U.S. patent application number 17/402459 was filed with the patent office on 2022-07-07 for dual targeting sirna agents.
This patent application is currently assigned to Alnylam Pharmaceuticals, Inc.. The applicant listed for this patent is Alnylam Pharmaceuticals, Inc.. Invention is credited to Klaus Charisse, Kevin Fitzgerald, Maria Frank-Kamenetsky.
Application Number | 20220213475 17/402459 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220213475 |
Kind Code |
A1 |
Fitzgerald; Kevin ; et
al. |
July 7, 2022 |
DUAL TARGETING siRNA AGENTS
Abstract
The invention relates to dual targeting siRNA agents targeting a
PCSK9 gene and a second gene, and methods of using dual targeting
siRNA agents to inhibit expression of PCSK9 and to treat PCSK9
related disorders, e.g., hyperlipidemia.
Inventors: |
Fitzgerald; Kevin;
(Brookline, MA) ; Frank-Kamenetsky; Maria;
(Brookline, MA) ; Charisse; Klaus; (Acton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alnylam Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Alnylam Pharmaceuticals,
Inc.
Cambridge
MA
|
Appl. No.: |
17/402459 |
Filed: |
August 13, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16803738 |
Feb 27, 2020 |
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17402459 |
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16521422 |
Jul 24, 2019 |
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16803738 |
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15724175 |
Oct 3, 2017 |
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16521422 |
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14885342 |
Oct 16, 2015 |
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15724175 |
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13497226 |
Oct 10, 2012 |
9187746 |
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PCT/US2010/049868 |
Sep 22, 2010 |
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14885342 |
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61313584 |
Mar 12, 2010 |
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61244859 |
Sep 22, 2009 |
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International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/713 20060101 A61K031/713; C07H 21/02 20060101
C07H021/02 |
Claims
1. A dual targeting siRNA agent comprising a first dsRNA targeting
a PCSK9 gene and a second dsRNA targeting a second gene, wherein
the first dsRNA and the second dsRNA are linked with a covalent
linker.
2. A dual targeting siRNA agent comprising a first dsRNA AD-10792
targeting a PCSK9 gene and a second dsRNA AD-18038 targeting an
XBP-1 gene, wherein the AD-10792 sense strand and the AD-18038
sense strand are covalently linked with a disulfide linker.
3. The dual targeting siRNA agent of claim 1, wherein the second
gene is selected from the group consisting of XBP-1, PCSK9, PCSK5,
ApoC3, SCAP, and MIG12.
4. (canceled)
5. The dual targeting siRNA agent of claim 1, wherein the first
dsRNA comprises at least 15 contiguous nucleotides of an antisense
strand of one of Tables 1, 2, or 4-8, or comprises an antisense
strand of one of Tables 1, 2, or 4-8, or comprises a sense strand
and an antisense strand of one of Tables 1, 2, or 4-8.
6. The dual targeting siRNA agent of claim 1, wherein the first
dsRNA comprises AD-9680 or AD-10792.
7. The dual targeting siRNA agent of claim 1, wherein the second
dsRNA comprises at least 15 contiguous nucleotides of an antisense
strand of one of Tables 3 or 9-13, or comprises an antisense strand
of one of Tables 3 or 9-13, or comprises a sense strand and an
antisense strand of one of Tables 3 or 9-13.
8. The dual targeting siRNA agent of claim 1, wherein the second
dsRNA comprises AD-18038.
9. The dual targeting siRNA agent of claim 1, wherein the first and
second dsRNA comprises at least one modified nucleotide.
10. The dual targeting siRNA agent of claim 9, 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.
11. The dual targeting siRNA agent of claim 9, 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, a 2'-amino-modified nucleotide, a
2'-alkyl-modified nucleotide, a morpholino nucleotide, a
phosphoramidate, and a non-natural base comprising nucleotide.
12. The dual targeting siRNA agent of claim 1, wherein each strand
of each dsRNA is 19-23 bases in length.
13. The dual targeting siRNA agent of claim 1, (a) wherein the
first and second dsRNAs are linked with a disulfide linker; (b)
wherein the covalent linker links the sense strand of the first
dsRNA to the sense strand of the second dsRNA; and/or (c) wherein
the covalent linker links the antisense strand of the first dsRNA
to the antisense strand of the second dsRNA.
14. (canceled)
15. (canceled)
16. The dual targeting siRNA agent of claim 1, further comprising a
ligand.
17. The dual targeting siRNA agent of claim 1, (a) wherein
administration of the dual targeting siRNA agent to a cell inhibits
expression of the PCSK9 gene and the second gene at a level
equivalent to inhibition of expression of both genes obtained by
the administration of each siRNA individually; or (b) wherein
administration of the dual targeting siRNA agent to a subject
results in a greater reduction of total serum cholesterol that that
obtained by administration of each siRNA individually.
18. (canceled)
19. A pharmaceutical composition comprising the dual targeting
siRNA agent of claim 1 and a pharmaceutical carrier.
20. The pharmaceutical composition of claim 19, wherein the
pharmaceutical carrier is (a) a lipid formulation; (b) a lipid
formulation comprising cationic lipid DLinDMA or cationic lipid
XTC; (c) a lipid formulation described in Table A: or (d) a lipid
formulation comprising XTC/DSPC/Cholesterol/PEG-DMG at % mol ratios
of 50/10/38.5/1.5.
21-23. (canceled)
24. A method of inhibiting expression of the PCSK9 gene and a
second gene in a cell, the method comprising (a) introducing into
the cell the dual targeting siRNA agent of claim 1; and (b)
maintaining the cell produced in step (a) for a time sufficient to
obtain degradation of the mRNA transcript of the PCSK9 gene and the
second gene, thereby inhibiting expression of the PCSK9 gene and
the second gene in the cell.
25. A method of treating a disorder mediated by PCSK9 expression
comprising administering to a subject in need of such treatment a
therapeutically effective amount of the pharmaceutical composition
of claim 19.
26. The method of claim 25, wherein the disorder is
hyperlipidemia.
27. A method of reducing total serum cholesterol in a subject
comprising administering to the subject a therapeutically effective
amount of the pharmaceutical composition of claim 19.
28. An isolated cell comprising the dual targeting siRNA agent of
claim 1.
29. A vector encoding at least one strand of the first dsRNA and at
least one strand of the second dsRNA of the dual targeting siRNA
agent of claim 1.
30. A cell comprising the vector of claim 29.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/803,738, filed Feb. 27, 2020, which is a continuation of
U.S. application Ser. No. 16/521,422, filed Jul. 24, 2019
(abandoned), which is a continuation of U.S. application Ser. No.
15/724,175, filed Oct. 3, 2017 (abandoned), which is a continuation
of U.S. application Ser. No. 14/885,342, filed Oct. 16, 2015
(abandoned), which is a continuation of U.S. application Ser. No.
13/497,226, with a 371(c) filing date of Oct. 10, 2012, now U.S.
Pat. No. 9,187,746, issued Nov. 17, 2015, which is a National Stage
of International Application No. PCT/US2010/049868, filed Sep. 22,
2010, which claims the benefit of U.S. Provisional Application No.
61/244,859, filed Sep. 22, 2009, and claims the benefit of U.S.
Provisional Application No. 61/313,584, filed Mar. 12, 2010, all of
which are hereby incorporated in their entirety by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes a Sequence Listing with 4166
sequences submitted electronically as a text file named
AYL108C4_sequencelisting.txt, created on Feb. 20, 2020, with a size
of 1,351,680 bytes. The sequence listing is incorporated by
reference.
FIELD OF THE INVENTION
[0003] The invention relates to a composition of two covalently
linked siRNAs, e.g., a dual targeting siRNA agent. At least one
siRNA is a dsRNA that targets a PCSK9 gene. The covalently linked
siRNA agent is used in methods of inhibition of PCSK9 gene
expression and methods of treatment of pathological conditions
associated with PCSK9 gene expression, e.g., hyperlipidemia.
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 SIP/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) 30 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) J. 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] X-box binding protein 1 (XBP-1) is a basic leucine zipper
transcription factor that is involved in the cellular unfolded
protein response (UPR). XBP-1 is known to be active in the
endoplasmic reticulum (ER). The ER consists of a system of folded
membranes and tubules in the cytoplasm of cells. Proteins and
lipids are manufactured and processed in the ER. When unusual
demands are placed on the ER, "ER stress" occurs. ER stress can be
triggered by a viral infection, gene mutations, exposure to toxins,
aggregation of improperly folded proteins or a shortage of
intracellular nutrients. The result can be Type II diabetes,
metabolic syndrome, a neurological disorder or cancer.
[0009] Two XBP-1 isoforms are known to exist in cells: spliced
XBP-1S and unspliced XBP-1U. Both isoforms of XBP-1 bind to the
21-bp Tax-responsive element of the human T-lymphotropic virus type
1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate
HTLV-1 transcription. HTLV-1 is associated with a rare form of
blood dyscrasia known as Adult T-cell Leukemia/lymphoma (ATLL) and
a myelopathy, tropical spastic paresis.
[0010] 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.)
disclosed 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.
[0011] A description of siRNA targeting PCSK9 can be found in U.S.
patent application Ser. No. 11/746,864 filed on May 10, 2007 (now
U.S. Pat. No. 7,605,251) and International Patent Application No.
PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161).
Additional disclosure can be found in U.S. patent application Ser.
No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066)
and International Patent Application No. PCT/US2009/032743 filed
Jan. 30, 2009 (published as WO 2009/134487).
[0012] A description of siRNA targeting XPB-1 can be found in U.S.
patent application Ser. No. 12/425,811 filed on Apr. 17, 2009 and
published as US 2009-0275638.
[0013] Dual targeting siRNAs can be found in International patent
application publication no. WO/2007/091269.
SUMMARY OF THE INVENTION
[0014] Described herein are dual targeting siRNA agent in which a
first siRNA targeting PCSK9 is covalently joined to a second siRNA
targeting a gene implicated in cholesterol metabolism, e.g., XBP-1.
The two siRNAs are covalently linked via, e.g., a disulfide
linker.
[0015] Accordingly one aspect of the invention is a dual targeting
siRNA agent having a first dsRNA targeting a PCSK9 gene and a
second dsRNA targeting a second gene, wherein the first dsRNA and
the second dsRNA are linked with a covalent linker. The second gene
is can be, e.g., XBP-1, PCSK9, PCSK5, ApoC3, SCAP, or MIG12. In one
embodiment, the second gene is XBP-1. Each dsRNA is 30 nucleotides
or less in length. In general, each strand of each dsRNA is 19-23
bases in length.
[0016] In one embodiment, the dual targeting siRNA agent comprising
a first dsRNA AD-10792 targeting a PCSK9 gene and a second dsRNA
AD-18038 targeting an XBP-1 gene, wherein AD-10792 sense strand and
AD-18038 sense strand are covalently linked with a disulfide
linker.
[0017] The first dsRNA of the dual targeting siRNA agent targets a
PCSK9 gene. In one aspect, the first dsRNA includes at least 15
contiguous nucleotides of an antisense strand of one of Tables 1,
2, or 4-8, or includes an antisense strand of one of Tables 1, 2,
or 4-8, or includes a sense strand and an antisense strand of one
of Tables 1, 2, or 4-8. The first dsRNA can be AD-9680 or
AD-10792.
[0018] In some embodiments, the second dsRNA target XBP-1. In one
aspect, the second dsRNA includes at least 15 contiguous
nucleotides of an antisense strand of one of Tables 3 or 9-13, or
includes an antisense strand of one of Tables 3 or 9-13, or
includes a sense strand and an antisense strand of one of Tables 3
or 9-13. For example, the second dsRNA can be AD-18038.
[0019] Either the first and second dsRNA can include at least one
modified nucleotide, e.g., a 2'-O-methyl modified nucleotide, a
nucleotide comprising a 5'-phosphorothioate group, a terminal
nucleotide linked to a cholesteryl derivative or dodecanoic acid
bisdecylamide group, a 2'-deoxy-2'-fluoro modified nucleotide, a
2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified
nucleotide, a morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide. In some embodiments, the
first and second dsRNAs include "endo-light" modification with
2'-O-methyl modified nucleotides and nucleotides comprising a
5'-phosphorothioate group.
[0020] The first and second dsRNAs are linked with a covalent
linker. In some embodiments, the linker is a disulfide linker.
Various combinations of strands can be linked, e.g., the first and
second dsRNA sense strands are covalently linked or, e.g., the
first and second dsRNA antisense strands are covalently linked. In
some embodiments, any of the dual targeting siRNA agents of the
invention include a ligand.
[0021] Also included in the invention are isolated cells having and
vectors encoding the dual targeting siRNA agent described
herein.
[0022] In one aspect, administration of the dual targeting siRNA
agent to a cell inhibits expression of the PCSK9 gene and the
second gene at a level equivalent to inhibition of expression of
both genes using administration of each siRNA individually. In
another aspect, administration of the dual targeting siRNA agent to
a subject results in a greater reduction of total serum cholesterol
that that obtained by administration of each siRNA alone.
[0023] The invention also includes a pharmaceutical composition
comprising the dual targeting siRNA agents described herein and a
pharmaceutical carrier. In one embodiment, the pharmaceutical
carrier is a lipid formulation, e.g., a lipid formulation including
cationic lipid DLinDMA or cationic lipid XTC. Examples of lipid
formulations described in (but not limited to) Table A, below. The
lipid formulation can be XTC/DSPC/Cholesterol/PEG-DMG at % mol
ratios of 50/10/38.5/1.5.
[0024] Another aspect of the invention includes methods of using
the dual targeting siRNA agents described herein. In one
embodiment, the invention is a method of inhibiting expression of
the PCSK9 gene and a second gene in a cell, the method comprising
(a) introducing into the cell the any of the dual targeting siRNA
agents and (b) maintaining the cell produced in step (a) for a time
sufficient to obtain degradation of the mRNA transcript of the
PCSK9 gene and the second gene, thereby inhibiting expression of
the PCSK9 gene and the second gene in the cell.
[0025] In another embodiment, the invention includes methods of
treating a disorder mediated by PCSK9 expression with the step of
administering to a subject in need of such treatment a
therapeutically effective amount of the pharmaceutical compositions
described herein. In one aspect, the disorder is hyperlipidemia. In
still another embodiment, the invention includes methods of
reducing total serum cholesterol in a subject comprising
administering to the subject a therapeutically effective amount of
the pharmaceutical compositions described herein.
[0026] The details of various embodiments of the invention are set
forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and the drawings, and from the claims.
DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1A is a graph showing the effect on PCSK9 mRNA levels
in primary mouse hepatocytes following treatment with a dual
targeting siRNA, AD-23426. AD-23426 was as effective at reducing
mRNA expression as each single gene target siRNA. AD-10792: PCSK9
siRNA. AD-18038: XBP-1 siRNA. Lipo2000: control transfection agent
only FIG. 1Bis a graph showing the effect on XBP-1 mRNA levels in
primary mouse hepatocytes following treatment with a dual targeting
siRNA, AD-23426. AD-23426 was as effective at reducing mRNA
expression as each single gene target siRNA. AD-10792: PCSK9 siRNA.
AD-18038: XBP-1 siRNA. Lipo2000: control transfection agent
only.
[0028] FIG. 2 is a graph showing the effect on PCSK9 and XBP-1 mRNA
levels in mice following treatment with a dual targeting siRNA,
AD-23426. LNP09 (lipid) formulated siRNA was administered to mice
as described. AD-23426 was as effective at reducing mRNA expression
as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038:
XBP-1 siRNA.
[0029] FIG. 3 is a graph showing the effect on serum cholesterol
levels in mice following treatment with a dual targeting siRNA,
AD-23426. LNP09 (lipid) formulated siRNA was administered to mice
as described. AD-23426 was more effective at reducing serum
cholesterol compared to each single gene target siRNA. AD-10792:
PCSK9 siRNA. AD-18038: XBP-1 siRNA.
[0030] FIG. 4A is a graph showing the effect on IFN-.alpha., in
human PBMC following treatment with a dual targeting siRNA,
AD-23426. FIG. 4B is a graph showing the effect on TNF-.alpha. in
human PBMC following treatment with a dual targeting siRNA,
AD-23426. DOTAP and LNP09 (lipid) formulated siRNAs was
administered huPBMC as described below. AD-23426 did not induce
IFN-.alpha. or TNF-.alpha..
DETAILED DESCRIPTION OF THE INVENTION
[0031] 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 dual targeting siRNA to
silence the PCSK9 gene.
[0032] The invention provides compositions and methods for
inhibiting the expression of the PCSK9 gene in a subject using two
siRNA, e.g., a dual targeting siRNA. 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.
[0033] The dual targeting siRNA agents target a PCSK9 gene and at
least one other gene. The other gene can be another region of the
PCSK9 gene, or can be another gene, e.g., XBP-1.
[0034] The dual targeting siRNA agents have the advantage of lower
toxicity, lower off-target effects, and lower effective
concentration compared to individual siRNAs.
[0035] The use of the dual targeting siRNA 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 it was demonstrated that inhibiting
both a PCSK9 gene and an XBP-1 gene using a dual targeting siRNA is
at least as effective at inhibiting their corresponding targets as
the use of single siRNAs. It was also demonstrated that
administration of a dual targeting siRNA results in a synergistic
lowering of total serum cholesterol. Thus, reduction of total serum
cholesterol is enhanced with a dual targeting siRNA compared to a
single target siRNA.
Definitions
[0036] 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.
[0037] "G," "C," "A," "T" and "U" each generally stand for a
nucleotide that contains guanine, cytosine, adenine, thymidine 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" 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 dsRNA featured in the invention by a
nucleotide containing, for example, inosine. In another example,
adenine and cytosine anywhere in the oligonucleotide can be
replaced with guanine and uracil, respectively to form G-U Wobble
base pairing with the target mRNA. Sequences containing such
replacement moieties are suitable for the compositions and methods
featured in the invention.
[0038] The term "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.
[0039] The term "XBP-1" refers to -Box Protein 1, which is also
known as Tax-responsive element-binding protein 5, TREB5, and XBP2.
XBP-1 sequence can be found as NCBI GeneID:7494 and RefSeq ID
number: NM_005080 (human) and NM_013842 (mouse). A dsRNA featured
in the invention can target a specific XBP-1 isoform, e.g., the
spliced form (XBP-1S) or the unspliced form (XBP-1U), or a dsRNA
featured in the invention can target both isoforms by binding to a
common region of the mRNA transcript.
[0040] The term "PCSK5" refers to the Proprotein convertase
subtilisin/kexin type 5 gene, mRNA or protein belonging to the
subtilisin-like proprotein convertase family.
[0041] The term "ApoC3" refers to the Apolipoprotein C-III protein
gene, mRNA or protein, and is a very low density lipoprotein
(VLDL).
[0042] The term "SCAP" refers to the SREBP cleavage-activating
protein gene, mRNA or protein. SCAP is a regulatory protein that is
required for the proteolytic cleavage of the sterol regulatory
element binding protein (SREBP). Example of siRNA targeting SCAP
are described in U.S. patent application Ser. No. 11/857,120, filed
on Sep. 18, 2007, published as US 20090093426. This application and
the siRNA sequences described therein are incorporated by reference
for all purposes.
[0043] The term "MIG12" is a gene also known as TMSB10 and TB10
refers to the thymosin beta 10 gene. Example of siRNA targeting
MIG12 are described International patent application no.
PCT/US10/25444, filed on Feb. 25, 2010, published as WO/20XX/XXXXXX
This application and the siRNA sequences described therein are
incorporated by reference for all purposes.
[0044] As used herein, the term "iRNA" refers to an agent that
contains RNA and which mediates the targeted cleavage of an RNA
transcript via an RNA-induced silencing complex (RISC) pathway. The
term iRNA includes siRNA.
[0045] As described in more detail below, the term "siRNA" and
"siRNA agent" refers to a dsRNA that mediates the targeted cleavage
of an RNA transcript via an RNA-induced silencing complex (RISC)
pathway.
[0046] A "double-stranded RNA" or "dsRNA," as used herein, refers
to an RNA molecule or complex of molecules having a hybridized
duplex region that comprises two anti-parallel and substantially
complementary nucleic acid strands, which will be referred to as
having "sense" and "antisense" orientations with respect to a
target RNA.
[0047] The term "dual targeting siRNA agent" refers to a
composition of two siRNAs, e.g., two dsRNAs. One dsRNA includes an
antisense strand with a first region of complementarity to a first
target gene, e.g., PCSK9. The second dsRNA include an antisense
strand with a second region of complementarity to a second target
gene. In some embodiments, the first and second target genes are
identical, e.g., both are PCSK9 and each dsRNA targets a different
region of PCSK9. In other embodiments, the first and second target
genes are different, e.g., the first dsRNA targets PCSK9 and the
second dsRNA targets a different gene, e.g., XBP-1.
[0048] "Covalent linker" refers to a molecule for covalently
joining two molecules, e.g., two dsRNAs. As described in more
detail below, the term includes, e.g., a nucleic acid linker, a
peptide linker, and the like and includes disulfide linkers.
[0049] The term "target gene" refers to a gene of interest, e.g.,
PCSK9 or a second gene, e.g., XBP-1, targeted by an siRNA of the
invention for inhibition of expression.
[0050] As described in more detail below, "target sequence" refers
to a contiguous portion of the nucleotide sequence of an mRNA
molecule formed during the transcription of a target gene,
including mRNA that is a product of RNA processing of a primary
transcription product. The target portion of the sequence will be
at least long enough to serve as a substrate for iRNA-directed
cleavage at or near that portion. For example, the target sequence
will generally be from 9-36 nucleotides in length, e.g., 15-30
nucleotides in length, including all sub-ranges therebetween.
[0051] 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.
[0052] 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.
[0053] Complementary sequences within an iRNA, e.g., within a dsRNA
as described herein, include base-pairing of the oligonucleotide or
polynucleotide comprising a first nucleotide sequence to an
oligonucleotide or polynucleotide comprising a second nucleotide
sequence over the entire length of one or both nucleotide
sequences. Such sequences can be referred to as "fully
complementary" with respect to each other herein. 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 5, 4, 1 or 2 mismatched base pairs upon hybridization for a
duplex up to 30 base pairs, while retaining the ability to
hybridize under the conditions most relevant to their ultimate
application, e.g., inhibition of gene expression via a RISC
pathway. 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 comprising
one oligonucleotide 21 nucleotides in length and another
oligonucleotide 23 nucleotides in length, wherein the longer
oligonucleotide comprises a sequence of 21 nucleotides that is
fully complementary to the shorter oligonucleotide, may yet be
referred to as "fully complementary" for the purposes described
herein.
[0054] "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 are not limited to, G:U Wobble or Hoogstein base pairing.
[0055] 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 an iRNA agent and a
target sequence, as will be understood from the context of their
use.
[0056] As used herein, a polynucleotide that 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 the target gene (e.g., an mRNA
encoding PCSK9 or a second gene, e.g., XBP-1). 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.
[0057] The skilled artisan will recognize that the term "RNA
molecule" or "ribonucleic acid molecule" encompasses not only RNA
molecules as expressed or found in nature, but also analogs and
derivatives of RNA comprising one or more
ribonucleotide/ribonucleoside analogs or derivatives as described
herein or as known in the art. Strictly speaking, a
"ribonucleoside" includes a nucleoside base and a ribose sugar, and
a "ribonucleotide" is a ribonucleoside with one, two or three
phosphate moieties. However, the terms "ribonucleoside" and
"ribonucleotide" can be considered to be equivalent as used herein.
The RNA can be modified in the nucleobase structure or in the
ribose-phosphate backbone structure, e.g., as described herein
below. However, the molecules comprising ribonucleoside analogs or
derivatives must retain the ability to form a duplex. As
non-limiting examples, an RNA molecule can also include at least
one modified ribonucleoside including but not limited to a
2'-O-methyl modified nucleotide, a nucleoside comprising a 5'
phosphorothioate group, a terminal nucleoside linked to a
cholesteryl derivative or dodecanoic acid bisdecylamide group, a
locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro
modified nucleoside, a 2'-amino-modified nucleoside,
2'-alkyl-modified nucleoside, morpholino nucleoside, a
phosphoramidate or a non-natural base comprising nucleoside, or any
combination thereof. Alternatively, an RNA molecule can comprise at
least two modified ribonucleosides, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 15, at least 20 or more, up to the entire length of
the dsRNA molecule. The modifications need not be the same for each
of such a plurality of modified ribonucleosides in an RNA molecule.
In one embodiment, modified RNAs contemplated for use in methods
and compositions described herein are peptide nucleic acids (PNAs)
that have the ability to form the required duplex structure and
that permit or mediate the specific degradation of a target RNA via
a RISC pathway.
[0058] In one aspect, a modified ribonucleoside includes a
deoxyribonucleoside. In such an instance, an iRNA agent can
comprise one or more deoxynucleosides, including, for example, a
deoxynucleoside overhang(s), or one or more deoxynucleosides within
the double stranded portion of a dsRNA. However, it is self evident
that under no circumstances is a double stranded DNA molecule
encompassed by the term "iRNA."
[0059] As used herein, the term "nucleotide overhang" refers to at
least one unpaired nucleotide that protrudes from the duplex
structure of an iRNA, e.g., a dsRNA. For example, when a 3'-end of
one strand of a dsRNA extends beyond the 5'-end of the other
strand, or vice versa, there is a nucleotide overhang. A dsRNA can
comprise an overhang of at least one nucleotide; alternatively the
overhang can comprise at least two nucleotides, at least three
nucleotides, at least four nucleotides, at least five nucleotides
or more. A nucleotide overhang can comprise or consist of a
nucleotide/nucleoside analog, including a
deoxynucleotide/nucleoside. The overhang(s) may be on the sense
strand, the antisense strand or any combination thereof.
Furthermore, the nucleotide(s) of an overhang can be present on the
5' end, 3' end or both ends of either an antisense or sense strand
of a dsRNA. One or more of the nucleotides in the overhang can be
replaced with a nucleoside thiophosphate.
[0060] The terms "blunt" or "blunt ended" as used herein in
reference to a dsRNA mean that there are no unpaired nucleotides or
nucleotide analogs at a given terminal end of a dsRNA, i.e., no
nucleotide overhang. One or both ends of a dsRNA can be blunt.
Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt
ended. To be clear, a "blunt ended" dsRNA is a dsRNA that is blunt
at both ends, i.e., no nucleotide overhang at either end of the
molecule. Most often such a molecule will be double-stranded over
its entire length.
[0061] The term "antisense strand" or "guide strand" refers to the
strand of an iRNA, e.g., 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 5, 4, 3, or 2 nucleotides of the
5' and/or 3' terminus.
[0062] The term "sense strand" or "passenger strand" as used
herein, refers to the strand of an iRNA that includes a region that
is substantially complementary to a region of the antisense strand
as that term is defined herein.
[0063] As used herein, the term "SNALP" refers to a stable nucleic
acid-lipid particle. A SNALP represents a vesicle of lipids coating
a reduced aqueous interior comprising a nucleic acid such as an
iRNA or a plasmid from which an iRNA is transcribed. SNALPs are
described, e.g., in U.S. Patent Application Publication Nos.
20060240093, 20070135372, and in International Application No. WO
2009082817. These applications are incorporated herein by reference
in their entirety.
[0064] "Introducing into a cell," when referring to an iRNA, means
facilitating or effecting uptake or absorption into the cell, as is
understood by those skilled in the art. Absorption or uptake of an
iRNA 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; an iRNA may also be
"introduced into a cell," wherein the cell is part of a living
organism. In such an instance, introduction into the cell will
include the delivery to the organism. For example, for in vivo
delivery, iRNA can be injected into a tissue site or administered
systemically. In vivo delivery can also be by a beta-glucan
delivery system, such as those described in U.S. Pat. Nos.
5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781,
which are hereby incorporated by reference in their entirety. In
vitro introduction into a cell includes methods known in the art
such as electroporation and lipofection. Further approaches are
described herein below or known in the art.
[0065] As used herein, the term "modulate the expression of,"
refers to at an least partial "inhibition" or partial "activation"
of target gene expression in a cell treated with an iRNA
composition as described herein compared to the expression of the
target gene in an untreated cell.
[0066] The terms "activate," "enhance," "up-regulate the expression
of," "increase the expression of," and the like, in so far as they
refer to a target gene, herein refer to the at least partial
activation of the expression of a target gene, as manifested by an
increase in the amount of target mRNA, which may be isolated from
or detected in a first cell or group of cells in which a target
gene is transcribed and which has or have been treated such that
the expression of a target gene is increased, 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).
[0067] In one embodiment, expression of a target gene is activated
by at least about 10%, 15%, 25%, 30%, 35%, 40%, 45%, or 50% by
administration of an iRNA as described herein. In some embodiments,
a target gene is activated by at least about 60%, 70%, or 80% by
administration of an iRNA featured in the invention. In some
embodiments, expression of a target gene is activated by at least
about 85%, 90%, or 95% or more by administration of an iRNA as
described herein. In some embodiments, the target gene expression
is increased by at least 1-fold, at least 2-fold, at least 5-fold,
at least 10-fold, at least 50-fold, at least 100-fold, at least
500-fold, at least 1000 fold or more in cells treated with an iRNA
as described herein compared to the expression in an untreated
cell. Activation of expression by small dsRNAs is described, for
example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A.
103:17337-42, and in US20070111963 and US2005226848, each of which
is incorporated herein by reference.
[0068] The terms "silence," "inhibit the expression of,"
"down-regulate the expression of," "suppress the expression of,"
and the like, in so far as they refer to a target gene, herein
refer to the at least partial suppression of the expression of a
target gene, as manifested by a reduction of the amount of target
mRNA which may be isolated from or detected in a first cell or
group of cells in which a target gene is transcribed and which has
or have been treated such that the expression of target 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 .times. .times. in .times. .times. control .times. .times.
cells ) - ( mRNA .times. .times. in .times. .times. treated .times.
.times. cells ) ( mRNA .times. .times. in .times. .times. control
.times. .times. cells ) 100 .times. % ##EQU00001##
[0069] Alternatively, the degree of inhibition may be given in
terms of a reduction of a parameter that is functionally linked to
target gene expression, e.g., the amount of protein encoded by a
target gene, or the number of cells displaying a certain phenotype,
e.g., lack of or decreased cytokine production. In principle,
target gene silencing may be determined in any cell expressing
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 iRNA inhibits the expression of the
target 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.
[0070] For example, in certain instances, expression of a target
gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, or 55% by administration of an iRNA featured in the
invention. In some embodiments, a target gene is suppressed by at
least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA
featured in the invention. In some embodiments, a target gene is
suppressed by at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more
by administration of an iRNA as described herein.
[0071] As used herein in the context of target gene expression, the
terms "treat," "treatment," and the like, refer to relief from or
alleviation of pathological processes mediated by target
expression. In the context of the present invention insofar as it
relates to any of the other conditions recited herein below (other
than pathological processes mediated by target expression), 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 or anticipated progression of
such condition.
[0072] By "lower" in the context of a disease marker or symptom is
meant a statistically significant decrease in such level. The
decrease can be, for example, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40% or
more, and is preferably down to a level accepted as within the
range of normal for an individual without such disorder.
[0073] As used herein, the phrase "therapeutically effective
amount" " refers to an amount that provides a therapeutic benefit
in the treatment or management of pathological processes mediated
by target gene expression, e.g., PCSK9 and/or a second gene, e.g.,
XBP-1, or an overt symptom of pathological processes mediated
target gene expression. The phrase "prophylactically effective
amount" refer to an amount that provides a therapeutic benefit in
the prevention of pathological processes mediated by target gene
expression or an overt symptom of pathological processes mediated
by target gene expression. 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, for example, the type of pathological processes
mediated by target gene expression, the patient's history and age,
the stage of pathological processes mediated by target gene
expression, and the administration of other agents that inhibit
pathological processes mediated by target gene expression.
[0074] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of an iRNA and a
pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of an
iRNA effective to produce the intended pharmacological or
therapeutic result. For example, if a given clinical treatment is
considered effective when there is at least a 10% 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 10% reduction in that parameter.
[0075] The term "pharmaceutically carrier" refers to a carrier for
administration of a therapeutic agent, e.g., a dual targeting siRNA
agent. Carriers are described in more detail below, and include
lipid formulations, e.g., LNP09 and SNALP formulations.
[0076] Double-Stranded Ribonucleic Acid (dsRNA)
[0077] Described herein are dual targeting siRNA agents, e.g.,
siRNAs that inhibit the expression of a PCSK9 gene and a second
gene. The dual targeting siRNA agent includes two siRNA covalently
linked via, e.g., a disulfide linker. The first siRNA targets a
first region of a PCSK9 gene. The second siRNA targets a second
gene, e.g., XBP-1, or, e.g., targets a second region of the PCSK9
gene.
[0078] 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, Applied Biosystems, Inc. Further descriptions of synthesis
are found below and in the examples.
[0079] Each siRNA is a dsRNA. A dsRNA includes two RNA strands that
are sufficiently complementary to hybridize to form a duplex
structure under conditions in which the dsRNA will be used. One
strand of a dsRNA (the antisense strand) includes 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 a target 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.
[0080] Where the duplex region is formed from two strands of a
single molecule, the molecule can have a duplex region separated by
a single stranded chain of nucleotides (herein referred to as a
"hairpin loop") between the 3'-end of one strand and the 5'-end of
the respective other strand forming the duplex structure. The
hairpin loop can comprise at least one unpaired nucleotide; in some
embodiments the hairpin loop can comprise at least 3, at least 4,
at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 20, at least 23 or more unpaired
nucleotides.
[0081] Where the two substantially complementary strands of a dsRNA
are comprised by separate RNA molecules, those molecules need not,
but can be covalently connected. Where the two strands are
connected covalently by means other than a hairpin loop, the
connecting structure is referred to as a "linker."
[0082] Generally, the duplex structure of the siRNA, e.g., dsRNA,
is between 15 and 30 inclusive, more generally between 18 and 25
inclusive, yet more generally between 19 and 24 inclusive, and most
generally between 19 and 21 base pairs in length, inclusive.
Considering a duplex between 9 and 36 base pairs, the duplex can be
any length in this range, for example, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, or 36 and any sub-range therein between, including, but
not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base
pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19
base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs,
18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base
pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23
base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs,
20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base
pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30
base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs,
21-23 base pairs, or 21-22 base pairs.
[0083] The two siRNAs in the dual targeting siRNA agent can have
duplex lengths that are identical or that differ.
[0084] The region of complementarity to the target sequence in an
siRNA is between 15 and 30 inclusive, more generally between 18 and
25 inclusive, yet more generally between 19 and 24 inclusive, and
most generally between 19 and 21 nucleotides in length, inclusive.
In some embodiments, the dsRNA is between 15 and 20 nucleotides in
length, inclusive, and in other embodiments, the dsRNA is between
25 and 30 nucleotides in length, inclusive. The region of
complementarity can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30 nucleotides in length. As non-limiting
examples, the target sequence can be from 15-30 nucleotides, 15-26
nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21
nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18
nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26
nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21
nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26
nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21
nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26
nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23
nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30
nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24
nucleotides, 21-23 nucleotides, or 21-22 nucleotides. In some
embodiments the target sequence is 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25 nucleotides.
[0085] The two siRNAs in the dual targeting siRNA agent can have
regions of complementarity that are identical in length or that
differ in length.
[0086] Any of the dsRNA, e.g., siRNA as described herein may
include one or more single-stranded nucleotide overhangs. In one
embodiment, at least one end of a dsRNA has a single-stranded
nucleotide overhang of 1 to 4, or 1 or 2 or 3 or 4 nucleotides.
dsRNAs having at least one nucleotide overhang have unexpectedly
superior inhibitory properties relative to their blunt-ended
counterparts. 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.
In another embodiment, one or more of the nucleotides in the
overhang is replaced with a nucleoside thiophosphate. The two
siRNAs in the dual targeting siRNA agent can have different or
identical overhangs as described by location, length, and
nucleotide.
[0087] The dual targeting siRNA agent includes at least a first
siRNA targeting a first region of a PCSK9 gene. In one embodiment,
a PCSK9 gene is a human PCSK9 gene. In another embodiment the PCSK9
gene is a mouse or a rat PCSK9 gene. Exemplary siRNA targeting
PCSK9 are described in U.S. patent application Ser. No. 11/746,864
filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and
International Patent Application No. PCT/US2007/068655 filed May
10, 2007 (published as WO 2007/134161). Additional disclosure can
be found in U.S. patent application Ser. No. 12/478,452 filed Jun.
4, 2009 (published as US 2010/0010066) and International Patent
Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as
WO 2009/134487). The sequences of the target, sense, and antisense
strands are incorporated by reference for all purposes.
[0088] Tables 1, 2, and 4-8 disclose sequences of the target, sense
strands, and antisense strands of PCSK9 targeting siRNA.
[0089] In one embodiment the first siRNA is AD-9680. The dsRNA
AD-9680 targets the human PCSK 9 gene at nucleotides 3530-3548 of a
human PCSK9 gene (accession number NM_174936).
TABLE-US-00001 TABLE 1 AD-9680 siRNA sequences SEQ ID Table 1:
AD-9680 Sequence 5' to 3' NO: Target sequence UUCUAGACCUGUUUUGCUU
4142 Sense strand UUCUAGACCUGUUUUGCUU 4143 Sense strand,
uucuAGAccuGuuuuGcuuTsT 4144 modified Antisense strand
AAGCAAAACAGGUCUAGAA 4145 Antisense strand, AAGcAAAAcAGGUCuAGAATsT
4146 modified
[0090] In another embodiment, the first siRNA is AD-10792. The
dsRNA AD-10792 targets the PCSK9 gene at nucleotides 1091-1109 of a
human PCSK9 gene (accession number NM 174936). AD-10792 is also
complementary to rodent PCSK9.
TABLE-US-00002 TABLE 2 AD-10792 siRNA sequences SEQ ID Table 2:
AD-10792 Sequence 5' to 3' NO: Target sequence GCCUGGAGUUUAUUCGGAA
4147 Sense strand GCCUGGAGUUUAUUCGGAA 4148 Sense strand,
GccuGGAGuuuAuucGGAATsT 4149 modified Antisense strand
UUCCGAAUAAACUCCAGGC 4150 Antisense strand, UUCCGAAuAAACUCcAGGCTsT
4151 modified
[0091] The second siRNA of the dual targeting siRNA agent targets a
second gene. In one embodiment, the second gene is PCSK9, and the
second siRNA target a region of PCSK9 that is different from the
region targeted by the first siRNA.
[0092] Alternatively, the second siRNA targets a different second
gene. Examples include genes that interact with PCSK9 and/or are
involved with lipid metabolism or cholesterol metabolism. For
example, the second target gene can be XBP-1, PCSK5, ApoC3, SCAP,
MIG12, HMG CoA Reductase, or IDOL (Inducible Degrader of the LDLR)
and the like. In one embodiment, the second gene is a human gene.
In another embodiment the second gene is a mouse or a rat gene.
[0093] In one embodiment, the second siRNA targets the XBP-1 gene.
Exemplary siRNA targeting XBP-1 can be found in U.S. patent
application Ser. No. 12/425,811 filed Apr. 17, 2009 (published as
US 2009-0275638). The sequences of the target, sense, and antisense
strands are incorporated by reference for all purposes.
[0094] Tables 3 and 9-13 disclose sequences of the target, sense
strands, and antisense strands of XBP-1 targeting siRNA.
[0095] In one embodiment the first siRNA is AD-18038. The dsRNA
AD-18038 targets the human XBP-1 gene at nucleotides 896-914 of a
human XBP-1 gene (accession number NM_001004210).
TABLE-US-00003 TABLE 3 AD-18038 siRNA sequences SEQ ID Table 3:
AD-18038 Sequence 5' to 3' NO: Target sequence CACCCUGAAUUCAUUGUCU
4153 Sense strand CACCCUGAAUUCAUUGUCU 4154 Sense strand,
cAcccuGAAuucAuuGucudTsdT 4155 modified Antisense strand
AGACAAUGAAUUCAGGGUG 4156 Antisense strand, AGAcAAUGAAUUcAGGGUGdTsdT
4157 modified
[0096] Additional dsRNA
[0097] A dsRNAs having a partial sequence of at least 15, 16, 17,
18, 19, 20, or more contiguous nucleotides from one of the
sequences in Tables 1-13, and differing in their ability to inhibit
the expression of a target gene by not more than 5, 10, 15, 20, 25,
or 30% inhibition from a dsRNA comprising the full sequence, are
contemplated according to the invention.
[0098] In addition, the RNAs provided in Tables 1-13 identify a
site in the target gene transcript that is susceptible to
RISC-mediated cleavage. As such, the present invention further
features iRNAs that target within one of such sequences. As used
herein, an iRNA is said to target within a particular site of an
RNA transcript if the iRNA promotes cleavage of the transcript
anywhere within that particular site. Such an iRNA will generally
include at least 15 contiguous nucleotides from one of the
sequences provided herein coupled to additional nucleotide
sequences taken from the region contiguous to the selected sequence
in a target gene.
[0099] While a target sequence is generally 15-30 nucleotides in
length, there is wide variation in the suitability of particular
sequences in this range for directing cleavage of any given target
RNA. Various software packages and the guidelines set out herein
provide guidance for the identification of optimal target sequences
for any given gene target, but an empirical approach can also be
taken in which a "window" or "mask" of a given size (as a
non-limiting example, 21 nucleotides) is literally or figuratively
(including, e.g., in silico) placed on the target RNA sequence to
identify sequences in the size range that may serve as target
sequences. By moving the sequence "window" progressively one
nucleotide upstream or downstream of an initial target sequence
location, the next potential target sequence can be identified,
until the complete set of possible sequences is identified for any
given target size selected. This process, coupled with systematic
synthesis and testing of the identified sequences (using assays as
described herein or as known in the art) to identify those
sequences that perform optimally can identify those RNA sequences
that, when targeted with an iRNA agent, mediate the best inhibition
of target gene expression. Thus, while the sequences identified,
for example, above represent effective target sequences, it is
contemplated that further optimization of inhibition efficiency can
be achieved by progressively "walking the window" one nucleotide
upstream or downstream of the given sequences to identify sequences
with equal or better inhibition characteristics.
[0100] Further, it is contemplated that for any sequence
identified, e.g., in Tables 1-13, further optimization could be
achieved by systematically either adding or removing nucleotides to
generate longer or shorter sequences and testing those and
sequences generated by walking a window of the longer or shorter
size up or down the target RNA from that point. Again, coupling
this approach to generating new candidate targets with testing for
effectiveness of iRNAs based on those target sequences in an
inhibition assay as known in the art or as described herein can
lead to further improvements in the efficiency of inhibition.
Further still, such optimized sequences can be adjusted by, e.g.,
the introduction of modified nucleotides as described herein or as
known in the art, addition or changes in overhang, or other
modifications as known in the art and/or discussed herein to
further optimize the molecule (e.g., increasing serum stability or
circulating half-life, increasing thermal stability, enhancing
transmembrane delivery, targeting to a particular location or cell
type, increasing interaction with silencing pathway enzymes,
increasing release from endosomes, etc.) as an expression
inhibitor.
[0101] An iRNA as described in Tables 1-13 can contain one or more
mismatches to the target sequence. In one embodiment, an iRNA as
described in Tables 1-13 contains no more than 3 mismatches. If the
antisense strand of the iRNA contains mismatches to a target
sequence, it is preferable that the area of mismatch not be located
in the center of the region of complementarity. If the antisense
strand of the iRNA contains mismatches to the target sequence, it
is preferable that the mismatch be restricted to be within the last
5 nucleotides from either the 5' or 3' end of the region of
complementarity. For example, for a 23 nucleotide iRNA agent RNA
strand which is complementary to a region of a PCSK9 gene, the RNA
strand generally does not contain any mismatch within the central
13 nucleotides. The methods described herein or methods known in
the art can be used to determine whether an iRNA containing a
mismatch to a target sequence is effective in inhibiting the
expression of a PCSK9 gene. Consideration of the efficacy of iRNAs
with mismatches in inhibiting expression of a PCSK9 gene is
important, especially if the particular region of complementarity
in a PCSK9 gene is known to have polymorphic sequence variation
within the population.
[0102] Covalent Linkage
[0103] The dual targeting siRNA agents of the invention include two
siRNAs joined via a covalent linker. Covalent linkers are
well-known to one of skill in the art and include, e.g., a nucleic
acid linker, a peptide linker, and the like.
[0104] The covalent linker joins the two siRNAs. The covalent
linker can join two sense strands, two antisense strands, one sense
and one antisense strand, two sense strands and one antisense
strand, two antisense strands and one sense strand, or two sense
and two antisense strands.
[0105] The covalent linker can include RNA and/or DNA and/or a
peptide. The linker can be single stranded, double stranded,
partially single strands, or partially double stranded. In some
embodiments the linker includes a disulfide bond. The linker can be
cleavable or non-cleavable.
[0106] The covalent linker can be, e.g.,
dTsdTuu=(5'-2'deoxythymidy1-3'-thiophosphate-5'-2'deoxythymidy
1-3'-phosphate-5'-uridy 1-3'-phosphate-5'-uridy 1-3'-phosphate);
rUsrU (a thiophosphate linker:
5'-uridy1-3'-thiophosphate-5'-uridy1-3'-phosphate); an rUrU linker;
dTsdTaa (aadTsdT,
5'-2'deoxythymidy1-3'-thiophosphate-5'-2'deoxythymidy1-3'-phosphate-5'-ad-
eny1-3'-phosphate-5'-adeny1-3'-phosphate); dTsdT
(5'-2'deoxythymidy1-3'-thiophosphate-5'-2'
deoxythymidy1-3'-phosphate);
dTsdTuu=uudTsdT=5'-2'deoxythymidy1-3'-thiophosphate-5'-2'deoxythymidy
1-3'-phosphate-5'-uridy1-3'-phosphate-5'-uridy1-3'-phosphate.
[0107] The covalent linker can be a polyRNA, such as
poly(5'-adenyl-3'-phosphate--AAAAAAAA) or
poly(5'-cytidyl-3'-phosphate-5'-uridyl-3'-phosphate--CUCUCUCU)),
e.g., X.sub.n single stranded poly RNA linker wherein n is an
integer from 2-50 inclusive, preferable 4-15 inclusive, most
preferably 7-8 inclusive. Modified nucleotides or a mixture of
nucleotides can also be present in said polyRNA linker. The
covalent linker can be a polyDNA, such as
poly(5'-2'deoxythymidy1-3'-phosphate--TTTTTTTT), e.g., wherein n is
an integer from 2-50 inclusive, preferable 4-15 inclusive, most
preferably 7-8 inclusive. Modified nucleotides or a mixture of
nucleotides can also be present in said polyDNA linker. a single
stranded polyDNA linker wherein n is an integer from 2-50
inclusive, preferable 4-15 inclusive, most preferably 7-8
inclusive. Modified nucleotides or a mixture of nucleotides can
also be present in said polyDNA linker.
[0108] The covalent linker can include a disulfide bond, optionally
a bis-hexyl-disulfide linker. In one embodiment, the disulfide
linker is
##STR00001##
[0109] The covalent linker can include a peptide bond, e.g.,
include amino acids. In one embodiment, the covalent linker is a
1-10 amino acid long linker, preferably comprising 4-5 amino acids,
optionally X-Gly-Phe-Gly-Y wherein X and Y represent any amino
acid.
[0110] The covalent linker can include HEG, a hexaethylenglycol
linker.
