U.S. patent application number 12/334224 was filed with the patent office on 2009-10-08 for rna interference mediated inhibition of platelet derived growth factor (pdgf) and platelet derived growth factor receptor (pdgfr) gene expression using short interfering nucleic acid (sina).
This patent application is currently assigned to Sirna Therapeutics, Inc.. Invention is credited to Leonid Beigelman, James McSwiggen.
Application Number | 20090253774 12/334224 |
Document ID | / |
Family ID | 46332079 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090253774 |
Kind Code |
A1 |
McSwiggen; James ; et
al. |
October 8, 2009 |
RNA INTERFERENCE MEDIATED INHIBITION OF PLATELET DERIVED GROWTH
FACTOR (PDGF) AND PLATELET DERIVED GROWTH FACTOR RECEPTOR (PDGFR)
GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
Abstract
This invention relates to compounds, compositions, and methods
useful for modulating platelet derived growth factor (PDGF) and/or
platelet derived growth factor receptor (PDGFr) gene expression
using short interfering nucleic acid (siNA) molecules. This
invention also relates to compounds, compositions, and methods
useful for modulating the expression and activity of other genes
involved in pathways of platelet derived growth factor (PDGF)
and/or platelet derived growth factor receptor (PDGFr) gene
expression and/or activity by RNA interference (RNAi) using small
nucleic acid 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 platelet
derived growth factor (PDGF) and/or platelet derived growth factor
receptor (PDGFr) genes, such as PDGF and/or PDGFr.
Inventors: |
McSwiggen; James; (Boulder,
CO) ; Beigelman; Leonid; (San Mateo, CA) |
Correspondence
Address: |
Sirna Therapeutics, Inc.
1700 Owens Street, 4th Floor
San Francisco
CA
94158
US
|
Assignee: |
Sirna Therapeutics, Inc.
San Francisco
CA
|
Family ID: |
46332079 |
Appl. No.: |
12/334224 |
Filed: |
December 12, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10923270 |
Aug 20, 2004 |
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12334224 |
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PCT/US03/03473 |
Feb 5, 2003 |
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10923270 |
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PCT/US04/16390 |
May 24, 2004 |
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PCT/US03/03473 |
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10826966 |
Apr 16, 2004 |
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PCT/US04/16390 |
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10757803 |
Jan 14, 2004 |
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10826966 |
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10720448 |
Nov 24, 2003 |
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10757803 |
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10693059 |
Oct 23, 2003 |
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10720448 |
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10444853 |
May 23, 2003 |
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10693059 |
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PCT/US03/05346 |
Feb 20, 2003 |
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10444853 |
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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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60358580 |
Feb 20, 2002 |
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60363124 |
Mar 11, 2002 |
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60363124 |
Mar 11, 2002 |
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60386782 |
Jun 6, 2002 |
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60386782 |
Jun 6, 2002 |
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60406784 |
Aug 29, 2002 |
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60406784 |
Aug 29, 2002 |
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60408378 |
Sep 5, 2002 |
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60408378 |
Sep 5, 2002 |
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60409293 |
Sep 9, 2002 |
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60409293 |
Sep 9, 2002 |
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60440129 |
Jan 15, 2003 |
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60440129 |
Jan 15, 2003 |
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60543480 |
Feb 10, 2004 |
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Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
C12N 2310/14 20130101;
C07H 21/02 20130101; C12N 15/1138 20130101; C12N 15/1136 20130101;
A61P 35/00 20180101; A61P 13/12 20180101 |
Class at
Publication: |
514/44.A ;
536/24.5 |
International
Class: |
A61K 31/7105 20060101
A61K031/7105; 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 Platelet Derived Growth Factor Receptor
(PDGFr) RNA sequence comprising SEQ ID NO: 749; (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 PDGFr 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. 10/923,270, filed Aug. 20, 2004, which is a
continuation-in-part of International Patent Application No.
PCT/US03/03473, filed Feb. 5, 2003. The parent application Ser. No.
10/923,270 is also a continuation-in-part of International Patent
Application No. PCT/US04/16390, filed May 24, 2004, which is a
continuation-in-part of U.S. patent application Ser. No.
10/826,966, filed Apr. 16, 2004 (now abandoned), which is
continuation-in-part of U.S. patent application Ser. No.
10/757,803, filed Jan. 14, 2004, which is a continuation-in-part of
U.S. patent application Ser. No. 10/720,448, filed Nov. 24, 2003
(now abandoned), which is a continuation-in-part of U.S. patent
application Ser. No. 10/693,059, filed Oct. 23, 2003, which is a
continuation-in-part of U.S. patent application Ser. No.
10/444,853, filed May 23, 2003, which is a continuation-in-part of
International Patent Application No. PCT/US03/05346, filed Feb. 20,
2003, and a continuation-in-part of International Patent
Application No. PCT/US03/05028, filed Feb. 20, 2003, both of which
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 parent application Ser. No. 10/923,270
also claims the benefit of U.S. Provisional Application No.
60/543,480, filed Feb. 10, 2004. 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
"SequenceListing46USCNT", created on Dec. 12, 2008, which is
182,897 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to compounds, compositions,
and methods for the study, diagnosis, and treatment of traits,
diseases and conditions that respond to the modulation of platelet
derived growth factor (PDGF) and platelet derived growth factor
receptor (PDGFr) gene expression and/or activity. The present
invention is also directed to compounds, compositions, and methods
relating to traits, diseases and conditions that respond to the
modulation of expression and/or activity of genes involved in
platelet derived growth factor (PDGF) and platelet derived growth
factor receptor (PDGFr) gene expression pathways or other cellular
processes that mediate the maintenance or development of such
traits, diseases and conditions. 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
platelet derived growth factor (PDGF) and/or platelet derived
growth factor receptor (PDGFr), such as PDGF and/or PDGFr gene
expression. Such small nucleic acid molecules are useful, for
example, in providing compositions for treatment of traits,
diseases and conditions that can respond to modulation of PDGF
and/or PDGFr expression in a subject, such as cancer, leukemia,
obliterative bronchiolitis, acute glomerulonephritis, stroke (CVA),
and/or inflammatory and proliferative traits, diseases, disorders,
or conditions.
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) (Zamore et al., 2000, Cell, 101, 25-33;
Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999,
Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129;
Sharp, 1999, Genes & Dev., 13:139-141; and Strauss, 1999,
Science, 286, 886). The corresponding process in plants (Heifetz et
al., International PCT Publication No. WO 99/61631) 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 through a mechanism that has yet to be fully
characterized. This mechanism appears to be different from other
known mechanisms involving double stranded RNA-specific
ribonucleases, such as 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 (see for example U.S. Pat. Nos.
6,107,094; 5,898,031; Clemens et al., 1997, J. Interferon &
Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8,
1189).
[0006] The presence of long dsRNAs in cells stimulates the activity
of a ribonuclease III enzyme referred to as dicer (Bass, 2000,
Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et
al., 2000, Nature, 404, 293). Dicer is involved in the processing
of the dsRNA into short pieces of dsRNA known as short interfering
RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000,
Cell, 101, 235; 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 (Zamore et al., 2000, Cell, 101, 25-33; 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. Bahramian and Zarbl, 1999, Molecular and Cellular Biology,
19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70,
describe RNAi mediated by dsRNA in mammalian systems. Hammond et
al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells
transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494 and
Tuschl et al., International PCT Publication No. WO 01/75164,
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 and
Tuschl et al., International PCT Publication No. WO 01/75164) 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 and Tuschl et al., International PCT
Publication No. WO 01/75164). 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 dsRNA molecules.
[0009] Parrish et al., 2000, Molecular Cell, 6, 1077-1087, 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 long (141
bp-488 bp) enzymatically synthesized or vector expressed 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 long (550 bp-714 bp),
enzymatically synthesized or vector expressed dsRNA molecules. Fire
et al., International PCT Publication No. WO 99/32619, describe
particular methods for introducing certain long dsRNA molecules
into cells for use in inhibiting gene expression in nematodes.
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 long
dsRNA molecules. Mello et al., International PCT Publication No. WO
01/29058, describe the identification of specific genes involved in
dsRNA-mediated RNAi. Pachuck et al., International PCT Publication
No. WO 00/63364, describe certain long (at least 200 nucleotide)
dsRNA constructs. 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 and 1998, PNAS, 95, 13959-13964, 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 expression
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,
1077-1087, describe specific chemically-modified dsRNA 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 long (over 250 bp), vector expressed 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 dsRNA. 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 (299 bp-1033 bp)
constructs that mediate RNAi. Martinez et al., 2002, Cell, 110,
563-574, describe certain single stranded siRNA constructs,
including certain 5'-phosphorylated single stranded siRNAs that
mediate RNA interference in Hela cells. Harborth et al., 2003,
Antisense & Nucleic Acid Drug Development, 13, 83-105, describe
certain chemically and structurally modified siRNA molecules. Chiu
and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically and
structurally modified siRNA molecules. Woolf et al., International
PCT Publication Nos. WO 03/064626 and WO 03/064625 describe certain
chemically modified dsRNA constructs.
SUMMARY OF THE INVENTION
[0012] This invention relates to compounds, compositions, and
methods useful for modulating platelet derived growth factor (PDGF)
and platelet derived growth factor receptor (PDGFr) gene expression
using short interfering nucleic acid (siNA) molecules. This
invention also relates to compounds, compositions, and methods
useful for modulating the expression and activity of other genes
involved in pathways of platelet derived growth factor (PDGF) and
platelet derived growth factor receptor (PDGFr) gene expression
and/or activity by RNA interference (RNAi) using small nucleic acid
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 platelet derived growth
factor (PDGF) and/or platelet derived growth factor receptor
(PDGFr) genes.
[0013] 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 PDGF and/or PDGFr 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, veterinary, diagnostic, target validation, genomic
discovery, genetic engineering, and pharmacogenomic
applications.
[0014] In one embodiment, the invention features one or more siNA
molecules and methods that independently or in combination modulate
the expression of PDGF and/or PDGFr genes encoding proteins, such
as proteins comprising PDGF and/or PDGFr associated with the
maintenance and/or development of cancer, leukemia, obliterative
bronchiolitis, acute glomerulonephritis, stroke (CVA), and/or
inflammatory and proliferative diseases, traits, conditions and/or
disorders, such as genes encoding sequences comprising those
sequences referred to by GenBank Accession Nos. shown in Table I,
referred to herein generally as PDGF and/or PDGFr. The description
below of the various aspects and embodiments of the invention is
provided with reference to exemplary PDGF and PDGFr genes referred
to herein as PDGF and PDGFr respectively. However, the various
aspects and embodiments are also directed to other PDGF and PDGFr
genes, such as homolog genes and transcript variants, and
polymorphisms (e.g., single nucleotide polymorphism, (SNPs))
associated with certain PDGF and PDGFr genes. As such, the various
aspects and embodiments are also directed to other genes that are
involved in PDGF and PDGFr mediated pathways of signal transduction
or gene expression that are involved, for example, in the
maintenance or development of diseases, traits, or conditions
described herein. These additional genes can be analyzed for target
sites using the methods described for PDGF and PDGFr genes herein.
Thus, the modulation of other genes and the effects of such
modulation of the other genes can be performed, determined, and
measured as described herein.
[0015] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a PDGFr gene, wherein said siNA molecule comprises
about 15 to about 28 base pairs.
[0016] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a PDGFr gene, wherein said siNA molecule comprises
about 15 to about 28 base pairs.
[0017] In one embodiment, the invention features a double stranded
short interfering nucleic acid (siNA) molecule that directs
cleavage of a PDGF and/or PDGFr RNA via RNA interference (RNAi),
wherein the double stranded siNA molecule comprises a first and a
second strand, each strand of the siNA molecule is about 15 to
about 30 nucleotides in length, the first strand of the siNA
molecule comprises nucleotide sequence having sufficient
complementarity to the PDGF and/or PDGFr RNA for the siNA molecule
to direct cleavage of the PDGF and/or PDGFr RNA via RNA
interference, and the second strand of said siNA molecule comprises
nucleotide sequence that is complementary to the first strand.
[0018] In one embodiment, the invention features a double stranded
short interfering nucleic acid (siNA) molecule that directs
cleavage of a PDGF and/or PDGFr RNA via RNA interference (RNAi),
wherein the double stranded siNA molecule comprises a first and a
second strand, each strand of the siNA molecule is about 18 to
about 23 nucleotides in length, the first strand of the siNA
molecule comprises nucleotide sequence having sufficient
complementarity to the PDGF and/or PDGFr RNA for the siNA molecule
to direct cleavage of the PDGF and/or PDGFr RNA via RNA
interference, and the second strand of said siNA molecule comprises
nucleotide sequence that is complementary to the first strand.
[0019] In one embodiment, the invention features a chemically
synthesized double stranded short interfering nucleic acid (siNA)
molecule that directs cleavage of a PDGF and/or PDGFr RNA via RNA
interference (RNAi), wherein each strand of the siNA molecule is
about 18 to about 28 nucleotides in length; and one strand of the
siNA molecule comprises nucleotide sequence having sufficient
complementarity to the PDGF and/or PDGFr RNA for the siNA molecule
to direct cleavage of the PDGF and/or PDGFr RNA via RNA
interference.
[0020] In one embodiment, the invention features a chemically
synthesized double stranded short interfering nucleic acid (siNA)
molecule that directs cleavage of a PDGF and/or PDGFr RNA via RNA
interference (RNAi), wherein each strand of the siNA molecule is
about 18 to about 23 nucleotides in length; and one strand of the
siNA molecule comprises nucleotide sequence having sufficient
complementarity to the PDGF and/or PDGFr RNA for the siNA molecule
to direct cleavage of the PDGF and/or PDGFr RNA via RNA
interference.
[0021] In one embodiment, the invention features a siNA molecule
that down-regulates expression of a PDGF gene, for example, wherein
the PDGF gene comprises PDGF encoding sequence. In one embodiment,
the invention features a siNA molecule that down-regulates
expression of a PDGF gene, for example, wherein the PDGF gene
comprises PDGF non-coding sequence or regulatory elements involved
in PDGF gene expression.
[0022] In one embodiment, the invention features a siNA molecule
that down-regulates expression of a PDGFr gene, for example,
wherein the PDGFr gene comprises PDGFr encoding sequence. In one
embodiment, the invention features a siNA molecule that
down-regulates expression of a PDGFr gene, for example, wherein the
PDGFr gene comprises PDGFr non-coding sequence or regulatory
elements involved in PDGFr gene expression.
[0023] In one embodiment, a siNA of the invention is used to
inhibit the expression of PDGF and/or PDGFr genes or a PDGF and/or
PDGFr gene family wherein the genes or gene family sequences share
sequence homology. Such homologous sequences can be identified as
is known in the art, for example using sequence alignments. siNA
molecules can be designed to target such homologous sequences, for
example using perfectly complementary sequences or by incorporating
non-canonical base pairs, for example mismatches and/or wobble base
pairs, that can provide additional target sequences. In instances
where mismatches are identified, non-canonical base pairs (for
example, mismatches and/or wobble bases) can be used to generate
siNA molecules that target more than one gene sequence. In a
non-limiting example, non-canonical base pairs such as UU and CC
base pairs are used to generate siNA molecules that are capable of
targeting sequences for differing PDGF and/or PDGFr targets that
share sequence homology. As such, one advantage of using siNAs of
the invention is that a single siNA can be designed to include
nucleic acid sequence that is complementary to the nucleotide
sequence that is conserved between the homologous genes. In this
approach, a single siNA can be used to inhibit expression of more
than one gene instead of using more than one siNA molecule to
target the different genes.
[0024] In one embodiment, the invention features a siNA molecule
having RNAi activity against PDGF RNA, wherein the siNA molecule
comprises a sequence complementary to any RNA having PDGF encoding
sequence, such as those sequences having GenBank Accession Nos.
shown in Table I. In another embodiment, the invention features a
siNA molecule having RNAi activity against PDGF RNA, wherein the
siNA molecule comprises a sequence complementary to an RNA having
variant PDGF encoding sequence, for example other mutant PDGF genes
not shown in Table I but known in the art to be associated with the
maintenance and/or development of cancer, leukemia, obliterative
bronchiolitis, acute glomerulonephritis, stroke (CVA), and/or
inflammatory and proliferative diseases, traits, conditions and/or
disorders. Chemical modifications as shown in Tables III and IV or
otherwise described herein can be applied to any siNA construct of
the invention. In another embodiment, a siNA molecule of the
invention includes a nucleotide sequence that can interact with
nucleotide sequence of a PDGF gene and thereby mediate silencing of
PDGF gene expression, for example, wherein the siNA mediates
regulation of PDGF gene expression by cellular processes that
modulate the chromatin structure or methylation patterns of the
PDGF gene and prevent transcription of the PDGF gene.
[0025] In one embodiment, the invention features a siNA molecule
having RNAi activity against PDGFr RNA, wherein the siNA molecule
comprises a sequence complementary to any RNA having PDGFr encoding
sequence, such as those sequences having GenBank Accession Nos.
shown in Table I. In another embodiment, the invention features a
siNA molecule having RNAi activity against PDGFr RNA, wherein the
siNA molecule comprises a sequence complementary to an RNA having
variant PDGFr encoding sequence, for example other mutant PDGFr
genes not shown in Table I but known in the art to be associated
with the maintenance and/or development of cancer, leukemia,
obliterative bronchiolitis, acute glomerulonephritis, stroke (CVA),
and/or inflammatory and proliferative diseases, traits, conditions
and/or disorders. Chemical modifications as shown in Tables III and
IV or otherwise described herein can be applied to any siNA
construct of the invention. In another embodiment, a siNA molecule
of the invention includes a nucleotide sequence that can interact
with nucleotide sequence of a PDGFr gene and thereby mediate
silencing of PDGFr gene expression, for example, wherein the siNA
mediates regulation of PDGFr gene expression by cellular processes
that modulate the chromatin structure or methylation patterns of
the PDGFr gene and prevent transcription of the PDGFr gene.
[0026] In one embodiment, siNA molecules of the invention are used
to down regulate or inhibit the expression of PDGF and/or PDGFr
proteins arising from PDGF and/or PDGFr haplotype polymorphisms
that are associated with a disease or condition, (e.g., cancer,
leukemia, obliterative bronchiolitis, acute glomerulonephritis,
stroke (CVA), and/or inflammatory and proliferative traits,
diseases, disorders, and/or conditions). Analysis of PDGF and/or
PDGFr genes, or PDGF and/or PDGFr protein or RNA levels can be used
to identify subjects with such polymorphisms or those subjects who
are at risk of developing traits, conditions, or diseases described
herein. These subjects are amenable to treatment, for example,
treatment with siNA molecules of the invention and any other
composition useful in treating diseases related to PDGF and/or
PDGFr gene expression. As such, analysis of PDGF and/or PDGFr
protein or RNA levels can be used to determine treatment type and
the course of therapy in treating a subject. Monitoring of PDGF
and/or PDGFr protein or RNA levels can be used to predict treatment
outcome and to determine the efficacy of compounds and compositions
that modulate the level and/or activity of certain PDGF and/or
PDGFr proteins associated with a trait, condition, or disease.
[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 or a portion thereof
encoding a PDGF and/or PDGFr protein. The siNA further comprises a
sense strand, wherein said sense strand comprises a nucleotide
sequence of a PDGF and/or PDGFr 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 PDGF and/or PDGFr
protein or a portion thereof. The siNA molecule further comprises a
sense region, wherein said sense region comprises a nucleotide
sequence of a PDGF and/or PDGFr gene or a portion thereof.
[0029] In another embodiment, the invention features a siNA
molecule comprising a nucleotide sequence in the antisense region
of the siNA molecule that is complementary to a nucleotide sequence
or portion of sequence of a PDGF and/or PDGFr 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 PDGF and/or PDGFr gene
sequence or a portion thereof.
[0030] In one embodiment, the antisense region of PDGF and/or PDGFr
siNA constructs comprises a sequence complementary to sequence
having any of SEQ ID NOs. 1-311 or 623-630. In one embodiment, the
antisense region of PDGF and/or PDGFr constructs comprises sequence
having any of SEQ ID NOs. 312-622, 639-646, 655-662, 671-678,
687-694, 703-726, 728, 730, 732, 735, 737, 739, 741, or 744. In
another embodiment, the sense region of PDGF and/or PDGFr
constructs comprises sequence having any of SEQ ID NOs. 1-311,
623-638, 647-654, 663-670, 679-686, 695-702, 727, 729, 731, 733,
734, 736, 738, 740, 742, or 743.
[0031] In one embodiment, a siNA molecule of the invention
comprises any of SEQ ID NOs. 1-744. The sequences shown in SEQ ID
NOs: 1-744 are not limiting. A siNA molecule of the invention can
comprise any contiguous PDGF and/or PDGFr sequence (e.g., about 15
to about 25 or more, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 or more contiguous PDGF and/or PDGFr nucleotides).
[0032] 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 siNA construct of the
invention.
[0033] In one embodiment of the invention a siNA molecule comprises
an antisense strand having about 15 to about 30 (e.g., about 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides, wherein the antisense strand is complementary to a RNA
sequence or a portion thereof encoding a PDGF and/or PDGFr protein,
and wherein said siNA further comprises a sense strand having about
15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, or 30) nucleotides, and wherein said sense
strand and said antisense strand are distinct nucleotide sequences
where at least about 15 nucleotides in each strand are
complementary to the other strand.
[0034] In another embodiment of the invention a siNA molecule of
the invention comprises an antisense region having about 15 to
about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30) nucleotides, wherein the antisense region is
complementary to a RNA sequence encoding a PDGF and/or PDGFr
protein, and wherein said siNA further comprises a sense region
having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein
said sense region and said antisense region are comprised in a
linear molecule where the sense region comprises at least about 15
nucleotides that are complementary to the antisense region.
[0035] In one embodiment, a siNA molecule of the invention has RNAi
activity that modulates expression of RNA encoded by a PDGF and/or
PDGFr gene. Because PDGF genes (e.g., PDGF superfamily) and PDGFr
(e.g., PDGFr superfamily) genes can share some degree of sequence
homology with each other, siNA molecules can be designed to target
a class of PDGF or PDGFr genes or alternately specific PDGF or
PDGFr genes (e.g., polymorphic variants) by selecting sequences
that are either shared amongst different PDGF or PDGFr targets or
alternatively that are unique for a specific PDGF or PDGFr target.
Therefore, in one embodiment, the siNA molecule can be designed to
target conserved regions of PDGF or PDGFr RNA sequences having
homology among several PDGF or PDGFr gene variants so as to target
a class of PDGF or PDGFr genes with one siNA molecule. Accordingly,
in one embodiment, the siNA molecule of the invention modulates the
expression of one or both PDGF or PDGFr alleles in a subject. In
another embodiment, the siNA molecule can be designed to target a
sequence that is unique to a specific PDGF or PDGFr RNA sequence
(e.g., a single PDGF or PDGFr allele or PDGF or PDGFr single
nucleotide polymorphism (SNP)) due to the high degree of
specificity that the siNA molecule requires to mediate RNAi
activity.
[0036] 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 duplex
nucleic acid molecules containing about 15 to about 30 base pairs
between oligonucleotides comprising about 15 to about 30 (e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30) nucleotides. In yet another embodiment, siNA molecules of
the invention comprise duplex nucleic acid molecules 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. In yet another embodiment, siNA molecules
of the invention comprise duplex nucleic acid molecules with blunt
ends, where both ends are blunt, or alternatively, where one of the
ends is blunt.
[0037] In one embodiment, the invention features one or more
chemically-modified siNA constructs having specificity for PDGF
and/or PDGFr expressing nucleic acid molecules, such as RNA
encoding a PDGF and/or PDGFr protein. In one embodiment, the
invention features a RNA based siNA molecule (e.g., a siNA
comprising 2'-OH nucleotides) having specificity for PDGF and/or
PDGFr expressing nucleic acid molecules that includes one or more
chemical modifications described herein. 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, (e.g., RNA based 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.
[0038] 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., about 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.
[0039] One aspect of the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that down-regulates
expression of a PDGF and/or PDGFr gene. In one embodiment, the
double stranded siNA molecule comprises one or more chemical
modifications and each strand of the double-stranded siNA is about
21 nucleotides long. In one embodiment, the double-stranded siNA
molecule does not contain any ribonucleotides. In another
embodiment, the double-stranded siNA molecule comprises one or more
ribonucleotides. In one embodiment, each strand of the
double-stranded siNA molecule independently comprises about 15 to
about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30) nucleotides, wherein each strand comprises
about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are
complementary to the nucleotides of the other strand. In one
embodiment, one of the strands of the double-stranded siNA molecule
comprises a nucleotide sequence that is complementary to a
nucleotide sequence or a portion thereof of the PDGF and/or PDGFr
gene, and the second strand of the double-stranded siNA molecule
comprises a nucleotide sequence substantially similar to the
nucleotide sequence of the PDGF and/or PDGFr gene or a portion
thereof.
[0040] In another embodiment, the invention features a
double-stranded short interfering nucleic acid (siNA) molecule that
down-regulates expression of a PDGF and/or PDGFr gene comprising an
antisense region, wherein the antisense region comprises a
nucleotide sequence that is complementary to a nucleotide sequence
of the PDGF and/or PDGFr gene or a portion thereof, and a sense
region, wherein the sense region comprises a nucleotide sequence
substantially similar to the nucleotide sequence of the PDGF and/or
PDGFr gene or a portion thereof. In one embodiment, the antisense
region and the sense region independently comprise about 15 to
about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30) nucleotides, wherein the antisense region
comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are
complementary to nucleotides of the sense region.
[0041] In another embodiment, the invention features a
double-stranded short interfering nucleic acid (siNA) molecule that
down-regulates expression of a PDGF and/or PDGFr gene comprising a
sense region and an antisense region, wherein the antisense region
comprises a nucleotide sequence that is complementary to a
nucleotide sequence of RNA encoded by the PDGF and/or PDGFr gene or
a portion thereof and the sense region comprises a nucleotide
sequence that is complementary to the antisense region.
[0042] In one embodiment, a siNA molecule of the invention
comprises blunt ends, i.e., ends that do not include any
overhanging nucleotides. For example, a siNA molecule comprising
modifications described herein (e.g., comprising nucleotides having
Formulae I-VII or siNA constructs comprising "Stab 00"-"Stab 32"
(Table. IV) or any combination thereof (see Table IV)) and/or any
length described herein can comprise blunt ends or ends with no
overhanging nucleotides.
