U.S. patent application number 12/201759 was filed with the patent office on 2009-01-22 for rna interference mediated inhibition of map kinase gene expression or expression of genes involved in map kinase pathway using short interfering nucleic acid (sina).
This patent application is currently assigned to Sirna Therapeutics, Inc.. Invention is credited to Leonid Beigelman, Bharat M. Chowrira, Peter Haeberli, James McSwiggen, Barry Polisky, Nassim Usman.
Application Number | 20090023676 12/201759 |
Document ID | / |
Family ID | 43446817 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090023676 |
Kind Code |
A1 |
McSwiggen; James ; et
al. |
January 22, 2009 |
RNA Interference Mediated Inhibition of MAP Kinase Gene Expression
or Expression of Genes Involved in MAP Kinase Pathway Using Short
Interfering Nucleic Acid (SiNA)
Abstract
The present invention concerns methods and reagents useful in
modulating MAP kinase gene expression in a variety of applications,
including use in therapeutic, diagnostic, target validation, and
genomic discovery applications. Specifically, the invention relates
to small nucleic acid molecules, such as short interfering nucleic
acid (siNA), short interfering RNA (siRNA), double-stranded RNA
(dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules
capable of mediating RNA interference (RNAi) against c-JUN, JNK,
p38, and ERK gene expression, useful in the treatment of cancer,
inflammation, obesity and insulin resistance (e.g. Type I and Type
II diabetes).
Inventors: |
McSwiggen; James; (Bothell,
WA) ; Beigelman; Leonid; (Brisbane, CA) ;
Usman; Nassim; (Lafayette, CO) ; Haeberli; Peter;
(Berthoud, CO) ; Chowrira; Bharat M.; (Louisville,
CO) ; Polisky; Barry; (Boulder, CO) |
Correspondence
Address: |
MCDONNELL, BOEHNEN, HULBERT AND BERGHOFF, LLP
300 SOUTH WACKER DRIVE, SUITE 3100
CHICAGO
IL
60606
US
|
Assignee: |
Sirna Therapeutics, Inc.
San Francisco
CA
|
Family ID: |
43446817 |
Appl. No.: |
12/201759 |
Filed: |
August 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10424339 |
Apr 25, 2003 |
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12201759 |
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PCT/US03/02510 |
Jan 28, 2003 |
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10424339 |
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PCT/US03/05346 |
Feb 20, 2003 |
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PCT/US03/02510 |
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PCT/US03/05028 |
Feb 20, 2003 |
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PCT/US03/05346 |
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60358580 |
Feb 20, 2002 |
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60363124 |
Mar 11, 2002 |
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60386782 |
Jun 6, 2002 |
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60406784 |
Aug 29, 2002 |
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60408378 |
Sep 5, 2002 |
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60409293 |
Sep 9, 2002 |
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60440129 |
Jan 15, 2003 |
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Current U.S.
Class: |
514/44R ;
536/24.5 |
Current CPC
Class: |
A61P 13/08 20180101;
C12N 2310/322 20130101; A61P 25/28 20180101; A61P 1/00 20180101;
A61P 9/00 20180101; A61P 31/12 20180101; A61P 31/10 20180101; A61P
37/04 20180101; A61K 38/00 20130101; C12N 2310/317 20130101; C12N
2310/332 20130101; A61P 37/00 20180101; A61P 27/02 20180101; A61P
29/00 20180101; A61P 31/16 20180101; A61P 35/00 20180101; A61P
19/00 20180101; A61P 25/16 20180101; A61P 1/16 20180101; A61P 31/18
20180101; C12N 2310/111 20130101; A61K 47/54 20170801; A61P 11/00
20180101; A61P 19/02 20180101; A61P 21/00 20180101; C12N 15/1131
20130101; A61P 17/00 20180101; A61P 25/02 20180101; A61P 37/06
20180101; C12N 15/111 20130101; C12N 15/8218 20130101; A61P 25/14
20180101; A61P 15/00 20180101; C07H 21/02 20130101; C12N 2310/14
20130101; C12N 2310/318 20130101; C12N 2310/53 20130101; A61P 13/10
20180101; A61P 13/12 20180101; Y02A 50/30 20180101; A61P 31/04
20180101; C12N 15/1138 20130101; C12N 2310/315 20130101; A61P 3/00
20180101; A61P 25/08 20180101; A61P 35/02 20180101; A61P 5/00
20180101; C12N 2320/51 20130101; A61P 43/00 20180101; A61P 31/20
20180101; A61P 37/08 20180101; A61P 1/04 20180101; A61P 9/10
20180101; A61P 41/00 20180101; A61P 3/10 20180101; A61P 17/02
20180101; A61P 25/00 20180101; C12N 2330/30 20130101; A61P 31/22
20180101; C12N 2310/321 20130101; Y02A 50/393 20180101; A61P 11/06
20180101; A61P 31/00 20180101; C12N 2310/344 20130101; A61P 31/14
20180101; A61P 27/16 20180101; C12N 2310/346 20130101; C12N
2310/321 20130101; C12N 2310/3521 20130101 |
Class at
Publication: |
514/44 ;
536/24.5 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; C07H 21/02 20060101 C07H021/02 |
Claims
1. A chemically modified nucleic acid molecule, wherein: (a) the
nucleic acid molecule comprises a sense strand and a separate
antisense strand, each strand having one or more pyrimidine
nucleotides and one or more purine nucleotides; (b) each strand of
the nucleic acid molecule is independently 18 to 27 nucleotides in
length; (c) an 18 to 27 nucleotide sequence of the antisense strand
is complementary to a human c-Jun RNA sequence comprising SEQ ID
NO:1715; (d) an 18 to 27 nucleotide sequence of the sense strand is
complementary to the antisense strand and comprises an 18 to 27
nucleotide sequence of the human RNA sequence; and (e) 50 percent
or more of the nucleotides in at least one strand comprise a
2'-sugar modification, wherein the 2'-sugar modification of any of
the pyrimidine nucleotides differs from the 2'-sugar modification
of any of the purine nucleotides.
2. The nucleic acid molecule of claim 1, wherein 50 percent or more
of the nucleotides in each strand comprise a 2'-sugar
modification.
3. The nucleic acid molecule of claim 1, wherein the 2'-sugar
modification is selected from the group consisting of
2'-deoxy-2'-fluoro, 2'-O-methyl, and 2'-deoxy.
4. The nucleic acid of claim 3, wherein the 2'-deoxy-2'-fluoro
sugar modification is a pyrimidine modification.
5. The nucleic acid of claim 3, wherein the 2'-deoxy sugar
modification is a pyrimidine modification.
6. The nucleic acid of claim 3, wherein the 2'-O-methyl sugar
modification is a pyrimidine modification.
7. The nucleic acid molecule of claim 4, wherein said pyrimidine
modification is in the sense strand, the antisense strand, or both
the sense strand and antisense strand.
8. The nucleic acid molecule of claim 6, wherein said pyrimidine
modification is in the sense strand, the antisense strand, or both
the sense strand and antisense strand.
9. The nucleic acid molecule of claim 3, wherein the 2'-deoxy sugar
modification is a purine modification.
10. The nucleic acid molecule of claim 3, wherein the 2'-O-methyl
sugar modification is a purine modification.
11. The nucleic acid molecule of claim 9, wherein the purine
modification is in the sense strand.
12. The nucleic acid molecule of claim 10, wherein the purine
modification is in the antisense strand.
13. The nucleic acid molecule of claim 1, wherein the nucleic acid
molecule comprises ribonucleotides.
14. The nucleic acid molecule of claim 1, wherein the sense strand
includes a terminal cap moiety at the 5'-end, the 3'-end, or both
of the 5'- and 3'-ends.
15. The nucleic acid molecule of claim 14, wherein the terminal cap
moiety is an inverted deoxy abasic moiety.
16. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule includes one or more phosphorothioate internucleotide
linkages.
17. The nucleic acid molecule of claim 16, wherein one of the
phosphorothioate internucleotide linkages is at the 3'-end of the
antisense strand.
18. The nucleic acid molecule of claim 1, wherein the 5'-end of the
antisense strand includes a terminal phosphate group.
19. The nucleic acid molecule of claim 1, wherein the sense strand,
the antisense strand, or both the sense strand and the antisense
strand include a 3'-overhang.
20. A composition comprising the nucleic acid molecule of claim 1,
in a pharmaceutically acceptable carrier or diluent.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/424,339, filed Apr. 25, 2003, which is a
continuation-in-part of International Patent Application No.
PCT/US03/02510, filed Jan. 28, 2003, and is a continuation-in-part
of International Patent Application No. PCT/US03/05346, filed Feb.
20, 2003, and is a continuation-in-part of International Patent
Application No. PCT/US03/05028, filed Feb. 20, 2003, which each
claim the benefit of U.S. Provisional Application No. 60/358,580
filed Feb. 20, 2002, U.S. Provisional Application No. 60/363,124
filed Mar. 11, 2002, U.S. Provisional Application No. 60/386,782
filed Jun. 6, 2002, U.S. Provisional Application No. 60/406,784
filed Aug. 29, 2002, U.S. Provisional Application No. 60/408,378
filed Sep. 5, 2002, U.S. Provisional Application No. 60/409,293
filed Sep. 9, 2002, and U.S. Provisional Application No. 60/440,129
filed Jan. 15, 2003. The instant application claims the benefit of
all the listed applications, which are hereby incorporated by
reference herein in their entireties, including the drawings.
SEQUENCE LISTING
[0002] The sequence listing submitted via EFS, in compliance with
37 CFR .sctn. 1.52(e)(5), is incorporated herein by reference. The
sequence listing text file submitted via EFS contains the file
"SequenceListing45USCNT", created on Aug. 27, 2008, which is
468,031 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention concerns compounds, compositions, and
methods for the study, diagnosis, and treatment of conditions and
diseases that respond to the modulation of mitogen activated
protein kinase (MAP kinase) gene expression and/or activity. The
present invention also concerns compounds, compositions, methods
relating to the modulation of expression or activity of genes
involved in the MAP kinase pathway. Specifically, the invention
relates to small nucleic acid molecules, such as short interfering
nucleic acid (siNA), short interfering RNA (siRNA), double-stranded
RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA)
molecules capable of mediating RNA interference (RNAi) against
genes involved in the Jun amino-terminal kinase (JNK), p38, and/or
ERK pathway, such as c-JUN. More specifically, the invention
relates to small nucleic acid molecules, such as short interfering
nucleic acid (siNA), short interfering RNA (siRNA), double-stranded
RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA)
molecules capable of mediating RNA interference (RNAi) against Jun
amino-terminal kinase (JNK), p38, ERK, and/or c-JUN genes.
BACKGROUND OF THE INVENTION
[0004] The following is a discussion of relevant art pertaining to
RNAi. The discussion is provided only for understanding of the
invention that follows. The summary is not an admission that any of
the work described below is prior art to the claimed invention.
[0005] RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806).
The corresponding process in plants is commonly referred to as
post-transcriptional gene silencing or RNA silencing and is also
referred to as quelling in fungi. The process of
post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent
the expression of foreign genes and is commonly shared by diverse
flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such
protection from foreign gene expression may have evolved in
response to the production of double-stranded RNAs (dsRNAs) derived
from viral infection or from the random integration of transposon
elements into a host genome via a cellular response that
specifically destroys homologous single-stranded RNA or viral
genomic RNA. The presence of dsRNA in cells triggers the RNAi
response though a mechanism that has yet to be fully characterized.
This mechanism appears to be different from the interferon response
that results from dsRNA-mediated activation of protein kinase PKR
and 2',5'-oligoadenylate synthetase resulting in non-specific
cleavage of mRNA by ribonuclease L.
[0006] The presence of long dsRNAs in cells stimulates the activity
of a ribonuclease III enzyme referred to as dicer. Dicer is
involved in the processing of the dsRNA into short pieces of dsRNA
known as short interfering RNAs (siRNAs) (Berstein et al., 2001,
Nature, 409, 363). Short interfering RNAs derived from dicer
activity are typically about 21 to about 23 nucleotides in length
and comprise about 19 base pair duplexes (Elbashir et al., 2001,
Genes Dev., 15, 188). Dicer has also been implicated in the
excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from
precursor RNA of conserved structure that are implicated in
translational control (Hutvagner et al., 2001, Science, 293, 834).
The RNAi response also features an endonuclease complex, commonly
referred to as an RNA-induced silencing complex (RISC), which
mediates cleavage of single-stranded RNA having sequence
complementary to the antisense strand of the siRNA duplex. Cleavage
of the target RNA takes place in the middle of the region
complementary to the antisense strand of the siRNA duplex (Elbashir
et al., 2001, Genes Dev., 15, 188).
[0007] RNAi has been studied in a variety of systems. Fire et al.,
1998, Nature, 391, 806, were the first to observe RNAi in C.
elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe
RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,
Nature, 404, 293, describe RNAi in Drosophila cells transfected
with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi
induced by introduction of duplexes of synthetic 21-nucleotide RNAs
in cultured mammalian cells including human embryonic kidney and
HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir
et al., 2001, EMBO J., 20, 6877) has revealed certain requirements
for siRNA length, structure, chemical composition, and sequence
that are essential to mediate efficient RNAi activity. These
studies have shown that 21-nucleotide siRNA duplexes are most
active when containing 3'-terminal dinucleotide overhangs.
Furthermore, complete substitution of one or both siRNA strands
with 2'-deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi
activity, whereas substitution of the 3'-terminal siRNA overhang
nucleotides with 2'-deoxy nucleotides (2'-H) was shown to be
tolerated. Single mismatch sequences in the center of the siRNA
duplex were also shown to abolish RNAi activity. In addition, these
studies also indicate that the position of the cleavage site in the
target RNA is defined by the 5'-end of the siRNA guide sequence
rather than the 3'-end of the guide sequence (Elbashir et al.,
2001, EMBO J., 20, 6877). Other studies have indicated that a
5'-phosphate on the target-complementary strand of a siRNA duplex
is required for siRNA activity and that ATP is utilized to maintain
the 5'-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell,
107, 309).
[0008] Studies have shown that replacing the 3'-terminal nucleotide
overhanging segments of a 21-mer siRNA duplex having two-nucleotide
3'-overhangs with deoxyribonucleotides does not have an adverse
effect on RNAi activity. Replacing up to four nucleotides on each
end of the siRNA with deoxyribonucleotides has been reported to be
well tolerated, whereas complete substitution with
deoxyribonucleotides results in no RNAi activity (Elbashir et al.,
2001, EMBO J., 20, 6877). In addition, Elbashir et al., supra, also
report that substitution of siRNA with 2'-O-methyl nucleotides
completely abolishes RNAi activity. Li et al., International PCT
Publication No. WO 00/44914, and Beach et al., International PCT
Publication No. WO 01/68836 preliminarily suggest that siRNA may
include modifications to either the phosphate-sugar backbone or the
nucleoside to include at least one of a nitrogen or sulfur
heteroatom, however, neither application postulates to what extent
such modifications would be tolerated in siRNA molecules, nor
provides any further guidance or examples of such modified siRNA.
Kreutzer et al., Canadian Patent Application No. 2,359,180, also
describe certain chemical modifications for use in dsRNA constructs
in order to counteract activation of double-stranded RNA-dependent
protein kinase PKR, specifically 2'-amino or 2'-methyl nucleotides,
and nucleotides containing a 2'-O or 4'-C methylene bridge.
However, Kreutzer et al. similarly fails to provide examples or
guidance as to what extent these modifications would be tolerated
in siRNA molecules.
[0009] Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested
certain chemical modifications targeting the unc-22 gene in C.
elegans using long (>25 nt) siRNA transcripts. The authors
describe the introduction of thiophosphate residues into these
siRNA transcripts by incorporating thiophosphate nucleotide analogs
with T7 and T3 RNA polymerase and observed that RNAs with two
phosphorothioate modified bases also had substantial decreases in
effectiveness as RNAi. Further, Parrish et al. reported that
phosphorothioate modification of more than two residues greatly
destabilized the RNAs in vitro such that interference activities
could not be assayed. Id. at 1081. The authors also tested certain
modifications at the 2'-position of the nucleotide sugar in the
long siRNA transcripts and found that substituting deoxynucleotides
for ribonucleotides produced a substantial decrease in interference
activity, especially in the case of Uridine to Thymidine and/or
Cytidine to deoxy-Cytidine substitutions. Id. In addition, the
authors tested certain base modifications, including substituting,
in sense and antisense strands of the siRNA, 4-thiouracil,
5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil,
and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil
substitution appeared to be tolerated, Parrish reported that
inosine produced a substantial decrease in interference activity
when incorporated in either strand. Parrish also reported that
incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the
antisense strand resulted in a substantial decrease in RNAi
activity as well.
[0010] The use of longer dsRNA has been described. For example,
Beach et al., International PCT Publication No. WO 01/68836,
describes specific methods for attenuating gene expression using
endogenously-derived dsRNA. Tuschl et al., International PCT
Publication No. WO 01/75164, describe a Drosophila in vitro RNAi
system and the use of specific siRNA molecules for certain
functional genomic and certain therapeutic applications; although
Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be
used to cure genetic diseases or viral infection due to the danger
of activating interferon response. Li et al., International PCT
Publication No. WO 00/44914, describe the use of specific dsRNAs
for attenuating the expression of certain target genes.
Zernicka-Goetz et al., International PCT Publication No. WO
01/36646, describe certain methods for inhibiting the expression of
particular genes in mammalian cells using certain dsRNA molecules.
Fire et al., International PCT Publication No. WO 99/32619,
describe particular methods for introducing certain dsRNA molecules
into cells for use in inhibiting gene expression. Plaetinck et al.,
International PCT Publication No. WO 00/01846, describe certain
methods for identifying specific genes responsible for conferring a
particular phenotype in a cell using specific dsRNA molecules.
Mello et al., International PCT Publication No. WO 01/29058,
describe the identification of specific genes involved in
dsRNA-mediated RNAi. Deschamps Depaillette et al., International
PCT Publication No. WO 99/07409, describe specific compositions
consisting of particular dsRNA molecules combined with certain
anti-viral agents. Waterhouse et al., International PCT Publication
No. 99/53050, describe certain methods for decreasing the
phenotypic expression of a nucleic acid in plant cells using
certain dsRNAs. Driscoll et al., International PCT Publication No.
WO 01/49844, describe specific DNA constructs for use in
facilitating gene silencing in targeted organisms.
[0011] Others have reported on various RNAi and gene-silencing
systems. For example, Parrish et al., 2000, Molecular Cell, 6,
1977-1087, describe specific chemically-modified siRNA constructs
targeting the unc-22 gene of C. elegans. Grossniklaus,
International PCT Publication No. WO 01/38551, describes certain
methods for regulating polycomb gene expression in plants using
certain dsRNAs. Churikov et al., International PCT Publication No.
WO 01/42443, describe certain methods for modifying genetic
characteristics of an organism using certain dsRNAs. Cogoni et al.,
International PCT Publication No. WO 01/53475, describe certain
methods for isolating a Neurospora silencing gene and uses thereof.
Reed et al., International PCT Publication No. WO 01/68836,
describe certain methods for gene silencing in plants. Honer et
al., International PCT Publication No. WO 01/70944, describe
certain methods of drug screening using transgenic nematodes as
Parkinson's Disease models using certain dsRNAs. Deak et al.,
International PCT Publication No. WO 01/72774, describe certain
Drosophila-derived gene products that may be related to RNAi in
Drosophila. Arndt et al., International PCT Publication No. WO
01/92513 describe certain methods for mediating gene suppression by
using factors that enhance RNAi. Tuschl et al., International PCT
Publication No. WO 02/44321, describe certain synthetic siRNA
constructs. Pachuk et al., International PCT Publication No. WO
00/63364, and Satishchandran et al., International PCT Publication
No. WO 01/04313, describe certain methods and compositions for
inhibiting the function of certain polynucleotide sequences using
certain dsRNAs. Echeverri et al., International PCT Publication No.
WO 02/38805, describe certain C. elegans genes identified via RNAi.
Kreutzer et al., International PCT Publications Nos. WO 02/055692,
WO 02/055693, and EP 1144623 B1 describes certain methods for
inhibiting gene expression using RNAi. Graham et al., International
PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501
describe certain vector expressed siRNA molecules. Fire et al.,
U.S. Pat. No. 6,506,559, describe certain methods for inhibiting
gene expression in vitro using certain long dsRNA (greater than 25
nucleotide) constructs that mediate RNAi.
SUMMARY OF THE INVENTION
[0012] This invention relates to compounds, compositions, and
methods useful for modulating the expression of genes associated
with mitogen activated protein kinase (MAP kinase) gene expression
pathways (see for example FIG. 12) by RNA interference (RNAi) using
short interfering nucleic acid (siNA) molecules. In particular, the
instant invention features small nucleic acid molecules, such as
short interfering nucleic acid (siNA), short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short
hairpin RNA (shRNA) molecules and methods used to modulate the
expression of MAP kinase genes, including c-JUN, JNK genes such as
JNK1 and JNK2, ERK genes such as ERK1 and ERK2, and p38 genes. A
siNA of the invention can be unmodified or chemically-modified. A
siNA of the instant invention can be chemically synthesized,
expressed from a vector or enzymatically synthesized. The instant
invention also features various chemically-modified synthetic short
interfering nucleic acid (siNA) molecules capable of modulating
telomerase gene expression or activity in cells by RNA interference
(RNAi). The use of chemically-modified siNA improves various
properties of native siNA molecules through increased resistance to
nuclease degradation in vivo and/or through improved cellular
uptake. Further, contrary to earlier published studies, siNA having
multiple chemical modifications retains its RNAi activity. The siNA
molecules of the instant invention provide useful reagents and
methods for a variety of therapeutic, diagnostic, target
validation, genomic discovery, genetic engineering, and
pharmacogenomic applications.
[0013] In one embodiment, the invention features one or more siNA
molecules and methods that independently or in combination modulate
the expression of gene(s) encoding MAP kinase proteins, such as
genes encoding sequences comprising those sequences referred to by
GenBank Accession Nos. shown in Table I, referred to herein
generally as MAP kinases. The description below of the various
aspects and embodiments of the invention is provided with reference
to exemplary MAP kinases such as JNK1 (also referred to as MAPK8,
for example Genbank Accession No. NM.sub.--002750), p38 (also
referred to as MAPK14, for example Genbank Accession No.
NM.sub.--139012), ERK2 (also referred to as MAPK1, for example
Genbank Accession No. NM.sub.--002745), and ERK1 (also referred to
as MAPK3, for example Genbank Accession XM.sub.--055766) genes.
However, the various aspects and embodiments are also directed to
other MAP kinases referred to by Accession number in Table 1 and
other genes involved in MAP kinase pathways such those genes
encoding c-JUN (for example Genbank Accession No. NM.sub.--002228),
TNF-alpha (for example Genbank Accession No. M10988), interleukins
such as IL-8 (for example Genbank Accession No. M68932), and
activating proteins such as AP-1 (for example Genbank Accession No.
NM.sub.--013277). The various aspects and embodiments are also
directed to other genes that are involved in the MAP kinase
pathways of gene expression. Those additional genes can be analyzed
for target sites using the methods described for MAP kinase genes
herein. Thus, the inhibition and the effects of such inhibition of
the other genes can be performed as described herein.
[0014] In one embodiment, the invention features a siNA molecule
that down-regulates expression of a MAP kinase gene, for example,
wherein the MAP kinase gene comprises MAP kinase encoding sequence
(e.g., c-JUN, JNK1, JNK2, p38, ERK1, or ERK2).
[0015] In one embodiment, the invention features a siNA molecule
having RNAi activity against MAP kinase RNA, wherein the siNA
molecule comprises a sequence complementary to an RNA having MAP
kinase encoding sequence, such as those sequences having MAP kinase
GenBank Accession Nos. shown in Table I. In another embodiment, the
invention features a siNA molecule having RNAi activity against MAP
kinase RNA, wherein the siNA molecule comprises a sequence
complementary to an RNA having other MAP kinase encoding sequence,
such as mutant MAP kinase genes, splice variants of MAP kinase
genes, and other MAP kinase ligands and receptors. Chemical
modifications as shown in Tables III and IV or otherwise described
herein can be applied to any siNA construct of the invention.
[0016] In another embodiment, the invention features a siNA
molecule having RNAi activity against a MAP kinase gene, wherein
the siNA molecule comprises nucleotide sequence complementary to
nucleotide sequence of a MAP kinase gene, such as those MAP kinase
sequences having GenBank Accession Nos. shown in Table I or other
MAP kinase encoding sequence, such as mutant MAP kinase genes,
splice variants of MAP kinase genes, and other MAP kinase ligands
and receptors. In another embodiment, a siNA molecule of the
invention includes nucleotide sequence that can interact with
nucleotide sequence of a MAP kinase gene and thereby mediate
silencing of MAP kinase gene expression, for example, wherein the
siNA mediates regulation of MAP kinase gene expression by cellular
processes that modulate the chromatin structure of the MAP kinase
gene and prevent transcription of the MAP kinase gene.
[0017] In another embodiment, the invention features a siNA
molecule comprising nucleotide sequence, for example, nucleotide
sequence in the antisense region of the siNA molecule, that is
complementary to a nucleotide sequence or portion of sequence of a
MAP kinase gene. In another embodiment, the invention features a
siNA molecule comprising a region, for example, the antisense
region of the siNA construct, complementary to a sequence
comprising a MAP kinase gene sequence or a portion thereof.
[0018] In one embodiment, the antisense region of ERK2 siNA
constructs can comprise a sequence complementary to sequence having
any of SEQ ID NOs. 1-163, or 1113-1116. The antisense region can
also comprise sequence having any of SEQ ID NOs. 164-326,
1133-1136, 1141-1144, or 1149-1152. In another embodiment, the
sense region of ERK2 siNA constructs can comprise sequence having
any of SEQ ID NOs. 1-163, 1113-1116, 1129-1132, 1137-1140, or
1145-1148.
[0019] In one embodiment, the antisense region of ERK1 siNA
constructs can comprise a sequence complementary to sequence having
any of SEQ ID NOs. 327-431, or 1117-1120. The antisense region can
also comprise sequence having any of SEQ ID NOs. 432-536,
1157-1160, 1165-1168, or 1173-1176. In another embodiment, the
sense region of ERK1 siNA constructs can comprise sequence having
any of SEQ ID NOs. 327-431, 1117-1120, 1153-1156, 1161-1164, or
1169-1172.
[0020] In one embodiment, the antisense region of JNK1 siNA
constructs can comprise a sequence complementary to sequence having
any of SEQ ID NOs. 537-615 or 1121-1124. The antisense region can
also comprise sequence having any of SEQ ID NOs. 616-694,
1181-1184, 1189-1192, 1197-1200, 1237, 1239, 1241, 1243, 1245, or
1246. In another embodiment, the sense region of JNK1 constructs
can comprise sequence having any of SEQ ID NOs. 537-615, 1121-1124,
1177-1180, 1185-1188, 1193-1196, 1236, 1238, 1240, 1242, or 1244.
The sense region can comprise a sequence of SEQ ID NO. 1225 and the
antisense region can comprise a sequence of SEQ ID NO. 1226. The
sense region can comprise a sequence of SEQ ID NO. 1227 and the
antisense region can comprise a sequence of SEQ ID NO. 1228. The
sense region can comprise a sequence of SEQ ID NO. 1229 and the
antisense region can comprise a sequence of SEQ ID NO. 1230. The
sense region can comprise a sequence of SEQ ID NO. 1231 and the
antisense region can comprise a sequence of SEQ ID NO. 1232. The
sense region can comprise a sequence of SEQ ID NO. 1233 and the
antisense region can comprise a sequence of SEQ ID NO. 1234. The
sense region can comprise a sequence of SEQ ID NO. 1231 and the
antisense region can comprise a sequence of SEQ ID NO. 1235.
[0021] In one embodiment, the antisense region of p38 siNA
constructs can comprise a sequence complementary to sequence having
any of SEQ ID NOs. 695-903 or 1125-1128. The antisense region can
also comprise sequence having any of SEQ ID NOs. 904-1112,
1205-1208, 1213-1216, or 1221-1224. In another embodiment, the
sense region of p38 siNA constructs can comprise sequence having
any of SEQ ID NOs. 695-903, 1125-1128, 1201-1204, 1209-1212, or
1217-1220.
[0022] In one embodiment, the antisense region of c-JUN siNA
constructs can comprise a sequence complementary to sequence having
any of SEQ ID NOs. 1247-1427 or 1609-1616. In one embodiment, the
antisense region of c-JUN siNA constructs can comprise sequence
having any of SEQ ID NOs. 1428-1608, 1625-1632, 1641-1648,
1657-1664, 1673-1680, 1698, 1700, 1702, 1705, 1707, 1709, 1711, or
1714. In another embodiment, the sense region of c-JUN siNA
constructs can comprise sequence having any of SEQ ID NOs.
1247-1427, 1609-1616, 1617-1624, 1633-1640, 1649-1656, 1665-1672,
1697, 1699, 1701, 1703, 1704, 1706, 1708, 1710, 1712, or 1713.
[0023] In one embodiment, a siNA molecule of the invention
comprises any of SEQ ID NOs. 1-1714. The sequences shown in SEQ ID
NOs: 1-1714 are not limiting. A siNA molecule of the invention can
comprise any contiguous MAP kinase sequence (e.g., about 19 to
about 25, or about 19, 20, 21, 22, 23, 24 or 25 contiguous MAP
kinase nucleotides).
[0024] In yet another embodiment, the invention features a siNA
molecule comprising a sequence, for example, the antisense sequence
of the siNA construct, complementary to a sequence or portion of
sequence comprising sequence represented by GenBank Accession Nos.
shown in Table I. Chemical modifications in Tables III and IV and
described herein can be applied to any siRNA construct of the
invention.
[0025] In one embodiment of the invention a siNA molecule comprises
an antisense strand having about 19 to about 29 nucleotides,
wherein the antisense strand is complementary to a RNA sequence
encoding a MAP kinase protein, and wherein said siNA further
comprises a sense strand having about 19 to about 29 (e.g., about
19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, and
wherein said sense strand and said antisense strand are distinct
nucleotide sequences with at least about 19 complementary
nucleotides.
[0026] In another embodiment of the invention a siNA molecule of
the invention comprises an antisense region having about 19 to
about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29)
nucleotides, wherein the antisense region is complementary to a RNA
sequence encoding a MAP kinase protein, and wherein said siNA
further comprises a sense region having about 19 to about 29
nucleotides, wherein said sense region and said antisense region
comprise a linear molecule with at least about 19 complementary
nucleotides.
[0027] In one embodiment of the invention a siNA molecule comprises
an antisense strand comprising a nucleotide sequence that is
complementary to a nucleotide sequence encoding a MAP kinase
protein or a portion thereof. The siNA further comprises a sense
strand, wherein said sense strand comprises a nucleotide sequence
of a MAP kinase gene or a portion thereof.
[0028] In another embodiment, a siNA molecule comprises an
antisense region comprising a nucleotide sequence that is
complementary to a nucleotide sequence encoding a MAP kinase
protein or a portion thereof. The siNA molecule further comprises a
sense region, wherein said sense region comprises a nucleotide
sequence of a MAP kinase gene or a portion thereof.
[0029] In one embodiment, a siNA molecule of the invention has RNAi
activity that modulates expression of RNA encoded by a MAP kinase
gene. Because MAP kinase genes can share some degree of sequence
homology with each other, siNA molecules can be designed to target
a class of MAP kinase genes (and associated receptor or ligand
genes) or alternately specific MAP kinase genes by selecting
sequences that are either shared amongst different MAP kinase
targets or alternatively that are unique for a specific MAP kinase
target. Therefore, in one embodiment, the siNA molecule can be
designed to target conserved regions of MAP kinase RNA sequence
having homology between several MAP kinase genes so as to target
several MAP kinase genes (e.g., different MAP kinase isoforms,
splice variants, mutant genes etc.) with one siNA molecule. In
another embodiment, the siNA molecule can be designed to target a
sequence that is unique to a specific MAP kinase RNA sequence due
to the high degree of specificity that the siNA molecule requires
to mediate RNAi activity.
[0030] In one embodiment, nucleic acid molecules of the invention
that act as mediators of the RNA interference gene silencing
response are double-stranded nucleic acid molecules. In another
embodiment, the siNA molecules of the invention consist of duplexes
containing about 19 base pairs between oligonucleotides comprising
about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24 or 25)
nucleotides. In yet another embodiment, siNA molecules of the
invention comprise duplexes with overhanging ends of about 1 to
about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about
21-nucleotide duplexes with about 19 base pairs and 3'-terminal
mononucleotide, dinucleotide, or trinucleotide overhangs.
[0031] In one embodiment, the invention features one or more
chemically-modified siNA constructs having specificity for MAP
kinase expressing nucleic acid molecules, such as RNA encoding a
MAP kinase protein. Non-limiting examples of such chemical
modifications include without limitation phosphorothioate
internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl
ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal
base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides,
and terminal glyceryl and/or inverted deoxy abasic residue
incorporation. These chemical modifications, when used in various
siNA constructs, are shown to preserve RNAi activity in cells while
at the same time, dramatically increasing the serum stability of
these compounds. Furthermore, contrary to the data published by
Parrish et al., supra, applicant demonstrates that multiple
(greater than one) phosphorothioate substitutions are
well-tolerated and confer substantial increases in serum stability
for modified siNA constructs.
[0032] In one embodiment, a siNA molecule of the invention
comprises modified nucleotides while maintaining the ability to
mediate RNAi. The modified nucleotides can be used to improve in
vitro or in vivo characteristics such as stability, activity,
and/or bioavailability. For example, a siNA molecule of the
invention can comprise modified nucleotides as a percentage of the
total number of nucleotides present in the siNA molecule. As such,
a siNA molecule of the invention can generally comprise about 5% to
about 100% modified nucleotides (e.g., 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
100% modified nucleotides). The actual percentage of modified
nucleotides present in a given siNA molecule will depend on the
total number of nucleotides present in the siNA. If the siNA
molecule is single stranded, the percent modification can be based
upon the total number of nucleotides present in the single stranded
siNA molecules. Likewise, if the siNA molecule is double stranded,
the percent modification can be based upon the total number of
nucleotides present in the sense strand, antisense strand, or both
the sense and antisense strands.
[0033] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein the siNA molecule
comprises one or more chemical modifications and each strand of the
double-stranded siNA is about 21 nucleotides long.
[0034] In one embodiment, a siNA molecule of the invention does not
contain any ribonucleotides. In another embodiment, a siNA molecule
of the invention comprises one or more ribonucleotides.
[0035] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein one of the strands of the
double-stranded siNA molecule comprises a nucleotide sequence that
is complementary to a nucleotide sequence of the MAP kinase gene or
a portion thereof, and wherein the second strand of the
double-stranded siNA molecule comprises a nucleotide sequence
substantially similar to the nucleotide sequence of the MAP kinase
gene or a portion thereof.
[0036] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein each strand of the siNA
molecule comprises about 19 to about 23 nucleotides, and wherein
each strand comprises at least about 19 nucleotides that are
complementary to the nucleotides of the other strand.
[0037] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein the siNA molecule
comprises an antisense region comprising a nucleotide sequence that
is complementary to a nucleotide sequence of the MAP kinase gene or
a portion thereof, and wherein the siNA further comprises a sense
region, wherein the sense region comprises a nucleotide sequence
substantially similar to the nucleotide sequence of the MAP kinase
gene or a portion thereof.
[0038] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein the antisense region and
the sense region each comprise about 19 to about 23 nucleotides,
and wherein the antisense region comprises at least about 19
nucleotides that are complementary to nucleotides of the sense
region.
[0039] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein the siNA molecule
comprises a sense region and an antisense region and wherein the
antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence of RNA encoded by the MAP
kinase gene or a portion thereof and the sense region comprises a
nucleotide sequence that is complementary to the antisense
region.
[0040] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein the siNA molecule is
assembled from two separate oligonucleotide fragments wherein one
fragment comprises the sense region and the second fragment
comprises the antisense region of the siNA molecule. The sense
region can be connected to the antisense region via a linker
molecule, such as a polynucleotide linker or a non-nucleotide
linker.
[0041] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein the siNA molecule
comprises a sense region and an antisense region and wherein the
antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence of RNA encoded by the MAP
kinase gene or a portion thereof and the sense region comprises a
nucleotide sequence that is complementary to the antisense region,
wherein the siNA molecule has one or more modified pyrimidine
and/or purine nucleotides. In one embodiment, the pyrimidine
nucleotides in the sense region are 2'-O-methylpyrimidine
nucleotides, or 2'-deoxy-2'-fluoro pyrimidine nucleotides and the
purine nucleotides present in the sense region are 2'-deoxy purine
nucleotides. In another embodiment, the pyrimidine nucleotides in
the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides and
the purine nucleotides present in the sense region are 2'-O-methyl
purine nucleotides. In another embodiment of any of the above
described siNA molecules, any nucleotides present in a
non-complementary region of the sense strand (e.g. overhang region)
are 2'-deoxy nucleotides.
[0042] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein the siNA molecule is
assembled from two separate oligonucleotide fragments wherein one
fragment comprises the sense region and the second fragment
comprises the antisense region of the siNA molecule, and wherein
the fragment comprising the sense region includes a terminal cap
moiety at the 5'-end, the 3'-end, or both of the 5' and 3' ends of
the fragment comprising the sense region. In one embodiment, the
terminal cap moiety is an inverted deoxy abasic moiety or glyceryl
moiety. In one embodiment, each of the two fragments of the siNA
molecule comprises about 21 nucleotides.
[0043] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein the siNA molecule
comprises a sense region and an antisense region and wherein the
antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence or a portion thereof of RNA
encoded by the MAP kinase gene and the sense region comprises a
nucleotide sequence that is complementary to the antisense region,
and wherein the purine nucleotides present in the antisense region
comprise 2'-deoxy-purine nucleotides. In another embodiment, the
purine nucleotides present in the antisense region comprise
2'-O-methyl purine nucleotides. In either of the above embodiments,
the antisense region comprises a phosphorothioate internucleotide
linkage at the 3' end of the antisense region. In an alternative
embodiment, the antisense region comprises a glyceryl modification
at the 3' end of the antisense region. In another embodiment of any
of the above described siNA molecules, any nucleotides present in a
non-complementary region of the antisense strand (e.g. overhang
region) are 2'-deoxy nucleotides.
[0044] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein the siNA molecule is
assembled from two separate oligonucleotide fragments each
comprising 21 nucleotides, wherein one fragment comprises the sense
region and the second fragment comprises the antisense region of
the siNA molecule, and wherein about 19 nucleotides of each
fragment of the siNA molecule are base-paired to the complementary
nucleotides of the other fragment of the siNA molecule and wherein
at least two 3' terminal nucleotides of each fragment of the siNA
molecule are not base-paired to the nucleotides of the other
fragment of the siNA molecule. In one embodiment, each of the two
3' terminal nucleotides of each fragment of the siNA molecule is a
2'-deoxy-pyrimidine, such as 2'-deoxy-thymidine. In another
embodiment, all 21 nucleotides of each fragment of the siNA
molecule are base-paired to the complementary nucleotides of the
other fragment of the siNA molecule. In another embodiment, about
19 nucleotides of the antisense region are base-paired to the
nucleotide sequence or a portion thereof of the RNA encoded by the
MAP kinase gene. In another embodiment, 21 nucleotides of the
antisense region are base-paired to the nucleotide sequence or a
portion thereof of the RNA encoded by the MAP kinase gene. In any
of the above embodiments, the 5'-end of the fragment comprising
said antisense region can optionally include a phosphate group.
[0045] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits the
expression of a MAP kinase RNA sequence (e.g., wherein said target
RNA sequence is encoded by a MAP kinase gene or a gene involved in
the MAP kinase pathway), wherein the siNA molecule does not contain
any ribonucleotides and wherein each strand of the double-stranded
siNA molecule is about 21 nucleotides long.
[0046] In one embodiment, the invention features a medicament
comprising a siNA molecule of the invention.
[0047] In one embodiment, the invention features an active
ingredient comprising a siNA molecule of the invention.
[0048] In one embodiment, the invention features the use of a
double-stranded short interfering nucleic acid (siNA) molecule to
down-regulate expression of a MAP kinase gene, wherein the siNA
molecule comprises one or more chemical modifications and each
strand of the double-stranded siNA is about 21 nucleotides
long.
[0049] The invention features a double-stranded short interfering
nucleic acid (siNA) molecule that inhibits expression of a MAP
kinase gene, wherein one of the strands of the double-stranded siNA
molecule is an antisense strand which comprises nucleotide sequence
that is complementary to nucleotide sequence of a MAP kinase RNA or
a portion thereof, the other strand is a sense strand which
comprises nucleotide sequence that is complementary to a nucleotide
sequence of the antisense strand and wherein a majority of the
pyrimidine nucleotides present in the double-stranded siNA molecule
comprises a sugar modification. In one embodiment, the nucleotide
sequence of the antisense strand of the double-stranded siNA
molecule is complementary to the nucleotide sequence of the MAP
kinase RNA which encodes a protein or a portion thereof. In one
embodiment, each strand of the siNA molecule comprises about 19 to
about 29 nucleotides, and each strand comprises at least about 19
nucleotides that are complementary to the nucleotides of the other
strand. In one embodiment, the siNA molecule is assembled from two
oligonucleotide fragments, wherein one fragment comprises the
nucleotide sequence of the antisense strand of the siNA molecule
and a second fragment comprises nucleotide sequence of the sense
region of the siNA molecule. In another embodiment, the sense
strand is connected to the antisense strand via a linker molecule,
such as a polynucleotide linker or a non-nucleotide linker. In one
embodiment, the pyrimidine nucleotides present in the sense strand
are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine
nucleotides present in the sense region are 2'-deoxy purine
nucleotides. In another embodiment, the pyrimidine nucleotides
present in the sense strand are 2'-deoxy-2'-fluoro pyrimidine
nucleotides and the purine nucleotides present in the sense region
are 2'-O-methyl purine nucleotides. In one embodiment, wherein the
sense strand comprises a 3'-end and a 5'-end, a terminal cap moiety
(e.g., an inverted deoxy abasic moiety) is present at the 5'-end,
the 3'-end, or both of the 5' and 3' ends of the sense strand. In
one embodiment, the antisense strand comprises one or more
2'-deoxy-2'-fluoro pyrimidine nucleotides and one or more
2'-O-methyl purine nucleotides. In one embodiment, the pyrimidine
nucleotides present in the antisense strand are 2'-deoxy-2'-fluoro
pyrimidine nucleotides and any purine nucleotides present in the
antisense strand are 2'-O-methyl purine nucleotides. In one
embodiment, the antisense strand comprises a phosphorothioate
internucleotide linkage at the 3' end of the antisense strand. In
another embodiment, the antisense strand comprises a glyceryl
modification at the 3' end. In another embodiment, the 5'-end of
the antisense strand optionally includes a phosphate group. In one
embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that down-regulates
expression of a MAP kinase gene, wherein one of the strands of the
double-stranded siNA molecule is an antisense strand which
comprises nucleotide sequence that is complementary to nucleotide
sequence of MAP kinase RNA or a portion thereof, the other strand
is a sense strand which comprises nucleotide sequence that is
complementary to a nucleotide sequence of the antisense strand and
wherein a majority of the pyrimidine nucleotides present in the
double-stranded siNA molecule comprises a sugar modification, and
wherein the nucleotide sequence of the antisense strand is
complementary to a nucleotide sequence of the 5'-untranslated
region or a portion thereof of the MAP kinase RNA. In another
embodiment, the nucleotide sequence of the antisense strand is
complementary to a nucleotide sequence of the MAP kinase RNA or a
portion thereof.
[0050] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits
expression of a MAP kinase gene, wherein one of the strands of the
double-stranded siNA molecule is an antisense strand which
comprises nucleotide sequence that is complementary to nucleotide
sequence of MAP kinase RNA or a portion thereof, the other strand
is a sense strand which comprises nucleotide sequence that is
complementary to a nucleotide sequence of the antisense strand and
wherein a majority of the pyrimidine nucleotides present in the
double-stranded siNA molecule comprises a sugar modification, and
wherein each of the two strands of the siNA molecule comprises 21
nucleotides. In one embodiment, about 19 nucleotides of each strand
of the siNA molecule are base-paired to the complementary
nucleotides of the other strand of the siNA molecule and at least
two 3' terminal nucleotides of each strand of the siNA molecule are
not base-paired to the nucleotides of the other strand of the siNA
molecule. In one embodiment, each of the two 3' terminal
nucleotides of each fragment of the siNA molecule are
2'-deoxy-pyrimidines, such as 2'-deoxy-thymidine. In another
embodiment, each strand of the siNA molecule is base-paired to the
complementary nucleotides of the other strand of the siNA molecule.
In one embodiment, about 19 nucleotides of the antisense strand are
base-paired to the nucleotide sequence of the MAP kinase RNA or a
portion thereof. In another embodiment, 21 nucleotides of the
antisense strand are base-paired to the nucleotide sequence of the
MAP kinase RNA or a portion thereof.
[0051] In one embodiment, the invention features a composition
comprising a siNA molecule of the invention and a pharmaceutically
acceptable carrier or diluent.
[0052] In one embodiment, the invention features the use of a
double-stranded short interfering nucleic acid (siNA) molecule that
inhibits expression of a MAP kinase gene, wherein one of the
strands of the double-stranded siNA molecule is an antisense strand
which comprises nucleotide sequence that is complementary to
nucleotide sequence of MAP kinase RNA or a portion thereof, the
other strand is a sense strand which comprises nucleotide sequence
that is complementary to a nucleotide sequence of the antisense
strand and wherein a majority of the pyrimidine nucleotides present
in the double-stranded siNA molecule comprises a sugar
modification.
[0053] In a non-limiting example, the introduction of
chemically-modified nucleotides into nucleic acid molecules
provides a powerful tool in overcoming potential limitations of in
vivo stability and bioavailability inherent to native RNA molecules
that are delivered exogenously. For example, the use of
chemically-modified nucleic acid molecules can enable a lower dose
of a particular nucleic acid molecule for a given therapeutic
effect since chemically-modified nucleic acid molecules tend to
have a longer half-life in serum. Furthermore, certain chemical
modifications can improve the bioavailability of nucleic acid
molecules by targeting particular cells or tissues and/or improving
cellular uptake of the nucleic acid molecule. Therefore, even if
the activity of a chemically-modified nucleic acid molecule is
reduced as compared to a native nucleic acid molecule, for example,
when compared to an all-RNA nucleic acid molecule, the overall
activity of the modified nucleic acid molecule can be greater than
that of the native molecule due to improved stability and/or
delivery of the molecule. Unlike native unmodified siNA,
chemically-modified siNA can also minimize the possibility of
activating interferon activity in humans.
[0054] In any of the embodiments of siNA molecules described
herein, the antisense region of a siNA molecule of the invention
can comprise a phosphorothioate internucleotide linkage at the
3'-end of said antisense region. In any of the embodiments of siNA
molecules described herein, the antisense region can comprise about
one to about five phosphorothioate internucleotide linkages at the
5'-end of said antisense region. In any of the embodiments of siNA
molecules described herein, the 3'-terminal nucleotide overhangs of
a siNA molecule of the invention can comprise ribonucleotides or
deoxyribonucleotides that are chemically-modified at a nucleic acid
sugar, base, or backbone. In any of the embodiments of siNA
molecules described herein, the 3'-terminal nucleotide overhangs
can comprise one or more universal base ribonucleotides. In any of
the embodiments of siNA molecules described herein, the 3'-terminal
nucleotide overhangs can comprise one or more acyclic
nucleotides.
[0055] One embodiment of the invention provides an expression
vector comprising a nucleic acid sequence encoding at least one
siNA molecule of the invention in a manner that allows expression
of the nucleic acid molecule. Another embodiment of the invention
provides a mammalian cell comprising such an expression vector. The
mammalian cell can be a human cell. The siNA molecule of the
expression vector can comprise a sense region and an antisense
region. The antisense region can comprise sequence complementary to
an RNA or DNA sequence encoding MAP kinase and the sense region can
comprise sequence complementary to the antisense region. The siNA
molecule can comprise two distinct strands having complementary
sense and antisense regions. The siNA molecule can comprise a
single strand having complementary sense and antisense regions.
[0056] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against a MAP kinase
inside a cell or reconstituted in vitro system, wherein the
chemical modification comprises one or more (e.g., about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbone
modified internucleotide linkage having Formula I:
##STR00001##
wherein each R1 and R2 is independently any nucleotide,
non-nucleotide, or polynucleotide which can be naturally-occurring
or chemically-modified, each X and Y is independently O, S, N,
alkyl, or substituted alkyl, each Z and W is independently O, S, N,
alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl,
and wherein W, X, Y, and Z are optionally not all O.
[0057] The chemically-modified internucleotide linkages having
Formula I, for example, wherein any Z, W, X, and/or Y independently
comprises a sulphur atom, can be present in one or both
oligonucleotide strands of the siNA duplex, for example, in the
sense strand, the antisense strand, or both strands. The siNA
molecules of the invention can comprise one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified
internucleotide linkages having Formula I at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of the sense strand, the
antisense strand, or both strands. For example, an exemplary siNA
molecule of the invention can comprise about 1 to about 5 or more
(e.g., about 1, 2, 3, 4, 5, or more) chemically-modified
internucleotide linkages having Formula I at the 5'-end of the
sense strand, the antisense strand, or both strands. In another
non-limiting example, an exemplary siNA molecule of the invention
can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more) pyrimidine nucleotides with chemically-modified
internucleotide linkages having Formula I in the sense strand, the
antisense strand, or both strands. In yet another non-limiting
example, an exemplary siNA molecule of the invention can comprise
one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
purine nucleotides with chemically-modified internucleotide
linkages having Formula I in the sense strand, the antisense
strand, or both strands. In another embodiment, a siNA molecule of
the invention having internucleotide linkage(s) of Formula I also
comprises a chemically-modified nucleotide or non-nucleotide having
any of Formulae I-VII.
[0058] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against a MAP kinase
inside a cell or reconstituted in vitro system, wherein the
chemical modification comprises one or more (e.g., about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides
having Formula II:
##STR00002##
[0059] wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is
independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,
F, Cl, Br, CN, CF3, OCF.sub.3, OCN, O-alkyl, S-alkyl, N-alkyl,
O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH,
O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl,
ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2,
O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted
silyl, or group having Formula I; R9 is O, S, CH2, S.dbd.O, CHF, or
CF2, and B is a nucleosidic base such as adenine, guanine, uracil,
cytosine, thymine, 2-aminoadenosine, 5-methylcytosine,
2,6-diaminopurine, or any other non-naturally occurring base that
can be complementary or non-complementary to target RNA or a
non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,
5-nitroindole, nebularine, pyridone, pyridinone, or any other
non-naturally occurring universal base that can be complementary or
non-complementary to target RNA.
[0060] The chemically-modified nucleotide or non-nucleotide of
Formula II can be present in one or both oligonucleotide strands of
the siNA duplex, for example in the sense strand, the antisense
strand, or both strands. The siNA molecules of the invention can
comprise one or more chemically-modified nucleotide or
non-nucleotide of Formula II at the 3'-end, the 5'-end, or both of
the 3' and 5'-ends of the sense strand, the antisense strand, or
both strands. For example, an exemplary siNA molecule of the
invention can comprise about 1 to about 5 or more (e.g., about 1,
2, 3, 4, 5, or more) chemically-modified nucleotides or
non-nucleotides of Formula II at the 5'-end of the sense strand,
the antisense strand, or both strands. In another non-limiting
example, an exemplary siNA molecule of the invention can comprise
about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more)
chemically-modified nucleotides or non-nucleotides of Formula II at
the 3'-end of the sense strand, the antisense strand, or both
strands.
[0061] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against a MAP kinase
inside a cell or reconstituted in vitro system, wherein the
chemical modification comprises one or more (e.g., about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides
having Formula III:
##STR00003##
wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is
independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,
F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl,
O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH,
O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl,
ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2,
O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted
silyl, or group having Formula I; R9 is O, S, CH2, S.dbd.O, CHF, or
CF2, and B is a nucleosidic base such as adenine, guanine, uracil,
cytosine, thymine, 2-aminoadenosine, 5-methylcytosine,
2,6-diaminopurine, or any other non-naturally occurring base that
can be employed to be complementary or non-complementary to target
RNA or a non-nucleosidic base such as phenyl, naphthyl,
3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or
any other non-naturally occurring universal base that can be
complementary or non-complementary to target RNA.
[0062] The chemically-modified nucleotide or non-nucleotide of
Formula III can be present in one or both oligonucleotide strands
of the siNA duplex, for example, in the sense strand, the antisense
strand, or both strands. The siNA molecules of the invention can
comprise one or more chemically-modified nucleotide or
non-nucleotide of Formula III at the 3'-end, the 5'-end, or both of
the 3' and 5'-ends of the sense strand, the antisense strand, or
both strands. For example, an exemplary siNA molecule of the
invention can comprise about 1 to about 5 or more (e.g., about 1,
2, 3, 4, 5, or more) chemically-modified nucleotide(s) or
non-nucleotide(s) of Formula III at the 5'-end of the sense strand,
the antisense strand, or both strands. In another non-limiting
example, an exemplary siNA molecule of the invention can comprise
about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more)
chemically-modified nucleotide or non-nucleotide of Formula III at
the 3'-end of the sense strand, the antisense strand, or both
strands.
[0063] In another embodiment, a siNA molecule of the invention
comprises a nucleotide having Formula II or III, wherein the
nucleotide having Formula II or III is in an inverted
configuration. For example, the nucleotide having Formula II or III
is connected to the siNA construct in a 3'-3',3'-2',2'-3', or 5'-5'
configuration, such as at the 3'-end, the 5'-end, or both of the 3'
and 5'-ends of one or both siNA strands.
[0064] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against a MAP kinase
inside a cell or reconstituted in vitro system, wherein the
chemical modification comprises a 5'-terminal phosphate group
having Formula IV:
##STR00004##
wherein each X and Y is independently O, S, N, alkyl, substituted
alkyl, or alkylhalo; wherein each Z and W is independently O, S, N,
alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or
alkylhalo; and wherein W, X, Y and Z are not all O.