[0111] Modifications
[0112] In yet another embodiment, at least one of the siRNA of the
dual targeting siRNA agent is chemically modified to enhance
stability or other beneficial characteristics. The nucleic acids
featured in 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. Modifications
include, for example, (a) end modifications, e.g., 5' end
modifications (phosphorylation, conjugation, inverted linkages,
etc.) 3' end modifications (conjugation, DNA nucleotides, inverted
linkages, etc.), (b) base modifications, e.g., replacement with
stabilizing bases, destabilizing bases, or bases that base pair
with an expanded repertoire of partners, removal of bases (abasic
nucleotides), or conjugated bases, (c) sugar modifications (e.g.,
at the 2' position or 4' position) or replacement of the sugar, as
well as (d) backbone modifications, including modification or
replacement of the phosphodiester linkages. Specific examples of
RNA compounds useful in this invention include, but are not limited
to RNAs containing modified backbones or no natural internucleoside
linkages. RNAs having modified backbones include, among others,
those that do not have a phosphorus atom in the backbone. For the
purposes of this specification, and as sometimes referenced in the
art, modified RNAs that do not have a phosphorus atom in their
internucleoside backbone can also be considered to be
oligonucleosides. In particular embodiments, the modified RNA will
have a phosphorus atom in its internucleoside backbone.
[0113] Modified RNA 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.
[0114] Representative U.S. patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,195; 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; 5,541,316; 5,550,111;
5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445;
6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199;
6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167;
6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933;
7,321,029; and U.S. Pat. No. RE39464, each of which is herein
incorporated by reference
[0115] Modified RNA backbones that do not include a phosphorus atom
therein have backbones that are formed by short chain alkyl or
cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or
cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic internucleoside linkages. These
include those having morpholino linkages (formed in part from the
sugar portion of a nucleoside); siloxane backbones; sulfide,
sulfoxide and sulfone backbones; 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.
[0116] Representative U.S. patents that teach 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; 5,64,562; 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;
5,608,046; 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.
[0117] In other RNA mimetics suitable or contemplated for use in
iRNAs, both the sugar and the internucleoside linkage, i.e., the
backbone, of the nucleotide units are replaced with novel groups.
The base units are maintained for hybridization with an appropriate
nucleic acid target compound. One such oligomeric compound, an RNA
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar backbone of an RNA is replaced with an amide
containing backbone, in particular an aminoethylglycine backbone.
The nucleobases are retained and are bound directly or indirectly
to aza nitrogen atoms of the amide portion of the backbone.
Representative U.S. patents that teach the preparation of PNA
compounds include, but are not limited to, U.S. Pat. Nos.
5,539,082; 5,714,331; and 5,719,262, each of which is herein
incorporated by reference. Further teaching of PNA compounds can be
found, for example, in Nielsen et al., Science, 1991, 254,
1497-1500.
[0118] Some embodiments featured in the invention include RNAs with
phosphorothioate backbones and oligonucleosides with heteroatom
backbones, and in particular --CH.sub.2--NH--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--[known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--N(CH.sub.3)--CH.sub.2--CH.sub.2--[wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above-referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above-referenced U.S. Pat. No. 5,602,240. In some
embodiments, the RNAs featured herein have morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0119] Modified RNAs may also contain one or more substituted sugar
moieties. The iRNAs, e.g., dsRNAs, featured herein can include one
of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-,
S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein
the alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Exemplary suitable modifications include
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2)..sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. In other embodiments, dsRNAs include one of
the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,
SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an iRNA, or a group for improving the
pharmacodynamic properties of an iRNA, and other substituents
having similar properties. In some embodiments, the modification
includes a 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also
known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.
Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another
exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples herein below, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples herein below.
[0120] Other modifications include 2'-methoxy (2'-OCH.sub.3),
2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and
2'-fluoro (2'-F). Similar modifications may also be made at other
positions on the RNA of an iRNA, particularly the 3' position of
the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs
and the 5' position of 5' terminal nucleotide. iRNAs may also have
sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative U.S. patents that teach the
preparation of such modified sugar structures include, but are not
limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain
of which are commonly owned with the instant application, and each
of which is herein incorporated by reference.
[0121] An iRNA may also include nucleobase (often referred to in
the art simply as "base") modifications or substitutions. As used
herein, "unmodified" or "natural" nucleobases include the purine
bases adenine (A) and guanine (G), and the pyrimidine bases thymine
(T), cytosine (C) and uracil (U). Modified nucleobases include
other synthetic and natural nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl
anal other 8-substituted adenines and guanines, 5-halo,
particularly 5-bromo, 5-trifluoromethyl and other 5-substituted
uracils and cytosines, 7-methylguanine and 7-methyladenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine
and 3-deazaguanine and 3-deazaadenine. Further nucleobases include
those disclosed in U.S. Pat. No. 3,687,808, those disclosed in
Modified Nucleosides in Biochemistry, Biotechnology and Medicine,
Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise
Encyclopedia Of Polymer Science And Engineering, pages 858-859,
Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed
by Englisch et al., Angewandte Chemie, International Edition, 1991,
30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA
Research and Applications, pages 289-302, Crooke, S. T. and Lebleu,
B., Ed., CRC Press, 1993. Certain of these nucleobases are
particularly useful for increasing the binding affinity of the
oligomeric compounds featured in the invention. These include
5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6
substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and
Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca
Raton, 1993, pp. 276-278) and are exemplary base substitutions,
even more particularly when combined with 2'-O-methoxyethyl sugar
modifications.
[0122] Representative U.S. patents that teach the preparation of
certain of the above noted modified nucleobases as well as other
modified nucleobases include, but are not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;
5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;
5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;
5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200;
6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062;
6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is
herein incorporated by reference, and U.S. Pat. No. 5,750,692, also
herein incorporated by reference.
[0123] The RNA of an iRNA can also be modified to include one or
more locked nucleic acids (LNA). A locked nucleic acid is a
nucleotide having a modified ribose moiety in which the ribose
moiety comprises an extra bridge connecting the 2' and 4' carbons.
This structure effectively "locks" the ribose in the 3'-endo
structural conformation. The addition of locked nucleic acids to
siRNAs has been shown to increase siRNA stability in serum, and to
reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids
Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther
6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research
31(12):3185-3193).
[0124] Representative U.S. Patents that teach the preparation of
locked nucleic acid nucleotides include, but are not limited to,
the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;
6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is
herein incorporated by reference in its entirety.
[0125] Potentially stabilizing modifications to the ends of RNA
molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol
(Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6),
N-(acetyl-4-hydroxyprolinol (Hyp-NHAc),
thymidine-2'-O-deoxythymidine (ether),
N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),
2'-docosanoyl-uridine-3'-phosphate, inverted base dT(idT) and
others. Disclosure of this modification can be found in U.S.
Provisional Patent Application No. 61/223,665 ("the '665
application"), filed Jul. 7, 2009, entitled "Oligonucleotide End
Caps" and International patent application no. PCT/US10/41214,
filed Jul. 7, 2010.
[0126] Ligands
[0127] Another modification of the RNA of an iRNA featured in the
invention involves chemically linking to the RNA one or more
ligands, moieties or conjugates that enhance the activity, cellular
distribution or cellular uptake of the iRNA. Such moieties include
but are not limited to lipid moieties such as a cholesterol moiety
(Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:
6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,
1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309;
Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a
thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,
20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl
residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118;
Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al.,
Biochimie, 1993, 75:49-54), a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995,
14:969-973), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra
et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an
octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke
et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
[0128] In one embodiment, a ligand alters the distribution,
targeting or lifetime of an iRNA agent into which it is
incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a selected target, e.g., molecule, cell or
cell type, compartment, e.g., a cellular or organ compartment,
tissue, organ or region of the body, as, e.g., compared to a
species absent such a ligand. Preferred ligands will not take part
in duplex pairing in a duplexed nucleic acid.
[0129] Ligands can include a naturally occurring substance, such as
a protein (e.g., human serum albumin (HSA), low-density lipoprotein
(LDL), or globulin); carbohydrate (e.g., a dextran, pullulan,
chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a
lipid. The ligand may also be a recombinant or synthetic molecule,
such as a synthetic polymer, e.g., a synthetic polyamino acid.
Examples of polyamino acids include polyamino acid is a polylysine
(PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic
acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer,
divinyl ether-maleic anhydride copolymer,
N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene
glycol (PEG), polyvinyl alcohol (PVA), polyurethane,
poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or
polyphosphazine. Example of polyamines include: polyethylenimine,
polylysine (PLL), spermine, spermidine, polyamine,
pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer
polyamine, arginine, amidine, protamine, cationic lipid, cationic
porphyrin, quaternary salt of a polyamine, or an alpha helical
peptide.
[0130] Ligands can also include targeting groups, e.g., a cell or
tissue targeting agent, e.g., a lectin, glycoprotein, lipid or
protein, e.g., an antibody, that binds to a specified cell type
such as a kidney cell. A targeting group can be a thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, Mucin
carbohydrate, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
multivalent fucose, glycosylated polyaminoacids, multivalent
galactose, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate,
vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.
[0131] Other examples of ligands include dyes, intercalating agents
(e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),
porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic
hydrocarbons (e.g., phenazine, dihydrophenazine), artificial
endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol,
cholic acid, adamantane acetic acid, 1-pyrene butyric acid,
dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl
group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol,
heptadecyl group, palmitic acid, myristic acid,
O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,
antennapedia peptide, Tat peptide), alkylating agents, phosphate,
amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino,
alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens
(e.g. biotin), transport/absorption facilitators (e.g., aspirin,
vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,
bisimidazole, histamine, imidazole clusters, acridine-imidazole
conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl,
HRP, or AP.
[0132] Ligands can be proteins, e.g., glycoproteins, or peptides,
e.g., molecules having a specific affinity for a co-ligand, or
antibodies e.g., an antibody, that binds to a specified cell type
such as a cancer cell, endothelial cell, or bone cell. Ligands may
also include hormones and hormone receptors. They can also include
non-peptidic species, such as lipids, lectins, carbohydrates,
vitamins, cofactors, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
or multivalent fucose. The ligand can be, for example, a
lipopolysaccharide, an activator of p38 MAP kinase, or an activator
of NF-.kappa.B.
[0133] The ligand can be a substance, e.g., a drug, which can
increase the uptake of the iRNA agent into the cell, for example,
by disrupting the cell's cytoskeleton, e.g., by disrupting the
cell's microtubules, microfilaments, and/or intermediate filaments.
The drug can be, for example, taxon, vincristine, vinblastine,
cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin,
swinholide A, indanocine, or myoservin.
[0134] In one ligand, the ligand is a lipid or lipid-based
molecule. Such a lipid or lipid-based molecule preferably binds a
serum protein, e.g., human serum albumin (HSA). An HSA binding
ligand allows for distribution of the conjugate to a target tissue,
e.g., a non-kidney target tissue of the body. For example, the
target tissue can be the liver, including parenchymal cells of the
liver. Other molecules that can bind HSA can also be used as
ligands. For example, neproxin or aspirin can be used. A lipid or
lipid-based ligand can (a) increase resistance to degradation of
the conjugate, (b) increase targeting or transport into a target
cell or cell membrane, and/or (c) can be used to adjust binding to
a serum protein, e.g., HSA.
[0135] A lipid based ligand can be used to modulate, e.g., control
the binding of the conjugate to a target tissue. For example, a
lipid or lipid-based ligand that binds to HSA more strongly will be
less likely to be targeted to the kidney and therefore less likely
to be cleared from the body. A lipid or lipid-based ligand that
binds to HSA less strongly can be used to target the conjugate to
the kidney.
[0136] In a preferred embodiment, the lipid based ligand binds HSA.
Preferably, it binds HSA with a sufficient affinity such that the
conjugate will be preferably distributed to a non-kidney tissue.
However, it is preferred that the affinity not be so strong that
the HSA-ligand binding cannot be reversed.
[0137] In another preferred embodiment, the lipid based ligand
binds HSA weakly or not at all, such that the conjugate will be
preferably distributed to the kidney. Other moieties that target to
kidney cells can also be used in place of or in addition to the
lipid based ligand. In another aspect, the ligand is a moiety,
e.g., a vitamin, which is taken up by a target cell, e.g., a
proliferating cell. These are particularly useful for treating
disorders characterized by unwanted cell proliferation, e.g., of
the malignant or non-malignant type, e.g., cancer cells. Exemplary
vitamins include vitamin A, E, and K. Other exemplary vitamins
include are B vitamin, e.g., folic acid, B12, riboflavin, biotin,
pyridoxal or other vitamins or nutrients taken up by cancer cells.
Also included are HSA and low density lipoprotein (LDL).
[0138] In another aspect, the ligand is a cell-permeation agent,
preferably a helical cell-permeation agent. Preferably, the agent
is amphipathic. An exemplary agent is a peptide such as tat or
antennopedia. If the agent is a peptide, it can be modified,
including a peptidylmimetic, invertomers, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. The helical
agent is preferably an alpha-helical agent, which preferably has a
lipophilic and a lipophobic phase.
[0139] The ligand can be a peptide or peptidomimetic. A
peptidomimetic (also referred to herein as an oligopeptidomimetic)
is a molecule capable of folding into a defined three-dimensional
structure similar to a natural peptide. The attachment of peptide
and peptidomimetics to iRNA agents can affect pharmacokinetic
distribution of the iRNA, such as by enhancing cellular recognition
and absorption. The peptide or peptidomimetic moiety can be about
5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40,
45, or 50 amino acids long.
[0140] A peptide or peptidomimetic can be, for example, a cell
permeation peptide, cationic peptide, amphipathic peptide, or
hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or
Phe). The peptide moiety can be a dendrimer peptide, constrained
peptide or crosslinked peptide. In another alternative, the peptide
moiety can include a hydrophobic membrane translocation sequence
(MTS). An exemplary hydrophobic MTS-containing peptide is RFGF
having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:4158).
An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID
NO:4159)) containing a hydrophobic MTS can also be a targeting
moiety. The peptide moiety can be a "delivery" peptide, which can
carry large polar molecules including peptides, oligonucleotides,
and protein across cell membranes. For example, sequences from the
HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:4160)) and the Drosophila
Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:4161)) have been
found to be capable of functioning as delivery peptides. A peptide
or peptidomimetic can be encoded by a random sequence of DNA, such
as a peptide identified from a phage-display library, or
one-bead-one-compound (OBOC) combinatorial library (Lam et al.,
Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic
tethered to a dsRNA agent via an incorporated monomer unit is a
cell targeting peptide such as an arginine-glycine-aspartic acid
(RGD)-peptide, or RGD mimic. A peptide moiety can range in length
from about 5 amino acids to about 40 amino acids. The peptide
moieties can have a structural modification, such as to increase
stability or direct conformational properties. Any of the
structural modifications described below can be utilized.
[0141] An RGD peptide moiety can be used to target a tumor cell,
such as an endothelial tumor cell or a breast cancer tumor cell
(Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide
can facilitate targeting of an dsRNA agent to tumors of a variety
of other tissues, including the lung, kidney, spleen, or liver
(Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the
RGD peptide will facilitate targeting of an iRNA agent to the
kidney. The RGD peptide can be linear or cyclic, and can be
modified, e.g., glycosylated or methylated to facilitate targeting
to specific tissues. For example, a glycosylated RGD peptide can
deliver a iRNA agent to a tumor cell expressing .alpha.v .sub.3
(Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).
[0142] A "cell permeation peptide" is capable of permeating a cell,
e.g., a microbial cell, such as a bacterial or fungal cell, or a
mammalian cell, such as a human cell. A microbial cell-permeating
peptide can be, for example, an .alpha.-helical linear peptide
(e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide
(e.g., .alpha.-defensin, .beta.-defensin or bactenecin), or a
peptide containing only one or two dominating amino acids (e.g.,
PR-39 or indolicidin). A cell permeation peptide can also include a
nuclear localization signal (NLS). For example, a cell permeation
peptide can be a bipartite amphipathic peptide, such as MPG, which
is derived from the fusion peptide domain of HIV-1 gp41 and the NLS
of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.
31:2717-2724, 2003).
[0143] Representative U.S. patents that teach the preparation of
RNA conjugates include, but are not limited to, U.S. Pat. Nos.
4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;
5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802;
5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;
4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941;
4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963;
5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469;
5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,
5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785;
5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726;
5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664;
6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of
which is herein incorporated by reference.
[0144] Chimeras
[0145] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an iRNA. The present
invention also includes iRNA compounds that are chimeric compounds.
"Chimeric" iRNA compounds or "chimeras," in the context of this
invention, are iRNA compounds, preferably dsRNAs, which contain two
or more chemically distinct regions, each made up of at least one
monomer unit, i.e., a nucleotide in the case of a dsRNA compound.
These iRNAs typically contain at least one region wherein the RNA
is modified so as to confer upon the iRNA increased resistance to
nuclease degradation, increased cellular uptake, and/or increased
binding affinity for the target nucleic acid. An additional region
of the iRNA may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of iRNA
inhibition of gene expression. Consequently, comparable results can
often be obtained with shorter iRNAs when chimeric dsRNAs are used,
compared to phosphorothioate deoxy dsRNAs hybridizing to the same
target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0146] Non-Ligand Groups
[0147] In certain instances, the RNA of an iRNA can be modified by
a non-ligand group. A number of non-ligand molecules have been
conjugated to iRNAs in order to enhance the activity, cellular
distribution or cellular uptake of the iRNA, and procedures for
performing such conjugations are available in the scientific
literature. Such non-ligand moieties have included lipid moieties,
such as cholesterol (Kubo, T. et al.. Biochem. Biophys. Res. Comm.,
2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.
Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan
et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol
(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic
chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et
al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990,
259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2d-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 RNA conjugates have been
listed above. Typical conjugation protocols involve the synthesis
of an RNAs 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 RNA still
bound to the solid support or following cleavage of the RNA, in
solution phase. Purification of the RNA conjugate by HPLC typically
affords the pure conjugate.
[0148] Delivery of iRNA
[0149] The delivery of an iRNA to a subject in need thereof can be
achieved in a number of different ways. In vivo delivery can be
performed directly by administering a composition comprising an
iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be
performed indirectly by administering one or more vectors that
encode and direct the expression of the iRNA. These alternatives
are discussed further below.
[0150] Direct Delivery
[0151] In general, any method of delivering a nucleic acid molecule
can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian
RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which
are incorporated herein by reference in their entireties). However,
there are three factors that are important to consider in order to
successfully deliver an iRNA molecule in vivo: (a) biological
stability of the delivered molecule, (2) preventing non-specific
effects, and (3) accumulation of the delivered molecule in the
target tissue. The non-specific effects of an iRNA can be minimized
by local administration, for example by direct injection or
implantation into a tissue (as a non-limiting example, a tumor) or
topically administering the preparation. Local administration to a
treatment site maximizes local concentration of the agent, limits
the exposure of the agent to systemic tissues that may otherwise be
harmed by the agent or that may degrade the agent, and permits a
lower total dose of the iRNA molecule to be administered. Several
studies have shown successful knockdown of gene products when an
iRNA is administered locally. For example, intraocular delivery of
a VEGF dsRNA by intravitreal injection in cynomolgus monkeys
(Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal
injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216)
were both shown to prevent neovascularization in an experimental
model of age-related macular degeneration. In addition, direct
intratumoral injection of a dsRNA in mice reduces tumor volume
(Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong
survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther.
14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA
interference has also shown success with local delivery to the CNS
by direct injection (Dom, G., et al. (2004) Nucleic Acids 32:e49;
Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al
(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004)
Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl.
Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J.
Neurophysiol. 93:594-602) and to the lungs by intranasal
administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484;
Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V.,
et al (2005) Nat. Med. 11:50-55). For administering an iRNA
systemically for the treatment of a disease, the RNA can be
modified or alternatively delivered using a drug delivery system;
both methods act to prevent the rapid degradation of the dsRNA by
endo- and exo-nucleases in vivo. Modification of the RNA or the
pharmaceutical carrier can also permit targeting of the iRNA
composition to the target tissue and avoid undesirable off-target
effects. iRNA molecules can be modified by chemical conjugation to
lipophilic groups such as cholesterol to enhance cellular uptake
and prevent degradation. For example, an iRNA directed against ApoB
conjugated to a lipophilic cholesterol moiety was injected
systemically into mice and resulted in knockdown of apoB mRNA in
both the liver and jejunum (Soutschek, J., et al (2004) Nature
432:173-178). Conjugation of an iRNA to an aptamer has been shown
to inhibit tumor growth and mediate tumor regression in a mouse
model of prostate cancer (McNamara, J O., et al (2006) Nat.
Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA
can be delivered using drug delivery systems such as a
nanoparticle, a dendrimer, a polymer, liposomes, or a cationic
delivery system. Positively charged cationic delivery systems
facilitate binding of an iRNA molecule (negatively charged) and
also enhance interactions at the negatively charged cell membrane
to permit efficient uptake of an iRNA by the cell. Cationic lipids,
dendrimers, or polymers can either be bound to an iRNA, or induced
to form a vesicle or micelle (see e.g., Kim SH., et al (2008)
Journal of Controlled Release 129(2):107-116) that encases an iRNA.
The formation of vesicles or micelles further prevents degradation
of the iRNA when administered systemically. Methods for making and
administering cationic-iRNA complexes are well within the abilities
of one skilled in the art (see e.g., Sorensen, D R., et al (2003)
J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer
Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens.
25:197-205, which are incorporated herein by reference in their
entirety). Some non-limiting examples of drug delivery systems
useful for systemic delivery of iRNAs include DOTAP (Sorensen, D
R., et al (2003), supra; Verma, U N., et al (2003), supra),
Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T
S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et
al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int
J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al
(2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006)
J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S.
(2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A.,
et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999)
Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a
complex with cyclodextrin for systemic administration. Methods for
administration and pharmaceutical compositions of iRNAs and
cyclodextrins can be found U.S. Pat. No. 7,427,605, which is herein
incorporated by reference in its entirety.
[0152] Vector Encoded dsRNAs
[0153] In another aspect, the dsRNAs of the invention can be
expressed from transcription units inserted into DNA or RNA vectors
(see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillem, 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). Expression can be transient (on the order of
hours to weeks) or sustained (weeks to months or longer), depending
upon the specific construct used and the target tissue or cell
type. These transgenes can be introduced as a linear construct, a
circular plasmid, or a viral vector, which can be an integrating or
non-integrating vector. 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).
[0154] The individual strand or strands of an iRNA can be
transcribed from a promoter on an expression vector. Where two
separate strands are to be expressed to generate, for example, a
dsRNA, two separate expression vectors can be co-introduced (e.g.,
by transfection or infection) into a target cell. Alternatively
each individual strand of a 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.
[0155] iRNA expression vectors are generally DNA plasmids or viral
vectors. Expression vectors compatible with eukaryotic cells,
preferably those compatible with vertebrate cells, can be used to
produce recombinant constructs for the expression of an iRNA as
described herein. Eukaryotic cell expression vectors are well known
in the art and are available from a number of commercial sources.
Typically, such vectors are provided containing convenient
restriction sites for insertion of the desired nucleic acid
segment. Delivery of iRNA 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.
[0156] iRNA expression plasmids can be 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 iRNA-mediated
knockdowns targeting different regions of a target RNA over a
period of a week or more are also contemplated by the invention.
Successful introduction of vectors 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 cells ex
vivo 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.
[0157] Viral vector systems which can be utilized with the methods
and compositions described herein include, but are not limited to,
(a) adenovirus vectors; (b) retrovirus vectors, including but not
limited to lentiviral vectors, moloney murine leukemia virus, etc.;
(c) adeno-associated virus vectors; (d) herpes simplex virus
vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g)
papilloma virus vectors; (h) picomavirus vectors; (i) pox virus
vectors such as an orthopox, e.g., vaccinia virus vectors or
avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or
gutless adenovirus. Replication-defective viruses can also be
advantageous. Different vectors will or will not become
incorporated into the cells' genome. The constructs can include
viral sequences for transfection, if desired. Alternatively, the
construct may be incorporated into vectors capable of episomal
replication, e.g. EPV and EBV vectors. Constructs for the
recombinant expression of an iRNA will generally require regulatory
elements, e.g., promoters, enhancers, etc., to ensure the
expression of the iRNA in target cells. Other aspects to consider
for vectors and constructs are further described below.
[0158] Vectors useful for the delivery of an iRNA will include
regulatory elements (promoter, enhancer, etc.) sufficient for
expression of the iRNA in the desired target cell or tissue. The
regulatory elements can be chosen to provide either constitutive or
regulated/inducible expression.
[0159] Expression of the iRNA can be precisely regulated, for
example, by using an inducible 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 dsRNA expression in cells or in mammals include, for
example, regulation by ecdysone, by estrogen, progesterone,
tetracycline, chemical inducers of dimerization, and
isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in
the art would be able to choose the appropriate regulatory/promoter
sequence based on the intended use of the iRNA transgene.
[0160] In a specific embodiment, viral vectors that contain nucleic
acid sequences encoding an iRNA can be used. For example, a
retroviral vector can be used (see Miller et al., Meth. Enzymol.
217:581-599 (1993)). These retroviral vectors contain the
components necessary for the correct packaging of the viral genome
and integration into the host cell DNA. The nucleic acid sequences
encoding an iRNA are cloned into one or more vectors, which
facilitates delivery of the nucleic acid into a patient. More
detail about retroviral vectors can be found, for example, in
Boesen et al., Biotherapy 6:291-302 (1994), which describes the use
of a retroviral vector to deliver the mdr1 gene to hematopoietic
stem cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of retroviral
vectors in gene therapy are: Clowes et al., J. Clin. Invest.
93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons
and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and
Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
Lentiviral vectors contemplated for use include, for example, the
HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557;
and 5,981,276, which are herein incorporated by reference.
[0161] Adenoviruses are also contemplated for use in delivery of
iRNAs. Adenoviruses are especially attractive vehicles, e.g., for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). A suitable AV vector for expressing an iRNA featured in 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.
[0162] Use of Adeno-associated virus (AAV) vectors is also
contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300
(1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can
be 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.
Suitable AAV vectors for expressing the dsRNA featured in 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. Nos. 5,252,479; 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.
[0163] Another preferred viral vector is a pox virus such as a
vaccinia virus, for example an attenuated vaccinia such as Modified
Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary
pox.
[0164] 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. For example, lentiviral vectors can be pseudotyped
with surface proteins from vesicular stomatitis virus (VSV),
rabies, Ebola, Mokola, and the like. AAV vectors can be made to
target different cells by engineering the vectors to express
different capsid protein serotypes; see, e.g., Rabinowitz J E et
al. (2002), J Virol 76:791-801, the entire disclosure of which is
herein incorporated by reference.
[0165] The pharmaceutical preparation of a vector can include the
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.
[0166] Pharmaceutical Compositions Containing iRNA
[0167] In one embodiment, the invention provides pharmaceutical
compositions containing a dual targeting siRNA agent, as described
herein, and a pharmaceutically acceptable carrier. The
pharmaceutical composition containing the siRNA is useful for
treating a disease or disorder associated with the expression or
activity of a target gene, such as pathological processes mediated
by PCSK9 expression. Such pharmaceutical compositions are
formulated based on the mode of delivery. One example is
compositions that are formulated for systemic administration via
parenteral delivery, e.g., by intravenous (IV) delivery. Another
example is compositions that are formulated for direct delivery
into the brain parenchyma, e.g., by infusion into the brain, such
as by continuous pump infusion.
[0168] The pharmaceutical compositions featured herein are
administered in dosages sufficient to inhibit expression of the
target genes. In general, a suitable dose of siRNA 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, 0.02 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, 0.1
mg/kg, 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, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9
mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8
mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg,
15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21
mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg,
28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34
mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg,
41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47
mg/kg, 48 mg/kg, 49 mg/kg, or 50 mg/kg per single dose.
[0169] The pharmaceutical composition may be administered once
daily, or the iRNA may be administered as two, three, or more
sub-doses at appropriate intervals throughout the day or even using
continuous infusion or delivery through a controlled release
formulation. In that case, the iRNA 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 iRNA 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.
[0170] The effect of a single dose of siRNA on PCSK9 levels can be
long lasting, such that subsequent doses are administered at not
more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or
4 week intervals.
[0171] 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
iRNAs 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.
[0172] 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
can be used for in vivo testing of iRNA, as well as for determining
a therapeutically effective dose. A suitable mouse model is, for
example, a mouse containing a transgene expressing human PCSK9.
[0173] The present invention also includes pharmaceutical
compositions and formulations that include the iRNA compounds
featured in the invention. The pharmaceutical compositions of the
present invention may be administered in a number of ways depending
upon whether local or systemic treatment is desired and upon the
area to be treated. Administration may be topical (e.g., by a
transdermal patch), pulmonary, e.g., by inhalation or insufflation
of powders or aerosols, including by nebulizer; intratracheal,
intranasal, epidermal and transdermal, oral or parenteral.
Parenteral administration includes intravenous, intraarterial,
subcutaneous, intraperitoneal or intramuscular injection or
infusion; subdermal, e.g., via an implanted device; or
intracranial, e.g., by intraparenchymal, intrathecal or
intraventricular, administration.
[0174] The iRNA can be delivered in a manner to target a particular
tissue, such as the liver (e.g., the hepatocytes of the liver).
[0175] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful. Suitable
topical formulations include those in which the iRNAs featured in
the invention are in admixture with a topical delivery agent such
as lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Suitable lipids and liposomes
include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl
choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and
cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, iRNAs may be complexed to lipids, in particular to
cationic lipids. Suitable fatty acids and esters include but are
not limited to arachidonic acid, oleic acid, eicosanoic acid,
lauric acid, caprylic acid, capric acid, myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or
a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,
diglyceride or pharmaceutically acceptable salt thereof. Topical
formulations are described in detail in U.S. Pat. No. 6,747,014,
which is incorporated herein by reference.
[0176] Liposomal Formulations
[0177] 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.
[0178] 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.
[0179] In order to traverse 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.
[0180] Further advantages of liposomes include; liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
[0181] 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.
[0182] 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.
[0183] 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
[0184] 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).
[0185] 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).
[0186] 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.
[0187] 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).
[0188] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome.TM. I
(glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether)
and Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
[0189] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.M1, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765).
[0190] Various liposomes comprising one or more glycolipids are
known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci.,
1987, 507, 64) reported the ability of monosialoganglioside
G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to
improve blood half-lives of liposomes. These findings were
expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A.,
1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to
Allen et al., disclose liposomes comprising (1) sphingomyelin and
(2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester.
U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes
comprising sphingomyelin. Liposomes comprising
1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499
(Lim et al).
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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).
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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).
[0200] Nucleic Acid Lipid Particles
[0201] In one embodiment, a dual targeting siRNA agent featured in
the invention is fully encapsulated in the lipid formulation, e.g.,
to form a nucleic acid-lipid particle, e.g., a SPLP, pSPLP, or
SNALP. As used herein, the term "SNALP" refers to a stable nucleic
acid-lipid particle, including SPLP. As used herein, the term
"SPLP" refers to a nucleic acid-lipid particle comprising plasmid
DNA encapsulated within a lipid vesicle. Nucleic acid-lipid
particles, e.g., SNALPs, typically contain a cationic lipid, a
non-cationic lipid, and a lipid that prevents aggregation of the
particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs 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). SPLPs include "pSPLP",
which include an encapsulated condensing agent-nucleic acid complex
as set forth in PCT Publication No. WO 00/03683.
[0202] 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. For example, the mean diameter of the particles can be
about 50 nm, 55 nm, 60 nn, 65 nn, 70 nm, 75 nm, 80 nm, 85 nm, 90
nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm,
140 nm, 145 nm, or 150 nm.
[0203] 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.
[0204] 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. The lipid to dsRNA ratio can be
about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 113:1, 14:1, 15:1,
16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1,
27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1,
38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1,
49:1, or 50:1.
[0205] The nucleic acid lipid particles 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,
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC),
(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tet-
rahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100),
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (MC3),
1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)ami-
no)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1,
e.g., C12-200), or a mixture thereof. The cationic lipid may
comprise from about 10 mol % to about 70 mol % or about 40 mol % of
the total lipid present in the particle. The cationic lipid may
comprise 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol
%, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70
mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol % of the
total lipid present in the particle. The cationic lipid may
comprise 57.1 mol % or 57.5 mol % of the total lipid present in the
particle.
[0206] In one embodiment, the compound
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC) can be
used to prepare lipid-siRNA nanoparticles. Synthesis of
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane 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 embodiment, the lipid-siRNA particle includes 40% 2,
2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%
Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of
63.0.+-.20 nm and a 0.027 siRNA/Lipid Ratio.
[0208] The nucleic acid lipid particle generally includes a
non-cationic lipid. The non-cationic lipid may be an anionic lipid
or a neutral lipid including, but not limited to,
distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or
a mixture thereof.
[0209] The non-cationic lipid may be from about 5 mol % to about 90
mol %, about 10 mol %, or about 58 mol % if cholesterol is
included, of the total lipid present in the particle. The
non-cationic lipid may be about 5 mol %, 6 mol %, 7 mol %, 7.5 mol
%, 7.7 mol %, 8 mol %. 9 mol %, 10 mol %, 11 mol %, 12 mol %, 13
mol %, 14 mol %, 15 mol %, 16 mol %, 17 mol %, 18 mol %, 19 mol %,
20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol
%, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85
mol %, 90 mol %, or 95 mol %.
[0210] The nucleic acid lipid particle generally includes a
conjugated lipid. 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 (C].sub.8). The conjugated
lipid can be PEG-DMG (PEG-didimyristoyl glycerol (C14-PEG, or
PEG-C14) (PEG with avg mol wt of 2000); PEG-DSG (PEG-distyryl
glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000); or
PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg
mol wt of 2000).
[0211] The conjugated lipid that prevents aggregation of particles
may be from 0 mol % to about 20 mol % or about 1.0, 2.0, 3.0, 4.0,
5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0
17.0, 18, 19.0 or 20.0 mol % of the total lipid present in the
particle.
[0212] In some embodiments, the nucleic acid-lipid particle further
includes cholesterol at, e.g., about 10 mol % to about 60 mol % or
about 48 mol % of the total lipid present in the particle. For
example, the nucleic acid-lipid particle further includes
cholesterol at about 5 mol %, 10 mol %, 15 mol %, 20 mol %, 25 mol
%, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or
60 mol %. The nucleic acid-lipid particle can include cholesterol
at about 31.5 mol %, 34.4 mol %, 35 mol %, 38.5 mol %, or 40 mol %
of the total lipid present in the particle.
[0213] LNP01
[0214] In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see
U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008,
which is herein incorporated by reference), Cholesterol
(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be
used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles).
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 dsRNA (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-dsRNA 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.
##STR00002##
[0215] LNP01 formulations are described, e.g., in International
Application Publication No. WO 2008/042973, which is hereby
incorporated by reference.
[0216] Exemplary Nucleic Acid Lipid Particles
[0217] Additional exemplary lipid-dsRNA formulations arm as
follows:
TABLE-US-00004 TABLE A cationic lipid/non-cationic
lipid/cholesterol/PEG-lipid conjugate Cationic Mol % ratios Lipid
Lipid:siRNA ratio SNALP DLinDMA DLinDMA/DPPC/Cholesterol/PEG-cDMA
(57.1/7.1/34.4/1.4) lipid:siRNA ~ 7:1 S-XTC XTC
XTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4 lipid:siRNA ~ 7:1
LNP05 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5
lipid:siRNA ~ 6:1 LNP06 XTC XTC/DSPC/Cholesterol/PEG-DMG
57.5/7.5/31.5/3.5 lipid:siRNA ~ 11:1 LNP07 XTC
XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~ 6:1 LNP08
XTC XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~ 11:1
LNP09 XTC XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA
10:1 LNP10 ALN100 ALN100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5
Lipid:siRNA 10:1 LNP11 MC3 MC-3/DSPC/Cholesterol/PEG-DMG
50/10/38.5/1.5 Lipid:siRNA 10:1 LNP12 C12-200
C12-200/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1
LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1
LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15
MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5
Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5
Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5
Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5
Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5
Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5
Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG
50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG
50/10/38.5/1.5 Lipid:siRNA: 10:1
[0218] SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane
(DLinDMA)) comprising formulations are described in International
Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby
incorporated by reference.
[0219] XTC comprising formulations are described, e.g., in U.S.
Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and
International patent application no. PCT/US10/22614, filed Jan. 29,
2010, which are hereby incorporated by reference.
[0220] 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.
[0221] ALN100, i.e., 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.
[0222] C12-200, i.e., Tech G1 comprising formulations are described
in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009, which
is hereby incorporated by reference.
[0223] Synthesis of Cationic Lipids.
[0224] 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.
[0225] "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.
[0226] "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.
[0227] "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.
[0228] "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.
[0229] "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
quaternized, 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.
[0230] 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, --ORx, --NRxRy, --NRxC(.dbd.O)Ry,
--NRxSO2Ry, --C(.dbd.O)Rx, --C(.dbd.O)ORx, --C(.dbd.O)NRxRy,
--SOnRx and --SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry 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,
--ORx, heterocycle, --NRxRy, --NRxC(.dbd.O)Ry, --NRxSO2Ry,
--C(.dbd.O)Rx, --C(.dbd.O)ORx, --C(.dbd.O)NRxRy, --SOnRx and
--SOnNRxRy.
[0231] "Halogen" means fluoro, chloro, bromo and iodo.
[0232] 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.
[0233] Synthesis of Formula A
[0234] In one embodiments, nucleic acid-lipid particles of the
invention are formulated using a cationic lipid of formula A; XTC
is a cationic lipid of formula A:
##STR00003##
[0235] 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.
[0236] 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.
##STR00004##
[0237] 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 X is anion counter ion selected from halogen, hydroxide,
phosphate, sulfate, or the like.
##STR00005##
[0238] 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.
[0239] Synthesis of MC3
[0240] 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).
[0241] Synthesis of ALNY-100
[0242] Synthesis of ketal 519 [ALNY-100] was performed using the
following scheme 3:
##STR00006##
[0243] Synthesis of 515:
[0244] 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 0C 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 0C 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).
[0245] Synthesis of 516:
[0246] 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 0C 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
NaHCO.sub.3solution (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%).
[0247] Synthesis of 517A and 517B:
[0248] 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
NaHCO.sub.3 (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
[0249] 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.
[0250] Synthesis of 518:
[0251] 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%.
[0252] General Procedure for the Synthesis of Compound 519:
[0253] 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.degree. 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.
[0254] General Synthesis of Nucleic Add Lipid Particles
[0255] 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 dsRNA concentration in the formulation, as well as the
entrapped fraction, is estimated using a dye exclusion assay. A
sample of the formulated dsRNA 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 dsRNA 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" dsRNA content (as measured by the signal in
the absence of surfactant) from the total dsRNA content. Percent
entrapped dsRNA is typically >85%. For SNALP formulation, 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.
[0256] Other Formulations
[0257] 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
acylcamitine, 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, polyomithine, 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, US
Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which
is incorporated herein by reference.
[0258] 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.
[0259] 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. Particularly preferred are
formulations that target the liver when treating hepatic disorders
such as hepatic carcinoma.
[0260] 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.
[0261] 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.
[0262] Additional Formulations
[0263] Emulsions
[0264] 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 (see e.g., Ansel's
Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV.,
Popovich NG., and Ansel HC., 2004, Lippincott Williams &
Wilkins (8th ed.), New York, N.Y.; 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.
[0265] 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 (see
e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery
Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott
Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0266] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (see e.g., Ansel's
Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV.,
Popovich NG., and Ansel HC., 2004, Lippincott Williams &
Wilkins (8th ed.), New York, N.Y.; 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 (see
e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery
Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott
Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
[0267] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0268] A large variety 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).
[0269] 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.
[0270] 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.
[0271] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (see e.g., Ansel's Pharmaceutical
Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG.,
and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.),
New York, N.Y.; 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 (see
e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery
Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott
Williams & Wilkins (8th ed.), New York, N.Y.; 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.
[0272] In one embodiment of the present invention, the compositions
of iRNAs 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 (see e.g., Ansel's
Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV.,
Popovich NG., and Ansel HC., 2004, Lippincott Williams &
Wilkins (8th ed.), New York, N.Y.; 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).
[0273] 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 (see
e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery
Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott
Williams & Wilkins (8th ed.), New York, N.Y.; 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.
[0274] 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.
[0275] 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 (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802;
7,157,099; 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 (see e.g., U.S.
Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099;
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 iRNAs. 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 iRNAs and nucleic acids from the gastrointestinal
tract, as well as improve the local cellular uptake of iRNAs and
nucleic acids.
[0276] 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
iRNAs 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.
[0277] Penetration Enhancers
[0278] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly iRNAs, 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.
[0279] 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 (see
e.g., Malmsten, M. Surfactants and polymers in drug delivery,
Informa Health Care, New York, N.Y., 2002; 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.
[0280] 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 iRNAs 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) (see e.g., Malmsten, M.
Surfactants and polymers in drug delivery, Informa Health Care, New
York, N.Y., 2002; 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).
[0281] 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, acylcamitines, acylcholines, C1-20
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.) (see e.g.,
Touitou, E., et al. Enhancement in Drug Delivery, CRC Press,
Danvers, Mass., 2006; 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).
[0282] Bile salts: The physiological role of bile includes the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug
delivery, Informa Health Care, New York, N.Y., 2002; 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) (see e.g., Malmsten, M.
Surfactants and polymers in drug delivery, Informa Health Care, New
York, N.Y., 2002; 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).
[0283] 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 iRNAs 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 (enaminesxsee e.g., Katdare, A. et al., Excipient
development for pharmaceutical, biotechnology, and drug delivery,
CRC Press, Danvers, Mass., 2006; 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).
[0284] 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
iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical
Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This
class of penetration enhancers include, 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).
[0285] Agents that enhance uptake of iRNAs 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. Examples of
commercially available transfection reagents include, for example
Lipofectamine.TM. (Invitrogen; Carlsbad, Calif.), Lipofectamine
2000.TM. (Invitrogen; Carlsbad, Calif.), 293Fectin.TM. (Invitrogen;
Carlsbad, Calif.), Cellfectin.TM. (Invitrogen; Carlsbad, Calif.),
DMRIE-CT.TM. (Invitrogen; Carlsbad, Calif.), FreeStyle.TM. MAX
(Invitrogen; Carlsbad, Calif.), Lipofectamine.TM. 2000 CD
(Invitrogen; Carlsbad, Calif.), Lipofectamine.TM. (Invitrogen;
Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.),
Oligofectamine.TM. (Invitrogen; Carlsbad, Calif.), Optifect.TM.
(Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent
(Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal
Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER
Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or
Fugene (Grenzacherstrasse, Switzerland), Transfectam.RTM. Reagent
(Promega; Madison, Wis.), TransFast.TM. Transfection Reagent
(Promega; Madison, Wis.), Tfx.TM.-20 Reagent (Promega; Madison,
Wis.), Tfx.TM.-50 Reagent (Promega; Madison, Wis.), DreamFect.TM.
(OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences;
Marseille, France), TransPass.sup.a D1 Transfection Reagent (New
England Biolabs; Ipswich, Mass., USA), LyoVec.TM./LipoGen.TM.
(Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent
(Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection
Reagent (Genlantis; San Diego, Calif., USA), GenePORTER
Transfection reagent (Genlantis; San Diego, Calif., USA),
GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif.,
USA), Cytofectin Transfection Reagent (Genlantis; San Diego,
Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San
Diego, Calif., USA), TroganPORTER.TM. transfection Reagent
(Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton,
Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR
(B-Bridge International; Mountain View, Calif., USA), SureFECTOR
(B-Bridge International; Mountain View, Calif., USA), or HiFect.TM.
(B-Bridge International, Mountain View, Calif., USA), among
others.
[0286] 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.
[0287] Carriers
[0288] 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
coadministration 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 extracirculatory 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 coadministered 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.
[0289] Excipients
[0290] 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).
[0291] 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.
[0292] 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.
[0293] 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.
[0294] Other Components
[0295] 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.
[0296] Aqueous suspensions may contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0297] In some embodiments, pharmaceutical compositions featured in
the invention include (a) one or more iRNA compounds and (b) one or
more biologic agents which function by a non-RNAi mechanism.
Examples of such biologics include, biologics that target one or
more of PD-1, PD-L1, or B7-H1 (CD80) (e.g., monoclonal antibodies
against PD-1, PD-L1, or B7-H1), or one or more recombinant
cytokines (e.g., IL6, IFN-.gamma., and TNF).
[0298] 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.
[0299] 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.
[0300] In addition to their administration, as discussed above, the
dual targeting siRNAs featured in the invention can be administered
in combination with other known agents effective in treatment of
pathological processes mediated by PCSK9 expression. In any event,
the administering physician can adjust the amount and timing of
iRNA administration on the basis of results observed using standard
measures of efficacy known in the art or described herein.
[0301] Method Using Dual Targeting siRNAs
[0302] In one aspect, the invention provides use of a dual
targeting siRNA agent 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. In some embodiments, PCSK9 expression is
decreased for an extended duration, e.g., at least one week, two
weeks, three weeks, or four weeks or longer. For example, in
certain instances, expression of the PCSK9 gene is suppressed by at
least about 5%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, or 50% by
administration of a dual targeting siRNA agent described herein. In
some embodiments, the PCSK9 gene is suppressed by at least about
60%, 70%, or 80% by administration of the dual targeting siRNA
agent. In some embodiments, the is suppressed by at least about
85%, 90%, or 95% by administration of the double-stranded
oligonucleotide.
[0303] 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
[0304] Therefore, the invention also relates to the use of a dual
targeting siRNA agent for the treatment of a PCSK9-mediated
disorder or disease. For example, a dual targeting siRNA agent is
used for treatment of a hyperlipidemia.
[0305] The effect of the decreased 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.
[0306] The method includes administering a dual targeting siRNA
agent to the subject 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.
[0307] The method includes administering a dual targeting siRNA
agent, 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.
[0308] In one embodiment, doses of dual targeting siRNA agent 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.
[0309] 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 mVdL, 300 mg/dL, or 400 mg/dL.
[0310] dual targeting siRNA agent
[0311] For example, a subject can be administered a therapeutic
amount of dual targeting siRNA agent, such as 0.5 mg/kg, 1.0 mg/kg,
1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dual targeting siRNA
agent can be administered by intravenous infusion over a period of
time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or
25 minute period. The administration is repeated, for example, on a
regular basis, such as biweekly (i.e., every two weeks) for one
month, two months, three months, four months or longer. After an
initial treatment regimen, the treatments can be administered on a
less frequent basis. For example, after administration biweekly for
three months, administration can be repeated once per month, for
six months or a year or longer. Administration of the dual
targeting siRNA agent can reduce PCSK9 levels, e.g., in a cell,
tissue, blood, urine or other compartment of the patient by at
least 10%, at least 15%, at least 20%, at least 25%., at least 30%,
at least 40%., at least 50%, at least 60%, at least 70%, at least
80% or at least 90% or more.
[0312] Before administration of a full dose of the iRNA, patients
can be administered a smaller dose, such as a 5% infusion reaction,
and monitored for adverse effects, such as an allergic reaction, or
for elevated lipid levels or blood pressure. In another example,
the patient can be monitored for unwanted immunostimulatory
effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)
levels.