[0043] In one embodiment, any siNA molecule of the invention can
comprise one or more blunt ends, i.e. where a blunt end does not
have any overhanging nucleotides. In one embodiment, the blunt
ended siNA molecule has a number of base pairs equal to the number
of nucleotides present in each strand of the siNA molecule. In
another embodiment, the siNA molecule comprises one blunt end, for
example wherein the 5'-end of the antisense strand and the 3'-end
of the sense strand do not have any overhanging nucleotides. In
another example, the siNA molecule comprises one blunt end, for
example wherein the 3'-end of the antisense strand and the 5'-end
of the sense strand do not have any overhanging nucleotides. In
another example, a siNA molecule comprises two blunt ends, for
example wherein the 3'-end of the antisense strand and the 5'-end
of the sense strand as well as the 5'-end of the antisense strand
and 3'-end of the sense strand do not have any overhanging
nucleotides. A blunt ended siNA molecule can comprise, for example,
from about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).
Other nucleotides present in a blunt ended siNA molecule can
comprise, for example, mismatches, bulges, loops, or wobble base
pairs to modulate the activity of the siNA molecule to mediate RNA
interference.
[0044] By "blunt ends" is meant symmetric termini or termini of a
double stranded siNA molecule having no overhanging nucleotides.
The two strands of a double stranded siNA molecule align with each
other without over-hanging nucleotides at the termini. For example,
a blunt ended siNA construct comprises terminal nucleotides that
are complementary between the sense and antisense regions of the
siNA molecule.
[0045] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a PDGF and/or PDGFr 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.
[0046] In one embodiment, the invention features double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a PDGF and/or PDGFr gene, wherein the siNA molecule
comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein
each strand of the siNA molecule comprises one or more chemical
modifications. In another embodiment, one of the strands of the
double-stranded siNA molecule comprises a nucleotide sequence that
is complementary to a nucleotide sequence of a PDGF and/or PDGFr
gene or a portion thereof, and the second strand of the
double-stranded siNA molecule comprises a nucleotide sequence
substantially similar to the nucleotide sequence or a portion
thereof of the PDGF and/or PDGFr gene. In another embodiment, one
of the strands of the double-stranded siNA molecule comprises a
nucleotide sequence that is complementary to a nucleotide sequence
of a PDGF and/or PDGFr gene or portion thereof, and the second
strand of the double-stranded siNA molecule comprises a nucleotide
sequence substantially similar to the nucleotide sequence or
portion thereof of the PDGF and/or PDGFr gene. In another
embodiment, each strand of the siNA molecule comprises about 15 to
about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30) nucleotides, and each strand comprises at
least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are
complementary to the nucleotides of the other strand. The PDGF
and/or PDGFr gene can comprise, for example, sequences referred to
in Table I.
[0047] In one embodiment, a siNA molecule of the invention
comprises no ribonucleotides. In another embodiment, a siNA
molecule of the invention comprises ribonucleotides.
[0048] In one embodiment, a siNA molecule of the invention
comprises an antisense region comprising a nucleotide sequence that
is complementary to a nucleotide sequence of a PDGF and/or PDGFr
gene or a portion thereof, and the siNA further comprises a sense
region comprising a nucleotide sequence substantially similar to
the nucleotide sequence of the PDGF and/or PDGFr gene or a portion
thereof. In another embodiment, the antisense region and the sense
region each comprise about 15 to about 30 (e.g. about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides
and the antisense region comprises at least about 15 to about 30
(e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, or 30) nucleotides that are complementary to nucleotides of the
sense region. The PDGF and/or PDGFr gene can comprise, for example,
sequences referred to in Table I. In another embodiment, the siNA
is a double stranded nucleic acid molecule, where each of the two
strands of the siNA molecule independently comprise about 15 to
about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40)
nucleotides, and where one of the strands of the siNA molecule
comprises at least about 15 (e.g. about 15, 16, 17, 18, 19, 20, 21,
22, 23, 24 or 25 or more) nucleotides that are complementary to the
nucleic acid sequence of the PDGF and/or PDGFr gene or a portion
thereof.
[0049] In one embodiment, a siNA molecule of the invention
comprises a sense region and an antisense region, wherein the
antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence of RNA encoded by a PDGF
and/or PDGFr gene, or a portion thereof, and the sense region
comprises a nucleotide sequence that is complementary to the
antisense region. In one embodiment, 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. In another embodiment, the
sense region is connected to the antisense region via a linker
molecule. In another embodiment, the sense region is connected to
the antisense region via a linker molecule, such as a nucleotide or
non-nucleotide linker. The PDGF and/or PDGFr gene can comprise, for
example, sequences referred in to Table I.
[0050] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a PDGF and/or PDGFr gene comprising a sense region
and an antisense region, wherein the antisense region comprises a
nucleotide sequence that is complementary to a nucleotide sequence
of RNA encoded by the PDGF and/or PDGFr gene or a portion thereof
and the sense region comprises a nucleotide sequence that is
complementary to the antisense region, and 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, 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'-deoxy purine nucleotides. In one embodiment, the pyrimidine
nucleotides in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides and the purine nucleotides present in the
antisense region are 2'-O-methyl or 2'-deoxy 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.
[0051] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a PDGF and/or PDGFr 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. 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 independently
comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In another
embodiment, each of the two fragments of the siNA molecule
independently comprise about 15 to about 40 (e.g. about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34,
35, 36, 37, 38, 39, or 40) nucleotides. In a non-limiting example,
each of the two fragments of the siNA molecule comprise about 21
nucleotides.
[0052] In one embodiment, the invention features a siNA molecule
comprising at least one modified nucleotide, wherein the modified
nucleotide is a 2'-deoxy-2'-fluoro nucleotide. The siNA can be, for
example, about 15 to about 40 nucleotides in length. In one
embodiment, all pyrimidine nucleotides present in the siNA are
2'-deoxy-2'-fluoro pyrimidine nucleotides. In one embodiment, the
modified nucleotides in the siNA include at least one
2'-deoxy-2'-fluoro cytidine or 2'-deoxy-2'-fluoro uridine
nucleotide. In another embodiment, the modified nucleotides in the
siNA include at least one 2'-fluoro cytidine and at least one
2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all
uridine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
uridine nucleotides. In one embodiment, all cytidine nucleotides
present in the siNA are 2'-deoxy-2'-fluoro cytidine nucleotides. In
one embodiment, all adenosine nucleotides present in the siNA are
2'-deoxy-2'-fluoro adenosine nucleotides. In one embodiment, all
guanosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
guanosine nucleotides. The siNA can further comprise at least one
modified internucleotidic linkage, such as phosphorothioate
linkage. In one embodiment, the 2'-deoxy-2'-fluoronucleotides are
present at specifically selected locations in the siNA that are
sensitive to cleavage by ribonucleases, such as locations having
pyrimidine nucleotides.
[0053] In one embodiment, the invention features a method of
increasing the stability of a siNA molecule against cleavage by
ribonucleases comprising introducing at least one modified
nucleotide into the siNA molecule, wherein the modified nucleotide
is a 2'-deoxy-2'-fluoro nucleotide. In one embodiment, all
pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
pyrimidine nucleotides. In one embodiment, the modified nucleotides
in the siNA include at least one 2'-deoxy-2'-fluoro cytidine or
2'-deoxy-2'-fluoro uridine nucleotide. In another embodiment, the
modified nucleotides in the siNA include at least one 2'-fluoro
cytidine and at least one 2'-deoxy-2'-fluoro uridine nucleotides.
In one embodiment, all uridine nucleotides present in the siNA are
2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all
cytidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
cytidine nucleotides. In one embodiment, all adenosine nucleotides
present in the siNA are 2'-deoxy-2'-fluoro adenosine
nucleotides.
[0054] In one embodiment, all guanosine nucleotides present in the
siNA are 2'-deoxy-2'-fluoro guanosine nucleotides. The siNA can
further comprise at least one modified internucleotidic linkage,
such as phosphorothioate linkage. In one embodiment, the
2'-deoxy-2'-fluoronucleotides are present at specifically selected
locations in the siNA that are sensitive to cleavage by
ribonucleases, such as locations having pyrimidine nucleotides.
[0055] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a PDGF and/or PDGFr gene comprising a sense region
and an antisense region, wherein the antisense region comprises a
nucleotide sequence that is complementary to a nucleotide sequence
of RNA encoded by the PDGF and/or PDGFr gene or a portion thereof
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 an alternative embodiment, the
purine nucleotides present in the antisense region comprise
2'-O-methyl purine nucleotides. In either of the above embodiments,
the antisense region can comprise a phosphorothioate
internucleotide linkage at the 3' end of the antisense region.
Alternatively, in either of the above embodiments, the antisense
region can comprise 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.
[0056] In one embodiment, the antisense region of a siNA molecule
of the invention comprises sequence complementary to a portion of a
PDGF and/or PDGFr transcript having sequence unique to a particular
PDGF and/or PDGFr disease related allele, such as sequence
comprising a single nucleotide polymorphism (SNP) associated with
the disease specific allele. As such, the antisense region of a
siNA molecule of the invention can comprise sequence complementary
to sequences that are unique to a particular allele to provide
specificity in mediating selective RNAi against the disease,
condition, or trait related allele.
[0057] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a PDGF and/or PDGFr 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. In another
embodiment, the siNA molecule is a double stranded nucleic acid
molecule, where each strand is about 21 nucleotides long and where
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, 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 another
embodiment, the siNA molecule is a double stranded nucleic acid
molecule, where each strand is about 19 nucleotide long and where
the nucleotides of each fragment of the siNA molecule are
base-paired to the complementary nucleotides of the other fragment
of the siNA molecule to form at least about 15 (e.g., 15, 16, 17,
18, or 19) base pairs, wherein one or both ends of the siNA
molecule are blunt ends. In one embodiment, each of the two 3'
terminal nucleotides of each fragment of the siNA molecule is a
2'-deoxy-pyrimidine nucleotide, such as a 2'-deoxy-thymidine. In
another embodiment, all 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, the
siNA molecule is a double stranded nucleic acid molecule of about
19 to about 25 base pairs having a sense region and an antisense
region, where about 19 nucleotides of the antisense region are
base-paired to the nucleotide sequence or a portion thereof of the
RNA encoded by the PDGF and/or PDGFr gene. In another embodiment,
about 21 nucleotides of the antisense region are base-paired to the
nucleotide sequence or a portion thereof of the RNA encoded by the
PDGF and/or PDGFr gene. In any of the above embodiments, the 5'-end
of the fragment comprising said antisense region can optionally
include a phosphate group.
[0058] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits the
expression of a PDGF and/or PDGFr RNA sequence (e.g., wherein said
target RNA sequence is encoded by a PDGF and/or PDGFr gene involved
in the PDGF and/or PDGFr pathway), wherein the siNA molecule does
not contain any ribonucleotides and wherein each strand of the
double-stranded siNA molecule is about 15 to about 30 nucleotides.
In one embodiment, the siNA molecule is 21 nucleotides in length.
Examples of non-ribonucleotide containing siNA constructs are
combinations of stabilization chemistries shown in Table IV in any
combination of Sense/Antisense chemistries, such as Stab 7/8, Stab
7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab
18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab
18/20, Stab 7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having
Stab 7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense or
antisense strands or any combination thereof).
[0059] In one embodiment, the invention features a chemically
synthesized double stranded RNA molecule that directs cleavage of a
PDGF and/or PDGFr RNA via RNA interference, wherein each strand of
said RNA molecule is about 15 to about 30 nucleotides in length;
one strand of the RNA molecule comprises nucleotide sequence having
sufficient complementarity to the PDGF and/or PDGFr RNA for the RNA
molecule to direct cleavage of the PDGF and/or PDGFr RNA via RNA
interference; and wherein at least one strand of the RNA molecule
optionally comprises one or more chemically modified nucleotides
described herein, such as without limitation deoxynucleotides,
2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides,
2'-O-methoxyethyl nucleotides etc.
[0060] In one embodiment, the invention features a medicament
comprising a siNA molecule of the invention.
[0061] In one embodiment, the invention features an active
ingredient comprising a siNA molecule of the invention.
[0062] In one embodiment, the invention features the use of a
double-stranded short interfering nucleic acid (siNA) molecule to
inhibit, down-regulate, or reduce expression of a PDGF and/or PDGFr
gene, wherein the siNA molecule comprises one or more chemical
modifications and each strand of the double-stranded siNA is
independently about 15 to about 30 or more (e.g., about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more)
nucleotides long. In one embodiment, the siNA molecule of the
invention is a double stranded nucleic acid molecule comprising one
or more chemical modifications, where each of the two fragments of
the siNA molecule independently comprise about 15 to about 40 (e.g.
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and
where one of the strands comprises at least 15 nucleotides that are
complementary to nucleotide sequence of PDGF and/or PDGFr encoding
RNA or a portion thereof. In a non-limiting example, each of the
two fragments of the siNA molecule comprise about 21 nucleotides.
In another embodiment, the siNA molecule is a double stranded
nucleic acid molecule comprising one or more chemical
modifications, where each strand is about 21 nucleotide long and
where 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, 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 another embodiment, the siNA molecule is a double
stranded nucleic acid molecule comprising one or more chemical
modifications, where each strand is about 19 nucleotide long and
where the nucleotides of each fragment of the siNA molecule are
base-paired to the complementary nucleotides of the other fragment
of the siNA molecule to form at least about 15 (e.g., 15, 16, 17,
18, or 19) base pairs, wherein one or both ends of the siNA
molecule are blunt ends. In one embodiment, each of the two 3'
terminal nucleotides of each fragment of the siNA molecule is a
2'-deoxy-pyrimidine nucleotide, such as a 2'-deoxy-thymidine. In
another embodiment, all 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, the
siNA molecule is a double stranded nucleic acid molecule of about
19 to about 25 base pairs having a sense region and an antisense
region and comprising one or more chemical modifications, where
about 19 nucleotides of the antisense region are base-paired to the
nucleotide sequence or a portion thereof of the RNA encoded by the
PDGF and/or PDGFr gene. In another embodiment, about 21 nucleotides
of the antisense region are base-paired to the nucleotide sequence
or a portion thereof of the RNA encoded by the PDGF and/or PDGFr
gene. In any of the above embodiments, the 5'-end of the fragment
comprising said antisense region can optionally include a phosphate
group.
[0063] In one embodiment, the invention features the use of a
double-stranded short interfering nucleic acid (siNA) molecule that
inhibits, down-regulates, or reduces expression of a PDGF and/or
PDGFr 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 PDGF and/or PDGFr
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.
[0064] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits,
down-regulates, or reduces expression of a PDGF and/or PDGFr 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 PDGF and/or PDGFr RNA or a
portion thereof, wherein 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.
[0065] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits,
down-regulates, or reduces expression of a PDGF and/or PDGFr 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 PDGF and/or PDGFr RNA that
encodes a protein or 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, each strand of the siNA molecule comprises about 15
to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides, wherein
each strand comprises at least about 15 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 one 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 a further 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 still another 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'-deoxy purine nucleotides. In another
embodiment, the antisense strand comprises one or more
2'-deoxy-2'-fluoro pyrimidine nucleotides and one or more
2'-O-methyl purine nucleotides. In another 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 a further embodiment the sense strand comprises a
3'-end and a 5'-end, wherein a terminal cap moiety (e.g., an
inverted deoxy abasic moiety or inverted deoxy nucleotide moiety
such as inverted thymidine) is present at the 5'-end, the 3'-end,
or both of the 5' and 3' ends of the sense strand. In another
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.
[0066] In any of the above-described embodiments of a
double-stranded short interfering nucleic acid (siNA) molecule that
inhibits expression of a PDGF and/or PDGFr gene, wherein a majority
of the pyrimidine nucleotides present in the double-stranded siNA
molecule comprises a sugar modification, each of the two strands of
the siNA molecule can comprise about 15 to about 30 or more (e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30 or more) nucleotides. In one embodiment, about 15 to about 30
or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30 or more) nucleotides of each strand of the
siNA molecule are base-paired to the complementary nucleotides of
the other strand of the siNA molecule. In another embodiment, about
15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each
strand of the siNA molecule are base-paired to the complementary
nucleotides of the other strand of the siNA molecule, wherein 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 another 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 one 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 15 to about 30 (e.g., about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides
of the antisense strand are base-paired to the nucleotide sequence
of the PDGF and/or PDGFr RNA or a portion thereof. In one
embodiment, about 18 to about 25 (e.g., about 18, 19, 20, 21, 22,
23, 24, or 25) nucleotides of the antisense strand are base-paired
to the nucleotide sequence of the PDGF and/or PDGFr RNA or a
portion thereof.
[0067] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits
expression of a PDGF and/or PDGFr 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 PDGF and/or PDGFr 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 5'-end of the antisense strand optionally includes a
phosphate group.
[0068] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits
expression of a PDGF and/or PDGFr 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 PDGF and/or PDGFr 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 or a portion thereof of the
antisense strand is complementary to a nucleotide sequence of the
untranslated region or a portion thereof of the PDGF and/or PDGFr
RNA.
[0069] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits
expression of a PDGF and/or PDGFr 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 PDGF and/or PDGFr RNA or a portion thereof, wherein the
other strand is a sense strand which comprises nucleotide sequence
that is complementary to a nucleotide sequence of the antisense
strand, 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 PDGF and/or PDGFr RNA
or a portion thereof that is present in the PDGF and/or PDGFr
RNA.
[0070] In one embodiment, the invention features a composition
comprising a siNA molecule of the invention in a pharmaceutically
acceptable carrier or diluent.
[0071] 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.
[0072] 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.
[0073] 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
a RNA or DNA sequence encoding PDGF and/or PDGFr 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.
[0074] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against PDGF and/or
PDGFr 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, aralkyl, or
acetyl and wherein W, X, Y, and Z are optionally not all O. In
another embodiment, a backbone modification of the invention
comprises a phosphonoacetate and/or thiophosphonoacetate
internucleotide linkage (see for example Sheehan et al., 2003,
Nucleic Acids Research, 31, 4109-4118).
[0075] 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.
[0076] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against PDGF and/or
PDGFr 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##
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-SH, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl,
alkyl-O-alkyl, ONO2, NO.sub.2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I or
II; 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.
[0077] 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 nucleotides or
non-nucleotides 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 anther 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.
[0078] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against PDGF and/or
PDGFr 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-SH, 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 or
II; 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.
[0079] 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 nucleotides or
non-nucleotides 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 anther 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.
[0080] 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.
[0081] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against PDGF and/or
PDGFr 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;
[0082] wherein each Z and W is independently O, S, N, alkyl,
substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo,
or acetyl; and wherein W, X, Y and Z are not all O.
[0083] 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.
[0084] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against PDGF and/or
PDGFr 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.
[0085] 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.
[0086] 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.
[0087] 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, 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.
[0088] 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.
[0089] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
having about 1 to about 5 or more (specifically about 1, 2, 3, 4, 5
or more) phosphorothioate internucleotide linkages in each strand
of the siNA molecule.
[0090] 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.
[0091] 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
independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in
length, wherein the duplex has about 15 to about 30 (e.g., about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
base pairs, 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 15 to about 30
(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30) base pairs, 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 to about
21 (e.g., 19, 20, or 21) 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.
[0092] In another embodiment, a siNA molecule of the invention
comprises a hairpin structure, wherein the siNA is about 25 to
about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50)
nucleotides in length having about 3 to about 25 (e.g., about 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25) base pairs, and wherein the siNA can include one or
more chemical modifications 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 25 to about 35
(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)
nucleotides that is chemically-modified with one or more chemical
modifications having any of Formulae I-VII or any combination
thereof, wherein the linear oligonucleotide forms a hairpin
structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or
25) base pairs and a 5'-terminal phosphate group that can be
chemically modified as described herein (for example a 5'-terminal
phosphate group having Formula IV). 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. In
one embodiment, a linear hairpin siNA molecule of the invention
comprises a loop portion comprising a non-nucleotide linker.
[0093] In another embodiment, a siNA molecule of the invention
comprises an asymmetric hairpin structure, wherein the siNA is
about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
or 50) nucleotides in length having about 3 to about 25 (e.g.,
about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can
include one or more chemical modifications 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 25 to
about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or
35) nucleotides that is chemically-modified with one or more
chemical modifications having any of Formulae I-VII or any
combination thereof, wherein the linear oligonucleotide forms an
asymmetric hairpin structure having about 3 to about 25 (e.g.,
about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25) base pairs and a 5'-terminal phosphate
group that can be chemically modified as described herein (for
example a 5'-terminal phosphate group having Formula IV). In one
embodiment, an asymmetric hairpin siNA molecule of the invention
contains a stem loop motif, wherein the loop portion of the siNA
molecule is biodegradable. In another embodiment, an asymmetric
hairpin siNA molecule of the invention comprises a loop portion
comprising a non-nucleotide linker.
[0094] In another embodiment, a siNA molecule of the invention
comprises an asymmetric double stranded structure having separate
polynucleotide strands comprising sense and antisense regions,
wherein the antisense region is about 15 to about 30 (e.g., about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides in length, wherein the sense region is about 3 to about
25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length,
wherein the sense region and the antisense region have at least 3
complementary nucleotides, and wherein the siNA can include one or
more chemical modifications 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 an asymmetric double stranded structure having separate
polynucleotide strands comprising sense and antisense regions,
wherein the antisense region is about 18 to about 23 (e.g., about
18, 19, 20, 21, 22, or 23) nucleotides in length and wherein the
sense region is about 3 to about 15 (e.g., about 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15) nucleotides in length, wherein the
sense region the antisense region have at least 3 complementary
nucleotides, and wherein the siNA can include one or more chemical
modifications comprising a structure having any of Formulae I-VII
or any combination thereof. In another embodiment, the asymmetric
double stranded siNA molecule can also have a 5'-terminal phosphate
group that can be chemically modified as described herein (for
example a 5'-terminal phosphate group having Formula IV).
[0095] 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 15 to about 30 (e.g., about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
base pairs, 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.
[0096] 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.
[0097] 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-SH, 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 or
II; R9 is O, S, CH2, S.dbd.O, CHF, or CF2.
[0098] 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-SH, 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 or
II; 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.
[0099] 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-SH,
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.
[0100] 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).
[0101] In another embodiment, a chemically modified nucleoside or
non-nucleoside (e.g. 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,
chemically modified nucleoside or non-nucleoside (e.g., 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 one embodiment, the chemically modified nucleoside or
non-nucleoside (e.g., a moiety having Formula V, VI or VII) is
present at the 5'-end and 3'-end of the sense strand and the 3'-end
of the antisense strand of a double stranded siNA molecule of the
invention. In one embodiment, the chemically modified nucleoside or
non-nucleoside (e.g., a moiety having Formula V, VI or VII) is
present at the terminal position of the 5'-end and 3'-end of the
sense strand and the 3'-end of the antisense strand of a double
stranded siNA molecule of the invention. In one embodiment, the
chemically modified nucleoside or non-nucleoside (e.g., a moiety
having Formula V, VI or VII) is present at the two terminal
positions of the 5'-end and 3'-end of the sense strand and the
3'-end of the antisense strand of a double stranded siNA molecule
of the invention. In one embodiment, the chemically modified
nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI
or VII) is present at the penultimate position of the 5'-end and
3'-end of the sense strand and the 3'-end of the antisense strand
of a double stranded siNA molecule of the invention. 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising a sense region, wherein 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 wherein 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).
[0106] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising a sense region, wherein 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 wherein 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.
[0107] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising a sense region, wherein 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 wherein 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).
[0108] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising a sense region, wherein 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), wherein 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), and wherein any nucleotides comprising a 3'-terminal
nucleotide overhang that are present in said sense region are
2'-deoxy nucleotides.
[0109] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising an antisense region, wherein 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).
[0110] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising an antisense region, wherein 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), 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), and wherein any nucleotides comprising a 3'-terminal
nucleotide overhang that are present in said antisense region are
2'-deoxy nucleotides.
[0111] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising an antisense region, wherein 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'-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).
[0112] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising an antisense region, wherein 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).
[0113] 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 PDGF and/or PDGFr inside a cell or reconstituted in vitro
system comprising a sense region, wherein 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 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 an antisense region, wherein 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 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). The sense region
and/or the antisense region can have 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 sense and/or antisense sequence. The sense
and/or antisense region can optionally further comprise a
3'-terminal nucleotide overhang having about 1 to about 4 (e.g.,
about 1, 2, 3, or 4) 2'-deoxynucleotides. The overhang nucleotides
can further comprise one or more (e.g., about 1, 2, 3, 4 or more)
phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate
internucleotide linkages. Non-limiting examples of these
chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III
and IV herein. In any of these described embodiments, the purine
nucleotides present in the sense region are alternatively
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 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). Also, in any of these embodiments, one or more purine
nucleotides present in the sense region are alternatively purine
ribonucleotides (e.g., wherein all purine nucleotides are purine
ribonucleotides or alternately a plurality of purine nucleotides
are purine ribonucleotides) and 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). Additionally, in any of these embodiments, one
or more purine nucleotides present in the sense region and/or
present in the antisense region are alternatively 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).
[0114] 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.
[0115] In one embodiment, the sense strand of a double stranded
siNA molecule of the invention comprises a terminal cap moiety,
(see for example FIG. 10) such as an inverted deoxyabasic moiety,
at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense
strand.
[0116] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid molecule (siNA)
capable of mediating RNA interference (RNAi) against PDGF and/or
PDGFr inside a cell or reconstituted in vitro system, wherein the
chemical modification comprises a conjugate covalently attached to
the chemically-modified siNA molecule. Non-limiting examples of
conjugates contemplated by the invention include conjugates and
ligands described in Vargeese et al., U.S. Ser. No. 10/427,160,
filed Apr. 30, 2003, incorporated by reference herein in its
entirety, including the drawings. 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, filed Jul. 22, 2002 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.
[0117] 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 about 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; Sun, 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.)
[0118] 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.
[0119] 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.
[0120] 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 comprising 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 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) 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.
[0121] 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 comprising a single stranded
polynucleotide having complementarity to a target nucleic acid
sequence, 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 comprises about 1 to about 4 or more (e.g.,
about 1, 2, 3, 4 or more) 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, 4 or more)
phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate
internucleotide linkages, and wherein the siNA optionally further
comprises a terminal phosphate group, such as a 5'-terminal
phosphate group. In any of these embodiments, any purine
nucleotides present in the antisense region are alternatively
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). Also, in any
of these embodiments, any purine nucleotides present in the siNA
(i.e., purine nucleotides present in the sense and/or antisense
region) can alternatively be locked nucleic acid (LNA) nucleotides
(e.g., wherein all purine nucleotides are LNA nucleotides or
alternately a plurality of purine nucleotides are LNA nucleotides).