[0065] In one embodiment, the invention features a siNA molecule
having a 5'-terminal phosphate group having Formula IV on the
target-complementary strand, for example, a strand complementary to
a target RNA, wherein the siNA molecule comprises an all RNA siNA
molecule. In another embodiment, the invention features a siNA
molecule having a 5'-terminal phosphate group having Formula IV on
the target-complementary strand wherein the siNA molecule also
comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide
3'-terminal nucleotide overhangs having about 1 to about 4 (e.g.,
about 1, 2, 3, or 4) deoxyribonucleotides on the 3'-end of one or
both strands. In another embodiment, a 5'-terminal phosphate group
having Formula IV is present on the target-complementary strand of
a siNA molecule of the invention, for example a siNA molecule
having chemical modifications having any of Formulae I-VII.
[0066] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against a MAP kinase
inside a cell or reconstituted in vitro system, wherein the
chemical modification comprises one or more phosphorothioate
internucleotide linkages. For example, in a non-limiting example,
the invention features a chemically-modified short interfering
nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more
phosphorothioate internucleotide linkages in one siNA strand. In
yet another embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA)
individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more
phosphorothioate internucleotide linkages in both siNA strands. The
phosphorothioate internucleotide linkages can be present in one or
both oligonucleotide strands of the siNA duplex, for example in the
sense strand, the antisense strand, or both strands. The siNA
molecules of the invention can comprise one or more
phosphorothioate internucleotide linkages at the 3'-end, the
5'-end, or both of the 3'- and 5'-ends of the sense strand, the
antisense strand, or both strands. For example, an exemplary siNA
molecule of the invention can comprise about 1 to about 5 or more
(e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate
internucleotide linkages at the 5'-end of the sense strand, the
antisense strand, or both strands. In another non-limiting example,
an exemplary siNA molecule of the invention can comprise one or
more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
pyrimidine phosphorothioate internucleotide linkages in the sense
strand, the antisense strand, or both strands. In yet another
non-limiting example, an exemplary siNA molecule of the invention
can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more) purine phosphorothioate internucleotide linkages in
the sense strand, the antisense strand, or both strands.
[0067] In one embodiment, the invention features a siNA molecule,
wherein the sense strand comprises one or more, for example, about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl,
2'-deoxy-2'-fluoro, and/or about one or more (e.g., about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides,
and optionally a terminal cap molecule at the 3'-end, the 5'-end,
or both of the 3'- and 5'-ends of the sense strand; and wherein the
antisense strand comprises about 1 to about 10 or more,
specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
phosphorothioate internucleotide linkages, and/or one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy,
2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified
nucleotides, and optionally a terminal cap molecule at the 3'-end,
the 5'-end, or both of the 3'- and 5'-ends of the antisense strand.
In another embodiment, one or more, for example about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense
and/or antisense siNA strand are chemically-modified with 2'-deoxy,
2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without
one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more, phosphorothioate internucleotide linkages and/or a terminal
cap molecule at the 3'-end, the 5'-end, or both of the 3'- and
5'-ends, being present in the same or different strand.
[0068] In another embodiment, the invention features a siNA
molecule, wherein the sense strand comprises about 1 to about 5,
specifically about 1, 2, 3, 4, or 5 phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3,
4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or
one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base
modified nucleotides, and optionally a terminal cap molecule at the
3-end, the 5'-end, or both of the 3'- and 5'-ends of the sense
strand; and wherein the antisense strand comprises about 1 to about
5 or more, specifically about 1, 2, 3, 4, 5, or more
phosphorothioate internucleotide linkages, and/or one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy,
2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified
nucleotides, and optionally a terminal cap molecule at the 3'-end,
the 5'-end, or both of the 3'- and 5'-ends of the antisense strand.
In another embodiment, one or more, for example about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense
and/or antisense siNA strand are chemically-modified with 2'-deoxy,
2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without
about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or
more phosphorothioate internucleotide linkages and/or a terminal
cap molecule at the 3'-end, the 5'-end, or both of the 3'- and
5'-ends, being present in the same or different strand.
[0069] In one embodiment, the invention features a siNA molecule,
wherein the sense strand comprises one or more, for example, about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate
internucleotide linkages, and/or about one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl,
2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 or more) universal base modified nucleotides, and
optionally a terminal cap molecule at the 3'-end, the 5'-end, or
both of the 3'- and 5'-ends of the sense strand; and wherein the
antisense strand comprises about 1 to about 10 or more,
specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
phosphorothioate internucleotide linkages, and/or one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy,
2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified
nucleotides, and optionally a terminal cap molecule at the 3'-end,
the 5'-end, or both of the 3'- and 5'-ends of the antisense strand.
In another embodiment, one or more, for example about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense
and/or antisense siNA strand are chemically-modified with 2'-deoxy,
2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without
one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or more
phosphorothioate internucleotide linkages and/or a terminal cap
molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends,
being present in the same or different strand.
[0070] In another embodiment, the invention features a siNA
molecule, wherein the sense strand comprises about 1 to about 5 or
more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl,
2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 or more) universal base modified nucleotides, and
optionally a terminal cap molecule at the 3'-end, the 5'-end, or
both of the 3'- and 5'-ends of the sense strand; and wherein the
antisense strand comprises about 1 to about 5 or more, specifically
about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide
linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or
one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)
universal base modified nucleotides, and optionally a terminal cap
molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends
of the antisense strand. In another embodiment, one or more, for
example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine
nucleotides of the sense and/or antisense siNA strand are
chemically-modified with 2'-deoxy, 2'-O-methyl and/or
2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 5,
for example about 1, 2, 3, 4, 5 or more phosphorothioate
internucleotide linkages and/or a terminal cap molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends, being present
in the same or different strand.
[0071] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
having about 1 to about 5, specifically about 1, 2, 3, 4, 5 or more
phosphorothioate internucleotide linkages in each strand of the
siNA molecule.
[0072] In another embodiment, the invention features a siNA
molecule comprising 2'-5' internucleotide linkages. The 2'-5'
internucleotide linkage(s) can be at the 3'-end, the 5'-end, or
both of the 3'- and 5'-ends of one or both siNA sequence strands.
In addition, the 2'-5' internucleotide linkage(s) can be present at
various other positions within one or both siNA sequence strands,
for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including
every internucleotide linkage of a pyrimidine nucleotide in one or
both strands of the siNA molecule can comprise a 2'-5'
internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more including every internucleotide linkage of a purine nucleotide
in one or both strands of the siNA molecule can comprise a 2'-5'
internucleotide linkage.
[0073] In another embodiment, a chemically-modified siNA molecule
of the invention comprises a duplex having two strands, one or both
of which can be chemically-modified, wherein each strand is about
18 to about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or
27) nucleotides in length, wherein the duplex has about 18 to about
23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein
the chemical modification comprises a structure having any of
Formulae I-VII. For example, an exemplary chemically-modified siNA
molecule of the invention comprises a duplex having two strands,
one or both of which can be chemically-modified with a chemical
modification having any of Formulae I-VII or any combination
thereof, wherein each strand consists of about 21 nucleotides, each
having a 2-nucleotide 3'-terminal nucleotide overhang, and wherein
the duplex has about 19 base pairs. In another embodiment, a siNA
molecule of the invention comprises a single stranded hairpin
structure, wherein the siNA is about 36 to about 70 (e.g., about
36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having
about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base
pairs, and wherein the siNA can include a chemical modification
comprising a structure having any of Formulae I-VII or any
combination thereof. For example, an exemplary chemically-modified
siNA molecule of the invention comprises a linear oligonucleotide
having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47,
48, 49, or 50) nucleotides that is chemically-modified with a
chemical modification having any of Formulae I-VII or any
combination thereof, wherein the linear oligonucleotide forms a
hairpin structure having about 19 base pairs and a 2-nucleotide
3'-terminal nucleotide overhang. In another embodiment, a linear
hairpin siNA molecule of the invention contains a stem loop motif,
wherein the loop portion of the siNA molecule is biodegradable. For
example, a linear hairpin siNA molecule of the invention is
designed such that degradation of the loop portion of the siNA
molecule in vivo can generate a double-stranded siNA molecule with
3'-terminal overhangs, such as 3'-terminal nucleotide overhangs
comprising about 2 nucleotides.
[0074] In another embodiment, a siNA molecule of the invention
comprises a circular nucleic acid molecule, wherein the siNA is
about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or
70) nucleotides in length having about 18 to about 23 (e.g., about
18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can
include a chemical modification, which comprises a structure having
any of Formulae I-VII or any combination thereof. For example, an
exemplary chemically-modified siNA molecule of the invention
comprises a circular oligonucleotide having about 42 to about 50
(e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides
that is chemically-modified with a chemical modification having any
of Formulae I-VII or any combination thereof, wherein the circular
oligonucleotide forms a dumbbell shaped structure having about 19
base pairs and 2 loops.
[0075] In another embodiment, a circular siNA molecule of the
invention contains two loop motifs, wherein one or both loop
portions of the siNA molecule is biodegradable. For example, a
circular siNA molecule of the invention is designed such that
degradation of the loop portions of the siNA molecule in vivo can
generate a double-stranded siNA molecule with 3'-terminal
overhangs, such as 3'-terminal nucleotide overhangs comprising
about 2 nucleotides.
[0076] In one embodiment, a siNA molecule of the invention
comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) abasic moiety, for example a compound having Formula
V:
##STR00005##
wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is
independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,
F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl,
O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH,
O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl,
ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2,
O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted
silyl, or group having Formula I; R9 is O, S, CH2, S.dbd.O, CHF, or
CF2.
[0077] In one embodiment, a siNA molecule of the invention
comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) inverted abasic moiety, for example a compound having
Formula VI:
##STR00006##
wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is
independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,
F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl,
O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH,
O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl,
ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2,
O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted
silyl, or group having Formula I; R9 is O, S, CH2, S.dbd.O, CHF, or
CF2, and either R2, R3, R8 or R13 serve as points of attachment to
the siNA molecule of the invention.
[0078] In another embodiment, a siNA molecule of the invention
comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) substituted polyalkyl moieties, for example a compound
having Formula VII:
##STR00007##
wherein each n is independently an integer from 1 to 12, each R1,
R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl
or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl,
N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl,
alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or a group having Formula I,
and R1, R2 or R3 serves as points of attachment to the siNA
molecule of the invention.
[0079] In another embodiment, the invention features a compound
having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups,
n=1, and R3 comprises 0 and is the point of attachment to the
3'-end, the 5'-end, or both of the 3' and 5'-ends of one or both
strands of a double-stranded siNA molecule of the invention or to a
single-stranded siNA molecule of the invention. This modification
is referred to herein as "glyceryl" (for example modification 6 in
FIG. 10).
[0080] In another embodiment, a moiety having any of Formula V, VI
or VII of the invention is at the 3'-end, the 5'-end, or both of
the 3' and 5'-ends of a siNA molecule of the invention. For
example, a moiety having Formula V, VI or VII can be present at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense
strand, the sense strand, or both antisense and sense strands of
the siNA molecule. In addition, a moiety having Formula VII can be
present at the 3'-end or the 5'-end of a hairpin siNA molecule as
described herein.
[0081] In another embodiment, a siNA molecule of the invention
comprises an abasic residue having Formula V or VI, wherein the
abasic residue having Formula VI or VI is connected to the siNA
construct in a 3'-3',3'-2',2'-3', or 5'-5' configuration, such as
at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of one or
both siNA strands.
[0082] In one embodiment, a siNA molecule of the invention
comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) locked nucleic acid (LNA) nucleotides, for example at the
5'-end, the 3'-end, both of the 5' and 3'-ends, or any combination
thereof, of the siNA molecule.
[0083] In another embodiment, a siNA molecule of the invention
comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) acyclic nucleotides, for example at the 5'-end, the
3'-end, both of the 5' and 3'-ends, or any combination thereof, of
the siNA molecule.
[0084] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention, wherein the chemically-modified siNA comprises a
sense region, where any (e.g., one or more or all) pyrimidine
nucleotides present in the sense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all)
purine nucleotides present in the sense region are 2'-deoxy purine
nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy
purine nucleotides or alternately a plurality of purine nucleotides
are 2'-deoxy purine nucleotides).
[0085] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention, wherein the chemically-modified siNA comprises a
sense region, where any (e.g., one or more or all) pyrimidine
nucleotides present in the sense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all)
purine nucleotides present in the sense region are 2'-deoxy purine
nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy
purine nucleotides or alternately a plurality of purine nucleotides
are 2'-deoxy purine nucleotides), wherein any nucleotides
comprising a 3'-terminal nucleotide overhang that are present in
said sense region are 2'-deoxy nucleotides.
[0086] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention, wherein the chemically-modified siNA comprises a
sense region, where any (e.g., one or more or all) pyrimidine
nucleotides present in the sense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all)
purine nucleotides present in the sense region are 2'-O-methyl
purine nucleotides (e.g., wherein all purine nucleotides are
2'-O-methyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-O-methyl purine nucleotides).
[0087] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention, wherein the chemically-modified siNA comprises a
sense region, where any (e.g., one or more or all) pyrimidine
nucleotides present in the sense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all)
purine nucleotides present in the sense region are 2'-O-methyl
purine nucleotides (e.g., wherein all purine nucleotides are
2'-O-methyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-O-methyl purine nucleotides), wherein any
nucleotides comprising a 3'-terminal nucleotide overhang that are
present in said sense region are 2'-deoxy nucleotides.
[0088] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention, wherein the chemically-modified siNA comprises an
antisense region, where any (e.g., one or more or all) pyrimidine
nucleotides present in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any (e.g., one or more or all)
purine nucleotides present in the antisense region are 2'-O-methyl
purine nucleotides (e.g., wherein all purine nucleotides are
2'-O-methyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-O-methyl purine nucleotides).
[0089] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention, wherein the chemically-modified siNA comprises an
antisense region, where any (e.g., one or more or all) pyrimidine
nucleotides present in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any (e.g., one or more or all)
purine nucleotides present in the antisense region are 2'-O-methyl
purine nucleotides (e.g., wherein all purine nucleotides are
2'-O-methyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-O-methyl purine nucleotides), wherein any
nucleotides comprising a 3'-terminal nucleotide overhang that are
present in said antisense region are 2'-deoxy nucleotides.
[0090] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention, wherein the chemically-modified siNA comprises an
antisense region, where any (e.g., one or more or all) pyrimidine
nucleotides present in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all)
purine nucleotides present in the antisense region are 2'-deoxy
purine nucleotides (e.g., wherein all purine nucleotides are
2'-deoxy purine nucleotides or alternately a plurality of purine
nucleotides are 2'-deoxy purine nucleotides).
[0091] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention, wherein the chemically-modified siNA comprises an
antisense region, where any (e.g., one or more or all) pyrimidine
nucleotides present in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all)
purine nucleotides present in the antisense region are 2'-O-methyl
purine nucleotides (e.g., wherein all purine nucleotides are
2'-O-methyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-O-methyl purine nucleotides).
[0092] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention capable of mediating RNA interference (RNAi)
against a MAP kinase inside a cell or reconstituted in vitro
system, wherein the chemically-modified siNA comprises a sense
region, where one or more pyrimidine nucleotides present in the
sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g.,
wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and
where one or more purine nucleotides present in the sense region
are 2'-deoxy purine nucleotides (e.g., wherein all purine
nucleotides are 2'-deoxy purine nucleotides or alternately a
plurality of purine nucleotides are 2'-deoxy purine nucleotides),
and inverted deoxy abasic modifications that are optionally present
at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the
sense region, the sense region optionally further comprising a
3'-terminal overhang having about 1 to about 4 (e.g., about 1, 2,
3, or 4) 2'-deoxyribonucleotides; and wherein the
chemically-modified short interfering nucleic acid molecule
comprises an antisense region, where one or more pyrimidine
nucleotides present in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein one or more purine nucleotides
present in the antisense region are 2'-O-methyl purine nucleotides
(e.g., wherein all purine nucleotides are 2'-O-methyl purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl purine nucleotides), and a terminal cap modification,
such as any modification described herein or shown in FIG. 10, that
is optionally present at the 3'-end, the 5'-end, or both of the 3'
and 5'-ends of the antisense sequence, the antisense region
optionally further comprising a 3'-terminal nucleotide overhang
having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxynucleotides, wherein the overhang nucleotides can further
comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate
internucleotide linkages. Non-limiting examples of these
chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III
and IV herein.
[0093] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention capable of mediating RNA interference (RNAi)
against a MAP kinase inside a cell or reconstituted in vitro
system, wherein the chemically-modified siNA comprises a sense
region, where one or more pyrimidine nucleotides present in the
sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g.,
wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and
where one or more purine nucleotides present in the sense region
are 2'-O-methyl purine nucleotides (e.g., wherein all purine
nucleotides are 2'-O-methyl purine nucleotides or alternately a
plurality of purine nucleotides are 2'-O-methyl purine
nucleotides), and inverted deoxy abasic modifications that are
optionally present at the 3'-end, the 5'-end, or both of the 3' and
5'-ends of the sense region, the sense region optionally further
comprising a 3'-terminal overhang having about 1 to about 4 (e.g.,
about 1, 2, 3, or 4) 2'-deoxyribonucleotides; and wherein the
chemically-modified short interfering nucleic acid molecule
comprises an antisense region, where one or more pyrimidine
nucleotides present in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein one or more purine nucleotides
present in the antisense region are 2'-O-methyl purine nucleotides
(e.g., wherein all purine nucleotides are 2'-O-methyl purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl purine nucleotides), and a terminal cap modification,
such as any modification described herein or shown in FIG. 10, that
is optionally present at the 3'-end, the 5'-end, or both of the 3'
and 5'-ends of the antisense sequence, the antisense region
optionally further comprising a 3'-terminal nucleotide overhang
having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxynucleotides, wherein the overhang nucleotides can further
comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate
internucleotide linkages. Non-limiting examples of these
chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III
and IV herein.
[0094] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention capable of mediating RNA interference (RNAi)
against a MAP kinase inside a cell or reconstituted in vitro
system, wherein the siNA comprises a sense region, where one or
more pyrimidine nucleotides present in the sense region are
2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides or alternately a plurality of pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and where one or
more purine nucleotides present in the sense region are purine
ribonucleotides (e.g., wherein all purine nucleotides are purine
ribonucleotides or alternately a plurality of purine nucleotides
are purine ribonucleotides), and inverted deoxy abasic
modifications that are optionally present at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of the sense region, the
sense region optionally further comprising a 3'-terminal overhang
having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxyribonucleotides; and wherein the siNA comprises an
antisense region, where one or more pyrimidine nucleotides present
in the antisense region are 2'-deoxy-2'-fluoro pyrimidine
nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present
in the antisense region are 2'-O-methyl purine nucleotides (e.g.,
wherein all purine nucleotides are 2'-O-methyl purine nucleotides
or alternately a plurality of purine nucleotides are 2'-O-methyl
purine nucleotides), and a terminal cap modification, such as any
modification described herein or shown in FIG. 10, that is
optionally present at the 3'-end, the 5'-end, or both of the 3' and
5'-ends of the antisense sequence, the antisense region optionally
further comprising a 3'-terminal nucleotide overhang having about 1
to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxynucleotides, wherein
the overhang nucleotides can further comprise one or more (e.g., 1,
2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting
examples of these chemically-modified siNAs are shown in FIGS. 4
and 5 and Tables III and IV herein.
[0095] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention capable of mediating RNA interference (RNAi)
against a MAP kinase inside a cell or reconstituted in vitro
system, wherein the chemically-modified siNA comprises a sense
region, where one or more pyrimidine nucleotides present in the
sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g.,
wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and for
example where one or more purine nucleotides present in the sense
region are selected from the group consisting of 2'-deoxy
nucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyethyl
nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides (e.g.,
wherein all purine nucleotides are selected from the group
consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA)
nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and
2'-O-methyl nucleotides or alternately a plurality of purine
nucleotides are selected from the group consisting of 2'-deoxy
nucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyethyl
nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides), and
wherein inverted deoxy abasic modifications are optionally present
at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the
sense region, the sense region optionally further comprising a
3'-terminal overhang having about 1 to about 4 (e.g., about 1, 2,
3, or 4) 2'-deoxyribonucleotides; and wherein the
chemically-modified short interfering nucleic acid molecule
comprises an antisense region, where one or more pyrimidine
nucleotides present in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein one or more purine nucleotides
present in the antisense region are selected from the group
consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA)
nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and
2'-O-methyl nucleotides (e.g., wherein all purine nucleotides are
selected from the group consisting of 2'-deoxy nucleotides, locked
nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides,
4'-thionucleotides, and 2'-O-methyl nucleotides or alternately a
plurality of purine nucleotides are selected from the group
consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA)
nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and
2'-O-methyl nucleotides), and a terminal cap modification, such as
any modification described herein or shown in FIG. 10, that is
optionally present at the 3'-end, the 5'-end, or both of the 3' and
5'-ends of the antisense sequence, the antisense region optionally
further comprising a 3'-terminal nucleotide overhang having about 1
to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxynucleotides, wherein
the overhang nucleotides can further comprise one or more (e.g., 1,
2, 3, or 4) phosphorothioate internucleotide linkages.
[0096] In another embodiment, any modified nucleotides present in
the siNA molecules of the invention, preferably in the antisense
strand of the siNA molecules of the invention, but also optionally
in the sense and/or both antisense and sense strands, comprise
modified nucleotides having properties or characteristics similar
to naturally occurring ribonucleotides. For example, the invention
features siNA molecules including modified nucleotides having a
Northern conformation (e.g., Northern pseudorotation cycle, see for
example Saenger, Principles of Nucleic Acid Structure,
Springer-Verlag ed., 1984). As such, chemically modified
nucleotides present in the siNA molecules of the invention,
preferably in the antisense strand of the siNA molecules of the
invention, but also optionally in the sense and/or both antisense
and sense strands, are resistant to nuclease degradation while at
the same time maintaining the capacity to mediate RNAi.
Non-limiting examples of nucleotides having a northern
configuration include locked nucleic acid (LNA) nucleotides (e.g.,
2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides);
2'-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl,
2'-deoxy-2'-fluoro nucleotides, 2'-deoxy-2'-chloro nucleotides,
2'-azido nucleotides, and 2'-O-methyl nucleotides.
[0097] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid molecule (siNA)
capable of mediating RNA interference (RNAi) against a MAP kinase
inside a cell or reconstituted in vitro system, wherein the
chemical modification comprises a conjugate covalently attached to
the chemically-modified siNA molecule. In another embodiment, the
conjugate is covalently attached to the chemically-modified siNA
molecule via a biodegradable linker. In one embodiment, the
conjugate molecule is attached at the 3'-end of either the sense
strand, the antisense strand, or both strands of the
chemically-modified siNA molecule. In another embodiment, the
conjugate molecule is attached at the 5'-end of either the sense
strand, the antisense strand, or both strands of the
chemically-modified siNA molecule. In yet another embodiment, the
conjugate molecule is attached both the 3'-end and 5'-end of either
the sense strand, the antisense strand, or both strands of the
chemically-modified siNA molecule, or any combination thereof. In
one embodiment, a conjugate molecule of the invention comprises a
molecule that facilitates delivery of a chemically-modified siNA
molecule into a biological system, such as a cell. In another
embodiment, the conjugate molecule attached to the
chemically-modified siNA molecule is a polyethylene glycol, human
serum albumin, or a ligand for a cellular receptor that can mediate
cellular uptake. Examples of specific conjugate molecules
contemplated by the instant invention that can be attached to
chemically-modified siNA molecules are described in Vargeese et
al., U.S. Ser. No. 10/201,394, incorporated by reference herein.
The type of conjugates used and the extent of conjugation of siNA
molecules of the invention can be evaluated for improved
pharmacokinetic profiles, bioavailability, and/or stability of siNA
constructs while at the same time maintaining the ability of the
siNA to mediate RNAi activity. As such, one skilled in the art can
screen siNA constructs that are modified with various conjugates to
determine whether the siNA conjugate complex possesses improved
properties while maintaining the ability to mediate RNAi, for
example, in animal models as are generally known in the art.
[0098] In one embodiment, the invention features a short
interfering nucleic acid (siNA) molecule of the invention, wherein
the siNA further comprises a nucleotide, non-nucleotide, or mixed
nucleotide/non-nucleotide linker that joins the sense region of the
siNA to the antisense region of the siNA. In one embodiment, a
nucleotide linker of the invention can be a linker of .gtoreq.2
nucleotides in length, for example 3, 4, 5, 6, 7, 8, 9, or 10
nucleotides in length. In another embodiment, the nucleotide linker
can be a nucleic acid aptamer. By "aptamer" or "nucleic acid
aptamer" as used herein is meant a nucleic acid molecule that binds
specifically to a target molecule wherein the nucleic acid molecule
has sequence that comprises a sequence recognized by the target
molecule in its natural setting. Alternately, an aptamer can be a
nucleic acid molecule that binds to a target molecule where the
target molecule does not naturally bind to a nucleic acid. The
target molecule can be any molecule of interest. For example, the
aptamer can be used to bind to a ligand-binding domain of a
protein, thereby preventing interaction of the naturally occurring
ligand with the protein. This is a non-limiting example and those
in the art will recognize that other embodiments can be readily
generated using techniques generally known in the art. (See, for
example, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and
Gold, 2000, J. Biotechnol., 74, 5; Sunday, 2000, Curr. Opin. Mol.
Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and
Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical
Chemistry, 45, 1628.)
[0099] In yet another embodiment, a non-nucleotide linker of the
invention comprises abasic nucleotide, polyether, polyamine,
polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other
polymeric compounds (e.g. polyethylene glycols such as those having
between 2 and 100 ethylene glycol units). Specific examples include
those described by Seela and Kaiser, Nucleic Acids Res. 1990,
18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz,
J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am.
Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993,
21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic
Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &
Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993,
34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al.,
International Publication No. WO 89/02439; Usman et al.,
International Publication No. WO 95/06731; Dudycz et al.,
International Publication No. WO 95/11910 and Ferentz and Verdine,
J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by
reference herein. A "non-nucleotide" further means any group or
compound that can be incorporated into a nucleic acid chain in the
place of one or more nucleotide units, including either sugar
and/or phosphate substitutions, and allows the remaining bases to
exhibit their enzymatic activity. The group or compound can be
abasic in that it does not contain a commonly recognized nucleotide
base, such as adenosine, guanine, cytosine, uracil or thymine, for
example at the C1 position of the sugar.
[0100] In one embodiment, the invention features a short
interfering nucleic acid (siNA) molecule capable of mediating RNA
interference (RNAi) inside a cell or reconstituted in vitro system,
wherein one or both strands of the siNA molecule that are assembled
from two separate oligonucleotides do not comprise any
ribonucleotides. For example, a siNA molecule can be assembled from
a single oligonucleotide where the sense and antisense regions of
the siNA comprise separate oligonucleotides that do not have any
ribonucleotides (e.g., nucleotides having a 2'-OH group) present in
the oligonucleotides. In another example, a siNA molecule can be
assembled from a single oligonucleotide where the sense and
antisense regions of the siNA are linked or circularized by a
nucleotide or non-nucleotide linker as described herein, wherein
the oligonucleotide does not have any ribonucleotides (e.g.,
nucleotides having a 2'-OH group) present in the oligonucleotide.
Applicant has surprisingly found that the presence of
ribonucleotides (e.g., nucleotides having a 2'-hydroxyl group)
within the siNA molecule is not required or essential to support
RNAi activity. As such, in one embodiment, all positions within the
siNA can include chemically modified nucleotides and/or
non-nucleotides such as nucleotides and or non-nucleotides having
Formula I, II, III, IV, V, VI, or VII or any combination thereof to
the extent that the ability of the siNA molecule to support RNAi
activity in a cell is maintained.
[0101] In one embodiment, a siNA molecule of the invention is a
single stranded siNA molecule that mediates RNAi activity in a cell
or reconstituted in vitro system, wherein the siNA molecule
comprises a single stranded polynucleotide having complementarity
to a target nucleic acid sequence. In another embodiment, the
single stranded siNA molecule of the invention comprises a
5'-terminal phosphate group. In another embodiment, the single
stranded siNA molecule of the invention comprises a 5'-terminal
phosphate group and a 3'-terminal phosphate group (e.g., a
2',3'-cyclic phosphate). In another embodiment, the single stranded
siNA molecule of the invention comprises about 19 to about 29
nucleotides. In yet another embodiment, the single stranded siNA
molecule of the invention comprises one or more chemically modified
nucleotides or non-nucleotides described herein. For example, all
the positions within the siNA molecule can include
chemically-modified nucleotides such as nucleotides having any of
Formulae I-VII, or any combination thereof to the extent that the
ability of the siNA molecule to support RNAi activity in a cell is
maintained.
[0102] In one embodiment, a siNA molecule of the invention is a
single stranded siNA molecule that mediates RNAi activity in a cell
or reconstituted in vitro system, wherein the siNA molecule
comprises a single stranded polynucleotide having complementarity
to a target nucleic acid sequence, and wherein one or more
pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present
in the antisense region are 2'-O-methyl purine nucleotides (e.g.,
wherein all purine nucleotides are 2'-O-methyl purine nucleotides
or alternately a plurality of purine nucleotides are 2'-O-methyl
purine nucleotides), and a terminal cap modification, such as any
modification described herein or shown in FIG. 10, that is
optionally present at the 3'-end, the 5'-end, or both of the 3' and
5'-ends of the antisense sequence, the siNA optionally further
comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal
2'-deoxynucleotides at the 3'-end of the siNA molecule, wherein the
terminal nucleotides can further comprise one or more (e.g., 1, 2,
3, or 4) phosphorothioate internucleotide linkages, and wherein the
siNA optionally further comprises a terminal phosphate group, such
as a 5'-terminal phosphate group.
[0103] In one embodiment, a siNA molecule of the invention is a
single stranded siNA molecule that mediates RNAi activity in a cell
or reconstituted in vitro system, wherein the siNA molecule
comprises a single stranded polynucleotide having complementarity
to a target nucleic acid sequence, and wherein one or more
pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present
in the siNA are 2'-O-methyl purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl purine nucleotides or
alternately a plurality of purine nucleotides are 2'-O-methyl
purine nucleotides), and a terminal cap modification, such as any
modification described herein or shown in FIG. 10, that is
optionally present at the 3'-end, the 5'-end, or both of the 3' and
5'-ends of the antisense sequence, the siNA optionally further
comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal
2'-deoxynucleotides at the 3'-end of the siNA molecule, wherein the
terminal nucleotides can further comprise one or more (e.g., 1, 2,
3, or 4) phosphorothioate internucleotide linkages, and wherein the
siNA optionally further comprises a terminal phosphate group, such
as a 5'-terminal phosphate group.
[0104] In one embodiment, a siNA molecule of the invention is a
single stranded siNA molecule that mediates RNAi activity in a cell
or reconstituted in vitro system, wherein the siNA molecule
comprises a single stranded polynucleotide having complementarity
to a target nucleic acid sequence, and wherein one or more
pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present
in the siNA are 2'-deoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-deoxy purine nucleotides or alternately a
plurality of purine nucleotides are 2'-deoxy purine nucleotides),
and a terminal cap modification, such as any modification described
herein or shown in FIG. 10, that is optionally present at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense
sequence, the siNA optionally further comprising about 1 to about 4
(e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the
3'-end of the siNA molecule, wherein the terminal nucleotides can
further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate
internucleotide linkages, and wherein the siNA optionally further
comprises a terminal phosphate group, such as a 5'-terminal
phosphate group.
[0105] In one embodiment, a siNA molecule of the invention is a
single stranded siNA molecule that mediates RNAi activity in a cell
or reconstituted in vitro system, wherein the siNA molecule
comprises a single stranded polynucleotide having complementarity
to a target nucleic acid sequence, and wherein one or more
pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present
in the siNA are locked nucleic acid (LNA) nucleotides (e.g.,
wherein all purine nucleotides are LNA nucleotides or alternately a
plurality of purine nucleotides are LNA nucleotides), and a
terminal cap modification, such as any modification described
herein or shown in FIG. 10, that is optionally present at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense
sequence, the siNA optionally further comprising about 1 to about 4
(e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the
3'-end of the siNA molecule, wherein the terminal nucleotides can
further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate
internucleotide linkages, and wherein the siNA optionally further
comprises a terminal phosphate group, such as a 5'-terminal
phosphate group.
[0106] In one embodiment, a siNA molecule of the invention is a
single stranded siNA molecule that mediates RNAi activity in a cell
or reconstituted in vitro system, wherein the siNA molecule
comprises a single stranded polynucleotide having complementarity
to a target nucleic acid sequence, and wherein one or more
pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present
in the siNA are 2'-methoxyethyl purine nucleotides (e.g., wherein
all purine nucleotides are 2'-methoxyethyl purine nucleotides or
alternately a plurality of purine nucleotides are 2'-methoxyethyl
purine nucleotides), and a terminal cap modification, such as any
modification described herein or shown in FIG. 10, that is
optionally present at the 3'-end, the 5'-end, or both of the 3' and
5'-ends of the antisense sequence, the siNA optionally further
comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal
2'-deoxynucleotides at the 3'-end of the siNA molecule, wherein the
terminal nucleotides can further comprise one or more (e.g., 1, 2,
3, or 4) phosphorothioate internucleotide linkages, and wherein the
siNA optionally further comprises a terminal phosphate group, such
as a 5'-terminal phosphate group.
[0107] In another embodiment, any modified nucleotides present in
the single stranded siNA molecules of the invention comprise
modified nucleotides having properties or characteristics similar
to naturally occurring ribonucleotides. For example, the invention
features siNA molecules including modified nucleotides having a
Northern conformation (e.g., Northern pseudorotation cycle, see for
example Saenger, Principles of Nucleic Acid Structure,
Springer-Verlag ed., 1984). As such, chemically modified
nucleotides present in the single stranded siNA molecules of the
invention are preferably resistant to nuclease degradation while at
the same time maintaining the capacity to mediate RNAi.
[0108] In one embodiment, the invention features a method for
modulating the expression of a MAP kinase gene within a cell
comprising: (a) synthesizing a siNA molecule of the invention,
which can be chemically-modified, wherein one of the siNA strands
comprises a sequence complementary to RNA of the MAP kinase gene;
and (b) introducing the siNA molecule into a cell under conditions
suitable to modulate the expression of the MAP kinase gene in the
cell.
[0109] In one embodiment, the invention features a method for
modulating the expression of a MAP kinase gene within a cell
comprising: (a) synthesizing a siNA molecule of the invention,
which can be chemically-modified, wherein one of the siNA strands
comprises a sequence complementary to RNA of the MAP kinase gene
and wherein the sense strand sequence of the siNA comprises a
sequence identical to the sequence of the target RNA; and (b)
introducing the siNA molecule into a cell under conditions suitable
to modulate the expression of the MAP kinase gene in the cell.
[0110] In another embodiment, the invention features a method for
modulating the expression of more than one MAP kinase gene within a
cell comprising: (a) synthesizing siNA molecules of the invention,
which can be chemically-modified, wherein one of the siNA strands
comprises a sequence complementary to RNA of the MAP kinase genes;
and (b) introducing the siNA molecules into a cell under conditions
suitable to modulate the expression of the MAP kinase genes in the
cell.
[0111] In another embodiment, the invention features a method for
modulating the expression of more than one MAP kinase gene within a
cell comprising: (a) synthesizing a siNA molecule of the invention,
which can be chemically-modified, wherein one of the siNA strands
comprises a sequence complementary to RNA of the MAP kinase gene
and wherein the sense strand sequence of the siNA comprises a
sequence identical to the sequence of the target RNA; and (b)
introducing the siNA molecules into a cell under conditions
suitable to modulate the expression of the MAP kinase genes in the
cell.
[0112] In one embodiment, siNA molecules of the invention are used
as reagents in ex vivo applications. For example, siNA reagents are
introduced into tissue or cells that are transplanted into a
subject for therapeutic effect. The cells and/or tissue can be
derived from an organism or subject that later receives the
explant, or can be derived from another organism or subject prior
to transplantation. The siNA molecules can be used to modulate the
expression of one or more genes in the cells or tissue, such that
the cells or tissue obtain a desired phenotype or are able to
perform a function when transplanted in vivo. In one embodiment,
certain target cells from a patient are extracted. These extracted
cells are contacted with siNAs targeting a specific nucleotide
sequence within the cells under conditions suitable for uptake of
the siNAs by these cells (e.g. using delivery reagents such as
cationic lipids, liposomes and the like or using techniques such as
electroporation to facilitate the delivery of siNAs into cells).
The cells are then reintroduced back into the same patient or other
patients. In one embodiment, the invention features a method of
modulating the expression of a MAP kinase gene in a tissue explant
comprising: (a) synthesizing a siNA molecule of the invention,
which can be chemically-modified, wherein one of the siNA strands
comprises a sequence complementary to RNA of the MAP kinase gene;
and (b) introducing the siNA molecule into a cell of the tissue
explant derived from a particular organism under conditions
suitable to modulate the expression of the MAP kinase gene in the
tissue explant. In another embodiment, the method further comprises
introducing the tissue explant back into the organism the tissue
was derived from or into another organism under conditions suitable
to modulate the expression of the MAP kinase gene in that
organism.
[0113] In one embodiment, the invention features a method of
modulating the expression of a MAP kinase gene in a tissue explant
comprising: (a) synthesizing a siNA molecule of the invention,
which can be chemically-modified, wherein one of the siNA strands
comprises a sequence complementary to RNA of the MAP kinase gene
and wherein the sense strand sequence of the siNA comprises a
sequence identical to the sequence of the target RNA; and (b)
introducing the siNA molecule into a cell of the tissue explant
derived from a particular organism under conditions suitable to
modulate the expression of the MAP kinase gene in the tissue
explant. In another embodiment, the method further comprises
introducing the tissue explant back into the organism the tissue
was derived from or into another organism under conditions suitable
to modulate the expression of the MAP kinase gene in that
organism.
[0114] In another embodiment, the invention features a method of
modulating the expression of more than one MAP kinase gene in a
tissue explant comprising: (a) synthesizing siNA molecules of the
invention, which can be chemically-modified, wherein one of the
siNA strands comprises a sequence complementary to RNA of the MAP
kinase genes; and (b) introducing the siNA molecules into a cell of
the tissue explant derived from a particular organism under
conditions suitable to modulate the expression of the MAP kinase
genes in the tissue explant. In another embodiment, the method
further comprises introducing the tissue explant back into the
organism the tissue was derived from or into another organism under
conditions suitable to modulate the expression of the MAP kinase
genes in that organism.
[0115] In one embodiment, the invention features a method of
modulating the expression of a MAP kinase gene in an organism
comprising: (a) synthesizing a siNA molecule of the invention,
which can be chemically-modified, wherein one of the siNA strands
comprises a sequence complementary to RNA of the MAP kinase gene;
and (b) introducing the siNA molecule into the organism under
conditions suitable to modulate the expression of the MAP kinase
gene in the organism.
[0116] In another embodiment, the invention features a method of
modulating the expression of more than one MAP kinase gene in an
organism comprising: (a) synthesizing siNA molecules of the
invention, which can be chemically-modified, wherein one of the
siNA strands comprises a sequence complementary to RNA of the MAP
kinase genes; and (b) introducing the siNA molecules into the
organism under conditions suitable to modulate the expression of
the MAP kinase genes in the organism.
[0117] In one embodiment, the invention features a method for
modulating the expression of a MAP kinase gene within a cell
comprising: (a) synthesizing a siNA molecule of the invention,
which can be chemically-modified, wherein the siNA comprises a
single stranded sequence having complementarity to RNA of the MAP
kinase gene; and (b) introducing the siNA molecule into a cell
under conditions suitable to modulate the expression of the MAP
kinase gene in the cell.
[0118] In another embodiment, the invention features a method for
modulating the expression of more than one MAP kinase gene within a
cell comprising: (a) synthesizing siNA molecules of the invention,
which can be chemically-modified, wherein the siNA comprises a
single stranded sequence having complementarity to RNA of the MAP
kinase gene; and (b) contacting the siNA molecule with a cell in
vitro or in vivo under conditions suitable to modulate the
expression of the MAP kinase genes in the cell.
[0119] In one embodiment, the invention features a method of
modulating the expression of a MAP kinase gene in a tissue explant
comprising: (a) synthesizing a siNA molecule of the invention,
which can be chemically-modified, wherein the siNA comprises a
single stranded sequence having complementarity to RNA of the MAP
kinase gene; and (b) contacting the siNA molecule with a cell of
the tissue explant derived from a particular organism under
conditions suitable to modulate the expression of the MAP kinase
gene in the tissue explant. In another embodiment, the method
further comprises introducing the tissue explant back into the
organism the tissue was derived from or into another organism under
conditions suitable to modulate the expression of the MAP kinase
gene in that organism.
[0120] In another embodiment, the invention features a method of
modulating the expression of more than one MAP kinase gene in a
tissue explant comprising: (a) synthesizing siNA molecules of the
invention, which can be chemically-modified, wherein the siNA
comprises a single stranded sequence having complementarity to RNA
of the MAP kinase gene; and (b) introducing the siNA molecules into
a cell of the tissue explant derived from a particular organism
under conditions suitable to modulate the expression of the MAP
kinase genes in the tissue explant. In another embodiment, the
method further comprises introducing the tissue explant back into
the organism the tissue was derived from or into another organism
under conditions suitable to modulate the expression of the MAP
kinase genes in that organism.
[0121] In one embodiment, the invention features a method of
modulating the expression of a MAP kinase gene in an organism
comprising: (a) synthesizing a siNA molecule of the invention,
which can be chemically-modified, wherein the siNA comprises a
single stranded sequence having complementarity to RNA of the MAP
kinase gene; and (b) introducing the siNA molecule into the
organism under conditions suitable to modulate the expression of
the MAP kinase gene in the organism.
[0122] In another embodiment, the invention features a method of
modulating the expression of more than one MAP kinase gene in an
organism comprising: (a) synthesizing siNA molecules of the
invention, which can be chemically-modified, wherein the siNA
comprises a single stranded sequence having complementarity to RNA
of the MAP kinase gene; and (b) introducing the siNA molecules into
the organism under conditions suitable to modulate the expression
of the MAP kinase genes in the organism.
[0123] In one embodiment, the invention features a method of
modulating the expression of a MAP kinase gene in an organism
comprising contacting the organism with a siNA molecule of the
invention under conditions suitable to modulate the expression of
the MAP kinase gene in the organism.
[0124] In another embodiment, the invention features a method of
modulating the expression of more than one MAP kinase gene in an
organism comprising contacting the organism with one or more siNA
molecules of the invention under conditions suitable to modulate
the expression of the MAP kinase genes in the organism.
[0125] The siNA molecules of the invention can be designed to
down-regulate or inhibit target (MAP kinase) gene expression
through RNAi targeting of a variety of RNA molecules. In one
embodiment, the siNA molecules of the invention are used to target
various RNAs corresponding to a target gene. Non-limiting examples
of such RNAs include messenger RNA (mRNA), alternate RNA splice
variants of target gene(s), post-transcriptionally modified RNA of
target gene(s), pre-mRNA of target gene(s), and/or RNA templates.
If alternate splicing produces a family of transcripts that are
distinguished by usage of appropriate exons, the instant invention
can be used to inhibit gene expression through the appropriate
exons to specifically inhibit or to distinguish among the functions
of gene family members. For example, a protein that contains an
alternatively spliced transmembrane domain can be expressed in both
membrane bound and secreted forms. Use of the invention to target
the exon containing the transmembrane domain can be used to
determine the functional consequences of pharmaceutical targeting
of membrane bound as opposed to the secreted form of the protein.
Non-limiting examples of applications of the invention relating to
targeting these RNA molecules include therapeutic pharmaceutical
applications, pharmaceutical discovery applications, molecular
diagnostic and gene function applications, and gene mapping, for
example using single nucleotide polymorphism mapping with siNA
molecules of the invention. Such applications can be implemented
using known gene sequences or from partial sequences available from
an expressed sequence tag (EST).
[0126] In another embodiment, the siNA molecules of the invention
are used to target conserved sequences corresponding to a gene
family or gene families such as MAP kinase family genes. As such,
siNA molecules targeting multiple MAP kinase targets can provide
increased therapeutic effect. In addition, siNA can be used to
characterize pathways of gene function in a variety of
applications. For example, the present invention can be used to
inhibit the activity of target gene(s) in a pathway to determine
the function of uncharacterized gene(s) in gene function analysis,
mRNA function analysis, or translational analysis. The invention
can be used to determine potential target gene pathways involved in
various diseases and conditions toward pharmaceutical development.
The invention can be used to understand pathways of gene expression
involved in, for example, the progression and/or maintenance of
cancer.
[0127] In one embodiment, siNA molecule(s) and/or methods of the
invention are used to down-regulate the expression of gene(s) that
encode RNA referred to by Genbank Accession, for example MAP kinase
genes encoding RNA sequence(s) referred to herein by Genbank
Accession number, for example, Genbank Accession Nos. shown in
Table I.
[0128] In one embodiment, the invention features a method
comprising: (a) generating a library of siNA constructs having a
predetermined complexity; and (b) assaying the siNA constructs of
(a) above, under conditions suitable to determine RNAi target sites
within the target RNA sequence. In one embodiment, the siNA
molecules of (a) have strands of a fixed length, for example, about
23 nucleotides in length. In another embodiment, the siNA molecules
of (a) are of differing length, for example having strands of about
19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25)
nucleotides in length. In one embodiment, the assay can comprise a
reconstituted in vitro siNA assay as described herein. In another
embodiment, the assay can comprise a cell culture system in which
target RNA is expressed. In another embodiment, fragments of target
RNA are analyzed for detectable levels of cleavage, for example by
gel electrophoresis, northern blot analysis, or RNAse protection
assays, to determine the most suitable target site(s) within the
target RNA sequence. The target RNA sequence can be obtained as is
known in the art, for example, by cloning and/or transcription for
in vitro systems, and by cellular expression in in vivo
systems.
[0129] In one embodiment, the invention features a method
comprising: (a) generating a randomized library of siNA constructs
having a predetermined complexity, such as of 4N, where N
represents the number of base paired nucleotides in each of the
siNA construct strands (eg. for a siNA construct having 21
nucleotide sense and antisense strands with 19 base pairs, the
complexity would be 419); and (b) assaying the siNA constructs of
(a) above, under conditions suitable to determine RNAi target sites
within the target MAP kinase RNA sequence. In another embodiment,
the siNA molecules of (a) have strands of a fixed length, for
example about 23 nucleotides in length. In yet another embodiment,
the siNA molecules of (a) are of differing length, for example
having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22,
23, 24, or 25) nucleotides in length. In one embodiment, the assay
can comprise a reconstituted in vitro siNA assay as described in
Example 7 herein. In another embodiment, the assay can comprise a
cell culture system in which target RNA is expressed. In another
embodiment, fragments of MAP kinase RNA are analyzed for detectable
levels of cleavage, for example by gel electrophoresis, northern
blot analysis, or RNAse protection assays, to determine the most
suitable target site(s) within the target MAP kinase RNA sequence.
The target MAP kinase RNA sequence can be obtained as is known in
the art, for example, by cloning and/or transcription for in vitro
systems, and by cellular expression in in vivo systems.
[0130] In another embodiment, the invention features a method
comprising: (a) analyzing the sequence of a RNA target encoded by a
target gene; (b) synthesizing one or more sets of siNA molecules
having sequence complementary to one or more regions of the RNA of
(a); and (c) assaying the siNA molecules of (b) under conditions
suitable to determine RNAi targets within the target RNA sequence.
In one embodiment, the siNA molecules of (b) have strands of a
fixed length, for example about 23 nucleotides in length. In
another embodiment, the siNA molecules of (b) are of differing
length, for example having strands of about 19 to about 25 (e.g.,
about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one
embodiment, the assay can comprise a reconstituted in vitro siNA
assay as described herein. In another embodiment, the assay can
comprise a cell culture system in which target RNA is expressed.
Fragments of target RNA are analyzed for detectable levels of
cleavage, for example by gel electrophoresis, northern blot
analysis, or RNAse protection assays, to determine the most
suitable target site(s) within the target RNA sequence. The target
RNA sequence can be obtained as is known in the art, for example,
by cloning and/or transcription for in vitro systems, and by
expression in in vivo systems.
[0131] By "target site" is meant a sequence within a target RNA
that is "targeted" for cleavage mediated by a siNA construct which
contains sequences within its antisense region that are
complementary to the target sequence.
[0132] By "detectable level of cleavage" is meant cleavage of
target RNA (and formation of cleaved product RNAs) to an extent
sufficient to discern cleavage products above the background of
RNAs produced by random degradation of the target RNA. Production
of cleavage products from 1-5% of the target RNA is sufficient to
detect above the background for most methods of detection.
[0133] In one embodiment, the invention features a composition
comprising a siNA molecule of the invention, which can be
chemically-modified, in a pharmaceutically acceptable carrier or
diluent. In another embodiment, the invention features a
pharmaceutical composition comprising siNA molecules of the
invention, which can be chemically-modified, targeting one or more
genes in a pharmaceutically acceptable carrier or diluent. In
another embodiment, the invention features a method for diagnosing
a disease or condition in a subject comprising administering to the
subject a composition of the invention under conditions suitable
for the diagnosis of the disease or condition in the subject. In
another embodiment, the invention features a method for treating or
preventing a disease or condition in a subject, comprising
administering to the subject a composition of the invention under
conditions suitable for the treatment or prevention of the disease
or condition in the subject, alone or in conjunction with one or
more other therapeutic compounds. In yet another embodiment, the
invention features a method for reducing or preventing tissue
rejection in a subject comprising administering to the subject a
composition of the invention under conditions suitable for the
reduction or prevention of tissue rejection in the subject.
[0134] In another embodiment, the invention features a method for
validating a MAP kinase gene target comprising: (a) synthesizing a
siNA molecule of the invention, which can be chemically-modified,
wherein one of the siNA strands includes a sequence complementary
to RNA of a MAP kinase target gene; (b) introducing the siNA
molecule into a cell, tissue, or organism under conditions suitable
for modulating expression of the MAP kinase target gene in the
cell, tissue, or organism; and (c) determining the function of the
gene by assaying for any phenotypic change in the cell, tissue, or
organism.
[0135] In another embodiment, the invention features a method for
validating a MAP kinase target comprising: (a) synthesizing a siNA
molecule of the invention, which can be chemically-modified,
wherein one of the siNA strands includes a sequence complementary
to RNA of a MAP kinase target gene; (b) introducing the siNA
molecule into a biological system under conditions suitable for
modulating expression of the MAP kinase target gene in the
biological system; and (c) determining the function of the gene by
assaying for any phenotypic change in the biological system.
[0136] By "biological system" is meant, material, in a purified or
unpurified form, from biological sources, including but not limited
to human, animal, plant, insect, bacterial, viral or other sources,
wherein the system comprises the components required for RNAi
activity. The term "biological system" includes, for example, a
cell, tissue, or organism, or extract thereof. The term biological
system also includes reconstituted RNAi systems that can be used in
an in vitro setting.
[0137] By "phenotypic change" is meant any detectable change to a
cell that occurs in response to contact or treatment with a nucleic
acid molecule of the invention (e.g., siNA). Such detectable
changes include, but are not limited to, changes in shape, size,
proliferation, motility, protein expression or RNA expression or
other physical or chemical changes as can be assayed by methods
known in the art. The detectable change can also include expression
of reporter genes/molecules such as Green Florescent Protein (GFP)
or various tags that are used to identify an expressed protein or
any other cellular component that can be assayed.
[0138] In one embodiment, the invention features a kit containing a
siNA molecule of the invention, which can be chemically-modified,
that can be used to modulate the expression of a MAP kinase target
gene in a biological system, including, for example, in a cell,
tissue, or organism. In another embodiment, the invention features
a kit containing more than one siNA molecule of the invention,
which can be chemically-modified, that can be used to modulate the
expression of more than one MAP kinase target gene in a biological
system, including, for example, in a cell, tissue, or organism.
[0139] In one embodiment, the invention features a cell containing
one or more siNA molecules of the invention, which can be
chemically-modified. In another embodiment, the cell containing a
siNA molecule of the invention is a mammalian cell. In yet another
embodiment, the cell containing a siNA molecule of the invention is
a human cell.
[0140] In one embodiment, the synthesis of a siNA molecule of the
invention, which can be chemically-modified, comprises: (a)
synthesis of two complementary strands of the siNA molecule; (b)
annealing the two complementary strands together under conditions
suitable to obtain a double-stranded siNA molecule. In another
embodiment, synthesis of the two complementary strands of the siNA
molecule is by solid phase oligonucleotide synthesis. In yet
another embodiment, synthesis of the two complementary strands of
the siNA molecule is by solid phase tandem oligonucleotide
synthesis.
[0141] In one embodiment, the invention features a method for
synthesizing a siNA duplex molecule comprising: (a) synthesizing a
first oligonucleotide sequence strand of the siNA molecule, wherein
the first oligonucleotide sequence strand comprises a cleavable
linker molecule that can be used as a scaffold for the synthesis of
the second oligonucleotide sequence strand of the siNA; (b)
synthesizing the second oligonucleotide sequence strand of siNA on
the scaffold of the first oligonucleotide sequence strand, wherein
the second oligonucleotide sequence strand further comprises a
chemical moiety than can be used to purify the siNA duplex; (c)
cleaving the linker molecule of (a) under conditions suitable for
the two siNA oligonucleotide strands to hybridize and form a stable
duplex; and (d) purifying the siNA duplex utilizing the chemical
moiety of the second oligonucleotide sequence strand. In one
embodiment, cleavage of the linker molecule in (c) above takes
place during deprotection of the oligonucleotide, for example,
under hydrolysis conditions using an alkylamine base such as
methylamine. In one embodiment, the method of synthesis comprises
solid phase synthesis on a solid support such as controlled pore
glass (CPG) or polystyrene, wherein the first sequence of (a) is
synthesized on a cleavable linker, such as a succinyl linker, using
the solid support as a scaffold. The cleavable linker in (a) used
as a scaffold for synthesizing the second strand can comprise
similar reactivity as the solid support derivatized linker, such
that cleavage of the solid support derivatized linker and the
cleavable linker of (a) takes place concomitantly. In another
embodiment, the chemical moiety of (b) that can be used to isolate
the attached oligonucleotide sequence comprises a trityl group, for
example a dimethoxytrityl group, which can be employed in a
trityl-on synthesis strategy as described herein. In yet another
embodiment, the chemical moiety, such as a dimethoxytrityl group,
is removed during purification, for example, using acidic
conditions.
