGP according to the procedure by Atsma et al. (see
J Lipid Res. 1991 January; 32(1):173-81.) In this method,
desialylated .alpha.1-acid glycoprotein (de-AGP) was added to 10 mM
iodine chloride, Na.sup.125I, and 1 M glycine in 0.25 M NaOH. After
incubation for 10 minutes at room temperature, .sup.125I-labeled
de-AGP was separated from free .sup.125I by concentrating the
mixture twice utilizing a 3 KDMWCO spin column. The protein was
tested for labeling efficiency and purity on a HPLC system equipped
with an Agilent SEC-3 column (7.8.times.300 mm) and a -RAM counter.
Competition experiments utilizing .sup.125I-labeled de-AGP and
various GalNAc-cluster containing ASOs were performed as follows.
Human HepG2 cells (10.sup.6 cells/ml) were plated on 6-well plates
in 2 ml of appropriate growth media. MEM media supplemented with
10% fetal bovine serum (FBS), 2 mM L-Glutamine and 10 mM HEPES was
used. Cells were incubated 16-20 hours @ 3TC with 5% and 10%
CO.sub.2 respectively. Cells were washed with media without FBS
prior to the experiment. Cells were incubated for 30 min
@37.degree. C. with 1 ml competition mix containing appropriate
growth media with 2% FBS, 10.sup.-8 M .sup.125I-labeled de-AGP and
GalNAc-cluster containing ASOs at concentrations ranging from
10.sup.-11 to 10.sup.-5 M. Non-specific binding was determined in
the presence of 10.sup.-2 M GalNAc sugar. Cells were washed twice
with media without FBS to remove unbound .sup.125I-labeled de-AGP
and competitor GalNAc ASO. Cells were lysed using Qiagen's RLT
buffer containing 1% -mercaptoethanol. Lysates were transferred to
round bottom assay tubes after a brief 10 min freeze/thaw cycle and
assayed on a .gamma.-counter. Non-specific binding was subtracted
before dividing .sup.125I protein counts by the value of the lowest
GalNAc-ASO concentration counts. The inhibition curves were fitted
according to a single site competition binding equation using a
nonlinear regression algorithm to calculate the binding affinities
(K.sub.D's).
[1037] The results in Table 110 were obtained from experiments
performed on five different days. Results for oligonucleotides
marked with superscript "a" are the average of experiments run on
two different days. The results show that the oligonucleotides
comprising a GalNAc conjugate group on the 5'-end bound the
asialoglycoprotein receptor on human HepG2 cells with 1.5 to
16-fold greater affinity than the oligonucleotides comprising a
GalNAc conjugate group on the 3'-end.
TABLE-US-00100 TABLE 110 Asialoglycoprotein receptor binding assay
results Oligonucleotide end ISIS to which GalNAc K.sub.D No. GalNAc
conjugate conjugate is attached (nM) 661161.sup.a GalNAc.sub.3-3 5'
3.7 666881.sup.a GalNAc.sub.3-10 5' 7.6 666981 GalNAc.sub.3-7 5'
6.0 670061 GalNAc.sub.3-13 5' 7.4 655861.sup.a GalNAc.sub.3-1 3'
11.6 677841.sup.a GalNAc.sub.3-19 3' 60.8
Example 100: Antisense Inhibition In Vivo by Oligonucleotides
Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo
[1038] The oligonucleotides listed in Table 111a below were tested
in a single dose study for duration of action in mice.
TABLE-US-00101 TABLE 111a Modified ASOs targeting APO(a) ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 681251
GalNAC.sub.3-7.sub.a-.sub.o'T.sub.esG.sub.es.sup.mC.sub.esT.sub.es.-
sup.mC.sub.es.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.ds
GalNAc.sub.3-7a PO 847
T.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.esG.sub.esT.sub.esT.sub.es.su-
p.mC.sub.e 681257
GalNAC.sub.3-7.sub.a-.sub.o'T.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eo.-
sup.mC.sub.eo.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.ds
GalNAc.sub.3-7a PO 847
T.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eoG.sub.eoT.sub.esT.sub.es.su-
p.mC.sub.e
The structure of GalNAc.sub.3-7.sub.a was shown in Example 48.
Treatment
[1039] Female transgenic mice that express human Apo(a) were each
injected subcutaneously once per week, for a total of 6 doses, with
an oligonucleotide and dosage listed in Table 111b or with PBS.
Each treatment group consisted of 3 animals. Blood was drawn the
day before dosing to determine baseline levels of Apo(a) protein in
plasma and at 72 hours, 1 week, and 2 weeks following the first
dose. Additional blood draws will occur at 3 weeks, 4 weeks, 5
weeks, and 6 weeks following the first dose. Plasma Apo(a) protein
levels were measured using an ELISA. The results in Table 111b are
presented as the average percent of plasma Apo(a) protein levels
for each treatment group, normalized to baseline levels (% BL), The
results show that the oligonucleotides comprising a GalNAc
conjugate group exhibited potent reduction in Apo(a) expression.
This potent effect was observed for the oligonucleotide that
comprises full PS internucleoside linkages and the oligonucleotide
that comprises mixed PO and PS linkages.
TABLE-US-00102 TABLE 111b Apo(a) plasma protein levels Apo(a) at
Apo(a) at Apo(a) at ISIS Dosage 72 hours 1 week 3 weeks No. (mg/kg)
(% BL) (% BL) (% BL) PBS n/a 116 104 107 681251 0.3 97 108 93 1.0
85 77 57 3.0 54 49 11 10.0 23 15 4 681257 0.3 114 138 104 1.0 91 98
54 3.0 69 40 6 10.0 30 21 4
Example 101: Antisense Inhibition by Oligonucleotides Comprising a
GalNAc Cluster Linked Via a Stable Moiety
[1040] The oligonucleotides listed in Table 112 were tested for
inhibition of mouse APOC-III expression in vivo. C57Bl/6 mice were
each injected subcutaneously once with an oligonucleotide listed in
Table 112 or with PBS. Each treatment group consisted of 4 animals.
Each mouse treated with ISIS 440670 received a dose of 2, 6, 20, or
60 mg/kg. Each mouse treated with ISIS 680772 or 696847 received
0.6, 2, 6, or 20 mg/kg. The GalNAc conjugate group of ISIS 696847
is linked via a stable moiety, a phosphorothioate linkage instead
of a readily cleavable phosphodiester containing linkage. The
animals were sacrificed 72 hours after the dose. Liver APOC-III
mRNA levels were measured using real-time PCR. APOC-III mRNA levels
were normalized to cyclophilin mRNA levels according to standard
protocols. The results are presented in Table 112 as the average
percent of APOC-III mRNA levels for each treatment group relative
to the saline control group. The results show that the
oligonucleotides comprising a GalNAc conjugate group were
significantly more potent than the oligonucleotide lacking a
conjugate group. Furthermore, the oligonucleotide comprising a
GalNAc conjugate group linked to the oligonucleotide via a
cleavable moiety (ISIS 680772) was even more potent than the
oligonucleotide comprising a GalNAc conjugate group linked to the
oligonucleotide via a stable moiety (ISIS 696847).
TABLE-US-00103 TABLE 112 Modified oligonucleotides targeting mouse
APOC-III APOC-III SEQ ISIS Dosage mRNA ID No. Sequences (5' to 3')
CM (mg/kg) (% PBS) No. 440670
.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.d-
sA.sub.dsT.sub.dsT.sub.dsA.sub.ds n/a 2 92 849
G.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub-
.esA.sub.e 6 86 20 59 60 37 680772
GalNAc.sub.3-7.sub.a-.sub.o'.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.s-
ub.esT.sub.esT.sub.dsT.sub.dsA.sub.ds PO 0.6 79 849
T.sub.dsT.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.esA.s-
ub.esG.sub.es.sup.mC.sub.esA.sub.e 2 58 6 31 20 13 696847
GalNAc.sub.3-7.sub.a- n/a 0.6 83 849
.sub.s'.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.dsT.sub.-
dsA.sub.dsT.sub.ds (PS) 2 73
T.sub.dsA.sub.dsG.sub.dsG.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.esA.sub.esG.s-
ub.es.sup.mC.sub.esA.sub.e 6 40 20 28
The structure of GalNAc.sub.3-7.sub.a was shown in Example 48.
Example 102: Distribution in Liver of Antisense Oligonucleotides
Comprising a GalNAc Conjugate
[1041] The liver distribution of ISIS 353382 (see Table 36) that
does not comprise a GalNAc conjugate and ISIS 655861 (see Table 36)
that does comprise a GalNAc conjugate was evaluated. Male balb/c
mice were subcutaneously injected once with ISIS 353382 or 655861
at a dosage listed in Table 113. Each treatment group consisted of
3 animals except for the 18 mg/kg group for ISIS 655861, which
consisted of 2 animals. The animals were sacrificed 48 hours
following the dose to determine the liver distribution of the
oligonucleotides. In order to measure the number of antisense
oligonucleotide molecules per cell, a Ruthenium (II)
tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated
to an oligonucleotide probe used to detect the antisense
oligonucleotides. The results presented in Table 113 are the
average concentrations of oligonucleotide for each treatment group
in units of millions of oligonucleotide molecules per cell. The
results show that at equivalent doses, the oligonucleotide
comprising a GalNAc conjugate was present at higher concentrations
in the total liver and in hepatocytes than the oligonucleotide that
does not comprise a GalNAc conjugate. Furthermore, the
oligonucleotide comprising a GalNAc conjugate was present at lower
concentrations in non-parenchymal liver cells than the
oligonucleotide that does not comprise a GalNAc conjugate. And
while the concentrations of ISIS 655861 in hepatocytes and
non-parenchymal liver cells were similar per cell, the liver is
approximately 80% hepatocytes by volume. Thus, the majority of the
ISIS 655861 oligonucleotide that was present in the liver was found
in hepatocytes, whereas the majority of the ISIS 353382
oligonucleotide that was present in the liver was found in
non-parenchymal liver cells.
TABLE-US-00104 TABLE 113 Concentration Concentration Concentration
in non-parenchymal in whole liver in hepatocytes liver cells (mole-
(mole- (mole- ISIS Dosage cules*10{circumflex over ( )}6
cules*10{circumflex over ( )}6 cules*10{circumflex over ( )}6 No.
(mg/kg) per cell) per cell) per cell) 353382 3 9.7 1.2 37.2 10 17.3
4.5 34.0 20 23.6 6.6 65.6 30 29.1 11.7 80.0 60 73.4 14.8 98.0 90
89.6 18.5 119.9 655861 0.5 2.6 2.9 3.2 1 6.2 7.0 8.8 3 19.1 25.1
28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1
Example 103: Duration of Action In Vivo of Oligonucleotides
Targeting APOC-III Comprising a GalNAc.sub.3 Conjugate
[1042] The oligonucleotides listed in Table 114 below were tested
in a single dose study for duration of action in mice.
TABLE-US-00105 TABLE 114 Modified ASOs targeting APOC-III ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 304801
A.sub.esG.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.dsT.sub.d-
sT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.-
mC.sub.dsT.sub.esT.sub.es n/a n/a 821 T.sub.esA.sub.esT.sub.e
663084
GalNAC.sub.3-3.sub.a-.sub.o'A.sub.doA.sub.esG.sub.eo.sup.mC.sub.eoT-
.sub.eoT.sub.eo.sup.mC.sub.dsT.sub.dsT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.d-
s GalNAc.sub.3-3a A.sub.d 837
.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.eoT.sub.eoT.sub.esA.sub-
.esT.sub.e 679241
A.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.dsT.sub.d-
sT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsA.sub.dsG.sub.ds.sup.-
mC.sub.dsT.sub.eoT.sub.eo GalNAc.sub.3-19a A.sub.d 822
T.sub.esA.sub.esT.sub.eoA.sub.do'-GalNAc.sub.3-19.sub.a
The structure of GalNAc.sub.3-3.sub.a was shown in Example 39, and
GalNAc.sub.3-19.sub.a was shown in Example 70.
Treatment
[1043] Female transgenic mice that express human APOC-III were each
injected subcutaneously once with an oligonucleotide listed in
Table 114 or with PBS. Each treatment group consisted of 3 animals.
Blood was drawn before dosing to determine baseline and at 3, 7,
14, 21, 28, 35, and 42 days following the dose. Plasma triglyceride
and APOC-III protein levels were measured as described in Example
20. The results in Table 115 are presented as the average percent
of plasma triglyceride and APOC-III levels for each treatment
group, normalized to baseline levels. A comparison of the results
in Table 71 of example 79 with the results in Table 115 below show
that oligonucleotides comprising a mixture of phosphodiester and
phosphorothioate internucleoside linkages exhibited increased
duration of action than equivalent oligonucleotides comprising only
phosphorothioate internucleoside linkages.
TABLE-US-00106 TABLE 115 Plasma triglyceride and APOC-III protein
levels in transgenic mice Time point APOC-III ISIS Dosage (days
post- Triglycerides protein GalNAc.sub.3 No. (mg/kg) dose) (%
baseline) (% baseline) Cluster CM PBS n/a 3 96 101 n/a n/a 7 88 98
14 91 103 21 69 92 28 83 81 35 65 86 42 72 88 304801 30 3 42 46 n/a
n/a 7 42 51 14 59 69 21 67 81 28 79 76 35 72 95 42 82 92 663084 10
3 35 28 GalNAc.sub.3-3a A.sub.d 7 23 24 14 23 26 21 23 29 28 30 22
35 32 36 42 37 47 679241 10 3 38 30 GalNAc.sub.3-19a A.sub.d 7 31
28 14 30 22 21 36 34 28 48 34 35 50 45 42 72 64
Example 104: Synthesis of Oligonucleotides Comprising a 5'-GalNAc2
Conjugate
##STR00255## ##STR00256##
[1045] Compound 120 is commercially available, and the synthesis of
compound 126 is described in Example 49. Compound 120 (1 g, 2.89
mmol), HBTU (0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were
dissolved in DMF (10 mL. and N,N-diisopropylethylamine (1.75 mL,
10.1 mmol) were added. After about 5 min, aminohexanoic acid benzyl
ester (1.36 g, 3.46 mmol) was added to the reaction. After 3 h, the
reaction mixture was poured into 100 mL of 1 M NaHSO4 and extracted
with 2.times.50 mL ethyl acetate. Organic layers were combined and
washed with 3.times.40 mL sat NaHCO.sub.3 and 2.times. brine, dried
with Na.sub.2SO.sub.4, filtered and concentrated. The product was
purified by silica gel column chromatography (DCM:EA:Hex, 1:1:1) to
yield compound 231. LCMS and NMR were consistent with the
structure. Compounds 231 (1.34 g, 2.438 mmol) was dissolved in
dichloromethane (10 mL) and trifluoracetic acid (10 mL) was added.
After stirring at room temperature for 2 h, the reaction mixture
was concentrated under reduced pressure and co-evaporated with
toluene (3.times.10 mL). The residue was dried under reduced
pressure to yield compound 232 as the trifuloracetate salt. The
synthesis of compound 166 is described in Example 54. Compound 166
(3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution of
compound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) and
N,N-diisopropylethylamine (1.55 mL) was added. The reaction was
stirred at room temperature for 30 minutes, then poured into water
(80 mL) and the aqueous layer was extracted with EtOAc (2.times.100
mL). The organic phase was separated and washed with sat. aqueous
NaHCO.sub.3 (3.times.80 mL), 1 M NaHSO.sub.4 (3.times.80 mL) and
brine (2.times.80 mL), then dried (Na.sub.2SO.sub.4), filtered, and
concentrated. The residue was purified by silica gel column
chromatography to yield compound 233. LCMS and NMR were consistent
with the structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved
in methanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on
carbon (10 wt % Pd/C, wet, 0.07 g) was added, and the reaction
mixture was stirred under hydrogen atmosphere for 3 h. The reaction
mixture was filtered through a pad of Celite and concentrated to
yield the carboxylic acid. The carboxylic acid (1.32 g, 1.15 mmol,
cluster free acid) was dissolved in DMF (3.2 mL). To this
N,N-diisopropylehtylamine (0.3 mL, 1.73 mmol) and PFPTFA (0.30 mL,
1.73 mmol) were added. After 30 min stirring at room temperature
the reaction mixture was poured into water (40 mL) and extracted
with EtOAc (2.times.50 mL). A standard work-up was completed as
described above to yield compound 234. LCMS and NMR were consistent
with the structure. Oligonucleotide 235 was prepared using the
general procedure described in Example 46. The GalNAc2 cluster
portion (GalNAc2-24.sub.a) of the conjugate group GalNAc2-24 can be
combined with any cleavable moiety present on the oligonucleotide
to provide a variety of conjugate groups. The structure of
GalNAc2-24 (GalNAc2-24.sub.a-CM) is shown below:
##STR00257##
Example 105: Synthesis of Oligonucleotides Comprising a
GalNAc.sub.1-25 Conjugate
##STR00258##
[1047] The synthesis of compound 166 is described in Example 54.
Oligonucleotide 236 was prepared using the general procedure
described in Example 46.
[1048] Alternatively, oligonucleotide 236 was synthesized using the
scheme shown below, and compound 238 was used to form the
oligonucleotide 236 using procedures described in Example 10.
##STR00259##
The GalNAc.sub.1 cluster portion (GalNAc.sub.1-25.sub.a) of the
conjugate group GalNAc.sub.1-25 can be combined with any cleavable
moiety present on the oligonucleotide to provide a variety of
conjugate groups. The structure of GalNAc.sub.1-25
(GalNAc.sub.1-25.sub.a-CM) is shown below:
##STR00260##
Example 106: Antisense Inhibition In Vivo by Oligonucleotides
Targeting SRB-1 Comprising a 5% GalNAc2 or a 5'-GalNAc.sub.3
Conjugate
[1049] Oligonucleotides listed in Tables 116 and 117 were tested in
dose-dependent studies for antisense inhibition of SRB-1 in
mice.
Treatment
[1050] Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously once with 2, 7, or 20
mg/kg of ISIS No. 440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of
ISIS No. 686221, 686222, or 708561; or with saline. Each treatment
group consisted of 4 animals. The mice were sacrificed 72 hours
following the final administration. Liver SRB-1 mRNA levels were
measured using real-time PCR. SRB-1 mRNA levels were normalized to
cyclophilin mRNA levels according to standard protocols. The
antisense oligonucleotides lowered SRB-1 mRNA levels in a
dose-dependent manner, and the ED50 results are presented in Tables
116 and 117. Although previous studies showed that trivalent
GalNAc-conjugated oligonucleotides were significantly more potent
than divalent GalNAc-conjugated oligonucleotides, which were in
turn significantly more potent than monovalent GalNAc conjugated
oligonucleotides (see, e.g., Khorev et al., Bioorg. & Med.
Chem., Vol. 16, 5216-5231 (2008)), treatment with antisense
oligonucleotides comprising monovalent, divalent, and trivalent
GalNAc clusters lowered SRB-1 mRNA levels with similar potencies as
shown in Tables 116 and 117.
TABLE-US-00107 TABLE 116 Modified oligonucleotides targeting SRB-1
ISIS GalNAc ED.sub.50 SEQ No. Sequences (5' to 3') Cluster (mg/kg)
ID No 440762
T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.d-
sT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub.k
n/a 4.7 823 686221
GalNAc.sub.2-24.sub.a-.sub.o'A.sub.doT.sub.ks.sup.mC.sub.ksA.sub.ds-
G.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.2-24.sub.a 0.39 827
.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub.k 686222
GalNAc.sub.3-13.sub.a-.sub.o'A.sub.doT.sub.ks.sup.mC.sub.ksA.sub.ds-
G.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.3-13.sub.a 0.41 827
.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub.k
See Example 93 for table legend. The structure of GalNAc.sub.3-13a
was shown in Example 62, and the structure of GalNAc2-24a was shown
in Example 104.
TABLE-US-00108 TABLE 117 Modified oligonucleotides targeting SRB-1
ISIS GalNAc ED.sub.50 SEQ No. Sequences (5' to 3') Cluster (mg/kg)
ID No 440762
T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.d-
sT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub.k
n/a 5 823 708561
GalNAc.sub.1-25.sub.a-.sub.o'T.sub.ks.sup.mC.sub.ksA.sub.dsG.sub.ds-
T.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.1-25.sub.a 0.4 823
.sup.mC.sub.dsT.sub.dsT.sub.ks.sup.mC.sub.k
See Example 93 for table legend. The structure of GalNAc.sub.1-25a
was shown in Example 105.
[1051] The concentrations of the oligonucleotides in Tables 116 and
117 in liver were also assessed, using procedures described in
Example 75. The results shown in Tables 117a and 117b below are the
average total antisense oligonucleotide tissues levels for each
treatment group, as measured by UV in units of .mu.g
oligonucleotide per gram of liver tissue. The results show that the
oligonucleotides comprising a GalNAc conjugate group accumulated in
the liver at significantly higher levels than the same dose of the
oligonucleotide lacking a GalNAc conjugate group. Furthermore, the
antisense oligonucleotides comprising one, two, or three GalNAc
ligands in their respective conjugate groups all accumulated in the
liver at similar levels. This result is surprising in view of the
Khorev et al. literature reference cited above and is consistent
with the activity data shown in Tables 116 and 117 above.
TABLE-US-00109 TABLE 117a Liver concentrations of oligonucleotides
comprising a GalNAc.sub.2 or GalNAc.sub.3 conjugate group ISIS
Dosage [Antisense oligonucleotide] No. (mg/kg) (.mu.g/g) GalNAc
cluster CM 440762 2 2.1 n/a n/a 7 13.1 20 31.1 686221 0.2 0.9
GalNAc.sub.2-24a A.sub.d 0.6 2.7 2 12.0 6 26.5 686222 0.2 0.5
GalNAc.sub.3-13a A.sub.d 0.6 1.6 2 11.6 6 19.8
TABLE-US-00110 TABLE 117b Liver concentrations of oligonucleotides
comprising a GalNAc.sub.1 conjugate group ISIS Dosage [Antisense
oligonucleotide] No. (mg/kg) (.mu.g/g) GalNAc cluster CM 440762 2
2.3 n/a n/a 7 8.9 20 23.7 708561 0.2 0.4 GalNAc.sub.1-25.sub.a PO
0.6 1.1 2 5.9 6 23.7 20 53.9
Example 107: Synthesis of Oligonucleotides Comprising a
GalNAc.sub.1-26 or GalNAc.sub.1-27 Conjugate
##STR00261##
[1053] Oligonucleotide 239 is synthesized via coupling of compound
47 (see Example 15) to acid 64 (see Example 32) using HBTU and DIEA
in DMF. The resulting amide containing compound is phosphitylated,
then added to the 5'-end of an oligonucleotide using procedures
described in Example 10. The GalNAc.sub.1 cluster portion
(GalNAc.sub.1-26.sub.a) of the conjugate group GalNAc.sub.1-26 can
be combined with any cleavable moiety present on the
oligonucleotide to provide a variety of conjugate groups. The
structure of GalNAc.sub.1-26 (GalNAc.sub.1-26.sub.a-CM) is shown
below:
##STR00262##
[1054] In order to add the GalNAc.sub.1 conjugate group to the
3'-end of an oligonucleotide, the amide formed from the reaction of
compounds 47 and 64 is added to a solid support using procedures
described in Example 7. The oligonucleotide synthesis is then
completed using procedures described in Example 9 in order to form
oligonucleotide 240.
##STR00263##
The GalNAc.sub.1 cluster portion (GalNAc.sub.1-27.sub.a) of the
conjugate group GalNAc.sub.1-27 can be combined with any cleavable
moiety present on the oligonucleotide to provide a variety of
conjugate groups. The structure of GalNAc.sub.1-27
(GalNAc.sub.1-27.sub.a-CM) is shown below:
##STR00264##
Example 108: Antisense Inhibition In Vivo by Oligonucleotides
Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo
[1055] The oligonucleotides listed in Table 118 below were tested
in a single dose study in mice.
TABLE-US-00111 TABLE 118 Modified ASOs targeting APO(a) ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 494372
T.sub.esG.sub.es.sup.mC.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.dsG-
.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.dsT.sub.dsG.sub.ds.sup.mC.sub.ds
n/a n/a 847 T.sub.dsT.sub.esG.sub.esT.sub.esT.sub.es.sup.mC.sub.e
681251
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.esG.sub.es.sup.mC.sub.esT.sub.es.-
sup.mC.sub.es.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.ds
GalNAc.sub.3-7a PO 847
T.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.esG.sub.esT.sub.esT.sub.es.su-
p.mC.sub.e 681255
GalNAc.sub.3-3.sub.a-.sub.o'T.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eo.-
sup.mC.sub.eo.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.ds
GalNAc.sub.3-3a PO 847
T.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eoG.sub.eoT.sub.esT.sub.es.su-
p.mC.sub.e 681256
GalNAc.sub.3-10.sub.a-.sub.o'T.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eo-
.sup.mC.sub.eo.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.ds
GalNAc.sub.3-10a PO 847
T.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eoG.sub.eoT.sub.esT.sub.es.su-
p.mC.sub.e 681257
GalNAc.sub.3-7.sub.a-.sub.o'T.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eo.-
sup.mC.sub.eo.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.ds
GalNAc.sub.3-7a PO 847
T.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eoG.sub.eoT.sub.esT.sub.es.su-
p.mC.sub.e 681258
GalNAc.sub.3-13.sub.a-.sub.o'T.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eo-
.sup.mC.sub.eo.sup.mC.sub.dsG.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.ds
GalNAc.sub.3-13a PO 847
T.sub.dsG.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eoG.sub.eoT.sub.esT.sub.es.su-
p.mC.sub.e 681260
T.sub.esG.sub.eo.sup.mC.sub.eoT.sub.eo.sup.mC.sub.eo.sup.mC.sub.dsG-
.sub.dsT.sub.dsT.sub.dsG.sub.dsG.sub.dsT.sub.dsG.sub.ds.sup.mC.sub.dsT.sub-
.dsT.sub.eoG.sub.eo GalNAc.sub.3-19a A.sub.d 854
T.sub.esT.sub.es.sup.mC.sub.eoA.sub.do'-GalNAc.sub.3-19
The structure of GalNAc.sub.3-7.sub.a was shown in Example 48.
Treatment
[1056] Male transgenic mice that express human Apo(a) were each
injected subcutaneously once with an oligonucleotide and dosage
listed in Table 119 or with PBS. Each treatment group consisted of
4 animals. Blood was drawn the day before dosing to determine
baseline levels of Apo(a) protein in plasma and at 1 week following
the first dose. Additional blood draws will occur weekly for
approximately 8 weeks. Plasma Apo(a) protein levels were measured
using an ELISA. The results in Table 119 are presented as the
average percent of plasma Apo(a) protein levels for each treatment
group, normalized to baseline levels (% BL), The results show that
the antisense oligonucleotides reduced Apo(a) protein expression.
Furthermore, the oligonucleotides comprising a GalNAc conjugate
group exhibited even more potent reduction in Apo(a) expression
than the oligonucleotide that does not comprise a conjugate
group.
TABLE-US-00112 TABLE 119 Apo(a) plasma protein levels ISIS No.
Dosage (mg/kg) Apo(a) at 1 week (% BL) PBS n/a 143 494372 50 58
681251 10 15 681255 10 14 681256 10 17 681257 10 24 681258 10 22
681260 10 26
Example 109: Synthesis of Oligonucleotides Comprising a
GalNAc.sub.1-28 or GalNAc.sub.1-29 Conjugate
##STR00265##
[1058] Oligonucleotide 241 is synthesized using procedures similar
to those described in Example 71 to form the phosphoramidite
intermediate, followed by procedures described in Example 10 to
synthesize the oligonucleotide. The GalNAc.sub.1 cluster portion
(GalNAc.sub.1-28.sub.a) of the conjugate group GalNAc.sub.1-28 can
be combined with any cleavable moiety present on the
oligonucleotide to provide a variety of conjugate groups. The
structure of GalNAc.sub.1-28 (GalNAc.sub.1-28.sub.a-CM) is shown
below:
##STR00266##
[1059] In order to add the GalNAc.sub.1 conjugate group to the
3'-end of an oligonucleotide, procedures similar to those described
in Example 71 are used to form the hydroxyl intermediate, which is
then added to the solid support using procedures described in
Example 7. The oligonucleotide synthesis is then completed using
procedures described in Example 9 in order to form oligonucleotide
242.
##STR00267##
[1060] The GalNAc.sub.1 cluster portion (GalNAc.sub.1-29.sub.a) of
the conjugate group GalNAc.sub.1-29 can be combined with any
cleavable moiety present on the oligonucleotide to provide a
variety of conjugate groups. The structure of GalNAc.sub.1-29
(GalNAc.sub.1-29.sub.a-CM) is shown below:
##STR00268##
Example 110: Synthesis of Oligonucleotides Comprising a
GalNAc.sub.1-30 Conjugate
##STR00269##
[1062] Oligonucleotide 246 comprising a GalNAc.sub.1-30 conjugate
group, wherein Y is selected from O, S, a substituted or
unsubstituted C.sub.1-C.sub.10 alkyl, amino, substituted amino,
azido, alkenyl or alkynyl, is synthesized as shown above. The
GalNAc.sub.1 cluster portion (GalNAc.sub.1-30.sub.a) of the
conjugate group GalNAc.sub.1-30 can be combined with any cleavable
moiety to provide a variety of conjugate groups. In certain
embodiments, Y is part of the cleavable moiety. In certain
embodiments, Y is part of a stable moiety, and the cleavable moiety
is present on the oligonucleotide. The structure of
GalNAc.sub.1-30.sub.a is shown below:
##STR00270##
Example 111: Synthesis of Oligonucleotides Comprising a GalNAc2-31
or GalNAc2-32 Conjugate
##STR00271##
[1064] Oligonucleotide 250 comprising a GalNAc2-31 conjugate group,
wherein Y is selected from O, S, a substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, amino, substituted amino, azido, alkenyl or
alkynyl, is synthesized as shown above. The GalNAc2 cluster portion
(GalNAc.sub.2-31.sub.a) of the conjugate group GalNAc.sub.2-31 can
be combined with any cleavable moiety to provide a variety of
conjugate groups. In certain embodiments, the Y-containing group
directly adjacent to the 5'-end of the oligonucleotide is part of
the cleavable moiety. In certain embodiments, the Y-containing
group directly adjacent to the 5'-end of the oligonucleotide is
part of a stable moiety, and the cleavable moiety is present on the
oligonucleotide. The structure of GalNAc.sub.2-31.sub.a is shown
below:
##STR00272##
[1065] The synthesis of an oligonucleotide comprising a
GalNAc.sub.2-32 conjugate is shown below.
##STR00273##
[1066] Oligonucleotide 252 comprising a GalNAc.sub.2-32 conjugate
group, wherein Y is selected from O, S, a substituted or
unsubstituted C.sub.1-C.sub.10 alkyl, amino, substituted amino,
azido, alkenyl or alkynyl, is synthesized as shown above. The
GalNAc.sub.2 cluster portion (GalNAc.sub.2-32.sub.a) of the
conjugate group GalNAc.sub.2-32 can be combined with any cleavable
moiety to provide a variety of conjugate groups. In certain
embodiments, the Y-containing group directly adjacent to the 5'-end
of the oligonucleotide is part of the cleavable moiety. In certain
embodiments, the Y-containing group directly adjacent to the 5'-end
of the oligonucleotide is part of a stable moiety, and the
cleavable moiety is present on the oligonucleotide. The structure
of GalNAc.sub.2-32.sub.a is shown below:
##STR00274##
Example 112: Modified Oligonucleotides Comprising a GalNAc.sub.1
Conjugate
[1067] The oligonucleotides in Table 120 targeting SRB-1 were
synthesized with a GalNAc.sub.1 conjugate group in order to further
test the potency of oligonucleotides comprising conjugate groups
that contain one GalNAc ligand.
TABLE-US-00113 TABLE 120 ISIS GalNAc SEQ No. Sequences (5' to 3')
Cluster CM ID No. 711461
GalNAc.sub.1-25.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.es-
T.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds
GalNAc.sub.1-25.sub.a A.sub.d 831
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 711462
GalNAc.sub.1-25.sub.a-.sub.o'G.sub.es.sup.mC.sub.esT.sub.esT.sub.es-
.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds
GalNAc.sub.1-25.sub.a PO 829
A.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub-
.es.sup.mC.sub.esT.sub.esT.sub.e 711463
GalNAc.sub.1-25.sub.a-.sub.o'G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo-
.sup.mC.sub.eoA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds
GalNAc.sub.1-25.sub.a PO 829
A.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub-
.eo.sup.mC.sub.esT.sub.esT.sub.e 711465
GalNAc.sub.1-26.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.es-
T.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds
GalNAc.sub.1-26.sub.a A.sub.d 831
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 711466
GalNAc.sub.1-26.sub.a-.sub.o'G.sub.es.sup.mC.sub.esT.sub.esT.sub.es-
.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds
GalNAc.sub.1-26.sub.a PO 829
A.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub-
.es.sup.mC.sub.esT.sub.esT.sub.e 711467
GalNAc.sub.1-26.sub.a-.sub.o'G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo-
.sup.mC.sub.eoA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds
GalNAc.sub.1-26.sub.a PO 829
A.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub-
.eo.sup.mC.sub.esT.sub.esT.sub.e 711468
GalNAc.sub.1-28.sub.a-.sub.o'A.sub.doG.sub.es.sup.mC.sub.esT.sub.es-
T.sub.es.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds
GalNAc.sub.1-28.sub.a A.sub.d 831
.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub-
.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e 711469
GalNAc.sub.1-28.sub.a-.sub.o'G.sub.es.sup.mC.sub.esT.sub.esT.sub.es-
.sup.mC.sub.esA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds
GalNAc.sub.1-28.sub.a PO 829
A.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub-
.es.sup.mC.sub.esT.sub.esT.sub.e 711470
GalNAc.sub.1-28.sub.a-.sub.o'G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo-
.sup.mC.sub.eoA.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.ds
GalNAc.sub.1-28.sub.a PO 829
A.sub.dsT.sub.dsG.sub.dsA.sub.ds.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub-
.eo.sup.mC.sub.esT.sub.esT.sub.e 713844
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.1-27.sub.a PO 829
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e-
o'-GalNAc.sub.1-27.sub.a 713845
G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.1-27.sub.a PO 829
.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT.sub.e-
o'-GalNAc.sub.1-27.sub.a 713846
G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.1-27.sub.a A.sub.d 830
.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT.sub.e-
oA.sub.do'-GalNAc.sub.1-27.sub.a 713847
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.1-29.sub.a PO 829
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e-
o'-GalNAc.sub.1-29.sub.a 713848
G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.1-29.sub.a PO 829
.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT.sub.e-
o'-GalNAc.sub.1-29.sub.a 713849
G.sub.es.sup.mC.sub.esT.sub.esT.sub.es.sup.mC.sub.esA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.1-29.sub.a A.sub.d 830
.sup.mC.sub.dsT.sub.dsT.sub.es.sup.mC.sub.es.sup.mC.sub.esT.sub.esT.sub.e-
oA.sub.do'-GalNAc.sub.1-29.sub.a 713850
G.sub.es.sup.mC.sub.eoT.sub.eoT.sub.eo.sup.mC.sub.eoA.sub.dsG.sub.d-
sT.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.dsG.sub.dsA.sub.ds
GalNAc.sub.1-29.sub.a A.sub.d 830
.sup.mC.sub.dsT.sub.dsT.sub.eo.sup.mC.sub.eo.sup.mC.sub.esT.sub.esT.sub.e-
oA.sub.do'-GalNAc.sub.1-29.sub.a
Example 113: Antisense Inhibition In Vivo by Oligonucleotides
Targeting CFB
[1068] The oligonucleotides listed in Table 121 were tested in a
dose-dependent study for antisense inhibition of human Complement
Factor B (CFB) in mice.
TABLE-US-00114 TABLE 121 Modified ASOs targeting CFB ISIS
GalNAc.sub.3 SEQ No. Sequences (5' to 3') Cluster CM ID No. 588540
A.sub.esT.sub.es.sup.mC.sub.es.sup.mC.sub.es.sup.mC.sub.esA.sub.ds.-
sup.mC.sub.dsG.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds n/a
n/a 440
.sup.mC.sub.dsT.sub.dsG.sub.dsT.sub.ds.sup.mC.sub.es.sup.mC.sub.esA.sub.e-
sG.sub.es.sup.mCe 687301
GalNAc.sub.3-3.sub.a-.sub.o'A.sub.esT.sub.es.sup.mC.sub.es.sup.mC.s-
ub.es.sup.mC.sub.esA.sub.ds.sup.mC.sub.dsG.sub.ds GalNAc.sub.3-3a
PO 440
.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsT.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.es.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.e
The structure of GalNAc.sub.3-3.sub.a was shown previously in
Example 39.
Treatment
[1069] Transgenic mice that express human CFB (Jackson Laboratory,
Bar Harbor, Me.) were injected subcutaneously once per week for 3
weeks (a total of 4 doses) with an oligonucleotide listed in Table
122 or with saline. The four treatment groups that received ISIS
No. 588540 were given 6, 12, 25, or 50 mg/kg per dose. The four
treatment groups that received ISIS No. 687301 were given 0.25,
0.5, 2, or 6 mg/kg per dose. Each treatment group consisted of 4
animals. The mice were sacrificed 2 days following the final
administration to determine the liver and kidney human CFB and
cyclophilin mRNA levels using real-time PCR according to standard
protocols. The CFB mRNA levels were normalized to the cyclophilin
levels, and the averages for each treatment group were used to
determine the dose that achieved 50% inhibition of the human CFB
transcript expression (ED.sub.50). The results are the averages of
four experiments completed with two different primer probe sets and
are shown in Table 122.
TABLE-US-00115 TABLE 122 Potencies of oligonucleotides targeting
human CFB in vivo ISIS ED.sub.50 in liver ED.sub.50 in kidney
GalNAc.sub.3 No. (mg/kg) (mg/kg) Cluster CM 588540 7.9 11.7 n/a n/a
687301 0.49 0.35 GalNAc.sub.3-3a PO
[1070] Liver transaminase levels, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), in serum were measured
relative to saline injected mice using standard protocols. Total
bilirubin, BUN, and body weights were also evaluated. The results
show that there were no significant changes in any of the treatment
groups relative to the saline treated group (data not shown),
indicating that both oligonucleotides were very well tolerated.
Example 114: Antisense Inhibition In Vivo by Oligonucleotides
Targeting CFB
[1071] The oligonucleotides listed in Table 123 were tested in a
dose-dependent study for antisense inhibition of human CFB in
mice.
Treatment
[1072] Transgenic mice that express human CFB (Jackson Laboratory,
Bar Harbor, Me.) were injected subcutaneously once with 0.6, 1, 6,
or 18 mg/kg of an oligonucleotide listed in Table 123 or with
saline. Each treatment group consisted of 4 or 5 animals. The mice
were sacrificed 72 hours following the dose to determine the liver
human CFB and cyclophilin mRNA levels using real-time PCR according
to standard protocols. The CFB mRNA levels were normalized to the
cyclophilin levels, and the averages for each treatment group were
used to determine the dose that achieved 50% inhibition of the
human CFB transcript expression (ED.sub.50).sub.n The results are
shown in Table 123.
TABLE-US-00116 TABLE 123 Modified ASOs targeting CFB ED.sub.50 in
ISIS GalNAc.sub.3 liver SEQ No. Sequences (5' to 3') Cluster CM
(mg/kg) ID No. 696844
GalNAc.sub.3-7.sub.a-.sub.o'A.sub.esT.sub.es.sup.mC.sub.es.sup.mC.s-
ub.es.sup.mC.sub.esA.sub.ds.sup.mC.sub.dsG.sub.ds GalNAc.sub.3-7a
PO 0.86 440
.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsT.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.es.sup.mC.sub.esA.sub.esG.sub.es.sup.mC.sub.e
696845
GalNAc.sub.3-7.sub.a-.sub.o'A.sub.esT.sub.eo.sup.mC.sub.eo.sup.mC.s-
ub.eo.sup.mC.sub.eoA.sub.ds.sup.mC.sub.dsG.sub.ds GalNAc.sub.3-7a
PO 0.71 440
.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsT.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.eo.sup.mC.sub.eoA.sub.esG.sub.es.sup.mC.sub.e
698969
GalNAc.sub.3-7.sub.a-.sub.o'A.sub.esT.sub.eo.sup.mC.sub.eo.sup.mC.s-
ub.eo.sup.mC.sub.esA.sub.ds.sup.mC.sub.dsG.sub.ds GalNAc.sub.3-7a
PO 0.51 440
.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsT.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.eo.sup.mC.sub.eoA.sub.esG.sub.es.sup.mC.sub.e
698970
GalNAc.sub.3-7.sub.a-.sub.o'A.sub.esT.sub.es.sup.mC.sub.eo.sup.mC.s-
ub.eo.sup.mC.sub.eoA.sub.ds.sup.mC.sub.dsG.sub.ds GalNAc.sub.3-7a
PO 0.55 440
.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.ds.sup.mC.sub.dsT.sub.dsG.sub.dsT-
.sub.ds.sup.mC.sub.eo.sup.mC.sub.eoA.sub.esG.sub.es.sup.mC.sub.e
The structure of GalNAc.sub.3-7.sub.a was shown previously in
Example 48.
Example 115: Antisense Inhibition of Human Complement Factor B
(CFB) in HepG2 Cells by MOE Gapmers
[1073] Antisense oligonucleotides were designed targeting human
Complement Factor B (CFB) nucleic acid and were tested for their
effects on CFB mRNA in vitro. The antisense oligonucleotides were
tested in a series of experiments that had similar culture
conditions. The results for each experiment are presented in
separate tables shown below. Cultured HepG2 cells at a density of
20,000 cells per well were transfected using electroporation with
4,500 nM antisense oligonucleotide. After a treatment period of
approximately 24 hours, RNA was isolated from the cells and CFB
mRNA levels were measured by quantitative real-time PCR. Human
primer probe set RTS3459 (forward sequence AGTCTCTGTGGCATGGTTTGG,
designated herein as SEQ ID NO: 810; reverse sequence
GGGCGAATGACTGAGATCTTG, designated herein as SEQ ID NO: 811; probe
sequence TACCGATTACCACAAGCAACCATGGCA, designated herein as SEQ ID
NO: 812) was used to measure mRNA levels. CFB mRNA levels were
adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of CFB,
relative to untreated control cells.
[1074] The newly designed chimeric antisense oligonucleotides in
the Tables below were designed as 5-10-5 MOE gapmers. The 5-10-5
MOE gapmers are 20 nucleosides in length, wherein the central gap
segment comprises often 2'-deoxynucleosides and is flanked by wing
segments on the 5' direction and the 3' direction comprising five
nucleosides each. Each nucleoside in the 5' wing segment and each
nucleoside in the 3' wing segment has a 2'-MOE modification. The
internucleoside linkages throughout each gapmer are
phosphorothioate (P.dbd.S) linkages. All cytosine residues
throughout each gapmer are 5-methylcytosines. "Start site"
indicates the 5'-most nucleoside to which the gapmer is targeted in
the human gene sequence. "Stop site" indicates the 3'-most
nucleoside to which the gapmer is targeted human gene sequence.
Each gapmer listed in the Tables below is targeted to either the
human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK
Accession No. NM_001710.5) or the human CFB genomic sequence,
designated herein as SEQ ID NO: 2 (GENBANK Accession No.
NT_007592.15 truncated from nucleotides 31852000 to 31861000), or
both. `n/a` indicates that the antisense oligonucleotide does not
target that particular gene sequence with 100% complementarity.
TABLE-US-00117 TABLE 124 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi- start
stop ID NO site site Region Sequence bition site site NO: 532608 20
39 Exon 1 GCTGAGCTGCCAGTCAAGGA 36 1741 1760 6 532609 26 45 Exon 1
GGCCCCGCTGAGCTGCCAGT 16 1747 1766 7 532610 45 64 Exon 1
CGGAACATCCAAGCGGGAGG 11 1766 1785 8 532611 51 70 Exon 1
CTTTCCCGGAACATCCAAGC 26 1772 1791 9 532612 100 119 Exon 1
ATCTGTGTTCTGGCACCTGC 25 1821 1840 10 532613 148 167 Exon 1
GTCACATTCCCTTCCCCTGC 39 1869 1888 11 532614 154 173 Exon 1
GACCTGGTCACATTCCCTTC 71 1875 1894 12 532615 160 179 Exon 1
GACCTAGACCTGGTCACATT 35 1881 1900 13 532616 166 185 Exon 1
ACTCCAGACCTAGACCTGGT 39 1887 1906 14 532617 172 191 Exon 1
GCTGAAACTCCAGACCTAGA 27 1893 1912 15 532618 178 197 Exon 1
GTCCAAGCTGAAACTCCAGA 29 1899 1918 16 532619 184 203 Exon 1
CTCAGTGTCCAAGCTGAAAC 21 1905 1924 17 532620 246 265 Exon 1
AGGAGAGAAGCTGGGCCTGG 31 1967 1986 18 532621 252 271 Exon 1
GAAGGCAGGAGAGAAGCTGG 25 1973 1992 19 532622 336 355 Exon 1-2
GTGGTGGTCACACCTCCAGA 28 n/a n/a 20 Junction 532623 381 400 Exon 2
CCCTCCAGAGAGCAGGATCC 22 2189 2208 21 532624 387 406 Exon 2
TCTACCCCCTCCAGAGAGCA 37 2195 2214 22 532625 393 412 Exon 2
TTGATCTCTACCCCCTCCAG 30 2201 2220 23 532626 417 436 Exon 2
TGGAGAAGTCGGAAGGAGCC 35 2225 2244 24 532627 423 442 Exon 2
CCCTCTTGGAGAAGTCGGAA 37 2231 2250 25 532628 429 448 Exon 2
GCCTGGCCCTCTTGGAGAAG 0 2237 2256 26 532629 435 454 Exon 2
TCCAGTGCCTGGCCCTCTTG 26 2243 2262 27 532630 458 477 Exon 2
AGAAGCCAGAAGGACACACG 30 2266 2285 28 532631 464 483 Exon 2
ACGGGTAGAAGCCAGAAGGA 43 2272 2291 29 532632 480 499 Exon 2
CGTGTCTGCACAGGGTACGG 57 2288 2307 30 532633 513 532 Exon 2
AGGGTGCTCCAGGACCCCGT 27 2321 2340 31 532634 560 579 Exon 2-3
TTGCTCTGCACTCTGCCTTC 41 n/a n/a 32 Junction 532635 600 619 Exon 3
TATTCCCCGTTCTCGAAGTC 67 2808 2827 33 532636 626 645 Exon 3
CATTGTAGTAGGGAGACCGG 24 2834 2853 34 532637 632 651 Exon 3
CACTCACATTGTAGTAGGGA 49 2840 2859 35 532638 638 657 Exon 3
TCTCATCACTCACATTGTAG 50 2846 2865 36 532639 644 663 Exon 3
AAGAGATCTCATCACTCACA 52 2852 2871 37 532640 650 669 Exon 3
AGTGGAAAGAGATCTCATCA 34 2858 2877 38 532641 656 675 Exon 3
CATAGCAGTGGAAAGAGATC 32 2864 2883 39 532642 662 681 Exon 3
AACCGTCATAGCAGTGGAAA 45 2870 2889 40 532643 668 687 Exon 3
GAGTGTAACCGTCATAGCAG 36 2876 2895 41 532644 674 693 Exon 3
CCCGGAGAGTGTAACCGTCA 30 2882 2901 42 532645 680 699 Exon 3
CAGAGCCCCGGAGAGTGTAA 27 2888 2907 43 532646 686 705 Exon 3
GATTGGCAGAGCCCCGGAGA 20 2894 2913 44 532647 692 711 Exon 3
AGGTGCGATTGGCAGAGCCC 28 2900 2919 45 532648 698 717 Exon 3
CTTGGCAGGTGCGATTGGCA 24 2906 2925 46 532649 704 723 Exon 3
CATTCACTTGGCAGGTGCGA 28 2912 2931 47 532650 729 748 Exon 3
ATCGCTGTCTGCCCACTCCA 44 2937 2956 48 532651 735 754 Exon 3
TCACAGATCGCTGTCTGCCC 44 2943 2962 49 532652 741 760 Exon 3
CCGTTGTCACAGATCGCTGT 27 2949 2968 50 532653 747 766 Exon 3-4
CCCGCTCCGTTGTCACAGAT 28 n/a n/a 51 Junction 532654 753 772 Exon 3-4
CAGTACCCCGCTCCGTTGTC 13 n/a n/a 52 Junction 532655 759 778 Exon 3-4
TTGGAGCAGTACCCCGCTCC 8 n/a n/a 53 Junction 532656 789 808 Exon 4
ACCTTCCTTGTGCCAATGGG 40 3152 3171 54 532657 795 814 Exon 4
CTGCCCACCTTCCTTGTGCC 41 3158 3177 55 532658 818 837 Exon 4
CGCTGTCTTCAAGGCGGTAC 33 3181 3200 56 532659 835 854 Exon 4
GCTGCAGTGGTAGGTGACGC 32 3198 3217 57 532660 841 860 Exon 4
CCCCCGGCTGCAGTGGTAGG 17 3204 3223 58 532661 847 866 Exon 4
GGTAAGCCCCCGGCTGCAGT 28 3210 3229 59 532662 853 872 Exon 4
ACGCAGGGTAAGCCCCCGGC 13 3216 3235 60 532663 859 878 Exon 4
GGAGCCACGCAGGGTAAGCC 33 3222 3241 61 532664 866 885 Exon 4
GCCGCTGGGAGCCACGCAGG 10 3229 3248 62 532665 891 910 Exon 4
CAAGAGCCACCTTCCTGACA 17 3254 3273 63 532666 897 916 Exon 4
CCGCTCCAAGAGCCACCTTC 25 3260 3279 64 532667 903 922 Exon 4
TCCGTCCCGCTCCAAGAGCC 29 3266 3285 65 532668 909 928 Exon 4
GAAGGCTCCGTCCCGCTCCA 14 3272 3291 66 532669 915 934 Exon 4