Also, in any of these embodiments, any purine nucleotides present
in the siNA are alternatively 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). 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.
[0122] In one embodiment, a siNA molecule of the invention
comprises chemically modified nucleotides or non-nucleotides (e.g.,
having any of Formulae I-VII, such as 2'-deoxy, 2'-deoxy-2'-fluoro,
or 2'-O-methyl nucleotides) at alternating positions within one or
more strands or regions of the siNA molecule. For example, such
chemical modifications can be introduced at every other position of
a RNA based siNA molecule, starting at either the first or second
nucleotide from the 3'-end or 5'-end of the siNA. In a non-limiting
example, a double stranded siNA molecule of the invention in which
each strand of the siNA is 21 nucleotides in length is featured
wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of each
strand are chemically modified (e.g., with compounds having any of
Formulae I-VII, such as such as 2'-deoxy, 2'-deoxy-2'-fluoro, or
2'-O-methyl nucleotides). In another non-limiting example, a double
stranded siNA molecule of the invention in which each strand of the
siNA is 21 nucleotides in length is featured wherein positions 2,
4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand are chemically
modified (e.g., with compounds having any of Formulae I-VII, such
as such as 2'-deoxy, 2'-deoxy-2'-fluoro, or 2'-O-methyl
nucleotides). Such siNA molecules can further comprise terminal cap
moieties and/or backbone modifications as described herein.
[0123] In one embodiment, the invention features a method for
modulating the expression of a PDGF and/or PDGFr 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 PDGF and/or PDGFr
gene; and (b) introducing the siNA molecule into a cell under
conditions suitable to modulate the expression of the PDGF and/or
PDGFr gene in the cell.
[0124] In one embodiment, the invention features a method for
modulating the expression of a PDGF and/or PDGFr 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 PDGF and/or PDGFr
gene and wherein the sense strand sequence of the siNA comprises a
sequence identical or substantially similar 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 PDGF and/or
PDGFr gene in the cell.
[0125] In another embodiment, the invention features a method for
modulating the expression of more than one PDGF and/or PDGFr 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 PDGF
and/or PDGFr genes; and (b) introducing the siNA molecules into a
cell under conditions suitable to modulate the expression of the
PDGF and/or PDGFr genes in the cell.
[0126] In another embodiment, the invention features a method for
modulating the expression of two or more PDGF and/or PDGFr genes
within a cell comprising: (a) synthesizing one or more siNA
molecules of the invention, which can be chemically-modified,
wherein the siNA strands comprise sequences complementary to RNA of
the PDGF and/or PDGFr genes and wherein the sense strand sequences
of the siNAs comprise sequences identical or substantially similar
to the sequences of the target RNAs; and (b) introducing the siNA
molecules into a cell under conditions suitable to modulate the
expression of the PDGF and/or PDGFr genes in the cell.
[0127] In another embodiment, the invention features a method for
modulating the expression of more than one PDGF and/or PDGFr 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 PDGF
and/or PDGFr gene and wherein the sense strand sequence of the siNA
comprises a sequence identical or substantially similar to the
sequences of the target RNAs; and (b) introducing the siNA molecule
into a cell under conditions suitable to modulate the expression of
the PDGF and/or PDGFr genes in the cell.
[0128] 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 PDGF and/or PDGFr 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 PDGF
and/or PDGFr 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 PDGF and/or
PDGFr 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 PDGF and/or
PDGFr gene in that organism.
[0129] In one embodiment, the invention features a method of
modulating the expression of a PDGF and/or PDGFr 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 PDGF
and/or PDGFr gene and wherein the sense strand sequence of the siNA
comprises a sequence identical or substantially similar 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 PDGF and/or PDGFr 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 PDGF and/or PDGFr gene in that organism.
[0130] In another embodiment, the invention features a method of
modulating the expression of more than one PDGF and/or PDGFr 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 PDGF
and/or PDGFr 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 PDGF and/or
PDGFr 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 PDGF
and/or PDGFr genes in that organism.
[0131] In one embodiment, the invention features a method of
modulating the expression of a PDGF and/or PDGFr gene in a subject
or 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 PDGF
and/or PDGFr gene; and (b) introducing the siNA molecule into the
subject or organism under conditions suitable to modulate the
expression of the PDGF and/or PDGFr gene in the subject or
organism. The level of PDGF and/or PDGFr protein or RNA can be
determined using various methods well-known in the art.
[0132] In another embodiment, the invention features a method of
modulating the expression of more than one PDGF and/or PDGFr gene
in a subject or 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 PDGF and/or PDGFr genes; and (b) introducing the siNA
molecules into the subject or organism under conditions suitable to
modulate the expression of the PDGF and/or PDGFr genes in the
subject or organism. The level of PDGF and/or PDGFr protein or RNA
can be determined as is known in the art.
[0133] In one embodiment, the invention features a method for
modulating the expression of a PDGF and/or PDGFr 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 PDGF
and/or PDGFr gene; and (b) introducing the siNA molecule into a
cell under conditions suitable to modulate the expression of the
PDGF and/or PDGFr gene in the cell.
[0134] In another embodiment, the invention features a method for
modulating the expression of more than one PDGF and/or PDGFr 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 PDGF and/or PDGFr gene; and (b) contacting the cell in vitro
or in vivo with the siNA molecule under conditions suitable to
modulate the expression of the PDGF and/or PDGFr genes in the
cell.
[0135] In one embodiment, the invention features a method of
modulating the expression of a PDGF and/or PDGFr 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 PDGF and/or PDGFr gene; and (b) contacting a cell of the
tissue explant derived from a particular subject or organism with
the siNA molecule under conditions suitable to modulate the
expression of the PDGF and/or PDGFr gene in the tissue explant. In
another embodiment, the method further comprises introducing the
tissue explant back into the subject or organism the tissue was
derived from or into another subject or organism under conditions
suitable to modulate the expression of the PDGF and/or PDGFr gene
in that subject or organism.
[0136] In another embodiment, the invention features a method of
modulating the expression of more than one PDGF and/or PDGFr 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 PDGF and/or PDGFr gene; and (b) introducing the siNA
molecules into a cell of the tissue explant derived from a
particular subject or organism under conditions suitable to
modulate the expression of the PDGF and/or PDGFr genes in the
tissue explant. In another embodiment, the method further comprises
introducing the tissue explant back into the subject or organism
the tissue was derived from or into another subject or organism
under conditions suitable to modulate the expression of the PDGF
and/or PDGFr genes in that subject or organism.
[0137] In one embodiment, the invention features a method of
modulating the expression of a PDGF and/or PDGFr gene in a subject
or 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 PDGF and/or PDGFr gene; and (b) introducing the siNA
molecule into the subject or organism under conditions suitable to
modulate the expression of the PDGF and/or PDGFr gene in the
subject or organism.
[0138] In another embodiment, the invention features a method of
modulating the expression of more than one PDGF and/or PDGFr gene
in a subject or 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 PDGF and/or PDGFr gene; and (b)
introducing the siNA molecules into the subject or organism under
conditions suitable to modulate the expression of the PDGF and/or
PDGFr genes in the subject or organism.
[0139] In one embodiment, the invention features a method of
modulating the expression of a PDGF and/or PDGFr gene in a subject
or organism comprising contacting the subject or organism with a
siNA molecule of the invention under conditions suitable to
modulate the expression of the PDGF and/or PDGFr gene in the
subject or organism.
[0140] In one embodiment, the invention features a method for
treating or preventing cancer in a subject or organism comprising
contacting the subject or organism with a siNA molecule of the
invention under conditions suitable to modulate the expression of
the PDGF and/or PDGFr gene in the subject or organism.
[0141] In one embodiment, the invention features a method for
treating or preventing leukemia in a subject or organism comprising
contacting the subject or organism with a siNA molecule of the
invention under conditions suitable to modulate the expression of
the PDGF and/or PDGFr gene in the subject or organism.
[0142] In one embodiment, the invention features a method for
treating or preventing obliterative bronchiolitis in a subject or
organism comprising contacting the subject or organism with a siNA
molecule of the invention under conditions suitable to modulate the
expression of the PDGF and/or PDGFr gene in the subject or
organism.
[0143] In one embodiment, the invention features a method for
treating or preventing acute glomerulonephritis in a subject or
organism comprising contacting the subject or organism with a siNA
molecule of the invention under conditions suitable to modulate the
expression of the PDGF and/or PDGFr gene in the subject or
organism.
[0144] In one embodiment, the invention features a method for
treating or preventing a stroke (CVA) in a subject or organism
comprising contacting the subject or organism with a siNA molecule
of the invention under conditions suitable to modulate the
expression of the PDGF and/or PDGFr gene in the subject or
organism.
[0145] In one embodiment, the invention features a method for
treating or preventing an inflammatory disease, disorder, and/or
condition in a subject or organism comprising contacting the
subject or organism with a siNA molecule of the invention under
conditions suitable to modulate the expression of the PDGF and/or
PDGFr gene in the subject or organism.
[0146] In one embodiment, the invention features a method for
treating or preventing a proliferative disease, disorder, and/or
condition in a subject or organism comprising contacting the
subject or organism with a siNA molecule of the invention under
conditions suitable to modulate the expression of the PDGF and/or
PDGFr gene in the subject or organism.
[0147] In another embodiment, the invention features a method of
modulating the expression of more than one PDGF and/or PDGFr genes
in a subject or organism comprising contacting the subject or
organism with one or more siNA molecules of the invention under
conditions suitable to modulate the expression of the PDGF and/or
PDGFr genes in the subject or organism.
[0148] The siNA molecules of the invention can be designed to down
regulate or inhibit target (e.g., PDGF and/or PDGFr) 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).
[0149] 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 PDGF and/or PDGFr family genes. As
such, siNA molecules targeting multiple PDGF and/or PDGFr 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 cancer, leukemia, obliterative
bronchiolitis, acute glomerulonephritis, stroke (CVA), and/or
inflammatory and proliferative traits, diseases, disorders, and/or
conditions.
[0150] 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, PDGF
and/or PDGFr genes encoding RNA sequence(s) referred to herein by
Genbank Accession number, for example, Genbank Accession Nos. shown
in Table I.
[0151] 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
15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, or 30) 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.
[0152] In one embodiment, the invention features a method
comprising: (a) generating a randomized library of siNA constructs
having a predetermined complexity, such as of 4.sup.N, 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 4.sup.19); and (b) assaying the siNA constructs
of (a) above, under conditions suitable to determine RNAi target
sites within the target PDGF and/or PDGFr 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 15 to about 30 (e.g., about 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides in length. In one embodiment, the assay can comprise a
reconstituted in vitro siNA assay as described in Example 6 herein.
In another embodiment, the assay can comprise a cell culture system
in which target RNA is expressed. In another embodiment, fragments
of PDGF and/or PDGFr 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 PDGF and/or PDGFr RNA
sequence. The target PDGF and/or PDGFr 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.
[0153] 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 15 to about 30 (e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30) 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.
[0154] 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.
[0155] 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.
[0156] 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 treating or preventing cancer,
leukemia, obliterative bronchiolitis, acute glomerulonephritis,
stroke (CVA), and/or inflammatory and proliferative diseases,
traits, conditions and/or disorders in a subject or organism
comprising administering to the subject a composition of the
invention under conditions suitable for the treatment or prevention
of cancer, leukemia, obliterative bronchiolitis, acute
glomerulonephritis, stroke (CVA), and/or inflammatory and
proliferative diseases, traits, conditions and/or disorders in the
subject or organism.
[0157] In another embodiment, the invention features a method for
validating a PDGF and/or PDGFr 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 PDGF and/or PDGFr target gene;
(b) introducing the siNA molecule into a cell, tissue, subject, or
organism under conditions suitable for modulating expression of the
PDGF and/or PDGFr target gene in the cell, tissue, subject, or
organism; and (c) determining the function of the gene by assaying
for any phenotypic change in the cell, tissue, subject, or
organism.
[0158] In another embodiment, the invention features a method for
validating a PDGF and/or PDGFr 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 PDGF and/or PDGFr target gene; (b) introducing the siNA
molecule into a biological system under conditions suitable for
modulating expression of the PDGF and/or PDGFr target gene in the
biological system; and (c) determining the function of the gene by
assaying for any phenotypic change in the biological system.
[0159] By "biological system" is meant, material, in a purified or
unpurified form, from biological sources, including but not limited
to human or animal, wherein the system comprises the components
required for RNAi activity. The term "biological system" includes,
for example, a cell, tissue, subject, or organism, or extract
thereof. The term biological system also includes reconstituted
RNAi systems that can be used in an in vitro setting.
[0160] 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.
[0161] 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 PDGF and/or PDGFr
target gene in a biological system, including, for example, in a
cell, tissue, subject, 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 PDGF and/or PDGFr
target gene in a biological system, including, for example, in a
cell, tissue, subject, or organism.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] In one embodiment, the invention features siNA constructs
that mediate RNAi against PDGF and/or PDGFr, 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.
[0170] 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.
[0171] In another embodiment, the invention features a method for
generating siNA molecules with improved toxicologic profiles (e.g.,
have attenuated or no immunostimulatory properties) comprising (a)
introducing nucleotides having any of Formula I-VII (e.g., siNA
motifs referred to in Table IV) 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
toxicologic profiles.
[0172] In another embodiment, the invention features a method for
generating siNA molecules that do not stimulate an interferon
response (e.g., no interferon response or attenuated interferon
response) in a cell, subject, or organism, comprising (a)
introducing nucleotides having any of Formula I-VII (e.g., siNA
motifs referred to in Table IV) or any combination thereof into a
siNA molecule, and (b) assaying the siNA molecule of step (a) under
conditions suitable for isolating siNA molecules that do not
stimulate an interferon response.
[0173] By "improved toxicologic profile", is meant that the
chemically modified siNA construct exhibits decreased toxicity in a
cell, subject, or organism compared to an unmodified siNA or siNA
molecule having fewer modifications or modifications that are less
effective in imparting improved toxicology. In a non-limiting
example, siNA molecules with improved toxicologic profiles are
associated with a decreased or attenuated immunostimulatory
response in a cell, subject, or organism compared to an unmodified
siNA or siNA molecule having fewer modifications or modifications
that are less effective in imparting improved toxicology. In one
embodiment, a siNA molecule with an improved toxicological profile
comprises no ribonucleotides. In one embodiment, a siNA molecule
with an improved toxicological profile comprises less than 5
ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In one
embodiment, a siNA molecule with an improved toxicological profile
comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab
17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26,
Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32 or any
combination thereof (see Table IV). In one embodiment, the level of
immunostimulatory response associated with a given siNA molecule
can be measured as is known in the art, for example by determining
the level of PKR/interferon response, proliferation, B-cell
activation, and/or cytokine production in assays to quantitate the
immunostimulatory response of particular siNA molecules (see, for
example, Leifer et al., 2003, J. Immunother. 26, 313-9; and U.S.
Pat. No. 5,968,909, incorporated in its entirety by reference).
[0174] In one embodiment, the invention features siNA constructs
that mediate RNAi against PDGF and/or PDGFr, 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.
[0175] 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.
[0176] In one embodiment, the invention features siNA constructs
that mediate RNAi against PDGF and/or PDGFr, 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.
[0177] In one embodiment, the invention features siNA constructs
that mediate RNAi against PDGF and/or PDGFr, 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.
[0178] 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.
[0179] 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.
[0180] In one embodiment, the invention features siNA constructs
that mediate RNAi against PDGF and/or PDGFr, 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.
[0181] 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.
[0182] In one embodiment, the invention features
chemically-modified siNA constructs that mediate RNAi against PDGF
and/or PDGFr 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.
[0183] In another embodiment, the invention features a method for
generating siNA molecules with improved RNAi activity against PDGF
and/or PDGFr 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.
[0184] In yet another embodiment, the invention features a method
for generating siNA molecules with improved RNAi activity against
PDGF and/or PDGFr 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.
[0185] In yet another embodiment, the invention features a method
for generating siNA molecules with improved RNAi activity against
PDGF and/or PDGFr 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.
[0186] In one embodiment, the invention features siNA constructs
that mediate RNAi against PDGF and/or PDGFr, wherein the siNA
construct comprises one or more chemical modifications described
herein that modulates the cellular uptake of the siNA
construct.
[0187] In another embodiment, the invention features a method for
generating siNA molecules against PDGF and/or PDGFr 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.
[0188] In one embodiment, the invention features siNA constructs
that mediate RNAi against PDGF and/or PDGFr, 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.
[0189] 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.
[0190] In one embodiment, the invention features a double stranded
short interfering nucleic acid (siNA) molecule that comprises a
first nucleotide sequence complementary to a target RNA sequence or
a portion thereof, and a second sequence having complementarity to
said first sequence, wherein said second sequence is chemically
modified in a manner that it can no longer act as a guide sequence
for efficiently mediating RNA interference and/or be recognized by
cellular proteins that facilitate RNAi.
[0191] In one embodiment, the invention features a double stranded
short interfering nucleic acid (siNA) molecule that comprises a
first nucleotide sequence complementary to a target RNA sequence or
a portion thereof, and a second sequence having complementarity to
said first sequence, wherein the second sequence is designed or
modified in a manner that prevents its entry into the RNAi pathway
as a guide sequence or as a sequence that is complementary to a
target nucleic acid (e.g., RNA) sequence. Such design or
modifications are expected to enhance the activity of siNA and/or
improve the specificity of siNA molecules of the invention. These
modifications are also expected to minimize any off-target effects
and/or associated toxicity.
[0192] In one embodiment, the invention features a double stranded
short interfering nucleic acid (siNA) molecule that comprises a
first nucleotide sequence complementary to a target RNA sequence or
a portion thereof, and a second sequence having complementarity to
said first sequence, wherein said second sequence is incapable of
acting as a guide sequence for mediating RNA interference.
[0193] In one embodiment, the invention features a double stranded
short interfering nucleic acid (siNA) molecule that comprises a
first nucleotide sequence complementary to a target RNA sequence or
a portion thereof, and a second sequence having complementarity to
said first sequence, wherein said second sequence does not have a
terminal 5'-hydroxyl (5'-OH) or 5'-phosphate group.
[0194] In one embodiment, the invention features a double stranded
short interfering nucleic acid (siNA) molecule that comprises a
first nucleotide sequence complementary to a target RNA sequence or
a portion thereof, and a second sequence having complementarity to
said first sequence, wherein said second sequence comprises a
terminal cap moiety at the 5'-end of said second sequence. In one
embodiment, the terminal cap moiety comprises an inverted abasic,
inverted deoxy abasic, inverted nucleotide moiety, a group shown in
FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other
group that prevents RNAi activity in which the second sequence
serves as a guide sequence or template for RNAi.
[0195] In one embodiment, the invention features a double stranded
short interfering nucleic acid (siNA) molecule that comprises a
first nucleotide sequence complementary to a target RNA sequence or
a portion thereof, and a second sequence having complementarity to
said first sequence, wherein said second sequence comprises a
terminal cap moiety at the 5'-end and 3'-end of said second
sequence. In one embodiment, each terminal cap moiety individually
comprises an inverted abasic, inverted deoxy abasic, inverted
nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl
group, a heterocycle, or any other group that prevents RNAi
activity in which the second sequence serves as a guide sequence or
template for RNAi.
[0196] In one embodiment, the invention features a method for
generating siNA molecules of the invention with improved
specificity for down regulating or inhibiting the expression of a
target nucleic acid (e.g., a DNA or RNA such as a gene or its
corresponding RNA), comprising (a) introducing one or more chemical
modifications 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 specificity. In
another embodiment, the chemical modification used to improve
specificity comprises terminal cap modifications at the 5'-end,
3'-end, or both 5' and 3'-ends of the siNA molecule. The terminal
cap modifications can comprise, for example, structures shown in
FIG. 10 (e.g. inverted deoxyabasic moieties) or any other chemical
modification that renders a portion of the siNA molecule (e.g. the
sense strand) incapable of mediating RNA interference against an
off target nucleic acid sequence. In a non-limiting example, a siNA
molecule is designed such that only the antisense sequence of the
siNA molecule can serve as a guide sequence for RISC mediated
degradation of a corresponding target RNA sequence. This can be
accomplished by rendering the sense sequence of the siNA inactive
by introducing chemical modifications to the sense strand that
preclude recognition of the sense strand as a guide sequence by
RNAi machinery. In one embodiment, such chemical modifications
comprise any chemical group at the 5'-end of the sense strand of
the siNA, or any other group that serves to render the sense strand
inactive as a guide sequence for mediating RNA interference. These
modifications, for example, can result in a molecule where the
5'-end of the sense strand no longer has a free 5'-hydroxyl (5'-OH)
or a free 5'-phosphate group (e.g., phosphate, diphosphate,
triphosphate, cyclic phosphate etc.). Non-limiting examples of such
siNA constructs are described herein, such as "Stab 9/10", "Stab
7/8", "Stab 7/19", "Stab 17/22", "Stab 23/24", "Stab 24/25", and
"Stab 24/26" (e.g., any siNA having Stab 7, 9, 17, 23, or 24 sense
strands) chemistries and variants thereof (see Table IV) wherein
the 5'-end and 3'-end of the sense strand of the siNA do not
comprise a hydroxyl group or phosphate group.
[0197] In one embodiment, the invention features a method for
generating siNA molecules of the invention with improved
specificity for down regulating or inhibiting the expression of a
target nucleic acid (e.g., a DNA or RNA such as a gene or its
corresponding RNA), comprising introducing one or more chemical
modifications into the structure of a siNA molecule that prevent a
strand or portion of the siNA molecule from acting as a template or
guide sequence for RNAi activity. In one embodiment, the inactive
strand or sense region of the siNA molecule is the sense strand or
sense region of the siNA molecule, i.e. the strand or region of the
siNA that does not have complementarity to the target nucleic acid
sequence. In one embodiment, such chemical modifications comprise
any chemical group at the 5'-end of the sense strand or region of
the siNA that does not comprise a 5'-hydroxyl (5'-OH) or
5'-phosphate group, or any other group that serves to render the
sense strand or sense region inactive as a guide sequence for
mediating RNA interference. Non-limiting examples of such siNA
constructs are described herein, such as "Stab 9/10", "Stab 7/8",
"Stab 7/19", "Stab 17/22", "Stab 23/24", "Stab 24/25", and "Stab
24/26" (e.g., any siNA having Stab 7, 9, 17, 23, or 24 sense
strands) chemistries and variants thereof (see Table IV) wherein
the 5'-end and 3'-end of the sense strand of the siNA do not
comprise a hydroxyl group or phosphate group.
[0198] In one embodiment, the invention features a method for
screening siNA molecules that are active in mediating RNA
interference against a target nucleic acid sequence comprising (a)
generating a plurality of unmodified siNA molecules, (b) screening
the siNA molecules of step (a) under conditions suitable for
isolating siNA molecules that are active in mediating RNA
interference against the target nucleic acid sequence, and (c)
introducing chemical modifications (e.g. chemical modifications as
described herein or as otherwise known in the art) into the active
siNA molecules of (b).
[0199] In one embodiment, the method further comprises re-screening
the chemically modified siNA molecules of step (c) under conditions
suitable for isolating chemically modified siNA molecules that are
active in mediating RNA interference against the target nucleic
acid sequence.
[0200] In one embodiment, the invention features a method for
screening chemically modified siNA molecules that are active in
mediating RNA interference against a target nucleic acid sequence
comprising (a) generating a plurality of chemically modified siNA
molecules (e.g. siNA molecules as described herein or as otherwise
known in the art), and (b) screening the siNA molecules of step (a)
under conditions suitable for isolating chemically modified siNA
molecules that are active in mediating RNA interference against the
target nucleic acid sequence.
[0201] The term "ligand" refers to any compound or molecule, such
as a drug, peptide, hormone, or neurotransmitter, that is capable
of interacting with another compound, such as a receptor, either
directly or indirectly. The receptor that interacts with a ligand
can be present on the surface of a cell or can alternately be an
intercellular receptor. Interaction of the ligand with the receptor
can result in a biochemical reaction, or can simply be a physical
interaction or association.
[0202] 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,
nanoparticles, receptors, ligands, and others.
[0203] 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.
[0204] 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).
[0205] 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.
[0206] 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 or viral replication,
for example by mediating RNA interference "RNAi" or gene silencing
in a sequence-specific manner; see for example Zamore et al., 2000,
Cell, 101, 25-33; 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 and III 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 15 to about 30, e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or
30 base pairs; the antisense strand comprises nucleotide sequence
that is complementary to nucleotide sequence in a target nucleic
acid molecule or a portion thereof and the sense strand comprises
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof (e.g., about 15 to about 25 or more
nucleotides of the siNA molecule are complementary to the target
nucleic acid 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 duplex,
asymmetric duplex, hairpin or asymmetric hairpin secondary
structure, having self-complementary sense and antisense regions,
wherein the antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a separate target nucleic
acid molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The 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 embodiments, 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 and 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
or methylation pattern to alter gene expression (see, for example,
Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al.,
2004, Science, 303, 669-672; Allshire, 2002, Science, 297,
1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,
2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,
2232-2237).
[0207] In one embodiment, a siNA molecule of the invention is a
duplex forming oligonucleotide "DFO", (see for example FIGS. 14-15
and Vaish et al., U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and
International PCT Application No. US04/16390, filed May 24,
2004).
[0208] In one embodiment, a siNA molecule of the invention is a
multifunctional siNA, (see for example FIGS. 16-21 and Jadhav et
al., U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and International
PCT Application No. US04/16390, filed May 24, 2004). The
multifunctional siNA of the invention can comprise sequence
targeting, for example, two regions of PDGF and/or PDGFr RNA (see
for example target sequences in Tables II and III).
[0209] By "asymmetric hairpin" as used herein is meant a linear
siNA molecule comprising an antisense region, a loop portion that
can comprise nucleotides or non-nucleotides, and a sense region
that comprises fewer nucleotides than the antisense region to the
extent that the sense region has enough complementary nucleotides
to base pair with the antisense region and form a duplex with loop.