[0313] A treatment or preventive effect is evident when there is a
statistically significant improvement in one or more parameters of
disease status, or by a failure to worsen or to develop symptoms
where they would otherwise be anticipated. As an example, a
favorable change of at least 10% in a measurable parameter of
disease, and preferably at least 20%, 30%, 40%, 50% or more can be
indicative of effective treatment. Efficacy for a given dual
targeting siRNA agent drug or formulation of that drug can also be
judged using an experimental animal model for the given disease as
known in the art. When using an experimental animal model, efficacy
of treatment is evidenced when a statistically significant
reduction in a marker or symptom is observed.
[0314] Additional Agents
[0315] In further embodiments, administration of a dual targeting
siRNA agent is administered in combination an additional
therapeutic agent. The dual targeting siRNA agent and an additional
therapeutic agent can be administered in combination in the same
composition, e.g., parenterally, or the additional therapeutic
agent can be administered as part of a separate composition or by
another method described herein.
[0316] Examples of additional therapeutic agents include those
known to treat an agent known to treat a lipid disorders, such as
hypercholesterolemia, atherosclerosis or dyslipidemia. For example,
a dual targeting siRNA agent 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
(Sanof{dot over ()}-Synthelabo/Bristol-Myers Squibb's Plavix), and
ticlopidine (e.g., Sanof{dot over ()}-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 Pharmaceuticals/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-Al (ABCAl) (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 Pharmaceuticals)
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).
[0317] In one embodiment, a dual targeting siRNA agent is
administered in combination with an ezetimibe/simvastatin
combination (e.g., Vytorin.RTM. (Merck/Schering-Plough
Pharmaceuticals)).
[0318] In one embodiment, the dual targeting siRNA agent is
administered to the patient, and then the additional therapeutic
agent is administered to the patient (or vice versa). In another
embodiment, the dual targeting siRNA agent and the additional
therapeutic agent are administered at the same time.
[0319] 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 dual targeting siRNA agent described herein. The
method includes, optionally, providing the end user with one or
more doses of the dual targeting siRNA agent, and instructing the
end user to administer the dual targeting siRNA agent on a regimen
described herein, thereby instructing the end user.
[0320] Identification of Patients
[0321] In one 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 dual targeting siRNA agent in an
amount sufficient to lower the patient's LDL levels or ApoB levels,
e.g., without substantially lowering HDL levels.
[0322] Genetic predisposition plays a role in the development of
target gene associated diseases, e.g., hyperlipidemia. Therefore, a
patient in need of a dual targeting siRNA agent can be identified
by taking a family history, or, for example, screening for one or
more genetic markers or variants. A healthcare provider, such as a
doctor, nurse, or family member, can take a family history before
prescribing or administering a dual targeting siRNA agent. For
example, a DNA test may also be performed on the patient to
identify a mutation in the PCSK9 gene, before a PCSK9 dsRNA is
administered to the patient.
[0323] 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 iRNAs and
methods featured in the invention, suitable methods and materials
are described below. All publications, patent applications,
patents, and other references mentioned herein am incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control. In addition,
the materials, methods, and examples am illustrative only and not
intended to be limiting.
EXAMPLES
Example 1. iRNA Synthesis
[0324] Source of Reagents
[0325] 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.
[0326] Oligonucleotide Synthesis.
[0327] All oligonucleotides are synthesized on an AKTAoligopilot
synthesizer. Commercially available controlled pore glass solid
support (dT-CPG, 500{acute over (.ANG.)}, Prime Synthesis) and RNA
phosphoramidites with standard protecting groups,
5'-O-dimethoxytrityl
N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O--N,N'-diisopropyl-2-cya-
noethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-O--N,N-
'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethylsilyl-guanosine-3'-O--
-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O--N,N'-diisoprop-
yl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies)
were used for the oligonucleotide synthesis. The 2'-F
phosphoramidites,
5'-O-dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3'-O--N,N'-diisopropyl-2-
-cyanoethyl-phosphoramidite and
5'-O-dimethoxytrityl-2'-fluro-uridine-3'-O--N,N'-diisopropyl-2-cyanoethyl-
-phosphoramidite are purchased from (Promega). All phosphoramidites
are used at a concentration of 0.2M in acetonitrile (CH.sub.3CN)
except for guanosine which is used at 0.2M concentration in 10%
THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The
activator is 5-ethyl thiotetrazole (0.75M, American International
Chemicals); for the PO-oxidation iodine/water/pyridine is used and
for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is
used.
[0328] 3'-ligand conjugated strands are synthesized using solid
support containing the corresponding ligand. For example, the
introduction of cholesterol unit in the sequence is performed from
a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is
tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage
to obtain a hydroxyprolinol-cholesterol moiety. 5'-end Cy-3 and
Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the
corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from
Biosearch Technologies. Conjugation of ligands to 5'-end and or
internal position is achieved by using appropriately protected
ligand-phosphoramidite building block. An extended 15 min coupling
of 0.1 M solution of phosphoramidite in anhydrous CH.sub.3CN in the
presence of 5-(ethylthio)-1H-tetrazole activator to a
solid-support-bound oligonucleotide. Oxidation of the
internucleotide phosphite to the phosphate is carried out using
standard iodine-water as reported (1) or by treatment with
tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min
oxidation wait time conjugated oligonucleotide. Phosphorothioate is
introduced by the oxidation of phosphite to phosphorothioate by
using a sulfur transfer reagent such as DDTT (purchased from AM
Chemicals), PADS and or Beaucage reagent. The cholesterol
phosphoramidite is synthesized in house and used at a concentration
of 0.1 M in dichloromethane. Coupling time for the cholesterol
phosphoramidite is 16 minutes.
[0329] Deprotection I (Nucleobase Deprotection)
[0330] After completion of synthesis, the support is transferred to
a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from
the support with simultaneous deprotection of base and phosphate
groups with 80 mL of a mixture of ethanolic ammonia [ammonia:
ethanol (3:1)] for 6.5 h at 55.degree. C. The bottle is cooled
briefly on ice and then the ethanolic ammonia mixture is filtered
into a new 250-mL bottle. The CPG is washed with 2.times.40 mL
portions of ethanol/water (1:1 v/v). The volume of the mixture is
then reduced to .about.30 mL by roto-vap. The mixture is then
frozen on dry ice and dried under vacuum on a speed vac.
[0331] Deprotection II (Removal of 2'-TBDMS Group)
[0332] The dried residue is resuspended in 26 mL of triethylamine,
triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO
(3:4:6) and heated at 60.degree. C. for 90 minutes to remove the
tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The
reaction is then quenched with 50 mL of 20 mM sodium acetate and
the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer
until purification.
[0333] Analysis
[0334] The oligonucleotides are analyzed by high-performance liquid
chromatography (HPLC) prior to purification and selection of buffer
and column depends on nature of the sequence and or conjugated
ligand.
[0335] HPLC Purification
[0336] The ligand-conjugated oligonucleotides are purified by
reverse-phase preparative HPLC. The unconjugated oligonucleotides
are purified by anion-exchange HPLC on a TSK gel column packed in
house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10%
CH.sub.3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10%
CH.sub.3CN, 1M NaBr (buffer B). Fractions containing full-length
oligonucleotides are pooled, desalted, and lyophilized.
Approximately 0.15 OD of desalted oligonucleotides are diluted in
water to 150 .mu.L and then pipetted into special vials for CGE and
LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.
[0337] iRNA Preparation
[0338] For the general preparation of iRNA, equimolar amounts of
sense and antisense strand are heated in 1.times.PBS at 95.degree.
C. for 5 min and slowly cooled to room temperature. Integrity of
the duplex is confirmed by HPLC analysis.
[0339] Nucleic acid sequences are represented below using standard
nomenclature, and specifically the abbreviations of Table B.
TABLE-US-00005 TABLE B Abbreviations of nucleotide monomers used in
nucleic acid sequence representation. It will be understood that
these monomers, when present in an oligonucleotide, are mutually
linked by 5'-3'-phosphodiester bonds. Abbreviation Nucleotide (s) A
adenosine C cytidine G guanosine U uridine N any nucleotide (G, A,
C, T or U) a 2'-O-methyladenosine c 2'-O-methylcytidine g
2'-O-methylguanosine u 2'-O-methyluridine dT , T 2'-deoxythymidine
s phosphorothioate linkage
Example 2. PCSK9 siRNA Design, Synthesis, and Screening
[0340] A description of the design, synthesis, and assays using
PCSK9 siRNA can be found in detail in U.S. patent application Ser.
No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251)
and International Patent Application No. PCT/US2007/068655 filed
May 10, 2007 (published as WO 2007/134161) and in U.S. patent
application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US
2010/0010066) and International Patent Application No.
PCT/US2009/032743 filed Jan. 30, 2009 (published as WO
2009/134487). All are incorporated by reference in their entirety
for all purposes.
[0341] The sequences of siRNA targeting a PCSK9 gene are described
in Table 1 and Table 2 above, and Tables 4-8 below.
Example 3. XBP-1 siRNA Design, Synthesis, and Screening
[0342] A description of the design, synthesis, and assays using
XBP-1 siRNA can be found in detail in U.S. patent application Ser.
No. 12/425,811 filed on Apr. 17, 2009 and published as US
2009-0275638. This application is incorporated by reference in its
entirety for all purposes. The sequences of siRNA targeting a XBP-1
gene are described in Table 3 above, and Tables 9-13 below.
Example 4. A Dual Targeting siRNA Agent
[0343] A dual targeting siRNA agent was synthesized. The sense and
antisense strands for AD-10792 (target gene is PCSK9, see Table 2))
and AD-18038 (target gene is XBP-1, see Table 3) were synthesized.
The two sense strands were covalently bound using a disulfide
linker "Q51" with the structure shown below.
##STR00007##
[0344] The resulting dual sense strand was hybridized to the
corresponding antisense strands to create a 42 mer dual targeting
siRNA agent "AD-23426" (SEQ ID NOS 4162-4165, respectively, in
order of appearance):
TABLE-US-00006 GccuGGAGuuuAuucGGAAdTsdTQ51cAcccuGAAuucAuuGucudTsdT
dTsdTCGGAcCUCAAAuAAGCCUU dTsdTGUGGGAcUUAAGUAAcAGA
Example 5. Inhibition of PCSK9 and Xbp-1 mRNA Levels by the
PCSK9-Xbp1 Dual Targeting siRNA in Primary Mouse Hepatocytes
[0345] Primary mouse hepatocytes were transfected with dual
targeting AD-23426 or individual siRNAs (AD-10792 and AD-18038) in
lipofectamine 2000 (Invitrogen protocol). 48 hours after
transfection cells were harvested and lysed. PCSK9, Xbp-1 and GAPDH
transcripts were measured via bDNA in cell lysates prepared
according to manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to
GAPDH ratios were normalized to control (luciferase) and
graphed.
[0346] As shown in FIG. 1, the dual targeting siRNA was at least as
effective at inhibiting their corresponding target gene as the
single siRNAs.
Example 6. Inhibition of PCSK9 and Xbp-1 mRNA Levels and Reduction
of Total Serum Cholesterol by the PCSK9-Xbp1 Dual Targeting siRNA
in Mice
[0347] The dual targeting AD-23426 was formulated in an LNP09
formulation: XTC/DSPC/Cholesterol/PEG-DMG in a % mol ratio of
50/10/38.5/1.5 with a lipid:siRNA ratio of about 10:1. The
LNP09-AD-23426 was administered by tail vein injection into C57B6
mice at 6.0 mg/kg, 2.0 mg/kg and 0.6 mg/kg. LNP09 formulated single
siRNAs (AD-10792 and AD-18038) were administered each at 3.0 mg/kg,
1.0 mg/kg and 0.3 mg/kg. Livers and plasma were harvested 72 hours
post-injection (5 animals per group).
[0348] PCSK9, Xbp-1 and GAPDH transcript levels were measured via
bDNA in livers prepared according to the manufacturer's protocol.
PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control
(luciferase) and graphed. The results are shown in FIG. 2.
[0349] Total cholesterol was measure in serum according to
manufacturer's instructions using a cholesterol kit from WAKO
Tex.
[0350] The results demonstrate that the dual targeting siRNAs were
at least as effective at inhibiting their corresponding target as
single siRNAs in vivo. The results also show that the dual
targeting construct has an additive effect compared to the single
siRNAs at reducing total serum cholesterol.
Example 7: No Induction of IFN-.alpha. and TNF-.alpha. in
HuPBMC
[0351] The effect of a dual targeting siRNA, AD-23426, on
IFN-.alpha. and TNF-.alpha. in human PBMC was investigated.
[0352] Whole Blood anti-coagulated with Sodium Heparin was obtained
from healthy donors at Research Blood Components, Inc (Boston,
Mass.). Peripheral blood mononuclear cells (PBMC) were isolated by
standard Ficoll-Hypaque density centrifugation. Isolated PBMC were
seeded at 1.times.10.sup.5cells/well in 96 well plates and cultured
in RPMI 1640 GlutaMax Medium (Invitrogen) supplemented with 10%
heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic
(Invitrogen). siRNAs were transfected using DOTAP Transfection
Reagent (Roche Applied Science). DOTAP was first diluted in
Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal
volume of Opti-MEM containing the siRNA. siRNA/transfection reagent
complexes were incubated for 15 minutes at room temperature prior
to being added to PBMC. siRNAs were transfected at final
concentrations of 266 nM, 133 nM or 67 nM using 16 .mu.g/ml, 8
.mu.g/ml or 4 .mu.g/ml DOTAP, respectively. The ratio of siRNA to
DOTAP is 16.5 .mu.mol/.mu.g. Transfected PBMC were incubated at
37.degree. C., 5% CO.sub.2 for 24 hrs after which supernatants were
harvested and stored at -80.degree. C. until analysis. Quantitative
cytokine analysis was done using commercially available Instant
ELISA Kits for IFN-.alpha., (BMS216INST) and TNF-.alpha.
(BMS223INST); both from Bender MedSystems (Vienna, Austria).
[0353] LNP09 and DOTAP formulated siRNAs were administered. Control
siRNAs were AD-1730, AD-1955, AD-6248, AD-18889, AD-5048, and
AD-18221. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.
[0354] The results are shown in FIG. 4. AD-23426 did not induce
production of IFN-.alpha. and TNF-.alpha., similar to the result
obtained with the single target gene siRNAs. As expected,
unmodified siRNAs (AD-5048 and AD-18889) induced production of both
IFN-.alpha. and TNF-.alpha.. These results demonstrate that a dual
targeting siRNA does not induce an immune response.
Example 8. Reduction of Total Serum Cholesterol with PCSK9-Xbp1
Dual Targeting siRNA Humans
[0355] A human subject is treated with a pharmaceutical
composition, e.g., a nucleic acid-lipid particle having a dual
targeting siRNA agent.
[0356] At time zero, a suitable first dose of the pharmaceutical
composition is subcutaneously administered to the subject. The
composition is formulated as described herein. After a period of
time, the subject's condition is evaluated, e.g., by measurement of
total serum cholesterol. 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.
[0357] After treatment, the subject's condition is compared to the
condition existing prior to the treatment, or relative to the
condition of a similarly afflicted but untreated subject.
[0358] 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-00007 TABLE 4 Sequences of siRNA targeted to PCSK9 SEQ
Antisense SEQ Sense strand ID strand ID *Target (5'-3').sup.1 NO:
(5'-3').sup.1 NO: Duplex # 2-20 AGCGACGUCGAGGCGCUCATT 1
UGAGCGCCUCGAC 2 AD-15220 GUCGCUTT 15-33 CGCUCAUGGUUGCAGGCGGTT 3
CCGCCUGCAACCA 4 AD-15275 UGAGCGTT 16-34 GCUCAUGGUUGCAGGCGGGTT 5
CCCGCCUGCAACC 6 AD-15301 AUGAGCTT 30-48 GCGGGCGCCGCCGUUCAGUTT 7
ACUGAACGGCGGC 8 AD-15276 GCCCGCTT 31-49 CGGGCGCCGCCGUUCAGUUTT 9
AACUGAACGGCGG 10 AD-15302 CGCCCGTT 32-50 GGGCGCCGCCGUUCAGUUCTT 11
GAACUGAACGGCG 12 AD-15303 GCGCCCTT 40-58 CCGUUCAGUUCAGGGUCUGTT 13
CAGACCCUGAACU 14 AD-15221 GAACGGTT 43-61 UUCAGUUCAGGGUCUGAGCTT 15
GCUCAGACCCUGA 16 AD-15413 ACUGAATT 82-100 GUGAGACUGGCUCGGGCGGTT 17
CCGCCCGAGCCAG 18 AD-15304 UCUCACTT 100-118 GGCCGGGACGCGUCGUUGCTT 19
GCAACGACGCGUC 20 AD-15305 CCGGCCTT 101-119 GCCGGGACGCGUCGUUGCATT 21
UGCAACGACGCGU 22 AD-15306 CCCGGCTT 102-120 CCGGGACGCGUCGUUGCAGTT 23
CUGCAACGACGCG 24 AD-15307 UCCCGGTT 105-123 GGACGCGUCGUUGCAGCAGTT 25
CUGCUGCAACGAC 26 AD-15277 GCGUCCTT 135-153 UCCCAGCCAGGAUUCCGCGTsT
27 CGCGGAAUCCUGG 28 AD-9526 CUGGGATsT 135-153
ucccAGccAGGAuuccGcGTsT 29 CGCGGAAUCCUGG 30 AD-9652 CUGGGATsT
136-154 CCCAGCCAGGAUUCCGCGCTsT 31 GCGCGGAAUCCUG 32 AD-9519
GCUGGGTsT 136-154 cccAGccAGGAuuccGcGcTsT 33 GCGCGGAAUCCUG 34
AD-9645 GCUGGGTsT 138-156 CAGCCAGGAUUCCGCGCGCTsT 35 GCGCGCGGAAUCC
36 AD-9523 UGGCUGTsT 138-156 cAGccAGGAuuccGcGcGcTsT 37
GCGCGCGGAAUCC 38 AD-9649 UGGCUGTsT 185-203 AGCUCCUGCACAGUCCUCCTsT
39 GGAGGACUGUGCA 40 AD-9569 GGAGCUTsT 185-203
AGcuccuGcAcAGuccuccTsT 41 GGAGGACUGUGcA 42 AD-9695 GGAGCUTsT
205-223 CACCGCAAGGCUCAAGGCGTT 43 CGCCUUGAGCCUU 44 AD-15222 GCGGUGTT
208-226 CGCAAGGCUCAAGGCGCCGTT 45 CGGCGCCUUGAGC 46 AD-15278 CUUGCGTT
210-228 CAAGGCUCAAGGCGCCGCCTT 47 GGCGGCGCCUUGA 48 AD-15178 GCCUUGTT
232-250 GUGGACCGCGCACGGCCUCTT 49 GAGGCCGUGCGCG 50 AD-15308 GUCCACTT
233-251 UGGACCGCGCACGGCCUCUTT 51 AGAGGCCGUGCGC 52 AD-15223 GGUCCATT
234-252 GGACCGCGCACGGCCUCUATT 53 UAGAGGCCGUGCG 54 AD-15309 CGGUCCTT
235-253 GACCGCGCACGGCCUCUAGTT 55 CUAGAGGCCGUGC 56 AD-15279 GCGGUCTT
236-254 ACCGCGCACGGCCUCUAGGTT 57 CCUAGAGGCCGUG 58 AD-15194 CGCGGUTT
237-255 CCGCGCACGGCCUCUAGGUTT 59 ACCUAGAGGCCGU 60 AD-15310 GCGCGGTT
238-256 CGCGCACGGCCUCUAGGUCTT 61 GACCUAGAGGCCG 62 AD-15311 UGCGCGTT
239-257 GCGCACGGCCUCUAGGUCUTT 63 AGACCUAGAGGCC 64 AD-15392 GUGCGCTT
240-258 CGCACGGCCUCUAGGUCUCTT 65 GAGACCUAGAGGC 66 AD-15312 CGUGCGTT
248-266 CUCUAGGUCUCCUCGCCAGTT 67 CUGGCGAGGAGAC 68 AD-15313 CUAGAGTT
249-267 UCUAGGUCUCCUCGCCAGGTT 69 CCUGGCGAGGAGA 70 AD-15280 CCUAGATT
250-268 CUAGGUCUCCUCGCCAGGATT 71 UCCUGGCGAGGAG 72 AD-15267 ACCUAGTT
252-270 AGGUCUCCUCGCCAGGACATT 73 UGUCCUGGCGAGG 74 AD-15314 AGACCUTT
258-276 CCUCGCCAGGACAGCAACCTT 75 GGUUGCUGUCCUG 76 AD-15315 GCGAGGTT
300-318 CGUCAGCUCCAGGCGGUCCTsT 77 GGACCGCCUGGAG 78 AD-9624
CUGACGTsT 300-318 cGucAGcuccAGGcGGuccTsT 79 GGACCGCCUGGAG 80
AD-9750 CUGACGTsT 301-319 GUCAGCUCCAGGCGGUCCUTsT 81 AGGACCGCCUGGA
82 AD-9623 GCUGACTsT 301-319 GucAGcuccAGGcGGuccuTsT 83
AGGACCGCCUGGA 84 AD-9749 GCUGACTsT 370-388 GGCGCCCGUGCGCAGGAGGTT 85
CCUCCUGCGCACG 86 AD-15384 GGCGCCTT 408-426 GGAGCUGGUGCUAGCCUUGTsT
87 CAAGGCUAGCACC 88 AD-9607 AGCUCCTsT 408-426
GGAGcuGGuGcuAGccuuGTsT 89 cAAGGCuAGcACc 90 AD-9733 AGCUCCTsT
411-429 GCUGGUGCUAGCCUUGCGUTsT 91 ACGCAAGGCUAGC 92 AD-9524
ACCAGCTsT 411-429 GcuGGuGcuAGccuuGcGuTsT 93 ACGcAAGGCuAGc 94
AD-9650 ACcAGCTsT 412-430 CUGGUGCUAGCCUUGCGUUTsT 95 AACGCAAGGCUAG
96 AD-9520 CACCAGTsT 412-430 CUGGUGCUAGCCUUGCGUUTsT 97
AACGCAAGGCUAG 98 AD-9520 CACCAGTsT 412-430 cuGGuGcuAGccuuGcGuuTsT
99 AACGcAAGGCuAG 100 AD-9646 cACcAGTsT 416-434
UGCUAGCCUUGCGUUCCGATsT 101 UCGGAACGCAAGG 102 AD-9608 CUAGCATsT
416-434 uGcuAGccuuGcGuuccGATsT 103 UCGGAACGcAAGG 104 AD-9734
CuAGcATsT 419-437 UAGCCUUGCGUUCCGAGGATsT 105 UCCUCGGAACGCA 106
AD-9546 AGGCUATsT 419-437 uAGccuuGcGuuccGAGGATsT 107 UCCUCGGAACGcA
108 AD-9672 AGGCuATsT 439-457 GACGGCCUGGCCGAAGCACTT 109
GUGCUUCGGCCAG 110 AD-15385 GCCGUCTT 447-465 GGCCGAAGCACCCGAGCACTT
111 GUGCUCGGGUGCU 112 AD-15393 UCGGCCTT 448-466
GCCGAAGCACCCGAGCACGTT 113 CGUGCUCGGGUGC 114 AD-15316 UUCGGCTT
449-467 CCGAAGCACCCGAGCACGGTT 115 CCGUGCUCGGGUG 116 AD-15317
CUUCGGTT 458-476 CCGAGCACGGAACCACAGCTT 117 GCUGUGGUUCCGU 118
AD-15318 GCUCGGTT 484-502 CACCGCUGCGCCAAGGAUCTT 119 GAUCCUUGGCGCA
120 AD-15195 GCGGUGTT 486-504 CCGCUGCGCCAAGGAUCCGTT 121
CGGAUCCUUGGCG 122 AD-15224 CAGCGGTT 487-505 CGCUGCGCCAAGGAUCCGUTT
123 ACGGAUCCUUGGC 124 AD-15188 GCAGCGTT 489-507
CUGCGCCAAGGAUCCGUGGTT 125 CCACGGAUCCUUG 126 AD-15225 GCGCAGTT
500-518 AUCCGUGGAGGUUGCCUGGTT 127 CCAGGCAACCUCC 128 AD-15281
ACGGAUTT 509-527 GGUUGCCUGGCACCUACGUTT 129 ACGUAGGUGCCAG 130
AD-15282 GCAACCTT 542-560 AGGAGACCCACCUCUCGCATT 131 UGCGAGAGGUGGG
132 AD-15319 UCUCCUTT 543-561 GGAGACCCACCUCUCGCAGTT 133
CUGCGAGAGGUGG 134 AD-15226 GUCUCCTT 544-562 GAGACCCACCUCUCGCAGUTT
135 ACUGCGAGAGGUG 136 AD-15271 GGUCUCTT 549-567
CCACCUCUCGCAGUCAGAGTT 137 CUCUGACUGCGAG 138 AD-15283 AGGUGGTT
552-570 CCUCUCGCAGUCAGAGCGCTT 139 GCGCUCUGACUGC 140 AD-15284
GAGAGGTT 553-571 CUCUCGCAGUCAGAGCGCATT 141 UGCGCUCUGACUG 142
AD-15189 CGAGAGTT 554-572 UCUCGCAGUCAGAGCGCACTT 143 GUGCGCUCUGACU
144 AD-15227 GCGAGATT 555-573 CUCGCAGUCAGAGCGCACUTsT 145
AGUGCGCUCUGAC 146 AD-9547 UGCGAGTsT 555-573 cucGcAGucAGAGcGcAcuTsT
147 AGUGCGCUCUGAC 148 AD-9673 UGCGAGTsT 558-576
GCAGUCAGAGCGCACUGCCTsT 149 GGCAGUGCGCUCU 150 AD-9548 GACUGCTsT
558-576 GcAGucAGAGcGcAcuGccTsT 151 GGcAGUGCGCUCU 152 AD-9674
GACUGCTsT 606-624 GGGAUACCUCACCAAGAUCTsT 153 GAUCUUGGUGAGG 154
AD-9529 UAUCCCTsT 606-624 GGGAuAccucAccAAGAucTsT 155 GAUCUUGGUGAGG
156 AD-9655 uAUCCCTsT 659-677 UGGUGAAGAUGAGUGGCGATsT 157
UCGCCACUCAUCU 158 AD-9605 UCACCATsT 659-677 uGGuGAAGAuGAGuGGcGATsT
159 UCGCcACUcAUCU 160 AD-9731 UcACcATsT 663-681
GAAGAUGAGUGGCGACCUGTsT 161 CAGGUCGCCACUC 162 AD-9596 AUCUUCTsT
663-681 GAAGAuGAGuGGcGAccuGTsT 163 cAGGUCGCcACUc 164 AD-9722
AUCUUCTsT 704-722 CCCAUGUCGACUACAUCGATsT 165 UCGAUGUAGUCGA 166
AD-9583 CAUGGGTsT 704-722 cccAuGucGAcuAcAucGATsT 167 UCGAUGuAGUCGA
168 AD-9709 cAUGGGTsT 718-736 AUCGAGGAGGACUCCUCUGTsT 169
CAGAGGAGUCCUC 170 AD-9579 CUCGAUTsT 718-736 AucGAGGAGGAcuccucuGTsT
171 cAGAGGAGUCCUC 172 AD-9705 CUCGAUTsT 758-776
GGAACCUGGAGCGGAUUACTT 173 GUAAUCCGCUCCA 174 AD-15394 GGUUCCTT
759-777 GAACCUGGAGCGGAUUACCTT 175 GGUAAUCCGCUCC 176 AD-15196
AGGUUCTT 760-778 AACCUGGAGCGGAUUACCCTT 177 GGGUAAUCCGCUC 178
AD-15197 CAGGUUTT 777-795 CCCUCCACGGUACCGGGCGTT 179 CGCCCGGUACCGU
180 AD-15198 GGAGGGTT 782-800 CACGGUACCGGGCGGAUGATsT 181
UCAUCCGCCCGGU 182 AD-9609 ACCGUGTsT 782-800 cAcGGuAccGGGcGGAuGATsT
183 UcAUCCGCCCGGu 184 AD-9735 ACCGUGTsT 783-801
ACGGUACCGGGCGGAUGAATsT 185 UUCAUCCGCCCGG 186 AD-9537 UACCGUTsT
783-801 AcGGuAccGGGcGGAuGAATsT 187 UUcAUCCGCCCGG 188 AD-9663
uACCGUTsT 784-802 CGGUACCGGGCGGAUGAAUTsT 189 AUUCAUCCGCCCG 190
AD-9528 GUACCGTsT 784-802 cGGuAccGGGcGGAuGAAuTsT 191 AUUcAUCCGCCCG
192 AD-9654 GuACCGTsT 785-803 GGUACCGGGCGGAUGAAUATsT 193
UAUUCAUCCGCCC 194 AD-9515 GGUACCTsT 785-803 GGuAccGGGcGGAuGAAuATsT
195 uAUUcAUCCGCCC 196 AD-9641 GGuACCTsT 786-804
GUACCGGGCGGAUGAAUACTsT 197 GUAUUCAUCCGCC 198 AD-9514 CGGUACTsT
786-804 GuAccGGGcGGAuGAAuAcTsT 199 GuAUUcAUCCGCC 200 AD-9640
CGGuACTsT 788-806 ACCGGGCGGAUGAAUACCATsT 201 UGGUAUUCAUCCG 202
AD-9530 CCCGGUTsT 788-806 AccGGGcGGAuGAAuAccATsT 203 UGGuAUUcAUCCG
204 AD-9656 CCCGGUTsT 789-807 CCGGGCGGAUGAAUACCAGTsT 205
CUGGUAUUCAUCC 206 AD-9538 GCCCGGTsT 789-807 ccGGGcGGAuGAAuAccAGTsT
207 CUGGuAUUcAUCC 208 AD-9664 GCCCGGTsT 825-843
CCUGGUGGAGGUGUAUCUCTsT 209 GAGAUACACCUCC 210 AD-9598 ACCAGGTsT
825-843 ccuGGuGGAGGuGuAucucTsT 211 GAGAuAcACCUCc 212 AD-9724
ACcAGGTsT 826-844 CUGGUGGAGGUGUAUCUCCTsT 213 GGAGAUACACCUC 214
AD-9625 CACCAGTsT 826-844 cuGGuGGAGGuGuAucuccTsT 215 GGAGAuAcACCUC
216 AD-9751 cACcAGTsT 827-845 UGGUGGAGGUGUAUCUCCUTsT 217
AGGAGAUACACCU 218 AD-9556 CCACCATsT 827-845 uGGuGGAGGuGuAucuccuTsT
219 AGGAGAuAcACCU 220 AD-9682 CcACcATsT 828-846
GGUGGAGGUGUAUCUCCUATsT 221 UAGGAGAUACACC 222 AD-9539 UCCACCTsT
828-846 GGuGGAGGuGuAucuccuATsT 223 uAGGAGAuAcACC 224 AD-9665
UCcACCTsT 831-849 GGAGGUGUAUCUCCUAGACTsT 225 GUCUAGGAGAUAC 226
AD-9517 ACCUCCTsT 831-849 GGAGGuGuAucuccuAGAcTsT 227 GUCuAGGAGAuAc
228 AD-9643 ACCUCCTsT 833-851 AGGUGUAUCUCCUAGACACTsT 229
GUGUCUAGGAGAU 230 AD-9610 ACACCUTsT 833-851 AGGuGuAucuccuAGAcAcTsT
231 GUGUCuAGGAGAu 232 AD-9736 AcACCUTsT 833-851
AfgGfuGfuAfuCfuCfcUfaG 233 P*gUfgUfcUfaG 234 AD-14681 faCfaCfTsT
fgAfgAfuAfcAf cCfuTsT 833-851 AGGUfGUfAUfCfUfCfCfUfA 235
GUfGUfCfUfAGG 236 AD-14691 GACfACfTsT AGAUfACfACfCf UfTsT 833-851
AgGuGuAuCuCcUaGaCaCTsT 237 P*gUfgUfcUfaG 238 AD-14701 fgAfgAfuAfcAf
cCfuTsT 833-851 AgGuGuAuCuCcUaGaCaCTsT 239 GUfGUfCfUfAGG 240
AD-14711 AGAUfACfACfCf UfTsT 833-851 AfgGfuGfuAfuCfuCfcUfaG 241
GUGUCuaGGagAU 242 AD-14721 faCfaCfTsT ACAccuTsT 833-851
AGGUfGUfAUfCfUfCfCfUfA 243 GUGUCuaGGagAU 244 AD-14731 GACfACfTsT
ACAccuTsT 833-851 AgGuGuAuCuCcUaGaCaCTsT 245 GUGUCuaGGagAU 246
AD-14741 ACAccuTsT 833-851 GfcAfcCfcUfcAfuAfgGfcC 247 P*uCfcAfgGfcC
248 AD-15087 fuGfgAfTsT fuAfuGfaGfgGf uGfcTsT 833-851
GCfACfCfCfUfCfAUfAGGCf 249 UfCfCfAGGCfCf 250 AD-15097 CfUfGGATsT
UfAUfGAGGGUfG CfTsT 833-851 GcAcCcUcAuAgGcCuGgATsT 251
P*uCfcAfgGfcC 252 AD-15107 fuAfuGfaGfgGf uGfcTsT 833-851
GcAcCcUcAuAgGcCuGgATsT 253 UfCfCfAGGCfCf 254 AD-15117 UfAUfGAGGGUfG
CfTsT 833-851 GfcAfcCfcUfcAfuAfgGfcC 255 UCCAGgcCUauGA 256 AD-15127
fuGfgAfTsT GGGugcTsT 833-851 GCfACfCfCfUfCfAUfAGGCf 257
UCCAGgcCUauGA 258 AD-15137 CfUfGGATsT GGGugcTsT 833-851
GcAcCcUcAuAgGcCuGgATsT 259 UCCAGgcCUauGA 260 AD-15147 GGGugcTsT
836-854 UGUAUCUCCUAGACACCAGTsT 261 CUGGUGUCUAGGA 262 AD-9516
GAUACATsT 836-854 uGuAucuccuAGAcAccAGTsT 263 CUGGUGUCuAGGA 264
AD-9642 GAuAcATsT 840-858 UCUCCUAGACACCAGCAUATsT 265 UAUGCUGGUGUCU
266 AD-9562 AGGAGATsT 840-858 ucuccuAGAcAccAGcAuATsT 267
uAUGCUGGUGUCu 268 AD-9688 AGGAGATsT 840-858 UfcUfcCfuAfgAfcAfcCfaG
269 P*uAfuGfcUfgG 270 AD-14677 fcAfuAfTsT fuGfuCfuAfgGf aGfaTsT
840-858 UfCfUfCfCfUfAGACfACfCf 271 UfAUfGCfUfGGU 272 AD-14687
AGCfAUfATsT fGUfCfUfAGGAG ATsT 840-858 UcUcCuAgAcAcCaGcAuATsT 273
