U.S. patent application number 15/060492 was filed with the patent office on 2017-01-05 for lipid formulated dsrna targeting the pcsk9 gene.
The applicant listed for this patent is Alnylam Pharmaceuticals, Inc.. Invention is credited to Akin Akinc, Kevin Fitzgerald, Gregory Hinkle, Stuart Milstein.
Application Number | 20170000815 15/060492 |
Document ID | / |
Family ID | 43357015 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170000815 |
Kind Code |
A1 |
Fitzgerald; Kevin ; et
al. |
January 5, 2017 |
LIPID FORMULATED DSRNA TARGETING THE PCSK9 GENE
Abstract
This invention relates to composition and methods using lipid
formulated siRNA targeted to a PCSK9 gene.
Inventors: |
Fitzgerald; Kevin;
(Brookline, MA) ; Hinkle; Gregory; (Plymouth,
MA) ; Akinc; Akin; (Needham, MA) ; Milstein;
Stuart; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alnylam Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
43357015 |
Appl. No.: |
15/060492 |
Filed: |
March 3, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14058052 |
Oct 18, 2013 |
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15060492 |
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13568898 |
Aug 7, 2012 |
8598139 |
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14058052 |
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12816207 |
Jun 15, 2010 |
8273869 |
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13568898 |
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61187169 |
Jun 15, 2009 |
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61218350 |
Jun 18, 2009 |
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61244790 |
Sep 22, 2009 |
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61285598 |
Dec 11, 2009 |
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61293474 |
Jan 8, 2010 |
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61313578 |
Mar 12, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61P 3/06 20180101; A61K 47/545 20170801; C12N 2310/3515 20130101;
A61K 31/713 20130101; A61K 47/55 20170801; A61P 35/00 20180101;
C12N 2310/315 20130101; A61K 9/1272 20130101; C12N 15/1137
20130101; C12N 2310/322 20130101; A61K 9/1273 20130101; A61K 9/0019
20130101; A61K 47/6911 20170801; A61P 9/10 20180101; A61P 43/00
20180101; C12N 2310/321 20130101 |
International
Class: |
A61K 31/713 20060101
A61K031/713; A61K 47/48 20060101 A61K047/48; A61K 38/17 20060101
A61K038/17; C12N 15/113 20060101 C12N015/113 |
Claims
1. A composition comprising a nucleic acid lipid particle
comprising a double-stranded ribonucleic acid (dsRNA) for
inhibiting the expression of a human PCSK9 gene in a cell, wherein:
the nucleic acid lipid particle comprises a lipid formulation
comprising 45-65 mol % of a cationic lipid, 5 mol % to about 10 mol
%, of a non-cationic lipid, 25-40 mol % of a sterol, and 0.5-5 mol
% of a PEG or PEG-modified lipid, the dsRNA consists of a sense
strand and an antisense strand, and the sense strand comprises a
first sequence and the antisense strand comprises a second sequence
complementary to at least 15 contiguous nucleotides of a nucleotide
sequence of a target sequence of a dsRNA found in Table 1a, Table
2a, Table 5a, Table 6, Table 7, Table 8, wherein the first sequence
is complementary to the second sequence and wherein the dsRNA is
between 15 and 30 base pairs in length.
2. The composition of claim 1, wherein the cationic lipid comprises
MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate).
3. The composition of claim 2, wherein the cationic lipid comprises
MC3 and the lipid formulation is selected from the group consisting
of: TABLE-US-00025 LNP11 MC3/DSPC/Cholesterol/PEG-DMG
50/10/38.5/1.5 LNP14 MC3/DSPC/Cholesterol/PEG-DMG 40/15/40/5 LNP15
MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 LNP16
MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP17
MC3/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 LNP18
MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP19
MC3/DSPC/Cholesterol/PEG-DMG 50/10/35/5 LNP20
MC3/DSPC/Cholesterol/PEG-DPG 50/10/38.5/1.5
4.-9. (canceled)
10. The composition of claim 1, wherein the sense strand comprises
SEQ ID NO:1227 and the antisense strand comprises SEQ ID
NO:1228.
11. (canceled)
12. (canceled)
13. The composition of claim 1, wherein the dsRNA comprises at
least one modified nucleotide.
14. The composition of claim 13, wherein the modified nucleotide is
chosen from the group of: a 2'-O-methyl modified nucleotide, a
nucleotide comprising a 5'-phosphorothioate group, and a terminal
nucleotide linked to a cholesteryl derivative or dodecanoic acid
bisdecylamide group.
15. The composition of claim 13, wherein the modified nucleotide is
chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide,
a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified
nucleotide, morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide.
16. The composition of claim 1, wherein the dsRNA comprises at
least one 2'-O-methyl modified ribonucleotide and at least one
nucleotide comprising a 5'-phosphorothioate group.
17. The composition of claim 1, wherein each strand of the dsRNA is
19-23 bases in length.
18. The composition of claim 1, wherein each strand of the dsRNA is
21-23 bases in length.
19. The composition of claim 1, wherein each strand of the dsRNA is
21 bases in length.
20. The composition of claim 1, further comprising a
lipoprotein.
21. The composition of claim 1, further comprising apolipoprotein E
(ApoE).
22. The composition of claim 21, wherein the dsRNA is conjugated to
a lipophile.
23. The composition of claim 22, wherein the lipophile is
cholesterol.
24. The composition of claim 21, wherein the ApoE is reconstituted
with at least one amphiphilic agent.
25. The composition of claim 24, wherein the amphiphilic agent is a
phospholipid.
26. The composition of claim 24, wherein the amphilic agent is a
phospholipid selected from the group consisting of dimyristoyl
phosphatidyl choline (DMPC), dioleoylphosphatidylethanolamine
(DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg
phosphatidylcholine (EPC), di stearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG), -phosphatidylethanolamine
(POPE), dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), and
combinations thereof.
27. The composition of claim 25, wherein the ApoE is reconstituted
high density lipoprotein (rHDL).
28.-33. (canceled)
34. A method for inhibiting the expression of PCSK9 in a cell
comprising administering the composition of claim 1 to the
cell.
35.-43. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of pending U.S. patent
application Ser. No. 14/058,052 filed Oct. 18, 2013, which is a
continuation of U.S. patent application Ser. No. 13/568,898, filed
Aug. 7, 2012, now U.S. Pat. No. 8,598,139, issued Dec. 3, 2013,
which is a continuation of U.S. patent application Ser. No.
12/816,207, filed Jun. 15, 2010, now U.S. Pat. No. 8,273,869,
issued Sep. 25, 2012, which claims the benefit of U.S. Provisional
Application Ser. No. 61/187,169, filed Jun. 15, 2009; and U.S.
Provisional Application Ser. No. 61/218,350, filed Jun. 18, 2009;
and U.S. Provisional Application Ser. No. 61/244,790, filed Sep.
22, 2009; and U.S. Provisional Application Ser. No. 61/285,598,
filed Dec. 11, 2009; and U.S. Provisional Application Ser. No.
61/293,474, filed Jan. 8, 2010; and U.S. Provisional Application
Ser. No. 61/313,578, filed Mar. 12, 2010, all of which are
incorporated herein by reference, in their entirety, for all
purposes.
FIELD OF THE INVENTION
[0002] This invention relates to compositions comprising lipid
formulated dsRNA targeting a PCSK9 gene and methods for treating
diseases caused by PCSK9 gene expression.
REFERENCE TO A SEQUENCE LISTING
[0003] This application includes a Sequence Listing submitted
electronically as a text file named 33334_US_sequencelisting.txt,
created on Feb. 29, 2016, with a size of 565,248 bytes, with 1730
sequences. The sequence listing is incorporated by reference.
BACKGROUND OF THE INVENTION
[0004] Proprotein convertase subtilisin kexin 9 (PCSK9) is a member
of the subtilisin serine protease family. The other eight mammalian
subtilisin proteases, PCSK1-PCSK8 (also called PC1/3, PC2, furin,
PC4, PC5/6, PACE4, PC7, and S1P/SKI-1) are proprotein convertases
that process a wide variety of proteins in the secretory pathway
and play roles in diverse biological processes (Bergeron, F. (2000)
J. Mol. Endocrinol. 24, 1-22, Gensberg, K., (1998) Semin. Cell Dev.
Biol. 9, 11-17, Seidah, N. G. (1999) Brain Res. 848, 45-62, Taylor,
N. A., (2003) FASEB J. 17, 1215-1227, and Zhou, A., (1999) J. Biol.
Chem. 274, 20745-20748). PCSK9 has been proposed to play a role in
cholesterol metabolism. PCSK9 mRNA expression is down-regulated by
dietary cholesterol feeding in mice (Maxwell, K. N., (2003) J.
Lipid Res. 44, 2109-2119), up-regulated by statins in HepG2 cells
(Dubuc, G., (2004) Arterioscler. Thromb. Vasc. Biol. 24,
1454-1459), and up-regulated in sterol regulatory element binding
protein (SREBP) transgenic mice (Horton, J. D., (2003) Proc. Natl.
Acad. Sci. USA 100, 12027-12032), similar to the cholesterol
biosynthetic enzymes and the low-density lipoprotein receptor
(LDLR). Furthermore, PCSK9 missense mutations have been found to be
associated with a form of autosomal dominant hypercholesterolemia
(Hchola3) (Abifadel, M., et al. (2003) Nat. Genet. 34, 154-156,
Timms, K. M., (2004) Hum. Genet. 114, 349-353, Leren, T. P. (2004)
Clin. Genet. 65, 419-422). PCSK9 may also play a role in
determining LDL cholesterol levels in the general population,
because single-nucleotide polymorphisms (SNPs) have been associated
with cholesterol levels in a Japanese population (Shioji, K.,
(2004) J. Hum. Genet. 49, 109-114).
[0005] Autosomal dominant hypercholesterolemias (ADHs) are
monogenic diseases in which patients exhibit elevated total and LDL
cholesterol levels, tendon xanthomas, and premature atherosclerosis
(Rader, D. J., (2003) 1 Clin. Invest. 111, 1795-1803). The
pathogenesis of ADHs and a recessive form, autosomal recessive
hypercholesterolemia (ARH) (Cohen, J. C., (2003) Curr. Opin.
Lipidol. 14, 121-127), is due to defects in LDL uptake by the
liver. ADH may be caused by LDLR mutations, which prevent LDL
uptake, or by mutations in the protein on LDL, apolipoprotein B,
which binds to the LDLR. ARH is caused by mutations in the ARH
protein that are necessary for endocytosis of the LDLR-LDL complex
via its interaction with clathrin. Therefore, if PCSK9 mutations
are causative in Hchola3 families, it seems likely that PCSK9 plays
a role in receptor-mediated LDL uptake.
[0006] Overexpression studies point to a role for PCSK9 in
controlling LDLR levels and, hence, LDL uptake by the liver
(Maxwell, K. N. (2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105,
Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875, Park,
S. W., (2004) J. Biol. Chem. 279, 50630-50638). Adenoviral-mediated
overexpression of mouse or human PCSK9 for 3 or 4 days in mice
results in elevated total and LDL cholesterol levels; this effect
is not seen in LDLR knockout animals (Maxwell, K. N. (2004) Proc.
Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004)
J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem.
279, 50630-50638). In addition, PCSK9 overexpression results in a
severe reduction in hepatic LDLR protein, without affecting LDLR
mRNA levels, SREBP protein levels, or SREBP protein nuclear to
cytoplasmic ratio.
[0007] Loss of function mutations in PCSK9 have been designed in
mouse models (Rashid et al., (2005) PNAS, 102, 5374-5379), and
identified in human individuals (Cohen et al. (2005) Nature
Genetics 37:161-165). In both cases loss of PCSK9 function lead to
lowering of total and LDLc cholesterol. In a retrospective outcome
study over 15 years, loss of one copy of PCSK9 was shown to shift
LDLc levels lower and to lead to an increased risk-benefit
protection from developing cardiovascular heart disease (Cohen et
al., (2006)N Engl. J. Med., 354:1264-1272).
[0008] Recently, double-stranded RNA molecules (dsRNA) have been
shown to block gene expression in a highly conserved regulatory
mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et
al.) discloses the use of a dsRNA of at least 25 nucleotides in
length to inhibit the expression of genes in C. elegans. dsRNA has
also been shown to degrade target RNA in other organisms, including
plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631,
Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr.
Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer;
and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has
now become the focus for the development of a new class of
pharmaceutical agents for treating disorders that are caused by the
aberrant or unwanted regulation of a gene.
[0009] A description of siRNA targeting PCSK9 can be found in U.S.
Pat. No. 7,605,251 and WO 2007/134161. Additional disclosure can be
found in U.S. Patent Publication No. 2010/0010066 and WO
2009/134487
SUMMARY OF THE INVENTION
[0010] As described in more detail below, disclosed herein are
compositions comprising lipid formulated siRNA targeting PCSK9,
e.g., MC3 formulated siRNA targeting PCSK9. Also disclosed are
methods of using the compositions for inhibition of PCSK9
expression and for treatment of pathologies related to PCSK9
expression, e.g., hyperlipidemia
[0011] Accordingly, one aspect of the invention is a compositing
comprising a nucleic acid lipid particle comprising a
double-stranded ribonucleic acid (dsRNA) for inhibiting the
expression of a human PCSK9 gene in a cell, wherein the nucleic
acid lipid particle comprises a lipid formulation comprising 45-65
mol % of a cationic lipid, 5 mol % to about 10 mol %, of a
non-cationic lipid, 25-40 mol % of a sterol, and 0.5-5 mol % of a
PEG or PEG-modified lipid, the dsRNA consists of a sense strand and
an antisense strand, and the sense strand comprises a first
sequence and the antisense strand comprises a second sequence
complementary to at least 15 contiguous nucleotides of SEQ ID
NO:1523 (5'-UUCUAGACCUGUUUUGCUU-3'), wherein the first sequence is
complementary to the second sequence and wherein the dsRNA is
between 15 and 30 base pairs in length.
[0012] As described herein the composition includes a cationic
lipid. In one embodiment, the cationic lipid comprises MC3
(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate. For example, the lipid formulation can
be selected from the following:
TABLE-US-00001 LNP11 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5
LNP14 MC3/DSPC/Cholesterol/PEG-DMG 40/15/40/5 LNP15
MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 LNP16
MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP17
MC3/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 LNP18
MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP19
MC3/DSPC/Cholesterol/PEG-DMG 50/10/35/5 LNP20
MC3/DSPC/Cholesterol/PEG-DPG 50/10/38.5/1.5
[0013] In other embodiments, the cationic lipid comprises formula A
wherein formula A is
##STR00001##
[0014] where R1 and R2 are independently alkyl, alkenyl or alkynyl,
each can be optionally substituted, and R3 and R4 are independently
lower alkyl or R3 and R4 can be taken together to form an
optionally substituted heterocyclic ring. In some embodiments the
cationic lipid comprises formula A and is XTC
(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). The lipid
formulation can include the cationic lipid XTC, the non-cationic
lipid DSPC, the sterol cholesterol and the PEG lipid PEG-DMG. In
other embodiments the cationic lipid comprises XTC and the
formulation is selected from the group consisting of:
TABLE-US-00002 LNP05 XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5
LNP06 XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 LNP07
XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, LNP08
XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5 LNP09
XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP13
XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP22
XTC/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5
[0015] In still further embodiments, the cationic lipid comprises
ALNY-100
((3aR,5s,6aS)--N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahyd-
ro-3aH-cyclopenta[d][1,3]dioxol-5-amine)). For example, the
cationic lipid comprises ALNY-100 and the formulation consists of
ALNY-100/DSPC/Cholesterol/PEG-DMG in a ratio of 50/10/38.5/1.5
[0016] The composition includes a dsRNA targeting PCSK9. In some
embodiments, the sense strand comprises SEQ ID NO:1227 and the
antisense strand comprises SEQ ID NO:1228. In other embodiments,
the sense strand consists of SEQ ID NO:1227 and the antisense
strand consists of SEQ ID NO:1228. One or both strands can be
modified, e.g., each strand is modified as follows to include a
2'-O-methyl ribonucleotide as indicated by a lower case letter "c"
or "u" and a phosphorothioate as indicated by a lower case letter
"s": the dsRNA consists of a sense strand consisting of
TABLE-US-00003 SEQ ID NO: 1229 (5'-uucuAGAccuGuuuuGcuuTsT-3')
[0017] and an antisense strand consisting of
TABLE-US-00004 SEQ ID NO: 1230 (5'-AAGcAAAAcAGGUCuAGAATsT-3').
[0018] In other embodiments, the dsRNA comprises at least one
modified nucleotide, e.g., a modified nucleotide chosen from the
group of: a 2'-O-methyl modified nucleotide, a nucleotide
comprising a 5'-phosphorothioate group, and a terminal nucleotide
linked to a cholesteryl derivative or dodecanoic acid bisdecylamide
group, and/or, e.g., the modified nucleotide is chosen from the
group of: a 2'-deoxy-2'-fluoro modified nucleotide, a
2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified
nucleotide, morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide. In one embodiment, dsRNA
comprises at least one 2'-O-methyl modified ribonucleotide and at
least one nucleotide comprising a 5'-phosphorothioate group.
[0019] The compositions include a dsRNA between 15 and 30 base
pairs in length. In one embodiment, each strand of the dsRNA is
19-23 bases in length, or, e.g., 21-23 bases in length, or, e.g. 21
bases in length.
[0020] In one aspect, the compositions include a lipoprotein, e.g.,
apolipoprotein E (ApoE). In some embodiments, the compositions
include a lipoprotein and the dsRNA is conjugated to a lipophile,
e.g., a cholesterol. The ApoE can be reconstituted with at least
one amphiphilic agent, e.g., a phospholipid, e.g., a phospholipid
selected from the group consisting of dimyristoyl phosphatidyl
choline (DMPC), dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG), -phosphatidylethanolamine
(POPE), dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), and
combinations thereof. In some embodiments, the ApoE is
reconstituted high density lipoprotein (rHDL).
[0021] The compositions, e.g., lipid formulated dsRNA targeting
PCSK9, can be administered to a cell or subject, e.g., a primate,
e.g., a human. In one aspect, administration of the compositions
inhibits expression of PCSK9 by at least 40% compared to
administration of a control and/or reduces PCSK9 protein levels in
the mammal compared to administration of a control, and/or reduces
LDLc levels in a mammal compared to administration of a control
and/or reduces both PCSK9 hepatic mRNA and total serum cholesterol
at a dosage of less than 0.1 mg/kg compared to administration of a
control and/or reduces PCSK9 hepatic mRNA at an ED50 of about 0.2
mg/kg and reduces total serum cholesterol with an ED50 of about
0.08 mg/kg compared to administration of a control and/or reduces
serum LDL particle numbers by more than 90% or reduces serum HDL
particle numbers by more than 75% compared to administration of a
control.
[0022] The invention also provides methods comprising administering
the lipid formulated PCSK9 targeted dsRNA compositions described
herein. In one embodiment, the invention includes a method for
inhibiting the expression of PCSK9 in a cell comprising
administering the compositions to the cell. In another embodiment,
the invention includes a method for reducing LDLc levels in a
mammal in need of treatment comprising administering the
compositions to the mammal.
[0023] As described in more detail below, the methods can include
any appropriate dosage, e.g., between 0.25 mg/kg and 4 mg/kg dsRNA,
or e.g., at about 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg/kg dsRNA.
[0024] Also described herein are methods for inhibiting expression
of a PCSK9 gene in a subject, comprising administering to the
subject the lipid formulated PCSK9 targeted dsRNA compositions
described herein at a first dose of about 3 mg/kg followed by
administering at least one subsequent dose once a week, wherein the
subsequent dose is lower than the first dose. The subject can be,
e.g., a rat or a non-human primate or a human. The subsequent dose
can be about 1.0 mg/kg or about 0.3 mg/kg. In some embodiments, the
subsequent dose is administered once a week for four weeks. In some
embodiments, administration of the first dose decreases total
cholesterol levels by about 15-60%.
[0025] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The prefixes "AD-" "DP-" and "AL-DP-" are used
interchangeably e.g., AL-DP-9327 and AD-9237.
[0027] FIG. 1 shows the structure of the ND-98 lipid.
[0028] FIG. 2 shows the results of the in vivo screen of 16 mouse
specific (AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs directed
against different ORF regions of PCSK9 mRNA (having the first
nucleotide corresponding to the ORF position indicated on the
graph) in C57/BL6 mice (5 animals/group). The ratio of PCSK9 mRNA
to GAPDH mRNA in liver lysates was averaged over each treatment
group and compared to a control group treated with PBS or a control
group treated with an unrelated siRNA (blood coagulation factor
VII).
[0029] FIG. 3 shows the results of the in vivo screen of 16
human/mouse/rat cross-reactive (AL-DP-9311 through AL-DP-9326)
PCSK9 siRNAs directed against different ORF regions of PCSK9 mRNA
(having the first nucleotide corresponding to the ORF position
indicated on the graph) in C57/BL6 mice (5 animals/group). The
ratio of PCSK9 mRNA to GAPDH mRNA in liver lysates was averaged
over each treatment group and compared to a control group treated
with PBS or a control group treated with an unrelated siRNA (blood
coagulation factor VII).
[0030] FIG. 4 shows the results of the in vivo screen of 16 mouse
specific (AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs in C57/BL6
mice (5 animals/group). Total serum cholesterol levels were
averaged over each treatment group and compared to a control group
treated with PBS or a control group treated with an unrelated siRNA
(blood coagulation factor VII).
[0031] FIG. 5 shows the results of the in vivo screen of 16
human/mouse/rat cross-reactive (AL-DP-9311 through AL-DP-9326)
PCSK9 siRNAs in C57/BL6 mice (5 animals/group). Total serum
cholesterol levels were averaged over each treatment group and
compared to a control group treated with PBS or a control group
treated with an unrelated siRNA (blood coagulation factor VII).
[0032] FIG. 6A and FIG. 6B compare in vitro and in vivo results,
respectively, for silencing PCSK9.
[0033] FIG. 7A and FIG. 7B are examples of in vitro results for
silencing PCSK9 using monkey primary hepatocytes.
[0034] FIG. 7C shows results for silencing of PCSK9 in monkey
primary hepatocytes using AL-DP-9680 and chemically modified
version of AL-DP-9680.
[0035] FIG. 8 shows in vivo activity of LNP-01 formulated siRNAs to
PCSK-9.
[0036] FIG. 9A and FIG. 9B show in vivo activity of LNP-01
Formulated chemically modified 9314 and derivatives with chemical
modifications such as AD-10792, AD-12382, AD-12384, AD-12341 at
different times post a single dose in mice.
[0037] FIG. 10A shows the effect of PCSK9 siRNAs on PCSK9
transcript levels and total serum cholesterol levels in rats after
a single dose of formulated AD-10792. FIG. 10B shows the effect of
PCSK9 siRNAs on serum total cholesterol levels in the experiment as
FIG. 10A. A single dose of formulated AD-10792 results in an
.about.60% lowering of total cholesterol in the rats that returns
to baseline by .about.3-4 weeks. FIG. 10C shows the effect of PCSK9
siRNAs on hepatic cholesterol and triglyceride levels in the same
experiment as FIG. 10A.
[0038] FIG. 11 is a Western blot showing that liver LDL receptor
levels were upregulated following administration of PCSK9 siRNAs in
rat.
[0039] FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D show the effects
of PCSK9 siRNAs on LDLc and ApoB protein levels, total
cholesterol/HDLc ratios, and PCSK9 protein levels, respectively, in
nonhuman primates following a single dose of formulated AD-10792 or
AD-9680.
[0040] FIG. 13A is a graph showing that unmodified siRNA-AD-A1A
(AD-9314), but not 2'OMe modified siRNA-AD-1A2 (AD-10792), induced
IFN-alpha in human primary blood monocytes. FIG. 13B is a graph
showing that unmodified siRNA-AD-A1A (AD-9314), but not 2'OMe
modified siRNA-AD-1A2 (AD-10792), also induced TNF-alpha in human
primary blood monocytes.
[0041] FIG. 14A is a graph showing that the PCSK9 siRNA
siRNA-AD-1A2 (a.k.a. LNP-PCS-A2 or a.k.a. "formulated AD-10792")
decreased PCSK9 mRNA levels in mice liver in a dose-dependent
manner. FIG. 14B is a graph showing that single administration of 5
mg/kg siRNA-AD-1A2 decreased serum total cholesterol levels in mice
within 48 hours.
[0042] FIG. 15A is a graph showing that PCSK9 siRNAs targeting
human and monkey PCSK9 (LNP-PCS-C2) (a.k.a. "formulated AD-9736"),
and PCSK9 siRNAs targeting mouse PCSK9 (LNP-PCS-A2) (a.k.a.
"formulated AD-10792"), reduced liver PCSK9 levels in transgenic
mice expressing human PCSK9. FIG. 15B is a graph showing that
LNP-PCS-C2 and LNP-PCS-A2 reduced plasma PCSK9 levels in the same
transgenic mice.
[0043] FIG. 16 shows the structure of an siRNA conjugated to
Chol-p-(GalNAc)3 via phosphate linkage at the 3' end.
[0044] FIG. 17 shows the structure of an siRNA conjugated to
LCO(GalNAc)3 (a (GalNAc)3-3'-Lithocholic-oleoyl siRNA
Conjugate).
[0045] FIG. 18 is a graph showing the results of conjugated siRNAs
on PCSK9 transcript levels and total serum cholesterol in mice.
[0046] FIG. 19 is a graph showing the results of lipid formulated
siRNAs on PCSK9 transcript levels and total serum cholesterol in
rats.
[0047] FIG. 20 is a graph showing the results of siRNA transfection
on PCSK9 transcript levels in HeLa cells using AD-9680 and
variations of AD-9680 as described in Table 6.
[0048] FIG. 21 is a graph showing the results of siRNA transfection
on PCSK9 transcript levels in HeLa cells using AD-14676 and
variations of AD-14676 as described in Table 6.
[0049] FIG. 22 is a graph with the results of PCSK9 targeted siRNA
transfection of Hep3B cells and the effects on PCSK9 and off-target
gene levels.
[0050] FIG. 23 shows the results of treatment in rats with a
maintenance dose of PCSK9 targeted siRNA.
[0051] FIG. 24 is a dose response curve of treatment of HeLa cells
with modified siRNAs.
[0052] FIG. 25 is a graph of average IC50 of siRNA vs. target
position in human PCSK9 transcript. The large blue dot indicates
the IC50 and location of AD-9680.
[0053] FIG. 26 is a graph with the results of administration of
rEHDL formulated cholesterol conjugated siRNA.
[0054] FIG. 27A is a graph with results of administration of second
generation LNP formulated PCSK9 targeted siRNA (AD-9680 in LNP11)
to non-human primates, demonstrating a reduction in both PCSK9
protein and LDLc levels. LDLc: low density lipoprotein cholesterol;
mpk: mg per kg.
[0055] FIG. 27B is a bar graph showing dose dependent PCSK mRNA
silencing in non-human primates after treatment with LNP formulated
siRNA targeting PCSK9.
[0056] FIG. 27C is a graph with the results of administration of
second generation LNP formulated PCSK9 targeted siRNA (AD-9680) to
non-human primates, demonstrating a no change in HDLc levels.
[0057] FIG. 28 illustrates the chemical structures of the cationic
lipids MC3 and ALNY-100.
[0058] FIG. 29 is a graph of effects on PCSK9 mRNA and serum
cholesterol levels in rats after administration of LNP-09
formulated AD-10792, an siRNA targeting rodent PCSK9.
[0059] FIG. 30 are graphs of the effects on PCSK9 mRNA and LDL/HDL
particle numbers in CETP/ApoB tg mice after administration of
LNP-09 formulated AD-10792, an siRNA targeting rodent PCSK9.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The invention provides a solution to the problem of treating
diseases that can be modulated by the down regulation of the PCSK9
gene, such as hyperlipidemia, by using double-stranded ribonucleic
acid (dsRNA) to silence the PCSK9 gene.
[0061] The invention provides compositions and methods for
inhibiting the expression of the PCSK9 gene in a subject using a
dsRNA. The invention also provides compositions and methods for
treating pathological conditions and diseases, such as
hyperlipidemia, that can be modulated by down regulating the
expression of the PCSK9 gene. dsRNA directs the sequence-specific
degradation of mRNA through a process known as RNA interference
(RNAi).
[0062] The dsRNA useful for the compositions and methods of an
invention include an RNA strand (the antisense strand) having a
region that is less than 30 nucleotides in length, generally 19-24
nucleotides in length, and is substantially complementary to at
least part of an mRNA transcript of the PCSK9 gene. The use of
these dsRNAs enables the targeted degradation of an mRNA that is
involved in the regulation of the LDL Receptor and circulating
cholesterol levels. Using cell-based and animal assays, the present
inventors have demonstrated that very low dosages of these dsRNAs
can specifically and efficiently mediate RNAi, resulting in
significant inhibition of expression of the PCSK9 gene. Thus,
methods and compositions including these dsRNAs are useful for
treating pathological processes that can be mediated by down
regulating PCSK9, such as in the treatment of hyperlipidemia.
[0063] The following detailed description discloses how to make and
use the dsRNA and compositions containing dsRNA to inhibit the
expression of the target PCSK9 gene, as well as compositions and
methods for treating diseases that can be modulated by down
regulating the expression of PCSK9, such as hyperlipidemia. The
pharmaceutical compositions of the invention include a dsRNA having
an antisense strand having a region of complementarity that is less
than 30 nucleotides in length, generally 19-24 nucleotides in
length, and that is substantially complementary to at least part of
an RNA transcript of the PCSK9 gene, together with a
pharmaceutically acceptable carrier.
[0064] Accordingly, certain aspects of the invention provide
pharmaceutical compositions including the dsRNA that targets PCSK9
together with a pharmaceutically acceptable carrier, methods of
using the compositions to inhibit expression of the PCSK9 gene, and
methods of using the pharmaceutical compositions to treat diseases
by down regulating the expression of PCSK9.
DEFINITIONS
[0065] For convenience, the meaning of certain terms and phrases
used in the specification, examples, and appended claims, are
provided below. If there is an apparent discrepancy between the
usage of a term in other parts of this specification and its
definition provided in this section, the definition in this section
shall prevail.
[0066] "G," "C," "A" and "U" each generally stand for a nucleotide
that contains guanine, cytosine, adenine, and uracil as a base,
respectively. "T" and "dT" are used interchangeably herein and
refer to a deoxyribonucleotide wherein the nucleobase is thymine,
e.g., deoxyribothymine. However, it will be understood that the
term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can
also refer to a modified nucleotide, as further detailed below, or
a surrogate replacement moiety. The skilled person is well aware
that guanine, cytosine, adenine, and uracil may be replaced by
other moieties without substantially altering the base pairing
properties of an oligonucleotide comprising a nucleotide bearing
such replacement moiety. For example, without limitation, a
nucleotide comprising inosine as its base may base pair with
nucleotides containing adenine, cytosine, or uracil. Hence,
nucleotides containing uracil, guanine, or adenine may be replaced
in the nucleotide sequences of the invention by a nucleotide
containing, for example, inosine. Sequences comprising such
replacement moieties are embodiments of the invention.
[0067] As used herein, "PCSK9" refers to the proprotein convertase
subtilisin kexin 9 gene or protein (also known as FH3, HCHOLA3,
NARC-1, NARC1). Examples of mRNA sequences to PCSK9 include but are
not limited to the following: human: NM_174936; mouse: NM_153565,
and rat: NM_199253. Additional examples of PCSK9 mRNA sequences are
readily available using, e.g., GenBank.
[0068] As used herein, "target sequence" refers to a contiguous
portion of the nucleotide sequence of an mRNA molecule formed
during the transcription of the PCSK9 gene, including mRNA that is
a product of RNA processing of a primary transcription product.
[0069] As used herein, the term "strand comprising a sequence"
refers to an oligonucleotide comprising a chain of nucleotides that
is described by the sequence referred to using the standard
nucleotide nomenclature.
[0070] As used herein, and unless otherwise indicated, the term
"complementary," when used to describe a first nucleotide sequence
in relation to a second nucleotide sequence, refers to the ability
of an oligonucleotide or polynucleotide comprising the first
nucleotide sequence to hybridize and form a duplex structure under
certain conditions with an oligonucleotide or polynucleotide
comprising the second nucleotide sequence, as will be understood by
the skilled person. Such conditions can, for example, be stringent
conditions, where stringent conditions may include: 400 mM NaCl, 40
mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for
12-16 hours followed by washing. Other conditions, such as
physiologically relevant conditions as may be encountered inside an
organism, can apply. The skilled person will be able to determine
the set of conditions most appropriate for a test of
complementarity of two sequences in accordance with the ultimate
application of the hybridized nucleotides.
[0071] This includes base-pairing of the oligonucleotide or
polynucleotide having the first nucleotide sequence to the
oligonucleotide or polynucleotide having the second nucleotide
sequence over the entire length of the first and second nucleotide
sequences. Such sequences can be referred to as "fully
complementary" with respect to each other. However, where a first
sequence is referred to as "substantially complementary" with
respect to a second sequence herein, the two sequences can be fully
complementary, or they may form one or more, but generally not more
than 4, 3 or 2 mismatched base pairs upon hybridization, while
retaining the ability to hybridize under the conditions most
relevant to their ultimate application. However, where two
oligonucleotides are designed to form, upon hybridization, one or
more single stranded overhangs, such overhangs shall not be
regarded as mismatches with regard to the determination of
complementarity. For example, a dsRNA having one oligonucleotide 21
nucleotides in length and another oligonucleotide 23 nucleotides in
length, wherein the longer oligonucleotide has a sequence of 21
nucleotides that is fully complementary to the shorter
oligonucleotide, may yet be referred to as "fully
complementary."
[0072] "Complementary" sequences, as used herein, may also include,
or be formed entirely from, non-Watson-Crick base pairs and/or base
pairs formed from non-natural and modified nucleotides, in as far
as the above requirements with respect to their ability to
hybridize are fulfilled. Such non-Watson-Crick base pairs includes,
but not limited to, G:U Wobble or Hoogstein base pairing.
[0073] The terms "complementary", "fully complementary" and
"substantially complementary" herein may be used with respect to
the base matching between the sense strand and the antisense strand
of a dsRNA, or between the antisense strand of a dsRNA and a target
sequence, as will be understood from the context of their use.
[0074] As used herein, a polynucleotide which is "substantially
complementary to at least part of" a messenger RNA (mRNA) refers to
a polynucleotide that is substantially complementary to a
contiguous portion of the mRNA of interest (e.g., encoding PCSK9)
including a 5'' UTR, an open reading frame (ORF), or a 3' UTR. For
example, a polynucleotide is complementary to at least a part of a
PCSK9 mRNA if the sequence is substantially complementary to a
non-interrupted portion of an mRNA encoding PCSK9.
[0075] The term "double-stranded RNA" or "dsRNA", as used herein,
refers a duplex structure comprising two anti-parallel and
substantially complementary, as defined above, nucleic acid
strands. In general, the majority of nucleotides of each strand are
ribonucleotides, but as described in detail herein, each or both
strands can also include at least one non-ribonucleotide, e.g., a
deoxyribonucleotide and/or a modified nucleotide. In addition, as
used in this specification, "dsRNA" may include chemical
modifications to ribonucleotides, including substantial
modifications at multiple nucleotides and including all types of
modifications disclosed herein or known in the art. Any such
modifications, as used in an siRNA type molecule, are encompassed
by "dsRNA" for the purposes of this specification and claims.
[0076] The two strands forming the duplex structure may be
different portions of one larger RNA molecule, or they may be
separate RNA molecules. Where separate RNA molecules, such dsRNA
are often referred to in the literature as siRNA ("short
interfering RNA"). Where the two strands are part of one larger
molecule, and therefore are connected by an uninterrupted chain of
nucleotides between the 3'-end of one strand and the 5'end of the
respective other strand forming the duplex structure, the
connecting RNA chain is referred to as a "hairpin loop", "short
hairpin RNA" or "shRNA". Where the two strands are connected
covalently by means other than an uninterrupted chain of
nucleotides between the 3'-end of one strand and the 5' end of the
respective other strand forming the duplex structure, the
connecting structure is referred to as a "linker". The RNA strands
may have the same or a different number of nucleotides. The maximum
number of base pairs is the number of nucleotides in the shortest
strand of the dsRNA minus any overhangs that are present in the
duplex. In addition to the duplex structure, a dsRNA may comprise
one or more nucleotide overhangs. In general, the majority of
nucleotides of each strand are ribonucleotides, but as described in
detail herein, each or both strands can also include at least one
non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified
nucleotide. In addition, as used in this specification, "dsRNA" may
include chemical modifications to ribonucleotides, including
substantial modifications at multiple nucleotides and including all
types of modifications disclosed herein or known in the art. Any
such modifications, as used in an siRNA type molecule, are
encompassed by "dsRNA" for the purposes of this specification and
claims.
[0077] As used herein, a "nucleotide overhang" refers to the
unpaired nucleotide or nucleotides that protrude from the duplex
structure of a dsRNA when a 3'-end of one strand of the dsRNA
extends beyond the 5'-end of the other strand, or vice versa.
"Blunt" or "blunt end" means that there are no unpaired nucleotides
at that end of the dsRNA, i.e., no nucleotide overhang. A "blunt
ended" dsRNA is a dsRNA that is double-stranded over its entire
length, i.e., no nucleotide overhang at either end of the molecule.
For clarity, chemical caps or non-nucleotide chemical moieties
conjugated to the 3' end or 5' end of an siRNA are not considered
in determining whether an siRNA has an overhang or is blunt
ended.
[0078] The term "antisense strand" refers to the strand of a dsRNA
which includes a region that is substantially complementary to a
target sequence. As used herein, the term "region of
complementarity" refers to the region on the antisense strand that
is substantially complementary to a sequence, for example a target
sequence, as defined herein. Where the region of complementarity is
not fully complementary to the target sequence, the mismatches may
be in the internal or terminal regions of the molecule. Generally
the most tolerated mismatches are in the terminal regions, e.g.,
within 6, 5, 4, 3, or 2 nucleotides of the 5' and/or 3'
terminus.
[0079] The term "sense strand," as used herein, refers to the
strand of a dsRNA that includes a region that is substantially
complementary to a region of the antisense strand.
[0080] "Introducing into a cell", when referring to a dsRNA, means
facilitating uptake or absorption into the cell, as is understood
by those skilled in the art. Absorption or uptake of dsRNA can
occur through unaided diffusive or active cellular processes, or by
auxiliary agents or devices. The meaning of this term is not
limited to cells in vitro; a dsRNA may also be "introduced into a
cell", wherein the cell is part of a living organism. In such
instance, introduction into the cell will include the delivery to
the organism. For example, for in vivo delivery, dsRNA can be
injected into a tissue site or administered systemically. In vitro
introduction into a cell includes methods known in the art such as
electroporation and lipofection.
[0081] The terms "silence," "inhibit the expression of,"
"down-regulate the expression of," "suppress the expression of,"
and the like, in as far as they refer to the PCSK9 gene, herein
refer to the at least partial suppression of the expression of the
PCSK9 gene, as manifested by a reduction of the amount of PCSK9
mRNA which may be isolated from a first cell or group of cells in
which the PCSK9 gene is transcribed and which has or have been
treated such that the expression of the PCSK9 gene is inhibited, as
compared to a second cell or group of cells substantially identical
to the first cell or group of cells but which has or have not been
so treated (control cells). The degree of inhibition is usually
expressed in terms of
( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in
control cells ) 100 % ##EQU00001##
[0082] Alternatively, the degree of inhibition may be given in
terms of a reduction of a parameter that is functionally linked to
PCSK9 gene expression, e.g. the amount of protein encoded by the
PCSK9 gene which is produced by a cell, or the number of cells
displaying a certain phenotype. In principle, target gene silencing
can be determined in any cell expressing the target, either
constitutively or by genomic engineering, and by any appropriate
assay. However, when a reference is needed in order to determine
whether a given dsRNA inhibits the expression of the PCSK9 gene by
a certain degree and therefore is encompassed by the instant
invention, the assays provided in the Examples below shall serve as
such reference.
[0083] As used herein in the context of PCSK9 expression, the terms
"treat", "treatment", and the like, refer to relief from or
alleviation of pathological processes which can be mediated by down
regulating the PCSK9 gene. In the context of the present invention
insofar as it relates to any of the other conditions recited herein
below (other than pathological processes which can be mediated by
down regulating the PCSK9 gene), the terms "treat", "treatment",
and the like mean to relieve or alleviate at least one symptom
associated with such condition, or to slow or reverse the
progression of such condition. For example, in the context of
hyperlipidemia, treatment will involve a decrease in serum lipid
levels.
[0084] As used herein, the phrases "therapeutically effective
amount" and "prophylactically effective amount" refer to an amount
that provides a therapeutic benefit in the treatment, prevention,
or management of pathological processes that can be mediated by
down regulating the PCSK9 gene or an overt symptom of pathological
processes which can be mediated by down regulating the PCSK9 gene.
The specific amount that is therapeutically effective can be
readily determined by an ordinary medical practitioner, and may
vary depending on factors known in the art, such as, e.g., the type
of pathological processes that can be mediated by down regulating
the PCSK9 gene, the patient's history and age, the stage of
pathological processes that can be mediated by down regulating
PCSK9 gene expression, and the administration of other
anti-pathological processes that can be mediated by down regulating
PCSK9 gene expression.
[0085] As used herein, a "pharmaceutical composition" includes a
pharmacologically effective amount of a dsRNA and a
pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of an
RNA effective to produce the intended pharmacological, therapeutic
or preventive result. For example, if a given clinical treatment is
considered effective when there is at least a 25% reduction in a
measurable parameter associated with a disease or disorder, a
therapeutically effective amount of a drug for the treatment of
that disease or disorder is the amount necessary to effect at least
a 25% reduction in that parameter.
[0086] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent. Such carriers
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof and are
described in more detail below. The term specifically excludes cell
culture medium.
[0087] As used herein, a "transformed cell" is a cell into which a
vector has been introduced from which a dsRNA molecule may be
expressed.
[0088] Double-Stranded Ribonucleic Acid (dsRNA)
[0089] As described in more detail below, the invention provides
methods and composition having double-stranded ribonucleic acid
(dsRNA) molecules for inhibiting the expression of the PCSK9 gene
in a cell or mammal, wherein the dsRNA includes an antisense strand
having a region of complementarity that is complementary to at
least a part of an mRNA formed in the expression of the PCSK9 gene,
and wherein the region of complementarity is less than 30
nucleotides in length, generally 19-24 nucleotides in length. In
some embodiments, the dsRNA, upon contact with a cell expressing
the PCSK9 gene, inhibits the expression of said PCSK9 gene, e.g.,
as measured such as by an assay described herein. For example,
expression of a PCSK9 gene in cell culture, such as in HepB3 cells,
can be assayed by measuring PCSK9 mRNA levels, such as by bDNA or
TaqMan assay, or by measuring protein levels, such as by ELISA
assay. The dsRNA of the invention can further include one or more
single-stranded nucleotide overhangs.
[0090] The dsRNA can be synthesized by standard methods known in
the art as further discussed below, e.g., by use of an automated
DNA synthesizer, such as are commercially available from, for
example, Biosearch, Applied Biosystems, Inc. The dsRNA includes two
nucleic acid strands that are sufficiently complementary to
hybridize to form a duplex structure. One strand of the dsRNA (the
antisense strand) can have a region of complementarity that is
substantially complementary, and generally fully complementary, to
a target sequence, derived from the sequence of an mRNA formed
during the expression of the PCSK9 gene. The other strand (the
sense strand) includes a region that is complementary to the
antisense strand, such that the two strands hybridize and form a
duplex structure when combined under suitable conditions.
Generally, the duplex structure is between 15 and 30, or between 25
and 30, or between 18 and 25, or between 19 and 24, or between 19
and 21, or 19, 20, or 21 base pairs in length. In one embodiment
the duplex is 19 base pairs in length. In another embodiment the
duplex is 21 base pairs in length. When two different siRNAs are
used in combination, the duplex lengths can be identical or can
differ.
[0091] Each strand of the dsRNA of invention is generally between
15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or
25 nucleotides in length. In other embodiments, each is strand is
25-30 nucleotides in length. Each strand of the duplex can be the
same length or of different lengths. When two different siRNAs are
used in combination, the lengths of each strand of each siRNA can
be identical or can differ.
[0092] The dsRNA of the invention can include one or more
single-stranded overhang(s) of one or more nucleotides. In one
embodiment, at least one end of the dsRNA has a single-stranded
nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In
another embodiment, the antisense strand of the dsRNA has 1-10
nucleotides overhangs each at the 3' end and the 5' end over the
sense strand. In further embodiments, the sense strand of the dsRNA
has 1-10 nucleotides overhangs each at the 3' end and the 5' end
over the antisense strand.
[0093] A dsRNAs having at least one nucleotide overhang can have
unexpectedly superior inhibitory properties than the blunt-ended
counterpart. In some embodiments the presence of only one
nucleotide overhang strengthens the interference activity of the
dsRNA, without affecting its overall stability. A dsRNA having only
one overhang has proven particularly stable and effective in vivo,
as well as in a variety of cells, cell culture mediums, blood, and
serum. Generally, the single-stranded overhang is located at the
3'-terminal end of the antisense strand or, alternatively, at the
3'-terminal end of the sense strand. The dsRNA can also have a
blunt end, generally located at the 5'-end of the antisense strand.
Such dsRNAs can have improved stability and inhibitory activity,
thus allowing administration at low dosages, i.e., less than 5
mg/kg body weight of the recipient per day. Generally, the
antisense strand of the dsRNA has a nucleotide overhang at the
3'-end, and the 5'-end is blunt. In another embodiment, one or more
of the nucleotides in the overhang is replaced with a nucleoside
thiophosphate.
[0094] The dsRNA can be synthesized by standard methods known in
the art as further discussed below, e.g., by use of an automated
DNA synthesizer, such as are commercially available from, for
example, Biosearch, Applied Biosystems, Inc. In one embodiment, the
PCSK9 gene is a human PCSK9 gene. In other embodiments, the
antisense strand of the dsRNA includes a first strand selected from
the sense sequences of Table 1a, Table 2a, and Table 5a, and a
second strand selected from the antisense sequences of Table 1a,
Table 2a, and Table 5a. Alternative antisense agents that target
elsewhere in the target sequence provided in Table 1a, Table 2a,
and Table 5a, can readily be determined using the target sequence
and the flanking PCSK9 sequence.
[0095] For example, the dsRNA AD-9680 (from Table 1a) targets the
PCSK 9 gene at 3530-3548; therefore the target sequence is as
follows: 5' UUCUAGACCUGUUUUGCUU 3' (SEQ ID NO:1523). The dsRNA
AD-10792 (from Table 1a) targets the PCSK9 gene at 1091-1109;
therefore the target sequence is as follows: 5' GCCUGGAGUUUAUUCGGAA
3' (SEQ ID NO:1524). Included in the invention are dsRNAs that have
regions of complementarity to SEQ ID NO:1523 and SEQ ID
NO:1524.
[0096] In further embodiments, the dsRNA includes at least one
nucleotide sequence selected from the groups of sequences provided
in Table 1a, Table 2a, and Table 5a. In other embodiments, the
dsRNA includes at least two sequences selected from this group,
where one of the at least two sequences is complementary to another
of the at least two sequences, and one of the at least two
sequences is substantially complementary to a sequence of an mRNA
generated in the expression of the PCSK9 gene. Generally, the dsRNA
includes two oligonucleotides, where one oligonucleotide is
described as the sense strand in Table 1a, Table 2a, and Table 5a
and the second oligonucleotide is described as the antisense strand
in Table 1a, Table 2a, and Table 5a
[0097] The skilled person is well aware that dsRNAs having a duplex
structure of between 20 and 23, but specifically 21, base pairs
have been hailed as particularly effective in inducing RNA
interference (Elbashir et al., EMBO 2001, 20:6877-6888). However,
others have found that shorter or longer dsRNAs can be effective as
well. In the embodiments described above, by virtue of the nature
of the oligonucleotide sequences provided in Table 1a, Table 2a,
and Table 5a, the dsRNAs of the invention can include at least one
strand of a length of minimally 21 nt. It can be reasonably
expected that shorter dsRNAs having one of the sequences of Table
1a, Table 2a, and Table 5a minus only a few nucleotides on one or
both ends may be similarly effective as compared to the dsRNAs
described above. Hence, dsRNAs having a partial sequence of at
least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from
one of the sequences of Table 1a, Table 2a, and Table 5a, and
differing in their ability to inhibit the expression of the PCSK9
gene in a FACS assay as described herein below by not more than 5,
10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full
sequence, are contemplated by the invention. Further dsRNAs that
cleave within the target sequence provided in Table 1a, Table 2a,
and Table 5a can readily be made using the PCSK9 sequence and the
target sequence provided.
[0098] In addition, the RNAi agents provided in Table 1a, Table 2a,
and Table 5a identify a site in the PCSK9 mRNA that is susceptible
to RNAi based cleavage. As such the present invention further
includes RNAi agents that target within the sequence targeted by
one of the agents of the present invention. As used herein a second
RNAi agent is said to target within the sequence of a first RNAi
agent if the second RNAi agent cleaves the message anywhere within
the mRNA that is complementary to the antisense strand of the first
RNAi agent. Such a second agent will generally consist of at least
15 contiguous nucleotides from one of the sequences provided in
Table 1a, Table 2a, and Table 5a coupled to additional nucleotide
sequences taken from the region contiguous to the selected sequence
in the PCSK9 gene. For example, the last 15 nucleotides of SEQ ID
NO:1 (minus the added AA sequences) combined with the next 6
nucleotides from the target PCSK9 gene produces a single strand
agent of 21 nucleotides that is based on one of the sequences
provided in Table 1a, Table 2a, and Table 5a.
[0099] The dsRNA of the invention can contain one or more
mismatches to the target sequence. In one embodiment, the dsRNA of
the invention contains no more than 1, no more than 2, or no more
than 3 mismatches. In one embodiment, the antisense strand of the
dsRNA contains mismatches to the target sequence, and the area of
mismatch is not located in the center of the region of
complementarity. In another embodiment, the antisense strand of the
dsRNA contains mismatches to the target sequence and the mismatch
is restricted to 5 nucleotides from either end, for example 5, 4,
3, 2, or 1 nucleotide from either the 5' or 3' end of the region of
complementarity. For example, for a 23 nucleotide dsRNA strand
which is complementary to a region of the PCSK9 gene, the dsRNA
does not contain any mismatch within the central 13 nucleotides.
The methods described within the invention can be used to determine
whether a dsRNA containing a mismatch to a target sequence is
effective in inhibiting the expression of the PCSK9 gene.
Consideration of the efficacy of dsRNAs with mismatches in
inhibiting expression of the PCSK9 gene is important, especially if
the particular region of complementarity in the PCSK9 gene is known
to have polymorphic sequence variation within the population.
[0100] In one embodiment, at least one end of the dsRNA has a
single-stranded nucleotide overhang of 1 to 4, generally 1 or 2
nucleotides. dsRNAs having at least one nucleotide overhang have
unexpectedly superior inhibitory properties than their blunt-ended
counterparts. Moreover, the present inventors have discovered that
the presence of only one nucleotide overhang strengthens the
interference activity of the dsRNA, without affecting its overall
stability. dsRNA having only one overhang has proven particularly
stable and effective in vivo, as well as in a variety of cells,
cell culture mediums, blood, and serum. Generally, the
single-stranded overhang is located at the 3'-terminal end of the
antisense strand or, alternatively, at the 3'-terminal end of the
sense strand. The dsRNA may also have a blunt end, generally
located at the 5'-end of the antisense strand. Such dsRNAs have
improved stability and inhibitory activity, thus allowing
administration at low dosages, i.e., less than 5 mg/kg body weight
of the recipient per day. Generally, the antisense strand of the
dsRNA has a nucleotide overhang at the 3'-end, and the 5'-end is
blunt. In another embodiment, one or more of the nucleotides in the
overhang is replaced with a nucleoside thiophosphate.
[0101] Chemical Modifications and Conjugates
[0102] In yet another embodiment, the dsRNA is chemically modified
to enhance stability. The nucleic acids of the invention may be
synthesized and/or modified by methods well established in the art,
such as those described in "Current protocols in nucleic acid
chemistry", Beaucage, S. L. et al. (Edrs.), John Wiley & Sons,
Inc., New York, N.Y., USA, which is hereby incorporated herein by
reference. Chemical modifications may include, but are not limited
to 2' modifications, modifications at other sites of the sugar or
base of an oligonucleotide, introduction of non-natural bases into
the oligonucleotide chain, covalent attachment to a ligand or
chemical moiety, and replacement of internucleotide phosphate
linkages with alternate linkages such as thiophosphates. More than
one such modification may be employed.