For example, an asymmetric hairpin siNA molecule of the invention
can comprise an antisense region having length sufficient to
mediate RNAi in a cell or in vitro system (e.g. about 15 to about
30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30 nucleotides) and a loop region comprising about 4 to
about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides,
and a sense region having about 3 to about 25 (e.g., about 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25) nucleotides that are complementary to the antisense
region. The asymmetric hairpin siNA molecule can also comprise a
5'-terminal phosphate group that can be chemically modified. The
loop portion of the asymmetric hairpin siNA molecule can comprise
nucleotides, non-nucleotides, linker molecules, or conjugate
molecules as described herein.
[0210] By "asymmetric duplex" as used herein is meant a siNA
molecule having two separate strands comprising a sense region and
an antisense region, wherein the sense region comprises fewer
nucleotides than the antisense region to the extent that the sense
region has enough complementary nucleotides to base pair with the
antisense region and form a duplex. For example, an asymmetric
duplex siNA molecule of the invention can comprise an antisense
region having length sufficient to mediate RNAi in a cell or in
vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and
a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25) nucleotides that are complementary to the antisense
region.
[0211] 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.
[0212] 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. In one
embodiment, inhibition, down regulation, or reduction of gene
expression is associated with post transcriptional silencing, such
as RNAi mediated cleavage of a target nucleic acid molecule (e.g.
RNA) or inhibition of translation. In one embodiment, inhibition,
down regulation, or reduction of gene expression is associated with
pretranscriptional silencing.
[0213] 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. A gene or
target gene can also encode a functional RNA (fRNA) or non-coding
RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA),
small nuclear RNA (snRNA), short interfering RNA (siRNA), small
nuclear RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and
precursor RNAs thereof. Such non-coding RNAs can serve as target
nucleic acid molecules for siNA mediated RNA interference in
modulating the activity of fRNA or ncRNA involved in functional or
regulatory cellular processes. Aberrant fRNA or ncRNA activity
leading to disease can therefore be modulated by siNA molecules of
the invention. siNA molecules targeting fRNA and ncRNA can also be
used to manipulate or alter the genotype or phenotype of a subject,
organism or cell, by intervening in cellular processes such as
genetic imprinting, transcription, translation, or nucleic acid
processing (e.g., transamination, methylation etc.). 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. For a review, see for example
Snyder and Gerstein, 2003, Science, 300, 258-260.
[0214] By "non-canonical base pair" is meant any non-Watson Crick
base pair, such as mismatches and/or wobble base pairs, including
flipped mismatches, single hydrogen bond mismatches, trans-type
mismatches, triple base interactions, and quadruple base
interactions. Non-limiting examples of such non-canonical base
pairs include, but are not limited to, AC reverse Hoogsteen, AC
wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC
2-carbonyl-amino(H1)-N-3-amino(H2), GA sheared, UC
4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU
Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC
N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA
N7-N1 amino-carbonyl, GA+ carbonyl-amino N7-N1, GG N1-carbonyl
symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC
N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU
4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino
2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU
N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1,
GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC
carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG
carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU
carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU
imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU
imino-4-carbonyl, AC C2-H--N3, GA carbonyl-C2-H, UU
imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC
imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and
GU imino amino-2-carbonyl base pairs.
[0215] By "PDGF" as used herein is meant any platelet derived
growth factor protein, peptide, or polypeptide having any platelet
derived growth factor activity, such as encoded by PDGF Genbank
Accession Nos. shown in Table I. The term PDGF also refers to
nucleic acid sequences encoding any platelet derived growth factor
protein, peptide, or polypeptide having platelet derived growth
factor activity. The term "PDGF" is also meant to include other
platelet derived growth factor encoding sequence, such as other
PDGF isoforms, mutant PDGF genes, splice variants of PDGF genes,
and PDGF gene polymorphisms.
[0216] By "PDGFr" as used herein is meant any platelet derived
growth factor receptor protein, peptide, or polypeptide having any
platelet derived growth factor receptor activity, such as encoded
by PDGFr Genbank Accession Nos. shown in Table I. The term PDGFr
also refers to nucleic acid sequences encoding any platelet derived
growth factor receptor protein, peptide, or polypeptide having
PDGFr activity. The term "PDGFr" is also meant to include other
platelet derived growth factor receptor encoding sequence, such as
other PDGFr isoforms, mutant PDGFr genes, splice variants of PDGFr
genes, and PDGFr gene polymorphisms.
[0217] By "homologous sequence" is meant, a nucleotide sequence
that is shared by one or more polynucleotide sequences, such as
genes, gene transcripts and/or non-coding polynucleotides. For
example, a homologous sequence can be a nucleotide sequence that is
shared by two or more genes encoding related but different
proteins, such as different members of a gene family, different
protein epitopes, different protein isoforms or completely
divergent genes, such as a cytokine and its corresponding
receptors. A homologous sequence can be a nucleotide sequence that
is shared by two or more non-coding polynucleotides, such as
noncoding DNA or RNA, regulatory sequences, introns, and sites of
transcriptional control or regulation. Homologous sequences can
also include conserved sequence regions shared by more than one
polynucleotide sequence. Homology does not need to be perfect
homology (e.g., 100%), as partially homologous sequences are also
contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%,
95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,
82%, 81%, 80% etc.).
[0218] By "conserved sequence region" is meant, a nucleotide
sequence of one or more regions in a polynucleotide does not vary
significantly between generations or from one biological system,
subject, or organism to another biological system, subject, or
organism. The polynucleotide can include both coding and non-coding
DNA and RNA.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] 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, or 10 nucleotides out
of a total of 10 nucleotides in the first oligonucleotide being
based paired to a second nucleic acid sequence having 10
nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100%
complementary respectively). "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. In one embodiment, a siNA molecule of
the invention comprises about 15 to about 30 or more (e.g., about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
or more) nucleotides that are complementary to one or more target
nucleic acid molecules or a portion thereof.
[0223] In one embodiment, siNA molecules of the invention that down
regulate or reduce PDGF and/or PDGFr gene expression are used for
preventing or treating cancer, leukemia, obliterative
bronchiolitis, acute glomerulonephritis, stroke (CVA), and/or
inflammatory and proliferative diseases, traits, conditions and/or
disorders in a subject or organism.
[0224] In one embodiment, the siNA molecules of the invention are
used to treat cancer, leukemia, obliterative bronchiolitis, acute
glomerulonephritis, stroke (CVA), and/or inflammatory and
proliferative diseases, traits, conditions and/or disorders in a
subject or organism.
[0225] By "proliferative disease" or "cancer" as used herein is
meant, any disease, condition, trait, genotype or phenotype
characterized by unregulated cell growth or replication as is known
in the art; including leukemias, for example, acute myelogenous
leukemia (AML), chronic myelogenous leukemia (CML), acute
lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS
related cancers such as Kaposi's sarcoma; breast cancers; bone
cancers such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma,
Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas;
Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade
Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas,
and Metastatic brain cancers; cancers of the head and neck
including various lymphomas such as mantle cell lymphoma,
non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal
carcinoma, gallbladder and bile duct cancers, cancers of the retina
such as retinoblastoma, cancers of the esophagus, gastric cancers,
multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer,
testicular cancer, endometrial cancer, melanoma, colorectal cancer,
lung cancer, bladder cancer, prostate cancer, lung cancer
(including non-small cell lung carcinoma), pancreatic cancer,
sarcomas, Wilms' tumor, cervical cancer, head and neck cancer, skin
cancers, nasopharyngeal carcinoma, liposarcoma, epithelial
carcinoma, renal cell carcinoma, gallbladder adeno carcinoma,
parotid adenocarcinoma, endometrial sarcoma, multidrug resistant
cancers; and proliferative diseases and conditions, such as
neovascularization associated with tumor angiogenesis, macular
degeneration (e.g., wet/dry AMD), corneal neovascularization,
diabetic retinopathy, neovascular glaucoma, myopic degeneration and
other proliferative diseases and conditions such as restenosis and
polycystic kidney disease, and any other cancer or proliferative
disease, condition, trait, genotype or phenotype that can respond
to the modulation of disease related gene expression in a cell or
tissue, alone or in combination with other therapies.
[0226] By "leukemia" as used herein is meant any disease, disorder,
condition, trait, genotype or phenotype characterized by, for
example, the overproduction of immature atypical leukocytes, such
as acute myelogenous leukemia (AML), chronic myelogenous leukemia
(CML), acute lymphocytic leukemia (ALL), and chronic lympocytic
leukemia, and any other leukemia that can respond to the modulation
of disease related gene expression in a cell or tissue, alone or in
combination with other therapies.
[0227] By "inflammatory disease" or "inflammatory condition" as
used herein is meant any disease, condition, trait, genotype or
phenotype characterized by an inflammatory or allergic process as
is known in the art, such as inflammation, acute inflammation,
chronic inflammation, respiratory disease, atherosclerosis,
restenosis, asthma, allergic rhinitis, atopic dermatitis, septic
shock, rheumatoid arthritis, inflammatory bowl disease,
inflammatory pelvic disease, pain, ocular inflammatory disease,
celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency,
Familial eosinophilia (FE), autosomal recessive spastic ataxia,
laryngeal inflammatory disease; Tuberculosis, Chronic
cholecystitis, Bronchiectasis, Silicosis and other pneumoconioses,
and any other inflammatory disease, condition, trait, genotype or
phenotype that can respond to the modulation of disease related
gene expression in a cell or tissue, alone or in combination with
other therapies.
[0228] In one embodiment of the present invention, each sequence of
a siNA molecule of the invention is independently about 15 to about
30 nucleotides in length, in specific embodiments about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides
in length. In another embodiment, the siNA duplexes of the
invention independently comprise about 15 to about 30 base pairs
(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30). In another embodiment, one or more strands of the
siNA molecule of the invention independently comprises about 15 to
about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30) that are complementary to a
target nucleic acid molecule. 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., about 38,
39, 40, 41, 42, 43, or 44) nucleotides in length and comprising
about 15 to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25) base pairs. Exemplary siNA molecules of the
invention are shown in Table II. Exemplary synthetic siNA molecules
of the invention are shown in Table III and/or FIGS. 4-5.
[0229] 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.
[0230] 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 direct dermal application,
transdermal application, or injection, 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.
[0231] 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.
[0232] 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 D-ribofuranose
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.
[0233] 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.
[0234] 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.
[0235] The term "phosphonoacetate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z and/or W
comprise an acetyl or protected acetyl group.
[0236] The term "thiophosphonoacetate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z comprises an
acetyl or protected acetyl group and W comprises a sulfur atom or
alternately W comprises an acetyl or protected acetyl group and Z
comprises a sulfur atom.
[0237] 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).
[0238] 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.
[0239] The nucleic acid molecules of the instant invention,
individually, or in combination or in conjunction with other drugs,
can be used to for preventing or treating cancer, leukemia,
obliterative bronchiolitis, acute glomerulonephritis, stroke (CVA),
and/or inflammatory and proliferative diseases, traits, conditions
and/or disorders in a subject or organism.
[0240] For example, 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.
[0241] In a further embodiment, the siNA molecules can be used in
combination with other known treatments to prevent or cancer,
leukemia, obliterative bronchiolitis, acute glomerulonephritis,
stroke (CVA), and/or inflammatory and proliferative diseases,
traits, conditions and/or disorders in a subject or organism. For
example, the described molecules could be used in combination with
one or more known compounds, treatments, or procedures to prevent
or treat cancer, leukemia, obliterative bronchiolitis, acute
glomerulonephritis, stroke (CVA), and/or inflammatory and
proliferative diseases, traits, conditions and/or disorders in a
subject or organism as are known in the art.
[0242] 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.
[0243] In another embodiment, the invention features a mammalian
cell, for example, a human cell, including an expression vector of
the invention.
[0244] 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.
[0245] 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.
[0246] 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.
[0247] By "vectors" is meant any nucleic acid- and/or viral-based
technique used to deliver a desired nucleic acid.
[0248] 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
[0249] 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.
[0250] 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.
[0251] 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.
[0252] 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.
[0253] 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 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",
optionally connects the (N N) nucleotides in the antisense
strand.
[0254] 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",
optionally connects the (N N) nucleotides in the sense and
antisense strand.
[0255] 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",
optionally connects the (N N) nucleotides in the antisense
strand.
[0256] 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, 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",
optionally connects the (N N) nucleotides in the antisense
strand.
[0257] 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", optionally connects the (N N)
nucleotides in the antisense strand.
[0258] 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 having one 3'-terminal phosphorothioate
internucleotide linkage 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",
optionally 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.
[0259] 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 PDGFr siNA
sequence. Such chemical modifications can be applied to any PDGF
and/or PDGFr sequence and/or PDGF and/or PDGFr polymorphism
sequence.
[0260] 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.
[0261] FIG. 7A-C is a diagrammatic representation of a scheme
utilized in generating an expression cassette to generate siNA
hairpin constructs.
[0262] 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 PDGF and/or PDGFr 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.
[0263] 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 PDGF and/or PDGFr target sequence and
having self-complementary sense and antisense regions.
[0264] 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.
[0265] FIG. 8A-C is a diagrammatic representation of a scheme
utilized in generating an expression cassette to generate
double-stranded siNA constructs.
[0266] 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 PDGF and/or PDGFr 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).
[0267] FIG. 8B: The synthetic construct is then extended by DNA
polymerase to generate a hairpin structure having
self-complementary sequence.
[0268] 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.
[0269] 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.
[0270] 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.
[0271] 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.
[0272] FIG. 9D: Cells are sorted based on phenotypic change that is
associated with modulation of the target nucleic acid sequence.
[0273] FIG. 9E: The siNA is isolated from the sorted cells and is
sequenced to identify efficacious target sites within the target
nucleic acid sequence.
[0274] 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.
[0275] 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'-mofications, 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.
[0276] FIG. 12 shows non-limiting examples of phosphorylated siNA
molecules of the invention, including linear and duplex constructs
and asymmetric derivatives thereof.
[0277] FIG. 13 shows non-limiting examples of chemically modified
terminal phosphate groups of the invention.
[0278] FIG. 14A shows a non-limiting example of methodology used to
design self complementary DFO constructs utilizing palindrome
and/or repeat nucleic acid sequences that are identified in a
target nucleic acid sequence. (i) A palindrome or repeat sequence
is identified in a nucleic acid target sequence. (ii) A sequence is
designed that is complementary to the target nucleic acid sequence
and the palindrome sequence. (iii) An inverse repeat sequence of
the non-palindrome/repeat portion of the complementary sequence is
appended to the 3'-end of the complementary sequence to generate a
self complementary DFO molecule comprising sequence complementary
to the nucleic acid target. (iv) The DFO molecule can self-assemble
to form a double stranded oligonucleotide. FIG. 14B shows a
non-limiting representative example of a duplex forming
oligonucleotide sequence. FIG. 14C shows a non-limiting example of
the self assembly schematic of a representative duplex forming
oligonucleotide sequence. FIG. 14D shows a non-limiting example of
the self assembly schematic of a representative duplex forming
oligonucleotide sequence followed by interaction with a target
nucleic acid sequence resulting in modulation of gene
expression.
[0279] FIG. 15 shows a non-limiting example of the design of self
complementary DFO constructs utilizing palindrome and/or repeat
nucleic acid sequences that are incorporated into the DFO
constructs that have sequence complementary to any target nucleic
acid sequence of interest. Incorporation of these palindrome/repeat
sequences allow the design of DFO constructs that form duplexes in
which each strand is capable of mediating modulation of target gene
expression, for example by RNAi. First, the target sequence is
identified. A complementary sequence is then generated in which
nucleotide or non-nucleotide modifications (shown as X or Y) are
introduced into the complementary sequence that generate an
artificial palindrome (shown as XYXYXY in the Figure). An inverse
repeat of the non-palindrome/repeat complementary sequence is
appended to the 3'-end of the complementary sequence to generate a
self complementary DFO comprising sequence complementary to the
nucleic acid target. The DFO can self-assemble to form a double
stranded oligonucleotide.
[0280] FIG. 16 shows non-limiting examples of multifunctional siNA
molecules of the invention comprising two separate polynucleotide
sequences that are each capable of mediating RNAi directed cleavage
of differing target nucleic acid sequences. FIG. 16A shows a
non-limiting example of a multifunctional siNA molecule having a
first region that is complementary to a first target nucleic acid
sequence (complementary region 1) and a second region that is
complementary to a second target nucleic acid sequence
(complementary region 2), wherein the first and second
complementary regions are situated at the 3'-ends of each
polynucleotide sequence in the multifunctional siNA. The dashed
portions of each polynucleotide sequence of the multifunctional
siNA construct have complementarity with regard to corresponding
portions of the siNA duplex, but do not have complementarity to the
target nucleic acid sequences. FIG. 16B shows a non-limiting
example of a multifunctional siNA molecule having a first region
that is complementary to a first target nucleic acid sequence
(complementary region 1) and a second region that is complementary
to a second target nucleic acid sequence (complementary region 2),
wherein the first and second complementary regions are situated at
the 5'-ends of each polynucleotide sequence in the multifunctional
siNA. The dashed portions of each polynucleotide sequence of the
multifunctional siNA construct have complementarity with regard to
corresponding portions of the siNA duplex, but do not have
complementarity to the target nucleic acid sequences.
[0281] FIG. 17 shows non-limiting examples of multifunctional siNA
molecules of the invention comprising a single polynucleotide
sequence comprising distinct regions that are each capable of
mediating RNAi directed cleavage of differing target nucleic acid
sequences. FIG. 17A shows a non-limiting example of a
multifunctional siNA molecule having a first region that is
complementary to a first target nucleic acid sequence
(complementary region 1) and a second region that is complementary
to a second target nucleic acid sequence (complementary region 2),
wherein the second complementary region is situated at the 3'-end
of the polynucleotide sequence in the multifunctional siNA. The
dashed portions of each polynucleotide sequence of the
multifunctional siNA construct have complementarity with regard to
corresponding portions of the siNA duplex, but do not have
complementarity to the target nucleic acid sequences. FIG. 17B
shows a non-limiting example of a multifunctional siNA molecule
having a first region that is complementary to a first target
nucleic acid sequence (complementary region 1) and a second region
that is complementary to a second target nucleic acid sequence
(complementary region 2), wherein the first complementary region is
situated at the 5'-end of the polynucleotide sequence in the
multifunctional siNA. The dashed portion's of each polynucleotide
sequence of the multifunctional siNA construct have complementarity
with regard to corresponding portions of the siNA duplex, but do
not have complementarity to the target nucleic acid sequences. In
one embodiment, these multifunctional siNA constructs are processed
in vivo or in vitro to generate multifunctional siNA constructs as
shown in FIG. 16.
[0282] FIG. 18 shows non-limiting examples of multifunctional siNA
molecules of the invention comprising two separate polynucleotide
sequences that are each capable of mediating RNAi directed cleavage
of differing target nucleic acid sequences and wherein the
multifunctional siNA construct further comprises a self
complementary, palindrome, or repeat region, thus enabling shorter
bifunctional siNA constructs that can mediate RNA interference
against differing target nucleic acid sequences. FIG. 18A shows a
non-limiting example of a multifunctional siNA molecule having a
first region that is complementary to a first target nucleic acid
sequence (complementary region 1) and a second region that is
complementary to a second target nucleic acid sequence
(complementary region 2), wherein the first and second
complementary regions are situated at the 3'-ends of each
polynucleotide sequence in the multifunctional siNA, and wherein
the first and second complementary regions further comprise a self
complementary, palindrome, or repeat region. The dashed portions of
each polynucleotide sequence of the multifunctional siNA construct
have complementarity with regard to corresponding portions of the
siNA duplex, but do not have complementarity to the target nucleic
acid sequences. FIG. 18B shows a non-limiting example of a
multifunctional siNA molecule having a first region that is
complementary to a first target nucleic acid sequence
(complementary region 1) and a second region that is complementary
to a second target nucleic acid sequence (complementary region 2),
wherein the first and second complementary regions are situated at
the 5'-ends of each polynucleotide sequence in the multifunctional
siNA, and wherein the first and second complementary regions
further comprise a self complementary, palindrome, or repeat
region. The dashed portions of each polynucleotide sequence of the
multifunctional siNA construct have complementarity with regard to
corresponding portions of the siNA duplex, but do not have
complementarity to the target nucleic acid sequences.
[0283] FIG. 19 shows non-limiting examples of multifunctional siNA
molecules of the invention comprising a single polynucleotide
sequence comprising distinct regions that are each capable of
mediating RNAi directed cleavage of differing target nucleic acid
sequences and wherein the multifunctional siNA construct further
comprises a self complementary, palindrome, or repeat region, thus
enabling shorter bifunctional siNA constructs that can mediate RNA
interference against differing target nucleic acid sequences. FIG.
19A shows a non-limiting example of a multifunctional siNA molecule
having a first region that is complementary to a first target
nucleic acid sequence (complementary region 1) and a second region
that is complementary to a second target nucleic acid sequence
(complementary region 2), wherein the second complementary region
is situated at the 3'-end of the polynucleotide sequence in the
multifunctional siNA, and wherein the first and second
complementary regions further comprise a self complementary,
palindrome, or repeat region. The dashed portions of each
polynucleotide sequence of the multifunctional siNA construct have
complementarity with regard to corresponding portions of the siNA
duplex, but do not have complementarity to the target nucleic acid
sequences. FIG. 19B shows a non-limiting example of a
multifunctional siNA molecule having a first region that is
complementary to a first target nucleic acid sequence
(complementary region 1) and a second region that is complementary
to a second target nucleic acid sequence (complementary region 2),
wherein the first complementary region is situated at the 5'-end of
the polynucleotide sequence in the multifunctional siNA, and
wherein the first and second complementary regions further comprise
a self complementary, palindrome, or repeat region. The dashed
portions of each polynucleotide sequence of the multifunctional
siNA construct have complementarity with regard to corresponding
portions of the siNA duplex, but do not have complementarity to the
target nucleic acid sequences. In one embodiment, these
multifunctional siNA constructs are processed in vivo or in vitro
to generate multifunctional siNA constructs as shown in FIG.
18.
[0284] FIG. 20 shows a non-limiting example of how multifunctional
siNA molecules of the invention can target two separate target
nucleic acid molecules, such as separate RNA molecules encoding
differing proteins, for example, a cytokine and its corresponding
receptor, differing viral strains, a virus and a cellular protein
involved in viral infection or replication, or differing proteins
involved in a common or divergent biologic pathway that is
implicated in the maintenance of progression of disease. Each
strand of the multifunctional siNA construct comprises a region
having complementarity to separate target nucleic acid molecules.
The multifunctional siNA molecule is designed such that each strand
of the siNA can be utilized by the RISC to initiate RNA
interference mediated cleavage of its corresponding target. These
design parameters can include destabilization of each end of the
siNA construct (see for example Schwarz et al., 2003, Cell, 115,
199-208). Such destabilization can be accomplished for example by
using guanosine-cytidine base pairs, alternate base pairs (e.g.,
wobbles), or destabilizing chemically modified nucleotides at
terminal nucleotide positions as is known in the art.
[0285] FIG. 21 shows a non-limiting example of how multifunctional
siNA molecules of the invention can target two separate target
nucleic acid sequences within the same target nucleic acid
molecule, such as alternate coding regions of a RNA, coding and
non-coding regions of a RNA, or alternate splice variant regions of
a RNA. Each strand of the multifunctional siNA construct comprises
a region having complementarity to the separate regions of the
target nucleic acid molecule. The multifunctional siNA molecule is
designed such that each strand of the siNA can be utilized by the
RISC to initiate RNA interference mediated cleavage of its
corresponding target region. These design parameters can include
destabilization of each end of the siNA construct (see for example
Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can
be accomplished for example by using guanosine-cytidine base pairs,
alternate base pairs (e.g., wobbles), or destabilizing chemically
modified nucleotides at terminal nucleotide positions as is known
in the art.
DETAILED DESCRIPTION OF THE INVENTION
Mechanism of Action of Nucleic Acid Molecules of the Invention
[0286] 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 limiting only to
siRNA 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.
[0287] 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.
[0288] 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.
[0289] 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
[0290] 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.
[0291] 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 min 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 Biosystems, Inc.). 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.
[0292] 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:H2O/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.
[0293] 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 min
coupling step for alkylsilyl protected nucleotides and a 2.5 min
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 I.sub.2, 49 mM pyridine,
9% water in THF (PerSeptive Biosystems, Inc.). 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.
[0294] 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 min. 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:H2O/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 h, the oligomer is quenched with 1.5 M
NH.sub.4HCO.sub.3.
[0295] 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.
[0296] For purification of the trityl-on oligomers, the quenched
NH.sub.4HCO.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.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] 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.
[0304] 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.
[0305] 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.
[0306] 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.
[0307] 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).
[0308] 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.
[0309] 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.
[0310] The term "biodegradable" as used herein, refers to
degradation in a biological system, for example, enzymatic
degradation or chemical degradation.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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.
[0315] Use of the nucleic acid-based molecules of the invention
will lead to better treatments 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.
[0316] 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.
[0317] 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
may help in delivery and/or localization within a cell. The cap may
be present at the 5'-terminus (5'-cap) or at the 3'-terminal
(3'-cap) or may be present on both termini. In non-limiting
examples, the 5'-cap includes, but is 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. Non-limiting
examples of cap moieties are shown in FIG. 10.
[0318] 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).
[0319] 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.
[0320] 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.
[0321] 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.
[0322] 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.
[0323] 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.
[0324] 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.
[0325] 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.
[0326] 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.
[0327] 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.
[0328] 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
[0329] A siNA molecule of the invention can be adapted for use to
prevent or treat cancer, leukemia, obliterative bronchiolitis,
acute glomerulonephritis, stroke (CVA), and/or inflammatory and
proliferative diseases, traits, conditions and/or disorders, and/or
any other trait, disease, disorder or condition that is related to
or will respond to the levels of PDGF and/or PDGFr 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; Wang et al., International PCT
publication Nos. WO 03/47518 and WO 03/46185),
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.
[0330] In another embodiment, the nucleic acid molecules of the
invention can also be formulated or complexed with
polyethyleneimine and derivatives thereof, such as
polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine
(PEI-PEG-GAL) or
polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine
(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acid
molecules of the invention are formulated as described in United
States Patent Application Publication No. 20030077829, incorporated
by reference herein in its entirety.