TGGCAGGAAGGCTCCGTCCC 18 3278 3297 67 532670 921 940 Exon 4-5
GAGTCTTGGCAGGAAGGCTC 20 n/a n/a 68 Junction 532671 927 946 Exon 4-5
ATGAAGGAGTCTTGGCAGGA 14 n/a n/a 69 Junction 532672 956 975 Exon 5
CTTCGGCCACCTCTTGAGGG 45 3539 3558 70 532673 962 981 Exon 5
GGAAAGCTTCGGCCACCTCT 37 3545 3564 71 532674 968 987 Exon 5
AAGACAGGAAAGCTTCGGCC 28 3551 3570 72 532675 974 993 Exon 5
TCAGGGAAGACAGGAAAGCT 16 3557 3576 73 532676 996 1015 Exon 5
TCGACTCCTTCTATGGTCTC 31 3579 3598 74 532677 1033 1052 Exon 5-6
CTTCTGTTGTTCCCCTGGGC 36 n/a n/a 75 Junction 532678 1068 1087 Exon 6
TTCATGGAGCCTGAAGGGTC 19 3752 3771 76 532679 1074 1093 Exon 6
TAGATGTTCATGGAGCCTGA 24 3758 3777 77 532680 1080 1099 Exon 6
ACCAGGTAGATGTTCATGGA 13 3764 3783 78 532681 1086 1105 Exon 6
TCTAGCACCAGGTAGATGTT 20 3770 3789 79 532682 1092 1111 Exon 6
GATCCATCTAGCACCAGGTA 33 3776 3795 80 532683 1098 1117 Exon 6
CTGTCTGATCCATCTAGCAC 44 3782 3801 81 532684 1104 1123 Exon 6
CCAATGCTGTCTGATCCATC 29 3788 3807 82 532685 1129 1148 Exon 6
TTTGGCTCCTGTGAAGTTGC 40 3813 3832 83
TABLE-US-00118 TABLE 125 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi- start
stop ID NO site site Region Sequence bition site site NO: 532686
1135 1154 Exon 6 ACACTTTTTGGCTCCTGTGA 91 3819 3838 84 532687 1141
1160 Exon 6 GACTAGACACTTTTTGGCTC 77 3825 3844 85 532688 1147 1166
Exon 6 TAAGTTGACTAGACACTTTT 70 3831 3850 86 532689 1153 1172 Exon 6
CTCAATTAAGTTGACTAGAC 61 3837 3856 87 532690 1159 1178 Exon 6-7
CACCTTCTCAATTAAGTTGA 57 3843 3862 88 Junction 532691 1165 1184 Exon
6-7 ACTTGCCACCTTCTCAATTA 56 n/a n/a 89 Junction 532692 1171 1190
Exon 6-7 ACCATAACTTGCCACCTTCT 56 n/a n/a 90 Junction 532693 1177
1196 Exon 7 CTTCACACCATAACTTGCCA 56 4153 4172 91 532694 1183 1202
Exon 7 TCTTGGCTTCACACCATAAC 55 4159 4178 92 532695 1208 1227 Exon 7
ATGTGGCATATGTCACTAGA 55 4184 4203 93 532696 1235 1254 Exon 7
CAGACACTTTGACCCAAATT 55 4211 4230 94 532697 1298 1317 Exon 7-8
GGTCTTCATAATTGATTTCA 53 n/a n/a 95 Junction 532698 1304 1323 Exon
7-8 ACTTGTGGTCTTCATAATTG 53 n/a n/a 96 Junction 532699 1310 1329
Exon 7-8 ACTTCAACTTGTGGTCTTCA 52 n/a n/a 97 Junction 532700 1316
1335 Exon 8 TCCCTGACTTCAACTTGTGG 52 4609 4628 98 532701 1322 1341
Exon 8 TGTTAGTCCCTGACTTCAAC 52 4615 4634 99 532702 1328 1347 Exon 8
TCTTGGTGTTAGTCCCTGAC 51 4621 4640 100 532703 1349 1368 Exon 8
TGTACACTGCCTGGAGGGCC 51 4642 4661 101 532704 1355 1374 Exon 8
TCATGCTGTACACTGCCTGG 51 4648 4667 102 532705 1393 1412 Exon 8
GTTCCAGCCTTCAGGAGGGA 50 4686 4705 103 532706 1399 1418 Exon 8
GGTGCGGTTCCAGCCTTCAG 50 4692 4711 104 532707 1405 1424 Exon 8
ATGGCGGGTGCGGTTCCAGC 50 4698 4717 105 532708 1411 1430 Exon 8
GATGACATGGCGGGTGCGGT 49 4704 4723 106 532709 1417 1436 Exon 8
GAGGATGATGACATGGCGGG 49 4710 4729 107 532710 1443 1462 Exon 8-9
CCCATGTTGTGCAATCCATC 48 n/a n/a 108 Junction 532711 1449 1468 Exon
9 TCCCCGCCCATGTTGTGCAA 48 5023 5042 109 532712 1455 1474 Exon 9
ATTGGGTCCCCGCCCATGTT 48 5029 5048 110 532713 1461 1480 Exon 9
ACAGTAATTGGGTCCCCGCC 48 5035 5054 111 532714 1467 1486 Exon 9
TCAATGACAGTAATTGGGTC 47 5041 5060 112 532715 1473 1492 Exon 9
ATCTCATCAATGACAGTAAT 47 5047 5066 113 532716 1479 1498 Exon 9
TCCCGGATCTCATCAATGAC 46 5053 5072 114 532717 1533 1552 Exon 9-10
ACATCCAGATAATCCTCCCT 46 n/a n/a 115 Junction 532718 1539 1558 Exon
9-10 ACATAGACATCCAGATAATC 46 n/a n/a 116 Junction 532719 1545 1564
Exon 9-10 CCAAACACATAGACATCCAG 46 n/a n/a 117 Junction 532720 1582
1601 Exon 10 AGCATTGATGTTCACTTGGT 46 5231 5250 118 532721 1588 1607
Exon 10 AGCCAAAGCATTGATGTTCA 45 5237 5256 119 532722 1594 1613 Exon
10 CTTGGAAGCCAAAGCATTGA 45 5243 5262 120 532723 1600 1619 Exon 10
GTCTTTCTTGGAAGCCAAAG 45 5249 5268 121 532724 1606 1625 Exon 10
CTCATTGTCTTTCTTGGAAG 44 5255 5274 122 532725 1612 1631 Exon 10
ATGTTGCTCATTGTCTTTCT 44 5261 5280 123 532726 1618 1637 Exon 10
GAACACATGTTGCTCATTGT 44 5267 5286 124 532727 1624 1643 Exon 10
GACTTTGAACACATGTTGCT 43 5273 5292 125 532728 1630 1649 Exon 10
ATCCTTGACTTTGAACACAT 43 5279 5298 126 532729 1636 1655 Exon 10
TTCCATATCCTTGACTTTGA 43 5285 5304 127 532730 1642 1661 Exon 10
CAGGTTTTCCATATCCTTGA 42 5291 5310 128 532731 1686 1705 Exon 11
CTCAGAGACTGGCTTTCATC 42 5827 5846 129 532732 1692 1711 Exon 11
CAGAGACTCAGAGACTGGCT 42 5833 5852 130 516252 1698 1717 Exon 11
ATGCCACAGAGACTCAGAGA 42 5839 5858 131 532733 1704 1723 Exon 11
CAAACCATGCCACAGAGACT 41 5845 5864 132 532734 1710 1729 Exon 11
TGTTCCCAAACCATGCCACA 41 5851 5870 133 532735 1734 1753 Exon 11
TTGTGGTAATCGGTACCCTT 41 5875 5894 134 532736 1740 1759 Exon 11
GGTTGCTTGTGGTAATCGGT 40 5881 5900 135 532737 1746 1765 Exon 11
TGCCATGGTTGCTTGTGGTA 40 5887 5906 136 532738 1752 1771 Exon 11
TTGGCCTGCCATGGTTGCTT 40 5893 5912 137 532739 1758 1777 Exon 11
GAGATCTTGGCCTGCCATGG 38 5899 5918 138 532740 1803 1822 Exon 12
ACAGCCCCCATACAGCTCTC 38 6082 6101 139 532741 1809 1828 Exon 12
GACACCACAGCCCCCATACA 38 6088 6107 140 532742 1815 1834 Exon 12
TACTCAGACACCACAGCCCC 38 6094 6113 141 532743 1821 1840 Exon 12
ACAAAGTACTCAGACACCAC 37 6100 6119 142 532744 1827 1846 Exon 12
GTCAGCACAAAGTACTCAGA 37 6106 6125 143 532745 1872 1891 Exon 12
TTGATTGAGTGTTCCTTGTC 36 6151 6170 144 532746 1878 1897 Exon 12
CTGACCTTGATTGAGTGTTC 35 6157 6176 145 532747 1909 1928 Exon 13
TATCTCCAGGTCCCGCTTCT 35 6403 6422 146 532748 1967 1986 Exon 13
GAATTCCTGCTTCTTTTTTC 32 6461 6480 147 532749 1973 1992 Exon 13
ATTCAGGAATTCCTGCTTCT 32 6467 6486 148 532750 1979 1998 Exon 13
CATAAAATTCAGGAATTCCT 32 6473 6492 149 532751 1985 2004 Exon 13
CATAGTCATAAAATTCAGGA 31 6479 6498 150 532752 2006 2025 Exon 13
TGAGCTTGATCAGGGCAACG 30 6500 6519 151 532753 2012 2031 Exon 13
TATTCTTGAGCTTGATCAGG 30 6506 6525 152 532754 2048 2067 Exon 13-14
GACAAATGGGCCTGATAGTC 30 n/a n/a 153 Junction 532755 2070 2089 Exon
14 GTTGTTCCCTCGGTGCAGGG 29 6659 6678 154 532756 2076 2095 Exon 14
GCTCGAGTTGTTCCCTCGGT 28 6665 6684 155 532757 2082 2101 Exon 14
CTCAAAGCTCGAGTTGTTCC 28 6671 6690 156 532758 2088 2107 Exon 14
GGAAGCCTCAAAGCTCGAGT 25 6677 6696 157 532759 2094 2113 Exon 14
GTTGGAGGAAGCCTCAAAGC 23 6683 6702 158 532760 2100 2119 Exon 14
GTGGTAGTTGGAGGAAGCCT 23 6689 6708 159 532761 2106 2125 Exon 14
TGGCAAGTGGTAGTTGGAGG 18 6695 6714 160 532762 2112 2131 Exon 14
TGTTGCTGGCAAGTGGTAGT 14 6701 6720 161
TABLE-US-00119 TABLE 126 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi- start
stop ID NO site site Region Sequence bition site site NO: 532812
n/a n/a Exon 1 TCCAGCTCACTCCCCTGTTG 19 1593 1612 162 532813 n/a n/a
Exon 1 TAAGGATCCAGCTCACTCCC 40 1599 1618 163 532814 n/a n/a Exon 1
CAGAAATAAGGATCCAGCTC 39 1605 1624 164 532815 n/a n/a Exon 1
AGGGACCAGAAATAAGGATC 0 1611 1630 165 532816 n/a n/a Exon 1
CCACTTAGGGACCAGAAATA 27 1617 1636 166 532817 n/a n/a Exon 1
TCCAGGACTCTCCCCTTCAG 39 1682 1701 167 532818 n/a n/a Exon 1
AAGTCCCACCCTTTGCTGCC 15 1707 1726 168 532819 n/a n/a Exon 1
CTGCAGAAGTCCCACCCTTT 26 1713 1732 169 532820 n/a n/a Exon 1
CAGAAACTGCAGAAGTCCCA 8 1719 1738 170 532821 n/a n/a Exon 2-
AACCTCTGCACTCTGCCTTC 39 2368 2387 171 Intron 2 532822 n/a n/a Exon
2- CCCTCAAACCTCTGCACTCT 3 2374 2393 172 Intron 2 532823 n/a n/a
Exon 2- TCATTGCCCTCAAACCTCTG 19 2380 2399 173 Intron 2 532824 n/a
n/a Intron 2 CCACACTCATTGCCCTCAAA 37 2386 2405 174 532825 n/a n/a
Intron 2 CACTGCCCACACTCATTGCC 23 2392 2411 175 532826 n/a n/a
Intron 2 TTAGGCCACTGCCCACACTC 15 2398 2417 176 532827 n/a n/a
Intron 2 CTAGTCCTGACCTTGCTGCC 28 2436 2455 177 532828 n/a n/a
Intron 2 CTCATCCTAGTCCTGACCTT 25 2442 2461 178 532829 n/a n/a
Intron 2 CCTAGTCTCATCCTAGTCCT 23 2448 2467 179 532830 n/a n/a
Intron 2 ACCCTGCCTAGTCTCATCCT 30 2454 2473 180 532831 n/a n/a
Intron 2 CTTGTCACCCTGCCTAGTCT 34 2460 2479 181 532832 n/a n/a
Intron 2 GCCCACCTTGTCACCCTGCC 36 2466 2485 182 532833 n/a n/a
Intron 2 CCTAAAACTGCTCCTACTCC 9 2492 2511 183 532834 n/a n/a Intron
4 GAGTCAGAAATGAGGTCAAA 19 3494 3513 184 532835 n/a n/a Intron
CCCTACTCCCATTTCACCTT 16 5971 5990 185 11 532836 n/a n/a Intron 8-
TGTTGTGCAATCCTGCAGAA 25 5013 5032 186 Exon 9 532837 n/a n/a Intron
1 AAAGGCTGATGAAGCCTGGC 18 2123 2142 187 532838 n/a n/a Intron 7
CCTTTGACCACAAAGTGGCC 21 4461 4480 188 532839 n/a n/a Intron
AGGTACCACCTCTTTGTGGG 29 6362 6381 189 12 532840 n/a n/a Intron 1-
TGGTGGTCACACCTGAAGAG 34 2143 2162 190 Exon 2 532763 2133 2152 Exon
GCAGGGAGCAGCTCTTCCTT 40 n/a n/a 191 14-15 Junction 532764 2139 2158
Exon 15 TCCTGTGCAGGGAGCAGCTC 28 6927 6946 192 532765 2145 2164 Exon
15 TTGATATCCTGTGCAGGGAG 41 6933 6952 193 532766 2151 2170 Exon 15
AGAGCTTTGATATCCTGTGC 36 6939 6958 194 532767 2157 2176 Exon 15
ACAAACAGAGCTTTGATATC 33 6945 6964 195 532768 2163 2182 Exon 15
TCAGACACAAACAGAGCTTT 41 6951 6970 196 532769 2169 2188 Exon 15
TCCTCCTCAGACACAAACAG 49 6957 6976 197 532770 2193 2212 Exon 15
ACCTCCTTCCGAGTCAGCTT 61 6981 7000 198 532771 2199 2218 Exon 15
ATGTAGACCTCCTTCCGAGT 39 6987 7006 199 532772 2205 2224 Exon 15
TTCTTGATGTAGACCTCCTT 30 6993 7012 200 532773 2211 2230 Exon 15
TCCCCATTCTTGATGTAGAC 31 6999 7018 201 532774 2217 2236 Exon
TTCTTATCCCCATTCTTGAT 36 n/a n/a 202 15-16 Junction 532775 2223 2242
Exon CTGCCTTTCTTATCCCCATT 56 n/a n/a 203 15-16 Junction 532776 2229
2248 Exon TCACAGCTGCCTTTCTTATC 33 n/a n/a 204 15-16 Junction 532777
2235 2254 Exon 16 TCTCTCTCACAGCTGCCTTT 38 7119 7138 205 532778 2241
2260 Exon 16 TGAGCATCTCTCTCACAGCT 51 7125 7144 206 532779 2247 2266
Exon 16 GCATATTGAGCATCTCTCTC 39 7131 7150 207 532780 2267 2286 Exon
16 TGACTTTGTCATAGCCTGGG 56 7151 7170 208 532781 2273 2292 Exon 16
TGTCCTTGACTTTGTCATAG 36 7157 7176 209 532782 2309 2328 Exon 16
CAGTACAAAGGAACCGAGGG 30 7193 7212 210 532783 2315 2334 Exon 16
CTCCTCCAGTACAAAGGAAC 21 7199 7218 211 532784 2321 2340 Exon 16
GACTCACTCCTCCAGTACAA 31 7205 7224 212 532785 2327 2346 Exon 16
CATAGGGACTCACTCCTCCA 30 7211 7230 213 532786 2333 2352 Exon 16
GGTCAGCATAGGGACTCACT 31 7217 7236 214 532787 2352 2371 Exon
TCACCTCTGCAAGTATTGGG 42 7236 7255 215 16-17 Junction 532788 2358
2377 Exon CCAGAATCACCTCTGCAAGT 32 n/a n/a 216 16-17 Junction 532789
2364 2383 Exon GGGCCGCCAGAATCACCTCT 35 n/a n/a 217 16-17 Junction
532790 2382 2401 Exon 17 CTCTTGTGAACTATCAAGGG 33 7347 7366 218
532791 2388 2407 Exon 17 CGACTTCTCTTGTGAACTAT 52 7353 7372 219
532792 2394 2413 Exon 17 ATGAAACGACTTCTCTTGTG 16 7359 7378 220
532793 2400 2419 Exon ACTTGAATGAAACGACTTCT 45 7365 7384 221 17-18
Junction 532794 2406 2425 Exon ACACCAACTTGAATGAAACG 18 n/a n/a 222
17-18 Junction 532795 2427 2446 Exon 18 TCCACTACTCCCCAGCTGAT 30
7662 7681 223 532796 2433 2452 Exon 18 CAGACATCCACTACTCCCCA 38 7668
7687 224 532797 2439 2458 Exon 18 TTTTTGCAGACATCCACTAC 35 7674 7693
225 532798 2445 2464 Exon 18 TTCTGGTTTTTGCAGACATC 45 7680 7699 226
532799 2451 2470 Exon 18 TGCCGCTTCTGGTTTTTGCA 47 7686 7705 227
532800 2457 2476 Exon 18 TGCTTTTGCCGCTTCTGGTT 61 7692 7711 228
532801 2463 2482 Exon 18 GGTACCTGCTTTTGCCGCTT 47 7698 7717 229
532802 2469 2488 Exon 18 TGAGCAGGTACCTGCTTTTG 31 7704 7723 230
532803 2517 2536 Exon 18 TTCAGCCAGGGCAGCACTTG 41 7752 7771 231
532804 2523 2542 Exon 18 TTCTCCTTCAGCCAGGGCAG 44 7758 7777 232
532805 2529 2548 Exon 18 TGGAGTTTCTCCTTCAGCCA 46 7764 7783 233
532806 2535 2554 Exon 18 TCATCTTGGAGTTTCTCCTT 49 7770 7789 234
532807 2541 2560 Exon 18 AAATCCTCATCTTGGAGTTT 30 7776 7795 235
532808 2547 2566 Exon 18 AAACCCAAATCCTCATCTTG 20 7782 7801 236
532809 2571 2590 Exon 18 GTCCAGCAGGAAACCCCTTA 65 7806 7825 237
532810 2577 2596 Exon 18 GCCCCTGTCCAGCAGGAAAC 74 7812 7831 238
532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 96 7834 7853 239
TABLE-US-00120 TABLE 127 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi- start
stop ID NO site site Region Sequence bition site site NO: 532841
n/a n/a Intron 6- AACTTGCCACCTGTGGGTGA 4142 4161 11 240 Exon 7
532842 n/a n/a Exon 15- TCACCTTATCCCCATTCTTG 7007 7026 16 241
Intron 15 532843 n/a n/a Intron 11 TCAACTTTCACAAACCACCA 6015 6034
19 242 532844 n/a n/a Intron 16- CCGCCAGAATCACCTGCAAG 7326 7345 33
243 Exon 17 532845 n/a n/a Intron 10 AGGAGGAATGAAGAAGGCTT 5431 5450
29 244 532846 n/a n/a Intron 13 GCCTTTCCTCAGGGATCTGG 6561 6580 26
245 532847 n/a n/a Intron 4 AAATGTCTGGGAGTGTCAGG 3477 3496 18 246
532848 n/a n/a Intron 15 GCCTAGAGTGCCTCCTTAGG 7038 7057 20 247
532849 n/a n/a Intron 17 GGCATCTCCCCAGATAGGAA 7396 7415 16 248
532850 n/a n/a Intron 6 AGGGAGCTAGTCCTGGAAGA 3906 3925 14 249
532851 n/a n/a Intron 1- ACACCTGAAGAGAAAGGCTG 2135 2154 6 250 Exon
2 532852 n/a n/a Intron 7 CCCTTTGACCACAAAGTGGC 4462 4481 25 251
532853 n/a n/a Intron 7 GCCCTCAAGGTAGTCTCATG 4354 4373 26 252
532854 n/a n/a Intron 6 AAGGGAAGGAGGACAGAATA 3977 3996 18 253
532855 n/a n/a Intron 1 AAAGGCCAAGGAGGGATGCT 2099 2118 9 254 532856
n/a n/a Exon 8- AGAGGTCCCTTCTGACCATC 4736 4755 4 255 Intron 8
532857 n/a n/a Intron 8 GCTGGGACAGGAGAGAGGTC 4749 4768 0 256 532858
n/a n/a Intron 4 TCAAATGTCTGGGAGTGTCA 3479 3498 13 257 532859 n/a
n/a Intron 10 AGAAGGAGAATGTGCTGAAA 5801 5820 20 258 532860 n/a n/a
Intron 17 TGCTGACCACTTGGCATCTC 7408 7427 20 259 532861 n/a n/a
Intron 11 CAACTTTCACAAACCACCAT 6014 6033 18 260 532862 n/a n/a
Intron 10 AGCTCTGTGATTCTAAGGTT 5497 5516 15 261 532863 n/a n/a
Intron 6- CCACCTGTGGGTGAGGAGAA 4136 4155 16 262 Exon 7 532864 n/a
n/a Exon 17- GAGGACTCACTTGAATGAAA 7373 7392 21 263 Intron 17 532865
n/a n/a Intron 6 TGGAATGATCAGGGAGCTAG 3916 3935 30 264 532866 n/a
n/a Intron 5 GTCCCTTCTCCATTTTCCCC 3659 3678 26 265 532867 n/a n/a
Intron 7 TCAACTTTTTAAGTTAATCA 4497 4516 14 266 532868 n/a n/a
Intron 6 GGGTGAGGAGAACAAGGCGC 4128 4147 21 267 532869 n/a n/a
Intron 7 CTTCCAAGCCATCTTTTAAC 4553 4572 5 268 532870 n/a n/a Exon
17- AGGACTCACTTGAATGAAAC 7372 7391 18 269 Intron 17 532871 n/a n/a
Intron 10 TTCCAGGCAACTAGAGCTTC 5412 5431 15 270 532872 n/a n/a Exon
1 CAGAGTCCAGCCACTGTTTG 1557 1576 13 271 532873 n/a n/a Intron 17-
CCAACCTGCAGAGGCAGTGG 7638 7657 23 272 Exon 18 532874 n/a n/a Intron
16 TGCAAGGAGAGGAGAAGCTG 7312 7331 10 273 532875 n/a n/a Exon 9-
CTAGGCAGGTTACTCACCCA 5120 5139 21 274 Intron 9 532876 n/a n/a
Intron 6- CACCATAACTTGCCACCTGT 4148 4167 41 275 Exon 7 532877 n/a
n/a Intron 12 TAGGTACCACCTCTTTGTGG 6363 6382 27 276 532878 n/a n/a
Intron 11 CTTGACCTCACCTCCCCCAA 5954 5973 13 277 532879 n/a n/a
Intron 12 CCACCTCTTTGTGGGCAGCT 6357 6376 33 278 532880 n/a n/a
Intron 11 TTCACAAACCACCATCTCTT 6009 6028 8 279 532881 n/a n/a Exon
3- TTCTCACCTCCGTTGTCACA 2958 2977 17 280 Intron 3 532882 n/a n/a
Intron 12 GAAAGTGGGAGGTGTTGCCT 6225 6244 19 281 532883 n/a n/a
Intron 1 ACAGCAGGAAGGGAAGGTTA 2075 2094 34 282 532884 n/a n/a
Intron 17 CATGCTGACCACTTGGCATC 7410 7429 18 283 532885 n/a n/a Exon
4- GGTCACCTTGGCAGGAAGGC 3286 3305 0 284 Intron 4 532886 n/a n/a
Intron 8 GTATAGTGTTACAAGTGGAC 4804 4823 13 285 532887 n/a n/a
Intron 7 GGACTTCCCTTTGACCACAA 4468 4487 18 286 532888 n/a n/a
Intron 11 TCACCTTGACCTCACCTCCC 5958 5977 20 287 532889 n/a n/a
Intron 15 TAGAGTGCCTCCTTAGGATG 7035 7054 27 288 532890 n/a n/a
Intron 7 TGACTTCAACTTGTGGTCTG 4605 4624 16 289 532891 n/a n/a
Intron 10 CAGAGAAGGAGAATGTGCTG 5804 5823 25 290 532892 n/a n/a
Intron 14- AGGGAGCAGCTCTTCCTCTG 6919 6938 47 291 Exon 15 532893 n/a
n/a Intron 5- TGTTCCCCTGGGTGCCAGGA 3710 3729 24 292 Exon 6 532894
n/a n/a Intron 10 GGCCTGGCTGTTTTCAAGCC 5612 5631 15 293 532895 n/a
n/a Intron 10- GACTGGCTTTCATCTGGCAG 5821 5840 25 294 Exon 11 532896
n/a n/a Intron 10 GAAGGCTTTCCAGGCAACTA 5419 5438 19 295 532897 n/a
n/a Exon 17- TCACTTGAATGAAACGACTT 7367 7386 11 296 Intron 17 532898
n/a n/a Intron 1 GGCCCCAAAAGGCCAAGGAG 2106 2125 5 297 532899 n/a
n/a Intron 16- AATCACCTGCAAGGAGAGGA 7319 7338 19 298 Exon 17 532900
n/a n/a Intron 12 GACCTTCAGTTGCATCCTTA 6183 6202 25 299 532901 n/a
n/a Intron 1 TGATGAAGCCTGGCCCCAAA 2117 2136 0 300 532902 n/a n/a
Intron 12 TAGAAAGTGGGAGGTGTTGC 6227 6246 0 301 532903 n/a n/a
Intron 12 CCCATCCCTGACTGGTCTGG 6295 6314 14 302 532904 n/a n/a
Intron 8 CCATGGGTATAGTGTTACAA 4810 4829 13 303 532905 n/a n/a
Intron 2 GTGTTCTCTTGACTTCCAGG 2586 2605 23 304 532906 n/a n/a
Intron 13 GGCCTGCTCCTCACCCCAGT 6597 6616 27 305 532907 n/a n/a
Intron 10 GAGGCCTGGCTGTTTTCAAG 5614 5633 32 306 532908 n/a n/a Exon
1 GACTCTCCCCTTCAGTACCT 1677 1696 16 307 532909 n/a n/a Intron 8
CATGGGTATAGTGTTACAAG 4809 4828 10 308 532910 n/a n/a Intron 10
GAAGGAGAATGTGCTGAAAA 5800 5819 0 309 532911 n/a n/a Intron 7
TCACCTGGTCTTCCAAGCCA 4562 4581 0 310 532912 n/a n/a Intron 17
CTCCCCAGATAGGAAAGGGA 7391 7410 0 311 532913 n/a n/a Exon 17-
GGACTCACTTGAATGAAACG 7371 7390 0 312 Intron 17 532914 n/a n/a
Intron 16- GGCCGCCAGAATCACCTGCA 7328 7347 30 313 Exon 17 532915 n/a
n/a Exon 17- CTCACTTGAATGAAACGACT 7368 7387 22 314 Intron 17 532916
n/a n/a Intron 13 CTTTCCCAGCCTTTCCTCAG 6569 6588 28 315 532918 n/a
n/a Intron 12 AGAAAGTGGGAGGTGTTGCC 6226 6245 3 316 532917 2604 2623
Exon 18 GTCGCAGCTGTTTTAATTCA 7839 7858 90 317
TABLE-US-00121 TABLE 128 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi- start
stop ID NO site site Region Sequence bition site site NO: 532919
n/a n/a Exon 1 CCAGGACTCTCCCCTTCAGT 1681 1700 4 318 532920 n/a n/a
Intron 6 AGGGAAGGAGGACAGAATAG 3976 3995 25 319 532921 n/a n/a
Intron 4 GAAATGAGGTCAAATGTCTG 3488 3507 30 320 532922 n/a n/a
Intron 4 GGAGAGTCAGAAATGAGGTC 3497 3516 25 321 532923 n/a n/a
Intron 12 GTAGAAAGTGGGAGGTGTTG 6228 6247 26 322 532924 n/a n/a
Intron 10 TAGAAAGATCTCTGAAGTGC 5521 5540 24 323 532925 n/a n/a
Intron 13 CTGCTCCTCACCCCAGTCCT 6594 6613 26 324 532926 n/a n/a
Intron 11 CTACTGGGATTCTGTGCTTA 5927 5946 30 325 532927 n/a n/a
Intron 1 CCCAAAAGGCCAAGGAGGGA 2103 2122 13 326 532928 n/a n/a
Intron 17 TGACCACTTGGCATCTCCCC 7405 7424 27 327 532929 n/a n/a
Intron 16- CCTGCAAGGAGAGGAGAAGC 7314 7333 29 328 Exon 17 532930 n/a
n/a Exon 16- CTCTCACCTCTGCAAGTATT 7239 7258 44 329 Intron 16 532931
n/a n/a Intron 1 CCCCAAAAGGCCAAGGAGGG 2104 2123 21 330 532932 n/a
n/a Intron 7 GTCTTCCAAGCCATCTTTTA 4555 4574 20 331 532933 n/a n/a
Intron 8 GTTACAAGTGGACTTAAGGG 4797 4816 30 332 532934 n/a n/a
Intron 8- CCCATGTTGTGCAATCCTGC 5017 5036 30 333 Exon 9 532935 n/a
n/a Intron 15 GAGGTGGGAAGCATGGAGAA 7091 7110 17 334 532936 n/a n/a
Intron 14 TGCTCCCACCACTGTCATCT 6874 6893 21 335 532937 n/a n/a Exon
9- AGGCAGGTTACTCACCCAGA 5118 5137 18 336 Intron 9 532938 n/a n/a
Intron 11 TACTGGGATTCTGTGCTTAC 5926 5945 15 337 532939 n/a n/a
Intron 13 GCCTTTCCCAGCCTTTCCTC 6571 6590 27 338 532940 n/a n/a
Intron 8- GTGCAATCCTGCAGAAGAGA 5009 5028 21 339 Exon 9 532941 n/a
n/a Intron 8 ACAGGAGAGAGGTCCCTTCT 4743 4762 20 340 532942 n/a n/a
Intron 10 CCCAAAAGGAGAAAGGGAAA 5717 5736 14 341 532943 n/a n/a
Intron 2 AAGCCCAGGGTAAATGCTTA 2557 2576 32 342 532944 n/a n/a
Intron 1 GATGAAGCCTGGCCCCAAAA 2116 2135 22 343 532945 n/a n/a
Intron 10 TGGCAGAGAAGGAGAATGTG 5807 5826 22 344 532946 n/a n/a
Intron 13 TTCCCAGCCTTTCCTCAGGG 6567 6586 35 345 532947 n/a n/a
Intron 10 GGCAGAGAAGGAGAATGTGC 5806 5825 30 346 532948 n/a n/a
Intron 10 ACAGTGCCAGGAAACAAGAA 5471 5490 25 347 532949 n/a n/a Exon
9- TAGGCAGGTTACTCACCCAG 5119 5138 22 348 Intron 9 532950 n/a n/a
Intron 2 TTCTCTTGACTTCCAGGGCT 2583 2602 22 349 532951 n/a n/a
Intron 13 CCTGCTCCTCACCCCAGTCC 6595 6614 16 350 532953 n/a n/a
Intron 7 TCCCACTAACCTCCATTGCC 4422 4441 14 351 532954 n/a n/a
Intron 7 TTCCCTTTGACCACAAAGTG 4464 4483 16 352 532955 n/a n/a
Intron 9 CTGGGTCCTAGGCAGGTTAC 5127 5146 30 353 532956 n/a n/a
Intron 10 TCCAGGCAACTAGAGCTTCA 5411 5430 20 354 532957 n/a n/a
Intron 8- GCCCATGTTGTGCAATCCTG 5018 5037 45 355 Exon 9 532958 n/a
n/a Intron 7 GGTTCCCACTAACCTCCATT 4425 4444 18 356 532959 n/a n/a
Intron 3 AGGTAGAGAGCAAGAGTTAC 3052 3071 26 357 532960 n/a n/a
Intron 7 CCACTAACCTCCATTGCCCA 4420 4439 10 358 532961 n/a n/a
Intron 11 TCACAAACCACCATCTCTTA 6008 6027 40 359 532962 n/a n/a Exon
9- TACTCACCCAGATAATCCTC 5110 5129 27 360 Intron 9 532963 n/a n/a
Intron 13 TGCTCCTCACCCCAGTCCTC 6593 6612 24 361 532964 n/a n/a
Intron 15- TCTCACAGCTGCCTTTCTGT 7115 7134 25 362 Exon 16 532965 n/a
n/a Exon 17- GAAAGGGAGGACTCACTTGA 7379 7398 11 363 Intron 17 532966
n/a n/a Intron 7 CCATCTTTTAACCCCAGAGA 4545 4564 18 364 532967 n/a
n/a Intron 13 TCCTCACCCCAGTCCTCCAG 6590 6609 27 365 532968 n/a n/a
Intron 10 CTGGCAGAGAAGGAGAATGT 5808 5827 15 366 532969 n/a n/a
Intron 17 TCTCCCCAGATAGGAAAGGG 7392 7411 23 367 532970 n/a n/a
Intron 14 ACTTCAGCTGCTCCCACCAC 6882 6901 18 368 532971 n/a n/a
Intron 1 GACAGCAGGAAGGGAAGGTT 2076 2095 13 369 532972 n/a n/a
Intron 13- GGAGACAAATGGGCCTATAA 6640 6659 33 370 Exon 14 532973 n/a
n/a Intron 14 CTGCTCCCACCACTGTCATC 6875 6894 11 371 532974 n/a n/a
Intron 10 AGGAATGAAGAAGGCTTTCC 5428 5447 21 372 532975 n/a n/a
Intron 14 GGGATCTCATCCTTATCCTC 6741 6760 31 373 532976 n/a n/a
Intron 9 GTGCTGGGTCCTAGGCAGGT 5130 5149 16 374 532977 n/a n/a
Intron 1 CAAAAGGCCAAGGAGGGATG 2101 2120 14 375 532978 n/a n/a
Intron 17 CCATGCTGACCACTTGGCAT 7411 7430 20 376 532979 n/a n/a
Intron 8 GGAGGCTGGGACAGGAGAGA 4753 4772 25 377 532980 n/a n/a
Intron 14- GGAGCAGCTCTTCCTCTGGA 6917 6936 36 378 Exon 15 532981 n/a
n/a Exon 3- TCTCACCTCCGTTGTCACAG 2957 2976 20 379 Intron 3 532982
n/a n/a Intron 13 CAGTCCTCCAGCCTTTCCCA 6581 6600 21 380 532983 n/a
n/a Intron 13 AGTCCTCCAGCCTTTCCCAG 6580 6599 22 381 532984 n/a n/a
Intron 4- TGAAGGAGTCTGGGAGAGTC 3509 3528 12 382 Exon 5 532985 n/a
n/a Intron 16- CAGAATCACCTGCAAGGAGA 7322 7341 20 383 Exon 17 532986
n/a n/a Exon 17- TAGGAAAGGGAGGACTCACT 7382 7401 3 384 Intron 17
532987 n/a n/a Exon 4- ACCTTGGCAGGAAGGCTCCG 3282 3301 12 385 Intron
4 532988 n/a n/a Intron 13- GAGACAAATGGGCCTATAAA 6639 6658 15 386
Exon 14 532989 n/a n/a Intron 1 CTGAAGAGAAAGGCTGATGA 2131 2150 17
387 532990 n/a n/a Intron 6 AATGATCAGGGAGCTAGTCC 3913 3932 30 388
532991 n/a n/a Intron 17 CTTAGCTGACCTAAAGGAAT 7557 7576 22 389
532992 n/a n/a Intron 8 TGGGTATAGTGTTACAAGTG 4807 4826 17 390
532993 n/a n/a Intron 1 TGAAGAGAAAGGCTGATGAA 2130 2149 19 391
532994 n/a n/a Intron 8 GTGTTACAAGTGGACTTAAG 4799 4818 25 392
532995 n/a n/a Intron 6 ACCTGTGGGTGAGGAGAACA 4134 4153 24 393
532996 n/a n/a Exon 9- TCACCCAGATAATCCTCCCT 5107 5126 36 394 Intron
9 532952 2608 2627 Exon 18 TGTTGTCGCAGCTGTTTTAA 7843 7862 90
395
Example 116: Antisense Inhibition of Human Complement Factor B
(CFB) in HepG2 Cells by MOE Gapmers
[1075] Additional antisense oligonucleotides were designed
targeting human Complement Factor B (CFB) nucleic acid and were
tested for their effects on CFB mRNA in vitro. Cultured HepG2 cells
at a density of 20,000 cells per well were transfected using
electroporation with 4,500 nM antisense oligonucleotide. After a
treatment period of approximately 24 hours, RNA was isolated from
the cells and CFB mRNA levels were measured by quantitative
real-time PCR. Human primer probe set RTS3460_MGB (forward sequence
CGAAGCAGCTCAATGAAATCAA, designated herein as SEQ ID NO: 813;
reverse sequence TGCCTGGAGGGCCTTCTT, designated herein as SEQ ID
NO: 814; probe sequence AGACCACAAGTTGAAGTC, designated herein as
SEQ ID NO: 815) was used to measure mRNA levels. CFB mRNA levels
were adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of CFB,
relative to untreated control cells.
[1076] The newly designed chimeric antisense oligonucleotides in
the Tables below were designed as 5-10-5 MOE gapmers. The 5-10-5
MOE gapmers are 20 nucleosides in length, wherein the central gap
segment comprises often 2'-deoxynucleosides and is flanked by wing
segments on the 5' direction and the 3' direction comprising five
nucleosides each. Each nucleoside in the 5' wing segment and each
nucleoside in the 3' wing segment has a 2'-MOE modification. The
internucleoside linkages throughout each gapmer are
phosphorothioate (P.dbd.S) linkages. All cytosine residues
throughout each gapmer are 5-methylcytosines. "Start site"
indicates the 5'-most nucleoside to which the gapmer is targeted in
the human gene sequence. "Stop site" indicates the 3'-most
nucleoside to which the gapmer is targeted human gene sequence.
Each gapmer listed in the Tables below is targeted to either the
human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK
Accession No. NM_001710.5) or the human CFB genomic sequence,
designated herein as SEQ ID NO: 2 (GENBANK Accession No.
NT_007592.15 truncated from nucleotides 31852000 to 31861000), or
both. `n/a` indicates that the antisense oligonucleotide does not
target that particular gene sequence with 100% complementarity.
TABLE-US-00122 TABLE 129 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi- start
stop ID NO site site Region Sequence bition site site NO: 532686
1135 1154 Exon 6 ACACTTTTTGGCTCCTGTGA 48 3819 3838 84 532687 1141
1160 Exon 6 GACTAGACACTTTTTGGCTC 63 3825 3844 85 532688 1147 1166
Exon 6 TAAGTTGACTAGACACTTTT 47 3831 3850 86 532689 1153 1172 Exon 6
CTCAATTAAGTTGACTAGAC 57 3837 3856 87 532690 1159 1178 Exon 6-7
CACCTTCTCAATTAAGTTGA 49 3843 3862 88 Junction 532691 1165 1184 Exon
6-7 ACTTGCCACCTTCTCAATTA 33 n/a n/a 89 Junction 532692 1171 1190
Exon 6-7 ACCATAACTTGCCACCTTCT 67 n/a n/a 90 Junction 532693 1177
1196 Exon 7 CTTCACACCATAACTTGCCA 56 4153 4172 91 532694 1183 1202
Exon 7 TCTTGGCTTCACACCATAAC 50 4159 4178 92 532695 1208 1227 Exon 7
ATGTGGCATATGTCACTAGA 53 4184 4203 93 532696 1235 1254 Exon 7
CAGACACTTTGACCCAAATT 52 4211 4230 94 532697 1298 1317 Exon 7-8
GGTCTTCATAATTGATTTCA 59 n/a n/a 95 Juncion 532698 1304 1323 Exon
7-8 ACTTGTGGTCTTCATAATTG 52 n/a n/a 96 Juncion 532699 1310 1329
Exon 7-8 ACTTCAACTTGTGGTCTTCA 85 n/a n/a 97 Juncion 532700 1316
1335 Exon 8 TCCCTGACTTCAACTTGTGG 96 4609 4628 98 532701 1322 1341
Exon 8 TGTTAGTCCCTGACTTCAAC 56 4615 4634 99 532702 1328 1347 Exon 8
TCTTGGTGTTAGTCCCTGAC 86 4621 4640 100 532703 1349 1368 Exon 8
TGTACACTGCCTGGAGGGCC 35 4642 4661 101 532704 1355 1374 Exon 8
TCATGCTGTACACTGCCTGG 12 4648 4667 102 532705 1393 1412 Exon 8
GTTCCAGCCTTCAGGAGGGA 27 4686 4705 103 532706 1399 1418 Exon 8
GGTGCGGTTCCAGCCTTCAG 67 4692 4711 104 532707 1405 1424 Exon 8
ATGGCGGGTGCGGTTCCAGC 26 4698 4717 105 532708 1411 1430 Exon 8
GATGACATGGCGGGTGCGGT 28 4704 4723 106 532709 1417 1436 Exon 8
GAGGATGATGACATGGCGGG 6 4710 4729 107 532710 1443 1462 Exon 8-9
CCCATGTTGTGCAATCCATC 35 n/a n/a 108 Junction 532711 1449 1468 Exon
9 TCCCCGCCCATGTTGTGCAA 28 5023 5042 109 532712 1455 1474 Exon 9
ATTGGGTCCCCGCCCATGTT 19 5029 5048 110 532713 1461 1480 Exon 9
ACAGTAATTGGGTCCCCGCC 29 5035 5054 111 532714 1467 1486 Exon 9
TCAATGACAGTAATTGGGTC 49 5041 5060 112 532715 1473 1492 Exon 9
ATCTCATCAATGACAGTAAT 45 5047 5066 113 532716 1479 1498 Exon 9
TCCCGGATCTCATCAATGAC 54 5053 5072 114 532717 1533 1552 Exon 9-
ACATCCAGATAATCCTCCCT 22 n/a n/a 115 10 Junction 532718 1539 1558
Exon 9- ACATAGACATCCAGATAATC 8 n/a n/a 116 10 Junction 532719 1545
1564 Exon 9- CCAAACACATAGACATCCAG 30 n/a n/a 117 10 Junction 532720
1582 1601 Exon 10 AGCATTGATGTTCACTTGGT 62 5231 5250 118 532721 1588
1607 Exon 10 AGCCAAAGCATTGATGTTCA 46 5237 5256 119 532722 1594 1613
Exon 10 CTTGGAAGCCAAAGCATTGA 35 5243 5262 120 532723 1600 1619 Exon
10 GTCTTTCTTGGAAGCCAAAG 43 5249 5268 121 532724 1606 1625 Exon 10
CTCATTGTCTTTCTTGGAAG 40 5255 5274 122 532725 1612 1631 Exon 10
ATGTTGCTCATTGTCTTTCT 49 5261 5280 123 532726 1618 1637 Exon 10
GAACACATGTTGCTCATTGT 68 5267 5286 124 532727 1624 1643 Exon 10
GACTTTGAACACATGTTGCT 54 5273 5292 125 532728 1630 1649 Exon 10
ATCCTTGACTTTGAACACAT 61 5279 5298 126 532729 1636 1655 Exon 10
TTCCATATCCTTGACTTTGA 55 5285 5304 127 532730 1642 1661 Exon 10
CAGGTTTTCCATATCCTTGA 51 5291 5310 440 532731 1686 1705 Exon 10-
CTCAGAGACTGGCTTTCATC 41 5827 5846 129 11 Junction 532732 1692 1711
Exon 11 CAGAGACTCAGAGACTGGCT 59 5833 5852 130 516252 1698 1717 Exon
11 ATGCCACAGAGACTCAGAGA 57 5839 5858 131 532733 1704 1723 Exon 11
CAAACCATGCCACAGAGACT 34 5845 5864 132 532734 1710 1729 Exon 11
TGTTCCCAAACCATGCCACA 51 5851 5870 133 532735 1734 1753 Exon 11
TTGTGGTAATCGGTACCCTT 50 5875 5894 134 532736 1740 1759 Exon 11
GGTTGCTTGTGGTAATCGGT 64 5881 5900 135 532737 1746 1765 Exon 11
TGCCATGGTTGCTTGTGGTA 40 5887 5906 136 532738 1752 1771 Exon 11
TTGGCCTGCCATGGTTGCTT 49 5893 5912 137 532739 1758 1777 Exon 11
GAGATCTTGGCCTGCCATGG 47 5899 5918 138 532740 1803 1822 Exon 12
ACAGCCCCCATACAGCTCTC 48 6082 6101 139 532741 1809 1828 Exon 12
GACACCACAGCCCCCATACA 40 6088 6107 140 532742 1815 1834 Exon 12
TACTCAGACACCACAGCCCC 33 6094 6113 141 532743 1821 1840 Exon 12
ACAAAGTACTCAGACACCAC 39 6100 6119 142 532744 1827 1846 Exon 12
GTCAGCACAAAGTACTCAGA 45 6106 6125 143 532745 1872 1891 Exon 12
TTGATTGAGTGTTCCTTGTC 42 6151 6170 144 532746 1878 1897 Exon 12
CTGACCTTGATTGAGTGTTC 53 6157 6176 145 532747 1909 1928 Exon 13
TATCTCCAGGTCCCGCTTCT 31 6403 6422 146 532748 1967 1986 Exon 13
GAATTCCTGCTTCTTTTTTC 30 6461 6480 147 532749 1973 1992 Exon 13
ATTCAGGAATTCCTGCTTCT 40 6467 6486 148 532750 1979 1998 Exon 13
CATAAAATTCAGGAATTCCT 45 6473 6492 149 532751 1985 2004 Exon 13
CATAGTCATAAAATTCAGGA 43 6479 6498 150 532752 2006 2025 Exon 13
TGAGCTTGATCAGGGCAACG 61 6500 6519 151 532753 2012 2031 Exon 13
TATTCTTGAGCTTGATCAGG 47 6506 6525 152 532754 2048 2067 Exon 13-
GACAAATGGGCCTGATAGTC 35 n/a n/a 153 14 Junction 532755 2070 2089
Exon 14 GTTGTTCCCTCGGTGCAGGG 43 6659 6678 154 532756 2076 2095 Exon
14 GCTCGAGTTGTTCCCTCGGT 51 6665 6684 155 532757 2082 2101 Exon 14
CTCAAAGCTCGAGTTGTTCC 36 6671 6690 156 532758 2088 2107 Exon 14
GGAAGCCTCAAAGCTCGAGT 54 6677 6696 157 532759 2094 2113 Exon 14
GTTGGAGGAAGCCTCAAAGC 52 6683 6702 158 532760 2100 2119 Exon 14
GTGGTAGTTGGAGGAAGCCT 22 6689 6708 159 532761 2106 2125 Exon 14
TGGCAAGTGGTAGTTGGAGG 34 6695 6714 160 532762 2112 2131 Exon 14
TGTTGCTGGCAAGTGGTAGT 52 6701 6720 161
Example 117: Antisense Inhibition of Human Complement Factor B
(CFB) in HepG2 Cells by MOE Gapmers
[1077] Additional antisense oligonucleotides were designed
targeting human Complement Factor B (CFB) nucleic acid and were
tested for their effects on CFB mRNA in vitro. The antisense
oligonucleotides were tested in a series of experiments that had
similar culture conditions. The results for each experiment are
presented in separate tables shown below. Cultured HepG2 cells at a
density of 20,000 cells per well were transfected using
electroporation with 5,000 nM antisense oligonucleotide. After a
treatment period of approximately 24 hours, RNA was isolated from
the cells and CFB mRNA levels were measured by quantitative
real-time PCR. Human primer probe set RTS3459 was used to measure
mRNA levels. CFB mRNA levels were adjusted according to total RNA
content, as measured by RIBOGREEN.RTM.. Results are presented as
percent inhibition of CFB, relative to untreated control cells.
[1078] The newly designed chimeric antisense oligonucleotides in
the Tables below were designed as 5-10-5 MOE gapmers. The gapmers
are 20 nucleosides in length, wherein the central gap segment
comprises of ten 2'-deoxynucleosides and is flanked by wing
segments on the 5' direction and the 3' direction comprising five
nucleosides each. Each nucleoside in the 5' wing segment and each
nucleoside in the 3' wing segment has a 2'-MOE modification. The
internucleoside linkages throughout each gapmer are
phosphorothioate (P.dbd.S) linkages. All cytosine residues
throughout each gapmer are 5-methylcytosines. "Start site"
indicates the 5'-most nucleoside to which the gapmer is targeted in
the human gene sequence. "Stop site" indicates the 3'-most
nucleoside to which the gapmer is targeted human gene sequence.
Each gapmer listed in the Tables below is targeted to either the
human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK
Accession No. NM_001710.5) or the human CFB genomic sequence,
designated herein as SEQ ID NO: 2 (GENBANK Accession No.
NT_007592.15 truncated from nucleotides 31852000 to 31861000), or
both. `n/a` indicates that the antisense oligonucleotide does not
target that particular gene sequence with 100% complementarity. In
case the sequence alignment for a target gene in a particular table
is not shown, it is understood that none of the oligonucleotides
presented in that table align with 100% complementarity with that
target gene.
TABLE-US-00123 TABLE 130 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 SEQ ID SEQ ID NO: 1 NO: 1 % SEQ ISIS
start stop Target inhi- ID NO site site Region Sequence bition NO:
588570 150 169 Exon 1 TGGTCACATTCCCTTCCCCT 54 396 588571 152 171
Exon 1 CCTGGTCACATTCCCTTCCC 63 397 532614 154 173 Exon 1
GACCTGGTCACATTCCCTTC 64 12 588572 156 175 Exon 1
TAGACCTGGTCACATTCCCT 62 398 588573 158 177 Exon 1
CCTAGACCTGGTCACATTCC 53 399 588566 2189 2208 Exon 15
CCTTCCGAGTCAGCTTTTTC 60 400 588567 2191 2210 Exon 15
CTCCTTCCGAGTCAGCTTTT 61 401 532770 2193 2212 Exon 15
ACCTCCTTCCGAGTCAGCTT 77 198 588568 2195 2214 Exon 15
AGACCTCCTTCCGAGTCAGC 72 402 588569 2197 2216 Exon 15
GTAGACCTCCTTCCGAGTCA 46 403 588574 2453 2472 Exon 18
TTTGCCGCTTCTGGTTTTTG 46 404 588575 2455 2474 Exon 18
CTTTTGCCGCTTCTGGTTTT 41 405 532800 2457 2476 Exon 18
TGCTTTTGCCGCTTCTGGTT 69 228 588576 2459 2478 Exon 18
CCTGCTTTTGCCGCTTCTGG 61 406 588577 2461 2480 Exon 18
TACCTGCTTTTGCCGCTTCT 51 407 516350 2550 2569 Exon 18
AGAAAACCCAAATCCTCATC 71 408 588509 2551 2570 Exon 18
TAGAAAACCCAAATCCTCAT 58 409 588510 2552 2571 Exon 18
ATAGAAAACCCAAATCCTCA 57 410 588511 2553 2572 Exon 18
TATAGAAAACCCAAATCCTC 57 411 588512 2554 2573 Exon 18
TTATAGAAAACCCAAATCCT 44 412 588513 2555 2574 Exon 18
CTTATAGAAAACCCAAATCC 37 413 588514 2556 2575 Exon 18
CCTTATAGAAAACCCAAATC 50 414 588515 2557 2576 Exon 18
CCCTTATAGAAAACCCAAAT 45 415 588516 2558 2577 Exon 18
CCCCTTATAGAAAACCCAAA 60 416 588517 2559 2578 Exon 18
ACCCCTTATAGAAAACCCAA 67 417 588518 2560 2579 Exon 18
AACCCCTTATAGAAAACCCA 57 418 588519 2561 2580 Exon 18
AAACCCCTTATAGAAAACCC 61 419 588520 2562 2581 Exon 18
GAAACCCCTTATAGAAAACC 27 420 588521 2563 2582 Exon 18
GGAAACCCCTTATAGAAAAC 25 421 588522 2564 2583 Exon 18
AGGAAACCCCTTATAGAAAA 36 422 588523 2565 2584 Exon 18
CAGGAAACCCCTTATAGAAA 36 423 588524 2566 2585 Exon 18
GCAGGAAACCCCTTATAGAA 46 424 588525 2567 2586 Exon 18
AGCAGGAAACCCCTTATAGA 38 425 588526 2568 2587 Exon 18
CAGCAGGAAACCCCTTATAG 47 426 588527 2569 2588 Exon 18
CCAGCAGGAAACCCCTTATA 68 427 588528 2570 2589 Exon 18
TCCAGCAGGAAACCCCTTAT 63 428 532809 2571 2590 Exon 18
GTCCAGCAGGAAACCCCTTA 85 237 588529 2572 2591 Exon 18
TGTCCAGCAGGAAACCCCTT 76 429 588530 2573 2592 Exon 18
CTGTCCAGCAGGAAACCCCT 74 430 588531 2574 2593 Exon 18
CCTGTCCAGCAGGAAACCCC 75 431 588532 2575 2594 Exon 18
CCCTGTCCAGCAGGAAACCC 73 432 588533 2576 2595 Exon 18
CCCCTGTCCAGCAGGAAACC 82 433 532810 2577 2596 Exon 18
GCCCCTGTCCAGCAGGAAAC 88 238 588534 2578 2597 Exon 18
CGCCCCTGTCCAGCAGGAAA 86 434 588535 2579 2598 Exon 18
ACGCCCCTGTCCAGCAGGAA 86 435 588536 2580 2599 Exon 18
CACGCCCCTGTCCAGCAGGA 93 436 588537 2581 2600 Exon 18
CCACGCCCCTGTCCAGCAGG 92 437 588538 2582 2601 Exon 18
CCCACGCCCCTGTCCAGCAG 94 438 588539 2583 2602 Exon 18
TCCCACGCCCCTGTCCAGCA 96 439 588540 2584 2603 Exon 18
ATCCCACGCCCCTGTCCAGC 88 440 588541 2585 2604 Exon 18
AATCCCACGCCCCTGTCCAG 79 441 588542 2586 2605 Exon 18
CAATCCCACGCCCCTGTCCA 83 442 588543 2587 2606 Exon 18
TCAATCCCACGCCCCTGTCC 86 443 588544 2588 2607 Exon 18
TTCAATCCCACGCCCCTGTC 90 444 588545 2589 2608 Exon 18
ATTCAATCCCACGCCCCTGT 92 445 588546 2590 2609 Exon 18
AATTCAATCCCACGCCCCTG 92 446 588547 2591 2610 Exon 18
TAATTCAATCCCACGCCCCT 88 447 588548 2592 2611 Exon 18
TTAATTCAATCCCACGCCCC 93 448 588549 2593 2612 Exon 18
TTTAATTCAATCCCACGCCC 88 449 588550 2594 2613 Exon 18
TTTTAATTCAATCCCACGCC 89 450 588551 2595 2614 Exon 18
GTTTTAATTCAATCCCACGC 94 451 588552 2596 2615 Exon 18
TGTTTTAATTCAATCCCACG 93 452 588553 2597 2616 Exon 18
CTGTTTTAATTCAATCCCAC 96 453 588554 2598 2617 Exon 18
GCTGTTTTAATTCAATCCCA 98 454 532811 2599 2618 Exon 18
AGCTGTTTTAATTCAATCCC 97 239 532811 2599 2618 Exon 18
AGCTGTTTTAATTCAATCCC 95 239 588555 2600 2619 Exon 18
CAGCTGTTTTAATTCAATCC 93 455 588556 2601 2620 Exon 18
GCAGCTGTTTTAATTCAATC 96 456 588557 2602 2621 Exon 18
CGCAGCTGTTTTAATTCAAT 98 457 588558 2603 2622 Exon 18
TCGCAGCTGTTTTAATTCAA 95 458 532917 2604 2623 Exon 18
GTCGCAGCTGTTTTAATTCA 97 317 588559 2605 2624 Exon 18
TGTCGCAGCTGTTTTAATTC 95 459 588560 2606 2625 Exon 18
TTGTCGCAGCTGTTTTAATT 92 460 588561 2607 2626 Exon 18
GTTGTCGCAGCTGTTTTAAT 93 461 532952 2608 2627 Exon 18
TGTTGTCGCAGCTGTTTTAA 88 395 588562 2609 2628 Exon 18/
TTGTTGTCGCAGCTGTTTTA 90 462 Repeat 588563 2610 2629 Exon 18/
TTTGTTGTCGCAGCTGTTTT 89 463 Repeat 588564 2611 2630 Exon 18/
TTTTGTTGTCGCAGCTGTTT 92 464 Repeat 588565 2612 2631 Exon 18/
TTTTTGTTGTCGCAGCTGTT 88 465 Repeat
TABLE-US-00124 TABLE 131 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID
SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi-
start stop ID NO site site Region Sequence bition site site NO:
588685 n/a n/a Exon 1 GGATCCAGCTCACTCCCCTG 48 1596 1615 466 588686
n/a n/a Exon 1 AAATAAGGATCCAGCTCACT 29 1602 n/a 467 588688 n/a n/a
Exon 1 GACCAGAAATAAGGATCCAG 58 1608 1627 468 588690 n/a n/a Exon 1
CTTAGGGACCAGAAATAAGG 45 1614 1633 469 588692 n/a n/a Exon 1
CACCCACTTAGGGACCAGAA 36 1620 1639 470 588694 n/a n/a Exon 1
ACCACCCACTTAGGGACCAG 47 1622 1641 471 588696 n/a n/a Exon 1
AGGTCCAGGACTCTCCCCTT 96 1685 1704 472 588698 n/a n/a Exon 1
AAGGTCCAGGACTCTCCCCT 96 1686 1705 473 588700 n/a n/a Exon 1
AAACTGCAGAAGTCCCACCC 2 1716 1735 474 588586 30 49 Exon 1
GGAGGGCCCCGCTGAGCTGC 59 1751 1770 475 588587 48 67 Exon 1
TCCCGGAACATCCAAGCGGG 45 1769 1788 476 588588 56 75 Exon 1
CATCACTTTCCCGGAACATC 39 1777 n/a 477 588589 151 170 Exon 1
CTGGTCACATTCCCTTCCCC 29 1872 1891 478 588590 157 176 Exon 1
CTAGACCTGGTCACATTCCC 47 1878 1897 479 588591 339 358 Exon 1-2
GGAGTGGTGGTCACACCTCC 44 n/a n/a 480 Junction 588592 384 403 Exon 2
ACCCCCTCCAGAGAGCAGGA 43 2192 2211 481 588593 390 409 Exon 2
ATCTCTACCCCCTCCAGAGA 34 2198 2217 482 588594 467 486 Exon 2
GGTACGGGTAGAAGCCAGAA 17 2275 2294 483 588595 671 690 Exon 3
GGAGAGTGTAACCGTCATAG 37 2879 2898 484 588596 689 708 Exon 3
TGCGATTGGCAGAGCCCCGG 18 2897 2916 485 588597 695 714 Exon 3
GGCAGGTGCGATTGGCAGAG 32 2903 2922 486 588598 707 726 Exon 3
GGCCATTCACTTGGCAGGTG 45 2915 2934 487 588599 738 757 Exon 3
TTGTCACAGATCGCTGTCTG 52 2946 2965 488 588600 924 943 Exon 4-5
AAGGAGTCTTGGCAGGAAGG 39 n/a n/a 489 Junction 588601 931 950 Exon
4-5 GTACATGAAGGAGTCTTGGC 37 n/a n/a 490 Junction 588602 959 978
Exon 5 AAGCTTCGGCCACCTCTTGA 21 3542 3561 491 588603 1089 1108 Exon
6 CCATCTAGCACCAGGTAGAT 22 3773 3792 492 588604 1108 1127 Exon 6
GGCCCCAATGCTGTCTGATC 21 3792 3811 493 588606 1150 1169 Exon 6
AATTAAGTTGACTAGACACT 56 3834 3853 494 588608 1162 1181 Exon 6-7
TGCCACCTTCTCAATTAAGT 50 19 495 Junction 588578 1167 1186 Exon 6-7
TAACTTGCCACCTTCTCAAT 23 n/a n/a 496 Junction 588579 1169 1188 Exon
6-7 CATAACTTGCCACCTTCTCA 23 n/a n/a 497 Junction 532692 1171 1190
Exon 6-7 ACCATAACTTGCCACCTTCT 15 n/a n/a 90 Junction 588580 1173
1192 Exon 6-7 ACACCATAACTTGCCACCTT 16 n/a n/a 498 Junction 588581
1175 1194 Exon 6-7 TCACACCATAACTTGCCACC 14 4151 4170 499 Junction
588610 1319 1338 Exon 8 TAGTCCCTGACTTCAACTTG 50 4612 4631 500
588612 1325 1344 Exon 8 TGGTGTTAGTCCCTGACTTC 47 4618 4637 501
588614 1396 1415 Exon 8 GCGGTTCCAGCCTTCAGGAG 47 4689 4708 502
588616 1421 1440 Exon 8 TCATGAGGATGATGACATGG 51 4714 4733 503
588618 1446 1465 Exon 9 CCGCCCATGTTGTGCAATCC 18 5020 5039 504
588620 1458 1477 Exon 9 GTAATTGGGTCCCCGCCCAT 40 5032 5051 505
588623 1482 1501 Exon 9 AAGTCCCGGATCTCATCAAT 40 5056 5075 506
588624 1542 1561 Exon 9-10 AACACATAGACATCCAGATA 45 n/a n/a 507
Junction 588626 1585 1604 Exon 10 CAAAGCATTGATGTTCACTT 43 5234 5253
508 588628 1621 1640 Exon 10 TTTGAACACATGTTGCTCAT 45 5270 5289 509
588631 1646 1665 Exon 10 CTTCCAGGTTTTCCATATCC 53 5295 5314 510
588632 1647 1666 Exon 10 TCTTCCAGGTTTTCCATATC 56 5296 5315 511
588634 1689 1708 Exon 11 AGACTCAGAGACTGGCTTTC 35 5830 5849 512
588636 1749 1768 Exon 11 GCCTGCCATGGTTGCTTGTG 55 5890 5909 513
588638 1763 1782 Exon 11 TGACTGAGATCTTGGCCTGC 78 5904 5923 514
588640 1912 1931 Exon 13 TTCTATCTCCAGGTCCCGCT 95 6406 6425 515
588642 1982 2001 Exon 13 AGTCATAAAATTCAGGAATT 44 6476 6495 516
588645 2073 2092 Exon 14 CGAGTTGTTCCCTCGGTGCA 40 6662 6681 517
588646 2085 2104 Exon 14 AGCCTCAAAGCTCGAGTTGT 57 6674 6693 518
588648 2091 2110 Exon 14 GGAGGAAGCCTCAAAGCTCG 48 6680 6699 519
588651 2097 2116 Exon 14 GTAGTTGGAGGAAGCCTCAA 40 6686 6705 520
588652 2103 2122 Exon 14 CAAGTGGTAGTTGGAGGAAG 43 6692 6711 521
588654 2166 2185 Exon 15 TCCTCAGACACAAACAGAGC 13 6954 6973 522
588656 2172 2191 Exon 15 TTCTCCTCCTCAGACACAAA 55 6960 6979 523
588658 2196 2215 Exon 15 TAGACCTCCTTCCGAGTCAG 44 6984 7003 524
588660 2202 2221 Exon 15 TTGATGTAGACCTCCTTCCG 50 6990 7009 525
588582 2219 2238 Exon 15-16 CTTTCTTATCCCCATTCTTG 19 n/a n/a 526
Junction 588583 2221 2240 Exon 15-16 GCCTTTCTTATCCCCATTCT 14 n/a
n/a 527 Junction 532775 2223 2242 Exon 15-16 CTGCCTTTCTTATCCCCATT 3
n/a n/a 203 Junction 588584 2225 2244 Exon 15-16
AGCTGCCTTTCTTATCCCCA 18 n/a n/a 528 Junction 588662 2226 2245 Exon
15-16 CAGCTGCCTTTCTTATCCCC 27 n/a n/a 529 Junction 588585 2227 2246
Exon 15-16 ACAGCTGCCTTTCTTATCCC 59 n/a n/a 530 Junction 588664 2238
2257 Exon 16 GCATCTCTCTCACAGCTGCC 49 7122 7141 531 588666 2276 2295
Exon 16 AGATGTCCTTGACTTTGTCA 41 7160 7179 532 588668 2330 2349 Exon
16 CAGCATAGGGACTCACTCCT 41 7214 7233 533 588670 2361 2380 Exon
16-17 CCGCCAGAATCACCTCTGCA 43 n/a n/a 534 Junction 588672 2397 2416
Exon 17 TGAATGAAACGACTTCTCTT 52 7362 7381 535 588674 2430 2449 Exon
18 ACATCCACTACTCCCCAGCT 39 7665 7684 536 588676 2448 2467 Exon 18
CGCTTCTGGTTTTTGCAGAC 69 7683 7702 537 588678 2454 2473 Exon 18
TTTTGCCGCTTCTGGTTTTT 46 7689 7708 538 588680 2466 2485 Exon 18
GCAGGTACCTGCTTTTGCCG 47 7701 7720 539 588682 2532 2551 Exon 18
TCTTGGAGTTTCTCCTTCAG 58 7767 7786 540 532811 2599 2618 Exon 18
AGCTGTTTTAATTCAATCCC 10 7834 7853 239 532917 2604 2623 Exon 18
GTCGCAGCTGTTTTAATTCA 11 7839 7858 317
Example 118: Antisense Inhibition of Human Complement Factor B
(CFB) in HepG2 Cells by MOE Gapmers
[1079] Antisense oligonucleotides were designed targeting human
Complement Factor B (CFB) nucleic acid and were tested for their
effects on CFB mRNA in vitro. The antisense oligonucleotides were
tested in a series of experiments that had similar culture
conditions. The results for each experiment are presented in
separate tables shown below. Cultured HepG2 cells at a density of
20,000 cells per well were transfected using electroporation with
3,000 nM antisense oligonucleotide. After a treatment period of
approximately 24 hours, RNA was isolated from the cells and CFB
mRNA levels were measured by quantitative real-time PCR. Human
primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA
levels were adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of CFB,
relative to untreated control cells.
[1080] The newly designed chimeric antisense oligonucleotides in
the Tables below were designed as 4-8-5 MOE, 5-9-5 MOE, 5-10-5 MOE,
3-10-4 MOE, 3-10-7 MOE, 6-7-6-MOE, 6-8-6 MOE, or 5-7-5 MOE gapmers,
or as deoxy, MOE, and (S)-cEt oligonucleotides.
[1081] The 4-8-5 MOE gapmers are 17 nucleosides in length, wherein
the central gap segment comprises of eight 2'-deoxynucleosides and
is flanked by wing segments on the 5' direction and the 3'
direction comprising four and five nucleosides respectively. The
5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central
gap segment comprises of nine 2'-deoxynucleosides and is flanked by
wing segments on the 5' direction and the 3' direction comprising
five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in
length, wherein the central gap segment comprises of ten
2'-deoxynucleosides and is flanked by wing segments on the 5'
direction and the 3' direction comprising five nucleosides each.
The 5-7-5 MOE gapmers are 17 nucleosides in length, wherein the
central gap segment comprises of seven 2'-deoxynucleosides and is
flanked by wing segments on the 5' direction and the 3' direction
comprising five nucleosides each. The 3-10-4 MOE gapmers are 17
nucleosides in length, wherein the central gap segment comprises
often 2'-deoxynucleosides and is flanked by wing segments on the 5'
direction and the 3' direction comprising three and four
nucleosides respectively. The 3-10-7 MOE gapmers are 20 nucleosides
in length, wherein the central gap segment comprises of ten
2'-deoxynucleosides and is flanked by wing segments on the 5'
direction and the 3' direction comprising three and seven
nucleosides respectively. The 6-7-6 MOE gapmers are 19 nucleosides
in length, wherein the central gap segment comprises of seven
2'-deoxynucleosides and is flanked by wing segments on the 5'
direction and the 3' direction comprising six nucleosides each. The
6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central
gap segment comprises of eight 2'-deoxynucleosides and is flanked
by wing segments on the 5' direction and the 3' direction
comprising six nucleosides each. The internucleoside linkages
throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All
cytosine residues throughout each gapmer are 5-methylcytosines.
[1082] The deoxy, MOE and (S)-cEt oligonucleotides are 16
nucleosides in length wherein the nucleoside have either a MOE
sugar modification, an (S)-cEt sugar modification, or a deoxy
modification. The `Chemistry` column describes the sugar
modifications of each oligonucleotide. `k` indicates an (S)-cEt
sugar modification; `d` indicates deoxyribose; and `e` indicates a
MOE modification.
[1083] "Start site" indicates the 5'-most nucleoside to which the
gapmer is targeted in the human gene sequence. "Stop site"
indicates the 3'-most nucleoside to which the gapmer is targeted
human gene sequence. Each gapmer listed in the Tables below is
targeted to either the human CFB mRNA, designated herein as SEQ ID
NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic
sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No.
NT_007592.15 truncated from nucleotides 31852000 to 31861000), or
both. `n/a` indicates that the antisense oligonucleotide does not
target that particular gene sequence with 100% complementarity.