[0103] Chemical linking of the two separate dsRNA strands may be
achieved by any of a variety of well-known techniques, for example
by introducing covalent, ionic or hydrogen bonds; hydrophobic
interactions, van der Waals or stacking interactions; by means of
metal-ion coordination, or through use of purine analogues.
Generally, the chemical groups that can be used to modify the dsRNA
include, without limitation, methylene blue; bifunctional groups,
generally bis-(2-chloroethyl)amine;
N-acetyl-N'-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and
psoralen. In one embodiment, the linker is a hexa-ethylene glycol
linker. In this case, the dsRNA are produced by solid phase
synthesis and the hexa-ethylene glycol linker is incorporated
according to standard methods (e.g., Williams, D. J., and K. B.
Hall, Biochem. (1996) 35:14665-14670). In a particular embodiment,
the 5'-end of the antisense strand and the 3'-end of the sense
strand are chemically linked via a hexaethylene glycol linker. In
another embodiment, at least one nucleotide of the dsRNA comprises
a phosphorothioate or phosphorodithioate groups. The chemical bond
at the ends of the dsRNA is generally formed by triple-helix bonds.
Table 1a, Table 2a, and Table 5a provides examples of modified RNAi
agents of the invention.
[0104] In yet another embodiment, the nucleotides at one or both of
the two single strands may be modified to prevent or inhibit the
degradation activities of cellular enzymes, such as, for example,
without limitation, certain nucleases. Techniques for inhibiting
the degradation activity of cellular enzymes against nucleic acids
are known in the art including, but not limited to, 2'-amino
modifications, 2'-amino sugar modifications, 2'-F sugar
modifications, 2'-F modifications, 2'-alkyl sugar modifications,
uncharged backbone modifications, morpholino modifications,
2'-O-methyl modifications, and phosphoramidate (see, e.g., Wagner,
Nat. Med. (1995) 1:1116-8). Thus, at least one 2'-hydroxyl group of
the nucleotides on a dsRNA is replaced by a chemical group,
generally by a 2'-amino or a 2'-methyl group. Also, at least one
nucleotide may be modified to form a locked nucleotide. Such locked
nucleotide contains a methylene bridge that connects the 2'-oxygen
of ribose with the 4'-carbon of ribose. Oligonucleotides containing
the locked nucleotide are described in Koshkin, A. A., et al.,
Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al.,
Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a locked
nucleotide into an oligonucleotide improves the affinity for
complementary sequences and increases the melting temperature by
several degrees (Braasch, D. A. and D. R. Corey, Chem. Biol.
(2001), 8:1-7).
[0105] Conjugating a ligand to a dsRNA can enhance its cellular
absorption as well as targeting to a particular tissue or uptake by
specific types of cells such as liver cells. In certain instances,
a hydrophobic ligand is conjugated to the dsRNA to facilitate
direct permeation of the cellular membrane and or uptake across the
liver cells. Alternatively, the ligand conjugated to the dsRNA is a
substrate for receptor-mediated endocytosis. These approaches have
been used to facilitate cell permeation of antisense
oligonucleotides as well as dsRNA agents. For example, cholesterol
has been conjugated to various antisense oligonucleotides resulting
in compounds that are substantially more active compared to their
non-conjugated analogs. See M. Manoharan Antisense & Nucleic
Acid Drug Development 2002, 12, 103. Other lipophilic compounds
that have been conjugated to oligonucleotides include 1-pyrene
butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One
example of a ligand for receptor-mediated endocytosis is folic
acid. Folic acid enters the cell by folate-receptor-mediated
endocytosis. dsRNA compounds bearing folic acid would be
efficiently transported into the cell via the
folate-receptor-mediated endocytosis. Li and coworkers report that
attachment of folic acid to the 3'-terminus of an oligonucleotide
resulted in an 8-fold increase in cellular uptake of the
oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res.
1998, 15, 1540. Other ligands that have been conjugated to
oligonucleotides include polyethylene glycols, carbohydrate
clusters, cross-linking agents, porphyrin conjugates, delivery
peptides and lipids such as cholesterol and cholesterylamine.
Examples of carbohydrate clusters include Chol-p-(GalNAc).sub.3
(N-acetyl galactosamine cholesterol) and LCO(GalNAc).sub.3
(N-acetyl galactosamine-3'-Lithocholic-oleoyl.
[0106] In certain instances, conjugation of a cationic ligand to
oligonucleotides results in improved resistance to nucleases.
Representative examples of cationic ligands are propylammonium and
dimethylpropylammonium. Interestingly, antisense oligonucleotides
were reported to retain their high binding affinity to mRNA when
the cationic ligand was dispersed throughout the oligonucleotide.
See M. Manoharan Antisense & Nucleic Acid Drug Development
2002, 12, 103 and references therein.
[0107] In some cases, a ligand can be multifunctional and/or a
dsRNA can be conjugated to more than one ligand. For example, the
dsRNA can be conjugated to one ligand for improved uptake and to a
second ligand for improved release.
[0108] The ligand-conjugated dsRNA of the invention may be
synthesized by the use of a dsRNA that bears a pendant reactive
functionality, such as that derived from the attachment of a
linking molecule onto the dsRNA. This reactive oligonucleotide may
be reacted directly with commercially-available ligands, ligands
that are synthesized bearing any of a variety of protecting groups,
or ligands that have a linking moiety attached thereto. The methods
of the invention facilitate the synthesis of ligand-conjugated
dsRNA by the use of, in some embodiments, nucleoside monomers that
have been appropriately conjugated with ligands and that may
further be attached to a solid-support material. Such
ligand-nucleoside conjugates, optionally attached to a
solid-support material, are prepared according to certain
embodiments of the methods described herein via reaction of a
selected serum-binding ligand with a linking moiety located on the
5' position of a nucleoside or oligonucleotide. In certain
instances, a dsRNA bearing an aralkyl ligand attached to the
3'-terminus of the dsRNA is prepared by first covalently attaching
a monomer building block to a controlled-pore-glass support via a
long-chain aminoalkyl group. Then, nucleotides are bonded via
standard solid-phase synthesis techniques to the monomer
building-block bound to the solid support. The monomer building
block may be a nucleoside or other organic compound that is
compatible with solid-phase synthesis.
[0109] The dsRNA used in the conjugates of the invention may be
conveniently and routinely made through the well-known technique of
solid-phase synthesis. Equipment for such synthesis is sold by
several vendors including, for example, Applied Biosystems (Foster
City, Calif.). Any other means for such synthesis known in the art
may additionally or alternatively be employed. It is also known to
use similar techniques to prepare other oligonucleotides, such as
the phosphorothioates and alkylated derivatives.
[0110] Synthesis
[0111] Teachings regarding the synthesis of particular modified
oligonucleotides may be found in the following U.S. patents: U.S.
Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated
oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for
the preparation of oligonucleotides having chiral phosphorus
linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to
oligonucleotides having modified backbones; U.S. Pat. No.
5,386,023, drawn to backbone-modified oligonucleotides and the
preparation thereof through reductive coupling; U.S. Pat. No.
5,457,191, drawn to modified nucleobases based on the 3-deazapurine
ring system and methods of synthesis thereof; U.S. Pat. No.
5,459,255, drawn to modified nucleobases based on N-2 substituted
purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing
oligonucleotides having chiral phosphorus linkages; U.S. Pat. No.
5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,
drawn to oligonucleotides having .beta.-lactam backbones; U.S. Pat.
No. 5,571,902, drawn to methods and materials for the synthesis of
oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides
having alkylthio groups, wherein such groups may be used as linkers
to other moieties attached at any of a variety of positions of the
nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to
oligonucleotides having phosphorothioate linkages of high chiral
purity; U.S. Pat. No. 5,506,351, drawn to processes for the
preparation of 2'-O-alkyl guanosine and related compounds,
including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469,
drawn to oligonucleotides having N-2 substituted purines; U.S. Pat.
No. 5,587,470, drawn to oligonucleotides having 3-deazapurines;
U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to
conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos.
5,602,240, and 5,610,289, drawn to backbone-modified
oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255,
drawn to, inter alia, methods of synthesizing
2'-fluoro-oligonucleotides.
[0112] In the ligand-conjugated dsRNA and ligand-molecule bearing
sequence-specific linked nucleosides of the invention, the
oligonucleotides and oligonucleosides may be assembled on a
suitable DNA synthesizer utilizing standard nucleotide or
nucleoside precursors, or nucleotide or nucleoside conjugate
precursors that already bear the linking moiety, ligand-nucleotide
or nucleoside-conjugate precursors that already bear the ligand
molecule, or non-nucleoside ligand-bearing building blocks.
[0113] When using nucleotide-conjugate precursors that already bear
a linking moiety, the synthesis of the sequence-specific linked
nucleosides is typically completed, and the ligand molecule is then
reacted with the linking moiety to form the ligand-conjugated
oligonucleotide. Oligonucleotide conjugates bearing a variety of
molecules such as steroids, vitamins, lipids and reporter
molecules, has previously been described (see Manoharan et al., PCT
Application WO 93/07883). In one embodiment, the oligonucleotides
or linked nucleosides featured in the invention are synthesized by
an automated synthesizer using phosphoramidites derived from
ligand-nucleoside conjugates in addition to the standard
phosphoramidites and non-standard phosphoramidites that are
commercially available and routinely used in oligonucleotide
synthesis.
[0114] The incorporation of a 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl,
2'-O-allyl, 2'-O-aminoalkyl or 2'-deoxy-2'-fluoro group in
nucleosides of an oligonucleotide confers enhanced hybridization
properties to the oligonucleotide. Further, oligonucleotides
containing phosphorothioate backbones have enhanced nuclease
stability. Thus, functionalized, linked nucleosides of the
invention can be augmented to include either or both a
phosphorothioate backbone or a 2'-O-methyl, 2'-O-ethyl,
2'-O-propyl, 2'-O-aminoalkyl, 2'-O-allyl or 2'-deoxy-2'-fluoro
group. A summary listing of some of the oligonucleotide
modifications known in the art is found at, for example, PCT
Publication WO 200370918.
[0115] In some embodiments, functionalized nucleoside sequences of
the invention possessing an amino group at the 5'-terminus are
prepared using a DNA synthesizer, and then reacted with an active
ester derivative of a selected ligand. Active ester derivatives are
well known to those skilled in the art. Representative active
esters include N-hydrosuccinimide esters, tetrafluorophenolic
esters, pentafluorophenolic esters and pentachlorophenolic esters.
The reaction of the amino group and the active ester produces an
oligonucleotide in which the selected ligand is attached to the
5'-position through a linking group. The amino group at the
5'-terminus can be prepared utilizing a 5'-Amino-Modifier C6
reagent. In one embodiment, ligand molecules may be conjugated to
oligonucleotides at the 5'-position by the use of a
ligand-nucleoside phosphoramidite wherein the ligand is linked to
the 5'-hydroxy group directly or indirectly via a linker. Such
ligand-nucleoside phosphoramidites are typically used at the end of
an automated synthesis procedure to provide a ligand-conjugated
oligonucleotide bearing the ligand at the 5'-terminus.
[0116] Examples of modified internucleoside linkages or backbones
include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free-acid forms are also
included.
[0117] Representative United States patents relating to the
preparation of the above phosphorus-atom-containing linkages
include, but are not limited to, U.S. Pat. Nos. 3,687,808;
4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;
5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;
5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050;
and 5,697,248, each of which is herein incorporated by
reference.
[0118] Examples of modified internucleoside linkages or backbones
that do not include a phosphorus atom therein (i.e.,
oligonucleosides) have backbones that are formed by short chain
alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl
or cycloalkyl intersugar linkages, or one or more short chain
heteroatomic or heterocyclic intersugar linkages. These include
those having morpholino linkages (formed in part from the sugar
portion of a nucleoside); siloxane backbones; sulfide, sulfoxide
and sulfone backbones;
[0119] formacetyl and thioformacetyl backbones; methylene
formacetyl and thioformacetyl backbones; alkene containing
backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH.sub.2
component parts.
[0120] Representative United States patents relating to the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, each of which is herein incorporated by reference.
[0121] In certain instances, the oligonucleotide may be modified by
a non-ligand group. A number of non-ligand molecules have been
conjugated to oligonucleotides in order to enhance the activity,
cellular distribution or cellular uptake of the oligonucleotide,
and procedures for performing such conjugations are available in
the scientific literature. Such non-ligand moieties have included
lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl.
Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al.,
Bioorg. Med. Chem. Left., 1994, 4:1053), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765),
a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,
20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues
(Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al.,
FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993,
75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al.,
Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene
glycol chain (Manoharan et al., Nucleosides & Nucleotides,
1995, 14:969), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et
al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States
patents that teach the preparation of such oligonucleotide
conjugates have been listed above. Typical conjugation protocols
involve the synthesis of oligonucleotides bearing an aminolinker at
one or more positions of the sequence. The amino group is then
reacted with the molecule being conjugated using appropriate
coupling or activating reagents. The conjugation reaction may be
performed either with the oligonucleotide still bound to the solid
support or following cleavage of the oligonucleotide in solution
phase. Purification of the oligonucleotide conjugate by HPLC
typically affords the pure conjugate. The use of a cholesterol
conjugate is particularly preferred since such a moiety can
increase targeting liver cells, a site of PCSK9 expression.
[0122] Vector Encoded RNAi Agents
[0123] In another aspect of the invention, PCSK9 specific dsRNA
molecules that modulate PCSK9 gene expression activity are
expressed from transcription units inserted into DNA or RNA vectors
(see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A.,
et al., International PCT Publication No. WO 00/22113, Conrad,
International PCT Publication No. WO 00/22114, and Conrad, U.S.
Pat. No. 6,054,299). These transgenes can be introduced as a linear
construct, a circular plasmid, or a viral vector, which can be
incorporated and inherited as a transgene integrated into the host
genome. The transgene can also be constructed to permit it to be
inherited as an extrachromosomal plasmid (Gassmann, et al., Proc.
Natl. Acad. Sci. USA (1995) 92:1292).
[0124] The individual strands of a dsRNA can be transcribed by
promoters on two separate expression vectors and co-transfected
into a target cell. Alternatively each individual strand of the
dsRNA can be transcribed by promoters both of which are located on
the same expression plasmid. In one embodiment, a dsRNA is
expressed as an inverted repeat joined by a linker polynucleotide
sequence such that the dsRNA has a stem and loop structure.
[0125] The recombinant dsRNA expression vectors are generally DNA
plasmids or viral vectors. dsRNA expressing viral vectors can be
constructed based on, but not limited to, adeno-associated virus
(for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol.
(1992) 158:97-129)); adenovirus (see, for example, Berkner, et al.,
BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science
252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or
alphavirus as well as others known in the art. Retroviruses have
been used to introduce a variety of genes into many different cell
types, including epithelial cells, in vitro and/or in vivo (see,
e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and
Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et
al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et
al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al.,
1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991,
Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991,
Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad.
Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy
3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA
89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S.
Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO
89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345;
and PCT Application WO 92/07573). Recombinant retroviral vectors
capable of transducing and expressing genes inserted into the
genome of a cell can be produced by transfecting the recombinant
retroviral genome into suitable packaging cell lines such as PA317
and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone
et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant
adenoviral vectors can be used to infect a wide variety of cells
and tissues in susceptible hosts (e.g., rat, hamster, dog, and
chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and
also have the advantage of not requiring mitotically active cells
for infection.
[0126] Any viral vector capable of accepting the coding sequences
for the dsRNA molecule(s) to be expressed can be used, for example
vectors derived from adenovirus (AV); adeno-associated virus (AAV);
retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine
leukemia virus); herpes virus, and the like. The tropism of viral
vectors can be modified by pseudotyping the vectors with envelope
proteins or other surface antigens from other viruses, or by
substituting different viral capsid proteins, as appropriate.
[0127] For example, lentiviral vectors of the invention can be
pseudotyped with surface proteins from vesicular stomatitis virus
(VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the
invention can be made to target different cells by engineering the
vectors to express different capsid protein serotypes. For example,
an AAV vector expressing a serotype 2 capsid on a serotype 2 genome
is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2
vector can be replaced by a serotype 5 capsid gene to produce an
AAV 2/5 vector. Techniques for constructing AAV vectors which
express different capsid protein serotypes are within the skill in
the art; see, e.g., Rabinowitz J E et al. (2002), J. Virol
76:791-801, the entire disclosure of which is herein incorporated
by reference.
[0128] Selection of recombinant viral vectors suitable for use in
the invention, methods for inserting nucleic acid sequences for
expressing the dsRNA into the vector, and methods of delivering the
viral vector to the cells of interest are within the skill in the
art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310;
Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990),
Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30;
and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire
disclosures of which are herein incorporated by reference.
[0129] Preferred viral vectors are those derived from AV and AAV.
In a particularly preferred embodiment, the dsRNA of the invention
is expressed as two separate, complementary single-stranded RNA
molecules from a recombinant AAV vector having, for example, either
the U6 or H1 RNA promoters, or the cytomegalovirus (CMV)
promoter.
[0130] A suitable AV vector for expressing the dsRNA of the
invention, a method for constructing the recombinant AV vector, and
a method for delivering the vector into target cells, are described
in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
[0131] Suitable AAV vectors for expressing the dsRNA of the
invention, methods for constructing the recombinant AV vector, and
methods for delivering the vectors into target cells are described
in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et
al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J.
Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No.
5,139,941; International Patent Application No. WO 94/13788; and
International Patent Application No. WO 93/24641, the entire
disclosures of which are herein incorporated by reference.
[0132] The promoter driving dsRNA expression in either a DNA
plasmid or viral vector of the invention may be a eukaryotic RNA
polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g.
CMV early promoter or actin promoter or U1 snRNA promoter) or
generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA
promoter) or a prokaryotic promoter, for example the T7 promoter,
provided the expression plasmid also encodes T7 RNA polymerase
required for transcription from a T7 promoter. The promoter can
also direct transgene expression to the pancreas (see, e.g., the
insulin regulatory sequence for pancreas (Bucchini et al., 1986,
Proc. Natl. Acad. Sci. USA 83:2511-2515)).
[0133] In addition, expression of the transgene can be precisely
regulated, for example, by using an inducible regulatory sequence
and expression systems such as a regulatory sequence that is
sensitive to certain physiological regulators, e.g., circulating
glucose levels, or hormones (Docherty et al., 1994, FASEB J.
8:20-24). Such inducible expression systems, suitable for the
control of transgene expression in cells or in mammals include
regulation by ecdysone, by estrogen, progesterone, tetracycline,
chemical inducers of dimerization, and
isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in
the art would be able to choose the appropriate regulatory/promoter
sequence based on the intended use of the dsRNA transgene.
[0134] Generally, recombinant vectors capable of expressing dsRNA
molecules are delivered as described below, and persist in target
cells. Alternatively, viral vectors can be used that provide for
transient expression of dsRNA molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the dsRNAs
bind to target RNA and modulate its function or expression.
Delivery of dsRNA expressing vectors can be systemic, such as by
intravenous or intramuscular administration, by administration to
target cells ex-planted from the patient followed by reintroduction
into the patient, or by any other means that allows for
introduction into a desired target cell.
[0135] dsRNA expression DNA plasmids are typically transfected into
target cells as a complex with cationic lipid carriers (e.g.
Oligofectamine) or non-cationic lipid-based carriers (e.g.
Transit-TKO.TM.). Multiple lipid transfections for dsRNA-mediated
knockdowns targeting different regions of a single PCSK9 gene or
multiple PCSK9 genes over a period of a week or more are also
contemplated by the invention. Successful introduction of the
vectors of the invention into host cells can be monitored using
various known methods. For example, transient transfection. can be
signaled with a reporter, such as a fluorescent marker, such as
Green Fluorescent Protein (GFP). Stable transfection of ex vivo
cells can be ensured using markers that provide the transfected
cell with resistance to specific environmental factors (e.g.,
antibiotics and drugs), such as hygromycin B resistance.
[0136] The PCSK9 specific dsRNA molecules can also be inserted into
vectors and used as gene therapy vectors for human patients. Gene
therapy vectors can be delivered to a subject by, for example,
intravenous injection, local administration (see U.S. Pat. No.
5,328,470) or by stereotactic injection (see e.g., Chen et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can include a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
[0137] Pharmaceutical Compositions Containing dsRNA
[0138] In one embodiment, the invention provides pharmaceutical
compositions containing a dsRNA, as described herein, and a
pharmaceutically acceptable carrier and methods of administering
the same. The pharmaceutical composition containing the dsRNA is
useful for treating a disease or disorder associated with the
expression or activity of a PCSK9 gene, such as pathological
processes mediated by PCSK9 expression, e.g., hyperlipidemia. Such
pharmaceutical compositions are formulated based on the mode of
delivery.
[0139] Dosage
[0140] The pharmaceutical compositions featured herein are
administered in dosages sufficient to inhibit expression of PCSK9
genes. In general, a suitable dose of dsRNA will be in the range of
0.01 to 200.0 milligrams per kilogram body weight of the recipient
per day, generally in the range of 1 to 50 mg per kilogram body
weight per day. For example, the dsRNA can be administered at 0.01
mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 3.0
mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50
mg/kg per single dose.
[0141] In another embodiment, the dosage is between 0.01 and 0.2
mg/kg. For example, the dsRNA can be administered at a dose of 0.01
mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg,
0.07 mg/kg 0.08 mg/kg 0.09 mg/kg, 0.10 mg/kg, 0.11 mg/kg, 0.12
mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg,
0.18 mg/kg, 0.19 mg/kg, or 0.20 mg/kg.
[0142] In one embodiment, the dosage is between 0.2 mg/kg and 1.5
mg/kg. For example, the dsRNA can be administered at a dose of 0.2
mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8
mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4
mg/kg, or 1.5 mg/kg.
[0143] The dsRNA can be administered at a dose of 0.03, 0.1, 0.3,
or 1.3, or 3.0 mg/kg.
[0144] The pharmaceutical composition can be administered once
daily, or the dsRNA may be administered as two, three, or more
sub-doses at appropriate intervals throughout the day. The effect
of a single dose on PCSK9 levels is long lasting, such that
subsequent doses are administered at not more than 7 day intervals,
or at not more than 1, 2, 3, or 4 week intervals.
[0145] In one embodiment the lipid formulated PCSK9 targeted dsRNA
is administered at a first dose of about 3 mg/kg followed by
administering at least one subsequent dose once a week, wherein the
subsequent dose is lower than the first dose, e.g., the subsequent
dose is about 1.0 mg/kg or about 0.3 mg/kg. The subsequent dose can
be administered, e.g., once a week for four weeks.
[0146] In some embodiments the dsRNA is administered using
continuous infusion or delivery through a controlled release
formulation. In that case, the dsRNA contained in each sub-dose
must be correspondingly smaller in order to achieve the total daily
dosage. The dosage unit can also be compounded for delivery over
several days, e.g., using a conventional sustained release
formulation which provides sustained release of the dsRNA over a
several day period. Sustained release formulations are well known
in the art and are particularly useful for delivery of agents at a
particular site, such as could be used with the agents of the
present invention. In this embodiment, the dosage unit contains a
corresponding multiple of the daily dose.
[0147] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a composition
can include a single treatment or a series of treatments. Estimates
of effective dosages and in vivo half-lives for the individual
dsRNAs encompassed by the invention can be made using conventional
methodologies or on the basis of in vivo testing using an
appropriate animal model, as described elsewhere herein.
[0148] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases, such as
pathological processes mediated by PCSK9 expression. Such models
are used for in vivo testing of dsRNA, as well as for determining a
therapeutically effective dose. A suitable mouse model is, for
example, a mouse containing a plasmid expressing human PCSK9.
Another suitable mouse model is a transgenic mouse carrying a
transgene that expresses human PCSK9.
[0149] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds that exhibit
high therapeutic indices are preferred.
[0150] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of compositions featured in the invention lies
generally within a range of circulating concentrations that include
the ED50 with little or no toxicity. The dosage may vary within
this range depending upon the dosage form employed and the route of
administration utilized. For any compound used in the methods
featured in the invention, the therapeutically effective dose can
be estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range of the compound or, when appropriate, of the
polypeptide product of a target sequence (e.g., achieving a
decreased concentration of the polypeptide) that includes the IC50
(i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0151] In addition to their administration, as discussed above, the
dsRNAs featured in the invention can be administered in combination
with other known agents effective in treatment of pathological
processes mediated by target gene expression. In any event, the
administering physician can adjust the amount and timing of dsRNA
administration on the basis of results observed using standard
measures of efficacy known in the art or described herein.
[0152] Administration
[0153] The pharmaceutical compositions of the present invention may
be administered in a number of ways depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Administration may be topical, pulmonary, e.g., by inhalation or
insufflation of powders or aerosols, including by nebulizer;
intratracheal, intranasal, epidermal and transdermal, and
subdermal, oral or parenteral, e.g., subcutaneous.
[0154] Typically, when treating a mammal with hyperlipidemia, the
dsRNA molecules are administered systemically via parental means.
Parenteral administration includes intravenous, intra-arterial,
subcutaneous, intraperitoneal or intramuscular injection or
infusion; or intracranial, e.g., intraparenchymal, intrathecal or
intraventricular, administration. For example, dsRNAs, conjugated
or unconjugate or formulated with or without liposomes, can be
administered intravenously to a patient. For such, a dsRNA molecule
can be formulated into compositions such as sterile and non-sterile
aqueous solutions, non-aqueous solutions in common solvents such as
alcohols, or solutions in liquid or solid oil bases. Such solutions
also can contain buffers, diluents, and other suitable additives.
For parenteral, intrathecal, or intraventricular administration, a
dsRNA molecule can be formulated into compositions such as sterile
aqueous solutions, which also can contain buffers, diluents, and
other suitable additives (e.g., penetration enhancers, carrier
compounds, and other pharmaceutically acceptable carriers).
Formulations are described in more detail herein.
[0155] The dsRNA can be delivered in a manner to target a
particular tissue, such as the liver (e.g., the hepatocytes of the
liver).
[0156] Formulations
[0157] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0158] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0159] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids. In one aspect are formulations that
target the liver when treating hepatic disorders such as
hyperlipidemia.
[0160] In addition, dsRNA that target the PCSK9 gene can be
formulated into compositions containing the dsRNA admixed,
encapsulated, conjugated, or otherwise associated with other
molecules, molecular structures, or mixtures of nucleic acids. For
example, a composition containing one or more dsRNA agents that
target the PCSK9 gene can contain other therapeutic agents, such as
other cancer therapeutics or one or more dsRNA compounds that
target non-PCSK9 genes.
[0161] Oral, Parenteral, Topical, and Biologic Formulations
[0162] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. In some embodiments, oral formulations are those
in which dsRNAs featured in the invention are administered in
conjunction with one or more penetration enhancers surfactants and
chelators. Suitable surfactants include fatty acids and/or esters
or salts thereof, bile acids and/or salts thereof. Suitable bile
acids/salts include chenodeoxycholic acid (CDCA) and
ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic
acid, deoxycholic acid, glucholic acid, glycholic acid,
glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid,
sodium tauro-24,25-dihydro-fusidate and sodium
glycodihydrofusidate. Suitable fatty acids include arachidonic
acid, undecanoic acid, oleic acid, lauric acid, caprylic acid,
capric acid, myristic acid, palmitic acid, stearic acid, linoleic
acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an
acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or
a pharmaceutically acceptable salt thereof (e.g., sodium). In some
embodiments, combinations of penetration enhancers are used, for
example, fatty acids/salts in combination with bile acids/salts.
One exemplary combination is the sodium salt of lauric acid, capric
acid and UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
dsRNAs featured in the invention may be delivered orally, in
granular form including sprayed dried particles, or complexed to
form micro or nanoparticles. dsRNA complexing agents include
poly-amino acids; polyimines; polyacrylates; polyalkylacrylates,
polyoxethanes, polyalkylcyanoacrylates; cationized gelatins,
albumins, starches, acrylates, polyethyleneglycols (PEG) and
starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,
pollulans, celluloses and starches. Suitable complexing agents
include chitosan, N-trimethylchitosan, poly-L-lysine,
polyhistidine, polyornithine, polyspermines, protamine,
polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE),
polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate),
poly(ethylcyanoacrylate), poly(butylcyanoacrylate),
poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate),
DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide,
DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG). Oral formulations for dsRNAs and their
preparation are described in detail in U.S. Pat. No. 6,887,906,
U.S. Patent Publication. No. 20030027780, and U.S. Pat. No.
6,747,014, each of which is incorporated herein by reference.
[0163] Compositions and formulations for parenteral,
intraparenchymal (into the brain), intrathecal, intraventricular or
intrahepatic administration may include sterile aqueous solutions
which may also contain buffers, diluents and other suitable
additives such as, but not limited to, penetration enhancers,
carrier compounds and other pharmaceutically acceptable carriers or
excipients.
[0164] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Suitable topical formulations include those in which the dsRNAs
featured in the invention are in admixture with a topical delivery
agent such as lipids, liposomes, fatty acids, fatty acid esters,
steroids, chelating agents and surfactants. Suitable lipids and
liposomes include neutral (e.g., dioleoylphosphatidyl DOPE
ethanolamine, dimyristoylphosphatidyl choline DMPC,
distearolyphosphatidyl choline) negative (e.g.,
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,
dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl
ethanolamine DOTMA). dsRNAs featured in the invention may be
encapsulated within liposomes or may form complexes thereto, in
particular to cationic liposomes. Alternatively, dsRNAs may be
complexed to lipids, in particular to cationic lipids. Suitable
fatty acids and esters include but are not limited to arachidonic
acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid,
capric acid, myristic acid, palmitic acid, stearic acid, linoleic
acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an
acylcarnitine, an acylcholine, or a C.sub.1-10 alkyl ester (e.g.,
isopropylmyristate IPM), monoglyceride, diglyceride or
pharmaceutically acceptable salt thereof. Topical formulations are
described in detail in U.S. Pat. No. 6,747,014, which is
incorporated herein by reference. In addition, dsRNA molecules can
be administered to a mammal as biologic or abiologic means as
described in, for example, U.S. Pat. No. 6,271,359. Abiologic
delivery can be accomplished by a variety of methods including,
without limitation, (1) loading liposomes with a dsRNA acid
molecule provided herein and (2) complexing a dsRNA molecule with
lipids or liposomes to form nucleic acid-lipid or nucleic
acid-liposome complexes. The liposome can be composed of cationic
and neutral lipids commonly used to transfect cells in vitro.
Cationic lipids can complex (e.g., charge-associate) with
negatively charged nucleic acids to form liposomes. Examples of
cationic liposomes include, without limitation, lipofectin,
lipofectamine, lipofectace, and DOTAP. Procedures for forming
liposomes are well known in the art. Liposome compositions can be
formed, for example, from phosphatidylcholine, dimyristoyl
phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl
phosphatidylglycerol, or dioleoyl phosphatidylethanolamine.
Numerous lipophilic agents are commercially available, including
Lipofectin.TM. (Invitrogen/Life Technologies, Carlsbad, Calif.) and
Effectene (Qiagen, Valencia, Calif.). In addition, systemic
delivery methods can be optimized using commercially available
cationic lipids such as DDAB or DOTAP, each of which can be mixed
with a neutral lipid such as DOPE or cholesterol. In some cases,
liposomes such as those described by Templeton et al. (Nature
Biotechnology, 15: 647-652 (1997)) can be used. In other
embodiments, polycations such as polyethyleneimine can be used to
achieve delivery in vivo and ex vivo (Boletta et al., J. Am Soc.
Nephrol. 7: 1728 (1996)). Additional information regarding the use
of liposomes to deliver nucleic acids can be found in U.S. Pat. No.
6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al.
2005. Nat Biotechnol. 23(8):1002-7.
[0165] Biologic delivery can be accomplished by a variety of
methods including, without limitation, the use of viral vectors.
For example, viral vectors (e.g., adenovirus and herpes virus
vectors) can be used to deliver dsRNA molecules to liver cells.
Standard molecular biology techniques can be used to introduce one
or more of the dsRNAs provided herein into one of the many
different viral vectors previously developed to deliver nucleic
acid to cells. These resulting viral vectors can be used to deliver
the one or more dsRNAs to cells by, for example, infection.
[0166] Liposomal Formulations
[0167] There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles, such as liposomes, have attracted great
interest because of their specificity and the duration of action
they offer from the standpoint of drug delivery. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0168] Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous
interior. The aqueous portion contains the composition to be
delivered. Cationic liposomes possess the advantage of being able
to fuse to the cell wall. Non-cationic liposomes, although not able
to fuse as efficiently with the cell wall, are taken up by
macrophages in vivo.
[0169] In order to cross intact mammalian skin, lipid vesicles must
pass through a series of fine pores, each with a diameter less than
50 nm, under the influence of a suitable transdermal gradient.
Therefore, it is desirable to use a liposome which is highly
deformable and able to pass through such fine pores.
[0170] Further advantages of liposomes include: liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; and liposomes can protect encapsulated drugs in their
internal compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
[0171] Liposomes are useful for the transfer and delivery of active
ingredients to the site of action. Because the liposomal membrane
is structurally similar to biological membranes, when liposomes are
applied to a tissue, the liposomes start to merge with the cellular
membranes and as the merging of the liposome and cell progresses,
the liposomal contents are emptied into the cell where the active
agent may act.
[0172] Liposomal formulations have been the focus of extensive
investigation as the mode of delivery for many drugs. There is
growing evidence that for topical administration, liposomes present
several advantages over other formulations. Such advantages include
reduced side-effects related to high systemic absorption of the
administered drug, increased accumulation of the administered drug
at the desired target, and the ability to administer a wide variety
of drugs, both hydrophilic and hydrophobic, into the skin.
[0173] Several reports have detailed the ability of liposomes to
deliver agents including high-molecular weight DNA into the skin.
Compounds including analgesics, antibodies, hormones and
high-molecular weight DNAs have been administered to the skin. The
majority of applications resulted in the targeting of the upper
epidermis
[0174] Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. The positively
charged DNA/liposome complex binds to the negatively charged cell
surface and is internalized in an endosome. Due to the acidic pH
within the endosome, the liposomes are ruptured, releasing their
contents into the cell cytoplasm (Wang et al., Biochem. Biophys.
Res. Commun., 1987, 147, 980-985).
[0175] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. Nevertheless, some DNA is entrapped within the
aqueous interior of these liposomes. pH-sensitive liposomes have
been used to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture. Expression of the exogenous gene was
detected in the target cells (Zhou et al., Journal of Controlled
Release, 1992, 19, 269-274).
[0176] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0177] Several studies have assessed the topical delivery of
liposomal drug formulations to the skin. Application of liposomes
containing interferon to guinea pig skin resulted in a reduction of
skin herpes sores while delivery of interferon via other means
(e.g., as a solution or as an emulsion) were ineffective (Weiner et
al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
[0178] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome I (glyceryl
dilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and
Novasome II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al., S. T. P. Pharma. Sci., 1994, 4, 6,
466).
[0179] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.mi, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765).
[0180] Various liposomes comprising one or more glycolipids are
known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci.,
1987, 507, 64) reported the ability of monosialoganglioside
G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to
improve blood half-lives of liposomes. These findings were
expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A.,
1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to
Allen et al., disclose liposomes comprising (1) sphingomyelin and
(2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester.
U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes
comprising sphingomyelin. Liposomes comprising
1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499
(Lim et al.).
[0181] Many liposomes comprising lipids derivatized with one or
more hydrophilic polymers, and methods of preparation thereof, are
known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53,
2778) described liposomes comprising a nonionic detergent,
2C.sub.1215G, that contains a PEG moiety. Illum et al. (FEBS Lett.,
1984, 167, 79) noted that hydrophilic coating of polystyrene
particles with polymeric glycols results in significantly enhanced
blood half-lives. Synthetic phospholipids modified by the
attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)
are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899).
Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments
demonstrating that liposomes comprising phosphatidylethanolamine
(PE) derivatized with PEG or PEG stearate have significant
increases in blood circulation half-lives. Blume et al. (Biochimica
et Biophysica Acta, 1990, 1029, 91) extended such observations to
other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from
the combination of distearoylphosphatidylethanolamine (DSPE) and
PEG. Liposomes having covalently bound PEG moieties on their
external surface are described in European Patent No. EP 0 445 131
B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20
mole percent of PE derivatized with PEG, and methods of use
thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556
and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and
European Patent No. EP 0 496 813 B1). Liposomes comprising a number
of other lipid-polymer conjugates are disclosed in WO 91/05545 and
U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073
(Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids
are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935
(Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.)
describe PEG-containing liposomes that can be further derivatized
with functional moieties on their surfaces.
[0182] A number of liposomes comprising nucleic acids are known in
the art. WO 96/40062 to Thierry et al. discloses methods for
encapsulating high molecular weight nucleic acids in liposomes.
U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded
liposomes and asserts that the contents of such liposomes may
include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes
certain methods of encapsulating oligodeoxynucleotides in
liposomes. WO 97/04787 to Love et al. discloses liposomes
comprising dsRNAs targeted to the raf gene.
[0183] Transfersomes are yet another type of liposomes and are
highly deformable lipid aggregates which are attractive candidates
for drug delivery vehicles. Transfersomes may be described as lipid
droplets which are so highly deformable that they are easily able
to penetrate through pores which are smaller than the droplet.
Transfersomes are adaptable to the environment in which they are
used, e.g., they are self-optimizing (adaptive to the shape of
pores in the skin), self-repairing, frequently reach their targets
without fragmenting, and often self-loading. To make transfersomes,
it is possible to add surface edge-activators, usually surfactants,
to a standard liposomal composition. Transfersomes have been used
to deliver serum albumin to the skin. The transfersome-mediated
delivery of serum albumin has been shown to be as effective as
subcutaneous injection of a solution containing serum albumin.
[0184] Surfactants find wide application in formulations such as
emulsions (including microemulsions) and liposomes. The most common
way of classifying and ranking the properties of the many different
types of surfactants, both natural and synthetic, is by the use of
the hydrophile/lipophile balance (HLB). The nature of the
hydrophilic group (also known as the "head") provides the most
useful means for categorizing the different surfactants used in
formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0185] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical and cosmetic products and are usable
over a wide range of pH values. In general their HLB values range
from 2 to about 18 depending on their structure. Nonionic
surfactants include nonionic esters such as ethylene glycol esters,
propylene glycol esters, glyceryl esters, polyglyceryl esters,
sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
alkanolamides and ethers such as fatty alcohol ethoxylates,
propoxylated alcohols, and ethoxylated/propoxylated block polymers
are also included in this class. The polyoxyethylene surfactants
are the most popular members of the nonionic surfactant class.
[0186] If the surfactant molecule carries a negative charge when it
is dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
[0187] If the surfactant molecule carries a positive charge when it
is dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
[0188] If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0189] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0190] Nucleic Acid Lipid Particles
[0191] In one embodiment, a dsRNA featured in the invention is
fully encapsulated in the lipid formulation, e.g., to form a
nucleic acid-lipid particle, e.g., Nucleic acid-lipid particles
typically contain a cationic lipid, a non-cationic lipid, a sterol,
and a lipid that prevents aggregation of the particle (e.g., a
PEG-lipid conjugate). Nucleic acid-lipid particles are extremely
useful for systemic applications, as they exhibit extended
circulation lifetimes following intravenous (i.v.) injection and
accumulate at distal sites (e.g., sites physically separated from
the administration site). In addition, the nucleic acids when
present in the nucleic acid-lipid particles of the present
invention are resistant in aqueous solution to degradation with a
nuclease. Nucleic acid-lipid particles and their method of
preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567;
5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No.
WO 96/40964.
[0192] Nucleic acid-lipid particles can further include one or more
additional lipids and/or other components such as cholesterol.
Other lipids may be included in the liposome compositions for a
variety of purposes, such as to prevent lipid oxidation or to
attach ligands onto the liposome surface. Any of a number of lipids
may be present, including amphipathic, neutral, cationic, and
anionic lipids. Such lipids can be used alone or in combination.
Specific examples of additional lipid components that may be
present are described herein.
[0193] Additional components that may be present in a nucleic
acid-lipid particle include bilayer stabilizing components such as
polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides,
proteins, detergents, lipid-derivatives, such as PEG coupled to
phosphatidylethanolamine and PEG conjugated to ceramides (see, U.S.
Pat. No. 5,885,613).
[0194] A nucleic acid-lipid particle can include one or more of a
second amino lipid or cationic lipid, a neutral lipid, a sterol,
and a lipid selected to reduce aggregation of lipid particles
during formation, which may result from steric stabilization of
particles which prevents charge-induced aggregation during
formation.
[0195] Nucleic acid-lipid particles include, e.g., a SPLP, pSPLP,
and SNALP. The term "SNALP" refers to a stable nucleic acid-lipid
particle, including SPLP. The term "SPLP" refers to a nucleic
acid-lipid particle comprising plasmid DNA encapsulated within a
lipid vesicle. SPLPs include "pSPLP," which include an encapsulated
condensing agent-nucleic acid complex as set forth in PCT
Publication No. WO 00/03683.
[0196] The particles of the present invention typically have a mean
diameter of about 50 nm to about 150 nm, more typically about 60 nm
to about 130 nm, more typically about 70 nm to about 110 nm, most
typically about 70 nm to about 90 nm, and are substantially
nontoxic
[0197] In one embodiment, the lipid to drug ratio (mass/mass ratio)
(e.g., lipid to dsRNA ratio) will be in the range of from about 1:1
to about 50:1, from about 1:1 to about 25:1, from about 3:1 to
about 15:1, from about 4:1 to about 10:1, from about 5:1 to about
9:1, or about 6:1 to about 9:1, or about 6:1, 7:1, 8:1, 9:1, 10:1,
11:1, 12:1, or 33:1.
[0198] Cationic Lipids
[0199] The nucleic acid-lipid particles of the invention typically
include a cationic lipid. The cationic lipid may be, for example,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N--(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N--(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),
1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),
1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane
(DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA) or analogs thereof,
(3aR,5s,6aS)--N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydr-
o-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALNY-100),
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (MC3), or a mixture thereof.
[0200] Other cationic lipids, which carry a net positive charge at
about physiological pH, in addition to those specifically described
above, may also be included in lipid particles of the invention.
Such cationic lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy)propyl-N,N--N-triethylammonium chloride
("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP"); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt
("DOTAP.Cl");
3.beta.-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Chol"),
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
ammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl
carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine
("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), N,
N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"). Additionally, a number of commercial
preparations of cationic lipids can be used, such as, e.g.,
LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL),
and LIPOFECTAMINE (comprising DOSPA and DOPE, available from
GIBCO/BRL). In particular embodiments, a cationic lipid is an amino
lipid.
[0201] As used herein, the term "amino lipid" is meant to include
those lipids having one or two fatty acid or fatty alkyl chains and
an amino head group (including an alkylamino or dialkylamino group)
that may be protonated to form a cationic lipid at physiological
pH.
[0202] Other amino lipids would include those having alternative
fatty acid groups and other dialkylamino groups, including those in
which the alkyl substituents are different (e.g.,
N-ethyl-N-methylamino-, N-propyl-N-ethylamino- and the like). For
those embodiments in which R.sup.11 and R.sup.12 are both long
chain alkyl or acyl groups, they can be the same or different. In
general, amino lipids having less saturated acyl chains are more
easily sized, particularly when the complexes must be sized below
about 0.3 microns, for purposes of filter sterilization. Amino
lipids containing unsaturated fatty acids with carbon chain lengths
in the range of C.sub.14 to C.sub.22 are preferred. Other scaffolds
can also be used to separate the amino group and the fatty acid or
fatty alkyl portion of the amino lipid. Suitable scaffolds are
known to those of skill in the art.
[0203] In certain embodiments, amino or cationic lipids of the
invention have at least one protonatable or deprotonatable group,
such that the lipid is positively charged at a pH at or below
physiological pH (e.g. pH 7.4), and neutral at a second pH,
preferably at or above physiological pH. It will, of course, be
understood that the addition or removal of protons as a function of
pH is an equilibrium process, and that the reference to a charged
or a neutral lipid refers to the nature of the predominant species
and does not require that all of the lipid be present in the
charged or neutral form. Lipids that have more than one
protonatable or deprotonatable group, or which are zwiterrionic,
are not excluded from use in the invention.
[0204] In certain embodiments, protonatable lipids according to the
invention have a pKa of the protonatable group in the range of
about 4 to about 11. Most preferred is pKa of about 4 to about 7,
because these lipids will be cationic at a lower pH formulation
stage, while particles will be largely (though not completely)
surface neutralized at physiological pH around pH 7.4. One of the
benefits of this pKa is that at least some nucleic acid associated
with the outside surface of the particle will lose its
electrostatic interaction at physiological pH and be removed by
simple dialysis; thus greatly reducing the particle's
susceptibility to clearance.
[0205] One example of a cationic lipid is
1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA). Synthesis
and preparation of nucleic acid-lipid particles including DLinDMA
is described in International application number PCT/CA2009/00496,
filed Apr. 15, 2009.
[0206] In one embodiment, the cationic lipid XTC
(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) is used to
prepare nucleic acid-lipid particles. Synthesis of XTC is described
in U.S. provisional patent application No. 61/107,998 filed on Oct.
23, 2008, which is herein incorporated by reference.
[0207] In one aspect, the cationic lipids have the structure
##STR00002##
[0208] and salts or isomers thereof, wherein R.sub.1 and R.sub.2
are each independently for each occurrence optionally substituted
C.sub.10-C.sub.30 alkyl, optionally substituted C.sub.10-C.sub.30
alkenyl, optionally substituted C.sub.10-C.sub.30 alkynyl,
optionally substituted C.sub.10-C.sub.30 acyl, or -linker-ligand;
R.sub.3 is H, optionally substituted C.sub.1-C.sub.10 alkyl,
optionally substituted C.sub.2-C.sub.10 alkenyl, optionally
substituted C.sub.2-C.sub.10 alkynyl, alkylhetrocycle,
alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,
alkylphosphonates, alkylamines, hydroxyalkyls, .omega.-aminoalkyls,
.omega.-(substituted)aminoalkyls, .omega.-phosphoalkyls,
.omega.-thiophosphoalkyls, optionally substituted polyethylene
glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),
heteroaryl, heterocycle, or linker-ligand; and E is C(O)O or OC(O).
Synthesis and use of this lipid family is described in WO
2010/054401 (PCTUS2009/063927 filed on Nov. 10, 2009. The cationic
lipid MC3 is one embodiment of this family of cationic lipids.
[0209] In another embodiment, the cationic lipid MC3
((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate), (e.g., DLin-M-C3-DMA) is used to
prepare nucleic acid-lipid particles. Synthesis of MC3 and MC3
comprising formulations are described, e.g., in U.S. Provisional
Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser.
No. 61/185,800, filed Jun. 10, 2009, and U.S. patent application
Ser. No. 12/813,448 filed on Jun. 10, 2010, which are hereby
incorporated by reference.
[0210] In another embodiment, the cationic lipid ALNY-100
((3aR,5s,6aS)--N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahyd-
ro-3aH-cyclopenta[d][1,3]dioxol-5-amine) is used to prepare nucleic
acid-lipid particles. Synthesis of ALNY-100 is described in
International patent application number PCT/US09/63933 filed on
Nov. 10, 2009, which is herein incorporated by reference.
[0211] FIG. 28 illustrates the structures of ALNY-100 and MC3.
[0212] The cationic lipid may comprise from about 20 mol % to about
70 mol % or about 45-65 mol % or about 10, 20, 30, 40, 50, 60, or
70 mol % of the total lipid present in the particle.
[0213] Non-Cationic Lipids
[0214] The nucleic acid-lipid particles of the invention can
include a non-cationic lipid. The non-cationic lipid may be an
anionic lipid or a neutral lipid. Examples include but not limited
to, distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or
a mixture thereof.
[0215] Anionic lipids suitable for use in lipid particles of the
invention include, but are not limited to, phosphatidylglycerol,
cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid,
N-dodecanoyl phosphatidylethanoloamine, N-succinyl
phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine,
lysylphosphatidylglycerol, and other anionic modifying groups
joined to neutral lipids.
[0216] Neutral lipids, when present in the lipid particle, can be
any of a number of lipid species which exist either in an uncharged
or neutral zwitterionic form at physiological pH. Such lipids
include, for example diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin,
dihydrosphingomyelin, cephalin, and cerebrosides. The selection of
neutral lipids for use in the particles described herein is
generally guided by consideration of, e.g., liposome size and
stability of the liposomes in the bloodstream. Preferably, the
neutral lipid component is a lipid having two acyl groups, (i.e.,
diacylphosphatidylcholine and diacylphosphatidylethanolamine).
Lipids having a variety of acyl chain groups of varying chain
length and degree of saturation are available or may be isolated or
synthesized by well-known techniques. In one group of embodiments,
lipids containing saturated fatty acids with carbon chain lengths
in the range of C.sub.14 to C.sub.22 are preferred. In another
group of embodiments, lipids with mono- or di-unsaturated fatty
acids with carbon chain lengths in the range of C.sub.14 to
C.sub.22 are used. Additionally, lipids having mixtures of
saturated and unsaturated fatty acid chains can be used.
Preferably, the neutral lipids used in the invention are DOPE,
DSPC, POPC, or any related phosphatidylcholine. The neutral lipids
useful in the invention may also be composed of sphingomyelin,
dihydrosphingomyeline, or phospholipids with other head groups,
such as serine and inositol.
[0217] In one embodiment the non-cationic lipid is
distearoylphosphatidylcholine (DSPC). In another embodiment the
non-cationic lipid is dipalmitoylphosphatidylcholine (DPPC).
[0218] The non-cationic lipid may be from about 5 mol % to about 90
mol %, about 5 mol % to about 10 mol %, about 10 mol %, or about 58
mol % if cholesterol is included, of the total lipid present in the
particle.
[0219] Conjugated Lipids
[0220] Conjugated lipids can be used in nucleic acid-lipid particle
to prevent aggregation, including polyethylene glycol
(PEG)-modified lipids, monosialoganglioside Gml, and polyamide
oligomers ("PAO") such as (described in U.S. Pat. No. 6,320,017).
Other compounds with uncharged, hydrophilic, steric-barrier
moieties, which prevent aggregation during formulation, like PEG,
Gml or ATTA, can also be coupled to lipids for use as in the
methods and compositions of the invention. ATTA-lipids are
described, e.g., in U.S. Pat. No. 6,320,017, and PEG-lipid
conjugates are described, e.g., in U.S. Pat. Nos. 5,820,873,
5,534,499 and 5,885,613. Typically, the concentration of the lipid
component selected to reduce aggregation is about 1 to 15% (by mole
percent of lipids).
[0221] Specific examples of PEG-modified lipids (or
lipid-polyoxyethylene conjugates) that are useful in the invention
can have a variety of "anchoring" lipid portions to secure the PEG
portion to the surface of the lipid vesicle. Examples of suitable
PEG-modified lipids include PEG-modified phosphatidylethanolamine
and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or
PEG-CerC20) which are described in co-pending U.S. Ser. No.
08/486,214, incorporated herein by reference, PEG-modified
dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines.
Particularly preferred are PEG-modified diacylglycerols and
dialkylglycerols.
[0222] In embodiments where a sterically-large moiety such as PEG
or ATTA are conjugated to a lipid anchor, the selection of the
lipid anchor depends on what type of association the conjugate is
to have with the lipid particle. It is well known that mePEG
(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remain
associated with a liposome until the particle is cleared from the
circulation, possibly a matter of days. Other conjugates, such as
PEG-CerC20 have similar staying capacity. PEG-CerC14, however,
rapidly exchanges out of the formulation upon exposure to serum,
with a T.sub.1/2 less than 60 minutes in some assays. As
illustrated in U.S. patent application Ser. No. 08/486,214, at
least three characteristics influence the rate of exchange: length
of acyl chain, saturation of acyl chain, and size of the
steric-barrier head group. Compounds having suitable variations of
these features may be useful for the invention. For some
therapeutic applications, it may be preferable for the PEG-modified
lipid to be rapidly lost from the nucleic acid-lipid particle in
vivo and hence the PEG-modified lipid will possess relatively short
lipid anchors. In other therapeutic applications, it may be
preferable for the nucleic acid-lipid particle to exhibit a longer
plasma circulation lifetime and hence the PEG-modified lipid will
possess relatively longer lipid anchors. Exemplary lipid anchors
include those having lengths of from about C.sub.14 to about
C.sub.22, preferably from about C.sub.14 to about C.sub.16. In some
embodiments, a PEG moiety, for example an mPEG-NH.sub.2, has a size
of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 Daltons.