[0331] In one embodiment, a siNA molecule of the invention is
complexed with membrane disruptive agents such as those described
in U.S. Patent Application Publication No. 20010007666,
incorporated by reference herein in its entirety including the
drawings. In another embodiment, the membrane disruptive agent or
agents and the siNA molecule are also complexed with a cationic
lipid or helper lipid molecule, such as those lipids described in
U.S. Pat. No. 6,235,310, incorporated by reference herein in its
entirety including the drawings.
[0332] In one embodiment, a siNA molecule of the invention is
complexed with delivery systems as described in U.S. Patent
Application Publication No. 2003077829 and International PCT
Publication Nos. WO 00/03683 and WO 02/087541, all incorporated by
reference herein in their entirety including the drawings.
[0333] In one embodiment, the nucleic acid molecules of the
invention are administered via pulmonary delivery, such as by
inhalation of an aerosol or spray dried formulation administered by
an inhalation device or nebulizer, providing rapid local uptake of
the nucleic acid molecules into relevant pulmonary tissues. Solid
particulate compositions containing respirable dry particles of
micronized nucleic acid compositions can be prepared by grinding
dried or lyophilized nucleic acid compositions, and then passing
the micronized composition through, for example, a 400 mesh screen
to break up or separate out large agglomerates. A solid particulate
composition comprising the nucleic acid compositions of the
invention can optionally contain a dispersant which serves to
facilitate the formation of an aerosol as well as other therapeutic
compounds. A suitable dispersant is lactose, which can be blended
with the nucleic acid compound in any suitable ratio, such as a 1
to 1 ratio by weight.
[0334] Aerosols of liquid particles comprising a nucleic acid
composition of the invention can be produced by any suitable means,
such as with a nebulizer (see for example U.S. Pat. No. 4,501,729).
Nebulizers are commercially available devices which transform
solutions or suspensions of an active ingredient into a therapeutic
aerosol mist either by means of acceleration of a compressed gas,
typically air or oxygen, through a narrow venturi orifice or by
means of ultrasonic agitation. Suitable formulations for use in
nebulizers comprise the active ingredient in a liquid carrier in an
amount of up to 40% w/w preferably less than 20% w/w of the
formulation. The carrier is typically water or a dilute aqueous
alcoholic solution, preferably made isotonic with body fluids by
the addition of, for example, sodium chloride or other suitable
salts. Optional additives include preservatives if the formulation
is not prepared sterile, for example, methyl hydroxybenzoate,
anti-oxidants, flavorings, volatile oils, buffering agents and
emulsifiers and other formulation surfactants. The aerosols of
solid particles comprising the active composition and surfactant
can likewise be produced with any solid particulate aerosol
generator. Aerosol generators for administering solid particulate
therapeutics to a subject produce particles which are respirable,
as explained above, and generate a volume of aerosol containing a
predetermined metered dose of a therapeutic composition at a rate
suitable for human administration. One illustrative type of solid
particulate aerosol generator is an insufflator. Suitable
formulations for administration by insufflation include finely
comminuted powders which can be delivered by means of an
insufflator. In the insufflator, the powder, e.g., a metered dose
thereof effective to carry out the treatments described herein, is
contained in capsules or cartridges, typically made of gelatin or
plastic, which are either pierced or opened in situ and the powder
delivered by air drawn through the device upon inhalation or by
means of a manually-operated pump. The powder employed in the
insufflator consists either solely of the active ingredient or of a
powder blend comprising the active ingredient, a suitable powder
diluent, such as lactose, and an optional surfactant. The active
ingredient typically comprises from 0.1 to 100 w/w of the
formulation. A second type of illustrative aerosol generator
comprises a metered dose inhaler. Metered dose inhalers are
pressurized aerosol dispensers, typically containing a suspension
or solution formulation of the active ingredient in a liquified
propellant. During use these devices discharge the formulation
through a valve adapted to deliver a metered volume to produce a
fine particle spray containing the active ingredient. Suitable
propellants include certain chlorofluorocarbon compounds, for
example, dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane and mixtures thereof. The formulation can
additionally contain one or more co-solvents, for example, ethanol,
emulsifiers and other formulation surfactants, such as oleic acid
or sorbitan trioleate, anti-oxidants and suitable flavoring agents.
Other methods for pulmonary delivery are described in, for example
US Patent Application No. 20040037780, and U.S. Pat. Nos.
6,592,904; 6,582,728; 6,565,885.
[0335] In one embodiment, nucleic acid molecules of the invention
are administered to the central nervous system (CNS) or peripheral
nervous system (PNS). Experiments have demonstrated the efficient
in vivo uptake of nucleic acids by neurons. As an example of local
administration of nucleic acids to nerve cells, Sommer et al.,
1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in
which a 15 mer phosphorothioate antisense nucleic acid molecule to
c-fos is administered to rats via microinjection into the brain.
Antisense molecules labeled with
tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein
isothiocyanate (FITC) were taken up by exclusively by neurons
thirty minutes post-injection. A diffuse cytoplasmic staining and
nuclear staining was observed in these cells. As an example of
systemic administration of nucleic acid to nerve cells, Epa et al.,
2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo
mouse study in which beta-cyclodextrin-adamantane-oligonucleotide
conjugates were used to target the p75 neurotrophin receptor in
neuronally differentiated PC12 cells. Following a two week course
of IP administration, pronounced uptake of p75 neurotrophin
receptor antisense was observed in dorsal root ganglion (DRG)
cells. In addition, a marked and consistent down-regulation of p75
was observed in DRG neurons. Additional approaches to the targeting
of nucleic acid to neurons are described in Broaddus et al., 1998,
J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. Pharmocol.,
340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304;
Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999,
BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1),
83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid
molecules of the invention are therefore amenable to delivery to
and uptake by cells in the CNS and/or PNS.
[0336] The delivery of nucleic acid molecules of the invention to
the CNS is provided by a variety of different strategies.
Traditional approaches to CNS delivery that can be used include,
but are not limited to, intrathecal and intracerebroventricular
administration, implantation of catheters and pumps, direct
injection or perfusion at the site of injury or lesion, injection
into the brain arterial system, or by chemical or osmotic opening
of the blood-brain barrier. Other approaches can include the use of
various transport and carrier systems, for example though the use
of conjugates and biodegradable polymers. Furthermore, gene therapy
approaches, for example as described in Kaplitt et al., U.S. Pat.
No. 6,180,613 and Davidson, WO 04/013280, can be used to express
nucleic acid molecules in the CNS.
[0337] In one embodiment, delivery systems of the invention
include, for example, aqueous and nonaqueous gels, creams, multiple
emulsions, microemulsions, liposomes, ointments, aqueous and
nonaqueous solutions, lotions, aerosols, hydrocarbon bases and
powders, and can contain excipients such as solubilizers,
permeation enhancers (e.g., fatty acids, fatty acid esters, fatty
alcohols and amino acids), and hydrophilic polymers (e.g.,
polycarbophil and polyvinylpyrolidone). In one embodiment, the
pharmaceutically acceptable carrier is a liposome or a transdermal
enhancer. Examples of liposomes which can be used in this invention
include the following: (1) CellFectin, 1:1.5 (M/M) liposome
formulation of the cationic lipid
N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and
dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2)
Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid
and DOPE (Glen Research); (3) DOTAP
(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)
(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome
formulation of the polycationic lipid DOSPA and the neutral lipid
DOPE (GIBCO BRL).
[0338] In one embodiment, delivery systems of the invention include
patches, tablets, suppositories, pessaries, gels and creams, and
can contain excipients such as solubilizers and enhancers (e.g.,
propylene glycol, bile salts and amino acids), and other vehicles
(e.g., polyethylene glycol, fatty acid esters and derivatives, and
hydrophilic polymers such as hydroxypropylmethylcellulose and
hyaluronic acid).
[0339] In one embodiment, siNA molecules of the invention are
formulated or complexed with polyethylenimine (e.g., linear or
branched PEI) and/or polyethylenimine derivatives, including for
example grafted PEIs such as galactose PEI, cholesterol PEI,
antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI)
derivatives thereof (see for example Ogris et al., 2001, AAPA
PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14,
840-847; Kunath et al., 2002, Pharmaceutical Research, 19, 810-817;
Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et
al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002,
Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of
Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNAS USA,
96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,
60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry,
274, 19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99,
14640-14645; and Sagara, U.S. Pat. No. 6,586,524, incorporated by
reference herein.
[0340] In one embodiment, a siNA molecule of the invention
comprises a bioconjugate, for example a nucleic acid conjugate as
described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr.
30, 2003; U.S. Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S.
Pat. No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat. No.
5,214,136; U.S. Pat. No. 5,138,045, all incorporated by reference
herein.
[0341] 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 to 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 creams, gels, sprays, oils and other
suitable compositions for topical, dermal, or transdermal
administration as is known in the art.
[0342] 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.
[0343] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
e.g., systemic or local 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.
[0344] In one embodiment, siNA molecules of the invention are
administered to a subject by systemic administration in a
pharmaceutically acceptable composition or formulation. 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.
[0345] By "pharmaceutically acceptable formulation" or
"pharmaceutically acceptable composition" 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); biodegradable polymers, such as
poly (DL-lactide-coglycolide) microspheres for sustained release
delivery (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58); and
loaded nanoparticles, such as those made of polybutylcyanoacrylate.
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.
[0346] 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.
[0347] 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.
[0348] 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.
[0349] 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.
[0350] 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 monosterate or glyceryl distearate can be
employed.
[0351] 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.
[0352] 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.
[0353] 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
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 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.
[0358] 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.
[0359] 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.
[0360] 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.
[0361] 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.
[0362] 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.
[0363] 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.
[0364] 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 a 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.
[0365] 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).
[0366] 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).
[0367] 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).
[0368] 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).
[0369] 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.
[0370] 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.
[0371] 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.
PDGF/PDGFr Biology and Biochemistry
[0372] The following discussion is adapted from R&D Systems
Mini-Reviews and Tech Notes, Cytokine Mini-Reviews, Platelet
Derived Growth Factor, Copyright .COPYRGT.2002 R&D Systems.
Historically, it has been a goal of tissue culture researchers to
identify substances that provide universal growth or maintenance
factor characteristics for various cell lines and isolates. Early
tissue culture work demonstrated the superiority of serum over
plasma in stimulating the proliferation of fibroblasts in vitro.
These observations suggested that a factor released from platelets
during degranulation was probably responsible for the stimulatory
activity. Subsequent investigations clearly demonstrated that a
certain factor released from platelets upon clotting was capable of
promoting the growth of various types of cells. This factor was
subsequently purified from platelets and given the name
platelet-derived growth factor (PDGF). PDGF is now known to be
produced by a number of cell types besides platelets and it has
been found to be a mitogen for almost all mesenchymally-derived
cells, such as blood, muscle, bone/cartilage, and connective tissue
cells.
[0373] Three forms of PDGF have been identified to date. Each form
consists of a 30 kDa homo- or heterodimeric combination of two
genetically distinct, but structurally related, polypeptide chains
which are designated A and B chains, respectively. Although
considerable work has been done on the primary structure of each of
the chains of human PDGF, the process has been complicated by the
fact that each is synthesized as a propeptide, that splice variants
exist for the A chain, and that C-terminal proteolytic processing
apparently occurs for the B chain and possibly the A chain as
well.
[0374] The PDGF A chain is the product of a seven exon chromosomal
7 gene that gives rise to one of two distinct splice variants. The
"long" variant, a prepropeptide of 211 amino acid residues, is
synthesized with a signal peptide of 20 amino acid residues, a
propeptide sequence of 66 amino acid residues, and a mature chain
of 125 amino acid residues. In contrast, the "short" 196 amino acid
residue variant shows a 20 amino acid residue signal sequence, a 66
amino acid residue propeptide, and a 16-18 kDa, 110 amino acid
residue mature form. The difference between the long and short
results from alternative exon usage, with the extended form
utilizing exon 6 (18 amino acid residues), but not exon 7, and the
short form utilizing exon 7 (3 amino acid residues), but not exon
6. The difference between exon 6 utilization and exon 7 utilization
is not, however, limited to length. Within the 18 amino acid
residues of exon 6 lies an approximately 10 amino acid residue
sequence that signals cell retention. Failure to remove this
carboxyterminal peptide results in a failure to release freely
circulating PDGF. Retention under these circumstances implies
binding to either cell-surface glycosaminoglycans or intercellular
matrix. The short version contains no retention sequence and is
secreted into the circulatory system. It is presently unclear
whether any C-terminal processing of A chains occurs, but the short
variant's 110 amino acid residue mature peptide terminates with an
arginine residue. This suggests the possibility, as is the case for
the B chain, of a carboxypeptidase-mediated C-terminal truncation
to 109 amino acid residues with equilization of A and B chain
lengths for dimerization. No definitive mechanism for C-terminus
processing of the long form of the A chain has been elucidated and
it is not presently clear if this form is secreted. One potential
N-linked glycosylation site exists in the mature A chain, but not
the B chain, and it is suggested to be utilized. Normal cells such
as endothelial cell, macrophages, and fibroblasts are known to
concurrently express both types of A chain, with the short version
being the most abundant.
[0375] The PDGF B chain is the product of a six exon gene on
chromosome 22. The B chain gene is known to be identical to the
human c-sis gene, the normal human cell counterpart to the monkey
v-sis (simian sarcoma) virus gene. The protein coded for by c-sis
is a 27 kDa, 241 amino acid residue prepropeptide with a 20 amino
acid residue signal sequence, 61 amino acid residue propeptide, and
a 16 kDa, 160 amino acid residue "mature" polypeptide. C-terminal
cleavage of the mature B chain is believed to occur, resulting in a
final mature product of 12 kDa and 109 amino acid residues. This is
proposed to occur in two stages with a trypsin-like cleavage of
residues 111 to 160, followed by a carboxypeptidase cleavage of the
remaining arginine at residue 110. As with the long form of chain
A, a particular retention sequence approximately 10 amino acid
residues in length has also been identified in the B chain
C-terminus. Failure to remove this peptide also results in B chain
glycosaminoglycan retention. Dimerization of the A and B chains
involves two interchain disulfide bonds, and each chain overlaps
the other with a 6 or 7 amino acid residue extension at either end.
Within the 103 overlapping amino acid residues, the two chains
exhibit about 50% sequence identity.
[0376] Cells known to express PDGF are diverse. Cells that are
reported to express the A chain protein (both long and short
variants) include fibroblasts, endothelial cells, osteoblasts,
platelets, vascular smooth muscle cells, macrophages and Langerhans
cells, and fetal fibroblasts. Cells producing B chain protein
include fetal fibroblasts, endothelial cells, platelets,
macrophages, neurons and breast ductal epithelium. A number of cell
types have also been shown to express mRNA for the PDGF chains. In
particular, A chain mRNA has been found in type I astrocytes,
embryonic endodermal respiratory epithelium, renal mesangial cells,
and osteoclasts and chrondrocytes, while B chain mRNA has been
localized to embryonic endodermal respiratory epithelium, renal
mesangial cells and osteoblasts.
[0377] As with many growth factors, PDGF is now considered to be a
member of a larger family of factors. In addition to PDGF, this
family includes the homodimeric factors VEGF (vascular endothelial
growth factor) and PIGF (placental growth factor), VEGF/PIGF
heterodimers, and CTGF (connective tissue growth factor), a
PDGF-like factor secreted by human vascular endothelial cells and
fibroblasts. Relative to the PDGF isoforms, VEGF shows distant
analogy to PDGF-BB while PIGF corresponds to PDGF-AA. CTGF shows
little amino acid identity with PDGF A or B, but reacts with
anitsera produced against PDGF. Recently, the status of PDGF has
been re-evaluated based on analysis of its 3-dimensional structure.
Along with NGF, TGF-beta and glycoprotein hormones (human chorionic
gonadotrophic), PDGF is now classified as a member of the
cysteine-knot growth factor superfamily. Each member of this group
occurs as a dimer and is characterized by six cysteines which link
together to form a "molecular knot". The existence of this knot is
only revealed by 3-D analysis, making the criteria for admission to
this family unique among superfamilies.
[0378] An association is known to exist between alpha-2
macroglobulin (alpha-2M) and the B chain-containing PDGF forms, AB
and BB. alpha-2M is a circulating 720 kDa homotetrameric
glycoprotein produced by hepatocytes, macrophages and astrocytes
whose most widely reported function is that of a scavenger of
proteases. Although PDGF does not interact with the region
associated with protease entrapment, it does bind to other alpha-2M
sites not influenced by activation. PDGF-BB has been noted to bind
to both fast and slow alpha-2M and does so principally in a
noncovalent manner. Significantly, the binding is reversible, and
PDGF dissociation is suggested to occur at either low pH or when
equilibrium kinetics favor dissociation, such as might be the case
when PDGF is removed from circulation by binding to its own
receptors. Functionally, it is not clear what the role is for B
chain binding to alpha-2M. PDGF binding to the slow form seems to
result in its storage, as the alpha-2M receptor binding motif(s)
are not exposed, and the PDGF-alpha-2M complex simply circulates.
On the other hand, binding to fast or activated alpha-2M results in
its rapid clearance via alpha-2M receptors, bringing the PDGF
molecule close to its own receptors and perhaps facilitating a
secondary PDGF-PDGFR interaction.
[0379] Two distinct human PDGF receptor transmembrane binding
proteins have been identified, a 170 kDa, 1066 amino acid residue
alpha-receptor (PDGFR alpha) and a 190 kDa, 1074 amino acid residue
beta-receptor (PDGFR beta). The two receptor proteins are
structurally related and consist of an extracellular portion
containing five immunoglobulin-like domains, a single transmembrane
region, and an intracellular portion with a protein-tyrosine kinase
domain. A functional PDGF receptor is formed when the two chains of
a dimeric PDGF molecule each bind one of the above receptor
molecules, resulting in their approximation, dimerization and
activation. Between the two proteins, there is 44% overall sequence
identity. Within the extracellular domain, 30% of the amino acid
residues are identical. In addition, a 90 kDa soluble form of PDGFR
alpha, consisting of the extracellular segment of the
alpha-receptor, has been found in cell culture medium and in human
plasma. The above two transmembrane receptors share characteristics
with other growth factor receptors, such as the M-CSF receptor,
c-kit, and the FGF receptor family. High-affinity binding of PDGF
involves dimerization of the receptors, forming either homodimers
or heterodimers with the alpha and beta receptors/chains. Although
it appears that each subunit of dimeric PDGF binds to one receptor
monomer, it is unclear if these PDGF subunits need to be covalently
linked. Recent evidence suggests noncovalently linked B chains are
able to activate the PDGFR.
[0380] PDGFR alpha binds each of the three forms of PDGF dimers
with high affinity. Although PDGFR beta binds both PDGF-BB and
PDGF-AB with high affinity, it has no reported binding to PDGF-AA.
The apparent high-affinity binding of the AB dimer to the
beta-receptor must be interpreted with caution, however. Although
PDGF-AB can bind to mutant 3T3 cells displaying only
beta-receptors, it requires 100-fold more PDGF-AB to dimerize the
beta-receptors and activate the cells than is required for cells
also displaying alpha-receptors. Cells known to express only
alpha-receptors include oligodendroglial progenitors, liver
endothelial cells and mesothelium, and platelets. Cells expressing
only beta-receptors include CNS capillary endothelium, neurons and
Ito (fat storing) cells of the liver, plus monocytes/macrophages.
Cells showing coincident expression of alpha and beta receptors
include smooth muscle cells, fibroblasts, and Schwann cells.
[0381] Receptor binding by PDGF is known to activate intracellular
tyrosine kinase, leading to autophosphorylation of the cytoplasmic
domain of the receptor as well as phosphorylation of other
intracellular substrates. This reaction is described as one in
trans, i.e., the two receptor molecules of the receptor dimer
phosphorylate each other. Specific substrates identified with the
beta-receptor include Src, GTPase Activating Protein (GAP),
phospholypase Cg (PLCg) and phosphotidylinositol 3-phosphate. Both
PLCg and GAP seem to bind with different affinities to the a- and
beta-receptors, suggesting that the particular response of a cell
depends on the type of receptor it expresses and the type of PDGF
dimer to which it is exposed. In addition to the above, a
non-tyrosine phosphorylation-associated signal transduction pathway
can also be activated that involves the zinc finger protein erg-1
(early growth response gene 1).
[0382] Because there are differences between cells relative to the
amounts of alpha- and beta-receptors that they express, and because
of the variability in PDGF isomer binding to receptors, there is a
large range of possibilities for biological responses by PDGF. This
is reflected in at least four experimental systems where different
isoforms of PDGF elicit different results. Vascular smooth muscle
cells (SMC) and fibroblasts are both known to express both the
alpha- and beta-receptors. In SMC, PDGF-AA initiates cellular
hypertrophy (increased protein synthesis), while BB induces
hyperplasia (mitosis). In fibroblasts, the BB isoform initiates
chemotaxis, while AA inhibits chemotaxis. In dopaminergic neurons,
PDGF-AA promotes embryonic neuron fiber development, while BB
serves only as a survival or maintenance factor. Finally, within
the developing lung, the BB isoform regulates the growth and number
of respiratory tubule epithelial cells, while the AA isoform
directs the actual formation of branches arising from the
respiratory tubules.
[0383] In general, PDGF isoforms are potent mitogens for connective
tissue cells, including dermal fibroblasts, arterial smooth muscle
cells, chondrocytes and some epithelial and endothelial cells. In
addition to its activity as a mitogen, PDGF is chemotactic for
fibroblasts and smooth muscle cells, cells which also respond
mitogenically to PDGF, and for neutrophils and mononuclear cells,
cells for which PDGF is not a mitogen. There is a considerable body
of evidence to indicate that PDGF derived from macrophages, acting
as a chemotactic and mitogenic agent for smooth muscle cells,
contributes to the myointimal thickening of arterial walls
characteristic of atherosclerosis. Other reported activities for
PDGF include the stimulation of granule release by neutrophils and
monocytes, the facilitation of steroid synthesis by Leydig cells,
stimulation of neutrophil phagocytosis, modulation of
thrombospondin expression and secretion, upregulation of ICAM-1 in
vascular smooth muscle cells, and the transient induction of T cell
IL-2 secretion, accompanied by a down-regulation of IL-4 and
IFN-gamma production, which allow clonal expansion of
antigen-activated B and T helper lymphocytes prior to
differentiation. PDGF also appears to be ubiquitous in neurons
throughout the CNS, where it is suggested to play an important role
in neuron survival and regeneration, and in mediation of glial cell
proliferation, differentiation and migration.
[0384] The use of small interfering nucleic acid molecules
targeting PDGF and its receptors therefore provides a class of
novel therapeutic agents that can be used in the treatment of
cancers, proliferative diseases (e.g., restenosis), inflammatory
disease, or any other disease or condition that responds to
modulation of PDGF and PDGFr genes.
EXAMPLES
[0385] 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
[0386] 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.
[0387] 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.
[0388] 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.
[0389] 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 H2O, and 2 CV 50 mM NaOAc. The sample is loaded
and then washed with 1 CV H2O 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 H2O 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 H20 followed by 1 CV 1M NaCl and additional
H2O. The siNA duplex product is then eluted, for example, using 1
CV 20% aqueous CAN.
[0390] 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
[0391] 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
[0392] The following non-limiting steps can be used to carry out
the selection of siNAs targeting a given gene sequence or
transcript. [0393] 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.
[0394] 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. [0395] 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. [0396] 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. [0397] 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.
[0398] 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. [0399] 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.
[0400] 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. [0401] 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. [0402] 10.
Other design considerations can be used when selecting target
nucleic acid sequences, see, for example, Reynolds et al., 2004,
Nature Biotechnology Advanced Online Publication, 1 Feb. 2004,
doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research,
32, doi:10.1093/nar/gkh247.
[0403] In an alternate approach, a pool of siNA constructs specific
to a PDGF and/or PDGFr target sequence is used to screen for target
sites in cells expressing PDGF and/or PDGFr RNA, such as such human
aortic smooth muscle cells (e.g., HASMC), HeLa cells, or A549
cells. The general strategy used in this approach is shown in FIG.
9. A non-limiting example of such is a pool comprising sequences
having any of SEQ ID NOS 1-744. Cells expressing PDGF and/or PDGFr
(e.g., HASMC, HeLa cells, or A549 cells) are transfected with the
pool of siNA constructs and cells that demonstrate a phenotype
associated with PDGF and/or PDGFr 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 PDGF and/or PDGFr mRNA
levels or decreased PDGF and/or PDGFr protein expression), are
sequenced to determine the most suitable target site(s) within the
target PDGF and/or PDGFr RNA sequence.
Example 4
PDGF and/or PDGFr Targeted siNA Design
[0404] siNA target sites were chosen by analyzing sequences of the
PDGF and/or PDGFr 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.
[0405] 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
[0406] 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. 5,889,136; 6,008,400;
6,111,086 all incorporated by reference herein in their
entirety).
[0407] 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).
[0408] 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.
[0409] 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 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
[0410] An in vitro assay that recapitulates RNAi in a cell-free
system is used to evaluate siNA constructs targeting PDGF and/or
PDGFr 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 PDGF
and/or PDGFr 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 PDGF and/or PDGFr 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.
[0411] 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 G50 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 IMAGER.RTM.
(autoradiography) quantitation of bands representing intact control
RNA or RNA from control reactions without siNA and the cleavage
products generated by the assay.
[0412] In one embodiment, this assay is used to determine target
sites in the PDGF and/or PDGFr RNA target for siNA mediated RNAi
cleavage, wherein a plurality of siNA constructs are screened for
RNAi mediated cleavage of the PDGF and/or PDGFr 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 PDGF and/or PDGFr Target RNA
[0413] siNA molecules targeted to the human PDGF and/or PDGFr 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 PDGF and/or PDGFr RNA are given in
Tables II and III.
[0414] Two formats are used to test the efficacy of siNAs targeting
PDGF and/or PDGFr. First, the reagents are tested in cell culture
using, for example, HASMC, HeLa cells, or A549 cells to determine
the extent of RNA and protein inhibition. siNA reagents (e.g.; see
Tables II and III) are selected against the PDGF and/or PDGFr
target as described herein. RNA inhibition is measured after
delivery of these reagents by a suitable transfection agent to, for
example, HASMC, HeLa cells, or A549 cells. Relative amounts of
target RNA are measured versus actin using real-time PCR monitoring
of amplification (e.g., 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
[0415] Cells such as HASMC, HeLa cells, or A549 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 (Bio
Whittaker) at 37.degree. C. for 30 minutes 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.RTM. (Real-Time PCR Monitoring of Amplification) and
Lightcycler Quantification of mRNA
[0416] 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.RTM. analysis
(real-time PCR monitoring of amplification), 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 .mu.M each
dATP, dCTP, dGTP, and dTTP, IOU RNase Inhibitor (Promega), 1.25 U
AMPLITAQ GOLD.RTM. (DNA polymerase) (PE-Applied Biosystems) and 10
U M-MLV Reverse Transcriptase (Promega). The thermal cycling
conditions can consist of 30 minutes at 48.degree. C., 10 minutes
at 95.degree. C., followed by 40 cycles of 15 seconds at 95.degree.