[0142] In a further embodiment, the method for siNA synthesis is a
solution phase synthesis or hybrid phase synthesis wherein both
strands of the siNA duplex are synthesized in tandem using a
cleavable linker attached to the first sequence which acts a
scaffold for synthesis of the second sequence. Cleavage of the
linker under conditions suitable for hybridization of the separate
siNA sequence strands results in formation of the double-stranded
siNA molecule.
[0143] In another embodiment, the invention features a method for
synthesizing a siNA duplex molecule comprising: (a) synthesizing
one oligonucleotide sequence strand of the siNA molecule, wherein
the sequence comprises a cleavable linker molecule that can be used
as a scaffold for the synthesis of another oligonucleotide
sequence; (b) synthesizing a second oligonucleotide sequence having
complementarity to the first sequence strand on the scaffold of
(a), wherein the second sequence comprises the other strand of the
double-stranded siNA molecule and wherein the second sequence
further comprises a chemical moiety than can be used to isolate the
attached oligonucleotide sequence; (c) purifying the product of (b)
utilizing the chemical moiety of the second oligonucleotide
sequence strand under conditions suitable for isolating the
full-length sequence comprising both siNA oligonucleotide strands
connected by the cleavable linker and under conditions suitable for
the two siNA oligonucleotide strands to hybridize and form a stable
duplex. In one embodiment, cleavage of the linker molecule in (c)
above takes place during deprotection of the oligonucleotide, for
example under hydrolysis conditions. In another embodiment,
cleavage of the linker molecule in (c) above takes place after
deprotection of the oligonucleotide. In another embodiment, the
method of synthesis comprises solid phase synthesis on a solid
support such as controlled pore glass (CPG) or polystyrene, wherein
the first sequence of (a) is synthesized on a cleavable linker,
such as a succinyl linker, using the solid support as a scaffold.
The cleavable linker in (a) used as a scaffold for synthesizing the
second strand can comprise similar reactivity or differing
reactivity as the solid support derivatized linker, such that
cleavage of the solid support derivatized linker and the cleavable
linker of (a) takes place either concomitantly or sequentially. In
one embodiment, the chemical moiety of (b) that can be used to
isolate the attached oligonucleotide sequence comprises a trityl
group, for example a dimethoxytrityl group.
[0144] In another embodiment, the invention features a method for
making a double-stranded siNA molecule in a single synthetic
process comprising: (a) synthesizing an oligonucleotide having a
first and a second sequence, wherein the first sequence is
complementary to the second sequence, and the first oligonucleotide
sequence is linked to the second sequence via a cleavable linker,
and wherein a terminal 5'-protecting group, for example, a
5'-O-dimethoxytrityl group (5'-O-DMT) remains on the
oligonucleotide having the second sequence; (b) deprotecting the
oligonucleotide whereby the deprotection results in the cleavage of
the linker joining the two oligonucleotide sequences; and (c)
purifying the product of (b) under conditions suitable for
isolating the double-stranded siNA molecule, for example using a
trityl-on synthesis strategy as described herein.
[0145] In another embodiment, the method of synthesis of siNA
molecules of the invention comprises the teachings of Scaringe et
al., U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086,
incorporated by reference herein in their entirety.
[0146] In one embodiment, the invention features siNA constructs
that mediate RNAi against a MAP kinase, wherein the siNA construct
comprises one or more chemical modifications, for example, one or
more chemical modifications having any of Formulae I-VII or any
combination thereof that increases the nuclease resistance of the
siNA construct.
[0147] In another embodiment, the invention features a method for
generating siNA molecules with increased nuclease resistance
comprising (a) introducing nucleotides having any of Formula I-VII
or any combination thereof into a siNA molecule, and (b) assaying
the siNA molecule of step (a) under conditions suitable for
isolating siNA molecules having increased nuclease resistance.
[0148] In one embodiment, the invention features siNA constructs
that mediate RNAi against a MAP kinase, wherein the siNA construct
comprises one or more chemical modifications described herein that
modulates the binding affinity between the sense and antisense
strands of the siNA construct.
[0149] In another embodiment, the invention features a method for
generating siNA molecules with increased binding affinity between
the sense and antisense strands of the siNA molecule comprising (a)
introducing nucleotides having any of Formula I-VII or any
combination thereof into a siNA molecule, and (b) assaying the siNA
molecule of step (a) under conditions suitable for isolating siNA
molecules having increased binding affinity between the sense and
antisense strands of the siNA molecule.
[0150] In one embodiment, the invention features siNA constructs
that mediate RNAi against a MAP kinase, wherein the siNA construct
comprises one or more chemical modifications described herein that
modulates the binding affinity between the antisense strand of the
siNA construct and a complementary target RNA sequence within a
cell.
[0151] In one embodiment, the invention features siNA constructs
that mediate RNAi against a MAP kinase, wherein the siNA construct
comprises one or more chemical modifications described herein that
modulates the binding affinity between the antisense strand of the
siNA construct and a complementary target DNA sequence within a
cell.
[0152] In another embodiment, the invention features a method for
generating siNA molecules with increased binding affinity between
the antisense strand of the siNA molecule and a complementary
target RNA sequence comprising (a) introducing nucleotides having
any of Formula I-VII or any combination thereof into a siNA
molecule, and (b) assaying the siNA molecule of step (a) under
conditions suitable for isolating siNA molecules having increased
binding affinity between the antisense strand of the siNA molecule
and a complementary target RNA sequence.
[0153] In another embodiment, the invention features a method for
generating siNA molecules with increased binding affinity between
the antisense strand of the siNA molecule and a complementary
target DNA sequence comprising (a) introducing nucleotides having
any of Formula I-VII or any combination thereof into a siNA
molecule, and (b) assaying the siNA molecule of step (a) under
conditions suitable for isolating siNA molecules having increased
binding affinity between the antisense strand of the siNA molecule
and a complementary target DNA sequence.
[0154] In one embodiment, the invention features siNA constructs
that mediate RNAi against a MAP kinase, wherein the siNA construct
comprises one or more chemical modifications described herein that
modulate the polymerase activity of a cellular polymerase capable
of generating additional endogenous siNA molecules having sequence
homology to the chemically-modified siNA construct.
[0155] In another embodiment, the invention features a method for
generating siNA molecules capable of mediating increased polymerase
activity of a cellular polymerase capable of generating additional
endogenous siNA molecules having sequence homology to a
chemically-modified siNA molecule comprising (a) introducing
nucleotides having any of Formula I-VII or any combination thereof
into a siNA molecule, and (b) assaying the siNA molecule of step
(a) under conditions suitable for isolating siNA molecules capable
of mediating increased polymerase activity of a cellular polymerase
capable of generating additional endogenous siNA molecules having
sequence homology to the chemically-modified siNA molecule.
[0156] In one embodiment, the invention features
chemically-modified siNA constructs that mediate RNAi against a MAP
kinase in a cell, wherein the chemical modifications do not
significantly effect the interaction of siNA with a target RNA
molecule, DNA molecule and/or proteins or other factors that are
essential for RNAi in a manner that would decrease the efficacy of
RNAi mediated by such siNA constructs.
[0157] In another embodiment, the invention features a method for
generating siNA molecules with improved RNAi activity against MAP
kinase comprising (a) introducing nucleotides having any of Formula
I-VII or any combination thereof into a siNA molecule, and (b)
assaying the siNA molecule of step (a) under conditions suitable
for isolating siNA molecules having improved RNAi activity.
[0158] In yet another embodiment, the invention features a method
for generating siNA molecules with improved RNAi activity against a
MAP kinase target RNA comprising (a) introducing nucleotides having
any of Formula I-VII or any combination thereof into a siNA
molecule, and (b) assaying the siNA molecule of step (a) under
conditions suitable for isolating siNA molecules having improved
RNAi activity against the target RNA.
[0159] In yet another embodiment, the invention features a method
for generating siNA molecules with improved RNAi activity against a
MAP kinase target DNA comprising (a) introducing nucleotides having
any of Formula I-VII or any combination thereof into a siNA
molecule, and (b) assaying the siNA molecule of step (a) under
conditions suitable for isolating siNA molecules having improved
RNAi activity against the target DNA.
[0160] In one embodiment, the invention features siNA constructs
that mediate RNAi against a MAP kinase, wherein the siNA construct
comprises one or more chemical modifications described herein that
modulates the cellular uptake of the siNA construct.
[0161] In another embodiment, the invention features a method for
generating siNA molecules against MAP kinase with improved cellular
uptake comprising (a) introducing nucleotides having any of Formula
I-VII or any combination thereof into a siNA molecule, and (b)
assaying the siNA molecule of step (a) under conditions suitable
for isolating siNA molecules having improved cellular uptake.
[0162] In one embodiment, the invention features siNA constructs
that mediate RNAi against a MAP kinase, wherein the siNA construct
comprises one or more chemical modifications described herein that
increases the bioavailability of the siNA construct, for example,
by attaching polymeric conjugates such as polyethyleneglycol or
equivalent conjugates that improve the pharmacokinetics of the siNA
construct, or by attaching conjugates that target specific tissue
types or cell types in vivo. Non-limiting examples of such
conjugates are described in Vargeese et al., U.S. Ser. No.
10/201,394 incorporated by reference herein.
[0163] In one embodiment, the invention features a method for
generating siNA molecules of the invention with improved
bioavailability comprising (a) introducing a conjugate into the
structure of a siNA molecule, and (b) assaying the siNA molecule of
step (a) under conditions suitable for isolating siNA molecules
having improved bioavailability. Such conjugates can include
ligands for cellular receptors, such as peptides derived from
naturally occurring protein ligands; protein localization
sequences, including cellular ZIP code sequences; antibodies;
nucleic acid aptamers; vitamins and other co-factors, such as
folate and N-acetylgalactosamine; polymers, such as
polyethyleneglycol (PEG); phospholipids; cholesterol; polyamines,
such as spermine or spermidine; and others.
[0164] In another embodiment, the invention features a method for
generating siNA molecules of the invention with improved
bioavailability comprising (a) introducing an excipient formulation
to a siNA molecule, and (b) assaying the siNA molecule of step (a)
under conditions suitable for isolating siNA molecules having
improved bioavailability. Such excipients include polymers such as
cyclodextrins, lipids, cationic lipids, polyamines, phospholipids,
and others.
[0165] In another embodiment, the invention features a method for
generating siNA molecules of the invention with improved
bioavailability comprising (a) introducing nucleotides having any
of Formulae I-VII or any combination thereof into a siNA molecule,
and (b) assaying the siNA molecule of step (a) under conditions
suitable for isolating siNA molecules having improved
bioavailability.
[0166] In another embodiment, polyethylene glycol (PEG) can be
covalently attached to siNA compounds of the present invention. The
attached PEG can be any molecular weight, preferably from about
2,000 to about 50,000 daltons (Da).
[0167] The present invention can be used alone or as a component of
a kit having at least one of the reagents necessary to carry out
the in vitro or in vivo introduction of RNA to test samples and/or
subjects. For example, preferred components of the kit include a
siNA molecule of the invention and a vehicle that promotes
introduction of the siNA into cells of interest as described herein
(e.g., using lipids and other methods of transfection known in the
art, see for example Beigelman et al, U.S. Pat. No. 6,395,713). The
kit can be used for target validation, such as in determining gene
function and/or activity, or in drug optimization, and in drug
discovery (see for example Usman et al., U.S. Ser. No. 60/402,996).
Such a kit can also include instructions to allow a user of the kit
to practice the invention.
[0168] The term "short interfering nucleic acid", "siNA", "short
interfering RNA", "siRNA", "short interfering nucleic acid
molecule", "short interfering oligonucleotide molecule", or
"chemically-modified short interfering nucleic acid molecule" as
used herein refers to any nucleic acid molecule capable of
inhibiting or down regulating gene expression, for example by
mediating RNA interference "RNAi" or gene silencing in a
sequence-specific manner; see for example Bass, 2001, Nature, 411,
428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer
et al., International PCT Publication No. WO 00/44895;
Zernicka-Goetz et al., International PCT Publication No. WO
01/36646; Fire, International PCT Publication No. WO 99/32619;
Plaetinck et al., International PCT Publication No. WO 00/01846;
Mello and Fire, International PCT Publication No. WO 01/29058;
Deschamps-Depaillette, International PCT Publication No. WO
99/07409; and Li et al., International PCT Publication No. WO
00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,
2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297,
2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;
Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al.,
2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,
1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).
Non limiting examples of siNA molecules of the invention are shown
in FIGS. 4-6, and Tables II, III, and IV herein. For example the
siNA can be a double-stranded polynucleotide molecule comprising
self-complementary sense and antisense regions, wherein the
antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The siNA can be assembled from two
separate oligonucleotides, where one strand is the sense strand and
the other is the antisense strand, wherein the antisense and sense
strands are self-complementary (i.e. each strand comprises
nucleotide sequence that is complementary to nucleotide sequence in
the other strand; such as where the antisense strand and sense
strand form a duplex or double stranded structure, for example
wherein the double stranded region is about 19 base pairs); the
antisense strand comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense strand comprises
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. Alternatively, the siNA is assembled
from a single oligonucleotide, where the self-complementary sense
and antisense regions of the siNA are linked by means of a nucleic
acid based or non-nucleic acid-based linker(s). The siNA can be a
polynucleotide with a hairpin secondary structure, having
self-complementary sense and antisense regions, wherein the
antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a separate target nucleic
acid molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The siNA can be a circular
single-stranded polynucleotide having two or more loop structures
and a stem comprising self-complementary sense and antisense
regions, wherein the antisense region comprises nucleotide sequence
that is complementary to nucleotide sequence in a target nucleic
acid molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof, and wherein the circular
polynucleotide can be processed either in vivo or in vitro to
generate an active siNA molecule capable of mediating RNAi. The
siNA can also comprise a single stranded polynucleotide having
nucleotide sequence complementary to nucleotide sequence in a
target nucleic acid molecule or a portion thereof (for example,
where such siNA molecule does not require the presence within the
siNA molecule of nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof), wherein the single
stranded polynucleotide can further comprise a terminal phosphate
group, such as a 5'-phosphate (see for example Martinez et al.,
2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell,
10, 537-568), or 5',3'-diphosphate. In certain embodiment, the siNA
molecule of the invention comprises separate sense and antisense
sequences or regions, wherein the sense and antisense regions are
covalently linked by nucleotide or non-nucleotide linkers molecules
as is known in the art, or are alternately non-covalently linked by
ionic interactions, hydrogen bonding, van der waals interactions,
hydrophobic interactions, and/or stacking interactions. In certain
embodiments, the siNA molecules of the invention comprise
nucleotide sequence that is complementary to nucleotide sequence of
a target gene. In another embodiment, the siNA molecule of the
invention interacts with nucleotide sequence of a target gene in a
manner that causes inhibition of expression of the target gene. As
used herein, siNA molecules need not be limited to those molecules
containing only RNA, but further encompasses chemically-modified
nucleotides and non-nucleotides. In certain embodiments, the short
interfering nucleic acid molecules of the invention lack 2'-hydroxy
(2'-OH) containing nucleotides. Applicant describes in certain
embodiments short interfering nucleic acids that do not require the
presence of nucleotides having a 2'-hydroxy group for mediating
RNAi and as such, short interfering nucleic acid molecules of the
invention optionally do not include any ribonucleotides (e.g.,
nucleotides having a 2'-OH group). Such siNA molecules that do not
require the presence of ribonucleotides within the siNA molecule to
support RNAi can however have an attached linker or linkers or
other attached or associated groups, moieties, or chains containing
one or more nucleotides with 2'-OH groups. Optionally, siNA
molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40,
or 50% of the nucleotide positions. The modified short interfering
nucleic acid molecules of the invention can also be referred to as
short interfering modified oligonucleotides "siMON." As used
herein, the term siNA is meant to be equivalent to other terms used
to describe nucleic acid molecules that are capable of mediating
sequence specific RNAi, for example short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA
(shRNA), short interfering oligonucleotide, short interfering
nucleic acid, short interfering modified oligonucleotide,
chemically-modified siRNA, post-transcriptional gene silencing RNA
(ptgsRNA), and others. In addition, as used herein, the term RNAi
is meant to be equivalent to other terms used to describe sequence
specific RNA interference, such as post transcriptional gene
silencing, translational inhibition, or epigenetics. For example,
siNA molecules of the invention can be used to epigenetically
silence genes at both the post-transcriptional level or the
pre-transcriptional level. In a non-limiting example, epigenetic
regulation of gene expression by siNA molecules of the invention
can result from siNA mediated modification of chromatin structure
to alter gene expression (see, for example, Allshire, 2002,
Science, 297, 1818-1819; Volpe et al., 2002, Science, 297,
1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et
al., 2002, Science, 297, 2232-2237).
[0169] By "modulate" is meant that the expression of the gene, or
level of RNA molecule or equivalent RNA molecules encoding one or
more proteins or protein subunits, or activity of one or more
proteins or protein subunits is up regulated or down regulated,
such that expression, level, or activity is greater than or less
than that observed in the absence of the modulator. For example,
the term "modulate" can mean "inhibit," but the use of the word
"modulate" is not limited to this definition.
[0170] By "inhibit", "down-regulate", or "reduce", it is meant that
the expression of the gene, or level of RNA molecules or equivalent
RNA molecules encoding one or more proteins or protein subunits, or
activity of one or more proteins or protein subunits, is reduced
below that observed in the absence of the nucleic acid molecules
(e.g., siNA) of the invention. In one embodiment, inhibition,
down-regulation or reduction with an siNA molecule is below that
level observed in the presence of an inactive or attenuated
molecule. In another embodiment, inhibition, down-regulation, or
reduction with siNA molecules is below that level observed in the
presence of, for example, an siNA molecule with scrambled sequence
or with mismatches. In another embodiment, inhibition,
down-regulation, or reduction of gene expression with a nucleic
acid molecule of the instant invention is greater in the presence
of the nucleic acid molecule than in its absence.
[0171] By "gene" or "target gene" is meant, a nucleic acid that
encodes an RNA, for example, nucleic acid sequences including, but
not limited to, structural genes encoding a polypeptide. The target
gene can be a gene derived from a cell, an endogenous gene, a
transgene, or exogenous genes such as genes of a pathogen, for
example a virus, which is present in the cell after infection
thereof. The cell containing the target gene can be derived from or
contained in any organism, for example a plant, animal, protozoan,
virus, bacterium, or fungus. Non-limiting examples of plants
include monocots, dicots, or gymnosperms. Non-limiting examples of
animals include vertebrates or invertebrates. Non-limiting examples
of fungi include molds or yeasts.
[0172] By "MAP kinase" is meant, any mitogen activated protein
kinase (MAP kinase) polypeptide, protein and/or a polynucleotide
encoding a MAP kinase protein (such as polynucleotides referred to
by Genbank Accession number in Table I or any other MAP kinase
transcript derived from a MAP kinase gene, e.g., c-JUN, ERK1, ERK2,
JNK1, JNK2, and/or p38). As used herein, the term "MAP kinase gene"
is meant to refer to any polynucleotide included in a group of MAP
kinase genes, such as c-JUN, ERK1, ERK2, JNK1, JNK2, and/or
p38).
[0173] By "MAP kinase protein" is meant, any mitogen activated
protein kinase (MAP kinase) peptide or protein or a component
thereof, wherein the peptide or protein is encoded by a MAP kinase
gene (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38).
[0174] By "highly conserved sequence region" is meant a nucleotide
sequence of one or more regions in a target gene does not vary
significantly from one generation to the other or from one
biological system to the other.
[0175] By "sense region" is meant a nucleotide sequence of a siNA
molecule having complementarity to an antisense region of the siNA
molecule. In addition, the sense region of a siNA molecule can
comprise a nucleic acid sequence having homology with a target
nucleic acid sequence.
[0176] By "antisense region" is meant a nucleotide sequence of a
siNA molecule having complementarity to a target nucleic acid
sequence. In addition, the antisense region of a siNA molecule can
optionally comprise a nucleic acid sequence having complementarity
to a sense region of the siNA molecule.
[0177] By "target nucleic acid" is meant any nucleic acid sequence
whose expression or activity is to be modulated. The target nucleic
acid can be DNA or RNA.
[0178] By "complementarity" is meant that a nucleic acid can form
hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick or other non-traditional types. In
reference to the nucleic molecules of the present invention, the
binding free energy for a nucleic acid molecule with its
complementary sequence is sufficient to allow the relevant function
of the nucleic acid to proceed, e.g., RNAi activity. Determination
of binding free energies for nucleic acid molecules is well known
in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol.
LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA
83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.
109:3783-3785). A percent complementarity indicates the percentage
of contiguous residues in a nucleic acid molecule that can form
hydrogen bonds (e.g., Watson-Crick base pairing) with a second
nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%,
60%, 70%, 80%, 90%, and 100% complementary). "Perfectly
complementary" means that all the contiguous residues of a nucleic
acid sequence will hydrogen bond with the same number of contiguous
residues in a second nucleic acid sequence.
[0179] The siRNA molecules of the invention represent a novel
therapeutic approach to treat a variety of pathologic indications
or other conditions, including oncology and proliferation related
indications and conditions such as multidrug resistant cancers,
breast cancer, cancers of the head and neck including various
lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma,
adenoma, squamous cell carcinoma, laryngeal carcinoma, cancers of
the retina, cancers of the esophagus, multiple myeloma, ovarian
cancer, melanoma, colorectal cancer, hepatocellular carcinoma, lung
cancer, bladder cancer, pancreatic cancer, prostate cancer,
glioblastoma; obesity and insulin resistance (e.g. type I and II
diabetes); inflammatory disorders such as asthma, septic shock,
rheumatoid arthritis, psoriasis, inflammatory bowl syndrome and any
other diseases or conditions that are related to or will respond to
the levels of MAP kinase in a cell or tissue, alone or in
combination with other therapies. The reduction of MAP kinase
expression (specifically MAP kinase gene RNA levels) and thus
reduction in the level of the respective protein relieves, to some
extent, the symptoms of the disease or condition.
[0180] In one embodiment of the present invention, each sequence of
a siNA molecule of the invention is independently about 18 to about
24 nucleotides in length, in specific embodiments about 18, 19, 20,
21, 22, 23, or 24 nucleotides in length. In another embodiment, the
siNA duplexes of the invention independently comprise about 17 to
about 23 base pairs (e.g., about 17, 18, 19, 20, 21, 22 or 23). In
yet another embodiment, siNA molecules of the invention comprising
hairpin or circular structures are about 35 to about 55 (e.g.,
about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to
about 44 (e.g., 38, 39, 40, 41, 42, 43 or 44) nucleotides in length
and comprising about 16 to about 22 (e.g., about 16, 17, 18, 19,
20, 21 or 22) base pairs. Exemplary siNA molecules of the invention
are shown in Table II. Exemplary synthetic siNA molecules of the
invention are shown in Tables III and IV and/or FIGS. 4-5.
[0181] As used herein "cell" is used in its usual biological sense,
and does not refer to an entire multicellular organism, e.g.,
specifically does not refer to a human. The cell can be present in
an organism, e.g., birds, plants and mammals such as humans, cows,
sheep, apes, monkeys, swine, dogs, and cats. The cell can be
prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian
or plant cell). The cell can be of somatic or germ line origin,
totipotent or pluripotent, dividing or non-dividing. The cell can
also be derived from or can comprise a gamete or embryo, a stem
cell, or a fully differentiated cell.
[0182] The siNA molecules of the invention are added directly, or
can be complexed with cationic lipids, packaged within liposomes,
or otherwise delivered to target cells or tissues. The nucleic acid
or nucleic acid complexes can be locally administered to relevant
tissues ex vivo, or in vivo through injection, infusion pump or
stent, with or without their incorporation in biopolymers. In
particular embodiments, the nucleic acid molecules of the invention
comprise sequences shown in Tables II-III and/or FIGS. 4-5.
Examples of such nucleic acid molecules consist essentially of
sequences defined in these tables and figures. Furthermore, the
chemically modified constructs described in Table IV can be applied
to any siNA sequence of the invention.
[0183] In another aspect, the invention provides mammalian cells
containing one or more siNA molecules of this invention. The one or
more siNA molecules can independently be targeted to the same or
different sites.
[0184] By "RNA" is meant a molecule comprising at least one
ribonucleotide residue. By "ribonucleotide" is meant a nucleotide
with a hydroxyl group at the 2' position of a
.beta.-D-ribo-furanose moiety. The terms include double-stranded
RNA, single-stranded RNA, isolated RNA such as partially purified
RNA, essentially pure RNA, synthetic RNA, recombinantly produced
RNA, as well as altered RNA that differs from naturally occurring
RNA by the addition, deletion, substitution and/or alteration of
one or more nucleotides. Such alterations can include addition of
non-nucleotide material, such as to the end(s) of the siNA or
internally, for example at one or more nucleotides of the RNA.
Nucleotides in the RNA molecules of the instant invention can also
comprise non-standard nucleotides, such as non-naturally occurring
nucleotides or chemically synthesized nucleotides or
deoxynucleotides. These altered RNAs can be referred to as analogs
or analogs of naturally-occurring RNA.
[0185] By "subject" is meant an organism, which is a donor or
recipient of explanted cells or the cells themselves. "Subject"
also refers to an organism to which the nucleic acid molecules of
the invention can be administered. A subject can be a mammal or
mammalian cells, including a human or human cells.
[0186] The term "phosphorothioate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z and/or W
comprise a sulfur atom. Hence, the term phosphorothioate refers to
both phosphorothioate and phosphorodithioate internucleotide
linkages.
[0187] The term "universal base" as used herein refers to
nucleotide base analogs that form base pairs with each of the
natural DNA/RNA bases with little discrimination between them.
Non-limiting examples of universal bases include C-phenyl,
C-naphthyl and other aromatic derivatives, inosine, azole
carboxamides, and nitroazole derivatives such as 3-nitropyrrole,
4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art
(see for example Loakes, 2001, Nucleic Acids Research, 29,
2437-2447).
[0188] The term "acyclic nucleotide" as used herein refers to any
nucleotide having an acyclic ribose sugar, for example where any of
the ribose carbons (C1, C2, C3, C4, or C5), are independently or in
combination absent from the nucleotide.
[0189] The nucleic acid molecules of the instant invention,
individually, or in combination or in conjunction with other drugs,
can be used to treat diseases or conditions discussed herein (e.g.,
cancers and other proliferative conditions). For example, to treat
a particular disease or condition, the siNA molecules can be
administered to a subject or can be administered to other
appropriate cells evident to those skilled in the art, individually
or in combination with one or more drugs under conditions suitable
for the treatment.
[0190] In a further embodiment, the siNA molecules can be used in
combination with other known treatments to treat conditions or
diseases discussed above. For example, the described molecules
could be used in combination with one or more known therapeutic
agents to treat a disease or condition. Non-limiting examples of
other therapeutic agents that can be readily combined with a siNA
molecule of the invention are enzymatic nucleic acid molecules,
allosteric nucleic acid molecules, antisense, decoy, or aptamer
nucleic acid molecules, antibodies such as monoclonal antibodies,
small molecules, and other organic and/or inorganic compounds
including metals, salts and ions.
[0191] In one embodiment, the invention features an expression
vector comprising a nucleic acid sequence encoding at least one
siNA molecule of the invention, in a manner which allows expression
of the siNA molecule. For example, the vector can contain
sequence(s) encoding both strands of a siNA molecule comprising a
duplex. The vector can also contain sequence(s) encoding a single
nucleic acid molecule that is self-complementary and thus forms a
siNA molecule. Non-limiting examples of such expression vectors are
described in Paul et al., 2002, Nature Biotechnology, 19, 505;
Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et
al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002,
Nature Medicine, advance online publication doi:10.1038/nm725.
[0192] In another embodiment, the invention features a mammalian
cell, for example, a human cell, including an expression vector of
the invention.
[0193] In yet another embodiment, the expression vector of the
invention comprises a sequence for a siNA molecule having
complementarity to a RNA molecule referred to by a Genbank
Accession numbers, for example Genbank Accession Nos. shown in
Table I.
[0194] In one embodiment, an expression vector of the invention
comprises a nucleic acid sequence encoding two or more siNA
molecules, which can be the same or different.
[0195] In another aspect of the invention, siNA molecules that
interact with target RNA molecules and down-regulate gene encoding
target RNA molecules (for example target RNA molecules referred to
by Genbank Accession numbers herein) are expressed from
transcription units inserted into DNA or RNA vectors. The
recombinant vectors can be DNA plasmids or viral vectors. siNA
expressing viral vectors can be constructed based on, but not
limited to, adeno-associated virus, retrovirus, adenovirus, or
alphavirus. The recombinant vectors capable of expressing the siNA
molecules can be delivered as described herein, and persist in
target cells. Alternatively, viral vectors can be used that provide
for transient expression of siNA molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the siNA
molecules bind and down-regulate gene function or expression via
RNA interference (RNAi). Delivery of siNA expressing vectors can be
systemic, such as by intravenous or intramuscular administration,
by administration to target cells ex-planted from a subject
followed by reintroduction into the subject, or by any other means
that would allow for introduction into the desired target cell.
[0196] By "vectors" is meant any nucleic acid- and/or viral-based
technique used to deliver a desired nucleic acid.
[0197] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0198] FIG. 1 shows a non-limiting example of a scheme for the
synthesis of siNA molecules. The complementary siNA sequence
strands, strand 1 and strand 2, are synthesized in tandem and are
connected by a cleavable linkage, such as a nucleotide succinate or
abasic succinate, which can be the same or different from the
cleavable linker used for solid phase synthesis on a solid support.
The synthesis can be either solid phase or solution phase, in the
example shown, the synthesis is a solid phase synthesis. The
synthesis is performed such that a protecting group, such as a
dimethoxytrityl group, remains intact on the terminal nucleotide of
the tandem oligonucleotide. Upon cleavage and deprotection of the
oligonucleotide, the two siNA strands spontaneously hybridize to
form a siNA duplex, which allows the purification of the duplex by
utilizing the properties of the terminal protecting group, for
example by applying a trityl on purification method wherein only
duplexes/oligonucleotides with the terminal protecting group are
isolated.
[0199] FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA
duplex synthesized by a method of the invention. The two peaks
shown correspond to the predicted mass of the separate siNA
sequence strands. This result demonstrates that the siNA duplex
generated from tandem synthesis can be purified as a single entity
using a simple trityl-on purification methodology.
[0200] FIG. 3 shows a non-limiting proposed mechanistic
representation of target RNA degradation involved in RNAi.
Double-stranded RNA (dsRNA), which is generated by RNA-dependent
RNA polymerase (RdRP) from foreign single-stranded RNA, for example
viral, transposon, or other exogenous RNA, activates the DICER
enzyme that in turn generates siNA duplexes. Alternately, synthetic
or expressed siNA can be introduced directly into a cell by
appropriate means. An active siNA complex forms which recognizes a
target RNA, resulting in degradation of the target RNA by the RISC
endonuclease complex or in the synthesis of additional RNA by
RNA-dependent RNA polymerase (RdRP), which can activate DICER and
result in additional siNA molecules, thereby amplifying the RNAi
response.
[0201] FIG. 4A-F shows non-limiting examples of chemically-modified
siNA constructs of the present invention. In the figure, N stands
for any nucleotide (adenosine, guanosine, cytosine, uridine, or
optionally thymidine, for example thymidine can be substituted in
the overhanging regions designated by parenthesis (N N). Various
modifications are shown for the sense and antisense strands of the
siNA constructs.
[0202] FIG. 4A: The sense strand comprises 21 nucleotides wherein
the two terminal 3'-nucleotides are optionally base paired and
wherein all nucleotides present are ribonucleotides except for (N
N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications
described herein. The antisense strand comprises 21 nucleotides,
optionally having a 3'-terminal glyceryl moiety and wherein the two
terminal 3'-nucleotides are optionally complementary to the target
RNA sequence, and wherein all nucleotides present are
ribonucleotides except for (N N) nucleotides, which can comprise
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. A modified internucleotide
linkage, such as a phosphorothioate, phosphorodithioate or other
modified internucleotide linkage as described herein, shown as "s"
connects the (N N) nucleotides in the antisense strand.
[0203] FIG. 4B: The sense strand comprises 21 nucleotides wherein
the two terminal 3'-nucleotides are optionally base paired and
wherein all pyrimidine nucleotides that may be present are
2'-deoxy-2'-fluoro modified nucleotides and all purine nucleotides
that may be present are 2'-O-methyl modified nucleotides except for
(N N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications
described herein. The antisense strand comprises 21 nucleotides,
optionally having a 3'-terminal glyceryl moiety and wherein the two
terminal 3'-nucleotides are optionally complementary to the target
RNA sequence, and wherein all pyrimidine nucleotides that may be
present are 2'-deoxy-2'-fluoro modified nucleotides and all purine
nucleotides that may be present are 2'-O-methyl modified
nucleotides except for (N N) nucleotides, which can comprise
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. A modified internucleotide
linkage, such as a phosphorothioate, phosphorodithioate or other
modified internucleotide linkage as described herein, shown as "s"
connects the (N N) nucleotides in the sense and antisense
strand.
[0204] FIG. 4C: The sense strand comprises 21 nucleotides having
5'- and 3'-terminal cap moieties wherein the two terminal
3'-nucleotides are optionally base paired and wherein all
pyrimidine nucleotides that may be present are 2'-O-methyl or
2'-deoxy-2'-fluoro modified nucleotides except for (N N)
nucleotides, which can comprise ribonucleotides, deoxynucleotides,
universal bases, or other chemical modifications described herein.
The antisense strand comprises 21 nucleotides, optionally having a
3'-terminal glyceryl moiety and wherein the two terminal
3'-nucleotides are optionally complementary to the target RNA
sequence, and wherein all pyrimidine nucleotides that may be
present are 2'-deoxy-2'-fluoro modified nucleotides except for (N
N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications
described herein. A modified internucleotide linkage, such as a
phosphorothioate, phosphorodithioate or other modified
internucleotide linkage as described herein, shown as "s" connects
the (N N) nucleotides in the antisense strand.
[0205] FIG. 4D: The sense strand comprises 21 nucleotides having
5'- and 3'-terminal cap moieties wherein the two terminal
3'-nucleotides are optionally base paired and wherein all
pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro
modified nucleotides except for (N N) nucleotides, which can
comprise ribonucleotides, deoxynucleotides, universal bases, or
other chemical modifications described herein and wherein and all
purine nucleotides that may be present are 2'-deoxy nucleotides.
The antisense strand comprises 21 nucleotides, optionally having a
3'-terminal glyceryl moiety and wherein the two terminal
3'-nucleotides are optionally complementary to the target RNA
sequence, and wherein all pyrimidine nucleotides that may be
present are 2'-deoxy-2'-fluoro modified nucleotides and all purine
nucleotides that may be present are 2'-O-methyl modified
nucleotides except for (N N) nucleotides, which can comprise
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. A modified internucleotide
linkage, such as a phosphorothioate, phosphorodithioate or other
modified internucleotide linkage as described herein, shown as "s"
connects the (N N) nucleotides in the antisense strand.
[0206] FIG. 4E: The sense strand comprises 21 nucleotides having
5'- and 3'-terminal cap moieties wherein the two terminal
3'-nucleotides are optionally base paired and wherein all
pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro
modified nucleotides except for (N N) nucleotides, which can
comprise ribonucleotides, deoxynucleotides, universal bases, or
other chemical modifications described herein. The antisense strand
comprises 21 nucleotides, optionally having a 3'-terminal glyceryl
moiety and wherein the two terminal 3'-nucleotides are optionally
complementary to the target RNA sequence, and wherein all
pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro
modified nucleotides and all purine nucleotides that may be present
are 2'-O-methyl modified nucleotides except for (N N) nucleotides,
which can comprise ribonucleotides, deoxynucleotides, universal
bases, or other chemical modifications described herein. A modified
internucleotide linkage, such as a phosphorothioate,
phosphorodithioate or other modified internucleotide linkage as
described herein, shown as "s" connects the (N N) nucleotides in
the antisense strand.
[0207] FIG. 4F: The sense strand comprises 21 nucleotides having
5'- and 3'-terminal cap moieties wherein the two terminal
3'-nucleotides are optionally base paired and wherein all
pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro
modified nucleotides except for (N N) nucleotides, which can
comprise ribonucleotides, deoxynucleotides, universal bases, or
other chemical modifications described herein and wherein and all
purine nucleotides that may be present are 2'-deoxy nucleotides.
The antisense strand comprises 21 nucleotides, optionally having a
3'-terminal glyceryl moiety and wherein the two terminal
3'-nucleotides are optionally complementary to the target RNA
sequence, and wherein all pyrimidine nucleotides that may be
present are 2'-deoxy-2'-fluoro modified nucleotides and all purine
nucleotides that may be present are 2'-deoxy nucleotides except for
(N N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications
described herein. A modified internucleotide linkage, such as a
phosphorothioate, phosphorodithioate or other modified
internucleotide linkage as described herein, shown as "s" connects
the (N N) nucleotides in the antisense strand. The antisense strand
of constructs A-F comprise sequence complementary to any target
nucleic acid sequence of the invention. Furthermore, when a
glyceryl moiety (L) is present at the 3'-end of the antisense
strand for any construct shown in FIG. 4 A-F, the modified
internucleotide linkage is optional.
[0208] FIG. 5A-F shows non-limiting examples of specific
chemically-modified siNA sequences of the invention. A-F applies
the chemical modifications described in FIG. 4A-F to a c-JUN siNA
sequence. Such chemical modifications can be applied to any
sequence herein, such as any MAP kinase sequence.
[0209] FIG. 6 shows non-limiting examples of different siNA
constructs of the invention. The examples shown (constructs 1, 2,
and 3) have 19 representative base pairs; however, different
embodiments of the invention include any number of base pairs
described herein. Bracketed regions represent nucleotide overhangs,
for example comprising about 1, 2, 3, or 4 nucleotides in length,
preferably about 2 nucleotides. Constructs 1 and 2 can be used
independently for RNAi activity. Construct 2 can comprise a
polynucleotide or non-nucleotide linker, which can optionally be
designed as a biodegradable linker. In one embodiment, the loop
structure shown in construct 2 can comprise a biodegradable linker
that results in the formation of construct 1 in vivo and/or in
vitro. In another example, construct 3 can be used to generate
construct 2 under the same principle wherein a linker is used to
generate the active siNA construct 2 in vivo and/or in vitro, which
can optionally utilize another biodegradable linker to generate the
active siNA construct 1 in vivo and/or in vitro. As such, the
stability and/or activity of the siNA constructs can be modulated
based on the design of the siNA construct for use in vivo or in
vitro and/or in vitro.
[0210] FIG. 7A-C is a diagrammatic representation of a scheme
utilized in generating an expression cassette to generate siNA
hairpin constructs.
[0211] FIG. 7A: A DNA oligomer is synthesized with a 5'-restriction
site (R1) sequence followed by a region having sequence identical
(sense region of siNA) to a predetermined MAP kinase target
sequence, wherein the sense region comprises, for example, about
19, 20, 21, or 22 nucleotides (N) in length, which is followed by a
loop sequence of defined sequence (X), comprising, for example,
about 3 to about 10 nucleotides.
[0212] FIG. 7B: The synthetic construct is then extended by DNA
polymerase to generate a hairpin structure having
self-complementary sequence that will result in a siNA transcript
having specificity for a MAP kinase target sequence and having
self-complementary sense and antisense regions.
[0213] FIG. 7C: The construct is heated (for example to about
95.degree. C.) to linearize the sequence, thus allowing extension
of a complementary second DNA strand using a primer to the
3'-restriction sequence of the first strand. The double-stranded
DNA is then inserted into an appropriate vector for expression in
cells. The construct can be designed such that a 3'-terminal
nucleotide overhang results from the transcription, for example by
engineering restriction sites and/or utilizing a poly-U termination
region as described in Paul et al., 2002, Nature Biotechnology, 29,
505-508.
[0214] FIG. 8A-C is a diagrammatic representation of a scheme
utilized in generating an expression cassette to generate
double-stranded siNA constructs.
[0215] FIG. 8A: A DNA oligomer is synthesized with a 5'-restriction
(R1) site sequence followed by a region having sequence identical
(sense region of siNA) to a predetermined MAP kinase target
sequence, wherein the sense region comprises, for example, about
19, 20, 21, or 22 nucleotides (N) in length, and which is followed
by a 3'-restriction site (R2) which is adjacent to a loop sequence
of defined sequence (X).
[0216] FIG. 8B: The synthetic construct is then extended by DNA
polymerase to generate a hairpin structure having
self-complementary sequence.
[0217] FIG. 8C: The construct is processed by restriction enzymes
specific to R1 and R2 to generate a double-stranded DNA which is
then inserted into an appropriate vector for expression in cells.
The transcription cassette is designed such that a U6 promoter
region flanks each side of the dsDNA which generates the separate
sense and antisense strands of the siNA. Poly T termination
sequences can be added to the constructs to generate U overhangs in
the resulting transcript.
[0218] FIG. 9A-E is a diagrammatic representation of a method used
to determine target sites for siNA mediated RNAi within a
particular target nucleic acid sequence, such as messenger RNA.
[0219] FIG. 9A: A pool of siNA oligonucleotides are synthesized
wherein the antisense region of the siNA constructs has
complementarity to target sites across the target nucleic acid
sequence, and wherein the sense region comprises sequence
complementary to the antisense region of the siNA.
[0220] FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are
inserted into vectors such that (FIG. 9C) transfection of a vector
into cells results in the expression of the siNA.
[0221] FIG. 9D: Cells are sorted based on phenotypic change that is
associated with modulation of the target nucleic acid sequence.
[0222] FIG. 9E: The siNA is isolated from the sorted cells and is
sequenced to identify efficacious target sites within the target
nucleic acid sequence.
[0223] FIG. 10 shows non-limiting examples of different
stabilization chemistries (1-10) that can be used, for example, to
stabilize the 3'-end of siNA sequences of the invention, including
(1) [3-3']-inverted deoxyribose; (2) deoxyribonucleotide; (3)
[5'-3']-3'-deoxyribonucleotide; (4) [5'-3']-ribonucleotide; (5)
[5'-3']-3'-O-methyl ribonucleotide; (6) 3'-glyceryl; (7)
[3'-5']-3'-deoxyribonucleotide; (8) [3'-3']-deoxyribonucleotide;
(9) [5'-2']-deoxyribonucleotide; and (10)
[5-3']-dideoxyribonucleotide. In addition to modified and
unmodified backbone chemistries indicated in the figure, these
chemistries can be combined with different backbone modifications
as described herein, for example, backbone modifications having
Formula I. In addition, the 2'-deoxy nucleotide shown 5' to the
terminal modifications shown can be another modified or unmodified
nucleotide or non-nucleotide described herein, for example
modifications having any of Formulae I-VII or any combination
thereof.
[0224] FIG. 11 shows a non-limiting example of a strategy used to
identify chemically modified siNA constructs of the invention that
are nuclease resistance while preserving the ability to mediate
RNAi activity. Chemical modifications are introduced into the siNA
construct based on educated design parameters (e.g. introducing
2'-modifications, base modifications, backbone modifications,
terminal cap modifications etc). The modified construct in tested
in an appropriate system (e.g. human serum for nuclease resistance,
shown, or an animal model for PK/delivery parameters). In parallel,
the siNA construct is tested for RNAi activity, for example in a
cell culture system such as a luciferase reporter assay). Lead siNA
constructs are then identified which possess a particular
characteristic while maintaining RNAi activity, and can be further
modified and assayed once again. This same approach can be used to
identify siNA-conjugate molecules with improved pharmacokinetic
profiles, delivery, and RNAi activity.
[0225] FIG. 12 shows a non-limiting example of reduction of p38
mRNA in A549 cells mediated by siNAs that target p38 mRNA. A549
cells were transfected with 0.25 ug/well of lipid complexed with 25
nM siNA. A screen of siNA constructs comprising ribonucleotides and
3'-terminal dithymidine caps was compared to untreated cells,
scrambled siNA control constructs (Scram1 and Scram2), and cells
transfected with lipid alone (transfection control). As shown in
the figure, the siNA constructs significantly reduce p38 RNA
expression.
[0226] FIG. 13 shows a non-limiting example of reduction of JNK1
mRNA in A549 cells mediated by siNAs that target JNK1 mRNA. A549
cells were transfected with 0.25 ug/well of lipid complexed with 25
nM siNA. A screen of siNA constructs comprising ribonucleotides and
3'-terminal dithymidine caps was compared to untreated cells,
scrambled siNA control constructs (Scram1 and Scram2), and cells
transfected with lipid alone (transfection control). As shown in
the figure, the siNA constructs significantly reduce JNK1 RNA
expression.
DETAILED DESCRIPTION OF THE INVENTION
Mechanism of Action of Nucleic Acid Molecules of the Invention
[0227] The discussion that follows discusses the proposed mechanism
of RNA interference mediated by short interfering RNA as is
presently known, and is not meant to be limiting and is not an
admission of prior art. Applicant demonstrates herein that
chemically-modified short interfering nucleic acids possess similar
or improved capacity to mediate RNAi as do siRNA molecules and are
expected to possess improved stability and activity in vivo;
therefore, this discussion is not meant to be limited to siRNA only
and can be applied to siNA as a whole. By "improved capacity to
mediate RNAi" or "improved RNAi activity" is meant to include RNAi
activity measured in vitro and/or in vivo where the RNAi activity
is a reflection of both the ability of the siNA to mediate RNAi and
the stability of the siNAs of the invention. In this invention, the
product of these activities can be increased in vitro and/or in
vivo compared to an all RNA siRNA or a siNA containing a plurality
of ribonucleotides. In some cases, the activity or stability of the
siNA molecule can be decreased (i.e., less than ten-fold), but the
overall activity of the siNA molecule is enhanced in vitro and/or
in vivo.
[0228] RNA interference refers to the process of sequence specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806).
The corresponding process in plants is commonly referred to as
post-transcriptional gene silencing or RNA silencing and is also
referred to as quelling in fungi. The process of
post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent
the expression of foreign genes which is commonly shared by diverse
flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such
protection from foreign gene expression may have evolved in
response to the production of double-stranded RNAs (dsRNAs) derived
from viral infection or the random integration of transposon
elements into a host genome via a cellular response that
specifically destroys homologous single-stranded RNA or viral
genomic RNA. The presence of dsRNA in cells triggers the RNAi
response though a mechanism that has yet to be fully characterized.
This mechanism appears to be different from the interferon response
that results from dsRNA-mediated activation of protein kinase PKR
and 2',5'-oligoadenylate synthetase resulting in non-specific
cleavage of mRNA by ribonuclease L.
[0229] The presence of long dsRNAs in cells stimulates the activity
of a ribonuclease III enzyme referred to as Dicer. Dicer is
involved in the processing of the dsRNA into short pieces of dsRNA
known as short interfering RNAs (siRNAs) (Berstein et al., 2001,
Nature, 409, 363). Short interfering RNAs derived from Dicer
activity are typically about 21 to about 23 nucleotides in length
and comprise about 19 base pair duplexes. Dicer has also been
implicated in the excision of 21- and 22-nucleotide small temporal
RNAs (stRNAs) from precursor RNA of conserved structure that are
implicated in translational control (Hutvagner et al., 2001,
Science, 293, 834). The RNAi response also features an endonuclease
complex containing a siRNA, commonly referred to as an RNA-induced
silencing complex (RISC), which mediates cleavage of
single-stranded RNA having sequence homologous to the siRNA.
Cleavage of the target RNA takes place in the middle of the region
complementary to the guide sequence of the siRNA duplex (Elbashir
et al., 2001, Genes Dev., 15, 188). In addition, RNA interference
can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene
silencing, presumably though cellular mechanisms that regulate
chromatin structure and thereby prevent transcription of target
gene sequences (see for example Allshire, 2002, Science, 297,
1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,
2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,
2232-2237). As such, siNA molecules of the invention can be used to
mediate gene silencing via interaction with RNA transcripts or
alternately by interaction with particular gene sequences, wherein
such interaction results in gene silencing either at the
transcriptional level or post-transcriptional level.
[0230] RNAi has been studied in a variety of systems. Fire et al.,
1998, Nature, 391, 806, were the first to observe RNAi in C.
elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe
RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,
Nature, 404, 293, describe RNAi in Drosophila cells transfected
with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi
induced by introduction of duplexes of synthetic 21-nucleotide RNAs
in cultured mammalian cells including human embryonic kidney and
HeLa cells. Recent work in Drosophila embryonic lysates has
revealed certain requirements for siRNA length, structure, chemical
composition, and sequence that are essential to mediate efficient
RNAi activity. These studies have shown that 21 nucleotide siRNA
duplexes are most active when containing two 2-nucleotide
3'-terminal nucleotide overhangs. Furthermore, substitution of one
or both siRNA strands with 2'-deoxy or 2'-O-methyl nucleotides
abolishes RNAi activity, whereas substitution of 3'-terminal siRNA
nucleotides with deoxy nucleotides was shown to be tolerated.
Mismatch sequences in the center of the siRNA duplex were also
shown to abolish RNAi activity. In addition, these studies also
indicate that the position of the cleavage site in the target RNA
is defined by the 5'-end of the siRNA guide sequence rather than
the 3'-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other
studies have indicated that a 5'-phosphate on the
target-complementary strand of a siRNA duplex is required for siRNA
activity and that ATP is utilized to maintain the 5'-phosphate
moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309);
however, siRNA molecules lacking a 5'-phosphate are active when
introduced exogenously, suggesting that 5'-phosphorylation of siRNA
constructs may occur in vivo.
Synthesis of Nucleic Acid Molecules
[0231] Synthesis of nucleic acids greater than 100 nucleotides in
length is difficult using automated methods, and the therapeutic
cost of such molecules is prohibitive. In this invention, small
nucleic acid motifs ("small" refers to nucleic acid motifs no more
than 100 nucleotides in length, preferably no more than 80
nucleotides in length, and most preferably no more than 50
nucleotides in length; e.g., individual siNA oligonucleotide
sequences or siNA sequences synthesized in tandem) are preferably
used for exogenous delivery. The simple structure of these
molecules increases the ability of the nucleic acid to invade
targeted regions of protein and/or RNA structure. Exemplary
molecules of the instant invention are chemically synthesized, and
others can similarly be synthesized.
[0232] Oligonucleotides (e.g., certain modified oligonucleotides or
portions of oligonucleotides lacking ribonucleotides) are
synthesized using protocols known in the art, for example as
described in Caruthers et al., 1992, Methods in Enzymology 211,
3-19, Thompson et al., International PCT Publication No. WO
99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684,
Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al.,
1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No.
6,001,311. All of these references are incorporated herein by
reference. The synthesis of oligonucleotides makes use of common
nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
In a non-limiting example, small scale syntheses are conducted on a
394 Applied Biosystems, Inc. synthesizer using a 0.2 .mu.mol scale
protocol with a 2.5 minute coupling step for 2'-O-methylated
nucleotides and a 45 second coupling step for 2'-deoxy nucleotides
or 2'-deoxy-2'-fluoro nucleotides. Table V outlines the amounts and
the contact times of the reagents used in the synthesis cycle.
Alternatively, syntheses at the 0.2 .mu.mol scale can be performed
on a 96-well plate synthesizer, such as the instrument produced by
Protogene (Palo Alto, Calif.) with minimal modification to the
cycle. A 33-fold excess (60 .mu.L of 0.11 M=6.6 .mu.mol) of
2'-O-methyl phosphoramidite and a 105-fold excess of S-ethyl
tetrazole (60 .mu.L of 0.25 M=15 .mu.mol) can be used in each
coupling cycle of 2'-O-methyl residues relative to polymer-bound
5'-hydroxyl. A 22-fold excess (40 .mu.L of 0.11 M=4.4 .mu.mol) of
deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40
.mu.L of 0.25 M=10 .mu.mol) can be used in each coupling cycle of
deoxy residues relative to polymer-bound 5'-hydroxyl. Average
coupling yields on the 394 Applied Biosystems, Inc. synthesizer,
determined by colorimetric quantitation of the trityl fractions,
are typically 97.5-99%. Other oligonucleotide synthesis reagents
for the 394 Applied Biosystems, Inc. synthesizer include the
following: detritylation solution is 3% TCA in methylene chloride
(ABI); capping is performed with 16% N-methyl imidazole in THF
(ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and
oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF
(PERSEPTIVE.TM.). Burdick & Jackson Synthesis Grade
acetonitrile is used directly from the reagent bottle.
S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from
the solid obtained from American International Chemical, Inc.
Alternately, for the introduction of phosphorothioate linkages,
Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in
acetonitrile) is used.
[0233] Deprotection of the DNA-based oligonucleotides is performed
as follows: the polymer-bound trityl-on oligoribonucleotide is
transferred to a 4 mL glass screw top vial and suspended in a
solution of 40% aqueous methylamine (1 mL) at 65.degree. C. for 10
minutes. After cooling to -20.degree. C., the supernatant is
removed from the polymer support. The support is washed three times
with 1.0 mL of EtOH:MeCN:H.sub.2O/3:1:1, vortexed and the
supernatant is then added to the first supernatant. The combined
supernatants, containing the oligoribonucleotide, are dried to a
white powder.
[0234] The method of synthesis used for RNA including certain siNA
molecules of the invention follows the procedure as described in
Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al.,
1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995,
Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol.