[0223] It should be noted that aggregation preventing compounds do
not necessarily require lipid conjugation to function properly.
Free PEG or free ATTA in solution may be sufficient to prevent
aggregation. If the particles are stable after formulation, the PEG
or ATTA can be dialyzed away before administration to a
subject.
[0224] The conjugated lipid that inhibits aggregation of particles
may be, for example, a polyethyleneglycol (PEG)-lipid including,
without limitation, a PEG-diacylglycerol (DAG), a
PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide
(Cer), or a mixture thereof. The PEG-DAA conjugate may be, for
example, a PEG-dilauryloxypropyl (Ci.sub.2), a
PEG-dimyristyloxypropyl (Ci.sub.4), a PEG-dipalmityloxypropyl
(Ci.sub.6), or a PEG-distearyloxypropyl (Ci.sub.8). Additional
conjugated lipids include polyethylene glycol-didimyristoyl
glycerol (C14-PEG or PEG-C14, where PEG has an average molecular
weight of 2000 Da) (PEG-DMG);
(R)-2,3-bis(octadecyloxy)propyl1-(methoxy poly(ethylene
glycol)2000)propylcarbamate) (PEG-DSG);
PEG-carbamoyl-1,2-dimyristyloxypropylamine, in which PEG has an
average molecular weight of 2000 Da (PEG-cDMA);
N-Acetylgalactosamine-((R)-2,3-bis(octadecyloxy)propyl1-(methoxy
poly(ethylene glycol)2000)propylcarbamate)) (GalNAc-PEG-DSG); and
polyethylene glycol-dipalmitoylglycerol (PEG-DPG).
[0225] In one embodiment the conjugated lipid is PEG-DMG. In
another embodiment the conjugated lipid is PEG-cDMA. In still
another embodiment the conjugated lipid is PEG-DPG. Alternatively
the conjugated lipid is GalNAc-PEG-DSG.
[0226] The conjugated lipid that prevents aggregation of particles
may be from 0 mol % to about 20 mol % or about 0.5 to about 5.0 mol
% or about 2 mol % of the total lipid present in the particle.
[0227] The sterol component of the lipid mixture, when present, can
be any of those sterols conventionally used in the field of
liposome, lipid vesicle or lipid particle preparation. A preferred
sterol is cholesterol.
[0228] In some embodiments, the nucleic acid-lipid particle further
includes a sterol, e.g., a cholesterol at, e.g., about 10 mol % to
about 60 mol % or about 25 to about 40 mol % or about 48 mol % of
the total lipid present in the particle.
[0229] Lipoproteins
[0230] In one embodiment, the formulations of the invention further
comprise an apolipoprotein. As used herein, the term
"apolipoprotein" or "lipoprotein" refers to apolipoproteins known
to those of skill in the art and variants and fragments thereof and
to apolipoprotein agonists, analogues or fragments thereof
described below.
[0231] Suitable apolipoproteins include, but are not limited to,
ApoA-I, ApoA-II, ApoA-IV, ApoA-V and ApoE, and active polymorphic
forms, isoforms, variants and mutants as well as fragments or
truncated forms thereof. In certain embodiments, the apolipoprotein
is a thiol containing apolipoprotein. "Thiol containing
apolipoprotein" refers to an apolipoprotein, variant, fragment or
isoform that contains at least one cysteine residue. The most
common thiol containing apolipoproteins are ApoA-I Milano
(ApoA-I.sub.M) and ApoA-I Paris (ApoA-Ip) which contain one
cysteine residue (Jia et al., 2002, Biochem. Biophys. Res. Comm.
297: 206-13; Bielicki and Oda, 2002, Biochemistry 41: 2089-96).
ApoA-II, ApoE2 and ApoE3 are also thiol containing apolipoproteins.
Isolated ApoE and/or active fragments and polypeptide analogues
thereof, including recombinantly produced forms thereof, are
described in U.S. Pat. Nos. 5,672,685; 5,525,472; 5,473,039;
5,182,364; 5,177,189; 5,168,045; 5,116,739; the disclosures of
which are herein incorporated by reference. ApoE3 is disclosed in
Weisgraber, et al., "Human E apoprotein heterogeneity:
cysteine-arginine interchanges in the amino acid sequence of the
apo-E isoforms," J. Biol. Chem. (1981) 256: 9077-9083; and Rall, et
al., "Structural basis for receptor binding heterogeneity of
apolipoprotein E from type III hyperlipoproteinemic subjects,"
Proc. Nat. Acad. Sci. (1982) 79: 4696-4700. (See also GenBank
accession number K00396.)
[0232] In certain embodiments, the apolipoprotein can be in its
mature form, in its preproapolipoprotein form or in its
proapolipoprotein form. Homo- and heterodimers (where feasible) of
pro- and mature ApoA-I (Duverger et al., 1996, Arterioscler.
Thromb. Vasc. Biol. 16(12):1424-29), ApoA-I Milano (Klon et al.,
2000, Biophys. J. 79:(3)1679-87; Franceschini et al., 1985, J.
Biol. Chem. 260: 1632-35), ApoA-I Paris (Daum et al., 1999, J. Mol.
Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol. Chem.
260(14):8637-46; Shelness et al., 1984, J. Biol. Chem.
259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem.
201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem.
258(14):8993-9000) can also be utilized within the scope of the
invention.
[0233] In certain embodiments, the apolipoprotein can be a
fragment, variant or isoform of the apolipoprotein. The term
"fragment" refers to any apolipoprotein having an amino acid
sequence shorter than that of a native apolipoprotein and which
fragment retains the activity of native apolipoprotein, including
lipid binding properties. By "variant" is meant substitutions or
alterations in the amino acid sequences of the apolipoprotein,
which substitutions or alterations, e.g., additions and deletions
of amino acid residues, do not abolish the activity of native
apolipoprotein, including lipid binding properties. Thus, a variant
can comprise a protein or peptide having a substantially identical
amino acid sequence to a native apolipoprotein provided herein in
which one or more amino acid residues have been conservatively
substituted with chemically similar amino acids. Examples of
conservative substitutions include the substitution of at least one
hydrophobic residue such as isoleucine, valine, leucine or
methionine for another. Likewise, the present invention
contemplates, for example, the substitution of at least one
hydrophilic residue such as, for example, between arginine and
lysine, between glutamine and asparagine, and between glycine and
serine (see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The
term "isoform" refers to a protein having the same, greater or
partial function and similar, identical or partial sequence, and
may or may not be the product of the same gene and usually tissue
specific (see Weisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson
and Powers 1991, J. Lipid Res. 32(9):1529-35; Lackner et al., 1985,
J. Biol. Chem. 260(2):703-6; Hoeg et al., 1986, J. Biol. Chem.
261(9):3911-4; Gordon et al., 1984, J. Biol. Chem. 259(1):468-74;
Powell et al., 1987, Cell 50(6):831-40; Aviram et al., 1998,
Arterioscler. Thromb. Vase. Biol. 18(10):1617-24; Aviram et al.,
1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, Drug
Metab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol.
Chem. 275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.
260(4):2258-64; Widler et al., 1980, J. Biol. Chem.
255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8;
Sacre et al., 2003, FEBS Lett. 540(1-3):181-7; Weers, et al., 2003,
Biophys. Chem. 100(1-3):481-92; Gong et al., 2002, J. Biol. Chem.
277(33):29919-26; Ohta et al., 1984, J. Biol. Chem.
259(23):14888-93 and U.S. Pat. No. 6,372,886).
[0234] In certain embodiments, the methods and compositions of the
present invention include the use of a chimeric construction of an
apolipoprotein. For example, a chimeric construction of an
apolipoprotein can be comprised of an apolipoprotein domain with
high lipid binding capacity associated with an apolipoprotein
domain containing ischemia reperfusion protective properties. A
chimeric construction of an apolipoprotein can be a construction
that includes separate regions within an apolipoprotein (i.e.,
homologous construction) or a chimeric construction can be a
construction that includes separate regions between different
apolipoproteins (i.e., heterologous constructions). Compositions
comprising a chimeric construction can also include segments that
are apolipoprotein variants or segments designed to have a specific
character (e.g., lipid binding, receptor binding, enzymatic, enzyme
activating, antioxidant or reduction-oxidation property) (see
Weisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers
1991, J. Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol.
Chem. 260(2):703-6; Hoeg et al., 1986, J. Biol. Chem.
261(9):3911-4; Gordon et al., 1984, J. Biol. Chem. 259(1):468-74;
Powell et al., 1987, Cell 50(6):831-40; Aviram et al., 1998,
Arterioscler. Thromb. Vasc. Biol. 18(10):1617-24; Aviram et al.,
1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, Drug
Metab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol.
Chem. 275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.
260(4):2258-64; Widler et al., 1980, J. Biol. Chem.
255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8;
Sorenson et al., 1999, Arterioscler. Thromb. Vasc. Biol.
19(9):2214-25; Palgunachari 1996, Arterioscler. Throb. Vasc. Biol.
16(2):328-38: Thurberg et al., J. Biol. Chem. 271(11):6062-70; Dyer
1991, J. Biol. Chem. 266(23):150009-15; Hill 1998, J. Biol. Chem.
273(47):30979-84).
[0235] Apolipoproteins utilized in the invention also include
recombinant, synthetic, semi-synthetic or purified apolipoproteins.
Methods for obtaining apolipoproteins or equivalents thereof,
utilized by the invention are well-known in the art. For example,
apolipoproteins can be separated from plasma or natural products
by, for example, density gradient centrifugation or immunoaffinity
chromatography, or produced synthetically, semi-synthetically or
using recombinant DNA techniques known to those of the art (see,
e.g., Mulugeta et al., 1998, J. Chromatogr. 798(1-2): 83-90; Chung
et al., 1980, J. Lipid Res. 21(3):284-91; Cheung et al., 1987, J.
Lipid Res. 28(8):913-29; Persson, et al., 1998, J. Chromatogr.
711:97-109; U.S. Pat. Nos. 5,059,528, 5,834,596, 5,876,968 and
5,721,114; and PCT Publications WO 86/04920 and WO 87/02062).
[0236] Apolipoproteins utilized in the invention further include
apolipoprotein agonists such as peptides and peptide analogues that
mimic the activity of ApoA-I, ApoA-I Milano (ApoA-I.sub.M), ApoA-I
Paris (ApoA-I.sub.P), ApoA-II, ApoA-IV, and ApoE. For example, the
apolipoprotein can be any of those described in U.S. Pat. Nos.
6,004,925, 6,037,323, 6,046,166, and 5,840,688, the contents of
which are incorporated herein by reference in their entireties.
[0237] Apolipoprotein agonist peptides or peptide analogues can be
synthesized or manufactured using any technique for peptide
synthesis known in the art including, e.g., the techniques
described in U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166. For
example, the peptides may be prepared using the solid-phase
synthetic technique initially described by Merrifield (1963, J. Am.
Chem. Soc. 85:2149-2154). Other peptide synthesis techniques may be
found in Bodanszky et al., Peptide Synthesis, John Wiley &
Sons, 2d Ed., (1976) and other references readily available to
those skilled in the art. A summary of polypeptide synthesis
techniques can be found in Stuart and Young, Solid Phase Peptide.
Synthesis, Pierce Chemical Company, Rockford, Ill., (1984).
Peptides may also be synthesized by solution methods as described
in The Proteins, Vol. II, 3d Ed., Neurath et al., Eds., p. 105-237,
Academic Press, New York, N.Y. (1976). Appropriate protective
groups for use in different peptide syntheses are described in the
above-mentioned texts as well as in McOmie, Protective Groups in
Organic Chemistry, Plenum Press, New York, N.Y. (1973). The
peptides of the present invention might also be prepared by
chemical or enzymatic cleavage from larger portions of, for
example, apolipoprotein A-I.
[0238] In certain embodiments, the apolipoprotein can be a mixture
of apolipoproteins. In one embodiment, the apolipoprotein can be a
homogeneous mixture, that is, a single type of apolipoprotein. In
another embodiment, the apolipoprotein can be a heterogeneous
mixture of apolipoproteins, that is, a mixture of two or more
different apolipoproteins. Embodiments of heterogeneous mixtures of
apolipoproteins can comprise, for example, a mixture of an
apolipoprotein from an animal source and an apolipoprotein from a
semi-synthetic source. In certain embodiments, a heterogeneous
mixture can comprise, for example, a mixture of ApoA-I and ApoA-I
Milano. In certain embodiments, a heterogeneous mixture can
comprise, for example, a mixture of ApoA-I Milano and ApoA-I Paris.
Suitable mixtures for use in the methods and compositions of the
invention will be apparent to one of skill in the art.
[0239] If the apolipoprotein is obtained from natural sources, it
can be obtained from a plant or animal source. If the
apolipoprotein is obtained from an animal source, the
apolipoprotein can be from any species. In certain embodiments, the
apolipoprotien can be obtained from an animal source. In certain
embodiments, the apolipoprotein can be obtained from a human
source. In preferred embodiments of the invention, the
apolipoprotein is derived from the same species as the individual
to which the apolipoprotein is administered.
[0240] Other Components
[0241] In numerous embodiments, amphipathic lipids are included in
lipid particles of the invention. "Amphipathic lipids" refer to any
suitable material, wherein the hydrophobic portion of the lipid
material orients into a hydrophobic phase, while the hydrophilic
portion orients toward the aqueous phase. Such compounds include,
but are not limited to, phospholipids, aminolipids, and
sphingolipids. Representative phospholipids include sphingomyelin,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyloleoyl
phosphatdylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or
dilinoleylphosphatidylcholine. Other phosphorus-lacking compounds,
such as sphingolipids, glycosphingolipid families, diacylglycerols,
and .beta.-acyloxyacids, can also be used. Additionally, such
amphipathic lipids can be readily mixed with other lipids, such as
triglycerides and sterols.
[0242] Also suitable for inclusion in the lipid particles of the
invention are programmable fusion lipids. Such lipid particles have
little tendency to fuse with cell membranes and deliver their
payload until a given signal event occurs. This allows the lipid
particle to distribute more evenly after injection into an organism
or disease site before it starts fusing with cells. The signal
event can be, for example, a change in pH, temperature, ionic
environment, or time. In the latter case, a fusion delaying or
"cloaking" component, such as an ATTA-lipid conjugate or a
PEG-lipid conjugate, can simply exchange out of the lipid particle
membrane over time. Exemplary lipid anchors include those having
lengths of from about C.sub.14 to about C.sub.22, preferably from
about C.sub.14 to about C.sub.16. In some embodiments, a PEG
moiety, for example an mPEG-NH.sub.2, has a size of about 1000,
2000, 5000, 10,000, 15,000 or 20,000 Daltons.
[0243] A lipid particle conjugated to a nucleic acid agent can also
include a targeting moiety, e.g., a targeting moiety that is
specific to a cell type or tissue. Targeting of lipid particles
using a variety of targeting moieties, such as ligands, cell
surface receptors, glycoproteins, vitamins (e.g., riboflavin) and
monoclonal antibodies, has been previously described (see, e.g.,
U.S. Pat. Nos. 4,957,773 and 4,603,044). The targeting moieties can
include the entire protein or fragments thereof. Targeting
mechanisms generally require that the targeting agents be
positioned on the surface of the lipid particle in such a manner
that the targeting moiety is available for interaction with the
target, for example, a cell surface receptor. A variety of
different targeting agents and methods are known and available in
the art, including those described, e.g., in Sapra, P. and Allen, T
M, Prog. Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J.
Liposome Res. 12:1-3, (2002).
[0244] The use of lipid particles, i.e., liposomes, with a surface
coating of hydrophilic polymer chains, such as polyethylene glycol
(PEG) chains, for targeting has been proposed (Allen, et al.,
Biochimica et Biophysica Acta 1237: 99-108 (1995); DeFrees, et al.,
Journal of the American Chemistry Society 118: 6101-6104 (1996);
Blume, et al., Biochimica et Biophysica Acta 1149: 180-184 (1993);
Klibanov, et al., Journal of Liposome Research 2: 321-334 (1992);
U.S. Pat. No. 5,013,556; Zalipsky, Bioconjugate Chemistry 4:
296-299 (1993); Zalipsky, FEBS Letters 353: 71-74 (1994); Zalipsky,
in Stealth Liposomes Chapter 9 (Lasic and Martin, Eds) CRC Press,
Boca Raton Fla. (1995). In one approach, a ligand, such as an
antibody, for targeting the lipid particle is linked to the polar
head group of lipids forming the lipid particle. In another
approach, the targeting ligand is attached to the distal ends of
the PEG chains forming the hydrophilic polymer coating (Klibanov,
et al., Journal of Liposome Research 2: 321-334 (1992); Kirpotin et
al., FEBS Letters 388: 115-118 (1996)).
[0245] Standard methods for coupling the target agents can be used.
For example, phosphatidylethanolamine, which can be activated for
attachment of target agents, or derivatized lipophilic compounds,
such as lipid-derivatized bleomycin, can be used. Antibody-targeted
liposomes can be constructed using, for instance, liposomes that
incorporate protein A (see, Renneisen, et al., J. Bio. Chem.,
265:16337-16342 (1990) and Leonetti, et al., Proc. Natl. Acad. Sci.
(USA), 87:2448-2451 (1990). Other examples of antibody conjugation
are disclosed in U.S. Pat. No. 6,027,726, the teachings of which
are incorporated herein by reference. Examples of targeting
moieties can also include other proteins, specific to cellular
components, including antigens associated with neoplasms or tumors.
Proteins used as targeting moieties can be attached to the
liposomes via covalent bonds (see, Heath, Covalent Attachment of
Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic
Press, Inc. 1987)). Other targeting methods include the
biotin-avidin system.
[0246] Production of Nucleic Acid-Lipid Particles
[0247] In one embodiment, the nucleic acid-lipid particle
formulations of the invention are produced via an extrusion method
or an in-line mixing method.
[0248] The extrusion method (also referred to as preformed method
or batch process) is a method where the empty liposomes (i.e. no
nucleic acid) are prepared first, followed by the addition of
nucleic acid to the empty liposome. Extrusion of liposome
compositions through a small-pore polycarbonate membrane or an
asymmetric ceramic membrane results in a relatively well-defined
size distribution. Typically, the suspension is cycled through the
membrane one or more times until the desired liposome complex size
distribution is achieved. The liposomes may be extruded through
successively smaller-pore membranes, to achieve a gradual reduction
in liposome size. In some instances, the lipid-nucleic acid
compositions which are formed can be used without any sizing. These
methods are disclosed in the U.S. Pat. No. 5,008,050; U.S. Pat. No.
4,927,637; U.S. Pat. No. 4,737,323; Biochim Biophys Acta. 1979 Oct.
19; 557(1):9-23; Biochim Biophys Acta. 1980 Oct. 2; 601(3):559-7;
Biochim Biophys Acta. 1986 Jun. 13; 858(1):161-8; and Biochim.
Biophys. Acta 1985 812, 55-65, which are hereby incorporated by
reference in their entirety.
[0249] The in-line mixing method is a method wherein both the
lipids and the nucleic acid are added in parallel into a mixing
chamber. The mixing chamber can be a simple T-connector or any
other mixing chamber that is known to one skill in the art. These
methods are disclosed in U.S. Pat. No. 6,534,018 and U.S. Pat. No.
6,855,277; US publication 2007/0042031 and Pharmaceuticals
Research, Vol. 22, No. 3, March 2005, p. 362-372, which are hereby
incorporated by reference in their entirety.
[0250] It is further understood that the formulations of the
invention can be prepared by any methods known to one of ordinary
skill in the art.
[0251] Characterization of Nucleic Acid-Lipid Particles
[0252] Formulations prepared by either the standard or
extrusion-free method can be characterized in similar manners. For
example, formulations are typically characterized by visual
inspection. They should be whitish translucent solutions free from
aggregates or sediment. Particle size and particle size
distribution of lipid-nanoparticles can be measured by light
scattering using, for example, a Malvern Zetasizer Nano ZS
(Malvern, USA). Particles should be about 20-300 nm, such as 40-100
nm in size. The particle size distribution should be unimodal. The
total siRNA concentration in the formulation, as well as the
entrapped fraction, is estimated using a dye exclusion assay. A
sample of the formulated siRNA can be incubated with an RNA-binding
dye, such as Ribogreen (Molecular Probes) in the presence or
absence of a formulation disrupting surfactant, e.g., 0.5%
Triton-X100. The total siRNA in the formulation can be determined
by the signal from the sample containing the surfactant, relative
to a standard curve. The entrapped fraction is determined by
subtracting the "free" siRNA content (as measured by the signal in
the absence of surfactant) from the total siRNA content. Percent
entrapped siRNA is typically >85%. In one embodiment, the
formulations of the invention are entrapped by at least 75%, at
least 80% or at least 90%.
[0253] For nucleic acid-lipid particle formulations, the particle
size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60
nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100
nm, at least 110 nm, and at least 120 nm. The suitable range is
typically about at least 50 nm to about at least 110 nm, about at
least 60 nm to about at least 100 nm, or about at least 80 nm to
about at least 90 nm.
[0254] Formulations of Nucleic Acid-Lipid Particles
[0255] LNP01
[0256] One example of synthesis of a nucleic acid-lipid particle is
as follows. Nucleic acid-lipid particles are synthesized using the
lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol
(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids). This
nucleic acid-lipid particle is sometimes referred to as a LNP01
particle. Stock solutions of each in ethanol can be prepared as
follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16,
100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock
solutions can then be combined in a, e.g., 42:48:10 molar ratio.
The combined lipid solution can be mixed with aqueous siRNA (e.g.,
in sodium acetate pH 5) such that the final ethanol concentration
is about 35-45% and the final sodium acetate concentration is about
100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously
upon mixing. Depending on the desired particle size distribution,
the resultant nanoparticle mixture can be extruded through a
polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a
thermobarrel extruder, such as Lipex Extruder (Northern Lipids,
Inc). In some cases, the extrusion step can be omitted. Ethanol
removal and simultaneous buffer exchange can be accomplished by,
for example, dialysis or tangential flow filtration. Buffer can be
exchanged with, for example, phosphate buffered saline (PBS) at
about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about
pH 7.2, about pH 7.3, or about pH 7.4.
##STR00003##
[0257] LNP01 formulations are described, e.g., in International
Application Publication No. WO 2008/042973, which is hereby
incorporated by reference.
[0258] Additional exemplary nucleic acid-lipid particle
formulations are described in the following table. It is to be
understood that the name of the nucleic acid-lipid particle in the
table is not meant to be limiting. For example, as used herein, the
term SNALP refers to formulations that include the cationic lipid
DLinDMA.
TABLE-US-00005 cationic lipid/non-cationic
lipid/cholesterol/PEG-lipid conjugate mol % ratio Name Lipid:siRNA
ratio SNALP DLinDMA/DPPC/Cholesterol/PEG-cDMA (57.1/7.1/34.4/1.4)
lipid:siRNA ~7:1 LNP-S-X XTC/DPPC/Cholesterol/PEG-cDMA
57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP05
XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1
LNP06 XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA
~11:1 LNP07 XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA
~6:1 LNP08 XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA
~11:1 LNP09 XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA
~10:1 LNP10 ALNY-100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5
lipid:siRNA ~10:1 LNP11 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5
lipid:siRNA ~10:1 LNP13 XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5
lipid:siRNA ~33:1 LNP14 MC3/DSPC/Cholesterol/PEG-DMG 40/15/40/5
lipid:siRNA ~11:1 LNP15 MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG
50/10/35/4.5/0.5 lipid:siRNA ~11:1 LNP16
MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~7:1 LNP17
MC3/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP18
MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~12:1 LNP19
MC3/DSPC/Cholesterol/PEG-DMG 50/10/35/5 lipid:siRNA ~8:1 LNP20
MC3/DSPC/Cholesterol/PEG-DPG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP22
XTC/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 lipid:siRNA ~10:1
[0259] XTC comprising formulations are described, e.g., in U.S.
Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, which is
hereby incorporated by reference.
[0260] MC3 comprising formulations are described, e.g., in U.S.
Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S.
Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, which are
hereby incorporated by reference.
[0261] ALNY-100 comprising formulations are described, e.g.,
International patent application number PCT/US09/63933, filed on
Nov. 10, 2009, which is hereby incorporated by reference.
[0262] Additional representative formulations delineated in Tables
11 and 12. Lipid refers to a cationic lipid.
TABLE-US-00006 TABLE 11 Composition of exemplary nucleic acid-lipid
particle (mole %) prepared via extrusion methods. PEG Lipid/ Lipid
(mol %) DSPC (mol %) Chol (mol %) (mol %) siRNA 20 30 40 10 2.13 20
30 40 10 2.35 20 30 40 10 2.37 20 30 40 10 3.23 20 30 40 10 3.91 30
20 40 10 2.89 30 20 40 10 3.34 30 20 40 10 3.34 30 20 40 10 4.10 30
20 40 10 5.64 40 10 40 10 3.02 40 10 40 10 3.35 40 10 40 10 3.74 40
10 40 10 5.80 40 10 40 10 8.00 45 5 40 10 3.27 45 5 40 10 3.30 45 5
40 10 4.45 45 5 40 10 7.00 45 5 40 10 9.80 50 0 40 10 27.03 20 35
40 5 3.00 20 35 40 5 3.32 20 35 40 5 3.05 20 35 40 5 3.67 20 35 40
5 4.71 30 25 40 5 2.47 30 25 40 5 2.98 30 25 40 5 3.29 30 25 40 5
4.99 30 25 40 5 7.15 40 15 40 5 2.79 40 15 40 5 3.29 40 15 40 5
4.33 40 15 40 5 7.05 40 15 40 5 9.63 45 10 40 5 2.44 45 10 40 5
3.21 45 10 40 5 4.29 45 10 40 5 6.50 45 10 40 5 8.67 20 35 40 5
4.10 20 35 40 5 4.83 30 25 40 5 3.86 30 25 40 5 5.38 30 25 40 5
7.07 40 15 40 5 3.85 40 15 40 5 4.88 40 15 40 5 7.22 40 15 40 5
9.75 45 10 40 5 2.83 45 10 40 5 3.85 45 10 40 5 4.88 45 10 40 5
7.05 45 10 40 5 9.29 45 20 30 5 4.01 45 20 30 5 3.70 50 15 30 5
4.75 50 15 30 5 3.80 55 10 30 5 3.85 55 10 30 5 4.13 60 5 30 5 5.09
60 5 30 5 4.67 65 0 30 5 4.75 65 0 30 5 6.06 56.5 10 30 3.5 3.70
56.5 10 30 3.5 3.56 57.5 10 30 2.5 3.48 57.5 10 30 2.5 3.20 58.5 10
30 1.5 3.24 58.5 10 30 1.5 3.13 59.5 10 30 0.5 3.24 59.5 10 30 0.5
3.03 45 10 40 5 7.57 45 10 40 5 7.24 45 10 40 5 7.48 45 10 40 5
7.84 65 0 30 5 4.01 60 5 30 5 3.70 55 10 30 5 3.65 50 10 35 5 3.43
50 15 30 5 3.80 45 15 35 5 3.70 45 20 30 5 3.75 45 25 25 5 3.85 55
10 32.5 2.5 3.61 60 10 27.5 2.5 3.65 60 10 25 5 4.07 55 5 38.5 1.5
3.75 60 10 28.5 1.5 3.43 55 10 33.5 1.5 3.48 60 5 33.5 1.5 3.43 55
5 37.5 2.5 3.75 60 5 32.5 2.5 4.52 60 5 32.5 2.5 3.52 45 15 (DMPC)
35 5 3.20 45 15 (DPPC) 35 5 3.43 45 15 (DOPC) 35 5 4.52 45 15
(POPC) 35 5 3.85 55 5 37.5 2.5 3.96 55 10 32.5 2.5 3.56 60 5 32.5
2.5 3.80 60 10 27.5 2.5 3.75 60 5 30 5 4.19 60 5 33.5 1.5 3.48 60 5
33.5 1.5 6.64 60 5 30 5 3.90 60 5 30 5 4.65 60 5 30 5 5.88 60 5 30
5 7.51 60 5 30 5 9.51 60 5 30 5 11.06 62.5 2.5 50 5 6.63 45 15 35 5
3.31 45 15 35 5 6.80 60 5 25 10 6.48 60 5 32.5 2.5 3.43 60 5 30 5
3.90 60 5 30 5 7.61 45 15 35 5 3.13 45 15 35 5 6.42 60 5 25 10 6.48
60 5 32.5 2.5 3.03 60 5 30 5 3.43 60 5 30 5 6.72 60 5 30 5 4.13 70
5 20 5 5.48 80 5 10 5 5.94 90 5 0 5 9.50 60 5 30 5 C12PEG 3.85 60 5
30 5 3.70 60 5 30 5 C16PEG 3.80 60 5 30 5 4.19 60 5 29 5 4.07 60 5
30 5 3.56 60 5 30 5 3.39 60 5 30 5 3.96 60 5 30 5 4.01 60 5 30 5
4.07 60 5 30 5 4.25 60 5 30 5 3.80 60 5 30 5 3.31 60 5 30 5 4.83 60
5 30 5 4.67 60 5 30 5 3.96 57.5 7.5 33.5 1.5 3.39 57.5 7.5 32.5 2.5
3.39 57.5 7.5 31.5 3.5 3.52 57.5 7.5 30 5 4.19 60 5 30 5 3.96 60 5
30 5 3.96 60 5 30 5 3.56 60 5 33.5 1.5 3.52 60 5 25 10 5.18 60 5
(DPPC) 30 5 4.25 60 5 32.5 2.5 3.70 57.5 7.5 31.5 3.5 3.06 57.5 7.5
31.5 3.5 3.65 57.5 7.5 31.5 3.5 4.70 57.5 7.5 31.5 3.5 6.56
TABLE-US-00007 TABLE 12 Composition of exemplary nucleic acid-lipid
particles prepared via in- line mixing Lipid DSPC (mol %) (mol %)
Chol (mol %) PEG (mol %) Lipid A/siRNA 55 5 37.5 2.5 3.96 55 10
32.5 2.5 3.56 60 5 32.5 2.5 3.80 60 10 27.5 2.5 3.75 60 5 30 5 4.19
60 5 33.5 1.5 3.48 60 5 33.5 1.5 6.64 60 5 25 10 6.79 60 5 32.5 2.5
3.96 60 5 34 1 3.75 60 5 34.5 0.5 3.28 50 5 40 5 3.96 60 5 30 5
4.75 70 5 20 5 5.00 80 5 10 5 5.18 60 5 30 5 13.60 60 5 30 5 14.51
60 5 30 5 6.20 60 5 30 5 4.60 60 5 30 5 6.20 60 5 30 5 5.82 40 5 54
1 3.39 40 7.5 51.5 1 3.39 40 10 49 1 3.39 50 5 44 1 3.39 50 7.5
41.5 1 3.43 50 10 39 1 3.35 60 5 34 1 3.52 60 7.5 31.5 1 3.56 60 10
29 1 3.80 70 5 24 1 3.70 70 7.5 21.5 1 4.13 70 10 19 1 3.85 60 5 34
1 3.52 60 5 34 1 3.70 60 5 34 1 3.52 60 7.5 27.5 5 5.18 60 7.5 29
3.5 4.45 60 5 31.5 3.5 4.83 60 7.5 31 1.5 3.48 57.5 7.5 30 5 4.75
57.5 7.5 31.5 3.5 4.83 57.5 5 34 3.5 4.67 57.5 7.5 33.5 1.5 3.43 55
7.5 32.5 5 4.38 55 7.5 34 3.5 4.13 55 5 36.5 3.5 4.38 55 7.5 36 1.5
3.35
[0263] Synthesis of Cationic Lipids.
[0264] Any of the compounds, e.g., cationic lipids and the like,
used in the nucleic acid-lipid particles of the invention may be
prepared by known organic synthesis techniques, including the
methods described in more detail in the Examples. All substituents
are as defined below unless indicated otherwise.
[0265] "Alkyl" means a straight chain or branched, noncyclic or
cyclic, saturated aliphatic hydrocarbon containing from 1 to 24
carbon atoms. Representative saturated straight chain alkyls
include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and
the like; while saturated branched alkyls include isopropyl,
sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
Representative saturated cyclic alkyls include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and the like; while
unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl,
and the like.
[0266] "Alkenyl" means an alkyl, as defined above, containing at
least one double bond between adjacent carbon atoms. Alkenyls
include both cis and trans isomers. Representative straight chain
and branched alkenyls include ethylenyl, propylenyl, 1-butenyl,
2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and
the like.
[0267] "Alkynyl" means any alkyl or alkenyl, as defined above,
which additionally contains at least one triple bond between
adjacent carbons. Representative straight chain and branched
alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl,
1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
[0268] "Acyl" means any alkyl, alkenyl, or alkynyl wherein the
carbon at the point of attachment is substituted with an oxo group,
as defined below. For example, --C(.dbd.O)alkyl,
--C(.dbd.O)alkenyl, and --C(.dbd.O)alkynyl are acyl groups.
[0269] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to
10-membered bicyclic, heterocyclic ring which is either saturated,
unsaturated, or aromatic, and which contains from 1 or 2
heteroatoms independently selected from nitrogen, oxygen and
sulfur, and wherein the nitrogen and sulfur heteroatoms may be
optionally oxidized, and the nitrogen heteroatom may be optionally
quatemized, including bicyclic rings in which any of the above
heterocycles are fused to a benzene ring. The heterocycle may be
attached via any heteroatom or carbon atom. Heterocycles include
heteroaryls as defined below. Heterocycles include morpholinyl,
pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,
hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
[0270] The terms "optionally substituted alkyl", "optionally
substituted alkenyl", "optionally substituted alkynyl", "optionally
substituted acyl", and "optionally substituted heterocycle" means
that, when substituted, at least one hydrogen atom is replaced with
a substituent. In the case of an oxo substituent (.dbd.O) two
hydrogen atoms are replaced. In this regard, substituents include
oxo, halogen, heterocycle, --CN, --OR.sup.x, --NR.sup.xR.sup.y,
--NR.sup.xC(.dbd.O)R.sup.y, --NR.sup.xSO.sub.2R.sup.y,
--C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO--R.sup.x and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1 or 2, R.sup.x and
R.sup.y are the same or different and independently hydrogen, alkyl
or heterocycle, and each of said alkyl and heterocycle substituents
may be further substituted with one or more of oxo, halogen, --OH,
--CN, alkyl, --OR', heterocycle, --NR.sup.xR.sup.y,
--NR.sup.xC(.dbd.O)R.sup.y, --NR.sup.xSO.sub.2R.sup.y,
--C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO--R.sup.x and
--SO.sub.nNR.sup.xR.sup.y.
[0271] "Halogen" means fluoro, chloro, bromo and iodo.
[0272] In some embodiments, the methods of the invention may
require the use of protecting groups. Protecting group methodology
is well known to those skilled in the art (see, for example,
PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al.,
Wiley-Interscience, New York City, 1999). Briefly, protecting
groups within the context of this invention are any group that
reduces or eliminates unwanted reactivity of a functional group. A
protecting group can be added to a functional group to mask its
reactivity during certain reactions and then removed to reveal the
original functional group. In some embodiments an "alcohol
protecting group" is used. An "alcohol protecting group" is any
group which decreases or eliminates unwanted reactivity of an
alcohol functional group. Protecting groups can be added and
removed using techniques well known in the art.
[0273] Synthesis of MC3
[0274] Preparation of DLin-M-C3-DMA (i.e.,
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate) was as follows. A solution of
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),
4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),
4-N,N-dimethylaminopyridine (0.61 g) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53
g) in dichloromethane (5 mL) was stirred at room temperature
overnight. The solution was washed with dilute hydrochloric acid
followed by dilute aqueous sodium bicarbonate. The organic
fractions were dried over anhydrous magnesium sulphate, filtered
and the solvent removed on a rotovap. The residue was passed down a
silica gel column (20 g) using a 1-5% methanol/dichloromethane
elution gradient. Fractions containing the purified product were
combined and the solvent removed, yielding a colorless oil (0.54
g). Further description is provided in WO 2010/054401
(PCTUS2009/063927 filed on Nov. 10, 2009 and U.S. patent
application Ser. No. 12/813,448 filed on Jun. 10, 2010.
[0275] Synthesis of Formula A
[0276] In one embodiment, nucleic acid-lipid particles of the
invention are formulated using a cationic lipid of formula A:
##STR00004##
[0277] where R1 and R2 are independently alkyl, alkenyl or alkynyl,
each can be optionally substituted, and R3 and R4 are independently
lower alkyl or R3 and R4 can be taken together to form an
optionally substituted heterocyclic ring. In some embodiments, the
cationic lipid is XTC
(2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane). In general,
the lipid of formula A above may be made by the following Reaction
Schemes 1 or 2, wherein all substituents are as defined above
unless indicated otherwise.
##STR00005##
[0278] Lipid A, where R.sub.1 and R.sub.2 are independently alkyl,
alkenyl or alkynyl, each can be optionally substituted, and R.sub.3
and R.sub.4 are independently lower alkyl or R.sub.3 and R.sub.4
can be taken together to form an optionally substituted
heterocyclic ring, can be prepared according to Scheme 1. Ketone 1
and bromide 2 can be purchased or prepared according to methods
known to those of ordinary skill in the art. Reaction of 1 and 2
yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of
formula A. The lipids of formula A can be converted to the
corresponding ammonium salt with an organic salt of formula 5,
where Xis anion counter ion selected from halogen, hydroxide,
phosphate, sulfate, or the like.
##STR00006##
[0279] Alternatively, the ketone 1 starting material can be
prepared according to Scheme 2. Grignard reagent 6 and cyanide 7
can be purchased or prepared according to methods known to those of
ordinary skill in the art. Reaction of 6 and 7 yields ketone 1.
Conversion of ketone 1 to the corresponding lipids of formula A is
as described in Scheme 1.
[0280] Synthesis of ALNY-100
[0281] Synthesis of ketal 519 [ALNY-100] was performed using the
following scheme 3:
##STR00007##
[0282] Synthesis of 515:
[0283] To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in
200 ml anhydrous THF in a two neck RBF (1 L), was added a solution
of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0.degree. C.
under nitrogen atmosphere. After complete addition, reaction
mixture was warmed to room temperature and then heated to reflux
for 4 h. Progress of the reaction was monitored by TLC. After
completion of reaction (by TLC) the mixture was cooled to 0.degree.
C. and quenched with careful addition of saturated Na2SO4 solution.
Reaction mixture was stirred for 4 h at room temperature and
filtered off. Residue was washed well with THF. The filtrate and
washings were mixed and diluted with 400 mL dioxane and 26 mL conc.
HCl and stirred for 20 minutes at room temperature. The
volatilities were stripped off under vacuum to furnish the
hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR
(DMSO, 400 MHz): .delta.=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m,
1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
[0284] Synthesis of 516:
[0285] To a stirred solution of compound 515 in 100 mL dry DCM in a
250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and
cooled to 0.degree. C. under nitrogen atmosphere. After a slow
addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007
mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room
temperature. After completion of the reaction (2-3 h by TLC)
mixture was washed successively with 1N HCl solution (1.times.100
mL) and saturated NaHCO3 solution (1.times.50 mL). The organic
layer was then dried over anhyd. Na2SO4 and the solvent was
evaporated to give crude material which was purified by silica gel
column chromatography to get 516 as sticky mass. Yield: 11 g (89%).
1H-NMR (CDCl3, 400 MHz): .delta.=7.36-7.27 (m, 5H), 5.69 (s, 2H),
5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25
(m, 2H). LC-MS [M+H]-232.3 (96.94%).
[0286] Synthesis of 517A and 517B:
[0287] The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a
solution of 220 mL acetone and water (10:1) in a single neck 500 mL
RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492
mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108
mol) in tert-butanol at room temperature. After completion of the
reaction (.about.3 h), the mixture was quenched with addition of
solid Na2SO3 and resulting mixture was stirred for 1.5 h at room
temperature. Reaction mixture was diluted with DCM (300 mL) and
washed with water (2.times.100 mL) followed by saturated NaHCO3
(1.times.50 mL) solution, water (1.times.30 mL) and finally with
brine (lx 50 mL). Organic phase was dried over an.Na2SO4 and
solvent was removed in vacuum. Silica gel column chromatographic
purification of the crude material was afforded a mixture of
diastereomers, which were separated by prep HPLC. Yield:--6 g
crude
[0288] 517A--Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400
MHz): .delta.=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H),
4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m,
4H). LC-MS--[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%.
Stereochemistry confirmed by X-ray.
[0289] Synthesis of 518:
[0290] Using a procedure analogous to that described for the
synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained
as a colorless oil. 1H-NMR (CDCl3, 400 MHz): .delta.=7.35-7.33 (m,
4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m,
1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H),
1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m,
36H), 0.87 (m, 6H). HPLC-98.65%.
[0291] General Procedure for the Synthesis of Compound 519:
[0292] A solution of compound 518 (1 eq) in hexane (15 mL) was
added in a drop-wise fashion to an ice-cold solution of LAH in THF
(1 M, 2 eq). After complete addition, the mixture was heated at
40.quadrature.C over 0.5 h then cooled again on an ice bath. The
mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then
filtered through celite and reduced to an oil. Column
chromatography provided the pure 519 (1.3 g, 68%) which was
obtained as a colorless oil. 13C NMR .quadrature.=130.2, 130.1
(.times.2), 127.9 (.times.3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4,
31.5, 29.9 (.times.2), 29.7, 29.6 (.times.2), 29.5 (.times.3), 29.3
(.times.2), 27.2 (.times.3), 25.6, 24.5, 23.3, 226, 14.1;
Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.
654.6. Found 654.6.
[0293] Therapeutic Agent-Lipid Particle Compositions and
Formulations
[0294] The invention includes compositions comprising a lipid
particle of the invention and an active agent, wherein the active
agent is associated with the lipid particle. In particular
embodiments, the active agent is a therapeutic agent. In particular
embodiments, the active agent is encapsulated within an aqueous
interior of the lipid particle. In other embodiments, the active
agent is present within one or more lipid layers of the lipid
particle. In other embodiments, the active agent is bound to the
exterior or interior lipid surface of a lipid particle.
[0295] "Fully encapsulated" as used herein indicates that the
nucleic acid in the particles is not significantly degraded after
exposure to serum or a nuclease assay that would significantly
degrade free DNA. In a fully encapsulated system, preferably less
than 25% of particle nucleic acid is degraded in a treatment that
would normally degrade 100% of free nucleic acid, more preferably
less than 10% and most preferably less than 5% of the particle
nucleic acid is degraded. Alternatively, full encapsulation may be
determined by an Oligreen.RTM. assay. Oligreen.RTM. is an
ultra-sensitive fluorescent nucleic acid stain for quantitating
oligonucleotides and single-stranded DNA in solution (available
from Invitrogen Corporation, Carlsbad, Calif.). Fully encapsulated
also suggests that the particles are serum stable, that is, that
they do not rapidly decompose into their component parts upon in
vivo administration.
[0296] Active agents, as used herein, include any molecule or
compound capable of exerting a desired effect on a cell, tissue,
organ, or subject. Such effects may be biological, physiological,
or cosmetic, for example. Active agents may be any type of molecule
or compound, including e.g., nucleic acids, peptides and
polypeptides, including, e.g., antibodies, such as, e.g.,
polyclonal antibodies, monoclonal antibodies, antibody fragments;
humanized antibodies, recombinant antibodies, recombinant human
antibodies, and Primatized.TM. antibodies, cytokines, growth
factors, apoptotic factors, differentiation-inducing factors, cell
surface receptors and their ligands; hormones; and small molecules,
including small organic molecules or compounds.
[0297] In one embodiment, the active agent is a therapeutic agent,
or a salt or derivative thereof. Therapeutic agent derivatives may
be therapeutically active themselves or they may be prodrugs, which
become active upon further modification. Thus, in one embodiment, a
therapeutic agent derivative retains some or all of the therapeutic
activity as compared to the unmodified agent, while in another
embodiment, a therapeutic agent derivative lacks therapeutic
activity.
[0298] In various embodiments, therapeutic agents include any
therapeutically effective agent or drug, such as anti-inflammatory
compounds, anti-depressants, stimulants, analgesics, antibiotics,
birth control medication, antipyretics, vasodilators,
anti-angiogenics, cytovascular agents, signal transduction
inhibitors, cardiovascular drugs, e.g., anti-arrhythmic agents,
vasoconstrictors, hormones, and steroids.
[0299] In certain embodiments, the therapeutic agent is an oncology
drug, which may also be referred to as an anti-tumor drug, an
anti-cancer drug, a tumor drug, an antineoplastic agent, or the
like. Examples of oncology drugs that may be used according to the
invention include, but are not limited to, adriamycin, alkeran,
allopurinol, altretamine, amifostine, anastrozole, araC, arsenic
trioxide, azathioprine, bexarotene, biCNU, bleomycin, busulfan
intravenous, busulfan oral, capecitabine (Xeloda), carboplatin,
carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine,
cyclosporin A, cytarabine, cytosine arabinoside, daunorubicin,
cytoxan, daunorubicin, dexamethasone, dexrazoxane, dodetaxel,
doxorubicin, doxorubicin, DTIC, epirubicin, estramustine, etoposide
phosphate, etoposide and VP-16, exemestane, FK506, fludarabine,
fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab-ozogamicin,
goserelin acetate, hydrea, hydroxyurea, idarubicin, ifosfamide,
imatinib mesylate, interferon, irinotecan (Camptostar, CPT-111),
letrozole, leucovorin, leustatin, leuprolide, levamisole,
litretinoin, megastrol, melphalan, L-PAM, mesna, methotrexate,
methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen
mustard, paclitaxel, pamidronate, Pegademase, pentostatin, porfimer
sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen,
taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin),
toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine,
vincristine, VP16, and vinorelbine. Other examples of oncology
drugs that may be used according to the invention are ellipticin
and ellipticin analogs or derivatives, epothilones, intracellular
kinase inhibitors and camptothecins.
[0300] Additional Formulations
[0301] Emulsions
[0302] The compositions of the present invention may be prepared
and formulated as emulsions. Emulsions are typically heterogeneous
systems of one liquid dispersed in another in the form of droplets
usually exceeding 0.1 .mu.m in diameter (Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p.
335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often
biphasic systems comprising two immiscible liquid phases intimately
mixed and dispersed with each other. In general, emulsions may be
of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
When an aqueous phase is finely divided into and dispersed as
minute droplets into a bulk oily phase, the resulting composition
is called a water-in-oil (w/o) emulsion. Alternatively, when an
oily phase is finely divided into and dispersed as minute droplets
into a bulk aqueous phase, the resulting composition is called an
oil-in-water (o/w) emulsion. Emulsions may contain additional
components in addition to the dispersed phases, and the active drug
which may be present as a solution in either the aqueous phase,
oily phase or itself as a separate phase. Pharmaceutical excipients
such as emulsifiers, stabilizers, dyes, and anti-oxidants may also
be present in emulsions as needed. Pharmaceutical emulsions may
also be multiple emulsions that are comprised of more than two
phases such as, for example, in the case of oil-in-water-in-oil
(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex
formulations often provide certain advantages that simple binary
emulsions do not. Multiple emulsions in which individual oil
droplets of an o/w emulsion enclose small water droplets constitute
a w/o/w emulsion. Likewise a system of oil droplets enclosed in
globules of water stabilized in an oily continuous phase provides
an o/w/o emulsion.
[0303] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0304] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (Rieger, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
Surfactants are typically amphiphilic and comprise a hydrophilic
and a hydrophobic portion. The ratio of the hydrophilic to the
hydrophobic nature of the surfactant has been termed the
hydrophile/lipophile balance (HLB) and is a valuable tool in
categorizing and selecting surfactants in the preparation of
formulations. Surfactants may be classified into different classes
based on the nature of the hydrophilic group: nonionic, anionic,
cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0305] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, non-swelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0306] Large varieties of non-emulsifying materials are also
included in emulsion formulations and contribute to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0307] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0308] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that may
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0309] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture has
been reviewed in the literature (Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for
oral delivery have been very widely used because of ease of
formulation, as well as efficacy from an absorption and
bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base
laxatives, oil-soluble vitamins and high fat nutritive preparations
are among the materials that have commonly been administered orally
as o/w emulsions.
[0310] In one embodiment of the present invention, the compositions
of dsRNAs and nucleic acids are formulated as microemulsions. A
microemulsion may be defined as a system of water, oil and
amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution (Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically
microemulsions are systems that are prepared by first dispersing an
oil in an aqueous surfactant solution and then adding a sufficient
amount of a fourth component, generally an intermediate
chain-length alcohol to form a transparent system. Therefore,
microemulsions have also been described as thermodynamically
stable, isotropically clear dispersions of two immiscible liquids
that are stabilized by interfacial films of surface-active
molecules (Leung and Shah, in: Controlled Release of Drugs:
Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
[0311] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0312] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750),
decaglycerol decaoleate (DAO750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized C8-C10
glycerides, vegetable oils and silicone oil.
[0313] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13,
205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form
spontaneously when their components are brought together at ambient
temperature. This may be particularly advantageous when formulating
thermolabile drugs, peptides or dsRNAs. Microemulsions have also
been effective in the transdermal delivery of active components in
both cosmetic and pharmaceutical applications. It is expected that
the microemulsion compositions and formulations of the present
invention will facilitate the increased systemic absorption of
dsRNAs and nucleic acids from the gastrointestinal tract, as well
as improve the local cellular uptake of dsRNAs and nucleic
acids.
[0314] Microemulsions of the present invention may also contain
additional components and additives such as sorbitan monostearate
(Grill 3), Labrasol, and penetration enhancers to improve the
properties of the formulation and to enhance the absorption of the
dsRNAs and nucleic acids of the present invention. Penetration
enhancers used in the microemulsions of the present invention may
be classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
[0315] Penetration Enhancers
[0316] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly dsRNAs, to the skin of animals. Most drugs are
present in solution in both ionized and nonionized forms. However,
usually only lipid soluble or lipophilic drugs readily cross cell
membranes. It has been discovered that even non-lipophilic drugs
may cross cell membranes if the membrane to be crossed is treated
with a penetration enhancer. In addition to aiding the diffusion of
non-lipophilic drugs across cell membranes, penetration enhancers
also enhance the permeability of lipophilic drugs.
[0317] Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.
92). Each of the above mentioned classes of penetration enhancers
are described below in greater detail.
[0318] Surfactants: In connection with the present invention,
surfactants (or "surface-active agents") are chemical entities
which, when dissolved in an aqueous solution, reduce the surface
tension of the solution or the interfacial tension between the
aqueous solution and another liquid, with the result that
absorption of dsRNAs through the mucosa is enhanced. In addition to
bile salts and fatty acids, these penetration enhancers include,
for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether
and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews
in Therapeutic Drug Carrier Systems, 1991, p. 92); and
perfluorochemical emulsions, such as FC-43. Takahashi et al., J.
Pharm. Pharmacol., 1988, 40, 252).
[0319] Fatty acids: Various fatty acids and their derivatives which
act as penetration enhancers include, for example, oleic acid,
lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,
caprylic acid, arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and di-glycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm.
Pharmacol., 1992, 44, 651-654).
[0320] Bile salts: The physiological role of bile includes the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.
Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural
bile salts, and their synthetic derivatives, act as penetration
enhancers. Thus the term "bile salts" includes any of the naturally
occurring components of bile as well as any of their synthetic
derivatives. Suitable bile salts include, for example, cholic acid
(or its pharmaceutically acceptable sodium salt, sodium cholate),
dehydrocholic acid (sodium dehydrocholate), deoxycholic acid
(sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.
Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990,
79, 579-583).
[0321] Chelating Agents: Chelating agents, as used in connection
with the present invention, can be defined as compounds that remove
metallic ions from solution by forming complexes therewith, with
the result that absorption of dsRNAs through the mucosa is
enhanced. With regards to their use as penetration enhancers in the
present invention, chelating agents have the added advantage of
also serving as DNase inhibitors, as most characterized DNA
nucleases require a divalent metal ion for catalysis and are thus
inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,
315-339). Suitable chelating agents include but are not limited to
disodium ethylenediaminetetraacetate (EDTA), citric acid,
salicylates (e.g., sodium salicylate, 5-methoxysalicylate and
homovanilate), N-acyl derivatives of collagen, laureth-9 and
N-amino acyl derivatives of beta-diketones (enamines)(Lee et al.,
Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page
92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14,
43-51).