P*uAfuGfcUfgG 274 AD-14697 fuGfuCfuAfgGf aGfaTsT 840-858
UcUcCuAgAcAcCaGcAuATsT 275 UfAUfGCfUfGGU 276 AD-14707 fGUfCfUfAGGAG
ATsT 840-858 UfcUfcCfuAafAfcAfcCfaG 277 UAUGCugGUguCU 278 AD-14717
fcAfuAfTsT AGGagaTsT 840-858 UfCfUfCfCfUfAGACfACfCf 279
UAUGCugGUguCU 280 AD-14727 AGCfAUfATsT AGGagaTsT 840-858
UcUcCuAgAcAcCaGcAuATsT 281 UAUGCugGUguCU 282 AD-14737 AGGagaTsT
840-858 AfgGfcCfuGfgAfgUfuUfaU 283 P*cCfgAfaUfaA 284 AD-15083
fuCfgGfTsT faCfuCfcAfgGf cCfuTsT 840-858 AGGCfCfUfGGAGUfUfUfAUf 285
CfCfGAAUfAAAC 286 AD-15093 UfCfGGTsT fUfCfCfAGGCfC fUfTsT 840-858
AgGcCuGgAgUuUaUuCgGTsT 287 P*cCfgAfaUfaA 288 AD-15103 faCfuCfcAfgGf
cCfuTsT 840-858 AgGcCuGgAgUuUaUuCgGTsT 289 CfCfGAAUfAAAC 290
AD-15113 fUfCfCfAGGCfC fUfTsT 840-858 AfgGfcCfuGfgAfgUfuUfaU 291
CCGAAuaAAcuCC 292 AD-15123 fuCfgGfTsT AGGccuTsT 840-858
AGGCfCfUfGGAGUfUfUfAUf 293 CCGAAuaAAcuCC 294 AD-15133 UfCfGGTsT
AGGccuTsT 840-858 AgGcCuGgAgUuUaUuCgGTsT 295 CCGAAuaAAcuCC 296
AD-15143 AGGccuTsT 841-859 CUCCUAGACACCAGCAUACTsT 297 GUAUGCUGGUGUC
298 AD-9521 UAGGAGTsT 841-859 cuccuAGAcAccAGcAuAcTsT 299
GuAUGCUGGUGUC 300 AD-9647 uAGGAGTsT 842-860 UCCUAGACACCAGCAUACATsT
301 UGUAUGCUGGUGU 302 AD-9611 CUAGGATsT 842-860
uccuAGAcAccAGcAuAcATsT 303 UGuAUGCUGGUGU 304 AD-9737 CuAGGATsT
843-861 CCUAGACACCAGCAUACAGTsT 305 CUGUAUGCUGGUG 306 AD-9592
UCUAGGTsT 843-861 ccuAGAcAccAGcAuAcAGTsT 307 CUGuAUGCUGGUG 308
AD-9718 UCuAGGTsT 847-865 GACACCAGCAUACAGAGUGTsT 309 CACUCUGUAUGCU
310 AD-9561 GGUGUCTsT 847-865 GAcAccAGcAuAcAGAGuGTsT 311
cACUCUGuAUGCU 312 AD-9687 GGUGUCTsT 855-873 CAUACAGAGUGACCACCGGTsT
313 CCGGUGGUCACUC 314 AD-9636 UGUAUGTsT 855-873
cAuAcAGAGuGAccAccGGTsT 315 CCGGUGGUcACUC 316 AD-9762 UGuAUGTsT
860-878 AGAGUGACCACCGGGAAAUTsT 317 AUUUCCCGGUGGU 318 AD-9540
CACUCUTsT
860-878 AGAGuGAccAccGGGAAAuTsT 319 AUUUCCCGGUGGU 320 AD-9666
cACUCUTsT 861-879 GAGUGACCACCGGGAAAUCTsT 321 GAUUUCCCGGUGG 322
AD-9535 UCACUCTsT 861-879 GAGuGAccAccGGGAAAucTsT 323 GAUUUCCCGGUGG
324 AD-9661 UcACUCTsT 863-881 GUGACCACCGGGAAAUCGATsT 325
UCGAUUUCCCGGU 326 AD-9559 GGUCACTsT 863-881 GuGAccAccGGGAAAucGATsT
327 UCGAUUUCCCGGU 328 AD-9685 GGUcACTsT 865-883
GACCACCGGGAAAUCGAGGTsT 329 CCUCGAUUUCCCG 330 AD-9533 GUGGUCTsT
865-883 GAccAccGGGAAAucGAGGTsT 331 CCUCGAUUUCCCG 332 AD-9659
GUGGUCTsT 866-884 ACCACCGGGAAAUCGAGGGTsT 333 CCCUCGAUUUCCC 334
AD-9612 GGUGGUTsT 866-884 AccAccGGGAAAucGAGGGTsT 335 CCCUCGAUUUCCC
336 AD-9738 GGUGGUTsT 867-885 CCACCGGGAAAUCGAGGGCTsT 337
GCCCUCGAUUUCC 338 AD-9557 CGGUGGTsT 867-885 ccAccGGGAAAucGAGGGcTsT
339 GCCCUCGAUUUCC 340 AD-9683 CGGUGGTsT 875-893
AAAUCGAGGGCAGGGUCAUTsT 341 AUGACCCUGCCCU 342 AD-9531 CGAUUUTsT
875-893 AAAucGAGGGcAGGGucAuTsT 343 AUGACCCUGCCCU 344 AD-9657
CGAUUUTsT 875-893 AfaAfuCfgAfgGfgCfaGfgG 345 P*aUfgAfcCfcU 346
AD-14673 fuCfaUfTsT fgCfcCfuCfgAf uUfuTsT 875-893
AAAUfCfGAGGGCfAGGGUfCf 347 AUfGACfCfCfUf 348 AD-14683 AUfTsT
GCfCfCfUfCfGA UfUfUfTsT 875-893 AaAuCgAgGgCaGgGuCaUTsT 349
P*aUfgAfcCfcU 350 AD-14693 fgCfcCfuCfgAf uUfuTsT 875-893
AaAuCgAgGgCaGgGuCaUTsT 351 AUfGACfCfCfUf 352 AD-14703 GCfCfCfUfCfGA
UfUfUfTsT 875-893 AfaAfuCfgAfgGfgCfaGfgG 353 AUGACccUGccCU 354
AD-14713 fuCfaUfTsT CGAuuuTsT 875-893 AAAUfCfGAGGGCfAGGGUfCf 355
AUGACccUGccCU 356 AD-14723 AUfTsT CGAuuuTsT 875-893
AaAuCgAgGgCaGgGuCaUTsT 357 AUGACccUGccCU 358 AD-14733 CGAuuuTsT
875-893 CfgGfcAfcCfcUfcAfuAfgG 359 P*cAfgGfcCfuA 360 AD-15079
fcCfuGfTsT fuGfaGfgGfuGf cCfgTsT 875-893 CfGGCfACfCfCfUfCfAUfAG 361
CfAGGCfCfUfAU 362 AD-15089 GCfCfUfGTsT fGAGGGUfGCfCf GTsT 875-893
CgGcAcCcUcAuAgGcCuGTsT 363 P*cAfgGfcCfuA 364 AD-15099 fuGfaGfgGfuGf
cCfgTsT 875-893 CgGcAcCcUcAuAgGcCuGTsT 365 CfAGGCfCfUfAU 366
AD-15109 fGAGGGUfGCfCf GTsT 875-893 CfgGfcAfcCfcUfcAfuAfgG 367
CAGGCcuAUgaGG 368 AD-15119 fcCfuGfTsT GUGccgTsT 875-893
CfGGCfACfCfCfUfCfAUfAG 369 CAGGCcuAUgaGG 370 AD-15129 GCfCfUfGTsT
GUGccgTsT 875-893 CgGcAcCcUcAuAgGcCuGTsT 371 CAGGCcuAUgaGG 372
AD-15139 GUGccgTsT 877-895 AUCGAGGGCAGGGUCAUGGTsT 373 CCAUGACCCUGCC
374 AD-9542 CUCGAUTsT 877-895 AucGAGGGcAGGGucAuGGTsT 375
CcAUGACCCUGCC 376 AD-9668 CUCGAUTsT 878-896 cGAGGGcAGGGucAuGGucTsT
377 GACcAUGACCCUG 378 AD-9739 CCCUCGTsT 880-898
GAGGGCAGGGUCAUGGUCATsT 379 UGACCAUGACCCU 380 AD-9637 GCCCUCTsT
880-898 GAGGGcAGGGucAuGGucATsT 381 UGACcAUGACCCU 382 AD-9763
GCCCUCTsT 882-900 GGGCAGGGUCAUGGUCACCTsT 383 GGUGACCAUGACC 384
AD-9630 CUGCCCTsT 882-900 GGGcAGGGucAuGGucAccTsT 385 GGUGACcAUGACC
386 AD-9756 CUGCCCTsT 885-903 CAGGGUCAUGGUCACCGACTsT 387
GUCGGUGACCAUG 388 AD-9593 ACCCUGTsT 885-903 cAGGGucAuGGucAccGAcTsT
389 GUCGGUGACcAUG 390 AD-9719 ACCCUGTsT 886-904
AGGGUCAUGGUCACCGACUTsT 391 AGUCGGUGACCAU 392 AD-9601 GACCCUTsT
886-904 AGGGucAuGGucAccGAcuTsT 393 AGUCGGUGACcAU 394 AD-9727
GACCCUTsT 892-910 AUGGUCACCGACUUCGAGATsT 395 UCUCGAAGUCGGU 396
AD-9573 GACCAUTsT 892-910 AuGGucAccGAcuucGAGATsT 397 UCUCGAAGUCGGU
398 AD-9699 GACcAUTsT 899-917 CCGACUUCGAGAAUGUGCCTT 399
GGCACAUUCUCGA 400 AD-15228 AGUCGGTT 921-939 GGAGGACGGGACCCGCUUCTT
401 GAAGCGGGUCCCG 402 AD-15395 UCCUCCTT 993- CAGCGGCCGGGAUGCCGGCTsT
403 GCCGGCAUCCCGG 404 AD-9602 1011 CCGCUGTsT 993-
cAGcGGccGGGAuGccGGcTsT 405 GCCGGcAUCCCGG 406 AD-9728 1011 CCGCUGTsT
1020- GGGUGCCAGCAUGCGCAGCTT 407 GCUGCGCAUGCUG 408 AD-15386 1038
GCACCCTT 1038- CCUGCGCGUGCUCAACUGCTsT 409 GCAGUUGAGCACG 410 AD-9580
1056 CGCAGGTsT 1038- ccuGcGcGuGcucAAcuGcTsT 411 GcAGUUGAGcACG 412
AD-9706 1056 CGcAGGTsT 1040- UGCGCGUGCUCAACUGCCATsT 413
UGGCAGUUGAGCA 414 AD-9581 1058 CGCGCATsT 1040-
uGcGcGuGcucAAcuGccATsT 415 UGGcAGUUGAGcA 416 AD-9707 1058 CGCGcATsT
1042- CGCGUGCUCAACUGCCAAGTsT 417 CUUGGCAGUUGAG 418 AD-9543 1060
CACGCGTsT 1042- cGcGuGcucAAcuGccAAGTsT 419 CUUGGcAGUUGAG 420
AD-9669 1060 cACGCGTsT 1053- CUGCCAAGGGAAGGGCACGTsT 421
CGUGCCCUUCCCU 422 AD-9574 1071 UGGCAGTsT 1053-
cuGccAAGGGAAGGGcAcGTsT 423 CGUGCCCUUCCCU 424 AD-9700 1071 UGGcAGTsT
1057- CAAGGGAAGGGCACGGUUATT 425 UAACCGUGCCCUU 426 AD-15320 1075
CCCUUGTT 1058- AAGGGAAGGGCACGGUUAGTT 427 CUAACCGUGCCCU 428 AD-15321
1076 UCCCUUTT 1059- AGGGAAGGGCACGGUUAGCTT 429 GCUAACCGUGCCC 430
AD-15199 1077 UUCCCUTT 1060- GGGAAGGGCACGGUUAGCGTT 431
CGCUAACCGUGCC 432 AD-15167 1078 CUUCCCTT 1061-
GGAAGGGCACGGUUAGCGGTT 433 CCGCUAACCGUGC 434 AD-15164 1079 CCUUCCTT
1062- GAAGGGCACGGUUAGCGGCTT 435 GCCGCUAACCGUG 436 AD-15166 1080
CCCUUCTT 1063- AAGGGCACGGUUAGCGGCATT 437 UGCCGCUAACCGU 438 AD-15322
1081 GCCCUUTT 1064- AGGGCACGGUUAGCGGCACTT 439 GUGCCGCUAACCG 440
AD-15200 1082 UGCCCUTT 1068- CACGGUUAGCGGCACCCUCTT 441
GAGGGUGCCGCUA 442 AD-15213 1086 ACCGUGTT 1069-
ACGGUUAGCGGCACCCUCATT 443 UGAGGGUGCCGCU 444 AD-15229 1087 AACCGUTT
1072- GUUAGCGGCACCCUCAUAGTT 445 CUAUGAGGGUGCC 446 AD-15215 1090
GCUAACTT 1073- UUAGCGGCACCCUCAUAGGTT 447 CCUAUGAGGGUGC 448 AD-15214
1091 CGCUAATT 1076- GCGGCACCCUCAUAGGCCUTsT 449 AGGCCUAUGAGGG 450
AD-9315 1094 UGCCGCTsT 1079- GCACCCUCAUAGGCCUGGATsT 451
UCCAGGCCUAUGA 452 AD-9326 1097 GGGUGCTsT 1085-
UCAUAGGCCUGGAGUUUAUTsT 453 AUAAACUCCAGGC 454 AD-9318 1103 CUAUGATsT
1090- GGCCUGGAGUUUAUUCGGATsT 455 UCCGAAUAAACUC 456 AD-9323 1108
CAGGCCTsT 1091- GCCUGGAGUUUAUUCGGAATsT 457 UUCCGAAUAAACU 458
AD-9314 1109 CCAGGCTsT 1091- GccuGGAGuuuAuucGGAATsT 459
UUCCGAAuAAACU 460 AD-10792 1109 CcAGGCTsT 1091-
GccuGGAGuuuAuucGGAATsT 461 UUCCGAAUAACUC 462 AD-10796 1109 CAGGCTsT
1093- CUGGAGUUUAUUCGGAAAATsT 463 UUUUCCGAAUAAA 464 AD-9638 1111
CUCCAGTsT 1093- cuGGAGuuuAuucGGAAAATsT 465 UUUUCCGAAuAAA 466
AD-9764 1111 CUCcAGTsT 1095- GGAGUUUAUUCGGAAAAGCTsT 467
GCUUUUCCGAAUA 468 AD-9525 1113 AACUCCTsT 1095-
GGAGuuuAuucGGAAAAGcTsT 469 GCUUUUCCGAAuA 470 AD-9651 1113 AACUCCTsT
1096- GAGUUUAUUCGGAAAAGCCTsT 471 GGCUUUUCCGAAU 472 AD-9560 1114
AAACUCTsT 1096- GAGuuuAuucGGAAAAGccTsT 473 GGCUUUUCCGAAu 474
AD-9686 1114 AAACUCTsT 1100- UUAUUCGGAAAAGCCAGCUTsT 475
AGCUGGCUUUUCC 476 AD-9536 1118 GAAUAATsT 1100-
uuAuucGGAAAAGccAGcuTsT 477 AGCUGGCUUUUCC 478 AD-9662 1118 GAAuAATsT
1154- CCCUGGCGGGUGGGUACAGTsT 479 CUGUACCCACCCG 480 AD-9584 1172
CCAGGGTsT
1154- cccuGGcGGGuGGGuAcAGTsT 481 CUGuACCcACCCG 482 AD-9710 1172
CcAGGGTsT 1155- CCUGGCGGGUGGGUACAGCTT 483 GCUGUACCCACCC 484
AD-15323 1173 GCCAGGTT 1157- UGGCGGGUGGGUACAGCCGTsT 485
CGGCUGUACCCAC 486 AD-9551 1175 CCGCCATsT 1157-
uGGcGGGuGGGuAcAGccGTsT 487 CGGCUGuACCcAC 488 AD-9677 1175 CCGCcATsT
1158- GGCGGGUGGGUACAGCCGCTT 489 GCGGCUGUACCCA 490 AD-15230 1176
CCCGCCTT 1162- GGUGGGUACAGCCGCGUCCTT 491 GGACGCGGCUGUA 492 AD-15231
1180 CCCACCTT 1164- UGGGUACAGCCGCGUCCUCTT 493 GAGGACGCGGCUG 494
AD-15285 1182 UACCCATT 1172- GCCGCGUCCUCAACGCCGCTT 495
GCGGCGUUGAGGA 496 AD-15396 1190 CGCGGCTT 1173-
CCGCGUCCUCAACGCCGCCTT 497 GGCGGCGUUGAGG 498 AD-15397 1191 ACGCGGTT
1216- GUCGUGCUGGUCACCGCUGTsT 499 CAGCGGUGACCAG 500 AD-9600 1234
CACGACTsT 1216- GucGuGcuGGucAccGcuGTsT 501 cAGCGGUGACcAG 502
AD-9726 1234 cACGACTsT 1217- UCGUGCUGGUCACCGCUGCTsT 503
GCAGCGGUGACCA 504 AD-9606 1235 GCACGATsT 1217-
ucGuGcuGGucAccGcuGcTsT 505 GcAGCGGUGACcA 506 AD-9732 1235 GcACGATsT
1223- UGGUCACCGCUGCCGGCAATsT 507 UUGCCGGCAGCGG 508 AD-9633 1241
UGACCATsT 1223- uGGucAccGcuGccGGcAATsT 509 UUGCCGGcAGCGG 510
AD-9759 1241 UGACcATsT 1224- GGUCACCGCUGCCGGCAACTsT 511
GUUGCCGGCAGCG 512 AD-9588 1242 GUGACCTsT 1224-
GGucAccGcuGccGGcAAcTsT 513 GUUGCCGGcAGCG 514 AD-9714 1242 GUGACCTsT
1227- CACCGCUGCCGGCAACUUCTsT 515 GAAGUUGCCGGCA 516 AD-9589 1245
GCGGUGTsT 1227- cAccGcuGccGGcAAcuucTsT 517 GAAGUUGCCGGcA 518
AD-9715 1245 GCGGUGTsT 1229- CCGCUGCCGGCAACUUCCGTsT 519
CGGAAGUUGCCGG 520 AD-9575 1247 CAGCGGTsT 1229-
ccGcuGccGGcAAcuuccGTsT 521 CGGAAGUUGCCGG 522 AD-9701 1247 cAGCGGTsT
1230- CGCUGCCGGCAACUUCCGGTsT 523 CCGGAAGUUGCCG 524 AD-9563 1248
GCAGCGTsT 1230- cGcuGccGGcAAcuuccGGTsT 525 CCGGAAGUUGCCG 526
AD-9689 1248 GcAGCGTsT 1231- GCUGCCGGCAACUUCCGGGTsT 527
CCCGGAAGUUGCC 528 AD-9594 1249 GGCAGCTsT 1231-
GcuGccGGcAAcuuccGGGTsT 529 CCCGGAAGUUGCC 530 AD-9720 1249 GGcAGCTsT
1236- CGGCAACUUCCGGGACGAUTsT 531 AUCGUCCCGGAAG 532 AD-9585 1254
UUGCCGTsT 1236- cGGcAAcuuccGGGAcGAuTsT 533 AUCGUCCCGGAAG 534
AD-9711 1254 UUGCCGTsT 1237- GGCAACUUCCGGGACGAUGTsT 535
CAUCGUCCCGGAA 536 AD-9614 1255 GUUGCCTsT 1237-
GGcAAcuuccGGGAcGAuGTsT 537 cAUCGUCCCGGAA 538 AD-9740 1255 GUUGCCTsT
1243- UUCCGGGACGAUGCCUGCCTsT 539 GGCAGGCAUCGUC 540 AD-9615 1261
CCGGAATsT 1243- uuccGGGAcGAuGccuGccTsT 541 GGcAGGcAUCGUC 542
AD-9741 1261 CCGGAATsT 1248- GGACGAUGCCUGCCUCUACTsT 543
GUAGAGGCAGGCA 544 AD-9534 1266 UCGUCCTsT 1248-
GGACGAUGCCUGCCUCUACTsT 545 GUAGAGGCAGGCA 546 AD-9534 1266 UCGUCCTsT
1248- GGAcGAuGccuGccucuAcTsT 547 GuAGAGGcAGGcA 548 AD-9660 1266
UCGUCCTsT 1279- GCUCCCGAGGUCAUCACAGTT 549 CUGUGAUGACCUC 550
AD-15324 1297 GGGAGCTT 1280- CUCCCGAGGUCAUCACAGUTT 551
ACUGUGAUGACCU 552 AD-15232 1298 CGGGAGTT 1281-
UCCCGAGGUCAUCACAGUUTT 553 AACUGUGAUGACC 554 AD-15233 1299 UCGGGATT
1314- CCAAGACCAGCCGGUGACCTT 555 GGUCACCGGCUGG 556 AD-15234 1332
UCUUGGTT 1315- CAAGACCAGCCGGUGACCCTT 557 GGGUCACCGGCUG 558 AD-15286
1333 GUCUUGTT 1348- ACCAACUUUGGCCGCUGUGTsT 559 CACAGCGGCCAAA 560
AD-9590 1366 GUUGGUTsT 1348- AccAAcuuuGGccGcuGuGTsT 561
cAcAGCGGCcAAA 562 AD-9716 1366 GUUGGUTsT 1350-
CAACUUUGGCCGCUGUGUGTsT 563 CACACAGCGGCCA 564 AD-9632 1368 AAGUUGTsT
1350- cAAcuuuGGccGcuGuGuGTsT 565 cAcAcAGCGGCcA 566 AD-9758 1368
AAGUUGTsT 1360- CGCUGUGUGGACCUCUUUGTsT 567 CAAAGAGGUCCAC 568
AD-9567 1378 ACAGCGTsT 1360- cGcuGuGuGGAccucuuuGTsT 569
cAAAGAGGUCcAc 570 AD-9693 1378 AcAGCGTsT 1390-
GACAUCAUUGGUGCCUCCATsT 571 UGGAGGCACCAAU 572 AD-9586 1408 GAUGUCTsT
1390- GAcAucAuuGGuGccuccATsT 573 UGGAGGcACcAAU 574 AD-9712 1408
GAUGUCTsT 1394- UCAUUGGUGCCUCCAGCGATsT 575 UCGCUGGAGGCAC 576
AD-9564 1412 CAAUGATsT 1394- ucAuuGGuGccuccAGcGATsT 577
UCGCUGGAGGcAC 578 AD-9690 1412 cAAUGATsT 1417-
AGCACCUGCUUUGUGUCACTsT 579 GUGACACAAAGCA 580 AD-9616 1435 GGUGCUTsT
1417- AGcAccuGcuuuGuGucAcTsT 581 GUGAcAcAAAGcA 582 AD-9742 1435
GGUGCUTsT 1433- CACAGAGUGGGACAUCACATT 583 UGUGAUGUCCCAC 584
AD-15398 1451 UCUGUGTT 1486- AUGCUGUCUGCCGAGCCGGTsT 585
CCGGCUCGGCAGA 586 AD-9617 1504 CAGCAUTsT 1486-
AuGcuGucuGccGAGccGGTsT 587 CCGGCUCGGcAGA 588 AD-9743 1504 cAGcAUTsT
1491- GUCUGCCGAGCCGGAGCUCTsT 589 GAGCUCCGGCUCG 590 AD-9635 1509
GCAGACTsT 1491- GucuGccGAGccGGAGcucTsT 591 GAGCUCCGGCUCG 592
AD-9761 1509 GcAGACTsT 1521- GUUGAGGCAGAGACUGAUCTsT 593
GAUCAGUCUCUGC 594 AD-9568 1539 CUCAACTsT 1521-
GuuGAGGcAGAGAcuGAucTsT 595 GAUcAGUCUCUGC 596 AD-9694 1539 CUcAACTsT
1527- GCAGAGACUGAUCCACUUCTsT 597 GAAGUGGAUCAGU 598 AD-9576 1545
CUCUGCTsT 1527- GcAGAGAcuGAuccAcuucTsT 599 GAAGUGGAUcAGU 600
AD-9702 1545 CUCUGCTsT 1529- AGAGACUGAUCCACUUCUCTsT 601
GAGAAGUGGAUCA 602 AD-9627 1547 GUCUCUTsT 1529-
AGAGAcuGAuccAcuucucTsT 603 GAGAAGUGGAUcA 604 AD-9753 1547 GUCUCUTsT
1543- UUCUCUGCCAAAGAUGUCATsT 605 UGACAUCUUUGGC 606 AD-9628 1561
AGAGAATsT 1543- uucucuGccAAAGAuGucATsT 607 UGAcAUCUUUGGc 608
AD-9754 1561 AGAGAATsT 1545- CUCUGCCAAAGAUGUCAUCTsT 609
GAUGACAUCUUUG 610 AD-9631 1563 GCAGAGTsT 1545-
cucuGccAAAGAuGucAucTsT 611 GAUGAcAUCUUUG 612 AD-9757 1563 GcAGAGTsT
1580- CUGAGGACCAGCGGGUACUTsT 613 AGUACCCGCUGGU 614 AD-9595 1598
CCUCAGTsT 1580- cuGAGGAccAGcGGGuAcuTsT 615 AGuACCCGCUGGU 616
AD-9721 1598 CCUcAGTsT 1581- UGAGGACCAGCGGGUACUGTsT 617
CAGUACCCGCUGG 618 AD-9544 1599 UCCUCATsT 1581-
uGAGGAccAGcGGGuAcuGTsT 619 cAGuACCCGCUGG 620 AD-9670 1599 UCCUcATsT
1666- ACUGUAUGGUCAGCACACUTT 621 AGUGUGCUGACCA 622 AD-15235 1684
UACAGUTT 1668- UGUAUGGUCAGCACACUCGTT 623 CGAGUGUGCUGAC 624 AD-15236
1686 CAUACATT 1669- GUAUGGUCAGCACACUCGGTT 625 CCGAGUGUGCUGA 626
AD-15168 1687 CCAUACTT 1697- GGAUGGCCACAGCCGUCGCTT 627
GCGACGGCUGUGG 628 AD-15174 1715 CCAUCCTT 1698-
GAUGGCCACAGCCGUCGCCTT 629 GGCGACGGCUGUG 630 AD-15325 1716 GCCAUCTT
1806- CAAGCUGGUCUGCCGGGCCTT 631 GGCCCGGCAGACC 632 AD-15326 1824
AGCUUGTT 1815- CUGCCGGGCCCACAACGCUTsT 633 AGCGUUGUGGGCC 634 AD-9570
1833 CGGCAGTsT 1815- cuGccGGGcccAcAAcGcuTsT 635 AGCGUUGUGGGCC 636
AD-9696 1833 CGGcAGTsT 1816- UGCCGGGCCCACAACGCUUTsT 637
AAGCGUUGUGGGC 638 AD-9566 1834 CCGGCATsT 1816-
uGccGGGcccAcAAcGcuuTsT 639 AAGCGUUGUGGGC 640 AD-9692 1834 CCGGcATsT
1818- CCGGGCCCACAACGCUUUUTsT 641 AAAAGCGUUGUGG 642 AD-9532 1836
GCCCGGTsT 1818- ccGGGcccAcAAcGcuuuuTsT 643 AAAAGCGUUGUGG 644
AD-9658 1836 GCCCGGTsT 1820- GGGCCCACAACGCUUUUGGTsT 645
CCAAAAGCGUUGU 646 AD-9549 1838 GGGCCCTsT 1820-
GGGcccAcAAcGcuuuuGGTsT 647 CcAAAAGCGUUGU 648 AD-9675 1838
GGGCCCTsT
1840- GGUGAGGGUGUCUACGCCATsT 649 UGGCGUAGACACC 650 AD-9541 1858
CUCACCTsT 1840- GGuGAGGGuGucuAcGccATsT 651 UGGCGuAGAcACC 652
AD-9667 1858 CUcACCTsT 1843- GAGGGUGUCUACGCCAUUGTsT 653
CAAUGGCGUAGAC 654 AD-9550 1861 ACCCUCTsT 1843-
GAGGGuGucuAcGccAuuGTsT 655 cAAUGGCGuAGAc 656 AD-9676 1861 ACCCUCTsT
1861- GCCAGGUGCUGCCUGCUACTsT 657 GUAGCAGGCAGCA 658 AD-9571 1879
CCUGGCTsT 1861- GccAGGuGcuGccuGcuAcTsT 659 GuAGcAGGcAGcA 660
AD-9697 1879 CCUGGCTsT 1862- CCAGGUGCUGCCUGCUACCTsT 661
GGUAGCAGGCAGC 662 AD-9572 1880 ACCUGGTsT 1862-
ccAGGuGcuGccuGcuAccTsT 663 GGuAGcAGGcAGc 664 AD-9698 1880 ACCUGGTsT
2008- ACCCACAAGCCGCCUGUGCTT 665 GCACAGGCGGCUU 666 AD-15327 2026
GUGGGUTT 2023- GUGCUGAGGCCACGAGGUCTsT 667 GACCUCGUGGCCU 668 AD-9639
2041 CAGCACTsT 2023- GuGcuGAGGccAcGAGGucTsT 669 GACCUCGUGGCCU 670
AD-9765 2041 cAGcACTsT 2024- UGCUGAGGCCACGAGGUCATsT 671
UGACCUCGUGGCC 672 AD-9518 2042 UCAGCATsT 2024-
UGCUGAGGCCACGAGGUCATsT 673 UGACCUCGUGGCC 674 AD-9518 2042 UCAGCATsT
2024- uGcuGAGGccAcGAGGucATsT 675 UGACCUCGUGGCC 676 AD-9644 2042
UcAGcATsT 2024- UfgCfuGfaGfgCfcAfcGfaG 677 P*uGfaCfcUfcG 678
AD-14672 2042 fgUfcAfTsT fuGfgCfcUfcAf gCfaTsT 2024-
UfGCfUfGAGGCfCfACfGAGG 679 UfGACfCfUfCfG 680 AD-14682 2042 UfCfATsT
UfGGCfCfUfCfA GCfATsT 2024- UgCuGaGgCcAcGaGgUcATsT 681
P*uGfaCfcUfcG 682 AD-14692 2042 fuGfgCfcUfcAf gCfaTsT 2024-
UgCuGaGgCcAcGaGgUcATsT 683 UfGACfCfUfCfG 684 AD-14702 2042
UfGGCfCfUfCfA GCfATsT 2024- UfgCfuGfaGfgCfcAfcGfaG 685
UGACCucGUggCC 686 AD-14712 2042 fgUfcAfTsT UCAgcaTsT 2024-
UfGCfUfGAGGCfCfACfGAGG 687 UGACCucGUggCC 688 AD-14722 2042 UfCfATsT
UCAgcaTsT 2024- UgCuGaGgCcAcGaGgUcATsT 689 UGACCucGUggCC 690
AD-14732 2042 UCAgcaTsT 2024- GfuGfgUfcAfgCfgGfcCfgG 691
P*cAfuCfcCfgG 692 AD-15078 2042 fgAfuGfTsT fcCfgCfuGfaCf cAfcTsT
2024- GUfGGUfCfAGCfGGCfCfGGG 693 CfAUfCfCfCfGG 694 AD-15088 2042
AUfGTsT CfCfGCfUfGACf CfACfTsT 2024- GuGgUcAgCgGcCgGgAuGTsT 695
P*cAfuCfcCfgG 696 AD-15098 2042 fcCfgCfuGfaCf cAfcTsT 2024-
GuGgUcAgCgGcCgGgAuGTsT 697 CfAUfCfCfCfGG 698 AD-15108 2042
CfCfGCfUfGACf CfACfTsT 2024- GfuGfgUfcAfgCfgGfcCfgG 699
CAUCCcgGCcgCU 700 AD-15118 2042 fgAfuGfTsT GACcacTsT 2024-
GUfGGUfCfAGCfGGCfCfGGG 701 CAUCCcgGCcgCU 702 AD-15128 2042 AUfGTsT
GACcacTsT 2024- GuGgUcAgCgGcCgGgAuGTsT 703 CAUCCcgGCcgCU 704
AD-15138 2042 GACcacTsT 2030- GGCCACGAGGUCAGCCCAATT 705
UUGGGCUGACCUC 706 AD-15237 2048 GUGGCCTT 2035-
CGAGGUCAGCCCAACCAGUTT 707 ACUGGUUGGGCUG 708 AD-15287 2053 ACCUCGTT
2039- GUCAGCCCAACCAGUGCGUTT 709 ACGCACUGGUUGG 710 AD-15238 2057
GCUGACTT 2041- CAGCCCAACCAGUGCGUGGTT 711 CCACGCACUGGUU 712 AD-15328
2059 GGGCUGTT 2062- CACAGGGAGGCCAGCAUCCTT 713 GGAUGCUGGCCUC 714
AD-15399 2080 CCUGUGTT 2072- CCAGCAUCCACGCUUCCUGTsT 715
CAGGAAGCGUGGA 716 AD-9582 2090 UGCUGGTsT 2072-
ccAGcAuccAcGcuuccuGTsT 717 cAGGAAGCGUGGA 718 AD-9708 2090 UGCUGGTsT
2118- AGUCAAGGAGCAUGGAAUCTsT 719 GAUUCCAUGCUCC 720 AD-9545 2136
UUGACUTsT 2118- AGucAAGGAGcAuGGAAucTsT 721 GAUUCcAUGCUCC 722
AD-9671 2136 UUGACUTsT 2118- AfgUfcAfaGfgAfgCfaUfgG 723
P*gAfuUfcCfaU 724 AD-14674 2136 faAfuCfTsT fgCfuCfcUfuGf aCfuTsT
2118- AGUfCfAAGGAGCfAUfGGAAU 725 GAUfUfCfCfAUf 726 AD-14684 2136
fCfTsT GCfUfCfCfUfUf GACfUfTsT 2118- AgUcAaGgAgCaUgGaAuCTsT 727
P*gAfuUfcCfaU 728 AD-14694 2136 fgCfuCfcUfuGf aCfuTsT 2118-
AgUcAaGgAgCaUgGaAuCTsT 729 GAUfUfCfCfAUf 730 AD-14704 2136
GCfUfCfCfUfUf GACfUfTsT 2118- AfgUfcAfaGfgAfgCfaUfgG 731
GAUUCcaUGcuCC 732 AD-14714 2136 faAfuCfTsT UUGacuTsT 2118-
AGUfCfAAGGAGCfAUfGGAAU 733 GAUUCcaUGcuCC 734 AD-14724 2136 fCfTsT
UUGacuTsT 2118- AgUcAaGgAgCaUgGaAuCTsT 735 GAUUCcaUGcuCC 736
AD-14734 2136 UUGacuTsT 2118- GfcGfgCfaCfcCfuCfaUfaG 737
P*aGfgCfcUfaU 738 AD-15080 2136 fgCfcUfTsT fgAfgGfgUfgCf cGfcTsT
2118- GCfGGCfACfCfCfUfCfAUfA 739 AGGCfCfUfAUfG 740 AD-15090 2136
GGCfCfUfTsT AGGGUfGCfCfGC fTsT 2118- GcGgCaCcCuCaUaGgCcUTsT 741
P*aGfgCfcUfaU 742 AD-15100 2136 fgAfgGfgUfgCf cGfcTsT 2118-
GcGgCaCcCuCaUaGgCcUTsT 743 AGGCfCfUfAUfG 744 AD-15110 2136
AGGGUfGCfCfGC fTsT 2118- GfcGfgCfaCfcCfuCfaUfaG 745 AGGCCuaUGagGG
746 AD-15120 2136 fgCfcUfTsT UGCcgcTsT 2118- GCfGGCfACfCfCfUfCfAUfA
747 AGGCCuaUGagGG 748 AD-15130 2136 GGCfCfUfTsT UGCcgcTsT 2118-
GcGgCaCcCuCaUaGgCcUTsT 749 AGGCCuaUGagGG 750 AD-15140 2136
UGCcgcTsT 2122- AAGGAGCAUGGAAUCCCGGTsT 751 CCGGGAUUCCAUG 752
AD-9522 2140 CUCCUUTsT 2122- AAGGAGcAuGGAAucccGGTsT 753
CCGGGAUUCcAUG 754 AD-9648 2140 CUCCUUTsT 2123-
AGGAGCAUGGAAUCCCGGCTsT 755 GCCGGGAUUCCAU 756 AD-9552 2141 GCUCCUTsT
2123- AGGAGcAuGGAAucccGGcTsT 757 GCCGGGAUUCcAU 758 AD-9678 2141
GCUCCUTsT 2125- GAGCAUGGAAUCCCGGCCCTsT 759 GGGCCGGGAUUCC 760
AD-9618 2143 AUGCUCTsT 2125- GAGcAuGGAAucccGGcccTsT 761
GGGCCGGGAUUCc 762 AD-9744 2143 AUGCUCTsT 2230-
GCCUACGCCGUAGACAACATT 763 UGUUGUCUACGGC 764 AD-15239 2248 GUAGGCTT
2231- CCUACGCCGUAGACAACACTT 765 GUGUUGUCUACGG 766 AD-15212 2249
CGUAGGTT 2232- CUACGCCGUAGACAACACGTT 767 CGUGUUGUCUACG 768 AD-15240
2250 GCGUAGTT 2233- UACGCCGUAGACAACACGUTT 769 ACGUGUUGUCUAC 770
AD-15177 2251 GGCGUATT 2235- CGCCGUAGACAACACGUGUTT 771
ACACGUGUUGUCU 772 AD-15179 2253 ACGGCGTT 2236-
GCCGUAGACAACACGUGUGTT 773 CACACGUGUUGUC 774 AD-15180 2254 UACGGCTT
2237- CCGUAGACAACACGUGUGUTT 775 ACACACGUGUUGU 776 AD-15241 2255
CUACGGTT 2238- CGUAGACAACACGUGUGUATT 777 UACACACGUGUUG 778 AD-15268
2256 UCUACGTT 2240- UAGACAACACGUGUGUAGUTT 779 ACUACACACGUGU 780
AD-15242 2258 UGUCUATT 2241- AGACAACACGUGUGUAGUCTT 781
GACUACACACGUG 782 AD-15216 2259 UUGUCUTT 2242-
GACAACACGUGUGUAGUCATT 783 UGACUACACACGU 784 AD-15176 2260 GUUGUCTT
2243- ACAACACGUGUGUAGUCAGTT 785 CUGACUACACACG 786 AD-15181 2261
UGUUGUTT 2244- CAACACGUGUGUAGUCAGGTT 787 CCUGACUACACAC 788 AD-15243
2262 GUGUUGTT 2247- CACGUGUGUAGUCAGGAGCTT 789 GCUCCUGACUACA 790
AD-15182 2265 CACGUGTT 2248- ACGUGUGUAGUCAGGAGCCTT 791
GGCUCCUGACUAC 792 AD-15244 2266 ACACGUTT 2249-
CGUGUGUAGUCAGGAGCCGTT 793 CGGCUCCUGACUA 794 AD-15387 2267 CACACGTT
2251- UGUGUAGUCAGGAGCCGGGTT 795 CCCGGCUCCUGAC 796 AD-15245 2269
UACACATT 2257- GUCAGGAGCCGGGACGUCATsT 797 UGACGUCCCGGCU 798 AD-9555
2275 CCUGACTsT 2257- GucAGGAGccGGGAcGucATsT 799 UGACGUCCCGGCU 800
AD-9681 2275 CCUGACTsT 2258- UCAGGAGCCGGGACGUCAGTsT 801
CUGACGUCCCGGC 802 AD-9619 2276 UCCUGATsT 2258-
ucAGGAGccGGGAcGucAGTsT 803 CUGACGUCCCGGC 804 AD-9745 2276
UCCUGATsT
2259- CAGGAGCCGGGACGUCAGCTsT 805 GCUGACGUCCCGG 806 AD-9620 2277
CUCCUGTsT 2259- cAGGAGccGGGAcGucAGcTsT 807 GCUGACGUCCCGG 808
AD-9746 2277 CUCCUGTsT 2263- AGCCGGGACGUCAGCACUATT 809
UAGUGCUGACGUC 810 AD-15288 2281 CCGGCUTT 2265-
CCGGGACGUCAGCACUACATT 811 UGUAGUGCUGACG 812 AD-15246 2283 UCCCGGTT
2303- CCGUGACAGCCGUUGCCAUTT 813 AUGGCAACGGCUG 814 AD-15289 2321
UCACGGTT 2317- GCCAUCUGCUGCCGGAGCCTsT 815 GGCUCCGGCAGCA 816 AD-9324
2335 GAUGGCTsT 2375- CCCAUCCCAGGAUGGGUGUTT 817 ACACCCAUCCUGG 818
AD-15329 2393 GAUGGGTT 2377- CAUCCCAGGAUGGGUGUCUTT 819
AGACACCCAUCCU 820 AD-15330 2395 GGGAUGTT 2420- AGCUUMAAAUGGUUCCGATT
821 UCGGAACCAUUUU 822 AD-15169 2438 AAAGCUTT 2421-
GCUUUAAAAUGGUUCCGACTT 823 GUCGGAACCAUUU 824 AD-15201 2439 UAAAGCTT
2422- CUUMAAAUGGUUCCGACUTT 825 AGUCGGAACCAUU 826 AD-15331 2440
UUAAAGTT 2423- UUUAAAAUGGUUCCGACUUTT 827 AAGUCGGAACCAU 828 AD-15190
2441 UUUAAATT 2424- UUAAAAUGGUUCCGACUUGTT 829 CAAGUCGGAACCA 830
AD-15247 2442 UUUUAATT 2425- UAAAAUGGUUCCGACUUGUTT 831
ACAAGUCGGAACC 832 AD-15248 2443 AUUUUATT 2426-
AAAAUGGUUCCGACUUGUCTT 833 GACAAGUCGGAAC 834 AD-15175 2444 CAUUUUTT
2427- AAAUGGUUCCGACUUGUCCTT 835 GGACAAGUCGGAA 836 AD-15249 2445
CCAUUUTT 2428- AAUGGUUCCGACUUGUCCCTT 837 GGGACAAGUCGGA 838 AD-15250
2446 ACCAUUTT 2431- GGUUCCGACUUGUCCCUCUTT 839 AGAGGGACAAGUC 840
AD-15400 2449 GGAACCTT 2457- CUCCAUGGCCUGGCACGAGTT 841
CUCGUGCCAGGCC 842 AD-15332 2475 AUGGAGTT 2459-
CCAUGGCCUGGCACGAGGGTT 843 CCCUCGUGCCAGG 844 AD-15388 2477 CCAUGGTT
2545- GAACUCACUCACUCUGGGUTT 845 ACCCAGAGUGAGU 846 AD-15333 2563
GAGUUCTT 2549- UCACUCACUCUGGGUGCCUTT 847 AGGCACCCAGAGU 848 AD-15334
2567 GAGUGATT 2616- UUUCACCAUUCAAACAGGUTT 849 ACCUGUUUGAAUG 850
AD-15335 2634 GUGAAATT 2622- CAUUCAAACAGGUCGAGCUTT 851
AGCUCGACCUGUU 852 AD-15183 2640 UGAAUGTT 2623-
AUUCAAACAGGUCGAGCUGTT 853 CAGCUCGACCUGU 854 AD-15202 2641 UUGAAUTT
2624- UUCAAACAGGUCGAGCUGUTT 855 ACAGCUCGACCUG 856 AD-15203 2642
UUUGAATT 2625- UCAAACAGGUCGAGCUGUGTT 857 CACAGCUCGACCU 858 AD-15272
2643 GUUUGATT 2626- CAAACAGGUCGAGCUGUGCTT 859 GCACAGCUCGACC 860
AD-15217 2644 UGUUUGTT 2627- AAACAGGUCGAGCUGUGCUTT 861
AGCACAGCUCGAC 862 AD-15290 2645 CUGUUUTT 2628-
AACAGGUCGAGCUGUGCUCTT 863 GAGCACAGCUCGA 864 AD-15218 2646 CCUGUUTT
2630- CAGGUCGAGCUGUGCUCGGTT 865 CCGAGCACAGCUC 866 AD-15389 2648
GACCUGTT 2631- AGGUCGAGCUGUGCUCGGGTT 867 CCCGAGCACAGCU 868 AD-15336
2649 CGACCUTT 2633- GUCGAGCUGUGCUCGGGUGTT 869 CACCCGAGCACAG 870
AD-15337 2651 CUCGACTT 2634- UCGAGCUGUGCUCGGGUGCTT 871
GCACCCGAGCACA 872 AD-15191 2652 GCUCGATT 2657-
AGCUGCUCCCAAUGUGCCGTT 873 CGGCACAUUGGGA 874 AD-15390 2675 GCAGCUTT
2658- GCUGCUCCCAAUGUGCCGATT 875 UCGGCACAUUGGG 876 AD-15338 2676
AGCAGCTT 2660- UGCUCCCAAUGUGCCGAUGTT 877 CAUCGGCACAUUG 878 AD-15204
2678 GGAGCATT 2663- UCCCAAUGUGCCGAUGUCCTT 879 GGACAUCGGCACA 880
AD-15251 2681 UUGGGATT 2665- CCAAUGUGCCGAUGUCCGUTT 881
ACGGACAUCGGCA 882 AD-15205 2683 CAUUGGTT 2666-
CAAUGUGCCGAUGUCCGUGTT 883 CACGGACAUCGGC 884 AD-15171 2684 ACAUUGTT
2667- AAUGUGCCGAUGUCCGUGGTT 885 CCACGGACAUCGG 886 AD-15252 2685
CACAUUTT 2673- CCGAUGUCCGUGGGCAGAATT 887 UUCUGCCCACGGA 888 AD-15339
2691 CAUCGGTT 2675- GAUGUCCGUGGGCAGAAUGTT 889 CAUUCUGCCCACG 890
AD-15253 2693 GACAUCTT 2678- GUCCGUGGGCAGAAUGACUTT 891
AGUCAUUCUGCCC 892 AD-15340 2696 ACGGACTT 2679-
UCCGUGGGCAGAAUGACUUTT 893 AAGUCAUUCUGCC 894 AD-15291 2697 CACGGATT
2683- UGGGCAGAAUGACUUDUAUTT 895 AUAAAAGUCAUUC 896 AD-15341 2701
UGCCCATT 2694- ACUUUUAUUGAGCUCUUGUTT 897 ACAAGAGCUCAAU 898 AD-15401
2712 AAAAGUTT 2700- AUUGAGCUCUUGUUCCGUGTT 899 CACGGAACAAGAG 900
AD-15342 2718 CUCAAUTT 2704- AGCUCUUGUUCCGUGCCAGTT 901
CUGGCACGGAACA 902 AD-15343 2722 AGAGCUTT 2705-
GCUCUUGUUCCGUGCCAGGTT 903 CCUGGCACGGAAC 904 AD-15292 2723 AAGAGCTT
2710- UGUUCCGUGCCAGGCAUUCTT 905 GAAUGCCUGGCAC 906 AD-15344 2728
GGAACATT 2711- GUUCCGUGCCAGGCAUUCATT 907 UGAAUGCCUGGCA 908 AD-15254
2729 CGGAACTT 2712- UUCCGUGCCAGGCAUUCAATT 909 UUGAAUGCCUGGC 910
AD-15345 2730 ACGGAATT 2715- CGUGCCAGGCAUUCAAUCCTT 911
GGAUUGAAUGCCU 912 AD-15206 2733 GGCACGTT 2716-
GUGCCAGGCAUUCAAUCCUTT 913 AGGAUUGAAUGCC 914 AD-15346 2734 UGGCACTT
2728- CAAUCCUCAGGUCUCCACCTT 915 GGUGGAGACCUGA 916 AD-15347 2746
GGAUUGTT 2743- CACCAAGGAGGCAGGAUUCTsT 917 GAAUCCUGCCUCC 918 AD-9577
2761 UUGGUGTsT 2743- cAccAAGGAGGcAGGAuucTsT 919 GAAUCCUGCCUCC 920
AD-9703 2761 UUGGUGTsT 2743- CfaCfcAfaGfgAfgGfcAfgG 921
P*gAfaUfcCfuG 922 AD-14678 2761 faUfuCfTsT fcCfuCfcUfuGf gUfgTsT
2743- CfACfCfAAGGAGGCfAGGAUf 923 GAAUfCfCfUfGC 924 AD-14688 2761
UfCfTsT fCfUfCfCfUfUf GGUfGTsT 2743- CaCcAaGgAgGcAgGaUuCTsT 925
P*gAfaUfcCfuG 926 AD-14698 2761 fcCfuCfcUfuGf gUfgTsT 2743-
CaCcAaGgAgGcAgGaUuCTsT 927 GAAUfCfCfUfGC 928 AD-14708 2761
fCfUfCfCfUfUf GGUfGTsT 2743- CfaCfcAfaGfgAfgGfcAfgG 929
GAAUCcuGCcuCC 930 AD-14718 2761 faUfuCfTsT UUGgugTsT 2743-
CfACfCfAAGGAGGCfAGGAUf 931 GAAUCcuGCcuCC 932 AD-14728 2761 UfCfTsT
UUGgugTsT 2743- CaCcAaGgAgGcAgGaUuCTsT 933 GAAUCcuGCcuCC 934
AD-14738 2761 UUGgugTsT 2743- GfgCfcUfgGfaGfuUfuAfuU 935
P*uCfcGfaAfuA 936 AD-15084 2761 fcGfgAfTsT faAfcUfcCfaGf gCfcTsT
2743- GGCfCfUfGGAGUfUfUfAUfU 937 UfCfCfGAAUfAA 938 AD-15094 2761
fCfGGATsT ACfUfCfCfAGGC fCfTsT 2743- GgCcUgGaGuUuAuUcGgATsT 939
P*uCfcGfaAfuA 940 AD-15104 2761 faAfcUfcCfaGf gCfcTsT 2743-
GgCcUgGaGuUuAuUcGgATsT 941 UfCfCfGAAUfAA 942 AD-15114 2761
ACfUfCfCfAGGC fCfTsT 2743- GfgCfcUfgGfaGfuUfuAfuU 943 UCCGAauAAacUC
944 AD-15124 2761 fcGfgAfTsT CAGgccTsT 2743- GGCfCfUfGGAGUfUfUfAUfU
945 UCCGAauAAacUC 946 AD-15134 2761 fCfGGATsT CAGgccTsT 2743-
GgCcUgGaGuUuAuUcGgATsT 947 UCCGAauAAacUC 948 AD-15144 2761
CAGgccTsT 2753- GCAGGAUUCUUCCCAUGGATT 949 UCCAUGGGAAGAA 950
AD-15391 2771 UCCUGCTT 2794- UGCAGGGACAAACAUCGUUTT 951
AACGAUGUUUGUC 952 AD-15348 2812 CCUGCATT 2795-
GCAGGGACAAACAUCGUUGTT 953 CAACGAUGUUUGU 954 AD-15349 2813 CCCUGCTT
2797- AGGGACAAACAUCGUUGGGTT 955 CCCAACGAUGUUU 956 AD-15170 2815
GUCCCUTT 2841- CCCUCAUCUCCAGCUAACUTT 957 AGUUAGCUGGAGA 958 AD-15350
2859 UGAGGGTT 2845- CAUCUCCAGCUAACUGUGGTT 959 CCACAGUUAGCUG 960
AD-15402 2863 GAGAUGTT 2878- GCUCCCUGAUUAAUGGAGGTT 961
CCUCCAUUAAUCA 962 AD-15293 2896 GGGAGCTT 2881-
CCCUGAUUAAUGGAGGCUUTT 963 AAGCCUCCAUUAA 964 AD-15351 2899 UCAGGGTT
2882- CCUGAUUAAUGGAGGCUUATT 965 UAAGCCUCCAUUA 966 AD-15403 2900
AUCAGGTT
2884- UGAUUAAUGGAGGCUUAGCTT 967 GCUAAGCCUCCAU 968 AD-15404 2902
UAAUCATT 2885- GAUUAAUGGAGGCUUAGCUTT 969 AGCUAAGCCUCCA 970 AD-15207
2903 UUAAUCTT 2886- AUUAAUGGAGGCUUAGCUUTT 971 AAGCUAAGCCUCC 972
AD-15352 2904 AUUAAUTT 2887- UUAAUGGAGGCUUAGCUUUTT 973
AAAGCUAAGCCUC 974 AD-15255 2905 CAUUAATT 2903-