C. and 1 minute 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/rxn) and normalizing to
.beta.-actin or GAPDH mRNA in parallel TAQMAN.RTM. reactions
(real-time PCR monitoring of amplification). 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 lightcycler. A standard curve is generated for each
primer pair using control cRNA. Values are represented as relative
expression to GAPDH in each sample.
Western Blotting
[0417] 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 PDGF and/or PDGFr
Gene Expression
Cell Culture
[0418] There are numerous cell culture systems that can be used to
analyze reduction of PDGF and/or PDGFr levels either directly or
indirectly by measuring downstream effects. For example, HASMC,
HeLa, or A549 cells can be used in cell culture experiments to
assess the efficacy of nucleic acid molecules of the invention. As
such, HASMC, HeLa, or A549 cells treated with nucleic acid
molecules of the invention (e.g., siNA) targeting PDGF and/or PDGFr
RNA would be expected to have decreased PDGF and/or PDGFr
expression capacity following stimulation with pro-inflammatory
cytokines compared to matched control nucleic acid molecules having
a scrambled or inactive sequence. In a non-limiting example, HASMC,
HeLa, or A549 cells are cultured and PDGF and/or PDGFr expression
is quantified, for example by time-resolved immunofluorometric
assay. PDGF and/or PDGFr 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
PDGF and/or PDGFr protein and RNA levels are quantitated. Dose
response assays are then performed to establish dose dependent
inhibition of PDGF and/or PDGFr expression.
[0419] 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
[0420] Evaluating the efficacy of anti-PDGF and/or PDGFr agents in
animal models is an important prerequisite to human clinical
trials. Barisoni et al., 1995, Am J. Pathol., 147, 1728-35 describe
a transgenic mouse model of polycystic kidney disease. Adult
polycystic kidney disease is believed to be the most frequent (
1/500) inherited genetic disorder in humans. Barisoni et al., supra
generated a genetic model of the disease in transgenic mice by
introducing a deregulated proto-oncogene c-myc specifically
expressed in the kidney. All transgenic lines produced develop
adult polycystic kidney disease in a reproducible manner. The
clinical phenotype observed in mice is present at birth and leads
to renal insufficiency in adulthood. Barisoni et al., supra
determined that abnormal proliferation and programmed cell death
are responsible for cystogenesis in polycystic kidney disease.
Furthermore, this phenomena is controlled by a specific c-myc
mechanism independent of the p53 pathway. A similar mechanism also
prevails in human autosomal dominant polycystic kidney disease.
Therefore, this murine model provides a useful model to understand
the polycystic kidney disease pathogenesis and can be used to
evaluate potential therapeutic agents such as siNA molecules of the
invention.
[0421] Other animal models known in the art can be used to evaluate
siNA molecules of the invention targeting PDGF and PDGFr for other
disease conditions, see for example Karas et al., 1992, Coronary
intimal proliferation after balloon injury and stenting in swine:
An animal model of restenosis. J Am Coll Cardiol. 20, 467-474;
Hele, 2001, The heterotopic tracheal allograft as an animal model
of obliterative bronchiolitis. Respir. Res., 2, 169-183; Floege et
al. 1999, Am. J. Pathol., 154, 169 (animal model of acute
glomerulonephritis). Similarly, using various animal models of
oncology known in the art, animals treated with siNA molecules of
the invention targeting PDGF and/or PDGFr RNA can be evaluated for
clinical response (e.g., decreased tumor size/metastasis) and/or
decreased levels of Myc RNA or protein.
Example 9
RNAi Mediated Inhibition of PDGF and/or PDGFr Expression
[0422] siNA constructs (Table III) are tested for efficacy in
reducing PDGF and/or PDGFr RNA expression in, for example, HASMC,
HeLa cells, or 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 is determined.
Example 10
Indications
[0423] The present body of knowledge in PDGF and PDGFr research
indicates the need for methods and compounds that can regulate PDGF
and PDGFr gene product expression for research, diagnostic, and
therapeutic use. As described herein, the nucleic acid molecules of
the present invention can be used to treat leukemias, including
acute myelogenous leukemia (AML), chronic myelogenous leukemia
(CML), Acute lymphocytic leukemia (ALL), and chronic lymphocytic
leukemia; ovarian cancer, breast cancer, cancers of the head and
neck, lymphomas, such as mantle cell lymphoma, non-Hodgkin's
lymphoma, and Burkitt's lymphoma, adenoma, squamous cell carcinoma,
laryngeal carcinoma, multiple myeloma, melanoma, colorectal cancer,
prostate cancer, and inflammatory and proliferative diseases such
as restenosis, polycystic kidney disease, obliterative
bronchiolitis, acute glomerulonephritis, stroke (CVA), and any
other diseases or conditions that are related to or will respond to
the levels of PDGF and/or PDGFr in a cell or tissue, alone or in
combination with other therapies.
[0424] The use of radiation treatments and chemotherapeutics such
as Gemcytabine and cyclophosphamide are non-limiting examples of
chemotherapeutic agents that can be combined with or used in
conjunction with the nucleic acid molecules (e.g. siNA molecules)
of the instant invention. Those skilled in the art will recognize
that other anti-cancer and/or antiproliferative 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. 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 11
Diagnostic Uses
[0425] 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 can 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).
[0426] 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 RNAs 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.
[0427] 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.
[0428] 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.
[0429] 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.
[0430] 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.
[0431] 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 PDGFr and PDGF Accession Numbers NM_002609
Homo sapiens platelet-derived growth factor receptor, beta
polypeptide (PDGFRB), mRNA gi|15451788|ref|NM_002609.2|[15451788]
NM_006206 Homo sapiens platelet-derived growth factor receptor,
alpha polypeptide (PDGFRA), mRNA
gi|15451787|ref|NM_006206.2|[15451787] 1: BD166138 Platelet-derived
growth factor receptors
gi|27871950|dbj|BD166138.1||pat|JP|2002186490|3[27871950] BD166137
Platelet-derived growth factor receptors
gi|27871949|dbj|BD166137.1||pat|JP|2002186490|2[27871949] BD166136
Platelet-derived growth factor receptors
gi|27871948|dbj|BD166136.1||pat|JP|2002186490|1[27871948] NM_033016
Homo sapiens platelet-derived growth factor beta polypeptide
(simian sarcoma viral (v-sis) oncogene homolog) (PDGFB), transcript
variant 2, mRNA gi|15451785|ref|NM_033016.1|[15451785] NM_002608
Homo sapiens platelet-derived growth factor beta polypeptide
(simian sarcoma viral (v-sis) oncogene homolog) (PDGFB), transcript
variant 1, mRNA gi|4505680|ref|NM_002608.1|[4505680] M59423 Human
platelet-derived growth factor A-chain (PDGF) gene, 5' end and
promoter region gi|189877|gb|M59423.1|HUMPGDF[189877] Y14326 Homo
sapiens platelet derived growth factor, B-chain 5'UTR
gi|2832416|emb|Y14326.1|HSPDGFBC[2832416] X83705 H. sapiens mRNA
for c-sis proto-oncogene gi|951023|emb|X83705.1|HSRNASIS[951023]
X00562 Human proto-oncogene c-sis fragment for PDGF B chain
precursor (platelet-derived growth factor)
gi|36477|emb|X00562.1|HSSISB5[36477] X00561 Human proto-oncogene
c-sis fragment for PDGF B chain precursor (platelet-derived growth
factor) gi|36474|emb|X00561.1|HSSISB4[36474] X00560 Human
proto-oncogene c-sis fragment for PDGF B chain precursor
(platelet-derived growth factor)
gi|36472|emb|X00560.1|HSSISB3[36472] X00559 Human proto-oncogene
c-sis fragment for PDGF B chain precursor (platelet-derived growth
factor) gi|36470|emb|X00559.1|HSSISB2[36470] X00556 Human
proto-oncogene c-sis fragment for PDGF B chain precursor
(platelet-derived growth factor)
gi|36468|emb|X00556.1|HSSISB1[36468] X02811 Human mRNA for
platelet-derived growth factor B chain (PDGF-B)
gi|35371|emb|X02811.1|HSPDGFB[35371] X03795 Human mRNA for platelet
derived growth factor A-chain (PDGF-A)
gi|35365|emb|X03795.1|HSPDGFAR[35365] X06374 Human mRNA for
platelet-derived growth factor PDGF-A
gi|35363|emb|X06374.1|HSPDGFA[35363] AF417590 Homo sapiens
platelet-derived growth factor A chain (PDGFA) gene, exon 1 and
partial sequence gi|16033732|gb|AF417590.1|AF417590[16033732]
NM_006206 Homo sapiens platelet-derived growth factor receptor,
alpha polypeptide (PDGFRA), mRNA
gi|15451787|ref|NM_006206.2|[15451787] AF244813 Homo sapiens
platelet-derived growth factor C mRNA, complete cds
gi|8886883|gb|AF244813.1|AF244813[8886883] NM_002607 Homo sapiens
platelet-derived growth factor alpha polypeptide (PDGFA),
transcript variant 1, mRNA
gi|15208657|ref|NM_002607.2|[15208657]
TABLE-US-00002 TABLE II PDGFRB siNA AND TARGET SEQUENCES PDGFRB
NM_002609.2 Seq Seq Seq Pos Seq ID UPos Upper seq ID LPos Lower seq
ID 3 CCCCUCAGCCCUGCUGCCC 1 3 CCCCUCAGCCCUGCUGCCC 1 21
GGGCAGCAGGGCUGAGGGG 312 21 CAGCACGAGCCUGUGCUCG 2 21
CAGCACGAGCCUGUGCUCG 2 39 CGAGCACAGGCUCGUGCUG 313 39
GCCCUGCCCAACGCAGACA 3 39 GCCCUGCCCAACGCAGACA 3 57
UGUCUGCGUUGGGCAGGGC 314 57 AGCCAGACCCAGGGCGGCC 4 57
AGCCAGACCCAGGGCGGCC 4 75 GGCCGCCCUGGGUCUGGCU 315 75
CCCUCUGGCGGCUCUGCUC 5 75 CCCUCUGGCGGCUCUGCUC 5 93
GAGCAGAGCCGCCAGAGGG 316 93 CCUCCCGAAGGAUGCUUGG 6 93
CCUCCCGAAGGAUGCUUGG 6 111 CCAAGCAUCCUUCGGGAGG 317 111
GGGAGUGAGGCGAAGCUGG 7 111 GGGAGUGAGGCGAAGCUGG 7 129
CCAGCUUCGCCUCACUCCC 318 129 GGCGCUCCUCUCCCCUACA 8 129
GGCGCUCCUCUCCCCUACA 8 147 UGUAGGGGAGAGGAGCGCC 319 147
AGCAGCCCCCUUCCUCCAU 9 147 AGCAGCCCCCUUCCUCCAU 9 165
AUGGAGGAAGGGGGCUGCU 320 165 UCCCUCUGUUCUCCUGAGC 10 165
UCCCUCUGUUCUCCUGAGC 10 183 GCUCAGGAGAACAGAGGGA 321 183
CCUUCAGGAGCCUGCACCA 11 183 CCUUCAGGAGCCUGCACCA 11 201
UGGUGCAGGCUCCUGAAGG 322 201 AGUCCUGCCUGUCCUUCUA 12 201
AGUCCUGCCUGUCCUUCUA 12 219 UAGAAGGACAGGCAGGACU 323 219
ACUCAGCUGUUACCCACUC 13 219 ACUCAGCUGUUACCCACUC 13 237
GAGUGGGUAACAGCUGAGU 324 237 CUGGGACCAGCAGUCUUUC 14 237
CUGGGACCAGCAGUCUUUC 14 255 GAAAGACUGCUGGUCCCAG 325 255
CUGAUAACUGGGAGAGGGC 15 255 CUGAUAACUGGGAGAGGGC 15 273
GCCCUCUCCCAGUUAUCAG 326 273 CAGUAAGGAGGACUUCCUG 16 273
CAGUAAGGAGGACUUCCUG 16 291 CAGGAAGUCCUCCUUACUG 327 291
GGAGGGGGUGACUGUCCAG 17 291 GGAGGGGGUGACUGUCCAG 17 309
CUGGACAGUCACCCCCUCC 328 309 GAGCCUGGAACUGUGCCCA 18 309
GAGCCUGGAACUGUGCCCA 18 327 UGGGCACAGUUCCAGGCUC 329 327
ACACCAGAAGCCAUCAGCA 19 327 ACACCAGAAGCCAUCAGCA 19 345
UGCUGAUGGCUUCUGGUGU 330 345 AGCAAGGACACCAUGCGGC 20 345
AGCAAGGACACCAUGCGGC 20 363 GCCGCAUGGUGUCCUUGCU 331 363
CUUCCGGGUGCGAUGCCAG 21 363 CUUCCGGGUGCGAUGCCAG 21 381
CUGGCAUCGCACCCGGAAG 332 381 GCUCUGGCCCUCAAAGGCG 22 381
GCUCUGGCCCUCAAAGGCG 22 399 CGCCUUUGAGGGCCAGAGC 333 399
GAGCUGCUGUUGCUGUCUC 23 399 GAGCUGCUGUUGCUGUCUC 23 417
GAGACAGCAACAGCAGCUC 334 417 CUCCUGUUACUUCUGGAAC 24 417
CUCCUGUUACUUCUGGAAC 24 435 GUUCCAGAAGUAACAGGAG 335 435
CCACAGAUCUCUCAGGGCC 25 435 CCACAGAUCUCUCAGGGCC 25 453
GGCCCUGAGAGAUCUGUGG 336 453 CUGGUCGUCACACCCCCGG 26 453
CUGGUCGUCACACCCCCGG 26 471 CCGGGGGUGUGACGACCAG 337 471
GGGCCAGAGCUUGUCCUCA 27 471 GGGCCAGAGCUUGUCCUCA 27 489
UGAGGACAAGCUCUGGCCC 338 489 AAUGUCUCCAGCACCUUCG 28 489
AAUGUCUCCAGCACCUUCG 28 507 CGAAGGUGCUGGAGACAUU 339 507
GUUCUGACCUGCUCGGGUU 29 507 GUUCUGACCUGCUCGGGUU 29 525
AACCCGAGCAGGUCAGAAC 340 525 UCAGCUCCGGUGGUGUGGG 30 525
UCAGCUCCGGUGGUGUGGG 30 543 CCCACACCACCGGAGCUGA 341 543
GAACGGAUGUCCCAGGAGC 31 543 GAACGGAUGUCCCAGGAGC 31 561
GCUCCUGGGACAUCCGUUC 342 561 CCCCCACAGGAAAUGGCCA 32 561
CCCCCACAGGAAAUGGCCA 32 579 UGGCCAUUUCCUGUGGGGG 343 579
AAGGCCCAGGAUGGCACCU 33 579 AAGGCCCAGGAUGGCACCU 33 597
AGGUGCCAUCCUGGGCCUU 344 597 UUCUCCAGCGUGCUCACAC 34 597
UUCUCCAGCGUGCUCACAC 34 615 GUGUGAGCACGCUGGAGAA 345 615
CUGACCAACCUCACUGGGC 35 615 CUGACCAACCUCACUGGGC 35 633
GCCCAGUGAGGUUGGUCAG 346 633 CUAGACACGGGAGAAUACU 36 633
CUAGACACGGGAGAAUACU 36 651 AGUAUUCUCCCGUGUCUAG 347 651
UUUUGCACCCACAAUGACU 37 651 UUUUGCACCCACAAUGACU 37 669
AGUCAUUGUGGGUGCAAAA 348 669 UCCCGUGGACUGGAGACCG 38 669
UCCCGUGGACUGGAGACCG 38 687 CGGUCUCCAGUCCACGGGA 349 687
GAUGAGCGGAAACGGCUCU 39 687 GAUGAGCGGAAACGGCUCU 39 705
AGAGCCGUUUCCGCUCAUC 350 705 UACAUCUUUGUGCCAGAUC 40 705
UACAUCUUUGUGCCAGAUC 40 723 GAUCUGGCACAAAGAUGUA 351 723
CCCACCGUGGGCUUCCUCC 41 723 CCCACCGUGGGCUUCCUCC 41 741
GGAGGAAGCCCACGGUGGG 352 741 CCUAAUGAUGCCGAGGAAC 42 741
CCUAAUGAUGCCGAGGAAC 42 759 GUUCCUCGGCAUCAUUAGG 353 759
CUAUUCAUCUUUCUCACGG 43 759 CUAUUCAUCUUUCUCACGG 43 777
CCGUGAGAAAGAUGAAUAG 354 777 GAAAUAACUGAGAUCACCA 44 777
GAAAUAACUGAGAUCACCA 44 795 UGGUGAUCUCAGUUAUUUC 355 795
AUUCCAUGCCGAGUAACAG 45 795 AUUCCAUGCCGAGUAACAG 45 813
CUGUUACUCGGCAUGGAAU 356 813 GACCCACAGCUGGUGGUGA 46 813
GACCCACAGCUGGUGGUGA 46 831 UCACCACCAGCUGUGGGUC 357 831
ACACUGCACGAGAAGAAAG 47 831 ACACUGCACGAGAAGAAAG 47 849
CUUUCUUCUCGUGCAGUGU 358 849 GGGGACGUUGCACUGCCUG 48 849
GGGGACGUUGCACUGCCUG 48 867 CAGGCAGUGCAACGUCCCC 359 867
GUCCCCUAUGAUCACCAAC 49 867 GUCCCCUAUGAUCACCAAC 49 885
GUUGGUGAUCAUAGGGGAC 360 885 CGUGGCUUUUCUGGUAUCU 50 885
CGUGGCUUUUCUGGUAUCU 50 903 AGAUACCAGAAAAGCCACG 361 903
UUUGAGGACAGAAGCUACA 51 903 UUUGAGGACAGAAGCUACA 51 921
UGUAGCUUCUGUCCUCAAA 362 921 AUCUGCAAAACCACCAUUG 52 921
AUCUGCAAAACCACCAUUG 52 939 CAAUGGUGGUUUUGCAGAU 363 939
GGGGACAGGGAGGUGGAUU 53 939 GGGGACAGGGAGGUGGAUU 53 957
AAUCCACCUCCCUGUCCCC 364 957 UCUGAUGCCUACUAUGUCU 54 957
UCUGAUGCCUACUAUGUCU 54 975 AGACAUAGUAGGCAUCAGA 365 975
UACAGACUCCAGGUGUCAU 55 975 UACAGACUCCAGGUGUCAU 55 993
AUGACACCUGGAGUCUGUA 366 993 UCCAUCAACGUCUCUGUGA 56 993
UCCAUCAACGUCUCUGUGA 56 1011 UCACAGAGACGUUGAUGGA 367 1011
AACGCAGUGCAGACUGUGG 57 1011 AACGCAGUGCAGACUGUGG 57 1029
CCACAGUCUGCACUGCGUU 368 1029 GUCCGCCAGGGUGAGAACA 58 1029
GUCCGCCAGGGUGAGAACA 58 1047 UGUUCUCACCCUGGCGGAC 369 1047
AUCACCCUCAUGUGCAUUG 59 1047 AUCACCCUCAUGUGCAUUG 59 1065
CAAUGCACAUGAGGGUGAU 370 1065 GUGAUCGGGAAUGAGGUGG 60 1065
GUGAUCGGGAAUGAGGUGG 60 1083 CCACCUCAUUCCCGAUCAC 371 1083
GUCAACUUCGAGUGGACAU 61 1083 GUCAACUUCGAGUGGACAU 61 1101
AUGUCCACUCGAAGUUGAC 372 1101 UACCCCCGCAAAGAAAGUG 62 1101
UACCCCCGCAAAGAAAGUG 62 1119 CACUUUCUUUGCGGGGGUA 373 1119
GGGCGGCUGGUGGAGCCGG 63 1119 GGGCGGCUGGUGGAGCCGG 63 1137
CCGGCUCCACCAGCCGCCC 374 1137 GUGACUGACUUCCUCUUGG 64 1137
GUGACUGACUUCCUCUUGG 64 1155 CCAAGAGGAAGUCAGUCAC 375 1155
GAUAUGCCUUACCACAUCC 65 1155 GAUAUGCCUUACCACAUCC 65 1173
GGAUGUGGUAAGGCAUAUC 376 1173 CGCUCCAUCCUGCACAUCC 66 1173
CGCUCCAUCCUGCACAUCC 66 1191 GGAUGUGCAGGAUGGAGCG 377 1191
CCCAGUGCCGAGUUAGAAG 67 1191 CCCAGUGCCGAGUUAGAAG 67 1209
CUUCUAACUCGGCACUGGG 378 1209 GACUCGGGGACCUACACCU 68 1209
GACUCGGGGACCUACACCU 68 1227 AGGUGUAGGUCCCCCAGUC 379 1227
UGCAAUGUGACGGAGAGUG 69 1227 UGCAAUGUGACGGAGAGUG 69 1245
CACUCUCCGUCACAUUGCA 380 1245 GUGAAUGACCAUCAGGAUG 70 1245
GUGAAUGACCAUCAGGAUG 70 1263 CAUCCUGAUGGUCAUUCAC 381 1263
GAAAAGGCCAUCAACAUCA 71 1263 GAAAAGGCCAUCAACAUCA 71 1281
UGAUGUUGAUGGCCUUUUC 382 1281 ACCGUGGUUGAGAGCGGCU 72 1281
ACCGUGGUUGAGAGCGGCU 72 1299 AGCCGCUCUCAACCACGGU 383 1299
UACGUGCGGCUCCUGGGAG 73 1299 UACGUGCGGCUCCUGGGAG 73 1317
CUCCCAGGAGCCGCACGUA 384 1317 GAGGUGGGCACACUACAAU 74 1317
GAGGUGGGCACACUACAAU 74 1335 AUUGUAGUGUGCCCACCUC 385 1335