[0322] Non-chelating non-surfactants: As used herein, non-chelating
non-surfactant penetration enhancing compounds can be defined as
compounds that demonstrate insignificant activity as chelating
agents or as surfactants but that nonetheless enhance absorption of
dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews
in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of
penetration enhancers includes, for example, unsaturated cyclic
ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
[0323] Agents that enhance uptake of dsRNAs at the cellular level
may also be added to the pharmaceutical and other compositions of
the present invention. For example, cationic lipids, such as
lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic
glycerol derivatives, and polycationic molecules, such as
polylysine (Lollo et al., PCT Application WO 97/30731), are also
known to enhance the cellular uptake of dsRNAs.
[0324] Other agents may be utilized to enhance the penetration of
the administered nucleic acids, including glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and
terpenes such as limonene and menthone.
[0325] Carriers
[0326] dsRNAs of the present invention can be formulated in a
pharmaceutically acceptable carrier or diluent. A "pharmaceutically
acceptable carrier" (also referred to herein as an "excipient") is
a pharmaceutically acceptable solvent, suspending agent, or any
other pharmacologically inert vehicle. Pharmaceutically acceptable
carriers can be liquid or solid, and can be selected with the
planned manner of administration in mind so as to provide for the
desired bulk, consistency, and other pertinent transport and
chemical properties. Typical pharmaceutically acceptable carriers
include, by way of example and not limitation: water; saline
solution; binding agents (e.g., polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose and other
sugars, gelatin, or calcium sulfate); lubricants (e.g., starch,
polyethylene glycol, or sodium acetate); disintegrates (e.g.,
starch or sodium starch glycolate); and wetting agents (e.g.,
sodium lauryl sulfate).
[0327] Certain compositions of the present invention also
incorporate carrier compounds in the formulation. As used herein,
"carrier compound" or "carrier" can refer to a nucleic acid, or
analog thereof, which is inert (i.e., does not possess biological
activity per se) but is recognized as a nucleic acid by in vivo
processes that reduce the bioavailability of a nucleic acid having
biological activity by, for example, degrading the biologically
active nucleic acid or promoting its removal from circulation. The
co-administration of a nucleic acid and a carrier compound,
typically with an excess of the latter substance, can result in a
substantial reduction of the amount of nucleic acid recovered in
the liver, kidney or other extra-circulatory reservoirs, presumably
due to competition between the carrier compound and the nucleic
acid for a common receptor. For example, the recovery of a
partially phosphorothioate dsRNA in hepatic tissue can be reduced
when it is co-administered with polyinosinic acid, dextran sulfate,
polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA
& Nucl. Acid Drug Dev., 1996, 6, 177-183.
[0328] Excipients
[0329] In contrast to a carrier compound, a "pharmaceutical
carrier" or "excipient" is a pharmaceutically acceptable solvent,
suspending agent or any other pharmacologically inert vehicle for
delivering one or more nucleic acids to an animal. The excipient
may be liquid or solid and is selected, with the planned manner of
administration in mind, so as to provide for the desired bulk,
consistency, etc., when combined with a nucleic acid and the other
components of a given pharmaceutical composition. Typical
pharmaceutical carriers include, but are not limited to, binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and
other sugars, microcrystalline cellulose, pectin, gelatin, calcium
sulfate, ethyl cellulose, polyacrylates or calcium hydrogen
phosphate, etc.); lubricants (e.g., magnesium stearate, talc,
silica, colloidal silicon dioxide, stearic acid, metallic
stearates, hydrogenated vegetable oils, corn starch, polyethylene
glycols, sodium benzoate, sodium acetate, etc.); disintegrants
(e.g., starch, sodium starch glycolate, etc.); and wetting agents
(e.g., sodium lauryl sulphate, etc).
[0330] Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can also be used to
formulate the compositions of the present invention. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the
like.
[0331] Formulations for topical administration of nucleic acids may
include sterile and non-sterile aqueous solutions, non-aqueous
solutions in common solvents such as alcohols, or solutions of the
nucleic acids in liquid or solid oil bases. The solutions may also
contain buffers, diluents and other suitable additives.
Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can be used.
[0332] Suitable pharmaceutically acceptable excipients include, but
are not limited to, water, salt solutions, alcohol, polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate, talc,
silicic acid, viscous paraffin, hydroxymethylcellulose,
polyvinylpyrrolidone and the like.
[0333] Other Components
[0334] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional,
compatible, pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the compositions of the present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention. The formulations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
[0335] Aqueous suspensions may contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0336] Combination Therapy
[0337] In one aspect, a composition of the invention can be used in
combination therapy. The term "combination therapy" includes the
administration of the subject compounds in further combination with
other biologically active ingredients (such as, but not limited to,
a second and different antineoplastic agent) and non-drug therapies
(such as, but not limited to, surgery or radiation treatment). For
instance, the compounds of the invention can be used in combination
with other pharmaceutically active compounds, preferably compounds
that are able to enhance the effect of the compounds of the
invention. The compounds of the invention can be administered
simultaneously (as a single preparation or separate preparation) or
sequentially to the other drug therapy. In general, a combination
therapy envisions administration of two or more drugs during a
single cycle or course of therapy.
[0338] In one aspect of the invention, the subject compounds may be
administered in combination with one or more separate agents that
modulate protein kinases involved in various disease states.
Examples of such kinases may include, but are not limited to:
serine/threonine specific kinases, receptor tyrosine specific
kinases and non-receptor tyrosine specific kinases.
Serine/threonine kinases include mitogen activated protein kinases
(MAPK), meiosis specific kinase (MEK), RAF and aurora kinase.
Examples of receptor kinase families include epidermal growth
factor receptor (EGFR) (e.g., HER2/neu, HER3, HERO, ErbB, ErbB2,
ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF)
receptor (e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF,
KGF-R); hepatocyte growth/scatter factor receptor (HGFR) (e.g.,
MET, RON, SEA, SEX); insulin receptor (e.g. IGFI-R); Eph (e.g.
CEKS, CEK8, EBK, ECK, EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2,
MDKS, SEK); AxI (e.g. Mer/Nyk, Rse); RET; and platelet-derived
growth factor receptor (PDGFR) (e.g. PDGF.alpha.-R, PDG.beta.-R,
CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1, FLT3/FLK2/STK-1).
Non-receptor tyrosine kinase families include, but are not limited
to, BCR-ABL (e.g. p43.sup.abl ARG); BTK (e.g. ITK/EMT, TEC); CSK,
FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.
[0339] In another aspect of the invention, the subject compounds
may be administered in combination with one or more agents that
modulate non-kinase biological targets or processes. Such targets
include histone deacetylases (HDAC), DNA methyltransferase (DNMT),
heat shock proteins (e.g., HSP90), and proteosomes.
[0340] In one embodiment, subject compounds may be combined with
antineoplastic agents (e.g. small molecules, monoclonal antibodies,
antisense RNA, and fusion proteins) that inhibit one or more
biological targets such as Zolinza, Tarceva, Iressa, Tykerb,
Gleevec, Sutent, Sprycel, Nexavar, Sorafenib, CNF2024, RG108,
BMS387032, Affmitak, Avastin, Herceptin, Erbitux, AG24322,
PD325901, ZD6474, PD 184322, Obatodax, ABT737 and AEE788. Such
combinations may enhance therapeutic efficacy over efficacy
achieved by any of the agents alone and may prevent or delay the
appearance of resistant mutational variants.
[0341] In certain preferred embodiments, the compounds of the
invention are administered in combination with a chemotherapeutic
agent. Chemotherapeutic agents encompass a wide range of
therapeutic treatments in the field of oncology. These agents are
administered at various stages of the disease for the purposes of
shrinking tumors, destroying remaining cancer cells left over after
surgery, inducing remission, maintaining remission and/or
alleviating symptoms relating to the cancer or its treatment.
Examples of such agents include, but are not limited to, alkylating
agents such as mustard gas derivatives (Mechlorethamine,
cylophosphamide, chlorambucil, melphalan, ifosfamide),
ethylenimines (thiotepa, hexamethylmelanine), Alkylsulfonates
(Busulfan), Hydrazines and Triazines (Altretamine, Procarbazine,
Dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine
and Streptozocin), Ifosfamide and metal salts (Carboplatin,
Cisplatin, and Oxaliplatin); plant alkaloids such as
Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxel
and Docetaxel), Vinca alkaloids (Vincristine, Vinblastine,
Vindesine and Vinorelbine), and Camptothecan analogs (Irinotecan
and Topotecan); anti-tumor antibiotics such as Chromomycins
(Dactinomycin and Plicamycin), Anthracyclines (Doxorubicin,
Daunorubicin, Epirubicin, Mitoxantrone, Valrubicin and Idarubicin),
and miscellaneous antibiotics such as Mitomycin, Actinomycin and
Bleomycin; anti-metabolites such as folic acid antagonists
(Methotrexate, Pemetrexed, Raltitrexed, Aminopterin), pyrimidine
antagonists (5-Fluorouracil, Floxuridine, Cytarabine, Capecitabine,
and Gemcitabine), purine antagonists (6-Mercaptopurine and
6-Thioguanine) and adenosine deaminase inhibitors (Cladribine,
Fludarabine, Mercaptopurine, Clofarabine, Thioguanine, Nelarabine
and Pentostatin); topoisomerase inhibitors such as topoisomerase I
inhibitors (Ironotecan, topotecan) and topoisomerase II inhibitors
(Amsacrine, etoposide, etoposide phosphate, teniposide); monoclonal
antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab,
Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab,
Tositumomab, Bevacizumab); and miscellaneous anti-neoplasties such
as ribonucleotide reductase inhibitors (Hydroxyurea);
adrenocortical steroid inhibitor (Mitotane); enzymes (Asparaginase
and Pegaspargase); anti-microtubule agents (Estramustine); and
retinoids (Bexarotene, Isotretinoin, Tretinoin (ATRA). In certain
preferred embodiments, the compounds of the invention are
administered in combination with a chemoprotective agent.
Chemoprotective agents act to protect the body or minimize the side
effects of chemotherapy. Examples of such agents include, but are
not limited to, amfostine, mesna, and dexrazoxane.
[0342] In one aspect of the invention, the subject compounds are
administered in combination with radiation therapy. Radiation is
commonly delivered internally (implantation of radioactive material
near cancer site) or externally from a machine that employs photon
(x-ray or gamma-ray) or particle radiation. Where the combination
therapy further comprises radiation treatment, the radiation
treatment may be conducted at any suitable time so long as a
beneficial effect from the co-action of the combination of the
therapeutic agents and radiation treatment is achieved. For
example, in appropriate cases, the beneficial effect is still
achieved when the radiation treatment is temporally removed from
the administration of the therapeutic agents, perhaps by days or
even weeks.
[0343] It will be appreciated that compounds of the invention can
be used in combination with an immunotherapeutic agent. One form of
immunotherapy is the generation of an active systemic
tumor-specific immune response of host origin by administering a
vaccine composition at a site distant from the tumor. Various types
of vaccines have been proposed, including isolated tumor-antigen
vaccines and anti-idiotype vaccines. Another approach is to use
tumor cells from the subject to be treated, or a derivative of such
cells (reviewed by Schirrmacher et al. (1995) J. Cancer Res. Clin.
Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr. et al. claim
a method for treating a resectable carcinoma to prevent recurrence
or metastases, comprising surgically removing the tumor, dispersing
the cells with collagenase, irradiating the cells, and vaccinating
the patient with at least three consecutive doses of about 10.sup.7
cells.
[0344] It will be appreciated that the compounds of the invention
may advantageously be used in conjunction with one or more
adjunctive therapeutic agents. Examples of suitable agents for
adjunctive therapy include steroids, such as corticosteroids
(amcinonide, betamethasone, betamethasone dipropionate,
betamethasone valerate, budesonide, clobetasol, clobetasol acetate,
clobetasol butyrate, clobetasol 17-propionate, cortisone,
deflazacort, desoximetasone, diflucortolone valerate,
dexamethasone, dexamethasone sodium phosphate, desonide, furoate,
fluocinonide, fluocinolone acetonide, halcinonide, hydrocortisone,
hydrocortisone butyrate, hydrocortisone sodium succinate,
hydrocortisone valerate, methyl prednisolone, mometasone,
prednicarbate, prednisolone, triamcinolone, triamcinolone
acetonide, and halobetasol proprionate); a SHTi agonist, such as a
triptan (e.g. sumatriptan or naratriptan); an adenosine A1 agonist;
an EP ligand; an NMDA modulator, such as a glycine antagonist; a
sodium channel blocker (e.g. lamotrigine); a substance P antagonist
(e.g. an NKi antagonist); a cannabinoid; acetaminophen or
phenacetin; a 5-lipoxygenase inhibitor; a leukotriene receptor
antagonist; a DMARD (e.g. methotrexate); gabapentin and related
compounds; a tricyclic antidepressant (e.g. amitryptilline); a
neurone stabilizing antiepileptic drug; a mono-aminergic uptake
inhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor;
a nitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOS
inhibitor; an inhibitor of the release, or action, of tumour
necrosis factor .alpha.; an antibody therapy, such as a monoclonal
antibody therapy; an antiviral agent, such as a nucleoside
inhibitor (e.g. lamivudine) or an immune system modulator (e.g.
interferon); an opioid analgesic; a local anaesthetic; a stimulant,
including caffeine; an H2-antagonist (e.g. ranitidine); a proton
pump inhibitor (e.g. omeprazole); an antacid (e.g. aluminium or
magnesium hydroxide; an antiflatulent (e.g. simethicone); a
decongestant (e.g. phenylephrine, phenylpropanolamine,
pseudoephedrine, oxymetazoline, epinephrine, naphazoline,
xylometazoline, propylhexedrine, or levo-desoxyephedrine); an
antitussive (e.g. codeine, hydrocodone, carmiphen, carbetapentane,
or dextramethorphan); a diuretic; or a sedating or non-sedating
antihistamine.
[0345] The compounds of the invention can be co-administered with
siRNA that target other genes. For example, a compound of the
invention can be co-administered with an siRNA targeted to a c-Myc
gene. In one example, AD-12115 can be co-administered with a c-Myc
siRNA. Examples of c-Myc targeted siRNAs are disclosed in U.S.
patent application Ser. No. 12/373,039 which is herein incorporated
by reference.
[0346] Methods of Preparing Lipid Particles
[0347] The methods and compositions of the invention make use of
certain cationic lipids, the synthesis, preparation and
characterization of which is described below and in the
accompanying Examples. In addition, the present invention provides
methods of preparing lipid particles, including those associated
with a therapeutic agent, e.g., a nucleic acid. In the methods
described herein, a mixture of lipids is combined with a buffered
aqueous solution of nucleic acid to produce an intermediate mixture
containing nucleic acid encapsulated in lipid particles wherein the
encapsulated nucleic acids are present in a nucleic acid/lipid
ratio of about 3 wt % to about 25 wt %, preferably 5 to 15 wt %.
The intermediate mixture may optionally be sized to obtain
lipid-encapsulated nucleic acid particles wherein the lipid
portions are unilamellar vesicles, preferably having a diameter of
30 to 150 nm, more preferably about 40 to 90 nm. The pH is then
raised to neutralize at least a portion of the surface charges on
the lipid-nucleic acid particles, thus providing an at least
partially surface-neutralized lipid-encapsulated nucleic acid
composition.
[0348] As described above, several of these cationic lipids are
amino lipids that are charged at a pH below the pK.sub.a of the
amino group and substantially neutral at a pH above the pK.sub.a.
These cationic lipids are termed titratable cationic lipids and can
be used in the formulations of the invention using a two-step
process. First, lipid vesicles can be formed at the lower pH with
titratable cationic lipids and other vesicle components in the
presence of nucleic acids. In this manner, the vesicles will
encapsulate and entrap the nucleic acids. Second, the surface
charge of the newly formed vesicles can be neutralized by
increasing the pH of the medium to a level above the pK.sub.a of
the titratable cationic lipids present, i.e., to physiological pH
or higher. Particularly advantageous aspects of this process
include both the facile removal of any surface adsorbed nucleic
acid and a resultant nucleic acid delivery vehicle which has a
neutral surface. Liposomes or lipid particles having a neutral
surface are expected to avoid rapid clearance from circulation and
to avoid certain toxicities which are associated with cationic
liposome preparations. Additional details concerning these uses of
such titratable cationic lipids in the formulation of nucleic
acid-lipid particles are provided in U.S. Pat. No. 6,287,591 and
U.S. Pat. No. 6,858,225, incorporated herein by reference.
[0349] It is further noted that the vesicles formed in this manner
provide formulations of uniform vesicle size with high content of
nucleic acids. Additionally, the vesicles have a size range of from
about 30 to about 150 nm, more preferably about 30 to about 90
nm.
[0350] Without intending to be bound by any particular theory, it
is believed that the very high efficiency of nucleic acid
encapsulation is a result of electrostatic interaction at low pH.
At acidic pH (e.g. pH 4.0) the vesicle surface is charged and binds
a portion of the nucleic acids through electrostatic interactions.
When the external acidic buffer is exchanged for a more neutral
buffer (e.g., pH 7.5) the surface of the lipid particle or liposome
is neutralized, allowing any external nucleic acid to be removed.
More detailed information on the formulation process is provided in
various publications (e.g., U.S. Pat. No. 6,287,591 and U.S. Pat.
No. 6,858,225).
[0351] In view of the above, the present invention provides methods
of preparing lipid/nucleic acid formulations. In the methods
described herein, a mixture of lipids is combined with a buffered
aqueous solution of nucleic acid to produce an intermediate mixture
containing nucleic acid encapsulated in lipid particles, e.g.,
wherein the encapsulated nucleic acids are present in a nucleic
acid/lipid ratio of about 10 wt % to about 20 wt %. The
intermediate mixture may optionally be sized to obtain
lipid-encapsulated nucleic acid particles wherein the lipid
portions are unilamellar vesicles, preferably having a diameter of
30 to 150 nm, more preferably about 40 to 90 nm. The pH is then
raised to neutralize at least a portion of the surface charges on
the lipid-nucleic acid particles, thus providing an at least
partially surface-neutralized lipid-encapsulated nucleic acid
composition.
[0352] In certain embodiments, the mixture of lipids includes at
least two lipid components: a first amino lipid component of the
present invention that is selected from among lipids which have a
pKa such that the lipid is cationic at pH below the pKa and neutral
at pH above the pKa, and a second lipid component that is selected
from among lipids that prevent particle aggregation during
lipid-nucleic acid particle formation. In particular embodiments,
the amino lipid is a novel cationic lipid of the present
invention.
[0353] In preparing the nucleic acid-lipid particles of the
invention, the mixture of lipids is typically a solution of lipids
in an organic solvent. This mixture of lipids can then be dried to
form a thin film or lyophilized to form a powder before being
hydrated with an aqueous buffer to form liposomes. Alternatively,
in a preferred method, the lipid mixture can be solubilized in a
water miscible alcohol, such as ethanol, and this ethanolic
solution added to an aqueous buffer resulting in spontaneous
liposome formation. In most embodiments, the alcohol is used in the
form in which it is commercially available. For example, ethanol
can be used as absolute ethanol (100%), or as 95% ethanol, the
remainder being water. This method is described in more detail in
U.S. Pat. No. 5,976,567).
[0354] In accordance with the invention, the lipid mixture is
combined with a buffered aqueous solution that may contain the
nucleic acids. The buffered aqueous solution of is typically a
solution in which the buffer has a pH of less than the pK.sub.a of
the protonatable lipid in the lipid mixture. Examples of suitable
buffers include citrate, phosphate, acetate, and MES. A
particularly preferred buffer is citrate buffer. Preferred buffers
will be in the range of 1-1000 mM of the anion, depending on the
chemistry of the nucleic acid being encapsulated, and optimization
of buffer concentration may be significant to achieving high
loading levels (see, e.g., U.S. Pat. No. 6,287,591 and U.S. Pat.
No. 6,858,225). Alternatively, pure water acidified to pH 5-6 with
chloride, sulfate or the like may be useful. In this case, it may
be suitable to add 5% glucose, or another non-ionic solute which
will balance the osmotic potential across the particle membrane
when the particles are dialyzed to remove ethanol, increase the pH,
or mixed with a pharmaceutically acceptable carrier such as normal
saline. The amount of nucleic acid in buffer can vary, but will
typically be from about 0.01 mg/mL to about 200 mg/mL, more
preferably from about 0.5 mg/mL to about 50 mg/mL.
[0355] The mixture of lipids and the buffered aqueous solution of
therapeutic nucleic acids is combined to provide an intermediate
mixture. The intermediate mixture is typically a mixture of lipid
particles having encapsulated nucleic acids. Additionally, the
intermediate mixture may also contain some portion of nucleic acids
which are attached to the surface of the lipid particles (liposomes
or lipid vesicles) due to the ionic attraction of the
negatively-charged nucleic acids and positively-charged lipids on
the lipid particle surface (the amino lipids or other lipid making
up the protonatable first lipid component are positively charged in
a buffer having a pH of less than the pK.sub.a of the protonatable
group on the lipid). In one group of preferred embodiments, the
mixture of lipids is an alcohol solution of lipids and the volumes
of each of the solutions are adjusted so that upon combination, the
resulting alcohol content is from about 20% by volume to about 45%
by volume. The method of combining the mixtures can include any of
a variety of processes, often depending upon the scale of
formulation produced. For example, when the total volume is about
10-20 mL or less, the solutions can be combined in a test tube and
stirred together using a vortex mixer. Large-scale processes can be
carried out in suitable production scale glassware.
[0356] Optionally, the lipid-encapsulated therapeutic agent (e.g.,
nucleic acid) complexes which are produced by combining the lipid
mixture and the buffered aqueous solution of therapeutic agents
(nucleic acids) can be sized to achieve a desired size range and
relatively narrow distribution of lipid particle sizes. Preferably,
the compositions provided herein will be sized to a mean diameter
of from about 70 to about 200 nm, more preferably about 90 to about
130 nm. Several techniques are available for sizing liposomes to a
desired size. One sizing method is described in U.S. Pat. No.
4,737,323, incorporated herein by reference. Sonicating a liposome
suspension either by bath or probe sonication produces a
progressive size reduction down to small unilamellar vesicles
(SUVs) less than about 0.05 microns in size. Homogenization is
another method which relies on shearing energy to fragment large
liposomes into smaller ones. In a typical homogenization procedure,
multilamellar vesicles are recirculated through a standard emulsion
homogenizer until selected liposome sizes, typically between about
0.1 and 0.5 microns, are observed. In both methods, the particle
size distribution can be monitored by conventional laser-beam
particle size determination. For certain methods herein, extrusion
is used to obtain a uniform vesicle size.
[0357] Extrusion of liposome compositions through a small-pore
polycarbonate membrane or an asymmetric ceramic membrane results in
a relatively well-defined size distribution. Typically, the
suspension is cycled through the membrane one or more times until
the desired liposome complex size distribution is achieved. The
liposomes may be extruded through successively smaller-pore
membranes, to achieve a gradual reduction in liposome size. In some
instances, the lipid-nucleic acid compositions which are formed can
be used without any sizing.
[0358] In particular embodiments, methods of the present invention
further comprise a step of neutralizing at least some of the
surface charges on the lipid portions of the lipid-nucleic acid
compositions. By at least partially neutralizing the surface
charges, unencapsulated nucleic acid is freed from the lipid
particle surface and can be removed from the composition using
conventional techniques. Preferably, unencapsulated and surface
adsorbed nucleic acids are removed from the resulting compositions
through exchange of buffer solutions. For example, replacement of a
citrate buffer (pH about 4.0, used for forming the compositions)
with a HEPES-buffered saline (HBS pH about 7.5) solution, results
in the neutralization of liposome surface and nucleic acid release
from the surface. The released nucleic acid can then be removed via
chromatography using standard methods, and then switched into a
buffer with a pH above the pKa of the lipid used.
[0359] Optionally the lipid vesicles (i.e., lipid particles) can be
formed by hydration in an aqueous buffer and sized using any of the
methods described above prior to addition of the nucleic acid. As
described above, the aqueous buffer should be of a pH below the pKa
of the amino lipid. A solution of the nucleic acids can then be
added to these sized, preformed vesicles. To allow encapsulation of
nucleic acids into such "pre-formed" vesicles the mixture should
contain an alcohol, such as ethanol. In the case of ethanol, it
should be present at a concentration of about 20% (w/w) to about
45% (w/w). In addition, it may be necessary to warm the mixture of
pre-formed vesicles and nucleic acid in the aqueous buffer-ethanol
mixture to a temperature of about 25.degree. C. to about 50.degree.
C. depending on the composition of the lipid vesicles and the
nature of the nucleic acid. It will be apparent to one of ordinary
skill in the art that optimization of the encapsulation process to
achieve a desired level of nucleic acid in the lipid vesicles will
require manipulation of variable such as ethanol concentration and
temperature. Examples of suitable conditions for nucleic acid
encapsulation are provided in the Examples. Once the nucleic acids
are encapsulated within the preformed vesicles, the external pH can
be increased to at least partially neutralize the surface charge.
Unencapsulated and surface adsorbed nucleic acids can then be
removed as described above.
[0360] Methods for Inhibiting Expression of the PCSK9 Gene
[0361] In yet another aspect, the invention provides a method for
inhibiting the expression of the PCSK9 gene in a mammal. The method
includes administering a composition of the invention to the mammal
such that expression of the target PCSK9 gene is decreased for an
extended duration, e.g., at least one week, two weeks, three weeks,
or four weeks or longer.
[0362] For example, in certain instances, expression of the PCSK9
gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, or 50% by administration of a double-stranded
oligonucleotide described herein. In some embodiments, the PCSK9
gene is suppressed by at least about 60%, 70%, or 80% by
administration of the double-stranded oligonucleotide. In some
embodiments, the PCSK9 gene is suppressed by at least about 85%,
90%, or 95% by administration of the double-stranded
oligonucleotide. Table 1b, Table 2b, and Table 5b provide a wide
range of values for inhibition of expression obtained in an in
vitro assay using various PCSK9 dsRNA molecules at various
concentrations.
[0363] The effect of the decreased target PCSK9 gene preferably
results in a decrease in LDLc (low density lipoprotein cholesterol)
levels in the blood, and more particularly in the serum, of the
mammal. In some embodiments, LDLc levels are decreased by at least
10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to
pretreatment levels.
[0364] The method includes administering a composition containing a
dsRNA, where the dsRNA has a nucleotide sequence that is
complementary to at least a part of an RNA transcript of the PCSK9
gene of the mammal to be treated. When the organism to be treated
is a mammal such as a human, the composition can be administered by
any means known in the art including, but not limited to oral or
parenteral routes, including intravenous, intramuscular,
subcutaneous, transdermal, and airway (aerosol) administration. In
some embodiments, the compositions are administered by intravenous
infusion or injection.
[0365] The methods and compositions described herein can be used to
treat diseases and conditions that can be modulated by down
regulating PCSK9 gene expression. For example, the compositions
described herein can be used to treat hyperlipidemia and other
forms of lipid imbalance such as hypercholesterolemia,
hypertriglyceridemia and the pathological conditions associated
with these disorders such as heart and circulatory diseases. In
some embodiments, a patient treated with a PCSK9 dsRNA is also
administered a non-dsRNA therapeutic agent, such as an agent known
to treat lipid disorders.
[0366] In one aspect, the invention provides a method of inhibiting
the expression of the PCSK9 gene in a subject, e.g., a human. The
method includes administering a first single dose of dsRNA, e.g., a
dose sufficient to depress levels of PCSK9 mRNA for at least 5,
more preferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally,
administering a second single dose of dsRNA, wherein the second
single dose is administered at least 5, more preferably 7, 10, 14,
21, 25, 30 or 40 days after the first single dose is administered,
thereby inhibiting the expression of the PCSK9 gene in a
subject.
[0367] In one embodiment, doses of dsRNA are administered not more
than once every four weeks, not more than once every three weeks,
not more than once every two weeks, or not more than once every
week. In another embodiment, the administrations can be maintained
for one, two, three, or six months, or one year or longer.
[0368] In another embodiment, administration can be provided when
Low Density Lipoprotein cholesterol (LDLc) levels reach or surpass
a predetermined minimal level, such as greater than 70 mg/dL, 130
mg/dL, 150 mg/dL, 200 mg/dL, 300 mg/dL, or 400 mg/dL.
[0369] In one embodiment, the subject is selected, at least in
part, on the basis of needing (as opposed to merely selecting a
patient on the grounds of who happens to be in need of) LDL
lowering, LDL lowering without lowering of HDL, ApoB lowering, or
total cholesterol lowering without HDL lowering.
[0370] In one embodiment, the dsRNA does not activate the immune
system, e.g., it does not increase cytokine levels, such as
TNF-alpha or IFN-alpha levels. For example, when measured by an
assay, such as an in vitro PBMC assay, such as described herein,
the increase in levels of TNF-alpha or IFN-alpha, is less than 30%,
20%, or 10% of control cells treated with a control dsRNA, such as
a dsRNA that does not target PCSK9.
[0371] In one aspect, the invention provides a method for treating,
preventing or managing a disorder, pathological process or symptom,
which, for example, can be mediated by down regulating PCSK9 gene
expression in a subject, such as a human subject. In one
embodiment, the disorder is hyperlipidemia. The method includes
administering a first single dose of dsRNA, e.g., a dose sufficient
to depress levels of PCSK9 mRNA for at least 5, more preferably 7,
10, 14, 21, 25, 30 or 40 days; and optionally, administering a
second single dose of dsRNA, wherein the second single dose is
administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or
40 days after the first single dose is administered, thereby
inhibiting the expression of the PCSK9 gene in a subject.
[0372] In another embodiment, a composition containing a dsRNA
featured in the invention, i.e., a dsRNA targeting PCSK9, is
administered with a non-dsRNA therapeutic agent, such as an agent
known to treat a lipid disorders, such as hypercholesterolemia,
atherosclerosis or dyslipidemia. For example, a dsRNA featured in
the invention can be administered with, e.g., an HMG-CoA reductase
inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant,
niacin, an antiplatelet agent, an angiotensin converting enzyme
inhibitor, an angiotensin II receptor antagonist (e.g., losartan
potassium, such as Merck & Co.'s Cozaar.RTM.), an acylCoA
cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol
absorption inhibitor, a cholesterol ester transfer protein (CETP)
inhibitor, a microsomal triglyceride transfer protein (MTTP)
inhibitor, a cholesterol modulator, a bile acid modulator, a
peroxisome proliferation activated receptor (PPAR) agonist, a
gene-based therapy, a composite vascular protectant (e.g.,
AGI-1067, from Atherogenics), a glycoprotein IIb/IIIa inhibitor,
aspirin or an aspirin-like compound, an IBAT inhibitor (e.g.,
S-8921, from Shionogi), a squalene synthase inhibitor, or a
monocyte chemoattractant protein (MCP)-I inhibitor. Exemplary
HMG-CoA reductase inhibitors include atorvastatin (Pfizer's
Lipitor.RTM./Tahor/Sortis/Torvast/Cardyl), pravastatin
(Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav),
simvastatin (Merck's Zocor.RTM./Sinvacor, Boehringer Ingelheim's
Denan, Banyu's Lipovas), lovastatin (Merck's Mevacor/Mevinacor,
Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler), fluvastatin
(Novartis' Lescol.RTM./Locol/Lochol, Fujisawa's Cranoc, Solvay's
Digaril), cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol),
rosuvastatin (AstraZeneca's Crestor.RTM.), and pitivastatin
(itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo,
and Novartis). Exemplary fibrates include, e.g., bezafibrate (e.g.,
Roche's Befizal.RTM./Cedur.RTM./Bezalip.RTM., Kissei's Bezatol),
clofibrate (e.g., Wyeth's Atromid-S.RTM.), fenofibrate (e.g.,
Fournier's Lipidil/Lipantil, Abbott's Tricor.RTM., Takeda's
Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid/Lipur) and
ciprofibrate (Sanofi-Synthelabo's Modalim.RTM.). Exemplary bile
acid sequestrants include, e.g., cholestyramine (Bristol-Myers
Squibb's Questran.RTM. and Questran Light.TM.), colestipol (e.g.,
Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's
WelChol.TM.). Exemplary niacin therapies include, e.g., immediate
release formulations, such as Aventis' Nicobid, Upsher-Smith's
Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Niacin
extended release formulations include, e.g., Kos Pharmaceuticals'
Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet
agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel
(Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine
(e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Other
aspirin-like compounds useful in combination with a dsRNA targeting
PCSK9 include, e.g., Asacard (slow-release aspirin, by Pharmacia)
and Pamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplary
angiotensin-converting enzyme inhibitors include, e.g., ramipril
(e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s
Vasotec). Exemplary acyl CoA cholesterol acetyltransferase (ACAT)
inhibitors include, e.g., avasimibe (Pfizer), eflucimibe
(BioM{acute over (.epsilon.)}rieux Pierre Fabre/Eli Lilly), CS-505
(Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterol
absorption inhibitors include, e.g., ezetimibe
(Merck/Schering-Plough Pharmaceuticals Zetia.RTM.) and Pamaqueside
(Pfizer). Exemplary CETP inhibitors include, e.g., Torcetrapib
(also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and
CETi-I (Avant Immunotherapeutics). Exemplary microsomal
triglyceride transfer protein (MTTP) inhibitors include, e.g.,
implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer).
Other exemplary cholesterol modulators include, e.g., NO-1886
(Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433
(Wyeth-Ayerst). Exemplary bile acid modulators include, e.g.,
HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group),
BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and
AZD-7806 (AstraZeneca). Exemplary peroxisome proliferation
activated receptor (PPAR) agonists include, e.g., tesaglitazar
(AZ-242) (AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson
& Johnson), GW-409544 (Ligand
Pharniaceuticals/GlaxoSmithKline), GW-501516 (Ligand
Pharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals
and Eli Lilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly),
LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck
and Kyorin). Exemplary gene-based therapies include, e.g.,
AdGWEGF121.10 (GenVec), ApoAl (UCB Pharma/Groupe Fournier), EG-004
(Trinam) (Ark Therapeutics), and ATP-binding cassette
transporter-A1 (ABCA1) (CV Therapeutics/Incyte, Aventis, Xenon).
Exemplary Glycoprotein Ilb/IIIa inhibitors include, e.g., roxifiban
(also called DMP754, Bristol-Myers Squibb), Gantofiban (Merck
KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals).
Exemplary squalene synthase inhibitors include, e.g., BMS-1884941
(Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer),
CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I
inhibitor is, e.g., RS-504393 (Roche Bioscience). The
anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the
nicotinic acid derivative Nyclin (Yamanouchi Pharmacuticals) are
also appropriate for administering in combination with a dsRNA
featured in the invention. Exemplary combination therapies suitable
for administration with a dsRNA targeting PCSK9 include, e.g.,
advicor (Niacin/lovastatin from Kos Pharmaceuticals),
amlodipine/atorvastatin (Pfizer), and ezetimibe/simvastatin (e.g.,
Vytorin.RTM. 10/10, 10/20, 10/40, and 10/80 tablets by
Merck/Schering-Plough Pharmaceuticals). Agents for treating
hypercholesterolemia, and suitable for administration in
combination with a dsRNA targeting PCSK9 include, e.g., lovastatin,
niacin Altoprev.RTM. Extended-Release Tablets (Andrx Labs),
lovastatin Caduet.RTM. Tablets (Pfizer), amlodipine besylate,
atorvastatin calcium Crestor.RTM. Tablets (AstraZeneca),
rosuvastatin calcium Lescol.RTM. Capsules (Novartis), fluvastatin
sodium Lescol.RTM. (Reliant, Novartis), fluvastatin sodium
Lipitor.RTM. Tablets (Parke-Davis), atorvastatin calcium
Lofibra.RTM. Capsules (Gate), Niaspan Extended-Release Tablets
(Kos), niacin Pravachol Tablets (Bristol-Myers Squibb), pravastatin
sodium TriCor.RTM. Tablets (Abbott), fenofibrate Vytorin.RTM. 10/10
Tablets (Merck/Schering-Plough Pharmaceuticals), ezetimibe,
simvastatin WelChol.TM. Tablets (Sankyo), colesevelam hydrochloride
Zetia.RTM. Tablets (Schering), ezetimibe Zetia.RTM. Tablets
(Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor.RTM.
Tablets (Merck).
[0373] In one embodiment, a dsRNA targeting PCSK9 is administered
in combination with an ezetimibe/simvastatin combination (e.g.,
Vytorin.RTM. (Merck/Schering-Plough Pharmaceuticals)).
[0374] In one embodiment, the PCSK9 dsRNA is administered to the
patient, and then the non-dsRNA agent is administered to the
patient (or vice versa). In another embodiment, the PCSK9 dsRNA and
the non-dsRNA therapeutic agent are administered at the same
time.
[0375] In another aspect, the invention features, a method of
instructing an end user, e.g., a caregiver or a subject, on how to
administer a dsRNA described herein. The method includes,
optionally, providing the end user with one or more doses of the
dsRNA, and instructing the end user to administer the dsRNA on a
regimen described herein, thereby instructing the end user.
[0376] In yet another aspect, the invention provides a method of
treating a patient by selecting a patient on the basis that the
patient is in need of LDL lowering, LDL lowering without lowering
of HDL, ApoB lowering, or total cholesterol lowering. The method
includes administering to the patient a dsRNA targeting PCSK9 in an
amount sufficient to lower the patient's LDL levels or ApoB levels,
e.g., without substantially lowering HDL levels.
[0377] In another aspect, the invention provides a method of
treating a patient by selecting a patient on the basis that the
patient is in need of lowered ApoB levels, and administering to the
patient a dsRNA targeting PCSK9 in an amount sufficient to lower
the patient's ApoB levels. In one embodiment, the amount of PCSK9
is sufficient to lower LDL levels as well as ApoB levels. In
another embodiment, administration of the PCSK9 dsRNA does not
affect the level of HDL cholesterol in the patient.
[0378] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
Gene Walking of the PCSK9 Gene
[0379] siRNA Design was Carried Out to Identify in Two Separate
Selections
[0380] a) siRNAs targeting PCSK9 human and either mouse or rat mRNA
and
[0381] b) all human reactive siRNAs with predicted specificity to
the target gene PCSK9.
[0382] mRNA sequences to human, mouse and rat PCSK9 were used:
Human sequence NM_174936.2 was used as reference sequence during
the complete siRNA selection procedure.
[0383] 19 mer stretches conserved in human and mouse, and human and
rat PCSK9 mRNA sequences were identified in the first step,
resulting in the selection of siRNAs cross-reactive to human and
mouse, and siRNAs cross-reactive to human and rat targets
[0384] SiRNAs specifically targeting human PCSK9 were identified in
a second selection. All potential 19mer sequences of human PCSK9
were extracted and defined as candidate target sequences. Sequences
cross-reactive to human, monkey, and those cross-reactive to mouse,
rat, human and monkey are all listed in Tables 1a and 2a.
Chemically modified versions of those sequences and their activity
in both in vitro and in vivo assays are also listed in Tables 1a
and 2a. The data is described in the examples and in FIGS. 2-8.
[0385] In order to rank candidate target sequences and their
corresponding siRNAs and select appropriate ones, their predicted
potential for interacting with irrelevant targets (off-target
potential) was taken as a ranking parameter. siRNAs with low
off-target potential were defined as preferable and assumed to be
more specific in vivo.
[0386] For predicting siRNA-specific off-target potential, the
following assumptions were made:
[0387] 1) positions 2 to 9 (counting 5' to 3') of a strand (seed
region) may contribute more to off-target potential than rest of
sequence (non-seed and cleavage site region)
[0388] 2) positions 10 and 11 (counting 5' to 3') of a strand
(cleavage site region) may contribute more to off-target potential
than non-seed region
[0389] 3) positions 1 and 19 of each strand are not relevant for
off-target interactions
[0390] 4) an off-target score can be calculated for each gene and
each strand, based on complementarity of siRNA strand sequence to
the gene's sequence and position of mismatches
[0391] 5) number of predicted off-targets as well as highest
off-target score must be considered for off-target potential
[0392] 6) off-target scores are to be considered more relevant for
off-target potential than numbers of off-targets
[0393] 7) assuming potential abortion of sense strand activity by
internal modifications introduced, only off-target potential of
antisense strand will be relevant
[0394] To identify potential off-target genes, 19mer candidate
sequences were subjected to a homology search against publically
available human mRNA sequences.
[0395] The following off-target properties for each 19mer input
sequence were extracted for each off-target gene to calculate the
off-target score:
[0396] Number of mismatches in non-seed region
[0397] Number of mismatches in seed region
[0398] Number of mismatches in cleavage site region
[0399] The off-target score was calculated for considering
assumption 1 to 3 as follows:
Off-target score=number of seed mismatches*10+number of cleavage
site mismatches*1.2+number of non-seed mismatches*1
[0400] The most relevant off-target gene for each siRNA
corresponding to the input 19mer sequence was defined as the gene
with the lowest off-target score. Accordingly, the lowest
off-target score was defined as the relevant off-target score for
each siRNA.
Example 2
dsRNA Synthesis
[0401] Source of Reagents
[0402] Where the source of a reagent is not specifically given
herein, such reagent may be obtained from any supplier of reagents
for molecular biology at a quality/purity standard for application
in molecular biology.
[0403] siRNA Synthesis
[0404] Single-stranded RNAs were produced by solid phase synthesis
on a scale of 1 .mu.mole using an Expedite 8909 synthesizer
(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany)
and controlled pore glass (CPG, 500 .ANG., Proligo Biochemie GmbH,
Hamburg, Germany) as solid support. RNA and RNA containing
2'-O-methyl nucleotides were generated by solid phase synthesis
employing the corresponding phosphoramidites and 2'-O-methyl
phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg,
Germany). These building blocks were incorporated at selected sites
within the sequence of the oligoribonucleotide chain using standard
nucleoside phosphoramidite chemistry such as described in Current
protocols in nucleic acid chemistry, Beaucage, S. L. et al.
(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA.
Phosphorothioate linkages were introduced by replacement of the
iodine oxidizer solution with a solution of the Beaucage reagent
(Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further
ancillary reagents were obtained from Mallinckrodt Baker
(Griesheim, Germany).
[0405] Deprotection and purification of the crude
oligoribonucleotides by anion exchange HPLC were carried out
according to established procedures. Yields and concentrations were
determined by UV absorption of a solution of the respective RNA at
a wavelength of 260 nm using a spectral photometer (DU 640B,
Beckman Coulter GmbH, Unterschlei.beta.heim, Germany). Double
stranded RNA was generated by mixing an equimolar solution of
complementary strands in annealing buffer (20 mM sodium phosphate,
pH 6.8; 100 mM sodium chloride), heated in a water bath at
85-90.degree. C. for 3 minutes and cooled to room temperature over
a period of 3-4 hours. The annealed RNA solution was stored at
-20.degree. C. until use.
[0406] Conjugated siRNAs
[0407] For the synthesis of 3'-cholesterol-conjugated siRNAs
(herein referred to as -Chol-3'), an appropriately modified solid
support was used for RNA synthesis. The modified solid support was
prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA
##STR00008##
[0409] A 4.7 M aqueous solution of sodium hydroxide (50 ml) was
added into a stirred, ice-cooled solution of ethyl glycinate
hydrochloride (32.19 g, 0.23 mole) in water (50 ml). Then, ethyl
acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred
at room temperature until completion of the reaction was
ascertained by TLC. After 19 h the solution was partitioned with
dichloromethane (3.times.100 ml). The organic layer was dried with
anhydrous sodium sulfate, filtered and evaporated. The residue was
distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl-
]-amino}-propionic acid ethyl ester AB
##STR00009##
[0411] Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was
dissolved in dichloromethane (50 ml) and cooled with ice.
Diisopropylcarbodiimde (3.25 g, 3.99 ml, 25.83 mmol) was added to
the solution at 0.degree. C. It was then followed by the addition
of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and
dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was
brought to room temperature and stirred further for 6 h. Completion
of the reaction was ascertained by TLC. The reaction mixture was
concentrated under vacuum and ethyl acetate was added to
precipitate diisopropyl urea. The suspension was filtered. The
filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium
bicarbonate and water. The combined organic layer was dried over
sodium sulfate and concentrated to give the crude product which was
purified by column chromatography (50% EtOAC/Hexanes) to yield
11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid
ethyl ester AC
##STR00010##
[0413]
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-he-
xanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol)
was dissolved in 20% piperidine in dimethylformamide at 0.degree.
C. The solution was continued stirring for 1 h. The reaction
mixture was concentrated under vacuum, water was added to the
residue, and the product was extracted with ethyl acetate. The
crude product was purified by conversion into its hydrochloride
salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,1-
5,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-h-
exanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester
AD
##STR00011##
[0415] The hydrochloride salt of
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid
ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane.
The suspension was cooled to 0.degree. C. on ice. To the suspension
diisopropylethylamine (3.87 g, 5.2 ml, 30 mmol) was added. To the
resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol)
was added. The reaction mixture was stirred overnight. The reaction
mixture was diluted with dichloromethane and washed with 10%
hydrochloric acid. The product was purified by flash chromatography
(10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15-
,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxyli-
c acid ethyl ester AE
##STR00012##
[0417] Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 ml
of dry toluene. The mixture was cooled to 0.degree. C. on ice and 5
g (6.6 mmol) of diester AD was added slowly with stirring within 20
mins. The temperature was kept below 5.degree. C. during the
addition. The stirring was continued for 30 mins at 0.degree. C.
and 1 ml of glacial acetic acid was added, immediately followed by
4 g of NaH.sub.2PO.sub.4.H.sub.2O in 40 ml of water The resultant
mixture was extracted twice with 100 ml of dichloromethane each and
the combined organic extracts were washed twice with 10 ml of
phosphate buffer each, dried, and evaporated to dryness. The
residue was dissolved in 60 ml of toluene, cooled to 0.degree. C.
and extracted with three 50 ml portions of cold pH 9.5 carbonate
buffer. The aqueous extracts were adjusted to pH 3 with phosphoric
acid, and extracted with five 40 ml portions of chloroform which
were combined, dried and evaporated to dryness. The residue was
purified by column chromatography using 25% ethylacetate/hexane to
afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic
acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF
##STR00013##
[0419] Methanol (2 ml) was added dropwise over a period of 1 h to a
refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium
borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 ml). Stirring
was continued at reflux temperature for 1 h. After cooling to room
temperature, 1 N HCl (12.5 ml) was added, the mixture was extracted
with ethylacetate (3.times.40 ml). The combined ethylacetate layer
was dried over anhydrous sodium sulfate and concentrated under
vacuum to yield the product which was purified by column
chromatography (10% MeOH/CHCl.sub.3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-
-yl}-6-oxo-hexyl)-carbamic acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG
##STR00014##
[0421] Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with
pyridine (2.times.5 ml) in vacuo. Anhydrous pyridine (10 ml) and
4,4'-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with
stirring. The reaction was carried out at room temperature
overnight. The reaction was quenched by the addition of methanol.
The reaction mixture was concentrated under vacuum and to the
residue dichloromethane (50 ml) was added. The organic layer was
washed with 1M aqueous sodium bicarbonate. The organic layer was
dried over anhydrous sodium sulfate, filtered and concentrated. The
residual pyridine was removed by evaporating with toluene. The
crude product was purified by column chromatography (2%
MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl.sub.3) (1.75 g, 95%).
Succinic acid
mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimet-
hyl-hexyl)-10,13-dimethyl
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H
cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)
ester AH
##STR00015##
[0423] Compound AG (1.0 g, 1.05 mmol) was mixed with succinic
anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and
dried in a vacuum at 40.degree. C. overnight. The mixture was
dissolved in anhydrous dichloroethane (3 ml), triethylamine (0.318
g, 0.440 ml, 3.15 mmol) was added and the solution was stirred at
room temperature under argon atmosphere for 16 h. It was then
diluted with dichloromethane (40 ml) and washed with ice cold
aqueous citric acid (5 wt %, 30 ml) and water (2.times.20 ml). The
organic phase was dried over anhydrous sodium sulfate and
concentrated to dryness. The residue was used as such for the next
step.
[0424] Cholesterol Derivatised CPG AI
##STR00016##
[0425] Succinate AH (0.254 g, 0.242 mmol) was dissolved in a
mixture of dichloromethane/acetonitrile (3:2, 3 ml). To that
solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 ml),
2,2'-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in
acetonitrile/dichloroethane (3:1, 1.25 ml) were added successively.
To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol)
in acetonitrile (0.6 ml) was added. The reaction mixture turned
bright orange in color. The solution was agitated briefly using a
wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG)
(1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The
CPG was filtered through a sintered funnel and washed with
acetonitrile, dichloromethane and ether successively. Unreacted
amino groups were masked using acetic anhydride/pyridine. The
achieved loading of the CPG was measured by taking UV measurement
(37 mM/g).
[0426] The synthesis of siRNAs bearing a 5'-12-dodecanoic acid
bisdecylamide group (herein referred to as "5'-C32-") or a
5'-cholesteryl derivative group (herein referred to as "5'-Chol-")
was performed as described in WO 2004/065601, except that, for the
cholesteryl derivative, the oxidation step was performed using the
Beaucage reagent in order to introduce a phosphorothioate linkage
at the 5'-end of the nucleic acid oligomer.
[0427] Synthesis of dsRNAs conjugated to Chol-p-(GalNAc).sub.3
(N-acetyl galactosamine-cholesterol) (FIG. 16) and
LCO(GalNAc).sub.3 (N-acetyl galactosamine-3'-Lithocholic-oleoyl)
(FIG. 17) is described in U.S. patent application Ser. No.
12/328,528, filed on Dec. 4, 2008, which is hereby incorporated by
reference.
Example 3
PCSK9 siRNA Screening in HuH7, HepG2, HeLa and Primary Monkey
Hepatocytes Discovers Highly Active Sequences
[0428] HuH-7cells were obtained from JCRB Cell Bank (Japanese
Collection of Research Bioresources) (Shinjuku, Japan, cat. No.:
JCRB0403) Cells were cultured in Dulbecco's MEM (Biochrom AG,
Berlin, Germany, cat. No. F0435) supplemented to contain 10% fetal
calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115),
Penicillin 100 U/ml, Streptomycin 100 .mu.g/ml (Biochrom AG,
Berlin, Germany, cat. No. A2213) and 2 mM L-Glutamin (Biochrom AG,
Berlin, Germany, cat. No K0282) at 37.degree. C. in an atmosphere
with 5% CO.sub.2 in a humidified incubator (Heraeus HERAcell,
Kendro Laboratory Products, Langenselbold, Germany). HepG2 and HeLa
cells were obtained from American Type Culture Collection
(Rockville, Md., cat. No. HB-8065) and cultured in MEM (Gibco
Invitrogen, Karlsruhe, Germany, cat. No. 21090-022) supplemented to
contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany,
cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100 .mu.g/ml
(Biochrom AG, Berlin, Germany, cat. No. A2213), lx Non Essential
Amino Acids (Biochrom AG, Berlin, Germany, cat. No. K-0293), and 1
mM Sodium Pyruvate (Biochrom AG, Berlin, Germany, cat. No. L-0473)
at 37.degree. C. in an atmosphere with 5% CO.sub.2 in a humidified
incubator (Heraeus HERAcell, Kendro Laboratory Products,
Langenselbold, Germany).
[0429] For transfection with siRNA, HuH7, HepG2, or HeLa cells were
seeded at a density of 2.0.times.10.sup.4 cells/well in 96-well
plates and transfected directly. Transfection of siRNA (30 nM for
single dose screen) was carried out with lipofectamine 2000
(Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as
described by the manufacturer.
[0430] 24 hours after transfection HuH7 and HepG2 cells were lysed
and PCSK9 mRNA levels were quantified with the Quantigene Explore
Kit (Genosprectra, Dumbarton Circle Fremont, USA, cat. No.
QG-000-02) according to the protocol. PCSK9 mRNA levels were
normalized to GAP-DH mRNA. For each siRNA eight individual
datapoints were collected. siRNA duplexes unrelated to PCSK9 gene
were used as control. The activity of a given PCSK9 specific siRNA
duplex was expressed as percent PCSK9 mRNA concentration in treated
cells relative to PCSK9 mRNA concentration in cells treated with
the control siRNA duplex.
[0431] Primary cynomolgus monkey hepatocytes (cryopreserved) were
obtained from In vitro Technologies, Inc. (Baltimore, Md., USA, cat
No MO0305) and cultured in InVitroGRO CP Medium (cat No Z99029) at
37.degree. C. in an atmosphere with 5% CO.sub.2 in a humidified
incubator.
[0432] For transfection with siRNA, primary cynomolgus monkey cells
were seeded on Collagen coated plates (Fisher Scientific, cat. No.
08-774-5) at a density of 3.5.times.104 cells/well in 96-well
plates and transfected directly. Transfection of siRNA (eight
2-fold dilution series starting from 30 nM) in duplicates was
carried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe,
Germany, cat. No. 11668-019) as described by the manufacturer.