UUUCUGGAUGGCAUCUAGCTsT 975 GCUAGAUGCCAUC 976 AD-9603 2921 CAGAAATsT
2903- uuucuGGAuGGcAucuAGcTsT 977 GCuAGAUGCcAUC 978 AD-9729 2921
cAGAAATsT 2904- UUCUGGAUGGCAUCUAGCCTsT 979 GGCUAGAUGCCAU 980
AD-9599 2922 CCAGAATsT 2904- uucuGGAuGGcAucuAGccTsT 981
GGCuAGAUGCcAU 982 AD-9725 2922 CcAGAATsT 2905-
UCUGGAUGGCAUCUAGCCATsT 983 UGGCUAGAUGCCA 984 AD-9621 2923 UCCAGATsT
2905- ucuGGAuGGcAucuAGccATsT 985 UGGCuAGAUGCcA 986 AD-9747 2923
UCcAGATsT 2925- AGGCUGGAGACAGGUGCGCTT 987 GCGCACCUGUCUC 988
AD-15405 2943 CAGCCUTT 2926- GGCUGGAGACAGGUGCGCCTT 989
GGCGCACCUGUCU 990 AD-15353 2944 CCAGCCTT 2927-
GCUGGAGACAGGUGCGCCCTT 991 GGGCGCACCUGUC 992 AD-15354 2945 UCCAGCTT
2972- UUCCUGAGCCACCUUUACUTT 993 AGUAAAGGUGGCU 994 AD-15406 2990
CAGGAATT 2973- UCCUGAGCCACCUUUACUCTT 995 GAGUAAAGGUGGC 996 AD-15407
2991 UCAGGATT 2974- CCUGAGCCACCUUUACUCUTT 997 AGAGUAAAGGUGG 998
AD-15355 2992 CUCAGGTT 2976- UGAGCCACCUUUACUCUGCTT 999
GCAGAGUAAAGGU 1000 AD-15356 2994 GGCUCATT 2978-
AGCCACCUUUACUCUGCUCTT 1001 GAGCAGAGUAAAG 1002 AD-15357 2996
GUGGCUTT 2981- CACCUUUACUCUGCUCUAUTT 1003 AUAGAGCAGAGUA 1004
AD-15269 2999 AAGGUGTT 2987- UACUCUGCUCUAUGCCAGGTsT 1005
CCUGGCAUAGAGC 1006 AD-9565 3005 AGAGUATsT 2987-
uAcucuGcucuAuGccAGGTsT 1007 CCUGGcAuAGAGc 1008 AD-9691 3005
AGAGuATsT 2998- AUGCCAGGCUGUGCUAGCATT 1009 UGCUAGCACAGCC 1010
AD-15358 3016 UGGCAUTT 3003- AGGCUGUGCUAGCAACACCTT 1011
GGUGUUGCUAGCA 1012 AD-15359 3021 CAGCCUTT 3006-
CUGUGCUAGCAACACCCAATT 1013 UUGGGUGUUGCUA 1014 AD-15360 3024
GCACAGTT 3010- GCUAGCAACACCCAAAGGUTT 1015 ACCUUUGGGUGUU 1016
AD-15219 3028 GCUAGCTT 3038- GGAGCCAUCACCUAGGACUTT 1017
AGUCCUAGGUGAU 1018 AD-15361 3056 GGCUCCTT 3046-
CACCUAGGACUGACUCGGCTT 1019 GCCGAGUCAGUCC 1020 AD-15273 3064
UAGGUGTT 3051- AGGACUGACUCGGCAGUGUTT 1021 ACACUGCCGAGUC 1022
AD-15362 3069 AGUCCUTT 3052- GGACUGACUCGGCAGUGUGTT 1023
CACACUGCCGAGU 1024 AD-15192 3070 CAGUCCTT 3074-
UGGUGCAUGCACUGUCUCATT 1025 UGAGACAGUGCAU 1026 AD-15256 3092
GCACCATT 3080- AUGCACUGUCUCAGCCAACTT 1027 GUUGGCUGAGACA 1028
AD-15363 3098 GUGCAUTT 3085- CUGUCUCAGCCAACCCGCUTT 1029
AGCGGGUUGGCUG 1030 AD-15364 3103 AGACAGTT 3089-
CUCAGCCAACCCGCUCCACTsT 1031 GUGGAGCGGGUUG 1032 AD-9604 3107
GCUGAGTsT 3089- cucAGccAAcccGcuccAcTsT 1033 GUGGAGCGGGUUG 1034
AD-9730 3107 GCUGAGTsT 3093- GCCAACCCGCUCCACUACCTsT 1035
GGUAGUGGAGCGG 1036 AD-9527 3111 GUUGGCTsT 3093-
GccAAcccGcuccAcuAccTsT 1037 GGuAGUGGAGCGG 1038 AD-9653 3111
GUUGGCTsT 3096- AACCCGCUCCACUACCCGGTT 1039 CCGGGUAGUGGAG 1040
AD-15365 3114 CGGGUUTT 3099- CCGCUCCACUACCCGGCAGTT 1041
CUGCCGGGUAGUG 1042 AD-15294 3117 GAGCGGTT 3107-
CUACCCGGCAGGGUACACATT 1043 UGUGUACCCUGCC 1044 AD-15173 3125
GGGUAGTT 3108- UACCCGGCAGGGUACACAUTT 1045 AUGUGUACCCUGC 1046
AD-15366 3126 CGGGUATT 3109- ACCCGGCAGGGUACACAUUTT 1047
AAUGUGUACCCUG 1048 AD-15367 3127 CCGGGUTT 3110-
CCCGGCAGGGUACACAUUCTT 1049 GAAUGUGUACCCU 1050 AD-15257 3128
GCCGGGTT 3112- CGGCAGGGUACACAUUCGCTT 1051 GCGAAUGUGUACC 1052
AD-15184 3130 CUGCCGTT 3114- GCAGGGUACACAUUCGCACTT 1053
GUGCGAAUGUGUA 1054 AD-15185 3132 CCCUGCTT 3115-
CAGGGUACACAUUCGCACCTT 1055 GGUGCGAAUGUGU 1056 AD-15258 3133
ACCCUGTT 3116- AGGGUACACAUUCGCACCCTT 1057 GGGUGCGAAUGUG 1058
AD-15186 3134 UACCCUTT 3196- GGAACUGAGCCAGAAACGCTT 1059
GCGUUUCUGGCUC 1060 AD-15274 3214 AGUUCCTT 3197-
GAACUGAGCCAGAAACGCATT 1061 UGCGUUUCUGGCU 1062 AD-15368 3215
CAGUUCTT 3198- AACUGAGCCAGAAACGCAGTT 1063 CUGCGUUUCUGGC 1064
AD-15369 3216 UCAGUUTT 3201- UGAGCCAGAAACGCAGAUUTT 1065
AAUCUGCGUUUCU 1066 AD-15370 3219 GGCUCATT 3207-
AGAAACGCAGAUUGGGCUGTT 1067 CAGCCCAAUCUGC 1068 AD-15259 3225
GUUUCUTT 3210- AACGCAGAUUGGGCUGGCUTT 1069 AGCCAGCCCAAUC 1070
AD-15408 3228 UGCGUUTT 3233- AGCCAAGCCUCUUCUUACUTsT 1071
AGUAAGAAGAGGC 1072 AD-9597 3251 UUGGCUTsT 3233-
AGccAAGccucuucuuAcuTsT 1073 AGuAAGAAGAGGC 1074 AD-9723 3251
UUGGCUTsT 3233- AfgCfcAfaGfcCfuCfuUfcU 1075 P*aGfuAfaGfaA 1076
AD-14680 3251 fuAfcUfTsT fgAfgGfcUfuGf gCfuTsT 3233-
AGCfCfAAGCfCfUfCfUfUfC 1077 AGUfAAGAAGAGG 1078 AD-14690 3251
fUfUfACfUfTsT CfUfUfGGCfUfT sT 3233- AgCcAaGcCuCuUcUuAcUTsT 1079
P*aGfuAfaGfaA 1080 AD-14700 3251 fgAfgGfcUfuGf gCfuTsT 3233-
AgCcAaGcCuCuUcUuAcUTsT 1081 AGUfAAGAAGAGG 1082 AD-14710 3251
CfUfUfGGCfUfT sT 3233- AfgCfcAfaGfcCfuCfuUfcU 1083 AGUAAgaAGagGC
1084 AD-14720 3251 fuAfcUfTsT UUGgcuTsT 3233-
AGCfCfAAGCfCfUfCfUfUfC 1085 AGUAAgaAGagGC 1086 AD-14730 3251
fUfUfACfUfTsT UUGgcuTsT 3233- AgCcAaGcCuCuUcUuAcUTsT 1087
AGUAAgaAGagGC 1088 AD-14740 3251 UUGgcuTsT 3233-
UfgGfuUfcCfcUfgAfgGfaC 1089 P*gCfuGfgUfcC 1090 AD-15086 3251
fcAfgCfTsT fuCfaGfgGfaAf cCfaTsT 3233- UfGGUfUfCfCfCfUfGAGGAC 1091
GCfUfGGUfCfCf 1092 AD-15096 3251 fCfAGCfTsT UfCfAGGGAACfC fATsT
3233- UgGuUcCcUgAgGaCcAgCTsT 1093 P*gCfuGfgUfcC 1094 AD-15106 3251
fuCfaGfgGfaAf cCfaTsT 3233- UgGuUcCcUgAgGaCcAgCTsT 1095
GCfUfGGUfCfCf 1096 AD-15116 3251 UfCfAGGGAACfC fATsT 3233-
UfgGfuUfcCfcUfgAfgGfaC 1097 GCUGGucCUcaGG 1098 AD-15126 3251
fcAfgCfTsT GAAccaTsT 3233- UfGGUfUfCfCfCfUfGAGGAC 1099
GCUGGucCUcaGG 1100 AD-15136 3251 fCfAGCfTsT GAAccaTsT 3233-
UgGuUcCcUgAgGaCcAgCTsT 1101 GCUGGucCUcaGG 1102 AD-15146 3251
GAAccaTsT 3242- UCUUCUUACUUCACCCGGCTT 1103 GCCGGGUGAAGUA 1104
AD-15260 3260 AGAAGATT 3243- CUUCUUACUUCACCCGGCUTT 1105
AGCCGGGUGAAGU 1106 AD-15371 3261 AAGAAGTT 3244-
UUCUUACUUCACCCGGCUGTT 1107 CAGCCGGGUGAAG 1108 AD-15372 3262
UAAGAATT 3262- GGGCUCCUCAUUUUUACGGTT 1109 CCGUAAAAAUGAG 1110
AD-15172 3280 GAGCCCTT 3263- GGCUCCUCAUUUUUACGGGTT 1111
CCCGUAAAAAUGA 1112 AD-15295 3281 GGAGCCTT 3264-
GCUCCUCAUUUUUACGGGUTT 1113 ACCCGUAAAAAUG 1114 AD-15373 3282
AGGAGCTT 3265- CUCCUCAUUUUUACGGGUATT 1115 UACCCGUAAAAAU 1116
AD-15163 3283 GAGGAGTT 3266- UCCUCAUUUUUACGGGUAATT 1117
UUACCCGUAAAAA 1118 AD-15165 3284 UGAGGATT 3267-
CCUCAUUUUUACGGGUAACTT 1119 GUUACCCGUAAAA 1120 AD-15374 3285
AUGAGGTT 3268- CUCAUUUUUACGGGUAACATT 1121 UGUUACCCGUAAA 1122
AD-15296 3286 AAUGAGTT 3270- CAUUUUUACGGGUAACAGUTT 1123
ACUGUUACCCGUA 1124 AD-15261 3288 AAAAUGTT 3271-
AUUUUUACGGGUAACAGUGTT 1125 CACUGUUACCCGU 1126 AD-15375 3289
AAAAAUTT 3274- UUUACGGGUAACAGUGAGGTT 1127 CCUCACUGUUACC 1128
AD-15262 3292 CGUAAATT
3308- CAGACCAGGAAGCUCGGUGTT 1129 CACCGAGCUUCCU 1130 AD-15376 3326
GGUCUGTT 3310- GACCAGGAAGCUCGGUGAGTT 1131 CUCACCGAGCUUC 1132
AD-15377 3328 CUGGUCTT 3312- CCAGGAAGCUCGGUGAGUGTT 1133
CACUCACCGAGCU 1134 AD-15409 3330 UCCUGGTT 3315-
GGAAGCUCGGUGAGUGAUGTT 1135 CAUCACUCACCGA 1136 AD-15378 3333
GCUUCCTT 3324- GUGAGUGAUGGCAGAACGATT 1137 UCGUUCUGCCAUC 1138
AD-15410 3342 ACUCACTT 3326- GAGUGAUGGCAGAACGAUGTT 1139
CAUCGUUCUGCCA 1140 AD-15379 3344 UCACUCTT 3330-
GAUGGCAGAACGAUGCCUGTT 1141 CAGGCAUCGUUCU 1142 AD-15187 3348
GCCAUCTT 3336- AGAACGAUGCCUGCAGGCATT 1143 UGCCUGCAGGCAU 1144
AD-15263 3354 CGUUCUTT 3339- ACGAUGCCUGCAGGCAUGGTT 1145
CCAUGCCUGCAGG 1146 AD-15264 3357 CAUCGUTT 3348-
GCAGGCAUGGAACUUUUUCTT 1147 GAAAAAGUUCCAU 1148 AD-15297 3366
GCCUGCTT 3356- GGAACUUUUUCCGUUAUCATT 1149 UGAUAACGGAAAA 1150
AD-15208 3374 AGUUCCTT 3357- GAACUUUUUCCGUUAUCACTT 1151
GUGAUAACGGAAA 1152 AD-15209 3375 AAGUUCTT 3358-
AACUUUUUCCGUUAUCACCTT 1153 GGUGAUAACGGAA 1154 AD-15193 3376
AAAGUUTT 3370- UAUCACCCAGGCCUGAUUCTT 1155 GAAUCAGGCCUGG 1156
AD-15380 3388 GUGAUATT 3378- AGGCCUGAUUCACUGGCCUTT 1157
AGGCCAGUGAAUC 1158 AD-15298 3396 AGGCCUTT 3383-
UGAUUCACUGGCCUGGCGGTT 1159 CCGCCAGGCCAGU 1160 AD-15299 3401
GAAUCATT 3385- AUUCACUGGCCUGGCGGAGTT 1161 CUCCGCCAGGCCA 1162
AD-15265 3403 GUGAAUTT 3406- GCUUCUAAGGCAUGGUCGGTT 1163
CCGACCAUGCCUU 1164 AD-15381 3424 AGAAGCTT 3407-
CUUCUAAGGCAUGGUCGGGTT 1165 CCCGACCAUGCCU 1166 AD-15210 3425
UAGAAGTT 3429- GAGGGCCAACAACUGUCCCTT 1167 GGGACAGUUGUUG 1168
AD-15270 3447 GCCCUCTT 3440- ACUGUCCCUCCUUGAGCACTsT 1169
GUGCUCAAGGAGG 1170 AD-9591 3458 GACAGUTsT 3440-
AcuGucccuccuuGAGcAcTsT 1171 GUGCUcAAGGAGG 1172 AD-9717 3458
GAcAGUTsT 3441- CUGUCCCUCCUUGAGCACCTsT 1173 GGUGCUCAAGGAG 1174
AD-9622 3459 GGACAGTsT 3441- cuGucccuccuuGAGcAccTsT 1175
GGUGCUcAAGGAG 1176 AD-9748 3459 GGAcAGTsT 3480-
ACAUUUAUCUUUUGGGUCUTsT 1177 AGACCCAAAAGAU 1178 AD-9587 3498
AAAUGUTsT 3480- AcAuuuAucuuuuGGGucuTsT 1179 AGACCcAAAAGAu 1180
AD-9713 3498 AAAUGUTsT 3480- AfcAfuUfuAfuCfuUfuUfgG 1181
P*aGfaCfcCfaA 1182 AD-14679 3498 fgUfcUfTsT faAfgAfuAfaAf uGfuTsT
3480- ACfAUfUfUfAUfCfUfUfUfU 1183 AGACfCfCfAAAA 1184 AD-14689 3498
fGGGUfCfUfTsT GAUfAAAUfGUfT sT 3480- AcAuUuAuCuUuUgGgUcUTsT 1185
P*aGfaCfcCfaA 1186 AD-14699 3498 faAfgAfuAfaAf uGfuTsT 3480-
AcAuUuAuCuUuUgGgUcUTsT 1187 AGACfCfCfAAAA 1188 AD-14709 3498
GAUfAAAUfGUfT sT 3480- AfcAfuUfuAfuCfuUfuUfgG 1189 AGACCcaAAagAU
1190 AD-14719 3498 fgUfcUfTsT AAAuguTsT 3480-
ACfAUfUfUfAUfCfUfUfUfU 1191 AGACCcaAAagAU 1192 AD-14729 3498
fGGGUfCfUfTsT AAAuguTsT 3480- AcAuUuAuCuUuUgGgUcUTsT 1193
AGACCcaAAagAU 1194 AD-14739 3498 AAAuguTsT 3480-
GfcCfaUfcUfgCfuGfcCfgG 1195 P*gGfcUfcCfgG 1196 AD-15085 3498
faGfcCfTsT fcAfgCfaGfaUf gGfcTsT 3480- GCfCfAUfCfUfGCfUfGCfCf 1197
GGCfUfCfCfGGC 1198 AD-15095 3498 GGAGCfCfTsT fAGCfAGAUfGGC fTsT
3480- GcCaUcUgCuGcCgGaGcCTsT 1199 P*gGfcUfcCfgG 1200 AD-15105 3498
fcAfgCfaGfaUf gGfcTsT 3480- GcCaUcUgCuGcCgGaGcCTsT 1201
GGCfUfCfCfGGC 1202 AD-15115 3498 fAGCfAGAUfGGC fTsT 3480-
GfcCfaUfcUfgCfuGfcCfgG 1203 GGCUCauGCagCA 1204 AD-15125 3498
faGfcCfTsT GAUggcTsT 3480- GCfCfAUfCfUfGCfUfGCfCf 1205
GGCUCauGCagCA 1206 AD-15135 3498 GGAGCfCfTsT GAUggcTsT 3480-
GcCaUcUgCuGcCgGaGcCTsT 1207 GGCUCauGCagCA 1208 AD-15145 3498
GAUggcTsT 3481- CAUUUAUCUUUUGGGUCUGTsT 1209 CAGACCCAAAAGA 1210
AD-9578 3499 UAAAUGTsT 3481- cAuuuAucuuuuGGGucuGTsT 1211
cAGACCcAAAAGA 1212 AD-9704 3499 uAAAUGTsT 3485-
UAUCUUUUGGGUCUGUCCUTsT 1213 AGGACAGACCCAA 1214 AD-9558 3503
AAGAUATsT 3485- uAucuuuuGGGucuGuccuTsT 1215 AGGAcAGACCcAA 1216
AD-9684 3503 AAGAuATsT 3504- CUCUGUUGCCUUUUUACAGTsT 1217
CUGUAAAAAGGCA 1218 AD-9634 3522 ACAGAGTsT 3504-
cucuGuuGccuuuuuAcAGTsT 1219 CUGuAAAAAGGcA 1220 AD-9760 3522
AcAGAGTsT 3512- CCUUUUUACAGCCAACUUUTT 1221 AAAGUUGGCUGUA 1222
AD-15411 3530 AAAAGGTT 3521- AGCCAACUUUUCUAGACCUTT 1223
AGGUCUAGAAAAG 1224 AD-15266 3539 UUGGCUTT 3526-
ACUUUUCUAGACCUGUUUUTT 1225 AAAACAGGUCUAG 1226 AD-15382 3544
AAAAGUTT 3530- UUCUAGACCUGUUUUGCUUTsT 1227 AAGCAAAACAGGU 1228
AD-9554 3548 CUAGAATsT 3530- uucuAGAccuGuuuuGcuuTsT 1229
AAGcAAAAcAGGU 1230 AD-9680 3548 CuAGAATsT 3530-
UfuCfuAfgAfcCfuGfuUfuU 1231 P*aAfgCfaAfaA 1232 AD-14676 3548
fgCfuUfTsT fcAfgGfuCfuAf gAfaTsT 3530- UfUfCfUfAGACfCfUfGUfUf 1233
AAGCfAAAACfAG 1234 AD-14686 3548 UfUfGCfUfUfTsT GUfCfUfAGAATs T
3530- UuCuAgAcCuGuUuUgCuUTsT 1235 P*aAfgCfaAfaA 1236 AD-14696 3548
fcAfgGfuCfuAf gAfaTsT 3530- UuCuAgAcCuGuUuUgCuUTsT 1237
AAGCfAAAACfAG 1238 AD-14706 3548 GUfCfUfAGAATs T 3530-
UfuCfuAfgAfcCfuGfuUfuU 1239 AAGcAaaACagGU 1240 AD-14716 3548
ffCfuUfTsT CUAgaaTsT 3530- UfUfCfUfAGACfCfUfGUfUf 1241
AAGcAaaACagGU 1242 AD-14726 3548 UfUfGCfUfUfTsT CUAgaaTsT 3530-
UuCuAgAcCuGuUuUgCuUTsT 1243 AAGcAaaACagGU 1244 AD-14736 3548
CUAgaaTsT 3530- CfaUfaGfgCfcUfgGfaGfuU 1245 P*aAfuAfaAfcU 1246
AD-15082 3548 fuAfuUfTsT fcCfaGfgCfcUf aUfgTsT 3530-
CfAUfAGGCfCfUfGGAGUfUf 1247 AAUfAAACfUfCf 1248 AD-15092 3548
UfAUfUfTsT CfAGGCfCfUfAU fGTsT 3530- CaUaGgCcUgGaGuUuAuUTsT 1249
P*aAfuAfaAfcU 1250 AD-15102 3548 fcCfaGfgCfcUf aUfgTsT 3530-
CaUaGgCcUgGaGuUuAuUTsT 1251 AAUfAAACfUfCf 1252 AD-15112 3548
CfAGGCfCfUfAU fGTsT 3530- CfaUfaGfgCfcUfgGfaGfuU 1253 AAUAAacUCcaGG
1254 AD-15122 3548 fuAfuUfTsT CCUaugTsT 3530-
CfAUfAGGCfCfUfGGAGUfUf 1255 AAUAAacUCcaGG 1256 AD-15132 3548
UfAUfUfTsT CCUaugTsT 3530- CaUaGgCcUgGaGuUuAuUTsT 1257
AAUAAacUCcaGG 1258 AD-15142 3548 CCUaugTsT 3531-
UCUAGACCUGUUUUGCUUUTsT 1259 AAAGCAAAACAGG 1260 AD-9553 3549
UCUAGATsT 3531- ucuAGAccuGuuuuGcuuuTsT 1261 AAAGcAAAAcAGG 1262
AD-9679 3549 UCuAGATsT 3531- UfcUfaGfaCfcUfgUfuUfuG 1263
P*aAfaGfcAfaA 1264 AD-14675 3549 fcUfuUfTsT faCfaGfgUfcUf aGfaTsT
3531- UfCfUfAGACfCfUfGUfUfUf 1265 AAAGCfAAAACfA 1266 AD-14685 3549
UfGCfUfUfUfTsT GGUfCfUfAGATs T 3531- UcUaGaCcUgUuUuGcUuUTsT 1267
P*aAfaGfcAfaA 1268 AD-14695 3549 faCfaGfgUfcUf aGfaTsT 3531-
UcUaGaCcUgUuUuGcUuUTsT 1269 AAAGCfAAAACfA 1270 AD-14705 3549
GGUfCfUfAGATs T 3531- UfcUfaGfaCfcUfgUfuUfuG 1271 AAAGCaaAAcaGG
1272 AD-14715 3549 fcUfuUfTsT UCUagaTsT 3531-
UfCfUfAGACfCfUfGUfUfUf 1273 AAAGCaaAAcaGG 1274 AD-14725 3549
UfGCfUfUfUfTsT UCUagaTsT 3531- UcUaGaCcUgUuUuGcUuUTsT 1275
AAAGCaaAAcaGG 1276 AD-14735 3549 UCUagaTsT 3531-
UfcAfuAfgGfcCfuGfgAfgU 1277 P*aUfaAfaCfuC 1278 AD-15081 3549
fuUfaUfTsT fcAfgGfcCfuAf uGfaTsT 3531- UfCfAUfAGGCfCfUfGGAGUf 1297
AUfAAACfUfCfC 1280 AD-15091 3549 UfUfAUfTsT fAGGCfCfUfAUf GATsT
3531- UcAuAgGcCuGgAgUuUaUTsT 1281 P*aUfaAfaCfuC 1282 AD-15101
3549 fcAfgGfcCfuAf uGfaTsT 3531- UcAuAgGcCuGgAgUuUaUTsT 1283
AUfAAACfUfCfC 1284 AD-15111 3549 fAGGCfCfUfAUf GATsT 3531-
UfcAfuAfgGfcCfuGfgAfgU 1285 AUAAAcuCCagGC 1286 AD-15121 3549
fuUfaUfTsT CUAugaTsT 3531- UfCfAUfAGGCfCfUfGGAGUf 1287
AUAAAcuCCagGC 1288 AD-15131 3549 UfUfAUfTsT CUAugaTsT 3531-
UcAuAgGcCuGgAgUuUaUTsT 1289 AUAAAcuCCagGC 1290 AD-15141 3549
CUAugaTsT 3557- UGAAGAUAUUUAUUCUGGGTsT 1291 CCCAGAAUAAAUA 1292
AD-9626 3575 UCUUCATsT 3557- uGAAGAuAuuuAuucuGGGTsT 1293
CCcAGAAuAAAuA 1294 AD-9752 3575 UCUUcATsT 3570-
UCUGGGUUUUGUAGCAUUUTsT 1295 AAAUGCUACAAAA 1296 AD-9629 3588
CCCAGATsT 3570- ucuGGGuuuuGuAGcAuuuTsT 1297 AAAUGCuAcAAAA 1298
AD-9755 3588 CCcAGATsT 3613- AUAAAAACAAACAAACGUUTT 1299
AACGUUUGUUUGU 1300 AD-15412 3631 UUUUAUTT 3617-
AAACAAACAAACGUUGUCCTT 1301 GGACAACGUUUGU 1302 AD-15211 3635
UUGUUUTT 3618- AACAAACAAACGUUGUCCUTT 1303 AGGACAACGUUUG 1304
AD-15300 3636 UUUGUUTT *Target: target in human PCSK9 gene, access.
# NM_174936 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-00008 TABLE 5 Sequences of modified dsRNA targeted to
PCSK9 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. Sense strand sequence SEQ ID Antisense-strand
SEQ ID Duplex # (5'-3').sup.1 NO: sequence (5'-3').sup.1 NO:
AD-10792 GccuGGAGuuuAuucGGAATsT 1305 UUCCGAAuAAACUCcAGGCT 1306 sT
AD-10793 GccuGGAGuuuAuucGGAATsT 1307 uUcCGAAuAAACUccAGGCT 1308 sT
AD-10796 GccuGGAGuuuAuucGGAATsT 1309 UUCCGAAUAAACUCCAGGCT 1310 sT
AD-12038 GccuGGAGuuuAuucGGAATsT 1311 uUCCGAAUAAACUCCAGGCT 1312 sT
AD-12039 GccuGGAGuuuAuucGGAATsT 1313 UuCCGAAUAAACUCCAGGCT 1314 sT
AD-12040 GccuGGAGuuuAuucGGAATsT 1315 UUcCGAAUAAACUCCAGGCT 1316 sT
AD-12041 GccuGGAGuuuAuucGGAATsT 1317 UUCcGAAUAAACUCCAGGCT 1318 sT
AD-12042 GCCUGGAGUUUAUUCGGAATsT 1319 uUCCGAAUAAACUCCAGGCT 1320 sT
AD-12043 GCCUGGAGUUUAUUCGGAATsT 1321 UuCCGAAUAAACUCCAGGCT 1322 sT
AD-12044 GCCUGGAGUUUAUUCGGAATsT 1323 UUcCGAAUAAACUCCAGGCT 1324 sT
AD-12045 GCCUGGAGUUUAUUCGGAATsT 1325 UUCcGAAUAAACUCCAGGCT 1326 sT
AD-12046 GccuGGAGuuuAuucGGAA 1327 UUCCGAAUAAACUCCAGGCs 1328 csu
AD-12047 GccuGGAGuuuAuucGGAAA 1329 UUUCCGAAUAAACUCCAGGC 1330 scsu
AD-12048 GccuGGAGuuuAuucGGAAAA 1331 UUUUCCGAAUAAACUCCAGG 1332 Cscsu
AD-12049 GccuGGAGuuuAuucGGAAAAG 1333 CUUUUCCGAAUAAACUCCAG 1334
GCscsu AD-12050 GccuGGAGuuuAuucGGAATTab 1335 UUCCGAAUAAACUCCAGGCT
1336 Tab AD-12051 GccuGGAGuuuAuucGGAAATTab 1337
UUUCCGAAuAAACUCCAGGC 1338 TTab AD-12052 GccuGGAGuuuAuucGGAAAATTab
1339 UUUUCCGAAUAAACUCCAGG 1340 CTTab AD-12053
GccuGGAGuuuAuucGGAAAAGTTab 1341 CUUUUCCGAAUAAACUCCAG 1342 GCTTab
AD-12054 GCCUGGAGUUUAUUCGGAATsT 1343 UUCCGAAUAAACUCCAGGCs 1344 csu
AD-12055 GccuGGAGuuuAuucGGAATsT 1345 UUCCGAAUAAACUCCAGGCs 1346 csu
AD-12056 GcCuGgAgUuUaUuCgGaA 1347 UUCCGAAUAAACUCCAGGCT 1348 Tab
AD-12057 GcCuGgAgUuUaUuCgGaA 1349 UUCCGAAUAAACUCCAGGCT 1350 sT
AD-12058 GcCuGgAgUuUaUuCgGaA 1351 UUCCGAAuAAACUCcAGGCT 1352 sT
AD-12059 GcCuGgAgUuUaUuCgGaA 1353 uUcCGAAuAAACUccAGGCT 1354 sT
AD-12060 GcCuGgAgUuUaUuCgGaA 1355 UUCCGaaUAaaCUCCAggc 1356 AD-12061
GcCuGgnAgUuUaUuCgGaATsT 1357 UUCCGaaUAaaCUCCAggcT 1358 sT AD-12062
GcCuGgAgUuUaUuCgGaATTab 1359 UUCCGaaUAaaCUCCAggcT 1360 Tab AD-12063
GcCuGgAgUuUaUuCgGaA 1361 UUCCGaaUAaaCUCCAggcs 1362 csu AD-12064
GcCuGgnAgUuUaUuCgGaATsT 1363 UUCCGAAuAAACUCcAGGCT 1364 sT AD-12065
GcCuGgAgUuUaUuCgGaATTab 1365 UUCCGAAuAAACUCcAGGCT 1366 Tab AD-12066
GcCuGgAgUuUaUuCgGaA 1367 UUCCGAAuAAACUCcAGGCs 1368 csu AD-12067
GcCuGgnAgUuUaUuCgGaATsT 1369 UUCCGAAUAAACUCCAGGCT 1370 sT AD-12068
GcCuGgAgUuUaUuCgGaATTab 1371 UUCCGAAUAAACUCCAGGCT 1372 Tab AD-12069
GcCuGgAgUuUaUuCgGaA 1373 UUCCGAAUAAACUCCAGGCs 1374 csu AD-12338
GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1375 P*uUfcCfgAfaUfaAfaCf 1376 f
uCfcAfgGfc AD-12339 GcCuGgAgUuUaUuCgGaA 1377 P*uUfcCfgAfaUfaAfaCf
1378 uCfcAfgGfc AD-12340 GccuGGAGuuuAuucGGAA 1379
P*uUfcCfgAfaUfaAfaCf 1380 uCfcAfgGfc AD-12341
GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1381 P*uUfcCfgAfaUfaAfaCf 1382 fTsT
uCfcAfgGfcTsT AD-12342 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1383
UUCCGAAuAAACUCcAGGCT 1384 fTsT sT AD-12343
GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1385 uUcCGAAuAAACUccAGGCT 1386 fTsT sT
AD-12344 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1387 UUCCGAAUAAACUCCAGGCT
1388 fTsT sT AD-12345 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1389
UUCCGAAUAAACUCCAGGCs 1390 fTsT csu AD-12346
GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1391 UUCCGaaUAaaCUCCAggcs 1392 fTsT
csu AD-12347 GCCUGGAGUUUAUUCGGAATsT 1393 P*uUfcCfgAfaUfaAfaCf 1394
uCfcAfgGfcTsT AD-12348 GccuGGAGuuuAuucGGAATsT 1395
P*uUfcCfgAfaUfaAfaCf 1396 uCfcAfgGfcTsT AD-12349
GcCuGgnAgUuUaUuCgGaATsT 1397 P*uUfcCfgAfaUfaAfaCf 1398
uCfcAfgGfcTsT AD-12350 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1399
P*uUfcCfgAfaUfaAfaCf 1400 fTTab uCfcAfgGfcTTab AD-12351
GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1401 P*uUfcCfgAfaUfaAfaCf 1402 f
uCfcAfgGfcsCfsu AD-12352 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1403
UUCCGaaUAaaCUCCAggcs 1404 f csu AD-12354
GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1405 UUCCGAAUAAACUCCAGGCs 1406 f csu
AD-12355 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1407 UUCCGAAuAAACUCcAGGCT
1408 f sT AD-12356 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1409
uUcCGAAuAAACUccAGGCT 1410 f sT AD-12357
GmocCmouGmogAm02gUmouUmoaUmo 1411 UUCCGaaUAaaCUCCAggc 1412
uCmogGmoaA AD-12358 GmocCmouGmogAm02gUmouUmoaUmo 1413
P*uUfcCfgAfaUfaAfaCf 1414 uCmogGmoaA uCfcAfgGfc AD-12359
GmocCmouGmogAm02gUmouUmoaUmo 1415 P*uUfcCfgAfaUfaAfaCf 1416
uCmogGmoaA uCfcAfgGfcsCfsu AD-12360 GmocCmouGmogAm02gUmouUmoaUmo
1417 UUCCGAAUAAACUCCAGGCs 1418 uCmogGmoaA csu AD-12361
GmocCmouGmogAm02gUmouUmoaUmo 1419 UUCCGAAuAAACUCcAGGCT 1420
uCmogGmoaA sT AD-12362 GmocCmouGmogAm02gUmouUmoaUmo 1421
uUcCGAAuAAACUccAGGCT 1422 uCmogGmoaA sT AD-12363
GmocCmouGmogAm02gUmouUmoaUmo 1423 UUCCGaaUAaaCUCCAggcs 1424
uCmogGmoaA csu AD-12364 GmocCmouGmogAmogUmouUmoaUmou 1425
UUCCGaaUAaaCUCCAggcT 1426 CmogGmoaATsT sT AD-12365
GmocCmouGmogAmogUmouUmoaUmou 1427 UUCCGAAuAAACUCcAGGCT 1428
CmogGmoaATsT sT AD-12366 GmocCmouGmogAmogUmouUmoaUmou 1429
UUCCGAAUAAACUCCAGGCT 1430 CmogGmoaATsT sT AD-12367
GmocmocmouGGAGmoumoumouAmoum 1431 UUCCGaaUAaaCUCCAggcT 1432
oumocGGAATsT sT AD-12368 GmocmocmouGGAGmoumoumouAmoum 1433
UUCCGAAuAAACUCcAGGCT 1434 oumocGGAATsT sT AD-12369
GmocmocmouGGAGmoumoumouAmoum 1435 UUCCGAAUAAACUCCAGGCT 1436
oumocGGAATsT sT AD-12370 GmocmocmouGGAGmoumoumouAmoum 1437
P*UfUfCfCfGAAUfAAACf 1438 oumocGGAATsT UfCfCfAGGCfTsT AD-12371
GmocmocmouGGAGmoumoumouAmoum 1439 P*UfUfCfCfGAAUfAAACf 1440
oumocGGAATsT UfCfCfAGGCfsCfsUf AD-12372
GmocmocmouGGAGmoumoumouAmoum 1441 P*uUfcCfgAfaUfaAfaCf 1442
oumocGGAATsT uCfcAfgGfcsCfsu AD-12373 GmocmocmouGGAGmoumoumouAmoum
1443 UUCCGAAUAAACUCCAGGCT 1444 oumocGGAATsT sT AD-12374
GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1445 UfUfCfCfGAAUfAAACfUf 1446 TsT
CfCfAGGCfTsT AD-12375 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1447
UUCCGAAUAAACUCCAGGCT 1448 TsT sT AD-12377
GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1449 uUcCGAAuAAACUccAGGCT 1450 TsT sT
AD-12378 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1451 UUCCGaaUAaaCUCCAggcs
1452 TsT csu AD-12379 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1453
UUCCGAAUAAACUCCAGGCs 1454 TsT csu AD-12380
GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1455 P*uUfcCfgAfaUfaAfaCf 1456 TsT
uCfcAfgGfcsCfsu AD-12381 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1457
P*uUfcCfgAfaUfaAfaCf 1458 TsT uCfcAfgGfcTsT AD-12382
GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1459 P*UfUfCfCfGAAUfAAACf 1460 TsT
UfCfCfAGGCfTsT
AD-12383 GCCUGGAGUUUAUUCGGAATsT 1461 P*UfUfCfCfGAAUfAAACf 1462
UfCfCfAGGCfTsT AD-12384 GccuGGAGuuuAuucGGAATsT 1463
P*UfUfCfCfGAAUfAAACf 1464 UfCfCfAGGCfTsT AD-12385
GcCuGgnAgUuUaUuCgGaATsT 1465 P*UfUfCfCfGAAUfAAACf 1466
UfCfCfAGGCfTsT AD-12386 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1467
P*UfUfCfCfGAAUfAAACf 1468 f UfCfCfAGGCfTsT AD-12387
GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1469 UfUfCfCfGAAUfAAACfUf 1470 A
CfCfAGGCfsCfsUf AD-12388 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1471
P*uUfcCfgAfaUfaAfaCf 1472 A uCfcAfgGfc AD-12389
GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1473 P*uUfcCfgAfaUfaAfaCf 1474 A
uCfcAfgGfcsCfsu AD-12390 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1475
UUCCGAAUAAACUCCAGGCs 1476 A csu AD-12391
GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1477 UUCCGaaUAaaCUCCAggc 1478 A
AD-12392 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1479 UUCCGAAUAAACUCCAGGCT
1480 A sT AD-12393 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1481
UUCCGAAuAAACUCcAGGCT 1482 A sT AD-12394
GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1483 uUcCGAAuAAACUccAGGCT 1484 A sT
AD-12395 GmocCmouGmogAmogUmouUmoaUmou 1485 P*UfUfCfCfGAAUfAAACf
1486 CmogGmoaATsT UfCfCfAGGCfsCfsUf AD-12396
GmocCmouGmogAm02gUmouUmoaUmo 1487 P*UfUfCfCfGAAUfAAACf 1488
uCmogGmoaA UfCfCfAGGCfsCfsUf AD-12397 GfcCfuGfgAfgUfuUfaUfuCfgGfaA
1489 P*UfUfCfCfGAAUfAAACf 1490 f UfCfCfAGGCfsCfsUf AD-12398
GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1491 P*UfUfCfCfGAAUfAAACf 1492 fTsT
UfCfCfAGGCfsCfsUf AD-12399 GcCuGgnAgUuUaUuCgGaATsT 1493
P*UfUfCfCfGAAUfAAACf 1494 UfCfCfAGGCfsCfsUf AD-12400
GCCUGGAGUUUAUUCGGAATsT 1495 P*UfUfCfCfGAAUfAAACf 1496
UfCfCfAGGCfsCfsUf AD-12401 GccuGGAGuuuAuucGGAATsT 1497
P*UfUfCfCfGAAUfAAACf 1498 UfCfCfAGGCfsCfsUf AD-12402
GccuGGAGuuuAuucGGAA 1499 P*UfUfCfCfGAAUfAAACf 1500
UfCfCfAGGCfsCfsUf AD-12403 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1501
P*UfUfCfCfGAAUfAAACf 1502 A UfCfCfAGGCfsCfsUf AD-9314
GCCUGGAGUUUAUUCGGAATsT 1503 UUCCGAAUAAACUCCAGGCT 1504 sT AD-10794
ucAuAGGccuGGAGuuuAudTsdT 1525 AuAAACUCcAGGCCuAUGAd 1526 TsdT
AD-10795 ucAuAGGccuGGAGuuuAudTsdT 1527 AuAAACUccAGGcCuAuGAd 1528
TsdT AD-10797 ucAuAGGccuGGAGuuuAudTsdT 1529 AUAAACUCCAGGCCUAUGAd
1530 TsdT
TABLE-US-00009 TABLE 6 dsRNA targeted to PCSK9: mismatches and
modifications 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' phospho-
diester 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 Duplex # Strand SEQ ID 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
UfuCfuAfgAfcY1uGfuUfuUfgCfuUfTsT 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
TABLE-US-00010 TABLE 7 Sequences of unmodified siRNA flanking
AD-9680 *Target: target in human PCSK9 gene, access. # NM_174936 U,
C, A, G: corresponding ribonucleotide; dT: deoxythymidine; 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. Duplex # Strand
Sequence (5' to 3') *Target 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 UAAABAUCUUCAAGUUACAdTsdT 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-00011 TABLE 8 Sequences of modified siRNA flanking AD-9680
*Target: 5' nutlcoetide of target sequence in human PCSK9 gene,
access. # NM_174936 U, C, A, G: corresponding ribonucleotide; dT:
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 phosphoro- thiodiester
groups; unless denoted by prefix "P*", oligonucleotides are devoid
of a 5'-phosphate group on the 5'-most nucleotide; all oligo-
nucleotides bear 3'-OH on the 3'-most nucleotide Duplex # Strand
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-00012 TABLE 9 Sequences of XBP-1 dsRNAs *Target refers to
target gene and location of target sequence. NM_001004210 is the
gene for rat XBP-1. XM_001103095 is the sequence for Macaca mulatta
(rhesus monkey) XBP-1. SEQ ID SEQ ID Target* NO sense (5'-3') NO
antisense (5'-3') NM_001004210 1729 CCCAGCUGAUUAGUGUCUA 1753
UAGACACUAAUCAGCUGGG 1128-1146 NM_001004210 1730 CCAGCUGAUUAGUGUCUAA
1754 UUAGACACUAAUCAGCUGG 1129-1147 NM_001004210 1731
CUCCCAGAGGUCUACCCAG 1755 CUGGGUAGACCUCUGGGAG 677-695 NM_001004210
1732 GAUCACCCUGAAUUCAUUG 1756 CAAUGAAUUCAGGGUGAUC 893-911
NM_001004210 1733 UCACCCUGAAUUCAUUGUC 1757 GACAAUGAAUUCAGGGUGA
895-913 NM_001004210 1734 CCCCAGCUGAUUAGUGUCU 1758
AGACACUAAUCAGCUGGGG 1127-1145 NM_001004210 1735 AUCACCCUGAAUUCAUUGU
1759 ACAAUGAAUUCAGGGUGAU 894-912 NM_001004210 1736
CAUUUAUUUAAAACUACCC 1760 GGGUAGUUUUAAAUAAAUG 1760-1778 NM_001004210
1737 ACUGAAAAACAGAGUAGCA 1761 UGCUACUCUGUUUUUCAGU 215-233
NM_001004210 1738 CCAUUUAUUUAAAACUACC 1762 GGUAGUUUUAAAUAAAUGG
1759-1777 NM_001004210 1739 UUGAGAACCAGGAGUUAAG 1763
CUUAACUCCUGGUUCUCAA 367-385 NM_001004210 1740 CACCCUGAAUUCAUUGUCU
1764 AGACAAUGAAUUCAGGGUG 896-914 NM_001004210 1741
AACUGAAAAACAGAGUAGC 1765 GCUACUCUGUUUUUCAGUU 214-232 NM_001004210
1742 CUGAAAAACAGAGUAGCAG 1766 CUGCUACUCUGUUUUUCAG 216-234
XM_001103095 1743 AGAAAAUCAGCUUUUACGA 1767 UCGUAAAAGCUGAUUUUCU
387-405 XM_001103095 1744 UCCCCAGCUGAUUAGUGUC 1768
GACACUAAUCAGCUGGGGA 1151-1169 XM_001103095 1745 UACUUAUUAUGUAAGGGUC
1769 GACCCUUACAUAAUAAGUA 1466-1484 XM_001103095 1746
UAUCUUAAAAGGGUGGUAG 1770 CUACCACCCUUUUAAGAUA 1435-1453 XM_001103095
1747 CCAUGGAUUCUGGCGGUAU 1771 AUACCGCCAGAAUCCAUGG 577-595
XM_001103095 1748 UUAAUGAACUAAUUCGUUU 1772 AAACGAAUUAGUUCAUUAA
790-808 XM_001103095 1749 AGGGUCAUUAGACAAAUGU 1773
ACAUUUGUCUAAUGACCCU 1479-1497 XM_001103095 1750 UGAACUAAUUCGUUUUGAC
1774 GUCAAAACGAAUUAGUUCA 794-812 XM_001103095 1751
UUCCCCAGCUGAUUAGUGU 1775 ACACUAAUCAGCUGGGGAA 1150-1168 XM_001103095
1752 UAUGUAAGGGUCAUUAGAC 1776 GUCUAAUGACCCUUACAUA 1473-1491
TABLE-US-00013 TABLE 10 Target gene name and target sequence
location for dsRNA targeting XBP-1 Duplex # Target gene and
location of target sequence AD18027 NM_001004210_1128-1146 AD18028
NM_001004210_1129-1147 AD18029 NM_001004210_677-695 AD18030
NM_001004210_893-911 AD18031 NM_001004210_895-913 AD18032
NM_001004210_1127-1145 AD18033 NM_001004210_894-912 AD18034
NM_001004210_1760-1778 AD18035 NM_001004210_215-233 AD18036
NM_001004210_1759-1777 AD18037 NM_001004210_367-385 AD18038
NM_001004210_896-914 AD18039 NM_001004210_214-232 AD18040
NM_001004210_216-234 AD18041 XM_001103095_387-405 AD18042
XM_001103095_1151-1169 AD18043 XM_001103095_1466-1484 AD18044
XM_001103095_1435-1453 AD18045 XM_001103095_577-595 AD18046
XM_001103095_790-808 AD18047 XM_001103095_1479-1497 AD18048
XM_001103095_794-812 AD18049 XM_001103095_1150-1168 AD18050
XM_001103095_1473-1491 *Target refers to target gene and location
of target sequence. NM_001004210 is the gene for rat XBP-1.
XM_001103095 is the sequence for Macaca mulatta (rhesus monkey)
XBP-1.