TABLE-US-00125 TABLE 132 Inhibition of CFB mRNA by deoxy, MOE and
(S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ
ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS Start
stop Target inhi- Start Stop ID NO site site region Sequence bition
site site Motif NO: 532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC
10 7834 7853 eeeeeddddddddddeeeee 239 588884 48 63 Exon 1
GGAACATCCAAGCGGG 79 1769 1784 eekdddddddddddke 541 588872 154 169
Exon 1 TGGTCACATTCCCTTC 91 1875 1890 eekdddddddddddke 542 588873
156 171 Exon 1 CCTGGTCACATTCCCT 91 1877 1892 eekdddddddddddke 543
588874 158 173 Exon 1 GACCTGGTCACATTCC 91 1879 1894
eekdddddddddddke 544 588878 1171 1186 Exon 6-7 TAACTTGCCACCTTCT 92
n/a n/a eekdddddddddddke 545 Junction 588879 1173 1188 Exon 6-7
CATAACTTGCCACCTT 94 n/a n/a eekdddddddddddke 546 Junction 588880
1175 1190 Exon 6-7 ACCATAACTTGCCACC 89 4151 4166 eekdddddddddddke
547 Junction 588869 2193 2208 Exon 15 CCTTCCGAGTCAGCTT 17 6981 6996
eekdddddddddddke 548 588870 2195 2210 Exon 15 CTCCTTCCGAGTCAGC 78
6983 6998 eekdddddddddddke 549 588871 2197 2212 Exon 15
ACCTCCTTCCGAGTCA 80 6985 7000 eekdddddddddddke 550 588881 2223 2238
Exon 15- CTTTCTTATCCCCATT 93 n/a n/a eekdddddddddddke 551 16
Junction 588882 2225 2240 Exon 15- GCCTTTCTTATCCCCA 88 n/a n/a
eekdddddddddddke 552 16 Junction 588883 2227 2242 Exon 15-
CTGCCTTTCTTATCCC 90 n/a n/a eekdddddddddddke 553 16 Junction 588875
2457 2472 Exon 18 TTTGCCGCTTCTGGTT 81 7692 7707 eekdddddddddddke
554 588876 2459 2474 Exon 18 CTTTTGCCGCTTCTGG 95 7694 7709
eekdddddddddddke 555 588877 2461 2476 Exon 18 TGCTTTTGCCGCTTCT 91
7696 7711 eekdddddddddddke 556 588807 2551 2566 Exon 18
AAACCCAAATCCTCAT 82 7786 7801 eekdddddddddddke 557 588808 2553 2568
Exon 18 GAAAACCCAAATCCTC 69 7788 7803 eekdddddddddddke 558 588809
2555 2570 Exon 18 TAGAAAACCCAAATCC 51 7790 7805 eekdddddddddddke
559 588810 2556 2571 Exon 18 ATAGAAAACCCAAATC 23 7791 7806
eekdddddddddddke 560 588811 2559 2574 Exon 18 CTTATAGAAAACCCAA 13
7794 7809 eekdddddddddddke 561 588812 2560 2575 Exon 18
CCTTATAGAAAACCCA 29 7795 7810 eekdddddddddddke 562 588813 2561 2576
Exon 18 CCCTTATAGAAAACCC 53 7796 7811 eekdddddddddddke 563 588814
2562 2577 Exon 18 CCCCTTATAGAAAACC 86 7797 7812 eekdddddddddddke
564 588815 2563 2578 Exon 18 ACCCCTTATAGAAAAC 76 7798 7813
eekdddddddddddke 565 588816 2564 2579 Exon 18 AACCCCTTATAGAAAA 33
7799 7814 eekdddddddddddke 566 588817 2565 2580 Exon 18
AAACCCCTTATAGAAA 48 7800 7815 eekdddddddddddke 567 588818 2566 2581
Exon 18 GAAACCCCTTATAGAA 44 7801 7816 eekdddddddddddke 568 588819
2567 2582 Exon 18 GGAAACCCCTTATAGA 74 7802 7817 eekdddddddddddke
569 588820 2568 2583 Exon 18 AGGAAACCCCTTATAG 68 7803 7818
eekdddddddddddke 570 588821 2569 2584 Exon 18 CAGGAAACCCCTTATA 45
7804 7819 eekdddddddddddke 571 588822 2570 2585 Exon 18
GCAGGAAACCCCTTAT 50 7805 7820 eekdddddddddddke 572 588823 2571 2586
Exon 18 AGCAGGAAACCCCTTA 54 7806 7821 eekdddddddddddke 573 588824
2572 2587 Exon 18 CAGCAGGAAACCCCTT 35 7807 7822 eekdddddddddddke
574 588825 2573 2588 Exon 18 CCAGCAGGAAACCCCT 11 7808 7823
eekdddddddddddke 575 588826 2574 2589 Exon 18 TCCAGCAGGAAACCCC 19
7809 7824 eekdddddddddddke 576 588827 2575 2590 Exon 18
GTCCAGCAGGAAACCC 42 7810 7825 eekdddddddddddke 577 588828 2576 2591
Exon 18 TGTCCAGCAGGAAACC 0 7811 7826 eekdddddddddddke 578 588829
2577 2592 Exon 18 CTGTCCAGCAGGAAAC 49 7812 7827 eekdddddddddddke
579 588830 2578 2593 Exon 18 CCTGTCCAGCAGGAAA 11 7813 7828
eekdddddddddddke 580 588831 2579 2594 Exon 18 CCCTGTCCAGCAGGAA 20
7814 7829 eekdddddddddddke 581 588832 2580 2595 Exon 18
CCCCTGTCCAGCAGGA 19 7815 7830 eekdddddddddddke 582 588833 2581 2596
Exon 18 GCCCCTGTCCAGCAGG 12 7816 7831 eekdddddddddddke 583 588834
2582 2597 Exon 18 CGCCCCTGTCCAGCAG 10 7817 7832 eekdddddddddddke
584 588835 2583 2598 Exon 18 ACGCCCCTGTCCAGCA 13 7818 7833
eekdddddddddddke 585 588836 2584 2599 Exon 18 CACGCCCCTGTCCAGC 13
7819 7834 eekdddddddddddke 586 588837 2585 2600 Exon 18
CCACGCCCCTGTCCAG 39 7820 7835 eekdddddddddddke 587 588838 2586 2601
Exon 18 CCCACGCCCCTGTCCA 54 7821 7836 eekdddddddddddke 588 588839
2587 2602 Exon 18 TCCCACGCCCCTGTCC 51 7822 7837 eekdddddddddddke
589 588840 2588 2603 Exon 18 ATCCCACGCCCCTGTC 65 7823 7838
eekdddddddddddke 590 588841 2589 2604 Exon 18 AATCCCACGCCCCTGT 59
7824 7839 eekdddddddddddke 591 588842 2590 2605 Exon 18
CAATCCCACGCCCCTG 70 7825 7840 eekdddddddddddke 592 588843 2591 2606
Exon 18 TCAATCCCACGCCCCT 0 7826 7841 eekdddddddddddke 593 588844
2592 2607 Exon 18 TTCAATCCCACGCCCC 48 7827 7842 eekdddddddddddke
594 588845 2593 2608 Exon 18 ATTCAATCCCACGCCC 46 7828 7843
eekdddddddddddke 595 588846 2594 2609 Exon 18 AATTCAATCCCACGCC 67
7829 7844 eekdddddddddddke 596 588847 2595 2610 Exon 18
TAATTCAATCCCACGC 75 7830 7845 eekdddddddddddke 597 588848 2596 2611
Exon 18 TTAATTCAATCCCACG 76 7831 7846 eekdddddddddddke 598 588849
2597 2612 Exon 18 TTTAATTCAATCCCAC 94 7832 7847 eekdddddddddddke
599 588850 2598 2613 Exon 18 TTTTAATTCAATCCCA 91 7833 7848
eekdddddddddddke 600 588851 2599 2614 Exon 18 GTTTTAATTCAATCCC 91
7834 7849 eekdddddddddddke 601 588852 2600 2615 Exon 18
TGTTTTAATTCAATCC 78 7835 7850 eekdddddddddddke 602 588853 2601 2616
Exon 18 CTGTTTTAATTCAATC 81 7836 7851 eekdddddddddddke 603 588854
2602 2617 Exon 18 GCTGTTTTAATTCAAT 63 7837 7852 eekdddddddddddke
604 588855 2603 2618 Exon 18 AGCTGTTTTAATTCAA 65 7838 7853
eekdddddddddddke 605 588856 2604 2619 Exon 18 CAGCTGTTTTAATTCA 76
7839 7854 eekdddddddddddke 606 588857 2605 2620 Exon 18
GCAGCTGTTTTAATTC 89 7840 7855 eekdddddddddddke 607 588858 2606 2621
Exon 18 CGCAGCTGTTTTAATT 89 7841 7856 eekdddddddddddke 608 588859
2607 2622 Exon 18 TCGCAGCTGTTTTAAT 89 7842 7857 eekdddddddddddke
609 588860 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 76 7843 7858
eekdddddddddddke 610 588861 2609 2624 Exon 18 TGTCGCAGCTGTTTTA 87
7844 7859 eekdddddddddddke 611 588862 2610 2625 Exon 18
TTGTCGCAGCTGTTTT 85 7845 7860 eekdddddddddddke 612 588863 2611 2626
Exon 18 GTTGTCGCAGCTGTTT 87 7846 7861 eekdddddddddddke 613 588864
2612 2627 Exon 18 TGTTGTCGCAGCTGTT 67 7847 7862 eekdddddddddddke
614 588865 2613 2628 Exon 18 TTGTTGTCGCAGCTGT 51 n/a n/a
eekdddddddddddke 615 588866 2614 2629 Exon 18 TTTGTTGTCGCAGCTG 95
n/a n/a eekdddddddddddke 616 588867 2615 2630 Exon 18
TTTTGTTGTCGCAGCT 92 n/a n/a eekdddddddddddke 617
588868 2616 2631 Exon 18 TTTTTGTTGTCGCAGC 66 n/a n/a
eekdddddddddddke 618
TABLE-US-00126 TABLE 133 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID
SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS Start stop Target inhi-
Start Stop ID NO site site region Sequence bition site site NO:
588685 n/a n/a Exon 1 GGATCCAGCTCACTCCCCTG 14 1596 1615 466 588686
n/a n/a Exon 1 AAATAAGGATCCAGCTCACT 2 1602 1621 467 588688 n/a n/a
Exon 1 GACCAGAAATAAGGATCCAG 3 1608 1627 468 588690 n/a n/a Exon 1
CTTAGGGACCAGAAATAAGG 10 1614 1633 469 588692 n/a n/a Exon 1
CACCCACTTAGGGACCAGAA 23 1620 1639 470 588694 n/a n/a Exon 1
ACCACCCACTTAGGGACCAG 23 1622 1641 471 588696 n/a n/a Exon 1
AGGTCCAGGACTCTCCCCTT 15 1685 1704 472 588698 n/a n/a Exon 1
AAGGTCCAGGACTCTCCCCT 19 1686 1705 473 588700 n/a n/a Exon 1
AAACTGCAGAAGTCCCACCC 16 1716 1735 474 588586 30 49 Exon 1
GGAGGGCCCCGCTGAGCTGC 11 1751 1770 475 588587 48 67 Exon 1
TCCCGGAACATCCAAGCGGG 14 1769 1788 476 588588 56 75 Exon 1
CATCACTTTCCCGGAACATC 18 1777 1796 477 588589 151 170 Exon 1
CTGGTCACATTCCCTTCCCC 59 1872 1891 478 588590 157 176 Exon 1
CTAGACCTGGTCACATTCCC 59 1878 1897 479 588591 339 358 Exon 1-2
GGAGTGGTGGTCACACCTCC 45 n/a n/a 480 Junction 588592 384 403 Exon 2
ACCCCCTCCAGAGAGCAGGA 39 2192 2211 481 588593 390 409 Exon 2
ATCTCTACCCCCTCCAGAGA 29 2198 2217 482 588594 467 486 Exon 2
GGTACGGGTAGAAGCCAGAA 47 2275 2294 483 588595 671 690 Exon 3
GGAGAGTGTAACCGTCATAG 44 2879 2898 484 588596 689 708 Exon 3
TGCGATTGGCAGAGCCCCGG 43 2897 2916 638 588597 695 714 Exon 3
GGCAGGTGCGATTGGCAGAG 34 2903 2922 486 588598 707 726 Exon 3
GGCCATTCACTTGGCAGGTG 17 2915 2934 487 588599 738 757 Exon 3
TTGTCACAGATCGCTGTCTG 37 2946 2965 488 588600 924 943 Exon 3-4
AAGGAGTCTTGGCAGGAAGG 18 n/a n/a 489 Junction 588601 931 950 Exon
3-4 GTACATGAAGGAGTCTTGGC 32 n/a n/a 490 Junction 588602 959 978
Exon 5 AAGCTTCGGCCACCTCTTGA 45 3542 3561 491 588603 1089 1108 Exon
6 CCATCTAGCACCAGGTAGAT 52 3773 3792 492 588604 1108 1127 Exon 6
GGCCCCAATGCTGTCTGATC 39 3792 3811 493 588606 1150 1169 Exon 6
AATTAAGTTGACTAGACACT 37 3834 3853 494 588608 1162 1181 Exon 6-7
TGCCACCTTCTCAATTAAGT 21 n/a n/a 648 Junction 588578 1167 1186 Exon
6-7 TAACTTGCCACCTTCTCAAT 22 n/a n/a 496 Junction 588579 1169 1188
Exon 6-7 CATAACTTGCCACCTTCTCA 21 n/a n/a 497 Junction 532692 1171
1190 Exon 6-7 ACCATAACTTGCCACCTTCT 56 n/a n/a 90 Junction 588580
1173 1192 Exon 6-7 ACACCATAACTTGCCACCTT 50 n/a n/a 498 Junction
588581 1175 1194 Exon 7 TCACACCATAACTTGCCACC 50 4151 4170 499
588610 1319 1338 Exon 8 TAGTCCCTGACTTCAACTTG 47 4612 4631 500
588612 1325 1344 Exon 8 TGGTGTTAGTCCCTGACTTC 47 4618 4637 501
588614 1396 1415 Exon 8 GCGGTTCCAGCCTTCAGGAG 51 4689 4708 502
588616 1421 1440 Exon 8 TCATGAGGATGATGACATGG 18 4714 4733 503
588618 1446 1465 Exon 9 CCGCCCATGTTGTGCAATCC 40 5020 5039 504
588620 1458 1477 Exon 9 GTAATTGGGTCCCCGCCCAT 40 5032 5051 505
588623 1482 1501 Exon 9 AAGTCCCGGATCTCATCAAT 45 5056 5075 506
588624 1542 1561 Exon 9-10 AACACATAGACATCCAGATA 43 n/a n/a 507
Junction 588626 1585 1604 Exon 10 CAAAGCATTGATGTTCACTT 45 5234 5253
508 588628 1621 1640 Exon 10 TTTGAACACATGTTGCTCAT 53 5270 5289 509
588631 1646 1665 Exon 10 CTTCCAGGTTTTCCATATCC 56 5295 5314 510
588632 1647 1666 Exon 10 TCTTCCAGGTTTTCCATATC 35 5296 5315 511
588634 1689 1708 Exon 11 AGACTCAGAGACTGGCTTTC 55 5830 5849 512
588636 1749 1768 Exon 11 GCCTGCCATGGTTGCTTGTG 78 5890 5909 513
588638 1763 1782 Exon 11 TGACTGAGATCTTGGCCTGC 95 5904 5923 514
588640 1912 1931 Exon 13 TTCTATCTCCAGGTCCCGCT 44 6406 6425 515
588642 1982 2001 Exon 13 AGTCATAAAATTCAGGAATT 40 6476 6495 516
588645 2073 2092 Exon 14 CGAGTTGTTCCCTCGGTGCA 57 6662 6681 517
588646 2085 2104 Exon 14 AGCCTCAAAGCTCGAGTTGT 48 6674 6693 518
588648 2091 2110 Exon 14 GGAGGAAGCCTCAAAGCTCG 40 6680 6699 519
588651 2097 2116 Exon 14 GTAGTTGGAGGAAGCCTCAA 43 6686 6705 520
588652 2103 2122 Exon 14 CAAGTGGTAGTTGGAGGAAG 13 6692 6711 521
588654 2166 2185 Exon 15 TCCTCAGACACAAACAGAGC 55 6954 6973 522
588656 2172 2191 Exon 15 TTCTCCTCCTCAGACACAAA 44 6960 6979 523
588658 2196 2215 Exon 15 TAGACCTCCTTCCGAGTCAG 50 6984 7003 524
588660 2202 2221 Exon 15 TTGATGTAGACCTCCTTCCG 27 6990 7009 525
588582 2219 2238 Exon 15- CTTTCTTATCCCCATTCTTG 49 n/a n/a 526 16
Junction 588583 2221 2240 Exon 15- GCCTTTCTTATCCCCATTCT 41 n/a n/a
527 16 Junction 532775 2223 2242 Exon 15- CTGCCTTTCTTATCCCCATT 41
n/a n/a 203 16 Junction 588584 2225 2244 Exon 15-
AGCTGCCTTTCTTATCCCCA 43 n/a n/a 528 16 Junction 588662 2226 2245
Exon 15- CAGCTGCCTTTCTTATCCCC 52 n/a n/a 529 16 Junction 588585
2227 2246 Exon 15- ACAGCTGCCTTTCTTATCCC 39 n/a n/a 530 16 Junction
588664 2238 2257 Exon 16 GCATCTCTCTCACAGCTGCC 69 7122 7141 531
588666 2276 2295 Exon 16 AGATGTCCTTGACTTTGTCA 46 7160 7179 532
588668 2330 2349 Exon 16 CAGCATAGGGACTCACTCCT 47 7214 7233 533
588670 2361 2380 Exon 16- CCGCCAGAATCACCTCTGCA 58 n/a n/a 534 17
Junction 588672 2397 2416 Exon 17 TGAATGAAACGACTTCTCTT 48 7362 7381
535 588674 2430 2449 Exon 18 ACATCCACTACTCCCCAGCT 29 7665 7684 536
588676 2448 2467 Exon 18 CGCTTCTGGTTTTTGCAGAC 58 7683 7702 537
588678 2454 2473 Exon 18 TTTTGCCGCTTCTGGTTTTT 45 7689 7708 538
588680 2466 2485 Exon 18 GCAGGTACCTGCTTTTGCCG 36 7701 7720 539
588682 2532 2551 Exon 18 TCTTGGAGTTTCTCCTTCAG 47 7767 7786 540
532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 96 7834 7853 239
532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 96 7839 7858 317
TABLE-US-00127 TABLE 134 Inhibition of CFB mRNA by MOE gapmers
targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS Start stop Target inhi- Start
Stop ID NO site site region Sequence bition site site Motif NO:
598973 2552 2568 Exon 18 GAAAACCCAAATCCTCA 40 7787 7803 3-10-4 619
599036 2552 2568 Exon 18 GAAAACCCAAATCCTCA 18 7787 7803 5-7-5 619
598974 2553 2569 Exon 18 AGAAAACCCAAATCCTC 28 7788 7804 3-10-4 620
599037 2553 2569 Exon 18 AGAAAACCCAAATCCTC 19 7788 7804 5-7-5 620
598975 2554 2570 Exon 18 TAGAAAACCCAAATCCT 15 7789 7805 3-10-4 621
599038 2554 2570 Exon 18 TAGAAAACCCAAATCCT 32 7789 7805 5-7-5 621
598976 2555 2571 Exon 18 ATAGAAAACCCAAATCC 12 7790 7806 3-10-4 622
599039 2555 2571 Exon 18 ATAGAAAACCCAAATCC 7 7790 7806 5-7-5 622
598977 2557 2573 Exon 18 TTATAGAAAACCCAAAT 13 7792 7808 3-10-4 623
599040 2557 2573 Exon 18 TTATAGAAAACCCAAAT 13 7792 7808 5-7-5 623
598978 2558 2574 Exon 18 CTTATAGAAAACCCAAA 0 7793 7809 3-10-4 624
599041 2558 2574 Exon 18 CTTATAGAAAACCCAAA 0 7793 7809 5-7-5 624
598979 2559 2575 Exon 18 CCTTATAGAAAACCCAA 8 7794 7810 3-10-4 625
599042 2559 2575 Exon 18 CCTTATAGAAAACCCAA 19 7794 7810 5-7-5 625
598980 2560 2576 Exon 18 CCCTTATAGAAAACCCA 42 7795 7811 3-10-4 626
599043 2560 2576 Exon 18 CCCTTATAGAAAACCCA 10 7795 7811 5-7-5 626
598981 2561 2577 Exon 18 CCCCTTATAGAAAACCC 20 7796 7812 3-10-4 627
599044 2561 2577 Exon 18 CCCCTTATAGAAAACCC 12 7796 7812 5-7-5 627
598982 2562 2578 Exon 18 ACCCCTTATAGAAAACC 10 7797 7813 3-10-4 628
599045 2562 2578 Exon 18 ACCCCTTATAGAAAACC 3 7797 7813 5-7-5 628
598983 2563 2579 Exon 18 AACCCCTTATAGAAAAC 0 7798 7814 3-10-4 629
599046 2563 2579 Exon 18 AACCCCTTATAGAAAAC 18 7798 7814 5-7-5 629
598984 2564 2580 Exon 18 AAACCCCTTATAGAAAA 0 7799 7815 3-10-4 630
599047 2564 2580 Exon 18 AAACCCCTTATAGAAAA 7 7799 7815 5-7-5 630
598985 2565 2581 Exon 18 GAAACCCCTTATAGAAA 0 7800 7816 3-10-4 631
599048 2565 2581 Exon 18 GAAACCCCTTATAGAAA 9 7800 7816 5-7-5 631
598986 2566 2582 Exon 18 GGAAACCCCTTATAGAA 0 7801 7817 3-10-4 632
599049 2566 2582 Exon 18 GGAAACCCCTTATAGAA 18 7801 7817 5-7-5 632
598988 2567 2583 Exon 18 AGGAAACCCCTTATAGA 0 7802 7818 3-10-4 633
599050 2567 2583 Exon 18 AGGAAACCCCTTATAGA 8 7802 7818 5-7-5 633
598989 2568 2584 Exon 18 CAGGAAACCCCTTATAG 0 7803 7819 3-10-4 634
598990 2569 2585 Exon 18 GCAGGAAACCCCTTATA 8 7804 7820 3-10-4 635
598991 2570 2586 Exon 18 AGCAGGAAACCCCTTAT 25 7805 7821 3-10-4 636
598992 2571 2587 Exon 18 CAGCAGGAAACCCCTTA 12 7806 7822 3-10-4 637
598993 2572 2588 Exon 18 CCAGCAGGAAACCCCTT 37 7807 7823 3-10-4 638
598994 2573 2589 Exon 18 TCCAGCAGGAAACCCCT 29 7808 7824 3-10-4 639
598995 2574 2590 Exon 18 GTCCAGCAGGAAACCCC 42 7809 7825 3-10-4 640
598996 2575 2591 Exon 18 TGTCCAGCAGGAAACCC 36 7810 7826 3-10-4 641
598997 2576 2592 Exon 18 CTGTCCAGCAGGAAACC 18 7811 7827 3-10-4 642
598998 2577 2593 Exon 18 CCTGTCCAGCAGGAAAC 27 7812 7828 3-10-4 643
598999 2578 2594 Exon 18 CCCTGTCCAGCAGGAAA 61 7813 7829 3-10-4 644
599000 2580 2596 Exon 18 GCCCCTGTCCAGCAGGA 71 7815 7831 3-10-4 645
599001 2581 2597 Exon 18 CGCCCCTGTCCAGCAGG 80 7816 7832 3-10-4 646
599002 2582 2598 Exon 18 ACGCCCCTGTCCAGCAG 68 7817 7833 3-10-4 647
599003 2583 2599 Exon 18 CACGCCCCTGTCCAGCA 71 7818 7834 3-10-4 648
599004 2584 2600 Exon 18 CCACGCCCCTGTCCAGC 76 7819 7835 3-10-4 649
599005 2585 2601 Exon 18 CCCACGCCCCTGTCCAG 70 7820 7836 3-10-4 650
599006 2586 2602 Exon 18 TCCCACGCCCCTGTCCA 65 7821 7837 3-10-4 651
599007 2587 2603 Exon 18 ATCCCACGCCCCTGTCC 60 7822 7838 3-10-4 652
599008 2588 2604 Exon 18 AATCCCACGCCCCTGTC 72 7823 7839 3-10-4 653
599009 2589 2605 Exon 18 CAATCCCACGCCCCTGT 79 7824 7840 3-10-4 654
599010 2590 2606 Exon 18 TCAATCCCACGCCCCTG 73 7825 7841 3-10-4 655
599011 2591 2607 Exon 18 TTCAATCCCACGCCCCT 79 7826 7842 3-10-4 656
599012 2592 2608 Exon 18 ATTCAATCCCACGCCCC 67 7827 7843 3-10-4 657
599013 2593 2609 Exon 18 AATTCAATCCCACGCCC 65 7828 7844 3-10-4 658
599014 2594 2610 Exon 18 TAATTCAATCCCACGCC 74 7829 7845 3-10-4 659
599015 2595 2611 Exon 18 TTAATTCAATCCCACGC 71 7830 7846 3-10-4 660
599016 2596 2612 Exon 18 TTTAATTCAATCCCACG 48 7831 7847 3-10-4 661
599017 2597 2613 Exon 18 TTTTAATTCAATCCCAC 34 7832 7848 3-10-4 662
599018 2598 2614 Exon 18 GTTTTAATTCAATCCCA 56 7833 7849 3-10-4 663
599019 2599 2615 Exon 18 TGTTTTAATTCAATCCC 60 7834 7850 3-10-4 664
599020 2600 2616 Exon 18 CTGTTTTAATTCAATCC 0 7835 7851 3-10-4 665
599021 2601 2617 Exon 18 GCTGTTTTAATTCAATC 33 7836 7852 3-10-4 666
599022 2602 2618 Exon 18 AGCTGTTTTAATTCAAT 17 7837 7853 3-10-4 667
599023 2603 2619 Exon 18 CAGCTGTTTTAATTCAA 52 7838 7854 3-10-4 668
532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 86 7839 7858 5-10-5
317 599024 2604 2620 Exon 18 GCAGCTGTTTTAATTCA 88 7839 7855 3-10-4
669 599025 2605 2621 Exon 18 CGCAGCTGTTTTAATTC 85 7840 7856 3-10-4
670 599026 2606 2622 Exon 18 TCGCAGCTGTTTTAATT 69 7841 7857 3-10-4
671 599027 2607 2623 Exon 18 GTCGCAGCTGTTTTAAT 77 7842 7858 3-10-4
672 599028 2608 2624 Exon 18 TGTCGCAGCTGTTTTAA 73 7843 7859 3-10-4
673 599029 2609 2625 Exon 18 TTGTCGCAGCTGTTTTA 78 7844 7860 3-10-4
674 599030 2610 2626 Exon 18 GTTGTCGCAGCTGTTTT 75 7845 7861 3-10-4
675 599031 2611 2627 Exon 18 TGTTGTCGCAGCTGTTT 77 7846 7862 3-10-4
676 599032 2612 2628 Exon 18/ TTGTTGTCGCAGCTGTT 79 n/a n/a 3-10-4
677 Repeat 599033 2613 2629 Exon 18/ TTTGTTGTCGCAGCTGT 80 n/a n/a
3-10-4 678 Repeat 599034 2614 2630 Exon 18/ TTTTGTTGTCGCAGCTG 78
n/a n/a 3-10-4 679 Repeat 599035 2615 2631 Exon 18/
TTTTTGTTGTCGCAGCT 63 n/a n/a 3-10-4 680 Repeat
TABLE-US-00128 TABLE 135 Inhibition of CFB mRNA by MOE gapmers
targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS Start stop Target inhi- Start
Stop ID NO site site region Sequence bition site site Motif NO:
599098 2552 2568 Exon 18 GAAAACCCAAATCCTCA 57 7787 7803 4-8-5 619
599099 2553 2569 Exon 18 AGAAAACCCAAATCCTC 33 7788 7804 4-8-5 620
599100 2554 2570 Exon 18 TAGAAAACCCAAATCCT 32 7789 7805 4-8-5 621
599101 2555 2571 Exon 18 ATAGAAAACCCAAATCC 47 7790 7806 4-8-5 622
599102 2557 2573 Exon 18 TTATAGAAAACCCAAAT 59 7792 7808 4-8-5 623
599103 2558 2574 Exon 18 CTTATAGAAAACCCAAA 10 7793 7809 4-8-5 624
599104 2559 2575 Exon 18 CCTTATAGAAAACCCAA 3 7794 7810 4-8-5 625
599105 2560 2576 Exon 18 CCCTTATAGAAAACCCA 45 7795 7811 4-8-5 626
599106 2561 2577 Exon 18 CCCCTTATAGAAAACCC 49 7796 7812 4-8-5 627
599107 2562 2578 Exon 18 ACCCCTTATAGAAAACC 35 7797 7813 4-8-5 628
599108 2563 2579 Exon 18 AACCCCTTATAGAAAAC 17 7798 7814 4-8-5 629
599109 2564 2580 Exon 18 AAACCCCTTATAGAAAA 36 7799 7815 4-8-5 630
599110 2565 2581 Exon 18 GAAACCCCTTATAGAAA 20 7800 7816 4-8-5 631
599111 2566 2582 Exon 18 GGAAACCCCTTATAGAA 20 7801 7817 4-8-5 632
599112 2567 2583 Exon 18 AGGAAACCCCTTATAGA 15 7802 7818 4-8-5 633
599113 2568 2584 Exon 18 CAGGAAACCCCTTATAG 19 7803 7819 4-8-5 634
599051 2568 2584 Exon 18 CAGGAAACCCCTTATAG 26 7803 7819 5-7-5 634
599114 2569 2585 Exon 18 GCAGGAAACCCCTTATA 18 7804 7820 4-8-5 635
599052 2569 2585 Exon 18 GCAGGAAACCCCTTATA 21 7804 7820 5-7-5 635
599115 2570 2586 Exon 18 AGCAGGAAACCCCTTAT 31 7805 7821 4-8-5 636
599053 2570 2586 Exon 18 AGCAGGAAACCCCTTAT 25 7805 7821 5-7-5 636
599116 2571 2587 Exon 18 CAGCAGGAAACCCCTTA 39 7806 7822 4-8-5 637
599054 2571 2587 Exon 18 CAGCAGGAAACCCCTTA 36 7806 7822 5-7-5 637
599117 2572 2588 Exon 18 CCAGCAGGAAACCCCTT 46 7807 7823 4-8-5 638
599055 2572 2588 Exon 18 CCAGCAGGAAACCCCTT 22 7807 7823 5-7-5 638
599118 2573 2589 Exon 18 TCCAGCAGGAAACCCCT 40 7808 7824 4-8-5 639
599056 2573 2589 Exon 18 TCCAGCAGGAAACCCCT 32 7808 7824 5-7-5 639
599119 2574 2590 Exon 18 GTCCAGCAGGAAACCCC 50 7809 7825 4-8-5 640
599057 2574 2590 Exon 18 GTCCAGCAGGAAACCCC 46 7809 7825 5-7-5 640
599120 2575 2591 Exon 18 TGTCCAGCAGGAAACCC 30 7810 7826 4-8-5 641
599058 2575 2591 Exon 18 TGTCCAGCAGGAAACCC 52 7810 7826 5-7-5 641
599121 2576 2592 Exon 18 CTGTCCAGCAGGAAACC 31 7811 7827 4-8-5 642
599059 2576 2592 Exon 18 CTGTCCAGCAGGAAACC 24 7811 7827 5-7-5 642
599122 2577 2593 Exon 18 CCTGTCCAGCAGGAAAC 23 7812 7828 4-8-5 643
599060 2577 2593 Exon 18 CCTGTCCAGCAGGAAAC 37 7812 7828 5-7-5 643
599123 2578 2594 Exon 18 CCCTGTCCAGCAGGAAA 51 7813 7829 4-8-5 644
599061 2578 2594 Exon 18 CCCTGTCCAGCAGGAAA 34 7813 7829 5-7-5 644
599124 2580 2596 Exon 18 GCCCCTGTCCAGCAGGA 56 7815 7831 4-8-5 645
599062 2580 2596 Exon 18 GCCCCTGTCCAGCAGGA 51 7815 7831 5-7-5 645
599125 2581 2597 Exon 18 CGCCCCTGTCCAGCAGG 70 7816 7832 4-8-5 646
599063 2581 2597 Exon 18 CGCCCCTGTCCAGCAGG 56 7816 7832 5-7-5 646
599126 2582 2598 Exon 18 ACGCCCCTGTCCAGCAG 76 7817 7833 4-8-5 647
599064 2582 2598 Exon 18 ACGCCCCTGTCCAGCAG 61 7817 7833 5-7-5 647
599127 2583 2599 Exon 18 CACGCCCCTGTCCAGCA 67 7818 7834 4-8-5 648
599065 2583 2599 Exon 18 CACGCCCCTGTCCAGCA 64 7818 7834 5-7-5 648
599066 2584 2600 Exon 18 CCACGCCCCTGTCCAGC 40 7819 7835 5-7-5 649
599067 2585 2601 Exon 18 CCCACGCCCCTGTCCAG 37 7820 7836 5-7-5 650
599068 2586 2602 Exon 18 TCCCACGCCCCTGTCCA 31 7821 7837 5-7-5 651
599069 2587 2603 Exon 18 ATCCCACGCCCCTGTCC 39 7822 7838 5-7-5 652
599070 2588 2604 Exon 18 AATCCCACGCCCCTGTC 59 7823 7839 5-7-5 653
599071 2589 2605 Exon 18 CAATCCCACGCCCCTGT 63 7824 7840 5-7-5 657
599072 2590 2606 Exon 18 TCAATCCCACGCCCCTG 74 7825 7841 5-7-5 655
599073 2591 2607 Exon 18 TTCAATCCCACGCCCCT 53 7826 7842 5-7-5 656
599074 2592 2608 Exon 18 ATTCAATCCCACGCCCC 56 7827 7843 5-7-5 657
599075 2593 2609 Exon 18 AATTCAATCCCACGCCC 49 7828 7844 5-7-5 658
599076 2594 2610 Exon 18 TAATTCAATCCCACGCC 54 7829 7845 5-7-5 659
599077 2595 2611 Exon 18 TTAATTCAATCCCACGC 79 7830 7846 5-7-5 660
599078 2596 2612 Exon 18 TTTAATTCAATCCCACG 67 7831 7847 5-7-5 661
599079 2597 2613 Exon 18 TTTTAATTCAATCCCAC 69 7832 7848 5-7-5 662
599080 2598 2614 Exon 18 GTTTTAATTCAATCCCA 79 7833 7849 5-7-5 663
599081 2599 2615 Exon 18 TGTTTTAATTCAATCCC 57 7834 7850 5-7-5 664
599082 2600 2616 Exon 18 CTGTTTTAATTCAATCC 50 7835 7851 5-7-5 665
599083 2601 2617 Exon 18 GCTGTTTTAATTCAATC 67 7836 7852 5-7-5 666
599084 2602 2618 Exon 18 AGCTGTTTTAATTCAAT 60 7837 7853 5-7-5 667
599085 2603 2619 Exon 18 CAGCTGTTTTAATTCAA 71 7838 7854 5-7-5 668
532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 82 7839 7858 5-10-5
317 599086 2604 2620 Exon 18 GCAGCTGTTTTAATTCA 81 7839 7855 5-7-5
669 599087 2605 2621 Exon 18 CGCAGCTGTTTTAATTC 88 7840 7856 5-7-5
670 599088 2606 2622 Exon 18 TCGCAGCTGTTTTAATT 84 7841 7857 5-7-5
671 599089 2607 2623 Exon 18 GTCGCAGCTGTTTTAAT 81 7842 7858 5-7-5
672 599090 2608 2624 Exon 18 TGTCGCAGCTGTTTTAA 77 7843 7859 5-7-5
673 599091 2609 2625 Exon 18 TTGTCGCAGCTGTTTTA 74 7844 7860 5-7-5
674 599092 2610 2626 Exon 18 GTTGTCGCAGCTGTTTT 66 7845 7861 5-7-5
675 599093 2611 2627 Exon 18 TGTTGTCGCAGCTGTTT 89 7846 7862 5-7-5
676 599094 2612 2628 Exon 18/ TTGTTGTCGCAGCTGTT 82 n/a n/a 5-7-5
677 Repeat 599095 2613 2629 Exon 18/ TTTGTTGTCGCAGCTGT 87 n/a n/a
5-7-5 678 Repeat 599096 2614 2630 Exon 18/ TTTTGTTGTCGCAGCTG 85 n/a
n/a 5-7-5 679 Repeat 599097 2615 2631 Exon 18/ TTTTTGTTGTCGCAGCT 78
n/a n/a 5-7-5 680 Repeat
TABLE-US-00129 TABLE 136 Inhibition of CFB mRNA by MOE gapmers
targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS Start stop Target inhi- Start
Stop ID NO site site region Sequence bition site site Motif NO:
599510 2552 2570 Exon 18 TAGAAAACCCAAATCCTCA 45 7787 7805 5-9-5 681
599331 2553 2571 Exon 18 ATAGAAAACCCAAATCCTC 46 7788 7806 5-9-5 682
599332 2554 2572 Exon 18 TATAGAAAACCCAAATCCT 38 7789 7807 5-9-5 683
599333 2556 2574 Exon 18 CTTATAGAAAACCCAAATC 1 7791 7809 5-9-5 684
599334 2557 2575 Exon 18 CCTTATAGAAAACCCAAAT 5 7792 7810 5-9-5 685
599335 2558 2576 Exon 18 CCCTTATAGAAAACCCAAA 34 7793 7811 5-9-5 686
599336 2559 2577 Exon 18 CCCCTTATAGAAAACCCAA 40 7794 7812 5-9-5 687
599337 2560 2578 Exon 18 ACCCCTTATAGAAAACCCA 39 7795 7813 5-9-5 688
599338 2561 2579 Exon 18 AACCCCTTATAGAAAACCC 57 7796 7814 5-9-5 689
599339 2562 2580 Exon 18 AAACCCCTTATAGAAAACC 26 7797 7815 5-9-5 690
599281 2562 2580 Exon 18 AAACCCCTTATAGAAAACC 15 7797 7815 6-7-6 690
599340 2563 2581 Exon 18 GAAACCCCTTATAGAAAAC 17 7798 7816 5-9-5 691
599282 2563 2581 Exon 18 GAAACCCCTTATAGAAAAC 12 7798 7816 6-7-6 691
599341 2564 2582 Exon 18 GGAAACCCCTTATAGAAAA 23 7799 7817 5-9-5 692
599283 2564 2582 Exon 18 GGAAACCCCTTATAGAAAA 18 7799 7817 6-7-6 692
599342 2565 2583 Exon 18 AGGAAACCCCTTATAGAAA 10 7800 7818 5-9-5 693
599284 2565 2583 Exon 18 AGGAAACCCCTTATAGAAA 14 7800 7818 6-7-6 693
599343 2566 2584 Exon 18 CAGGAAACCCCTTATAGAA 10 7801 7819 5-9-5 694
599285 2566 2584 Exon 18 CAGGAAACCCCTTATAGAA 13 7801 7819 6-7-6 694
599344 2567 2585 Exon 18 GCAGGAAACCCCTTATAGA 22 7802 7820 5-9-5 695
599286 2567 2585 Exon 18 GCAGGAAACCCCTTATAGA 31 7802 7820 6-7-6 695
599345 2568 2586 Exon 18 AGCAGGAAACCCCTTATAG 19 7803 7821 5-9-5 696
599287 2568 2586 Exon 18 AGCAGGAAACCCCTTATAG 12 7803 7821 6-7-6 696
599346 2569 2587 Exon 18 CAGCAGGAAACCCCTTATA 30 7804 7822 5-9-5 697
599288 2569 2587 Exon 18 CAGCAGGAAACCCCTTATA 28 7804 7822 6-7-6 697
599347 2570 2588 Exon 18 CCAGCAGGAAACCCCTTAT 46 7805 7823 5-9-5 698
599289 2570 2588 Exon 18 CCAGCAGGAAACCCCTTAT 32 7805 7823 6-7-6 698
599348 2571 2589 Exon 18 TCCAGCAGGAAACCCCTTA 44 7806 7824 5-9-5 699
599290 2571 2589 Exon 18 TCCAGCAGGAAACCCCTTA 24 7806 7824 6-7-6 699
599349 2572 2590 Exon 18 GTCCAGCAGGAAACCCCTT 60 7807 7825 5-9-5 700
599291 2572 2590 Exon 18 GTCCAGCAGGAAACCCCTT 38 7807 7825 6-7-6 700
599350 2573 2591 Exon 18 TGTCCAGCAGGAAACCCCT 49 7808 7826 5-9-5 701
599292 2573 2591 Exon 18 TGTCCAGCAGGAAACCCCT 35 7808 7826 6-7-6 701
599351 2575 2593 Exon 18 CCTGTCCAGCAGGAAACCC 46 7810 7828 5-9-5 702
599293 2575 2593 Exon 18 CCTGTCCAGCAGGAAACCC 12 7810 7828 6-7-6 702
599352 2576 2594 Exon 18 CCCTGTCCAGCAGGAAACC 49 7811 7829 5-9-5 703
599294 2576 2594 Exon 18 CCCTGTCCAGCAGGAAACC 38 7811 7829 6-7-6 703
599353 2577 2595 Exon 18 CCCCTGTCCAGCAGGAAAC 64 7812 7830 5-9-5 704
599295 2577 2595 Exon 18 CCCCTGTCCAGCAGGAAAC 33 7812 7830 6-7-6 704
599354 2578 2596 Exon 18 GCCCCTGTCCAGCAGGAAA 56 7813 7831 5-9-5 705
599296 2578 2596 Exon 18 GCCCCTGTCCAGCAGGAAA 13 7813 7831 6-7-6 705
599355 2580 2598 Exon 18 ACGCCCCTGTCCAGCAGGA 81 7815 7833 5-9-5 706
599297 2580 2598 Exon 18 ACGCCCCTGTCCAGCAGGA 57 7815 7833 6-7-6 706
599356 2581 2599 Exon 18 CACGCCCCTGTCCAGCAGG 64 7816 7834 5-9-5 707
599298 2581 2599 Exon 18 CACGCCCCTGTCCAGCAGG 39 7816 7834 6-7-6 707
599299 2582 2600 Exon 18 CCACGCCCCTGTCCAGCAG 55 7817 7835 6-7-6 708
599300 2583 2601 Exon 18 CCCACGCCCCTGTCCAGCA 45 7818 7836 6-7-6 709
599301 2584 2602 Exon 18 TCCCACGCCCCTGTCCAGC 39 7819 7837 6-7-6 710
599302 2585 2603 Exon 18 ATCCCACGCCCCTGTCCAG 27 7820 7838 6-7-6 711
599303 2586 2604 Exon 18 AATCCCACGCCCCTGTCCA 35 7821 7839 6-7-6 712
599304 2587 2605 Exon 18 CAATCCCACGCCCCTGTCC 16 7822 7840 6-7-6 713
599305 2588 2606 Exon 18 TCAATCCCACGCCCCTGTC 41 7823 7841 6-7-6 714
599306 2589 2607 Exon 18 TTCAATCCCACGCCCCTGT 70 7824 7842 6-7-6 715
599307 2590 2608 Exon 18 ATTCAATCCCACGCCCCTG 66 7825 7843 6-7-6 716
599308 2591 2609 Exon 18 AATTCAATCCCACGCCCCT 68 7826 7844 6-7-6 717
599309 2592 2610 Exon 18 TAATTCAATCCCACGCCCC 52 7827 7845 6-7-6 718
599310 2593 2611 Exon 18 TTAATTCAATCCCACGCCC 39 7828 7846 6-7-6 719
599311 2594 2612 Exon 18 TTTAATTCAATCCCACGCC 83 7829 7847 6-7-6 720
599312 2595 2613 Exon 18 TTTTAATTCAATCCCACGC 72 7830 7848 6-7-6 721
599313 2596 2614 Exon 18 GTTTTAATTCAATCCCACG 86 7831 7849 6-7-6 722
599314 2597 2615 Exon 18 TGTTTTAATTCAATCCCAC 91 7832 7850 6-7-6 723
599315 2598 2616 Exon 18 CTGTTTTAATTCAATCCCA 71 7833 7851 6-7-6 724
599316 2599 2617 Exon 18 GCTGTTTTAATTCAATCCC 89 7834 7852 6-7-6 725
599317 2600 2618 Exon 18 AGCTGTTTTAATTCAATCC 87 7835 7853 6-7-6 726
599318 2601 2619 Exon 18 CAGCTGTTTTAATTCAATC 81 7836 7854 6-7-6 727
599319 2602 2620 Exon 18 GCAGCTGTTTTAATTCAAT 75 7837 7855 6-7-6 728
599320 2603 2621 Exon 18 CGCAGCTGTTTTAATTCAA 84 7838 7856 6-7-6 729
532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 92 7839 7858 5-10-5
317 599321 2604 2622 Exon 18 TCGCAGCTGTTTTAATTCA 90 7839 7857 6-7-6
730 599322 2605 2623 Exon 18 GTCGCAGCTGTTTTAATTC 89 7840 7858 6-7-6
731 599323 2606 2624 Exon 18 TGTCGCAGCTGTTTTAATT 81 7841 7859 6-7-6
732 599324 2607 2625 Exon 18 TTGTCGCAGCTGTTTTAAT 68 7842 7860 6-7-6
733 599325 2608 2626 Exon 18 GTTGTCGCAGCTGTTTTAA 71 7843 7861 6-7-6
734 599326 2609 2627 Exon 18 TGTTGTCGCAGCTGTTTTA 52 7844 7862 6-7-6
735 599327 2610 2628 Exon 18/ TTGTTGTCGCAGCTGTTTT 88 n/a n/a 6-7-6
736 Repeat 599328 2611 2629 Exon 18/ TTTGTTGTCGCAGCTGTTT 87 n/a n/a
6-7-6 737 Repeat 599329 2612 2630 Exon 18/ TTTTGTTGTCGCAGCTGTT 84
n/a n/a 6-7-6 738 Repeat 599330 2613 2631 Exon 18/
TTTTTGTTGTCGCAGCTGT 87 n/a n/a 6-7-6 739 Repeat
TABLE-US-00130 TABLE 137 Inhibition of CFB mRNA by MOE gapmers
targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS Start stop Target inhi- Start
Stop ID NO site site region Sequence bition site site Motif NO:
599512 2552 2571 Exon 18 ATAGAAAACCCAAATCCTCA 74 7787 7806 3-10-7
410 599449 2553 2572 Exon 18 TATAGAAAACCCAAATCCTC 43 7788 7807
3-10-7 411 599450 2554 2573 Exon 18 TTATAGAAAACCCAAATCCT 51 7789
7808 3-10-7 412 599451 2555 2574 Exon 18 CTTATAGAAAACCCAAATCC 35
7790 7809 3-10-7 413 599452 2556 2575 Exon 18 CCTTATAGAAAACCCAAATC
34 7791 7810 3-10-7 414 599453 2557 2576 Exon 18
CCCTTATAGAAAACCCAAAT 44 7792 7811 3-10-7 415 599454 2558 2577 Exon
18 CCCCTTATAGAAAACCCAAA 54 7793 7812 3-10-7 416 599455 2559 2578
Exon 18 ACCCCTTATAGAAAACCCAA 53 7794 7813 3-10-7 417 599456 2560
2579 Exon 18 AACCCCTTATAGAAAACCCA 69 7795 7814 3-10-7 418 599457
2561 2580 Exon 18 AAACCCCTTATAGAAAACCC 46 7796 7815 3-10-7 419
599458 2562 2581 Exon 18 GAAACCCCTTATAGAAAACC 0 7797 7816 3-10-7
420 599459 2563 2582 Exon 18 GGAAACCCCTTATAGAAAAC 12 7798 7817
3-10-7 421 599460 2564 2583 Exon 18 AGGAAACCCCTTATAGAAAA 17 7799
7818 3-10-7 422 599461 2565 2584 Exon 18 CAGGAAACCCCTTATAGAAA 24
7800 7819 3-10-7 423 599462 2566 2585 Exon 18 GCAGGAAACCCCTTATAGAA
33 7801 7820 3-10-7 424 599463 2567 2586 Exon 18
AGCAGGAAACCCCTTATAGA 38 7802 7821 3-10-7 425 599464 2568 2587 Exon
18 CAGCAGGAAACCCCTTATAG 33 7803 7822 3-10-7 426 599465 2569 2588
Exon 18 CCAGCAGGAAACCCCTTATA 49 7804 7823 3-10-7 427 599466 2570
2589 Exon 18 TCCAGCAGGAAACCCCTTAT 45 7805 7824 3-10-7 428 599467
2571 2590 Exon 18 GTCCAGCAGGAAACCCCTTA 60 7806 7825 3-10-7 237
599468 2572 2591 Exon 18 TGTCCAGCAGGAAACCCCTT 61 7807 7826 3-10-7
429 599469 2573 2592 Exon 18 CTGTCCAGCAGGAAACCCCT 52 7808 7827
3-10-7 430 599470 2574 2593 Exon 18 CCTGTCCAGCAGGAAACCCC 45 7809
7828 3-10-7 431 599471 2575 2594 Exon 18 CCCTGTCCAGCAGGAAACCC 67
7810 7829 3-10-7 432 599472 2576 2595 Exon 18 CCCCTGTCCAGCAGGAAACC
79 7811 7830 3-10-7 433 599473 2577 2596 Exon 18
GCCCCTGTCCAGCAGGAAAC 72 7812 7831 3-10-7 238 599474 2578 2597 Exon
18 CGCCCCTGTCCAGCAGGAAA 87 7813 7832 3-10-7 434 599475 2579 2598
Exon 18 ACGCCCCTGTCCAGCAGGAA 76 7814 7833 3-10-7 435 599476 2580
2599 Exon 18 CACGCCCCTGTCCAGCAGGA 81 7815 7834 3-10-7 436 599477
2581 2600 Exon 18 CCACGCCCCTGTCCAGCAGG 83 7816 7835 3-10-7 437
599478 2582 2601 Exon 18 CCCACGCCCCTGTCCAGCAG 72 7817 7836 3-10-7
438 599479 2583 2602 Exon 18 TCCCACGCCCCTGTCCAGCA 81 7818 7837
3-10-7 439 599480 2584 2603 Exon 18 ATCCCACGCCCCTGTCCAGC 77 7819
7838 3-10-7 440 599481 2585 2604 Exon 18 AATCCCACGCCCCTGTCCAG 83
7820 7839 3-10-7 441 599482 2586 2605 Exon 18 CAATCCCACGCCCCTGTCCA
87 7821 7840 3-10-7 442 599483 2587 2606 Exon 18
TCAATCCCACGCCCCTGTCC 90 7822 7841 3-10-7 443 599484 2588 2607 Exon
18 TTCAATCCCACGCCCCTGTC 72 7823 7842 3-10-7 444 599485 2589 2608
Exon 18 ATTCAATCCCACGCCCCTGT 82 7824 7843 3-10-7 445 599486 2590
2609 Exon 18 AATTCAATCCCACGCCCCTG 84 7825 7844 3-10-7 446 599487
2591 2610 Exon 18 TAATTCAATCCCACGCCCCT 84 7826 7845 3-10-7 447
599488 2592 2611 Exon 18 TTAATTCAATCCCACGCCCC 87 7827 7846 3-10-7
448 599489 2593 2612 Exon 18 TTTAATTCAATCCCACGCCC 87 7828 7847
3-10-7 449 599490 2594 2613 Exon 18 TTTTAATTCAATCCCACGCC 86 7829
7848 3-10-7 450 599491 2595 2614 Exon 18 GTTTTAATTCAATCCCACGC 87
7830 7849 3-10-7 451 599492 2596 2615 Exon 18 TGTTTTAATTCAATCCCACG
88 7831 7850 3-10-7 452 599493 2597 2616 Exon 18
CTGTTTTAATTCAATCCCAC 75 7832 7851 3-10-7 453 599433 2597 2616 Exon
18 CTGTTTTAATTCAATCCCAC 89 7832 7851 6-8-6 453 599494 2598 2617
Exon 18 GCTGTTTTAATTCAATCCCA 90 7833 7852 3-10-7 454 599434 2598
2617 Exon 18 GCTGTTTTAATTCAATCCCA 89 7833 7852 6-8-6 454 599495
2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 88 7834 7853 3-10-7 239
599435 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 91 7834 7853 6-8-6
239 599496 2600 2619 Exon 18 CAGCTGTTTTAATTCAATCC 89 7835 7854
3-10-7 455 599436 2600 2619 Exon 18 CAGCTGTTTTAATTCAATCC 89 7835
7854 6-8-6 455 599497 2601 2620 Exon 18 GCAGCTGTTTTAATTCAATC 89
7836 7855 3-10-7 456 599437 2601 2620 Exon 18 GCAGCTGTTTTAATTCAATC
91 7836 7855 6-8-6 456 599498 2602 2621 Exon 18
CGCAGCTGTTTTAATTCAAT 88 7837 7856 3-10-7 457 599438 2602 2621 Exon
18 CGCAGCTGTTTTAATTCAAT 90 7837 7856 6-8-6 457 599499 2603 2622
Exon 18 TCGCAGCTGTTTTAATTCAA 81 7838 7857 3-10-7 458 599439 2603
2622 Exon 18 TCGCAGCTGTTTTAATTCAA 88 7838 7857 6-8-6 458 532917
2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 90 7839 7858 5-10-5 317
599500 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 88 7839 7858 3-10-7
317 599440 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 88 7839 7858
6-8-6 317 599501 2605 2624 Exon 18 TGTCGCAGCTGTTTTAATTC 78 7840
7859 3-10-7 459 599441 2605 2624 Exon 18 TGTCGCAGCTGTTTTAATTC 90
7840 7859 6-8-6 459 599502 2606 2625 Exon 18 TTGTCGCAGCTGTTTTAATT
87 7841 7860 3-10-7 460 599442 2606 2625 Exon 18
TTGTCGCAGCTGTTTTAATT 76 7841 7860 6-8-6 460 599503 2607 2626 Exon
18 GTTGTCGCAGCTGTTTTAAT 83 7842 7861 3-10-7 461 599443 2607 2626
Exon 18 GTTGTCGCAGCTGTTTTAAT 77 7842 7861 6-8-6 461 599504 2608
2627 Exon 18 TGTTGTCGCAGCTGTTTTAA 89 7843 7862 3-10-7 395 599444
2608 2627 Exon 18 TGTTGTCGCAGCTGTTTTAA 69 7843 7862 6-8-6 395
599505 2609 2628 Exon 19/ TTGTTGTCGCAGCTGTTTTA 83 n/a n/a 3-10-7
462 Repeat 599445 2609 2628 Exon 19/ TTGTTGTCGCAGCTGTTTTA 85 n/a
n/a 6-8-6 462 Repeat 599506 2610 2629 Exon 19/ TTTGTTGTCGCAGCTGTTTT
89 n/a n/a 3-10-7 463 Repeat 599446 2610 2629 Exon 19/
TTTGTTGTCGCAGCTGTTTT 85 n/a n/a 6-8-6 463 Repeat 599507 2611 2630
Exon 19/ TTTTGTTGTCGCAGCTGTTT 82 n/a n/a 3-10-7 464 Repeat 599447
2611 2630 Exon 19/ TTTTGTTGTCGCAGCTGTTT 83 n/a n/a 6-8-6 464 Repeat
599508 2612 2631 Exon 19/ TTTTTGTTGTCGCAGCTGTT 90 n/a n/a 3-10-7
465 Repeat 599448 2612 2631 Exon 19/ TTTTTGTTGTCGCAGCTGTT 87 n/a
n/a 6-8-6 465 Repeat
Example 119: Antisense Inhibition of Human Complement Factor B
(CFB) in HepG2 Cells by MOE Gapmers
[1084] Additional antisense oligonucleotides were designed
targeting human Complement Factor B (CFB) nucleic acid and were
tested for their effects on CFB mRNA in vitro. The antisense
oligonucleotides were tested in a series of experiments that had
similar culture conditions. The results for each experiment are
presented in separate tables shown below. Cultured HepG2 cells at a
density of 20,000 cells per well were transfected using
electroporation with 2,000 nM antisense oligonucleotide. After a
treatment period of approximately 24 hours, RNA was isolated from
the cells and CFB mRNA levels were measured by quantitative
real-time PCR. Human primer probe set RTS3459 was used to measure
mRNA levels. CFB mRNA levels were adjusted according to total RNA
content, as measured by RIBOGREEN.RTM.. Results are presented as
percent inhibition of CFB, relative to untreated control cells.
[1085] The newly designed chimeric antisense oligonucleotides in
the Tables below were designed as 4-8-5 MOE, 5-8-5 MOE, 5-9-5 MOE,
5-10-5 MOE, 6-7-6-MOE, 3-10-5 MOE, or 6-8-6 MOE gapmers.
[1086] The 4-8-5 MOE gapmers are 17 nucleosides in length, wherein
the central gap segment comprises of eight 2'-deoxynucleosides and
is flanked by wing segments on the 5' direction and the 3'
direction comprising four and five nucleosides respectively. The
5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central
gap segment comprises of eight 2'-deoxynucleosides and is flanked
by wing segments on the 5' direction and the 3' direction
comprising five nucleosides each. The 5-9-5 MOE gapmers are 19
nucleosides in length, wherein the central gap segment comprises of
nine 2'-deoxynucleosides and is flanked by wing segments on the 5'
direction and the 3' direction comprising five nucleosides each.
The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the
central gap segment comprises of ten 2'-deoxynucleosides and is
flanked by wing segments on the 5' direction and the 3' direction
comprising five nucleosides each. The 3-10-5 MOE gapmers are 18
nucleosides in length, wherein the central gap segment comprises
often 2'-deoxynucleosides and is flanked by wing segments on the 5'
direction and the 3' direction comprising three and five
nucleosides respectively. The 6-7-6 MOE gapmers are 19 nucleosides
in length, wherein the central gap segment comprises of seven
2'-deoxynucleosides and is flanked by wing segments on the 5'
direction and the 3' direction comprising six nucleosides each. The
6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central
gap segment comprises of eight 2'-deoxynucleosides and is flanked
by wing segments on the 5' direction and the 3' direction
comprising six nucleosides each. Each nucleoside in the 5' wing
segment and each nucleoside in the 3' wing segment has a 2'-MOE
modification. The internucleoside linkages throughout each gapmer
are phosphorothioate (P.dbd.S) linkages. All cytosine residues
throughout each gapmer are 5-methylcytosines.
[1087] "Start site" indicates the 5'-most nucleoside to which the
gapmer is targeted in the human gene sequence. "Stop site"
indicates the 3'-most nucleoside to which the gapmer is targeted
human gene sequence. Each gapmer listed in the Tables below is
targeted to either the human CFB mRNA, designated herein as SEQ ID
NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic
sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No.
NT_007592.15 truncated from nucleotides 31852000 to 31861000), or
both. `n/a` indicates that the antisense oligonucleotide does not
target that particular gene sequence with 100% complementarity.