Bio., 74, 59, and makes use of common nucleic acid protecting and
coupling groups, such as dimethoxytrityl at the 5'-end, and
phosphoramidites at the 3'-end. In a non-limiting example, small
scale syntheses are conducted on a 394 Applied Biosystems, Inc.
synthesizer using a 0.2 .mu.mol scale protocol with a 7.5 minute
coupling step for alkylsilyl protected nucleotides and a 2.5 minute
coupling step for 2'-O-methylated nucleotides. Table V outlines the
amounts and the contact times of the reagents used in the synthesis
cycle. Alternatively, syntheses at the 0.2 .mu.mol scale can be
done on a 96-well plate synthesizer, such as the instrument
produced by Protogene (Palo Alto, Calif.) with minimal modification
to the cycle. A 33-fold excess (60 .mu.L of 0.11 M=6.6 .mu.mol) of
2'-O-methyl phosphoramidite and a 75-fold excess of S-ethyl
tetrazole (60 .mu.L of 0.25 M=15 .mu.mol) can be used in each
coupling cycle of 2'-O-methyl residues relative to polymer-bound
5'-hydroxyl. A 66-fold excess (120 .mu.L of 0.11 M=13.2 .mu.mol) of
alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess
of S-ethyl tetrazole (120 .mu.L of 0.25 M=30 .mu.mol) can be used
in each coupling cycle of ribo residues relative to polymer-bound
5'-hydroxyl. Average coupling yields on the 394 Applied Biosystems,
Inc. synthesizer, determined by colorimetric quantitation of the
trityl fractions, are typically 97.5-99%. Other oligonucleotide
synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer
include the following: detritylation solution is 3% TCA in
methylene chloride (ABI); capping is performed with 16% N-methyl
imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in
THF (ABI); oxidation solution is 16.9 mM 12, 49 mM pyridine, 9%
water in THF (PERSEPTIVE.TM.). Burdick & Jackson Synthesis
Grade acetonitrile is used directly from the reagent bottle.
S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from
the solid obtained from American International Chemical, Inc.
Alternately, for the introduction of phosphorothioate linkages,
Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in
acetonitrile) is used.
[0235] Deprotection of the RNA is performed using either a two-pot
or one-pot protocol. For the two-pot protocol, the polymer-bound
trityl-on oligoribonucleotide is transferred to a 4 mL glass screw
top vial and suspended in a solution of 40% aq. methylamine (1 mL)
at 65.degree. C. for 10 minutes. After cooling to -20.degree. C.,
the supernatant is removed from the polymer support. The support is
washed three times with 1.0 mL of EtOH:MeCN:H.sub.2O/3:1:1,
vortexed and the supernatant is then added to the first
supernatant. The combined supernatants, containing the
oligoribonucleotide, are dried to a white powder. The base
deprotected oligoribonucleotide is resuspended in anhydrous
TEA/HF/NMP solution (300 .mu.L of a solution of 1.5 mL
N-methylpyrrolidinone, 750 .mu.L TEA and 1 mL TEA.3HF to provide a
1.4 M HF concentration) and heated to 65.degree. C. After 1.5
hours, the oligomer is quenched with 1.5 M NH.sub.4HCO.sub.3.
[0236] Alternatively, for the one-pot protocol, the polymer-bound
trityl-on oligoribonucleotide is transferred to a 4 mL glass screw
top vial and suspended in a solution of 33% ethanolic
methylamine/DMSO: 1/1 (0.8 mL) at 65.degree. C. for 15 minutes. The
vial is brought to room temperature. TEA.3HF (0.1 mL) is added and
the vial is heated at 65.degree. C. for 15 minutes. The sample is
cooled at -20.degree. C. and then quenched with 1.5 M
NH.sub.4HCO.sub.3.
[0237] For purification of the trityl-on oligomers, the quenched
NH.sub.4.HCO.sub.3 solution is loaded onto a C-18 containing
cartridge that had been prewashed with acetonitrile followed by 50
mM TEAA. After washing the loaded cartridge with water, the RNA is
detritylated with 0.5% TFA for 13 minutes. The cartridge is then
washed again with water, salt exchanged with 1 M NaCl and washed
with water again. The oligonucleotide is then eluted with 30%
acetonitrile.
[0238] The average stepwise coupling yields are typically >98%
(Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of
ordinary skill in the art will recognize that the scale of
synthesis can be adapted to be larger or smaller than the example
described above including but not limited to 96-well format.
[0239] Alternatively, the nucleic acid molecules of the present
invention can be synthesized separately and joined together
post-synthetically, for example, by ligation (Moore et al., 1992,
Science 256, 9923; Draper et al., International PCT publication No.
WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19,
4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951;
Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by
hybridization following synthesis and/or deprotection.
[0240] The siNA molecules of the invention can also be synthesized
via a tandem synthesis methodology as described in Example 1
herein, wherein both siNA strands are synthesized as a single
contiguous oligonucleotide fragment or strand separated by a
cleavable linker which is subsequently cleaved to provide separate
siNA fragments or strands that hybridize and permit purification of
the siNA duplex. The linker can be a polynucleotide linker or a
non-nucleotide linker. The tandem synthesis of siNA as described
herein can be readily adapted to both multiwell/multiplate
synthesis platforms such as 96 well or similarly larger multi-well
platforms. The tandem synthesis of siNA as described herein can
also be readily adapted to large scale synthesis platforms
employing batch reactors, synthesis columns and the like.
[0241] A siNA molecule can also be assembled from two distinct
nucleic acid strands or fragments wherein one fragment includes the
sense region and the second fragment includes the antisense region
of the RNA molecule.
[0242] The nucleic acid molecules of the present invention can be
modified extensively to enhance stability by modification with
nuclease resistant groups, for example, 2'-amino, 2'-C-allyl,
2'-fluoro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren,
1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31,
163). siNA constructs can be purified by gel electrophoresis using
general methods or can be purified by high pressure liquid
chromatography (HPLC; see Wincott et al., supra, the totality of
which is hereby incorporated herein by reference) and re-suspended
in water.
[0243] In another aspect of the invention, siNA molecules of the
invention are expressed from transcription units inserted into DNA
or RNA vectors. The recombinant vectors can be DNA plasmids or
viral vectors. siNA expressing viral vectors can be constructed
based on, but not limited to, adeno-associated virus, retrovirus,
adenovirus, or alphavirus. The recombinant vectors capable of
expressing the siNA molecules can be delivered as described herein,
and persist in target cells. Alternatively, viral vectors can be
used that provide for transient expression of siNA molecules.
Optimizing Activity of the Nucleic Acid Molecule of the
Invention.
[0244] Chemically synthesizing nucleic acid molecules with
modifications (base, sugar and/or phosphate) can prevent their
degradation by serum ribonucleases, which can increase their
potency (see e.g., Eckstein et al., International Publication No.
WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al.,
1991, Science 253, 314; Usman and Cedergren, 1992, Trends in
Biochem. Sci. 17, 334; Usman et al., International Publication No.
WO 93/15187; and Rossi et al., International Publication No. WO
91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat.
No. 6,300,074; and Burgin et al., supra; all of which are
incorporated by reference herein). All of the above references
describe various chemical modifications that can be made to the
base, phosphate and/or sugar moieties of the nucleic acid molecules
described herein. Modifications that enhance their efficacy in
cells, and removal of bases from nucleic acid molecules to shorten
oligonucleotide synthesis times and reduce chemical requirements
are desired.
[0245] There are several examples in the art describing sugar, base
and phosphate modifications that can be introduced into nucleic
acid molecules with significant enhancement in their nuclease
stability and efficacy. For example, oligonucleotides are modified
to enhance stability and/or enhance biological activity by
modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide
base modifications (for a review see Usman and Cedergren, 1992,
TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163;
Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification
of nucleic acid molecules have been extensively described in the
art (see Eckstein et al., International Publication PCT No. WO
92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al.
Science, 1991, 253, 314-317; Usman and Cedergren, Trends in
Biochem. Sci., 1992, 17, 334-339; Usman et al. International
Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711
and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman
et al., International PCT publication No. WO 97/26270; Beigelman et
al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No.
5,627,053; Woolf et al., International PCT Publication No. WO
98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed
on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39,
1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences),
48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,
99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010;
all of the references are hereby incorporated in their totality by
reference herein). Such publications describe general methods and
strategies to determine the location of incorporation of sugar,
base and/or phosphate modifications and the like into nucleic acid
molecules without modulating catalysis, and are incorporated by
reference herein. In view of such teachings, similar modifications
can be used as described herein to modify the siNA nucleic acid
molecules of the instant invention so long as the ability of siNA
to promote RNAi is cells is not significantly inhibited.
[0246] While chemical modification of oligonucleotide
internucleotide linkages with phosphorothioate, phosphorodithioate,
and/or 5'-methylphosphonate linkages improves stability, excessive
modifications can cause some toxicity or decreased activity.
Therefore, when designing nucleic acid molecules, the amount of
these internucleotide linkages should be minimized. The reduction
in the concentration of these linkages should lower toxicity,
resulting in increased efficacy and higher specificity of these
molecules.
[0247] Short interfering nucleic acid (siNA) molecules having
chemical modifications that maintain or enhance activity are
provided. Such a nucleic acid is also generally more resistant to
nucleases than an unmodified nucleic acid. Accordingly, the in
vitro and/or in vivo activity should not be significantly lowered.
In cases in which modulation is the goal, therapeutic nucleic acid
molecules delivered exogenously should optimally be stable within
cells until translation of the target RNA has been modulated long
enough to reduce the levels of the undesirable protein. This period
of time varies between hours to days depending upon the disease
state. Improvements in the chemical synthesis of RNA and DNA
(Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et
al., 1992, Methods in Enzymology 211, 3-19 (incorporated by
reference herein)) have expanded the ability to modify nucleic acid
molecules by introducing nucleotide modifications to enhance their
nuclease stability, as described above.
[0248] In one embodiment, nucleic acid molecules of the invention
include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more) G-clamp nucleotides. A G-clamp nucleotide is a modified
cytosine analog wherein the modifications confer the ability to
hydrogen bond both Watson-Crick and Hoogsteen faces of a
complementary guanine within a duplex, see for example Lin and
Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single
G-clamp analog substitution within an oligonucleotide can result in
substantially enhanced helical thermal stability and mismatch
discrimination when hybridized to complementary oligonucleotides.
The inclusion of such nucleotides in nucleic acid molecules of the
invention results in both enhanced affinity and specificity to
nucleic acid targets, complementary sequences, or template strands.
In another embodiment, nucleic acid molecules of the invention
include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more) LNA "locked nucleic acid" nucleotides such as a 2',4'-C
methylene bicyclo nucleotide (see for example Wengel et al.,
International PCT Publication No. WO 00/66604 and WO 99/14226).
[0249] In another embodiment, the invention features conjugates
and/or complexes of siNA molecules of the invention. Such
conjugates and/or complexes can be used to facilitate delivery of
siNA molecules into a biological system, such as a cell. The
conjugates and complexes provided by the instant invention can
impart therapeutic activity by transferring therapeutic compounds
across cellular membranes, altering the pharmacokinetics, and/or
modulating the localization of nucleic acid molecules of the
invention. The present invention encompasses the design and
synthesis of novel conjugates and complexes for the delivery of
molecules, including, but not limited to, small molecules, lipids,
cholesterol, phospholipids, nucleosides, nucleotides, nucleic
acids, antibodies, toxins, negatively charged polymers and other
polymers, for example, proteins, peptides, hormones, carbohydrates,
polyethylene glycols, or polyamines, across cellular membranes. In
general, the transporters described are designed to be used either
individually or as part of a multi-component system, with or
without degradable linkers. These compounds are expected to improve
delivery and/or localization of nucleic acid molecules of the
invention into a number of cell types originating from different
tissues, in the presence or absence of serum (see Sullenger and
Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules
described herein can be attached to biologically active molecules
via linkers that are biodegradable, such as biodegradable nucleic
acid linker molecules.
[0250] The term "biodegradable linker" as used herein, refers to a
nucleic acid or non-nucleic acid linker molecule that is designed
as a biodegradable linker to connect one molecule to another
molecule, for example, a biologically active molecule to a siNA
molecule of the invention or the sense and antisense strands of a
siNA molecule of the invention. The biodegradable linker is
designed such that its stability can be modulated for a particular
purpose, such as delivery to a particular tissue or cell type. The
stability of a nucleic acid-based biodegradable linker molecule can
be modulated by using various chemistries, for example combinations
of ribonucleotides, deoxyribonucleotides, and chemically-modified
nucleotides, such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino,
2'-C-allyl, 2'-O-allyl, and other 2'-modified or base modified
nucleotides. The biodegradable nucleic acid linker molecule can be
a dimer, trimer, tetramer or longer nucleic acid molecule, for
example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or
can comprise a single nucleotide with a phosphorus-based linkage,
for example, a phosphoramidate or phosphodiester linkage. The
biodegradable nucleic acid linker molecule can also comprise
nucleic acid backbone, nucleic acid sugar, or nucleic acid base
modifications.
[0251] The term "biodegradable" as used herein, refers to
degradation in a biological system, for example enzymatic
degradation or chemical degradation.
[0252] The term "biologically active molecule" as used herein,
refers to compounds or molecules that are capable of eliciting or
modifying a biological response in a system. Non-limiting examples
of biologically active siNA molecules either alone or in
combination with other molecules contemplated by the instant
invention include therapeutically active molecules such as
antibodies, cholesterol, hormones, antivirals, peptides, proteins,
chemotherapeutics, small molecules, vitamins, co-factors,
nucleosides, nucleotides, oligonucleotides, enzymatic nucleic
acids, antisense nucleic acids, triplex forming oligonucleotides,
2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and
analogs thereof. Biologically active molecules of the invention
also include molecules capable of modulating the pharmacokinetics
and/or pharmacodynamics of other biologically active molecules, for
example, lipids and polymers such as polyamines, polyamides,
polyethylene glycol and other polyethers.
[0253] The term "phospholipid" as used herein, refers to a
hydrophobic molecule comprising at least one phosphorus group. For
example, a phospholipid can comprise a phosphorus-containing group
and saturated or unsaturated alkyl group, optionally substituted
with OH, COOH, oxo, amine, or substituted or unsubstituted aryl
groups.
[0254] Therapeutic nucleic acid molecules (e.g., siNA molecules)
delivered exogenously optimally are stable within cells until
reverse transcription of the RNA has been modulated long enough to
reduce the levels of the RNA transcript. The nucleic acid molecules
are resistant to nucleases in order to function as effective
intracellular therapeutic agents. Improvements in the chemical
synthesis of nucleic acid molecules described in the instant
invention and in the art have expanded the ability to modify
nucleic acid molecules by introducing nucleotide modifications to
enhance their nuclease stability as described above.
[0255] In yet another embodiment, siNA molecules having chemical
modifications that maintain or enhance enzymatic activity of
proteins involved in RNAi are provided. Such nucleic acids are also
generally more resistant to nucleases than unmodified nucleic
acids. Thus, in vitro and/or in vivo the activity should not be
significantly lowered.
[0256] Use of the nucleic acid-based molecules of the invention
will lead to better treatment of the disease progression by
affording the possibility of combination therapies (e.g., multiple
siNA molecules targeted to different genes; nucleic acid molecules
coupled with known small molecule modulators; or intermittent
treatment with combinations of molecules, including different
motifs and/or other chemical or biological molecules). The
treatment of subjects with siNA molecules can also include
combinations of different types of nucleic acid molecules, such as
enzymatic nucleic acid molecules (ribozymes), allozymes, antisense,
2,5-A oligoadenylate, decoys, and aptamers.
[0257] In another aspect a siNA molecule of the invention comprises
one or more 5' and/or a 3'-cap structure, for example on only the
sense siNA strand, the antisense siNA strand, or both siNA
strands.
[0258] By "cap structure" is meant chemical modifications, which
have been incorporated at either terminus of the oligonucleotide
(see, for example, Adamic et al., U.S. Pat. No. 5,998,203,
incorporated by reference herein). These terminal modifications
protect the nucleic acid molecule from exonuclease degradation, and
can help in delivery and/or localization within a cell. The cap can
be present at the 5'-terminus (5'-cap) or at the 3'-terminal
(3'-cap) or can be present on both termini. Non-limiting examples
of the 5'-cap include, but are not limited to, glyceryl, inverted
deoxy abasic residue (moiety); 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide;
carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide;
L-nucleotides; alpha-nucleotides; modified base nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl
nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted
nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol
phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate;
or bridging or non-bridging methylphosphonate moiety.
[0259] Non-limiting examples of the 3'-cap include, but are not
limited to, glyceryl, inverted deoxy abasic residue (moiety),
4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide;
4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl
phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or non
bridging methylphosphonate and 5'-mercapto moieties (for more
details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
[0260] By the term "non-nucleotide" is meant any group or compound
which can be incorporated into a nucleic acid chain in the place of
one or more nucleotide units, including either sugar and/or
phosphate substitutions, and allows the remaining bases to exhibit
their enzymatic activity. The group or compound is abasic in that
it does not contain a commonly recognized nucleotide base, such as
adenosine, guanine, cytosine, uracil or thymine and therefore lacks
a base at the 1'-position.
[0261] An "alkyl" group refers to a saturated aliphatic
hydrocarbon, including straight-chain, branched-chain, and cyclic
alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More
preferably, it is a lower alkyl of from 1 to 7 carbons, more
preferably 1 to 4 carbons. The alkyl group can be substituted or
unsubstituted. When substituted the substituted group(s) is
preferably, hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or
N(CH.sub.3).sub.2, amino, or SH. The term also includes alkenyl
groups that are unsaturated hydrocarbon groups containing at least
one carbon-carbon double bond, including straight-chain,
branched-chain, and cyclic groups. Preferably, the alkenyl group
has 1 to 12 carbons. More preferably, it is a lower alkenyl of from
1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group
may be substituted or unsubstituted. When substituted the
substituted group(s) is preferably, hydroxyl, cyano, alkoxy,
.dbd.O, .dbd.S, NO.sub.2, halogen, N(CH.sub.3).sub.2, amino, or SH.
The term "alkyl" also includes alkynyl groups that have an
unsaturated hydrocarbon group containing at least one carbon-carbon
triple bond, including straight-chain, branched-chain, and cyclic
groups. Preferably, the alkynyl group has 1 to 12 carbons. More
preferably, it is a lower alkynyl of from 1 to 7 carbons, more
preferably 1 to 4 carbons. The alkynyl group may be substituted or
unsubstituted. When substituted the substituted group(s) is
preferably, hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or
N(CH.sub.3).sub.2, amino or SH.
[0262] Such alkyl groups can also include aryl, alkylaryl,
carbocyclic aryl, heterocyclic aryl, amide and ester groups. An
"aryl" group refers to an aromatic group that has at least one ring
having a conjugated pi electron system and includes carbocyclic
aryl, heterocyclic aryl and biaryl groups, all of which may be
optionally substituted. The preferred substituent(s) of aryl groups
are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl,
alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to
an alkyl group (as described above) covalently joined to an aryl
group (as described above). Carbocyclic aryl groups are groups
wherein the ring atoms on the aromatic ring are all carbon atoms.
The carbon atoms are optionally substituted. Heterocyclic aryl
groups are groups having from 1 to 3 heteroatoms as ring atoms in
the aromatic ring and the remainder of the ring atoms are carbon
atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,
and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl
pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all
optionally substituted. An "amide" refers to an --C(O)--NH--R,
where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester"
refers to an --C(O)--OR, where R is either alkyl, aryl, alkylaryl
or hydrogen.
[0263] By "nucleotide" as used herein is as recognized in the art
to include natural bases (standard), and modified bases well known
in the art. Such bases are generally located at the 1' position of
a nucleotide sugar moiety. Nucleotides generally comprise a base,
sugar and a phosphate group. The nucleotides can be unmodified or
modified at the sugar, phosphate and/or base moiety, (also referred
to interchangeably as nucleotide analogs, modified nucleotides,
non-natural nucleotides, non-standard nucleotides and other; see,
for example, Usman and McSwiggen, supra; Eckstein et al.,
International PCT Publication No. WO 92/07065; Usman et al.,
International PCT Publication No. WO 93/15187; Uhlman & Peyman,
supra, all are hereby incorporated by reference herein). There are
several examples of modified nucleic acid bases known in the art as
summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
Some of the non-limiting examples of base modifications that can be
introduced into nucleic acid molecules include, inosine, purine,
pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,
2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,
naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),
5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,
5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
6-methyluridine), propyne, and others (Burgin et al., 1996,
Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modified
bases" in this aspect is meant nucleotide bases other than adenine,
guanine, cytosine and uracil at 1' position or their
equivalents.
[0264] In one embodiment, the invention features modified siNA
molecules, with phosphate backbone modifications comprising one or
more phosphorothioate, phosphorodithioate, methylphosphonate,
phosphotriester, morpholino, amidate carbamate, carboxymethyl,
acetamidate, polyamide, sulfonate, sulfonamide, sulfamate,
formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a
review of oligonucleotide backbone modifications, see Hunziker and
Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in
Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994,
Novel Backbone Replacements for Oligonucleotides, in Carbohydrate
Modifications in Antisense Research, ACS, 24-39.
[0265] By "abasic" is meant sugar moieties lacking a base or having
other chemical groups in place of a base at the 1' position, see
for example Adamic et al., U.S. Pat. No. 5,998,203.
[0266] By "unmodified nucleoside" is meant one of the bases
adenine, cytosine, guanine, thymine, or uracil joined to the 1'
carbon of .beta.-D-ribo-furanose.
[0267] By "modified nucleoside" is meant any nucleotide base which
contains a modification in the chemical structure of an unmodified
nucleotide base, sugar and/or phosphate. Non-limiting examples of
modified nucleotides are shown by Formulae I-VII and/or other
modifications described herein.
[0268] In connection with 2'-modified nucleotides as described for
the present invention, by "amino" is meant 2'-NH.sub.2 or
2'-O--NH.sub.2, which can be modified or unmodified. Such modified
groups are described, for example, in Eckstein et al., U.S. Pat.
No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878,
which are both incorporated by reference in their entireties.
[0269] Various modifications to nucleic acid siNA structure can be
made to enhance the utility of these molecules. Such modifications
will enhance shelf-life, half-life in vitro, stability, and ease of
introduction of such oligonucleotides to the target site, e.g., to
enhance penetration of cellular membranes, and confer the ability
to recognize and bind to targeted cells.
Administration of Nucleic Acid Molecules
[0270] A siNA molecule of the invention can be adapted for use to
treat, for example, oncology and proliferation related indications
and conditions such as multidrug resistant cancers, breast cancer,
cancers of the head and neck including various lymphomas such as
mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell
carcinoma, laryngeal carcinoma, cancers of the retina, cancers of
the esophagus, multiple myeloma, ovarian cancer, melanoma,
colorectal cancer, hepatocellular carcinoma, lung cancer, bladder
cancer, pancreatic cancer, prostate cancer, glioblastoma; obesity
and insulin resistance (e.g. type I and II diabetes); inflammatory
disorders such as asthma, septic shock, rheumatoid arthritis,
psoriasis, inflammatory bowl syndrome and any other diseases or
conditions that are related to or will respond to the levels of MAP
kinase in a cell or tissue, alone or in combination with other
therapies. For example, a siNA molecule can comprise a delivery
vehicle, including liposomes, for administration to a subject,
carriers and diluents and their salts, and/or can be present in
pharmaceutically acceptable formulations. Methods for the delivery
of nucleic acid molecules are described in Akhtar et al., 1992,
Trends Cell Bio., 2, 139; Delivery Strategies for Antisense
Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al.,
1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999,
Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS
Symp. Ser., 752, 184-192, all of which are incorporated herein by
reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan
et al., PCT WO 94/02595 further describe the general methods for
delivery of nucleic acid molecules. These protocols can be utilized
for the delivery of virtually any nucleic acid molecule. Nucleic
acid molecules can be administered to cells by a variety of methods
known to those of skill in the art, including, but not restricted
to, encapsulation in liposomes, by iontophoresis, or by
incorporation into other vehicles, such as biodegradable polymers,
hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,
Bioconjugate Chem., 10, 1068-1074), poly(lactic-co-glycolic)acid
(PLGA) and PLCA microspheres (see for example U.S. Pat. No.
6,447,796 and US Patent Application Publication No. US 2002130430),
biodegradable nanocapsules, and bioadhesive microspheres, or by
proteinaceous vectors (O'Hare and Normand, International PCT
Publication No. WO 00/53722). Alternatively, the nucleic
acid/vehicle combination is locally delivered by direct injection
or by use of an infusion pump. Direct injection of the nucleic acid
molecules of the invention, whether subcutaneous, intramuscular, or
intradermal, can take place using standard needle and syringe
methodologies, or by needle-free technologies such as those
described in Conry et al., 1999, Clin. Cancer Res., 5, 2330-2337
and Barry et al., International PCT Publication No. WO 99/31262.
The molecules of the instant invention can be used as
pharmaceutical agents. Pharmaceutical agents prevent, modulate the
occurrence, or treat (alleviate a symptom to some extent,
preferably all of the symptoms) of a disease state in a
subject.
[0271] Thus, the invention features a pharmaceutical composition
comprising one or more nucleic acid(s) of the invention in an
acceptable carrier, such as a stabilizer, buffer, and the like. The
polynucleotides of the invention can be administered (e.g., RNA,
DNA or protein) and introduced into a subject by any standard
means, with or without stabilizers, buffers, and the like, to form
a pharmaceutical composition. When it is desired to use a liposome
delivery mechanism, standard protocols for formation of liposomes
can be followed. The compositions of the present invention can also
be formulated and used as tablets, capsules or elixirs for oral
administration, suppositories for rectal administration, sterile
solutions, suspensions for injectable administration, and the other
compositions known in the art.
[0272] The present invention also includes pharmaceutically
acceptable formulations of the compounds described. These
formulations include salts of the above compounds, e.g., acid
addition salts, for example, salts of hydrochloric, hydrobromic,
acetic acid, and benzene sulfonic acid.
[0273] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
e.g., systemic administration, into a cell or subject, including
for example a human. Suitable forms, in part, depend upon the use
or the route of entry, for example oral, transdermal, or by
injection. Such forms should not prevent the composition or
formulation from reaching a target cell (i.e., a cell to which the
negatively charged nucleic acid is desirable for delivery). For
example, pharmacological compositions injected into the blood
stream should be soluble. Other factors are known in the art, and
include considerations such as toxicity and forms that prevent the
composition or formulation from exerting its effect.
[0274] By "systemic administration" is meant in vivo systemic
absorption or accumulation of drugs in the blood stream followed by
distribution throughout the entire body. Administration routes that
lead to systemic absorption include, without limitation:
intravenous, subcutaneous, intraperitoneal, inhalation, oral,
intrapulmonary and intramuscular. Each of these administration
routes exposes the siNA molecules of the invention to an accessible
diseased tissue. The rate of entry of a drug into the circulation
has been shown to be a function of molecular weight or size. The
use of a liposome or other drug carrier comprising the compounds of
the instant invention can potentially localize the drug, for
example, in certain tissue types, such as the tissues of the
reticular endothelial system (RES). A liposome formulation that can
facilitate the association of drug with the surface of cells, such
as, lymphocytes and macrophages is also useful. This approach can
provide enhanced delivery of the drug to target cells by taking
advantage of the specificity of macrophage and lymphocyte immune
recognition of abnormal cells, such as cells producing excess MAP
kinase.
[0275] By "pharmaceutically acceptable formulation" is meant a
composition or formulation that allows for the effective
distribution of the nucleic acid molecules of the instant invention
in the physical location most suitable for their desired activity.
Non-limiting examples of agents suitable for formulation with the
nucleic acid molecules of the instant invention include:
P-glycoprotein inhibitors (such as Pluronic P85), which can enhance
entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999,
Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such
as poly (DL-lactide-coglycolide) microspheres for sustained release
delivery after intracerebral implantation (Emerich, D F et al,
1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.);
and loaded nanoparticles, such as those made of
polybutylcyanoacrylate, which can deliver drugs across the blood
brain barrier and can alter neuronal uptake mechanisms (Prog
Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other
non-limiting examples of delivery strategies for the nucleic acid
molecules of the instant invention include material described in
Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al.,
1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA.,
92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107;
Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916;
and Tyler et al., 1999, PNAS USA., 96, 7053-7058.
[0276] The invention also features the use of the composition
comprising surface-modified liposomes containing poly (ethylene
glycol) lipids (PEG-modified, or long-circulating liposomes or
stealth liposomes). These formulations offer a method for
increasing the accumulation of drugs in target tissues. This class
of drug carriers resists opsonization and elimination by the
mononuclear phagocytic system (MPS or RES), thereby enabling longer
blood circulation times and enhanced tissue exposure for the
encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627;
Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such
liposomes have been shown to accumulate selectively in tumors,
presumably by extravasation and capture in the neovascularized
target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et
al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The
long-circulating liposomes enhance the pharmacokinetics and
pharmacodynamics of DNA and RNA, particularly compared to
conventional cationic liposomes which are known to accumulate in
tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,
24864-24870; Choi et al., International PCT Publication No. WO
96/10391; Ansell et al., International PCT Publication No. WO
96/10390; Holland et al., International PCT Publication No. WO
96/10392). Long-circulating liposomes are also likely to protect
drugs from nuclease degradation to a greater extent compared to
cationic liposomes, based on their ability to avoid accumulation in
metabolically aggressive MPS tissues such as the liver and
spleen.
[0277] The present invention also includes compositions prepared
for storage or administration that include a pharmaceutically
effective amount of the desired compounds in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated
by reference herein. For example, preservatives, stabilizers, dyes
and flavoring agents can be provided. These include sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In
addition, antioxidants and suspending agents can be used.
[0278] A pharmaceutically effective dose is that dose required to
prevent, inhibit the occurrence, or treat (alleviate a symptom to
some extent, preferably all of the symptoms) of a disease state.
The pharmaceutically effective dose depends on the type of disease,
the composition used, the route of administration, the type of
mammal being treated, the physical characteristics of the specific
mammal under consideration, concurrent medication, and other
factors that those skilled in the medical arts will recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body
weight/day of active ingredients is administered dependent upon
potency of the negatively charged polymer.
[0279] The nucleic acid molecules of the invention and formulations
thereof can be administered orally, topically, parenterally, by
inhalation or spray, or rectally in dosage unit formulations
containing conventional non-toxic pharmaceutically acceptable
carriers, adjuvants and/or vehicles. The term parenteral as used
herein includes percutaneous, subcutaneous, intravascular (e.g.,
intravenous), intramuscular, or intrathecal injection or infusion
techniques and the like. In addition, there is provided a
pharmaceutical formulation comprising a nucleic acid molecule of
the invention and a pharmaceutically acceptable carrier. One or
more nucleic acid molecules of the invention can be present in
association with one or more non-toxic pharmaceutically acceptable
carriers and/or diluents and/or adjuvants, and if desired other
active ingredients. The pharmaceutical compositions containing
nucleic acid molecules of the invention can be in a form suitable
for oral use, for example, as tablets, troches, lozenges, aqueous
or oily suspensions, dispersible powders or granules, emulsion,
hard or soft capsules, or syrups or elixirs.
[0280] Compositions intended for oral use can be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions can contain one
or more such sweetening agents, flavoring agents, coloring agents
or preservative agents in order to provide pharmaceutically elegant
and palatable preparations. Tablets contain the active ingredient
in admixture with non-toxic pharmaceutically acceptable excipients
that are suitable for the manufacture of tablets. These excipients
can be, for example, inert diluents; such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia; and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets can be uncoated or they can be
coated by known techniques. In some cases such coatings can be
prepared by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monostearate or glyceryl distearate can be
employed.
[0281] Formulations for oral use can also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0282] Aqueous suspensions contain the active materials in a
mixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0283] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions can contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
and flavoring agents can be added to provide palatable oral
preparations. These compositions can be preserved by the addition
of an anti-oxidant such as ascorbic acid
[0284] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents or suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, can also be present.
[0285] Pharmaceutical compositions of the invention can also be in
the form of oil-in-water emulsions. The oily phase can be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents can be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions can also contain sweetening and flavoring
agents.
[0286] Syrups and elixirs can be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations can also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions can be in the form of a sterile injectable aqueous or
oleaginous suspension. This suspension can be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents that have been mentioned above. The sterile
injectable preparation can also be a sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that can be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil can be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0287] The nucleic acid molecules of the invention can also be
administered in the form of suppositories, e.g., for rectal
administration of the drug. These compositions can be prepared by
mixing the drug with a suitable non-irritating excipient that is
solid at ordinary temperatures but liquid at the rectal temperature
and will therefore melt in the rectum to release the drug. Such
materials include cocoa butter and polyethylene glycols.
[0288] Nucleic acid molecules of the invention can be administered
parenterally in a sterile medium. The drug, depending on the
vehicle and concentration used, can either be suspended or
dissolved in the vehicle. Advantageously, adjuvants such as local
anesthetics, preservatives and buffering agents can be dissolved in
the vehicle.
[0289] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
of the above-indicated conditions (about 0.5 mg to about 7 g per
subject per day). The amount of active ingredient that can be
combined with the carrier materials to produce a single dosage form
varies depending upon the host treated and the particular mode of
administration. Dosage unit forms generally contain between from
about 1 mg to about 500 mg of an active ingredient.
[0290] It is understood that the specific dose level for any
particular subject depends upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, sex, diet, time of administration, route of
administration, and rate of excretion, drug combination and the
severity of the particular disease undergoing therapy.
[0291] For administration to non-human animals, the composition can
also be added to the animal feed or drinking water. It can be
convenient to formulate the animal feed and drinking water
compositions so that the animal takes in a therapeutically
appropriate quantity of the composition along with its diet. It can
also be convenient to present the composition as a premix for
addition to the feed or drinking water.
[0292] The nucleic acid molecules of the present invention can also
be administered to a subject in combination with other therapeutic
compounds to increase the overall therapeutic effect. The use of
multiple compounds to treat an indication can increase the
beneficial effects while reducing the presence of side effects.
[0293] In one embodiment, the invention comprises compositions
suitable for administering nucleic acid molecules of the invention
to specific cell types. For example, the asialoglycoprotein
receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432)
is unique to hepatocytes and binds branched galactose-terminal
glycoproteins, such as asialoorosomucoid (ASOR). In another
example, the folate receptor is overexpressed in many cancer cells.
Binding of such glycoproteins, synthetic glycoconjugates, or
folates to the receptor takes place with an affinity that strongly
depends on the degree of branching of the oligosaccharide chain,
for example, triatennary structures are bound with greater affinity
than biatenarry or monoatennary chains (Baenziger and Fiete, 1980,
Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257,
939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328,
obtained this high specificity through the use of
N-acetyl-D-galactosamine as the carbohydrate moiety, which has
higher affinity for the receptor, compared to galactose. This
"clustering effect" has also been described for the binding and
uptake of mannosyl-terminating glycoproteins or glycoconjugates
(Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of
galactose, galactosamine, or folate based conjugates to transport
exogenous compounds across cell membranes can provide a targeted
delivery approach to, for example, the treatment of liver disease,
cancers of the liver, or other cancers. The use of bioconjugates
can also provide a reduction in the required dose of therapeutic
compounds required for treatment. Furthermore, therapeutic
bioavailability, pharmacodynamics, and pharmacokinetic parameters
can be modulated through the use of nucleic acid bioconjugates of
the invention. Non-limiting examples of such bioconjugates are
described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Aug.
13, 2001; and Matulic-Adamic et al., U.S. Ser. No. 60/362,016,
filed Mar. 6, 2002.
[0294] Alternatively, certain siNA molecules of the instant
invention can be expressed within cells from eukaryotic promoters
(e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and
Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et
al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet
et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992,
J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65,
5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89,
10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver
et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995,
Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,
45. Those skilled in the art realize that any nucleic acid can be
expressed in eukaryotic cells from the appropriate DNA/RNA vector.
The activity of such nucleic acids can be augmented by their
release from the primary transcript by an enzymatic nucleic acid
(Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO
94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6;
Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et
al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994,
J. Biol. Chem., 269, 25856.
[0295] In another aspect of the invention, RNA molecules of the
present invention can be expressed from transcription units (see
for example Couture et al., 1996, TIG., 12, 510) inserted into DNA
or RNA vectors. The recombinant vectors can be DNA plasmids or
viral vectors. siNA expressing viral vectors can be constructed
based on, but not limited to, adeno-associated virus, retrovirus,
adenovirus, or alphavirus. In another embodiment, pol III based
constructs are used to express nucleic acid molecules of the
invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and
6,146,886). The recombinant vectors capable of expressing the siNA
molecules can be delivered as described above, and persist in
target cells. Alternatively, viral vectors can be used that provide
for transient expression of nucleic acid molecules. Such vectors
can be repeatedly administered as necessary. Once expressed, the
siNA molecule interacts with the target mRNA and generates an RNAi
response. Delivery of siNA molecule expressing vectors can be
systemic, such as by intravenous or intramuscular administration,
by administration to target cells ex-planted from a subject
followed by reintroduction into the subject, or by any other means
that would allow for introduction into the desired target cell (for
a review see Couture et al., 1996, TIG., 12, 510).
[0296] In one aspect the invention features an expression vector
comprising a nucleic acid sequence encoding at least one siNA
molecule of the instant invention. The expression vector can encode
one or both strands of a siNA duplex, or a single
self-complementary strand that self hybridizes into a siNA duplex.
The nucleic acid sequences encoding the siNA molecules of the
instant invention can be operably linked in a manner that allows
expression of the siNA molecule (see for example Paul et al., 2002,
Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature
Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19,
500; and Novina et al., 2002, Nature Medicine, advance online
publication doi:10.1038/nm725).
[0297] In another aspect, the invention features an expression
vector comprising: a) a transcription initiation region (e.g.,
eukaryotic pol I, II or III initiation region); b) a transcription
termination region (e.g., eukaryotic pol I, II or III termination
region); and c) a nucleic acid sequence encoding at least one of
the siNA molecules of the instant invention, wherein said sequence
is operably linked to said initiation region and said termination
region in a manner that allows expression and/or delivery of the
siNA molecule. The vector can optionally include an open reading
frame (ORF) for a protein operably linked on the 5' side or the
3'-side of the sequence encoding the siNA of the invention and/or
an intron (intervening sequences).
[0298] Transcription of the siNA molecule sequences can be driven
from a promoter for eukaryotic RNA polymerase I (pol I), RNA
polymerase II (pol II), or RNA polymerase III (pol III).
Transcripts from pol II or pol III promoters are expressed at high
levels in all cells; the levels of a given pol II promoter in a
given cell type depends on the nature of the gene regulatory
sequences (enhancers, silencers, etc.) present nearby. Prokaryotic
RNA polymerase promoters are also used, providing that the
prokaryotic RNA polymerase enzyme is expressed in the appropriate
cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87,
6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber
et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol.
Cell. Biol., 10, 4529-37). Several investigators have demonstrated
that nucleic acid molecules expressed from such promoters can
function in mammalian cells (e.g. Kashani-Sabet et al., 1992,
Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl.
Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res.,
20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90,
6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et
al., 1993, Proc. Natl. Acad. Sci. U.S. A, 90, 8000-4; Thompson et
al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,
1993, Science, 262, 1566). More specifically, transcription units
such as the ones derived from genes encoding U6 small nuclear
(snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in
generating high concentrations of desired RNA molecules such as
siNA in cells (Thompson et al., supra; Couture and Stinchcomb,
1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830;
Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene
Ther., 4, 45; Beigelman et al., International PCT Publication No.
WO 96/18736. The above siNA transcription units can be incorporated
into a variety of vectors for introduction into mammalian cells,
including but not restricted to, plasmid DNA vectors, viral DNA
vectors (such as adenovirus or adeno-associated virus vectors), or
viral RNA vectors (such as retroviral or alphavirus vectors) (for a
review see Couture and Stinchcomb, 1996, supra).
[0299] In another aspect the invention features an expression
vector comprising a nucleic acid sequence encoding at least one of
the siNA molecules of the invention in a manner that allows
expression of that siNA molecule. The expression vector comprises
in one embodiment; a) a transcription initiation region; b) a
transcription termination region; and c) a nucleic acid sequence
encoding at least one strand of the siNA molecule, wherein the
sequence is operably linked to the initiation region and the
termination region in a manner that allows expression and/or
delivery of the siNA molecule.
[0300] In another embodiment the expression vector comprises: a) a
transcription initiation region; b) a transcription termination
region; c) an open reading frame; and d) a nucleic acid sequence
encoding at least one strand of a siNA molecule, wherein the
sequence is operably linked to the 3'-end of the open reading frame
and wherein the sequence is operably linked to the initiation
region, the open reading frame and the termination region in a
manner that allows expression and/or delivery of the siNA molecule.
In yet another embodiment, the expression vector comprises: a) a
transcription initiation region; b) a transcription termination
region; c) an intron; and d) a nucleic acid sequence encoding at
least one siNA molecule, wherein the sequence is operably linked to
the initiation region, the intron and the termination region in a
manner which allows expression and/or delivery of the nucleic acid
molecule.
[0301] In another embodiment, the expression vector comprises: a) a
transcription initiation region; b) a transcription termination
region; c) an intron; d) an open reading frame; and e) a nucleic
acid sequence encoding at least one strand of a siNA molecule,
wherein the sequence is operably linked to the 3'-end of the open
reading frame and wherein the sequence is operably linked to the
initiation region, the intron, the open reading frame and the
termination region in a manner which allows expression and/or
delivery of the siNA molecule.
MAP Kinase Biology and Biochemistry
[0302] The mitogen-activated protein kinases (MAPKs) have been at
the forefront of a rapid advance in the understanding of cellular
events in growth factor and cytokine receptor signaling. The MAP
kinases (also referred to as extracellular signal-regulated protein
kinases, or ERKs) are the terminal enzymes in a three-kinase
cascade. The reiteration of three-kinase cascades for related but
distinct signaling pathways gave rise to the concept of a MAPK
pathway as a modular, multifunctional signaling element that acts
sequentially within one pathway, where each enzyme phosphorylates
and thereby activates the next member in the sequence. A typical
MAPK pathway thus consists of three protein kinases: a MAPK kinase
kinase (or MEKK) that activates a MAPK kinase (or MEK) which, in
turn, activates a MAPK/ERK enzyme. Each of the MAPK/ERK, JNK and
p38 cascades consists of a three-enzyme module that includes MEKK,
MEK and an ERK or MAPK superfamily member. A variety of
extracellular signals triggers initial events upon association with
their respective cell surface receptors and this signal is then
transmitted to the interior of the cell where it activates the
appropriate cascades (see for example FIG. 12).
[0303] The identification of distinct MAPK cascades that are
conserved across all eukaryotes indicates that the MAPK module has
been adapted for interpretation of a diverse array of extracellular
signals. Although mitogen activation of the MAPK subfamily (e.g.,
ERK1 and ERK2) has dominated efforts to understand MAPK signaling,
increasing appreciation of the role of the stress-activated
kinases, JNK and p38, illustrates the diverse nature of the MAPK
superfamily of enzymes. Although sequence similarities among
components of the individual MAPK modules used for activation of
ERK1/2, JNKs and p38 are considerable, the fidelity that is
maintained in order to translate specific extracellular signals
into discrete physiological responses illustrates the selective
adaptation of each MAPK pathway. The MAPK superfamily of enzymes is
a critical component cellular regulative processes that coordinates
incoming signals generated by a variety of extracellular and
intracellular mediators. Specific phosphorylation and activation of
enzymes in the MAPK pathway transmits the signal down the cascade,
resulting in phosphorylation of many proteins with substantial
regulatory functions throughout the cell, including other protein
kinases, transcription factors, cytoskeletal proteins and other
enzymes. The diversity of signals that culminates in MAPK
activation indicates that these enzymes are not dedicated to
regulation of any single growth factor, hormone or cytokine system.
Instead, MAPKs--like cAMP-dependent protein kinase (PKA) and
Ca.sup.2+- and phospholipid-dependent protein kinases (PKC) serve
many signaling purposes. Because activation of the MAPK pathways
are triggered to varying extents by a large number of receptor
systems, temporal and spatial differences are critical to
determining ligand- and cell-type-specific functions.
[0304] Following activation of cells with an appropriate
extracellular stimuli, the signal is transmitted to the canonical
MAPK module comprising three protein kinases. The progression of
events for each enzyme cascade is the same, although specific
isoforms of each enzyme appear to confer the required specificity
within each pathway. The first enzyme in the module is a MEKK
enzyme, of which Raf and its isoforms are one example. The MEKK
enzymes comprise Ser/Thr protein kinases that activate the MEK
enzymes by phosphorylating two serine or threonine residues within
a Ser-X-X-X-Ser/Thr motif. Once activated, the MEK enzymes, which
are hybrid function Ser/Thr/Tyr protein kinases, phosphorylate the
MAPK/ERK enzymes on Thr and Tyr residues within the Thr-X-Tyr (TXY)
consensus sequence. A critical and common feature of the MAPK
superfamily of enzymes is that they are activated upon dual
phosphorylation within a TXY consensus sequence present in the
activation loop of the catalytic domain. The central amino acid
differs for each MAPK superfamily member, corresponding to Glu for
ERK1/2, Gly for p38/HOG and Pro for JNK/SAPK, although MEK
specificity is not limited to these particular residues.
Phosphorylation at only one of the two positions does not appear to
activate the enzyme, although it may prime the kinase domain for
receipt of the second phosphorylation event.
[0305] ERK1 and ERK2 were the first members of the MAPK superfamily
whose cDNAs were cloned and the signaling cascades that lead to
their activation characterized. Potent activation of ERK1 and ERK2
can be initiated through activation of transmembrane receptors with
intrinsic protein tyrosine kinase (PTK) activity. Binding of
extracellular ligands to their respective cell surface receptors
results in receptor autophosphorylation and enhanced PTK activity.
The subsequent association of the Src homology 2 (SH2) domains of
adaptor proteins such as Grb2 and Shc with the autophosphorylated
receptors, or with additional docking proteins, provides the
molecular interactions that bring the required signal transduction
molecules into close proximity with each other. Receptors without
intrinsic PTK activity but which comprise sites for tyrosine
phosphorylation can also activate the cascade via association of
their phosphotyrosine residues with adaptor molecules. For example,
the SH3 domain of Grb2 binds a proline-rich region of the guanine
nucleotide-exchange protein SOS which, in turn, increases the
association of Ras with GTP. The GTP-bound form of Ras binds to Raf
(a MAPK kinase) isoforms, including C-Raf-1, B-Raf and A-Raf. This
action targets Raf to the membrane, where its protein kinase
activity is increased by phosphorylation. MAPK kinases (MEK1 and
MEK2), are phosphorylated and activated by Raf. MEK1 and MEK2 are
dual-specificity protein kinases that dually phosphorylate the ERK
enzymes (corresponding to Thr.sup.183 and Tyr.sup.185 of p42ERK2),
thereby increasing their enzymatic activity by approximately
1,000-fold over the activity found with the basal or
monophosphorylated forms. Phosphorylation of these residues causes
closure of the kinase active site and induces conformational
changes necessary for high activity.
[0306] MAPK mutants, lacking either a lysine required for catalytic
activity or the prerequisite TXY phosphorylation sites, can inhibit
signaling by the native enzymes in cells. In the case of ERK1 and
ERK2, these mutants have been used with repeated success. For
example, mutant ERK2 completely blocks proliferation in response to
epidermal growth factor (EGF) and v-Raf, and partially blocks
induction by serum or small t antigen. ERK1 antisense mRNA and an
ERK1 phosphorylation site mutants interfere with thrombin-induced
transcription as well as serum-dependent proliferation. These
findings suggest an essential role in proliferation and
transformation for the ERK/MAPK pathway.
[0307] The JNK/SAPK and p38/HOG pathways are activated by
ultraviolet light, cytokines, osmotic shock, inhibitors of DNA,
RNA, and protein synthesis, and to a lesser extent by certain
growth factors. This spectrum of regulators suggests that the
enzymes are transducers of a variety of cellular stress responses.
In contrast to activation of ERK1 and ERK2, upstream signal
transduction mechanisms for the JNK and p38 cascades are less well
understood. When transfected into mammalian cells, a diverse group
of protein kinases including the mixed lineage kinases (MLKs) and
relatives of the yeast Step 20p, such as the p21-activated kinases
(PAKs) and germinal center kinase (GCK), cause activation of
JNK/SAPK. Similarly, GTP-bound forms of the small GTP-binding
proteins, Rac and Cdc42, activate the JNK/SAPK pathway and, to a
lesser extent, the p38 pathway. Direct activation of both pathways
by PAKs also has been demonstrated, suggesting that PAKs can be the
relevant effectors for these small G proteins. The PAKs are
homologs of the yeast kinases Step 20p and Shk1, enzymes upstream
of the MAPK modules in yeast pheromone response pathways. Both
yeast and mammalian protein kinases contain a binding site for
Rac/Cdc42 and share the property of being activated in vitro
through association with these small G proteins when in their
GTP-bound states. In yeast, Step 20p is thought to phosphorylate
and activate the MEKK isoform Ste11p, suggesting that MEKKs may be
PAK targets. This summary of MAP kinase pathways has been adapted
from Cobb and Schaefer, 1996, Promega Notes Magazine Number 59,
page 37.
[0308] The regulation of c-Jun transcriptional activity by Jun
N-terminal kinase (JNK), ERK1, ERK2, and p38 kinases has become a
paradigm for the understanding of how mitogen-activated protein
(MAP) kinase signaling pathways elicit specific changes in gene
transcription through selective phosphorylation of nuclear
transcription factors. Selective phosphorylation of c-Jun by JNK is
detected by a specific docking motif in c-Jun, the delta region,
which enables JNK to physically interact with c-Jun. Analogous MAP
kinase docking motifs have subsequently been found in several other
transcription factors, indicating that this is a general mechanism
for ensuring the specificity of signal transduction. Furthermore,
genetic and biochemical studies in mice, flies and cultured cells
have provided evidence that signals relayed by JNK through c-Jun
regulate a wide range of cellular processes including cell
proliferation, tumorigenesis, apoptosis and embryonic development.
Despite these advances, in most cases, the genes or programs of
gene expression downstream of JNK and c-Jun, which control these
processes, have yet to be defined. One important process that is
associated with JNK gene expression is the development of insulin
resistance in obese subjects.
[0309] Obesity is closely associated with insulin resistance and
establishes the leading risk factor for type 2 diabetes mellitus in
mammals. The c-Jun amino-terminal kinases (JNKs) can interfere with
insulin activity in cultured cells and are activated by
inflammatory cytokines and free fatty acids molecules that have
been implicated in the development of type 2 diabetes. Hirosumi et
al, 2002, Nature, 420, 333-336, demonstrate that JNK activity is
abnormally elevated in obesity. Furthermore, Hirosumi et al, supra
have shown that an absence of JNK1 results in decreased adiposity
with significantly improved insulin sensitivity and enhanced
insulin receptor capacity in two different models of mouse obesity.
Thus, JNK is a crucial mediator of obesity and insulin resistance
and as such, provides a potential target for nucleic acid based
therapeutics that modulate JNK gene expression.
[0310] The transcription factor and oncogene, c-JUN, is implicated
in several critical cell processes including cell proliferation,
cell survival, and oncogenic transformation. Although it is broadly
expressed in a wide variety of cell types, it plays an especially
important role in hepatocytes. However, the precise role played by
c-JUN in hepatocytes seems to depend on the differentiation state
of this cell type. Adult differentiated hepatocytes depend on c-JUN
for progression through the cell cycle. Deletion of c-JUN reduces
the proliferation capacity of hepatocytes following partial
hepatectomy. c-JUN is thought to be major component in the
development of human hepatocellular carcinoma (HCC). HCC is the
most common form of primary liver cancer. Chronic HCV infection is
a major risk factor for HCC.
[0311] The role of c-JUN in liver cancer has recently been
investigated (Eferl et al., 2003, Cell, 112, 181). These
investigators deleted c-JUN and then induced liver cancer by
chemical carcinogenesis. They observed that deletion of c-JUN
dramatically interfered with liver tumor formation. Animal survival
was markedly worse in c-JUN wild-type animals relative to deletion
mutants. In particular, the number of apoptotic cells increased
about five fold in tumors in the c-JUN deletion strain relative to
the wild-type animals. Importantly, levels of the pro-apoptotic
gene products such as p53 and noxa were elevated in the c-JUN
deletion strain. c-JUN is likely to antagonize other pro-apoptotic
genes such as TNF-a. Thus, by blocking p53 and its large family of
dependent genes, c-JUN seems to promote tumor formation. Since a
large fraction of chronically infected HCV patients develop
hepatocellular carcinoma, c-JUN provides an attractive target for
treating HCV infected patients to prevent or ameliorate
hepatocellular carcinoma.
[0312] Based upon the current understanding of MAP kinase pathways,
the modulation of MAP kinase pathways is instrumental in the
development of new therapeutics in, for example, the fields of
inflammation, oncology, and metabolism. As such, modulation of a
specific MAP kinase pathway using small interfering nucleic acid
(siNA) mediated RNAi represents a novel approach to the treatment
and study of diseases and conditions related to a specific MAP
kinase activity and/or gene expression.
EXAMPLES
[0313] The following are non-limiting examples showing the
selection, isolation, synthesis and activity of nucleic acids of
the instant invention.
Example 1
Tandem Synthesis of siNA Constructs
[0314] Exemplary siNA molecules of the invention are synthesized in
tandem using a cleavable linker, for example, a succinyl-based
linker. Tandem synthesis as described herein is followed by a
one-step purification process that provides RNAi molecules in high
yield. This approach is highly amenable to siNA synthesis in
support of high throughput RNAi screening, and can be readily
adapted to multi-column or multi-well synthesis platforms.
[0315] After completing a tandem synthesis of a siNA oligo and its
complement in which the 5'-terminal dimethoxytrityl (5'-O-DMT)
group remains intact (trityl on synthesis), the oligonucleotides
are deprotected as described above. Following deprotection, the
siNA sequence strands are allowed to spontaneously hybridize. This
hybridization yields a duplex in which one strand has retained the
5'-O-DMT group while the complementary strand comprises a terminal
5'-hydroxyl. The newly formed duplex behaves as a single molecule
during routine solid-phase extraction purification (Trityl-On
purification) even though only one molecule has a dimethoxytrityl
group. Because the strands form a stable duplex, this
dimethoxytrityl group (or an equivalent group, such as other trityl
groups or other hydrophobic moieties) is all that is required to
purify the pair of oligos, for example, by using a C18
cartridge.