TABLE-US-00014 TABLE 11 Sequences of dsRNA targeting XBP-1, with
Endolight chemistry modifications U, C, A, G: corresponding
ribonucleotide; dT: deoxythymidine; u, c, a, g: corresponding
2'-O-methyl 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. SEQ SEQ ID ID Duplex # NO Sense
(5'-3') NO Antisense (5'-3') AD18027 4166 cccAGcuGAuuAGuGucuAdTsdT
1800 uAGAcACuAAUcAGCUGGGdTsdT AD18028 1777 ccAGcuGAuuAGuGucuAAdTsdT
1801 UuAGAcACuAAUcAGCUGGdTsdT AD18029 1778 cucccAGAGGucuAcccAGdTsdT
1802 CUGGGuAGACCUCUGGGAGdTsdT AD18030 1779 GAucAcccuGAAuucAuuGdTsdT
1803 cAAUGAAUUcAGGGUGAUCdTsdT AD18031 1780 ucAcccuGAAuucAuuGucdTsdT
1804 GAcAAUGAAUUcAGGGUGAdTsdT AD18032 1781 ccccAGcuGAuuAGuGucudTsdT
1805 AGAcACuAAUcAGCUGGGGdTsdT AD18033 1782 AucAcccuGAAuucAuuGudTsdT
1806 AcAAUGAAUUcAGGGUGAUdTsdT AD18034 1783 cAuuuAuuuAAAAcuAcccdTsdT
1807 GGGuAGUUUuAAAuAAAUGdTsdT AD18035 1784 AcuGAAAAAcAGAGuAGcAdTsdT
1808 UGCuACUCUGUUUUUcAGUdTsdT AD18036 1785 ccAuuuAuuuAAAAcuAccdTsdT
1809 GGuAGUUUuAAAuAAAUGGdTsdT AD18037 1786 uuGAGAAccAGGAGuuAAGdTsdT
1810 CUuAACUCCUGGUUCUcAAdTsdT AD18038 1787 cAcccuGAAuucAuuGucudTsdT
1811 AGAcAAUGAAUUcAGGGUGdTsdT AD18039 1788 AAcuGAAAAAcAGAGuAGcdTsdT
1812 GCuACUCUGUUUUUcAGUUdTsdT AD18040 1789 cuGAAAAAcAGAGuAGcAGdTsdT
1813 CUGCuACUCUGUUUUUcAGdTsdT AD18041 1790 AGAAAAucAGcuuuuAcGAdTsdT
1814 UCGuAAAAGCUGAUUUUCUdTsdT AD18042 1791 uccccAGcuGAuuAGuGucdTsdT
1815 GAcACuAAUcAGCUGGGGAdTsdT AD18043 1792 uAcuuAuuAuGuAAGGGucdTsdT
1816 GACCCUuAcAuAAuAAGuAdTsdT AD18044 1793 uAucuuAAAAGGGuGGuAGdTsdT
1817 CuACcACCCUUUuAAGAuAdTsdT AD18045 1794 ccAuGGAuucuGGcGGuAudTsdT
1818 AuACCGCcAGAAUCcAUGGdTsdT AD18046 1795 uuAAuGAAcuAAuucGuuudTsdT
1819 AAACGAAUuAGUUcAUuAAdTsdT AD18047 1796 AGGGucAuuAGAcAAAuGudTsdT
1820 AcAUUUGUCuAAUGACCCUdTsdT AD18048 1797 uGAAcuAAuucGuuuuGAcdTsdT
1821 GUcAAAACGAAUuAGUUcAdTsdT AD18049 1798 uuccccAGcuGAuuAGuGudTsdT
1822 AcACuAAUcAGCUGGGGAAdTsdT AD18050 1799 uAuGuAAGGGucAuuAGAcdTsdT
1823 GUCuAAUGACCCUuAcAuAdTsdT
TABLE-US-00015 TABLE 12 Sequences of dsRNA targeting both human and
rhesus monkey XBP-1. *Target refers location of target sequence in
NM_005080 (human XBP-1 mRNA). Sense and antisense sequences are
described with optional dinucleotide (NN) overhangs. SEQ ID SEQ ID
Target* sense (5'-3') NO antisense (5'-3') NO 100-118
CUGCUUCUGUCGGGGCAGCNN 1824 GCUGCCCCGACAGAAGCAGNN 28 1011-1029
GAGCUGGGUAUCUCAAAUCNN 1825 GAUUUGAGAUACCCAGCUCNN 28 101-119
UGCUUCUGUCGGGGCAGCCNN 1826 GGCUGCCCCGACAGAAGCANN 28 1012-1030
AGCUGGGUAUCUCAAAUCUNN 1827 AGAUUUGAGAUACCCAGCUNN 28 1013-1031
GCUGGGUAUCUCAAAUCUGNN 1828 CAGAUUUGAGAUACCCAGCNN 28 1014-1032
CUGGGUAUCUCAAAUCUGCNN 1829 GCAGAUUUGAGAUACCCAGNN 28 1015-1033
UGGGUAUCUCAAAUCUGCUNN 1830 AGCAGAUUUGAGAUACCCANN 28 1016-1034
GGGUAUCUCAAAUCUGCUUNN 1831 AAGCAGAUUUGAGAUACCCNN 28 1017-1035
GGUAUCUCAAAUCUGCUUUNN 1832 AAAGCAGAUUUGAGAUACCNN 28 1018-1036
GUAUCUCAAAUCUGCUUUCNN 1833 GAAAGCAGAUUUGAGAUACNN 28 1019-1037
UAUCUCAAAUCUGCUUUCANN 1834 UGAAAGCAGAUUUGAGAUANN 28 1020-1038
AUCUCAAAUCUGCUUUCAUNN 1835 AUGAAAGCAGAUUUGAGAUNN 28 1021-1039
UCUCAAAUCUGCUUUCAUCNN 1836 GAUGAAAGCAGAUUUGAGANN 28 102-120
GCUUCUGUCGGGGCAGCCCNN 1837 GGGCUGCCCCGACAGAAGCNN 28 1022-1040
CUCAAAUCUGCUUUCAUCCNN 1838 GGAUGAAAGCAGAUUUGAGNN 28 1023-1041
UCAAAUCUGCUUUCAUCCANN 1839 UGGAUGAAAGCAGAUUUGANN 28 1024-1042
CAAAUCUGCUUUCAUCCAGNN 1840 CUGGAUGAAAGCAGAUUUGNN 28 1025-1043
AAAUCUGCUUUCAUCCAGCNN 1841 GCUGGAUGAAAGCAGAUUUNN 29 1026-1044
AAUCUGCUUUCAUCCAGCCNN 1842 GGCUGGAUGAAAGCAGAUUNN 29 1027-1045
AUCUGCUUUCAUCCAGCCANN 1843 UGGCUGGAUGAAAGCAGAUNN 29 1028-1046
UCUGCUUUCAUCCAGCCACNN 1844 GUGGCUGGAUGAAAGCAGANN 29 1029-1047
CUGCUUUCAUCCAGCCACUNN 1845 AGUGGCUGGAUGAAAGCAGNN 29 1030-1048
UGCUUUCAUCCAGCCACUGNN 1846 CAGUGGCUGGAUGAAAGCANN 29 1031-1049
GCUUUCAUCCAGCCACUGCNN 1847 GCAGUGGCUGGAUGAAAGCNN 29 103-121
CUUCUGUCGGGGCAGCCCGNN 1848 CGGGCUGCCCCGACAGAAGNN 29 1032-1050
CUUUCAUCCAGCCACUGCCNN 1849 GGCAGUGGCUGGAUGAAAGNN 29 1033-1051
UUUCAUCCAGCCACUGCCCNN 1850 GGGCAGUGGCUGGAUGAAANN 29 104-122
UUCUGUCGGGGCAGCCCGCNN 1851 GCGGGCUGCCCCGACAGAANN 29 105-123
UCUGUCGGGGCAGCCCGCCNN 1852 GGCGGGCUGCCCCGACAGANN 29 1056-1074
CCAUCUUCCUGCCUACUGGNN 1853 CCAGUAGGCAGGAAGAUGGNN 29 1057-1075
CAUCUUCCUGCCUACUGGANN 1854 UCCAGUAGGCAGGAAGAUGNN 29 1058-1076
AUCUUCCUGCCUACUGGAUNN 1855 AUCCAGUAGGCAGGAAGAUNN 29 1059-1077
UCUUCCUGCCUACUGGAUGNN 1856 CAUCCAGUAGGCAGGAAGANN 29 1060-1078
CUUCCUGCCUACUGGAUGCNN 1857 GCAUCCAGUAGGCAGGAAGNN 29 1061-1079
UUCCUGCCUACUGGAUGCUNN 1858 AGCAUCCAGUAGGCAGGAANN 29 106-124
CUGUCGGGGCAGCCCGCCUNN 1859 AGGCGGGCUGCCCCGACAGNN 29 1062-1080
UCCUGCCUACUGGAUGCUUNN 1860 AAGCAUCCAGUAGGCAGGANN 29 1063-1081
CCUGCCUACUGGAUGCUUANN 1861 UAAGCAUCCAGUAGGCAGGNN 29 1064-1082
CUGCCUACUGGAUGCUUACNN 1862 GUAAGCAUCCAGUAGGCAGNN 29 1065-1083
UGCCUACUGGAUGCUUACANN 1863 UGUAAGCAUCCAGUAGGCANN 29 1066-1084
GCCUACUGGAUGCUUACAGNN 1864 CUGUAAGCAUCCAGUAGGCNN 29 1067-1085
CCUACUGGAUGCUUACAGUNN 1865 ACUGUAAGCAUCCAGUAGGNN 29 1068-1086
CUACUGGAUGCUUACAGUGNN 1866 CACUGUAAGCAUCCAGUAGNN 29 1069-1087
UACUGGAUGCUUACAGUGANN 1867 UCACUGUAAGCAUCCAGUANN 29 1070-1088
ACUGGAUGCUUACAGUGACNN 1868 GUCACUGUAAGCAUCCAGUNN 29 1071-1089
CUGGAUGCUUACAGUGACUNN 1869 AGUCACUGUAAGCAUCCAGNN 29 107-125
UGUCGGGGCAGCCCGCCUCNN 1870 GAGGCGGGCUGCCCCGACANN 29 1072-1090
UGGAUGCUUACAGUGACUGNN 1871 CAGUCACUGUAAGCAUCCANN 29 1073-1091
GGAUGCUUACAGUGACUGUNN 1872 ACAGUCACUGUAAGCAUCCNN 29 1074-1092
GAUGCUUACAGUGACUGUGNN 1873 CACAGUCACUGUAAGCAUCNN 29 1075-1093
AUGCUUACAGUGACUGUGGNN 1874 CCACAGUCACUGUAAGCAUNN 29 1076-1094
UGCUUACAGUGACUGUGGANN 1875 UCCACAGUCACUGUAAGCANN 29 1077-1095
GCUUACAGUGACUGUGGAUNN 1876 AUCCACAGUCACUGUAAGCNN 29 1078-1096
CUUACAGUGACUGUGGAUANN 1877 UAUCCACAGUCACUGUAAGNN 29 108-126
GUCGGGGCAGCCCGCCUCCNN 1878 GGAGGCGGGCUGCCCCGACNN 29 109-127
UCGGGGCAGCCCGCCUCCGNN 1879 CGGAGGCGGGCUGCCCCGANN 29 110-128
CGGGGCAGCCCGCCUCCGCNN 1880 GCGGAGGCGGGCUGCCCCGNN 29 111-129
GGGGCAGCCCGCCUCCGCCNN 1881 GGCGGAGGCGGGCUGCCCCNN 29 1116-1134
UUCAGUGACAUGUCCUCUCNN 1882 GAGAGGACAUGUCACUGAANN 29 112-130
GGGCAGCCCGCCUCCGCCGNN 1883 CGGCGGAGGCGGGCUGCCCNN 29 113-131
GGCAGCCCGCCUCCGCCGCNN 1884 GCGGCGGAGGCGGGCUGCCNN 29 1136-1154
GCUUGGUGUAAACCAUUCUNN 1885 AGAAUGGUUUACACCAAGCNN 29 1137-1155
CUUGGUGUAAACCAUUCUUNN 1886 AAGAAUGGUUUACACCAAGNN 29 1138-1156
UUGGUGUAAACCAUUCUUGNN 1887 CAAGAAUGGUUUACACCAANN 29 1139-1157
UGGUGUAAACCAUUCUUGGNN 1888 CCAAGAAUGGUUUACACCANN 29 1140-1158
GGUGUAAACCAUUCUUGGGNN 1889 CCCAAGAAUGGUUUACACCNN 29 1141-1159
GUGUAAACCAUUCUUGGGANN 1890 UCCCAAGAAUGGUUUACACNN 29 114-132
GCAGCCCGCCUCCGCCGCCNN 1891 GGCGGCGGAGGCGGGCUGCNN 29 1142-1160
UGUAAACCAUUCUUGGGAGNN 1892 CUCCCAAGAAUGGUUUACANN 29 1143-1161
GUAAACCAUUCUUGGGAGGNN 1893 CCUCCCAAGAAUGGUUUACNN 29 1144-1162
UAAACCAUUCUUGGGAGGANN 1894 UCCUCCCAAGAAUGGUUUANN 29 1145-1163
AAACCAUUCUUGGGAGGACNN 1895 GUCCUCCCAAGAAUGGUUUNN 29 1146-1164
AACCAUUCUUGGGAGGACANN 1896 UGUCCUCCCAAGAAUGGUUNN 29 1147-1165
ACCAUUCUUGGGAGGACACNN 1897 GUGUCCUCCCAAGAAUGGUNN 29 1148-1166
CCAUUCUUGGGAGGACACUNN 1898 AGUGUCCUCCCAAGAAUGGNN 29 1149-1167
CAUUCUUGGGAGGACACUUNN 1899 AAGUGUCCUCCCAAGAAUGNN 29 1150-1168
AUUCUUGGGAGGACACUUUNN 1900 AAAGUGUCCUCCCAAGAAUNN 29 1151-1169
UUCUUGGGAGGACACUUUUNN 1901 AAAAGUGUCCUCCCAAGAANN 29 115-133
CAGCCCGCCUCCGCCGCCGNN 1902 CGGCGGCGGAGGCGGGCUGNN 29 1152-1170
UCUUGGGAGGACACUUUUGNN 1903 CAAAAGUGUCCUCCCAAGANN 29 1153-1171
CUUGGGAGGACACUUUUGCNN 1904 GCAAAAGUGUCCUCCCAAGNN 29 1154-1172
UUGGGAGGACACUUUUGCCNN 1905 GGCAAAAGUGUCCUCCCAANN 29 1155-1173
UGGGAGGACACUUUUGCCANN 1906 UGGCAAAAGUGUCCUCCCANN 29 1156-1174
GGGAGGACACUUUUGCCAANN 1907 UUGGCAAAAGUGUCCUCCCNN 29 1157-1175
GGAGGACACUUUUGCCAAUNN 1908 AUUGGCAAAAGUGUCCUCCNN 29 1158-1176
GAGGACACUUUUGCCAAUGNN 1909 CAUUGGCAAAAGUGUCCUCNN 29 1159-1177
AGGACACUUUUGCCAAUGANN 1910 UCAUUGGCAAAAGUGUCCUNN 29 1160-1178
GGACACUUUUGCCAAUGAANN 1911 UUCAUUGGCAAAAGUGUCCNN 29 1161-1179
GACACUUUUGCCAAUGAACNN 1912 GUUCAUUGGCAAAAGUGUCNN 29 116-134
AGCCCGCCUCCGCCGCCGGNN 1913 CCGGCGGCGGAGGCGGGCUNN 29 1162-1180
ACACUUUUGCCAAUGAACUNN 1914 AGUUCAUUGGCAAAAGUGUNN 29 117-135
GCCCGCCUCCGCCGCCGGANN 1915 UCCGGCGGCGGAGGCGGGCNN 29 118-136
CCCGCCUCCGCCGCCGGAGNN 1916 CUCCGGCGGCGGAGGCGGGNN 29 1182-1200
UUUCCCCAGCUGAUUAGUGNN 1917 CACUAAUCAGCUGGGGAAANN 29 1183-1201
UUCCCCAGCUGAUUAGUGUNN 1918 ACACUAAUCAGCUGGGGAANN 29 1184-1202
UCCCCAGCUGAUUAGUGUCNN 1919 GACACUAAUCAGCUGGGGANN 29 1185-1203
CCCCAGCUGAUUAGUGUCUNN 1920 AGACACUAAUCAGCUGGGGNN 29 1186-1204
CCCAGCUGAUUAGUGUCUANN 1921 UAGACACUAAUCAGCUGGGNN 29 1187-1205
CCAGCUGAUUAGUGUCUAANN 1922 UUAGACACUAAUCAGCUGGNN 29 1188-1206
CAGCUGAUUAGUGUCUAAGNN 1923 CUUAGACACUAAUCAGCUGNN 29 1189-1207
AGCUGAUUAGUGUCUAAGGNN 1924 CCUUAGACACUAAUCAGCUNN 29 1190-1208
GCUGAUUAGUGUCUAAGGANN 1925 UCCUUAGACACUAAUCAGCNN 29 1191-1209
CUGAUUAGUGUCUAAGGAANN 1926 UUCCUUAGACACUAAUCAGNN 29 119-137
CCGCCUCCGCCGCCGGAGCNN 1927 GCUCCGGCGGCGGAGGCGGNN 29 1192-1210
UGAUUAGUGUCUAAGGAAUNN 1928 AUUCCUUAGACACUAAUCANN 29 1193-1211
GAUUAGUGUCUAAGGAAUGNN 1929 CAUUCCUUAGACACUAAUCNN 29 1194-1212
AUUAGUGUCUAAGGAAUGANN 1930 UCAUUCCUUAGACACUAAUNN 29 1195-1213
UUAGUGUCUAAGGAAUGAUNN 1931 AUCAUUCCUUAGACACUAANN 29 1196-1214
UAGUGUCUAAGGAAUGAUCNN 1932 GAUCAUUCCUUAGACACUANN 29 1197-1215
AGUGUCUAAGGAAUGAUCCNN 1933 GGAUCAUUCCUUAGACACUNN 29 1198-1216
GUGUCUAAGGAAUGAUCCANN 1934 UGGAUCAUUCCUUAGACACNN 29 120-138
CGCCUCCGCCGCCGGAGCCNN 1935 GGCUCCGGCGGCGGAGGCGNN 29 121-139
GCCUCCGCCGCCGGAGCCCNN 1936 GGGCUCCGGCGGCGGAGGCNN 29 1218-1236
UACUGUUGCCCUUUUCCUUNN 1937 AAGGAAAAGGGCAACAGUANN 29 1219-1237
ACUGUUGCCCUUUUCCUUGNN 1938 CAAGGAAAAGGGCAACAGUNN 29 1220-1238
CUGUUGCCCUUUUCCUUGANN 1939 UCAAGGAAAAGGGCAACAGNN 29 1221-1239
UGUUGCCCUUUUCCUUGACNN 1940 GUCAAGGAAAAGGGCAACANN 29 122-140
CCUCCGCCGCCGGAGCCCCNN 1941 GGGGCUCCGGCGGCGGAGGNN 30 1222-1240
GUUGCCCUUUUCCUUGACUNN 1942 AGUCAAGGAAAAGGGCAACNN 30 1223-1241
UUGCCCUUUUCCUUGACUANN 1943 UAGUCAAGGAAAAGGGCAANN 30
1224-1242 UGCCCUUUUCCUUGACUAUNN 1944 AUAGUCAAGGAAAAGGGCANN 30
1225-1243 GCCCUUUUCCUUGACUAUUNN 1945 AAUAGUCAAGGAAAAGGGCNN 30
1226-1244 CCCUUUUCCUUGACUAUUANN 1946 UAAUAGUCAAGGAAAAGGGNN 30
1227-1245 CCUUUUCCUUGACUAUUACNN 1947 GUAAUAGUCAAGGAAAAGGNN 30
1228-1246 CUUUUCCUUGACUAUUACANN 1948 UGUAAUAGUCAAGGAAAAGNN 30
1229-1247 UUUUCCUUGACUAUUACACNN 1949 GUGUAAUAGUCAAGGAAAANN 30
1230-1248 UUUCCUUGACUAUUACACUNN 1950 AGUGUAAUAGUCAAGGAAANN 30
1231-1249 UUCCUUGACUAUUACACUGNN 1951 CAGUGUAAUAGUCAAGGAANN 30
123-141 CUCCGCCGCCGGAGCCCCGNN 1952 CGGGGCUCCGGCGGCGGAGNN 30
1232-1250 UCCUUGACUAUUACACUGCNN 1953 GCAGUGUAAUAGUCAAGGANN 30
1233-1251 CCUUGACUAUUACACUGCCNN 1954 GGCAGUGUAAUAGUCAAGGNN 30
1234-1252 CUUGACUAUUACACUGCCUNN 1955 AGGCAGUGUAAUAGUCAAGNN 30
1235-1253 UUGACUAUUACACUGCCUGNN 1956 CAGGCAGUGUAAUAGUCAANN 30
1236-1254 UGACUAUUACACUGCCUGGNN 1957 CCAGGCAGUGUAAUAGUCANN 30
1237-1255 GACUAUUACACUGCCUGGANN 1958 UCCAGGCAGUGUAAUAGUCNN 30
1238-1256 ACUAUUACACUGCCUGGAGNN 1959 CUCCAGGCAGUGUAAUAGUNN 30
1239-1257 CUAUUACACUGCCUGGAGGNN 1960 CCUCCAGGCAGUGUAAUAGNN 30
1240-1258 UAUUACACUGCCUGGAGGANN 1961 UCCUCCAGGCAGUGUAAUANN 30
1241-1259 AUUACACUGCCUGGAGGAUNN 1962 AUCCUCCAGGCAGUGUAAUNN 30
124-142 UCCGCCGCCGGAGCCCCGGNN 1963 CCGGGGCUCCGGCGGCGGANN 30
1242-1260 UUACACUGCCUGGAGGAUANN 1964 UAUCCUCCAGGCAGUGUAANN 30
1243-1261 UACACUGCCUGGAGGAUAGNN 1965 CUAUCCUCCAGGCAGUGUANN 30
1244-1262 ACACUGCCUGGAGGAUAGCNN 1966 GCUAUCCUCCAGGCAGUGUNN 30
1245-1263 CACUGCCUGGAGGAUAGCANN 1967 UGCUAUCCUCCAGGCAGUGNN 30
1246-1264 ACUGCCUGGAGGAUAGCAGNN 1968 CUGCUAUCCUCCAGGCAGUNN 30
125-143 CCGCCGCCGGAGCCCCGGCNN 1969 GCCGGGGCTCCGGCGGCGGNN 30 126-144
CGCCGCCGGAGCCCCGGCCNN 1970 GGCCGGGGCTCCGGCGGCGNN 30 127-145
GCCGCCGGAGCCCCGGCCGNN 1971 CGGCCGGGGCTCCGGCGGCNN 30 1280-1298
CUUCAUUCAAAAAGCCAAANN 1972 UUUGGCUUUUUGAAUGAAGNN 30 1281-1299
UUCAUUCAAAAAGCCAAAANN 1973 UUUUGGCUUUUUGAAUGAANN 30 128-146
CCGCCGGAGCCCCGGCCGGNN 1974 CCGGCCGGGGCTCCGGCGGNN 30 1282-1300
UCAUUCAAAAAGCCAAAAUNN 1975 AUUUUGGCUUUUUGAAUGANN 30 1283-1301
CAUUCAAAAAGCCAAAAUANN 1976 UAUUUUGGCUUUUUGAAUGNN 30 1284-1302
AUUCAAAAAGCCAAAAUAGNN 1977 CUAUUUUGGCUUUUUGAAUNN 30 1285-1303
UUCAAAAAGCCAAAAUAGANN 1978 UCUAUUUUGGCUUUUUGAANN 30 1286-1304
UCAAAAAGCCAAAAUAGAGNN 1979 CUCUAUUUUGGCUUUUUGANN 30 1287-1305
CAAAAAGCCAAAAUAGAGANN 1980 UCUCUAUUUUGGCUUUUUGNN 30 1288-1306
AAAAAGCCAAAAUAGAGAGNN 1981 CUCUCUAUUUUGGCUUUUUNN 30 1289-1307
AAAAGCCAAAAUAGAGAGUNN 1982 ACUCUCUAUUUUGGCUUUUNN 30 1290-1308
AAAGCCAAAAUAGAGAGUANN 1983 UACUCUCUAUUUUGGCUUUNN 30 129-147
CGCCGGAGCCCCGGCCGGCNN 1984 GCCGGCCGGGGCTCCGGCGNN 30 130-148
GCCGGAGCCCCGGCCGGCCNN 1985 GGCCGGCCGGGGCTCCGGCNN 30 1310-1328
ACAGUCCUAGAGAAUUCCUNN 1986 AGGAAUUCUCUAGGACUGUNN 30 131-149
CCGGAGCCCCGGCCGGCCANN 1987 TGGCCGGCCGGGGCTCCGGNN 30 132-150
CGGAGCCCCGGCCGGCCAGNN 1988 CTGGCCGGCCGGGGCTCCGNN 30 1330-1348
UAUUUGUUCAGAUCUCAUANN 1989 UAUGAGAUCUGAACAAAUANN 30 1331-1349
AUUUGUUCAGAUCUCAUAGNN 1990 CUAUGAGAUCUGAACAAAUNN 30 133-151
GGAGCCCCGGCCGGCCAGGNN 1991 CCTGGCCGGCCGGGGCTCCNN 30 1332-1350
UUUGUUCAGAUCUCAUAGANN 1992 UCUAUGAGAUCUGAACAAANN 30 1333-1351
UUGUUCAGAUCUCAUAGAUNN 1993 AUCUAUGAGAUCUGAACAANN 30 1334-1352
UGUUCAGAUCUCAUAGAUGNN 1994 CAUCUAUGAGAUCUGAACANN 30 1335-1353
GUUCAGAUCUCAUAGAUGANN 1995 UCAUCUAUGAGAUCUGAACNN 30 134-152
GAGCCCCGGCCGGCCAGGCNN 1996 GCCTGGCCGGCCGGGGCTCNN 30 135-153
AGCCCCGGCCGGCCAGGCCNN 1997 GGCCTGGCCGGCCGGGGCTNN 30 136-154
GCCCCGGCCGGCCAGGCCCNN 1998 GGGCCTGGCCGGCCGGGGCNN 30 1365-1383
UGUCUUUUGACAUCCAGCANN 1999 UGCUGGAUGUCAAAAGACANN 30 1366-1384
GUCUUUUGACAUCCAGCAGNN 2000 CUGCUGGAUGUCAAAAGACNN 30 1367-1385
UCUUUUGACAUCCAGCAGUNN 2001 ACUGCUGGAUGUCAAAAGANN 30 1368-1386
CUUUUGACAUCCAGCAGUCNN 2002 GACUGCUGGAUGUCAAAAGNN 30 1369-1387
UUUUGACAUCCAGCAGUCCNN 2003 GGACUGCUGGAUGUCAAAANN 30 1370-1388
UUUGACAUCCAGCAGUCCANN 2004 UGGACUGCUGGAUGUCAAANN 30 1371-1389
UUGACAUCCAGCAGUCCAANN 2005 UUGGACUGCUGGAUGUCAANN 30 137-155
CCCCGGCCGGCCAGGCCCUNN 2006 AGGGCCUGGCCGGCCGGGGNN 30 138-156
CCCGGCCGGCCAGGCCCUGNN 2007 CAGGGCCUGGCCGGCCGGGNN 30 1391-1409
GUAUUGAGACAUAUUACUGNN 2008 CAGUAAUAUGUCUCAAUACNN 30 139-157
CCGGCCGGCCAGGCCCUGCNN 2009 GCAGGGCCUGGCCGGCCGGNN 30 140-158
CGGCCGGCCAGGCCCUGCCNN 2010 GGCAGGGCCUGGCCGGCCGNN 30 141-159
GGCCGGCCAGGCCCUGCCGNN 2011 CGGCAGGGCCUGGCCGGCCNN 30 1414-1432
UAAGAAAUAUUACUAUAAUNN 2012 AUUAUAGUAAUAUUUCUUANN 30 1415-1433
AAGAAAUAUUACUAUAAUUNN 2013 AAUUAUAGUAAUAUUUCUUNN 30 1416-1434
AGAAAUAUUACUAUAAUUGNN 2014 CAAUUAUAGUAAUAUUUCUNN 30 1417-1435
GAAAUAUUACUAUAAUUGANN 2015 UCAAUUAUAGUAAUAUUUCNN 30 1418-1436
AAAUAUUACUAUAAUUGAGNN 2016 CUCAAUUAUAGUAAUAUUUNN 30 1419-1437
AAUAUUACUAUAAUUGAGANN 2017 UCUCAAUUAUAGUAAUAUUNN 30 1420-1438
AUAUUACUAUAAUUGAGAANN 2018 UUCUCAAUUAUAGUAAUAUNN 30 1421-1439
UAUUACUAUAAUUGAGAACNN 2019 GUUCUCAAUUAUAGUAAUANN 30 142-160
GCCGGCCAGGCCCUGCCGCNN 2020 GCGGCAGGGCCUGGCCGGCNN 30 1422-1440
AUUACUAUAAUUGAGAACUNN 2021 AGUUCUCAAUUAUAGUAAUNN 30 1423-1441
UUACUAUAAUUGAGAACUANN 2022 UAGUUCUCAAUUAUAGUAANN 30 1424-1442
UACUAUAAUUGAGAACUACNN 2023 GUAGUUCUCAAUUAUAGUANN 30 1425-1443
ACUAUAAUUGAGAACUACANN 2024 UGUAGUUCUCAAUUAUAGUNN 30 1426-1444
CUAUAAUUGAGAACUACAGNN 2025 CUGUAGUUCUCAAUUAUAGNN 30 1427-1445
UAUAAUUGAGAACUACAGCNN 2026 GCUGUAGUUCUCAAUUAUANN 30 1428-1446
AUAAUUGAGAACUACAGCUNN 2027 AGCUGUAGUUCUCAAUUAUNN 30 1429-1447
UAAUUGAGAACUACAGCUUNN 2028 AAGCUGUAGUUCUCAAUUANN 30 1430-1448
AAUUGAGAACUACAGCUUUNN 2029 AAAGCUGUAGUUCUCAAUUNN 30 1431-1449
AUUGAGAACUACAGCUUUUNN 2030 AAAAGCUGUAGUUCUCAAUNN 30 143-161
CCGGCCAGGCCCUGCCGCUNN 2031 AGCGGCAGGGCCUGGCCGGNN 30 1432-1450
UUGAGAACUACAGCUUUUANN 2032 UAAAAGCUGUAGUUCUCAANN 30 1433-1451
UGAGAACUACAGCUUUUAANN 2033 UUAAAAGCUGUAGUUCUCANN 30 1434-1452
GAGAACUACAGCUUUUAAGNN 2034 CUUAAAAGCUGUAGUUCUCNN 30 1435-1453
AGAACUACAGCUUUUAAGANN 2035 UCUUAAAAGCUGUAGUUCUNN 30 1436-1454
GAACUACAGCUUUUAAGAUNN 2036 AUCUUAAAAGCUGUAGUUCNN 30 1437-1455
AACUACAGCUUUUAAGAUUNN 2037 AAUCUUAAAAGCUGUAGUUNN 30 1438-1456
ACUACAGCUUUUAAGAUUGNN 2038 CAAUCUUAAAAGCUGUAGUNN 30 1439-1457
CUACAGCUUUUAAGAUUGUNN 2039 ACAAUCUUAAAAGCUGUAGNN 30 1440-1458
UACAGCUUUUAAGAUUGUANN 2040 UACAAUCUUAAAAGCUGUANN 30 1441-1459
ACAGCUUUUAAGAUUGUACNN 2041 GUACAAUCUUAAAAGCUGUNN 31 144-162
CGGCCAGGCCCUGCCGCUCNN 2042 GAGCGGCAGGGCCUGGCCGNN 31 1442-1460
CAGCUUUUAAGAUUGUACUNN 2043 AGUACAAUCUUAAAAGCUGNN 31 1443-1461
AGCUUUUAAGAUUGUACUUNN 2044 AAGUACAAUCUUAAAAGCUNN 31 1444-1462
GCUUUUAAGAUUGUACUUUNN 2045 AAAGUACAAUCUUAAAAGCNN 31 1445-1463
CUUUUAAGAUUGUACUUUUNN 2046 AAAAGUACAAUCUUAAAAGNN 31 1446-1464
UUUUAAGAUUGUACUUUUANN 2047 UAAAAGUACAAUCUUAAAANN 31 1447-1465
UUUAAGAUUGUACUUUUAUNN 2048 AUAAAAGUACAAUCUUAAANN 31 1448-1466
UUAAGAUUGUACUUUUAUCNN 2049 GAUAAAAGUACAAUCUUAANN 31 1449-1467
UAAGAUUGUACUUUUAUCUNN 2050 AGAUAAAAGUACAAUCUUANN 31 1450-1468
AAGAUUGUACUUUUAUCUUNN 2051 AAGAUAAAAGUACAAUCUUNN 31 1451-1469
AGAUUGUACUUUUAUCUUANN 2052 UAAGAUAAAAGUACAAUCUNN 31 145-163
GGCCAGGCCCUGCCGCUCANN 2053 UGAGCGGCAGGGCCUGGCCNN 31 1452-1470
GAUUGUACUUUUAUCUUAANN 2054 UUAAGAUAAAAGUACAAUCNN 31 1453-1471
AUUGUACUUUUAUCUUAAANN 2055 UUUAAGAUAAAAGUACAAUNN 31 1454-1472
UUGUACUUUUAUCUUAAAANN 2056 UUUUAAGAUAAAAGUACAANN 31 1455-1473
UGUACUUUUAUCUUAAAAGNN 2057 CUUUUAAGAUAAAAGUACANN 31 1456-1474
GUACUUUUAUCUUAAAAGGNN 2058 CCUUUUAAGAUAAAAGUACNN 31 1457-1475
UACUUUUAUCUUAAAAGGGNN 2059 CCCUUUUAAGAUAAAAGUANN 31 1458-1476
ACUUUUAUCUUAAAAGGGUNN 2060 ACCCUUUUAAGAUAAAAGUNN 31 1459-1477
CUUUUAUCUUAAAAGGGUGNN 2061 CACCCUUUUAAGAUAAAAGNN 31 1460-1478
UUUUAUCUUAAAAGGGUGGNN 2062 CCACCCUUUUAAGAUAAAANN 31 1461-1479
UUUAUCUUAAAAGGGUGGUNN 2063 ACCACCCUUUUAAGAUAAANN 31 146-164
GCCAGGCCCUGCCGCUCAUNN 2064 AUGAGCGGCAGGGCCUGGCNN 31 1462-1480
UUAUCUUAAAAGGGUGGUANN 2065 UACCACCCUUUUAAGAUAANN 31 1463-1481
UAUCUUAAAAGGGUGGUAGNN 2066 CUACCACCCUUUUAAGAUANN 31 1464-1482
AUCUUAAAAGGGUGGUAGUNN 2067 ACUACCACCCUUUUAAGAUNN 31 1465-1483
UCUUAAAAGGGUGGUAGUUNN 2068 AACUACCACCCUUUUAAGANN 31 1466-1484
CUUAAAAGGGUGGUAGUUUNN 2069 AAACUACCACCCUUUUAAGNN 31
147-165 CCAGGCCCUGCCGCUCAUGNN 2070 CAUGAGCGGCAGGGCCUGGNN 31 148-166
CAGGCCCUGCCGCUCAUGGNN 2071 CCAUGAGCGGCAGGGCCUGNN 31 1486-1504
CCCUAAAAUACUUAUUAUGNN 2072 CAUAAUAAGUAUUUUAGGGNN 31 1487-1505
CCUAAAAUACUUAUUAUGUNN 2073 ACAUAAUAAGUAUUUUAGGNN 31 1488-1506
CUAAAAUACUUAUUAUGUANN 2074 UACAUAAUAAGUAUUUUAGNN 31 1489-1507
UAAAAUACUUAUUAUGUAANN 2075 UUACAUAAUAAGUAUUUUANN 31 1490-1508
AAAAUACUUAUUAUGUAAGNN 2076 CUUACAUAAUAAGUAUUUUNN 31 1491-1509
AAAUACUUAUUAUGUAAGGNN 2077 CCUUACAUAAUAAGUAUUUNN 31 149-167
AGGCCCUGCCGCUCAUGGUNN 2078 ACCAUGAGCGGCAGGGCCUNN 31 1492-1510
AAUACUUAUUAUGUAAGGGNN 2079 CCCUUACAUAAUAAGUAUUNN 31 1493-1511
AUACUUAUUAUGUAAGGGUNN 2080 ACCCUUACAUAAUAAGUAUNN 31 1494-1512
UACUUAUUAUGUAAGGGUCNN 2081 GACCCUUACAUAAUAAGUANN 31 1495-1513
ACUUAUUAUGUAAGGGUCANN 2082 UGACCCUUACAUAAUAAGUNN 31 1496-1514
CUUAUUAUGUAAGGGUCAUNN 2083 AUGACCCUUACAUAAUAAGNN 31 1497-1515
UUAUUAUGUAAGGGUCAUUNN 2084 AAUGACCCUUACAUAAUAANN 31 1498-1516
UAUUAUGUAAGGGUCAUUANN 2085 UAAUGACCCUUACAUAAUANN 31 1499-1517
AUUAUGUAAGGGUCAUUAGNN 2086 CUAAUGACCCUUACAUAAUNN 31 1500-1518
UUAUGUAAGGGUCAUUAGANN 2087 UCUAAUGACCCUUACAUAANN 31 1501-1519
UAUGUAAGGGUCAUUAGACNN 2088 GUCUAAUGACCCUUACAUANN 31 150-168
GGCCCUGCCGCUCAUGGUGNN 2089 CACCAUGAGCGGCAGGGCCNN 31 1502-1520
AUGUAAGGGUCAUUAGACANN 2090 UGUCUAAUGACCCUUACAUNN 31 1503-1521
UGUAAGGGUCAUUAGACAANN 2091 UUGUCUAAUGACCCUUACANN 31 1504-1522
GUAAGGGUCAUUAGACAAANN 2092 UUUGUCUAAUGACCCUUACNN 31 1505-1523
UAAGGGUCAUUAGACAAAUNN 2093 AUUUGUCUAAUGACCCUUANN 31 1506-1524
AAGGGUCAUUAGACAAAUGNN 2094 CAUUUGUCUAAUGACCCUUNN 31 1507-1525
AGGGUCAUUAGACAAAUGUNN 2095 ACAUUUGUCUAAUGACCCUNN 31 1508-1526
GGGUCAUUAGACAAAUGUCNN 2096 GACAUUUGUCUAAUGACCCNN 31 1509-1527
GGUCAUUAGACAAAUGUCUNN 2097 AGACAUUUGUCUAAUGACCNN 31 1510-1528
GUCAUUAGACAAAUGUCUUNN 2098 AAGACAUUUGUCUAAUGACNN 31 1511-1529
UCAUUAGACAAAUGUCUUGNN 2099 CAAGACAUUUGUCUAAUGANN 31 151-169
GCCCUGCCGCUCAUGGUGCNN 2100 GCACCAUGAGCGGCAGGGCNN 31 1512-1530
CAUUAGACAAAUGUCUUGANN 2101 UCAAGACAUUUGUCUAAUGNN 31 1513-1531
AUUAGACAAAUGUCUUGAANN 2102 UUCAAGACAUUUGUCUAAUNN 31 1514-1532
UUAGACAAAUGUCUUGAAGNN 2103 CUUCAAGACAUUUGUCUAANN 31 1515-1533
UAGACAAAUGUCUUGAAGUNN 2104 ACUUCAAGACAUUUGUCUANN 31 1516-1534
AGACAAAUGUCUUGAAGUANN 2105 UACUUCAAGACAUUUGUCUNN 31 1517-1535
GACAAAUGUCUUGAAGUAGNN 2106 CUACUUCAAGACAUUUGUCNN 31 1518-1536
ACAAAUGUCUUGAAGUAGANN 2107 UCUACUUCAAGACAUUUGUNN 31 152-170
CCCUGCCGCUCAUGGUGCCNN 2108 GGCACCAUGAGCGGCAGGGNN 31 153-171
CCUGCCGCUCAUGGUGCCANN 2109 UGGCACCAUGAGCGGCAGGNN 31 1541-1559
GAAUUUAUGAAUGGUUCUUNN 2110 AAGAACCAUUCAUAAAUUCNN 31 154-172
CUGCCGCUCAUGGUGCCAGNN 2111 CUGGCACCAUGAGCGGCAGNN 31 1542-1560
AAUUUAUGAAUGGUUCUUUNN 2112 AAAGAACCAUUCAUAAAUUNN 31 1543-1561
AUUUAUGAAUGGUUCUUUANN 2113 UAAAGAACCAUUCAUAAAUNN 31 1544-1562
UUUAUGAAUGGUUCUUUAUNN 2114 AUAAAGAACCAUUCAUAAANN 31 1545-1563
UUAUGAAUGGUUCUUUAUCNN 2115 GAUAAAGAACCAUUCAUAANN 31 1546-1564
UAUGAAUGGUUCUUUAUCANN 2116 UGAUAAAGAACCAUUCAUANN 31 1547-1565
AUGAAUGGUUCUUUAUCAUNN 2117 AUGAUAAAGAACCAUUCAUNN 31 1548-1566
UGAAUGGUUCUUUAUCAUUNN 2118 AAUGAUAAAGAACCAUUCANN 31 1549-1567
GAAUGGUUCUUUAUCAUUUNN 2119 AAAUGAUAAAGAACCAUUCNN 31 1550-1568
AAUGGUUCUUUAUCAUUUCNN 2120 GAAAUGAUAAAGAACCAUUNN 31 1551-1569
AUGGUUCUUUAUCAUUUCUNN 2121 AGAAAUGAUAAAGAACCAUNN 31 155-173
UGCCGCUCAUGGUGCCAGCNN 2122 GCUGGCACCAUGAGCGGCANN 31 1552-1570
UGGUUCUUUAUCAUUUCUCNN 2123 GAGAAAUGAUAAAGAACCANN 31 1553-1571
GGUUCUUUAUCAUUUCUCUNN 2124 AGAGAAAUGAUAAAGAACCNN 31 1554-1572
GUUCUUUAUCAUUUCUCUUNN 2125 AAGAGAAAUGAUAAAGAACNN 31 1555-1573
UUCUUUAUCAUUUCUCUUCNN 2126 GAAGAGAAAUGAUAAAGAANN 31 1556-1574
UCUUUAUCAUUUCUCUUCCNN 2127 GGAAGAGAAAUGAUAAAGANN 31 1557-1575
CUUUAUCAUUUCUCUUCCCNN 2128 GGGAAGAGAAAUGAUAAAGNN 31 1558-1576
UUUAUCAUUUCUCUUCCCCNN 2129 GGGGAAGAGAAAUGAUAAANN 31 1559-1577
UUAUCAUUUCUCUUCCCCCNN 2130 GGGGGAAGAGAAAUGAUAANN 31 1560-1578
UAUCAUUUCUCUUCCCCCUNN 2131 AGGGGGAAGAGAAAUGAUANN 31 1561-1579
AUCAUUUCUCUUCCCCCUUNN 2132 AAGGGGGAAGAGAAAUGAUNN 31 156-174
GCCGCUCAUGGUGCCAGCCNN 2133 GGCUGGCACCAUGAGCGGCNN 31 1562-1580
UCAUUUCUCUUCCCCCUUUNN 2134 AAAGGGGGAAGAGAAAUGANN 31 1563-1581
CAUUUCUCUUCCCCCUUUUNN 2135 AAAAGGGGGAAGAGAAAUGNN 31 1564-1582
AUUUCUCUUCCCCCUUUUUNN 2136 AAAAAGGGGGAAGAGAAAUNN 31 1565-1583
UUUCUCUUCCCCCUUUUUGNN 2137 CAAAAAGGGGGAAGAGAAANN 31 1566-1584
UUCUCUUCCCCCUUUUUGGNN 2138 CCAAAAAGGGGGAAGAGAANN 31 1567-1585
UCUCUUCCCCCUUUUUGGCNN 2139 GCCAAAAAGGGGGAAGAGANN 31 1568-1586
CUCUUCCCCCUUUUUGGCANN 2140 UGCCAAAAAGGGGGAAGAGNN 31 1569-1587
UCUUCCCCCUUUUUGGCAUNN 2141 AUGCCAAAAAGGGGGAAGANN 32 1570-1588
CUUCCCCCUUUUUGGCAUCNN 2142 GAUGCCAAAAAGGGGGAAGNN 32 1571-1589
UUCCCCCUUUUUGGCAUCCNN 2143 GGAUGCCAAAAAGGGGGAANN 32 157-175
CCGCUCAUGGUGCCAGCCCNN 2144 GGGCUGGCACCAUGAGCGGNN 32 1572-1590
UCCCCCUUUUUGGCAUCCUNN 2145 AGGAUGCCAAAAAGGGGGANN 32 1573-1591
CCCCCUUUUUGGCAUCCUGNN 2146 CAGGAUGCCAAAAAGGGGGNN 32 1574-1592
CCCCUUUUUGGCAUCCUGGNN 2147 CCAGGAUGCCAAAAAGGGGNN 32 1575-1593
CCCUUUUUGGCAUCCUGGCNN 2148 GCCAGGAUGCCAAAAAGGGNN 32 1576-1594
CCUUUUUGGCAUCCUGGCUNN 2149 AGCCAGGAUGCCAAAAAGGNN 32 1577-1595
CUUUUUGGCAUCCUGGCUUNN 2150 AAGCCAGGAUGCCAAAAAGNN 32 1578-1596
UUUUUGGCAUCCUGGCUUGNN 2151 CAAGCCAGGAUGCCAAAAANN 32 1579-1597
UUUUGGCAUCCUGGCUUGCNN 2152 GCAAGCCAGGAUGCCAAAANN 32 1580-1598
UUUGGCAUCCUGGCUUGCCNN 2153 GGCAAGCCAGGAUGCCAAANN 32 1581-1599
UUGGCAUCCUGGCUUGCCUNN 2154 AGGCAAGCCAGGAUGCCAANN 32 158-176
CGCUCAUGGUGCCAGCCCANN 2155 UGGGCUGGCACCAUGAGCGNN 32 1582-1600
UGGCAUCCUGGCUUGCCUCNN 2156 GAGGCAAGCCAGGAUGCCANN 32 1583-1601
GGCAUCCUGGCUUGCCUCCNN 2157 GGAGGCAAGCCAGGAUGCCNN 32 1584-1602
GCAUCCUGGCUUGCCUCCANN 2158 UGGAGGCAAGCCAGGAUGCNN 32 1585-1603
CAUCCUGGCUUGCCUCCAGNN 2159 CUGGAGGCAAGCCAGGAUGNN 32 1586-1604
AUCCUGGCUUGCCUCCAGUNN 2160 ACUGGAGGCAAGCCAGGAUNN 32 1587-1605
UCCUGGCUUGCCUCCAGUUNN 2161 AACUGGAGGCAAGCCAGGANN 32 1588-1606
CCUGGCUUGCCUCCAGUUUNN 2162 AAACUGGAGGCAAGCCAGGNN 32 1589-1607
CUGGCUUGCCUCCAGUUUUNN 2163 AAAACUGGAGGCAAGCCAGNN 32 1590-1608
UGGCUUGCCUCCAGUUUUANN 2164 UAAAACUGGAGGCAAGCCANN 32 1591-1609
GGCUUGCCUCCAGUUUUAGNN 2165 CUAAAACUGGAGGCAAGCCNN 32 159-177
GCUCAUGGUGCCAGCCCAGNN 2166 CUGGGCUGGCACCAUGAGCNN 32 1592-1610
GCUUGCCUCCAGUUUUAGGNN 2167 CCUAAAACUGGAGGCAAGCNN 32 1593-1611
CUUGCCUCCAGUUUUAGGUNN 2168 ACCUAAAACUGGAGGCAAGNN 32 1594-1612
UUGCCUCCAGUUUUAGGUCNN 2169 GACCUAAAACUGGAGGCAANN 32 1595-1613
UGCCUCCAGUUUUAGGUCCNN 2170 GGACCUAAAACUGGAGGCANN 32 160-178
CUCAUGGUGCCAGCCCAGANN 2171 UCUGGGCUGGCACCAUGAGNN 32 161-179
UCAUGGUGCCAGCCCAGAGNN 2172 CUCUGGGCUGGCACCAUGANN 32 1615-1633
UUAGUUUGCUUCUGUAAGCNN 2173 GCUUACAGAAGCAAACUAANN 32 1616-1634
UAGUUUGCUUCUGUAAGCANN 2174 UGCUUACAGAAGCAAACUANN 32 1617-1635
AGUUUGCUUCUGUAAGCAANN 2175 UUGCUUACAGAAGCAAACUNN 32 162-180
CAUGGUGCCAGCCCAGAGANN 2176 UCUCUGGGCUGGCACCAUGNN 32 163-181
AUGGUGCCAGCCCAGAGAGNN 2177 CUCUCUGGGCUGGCACCAUNN 32 1639-1657
GAACACCUGCUGAGGGGGCNN 2178 GCCCCCUCAGCAGGUGUUCNN 32 1640-1658
AACACCUGCUGAGGGGGCUNN 2179 AGCCCCCUCAGCAGGUGUUNN 32 1641-1659
ACACCUGCUGAGGGGGCUCNN 2180 GAGCCCCCUCAGCAGGUGUNN 32 164-182
UGGUGCCAGCCCAGAGAGGNN 2181 CCUCUCUGGGCUGGCACCANN 32 1642-1660
CACCUGCUGAGGGGGCUCUNN 2182 AGAGCCCCCUCAGCAGGUGNN 32 1643-1661
ACCUGCUGAGGGGGCUCUUNN 2183 AAGAGCCCCCUCAGCAGGUNN 32 1644-1662