TABLE-US-00131 TABLE 138 Inhibition of CFB mRNA by MOE gapmers
targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS Start stop Target inhi- Start
Stop ID NO site site region Sequence bition site site Motif NO:
599160 2560 2577 Exon 18 CCCCTTATAGAAAACCCA 26 7795 7812 5-8-5 740
599161 2561 2578 Exon 18 ACCCCTTATAGAAAACCC 20 7796 7813 5-8-5 741
599162 2562 2579 Exon 18 AACCCCTTATAGAAAACC 12 7797 7814 5-8-5 742
599163 2563 2580 Exon 18 AAACCCCTTATAGAAAAC 11 7798 7815 5-8-5 743
599164 2564 2581 Exon 18 GAAACCCCTTATAGAAAA 11 7799 7816 5-8-5 744
599165 2566 2583 Exon 18 AGGAAACCCCTTATAGAA 0 7801 7818 5-8-5 745
599166 2567 2584 Exon 18 CAGGAAACCCCTTATAGA 12 7802 7819 5-8-5 746
599167 2568 2585 Exon 18 GCAGGAAACCCCTTATAG 14 7803 7820 5-8-5 747
599168 2569 2586 Exon 18 AGCAGGAAACCCCTTATA 16 7804 7821 5-8-5 748
599169 2570 2587 Exon 18 CAGCAGGAAACCCCTTAT 24 7805 7822 5-8-5 749
599170 2571 2588 Exon 18 CCAGCAGGAAACCCCTTA 37 7806 7823 5-8-5 750
599171 2572 2589 Exon 18 TCCAGCAGGAAACCCCTT 30 7807 7824 5-8-5 751
599172 2573 2590 Exon 18 GTCCAGCAGGAAACCCCT 43 7808 7825 5-8-5 752
599173 2574 2591 Exon 18 TGTCCAGCAGGAAACCCC 47 7809 7826 5-8-5 753
599174 2575 2592 Exon 18 CTGTCCAGCAGGAAACCC 27 7810 7827 5-8-5 754
599175 2576 2593 Exon 18 CCTGTCCAGCAGGAAACC 30 7811 7828 5-8-5 755
599176 2577 2594 Exon 18 CCCTGTCCAGCAGGAAAC 34 7812 7829 5-8-5 756
599177 2578 2595 Exon 18 CCCCTGTCCAGCAGGAAA 41 7813 7830 5-8-5 757
599178 2580 2597 Exon 18 CGCCCCTGTCCAGCAGGA 67 7815 7832 5-8-5 758
599179 2581 2598 Exon 18 ACGCCCCTGTCCAGCAGG 61 7816 7833 5-8-5 759
599180 2582 2599 Exon 18 CACGCCCCTGTCCAGCAG 62 7817 7834 5-8-5 760
599181 2583 2600 Exon 18 CCACGCCCCTGTCCAGCA 63 7818 7835 5-8-5 761
599128 2584 2600 Exon 18 CCACGCCCCTGTCCAGC 55 7819 7835 4-8-5 649
599182 2584 2601 Exon 18 CCCACGCCCCTGTCCAGC 58 7819 7836 5-8-5 762
599129 2585 2601 Exon 18 CCCACGCCCCTGTCCAG 41 7820 7836 4-8-5 650
599183 2585 2602 Exon 18 TCCCACGCCCCTGTCCAG 43 7820 7837 5-8-5 763
599130 2586 2602 Exon 18 TCCCACGCCCCTGTCCA 46 7821 7837 4-8-5 651
599184 2586 2603 Exon 18 ATCCCACGCCCCTGTCCA 32 7821 7838 5-8-5 764
599131 2587 2603 Exon 18 ATCCCACGCCCCTGTCC 30 7822 7838 4-8-5 652
599185 2587 2604 Exon 18 AATCCCACGCCCCTGTCC 35 7822 7839 5-8-5 765
599132 2588 2604 Exon 18 AATCCCACGCCCCTGTC 52 7823 7839 4-8-5 653
599186 2588 2605 Exon 18 CAATCCCACGCCCCTGTC 55 7823 7840 5-8-5 766
599133 2589 2605 Exon 18 CAATCCCACGCCCCTGT 66 7824 7840 4-8-5 654
599187 2589 2606 Exon 18 TCAATCCCACGCCCCTGT 72 7824 7841 5-8-5 767
599134 2590 2606 Exon 18 TCAATCCCACGCCCCTG 80 7825 7841 4-8-5 655
599188 2590 2607 Exon 18 TTCAATCCCACGCCCCTG 92 7825 7842 5-8-5 768
599135 2591 2607 Exon 18 TTCAATCCCACGCCCCT 61 7826 7842 4-8-5 656
599189 2591 2608 Exon 18 ATTCAATCCCACGCCCCT 52 7826 7843 5-8-5 769
599136 2592 2608 Exon 18 ATTCAATCCCACGCCCC 68 7827 7843 4-8-5 657
599190 2592 2609 Exon 18 AATTCAATCCCACGCCCC 62 7827 7844 5-8-5 770
599137 2593 2609 Exon 18 AATTCAATCCCACGCCC 51 7828 7844 4-8-5 658
599191 2593 2610 Exon 18 TAATTCAATCCCACGCCC 54 7828 7845 5-8-5 771
599138 2594 2610 Exon 18 TAATTCAATCCCACGCC 71 7829 7845 4-8-5 659
599192 2594 2611 Exon 18 TTAATTCAATCCCACGCC 66 7829 7846 5-8-5 772
599139 2595 2611 Exon 18 TTAATTCAATCCCACGC 80 7830 7846 4-8-5 660
599193 2595 2612 Exon 18 TTTAATTCAATCCCACGC 74 7830 7847 5-8-5 773
599140 2596 2612 Exon 18 TTTAATTCAATCCCACG 66 7831 7847 4-8-5 786
599194 2596 2613 Exon 18 TTTTAATTCAATCCCACG 66 7831 7848 5-8-5 774
599141 2597 2613 Exon 18 TTTTAATTCAATCCCAC 63 7832 7848 4-8-5 662
599195 2597 2614 Exon 18 GTTTTAATTCAATCCCAC 86 7832 7849 5-8-5 775
599142 2598 2614 Exon 18 GTTTTAATTCAATCCCA 69 7833 7849 4-8-5 663
599196 2598 2615 Exon 18 TGTTTTAATTCAATCCCA 82 7833 7850 5-8-5 776
599143 2599 2615 Exon 18 TGTTTTAATTCAATCCC 59 7834 7850 4-8-5 664
599197 2599 2616 Exon 18 CTGTTTTAATTCAATCCC 79 7834 7851 5-8-5 777
599144 2600 2616 Exon 18 CTGTTTTAATTCAATCC 52 7835 7851 4-8-5 665
599198 2600 2617 Exon 18 GCTGTTTTAATTCAATCC 86 7835 7852 5-8-5 778
599145 2601 2617 Exon 18 GCTGTTTTAATTCAATC 53 7836 7852 4-8-5 666
599199 2601 2618 Exon 18 AGCTGTTTTAATTCAATC 72 7836 7853 5-8-5 779
599146 2602 2618 Exon 18 AGCTGTTTTAATTCAAT 42 7837 7853 4-8-5 667
599200 2602 2619 Exon 18 CAGCTGTTTTAATTCAAT 76 7837 7854 5-8-5 780
599147 2603 2619 Exon 18 CAGCTGTTTTAATTCAA 55 7838 7854 4-8-5 668
599201 2603 2620 Exon 18 GCAGCTGTTTTAATTCAA 87 7838 7855 5-8-5 781
532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 93 7839 7858 5-10-5
317 599148 2604 2620 Exon 18 GCAGCTGTTTTAATTCA 84 7839 7855 4-8-5
669 599202 2604 2621 Exon 18 CGCAGCTGTTTTAATTCA 89 7839 7856 5-8-5
782 599149 2605 2621 Exon 18 CGCAGCTGTTTTAATTC 92 7840 7856 4-8-5
670 599203 2605 2622 Exon 18 TCGCAGCTGTTTTAATTC 90 7840 7857 5-8-5
783 599150 2606 2622 Exon 18 TCGCAGCTGTTTTAATT 75 7841 7857 4-8-5
671 599151 2607 2623 Exon 18 GTCGCAGCTGTTTTAAT 80 7842 7858 4-8-5
672 599152 2608 2624 Exon 18 TGTCGCAGCTGTTTTAA 76 7843 7859 4-8-5
673 599153 2609 2625 Exon 18 TTGTCGCAGCTGTTTTA 56 7844 7860 4-8-5
674 599154 2610 2626 Exon 18 GTTGTCGCAGCTGTTTT 85 7845 7861 4-8-5
675 599155 2611 2627 Exon 18 TGTTGTCGCAGCTGTTT 89 7846 7862 4-8-5
676 599156 2612 2628 Exon 18/ TTGTTGTCGCAGCTGTT 83 n/a n/a 4-8-5
813 Repeat 599157 2613 2629 Exon 18/ TTTGTTGTCGCAGCTGT 78 n/a n/a
4-8-5 678 Repeat 599158 2614 2630 Exon 18/ TTTTGTTGTCGCAGCTG 83 n/a
n/a 4-8-5 679 Repeat 599159 2615 2631 Exon 18/ TTTTTGTTGTCGCAGCT 65
n/a n/a 4-8-5 680 Repeat 599204 2606 2623 Exon 18
GTCGCAGCTGTTTTAATT 83 7841 7858 5-8-5 784
TABLE-US-00132 TABLE 139 Inhibition of CFB mRNA by MOE gapmers
targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS Start stop Target inhi- Start
Stop ID NO site site region Sequence bition site site Motif NO:
599509 2552 2570 Exon 18 TAGAAAACCCAAATCCTCA 45 7787 7805 6-7-6 681
599213 2553 2570 Exon 18 TAGAAAACCCAAATCCTC 89 7788 7805 3-10-5 785
599273 2553 2571 Exon 18 ATAGAAAACCCAAATCCTC 85 7788 7806 6-7-6 682
599214 2554 2571 Exon 18 ATAGAAAACCCAAATCCT 79 7789 7806 3-10-5 786
599274 2554 2572 Exon 18 TATAGAAAACCCAAATCCT 75 7789 7807 6-7-6 683
599215 2555 2572 Exon 18 TATAGAAAACCCAAATCC 81 7790 7807 3-10-5 787
599216 2556 2573 Exon 18 TTATAGAAAACCCAAATC 87 7791 7808 3-10-5 788
599275 2556 2574 Exon 18 CTTATAGAAAACCCAAATC 84 7791 7809 6-7-6 684
599217 2557 2574 Exon 18 CTTATAGAAAACCCAAAT 84 7792 7809 3-10-5 789
599276 2557 2575 Exon 18 CCTTATAGAAAACCCAAAT 68 7792 7810 6-7-6 685
599218 2558 2575 Exon 18 CCTTATAGAAAACCCAAA 82 7793 7810 3-10-5 790
599277 2558 2576 Exon 18 CCCTTATAGAAAACCCAAA 82 7793 7811 6-7-6 686
599219 2559 2576 Exon 18 CCCTTATAGAAAACCCAA 81 7794 7811 3-10-5 791
599278 2559 2577 Exon 18 CCCCTTATAGAAAACCCAA 84 7794 7812 6-7-6 687
599220 2560 2577 Exon 18 CCCCTTATAGAAAACCCA 92 7795 7812 3-10-5 740
599279 2560 2578 Exon 18 ACCCCTTATAGAAAACCCA 92 7795 7813 6-7-6 688
599221 2561 2578 Exon 18 ACCCCTTATAGAAAACCC 93 7796 7813 3-10-5 741
599280 2561 2579 Exon 18 AACCCCTTATAGAAAACCC 90 7796 7814 6-7-6 689
599222 2562 2579 Exon 18 AACCCCTTATAGAAAACC 95 7797 7814 3-10-5 742
599223 2563 2580 Exon 18 AAACCCCTTATAGAAAAC 93 7798 7815 3-10-5 743
599224 2564 2581 Exon 18 GAAACCCCTTATAGAAAA 90 7799 7816 3-10-5 744
599225 2566 2583 Exon 18 AGGAAACCCCTTATAGAA 93 7801 7818 3-10-5 745
599226 2567 2584 Exon 18 CAGGAAACCCCTTATAGA 95 7802 7819 3-10-5 746
599227 2568 2585 Exon 18 GCAGGAAACCCCTTATAG 94 7803 7820 3-10-5 747
599228 2569 2586 Exon 18 AGCAGGAAACCCCTTATA 96 7804 7821 3-10-5 748
599229 2570 2587 Exon 18 CAGCAGGAAACCCCTTAT 92 7805 7822 3-10-5 749
599230 2571 2588 Exon 18 CCAGCAGGAAACCCCTTA 88 7806 7823 3-10-5 750
599231 2572 2589 Exon 18 TCCAGCAGGAAACCCCTT 83 7807 7824 3-10-5 751
599232 2573 2590 Exon 18 GTCCAGCAGGAAACCCCT 89 7808 7825 3-10-5 752
599233 2574 2591 Exon 18 TGTCCAGCAGGAAACCCC 83 7809 7826 3-10-5 753
599234 2575 2592 Exon 18 CTGTCCAGCAGGAAACCC 88 7810 7827 3-10-5 754
599235 2576 2593 Exon 18 CCTGTCCAGCAGGAAACC 91 7811 7828 3-10-5 755
599236 2577 2594 Exon 18 CCCTGTCCAGCAGGAAAC 90 7812 7829 3-10-5 756
599237 2578 2595 Exon 18 CCCCTGTCCAGCAGGAAA 34 7813 7830 3-10-5 757
599238 2580 2597 Exon 18 CGCCCCTGTCCAGCAGGA 14 7815 7832 3-10-5 758
599239 2581 2598 Exon 18 ACGCCCCTGTCCAGCAGG 10 7816 7833 3-10-5 759
599240 2582 2599 Exon 18 CACGCCCCTGTCCAGCAG 26 7817 7834 3-10-5 760
599241 2583 2600 Exon 18 CCACGCCCCTGTCCAGCA 11 7818 7835 3-10-5 761
599242 2584 2601 Exon 18 CCCACGCCCCTGTCCAGC 24 7819 7836 3-10-5 762
599243 2585 2602 Exon 18 TCCCACGCCCCTGTCCAG 23 7820 7837 3-10-5 763
599244 2586 2603 Exon 18 ATCCCACGCCCCTGTCCA 29 7821 7838 3-10-5 764
599245 2587 2604 Exon 18 AATCCCACGCCCCTGTCC 11 7822 7839 3-10-5 765
599246 2588 2605 Exon 18 CAATCCCACGCCCCTGTC 0 7823 7840 3-10-5 766
599247 2589 2606 Exon 18 TCAATCCCACGCCCCTGT 21 7824 7841 3-10-5 767
599248 2590 2607 Exon 18 TTCAATCCCACGCCCCTG 0 7825 7842 3-10-5 768
599249 2591 2608 Exon 18 ATTCAATCCCACGCCCCT 9 7826 7843 3-10-5 769
599250 2592 2609 Exon 18 AATTCAATCCCACGCCCC 4 7827 7844 3-10-5 770
599251 2593 2610 Exon 18 TAATTCAATCCCACGCCC 12 7828 7845 3-10-5 771
599252 2594 2611 Exon 18 TTAATTCAATCCCACGCC 2 7829 7846 3-10-5 772
599253 2595 2612 Exon 18 TTTAATTCAATCCCACGC 28 7830 7847 3-10-5 773
599254 2596 2613 Exon 18 TTTTAATTCAATCCCACG 27 7831 7848 3-10-5 774
599255 2597 2614 Exon 18 GTTTTAATTCAATCCCAC 38 7832 7849 3-10-5 775
599256 2598 2615 Exon 18 TGTTTTAATTCAATCCCA 36 7833 7850 3-10-5 776
599257 2599 2616 Exon 18 CTGTTTTAATTCAATCCC 48 7834 7851 3-10-5 777
599258 2600 2617 Exon 18 GCTGTTTTAATTCAATCC 19 7835 7852 3-10-5 778
599259 2601 2618 Exon 18 AGCTGTTTTAATTCAATC 36 7836 7853 3-10-5 779
599260 2602 2619 Exon 18 CAGCTGTTTTAATTCAAT 58 7837 7854 3-10-5 780
599261 2603 2620 Exon 18 GCAGCTGTTTTAATTCAA 35 7838 7855 3-10-5 781
532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 96 7839 7858 5-10-5
317 599262 2604 2621 Exon 18 CGCAGCTGTTTTAATTCA 52 7839 7856 3-10-5
782 599263 2605 2622 Exon 18 TCGCAGCTGTTTTAATTC 66 7840 7857 3-10-5
783 599264 2606 2623 Exon 18 GTCGCAGCTGTTTTAATT 48 7841 7858 3-10-5
784 599265 2607 2624 Exon 18 TGTCGCAGCTGTTTTAAT 46 7842 7859 3-10-5
792 599205 2607 2624 Exon 18 TGTCGCAGCTGTTTTAAT 83 7842 7859 5-8-5
792 599266 2608 2625 Exon 18 TTGTCGCAGCTGTTTTAA 76 7843 7860 3-10-5
793 599206 2608 2625 Exon 18 TTGTCGCAGCTGTTTTAA 90 7843 7860 5-8-5
793 599267 2609 2626 Exon 18 GTTGTCGCAGCTGTTTTA 53 7844 7861 3-10-5
794 599207 2609 2626 Exon 18 GTTGTCGCAGCTGTTTTA 82 7844 7861 5-8-5
794 599268 2610 2627 Exon 18 TGTTGTCGCAGCTGTTTT 58 7845 7862 3-10-5
795 599208 2610 2627 Exon 18 TGTTGTCGCAGCTGTTTT 70 7845 7862 5-8-5
795 599269 2611 2628 Exon 18/ TTGTTGTCGCAGCTGTTT 38 n/a n/a 3-10-5
796 Repeat 599209 2611 2628 Exon 18/ TTGTTGTCGCAGCTGTTT 50 n/a n/a
5-8-5 796 Repeat 599270 2612 2629 Exon 18/ TTTGTTGTCGCAGCTGTT 46
n/a n/a 3-10-5 797 Repeat 599210 2612 2629 Exon 18/
TTTGTTGTCGCAGCTGTT 76 n/a n/a 5-8-5 797 Repeat 599271 2613 2630
Exon 18/ TTTTGTTGTCGCAGCTGT 64 n/a n/a 3-10-5 798 Repeat 599211
2613 2630 Exon 18/ TTTTGTTGTCGCAGCTGT 78 n/a n/a 5-8-5 798 Repeat
599272 2614 2631 Exon 18/ TTTTTGTTGTCGCAGCTG 89 n/a n/a 3-10-5 799
Repeat 599212 2614 2631 Exon 18/ TTTTTGTTGTCGCAGCTG 84 n/a n/a
5-8-5 799 Repeat
TABLE-US-00133 TABLE 140 Inhibition of CFB mRNA by MOE gapmers
targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS Start stop Target inhi- Start
Stop ID NO site site region Sequence bition site site Motif NO:
599511 2552 2571 Exon 18 ATAGAAAACCCAAATCCTCA 38 7787 7806 6-8-6
410 599389 2553 2572 Exon 18 TATAGAAAACCCAAATCCTC 80 7788 7807
6-8-6 411 599390 2554 2573 Exon 18 TTATAGAAAACCCAAATCCT 92 7789
7808 6-8-6 412 599391 2555 2574 Exon 18 CTTATAGAAAACCCAAATCC 90
7790 7809 6-8-6 413 599392 2556 2575 Exon 18 CCTTATAGAAAACCCAAATC
87 7791 7810 6-8-6 414 599393 2557 2576 Exon 18
CCCTTATAGAAAACCCAAAT 87 7792 7811 6-8-6 415 599394 2558 2577 Exon
18 CCCCTTATAGAAAACCCAAA 74 7793 7812 6-8-6 416 599395 2559 2578
Exon 18 ACCCCTTATAGAAAACCCAA 78 7794 7813 6-8-6 417 599396 2560
2579 Exon 18 AACCCCTTATAGAAAACCCA 77 7795 7814 6-8-6 418 599397
2561 2580 Exon 18 AAACCCCTTATAGAAAACCC 89 7796 7815 6-8-6 419
599398 2562 2581 Exon 18 GAAACCCCTTATAGAAAACC 90 7797 7816 6-8-6
420 599399 2563 2582 Exon 18 GGAAACCCCTTATAGAAAAC 91 7798 7817
6-8-6 421 599400 2564 2583 Exon 18 AGGAAACCCCTTATAGAAAA 88 7799
7818 6-8-6 422 599401 2565 2584 Exon 18 CAGGAAACCCCTTATAGAAA 85
7800 7819 6-8-6 423 599402 2566 2585 Exon 18 GCAGGAAACCCCTTATAGAA
77 7801 7820 6-8-6 424 599403 2567 2586 Exon 18
AGCAGGAAACCCCTTATAGA 85 7802 7821 6-8-6 425 599404 2568 2587 Exon
18 CAGCAGGAAACCCCTTATAG 90 7803 7822 6-8-6 426 599405 2569 2588
Exon 18 CCAGCAGGAAACCCCTTATA 89 7804 7823 6-8-6 427 599406 2570
2589 Exon 18 TCCAGCAGGAAACCCCTTAT 72 7805 7824 6-8-6 428 599407
2571 2590 Exon 18 GTCCAGCAGGAAACCCCTTA 87 7806 7825 6-8-6 237
599408 2572 2591 Exon 18 TGTCCAGCAGGAAACCCCTT 87 7807 7826 6-8-6
429 599409 2573 2592 Exon 18 CTGTCCAGCAGGAAACCCCT 83 7808 7827
6-8-6 430 599410 2574 2593 Exon 18 CCTGTCCAGCAGGAAACCCC 88 7809
7828 6-8-6 431 599411 2575 2594 Exon 18 CCCTGTCCAGCAGGAAACCC 45
7810 7829 6-8-6 432 599412 2576 2595 Exon 18 CCCCTGTCCAGCAGGAAACC
66 7811 7830 6-8-6 433 599413 2577 2596 Exon 18
GCCCCTGTCCAGCAGGAAAC 92 7812 7831 6-8-6 238 599414 2578 2597 Exon
18 CGCCCCTGTCCAGCAGGAAA 92 7813 7832 6-8-6 434 599415 2579 2598
Exon 18 ACGCCCCTGTCCAGCAGGAA 87 7814 7833 6-8-6 435 599416 2580
2599 Exon 18 CACGCCCCTGTCCAGCAGGA 91 7815 7834 6-8-6 436 599417
2581 2600 Exon 18 CCACGCCCCTGTCCAGCAGG 84 7816 7835 6-8-6 437
599357 2582 2600 Exon 18 CCACGCCCCTGTCCAGCAG 88 7817 7835 5-9-5 708
599418 2582 2601 Exon 18 CCCACGCCCCTGTCCAGCAG 85 7817 7836 6-8-6
438 599358 2583 2601 Exon 18 CCCACGCCCCTGTCCAGCA 86 7818 7836 5-9-5
709 599419 2583 2602 Exon 18 TCCCACGCCCCTGTCCAGCA 91 7818 7837
6-8-6 833 599359 2584 2602 Exon 18 TCCCACGCCCCTGTCCAGC 85 7819 7837
5-9-5 834 599420 2584 2603 Exon 18 ATCCCACGCCCCTGTCCAGC 91 7819
7838 6-8-6 440 599360 2585 2603 Exon 18 ATCCCACGCCCCTGTCCAG 89 7820
7838 5-9-5 711 599421 2585 2604 Exon 18 AATCCCACGCCCCTGTCCAG 87
7820 7839 6-8-6 441 599361 2586 2604 Exon 18 AATCCCACGCCCCTGTCCA 89
7821 7839 5-9-5 712 599422 2586 2605 Exon 18 CAATCCCACGCCCCTGTCCA
90 7821 7840 6-8-6 442 599362 2587 2605 Exon 18 CAATCCCACGCCCCTGTCC
94 7822 7840 5-9-5 713 599423 2587 2606 Exon 18
TCAATCCCACGCCCCTGTCC 85 7822 7841 6-8-6 841 599363 2588 2606 Exon
18 TCAATCCCACGCCCCTGTC 88 7823 7841 5-9-5 714 599424 2588 2607 Exon
18 TTCAATCCCACGCCCCTGTC 88 7823 7842 6-8-6 444 599364 2589 2607
Exon 18 TTCAATCCCACGCCCCTGT 88 7824 7842 5-9-5 715 599425 2589 2608
Exon 18 ATTCAATCCCACGCCCCTGT 68 7824 7843 6-8-6 445 599365 2590
2608 Exon 18 ATTCAATCCCACGCCCCTG 48 7825 7843 5-9-5 716 599426 2590
2609 Exon 18 AATTCAATCCCACGCCCCTG 55 7825 7844 6-8-6 446 599366
2591 2609 Exon 18 AATTCAATCCCACGCCCCT 28 7826 7844 5-9-5 717 599427
2591 2610 Exon 18 TAATTCAATCCCACGCCCCT 13 7826 7845 6-8-6 849
599367 2592 2610 Exon 18 TAATTCAATCCCACGCCCC 21 7827 7845 5-9-5 718
599428 2592 2611 Exon 18 TTAATTCAATCCCACGCCCC 39 7827 7846 6-8-6
448 599368 2593 2611 Exon 18 TTAATTCAATCCCACGCCC 20 7828 7846 5-9-5
719 599429 2593 2612 Exon 18 TTTAATTCAATCCCACGCCC 18 7828 7847
6-8-6 449 599369 2594 2612 Exon 18 TTTAATTCAATCCCACGCC 78 7829 7847
5-9-5 720 599430 2594 2613 Exon 18 TTTTAATTCAATCCCACGCC 24 7829
7848 6-8-6 450 599370 2595 2613 Exon 18 TTTTAATTCAATCCCACGC 25 7830
7848 5-9-5 721 599431 2595 2614 Exon 18 GTTTTAATTCAATCCCACGC 30
7830 7849 6-8-6 451 599371 2596 2614 Exon 18 GTTTTAATTCAATCCCACG 84
7831 7849 5-9-5 722 599432 2596 2615 Exon 18 TGTTTTAATTCAATCCCACG
29 7831 7850 6-8-6 452 599372 2597 2615 Exon 18 TGTTTTAATTCAATCCCAC
83 7832 7850 5-9-5 723 599373 2598 2616 Exon 18 CTGTTTTAATTCAATCCCA
81 7833 7851 5-9-5 724 599374 2599 2617 Exon 18 GCTGTTTTAATTCAATCCC
26 7834 7852 5-9-5 725 599375 2600 2618 Exon 18 AGCTGTTTTAATTCAATCC
26 7835 7853 5-9-5 726 599376 2601 2619 Exon 18 CAGCTGTTTTAATTCAATC
62 7836 7854 5-9-5 727 599377 2602 2620 Exon 18 GCAGCTGTTTTAATTCAAT
21 7837 7855 5-9-5 728 599378 2603 2621 Exon 18 CGCAGCTGTTTTAATTCAA
90 7838 7856 5-9-5 729 532917 2604 2623 Exon 18
GTCGCAGCTGTTTTAATTCA 95 7839 7858 5-10-5 867 599379 2604 2622 Exon
18 TCGCAGCTGTTTTAATTCA 88 7839 7857 5-9-5 730 599380 2605 2623 Exon
18 GTCGCAGCTGTTTTAATTC 37 7840 7858 5-9-5 869 599381 2606 2624 Exon
18 TGTCGCAGCTGTTTTAATT 33 7841 7859 5-9-5 732 599382 2607 2625 Exon
18 TTGTCGCAGCTGTTTTAAT 81 7842 7860 5-9-5 733 599383 2608 2626 Exon
18 GTTGTCGCAGCTGTTTTAA 54 7843 7861 5-9-5 734 599384 2609 2627 Exon
18 TGTTGTCGCAGCTGTTTTA 85 7844 7862 5-9-5 873 599385 2610 2628 Exon
18/ TTGTTGTCGCAGCTGTTTT 59 n/a n/a 5-9-5 736 Repeat 599386 2611
2629 Exon 18/ TTTGTTGTCGCAGCTGTTT 81 n/a n/a 5-9-5 737 Repeat
599387 2612 2630 Exon 18/ TTTTGTTGTCGCAGCTGTT 80 n/a n/a 5-9-5 738
Repeat 599388 2613 2631 Exon 18/ TTTTTGTTGTCGCAGCTGT 84 n/a n/a
5-9-5 739 Repeat
Example 120: Antisense Inhibition of Human Complement Factor B
(CFB) in HepG2 Cells by MOE Gapmers
[1088] Additional antisense oligonucleotides were designed
targeting human Complement Factor B (CFB) nucleic acid and were
tested for their effects on CFB mRNA in vitro. Cultured HepG2 cells
at a density of 20,000 cells per well were transfected using
electroporation with 1,000 nM antisense oligonucleotide. After a
treatment period of approximately 24 hours, RNA was isolated from
the cells and CFB mRNA levels were measured by quantitative
real-time PCR. Human primer probe set RTS3459 was used to measure
mRNA levels. CFB mRNA levels were adjusted according to total RNA
content, as measured by RIBOGREEN.RTM.. Results are presented as
percent inhibition of CFB, relative to untreated control cells.
[1089] The newly designed chimeric antisense oligonucleotides in
the Tables below were designed deoxy, MOE and (S)-cEt
oligonucleotides. The deoxy, MOE and (S)-cEt oligonucleotides are
16 nucleosides in length wherein the nucleoside have either a MOE
sugar modification, an (S)-cEt sugar modification, or a deoxy
modification. The `Chemistry` column describes the sugar
modifications of each oligonucleotide. `k` indicates an (S)-cEt
sugar modification; `d` indicates deoxyribose; and `e` indicates a
MOE modification.
[1090] "Start site" indicates the 5'-most nucleoside to which the
gapmer is targeted in the human gene sequence. "Stop site"
indicates the 3'-most nucleoside to which the gapmer is targeted
human gene sequence. Each gapmer listed in the Tables below is
targeted to either the human CFB mRNA, designated herein as SEQ ID
NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic
sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No.
NT_007592.15 truncated from nucleotides 31852000 to 31861000), or
both. `n/a` indicates that the antisense oligonucleotide does not
target that particular gene sequence with 100% complementarity.
TABLE-US-00134 TABLE 141 Inhibition of CFB mRNA by deoxy, MOE and
(S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ
ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start
stop Target inhi- start stop ID NO site site region Sequence bition
site site Motif NO: 599513 2551 2566 Exon 18 AAACCCAAATCCTCAT 11
7786 7801 ekkeekkddddddddk 557 599514 2553 2568 Exon 18
GAAAACCCAAATCCTC 13 7788 7803 ekkeekkddddddddk 801 599515 2555 2570
Exon 18 TAGAAAACCCAAATCC 54 7790 7805 ekkeekkddddddddk 559 599516
2559 2574 Exon 18 CTTATAGAAAACCCAA 16 7794 7809 ekkeekkddddddddk
561 599517 2560 2575 Exon 18 CCTTATAGAAAACCCA 29 7795 7810
ekkeekkddddddddk 562 599518 2561 2576 Exon 18 CCCTTATAGAAAACCC 55
7796 7811 ekkeekkddddddddk 563 599519 2562 2577 Exon 18
CCCCTTATAGAAAACC 31 7797 7812 ekkeekkddddddddk 564 599520 2563 2578
Exon 18 ACCCCTTATAGAAAAC 14 7798 7813 ekkeekkddddddddk 565 599521
2564 2579 Exon 18 AACCCCTTATAGAAAA 9 7799 7814 ekkeekkddddddddk 566
599522 2565 2580 Exon 18 AAACCCCTTATAGAAA 8 7800 7815
ekkeekkddddddddk 567 599523 2566 2581 Exon 18 GAAACCCCTTATAGAA 6
7801 7816 ekkeekkddddddddk 568 599524 2567 2582 Exon 18
GGAAACCCCTTATAGA 14 7802 7817 ekkeekkddddddddk 569 599525 2568 2583
Exon 18 AGGAAACCCCTTATAG 6 7803 7818 ekkeekkddddddddk 570 599526
2569 2584 Exon 18 CAGGAAACCCCTTATA 16 7804 7819 ekkeekkddddddddk
571 599527 2570 2585 Exon 18 GCAGGAAACCCCTTAT 0 7805 7820
ekkeekkddddddddk 572 599528 2571 2586 Exon 18 AGCAGGAAACCCCTTA 6
7806 7821 ekkeekkddddddddk 573 599529 2572 2587 Exon 18
CAGCAGGAAACCCCTT 6 7807 7822 ekkeekkddddddddk 574 599530 2574 2589
Exon 18 TCCAGCAGGAAACCCC 29 7809 7824 ekkeekkddddddddk 576 599531
2575 2590 Exon 18 GTCCAGCAGGAAACCC 64 7810 7825 ekkeekkddddddddk
577 599532 2576 2591 Exon 18 TGTCCAGCAGGAAACC 43 7811 7826
ekkeekkddddddddk 578 599533 2577 2592 Exon 18 CTGTCCAGCAGGAAAC 25
7812 7827 ekkeekkddddddddk 820 599534 2578 2593 Exon 18
CCTGTCCAGCAGGAAA 12 7813 7828 ekkeekkddddddddk 580 599535 2580 2595
Exon 18 CCCCTGTCCAGCAGGA 16 7815 7830 ekkeekkddddddddk 582 599536
2582 2597 Exon 18 CGCCCCTGTCCAGCAG 27 7817 7832 ekkeekkddddddddk
584 599537 2583 2598 Exon 18 ACGCCCCTGTCCAGCA 35 7818 7833
ekkeekkddddddddk 585 599538 2584 2599 Exon 18 CACGCCCCTGTCCAGC 26
7819 7834 ekkeekkddddddddk 586 599539 2585 2600 Exon 18
CCACGCCCCTGTCCAG 33 7820 7835 ekkeekkddddddddk 587 599540 2586 2601
Exon 18 CCCACGCCCCTGTCCA 27 7821 7836 ekkeekkddddddddk 588 599541
2587 2602 Exon 18 TCCCACGCCCCTGTCC 52 7822 7837 ekkeekkddddddddk
589 599542 2588 2603 Exon 18 ATCCCACGCCCCTGTC 16 7823 7838
ekkeekkddddddddk 590 599543 2589 2604 Exon 18 AATCCCACGCCCCTGT 19
7824 7839 ekkeekkddddddddk 591 599544 2590 2605 Exon 18
CAATCCCACGCCCCTG 33 7825 7840 ekkeekkddddddddk 831 599545 2591 2606
Exon 18 TCAATCCCACGCCCCT 24 7826 7841 ekkeekkddddddddk 593 599546
2592 2607 Exon 18 TTCAATCCCACGCCCC 54 7827 7842 ekkeekkddddddddk
594 599547 2593 2608 Exon 18 ATTCAATCCCACGCCC 87 7828 7843
ekkeekkddddddddk 595 599548 2594 2609 Exon 18 AATTCAATCCCACGCC 79
7829 7844 ekkeekkddddddddk 596 599549 2595 2610 Exon 18
TAATTCAATCCCACGC 62 7830 7845 ekkeekkddddddddk 597 599550 2596 2611
Exon 18 TTAATTCAATCCCACG 52 7831 7846 ekkeekkddddddddk 598 599551
2597 2612 Exon 18 TTTAATTCAATCCCAC 27 7832 7847 ekkeekkddddddddk
599 599577 2597 2613 Exon 18 TTTTAATTCAATCCCAC 90 7832 7848
eeekkddddddddkeee 662 599552 2598 2613 Exon 18 TTTTAATTCAATCCCA 92
7833 7848 ekkeekkddddddddk 600 599578 2598 2614 Exon 18
GTTTTAATTCAATCCCA 88 7833 7849 eeekkddddddddkeee 663 599553 2599
2614 Exon 18 GTTTTAATTCAATCCC 91 7834 7849 ekkeekkddddddddk 601
599579 2599 2615 Exon 18 TGTTTTAATTCAATCCC 79 7834 7850
eeekkddddddddkeee 664 599554 2600 2615 Exon 18 TGTTTTAATTCAATCC 90
7835 7850 ekkeekkddddddddk 602 599580 2600 2616 Exon 18
CTGTTTTAATTCAATCC 79 7835 7851 eeekkddddddddkeee 665 599555 2601
2616 Exon 18 CTGTTTTAATTCAATC 79 7836 7851 ekkeekkddddddddk 846
599581 2601 2617 Exon 18 GCTGTTTTAATTCAATC 90 7836 7852
eeekkddddddddkeee 666 599556 2602 2617 Exon 18 GCTGTTTTAATTCAAT 47
7837 7852 ekkeekkddddddddk 604 599582 2602 2618 Exon 18
AGCTGTTTTAATTCAAT 89 7837 7853 eeekkddddddddkeee 849 599557 2603
2618 Exon 18 AGCTGTTTTAATTCAA 67 7838 7853 ekkeekkddddddddk 850
599583 2603 2619 Exon 18 CAGCTGTTTTAATTCAA 49 7838 7854
eeekkddddddddkeee 668 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAAT 78
7839 7858 eeeeeddddddddddeee 317 TCA ee 599558 2604 2619 Exon 18
CAGCTGTTTTAATTCA 80 7839 7854 ekkeekkddddddddk 606 599584 2604 2620
Exon 18 GCAGCTGTTTTAATTCA 66 7839 7855 eeekkddddddddkeee 669 599559
2605 2620 Exon 18 GCAGCTGTTTTAATTC 38 7840 7855 ekkeekkddddddddk
607 599585 2605 2621 Exon 18 CGCAGCTGTTTTAATTC 80 7840 7856
eeekkddddddddkeee 670 599560 2606 2621 Exon 18 CGCAGCTGTTTTAATT 16
7841 7856 ekkeekkddddddddk 608 599586 2606 2622 Exon 18
TCGCAGCTGTTTTAATT 78 7841 7857 eeekkddddddddkeee 671 599561 2607
2622 Exon 18 TCGCAGCTGTTTTAAT 58 7842 7857 ekkeekkddddddddk 609
599587 2607 2623 Exon 18 GTCGCAGCTGTTTTAAT 81 7842 7858
eeekkddddddddkeee 672 588860 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 92
7843 7858 eekdddddddddddke 610 599562 2608 2623 Exon 18
GTCGCAGCTGTTTTAA 78 7843 7858 ekkeekkddddddddk 610 599588 2608 2624
Exon 18 TGTCGCAGCTGTTTTAA 81 7843 7859 eeekkddddddddkeee 673 599563
2609 2624 Exon 18 TGTCGCAGCTGTTTTA 86 7844 7859 ekkeekkddddddddk
611 599589 2609 2625 Exon 18 TTGTCGCAGCTGTTTTA 75 7844 7860
eeekkddddddddkeee 674 599564 2610 2625 Exon 18 TTGTCGCAGCTGTTTT 75
7845 7860 ekkeekkddddddddk 612 599590 2610 2626 Exon 18
GTTGTCGCAGCTGTTTT 88 7845 7861 eeekkddddddddkeee 675 599565 2611
2626 Exon 18 GTTGTCGCAGCTGTTT 65 7846 7861 ekkeekkddddddddk 613
599591 2611 2627 Exon 18 TGTTGTCGCAGCTGTTT 94 7846 7862
eeekkddddddddkeee 676 599566 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 72
7847 7862 ekkeekkddddddddk 614 599592 2612 2628 Exon 18/
TTGTTGTCGCAGCTGTT 90 n/a n/a eeekkddddddddkeee 677 Repeat 599567
2613 2628 Exon 18/ TTGTTGTCGCAGCTGT 82 n/a n/a ekkeekkddddddddk 615
Repeat 599593 2613 2629 Exon 18/ TTTGTTGTCGCAGCTGT 95 n/a n/a
eeekkddddddddkeee 678 Repeat 599568 2614 2629 Exon 18/
TTTGTTGTCGCAGCTG 92 n/a n/a ekkeekkddddddddk 616 Repeat 599594 2614
2630 Exon 18/ TTTTGTTGTCGCAGCTG 86 n/a n/a eeekkddddddddkeee 679
Repeat 599569 2615 2630 Exon 18/ TTTTGTTGTCGCAGCT 89 n/a n/a
ekkeekkddddddddk 617 Repeat 599595 2615 2631 Exon 18/
TTTTTGTTGTCGCAGCT 76 n/a n/a eeekkddddddddkeee 680
Repeat 599570 2616 2631 Exon 18/ TTTTTGTTGTCGCAGC 95 n/a n/a
ekkeekkddddddddk 618 Repeat
Example 121: Antisense Inhibition of Human Complement Factor B
(CFB) in HepG2 Cells by MOE Gapmers
[1091] Additional antisense oligonucleotides were designed
targeting human Complement Factor B (CFB) nucleic acid and were
tested for their effects on CFB mRNA in vitro. The antisense
oligonucleotides were tested in a series of experiments that had
similar culture conditions. The results for each experiment are
presented in separate tables shown below. Cultured HepG2 cells at a
density of 20,000 cells per well were transfected using
electroporation with 500 nM antisense oligonucleotide. After a
treatment period of approximately 24 hours, RNA was isolated from
the cells and CFB mRNA levels were measured by quantitative
real-time PCR. Human primer probe set RTS3459 was used to measure
mRNA levels. CFB mRNA levels were adjusted according to total RNA
content, as measured by RIBOGREEN.RTM.. Results are presented as
percent inhibition of CFB, relative to untreated control cells.
[1092] The newly designed chimeric antisense oligonucleotides in
the Tables below were designed as deoxy, MOE and (S)-cEt
oligonucleotides, or as 5-8-5 MOE, 5-9-5 MOE, 5-10-5 MOE,
6-7-6-MOE, 3-10-5 MOE, or 6-8-6 MOE gapmers.
[1093] The deoxy, MOE and (S)-cEt oligonucleotides are 16
nucleosides in length wherein the nucleoside have either a MOE
sugar modification, an (S)-cEt sugar modification, or a deoxy
modification. The `Chemistry` column describes the sugar
modifications of each oligonucleotide. `k` indicates an (S)-cEt
sugar modification; `d` indicates deoxyribose; and `e` indicates a
MOE modification.
[1094] The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein
the central gap segment comprises of eight 2'-deoxynucleosides and
is flanked by wing segments on the 5' direction and the 3'
direction comprising five nucleosides each. The 5-9-5 MOE gapmers
are 19 nucleosides in length, wherein the central gap segment
comprises of nine 2'-deoxynucleosides and is flanked by wing
segments on the 5' direction and the 3' direction comprising five
nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in
length, wherein the central gap segment comprises of ten
2'-deoxynucleosides and is flanked by wing segments on the 5'
direction and the 3' direction comprising five nucleosides each.
The 3-10-5 MOE gapmers are 18 nucleosides in length, wherein the
central gap segment comprises of ten 2'-deoxynucleosides and is
flanked by wing segments on the 5' direction and the 3' direction
comprising three and five nucleosides respectively. The 6-7-6 MOE
gapmers are 19 nucleosides in length, wherein the central gap
segment comprises of seven 2'-deoxynucleosides and is flanked by
wing segments on the 5' direction and the 3' direction comprising
six nucleosides each. The 6-8-6 MOE gapmers are 20 nucleosides in
length, wherein the central gap segment comprises of eight
2'-deoxynucleosides and is flanked by wing segments on the 5'
direction and the 3' direction comprising six nucleosides each.
Each nucleoside in the 5' wing segment and each nucleoside in the
3' wing segment has a 2'-MOE modification. The internucleoside
linkages throughout each gapmer are phosphorothioate (P.dbd.S)
linkages. All cytosine residues throughout each gapmer are
5-methylcytosines.
[1095] "Start site" indicates the 5'-most nucleoside to which the
gapmer is targeted in the human gene sequence. "Stop site"
indicates the 3'-most nucleoside to which the gapmer is targeted
human gene sequence. Each gapmer listed in the Tables below is
targeted to either the human CFB mRNA, designated herein as SEQ ID
NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic
sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No.
NT_007592.15 truncated from nucleotides 31852000 to 31861000), or
both. `n/a` indicates that the antisense oligonucleotide does not
target that particular gene sequence with 100% complementarity.
TABLE-US-00135 TABLE 142 Inhibition of CFB mRNA by deoxy, MOE and
(S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ
ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start
stop Target inhi- start stop ID NO site site region Sequence bition
site site Motif NO: 601152 2551 2566 Exon 18 AAACCCAAATCCTCAT 22
7786 7801 eekkdddddddddkee 557 601218 2551 2566 Exon 18
AAACCCAAATCCTCAT 21 7786 7801 edkkdddddddddeee 557 601153 2552 2567
Exon 18 AAAACCCAAATCCTCA 27 7787 7802 eekkdddddddddkee 800 601219
2552 2567 Exon 18 AAAACCCAAATCCTCA 19 7787 7802 edkkdddddddddeee
800 601154 2553 2568 Exon 18 GAAAACCCAAATCCTC 23 7788 7803
eekkdddddddddkee 558 601220 2553 2568 Exon 18 GAAAACCCAAATCCTC 24
7788 7803 edkkdddddddddeee 558 601155 2554 2569 Exon 18
AGAAAACCCAAATCCT 20 7789 7804 eekkdddddddddkee 801 601221 2554 2569
Exon 18 AGAAAACCCAAATCCT 0 7789 7804 edkkdddddddddeee 801 601156
2555 2570 Exon 18 TAGAAAACCCAAATCC 11 7790 7805 eekkdddddddddkee
559 601222 2555 2570 Exon 18 TAGAAAACCCAAATCC 23 7790 7805
edkkdddddddddeee 559 601157 2556 2571 Exon 18 ATAGAAAACCCAAATC 9
7791 7806 eekkdddddddddkee 560 601223 2556 2571 Exon 18
ATAGAAAACCCAAATC 0 7791 7806 edkkdddddddddeee 560 601158 2557 2572
Exon 18 TATAGAAAACCCAAAT 0 7792 7807 eekkdddddddddkee 802 601224
2557 2572 Exon 18 TATAGAAAACCCAAAT 0 7792 7807 edkkdddddddddeee 802
601159 2558 2573 Exon 18 TTATAGAAAACCCAAA 2 7793 7808
eekkdddddddddkee 803 601225 2558 2573 Exon 18 TTATAGAAAACCCAAA 0
7793 7808 edkkdddddddddeee 803 601160 2559 2574 Exon 18
CTTATAGAAAACCCAA 0 7794 7809 eekkdddddddddkee 561 601226 2559 2574
Exon 18 CTTATAGAAAACCCAA 0 7794 7809 edkkdddddddddeee 561 601161
2560 2575 Exon 18 CCTTATAGAAAACCCA 1 7795 7810 eekkdddddddddkee 562
601227 2560 2575 Exon 18 CCTTATAGAAAACCCA 14 7795 7810
edkkdddddddddeee 562 601162 2561 2576 Exon 18 CCCTTATAGAAAACCC 9
7796 7811 eekkdddddddddkee 563 601228 2561 2576 Exon 18
CCCTTATAGAAAACCC 9 7796 7811 edkkdddddddddeee 563 601163 2562 2577
Exon 18 CCCCTTATAGAAAACC 0 7797 7812 eekkdddddddddkee 564 601164
2563 2578 Exon 18 ACCCCTTATAGAAAAC 3 7798 7813 eekkdddddddddkee 565
601165 2564 2579 Exon 18 AACCCCTTATAGAAAA 0 7799 7814
eekkdddddddddkee 566 601166 2565 2580 Exon 18 AAACCCCTTATAGAAA 0
7800 7815 eekkdddddddddkee 567 601167 2566 2581 Exon 18
GAAACCCCTTATAGAA 0 7801 7816 eekkdddddddddkee 568 601168 2567 2582
Exon 18 GGAAACCCCTTATAGA 0 7802 7817 eekkdddddddddkee 569 601169
2568 2583 Exon 18 AGGAAACCCCTTATAG 0 7803 7818 eekkdddddddddkee 570
601170 2569 2584 Exon 18 CAGGAAACCCCTTATA 10 7804 7819
eekkdddddddddkee 571 601171 2570 2585 Exon 18 GCAGGAAACCCCTTAT 9
7805 7820 eekkdddddddddkee 572 601172 2571 2586 Exon 18
AGCAGGAAACCCCTTA 15 7806 7821 eekkdddddddddkee 573 601173 2572 2587
Exon 18 CAGCAGGAAACCCCTT 29 7807 7822 eekkdddddddddkee 574 601174
2573 2588 Exon 18 CCAGCAGGAAACCCCT 25 7808 7823 eekkdddddddddkee
575 601175 2574 2589 Exon 18 TCCAGCAGGAAACCCC 15 7809 7824
eekkdddddddddkee 576 601176 2575 2590 Exon 18 GTCCAGCAGGAAACCC 18
7810 7825 eekkdddddddddkee 577 601177 2576 2591 Exon 18
TGTCCAGCAGGAAACC 10 7811 7826 eekkdddddddddkee 578 601178 2577 2592
Exon 18 CTGTCCAGCAGGAAAC 11 7812 7827 eekkdddddddddkee 579 601179
2578 2593 Exon 18 CCTGTCCAGCAGGAAA 19 7813 7828 eekkdddddddddkee
580 601180 2579 2594 Exon 18 CCCTGTCCAGCAGGAA 7 7814 7829
eekkdddddddddkee 581 601181 2580 2595 Exon 18 CCCCTGTCCAGCAGGA 3
7815 7830 eekkdddddddddkee 582 601182 2581 2596 Exon 18
GCCCCTGTCCAGCAGG 0 7816 7831 eekkdddddddddkee 583 601183 2582 2597
Exon 18 CGCCCCTGTCCAGCAG 4 7817 7832 eekkdddddddddkee 584 601184
2583 2598 Exon 18 ACGCCCCTGTCCAGCA 14 7818 7833 eekkdddddddddkee
585 601185 2584 2599 Exon 18 CACGCCCCTGTCCAGC 26 7819 7834
eekkdddddddddkee 586 601186 2585 2600 Exon 18 CCACGCCCCTGTCCAG 8
7820 7835 eekkdddddddddkee 587 601187 2586 2601 Exon 18
CCCACGCCCCTGTCCA 18 7821 7836 eekkdddddddddkee 588 601188 2587 2602
Exon 18 TCCCACGCCCCTGTCC 20 7822 7837 eekkdddddddddkee 589 601189
2588 2603 Exon 18 ATCCCACGCCCCTGTC 12 7823 7838 eekkdddddddddkee
590 601190 2589 2604 Exon 18 AATCCCACGCCCCTGT 33 7824 7839
eekkdddddddddkee 591 601191 2590 2605 Exon 18 CAATCCCACGCCCCTG 52
7825 7840 eekkdddddddddkee 592 601192 2591 2606 Exon 18
TCAATCCCACGCCCCT 46 7826 7841 eekkdddddddddkee 593 601193 2592 2607
Exon 18 TTCAATCCCACGCCCC 30 7827 7842 eekkdddddddddkee 594 601194
2593 2608 Exon 18 ATTCAATCCCACGCCC 41 7828 7843 eekkdddddddddkee
595 601195 2594 2609 Exon 18 AATTCAATCCCACGCC 40 7829 7844
eekkdddddddddkee 596 601196 2595 2610 Exon 18 TAATTCAATCCCACGC 71
7830 7845 eekkdddddddddkee 597 601197 2596 2611 Exon 18
TTAATTCAATCCCACG 42 7831 7846 eekkdddddddddkee 598 601198 2597 2612
Exon 18 TTTAATTCAATCCCAC 63 7832 7847 eekkdddddddddkee 599 601199
2598 2613 Exon 18 TTTTAATTCAATCCCA 51 7833 7848 eekkdddddddddkee
600 601200 2599 2614 Exon 18 GTTTTAATTCAATCCC 65 7834 7849
eekkdddddddddkee 601 601201 2600 2615 Exon 18 TGTTTTAATTCAATCC 49
7835 7850 eekkdddddddddkee 602 601202 2601 2616 Exon 18
CTGTTTTAATTCAATC 33 7836 7851 eekkdddddddddkee 603 601203 2602 2617
Exon 18 GCTGTTTTAATTCAAT 63 7837 7852 eekkdddddddddkee 604 601204
2603 2618 Exon 18 AGCTGTTTTAATTCAA 69 7838 7853 eekkdddddddddkee
605 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATT 73 7839 7858
eeeeeddddddddddeeee 317 CA e 601205 2604 2619 Exon 18
CAGCTGTTTTAATTCA 51 7839 7854 eekkdddddddddkee 606 601206 2605 2620
Exon 18 GCAGCTGTTTTAATTC 43 7840 7855 eekkdddddddddkee 607 601207
2606 2621 Exon 18 CGCAGCTGTTTTAATT 52 7841 7856 eekkdddddddddkee
608 601208 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 61 7842 7857
eekkdddddddddkee 609 588860 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 75
7843 7858 eekdddddddddddke 610 601209 2608 2623 Exon 18
GTCGCAGCTGTTTTAA 73 7843 7858 eekkdddddddddkee 610 601210 2609 2624
Exon 18 TGTCGCAGCTGTTTTA 80 7844 7859 eekkdddddddddkee 611 601211
2610 2625 Exon 18 TTGTCGCAGCTGTTTT 64 7845 7860 eekkdddddddddkee
612 601212 2611 2626 Exon 18 GTTGTCGCAGCTGTTT 86 7846 7861
eekkdddddddddkee 613 601213 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 87
7847 7862 eekkdddddddddkee 614 601214 2613 2628 Exon 18/
TTGTTGTCGCAGCTGT 84 n/a n/a eekkdddddddddkee 615 Repeat 601215 2614
2629 Exon 18/ TTTGTTGTCGCAGCTG 78 n/a n/a eekkdddddddddkee 616
Repeat 601216 2615 2630 Exon 18/ TTTTGTTGTCGCAGCT 73 n/a n/a
eekkdddddddddkee 617 Repeat 601217 2616 2631 Exon 18/
TTTTTGTTGTCGCAGC 66 n/a n/a eekkdddddddddkee 618
Repeat
TABLE-US-00136 TABLE 143 Inhibition of CFB mRNA by deoxy, MOE and
(S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ
ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start
stop Target inhi- start stop ID NO site site region Sequence bition
site site Motif NO: 601284 2551 2566 Exon 18 AAACCCAAATCCTCAT 8
7786 7801 ekkdddddddddkeee 557 601285 2552 2567 Exon 18
AAAACCCAAATCCTCA 15 7787 7802 ekkdddddddddkeee 800 601286 2553 2568
Exon 18 GAAAACCCAAATCCTC 21 7788 7803 ekkdddddddddkeee 558 601287
2554 2569 Exon 18 AGAAAACCCAAATCCT 9 7789 7804 ekkdddddddddkeee 801
601288 2555 2570 Exon 18 TAGAAAACCCAAATCC 0 7790 7805
ekkdddddddddkeee 559 601289 2556 2571 Exon 18 ATAGAAAACCCAAATC 40
7791 7806 ekkdddddddddkeee 560 601290 2557 2572 Exon 18
TATAGAAAACCCAAAT 16 7792 7807 ekkdddddddddkeee 802 601291 2558 2573
Exon 18 TTATAGAAAACCCAAA 15 7793 7808 ekkdddddddddkeee 803 601292
2559 2574 Exon 18 CTTATAGAAAACCCAA 5 7794 7809 ekkdddddddddkeee 561
601293 2560 2575 Exon 18 CCTTATAGAAAACCCA 15 7795 7810
ekkdddddddddkeee 562 601294 2561 2576 Exon 18 CCCTTATAGAAAACCC 3
7796 7811 ekkdddddddddkeee 563 601229 2562 2577 Exon 18
CCCCTTATAGAAAACC 15 7797 7812 edkddddddddddeee 564 601295 2562 2577
Exon 18 CCCCTTATAGAAAACC 5 7797 7812 ekkdddddddddkeee 564 601230
2563 2578 Exon 18 ACCCCTTATAGAAAAC 14 7798 7813 edkkdddddddddeee
565 601296 2563 2578 Exon 18 ACCCCTTATAGAAAAC 0 7798 7813
ekkdddddddddkeee 565 601231 2564 2579 Exon 18 AACCCCTTATAGAAAA 14
7799 7814 edkkdddddddddeee 566 601297 2564 2579 Exon 18
AACCCCTTATAGAAAA 14 7799 7814 ekkdddddddddkeee 566 601232 2565 2580
Exon 18 AAACCCCTTATAGAAA 15 7800 7815 edkddddddddddeee 567 601298
2565 2580 Exon 18 AAACCCCTTATAGAAA 7 7800 7815 ekkdddddddddkeee 567
601233 2566 2581 Exon 18 GAAACCCCTTATAGAA 0 7801 7816
edkddddddddddeee 568 601299 2566 2581 Exon 18 GAAACCCCTTATAGAA 0
7801 7816 ekkdddddddddkeee 568 601234 2567 2582 Exon 18
GGAAACCCCTTATAGA 0 7802 7817 edkddddddddddeee 569 601300 2567 2582
Exon 18 GGAAACCCCTTATAGA 9 7802 7817 ekkdddddddddkeee 569 601235
2568 2583 Exon 18 AGGAAACCCCTTATAG 3 7803 7818 edkddddddddddeee 570
601301 2568 2583 Exon 18 AGGAAACCCCTTATAG 14 7803 7818
ekkdddddddddkeee 570 601236 2569 2584 Exon 18 CAGGAAACCCCTTATA 0
7804 7819 edkkdddddddddeee 571 601302 2569 2584 Exon 18
CAGGAAACCCCTTATA 0 7804 7819 ekkdddddddddkeee 571 601237 2570 2585
Exon 18 GCAGGAAACCCCTTAT 16 7805 7820 edkkdddddddddeee 572 601303
2570 2585 Exon 18 GCAGGAAACCCCTTAT 16 7805 7820 ekkdddddddddkeee
572 601238 2571 2586 Exon 18 AGCAGGAAACCCCTTA 11 7806 7821
edkkdddddddddeee 573 601304 2571 2586 Exon 18 AGCAGGAAACCCCTTA 10
7806 7821 ekkdddddddddkeee 573 601239 2572 2587 Exon 18
CAGCAGGAAACCCCTT 21 7807 7822 edkkdddddddddeee 574 601305 2572 2587
Exon 18 CAGCAGGAAACCCCTT 7 7807 7822 ekkdddddddddkeee 574 601240
2573 2588 Exon 18 CCAGCAGGAAACCCCT 6 7808 7823 edkkdddddddddeee 575
601241 2574 2589 Exon 18 TCCAGCAGGAAACCCC 10 7809 7824
edkkdddddddddeee 576 601242 2575 2590 Exon 18 GTCCAGCAGGAAACCC 19
7810 7825 edkkdddddddddeee 577 601243 2576 2591 Exon 18
TGTCCAGCAGGAAACC 10 7811 7826 edkkdddddddddeee 578 601244 2577 2592
Exon 18 CTGTCCAGCAGGAAAC 28 7812 7827 edkkdddddddddeee 579 601245
2578 2593 Exon 18 CCTGTCCAGCAGGAAA 5 7813 7828 edkkdddddddddeee 580
601246 2579 2594 Exon 18 CCCTGTCCAGCAGGAA 18 7814 7829
edkkdddddddddeee 581 601247 2580 2595 Exon 18 CCCCTGTCCAGCAGGA 4
7815 7830 edkkdddddddddeee 582 601248 2581 2596 Exon 18
GCCCCTGTCCAGCAGG 6 7816 7831 edkkdddddddddeee 583 601249 2582 2597
Exon 18 CGCCCCTGTCCAGCAG 18 7817 7832 edkkdddddddddeee 584 601250
2583 2598 Exon 18 ACGCCCCTGTCCAGCA 26 7818 7833 edkkdddddddddeee
585 601251 2584 2599 Exon 18 CACGCCCCTGTCCAGC 27 7819 7834
edkkdddddddddeee 586 601252 2585 2600 Exon 18 CCACGCCCCTGTCCAG 21
7820 7835 edkkdddddddddeee 587 601253 2586 2601 Exon 18
CCCACGCCCCTGTCCA 0 7821 7836 edkkdddddddddeee 588 601254 2587 2602
Exon 18 TCCCACGCCCCTGTCC 31 7822 7837 edkkdddddddddeee 589 601255
2588 2603 Exon 18 ATCCCACGCCCCTGTC 3 7823 7838 edkkdddddddddeee 590
601256 2589 2604 Exon 18 AATCCCACGCCCCTGT 21 7824 7839
edkkdddddddddeee 591 601257 2590 2605 Exon 18 CAATCCCACGCCCCTG 47
7825 7840 edkkdddddddddeee 592 601258 2591 2606 Exon 18
TCAATCCCACGCCCCT 48 7826 7841 edkkdddddddddeee 593 601259 2592 2607
Exon 18 TTCAATCCCACGCCCC 38 7827 7842 edkkdddddddddeee 594 601260
2593 2608 Exon 18 ATTCAATCCCACGCCC 33 7828 7843 edkkdddddddddeee
595 601261 2594 2609 Exon 18 AATTCAATCCCACGCC 17 7829 7844
edkkdddddddddeee 596 601262 2595 2610 Exon 18 TAATTCAATCCCACGC 40
7830 7845 edkkdddddddddeee 597 601263 2596 2611 Exon 18
TTAATTCAATCCCACG 31 7831 7846 edkkdddddddddeee 598 601264 2597 2612
Exon 18 TTTAATTCAATCCCAC 72 7832 7847 edkkdddddddddeee 599 601265
2598 2613 Exon 18 TTTTAATTCAATCCCA 48 7833 7848 edkkdddddddddeee
600 601266 2599 2614 Exon 18 GTTTTAATTCAATCCC 64 7834 7849
edkkdddddddddeee 601 601267 2600 2615 Exon 18 TGTTTTAATTCAATCC 43
7835 7850 edkkdddddddddeee 602 601268 2601 2616 Exon 18
CTGTTTTAATTCAATC 44 7836 7851 edkkdddddddddeee 603 601269 2602 2617
Exon 18 GCTGTTTTAATTCAAT 66 7837 7852 edkkdddddddddeee 604 601270
2603 2618 Exon 18 AGCTGTTTTAATTCAA 47 7838 7853 edkkdddddddddeee
605 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATT 3 7839 7858
eeeeeddddddddddeeee 317 CA e 601271 2604 2619 Exon 18
CAGCTGTTTTAATTCA 26 7839 7854 edkkdddddddddeee 606 601272 2605 2620
Exon 18 GCAGCTGTTTTAATTC 33 7840 7855 edkkdddddddddeee 607 601273
2606 2621 Exon 18 CGCAGCTGTTTTAATT 34 7841 7856 edkkdddddddddeee
608 601274 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 39 7842 7857
edkkdddddddddeee 609 588860 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 72
7843 7858 eekdddddddddddke 610 601275 2608 2623 Exon 18
GTCGCAGCTGTTTTAA 65 7843 7858 edkkdddddddddeee 610 601276 2609 2624
Exon 18 TGTCGCAGCTGTTTTA 65 7844 7859 edkkdddddddddeee 611 601277
2610 2625 Exon 18 TTGTCGCAGCTGTTTT 51 7845 7860 edkkdddddddddeee
612 601278 2611 2626 Exon 18 GTTGTCGCAGCTGTTT 78 7846 7861
edkkdddddddddeee 613 601279 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 79
7847 7862 edkkdddddddddeee 614 601280 2613 2628 Exon 18/
TTGTTGTCGCAGCTGT 70 n/a n/a edkkdddddddddeee 615 Repeat 601281 2614
2629 Exon 18/ TTTGTTGTCGCAGCTG 78 n/a n/a edkkdddddddddeee 616
Repeat 601282 2615 2630 Exon 18/ TTTTGTTGTCGCAGCT 68 n/a n/a
edkkdddddddddeee 617 Repeat 601283 2616 2631 Exon 18/
TTTTTGTTGTCGCAGC 61 n/a n/a edkkdddddddddeee 618
Repeat
TABLE-US-00137 TABLE 144 Inhibition of CFB mRNA by deoxy, MOE and
(S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ
ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start
stop Target inhi- start stop ID NO site site region Sequence bition
site site Motif NO: 601306 2573 2588 Exon 18 CCAGCAGGAAACCCCT 22
7808 7823 ekkdddddddddkeee 575 601307 2574 2589 Exon 18
TCCAGCAGGAAACCCC 22 7809 7824 ekkdddddddddkeee 576 601308 2575 2590
Exon 18 GTCCAGCAGGAAACCC 33 7810 7825 ekkdddddddddkeee 577 601309
2576 2591 Exon 18 TGTCCAGCAGGAAACC 33 7811 7826 ekkdddddddddkeee
578 601310 2577 2592 Exon 18 CTGTCCAGCAGGAAAC 28 7812 7827
ekkdddddddddkeee 579 601311 2578 2593 Exon 18 CCTGTCCAGCAGGAAA 33
7813 7828 ekkdddddddddkeee 580 601312 2579 2594 Exon 18
CCCTGTCCAGCAGGAA 13 7814 7829 ekkdddddddddkeee 581 601313 2580 2595
Exon 18 CCCCTGTCCAGCAGGA 32 7815 7830 ekkdddddddddkeee 582 601314
2581 2596 Exon 18 GCCCCTGTCCAGCAGG 0 7816 7831 ekkdddddddddkeee 583
601315 2582 2597 Exon 18 CGCCCCTGTCCAGCAG 36 7817 7832
ekkdddddddddkeee 584 601316 2583 2598 Exon 18 ACGCCCCTGTCCAGCA 39
7818 7833 ekkdddddddddkeee 585 601317 2584 2599 Exon 18
CACGCCCCTGTCCAGC 33 7819 7834 ekkdddddddddkeee 586 601356 2584 2599
Exon 18 CACGCCCCTGTCCAGC 27 7819 7834 kdkdddddddddeeee 586 601318
2585 2600 Exon 18 CCACGCCCCTGTCCAG 35 7820 7835 ekkdddddddddkeee
587 601357 2585 2600 Exon 18 CCACGCCCCTGTCCAG 26 7820 7835
kdkdddddddddeeee 587 601319 2586 2601 Exon 18 CCCACGCCCCTGTCCA 33
7821 7836 ekkdddddddddkeee 588 601358 2586 2601 Exon 18
CCCACGCCCCTGTCCA 26 7821 7836 kdkdddddddddeeee 588 601320 2587 2602
Exon 18 TCCCACGCCCCTGTCC 25 7822 7837 ekkdddddddddkeee 589 601359
2587 2602 Exon 18 TCCCACGCCCCTGTCC 23 7822 7837 kdkdddddddddeeee
589 601321 2588 2603 Exon 18 ATCCCACGCCCCTGTC 50 7823 7838
ekkdddddddddkeee 590 601360 2588 2603 Exon 18 ATCCCACGCCCCTGTC 33
7823 7838 kdkdddddddddeeee 590 601322 2589 2604 Exon 18
AATCCCACGCCCCTGT 52 7824 7839 ekkdddddddddkeee 591 601361 2589 2604
Exon 18 AATCCCACGCCCCTGT 48 7824 7839 kdkdddddddddeeee 591 601323
2590 2605 Exon 18 CAATCCCACGCCCCTG 67 7825 7840 ekkdddddddddkeee
592 601362 2590 2605 Exon 18 CAATCCCACGCCCCTG 51 7825 7840
kdkdddddddddeeee 592 601324 2591 2606 Exon 18 TCAATCCCACGCCCCT 42
7826 7841 ekkdddddddddkeee 593 601363 2591 2606 Exon 18
TCAATCCCACGCCCCT 42 7826 7841 kdkdddddddddeeee 593 601325 2592 2607
Exon 18 TTCAATCCCACGCCCC 52 7827 7842 ekkdddddddddkeee 594 601364
2592 2607 Exon 18 TTCAATCCCACGCCCC 48 7827 7842 kdkdddddddddeeee
594 601326 2593 2608 Exon 18 ATTCAATCCCACGCCC 27 7828 7843
ekkdddddddddkeee 595 601365 2593 2608 Exon 18 ATTCAATCCCACGCCC 36
7828 7843 kdkdddddddddeeee 595 601327 2594 2609 Exon 18
AATTCAATCCCACGCC 66 7829 7844 ekkdddddddddkeee 596 601366 2594 2609
Exon 18 AATTCAATCCCACGCC 49 7829 7844 kdkdddddddddeeee 596 601328
2595 2610 Exon 18 TAATTCAATCCCACGC 55 7830 7845 ekkdddddddddkeee
597 601367 2595 2610 Exon 18 TAATTCAATCCCACGC 57 7830 7845
kdkdddddddddeeee 597 601329 2596 2611 Exon 18 TTAATTCAATCCCACG 69
7831 7846 ekkdddddddddkeee 598 601368 2596 2611 Exon 18
TTAATTCAATCCCACG 68 7831 7846 kdkdddddddddeeee 598 601330 2597 2612
Exon 18 TTTAATTCAATCCCAC 58 7832 7847 ekkdddddddddkeee 599 601369
2597 2612 Exon 18 TTTAATTCAATCCCAC 65 7832 7847 kdkdddddddddeeee
599 601331 2598 2613 Exon 18 TTTTAATTCAATCCCA 45 7833 7848
ekkdddddddddkeee 600 601370 2598 2613 Exon 18 TTTTAATTCAATCCCA 42
7833 7848 kdkdddddddddeeee 600 601332 2599 2614 Exon 18
GTTTTAATTCAATCCC 84 7834 7849 ekkdddddddddkeee 601 601371 2599 2614
Exon 18 GTTTTAATTCAATCCC 79 7834 7849 kdkdddddddddeeee 601 601333
2600 2615 Exon 18 TGTTTTAATTCAATCC 61 7835 7850 ekkdddddddddkeee
602 601372 2600 2615 Exon 18 TGTTTTAATTCAATCC 71 7835 7850
kdkdddddddddeeee 602 601334 2601 2616 Exon 18 CTGTTTTAATTCAATC 61
7836 7851 ekkdddddddddkeee 603 601373 2601 2616 Exon 18
CTGTTTTAATTCAATC 57 7836 7851 kdkdddddddddeeee 603 601335 2602 2617
Exon 18 GCTGTTTTAATTCAAT 73 7837 7852 ekkdddddddddkeee 604 601374
2602 2617 Exon 18 GCTGTTTTAATTCAAT 66 7837 7852 kdkdddddddddeeee
604 601336 2603 2618 Exon 18 AGCTGTTTTAATTCAA 64 7838 7853
ekkdddddddddkeee 605 601375 2603 2618 Exon 18 AGCTGTTTTAATTCAA 61
7838 7853 kdkdddddddddeeee 605 532917 2604 2623 Exon 18
GTCGCAGCTGTTTTAATT 66 7839 7858 eeeeedddddddddde 317 CA eeee 601337
2604 2619 Exon 18 CAGCTGTTTTAATTCA 53 7839 7854 ekkdddddddddkeee
606 601376 2604 2619 Exon 18 CAGCTGTTTTAATTCA 39 7839 7854
kdkdddddddddeeee 606 601338 2605 2620 Exon 18 GCAGCTGTTTTAATTC 67
7840 7855 ekkdddddddddkeee 607 601377 2605 2620 Exon 18
GCAGCTGTTTTAATTC 67 7840 7855 kdkdddddddddeeee 607 601339 2606 2621
Exon 18 CGCAGCTGTTTTAATT 63 7841 7856 ekkdddddddddkeee 608 601378
2606 2621 Exon 18 CGCAGCTGTTTTAATT 60 7841 7856 kdkdddddddddeeee
608 601340 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 40 7842 7857
ekkdddddddddkeee 609 601379 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 36
7842 7857 kdkdddddddddeeee 609 588860 2608 2623 Exon 18
GTCGCAGCTGTTTTAA 84 7843 7858 eekdddddddddddke 610 601341 2608 2623