[0316] Standard phosphoramidite synthesis chemistry is used up to
the point of introducing a tandem linker, such as an inverted deoxy
abasic succinate or glyceryl succinate linker (see FIG. 1) or an
equivalent cleavable linker. A non-limiting example of linker
coupling conditions that can be used includes a hindered base such
as diisopropylethylamine (DIPA) and/or DMAP in the presence of an
activator reagent such as
Bromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After
the linker is coupled, standard synthesis chemistry is utilized to
complete synthesis of the second sequence leaving the terminal the
5'-O-DMT intact. Following synthesis, the resulting oligonucleotide
is deprotected according to the procedures described herein and
quenched with a suitable buffer, for example with 50 mM NaOAc or
1.5M NH.sub.4H.sub.2CO.sub.3.
[0317] Purification of the siNA duplex can be readily accomplished
using solid phase extraction, for example using a Waters C18 SepPak
Ig cartridge conditioned with 1 column volume (CV) of acetonitrile,
2 CV H.sub.2O, and 2 CV 50 mM NaOAc. The sample is loaded and then
washed with 1 CV H.sub.2O or 50 mM NaOAc. Failure sequences are
eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl).
The column is then washed, for example with 1 CV H.sub.2O followed
by on-column detritylation, for example by passing 1 CV of 1%
aqueous trifluoroacetic acid (TFA) over the column, then adding a
second CV of 1% aqueous TFA to the column and allowing to stand for
approximately 10 minutes. The remaining TFA solution is removed and
the column washed with H.sub.2O followed by 1 CV 1M NaCl and
additional H.sub.2O. The siNA duplex product is then eluted, for
example, using 1 CV 20% aqueous CAN.
[0318] FIG. 2 provides an example of MALDI-TOF mass spectrometry
analysis of a purified siNA construct in which each peak
corresponds to the calculated mass of an individual siNA strand of
the siNA duplex. The same purified siNA provides three peaks when
analyzed by capillary gel electrophoresis (CGE), one peak
presumably corresponding to the duplex siNA, and two peaks
presumably corresponding to the separate siNA sequence strands. Ion
exchange HPLC analysis of the same siNA contract only shows a
single peak. Testing of the purified siNA construct using a
luciferase reporter assay described below demonstrated the same
RNAi activity compared to siNA constructs generated from separately
synthesized oligonucleotide sequence strands.
Example 2
Identification of Potential siNA Target Sites in Any RNA
Sequence
[0319] The sequence of an RNA target of interest, such as a viral
or human mRNA transcript, is screened for target sites, for example
by using a computer folding algorithm. In a non-limiting example,
the sequence of a gene or RNA gene transcript derived from a
database, such as Genbank, is used to generate siNA targets having
complementarity to the target. Such sequences can be obtained from
a database, or can be determined experimentally as known in the
art. Target sites that are known, for example, those target sites
determined to be effective target sites based on studies with other
nucleic acid molecules, for example ribozymes or antisense, or
those targets known to be associated with a disease or condition
such as those sites containing mutations or deletions, can be used
to design siNA molecules targeting those sites. Various parameters
can be used to determine which sites are the most suitable target
sites within the target RNA sequence. These parameters include but
are not limited to secondary or tertiary RNA structure, the
nucleotide base composition of the target sequence, the degree of
homology between various regions of the target sequence, or the
relative position of the target sequence within the RNA transcript.
Based on these determinations, any number of target sites within
the RNA transcript can be chosen to screen siNA molecules for
efficacy, for example by using in vitro RNA cleavage assays, cell
culture, or animal models. In a non-limiting example, anywhere from
1 to 1000 target sites are chosen within the transcript based on
the size of the siNA construct to be used. High throughput
screening assays can be developed for screening siNA molecules
using methods known in the art, such as with multi-well or
multi-plate assays to determine efficient reduction in target gene
expression.
Example 3
Selection of siNA Molecule Target Sites in a RNA
[0320] The following non-limiting steps can be used to carry out
the selection of siNAs targeting a given gene sequence or
transcript.
[0321] 1. The target sequence is parsed in silico into a list of
all fragments or subsequences of a particular length, for example
23 nucleotide fragments, contained within the target sequence. This
step is typically carried out using a custom Perl script, but
commercial sequence analysis programs such as Oligo, MacVector, or
the GCG Wisconsin Package can be employed as well.
[0322] 2. In some instances the siNAs correspond to more than one
target sequence; such would be the case for example in targeting
different transcripts of the same gene, targeting different
transcripts of more than one gene, or for targeting both the human
gene and an animal homolog. In this case, a subsequence list of a
particular length is generated for each of the targets, and then
the lists are compared to find matching sequences in each list. The
subsequences are then ranked according to the number of target
sequences that contain the given subsequence; the goal is to find
subsequences that are present in most or all of the target
sequences. Alternately, the ranking can identify subsequences that
are unique to a target sequence, such as a mutant target sequence.
Such an approach would enable the use of siNA to target
specifically the mutant sequence and not effect the expression of
the normal sequence.
[0323] 3. In some instances the siNA subsequences are absent in one
or more sequences while present in the desired target sequence;
such would be the case if the siNA targets a gene with a paralogous
family member that is to remain untargeted. As in case 2 above, a
subsequence list of a particular length is generated for each of
the targets, and then the lists are compared to find sequences that
are present in the target gene but are absent in the untargeted
paralog.
[0324] 4. The ranked siNA subsequences can be further analyzed and
ranked according to GC content. A preference can be given to sites
containing 30-70% GC, with a further preference to sites containing
40-60% GC.
[0325] 5. The ranked siNA subsequences can be further analyzed and
ranked according to self-folding and internal hairpins. Weaker
internal folds are preferred; strong hairpin structures are to be
avoided.
[0326] 6. The ranked siNA subsequences can be further analyzed and
ranked according to whether they have runs of GGG or CCC in the
sequence. GGG (or even more Gs) in either strand can make
oligonucleotide synthesis problematic and can potentially interfere
with RNAi activity, so it is avoided whenever better sequences are
available. CCC is searched in the target strand because that will
place GGG in the antisense strand.
[0327] 7. The ranked siNA subsequences can be further analyzed and
ranked according to whether they have the dinucleotide UU (uridine
dinucleotide) on the 3'-end of the sequence, and/or AA on the
5'-end of the sequence (to yield 3' UU on the antisense sequence).
These sequences allow one to design siNA molecules with terminal TT
thymidine dinucleotides.
[0328] 8. Four or five target sites are chosen from the ranked list
of subsequences as described above. For example, in subsequences
having 23 nucleotides, the right 21 nucleotides of each chosen
23-mer subsequence are then designed and synthesized for the upper
(sense) strand of the siNA duplex, while the reverse complement of
the left 21 nucleotides of each chosen 23-mer subsequence are then
designed and synthesized for the lower (antisense) strand of the
siNA duplex (see Tables II and III). If terminal TT residues are
desired for the sequence (as described in paragraph 7), then the
two 3' terminal nucleotides of both the sense and antisense strands
are replaced by TT prior to synthesizing the oligos.
[0329] 9. The siNA molecules are screened in an in vitro, cell
culture or animal model system to identify the most active siNA
molecule or the most preferred target site within the target RNA
sequence.
[0330] In an alternate approach, a pool of siNA constructs specific
to a MAP kinase target sequence is used to screen for target sites
in cells expressing MAP kinase (e.g., c-JUN) RNA, such as human
kidney fibroblast (e.g., 293 cells), HeLa, or HepG2 cells. The
general strategy used in this approach is shown in FIG. 9. A
non-limiting example of such as pool is a pool comprising sequences
having sense sequences comprising SEQ ID NOs. 1247-1427 and
antisense sequences comprising SEQ ID NOs. 1428-1608 respectively.
293, HeLa, or HepG2 cells expressing MAP kinase (e.g., c-JUN) are
transfected with the pool of siNA constructs and cells that
demonstrate a phenotype associated with MAP kinase (e.g., c-JUN)
inhibition are sorted. The pool of siNA constructs can be expressed
from transcription cassettes inserted into appropriate vectors (see
for example FIG. 7 and FIG. 8). The siNA from cells demonstrating a
positive phenotypic change (e.g., decreased proliferation,
decreased MAP kinase (e.g., c-JUN) mRNA levels or decreased MAP
kinase (e.g., c-JUN) protein expression), are sequenced to
determine the most suitable target site(s) within the target MAP
kinase (e.g., c-JUN) RNA sequence.
Example 4
MAP Kinase Targeted siNA Design
[0331] siNA target sites were chosen by analyzing sequences of the
MAP kinase (e.g., c-JUN) RNA target and optionally prioritizing the
target sites on the basis of folding (structure of any given
sequence analyzed to determine siNA accessibility to the target),
by using a library of siNA molecules as described in Example 3, or
alternately by using an in vitro siNA system as described in
Example 6 herein. siNA molecules were designed that could bind each
target and are optionally individually analyzed by computer folding
to assess whether the siNA molecule can interact with the target
sequence. Varying the length of the siNA molecules can be chosen to
optimize activity. Generally, a sufficient number of complementary
nucleotide bases are chosen to bind to, or otherwise interact with,
the target RNA, but the degree of complementarity can be modulated
to accommodate siNA duplexes or varying length or base composition.
By using such methodologies, siNA molecules can be designed to
target sites within any known RNA sequence, for example those RNA
sequences corresponding to the any gene transcript.
[0332] Chemically modified siNA constructs are designed to provide
nuclease stability for systemic administration in vivo and/or
improved pharmacokinetic, localization, and delivery properties
while preserving the ability to mediate RNAi activity. Chemical
modifications as described herein are introduced synthetically
using synthetic methods described herein and those generally known
in the art. The synthetic siNA constructs are then assayed for
nuclease stability in serum and/or cellular/tissue extracts (e.g.
liver extracts). The synthetic siNA constructs are also tested in
parallel for RNAi activity using an appropriate assay, such as a
luciferase reporter assay as described herein or another suitable
assay that can quantity RNAi activity. Synthetic siNA constructs
that possess both nuclease stability and RNAi activity can be
further modified and re-evaluated in stability and activity assays.
The chemical modifications of the stabilized active siNA constructs
can then be applied to any siNA sequence targeting any chosen RNA
and used, for example, in target screening assays to pick lead siNA
compounds for therapeutic development (see for example FIG.
11).
Example 5
Chemical Synthesis and Purification of siNA
[0333] siNA molecules can be designed to interact with various
sites in the RNA message, for example, target sequences within the
RNA sequences described herein. The sequence of one strand of the
siNA molecule(s) is complementary to the target site sequences
described above. The siNA molecules can be chemically synthesized
using methods described herein. Inactive siNA molecules that are
used as control sequences can be synthesized by scrambling the
sequence of the siNA molecules such that it is not complementary to
the target sequence. Generally, siNA constructs can by synthesized
using solid phase oligonucleotide synthesis methods as described
herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683;
5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117;
6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400;
6,111,086 all incorporated by reference herein in their
entirety).
[0334] In a non-limiting example, RNA oligonucleotides are
synthesized in a stepwise fashion using the phosphoramidite
chemistry as is known in the art. Standard phosphoramidite
chemistry involves the use of nucleosides comprising any of
5'-O-dimethoxytrityl, 2'-O-tert-butyldimethylsilyl,
3'-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and
exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4
acetyl cytidine, and N2-isobutyryl guanosine). Alternately,
2'-O-Silyl Ethers can be used in conjunction with acid-labile
2'-O-orthoester protecting groups in the synthesis of RNA as
described by Scaringe supra. Differing 2' chemistries can require
different protecting groups, for example 2'-deoxy-2'-amino
nucleosides can utilize N-phthaloyl protection as described by
Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference
herein in its entirety).
[0335] During solid phase synthesis, each nucleotide is added
sequentially (3'- to 5'-direction) to the solid support-bound
oligonucleotide. The first nucleoside at the 3'-end of the chain is
covalently attached to a solid support (e.g., controlled pore glass
or polystyrene) using various linkers. The nucleotide precursor, a
ribonucleoside phosphoramidite, and activator are combined
resulting in the coupling of the second nucleoside phosphoramidite
onto the 5'-end of the first nucleoside. The support is then washed
and any unreacted 5'-hydroxyl groups are capped with a capping
reagent such as acetic anhydride to yield inactive 5'-acetyl
moieties. The trivalent phosphorus linkage is then oxidized to a
more stable phosphate linkage. At the end of the nucleotide
addition cycle, the 5'-O-protecting group is cleaved under suitable
conditions (e.g., acidic conditions for trityl-based groups and
Fluoride for silyl-based groups). The cycle is repeated for each
subsequent nucleotide.
[0336] Modification of synthesis conditions can be used to optimize
coupling efficiency, for example by using differing coupling times,
differing reagent/phosphoramidite concentrations, differing contact
times, differing solid supports and solid support linker
chemistries depending on the particular chemical composition of the
siNA to be synthesized. Deprotection and purification of the siNA
can be performed as is generally described in Deprotection and
purification of the siNA can be performed as is generally described
in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat. No. 6,353,098,
U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No.
6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or
Scaringe supra, incorporated by reference herein in their
entireties. Additionally, deprotection conditions can be modified
to provide the best possible yield and purity of siNA constructs.
For example, applicant has observed that oligonucleotides
comprising 2'-deoxy-2'-fluoro nucleotides can degrade under
inappropriate deprotection conditions. Such oligonucleotides are
deprotected using aqueous methylamine at about 35.degree. C. for 30
minutes. If the 2'-deoxy-2'-fluoro containing oligonucleotide also
comprises ribonucleotides, after deprotection with aqueous
methylamine at about 35.degree. C. for 30 minutes, TEA-HF is added
and the reaction maintained at about 65.degree. C. for an
additional 15 minutes.
Example 6
RNAi In Vitro Assay to Assess siNA Activity
[0337] An in vitro assay that recapitulates RNAi in a cell-free
system is used to evaluate siNA constructs targeting MAP kinase
(e.g., c-JUN) RNA targets. The assay comprises the system described
by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and
Zamore et al., 2000, Cell, 101, 25-33 adapted for use with MAP
kinase target RNA. A Drosophila extract derived from syncytial
blastoderm is used to reconstitute RNAi activity in vitro. Target
RNA is generated via in vitro transcription from an appropriate MAP
kinase expressing plasmid using T7 RNA polymerase or via chemical
synthesis as described herein. Sense and antisense siNA strands
(for example 20 uM each) are annealed by incubation in buffer (such
as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM
magnesium acetate) for 1 minute at 90.degree. C. followed by 1 hour
at 37.degree. C., then diluted in lysis buffer (for example 100 mM
potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium
acetate). Annealing can be monitored by gel electrophoresis on an
agarose gel in TBE buffer and stained with ethidium bromide. The
Drosophila lysate is prepared using zero to two-hour-old embryos
from Oregon R flies collected on yeasted molasses agar that are
dechorionated and lysed. The lysate is centrifuged and the
supernatant isolated. The assay comprises a reaction mixture
containing 50% lysate [vol/vol], RNA (10-50 .mu.M final
concentration), and 10% [vol/vol] lysis buffer containing siNA (10
nM final concentration). The reaction mixture also contains 10 mM
creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um GTP,
100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin
(Promega), and 100 uM of each amino acid. The final concentration
of potassium acetate is adjusted to 100 mM. The reactions are
pre-assembled on ice and preincubated at 25.degree. C. for 10
minutes before adding RNA, then incubated at 25.degree. C. for an
additional 60 minutes. Reactions are quenched with 4 volumes of
1.25.times. Passive Lysis Buffer (Promega). Target RNA cleavage is
assayed by RT-PCR analysis or other methods known in the art and
are compared to control reactions in which siNA is omitted from the
reaction.
[0338] Alternately, internally-labeled target RNA for the assay is
prepared by in vitro transcription in the presence of
[alpha-.sup.32P] CTP, passed over a G 50 Sephadex column by spin
chromatography and used as target RNA without further purification.
Optionally, target RNA is 5'-.sup.32P-end labeled using T4
polynucleotide kinase enzyme. Assays are performed as described
above and target RNA and the specific RNA cleavage products
generated by RNAi are visualized on an autoradiograph of a gel. The
percentage of cleavage is determined by Phosphor Inager.RTM.
quantitation of bands representing intact control RNA or RNA from
control reactions without siNA and the cleavage products generated
by the assay.
[0339] In one embodiment, this assay is used to determine target
sites the MAP kinase RNA target for siNA mediated RNAi cleavage,
wherein a plurality of siNA constructs are screened for RNAi
mediated cleavage of the MAP kinase RNA target, for example, by
analyzing the assay reaction by electrophoresis of labeled target
RNA, or by northern blotting, as well as by other methodology well
known in the art.
Example 7
Nucleic Acid Inhibition of MAP Kinase Target RNA In Vivo
[0340] siNA molecules targeted to the human MAP kinase (e.g.,
c-JUN) RNA are designed and synthesized as described above. These
nucleic acid molecules can be tested for cleavage activity in vivo,
for example, using the following procedure. The target sequences
and the nucleotide location within the MAP kinase (e.g., c-JUN) RNA
are given in Table II and III.
[0341] Two formats are used to test the efficacy of siNAs targeting
MAP kinase (e.g., c-JUN). First, the reagents are tested in cell
culture, for example using cultured human kidney fibroblast cells
(e.g., 293, HeLa, or HepG2 cells), to determine the extent of RNA
and protein inhibition. siNA reagents (e.g.; see Tables II and III)
are selected against the MAP kinase (e.g., c-JUN) target as
described herein. RNA inhibition is measured after delivery of
these reagents by a suitable transfection agent to, for example,
293, HeLa, or HepG2 cells. Relative amounts of target RNA are
measured versus actin using real-time PCR monitoring of
amplification (eg., ABI 7700 Taqman.RTM.). A comparison is made to
a mixture of oligonucleotide sequences made to unrelated targets or
to a randomized siNA control with the same overall length and
chemistry, but randomly substituted at each position. Primary and
secondary lead reagents are chosen for the target and optimization
performed. After an optimal transfection agent concentration is
chosen, a RNA time-course of inhibition is performed with the lead
siNA molecule. In addition, a cell-plating format can be used to
determine RNA inhibition.
Delivery of siNA to Cells
[0342] Cells (e.g., 293, HeLa, or HepG2 cells) are seeded, for
example, at 1.times.10.sup.5 cells per well of a six-well dish in
EGM-2 (BioWhittaker) the day before transfection. siNA (final
concentration, for example 20 nM) and cationic lipid (e.g., final
concentration 2 .mu.g/ml) are complexed in EGM basal media
(BioWhittaker) at 37.degree. C. for 30 mins in polystyrene tubes.
Following vortexing, the complexed siNA is added to each well and
incubated for the times indicated. For initial optimization
experiments, cells are seeded, for example, at 1.times.10.sup.3 in
96 well plates and siNA complex added as described. Efficiency of
delivery of siNA to cells is determined using a fluorescent siNA
complexed with lipid. Cells in 6-well dishes are incubated with
siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde
for 15 minutes at room temperature. Uptake of siNA is visualized
using a fluorescent microscope.
Taqman and Lightcycler Quantification of mRNA
[0343] Total RNA is prepared from cells following siNA delivery,
for example using Qiagen RNA purification kits for 6-well or Rneasy
extraction kits for 96-well assays. For Taqman analysis,
dual-labeled probes are synthesized with the reporter dye, FAM or
JOE, covalently linked at the 5'-end and the quencher dye TAMRA
conjugated to the 3'-end. One-step RT-PCR amplifications are
performed on, for example, an ABI PRISM 7700 Sequence Detector
using 50 .mu.l reactions consisting of 10 .mu.l total RNA, 100 nM
forward primer, 900 nM reverse primer, 100 nM probe, 1.times.TaqMan
PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl.sub.2, 300
mM each dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega),
1.25U AmpliTaq Gold (PE-Applied Biosystems) and 10U M-MLV Reverse
Transcriptase (Promega). The thermal cycling conditions can consist
of 30 min at 48.degree. C., 10 min at 95.degree. C., followed by 40
cycles of 15 sec at 95.degree. C. and 1 min at 60.degree. C.
Quantitation of mRNA levels is determined relative to standards
generated from serially diluted total cellular RNA (300, 100, 33,
11 ng/r.times.n) and normalizing to .beta.-actin or GAPDH mRNA in
parallel TaqMan reactions. For each gene of interest an upper and
lower primer and a fluorescently labeled probe are designed. Real
time incorporation of SYBR Green I dye into a specific PCR product
can be measured in glass capillary tubes using a lightcyler. A
standard curve is generated for each primer pair using control c
RNA allularnd values are represented as relative expression to
GAPDH in each sample.
Western Blotting
[0344] Nuclear extracts can be prepared using a standard micro
preparation technique (see for example Andrews and Faller, 1991,
Nucleic Acids Research, 19, 2499). Protein extracts from
supernatants are prepared, for example using TCA precipitation. An
equal volume of 20% TCA is added to the cell supernatant, incubated
on ice for 1 hour and pelleted by centrifugation for 5 minutes.
Pellets are washed in acetone, dried and resuspended in water.
Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear
extracts) or 4-12% Tris-Glycine (supernatant extracts)
polyacrylamide gel and transferred onto nitro-cellulose membranes.
Non-specific binding can be blocked by incubation, for example,
with 5% non-fat milk for 1 hour followed by primary antibody for 16
hour at 4.degree. C. Following washes, the secondary antibody is
applied, for example (1:10,000 dilution) for 1 hour at room
temperature and the signal detected with SuperSignal reagent
(Pierce).
Example 8
Models Useful to Evaluate the Down-Regulation of MAP Kinase Gene
(e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38) expression
Cell Culture
[0345] There are numerous cell culture systems that can be used to
analyze reduction of MAP kinase levels either directly or
indirectly by measuring downstream effects. For example, cultured
human kidney fibroblast cells (e.g., 293 cells), HeLa, or HepG2
cells can be used in cell culture experiments to assess the
efficacy of nucleic acid molecules of the invention. As such, cells
treated with nucleic acid molecules of the invention (e.g., siNA)
targeting MAP kinase RNA would be expected to have decreased MAP
kinase expression capacity compared to matched control nucleic acid
molecules having a scrambled or inactive sequence. In a
non-limiting example, 293, HeLa, or HepG2 cells are cultured and
MAP kinase expression is quantified, for example by time-resolved
immuno fluorometric assay. MAP kinase messenger-RNA expression is
quantitated with RT-PCR in cultured cells. Untreated cells are
compared to cells treated with siNA molecules transfected with a
suitable reagent, for example a cationic lipid such as
lipofectamine, and MAP kinase protein and RNA levels are
quantitated. Dose response assays are then performed to establish
dose dependent inhibition of MAP kinase expression. In another
non-limiting example, cell culture experiments are carried out as
described by Aguirre et al., 2000, J. Biol. Chem., 275,
9047-9054.
[0346] In several cell culture systems, cationic lipids have been
shown to enhance the bioavailability of oligonucleotides to cells
in culture (Bennet, et al., 1992, Mol. Pharmacology, 41,
1023-1033). In one embodiment, siNA molecules of the invention are
complexed with cationic lipids for cell culture experiments. siNA
and cationic lipid mixtures are prepared in serum-free DMEM
immediately prior to addition to the cells. DMEM plus additives are
warmed to room temperature (about 20-25.degree. C.) and cationic
lipid is added to the final desired concentration and the solution
is vortexed briefly. siNA molecules are added to the final desired
concentration and the solution is again vortexed briefly and
incubated for 10 minutes at room temperature. In dose response
experiments, the RNA/lipid complex is serially diluted into DMEM
following the 10 minute incubation.
Animal Models
[0347] Evaluating the efficacy of anti-MAP kinase agents in animal
models is an important prerequisite to human clinical trials.
Obesity and type 2 diabetes are the most prevalent and serious
metabolic diseases in that they affect more than 50% of adults in
the USA. These conditions are associated with a chronic
inflammatory response characterized by abnormal inflammatory
cytokine production, increased acute-phase reactants and other
stress-induced molecules. Many of these alterations seem to be
initiated and to reside within adipose tissue. Elevated production
of tumor necrosis factor (TNF)-alpha by adipose tissue decreases
sensitivity to insulin and has been detected in several
experimental obesity models and obese humans. Free fatty acids
(FFAs) are also implicated in the etiology of obesity-induced
insulin resistance and diabetes. Because both TNF-alpha and FFAs
are potent MAP kinase activators, Hirosumi et al., 2002, Nature,
420, 333-336 determined whether obesity is associated with
alterations in stress-activated and inflammatory responses through
this pathway and whether MAP kinases are causally linked to
aberrant metabolic control in this state. In this study, Hirosumi
et al., describe dietary and genetic (ob/ob) mouse models of
obesity useful in evaluating MAP kinase gene expression. Such
transgenic mice are useful as models for obesity and insulin
resistance and can be used to identify nucleic acid molecules of
the invention that modulate MAP kinase gene (e.g., ERK1, ERK2,
JNK1, JNK2, and/or p38) expression and gene function toward
therapeutic use in treating obesity and insulin resistance (e.g.
type I and II diabetes).
[0348] The role of c-JUN in liver cancer has recently been
investigated (Eferl et al., 2003, Cell, 112, 181). These
investigators deleted c-JUN and then induced liver cancer by
chemical carcinogenesis. They observed that deletion of c-JUN
dramatically interfered with liver tumor formation. Animal survival
was markedly worse in c-JUN wild-type animals relative to deletion
mutants. In particular, the number of apoptotic cells increased
about five fold in tumors in the c-JUN deletion strain relative to
the wild-type animals. Importantly, levels of the pro-apoptotic
gene products such as p53 and noxa were elevated in the c-JUN
deletion strain. c-JUN is likely to antagonize other pro-apoptotic
genes such as TNF-a. Thus, by blocking p53 and its large family of
dependent genes, c-JUN seems to promote tumor formation. Since a
large fraction of chronically infected HCV patients develop
hepatocellular carcinoma, c-JUN provides an attractive target for
treating HCV infected patients to prevent or ameliorate
hepatocellular carcinoma. The animal model described by Eferl et
al., supra, can be used to evaluate siNA molecules of the invention
for efficacy in inhibiting c-JUN expression in liver toward
therapeutic use in preventing and/or treating hepatocellular
carcinoma in human subjects.
[0349] Because mitogen activated protein kinases (MAP kinases) are
constituents of numerous signal transduction pathways, and are
activated by protein kinase cascades, intense efforts are under way
to develop and evaluate compounds that target components of MAPK
pathways. Several of these inhibitors are effective in animal
models of disease and have advanced to clinical trials for the
treatment of inflammatory diseases, metabolic diseases, autoimmune
diseases and cancer. The clinical utility of specifically targeting
MAP kinase genes (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38)
can be studied in animal models and clinical studies of
inflammatory diseases, metabolic diseases, autoimmune diseases and
cancer (see for example English et al., 2002, Trends in
Pharmacological Sciences, 23, 40-45).
Example 9
RNAi Mediated Inhibition of p38 RNA Expression
[0350] siNA constructs are tested for efficacy in reducing p38 RNA
expression in, for example in A549 cells. Cells are plated
approximately 24 hours before transfection in 96-well plates at
5,000-7,500 cells/well, 100 .mu.l/well, such that at the time of
transfection cells are 70-90% confluent. For transfection, annealed
siNAs are mixed with the transfection reagent (Lipofectamine 2000,
Invitrogen) in a volume of 50 .mu.l/well and incubated for 20
minutes at room temperature. The siNA transfection mixtures are
added to cells to give a final siNA concentration of 25 nM in a
volume of 150 .mu.l. Each siNA transfection mixture is added to 3
wells for triplicate siNA treatments. Cells are incubated at
37.degree. for 24 hours in the continued presence of the siNA
transfection mixture. At 24 hours, RNA is prepared from each well
of treated cells. The supernatants with the transfection mixtures
are first removed and discarded, then the cells are lysed and RNA
prepared from each well. Target gene expression following treatment
is evaluated by RT-PCR for the target gene and for a control gene
(36B4, an RNA polymerase subunit) for normalization. The triplicate
data is averaged and the standard deviations determined for each
treatment. Normalized data are graphed and the percent reduction of
target mRNA by active siNAs in comparison to their respective
inverted control siNAs was determined.
[0351] In a non-limiting example, siNA constructs were screened for
activity (see FIG. 12) and compared to untreated cells, scrambled
siNA control constructs (Scram1 and Scram2), and cells transfected
with lipid alone (transfection control). As shown in FIG. 12, the
siNA constructs significantly reduce p38 RNA expression. Leads
generated from such a screen are then further assayed. In a
non-limiting example, siNA constructs comprising chemical
modifications described herein (e.g., having modifications
comprising Formulae I-VII and/or those modifications described in
Table IV are assayed for activity. These siNA constructs are
compared to appropriate matched chemistry inverted controls. In
addition, the siNA constructs are also compared to untreated cells,
cells transfected with lipid and scrambled siNA constructs, and
cells transfected with lipid alone (transfection control).
Example 10
RNAi Mediated Inhibition of p38 RNA Expression
[0352] siNA constructs are tested for efficacy in reducing JNK1 RNA
expression in, for example in A549 cells. Cells are plated
approximately 24 hours before transfection in 96-well plates at
5,000-7,500 cells/well, 100 .mu.l/well, such that at the time of
transfection cells are 70-90% confluent. For transfection, annealed
siNAs are mixed with the transfection reagent (Lipofectamine 2000,
Invitrogen) in a volume of 50 .mu.l/well and incubated for 20
minutes at room temperature. The siNA transfection mixtures are
added to cells to give a final siNA concentration of 25 nM in a
volume of 150 .mu.l. Each siNA transfection mixture is added to 3
wells for triplicate siNA treatments. Cells are incubated at
37.degree. for 24 hours in the continued presence of the siNA
transfection mixture. At 24 hours, RNA is prepared from each well
of treated cells. The supernatants with the transfection mixtures
are first removed and discarded, then the cells are lysed and RNA
prepared from each well. Target gene expression following treatment
is evaluated by RT-PCR for the target gene and for a control gene
(36B4, an RNA polymerase subunit) for normalization. The triplicate
data is averaged and the standard deviations determined for each
treatment. Normalized data are graphed and the percent reduction of
target mRNA by active siNAs in comparison to their respective
inverted control siNAs was determined.
[0353] In a non-limiting example, siNA constructs were screened for
activity (see FIG. 13) and compared to untreated cells, scrambled
siNA control constructs (Scram1 and Scram2), and cells transfected
with lipid alone (transfection control). As shown in FIG. 13, the
siNA constructs significantly reduce p38 RNA expression. Leads
generated from such a screen are then further assayed. In a
non-limiting example, siNA constructs comprise chemical
modifications described herein (e.g., having modifications
comprising Formulae I-VII and/or those modifications described in
Table IV are assayed for activity). These siNA constructs are
compared to appropriate matched chemistry inverted controls. In
addition, the siNA constructs are also compared to untreated cells,
cells transfected with lipid and scrambled siNA constructs, and
cells transfected with lipid alone (transfection control).
Example 11
Indications
[0354] The present body of knowledge in MAP kinase research
indicates the need for methods and compounds that can regulate MAP
kinase gene (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38)
product expression for research, diagnostic, and therapeutic use.
As described herein, the nucleic acid molecules of the present
invention can be used to treat obesity and insulin resistance (e.g.
type I and II diabetes), oncology and proliferation related
indications and conditions, including cancers of the lung, bladder,
colon, breast, prostate, retina, larynx, esophagus, liver (e.g.,
hepatocellular carcinoma), and ovary, along with lymphomas,
melanomas and glioblastomas, inflammatory disorders such as asthma,
septic shock, rheumatoid arthritis, psoriasis, inflammatory bowl
syndrome and any other disease that responds to modulation of MAP
kinase expression.
[0355] Troglitazone, insulin, and PTP-1B modulators are
non-limiting examples of pharmaceutical agents that can be combined
with or used in conjunction with the nucleic acid molecules (e.g.
siNA molecules) of the instant invention for treating obesity and
diabetes. The use of radiation treatments and chemotherapeutics
such as Gemcytabine and cyclophosphamide are non-limiting examples
of chemotherapeutic agents that can also be combined with or used
in conjunction with the nucleic acid molecules (e.g. siNA
molecules) of the instant invention for oncology therapeutic
applications. Those skilled in the art will recognize that other
anti-cancer compounds and therapies can be similarly be readily
combined with the nucleic acid molecules of the instant invention
(e.g. siNA molecules) and are hence within the scope of the instant
invention. Such compounds and therapies are well known in the art
(see for example Cancer: Principles and Practice of Oncology,
Volumes 1 and 2, eds Devita, V. T., Hellman, S., and Rosenberg, S.
A., J.B. Lippincott Company, Philadelphia, USA; incorporated herein
by reference) and include, without limitations, folates,
antifolates, pyrimidine analogs, fluoropyrimidines, purine analogs,
adenosine analogs, topoisomerase I inhibitors, anthrapyrazoles,
retinoids, antibiotics, anthacyclins, platinum analogs, alkylating
agents, nitrosoureas, plant derived compounds such as vinca
alkaloids, epipodophyllotoxins, tyrosine kinase inhibitors, taxols,
radiation therapy, surgery, nutritional supplements, gene therapy,
radiotherapy, for example 3D-CRT, immunotoxin therapy, for example
ricin, and monoclonal antibodies. Specific examples of
chemotherapeutic compounds that can be combined with or used in
conjunction with the nucleic acid molecules of the invention
include, but are not limited to, Paclitaxel; Docetaxel;
Methotrexate; Doxorubin; Edatrexate; Vinorelbine; Tamoxifen;
Leucovorin; 5-fluoro uridine (5-FU); Ionotecan; Cisplatin;
Carboplatin; Amsacrine; Cytarabine; Bleomycin; Mitomycin C;
Dactinomycin; Mithramycin; Hexamethylmelamine; Dacarbazine;
L-asperginase; Nitrogen mustard; Melphalan, Chlorambucil; Busulfan;
Ifosfamide; 4-hydroperoxycyclophosphamide, Thiotepa; Irinotecan
(CAMPTOSAR.RTM., CPT-11, Camptothecin-11, Campto) Tamoxifen,
Herceptin; IMC C225; ABX-EGF: and combinations thereof are
non-limiting examples of compounds and/or methods that can be
combined with or used in conjunction with the nucleic acid
molecules (e.g. siNA) of the instant invention. In addition,
treatment of HCV infected subjects with siNA molecules of the
invention targeting c-JUN or other MAP kinases involved in the
maintenance or development of hepatocellular carcinoma can be
combined with anti-viral compounds, such as siNA molecules
targeting HCV RNA or other antiviral compounds known in the art
(e.g., interferons, nucleoside analogs etc.). Those skilled in the
art will recognize that other drug compounds and therapies can be
similarly be readily combined with the nucleic acid molecules of
the instant invention (e.g., siNA molecules) are hence within the
scope of the instant invention.
Example 12
Diagnostic Uses
[0356] The siNA molecules of the invention can be used in a variety
of diagnostic applications, such as in the identification of
molecular targets (e.g., RNA) in a variety of applications, for
example, in clinical, industrial, environmental, agricultural
and/or research settings. Such diagnostic use of siNA molecules
involves utilizing reconstituted RNAi systems, for example, using
cellular lysates or partially purified cellular lysates. siNA
molecules of this invention can be used as diagnostic tools to
examine genetic drift and mutations within diseased cells or to
detect the presence of endogenous or exogenous, for example viral,
RNA in a cell. The close relationship between siNA activity and the
structure of the target RNA allows the detection of mutations in
any region of the molecule, which alters the base-pairing and
three-dimensional structure of the target RNA. By using multiple
siNA molecules described in this invention, one ca MAP nucleotide
changes, which are important to RNA structure and function in
vitro, as well as in cells and tissues. Cleavage of target RNAs
with siNA molecules can be used to inhibit gene expression and
define the role of specified gene products in the progression of
disease or infection. In this manner, other genetic targets can be
defined as important mediators of the disease. These experiments
will lead to better treatment of the disease progression by
affording the possibility of combination therapies (e.g., multiple
siNA molecules targeted to different genes, siNA molecules coupled
with known small molecule inhibitors, or intermittent treatment
with combinations siNA molecules and/or other chemical or
biological molecules). Other in vitro uses of siNA molecules of
this invention are well known in the art, and include detection of
the presence of mRNAs associated with a disease, infection, or
related condition. Such RNA is detected by determining the presence
of a cleavage product after treatment with a siNA using standard
methodologies, for example, fluorescence resonance emission
transfer (FRET).
[0357] In a specific example, siNA molecules that cleave only
wild-type or mutant forms of the target RNA are used for the assay.
The first siNA molecules (i.e., those that cleave only wild-type
forms of target RNA) are used to identify wild-type RNA present in
the sample and the second siNA molecules (i.e., those that cleave
only mutant forms of target RNA) are used to identify mutant RNA in
the sample. As reaction controls, synthetic substrates of both
wild-type and mutant RNA are cleaved by both siNA molecules to
demonstrate the relative siNA efficiencies in the reactions and the
absence of cleavage of the "non-targeted" RNA species. The cleavage
products from the synthetic substrates also serve to generate size
markers for the analysis of wild-type and mutant RNAs in the sample
population. Thus, each analysis requires two siNA molecules, two
substrates and one unknown sample, which is combined into six
reactions. The presence of cleavage products is determined using an
RNase protection assay so that full-length and cleavage fragments
of each RNA can be analyzed in one lane of a polyacrylamide gel. It
is not absolutely required to quantify the results to gain insight
into the expression of mutant RNAs and putative risk of the desired
phenotypic changes in target cells. The expression of mRNA whose
protein product is implicated in the development of the phenotype
(i.e., disease related or infection related) is adequate to
establish risk. If probes of comparable specific activity are used
for both transcripts, then a qualitative comparison of RNA levels
is adequate and decreases the cost of the initial diagnosis. Higher
mutant form to wild-type ratios are correlated with higher risk
whether RNA levels are compared qualitatively or
quantitatively.
[0358] All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the invention pertains. All references cited in this
disclosure are incorporated by reference to the same extent as if
each reference had been incorporated by reference in its entirety
individually.
[0359] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The methods and compositions described herein as presently
representative of preferred embodiments are exemplary and are not
intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art,
which are encompassed within the spirit of the invention, are
defined by the scope of the claims.
[0360] It will be readily apparent to one skilled in the art that
varying substitutions and modifications can be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. Thus, such additional embodiments are
within the scope of the present invention and the following claims.
The present invention teaches one skilled in the art to test
various combinations and/or substitutions of chemical modifications
described herein toward generating nucleic acid constructs with
improved activity for mediating RNAi activity. Such improved
activity can comprise improved stability, improved bioavailability,
and/or improved activation of cellular responses mediating RNAi.
Therefore, the specific embodiments described herein are not
limiting and one skilled in the art can readily appreciate that
specific combinations of the modifications described herein can be
tested without undue experimentation toward identifying siNA
molecules with improved RNAi activity.
[0361] The invention illustratively described herein suitably can
be practiced in the absence of any element or elements, limitation
or limitations that are not specifically disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by preferred embodiments, optional features,
modification and variation of the concepts herein disclosed may be
resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention as defined by the description and the appended
claims.
[0362] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
TABLE-US-00001 TABLE I MAP kinase Accession Numbers NM_002745 Homo
sapiens mitogen-activated protein kinase 1 (MAPK1), transcript
variant 1, mRNA. NM_138957 Homo sapiens mitogen-activated protein
kinase 1 (MAPK1), transcript variant 2, mRNA. X60188 Human ERK1
mRNA for protein serine/threonine kinase (MAPK3). XM_055766 Homo
sapiens mitogen-activated protein kinase 3 (MAPK3), mRNA NM_002747
Homo sapiens mitogen-activated protein kinase 4 (MAPK4), mRNA
XM_165662 Homo sapiens Mitogen-activated protein kinase 4
(Extracellular signal-regulated kinase 4) (ERK-4) (MAP kinase
isoform p63) (p63-MAPK) (LOC220131), mRNA NM_002748 Homo sapiens
mitogen-activated protein kinase 6 (MAPK6), mRNA. XM_166057 Homo
sapiens Mitogen-activated protein kinase 6 (Extracellular
signal-regulated kinase 3) (ERK-3) (MAP kinase isoform p97)
(p97-MAPK) (LOC220839), mRNA XM_035575 Homo sapiens
mitogen-activated protein kinase 6 (MAPK6), mRNA NM_139033 Homo
sapiens mitogen-activated protein kinase 7 (MAPK7), transcript
variant 1, mRNA NM_139032 Homo sapiens mitogen-activated protein
kinase 7 (MAPK7), transcript variant 2, mRNA NM_002749 Homo sapiens
mitogen-activated protein kinase 7 (MAPK7), transcript variant 3,
mRNA NM_139034 Homo sapiens mitogen-activated protein kinase 7
(MAPK7), transcript variant 4, mRNA NM_139049 Homo sapiens
mitogen-activated protein kinase 8 (MAPK8), transcript variant 1,
mRNA. NM_002750 Homo sapiens mitogen-activated protein kinase 8
(MAPK8), transcript variant 2, mRNA. NM_139046 Homo sapiens
mitogen-activated protein kinase 8 (MAPK8), transcript variant 3,
mRNA. NM_139047 Homo sapiens mitogen-activated protein kinase 8
(MAPK8), transcript variant 4, mRNA. NM_002752 Homo sapiens
mitogen-activated protein kinase 9 (MAPK9), transcript variant 1,
mRNA. NM_139068 Homo sapiens mitogen-activated protein kinase 9
(MAPK9), transcript variant 2, mRNA. NM_139069 Homo sapiens
mitogen-activated protein kinase 9 (MAPK9), transcript variant 3,
mRNA. NM_139070 Homo sapiens mitogen-activated protein kinase 9
(MAPK9), transcript variant 4, mRNA. NM_002753 Homo sapiens
mitogen-activated protein kinase 10 (MAPK10), transcript variant 1,
mRNA NM_138982 Homo sapiens mitogen-activated protein kinase 10
(MAPK10), transcript variant 2, mRNA NM_138980 Homo sapiens
mitogen-activated protein kinase 10 (MAPK10), transcript variant 3,
mRNA NM_138981 Homo sapiens mitogen-activated protein kinase 10
(MAPK10), transcript variant 4, mRNA NM_002751 Homo sapiens
mitogen-activated protein kinase 11 (MAPK11), transcript variant 1,
mRNA. NM_138993 Homo sapiens mitogen-activated protein kinase 11
(MAPK11), transcript variant 2, mRNA. NM_002969 Homo sapiens
mitogen-activated protein kinase 12 (MAPK12), mRNA. NM_002754 Homo
sapiens mitogen-activated protein kinase 13 (MAPK13), mRNA.