CCUGCUGAGGGGGCUCUUUNN 2184 AAAGAGCCCCCUCAGCAGGNN 32 1645-1663
CUGCUGAGGGGGCUCUUUCNN 2185 GAAAGAGCCCCCUCAGCAGNN 32 1646-1664
UGCUGAGGGGGCUCUUUCCNN 2186 GGAAAGAGCCCCCUCAGCANN 32 1647-1665
GCUGAGGGGGCUCUUUCCCNN 2187 GGGAAAGAGCCCCCUCAGCNN 32 1648-1666
CUGAGGGGGCUCUUUCCCUNN 2188 AGGGAAAGAGCCCCCUCAGNN 32 1649-1667
UGAGGGGGCUCUUUCCCUCNN 2189 GAGGGAAAGAGCCCCCUCANN 32 1650-1668
GAGGGGGCUCUUUCCCUCANN 2190 UGAGGGAAAGAGCCCCCUCNN 32 165-183
GGUGCCAGCCCAGAGAGGGNN 2191 CCCUCUCUGGGCUGGCACCNN 32 166-184
GUGCCAGCCCAGAGAGGGGNN 2192 CCCCUCUCUGGGCUGGCACNN 32 1670-1688
GUAUACUUCAAGUAAGAUCNN 2193 GAUCUUACUUGAAGUAUACNN 32 1671-1689
UAUACUUCAAGUAAGAUCANN 2194 UGAUCUUACUUGAAGUAUANN 32
167-185 UGCCAGCCCAGAGAGGGGCNN 2195 GCCCCUCUCUGGGCUGGCANN 32
1672-1690 AUACUUCAAGUAAGAUCAANN 2196 UUGAUCUUACUUGAAGUAUNN 32
1673-1691 UACUUCAAGUAAGAUCAAGNN 2197 CUUGAUCUUACUUGAAGUANN 32
1674-1692 ACUUCAAGUAAGAUCAAGANN 2198 UCUUGAUCUUACUUGAAGUNN 32
1675-1693 CUUCAAGUAAGAUCAAGAANN 2199 UUCUUGAUCUUACUUGAAGNN 32
1676-1694 UUCAAGUAAGAUCAAGAAUNN 2200 AUUCUUGAUCUUACUUGAANN 32
1677-1695 UCAAGUAAGAUCAAGAAUCNN 2201 GAUUCUUGAUCUUACUUGANN 32
1678-1696 CAAGUAAGAUCAAGAAUCUNN 2202 AGAUUCUUGAUCUUACUUGNN 32
1679-1697 AAGUAAGAUCAAGAAUCUUNN 2203 AAGAUUCUUGAUCUUACUUNN 32
1680-1698 AGUAAGAUCAAGAAUCUUUNN 2204 AAAGAUUCUUGAUCUUACUNN 32
1681-1699 GUAAGAUCAAGAAUCUUUUNN 2205 AAAAGAUUCUUGAUCUUACNN 32
1682-1700 UAAGAUCAAGAAUCUUUUGNN 2206 CAAAAGAUUCUUGAUCUUANN 32
1683-1701 AAGAUCAAGAAUCUUUUGUNN 2207 ACAAAAGAUUCUUGAUCUUNN 32
1684-1702 AGAUCAAGAAUCUUUUGUGNN 2208 CACAAAAGAUUCUUGAUCUNN 32
1685-1703 GAUCAAGAAUCUUUUGUGANN 2209 UCACAAAAGAUUCUUGAUCNN 32
1686-1704 AUCAAGAAUCUUUUGUGAANN 2210 UUCACAAAAGAUUCUUGAUNN 32
1687-1705 UCAAGAAUCUUUUGUGAAANN 2211 UUUCACAAAAGAUUCUUGANN 32
1707-1725 UAUAGAAAUUUACUAUGUANN 2212 UACAUAGUAAAUUUCUAUANN 32
1708-1726 AUAGAAAUUUACUAUGUAANN 2213 UUACAUAGUAAAUUUCUAUNN 32
1709-1727 UAGAAAUUUACUAUGUAAANN 2214 UUUACAUAGUAAAUUUCUANN 32
1710-1728 AGAAAUUUACUAUGUAAAUNN 2215 AUUUACAUAGUAAAUUUCUNN 32
1711-1729 GAAAUUUACUAUGUAAAUGNN 2216 CAUUUACAUAGUAAAUUUCNN 32
1712-1730 AAAUUUACUAUGUAAAUGCNN 2217 GCAUUUACAUAGUAAAUUUNN 32
1713-1731 AAUUUACUAUGUAAAUGCUNN 2218 AGCAUUUACAUAGUAAAUUNN 32
1714-1732 AUUUACUAUGUAAAUGCUUNN 2219 AAGCAUUUACAUAGUAAAUNN 32
1715-1733 UUUACUAUGUAAAUGCUUGNN 2220 CAAGCAUUUACAUAGUAAANN 32
1716-1734 UUACUAUGUAAAUGCUUGANN 2221 UCAAGCAUUUACAUAGUAANN 32
1717-1735 UACUAUGUAAAUGCUUGAUNN 2222 AUCAAGCAUUUACAUAGUANN 32
1718-1736 ACUAUGUAAAUGCUUGAUGNN 2223 CAUCAAGCAUUUACAUAGUNN 32
1719-1737 CUAUGUAAAUGCUUGAUGGNN 2224 CCAUCAAGCAUUUACAUAGNN 32
1720-1738 UAUGUAAAUGCUUGAUGGANN 2225 UCCAUCAAGCAUUUACAUANN 32
1721-1739 AUGUAAAUGCUUGAUGGAANN 2226 UUCCAUCAAGCAUUUACAUNN 32
1722-1740 UGUAAAUGCUUGAUGGAAUNN 2227 AUUCCAUCAAGCAUUUACANN 32
1723-1741 GUAAAUGCUUGAUGGAAUUNN 2228 AAUUCCAUCAAGCAUUUACNN 32
1724-1742 UAAAUGCUUGAUGGAAUUUNN 2229 AAAUUCCAUCAAGCAUUUANN 32
1725-1743 AAAUGCUUGAUGGAAUUUUNN 2230 AAAAUUCCAUCAAGCAUUUNN 32
1726-1744 AAUGCUUGAUGGAAUUUUUNN 2231 AAAAAUUCCAUCAAGCAUUNN 32
1727-1745 AUGCUUGAUGGAAUUUUUUNN 2232 AAAAAAUUCCAUCAAGCAUNN 32
1728-1746 UGCUUGAUGGAAUUUUUUCNN 2233 GAAAAAAUUCCAUCAAGCANN 32
1729-1747 GCUUGAUGGAAUUUUUUCCNN 2234 GGAAAAAAUUCCAUCAAGCNN 32
1730-1748 CUUGAUGGAAUUUUUUCCUNN 2235 AGGAAAAAAUUCCAUCAAGNN 32
1731-1749 UUGAUGGAAUUUUUUCCUGNN 2236 CAGGAAAAAAUUCCAUCAANN 32
1732-1750 UGAUGGAAUUUUUUCCUGCNN 2237 GCAGGAAAAAAUUCCAUCANN 32
1733-1751 GAUGGAAUUUUUUCCUGCUNN 2238 AGCAGGAAAAAAUUCCAUCNN 32
1734-1752 AUGGAAUUUUUUCCUGCUANN 2239 UAGCAGGAAAAAAUUCCAUNN 32
1735-1753 UGGAAUUUUUUCCUGCUAGNN 2240 CUAGCAGGAAAAAAUUCCANN 32
1736-1754 GGAAUUUUUUCCUGCUAGUNN 2241 ACUAGCAGGAAAAAAUUCCNN 33
1737-1755 GAAUUUUUUCCUGCUAGUGNN 2242 CACUAGCAGGAAAAAAUUCNN 33
1738-1756 AAUUUUUUCCUGCUAGUGUNN 2243 ACACUAGCAGGAAAAAAUUNN 33
1739-1757 AUUUUUUCCUGCUAGUGUANN 2244 UACACUAGCAGGAAAAAAUNN 33
1740-1758 UUUUUUCCUGCUAGUGUAGNN 2245 CUACACUAGCAGGAAAAAANN 33
1741-1759 UUUUUCCUGCUAGUGUAGCNN 2246 GCUACACUAGCAGGAAAAANN 33
1742-1760 UUUUCCUGCUAGUGUAGCUNN 2247 AGCUACACUAGCAGGAAAANN 33
1743-1761 UUUCCUGCUAGUGUAGCUUNN 2248 AAGCUACACUAGCAGGAAANN 33
1744-1762 UUCCUGCUAGUGUAGCUUCNN 2249 GAAGCUACACUAGCAGGAANN 33
1745-1763 UCCUGCUAGUGUAGCUUCUNN 2250 AGAAGCUACACUAGCAGGANN 33
1746-1764 CCUGCUAGUGUAGCUUCUGNN 2251 CAGAAGCUACACUAGCAGGNN 33
1747-1765 CUGCUAGUGUAGCUUCUGANN 2252 UCAGAAGCUACACUAGCAGNN 33
1748-1766 UGCUAGUGUAGCUUCUGAANN 2253 UUCAGAAGCUACACUAGCANN 33
1749-1767 GCUAGUGUAGCUUCUGAAANN 2254 UUUCAGAAGCUACACUAGCNN 33
1750-1768 CUAGUGUAGCUUCUGAAAGNN 2255 CUUUCAGAAGCUACACUAGNN 33
1751-1769 UAGUGUAGCUUCUGAAAGGNN 2256 CCUUUCAGAAGCUACACUANN 33
1752-1770 AGUGUAGCUUCUGAAAGGUNN 2257 ACCUUUCAGAAGCUACACUNN 33
1753-1771 GUGUAGCUUCUGAAAGGUGNN 2258 CACCUUUCAGAAGCUACACNN 33
1754-1772 UGUAGCUUCUGAAAGGUGCNN 2259 GCACCUUUCAGAAGCUACANN 33
1755-1773 GUAGCUUCUGAAAGGUGCUNN 2260 AGCACCUUUCAGAAGCUACNN 33
1756-1774 UAGCUUCUGAAAGGUGCUUNN 2261 AAGCACCUUUCAGAAGCUANN 33
1757-1775 AGCUUCUGAAAGGUGCUUUNN 2262 AAAGCACCUUUCAGAAGCUNN 33
1758-1776 GCUUCUGAAAGGUGCUUUCNN 2263 GAAAGCACCUUUCAGAAGCNN 33
1777-1795 UCCAUUUAUUUAAAACUACNN 2264 GUAGUUUUAAAUAAAUGGANN 33
1778-1796 CCAUUUAUUUAAAACUACCNN 2265 GGUAGUUUUAAAUAAAUGGNN 33
1779-1797 CAUUUAUUUAAAACUACCCNN 2266 GGGUAGUUUUAAAUAAAUGNN 33
1780-1798 AUUUAUUUAAAACUACCCANN 2267 UGGGUAGUUUUAAAUAAAUNN 33
1781-1799 UUUAUUUAAAACUACCCAUNN 2268 AUGGGUAGUUUUAAAUAAANN 33
1782-1800 UUAUUUAAAACUACCCAUGNN 2269 CAUGGGUAGUUUUAAAUAANN 33
1783-1801 UAUUUAAAACUACCCAUGCNN 2270 GCAUGGGUAGUUUUAAAUANN 33
1784-1802 AUUUAAAACUACCCAUGCANN 2271 UGCAUGGGUAGUUUUAAAUNN 33
1785-1803 UUUAAAACUACCCAUGCAANN 2272 UUGCAUGGGUAGUUUUAAANN 33
1786-1804 UUAAAACUACCCAUGCAAUNN 2273 AUUGCAUGGGUAGUUUUAANN 33
1787-1805 UAAAACUACCCAUGCAAUUNN 2274 AAUUGCAUGGGUAGUUUUANN 33
1788-1806 AAAACUACCCAUGCAAUUANN 2275 UAAUUGCAUGGGUAGUUUUNN 33
1789-1807 AAACUACCCAUGCAAUUAANN 2276 UUAAUUGCAUGGGUAGUUUNN 33
1790-1808 AACUACCCAUGCAAUUAAANN 2277 UUUAAUUGCAUGGGUAGUUNN 33
1791-1809 ACUACCCAUGCAAUUAAAANN 2278 UUUUAAUUGCAUGGGUAGUNN 33
1792-1810 CUACCCAUGCAAUUAAAAGNN 2279 CUUUUAAUUGCAUGGGUAGNN 33
1793-1811 UACCCAUGCAAUUAAAAGGNN 2280 CCUUUUAAUUGCAUGGGUANN 33
1794-1812 ACCCAUGCAAUUAAAAGGUNN 2281 ACCUUUUAAUUGCAUGGGUNN 33
1795-1813 CCCAUGCAAUUAAAAGGUANN 2282 UACCUUUUAAUUGCAUGGGNN 33
1796-1814 CCAUGCAAUUAAAAGGUACNN 2283 GUACCUUUUAAUUGCAUGGNN 33
1797-1815 CAUGCAAUUAAAAGGUACANN 2284 UGUACCUUUUAAUUGCAUGNN 33
1798-1816 AUGCAAUUAAAAGGUACAANN 2285 UUGUACCUUUUAAUUGCAUNN 33
1799-1817 UGCAAUUAAAAGGUACAAUNN 2286 AUUGUACCUUUUAAUUGCANN 33
1800-1818 GCAAUUAAAAGGUACAAUGNN 2287 CAUUGUACCUUUUAAUUGCNN 33
1801-1819 CAAUUAAAAGGUACAAUGCNN 2288 GCAUUGUACCUUUUAAUUGNN 33
1802-1820 AAUUAAAAGGUACAAUGCANN 2289 UGCAUUGUACCUUUUAAUUNN 33
187-205 AGCCCGGAGGCAGCGAGCGNN 2290 CGCTCGCTGCCTCCGGGCTNN 33 188-206
GCCCGGAGGCAGCGAGCGGNN 2291 CCGCTCGCTGCCTCCGGGCNN 33 189-207
CCCGGAGGCAGCGAGCGGGNN 2292 CCCGCTCGCTGCCTCCGGGNN 33 190-208
CCGGAGGCAGCGAGCGGGGNN 2293 CCCCGCTCGCTGCCTCCGGNN 33 191-209
CGGAGGCAGCGAGCGGGGGNN 2294 CCCCCGCTCGCTGCCTCCGNN 33 192-210
GGAGGCAGCGAGCGGGGGGNN 2295 CCCCCCGCTCGCTGCCTCCNN 33 193-211
GAGGCAGCGAGCGGGGGGCNN 2296 GCCCCCCGCTCGCTGCCTCNN 33 194-212
AGGCAGCGAGCGGGGGGCUNN 2297 AGCCCCCCGCUCGCUGCCUNN 33 195-213
GGCAGCGAGCGGGGGGCUGNN 2298 CAGCCCCCCGCUCGCUGCCNN 33 196-214
GCAGCGAGCGGGGGGCUGCNN 2299 GCAGCCCCCCGCUCGCUGCNN 33 197-215
CAGCGAGCGGGGGGCUGCCNN 2300 GGCAGCCCCCCGCUCGCUGNN 33 198-216
AGCGAGCGGGGGGCUGCCCNN 2301 GGGCAGCCCCCCGCUCGCUNN 33 199-217
GCGAGCGGGGGGCUGCCCCNN 2302 GGGGCAGCCCCCCGCUCGCNN 33 200-218
CGAGCGGGGGGCUGCCCCANN 2303 UGGGGCAGCCCCCCGCUCGNN 33 201-219
GAGCGGGGGGCUGCCCCAGNN 2304 CUGGGGCAGCCCCCCGCUCNN 33 202-220
AGCGGGGGGCUGCCCCAGGNN 2305 CCUGGGGCAGCCCCCCGCUNN 33 203-221
GCGGGGGGCUGCCCCAGGCNN 2306 GCCUGGGGCAGCCCCCCGCNN 33 204-222
CGGGGGGCUGCCCCAGGCGNN 2307 CGCCUGGGGCAGCCCCCCGNN 33 205-223
GGGGGGCUGCCCCAGGCGCNN 2308 GCGCCUGGGGCAGCCCCCCNN 33 206-224
GGGGGCUGCCCCAGGCGCGNN 2309 CGCGCCUGGGGCAGCCCCCNN 33 207-225
GGGGCUGCCCCAGGCGCGCNN 2310 GCGCGCCUGGGGCAGCCCCNN 33 208-226
GGGCUGCCCCAGGCGCGCANN 2311 UGCGCGCCUGGGGCAGCCCNN 33 209-227
GGCUGCCCCAGGCGCGCAANN 2312 UUGCGCGCCUGGGGCAGCCNN 33 210-228
GCUGCCCCAGGCGCGCAAGNN 2313 CUUGCGCGCCUGGGGCAGCNN 33 211-229
CUGCCCCAGGCGCGCAAGCNN 2314 GCUUGCGCGCCUGGGGCAGNN 33 212-230
UGCCCCAGGCGCGCAAGCGNN 2315 CGCUUGCGCGCCUGGGGCANN 33 247-265
CUGAGCCCCGAGGAGAAGGNN 2316 CCUUCUCCUCGGGGCUCAGNN 33 248-266
UGAGCCCCGAGGAGAAGGCNN 2317 GCCUUCUCCUCGGGGCUCANN 33 249-267
GAGCCCCGAGGAGAAGGCGNN 2318 CGCCTTCTCCTCGGGGCTCNN 33 250-268
AGCCCCGAGGAGAAGGCGCNN 2319 GCGCCTTCTCCTCGGGGCTNN 33 251-269
GCCCCGAGGAGAAGGCGCUNN 2320 AGCGCCUUCUCCUCGGGGCNN 33
252-270 CCCCGAGGAGAAGGCGCUGNN 2321 CAGCGCCUUCUCCUCGGGGNN 33 253-271
CCCGAGGAGAAGGCGCUGANN 2322 UCAGCGCCUUCUCCUCGGGNN 33 254-272
CCGAGGAGAAGGCGCUGAGNN 2323 CUCAGCGCCUUCUCCUCGGNN 33 255-273
CGAGGAGAAGGCGCUGAGGNN 2324 CCUCAGCGCCUUCUCCUCGNN 33 256-274
GAGGAGAAGGCGCUGAGGANN 2325 UCCUCAGCGCCUUCUCCUCNN 33 257-275
AGGAGAAGGCGCUGAGGAGNN 2326 CUCCUCAGCGCCUUCUCCUNN 33 258-276
GGAGAAGGCGCUGAGGAGGNN 2327 CCUCCUCAGCGCCUUCUCCNN 33 259-277
GAGAAGGCGCUGAGGAGGANN 2328 UCCUCCUCAGCGCCUUCUCNN 33 260-278
AGAAGGCGCUGAGGAGGAANN 2329 UUCCUCCUCAGCGCCUUCUNN 33 261-279
GAAGGCGCUGAGGAGGAAANN 2330 UUUCCUCCUCAGCGCCUUCNN 33 262-280
AAGGCGCUGAGGAGGAAACNN 2331 GUUUCCUCCUCAGCGCCUUNN 33 263-281
AGGCGCUGAGGAGGAAACUNN 2332 AGUUUCCUCCUCAGCGCCUNN 33 264-282
GGCGCUGAGGAGGAAACUGNN 2333 CAGUUUCCUCCUCAGCGCCNN 33 265-283
GCGCUGAGGAGGAAACUGANN 2334 UCAGUUUCCUCCUCAGCGCNN 33 266-284
CGCUGAGGAGGAAACUGAANN 2335 UUCAGUUUCCUCCUCAGCGNN 33 267-285
GCUGAGGAGGAAACUGAAANN 2336 UUUCAGUUUCCUCCUCAGCNN 33 268-286
CUGAGGAGGAAACUGAAAANN 2337 UUUUCAGUUUCCUCCUCAGNN 33 269-287
UGAGGAGGAAACUGAAAAANN 2338 UUUUUCAGUUUCCUCCUCANN 33 270-288
GAGGAGGAAACUGAAAAACNN 2339 GUUUUUCAGUUUCCUCCUCNN 33 271-289
AGGAGGAAACUGAAAAACANN 2340 UGUUUUUCAGUUUCCUCCUNN 33 272-290
GGAGGAAACUGAAAAACAGNN 2341 CUGUUUUUCAGUUUCCUCCNN 34 273-291
GAGGAAACUGAAAAACAGANN 2342 UCUGUUUUUCAGUUUCCUCNN 34 274-292
AGGAAACUGAAAAACAGAGNN 2343 CUCUGUUUUUCAGUUUCCUNN 34 275-293
GGAAACUGAAAAACAGAGUNN 2344 ACUCUGUUUUUCAGUUUCCNN 34 276-294
GAAACUGAAAAACAGAGUANN 2345 UACUCUGUUUUUCAGUUUCNN 34 277-295
AAACUGAAAAACAGAGUAGNN 2346 CUACUCUGUUUUUCAGUUUNN 34 278-296
AACUGAAAAACAGAGUAGCNN 2347 GCUACUCUGUUUUUCAGUUNN 34 279-297
ACUGAAAAACAGAGUAGCANN 2348 UGCUACUCUGUUUUUCAGUNN 34 280-298
CUGAAAAACAGAGUAGCAGNN 2349 CUGCUACUCUGUUUUUCAGNN 34 281-299
UGAAAAACAGAGUAGCAGCNN 2350 GCUGCUACUCUGUUUUUCANN 34 282-300
GAAAAACAGAGUAGCAGCUNN 2351 AGCUGCUACUCUGUUUUUCNN 34 283-301
AAAAACAGAGUAGCAGCUCNN 2352 GAGCUGCUACUCUGUUUUUNN 34 284-302
AAAACAGAGUAGCAGCUCANN 2353 UGAGCUGCUACUCUGUUUUNN 34 285-303
AAACAGAGUAGCAGCUCAGNN 2354 CUGAGCUGCUACUCUGUUUNN 34 286-304
AACAGAGUAGCAGCUCAGANN 2355 UCUGAGCUGCUACUCUGUUNN 34 287-305
ACAGAGUAGCAGCUCAGACNN 2356 GUCUGAGCUGCUACUCUGUNN 34 288-306
CAGAGUAGCAGCUCAGACUNN 2357 AGUCUGAGCUGCUACUCUGNN 34 289-307
AGAGUAGCAGCUCAGACUGNN 2358 CAGUCUGAGCUGCUACUCUNN 34 290-308
GAGUAGCAGCUCAGACUGCNN 2359 GCAGUCUGAGCUGCUACUCNN 34 291-309
AGUAGCAGCUCAGACUGCCNN 2360 GGCAGUCUGAGCUGCUACUNN 34 292-310
GUAGCAGCUCAGACUGCCANN 2361 UGGCAGUCUGAGCUGCUACNN 34 293-311
UAGCAGCUCAGACUGCCAGNN 2362 CUGGCAGUCUGAGCUGCUANN 34 294-312
AGCAGCUCAGACUGCCAGANN 2363 UCUGGCAGUCUGAGCUGCUNN 34 295-313
GCAGCUCAGACUGCCAGAGNN 2364 CUCUGGCAGUCUGAGCUGCNN 34 296-314
CAGCUCAGACUGCCAGAGANN 2365 UCUCUGGCAGUCUGAGCUGNN 34 297-315
AGCUCAGACUGCCAGAGAUNN 2366 AUCUCUGGCAGUCUGAGCUNN 34 298-316
GCUCAGACUGCCAGAGAUCNN 2367 GAUCUCUGGCAGUCUGAGCNN 34 299-317
CUCAGACUGCCAGAGAUCGNN 2368 CGAUCUCUGGCAGUCUGAGNN 34 300-318
UCAGACUGCCAGAGAUCGANN 2369 UCGAUCUCUGGCAGUCUGANN 34 301-319
CAGACUGCCAGAGAUCGAANN 2370 UUCGAUCUCUGGCAGUCUGNN 34 302-320
AGACUGCCAGAGAUCGAAANN 2371 UUUCGAUCUCUGGCAGUCUNN 34 303-321
GACUGCCAGAGAUCGAAAGNN 2372 CUUUCGAUCUCUGGCAGUCNN 34 304-322
ACUGCCAGAGAUCGAAAGANN 2373 UCUUUCGAUCUCUGGCAGUNN 34 305-323
CUGCCAGAGAUCGAAAGAANN 2374 UUCUUUCGAUCUCUGGCAGNN 34 325-343
GCUCGAAUGAGUGAGCUGGNN 2375 CCAGCUCACUCAUUCGAGCNN 34 326-344
CUCGAAUGAGUGAGCUGGANN 2376 UCCAGCUCACUCAUUCGAGNN 34 327-345
UCGAAUGAGUGAGCUGGAANN 2377 UUCCAGCUCACUCAUUCGANN 34 328-346
CGAAUGAGUGAGCUGGAACNN 2378 GUUCCAGCUCACUCAUUCGNN 34 329-347
GAAUGAGUGAGCUGGAACANN 2379 UGUUCCAGCUCACUCAUUCNN 34 330-348
AAUGAGUGAGCUGGAACAGNN 2380 CUGUUCCAGCUCACUCAUUNN 34 331-349
AUGAGUGAGCUGGAACAGCNN 2381 GCUGUUCCAGCUCACUCAUNN 34 332-350
UGAGUGAGCUGGAACAGCANN 2382 UGCUGUUCCAGCUCACUCANN 34 333-351
GAGUGAGCUGGAACAGCAANN 2383 UUGCUGUUCCAGCUCACUCNN 34 334-352
AGUGAGCUGGAACAGCAAGNN 2384 CUUGCUGUUCCAGCUCACUNN 34 335-353
GUGAGCUGGAACAGCAAGUNN 2385 ACUUGCUGUUCCAGCUCACNN 34 336-354
UGAGCUGGAACAGCAAGUGNN 2386 CACUUGCUGUUCCAGCUCANN 34 337-355
GAGCUGGAACAGCAAGUGGNN 2387 CCACUUGCUGUUCCAGCUCNN 34 338-356
AGCUGGAACAGCAAGUGGUNN 2388 ACCACUUGCUGUUCCAGCUNN 34 339-357
GCUGGAACAGCAAGUGGUANN 2389 UACCACUUGCUGUUCCAGCNN 34 340-358
CUGGAACAGCAAGUGGUAGNN 2390 CUACCACUUGCUGUUCCAGNN 34 341-359
UGGAACAGCAAGUGGUAGANN 2391 UCUACCACUUGCUGUUCCANN 34 342-360
GGAACAGCAAGUGGUAGAUNN 2392 AUCUACCACUUGCUGUUCCNN 34 343-361
GAACAGCAAGUGGUAGAUUNN 2393 AAUCUACCACUUGCUGUUCNN 34 344-362
AACAGCAAGUGGUAGAUUUNN 2394 AAAUCUACCACUUGCUGUUNN 34 345-363
ACAGCAAGUGGUAGAUUUANN 2395 UAAAUCUACCACUUGCUGUNN 34 346-364
CAGCAAGUGGUAGAUUUAGNN 2396 CUAAAUCUACCACUUGCUGNN 34 347-365
AGCAAGUGGUAGAUUUAGANN 2397 UCUAAAUCUACCACUUGCUNN 34 348-366
GCAAGUGGUAGAUUUAGAANN 2398 UUCUAAAUCUACCACUUGCNN 34 349-367
CAAGUGGUAGAUUUAGAAGNN 2399 CUUCUAAAUCUACCACUUGNN 34 350-368
AAGUGGUAGAUUUAGAAGANN 2400 UCUUCUAAAUCUACCACUUNN 34 351-369
AGUGGUAGAUUUAGAAGAANN 2401 UUCUUCUAAAUCUACCACUNN 34 352-370
GUGGUAGAUUUAGAAGAAGNN 2402 CUUCUUCUAAAUCUACCACNN 34 353-371
UGGUAGAUUUAGAAGAAGANN 2403 UCUUCUUCUAAAUCUACCANN 34 354-372
GGUAGAUUUAGAAGAAGAGNN 2404 CUCUUCUUCUAAAUCUACCNN 34 355-373
GUAGAUUUAGAAGAAGAGANN 2405 UCUCUUCUUCUAAAUCUACNN 34 356-374
UAGAUUUAGAAGAAGAGAANN 2406 UUCUCUUCUUCUAAAUCUANN 34 357-375
AGAUUUAGAAGAAGAGAACNN 2407 GUUCUCUUCUUCUAAAUCUNN 34 358-376
GAUUUAGAAGAAGAGAACCNN 2408 GGUUCUCUUCUUCUAAAUCNN 34 359-377
AUUUAGAAGAAGAGAACCANN 2409 UGGUUCUCUUCUUCUAAAUNN 34 360-378
UUUAGAAGAAGAGAACCAANN 2410 UUGGUUCUCUUCUUCUAAANN 34 361-379
UUAGAAGAAGAGAACCAAANN 2411 UUUGGUUCUCUUCUUCUAANN 34 362-380
UAGAAGAAGAGAACCAAAANN 2412 UUUUGGUUCUCUUCUUCUANN 34 363-381
AGAAGAAGAGAACCAAAAANN 2413 TTTTTGGTTCTCTTCTTCTNN 34 364-382
GAAGAAGAGAACCAAAAACNN 2414 GTTTTTGGTTCTCTTCTTCNN 34 365-383
AAGAAGAGAACCAAAAACUNN 2415 AGUUUUUGGUUCUCUUCUUNN 34 366-384
AGAAGAGAACCAAAAACUUNN 2416 AAGUUUUUGGUUCUCUUCUNN 34 367-385
GAAGAGAACCAAAAACUUUNN 2417 AAAGUUUUUGGUUCUCUUCNN 34 368-386
AAGAGAACCAAAAACUUUUNN 2418 AAAAGUUUUUGGUUCUCUUNN 34 369-387
AGAGAACCAAAAACUUUUGNN 2419 CAAAAGUUUUUGGUUCUCUNN 34 370-388
GAGAACCAAAAACUUUUGCNN 2420 GCAAAAGUUUUUGGUUCUCNN 34 371-389
AGAACCAAAAACUUUUGCUNN 2421 AGCAAAAGUUUUUGGUUCUNN 34 372-390
GAACCAAAAACUUUUGCUANN 2422 UAGCAAAAGUUUUUGGUUCNN 34 373-391
AACCAAAAACUUUUGCUAGNN 2423 CUAGCAAAAGUUUUUGGUUNN 34 374-392
ACCAAAAACUUUUGCUAGANN 2424 UCUAGCAAAAGUUUUUGGUNN 34 375-393
CCAAAAACUUUUGCUAGAANN 2425 UUCUAGCAAAAGUUUUUGGNN 34 376-394
CAAAAACUUUUGCUAGAAANN 2426 UUUCUAGCAAAAGUUUUUGNN 34 377-395
AAAAACUUUUGCUAGAAAANN 2427 UUUUCUAGCAAAAGUUUUUNN 34 378-396
AAAACUUUUGCUAGAAAAUNN 2428 AUUUUCUAGCAAAAGUUUUNN 34 379-397
AAACUUUUGCUAGAAAAUCNN 2429 GAUUUUCUAGCAAAAGUUUNN 34 380-398
AACUUUUGCUAGAAAAUCANN 2430 UGAUUUUCUAGCAAAAGUUNN 34 381-399
ACUUUUGCUAGAAAAUCAGNN 2431 CUGAUUUUCUAGCAAAAGUNN 34 382-400
CUUUUGCUAGAAAAUCAGCNN 2432 GCUGAUUUUCUAGCAAAAGNN 34 383-401
UUUUGCUAGAAAAUCAGCUNN 2433 AGCUGAUUUUCUAGCAAAANN 34 384-402
UUUGCUAGAAAAUCAGCUUNN 2434 AAGCUGAUUUUCUAGCAAANN 34 385-403
UUGCUAGAAAAUCAGCUUUNN 2435 AAAGCUGAUUUUCUAGCAANN 34 386-404
UGCUAGAAAAUCAGCUUUUNN 2436 AAAAGCUGAUUUUCUAGCANN 34 387-405
GCUAGAAAAUCAGCUUUUANN 2437 UAAAAGCUGAUUUUCUAGCNN 34 388-406
CUAGAAAAUCAGCUUUUACNN 2438 GUAAAAGCUGAUUUUCUAGNN 34 389-407
UAGAAAAUCAGCUUUUACGNN 2439 CGUAAAAGCUGAUUUUCUANN 34 390-408
AGAAAAUCAGCUUUUACGANN 2440 UCGUAAAAGCUGAUUUUCUNN 34 391-409
GAAAAUCAGCUUUUACGAGNN 2441 CUCGUAAAAGCUGAUUUUCNN 35 392-410
AAAAUCAGCUUUUACGAGANN 2442 UCUCGUAAAAGCUGAUUUUNN 35 393-411
AAAUCAGCUUUUACGAGAGNN 2443 CUCUCGUAAAAGCUGAUUUNN 35 394-412
AAUCAGCUUUUACGAGAGANN 2444 UCUCUCGUAAAAGCUGAUUNN 35 395-413
AUCAGCUUUUACGAGAGAANN 2445 UUCUCUCGUAAAAGCUGAUNN 35
396-414 UCAGCUUUUACGAGAGAAANN 2446 UUUCUCUCGUAAAAGCUGANN 35 397-415
CAGCUUUUACGAGAGAAAANN 2447 UUUUCUCUCGUAAAAGCUGNN 35 398-416
AGCUUUUACGAGAGAAAACNN 2448 GUUUUCUCUCGUAAAAGCUNN 35 399-417
GCUUUUACGAGAGAAAACUNN 2449 AGUUUUCUCUCGUAAAAGCNN 35 400-418
CUUUUACGAGAGAAAACUCNN 2450 GAGUUUUCUCUCGUAAAAGNN 35 401-419
UUUUACGAGAGAAAACUCANN 2451 UGAGUUUUCUCUCGUAAAANN 35 421-439
GGCCUUGUAGUUGAGAACCNN 2452 GGUUCUCAACUACAAGGCCNN 35 422-440
GCCUUGUAGUUGAGAACCANN 2453 UGGUUCUCAACUACAAGGCNN 35 423-441
CCUUGUAGUUGAGAACCAGNN 2454 CUGGUUCUCAACUACAAGGNN 35 424-442
CUUGUAGUUGAGAACCAGGNN 2455 CCUGGUUCUCAACUACAAGNN 35 425-443
UUGUAGUUGAGAACCAGGANN 2456 UCCUGGUUCUCAACUACAANN 35 426-444
UGUAGUUGAGAACCAGGAGNN 2457 CUCCUGGUUCUCAACUACANN 35 427-445
GUAGUUGAGAACCAGGAGUNN 2458 ACUCCUGGUUCUCAACUACNN 35 428-446
UAGUUGAGAACCAGGAGUUNN 2459 AACUCCUGGUUCUCAACUANN 35 429-447
AGUUGAGAACCAGGAGUUANN 2460 UAACUCCUGGUUCUCAACUNN 35 430-448
GUUGAGAACCAGGAGUUAANN 2461 UUAACUCCUGGUUCUCAACNN 35 431-449
UUGAGAACCAGGAGUUAAGNN 2462 CUUAACUCCUGGUUCUCAANN 35 432-450
UGAGAACCAGGAGUUAAGANN 2463 UCUUAACUCCUGGUUCUCANN 35 433-451
GAGAACCAGGAGUUAAGACNN 2464 GUCUUAACUCCUGGUUCUCNN 35 434-452
AGAACCAGGAGUUAAGACANN 2465 UGUCUUAACUCCUGGUUCUNN 35 435-453
GAACCAGGAGUUAAGACAGNN 2466 CUGUCUUAACUCCUGGUUCNN 35 436-454
AACCAGGAGUUAAGACAGCNN 2467 GCUGUCUUAACUCCUGGUUNN 35 437-455
ACCAGGAGUUAAGACAGCGNN 2468 CGCUGUCUUAACUCCUGGUNN 35 438-456
CCAGGAGUUAAGACAGCGCNN 2469 GCGCUGUCUUAACUCCUGGNN 35 44-62
GAGCUAUGGUGGUGGUGGCNN 2470 GCCACCACCACCAUAGCUCNN 35 45-63
AGCUAUGGUGGUGGUGGCANN 2471 UGCCACCACCACCAUAGCUNN 35 458-476
UGGGGAUGGAUGCCCUGGUNN 2472 ACCAGGGCAUCCAUCCCCANN 35 459-477
GGGGAUGGAUGCCCUGGUUNN 2473 AACCAGGGCAUCCAUCCCCNN 35 460-478
GGGAUGGAUGCCCUGGUUGNN 2474 CAACCAGGGCAUCCAUCCCNN 35 461-479
GGAUGGAUGCCCUGGUUGCNN 2475 GCAACCAGGGCAUCCAUCCNN 35 462-480
GAUGGAUGCCCUGGUUGCUNN 2476 AGCAACCAGGGCAUCCAUCNN 35 46-64
GCUAUGGUGGUGGUGGCAGNN 2477 CUGCCACCACCACCAUAGCNN 35 47-65
CUAUGGUGGUGGUGGCAGCNN 2478 GCUGCCACCACCACCAUAGNN 35 482-500
AAGAGGAGGCGGAAGCCAANN 2479 TTGGCTTCCGCCTCCTCTTNN 35 483-501
AGAGGAGGCGGAAGCCAAGNN 2480 CTTGGCTTCCGCCTCCTCTNN 35 484-502
GAGGAGGCGGAAGCCAAGGNN 2481 CCTTGGCTTCCGCCTCCTCNN 35 485-503
AGGAGGCGGAAGCCAAGGGNN 2482 CCCTTGGCTTCCGCCTCCTNN 35 486-504
GGAGGCGGAAGCCAAGGGGNN 2483 CCCCTTGGCTTCCGCCTCCNN 35 48-66
UAUGGUGGUGGUGGCAGCCNN 2484 GGCUGCCACCACCACCAUANN 35 487-505
GAGGCGGAAGCCAAGGGGANN 2485 TCCCCTTGGCTTCCGCCTCNN 35 488-506
AGGCGGAAGCCAAGGGGAANN 2486 TTCCCCTTGGCTTCCGCCTNN 35 489-507
GGCGGAAGCCAAGGGGAAUNN 2487 AUUCCCCUUGGCUUCCGCCNN 35 490-508
GCGGAAGCCAAGGGGAAUGNN 2488 CAUUCCCCUUGGCUUCCGCNN 35 49-67
AUGGUGGUGGUGGCAGCCGNN 2489 CGGCUGCCACCACCACCAUNN 35 50-68
UGGUGGUGGUGGCAGCCGCNN 2490 GCGGCUGCCACCACCACCANN 35 510-528
AGUGAGGCCAGUGGCCGGGNN 2491 CCCGGCCACUGGCCUCACUNN 35 511-529
GUGAGGCCAGUGGCCGGGUNN 2492 ACCCGGCCACUGGCCUCACNN 35 512-530
UGAGGCCAGUGGCCGGGUCNN 2493 GACCCGGCCACUGGCCUCANN 35 513-531
GAGGCCAGUGGCCGGGUCUNN 2494 AGACCCGGCCACUGGCCUCNN 35 514-532
AGGCCAGUGGCCGGGUCUGNN 2495 CAGACCCGGCCACUGGCCUNN 35 515-533
GGCCAGUGGCCGGGUCUGCNN 2496 GCAGACCCGGCCACUGGCCNN 35 516-534
GCCAGUGGCCGGGUCUGCUNN 2497 AGCAGACCCGGCCACUGGCNN 35 517-535
CCAGUGGCCGGGUCUGCUGNN 2498 CAGCAGACCCGGCCACUGGNN 35 518-536
CAGUGGCCGGGUCUGCUGANN 2499 UCAGCAGACCCGGCCACUGNN 35 519-537
AGUGGCCGGGUCUGCUGAGNN 2500 CUCAGCAGACCCGGCCACUNN 35 520-538
GUGGCCGGGUCUGCUGAGUNN 2501 ACUCAGCAGACCCGGCCACNN 35 521-539
UGGCCGGGUCUGCUGAGUCNN 2502 GACUCAGCAGACCCGGCCANN 35 522-540
GGCCGGGUCUGCUGAGUCCNN 2503 GGACUCAGCAGACCCGGCCNN 35 523-541
GCCGGGUCUGCUGAGUCCGNN 2504 CGGACUCAGCAGACCCGGCNN 35 524-542
CCGGGUCUGCUGAGUCCGCNN 2505 GCGGACUCAGCAGACCCGGNN 35 525-543
CGGGUCUGCUGAGUCCGCANN 2506 UGCGGACUCAGCAGACCCGNN 35 526-544
GGGUCUGCUGAGUCCGCAGNN 2507 CUGCGGACUCAGCAGACCCNN 35 574-592
GUGCAGGCCCAGUUGUCACNN 2508 GUGACAACUGGGCCUGCACNN 35 575-593
UGCAGGCCCAGUUGUCACCNN 2509 GGUGACAACUGGGCCUGCANN 35 576-594
GCAGGCCCAGUUGUCACCCNN 2510 GGGUGACAACUGGGCCUGCNN 35 577-595
CAGGCCCAGUUGUCACCCCNN 2511 GGGGUGACAACUGGGCCUGNN 35 578-596
AGGCCCAGUUGUCACCCCUNN 2512 AGGGGUGACAACUGGGCCUNN 35 579-597
GGCCCAGUUGUCACCCCUCNN 2513 GAGGGGUGACAACUGGGCCNN 35 580-598
GCCCAGUUGUCACCCCUCCNN 2514 GGAGGGGUGACAACUGGGCNN 35 581-599
CCCAGUUGUCACCCCUCCANN 2515 UGGAGGGGUGACAACUGGGNN 35 582-600
CCAGUUGUCACCCCUCCAGNN 2516 CUGGAGGGGUGACAACUGGNN 35 583-601
CAGUUGUCACCCCUCCAGANN 2517 UCUGGAGGGGUGACAACUGNN 35 584-602
AGUUGUCACCCCUCCAGAANN 2518 UUCUGGAGGGGUGACAACUNN 35 585-603
GUUGUCACCCCUCCAGAACNN 2519 GUUCUGGAGGGGUGACAACNN 35 586-604
UUGUCACCCCUCCAGAACANN 2520 UGUUCUGGAGGGGUGACAANN 35 587-605
UGUCACCCCUCCAGAACAUNN 2521 AUGUUCUGGAGGGGUGACANN 35 588-606
GUCACCCCUCCAGAACAUCNN 2522 GAUGUUCUGGAGGGGUGACNN 35 589-607
UCACCCCUCCAGAACAUCUNN 2523 AGAUGUUCUGGAGGGGUGANN 35 590-608
CACCCCUCCAGAACAUCUCNN 2524 GAGAUGUUCUGGAGGGGUGNN 35 591-609
ACCCCUCCAGAACAUCUCCNN 2525 GGAGAUGUUCUGGAGGGGUNN 35 592-610
CCCCUCCAGAACAUCUCCCNN 2526 GGGAGAUGUUCUGGAGGGGNN 35 593-611
CCCUCCAGAACAUCUCCCCNN 2527 GGGGAGAUGUUCUGGAGGGNN 35 594-612
CCUCCAGAACAUCUCCCCANN 2528 UGGGGAGAUGUUCUGGAGGNN 35 595-613
CUCCAGAACAUCUCCCCAUNN 2529 AUGGGGAGAUGUUCUGGAGNN 35 596-614
UCCAGAACAUCUCCCCAUGNN 2530 CAUGGGGAGAUGUUCUGGANN 35 597-615
CCAGAACAUCUCCCCAUGGNN 2531 CCAUGGGGAGAUGUUCUGGNN 35 598-616
CAGAACAUCUCCCCAUGGANN 2532 UCCAUGGGGAGAUGUUCUGNN 35 599-617
AGAACAUCUCCCCAUGGAUNN 2533 AUCCAUGGGGAGAUGUUCUNN 35 600-618
GAACAUCUCCCCAUGGAUUNN 2534 AAUCCAUGGGGAGAUGUUCNN 35 601-619
AACAUCUCCCCAUGGAUUCNN 2535 GAAUCCAUGGGGAGAUGUUNN 35 602-620
ACAUCUCCCCAUGGAUUCUNN 2536 AGAAUCCAUGGGGAGAUGUNN 35 603-621
CAUCUCCCCAUGGAUUCUGNN 2537 CAGAAUCCAUGGGGAGAUGNN 35 604-622
AUCUCCCCAUGGAUUCUGGNN 2538 CCAGAAUCCAUGGGGAGAUNN 35 605-623
UCUCCCCAUGGAUUCUGGCNN 2539 GCCAGAAUCCAUGGGGAGANN 35 606-624
CUCCCCAUGGAUUCUGGCGNN 2540 CGCCAGAAUCCAUGGGGAGNN 35 607-625
UCCCCAUGGAUUCUGGCGGNN 2541 CCGCCAGAAUCCAUGGGGANN 36 608-626
CCCCAUGGAUUCUGGCGGUNN 2542 ACCGCCAGAAUCCAUGGGGNN 36 609-627
CCCAUGGAUUCUGGCGGUANN 2543 UACCGCCAGAAUCCAUGGGNN 36 610-628
CCAUGGAUUCUGGCGGUAUNN 2544 AUACCGCCAGAAUCCAUGGNN 36 611-629
CAUGGAUUCUGGCGGUAUUNN 2545 AAUACCGCCAGAAUCCAUGNN 36 612-630
AUGGAUUCUGGCGGUAUUGNN 2546 CAAUACCGCCAGAAUCCAUNN 36 613-631
UGGAUUCUGGCGGUAUUGANN 2547 UCAAUACCGCCAGAAUCCANN 36 614-632
GGAUUCUGGCGGUAUUGACNN 2548 GUCAAUACCGCCAGAAUCCNN 36 615-633
GAUUCUGGCGGUAUUGACUNN 2549 AGUCAAUACCGCCAGAAUCNN 36 616-634
AUUCUGGCGGUAUUGACUCNN 2550 GAGUCAAUACCGCCAGAAUNN 36 617-635
UUCUGGCGGUAUUGACUCUNN 2551 AGAGUCAAUACCGCCAGAANN 36 618-636
UCUGGCGGUAUUGACUCUUNN 2552 AAGAGUCAAUACCGCCAGANN 36 619-637
CUGGCGGUAUUGACUCUUCNN 2553 GAAGAGUCAAUACCGCCAGNN 36 620-638
UGGCGGUAUUGACUCUUCANN 2554 UGAAGAGUCAAUACCGCCANN 36 621-639
GGCGGUAUUGACUCUUCAGNN 2555 CUGAAGAGUCAAUACCGCCNN 36 622-640
GCGGUAUUGACUCUUCAGANN 2556 UCUGAAGAGUCAAUACCGCNN 36 623-641
CGGUAUUGACUCUUCAGAUNN 2557 AUCUGAAGAGUCAAUACCGNN 36 624-642
GGUAUUGACUCUUCAGAUUNN 2558 AAUCUGAAGAGUCAAUACCNN 36 625-643
GUAUUGACUCUUCAGAUUCNN 2559 GAAUCUGAAGAGUCAAUACNN 36 626-644
UAUUGACUCUUCAGAUUCANN 2560 UGAAUCUGAAGAGUCAAUANN 36 627-645
AUUGACUCUUCAGAUUCAGNN 2561 CUGAAUCUGAAGAGUCAAUNN 36 628-646
UUGACUCUUCAGAUUCAGANN 2562 UCUGAAUCUGAAGAGUCAANN 36 629-647
UGACUCUUCAGAUUCAGAGNN 2563 CUCUGAAUCUGAAGAGUCANN 36 630-648
GACUCUUCAGAUUCAGAGUNN 2564 ACUCUGAAUCUGAAGAGUCNN 36 631-649
ACUCUUCAGAUUCAGAGUCNN 2565 GACUCUGAAUCUGAAGAGUNN 36 632-650
CUCUUCAGAUUCAGAGUCUNN 2566 AGACUCUGAAUCUGAAGAGNN 36 633-651
UCUUCAGAUUCAGAGUCUGNN 2567 CAGACUCUGAAUCUGAAGANN 36 634-652
CUUCAGAUUCAGAGUCUGANN 2568 UCAGACUCUGAAUCUGAAGNN 36 635-653
UUCAGAUUCAGAGUCUGAUNN 2569 AUCAGACUCUGAAUCUGAANN 36 636-654
UCAGAUUCAGAGUCUGAUANN 2570 UAUCAGACUCUGAAUCUGANN 36 637-655
CAGAUUCAGAGUCUGAUAUNN 2571 AUAUCAGACUCUGAAUCUGNN 36
638-656 AGAUUCAGAGUCUGAUAUCNN 2572 GAUAUCAGACUCUGAAUCUNN 36 639-657
GAUUCAGAGUCUGAUAUCCNN 2573 GGAUAUCAGACUCUGAAUCNN 36 640-658
AUUCAGAGUCUGAUAUCCUNN 2574 AGGAUAUCAGACUCUGAAUNN 36 641-659
UUCAGAGUCUGAUAUCCUGNN 2575 CAGGAUAUCAGACUCUGAANN 36 642-660
UCAGAGUCUGAUAUCCUGUNN 2576 ACAGGAUAUCAGACUCUGANN 36 643-661
CAGAGUCUGAUAUCCUGUUNN 2577 AACAGGAUAUCAGACUCUGNN 36 644-662
AGAGUCUGAUAUCCUGUUGNN 2578 CAACAGGAUAUCAGACUCUNN 36 645-663
GAGUCUGAUAUCCUGUUGGNN 2579 CCAACAGGAUAUCAGACUCNN 36 646-664
AGUCUGAUAUCCUGUUGGGNN 2580 CCCAACAGGAUAUCAGACUNN 36 647-665
GUCUGAUAUCCUGUUGGGCNN 2581 GCCCAACAGGAUAUCAGACNN 36 648-666
UCUGAUAUCCUGUUGGGCANN 2582 UGCCCAACAGGAUAUCAGANN 36 649-667
CUGAUAUCCUGUUGGGCAUNN 2583 AUGCCCAACAGGAUAUCAGNN 36 650-668
UGAUAUCCUGUUGGGCAUUNN 2584 AAUGCCCAACAGGAUAUCANN 36 651-669
GAUAUCCUGUUGGGCAUUCNN 2585 GAAUGCCCAACAGGAUAUCNN 36 652-670
AUAUCCUGUUGGGCAUUCUNN 2586 AGAAUGCCCAACAGGAUAUNN 36 653-671
UAUCCUGUUGGGCAUUCUGNN 2587 CAGAAUGCCCAACAGGAUANN 36 654-672
AUCCUGUUGGGCAUUCUGGNN 2588 CCAGAAUGCCCAACAGGAUNN 36 655-673