UUUGCUGAGCUGCAUCGGA 75 1335 UUUGCUGAGCUGCAUCGGA 75 1353
UCCGAUGCAGCUCAGCAAA 386 1353 AGCCGGACACUGCAGGUAG 76 1353
AGCCGGACACUGCAGGUAG 76 1371 CUACCUGCAGUGUCCGGCU 387 1371
GUGUUCGAGGCCUACCCAC 77 1371 GUGUUCGAGGCCUACCCAC 77 1389
GUGGGUAGGCCUCGAACAC 388 1389 CCGCCCACUGUCCUGUGGU 78 1389
CCGCCCACUGUCCUGUGGU 78 1407 ACCACAGGACAGUGGGCGG 389 1407
UUCAAAGACAACCGCACCC 79 1407 UUCAAAGACAACCGCACCC 79 1425
GGGUGCGGUUGUCUUUGAA 390 1425 CUGGGCGACUCCAGCGCUG 80 1425
CUGGGCGACUCCAGCGCUG 80 1443 CAGCGCUGGAGUCGCCCAG 391 1443
GGCGAAAUCGCCCUGUCCA 81 1443 GGCGAAAUCGCCCUGUCCA 81 1461
UGGACAGGGCGAUUUCGCC 392
1461 ACGCGCAACGUGUCGGAGA 82 1461 ACGCGCAACGUGUCGGAGA 82 1479
UCUCCGACACGUUGCGCGU 393 1479 ACCCGGUAUGUGUCAGAGC 83 1479
ACCCGGUAUGUGUCAGAGC 83 1497 GCUCUGACACAUACCGGGU 394 1497
CUGACACUGGUUCGCGUGA 84 1497 CUGACACUGGUUCGCGUGA 84 1515
UCACGCGAACCAGUGUCAG 395 1515 AAGGUGGCAGAGGCUGGCC 85 1515
AAGGUGGCAGAGGCUGGCC 85 1533 GGCCAGCCUCUGCCACCUU 396 1533
CACUACACCAUGCGGGCCU 86 1533 CACUACACCAUGCGGGCCU 86 1551
AGGCCCGCAUGGUGUAGUG 397 1551 UUCCAUGAGGAUGCUGAGG 87 1551
UUCCAUGAGGAUGCUGAGG 87 1569 CCUCAGCAUCCUCAUGGAA 398 1569
GUCCAGCUCUCCUUCCAGC 88 1569 GUCCAGCUCUCCUUCCAGC 88 1587
GCUGGAAGGAGAGCUGGAC 399 1587 CUACAGAUCAAUGUCCCUG 89 1587
CUACAGAUCAAUGUCCCUG 89 1605 CAGGGACAUUGAUCUGUAG 400 1605
GUCCGAGUGCUGGAGCUAA 90 1605 GUCCGAGUGCUGGAGCUAA 90 1623
UUAGCUCCAGCACUCGGAC 401 1623 AGUGAGAGCCACCCUGACA 91 1623
AGUGAGAGCCACCCUGACA 91 1641 UGUCAGGGUGGCUCUCACU 402 1641
AGUGGGGAACAGACAGUCC 92 1641 AGUGGGGAACAGACAGUCC 92 1659
GGACUGUCUGUUCCCCACU 403 1659 CGCUGUCGUGGCCGGGGCA 93 1659
CGCUGUCGUGGCCGGGGCA 93 1677 UGCCCCGGCCACGACAGCG 404 1677
AUGCCCCAGCCGAACAUCA 94 1677 AUGCCCCAGCCGAACAUCA 94 1695
UGAUGUUCGGCUGGGGCAU 405 1695 AUCUGGUCUGCCUGCAGAG 95 1695
AUCUGGUCUGCCUGCAGAG 95 1713 CUCUGCAGGCAGACCAGAU 406 1713
GACCUCAAAAGGUGUCCAC 96 1713 GACCUCAAAAGGUGUCCAC 96 1731
GUGGACACCUUUUGAGGUC 407 1731 CGUGAGCUGCCGCCCACGC 97 1731
CGUGAGCUGCCGCCCACGC 97 1749 GCGUGGGCGGCAGCUCACG 408 1749
CUGCUGGGGAACAGUUCCG 98 1749 CUGCUGGGGAACAGUUCCG 98 1767
CGGAACUGUUCCCCAGCAG 409 1767 GAAGAGGAGAGCCAGCUGG 99 1767
GAAGAGGAGAGCCAGCUGG 99 1785 CCAGCUGGCUCUCCUCUUC 410 1785
GAGACUAACGUGACGUACU 100 1785 GAGACUAACGUGACGUACU 100 1803
AGUACGUCACGUUAGUCUC 411 1803 UGGGAGGAGGAGCAGGAGU 101 1803
UGGGAGGAGGAGCAGGAGU 101 1821 ACUCCUGGUCCUCCUCCCA 412 1821
UUUGAGGUGGUGAGCACAC 102 1821 UUUGAGGUGGUGAGCACAC 102 1839
GUGUGCUCACCACCUCAAA 413 1839 CUGCGUCUGCAGCACGUGG 103 1839
CUGCGUCUGCAGCACGUGG 103 1857 CCACGUGCUGCAGACGCAG 414 1857
GAUCGGCCACUGUCGGUGC 104 1857 GAUCGGCCACUGUCGGUGC 104 1875
GCACCGACAGUGGCCGAUC 415 1875 CGCUGCACGCUGCGCAACG 105 1875
CGCUGCACGCUGCGCAACG 105 1893 CGUUGCGCAGCGUGCAGCG 416 1893
GCUGUGGGCCAGGACACGC 106 1893 GCUGUGGGCCAGGACACGC 106 1911
GCGUGUCCUGGCCCACAGC 417 1911 CAGGAGGUCAUCGUGGUGC 107 1911
CAGGAGGUCAUCGUGGUGC 107 1929 GCACCACGAUGACCUCCUG 418 1929
CCACACUCCUUGCCCUUUA 108 1929 CCACACUCCUUGCCCUUUA 108 1947
UAAAGGGCAAGGAGUGUGG 419 1947 AAGGUGGUGGUGAUCUCAG 109 1947
AAGGUGGUGGUGAUCUCAG 109 1965 CUGAGAUCACCACCACCUU 420 1965
GCCAUCCUGGCCCUGGUGG 110 1965 GCCAUCCUGGCCCUGGUGG 110 1983
CCACCAGGGCCAGGAUGGC 421 1983 GUGCUCACCAUCAUCUCCC 111 1983
GUGCUCACCAUCAUCUCCC 111 2001 GGGAGAUGAUGGUGAGCAC 422 2001
CUUAUCAUCCUCAUCAUGC 112 2001 CUUAUCAUCCUCAUCAUGC 112 2019
GCAUGAUGAGGAUGAUAAG 423 2019 CUUUGGCAGAAGAAGCCAC 113 2019
CUUUGGCAGAAGAAGCCAC 113 2037 GUGGCUUCUUCUGCCAAAG 424 2037
CGUUACGAGAUCCGAUGGA 114 2037 CGUUACGAGAUCCGAUGGA 114 2055
UCCAUCGGAUCUCGUAACG 425 2055 AAGGUGAUUGAGUCUGUGA 115 2055
AAGGUGAUUGAGUCUGUGA 115 2073 UCACAGACUCAAUCACCUU 426 2073
AGCUCUGACGGCCAUGAGU 116 2073 AGCUCUGACGGCCAUGAGU 116 2091
ACUCAUGGCCGUCAGAGCU 427 2091 UACAUCUACGUGGACCCCA 117 2091
UACAUCUACGUGGACCCCA 117 2109 UGGGGUCCACGUAGAUGUA 428 2109
AUGCAGCUGCCCUAUGACU 118 2109 AUGCAGCUGCCCUAUGACU 118 2127
AGUCAUAGGGCAGCUGCAU 429 2127 UCCACGUGGGAGCUGCCGC 119 2127
UCCACGUGGGAGCUGCCGC 119 2145 GCGGCAGCUCCCACGUGGA 430 2145
CGGGACCAGCUUGUGCUGG 120 2145 CGGGACCAGCUUGUGCUGG 120 2163
CCAGCACAAGCUGGUCCCG 431 2163 GGACGCACCCUCGGCUCUG 121 2163
GGACGCACCCUCGGCUCUG 121 2181 CAGAGCCGAGGGUGCGUCC 432 2181
GGGGCCUUUGGGCAGGUGG 122 2181 GGGGCCUUUGGGCAGGUGG 122 2199
CCACCUGCCCAAAGGCCCC 433 2199 GUGGAGGCCACGGCUCAUG 123 2199
GUGGAGGCCACGGCUCAUG 123 2217 CAUGAGCCGUGGCCUCCAC 434 2217
GGCCUGAGCCAUUCUCAGG 124 2217 GGCCUGAGCCAUUCUCAGG 124 2235
CCUGAGAAUGGCUCAGGCC 435 2235 GCCACGAUGAAAGUGGCCG 125 2235
GCCACGAUGAAAGUGGCCG 125 2253 CGGCCACUUUCAUCGUGGC 436 2253
GUCAAGAUGCUUAAAUCCA 126 2253 GUCAAGAUGCUUAAAUCCA 126 2271
UGGAUUUAAGCAUCUUGAC 437 2271 ACAGCCCGCAGCAGUGAGA 127 2271
ACAGCCCGCAGCAGUGAGA 127 2289 UCUCACUGCUGCGGGCUGU 438 2289
AAGCAAGCCCUUAUGUCGG 128 2289 AAGCAAGCCCUUAUGUCGG 128 2307
CCGACAUAAGGGCUUGCUU 439 2307 GAGCUGAAGAUCAUGAGUC 129 2307
GAGCUGAAGAUCAUGAGUC 129 2325 GACUCAUGAUCUUCAGCUC 440 2325
CACCUUGGGCCCCACCUGA 130 2325 CACCUUGGGCCCCACCUGA 130 2343
UCAGGUGGGGCCCAAGGUG 441 2343 AACGUGGUCAACCUGUUGG 131 2343
AACGUGGUCAACCUGUUGG 131 2361 CCAACAGGUUGACCACGUU 442 2361
GGGGCCUGCACCAAAGGAG 132 2361 GGGGCCUGCACCAAAGGAG 132 2379
CUCCUUUGGUGCAGGCCCC 443 2379 GGACCCAUCUAUAUCAUCA 133 2379
GGACCCAUCUAUAUCAUCA 133 2397 UGAUGAUAUAGAUGGGUCC 444 2397
ACUGAGUACUGCCGCUACG 134 2397 ACUGAGUACUGCCGCUACG 134 2415
CGUAGCGGCAGUACUCAGU 445 2415 GGAGACCUGGUGGACUACC 135 2415
GGAGACCUGGUGGACUACC 135 2433 GGUAGUCCACCAGGUCUCC 446 2433
CUGCACCGCAACAAACACA 136 2433 CUGCACCGCAACAAACACA 136 2451
UGUGUUUGUUGCGGUGCAG 447 2451 ACCUUCCUGCAGCACCACU 137 2451
ACCUUCCUGCAGCACCACU 137 2469 AGUGGUGCUGCAGGAAGGU 448 2469
UCCGACAAGCGCCGCCCGC 138 2469 UCCGACAAGCGCCGCCCGC 138 2487
GCGGGCGGCGCUUGUCGGA 449 2487 CCCAGCGCGGAGCUCUACA 139 2487
CCCAGCGCGGAGCUCUACA 139 2505 UGUAGAGCUCCGCGCUGGG 450 2505
AGCAAUGCUCUGCCCGUUG 140 2505 AGCAAUGCUCUGCCCGUUG 140 2523
CAACGGGCAGAGCAUUGCU 451 2523 GGGCUCCCCCUGCCCAGCC 141 2523
GGGCUCCCCCUGCCCAGCC 141 2541 GGCUGGGCAGGGGGAGCCC 452 2541
CAUGUGUCCUUGACCGGGG 142 2541 CAUGUGUCCUUGACCGGGG 142 2559
CCCCGGUCAAGGACACAUG 453 2559 GAGAGCGACGGUGGCUACA 143 2559
GAGAGCGACGGUGGCUACA 143 2577 UGUAGCCACCGUCGCUCUC 454 2577
AUGGACAUGAGCAAGGACG 144 2577 AUGGACAUGAGCAAGGACG 144 2595
CGUCCUUGCUCAUGUCCAU 455 2595 GAGUCGGUGGACUAUGUGC 145 2595
GAGUCGGUGGACUAUGUGC 145 2613 GCACAUAGUCCACCGACUC 456 2613
CCCAUGCUGGACAUGAAAG 146 2613 CCCAUGCUGGACAUGAAAG 146 2631
CUUUCAUGUCCAGCAUGGG 457 2631 GGAGACGUCAAAUAUGCAG 147 2631
GGAGACGUCAAAUAUGCAG 147 2649 CUGCAUAUUUGACGUCUCC 458 2649
GACAUCGAGUCCUCCAACU 148 2649 GACAUCGAGUCCUCCAACU 148 2667
AGUUGGAGGACUCGAUGUC 459 2667 UACAUGGCCCCUUACGAUA 149 2667
UACAUGGCCCCUUACGAUA 149 2685 UAUCGUAAGGGGCCAUGUA 460 2685
AACUACGUUCCCUCUGCCC 150 2685 AACUACGUUCCCUCUGCCC 150 2703
GGGCAGAGGGAACGUAGUU 461 2703 CCUGAGAGGACCUGCCGAG 151 2703
CCUGAGAGGACCUGCCGAG 151 2721 CUCGGCAGGUCCUCUCAGG 462 2721
GCAACUUUGAUCAACGAGU 152 2721 GCAACUUUGAUCAACGAGU 152 2739
ACUCGUUGAUCAAAGUUGC 463 2739 UCUCCAGUGCUAAGCUACA 153 2739
UCUCCAGUGCUAAGCUACA 153 2757 UGUAGCUUAGCACUGGAGA 464 2757
AUGGACCUCGUGGGCUUCA 154 2757 AUGGACCUCGUGGGCUUCA 154 2775
UGAAGCCCACGAGGUCCAU 465 2775 AGCUACCAGGUGGCCAAUG 155 2775
AGCUACCAGGUGGCCAAUG 155 2793 CAUUGGCCACCUGGUAGCU 466 2793
GGCAUGGAGUUUCUGGCCU 156 2793 GGCAUGGAGUUUCUGGCCU 156 2811
AGGCCAGAAACUCCAUGCC 467 2811 UCCAAGAACUGCGUCCACA 157 2811
UCCAAGAACUGCGUCCACA 157 2829 UGUGGACGCAGUUCUUGGA 468 2829
AGAGACCUGGCGGCUAGGA 158 2829 AGAGACCUGGCGGCUAGGA 158 2847
UCCUAGCCGCCAGGUCUCU 469 2847 AACGUGCUCAUCUGUGAAG 159 2847
AACGUGCUCAUCUGUGAAG 159 2865 CUUCACAGAUGAGCACGUU 470 2865
GGCAAGCUGGUCAAGAUCU 160 2865 GGCAAGCUGGUCAAGAUCU 160 2883
AGAUCUUGACCAGCUUGCC 471 2883 UGUGACUUUGGCCUGGCUC 161 2883
UGUGACUUUGGCCUGGCUC 161 2901 GAGCCAGGCCAAAGUCACA 472 2901
CGAGACAUCAUGCGGGACU 162 2901 CGAGACAUCAUGCGGGACU 162 2919
AGUCCCGGAUGAUGUCUCG 473 2919 UCGAAUUACAUCUCCAAAG 163 2919
UCGAAUUACAUCUCCAAAG 163 2937 CUUUGGAGAUGUAAUUCGA 474 2937
GGCAGCACCUUUUUGCCUU 164 2937 GGCAGCACCUUUUUGCCUU 164 2955
AAGGCAAAAAGGUGCUGCC 475 2955 UUAAAGUGGAUGGCUCCGG 165 2955
UUAAAGUGGAUGGCUCCGG 165 2973 CCGGAGCCAUCCACUUUAA 476
2973 GAGAGCAUCUUCAACAGCC 166 2973 GAGAGCAUCUUCAACAGCC 166 2991
GGCUGUUGAAGAUGCUCUC 477 2991 CUCUACACCACCCUGAGCG 167 2991
CUCUACACCACCCUGAGCG 167 3009 CGCUCAGGGUGGUGUAGAG 478 3009
GACGUGUGGUCCUUCGGGA 166 3009 GACGUGUGGUCCUUCGGGA 168 3027
UCCCGAAGGACCACACGUC 479 3027 AUCCUGCUCUGGGAGAUCU 169 3027
AUCCUGCUCUGGGAGAUCU 169 3045 AGAUCUCCCAGAGCAGGAU 480 3045
UUCACCUUGGGUGGCACCC 170 3045 UUCACCUUGGGUGGCACCC 170 3063
GGGUGCCACCCAAGGUGAA 481 3063 CCUUACCCAGAGCUGCCCA 171 3063
CCUUACCCAGAGCUGCCCA 171 3081 UGGGCAGCUCUGGGUAAGG 482 3081
AUGAACGAGCAGUUCUACA 172 3081 AUGAACGAGCAGUUCUACA 172 3099
UGUAGAACUGCUCGUUCAU 483 3099 AAUGCCAUCAAACGGGGUU 173 3099
AAUGCCAUCAAACGGGGUU 173 3117 AACCCCGUUUGAUGGCAUU 484 3117
UACCGCAUGGCCCAGCCUG 174 3117 UACCGCAUGGCCCAGCCUG 174 3135
CAGGCUGGGCCAUGCGGUA 485 3135 GCCCAUGCCUCCGACGAGA 175 3135
GCCCAUGCCUCCGACGAGA 175 3153 UCUCGUCGGAGGCAUGGGC 486 3153
AUCUAUGAGAUCAUGCAGA 176 3153 AUCUAUGAGAUCAUGCAGA 176 3171
UCUGCAUGAUCUCAUAGAU 487 3171 AAGUGCUGGGAAGAGAAGU 177 3171
AAGUGCUGGGAAGAGAAGU 177 3189 ACUUCUCUUCCCAGCACUU 488 3189
UUUGAGAUUCGGCCCCCCU 178 3189 UUUGAGAUUCGGCCCCCCU 178 3207
AGGGGGGCCGAAUCUCAAA 489 3207 UUCUCCCAGCUGGUGCUGC 179 3207
UUCUCCCAGCUGGUGCUGC 179 3225 GCAGCACCAGCUGGGAGAA 490 3225
CUUCUCGAGAGACUGUUGG 180 3225 CUUCUCGAGAGACUGUUGG 180 3243
CCAACAGUCUCUCGAGAAG 491 3243 GGCGAAGGUUACAAAAAGA 181 3243
GGCGAAGGUUACAAAAAGA 181 3261 UCUUUUUGUAACCUUCGCC 492 3261
AAGUACCAGCAGGUGGAUG 182 3261 AAGUACCAGCAGGUGGAUG 182 3279
CAUCCACCUGCUGGUACUU 493 3279 GAGGAGUUUCUGAGGAGUG 183 3279
GAGGAGUUUCUGAGGAGUG 183 3297 CACUCCUCAGAAACUCCUC 494 3297
GACCACCCAGCCAUCCUUC 184 3297 GACCACCCAGCCAUCCUUC 184 3315
GAAGGAUGGCUGGGUGGUC 495 3315 CGGUCCCAGGCCCGCUUGC 185 3315
CGGUCCCAGGCCCGCUUGC 185 3333 GCAAGCGGGCCUGGGACCG 496 3333
CCUGGGUUCCAUGGCCUCC 186 3333 CCUGGGUUCCAUGGCCUCC 186 3351
GGAGGCCAUGGAACCCAGG 497 3351 CGAUCUCCCCUGGACACCA 187 3351
CGAUCUCCCCUGGACACCA 187 3369 UGGUGUCCAGGGGAGAUCG 498 3369
AGCUCCGUCCUCUAUACUG 188 3369 AGCUCCGUCCUCUAUACUG 188 3387
CAGUAUAGAGGACGGAGCU 499 3387 GCCGUGCAGCCCAAUGAGG 189 3387
GCCGUGCAGCCCAAUGAGG 189 3405 CCUCAUUGGGCUGCACGGC 500 3405
GGUGACAACGACUAUAUCA 190 3405 GGUGACAACGACUAUAUCA 190 3423
UGAUAUAGUCGUUGUCACC 501 3423 AUCCCCCUGCCUGACCCCA 191 3423
AUCCCCCUGCCUGACCCCA 191 3441 UGGGGUCAGGCAGGGGGAU 502 3441
AAACCCGAGGUUGCUGACG 192 3441 AAACCCGAGGUUGCUGACG 192 3459
CGUCAGCAACCUCGGGUUU 503 3459 GAGGGCCCACUGGAGGGUU 193 3459
GAGGGCCCACUGGAGGGUU 193 3477 AACCCUCCAGUGGGCCCUC 504 3477
UCCCCCAGCCUAGCCAGCU 194 3477 UCCCCCAGCCUAGCCAGCU 194 3495
AGCUGGCUAGGCUGGGGGA 505 3495 UCCACCCUGAAUGAAGUCA 195 3495
UCCACCCUGAAUGAAGUCA 195 3513 UGACUUCAUUCAGGGUGGA 506 3513
AACACCUCCUCAACCAUCU 196 3513 AACACCUCCUCAACCAUCU 196 3531
AGAUGGUUGAGGAGGUGUU 507 3531 UCCUGUGACAGCCCCCUGG 197 3531
UCCUGUGACAGCCCCCUGG 197 3549 CCAGGGGGCUGUCACAGGA 508 3549
GAGCCCCAGGACGAACCAG 198 3549 GAGCCCCAGGACGAACCAG 198 3567
CUGGUUCGUCCUGGGGCUC 509 3567 GAGCCAGAGCCCCAGCUUG 199 3567
GAGCCAGAGCCCCAGCUUG 199 3585 CAAGCUGGGGCUCUGGCUC 510 3585
GAGCUCCAGGUGGAGCCGG 200 3585 GAGCUCCAGGUGGAGCCGG 200 3663
CCGGCUCCACCUGGAGCUC 511 3603 GAGCCAGAGCUGGAACAGU 201 3603
GAGCCAGAGCUGGAACAGU 201 3621 ACUGUUCCAGCUCUGGCUC 512 3621
UUGCCGGAUUCGGGGUGCC 202 3621 UUGCCGGAUUCGGGGUGCC 202 3639
GGCACCCCGAAUCCGGCAA 513 3639 CCUGCGCCUCGGGCGGAAG 203 3639
CCUGCGCCUCGGGCGGAAG 203 3657 CUUCCGCCCGAGGCGCAGG 514 3657
GCAGAGGAUAGCUUCCUGU 204 3657 GCAGAGGAUAGCUUCCUGU 204 3675
ACAGGAAGCUAUCCUCUGC 515 3675 UAGGGGGCUGGCCCCUACC 205 3675
UAGGGGGCUGGCCCCUACC 205 3693 GGUAGGGGCCAGCCCCCUA 516 3693
CCUGCCCUGCCUGAAGCUC 206 3693 CCUGCCCUGCCUGAAGCUC 206 3711
GAGCUUCAGGCAGGGCAGG 517 3711 CCCCCCCUGCCAGCACCCA 207 3711
CCCCCCCUGCCAGCACCCA 207 3729 UGGGUGCUGGCAGGGGGGG 518 3729
AGCAUCUCCUGGCCUGGCC 208 3729 AGCAUCUCCUGGCCUGGCC 208 3747
GGCCAGGCCAGGAGAUGCU 519 3747 CUGACCGGGCUUCCUGUCA 209 3747
CUGACCGGGCUUCCUGUCA 209 3765 UGACAGGAAGCCCGGUCAG 520 3765
AGCCAGGCUGCCCUUAUCA 210 3765 AGCCAGGCUGCCCUUAUCA 210 3783
UGAUAAGGGCAGCCUGGCU 521 3783 AGCUGUCCCCUUCUGGAAG 211 3783
AGCUGUCCCCUUCUGGAAG 211 3801 CUUCCAGAAGGGGACAGCU 522 3801
GCUUUCUGCUCCUGACGUG 212 3801 GCUUUCUGCUCCUGACGUG 212 3819
CACGUCAGGAGCAGAAAGC 523 3819 GUUGUGCCCCAAACCCUGG 213 3819
GUUGUGCCCCAAACCCUGG 213 3837 CCAGGGUUUGGGGCACAAC 524 3837
GGGCUGGCUUAGGAGGCAA 214 3837 GGGCUGGCUUAGGAGGCAA 214 3855
UUGCCUCCUAAGCCAGCCC 525 3855 AGAAAACUGCAGGGGCCGU 215 3855
AGAAAACUGCAGGGGCCGU 215 3873 ACGGCCCCUGCAGUUUUCU 526 3873
UGACCAGCCCUCUGCCUCC 216 3873 UGACCAGCCCUCUGCCUCC 216 3891
GGAGGCAGAGGGCUGGUCA 527 3891 CAGGGAGGCCAACUGACUC 217 3891
CAGGGAGGCCAACUGACUC 217 3909 GAGUCAGUUGGCCUCCCUG 528 3909
CUGAGCCAGGGUUCCCCCA 218 3909 CUGAGCCAGGGUUCCCCCA 218 3927
UGGGGGAACCCUGGCUCAG 529 3927 AGGGAACUCAGUUUUCCCA 219 3927
AGGGAACUCAGUUUUCCCA 219 3945 UGGGAAAACUGAGUUCCCU 530 3945
AUAUGUAAGAUGGGAAAGU 220 3945 AUAUGUAAGAUGGGAAAGU 220 3963
ACUUUCCCAUCUUACAUAU 531 3963 UUAGGCUUGAUGACCCAGA 221 3963
UUAGGCUUGAUGACCCAGA 221 3981 UCUGGGUCAUCAAGCCUAA 532 3981
AAUCUAGGAUUCUCUCCCU 222 3981 AAUCUAGGAUUCUCUCCCU 222 3999
AGGGAGAGAAUCCUAGAUU 533 3999 UGGCUGACAGGUGGGGAGA 223 3999
UGGCUGACAGGUGGGGAGA 223 4017 UCUCCCCACCUGUCAGCCA 534 4017
ACCGAAUCCCUCCCUGGGA 224 4017 ACCGAAUCCCUCCCUGGGA 224 4035
UCCCAGGGAGGGAUUCGGU 535 4035 AAGAUUCUUGGAGUUACUG 225 4035
AAGAUUCUUGGAGUUACUG 225 4053 CAGUAACUCCAAGAAUCUU 536 4053
GAGGUGGUAAAUUAACUUU 226 4053 GAGGUGGUAAAUUAACUUU 226 4071
AAAGUUAAUUUACCACCUC 537 4071 UUUUCUGUUCAGCCAGCUA 227 4071
UUUUCUGUUCAGCCAGCUA 227 4089 UAGCUGGCUGAACAGAAAA 538 4089
ACCCCUCAAGGAAUCAUAG 228 4089 ACCCCUCAAGGAAUCAUAG 228 4107
CUAUGAUUCCUUGAGGGGU 539 4107 GCUCUCUCCUCGCACUUUU 229 4107
GCUCUCUCCUCGCACUUUU 229 4125 AAAAGUGCGAGGAGAGAGC 540 4125
UUAUCCACCCAGGAGCUAG 230 4125 UUAUCCACCCAGGAGCUAG 230 4143
CUAGCUCCUGGGUGGAUAA 541 4143 GGGAAGAGACCCUAGCCUC 231 4143
GGGAAGAGACCCUAGCCUC 231 4161 GAGGCUAGGGUCUCUUCCC 542 4161
CCCUGGCUGCUGGCUGAGC 232 4161 CCCUGGCUGCUGGCUGAGC 232 4179
GCUCAGCCAGCAGCCAGGG 543 4179 CUAGGGCCUAGCCUUGAGC 233 4179
CUAGGGCCUAGCCUUGAGC 233 4197 GCUCAAGGCUAGGCCCUAG 544 4197
CAGUGUUGCCUCAUCCAGA 234 4197 CAGUGUUGCCUCAUCCAGA 234 4215
UCUGGAUGAGGCAACACUG 545 4215 AAGAAAGCCAGUCUCCUCC 235 4215
AAGAAAGCCAGUCUCCUCC 235 4233 GGAGGAGACUGGCUUUCUU 546 4233
CCUAUGAUGCCAGUCCCUG 236 4233 CCUAUGAUGCCAGUCCCUG 236 4251
CAGGGACUGGCAUCAUAGG 547 4251 GCGUUCCCUGGCCCGAGCU 237 4251
GCGUUCCCUGGCCCGAGCU 237 4269 AGCUCGGGCCAGGGAACGC 548 4269
UGGUCUGGGGCCAUUAGGC 238 4269 UGGUCUGGGGCCAUUAGGC 238 4287
GCCUAAUGGCCCCAGACCA 549 4287 CAGCCUAAUUAAUGCUGGA 239 4287
CAGCCUAAUUAAUGCUGGA 239 4305 UCCAGCAUUAAUUAGGCUG 550 4305
AGGCUGAGCCAAGUACAGG 240 4305 AGGCUGAGCCAAGUACAGG 240 4323
CCUGUACUUGGCUCAGCCU 551 4323 GACACCCCCAGCCUGCAGC 241 4323
GACACCCCCAGCCUGCAGC 241 4341 GCUGCAGGCUGGGGGUGUC 552 4341
CCCUUGCCCAGGGCACUUG 242 4341 CCCUUGCCCAGGGCACUUG 242 4359
CAAGUGCCCUGGGCAAGGG 553 4359 GGAGCACACGCAGCCAUAG 243 4359
GGAGCACACGCAGCCAUAG 243 4377 CUAUGGCUGCGUGUGCUCC 554 4377
GCAAGUGCCUGUGUCCCUG 244 4377 GCAAGUGCCUGUGUCCCUG 244 4395
CAGGGACACAGGCACUUGC 555 4395 GUCCUUCAGGCCCAUCAGU 245 4395
GUCCUUCAGGCCCAUCAGU 245 4413 ACUGAUGGGCCUGAAGGAC 556 4413
UCCUGGGGCUUUUUCUUUA 246 4413 UCCUGGGGCUUUUUCUUUA 246 4431
UAAAGAAAAAGCCCCAGGA 557 4431 AUCACCCUCAGUCUUAAUC 247 4431
AUCACCCUCAGUCUUAAUC 247 4449 GAUUAAGACUGAGGGUGAU 558 4449
CCAUCCACCAGAGUCUAGA 248 4449 CCAUCCACCAGAGUCUAGA 248 4467
UCUAGACUCUGGUGGAUGG 559 4467 AAGGCCAGACGGGCCCCGC 249 4467
AAGGCCAGACGGGCCCCGC 249 4485
GCGGGGCCCGUCUGGCCUU 560 4485 CAUCUGUGAUGAGAAUGUA 250 4485
CAUCUGUGAUGAGAAUGUA 250 4503 UACAUUCUCAUCACAGAUG 561 4503
AAAUGUGCCAGUGUGGAGU 251 4503 AAAUGUGCCAGUGUGGAGU 251 4521
ACUCCACACUGGCACAUUU 562 4521 UGGCCACGUGUGUGUGCCA 252 4521