NM_001315 Homo sapiens mitogen-activated protein kinase 14
(MAPK14), transcript variant 1, mRNA. NM_139012 Homo sapiens
mitogen-activated protein kinase 14 (MAPK14), transcript variant 2,
mRNA. NM_139013 Homo sapiens mitogen-activated protein kinase 14
(MAPK14), transcript variant 3, mRNA. NM_139014 Homo sapiens
mitogen-activated protein kinase 14 (MAPK14), transcript variant 4,
mRNA. NM_002755 Homo sapiens mitogen-activated protein kinase
kinase 1 (MAP2K1), mRNA NM_030662 Homo sapiens mitogen-activated
protein kinase kinase 2 (MAP2K2), mRNA NM_002756 Homo sapiens
mitogen-activated protein kinase kinase 3 (MAP2K3), transcript
variant A, mRNA NM_145109 Homo sapiens mitogen-activated protein
kinase kinase 3 (MAP2K3), transcript variant B, mRNA NM_145110 Homo
sapiens mitogen-activated protein kinase kinase 3 (MAP2K3),
transcript variant C, mRNA XM_008654 Homo sapiens mitogen-activated
protein kinase kinase 4 (MAP2K4), mRNA NM_003010 Homo sapiens
mitogen-activated protein kinase kinase 4 (MAP2K4), mRNA NM_145160
Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5),
transcript variant A, mRNA NM_002757 Homo sapiens mitogen-activated
protein kinase kinase 5 (MAP2K5), transcript variant B, mRNA
NM_145161 Homo sapiens mitogen-activated protein kinase kinase 5
(MAP2K5), transcript variant C, mRNA NM_145162 Homo sapiens
mitogen-activated protein kinase kinase 5 (MAP2K5), transcript
variant D, mRNA XM_113313 Homo sapiens mitogen-activated protein
kinase kinase 6 (MAP2K6), mRNA NM_002758 Homo sapiens
mitogen-activated protein kinase kinase 6 (MAP2K6), transcript
variant 1, mRNA NM_031988 Homo sapiens mitogen-activated protein
kinase kinase 6 (MAP2K6), transcript variant 2, mRNA NM_005043 Homo
sapiens mitogen-activated protein kinase kinase 7 (MAP2K7),
transcript variant A, mRNA NM_145185 Homo sapiens mitogen-activated
protein kinase kinase 7 (MAP2K7), transcript variant B, mRNA
NM_145329 Homo sapiens mitogen-activated protein kinase kinase 7
(MAP2K7), transcript variant C, mRNA AF042838 Homo sapiens
mitogen-activated protein kinase kinase kinase 1 (MAP3K1), mRNA
NM_006609 Homo sapiens mitogen-activated protein kinase kinase
kinase 2 (MAP3K2), mRNA NM_002401 Homo sapiens mitogen-activated
protein kinase kinase kinase 3 (MAP3K3), mRNA NM_005922 Homo
sapiens mitogen-activated protein kinase kinase kinase 4 (MAP3K4),
transcript variant 1, mRNA NM_006724 Homo sapiens mitogen-activated
protein kinase kinase kinase 4 (MAP3K4), transcript variant 2, mRNA
NM_005923 Homo sapiens mitogen-activated protein kinase kinase
kinase 5 (MAP3K5), mRNA NM_004672 Homo sapiens mitogen-activated
protein kinase kinase kinase 6 (MAP3K6), mRNA NM_003188 Homo
sapiens mitogen-activated protein kinase kinase kinase 7 (MAP3K7),
mRNA NM_005204 Homo sapiens mitogen-activated protein kinase kinase
kinase 8 (MAP3K8), mRNA AF251442 Homo sapiens mitogen-activated
protein kinase kinase kinase 9 (MAP3K9), mRNA NM_002446 Homo
sapiens mitogen-activated protein kinase kinase kinase 10
(MAP3K10), mRNA NM_002419 Homo sapiens mitogen-activated protein
kinase kinase kinase 11 (MAP3K11), mRNA NM_006301 Homo sapiens
mitogen-activated protein kinase kinase kinase 12 (MAP3K12), mRNA
NM_004721 Homo sapiens mitogen-activated protein kinase kinase
kinase 13 (MAP3K13), mRNA NM_003954 Homo sapiens mitogen-activated
protein kinase kinase kinase 14 (MAP3K14), mRNA NM_007181 Homo
sapiens mitogen-activated protein kinase kinase kinase kinase 1
(MAP4K1), mRNA NM_004579 Homo sapiens mitogen-activated protein
kinase kinase kinase kinase 2 (MAP4K2), mRNA NM_003618 Homo sapiens
mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3),
mRNA NM_004834 Homo sapiens mitogen-activated protein kinase kinase
kinase kinase 4 (MAP4K4), mRNA NM_006575 Homo sapiens
mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5),
mRNA NM_003668 Homo sapiens mitogen-activated protein
kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 1,
mRNA NM_139078 Homo sapiens mitogen-activated protein
kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 2,
mRNA NM_004635 Homo sapiens mitogen-activated protein
kinase-activated protein kinase 3 (MAPKAPK3), mRNA NM_004759 Homo
sapiens mitogen-activated protein kinase-activated protein kinase 2
(MAPKAPK2), transcript variant 1, mRNA NM_032960 Homo sapiens
mitogen-activated protein kinase-activated protein kinase 2
(MAPKAPK2), transcript variant 2, mRNA NM_005373 Homo sapiens
myeloproliferative leukemia virus oncogene (MPL), mRNA NM_016848
Homo sapiens neuronal Shc (SHC3), mRNA NM_002649 Homo sapiens
phosphoinositide-3-kinase, catalytic, gamma polypeptide (PIK3CG),
mRNA NM_021003 Homo sapiens protein phosphatase 1A (formerly 2C),
magnesium-dependent, alpha isoform (PPM1A), mRNA NM_003942 Homo
sapiens ribosomal protein S6 kinase, 90 kD, polypeptide 4
(RPS6KA4), mRNA NM_004755 Homo sapiens ribosomal protein S6 kinase,
90 kD, polypeptide 5 (RPS6KA5), mRNA NM_002228 Homo sapiens v-jun
sarcoma virus 17 oncogene homolog (avian) (JUN), mRNA
TABLE-US-00002 TABLE II MAP kinase siNA and Target Sequences Seq
Seq Seq Pos Target Sequence ID UPos Upper seq ID LPos Lower seq ID
NM_002745 (MAPK1/ERK2) 3 CCCUCCCUCCGCCCGCCCG 1 3
CCCUCCCUCCGCCCGCCCG 1 21 CGGGCGGGCGGAGGGAGGG 164 21
GCCGGCCCGCCCGUCAGUC 2 21 GCCGGCCCGCCCGUCAGUC 2 39
GACUGACGGGCGGGCCGGC 165 39 CUGGCAGGCAGGCAGGCAA 3 39
CUGGCAGGCAGGCAGGCAA 3 57 UUGCCUGCCUGCCUGCCAG 166 57
AUCGGUCCGAGUGGCUGUC 4 57 AUCGGUCCGAGUGGCUGUC 4 75
GACAGCCACUCGGACCGAU 167 75 CGGCUCUUCAGCUCUCCCG 5 75
CGGCUCUUCAGCUCUCCCG 5 93 CGGGAGAGCUGAAGAGCCG 168 93
GCUCGGCGUCUUCCUUCCU 6 93 GCUCGGCGUCUUCCUUCCU 6 111
AGGAAGGAAGACGCCGAGC 169 111 UCCUCCCGGUCAGCGUCGG 7 111
UCCUCCCGGUCAGCGUCGG 7 129 CCGACGCUGACCGGGAGGA 170 129
GCGGCUGCACCGGCGGCGG 8 129 GCGGCUGCACCGGCGGCGG 8 147
CCGCCGCCGGUGCAGCCGC 171 147 GCGCAGUCCCUGCGGGAGG 9 147
GCGCAGUCCCUGCGGGAGG 9 165 CCUCCCGCAGGGACUGCGC 172 165
GGGCGACAAGAGCUGAGCG 10 165 GGGCGACAAGAGCUGAGCG 10 183
CGCUCAGCUCUUGUCGCCC 173 183 GGCGGCCGCCGAGCGUCGA 11 183
GGCGGCCGCCGAGCGUCGA 11 201 UCGACGCUCGGCGGCCGCC 174 201
AGCUCAGCGCGGCGGAGGC 12 201 AGCUCAGCGCGGCGGAGGC 12 219
GCCUCCGCCGCGCUGAGCU 175 219 CGGCGGCGGCCCGGCAGCC 13 219
CGGCGGCGGCCCGGCAGCC 13 237 GGCUGCCGGGCCGCCGCCG 176 237
CAACAUGGCGGCGGCGGCG 14 237 CAACAUGGCGGCGGCGGCG 14 255
CGCCGCCGCCGCCAUGUUG 177 255 GGCGGCGGGCGCGGGCCCG 15 255
GGCGGCGGGCGCGGGCCCG 15 273 CGGGCCCGCGCCCGCCGCC 178 273
GGAGAUGGUCCGCGGGCAG 16 273 GGAGAUGGUCCGCGGGCAG 16 291
CUGCCCGCGGACCAUCUCC 179 291 GGUGUUCGACGUGGGGCCG 17 291
GGUGUUCGACGUGGGGCCG 17 309 CGGCCCCACGUCGAACACC 180 309
GCGCUACACCAACCUCUCG 18 309 GCGCUACACCAACCUCUCG 18 327
CGAGAGGUUGGUGUAGCGC 181 327 GUACAUCGGCGAGGGCGCC 19 327
GUACAUCGGCGAGGGCGCC 19 345 GGCGCCCUCGCCGAUGUAC 182 345
CUACGGCAUGGUGUGCUCU 20 345 CUACGGCAUGGUGUGCUCU 20 363
AGAGCACACCAUGCCGUAG 183 363 UGCUUAUGAUAAUGUCAAC 21 363
UGCUUAUGAUAAUGUCAAC 21 381 GUUGACAUUAUCAUAAGCA 184 381
CAAAGUUCGAGUAGCUAUC 22 381 CAAAGUUCGAGUAGCUAUC 22 399
GAUAGCUACUCGAACUUUG 185 399 CAAGAAAAUCAGCCCCUUU 23 399
CAAGAAAAUCAGCCCCUUU 23 417 AAAGGGGCUGAUUUUCUUG 186 417
UGAGCACCAGACCUACUGC 24 417 UGAGCACCAGACCUACUGC 24 435
GCAGUAGGUCUGGUGCUCA 187 435 CCAGAGAACCCUGAGGGAG 25 435
CCAGAGAACCCUGAGGGAG 25 453 CUCCCUCAGGGUUCUCUGG 188 453
GAUAAAAAUCUUACUGCGC 26 453 GAUAAAAAUCUUACUGCGC 26 471
GCGCAGUAAGAUUUUUAUC 189 471 CUUCAGACAUGAGAACAUC 27 471
CUUCAGACAUGAGAACAUC 27 489 GAUGUUCUCAUGUCUGAAG 190 489
CAUUGGAAUCAAUGACAUU 28 489 CAUUGGAAUCAAUGACAUU 28 507
AAUGUCAUUGAUUCCAAUG 191 507 UAUUCGAGCACCAACCAUC 29 507
UAUUCGAGCACCAACCAUC 29 525 GAUGGUUGGUGCUCGAAUA 192 525
CGAGCAAAUGAAAGAUGUA 30 525 CGAGCAAAUGAAAGAUGUA 30 543
UACAUCUUUCAUUUGCUCG 193 543 AUAUAUAGUACAGGACCUC 31 543
AUAUAUAGUACAGGACCUC 31 561 GAGGUCCUGUACUAUAUAU 194 561
CAUGGAAACAGAUCUUUAC 32 561 CAUGGAAACAGAUCUUUAC 32 579
GUAAAGAUCUGUUUCCAUG 195 579 CAAGCUCUUGAAGACACAA 33 579
CAAGCUCUUGAAGACACAA 33 597 UUGUGUCUUCAAGAGCUUG 196 597
ACACCUCAGCAAUGACCAU 34 597 ACACCUCAGCAAUGACCAU 34 615
AUGGUCAUUGCUGAGGUGU 197 615 UAUCUGCUAUUUUCUCUAC 35 615
UAUCUGCUAUUUUCUCUAC 35 633 GUAGAGAAAAUAGCAGAUA 198 633
CCAGAUCCUCAGAGGGUUA 36 633 CCAGAUCCUCAGAGGGUUA 36 651
UAACCCUCUGAGGAUCUGG 199 651 AAAAUAUAUCCAUUCAGCU 37 651
AAAAUAUAUCCAUUCAGCU 37 669 AGCUGAAUGGAUAUAUUUU 200 669
UAACGUUCUGCACCGUGAC 38 669 UAACGUUCUGCACCGUGAC 38 687
GUCACGGUGCAGAACGUUA 201 687 CCUCAAGCCUUCCAACCUG 39 687
CCUCAAGCCUUCCAACCUG 39 705 CAGGUUGGAAGGCUUGAGG 202 705
GCUGCUCAACACCACCUGU 40 705 GCUGCUCAACACCACCUGU 40 723
ACAGGUGGUGUUGAGCAGC 203 723 UGAUCUCAAGAUCUGUGAC 41 723
UGAUCUCAAGAUCUGUGAC 41 741 GUCACAGAUCUUGAGAUCA 204 741
CUUUGGCCUGGCCCGUGUU 42 741 CUUUGGCCUGGCCCGUGUU 42 759
AACACGGGCCAGGCCAAAG 205 759 UGCAGAUCCAGACCAUGAU 43 759
UGCAGAUCCAGACCAUGAU 43 777 AUCAUGGUCUGGAUCUGCA 206 777
UCACACAGGGUUCCUGACA 44 777 UCACACAGGGUUCCUGACA 44 795
UGUCAGGAACCCUGUGUGA 207 795 AGAAUAUGUGGCCACACGU 45 795
AGAAUAUGUGGCCACACGU 45 813 ACGUGUGGCCACAUAUUCU 208 813
UUGGUACAGGGCUCCAGAA 46 813 UUGGUACAGGGCUCCAGAA 46 831
UUCUGGAGCCCUGUACCAA 209 831 AAUUAUGUUGAAUUCCAAG 47 831
AAUUAUGUUGAAUUCCAAG 47 849 CUUGGAAUUCAACAUAAUU 210 849
GGGCUACACCAAGUCCAUU 48 849 GGGCUACACCAAGUCCAUU 48 867
AAUGGACUUGGUGUAGCCC 211 867 UGAUAUUUGGUCUGUAGGC 49 867
UGAUAUUUGGUCUGUAGGC 49 885 GCCUACAGACCAAAUAUCA 212 885
CUGCAUUCUGGCAGAAAUG 50 885 CUGCAUUCUGGCAGAAAUG 50 903
CAUUUCUGCCAGAAUGCAG 213 903 GCUUUCUAACAGGCCCAUC 51 903
GCUUUCUAACAGGCCCAUC 51 921 GAUGGGCCUGUUAGAAAGC 214 921
CUUUCCAGGGAAGCAUUAU 52 921 CUUUCCAGGGAAGCAUUAU 52 939
AUAAUGCUUCCCUGGAAAG 215 939 UCUUGACCAGCUGAAACAC 53 939
UCUUGACCAGCUGAAACAC 53 957 GUGUUUCAGCUGGUCAAGA 216 957
CAUUUUGGGUAUUCUUGGA 54 957 CAUUUUGGGUAUUCUUGGA 54 975
UCCAAGAAUACCCAAAAUG 217 975 AUCCCCAUCACAAGAAGAC 55 975
AUCCCCAUCACAAGAAGAC 55 993 GUCUUCUUGUGAUGGGGAU 218 993
CCUGAAUUGUAUAAUAAAU 56 993 CCUGAAUUGUAUAAUAAAU 56 1011
AUUUAUUAUACAAUUCAGG 219 1011 UUUAAAAGCUAGGAACUAU 57 1011
UUUAAAAGCUAGGAACUAU 57 1029 AUAGUUCCUAGCUUUUAAA 220 1029
UUUGCUUUCUCUUCCACAC 58 1029 UUUGCUUUCUCUUCCACAC 58 1047
GUGUGGAAGAGAAAGCAAA 221 1047 CAAAAAUAAGGUGCCAUGG 59 1047
CAAAAAUAAGGUGCCAUGG 59 1065 CCAUGGCACCUUAUUUUUG 222 1065
GAACAGGCUGUUCCCAAAU 60 1065 GAACAGGCUGUUCCCAAAU 60 1083
AUUUGGGAACAGCCUGUUC 223 1083 UGCUGACUCCAAAGCUCUG 61 1083
UGCUGACUCCAAAGCUCUG 61 1101 CAGAGCUUUGGAGUCAGCA 224 1101
GGACUUAUUGGACAAAAUG 62 1101 GGACUUAUUGGACAAAAUG 62 1119
CAUUUUGUCCAAUAAGUCC 225 1119 GUUGACAUUCAACCCACAC 63 1119
GUUGACAUUCAACCCACAC 63 1137 GUGUGGGUUGAAUGUCAAC 226 1137
CAAGAGGAUUGAAGUAGAA 64 1137 CAAGAGGAUUGAAGUAGAA 64 1155
UUCUACUUCAAUCCUCUUG 227 1155 ACAGGCUCUGGCCCACCCA 65 1155
ACAGGCUCUGGCCCACCCA 65 1173 UGGGUGGGCCAGAGCCUGU 228 1173
AUAUCUGGAGCAGUAUUAC 66 1173 AUAUCUGGAGCAGUAUUAC 66 1191
GUAAUACUGCUCCAGAUAU 229 1191 CGACCCGAGUGACGAGCCC 67 1191
CGACCCGAGUGACGAGCCC 67 1209 GGGCUCGUCACUCGGGUCG 230 1209
CAUCGCCGAAGCACCAUUC 68 1209 CAUCGCCGAAGCACCAUUC 68 1227
GAAUGGUGCUUCGGCGAUG 231 1227 CAAGUUCGACAUGGAAUUG 69 1227
CAAGUUCGACAUGGAAUUG 69 1245 CAAUUCCAUGUCGAACUUG 232 1245
GGAUGACUUGCCUAAGGAA 70 1245 GGAUGACUUGCCUAAGGAA 70 1263
UUCCUUAGGCAAGUCAUCC 233 1263 AAAGCUCAAAGAACUAAUU 71 1263
AAAGCUCAAAGAACUAAUU 71 1281 AAUUAGUUCUUUGAGCUUU 234 1281
UUUUGAAGAGACUGCUAGA 72 1281 UUUUGAAGAGACUGCUAGA 72 1299
UCUAGCAGUCUCUUCAAAA 235 1299 AUUCCAGCCAGGAUACAGA 73 1299
AUUCCAGCCAGGAUACAGA 73 1317 UCUGUAUCCUGGCUGGAAU 236 1317
AUCUUAAAUUUGUCAGGAC 74 1317 AUCUUAAAUUUGUCAGGAC 74 1335
GUCCUGACAAAUUUAAGAU 237 1335 CAAGGGCUCAGAGGACUGG 75 1335
CAAGGGCUCAGAGGACUGG 75 1353 CCAGUCCUCUGAGCCCUUG 238 1353
GACGUGCUCAGACAUCGGU 76 1353 GACGUGCUCAGACAUCGGU 76 1371
ACCGAUGUCUGAGCACGUC 239 1371 UGUUCUUCUUCCCAGUUCU 77 1371
UGUUCUUCUUCCCAGUUCU 77 1389 AGAACUGGGAAGAAGAACA 240 1389
UUGACCCCUGGUCCUGUCU 78 1389 UUGACCCCUGGUCCUGUCU 78 1407
AGACAGGACCAGGGGUCAA 241 1407 UCCAGCCCGUCUUGGCUUA 79 1407
UCCAGCCCGUCUUGGCUUA 79 1425 UAAGCCAAGACGGGCUGGA 242 1425
AUCCACUUUGACUCCUUUG 80 1425 AUCCACUUUGACUCCUUUG 80 1443
CAAAGGAGUCAAAGUGGAU 243 1443 GAGCCGUUUGGAGGGGCGG 81 1443
GAGCCGUUUGGAGGGGCGG 81 1461
CCGCCCCUCCAAACGGCUC 244 1461 GUUUCUGGUAGUUGUGGCU 82 1461
GUUUCUGGUAGUUGUGGCU 82 1479 AGCCACAACUACCAGAAAC 245 1479
UUUUAUGCUUUCAAAGAAU 83 1479 UUUUAUGCUUUCAAAGAAU 83 1497
AUUCUUUGAAAGCAUAAAA 246 1497 UUUCUUCAGUCCAGAGAAU 84 1497
UUUCUUCAGUCCAGAGAAU 84 1515 AUUCUCUGGACUGAAGAAA 247 1515
UUCCUCCUGGCAGCCCUGU 85 1515 UUCCUCCUGGCAGCCCUGU 85 1533
ACAGGGCUGCCAGGAGGAA 248 1533 UGUGUGUCACCCAUUGGUG 86 1533
UGUGUGUCACCCAUUGGUG 86 1551 CACCAAUGGGUGACACACA 249 1551
GACCUGCGGCAGUAUGUAC 87 1551 GACCUGCGGCAGUAUGUAC 87 1569
GUACAUACUGCCGCAGGUC 250 1569 CUUCAGUGCACCUUACUGC 88 1569
CUUCAGUGCACCUUACUGC 88 1587 GCAGUAAGGUGCACUGAAG 251 1587
CUUACUGUUGCUUUAGUCA 89 1587 CUUACUGUUGCUUUAGUCA 89 1605
UGACUAAAGCAACAGUAAG 252 1605 ACUAAUUGCUUUCUGGUUU 90 1605
ACUAAUUGCUUUCUGGUUU 90 1623 AAACCAGAAAGCAAUUAGU 253 1623
UGAAAGAUGCAGUGGUUCC 91 1623 UGAAAGAUGCAGUGGUUCC 91 1641
GGAACCACUGCAUCUUUCA 254 1641 CUCCCUCUCCUGAAUCCUU 92 1641
CUCCCUCUCCUGAAUCCUU 92 1659 AAGGAUUCAGGAGAGGGAG 255 1659
UUUCUACAUGAUGCCCUGC 93 1659 UUUCUACAUGAUGCCCUGC 93 1677
GCAGGGCAUCAUGUAGAAA 256 1677 CUGACCAUGCAGCCGCACC 94 1677
CUGACCAUGCAGCCGCACC 94 1695 GGUGCGGCUGCAUGGUCAG 257 1695
CAGAGAGAGAUUCUUCCCC 95 1695 CAGAGAGAGAUUCUUCCCC 95 1713
GGGGAAGAAUCUCUCUCUG 258 1713 CAAUUGGCUCUAGUCACUG 96 1713
CAAUUGGCUCUAGUCACUG 96 1731 CAGUGACUAGAGCCAAUUG 259 1731
GGCAUCUCACUUUAUGAUA 97 1731 GGCAUCUCACUUUAUGAUA 97 1749
UAUCAUAAAGUGAGAUGCC 260 1749 AGGGAAGGCUACUACCUAG 98 1749
AGGGAAGGCUACUACCUAG 98 1767 CUAGGUAGUAGCCUUCCCU 261 1767
GGGCACUUUAAGUCAGUGA 99 1767 GGGCACUUUAAGUCAGUGA 99 1785
UCACUGACUUAAAGUGCCC 262 1785 ACAGCCCCUUAUUUGCACU 100 1785
ACAGCCCCUUAUUUGCACU 100 1803 AGUGCAAAUAAGGGGCUGU 263 1803
UUCACCUUUUGACCAUAAC 101 1803 UUCACCUUUUGACCAUAAC 101 1821
GUUAUGGUCAAAAGGUGAA 264 1821 CUGUUUCCCCAGAGCAGGA 102 1821
CUGUUUCCCCAGAGCAGGA 102 1839 UCCUGCUCUGGGGAAACAG 265 1839
AGCUUGUGGAAAUACCUUG 103 1839 AGCUUGUGGAAAUACCUUG 103 1857
CAAGGUAUUUCCACAAGCU 266 1857 GGCUGAUGUUGCAGCCUGC 104 1857
GGCUGAUGUUGCAGCCUGC 104 1875 GCAGGCUGCAACAUCAGCC 267 1875
CAGCAAGUGCUUCCGUCUC 105 1875 CAGCAAGUGCUUCCGUCUC 105 1893
GAGACGGAAGCACUUGCUG 268 1893 CCGGAAUCCUUGGGGAGCA 106 1893
CCGGAAUCCUUGGGGAGCA 106 1911 UGCUCCCCAAGGAUUCCGG 269 1911
ACUUGUCCACGUCUUUUCU 107 1911 ACUUGUCCACGUCUUUUCU 107 1929
AGAAAAGACGUGGACAAGU 270 1929 UCAUAUCAUGGUAGUCACU 108 1929
UCAUAUCAUGGUAGUCACU 108 1947 AGUGACUACCAUGAUAUGA 271 1947
UAACAUAUAUAAGGUAUGU 109 1947 UAACAUAUAUAAGGUAUGU 109 1965
ACAUACCUUAUAUAUGUUA 272 1965 UGCUAUUGGCCCAGCUUUU 110 1965
UGCUAUUGGCCCAGCUUUU 110 1983 AAAAGCUGGGCCAAUAGCA 273 1983
UAGAAAAUGCAGUCAUUUU 111 1983 UAGAAAAUGCAGUCAUUUU 111 2001
AAAAUGACUGCAUUUUCUA 274 2001 UUCUAAAUAAAAAGGAAGU 112 2001
UUCUAAAUAAAAAGGAAGU 112 2019 ACUUCCUUUUUAUUUAGAA 275 2019
UACUGCACCCAGCAGUGUC 113 2019 UACUGCACCCAGCAGUGUC 113 2037
GACACUGCUGGGUGCAGUA 276 2037 CACUCUGUAGUUACUGUGG 114 2037
CACUCUGUAGUUACUGUGG 114 2055 CCACAGUAACUACAGAGUG 277 2055
GUCACUUGUACCAUAUAGA 115 2055 GUCACUUGUACCAUAUAGA 115 2073
UCUAUAUGGUACAAGUGAC 278 2073 AGGUGUAACACUUGUCAAG 116 2073
AGGUGUAACACUUGUCAAG 116 2091 CUUGACAAGUGUUACACCU 279 2091
GAAGCGUUAUGUGCAGUAC 117 2091 GAAGCGUUAUGUGCAGUAC 117 2109
GUACUGCACAUAACGCUUC 280 2109 CUUAAUGUUUGUAAGACUU 118 2109
CUUAAUGUUUGUAAGACUU 118 2127 AAGUCUUACAAACAUUAAG 281 2127
UACAAAAAAAGAUUUAAAG 119 2127 UACAAAAAAAGAUUUAAAG 119 2145
CUUUAAAUCUUUUUUUGUA 282 2145 GUGGCAGCUUCACUCGACA 120 2145
GUGGCAGCUUCACUCGACA 120 2163 UGUCGAGUGAAGCUGCCAC 283 2163
AUUUGGUGAGAGAAGUACA 121 2163 AUUUGGUGAGAGAAGUACA 121 2181
UGUACUUCUCUCACCAAAU 284 2181 AAAGGUUGCAGUGCUGAGC 122 2181
AAAGGUUGCAGUGCUGAGC 122 2199 GCUCAGCACUGCAACCUUU 285 2199
CUGUGGGCGGUUUCUGGGG 123 2199 CUGUGGGCGGUUUCUGGGG 123 2217
CCCCAGAAACCGCCCACAG 286 2217 GAUGUCCCAGGGUGGAACU 124 2217
GAUGUCCCAGGGUGGAACU 124 2235 AGUUCCACCCUGGGACAUC 287 2235
UCCACAUGCUGGUGCAUAU 125 2235 UCCACAUGCUGGUGCAUAU 125 2253
AUAUGCACCAGCAUGUGGA 288 2253 UACGCCCUUGAGCUACUUC 126 2253
UACGCCCUUGAGCUACUUC 126 2271 GAAGUAGCUCAAGGGCGUA 289 2271
CAAAUGUGGUUUAUACCUC 127 2271 CAAAUGUGGUUUAUACCUC 127 2289
GAGGUAUAAACCACAUUUG 290 2289 CGCAGAUACAAGAAUCUUU 128 2289
CGCAGAUACAAGAAUCUUU 128 2307 AAAGAUUCUUGUAUCUGCG 291 2307
UAUGAAUAUACAAUUCUUU 129 2307 UAUGAAUAUACAAUUCUUU 129 2325
AAAGAAUUGUAUAUUCAUA 292 2325 UUUCCUUCUACAGCUUAGC 130 2325
UUUCCUUCUACAGCUUAGC 130 2343 GCUAAGCUGUAGAAGGAAA 293 2343
CUCCGUCUUUUCAACCACG 131 2343 CUCCGUCUUUUCAACCACG 131 2361
CGUGGUUGAAAAGACGGAG 294 2361 GAACAUUUAAAACCCGACC 132 2361
GAACAUUUAAAACCCGACC 132 2379 GGUCGGGUUUUAAAUGUUC 295 2379
CUACUAGCACUGUUCUGUC 133 2379 CUACUAGCACUGUUCUGUC 133 2397
GACAGAACAGUGCUAGUAG 296 2397 CCUCAAGUACUCAAAUAUU 134 2397
CCUCAAGUACUCAAAUAUU 134 2415 AAUAUUUGAGUACUUGAGG 297 2415
UUCUGAUACUGCUGAGUCA 135 2415 UUCUGAUACUGCUGAGUCA 135 2433
UGACUCAGCAGUAUCAGAA 298 2433 AGACUGUCAGAAAAAGCUA 136 2433
AGACUGUCAGAAAAAGCUA 136 2451 UAGCUUUUUCUGACAGUCU 299 2451
AGCACUAACUCGUGUUUGG 137 2451 AGCACUAACUCGUGUUUGG 137 2469
CCAAACACGAGUUAGUGCU 300 2469 GAGCUCUAUCCAUAUUUUA 138 2469
GAGCUCUAUCCAUAUUUUA 138 2487 UAAAAUAUGGAUAGAGCUC 301 2487
ACUGAUCUCUUUAAGUAUU 139 2487 ACUGAUCUCUUUAAGUAUU 139 2505
AAUACUUAAAGAGAUCAGU 302 2505 UUGUUCCUGCCACUGUGUA 140 2505
UUGUUCCUGCCACUGUGUA 140 2523 UACACAGUGGCAGGAACAA 303 2523
ACUGUGGAGUUGACUCGGU 141 2523 ACUGUGGAGUUGACUCGGU 141 2541
ACCGAGUCAACUCCACAGU 304 2541 UGUUCUGUCCCAGUGCGGU 142 2541
UGUUCUGUCCCAGUGCGGU 142 2559 ACCGCACUGGGACAGAACA 305 2559
UGCCUCCUCUUGACUUCCC 143 2559 UGCCUCCUCUUGACUUCCC 143 2577
GGGAAGUCAAGAGGAGGCA 306 2577 CCACUGCUCUCUGUGGUGA 144 2577
CCACUGCUCUCUGUGGUGA 144 2595 UCACCACAGAGAGCAGUGG 307 2595
AGAAAUUUGCCUUGUUCAA 145 2595 AGAAAUUUGCCUUGUUCAA 145 2613
UUGAACAAGGCAAAUUUCU 308 2613 AUAAUUACUGUACCCUCGC 146 2613
AUAAUUACUGUACCCUCGC 146 2631 GCGAGGGUACAGUAAUUAU 309 2631
CAUGACUGUUACAGCUUUC 147 2631 CAUGACUGUUACAGCUUUC 147 2649
GAAAGCUGUAACAGUCAUG 310 2649 CUGUGCAGAGAUGACUGUC 148 2649
CUGUGCAGAGAUGACUGUC 148 2667 GACAGUCAUCUCUGCACAG 311 2667
CCAAGUGCCACAUGCCUAC 149 2667 CCAAGUGCCACAUGCCUAC 149 2685
GUAGGCAUGUGGCACUUGG 312 2685 CGAUUGAAAUGAAAACUCU 150 2685
CGAUUGAAAUGAAAACUCU 150 2703 AGAGUUUUCAUUUCAAUCG 313 2703
UAUUGUUACCUCUGAGUUG 151 2703 UAUUGUUACCUCUGAGUUG 151 2721
CAACUCAGAGGUAACAAUA 314 2721 GUGUUCCACGGAAAAUGCU 152 2721
GUGUUCCACGGAAAAUGCU 152 2739 AGCAUUUUCCGUGGAACAC 315 2739
UAUCCAGCAGAUCAUUUAG 153 2739 UAUCCAGCAGAUCAUUUAG 153 2757
CUAAAUGAUCUGCUGGAUA 316 2757 GGAAAAAUAAUUCUAUUUU 154 2757
GGAAAAAUAAUUCUAUUUU 154 2775 AAAAUAGAAUUAUUUUUCC 317 2775
UUAGCUUUUCAUUUCUCAG 155 2775 UUAGCUUUUCAUUUCUCAG 155 2793
CUGAGAAAUGAAAAGCUAA 318 2793 GCUGUCCUUUUUUCUUGUU 156 2793
GCUGUCCUUUUUUCUUGUU 156 2811 AACAAGAAAAAAGGACAGC 319 2811
UUGAUUUUUGACAGCAAUG 157 2811 UUGAUUUUUGACAGCAAUG 157 2829
CAUUGCUGUCAAAAAUCAA 320 2829 GGAGAAUGGGUUAUAUAAA 158 2829
GGAGAAUGGGUUAUAUAAA 158 2847 UUUAUAUAACCCAUUCUCC 321 2847
AGACUGCCUGCUAAUAUGA 159 2847 AGACUGCCUGCUAAUAUGA 159 2865
UCAUAUUAGCAGGCAGUCU 322 2865 AACAGAAAUGCAUUUGUAA 160 2865
AACAGAAAUGCAUUUGUAA 160 2883 UUACAAAUGCAUUUCUGUU 323 2883
AUUCAUGAAAAUAAAUGUA 161 2883 AUUCAUGAAAAUAAAUGUA 161 2901
UACAUUUAUUUUCAUGAAU 324 2901 ACAUCUUCUAUCUUCAAAA 162 2901
ACAUCUUCUAUCUUCAAAA 162 2919 UUUUGAAGAUAGAAGAUGU 325 2913
UUCAAAAAAAAAAAAAAAA 163 2913 UUCAAAAAAAAAAAAAAAA 163 2931
UUUUUUUUUUUUUUUUGAA 326 Seq Seq Seq Pos Target Sequence ID UPos
Upper seq ID LPos Lower seq ID
XM_055766.6 (MAPK3/ERK1) 3 CGGGGCCUCGGGCGGGGCC 327 3
CGGGGCCUCGGGCGGGGCC 327 21 GGCCCCGCCCGAGGCCCCG 432 21
CGCCGUGGGGAGGAGGGCG 328 21 CGCCGUGGGGAGGAGGGCG 328 39
CGCCCUCCUCCCCACGGCG 433 39 GGUGGGAGGGGAGGAGUGG 329 39
GGUGGGAGGGGAGGAGUGG 329 57 CCACUCCUCCCCUCCCACC 434 57
GAGAUGGCGGCGGCGGCGG 330 57 GAGAUGGCGGCGGCGGCGG 330 75
CCGCCGCCGCCGCCAUCUC 435 75 GCUCAGGGGGGCGGGGGCG 331 75
GCUCAGGGGGGCGGGGGCG 331 93 CGCCCCCGCCCCCCUGAGC 436 93
GGGGAGCCCCGUAGAACCG 332 93 GGGGAGCCCCGUAGAACCG 332 111
CGGUUCUACGGGGCUCCCC 437 111 GAGGGGGUCGGCCCGGGGG 333 111
GAGGGGGUCGGCCCGGGGG 333 129 CCCCCGGGCCGACCCCCUC 438 129
GUCCCGGGGGAGGUGGAGA 334 129 GUCCCGGGGGAGGUGGAGA 334 147
UCUCCACCUCCCCCGGGAC 439 147 AUGGUGAAGGGGCAGCCGU 335 147
AUGGUGAAGGGGCAGCCGU 335 165 ACGGCUGCCCCUUCACCAU 440 165
UUCGACGUGGGCCCGCGCU 336 165 UUCGACGUGGGCCCGCGCU 336 183
AGCGCGGGCCCACGUCGAA 441 183 UACACGCAGUUGCAGUACA 337 183
UACACGCAGUUGCAGUACA 337 201 UGUACUGCAACUGCGUGUA 442 201
AUCGGCGAGGGCGCGUACG 338 201 AUCGGCGAGGGCGCGUACG 338 219
CGUACGCGCCCUCGCCGAU 443 219 GGCAUGGUCAGCUCGGCCU 339 219
GGCAUGGUCAGCUCGGCCU 339 237 AGGCCGAGCUGACCAUGCC 444 237
UAUGACCACGUGCGCAAGA 340 237 UAUGACCACGUGCGCAAGA 340 255
UCUUGCGCACGUGGUCAUA 445 255 ACUCGCGUGGCCAUCAAGA 341 255
ACUCGCGUGGCCAUCAAGA 341 273 UCUUGAUGGCCACGCGAGU 446 273
AAGAUCAGCCCCUUCGAAC 342 273 AAGAUCAGCCCCUUCGAAC 342 291
GUUCGAAGGGGCUGAUCUU 447 291 CAUCAGACCUACUGCCAGC 343 291
CAUCAGACCUACUGCCAGC 343 309 GCUGGCAGUAGGUCUGAUG 448 309
CGCACGCUCCGGGAGAUCC 344 309 CGCACGCUCCGGGAGAUCC 344 327
GGAUCUCCCGGAGCGUGCG 449 327 CAGAUCCUGCUGCGCUUCC 345 327
CAGAUCCUGCUGCGCUUCC 345 345 GGAAGCGCAGCAGGAUCUG 450 345
CGCCAUGAGAAUGUCAUCG 346 345 CGCCAUGAGAAUGUCAUCG 346 363
CGAUGACAUUCUCAUGGCG 451 363 GGCAUCCGAGACAUUCUGC 347 363
GGCAUCCGAGACAUUCUGC 347 381 GCAGAAUGUCUCGGAUGCC 452 381
CGGGCGUCCACCCUGGAAG 348 381 CGGGCGUCCACCCUGGAAG 348 399
CUUCCAGGGUGGACGCCCG 453 399 GCCAUGAGAGAUGUCUACA 349 399
GCCAUGAGAGAUGUCUACA 349 417 UGUAGACAUCUCUCAUGGC 454 417
AUUGUGCAGGACCUGAUGG 350 417 AUUGUGCAGGACCUGAUGG 350 435
CCAUCAGGUCCUGCACAAU 455 435 GAGACUGACCUGUACAAGU 351 435
GAGACUGACCUGUACAAGU 351 453 ACUUGUACAGGUCAGUCUC 456 453
UUGCUGAAAAGCCAGCAGC 352 453 UUGCUGAAAAGCCAGCAGC 352 471
GCUGCUGGCUUUUCAGCAA 457 471 CUGAGCAAUGACCAUAUCU 353 471
CUGAGCAAUGACCAUAUCU 353 489 AGAUAUGGUCAUUGCUCAG 458 489
UGCUACUUCCUCUACCAGA 354 489 UGCUACUUCCUCUACCAGA 354 507
UCUGGUAGAGGAAGUAGCA 459 507 AUCCUGCGGGGCCUCAAGU 355 507
AUCCUGCGGGGCCUCAAGU 355 525 ACUUGAGGCCCCGCAGGAU 460 525
UACAUCCACUCCGCCAACG 356 525 UACAUCCACUCCGCCAACG 356 543
CGUUGGCGGAGUGGAUGUA 461 543 GUGCUCCACCGAGAUCUAA 357 543
GUGCUCCACCGAGAUCUAA 357 561 UUAGAUCUCGGUGGAGCAC 462 561
AAGCCCUCCAACCUGCUCA 358 561 AAGCCCUCCAACCUGCUCA 358 579
UGAGCAGGUUGGAGGGCUU 463 579 AUCAACACCACCUGCGACC 359 579
AUCAACACCACCUGCGACC 359 597 GGUCGCAGGUGGUGUUGAU 464 597
CUUAAGAUUUGUGAUUUCG 360 597 CUUAAGAUUUGUGAUUUCG 360 615
CGAAAUCACAAAUCUUAAG 465 615 GGCCUGGCCCGGAUUGCCG 361 615
GGCCUGGCCCGGAUUGCCG 361 633 CGGCAAUCCGGGCCAGGCC 466 633
GAUCCUGAGCAUGACCACA 362 633 GAUCCUGAGCAUGACCACA 362 651
UGUGGUCAUGCUCAGGAUC 467 651 ACCGGCUUCCUGACGGAGU 363 651
ACCGGCUUCCUGACGGAGU 363 669 ACUCCGUCAGGAAGCCGGU 468 669
UAUGUGGCUACGCGCUGGU 364 669 UAUGUGGCUACGCGCUGGU 364 687
ACCAGCGCGUAGCCACAUA 469 687 UACCGGGCCCCAGAGAUCA 365 687
UACCGGGCCCCAGAGAUCA 365 705 UGAUCUCUGGGGCCCGGUA 470 705
AUGCUGAACUCCAAGGGCU 366 705 AUGCUGAACUCCAAGGGCU 366 723
AGCCCUUGGAGUUCAGCAU 471 723 UAUACCAAGUCCAUCGACA 367 723
UAUACCAAGUCCAUCGACA 367 741 UGUCGAUGGACUUGGUAUA 472 741
AUCUGGUCUGUGGGCUGCA 368 741 AUCUGGUCUGUGGGCUGCA 368 759
UGCAGCCCACAGACCAGAU 473 759 AUUCUGGCUGAGAUGCUCU 369 759
AUUCUGGCUGAGAUGCUCU 369 777 AGAGCAUCUCAGCCAGAAU 474 777
UCUAACCGGCCCAUCUUCC 370 777 UCUAACCGGCCCAUCUUCC 370 795
GGAAGAUGGGCCGGUUAGA 475 795 CCUGGCAAGCACUACCUGG 371 795
CCUGGCAAGCACUACCUGG 371 813 CCAGGUAGUGCUUGCCAGG 476 813
GAUCAGCUCAACCACAUUC 372 813 GAUCAGCUCAACCACAUUC 372 831
GAAUGUGGUUGAGCUGAUC 477 831 CUGGGCAUCCUGGGCUCCC 373 831
CUGGGCAUCCUGGGCUCCC 373 849 GGGAGCCCAGGAUGCCCAG 478 849
CCAUCCCAGGAGGACCUGA 374 849 CCAUCCCAGGAGGACCUGA 374 867
UCAGGUCCUCCUGGGAUGG 479 867 AAUUGUAUCAUCAACAUGA 375 867
AAUUGUAUCAUCAACAUGA 375 885 UCAUGUUGAUGAUACAAUU 480 885
AAGGCCCGAAACUACCUAC 376 885 AAGGCCCGAAACUACCUAC 376 903
GUAGGUAGUUUCGGGCCUU 481 903 CAGUCUCUGCCCUCCAAGA 377 903
CAGUCUCUGCCCUCCAAGA 377 921 UCUUGGAGGGCAGAGACUG 482 921
ACCAAGGUGGCUUGGGCCA 378 921 ACCAAGGUGGCUUGGGCCA 378 939
UGGCCCAAGCCACCUUGGU 483 939 AAGCUUUUCCCCAAGUCAG 379 939
AAGCUUUUCCCCAAGUCAG 379 957 CUGACUUGGGGAAAAGCUU 484 957
GACUCCAAAGCCCUUGACC 380 957 GACUCCAAAGCCCUUGACC 380 975
GGUCAAGGGCUUUGGAGUC 485 975 CUGCUGGACCGGAUGUUAA 381 975
CUGCUGGACCGGAUGUUAA 381 993 UUAACAUCCGGUCCAGCAG 486 993
ACCUUUAACCCCAAUAAAC 382 993 ACCUUUAACCCCAAUAAAC 382 1011
GUUUAUUGGGGUUAAAGGU 487 1011 CGGAUCACAGUGGAGGAAG 383 1011
CGGAUCACAGUGGAGGAAG 383 1029 CUUCCUCCACUGUGAUCCG 488 1029
GCGCUGGCUCACCCCUACC 384 1029 GCGCUGGCUCACCCCUACC 384 1047
GGUAGGGGUGAGCCAGCGC 489 1047 CUGGAGCAGUACUAUGACC 385 1047
CUGGAGCAGUACUAUGACC 385 1065 GGUCAUAGUACUGCUCCAG 490 1065
CCGACGGAUGAGCCAGUGG 386 1065 CCGACGGAUGAGCCAGUGG 386 1083
CCACUGGCUCAUCCGUCGG 491 1083 GCCGAGGAGCCCUUCACCU 387 1083
GCCGAGGAGCCCUUCACCU 387 1101 AGGUGAAGGGCUCCUCGGC 492 1101
UUCGCCAUGGAGCUGGAUG 388 1101 UUCGCCAUGGAGCUGGAUG 388 1119
CAUCCAGCUCCAUGGCGAA 493 1119 GACCUACCUAAGGAGCGGC 389 1119
GACCUACCUAAGGAGCGGC 389 1137 GCCGCUCCUUAGGUAGGUC 494 1137
CUGAAGGAGCUCAUCUUCC 390 1137 CUGAAGGAGCUCAUCUUCC 390 1155
GGAAGAUGAGCUCCUUCAG 495 1155 CAGGAGACAGCACGCUUCC 391 1155
CAGGAGACAGCACGCUUCC 391 1173 GGAAGCGUGCUGUCUCCUG 496 1173
CAGCCCGGAGUGCUGGAGG 392 1173 CAGCCCGGAGUGCUGGAGG 392 1191
CCUCCAGCACUCCGGGCUG 497 1191 GCCCCCUAGCCCAGACAGA 393 1191
GCCCCCUAGCCCAGACAGA 393 1209 UCUGUCUGGGCUAGGGGGC 498 1209
ACAUCUCUGCACCCUGGGG 394 1209 ACAUCUCUGCACCCUGGGG 394 1227
CCCCAGGGUGCAGAGAUGU 499 1227 GCCUGGAACAGAACUGGCA 395 1227
GCCUGGAACAGAACUGGCA 395 1245 UGCCAGUUCUGUUCCAGGC 500 1245
AAAGAGGCAAGAGGUCACU 396 1245 AAAGAGGCAAGAGGUCACU 396 1263
AGUGACCUCUUGCCUCUUU 501 1263 UGAGGGCCUCUGUCACCCA 397 1263
UGAGGGCCUCUGUCACCCA 397 1281 UGGGUGACAGAGGCCCUCA 502 1281
AGGACCUGCCUCCUGCCUG 398 1281 AGGACCUGCCUCCUGCCUG 398 1299
CAGGCAGGAGGCAGGUCCU 503 1299 GCCCCUCUCCCGCCAGACU 399 1299
GCCCCUCUCCCGCCAGACU 399 1317 AGUCUGGCGGGAGAGGGGC 504 1317
UGUUAGAAAAUGGACACUG 400 1317 UGUUAGAAAAUGGACACUG 400 1335
CAGUGUCCAUUUUCUAACA 505 1335 GUGCCCAGCCCGGACCUUG 401 1335
GUGCCCAGCCCGGACCUUG 401 1353 CAAGGUCCGGGCUGGGCAC 506 1353
GGCAGCCCAGGCCGGGGUG 402 1353 GGCAGCCCAGGCCGGGGUG 402 1371
CACCCCGGCCUGGGCUGCC 507 1371 GGAGCAUGGGCCUGGCCAC 403 1371
GGAGCAUGGGCCUGGCCAC 403 1389 GUGGCCAGGCCCAUGCUCC 508 1389
CCUCUCUCCUUUGCUGAGG 404 1389 CCUCUCUCCUUUGCUGAGG 404 1407
CCUCAGCAAAGGAGAGAGG 509 1407 GCCUCCAGCUUCAGGCAGG 405 1407
GCCUCCAGCUUCAGGCAGG 405 1425 CCUGCCUGAAGCUGGAGGC 510 1425
GCCAAGGCCUUCUCCUCCC 406 1425 GCCAAGGCCUUCUCCUCCC 406 1443
GGGAGGAGAAGGCCUUGGC 511 1443 CCACCCGCCCUCCCCACGG 407 1443
CCACCCGCCCUCCCCACGG 407 1461 CCGUGGGGAGGGCGGGUGG 512 1461
GGGCCUCGGGACCUCAGGU 408 1461 GGGCCUCGGGACCUCAGGU 408 1479
ACCUGAGGUCCCGAGGCCC 513 1479 UGGCCCCAGUUCAAUCUCC 409 1479
UGGCCCCAGUUCAAUCUCC 409 1497 GGAGAUUGAACUGGGGCCA 514 1497
CCGCUGCUGCUGCUGCGCC 410 1497 CCGCUGCUGCUGCUGCGCC 410 1515
GGCGCAGCAGCAGCAGCGG 515 1515 CCUUACCUUCCCCAGCGUC 411 1515
CCUUACCUUCCCCAGCGUC 411 1533 GACGCUGGGGAAGGUAAGG 516 1533
CCCAGUCUCUGGCAGUUCU 412 1533 CCCAGUCUCUGGCAGUUCU 412 1551
AGAACUGCCAGAGACUGGG 517 1551 UGGAAUGGAAGGGUUCUGG 413 1551
UGGAAUGGAAGGGUUCUGG 413 1569 CCAGAACCCUUCCAUUCCA 518 1569
GCUGCCCCAACCUGCUGAA 414 1569 GCUGCCCCAACCUGCUGAA 414 1587
UUCAGCAGGUUGGGGCAGC 519 1587 AGGGCAGAGGUGGAGGGUG 415 1587
AGGGCAGAGGUGGAGGGUG 415 1605 CACCCUCCACCUCUGCCCU 520 1605
GGGGGGCGCUGAGUAGGGA 416 1605 GGGGGGCGCUGAGUAGGGA 416 1623
UCCCUACUCAGCGCCCCCC 521 1623 ACUCAGGGCCAUGCCUGCC 417 1623
ACUCAGGGCCAUGCCUGCC 417 1641 GGCAGGCAUGGCCCUGAGU 522 1641
CCCCCUCAUCUCAUUCAAA 418 1641 CCCCCUCAUCUCAUUCAAA 418 1659
UUUGAAUGAGAUGAGGGGG 523 1659 ACCCCACCCUAGUUUCCCU 419 1659
ACCCCACCCUAGUUUCCCU 419 1677 AGGGAAACUAGGGUGGGGU 524 1677
UGAAGGAACAUUCCUUAGU 420 1677 UGAAGGAACAUUCCUUAGU 420 1695
ACUAAGGAAUGUUCCUUCA 525 1695 UCUCAAGGGCUAGCAUCCC 421 1695
UCUCAAGGGCUAGCAUCCC 421 1713 GGGAUGCUAGCCCUUGAGA 526 1713
CUGAGGAGCCAGGCCGGGC 422 1713 CUGAGGAGCCAGGCCGGGC 422 1731
GCCCGGCCUGGCUCCUCAG 527 1731 CCGAAUCCCCUCCCUGUCA 423 1731
CCGAAUCCCCUCCCUGUCA 423 1749 UGACAGGGAGGGGAUUCGG 528 1749
AAAGCUGUCACUUCGCGUG 424 1749 AAAGCUGUCACUUCGCGUG 424 1767
CACGCGAAGUGACAGCUUU 529 1767 GCCCUCGCUGCUUCUGUGU 425 1767
GCCCUCGCUGCUUCUGUGU 425 1785 ACACAGAAGCAGCGAGGGC 530 1785
UGUGGUGAGCAGAAGUGGA 426 1785 UGUGGUGAGCAGAAGUGGA 426 1803
UCCACUUCUGCUCACCACA 531 1803 AGCUGGGGGGCGUGGAGAG 427 1803
AGCUGGGGGGCGUGGAGAG 427 1821 CUCUCCACGCCCCCCAGCU 532 1821
GCCCGGCGCCCCUGCCACC 428 1821 GCCCGGCGCCCCUGCCACC 428 1839
GGUGGCAGGGGCGCCGGGC 533 1839 CUCCCUGACCCGUCUAAUA 429 1839
CUCCCUGACCCGUCUAAUA 429 1857 UAUUAGACGGGUCAGGGAG 534 1857
AUAUAAAUAUAGAGAUGUG 430 1857 AUAUAAAUAUAGAGAUGUG 430 1875
CACAUCUCUAUAUUUAUAU 535 1865 AUAGAGAUGUGUCUAUGGC 431 1865
AUAGAGAUGUGUCUAUGGC 431 1883 GCCAUAGACACAUCUCUAU 536 ID Seq Seq Pos
Target Sequence Seq UPos Upper seq ID LPos Lower seq ID NM_002750
(MAPK8/JNK1) 3 UAAUUGCUUGCCAUCAUGA 537 3 UAAUUGCUUGCCAUCAUGA 537 21
UCAUGAUGGCAAGCAAUUA 616 21 AGCAGAAGCAAGCGUGACA 538 21
AGCAGAAGCAAGCGUGACA 538 39 UGUCACGCUUGCUUCUGCU 617 39
AACAAUUUUUAUAGUGUAG 539 39 AACAAUUUUUAUAGUGUAG 539 57
CUACACUAUAAAAAUUGUU 618 57 GAGAUUGGAGAUUCUACAU 540 57
GAGAUUGGAGAUUCUACAU 540 75 AUGUAGAAUCUCCAAUCUC 619 75
UUCACAGUCCUGAAACGAU 541 75 UUCACAGUCCUGAAACGAU 541 93
AUCGUUUCAGGACUGUGAA 620 93 UAUCAGAAUUUAAAACCUA 542 93
UAUCAGAAUUUAAAACCUA 542 111 UAGGUUUUAAAUUCUGAUA 621 111
AUAGGCUCAGGAGCUCAAG 543 111 AUAGGCUCAGGAGCUCAAG 543 129
CUUGAGCUCCUGAGCCUAU 622 129 GGAAUAGUAUGCGCAGCUU 544 129
GGAAUAGUAUGCGCAGCUU 544 147 AAGCUGCGCAUACUAUUCC 623 147
UAUGAUGCCAUUCUUGAAA 545 147 UAUGAUGCCAUUCUUGAAA 545 165
UUUCAAGAAUGGCAUCAUA 624 165 AGAAAUGUUGCAAUCAAGA 546 165
AGAAAUGUUGCAAUCAAGA 546 183 UCUUGAUUGCAACAUUUCU 625 183
AAGCUAAGCCGACCAUUUC 547 183 AAGCUAAGCCGACCAUUUC 547 201
GAAAUGGUCGGCUUAGCUU 626 201 CAGAAUCAGACUCAUGCCA 548 201
CAGAAUCAGACUCAUGCCA 548 219 UGGCAUGAGUCUGAUUCUG 627 219
AAGCGGGCCUACAGAGAGC 549 219 AAGCGGGCCUACAGAGAGC 549 237
GCUCUCUGUAGGCCCGCUU 628 237 CUAGUUCUUAUGAAAUGUG 550 237
CUAGUUCUUAUGAAAUGUG 550 255 CACAUUUCAUAAGAACUAG 629 255
GUUAAUCACAAAAAUAUAA 551 255 GUUAAUCACAAAAAUAUAA 551 273
UUAUAUUUUUGUGAUUAAC 630 273 AUUGGCCUUUUGAAUGUUU 552 273
AUUGGCCUUUUGAAUGUUU 552 291 AAACAUUCAAAAGGCCAAU 631 291
UUCACACCACAGAAAUCCC 553 291 UUCACACCACAGAAAUCCC 553 309
GGGAUUUCUGUGGUGUGAA 632 309 CUAGAAGAAUUUCAAGAUG 554 309
CUAGAAGAAUUUCAAGAUG 554 327 CAUCUUGAAAUUCUUCUAG 633 327
GUUUACAUAGUCAUGGAGC 555 327 GUUUACAUAGUCAUGGAGC 555 345
GCUCCAUGACUAUGUAAAC 634 345 CUCAUGGAUGCAAAUCUUU 556 345
CUCAUGGAUGCAAAUCUUU 556 363 AAAGAUUUGCAUCCAUGAG 635 363
UGCCAAGUGAUUCAGAUGG 557 363 UGCCAAGUGAUUCAGAUGG 557 381
CCAUCUGAAUCACUUGGCA 636 381 GAGCUAGAUCAUGAAAGAA 558 381
GAGCUAGAUCAUGAAAGAA 558 399 UUCUUUCAUGAUCUAGCUC 637 399
AUGUCCUACCUUCUCUAUC 559 399 AUGUCCUACCUUCUCUAUC 559 417
GAUAGAGAAGGUAGGACAU 638 417 CAGAUGCUGUGUGGAAUCA 560 417
CAGAUGCUGUGUGGAAUCA 560 435 UGAUUCCACACAGCAUCUG 639 435
AAGCACCUUCAUUCUGCUG 561 435 AAGCACCUUCAUUCUGCUG 561 453
CAGCAGAAUGAAGGUGCUU 640 453 GGAAUUAUUCAUCGGGACU 562 453
GGAAUUAUUCAUCGGGACU 562 471 AGUCCCGAUGAAUAAUUCC 641 471
UUAAAGCCCAGUAAUAUAG 563 471 UUAAAGCCCAGUAAUAUAG 563 489
CUAUAUUACUGGGCUUUAA 642 489 GUAGUAAAAUCUGAUUGCA 564 489
GUAGUAAAAUCUGAUUGCA 564 507 UGCAAUCAGAUUUUACUAC 643 507
ACUUUGAAGAUUCUUGACU 565 507 ACUUUGAAGAUUCUUGACU 565 525
AGUCAAGAAUCUUCAAAGU 644 525 UUCGGUCUGGCCAGGACUG 566 525