Exon 18 GTCGCAGCTGTTTTAA 74 7843 7858 ekkdddddddddkeee 610 601380
2608 2623 Exon 18 GTCGCAGCTGTTTTAA 78 7843 7858 kdkdddddddddeeee
610 601342 2609 2624 Exon 18 TGTCGCAGCTGTTTTA 68 7844 7859
ekkdddddddddkeee 611 601381 2609 2624 Exon 18 TGTCGCAGCTGTTTTA 66
7844 7859 kdkdddddddddeeee 611 601343 2610 2625 Exon 18
TTGTCGCAGCTGTTTT 71 7845 7860 ekkdddddddddkeee 612 601382 2610 2625
Exon 18 TTGTCGCAGCTGTTTT 84 7845 7860 kdkdddddddddeeee 612 601344
2611 2626 Exon 18 GTTGTCGCAGCTGTTT 87 7846 7861 ekkdddddddddkeee
613 601383 2611 2626 Exon 18 GTTGTCGCAGCTGTTT 85 7846 7861
kdkdddddddddeeee 613 601345 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 82
7847 7862 ekkdddddddddkeee 614 601384 2612 2627 Exon 18
TGTTGTCGCAGCTGTT 79 7847 7862 kdkdddddddddeeee 614 601346 2613 2628
Exon 18/ TTGTTGTCGCAGCTGT 73 n/a n/a ekkdddddddddkeee 615 Repeat
601385 2613 2628 Exon 18/ TTGTTGTCGCAGCTGT 84 n/a n/a
kdkdddddddddeeee 615 Repeat 601347 2614 2629 Exon 18/
TTTGTTGTCGCAGCTG 70 n/a n/a ekkdddddddddkeee 616 Repeat 601386 2614
2629 Exon 18/ TTTGTTGTCGCAGCTG 71 n/a n/a kdkdddddddddeeee 616
Repeat 601348 2615 2630 Exon 18/ TTTTGTTGTCGCAGCT 71 n/a n/a
ekkdddddddddkeee 617 Repeat 601387 2615 2630 Exon 18/
TTTTGTTGTCGCAGCT 76 n/a n/a kdkdddddddddeeee 617 Repeat 601349 2616
2631 Exon 18/ TTTTTGTTGTCGCAGC 71 n/a n/a ekkdddddddddkeee 618
Repeat 601388 2616 2631 Exon 18/ TTTTTGTTGTCGCAGC 67 n/a n/a
kdkdddddddddeeee 618 Repeat
TABLE-US-00138 TABLE 145 Inhibition of CFB mRNA by MOE gapmers
targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi- start
stop ID NO site site region Sequence bition site site Motif NO:
599357 2582 2600 Exon 18 CCACGCCCCTGTCCAGCAG 26 7817 7835 5-9-5 708
599358 2583 2601 Exon 18 CCCACGCCCCTGTCCAGCA 22 7818 7836 5-9-5 709
599359 2584 2602 Exon 18 TCCCACGCCCCTGTCCAGC 13 7819 7837 5-9-5 710
599360 2585 2603 Exon 18 ATCCCACGCCCCTGTCCAG 7 7820 7838 5-9-5 711
599361 2586 2604 Exon 18 AATCCCACGCCCCTGTCCA 11 7821 7839 5-9-5 712
599362 2587 2605 Exon 18 CAATCCCACGCCCCTGTCC 14 7822 7840 5-9-5 713
599363 2588 2606 Exon 18 TCAATCCCACGCCCCTGTC 17 7823 7841 5-9-5 714
599364 2589 2607 Exon 18 TTCAATCCCACGCCCCTGT 20 7824 7842 5-9-5 715
599365 2590 2608 Exon 18 ATTCAATCCCACGCCCCTG 22 7825 7843 5-9-5 716
599366 2591 2609 Exon 18 AATTCAATCCCACGCCCCT 13 7826 7844 5-9-5 717
599367 2592 2610 Exon 18 TAATTCAATCCCACGCCCC 11 7827 7845 5-9-5 718
599368 2593 2611 Exon 18 TTAATTCAATCCCACGCCC 10 7828 7846 5-9-5 719
599369 2594 2612 Exon 18 TTTAATTCAATCCCACGCC 19 7829 7847 5-9-5 720
599370 2595 2613 Exon 18 TTTTAATTCAATCCCACGC 23 7830 7848 5-9-5 721
599371 2596 2614 Exon 18 GTTTTAATTCAATCCCACG 4 7831 7849 5-9-5 722
599372 2597 2615 Exon 18 TGTTTTAATTCAATCCCAC 16 7832 7850 5-9-5 723
599373 2598 2616 Exon 18 CTGTTTTAATTCAATCCCA 3 7833 7851 5-9-5 724
599374 2599 2617 Exon 18 GCTGTTTTAATTCAATCCC 10 7834 7852 5-9-5 725
599375 2600 2618 Exon 18 AGCTGTTTTAATTCAATCC 17 7835 7853 5-9-5 726
599376 2601 2619 Exon 18 CAGCTGTTTTAATTCAATC 18 7836 7854 5-9-5 727
599377 2602 2620 Exon 18 GCAGCTGTTTTAATTCAAT 22 7837 7855 5-9-5 728
599378 2603 2621 Exon 18 CGCAGCTGTTTTAATTCAA 11 7838 7856 5-9-5 729
599511 2552 2571 Exon 18 ATAGAAAACCCAAATCCTCA 7 7787 7806 6-8-6 410
599389 2553 2572 Exon 18 TATAGAAAACCCAAATCCTC 22 7788 7807 6-8-6
411 599390 2554 2573 Exon 18 TTATAGAAAACCCAAATCCT 21 7789 7808
6-8-6 412 599391 2555 2574 Exon 18 CTTATAGAAAACCCAAATCC 27 7790
7809 6-8-6 413 599392 2556 2575 Exon 18 CCTTATAGAAAACCCAAATC 30
7791 7810 6-8-6 414 599393 2557 2576 Exon 18 CCCTTATAGAAAACCCAAAT
30 7792 7811 6-8-6 415 599394 2558 2577 Exon 18
CCCCTTATAGAAAACCCAAA 28 7793 7812 6-8-6 416 599395 2559 2578 Exon
18 ACCCCTTATAGAAAACCCAA 23 7794 7813 6-8-6 417 599396 2560 2579
Exon 18 AACCCCTTATAGAAAACCCA 53 7795 7814 6-8-6 418 599397 2561
2580 Exon 18 AAACCCCTTATAGAAAACCC 33 7796 7815 6-8-6 419 599398
2562 2581 Exon 18 GAAACCCCTTATAGAAAACC 58 7797 7816 6-8-6 420
599399 2563 2582 Exon 18 GGAAACCCCTTATAGAAAAC 23 7798 7817 6-8-6
421 599400 2564 2583 Exon 18 AGGAAACCCCTTATAGAAAA 54 7799 7818
6-8-6 422 599401 2565 2584 Exon 18 CAGGAAACCCCTTATAGAAA 30 7800
7819 6-8-6 423 599402 2566 2585 Exon 18 GCAGGAAACCCCTTATAGAA 25
7801 7820 6-8-6 424 599403 2567 2586 Exon 18 AGCAGGAAACCCCTTATAGA
17 7802 7821 6-8-6 425 599404 2568 2587 Exon 18
CAGCAGGAAACCCCTTATAG 20 7803 7822 6-8-6 426 599405 2569 2588 Exon
18 CCAGCAGGAAACCCCTTATA 12 7804 7823 6-8-6 427 599406 2570 2589
Exon 18 TCCAGCAGGAAACCCCTTAT 51 7805 7824 6-8-6 428 599407 2571
2590 Exon 18 GTCCAGCAGGAAACCCCTTA 39 7806 7825 6-8-6 237 599408
2572 2591 Exon 18 TGTCCAGCAGGAAACCCCTT 53 7807 7826 6-8-6 429
599409 2573 2592 Exon 18 CTGTCCAGCAGGAAACCCCT 65 7808 7827 6-8-6
430 599410 2574 2593 Exon 18 CCTGTCCAGCAGGAAACCCC 56 7809 7828
6-8-6 431 599411 2575 2594 Exon 18 CCCTGTCCAGCAGGAAACCC 60 7810
7829 6-8-6 432 599412 2576 2595 Exon 18 CCCCTGTCCAGCAGGAAACC 61
7811 7830 6-8-6 433 599413 2577 2596 Exon 18 GCCCCTGTCCAGCAGGAAAC
40 7812 7831 6-8-6 238 599414 2578 2597 Exon 18
CGCCCCTGTCCAGCAGGAAA 41 7813 7832 6-8-6 434 599415 2579 2598 Exon
18 ACGCCCCTGTCCAGCAGGAA 37 7814 7833 6-8-6 435 599416 2580 2599
Exon 18 CACGCCCCTGTCCAGCAGGA 54 7815 7834 6-8-6 436 599417 2581
2600 Exon 18 CCACGCCCCTGTCCAGCAGG 36 7816 7835 6-8-6 437 599418
2582 2601 Exon 18 CCCACGCCCCTGTCCAGCAG 53 7817 7836 6-8-6 438
599419 2583 2602 Exon 18 TCCCACGCCCCTGTCCAGCA 54 7818 7837 6-8-6
439 599420 2584 2603 Exon 18 ATCCCACGCCCCTGTCCAGC 50 7819 7838
6-8-6 440 599421 2585 2604 Exon 18 AATCCCACGCCCCTGTCCAG 48 7820
7839 6-8-6 441 599422 2586 2605 Exon 18 CAATCCCACGCCCCTGTCCA 55
7821 7840 6-8-6 442 599423 2587 2606 Exon 18 TCAATCCCACGCCCCTGTCC
75 7822 7841 6-8-6 443 599424 2588 2607 Exon 18
TTCAATCCCACGCCCCTGTC 69 7823 7842 6-8-6 444 599425 2589 2608 Exon
18 ATTCAATCCCACGCCCCTGT 77 7824 7843 6-8-6 445 599426 2590 2609
Exon 18 AATTCAATCCCACGCCCCTG 60 7825 7844 6-8-6 446 599427 2591
2610 Exon 18 TAATTCAATCCCACGCCCCT 72 7826 7845 6-8-6 447 599428
2592 2611 Exon 18 TTAATTCAATCCCACGCCCC 81 7827 7846 6-8-6 448
599429 2593 2612 Exon 18 TTTAATTCAATCCCACGCCC 68 7828 7847 6-8-6
449 599430 2594 2613 Exon 18 TTTTAATTCAATCCCACGCC 58 7829 7848
6-8-6 450 599431 2595 2614 Exon 18 GTTTTAATTCAATCCCACGC 70 7830
7849 6-8-6 451 599432 2596 2615 Exon 18 TGTTTTAATTCAATCCCACG 85
7831 7850 6-8-6 452 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA
85 7839 7858 5-10-5 317 599379 2604 2622 Exon 18
TCGCAGCTGTTTTAATTCA 73 7839 7857 5-9-5 730 599380 2605 2623 Exon 18
GTCGCAGCTGTTTTAATTC 77 7840 7858 5-9-5 731 599381 2606 2624 Exon 18
TGTCGCAGCTGTTTTAATT 69 7841 7859 5-9-5 732 599382 2607 2625 Exon 18
TTGTCGCAGCTGTTTTAAT 58 7842 7860 5-9-5 733 599383 2608 2626 Exon 18
GTTGTCGCAGCTGTTTTAA 52 7843 7861 5-9-5 734 599384 2609 2627 Exon 18
TGTTGTCGCAGCTGTTTTA 63 7844 7862 5-9-5 735 599385 2610 2628 Exon
18/ TTGTTGTCGCAGCTGTTTT 53 n/a n/a 5-9-5 736 Repeat 599386 2611
2629 Exon 18/ TTTGTTGTCGCAGCTGTTT 63 n/a n/a 5-9-5 737 Repeat
599387 2612 2630 Exon 18/ TTTTGTTGTCGCAGCTGTT 64 n/a n/a 5-9-5 438
Repeat 599388 2613 2631 Exon 18/ TTTTTGTTGTCGCAGCTGT 66 n/a n/a
5-9-5 739 Repeat
TABLE-US-00139 TABLE 146 Inhibition of CFB mRNA by MOE gapmers
targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID
NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi- start
stop ID NO site site region Sequence bition site site Motif NO:
599213 2553 2570 Exon 18 TAGAAAACCCAAATCCTC 0 7788 7805 3-10-5 785
599214 2554 2571 Exon 18 ATAGAAAACCCAAATCCT 0 7789 7806 3-10-5 786
599215 2555 2572 Exon 18 TATAGAAAACCCAAATCC 36 7790 7807 3-10-5 787
599216 2556 2573 Exon 18 TTATAGAAAACCCAAATC 8 7791 7808 3-10-5 788
599217 2557 2574 Exon 18 CTTATAGAAAACCCAAAT 5 7792 7809 3-10-5 789
599218 2558 2575 Exon 18 CCTTATAGAAAACCCAAA 0 7793 7810 3-10-5 790
599219 2559 2576 Exon 18 CCCTTATAGAAAACCCAA 8 7794 7811 3-10-5 791
599220 2560 2577 Exon 18 CCCCTTATAGAAAACCCA 0 7795 7812 3-10-5 740
599221 2561 2578 Exon 18 ACCCCTTATAGAAAACCC 54 7796 7813 3-10-5 741
599222 2562 2579 Exon 18 AACCCCTTATAGAAAACC 3 7797 7814 3-10-5 742
599223 2563 2580 Exon 18 AAACCCCTTATAGAAAAC 0 7798 7815 3-10-5 743
599224 2564 2581 Exon 18 GAAACCCCTTATAGAAAA 0 7799 7816 3-10-5 744
599225 2566 2583 Exon 18 AGGAAACCCCTTATAGAA 60 7801 7818 3-10-5 745
599226 2567 2584 Exon 18 CAGGAAACCCCTTATAGA 0 7802 7819 3-10-5 746
599227 2568 2585 Exon 18 GCAGGAAACCCCTTATAG 37 7803 7820 3-10-5 747
599228 2569 2586 Exon 18 AGCAGGAAACCCCTTATA 0 7804 7821 3-10-5 748
599229 2570 2587 Exon 18 CAGCAGGAAACCCCTTAT 39 7805 7822 3-10-5 749
599230 2571 2588 Exon 18 CCAGCAGGAAACCCCTTA 10 7806 7823 3-10-5 750
599231 2572 2589 Exon 18 TCCAGCAGGAAACCCCTT 16 7807 7824 3-10-5 751
599232 2573 2590 Exon 18 GTCCAGCAGGAAACCCCT 9 7808 7825 3-10-5 752
599233 2574 2591 Exon 18 TGTCCAGCAGGAAACCCC 44 7809 7826 3-10-5 753
599234 2575 2592 Exon 18 CTGTCCAGCAGGAAACCC 14 7810 7827 3-10-5 754
599235 2576 2593 Exon 18 CCTGTCCAGCAGGAAACC 0 7811 7828 3-10-5 755
599236 2577 2594 Exon 18 CCCTGTCCAGCAGGAAAC 43 7812 7829 3-10-5 756
599237 2578 2595 Exon 18 CCCCTGTCCAGCAGGAAA 0 7813 7830 3-10-5 757
599238 2580 2597 Exon 18 CGCCCCTGTCCAGCAGGA 9 7815 7832 3-10-5 758
599239 2581 2598 Exon 18 ACGCCCCTGTCCAGCAGG 36 7816 7833 3-10-5 759
599240 2582 2599 Exon 18 CACGCCCCTGTCCAGCAG 11 7817 7834 3-10-5 760
599241 2583 2600 Exon 18 CCACGCCCCTGTCCAGCA 51 7818 7835 3-10-5 761
599242 2584 2601 Exon 18 CCCACGCCCCTGTCCAGC 7 7819 7836 3-10-5 762
599243 2585 2602 Exon 18 TCCCACGCCCCTGTCCAG 47 7820 7837 3-10-5 763
599244 2586 2603 Exon 18 ATCCCACGCCCCTGTCCA 37 7821 7838 3-10-5 764
599245 2587 2604 Exon 18 AATCCCACGCCCCTGTCC 35 7822 7839 3-10-5 765
599246 2588 2605 Exon 18 CAATCCCACGCCCCTGTC 21 7823 7840 3-10-5 766
599247 2589 2606 Exon 18 TCAATCCCACGCCCCTGT 61 7824 7841 3-10-5 767
599248 2590 2607 Exon 18 TTCAATCCCACGCCCCTG 51 7825 7842 3-10-5 768
599249 2591 2608 Exon 18 ATTCAATCCCACGCCCCT 58 7826 7843 3-10-5 769
599250 2592 2609 Exon 18 AATTCAATCCCACGCCCC 49 7827 7844 3-10-5 770
599251 2593 2610 Exon 18 TAATTCAATCCCACGCCC 46 7828 7845 3-10-5 771
599252 2594 2611 Exon 18 TTAATTCAATCCCACGCC 32 7829 7846 3-10-5 772
599253 2595 2612 Exon 18 TTTAATTCAATCCCACGC 23 7830 7847 3-10-5 773
599254 2596 2613 Exon 18 TTTTAATTCAATCCCACG 0 7831 7848 3-10-5 774
599255 2597 2614 Exon 18 GTTTTAATTCAATCCCAC 61 7832 7849 3-10-5 775
599256 2598 2615 Exon 18 TGTTTTAATTCAATCCCA 64 7833 7850 3-10-5 776
599257 2599 2616 Exon 18 CTGTTTTAATTCAATCCC 66 7834 7851 3-10-5 777
599258 2600 2617 Exon 18 GCTGTTTTAATTCAATCC 59 7835 7852 3-10-5 778
599259 2601 2618 Exon 18 AGCTGTTTTAATTCAATC 40 7836 7853 3-10-5 779
599260 2602 2619 Exon 18 CAGCTGTTTTAATTCAAT 38 7837 7854 3-10-5 780
599261 2603 2620 Exon 18 GCAGCTGTTTTAATTCAA 54 7838 7855 3-10-5 781
599509 2552 2570 Exon 18 TAGAAAACCCAAATCCTCA 54 7787 7805 6-7-6 681
599273 2553 2571 Exon 18 ATAGAAAACCCAAATCCTC 0 7788 7806 6-7-6 682
599274 2554 2572 Exon 18 TATAGAAAACCCAAATCCT 57 7789 7807 6-7-6 683
599275 2556 2574 Exon 18 CTTATAGAAAACCCAAATC 0 7791 7809 6-7-6 684
599276 2557 2575 Exon 18 CCTTATAGAAAACCCAAAT 44 7792 7810 6-7-6 685
599277 2558 2576 Exon 18 CCCTTATAGAAAACCCAAA 0 7793 7811 6-7-6 686
599278 2559 2577 Exon 18 CCCCTTATAGAAAACCCAA 0 7794 7812 6-7-6 687
599279 2560 2578 Exon 18 ACCCCTTATAGAAAACCCA 20 7795 7813 6-7-6 688
599280 2561 2579 Exon 18 AACCCCTTATAGAAAACCC 70 7796 7814 6-7-6 689
532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 85 7839 7858 5-10-5
317 599262 2604 2621 Exon 18 CGCAGCTGTTTTAATTCA 49 7839 7856 3-10-5
782 599263 2605 2622 Exon 18 TCGCAGCTGTTTTAATTC 49 7840 7857 3-10-5
783 599264 2606 2623 Exon 18 GTCGCAGCTGTTTTAATT 62 7841 7858 3-10-5
784 599265 2607 2624 Exon 18 TGTCGCAGCTGTTTTAAT 63 7842 7859 3-10-5
792 599266 2608 2625 Exon 18 TTGTCGCAGCTGTTTTAA 41 7843 7860 3-10-5
793 599267 2609 2626 Exon 18 GTTGTCGCAGCTGTTTTA 52 7844 7861 3-10-5
794 599268 2610 2627 Exon 18 TGTTGTCGCAGCTGTTTT 51 7845 7862 3-10-5
795 599269 2611 2628 Exon 18/ TTGTTGTCGCAGCTGTTT 58 n/a n/a 3-10-5
796 Repeat 599270 2612 2629 Exon 18/ TTTGTTGTCGCAGCTGTT 69 n/a n/a
3-10-5 797 Repeat 599271 2613 2630 Exon 18/ TTTTGTTGTCGCAGCTGT 69
n/a n/a 3-10-5 798 Repeat 599272 2614 2631 Exon 18/
TTTTTGTTGTCGCAGCTG 72 n/a n/a 3-10-5 799 Repeat 599205 2607 2624
Exon 18 TGTCGCAGCTGTTTTAAT 54 7842 7859 5-8-5 792 599206 2608 2625
Exon 18 TTGTCGCAGCTGTTTTAA 62 7843 7860 5-8-5 793 599207 2609 2626
Exon 18 GTTGTCGCAGCTGTTTTA 62 7844 7861 5-8-5 794 599208 2610 2627
Exon 18 TGTTGTCGCAGCTGTTTT 66 7845 7862 5-8-5 795 599209 2611 2628
Exon 18/ TTGTTGTCGCAGCTGTTT 60 n/a n/a 5-8-5 796 Repeat 599210 2612
2629 Exon 18/ TTTGTTGTCGCAGCTGTT 62 n/a n/a 5-8-5 797 Repeat 599211
2613 2630 Exon 18/ TTTTGTTGTCGCAGCTGT 65 n/a n/a 5-8-5 798 Repeat
599212 2614 2631 Exon 18/ TTTTTGTTGTCGCAGCTG 67 n/a n/a 5-8-5 799
Repeat
TABLE-US-00140 TABLE 147 Inhibition of CFB mRNA by 5-10-5 MOE
gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID
SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISIS start stop Target inhi-
start stop ID NO site site region Sequence bition site site NO:
588570 150 169 Exon 1 TGGTCACATTCCCTTCCCCT 72 1871 1890 396 588571
152 171 Exon 1 CCTGGTCACATTCCCTTCCC 80 1873 1892 397 532614 154 173
Exon 1 GACCTGGTCACATTCCCTTC 65 1875 1894 12 588572 156 175 Exon 1
TAGACCTGGTCACATTCCCT 74 1877 1896 398 588573 158 177 Exon 1
CCTAGACCTGGTCACATTCC 72 1879 1898 399 588566 2189 2208 Exon 15
CCTTCCGAGTCAGCTTTTTC 66 6977 6996 400 588567 2191 2210 Exon 15
CTCCTTCCGAGTCAGCTTTT 66 6979 6998 401 532770 2193 2212 Exon 15
ACCTCCTTCCGAGTCAGCTT 64 6981 7000 198 588568 2195 2214 Exon 15
AGACCTCCTTCCGAGTCAGC 78 6983 7002 402 588569 2197 2216 Exon 15
GTAGACCTCCTTCCGAGTCA 74 6985 7004 403 588574 2453 2472 Exon 18
TTTGCCGCTTCTGGTTTTTG 71 7688 7707 404 588575 2455 2474 Exon 18
CTTTTGCCGCTTCTGGTTTT 72 7690 7709 405 532800 2457 2476 Exon 18
TGCTTTTGCCGCTTCTGGTT 71 7692 7711 228 588576 2459 2478 Exon 18
CCTGCTTTTGCCGCTTCTGG 59 7694 7713 406 588577 2461 2480 Exon 18
TACCTGCTTTTGCCGCTTCT 76 7696 7715 407 516350 2550 2569 Exon 18
AGAAAACCCAAATCCTCATC 58 7785 7804 408 588509 2551 2570 Exon 18
TAGAAAACCCAAATCCTCAT 6 7786 7805 409 588510 2552 2571 Exon 18
ATAGAAAACCCAAATCCTCA 10 7787 7806 410 588511 2553 2572 Exon 18
TATAGAAAACCCAAATCCTC 9 7788 7807 411 588512 2554 2573 Exon 18
TTATAGAAAACCCAAATCCT 80 7789 7808 412 588513 2555 2574 Exon 18
CTTATAGAAAACCCAAATCC 70 7790 7809 413 588514 2556 2575 Exon 18
CCTTATAGAAAACCCAAATC 71 7791 7810 414 588515 2557 2576 Exon 18
CCCTTATAGAAAACCCAAAT 78 7792 7811 415 588516 2558 2577 Exon 18
CCCCTTATAGAAAACCCAAA 72 7793 7812 416 588517 2559 2578 Exon 18
ACCCCTTATAGAAAACCCAA 80 7794 7813 417 588518 2560 2579 Exon 18
AACCCCTTATAGAAAACCCA 80 7795 7814 418 588519 2561 2580 Exon 18
AAACCCCTTATAGAAAACCC 62 7796 7815 419 588520 2562 2581 Exon 18
GAAACCCCTTATAGAAAACC 59 7797 7816 420 588521 2563 2582 Exon 18
GGAAACCCCTTATAGAAAAC 40 7798 7817 421 588522 2564 2583 Exon 18
AGGAAACCCCTTATAGAAAA 66 7799 7818 422 588523 2565 2584 Exon 18
CAGGAAACCCCTTATAGAAA 63 7800 7819 423 588524 2566 2585 Exon 18
GCAGGAAACCCCTTATAGAA 70 7801 7820 424 588525 2567 2586 Exon 18
AGCAGGAAACCCCTTATAGA 67 7802 7821 425 588526 2568 2587 Exon 18
CAGCAGGAAACCCCTTATAG 0 7803 7822 426 588527 2569 2588 Exon 18
CCAGCAGGAAACCCCTTATA 11 7804 7823 427 588528 2570 2589 Exon 18
TCCAGCAGGAAACCCCTTAT 15 7805 7824 428 532809 2571 2590 Exon 18
GTCCAGCAGGAAACCCCTTA 75 7806 7825 237 588529 2572 2591 Exon 18
TGTCCAGCAGGAAACCCCTT 16 7807 7826 429 588530 2573 2592 Exon 18
CTGTCCAGCAGGAAACCCCT 16 7808 7827 430 588531 2574 2593 Exon 18
CCTGTCCAGCAGGAAACCCC 19 7809 7828 431 588532 2575 2594 Exon 18
CCCTGTCCAGCAGGAAACCC 15 7810 7829 432 588533 2576 2595 Exon 18
CCCCTGTCCAGCAGGAAACC 29 7811 7830 433 532810 2577 2596 Exon 18
GCCCCTGTCCAGCAGGAAAC 74 7812 7831 238 588534 2578 2597 Exon 18
CGCCCCTGTCCAGCAGGAAA 21 7813 7832 434 588535 2579 2598 Exon 18
ACGCCCCTGTCCAGCAGGAA 16 7814 7833 435 588536 2580 2599 Exon 18
CACGCCCCTGTCCAGCAGGA 0 7815 7834 436 588537 2581 2600 Exon 18
CCACGCCCCTGTCCAGCAGG 8 7816 7835 437 588538 2582 2601 Exon 18
CCCACGCCCCTGTCCAGCAG 10 7817 7836 438 588539 2583 2602 Exon 18
TCCCACGCCCCTGTCCAGCA 23 7818 7837 439 588540 2584 2603 Exon 18
ATCCCACGCCCCTGTCCAGC 16 7819 7838 440 588541 2585 2604 Exon 18
AATCCCACGCCCCTGTCCAG 16 7820 7839 441 588542 2586 2605 Exon 18
CAATCCCACGCCCCTGTCCA 12 7821 7840 442 588543 2587 2606 Exon 18
TCAATCCCACGCCCCTGTCC 26 7822 7841 443 588544 2588 2607 Exon 18
TTCAATCCCACGCCCCTGTC 26 7823 7842 444 588545 2589 2608 Exon 18
ATTCAATCCCACGCCCCTGT 31 7824 7843 445 588546 2590 2609 Exon 18
AATTCAATCCCACGCCCCTG 22 7825 7844 446 588547 2591 2610 Exon 18
TAATTCAATCCCACGCCCCT 12 7826 7845 447 588548 2592 2611 Exon 18
TTAATTCAATCCCACGCCCC 20 7827 7846 448 588549 2593 2612 Exon 18
TTTAATTCAATCCCACGCCC 26 7828 7847 449 588550 2594 2613 Exon 18
TTTTAATTCAATCCCACGCC 32 7829 7848 450 588551 2595 2614 Exon 18
GTTTTAATTCAATCCCACGC 48 7830 7849 451 588552 2596 2615 Exon 18
TGTTTTAATTCAATCCCACG 57 7831 7850 452 588553 2597 2616 Exon 18
CTGTTTTAATTCAATCCCAC 49 7832 7851 453 588554 2598 2617 Exon 18
GCTGTTTTAATTCAATCCCA 64 7833 7852 454 532811 2599 2618 Exon 18
AGCTGTTTTAATTCAATCCC 78 7834 7853 239 588555 2600 2619 Exon 18
CAGCTGTTTTAATTCAATCC 48 7835 7854 455 588556 2601 2620 Exon 18
GCAGCTGTTTTAATTCAATC 55 7836 7855 456 588557 2602 2621 Exon 18
CGCAGCTGTTTTAATTCAAT 51 7837 7856 457 588558 2603 2622 Exon 18
TCGCAGCTGTTTTAATTCAA 51 7838 7857 458 532917 2604 2623 Exon 18
GTCGCAGCTGTTTTAATTCA 82 7839 7858 317 588559 2605 2624 Exon 18
TGTCGCAGCTGTTTTAATTC 58 7840 7859 459 588560 2606 2625 Exon 18
TTGTCGCAGCTGTTTTAATT 72 7841 7860 460 588561 2607 2626 Exon 18
GTTGTCGCAGCTGTTTTAAT 75 7842 7861 461 532952 2608 2627 Exon 18
TGTTGTCGCAGCTGTTTTAA 39 7843 7862 395 588562 2609 2628 Exon 18/
TTGTTGTCGCAGCTGTTTTA 53 n/a n/a 462 Repeat 588563 2610 2629 Exon
18/ TTTGTTGTCGCAGCTGTTTT 62 n/a n/a 463 Repeat 588564 2611 2630
Exon 18/ TTTTGTTGTCGCAGCTGTTT 63 n/a n/a 464 Repeat 588565 2612
2631 Exon 18/ TTTTTGTTGTCGCAGCTGTT 64 n/a n/a 465 Repeat
Example 122: Dose-Dependent Antisense Inhibition of Human CFB in
HepG2 Cells by 5-10-5 MOE Gapmers
[1096] Gapmers from studies described above exhibiting in vitro
inhibition of CFB mRNA were selected and tested at various doses in
HepG2 cells. Cells were plated at a density of 20,000 cells per
well and transfected using electroporation with 0.313 .mu.M, 0.625
.mu.M, 1.25 .mu.M, 2.50 .mu.M, 5.00 .mu.M, or 10.00 .mu.M
concentrations of antisense oligonucleotide, as specified in the
Table below. After a treatment period of approximately 16 hours,
RNA was isolated from the cells and CFB mRNA levels were measured
by quantitative real-time PCR. Human primer probe set RTS3459 was
used to measure mRNA levels. CFB mRNA levels were adjusted
according to total RNA content, as measured by RIBOGREEN.RTM..
Results are presented as percent inhibition of CFB, relative to
untreated control cells.
[1097] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented. CFB mRNA levels were
reduced in a dose-dependent manner in antisense oligonucleotide
treated cells.
TABLE-US-00141 TABLE 148 ISIS 0.313 0.625 1.25 2.50 5.00 10.00
IC.sub.50 No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 532614 7
13 43 72 65 71 2.2 532635 12 0 3 28 0 0 >10 532692 26 0 12 52 55
74 3.7 532770 21 18 32 73 64 88 1.8 532775 8 0 26 35 47 59 6.2
532800 0 5 30 65 50 75 3.1 532809 12 30 28 40 46 66 4.6 532810 28
44 32 69 84 95 1.2 532811 66 83 90 94 97 99 <0.3 532917 64 85 88
96 97 99 <0.3 532952 50 53 68 80 91 94 0.4
Example 123: Dose-Dependent Antisense Inhibition of Human CFB in
HepG2 Cells
[1098] Gapmers from studies described above exhibiting in vitro
inhibition of CFB mRNA were selected and tested at various doses in
HepG2 cells. The antisense oligonucleotides were tested in a number
of experiments with similar culture conditions. The results for
each experiment are presented in separate tables shown below. Cells
were plated at a density of 20,000 cells per well and transfected
using electroporation with 0.08 .mu.M, 0.25 .mu.M, 0.74 .mu.M, 2.22
.mu.M, 6.67 .mu.M, and 20.00 .mu.M concentrations of antisense
oligonucleotide, as specified in the Table below. After a treatment
period of approximately 16 hours, RNA was isolated from the cells
and CFB mRNA levels were measured by quantitative real-time PCR.
Human primer probe set RTS3459 was used to measure mRNA levels. CFB
mRNA levels were adjusted according to total RNA content, as
measured by RIBOGREEN.RTM.. Results are presented as percent
inhibition of CFB, relative to untreated control cells.
[1099] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented. CFB mRNA levels were
reduced in a dose-dependent manner in antisense oligonucleotide
treated cells.
TABLE-US-00142 TABLE 149 ISIS 0.08 0.25 0.74 2.22 6.67 20.00
IC.sub.50 No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 532811 19
53 81 87 96 97 0.2 588834 7 42 64 92 98 98 0.5 588835 11 30 66 89
97 97 0.5 588836 14 40 61 91 97 97 0.5 588837 6 39 67 89 96 97 0.5
588838 0 27 41 81 87 97 1.0 588842 17 51 68 86 93 95 0.3 588843 21
38 72 90 95 96 0.4 588870 9 31 56 88 95 97 0.6 588871 14 25 47 79
93 97 0.7 588872 18 28 59 84 92 97 0.6
TABLE-US-00143 TABLE 150 ISIS 0.08 0.25 0.74 2.22 6.67 20.00
IC.sub.50 No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 532811 31
70 89 94 97 97 0.1 588844 31 60 77 91 95 96 0.1 588846 32 52 78 89
95 97 0.2 588847 22 52 77 91 95 97 0.2 588848 20 40 73 91 96 98 0.3
588851 40 52 82 94 97 97 0.1 588854 17 55 59 84 94 96 0.4 588855 10
32 56 82 93 96 0.6 588856 13 46 75 90 96 97 0.3 588857 11 52 73 94
96 97 0.3 588858 19 48 75 94 97 98 0.3
TABLE-US-00144 TABLE 151 ISIS 0.08 0.25 0.74 2.22 6.67 20.00
IC.sub.50 No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 532811 42
66 88 96 97 98 0.1 588859 18 46 66 90 96 97 0.4 588860 55 80 94 97
97 97 <0.1 588861 24 61 86 93 96 97 0.2 588862 25 64 85 94 96 98
0.1 588863 50 73 89 96 96 98 <0.1 588864 52 80 92 96 98 98
<0.1 588865 46 72 91 96 96 99 <0.1 588866 47 76 88 96 97 98
<0.1 588867 43 69 83 92 96 99 0.1 588868 43 56 65 84 93 97
0.1
TABLE-US-00145 TABLE 152 ISIS 0.08 0.25 0.74 2.22 6.67 20.00
IC.sub.50 No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 532810 0
14 38 72 89 96 1.2 532811 18 54 79 93 96 97 0.3 532952 19 34 73 87
94 96 0.4 588534 17 13 44 77 93 97 0.9 588544 12 43 69 86 89 93 0.4
588545 17 55 67 86 91 93 0.3 588546 10 32 67 85 91 93 0.6 588552 27
54 76 90 94 97 0.2 588553 32 68 87 93 95 97 0.1 588560 16 54 76 90
94 96 0.3 588561 18 45 68 85 93 96 0.4
TABLE-US-00146 TABLE 153 ISIS 0.08 0.25 0.74 2.22 6.67 20.00
IC.sub.50 No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 532811 22
60 82 94 97 98 0.2 588536 2 38 65 90 96 97 0.6 588537 12 38 63 87
94 97 0.5 588547 19 35 61 86 93 97 0.5 588548 19 36 75 88 95 96 0.4
588554 0 76 92 95 97 97 <0.1 588555 31 61 89 96 97 98 0.1 588556
33 56 82 95 94 97 0.1 588562 12 39 71 87 94 97 0.4 588563 25 48 72
86 94 96 0.3 588564 15 33 63 89 91 97 0.5
TABLE-US-00147 TABLE 154 ISIS 0.08 0.25 0.74 2.22 6.67 20.00
IC.sub.50 No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 532811 39
68 86 96 98 98 0.1 588538 0 40 82 94 97 98 0.3 588539 34 65 88 95
98 98 0.1 588540 30 51 81 91 97 98 0.2 588549 31 57 82 95 96 98 0.1
588550 34 65 88 96 98 98 0.1 588551 47 66 87 96 98 99 <0.1
588557 40 84 95 98 98 98 <0.1 588558 45 73 93 97 98 99 <0.1
588559 51 69 83 96 98 99 <0.1 588565 19 56 81 92 96 98 0.2
Example 124: Dose-Dependent Antisense Inhibition of Human CFB in
HepG2 Cells
[1100] Gapmers from studies described above exhibiting in vitro
inhibition of CFB mRNA were selected and tested at various doses in
HepG2 cells. The antisense oligonucleotides were tested in a number
of experiments with similar culture conditions. The results for
each experiment are presented in separate tables shown below. Cells
were plated at a density of 20,000 cells per well and transfected
using electroporation with 0.06 .mu.M, 0.25 .mu.M, 1.00 .mu.M, and
4.00 .mu.M concentrations of antisense oligonucleotide, as
specified in the Table below. After a treatment period of
approximately 16 hours, RNA was isolated from the cells and CFB
mRNA levels were measured by quantitative real-time PCR. Human
primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA
levels were adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of CFB,
relative to untreated control cells.
[1101] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented. CFB mRNA levels were
reduced in a dose-dependent manner in antisense oligonucleotide
treated cells.
TABLE-US-00148 TABLE 155 ISIS 0.06 0.25 1.00 4.00 IC.sub.50 No
.mu.M .mu.M .mu.M .mu.M (.mu.M) 532917 31 58 87 92 0.2 588860 18 50
79 93 0.3 599001 16 28 69 90 0.5 599024 14 32 74 90 0.4 599025 0 31
56 92 0.7 599032 28 44 62 88 0.3 599033 28 46 80 92 0.2 599077 8 20
59 80 0.8 599080 9 33 48 76 0.9 599086 7 22 53 83 0.8 599087 21 31
74 87 0.4 599088 13 37 69 82 0.5 599089 3 36 55 79 0.7 599093 25 59
79 88 0.2 599094 19 29 75 89 0.4 599095 29 43 67 87 0.3 599096 23
51 70 88 0.3 599149 20 53 82 92 0.3 599188 0 21 62 85 0.8
TABLE-US-00149 TABLE 156 ISIS 0.06 0.25 1.00 4.00 IC.sub.50 No
.mu.M .mu.M .mu.M .mu.M (.mu.M) 532917 0 42 81 91 0.4 588860 17 49
74 92 0.3 599155 29 52 67 87 0.3 599198 3 25 64 89 0.6 599201 13 26
67 91 0.5 599202 0 44 72 87 0.5 599203 22 41 75 88 0.3 599314 12 34
71 84 0.5 599316 7 37 66 88 0.5 599317 8 1 54 83 1.0 599321 8 33 70
85 0.5 599322 24 38 66 87 0.4 599327 22 32 66 89 0.4 599328 0 31 59
88 0.7 599330 5 43 67 84 0.5 599374 23 42 80 91 0.3 599378 21 57 80
93 0.2 599380 23 56 82 93 0.2 599432 17 37 73 93 0.4
TABLE-US-00150 TABLE 157 ISIS 0.06 0.25 1.00 4.00 IC.sub.50 No
.mu.M .mu.M .mu.M .mu.M (.mu.M) 532917 23 65 76 93 0.2 588860 17 60
76 90 0.3 601282 48 68 81 88 0.1 601269 18 59 80 94 0.2 601276 34
64 81 91 0.1 601275 14 39 78 90 0.4 601344 52 84 92 94 <0.06
601383 53 81 86 94 <0.06 601382 41 76 88 94 0.1 601385 52 74 89
91 <0.06 601332 41 69 86 94 0.1 601345 36 75 86 95 0.1 601371 34
72 91 93 0.1 601384 50 78 91 95 <0.06 601380 28 57 83 92 0.2
601387 48 61 82 88 0.1 601341 28 65 83 91 0.2 601346 31 69 82 93
0.1 601335 24 56 85 92 0.2
TABLE-US-00151 TABLE 158 ISIS 0.06 0.25 1.00 4.00 IC.sub.50 No
.mu.M .mu.M .mu.M .mu.M (.mu.M) 532917 31 66 86 93 0.1 588860 28 62
85 94 0.2 599208 24 50 71 89 0.3 599261 31 49 81 94 0.2 599267 41
48 80 88 0.2 599268 28 56 75 92 0.2 599313 14 24 71 92 0.5 599441
24 57 80 87 0.2 599494 13 55 86 94 0.3 599552 30 69 93 95 0.1
599553 34 71 93 96 0.1 599554 30 74 93 96 0.1 599568 40 77 90 97
0.1 599570 61 82 93 96 <0.06 599577 18 62 81 93 0.2 599581 27 60
80 94 0.2 599591 49 74 93 96 <0.06 599592 46 76 90 94 0.1 599593
44 72 91 95 0.1
TABLE-US-00152 TABLE 159 ISIS 0.06 0.25 1.00 4.00 IC.sub.50 No
.mu.M .mu.M .mu.M .mu.M (.mu.M) 532917 25 56 84 92 0.2 588860 11 51
80 92 0.3 599547 23 60 82 90 0.2 599569 42 73 85 88 0.1 599578 29
49 82 89 0.2 599582 21 56 78 91 0.2 599590 24 62 80 90 0.2 601209
21 49 85 88 0.3 601210 34 64 86 92 0.1 601212 46 68 88 90 0.1
601213 54 80 90 92 <0.06 601214 38 77 88 95 0.1 601215 42 64 85
92 0.1 601216 45 57 76 89 0.1 601264 29 58 86 95 0.2 601278 51 82
83 93 <0.06 601279 44 80 92 96 0.1 601280 44 73 87 94 0.1 601281
51 80 91 94 <0.06
Example 125: Dose-Dependent Antisense Inhibition of Human CFB in
HepG2 Cells
[1102] Gapmers from studies described above exhibiting in vitro
inhibition of CFB mRNA were selected and tested at various doses in
HepG2 cells. Additionally, a deoxy, MOE and (S)-cEt
oligonucleotide, ISIS 594430, was designed with the same sequence
(CTCCTTCCGAGTCAGC, SEQ ID NO: 549) and target region (target start
site 2195 of SEQ ID NO: 1 and target start site 6983 of SED ID NO:
2) as ISIS 588870, another deoxy, MOE, and (S)-cEt oligonucleotide.
ISIS 594430 is a 3-10-3 (S)-cEt gapmer.
[1103] Cells were plated at a density of 20,000 cells per well and
transfected using electroporation with 0.01 .mu.M, 0.04 .mu.M, 0.12
.mu.M, 0.37 .mu.M, 1.11 .mu.M, 3.33 .mu.M, and 10.00 .mu.M
concentrations of antisense oligonucleotide, as specified in the
Table below. After a treatment period of approximately 16 hours,
RNA was isolated from the cells and CFB mRNA levels were measured
by quantitative real-time PCR. Human primer probe set RTS3459 was
used to measure mRNA levels. CFB mRNA levels were adjusted
according to total RNA content, as measured by RIBOGREEN.RTM..
Results are presented as percent inhibition of CFB, relative to
untreated control cells.
[1104] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented. CFB mRNA levels were
reduced in a dose-dependent manner in antisense oligonucleotide
treated cells.
TABLE-US-00153 TABLE 160 ISIS 0.01 0.04 0.12 0.37 1.11 3.33 10.00
IC.sub.50 No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M)
588536 0 0 0 5 45 73 94 1.4 588548 0 0 0 19 52 78 90 1.2 588553 0 0
9 42 76 85 94 0.6 588555 0 52 23 58 78 83 95 0.3 588847 4 1 18 45
67 84 96 0.5 588848 0 3 13 38 67 83 95 0.6 594430 0 0 10 34 50 55
84 1.4
Example 126: Tolerability of MOE Gapmers Targeting Human CFB in CD1
Mice
[1105] CD1.RTM. mice (Charles River, Mass.) are a multipurpose mice
model, frequently utilized for safety and efficacy testing. The
mice were treated with ISIS antisense oligonucleotides selected
from studies described above and evaluated for changes in the
levels of various plasma chemistry markers.
Study 1 (with 5-10-5 MOE Gapmers)
[1106] Groups of seven-week old male CD1 mice were injected
subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS
oligonucleotide. A group of male CD1 mice was injected
subcutaneously once a week for 6 weeks with PBS. One group of mice
was injected with subcutaneously once a week for 6 weeks with 100
mg/kg of control oligonucleotide ISIS 141923 (CCTTCCCTGAAGGTTCCTCC,
designated herein as SEQ ID NO: 809, 5-10-5 MOE gapmer with no
known murine target). Mice were euthanized 48 hours after the last
dose, and organs and plasma were harvested for further
analysis.
Plasma Chemistry Markers
[1107] To evaluate the effect of ISIS oligonucleotides on liver and
kidney function, plasma levels of transaminases, and BUN were
measured using an automated clinical chemistry analyzer (Hitachi
Olympus AU400e, Melville, N.Y.). The results are presented in the
Table below. ISIS oligonucleotides that caused changes in the
levels of any of the liver or kidney function markers outside the
expected range for antisense oligonucleotides were excluded in
further studies.
TABLE-US-00154 TABLE 161 Plasma chemistry markers in CD1 mice
plasma on day 40 ALT (IU/L) AST (IU/L) BUN (mg/dL) PBS 25 46 20
ISIS 532614 513 407 22 ISIS 532692 131 130 24 ISIS 532770 36 53 25
ISIS 532775 193 158 23 ISIS 532800 127 110 25 ISIS 532809 36 42 22
ISIS 532810 229 286 26 ISIS 532811 197 183 21 ISIS 532917 207 204
27 ISIS 532952 246 207 25 ISIS 141923 39 67 23
Weights Body weights of the mice were measured on day 40 before
sacrificing the mice. Weights of organs, liver, kidney, and spleen
were also measured after the mice were sacrificed. The results are
presented in the Table below. ISIS oligonucleotides that caused
changes in the weights outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00155 TABLE 162 Weights (g) of CD1 mice on day 40 Body
Kidney Liver Spleen PBS 44 0.8 2.0 0.1 ISIS 532614 43 0.7 4.3 0.2
ISIS 532692 42 0.7 2.6 0.2 ISIS 532770 42 0.6 2.3 0.2 ISIS 532775
42 0.7 2.5 0.2 ISIS 532800 43 0.6 2.8 0.3 ISIS 532809 42 0.6 2.2
0.1 ISIS 532810 43 0.6 2.3 0.2 ISIS 532811 41 0.7 2.4 0.2 ISIS
532917 42 0.7 3.0 0.2 ISIS 532952 44 0.8 2.5 0.3 ISIS 141923 41 0.6
2.0 0.1
Study 2 (with 5-10-5 MOE Gapmers)
[1108] Groups of six- to eight-week old male CD1 mice were injected
subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS
oligonucleotide. Two groups of male CD1 mice were injected
subcutaneously once a week for 6 weeks with PBS. One group of mice
was injected with subcutaneously once a week for 6 weeks with 100
mg/kg of control oligonucleotide ISIS 141923. Mice were euthanized
48 hours after the last dose, and organs and plasma were harvested
for further analysis.
Plasma Chemistry Markers
[1109] To evaluate the effect of ISIS oligonucleotides on liver and
kidney function, plasma levels of transaminases, albumin, and BUN
were measured using an automated clinical chemistry analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). The results are presented
in the Table below. ISIS oligonucleotides that caused changes in
the levels of any of the liver or kidney function markers outside
the expected range for antisense oligonucleotides were excluded in
further studies.
TABLE-US-00156 TABLE 163 Plasma chemistry markers in CD1 mice
plasma on day 45 ALT AST Albumin BUN (IU/L) (IU/L) (g/dL) (mg/dL)
PBS 39 53 2.9 29 PBS 50 97 2.9 30 ISIS 141923 163 174 4.1 25 ISIS
532810 321 297 2.5 26 ISIS 532952 182 199 2.7 27 ISIS 588534 276
248 2.6 29 ISIS 588536 48 60 2.9 31 ISIS 588537 72 79 4.0 25 ISIS
588538 63 67 4.5 29 ISIS 588539 238 177 3.9 28 ISIS 588545 496 256
4.4 24 ISIS 588547 323 210 4.4 25 ISIS 588548 61 63 4.2 27 ISIS
588549 127 132 4.1 23 ISIS 588551 302 282 4.2 22 ISIS 588552 76 98
4.0 30 ISIS 588558 1066 521 3.9 27 ISIS 588559 76 94 4.1 26 ISIS
588561 502 500 4.4 26 ISIS 588563 50 99 4.4 28
Weights
[1110] Body weights of the mice were measured on day 42. Weights of
organs, liver, kidney, and spleen were also measured after the mice
were sacrificed on day 45. The results are presented in the Table
below. ISIS oligonucleotides that caused changes in the weights
outside the expected range for antisense oligonucleotides were
excluded in further studies.
TABLE-US-00157 TABLE 164 Weights (g) of CD1 mice on day 40 Body
Kidney Liver Spleen PBS 44 0.7 2.4 0.1 PBS 43 0.7 2.4 0.2 ISIS
141923 43 0.6 2.4 0.2 ISIS 532810 41 0.6 1.9 0.1 ISIS 532952 43 0.6
2.4 0.2 ISIS 588534 44 0.7 2.8 0.2 ISIS 588536 43 0.7 2.7 0.2 ISIS
588537 43 0.7 2.4 0.2 ISIS 588538 44 0.7 2.8 0.2 ISIS 588539 44 0.6
2.7 0.2 ISIS 588545 44 0.8 3.3 0.3 ISIS 588547 42 0.6 3.3 0.3 ISIS
588548 43 0.6 2.8 0.2 ISIS 588549 42 0.6 2.8 0.3 ISIS 588551 39 0.6
2.2 0.2 ISIS 588552 41 0.6 2.2 0.2 ISIS 588558 44 0.7 3.3 0.3 ISIS
588559 43 0.6 2.7 0.3 ISIS 588561 40 0.7 2.4 0.3 ISIS 588563 41 0.7
2.4 0.2
Study 3 (with 5-10-5 MOE Gapmers)
[1111] Groups of six- to eight-week old male CD1 mice were injected
subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS
oligonucleotide. Two groups of male CD1 mice were injected
subcutaneously once a week for 6 weeks with PBS. Mice were
euthanized 48 hours after the last dose, and organs and plasma were
harvested for further analysis.
Plasma Chemistry Markers
[1112] To evaluate the effect of ISIS oligonucleotides on liver and
kidney function, plasma levels of transaminases, albumin, and BUN
were measured using an automated clinical chemistry analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). The results are presented
in the Table below. ISIS oligonucleotides that caused changes in
the levels of any of the liver or kidney function markers outside
the expected range for antisense oligonucleotides were excluded in
further studies.
TABLE-US-00158 TABLE 165 Plasma chemistry markers in CD1 mice
plasma on day 42 ALT AST Albumin BUN (IU/L) (IU/L) (g/dL) (mg/dL)
PBS 37 108 3.1 30 PBS 45 51 3.0 27 ISIS 588544 209 168 2.9 26 ISIS
588546 526 279 3.0 22 ISIS 588550 82 136 2.7 25 ISIS 588553 79 105
3.0 24 ISIS 588554 112 220 3.2 19 ISIS 588555 95 162 2.8 25 ISIS
588556 345 236 3.0 26 ISIS 588557 393 420 2.8 24 ISIS 588560 109
148 2.7 27 ISIS 588562 279 284 2.8 22 ISIS 588564 152 188 3.0 23
ISIS 588565 247 271 2.8 28
Weights Body weights of the mice were measured on day 42. Weights
of organs, liver, kidney, and spleen were also measured after the
mice were sacrificed on day 42. The results are presented in the
Table below. ISIS oligonucleotides that caused changes in the
weights outside the expected range for antisense oligonucleotides
were excluded in further studies.
TABLE-US-00159 TABLE 166 Weights (g) of CD1 mice on day 40 Body
Kidney Liver Spleen PBS 42 0.7 2.4 0.1 PBS 41 0.7 2.4 0.2 ISIS
588544 44 0.6 1.9 0.1 ISIS 588546 43 0.6 2.4 0.2 ISIS 588550 41 0.7
2.8 0.2 ISIS 588553 44 0.7 2.7 0.2 ISIS 588554 40 0.7 2.4 0.2 ISIS
588555 44 0.7 2.8 0.2 ISIS 588556 39 0.6 2.7 0.2 ISIS 588557 41 0.8
3.3 0.3 ISIS 588560 38 0.6 3.2 0.3 ISIS 588562 41 0.6 2.8 0.2 ISIS
588564 40 0.6 2.8 0.3 ISIS 588565 39 0.6 2.2 0.2
Study 4 (with (S) cEt Gapmers and Deoxy, MOE and (S)-cEt
Oligonucleotides)
[1113] Groups of ten-week old male CD1 mice were injected
subcutaneously once a week for 6 weeks with 50 mg/kg of ISIS
oligonucleotide from the studies described above. In addition, two
oligonucleotides, ISIS 594431 and ISIS 594432, were designed as
3-10-3 (S)-cEt gapmers and were also tested in this study. ISIS
594431 (ACCTCCTTCCGAGTCA, SEQ ID NO: 550) targets the same region
as ISIS 588871, a deoxy, MOE and (S)-cEt gapmer (target start site
2197 of SEQ ID NO: 1 and target start site 6985 of SEQ ID NO: 2).
ISIS 594432 (TGGTCACATTCCCTTC, SEQ ID NO: 542) targets the same
region as ISIS 588872 a deoxy, MOE and (S)-cEt gapmer (target start
site 154 of SEQ ID NO: 1 and target start site 1875 of SEQ ID NO:
2).
[1114] Two groups of male CD1 mice were injected subcutaneously
once a week for 6 weeks with PBS. Mice were euthanized 48 hours
after the last dose, and organs and plasma were harvested for
further analysis.
Plasma Chemistry Markers
[1115] To evaluate the effect of ISIS oligonucleotides on liver and
kidney function, plasma levels of transaminases, albumin,
creatinine, and BUN were measured using an automated clinical
chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The
results are presented in the Table below. ISIS oligonucleotides
that caused changes in the levels of any of the liver or kidney
function markers outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00160 TABLE 167 Plasma chemistry markers in CD1 mice
plasma on day 42 ALT AST Albumin Creatinine BUN Chemistry (IU/L)
(IU/L) (g/dL) (mg/dL) (mg/dL) PBS -- 71 77 2.7 0.2 29 PBS -- 30 36
2.7 0.2 26 ISIS 588834 Deoxy, MOE and (S)-cEt 436 510 2.8 0.2 25
ISIS 588835 Deoxy, MOE and (S)-cEt 70 98 3.0 0.2 27 ISIS 588836
Deoxy, MOE and (S)-cEt 442 312 2.7 0.2 27 ISIS 588846 Deoxy, MOE
and (S)-cEt 50 75 2.5 0.1 28 ISIS 588847 Deoxy, MOE and (S)-cEt 44
71 2.6 0.1 24 ISIS 588848 Deoxy, MOE and (S)-cEt 47 70 2.4 0.1 27
ISIS 588857 Deoxy, MOE and (S)-cEt 1287 655 2.7 0.2 26 ISIS 588858
Deoxy, MOE and (S)-cEt 1169 676 2.5 0.2 26 ISIS 588859 Deoxy, MOE
and (S)-cEt 1036 1300 3.2 0.2 25 ISIS 588861 Deoxy, MOE and (S)-cEt
749 466 3.1 0.1 24 ISIS 588862 Deoxy, MOE and (S)-cEt 1564 1283 2.9
0.2 22 ISIS 588863 Deoxy, MOE and (S)-cEt 477 362 2.8 0.1 23 ISIS
588864 Deoxy, MOE and (S)-cEt 118 165 2.9 0.2 27 ISIS 588866 Deoxy,
MOE and (S)-cEt 843 784 3.2 0.2 25 ISIS 594430 3-10-3 (S)-cEt 89 99
2.4 0.1 28 ISIS 594431 3-10-3 (S)-cEt 590 433 3.0 0.2 24 ISIS
594432 3-10-3 (S)-cEt 2595 2865 2.4 0.1 25
Weights
[1116] Body weights of the mice were measured on day 39. Weights of
organs, liver, kidney, and spleen were also measured after the mice
were sacrificed on day 42. The results are presented in the Table
below. ISIS oligonucleotides that caused changes in the weights
outside the expected range for antisense oligonucleotides were
excluded in further studies.
TABLE-US-00161 TABLE 168 Weights (g) of CD1 mice Chemistry Body
Kidney Liver Spleen PBS -- 37 0.6 2.1 0.1 PBS -- 45 0.7 2.5 0.2
ISIS 588834 Deoxy, MOE and (S)-cEt 40 0.6 3.2 0.2 ISIS 588835
Deoxy, MOE and (S)-cEt 38 0.7 2.8 0.3 ISIS 588836 Deoxy, MOE and
(S)-cEt 41 0.7 2.3 0.2 ISIS 588837 Deoxy, MOE and (S)-cEt 38 0.6
2.4 0.3 ISIS 588846 Deoxy, MOE and (S)-cEt 39 0.6 2.3 0.2 ISIS
588847 Deoxy, MOE and (S)-cEt 40 0.7 2.5 0.2 ISIS 588848 Deoxy, MOE
and (S)-cEt 43 0.7 2.6 0.3 ISIS 588857 Deoxy, MOE and (S)-cEt 39
0.6 3.3 0.2 ISIS 588858 Deoxy, MOE and (S)-cEt 37 0.6 3.4 0.2 ISIS
588859 Deoxy, MOE and (S)-cEt 41 0.7 2.5 0.3 ISIS 588861 Deoxy, MOE
and (S)-cEt 39 0.6 2.6 0.4 ISIS 588862 Deoxy, MOE and (S)-cEt 34
0.6 2.5 0.4 ISIS 588863 Deoxy, MOE and (S)-cEt 40 0.6 2.7 0.3 ISIS
588864 Deoxy, MOE and (S)-cEt 40 0.7 2.3 0.2 ISIS 588866 Deoxy, MOE
and (S)-cEt 45 0.7 3.0 0.2 ISIS 594430 3-10-3 (S)-cEt 39 0.6 2.2
0.2 ISIS 594431 3-10-3 (S)-cEt 36 0.6 3.2 0.2 ISIS 594432 3-10-3
(S)-cEt 31 0.4 1.9 0.1
Study 5 (with MOE Gapmers, (S) cEt Gapmers and Deoxy, MOE and
(S)-cEt Oligonucleotides)
[1117] Groups of eight- to nine-week old male CD1 mice were
injected subcutaneously once a week for 6 weeks with 50 mg/kg of
ISIS oligonucleotide. Two groups of male CD1 mice were injected
subcutaneously once a week for 6 weeks with PBS. Mice were
euthanized 48 hours after the last dose, and organs and plasma were
harvested for further analysis.
Plasma Chemistry Markers
[1118] To evaluate the effect of ISIS oligonucleotides on liver and
kidney function, plasma levels of transaminases, albumin,
creatinine, and BUN were measured using an automated clinical
chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The
results are presented in the Table below. ISIS oligonucleotides
that caused changes in the levels of any of the liver or kidney
function markers outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00162 TABLE 169 Plasma chemistry markers in CD1 mice
plasma on day 42 ALT AST Albumin Creatinine BUN Chemistry (IU/L)
(IU/L) (g/dL) (mg/dL) (mg/dL) PBS -- 33 84 2.9 0.2 28 PBS -- 32 65
2.5 0.1 27 ISIS 532692 5-10-5 MOE 363 281 3.0 0.2 30 ISIS 532770
5-10-5 MOE 69 100 2.9 0.1 28 ISIS 532775 5-10-5 MOE 371 333 2.6 0.1
29 ISIS 532800 5-10-5 MOE 104 106 2.7 0.1 31 ISIS 532809 5-10-5 MOE
69 127 2.8 0.1 26 ISIS 588540 5-10-5 MOE 66 110 2.8 0.1 26 ISIS
588838 3-10-3 (S)-cEt 391 330 2.9 0.1 25 ISIS 588842 Deoxy, MOE and
(S)-cEt 224 264 2.6 0.1 26 ISIS 588843 3-10-3 (S)-cEt 185 160 2.8
0.1 24 ISIS 588844 Deoxy, MOE and (S)-cEt 304 204 2.7 0.1 25 ISIS
588851 Deoxy, MOE and (S)-cEt 186 123 2.7 0.1 31 ISIS 588854 Deoxy,
MOE and (S)-cEt 1232 925 2.7 0.1 25 ISIS 588855 Deoxy, MOE and
(S)-cEt 425 321 2.7 0.1 28 ISIS 588856 Deoxy, MOE and (S)-cEt 78
101 2.4 0.1 31 ISIS 588865 Deoxy, MOE and (S)-cEt 126 145 2.5 0.1
23 ISIS 588867 Deoxy, MOE and (S)-cEt 108 112 2.5 0.1 32 ISIS
588868 Deoxy, MOE and (S)-cEt 61 124 2.5 0.1 28 ISIS 588870 Deoxy,
MOE and (S)-cEt 48 69 2.4 0.1 31 ISIS 588871 Deoxy, MOE and (S)-cEt
723 881 2.5 0.1 24 ISIS 588872 Deoxy, MOE and (S)-cEt 649 654 2.7
0.1 26
Weights
[1119] Body weights of the mice were measured on day 40. Weights of
organs, liver, kidney, and spleen were also measured after the mice
were sacrificed on day 42. The results are presented in the Table
below. ISIS oligonucleotides that caused changes in the weights
outside the expected range for antisense oligonucleotides were
excluded in further studies.
TABLE-US-00163 TABLE 170 Weights (g) of CD1 mice Chemistry Body
Kidney Liver Spleen PBS -- 46 0.7 2.3 0.2 PBS -- 44 0.7 2.3 0.2
ISIS 532692 5-10-5 MOE 44 0.6 2.8 0.2 ISIS 532770 5-10-5 MOE 43 0.6
2.2 0.2 ISIS 532775 5-10-5 MOE 43 0.6 2.8 0.2 ISIS 532800 5-10-5
MOE 47 0.7 2.9 0.2 ISIS 532809 5-10-5 MOE 44 0.7 2.6 0.2 ISIS
588540 5-10-5 MOE 44 0.7 2.5 0.2 ISIS 588838 3-10-3 (S)-cEt 45 0.7
3.1 0.2 ISIS 588842 Deoxy, MOE and (S)-cEt 41 0.6 2.6 0.2 ISIS
588843 3-10-3 (S)-cEt 43 0.7 2.9 0.2 ISIS 588844 Deoxy, MOE and
(S)-cEt 43 0.7 2.8 0.2 ISIS 588851 Deoxy, MOE and (S)-cEt 46 0.6
2.6 0.2 ISIS 588854 Deoxy, MOE and (S)-cEt 45 0.7 4.1 0.2 ISIS
588855 Deoxy, MOE and (S)-cEt 44 0.7 2.9 0.3 ISIS 588856 Deoxy, MOE
and (S)-cEt 44 0.7 3.2 0.2 ISIS 588865 Deoxy, MOE and (S)-cEt 45
0.7 2.6 0.3 ISIS 588867 Deoxy, MOE and (S)-cEt 46 0.7 3.2 0.3 ISIS
588868 Deoxy, MOE and (S)-cEt 42 0.7 2.9 0.3 ISIS 588870 Deoxy, MOE
and (S)-cEt 43 0.6 2.2 0.2 ISIS 588871 Deoxy, MOE and (S)-cEt 41
0.7 3.1 0.2 ISIS 588872 Deoxy, MOE and (S)-cEt 39 0.6 3.2 0.3
Study 6 (with Deoxy, MOE and (S)-cEt Oligonucleotides)
[1120] Groups of eight- to nine-week old male CD1 mice were
injected subcutaneously once a week for 6 weeks with 50 mg/kg of
deoxy, MOE, and (S)-cEt oligonucleotides. Two groups of male CD1
mice were injected subcutaneously once a week for 6 weeks with PBS.
Mice were euthanized 48 hours after the last dose, and organs and
plasma were harvested for further analysis.
Plasma Chemistry Markers
[1121] To evaluate the effect of ISIS oligonucleotides on liver and
kidney function, plasma levels of transaminases, albumin,
creatinine, bilirubin, and BUN were measured using an automated
clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,
N.Y.). The results are presented in the Table below. ISIS
oligonucleotides that caused changes in the levels of any of the
liver or kidney function markers outside the expected range for
antisense oligonucleotides were excluded in further studies.
TABLE-US-00164 TABLE 171 Plasma chemistry markers in CD1 mice
plasma on day 45 ALT AST Albumin Creatinine Bilirubin BUN (IU/L)
(IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 39 78 3.4 0.2 0.2 31 PBS
37 59 2.9 0.1 0.2 27 ISIS 599552 167 208 3.0 0.1 0.2 32 ISIS 599553
43 86 2.9 0.1 0.2 28 ISIS 599554 57 101 2.2 0.2 0.2 31 ISIS 599569
469 530 3.5 0.2 0.3 27 ISIS 599577 37 84 2.9 0.1 0.1 31 ISIS 599578
45 104 2.8 0.1 0.2 30 ISIS 599581 54 88 3.1 0.1 0.2 31 ISIS 599590
1741 1466 3.1 0.1 0.3 25 ISIS 599591 2230 1183 3.2 0.1 0.3 27 ISIS
601209 68 104 2.9 0.1 0.2 30 ISIS 601212 1795 968 3.2 0.1 0.3 22
ISIS 601215 424 385 3.1 0.1 0.4 25 ISIS 601216 90 125 2.9 0.1 0.2
29 ISIS 601276 946 366 2.9 0.1 0.5 31 ISIS 601282 831 540 3.3 0.2
0.2 32
Weights
[1122] Body weights of the mice were measured on day 40. Weights of
organs, liver, kidney, and spleen were also measured after the mice
were sacrificed on day 45. The results are presented in the Table
below. ISIS oligonucleotides that caused changes in the weights
outside the expected range for antisense oligonucleotides were
excluded in further studies.
TABLE-US-00165 TABLE 172 Weights (g) of CD1 mice Body Kidney Liver
Spleen PBS 40 0.7 2.1 0.2 PBS 42 0.8 2.3 0.2 ISIS 599552 38 0.6 2.3
0.2 ISIS 599553 39 0.7 2.2 0.2 ISIS 599554 39 0.7 2.4 0.2 ISIS
599569 39 0.7 2.2 0.2 ISIS 599577 41 0.7 2.5 0.2 ISIS 599578 37 0.6
2.0 0.2 ISIS 599581 40 0.6 2.5 0.2 ISIS 599590 34 0.6 3.5 0.2 ISIS
599591 38 0.8 2.7 0.2 ISIS 601209 42 0.7 2.6 0.3 ISIS 601212 38 0.6
2.9 0.2 ISIS 601215 36 0.7 2.6 0.2 ISIS 601216 42 0.6 2.7 0.2 ISIS
601276 42 0.7 3.2 0.2 ISIS 601282 38 0.7 3.2 0.2
Study 7 (with MOE Gapmers and Deoxy, MOE and (S)-cEt
Oligonucleotides)
[1123] Groups of eight- to nine-week old male CD1 mice were
injected subcutaneously once a week for 6 weeks with 100 mg/kg of
ISIS oligonucleotides. One group of male CD1 mice was injected
subcutaneously once a week for 6 weeks with PBS. Mice were
euthanized 48 hours after the last dose, and organs and plasma were
harvested for further analysis.