UCCUGUUGGGCAUUCUGGANN 2589 UCCAGAAUGCCCAACAGGANN 36 656-674
CCUGUUGGGCAUUCUGGACNN 2590 GUCCAGAAUGCCCAACAGGNN 36 657-675
CUGUUGGGCAUUCUGGACANN 2591 UGUCCAGAAUGCCCAACAGNN 36 658-676
UGUUGGGCAUUCUGGACAANN 2592 UUGUCCAGAAUGCCCAACANN 36 659-677
GUUGGGCAUUCUGGACAACNN 2593 GUUGUCCAGAAUGCCCAACNN 36 660-678
UUGGGCAUUCUGGACAACUNN 2594 AGUUGUCCAGAAUGCCCAANN 36 661-679
UGGGCAUUCUGGACAACUUNN 2595 AAGUUGUCCAGAAUGCCCANN 36 662-680
GGGCAUUCUGGACAACUUGNN 2596 CAAGUUGUCCAGAAUGCCCNN 36 663-681
GGCAUUCUGGACAACUUGGNN 2597 CCAAGUUGUCCAGAAUGCCNN 36 664-682
GCAUUCUGGACAACUUGGANN 2598 UCCAAGUUGUCCAGAAUGCNN 36 665-683
CAUUCUGGACAACUUGGACNN 2599 GUCCAAGUUGUCCAGAAUGNN 36 666-684
AUUCUGGACAACUUGGACCNN 2600 GGUCCAAGUUGUCCAGAAUNN 36 667-685
UUCUGGACAACUUGGACCCNN 2601 GGGUCCAAGUUGUCCAGAANN 36 668-686
UCUGGACAACUUGGACCCANN 2602 UGGGUCCAAGUUGUCCAGANN 36 669-687
CUGGACAACUUGGACCCAGNN 2603 CUGGGUCCAAGUUGUCCAGNN 36 670-688
UGGACAACUUGGACCCAGUNN 2604 ACUGGGUCCAAGUUGUCCANN 36 671-689
GGACAACUUGGACCCAGUCNN 2605 GACUGGGUCCAAGUUGUCCNN 36 672-690
GACAACUUGGACCCAGUCANN 2606 UGACUGGGUCCAAGUUGUCNN 36 673-691
ACAACUUGGACCCAGUCAUNN 2607 AUGACUGGGUCCAAGUUGUNN 36 674-692
CAACUUGGACCCAGUCAUGNN 2608 CAUGACUGGGUCCAAGUUGNN 36 675-693
AACUUGGACCCAGUCAUGUNN 2609 ACAUGACUGGGUCCAAGUUNN 36 676-694
ACUUGGACCCAGUCAUGUUNN 2610 AACAUGACUGGGUCCAAGUNN 36 677-695
CUUGGACCCAGUCAUGUUCNN 2611 GAACAUGACUGGGUCCAAGNN 36 678-696
UUGGACCCAGUCAUGUUCUNN 2612 AGAACAUGACUGGGUCCAANN 36 679-697
UGGACCCAGUCAUGUUCUUNN 2613 AAGAACAUGACUGGGUCCANN 36 680-698
GGACCCAGUCAUGUUCUUCNN 2614 GAAGAACAUGACUGGGUCCNN 36 681-699
GACCCAGUCAUGUUCUUCANN 2615 UGAAGAACAUGACUGGGUCNN 36 682-700
ACCCAGUCAUGUUCUUCAANN 2616 UUGAAGAACAUGACUGGGUNN 36 683-701
CCCAGUCAUGUUCUUCAAANN 2617 UUUGAAGAACAUGACUGGGNN 36 684-702
CCAGUCAUGUUCUUCAAAUNN 2618 AUUUGAAGAACAUGACUGGNN 36 685-703
CAGUCAUGUUCUUCAAAUGNN 2619 CAUUUGAAGAACAUGACUGNN 36 686-704
AGUCAUGUUCUUCAAAUGCNN 2620 GCAUUUGAAGAACAUGACUNN 36 687-705
GUCAUGUUCUUCAAAUGCCNN 2621 GGCAUUUGAAGAACAUGACNN 36 688-706
UCAUGUUCUUCAAAUGCCCNN 2622 GGGCAUUUGAAGAACAUGANN 36 689-707
CAUGUUCUUCAAAUGCCCUNN 2623 AGGGCAUUUGAAGAACAUGNN 36 690-708
AUGUUCUUCAAAUGCCCUUNN 2624 AAGGGCAUUUGAAGAACAUNN 36 691-709
UGUUCUUCAAAUGCCCUUCNN 2625 GAAGGGCAUUUGAAGAACANN 36 692-710
GUUCUUCAAAUGCCCUUCCNN 2626 GGAAGGGCAUUUGAAGAACNN 36 693-711
UUCUUCAAAUGCCCUUCCCNN 2627 GGGAAGGGCAUUUGAAGAANN 36 694-712
UCUUCAAAUGCCCUUCCCCNN 2628 GGGGAAGGGCAUUUGAAGANN 36 695-713
CUUCAAAUGCCCUUCCCCANN 2629 UGGGGAAGGGCAUUUGAAGNN 36 696-714
UUCAAAUGCCCUUCCCCAGNN 2630 CUGGGGAAGGGCAUUUGAANN 36 697-715
UCAAAUGCCCUUCCCCAGANN 2631 UCUGGGGAAGGGCAUUUGANN 36 698-716
CAAAUGCCCUUCCCCAGAGNN 2632 CUCUGGGGAAGGGCAUUUGNN 36 718-736
CUGCCAGCCUGGAGGAGCUNN 2633 AGCUCCUCCAGGCUGGCAGNN 36 719-737
UGCCAGCCUGGAGGAGCUCNN 2634 GAGCUCCUCCAGGCUGGCANN 36 720-738
GCCAGCCUGGAGGAGCUCCNN 2635 GGAGCUCCUCCAGGCUGGCNN 36 721-739
CCAGCCUGGAGGAGCUCCCNN 2636 GGGAGCUCCUCCAGGCUGGNN 36 722-740
CAGCCUGGAGGAGCUCCCANN 2637 UGGGAGCUCCUCCAGGCUGNN 36 723-741
AGCCUGGAGGAGCUCCCAGNN 2638 CUGGGAGCUCCUCCAGGCUNN 36 724-742
GCCUGGAGGAGCUCCCAGANN 2639 UCUGGGAGCUCCUCCAGGCNN 36 725-743
CCUGGAGGAGCUCCCAGAGNN 2640 CUCUGGGAGCUCCUCCAGGNN 36 726-744
CUGGAGGAGCUCCCAGAGGNN 2641 CCUCUGGGAGCUCCUCCAGNN 37 727-745
UGGAGGAGCUCCCAGAGGUNN 2642 ACCUCUGGGAGCUCCUCCANN 37 728-746
GGAGGAGCUCCCAGAGGUCNN 2643 GACCUCUGGGAGCUCCUCCNN 37 729-747
GAGGAGCUCCCAGAGGUCUNN 2644 AGACCUCUGGGAGCUCCUCNN 37 730-748
AGGAGCUCCCAGAGGUCUANN 2645 UAGACCUCUGGGAGCUCCUNN 37 731-749
GGAGCUCCCAGAGGUCUACNN 2646 GUAGACCUCUGGGAGCUCCNN 37 732-750
GAGCUCCCAGAGGUCUACCNN 2647 GGUAGACCUCUGGGAGCUCNN 37 733-751
AGCUCCCAGAGGUCUACCCNN 2648 GGGUAGACCUCUGGGAGCUNN 37 734-752
GCUCCCAGAGGUCUACCCANN 2649 UGGGUAGACCUCUGGGAGCNN 37 735-753
CUCCCAGAGGUCUACCCAGNN 2650 CUGGGUAGACCUCUGGGAGNN 37 736-754
UCCCAGAGGUCUACCCAGANN 2651 UCUGGGUAGACCUCUGGGANN 37 737-755
CCCAGAGGUCUACCCAGAANN 2652 UUCUGGGUAGACCUCUGGGNN 37 738-756
CCAGAGGUCUACCCAGAAGNN 2653 CUUCUGGGUAGACCUCUGGNN 37 739-757
CAGAGGUCUACCCAGAAGGNN 2654 CCUUCUGGGUAGACCUCUGNN 37 740-758
AGAGGUCUACCCAGAAGGANN 2655 UCCUUCUGGGUAGACCUCUNN 37 741-759
GAGGUCUACCCAGAAGGACNN 2656 GUCCUUCUGGGUAGACCUCNN 37 742-760
AGGUCUACCCAGAAGGACCNN 2657 GGUCCUUCUGGGUAGACCUNN 37 743-761
GGUCUACCCAGAAGGACCCNN 2658 GGGUCCUUCUGGGUAGACCNN 37 744-762
GUCUACCCAGAAGGACCCANN 2659 UGGGUCCUUCUGGGUAGACNN 37 745-763
UCUACCCAGAAGGACCCAGNN 2660 CUGGGUCCUUCUGGGUAGANN 37 746-764
CUACCCAGAAGGACCCAGUNN 2661 ACUGGGUCCUUCUGGGUAGNN 37 747-765
UACCCAGAAGGACCCAGUUNN 2662 AACUGGGUCCUUCUGGGUANN 37 748-766
ACCCAGAAGGACCCAGUUCNN 2663 GAACUGGGUCCUUCUGGGUNN 37 749-767
CCCAGAAGGACCCAGUUCCNN 2664 GGAACUGGGUCCUUCUGGGNN 37 750-768
CCAGAAGGACCCAGUUCCUNN 2665 AGGAACUGGGUCCUUCUGGNN 37 751-769
CAGAAGGACCCAGUUCCUUNN 2666 AAGGAACUGGGUCCUUCUGNN 37 752-770
AGAAGGACCCAGUUCCUUANN 2667 UAAGGAACUGGGUCCUUCUNN 37 753-771
GAAGGACCCAGUUCCUUACNN 2668 GUAAGGAACUGGGUCCUUCNN 37 754-772
AAGGACCCAGUUCCUUACCNN 2669 GGUAAGGAACUGGGUCCUUNN 37 755-773
AGGACCCAGUUCCUUACCANN 2670 UGGUAAGGAACUGGGUCCUNN 37 756-774
GGACCCAGUUCCUUACCAGNN 2671 CUGGUAAGGAACUGGGUCCNN 37 757-775
GACCCAGUUCCUUACCAGCNN 2672 GCUGGUAAGGAACUGGGUCNN 37 758-776
ACCCAGUUCCUUACCAGCCNN 2673 GGCUGGUAAGGAACUGGGUNN 37 759-777
CCCAGUUCCUUACCAGCCUNN 2674 AGGCUGGUAAGGAACUGGGNN 37 760-778
CCAGUUCCUUACCAGCCUCNN 2675 GAGGCUGGUAAGGAACUGGNN 37 761-779
CAGUUCCUUACCAGCCUCCNN 2676 GGAGGCUGGUAAGGAACUGNN 37 762-780
AGUUCCUUACCAGCCUCCCNN 2677 GGGAGGCUGGUAAGGAACUNN 37 763-781
GUUCCUUACCAGCCUCCCUNN 2678 AGGGAGGCUGGUAAGGAACNN 37 764-782
UUCCUUACCAGCCUCCCUUNN 2679 AAGGGAGGCUGGUAAGGAANN 37 765-783
UCCUUACCAGCCUCCCUUUNN 2680 AAAGGGAGGCUGGUAAGGANN 37 766-784
CCUUACCAGCCUCCCUUUCNN 2681 GAAAGGGAGGCUGGUAAGGNN 37 767-785
CUUACCAGCCUCCCUUUCUNN 2682 AGAAAGGGAGGCUGGUAAGNN 37 768-786
UUACCAGCCUCCCUUUCUCNN 2683 GAGAAAGGGAGGCUGGUAANN 37 769-787
UACCAGCCUCCCUUUCUCUNN 2684 AGAGAAAGGGAGGCUGGUANN 37 770-788
ACCAGCCUCCCUUUCUCUGNN 2685 CAGAGAAAGGGAGGCUGGUNN 37 771-789
CCAGCCUCCCUUUCUCUGUNN 2686 ACAGAGAAAGGGAGGCUGGNN 37 772-790
CAGCCUCCCUUUCUCUGUCNN 2687 GACAGAGAAAGGGAGGCUGNN 37 773-791
AGCCUCCCUUUCUCUGUCANN 2688 UGACAGAGAAAGGGAGGCUNN 37 774-792
GCCUCCCUUUCUCUGUCAGNN 2689 CUGACAGAGAAAGGGAGGCNN 37 775-793
CCUCCCUUUCUCUGUCAGUNN 2690 ACUGACAGAGAAAGGGAGGNN 37 776-794
CUCCCUUUCUCUGUCAGUGNN 2691 CACUGACAGAGAAAGGGAGNN 37 777-795
UCCCUUUCUCUGUCAGUGGNN 2692 CCACUGACAGAGAAAGGGANN 37 778-796
CCCUUUCUCUGUCAGUGGGNN 2693 CCCACUGACAGAGAAAGGGNN 37 779-797
CCUUUCUCUGUCAGUGGGGNN 2694 CCCCACUGACAGAGAAAGGNN 37 780-798
CUUUCUCUGUCAGUGGGGANN 2695 UCCCCACUGACAGAGAAAGNN 37 781-799
UUUCUCUGUCAGUGGGGACNN 2696 GUCCCCACUGACAGAGAAANN 37
782-800 UUCUCUGUCAGUGGGGACGNN 2697 CGUCCCCACUGACAGAGAANN 37 783-801
UCUCUGUCAGUGGGGACGUNN 2698 ACGUCCCCACUGACAGAGANN 37 784-802
CUCUGUCAGUGGGGACGUCNN 2699 GACGUCCCCACUGACAGAGNN 37 785-803
UCUGUCAGUGGGGACGUCANN 2700 UGACGUCCCCACUGACAGANN 37 786-804
CUGUCAGUGGGGACGUCAUNN 2701 AUGACGUCCCCACUGACAGNN 37 787-805
UGUCAGUGGGGACGUCAUCNN 2702 GAUGACGUCCCCACUGACANN 37 788-806
GUCAGUGGGGACGUCAUCANN 2703 UGAUGACGUCCCCACUGACNN 37 789-807
UCAGUGGGGACGUCAUCAGNN 2704 CUGAUGACGUCCCCACUGANN 37 790-808
CAGUGGGGACGUCAUCAGCNN 2705 GCUGAUGACGUCCCCACUGNN 37 791-809
AGUGGGGACGUCAUCAGCCNN 2706 GGCUGAUGACGUCCCCACUNN 37 792-810
GUGGGGACGUCAUCAGCCANN 2707 UGGCUGAUGACGUCCCCACNN 37 793-811
UGGGGACGUCAUCAGCCAANN 2708 UUGGCUGAUGACGUCCCCANN 37 794-812
GGGGACGUCAUCAGCCAAGNN 2709 CUUGGCUGAUGACGUCCCCNN 37 795-813
GGGACGUCAUCAGCCAAGCNN 2710 GCUUGGCUGAUGACGUCCCNN 37 796-814
GGACGUCAUCAGCCAAGCUNN 2711 AGCUUGGCUGAUGACGUCCNN 37 797-815
GACGUCAUCAGCCAAGCUGNN 2712 CAGCUUGGCUGAUGACGUCNN 37 798-816
ACGUCAUCAGCCAAGCUGGNN 2713 CCAGCUUGGCUGAUGACGUNN 37 799-817
CGUCAUCAGCCAAGCUGGANN 2714 UCCAGCUUGGCUGAUGACGNN 37 800-818
GUCAUCAGCCAAGCUGGAANN 2715 UUCCAGCUUGGCUGAUGACNN 37 801-819
UCAUCAGCCAAGCUGGAAGNN 2716 CUUCCAGCUUGGCUGAUGANN 37 802-820
CAUCAGCCAAGCUGGAAGCNN 2717 GCUUCCAGCUUGGCUGAUGNN 37 803-821
AUCAGCCAAGCUGGAAGCCNN 2718 GGCUUCCAGCUUGGCUGAUNN 37 804-822
UCAGCCAAGCUGGAAGCCANN 2719 UGGCUUCCAGCUUGGCUGANN 37 805-823
CAGCCAAGCUGGAAGCCAUNN 2720 AUGGCUUCCAGCUUGGCUGNN 37 806-824
AGCCAAGCUGGAAGCCAUUNN 2721 AAUGGCUUCCAGCUUGGCUNN 37 807-825
GCCAAGCUGGAAGCCAUUANN 2722 UAAUGGCUUCCAGCUUGGCNN 37 808-826
CCAAGCUGGAAGCCAUUAANN 2723 UUAAUGGCUUCCAGCUUGGNN 37 809-827
CAAGCUGGAAGCCAUUAAUNN 2724 AUUAAUGGCUUCCAGCUUGNN 37 810-828
AAGCUGGAAGCCAUUAAUGNN 2725 CAUUAAUGGCUUCCAGCUUNN 37 811-829
AGCUGGAAGCCAUUAAUGANN 2726 UCAUUAAUGGCUUCCAGCUNN 37 812-830
GCUGGAAGCCAUUAAUGAANN 2727 UUCAUUAAUGGCUUCCAGCNN 37 813-831
CUGGAAGCCAUUAAUGAACNN 2728 GUUCAUUAAUGGCUUCCAGNN 37 814-832
UGGAAGCCAUUAAUGAACUNN 2729 AGUUCAUUAAUGGCUUCCANN 37 815-833
GGAAGCCAUUAAUGAACUANN 2730 UAGUUCAUUAAUGGCUUCCNN 37 816-834
GAAGCCAUUAAUGAACUAANN 2731 UUAGUUCAUUAAUGGCUUCNN 37 817-835
AAGCCAUUAAUGAACUAAUNN 2732 AUUAGUUCAUUAAUGGCUUNN 37 818-836
AGCCAUUAAUGAACUAAUUNN 2733 AAUUAGUUCAUUAAUGGCUNN 37 819-837
GCCAUUAAUGAACUAAUUCNN 2734 GAAUUAGUUCAUUAAUGGCNN 37 820-838
CCAUUAAUGAACUAAUUCGNN 2735 CGAAUUAGUUCAUUAAUGGNN 37 821-839
CAUUAAUGAACUAAUUCGUNN 2736 ACGAAUUAGUUCAUUAAUGNN 37 822-840
AUUAAUGAACUAAUUCGUUNN 2737 AACGAAUUAGUUCAUUAAUNN 37 823-841
UUAAUGAACUAAUUCGUUUNN 2738 AAACGAAUUAGUUCAUUAANN 37 824-842
UAAUGAACUAAUUCGUUUUNN 2739 AAAACGAAUUAGUUCAUUANN 37 825-843
AAUGAACUAAUUCGUUUUGNN 2740 CAAAACGAAUUAGUUCAUUNN 37 826-844
AUGAACUAAUUCGUUUUGANN 2741 UCAAAACGAAUUAGUUCAUNN 38 827-845
UGAACUAAUUCGUUUUGACNN 2742 GUCAAAACGAAUUAGUUCANN 38 828-846
GAACUAAUUCGUUUUGACCNN 2743 GGUCAAAACGAAUUAGUUCNN 38 829-847
AACUAAUUCGUUUUGACCANN 2744 UGGUCAAAACGAAUUAGUUNN 38 830-848
ACUAAUUCGUUUUGACCACNN 2745 GUGGUCAAAACGAAUUAGUNN 38 831-849
CUAAUUCGUUUUGACCACANN 2746 UGUGGUCAAAACGAAUUAGNN 38 832-850
UAAUUCGUUUUGACCACAUNN 2747 AUGUGGUCAAAACGAAUUANN 38 833-851
AAUUCGUUUUGACCACAUANN 2748 UAUGUGGUCAAAACGAAUUNN 38 834-852
AUUCGUUUUGACCACAUAUNN 2749 AUAUGUGGUCAAAACGAAUNN 38 835-853
UUCGUUUUGACCACAUAUANN 2750 UAUAUGUGGUCAAAACGAANN 38 836-854
UCGUUUUGACCACAUAUAUNN 2751 AUAUAUGUGGUCAAAACGANN 38 837-855
CGUUUUGACCACAUAUAUANN 2752 UAUAUAUGUGGUCAAAACGNN 38 838-856
GUUUUGACCACAUAUAUACNN 2753 GUAUAUAUGUGGUCAAAACNN 38 839-857
UUUUGACCACAUAUAUACCNN 2754 GGUAUAUAUGUGGUCAAAANN 38 840-858
UUUGACCACAUAUAUACCANN 2755 UGGUAUAUAUGUGGUCAAANN 38 841-859
UUGACCACAUAUAUACCAANN 2756 UUGGUAUAUAUGUGGUCAANN 38 842-860
UGACCACAUAUAUACCAAGNN 2757 CUUGGUAUAUAUGUGGUCANN 38 843-861
GACCACAUAUAUACCAAGCNN 2758 GCUUGGUAUAUAUGUGGUCNN 38 844-862
ACCACAUAUAUACCAAGCCNN 2759 GGCUUGGUAUAUAUGUGGUNN 38 845-863
CCACAUAUAUACCAAGCCCNN 2760 GGGCUUGGUAUAUAUGUGGNN 38 846-864
CACAUAUAUACCAAGCCCCNN 2761 GGGGCUUGGUAUAUAUGUGNN 38 847-865
ACAUAUAUACCAAGCCCCUNN 2762 AGGGGCUUGGUAUAUAUGUNN 38 867-885
GUCUUAGAGAUACCCUCUGNN 2763 CAGAGGGUAUCUCUAAGACNN 38 868-886
UCUUAGAGAUACCCUCUGANN 2764 UCAGAGGGUAUCUCUAAGANN 38 869-887
CUUAGAGAUACCCUCUGAGNN 2765 CUCAGAGGGUAUCUCUAAGNN 38 870-888
UUAGAGAUACCCUCUGAGANN 2766 UCUCAGAGGGUAUCUCUAANN 38 871-889
UAGAGAUACCCUCUGAGACNN 2767 GUCUCAGAGGGUAUCUCUANN 38 872-890
AGAGAUACCCUCUGAGACANN 2768 UGUCUCAGAGGGUAUCUCUNN 38 873-891
GAGAUACCCUCUGAGACAGNN 2769 CUGUCUCAGAGGGUAUCUCNN 38 874-892
AGAUACCCUCUGAGACAGANN 2770 UCUGUCUCAGAGGGUAUCUNN 38 875-893
GAUACCCUCUGAGACAGAGNN 2771 CUCUGUCUCAGAGGGUAUCNN 38 876-894
AUACCCUCUGAGACAGAGANN 2772 UCUCUGUCUCAGAGGGUAUNN 38 877-895
UACCCUCUGAGACAGAGAGNN 2773 CUCUCUGUCUCAGAGGGUANN 38 878-896
ACCCUCUGAGACAGAGAGCNN 2774 GCUCUCUGUCUCAGAGGGUNN 38 879-897
CCCUCUGAGACAGAGAGCCNN 2775 GGCUCUCUGUCUCAGAGGGNN 38 880-898
CCUCUGAGACAGAGAGCCANN 2776 UGGCUCUCUGUCUCAGAGGNN 38 881-899
CUCUGAGACAGAGAGCCAANN 2777 UUGGCUCUCUGUCUCAGAGNN 38 882-900
UCUGAGACAGAGAGCCAAGNN 2778 CUUGGCUCUCUGUCUCAGANN 38 883-901
CUGAGACAGAGAGCCAAGCNN 2779 GCUUGGCUCUCUGUCUCAGNN 38 884-902
UGAGACAGAGAGCCAAGCUNN 2780 AGCUUGGCUCUCUGUCUCANN 38 885-903
GAGACAGAGAGCCAAGCUANN 2781 UAGCUUGGCUCUCUGUCUCNN 38 886-904
AGACAGAGAGCCAAGCUAANN 2782 UUAGCUUGGCUCUCUGUCUNN 38 887-905
GACAGAGAGCCAAGCUAAUNN 2783 AUUAGCUUGGCUCUCUGUCNN 38 888-906
ACAGAGAGCCAAGCUAAUGNN 2784 CAUUAGCUUGGCUCUCUGUNN 38 889-907
CAGAGAGCCAAGCUAAUGUNN 2785 ACAUUAGCUUGGCUCUCUGNN 38 890-908
AGAGAGCCAAGCUAAUGUGNN 2786 CACAUUAGCUUGGCUCUCUNN 38 891-909
GAGAGCCAAGCUAAUGUGGNN 2787 CCACAUUAGCUUGGCUCUCNN 38 892-910
AGAGCCAAGCUAAUGUGGUNN 2788 ACCACAUUAGCUUGGCUCUNN 38 893-911
GAGCCAAGCUAAUGUGGUANN 2789 UACCACAUUAGCUUGGCUCNN 38 894-912
AGCCAAGCUAAUGUGGUAGNN 2790 CUACCACAUUAGCUUGGCUNN 38 895-913
GCCAAGCUAAUGUGGUAGUNN 2791 ACUACCACAUUAGCUUGGCNN 38 896-914
CCAAGCUAAUGUGGUAGUGNN 2792 CACUACCACAUUAGCUUGGNN 38 897-915
CAAGCUAAUGUGGUAGUGANN 2793 UCACUACCACAUUAGCUUGNN 38 898-916
AAGCUAAUGUGGUAGUGAANN 2794 UUCACUACCACAUUAGCUUNN 38 899-917
AGCUAAUGUGGUAGUGAAANN 2795 UUUCACUACCACAUUAGCUNN 38 900-918
GCUAAUGUGGUAGUGAAAANN 2796 UUUUCACUACCACAUUAGCNN 38 901-919
CUAAUGUGGUAGUGAAAAUNN 2797 AUUUUCACUACCACAUUAGNN 38 902-920
UAAUGUGGUAGUGAAAAUCNN 2798 GAUUUUCACUACCACAUUANN 38 903-921
AAUGUGGUAGUGAAAAUCGNN 2799 CGAUUUUCACUACCACAUUNN 38 904-922
AUGUGGUAGUGAAAAUCGANN 2800 UCGAUUUUCACUACCACAUNN 38 905-923
UGUGGUAGUGAAAAUCGAGNN 2801 CUCGAUUUUCACUACCACANN 38 906-924
GUGGUAGUGAAAAUCGAGGNN 2802 CCUCGAUUUUCACUACCACNN 38 907-925
UGGUAGUGAAAAUCGAGGANN 2803 UCCUCGAUUUUCACUACCANN 38 908-926
GGUAGUGAAAAUCGAGGAANN 2804 UUCCUCGAUUUUCACUACCNN 38 909-927
GUAGUGAAAAUCGAGGAAGNN 2805 CUUCCUCGAUUUUCACUACNN 38 910-928
UAGUGAAAAUCGAGGAAGCNN 2806 GCUUCCUCGAUUUUCACUANN 38 911-929
AGUGAAAAUCGAGGAAGCANN 2807 UGCUUCCUCGAUUUUCACUNN 38 912-930
GUGAAAAUCGAGGAAGCACNN 2808 GUGCUUCCUCGAUUUUCACNN 38 913-931
UGAAAAUCGAGGAAGCACCNN 2809 GGUGCUUCCUCGAUUUUCANN 38 914-932
GAAAAUCGAGGAAGCACCUNN 2810 AGGUGCUUCCUCGAUUUUCNN 38 915-933
AAAAUCGAGGAAGCACCUCNN 2811 GAGGUGCUUCCUCGAUUUUNN 38 916-934
AAAUCGAGGAAGCACCUCUNN 2812 AGAGGUGCUUCCUCGAUUUNN 38 917-935
AAUCGAGGAAGCACCUCUCNN 2813 GAGAGGUGCUUCCUCGAUUNN 38 918-936
AUCGAGGAAGCACCUCUCANN 2814 UGAGAGGUGCUUCCUCGAUNN 38 919-937
UCGAGGAAGCACCUCUCAGNN 2815 CUGAGAGGUGCUUCCUCGANN 38 920-938
CGAGGAAGCACCUCUCAGCNN 2816 GCUGAGAGGUGCUUCCUCGNN 38 921-939
GAGGAAGCACCUCUCAGCCNN 2817 GGCUGAGAGGUGCUUCCUCNN 38 922-940
AGGAAGCACCUCUCAGCCCNN 2818 GGGCUGAGAGGUGCUUCCUNN 38 923-941
GGAAGCACCUCUCAGCCCCNN 2819 GGGGCUGAGAGGUGCUUCCNN 38 924-942
GAAGCACCUCUCAGCCCCUNN 2820 AGGGGCUGAGAGGUGCUUCNN 38 925-943
AAGCACCUCUCAGCCCCUCNN 2821 GAGGGGCUGAGAGGUGCUUNN 38 926-944
AGCACCUCUCAGCCCCUCANN 2822 UGAGGGGCUGAGAGGUGCUNN 38
927-945 GCACCUCUCAGCCCCUCAGNN 2823 CUGAGGGGCUGAGAGGUGCNN 38 928-946
CACCUCUCAGCCCCUCAGANN 2824 UCUGAGGGGCUGAGAGGUGNN 38 929-947
ACCUCUCAGCCCCUCAGAGNN 2825 CUCUGAGGGGCUGAGAGGUNN 38 930-948
CCUCUCAGCCCCUCAGAGANN 2826 UCUCUGAGGGGCUGAGAGGNN 38 931-949
CUCUCAGCCCCUCAGAGAANN 2827 UUCUCUGAGGGGCUGAGAGNN 38 932-950
UCUCAGCCCCUCAGAGAAUNN 2828 AUUCUCUGAGGGGCUGAGANN 38 933-951
CUCAGCCCCUCAGAGAAUGNN 2829 CAUUCUCUGAGGGGCUGAGNN 38 934-952
UCAGCCCCUCAGAGAAUGANN 2830 UCAUUCUCUGAGGGGCUGANN 38 935-953
CAGCCCCUCAGAGAAUGAUNN 2831 AUCAUUCUCUGAGGGGCUGNN 38 936-954
AGCCCCUCAGAGAAUGAUCNN 2832 GAUCAUUCUCUGAGGGGCUNN 38 937-955
GCCCCUCAGAGAAUGAUCANN 2833 UGAUCAUUCUCUGAGGGGCNN 38 938-956
CCCCUCAGAGAAUGAUCACNN 2834 GUGAUCAUUCUCUGAGGGGNN 38 939-957
CCCUCAGAGAAUGAUCACCNN 2835 GGUGAUCAUUCUCUGAGGGNN 38 940-958
CCUCAGAGAAUGAUCACCCNN 2836 GGGUGAUCAUUCUCUGAGGNN 38 941-959
CUCAGAGAAUGAUCACCCUNN 2837 AGGGUGAUCAUUCUCUGAGNN 38 942-960
UCAGAGAAUGAUCACCCUGNN 2838 CAGGGUGAUCAUUCUCUGANN 38 943-961
CAGAGAAUGAUCACCCUGANN 2839 UCAGGGUGAUCAUUCUCUGNN 38 944-962
AGAGAAUGAUCACCCUGAANN 2840 UUCAGGGUGAUCAUUCUCUNN 38 945-963
GAGAAUGAUCACCCUGAAUNN 2841 AUUCAGGGUGAUCAUUCUCNN 39 946-964
AGAAUGAUCACCCUGAAUUNN 2842 AAUUCAGGGUGAUCAUUCUNN 39 947-965
GAAUGAUCACCCUGAAUUCNN 2843 GAAUUCAGGGUGAUCAUUCNN 39 948-966
AAUGAUCACCCUGAAUUCANN 2844 UGAAUUCAGGGUGAUCAUUNN 39 949-967
AUGAUCACCCUGAAUUCAUNN 2845 AUGAAUUCAGGGUGAUCAUNN 39 950-968
UGAUCACCCUGAAUUCAUUNN 2846 AAUGAAUUCAGGGUGAUCANN 39 951-969
GAUCACCCUGAAUUCAUUGNN 2847 CAAUGAAUUCAGGGUGAUCNN 39 952-970
AUCACCCUGAAUUCAUUGUNN 2848 ACAAUGAAUUCAGGGUGAUNN 39 953-971
UCACCCUGAAUUCAUUGUCNN 2849 GACAAUGAAUUCAGGGUGANN 39 954-972
CACCCUGAAUUCAUUGUCUNN 2850 AGACAAUGAAUUCAGGGUGNN 39 955-973
ACCCUGAAUUCAUUGUCUCNN 2851 GAGACAAUGAAUUCAGGGUNN 39 956-974
CCCUGAAUUCAUUGUCUCANN 2852 UGAGACAAUGAAUUCAGGGNN 39 957-975
CCUGAAUUCAUUGUCUCAGNN 2853 CUGAGACAAUGAAUUCAGGNN 39 958-976
CUGAAUUCAUUGUCUCAGUNN 2854 ACUGAGACAAUGAAUUCAGNN 39 959-977
UGAAUUCAUUGUCUCAGUGNN 2855 CACUGAGACAAUGAAUUCANN 39 960-978
GAAUUCAUUGUCUCAGUGANN 2856 UCACUGAGACAAUGAAUUCNN 39 961-979
AAUUCAUUGUCUCAGUGAANN 2857 UUCACUGAGACAAUGAAUUNN 39 962-980
AUUCAUUGUCUCAGUGAAGNN 2858 CUUCACUGAGACAAUGAAUNN 39 963-981
UUCAUUGUCUCAGUGAAGGNN 2859 CCUUCACUGAGACAAUGAANN 39 964-982
UCAUUGUCUCAGUGAAGGANN 2860 UCCUUCACUGAGACAAUGANN 39 965-983
CAUUGUCUCAGUGAAGGAANN 2861 UUCCUUCACUGAGACAAUGNN 39 966-984
AUUGUCUCAGUGAAGGAAGNN 2862 CUUCCUUCACUGAGACAAUNN 39 967-985
UUGUCUCAGUGAAGGAAGANN 2863 UCUUCCUUCACUGAGACAANN 39 968-986
UGUCUCAGUGAAGGAAGAANN 2864 UUCUUCCUUCACUGAGACANN 39 969-987
GUCUCAGUGAAGGAAGAACNN 2865 GUUCUUCCUUCACUGAGACNN 39 970-988
UCUCAGUGAAGGAAGAACCNN 2866 GGUUCUUCCUUCACUGAGANN 39 971-989
CUCAGUGAAGGAAGAACCUNN 2867 AGGUUCUUCCUUCACUGAGNN 39 972-990
UCAGUGAAGGAAGAACCUGNN 2868 CAGGUUCUUCCUUCACUGANN 39 973-991
CAGUGAAGGAAGAACCUGUNN 2869 ACAGGUUCUUCCUUCACUGNN 39 974-992
AGUGAAGGAAGAACCUGUANN 2870 UACAGGUUCUUCCUUCACUNN 39 975-993
GUGAAGGAAGAACCUGUAGNN 2871 CUACAGGUUCUUCCUUCACNN 39 976-994
UGAAGGAAGAACCUGUAGANN 2872 UCUACAGGUUCUUCCUUCANN 39 977-995
GAAGGAAGAACCUGUAGAANN 2873 UUCUACAGGUUCUUCCUUCNN 39 978-996
AAGGAAGAACCUGUAGAAGNN 2874 CUUCUACAGGUUCUUCCUUNN 39 979-997
AGGAAGAACCUGUAGAAGANN 2875 UCUUCUACAGGUUCUUCCUNN 39 980-998
GGAAGAACCUGUAGAAGAUNN 2876 AUCUUCUACAGGUUCUUCCNN 39 981-999
GAAGAACCUGUAGAAGAUGNN 2877 CAUCUUCUACAGGUUCUUCNN 39 982-1000
AAGAACCUGUAGAAGAUGANN 2878 UCAUCUUCUACAGGUUCUUNN 39 983-1001
AGAACCUGUAGAAGAUGACNN 2879 GUCAUCUUCUACAGGUUCUNN 39 984-1002
GAACCUGUAGAAGAUGACCNN 2880 GGUCAUCUUCUACAGGUUCNN 39 985-1003
AACCUGUAGAAGAUGACCUNN 2881 AGGUCAUCUUCUACAGGUUNN 39 986-1004
ACCUGUAGAAGAUGACCUCNN 2882 GAGGUCAUCUUCUACAGGUNN 39
TABLE-US-00016 TABLE 13 Sequences of dsRNA targeting both mouse and
rhesus monkey XBP-1. *Target refers location of target sequence in
NM_013842 (Mus musculis XPB1 mRNA). Sense and antisense sequences
are described with optional dinucleotide (NN) overhangs. SEQ ID SEQ
ID *Target sense (5'-3') NO antisense (5'-3') NO 369-387
AGAAAACUCACGGCCUUGUNN 3942 ACAAGGCCGUGAGUUUUCUNN 4042 237-255
AACUGAAAAACAGAGUAGCNN 3943 GCUACUCUGUUUUUCAGUUNN 4043 491-509
GGGUCUGCUGAGUCCGCAGNN 3944 CUGCGGACUCAGCAGACCCNN 4044 917-935
AUCACCCUGAAUUCAUUGUNN 3945 ACAAUGAAUUCAGGGUGAUNN 4045 923-941
CUGAAUUCAUUGUCUCAGUNN 3946 ACUGAGACAAUGAAUUCAGNN 4046 702-720
CCCAGAGGUCUACCCAGAANN 3947 UUCUGGGUAGACCUCUGGGNN 4047 926-944
AAUUCAUUGUCUCAGUGAANN 3948 UUCACUGAGACAAUGAAUUNN 4048 391-409
UGAGAACCAGGAGUUAAGANN 3949 UCUUAACUCCUGGUUCUCANN 4049 775-793
AAGCUGGAAGCCAUUAAUGNN 3950 CAUUAAUGGCUUCCAGCUUNN 4050 1150-1168
CCCCAGCUGAUUAGUGUCUNN 3951 AGACACUAAUCAGCUGGGGNN 4051 776-794
AGCUGGAAGCCAUUAAUGANN 3952 UCAUUAAUGGCUUCCAGCUNN 4052 921-939
CCCUGAAUUCAUUGUCUCANN 3953 UGAGACAAUGAAUUCAGGGNN 4053 777-795
GCUGGAAGCCAUUAAUGAANN 3954 UUCAUUAAUGGCUUCCAGCNN 4054 539-557
GUGCAGGCCCAGUUGUCACNN 3955 GUGACAACUGGGCCUGCACNN 4055 731-749
CCUUACCAGCCUCCCUUUCNN 3956 GAAAGGGAGGCUGGUAAGGNN 4056 924-942
UGAAUUCAUUGUCUCAGUGNN 3957 CACUGAGACAAUGAAUUCANN 4057 1151-1169
CCCAGCUGAUUAGUGUCUANN 3958 UAGACACUAAUCAGCUGGGNN 4058 1152-1170
CCAGCUGAUUAGUGUCUAANN 3959 UUAGACACUAAUCAGCUGGNN 4059 1718-1736
ACUAUGUAAAUGCUUGAUGNN 3960 CAUCAAGCAUUUACAUAGUNN 4060 368-386
GAGAAAACUCACGGCCUUGNN 3961 CAAGGCCGUGAGUUUUCUCNN 4061 489-507
CCGGGUCUGCUGAGUCCGCNN 3962 GCGGACUCAGCAGACCCGGNN 4062 238-256
ACUGAAAAACAGAGUAGCANN 3963 UGCUACUCUGUUUUUCAGUNN 4063 240-258
UGAAAAACAGAGUAGCAGCNN 3964 GCUGCUACUCUGUUUUUCANN 4064 390-408
UUGAGAACCAGGAGUUAAGNN 3965 CUUAACUCCUGGUUCUCAANN 4065 487-505
GGCCGGGUCUGCUGAGUCCNN 3966 GGACUCAGCAGACCCGGCCNN 4066 741-759
CUCCCUUUCUCUGUCAGUGNN 3967 CACUGACAGAGAAAGGGAGNN 4067 918-936
UCACCCUGAAUUCAUUGUCNN 3968 GACAAUGAAUUCAGGGUGANN 4068 919-937
CACCCUGAAUUCAUUGUCUNN 3969 AGACAAUGAAUUCAGGGUGNN 4069 1130-1148
CUUUUGCCAAUGAACUUUUNN 3970 AAAAGUUCAUUGGCAAAAGNN 4070 1712-1730
AAAUUUACUAUGUAAAUGCNN 3971 GCAUUUACAUAGUAAAUUUNN 4071 1714-1732
AUUUACUAUGUAAAUGCUUNN 3972 AAGCAUUUACAUAGUAAAUNN 4072 1717-1735
UACUAUGUAAAUGCUUGAUNN 3973 AUCAAGCAUUUACAUAGUANN 4073 1719-1737
CUAUGUAAAUGCUUGAUGGNN 3974 CCAUCAAGCAUUUACAUAGNN 4074 1775-1793
CCAUUUAUUUAAAACUACCNN 3975 GGUAGUUUUAAAUAAAUGGNN 4075 1776-1794
CAUUUAUUUAAAACUACCCNN 3976 GGGUAGUUUUAAAUAAAUGNN 4076 239-257
CUGAAAAACAGAGUAGCAGNN 3977 CUGCUACUCUGUUUUUCAGNN 4077 347-365
CUAGAAAAUCAGCUUUUACNN 3978 GUAAAAGCUGAUUUUCUAGNN 4078 348-366
UAGAAAAUCAGCUUUUACGNN 3979 CGUAAAAGCUGAUUUUCUANN 4079 485-503
GUGGCCGGGUCUGCUGAGUNN 3980 ACUCAGCAGACCCGGCCACNN 4080 486-504
UGGCCGGGUCUGCUGAGUCNN 3981 GACUCAGCAGACCCGGCCANN 4081 488-506
GCCGGGUCUGCUGAGUCCGNN 3982 CGGACUCAGCAGACCCGGCNN 4082 540-558
UGCAGGCCCAGUUGUCACCNN 3983 GGUGACAACUGGGCCUGCANN 4083 703-721
CCAGAGGUCUACCCAGAAGNN 3984 CUUCUGGGUAGACCUCUGGNN 4084 705-723
AGAGGUCUACCCAGAAGGANN 3985 UCCUUCUGGGUAGACCUCUNN 4085 730-748
UCCUUACCAGCCUCCCUUUNN 3986 AAAGGGAGGCUGGUAAGGANN 4086 742-760
UCCCUUUCUCUGUCAGUGGNN 3987 CCACUGACAGAGAAAGGGANN 4087 744-762
CCUUUCUCUGUCAGUGGGGNN 3988 CCCCACUGACAGAGAAAGGNN 4088 767-785
CAUCAGCCAAGCUGGAAGCNN 3989 GCUUCCAGCUUGGCUGAUGNN 4089 771-789
AGCCAAGCUGGAAGCCAUUNN 3990 AAUGGCUUCCAGCUUGGCUNN 4090 916-934
GAUCACCCUGAAUUCAUUGNN 3991 CAAUGAAUUCAGGGUGAUCNN 4091 920-938
ACCCUGAAUUCAUUGUCUCNN 3992 GAGACAAUGAAUUCAGGGUNN 4092 922-940
CCUGAAUUCAUUGUCUCAGNN 3993 CUGAGACAAUGAAUUCAGGNN 4093 925-943
GAAUUCAUUGUCUCAGUGANN 3994 UCACUGAGACAAUGAAUUCNN 4094 1720-1738
UAUGUAAAUGCUUGAUGGANN 3995 UCCAUCAAGCAUUUACAUANN 4095 232-250
GAGGAAACUGAAAAACAGANN 3996 UCUGUUUUUCAGUUUCCUCNN 4096 236-254
AAACUGAAAAACAGAGUAGNN 3997 CUACUCUGUUUUUCAGUUUNN 4097 728-746
GUUCCUUACCAGCCUCCCUNN 3998 AGGGAGGCUGGUAAGGAACNN 4098 729-747
UUCCUUACCAGCCUCCCUUNN 3999 AAGGGAGGCUGGUAAGGAANN 4099 745-763
CUUUCUCUGUCAGUGGGGANN 4000 UCCCCACUGACAGAGAAAGNN 4100 766-784
UCAUCAGCCAAGCUGGAAGNN 4001 CUUCCAGCUUGGCUGAUGANN 4101 927-945
AUUCAUUGUCUCAGUGAAGNN 4002 CUUCACUGAGACAAUGAAUNN 4102 234-252
GGAAACUGAAAAACAGAGUNN 4003 ACUCUGUUUUUCAGUUUCCNN 4103 235-253
GAAACUGAAAAACAGAGUANN 4004 UACUCUGUUUUUCAGUUUCNN 4104 346-364
GCUAGAAAAUCAGCUUUUANN 4005 UAAAAGCUGAUUUUCUAGCNN 4105 490-508
CGGGUCUGCUGAGUCCGCANN 4006 UGCGGACUCAGCAGACCCGNN 4106 700-718
CUCCCAGAGGUCUACCCAGNN 4007 CUGGGUAGACCUCUGGGAGNN 4107 1715-1733
UUUACUAUGUAAAUGCUUGNN 4008 CAAGCAUUUACAUAGUAAANN 4108 734-752
UACCAGCCUCCCUUUCUCUNN 4009 AGAGAAAGGGAGGCUGGUANN 4109 773-791
CCAAGCUGGAAGCCAUUAANN 4010 UUAAUGGCUUCCAGCUUGGNN 4110 778-796
CUGGAAGCCAUUAAUGAACNN 4011 GUUCAUUAAUGGCUUCCAGNN 4111 779-797
UGGAAGCCAUUAAUGAACUNN 4012 AGUUCAUUAAUGGCUUCCANN 4112 1774-1792
UCCAUUUAUUUAAAACUACNN 4013 GUAGUUUUAAAUAAAUGGANN 4113 704-722
CAGAGGUCUACCCAGAAGGNN 4014 CCUUCUGGGUAGACCUCUGNN 4114 1716-1734
UUACUAUGUAAAUGCUUGANN 4015 UCAAGCAUUUACAUAGUAANN 4115 1713-1731
AAUUUACUAUGUAAAUGCUNN 4016 AGCAUUUACAUAGUAAAUUNN 4116 768-786
AUCAGCCAAGCUGGAAGCCNN 4017 GGCUUCCAGCUUGGCUGAUNN 4117 1129-1147
ACUUUUGCCAAUGAACUUUNN 4018 AAAGUUCAUUGGCAAAAGUNN 4118 389-407
GUUGAGAACCAGGAGUUAANN 4019 UUAACUCCUGGUUCUCAACNN 4119 701-719
UCCCAGAGGUCUACCCAGANN 4020 UCUGGGUAGACCUCUGGGANN 4120 706-724
GAGGUCUACCCAGAAGGACNN 4021 GUCCUUCUGGGUAGACCUCNN 4121 707-725
AGGUCUACCCAGAAGGACCNN 4022 GGUCCUUCUGGGUAGACCUNN 4122 727-745
AGUUCCUUACCAGCCUCCCNN 4023 GGGAGGCUGGUAAGGAACUNN 4123 733-751
UUACCAGCCUCCCUUUCUCNN 4024 GAGAAAGGGAGGCUGGUAANN 4124 736-754
CCAGCCUCCCUUUCUCUGUNN 4025 ACAGAGAAAGGGAGGCUGGNN 4125 738-756
AGCCUCCCUUUCUCUGUCANN 4026 UGACAGAGAAAGGGAGGCUNN 4126 743-761
CCCUUUCUCUGUCAGUGGGNN 4027 CCCACUGACAGAGAAAGGGNN 4127 769-787
UCAGCCAAGCUGGAAGCCANN 4028 UGGCUUCCAGCUUGGCUGANN 4128 772-790
GCCAAGCUGGAAGCCAUUANN 4029 UAAUGGCUUCCAGCUUGGCNN 4129 774-792
CAAGCUGGAAGCCAUUAAUNN 4030 AUUAAUGGCUUCCAGCUUGNN 4130 231-249
GGAGGAAACUGAAAAACAGNN 4031 CUGUUUUUCAGUUUCCUCCNN 4131 233-251
AGGAAACUGAAAAACAGAGNN 4032 CUCUGUUUUUCAGUUUCCUNN 4132 735-753
ACCAGCCUCCCUUUCUCUGNN 4033 CAGAGAAAGGGAGGCUGGUNN 4133 737-755
CAGCCUCCCUUUCUCUGUCNN 4034 GACAGAGAAAGGGAGGCUGNN 4134 739-757
GCCUCCCUUUCUCUGUCAGNN 4035 CUGACAGAGAAAGGGAGGCNN 4135 740-758
CCUCCCUUUCUCUGUCAGUNN 4036 ACUGACAGAGAAAGGGAGGNN 4136 746-764
UUUCUCUGUCAGUGGGGACNN 4037 GUCCCCACUGACAGAGAAANN 4137 770-788
CAGCCAAGCUGGAAGCCAUNN 4038 AUGGCUUCCAGCUUGGCUGNN 4138 26-44
GCUAUGGUGGUGGUGGCAGNN 4039 CUGCCACCACCACCAUAGCNN 4139 27-45
CUAUGGUGGUGGUGGCAGCNN 4040 GCUGCCACCACCACCAUAGNN 4140 732-750
CUUACCAGCCUCCCUUUCUNN 4041 AGAAAGGGAGGCUGGUAAGNN 4141
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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220213475A1).
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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220213475A1).
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