UUCGGUCUGGCCAGGACUG 566 543 CAGUCCUGGCCAGACCGAA 645 543
GCAGGAACGAGUUUUAUGA 567 543 GCAGGAACGAGUUUUAUGA 567 561
UCAUAAAACUCGUUCCUGC 646 561 AUGACGCCUUAUGUAGUGA 568 561
AUGACGCCUUAUGUAGUGA 568 579 UCACUACAUAAGGCGUCAU 647 579
ACUCGCUACUACAGAGCAC 569 579 ACUCGCUACUACAGAGCAC 569 597
GUGCUCUGUAGUAGCGAGU 648 597 CCCGAGGUCAUCCUUGGCA 570 597
CCCGAGGUCAUCCUUGGCA 570 615 UGCCAAGGAUGACCUCGGG 649 615
AUGGGCUACAAGGAAAACG 571 615 AUGGGCUACAAGGAAAACG 571 633
CGUUUUCCUUGUAGCCCAU 650 633 GUGGAUUUAUGGUCUGUGG 572 633
GUGGAUUUAUGGUCUGUGG 572 651 CCACAGACCAUAAAUCCAC 651 651
GGGUGCAUUAUGGGAGAAA 573 651 GGGUGCAUUAUGGGAGAAA 573 669
UUUCUCCCAUAAUGCACCC 652 669 AUGGUUUGCCACAAAAUCC 574 669
AUGGUUUGCCACAAAAUCC 574 687 GGAUUUUGUGGCAAACCAU 653 687
CUCUUUCCAGGAAGGGACU 575 687 CUCUUUCCAGGAAGGGACU 575 705
AGUCCCUUCCUGGAAAGAG 654 705 UAUAUUGAUCAGUGGAAUA 576 705
UAUAUUGAUCAGUGGAAUA 576 723 UAUUCCACUGAUCAAUAUA 655 723
AAAGUUAUUGAACAGCUUG 577 723 AAAGUUAUUGAACAGCUUG 577 741
CAAGCUGUUCAAUAACUUU 656 741 GGAACACCAUGUCCUGAAU 578 741
GGAACACCAUGUCCUGAAU 578 759 AUUCAGGACAUGGUGUUCC 657 759
UUCAUGAAGAAACUGCAAC 579 759 UUCAUGAAGAAACUGCAAC 579 777
GUUGCAGUUUCUUCAUGAA 658 777 CCAACAGUAAGGACUUACG 580 777
CCAACAGUAAGGACUUACG 580 795 CGUAAGUCCUUACUGUUGG 659 795
GUUGAAAACAGACCUAAAU 581 795 GUUGAAAACAGACCUAAAU 581 813
AUUUAGGUCUGUUUUCAAC 660 813 UAUGCUGGAUAUAGCUUUG 582 813
UAUGCUGGAUAUAGCUUUG 582 831 CAAAGCUAUAUCCAGCAUA 661 831
GAGAAACUCUUCCCUGAUG 583 831 GAGAAACUCUUCCCUGAUG 583 849
CAUCAGGGAAGAGUUUCUC 662 849 GUCCUUUUCCCAGCUGACU 584 849
GUCCUUUUCCCAGCUGACU 584 867 AGUCAGCUGGGAAAAGGAC 663 867
UCAGAACACAACAAACUUA 585 867 UCAGAACACAACAAACUUA 585 885
UAAGUUUGUUGUGUUCUGA 664 885 AAAGCCAGUCAGGCAAGGG 586 885
AAAGCCAGUCAGGCAAGGG 586 903 CCCUUGCCUGACUGGCUUU 665 903
GAUUUGUUAUCCAAAAUGC 587 903 GAUUUGUUAUCCAAAAUGC 587 921
GCAUUUUGGAUAACAAAUC 666 921 CUGGUAAUAGAUGCAUCUA 588 921
CUGGUAAUAGAUGCAUCUA 588 939 UAGAUGCAUCUAUUACCAG 667 939
AAAAGGAUCUCUGUAGAUG 589 939 AAAAGGAUCUCUGUAGAUG 589 957
CAUCUACAGAGAUCCUUUU 668 957 GAAGCUCUCCAACACCCGU 590 957
GAAGCUCUCCAACACCCGU 590 975 ACGGGUGUUGGAGAGCUUC 669 975
UACAUCAAUGUCUGGUAUG 591 975 UACAUCAAUGUCUGGUAUG 591 993
CAUACCAGACAUUGAUGUA 670 993 GAUCCUUCUGAAGCAGAAG 592 993
GAUCCUUCUGAAGCAGAAG 592 1011 CUUCUGCUUCAGAAGGAUC 671 1011
GCUCCACCACCAAAGAUCC 593 1011 GCUCCACCACCAAAGAUCC 593 1029
GGAUCUUUGGUGGUGGAGC 672 1029 CCUGACAAGCAGUUAGAUG 594 1029
CCUGACAAGCAGUUAGAUG 594 1047 CAUCUAACUGCUUGUCAGG 673 1047
GAAAGGGAACACACAAUAG 595 1047 GAAAGGGAACACACAAUAG 595 1065
CUAUUGUGUGUUCCCUUUC 674 1065 GAAGAGUGGAAAGAAUUGA 596 1065
GAAGAGUGGAAAGAAUUGA 596 1083 UCAAUUCUUUCCACUCUUC 675 1083
AUAUAUAAGGAAGUUAUGG 597 1083 AUAUAUAAGGAAGUUAUGG 597 1101
CCAUAACUUCCUUAUAUAU 676
1101 GACUUGGAGGAGAGAACCA 598 1101 GACUUGGAGGAGAGAACCA 598 1119
UGGUUCUCUCCUCCAAGUC 677 1119 AAGAAUGGAGUUAUACGGG 599 1119
AAGAAUGGAGUUAUACGGG 599 1137 CCCGUAUAACUCCAUUCUU 678 1137
GGGCAGCCCUCUCCUUUAG 600 1137 GGGCAGCCCUCUCCUUUAG 600 1155
CUAAAGGAGAGGGCUGCCC 679 1155 GCACAGGUGCAGCAGUGAU 601 1155
GCACAGGUGCAGCAGUGAU 601 1173 AUCACUGCUGCACCUGUGC 680 1173
UCAAUGGCUCUCAGCAUCC 602 1173 UCAAUGGCUCUCAGCAUCC 602 1191
GGAUGCUGAGAGCCAUUGA 681 1191 CAUCAUCAUCGUCGUCUGU 603 1191
CAUCAUCAUCGUCGUCUGU 603 1209 ACAGACGACGAUGAUGAUG 682 1209
UCAAUGAUGUGUCUUCAAU 604 1209 UCAAUGAUGUGUCUUCAAU 604 1227
AUUGAAGACACAUCAUUGA 683 1227 UGUCAACAGAUCCGACUUU 605 1227
UGUCAACAGAUCCGACUUU 605 1245 AAAGUCGGAUCUGUUGACA 684 1245
UGGCCUCUGAUACAGACAG 606 1245 UGGCCUCUGAUACAGACAG 606 1263
CUGUCUGUAUCAGAGGCCA 685 1263 GCAGUCUAGAAGCAGCAGC 607 1263
GCAGUCUAGAAGCAGCAGC 607 1281 GCUGCUGCUUCUAGACUGC 686 1281
CUGGGCCUCUGGGCUGCUG 608 1281 CUGGGCCUCUGGGCUGCUG 608 1299
CAGCAGCCCAGAGGCCCAG 687 1299 GUAGAUGACUACUUGGGCC 609 1299
GUAGAUGACUACUUGGGCC 609 1317 GGCCCAAGUAGUCAUCUAC 688 1317
CAUCGGGGGGUGGGAGGGA 610 1317 CAUCGGGGGGUGGGAGGGA 610 1335
UCCCUCCCACCCCCCGAUG 689 1335 AUGGGGAGUCGGUUAGUCA 611 1335
AUGGGGAGUCGGUUAGUCA 611 1353 UGACUAACCGACUCCCCAU 690 1353
AUUGAUAGAACUACUUUGA 612 1353 AUUGAUAGAACUACUUUGA 612 1371
UCAAAGUAGUUCUAUCAAU 691 1371 AAAACAAUUCAGUGGUCUU 613 1371
AAAACAAUUCAGUGGUCUU 613 1389 AAGACCACUGAAUUGUUUU 692 1389
UAUUUUUGGGUGAUUUUUC 614 1389 UAUUUUUGGGUGAUUUUUC 614 1407
GAAAAAUCACCCAAAAAUA 693 1397 GGUGAUUUUUCAAAAAAUG 615 1397
GGUGAUUUUUCAAAAAAUG 615 1415 CAUUUUUUGAAAAAUCACC 694 ID Seq Seq Pos
Target Sequence Seq UPos Upper seq ID LPos Lower seq ID NM_139012
(MAPK14/p38) 3 AACCGCGACCACUGGAGCC 695 3 AACCGCGACCACUGGAGCC 695 21
GGCUCCAGUGGUCGCGGUU 904 21 CUUAGCGGGCGCAGCAGCU 696 21
CUUAGCGGGCGCAGCAGCU 696 39 AGCUGCUGCGCCCGCUAAG 905 39
UGGAACGGGAGUACUGCGA 697 39 UGGAACGGGAGUACUGCGA 697 57
UCGCAGUACUCCCGUUCCA 906 57 ACGCAGCCCGGAGUCGGCC 698 57
ACGCAGCCCGGAGUCGGCC 698 75 GGCCGACUCCGGGCUGCGU 907 75
CUUGUAGGGGCGAAGGUGC 699 75 CUUGUAGGGGCGAAGGUGC 699 93
GCACCUUCGCCCCUACAAG 908 93 CAGGGAGAUCGCGGCGGGC 700 93
CAGGGAGAUCGCGGCGGGC 700 111 GCCCGCCGCGAUCUCCCUG 909 111
CGCAGUCUUGAGCGCCGGA 701 111 CGCAGUCUUGAGCGCCGGA 701 129
UCCGGCGCUCAAGACUGCG 910 129 AGCGCGUCCCUGCCCUUAG 702 129
AGCGCGUCCCUGCCCUUAG 702 147 CUAAGGGCAGGGACGCGCU 911 147
GCGGGGCUUGCCCCAGUCG 703 147 GCGGGGCUUGCCCCAGUCG 703 165
CGACUGGGGCAAGCCCCGC 912 165 GCAGGGGCACAUCCAGCCG 704 165
GCAGGGGCACAUCCAGCCG 704 183 CGGCUGGAUGUGCCCCUGC 913 183
GCUGCGGCUGACAGCAGCC 705 183 GCUGCGGCUGACAGCAGCC 705 201
GGCUGCUGUCAGCCGCAGC 914 201 CGCGCGCGCGGGAGUCUGC 706 201
CGCGCGCGCGGGAGUCUGC 706 219 GCAGACUCCCGCGCGCGCG 915 219
CGGGGUCGCGGCAGCCGCA 707 219 CGGGGUCGCGGCAGCCGCA 707 237
UGCGGCUGCCGCGACCCCG 916 237 ACCUGCGCGGGCGACCAGC 708 237
ACCUGCGCGGGCGACCAGC 708 255 GCUGGUCGCCCGCGCAGGU 917 255
CGCAAGGUCCCCGCCCGGC 709 255 CGCAAGGUCCCCGCCCGGC 709 273
GCCGGGCGGGGACCUUGCG 918 273 CUGGGCGGGCAGCAAGGGC 710 273
CUGGGCGGGCAGCAAGGGC 710 291 GCCCUUGCUGCCCGCCCAG 919 291
CCGGGGAGAGGGUGCGGGU 711 291 CCGGGGAGAGGGUGCGGGU 711 309
ACCCGCACCCUCUCCCCGG 920 309 UGCAGGCGGGGGCCCCACA 712 309
UGCAGGCGGGGGCCCCACA 712 327 UGUGGGGCCCCCGCCUGCA 921 327
AGGGCCACCUUCUUGCCCG 713 327 AGGGCCACCUUCUUGCCCG 713 345
CGGGCAAGAAGGUGGCCCU 922 345 GGCGGCUGCCGCUGGAAAA 714 345
GGCGGCUGCCGCUGGAAAA 714 363 UUUUCCAGCGGCAGCCGCC 923 363
AUGUCUCAGGAGAGGCCCA 715 363 AUGUCUCAGGAGAGGCCCA 715 381
UGGGCCUCUCCUGAGACAU 924 381 ACGUUCUACCGGCAGGAGC 716 381
ACGUUCUACCGGCAGGAGC 716 399 GCUCCUGCCGGUAGAACGU 925 399
CUGAACAAGACAAUCUGGG 717 399 CUGAACAAGACAAUCUGGG 717 417
CCCAGAUUGUCUUGUUCAG 926 417 GAGGUGCCCGAGCGUUACC 718 417
GAGGUGCCCGAGCGUUACC 718 435 GGUAACGCUCGGGCACCUC 927 435
CAGAACCUGUCUCCAGUGG 719 435 CAGAACCUGUCUCCAGUGG 719 453
CCACUGGAGACAGGUUCUG 928 453 GGCUCUGGCGCCUAUGGCU 720 453
GGCUCUGGCGCCUAUGGCU 720 471 AGCCAUAGGCGCCAGAGCC 929 471
UCUGUGUGUGCUGCUUUUG 721 471 UCUGUGUGUGCUGCUUUUG 721 489
CAAAAGCAGCACACACAGA 930 489 GACACAAAAACGGGGUUAC 722 489
GACACAAAAACGGGGUUAC 722 507 GUAACCCCGUUUUUGUGUC 931 507
CGUGUGGCAGUGAAGAAGC 723 507 CGUGUGGCAGUGAAGAAGC 723 525
GCUUCUUCACUGCCACACG 932 525 CUCUCCAGACCAUUUCAGU 724 525
CUCUCCAGACCAUUUCAGU 724 543 ACUGAAAUGGUCUGGAGAG 933 543
UCCAUCAUUCAUGCGAAAA 725 543 UCCAUCAUUCAUGCGAAAA 725 561
UUUUCGCAUGAAUGAUGGA 934 561 AGAACCUACAGAGAACUGC 726 561
AGAACCUACAGAGAACUGC 726 579 GCAGUUCUCUGUAGGUUCU 935 579
CGGUUACUUAAACAUAUGA 727 579 CGGUUACUUAAACAUAUGA 727 597
UCAUAUGUUUAAGUAACCG 936 597 AAACAUGAAAAUGUGAUUG 728 597
AAACAUGAAAAUGUGAUUG 728 615 CAAUCACAUUUUCAUGUUU 937 615
GGUCUGUUGGACGUUUUUA 729 615 GGUCUGUUGGACGUUUUUA 729 633
UAAAAACGUCCAACAGACC 938 633 ACACCUGCAAGGUCUCUGG 730 633
ACACCUGCAAGGUCUCUGG 730 651 CCAGAGACCUUGCAGGUGU 939 651
GAGGAAUUCAAUGAUGUGU 731 651 GAGGAAUUCAAUGAUGUGU 731 669
ACACAUCAUUGAAUUCCUC 940 669 UAUCUGGUGACCCAUCUCA 732 669
UAUCUGGUGACCCAUCUCA 732 687 UGAGAUGGGUCACCAGAUA 941 687
AUGGGGGCAGAUCUGAACA 733 687 AUGGGGGCAGAUCUGAACA 733 705
UGUUCAGAUCUGCCCCCAU 942 705 AACAUUGUGAAAUGUCAGA 734 705
AACAUUGUGAAAUGUCAGA 734 723 UCUGACAUUUCACAAUGUU 943 723
AAGCUUACAGAUGACCAUG 735 723 AAGCUUACAGAUGACCAUG 735 741
CAUGGUCAUCUGUAAGCUU 944 741 GUUCAGUUCCUUAUCUACC 736 741
GUUCAGUUCCUUAUCUACC 736 759 GGUAGAUAAGGAACUGAAC 945 759
CAAAUUCUCCGAGGUCUAA 737 759 CAAAUUCUCCGAGGUCUAA 737 777
UUAGACCUCGGAGAAUUUG 946 777 AAGUAUAUACAUUCAGCUG 738 777
AAGUAUAUACAUUCAGCUG 738 795 CAGCUGAAUGUAUAUACUU 947 795
GACAUAAUUCACAGGGACC 739 795 GACAUAAUUCACAGGGACC 739 813
GGUCCCUGUGAAUUAUGUC 948 813 CUAAAACCUAGUAAUCUAG 740 813
CUAAAACCUAGUAAUCUAG 740 831 CUAGAUUACUAGGUUUUAG 949 831
GCUGUGAAUGAAGACUGUG 741 831 GCUGUGAAUGAAGACUGUG 741 849
CACAGUCUUCAUUCACAGC 950 849 GAGCUGAAGAUUCUGGAUU 742 849
GAGCUGAAGAUUCUGGAUU 742 867 AAUCCAGAAUCUUCAGCUC 951 867
UUUGGACUGGCUCGGCACA 743 867 UUUGGACUGGCUCGGCACA 743 885
UGUGCCGAGCCAGUCCAAA 952 885 ACAGAUGAUGAAAUGACAG 744 885
ACAGAUGAUGAAAUGACAG 744 903 CUGUCAUUUCAUCAUCUGU 953 903
GGCUACGUGGCCACUAGGU 745 903 GGCUACGUGGCCACUAGGU 745 921
ACCUAGUGGCCACGUAGCC 954 921 UGGUACAGGGCUCCUGAGA 746 921
UGGUACAGGGCUCCUGAGA 746 939 UCUCAGGAGCCCUGUACCA 955 939
AUCAUGCUGAACUGGAUGC 747 939 AUCAUGCUGAACUGGAUGC 747 957
GCAUCCAGUUCAGCAUGAU 956 957 CAUUACAACCAGACAGUUG 748 957
CAUUACAACCAGACAGUUG 748 975 CAACUGUCUGGUUGUAAUG 957 975
GAUAUUUGGUCAGUGGGAU 749 975 GAUAUUUGGUCAGUGGGAU 749 993
AUCCCACUGACCAAAUAUC 958 993 UGCAUAAUGGCCGAGCUGU 750 993
UGCAUAAUGGCCGAGCUGU 750 1011 ACAGCUCGGCCAUUAUGCA 959 1011
UUGACUGGAAGAACAUUGU 751 1011 UUGACUGGAAGAACAUUGU 751 1029
ACAAUGUUCUUCCAGUCAA 960 1029 UUUCCUGGUACAGACCAUA 752 1029
UUUCCUGGUACAGACCAUA 752 1047 UAUGGUCUGUACCAGGAAA 961 1047
AUUGAUCAGUUGAAGCUCA 753 1047 AUUGAUCAGUUGAAGCUCA 753 1065
UGAGCUUCAACUGAUCAAU 962 1065 AUUUUAAGACUCGUUGGAA 754 1065
AUUUUAAGACUCGUUGGAA 754 1083 UUCCAACGAGUCUUAAAAU 963 1083
ACCCCAGGGGCUGAGCUUU 755 1083 ACCCCAGGGGCUGAGCUUU 755 1101
AAAGCUCAGCCCCUGGGGU 964 1101 UUGAAGAAAAUCUCCUCAG 756 1101
UUGAAGAAAAUCUCCUCAG 756 1119 CUGAGGAGAUUUUCUUCAA 965 1119
GAGUCUGCAAGAAACUAUA 757 1119 GAGUCUGCAAGAAACUAUA 757 1137
UAUAGUUUCUUGCAGACUC 966 1137 AUUCAGUCUUUGACUCAGA 758 1137
AUUCAGUCUUUGACUCAGA 758 1155 UCUGAGUCAAAGACUGAAU 967
1155 AUGCCGAAGAUGAACUUUG 759 1155 AUGCCGAAGAUGAACUUUG 759 1173
CAAAGUUCAUCUUCGGCAU 968 1173 GCGAAUGUAUUUAUUGGUG 760 1173
GCGAAUGUAUUUAUUGGUG 760 1191 CACCAAUAAAUACAUUCGC 969 1191
GCCAAUCCCCUGGCUGUCG 761 1191 GCCAAUCCCCUGGCUGUCG 761 1209
CGACAGCCAGGGGAUUGGC 970 1209 GACUUGCUGGAGAAGAUGC 762 1209
GACUUGCUGGAGAAGAUGC 762 1227 GCAUCUUCUCCAGCAAGUC 971 1227
CUUGUAUUGGACUCAGAUA 763 1227 CUUGUAUUGGACUCAGAUA 763 1245
UAUCUGAGUCCAAUACAAG 972 1245 AAGAGAAUUACAGCGGCCC 764 1245
AAGAGAAUUACAGCGGCCC 764 1263 GGGCCGCUGUAAUUCUCUU 973 1263
CAAGCCCUUGCACAUGCCU 765 1263 CAAGCCCUUGCACAUGCCU 765 1281
AGGCAUGUGCAAGGGCUUG 974 1281 UACUUUGCUCAGUACCACG 766 1281
UACUUUGCUCAGUACCACG 766 1299 CGUGGUACUGAGCAAAGUA 975 1299
GAUCCUGAUGAUGAACCAG 767 1299 GAUCCUGAUGAUGAACCAG 767 1317
CUGGUUCAUCAUCAGGAUC 976 1317 GUGGCCGAUCCUUAUGAUC 768 1317
GUGGCCGAUCCUUAUGAUC 768 1335 GAUCAUAAGGAUCGGCCAC 977 1335
CAGUCCUUUGAAAGCAGGG 769 1335 CAGUCCUUUGAAAGCAGGG 769 1353
CCCUGCUUUCAAAGGACUG 978 1353 GACCUCCUUAUAGAUGAGU 770 1353
GACCUCCUUAUAGAUGAGU 770 1371 ACUCAUCUAUAAGGAGGUC 979 1371
UGGAAAAGCCUGACCUAUG 771 1371 UGGAAAAGCCUGACCUAUG 771 1389
CAUAGGUCAGGCUUUUCCA 980 1389 GAUGAAGUCAUCAGCUUUG 772 1389
GAUGAAGUCAUCAGCUUUG 772 1407 CAAAGCUGAUGACUUCAUC 981 1407
GUGCCACCACCCCUUGACC 773 1407 GUGCCACCACCCCUUGACC 773 1425
GGUCAAGGGGUGGUGGCAC 982 1425 CAAGAAGAGAUGGAGUCCU 774 1425
CAAGAAGAGAUGGAGUCCU 774 1443 AGGACUCCAUCUCUUCUUG 983 1443
UGAGCACCUGGUUUCUGUU 775 1443 UGAGCACCUGGUUUCUGUU 775 1461
AACAGAAACCAGGUGCUCA 984 1461 UCUGUUGAUCCCACUUCAC 776 1461
UCUGUUGAUCCCACUUCAC 776 1479 GUGAAGUGGGAUCAACAGA 985 1479
CUGUGAGGGGAAGGCCUUU 777 1479 CUGUGAGGGGAAGGCCUUU 777 1497
AAAGGCCUUCCCCUCACAG 986 1497 UUCACGGGAACUCUCCAAA 778 1497
UUCACGGGAACUCUCCAAA 778 1515 UUUGGAGAGUUCCCGUGAA 987 1515
AUAUUAUUCAAGUGCCUCU 779 1515 AUAUUAUUCAAGUGCCUCU 779 1533
AGAGGCACUUGAAUAAUAU 988 1533 UUGUUGCAGAGAUUUCCUC 780 1533
UUGUUGCAGAGAUUUCCUC 780 1551 GAGGAAAUCUCUGCAACAA 989 1551
CCAUGGUGGAAGGGGGUGU 781 1551 CCAUGGUGGAAGGGGGUGU 781 1569
ACACCCCCUUCCACCAUGG 990 1569 UGCGUGCGUGUGCGUGCGU 782 1569
UGCGUGCGUGUGCGUGCGU 782 1587 ACGCACGCACACGCACGCA 991 1587
UGUUAGUGUGUGUGCAUGU 783 1587 UGUUAGUGUGUGUGCAUGU 783 1605
ACAUGCACACACACUAACA 992 1605 UGUGUGUCUGUCUUUGUGG 784 1605
UGUGUGUCUGUCUUUGUGG 784 1623 CCACAAAGACAGACACACA 993 1623
GGAGGGUAAGACAAUAUGA 785 1623 GGAGGGUAAGACAAUAUGA 785 1641
UCAUAUUGUCUUACCCUCC 994 1641 AACAAACUAUGAUCACAGU 786 1641
AACAAACUAUGAUCACAGU 786 1659 ACUGUGAUCAUAGUUUGUU 995 1659
UGACUUUACAGGAGGUUGU 787 1659 UGACUUUACAGGAGGUUGU 787 1677
ACAACCUCCUGUAAAGUCA 996 1677 UGGAUGCUCCAGGGCAGCC 788 1677
UGGAUGCUCCAGGGCAGCC 788 1695 GGCUGCCCUGGAGCAUCCA 997 1695
CUCCACCUUGCUCUUCUUU 789 1695 CUCCACCUUGCUCUUCUUU 789 1713
AAAGAAGAGCAAGGUGGAG 998 1713 UCUGAGAGUUGGCUCAGGC 790 1713
UCUGAGAGUUGGCUCAGGC 790 1731 GCCUGAGCCAACUCUCAGA 999 1731
CAGACAAGAGCUGCUGUCC 791 1731 CAGACAAGAGCUGCUGUCC 791 1749
GGACAGCAGCUCUUGUCUG 1000 1749 CUUUUAGGAAUAUGUUCAA 792 1749
CUUUUAGGAAUAUGUUCAA 792 1767 UUGAACAUAUUCCUAAAAG 1001 1767
AUGCAAAGUAAAAAAAUAU 793 1767 AUGCAAAGUAAAAAAAUAU 793 1785
AUAUUUUUUUACUUUGCAU 1002 1785 UGAAUUGUCCCCAAUCCCG 794 1785
UGAAUUGUCCCCAAUCCCG 794 1803 CGGGAUUGGGGACAAUUCA 1003 1803
GGUCAUGCUUUUGCCACUU 795 1803 GGUCAUGCUUUUGCCACUU 795 1821
AAGUGGCAAAAGCAUGACC 1004 1821 UUGGCUUCUCCUGUGACCC 796 1821
UUGGCUUCUCCUGUGACCC 796 1839 GGGUCACAGGAGAAGCCAA 1005 1839
CCACCUUGACGGUGGGGCG 797 1839 CCACCUUGACGGUGGGGCG 797 1857
CGCCCCACCGUCAAGGUGG 1006 1857 GUAGACUUGACAACAUCCC 798 1857
GUAGACUUGACAACAUCCC 798 1875 GGGAUGUUGUCAAGUCUAC 1007 1875
CACAGUGGCACGGAGAGAA 799 1875 CACAGUGGCACGGAGAGAA 799 1893
UUCUCUCCGUGCCACUGUG 1008 1893 AGGCCCAUACCUUCUGGUU 800 1893
AGGCCCAUACCUUCUGGUU 800 1911 AACCAGAAGGUAUGGGCCU 1009 1911
UGCUUCAGACCUGACACCG 801 1911 UGCUUCAGACCUGACACCG 801 1929
CGGUGUCAGGUCUGAAGCA 1010 1929 GUCCCUCAGUGAUACGUAC 802 1929
GUCCCUCAGUGAUACGUAC 802 1947 GUACGUAUCACUGAGGGAC 1011 1947
CAGCCAAAAAGGACCAACU 803 1947 CAGCCAAAAAGGACCAACU 803 1965
AGUUGGUCCUUUUUGGCUG 1012 1965 UGGCUUCUGUGCACUAGCC 804 1965
UGGCUUCUGUGCACUAGCC 804 1983 GGCUAGUGCACAGAAGCCA 1013 1983
CUGUGAUUAACUUGCUUAG 805 1983 CUGUGAUUAACUUGCUUAG 805 2001
CUAAGCAAGUUAAUCACAG 1014 2001 GUAUGGUUCUCAGAUCUUG 806 2001
GUAUGGUUCUCAGAUCUUG 806 2019 CAAGAUCUGAGAACCAUAC 1015 2019
GACAGUAUAUUUGAAACUG 807 2019 GACAGUAUAUUUGAAACUG 807 2037
CAGUUUCAAAUAUACUGUC 1016 2037 GUAAAUAUGUUUGUGCCUU 808 2037
GUAAAUAUGUUUGUGCCUU 808 2055 AAGGCACAAACAUAUUUAC 1017 2055
UAAAAGGAGAGAAGAAAGU 809 2055 UAAAAGGAGAGAAGAAAGU 809 2073
ACUUUCUUCUCUCCUUUUA 1018 2073 UGUAGAUAGUUAAAAGACU 810 2073
UGUAGAUAGUUAAAAGACU 810 2091 AGUCUUUUAACUAUCUACA 1019 2091
UGCAGCUGCUGAAGUUCUG 811 2091 UGCAGCUGCUGAAGUUCUG 811 2109
CAGAACUUCAGCAGCUGCA 1020 2109 GAGCCGGGCAAGUCGAGAG 812 2109
GAGCCGGGCAAGUCGAGAG 812 2127 CUCUCGACUUGCCCGGCUC 1021 2127
GGGCUGUUGGACAGCUGCU 813 2127 GGGCUGUUGGACAGCUGCU 813 2145
AGCAGCUGUCCAACAGCCC 1022 2145 UUGUGGGCCCGGAGUAAUC 814 2145
UUGUGGGCCCGGAGUAAUC 814 2163 GAUUACUCCGGGCCCACAA 1023 2163
CAGGCAGCCUUCAUAGGCG 815 2163 CAGGCAGCCUUCAUAGGCG 815 2181
CGCCUAUGAAGGCUGCCUG 1024 2181 GGUCAUGUGUGCAUGUGAG 816 2181
GGUCAUGUGUGCAUGUGAG 816 2199 CUCACAUGCACACAUGACC 1025 2199
GCACAUGCGUAUAUGUGCG 817 2199 GCACAUGCGUAUAUGUGCG 817 2217
CGCACAUAUACGCAUGUGC 1026 2217 GUCUCUCUUUCUCCCUCAC 818 2217
GUCUCUCUUUCUCCCUCAC 818 2235 GUGAGGGAGAAAGAGAGAC 1027 2235
CCCCCAGGUGUUGCCAUUU 819 2235 CCCCCAGGUGUUGCCAUUU 819 2253
AAAUGGCAACACCUGGGGG 1028 2253 UCUCUGCUUACCCUUCACC 820 2253
UCUCUGCUUACCCUUCACC 820 2271 GGUGAAGGGUAAGCAGAGA 1029 2271
CUUUGGUGCAGAGGUUUCU 821 2271 CUUUGGUGCAGAGGUUUCU 821 2289
AGAAACCUCUGCACCAAAG 1030 2289 UUGAAUAUCUGCCCCAGUA 822 2289
UUGAAUAUCUGCCCCAGUA 822 2307 UACUGGGGCAGAUAUUCAA 1031 2307
AGUCAGAAGCAGGUUCUUG 823 2307 AGUCAGAAGCAGGUUCUUG 823 2325
CAAGAACCUGCUUCUGACU 1032 2325 GAUGUCAUGUACUUCCUGU 824 2325
GAUGUCAUGUACUUCCUGU 824 2343 ACAGGAAGUACAUGACAUC 1033 2343
UGUACUCUUUAUUUCUAGC 825 2343 UGUACUCUUUAUUUCUAGC 825 2361
GCUAGAAAUAAAGAGUACA 1034 2361 CAGAGUGAGGAUGUGUUUU 826 2361
CAGAGUGAGGAUGUGUUUU 826 2379 AAAACACAUCCUCACUCUG 1035 2379
UGCACGUCUUGCUAUUUGA 827 2379 UGCACGUCUUGCUAUUUGA 827 2397
UCAAAUAGCAAGACGUGCA 1036 2397 AGCAUGCACAGCUGCUUGU 828 2397
AGCAUGCACAGCUGCUUGU 828 2415 ACAAGCAGCUGUGCAUGCU 1037 2415
UCCUGCUCUCUUCAGGAGG 829 2415 UCCUGCUCUCUUCAGGAGG 829 2433
CCUCCUGAAGAGAGCAGGA 1038 2433 GCCCUGGUGUCAGGCAGGU 830 2433
GCCCUGGUGUCAGGCAGGU 830 2451 ACCUGCCUGACACCAGGGC 1039 2451
UUUGCCAGUGAAGACUUCU 831 2451 UUUGCCAGUGAAGACUUCU 831 2469
AGAAGUCUUCACUGGCAAA 1040 2469 UUGGGUAGUUUAGAUCCCA 832 2469
UUGGGUAGUUUAGAUCCCA 832 2487 UGGGAUCUAAACUACCCAA 1041 2487
AUGUCACCUCAGCUGAUAU 833 2487 AUGUCACCUCAGCUGAUAU 833 2505
AUAUCAGCUGAGGUGACAU 1042 2505 UUAUGGCAAGUGAUAUCAC 834 2505
UUAUGGCAAGUGAUAUCAC 834 2523 GUGAUAUCACUUGCCAUAA 1043 2523
CCUCUCUUCAGCCCCUAGU 835 2523 CCUCUCUUCAGCCCCUAGU 835 2541
ACUAGGGGCUGAAGAGAGG 1044 2541 UGCUAUUCUGUGUUGAACA 836 2541
UGCUAUUCUGUGUUGAACA 836 2559 UGUUCAACACAGAAUAGCA 1045 2559
ACAAUUGAUACUUCAGGUG 837 2559 ACAAUUGAUACUUCAGGUG 837 2577
CACCUGAAGUAUCAAUUGU 1046 2577 GCUUUUGAUGUGAAAAUCA 838 2577
GCUUUUGAUGUGAAAAUCA 838 2595 UGAUUUUCACAUCAAAAGC 1047 2595
AUGAAAAGAGGAACAGGUG 839 2595 AUGAAAAGAGGAACAGGUG 839 2613
CACCUGUUCCUCUUUUCAU 1048 2613 GGAUGUAUAGCAUUUUUAU 840 2613
GGAUGUAUAGCAUUUUUAU 840 2631 AUAAAAAUGCUAUACAUCC 1049 2631
UUCAUGCCAUCUGUUUUCA 841 2631 UUCAUGCCAUCUGUUUUCA 841 2649
UGAAAACAGAUGGCAUGAA 1050 2649 AACCAACUAUUUUUGAGGA 842 2649
AACCAACUAUUUUUGAGGA 842 2667 UCCUCAAAAAUAGUUGGUU 1051
2667 AAUUAUCAUGGGAAAAGAC 843 2667 AAUUAUCAUGGGAAAAGAC 843 2685
GUCUUUUCCCAUGAUAAUU 1052 2685 CCAGGGCUUUUCCCAGGAA 844 2685
CCAGGGCUUUUCCCAGGAA 844 2703 UUCCUGGGAAAAGCCCUGG 1053 2703
AUAUCCCAAACUUCGGAAA 845 2703 AUAUCCCAAACUUCGGAAA 845 2721
UUUCCGAAGUUUGGGAUAU 1054 2721 ACAAGUUAUUCUCUUCACU 846 2721
ACAAGUUAUUCUCUUCACU 846 2739 AGUGAAGAGAAUAACUUGU 1055 2739
UCCCAAUAACUAAUGCUAA 847 2739 UCCCAAUAACUAAUGCUAA 847 2757
UUAGCAUUAGUUAUUGGGA 1056 2757 AGAAAUGCUGAAAAUCAAA 848 2757
AGAAAUGCUGAAAAUCAAA 848 2775 UUUGAUUUUCAGCAUUUCU 1057 2775
AGUAAAAAAUUAAAGCCCA 849 2775 AGUAAAAAAUUAAAGCCCA 849 2793
UGGGCUUUAAUUUUUUACU 1058 2793 AUAAGGCCAGAAACUCCUU 850 2793
AUAAGGCCAGAAACUCCUU 850 2811 AAGGAGUUUCUGGCCUUAU 1059 2811
UUUGCUGUCUUUCUCUAAA 851 2811 UUUGCUGUCUUUCUCUAAA 851 2829
UUUAGAGAAAGACAGCAAA 1060 2829 AUAUGAUUACUUUAAAAUA 852 2829
AUAUGAUUACUUUAAAAUA 852 2847 UAUUUUAAAGUAAUCAUAU 1061 2847
AAAAAAGUAACAAGGUGUC 853 2847 AAAAAAGUAACAAGGUGUC 853 2865
GACACCUUGUUACUUUUUU 1062 2865 CUUUUCCACUCCUAUGGAA 854 2865
CUUUUCCACUCCUAUGGAA 854 2883 UUCCAUAGGAGUGGAAAAG 1063 2883
AAAGGGUCUUCUUGGCAGC 855 2883 AAAGGGUCUUCUUGGCAGC 855 2901
GCUGCCAAGAAGACCCUUU 1064 2901 CUUAACAUUGACUUCUUGG 856 2901
CUUAACAUUGACUUCUUGG 856 2919 CCAAGAAGUCAAUGUUAAG 1065 2919
GUUUGGGGAGAAAUAAAUU 857 2919 GUUUGGGGAGAAAUAAAUU 857 2937
AAUUUAUUUCUCCCCAAAC 1066 2937 UUUGUUUCAGAAUUUUGUA 858 2937
UUUGUUUCAGAAUUUUGUA 858 2955 UACAAAAUUCUGAAACAAA 1067 2955
AUAUUGUAGGAAUCCCUUU 859 2955 AUAUUGUAGGAAUCCCUUU 859 2973
AAAGGGAUUCCUACAAUAU 1068 2973 UGAGAAUGUGAUUCCUUUU 860 2973
UGAGAAUGUGAUUCCUUUU 860 2991 AAAAGGAAUCACAUUCUCA 1069 2991
UGAUGGGGAGAAAGGGCAA 861 2991 UGAUGGGGAGAAAGGGCAA 861 3009
UUGCCCUUUCUCCCCAUCA 1070 3009 AAUUAUUUUAAUAUUUUGU 862 3009
AAUUAUUUUAAUAUUUUGU 862 3027 ACAAAAUAUUAAAAUAAUU 1071 3027
UAUUUUCAACUUUAUAAAG 863 3027 UAUUUUCAACUUUAUAAAG 863 3045
CUUUAUAAAGUUGAAAAUA 1072 3045 GAUAAAAUAUCCUCAGGGG 864 3045
GAUAAAAUAUCCUCAGGGG 864 3063 CCCCUGAGGAUAUUUUAUC 1073 3063
GUGGAGAAGUGUCGUUUUC 865 3063 GUGGAGAAGUGUCGUUUUC 865 3081
GAAAACGACACUUCUCCAC 1074 3081 CAUAACUUGCUGAAUUUCA 866 3081
CAUAACUUGCUGAAUUUCA 866 3099 UGAAAUUCAGCAAGUUAUG 1075 3099
AGGCAUUUUGUUCUACAUG 867 3099 AGGCAUUUUGUUCUACAUG 867 3117
CAUGUAGAACAAAAUGCCU 1076 3117 GAGGACUCAUAUAUUUAAG 868 3117
GAGGACUCAUAUAUUUAAG 868 3135 CUUAAAUAUAUGAGUCCUC 1077 3135
GCCUUUUGUGUAAUAAGAA 869 3135 GCCUUUUGUGUAAUAAGAA 869 3153
UUCUUAUUACACAAAAGGC 1078 3153 AAGUAUAAAGUCACUUCCA 870 3153
AAGUAUAAAGUCACUUCCA 870 3171 UGGAAGUGACUUUAUACUU 1079 3171
AGUGUUGGCUGUGUGACAG 871 3171 AGUGUUGGCUGUGUGACAG 871 3189
CUGUCACACAGCCAACACU 1080 3189 GAAUCUUGUAUUUGGGCCA 872 3189
GAAUCUUGUAUUUGGGCCA 872 3207 UGGCCCAAAUACAAGAUUC 1081 3207
AAGGUGUUUCCAUUUCUCA 873 3207 AAGGUGUUUCCAUUUCUCA 873 3225
UGAGAAAUGGAAACACCUU 1082 3225 AAUCAGUGCAGUGAUACAU 874 3225
AAUCAGUGCAGUGAUACAU 874 3243 AUGUAUCACUGCACUGAUU 1083 3243
UGUACUCCAGAGGGACAGG 875 3243 UGUACUCCAGAGGGACAGG 875 3261
CCUGUCCCUCUGGAGUACA 1084 3261 GGUGGACCCCCUGAGUCAA 876 3261
GGUGGACCCCCUGAGUCAA 876 3279 UUGACUCAGGGGGUCCACC 1085 3279
ACUGGAGCAAGAAGGAAGG 877 3279 ACUGGAGCAAGAAGGAAGG 877 3297
CCUUCCUUCUUGCUCCAGU 1086 3297 GAGGCAGACUGAUGGCGAU 878 3297
GAGGCAGACUGAUGGCGAU 878 3315 AUCGCCAUCAGUCUGCCUC 1087 3315
UUCCCUCUCACCCGGGACU 879 3315 UUCCCUCUCACCCGGGACU 879 3333
AGUCCCGGGUGAGAGGGAA 1088 3333 UCUCCCCCUUUCAAGGAAA 880 3333
UCUCCCCCUUUCAAGGAAA 880 3351 UUUCCUUGAAAGGGGGAGA 1089 3351
AGUGAACCUUUAAAGUAAA 881 3351 AGUGAACCUUUAAAGUAAA 881 3369
UUUACUUUAAAGGUUCACU 1090 3369 AGGCCUCAUCUCCUUUAUU 882 3369
AGGCCUCAUCUCCUUUAUU 882 3387 AAUAAAGGAGAUGAGGCCU 1091 3387
UGCAGUUCAAAUCCUCACC 883 3387 UGCAGUUCAAAUCCUCACC 883 3405
GGUGAGGAUUUGAACUGCA 1092 3405 CAUCCACAGCAAGAUGAAU 884 3405
CAUCCACAGCAAGAUGAAU 884 3423 AUUCAUCUUGCUGUGGAUG 1093 3423
UUUUAUCAGCCAUGUUUGG 885 3423 UUUUAUCAGCCAUGUUUGG 885 3441
CCAAACAUGGCUGAUAAAA 1094 3441 GUUGUAAAUGCUCGUGUGA 886 3441
GUUGUAAAUGCUCGUGUGA 886 3459 UCACACGAGCAUUUACAAC 1095 3459
AUUUCCUACAGAAAUACUG 887 3459 AUUUCCUACAGAAAUACUG 887 3477
CAGUAUUUCUGUAGGAAAU 1096 3477 GCUCUGAAUAUUUUGUAAU 888 3477
GCUCUGAAUAUUUUGUAAU 888 3495 AUUACAAAAUAUUCAGAGC 1097 3495
UAAAGGUCUUUGCACAUGU 889 3495 UAAAGGUCUUUGCACAUGU 889 3513
ACAUGUGCAAAGACCUUUA 1098 3513 UGACCACAUACGUGUUAGG 890 3513
UGACCACAUACGUGUUAGG 890 3531 CCUAACACGUAUGUGGUCA 1099 3531
GAGGCUGCAUGCUCUGGAA 891 3531 GAGGCUGCAUGCUCUGGAA 891 3549
UUCCAGAGCAUGCAGCCUC 1100 3549 AGCCUGGACUCUAAGCUGG 892 3549
AGCCUGGACUCUAAGCUGG 892 3567 CCAGCUUAGAGUCCAGGCU 1101 3567
GAGCUCUUGGAAGAGCUCU 893 3567 GAGCUCUUGGAAGAGCUCU 893 3585
AGAGCUCUUCCAAGAGCUC 1102 3585 UUCGGUUUCUGAGCAUAAU 894 3585
UUCGGUUUCUGAGCAUAAU 894 3603 AUUAUGCUCAGAAACCGAA 1103 3603
UGCUCCCAUCUCCUGAUUU 895 3603 UGCUCCCAUCUCCUGAUUU 895 3621
AAAUCAGGAGAUGGGAGCA 1104 3621 UCUCUGAACAGAAAACAAA 896 3621
UCUCUGAACAGAAAACAAA 896 3639 UUUGUUUUCUGUUCAGAGA 1105 3639
AAGAGAGAAUGAGGGAAAU 897 3639 AAGAGAGAAUGAGGGAAAU 897 3657
AUUUCCCUCAUUCUCUCUU 1106 3657 UUGCUAUUUUAUUUGUAUU 898 3657
UUGCUAUUUUAUUUGUAUU 898 3675 AAUACAAAUAAAAUAGCAA 1107 3675
UCAUGAACUUGGCUGUAAU 899 3675 UCAUGAACUUGGCUGUAAU 899 3693
AUUACAGCCAAGUUCAUGA 1108 3693 UCAGUUAUGCCGUAUAGGA 900 3693
UCAGUUAUGCCGUAUAGGA 900 3711 UCCUAUACGGCAUAACUGA 1109 3711
AUGUCAGACAAUACCACUG 901 3711 AUGUCAGACAAUACCACUG 901 3729
CAGUGGUAUUGUCUGACAU 1110 3729 GGUUAAAAUAAAGCCUAUU 902 3729
GGUUAAAAUAAAGCCUAUU 902 3747 AAUAGGCUUUAUUUUAACC 1111 3737
UAAAGCCUAUUUUUCAAAU 903 3737 UAAAGCCUAUUUUUCAAAU 903 3755
AUUUGAAAAAUAGGCUUUA 1112 Seq Seq Seq Pos Seq ID UPos Upper seq ID
LPos Lower seq ID hJUN NM_002228 3 AGUUGCACUGAGUGUGGCU 1247 3
AGUUGCACUGAGUGUGGCU 1247 21 AGCCACACUCAGUGCAACU 1428 21
UGAAGCAGCGAGGCGGGAG 1248 21 UGAAGCAGCGAGGCGGGAG 1248 39
CUCCCGCCUCGCUGCUUCA 1429 39 GUGGAGGUGCGCGGAGUCA 1249 39
GUGGAGGUGCGCGGAGUCA 1249 57 UGACUCCGCGCACCUCCAC 1430 57
AGGCAGACAGACAGACACA 1250 57 AGGCAGACAGACAGACACA 1250 75
UGUGUCUGUCUGUCUGCCU 1431 75 AGCCAGCCAGCCAGGUCGG 1251 75
AGCCAGCCAGCCAGGUCGG 1251 93 CCGACCUGGCUGGCUGGCU 1432 93
GCAGUAUAGUCCGAACUGC 1252 93 GCAGUAUAGUCCGAACUGC 1252 111
GCAGUUCGGACUAUACUGC 1433 111 CAAAUCUUAUUUUCUUUUC 1253 111
CAAAUCUUAUUUUCUUUUC 1253 129 GAAAAGAAAAUAAGAUUUG 1434 129
CACCUUCUCUCUAACUGCC 1254 129 CACCUUCUCUCUAACUGCC 1254 147
GGCAGUUAGAGAGAAGGUG 1435 147 CCAGAGCUAGCGCCUGUGG 1255 147
CCAGAGCUAGCGCCUGUGG 1255 165 CCACAGGCGCUAGCUCUGG 1436 165
GCUCCCGGGCUGGUGGUUC 1256 165 GCUCCCGGGCUGGUGGUUC 1256 183
GAACCACCAGCCCGGGAGC 1437 183 CGGGAGUGUCCAGAGAGCC 1257 183
CGGGAGUGUCCAGAGAGCC 1257 201 GGCUCUCUGGACACUCCCG 1438 201
CUUGUCUCCAGCCGGCCCC 1258 201 CUUGUCUCCAGCCGGCCCC 1258 219
GGGGCCGGCUGGAGACAAG 1439 219 CGGGAGGAGAGCCCUGCUG 1259 219
CGGGAGGAGAGCCCUGCUG 1259 237 CAGCAGGGCUCUCCUCCCG 1440 237
GCCCAGGCGCUGUUGACAG 1260 237 GCCCAGGCGCUGUUGACAG 1260 255
CUGUCAACAGCGCCUGGGC 1441 255 GCGGCGGAAAGCAGCGGUA 1261 255
GCGGCGGAAAGCAGCGGUA 1261 273 UACCGCUGCUUUCCGCCGC 1442 273
ACCCCACGCGCCCGCCGGG 1262 273 ACCCCACGCGCCCGCCGGG 1262 291
CCCGGCGGGCGCGUGGGGU 1443 291 GGGACGUCGGCGAGCGGCU 1263 291
GGGACGUCGGCGAGCGGCU 1263 309 AGCCGCUCGCCGACGUCCC 1444 309
UGCAGCAGCAAAGAACUUU 1264 309 UGCAGCAGCAAAGAACUUU 1264 327
AAAGUUCUUUGCUGCUGCA 1445 327 UCCCGGCGGGGAGGACCGG 1265 327
UCCCGGCGGGGAGGACCGG 1265 345 CCGGUCCUCCCCGCCGGGA 1446 345
GAGACAAGUGGCAGAGUCC 1266 345 GAGACAAGUGGCAGAGUCC 1266 363
GGACUCUGCCACUUGUCUC 1447 363 CCGGAGCGAACUUUUGCAA 1267 363
CCGGAGCGAACUUUUGCAA 1267 381 UUGCAAAAGUUCGCUCCGG 1448
381 AGCCUUUCCUGCGUCUUAG 1268 381 AGCCUUUCCUGCGUCUUAG 1268 399
CUAAGACGCAGGAAAGGCU 1449 399 GGCUUCUCCACGGCGGUAA 1269 399
GGCUUCUCCACGGCGGUAA 1269 417 UUACCGCCGUGGAGAAGCC 1450 417
AAGACCAGAAGGCGGCGGA 1270 417 AAGACCAGAAGGCGGCGGA 1270 435
UCCGCCGCCUUCUGGUCUU 1451 435 AGAGCCACGCAAGAGAAGA 1271 435
AGAGCCACGCAAGAGAAGA 1271 453 UCUUCUCUUGCGUGGCUCU 1452 453
AAGGACGUGCGCUCAGCUU 1272 453 AAGGACGUGCGCUCAGCUU 1272 471
AAGCUGAGCGCACGUCCUU 1453 471 UCGCUCGCACCGGUUGUUG 1273 471
UCGCUCGCACCGGUUGUUG 1273 489 CAACAACCGGUGCGAGCGA 1454 489
GAACUUGGGCGAGCGCGAG 1274 489 GAACUUGGGCGAGCGCGAG 1274 507
CUCGCGCUCGCCCAAGUUC 1455 507 GCCGCGGCUGCCGGGCGCC 1275 507
GCCGCGGCUGCCGGGCGCC 1275 525 GGCGCCCGGCAGCCGCGGC 1456 525
CCCCUCCCCCUAGCAGCGG 1276 525 CCCCUCCCCCUAGCAGCGG 1276 543
CCGCUGCUAGGGGGAGGGG 1457 543 GAGGAGGGGACAAGUCGUC 1277 543
GAGGAGGGGACAAGUCGUC 1277 561 GACGACUUGUCCCCUCCUC 1458 561
CGGAGUCCGGGCGGCCAAG 1278 561 CGGAGUCCGGGCGGCCAAG 1278 579
CUUGGCCGCCCGGACUCCG 1459 579 GACCCGCCGCCGGCCGGCC 1279 579
GACCCGCCGCCGGCCGGCC 1279 597 GGCCGGCCGGCGGCGGGUC 1460 597
CACUGCAGGGUCCGCACUG 1280 597 CACUGCAGGGUCCGCACUG 1280 615
CAGUGCGGACCCUGCAGUG 1461 615 GAUCCGCUCCGCGGGGAGA 1281 615
GAUCCGCUCCGCGGGGAGA 1281 633 UCUCCCCGCGGAGCGGAUC 1462 633
AGCCGCUGCUCUGGGAAGU 1282 633 AGCCGCUGCUCUGGGAAGU 1282 651
ACUUCCCAGAGCAGCGGCU 1463 651 UGAGUUCGCCUGCGGACUC 1283 651
UGAGUUCGCCUGCGGACUC 1283 669 GAGUCCGCAGGCGAACUCA 1464 669
CCGAGGAACCGCUGCGCCC 1284 669 CCGAGGAACCGCUGCGCCC 1284 687
GGGCGCAGCGGUUCCUCGG 1465 687 CGAAGAGCGCUCAGUGAGU 1285 687
CGAAGAGCGCUCAGUGAGU 1285 705 ACUCACUGAGCGCUCUUCG 1466 705
UGACCGCGACUUUUCAAAG 1286 705 UGACCGCGACUUUUCAAAG 1286 723
CUUUGAAAAGUCGCGGUCA 1467 723 GCCGGGUAGCGCGCGCGAG 1287 723
GCCGGGUAGCGCGCGCGAG 1287 741 CUCGCGCGCGCUACCCGGC 1468 741
GUCGACAAGUAAGAGUGCG 1288 741 GUCGACAAGUAAGAGUGCG 1288 759
CGCACUCUUACUUGUCGAC 1469 759 GGGAGGCAUCUUAAUUAAC 1289 759
GGGAGGCAUCUUAAUUAAC 1289 777 GUUAAUUAAGAUGCCUCCC 1470 777
CCCUGCGCUCCCUGGAGCG 1290 777 CCCUGCGCUCCCUGGAGCG 1290 795
CGCUCCAGGGAGCGCAGGG 1471 795 GAGCUGGUGAGGAGGGCGC 1291 795
GAGCUGGUGAGGAGGGCGC 1291 813 GCGCCCUCCUCACCAGCUC 1472 813
CAGCGGGGACGACAGCCAG 1292 813 CAGCGGGGACGACAGCCAG 1292 831
CUGGCUGUCGUCCCCGCUG 1473 831 GCGGGUGCGUGCGCUCUUA 1293 831
GCGGGUGCGUGCGCUCUUA 1293 849 UAAGAGCGCACGCACCCGC 1474 849
AGAGAAACUUUCCCUGUCA 1294 849 AGAGAAACUUUCCCUGUCA 1294 867
UGACAGGGAAAGUUUCUCU 1475 867 AAAGGCUCCGGGGGGCGCG 1295 867
AAAGGCUCCGGGGGGCGCG 1295 885 CGCGCCCCCCGGAGCCUUU 1476 885
GGGUGUCCCCCGCUUGCCA 1296 885 GGGUGUCCCCCGCUUGCCA 1296 903
UGGCAAGCGGGGGACACCC 1477 903 AGAGCCCUGUUGCGGCCCC 1297 903
AGAGCCCUGUUGCGGCCCC 1297 921 GGGGCCGCAACAGGGCUCU 1478 921
CGAAACUUGUGCGCGCACG 1298 921 CGAAACUUGUGCGCGCACG 1298 939
CGUGCGCGCACAAGUUUCG 1479 939 GCCAAACUAACCUCACGUG 1299 939
GCCAAACUAACCUCACGUG 1299 957 CACGUGAGGUUAGUUUGGC 1480 957
GAAGUGACGGACUGUUCUA 1300 957 GAAGUGACGGACUGUUCUA 1300 975
UAGAACAGUCCGUCACUUC 1481 975 AUGACUGCAAAGAUGGAAA 1301 975
AUGACUGCAAAGAUGGAAA 1301 993 UUUCCAUCUUUGCAGUCAU 1482 993
ACGACCUUCUAUGACGAUG 1302 993 ACGACCUUCUAUGACGAUG 1302 1011
CAUCGUCAUAGAAGGUCGU 1483 1011 GCCCUCAACGCCUCGUUCC 1303 1011
GCCCUCAACGCCUCGUUCC 1303 1029 GGAACGAGGCGUUGAGGGC 1484 1029
CUCCCGUCCGAGAGCGGAC 1304 1029 CUCCCGUCCGAGAGCGGAC 1304 1047
GUCCGCUCUCGGACGGGAG 1485 1047 CCUUAUGGCUACAGUAACC 1305 1047
CCUUAUGGCUACAGUAACC 1305 1065 GGUUACUGUAGCCAUAAGG 1486 1065
CCCAAGAUCCUGAAACAGA 1306 1065 CCCAAGAUCCUGAAACAGA 1306 1083
UCUGUUUCAGGAUCUUGGG 1487 1083 AGCAUGACCCUGAACCUGG 1307 1083
AGCAUGACCCUGAACCUGG 1307 1101 CCAGGUUCAGGGUCAUGCU 1488 1101
GCCGACCCAGUGGGGAGCC 1308 1101 GCCGACCCAGUGGGGAGCC 1308 1119
GGCUCCCCACUGGGUCGGC 1489 1119 CUGAAGCCGCACCUCCGCG 1309 1119
CUGAAGCCGCACCUCCGCG 1309 1137 CGCGGAGGUGCGGCUUCAG 1490 1137
GCCAAGAACUCGGACCUCC 1310 1137 GCCAAGAACUCGGACCUCC 1310 1155
GGAGGUCCGAGUUCUUGGC 1491 1155 CUCACCUCGCCCGACGUGG 1311 1155
CUCACCUCGCCCGACGUGG 1311 1173 CCACGUCGGGCGAGGUGAG 1492 1173
GGGCUGCUCAAGCUGGCGU 1312 1173 GGGCUGCUCAAGCUGGCGU 1312 1191
ACGCCAGCUUGAGCAGCCC 1493 1191 UCGCCCGAGCUGGAGCGCC 1313 1191
UCGCCCGAGCUGGAGCGCC 1313 1209 GGCGCUCCAGCUCGGGCGA 1494 1209
CUGAUAAUCCAGUCCAGCA 1314 1209 CUGAUAAUCCAGUCCAGCA 1314 1227
UGCUGGACUGGAUUAUCAG 1495 1227 AACGGGCACAUCACCACCA 1315 1227
AACGGGCACAUCACCACCA 1315 1245 UGGUGGUGAUGUGCCCGUU 1496 1245
ACGCCGACCCCCACCCAGU 1316 1245 ACGCCGACCCCCACCCAGU 1316 1263
ACUGGGUGGGGGUCGGCGU 1497 1263 UUCCUGUGCCCCAAGAACG 1317 1263
UUCCUGUGCCCCAAGAACG 1317 1281 CGUUCUUGGGGCACAGGAA 1498 1281
GUGACAGAUGAGCAGGAGG 1318 1281 GUGACAGAUGAGCAGGAGG 1318 1299
CCUCCUGCUCAUCUGUCAC 1499 1299 GGGUUCGCCGAGGGCUUCG 1319 1299
GGGUUCGCCGAGGGCUUCG 1319 1317 CGAAGCCCUCGGCGAACCC 1500 1317
GUGCGCGCCCUGGCCGAAC 1320 1317 GUGCGCGCCCUGGCCGAAC 1320 1335
GUUCGGCCAGGGCGCGCAC 1501 1335 CUGCACAGCCAGAACACGC 1321 1335
CUGCACAGCCAGAACACGC 1321 1353 GCGUGUUCUGGCUGUGCAG 1502 1353
CUGCCCAGCGUCACGUCGG 1322 1353 CUGCCCAGCGUCACGUCGG 1322 1371
CCGACGUGACGCUGGGCAG 1503 1371 GCGGCGCAGCCGGUCAACG 1323 1371
GCGGCGCAGCCGGUCAACG 1323 1389 CGUUGACCGGCUGCGCCGC 1504 1389
GGGGCAGGCAUGGUGGCUC 1324 1389 GGGGCAGGCAUGGUGGCUC 1324 1407
GAGCCACCAUGCCUGCCCC 1505 1407 CCCGCGGUAGCCUCGGUGG 1325 1407
CCCGCGGUAGCCUCGGUGG 1325 1425 CCACCGAGGCUACCGCGGG 1506 1425
GCAGGGGGCAGCGGCAGCG 1326 1425 GCAGGGGGCAGCGGCAGCG 1326 1443
CGCUGCCGCUGCCCCCUGC 1507 1443 GGCGGCUUCAGCGCCAGCC 1327 1443
GGCGGCUUCAGCGCCAGCC 1327 1461 GGCUGGCGCUGAAGCCGCC 1508 1461
CUGCACAGCGAGCCGCCGG 1328 1461 CUGCACAGCGAGCCGCCGG 1328 1479
CCGGCGGCUCGCUGUGCAG 1509 1479 GUCUACGCAAACCUCAGCA 1329 1479
GUCUACGCAAACCUCAGCA 1329 1497 UGCUGAGGUUUGCGUAGAC 1510 1497
AACUUCAACCCAGGCGCGC 1330 1497 AACUUCAACCCAGGCGCGC 1330 1515
GCGCGCCUGGGUUGAAGUU 1511 1515 CUGAGCAGCGGCGGCGGGG 1331 1515
CUGAGCAGCGGCGGCGGGG 1331 1533 CCCCGCCGCCGCUGCUCAG 1512 1533
GCGCCCUCCUACGGCGCGG 1332 1533 GCGCCCUCCUACGGCGCGG 1332 1551
CCGCGCCGUAGGAGGGCGC 1513 1551 GCCGGCCUGGCCUUUCCCG 1333 1551
GCCGGCCUGGCCUUUCCCG 1333 1569 CGGGAAAGGCCAGGCCGGC 1514 1569
GCGCAACCCCAGCAGCAGC 1334 1569 GCGCAACCCCAGCAGCAGC 1334 1587
GCUGCUGCUGGGGUUGCGC 1515 1587 CAGCAGCCGCCGCACCACC 1335 1587
CAGCAGCCGCCGCACCACC 1335 1605 GGUGGUGCGGCGGCUGCUG 1516 1605
CUGCCCCAGCAGAUGCCCG 1336 1605 CUGCCCCAGCAGAUGCCCG 1336 1623
CGGGCAUCUGCUGGGGCAG 1517 1623 GUGCAGCACCCGCGGCUGC 1337 1623