Plasma Chemistry Markers
[1124] To evaluate the effect of ISIS oligonucleotides on liver and
kidney function, plasma levels of transaminases, albumin,
creatinine, and BUN were measured using an automated clinical
chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The
results are presented in the Table below. ISIS oligonucleotides
that caused changes in the levels of any of the liver or kidney
function markers outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00166 TABLE 173 Plasma chemistry markers in CD1 mice
plasma on day 45 ALT AST Albumin Creatinine BUN Chemistry (IU/L)
(IU/L) (g/dL) (mg/dL) (mg/dL) PBS -- 120 102 2.7 0.2 26 ISIS 588842
Deoxy, MOE and (S)-cEt 177 164 2.7 0.1 23 ISIS 588843 Deoxy, MOE
and (S)-cEt 98 194 2.7 0.1 24 ISIS 588851 Deoxy, MOE and (S)-cEt 91
142 2.6 0.1 23 ISIS 588856 Deoxy, MOE and (S)-cEt 78 110 2.7 0.1 23
ISIS 599024 3-10-4 MOE 91 108 2.7 0.1 23 ISIS 599087 5-7-5 MOE 198
183 2.6 0.2 28 ISIS 599093 5-7-5 MOE 3285 2518 2.6 0.2 24 ISIS
599149 4-8-5 MOE 30 64 2.9 0.2 25 ISIS 599155 4-8-5 MOE 145 189 2.6
0.2 25 ISIS 599202 5-8-5 MOE 150 128 2.8 0.2 23 ISIS 599203 5-8-5
MOE 111 127 2.8 0.2 24 ISIS 599208 5-8-5 MOE 146 178 2.9 0.2 22
ISIS 599261 3-10-5 MOE 144 165 2.8 0.2 26 ISIS 599267 3-10-5 MOE 96
132 2.6 0.2 27 ISIS 599268 3-10-5 MOE 87 115 2.6 0.1 23 ISIS 599322
6-7-6 MOE 115 138 2.7 0.1 22 ISIS 599374 5-9-5 MOE 375 271 2.6 0.1
21 ISIS 599378 5-9-5 MOE 77 99 2.7 0.1 23 ISIS 599441 6-8-6 MOE 150
250 2.9 0.1 23
Weights
[1125] Body weights of the mice were measured on day 44. Weights of
organs, liver, kidney, and spleen were also measured after the mice
were sacrificed on day 49. The results are presented in the Table
below. ISIS oligonucleotides that caused changes in the weights
outside the expected range for antisense oligonucleotides were
excluded in further studies.
TABLE-US-00167 TABLE 174 Weights (g) of CD1 mice Chemistry Body
Kidney Liver Spleen PBS -- 39 0.6 1.9 0.1 ISIS 588842 Deoxy, MOE
and (S)-cEt 38 0.5 2.1 0.1 ISIS 588843 Deoxy, MOE and (S)-cEt 41
0.6 2.4 0.2 ISIS 588851 Deoxy, MOE and (S)-cEt 42 0.6 2.2 0.2 ISIS
588856 Deoxy, MOE and (S)-cEt 42 0.7 2.6 0.2 ISIS 599024 3-10-4 MOE
41 0.6 4.0 0.2 ISIS 599087 5-7-5 MOE 44 0.8 2.6 0.3 ISIS 599093
5-7-5 MOE 39 0.6 2.3 0.2 ISIS 599149 4-8-5 MOE 42 0.7 2.8 0.2 ISIS
599155 4-8-5 MOE 41 0.7 2.1 0.2 ISIS 599202 5-8-5 MOE 43 0.6 2.6
0.2 ISIS 599203 5-8-5 MOE 42 0.6 2.6 0.2 ISIS 599208 5-8-5 MOE 40
0.6 2.1 0.2 ISIS 599261 3-10-5 MOE 39 0.7 3.4 0.3 ISIS 599267
3-10-5 MOE 42 0.8 2.5 0.3 ISIS 599268 3-10-5 MOE 41 0.7 2.1 0.2
ISIS 599322 6-7-6 MOE 43 0.6 2.2 0.2 ISIS 599374 5-9-5 MOE 37 0.6
2.2 0.2 ISIS 599378 5-9-5 MOE 43 0.7 2.7 0.2 ISIS 599441 6-8-6 MOE
42 0.6 2.5 0.3
Study 8 (with MOE Gapmers, Deoxy, MOE and (S)-cEt Oligonucleotides,
and (S)-cEt Gapmers)
[1126] Groups of eight- to nine-week old male CD1 mice were
injected subcutaneously once a week for 6 weeks with 100 mg/kg of
MOE gapmers, or 50 mg/kg of deoxy, MOE and (S)-cEt oligonucleotides
or (S)-cEt gapmers. One group of male CD1 mice was injected
subcutaneously once a week for 6 weeks with PBS. Mice were
euthanized 48 hours after the last dose, and organs and plasma were
harvested for further analysis.
Plasma Chemistry Markers
[1127] To evaluate the effect of ISIS oligonucleotides on liver and
kidney function, plasma levels of transaminases, albumin,
creatinine, and BUN were measured using an automated clinical
chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The
results are presented in the Table below.
TABLE-US-00168 TABLE 175 Plasma chemistry markers in CD1 mice
plasma on day 43 Dose ALT AST Albumin Creatinine BUN Chemistry
(mg/kg/wk) (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) PBS -- -- 37 57 2.5
0.08 26 ISIS 532770 5-10-5 MOE 100 57 73 2.5 0.07 24 ISIS 532800
5-10-5 MOE 100 74 126 2.8 0.10 26 ISIS 532809 5-10-5 MOE 100 83 73
2.5 0.07 23 ISIS 588540 5-10-5 MOE 100 106 102 2.7 0.09 27 ISIS
588544 5-10-5 MOE 100 66 62 2.6 0.10 24 ISIS 588548 5-10-5 MOE 100
48 67 2.6 0.08 23 ISIS 588550 5-10-5 MOE 100 65 106 2.5 0.10 25
ISIS 588553 5-10-5 MOE 100 78 90 2.6 0.09 25 ISIS 588555 5-10-5 MOE
100 94 89 2.5 0.08 23 ISIS 588848 Deoxy, MOE 50 38 54 2.3 0.07 25
and (S)-cEt ISIS 594430 3-10-3 (S)-cEt 50 63 72 2.5 0.10 27
Weights
[1128] Body weights of the mice were measured on day 36. Weights of
organs, liver, kidney, and spleen were also measured after the mice
were sacrificed on day 43. The results for the organ weights were
expressed as a ratio to the body weights and normalized to the PBS
control ratio.
TABLE-US-00169 TABLE 176 Organ Weights/Body weight (BW) of CD1 mice
Dose Kidney/ Liver/ Spleen/ Chemistry (mg/kg/wk) BW BW BW PBS -- --
1.0 1.0 1.0 ISIS 532770 5-10-5 MOE 100 1.4 1.1 1.0 ISIS 532800
5-10-5 MOE 100 1.5 1.1 0.9 ISIS 532809 5-10-5 MOE 100 1.3 1.2 0.9
ISIS 588540 5-10-5 MOE 100 1.3 1.2 1.0 ISIS 588544 5-10-5 MOE 100
1.6 1.1 1.0 ISIS 588548 5-10-5 MOE 100 1.7 1.2 1.0 ISIS 588550
5-10-5 MOE 100 1.5 1.2 1.0 ISIS 588553 5-10-5 MOE 100 1.5 1.0 0.8
ISIS 588555 5-10-5 MOE 100 1.8 1.2 1.0 ISIS 588848 Deoxy, MOE 50
1.3 1.0 0.9 and (S)-cEt ISIS 594430 3-10-3 (S)-cEt 50 1.4 1.1
0.9
Cytokine Assays
[1129] Blood obtained from all mice groups were sent to Antech
Diagnostics for measurements of the various cytokine levels, such
as IL-6, MDC, MIP1.beta., IP-10, MCP1, MIP-1.alpha., and RANTES.
The results are presented in Table 54.
TABLE-US-00170 TABLE 177 Cytokine levels (pg/mL) in CD1 mice plasma
Chemistry IL-6 MDC MIP1.beta. IP-10 MCP1 MIP-1.alpha. RANTES PBS --
70 16 23 20 17 6 2 ISIS 532770 5-10-5 MOE 101 18 146 116 101 24 6
ISIS 532800 5-10-5 MOE 78 17 83 53 105 1 3 ISIS 532809 5-10-5 MOE
66 19 60 32 55 20 4 ISIS 588540 5-10-5 MOE 51 18 126 70 75 4 3 ISIS
588544 5-10-5 MOE 157 14 94 34 102 1 3 ISIS 588548 5-10-5 MOE 164
12 90 66 84 10 4 ISIS 588550 5-10-5 MOE 58 21 222 124 157 3 5 ISIS
588553 5-10-5 MOE 62 14 183 60 103 9 4 ISIS 588555 5-10-5 MOE 70 19
172 171 178 16 9 ISIS 588848 Deoxy, MOE 59 13 61 27 63 12 4 and
(S)-cEt ISIS 594430 3-10-3 (S)-cEt 48 14 56 38 85 10 3
Hematology Assays
[1130] Blood obtained from all mice groups were sent to Antech
Diagnostics for measurements of hematocrit (HCT), as well as of the
various blood cells, such as WBC, RBC, and platelets, and total
hemoglobin (Hb) content. The results are presented in Table 55.
TABLE-US-00171 TABLE 178 Hematology markers in CD1 mice plasma HCT
Hb WBC RBC Platelets Chemistry (%) (g/dL) (10.sup.3/.mu.L)
(10.sup.6/.mu.L) (10.sup.3/.mu.L) PBS -- 46 15 7 9 960 ISIS 532770
5-10-5 MOE 45 14 5 9 879 ISIS 532800 5-10-5 MOE 45 14 5 9 690 ISIS
532809 5-10-5 MOE 46 14 6 9 1005 ISIS 588540 5-10-5 MOE 49 15 6 10
790 ISIS 588544 5-10-5 MOE 36 11 7 7 899 ISIS 588548 5-10-5 MOE 46
14 6 9 883 ISIS 588550 5-10-5 MOE 42 13 8 8 721 ISIS 588553 5-10-5
MOE 45 14 6 9 719 ISIS 588555 5-10-5 MOE 43 13 8 9 838 ISIS 588848
Deoxy, MOE 40 15 8 10 840 and (S)-cEt ISIS 594430 3-10-3 (S)-cEt 45
14 8 9 993
Example 127: Tolerability of Antisense Oligonucleotides Targeting
Human CFB in Sprague-Dawley Rats
[1131] Sprague-Dawley rats are a multipurpose model used for safety
and efficacy evaluations. The rats were treated with ISIS antisense
oligonucleotides from the studies described in the Examples above
and evaluated for changes in the levels of various plasma chemistry
markers.
Study 1 (with 5-10-5 MOE Gapmers)
[1132] Male Sprague-Dawley rats, seven- to eight-week old, were
maintained on a 12-hour light/dark cycle and fed ad libitum with
Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats
each were injected subcutaneously once a week for 6 weeks with 100
mg/kg of 5-10-5 MOE gapmers. One control group of 6 rats was
injected subcutaneously once a week for 6 weeks with PBS. Forty
eight hours after the last dose, rats were euthanized and organs
and plasma were harvested for further analysis.
Liver Function
[1133] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma levels of transaminases were measured using an
automated clinical chemistry analyzer (Hitachi Olympus AU400e,
Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and
AST (aspartate transaminase) were measured and the results are
presented in the Table below expressed in IU/L. ISIS
oligonucleotides that caused changes in the levels of any markers
of liver function outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00172 TABLE 179 Liver function markers in Sprague-Dawley
rats ALT (IU/L) AST (IU/L) PBS 66 134 ISIS 588544 101 329 ISIS
588550 69 157 ISIS 588553 88 304 ISIS 588554 202 243 ISIS 588555 94
113 ISIS 588556 102 117 ISIS 588560 206 317 ISIS 588564 292 594
Kidney Function
[1134] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma levels of blood urea nitrogen (BUN) and creatinine
were measured using an automated clinical chemistry analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in
the Table below, expressed in mg/dL. ISIS oligonucleotides that
caused changes in the levels of any of the kidney function markers
outside the expected range for antisense oligonucleotides were
excluded in further studies.
TABLE-US-00173 TABLE 180 Kidney function markers (mg/dL) in
Sprague-Dawley rats BUN Creatinine PBS 18 3.5 ISIS 588544 21 3.1
ISIS 588550 21 3.0 ISIS 588553 22 2.8 ISIS 588554 23 3.0 ISIS
588555 22 3.5 ISIS 588556 21 3.2 ISIS 588560 26 2.4 ISIS 588564 24
2.7
Weights
[1135] Body weight measurements were taken on day 39. Liver, heart,
spleen and kidney weights were measured at the end of the study on
day 42, and are presented in the Table below. ISIS oligonucleotides
that caused any changes in organ weights outside the expected range
for antisense oligonucleotides were excluded from further
studies.
TABLE-US-00174 TABLE 181 Weights (g) Body Liver Spleen Kidney PBS
422 16 1.2 3.9 ISIS 588544 353 15 1.7 2.9 ISIS 588550 321 14 2.1
3.2 ISIS 588553 313 15 2.3 3.2 ISIS 588554 265 11 1.6 2.7 ISIS
588555 345 14 1.4 3.3 ISIS 588556 328 13 1.7 3.1 ISIS 588560 270 13
2.4 3.0 ISIS 588564 253 12 2.9 3.0
Study 2 (with Deoxy, MOE and (S)-cEt Oligonucleotides)
[1136] Male Sprague-Dawley rats, nine- to ten-week old, were
maintained on a 12-hour light/dark cycle and fed ad libitum with
Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats
each were injected subcutaneously once a week for 6 weeks with 100
mg/kg of deoxy, MOE, and (S)-cEt oligonucleotides. Two control
groups of 3 rats each were injected subcutaneously once a week for
6 weeks with PBS. Forty eight hours after the last dose, rats were
euthanized and organs and plasma were harvested for further
analysis.
Liver Function
[1137] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma levels of transaminases were measured on day 42
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma levels of ALT (alanine
transaminase) and AST (aspartate transaminase), and albumin were
measured and the results are presented in the Table below. ISIS
oligonucleotides that caused changes in the levels of any markers
of liver function outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00175 TABLE 182 Liver function markers in Sprague-Dawley
rats ALT (IU/L) AST (IU/L) Albumin (g/dL) PBS 55 150 3.4 PBS 64 91
3.5 ISIS 588554 52 92 3.2 ISIS 588835 971 844 4.1 ISIS 588842 317
359 3.8 ISIS 588843 327 753 2.9 ISIS 588846 70 111 3.2 ISIS 588847
65 100 3.0 ISIS 588864 91 109 3.0 ISIS 594430 85 106 3.7
Kidney Function
[1138] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma levels of blood urea nitrogen (BUN) and creatinine
were measured using an automated clinical chemistry analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in
the Table below, expressed in mg/dL. ISIS oligonucleotides that
caused changes in the levels of any of the kidney function markers
outside the expected range for antisense oligonucleotides were
excluded in further studies.
TABLE-US-00176 TABLE 183 Kidney function markers (mg/dL) in
Sprague-Dawley rats BUN Creatinine PBS 17 0.4 PBS 21 0.4 ISIS
588554 20 0.4 ISIS 588835 23 0.5 ISIS 588842 22 0.4 ISIS 588843 51
0.4 ISIS 588846 25 0.5 ISIS 588847 23 0.5 ISIS 588864 23 0.4 ISIS
594430 22 0.5
Weights
[1139] Body weight measurements were taken on day 39. Liver, heart,
spleen and kidney weights were measured at the end of the study on
day 42, and are presented in the Table below. ISIS oligonucleotides
that caused any changes in organ weights outside the expected range
for antisense oligonucleotides were excluded from further
studies.
TABLE-US-00177 TABLE 184 Weights (g) Body Liver Spleen Kidney PBS
466 16 0.9 3.8 PBS 485 16 0.9 3.6 ISIS 588554 393 15 2.3 2.6 ISIS
588835 387 16 1.0 3.3 ISIS 588842 414 22 1.5 3.7 ISIS 588843 427 20
2.5 4.2 ISIS 588846 366 16 2.1 3.3 ISIS 588847 402 15 1.6 3.1 ISIS
588864 364 15 2.1 3.8 ISIS 594430 420 16 1.2 3.6
Study 3 (with MOE Gapmers)
[1140] Male Sprague-Dawley rats, nine- to ten-week old, were
maintained on a 12-hour light/dark cycle and fed ad libitum with
Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats
each were injected subcutaneously once a week for 6 weeks with 100
mg/kg of MOE gapmers. One control group of 6 rats was injected
subcutaneously once a week for 6 weeks with PBS. Forty eight hours
after the last dose, rats were euthanized and organs and plasma
were harvested for further analysis.
Liver Function
[1141] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma levels of transaminases were measured on day 43
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma levels of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in the Table below expressed in IU/L.
ISIS oligonucleotides that caused changes in the levels of any
markers of liver function outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00178 TABLE 185 Liver function markers in Sprague-Dawley
rats ALT AST Albumin Chemistry (IU/L) (IU/L) (g/dL) PBS -- 52 110
3.7 ISIS 588563 5-10-5 MOE 175 291 2.9 ISIS 599024 3-10-4 MOE 139
173 1.4 ISIS 599093 5-7-5 MOE 116 238 2.6 ISIS 599149 4-8-5 MOE 232
190 3.4 ISIS 599155 4-8-5 MOE 108 215 2.5 ISIS 599202 5-8-5 MOE 65
86 3.5 ISIS 599203 5-8-5 MOE 71 97 3.1 ISIS 599208 5-8-5 MOE 257
467 1.9 ISIS 599261 3-10-5 MOE 387 475 1.5 ISIS 599267 3-10-5 MOE
201 337 2.7
Kidney Function
[1142] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma levels of blood urea nitrogen (BUN) and creatinine
were measured using an automated clinical chemistry analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in
the Table below, expressed in mg/dL. ISIS oligonucleotides that
caused changes in the levels of any of the kidney function markers
outside the expected range for antisense oligonucleotides were
excluded in further studies.
TABLE-US-00179 TABLE 186 Kidney function markers (mg/dL) in
Sprague-Dawley rats Chemistry BUN Creatinine PBS -- 16 0.3 ISIS
588563 5-10-5 MOE 26 0.4 ISIS 599024 3-10-4 MOE 135 1.2 ISIS 599093
5-7-5 MOE 29 0.4 ISIS 599149 4-8-5 MOE 23 0.4 ISIS 599155 4-8-5 MOE
29 0.4 ISIS 599202 5-8-5 MOE 19 0.4 ISIS 599203 5-8-5 MOE 22 0.4
ISIS 599208 5-8-5 MOE 26 0.3 ISIS 599261 3-10-5 MOE 228 1.6 ISIS
599267 3-10-5 MOE 24 0.4
Weights
[1143] Body weight measurements were taken on day 39. Liver, heart,
spleen and kidney weights were measured at the end of the study on
day 42, and are presented in the Table below. ISIS oligonucleotides
that caused any changes in organ weights outside the expected range
for antisense oligonucleotides were excluded from further
studies.
TABLE-US-00180 TABLE 187 Weights (g) Chemistry Body Liver Spleen
Kidney PBS -- 471 16 1.0 4.1 ISIS 588563 5-10-5 MOE 311 16 3.4 4.1
ISIS 599024 3-10-4 MOE 297 11 1.0 3.5 ISIS 599093 5-7-5 MOE 332 18
4.1 5.0 ISIS 599149 4-8-5 MOE 388 16 2.3 3.7 ISIS 599155 4-8-5 MOE
290 15 2.9 4.5 ISIS 599202 5-8-5 MOE 359 13 1.3 3.2 ISIS 599203
5-8-5 MOE 334 14 1.8 3.3 ISIS 599208 5-8-5 MOE 353 29 4.7 4.6 ISIS
599261 3-10-5 MOE 277 10 0.9 3.2 ISIS 599267 3-10-5 MOE 344 21 3.9
4.7
Study 4 (with MOE Gapmers)
[1144] Male Sprague-Dawley rats, nine- to ten-week old, were
maintained on a 12-hour light/dark cycle and fed ad libitum with
Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats
each were injected subcutaneously once a week for 6 weeks with 100
mg/kg of MOE gapmers. One control group of 6 rats was injected
subcutaneously once a week for 6 weeks with PBS. Forty eight hours
after the last dose, rats were euthanized and organs and plasma
were harvested for further analysis.
Liver Function
[1145] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma levels of transaminases were measured on day 42
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma levels of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in the Table below expressed in IU/L.
ISIS oligonucleotides that caused changes in the levels of any
markers of liver function outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00181 TABLE 188 Liver function markers in Sprague-Dawley
rats ALT AST Albumin Chemistry (IU/L) (IU/L) (g/dL) PBS -- 48 77
3.9 ISIS 532800 5-10-5 MOE 72 111 3.4 ISIS 532809 5-10-5 MOE 59 89
3.8 ISIS 588540 5-10-5 MOE 146 259 3.8 ISIS 599268 3-10-5 MOE 175
206 2.7 ISIS 599322 6-7-6 MOE 523 567 3.3 ISIS 599374 5-9-5 MOE 114
176 3.0 ISIS 599378 5-9-5 MOE 124 116 3.2 ISIS 599380 5-9-5 MOE 148
210 3.4 ISIS 599441 6-8-6 MOE 51 91 2.6
Kidney Function
[1146] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma levels of blood urea nitrogen (BUN) and creatinine
were measured using an automated clinical chemistry analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in
the Table below, expressed in mg/dL. ISIS oligonucleotides that
caused changes in the levels of any of the kidney function markers
outside the expected range for antisense oligonucleotides were
excluded in further studies.
TABLE-US-00182 TABLE 189 Kidney function markers (mg/dL) in
Sprague-Dawley rats Chemistry BUN Creatinine PBS -- 15 0.4 ISIS
532800 5-10-5 MOE 26 0.5 ISIS 532809 5-10-5 MOE 18 0.5 ISIS 588540
5-10-5 MOE 22 0.5 ISIS 599268 3-10-5 MOE 28 0.5 ISIS 599322 6-7-6
MOE 24 0.5 ISIS 599374 5-9-5 MOE 29 0.5 ISIS 599378 5-9-5 MOE 22
0.4 ISIS 599380 5-9-5 MOE 26 0.5 ISIS 599441 6-8-6 MOE 24 0.4
Weights
[1147] Body weight measurements were taken on day 39. Liver, heart,
spleen and kidney weights were measured at the end of the study on
day 42, and are presented in the Table below. ISIS oligonucleotides
that caused any changes in organ weights outside the expected range
for antisense oligonucleotides were excluded from further
studies.
TABLE-US-00183 TABLE 190 Weights (g) Chemistry Body Liver Spleen
Kidney PBS -- 502 16 0.9 3.7 ISIS 532800 5-10-5 MOE 376 16 2.0 3.4
ISIS 532809 5-10-5 MOE 430 16 1.4 3.4 ISIS 588540 5-10-5 MOE 391 16
1.8 3.5 ISIS 599268 3-10-5 MOE 332 16 3.6 3.6 ISIS 599322 6-7-6 MOE
348 13 2.1 3.4 ISIS 599374 5-9-5 MOE 302 12 2.0 3.3 ISIS 599378
5-9-5 MOE 332 11 1.1 2.8 ISIS 599380 5-9-5 MOE 350 11 1.5 3.3 ISIS
599441 6-8-6 MOE 368 16 2.5 3.3
Study 5 (with MOE Gapmers and Deoxy, MOE and (S)-cEt
Oligonucleotides)
[1148] Male Sprague-Dawley rats, nine- to ten-week old, were
maintained on a 12-hour light/dark cycle and fed ad libitum with
Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats
each were injected subcutaneously once a week for 6 weeks with 100
mg/kg of MOE gapmer or with 50 mg/kg of deoxy, MOE and (S)-cEt
oligonucleotides. One control group of 4 rats was injected
subcutaneously once a week for 6 weeks with PBS. Forty eight hours
after the last dose, rats were euthanized and organs and plasma
were harvested for further analysis.
Liver Function
[1149] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma levels of transaminases were measured on day 42
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma levels of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in the Table below expressed in IU/L.
ISIS oligonucleotides that caused changes in the levels of any
markers of liver function outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00184 TABLE 191 Liver function markers in Sprague-Dawley
rats ALT AST Albumin Chemistry (IU/L) (IU/L) (g/dL) PBS -- 49 74
3.3 ISIS 532770 5-10-5 MOE 95 132 3.3 ISIS 588851 Deoxy, MOE, and
(S)-cEt 47 72 3.1 ISIS 588856 Deoxy, MOE, and (S)-cEt 56 75 3.0
ISIS 588865 Deoxy, MOE, and (S)-cEt 62 84 2.9 ISIS 588867 Deoxy,
MOE, and (S)-cEt 73 214 2.9 ISIS 588868 Deoxy, MOE, and (S)-cEt 59
83 3.1 ISIS 588870 Deoxy, MOE, and (S)-cEt 144 144 3.4
Kidney Function
[1150] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma and urine levels of blood urea nitrogen (BUN) and
creatinine were measured using an automated clinical chemistry
analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are
presented in the Tables below, expressed in mg/dL. ISIS
oligonucleotides that caused changes in the levels of any of the
kidney function markers outside the expected range for antisense
oligonucleotides were excluded in further studies.
TABLE-US-00185 TABLE 192 Kidney function markers (mg/dL) in the
plasma of Sprague-Dawley rats Chemistry BUN Creatinine PBS -- 18
0.3 ISIS 532770 5-10-5 MOE 20 0.4 ISIS 588851 Deoxy, MOE, and
(S)-cEt 20 0.4 ISIS 588856 Deoxy, MOE, and (S)-cEt 22 0.4 ISIS
588865 Deoxy, MOE, and (S)-cEt 24 0.5 ISIS 588867 Deoxy, MOE, and
(S)-cEt 22 0.4 ISIS 588868 Deoxy, MOE, and (S)-cEt 19 0.4 ISIS
588870 Deoxy, MOE, and (S)-cEt 20 0.5
TABLE-US-00186 TABLE 193 Kidney function markers (mg/dL) in the
urine of Sprague-Dawley rats Chemistry Total protein Creatinine PBS
-- 80 92 ISIS 532770 5-10-5 MOE 466 69 ISIS 588851 Deoxy, MOE, and
(S)-cEt 273 64 ISIS 588856 Deoxy, MOE, and (S)-cEt 259 68 ISIS
588865 Deoxy, MOE, and (S)-cEt 277 67 ISIS 588867 Deoxy, MOE, and
(S)-cEt 337 68 ISIS 588868 Deoxy, MOE, and (S)-cEt 326 75 ISIS
588870 Deoxy, MOE, and (S)-cEt 388 82
Weights
[1151] Body weight measurements were taken on day 39. Liver, heart,
spleen and kidney weights were measured at the end of the study on
day 42, and are presented in the Table below. ISIS oligonucleotides
that caused any changes in organ weights outside the expected range
for antisense oligonucleotides were excluded from further
studies.
TABLE-US-00187 TABLE 194 Weights (g) Chemistry Body Liver Spleen
Kidney PBS -- 489 16 0.9 3.5 ISIS 532770 5-10-5 MOE 372 15 1.7 3.1
ISIS 588851 Deoxy, MOE, and 285 14 1.4 3.2 (S)-cEt ISIS 588856
Deoxy, MOE, and 415 15 1.1 3.3 (S)-cEt ISIS 588865 Deoxy, MOE, and
362 14 2.0 3.3 (S)-cEt ISIS 588867 Deoxy, MOE, and 406 15 2.4 3.4
(S)-cEt ISIS 588868 Deoxy, MOE, and 399 15 1.5 3.4 (S)-cEt ISIS
588870 Deoxy, MOE, and 446 14 1.4 3.3 (S)-cEt
Study 6 (with MOE Gapmers, Deoxy, MOE and (S)-cEt Oligonucleotides,
and (S)-cEt Gapmers)
[1152] Male rats were maintained on a 12-hour light/dark cycle and
fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4
rats each were injected subcutaneously once a week for 6 weeks with
100 mg/kg of MOE gapmers or with 50 mg/kg of deoxy, MOE and (S)-cEt
oligonucleotide or (S)-cEt gapmer. One control group of 4 rats was
injected subcutaneously once a week for 6 weeks with PBS. Forty
eight hours after the last dose, rats were euthanized and organs
and plasma were harvested for further analysis.
Liver Function
[1153] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma levels of transaminases were measured on day 42
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma levels of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in the Table below expressed in IU/L.
TABLE-US-00188 TABLE 195 Liver function markers Albu- Dose ALT AST
min Chemistry (mg/kg/wk) (IU/L) (IU/L) (g/dL) PBS -- -- 54 73 4.3
ISIS 532770 5-10-5 MOE 100 57 114 4.4 ISIS 532800 5-10-5 MOE 100
176 180 4.3 ISIS 532809 5-10-5 MOE 100 71 132 4.1 ISIS 588540
5-10-5 MOE 100 89 202 4.4 ISIS 588544 5-10-5 MOE 100 75 152 3.9
ISIS 588548 5-10-5 MOE 100 50 71 4.1 ISIS 588550 5-10-5 MOE 100 80
133 3.6 ISIS 588553 5-10-5 MOE 100 59 112 3.9 ISIS 588555 5-10-5
MOE 100 97 142 3.8 ISIS 588848 Deoxy, MOE 50 53 82 3.9 and (S)-cEt
ISIS 594430 3-10-3 (S)-cEt 50 198 172 4.4
Kidney Function
[1154] To evaluate the effect of ISIS oligonucleotides on kidney
function, urine levels of total protein and creatinine were
measured using an automated clinical chemistry analyzer (Hitachi
Olympus AU400e, Melville, N.Y.). Results are presented in the Table
below. ISIS oligonucleotides that caused changes in the levels of
any of the kidney function markers outside the expected range for
antisense oligonucleotides were excluded in further studies.
TABLE-US-00189 TABLE 196 Total protein/creatinine ratio in the
urine of rats Chemistry Dose (mg/kg/wk) P/C ratio PBS -- -- 1.1
ISIS 532770 5-10-5 MOE 100 8.3 ISIS 532800 5-10-5 MOE 100 6.5 ISIS
532809 5-10-5 MOE 100 6.1 ISIS 588540 5-10-5 MOE 100 10.1 ISIS
588544 5-10-5 MOE 100 7.9 ISIS 588548 5-10-5 MOE 100 6.6 ISIS
588550 5-10-5 MOE 100 7.6 ISIS 588553 5-10-5 MOE 100 7.0 ISIS
588555 5-10-5 MOE 100 6.2 ISIS 588848 Deoxy, MOE 50 5.2 and (S)-cEt
ISIS 594430 3-10-3 (S)-cEt 50 5.3
Weights
[1155] Body weight measurements were taken on day 39. Liver, heart,
spleen and kidney weights were measured at the end of the study on
day 42, and are presented in the Table below. The results for the
organ weights were expressed as a ratio to the body weights and
normalized to the PBS control ratio.
TABLE-US-00190 TABLE 197 Organ weights/Body weight (BW) ratios Dose
Spleen/ Liver/ Kidney/ Chemistry (mg/kg/wk) BW BW BW PBS -- -- 1.0
1.0 1.0 ISIS 532770 5-10-5 MOE 100 2.0 1.2 1.0 ISIS 532800 5-10-5
MOE 100 2.8 1.3 1.0 ISIS 532809 5-10-5 MOE 100 2.2 1.1 1.0 ISIS
588540 5-10-5 MOE 100 2.2 1.4 1.0 ISIS 588544 5-10-5 MOE 100 2.5
1.3 1.1 ISIS 588548 5-10-5 MOE 100 2.1 1.3 1.1 ISIS 588550 5-10-5
MOE 100 3.9 1.4 1.1 ISIS 588553 5-10-5 MOE 100 4.1 1.4 1.4 ISIS
588555 5-10-5 MOE 100 1.8 1.3 1.0 ISIS 588848 Deoxy, MOE 50 3.1 1.3
1.1 and (S)-cEt ISIS 594430 3-10-3 (S)-cEt 50 1.7 1.0 1.1
Example 128: Efficacy of Antisense Oligonucleotides Against CFB
mRNA in hCFB Mice
[1156] Selected compounds were tested for efficacy in human CFB
transgenic mice, founder line #6 The human CFB gene is located on
chromosome 6: position 31913721-31919861. A Fosmid
(ABC14-50933200C23) containing the CFB sequence was selected to
make transgenic mice expressing the human CFB gene. Cla I
(31926612) and Age I (31926815) restriction enzymes were used to
generate a 22,127 bp fragment containing the CFB gene for
pronuclear injection. DNA was confirmed by restriction enzyme
analysis using Pvu I. The 22,127 bp DNA fragment was injected into
C57BL/6NTac embryos. 6 positive founders were bred. Founder #6
expressed the liver human CFB mRNA and was crossbreed to the
3.sup.rd generation. Progeny from 3' generation mice were used to
evaluate human CFB ASOs for human CFB mRNA reduction.
Treatment
[1157] Groups of 3 mice each were injected subcutaneously twice a
week for the first week with 50 mg/kg of ISIS oligonucleotides,
followed by once a week dosing with 50 mg/kg of ISIS
oligonucleotides for an additional three weeks. One control group
of 4 mice was injected subcutaneously twice a week for 2 weeks for
the first week with PBS for the first week for an additional three
weeks. Forty eight hours after the last dose, mice were euthanized
and organs and plasma were harvested for further analysis.
RNA Analysis
[1158] At the end of the dosing period, RNA was extracted from the
liver and kidney for real-time PCR analysis of CFB mRNA levels.
Human CFB mRNA levels were measured using the human primer probe
set RTS3459. CFB mRNA levels were normalized to RIBOGREEN.RTM., and
also to the housekeeping gene, Cyclophilin. Results were calculated
as percent inhibition of CFB mRNA expression compared to the
control. All the antisense oligonucleotides effected inhibition of
human CFB mRNA levels in the liver.
TABLE-US-00191 TABLE 198 Percent reduction of CFB mRNA levels in
hCFB mice Normalized to Normalized to ISIS No RIBOGREEN Cyclophilin
532770 86 87 532800 88 87 532809 69 69 588540 95 94 588544 91 91
588548 78 77 588550 89 88 588553 94 94 588555 94 94 588848 83 82
594430 78 76
Example 129: In Vivo Antisense Inhibition of Murine CFB
[1159] Several antisense oligonucleotides were designed that were
targeted to murine CFB mRNA (GENBANK Accession No. NM_008198.2,
incorporated herein as SEQ ID NO: 5). The target start sites and
sequences of each oligonucleotide are described in the table below.
The chimeric antisense oligonucleotides in the table below were
designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in
length, wherein the central gap segment is comprised of 10
2'-deoxynucleosides and is flanked on both sides (in the 5' and 3'
directions) by wings comprising 5 nucleosides each. Each nucleoside
in the 5' wing segment and each nucleoside in the 3' wing segment
has a 2'-MOE modification. The internucleoside linkages throughout
each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine
residues throughout each gapmer are 5-methylcytosines.
TABLE-US-00192 TABLE 199 Gapmers targeting murine CFB Target Start
ISIS Site on SEQ SEQ ID No Sequence ID NO: 5 NO 516269
GCATAAGAGGGTACCAGCTG 2593 804 516272 GTCCTTTAGCCAGGGCAGCA 2642 805
516323 TCCACCCATGTTGTGCAAGC 1568 806 516330 CCACACCATGCCACAGAGAC
1826 807 516341 TTCCGAGTCAGGCTCTTCCC 2308 808
Treatment
[1160] Groups of four C57BL/6 mice each were injected with 50 mg/kg
of ISIS 516269, ISIS 516272, ISIS 516323, ISIS 516330, or ISIS
516341 administered weekly for 3 weeks. A control group of mice was
injected with phosphate buffered saline (PBS) administered weekly
for 3 weeks.
CFB RNA Analysis
[1161] At the end of the study, RNA was extracted from liver tissue
for real-time PCR analysis of CFB, using primer probe set RTS3430
(forward sequence GGGCAAACAGCAATTTGTGA, designated herein as SEQ ID
NO: 816; reverse sequence TGGCTACCCACCTTCCTTGT, designated herein
as SEQ ID NO: 817; probe sequence CTGGATACTGTCCCAATCCCGGTATTCCX,
designated herein as SEQ ID NO: 818). The mRNA levels were
normalized using RIBOGREEN.RTM.. As shown in the Table below, some
of the antisense oligonucleotides achieved reduction of murine CFB
over the PBS control. Results are presented as percent inhibition
of CFB, relative to control.
TABLE-US-00193 TABLE 200 Percent inhibition of murine CFB mRNA in
C57BL/6 mice ISIS No % 516269 29 516272 72 516323 77 516330 62
516341 72
Protein Analysis
[1162] CFB protein levels were measured in the kidney, liver,
plasma, and in the eye by western Blot using goat anti-CFB antibody
(Sigma Aldrich). Results are presented as percent inhibition of
CFB, relative to PBS control. `n/a` indicates that measurements
were not taken for that sample. As shown in the Table below,
antisense inhibition of CFB by ISIS oligonucleotides resulted in a
reduction of CFB protein in various tissues. As shown in the Table
below, systemic administration of ISIS oligonucleotides was
effective in reducing CFB levels in the eye.
TABLE-US-00194 TABLE 201 Percent inhibition of murine CFB protein
in C57BL/6 mice ISIS No Kidney Liver Plasma Eye 516269 20 58 n/a 70
516272 48 74 n/a 99 516323 73 85 90 93 516330 77 80 n/a n/a 516341
80 88 68 n/a
Example 130: Dose-Dependent Antisense Inhibition of Murine CFB
[1163] Groups of four C57BL/6 mice each were injected with 25
mg/kg, 50 mg/kg, or 100 mg/kg of ISIS 516272, and ISIS 516323
administered weekly for 6 weeks. Another two groups of mice were
injected with 100 mg/kg of ISIS 516330 or ISIS 516341 administered
weekly for 6 weeks. Two control groups of mice were injected with
phosphate buffered saline (PBS) administered weekly for 6
weeks.
CFB RNA Analysis
[1164] RNA was extracted from liver and kidney tissues for
real-time PCR analysis of CFB, using primer probe set RTS3430. The
mRNA levels were normalized using RIBOGREEN.RTM.. As shown in the
Table below, the antisense oligonucleotides achieved dose-dependent
reduction of murine CFB over the PBS control. Results are presented
as percent inhibition of CFB, relative to control.
TABLE-US-00195 TABLE 202 Percent inhibition of murine CFB mRNA in
C57BL/6 mice ISIS No Dose (mg/kg/wk) Liver Kidney 516272 25 39 32
50 73 36 100 87 42 516323 25 36 41 50 65 47 100 79 71 516330 100 85
45 516341 200 89 65
Protein Analysis
[1165] CFB protein levels were measured in the plasma by western
Blot using goat anti-CFB antibody (Sigma Aldrich). As shown in the
table below, antisense inhibition of CFB by the ISIS
oligonucleotides resulted in a reduction of CFB protein. Results
are presented as percent inhibition of CFB, relative to PBS
control. `n/a` indicates that measurements were not taken for that
sample.
[1166] CFB protein levels were also measured in the eye by Western
Blot. All treatment groups demonstrated an inhibition of CFB by
95%, with some sample measurements being below detection levels of
the assay.
TABLE-US-00196 TABLE 203 Percent inhibition of murine CFB protein
in C57BL/6 mice ISIS No Dose (mg/kg/wk) Liver 516272 25 32 50 70
100 83 516323 25 43 50 80 100 90 516330 100 n/a 516341 200 n/a
Example 131: Effect of Antisense Inhibition of CFB in the NZB/W F1
Mouse Model
[1167] The NZB/W F1 is the oldest classical model of lupus, where
the mice develop severe lupus-like phenotypes comparable to that of
lupus patients (Theofilopoulos, A. N. and Dixon, F. J. Advances in
Immunology, vol. 37, pp. 269-390, 1985).sub.n These lupus-like
phenotypes include lymphadenopathy, splenomegaly, elevated serum
antinuclear autoantibodies (ANA) including anti-dsDNA IgG, a
majority of which are IgG2a and IgG3, and immune complex-mediated
glomerulonephritis (GN) that becomes apparent at 5-6 months of age,
leading to kidney failure and death at 10-12 months of age.
Study 1
[1168] A study was conducted to demonstrate that treatment with
antisense oligonucleotides targeting CFB would improve renal
pathology in the mouse model. Female NZB/W F1 mice, 17 weeks old,
were purchased from Jackson Laboratories. Groups of 16 mice each
received doses of 100 .mu.g/kg/week of ISIS 516272 or ISIS 516323
for 20 weeks. Another group of 16 mice received doses of 100
.mu.g/kg/week of control oligonucleotide ISIS 141923 for 20 weeks.
Another group of 10 mice received doses of PBS for 20 weeks and
served as the control group to which all the other groups were
compared. Terminal endpoints were collected 48 hours after the last
dose was injected.
CFB RNA Analysis
[1169] RNA was extracted from liver and kidney tissue for real-time
PCR analysis of CFB, using primer probe set RTS3430. The mRNA
levels were normalized using RIBOGREEN.RTM.. As shown in the Table
below, some of the antisense oligonucleotides achieved reduction of
murine CFB over the PBS control. Results are presented as percent
inhibition of CFB, relative to control.
TABLE-US-00197 TABLE 204 Percent inhibition of murine CFB mRNA in
NZB/W F1 mice ISIS No Liver Kidney 516272 55 25 516323 63 43 141923
0 0
Proteinuria
[1170] Proteinuria is expected in 60% of animals in this mouse
model. The cumulative incidence of severe proteinuria was measured
by calculating the total protein to creatinine ratio using a
clinical analyzer. The results are presented in the table below and
demonstrate that treatment with antisense oligonucleotides
targeting CFB achieved reduction of proteinuria in the mice
compared to the PBS control and the control oligonucleotide treated
mice.
TABLE-US-00198 TABLE 205 Percent cumulative incidence of severe
proteinuria in NZB/W F1 mice % PBS 40 ISIS 516272 6 ISIS 516323 0
ISIS 141923 25
Survival
[1171] Survival of the mice was monitored by keeping count of the
mice at the start of treatment and then again at week 20. The
results are presented in the table below and demonstrate that
treatment with antisense oligonucleotides targeting CFB increased
survival in the mice compared to the PBS control and the control
oligonucleotide treated mice.
TABLE-US-00199 TABLE 206 Number of surviving mice and % survival
Week 1 Week 20 % survival at week 20 PBS 10 6 60 ISIS 516272 16 15
94 ISIS 516323 16 16 100 ISIS 141923 16 12 75
Glomerular Deposition
[1172] The amount of C3 deposition, as well as IgG deposition, in
the glomeruli of the kidneys was measured by immunohistochemistry
with an anti-C3 antibody. The results are presented in the table
below and demonstrate that treatment with antisense
oligonucleotides targeting CFB achieved reduction of both C3 and
IgG depositions in the kidney glomeruli compared to the PBS control
and the control oligonucleotide treated mice.
TABLE-US-00200 TABLE 207 Percent inhibition of glomerula deposition
in NZB/W F1 mice ISIS No C3 IgG 516272 45 20 516323 48 2 141923 0
0
Study 2
[1173] Female NZB/W F1 mice, 16 weeks old, were purchased from
Jackson Laboratories. A group of 10 mice received doses of 100
.mu.g/kg/week of ISIS 516323 for 12 weeks. Another group of 10 mice
received doses of 100 .mu.g/kg/week of control oligonucleotide ISIS
141923 for 12 weeks. Another group of 10 mice received doses of PBS
for 12 weeks and served as the control group to which all the other
groups were compared. Terminal endpoints were collected 48 hours
after the last dose was injected.
CFB RNA Analysis
[1174] RNA was extracted from liver and kidney tissue for real-time
PCR analysis of CFB, using primer probe set RTS3430. As shown in
the table below, treatment with ISIS 516323 achieved reduction of
murine CFB over the PBS control. Results are presented as percent
inhibition of CFB, relative to control.
TABLE-US-00201 TABLE 208 Percent inhibition of murine CFB mRNA in
NZB/W F1 mice ISIS No Liver Kidney 516323 75 46 141923 0 6
Proteinuria
[1175] The cumulative incidence of severe proteinuria was assessed
by measuring urine total protein to creatinine ratio, as well as by
measuring total microalbumin levels. The results are presented in
the tables below and demonstrate that treatment with antisense
oligonucleotides targeting CFB reduced proteinuria in the mice
compared to the PBS control and the control oligonucleotide treated
mice.
TABLE-US-00202 TABLE 209 Proteinuria in NZB/W F1 mice measured as
urine microalbumin levels (mg/dl) ISIS No Week 0 Week 6 Week 8 Week
10 516323 0 0 5.4 0.4 141923 0 8.28 8.6 5.6
TABLE-US-00203 TABLE 210 Proteinuria in NZB/W F1 mice measured as
total protein to creatinine ratio ISIS No Week 0 Week 6 Week 8 Week
10 516323 5.5 7.8 8.6 7.2 141923 6.9 10.0 13.5 7.2
Survival
[1176] Survival of the mice was monitored by keeping count of the
mice at the start of treatment and then again at week 12. The
results are presented in the table below and demonstrate that
treatment with antisense oligonucleotides targeting CFB increased
survival in the mice compared to the PBS control and the control
oligonucleotide treated mice.
TABLE-US-00204 TABLE 211 Number of surviving mice Week 1 Week 12
PBS 10 9 ISIS 516323 10 10 ISIS 141923 10 9
Example 132: Effect of Antisense Inhibition of CFB in the MRL Mouse
Model
[1177] The MRL/lpr lupus nephritis mouse model develops an SLE-like
phenotype characterized by lymphadenopathy due to an accumulation
of double negative (CD4.sup.- CD8.sup.-) and B220+ T-cells. These
mice display an accelerated mortality rate. In addition, the mice
have high concentrations of circulating immunoglobulins, which
included elevated levels of autoantibodies such as ANA, anti-ssDNA,
anti-dsDNA, anti-Sm, and rheumatoid factors, resulting in large
amounts of immune complexes (Andrews, B. et al., J. Exp. Med. 148:
1198-1215, 1978).
Treatment
[1178] A study was conducted to investigate whether treatment with
antisense oligonucleotides targeting CFB would reverse renal
pathology in the mouse model. Female MRL/lpr mice, 14 weeks old,
were purchased from Jackson Laboratories. A group of 10 mice
received doses of 50 .mu.g/kg/week of ISIS 516323 for 7 weeks.
Another group of 10 mice received doses of 50 .mu.g/kg/week of
control oligonucleotide ISIS 141923 for 7 weeks. Another group of
10 mice received doses of PBS for 7 weeks and served as the control
group to which all the other groups were compared. Terminal
endpoints were collected 48 hours after the last dose was
injected.
CFB RNA Analysis
[1179] RNA was extracted from liver tissue for real-time PCR
analysis of CFB, using primer probe set RTS3430. As shown in the
Table below, ISIS 516323 reduced CFB over the PBS control. Results
are presented as percent inhibition of CFB, relative to
control.
TABLE-US-00205 TABLE 212 Percent inhibition of murine CFB mRNA in
MRL/lpr mice ISIS No % 516323 68 141923 4
Renal Pathology
[1180] Renal pathology was evaluated by two methods. Histological
sections of the kidney were stained with Haematoxylin &Eosin.
The PBS control demonstrated presence of multiglomerular crescents
tubular casts, which is a symptom of glomerulosclerosis. In
contrast, the sections from mice treated with ISIS 516323 showed
absent crescents tubular casts with minimal bowman capsule fibrotic
changes, moderate to severe segmental mesangial cell expansion and
glomerular basement membrane thickening.
[1181] Accumulation of C3 in the kidney was also assessed by
immunohistochemistry with anti-C3 antibodies. The whole kidney C3
immunohistochemistry intensity score was calculated by intensity
scoring system, which was computed by capturing 10 glomeruli per
kidney and calculation the intensity of positive C3 staining. The
results are presented in the table below and demonstrate that
treatment with ISIS 516323 reduced renal C3 accumulation compared
to the control groups.
TABLE-US-00206 TABLE 213 Renal C3 accumulation in MRL/lpr mice
Whole kidney C3 C3 quantification (area/ intensity score total
area) % of average PBS PBS 2.5 100 ISIS 516323 1.6 68 ISIS 141923
2.2 99
Plasma C3 Levels
[1182] Reduction of CFB inhibits activation of the alternative
complement pathway, preventing C3 consumption and leading to an
apparent elevation of plasma C3 levels. Plasma C3 levels from
terminal bleed were measured by clinical analyzer. The results are
presented in the table below and demonstrate that treatment with
ISIS 516323 increased C3 levels (p<0.001) in the plasma compared
to the control groups.
TABLE-US-00207 TABLE 214 Plasma C3 levels (mg/dL) in MRL/lpr mice
ISIS No C3 516323 28 141923 16
The results indicate that treatment with antisense oligonucleotides
targeting CFB reverses renal pathology in the lupus mouse
model.
Example 133: Effect of Antisense Inhibition of CFB in the CFH Het
Mouse Model
[1183] CFH heterozygous (CFH Het, CFH.sup.+/-) mouse model express
a mutant Factor H protein in combination with the full-length mouse
protein (Pickering, M. C. et al., J. Exp. Med. 2007. 204:
1249-56).sub.n Renal histology remains normal in these mice up to
six months old.
Study 1
[1184] Groups of 8 CFH.sup.+/- mice, 6 weeks old, each received
doses of 75 mg/kg/week of ISIS 516323 or ISIS 516341 for 6 weeks.
Another group of 8 mice received doses of 75 mg/kg/week of control
oligonucleotide ISIS 141923 for 6 weeks. Another group of 8 mice
received doses of PBS for 6 weeks and served as the control group
to which all the other groups were compared. Terminal endpoints
were collected 48 hours after the last dose was injected.
CFB RNA Analysis
[1185] RNA was extracted from liver and kidney tissue for real-time
PCR analysis of CFB, using primer probe set RTS3430. As shown in
the Table below, the antisense oligonucleotides reduced CFB over
the PBS control. Results are presented as percent inhibition of
CFB, relative to control.
TABLE-US-00208 TABLE 215 Percent inhibition of murine CFB mRNA in
CFH.sup.+/- mice ISIS No Liver Kidney 516323 80 38 516341 90 44
141923 0 17
Plasma C3 levels
[1186] Reduction of CFB inhibits activation of the alternative
complement pathway, preventing C3 consumption and leading to an
apparent elevation of plasma C3 levels. Plasma C3 levels from
terminal plasma collection were measured by clinical analyzer. The
results are presented in the table below and demonstrate that
treatment with ISIS 516323 increased C3 to normal levels in the
plasma.
TABLE-US-00209 TABLE 216 Plasma C3 levels (mg/dL) in CFH.sup.+/-
mice ISIS No C3 516323 15 516341 17 141923 8
Study 2
[1187] Groups of 5 CFH.sup.+/- mice each received doses of 12.5
mg/kg/week, 25 mg/kg/week, 50 mg/kg/week, 75 mg/kg/week, or 100
mg/kg/week of ISIS 516323 or ISIS 516341 for 6 weeks. Another group
of 5 mice received doses of 75 .mu.g/kg/week of control
oligonucleotide ISIS 141923 for 6 weeks. Another group of 5 mice
received doses of PBS for 6 weeks and served as the control group
to which all the other groups were compared. Terminal endpoints
were collected 48 hours after the last dose was injected.
CFB RNA Analysis
[1188] RNA was extracted from liver and kidney tissue for real-time
PCR analysis of CFB, using primer probe set RTS3430. As shown in
the Table below, the antisense oligonucleotides reduced CFB over
the PBS control in a dose dependent manner. Results are presented
as percent inhibition of CFB, relative to control.
TABLE-US-00210 TABLE 217 Percent inhibition of murine CFB mRNA in
the liver of CFH.sup.+/- mice ISIS No Dose (mg/kg/week) % 516323
12.5 34 25 51 50 72 75 79 100 92 516341 12.5 38 25 57 50 89 75 92
100 90 141923 75 13
Plasma C3 levels
[1189] Reduction of CFB inhibits activation of the alternative
complement pathway, preventing C3 consumption and leading to an
apparent elevation of plasma C3 levels. Plasma C3 levels from
terminal plasma collection were measured by clinical analyzer. The
results are presented in the table below and demonstrate that
treatment with ISIS oligonucleotides targeting CFB increased C3
levels in the plasma.
TABLE-US-00211 TABLE 218 Plasma C3 levels (mg/dL) in CFH.sup.+/-
mice Dose (mg/kg/week) C3 PBS -- 10.1 516323 12.5 11.4 25 15.5 50
17.0 75 18.3 100 18.8 516341 12.5 12.1 25 16.3 50 18.6 75 22.1 100
19.1 141923 75 8.9
Example 134: Effect of ISIS Antisense Oligonucleotides Targeting
Human CFB in Cynomolgus Monkeys
[1190] Cynomolgus monkeys were treated with ISIS antisense
oligonucleotides selected from studies described in the Examples
above. Antisense oligonucleotide efficacy and tolerability, as well
as their pharmacokinetic profile in the liver and kidney, were
evaluated.
[1191] At the time this study was undertaken, the cynomolgus monkey
genomic sequence was not available in the National Center for
Biotechnology Information (NCBI) database; therefore
cross-reactivity with the cynomolgus monkey gene sequence could not
be confirmed. Instead, the sequences of the ISIS antisense
oligonucleotides used in the cynomolgus monkeys was compared to a
rhesus monkey sequence for homology. It is expected that ISIS
oligonucleotides with homology to the rhesus monkey sequence are
fully cross-reactive with the cynomolgus monkey sequence as well.
The human antisense oligonucleotides tested are cross-reactive with
the rhesus genomic sequence (GENBANK Accession No. NW_001116486.1
truncated from nucleotides 536000 to 545000, designated herein as
SEQ ID NO: 3). The greater the complementarity between the human
oligonucleotide and the rhesus monkey sequence, the more likely the
human oligonucleotide can cross-react with the rhesus monkey
sequence. The start and stop sites of each oligonucleotide targeted
to SEQ ID NO: 3 is presented in the Table below. "Start site"
indicates the 5'-most nucleotide to which the gapmer is targeted in
the rhesus monkey gene sequence. `Mismatches` indicates the number
of nucleobases in the human oligonucleotide that are mismatched
with the rhesus genomic sequence.
TABLE-US-00212 TABLE 219 Antisense oligonucleotides complementary
to the rhesus CFB genomic sequence (SEQ ID NO: 3) ISIS Target SEQ
No Start Site Mismatches Chemistry ID NO 532770 6788 0 5-10-5 MOE
198 532800 7500 0 5-10-5 MOE 228 532809 7614 0 5-10-5 MOE 237
588540 7627 1 5-10-5 MOE 440 588544 7631 1 5-10-5 MOE 444 588548
7635 1 5-10-5 MOE 448 588550 7637 1 5-10-5 MOE 450 588553 7640 1
5-10-5 MOE 453 588555 7643 0 5-10-5 MOE 455 588848 7639 1 Deoxy,
MOE and cEt 598 594430 6790 0 3-10-3 cEt 549
Treatment
[1192] Prior to the study, the monkeys were kept in quarantine for
at least a 30 day period, during which the animals were observed
daily for general health. The monkeys were 2-4 years old and
weighed between 2 and 4 kg. Eleven groups of 4-6 randomly assigned
male cynomolgus monkeys each were injected subcutaneously with ISIS
oligonucleotide or PBS at four sites on the back in a clockwise
rotation (i.e. left, top, right, and bottom), one site per dose.
The monkeys were given four loading doses of PBS or 40 mg/kg of
ISIS 532800, ISIS 532809, ISIS 588540, ISIS 588544, ISIS 588548,
ISIS 588550, ISIS 588553, ISIS 588555, ISIS 588848, or ISIS 594430
for the first week (days 1, 3, 5, and 7), and were subsequently
dosed once a week for 12 weeks (days 14, 21, 28, 35, 42, 49, 56,
63, 70, 77, and 84) with PBS or 40 mg/kg of ISIS oligonucleotide.
ISIS 532770 was tested in a separate study with similar conditions
with two male and two female cynomolgus monkeys in the group.
Hepatic Target Reduction
RNA Analysis
[1193] On day 86, liver and kidney samples were collected in
duplicate (approximately 250 mg each) for CFB mRNA analysis. The
samples were flash frozen in liquid nitrogen at necropsy within
approximately 10 minutes of sacrifice.
[1194] RNA was extracted from liver and kidney for real-time PCR
analysis of measurement of mRNA expression of CFB. Results are
presented as percent change of mRNA, relative to PBS control,
normalized with RIBOGREEN.RTM.. RNA levels were also normalized
with the house-keeping gene, Cyclophilin A. RNA levels were
measured with the primer probe sets RTS3459, described above, or
RTS4445_MGB (forward sequence CGAAGAAGCTCAGTGAAATCAA, designated
herein as SEQ ID NO: 819; reverse sequence TGCCTGGAGGGCCCTCTT,
designated herein as SEQ ID NO: 820; probe sequence
AGACCACAAGTTGAAGTC, designated herein as SEQ ID NO: 815).
[1195] As shown in the Tables below, treatment with ISIS antisense
oligonucleotides resulted in reduction of CFB mRNA in comparison to
the PBS control. Analysis of CFB mRNA levels revealed that several
of the ISIS oligonucleotides reduced CFB levels in liver and/or
kidney. Here `0` indicates that the expression levels were not
inhibited. `*` indicates that the oligonucleotide was tested in a
separate study with similar conditions.
TABLE-US-00213 TABLE 220 Percent inhibition of CFB mRNA in the
cynomolgus monkey liver relative to the PBS control ISIS RTS3459/
RTS3459/ RTS445_MGB/ RTS445_MGB/ No Cyclophilin A RIBOGREEN
Cyclophilin A RIBOGREEN 532770* 12 37 24 45 532800 54 45 56 46
588540 31 27 28 24 588548 68 67 68 67 588550 53 39 51 37 588553 74
59 74 59 588555 73 71 71 69 588848 9 0 6 0 594430 24 26 23 25
TABLE-US-00214 TABLE 221 Percent inhibition of CFB mRNA in the
cynomolgus monkey kidney relative to the PBS control ISIS RTS3459/
RTS3459/ RTS445_MGB/ RTS445_MGB/ No Cyclophilin A RIBOGREEN
Cyclophilin A RIBOGREEN 532770* 34 56 2 31 532800 36 30 43 37
588540 70 71 67 69 588548 83 84 82 83 588550 81 77 78 74 588553 86
84 86 85 588555 32 34 48 50 588848 89 91 87 90 594430 33 37 19
23
Protein Analysis
[1196] Approximately 1 mL of blood was collected from all available
animals at day 85 and placed in tubes containing the potassium salt
of EDTA. The blood samples were placed in wet-ice or Kryorack
immediately, and centrifuged (3000 rpm for 10 min at 4.degree. C.)
to obtain plasma (approximately 0.4 mL) within 60 minutes of
collection. Plasma levels of CFB were measured in the plasma by
radial immunodiffusion (RID), using a polyclonal anti-Factor B
antibody. The results are presented in the Table below. ISIS 532770
was tested in a separate study and plasma protein levels were
measured on day 91 or 92 in that group.
[1197] Analysis of plasma CFB revealed that several ISIS
oligonucleotides reduced protein levels in a sustained manner. ISIS
532770, which was tested in a separate study, reduced CFB protein
levels on day 91/92 by 50% compared to baseline values. The
reduction in plasma CFB protein levels correlates well with liver
CFB mRNA level reduction in the corresponding groups of
animals.
TABLE-US-00215 TABLE 222 Plasma protein levels (% baseline values)
in the cynomolgus monkey Day 1 Day 30 Day 58 Day 72 Day 86 PBS 113
115 95 83 86 ISIS 532800 117 68 52 39 34 ISIS 532809 104 121 100 80
71 ISIS 588540 108 72 61 40 38 ISIS 588544 118 74 53 33 29 ISIS
588548 110 41 28 20 16 ISIS 588550 104 64 54 38 37 ISIS 588553 97
42 35 18 16 ISIS 588555 107 35 37 18 18 ISIS 588848 116 95 92 69 71
ISIS 594430 104 64 59 45 46
Tolerability Studies
Body Weight Measurements
[1198] To evaluate the effect of ISIS oligonucleotides on the
overall health of the animals, body and organ weights were measured
and are presented in the Table below. `*` indicates that the
oligonucleotide was tested in a separate study with similar
conditions and is the average of the measurements from male and
female monkeys. The results indicate that effect of treatment with
antisense oligonucleotides on body and organ weights was within the
expected range for antisense oligonucleotides.
TABLE-US-00216 TABLE 223 Final body weights (g) in cynomolgus
monkey Day Day Day Day Day Day Day 1 14 28 42 56 70 84 PBS 2887
2953 3028 3094 3125 3143 3193 ISIS 532770* 2963 2947 2966 3050 3097
3138 3160 ISIS 532800 2886 2976 3072 3149 3220 3269 3265 ISIS
532809 2755 2836 2927 2983 3019 3071 3098 ISIS 588540 2779 2834
2907 2934 2981 3034 3057 ISIS 588544 2837 2896 3009 3064 3132 3163
3199 ISIS 588548 2694 2816 2882 2990 3073 3149 3161 ISIS 588550
2855 2988 3062 3188 3219 3282 3323 ISIS 588553 3033 3156 3256 3335
3379 3372 3442 ISIS 588555 2757 2863 2965 3022 3075 3088 3158 ISIS
588848 2850 3018 3032 3187 3230 3212 3291 ISIS 594430 2884 2963
2953 3149 3187 3204 3256
TABLE-US-00217 TABLE 224 Final organ weights (g) in cynomolgus
monkey Spleen Heart Kidney Liver PBS 2.8 11.6 11.9 55.8 ISIS
532770* 5.0 11.3 20.6 77.9 ISIS 532800 6.2 11.9 18.6 94.4 ISIS
588540 4.0 11.4 13.5 67.1 ISIS 588548 4.1 11.7 17.3 72.0 ISIS
588550 5.8 10.9 18.5 81.8 ISIS 588553 5.0 12.7 17.2 85.9 ISIS
588555 4.7 11.8 15.9 88.3 ISIS 588848 5.0 12.7 14.4 75.7 ISIS
594430 3.9 11.9 14.8 69.9
Liver Function
[1199] To evaluate the effect of ISIS oligonucleotides on hepatic
function, blood samples were collected from all the study groups.
The blood samples were collected from the cephalic, saphenous, or
femoral veins, 48 hours post-dosing. The monkeys were fasted
overnight prior to blood collection. Blood (1.5 mL) was collected
in tubes without anticoagulant for serum separation. The tubes were
kept at room temperature for a minimum of 90 minutes and then
centrifuged (approximately 3,000 rpm for 10 min) to obtain serum.
Levels of various liver function markers were measured using a
Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan).
[1200] Plasma levels of ALT and AST were measured and the results
are presented in the Table below, expressed in IU/L. Bilirubin, a
liver function marker, was similarly measured and is presented in
the Table below expressed in mg/dL. `*` indicates that the
oligonucleotide was tested in a separate study with similar
conditions and is the average of the measurements from male and
female monkeys. The results indicate that most of the antisense
oligonucleotides had no effect on liver function outside the
expected range for antisense oligonucleotides.
TABLE-US-00218 TABLE 225 Liver chemistry marker levels in
cynomolgus monkey plasma on day 86 ALT (IU/L) AST (IU/L) Bilirubin
(mg/dL) PBS 71 57 0.3 ISIS 532770* 59 58 0.1 ISIS 532800 65 86 0.1
ISIS 532809 35 58 0.1 ISIS 588540 70 88 0.2 ISIS 588544 55 97 0.2
ISIS 588548 61 85 0.2 ISIS 588550 94 84 0.2 ISIS 588553 44 65 0.2
ISIS 588555 63 84 0.2 ISIS 588848 69 65 0.2 ISIS 594430 86 53
0.2
Kidney Function
[1201] To evaluate the effect of ISIS oligonucleotides on kidney
function, blood samples were collected from all the study groups.
The blood samples were collected from the cephalic, saphenous, or
femoral veins, 48 hours post-dosing. The monkeys were fasted
overnight prior to blood collection. Blood was collected in tubes
without anticoagulant for serum separation. The tubes were kept at
room temperature for a minimum of 90 minutes and then centrifuged
(approximately 3,000 rpm for 10 min) to obtain serum. Levels of BUN
and creatinine were measured using a Toshiba 200FR NEO chemistry
analyzer (Toshiba Co., Japan). Results are presented in the Table
below, expressed in mg/dL. `*` indicates that the oligonucleotide
was tested in a separate study with similar conditions and is the
average of the measurements from male and female monkeys.