[0433] 16 hours after transfection medium was changed to fresh
InVitroGRO CP Medium with Torpedo Antibiotic Mix (In vitro
Technologies, Inc, cat. No Z99000) added.
[0434] 24 hours after medium change primary cynomolgus monkey cells
were lysed and PCSK9 mRNA levels were quantified with the
Quantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont,
USA, cat. No. QG-000-02) according to the protocol. PCSK9 mRNA
levels were normalized to GAPDH mRNA. Normalized PCSK9/GAPDH ratios
were then compared to PCSK9/GAPDH ratio of lipofectamine 2000 only
control.
[0435] Tables 1b and 2b (and FIG. 6A) summarize the results and
provide examples of in vitro screens in different cell lines at
different doses. Silencing of PCSK9 transcript was expressed as
percentage of remaining transcript at a given dose.
[0436] Highly active sequences are those with less than 70%
transcript remaining post treatment with a given siRNA at a dose
less than or equal to 100 nM. Very active sequences are those that
have less than 60% of transcript remaining after treatment with a
dose less than or equal to 100 nM. Active sequences are those that
have less than 90% transcript remaining after treatment with a high
dose (100 nM).
[0437] Examples of active siRNAs were also screened in vivo in
mouse in lipidoid formulations as described below. Active sequences
in vitro were also generally active in vivo (See FIGS. 6A and 6B
and example 4).
Example 4
In Vivo Efficacy Screen of PCSK9 siRNAs in Mice
[0438] 32 PCSK9 siRNAs formulated in LNP-01 liposomes were tested
in vivo in a mouse model. LNP01 is a lipidoid formulation formed
from cholesterol, mPEG2000-C14 Glyceride, and dsRNA. The LNP01
formulation is useful for delivering dsRNAs to the liver.
[0439] Formulation Procedure
[0440] The lipidoid LNP-01.4HCl (MW 1487) (FIG. 1), Cholesterol
(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were
used to prepare lipid-siRNA nanoparticles. Stock solutions of each
in ethanol were prepared: LNP-01, 133 mg/ml; Cholesterol, 25 mg/ml,
PEG-Ceramide C16, 100 mg/ml. LNP-01, Cholesterol, and PEG-Ceramide
C16 stock solutions were then combined in a 42:48:10 molar ratio.
Combined lipid solution was mixed rapidly with aqueous siRNA (in
sodium acetate pH 5) such that the final ethanol concentration was
35-45% and the final sodium acetate concentration was 100-300 mM.
Lipid-siRNA nanoparticles formed spontaneously upon mixing.
Depending on the desired particle size distribution, the resultant
nanoparticle mixture was in some cases extruded through a
polycarbonate membrane (100 nm cut-off) using a thermobarrel
extruder (Lipex Extruder, Northern Lipids, Inc). In other cases,
the extrusion step was omitted. Ethanol removal and simultaneous
buffer exchange was accomplished by either dialysis or tangential
flow filtration. Buffer was exchanged to phosphate buffered saline
(PBS) pH 7.2.
[0441] Characterization of Formulations
[0442] Formulations prepared by either the standard or
extrusion-free method are characterized in a similar manner.
Formulations are first characterized by visual inspection. They
should be whitish translucent solutions free from aggregates or
sediment. Particle size and particle size distribution of
lipid-nanoparticles are measured by dynamic light scattering using
a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be
20-300 nm, and ideally, 40-100 nm in size. The particle size
distribution should be unimodal. The total siRNA concentration in
the formulation, as well as the entrapped fraction, is estimated
using a dye exclusion assay. A sample of the formulated siRNA is
incubated with the RNA-binding dye Ribogreen (Molecular Probes) in
the presence or absence of a formulation disrupting surfactant,
0.5% Triton-X100. The total siRNA in the formulation is determined
by the signal from the sample containing the surfactant, relative
to a standard curve. The entrapped fraction is determined by
subtracting the "free" siRNA content (as measured by the signal in
the absence of surfactant) from the total siRNA content. Percent
entrapped siRNA is typically >85%.
[0443] Bolus Dosing
[0444] Bolus dosing of formulated siRNAs in C57/BL6 mice (5/group,
8-10 weeks old, Charles River Laboratories, MA) was performed by
tail vein injection using a 27 G needle. SiRNAs were formulated in
LNP-01 (and then dialyzed against PBS) at 0.5 mg/ml concentration
allowing the delivery of the 5 mg/kg dose in 10 .mu.l/g body
weight. Mice were kept under an infrared lamp for approximately 3
min prior to dosing to ease injection.
[0445] 48 hour post dosing mice were sacrificed by
CO.sub.2-asphyxiation. 0.2 ml blood was collected by retro-orbital
bleeding and the liver was harvested and frozen in liquid nitrogen.
Serum and livers were stored at -80.degree. C. .mu.l
[0446] Frozen livers were grinded using 6850 Freezer/Mill Cryogenic
Grinder (SPEX CentriPrep, Inc) and powders stored at -80.degree. C.
until analysis.
[0447] PCSK9 mRNA levels were detected using the branched-DNA
technology based kit from QuantiGene Reagent System (Genospectra)
according to the protocol. 10-20 mg of frozen liver powders was
lysed in 600 .mu.l of 0.16 .mu.g/ml Proteinase K (Epicentre,
#MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H)
at 65.degree. C. for 3 hours. Then 10 .mu.l of the lysates were
added to 90 .mu.l of Lysis Working Reagent (1 volume of stock Lysis
Mixture in two volumes of water) and incubated at 52.degree. C.
overnight on Genospectra capture plates with probe sets specific to
mouse PCSK9 and mouse GAPDH or cyclophilin B. Nucleic acid
sequences for Capture Extender (CE), Label Extender (LE) and
blocking (BL) probes were selected from the nucleic acid sequences
of PCSK9, GAPDH and cyclophilin B with the help of the QuantiGene
ProbeDesigner Software 2.0 (Genospectra, Fremont, Calif., USA, cat.
No. QG-002-02). Chemo luminescence was read on a Victor2-Light
(Perkin Elmer) as Relative light units. The ratio of PCSK9 mRNA to
GAPDH or cyclophilin B mRNA in liver lysates was averaged over each
treatment group and compared to a control group treated with PBS or
a control group treated with an unrelated siRNA (blood coagulation
factor VII).
[0448] Total serum cholesterol in mouse serum was measured using
the StanBio Cholesterol LiquiColor kit (StanBio Laboratory, Boerne,
Tex., USA) according to manufacturer's instructions. Measurements
were taken on a Victor2 1420 Multilabel Counter (Perkin Elmer) at
495 nm.
[0449] Results
[0450] At least 10 PCSK9 siRNAs showed more than 40% PCSK9 mRNA
knock down compared to a control group treated with PBS, while
control group treated with an unrelated siRNA (blood coagulation
factor VII) had no effect (FIGS. 2-3). Silencing of PCSK9
transcript also correlated with a lowering of total serum
cholesterol in these animals (FIGS. 4-5). The most efficacious
siRNAs with respect to knocking down PCSK9 mRNAs also showed the
most pronounced cholesterol lowering effects (compare FIGS. 2-3 and
FIGS. 4-5). In addition there was a strong correlation between
those molecules that were active in vitro and those active in vivo
(compare FIGS. 6A and 6B).
[0451] Sequences containing different chemical modifications were
also screened in vitro (Tables 1 and 2) and in vivo. As an example,
less modified sequences AD-9314 and AD-9318, and a more modified
versions of that sequence AD-9314 (AD-10792, AD-10793, and
AD-10796); AD-9318-(AD-10794, AD-10795, AD-10797) were tested both
in vitro (in primary monkey hepatocytes) or in vivo (AD-9314 and
AD-10792) formulated in LNP-01. FIG. 7 (also see Tables 1 and 2)
shows that the parent molecules AD-9314 and AD-9318 and the
modified versions were all active in vitro. FIG. 8 as an example
shows that both the parent AD-9314 and the more highly modified
AD-10792 sequences were active in vivo displaying 50-60% silencing
of endogenous PCSK9 in mice. FIG. 9 further exemplifies that
activity of other chemically modified versions of AD-9314 and
AD-0792.
[0452] AD-3511, a derivative of AD-10792, was as efficacious as
10792 (IC50 of .about.0.07-0.2 nM) (data not shown). The sequences
of the sense and antisense strands of AD-3511 are as follows:
TABLE-US-00008 Sense strand: SEQ ID NO: 1521
5'-GccuGGAGuuuAuucGGAAdTsdT Antisense strand: SEQ ID NO: 1522
5'-puUCCGAAuAAACUCcAGGCdTsdT
Example 5
PCSK9 Duration of Action Experiments in Rats and NHP
[0453] Rats
[0454] Rats were treated via tail vein injection with 5 mg/kg of
LNP01-10792 (Formulated ALDP-10792). Blood was drawn at the
indicated time points (see Table 3) and the amount of total
cholesterol compared to PBS treated animals was measured by
standard means. Total cholesterol levels decreased at day two
.about.60% and returned to baseline by day 28. These data show that
formulated versions of PCSK9 siRNAs lower cholesterol levels for
extended periods of time.
[0455] Monkeys
[0456] Cynomolgus monkeys were treated with LNP01 formulated dsRNA
and LDL-C levels were evaluated. A total of 19 cynomolgus monkeys
were assigned to dose groups. Beginning on Day -11, animals were
limit-fed twice-a-day according to the following schedule: feeding
at 9 a.m., feed removal at 10 a.m., feeding at 4 p.m., feed removal
at 5 p.m. On the first day of dosing all animals were dosed once
via 30-minute intravenous infusion. The animals were evaluated for
changes in clinical signs, body weight, and clinical pathology
indices, including direct LDL and HDL cholesterol.
[0457] Venipuncture through the femoral vein was used to collect
blood samples. Samples were collected prior to the morning feeding
(i.e., before 9 a.m.) and at approximately 4 hours (beginning at 1
p.m.) after the morning feeding on Days -3, -1, 3, 4, 5, and 7 for
Groups 1-7; on Day 14 for Groups 1, 4, and 6; on Days 18 and 21 for
Group 1; and on Day 21 for Groups 4 and 6. At least two 1.0 ml
samples were collected at each time point.
[0458] No anticoagulant was added to the 1.0 ml serum samples, and
the dry anticoagulant Ethylenediaminetetraacetic acid (K2) was
added to each 1.0 ml plasma sample. Serum samples were allowed to
stand at room temperature for at least 20 minutes to facilitate
coagulation and then the samples were placed on ice. Plasma samples
were placed on ice as soon as possible following sample collection.
Samples were transported to the clinical pathology lab within 30
minutes for further processing.
[0459] Blood samples were processed to serum or plasma as soon as
possible using a refrigerated centrifuge, per Testing Facility
Standard operating procedure. Each sample was split into 3
approximately equal volumes, quickly frozen in liquid nitrogen, and
placed at -70.degree. C. Each aliquot should have had a minimum of
approximately 50 .mu.L. If the total sample volume collected was
under 150 .mu.L, the residual sample volume went into the last
tube. Each sample was labeled with the animal number, dose group,
day of collection, date, nominal collection time, and study
number(s). Serum LDL cholesterol was measured directly per standard
procedures on a Beckman analyzer according to manufactures
instructions.
[0460] The results are shown in Table 4. LNP01-10792 and LNP01-9680
administered at 5 mg/kg decreased serum LDL cholesterol within 3 to
7 days following dose administration. Serum LDL cholesterol
returned to baseline levels by Day 14 in most animals receiving
LNP01-10792 and by Day 21 in animals receiving LNP01-9680. This
data demonstrated a greater than 21 day duration of action for
cholesterol lowering of LNP01 formulated ALDP-9680.
Example 6
PCSK9 siRNAs Cause Decreased PCSK mRNA in Liver Extracts, and Lower
Serum Cholesterol Levels in Mice and Rats
[0461] To test if acute silencing of the PCSK9 transcript by a
PCSK9 siRNA (and subsequent PCSK9 protein down-regulation), would
result in acutely lower total cholesterol levels, siRNA molecule
AD-1a2 (AD-10792) was formulated in an LNP01 lipidoid formulation.
Sequences and modifications of these dsRNAs are shown in Table 5a.
Liposomal formulated siRNA duplex AD-1a2 (LNP01-1a2) was injected
via tail vein in low volumes (.about.0.2 ml for mouse and
.about.1.0 ml for rats) at different doses into C57/BL6 mice or
Sprague Dawley rats.
[0462] In mice, livers were harvested 48 hours post-injection, and
levels of PCSK9 transcript were determined. In addition to liver,
blood was harvested and subjected to a total cholesterol analysis.
LNP01-1a2 displayed a clear dose response with maximal PCSK9
message suppression (.about.60-70%) as compared to a control siRNA
targeting luciferase (LNP01-ctrl) or PBS treated animals (FIG.
14A). The decrease of PCSK9 transcript at the highest dose
translated into a .about.30% lowering of total cholesterol in mice
(FIG. 14B). This level of cholesterol reduction is between that
reported for heterozygous and homozygous PCSK9 knock-out mice
(Rashid et al., Proc. Natl. Acad. Sci. USA 102:5374-9, 2005, epub
Apr. 1, 2005). Thus, lowering of PCSK9 transcript through an RNAi
mechanism is capable of acutely decreasing total cholesterol in
mice. Moreover the effect on the PCSK9 transcript persisted between
20-30 days, with higher doses displaying greater initial transcript
level reduction, and subsequently more persistent effects.
[0463] Down-modulation of total cholesterol in rats has been
historically difficult as cholesterol levels remain unchanged even
at high doses of HMG-CoA reductase inhibitors. Interestingly, as
compared to mice, rats appear to have a much higher level of PCSK9
basal transcript levels as measured by bDNA assays. Rats were dosed
with a single injection of LNP01-a2 via tail vein at 1, 2.5 and 5
mg/kg. Liver tissue and blood were harvested 72 hours
post-injection. LNP01-1a2 exhibited a clear dose response effect
with maximal 50-60% silencing of the PCSK9 transcript at the
highest dose, as compared to a control luciferase siRNA and PBS
(FIG. 10A). The mRNA silencing was associate with an acute
.about.50-60% decrease of serum total cholesterol (FIGS. 10A and
10B) lasting 10 days, with a gradual return to pre-dose levels by
.about.3 weeks (FIG. 10B). This result demonstrated that lowering
of PCSK9 via siRNA targeting had acute, potent and lasting effects
on total cholesterol in the rat model system. To confirm that the
transcript reduction observed was due to a siRNA mechanism, liver
extracts from treated or control animals were subjected to 5' RACE,
a method previously utilized to demonstrate that the predicted
siRNA cleavage event occurs (Zimmermann et al., Nature. 441:111-4,
2006, Epub 2006 Mar. 26). PCR amplification and detection of the
predicted site specific mRNA cleavage event was observed in animals
treated with LNP01-1a2, but not PBS or LNP01-ctrl control animals.
(Frank-Kamanetsky et al. (2008) PNAS 105:119715-11920) This result
demonstrated that the effects of LNP01-1a2 observed were due to
cleavage of the PCSK9 transcript via an siRNA specific
mechanism.
[0464] The mechanism by which PCSK9 impacts cholesterol levels has
been linked to the number of LDLRs on the cell surface. Rats (as
opposed to mice, NHP, and humans) control their cholesterol levels
through tight regulation of cholesterol synthesis and to a lesser
degree through the control of LDLR levels. To investigate whether
modulation of LDLR was occurring upon RNAi therapeutic targeting of
PCSK9, we quantified the liver LDLR levels (via western blotting)
in rats treated with 5 mg/kg LNP01-1a2. As shown in FIG. 11,
LNP01-1a2 treated animals had a significant (.about.3-5 fold
average) induction of LDLR levels 48 hours post a single dose of
LNP01-1a2 compared to PBS or LNP01-ctrl control siRNA treated
animals.
[0465] Assays were also performed to test whether reduction of
PCSK9 changes the levels of triglycerides and cholesterol in the
liver itself. Acute lowering of genes involved in VLDL assembly and
secretion such as microsomal triglyceride transfer protein (MTP) or
ApoB by genetic deletion, compounds, or siRNA inhibitors results in
increased liver triglycerides (see, e.g., Akdim et al., Curr. Opin.
Lipidol. 18:397-400, 2007). Increased clearance of plasma
cholesterol induced by PCSK9 silencing in the liver (and a
subsequent increase in liver LDLR levels) was not predicted to
result in accumulation of liver triglycerides. However, to address
this possibility, liver cholesterol and triglyceride concentrations
in livers of the treated or control animals were quantified. As
shown in FIG. 10C, there was no statistical difference in liver TG
levels or cholesterol levels of rats administered PCSK9 siRNAs
compared to the controls. These results indicated that PCSK9
silencing and subsequent cholesterol lowering is unlikely to result
in excess hepatic lipid accumulation.
Example 7
Additional Modifications to siRNAs do not Affect Silencing and
Duration of Cholesterol Reduction in Rats
[0466] Phosphorothioate modifications at the 3' ends of both sense
and antisense strands of a dsRNA can protect against exonucleases.
2'OMe and 2'F modifications in both the sense and antisense strands
of a dsRNA can protect against endonucleases. AD-1a2 (see Table 5b)
contains 2'OMe modifications on both the sense and antisense
strands. Experiments were performed to determine if the inherent
stability (as measured by siRNA stability in human serum) or the
degree or type of chemical modification (2'OMe versus 2'F or a
mixture) was related to either the observed rat efficacy or the
duration of silencing effects. Stability of siRNAs with the same
AD-1a2 core sequence, but containing different chemical
modifications were created and tested for activity in vitro in
primary Cyno monkey hepatocytes. A series of these molecules that
maintained similar activity as measured by in vitro IC50 values for
PCSK9 silencing (Table 5b), were then tested for their stability
against exo and endonuclease cleavage in human serum. Each duplex
was incubated in human serum at 37.degree. C. (a time course), and
subjected to HPLC analysis. The parent sequence AD-1a2 had a T1/2
of .about.7 hours in pooled human serum. Sequences AD-1a3, AD-1a5,
and AD-1a4, which were more heavily modified (see chemical
modifications in Table 5) all had T1/2's greater than 24 hours. To
test whether the differences in chemical modification or stability
resulted in changes in efficacy, AD-1a2, AD-1a3, AD-1a5, AD-1a4,
and an AD-control sequence were formulated and injected into rats.
Blood was collected from animals at various days post-dose, and
total cholesterol concentrations were measured. Previous
experiments had shown a very tight correlation between the lowering
of PCSK9 transcript levels and total cholesterol values in rats
treated with LNP01-1a2 (FIG. 10A). All four molecules were observed
to decrease total cholesterol by .about.60% day 2 post-dose (versus
PBS or control siRNA), and all of the molecules had equal effects
on total cholesterol levels displaying similar magnitude and
duration profiles. There was no statistical difference in the
magnitude of cholesterol lowering and the duration of effect
demonstrated by these molecules, regardless of their different
chemistries or stabilities in human serum.
Example 8
LNP01-1a2 and LNP01-3a1 Silence Human PCSK9 and Circulating Human
PCSK9 Protein in Transgenic Mice
[0467] The efficacy of LNP01-1a2 (i.e., PCS-A2 or AD-10792) and
another molecule, AD-3a1 (i.e., PCS-C2 or AD-9736) (which targets
only human and monkey PCSK9 message), to silence the human PCSK9
gene was tested in vivo. A line of transgenic mice expressing human
PCSK9 under the ApoE promoter was used (Lagace et al., J. Clin
Invest. 116:2995-3005, 2006). Specific PCR reagents and antibodies
were designed that detected the human but not the mouse transcripts
and protein respectively. Cohorts of the humanized mice were
injected with a single dose of LNP01-1a2 (a.k.a. LNP-PCS-A2) or
LNP01-3a1 (a.k.a. LNP-PCS-C2), and 48 hours later both livers and
blood were collected. A single dose of LNP01-1a2 or LNP01-3a1 was
able to decrease the human PCSK9 transcript levels by >70% (FIG.
15A), and this transcript down-regulation resulted in significantly
lower levels of circulating human PCSK9 protein as measured by
ELISA (FIG. 15B). These results demonstrated that both siRNAs were
capable of silencing the human transcript and subsequently reducing
the amount of circulating plasma human PCSK9 protein.
Example 9
Secreted PCSK9 Levels are Regulated by Diet in NHP
[0468] In mice, PCSK9 mRNA levels are regulated by the
transcription factor sterol regulatory element binding protein-2
and are reduced by fasting. In clinical practice, and standard NHP
studies, blood collection and cholesterol levels are measured after
an overnight fasting period. This is due in part to the potential
for changes in circulating TGs to interfere with the calculation of
LDLc values. Given the regulation of PCSK9 levels by fasting and
feeding behavior in mice, experiments were performed to understand
the effect of fasting and feeding in NHP.
[0469] Cyno monkeys were acclimated to a twice daily feeding
schedule during which food was removed after a one hour period.
Animals were fed from 9-10 am in the morning, after which food was
removed. The animals were next fed once again for an hour between 5
pm-6 pm with subsequent food removal. Blood was drawn after an
overnight fast (6 pm until 9 am the next morning), and again, 2 and
4 hours following the 9 am feeding. PCSK9 levels in blood plasma or
serum were determined by ELISA assay (see Methods). Interestingly,
circulating PCSK9 levels were found to be higher after the
overnight fasting and decreased 2 and 4 hours after feeding. This
data was consistent with rodent models where PCSK9 levels were
highly regulated by food intake. However, unexpectedly, the levels
of PCSK9 went down the first few hours post-feeding. This result
enabled a more carefully designed NHP experiment to probe the
efficacy of formulated AD-1a2 and another PCSK9 siRNA (AD-2a1) that
was highly active in primary Cyno hepatocytes.
Example 10
PCSK9 siRNAs Reduce Circulating LDLc, ApoB, and PCSK9, but not HDLc
in Non-Human Primates (NHPs)
[0470] siRNAs targeting PCSK9 acutely lowered both PCSK9 and total
cholesterol levels by 72 hours post-dose and lasted .about.21-30
days after a single dose in mice and rats. To extend these findings
to a species whose lipoprotein profiles most closely mimic that of
humans, further experiments were performed in the Cynomologous
(Cyno) monkey model.
[0471] siRNA 1 (LNP01-10792) and siRNA 2 (LNP-01-9680), both
targeting PCSK9 were administered to cynomologous monkeys. As shown
in FIG. 12, both siRNAs caused significant lipid lowering for up to
7 days post administration. siRNA 2 caused .about.50% lipid
lowering for at least 7 days post-administration, and .about.60%
lipid lowering at day 14 post-administration, and siRNA 1 caused
.about.60% LDLc lowering for at least 7 days.
[0472] Male Cynos were first pre-screened for those that had LDLc
of 40 mg/dl or higher. Chosen animals were then put on a fasted/fed
diet regime and acclimated for 11 days. At day -3 and -1 pre-dose,
serum was drawn at both fasted and 4 hours post-fed time points and
analyzed for total cholesterol (Tc), LDL (LDLc), HDL cholesterol
(HDLc) as well as triglycerides (TG), and PCSK9 plasma levels.
Animals were randomized based on their day -3 LDLc levels. On the
day of dosing (designated day 1), either 1 mg/kg or 5 mg/kg of
LNP01-1a2 and 5 mg/kg LNP01-2a1 were injected, along with PBS and 1
mg/kg LNP01-ctrl as controls. All doses were well tolerated with no
in-life findings. As the experiment progressed it became apparent
(based on LDLc lowering) that the lower dose was not efficacious.
We therefore dosed the PBS group animals on day 14 with 5 mg/kg
LNP01-ctrl control siRNA, which could then serve as an additional
control for the high dose groups of 5 mg/kg LNP01-1a2 and 5 mg/kg
LNP01-2a1. Initially blood was drawn from animals on days 3, 4, 5,
and 7 post-dose and Tc, HDLc, LDLc, and TGs concentrations were
measured. Additional blood draws from the LNP01-1a2, LNP01-2a1 high
dose groups were carried out at day 14 and day 21 post-dose (as the
LDLc levels had not returned to baseline by day 7).
[0473] As shown in FIG. 12A, a single dose of LNP01-1a2 or
LNP01-2a1 resulted in a statistically significant reduction of LDLc
beginning at day 3 post-dose that returned to baseline over
.about.14 days (for LNP01-1a2) and .about.21 days (LNP01-2a1). This
effect was not seen in either the PBS, the control siRNA groups, or
the 1 mg/kg treatment groups. LNP01-2a1 resulted in an average
lowering of LDLc of 56% 72 hours post-dose, with 1 of 4 animals
achieving nearly 70% LDLc, and all others achieving >50% LDLc
decrease, as compared to pre-dose levels, (see FIG. 12A. As
expected, the lowering of LDLc in the treated animals also
correlated with a reduction of circulating ApoB levels as measured
by serum ELISA (FIG. 12B). Interestingly, the degree of LDLc
lowering observed in this study of Cyno monkey was greater than
those that have been reported for high dose statins, as well as,
for other current standard of care compounds used for
hypercholesterolemia. The onset of action is also much more acute
than that of statins with effects being seen as early as 48 hours
post-dose.
[0474] Neither LNP01-1a2 nor LNP01-2a1 treatments resulted in a
lowering of HDLc. In fact, both molecules resulted (on average) in
a trend towards a decreased Tc/HDL ratio (FIG. 12C). In addition,
circulating triglyceride levels, and with the exception of one
animal, ALT and AST levels were not significantly impacted.
[0475] PCSK9 protein levels were also measured in treated and
control animals. As shown in FIG. 11, LNP01-1a2 and LNP01-2a1
treatment each resulted in trends toward decreased circulating
PCSK9 protein levels versus pre-dose. Specifically, the more active
siRNA LNP01-2a1 demonstrated significant reduction of circulating
PCSK9 protein versus both PBS (day 3-21) and LNP01-ctrl siRNA
control (day 4, day 7).
Example 11
Modified siRNA and Activation of Immune Responses in hPBMCs
[0476] siRNAs were tested for activation of the immune system in
primary human blood monocytes (hPBMC). Two control inducing
sequences and the unmodified parental compound AD-1a1 was found to
induce both IFN-alpha and TNF-alpha. However, chemically modified
versions of this sequence (AD-1a2, AD-1a3, AD-1a5, and AD-1a4) as
well as AD-2a1 were negative for both IFN-alpha and TNF-alpha
induction in these same assays (see Table 5, and FIGS. 13A and
13B). Thus chemical modifications are capable of dampening both
IFN-alpha and TNF-alpha responses to siRNA molecules. In addition,
neither AD-1a2, nor AD-2a1 activated IFN-alpha when formulated into
liposomes and tested in mice.
Example 12
Evaluation of siRNA Conjugates in Mice
[0477] AD-10792 was conjugated to GalNAc)3/Cholesterol (FIG. 16) or
GalNAc)3/LCO (FIG. 17). The sense strand was synthesized with the
conjugate on the 3' end. The conjugated siRNAs were assayed for
effects on PCSK9 transcript levels and total serum cholesterol in
mice using the methods described below.
[0478] Briefly, mice were dosed via tail injection with one of the
2 conjugated siRNAs or PBS on three consecutive days: day 0, day 1
and day 2 with a dosage of about 100, 50, 25 or 12.5 mg/kg. Each
dosage group included 6 mice. 24 hour post last dosing mice were
sacrificed and blood and liver samples were obtained, stored, and
processed to determine PCSK9 mRNA levels and total serum
cholesterol.
[0479] The results are shown in FIG. 18. Compared to control PBS,
both siRNA conjugates demonstrated activity with an ED50 of
3.times.50 mg/kg for GalNAc)3/Cholesterol conjugated AD-10792 and
3.times.100 mg/kg for GalNAc)3/LCO conjugated AD-10792. The results
indicate that Cholesterol conjugated siRNA with GalNAc are active
and capable of silencing PCSK9 in the liver resulting in
cholesterol lowering.
[0480] Bolus Dosing
[0481] Bolus dosing of formulated siRNAs in C57/BL6 mice (6/group,
8-10 weeks old, Charles River Laboratories, MA) was performed by
tail vein injection using a 27 G needle. SiRNAs were formulated in
LNP-01 (and then dialyzed against PBS) and diluted with PBS to
concentrations 1.0, 0.5, 0.25 and 0.125 mg/ml allowing the delivery
of 100; 50; 25 and 12.5 mg/kg doses in 10 .mu.l/g body weight. Mice
were kept under an infrared lamp for approximately 3 min prior to
dosing to ease injection.
[0482] 24 hour post last dose mice were sacrificed by
CO2-asphyxiation. 0.2 ml blood was collected by retro-orbital
bleeding and the liver was harvested and frozen in liquid nitrogen.
Serum and livers were stored at -80.degree. C. Frozen livers were
grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep,
Inc) and powders stored at -80.degree. C. until analysis.
[0483] PCSK9 mRNA levels were detected using the branched-DNA
technology based kit from QuantiGene Reagent System (Panomics, USA)
according to the protocol. 10-20 mg of frozen liver powders was
lysed in 600 .mu.l of 0.16 .mu.g/ml Proteinase K (Epicentre,
#MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H)
at 65.degree. C. for 3 hours. Then 10 .mu.l of the lysates were
added to 90 .mu.l of Lysis Working Reagent (1 volume of stock Lysis
Mixture in two volumes of water) and incubated at 52.degree. C.
overnight on Genospectra capture plates with probe sets specific to
mouse PCSK9 and mouse GAPDH. Probes sets for mouse PCSK9 and mouse
GAPDH were purchased from Panomics, USA. Chemo luminescence was
read on a Victor2-Light (Perkin Elmer) as Relative light units. The
ratio of PCSK9 mRNA to mGAPDH mRNA in liver lysates was averaged
over each treatment group and compared to a control group treated
with PBS or a control group treated with an unrelated siRNA (blood
coagulation factor VII).
[0484] Total serum cholesterol in mouse serum was measured using
the Total Cholesterol Assay (Wako, USA) according to manufacturer's
instructions. Measurements were taken on a Victor2 1420 Multilabel
Counter (Perkin Elmer) at 600 nm.
Example 13
Evaluation of Lipid Formulated siRNAs in Rats
[0485] Briefly, rats were dosed via tail injection with SNALP
formulated siRNAs or PBS with a single dosage of about 0.3, 1.0,
and 3.0 mg/kg of SNALP formulated AD-10792. Each dosage group
included 5 rats. 72 hour post dosing rats were sacrificed and blood
and liver samples were obtained, stored, and processed to determine
PCSK9 mRNA and total serum cholesterol levels. The results are
shown in FIG. 19. Compared to control PBS, SNALP formulated
AD-10792 (FIG. 19A) had an ED50 of about 1.0 mg/kg for both
lowering of PCSK9 transcript levels and total serum cholesterol
levels. These results show that administration of SNALP formulated
siRNA results in effective and efficient silencing of PCSK9 and
subsequent lowering of total cholesterol in vivo.
[0486] Bolus Dosing
[0487] Bolus dosing of formulated siRNAs in Sprague-Dawley rats
(5/group, 170-190 g body weight, Charles River Laboratories, MA)
was performed by tail vein injection using a 27 G needle. SiRNAs
were formulated in SNALP (and then dialyzed against PBS) and
diluted with PBS to concentrations 0.066; 0.2 and 0.6 mg/ml
allowing the delivery of 0.3; 1.0 and 3.0 mg/kg of SNALP formulated
AD-10792 in 5 .mu.l/g body weight. Rats were kept under an infrared
lamp for approximately 3 min prior to dosing to ease injection.
[0488] 72 hour post last dose rats were sacrificed by
CO2-asphyxiation. 0.2 ml blood was collected by retro-orbital
bleeding and the liver was harvested and frozen in liquid nitrogen.
Serum and livers were stored at -80.degree. C. Frozen livers were
grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep,
Inc) and powders stored at -80.degree. C. until analysis.
[0489] PCSK9 mRNA levels were detected using the branched-DNA
technology based kit from QuantiGene Reagent System (Panomics, USA)
according to the protocol. 10-20 mg of frozen liver powders was
lysed in 600 .mu.l of 0.16 .mu.g/ml Proteinase K (Epicentre,
#MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H)
at 65.degree. C. for 3 hours. Then 10 .mu.l of the lysates were
added to 90 .mu.l of Lysis Working Reagent (1 volume of stock Lysis
Mixture in two volumes of water) and incubated at 52.degree. C.
overnight on Genospectra capture plates with probe sets specific to
rat PCSK9 and rat GAPDH. Probes sets for rat PCSK9 and rat GAPDH
were purchased from Panomics, USA. Chemo luminescence was read on a
Victor2-Light (Perkin Elmer) as Relative light units. The ratio of
rat PCSK9 mRNA to rat GAPDH mRNA in liver lysates was averaged over
each treatment group and compared to a control group treated with
PBS or a control group treated with an unrelated siRNA (blood
coagulation factor VII).
[0490] Total serum cholesterol in rat serum was measured using the
Total Cholesterol Assay (Wako, USA) according to manufacturer's
instructions. Measurements were taken on a Victor2 1420 Multilabel
Counter (Perkin Elmer) at 600 nm.
Example 14
In Vitro Efficacy Screen in HeLa Cells of Mismatch Walk of AD-9680
and AD-14676
[0491] The effects of variations in sequence or modification on the
effectiveness of AD-9680, AD-14676, and AD-10792 were assayed in
HeLa cells. A number of variants were synthesized as shown in Table
6 and include adding DFT (2,4-Difluorotoluyl, a thymidine
triphosphate shape analog lacking Watson-Crick pairing); adding
single or combination mismatches; and testing two different
backbone chemistries: leading with a 2'-O methyl, or alternating
with 2'F.
[0492] Sequences of the 3 parent duplexes can be found in Table 1a
and are duplicated as follows:
TABLE-US-00009 SEQ SEQ target Sense strand ID Antisense strand ID
region (5' to 3') NO: (5' to 3') NO: Duplex 3530-
uucuAGAccuGuuuuGcuuTsT 1229 AAGcAAAAcAGGUCuAGAATsT 1230 AD- 3548
9680 3530- UfuCfuAfgAfcCfuGfuUfuUfg 1231 P- 1232 AD- 3548 CfuUfTsT
aAfgCfaAfaAfcAfgGfuCfuAfgA 14676 faTsT 1091- GccuGGAGuuuAuucGGAATsT
459 UUCCGAAuAAACUCcAGGCTsT 460 AD- 1109 10792
[0493] HeLa were plated in 96-well plates (8,000-10,000 cells/well)
in 100 .mu.l 10% fetal bovine serum in Dulbecco's Modified Eagle
Medium (DMEM). When the cells reached approximately 50% confluence
(.about.24 hours later) they were transfected with serial four-fold
dilutions of siRNA starting at 10 nM. 0.4 .mu.l of transfection
reagent Lipofectamine.TM. 2000 (Invitrogen Corporation, Carlsbad,
Calif.) was used per well and transfections were performed
according to the manufacturer's protocol. Namely, the siRNA:
Lipofectamine.TM. 2000 complexes were prepared as follows. The
appropriate amount of siRNA was diluted in Opti-MEM I Reduced Serum
Medium without serum and mixed gently. The Lipofectamine.TM. 2000
was mixed gently before use, then for each well of a 96 well plate
0.4 .mu.l was diluted in 25 .mu.l of Opti-MEM I Reduced Serum
Medium without serum and mixed gently and incubated for 5 minutes
at room temperature. After the 5 minute incubation, 1 .mu.l of the
diluted siRNA was combined with the diluted Lipofectamine.TM. 2000
(total volume is 26.4 .mu.l). The complex was mixed gently and
incubated for 20 minutes at room temperature to allow the siRNA:
Lipofectamine.TM. 2000 complexes to form. Then 100 .mu.l of 10%
fetal bovine serum in DMEM was added to each of the
siRNA:Lipofectamine.TM. 2000 complexes and mixed gently by rocking
the plate back and forth. 100 .mu.l of the above mixture was added
to each well containing the cells and the plates were incubated at
37.degree. C. in a CO2 incubator for 24 hours, then the culture
medium was removed and 100 .mu.l 10% fetal bovine serum in DMEM was
added.
[0494] 24 hours post medium change medium was removed, cells were
lysed and cell lysates assayed for PCSK9 mRNA silencing by bDNA
assay (Panomics, USA) following the manufacturer's protocol. Chemo
luminescence was read on a Victor2-Light (Perkin Elmer) as Relative
light units. The ratio of human PCSK9 mRNA to human GAPDH mRNA in
cell lysates was compared to that of cells treated with
Lipofectamine.TM. 2000 only control.
[0495] FIG. 20 is dose response curves of a series of compounds
related to AD-9680. FIG. 21 is a dose response curve of a series of
compounds related to AD-14676 The results show that DFTs or
mismatches in certain positions are able increase the activity (as
evidenced by lower IC50 values) of both parent compounds. FIG. 24
is a dose response curve comparing the efficiency of parent
duplexes AD-9680 and AD-10792 with modified duplexes wherein a DFT
is inserted at position 10 of the sense strand. This modification
improves the efficiency by about 2 fold in HeLa cells.
[0496] Without being bound by theory, it is hypothesized that
destabilization of the sense strand through the introduction of
mismatches, or DFT might result in quicker removal of the sense
strand.
Example 15
Lack of Off Target Effects in Hep3B Cells at High
Concentrations
[0497] A lipid formulated PCSK9 targeted siRNA (AD-9680) was
transfected into Hep3B cells at concentrations of 250 nM, 1 uM and
5 uM in triplicates using the reagent RNAiMAX (Invitrogen)
according to the manufacture's instruction: lul of transfection
reagent; reverse transfection protocol. Samples were collected 48
hrs post transfection. Total RNA was purified using MagMAX.TM.-96
Total RNA Isolation Kit (Ambion); cDNA was synthesized with High
Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (ABI)
from 13.5 .mu.l of RNA prep; ABI Gene Expression Taqman assays were
used; q-PCR reactions were set up according to manufacturer's
instruction using TaqMan.RTM. Gene Expression Master Mix (ABI) and
run on ABI 7900 machine. Delta delta Ct method was used to
calculate values. Samples were normalized to hGAPDH and calibrated
to mock transfection.
[0498] Transcript levels were measured for the following genes
having the closest homology to the target sequence: ORMDL2, BMP6,
TAPT1, MYEF2, LOC442252, RFT1, and PCSK9.
[0499] The results are shown in FIG. 22. No off target effects were
observed at high concentrations of dsRNA (PCS-B2=AD-9680).
TABLE-US-00010 AD-9680 S 1531 uucuAGAccuGuuuuGcuudTsdT AS 1532
AAGcAAAAcAGGUCuAGAAdTsdT
Example 16
Maintenance of Decrease in Total Cholesterol Levels in Rats by
Lower Dosage of AD-10792
[0500] Rats were treated with 3 mg/kg bolus dose of SNALP-DlinDMA
formulated AD-10792. At day 2, total serum cholesterol levels were
determined. This was followed by once a week dosing at 1.0 and 0.3
mg/kg for four weeks. Rats were bled one day prior to repeat dosing
and total serum cholesterol levels were determined. The negative
control was PBS.
[0501] The results are shown in the graph of FIG. 23. After 3 mg/kg
bolus dose, total cholesterol levels decreased by 60% and were
maintained at about 50% by repeated once a week 1.0 and 0.3 mg/kg
dosing and come back to pre dose levels after repeated dosing is
stopped.
[0502] A 10 fold lower (than EC50), once a week, maintenance dose
effectively maintains silencing with cholesterol levels returning
to baseline by 15 days post last injection. The initial does of
PCSK9 increased LDLR levels as reflected by the decrease in total
serum cholesterol. This increase in LDLR levels increased the
efficacy of the PCSK9 targeted siRNA as reflected by the lower
dosage of subsequent administration AD-10792.
Example 17
Assay of Effects on Cholesterol Levels in Rats after Administration
of Various Lipid Formulations of AD-10792
[0503] Rats were treated with four different lipid formulations of
AD-10792 including SNALP and LNP08, described herein. At day 3,
total serum cholesterol levels were determined. The experiment was
performed using the methods described herein. Administration of
LNP-08 formulated AD-10792 results in the lowest EC50 of 0.08 mg/kg
compared to LNP01 formulated (EC50 of 2.0 mg/kg) and SNALP
formulated (EC50 of 1.0 mg/kg). (data not shown).
Example 18
PCSK9 siRNA Filing Experiment
[0504] Bioinformatic Selection of PCSK9 Tiling Set
[0505] Sense and antisense oligomers were designed to target the
human PCSK9 transcript in the flanking regions immediately upstream
and downstream of the 19 base target region of ALN-PCSK9 (AD-9680).
We used the NCBI Refseq NM_174936.2 as the reference human
transcript for the PCSK9 gene. The antisense oligo of AD-9680
contains 19 contiguous bases complementary to the bases in the
region of NM_174936 from positions 3530 through 3548 relative to
the start of the mRNA. A set of siRNA molecules was designed to
each unique 19mer of the subset of the transcript sequence defined
by 10 bases upstream from the 5' end to 10 bases downstream from
the 3' end of the target region of AD-9680. With respect to the
NM_174936.2 transcript, the first base at the 5' position of the
sense oligo 19mer extends from positions 3520 to positions 3558
(Tables 7 and 8).
[0506] Synthesis of PCSK9 Tiling Sequences:
[0507] PCSK9 sequences were synthesized on MerMade 192 synthesizer.
Two sets of sequences were made. The first set contained no
chemical modifications (unmodified) and a second set was made with
endolight chemical modifications. In sequences containing endolight
chemical modification, all pyrimidines (cytosine and uridine) in
the sense strand were replaced with corresponding 2'-O-Methyl bases
(2' 0-Methyl C and 2'-O-Methyl U). In the antisense strand,
pyrimidines adjacent to (towards 5' position) ribo A nucleoside
were replaced with their corresponding 2-O-Methyl nucleosides. A
two base dTsdT extension at the 3' end of both sense and anti sense
sequences was introduced. The sequence file was converted to a text
file to make it compatible for loading in the MerMade 192 synthesis
software.
[0508] The synthesis of PCSK9 sequences used solid supported
oligonucleotide synthesis using phosphoramidite chemistry. The
synthesis of the above sequences was performed at 1 .mu.m scale in
96 well plates. The amidite solutions were prepared at 0.1 M
concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was
used as activator. The synthesized sequences were cleaved and
deprotected in 96 well plates, using methylamine in the first step
and Fluoride ion in the second step. The crude sequences thus
obtained were precipitated using acetone: ethanol mix and the
pellet were re-suspended in 0.2M sodium acetate buffer. Samples
from each sequence were analyzed by LC-MS and the resulting mass
data confirmed the identity of the sequences. A selected set of
samples were also analyzed by IEX chromatography. All sequences
were purified on AKTA explorer purification system using Source 15Q
column. A single peak corresponding to the full length sequence was
collected in the eluent and was subsequently analyzed for purity by
ion exchange chromatography. The purified sequences were desalted
on a Sephadex G25 column using AKTA purifier. The desalted PCSK9
sequences were analyzed for concentration and purity. The single
strands were then submitted for annealing.
[0509] In Vitro Screening of PCSK9 Tiling siRNAs:
[0510] Cell Culture and Transfection:
[0511] Hela cells (ATCC, Manassas, Va.) were grown to near
confluence at 37.degree. C. in an atmosphere of 5% CO.sub.2 in
Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10%
FBS, streptomycin, and glutamine (ATCC) before being released from
the plate by trypsinization. Reverse transfection was carried out
by adding 5 .mu.l of Opti-MEM to 5 .mu.l of siRNA duplexes per well
into a 96-well plate along with 10 .mu.l of Opti-MEM plus 0.2 .mu.l
of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat
#13778-150) and incubated at room temperature for 15 minutes. 80
.mu.l of complete growth media without antibiotic containing
2.0.times.10.sup.4 Hela cells were then added. Cells were incubated
for 24 hours prior to RNA purification. Experiments were performed
at 0.1 or 10 nM final duplex concentration. For dose response
screens, HeLa cells were transfected with siRNAs over seven,
ten-fold serial dilutions from 1 nM to 1 fM.
[0512] Total RNA was isolated using MagMAX-96 Total RNA Isolation
Kit (Applied Biosystem, Forer City Calif., part #: AM1830). Cells
were harvested and lysed in 140 .mu.l of Lysis/Binding Solution
then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer
(the mixing speed was the same throughout the process). Twenty
micro liters of magnetic beads and Lysis/Binding Enhancer mixture
were added into cell-lysate and mixed for 5 minutes. Magnetic beads
were captured using magnetic stand and the supernatant was removed
without disturbing the beads. After removing supernatant, magnetic
beads were washed with Wash Solution 1 (isopropanol added) and
mixed for 1 minute. Beads were capture again and supernatant
removed. Beads were then washed with 150 .mu.l Wash Solution 2
(Ethanol added), captured and supernatant was removed. 50 .mu.l of
DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then
added to the beads and they were mixed for 10 to 15 minutes. After
mixing, 100 .mu.l of RNA Rebinding Solution was added and mixed for
3 minutes. Supernatant was removed and magnetic beads were washed
again with 150 .mu.l Wash Solution 2 and mixed for 1 minute and
supernatant was removed completely. The magnetic beads were mixed
for 2 minutes to dry before RNA was eluted with 50 .mu.l of
water.
[0513] cDNA was synthesized using ABI High capacity cDNA reverse
transcription kit (Applied Biosystems, Foster City, Calif., Cat
#4368813). A master mix of 2 .mu.l 10.times. Buffer, 0.8 .mu.l
25.times.dNTPs, 2 .mu.l Random primers, 1 .mu.l Reverse
Transcriptase, 1 .mu.l RNase inhibitor and 3.2 .mu.l of H.sub.2O
per reaction were added into 10 .mu.l total RNA. cDNA was generated
using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.)
through the following steps: 25.degree. C. 10 min, 37.degree. C.
120 min, 85.degree. C. 5 sec, 4.degree. C. hold.
[0514] Real time PCR was performed as follows. 2 .mu.l of cDNA were
added to a master mix containing 1 .mu.l GAPDH TaqMan Probe
(Applied Biosystems Cat #4326317E), 1 .mu.l PCSK9 TaqMan probe
(Applied Biosystems cat # HS03037355_M1) and 10 .mu.l Roche Probes
Master Mix (Roche Cat #04887301001) per well in a LightCycler 480
384 well plate (Roche cat #0472974001). Real time PCR was done in a
LightCycler 480 Real Time PCR machine (Roche). Each duplex was
tested in two independent transfections and each transfections was
assayed in duplicate.
[0515] Real time data were analyzed using the .DELTA..DELTA. Ct
method. Each sample was normalized to GAPDH expression and
knockdown was assessed relative to cells transfected with the
non-targeting duplex AD-1955. IC50s were defined using a 4
parameter fit model in XLfit.
[0516] The data for the single dose experiments are shown in Table
9. Data are expressed as the percent of message remaining relative
to cells targeted with control AD-1955.
[0517] The data for the dose response screen is shown in Table 10.
Data are expressed as dose in pM that results in 50% inhibition
relative to AD-1955. Each dose response was repeated twice (Rep1
and Rep2). Average of the IC50s generated in the two dose response
screens is shown.
[0518] The average IC50 for siRNA flanking AD-9680 was plotted vs.
the starting position of the target region in the human PCSK9
transcript FIG. 25.
[0519] Thus, targeting nucleotide region 3520-3555 of PCSK9 with an
RNAi agent is highly effective at inhibiting PCSK9.
Example 19
ApoE3-Based Reconstituted HDL Complexed with dsRNAs Targeting
PCSK9
[0520] C57BL6 mice were administered 30 mg/kg rEHDL/chol-siPCSK9 by
intravenous administration (tail vein injection) in a single bolus
dose.
[0521] Chol-siPCSK9 (dsRNA Duplex AD-20583) has the following
sequence:
TABLE-US-00011 Sense: (SEQ ID NO: 1729) GccuGGAGuuuAuucGGAAdTsdTL10
Antisense: (SEQ ID NO:1730) PuUfcCfgAfaUfaAfaCfuCfcAfgGfcdTsdT
[0522] The structure of L10 is:
##STR00017##
[0523] After injection, mice were fasted overnight (.about.14
hours), and then sacrificed at 48 h post-injection. mRNA levels
from liver were determined by bDNA assay, and normalized to GAPDH
mRNA levels.
[0524] Results
[0525] The results of the bDNA assays are shown in FIG. 26, which
indicate that there was a significant reduction in PCSK9 following
administration of rEHDL/chol-siPCSK9, but not following
administration of uncomplexed siRNAs (chol-siPCSK9).
rEHDL/chol-siPCSK9 decreased PCSK9 mRNA levels by about 80%.
Example 20
LNP-11 Formulated siRNA in Non-Human Primates (NHPs)
[0526] An siRNA targeting PCSK9 (AD-9680) was formulated in a
LNP-11 formulation (described herein) and administered to
cynomologous monkeys. Control was AD-1955. The lipid formulated
siRNAs were administered via a 30 minute infusion on day 1 at
dosages of 0.03, 0.1, 0.3, and 1.0 mg/kg. Control was administered
at 1.0 mg/kg. On day 3, liver biopsies were performed for
measurement of PCSK9 transcript. Blood samples were collected on
days -3, -1, 3, 4, 5, 7, 9, 11, 12, 15, 22, 30, and 37 and PCSK9
protein levels and LDLc numbers and HDLc numbers were
determined.
[0527] The results are shown in FIG. 27A, FIG. 27B, and FIG.
27C.
[0528] As shown in FIG. 27A and FIG. 27B, administration resulted
in a rapid and durable dose dependent reduction in PCSK9 protein
levels and resulted in >50% reduction in LDLc (LDL cholesterol)
levels. These effects were very potent with ED50 dose levels
between 30 and 100 micrograms per kilogram. As shown in FIG. 27C,
administration resulted in no change in HDLc levels.
Example 21
Dose Response in Rats with LNP-09 Formulated PCSK9 dsRNA
[0529] The dsRNA AD-10792 (targeting rate PCSK9) was encapsulated
in a XTC containing formulation, e.g., a LNP09 formulation. LNP09
formulation was XTC/DSPC/Cholesterol/PEG-DMG at a % mol ratio of
50/10/38.5/1.5 and a lipid:siRNA ratio of 10:1.
[0530] Formulations were injected via tail vein, single dose (DRC)
into rats. Livers and plasma were harvested 72 hours post-injection
(5 animals per group). PCSK9 transcript levels were measured via
bDNA in livers prepared as manufacturer's protocol. GAPDH
transcript levels were also measured and the PCSK9 to GAPDH ratios
were normalized to those of PBS control and graphed. Total
cholesterol was measured in serum using cholesterol kit from WAKO
TX.
[0531] The results are shown in FIG. 29. With this formulation
PCSK9 silencing and total cholesterol lowering in rats was achieved
at doses <0.1 mg/kg. The ED.sub.50 for was 0.2 mg/kg for
lowering PCSK9 mRNA and 0.2 mg/kg and 0.08 for lowering serum
cholesterol.
Example 22
Treatment of Transgenic Mice with LNP-09 Formulated PCSK9 dsRNA
[0532] Transgenic mice that overexpress human CETP and ApoB100
(CETP/ApoB double humanized transgenic mice, Taconic Labs) more
closely mimic the LDL/HDL ratios found in man.
[0533] CETP/ApoB double humanized transgenic mice were purchased
from Taconic labs. Animals were injected through tail vein (single
injection) of 5 mg/kg of LNP09 formulated AD-10792 (standard
formulation procedure), or AD-1955 Luciferase control (4 animals
per group). Livers and plasma were harvested 72 hours
post-injection (5 animals per group) and liver PCSK9 mRNA, LDL
particle, and HDL particle number were determined.
[0534] PCSK9 transcript levels were measured via bDNA in livers
prepared according to manufacturer's protocol. GAPDH transcript
levels were also measured and the PCSK9 to GAPDH ratios were
graphed, normalized to those of PBS control. LDL and HDL particle
numbers/concentration were measured by NMR (Liposciences Inc.)
based on their SOP.
[0535] The results are shown in FIG. 30. Silencing of PCSK9 lowered
LDL particle concentrations .about.90%, while HDL levels were
modestly lower (as compared to those treated animals treated with
PBS controls). This demonstrates significant lowering of PCSK9
levels with subsequent LDLc lowering in these animals.
Example 23
Inhibition of PCSK9 Expression in Humans
[0536] A human subject is treated with a lipid formulated dsRNA
targeted to a PCSK9 gene, described herein, to inhibit expression
of the PCSK9 gene and lower cholesterol levels for an extended
period of time following a single dose. In one embodiment, the
lipid formulated dsRNA includes the lipid MC3.