GUGCAGCACCCGCGGCUGC 1337 1641 GCAGCCGCGGGUGCUGCAC 1518 1641
CAGGCCCUGAAGGAGGAGC 1338 1641 CAGGCCCUGAAGGAGGAGC 1338 1659
GCUCCUCCUUCAGGGCCUG 1519 1659 CCUCAGACAGUGCCCGAGA 1339 1659
CCUCAGACAGUGCCCGAGA 1339 1677 UCUCGGGCACUGUCUGAGG 1520 1677
AUGCCCGGCGAGACACCGC 1340 1677 AUGCCCGGCGAGACACCGC 1340 1695
GCGGUGUCUCGCCGGGCAU 1521 1695 CCCCUGUCCCCCAUCGACA 1341 1695
CCCCUGUCCCCCAUCGACA 1341 1713 UGUCGAUGGGGGACAGGGG 1522 1713
AUGGAGUCCCAGGAGCGGA 1342 1713 AUGGAGUCCCAGGAGCGGA 1342 1731
UCCGCUCCUGGGACUCCAU 1523 1731 AUCAAGGCGGAGAGGAAGC 1343 1731
AUCAAGGCGGAGAGGAAGC 1343 1749 GCUUCCUCUCCGCCUUGAU 1524 1749
CGCAUGAGGAACCGCAUCG 1344 1749 CGCAUGAGGAACCGCAUCG 1344 1767
CGAUGCGGUUCCUCAUGCG 1525 1767 GCUGCCUCCAAGUGCCGAA 1345 1767
GCUGCCUCCAAGUGCCGAA 1345 1785 UUCGGCACUUGGAGGCAGC 1526 1785
AAAAGGAAGCUGGAGAGAA 1346 1785 AAAAGGAAGCUGGAGAGAA 1346 1803
UUCUCUCCAGCUUCCUUUU 1527 1803 AUCGCCCGGCUGGAGGAAA 1347 1803
AUCGCCCGGCUGGAGGAAA 1347 1821 UUUCCUCCAGCCGGGCGAU 1528 1821
AAAGUGAAAACCUUGAAAG 1348 1821 AAAGUGAAAACCUUGAAAG 1348 1839
CUUUCAAGGUUUUCACUUU 1529 1839 GCUCAGAACUCGGAGCUGG 1349 1839
GCUCAGAACUCGGAGCUGG 1349 1857 CCAGCUCCGAGUUCUGAGC 1530 1857
GCGUCCACGGCCAACAUGC 1350 1857 GCGUCCACGGCCAACAUGC 1350 1875
GCAUGUUGGCCGUGGACGC 1531 1875 CUCAGGGAACAGGUGGCAC 1351 1875
CUCAGGGAACAGGUGGCAC 1351 1893 GUGCCACCUGUUCCCUGAG 1532
1893 CAGCUUAAACAGAAAGUCA 1352 1893 CAGCUUAAACAGAAAGUCA 1352 1911
UGACUUUCUGUUUAAGCUG 1533 1911 AUGAACCACGUUAACAGUG 1353 1911
AUGAACCACGUUAACAGUG 1353 1929 CACUGUUAACGUGGUUCAU 1534 1929
GGGUGCCAACUCAUGCUAA 1354 1929 GGGUGCCAACUCAUGCUAA 1354 1947
UUAGCAUGAGUUGGCACCC 1535 1947 ACGCAGCAGUUGCAAACAU 1355 1947
ACGCAGCAGUUGCAAACAU 1355 1965 AUGUUUGCAACUGCUGCGU 1536 1965
UUUUGAAGAGAGACCGUCG 1356 1965 UUUUGAAGAGAGACCGUCG 1356 1983
CGACGGUCUCUCUUCAAAA 1537 1983 GGGGGCUGAGGGGCAACGA 1357 1983
GGGGGCUGAGGGGCAACGA 1357 2001 UCGUUGCCCCUCAGCCCCC 1538 2001
AAGAAAAAAAAUAACACAG 1358 2001 AAGAAAAAAAAUAACACAG 1358 2019
CUGUGUUAUUUUUUUUCUU 1539 2019 GAGAGACAGACUUGAGAAC 1359 2019
GAGAGACAGACUUGAGAAC 1359 2037 GUUCUCAAGUCUGUCUCUC 1540 2037
CUUGACAAGUUGCGACGGA 1360 2037 CUUGACAAGUUGCGACGGA 1360 2055
UCCGUCGCAACUUGUCAAG 1541 2055 AGAGAAAAAAGAAGUGUCC 1361 2055
AGAGAAAAAAGAAGUGUCC 1361 2073 GGACACUUCUUUUUUCUCU 1542 2073
CGAGAACUAAAGCCAAGGG 1362 2073 CGAGAACUAAAGCCAAGGG 1362 2091
CCCUUGGCUUUAGUUCUCG 1543 2091 GUAUCCAAGUUGGACUGGG 1363 2091
GUAUCCAAGUUGGACUGGG 1363 2109 CCCAGUCCAACUUGGAUAC 1544 2109
GUUCGGUCUGACGGCGCCC 1364 2109 GUUCGGUCUGACGGCGCCC 1364 2127
GGGCGCCGUCAGACCGAAC 1545 2127 CCCAGUGUGCACGAGUGGG 1365 2127
CCCAGUGUGCACGAGUGGG 1365 2145 CCCACUCGUGCACACUGGG 1546 2145
GAAGGACUUGGUCGCGCCC 1366 2145 GAAGGACUUGGUCGCGCCC 1366 2163
GGGCGCGACCAAGUCCUUC 1547 2163 CUCCCUUGGCGUGGAGCCA 1367 2163
CUCCCUUGGCGUGGAGCCA 1367 2181 UGGCUCCACGCCAAGGGAG 1548 2181
AGGGAGCGGCCGCCUGCGG 1368 2181 AGGGAGCGGCCGCCUGCGG 1368 2199
CCGCAGGCGGCCGCUCCCU 1549 2199 GGCUGCCCCGCUUUGCGGA 1369 2199
GGCUGCCCCGCUUUGCGGA 1369 2217 UCCGCAAAGCGGGGCAGCC 1550 2217
ACGGGCUGUCCCCGCGCGA 1370 2217 ACGGGCUGUCCCCGCGCGA 1370 2235
UCGCGCGGGGACAGCCCGU 1551 2235 AACGGAACGUUGGACUUUC 1371 2235
AACGGAACGUUGGACUUUC 1371 2253 GAAAGUCCAACGUUCCGUU 1552 2253
CGUUAACAUUGACCAAGAA 1372 2253 CGUUAACAUUGACCAAGAA 1372 2271
UUCUUGGUCAAUGUUAACG 1553 2271 ACUGCAUGGACCUAACAUU 1373 2271
ACUGCAUGGACCUAACAUU 1373 2289 AAUGUUAGGUCCAUGCAGU 1554 2289
UCGAUCUCAUUCAGUAUUA 1374 2289 UCGAUCUCAUUCAGUAUUA 1374 2307
UAAUACUGAAUGAGAUCGA 1555 2307 AAAGGGGGGAGGGGGAGGG 1375 2307
AAAGGGGGGAGGGGGAGGG 1375 2325 CCCUCCCCCUCCCCCCUUU 1556 2325
GGGUUACAAACUGCAAUAG 1376 2325 GGGUUACAAACUGCAAUAG 1376 2343
CUAUUGCAGUUUGUAACCC 1557 2343 GAGACUGUAGAUUGCUUCU 1377 2343
GAGACUGUAGAUUGCUUCU 1377 2361 AGAAGCAAUCUACAGUCUC 1558 2361
UGUAGUACUCCUUAAGAAC 1378 2361 UGUAGUACUCCUUAAGAAC 1378 2379
GUUCUUAAGGAGUACUACA 1559 2379 CACAAAGCGGGGGGAGGGU 1379 2379
CACAAAGCGGGGGGAGGGU 1379 2397 ACCCUCCCCCCGCUUUGUG 1560 2397
UUGGGGAGGGGCGGCAGGA 1380 2397 UUGGGGAGGGGCGGCAGGA 1380 2415
UCCUGCCGCCCCUCCCCAA 1561 2415 AGGGAGGUUUGUGAGAGCG 1381 2415
AGGGAGGUUUGUGAGAGCG 1381 2433 CGCUCUCACAAACCUCCCU 1562 2433
GAGGCUGAGCCUACAGAUG 1382 2433 GAGGCUGAGCCUACAGAUG 1382 2451
CAUCUGUAGGCUCAGCCUC 1563 2451 GAACUCUUUCUGGCCUGCU 1383 2451
GAACUCUUUCUGGCCUGCU 1383 2469 AGCAGGCCAGAAAGAGUUC 1564 2469
UUUCGUUAACUGUGUAUGU 1384 2469 UUUCGUUAACUGUGUAUGU 1384 2487
ACAUACACAGUUAACGAAA 1565 2487 UACAUAUAUAUAUUUUUUA 1385 2487
UACAUAUAUAUAUUUUUUA 1385 2505 UAAAAAAUAUAUAUAUGUA 1566 2505
AAUUUGAUUAAAGCUGAUU 1386 2505 AAUUUGAUUAAAGCUGAUU 1386 2523
AAUCAGCUUUAAUCAAAUU 1567 2523 UACUGUCAAUAAACAGCUU 1387 2523
UACUGUCAAUAAACAGCUU 1387 2541 AAGCUGUUUAUUGACAGUA 1568 2541
UCAUGCCUUUGUAAGUUAU 1388 2541 UCAUGCCUUUGUAAGUUAU 1388 2559
AUAACUUACAAAGGCAUGA 1569 2559 UUUCUUGUUUGUUUGUUUG 1389 2559
UUUCUUGUUUGUUUGUUUG 1389 2577 CAAACAAACAAACAAGAAA 1570 2577
GGGUAUCCUGCCCAGUGUU 1390 2577 GGGUAUCCUGCCCAGUGUU 1390 2595
AACACUGGGCAGGAUACCC 1571 2595 UGUUUGUAAAUAAGAGAUU 1391 2595
UGUUUGUAAAUAAGAGAUU 1391 2613 AAUCUCUUAUUUACAAACA 1572 2613
UUGGAGCACUCUGAGUUUA 1392 2613 UUGGAGCACUCUGAGUUUA 1392 2631
UAAACUCAGAGUGCUCCAA 1573 2631 ACCAUUUGUAAUAAAGUAU 1393 2631
ACCAUUUGUAAUAAAGUAU 1393 2649 AUACUUUAUUACAAAUGGU 1574 2649
UAUAAUUUUUUUAUGUUUU 1394 2649 UAUAAUUUUUUUAUGUUUU 1394 2667
AAAACAUAAAAAAAUUAUA 1575 2667 UGUUUCUGAAAAUUCCAGA 1395 2667
UGUUUCUGAAAAUUCCAGA 1395 2685 UCUGGAAUUUUCAGAAACA 1576 2685
AAAGGAUAUUUAAGAAAAU 1396 2685 AAAGGAUAUUUAAGAAAAU 1396 2703
AUUUUCUUAAAUAUCCUUU 1577 2703 UACAAUAAACUAUUGGAAA 1397 2703
UACAAUAAACUAUUGGAAA 1397 2721 UUUCCAAUAGUUUAUUGUA 1578 2721
AGUACUCCCCUAACCUCUU 1398 2721 AGUACUCCCCUAACCUCUU 1398 2739
AAGAGGUUAGGGGAGUACU 1579 2739 UUUCUGCAUCAUCUGUAGA 1399 2739
UUUCUGCAUCAUCUGUAGA 1399 2757 UCUACAGAUGAUGCAGAAA 1580 2757
AUCCUAGUCUAUCUAGGUG 1400 2757 AUCCUAGUCUAUCUAGGUG 1400 2775
CACCUAGAUAGACUAGGAU 1581 2775 GGAGUUGAAAGAGUUAAGA 1401 2775
GGAGUUGAAAGAGUUAAGA 1401 2793 UCUUAACUCUUUCAACUCC 1582 2793
AAUGCUCGAUAAAAUCACU 1402 2793 AAUGCUCGAUAAAAUCACU 1402 2811
AGUGAUUUUAUCGAGCAUU 1583 2811 UCUCAGUGCUUCUUACUAU 1403 2811
UCUCAGUGCUUCUUACUAU 1403 2829 AUAGUAAGAAGCACUGAGA 1584 2829
UUAAGCAGUAAAAACUGUU 1404 2829 UUAAGCAGUAAAAACUGUU 1404 2847
AACAGUUUUUACUGCUUAA 1585 2847 UCUCUAUUAGACUUAGAAA 1405 2847
UCUCUAUUAGACUUAGAAA 1405 2865 UUUCUAAGUCUAAUAGAGA 1586 2865
AUAAAUGUACCUGAUGUAC 1406 2865 AUAAAUGUACCUGAUGUAC 1406 2883
GUACAUCAGGUACAUUUAU 1587 2883 CCUGAUGCUAUGUCAGGCU 1407 2883
CCUGAUGCUAUGUCAGGCU 1407 2901 AGCCUGACAUAGCAUCAGG 1588 2901
UUCAUACUCCACGCUCCCC 1408 2901 UUCAUACUCCACGCUCCCC 1408 2919
GGGGAGCGUGGAGUAUGAA 1589 2919 CCAGCGUAUCUAUAUGGAA 1409 2919
CCAGCGUAUCUAUAUGGAA 1409 2937 UUCCAUAUAGAUACGCUGG 1590 2937
AUUGCUUACCAAAGGCUAG 1410 2937 AUUGCUUACCAAAGGCUAG 1410 2955
CUAGCCUUUGGUAAGCAAU 1591 2955 GUGCGAUGUUUCAGGAGGC 1411 2955
GUGCGAUGUUUCAGGAGGC 1411 2973 GCCUCCUGAAACAUCGCAC 1592 2973
CUGGAGGAAGGGGGGUUGC 1412 2973 CUGGAGGAAGGGGGGUUGC 1412 2991
GCAACCCCCCUUCCUCCAG 1593 2991 CAGUGGAGAGGGACAGCCC 1413 2991
CAGUGGAGAGGGACAGCCC 1413 3009 GGGCUGUCCCUCUCCACUG 1594 3009
CACUGAGAAGUCAAACAUU 1414 3009 CACUGAGAAGUCAAACAUU 1414 3027
AAUGUUUGACUUCUCAGUG 1595 3027 UUCAAAGUUUGGAUUGCAU 1415 3027
UUCAAAGUUUGGAUUGCAU 1415 3045 AUGCAAUCCAAACUUUGAA 1596 3045
UCAAGUGGCAUGUGCUGUG 1416 3045 UCAAGUGGCAUGUGCUGUG 1416 3063
CACAGCACAUGCCACUUGA 1597 3063 GACCAUUUAUAAUGUUAGA 1417 3063
GACCAUUUAUAAUGUUAGA 1417 3081 UCUAACAUUAUAAAUGGUC 1598 3081
AAAUUUUACAAUAGGUGCU 1418 3081 AAAUUUUACAAUAGGUGCU 1418 3099
AGCACCUAUUGUAAAAUUU 1599 3099 UUAUUCUCAAAGCAGGAAU 1419 3099
UUAUUCUCAAAGCAGGAAU 1419 3117 AUUCCUGCUUUGAGAAUAA 1600 3117
UUGGUGGCAGAUUUUACAA 1420 3117 UUGGUGGCAGAUUUUACAA 1420 3135
UUGUAAAAUCUGCCACCAA 1601 3135 AAAGAUGUAUCCUUCCAAU 1421 3135
AAAGAUGUAUCCUUCCAAU 1421 3153 AUUGGAAGGAUACAUCUUU 1602 3153
UUUGGAAUCUUCUCUUUGA 1422 3153 UUUGGAAUCUUCUCUUUGA 1422 3171
UCAAAGAGAAGAUUCCAAA 1603 3171 ACAAUUCCUAGAUAAAAAG 1423 3171
ACAAUUCCUAGAUAAAAAG 1423 3189 CUUUUUAUCUAGGAAUUGU 1604 3189
GAUGGCCUUUGUCUUAUGA 1424 3189 GAUGGCCUUUGUCUUAUGA 1424 3207
UCAUAAGACAAAGGCCAUC 1605 3207 AAUAUUUAUAACAGCAUUC 1425 3207
AAUAUUUAUAACAGCAUUC 1425 3225 GAAUGCUGUUAUAAAUAUU 1606 3225
CUGUCACAAUAAAUGUAUU 1426 3225 CUGUCACAAUAAAUGUAUU 1426 3243
AAUACAUUUAUUGUGACAG 1607 3234 UAAAUGUAUUCAAAUACCA 1427 3234
UAAAUGUAUUCAAAUACCA 1427 3252 UGGUAUUUGAAUACAUUUA 1608 The 3-ends
of the Upper sequence and the Lower sequence of the siNA construct
can include an overhang sequence, for example about 1, 2, 3, or 4
nucleotides in length, preferably 2 nucleotides in length, wherein
the overhanging sequence of the lower sequence is optionally
complementary to a portion of the target sequence. The upper
sequence is also referred to as the sense strand, whereas the lower
sequence is also referred to as the antisense strand. The upper and
lower sequences in the Table can further comprise a chemical
modification having Formulae I-VII or any combination thereof.
TABLE-US-00003 TABLE III MAP Kinase Synthetic Modified siNA
constructs Target Seq Seq Pos Target ID Aliases Sequence ID
MAPK1/ERK2 3302 ACCAGACCUACUGCCAGAGAACC 1113 MAPK1:424U21 siRNA
sense CAGACCUACUGCCAGAGAATT 1129 3852 AUCACACAGGGUUCCUGACAGAA 1114
MAPK1:778U21 siRNA sense CACACAGGGUUCCUGACAGTT 1130 3892
UUGGCUCUAGUCACUGGCAUCUC 1115 MAPK1:1718U21 siRNA sense
GGCUCUAGUCACUGGCAUCTT 1131 3946 ACUGUGGAGUUGACUCGGUGUUC 1116
MAPK1:2525U21 siRNA sense UGUGGAGUUGACUCGGUGUTT 1132 3302
ACCAGACCUACUGCCAGAGAACC 1113 MAPK1:442L21 siRNA (424C)
UUCUCUGGCAGUAGGUCUGTT 1133 antisense 3852 AUCACACAGGGUUCCUGACAGAA
1114 MAPK1:796L21 siRNA (778C) CUGUCAGGAACCCUGUGUGTT 1134 antisense
3892 UUGGCUCUAGUCACUGGCAUCUC 1115 MAPK1:1736L21 siRNA (1718C)
GAUGCCAGUGACUAGAGCCTT 1135 antisense 3946 ACUGUGGAGUUGACUCGGUGUUC
1116 MAPK1:2543L21 siRNA (2525C) ACACCGAGUCAACUCCACATT 1136
antisense 3302 ACCAGACCUACUGCCAGAGAACC 1113 MAPK1:424U21 siRNA
stab04 B cAGAccuAcuGccAGAGAATT B 1137 sense RPI 30817 3852
AUCACACAGGGUUCCUGACAGAA 1114 MAPK1:778U21 siRNA stab04 B
cAcAcAGGGuuccuGAcAGTT B 1138 sense RPI 30818 3892
UUGGCUCUAGUCACUGGCAUCUC 1115 MAPK1:1718U21 siRNA stab04 B
GGcucuAGucAcuGGcAucTT B 1139 sense RPI 30819 3946
ACUGUGGAGUUGACUCGGUGUUC 1116 MAPK1:2525U21 siRNA stab04 B
uGuGGAGuuGAcucGGuGuTT B 1140 sense RPI 30820 3302
ACCAGACCUACUGCCAGAGAACC 1113 MAPK1:442L21 siRNA (424C)
uucucuGGcAGuAGGucuGTsT 1141 stab05 antisense RPI 30821 3852
AUCACACAGGGUUCCUGACAGAA 1114 MAPK1:796L21 siRNA (778C)
cuGucAGGAAcccuGuGuGTsT 1142 stab05 antisense RPI 30822 3892
UUGGCUCUAGUCACUGGCAUCUC 1115 MAPK1:1736L21 siRNA (1718C)
GAuGccAGuGAcuAGAGccTsT 1143 stab05 antisense RPI 30823 3946
ACUGUGGAGUUGACUCGGUGUUC 1116 MAPK1:2543L21 siRNA (2525C)
AcAccGAGucAAcuCCAcATsT 1144 stab05 antisense RPI 30824 3302
ACCAGACCUA0UGCCAGAGAACC 1113 MARK1:424U21 siRNA stab07 B
cAGAccuAcuGccAGAGAATT B 1145 sense 3852 AUCACACAGGGUUCCUGACAGAA
1114 MARK1:778U21 siRNA stabo7 B cAcAcAGGGuuccuGAcAGTT B 1146 sense
3892 UUGGCUCUAGUCACUGGCAUCUC 1115 MAPK1:1718U21 siRNA stabo7 B
GGcucuAGucAcuGGcAucTTB 1147 sense 3946 ACUGUGGAGUUGACUCGGUGUUC 1116
MARK1:2525U21 siRNA stabo7 B uGuGGAGuuGAcucGGuGuTT B 1148 sense
3302 ACCAGACCUACUGCCAGAGAACC 1113 MARK1:442L21 siRNA (424C)
uucucuGGcAGuAGGucuGTsT 1149 stab11 antisense 3852
AUCACACAGGGUUCCUGACAGAA 1114 MARK1:796L21 siRNA (778C)
cuGucAGGAAcccuGuGuGTsT 1150 stab11 antisense 3892
UUGGCUCUAGUCACUGGCAUCUC 1115 MARK1:1736L21 siRNA (1718C)
GAuGccAGuGAcuAGAGccTsT 1151 stab11 antisense 3946
ACUGUGGAGUUGACUCGGUGUUC 1116 MARK1:2543L21 siRNA (25250)
AcAccGAGucAAcuccAcATsT 1152 stab11 antisense Target Seq Seq Pos
Target ID Aliases Sequence ID MAPK3/ERK1 283
CCUUCGAACAUCAGACCUACUGC 1117 MAPK3:285U21 siRNA sense
UUCGAACAUCAGACCUACUTT 1153 709 UGAACUCCAAGGGCUAUACCAAG 1118
MAPK3:711U21 siRNA sense AACUCCAAGGGCUAUACCATT 1154 718
AGGGCUAUACCAAGUCCAUCGAC 1119 MAPK3:720U21 siRNA sense
GGCUAUACCAAGUCCAUCGTT 1155 1778 UUCUGUGUGUGGUGAGCAGAAGU 1120
MAPK3:1780U21 siRNA sense CUGUGUGUGGUGAGCAGAATT 1156 283
CCUUCGAACAUCAGACCUACUGC 1117 MAPK3:303L21 siRNA (285C)
AGUAGGUCUGAUGUUCGAATT 1157 antisense 709 UGAACUCCAAGGGCUAUACCAAG
1118 MAPK3:729L21 siRNA (711C) UGGUAUAGCCCUUGGAGUUTT 1158 antisense
718 AGGGCUAUACCAAGUCCAUCGAC 1119 MAPK3:738L21 siRNA (720C)
CGAUGGACUUGGUAUAGCCTT 1159 antisense 1778 UUCUGUGUGUGGUGAGCAGAAGU
1120 MAPK3:1798L21 siRNA (1780C) UUCUGCUCACCACACACAGTT 1160
antisense 283 CCUUCGAACAUCAGACCUACUGC 1117 MAPK3:285U21 siRNA
stab04 sense B uucGAAcAucAGAccuAcuTT B 1161 709
UGAACUCCAAGGGCUAUACCAAG 1118 MAPK3:711U21 siRNA stab04 sense B
AAcuccAAGGGcuAuAccATT B 1162 718 AGGGCUAUACCAAGUCCAUCGAC 1119
MAPK3:720U21 siRNA stab04 sense B GGcuAuAccAAGuccAucGTT B 1163 1778
UUCUGUGUGUGGUGAGCAGAAGU 1120 MAPK3:1780U21 siRNA stab04 sense B
cuGuGuGuGGuGAGcAGAATT B 1164 283 CCUUCGAACAUCAGACCUACUGC 1117
MAPK3:303L21 siRNA (285C) stab05 AGuAGGucuGAuGuucGAATsT 1165
antisense 709 UGAACUCCAAGGGCUAUACCAAG 1118 MAPK3:729L21 siRNA
(711C) stab05 uGGuAuAGcccuuGGAGuuTsT 1166 antisense 718
AGGGCUAUACCAAGUCCAUCGAC 1119 MAPK3:738L21 siRNA (720C) stab05
cGAuGGAcuuGGuAuAGccTsT 1167 antisense 1778 UUCUGUGUGUGGUGAGCAGAAGU
1120 MAPK3:1798L21 siRNA (1780C) uucuGcucAccAcAcAcAGTsT 1168 stab05
antisense 283 CCUUCGAACAUCAGACCUACUGC 1117 MAPK3:285U21 siRNA
stab07 sense B uucGAAcAucAGAccuAcuTT B 1169 709
UGAACUCCAAGGGCUAUACCAAG 1118 MAPK3:711U21 siRNA stab07 sense B
AAcuccAAGGGcuAuAccATT B 1170 718 AGGGCUAUACCAAGUCCAUCGAC 1119
MAPK3:720U21 siRNA stab07 sense B GGcuAuAccAAGuccAucGTT B 1171 1778
UUCUGUGUGUGGUGAGCAGAAGU 1120 MAPK3:1780U21 siRNA stab07 sense B
cuGuGuGuGGuGAGcAGAATT B 1172 283 CCUUCGAACAUCAGACCUACUGC 1117
MAPK3:303L21 siRNA (285C) stab11 AGuAGGucuGAuGuucGAATsT 1173
antisense 709 UGAACUCCAAGGGCUAUACCAAG 1118 MAPK3:729L21 siRNA
(711C) stab11 uGGuAuAGcccuuGGAGuuTsT 1174 antisense 718
AGGGCUAUACCAAGUCCAUCGAC 1119 MAPK3:738L21 siRNA (720C) stab11
cGAuGGAcuuGGuAuAGccTsT 1175 antisense 1778 UUCUGUGUGUGGUGAGCAGAAGU
1120 MAPK3:1798L21 siRNA (178C0) uucuGcucAccAcAcAcAGTsT 1176 stab11
antisense Target Seq Seq Pos Target ID RPI # Aliases Sequence ID
MAPK8/JNK1 733 AACAGCUUGGAACACCAUGUCCU 1121 31517 MAPK8:735U21
siRNA sense CAGCUUGGAACACCAUGUCTT 1177 853 UUUUCCCAGCUGACUCAGAACAC
1122 31518 MAPK8:855U21 siRNA sense UUCCCAGCUGACUCAGAACTT 1178 1224
CAAUGUCAACAGAUCCGACUUUG 1123 31519 MAPK8:1226U21 siRNA sense
AUGUCAACAGAUCCGACUUTT 1179 1242 CUUUGGCCUCUGAUACAGACAGC 1124 31520
MAPK8:1244U21 siRNA sense UUGGCCUCUGAUACAGACATT 1180 733
AACAGCUUGGAACACCAUGUCCU 1121 31521 MAPK8:753L21 siRNA (735C)
GACAUGGUGUUCCAAGCUGTT 1181 antisense 853 UUUUCCCAGCUGACUCAGAACAC
1122 31522 MAPK8:873L21 siRNA (855C) GUUCUGAGUCAGCUGGGAATT 1182
antisense 1224 CAAUGUCAACAGAUCCGACUUUG 1123 31523 MAPK8:1244L21
siRNA (1226C) AAGUCGGAUCUGUUGACAUTT 1183 antisense 1242
CUUUGGCCUCUGAUACAGACAGC 1124 31524 MAPK8:1262L21 siRNA (1244C)
UGUCUGUAUCAGAGGCCAATT 1184 antisense 733 AACAGCUUGGAACACCAUGUCCU
1121 MAPK8:735U21 siRNA stab4 B cAGcuuGGAAcAccAuGucTT B 1185 sense
853 UUUUCCCAGCUGACUCAGAACAC 1122 MAPK8:855U21 siRNA stab4 B
uucccAGcuGAcucAGAAcTT B 1186 sense 1224 CAAUGUCAACAGAUCCGACUUUG
1123 MAPK8:1226U21 siRNA stab4 BAuGucAAcAGAuccGAcuuTTB 1187 sense
1242 CUUUGGCCUCUGAUACAGACAGC 1124 MAPK8:1244U21 siRNA stab4 B
uuGGccucuGAuAcAGAcATT B 1188 sense 733 AACAGCUUGGAACACCAUGUCCU 1121
MAPK8:753L21 siRNA (735C) GAcAuGGuGuuccAAGcuGTsT 1189 stab5
antisense 853 UUUUCCCAGCUGACUCAGAACAC 1122 MAPK8:873L21 siRNA
(855C) GuucuGAGucAGcuGGGAATsT 1190 stab5 antisense 1224
CAAUGUCAACAGAUCCGACUUUG 1123 MAPK8:1244L21 siRNA (1226C)
AAGucGGAucuGuuGAcAuTsT 1191 stab5 antisense 1242
CUUUGGCCUCUGAUACAGACAGC 1124 MAPK8:1262L21 siRNA (1244C)
uGucuGuAucAGAGGccAATsT 1192 stab5 antisense 733
AACAGCUUGGAACACCAUGUCCU 1121 MAPK8:735U21 siRNA stab7
BcAGcuuGGAAcAccAuGucTTB 1193 sense 853 UUUUCCCAGCUGACUCAGAACAC 1122
MAPK8:855U21 siRNA stab7 B uucccAGcuGAcucAGAAcTT B 1194 sense 1224
CAAUGUCAACAGAUCCGACUUUG 1123 MAPK8:1226U21 siRNA stab7 B
AuGucAAcAGAuccGAcuuTT B 1195 sense 1242 CUUUGGCCUCUGAUACAGACAGC
1124 MAPK8:1244U21 siRNA stab7 B uuGGccucuGAuAcAGAcATT B 1196 sense
733 AACAGCUUGGAACACCAUGUCCU 1121 MAPK8:753L21 siRNA (735C)
GAcAuGGuGuuccAAGcuGTsT 1197 stab11 antisense 853
UUUUCCCAGCUGACUCAGAACAC 1122 MAPK8:873L21 siRNA (855C)
GuucuGAGucAGcuGGGAATsT 1198 stab11 antisense 1224
CAAUGUCAACAGAUCCGACUUUG 1123 MAPK8:1244L21 siRNA (1226C)
AAGucGGAucuGuuGAcAuTsT 1199 stab11 antisense 1242
CUUUGGCCUCUGAUACAGACAGC 1124 MAPK8:1262L21 siRNA (1244C)
uGucuGuAucAGAGGccAATsT 1200 stab11 antisense Target Seq Pos Target
ID RPI # Aliases Sequence ID MAPK14/p38 1278
GCCUACUUUGCUCAGUACCACGA 1125 31586 MAPK14:1280U21 siRNA sense
CUACUUUGCUCAGUACCACTT 1201 1609 UGUCUGUCUUUGUGGGAGGGUAA 1126 31587
MARK14:1611U21 siRNA sense UCUGUCUUUGUGGGAGGGUTT 1202 2882
AAAAGGGUCUUCUUGGCAGCUUA 1127 31588 MAPK14:2884U21 siRNA sense
AAGGGUCUUCUUGGCAGCUTT 1203 3554 GGACUCUAAGCUGGAGCUCUUGG 1128 31589
MAPK14:3556U21 siRNA sense ACUCUAAGCUGGAGCUCUUTT 1204 1278
GCCUACUUUGCUCAGUACCACGA 1125 31590 MAPK14:1298L21 siRNA (1280C)
GUGGUACUGAGCAAAGUAGTT 1205 antisense 1609 UGUCUGUCUUUGUGGGAGGGUAA
1126 31591 MAPK14:1629L21 siRNA (1611C) ACCCUCCCACAAAGACAGATT 1206
antisense 2882 AAAAGGGUCUUCUUGGCAGCUUA 1127 31592 MARK14:2902L21
siRNA (2884C) AGCUGCCAAGAAGACCCUUTT 1207 antisense 3554
GGACUCUAAGCUGGAGCUCUUGG 1128 31593 MAPK14:3574L21 siRNA (3556C)
AAGAGCUCCAGCUUAGAGUTT 1208 antisense 1278 GCCUACUUUGCUCAGUACCACGA
1125 MAPK14:1280U21 siRNA stab04 B cuAcuuuGcucAGuAccAcTT B 1209
sense 1609 UGUCUGUCUUUGUGGGAGGGUAA 1126 MARK14:1611U21 siRNA stab04
B ucuGucuuuGuGGGAGGGuTT B 1210 sense 2882 AAAAGGGUCUUCUUGGCAGCUUA
1127 MAPK14:2884U21 siRNA stab04 B AAGGGucuucuuGGcAGcuTT B 1211
sense 3554 GGACUCUAAGCUGGAGCUCUUGG 1128 MAPK14:3556U21 siRNA stab04
B AcucuAAGcuGGAGcucuuTT B 1212 sense 1278 GCCUACUUUGCUCAGUACCACGA
1125 MAPK14:1298L21 siRNA (1280C) GuGGuAcuGAGcAAAGuAGTsT 1213
stab05 antisense 1609 UGUCUGUCUUUGUGGGAGGGUAA 1126 MARK14:1629L21
siRNA (1611C) AcccucccAcAAAGAcAGATsT 1214 stab05 antisense 2882
AAAAGGGUCUUCUUGGCAGCUUA 1127 MAPK14:2902L21 siRNA (2884C)
AGcuGccAAGAAGAcccuuTsT 1215 stab05 antisense 3554
GGACUCUAAGCUGGAGCUCUUGG 1128 MARK14:3574L21 siRNA (3556C)
AAGAGcuccAGcuuAGAGuTsT 1216 stab05 antisense 1278
GCCUACUUUGCUCAGUACCACGA 1125 MAPK14:1280U21 siRNA stab07 B
cuAcuuuGcucAGuAccAcTT B 1217 sense 1609 UGUCUGUCUUUGUGGGAGGGUAA
1126 MARK14:1611U21 siRNA stab07 B ucuGucuuuGuGGGAGGGuTT B 1218
sense 2882 AAAAGGGUCUUCUUGGCAGCUUA 1127 MAPK14:2884U21 siRNA stab07
B AAGGGucuucuuGGcAGcuTT B 1219 sense 3554 GGACUCUAAGCUGGAGCUCUUGG
1128 MAPK14:3556U21 siRNA stab07 B AcucuAAGcuGGAGcucuuTT B 1220
sense 1278 GCCUACUUUGCUCAGUACCACGA 1125 MAPK14:1298L21 siRNA
(1280C) GuGGuAcuGAGcAAAGuAGTsT 1221 stab11 antisense 1609
UGUCUGUCUUUGUGGGAGGGUAA 1126 MARK14:1629L21 siRNA (1611C)
AcccucccAcAAAGAcAGATsT 1222 stab11 antisense 2882
AAAAGGGUCUUCUUGGCAGCUUA 1127 MAPK14:2902L21 siRNA (2884C)
AGcuGccAAGAAGAcccuuTsT 1223 stab11 antisense 3554
GGACUCUAAGCUGGAGCUCUUGG 1128 MAPK14:3574L21 siRNA (3556C)
AAGAGcuccAGcuuAGAGuTsT 1224 stab11 antisense Target Seq Seq Pos
Target ID RPI# Aliases Sequence ID c-JUN 1817
GGAAAAAGUGAAAACCUUGAAAG 1609 JUN:1819U21 siRNA sense
AAAAAGUGAAAACCUUGAATT 1617 1935 CAACUCAUGCUAACGCAGCAGUU 1610
JUN:1937U21 siRNA sense ACUCAUGCUAACGCAGCAGTT 1618 2259
CAUUGACCAAGAACUGCAUGGAC 1611 JUN:2261U21 siRNA sense
UUGACCAAGAACUGCAUGGTT 1619 2264 ACCAAGAACUGCAUGGACCUAAC 1612
JUN:2266U21 siRNA sense CAAGAACUGCAUGGACCUATT 1620 2269
GAACUGCAUGGACCUAACAUUCG 1613 JUN:2271U21 siRNA sense
ACUGCAUGGACCUAACAUUTT 1621 2270 AACUGCAUGGACCUAACAUUCGA 1614
JUN:2272U21 siRNA sense CUGCAUGGACCUAACAUUCTT 1622 2272
CUGCAUGGACCUAACAUUCGAUC 1615 JUN:2274U21 siRNA sense
GCAUGGACCUAACAUUCGATT 1623 2274 GCAUGGACCUAACAUUCGAUCUC 1616
JUN:2276U21 siRNA sense AUGGACCUAACAUUCGAUCTT 1624 1817
GGAAAAAGUGAAAACCUUGAAAG 1609 JUN:1837L21 siRNA (1819C)
UUCAAGGUUUUCACUUUUUTT 1625 antisense 1935 CAACUCAUGCUAACGCAGCAGUU
1610 JUN:1955L21 siRNA (1937C) CUGCUGCGUUAGCAUGAGUTT 1626 antisense
2259 CAUUGACCAAGAACUGCAUGGAC 1611 JUN:2279L21 siRNA (2261C)
CCAUGCAGUUCUUGGUCAATT 1627 antisense 2264 ACCAAGAACUGCAUGGACCUAAC
1612 JUN:2284L21 siRNA (2266C) UAGGUCCAUGCAGUUCUUGTT 1628 antisense
2269 GAACUGCAUGGACCUAACAUUCG 1613 JUN:2289L21 siRNA (2271C)
AAUGUUAGGUCCAUGCAGUTT 1629 antisense 2270 AACUGCAUGGACCUAACAUUCGA
1614 JUN:2290L21 siRNA (2272C) GAAUGUUAGGUCCAUGCAGTT 1630 antisense
2272 CUGCAUGGACCUAACAUUCGAUC 1615 JUN:2292L21 siRNA (2274C)
UCGAAUGUUAGGUCCAUGCTT 1631 antisense 2274 GCAUGGACCUAACAUUCGAUCUC
1616 JUN:2294L21 siRNA (2276C) GAUCGAAUGUUAGGUCCAUTT 1632 antisense
1817 GGAAAAAGUGAAAACCUUGAAAG 1609 JUN:1819U21 siRNA stab04 B
AAAAAGuGAAAAccuuGAATT B 1633 sense 1935 CAACUGAUGCUAACGCAGCAGUU
1610 JUN:1937U21 siRNA stab04 B AcucAuGcuAAcGcAGcAGTT B 1634 sense
2259 CAUUGACCAAGAACUGCAUGGAC 1611 JUN:2261U21 siRNA stab04 B
uuGAccAAGAAcuGcAuGGTT B 1635 sense 2264 ACCAAGAACUGCAUGGACCUAAC
1612 JUN:2266U21 siRNA stab04 B cAAGAAcuGcAuGGAccuATT B 1636 sense
2269 GAACUGCAUGGACCUAACAUUCG 1613 JUN:2271U21 siRNA stab04 B
AcuGcAuGGAccuAAcAuuTT B 1637 sense 2270 AACUGCAUGGACCUAACAUUCGA
1614 JUN:2272U21 siRNA stab04 B cuGcAuGGAccuAAcAuucTT B 1638 sense
2272 CUGCAUGGACCUAACAUUCGAUC 1615 JUN:2274U21 siRNA stab04 B
GcAuGGAccuAAcAuucGATT B 1639 sense 2274 GCAUGGACCUAACAUUCGAUCUC
1616 JUN:2276U21 siRNA stab04 B AuGGAccuAAcAuucGAucTT B 1640 sense
1817 GGAAAAAGUGAAAACCUUGAAAG 1609 JUN:1837L21 siRNA (1819C)
uucAAGGuuuucAcuuuuuTsT 1641 stab05 antisense 1935
CAACUCAUGCUAACGCAGCAGUU 1610 JUN:1955L21 siRNA (1937C)
cuGcuGcGuuAGcAuGAGuTsT 1642 stab05 antisense 2259
CAUUGACCAAGAACUGCAUGGAC 1611 JUN:2279L21 siRNA (2261C)
ccAuGcAGuucuuGGucAATsT 1643 stab05 antisense 2264
ACCAAGAACUGCAUGGACCUAAC 1612 JUN:2284L21 siRNA (2266C)
uAGGuccAuGcAGuucuuGTsT 1644 stab05 antisense 2269
GAACUGCAUGGACCUAACAUUCG 1613 JUN:2289L21 siRNA (2271C)
AAuGuuAGGuccAuGcAGuTsT 1645 stab05 antisense 2270
AACUGCAUGGACCUAACAUUCGA 1614 JUN:2290L21 siRNA (2272C)
GAAuGuuAGGuccAuGcAGTsT 1646 stab05 antisense 2272
CUGCAUGGACCUAACAUUCGAUC 1615 JUN:2292L21 siRNA (2274C)
ucGAAuGuuAGGuccAuGcTsT 1647 stab05 antisense
2274 GCAUGGACCUAACAUUCGAUCUG 1616 JUN:2294L21 siRNA (2276C)
GAucGAAuGuuAGGuccAuTsT 1648 stab05 antisense 1817
GGAAAAAGUGAAAACCUUGAAAG 1609 31818 JUN:1819U21 siRNA stab07 B
AAAAAGuGAAAAccuuGAATT B 1649 sense 1935 CAACUCAUGCUAACGCAGCAGUU
1610 31819 JUN:1937U21 siRNA stab07 B AcucAuGcuAAcGcAGcAGTT B 1650
sense 2259 CAUUGACCAAGAACUGCAUGGAC 1611 31820 JUN:2261U21 siRNA
stab07 B uuGAccAAGAAcuGcAuGGTT B 1651 sense 2264
ACCAAGAACUGCAUGGACCUAAC 1612 31821 JUN:2266U21 siRNA stab07 B
cAAGAAcuGcAuGGAccuATT B 1652 sense 2269 GAACUGCAUGGACCUAACAUUCG
1613 31822 JUN:2271U21 siRNA stab07 B AcuGcAuGGAccuAAcAuuTT B 1653
sense 2270 AACUGCAUGGACCUAACAUUCGA 1614 31823 JUN:2272U21 siRNA
stab07 B cuGcAuGGAccuAAcAuucTT B 1654 sense 2272
CUGCAUGGACCUAACAUUCGAUC 1615 31824 JUN:2274U21 siRNA stab07 B
GcAuGGAccuAAcAuucGATT B 1655 sense 2274 GCAUGGACCUAACAUUCGAUCUG
1616 31825 JUN:2276U21 siRNA stab07 B AuGGAccuAAcAuucGAucTT B 1656
sense 1817 GGAAAAAGUGAAAACCUUGAAAG 1609 JUN:1837L21 siRNA (1819C)
uucAAGGuuuucAcuuuuuTsT 1657 stab11 antisense 1935
CAACUCAUGCUAACGCAGCAGUU 1610 JUN:1955L21 siRNA (1937C)
cuGcuGcGuuAGcAuGAGuTsT 1658 stab11 antisense 2259
CAUUGACCAAGAACUGCAUGGAC 1611 JUN:2279L21 siRNA (2261C)
ccAucAGuucuuGGucAATsT 1659 stab11 antisense 2264
ACCAAGAACUGCAUGGACCUAAC 1612 JUN:2284L21 siRNA (2266C)
uAGGuccAuGcAGuucuuGTsT 1660 stab11 antisense 2269
GAACUGCAUGGACCUAACAUUCG 1613 JUN:2289L21 siRNA (2271C)
AAuGuuAGGuccAuGcAGuTsT 1661 stab11 antisense 2270
AACUGCAUGGACCUAACAUUCGA 1614 JUN:2290L21 siRNA (2272C)
GAAuGuuAGGuccAuGcAGTsT 1662 stab11 antisense 2272
CUGCAUGGACCUAACAUUCGAUC 1615 JUN:2292L21 siRNA (2274C)
ucGAAuGuuAGGuccAuGcTsT 1663 stab11 antisense 2274
GCAUGGACCUAACAUUCGAUCUC 1616 JUN:2294L21 siRNA (2276C)
GAucGAAuGuuAGGuccAuTsT 1664 stab11 antisense 1817
GGAAAAAGUGAAAACCUUGAAAG 1609 JUN:1819U21 siRNA stab08
AAAAAGuGAAAAccuuGAATsT 1665 sense 1935 CAACUCAUGCUAACGCAGCAGUU 1610
JUN:1937U21 siRNA stab08 AcucAuGcuAAcGcAGcAGTsT 1666 sense 2259
CAUUGACCAAGAACUGCAUGGAC 1611 JUN:2261U21 siRNA stab08
uuGAccAAGAAcuGcAuGGTsT 1667 sense 2264 ACCAAGAACUGCAUGGACCUAAC 1612
JUN:2266U21 siRNA stab08 cAAGAAcuGcAuGGAccuATsT 1668 sense 2269
GAACUGCAUGGACCUAACAUUCG 1613 JUN:2271U21 siRNA stab08
AcuGcAuGGAccuAAcAuuTsT 1669 sense 2270 AACUGCAUGGACCUAACAUUCGA 1614
JUN:2272U21 siRNA stab08 cuGcAuGGAccuAAcAuucTsT 1670 sense 2272
CUGCAUGGACCUAACAUUCGAUC 1615 JUN:2274U21 siRNA stabo8
GcAuGGAccuAAcAuucGATsT 1671 sense 2274 GCAUGGACCUAACAUUCGAUCUC 1616
JUN:2276U21 siRNA stabo8 AuGGAccuAAcAuucGAucTsT 1672 sense 1817
GGAAAAAGUGAAAACCUUGAAAG 1609 31826 JUN:1837L21 siRNA (1819C)
uucAAGGuuuucAcuuuuuTsT 1673 stab08 antisense 1935
CAACUCAUGCUAACGCAGCAGUU 1610 31827 JUN:1955L21 siRNA (1937C)
cuGcuGcGuuAGcAuGAGuTsT 1674 stab08 antisense 2259
CAUUGACCAAGAACUGCAUGGAC 1611 31828 JUN:2279L21 siRNA (2261C)
ccAuGcAGuucuuGGucAATsT 1675 stab08 antisense 2264
ACCAAGAACUGCAUGGACCUAAC 1612 31829 JUN:2284L21 siRNA (2266C)
uAGGuccAuGcAGuucuuGTsT 1676 stab08 antisense 2269
GAACUGCAUGGACCUAACAUUCG 1613 31830 JUN:2289L21 siRNA (2271C)
AAuGuuAGGuccAuGcAGuTsT 1677 stab08 antisense 2270
AACUGCAUGGACCUAACAUUCGA 1614 31831 JUN:2290L21 siRNA (2272C)
GAAuGuuAGGuccAuGcAGTsT 1678 stab08 antisense 2272
CUGCAUGGACCUAACAUUCGAUC 1615 31832 JUN:2292L21 siRNA (2274C)
ucGAAuGuuAGGuccAuGcTsT 1679 stab08 antisense 2274
GCAUGGACCUAACAUUCGAUCUC 1616 31833 JUN:2294L21 siRNA (2276C)
GAucGAAuGuuAGGuccAuTsT 1680 stab08 antisense 1817
GGAAAAAGUGAAAACCUUGAAAG 1609 31834 JUN:1819U21 siRNA inv stab07 B
AAGuuccAAAAGuGAAAAATT B 1681 sense 1935 CAACUCAUGCUAACGCAGCAGUU
1610 31835 JUN:1937U21 siRNA inv stab07 B GAcGAcGcAAucGuAcucATT B
1682 sense 2259 CAUUGACCAAGAACUGCAUGGAC 1611 31836 JUN:2261U21
siRNA inv stab07 B GGuAcGucAAGAAccAGuuTT B 1683 sense 2264
ACCAAGAACUGCAUGGACCUAAC 1612 31837 JUN:2266U21 siRNA inv stab07 B
AuccAGGuAcGucAAGAAcTT B 1684 sense 2269 GAACUGCAUGGACCUAACAUUCG
1613 31838 JUN:2271U21 siRNA inv stab07 B uuAcAAuccAGGuAcGucATT B
1685 sense 2270 AACUGCAUGGACCUAACAUUCGA 1614 31839 JUN:2272U21
siRNA inv stab07 B cuuAcAAuccAGGuAcGucTT B 1686 sense 2272
CUGCAUGGACCUAACAUUCGAUC 1615 31840 JUN:2274U21 siRNA inv stab07 B
AGcuuAcAAuccAGGuAcGTT B 1687 sense 2274 GCAUGGACCUAACAUUCGAUCUC
1616 31841 JUN:2276U21 siRNA inv stab07 B cuAGcuuAcAAuccAGGuATT B
1688 sense 1817 GGAAAAAGUGAAAACCUUGAAAG 1609 31842 JUN:1837L21
siRNA (1819C) uuuuucAcuuuuGGAAcuuTsT 1689 inv stab08 antisense 1935
CAACUCAUGCUAACGCAGCAGUU 1610 31843 JUN:1955L21 siRNA (1937C)
uGAGuAcGAuuGcGucGucTsT 1690 inv stab08 antisense 2259
CAUUGACCAAGAACUGCAUGGAC 1611 31844 JUN:2279L21 siRNA (2261C)
AAcuGGuucuuGAcGuAccTsT 1691 inv stab08 antisense 2264
ACCAAGAACUGCAUGGACCUAAC 1612 31845 JUN:2284L21 siRNA (2266C)
GuucuuGAcGuAccuGGAuTsT 1692 inv stab08 antisense 2269
GAACUGCAUGGACCUAACAUUCG 1613 31846 JUN:2289L21 siRNA (2271C)
uGAcGuAccuGGAuuGuAATsT 1693 inv stab08 antisense 2270
AACUGCAUGGACCUAACAUUCGA 1614 31847 JUN:2290L21 siRNA (2272C)
GAcGuAccuGGAuuGuAAGTsT 1694 inv stab08 antisense 2272
CUGCAUGGACCUAACAUUCGAUC 1615 31848 JUN:2292L21 siRNA (2274C)
cGuAccuGGAuuGuAAGcuTsT 1695 inv stab08 antisense 2274
GCAUGGACCUAACAUUCGAUCUC 1616 31849 JUN:2294L21 siRNA (2276C)
uAccuGGAuuGuAAGcuAGTsT 1696 inv stab08 antisense Uppercase =
ribonucleotide u,c = 2'-deoxy-2'-fluoro U, C A = 2'-O-methyl
Adenosine G = 2'-O-methyl Guanosine T = thymidine B = inverted
deoxy abasic s = phosphorothioate linkage A = deoxy Adenosine G =
deoxy Guanosine
TABLE-US-00004 TABLE IV Non-limiting examples of Stabilization
Chemistries for chemically modified siNA constructs Chemistry
pyrimidine Purine cap p = S Strand "Stab 1" Ribo Ribo -- 5 at
5'-end S/AS 1 at 3'-end "Stab 2" Ribo Ribo -- All linkages Usually
AS "Stab 3" 2'-fluoro Ribo -- 4 at 5'-end Usually S 4 at 3'-end
"Stab 4" 2'-fluoro Ribo 5' and 3'-ends -- Usually S "Stab 5"
2'-fluoro Ribo -- 1 at 3'-end Usually AS "Stab 6" 2'-O-Methyl Ribo
5' and 3'-ends -- Usually S "Stab 7" 2'-fluoro 2'-deoxy 5' and
3'-ends -- Usually S "Stab 8" 2'-fluoro 2'-O-Methyl -- 1 at 3'-end
S or AS "Stab 9" Ribo Ribo 5' and 3'-ends -- Usually S "Stab 10"
Ribo Ribo -- 1 at 3'-end Usually AS "Stab 11" 2'-fluoro 2'-deoxy --
1 at 3'-end Usually AS Stab 12 2'-fluoro LNA 5' and 3'-ends Usually
S "Stab 13" 2'-fluoro LNA 1 at 3'-end Usually AS "Stab 14"
2'-fluoro 2'-deoxy 2 at 5'-end Usually AS 1 at 3'-end "Stab 15"
2'-deoxy 2'-deoxy 2 at 5'-end Usually AS 1 at 3'-end "Stab 16 Ribo
2'-O-Methyl 5' and 3'-ends Usually S "Stab 17" 2'-O-Methyl
2'-O-Methyl 5' and 3'-ends Usually S "Stab 18" 2'-fluoro
2'-O-Methyl 1 at 3'-end Usually AS CAP = any terminal cap, see for
example FIG. 10. All Stab 1-18 chemistries can comprise 3'-terminal
thymidine (TT) residues All Stab 1-18 chemistries typically
comprise 21 nucleotides, but can vary as described herein. S =
sense strand AS = antisense strand
TABLE-US-00005 TABLE V A. 2.5 .mu.mol Synthesis Cycle ABI 394
Instrument Reagent Equivalents Amount Wait Time* DNA Wait Time*
2'-O-methyl Wait Time*RNA Phosphoramidites 6.5 163 .mu.L 45 sec 2.5
min 7.5 min S-Ethyl Tetrazole 23.8 238 .mu.L 45 sec 2.5 min 7.5 min
Acetic Anhydride 100 233 .mu.L 5 sec 5 sec 5 sec N-Methyl 186 233
.mu.L 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL 21 sec 21 sec 21
sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage 12.9 645 .mu.L
100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B. 0.2
.mu.mol Synthesis Cycle ABI 394 Instrument Reagent Equivalents
Amount Wait Time* DNA Wait Time* 2'-O-methyl Wait Time*RNA
Phosphoramidites 15 31 .mu.L 45 sec 233 sec 465 sec S-Ethyl
Tetrazole 38.7 31 .mu.L 45 sec 233 min 465 sec Acetic Anhydride 655
124 .mu.L 5 sec 5 sec 5 sec N-Methyl 1245 124 .mu.L 5 sec 5 sec 5
sec Imidazole TCA 700 732 .mu.L 10 sec 10 sec 10 sec Iodine 20.6
244 .mu.L 15 sec 15 sec 15 sec Beaucage 7.7 232 .mu.L 100 sec 300
sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 .mu.mol
Synthesis Cycle 96 well Instrument Equivalents: DNA/ Amount:
DNA/2'-O- Wait Time* 2'-O- Reagent 2'-O-methyl/Ribo methyl/Ribo
Wait Time* DNA methyl Wait Time* Ribo Phosphoramidites 22/33/66
40/60/120 .mu.L 60 sec 180 sec 360 sec S-Ethyl Tetrazole 70/105/210
40/60/120 .mu.L 60 sec 180 min 360 sec Acetic Anhydride 265/265/265
50/50/50 .mu.L 10 sec 10 sec 10 sec N-Methyl 502/502/502 50/50/50
.mu.L 10 sec 10 sec 10 sec Imidazole TCA 238/475/475 250/500/500
.mu.L 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 .mu.L 30 sec
30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 sec
Acetonitrile NA 1150/1150/1150 .mu.L NA NA NA Wait time does not
include contact time during delivery. Tandem synthesis utilizes
double coupling of linker molecule
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090023676A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090023676A1).
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