[1202] For urinalysis, fresh urine from all the animals was
collected in the morning using a clean cage pan on wet ice. Food
was removed overnight the day before urine collection but water was
supplied. Urine samples (approximately 1 mL) were analyzed for
protein to creatinine (P/C) ratio using a Toshiba 200FR NEO
automated chemistry analyzer (Toshiba Co., Japan). `n.d.` indicates
that the urine protein level was under the detection limit of the
analyzer.
[1203] The plasma and urine chemistry data indicate that most of
the ISIS oligonucleotides did not have any effect on the kidney
function outside the expected range for antisense
oligonucleotides.
TABLE-US-00219 TABLE 226 Renal chemistry marker levels (mg/dL) in
cynomolgus monkey plasma on day 86 BUN Creatinine Total protein PBS
28 0.9 8.0 ISIS 532770* 20 0.9 6.9 ISIS 532800 25 0.9 7.5 ISIS
532809 23 0.8 7.4 ISIS 588540 30 0.8 7.5 ISIS 588544 26 0.9 7.4
ISIS 588548 25 0.9 7.6 ISIS 588550 24 0.9 7.2 ISIS 588553 25 0.8
7.2 ISIS 588555 25 0.8 7.6 ISIS 588848 24 0.9 7.5 ISIS 594430 25
0.8 7.2
TABLE-US-00220 TABLE 227 Renal chemistry marker levels in
cynomolgus monkey urine on day 44 and day 86 Day 44 Day 86 PBS 0.03
n.d. ISIS 532800 0.01 n.d. ISIS 532809 0.01 n.d. ISIS 588540 0.03
n.d. ISIS 588544 0.01 0.09 ISIS 588548 0.01 0.01 ISIS 588550 0.04
0.01 ISIS 588553 0.05 n.d. ISIS 588555 0.03 0.03 ISIS 588848 0.09
n.d. ISIS 594430 0.03 n.d.
Hematology
[1204] To evaluate any effect of ISIS oligonucleotides in
cynomolgus monkeys on hematologic parameters, blood samples of
approximately 0.5 mL of blood was collected from each of the
available study animals in tubes containing K.sub.2-EDTA. Samples
were analyzed for red blood cell (RBC) count, white blood cells
(WBC) count, individual white blood cell counts, such as that of
monocytes, neutrophils, lymphocytes, as well as for platelet count,
hemoglobin content and hematocrit, using an ADVIA120 hematology
analyzer (Bayer, USA). The data is presented in the Tables below.
`*` indicates that the oligonucleotide was tested in a separate
study with similar conditions and is the average of the
measurements from male and female monkeys.
[1205] The data indicate the oligonucleotides did not cause any
changes in hematologic parameters outside the expected range for
antisense oligonucleotides at this dose.
TABLE-US-00221 TABLE 228 Blood cell counts in cynomolgus monkeys
RBC Platelets WBC Neutrophils Lymphocytes Monocytes
(.times.10.sup.6/.mu.L) (.times.10.sup.3/.mu.L)
(.times.10.sup.3/.mu.L) (% WBC) (% total) (% total) PBS 5.8 347 9.4
42.7 53.1 3.0 ISIS 532770* 5.4 386 10.8 22.3 71.7 3.3 ISIS 532800
5.6 360 13.1 29.5 61.1 6.5 ISIS 532809 5.2 400 11.5 56.6 38.2 2.5
ISIS 588540 5.5 367 11.7 50.9 42.7 2.1 ISIS 588544 5.2 373 14.3
56.6 37.6 4.3 ISIS 588548 5.1 373 9.7 40.4 54.3 3.9 ISIS 588550 6.1
343 9.9 32.1 61.7 4.6 ISIS 588553 5.2 424 9.3 41.7 53.2 3.6 ISIS
588555 5.1 411 9.6 45.1 49.7 3.5 ISIS 588848 5.7 370 10.0 39.8 55.8
3.1 ISIS 594430 5.7 477 10.6 47.3 47.8 3.6
TABLE-US-00222 TABLE 229 Hematologic parameters in cynomolgus
monkeys Hemoglobin (g/dL) HCT (%) PBS 14.1 46.6 ISIS 532770* 12.4
40.9 ISIS 532800 12.3 40.5 ISIS 532809 12.2 40.4 ISIS 588540 12.5
41.5 ISIS 588544 11.9 38.1 ISIS 588548 12.3 39.6 ISIS 588550 13.4
45.0 ISIS 588553 12.6 39.8 ISIS 588555 11.6 38.1 ISIS 588848 13.2
42.7 ISIS 594430 13.4 43.1
Measurement of Oligonucleotide Concentration
[1206] The concentration of the full-length oligonucleotide was
measured in the kidney and liver tissues. The method used is a
modification of previously published methods (Leeds et al., 1996;
Geary et al., 1999) which consist of a phenol-chloroform
(liquid-liquid) extraction followed by a solid phase extraction.
Tissue sample concentrations were calculated using calibration
curves, with a lower limit of quantitation (LLOQ) of approximately
1.14 .mu.g/g. The results are presented in the Table below,
expressed as .mu.gig liver or kidney tissue.
TABLE-US-00223 TABLE 230 Antisense oligonucleotide distribution
Kidney (.mu.g/g) Liver (.mu.g/g) Kidney/Liver ratio ISIS 532800
3881 1633 2.4 ISIS 588540 3074 1410 2.2 ISIS 588548 3703 1233 3.0
ISIS 588550 4242 860 4.9 ISIS 588553 3096 736 4.2 ISIS 588555 4147
1860 2.2 ISIS 588848 2235 738 3.0 ISIS 594430 1548 752 2.1
Example 135: 6 Week Efficacy Study of Unconjugated and
5'-THA-GalNAc3 Conjugated Antisense Oligonucleotides Targeted to
Human CFB in Transgenic Mice
[1207] Two antisense oligonucleotides having the same nucleobase
sequence: uncongugated antisense oligonucleotide ISIS 588540 and
5'-THA-GalNAc.sub.3-conjugated antisense oligonucleotide ISIS
696844, were tested in human CFB transgenic mice (hCFB-Tg
mice).
[1208] The mice were administered subcutaneously with ISIS 696844
at doses of 0.1, 1.25, 0.5, 2.0, 6.0, or 12.0 mg/kg/week or with
ISIS 588540 at doses of 2, 6, 12, 25, or 50 mg/kg/week for 6 weeks.
A control group of mice were administered subcutaneously with PBS
for 6 weeks. Mice were sacrificed 48 hours after the last dose.
Hepatic mRNA levels were analyzed by qRT-PCR.
Study 1
[1209] The results are presented in the Table below and demonstrate
that the 5'-THA-GalNAc.sub.3-conjugated antisense oligonucleotide
targeting CFB is more potent than the unconjugated antisense
oligonucleotide with the same sequence.
TABLE-US-00224 TABLE 231 Efficacy of antisense oligonucleotides
targeting CFB ED.sub.50 (mg/kg) ED.sub.75 (mg/kg) ISIS 588540 4.52
9.26 ISIS 696844 0.52 1.12
Study 2
[1210] Liver mRNA levels were measured with two different primer
probe sets targeting different regions of the mRNA and normalized
to either RIBOGREEN.RTM. (RGB) or Cyclophilin. The primer probe
sets were RTS3459, described above, and RTS3460 (forward sequence
CGAAGCAGCTCAATGAAATCAA, designated herein as SEQ ID NO: 813;
reverse sequence TGCCTGGAGGGCCTTCTT, designated herein as SEQ ID
NO: 814; probe sequence AGACCACAAGTTGAAGTC, designated herein as
SEQ ID NO: 815). The results are presented in the Table below and
demonstrate that the 5'-THA-GalNAc.sub.3-conjugated antisense
oligonucleotide targeting CFB is more potent than the unconjugated
antisense oligonucleotide with the same sequence, irrespective of
the primer probe set used.
TABLE-US-00225 TABLE 231 Efficacy of antisense oligonucleotides
targeting CFB ED.sub.50 ED.sub.50 ED.sub.50 ED.sub.50 ED.sub.75
ED.sub.75 ED.sub.75 ED.sub.75 RTS3459 RTS3460 RTS3459 RTS3460
RTS3459 RTS3460 RTS3459 RTS3460 (RGB) (RGB) (Cyclophilin)
(Cyclophilin) (RGB) (RGB) (Cyclophilin) (Cyclophilin) ISIS 588540
4.5 4.1 5.2 5.4 9.3 10.0 10.0 9.3 ISIS 696844 0.5 0.5 0.6 0.5 1.1
1.3 1.2 0.9
Sequence CWU 1
1
85412646DNAHomo sapiens 1gacttctgca gtttctgttt ccttgactgg
cagctcagcg gggccctccc gcttggatgt 60tccgggaaag tgatgtgggt aggacaggcg
gggcgagccg caggtgccag aacacagatt 120gtataaaagg ctgggggctg
gtggggagca ggggaaggga atgtgaccag gtctaggtct 180ggagtttcag
cttggacact gagccaagca gacaagcaaa gcaagccagg acacaccatc
240ctgccccagg cccagcttct ctcctgcctt ccaacgccat ggggagcaat
ctcagccccc 300aactctgcct gatgcccttt atcttgggcc tcttgtctgg
aggtgtgacc accactccat 360ggtctttggc ccggccccag ggatcctgct
ctctggaggg ggtagagatc aaaggcggct 420ccttccgact tctccaagag
ggccaggcac tggagtacgt gtgtccttct ggcttctacc 480cgtaccctgt
gcagacacgt acctgcagat ctacggggtc ctggagcacc ctgaagactc
540aagaccaaaa gactgtcagg aaggcagagt gcagagcaat ccactgtcca
agaccacacg 600acttcgagaa cggggaatac tggccccggt ctccctacta
caatgtgagt gatgagatct 660ctttccactg ctatgacggt tacactctcc
ggggctctgc caatcgcacc tgccaagtga 720atggccgatg gagtgggcag
acagcgatct gtgacaacgg agcggggtac tgctccaacc 780cgggcatccc
cattggcaca aggaaggtgg gcagccagta ccgccttgaa gacagcgtca
840cctaccactg cagccggggg cttaccctgc gtggctccca gcggcgaacg
tgtcaggaag 900gtggctcttg gagcgggacg gagccttcct gccaagactc
cttcatgtac gacacccctc 960aagaggtggc cgaagctttc ctgtcttccc
tgacagagac catagaagga gtcgatgctg 1020aggatgggca cggcccaggg
gaacaacaga agcggaagat cgtcctggac ccttcaggct 1080ccatgaacat
ctacctggtg ctagatggat cagacagcat tggggccagc aacttcacag
1140gagccaaaaa gtgtctagtc aacttaattg agaaggtggc aagttatggt
gtgaagccaa 1200gatatggtct agtgacatat gccacatacc ccaaaatttg
ggtcaaagtg tctgaagcag 1260acagcagtaa tgcagactgg gtcacgaagc
agctcaatga aatcaattat gaagaccaca 1320agttgaagtc agggactaac
accaagaagg ccctccaggc agtgtacagc atgatgagct 1380ggccagatga
cgtccctcct gaaggctgga accgcacccg ccatgtcatc atcctcatga
1440ctgatggatt gcacaacatg ggcggggacc caattactgt cattgatgag
atccgggact 1500tgctatacat tggcaaggat cgcaaaaacc caagggagga
ttatctggat gtctatgtgt 1560ttggggtcgg gcctttggtg aaccaagtga
acatcaatgc tttggcttcc aagaaagaca 1620atgagcaaca tgtgttcaaa
gtcaaggata tggaaaacct ggaagatgtt ttctaccaaa 1680tgatcgatga
aagccagtct ctgagtctct gtggcatggt ttgggaacac aggaagggta
1740ccgattacca caagcaacca tggcaggcca agatctcagt cattcgccct
tcaaagggac 1800acgagagctg tatgggggct gtggtgtctg agtactttgt
gctgacagca gcacattgtt 1860tcactgtgga tgacaaggaa cactcaatca
aggtcagcgt aggaggggag aagcgggacc 1920tggagataga agtagtccta
tttcacccca actacaacat taatgggaaa aaagaagcag 1980gaattcctga
attttatgac tatgacgttg ccctgatcaa gctcaagaat aagctgaaat
2040atggccagac tatcaggccc atttgtctcc cctgcaccga gggaacaact
cgagctttga 2100ggcttcctcc aactaccact tgccagcaac aaaaggaaga
gctgctccct gcacaggata 2160tcaaagctct gtttgtgtct gaggaggaga
aaaagctgac tcggaaggag gtctacatca 2220agaatgggga taagaaaggc
agctgtgaga gagatgctca atatgcccca ggctatgaca 2280aagtcaagga
catctcagag gtggtcaccc ctcggttcct ttgtactgga ggagtgagtc
2340cctatgctga ccccaatact tgcagaggtg attctggcgg ccccttgata
gttcacaaga 2400gaagtcgttt cattcaagtt ggtgtaatca gctggggagt
agtggatgtc tgcaaaaacc 2460agaagcggca aaagcaggta cctgctcacg
cccgagactt tcacatcaac ctctttcaag 2520tgctgccctg gctgaaggag
aaactccaag atgaggattt gggttttcta taaggggttt 2580cctgctggac
aggggcgtgg gattgaatta aaacagctgc gacaacaaaa aaaaaaaaaa 2640aaaaaa
264629001DNAHomo sapiens 2ggaggtgagg gtctcaggtt ggggatgctg
ggatccccct gtgacagctc ccagaatgtc 60tctcttcctt ctccaggtct ggctgctttc
tctctctgac gcgggtcacc cctcctccca 120agcctcacaa acctgctagg
tgtccctggg tctgcttatt ctttttttgt tgttattgag 180atggagtctt
gctctgtctc ccaggctgga gtgcagtggc acgacctcag ctcactgcaa
240cttctgcctc ctgggttcaa gcgattctcc tacttcagcc tcccgagtag
ctgagattac 300aggtgcccac caccacacca gctaattttt gtatttttag
tagagacggg atttcgccat 360gttggccagg atggtcttga actcctgacc
tcaagtgatc tgcctgcctc aacctcccaa 420agtgctgaga ttacaggcgt
gagccactgc acccacccgg gtctgcttat tctacccttc 480tctctggttc
cacccctgct gcagtggaca agctgtgccg aggttgtctc ccaagaaaaa
540accatgttcc ccaacttgac agatgtcagg gaggtggtga cagaccagtt
cctatgcagt 600gggacccagg aggatgagag tccctgcaag ggtgagtccc
tcaccatgcc tggattccca 660aggggaaggc cacctgtgtc tctgtggcca
gcatgcatgc cagaacacca gtccactgcc 720ctagatgaca ctgtctcctg
tcaccctttg ctggcaggag aatctggggg agcagttttc 780cttgagcgga
gattcaggtt ttttcaggtg agaaggtaga agcttgcagg acccaggggt
840tacaggatct cagccttgtt ggggggatga gggaggcctt tgagggatct
agggaggttg 900gggcttacag ttggggctgt ggcagcctcc cagccagttc
tctccttttc tccaggtggg 960tctggtgagc tggggtcttt acaacccctg
ccttggctct gctgacaaaa actcccgcaa 1020aagggcccct cgtagcaagg
tcccgccgcc acgagacttt cacatcaatc tcttccgcat 1080gcagccctgg
ctgaggcagc acctggggga tgtcctgaat tttttacccc tctagccatg
1140gccactgagc cctctgctgc cctgccagaa tctgccgccc ctccatcttc
tacctctgaa 1200tggccaccct tagaccctgt gatccatcct ctctcctagc
tgagtaaatc cgggtctcta 1260ggatgccaga ggcagcgcac acaagctggg
aaatcctcag ggctcctacc agcaggactg 1320cctcgctgcc ccacctcccg
ctccttggcc tgtccccaga ttccttccct ggttgacttg 1380actcatgctt
gtttcacttt cacatggaat ttcccagtta tgaaattaat aaaaatcaat
1440ggtttccaca tctctcagtg cctctatctg gaggccaggt agggctggcc
ttgggggagg 1500gggaggccag aatgactcca agagctacag gaaggcaggt
cagagacccc actggacaaa 1560cagtggctgg actctgcacc ataacacaca
atcaacaggg gagtgagctg gatccttatt 1620tctggtccct aagtgggtgg
tttgggctta ctggggagga gctaaggccg gagaggaggt 1680actgaagggg
agagtcctgg acctttggca gcaaagggtg ggacttctgc agtttctgtt
1740tccttgactg gcagctcagc ggggccctcc cgcttggatg ttccgggaaa
gtgatgtggg 1800taggacaggc ggggcgagcc gcaggtgcca gaacacagat
tgtataaaag gctgggggct 1860ggtggggagc aggggaaggg aatgtgacca
ggtctaggtc tggagtttca gcttggacac 1920tgagccaagc agacaagcaa
agcaagccag gacacaccat cctgccccag gcccagcttc 1980tctcctgcct
tccaacgcca tggggagcaa tctcagcccc caactctgcc tgatgccctt
2040tatcttgggc ctcttgtctg gaggtaagcg agggtaacct tcccttcctg
ctgtctccag 2100catccctcct tggccttttg gggccaggct tcatcagcct
ttctcttcag gtgtgaccac 2160cactccatgg tctttggccc ggccccaggg
atcctgctct ctggaggggg tagagatcaa 2220aggcggctcc ttccgacttc
tccaagaggg ccaggcactg gagtacgtgt gtccttctgg 2280cttctacccg
taccctgtgc agacacgtac ctgcagatct acggggtcct ggagcaccct
2340gaagactcaa gaccaaaaga ctgtcaggaa ggcagagtgc agaggtttga
gggcaatgag 2400tgtgggcagt ggcctaaggc agaaacaggg caggcggcag
caaggtcagg actaggatga 2460gactaggcag ggtgacaagg tgggctgacc
gggagtagga gcagttttag ggtggcaggc 2520ggaaaggggg caagaaaaag
cggagttaac ccttactaag catttaccct gggcttccag 2580gcagccctgg
aagtcaagag aacactcaga aatggggagg gagaagcagt ggaaatccat
2640atgggttgag gagtaggtaa gatgctgctt ctgcgggact gggaatgcgc
tgtttctcag 2700tgacatggtc tccgagacca ggagggatac acctaaggca
gcctttccct cttgatgact 2760tctacttgtc cccccttctc aaagcaatcc
actgtccaag accacacgac ttcgagaacg 2820gggaatactg gccccggtct
ccctactaca atgtgagtga tgagatctct ttccactgct 2880atgacggtta
cactctccgg ggctctgcca atcgcacctg ccaagtgaat ggccgatgga
2940gtgggcagac agcgatctgt gacaacggag gtgagaagca tcccctcccc
ctacattgct 3000gtctccctga cggcgcccag cccgaggagt gggcactcgg
ctccggacac tgtaactctt 3060gctctctacc ttgctcacgg ggcctcaggc
ttcagtgctt acctcgatgt ctcatacctc 3120tgcagcgggg tactgctcca
acccgggcat ccccattggc acaaggaagg tgggcagcca 3180gtaccgcctt
gaagacagcg tcacctacca ctgcagccgg gggcttaccc tgcgtggctc
3240ccagcggcga acgtgtcagg aaggtggctc ttggagcggg acggagcctt
cctgccaagg 3300tgacctttga cctgtacccc caggtcagat cctggtcttc
catcctactg tcttctctcc 3360ccacctcaac cctgctcttt cctcactttg
tttaaacctc cctgtacaac tatctcactt 3420ctgagccttt tataccctgg
aaacccatga tcccccgtct ctttggtcac tgtatccctg 3480acactcccag
acatttgacc tcatttctga ctctcccaga ctccttcatg tacgacaccc
3540ctcaagaggt ggccgaagct ttcctgtctt ccctgacaga gaccatagaa
ggagtcgatg 3600ctgaggatgg gcacggccca ggtttgaaga cagagaaggg
aggcagggca gggaactggg 3660ggaaaatgga gaagggacag aactgttaat
gctggagcct gagccactct cctggcaccc 3720aggggaacaa cagaagcgga
agatcgtcct ggacccttca ggctccatga acatctacct 3780ggtgctagat
ggatcagaca gcattggggc cagcaacttc acaggagcca aaaagtgtct
3840agtcaactta attgagaagg tggaatcctc ctatccctga actcggggga
atggaatctc 3900gctgatcttc caggactagc tccctgatca ttccagcccc
tctgaacaac agggccccag 3960gaaaatctcc aggtcctatt ctgtcctcct
tcccttttac ttgaagcagt ttcttgactg 4020gtaattcctc catgaacctc
agcccttgag cctcttactg agagcctccc tgtcccagca 4080aagtcgctga
aatctcccaa tcacagtatt ctattttcaa tgccatggcg ccttgttctc
4140ctcacccaca ggtggcaagt tatggtgtga agccaagata tggtctagtg
acatatgcca 4200cataccccaa aatttgggtc aaagtgtctg aagcagacag
cagtaatgca gactgggtca 4260cgaagcagct caatgaaatc aattatgaag
gtcagaggtt agggaatggt gggaggttca 4320ctttggggtc aggaggttca
gggtggaggg ggtcatgaga ctaccttgag ggcgacaggg 4380aggaccactt
tgtagtcaaa agttgaacag caggatcgtt gggcaatgga ggttagtggg
4440aacctgttgg gggctggaag ggccactttg tggtcaaagg gaagtccgtg
taatgatgat 4500taacttaaaa agttgaaaga tgtgggattt cagttgcaga
ttggtctctg gggttaaaag 4560atggcttgga agaccaggtg aggtgatggt
ctcttccctc tccacagacc acaagttgaa 4620gtcagggact aacaccaaga
aggccctcca ggcagtgtac agcatgatga gctggccaga 4680tgacgtccct
cctgaaggct ggaaccgcac ccgccatgtc atcatcctca tgactgatgg
4740tcagaaggga cctctctcct gtcccagcct ccccaccttc tcagaccagc
atgtggccct 4800taagtccact tgtaacacta tacccatggt tggggccctg
aatgtgactc atagctggct 4860gttcatctct cctgtgaccc ttcataagga
attcttccta agccctgtga tcaactatct 4920ctaacccttc ctcaacttgc
tcaccctgcc atgtgtatcc ctgcctttag ccagtttatc 4980ttccttatct
cctaccctca tggtcctgtc tcttctgcag gattgcacaa catgggcggg
5040gacccaatta ctgtcattga tgagatccgg gacttgctat acattggcaa
ggatcgcaaa 5100aacccaaggg aggattatct gggtgagtaa cctgcctagg
acccagcacc ccacttcctc 5160agggcttgga ccctcatcct tcctttttat
ccctcagatg tctatgtgtt tggggtcggg 5220cctttggtga accaagtgaa
catcaatgct ttggcttcca agaaagacaa tgagcaacat 5280gtgttcaaag
tcaaggatat ggaaaacctg gaagatgttt tctaccaaat gatcggtagg
5340gagatacaag ggaataaaga acacaactct cctcaggttc ccctgaagta
attcattctt 5400cctctacacc tgaagctcta gttgcctgga aagccttctt
cattcctcct tctctacctc 5460agtgtcacta ttcttgtttc ctggcactgt
tcacttaacc ttagaatcac agagctctga 5520gcacttcaga gatctttcta
tagtcctaca tttgacacgt ggaaacagaa gccaaaggag 5580gtcaagggac
agcaagttag caacaagggt gggcttgaaa acagccaggc ctctgacagc
5640ttgatcccaa gttctttccc ttttcagtcc accatagcag ttttctccta
acacgaggaa 5700acaaataccc gtggtctttc cctttctcct tttgggcctt
tgctccccat agactcctac 5760ccaaaaggct gctgccattt gggaatgaag
tgttccgagt tttcagcaca ttctccttct 5820ctgccagatg aaagccagtc
tctgagtctc tgtggcatgg tttgggaaca caggaagggt 5880accgattacc
acaagcaacc atggcaggcc aagatctcag tcattgtaag cacagaatcc
5940cagtagtggg gacttggggg aggtgaggtc aaggtgaaat gggagtaggg
gaaggaaaaa 6000atggccataa gagatggtgg tttgtgaaag ttgagctttc
cctctctact gttgtgtccc 6060cagcgccctt caaagggaca cgagagctgt
atgggggctg tggtgtctga gtactttgtg 6120ctgacagcag cacattgttt
cactgtggat gacaaggaac actcaatcaa ggtcagcgta 6180ggtaaggatg
caactgaagg tcctgggctg cacctatgct ctccaggcaa cacctcccac
6240tttctacaga tcctacactc cacccatcct caatgcagcc ccattccttg
caccccagac 6300cagtcaggga tgggggaaga cgtgaagtta ggaatgacac
ggggccagag gcaggaagct 6360gcccacaaag aggtggtacc tactctccta
cttcaggagg ggagaagcgg gacctggaga 6420tagaagtagt cctatttcac
cccaactaca acattaatgg gaaaaaagaa gcaggaattc 6480ctgaatttta
tgactatgac gttgccctga tcaagctcaa gaataagctg aaatatggcc
6540agactatcag gtgagagcgt ccagatccct gaggaaaggc tgggaaaggc
tggaggactg 6600gggtgaggag caggcctggt ttgctgttct ccttgtcctt
tataggccca tttgtctccc 6660ctgcaccgag ggaacaactc gagctttgag
gcttcctcca actaccactt gccagcaaca 6720aagtaagaca tacttggcaa
gaggataagg atgagatccc aagagacaag tggggcatga 6780gagggaggtg
caataggaag agatgatgcc tggcccagaa cctagctcta gaagggctta
6840ggggacatct actgagtgac aaaggcaatg gggagatgac agtggtggga
gcagctgaag 6900tgacgcagtc tattcgtcca gaggaagagc tgctccctgc
acaggatatc aaagctctgt 6960ttgtgtctga ggaggagaaa aagctgactc
ggaaggaggt ctacatcaag aatggggata 7020aggtgagaaa cgggcatcct
aaggaggcac tctaggcccc aatccttcct aagccacttc 7080tgttcattac
ttctccatgc ttcccacctc ccctacagaa aggcagctgt gagagagatg
7140ctcaatatgc cccaggctat gacaaagtca aggacatctc agaggtggtc
acccctcggt 7200tcctttgtac tggaggagtg agtccctatg ctgaccccaa
tacttgcaga ggtgagagaa 7260tgctctttgg ttgtgctaca agtgcccaag
gcccaacagt ccttttctct acagcttctc 7320ctctccttgc aggtgattct
ggcggcccct tgatagttca caagagaagt cgtttcattc 7380aagtgagtcc
tccctttcct atctggggag atgccaagtg gtcagcatgg gccccaaagc
7440aggaaagctc aatgcatgtg gctagtaatt cgaggtaggc agagcctgcc
tcaccttagg 7500accgcatgtc ttgcctgcgt gtgtcaagaa cgaggctgag
ctgggtccct agtctgattc 7560ctttaggtca gctaagacac aagcaggaac
agccatgctt ccaggattag gaattctact 7620gaatgatcca tggcacccca
ctgcctctgc aggttggtgt aatcagctgg ggagtagtgg 7680atgtctgcaa
aaaccagaag cggcaaaagc aggtacctgc tcacgcccga gactttcaca
7740tcaacctctt tcaagtgctg ccctggctga aggagaaact ccaagatgag
gatttgggtt 7800ttctataagg ggtttcctgc tggacagggg cgtgggattg
aattaaaaca gctgcgacaa 7860cacctgtgtt ccagatcctt ttggggcaag
ggagtgggga acaggcactg gccatgttgt 7920tacactgaga tcaaacctga
cagccgtttt taaaggttta accccaatcc caagtgctga 7980aaaaccagag
gctgagggag atgtgtaagc ttccacctca gtgttttact gagaccagca
8040ttggggcata tgaggcacaa ggaatccagc tctgttccct agaagccatc
cacaaggttt 8100tccttgtaga cgtcatcact gtagacaatc tgggtcctct
tgtcccggtg gcaaccctta 8160gggctgttct ggacagctag ggagggagga
gaggaacagt taaggtctaa aggagatcat 8220agaacagacc ctgaggctga
ctcctgacca cctcactcct ggccactggc ccctggaagc 8280ccagtttcca
cgctgccctc tggtggccag gatggcctgt cttccttagc tcctttgtgc
8340caacccatgg ccaagaaaag tataagtgga cattttgatg aatgttttgt
tcttagaaaa 8400atcccaaatg tcattgttga gacacgtgaa tgatattaac
ccactactta cagtcagtat 8460gtcagaagct aaaaactaga aaacctctgt
agcccttttt tgacatgctg gtcaattcta 8520gttcctttct tttgcctgaa
gggccactgt agctgagccc ttctttctgc tcactccttt 8580cccaggaaaa
tctactttca gggaaaatgg attattcaca ctaagaaatg ctactagctc
8640caccagaact cattcagggt gtagctttgg ccctcaccat tctctctcaa
gcctctagct 8700gtttcttccc cttcctcttt cctccctcca ccagacatgt
tactctcttc accccatcca 8760atggttccat ccccaccacc cttgagctac
agagaatctc tctcacccac tcccatcctg 8820tgatctctgt gcctcaacac
tgctggctac tccctctttc tcaaagtgtg tgtccttttg 8880cttcagtggc
ccaggcccct gcggtgctgc tcccagccct ccgacccctc ctcctgtctc
8940ctttgctaac gttaggctca acgttagcct aacatgtcag gacagctggg
gacatgtggg 9000g 900139001DNAMacaca mulatta 3gatggaatct tgctctgtct
cccacactgg agtgcagtgg cacgatcttg gctcactgcg 60acttctgcct cccagattta
agtgattctc ctacctcggc ctcccaagta gctgggatta 120taggtgcttg
ccaccacatg cagctaattt ttgtattttt agtagagaca ggattccgcc
180atgttggcca ggatggtctt gaactcctga cctcaagtga ttcgcccacc
tcagcctccc 240aaactgctgg gattacaggc gtgagccatt gcacccagtc
aggtctgctt attcttccct 300tctctctggt tccaccccta cggcagtgga
caagctgtgc cgaggtcgtc tcccaagaaa 360aaaccatgtt ccccaacttg
acagatgtca gggaggtggt gacagaccag tttctatgca 420gtgggaccca
ggaggatgag agtccctgca agggtgagtc cctcaccatg cctggattcc
480caaaggggaa ggccacctgt gtctctgtgg ccagggtgca tgccagaaca
ccagtccact 540gccctgtatg acgctgtctc ctgtcaccct ttgctggcag
gagaatctgg gggagcagtt 600ttccttgagc ggagattcag gttttttcag
gtgagaaggt ggaagcttgc aggacccagg 660ggttacagga tgtcagcctt
gttgggggga tgagggaggc ctttgaggga tctagggagg 720ttggggctta
cagctggagc tgtggcagcc tcccagccag ttctctcctt ttctccaggt
780gggtctggtg agctggggtc tttacaaccc ctgccttggc tctgctgaca
aaaactcccg 840caaaagggcc cctcgtagca aggtcccgcc gccacgagac
tttcacatca atctcttccg 900catgcagccc tggctgaggc agcacctggg
ggatgtcctg aattttttac ccctttagtc 960atggccactg agccctctgc
tgtcctgtta gaatccgccc cccctccatc ttctacctct 1020gaatgcccac
ccttagactc tgtgacccat gctgtctcct agttgagtaa atctgggtct
1080ctaggatgcc aggggcagcg cacacaagct gggaaatcct cagggctcct
accagcggga 1140ctgcctcgct gccccacctc ccgctccttg gcctgtcccc
aaattcctcc cctggttgac 1200ttgactcatg ctcatttcac tttcatatgg
aatttcccag ttatgaaatt aataaaaatc 1260agtggtttcc acatctgtct
gtgactctat ctggaggcca ggtagggctg gcctgggggg 1320aaggggaggc
cagaatgact ccaagagcca caggaaggca ggtcagagac cccactggac
1380aaacagtggc tggactctgt accataacac acaagcaaca ggggagtgag
ctggatcctt 1440atttctggtc cctaagtggg tggtctgggc ttgctgggga
ggagctgagg ccagaaggag 1500gtactgaagg ggagagtcct ggaccttggg
cagcaaaggg tgggacttct gcagtttctg 1560cttccttgac tggcagctca
gcggggccct cccgcttgga tgttccggga aagtgatgag 1620ggtaggacag
gcggggcaag ctgcaggtgc cagaacacag attgcataaa aggccgggag
1680ctggtggggg gcaggggaag ggaatgtgac caggtctagg tctggagttt
cagcttggac 1740actgagctaa gtagacaagc aaaacaagcc aggacacgcc
atcctgcccc aggcccagct 1800tctctcctgc cttctaacgc catggggagc
agtctcagcc cccagctcta cctgatgccc 1860ttcatcttgg gcctcttatc
tggaggtaag tgagggtaac cttcccttcc tgctgtcccc 1920agcatccctc
cttggccttt tggggccagg cttcatcagc ctttctcttc aggtgtgacc
1980accactccat tgtcttcggc ccagcctcaa ggatcctgct ctctggaggg
ggtagagatc 2040aaaggtggct ccttccgact tctccaagag ggccaggcac
tggaatacgt gtgtccttct 2100ggcttctacc cgtaccctgt gcagacacgt
acctgcagat ccacggggtc ctggagcacc 2160ctgcagactc aagatcgaaa
aactgtcaag aaggcagagt gcagaggttt gagggcaatg 2220agtgtgggca
gtggcctaag ggagaaacag ggcagatggc agcaaggtca ggactaggat
2280gagactaggc agggtgacaa ggtgggctga ccaggagtag gagcagtttt
agggttgtag 2340agggaaagga agggaaaaaa aaaggggagt taacctttag
taagcattta ccctgggctt 2400ccacgcagcc ctggaagtca agagaacact
cagcaatggg gagggaggag cagcggaaac 2460ccctatgggt tgaagggtag
gtaagatgca gcttctgcag gactgggaat gctctgtttc 2520tcagtgacct
ggtctctgag accaggaggg aaacacctaa ggcagccttt ccctcttaat
2580gacttctact tctcccctct tctcaaagca atccgctgtc cacgaccaca
ggacttcgag 2640aacggggaat accggccccg gtctccctac tacaatgtga
gtgatgagat ctctttccac 2700tgctatgacg gttacactct ccggggctct
gccaatcgca cctgccaagt gaatggccgg 2760tggagtgggc agacagcgat
ctgtgacaac ggaggtgaga agcatcctct ccccccacat 2820tgctgtctcc
ctgacagcgc ctagcctgag gagtgggcat ttgcccccgg acactgtaac
2880tcttgctctc taccttgccc tcggggcctc aggcttcagc gcttacctcc
atgtctcatg 2940cctctgcagc ggggtactgc tccaacccag gcatccccat
tggcacaagg aaggtgggca 3000gccggtaccg ccttgaagac agcgtcacct
accactgcag ccgggggctt accctgcgtg 3060gctcccagcg gcgaacatgt
caggaaggtg gctcttggag cgggacggag ccttcctgcc 3120aaggtgaccc
ttgacctgta cccccaggtc agatcctgat cttgcatcct actgtcttct
3180ctccccacct caaccctgct ctttcctcac ttcttttaaa ccttcctcta
gaactgtctc
3240acttctgagc cttttctacc ctggaaaccc acaatcccct gtctctttgg
tcactgtgtc 3300cctgacactc ccagacattt gacctcattt ctgactctcc
cagactcctt catgtacgac 3360acccctcaag aggtggccga agctttcctg
tcttccctga cggagaccat agaaggagtc 3420gatgccgagg atgggcacag
cccaggtttg aaggcagaga ggggaggcaa ggcagggaac 3480tgggggaaaa
tggagaaggg acaagataat cgttcatgct ggagcctgag tcactctcct
3540ggcacccagg ggaacaacag aagcggagga tcatcctaga cccttcaggc
tccatgaaca 3600tctacctggt gctagatgga tcagacagca ttggggccgg
caacttcaca ggagccaaaa 3660agtgtctagt caacttaatt gagaaggtgg
agtcctccta tccctgaact tgggggaatg 3720gaatcttgct gatcttccag
gactagctcc ctgatcattc cagcccctct gaaccgcagg 3780gccccaggaa
agtctccagg tcctattctg tcctccttcc cttgtacttg attcctccat
3840gaacctgtgc ttgagcctct tcctaagagc ctccctgtcc cagcaacgtt
gctgaagtct 3900cccaatcaca gtattctact ttcaatgcca tggcgccttg
ttctcctcac ccacaggtgg 3960caagttatgg tgtgaagcca agatatgctc
tagtgacata tgccacatac cccagaattt 4020gggtcaaagt gtctgaccaa
gagagcagca atgcagactg ggtcacgaag aagctcagtg 4080aaatcaatta
tgaaggtcag aggttaggga atggtgggag gttcactttg gggtcaggag
4140gttcaggagt gttgtgtgga gggggtcatg agactacctt gagggcaaca
gggggaccac 4200tttgtagtca aaggttgaac agcaggatca ttgggcaatg
gaggttagtg ggaacctgct 4260gagggctgga agggccactt tgtggtcaaa
gggaagtcca tatgatgatt aacttaaaaa 4320gttgaagatg tgagatttca
gttgcagatt ggtctctggg gttaaaagat ggcttggaag 4380accaggtgag
gcgatgctct cttccctccc cacagaccac aagttgaagt cagggactaa
4440caccaagagg gccctccagg cagtgtacag catgatgagt tggccagagg
acatccctcc 4500tgaaggctgg aaccgcaccc gccatgtcat catcctcatg
accgatggtc agaagggacc 4560tctctcctgt cccagcctcc ccaccttctc
agaccagcat gtggccctta agtccacttg 4620taacactata cccatggttg
gggccctgaa tgtgactcgt aactggctgt tcatctctcc 4680tgtgaccctt
cataaagaat tattcctaaa gccctgtgat caactacctc taacccttcc
4740tcaacttact caccctgcca cgtgtatcac tgcctctagc caatttatct
tatctcctac 4800cctcatggtc ccgtctcttc tgcaggattg cacaacatgg
gcggggaccc aattactgtc 4860attgatgaga tccgggactt gttatacatc
ggcaaggatc gtaaaaaccc gagggaggat 4920tatctgggtg agtaacctgc
ctaggaccca gcaccctact tcctcagggc ttggaccgtc 4980atccttcctt
tttctccctc agatgtctat gtgtttgggg ttggaccttt ggtggaccaa
5040gtgaacatca atgctttggc ttccaagaaa gacaatgagc aacatgtgtt
caaagtcaag 5100gatatggaaa acctggaaga cgttttcttc caaatgattg
gtaggcagac acaagggaat 5160caagaacgca actctcctca gcttcccctg
aaataattca ttcttcctct acccctgaag 5220ctctagttgc ctggaaagcc
ttcttcattc ctccttctct acctcagtat cactattctt 5280gtttcctggc
actgtttgct tcttaacctt agaatcacag agctctaggc acttcagaga
5340tctttctatt gtcctacatt tgacacatgt ggaaacaaag gccaaaggag
gtcaaggggc 5400agcaagctag caacagggct gggcttgaaa acagccaggc
ctctgatagc ttgatcccaa 5460gttctttccc ttttcactcc accacagcag
ttttctccta acacgaggaa acaaatacct 5520gtggcctttc cctttctcct
tttgggcctc tgccccccac agacttctac ccaaaggctg 5580ctgccgtttg
ggaatgaagt gttccaagtt ttcagcacat tctccttctc tgccagatga
5640aagccagtct ctgagtctct gtggcatggt ttgggaacac agcaagggta
ccgattacca 5700caagcaacca tggcaggcca agatctcagt cactgtaagc
acagaatccc agtagtgagg 5760acttggggga ggtgaggtca aggtgaaatg
ggagtagggg aagggcaaaa tggccgtaag 5820agatggtggt ttgtgaaagt
tgagttttcc ctttctactg ttctgttccc agcgcccttc 5880gaagggacat
gagagctgta tgggggctgt ggtgtctgag tactttgtgc tgacagcagc
5940acattgtttt actgtggacg acaaggaaca ctcaatcaag gtcagcgtgg
gtaaggatgc 6000aactgaaggt cccgggctgc acctacgccc tccaggcaac
acctcccact ttctacagat 6060cccacactcc actcatctgc aatgcagccc
catcccttgc accccagacc agtcagggat 6120ggggaagact tgaagttagg
aatgacatgg ggccagaggc aagaagctgc ccacaaagag 6180gtggtaccta
ttctcctact tcaagggaag aagcgggacc tggagataga aaaagtccta
6240tttcaccccg actacaacat tagcgagaaa aaagaagcag gaattcctga
attttatgac 6300tatgacgttg ccctgatcaa gctcaagaat aagttgaatt
atgacccgac tatcaggtga 6360gagcatccag atccctgagg aaaggctggg
aaaggctgga ggactggggt gaggagcagg 6420cctagtttgc tgttctttct
ccatccttta taggcccatt tgtctcccct gcaccgaggg 6480aacaactcga
gctttgaggc ttcctccaac taccacttgc cagcaacaga gtaagacata
6540ctagggggga ggataaggat gagatcccga gacaagtgag gcatgagagg
gagatgcaat 6600aggaagagac gatgcctggc ccagaaccta gcactaggaa
gggcttaggg gacatctgct 6660gagtgacaaa gtcaataggg agatgacagt
ggtgggagca gctgaagtga tgcagtctat 6720ttgtccagag gaagagctgc
tccctgcaca ggatatcaaa gctctgtttg tgtctgagga 6780ggagaagaag
ctgactcgga aggaggtcta catcaagaat ggggataagg tgagaaatgg
6840gcatcctaag gaggcactct aggccctaat ccttcctaag ccacctctgt
tcattacctt 6900tctccatgct tcccacctcc cctacagaaa ggcagctgtg
agagagatgc tcaatatgcc 6960ccaggctatg acaaagtcaa ggacatctcg
gaggtggtca cccctcggtt cctttgtact 7020ggaggagtga gtccctatgc
tgaccccaat acttgcagag gtgagagaac gctctctggt 7080tgtgctccaa
gtgcccgagg gccaagagtc cttttcccta cagcttctcc tctccttgca
7140ggtgattctg gcggcccctt gatagttcac aagagaagtc gtttcattca
agtgagtcct 7200ccctttccta tctggggaga tgccaagtgg tcagcatggg
ccccaaagca ggaaagcaca 7260atgcatgtgg ctagtaattc gaggtgggca
gagcctgcct cactttagga ctgcatgtct 7320ggcctgtgtg tgtcaagaat
gaggctgagc tgggtcccta gcctgattcc tttaggtcag 7380ctaagacaca
atcaggaaca gtcatgcttc caggattagg aattctatga atgatccatg
7440gcaccccact gcctctgcag gttggtgtca tcagctgggg agtagtggat
gtctgcaaaa 7500accagaagcg gcaaaagcag gtacctgctc acgcccgaga
ctttcacgtc aacctcttcc 7560aagtgctgcc ctggctgaag gagaaactcc
aagatgagga tttgggtttt ctctaagggg 7620tttcctgctg gacaggggcg
cgggattgaa ttaaaacagc tgcgacaaca cttgtgttcc 7680agatcctttt
ggggcaaggg agtggggaac gggcactggc catgttgtta cactgagatc
7740aaacctgaca cccattttta aaggcttaac cccaatccca agtgctgaaa
aaccagaggc 7800tgagggagat atgtaagctt ccacctcagt gttttactga
gaccagcatt ggggcatttg 7860aggcacaagg aatccagctc tgttccctag
aagccatcca caaggttttc cttgtagacg 7920tcatcactgt agacaatctg
ggtcctcttg tcccggtggc aacccttagg gctgttctgg 7980acagctaggg
agggaggaga ggaacagtta aggtctaaag gagatcatag atcagaccct
8040gaggctgact cctgaccacc tcagtcctgg ctgctggccc ctggaaaccc
agtttccacg 8100ctgccctctg gtggccagga tggcctgtct tccttagctc
ctttgtgcca acccatggcc 8160aaggagagtg taagtggaca ttttgatgaa
tgttttgttc ttagaaaaat cccaaatgtc 8220attgttgaga tatatgaatg
atattaaccc actacttata gtcagtatgt cagaagctaa 8280aaactagaaa
acctctgtag ccctttattg acatgctggt caactctagt tcctttcttt
8340tgcctgaaag gccactgttg ctctgagtcc ttctttctgc tcactccttt
cccaggaaaa 8400tctactttca ggtaaatggg ttactcatac taaggaatgc
tactagctcc accagaactc 8460atccagcatg tagctttggc cctcaccatt
ctctctcaag cctctagctg tttcttcccc 8520ttcctctttt cctccctcca
ccagacatgt tactctcttc accccatcca aagattccat 8580ccccaccacc
cttgacctag agagaatctc tcccacccac ttctcatcct gtgatctctg
8640taccttgaca ctgctggcta ctccctcttt ctcaaagcat gtgtcctttc
gcttcagtgg 8700cccaggcccc tctggtgctg ctcccagccc tctgacccct
cctcctgtct cctttgctaa 8760cgttaggctc aacgttagcc taacgtgtca
ggagagctgg agacacgtgg ggcgtaaggt 8820ggacagtcct gtttcctaac
atagtccctg agtattcctc aagtctagtc ctgggtcgtt 8880ttttttctcc
gaaatcagtc tccctcatga tcggggagcc accctgtgat gcagatgact
8940taatctatgt tttcattcct tacctcacac ctgagttcca gacccctaat
ttcaaatact 9000t 900144086DNAMacaca mulatta 4atagatatat tagcatcagg
gagacagggc aaaggttcca cccttcagct cagtccccag 60tccctgctta ttatttccct
aacagaagac catccccctt gccactccct gggttttctt 120ctctggcagc
aatgaagcag ctgctgagcc agctctggtt ttcgggaagt cagatgacct
180tttccctccc gcggctctct gcctctcgct gtccctaggg aggacaccat
ggacccactg 240atggttcttt tttgcctgct gttcctgtac ccaggtccgg
cagactcggc tacctcctgc 300cctcagaacg tgaatatctc tggtggcacc
ttcaccctca gccatggctg ggcccctggg 360agccttctca tctactcctg
tccccagggc ctgtacccat ccccagcgtc acggctgtgc 420aagagcagcg
gacagtggca gaccccaaga gccacccggt ctctgactaa ggcggtctgc
480aaacctggcc actgccccaa ccccggcatt tcgctgggcg cggtgcggac
aggctcccgc 540tttggtcatg gggacaaggt ccgctatcgc tgctcctcga
atcttgtgct cacggggtct 600gcggagcggg agtgccaggg caacggggtc
tggagtggaa cggagcccat ctgccgccag 660ccctactctt atgacttccc
tgaggacgtg gcccctgccc tgggcacctc cttctcccac 720atgcttgggg
ccaccaatcc cacccagagg acaaaggatc atgaaaatgg aactgggact
780aacacctatg cagccctaaa cagtgtctat ctcatgatga acaatcaaat
gcaactcctt 840ggcatgaaaa cgatggcctg gcaggaaatc cgacatgcca
tcatccttct gacagatgga 900aagtccaata tgggtggctc tcccaaaaca
gctgttgacc aaatcagaga gatcttgaat 960atcaaccaga agaggaatga
ctatctggac atctatgcca tcggggtggg caagctggat 1020gtggactgga
gagaactgaa tgagctgggg tccaagaagg atggcgagag gcatgccttc
1080attctgcagg acacaaaggc tctgcaccag gtctttgaac atatgctgga
tgtctccaag 1140ctcacagaca ccatctgcgg ggtggggaac atgtcagcaa
acgcctctga ccaagagagg 1200acaccctggc atgtcactat taagcccaag
agccaagaga cctgccgggg agccctcatc 1260tccgaccaat gggtcctgac
agcggctcac tgcttccgcg atggcaacga ccactcccta 1320tggagggtca
atgtgggaga ccccaaatcc cagtggggca aagaattcct tattgagaag
1380gcagtgattt ccccaggatt tgatgtcttt gccaaaaaga accagggaat
cctggagttc 1440tatggtgatg acatcgccct gctgaagctg gcccagaaag
taaagatgtc cacccatgcc 1500aggcccatct gccttccctg caccatggag
gccaatctgg ctctgcggag acctcaaggc 1560agcacctgta gggaccatga
gaatgaactg ctgaacaaac agagtgttcc tgctcatttt 1620gtcgccttga
atgggagcaa actgaacatt aaccttaaga tgggagtgga gtggacaagc
1680tgtgccgagg tcgtctccca agaaaaaacc atgttcccca acttgacaga
tgtcagggag 1740gtggtgacag accagtttct atgcagtggg acccaggagg
atgagagtcc ctgcaagggt 1800gtgaccacca ctccattgtc ttcggcccag
cctcaaggat cctgctctct ggagggggta 1860gagatcaaag gtggctcctt
ccgacttctc caagagggcc aggcactgga atacgtgtgt 1920ccttctggct
tctacccgta ccctgtgcag acacgtacct gcagatccac ggggtcctgg
1980agcaccctgc agactcaaga tcgaaaaact gtcaagaagg cagagtgcag
agcaatccgc 2040tgtccacgac cacaggactt cgagaacggg gaataccggc
cccggtctcc ctactacaat 2100gtgagtgatg agatctcttt ccactgctat
gacggttaca ctctccgggg ctctgccaat 2160cgcacctgcc aagtgaatgg
ccggtggagt gggcagacag cgatctgtga caacggagcg 2220gggtactgct
ccaacccagg catccccatt ggcacaagga aggtgggcag ccggtaccgc
2280cttgaagaca gcgtcaccta ccactgcagc cgggggctta ccctgcgtgg
ctcccagcgg 2340cgaacatgtc aggaaggtgg ctcttggagc gggacggagc
cttcctgcca agactccttc 2400atgtacgaca cccctcaaga ggtggccgaa
gctttcctgt cttccctgac ggagaccata 2460gaaggagtcg atgccgagga
tgggcacagc ccaggggaac aacagaagcg gaggatcatc 2520ctagaccctt
caggctccat gaacatctac ctggtgctag atggatcaga cagcattggg
2580gccggcaact tcacaggagc caaaaagtgt ctagtcaact taattgagaa
ggtggcaagt 2640tatggtgtga agccaagata tgctctagtg acatatgcca
cataccccag aatttgggtc 2700aaagtgtctg accaagagag cagcaatgca
gactgggtca cgaagaagct cagtgaaatc 2760aattatgaag accacaagtt
gaagtcaggg actaacacca agagggccct ccaggcagtg 2820tacagcatga
tgagttggcc agaggacatc cctcctgaag gctggaaccg cacccgccat
2880gtcatcatcc tcatgaccga tggattgcac aacatgggcg gggacccaat
tactgtcatt 2940gatgagatcc gggacttgtt atacatcggc aaggatcgta
aaaacccgag ggaggattat 3000ctggatgtct atgtgtttgg ggttggacct
ttggtggacc aagtgaacat caatgctttg 3060gcttccaaga aagacaatga
gcaacatgtg ttcaaagtca aggatatgga aaacctggaa 3120gacgttttct
tccaaatgat tgatgaaagc cagtctctga gtctctgtgg catggtttgg
3180gaacacagca agggtaccga ttaccacaag caaccatggc aggccaagat
ctcagtcact 3240cgcccttcga agggacatga gagctgtatg ggggctgtgg
tgtctgagta ctttgtgctg 3300acagcagcac attgttttac tgtggacgac
aaggaacact caatcaaggt cagcgtggga 3360gggaagaagc gggacctgga
gatagaaaaa gtcctatttc accccgacta caacattagc 3420gagaaaaaag
aagcaggaat tcctgaattt tatgactatg acgttgccct gatcaagctc
3480aagaataagt tgaattatga cccgactatc aggcccattt gtctcccctg
caccgaggga 3540acaactcgag ctttgaggct tcctccaact accacttgcc
agcaacagaa ggaagagctg 3600ctccctgcac aggatatcaa agctctgttt
gtgtctgagg aggagaagaa gctgactcgg 3660aaggaggtct acatcaagaa
tggggataag aaaggcagct gtgagagaga tgctcaatat 3720gccccaggct
atgacaaagt caaggacatc tcggaggtgg tcacccctcg gttcctttgt
3780actggaggag tgagtcccta tgctgacccc aatacttgca gaggtgattc
tggcggcccc 3840ttgatagttc acaagagaag tcgtttcatt caagttggtg
tcatcagctg gggagtagtg 3900gatgtctgca aaaaccagaa gcggcaaaag
caggtacctg ctcacgcccg agactttcac 3960gtcaacctct tccaagtgct
gccctggctg aaggagaaac tccaagatga ggatttgggt 4020tttctctaag
gggtttcctg ctggacaggg gcgcgggatt gaattaaaac agctgcgaca 4080acactt
408652767DNAMus musculus 5gctccatcac acagtccatg gaaagactga
tcttttaaat tgggggtagt ggaggtggtg 60gtctgtgctt gttaggaggg gtctgggggc
taagagggag ctttgaaagg gaagttctgg 120cccttggtca gtcaagggtg
gggctcacat agtttctgtt tcctcagttg gcagttcagc 180tggggccctc
cttcatgaat gttccgggaa gcagtggctg cgtgcgcagg gtaggctggc
240caggctgcag atgccagagc agattgcata aaaggttagg ggacagtggg
aaaggggtgt 300agccagatcc agcatttggg tttcagtttg gacaggaggt
caaataggca cccagagtga 360cctggagagg gctttgggcc actggactct
ctggtgcttt ccatgacaat ggagagcccc 420cagctctgcc tcgtcctctt
ggtcttaggc ttctcctctg gaggtgtgag cgcaactcca 480gtgcttgagg
cccggcccca agtctcctgc tctctggagg gagtagagat caaaggcggc
540tcctttcaac ttctccaagg cggtcaggcc ctggagtacc tatgtccctc
tggcttctac 600ccataccccg tgcagactcg aacctgcaga tccacaggct
cctggagcga cctgcagacc 660cgagaccaaa agattgtcca gaaggcggaa
tgcagagcaa tacgctgccc acgaccgcag 720gactttgaaa atggggaatt
ctggccccgg tcccccttct acaacctgag tgaccagatt 780tcttttcaat
gctatgatgg ttacgttctc cggggctctg ctaatcgcac ctgccaagag
840aatggccggt gggatgggca aacagcaatt tgtgatgatg gagctggata
ctgtcccaat 900cccggtattc ctattgggac aaggaaggtg ggtagccaat
accgccttga agacattgtt 960acttaccact gcagccgggg acttgtcctg
cgtggctccc agaagcgaaa gtgtcaagaa 1020ggtggctcat ggagtgggac
agagccttcc tgccaagatt ccttcatgta tgacagccct 1080caagaagtgg
ccgaagcatt cctatcctcc ctgacagaga ccatcgaagg agccgatgct
1140gaggatgggc acagcccagg agaacagcag aagaggaaga ttgtcctaga
cccctcgggc 1200tccatgaata tctacctggt gctagatgga tcagacagca
tcggaagcag caacttcaca 1260ggggctaagc ggtgcctcac caacttgatt
gagaaggtgg cgagttacgg ggtgaggcca 1320cgatatggtc tcctgacata
tgctacagtc cccaaagtgt tggtcagagt gtctgatgag 1380aggagtagcg
atgccgactg ggtcacagag aagctcaacc aaatcagtta tgaagaccac
1440aagctgaagt cagggaccaa caccaagagg gctctccagg ctgtgtatag
catgatgagc 1500tgggcagggg atgccccgcc tgaaggctgg aatagaaccc
gccatgtcat catcattatg 1560actgatggct tgcacaacat gggtggaaac
cctgtcactg tcattcagga catccgagcc 1620ttgctggaca tcggcaggga
tcccaaaaat cccagggagg attacctgga tgtgtatgtg 1680tttggggtcg
ggcctctggt ggactccgtg aacatcaatg ccttagcttc caaaaaggac
1740aatgagcatc atgtgtttaa agtcaaggat atggaagacc tggagaatgt
tttctaccaa 1800atgattgatg aaaccaaatc tctgagtctc tgtggcatgg
tgtgggagca taaaaaaggc 1860aacgattatc ataagcaacc atggcaagcc
aagatctcag tcactcgccc tctgaaagga 1920catgagacct gtatgggggc
cgtggtgtct gagtacttcg tgctgacagc agcgcactgc 1980ttcatggtgg
atgatcagaa acattccatc aaggtcagcg tggggggtca gaggcgggac
2040ctggagattg aagaggtcct gttccacccc aaatacaata ttaatgggaa
aaaggcagaa 2100gggatccctg agttctatga ttatgatgtg gccctagtca
agctcaagaa caagctcaag 2160tatggccaga ctctcaggcc catctgtctc
ccctgcacgg agggaaccac acgagccttg 2220aggcttcctc agacagccac
ctgcaagcag cacaaggaac agttgctccc tgtgaaggat 2280gtcaaagctc
tgtttgtatc tgagcaaggg aagagcctga ctcggaagga ggtgtacatc
2340aagaatgggg acaagaaagc cagttgtgag agagatgcta caaaggccca
aggctatgag 2400aaggtcaaag atgcctctga ggtggtcact ccacggttcc
tctgcacagg aggggtggat 2460ccctatgctg accccaacac atgcaaagga
gattccgggg gccctctcat tgttcacaag 2520agaagccgct tcattcaagt
tggtgtgatt agctggggag tagtagatgt ctgcagagac 2580cagaggcggc
aacagctggt accctcttat gcccgggact tccacatcaa cctcttccag
2640gtgctgccct ggctaaagga caagctcaaa gatgaggatt tgggttttct
ataaagagct 2700tcctgcaggg agagtgtgag gacagattaa agcagttaca
ataacaaaaa aaaaaaaaaa 2760aaaaaaa 2767620DNAArtificial
sequenceSynthetic Oligonucleotide 6gctgagctgc cagtcaagga
20720DNAArtificial sequenceSynthetic Oligonucleotide 7ggccccgctg
agctgccagt 20820DNAArtificial sequenceSynthetic Oligonucleotide
8cggaacatcc aagcgggagg 20920DNAArtificial sequenceSynthetic
Oligonucleotide 9ctttcccgga acatccaagc 201020DNAArtificial
sequenceSynthetic Oligonucleotide 10atctgtgttc tggcacctgc
201120DNAArtificial sequenceSynthetic Oligonucleotide 11gtcacattcc
cttcccctgc 201220DNAArtificial sequenceSynthetic Oligonucleotide
12gacctggtca cattcccttc 201320DNAArtificial sequenceSynthetic
Oligonucleotide 13gacctagacc tggtcacatt 201420DNAArtificial
sequenceSynthetic Oligonucleotide 14actccagacc tagacctggt
201520DNAArtificial sequenceSynthetic Oligonucleotide 15gctgaaactc
cagacctaga 201620DNAArtificial sequenceSynthetic Oligonucleotide
16gtccaagctg aaactccaga 201720DNAArtificial sequenceSynthetic
Oligonucleotide 17ctcagtgtcc aagctgaaac 201820DNAArtificial
sequenceSynthetic Oligonucleotide 18aggagagaag ctgggcctgg
201920DNAArtificial sequenceSynthetic Oligonucleotide 19gaaggcagga
gagaagctgg 202020DNAArtificial sequenceSynthetic Oligonucleotide
20gtggtggtca cacctccaga 202120DNAArtificial sequenceSynthetic
Oligonucleotide 21ccctccagag agcaggatcc 202220DNAArtificial
sequenceSynthetic Oligonucleotide 22tctaccccct ccagagagca
202320DNAArtificial sequenceSynthetic Oligonucleotide 23ttgatctcta
ccccctccag 202420DNAArtificial sequenceSynthetic Oligonucleotide
24tggagaagtc ggaaggagcc
202520DNAArtificial sequenceSynthetic Oligonucleotide 25ccctcttgga
gaagtcggaa 202620DNAArtificial sequenceSynthetic Oligonucleotide
26gcctggccct cttggagaag 202720DNAArtificial sequenceSynthetic
Oligonucleotide 27tccagtgcct ggccctcttg 202820DNAArtificial
sequenceSynthetic Oligonucleotide 28agaagccaga aggacacacg
202920DNAArtificial sequenceSynthetic Oligonucleotide 29acgggtagaa
gccagaagga 203020DNAArtificial sequenceSynthetic Oligonucleotide
30cgtgtctgca cagggtacgg 203120DNAArtificial sequenceSynthetic
Oligonucleotide 31agggtgctcc aggaccccgt 203220DNAArtificial
sequenceSynthetic Oligonucleotide 32ttgctctgca ctctgccttc
203320DNAArtificial sequenceSynthetic Oligonucleotide 33tattccccgt
tctcgaagtc 203420DNAArtificial sequenceSynthetic Oligonucleotide
34cattgtagta gggagaccgg 203520DNAArtificial sequenceSynthetic
Oligonucleotide 35cactcacatt gtagtaggga 203620DNAArtificial
sequenceSynthetic Oligonucleotide 36tctcatcact cacattgtag
203720DNAArtificial sequenceSynthetic Oligonucleotide 37aagagatctc
atcactcaca 203820DNAArtificial sequenceSynthetic Oligonucleotide
38agtggaaaga gatctcatca 203920DNAArtificial sequenceSynthetic
Oligonucleotide 39catagcagtg gaaagagatc 204020DNAArtificial
sequenceSynthetic Oligonucleotide 40aaccgtcata gcagtggaaa
204120DNAArtificial sequenceSynthetic Oligonucleotide 41gagtgtaacc
gtcatagcag 204220DNAArtificial sequenceSynthetic Oligonucleotide
42cccggagagt gtaaccgtca 204320DNAArtificial sequenceSynthetic
Oligonucleotide 43cagagccccg gagagtgtaa 204420DNAArtificial
sequenceSynthetic Oligonucleotide 44gattggcaga gccccggaga
204520DNAArtificial sequenceSynthetic Oligonucleotide 45aggtgcgatt
ggcagagccc 204620DNAArtificial sequenceSynthetic Oligonucleotide
46cttggcaggt gcgattggca 204720DNAArtificial sequenceSynthetic
Oligonucleotide 47cattcacttg gcaggtgcga 204820DNAArtificial
sequenceSynthetic Oligonucleotide 48atcgctgtct gcccactcca
204920DNAArtificial sequenceSynthetic Oligonucleotide 49tcacagatcg
ctgtctgccc 205020DNAArtificial sequenceSynthetic Oligonucleotide
50ccgttgtcac agatcgctgt 205120DNAArtificial sequenceSynthetic
Oligonucleotide 51cccgctccgt tgtcacagat 205220DNAArtificial
sequenceSynthetic Oligonucleotide 52cagtaccccg ctccgttgtc
205320DNAArtificial sequenceSynthetic Oligonucleotide 53ttggagcagt
accccgctcc 205420DNAArtificial sequenceSynthetic Oligonucleotide
54accttccttg tgccaatggg 205520DNAArtificial sequenceSynthetic
Oligonucleotide 55ctgcccacct tccttgtgcc 205620DNAArtificial
sequenceSynthetic Oligonucleotide 56cgctgtcttc aaggcggtac
205720DNAArtificial sequenceSynthetic Oligonucleotide 57gctgcagtgg
taggtgacgc 205820DNAArtificial sequenceSynthetic Oligonucleotide
58cccccggctg cagtggtagg 205920DNAArtificial sequenceSynthetic
Oligonucleotide 59ggtaagcccc cggctgcagt 206020DNAArtificial
sequenceSynthetic Oligonucleotide 60acgcagggta agcccccggc
206120DNAArtificial sequenceSynthetic Oligonucleotide 61ggagccacgc
agggtaagcc 206220DNAArtificial sequenceSynthetic Oligonucleotide
62gccgctggga gccacgcagg 206320DNAArtificial sequenceSynthetic
Oligonucleotide 63caagagccac cttcctgaca 206420DNAArtificial
sequenceSynthetic Oligonucleotide 64ccgctccaag agccaccttc
206520DNAArtificial sequenceSynthetic Oligonucleotide 65tccgtcccgc
tccaagagcc 206620DNAArtificial sequenceSynthetic Oligonucleotide
66gaaggctccg tcccgctcca 206720DNAArtificial sequenceSynthetic
Oligonucleotide 67tggcaggaag gctccgtccc 206820DNAArtificial
sequenceSynthetic Oligonucleotide 68gagtcttggc aggaaggctc
206920DNAArtificial sequenceSynthetic Oligonucleotide 69atgaaggagt
cttggcagga 207020DNAArtificial sequenceSynthetic Oligonucleotide
70cttcggccac ctcttgaggg 207120DNAArtificial sequenceSynthetic
Oligonucleotide 71ggaaagcttc ggccacctct 207220DNAArtificial
sequenceSynthetic Oligonucleotide 72aagacaggaa agcttcggcc
207320DNAArtificial sequenceSynthetic Oligonucleotide 73tcagggaaga
caggaaagct 207420DNAArtificial sequenceSynthetic Oligonucleotide
74tcgactcctt ctatggtctc 207520DNAArtificial sequenceSynthetic
Oligonucleotide 75cttctgttgt tcccctgggc 207620DNAArtificial
sequenceSynthetic Oligonucleotide 76ttcatggagc ctgaagggtc
207720DNAArtificial sequenceSynthetic Oligonucleotide 77tagatgttca
tggagcctga 207820DNAArtificial sequenceSynthetic Oligonucleotide
78accaggtaga tgttcatgga 207920DNAArtificial sequenceSynthetic
Oligonucleotide 79tctagcacca ggtagatgtt 208020DNAArtificial
sequenceSynthetic Oligonucleotide 80gatccatcta gcaccaggta
208120DNAArtificial sequenceSynthetic Oligonucleotide 81ctgtctgatc
catctagcac 208220DNAArtificial sequenceSynthetic Oligonucleotide
82ccaatgctgt ctgatccatc 208320DNAArtificial sequenceSynthetic
Oligonucleotide 83tttggctcct gtgaagttgc 208420DNAArtificial
sequenceSynthetic Oligonucleotide 84acactttttg gctcctgtga
208520DNAArtificial sequenceSynthetic Oligonucleotide 85gactagacac
tttttggctc 208620DNAArtificial sequenceSynthetic Oligonucleotide
86taagttgact agacactttt 208720DNAArtificial sequenceSynthetic
Oligonucleotide 87ctcaattaag ttgactagac 208820DNAArtificial
sequenceSynthetic Oligonucleotide 88caccttctca attaagttga
208920DNAArtificial sequenceSynthetic Oligonucleotide 89acttgccacc
ttctcaatta 209020DNAArtificial sequenceSynthetic Oligonucleotide
90accataactt gccaccttct 209120DNAArtificial sequenceSynthetic
Oligonucleotide 91cttcacacca taacttgcca 209220DNAArtificial
sequenceSynthetic Oligonucleotide 92tcttggcttc acaccataac
209320DNAArtificial sequenceSynthetic Oligonucleotide 93atgtggcata