[0537] A subject in need of treatment is selected or identified.
The subject can be in need of LDL lowering, LDL lowering without
lowering of HDL, ApoB lowering, or total cholesterol lowering. The
identification of the subject can occur in a clinical setting, or
elsewhere, e.g., in the subject's home through the subject's own
use of a self-testing kit.
[0538] At time zero, a suitable first dose of an anti-PCSK9 siRNA
is subcutaneously administered to the subject. The dsRNA is
formulated as described herein. After a period of time following
the first dose, e.g., 7 days, 14 days, and 21 days, the subject's
condition is evaluated, e.g., by measuring LDL, ApoB, and/or total
cholesterol levels. This measurement can be accompanied by a
measurement of PCSK9 expression in said subject, and/or the
products of the successful siRNA-targeting of PCSK9 mRNA. Other
relevant criteria can also be measured. The number and strength of
doses are adjusted according to the subject's needs.
[0539] After treatment, the subject's LDL, ApoB, or total
cholesterol levels are lowered relative to the levels existing
prior to the treatment, or relative to the levels measured in a
similarly afflicted but untreated subject.
[0540] Those skilled in the art are familiar with methods and
compositions in addition to those specifically set out in the
present disclosure which will allow them to practice this invention
to the full scope of the claims hereinafter appended.
TABLE-US-00012 TABLE 1a dsRNA sequences targeted to PCSK9 position
in human access. SEQ SEQ # ID Antisense- ID Duplex NM_174936 Sense
strand sequence (5'-3').sup.1 NO: strand sequence (5'-3').sup.1 NO:
name 2-20 AGCGACGUCGAGGCGCUCATT 1 UGAGCGCCUCGACGUCGCUTT 2 AD- 15220
15-33 CGCUCAUGGUUGCAGGCGGTT 3 CCGCCUGCAACCAUGAGCGTT 4 AD- 15275
16-34 GCUCAUGGUUGCAGGCGGGTT 5 CCCGCCUGCAACCAUGAGCTT 6 AD- 15301
30-48 GCGGGCGCCGCCGUUCAGUTT 7 ACUGAACGGCGGCGCCCGCTT a AD- 15276
31-49 CGGGCGCCGCCGUUCAGUUTT 9 AACUGAACGGCGGCGCCCGTT 10 AD- 15302
32-50 GGGCGCCGCCGUUCAGUUCTT 11 GAACUGAACGGCGGCGCCCTT 12 AD- 15303
40-58 CCGUUCAGUUCAGGGUCUGTT 13 CAGACCCUGAACUGAACGGTT 14 AD- 15221
43-61 UUCAGUUCAGGGUCUGAGCTT 15 GCUCAGACCCUGAACUGAATT 16 AD- 15413
82-100 GUGAGACUGGCUCGGGCGGTT 17 CCGCCCGAGCCAGUCUCACTT 18 AD- 15304
100-118 GGCCGGGACGCGUCGUUGCTT 19 GCAACGACGCGUCCCGGCCTT 20 AD- 15305
101-119 GCCGGGACGCGUCGUUGCATT 21 UGCAACGACGCGUCCCGGCTT 22 AD- 15306
102-120 CCGGGACGCGUCGUUGCAGTT 23 CUGCAACGACGCGUCCCGGTT 24 AD- 15307
105-123 GGACGCGUCGUUGCAGCAGTT 25 CUGCUGCAACGACGCGUCCTT 26 AD- 15277
135-153 UCCCAGCCAGGAUUCCGCGTsT 27 CGCGGAAUCCUGGCUGGGATsT 28 AD-
9526 135-153 ucccAGccAGGAuuccGcGTsT 29 CGCGGAAUCCUGGCUGGGATsT 30
AD- 9652 136-154 CCCAGCCAGGAUUCCGCGCTsT 31 GCGCGGAAUCCUGGCUGGGTsT
32 AD- 9519 136-154 cccAGccAGGAuuccGcGcTsT 33
GCGCGGAAUCCUGGCUGGGTsT 34 AD- 9645 138-156 CAGCCAGGAUUCCGCGCGCTsT
35 GCGCGCGGAAUCCUGGCUGTsT 36 AD- 9523 138-156
cAGccAGGAuuccGcGcGcTsT 37 GCGCGCGGAAUCCUGGCUGTsT 38 AD- 9649
185-203 AGCUCCUGCACAGUCCUCCTsT 39 GGAGGACUGUGCAGGAGCUTsT 40 AD-
9569 185-203 AGcuccuGcAcAGuccuccTsT 41 GGAGGACUGUGcAGGAGCUTsT 42
AD- 9695 205-223 CACCGCAAGGCUCAAGGCGTT 43 CGCCUUGAGCCUUGCGGUGTT 44
AD- 15222 208-226 CGCAAGGCUCAAGGCGCCGTT 45 CGGCGCCUUGAGCCUUGCGTT 46
AD- 15278 210-228 CAAGGCUCAAGGCGCCGCCTT 47 GGCGGCGCCUUGAGCCUUGTT 48
AD- 15178 232-250 GUGGACCGCGCACGGCCUCTT 49 GAGGCCGUGCGCGGUCCACTT 50
AD- 15308 233-251 UGGACCGCGCACGGCCUCUTT 51 AGAGGCCGUGCGCGGUCCATT 52
AD- 15223 234-252 GGACCGCGCACGGCCUCUATT 53 UAGAGGCCGUGCGCGGUCCTT 54
AD- 15309 235-253 GACCGCGCACGGCCUCUAGTT 55 CUAGAGGCCGUGCGCGGUCTT 56
AD- 15279 236-254 ACCGCGCACGGCCUCUAGGTT 57 CCUAGAGGCCGUGCGCGGUTT 58
AD- 15194 237-255 CCGCGCACGGCCUCUAGGUTT 59 ACCUAGAGGCCGUGCGCGGTT 60
AD- 15310 238-256 CGCGCACGGCCUCUAGGUCTT 61 GACCUAGAGGCCGUGCGCGTT 62
AD- 15311 239-257 GCGCACGGCCUCUAGGUCUTT 63 AGACCUAGAGGCCGUGCGCTT 64
AD- 15392 240-258 CGCACGGCCUCUAGGUCUCTT 65 GAGACCUAGAGGCCGUGCGTT 66
AD- 15312 248-266 CUCUAGGUCUCCUCGCCAGTT 67 CUGGCGAGGAGACCUAGAGTT 68
AD- 15313 249-267 UCUAGGUCUCCUCGCCAGGTT 69 CCUGGCGAGGAGACCUAGATT 70
AD- 15280 250-268 CUAGGUCUCCUCGCCAGGATT 71 UCCUGGCGAGGAGACCUAGTT 72
AD- 15267 252-270 AGGUCUCCUCGCCAGGACATT 73 UGUCCUGGCGAGGAGACCUTT 74
AD- 15314 258-276 CCUCGCCAGGACAGCAACCTT 75 GGUUGCUGUCCUGGCGAGGTT 76
AD- 15315 300-318 CGUCAGCUCCAGGCGGUCCTsT 77 GGACCGCCUGGAGCUGACGTsT
78 AD- 9624 300-318 cGucAGcuccAGGcGGuccTsT 79
GGACCGCCUGGAGCUGACGTsT 80 AD- 9750 301-319 GUCAGCUCCAGGCGGUCCUTsT
81 AGGACCGCCUGGAGCUGACTsT 82 AD- 9623 301-319
GucAGcuccAGGcGGuccuTsT 83 AGGACCGCCUGGAGCUGACTsT 84 AD- 9749
370-388 GGCGCCCGUGCGCAGGAGGTT 85 CCUCCUGCGCACGGGCGCCTT 86 AD- 15384
408-426 GGAGCUGGUGCUAGCCUUGTsT 87 CAAGGCUAGCACCAGCUCCTsT 88 AD-
9607 408-426 GGAGcuGGuGcuAGccuuGTsT 89 cAAGGCuAGcACcAGCUCCTsT 90
AD- 9733 411-429 GCUGGUGCUAGCCUUGCGUTsT 91 ACGCAAGGCUAGCACCAGCTsT
92 AD- 9524 411-429 GcuGGuGcuAGccuuGcGuTsT 93
ACGcAAGGCuAGcACcAGCTsT 94 AD- 9650 412-430 CUGGUGCUAGCCUUGCGUUTsT
95 AACGCAAGGCUAGCACCAGTsT 96 AD- 9520 412-430
CUGGUGCUAGCCUUGCGUUTsT 97 AACGCAAGGCUAGCACCAGTsT 98 AD- 9520
412-430 cuGGuGcuAGccuuGcGuuTsT 99 AACGcAAGGCuAGcACcAGTsT 100 AD-
9646 416-434 UGCUAGCCUUGCGUUCCGATsT 101 UCGGAACGCAAGGCUAGCATsT 102
AD- 9608 416-434 uGcuAGccuuGcGuuccGATsT 103 UCGGAACGcAAGGCuAGcATsT
104 AD- 9734 419-437 UAGCCUUGCGUUCCGAGGATsT 105
UCCUCGGAACGCAAGGCUATsT 106 AD- 9546 419-437 uAGc cuuGcGuuccGAGGATsT
107 UCCUCGGAACGcAAGGCuATsT 108 AD- 9672 439-457
GACGGCCUGGCCGAAGCACTT 109 GUGCUUCGGCCAGGCCGUCTT 110 AD- 15385
447-465 GGCCGAAGCACCCGAGCACTT 111 GUGCUCGGGUGCUUCGGCCTT 112 AD-
15393 448-466 GCCGAAGCACCCGAGCACGTT 113 CGUGCUCGGGUGCUUCGGCTT 114
AD- 15316 449-467 CCGAAGCACCCGAGCACGGTT 115 CCGUGCUCGGGUGCUUCGGTT
116 AD- 15317 458-476 CCGAGCACGGAACCACAGCTT 117
GCUGUGGUUCCGUGCUCGGTT 118 AD- 15318 484-502 CACCGCUGCGCCAAGGAUCTT
119 GAUCCUUGGCGCAGCGGUGTT 120 AD- 15195 486-504
CCGCUGCGCCAAGGAUCCGTT 121 CGGAUCCUUGGCGCAGCGGTT 122 AD- 15224
487-505 CGCUGCGCCAAGGAUCCGUTT 123 ACGGAUCCUUGGCGCAGCGTT 124 AD-
15188 489-507 CUGCGCCAAGGAUCCGUGGTT 125 CCACGGAUCCUUGGCGCAGTT 126
AD- 15225 500-518 AUCCGUGGAGGUUGCCUGGTT 127 CCAGGCAACCUCCACGGAUTT
128 AD- 15281 509-527 GGUUGCCUGGCACCUACGUTT 129
ACGUAGGUGCCAGGCAACCTT 130 AD- 15282 542-560 AGGAGACCCACCUCUCGCATT
131 UGCGAGAGGUGGGUCUCCUTT 132 AD- 15319 543-561
GGAGACCCACCUCUCGCAGTT 133 CUGCGAGAGGUGGGUCUCCTT 134 AD- 15226
544-562 GAGACCCACCUCUCGCAGUTT 135 ACUGCGAGAGGUGGGUCUCTT 136 AD-
15271 549-567 CCACCUCUCGCAGUCAGAGTT 137 CUCUGACUGCGAGAGGUGGTT 138
AD- 15283 552-570 CCUCUCGCAGUCAGAGCGCTT 139 GCGCUCUGACUGCGAGAGGTT
140 AD- 15284 553-571 CUCUCGCAGUCAGAGCGCATT 141
UGCGCUCUGACUGCGAGAGTT 142 AD- 15189 554-572 UCUCGCAGUCAGAGCGCACTT
143 GUGCGCUCUGACUGCGAGATT 144 AD- 15227 555-573
CUCGCAGUCAGAGCGCACUTsT 145 AGUGCGCUCUGACUGCGAGTsT 146 AD- 9547
555-573 cucGcAGucAGAGcGcAcuTsT 147 AGUGCGCUCUGACUGCGAGTsT 148 AD-
9673 558-576 GCAGUCAGAGCGCACUGCCTsT 149 GGCAGUGCGCUCUGACUGCTsT 150
AD- 9548 558-576 GcAGucAGAGcGcAcuGccTsT 151 GGcAGUGCGCUCUGACUGCTsT
152 AD- 9674 606-624 GGGAUACCUCACCAAGAUCTsT 153
GAUCUUGGUGAGGUAUCCCTsT 154 AD- 9529 606-624 GGGAuAccucAccAAGAucTsT
155 GAUCUUGGUGAGGuAUCCCTsT 156 AD- 9655 659-677
UGGUGAAGAUGAGUGGCGATsT 157 UCGCCACUCAUCUUCACCATsT 158 AD- 9605
659-677 uGGuGAAGAuGAGuGGcGATsT 159 UCGCcACUcAUCUUcACcATsT 160 AD-
9731
663-681 GAAGAUGAGUGGCGACCUGTsT 161 CAGGUCGCCACUCAUCUUCTsT 162 AD-
9596 663-681 GAAGAuGAGuGGcGAccuGTsT 163 cAGGUCGCcACUcAUCUUCTsT 164
AD- 9722 704-722 CCCAUGUCGACUACAUCGATsT 165 UCGAUGUAGUCGACAUGGGTsT
166 AD- 9583 704-722 cccAuGucGAcuAcAucGATsT 167
UCGAUGuAGUCGAcAUGGGTsT 168 AD- 9709 718-736 AUCGAGGAGGACUCCUCUGTsT
169 CAGAGGAGUCCUCCUCGAUTsT 170 AD- 9579 718-736
AucGAGGAGGAcuccucuGTsT 171 cAGAGGAGUCCUCCUCGAUTsT 172 AD- 9705
758-776 GGAACCUGGAGCGGAUUACTT 173 GUAAUCCGCUCCAGGUUCCTT 174 AD-
15394 759-777 GAACCUGGAGCGGAUUACCTT 175 GGUAAUCCGCUCCAGGUUCTT 176
AD- 15196 760-778 AACCUGGAGCGGAUUACCCTT 177 GGGUAAUCCGCUCCAGGUUTT
178 AD- 15197 777-795 CCCUCCACGGUACCGGGCGTT 179
CGCCCGGUACCGUGGAGGGTT 180 AD- 15198 782-800 CACGGUACCGGGCGGAUGATsT
181 UCAUCCGCCCGGUACCGUGTsT 182 AD- 9609 782-800
cAcGGuAccGGGcGGAuGATsT 183 UcAUCCGCCCGGuACCGUGTsT 184 AD- 9735
783-801 ACGGUACCGGGCGGAUGAATsT 185 UUCAUCCGCCCGGUACCGUTsT 186 AD-
9537 783-801 AcGGuAccGGGcGGAuGAATsT 187 UUcAUCCGCCCGGuACCGUTsT 188
AD- 9663 784-802 CGGUACCGGGCGGAUGAAUTsT 189 AUUCAUCCGCCCGGUACCGTsT
190 AD- 9528 784-802 cGGuAccGGGcGGAuGAAuTsT 191
AUUcAUCCGCCCGGuACCGTsT 192 AD- 9654 785-803 GGUACCGGGCGGAUGAAUATsT
193 UAUUCAUCCGCCCGGUACCTsT 194 AD- 9515 785-803
GGuAccGGGcGGAuGAAuATsT 195 uAUUcAUCCGCCCGGuACCTsT 196 AD- 9641
786-804 GUACCGGGCGGAUGAAUACTsT 197 GUAUUCAUCCGCCCGGUACTsT 198 AD-
9514 786-804 GuAccGGGcGGAuGAAuAcTsT 199 GuAUUcAUCCGCCCGGuACTsT 200
AD- 9640 788-806 ACCGGGCGGAUGAAUACCATsT 201 UGGUAUUCAUCCGCCCGGUTsT
202 AD- 9530 788-806 AccGGGcGGAuGAAuAccATsT 203
UGGuAUUcAUCCGCCCGGUTsT 204 AD- 9656 789-807 CCGGGCGGAUGAAUACCAGTsT
205 CUGGUAUUCAUCCGCCCGGTsT 206 AD- 9538 789-807
ccGGGcGGAuGAAuAccAGTsT 207 CUGGuAUUcAUCCGCCCGGTsT 208 AD- 9664
825-843 CCUGGUGGAGGUGUAUCUCTsT 209 GAGAUACACCUCCACCAGGTsT 210 AD-
9598 825-843 ccuGGuGGAGGuGuAucucTsT 211 GAGAuAcACCUCcACcAGGTsT 212
AD- 9724 826-844 CUGGUGGAGGUGUAUCUCCTsT 213 GGAGAUACACCUCCACCAGTsT
214 AD- 9625 826-844 cuGGuGGAGGuGuAucuccTsT 215
GGAGAuAcACCUCcACcAGTsT 216 AD- 9751 827-845 UGGUGGAGGUGUAUCUCCUTsT
217 AGGAGAUACACCUCCACCATsT 218 AD- 9556 827-845
uGGuGGAGGuGuAucuccuTsT 219 AGGAGAuAcACCUCcACcATsT 220 AD- 9682
828-846 GGUGGAGGUGUAUCUCCUATsT 221 UAGGAGAUACACCUCCACCTsT 222 AD-
9539 828-846 GGuGGAGGuGuAucuccuATsT 223 uAGGAGAuAcACCUCcACCTsT 224
AD- 9665 831-849 GGAGGUGUAUCUCCUAGACTsT 225 GUCUAGGAGAUACACCUCCTsT
226 AD- 9517 831-849 GGAGGuGuAucuccuAGAcTsT 227
GUCuAGGAGAuAcACCUCCTsT 228 AD- 9643 833-851 AGGUGUAUCUCCUAGACACTsT
229 GUGUCUAGGAGAUACACCUTsT 230 AD- 9610 833-851
AGGuGuAucuccuAGAcAcTsT 231 GUGUCuAGGAGAuAcACCUTsT 232 AD- 9736
833-851 AfgGfuGfuAfuCfuCfcUfaGfaCfaC 233 p- 234 AD- fTsT
gUfgUfcUfaGfgAfgAfuAfcAfcCfuTsT 14681 833-851
AGGUfGUfAUfCfUfCfCfUfAGACfAC 235 GUfGUfCfUfAGGAGAUfACfACfCfUfTsT
236 AD- fTsT 14691 833-851 AgGuGuAuCuCcUaGaCaCTsT 237 p- 238 AD-
gUfgUfcUfaGfgAfgAfuAfcAfcCfuTsT 14701 833-851
AgGuGuAuCuCcUaGaCaCTsT 239 GUfGUfCfUfAGGAGAUfACfACfCfUfTsT 240 AD-
14711 833-851 AfgGfuGfuAfuCfuCfcUfaGfaCfaC 241
GUGUCuaGGagAUACAccuTsT 242 AD- fTsT 14721 833-851
AGGUfGUfAUfCfUfCfCfUfAGACfAC 243 GUGUCuaGGagAUACAccuTsT 244 AD-
fTsT 14731 833-851 AgGuGuAuCuCcUaGaCaCTsT 245
GUGUCuaGGagAUACAccuTsT 246 AD- 14741 833-851
GfcAfcCfcUfcAfuAfgGfcCfuGfgA 247 p- 248 AD- fTsT
uCfcAfgGfcCfuAfuGfaGfgGfuGfcTsT 15087 833-851
GCfACfCfCfUfCfAUfAGGCfCfUfGG 249 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT
250 AD- ATsT 15097 833-851 GcAcCcUcAuAgGcCuGgATsT 251 p- 252 AD-
uCfcAfgGfcCfuAfuGfaGfgGfuGfcTsT 15107 833-851
GcAcCcUcAuAgGcCuGgATsT 253 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT 254 AD-
15117 833-851 GfcAfcCfcUfcAfuAfgGfcCfuGfgA 255
UCCAGgcCUauGAGGGugcTsT 256 AD- fTsT 15127 833-851
GCfACfCfCfUfCfAUfAGGCfCfUfGG 257 UCCAGgcCUauGAGGGugcTsT 258 AD-
ATsT 15137 833-851 GcAcCcUcAuAgGcCuGgATsT 259
UCCAGgcCUauGAGGGugcTsT 260 AD- 15147 836-854 UGUAUCUCCUAGACACCAGTsT
261 CUGGUGUCUAGGAGAUACATsT 262 AD- 9516 836-854
uGuAucuccuAGAcAccAGTsT 263 CUGGUGUCuAGGAGAuAcATsT 264 AD- 9642
840-858 UCUCCUAGACACCAGCAUATsT 265 UAUGCUGGUGUCUAGGAGATsT 266 AD-
9562 840-858 ucuccuAGAcAccAGcAuATsT 267 uAUGCUGGUGUCuAGGAGATsT 268
AD- 9688 840-858 UfcUfcCfuAfgAfcAfcCfaGfcAfuA 269 p- 270 AD- fTsT
uAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT 14677 840-858
UfCfUfCfCfUfAGACfACfCfAGCfAU 271 UfAUfGCfUfGGUfGUfCfUfAGGAGATsT 272
AD- fATsT 14687 840-858 UcUcCuAgAcAcCaGcAuATsT 273 p- 274 AD-
uAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT 14697 840-858
UcUcCuAgAcAcCaGcAuATsT 275 UfAUfGCfUfGGUfGUfCfUfAGGAGATsT 276 AD-
14707 840-858 UfcUfcCfuAafAfcAfcCfaGfcAfuA 277
UAUGCugGUguCUAGGagaTsT 278 AD- fTsT 14717 840-858
UfCfUfCfCfUfAGACfACfCfAGCfAU 279 UAUGCugGUguCUAGGagaTsT 280 AD-
fATsT 14727 840-858 UcUcCuAgAcAcCaGcAuATsT 281
UAUGCugGUguCUAGGagaTsT 282 AD- 14737 840-858
AfgGfcCfuGfgAfgUfuUfaUfuCfgG 283 p- 284 AD- fTsT
cCfgAfaUfaAfaCfuCfcAfgGfcCfuTsT 15083 840-858
AGGCfCfUfGGAGUfUfUfAUfUfCfGG 285 CfCfGAAUfAAACfUfCfCfAGGCfCfUfTs
286 AD- TsT T 15093 840-858 AgGcCuGgAgUuUaUuCgGTsT 287 p- 288 AD-
cCfgAfaUfaAfaCfuCfcAfgGfcCfuTsT 15103 840-858
AgGcCuGgAgUuUaUuCgGTsT 289 CfCfGAAUfAAACfUfCfCfAGGCfCfUfTs 290 AD-
T 15113 840-858 AfgGfcCfuGfgAfgUfuUfaUfuCfgG 291
CCGAAuaAAcuCCAGGccuTsT 292 AD- fTsT 15123 840-858
AGGCfCfUfGGAGUfUfUfAUfUfCfGG 293 CCGAAuaAAcuCCAGGccuTsT 294 AD- TsT
15133 840-858 AgGcCuGgAgUuUaUuCgGTsT 295 CCGAAuaAAcuCCAGGccuTsT 296
AD- 15143 841-859 CUCCUAGACACCAGCAUACTsT 297 GUAUGCUGGUGUCUAGGAGTsT
298 AD- 9521 841-859 cuccuAGAcAccAGcAuAcTsT 299
GuAUGCUGGUGUCuAGGAGTsT 300 AD- 9647 842-860 UCCUAGACACCAGCAUACATsT
301 UGUAUGCUGGUGUCUAGGATsT 302 AD- 9611 842-860
uccuAGAcAccAGcAuAcATsT 303 UGuAUGCUGGUGUCuAGGATsT 304 AD- 9737
843-861 CCUAGACACCAGCAUACAGTsT 305 CUGUAUGCUGGUGUCUAGGTsT 306 AD-
9592 843-861 ccuAGAcAccAGcAuAcAGTsT 307 CUGuAUGCUGGUGUCuAGGTsT 308
AD- 9718 847-865 GACACCAGCAUACAGAGUGTsT 309 CACUCUGUAUGCUGGUGUCTsT
310 AD- 9561 847-865 GAcAccAGcAuAcAGAGuGTsT 311
cACUCUGuAUGCUGGUGUCTsT 312 AD- 9687 855-873 CAUACAGAGUGACCACCGGTsT
313 CCGGUGGUCACUCUGUAUGTsT 314 AD- 9636 855-873
cAuAcAGAGuGAccAccGGTsT 315 CCGGUGGUcACUCUGuAUGTsT 316 AD- 9762
860-878 AGAGUGACCACCGGGAAAUTsT 317 AUUUCCCGGUGGUCACUCUTsT 318 AD-
9540 860-878 AGAGuGAccAccGGGAAAuTsT 319 AUUUCCCGGUGGUcACUCUTsT 320
AD- 9666 861-879 GAGUGACCACCGGGAAAUCTsT 321 GAUUUCCCGGUGGUCACUCTsT
322 AD- 9535
861-879 GAGuGAccAccGGGAAAucTsT 323 GAUUUCCCGGUGGUcACUCTsT 324 AD-
9661 863-881 GUGACCACCGGGAAAUCGATsT 325 UCGAUUUCCCGGUGGUCACTsT 326
AD- 9559 863-881 GuGAccAccGGGAAAucGATsT 327 UCGAUUUCCCGGUGGUcACTsT
328 AD- 9685 865-883 GACCACCGGGAAAUCGAGGTsT 329
CCUCGAUUUCCCGGUGGUCTsT 330 AD- 9533 865-883 GAccAccGGGAAAucGAGGTsT
331 CCUCGAUUUCCCGGUGGUCTsT 332 AD- 9659 866-884
ACCACCGGGAAAUCGAGGGTsT 333 CCCUCGAUUUCCCGGUGGUTsT 334 AD- 9612
866-884 AccAccGGGAAAucGAGGGTsT 335 CCCUCGAUUUCCCGGUGGUTsT 336 AD-
9738 867-885 CCACCGGGAAAUCGAGGGCTsT 337 GCCCUCGAUUUCCCGGUGGTsT 338
AD- 9557 867-885 ccAccGGGAAAucGAGGGcTsT 339 GCCCUCGAUUUCCCGGUGGTsT
340 AD- 9683 875-893 AAAUCGAGGGCAGGGUCAUTsT 341
AUGACCCUGCCCUCGAUUUTsT 342 AD- 9531 875-893 AAAucGAGGGcAGGGucAuTsT
343 AUGACCCUGCCCUCGAUUUTsT 344 AD- 9657 875-893
AfaAfuCfgAfgGfgCfaGfgGfuCfaU 345 p- 346 AD- fTsT
aUfgAfcCfcUfgCfcCfuCfgAfuUfuTsT 14673 875-893
AAAUfCfGAGGGCfAGGGUfCfAUfTsT 347 AUfGACfCfCfUfGCfCfCfUfCfGAUfUfU
348 AD- fTsT 14683 875-893 AaAuCgAgGgCaGgGuCaUTsT 349 p- 350 AD-
aUfgAfcCfcUfgCfcCfuCfgAfuUfuTsT 14693 875-893
AaAuCgAgGgCaGgGuCaUTsT 351 AUfGACfCfCfUfGCfCfCfUfCfGAUfUfU 352 AD-
fTsT 14703 875-893 AfaAfuCfgAfgGfgCfaGfgGfuCfaU 353
AUGACccUGccCUCGAuuuTsT 354 AD- fTsT 14713 875-893
AAAUfCfGAGGGCfAGGGUfCfAUfTsT 355 AUGACccUGccCUCGAuuuTsT 356 AD-
14723 875-893 AaAuCgAgGgCaGgGuCaUTsT 357 AUGACccUGccCUCGAuuuTsT 358
AD- 14733 875-893 CfgGfcAfcCfcUfcAfuAfgGfcCfuG 359 p- 360 AD- fTsT
cAfgGfcCfuAfuGfaGfgGfuGfcCfgTsT 15079 875-893
CfGGCfACfCfCfUfCfAUfAGGCfCfU 361 CfAGGCfCfUfAUfGAGGGUfGCfCfGTsT 362
AD- fGTsT 15089 875-893 CgGcAcCcUcAuAgGcCuGTsT 363 p- 364 AD-
cAfgGfcCfuAfuGfaGfgGfuGfcCfgTsT 15099 875-893
CgGcAcCcUcAuAgGcCuGTsT 365 CfAGGCfCfUfAUfGAGGGUfGCfCfGTsT 366 AD-
15109 875-893 CfgGfcAfcCfcUfcAfuAfgGfcCfuG 367
CAGGCcuAUgaGGGUGccgTsT 368 AD- fTsT 15119 875-893
CfGGCfACfCfCfUfCfAUfAGGCfCfU 369 CAGGCcuAUgaGGGUGccgTsT 370 AD-
fGTsT 15129 875-893 CgGcAcCcUcAuAgGcCuGTsT 371
CAGGCcuAUgaGGGUGccgTsT 372 AD- 15139 877-895 AUCGAGGGCAGGGUCAUGGTsT
373 CCAUGACCCUGCCCUCGAUTsT 374 AD- 9542 877-895
AucGAGGGcAGGGucAuGGTsT 375 CcAUGACCCUGCCCUCGAUTsT 376 AD- 9668
878-896 cGAGGGcAGGGucAuGGucTsT 377 GACcAUGACCCUGCCCUCGTsT 378 AD-
9739 880-898 GAGGGCAGGGUCAUGGUCATsT 379 UGACCAUGACCCUGCCCUCTsT 380
AD- 9637 880-898 GAGGGcAGGGucAuGGucATsT 381 UGACcAUGACCCUGCCCUCTsT
382 AD- 9763 882-900 GGGCAGGGUCAUGGUCACCTsT 383
GGUGACCAUGACCCUGCCCTsT 384 AD- 9630 882-900 GGGcAGGGucAuGGucAccTsT
385 GGUGACcAUGACCCUGCCCTsT 386 AD- 9756 885-903
CAGGGUCAUGGUCACCGACTsT 387 GUCGGUGACCAUGACCCUGTsT 388 AD- 9593
885-903 cAGGGucAuGGucAccGAcTsT 389 GUCGGUGACcAUGACCCUGTsT 390 AD-
9719 886-904 AGGGUCAUGGUCACCGACUTsT 391 AGUCGGUGACCAUGACCCUTsT 392
AD- 9601 886-904 AGGGucAuGGucAccGAcuTsT 393 AGUCGGUGACcAUGACCCUTsT
394 AD- 9727 892-910 AUGGUCACCGACUUCGAGATsT 395
UCUCGAAGUCGGUGACCAUTsT 396 AD- 9573 892-910 AuGGucAccGAcuucGAGATsT
397 UCUCGAAGUCGGUGACcAUTsT 398 AD- 9699 899-917
CCGACUUCGAGAAUGUGCCTT 399 GGCACAUUCUCGAAGUCGGTT 400 AD- 15228
921-939 GGAGGACGGGACCCGCUUCTT 401 GAAGCGGGUCCCGUCCUCCTT 402 AD-
15395 993-1011 CAGCGGCCGGGAUGCCGGCTsT 403 GCCGGCAUCCCGGCCGCUGTsT
404 AD- 9602 993-1011 cAGcGGccGGGAuGccGGcTsT 405
GCCGGcAUCCCGGCCGCUGTsT 406 AD- 9728 1020-1038 GGGUGCCAGCAUGCGCAGCTT
407 GCUGCGCAUGCUGGCACCCTT 408 AD- 15386 1038-1056
CCUGCGCGUGCUCAACUGCTsT 409 GCAGUUGAGCACGCGCAGGTsT 410 AD- 9580
1038-1056 ccuGcGcGuGcucAAcuGcTsT 411 GcAGUUGAGcACGCGcAGGTsT 412 AD-
9706 1040-1058 UGCGCGUGCUCAACUGCCATsT 413 UGGCAGUUGAGCACGCGCATsT
414 AD- 9581 1040-1058 uGcGcGuGcucAAcuGccATsT 415
UGGcAGUUGAGcACGCGcATsT 416 AD- 9707 1042-1060
CGCGUGCUCAACUGCCAAGTsT 417 CUUGGCAGUUGAGCACGCGTsT 418 AD- 9543
1042-1060 cGcGuGcucAAcuGccAAGTsT 419 CUUGGcAGUUGAGcACGCGTsT 420 AD-
9669 1053-1071 CUGCCAAGGGAAGGGCACGTsT 421 CGUGCCCUUCCCUUGGCAGTsT
422 AD- 9574 1053-1071 cuGccAAGGGAAGGGcAcGTsT 423
CGUGCCCUUCCCUUGGcAGTsT 424 AD- 9700 1057-1075 CAAGGGAAGGGCACGGUUATT
425 UAACCGUGCCCUUCCCUUGTT 426 AD- 15320 1058-1076
AAGGGAAGGGCACGGUUAGTT 427 CUAACCGUGCCCUUCCCUUTT 428 AD- 15321
1059-1077 AGGGAAGGGCACGGUUAGCTT 429 GCUAACCGUGCCCUUCCCUTT 430 AD-
15199 1060-1078 GGGAAGGGCACGGUUAGCGTT 431 CGCUAACCGUGCCCUUCCCTT 432
AD- 15167 1061-1079 GGAAGGGCACGGUUAGCGGTT 433 CCGCUAACCGUGCCCUUCCTT
434 AD- 15164 1062-1080 GAAGGGCACGGUUAGCGGCTT 435
GCCGCUAACCGUGCCCUUCTT 436 AD- 15166 1063-1081 AAGGGCACGGUUAGCGGCATT
437 UGCCGCUAACCGUGCCCUUTT 438 AD- 15322 1064-1082
AGGGCACGGUUAGCGGCACTT 439 GUGCCGCUAACCGUGCCCUTT 440 AD- 15200
1068-1086 CACGGUUAGCGGCACCCUCTT 441 GAGGGUGCCGCUAACCGUGTT 442 AD-
15213 1069-1087 ACGGUUAGCGGCACCCUCATT 443 UGAGGGUGCCGCUAACCGUTT 444
AD- 15229 1072-1090 GUUAGCGGCACCCUCAUAGTT 445 CUAUGAGGGUGCCGCUAACTT
446 AD- 15215 1073-1091 UUAGCGGCACCCUCAUAGGTT 447
CCUAUGAGGGUGCCGCUAATT 448 AD- 15214 1076-1094
GCGGCACCCUCAUAGGCCUTsT 449 AGGCCUAUGAGGGUGCCGCTsT 450 AD- 9315
1079-1097 GCACCCUCAUAGGCCUGGATsT 451 UCCAGGCCUAUGAGGGUGCTsT 452 AD-
9326 1085-1103 UCAUAGGCCUGGAGUUUAUTsT 453 AUAAACUCCAGGCCUAUGATsT
454 AD- 9318 1090-1108 GGCCUGGAGUUUAUUCGGATsT 455
UCCGAAUAAACUCCAGGCCTsT 456 AD- 9323 1091-1109
GCCUGGAGUUUAUUCGGAATsT 457 UUCCGAAUAAACUCCAGGCTsT 458 AD- 9314
1091-1109 GccuGGAGuuuAuucGGAATsT 459 UUCCGAAuAAACUCcAGGCTsT 460 AD-
10792 1091-1109 GccuGGAGuuuAuucGGAATsT 461 UUCCGAAUAACUCCAGGCTsT
462 AD- 10796 1093-1111 CUGGAGUUUAUUCGGAAAATsT 463
UUUUCCGAAUAAACUCCAGTsT 464 AD- 9638 1093-1111
cuGGAGuuuAuucGGAAAATsT 465 UUUUCCGAAuAAACUCcAGTsT 466 AD- 9764
1095-1113 GGAGUUUAUUCGGAAAAGCTsT 467 GCUUUUCCGAAUAAACUCCTsT 468 AD-
9525 1095-1113 GGAGuuuAuucGGAAAAGcTsT 469 GCUUUUCCGAAuAAACUCCTsT
470 AD- 9651 1096-1114 GAGUUUAUUCGGAAAAGCCTsT 471
GGCUUUUCCGAAUAAACUCTsT 472 AD- 9560 1096-1114
GAGuuuAuucGGAAAAGccTsT 473 GGCUUUUCCGAAuAAACUCTsT 474 AD- 9686
1100-1118 UUAUUCGGAAAAGCCAGCUTsT 475 AGCUGGCUUUUCCGAAUAATsT 476 AD-
9536 1100-1118 uuAuucGGAAAAGccAGcuTsT 477 AGCUGGCUUUUCCGAAuAATsT
478 AD- 9662 1154-1172 CCCUGGCGGGUGGGUACAGTsT 479
CUGUACCCACCCGCCAGGGTsT 480 AD- 9584 1154-1172
cccuGGcGGGuGGGuAcAGTsT 481 CUGuACCcACCCGCcAGGGTsT 482 AD- 9710
1155-1173 CCUGGCGGGUGGGUACAGCTT 483 GCUGUACCCACCCGCCAGGTT 484 AD-
15323 1157-1175 UGGCGGGUGGGUACAGCCGTsT 485 CGGCUGUACCCACCCGCCATsT
486 AD- 9551 1157-1175 uGGcGGGuGGGuAcAGccGTsT 487
CGGCUGuACCcACCCGCcATsT 488 AD-
9677 1158-1176 GGCGGGUGGGUACAGCCGCTT 489 GCGGCUGUACCCACCCGCCTT 490
AD- 15230 1162-1180 GGUGGGUACAGCCGCGUCCTT 491 GGACGCGGCUGUACCCACCTT
492 AD- 15231 1164-1182 UGGGUACAGCCGCGUCCUCTT 493
GAGGACGCGGCUGUACCCATT 494 AD- 15285 1172-1190 GCCGCGUCCUCAACGCCGCTT
495 GCGGCGUUGAGGACGCGGCTT 496 AD- 15396 1173-1191
CCGCGUCCUCAACGCCGCCTT 497 GGCGGCGUUGAGGACGCGGTT 498 AD- 15397
1216-1234 GUCGUGCUGGUCACCGCUGTsT 499 CAGCGGUGACCAGCACGACTsT 500 AD-
9600 1216-1234 GucGuGcuGGucAccGcuGTsT 501 cAGCGGUGACcAGcACGACTsT
502 AD- 9726 1217-1235 UCGUGCUGGUCACCGCUGCTsT 503
GCAGCGGUGACCAGCACGATsT 504 AD- 9606 1217-1235
ucGuGcuGGucAccGcuGcTsT 505 GcAGCGGUGACcAGcACGATsT 506 AD- 9732
1223-1241 UGGUCACCGCUGCCGGCAATsT 507 UUGCCGGCAGCGGUGACCATsT 508 AD-
9633 1223-1241 uGGucAccGcuGccGGcAATsT 509 UUGCCGGcAGCGGUGACcATsT
510 AD- 9759 1224-1242 GGUCACCGCUGCCGGCAACTsT 511
GUUGCCGGCAGCGGUGACCTsT 512 AD- 9588 1224-1242
GGucAccGcuGccGGcAAcTsT 513 GUUGCCGGcAGCGGUGACCTsT 514 AD- 9714
1227-1245 CACCGCUGCCGGCAACUUCTsT 515 GAAGUUGCCGGCAGCGGUGTsT 516 AD-
9589 1227-1245 cAccGcuGccGGcAAcuucTsT 517 GAAGUUGCCGGcAGCGGUGTsT
518 AD- 9715 1229-1247 CCGCUGCCGGCAACUUCCGTsT 519
CGGAAGUUGCCGGCAGCGGTsT 520 AD- 9575 1229-1247
ccGcuGccGGcAAcuuccGTsT 521 CGGAAGUUGCCGGcAGCGGTsT 522 AD- 9701
1230-1248 CGCUGCCGGCAACUUCCGGTsT 523 CCGGAAGUUGCCGGCAGCGTsT 524 AD-
9563 1230-1248 cGcuGccGGcAAcuuccGGTsT 525 CCGGAAGUUGCCGGcAGCGTsT
526 AD- 9689 1231-1249 GCUGCCGGCAACUUCCGGGTsT 527
CCCGGAAGUUGCCGGCAGCTsT 528 AD- 9594 1231-1249
GcuGccGGcAAcuuccGGGTsT 529 CCCGGAAGUUGCCGGcAGCTsT 530 AD- 9720
1236-1254 CGGCAACUUCCGGGACGAUTsT 531 AUCGUCCCGGAAGUUGCCGTsT 532 AD-
9585 1236-1254 cGGcAAcuuccGGGAcGAuTsT 533 AUCGUCCCGGAAGUUGCCGTsT
534 AD- 9711 1237-1255 GGCAACUUCCGGGACGAUGTsT 535
CAUCGUCCCGGAAGUUGCCTsT 536 AD- 9614 1237-1255
GGcAAcuuccGGGAcGAuGTsT 537 cAUCGUCCCGGAAGUUGCCTsT 538 AD- 9740
1243-1261 UUCCGGGACGAUGCCUGCCTsT 539 GGCAGGCAUCGUCCCGGAATsT 540 AD-
9615 1243-1261 uuccGGGAcGAuGccuGccTsT 541 GGcAGGcAUCGUCCCGGAATsT
542 AD- 9741 1248-1266 GGACGAUGCCUGCCUCUACTsT 543
GUAGAGGCAGGCAUCGUCCTsT 544 AD- 9534 1248-1266
GGACGAUGCCUGCCUCUACTsT 545 GUAGAGGCAGGCAUCGUCCTsT 546 AD- 9534
1248-1266 GGAcGAuGccuGccucuAcTsT 547 GuAGAGGcAGGcAUCGUCCTsT 548 AD-
9660 1279-1297 GCUCCCGAGGUCAUCACAGTT 549 CUGUGAUGACCUCGGGAGCTT 550
AD- 15324 1280-1298 CUCCCGAGGUCAUCACAGUTT 551 ACUGUGAUGACCUCGGGAGTT
552 AD- 15232 1281-1299 UCCCGAGGUCAUCACAGUUTT 553
AACUGUGAUGACCUCGGGATT 554 AD- 15233 1314-1332 CCAAGACCAGCCGGUGACCTT
555 GGUCACCGGCUGGUCUUGGTT 556 AD- 15234 1315-1333
CAAGACCAGCCGGUGACCCTT 557 GGGUCACCGGCUGGUCUUGTT 558 AD- 15286
1348-1366 ACCAACUUUGGCCGCUGUGTsT 559 CACAGCGGCCAAAGUUGGUTsT 560 AD-
9590 1348-1366 AccAAcuuuGGccGcuGuGTsT 561 cAcAGCGGCcAAAGUUGGUTsT
562 AD- 9716 1350-1368 CAACUUUGGCCGCUGUGUGTsT 563
CACACAGCGGCCAAAGUUGTsT 564 AD- 9632 1350-1368
cAAcuuuGGccGcuGuGuGTsT 565 cAcAcAGCGGCcAAAGUUGTsT 566 AD- 9758
1360-1378 CGCUGUGUGGACCUCUUUGTsT 567 CAAAGAGGUCCACACAGCGTsT 568 AD-
9567 1360-1378 cGcuGuGuGGAccucuuuGTsT 569 cAAAGAGGUCcAcAcAGCGTsT
570 AD- 9693 1390-1408 GACAUCAUUGGUGCCUCCATsT 571
UGGAGGCACCAAUGAUGUCTsT 572 AD- 9586 1390-1408
GAcAucAuuGGuGccuccATsT 573 UGGAGGcACcAAUGAUGUCTsT 574 AD- 9712
1394-1412 UCAUUGGUGCCUCCAGCGATsT 575 UCGCUGGAGGCACCAAUGATsT 576 AD-
9564 1394-1412 ucAuuGGuGccuccAGcGATsT 577 UCGCUGGAGGcACcAAUGATsT
578 AD- 9690 1417-1435 AGCACCUGCUUUGUGUCACTsT 579
GUGACACAAAGCAGGUGCUTsT 580 AD- 9616 1417-1435
AGcAccuGcuuuGuGucAcTsT 581 GUGAcAcAAAGcAGGUGCUTsT 582 AD- 9742
1433-1451 CACAGAGUGGGACAUCACATT 583 UGUGAUGUCCCACUCUGUGTT 584 AD-
15398 1486-1504 AUGCUGUCUGCCGAGCCGGTsT 585 CCGGCUCGGCAGACAGCAUTsT
586 AD- 9617 1486-1504 AuGcuGucuGccGAGccGGTsT 587
CCGGCUCGGcAGAcAGcAUTsT 588 AD- 9743 1491-1509
GUCUGCCGAGCCGGAGCUCTsT 589 GAGCUCCGGCUCGGCAGACTsT 590 AD- 9635
1491-1509 GucuGccGAGccGGAGcucTsT 591 GAGCUCCGGCUCGGcAGACTsT 592 AD-
9761 1521-1539 GUUGAGGCAGAGACUGAUCTsT 593 GAUCAGUCUCUGCCUCAACTsT
594 AD- 9568 1521-1539 GuuGAGGcAGAGAcuGAucTsT 595
GAUcAGUCUCUGCCUcAACTsT 596 AD- 9694 1527-1545
GCAGAGACUGAUCCACUUCTsT 597 GAAGUGGAUCAGUCUCUGCTsT 598 AD- 9576
1527-1545 GcAGAGAcuGAuccAcuucTsT 599 GAAGUGGAUcAGUCUCUGCTsT 600 AD-
9702 1529-1547 AGAGACUGAUCCACUUCUCTsT 601 GAGAAGUGGAUCAGUCUCUTsT
602 AD- 9627 1529-1547 AGAGAcuGAuccAcuucucTsT 603
GAGAAGUGGAUcAGUCUCUTsT 604 AD- 9753 1543-1561
UUCUCUGCCAAAGAUGUCATsT 605 UGACAUCUUUGGCAGAGAATsT 606 AD- 9628
1543-1561 uucucuGccAAAGAuGucATsT 607 UGAcAUCUUUGGcAGAGAATsT 608 AD-
9754 1545-1563 CUCUGCCAAAGAUGUCAUCTsT 609 GAUGACAUCUUUGGCAGAGTsT
610 AD- 9631 1545-1563 cucuGccAAAGAuGucAucTsT 611
GAUGAcAUCUUUGGcAGAGTsT 612 AD- 9757 1580-1598
CUGAGGACCAGCGGGUACUTsT 613 AGUACCCGCUGGUCCUCAGTsT 614 AD- 9595
1580-1598 cuGAGGAccAGcGGGuAcuTsT 615 AGuACCCGCUGGUCCUcAGTsT 616 AD-
9721 1581-1599 UGAGGACCAGCGGGUACUGTsT 617 CAGUACCCGCUGGUCCUCATsT
618 AD- 9544 1581-1599 uGAGGAccAGcGGGuAcuGTsT 619
cAGuACCCGCUGGUCCUcATsT 620 AD- 9670 1666-1684 ACUGUAUGGUCAGCACACUTT
621 AGUGUGCUGACCAUACAGUTT 622 AD- 15235 1668-1686
UGUAUGGUCAGCACACUCGTT 623 CGAGUGUGCUGACCAUACATT 624 AD- 15236
1669-1687 GUAUGGUCAGCACACUCGGTT 625 CCGAGUGUGCUGACCAUACTT 626 AD-
15168 1697-1715 GGAUGGCCACAGCCGUCGCTT 627 GCGACGGCUGUGGCCAUCCTT 628
AD- 15174 1698-1716 GAUGGCCACAGCCGUCGCCTT 629 GGCGACGGCUGUGGCCAUCTT
630 AD- 15325 1806-1824 CAAGCUGGUCUGCCGGGCCTT 631
GGCCCGGCAGACCAGCUUGTT 632 AD- 15326 1815-1833
CUGCCGGGCCCACAACGCUTsT 633 AGCGUUGUGGGCCCGGCAGTsT 634 AD- 9570
1815-1833 cuGccGGGcccAcAAcGcuTsT 635 AGCGUUGUGGGCCCGGcAGTsT 636 AD-
9696 1816-1834 UGCCGGGCCCACAACGCUUTsT 637 AAGCGUUGUGGGCCCGGCATsT
638 AD- 9566 1816-1834 uGccGGGcccAcAAcGcuuTsT 639
AAGCGUUGUGGGCCCGGcATsT 640 AD- 9692 1818-1836
CCGGGCCCACAACGCUUUUTsT 641 AAAAGCGUUGUGGGCCCGGTsT 642 AD- 9532
1818-1836 ccGGGcccAcAAcGcuuuuTsT 643 AAAAGCGUUGUGGGCCCGGTsT 644 AD-
9658 1820-1838 GGGCCCACAACGCUUUUGGTsT 645 CCAAAAGCGUUGUGGGCCCTsT
646 AD- 9549 1820-1838 GGGcccAcAAcGcuuuuGGTsT 647
CcAAAAGCGUUGUGGGCCCTsT 648 AD- 9675 1840-1858
GGUGAGGGUGUCUACGCCATsT 649 UGGCGUAGACACCCUCACCTsT 650 AD- 9541
1840-1858 GGuGAGGGuGucuAcGccATsT 651 UGGCGuAGAcACCCUcACCTsT 652 AD-
9667 1843-1861 GAGGGUGUCUACGCCAUUGTsT 653 CAAUGGCGUAGACACCCUCTsT
654 AD- 9550
1843-1861 GAGGGuGucuAcGccAuuGTsT 655 cAAUGGCGuAGAcACCCUCTsT 656 AD-
9676 1861-1879 GCCAGGUGCUGCCUGCUACTsT 657 GUAGCAGGCAGCACCUGGCTsT
658 AD- 9571 1861-1879G ccAGGuGcuGccuGcuAcTsT 659
GuAGcAGGcAGcACCUGGCTsT 660 AD- 9697 1862-1880
CCAGGUGCUGCCUGCUACCTsT 661 GGUAGCAGGCAGCACCUGGTsT 662 AD- 9572
1862-1880 ccAGGuGcuGccuGcuAccTsT 663 GGuAGcAGGcAGcACCUGGTsT 664 AD-
9698 2008-2026 ACCCACAAGCCGCCUGUGCTT 665 GCACAGGCGGCUUGUGGGUTT 666
AD- 15327 2023-2041 GUGCUGAGGCCACGAGGUCTsT 667
GACCUCGUGGCCUCAGCACTsT 668 AD- 9639 2023-2041
GuGcuGAGGccAcGAGGucTsT 669 GACCUCGUGGCCUcAGcACTsT 670 AD- 9765
2024-2042 UGCUGAGGCCACGAGGUCATsT 671 UGACCUCGUGGCCUCAGCATsT 672 AD-
9518 2024-2042 UGCUGAGGCCACGAGGUCATsT 673 UGACCUCGUGGCCUCAGCATsT
674 AD- 9518 2024-2042 uGcuGAGGccAcGAGGucATsT 675
UGACCUCGUGGCCUcAGcATsT 676 AD- 9644 2024-2042
UfgCfuGfaGfgCfcAfcGfaGfgUfCA 677 p- 678 AD- fTsT
uGfaCfcUfcGfuGfgCfcUfcAfgCfaTsT 14672 2024-2042
UfGCfUfGAGGCfCfACfGAGGUfCfAT 679 UfGACfCfUfCfGUfGGCfCfUfCfAGCfAT
680 AD- ST ST 14682 2024-2042 UgCuGaGgCcAcGaGgUcATsT 681 p- 682 AD-
uGfaCfcUfcGfuGfgCfcUfcAfgCfaTsT 14692 2024-2042
UgCuGaGgCcAcGaGgUcATsT 683 UfGACfCfUfCfGUfGGCfCfUfCfAGCfAT 684 AD-
sT 14702 2024-2042 UfgCfuGfaGfgCfcAfcGfaGfgUfCA 685
UGACCucGUggCCUCAgcaTsT 686 AD- fTsT 14712 2024-2042
UfGCfUfGAGGCfCfACfGAGGUfCfAT 687 UGACCucGUggCCUCAgcaTsT 688 AD- ST
14722 2024-2042 UgCuGaGgCcAcGaGgUcATsT 689 UGACCucGUggCCUCAgcaTsT
690 AD- 14732 2024-2042 GfuGfgUfcAfgCfgGfcCfgGfgAfuG 691 p- 692 AD-
fTsT cAfuCfcCfgGfcCfgCfuGfaCfcAfcTsT 15078 2024-2042
GUfGGUfCfAGCfGGCfCfGGGAUfGTs 693 CfAUfCfCfCfGGCfCfGCfUfGACfCfACf
694 AD- T TsT 15088 2024-2042 GuGgUcAgCgGcCgGgAuGTsT 695 p- 696 AD-
cAfuCfcCfgGfcCfgCfuGfaCfcAfcTsT 15098 2024-2042
GuGgUcAgCgGcCgGgAuGTsT 697 CfAUfCfCfCfGGCfCfGCfUfGACfCfACf 698 AD-
TsT 15108 2024-2042 GfuGfgUfcAfgCfgGfcCfgGfgAfuG 699
CAUCCcgGCcgCUGACcacTsT 700 AD- fTsT 15118 2024-2042
GUfGGUfCfAGCfGGCfCfGGGAUfGTs 701 CAUCCcgGCcgCUGACcacTsT 702 AD- T
15128 2024-2042 GuGgUcAgCgGcCgGgAuGTsT 703 CAUCCcgGCcgCUGACcacTsT
704 AD- 15138 2030-2048 GGCCACGAGGUCAGCCCAATT 705
UUGGGCUGACCUCGUGGCCTT 706 AD- 15237 2035-2053 CGAGGUCAGCCCAACCAGUTT
707 ACUGGUUGGGCUGACCUCGTT 708 AD- 15287 2039-2057
GUCAGCCCAACCAGUGCGUTT 709 ACGCACUGGUUGGGCUGACTT 710 AD- 15238
2041-2059 CAGCCCAACCAGUGCGUGGTT 711 CCACGCACUGGUUGGGCUGTT 712 AD-
15328 2062-2080 CACAGGGAGGCCAGCAUCCTT 713 GGAUGCUGGCCUCCCUGUGTT 714
AD- 15399 2072-2090 CCAGCAUCCACGCUUCCUGTsT 715
CAGGAAGCGUGGAUGCUGGTsT 716 AD- 9582 2072-2090
ccAGcAuccAcGcuuccuGTsT 717 cAGGAAGCGUGGAUGCUGGTsT 718 AD- 9708
2118-2136 AGUCAAGGAGCAUGGAAUCTsT 719 GAUUCCAUGCUCCUUGACUTsT 720 AD-
9545 2118-2136 AGucAAGGAGcAuGGAAucTsT 721 GAUUCcAUGCUCCUUGACUTsT
722 AD- 9671 2118-2136 AfgUfcAfaGfgAfgCfaUfgGfaAfuC 723 p- 724 AD-
fTsT gAfuUfcCfaUfgCfuCfcUfuGfaCfuTsT 14674 2118-2136
AGUfCfAAGGAGCfAUfGGAAUfCfTsT 725 GAUfUfCfCfAUfGCfUfCfCfUfUfGACfU
726 AD- fTsT 14684 2118-2136 AgUcAaGgAgCaUgGaAuCTsT 727 p- 728 AD-
gAfuUfcCfaUfgCfuCfcUfuGfaCfuTsT 14694 2118-2136
AgUcAaGgAgCaUgGaAuCTsT 729 GAUfUfCfCfAUfGCfUfCfCfUfUfGACfU 730 AD-
fTsT 14704 2118-2136 AfgUfcAfaGfgAfgCfaUfgGfaAfuC 731
GAUUCcaUGcuCCUUGacuTsT 732 AD- fTsT 14714 2118-2136
AGUfCfAAGGAGCfAUfGGAAUfCfTsT 733 GAUUCcaUGcuCCUUGacuTsT 734 AD-
14724 2118-2136 AgUcAaGgAgCaUgGaAuCTsT 735 GAUUCcaUGcuCCUUGacuTsT
736 AD- 14734 2118-2136 GfcGfgCfaCfcCfuCfaUfaGfgCfcU 737 p- 738 AD-
fTsT aGfgCfcUfaUfgAfgGfgUfgCfcGfcTsT 15080 2118-2136
GCfGGCfACfCfCfUfCfAUfAGGCfCf 739 AGGCfCfUfAUfGAGGGUfGCfCfGCfTsT 740
AD- UfTsT 15090 2118-2136 GcGgCaCcCuCaUaGgCcUTsT 741 p- 742 AD-
aGfgCfcUfaUfgAfgGfgUfgCfcGfcTsT 15100 2118-2136
GcGgCaCcCuCaUaGgCcUTsT 743 AGGCfCfUfAUfGAGGGUfGCfCfGCfTsT 744 AD-