tgtcactaga 209420DNAArtificial sequenceSynthetic Oligonucleotide
94cagacacttt gacccaaatt 209520DNAArtificial sequenceSynthetic
Oligonucleotide 95ggtcttcata attgatttca 209620DNAArtificial
sequenceSynthetic Oligonucleotide 96acttgtggtc ttcataattg
209720DNAArtificial sequenceSynthetic Oligonucleotide 97acttcaactt
gtggtcttca 209820DNAArtificial sequenceSynthetic Oligonucleotide
98tccctgactt caacttgtgg 209920DNAArtificial sequenceSynthetic
Oligonucleotide 99tgttagtccc tgacttcaac 2010020DNAArtificial
sequenceSynthetic Oligonucleotide 100tcttggtgtt agtccctgac
2010120DNAArtificial sequenceSynthetic Oligonucleotide
101tgtacactgc ctggagggcc 2010220DNAArtificial sequenceSynthetic
Oligonucleotide 102tcatgctgta cactgcctgg 2010320DNAArtificial
sequenceSynthetic Oligonucleotide 103gttccagcct tcaggaggga
2010420DNAArtificial sequenceSynthetic Oligonucleotide
104ggtgcggttc cagccttcag 2010520DNAArtificial sequenceSynthetic
Oligonucleotide 105atggcgggtg cggttccagc 2010620DNAArtificial
sequenceSynthetic Oligonucleotide 106gatgacatgg cgggtgcggt
2010720DNAArtificial sequenceSynthetic Oligonucleotide
107gaggatgatg acatggcggg 2010820DNAArtificial sequenceSynthetic
Oligonucleotide 108cccatgttgt gcaatccatc 2010920DNAArtificial
sequenceSynthetic Oligonucleotide 109tccccgccca tgttgtgcaa
2011020DNAArtificial sequenceSynthetic Oligonucleotide
110attgggtccc cgcccatgtt 2011120DNAArtificial sequenceSynthetic
Oligonucleotide 111acagtaattg ggtccccgcc 2011220DNAArtificial
sequenceSynthetic Oligonucleotide 112tcaatgacag taattgggtc
2011320DNAArtificial sequenceSynthetic Oligonucleotide
113atctcatcaa tgacagtaat 2011420DNAArtificial sequenceSynthetic
Oligonucleotide 114tcccggatct catcaatgac 2011520DNAArtificial
sequenceSynthetic Oligonucleotide 115acatccagat aatcctccct
2011620DNAArtificial sequenceSynthetic Oligonucleotide
116acatagacat ccagataatc 2011720DNAArtificial sequenceSynthetic
Oligonucleotide 117ccaaacacat agacatccag 2011820DNAArtificial
sequenceSynthetic Oligonucleotide 118agcattgatg ttcacttggt
2011920DNAArtificial sequenceSynthetic Oligonucleotide
119agccaaagca ttgatgttca 2012020DNAArtificial sequenceSynthetic
Oligonucleotide 120cttggaagcc aaagcattga 2012120DNAArtificial
sequenceSynthetic Oligonucleotide 121gtctttcttg gaagccaaag
2012220DNAArtificial sequenceSynthetic Oligonucleotide
122ctcattgtct ttcttggaag 2012320DNAArtificial sequenceSynthetic
Oligonucleotide 123atgttgctca ttgtctttct 2012420DNAArtificial
sequenceSynthetic Oligonucleotide 124gaacacatgt tgctcattgt
2012520DNAArtificial sequenceSynthetic Oligonucleotide
125gactttgaac acatgttgct 2012620DNAArtificial sequenceSynthetic
Oligonucleotide 126atccttgact ttgaacacat 2012720DNAArtificial
sequenceSynthetic Oligonucleotide 127ttccatatcc ttgactttga
2012820DNAArtificial sequenceSynthetic Oligonucleotide
128caggttttcc atatccttga 2012920DNAArtificial sequenceSynthetic
Oligonucleotide 129ctcagagact ggctttcatc 2013020DNAArtificial
sequenceSynthetic Oligonucleotide 130cagagactca gagactggct
2013120DNAArtificial sequenceSynthetic Oligonucleotide
131atgccacaga gactcagaga 2013220DNAArtificial sequenceSynthetic
Oligonucleotide 132caaaccatgc cacagagact 2013320DNAArtificial
sequenceSynthetic Oligonucleotide 133tgttcccaaa ccatgccaca
2013420DNAArtificial sequenceSynthetic Oligonucleotide
134ttgtggtaat cggtaccctt 2013520DNAArtificial sequenceSynthetic
Oligonucleotide 135ggttgcttgt ggtaatcggt 2013620DNAArtificial
sequenceSynthetic Oligonucleotide 136tgccatggtt gcttgtggta
2013720DNAArtificial sequenceSynthetic Oligonucleotide
137ttggcctgcc atggttgctt 2013820DNAArtificial sequenceSynthetic
Oligonucleotide 138gagatcttgg cctgccatgg 2013920DNAArtificial
sequenceSynthetic Oligonucleotide 139acagccccca tacagctctc
2014020DNAArtificial sequenceSynthetic Oligonucleotide
140gacaccacag cccccataca 2014120DNAArtificial sequenceSynthetic
Oligonucleotide 141tactcagaca ccacagcccc 2014220DNAArtificial
sequenceSynthetic Oligonucleotide 142acaaagtact cagacaccac
2014320DNAArtificial sequenceSynthetic Oligonucleotide
143gtcagcacaa agtactcaga 2014420DNAArtificial sequenceSynthetic
Oligonucleotide 144ttgattgagt gttccttgtc 2014520DNAArtificial
sequenceSynthetic Oligonucleotide 145ctgaccttga ttgagtgttc
2014620DNAArtificial sequenceSynthetic Oligonucleotide
146tatctccagg tcccgcttct 2014720DNAArtificial sequenceSynthetic
Oligonucleotide 147gaattcctgc ttcttttttc 2014820DNAArtificial
sequenceSynthetic Oligonucleotide 148attcaggaat tcctgcttct
2014920DNAArtificial sequenceSynthetic Oligonucleotide
149cataaaattc aggaattcct 2015020DNAArtificial sequenceSynthetic
Oligonucleotide 150catagtcata
aaattcagga 2015120DNAArtificial sequenceSynthetic Oligonucleotide
151tgagcttgat cagggcaacg 2015220DNAArtificial sequenceSynthetic
Oligonucleotide 152tattcttgag cttgatcagg 2015320DNAArtificial
sequenceSynthetic Oligonucleotide 153gacaaatggg cctgatagtc
2015420DNAArtificial sequenceSynthetic Oligonucleotide
154gttgttccct cggtgcaggg 2015520DNAArtificial sequenceSynthetic
Oligonucleotide 155gctcgagttg ttccctcggt 2015620DNAArtificial
sequenceSynthetic Oligonucleotide 156ctcaaagctc gagttgttcc
2015720DNAArtificial sequenceSynthetic Oligonucleotide
157ggaagcctca aagctcgagt 2015820DNAArtificial sequenceSynthetic
Oligonucleotide 158gttggaggaa gcctcaaagc 2015920DNAArtificial
sequenceSynthetic Oligonucleotide 159gtggtagttg gaggaagcct
2016020DNAArtificial sequenceSynthetic Oligonucleotide
160tggcaagtgg tagttggagg 2016120DNAArtificial sequenceSynthetic
Oligonucleotide 161tgttgctggc aagtggtagt 2016220DNAArtificial
sequenceSynthetic Oligonucleotide 162tccagctcac tcccctgttg
2016320DNAArtificial sequenceSynthetic Oligonucleotide
163taaggatcca gctcactccc 2016420DNAArtificial sequenceSynthetic
Oligonucleotide 164cagaaataag gatccagctc 2016520DNAArtificial
sequenceSynthetic Oligonucleotide 165agggaccaga aataaggatc
2016620DNAArtificial sequenceSynthetic Oligonucleotide
166ccacttaggg accagaaata 2016720DNAArtificial sequenceSynthetic
Oligonucleotide 167tccaggactc tccccttcag 2016820DNAArtificial
sequenceSynthetic Oligonucleotide 168aagtcccacc ctttgctgcc
2016920DNAArtificial sequenceSynthetic Oligonucleotide
169ctgcagaagt cccacccttt 2017020DNAArtificial sequenceSynthetic
Oligonucleotide 170cagaaactgc agaagtccca 2017120DNAArtificial
sequenceSynthetic Oligonucleotide 171aacctctgca ctctgccttc
2017220DNAArtificial sequenceSynthetic Oligonucleotide
172ccctcaaacc tctgcactct 2017320DNAArtificial sequenceSynthetic
Oligonucleotide 173tcattgccct caaacctctg 2017420DNAArtificial
sequenceSynthetic Oligonucleotide 174ccacactcat tgccctcaaa
2017520DNAArtificial sequenceSynthetic Oligonucleotide
175cactgcccac actcattgcc 2017620DNAArtificial sequenceSynthetic
Oligonucleotide 176ttaggccact gcccacactc 2017720DNAArtificial
sequenceSynthetic Oligonucleotide 177ctagtcctga ccttgctgcc
2017820DNAArtificial sequenceSynthetic Oligonucleotide
178ctcatcctag tcctgacctt 2017920DNAArtificial sequenceSynthetic
Oligonucleotide 179cctagtctca tcctagtcct 2018020DNAArtificial
sequenceSynthetic Oligonucleotide 180accctgccta gtctcatcct
2018120DNAArtificial sequenceSynthetic Oligonucleotide
181cttgtcaccc tgcctagtct 2018220DNAArtificial sequenceSynthetic
Oligonucleotide 182gcccaccttg tcaccctgcc 2018320DNAArtificial
sequenceSynthetic Oligonucleotide 183cctaaaactg ctcctactcc
2018420DNAArtificial sequenceSynthetic Oligonucleotide
184gagtcagaaa tgaggtcaaa 2018520DNAArtificial sequenceSynthetic
Oligonucleotide 185ccctactccc atttcacctt 2018620DNAArtificial
sequenceSynthetic Oligonucleotide 186tgttgtgcaa tcctgcagaa
2018720DNAArtificial sequenceSynthetic Oligonucleotide
187aaaggctgat gaagcctggc 2018820DNAArtificial sequenceSynthetic
Oligonucleotide 188cctttgacca caaagtggcc 2018920DNAArtificial
sequenceSynthetic Oligonucleotide 189aggtaccacc tctttgtggg
2019020DNAArtificial sequenceSynthetic Oligonucleotide
190tggtggtcac acctgaagag 2019120DNAArtificial sequenceSynthetic
Oligonucleotide 191gcagggagca gctcttcctt 2019220DNAArtificial
sequenceSynthetic Oligonucleotide 192tcctgtgcag ggagcagctc
2019320DNAArtificial sequenceSynthetic Oligonucleotide
193ttgatatcct gtgcagggag 2019420DNAArtificial sequenceSynthetic
Oligonucleotide 194agagctttga tatcctgtgc 2019520DNAArtificial
sequenceSynthetic Oligonucleotide 195acaaacagag ctttgatatc
2019620DNAArtificial sequenceSynthetic Oligonucleotide
196tcagacacaa acagagcttt 2019720DNAArtificial sequenceSynthetic
Oligonucleotide 197tcctcctcag acacaaacag 2019820DNAArtificial
sequenceSynthetic Oligonucleotide 198acctccttcc gagtcagctt
2019920DNAArtificial sequenceSynthetic Oligonucleotide
199atgtagacct ccttccgagt 2020020DNAArtificial sequenceSynthetic
Oligonucleotide 200ttcttgatgt agacctcctt 2020120DNAArtificial
sequenceSynthetic Oligonucleotide 201tccccattct tgatgtagac
2020220DNAArtificial sequenceSynthetic Oligonucleotide
202ttcttatccc cattcttgat 2020320DNAArtificial sequenceSynthetic
Oligonucleotide 203ctgcctttct tatccccatt 2020420DNAArtificial
sequenceSynthetic Oligonucleotide 204tcacagctgc ctttcttatc
2020520DNAArtificial sequenceSynthetic Oligonucleotide
205tctctctcac agctgccttt 2020620DNAArtificial sequenceSynthetic
Oligonucleotide 206tgagcatctc tctcacagct 2020720DNAArtificial
sequenceSynthetic Oligonucleotide 207gcatattgag catctctctc
2020820DNAArtificial sequenceSynthetic Oligonucleotide
208tgactttgtc atagcctggg 2020920DNAArtificial sequenceSynthetic
Oligonucleotide 209tgtccttgac tttgtcatag 2021020DNAArtificial
sequenceSynthetic Oligonucleotide 210cagtacaaag gaaccgaggg
2021120DNAArtificial sequenceSynthetic Oligonucleotide
211ctcctccagt acaaaggaac 2021220DNAArtificial sequenceSynthetic
Oligonucleotide 212gactcactcc tccagtacaa 2021320DNAArtificial
sequenceSynthetic Oligonucleotide 213catagggact cactcctcca
2021420DNAArtificial sequenceSynthetic Oligonucleotide
214ggtcagcata gggactcact 2021520DNAArtificial sequenceSynthetic
Oligonucleotide 215tcacctctgc aagtattggg 2021620DNAArtificial
sequenceSynthetic Oligonucleotide 216ccagaatcac ctctgcaagt
2021720DNAArtificial sequenceSynthetic Oligonucleotide
217gggccgccag aatcacctct 2021820DNAArtificial sequenceSynthetic
Oligonucleotide 218ctcttgtgaa ctatcaaggg 2021920DNAArtificial
sequenceSynthetic Oligonucleotide 219cgacttctct tgtgaactat
2022020DNAArtificial sequenceSynthetic Oligonucleotide
220atgaaacgac ttctcttgtg 2022120DNAArtificial sequenceSynthetic
Oligonucleotide 221acttgaatga aacgacttct 2022220DNAArtificial
sequenceSynthetic Oligonucleotide 222acaccaactt gaatgaaacg
2022320DNAArtificial sequenceSynthetic Oligonucleotide
223tccactactc cccagctgat 2022420DNAArtificial sequenceSynthetic
Oligonucleotide 224cagacatcca ctactcccca 2022520DNAArtificial
sequenceSynthetic Oligonucleotide 225tttttgcaga catccactac
2022620DNAArtificial sequenceSynthetic Oligonucleotide
226ttctggtttt tgcagacatc 2022720DNAArtificial sequenceSynthetic
Oligonucleotide 227tgccgcttct ggtttttgca 2022820DNAArtificial
sequenceSynthetic Oligonucleotide 228tgcttttgcc gcttctggtt
2022920DNAArtificial sequenceSynthetic Oligonucleotide
229ggtacctgct tttgccgctt 2023020DNAArtificial sequenceSynthetic
Oligonucleotide 230tgagcaggta cctgcttttg 2023120DNAArtificial
sequenceSynthetic Oligonucleotide 231ttcagccagg gcagcacttg
2023220DNAArtificial sequenceSynthetic Oligonucleotide
232ttctccttca gccagggcag 2023320DNAArtificial sequenceSynthetic
Oligonucleotide 233tggagtttct ccttcagcca 2023420DNAArtificial
sequenceSynthetic Oligonucleotide 234tcatcttgga gtttctcctt
2023520DNAArtificial sequenceSynthetic Oligonucleotide
235aaatcctcat cttggagttt 2023620DNAArtificial sequenceSynthetic
Oligonucleotide 236aaacccaaat cctcatcttg 2023720DNAArtificial
sequenceSynthetic Oligonucleotide 237gtccagcagg aaacccctta
2023820DNAArtificial sequenceSynthetic Oligonucleotide
238gcccctgtcc agcaggaaac 2023920DNAArtificial sequenceSynthetic
Oligonucleotide 239agctgtttta attcaatccc 2024020DNAArtificial
sequenceSynthetic Oligonucleotide 240aacttgccac ctgtgggtga
2024120DNAArtificial sequenceSynthetic Oligonucleotide
241tcaccttatc cccattcttg 2024220DNAArtificial sequenceSynthetic
Oligonucleotide 242tcaactttca caaaccacca 2024320DNAArtificial
sequenceSynthetic Oligonucleotide 243ccgccagaat cacctgcaag
2024420DNAArtificial sequenceSynthetic Oligonucleotide
244aggaggaatg aagaaggctt 2024520DNAArtificial sequenceSynthetic
Oligonucleotide 245gcctttcctc agggatctgg 2024620DNAArtificial
sequenceSynthetic Oligonucleotide 246aaatgtctgg gagtgtcagg
2024720DNAArtificial sequenceSynthetic Oligonucleotide
247gcctagagtg cctccttagg 2024820DNAArtificial sequenceSynthetic
Oligonucleotide 248ggcatctccc cagataggaa 2024920DNAArtificial
sequenceSynthetic Oligonucleotide 249agggagctag tcctggaaga
2025020DNAArtificial sequenceSynthetic Oligonucleotide
250acacctgaag agaaaggctg 2025120DNAArtificial sequenceSynthetic
Oligonucleotide 251ccctttgacc acaaagtggc 2025220DNAArtificial
sequenceSynthetic Oligonucleotide 252gccctcaagg tagtctcatg
2025320DNAArtificial sequenceSynthetic Oligonucleotide
253aagggaagga ggacagaata 2025420DNAArtificial sequenceSynthetic
Oligonucleotide 254aaaggccaag gagggatgct 2025520DNAArtificial
sequenceSynthetic Oligonucleotide 255agaggtccct tctgaccatc
2025620DNAArtificial sequenceSynthetic Oligonucleotide
256gctgggacag gagagaggtc 2025720DNAArtificial sequenceSynthetic
Oligonucleotide 257tcaaatgtct gggagtgtca 2025820DNAArtificial
sequenceSynthetic Oligonucleotide 258agaaggagaa tgtgctgaaa
2025920DNAArtificial sequenceSynthetic Oligonucleotide
259tgctgaccac ttggcatctc 2026020DNAArtificial sequenceSynthetic
Oligonucleotide 260caactttcac aaaccaccat 2026120DNAArtificial
sequenceSynthetic Oligonucleotide 261agctctgtga ttctaaggtt
2026220DNAArtificial sequenceSynthetic Oligonucleotide
262ccacctgtgg gtgaggagaa 2026320DNAArtificial sequenceSynthetic
Oligonucleotide 263gaggactcac ttgaatgaaa 2026420DNAArtificial
sequenceSynthetic Oligonucleotide 264tggaatgatc agggagctag
2026520DNAArtificial sequenceSynthetic Oligonucleotide
265gtcccttctc cattttcccc 2026620DNAArtificial sequenceSynthetic
Oligonucleotide 266tcaacttttt aagttaatca 2026720DNAArtificial
sequenceSynthetic Oligonucleotide 267gggtgaggag aacaaggcgc
2026820DNAArtificial sequenceSynthetic Oligonucleotide
268cttccaagcc atcttttaac 2026920DNAArtificial sequenceSynthetic
Oligonucleotide 269aggactcact tgaatgaaac 2027020DNAArtificial
sequenceSynthetic Oligonucleotide 270ttccaggcaa ctagagcttc
2027120DNAArtificial sequenceSynthetic Oligonucleotide
271cagagtccag ccactgtttg 2027220DNAArtificial sequenceSynthetic
Oligonucleotide 272ccaacctgca gaggcagtgg 2027320DNAArtificial
sequenceSynthetic Oligonucleotide 273tgcaaggaga ggagaagctg
2027420DNAArtificial sequenceSynthetic Oligonucleotide
274ctaggcaggt tactcaccca 2027520DNAArtificial sequenceSynthetic
Oligonucleotide 275caccataact tgccacctgt
2027620DNAArtificial sequenceSynthetic Oligonucleotide
276taggtaccac ctctttgtgg 2027720DNAArtificial sequenceSynthetic
Oligonucleotide 277cttgacctca cctcccccaa 2027820DNAArtificial
sequenceSynthetic Oligonucleotide 278ccacctcttt gtgggcagct
2027920DNAArtificial sequenceSynthetic Oligonucleotide
279ttcacaaacc accatctctt 2028020DNAArtificial sequenceSynthetic
Oligonucleotide 280ttctcacctc cgttgtcaca 2028120DNAArtificial
sequenceSynthetic Oligonucleotide 281gaaagtggga ggtgttgcct
2028220DNAArtificial sequenceSynthetic Oligonucleotide
282acagcaggaa gggaaggtta 2028320DNAArtificial sequenceSynthetic
Oligonucleotide 283catgctgacc acttggcatc 2028420DNAArtificial
sequenceSynthetic Oligonucleotide 284ggtcaccttg gcaggaaggc
2028520DNAArtificial sequenceSynthetic Oligonucleotide
285gtatagtgtt acaagtggac 2028620DNAArtificial sequenceSynthetic
Oligonucleotide 286ggacttccct ttgaccacaa 2028720DNAArtificial
sequenceSynthetic Oligonucleotide 287tcaccttgac ctcacctccc
2028820DNAArtificial sequenceSynthetic Oligonucleotide
288tagagtgcct ccttaggatg 2028920DNAArtificial sequenceSynthetic
Oligonucleotide 289tgacttcaac ttgtggtctg 2029020DNAArtificial
sequenceSynthetic Oligonucleotide 290cagagaagga gaatgtgctg
2029120DNAArtificial sequenceSynthetic Oligonucleotide
291agggagcagc tcttcctctg 2029220DNAArtificial sequenceSynthetic
Oligonucleotide 292tgttcccctg ggtgccagga 2029320DNAArtificial
sequenceSynthetic Oligonucleotide 293ggcctggctg ttttcaagcc
2029420DNAArtificial sequenceSynthetic Oligonucleotide
294gactggcttt catctggcag 2029520DNAArtificial sequenceSynthetic
Oligonucleotide 295gaaggctttc caggcaacta 2029620DNAArtificial
sequenceSynthetic Oligonucleotide 296tcacttgaat gaaacgactt
2029720DNAArtificial sequenceSynthetic Oligonucleotide
297ggccccaaaa ggccaaggag 2029820DNAArtificial sequenceSynthetic
Oligonucleotide 298aatcacctgc aaggagagga 2029920DNAArtificial
sequenceSynthetic Oligonucleotide 299gaccttcagt tgcatcctta
2030020DNAArtificial sequenceSynthetic Oligonucleotide
300tgatgaagcc tggccccaaa 2030120DNAArtificial sequenceSynthetic
Oligonucleotide 301tagaaagtgg gaggtgttgc 2030220DNAArtificial
sequenceSynthetic Oligonucleotide 302cccatccctg actggtctgg
2030320DNAArtificial sequenceSynthetic Oligonucleotide
303ccatgggtat agtgttacaa 2030420DNAArtificial sequenceSynthetic
Oligonucleotide 304gtgttctctt gacttccagg 2030520DNAArtificial
sequenceSynthetic Oligonucleotide 305ggcctgctcc tcaccccagt
2030620DNAArtificial sequenceSynthetic Oligonucleotide
306gaggcctggc tgttttcaag 2030720DNAArtificial sequenceSynthetic
Oligonucleotide 307gactctcccc ttcagtacct 2030820DNAArtificial
sequenceSynthetic Oligonucleotide 308catgggtata gtgttacaag
2030920DNAArtificial sequenceSynthetic Oligonucleotide
309gaaggagaat gtgctgaaaa 2031020DNAArtificial sequenceSynthetic
Oligonucleotide 310tcacctggtc ttccaagcca 2031120DNAArtificial
sequenceSynthetic Oligonucleotide 311ctccccagat aggaaaggga
2031220DNAArtificial sequenceSynthetic Oligonucleotide
312ggactcactt gaatgaaacg 2031320DNAArtificial sequenceSynthetic
Oligonucleotide 313ggccgccaga atcacctgca 2031420DNAArtificial
sequenceSynthetic Oligonucleotide 314ctcacttgaa tgaaacgact
2031520DNAArtificial sequenceSynthetic Oligonucleotide
315ctttcccagc ctttcctcag 2031620DNAArtificial sequenceSynthetic
Oligonucleotide 316agaaagtggg aggtgttgcc 2031720DNAArtificial
sequenceSynthetic Oligonucleotide 317gtcgcagctg ttttaattca
2031820DNAArtificial sequenceSynthetic Oligonucleotide
318ccaggactct ccccttcagt 2031920DNAArtificial sequenceSynthetic
Oligonucleotide 319agggaaggag gacagaatag 2032020DNAArtificial
sequenceSynthetic Oligonucleotide 320gaaatgaggt caaatgtctg
2032120DNAArtificial sequenceSynthetic Oligonucleotide
321ggagagtcag aaatgaggtc 2032220DNAArtificial sequenceSynthetic
Oligonucleotide 322gtagaaagtg ggaggtgttg 2032320DNAArtificial
sequenceSynthetic Oligonucleotide 323tagaaagatc tctgaagtgc
2032420DNAArtificial sequenceSynthetic Oligonucleotide
324ctgctcctca ccccagtcct 2032520DNAArtificial sequenceSynthetic
Oligonucleotide 325ctactgggat tctgtgctta 2032620DNAArtificial
sequenceSynthetic Oligonucleotide 326cccaaaaggc caaggaggga
2032720DNAArtificial sequenceSynthetic Oligonucleotide
327tgaccacttg gcatctcccc 2032820DNAArtificial sequenceSynthetic
Oligonucleotide 328cctgcaagga gaggagaagc 2032920DNAArtificial
sequenceSynthetic Oligonucleotide 329ctctcacctc tgcaagtatt
2033020DNAArtificial sequenceSynthetic Oligonucleotide
330ccccaaaagg ccaaggaggg 2033120DNAArtificial sequenceSynthetic
Oligonucleotide 331gtcttccaag ccatctttta 2033220DNAArtificial
sequenceSynthetic Oligonucleotide 332gttacaagtg gacttaaggg
2033320DNAArtificial sequenceSynthetic Oligonucleotide
333cccatgttgt gcaatcctgc 2033420DNAArtificial sequenceSynthetic
Oligonucleotide 334gaggtgggaa gcatggagaa 2033520DNAArtificial
sequenceSynthetic Oligonucleotide 335tgctcccacc actgtcatct
2033620DNAArtificial sequenceSynthetic Oligonucleotide
336aggcaggtta ctcacccaga 2033720DNAArtificial sequenceSynthetic
Oligonucleotide 337tactgggatt ctgtgcttac 2033820DNAArtificial
sequenceSynthetic Oligonucleotide 338gcctttccca gcctttcctc
2033920DNAArtificial sequenceSynthetic Oligonucleotide
339gtgcaatcct gcagaagaga 2034020DNAArtificial sequenceSynthetic
Oligonucleotide 340acaggagaga ggtcccttct 2034120DNAArtificial
sequenceSynthetic Oligonucleotide 341cccaaaagga gaaagggaaa
2034220DNAArtificial sequenceSynthetic Oligonucleotide
342aagcccaggg taaatgctta 2034320DNAArtificial sequenceSynthetic
Oligonucleotide 343gatgaagcct ggccccaaaa 2034420DNAArtificial
sequenceSynthetic Oligonucleotide 344tggcagagaa ggagaatgtg
2034520DNAArtificial sequenceSynthetic Oligonucleotide
345ttcccagcct ttcctcaggg 2034620DNAArtificial sequenceSynthetic
Oligonucleotide 346ggcagagaag gagaatgtgc 2034720DNAArtificial
sequenceSynthetic Oligonucleotide 347acagtgccag gaaacaagaa
2034820DNAArtificial sequenceSynthetic Oligonucleotide
348taggcaggtt actcacccag 2034920DNAArtificial sequenceSynthetic
Oligonucleotide 349ttctcttgac ttccagggct 2035020DNAArtificial
sequenceSynthetic Oligonucleotide 350cctgctcctc accccagtcc
2035120DNAArtificial sequenceSynthetic Oligonucleotide
351tcccactaac ctccattgcc 2035220DNAArtificial sequenceSynthetic
Oligonucleotide 352ttccctttga ccacaaagtg 2035320DNAArtificial
sequenceSynthetic Oligonucleotide 353ctgggtccta ggcaggttac
2035420DNAArtificial sequenceSynthetic Oligonucleotide
354tccaggcaac tagagcttca 2035520DNAArtificial sequenceSynthetic
Oligonucleotide 355gcccatgttg tgcaatcctg 2035620DNAArtificial
sequenceSynthetic Oligonucleotide 356ggttcccact aacctccatt
2035720DNAArtificial sequenceSynthetic Oligonucleotide
357aggtagagag caagagttac 2035820DNAArtificial sequenceSynthetic
Oligonucleotide 358ccactaacct ccattgccca 2035920DNAArtificial
sequenceSynthetic Oligonucleotide 359tcacaaacca ccatctctta
2036020DNAArtificial sequenceSynthetic Oligonucleotide
360tactcaccca gataatcctc 2036120DNAArtificial sequenceSynthetic
Oligonucleotide 361tgctcctcac cccagtcctc 2036220DNAArtificial
sequenceSynthetic Oligonucleotide 362tctcacagct gcctttctgt
2036320DNAArtificial sequenceSynthetic Oligonucleotide
363gaaagggagg actcacttga 2036420DNAArtificial sequenceSynthetic
Oligonucleotide 364ccatctttta accccagaga 2036520DNAArtificial
sequenceSynthetic Oligonucleotide 365tcctcacccc agtcctccag
2036620DNAArtificial sequenceSynthetic Oligonucleotide
366ctggcagaga aggagaatgt 2036720DNAArtificial sequenceSynthetic
Oligonucleotide 367tctccccaga taggaaaggg 2036820DNAArtificial
sequenceSynthetic Oligonucleotide 368acttcagctg ctcccaccac
2036920DNAArtificial sequenceSynthetic Oligonucleotide
369gacagcagga agggaaggtt 2037020DNAArtificial sequenceSynthetic
Oligonucleotide 370ggagacaaat gggcctataa 2037120DNAArtificial
sequenceSynthetic Oligonucleotide 371ctgctcccac cactgtcatc
2037220DNAArtificial sequenceSynthetic Oligonucleotide
372aggaatgaag aaggctttcc 2037320DNAArtificial sequenceSynthetic
Oligonucleotide 373gggatctcat ccttatcctc 2037420DNAArtificial
sequenceSynthetic Oligonucleotide 374gtgctgggtc ctaggcaggt
2037520DNAArtificial sequenceSynthetic Oligonucleotide
375caaaaggcca aggagggatg 2037620DNAArtificial sequenceSynthetic
Oligonucleotide 376ccatgctgac cacttggcat 2037720DNAArtificial
sequenceSynthetic Oligonucleotide 377ggaggctggg acaggagaga
2037820DNAArtificial sequenceSynthetic Oligonucleotide
378ggagcagctc ttcctctgga 2037920DNAArtificial sequenceSynthetic
Oligonucleotide 379tctcacctcc gttgtcacag 2038020DNAArtificial
sequenceSynthetic Oligonucleotide 380cagtcctcca gcctttccca
2038120DNAArtificial sequenceSynthetic Oligonucleotide
381agtcctccag cctttcccag 2038220DNAArtificial sequenceSynthetic
Oligonucleotide 382tgaaggagtc tgggagagtc 2038320DNAArtificial
sequenceSynthetic Oligonucleotide 383cagaatcacc tgcaaggaga
2038420DNAArtificial sequenceSynthetic Oligonucleotide
384taggaaaggg aggactcact 2038520DNAArtificial sequenceSynthetic
Oligonucleotide 385accttggcag gaaggctccg 2038620DNAArtificial
sequenceSynthetic Oligonucleotide 386gagacaaatg ggcctataaa
2038720DNAArtificial sequenceSynthetic Oligonucleotide
387ctgaagagaa aggctgatga 2038820DNAArtificial sequenceSynthetic
Oligonucleotide 388aatgatcagg gagctagtcc 2038920DNAArtificial
sequenceSynthetic Oligonucleotide 389cttagctgac ctaaaggaat
2039020DNAArtificial sequenceSynthetic Oligonucleotide
390tgggtatagt gttacaagtg 2039120DNAArtificial sequenceSynthetic
Oligonucleotide 391tgaagagaaa ggctgatgaa 2039220DNAArtificial
sequenceSynthetic Oligonucleotide 392gtgttacaag tggacttaag
2039320DNAArtificial sequenceSynthetic Oligonucleotide
393acctgtgggt gaggagaaca 2039420DNAArtificial sequenceSynthetic
Oligonucleotide 394tcacccagat aatcctccct 2039520DNAArtificial
sequenceSynthetic Oligonucleotide 395tgttgtcgca gctgttttaa
2039620DNAArtificial sequenceSynthetic Oligonucleotide
396tggtcacatt cccttcccct 2039720DNAArtificial sequenceSynthetic
Oligonucleotide 397cctggtcaca ttcccttccc 2039820DNAArtificial
sequenceSynthetic Oligonucleotide 398tagacctggt cacattccct
2039920DNAArtificial sequenceSynthetic Oligonucleotide
399cctagacctg gtcacattcc 2040020DNAArtificial sequenceSynthetic
Oligonucleotide 400ccttccgagt cagctttttc 2040120DNAArtificial
sequenceSynthetic Oligonucleotide 401ctccttccga
gtcagctttt 2040220DNAArtificial sequenceSynthetic Oligonucleotide
402agacctcctt ccgagtcagc 2040320DNAArtificial sequenceSynthetic
Oligonucleotide 403gtagacctcc ttccgagtca 2040420DNAArtificial
sequenceSynthetic Oligonucleotide 404tttgccgctt ctggtttttg
2040520DNAArtificial sequenceSynthetic Oligonucleotide
405cttttgccgc ttctggtttt 2040620DNAArtificial sequenceSynthetic
Oligonucleotide 406cctgcttttg ccgcttctgg 2040720DNAArtificial
sequenceSynthetic Oligonucleotide 407tacctgcttt tgccgcttct
2040820DNAArtificial sequenceSynthetic Oligonucleotide
408agaaaaccca aatcctcatc 2040920DNAArtificial sequenceSynthetic
Oligonucleotide 409tagaaaaccc aaatcctcat 2041020DNAArtificial
sequenceSynthetic Oligonucleotide 410atagaaaacc caaatcctca
2041120DNAArtificial sequenceSynthetic Oligonucleotide
411tatagaaaac ccaaatcctc 2041220DNAArtificial sequenceSynthetic
Oligonucleotide 412ttatagaaaa cccaaatcct 2041320DNAArtificial
sequenceSynthetic Oligonucleotide 413cttatagaaa acccaaatcc
2041420DNAArtificial sequenceSynthetic Oligonucleotide
414ccttatagaa aacccaaatc 2041520DNAArtificial sequenceSynthetic
Oligonucleotide 415cccttataga aaacccaaat 2041620DNAArtificial
sequenceSynthetic Oligonucleotide 416ccccttatag aaaacccaaa
2041720DNAArtificial sequenceSynthetic Oligonucleotide
417accccttata gaaaacccaa 2041820DNAArtificial sequenceSynthetic
Oligonucleotide 418aaccccttat agaaaaccca 2041920DNAArtificial
sequenceSynthetic Oligonucleotide 419aaacccctta tagaaaaccc
2042020DNAArtificial sequenceSynthetic Oligonucleotide
420gaaacccctt atagaaaacc 2042120DNAArtificial sequenceSynthetic
Oligonucleotide 421ggaaacccct tatagaaaac 2042220DNAArtificial
sequenceSynthetic Oligonucleotide 422aggaaacccc ttatagaaaa
2042320DNAArtificial sequenceSynthetic Oligonucleotide
423caggaaaccc cttatagaaa 2042420DNAArtificial sequenceSynthetic
Oligonucleotide 424gcaggaaacc ccttatagaa 2042520DNAArtificial
sequenceSynthetic Oligonucleotide 425agcaggaaac cccttataga
2042620DNAArtificial sequenceSynthetic Oligonucleotide
426cagcaggaaa ccccttatag 2042720DNAArtificial sequenceSynthetic
Oligonucleotide 427ccagcaggaa accccttata 2042820DNAArtificial
sequenceSynthetic Oligonucleotide 428tccagcagga aaccccttat
2042920DNAArtificial sequenceSynthetic Oligonucleotide
429tgtccagcag gaaacccctt 2043020DNAArtificial sequenceSynthetic
Oligonucleotide 430ctgtccagca ggaaacccct 2043120DNAArtificial
sequenceSynthetic Oligonucleotide 431cctgtccagc aggaaacccc
2043220DNAArtificial sequenceSynthetic Oligonucleotide
432ccctgtccag caggaaaccc 2043320DNAArtificial sequenceSynthetic
Oligonucleotide 433cccctgtcca gcaggaaacc 2043420DNAArtificial
sequenceSynthetic Oligonucleotide 434cgcccctgtc cagcaggaaa
2043520DNAArtificial sequenceSynthetic Oligonucleotide
435acgcccctgt ccagcaggaa 2043620DNAArtificial sequenceSynthetic
Oligonucleotide 436cacgcccctg tccagcagga 2043720DNAArtificial
sequenceSynthetic Oligonucleotide 437ccacgcccct gtccagcagg
2043820DNAArtificial sequenceSynthetic Oligonucleotide
438cccacgcccc tgtccagcag 2043920DNAArtificial sequenceSynthetic
Oligonucleotide 439tcccacgccc ctgtccagca 2044020DNAArtificial
sequenceSynthetic Oligonucleotide 440atcccacgcc cctgtccagc
2044120DNAArtificial sequenceSynthetic Oligonucleotide
441aatcccacgc ccctgtccag 2044220DNAArtificial sequenceSynthetic
Oligonucleotide 442caatcccacg cccctgtcca 2044320DNAArtificial
sequenceSynthetic Oligonucleotide 443tcaatcccac gcccctgtcc
2044420DNAArtificial sequenceSynthetic Oligonucleotide
444ttcaatccca cgcccctgtc 2044520DNAArtificial sequenceSynthetic
Oligonucleotide 445attcaatccc acgcccctgt 2044620DNAArtificial
sequenceSynthetic Oligonucleotide 446aattcaatcc cacgcccctg
2044720DNAArtificial sequenceSynthetic Oligonucleotide
447taattcaatc ccacgcccct 2044820DNAArtificial sequenceSynthetic
Oligonucleotide 448ttaattcaat cccacgcccc 2044920DNAArtificial
sequenceSynthetic Oligonucleotide 449tttaattcaa tcccacgccc
2045020DNAArtificial sequenceSynthetic Oligonucleotide
450ttttaattca atcccacgcc 2045120DNAArtificial sequenceSynthetic
Oligonucleotide 451gttttaattc aatcccacgc 2045220DNAArtificial
sequenceSynthetic Oligonucleotide 452tgttttaatt caatcccacg
2045320DNAArtificial sequenceSynthetic Oligonucleotide
453ctgttttaat tcaatcccac 2045420DNAArtificial sequenceSynthetic
Oligonucleotide 454gctgttttaa ttcaatccca 2045520DNAArtificial
sequenceSynthetic Oligonucleotide 455cagctgtttt aattcaatcc
2045620DNAArtificial sequenceSynthetic Oligonucleotide
456gcagctgttt taattcaatc 2045720DNAArtificial sequenceSynthetic
Oligonucleotide 457cgcagctgtt ttaattcaat 2045820DNAArtificial
sequenceSynthetic Oligonucleotide 458tcgcagctgt tttaattcaa
2045920DNAArtificial sequenceSynthetic Oligonucleotide
459tgtcgcagct gttttaattc 2046020DNAArtificial sequenceSynthetic
Oligonucleotide 460ttgtcgcagc tgttttaatt 2046120DNAArtificial
sequenceSynthetic Oligonucleotide 461gttgtcgcag ctgttttaat
2046220DNAArtificial sequenceSynthetic Oligonucleotide
462ttgttgtcgc agctgtttta 2046320DNAArtificial sequenceSynthetic
Oligonucleotide 463tttgttgtcg cagctgtttt 2046420DNAArtificial
sequenceSynthetic Oligonucleotide 464ttttgttgtc gcagctgttt
2046520DNAArtificial sequenceSynthetic Oligonucleotide
465tttttgttgt cgcagctgtt 2046620DNAArtificial sequenceSynthetic
Oligonucleotide 466ggatccagct cactcccctg 2046720DNAArtificial
sequenceSynthetic Oligonucleotide 467aaataaggat ccagctcact
2046820DNAArtificial sequenceSynthetic Oligonucleotide
468gaccagaaat aaggatccag 2046920DNAArtificial sequenceSynthetic
Oligonucleotide 469cttagggacc agaaataagg 2047020DNAArtificial
sequenceSynthetic Oligonucleotide 470cacccactta gggaccagaa
2047120DNAArtificial sequenceSynthetic Oligonucleotide
471accacccact tagggaccag 2047220DNAArtificial sequenceSynthetic
Oligonucleotide 472aggtccagga ctctcccctt 2047320DNAArtificial
sequenceSynthetic Oligonucleotide 473aaggtccagg actctcccct
2047420DNAArtificial sequenceSynthetic Oligonucleotide
474aaactgcaga agtcccaccc 2047520DNAArtificial sequenceSynthetic
Oligonucleotide 475ggagggcccc gctgagctgc 2047620DNAArtificial
sequenceSynthetic Oligonucleotide 476tcccggaaca tccaagcggg
2047720DNAArtificial sequenceSynthetic Oligonucleotide
477catcactttc ccggaacatc 2047820DNAArtificial sequenceSynthetic
Oligonucleotide 478ctggtcacat tcccttcccc 2047920DNAArtificial
sequenceSynthetic Oligonucleotide 479ctagacctgg tcacattccc
2048020DNAArtificial sequenceSynthetic Oligonucleotide
480ggagtggtgg tcacacctcc 2048120DNAArtificial sequenceSynthetic
Oligonucleotide 481accccctcca gagagcagga 2048220DNAArtificial
sequenceSynthetic Oligonucleotide 482atctctaccc cctccagaga
2048320DNAArtificial sequenceSynthetic Oligonucleotide
483ggtacgggta gaagccagaa 2048420DNAArtificial sequenceSynthetic
Oligonucleotide 484ggagagtgta accgtcatag 2048520DNAArtificial
sequenceSynthetic Oligonucleotide 485tgcgattggc agagccccgg
2048620DNAArtificial sequenceSynthetic Oligonucleotide
486ggcaggtgcg attggcagag 2048720DNAArtificial sequenceSynthetic
Oligonucleotide 487ggccattcac ttggcaggtg 2048820DNAArtificial
sequenceSynthetic Oligonucleotide 488ttgtcacaga tcgctgtctg
2048920DNAArtificial sequenceSynthetic Oligonucleotide
489aaggagtctt ggcaggaagg 2049020DNAArtificial sequenceSynthetic
Oligonucleotide 490gtacatgaag gagtcttggc 2049120DNAArtificial
sequenceSynthetic Oligonucleotide 491aagcttcggc cacctcttga
2049220DNAArtificial sequenceSynthetic Oligonucleotide
492ccatctagca ccaggtagat 2049320DNAArtificial sequenceSynthetic
Oligonucleotide 493ggccccaatg ctgtctgatc 2049420DNAArtificial
sequenceSynthetic Oligonucleotide 494aattaagttg actagacact
2049520DNAArtificial sequenceSynthetic Oligonucleotide
495tgccaccttc tcaattaagt 2049620DNAArtificial sequenceSynthetic
Oligonucleotide 496taacttgcca ccttctcaat 2049720DNAArtificial
sequenceSynthetic Oligonucleotide 497cataacttgc caccttctca
2049820DNAArtificial sequenceSynthetic Oligonucleotide
498acaccataac ttgccacctt 2049920DNAArtificial sequenceSynthetic
Oligonucleotide 499tcacaccata acttgccacc 2050020DNAArtificial
sequenceSynthetic Oligonucleotide 500tagtccctga cttcaacttg
2050120DNAArtificial sequenceSynthetic Oligonucleotide
501tggtgttagt ccctgacttc 2050220DNAArtificial sequenceSynthetic
Oligonucleotide 502gcggttccag ccttcaggag 2050320DNAArtificial
sequenceSynthetic Oligonucleotide 503tcatgaggat gatgacatgg
2050420DNAArtificial sequenceSynthetic Oligonucleotide
504ccgcccatgt tgtgcaatcc 2050520DNAArtificial sequenceSynthetic
Oligonucleotide 505gtaattgggt ccccgcccat 2050620DNAArtificial
sequenceSynthetic Oligonucleotide 506aagtcccgga tctcatcaat
2050720DNAArtificial sequenceSynthetic Oligonucleotide
507aacacataga catccagata 2050820DNAArtificial sequenceSynthetic
Oligonucleotide 508caaagcattg atgttcactt 2050920DNAArtificial
sequenceSynthetic Oligonucleotide 509tttgaacaca tgttgctcat
2051020DNAArtificial sequenceSynthetic Oligonucleotide
510cttccaggtt ttccatatcc 2051120DNAArtificial sequenceSynthetic
Oligonucleotide 511tcttccaggt tttccatatc 2051220DNAArtificial
sequenceSynthetic Oligonucleotide 512agactcagag actggctttc
2051320DNAArtificial sequenceSynthetic Oligonucleotide
513gcctgccatg gttgcttgtg 2051420DNAArtificial sequenceSynthetic
Oligonucleotide 514tgactgagat cttggcctgc 2051520DNAArtificial
sequenceSynthetic Oligonucleotide 515ttctatctcc aggtcccgct
2051620DNAArtificial sequenceSynthetic Oligonucleotide
516agtcataaaa ttcaggaatt 2051720DNAArtificial sequenceSynthetic
Oligonucleotide 517cgagttgttc cctcggtgca 2051820DNAArtificial
sequenceSynthetic Oligonucleotide 518agcctcaaag ctcgagttgt
2051920DNAArtificial sequenceSynthetic Oligonucleotide
519ggaggaagcc tcaaagctcg 2052020DNAArtificial sequenceSynthetic
Oligonucleotide 520gtagttggag gaagcctcaa 2052120DNAArtificial
sequenceSynthetic Oligonucleotide 521caagtggtag ttggaggaag
2052220DNAArtificial sequenceSynthetic Oligonucleotide
522tcctcagaca caaacagagc 2052320DNAArtificial sequenceSynthetic
Oligonucleotide 523ttctcctcct cagacacaaa 2052420DNAArtificial
sequenceSynthetic Oligonucleotide 524tagacctcct tccgagtcag
2052520DNAArtificial sequenceSynthetic Oligonucleotide
525ttgatgtaga cctccttccg 2052620DNAArtificial sequenceSynthetic
Oligonucleotide 526ctttcttatc cccattcttg
2052720DNAArtificial sequenceSynthetic Oligonucleotide
527gcctttctta tccccattct 2052820DNAArtificial sequenceSynthetic
Oligonucleotide 528agctgccttt cttatcccca 2052920DNAArtificial
sequenceSynthetic Oligonucleotide 529cagctgcctt tcttatcccc
2053020DNAArtificial sequenceSynthetic Oligonucleotide
530acagctgcct ttcttatccc 2053120DNAArtificial sequenceSynthetic
Oligonucleotide 531gcatctctct cacagctgcc 2053220DNAArtificial
sequenceSynthetic Oligonucleotide 532agatgtcctt gactttgtca
2053320DNAArtificial sequenceSynthetic Oligonucleotide
533cagcataggg actcactcct 2053420DNAArtificial sequenceSynthetic
Oligonucleotide 534ccgccagaat cacctctgca 2053520DNAArtificial
sequenceSynthetic Oligonucleotide 535tgaatgaaac gacttctctt
2053620DNAArtificial sequenceSynthetic Oligonucleotide
536acatccacta ctccccagct 2053720DNAArtificial sequenceSynthetic
Oligonucleotide 537cgcttctggt ttttgcagac 2053820DNAArtificial
sequenceSynthetic Oligonucleotide 538ttttgccgct tctggttttt
2053920DNAArtificial sequenceSynthetic Oligonucleotide
539gcaggtacct gcttttgccg 2054020DNAArtificial sequenceSynthetic
Oligonucleotide 540tcttggagtt tctccttcag 2054116DNAArtificial
sequenceSynthetic Oligonucleotide 541ggaacatcca agcggg
1654216DNAArtificial sequenceSynthetic Oligonucleotide
542tggtcacatt cccttc 1654316DNAArtificial sequenceSynthetic
Oligonucleotide 543cctggtcaca ttccct 1654416DNAArtificial
sequenceSynthetic Oligonucleotide 544gacctggtca cattcc
1654516DNAArtificial sequenceSynthetic Oligonucleotide
545taacttgcca ccttct 1654616DNAArtificial sequenceSynthetic
Oligonucleotide 546cataacttgc cacctt 1654716DNAArtificial
sequenceSynthetic Oligonucleotide 547accataactt gccacc
1654816DNAArtificial sequenceSynthetic Oligonucleotide
548ccttccgagt cagctt 1654916DNAArtificial sequenceSynthetic
Oligonucleotide 549ctccttccga gtcagc 1655016DNAArtificial
sequenceSynthetic Oligonucleotide 550acctccttcc gagtca
1655116DNAArtificial sequenceSynthetic Oligonucleotide
551ctttcttatc cccatt 1655216DNAArtificial sequenceSynthetic
Oligonucleotide 552gcctttctta tcccca 1655316DNAArtificial
sequenceSynthetic Oligonucleotide 553ctgcctttct tatccc
1655416DNAArtificial sequenceSynthetic Oligonucleotide
554tttgccgctt ctggtt 1655516DNAArtificial sequenceSynthetic
Oligonucleotide 555cttttgccgc ttctgg 1655616DNAArtificial
sequenceSynthetic Oligonucleotide 556tgcttttgcc gcttct
1655716DNAArtificial sequenceSynthetic Oligonucleotide
557aaacccaaat cctcat 1655816DNAArtificial sequenceSynthetic
Oligonucleotide 558gaaaacccaa atcctc 1655916DNAArtificial
sequenceSynthetic Oligonucleotide 559tagaaaaccc aaatcc
1656016DNAArtificial sequenceSynthetic Oligonucleotide
560atagaaaacc caaatc 1656116DNAArtificial sequenceSynthetic
Oligonucleotide 561cttatagaaa acccaa 1656216DNAArtificial
sequenceSynthetic Oligonucleotide 562ccttatagaa aaccca
1656316DNAArtificial sequenceSynthetic Oligonucleotide
563cccttataga aaaccc 1656416DNAArtificial sequenceSynthetic
Oligonucleotide 564ccccttatag aaaacc 1656516DNAArtificial
sequenceSynthetic Oligonucleotide 565accccttata gaaaac
1656616DNAArtificial sequenceSynthetic Oligonucleotide
566aaccccttat agaaaa 1656716DNAArtificial sequenceSynthetic
Oligonucleotide 567aaacccctta tagaaa 1656816DNAArtificial
sequenceSynthetic Oligonucleotide 568gaaacccctt atagaa
1656916DNAArtificial sequenceSynthetic Oligonucleotide
569ggaaacccct tataga 1657016DNAArtificial sequenceSynthetic
Oligonucleotide 570aggaaacccc ttatag 1657116DNAArtificial
sequenceSynthetic Oligonucleotide 571caggaaaccc cttata
1657216DNAArtificial sequenceSynthetic Oligonucleotide
572gcaggaaacc ccttat 1657316DNAArtificial sequenceSynthetic
Oligonucleotide 573agcaggaaac ccctta 1657416DNAArtificial
sequenceSynthetic Oligonucleotide 574cagcaggaaa cccctt
1657516DNAArtificial sequenceSynthetic Oligonucleotide
575ccagcaggaa acccct 1657616DNAArtificial sequenceSynthetic
Oligonucleotide 576tccagcagga aacccc 1657716DNAArtificial
sequenceSynthetic Oligonucleotide 577gtccagcagg aaaccc
1657816DNAArtificial sequenceSynthetic Oligonucleotide
578tgtccagcag gaaacc 1657916DNAArtificial sequenceSynthetic
Oligonucleotide 579ctgtccagca ggaaac 1658016DNAArtificial
sequenceSynthetic Oligonucleotide 580cctgtccagc aggaaa
1658116DNAArtificial sequenceSynthetic Oligonucleotide
581ccctgtccag caggaa 1658216DNAArtificial sequenceSynthetic
Oligonucleotide 582cccctgtcca gcagga 1658316DNAArtificial
sequenceSynthetic Oligonucleotide 583gcccctgtcc agcagg
1658416DNAArtificial sequenceSynthetic Oligonucleotide
584cgcccctgtc cagcag 1658516DNAArtificial sequenceSynthetic
Oligonucleotide 585acgcccctgt ccagca 1658616DNAArtificial
sequenceSynthetic Oligonucleotide 586cacgcccctg tccagc
1658716DNAArtificial sequenceSynthetic Oligonucleotide
587ccacgcccct gtccag 1658816DNAArtificial sequenceSynthetic
Oligonucleotide 588cccacgcccc tgtcca 1658916DNAArtificial
sequenceSynthetic Oligonucleotide 589tcccacgccc ctgtcc
1659016DNAArtificial sequenceSynthetic Oligonucleotide
590atcccacgcc cctgtc 1659116DNAArtificial sequenceSynthetic
Oligonucleotide 591aatcccacgc ccctgt 1659216DNAArtificial
sequenceSynthetic Oligonucleotide 592caatcccacg cccctg
1659316DNAArtificial sequenceSynthetic Oligonucleotide
593tcaatcccac gcccct 1659416DNAArtificial sequenceSynthetic
Oligonucleotide 594ttcaatccca cgcccc 1659516DNAArtificial
sequenceSynthetic Oligonucleotide 595attcaatccc acgccc
1659616DNAArtificial sequenceSynthetic Oligonucleotide
596aattcaatcc cacgcc 1659716DNAArtificial sequenceSynthetic
Oligonucleotide 597taattcaatc ccacgc 1659816DNAArtificial
sequenceSynthetic Oligonucleotide 598ttaattcaat cccacg
1659916DNAArtificial sequenceSynthetic Oligonucleotide
599tttaattcaa tcccac 1660016DNAArtificial sequenceSynthetic
Oligonucleotide 600ttttaattca atccca 1660116DNAArtificial
sequenceSynthetic Oligonucleotide 601gttttaattc aatccc
1660216DNAArtificial sequenceSynthetic Oligonucleotide
602tgttttaatt caatcc 1660316DNAArtificial sequenceSynthetic
Oligonucleotide 603ctgttttaat tcaatc 1660416DNAArtificial
sequenceSynthetic Oligonucleotide 604gctgttttaa ttcaat
1660516DNAArtificial sequenceSynthetic Oligonucleotide
605agctgtttta attcaa 1660616DNAArtificial sequenceSynthetic
Oligonucleotide 606cagctgtttt aattca 1660716DNAArtificial
sequenceSynthetic Oligonucleotide 607gcagctgttt taattc
1660816DNAArtificial sequenceSynthetic Oligonucleotide
608cgcagctgtt ttaatt 1660916DNAArtificial sequenceSynthetic
Oligonucleotide 609tcgcagctgt tttaat 1661016DNAArtificial
sequenceSynthetic Oligonucleotide 610gtcgcagctg ttttaa
1661116DNAArtificial sequenceSynthetic Oligonucleotide
611tgtcgcagct gtttta 1661216DNAArtificial sequenceSynthetic
Oligonucleotide 612ttgtcgcagc tgtttt 1661316DNAArtificial
sequenceSynthetic Oligonucleotide 613gttgtcgcag ctgttt
1661416DNAArtificial sequenceSynthetic Oligonucleotide
614tgttgtcgca gctgtt 1661516DNAArtificial sequenceSynthetic
Oligonucleotide 615ttgttgtcgc agctgt 1661616DNAArtificial
sequenceSynthetic Oligonucleotide 616tttgttgtcg cagctg
1661716DNAArtificial sequenceSynthetic Oligonucleotide
617ttttgttgtc gcagct 1661816DNAArtificial sequenceSynthetic
Oligonucleotide 618tttttgttgt cgcagc 1661917DNAArtificial
sequenceSynthetic Oligonucleotide 619gaaaacccaa atcctca
1762017DNAArtificial sequenceSynthetic Oligonucleotide
620agaaaaccca aatcctc 1762117DNAArtificial sequenceSynthetic
Oligonucleotide 621tagaaaaccc aaatcct 1762217DNAArtificial
sequenceSynthetic Oligonucleotide 622atagaaaacc caaatcc
1762317DNAArtificial sequenceSynthetic Oligonucleotide
623ttatagaaaa cccaaat 1762417DNAArtificial sequenceSynthetic
Oligonucleotide 624cttatagaaa acccaaa 1762517DNAArtificial
sequenceSynthetic Oligonucleotide 625ccttatagaa aacccaa
1762617DNAArtificial sequenceSynthetic Oligonucleotide
626cccttataga aaaccca 1762717DNAArtificial sequenceSynthetic
Oligonucleotide 627ccccttatag aaaaccc 1762817DNAArtificial
sequenceSynthetic Oligonucleotide 628accccttata gaaaacc
1762917DNAArtificial sequenceSynthetic Oligonucleotide
629aaccccttat agaaaac 1763017DNAArtificial sequenceSynthetic
Oligonucleotide 630aaacccctta tagaaaa 1763117DNAArtificial
sequenceSynthetic Oligonucleotide 631gaaacccctt atagaaa
1763217DNAArtificial sequenceSynthetic Oligonucleotide
632ggaaacccct tatagaa 1763317DNAArtificial sequenceSynthetic
Oligonucleotide 633aggaaacccc ttataga 1763417DNAArtificial
sequenceSynthetic Oligonucleotide 634caggaaaccc cttatag
1763517DNAArtificial sequenceSynthetic Oligonucleotide
635gcaggaaacc ccttata 1763617DNAArtificial sequenceSynthetic
Oligonucleotide 636agcaggaaac cccttat 1763717DNAArtificial
sequenceSynthetic Oligonucleotide 637cagcaggaaa cccctta
1763817DNAArtificial sequenceSynthetic Oligonucleotide
638ccagcaggaa acccctt 1763917DNAArtificial sequenceSynthetic
Oligonucleotide 639tccagcagga aacccct 1764017DNAArtificial
sequenceSynthetic Oligonucleotide 640gtccagcagg aaacccc
1764117DNAArtificial sequenceSynthetic Oligonucleotide
641tgtccagcag gaaaccc 1764217DNAArtificial sequenceSynthetic
Oligonucleotide 642ctgtccagca ggaaacc 1764317DNAArtificial
sequenceSynthetic Oligonucleotide 643cctgtccagc aggaaac
1764417DNAArtificial sequenceSynthetic Oligonucleotide
644ccctgtccag caggaaa 1764517DNAArtificial sequenceSynthetic
Oligonucleotide 645gcccctgtcc agcagga 1764617DNAArtificial
sequenceSynthetic Oligonucleotide 646cgcccctgtc cagcagg
1764717DNAArtificial sequenceSynthetic Oligonucleotide
647acgcccctgt ccagcag 1764817DNAArtificial sequenceSynthetic
Oligonucleotide 648cacgcccctg tccagca 1764917DNAArtificial
sequenceSynthetic Oligonucleotide 649ccacgcccct gtccagc
1765017DNAArtificial sequenceSynthetic Oligonucleotide
650cccacgcccc tgtccag 1765117DNAArtificial sequenceSynthetic
Oligonucleotide 651tcccacgccc ctgtcca 1765217DNAArtificial
sequenceSynthetic Oligonucleotide 652atcccacgcc
cctgtcc 1765317DNAArtificial sequenceSynthetic Oligonucleotide
653aatcccacgc ccctgtc 1765417DNAArtificial sequenceSynthetic
Oligonucleotide 654caatcccacg cccctgt 1765517DNAArtificial
sequenceSynthetic Oligonucleotide 655tcaatcccac gcccctg
1765617DNAArtificial sequenceSynthetic Oligonucleotide
656ttcaatccca cgcccct 1765717DNAArtificial sequenceSynthetic
Oligonucleotide 657attcaatccc acgcccc 1765817DNAArtificial
sequenceSynthetic Oligonucleotide 658aattcaatcc cacgccc
1765917DNAArtificial sequenceSynthetic Oligonucleotide
659taattcaatc ccacgcc 1766017DNAArtificial sequenceSynthetic
Oligonucleotide 660ttaattcaat cccacgc 1766117DNAArtificial
sequenceSynthetic Oligonucleotide 661tttaattcaa tcccacg
1766217DNAArtificial sequenceSynthetic Oligonucleotide
662ttttaattca atcccac 1766317DNAArtificial sequenceSynthetic
Oligonucleotide 663gttttaattc aatccca 1766417DNAArtificial
sequenceSynthetic Oligonucleotide 664tgttttaatt caatccc
1766517DNAArtificial sequenceSynthetic Oligonucleotide
665ctgttttaat tcaatcc 1766617DNAArtificial sequenceSynthetic
Oligonucleotide 666gctgttttaa ttcaatc 1766717DNAArtificial
sequenceSynthetic Oligonucleotide 667agctgtttta attcaat
1766817DNAArtificial sequenceSynthetic Oligonucleotide
668cagctgtttt aattcaa 1766917DNAArtificial sequenceSynthetic
Oligonucleotide 669gcagctgttt taattca 1767017DNAArtificial
sequen