15110 2118-2136 GfcGfgCfaCfcCfuCfaUfaGfgCfcU 745
AGGCCuaUGagGGUGCcgcTsT 746 AD- fTsT 15120 2118-2136
GCfGGCfACfCfCfUfCfAUfAGGCfCf 747 AGGCCuaUGagGGUGCcgcTsT 748 AD-
UfTsT 15130 2118-2136 GcGgCaCcCuCaUaGgCcUTsT 749
AGGCCuaUGagGGUGCcgcTsT 750 AD- 15140 2122-2140
AAGGAGCAUGGAAUCCCGGTsT 751 CCGGGAUUCCAUGCUCCUUTsT 752 AD- 9522
2122-2140 AAGGAGcAuGGAAucccGGTsT 753 CCGGGAUUCcAUGCUCCUUTsT 754 AD-
9648 2123-2141 AGGAGCAUGGAAUCCCGGCTsT 755 GCCGGGAUUCCAUGCUCCUTsT
756 AD- 9552 2123-2141 AGGAGcAuGGAAucccGGcTsT 757
GCCGGGAUUCcAUGCUCCUTsT 758 AD- 9678 2125-2143
GAGCAUGGAAUCCCGGCCCTsT 759 GGGCCGGGAUUCCAUGCUCTsT 760 AD- 9618
2125-2143 GAGcAuGGAAucccGGcccTsT 761 GGGCCGGGAUUCcAUGCUCTsT 762 AD-
9744 2230-2248 GCCUACGCCGUAGACAACATT 763 UGUUGUCUACGGCGUAGGCTT 764
AD- 15239 2231-2249 CCUACGCCGUAGACAACACTT 765 GUGUUGUCUACGGCGUAGGTT
766 AD- 15212 2232-2250 CUACGCCGUAGACAACACGTT 767
CGUGUUGUCUACGGCGUAGTT 768 AD- 15240 2233-2251 UACGCCGUAGACAACACGUTT
769 ACGUGUUGUCUACGGCGUATT 770 AD- 15177 2235-2253
CGCCGUAGACAACACGUGUTT 771 ACACGUGUUGUCUACGGCGTT 772 AD- 15179
2236-2254 GCCGUAGACAACACGUGUGTT 773 CACACGUGUUGUCUACGGCTT 774 AD-
15180 2237-2255 CCGUAGACAACACGUGUGUTT 775 ACACACGUGUUGUCUACGGTT 776
AD- 15241 2238-2256 CGUAGACAACACGUGUGUATT 777 UACACACGUGUUGUCUACGTT
778 AD- 15268 2240-2258 UAGACAACACGUGUGUAGUTT 779
ACUACACACGUGUUGUCUATT 780 AD- 15242 2241-2259 AGACAACACGUGUGUAGUCTT
781 GACUACACACGUGUUGUCUTT 782 AD- 15216 2242-2260
GACAACACGUGUGUAGUCATT 783 UGACUACACACGUGUUGUCTT 784 AD- 15176
2243-2261 ACAACACGUGUGUAGUCAGTT 785 CUGACUACACACGUGUUGUTT 786 AD-
15181 2244-2262 CAACACGUGUGUAGUCAGGTT 787 CCUGACUACACACGUGUUGTT 788
AD- 15243 2247-2265 CACGUGUGUAGUCAGGAGCTT 789 GCUCCUGACUACACACGUGTT
790 AD- 15182 2248-2266 ACGUGUGUAGUCAGGAGCCTT 791
GGCUCCUGACUACACACGUTT 792 AD- 15244 2249-2267 CGUGUGUAGUCAGGAGCCGTT
793 CGGCUCCUGACUACACACGTT 794 AD- 15387 2251-2269
UGUGUAGUCAGGAGCCGGGTT 795 CCCGGCUCCUGACUACACATT 796 AD- 15245
2257-2275 GUCAGGAGCCGGGACGUCATsT 797 UGACGUCCCGGCUCCUGACTsT 798 AD-
9555 2257-2275 GucAGGAGccGGGAcGucATsT 799 UGACGUCCCGGCUCCUGACTsT
800 AD- 9681 2258-2276 UCAGGAGCCGGGACGUCAGTsT 801
CUGACGUCCCGGCUCCUGATsT 802 AD- 9619 2258-2276
ucAGGAGccGGGAcGucAGTsT 803 CUGACGUCCCGGCUCCUGATsT 804 AD- 9745
2259-2277 CAGGAGCCGGGACGUCAGCTsT 805 GCUGACGUCCCGGCUCCUGTsT 806 AD-
9620 2259-2277 cAGGAGccGGGAcGucAGcTsT 807 GCUGACGUCCCGGCUCCUGTsT
808 AD- 9746 2263-2281 AGCCGGGACGUCAGCACUATT 809
UAGUGCUGACGUCCCGGCUTT 810 AD- 15288 2265-2283 CCGGGACGUCAGCACUACATT
811 UGUAGUGCUGACGUCCCGGTT 812 AD- 15246 2303-2321
CCGUGACAGCCGUUGCCAUTT 813 AUGGCAACGGCUGUCACGGTT 814 AD- 15289
2317-2335 GCCAUCUGCUGCCGGAGCCTsT 815 GGCUCCGGCAGCAGAUGGCTsT 816 AD-
9324
2375-2393 CCCAUCCCAGGAUGGGUGUTT 817 ACACCCAUCCUGGGAUGGGTT 818 AD-
15329 2377-2395 CAUCCCAGGAUGGGUGUCUTT 819 AGACACCCAUCCUGGGAUGTT 820
AD- 15330 2420-2438 AGCUUUAAAAUGGUUCCGATT 821 UCGGAACCAUUUUAAAGCUTT
822 AD- 15169 2421-2439 GCUUUAAAAUGGUUCCGACTT 823
GUCGGAACCAUUUUAAAGCTT 824 AD- 15201 2422-2440 CUUUAAAAUGGUUCCGACUTT
825 AGUCGGAACCAUUUUAAAGTT 826 AD- 15331 2423-2441
UUUAAAAUGGUUCCGACUUTT 827 AAGUCGGAACCAUUUUAAATT 828 AD- 15190
2424-2442 UUAAAAUGGUUCCGACUUGTT 829 CAAGUCGGAACCAUUUUAATT 830 AD-
15247 2425-2443 UAAAAUGGUUCCGACUUGUTT 831 ACAAGUCGGAACCAUUUUATT 832
AD- 15248 2426-2444 AAAAUGGUUCCGACUUGUCTT 833 GACAAGUCGGAACCAUUUUTT
834 AD- 15175 2427-2445 AAAUGGUUCCGACUUGUCCTT 835
GGACAAGUCGGAACCAUUUTT 836 AD- 15249 2428-2446 AAUGGUUCCGACUUGUCCCTT
837 GGGACAAGUCGGAACCAUUTT 838 AD- 15250 2431-2449
GGUUCCGACUUGUCCCUCUTT 839 AGAGGGACAAGUCGGAACCTT 840 AD- 15400
2457-2475 CUCCAUGGCCUGGCACGAGTT 841 CUCGUGCCAGGCCAUGGAGTT 842 AD-
15332 2459-2477 CCAUGGCCUGGCACGAGGGTT 843 CCCUCGUGCCAGGCCAUGGTT 844
AD- 15388 2545-2563 GAACUCACUCACUCUGGGUTT 845 ACCCAGAGUGAGUGAGUUCTT
846 AD- 15333 2549-2567 UCACUCACUCUGGGUGCCUTT 847
AGGCACCCAGAGUGAGUGATT 848 AD- 15334 2616-2634 UUUCACCAUUCAAACAGGUTT
849 ACCUGUUUGAAUGGUGAAATT 850 AD- 15335 2622-2640
CAUUCAAACAGGUCGAGCUTT 851 AGCUCGACCUGUUUGAAUGTT 852 AD- 15183
2623-2641 AUUCAAACAGGUCGAGCUGTT 853 CAGCUCGACCUGUUUGAAUTT 854 AD-
15202 2624-2642 UUCAAACAGGUCGAGCUGUTT 855 ACAGCUCGACCUGUUUGAATT 856
AD- 15203 2625-2643 UCAAACAGGUCGAGCUGUGTT 857 CACAGCUCGACCUGUUUGATT
858 AD- 15272 2626-2644 CAAACAGGUCGAGCUGUGCTT 859
GCACAGCUCGACCUGUUUGTT 860 AD- 15217 2627-2645 AAACAGGUCGAGCUGUGCUTT
861 AGCACAGCUCGACCUGUUUTT 862 AD- 15290 2628-2646
AACAGGUCGAGCUGUGCUCTT 863 GAGCACAGCUCGACCUGUUTT 864 AD- 15218
2630-2648 CAGGUCGAGCUGUGCUCGGTT 865 CCGAGCACAGCUCGACCUGTT 866 AD-
15389 2631-2649 AGGUCGAGCUGUGCUCGGGTT 867 CCCGAGCACAGCUCGACCUTT 868
AD- 15336 2633-2651 GUCGAGCUGUGCUCGGGUGTT 869 CACCCGAGCACAGCUCGACTT
870 AD- 15337 2634-2652 UCGAGCUGUGCUCGGGUGCTT 871
GCACCCGAGCACAGCUCGATT 872 AD- 15191 2657-2675 UCGAGCUGUGCUCGGGUGCTT
873 CGGCACAUUGGGAGCAGCUTT 874 AD- 15390 2658-2676
GCUGCUCCCAAUGUGCCGATT 875 UCGGCACAUUGGGAGCAGCTT 876 AD- 15338
2660-2678 UGCUCCCAAUGUGCCGAUGTT 877 CAUCGGCACAUUGGGAGCATT 878 AD-
15204 2663-2681 UCCCAAUGUGCCGAUGUCCTT 879 GGACAUCGGCACAUUGGGATT 880
AD- 15251 2665-2683 CCAAUGUGCCGAUGUCCGUTT 881 ACGGACAUCGGCACAUUGGTT
882 AD- 15205 2666-2684 CAAUGUGCCGAUGUCCGUGTT 883
CACGGACAUCGGCACAUUGTT 884 AD- 15171 2667-2685 AAUGUGCCGAUGUCCGUGGTT
885 CCACGGACAUCGGCACAUUTT 886 AD- 15252 2673-2691
CCGAUGUCCGUGGGCAGAATT 887 UUCUGCCCACGGACAUCGGTT 888 AD- 15339
2675-2693 GAUGUCCGUGGGCAGAAUGTT 889 CAUUCUGCCCACGGACAUCTT 890 AD-
15253 2678-2696 GUCCGUGGGCAGAAUGACUTT 891 AGUCAUUCUGCCCACGGACTT 892
AD- 15340 2679-2697 UCCGUGGGCAGAAUGACUUTT 893 AAGUCAUUCUGCCCACGGATT
894 AD- 15291 2683-2701 UGGGCAGAAUGACUUUUAUTT 895
AUAAAAGUCAUUCUGCCCATT 896 AD- 15341 2694-2712 ACUUUUAUUGAGCUCUUGUTT
897 ACAAGAGCUCAAUAAAAGUTT 898 AD- 15401 2700-2718
AUUGAGCUCUUGUUCCGUGTT 899 CACGGAACAAGAGCUCAAUTT 900 AD- 15342
2704-2722 AGCUCUUGUUCCGUGCCAGTT 901 CUGGCACGGAACAAGAGCUTT 902 AD-
15343 2705-2723 GCUCUUGUUCCGUGCCAGGTT 903 CCUGGCACGGAACAAGAGCTT 904
AD- 15292 2710-2728 UGUUCCGUGCCAGGCAUUCTT 905 GAAUGCCUGGCACGGAACATT
906 AD- 15344 2711-2729 GUUCCGUGCCAGGCAUUCATT 907
UGAAUGCCUGGCACGGAACTT 908 AD- 15254 2712-2730 UUCCGUGCCAGGCAUUCAATT
909 UUGAAUGCCUGGCACGGAATT 910 AD- 15345 2715-2733
CGUGCCAGGCAUUCAAUCCTT 911 GGAUUGAAUGCCUGGCACGTT 912 AD- 15206
2716-2734 GUGCCAGGCAUUCAAUCCUTT 913 AGGAUUGAAUGCCUGGCACTT 914 AD-
15346 2728-2746 CAAUCCUCAGGUCUCCACCTT 915 GGUGGAGACCUGAGGAUUGTT 916
AD- 15347 2743-2761 CACCAAGGAGGCAGGAUUCTsT 917
GAAUCCUGCCUCCUUGGUGTsT 918 AD- 9577 2743-2761
cAccAAGGAGGcAGGAuucTsT 919 GAAUCCUGCCUCCUUGGUGTsT 920 AD- 9703
2743-2761 CfaCfcAfaGfgAfgGfcAfgGfaUfuC 921 p- 922 AD- fTsT
gAfaUfcCfuGfcCfuCfcUfuGfgUfgTsT 14678 2743-2761
CfACfCfAAGGAGGCfAGGAUfUfCfTs 923 GAAUfCfCfUfGCfCfUfCfCfUfUfGGUfG
924 AD- T TsT 14688 2743-2761 CaCcAaGgAgGcAgGaUuCTsT 925 p- 926 AD-
gAfaUfcCfuGfcCfuCfcUfuGfgUfgTsT 14698 2743-2761
CaCcAaGgAgGcAgGaUuCTsT 927 GAAUfCfCfUfGCfCfUfCfCfUfUfGGUfG 928 AD-
TsT 14708 2743-2761 CfaCfcAfaGfgAfgGfcAfgGfaUfuC 929
GAAUCcuGCcuCCUUGgugTsT 930 AD- fTsT 14718 2743-2761
CfACfCfAAGGAGGCfAGGAUfUfCfTs 931 GAAUCcuGCcuCCUUGgugTsT 932 AD- T
14728 2743-2761 CaCcAaGgAgGcAgGaUuCTsT 933 GAAUCcuGCcuCCUUGgugTsT
934 AD- 14738 2743-2761 GfgCfcUfgGfaGfuUfuAfuUfcGfgA 935 p- 936 AD-
fTsT uCfcGfaAfuAfaAfcUfcCfaGfgCfcTsT 15084 2743-2761
GGCfCfUfGGAGUfUfUfAUfUfCfGGA 937 UfCfCfGAAUfAAACfUfCfCfAGGCfCfTs
938 AD- TsT T 15094 2743-2761 GgCcUgGaGuUuAuUcGgATsT 939 p- 940 AD-
uCfcGfaAfuAfaAfcUfcCfaGfgCfcTsT 15104 2743-2761
GgCcUgGaGuUuAuUcGgATsT 941 UfCfCfGAAUfAAACfUfCfCfAGGCfCfTs 942 AD-
T 15114 2743-2761 GfgCfcUfgGfaGfuUfuAfuUfcGfgA 943
UCCGAauAAacUCCAGgccTsT 944 AD- fTsT 15124 2743-2761
GGCfCfUfGGAGUfUfUfAUfUfCfGGA 945 UCCGAauAAacUCCAGgccTsT 946 AD- TsT
15134 2743-2761 GgCcUgGaGuUuAuUcGgATsT 947 UCCGAauAAacUCCAGgccTsT
948 AD- 15144 2753-2771 GCAGGAUUCUUCCCAUGGATT 949
UCCAUGGGAAGAAUCCUGCTT 950 AD- 15391 2794-2812 UGCAGGGACAAACAUCGUUTT
951 AACGAUGUUUGUCCCUGCATT 952 AD- 15348 2795-2813
GCAGGGACAAACAUCGUUGTT 953 CAACGAUGUUUGUCCCUGCTT 954 AD- 15349
2797-2815 AGGGACAAACAUCGUUGGGTT 955 CCCAACGAUGUUUGUCCCUTT 956 AD-
15170 2841-2859 CCCUCAUCUCCAGCUAACUTT 957 AGUUAGCUGGAGAUGAGGGTT 958
AD- 15350 2845-2863 CAUCUCCAGCUAACUGUGGTT 959 CCACAGUUAGCUGGAGAUGTT
960 AD- 15402 2878-2896 GCUCCCUGAUUAAUGGAGGTT 961
CCUCCAUUAAUCAGGGAGCTT 962 AD- 15293 2881-2899 CCCUGAUUAAUGGAGGCUUTT
963 AAGCCUCCAUUAAUCAGGGTT 964 AD- 15351 2882-2900
CCUGAUUAAUGGAGGCUUATT 965 UAAGCCUCCAUUAAUCAGGTT 966 AD- 15403
2884-2902 UGAUUAAUGGAGGCUUAGCTT 967 GCUAAGCCUCCAUUAAUCATT 968 AD-
15404 2885-2903 GAUUAAUGGAGGCUUAGCUTT 969 AGCUAAGCCUCCAUUAAUCTT 970
AD- 15207 2886-2904 AUUAAUGGAGGCUUAGCUUTT 971 AAGCUAAGCCUCCAUUAAUTT
972 AD- 15352 2887-2905 UUAAUGGAGGCUUAGCUUUTT 973
AAAGCUAAGCCUCCAUUAATT 974 AD- 15255 2903-2921
UUUCUGGAUGGCAUCUAGCTsT 975 GCUAGAUGCCAUCCAGAAATsT 976 AD- 9603
2903-2921 uuucuGGAuGGcAucuAGcTsT 977 GCuAGAUGCcAUCcAGAAATsT 978 AD-
9729 2904-2922 UUCUGGAUGGCAUCUAGCCTsT 979 GGCUAGAUGCCAUCCAGAATsT
980 AD- 9599 2904-2922 uucuGGAuGGcAucuAGccTsT 981
GGCuAGAUGCcAUCcAGAATsT 982 AD-
9725 2905-2923 UCUGGAUGGCAUCUAGCCATsT 983 UGGCUAGAUGCCAUCCAGATsT
984 AD- 9621 2905-2923 ucuGGAuGGcAucuAGccATsT 985
UGGCuAGAUGCcAUCcAGATsT 986 AD- 9747 2925-2943 AGGCUGGAGACAGGUGCGCTT
987 GCGCACCUGUCUCCAGCCUTT 988 AD- 15405 2926-2944
GGCUGGAGACAGGUGCGCCTT 989 GGCGCACCUGUCUCCAGCCTT 990 AD- 15353
2927-2945 GCUGGAGACAGGUGCGCCCTT 991 GGGCGCACCUGUCUCCAGCTT 992 AD-
15354 2972-2990 UUCCUGAGCCACCUUUACUTT 993 AGUAAAGGUGGCUCAGGAATT 994
AD- 15406 2973-2991 UCCUGAGCCACCUUUACUCTT 995 GAGUAAAGGUGGCUCAGGATT
996 AD- 15407 2974-2992 CCUGAGCCACCUUUACUCUTT 997
AGAGUAAAGGUGGCUCAGGTT 998 AD- 15355 2976-2994 UGAGCCACCUUUACUCUGCTT
999 GCAGAGUAAAGGUGGCUCATT 1000 AD- 15356 2978-2996
AGCCACCUUUACUCUGCUCTT 1001 GAGCAGAGUAAAGGUGGCUTT 1002 AD- 15357
2981-2999 CACCUUUACUCUGCUCUAUTT 1003 AUAGAGCAGAGUAAAGGUGTT 1004 AD-
15269 2987-3005 UACUCUGCUCUAUGCCAGGTsT 1005 CCUGGCAUAGAGCAGAGUATsT
1006 AD- 9565 2987-3005 uAcucuGcucuAuGccAGGTsT 1007
CCUGGcAuAGAGcAGAGuATsT 1008 AD- 9691 2998-3016
AUGCCAGGCUGUGCUAGCATT 1009 UGCUAGCACAGCCUGGCAUTT 1010 AD- 15358
3003-3021 AGGCUGUGCUAGCAACACCTT 1011 GGUGUUGCUAGCACAGCCUTT 1012 AD-
15359 3006-3024 CUGUGCUAGCAACACCCAATT 1013 UUGGGUGUUGCUAGCACAGTT
1014 AD- 15360 3010-3028 GCUAGCAACACCCAAAGGUTT 1015
ACCUUUGGGUGUUGCUAGCTT 1016 AD- 15219 3038-3056
GGAGCCAUCACCUAGGACUTT 1017 AGUCCUAGGUGAUGGCUCCTT 1018 AD- 15361
3046-3064 CACCUAGGACUGACUCGGCTT 1019 GCCGAGUCAGUCCUAGGUGTT 1020 AD-
15273 3051-3069 AGGACUGACUCGGCAGUGUTT 1021 ACACUGCCGAGUCAGUCCUTT
1022 AD- 15362 3052-3070 GGACUGACUCGGCAGUGUGTT 1023
CACACUGCCGAGUCAGUCCTT 1024 AD- 15192 3074-3092
UGGUGCAUGCACUGUCUCATT 1025 UGAGACAGUGCAUGCACCATT 1026 AD- 15256
3080-3098 AUGCACUGUCUCAGCCAACTT 1027 GUUGGCUGAGACAGUGCAUTT 1028 AD-
15363 3085-3103 CUGUCUCAGCCAACCCGCUTT 1029 AGCGGGUUGGCUGAGACAGTT
1030 AD- 15364 3089-3107 CUCAGCCAACCCGCUCCACTsT 1031
GUGGAGCGGGUUGGCUGAGTsT 1032 AD- 9604 3089-3107
cucAGccAAcccGcuccAcTsT 1033 GUGGAGCGGGUUGGCUGAGTsT 1034 AD- 9730
3093-3111 GCCAACCCGCUCCACUACCTsT 1035 GGUAGUGGAGCGGGUUGGCTsT 1036
AD- 9527 3093-3111 GccAAcccGcuccAcuAccTsT 1037
GGuAGUGGAGCGGGUUGGCTsT 1038 AD- 9653 3096-3114
AACCCGCUCCACUACCCGGTT 1039 CCGGGUAGUGGAGCGGGUUTT 1040 AD- 15365
3099-3117 CCGCUCCACUACCCGGCAGTT 1041 CUGCCGGGUAGUGGAGCGGTT 1042 AD-
15294 3107-3125 CUACCCGGCAGGGUACACATT 1043 UGUGUACCCUGCCGGGUAGTT
1044 AD- 15173 3108-3126 UACCCGGCAGGGUACACAUTT 1045
AUGUGUACCCUGCCGGGUATT 1046 AD- 15366 3109-3127
ACCCGGCAGGGUACACAUUTT 1047 AAUGUGUACCCUGCCGGGUTT 1048 AD- 15367
3110-3128 CCCGGCAGGGUACACAUUCTT 1049 GAAUGUGUACCCUGCCGGGTT 1050 AD-
15257 3112-3130 CGGCAGGGUACACAUUCGCTT 1051 GCGAAUGUGUACCCUGCCGTT
1052 AD- 15184 3114-3132 GCAGGGUACACAUUCGCACTT 1053
GUGCGAAUGUGUACCCUGCTT 1054 AD- 15185 3115-3133
CAGGGUACACAUUCGCACCTT 1055 GGUGCGAAUGUGUACCCUGTT 1056 AD- 15258
3116-3134 AGGGUACACAUUCGCACCCTT 1057 GGGUGCGAAUGUGUACCCUTT 1058 AD-
15186 3196-3214 GGAACUGAGCCAGAAACGCTT 1059 GCGUUUCUGGCUCAGUUCCTT
1060 AD- 15274 3197-3215 GAACUGAGCCAGAAACGCATT 1061
UGCGUUUCUGGCUCAGUUCTT 1062 AD- 15368 3198-3216
AACUGAGCCAGAAACGCAGTT 1063 CUGCGUUUCUGGCUCAGUUTT 1064 AD- 15369
3201-3219 UGAGCCAGAAACGCAGAUUTT 1065 AAUCUGCGUUUCUGGCUCATT 1066 AD-
15370 3207-3225 AGAAACGCAGAUUGGGCUGTT 1067 CAGCCCAAUCUGCGUUUCUTT
1068 AD- 15259 3210-3228 AACGCAGAUUGGGCUGGCUTT 1069
AGCCAGCCCAAUCUGCGUUTT 1070 AD- 15408 3233-3251
AGCCAAGCCUCUUCUUACUTsT 1071 AGUAAGAAGAGGCUUGGCUTsT 1072 AD- 9597
3233-3251 AGccAAGccucuucuuAcuTsT 1073 AGuAAGAAGAGGCUUGGCUTsT 1074
AD- 9723 3233-3251 AfgCfcAfaGfcCfuCfuUfcUfuAfcU 1075 p- 1076 AD-
fTsT aGfuAfaGfaAfgAfgGfcUfuGfgCfuTsT 14680 3233-3251
AGCfCfAAGCfCfUfCfUfUfCfUfUfA 1077 AGUfAAGAAGAGGCfUfUfGGCfUfTsT 1078
AD- CfUfTsT 14690 3233-3251 AgCcAaGcCuCuUcUuAcUTsT 1079 p- 1080 AD-
aGfuAfaGfaAfgAfgGfcUfuGfgCfuTsT 14700 3233-3251
AgCcAaGcCuCuUcUuAcUTsT 1081 AGUfAAGAAGAGGCfUfUfGGCfUfTsT 1082 AD-
14710 3233-3251 AfgCfcAfaGfcCfuCfuUfcUfuAfcU 1083
AGUAAgaAGagGCUUGgcuTsT 1084 AD- fTsT 14720 3233-3251
AGCfCfAAGCfCfUfCfUfUfCfUfUfA 1085 AGUAAgaAGagGCUUGgcuTsT 1086 AD-
CfUfTsT 14730 3233-3251 AgCcAaGcCuCuUcUuAcUTsT 1087
AGUAAgaAGagGCUUGgcuTsT 1088 AD- 14740 3233-3251
UfgGfuUfcCfcUfgAfgGfaCfcAfgC 1089 p- 1090 AD- fTsT
gCfuGfgUfcCfuCfaGfgGfaAfcCfaTsT 15086 3233-3251
UfGGUfUfCfCfCfUfGAGGACfCfAGC 1091 GCfUfGGUfCfCfUfCfAGGGAACfCfATsT
1092 AD- fTsT 15096 3233-3251 UgGuUcCcUgAgGaCcAgCTsT 1093 p- 1094
AD- gCfuGfgUfcCfuCfaGfgGfaAfcCfaTsT 15106 3233-3251
UgGuUcCcUgAgGaCcAgCTsT 1095 GCfUfGGUfCfCfUfCfAGGGAACfCfATsT 1096
AD- 15116 32333251- UfgGfuUfcCfcUfgAfgGfaCfcAfgC 1097
GCUGGucCUcaGGGAAccaTsT 1098 AD- fTsT 15126 3233-3251
UfGGUfUfCfCfCfUfGAGGACfCfAGC 1099 GCUGGucCUcaGGGAAccaTsT 1100 AD-
fTsT 15136 3233-3251 UgGuUcCcUgAgGaCcAgCTsT 1101
GCUGGucCUcaGGGAAccaTsT 1102 AD- 15146 3242-3260
UCUUCUUACUUCACCCGGCTT 1103 GCCGGGUGAAGUAAGAAGATT 1104 AD- 15260
3243-3261 CUUCUUACUUCACCCGGCUTT 1105 AGCCGGGUGAAGUAAGAAGTT 1106 AD-
15371 3244-3262 UUCUUACUUCACCCGGCUGTT 1107 CAGCCGGGUGAAGUAAGAATT
1108 AD- 15372 3262-3280 GGGCUCCUCAUUUUUACGGTT 1109
CCGUAAAAAUGAGGAGCCCTT 1110 AD- 15172 3263-3281
GGCUCCUCAUUUUUACGGGTT 1111 CCCGUAAAAAUGAGGAGCCTT 1112 AD- 15295
3264-3282 GCUCCUCAUUUUUACGGGUTT 1113 ACCCGUAAAAAUGAGGAGCTT 1114 AD-
15373 3265-3283 CUCCUCAUUUUUACGGGUATT 1115 UACCCGUAAAAAUGAGGAGTT
1116 AD- 15163 3266-3284 UCCUCAUUUUUACGGGUAATT 1117
UUACCCGUAAAAAUGAGGATT 1118 AD- 15165 3267-3285
CCUCAUUUUUACGGGUAACTT 1119 GUUACCCGUAAAAAUGAGGTT 1120 AD- 15374
3268-3286 CUCAUUUUUACGGGUAACATT 1121 UGUUACCCGUAAAAAUGAGTT 1122 AD-
15296 3270-3288 CAUUUUUACGGGUAACAGUTT 1123 ACUGUUACCCGUAAAAAUGTT
1124 AD- 15261 3271-3289 AUUUUUACGGGUAACAGUGTT 1125
CACUGUUACCCGUAAAAAUTT 1126 AD- 15375 3274-3292
UUUACGGGUAACAGUGAGGTT 1127 CCUCACUGUUACCCGUAAATT 1128 AD- 15262
3308-3326 CAGACCAGGAAGCUCGGUGTT 1129 CACCGAGCUUCCUGGUCUGTT 1130 AD-
15376 3310-3328 GACCAGGAAGCUCGGUGAGTT 1131 CUCACCGAGCUUCCUGGUCTT
1132 AD- 15377 3312-3330 CCAGGAAGCUCGGUGAGUGTT 1133
CACUCACCGAGCUUCCUGGTT 1134 AD- 15409 3315-3333
GGAAGCUCGGUGAGUGAUGTT 1135 CAUCACUCACCGAGCUUCCTT 1136 AD- 15378
3324-3342 GUGAGUGAUGGCAGAACGATT 1137 UCGUUCUGCCAUCACUCACTT 1138 AD-
15410 3326-3344 GAGUGAUGGCAGAACGAUGTT 1139 CAUCGUUCUGCCAUCACUCTT
1140 AD- 15379 3330-3348 GAUGGCAGAACGAUGCCUGTT 1141
CAGGCAUCGUUCUGCCAUCTT 1142 AD- 15187 3336-3354
AGAACGAUGCCUGCAGGCATT 1143 UGCCUGCAGGCAUCGUUCUTT 1144 AD-
15263 3339-3357 ACGAUGCCUGCAGGCAUGGTT 1145 CCAUGCCUGCAGGCAUCGUTT
1146 AD- 15264 3348-3366 GCAGGCAUGGAACUUUUUCTT 1147
GAAAAAGUUCCAUGCCUGCTT 1148 AD- 15297 3356-3374
GGAACUUUUUCCGUUAUCATT 1149 UGAUAACGGAAAAAGUUCCTT 1150 AD- 15208
3357-3375 GAACUUUUUCCGUUAUCACTT 1151 GUGAUAACGGAAAAAGUUCTT 1152 AD-
15209 3358-3376 AACUUUUUCCGUUAUCACCTT 1153 GGUGAUAACGGAAAAAGUUTT
1154 AD- 15193 3370-3388 UAUCACCCAGGCCUGAUUCTT 1155
GAAUCAGGCCUGGGUGAUATT 1156 AD- 15380 3378-3396
AGGCCUGAUUCACUGGCCUTT 1157 AGGCCAGUGAAUCAGGCCUTT 1158 AD- 15298
3383-3401 UGAUUCACUGGCCUGGCGGTT 1159 CCGCCAGGCCAGUGAAUCATT 1160 AD-
15299 3385-3403 AUUCACUGGCCUGGCGGAGTT 1161 CUCCGCCAGGCCAGUGAAUTT
1162 AD- 15265 3406-3424 GCUUCUAAGGCAUGGUCGGTT 1163
CCGACCAUGCCUUAGAAGCTT 1164 AD- 15381 3407-3425
CUUCUAAGGCAUGGUCGGGTT 1165 CCCGACCAUGCCUUAGAAGTT 1166 AD- 15210
3429-3447 GAGGGCCAACAACUGUCCCTT 1167 GGGACAGUUGUUGGCCCUCTT 1168 AD-
15270 3440-3458 ACUGUCCCUCCUUGAGCACTsT 1169 GUGCUCAAGGAGGGACAGUTsT
1170 AD- 9591 3440-3458 AcuGucccuccuuGAGcAcTsT 1171
GUGCUcAAGGAGGGAcAGUTsT 1172 AD- 9717 3441-3459
CUGUCCCUCCUUGAGCACCTsT 1173 GGUGCUCAAGGAGGGACAGTsT 1174 AD- 9622
3441-3459 cuGucccuccuuGAGcAccTsT 1175 GGUGCUcAAGGAGGGAcAGTsT 1176
AD- 9748 3480-3498 ACAUUUAUCUUUUGGGUCUTsT 1177
AGACCCAAAAGAUAAAUGUTsT 1178 AD- 9587 3480-3498
AcAuudAucuuuuGGGucuTsT 1179 AGACCcAAAAGAuAAAUGUTsT 1180 AD- 9713
3480-3498 AfcAfuUfuAfuCfuUfuUfgGfgUfCU 1181 p- 1182 AD- fTsT
aGfaCfcCfaAfaAfgAfuAfaAfuGfuTsT 14679 3480-3498
ACfAUfUfUfAUfCfUfUfUfUfGGGUf 1183 AGACfCfCfAAAAGAUfAAAUfGUfTsT 1184
AD- CfUfTsT 14689 3480-3498 AcAuUuAuCuUuUgGgUcUTsT 1185 P- 1186 AD-
aGfaCfcCfaAfaAfgAfuAfaAfuGfuTsT 14699 3480-3498
AcAuUuAuCuUuUgGgUcUTsT 1187 AGACfCfCfAAAAGAUfAAAUfGUfTsT 1188 AD-
14709 3480-3498 AfcAfuUfuAfuCfuUfuUfgGfgUfCU 1189
AGACCcaAAagAUAAAuguTsT 1190 AD- fTsT 14719 3480-3498
ACfAUfUfUfAUfCfUfUfUfUfGGGUf 1191 AGACCcaAAagAUAAAuguTsT 1192 AD-
CfUfTsT 14729 3480-3498 AcAuUuAuCuUuUgGgUcUTsT 1193
AGACCcaAAagAUAAAuguTsT 1194 AD- 14739 3480-3498
GfcCfaUfcUfgCfuGfcCfgGfaGfcC 1195 p- 1196 AD- fTsT
gGfcUfcCfgGfcAfgCfaGfaUfgGfcTsT 15085 3480-3498
GCfCfAUfCfUfGCfUfGCfCfGGAGCf 1197 GGCfUfCfCfGGCfAGCfAGAUfGGCfTsT
1198 AD- CfTsT 15095 3480-3498 GcCaUcUgCuGcCgGaGcCTsT 1199 P- 1200
AD- gGfcUfcCfgGfcAfgCfaGfaUfgGfcTsT 15105 3480-3498
GcCaUcUgCuGcCgGaGcCTsT 1201 GGCfUfCfCfGGCfAGCfAGAUfGGCfTsT 1202 AD-
15115 3480-3498 GfcCfaUfcUfgCfuGfcCfgGfaGfcC 1203
GGCUCauGCagCAGUggcTsT 1204 AD- fTsT 15125 3480-3498
GCfCfAUfCfUfGCfUfGCfCfGGAGCf 1205 GGCUCauGCagCAGAUggcTsT 1206 AD-
CfTsT 15135 3480-3498 GcCaUcUgCuGcCgGaGcCTsT 1207
GGCUCauGCagCAGAUggcTsT 1208 AD- 15145 3481-3499
CAUUUAUCUUUUGGGUCUGTsT 1209 CAGACCCAAAAGAUAAAUGTsT 1210 AD- 9578
3481-3499 cAuuuAucuuuuGGGucuGTsT 1211 cAGACCcAAAAGAuAAAUGTsT 1212
AD- 9704 3485-3503 UAUCUUUUGGGUCUGUCCUTsT 1213
AGGACAGACCCAAAAGAUATsT 1214 AD- 9558 3485-3503
uAucuuuuGGGucuGuccuTsT 1215 AGGAcAGACCcAAAAGAuATsT 1216 AD- 9684
3504-3522 CUCUGUUGCCUUUUUACAGTsT 1217 CUGUAAAAAGGCAACAGAGTsT 1218
AD- 9634 3504-3522 cucuGuuGccuuuuuAcAGTsT 1219
CUGuAAAAAGGcAAcAGAGTsT 1220 AD- 9760 3512-3530
CCUUUUUACAGCCAACUUUTT 1221 AAAGUUGGCUGUAAAAAGGTT 1222 AD- 15411
3521-3539 AGCCAACUUUUCUAGACCUTT 1223 AGGUCUAGAAAAGUUGGCUTT 1224 AD-
15266 3526-3544 ACUUUUCUAGACCUGUUUUTT 1225 AAAACAGGUCUAGAAAAGUTT
1226 AD- 15382 3530-3548 UUCUAGACUGUUUUGCUUTsT 1227
AAGCAAAACAGGUCUAGAATsT 1228 AD- 9554 3530-3548
uucuAGAccuGuuuuGcuuTsT 1229 AAGcAAAAcAGGUCuAGAATsT 1230 AD- 9680
3530-3548 UfuCfuAfgAfcCfuGfuUfuUfgCfuU 1231 p- 1232 AD- fTsT
aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT 14676 3530-3548
UfUfCfUfAGACfCfUfGUfUfUfUfGC 1233 AAGCfAAAACfAGGUfCfUfAGAATsT 1234
AD- fUfUfTsT 14686 3530-3548 UuCuAgAcCuGuUuUgCuUTsT 1235 p- 1236
AD- aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT 14696 3530-3548
UuCuAgAcCuGuUuUgCuUTsT 1237 AAGCfAAAACfAGGUfCfUfAGAATsT 1238 AD-
14706 3530-3548 UfuCfuAfgAfcCfuGfuUfuUffCfuU 1239
AAGcAaaACagGUCUAgaaTsT 1240 AD- fTsT 14716 3530-3548
UfUfCfUfAGACfCfUfGUfUfUfUfGC 1241 AAGcAaaACagGUCUAgaaTsT 1242 AD-
fUfUfTsT 14726 3530-3548 UuCuAgAcCuGuUuUgCuUTsT 1243
AAGcAaaACagGUCUAgaaTsT 1244 AD- 14736 3530-3548
CfaUfaGfgCfcUfgGfaGfuUfuAfuU 1245 p- 1246 AD- fTsT
aAfuAfaAfcUfcCfaGfgCfcUfaUfgTsT 15082 3530-3548
CfAUfAGGCfCfUfGGAGUfUfUfAUfU 1247 AAUfAAACfUfCfCfAGGCfCfUfAUfGTsT
1248 AD- fTsT 15092 3530-3548 CaUaGgCcUgGaGuUuAuUTsT 1249 P- 1250
AD- aAfuAfaAfcUfcCfaGfgCfcUfaUfgTsT 15102 3530-3548
CaUaGgCcUgGaGuUuAuUTsT 1251 AAUfAAACfUfCfCfAGGCfCfUfAUfGTsT 1252
AD- 15112 3530-3548 CfaUfaGfgCfcUfgGfaGfuUfuAfuU 1253
AAUAAacUCcaGGCCUaugTsT 1254 AD- fTsT 15122 3530-3548
CfAUfAGGCfCfUfGGAGUfUfUfAUfU 1255 AAUAAacUCcaGGCCUaugTsT 1256 AD-
fTsT 15132 3530-3548 CaUaGgCcUgGaGuUuAuUTsT 1257
AAUAAacUCcaGGCCUaugTsT 1258 AD- 15142 3531-3549
UCUAGACCUGUUUUGCUUUTsT 1259 AAAGCAAAACAGGUCUAGATsT 1260 AD- 9553
3531-3549 ucuAGAccuGuuuuGcuuuTsT 1261 AAAGcAAAAcAGGUCuAGATsT 1262
AD- 9679 3531-3549 UfcUfaGfaCfcUfgUfuUfuGfcUfuU 1263 p- 1264 AD-
fTsT aAfaGfcAfaAfaCfaGfgUfcUfaGfaTsT 14675 3531-3549
UfCfUfAGACfCfUfGUfUfUfUfGCfU 1265 AAAGCfAAAACfAGGUfCfUfAGATsT 1266
AD- fUfUfTsT 14685 3531-3549 UcUaGaCcUgUuUuGcUuUTsT 1267 P- 1268
AD- aAfaGfcAfaAfaCfaGfgUfcUfaGfaTsT 14695 3531-3549
UcUaGaCcUgUuUuGcUuUTsT 1269 AAAGCfAAAACfAGGUfCfUfAGATsT 1270 AD-
14705 3531-3549 UfcUfaGfaCfcUfgUfuUfuGfcUfuU 1271
AAAGCaaAAcaGGUCUagaTsT 1272 AD- fTsT 14715 3531-3549
UfCfUfAGACfCfUfGUfUfUfUfGCfU 1273 AAAGCaaAAcaGGUCUagaTsT 1274 AD-
fUfUfTsT 14725 3531-3549 UcUaGaCcUgUuUuGcUuUTsT 1275
AAAGCaaAAcaGGUCUagaTsT 1276 AD- 14735 3531-3549
UfcAfuAfgGfcCfuGfgAfgUfuUfaU 1277 p- 1279 AD- fTsT
aUfaAfaCfuCfcAfgGfcCfuAfuGfaTsT 15081 3531-3549
UfCfAUfAGGCfCfUfGGAGUfUfUfAU 1279 AUfAAACfUfCfCfAGGCfCfUfAUfGATsT
1280 AD- fTsT 15091 3531-3549 UcAuAgGcCuGgAgUuUaUTsT 1281 P- 1282
AD- aUfaAfaCfuCfcAfgGfcCfuAfuGfaTsT 15101 3531-3549
UcAuAgGcCuGgAgUuUaUTsT 1283 AUfAAACfUfCfCfAGGCfCfUfAUfGATsT 1284
AD- 15111 3531-3549 UfcAfuAfgGfcCfuGfgAfgUfuUfaU 1285
AUAAAcuCCagGCCUAugaTsT 1287 AD- fTsT 15121 3531-3549
UfCfAUfAGGCfCfUfGGAGUfUfUfAU 1287 AUAAAcuCCagGCCUAugaTsT 1288 AD-
fTsT 15131 3531-3549 UcAuAgGcCuGgAgUuUaUTsT 1289
AUAAAcuCCagGCCUAugaTsT 1290 AD- 15141 3557-3575
UGAAGAUAUUUAUUCUGGGTsT 1291 CCCAGAAUAAAUAUCUUCATsT 1292 AD- 9626
3557-3575 uGAAGAuAuuuAuucuGGGTsT 1293 CCcAGAAuAAAuAUCUUcATsT 1294
AD- 9752
3570-3588 CUGGGUUUUGUAGCAUUUTsT 1295 AAAUGCUACAAAACCCAGATsT 1296
AD- 9629 3570-3588 ucuGGGuuuuGuAGcAuuuTsT 1297
AAAUGCuAcAAAACCcAGATsT 1298 AD- 9755 3613-3631
AUAAAAACAAACAAACGUUTT 1299 AACGUUUGUUUGUUUUUAUTT 1300 AD- 15412
3617-3635 AAACAAACAAACGUUGUCCTT 1301 GGACAACGUUUGUUUGUUUTT 1302 AD-
15211 3618-3636 AACAAACAAACGUUGUCCUTT 1303 AGGACAACGUUUGUUUGUUTT
1304 AD- 15300 U, C, A, G: corresponding ribonucleotide; T:
deoxythymidine; u, c, a, g: corresponding 2'O-methyl
ribonucleotide; Uf, Cf, Af, Gf: corresponding 2'-deoxy-2'-fluoro
ribonucleotide; where nucleotides are written in sequence, they are
connected by 3'-5' phosphodiester groups; nucleotides with
interjected ''s'' are connected by 3'-O-5'-O phosphorothiodiester
groups; unless denoted by prefix ''p-'', oligonucleotides are
devoid of a 5'-phosphate group on the 5'-most nucleotide; all
oligonucleotides bear 3'-OH on the 3'-most nucleotide
TABLE-US-00013 TABLE 1b Screening of siRNAs targeted to PCSK9 Mean
percent remaining mRNA transcript at siRNA concentration/in cell
type IC50 in IC50 in Cynomolgous Duplex 100 nM/ 30 nM/ 3 nM/ 30 nM/
HepG2 monkey Hepatocyte name HepG2 HepG2 HepG2 HeLa [nM] [nM]s
AD-15220 35 AD-15275 56 AD-15301 70 AD-15276 42 AD-15302 32
AD-15303 37 AD-15221 30 AD-15413 61 AD-15304 70 AD-15305 36
AD-15306 20 AD-15307 38 AD-15277 50 AD-9526 74 89 AD-9652 97
AD-9519 78 AD-9645 66 AD-9523 55 AD-9649 60 AD-9569 112 AD-9695 102
AD-15222 75 AD-15278 78 AD-15178 83 AD-15308 84 AD-15223 67
AD-15309 34 AD-15279 44 AD-15194 63 AD-15310 42 AD-15311 30
AD-15392 18 AD-15312 21 AD-15313 19 AD-15280 81 AD-15267 82
AD-15314 32 AD-15315 74 AD-9624 94 AD-9750 96 AD-9623 43 66 AD-9749
105 AD-15384 48 AD-9607 32 28 0.20 AD-9733 78 73 AD-9524 23 28 0.07
AD-9650 91 90 AD-9520 23 32 AD-9520 23 AD-9646 97 108 AD-9608 37
AD-9734 91 AD-9546 32 AD-9672 57 AD-15385 54 AD-15393 31 AD-15316
37 AD-15317 37 AD-15318 63 AD-15195 45 AD-15224 57 AD-15188 42
AD-15225 51 AD-15281 89 AD-15282 75 AD-15319 61 AD-15226 56
AD-15271 25 AD-15283 25 AD-15284 64 AD-15189 17 AD-15227 62 AD-9547
31 29 0.20 AD-9673 56 57 AD-9548 54 60 AD-9674 36 57 AD-9529 60
AD-9655 140 AD-9605 27 31 0.27 AD-9731 31 31 0.32 AD-9596 37
AD-9722 76 AD-9583 42 AD-9709 104 AD-9579 113 AD-9705 81 AD-15394
32 AD-15196 72 AD-15197 85 AD-15198 71 AD-9609 66 71 AD-9735 115
AD-9537 145 AD-9663 102 AD-9528 113 AD-9654 107 AD-9515 49 AD-9641
92 AD-9514 57 AD-9640 89 AD-9530 75 AD-9656 77 AD-9538 79 80
AD-9664 53 AD-9598 69 83 AD-9724 127 AD-9625 58 88 AD-9751 60
AD-9556 46 AD-9682 38 AD-9539 56 63 AD-9665 83 AD-9517 36 AD-9643
40 AD-9610 36 34 0.04 AD-9736 22 29 0.04 0.5 AD-14681 33 AD-14691
27 AD-14701 32 AD-14711 33 AD-14721 22 AD-14731 21 AD-14741 22
AD-15087 37 AD-15097 51 AD-15107 26 AD-15117 28 AD-15127 33
AD-15137 54 AD-15147 52 AD-9516 94 AD-9642 105 AD-9562 46 51
AD-9688 26 34 4.20 AD-14677 38 AD-14687 52 AD-14697 35 AD-14707 58
AD-14717 42 AD-14727 50 AD-14737 32 AD-15083 16 AD-15093 24
AD-15103 11 AD-15113 34 AD-15123 19 AD-15133 15 AD-15143 16 AD-9521
50 AD-9647 62 AD-9611 48 AD-9737 68 AD-9592 46 55 AD-9718 78
AD-9561 64 AD-9687 84 AD-9636 42 41 2.10 AD-9762 9 28 0.40 0.5
AD-9540 45 AD-9666 81 AD-9535 48 73 AD-9661 83 AD-9559 35 AD-9685
77 AD-9533 100 AD-9659 88 AD-9612 122 AD-9738 83 AD-9557 75 96
AD-9683 48 AD-9531 31 32 0.53 AD-9657 23 29 0.66 0.5 AD-14673 81
AD-14683 56 AD-14693 56 AD-14703 68 AD-14713 55 AD-14723 24
AD-14733 34 AD-15079 85 AD-15089 54 AD-15099 70 AD-15109 67
AD-15119 67 AD-15129 57 AD-15139 69 AD-9542 160 AD-9668 92 AD-9739
109 AD-9637 56 83 AD-9763 79 AD-9630 82 AD-9756 63 AD-9593 55
AD-9719 115 AD-9601 111 AD-9727 118 AD-9573 36 42 1.60 AD-9699 32
36 2.50 AD-15228 26 AD-15395 53 AD-9602 126 AD-9728 94 AD-15386 45
AD-9580 112 AD-9706 86 AD-9581 35 AD-9707 81 AD-9543 51 AD-9669 97
AD-9574 74 AD-9700 AD-15320 26 AD-15321 34 AD-15199 64 AD-15167 86
AD-15164 41 AD-15166 43 AD-15322 64 AD-15200 46 AD-15213 27
AD-15229 44 AD-15215 49 AD-15214 101 AD-9315 15 32 0.98 AD-9326 35
51 AD-9318 14 37 0.40 AD-9323 14 33 AD-9314 11 22 0.04 AD-10792
0.10 0.10 AD-10796 0.1 0.1 AD-9638 101 AD-9764 112 AD-9525 53
AD-9651 58 AD-9560 97 AD-9686 111 AD-9536 157 AD-9662 81 AD-9584 52
68 AD-9710 111 AD-15323 62
AD-9551 91 AD-9677 62 AD-15230 52 AD-15231 25 AD-15285 36 AD-15396
27 AD-15397 56 AD-9600 112 AD-9726 95 AD-9606 107 AD-9732 105
AD-9633 56 75 AD-9759 111 AD-9588 66 AD-9714 106 AD-9589 67 85
AD-9715 113 AD-9575 120 AD-9701 100 AD-9563 103 AD-9689 81 AD-9594
80 95 AD-9720 92 AD-9585 83 AD-9711 122 AD-9614 100 AD-9740 198
AD-9615 116 AD-9741 130 AD-9534 32 30 AD-9534 32 AD-9660 89 79
AD-15324 46 AD-15232 19 AD-15233 25 AD-15234 59 AD-15286 109
AD-9590 122 AD-9716 114 AD-9632 34 AD-9758 96 AD-9567 41 AD-9693 50
AD-9586 81 104 AD-9712 107 AD-9564 120 AD-9690 92 AD-9616 74 84
AD-9742 127 AD-15398 24 AD-9617 111 AD-9743 104 AD-9635 73 90
AD-9761 15 33 0.5 AD-9568 76 AD-9694 52 AD-9576 47 AD-9702 79
AD-9627 69 AD-9753 127 AD-9628 141 AD-9754 89 AD-9631 80 AD-9757 78
AD-9595 31 32 AD-9721 87 70 AD-9544 68 AD-9670 67 AD-15235 25
AD-15236 73 AD-15168 100 AD-15174 92 AD-15325 81 AD-15326 65
AD-9570 35 42 AD-9696 77 AD-9566 38 AD-9692 78 AD-9532 100 AD-9658
102 AD-9549 50 AD-9675 78 AD-9541 43 AD-9667 73 AD-9550 36 AD-9676
100 AD-9571 27 32 AD-9697 74 89 AD-9572 47 53 AD-9698 73 AD-15327
82 AD-9639 30 35 AD-9765 82 74 AD-9518 31 35 0.60 AD-9518 31
AD-9644 35 37 2.60 0.5 AD-14672 26 AD-14682 27 AD-14692 22 AD-14702
19 AD-14712 25 AD-14722 18 AD-14732 32 AD-15078 86 AD-15088 97
AD-15098 74 AD-15108 67 AD-15118 76 AD-15128 86 AD-15138 74
AD-15237 30 AD-15287 30 AD-15238 36 AD-15328 35 AD-15399 47 AD-9582
37 AD-9708 81 AD-9545 31 43 AD-9671 15 33 2.50 AD-14674 16 AD-14684
26 AD-14694 18 AD-14704 27 AD-14714 20 AD-14724 18 AD-14734 18
AD-15080 29 AD-15090 23 AD-15100 26 AD-15110 23 AD-15120 20
AD-15130 20 AD-15140 19 AD-9522 59 AD-9648 78 AD-9552 80 AD-9678 76
AD-9618 90 AD-9744 91 AD-15239 38 AD-15212 19 AD-15240 43 AD-15177
59 AD-15179 13 AD-15180 15 AD-15241 14 AD-15268 42 AD-15242 21
AD-15216 28 AD-15176 35 AD-15181 35 AD-15243 22 AD-15182 42
AD-15244 31 AD-15387 23 AD-15245 18 AD-9555 34 AD-9681 55 AD-9619
42 61 AD-9745 56 AD-9620 44 77 AD-9746 89 AD-15288 19 AD-15246 16
AD-15289 37 AD-9324 59 67 AD-15329 103 AD-15330 62 AD-15169 22
AD-15201 6 AD-15331 14 AD-15190 47 AD-15247 61 AD-15248 22 AD-15175
45 AD-15249 51 AD-15250 96 AD-15400 12 AD-15332 22 AD-15388 30
AD-15333 20 AD-15334 96 AD-15335 75 AD-15183 16 AD-15202 41
AD-15203 39 AD-15272 49 AD-15217 16 AD-15290 15 AD-15218 13
AD-15389 13 AD-15336 40 AD-15337 19 AD-15191 33 AD-15390 25
AD-15338 9 AD-15204 33 AD-15251 76 AD-15205 14 AD-15171 16 AD-15252
58 AD-15339 20 AD-15253 15 AD-15340 18 AD-15291 17 AD-15341 11
AD-15401 13 AD-15342 30 AD-15343 21 AD-15292 16 AD-15344 20
AD-15254 18 AD-15345 18 AD-15206 15 AD-15346 16 AD-15347 62 AD-9577
33 31 AD-9703 17 26 1 AD-14678 22 AD-14688 23 AD-14698 23 AD-14708
14 AD-14718 31 AD-14728 25 AD-14738 31 AD-15084 19 AD-15094 11
AD-15104 16 AD-15114 15 AD-15124 11 AD-15134 12 AD-15144 9 AD-15391
7 AD-15348 13 AD-15349 8 AD-15170 40 AD-15350 14 AD-15402 27
AD-15293 27 AD-15351 14 AD-15403 11 AD-15404 38 AD-15207 15
AD-15352 23 AD-15255 31 AD-9603 123 AD-9729 56 AD-9599 139 AD-9725
38 AD-9621 77 AD-9747 63
AD-15405 32 AD-15353 39 AD-15354 49 AD-15406 35 AD-15407 39
AD-15355 18 AD-15356 50 AD-15357 54 AD-15269 23 AD-9565 74 AD-9691
49 AD-15358 12 AD-15359 24 AD-15360 13 AD-15219 19 AD-15361 24
AD-15273 36 AD-15362 31 AD-15192 20 AD-15256 19 AD-15363 33
AD-15364 24 AD-9604 35 49 AD-9730 85 AD-9527 45 AD-9653 86 AD-15365
62 AD-15294 30 AD-15173 12 AD-15366 21 AD-15367 11 AD-15257 18
AD-15184 50 AD-15185 12 AD-15258 73 AD-15186 36 AD-15274 19
AD-15368 7 AD-15369 17 AD-15370 19 AD-15259 38 AD-15408 52 AD-9597
23 21 0.04 AD-9723 12 26 0.5 AD-14680 15 AD-14690 18 AD-14700 15
AD-14710 15 AD-14720 18 AD-14730 18 AD-14740 17 AD-15086 85
AD-15096 70 AD-15106 71 AD-15116 73 AD-15126 71 AD-15136 56
AD-15146 72 AD-15260 79 AD-15371 24 AD-15372 52 AD-15172 27
AD-15295 22 AD-15373 11 AD-15163 18 AD-15165 13 AD-15374 23
AD-15296 13 AD-15261 20 AD-15375 90 AD-15262 72 AD-15376 14
AD-15377 19 AD-15409 17 AD-15378 18 AD-15410 8 AD-15379 11 AD-15187
36 AD-15263 18 AD-15264 75 AD-15297 21 AD-15208 6 AD-15209 28
AD-15193 131 AD-15380 88 AD-15298 43 AD-15299 99 AD-15265 95
AD-15381 18 AD-15210 40 AD-15270 83 AD-9591 75 95 AD-9717 105
AD-9622 94 AD-9748 103 AD-9587 63 49 AD-9713 22 25 0.5 AD-14679 19
AD-14689 24 AD-14699 19 AD-14709 21 AD-14719 24 AD-14729 23
AD-14739 24 AD-15085 74 AD-15095 60 AD-15105 33 AD-15115 30
AD-15125 54 AD-15135 51 AD-15145 49 AD-9578 49 61 AD-9704 111
AD-9558 66 AD-9684 63 AD-9634 29 30 AD-9760 14 27 AD-15411 5
AD-15266 23 AD-15382 12 AD-9554 23 24 AD-9680 12 22 0.1 0.1
AD-14676 12 .1 AD-14686 13 AD-14696 12 .1 AD-14706 18 .1 AD-14716
17 .1 AD-14726 16 .1 AD-14736 9 .1 AD-15082 27 AD-15092 28 AD-15102
19 AD-15112 17 AD-15122 56 AD-15132 39 AD-15142 46 AD-9553 27 22
0.02 AD-9679 17 21 0.1 AD-14675 11 AD-14685 19 AD-14695 12 AD-14705
16 AD-14715 19 AD-14725 19 AD-14735 19 AD-15081 30 AD-15091 16
AD-15101 16 AD-15111 11 AD-15121 19 AD-15131 17 AD-15141 18 AD-9626
97 68 AD-9752 28 33 AD-9629 23 24 AD-9755 28 29 0.5 AD-15412 21
AD-15211 73 AD-15300 41
TABLE-US-00014 TABLE 2a Sequences of modified dsRNA targeted to
PCSK9 SEQ SEQ Duplex ID ID number Sense strand sequence
(5'-3').sup.1 NO: Antisense-strand sequence (5'-3').sup.1 NO:
AD-10792 GccuGGAGuuuAuucGGAATsT 1305 UUCCGAAuAAACUCcAGGCTsT 1306
AD-10793 GccuGGAGuuuAuucGGAATsT 1307 uUcCGAAuAAACUccAGGCTsT 1308
AD-10796 GccuGGAGuuuAuucGGAATsT 1309 UUCCGAAUAAACUCCAGGCTsT 1310
AD-12038 GccuGGAGuuuAuucGGAATsT 1311 uUCCGAAUAAACUCCAGGCTsT 1312
AD-12039 GccuGGAGuuuAuucGGAATsT 1313 UuCCGAAUAAACUCCAGGCTsT 1314
AD-12040 GccuGGAGuuuAuucGGAATsT 1315 UUcCGAAUAAACUCCAGGCTsT 1316
AD-12041 GccuGGAGuuuAuucGGAATsT 1317 UUCcGAAUAAACUCCAGGCTsT 1318
AD-12042 GCCUGGAGUUUAUUCGGAATsT 1319 uUCCGAAUAAACUCCAGGCTsT 1320
AD-12043 GCCUGGAGUUUAUUCGGAATsT 1321 UuCCGAAUAAACUCCAGGCTsT 1322
AD-12044 GCCUGGAGUUUAUUCGGAATsT 1323 UUcCGAAUAAACUCCAGGCTsT 1324
AD-12045 GCCUGGAGUUUAUUCGGAATsT 1325 UUCcGAAUAAACUCCAGGCTsT 1326
AD-12046 GccuGGAGuuuAuucGGAA 1327 UUCCGAAUAAACUCCAGGCscsu 1328
AD-12047 GccuGGAGuuuAuucGGAAA 1329 UUUCCGAAUAAACUCCAGGCscsu 1330
AD-12048 GccuGGAGuuuAuucGGAAAA 1331 UUUUCCGAAUAAACUCCAGGCscsu 1332
AD-12049 GccuGGAGuuuAuucGGAAAAG 1333 CUUUUCCGAAUAAACUCCAGGCscsu
1334 AD-12050 GccuGGAGuuuAuucGGAATTab 1335 UUCCGAAUAAACUCCAGGCTTab
1336 AD-12051 GccuGGAGuuuAuucGGAAATTab 1337
UUUCCGAAuAAACUCCAGGCTTab 1338 AD-12052 GccuGGAGuuuAuucGGAAAATTab
1339 UUUUCCGAAUAAACUCCAGGCTTab 1340 AD-12053
GccuGGAGuuuAuucGGAAAAGTTab 1341 CUUUUCCGAAUAAACUCCAGGCTTab 1342
AD-12054 GCCUGGAGUUUAUUCGGAATsT 1343 UUCCGAAUAAACUCCAGGCscsu 1344
AD-12055 GccuGGAGuuuAuucGGAATsT 1345 UUCCGAAUAAACUCCAGGCscsu 1346
AD-12056 GcCuGgAgUuUaUuCgGaA 1347 UUCCGAAUAAACUCCAGGCTTab 1348
AD-12057 GcCuGgAgUuUaUuCgGaA 1349 UUCCGAAUAAACUCCAGGCTsT 1350
AD-12058 GcCuGgAgUuUaUuCgGaA 1351 UUCCGAAuAAACUCcAGGCTsT 1352
AD-12059 GcCuGgAgUuUaUuCgGaA 1353 uUcCGAAuAAACUccAGGCTsT 1354
AD-12060 GcCuGgAgUuUaUuCgGaA 1355 UUCCGaaUAaaCUCCAggc 1356 AD-12061
GcCuGgnAgUuUaUuCgGaATsT 1357 UUCCGaaUAaaCUCCAggcTsT 1358 AD-12062
GcCuGgAgUuUaUuCgGaATTab 1359 UUCCGaaUAaaCUCCAggcTTab 1360 AD-12063
GcCuGgAgUuUaUuCgGaA 1361 UUCCGaaUAaaCUCCAggcscsu 1362 AD-12064
GcCuGgnAgUuUaUuCgGaATsT 1363 UUCCGAAuAAACUCcAGGCTsT 1364 AD-12065
GcCuGgAgUuUaUuCgGaATTab 1365 UUCCGAAuAAACUCcAGGCTTab 1366 AD-12066
GcCuGgAgUuUaUuCgGaA 1367 UUCCGAAuAAACUCcAGGCscsu 1368 AD-12067
GcCuGgnAgUuUaUuCgGaATsT 1369 UUCCGAAUAAACUCCAGGCTsT 1370 AD-12068
GcCuGgAgUuUaUuCgGaATTab 1371 UUCCGAAUAAACUCCAGGCTTab 1372 AD-12069
GcCuGgAgUuUaUuCgGaA 1373 UUCCGAAUAAACUCCAGGCscsu 1374 AD-12338
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1375 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc
1376 AD-12339 GcCuGgAgUuUaUuCgGaA 1377
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1378 AD-12340 GccuGGAGuuuAuucGGAA
1379 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1380 AD-12341
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1381
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1382 AD-12342
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1383 UUCCGAAuAAACUCcAGGCTsT 1384
AD-12343 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1385
uUcCGAAuAAACUccAGGCTsT 1386 AD-12344
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1387 UUCCGAAUAAACUCCAGGCTsT 1388
AD-12345 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1389
UUCCGAAUAAACUCCAGGCscsu 1390 AD-12346
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1391 UUCCGaaUAaaCUCCAggcscsu 1392
AD-12347 GCCUGGAGUUUAUUCGGAATsT 1393
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1394 AD-12348
GccuGGAGuuuAuucGGAATsT 1395 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1396
AD-12349 GcCuGgnAgUuUaUuCgGaATsT 1397
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1398 AD-12350
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTTab 1399
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTTab 1400 AD-12351
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1401
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1402 AD-12352
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1403 UUCCGaaUAaaCUCCAggcscsu 1404
AD-12354 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1405 UUCCGAAUAAACUCCAGGCscsu
1406 AD-12355 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1407
UUCCGAAuAAACUCcAGGCTsT 1408 AD-12356 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf
1409 uUcCGAAuAAACUccAGGCTsT 1410 AD-12357
GmocCmouGmogAm02gUmouUmoaUmouCm 1411 UUCCGaaUAaaCUCCAggc 1412
ogGmoaA AD-12358 GmocCmouGmogAm02gUmouUmoaUmouCm 1413
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1414 ogGmoaA AD-12359
GmocCmouGmogAm02gUmouUmoaUmouCm 1415
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1416 ogGmoaA AD-12360
GmocCmouGmogAm02gUmouUmoaUmouCm 1417 UUCCGAAUAAACUCCAGGCscsu 1418
ogGmoaA AD-12361 GmocCmouGmogAm02gUmouUmoaUmouCm 1419
UUCCGAAuAAACUCcAGGCTsT 1420 ogGmoaA AD-12362
GmocCmouGmogAm02gUmouUmoaUmouCm 1421 uUcCGAAuAAACUccAGGCTsT 1422
ogGmoaA AD-12363 GmocCmouGmogAm02gUmouUmoaUmouCm 1423
UUCCGaaUAaaCUCCAggcscsu 1424 ogGmoaA AD-12364
GmocCmouGmogAmogUmouUmoaUmouCmo 1425 UUCCGaaUAaaCUCCAggcTsT 1426
gGmoaATsT AD-12365 GmocCmouGmogAmogUmouUmoaUmouCmo 1427
UUCCGAAuAAACUCcAGGCTsT 1428 gGmoaATsT AD-12366
GmocCmouGmogAmogUmouUmoaUmouCmo 1429 UUCCGAAUAAACUCCAGGCTsT 1430
gGmoaATsT AD-12367 GmocmocmouGGAGmoumoumouAmoumoum 1431
UUCCGaaUAaaCUCCAggcTsT 1432 ocGGAATsT AD-12368
GmocmocmouGGAGmoumoumouAmoumoum 1433 UUCCGAAuAAACUCcAGGCTsT 1434
ocGGAATsT AD-12369 GmocmocmouGGAGmoumoumouAmoumoum 1435
UUCCGAAUAAACUCCAGGCTsT 1436 ocGGAATsT AD-12370
GmocmocmouGGAGmoumoumouAmoumoum 1437
P-UfaCfCfGAAUfAAACMCfCfAGGCfTsT 1438 ocGGAATsT AD-12371
GmocmocmouGGAGmoumoumouAmoumoum 1439
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1440 ocGGAATsT AD -12372
GmocmocmouGGAGmoumoumouAmoumoum 1441
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1442 ocGGAATsT AD -12373
GmocmocmouGGAGmoumoumouAmoumoum 1443 UUCCGAAUAAACUCCAGGCTsT 1444
ocGGAATsT AD-12374 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1445
UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1446 AD-12375
GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1447 UUCCGAAUAAACUCCAGGCTsT 1448
AD-12377 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1449
uUcCGAAuAAACUccAGGCTsT 1450 AD-12378
GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1451 UUCCGaaUAaaCUCCAggcscsu 1452
AD-12379 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1453
UUCCGAAUAAACUCCAGGCscsu 1454 AD-12380
GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1455
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1456 AD-12381
GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1457
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1458 AD-12382
GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1459
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1460 AD-12383
GCCUGGAGUUUAUUCGGAATsT 1461 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1462
AD-12384 GccuGGAGuuuAuucGGAATsT 1463
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1464 AD-12385
GcCuGgnAgUuUaUuCgGaATsT 1465 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT
1466 AD-12386 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1467
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1468 AD-12387
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1469
UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1470 AD-12388
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1471 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc
1472 AD-12389 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1473
P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1474 AD-12390
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1475 UUCCGAAUAAACUCCAGGCscsu 1476
AD-12391 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1477 UUCCGaaUAaaCUCCAggc
1478 AD-12392 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1479
UUCCGAAUAAACUCCAGGCTsT 1480 AD-12393 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1481 UUCCGAAuAAACUCcAGGCTsT 1482 AD-12394
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1483 uUcCGAAuAAACUccAGGCTsT 1484
AD-12395 GmocCmouGmogAmogUmouUmoaUmouCmo 1485
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1486 gGmoaATsT AD-12396
GmocCmouGmogAm02gUmouUmoaUmouCm 1487
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1488 ogGmoaA AD-12397
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1489
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1490 AD-12398
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1491
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1492 AD-12399
GcCuGgnAgUuUaUuCgGaATsT 1493 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf
1494 AD-12400 GCCUGGAGUUUAUUCGGAATsT 1495
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1496 AD-12401
GccuGGAGuuuAuucGGAATsT 1497 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf
1498 AD-12402 GccuGGAGuuuAuucGGAA 1499
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1500 AD-12403
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1501
P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1502 AD-9314
GCCUGGAGUUUAUUCGGAATsT 1503 UUCCGAAUAAACUCCAGGCTsT 1504 AD-10794
ucAuAGGccuGGAGuuuAudTsdT 1525 AuAAACUCcAGGCCuAUGAdTsdT 1526
AD-10795 ucAuAGGccuGGAGuuuAudTsdT 1527 AuAAACUccAGGcCuAuGAdTsdT
1528 AD-10797 ucAuAGGccuGGAGuuuAudTsdT 1529
AUAAACUCCAGGCCUAUGAdTsdT 1530
[0541] U, C, A, G: corresponding ribonucleotide; T: deoxythymidine;
u, c, a, g: corresponding 2'-O-methyl ribonucleotide; Uf, Cf, Af,
Gf: corresponding 2'-deoxy-2'-fluoro ribonucleotide; moc, mou, mog,
moa: corresponding 2'-MOE nucleotide; where nucleotides are written
in sequence, they are connected by 3'-5' phosphodiester groups; ab:
3'-terminal abasic nucleotide; nucleotides with interjected "s" are
connected by 3'-O-5'-O phosphorothiodiester groups; unless denoted
by prefix "p-", oligonucleotides are devoid of a 5'-phosphate group
on the 5'-most nucleotide; all oligonucleotides bear 3'-OH on the
3'-most nucleotide
TABLE-US-00015 TABLE 2b Screening of dsRNAs targeted to PCSK9
Remaining mRNA in % of controls at Duplex number siRNA conc. of 30
nM AD-10792 15 AD-10793 32 AD-10796 13 AD-12038 13 AD-12039 29
AD-12040 10 AD-12041 11 AD-12042 12 AD-12043 13 AD-12044 7 AD-12045
8 AD-12046 13 AD-12047 17 AD-12048 43 AD-12049 34 AD-12050 16
AD-12051 31 AD-12052 81 AD-12053 46 AD-12054 8 AD-12055 13 AD-12056
11 AD-12057 8 AD-12058 9 AD-12059 23 AD-12060 10 AD-12061 7
AD-12062 10 AD-12063 19 AD-12064 15 AD-12065 16 AD-12066 20
AD-12067 17 AD-12068 18 AD-12069 13 AD-12338 15 AD-12339 14
AD-12340 19 AD-12341 12 AD-12342 13 AD-12343 24 AD-12344 9 AD-12345
12 AD-12346 13 AD-12347 11 AD-12348 8 AD-12349 11 AD-12350 17
AD-12351 11 AD-12352 11 AD-12354 11 AD-12355 9 AD-12356 25 AD-12357
56 AD-12358 29 AD-12359 30 AD-12360 15 AD-12361 20 AD-12362 51
AD-12363 11 AD-12364 25 AD-12365 18 AD-12366 23 AD-12367 42
AD-12368 40 AD-12369 26 AD-12370 68 AD-12371 60 AD-12372 60
AD-12373 55 AD-12374 9 AD-12375 16 AD-12377 88 AD-12378 6 AD-12379
6 AD-12380 8 AD-12381 10 AD-12382 7 AD-12383 7 AD-12384 8 AD-12385
8 AD-12386 11 AD-12387 13 AD-12388 19 AD-12389 16 AD-12390 17
AD-12391 21 AD-12392 28 AD-12393 17 AD-12394 75 AD-12395 55
AD-12396 59 AD-12397 20 AD-12398 11 AD-12399 13 AD-12400 12
AD-12401 13 AD-12402 14 AD-12403 4 AD-9314 9
TABLE-US-00016 TABLE 3 Cholesterol levels of rats treated with
LNP01-10792 Dosage of 5 mg/kg, n = 6 rats per group Day Total serum
cholesterol (relative to PBS control) 2 0.329 .+-. 0.035 4 0.350
.+-. 0.055 7 0.402 .+-. 0.09 9 0.381 .+-. 0.061 11 0.487 .+-. 0.028
14 0.587 .+-. 0.049 16 0.635 .+-. 0.107 18 0.704 .+-. 0.060 21
0.775 .+-. 0.102 28 0.815 .+-. 0.103
TABLE-US-00017 TABLE 4 Serum LDL-C levels of cynomolgus monkeys
treated with LNP formulated dsRNAs Serum LDL-C (relative to
pre-dose) Day 3 Day 4 Day 5 Day 7 Day 14 Day 21 PBS 1.053 .+-.
0.158 0.965 .+-. 0.074 1.033 .+-. 0.085 1.033 .+-. 0.157 1.009 .+-.
0.034 n = 3 LNP01-1955 1.027 .+-. 0.068 1.104 .+-. 0.114 n = 3
LNP01-10792 0.503 .+-. 0.055 0.596 .+-. 0.111 0.674 .+-. 0.139
0.644 .+-. 0.121 0.958 .+-. 0.165 1.111 .+-. 0.172 n = 5 LNP01-9680
0.542 .+-. 0.155 0.437 .+-. 0.076 0.505 .+-. 0.071 0.469 .+-. 0.066
0.596 .+-. 0.080 0.787 .+-. 0.138 n = 4
TABLE-US-00018 TABLE 5a Modified dsRNA targeted to PCSK9 Position
SEQ in human ID Name access.# Sense Antisense Sequence 5'-3' NO:
AD- 1091 unmodified unmodified GCCUGGAGUUUAUUCGGAAdTdT 1505 1a1
UUCCGAAUAAACUCCAGGCdTsdT 1506 AD- 1091 2'OMe 2'OMe
GccuGGAGuuuAuucGGAAdTsdT 1507 1a2 UUCCGAAuAAACUCcAGGCdTsdT 1508 AD-
1091 Alt 2'F, Alt 2'F, GfcCfuGfgAfgUfuUfaUfuCfgGfaAfdTdT 1509 1a3
2'OMe 2'OMe puUfcCfgAfaUfaAfaCfuCfcAfgGfcdTsdT 1510 AD- 1091 2'OMe
2'F all Py, GccuGGAGuuuAuucGGAAdTsdT 1511 1a4 5'Phosphate
PUfUfCfCfGAAUfAAACfUfCfCfAGGCfdTsdT 1512 AD- 1091 2'F 2'F all Py,
GCfCfUfGGAGUfUfUfAUfUfCfGGAAdTsdT 1513 1a5 5'Phosphate
PUfUfCfCfGAAUfAAACfUfCfCfAGGCfdTsdT 1514 AD-2a1 3530 2'OMe 2'OMe
uucuAGAccuGuuuuGcuudTsdT 1515 (3'UTR) AAGcAAAAcAGGUCuAGAAdTsdT 1516
AD-3a1 833 2'OMe 2'OMe AGGuGuAucuccuAGAcAcdTsdT 1517
GUGUCuAGGAGAuAcACCUdTsdT 1518 AD- N/A 2'OMe 2'OMe
cuuAcGcuGAGuAcuucGAdTsdT 1519 ctrl UCGAAGuACUcAGCGuAAGdTsdT 1520
(Luc.) U, C, A, G: corresponding ribonucleotide; T: deoxythymidine;
u, c, a, g: corresponding 2'-O-methyl ribonucleotide; Uf, Cf, Af,
Gf: corresponding 2'-deoxy-2'-fluoro ribonucleotide; where
nucleotides are written in sequence, they are connected by 3'-5'
phosphodiester groups; nucleotides with interjected ''s'' are
connected by 3'-O-5'-O phosphorothiodiester groups; unless denoted
by prefix ''p-'', oligonucleotides are devoid of a 5'-phosphate
group on the 5'-most nucleotide; all oligonucleotides bear 3'-OH on
the 3'-most nucleotide.
TABLE-US-00019 TABLE 5b Silencing activity of modified dsRNA in
monkey hepatocytes Primary Position in IFN-.alpha./ Cynomolgus
Monkey human TNF-.alpha. Hepatocytes Name access.# Induction Sense
Antisense ~IC50, nM AD-1a1 1091 Yes/Yes unmodified unmodified
0.07-0.2 AD-1a2 1091 No/No 2'OMe 2'OMe 0.07-0.2 AD-1a3 1091 No/No
Alt 2'F, Alt 2'F, 2'OMe 0.07-0.2 2'OMe AD-1a4 1091 No/No 2'OMe 2'F
all Py, 0.07-0.2 5'Phosphate AD-1a5 1091 No/No 2'F 2'F all Py,
0.07-0.2 5'Phosphate AD-2a1 3530 No/No 2'OMe 2'OMe 0.07-0.2 (3'UTR)
AD-3a1 833 No/No 2'OMe 2'OMe 0.1-0.3 AD-ctrl N/A No/No 2'OMe 2'OMe
N/A (Luc.)
TABLE-US-00020 TABLE 6 dsRNA targeted to PCSK9: mismatches and
modifications SEQ ID Duplex # Strand NO: Sequence 5' to 3' AD-9680
S 1531 uucuAGAccuGuuuuGcuudTsdT AS 1532 AAGcAAAAcAGGUCuAGAAdTsdT
AD-3267 S 1535 uucuAGAcCuGuuuuGcuuTsT AS 1536
AAGcAAAAcAGGUCuAGAATsT AD-3268 S 1537 uucuAGAccUGuuuuGcuuTsT AS
1538 AAGcAAAAcAGGUCuAGAATsT AD-3269 S 1539 uucuAGAcCUGuuuuGcuuTsT
AS 1540 AAGcAAAAcAGGUCuAGAATsT AD-3270 S 1541
uucuAGAcY1uGuuuuGcuuTsT AS 1542 AAGcAAAAcAGGUCuAGAATsT AD-3271 S
1543 uucuAGAcY1UGuuuuGcuuTsT AS 1544 AAGcAAAAcAGGUCuAGAATsT AD-3272
S 1545 uucuAGAccY1GuuuuGcuuTsT AS 1546 AAGcAAAAcAGGUCuAGAATsT
AD-3273 S 1547 uucuAGAcCY1GuuuuGcuuTsT AS 1548
AAGcAAAAcAGGUCuAGAATsT AD-3274 S 1549 uucuAGAccuY1uuuuGcuuTsT AS
1550 AAGcAAAAcAGGUCuAGAATsT AD-3275 S 1551 uucuAGAcCUY1uuuuGcuuTsT
AS 1552 AAGcAAAAcAGGUCuAGAATsT AD-14676 S 1553
UfuCfuAfgAfcCfuGfuUfuUfgCfuUfTsT AS 1554
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3276 S 1555
UfuCfuAfgAfcCuGfuUfuUfgCfuUfTsT AS 1556
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3277 S 1557
UfuCfuAfgAfcCfUGfuUfuUfgCfuUfTsT AS 1558
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3278 S 1559
UfuCfuAfgAfcCUGfuUfuUfgCfuUfTsT AS 1560
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3279 S 1561
UfuCfuAfgAfcYluGfuUfuUfgCfuUfTsT AS 1562
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3280 S 1563
UfuCfuAfgAfcY1UGfuUfuUfgCfuUfTsT AS 1564
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3281 S 1565
UfuCfuAfgAfcCfY1GfuUfuUfgCfuUfTsT AS 1566
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3282 S 1567
UfuCfuAfgAfcCY1GfuUfuUfgCfuUfTsT AS 1568
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3283 S 1569
UfuCfuAfgAfcCfuY1uUfuUfgCfuUfTsT AS 1570
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3284 S 1571
UfuCfuAfgAfcCUY1uUfuUfgCfuUfTsT AS 1572
p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-10792 S 459
GccuGGAGuuuAuucGGAATsT AS 460 UUCCGAAuAAACUCcAGGCTsT AD-3254 S 1573
GccuGGAGuY1uAuucGGAATsT AS 1574 UUCCGAAuAAACUCcAGGCTsT AD-3255 S
1575 GccuGGAGUY1uAuucGGAATsT AS 1576 UUCCGAAuAAACUCcAGGCTsT Strand:
S/Sense; AS/Antisense U, C, A, G: corresponding ribonucleotide; T:
deoxythymidine; u, c, a, g: corresponding 2'-O-methyl
ribonucleotide; Uf, Cf, Af, Gf: corresponding 2'-deoxy-2'-fluoro
ribonucleotide; Y1 corresponds to DFT difluorotoluyl ribo(or
deoxyribo)nucleotide; where nucleotides are written in sequence,
they are connected by 3'-5' phosphodiester groups; nucleotides with
interjected ''s'' are connected by 3'-O-5'-O phosphorothiodiester
groups; unless denoted by prefix ''p-'', oligonucleotides are
devoid of a 5'-phosphate group on the 5'-most nucleotide; all
oligonucleotides bear 3'-OH on the 3'-most nucleotide
TABLE-US-00021 TABLE 7 Sequences of unmodified siRNA flanking
AD-9680 Target Duplex Type Sequence (5' to 3') site SEQ ID NO:
AD-22169-b1 sense CAGCCAACUUUUCUAGACCdTsdT 3520 1577 antis
GGUCUAGAAAAGUUGGCUGdTsdT 3520 1578 AD-22170-b1 sense
AGCCAACUUUUCUAGACCUdTsdT 3521 1579 antis AGGUCUAGAAAAGUUGGCUdTsdT
3521 1580 AD-22171-b1 sense GCCAACUUUUCUAGACCUGdTsdT 3522 1581
antis CAGGUCUAGAAAAGUUGGCdTsdT 3522 1582 AD-22172-b1 sense
CCAACUUUUCUAGACCUGUdTsdT 3523 1583 antis ACAGGUCUAGAAAAGUUGGdTsdT
3523 1584 AD-22173-b1 sense CAACUUUUCUAGACCUGUUdTsdT 3524 1585
antis AACAGGUCUAGAAAAGUUGdTsdT 3524 1586 AD-22174-b1 sense
AACUUUUCUAGACCUGUUUdTsdT 3525 1587 antis AAACAGGUCUAGAAAAGUUdTsdT
3525 1588 AD-22175-b1 sense ACUUUUCUAGACCUGUUUUdTsdT 3526 1589
antis AAAACAGGUCUAGAAAAGUdTsdT 3526 1590 AD-22176-b1 sense
CUUUUCUAGACCUGUUUUGdTsdT 3527 1591 antis CAAAACAGGUCUAGAAAAGdTsdT
3527 1592 AD-22177-b1 sense UUUUCUAGACCUGUUUUGCdTsdT 3528 1593
antis GCAAAACAGGUCUAGAAAAdTsdT 3528 1594 AD-22178-b1 sense
UUUCUAGACCUGUUUUGCUdTsdT 3529 1595 antis AGCAAAACAGGUCUAGAAAdTsdT
3529 1596 AD-22179-b1 sense UCUAGACCUGUUUUGCUUUdTsdT 3531 1597
antis AAAGCAAAACAGGUCUAGAdTsdT 3531 1598 AD-22180-b1 sense
CUAGACCUGUUUUGCUUUUdTsdT 3532 1599 antis AAAAGCAAAACAGGUCUAGdTsdT
3532 1600 AD-22181-b1 sense UAGACCUGUUUUGCUUUUGdTsdT 3533 1601
antis CAAAAGCAAAACAGGUCUAdTsdT 3533 1602 AD-22182-b1 sense
AGACCUGUUUUGCUUUUGUdTsdT 3534 1603 antis ACAAAAGCAAAACAGGUCUdTsdT
3534 1604 AD-22183-b1 sense GACCUGUUUUGCUUUUGUAdTsdT 3535 1605
antis UACAAAAGCAAAACAGGUCdTsdT 3535 1606 AD-22184-b1 sense
ACCUGUUUUGCUUUUGUAAdTsdT 3536 1607 antis UUACAAAAGCAAAACAGGUdTsdT
3536 1608 AD-22185-b1 sense CCUGUUUUGCUUUUGUAACdTsdT 3537 1609
antis GUUACAAAAGCAAAACAGGdTsdT 3537 1610 AD-22186-b1 sense
CUGUUUUGCUUUUGUAACUdTsdT 3538 1611 antis AGUUACAAAAGCAAAACAGdTsdT
3538 1612 AD-22187-b1 sense UGUUUUGCUUUUGUAACUUdTsdT 3539 1613
antis AAGUUACAAAAGCAAAACAdTsdT 3539 1614 AD-22188-b1 sense
GUUUUGCUUUUGUAACUUGdTsdT 3540 1615 antis CAAGUUACAAAAGCAAAACdTsdT
3540 1616 AD-22189-b1 sense UUUUGCUUUUGUAACUUGAdTsdT 3541 1617
antis UCAAGUUACAAAAGCAAAAdTsdT 3541 1618 AD-22190-b1 sense
UUUGCUUUUGUAACUUGAAdTsdT 3542 1619 antis UUCAAGUUACAAAAGCAAAdTsdT
3542 1620 AD-22191-b1 sense UUGCUUUUGUAACUUGAAGdTsdT 3543 1621
antis CUUCAAGUUACAAAAGCAAdTsdT 3543 1622 AD-22192-b1 sense
UGCUUUUGUAACUUGAAGAdTsdT 3544 1623 antis UCUUCAAGUUACAAAAGCAdTsdT
3544 1624 AD-22193-b1 sense GCUUUUGUAACUUGAAGAUdTsdT 3545 1625
antis AUCUUCAAGUUACAAAAGCdTsdT 3545 1626 AD-22194-b1 sense
CUUUUGUAACUUGAAGAUAdTsdT 3546 1627 antis UAUCUUCAAGUUACAAAAGdTsdT
3546 1628 AD-22195-b1 sense UUUUGUAACUUGAAGAUAUdTsdT 3547 1629
antis AUAUCUUCAAGUUACAAAAdTsdT 3547 1630 AD-22196-b1 sense
UUUGUAACUUGAAGAUAUUdTsdT 3548 1631 antis AAUAUCUUCAAGUUACAAAdTsdT
3548 1632 AD-22197-b1 sense UUGUAACUUGAAGAUAUUUdTsdT 3549 1633
antis AAAUAUCUUCAAGUUACAAdTsdT 3549 1634 AD-22198-b1 sense
UGUAACUUGAAGAUAUUUAdTsdT 3550 1635 antis UAAAUAUCUUCAAGUUACAdTsdT
3550 1636 AD-22199-b1 sense GUAACUUGAAGAUAUUUAUdTsdT 3551 1637
antis AUAAAUAUCUUCAAGUUACdTsdT 3551 1638 AD-22200-b1 sense
UAACUUGAAGAUAUUUAUUdTsdT 3552 1639 antis AAUAAAUAUCUUCAAGUUAdTsdT
3552 1640 AD-22201-b1 sense AACUUGAAGAUAUUUAUUCdTsdT 3553 1641
antis GAAUAAAUAUCUUCAAGUUdTsdT 3553 1642 AD-22202-b1 sense
ACUUGAAGAUAUUUAUUCUdTsdT 3554 1643 antis AGAAUAAAUAUCUUCAAGUdTsdT
3554 1644 AD-22203-b1 sense CUUGAAGAUAUUUAUUCUGdTsdT 3555 1645
antis CAGAAUAAAUAUCUUCAAGdTsdT 3555 1646 AD-22204-b1 sense
UUGAAGAUAUUUAUUCUGGdTsdT 3556 1647 antis CCAGAAUAAAUAUCUUCAAdTsdT
3556 1648 AD-22205-b1 sense UGAAGAUAUUUAUUCUGGGdTsdT 3557 1649
antis CCCAGAAUAAAUAUCUUCAdTsdT 3557 1650 AD-22206-b1 sense
GAAGAUAUUUAUUCUGGGUdTsdT 3558 1651 antis ACCCAGAAUAAAUAUCUUCdTsdT
3558 1652
TABLE-US-00022 TABLE 8 Sequences of modified siRNA flanking AD-9680
Duplex Type Sequence (5' to 3') Target SEQ ID NO: AD-22098-b1 sense
cAGccAAcuuuucuAGAccdTsdT 3520 1653 antis GGUCuAGAAAAGUUGGCUGdTsdT
3520 1654 AD-22099-b1 sense AGccAAcuuuucuAGAccudTsdT 3521 1655
antis AGGUCuAGAAAAGUUGGCUdTsdT 3521 1656 AD-22100-b1 sense
GccAAcuuuucuAGAccuGdTsdT 3522 1657 antis cAGGUCuAGAAAAGUUGGCdTsdT
3522 1658 AD-22101-b1 sense ccAAcuuuucuAGAccuGudTsdT 3523 1659
antis AcAGGUCuAGAAAAGUUGGdTsdT 3523 1660 AD-22102-b1 sense
cAAcuuuucuAGAccuGuudTsdT 3524 1661 antis AAcAGGUCuAGAAAAGUUGdTsdT
3524 1662 AD-22103-b1 sense AAcuuuucuAGAccuGuuudTsdT 3525 1663
antis AAAcAGGUCuAGAAAAGUUdTsdT 3525 1664 AD-22104-b1 sense
AcuuuucuAGAccuGuuuudTsdT 3526 1665 antis AAAAcAGGUCuAGAAAAGUdTsdT
3526 1666 AD-22105-b1 sense cuuuucuAGAccuGuuuuGdTsdT 3527 1667
antis cAAAAcAGGUCuAGAAAAGdTsdT 3527 1668 AD-22106-b1 sense
uuuucuAGAccuGuuuuGcdTsdT 3528 1669 antis GcAAAAcAGGUCuAGAAAAdTsdT
3528 1670 AD-22107-b1 sense uuucuAGAccuGuuuuGcudTsdT 3529 1671
antis AGcAAAAcAGGUCuAGAAAdTsdT 3529 1672 AD-22108-b1 sense
ucuAGAccuGuuuuGcuuudTsdT 3531 1673 antis AAAGcAAAAcAGGUCuAGAdTsdT
3531 1674 AD-22109-b1 sense cuAGAccuGuuuuGcuuuudTsdT 3532 1675
antis AAAAGcAAAAcAGGUCuAGdTsdT 3532 1676 AD-22110-b1 sense
uAGAccuGuuuuGcuuuuGdTsdT 3533 1677 antis cAAAAGcAAAAcAGGUCuAdTsdT
3533 1678 AD-22111-b1 sense AGAccuGuuuuGcuuuuGudTsdT 3534 1679
antis AcAAAAGcAAAAcAGGUCUdTsdT 3534 1680 AD-22112-b1 sense
GAccuGuuuuGcuuuuGuAdTsdT 3535 1681 antis uAcAAAAGcAAAAcAGGUCdTsdT
3535 1682 AD-22113-b1 sense AccuGuuuuGcuuuuGuAAdTsdT 3536 1683
antis UuAcAAAAGcAAAAcAGGUdTsdT 3536 1684 AD-22114-b1 sense
ccuGuuuuGcuuuuGuAAcdTsdT 3537 1685 antis GUuAcAAAAGcAAAAcAGGdTsdT
3537 1686 AD-22115-b1 sense cuGuuuuGcuuuuGuAAcudTsdT 3538 1687
antis AGUuAcAAAAGcAAAAcAGdTsdT 3538 1688 sense
uGuuuuGcuuuuGuAAcuudTsdT 3539 1689 antis AAGUuAcAAAAGcAAAAcAdTsdT
3539 1690 AD-22116-b1 sense GuuuuGcuuuuGuAAcuuGdTsdT 3540 1691
antis cAAGUuAcAAAAGcAAAACdTsdT 3540 1692 AD-22117-b1 sense
uuuuGcuuuuGuAAcuuGAdTsdT 3541 1693 antis UcAAGUuAcAAAAGcAAAAdTsdT
3541 1694 AD-22118-b1 sense uuuGcuuuuGuAAcuuGAAdTsdT 3542 1695
antis UUcAAGUuAcAAAAGcAAAdTsdT 3542 1696 AD-22119-b1 sense
uuGcuuuuGuAAcuuGAAGdTsdT 3543 1697 antis CUUcAAGUuAcAAAAGcAAdTsdT
3543 1698 AD-22120-b1 sense uGcuuuuGuAAcuuGAAGAdTsdT 3544 1699
antis UCUUcAAGUuAcAAAAGcAdTsdT 3544 1700 AD-22121-b1 sense
GcuuuuGuAAcuuGAAGAudTsdT 3545 1701 antis AUCUUcAAGUuAcAAAAGCdTsdT
3545 1702 AD-22122-b1 sense cuuuuGuAAcuuGAAGAuAdTsdT 3546 1703
antis uAUCUUcAAGUuAcAAAAGdTsdT 3546 1704 AD-22123-b1 sense
uuuuGuAAcuuGAAGAuAudTsdT 3547 1705 antis AuAUCUUcAAGUuAcAAAAdTsdT
3547 1706 AD-22124-b1 sense uuuGuAAcuuGAAGAuAuudTsdT 3548 1707
antis AAuAUCUUcAAGUuAcAAAdTsdT 3548 1708 AD-22125-b1 sense
uuGuAAcuuGAAGAuAuuudTsdT 3549 1709 antis AAAuAUCUUcAAGUuAcAAdTsdT
3549 1710 AD-22126-b1 sense uGuAAcuuGAAGAuAuuuAdTsdT 3550 1711
antis uAAAuAUCUUcAAGUuAcAdTsdT 3550 1712 AD-22127-b1 sense
GuAAcuuGAAGAuAuuuAudTsdT 3551 1713 antis AuAAAuAUCUUcAAGUuACdTsdT
3551 1714 AD-22128-b1 sense uAAcuuGAAGAuAuuuAuudTsdT 3552 1715
antis AAuAAAuAUCUUcAAGUuAdTsdT 3552 1716 AD-22129-b1 sense
AAcuuGAAGAuAuuuAuucdTsdT 3553 1717 antis GAAuAAAuAUCUUcAAGUUdTsdT
3553 1718 AD-22130-b1 sense AcuuGAAGAuAuuuAuucudTsdT 3554 1719
antis AGAAuAAAuAUCUUcAAGUdTsdT 3554 1720 AD-22131-b1 sense
cuuGAAGAuAuuuAuucuGdTsdT 3555 1721 antis cAGAAuAAAuAUCUUcAAGdTsdT
3555 1722 AD-22132-b1 sense uuGAAGAuAuuuAuucuGGdTsdT 3556 1723
antis CcAGAAuAAAuAUCUUcAAdTsdT 3556 1724 AD-22133-b1 sense
uGAAGAuAuuuAuucuGGGdTsdT 3557 1725 antis CCcAGAAuAAAuAUCUUcAdTsdT
3557 1726 AD-22134-b1 sense GAAGAuAuuuAuucuGGGudTsdT 3558 1727
antis ACCcAGAAuAAAuAUCUUCdTsdT 3558 1728
TABLE-US-00023 TABLE 9 Single dose treatment of HeLa cells with
siRNA flanking AD-9680 % message % message remaining SD remaining
Duplex ID 0.1 nM 0.1 nM 10 nM SD 10 nM AD-22098-b1 10.6 1.9 9.2 3.7
AD-22098-b1 7.7 1.7 7.9 0.7 AD-22099-b1 21.3 4.5 27.4 7.2
AD-22099-b1 25.9 2.4 29.6 9.1 AD-22100-b1 58.6 9.6 35.8 11.1
AD-22100-b1 62.5 0.3 27.4 3.5 AD-22101-b1 21.9 3.8 12.9 1.4
AD-22101-b1 19.3 0.3 9.7 1.3 AD-22102-b1 6.6 0.1 7.7 3.3
AD-22103-b1 8.7 0.0 8.2 1.3 AD-22104-b1 7.6 0.2 8.5 2.8 AD-22105-b1
13.4 1.0 8.1 2.3 AD-22106-b1 59.1 0.4 35.4 4.6 AD-22107-b1 9.1 0.8
8.4 3.7 AD-22108-b1 8.8 0.9 6.2 1.7 AD-22109-b1 9.8 0.9 8.2 1.7
AD-22110-b1 24.8 1.7 15.3 5.9 AD-22111-b1 8.3 0.7 6.2 1.7
AD-22112-b1 15.1 0.0 10.3 2.9 AD-22113-b1 10.9 0.6 10.0 2.0
AD-22114-b1 8.9 1.1 7.3 1.3 AD-22115-b1 5.3 0.8 3.7 0.7 AD-22116-b1
58.1 0.4 34.5 7.3 AD-22117-b1 19.9 0.9 12.2 2.9 AD-22118-b1 5.3 0.0
4.4 1.0 AD-22119-b1 8.6 1.9 5.8 2.3 AD-22120-b1 7.2 0.8 5.8 2.4
AD-22121-b1 7.3 0.9 6.4 2.1 AD-22122-b1 32.5 2.5 18.1 6.3
AD-22123-b1 14.7 0.8 16.7 7.0 AD-22124-b1 12.8 1.9 10.5 5.3
AD-22125-b1 7.4 0.6 9.0 4.6 AD-22126-b1 12.8 0.4 16.4 7.3
AD-22127-b1 8.8 0.5 9.6 5.0 AD-22128-b1 9.9 0.2 12.4 5.9
AD-22129-b1 85.9 10.3 94.9 49.8 AD-22130-b1 5.6 1.0 6.2 4.1
AD-22131-b1 26.9 8.4 12.9 7.3 AD-22132-b1 78.5 18.5 67.5 34.1
AD-22133-b1 26.4 7.1 15.0 6.7 AD-22134-b1 26.9 0.1 22.4 6.5
AD-22169-b1 7.3 0.6 6.0 1.5 AD-22169-b1 7.0 1.1 6.1 1.3 AD-22170-b1
9.3 1.6 7.2 1.8 AD-22170-b1 9.7 1.1 11.2 1.0 AD-22171-b1 7.1 2.3
4.5 0.2 AD-22171-b1 6.5 1.9 4.4 2.8 AD-22172-b1 7.2 1.1 7.6 3.7
AD-22172-b1 7.0 0.4 7.0 2.4 AD-22173-b1 15.7 12.5 5.9 0.1
AD-22174-b1 8.9 2.7 6.4 0.9 AD-22175-b1 10.7 4.3 7.9 2.4
AD-22176-b1 9.6 0.8 8.4 3.1 AD-22177-b1 38.9 5.9 21.4 1.2
AD-22178-b1 6.5 0.5 5.6 0.9 AD-22179-b1 7.0 0.8 5.9 0.1 AD-22180-b1
7.3 3.7 7.2 1.6 AD-22181-b1 11.1 0.9 10.0 1.0 AD-22182-b1 5.4 1.4
4.0 1.5 AD-22183-b1 3.8 0.4 2.9 0.4 AD-22184-b1 5.1 0.2 3.7 0.7
AD-22185-b1 5.7 0.6 5.0 1.5 AD-22186-b1 5.3 0.3 5.7 1.0 AD-22187-b1
5.3 1.2 5.3 1.4 AD-22188-b1 12.6 2.6 11.6 0.2 AD-22189-b1 5.2 0.5
4.5 1.8 AD-22190-b1 4.7 1.3 3.4 1.1 AD-22191-b1 10.5 0.6 7.9 0.9
AD-22192-b1 6.9 2.2 5.8 3.5 AD-22193-b1 7.5 1.5 5.2 0.6 AD-22194-b1
8.0 1.4 6.5 1.9 AD-22195-b1 7.0 1.9 4.9 2.3 AD-22196-b1 5.4 0.0 3.8
0.9 AD-22197-b1 6.6 0.4 5.2 1.2 AD-22198-b1 7.3 0.8 8.5 2.4
AD-22199-b1 5.5 0.7 4.2 1.2 AD-22200-b1 11.0 0.5 12.5 3.1
AD-22201-b1 44.0 3.1 47.3 8.3 AD-22202-b1 9.0 1.2 7.2 0.9
AD-22203-b1 12.5 0.0 12.7 2.2 AD-22204-b1 57.1 5.2 50.2 10.2
AD-22205-b1 27.0 0.4 24.5 0.0 AD-22206-b1 13.9 1.1 11.4 1.3 AD-9680
7.1 ND 9.3 ND
TABLE-US-00024 TABLE 10 IC50 in HeLa cells using siRNA flanking
AD-9680 Rep1 Rep2 IC50 IC50 Average IC50 Duplex Name (pM) (pM) (pM)
AD-22098 6.0 6.7 6.4 AD-22099 25.0 37.8 31.4 AD-22101 66.5 81.9
74.2 AD-22102 2.3 1.5 1.9 AD-22103 6.3 1.2 3.8 AD-22104 2.2 1.4 1.8
AD-22105 13.3 0.1 6.7 AD-22107 2.2 0.9 1.6 AD-22108 2.3 2.0 2.1
AD-22109 5.5 6.3 5.9 AD-22110 59.1 42.2 50.7 AD-22111 9.1 8.2 8.7
AD-22112 25.8 31.0 28.4 AD-22113 4.2 4.4 4.3 AD-22114 6.9 4.0 5.5
AD-22115 3.0 2.2 2.6 AD-22117 56.0 37.6 46.8 AD-22118 2.9 1.7 2.3
AD-22119 6.7 0.0 3.4 AD-22120 2.0 1.2 1.6 AD-22121 2.1 4.1 3.1
AD-22122 203.3 156.3 179.8 AD-22123 33.1 50.7 41.9 AD-22124 18.8
13.1 15.9 AD-22125 3.3 2.6 3.0 AD-22126 17.9 18.5 18.2 AD-22127
11.1 4.3 7.7 AD-22128 14.6 3.3 8.9 AD-22130 1.7 0.3 1.0 AD-22131
172.5 59.6 116.0 AD-22133 94.6 57.2 75.9 AD-22134 113.0 81.3 97.2
AD-9680 3.8 2.4 3.1
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170000815A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170000815A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References