UGGCCACGUGUGUGUGCCA 252 4539 UGGCACACACACGUGGCCA 563 4539
AGUAUAUGGCCCUGGCUCU 253 4539 AGUAUAUGGCCCUGGCUCU 253 4557
AGAGCCAGGGCCAUAUACU 564 4557 UGCAUUGGACCUGCUAUGA 254 4557
UGCAUUGGACCUGCUAUGA 254 4575 UCAUAGCAGGUCCAAUGCA 565 4575
AGGCUUUGGAGGAAUCCCU 255 4575 AGGCUUUGGAGGAAUCCCU 255 4593
AGGGAUUCCUCCAAAGCCU 566 4593 UCACCCUCUCUGGGCCUCA 256 4593
UCACCCUCUCUGGGCCUCA 256 4611 UGAGGCCCAGAGAGGGUGA 567 4611
AGUUUCCCCUUCAAAAAAU 257 4611 AGUUUCCCCUUCAAAAAAU 257 4629
AUUUUUUGAAGGGGAAACU 568 4629 UGAAUAAGUCGGACUUAUU 258 4629
UGAAUAAGUCGGACUUAUU 258 4647 AAUAAGUCCGACUUAUUCA 569 4647
UAACUCUGAGUGCCUUGCC 259 4647 UAACUCUGAGUGCCUUGCC 259 4665
GGCAAGGCACUCAGAGUUA 570 4665 CAGCACUAACAUUCUAGAG 260 4665
CAGCACUAACAUUCUAGAG 260 4683 CUCUAGAAUGUUAGUGCUG 571 4683
GUAUUCCAGGUGGUUGCAC 261 4683 GUAUUCCAGGUGGUUGCAC 261 4701
GUGCAACCACCUGGAAUAC 572 4701 CAUUUGUCCAGAUGAAGCA 262 4701
CAUUUGUCCAGAUGAAGCA 262 4719 UGCUUCAUCUGGACAAAUG 573 4719
AAGGCCAUAUACCCUAAAC 263 4719 AAGGCCAUAUACCCUAAAC 263 4737
GUUUAGGGUAUAUGGCCUU 574 4737 CUUCCAUCCUGGGGGUCAG 264 4737
CUUCCAUCCUGGGGGUCAG 264 4755 CUGACCCCCAGGAUGGAAG 575 4755
GCUGGGCUCCUGGGAGAUU 265 4755 GCUGGGCUCCUGGGAGAUU 265 4773
AAUCUCCCAGGAGCCCAGC 576 4773 UCCAGAUCACACAUCACAC 266 4773
UCCAGAUCACACAUCACAC 266 4791 GUGUGAUGUGUGAUCUGGA 577 4791
CUCUGGGGACUCAGGAACC 267 4791 CUCUGGGGACUCAGGAACC 267 4809
GGUUCCUGAGUCCCCAGAG 578 4809 CAUGCCCCUUCCCCAGGCC 268 4809
CAUGCCCCUUCCCCAGGCC 268 4827 GGCCUGGGGAAGGGGCAUG 579 4827
CCCCAGCAAGUCUCAAGAA 269 4827 CCCCAGCAAGUCUCAAGAA 269 4845
UUCUUGAGACUUGCUGGGG 580 4845 ACACAGCUGCACAGGCCUU 270 4845
ACACAGCUGCACAGGCCUU 270 4863 AAGGCCUGUGCAGCUGUGU 581 4863
UGACUUAGAGUGACAGCCG 271 4863 UGACUUAGAGUGACAGCCG 271 4881
CGGCUGUCACUCUAAGUCA 582 4881 GGUGUCCUGGAAAGCCCCA 272 4881
GGUGUCCUGGAAAGCCCCA 272 4899 UGGGGCUUUCCAGGACACC 583 4899
AAGCAGCUGCCCCAGGGAC 273 4899 AAGCAGCUGCCCCAGGGAC 273 4917
GUCCCUGGGGCAGCUGCUU 584 4917 CAUGGGAAGACCACGGGAC 274 4917
CAUGGGAAGACCACGGGAC 274 4935 GUCCCGUGGUCUUCCCAUG 585 4935
CCUCUUUCACUACCCACGA 275 4935 CCUCUUUCACUACCCACGA 275 4953
UCGUGGGUAGUGAAAGAGG 586 4953 AUGACCUCCGGGGGUAUCC 276 4953
AUGACCUCCGGGGGUAUCC 276 4971 GGAUACCCCCGGAGGUCAU 587 4971
CUGGGCAAAAGGGACAAAG 277 4971 CUGGGCAAAAGGGACAAAG 277 4989
CUUUGUCCCUUUUGCCCAG 588 4989 GAGGGCAAAUGAGAUCACC 278 4989
GAGGGCAAAUGAGAUCACC 278 5007 GGUGAUCUCAUUUGCCCUC 589 5007
CUCCUGCAGCCCACCACUC 279 5007 CUCCUGCAGCCCACCACUC 279 5025
GAGUGGUGGGCUGCAGGAG 590 5025 CCAGCACCUGUGCCGAGGU 280 5025
CCAGCACCUGUGCCGAGGU 280 5043 ACCUCGGCACAGGUGCUGG 591 5043
UCUGCGUCGAAGACAGAAU 281 5043 UCUGCGUCGAAGACAGAAU 281 5061
AUUCUGUCUUCGACGCAGA 592 5061 UGGACAGUGAGGACAGUUA 282 5061
UGGACAGUGAGGACAGUUA 282 5079 UAACUGUCCUCACUGUCCA 593 5079
AUGUCUUGUAAAAGACAAG 283 5079 AUGUCUUGUAAAAGACAAG 283 5097
CUUGUCUUUUACAAGACAU 594 5097 GAAGCUUCAGAUGGUACCC 284 5097
GAAGCUUCAGAUGGUACCC 284 5115 GGGUACCAUCUGAAGCUUC 595 5115
CCAAGAAGGAUGUGAGAGG 285 5115 CCAAGAAGGAUGUGAGAGG 285 5133
CCUCUCACAUCCUUCUUGG 596 5133 GUGGCCGCUUGGAGUUUGC 286 5133
GUGGCCGCUUGGAGUUUGC 286 5151 GCAAACUCCAAGCGGCCAC 597 5151
CCCCUCACCCACCAGCUGC 287 5151 CCCCUCACCCACCAGCUGC 287 5169
GCAGCUGGUGGGUGAGGGG 598 5169 CCCCAUCCCUGAGGCAGCG 288 5169
CCCCAUCCCUGAGGCAGCG 288 5187 CGCUGCCUCAGGGAUGGGG 599 5187
GCUCCAUGGGGGUAUGGUU 289 5187 GCUCCAUGGGGGUAUGGUU 289 5205
AACCAUACCCCCAUGGAGC 600 5205 UUUGUCACUGCCCAGACCU 290 5205
UUUGUCACUGCCCAGACCU 290 5223 AGGUCUGGGCAGUGACAAA 601 5223
UAGCAGUGACAUCUCAUUG 291 5223 UAGCAGUGACAUCUCAUUG 291 5241
CAAUGAGAUGUCACUGCUA 602 5241 GUCCCCAGCCCAGUGGGCA 292 5241
GUCCCCAGCCCAGUGGGCA 292 5259 UGCCCACUGGGCUGGGGAC 603 5259
AUUGGAGGUGCCAGGGGAG 293 5259 AUUGGAGGUGCCAGGGGAG 293 5277
CUCCCCUGGCACCUCCAAU 604 5277 GUCAGGGUUGUAGCCAAGA 294 5277
GUCAGGGUUGUAGCCAAGA 294 5295 UCUUGGCUACAACCCUGAC 605 5295
ACGCCCCCGCACGGGGAGG 295 5295 ACGCCCCCGCACGGGGAGG 295 5313
CCUCCCCGUGCGGGGGCGU 606 5313 GGUUGGGAAGGGGGUGCAG 296 5313
GGUUGGGAAGGGGGUGCAG 296 5331 CUGCACCCCCUUCCCAACC 607 5331
GGAAGCUCAACCCCUCUGG 297 5331 GGAAGCUCAACCCCUCUGG 297 5349
CCAGAGGGGUUGAGCUUCC 608 5349 GGCACCAACCCUGCAUUGC 298 5349
GGCACCAACCCUGCAUUGC 298 5367 GCAAUGCAGGGUUGGUGCC 609 5367
CAGGUUGGCACCUUACUUC 299 5367 CAGGUUGGCACCUUACUUC 299 5385
GAAGUAAGGUGCCAACCUG 610 5385 CCCUGGGAUCCCCAGAGUU 300 5385
CCCUGGGAUCCCCAGAGUU 300 5403 AACUCUGGGGAUCCCAGGG 611 5403
UGGUCCAAGGAGGGAGAGU 301 5403 UGGUCCAAGGAGGGAGAGU 301 5421
ACUCUCCCUCCUUGGACCA 612 5421 UGGGUUCUCAAUACGGUAC 302 5421
UGGGUUCUCAAUACGGUAC 302 5439 GUACCGUAUUGAGAACCCA 613 5439
CCAAAGAUAUAAUCACCUA 303 5439 CCAAAGAUAUAAUCACCUA 303 5457
UAGGUGAUUAUAUCUUUGG 614 5457 AGGUUUACAAAUAUUUUUA 304 5457
AGGUUUACAAAUAUUUUUA 304 5475 UAAAAAUAUUUGUAAACCU 615 5475
AGGACUCACGUUAACUCAC 305 5475 AGGACUCACGUUAACUCAC 305 5493
GUGAGUUAACGUGAGUCCU 616 5493 CAUUUAUACAGCAGAAAUG 306 5493
CAUUUAUACAGCAGAAAUG 306 5511 CAUUUCUGCUGUAUAAAUG 617 5511
GCUAUUUUGUAUGCUGUUA 307 5511 GCUAUUUUGUAUGCUGUUA 307 5529
UAACAGCAUACAAAAUAGC 618 5529 AAGUUUUUCUAUCUGUGUA 308 5529
AAGUUUUUCUAUCUGUGUA 308 5547 UACACAGAUAGAAAAACUU 619 5547
ACUUUUUUUUAAGGGAAAG 309 5547 ACUUUUUUUUAAGGGAAAG 309 5565
CUUUCCCUUAAAAAAAAGU 620 5565 GAUUUUAAUAUUAAACCUG 310 5565
GAUUUUAAUAUUAAACCUG 310 5583 CAGGUUUAAUAUUAAAAUC 621 5578
AACCUGGUGCUUCUCACUC 311 5578 AACCUGGUGCUUCUCACUC 311 5596
GAGUGAGAAGCACCAGGUU 622 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, such as exemplary siNA constructs shown in FIGS. 4 and 5, or
having modifications described in Table IV or any combination
thereof.
TABLE-US-00003 TABLE III PDGFRB Synthetic Modified siNA Constructs
Target Seq Seq Pos Target ID Cmpd# Aliases Sequence ID 422
UGCCUGUCCUUCUACUCAGCUGU 623 31910 PDGFRB:208U21 sense siNA
CCUGUCCUUCUACUCAGCUTT 631 427 GGAGGUGGAUUCUGAUGCCUACU 624
PDGFRB:949U21 sense siNA AGGUGGAUUCUGAUGCCUATT 632 506
GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1325U21 sense siNA
CACACUACAAUUUGCUGAGTT 633 511 CGAGUGCUGGAGCUAAGUGAGAG 626
PDGFRB:1610U21 sense siNA AGUGCUGGAGCUAAGUGAGTT 634 616
CUCGAAUUACAUCUCCAAAGGCA 627 31911 PDGFRB:2920U21 sense siNA
CGAAUUACAUCUCCAAAGGTT 635 681 CUGCUAUGAGGCUUUGGAGGAAU 628 31912
PDGFRB:4569U21 sense siNA GCUAUGAGGCUUUGGAGGATT 636 751
GACAAAGAGGGCAAAUGAGAUCA 629 31913 PDGFRB:4985U21 sense siNA
CAAAGAGGGCAAAUGAGAUTT 637 815 AGGGAGAGUGGGUUCUCAAUACG 630
PDGFRB:5415U21 sense siNA GGAGAGUGGGUUCUCAAUATT 638 422
UGCCUGUCCUUCUACUCAGCUGU 623 31914 PDGFRB:226L21 antisense siNA
AGCUGAGUAGAAGGACAGGTT 639 (208C) 427 GGAGGUGGAUUCUGAUGCCUACU 624
PDGFRB:967L21 antisense siNA UAGGCAUCAGAAUCCACCUTT 640 (949C) 506
GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1343L21 antisense siNA
CUCAGCAAAUUGUAGUGUGTT 641 (1325C) 511 CGAGUGCUGGAGCUAAGUGAGAG 626
PDGFRB:1628L21 antisense siNA CUCACUUAGCUCCAGCACUTT 642 (1610C) 616
CUCGAAUUAGAUCUCCAAAGGCA 627 31915 PDGFRB:2938L21 antisense siNA
CCUUUGGAGAUGUAAUUCGTT 643 (2920C) 681 CUGCUAUGAGGCUUUGGAGGAAU 628
31916 PDGFRB:4587L21 antisense siNA UCCUCCAAAGCCUCAUAGCTT 644
(4569C) 751 GACAAAGAGGGCAAAUGAGAUCA 629 31917 PDGFRB:5003L21
antisense siNA AUCUCAUUUGCCCUCUUUGTT 645 (4985C) 815
AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNA
UAUUGAGAACCCACUCUCCTT 646 (5415C) 422 UGCCUGUCCUUCUACUCAGCUGU 623
PDGFRB:208U21 sense siNA stab04 B ccuGuccuucuAcucAGcuTT B 647 427
GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:949U21 sense siNA stab04 B
AGGuGGAuucuGAuGccuATT B 648 506 GGCACACUACAAUUUGCUGAGCU 625
PDGFRB:1325U21 sense siNA stab04 B cAcAcuAcAAuuuGcuGAGTT B 649 511
CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1610U21 sense siNA stab04 B
AGuGcuGGAGcuAAGuGAGTT B 650 616 CUCGAAUUACAUCUCCAAAGGCA 627
PDGFRB:2920U21 sense siNA stab04 B cGAAuuAcAucuccAAAGGTT B 651 681
CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4569U21 sense siNA stab04 B
GcuAuGAGGcuuuGGAGGATT B 652 751 GACAAAGAGGGCAAAUGAGAUCA 629
PDGFRB:4985U21 sense siNA stab04 B cAAAGAGGGcAAAuGAGAuTT B 653 815
AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5415U21 sense siNA stab04 B
GGAGAGuGGGuucucAAuATT B 654 422 UGCCUGUCCUUCUACUCAGCUGU 623
PDGFRB:226L21 antisense siNA AGcuGAGuAGAAGGAcAGGTsT 655 (208C)
stab05 427 GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNA
uAGGcAucAGAAuccAccuTsT 656 (949C) stab05 506
GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1343L21 antisense siNA
cucAGcAAAuuGuAGuGuGTsT 657 (1325C) stab05 511
CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNA
cucAcuuAGcuccAGcAcuTsT 658 (1610C) stab05 616
CUCGAAUUACAUCUCCAAAGGCA 627 PDGFRB:2938L21 antisense siNA
ccuuuGGAGAuGuAAuucGTsT 659 (2920C) stab05 681
CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4587L21 antisense siNA
uccuccAAAGccucAuAGcTsT 660 (4569C) stab05 751
GACAAAGAGGGCAAAUGAGAUCA 629 PDGFRB:5003L21 antisense siNA
AucucAuuuGcccucuuuGTsT 661 (4985C) stab05 815
AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNA
uAuuGAGAAcccAcucuccTsT 662 (5415C) stab05 422
UGCCUGUCCUUCUACUCAGCUGU 623 PDGFRB:208U21 sense siNA stab07 B
ccuGuccuucuAcucAGcuTT B 663 427 GGAGGUGGAUUCUGAUGCCUACU 624
PDGFRB:949U21 sense siNA stab07 B AGGuGGAuucuGAuGccuATT B 664 506
GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1325U21 sense siNA stab07 B
cAcAcuAcAAuuuGcuGAGTT B 665 511 CGAGUGCUGGAGCUAAGUGAGAG 626
PDGFRB:1610U21 sense siNA stab07 B AGuGcuGGAGcuAAGuGAGTT B 666 616
CUCGAAUUACAUCUCCAAAGGCA 627 PDGFRB:2920U21 sense siNA stab07 B
cGAAuuAcAucuccAAAGGTT B 667 681 CUGCUAUGAGGCUUUGGAGGAAU 628
PDGFRB:4569U21 sense siNA stab07 B GcuAuGAGGcuuuGGAGGATT B 668 751
GACAAAGAGGGCAAAUGAGAUCA 629 PDGFRB:4985U21 sense siNA stab07 B
cAAAGAGGGcAAAuGAGAuTT B 669 815 AGGGAGAGUGGGUUGUCAAUACG 630
PDGFRB:5415U21 sense siNA stab07 B GGAGAGuGGGuucucAAuATT B 670 422
UGCCUGUCCUUCUACUCAGCUGU 623 PDGFRB:226L21 antisense siNA
AGcuGAGuAGAAGGAcAGGTsT 671 (208C) stab11 427
GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNA
uAGGcAucAGAAuccAccuTsT 672 (949C) stab11 506
GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1343L21 antisense siNA
cucAGcAAAuuGuAGuGuGTsT 673 (1325C) stab11 511
CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNA
cucAcuuAGcuccAGcAcuTsT 674 (1610C) stab11 616
CUCGAAUUACAUCUCCAAAGGCA 627 PDGFRB:2938L21 antisense siNA
ccuuuGGAGAuGuAAuucGTsT 675 (2920C) stab11 681
CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4587L21 antisense siNA
uccuccAAAGccucAuAGcTsT 676 (4569C) stab11 751
GACAAAGAGGGCAAAUGAGAUCA 629 PDGFRB:5003L21 antisense siNA
AucucAuuuGcccucuuuGTsT 677 (4985C) stab11 815
AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNA
uAuuGAGAAcccAcucuccTsT 678 (5415C) stab11 422
UGCCUGUCCUUCUACUCAGCUGU 623 PDGFRB:208U21 sense siNA stab18 B
ccuGuccuucuAcucAGcuTT B 679 427 GGAGGUGGAUUCUGAUGCCUACU 624
PDGFRB:949U21 sense siNA stab18 B AGGuGGAuucuGAuGccuATT B 680 506
GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1325U21 sense siNA stab18 B
cAcAcuAcAAuuuGcuGAGTT B 681 511 CGAGUGCUGGAGCUAAGUGAGAG 626
PDGFRB:1610U21 sense siNA stab18 B AGuGcuGGAGcuAAGuGAGTT B 682 616
CUCGAAUUACAUCUCCAAAGGCA 627 PDGFRB:2920U21 sense siNA stab18 B
cGAAuuAcAucuccAAAGGTT B 683 681 CUGCUAUGAGGCUUUGGAGGAAU 628
PDGFRB:4569U21 sense siNA stab18 B GcuAuGAGGcuuuGGAGGATT B 684 751
GACAAAGAGGGCAAAUGAGAUCA 629 PDGFRB:4985U21 sense siNA stab18 B
cAAAGAGGGcAAAuGAGAuTT B 685 815 AGGGAGAGUGGGUUCUCAAUACG 630
PDGFRB:5415U21 sense siNA stab18 B GGAGAGuGGGuucucAAuATT B 686 422
UGCCUGUCCUUCUACUCAGCUGU 623 PDGFRB:226L21 antisense siNA
AGcuGAGuAGAAGGAcAGGTsT 687 (208C) stab08 427
GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNA
uAGGcAucAGAAuccAccuTsT 688 (949C) stab08 506
GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1343L21 antisense siNA
cucAGcAAAuuGuAGuGuGTsT 689 (1325C) stab08 511
CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNA
cucAcuuAGcuccAGcAcuTsT 690 (1610C) stab08 616
CUCGAAUUACAUCUCCAAAGGCA 627 PDGFRB:2938L21 antisense siNA
ccuuuGGAGAuGuAAuucGTsT 691 (2920C) stab08 681
CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4587L21 antisense siNA
uccuccAAAGccucAuAGcTsT 692 (4569C) stab08 751
GACAAAGAGGGCAAAUGAGAUCA 629 PDGFRB:5003L21 antisense siNA
AucucAuuuGcccucuuuGTsT 693 (4985C) stab08 815
AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNA
uAuuGAGAAcccAcucuccTsT 694 (5415C) stab08 422
UGCCUGUCCUUCUACUCAGCUGU 623 37092 PDGFRB:208U21 sense siNA stab09 B
CCUGUCCUUCUACUCAGCUTT B 695 427 GGAGGUGGAUUCUGAUGCCUACU 624 37093
PDGFRB:949U21 sense siNA stab09 B AGGUGGAUUCUGAUGCCUATT B 696 506
GGCACACUACAAUUUGCUGAGCU 625 37094 PDGFRB:1325U21 sense siNA stab09
B CACACUACAAUUUGCUGAGTT B 697 511 CGAGUGCUGGAGCUAAGUGAGAG 626 37095
PDGFRB:1610U21 sense siNA stab09 B AGUGCUGGAGCUAAGUGAGTT B 698 616
CUCGAAUUACAUCUCCAAAGGCA 627 37096 PDGFRB:2920U21 sense siNA stab09
B CGAAUUACAUCUCCAAAGGTT B 699 681 CUGCUAUGAGGCUUUGGAGGAAU 628 37097
PDGFRB:4569U21 sense siNA stab09 B GCUAUGAGGCUUUGGAGGATT B 700 751
GACAAAGAGGGCAAAUGAGAUCA 629 37098 PDGFRB:4985U21 sense siNA stab09
B CAAAGAGGGCAAAUGAGAUTT B 701
815 AGGGAGAGUGGGUUCUCAAUACG 630 37099 PDGFRB:5415U21 sense siNA
stab09 B GGAGAGUGGGUUCUCAAUATT B 702 422 UGCCUGUCCUUCUACUCAGCUGU
623 PDGFRB:226L21 antisense siNA AGCUGAGUAGAAGGACAGGTsT 703 (208C)
stab10 427 GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNA
UAGGCAUCAGAAUCCACCUTsT 704 (949C) stab10 506
GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1343L21 antisense siNA
CUCAGCAAAUUGUAGUGUGTsT 705 (1325C) stab10 511
CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNA
CUCACUUAGCUCCAGCACUTsT 706 (1610C) stab10 616
CUCGAAUUACAUCUC0AAAGGCA 627 PDGFRB:2938L21 antisense siNA
CCUUUGGAGAUGUAAUUCGTsT 707 (2920C) stab10 681
CUGCUAUGAGGCUUUGGAGGAAU 626 PDGFRB:4587L21 antisense siNA
UCCUCCAAAGCCUCAUAGCTsT 708 (4569C) stab10 751
GACAAAGAGGGCAAAUGAGAUCA 629 PDGFRB:5003L21 antisense siNA
AUCUCAUUUGCCCUCUUUGTsT 709 (4985C) stab10 815
AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNA
UAUUGAGAACCCACUCUCCTsT 710 (5415C) stab10 422
UGCCUGUCCUUCUACUCAGCUGU 623 PDGFRB:226L21 antisense siNA
AGcuGAGuAGAAGGAcAGGTT B 711 (208C) stab19 427
GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNA
uAGGcAucAGAAuCcAccuTT B 712 (949C) stab19 506
GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1343L21 antisense siNA
cucAGcAAAuuGuAGuGuGTT B 713 (1325C) stab19 511
CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNA
cucAcuuAGcuccAGcAcuTT B 714 (1610C) stab19 616
CUCGAAUUACAUCUCCAAAGGCA 627 PDGFRB:2938L21 antisense siNA
ccuuuGGAGAuGuAAuucGTT B 715 (2920C) stab19 681
CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4587L21 antisense siNA
uccuccAAAGccucAuAGcTT B 716 (4569C) stab19 751
GACAAAGAGGGCAAAUGAGAUCA 629 PDGFRB:5003L21 antisense siNA
AucucAuuuGcccucuuuGTT B 717 (4985C) stab19 815
AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNA
uAuuGAGAAcccAcucuccTT B 718 (5415C) stab19 422
UGCCUGUCCUUCUACUCAGCUGU 623 37100 PDGFRB:226L21 antisense siNA
AGCUGAGUAGAAGGACAGGTT B 719 (208C) stab22 427
GGAGGUGGAUUCUGAUGCCUACU 624 37101 PDGFRB:967L21 antisense siNA
UAGGCAUCAGAAUCCACCUTT B 720 (949C) stab22 506
GGCACACUACAAUUUGCUGAGCU 625 37102 PDGFRB:1343121 antisense siNA
CUCAGCAAAUUGUAGUGUGTT B 721 (1325C) stab22 511
CGAGUGCUGGAGCUAAGUGAGAG 626 37103 PDGFRB:1628L21 antisense siNA
CUCACUUAGCUCCAGCACUTT B 722 (1610C) stab22 616
CUCGAAUUACAUCUCCAAAGGCA 627 37104 PDGFRB:2938L21 antisense siNA
CCUUUGGAGAUGUAAUUCGTT B 723 (2920C) stab22 681
CUGCUAUGAGGCUUUGGAGGAAU 628 37105 PDGFRB:4587L21 antisense siNA
UCCUCCAAAGCCUCAUAGCTT B 724 (4569C) stab22 751
GACAAAGAGGGCAAAUGAGAUCA 629 37106 PDGFRB:5003L21 antisense siNA
AUCUCAUUUGCCCUCUUUGTT B 725 (4985C) stab22 815
AGGGAGAGUGGGUUCUCAAUACG 630 37107 PDGFRB:5433L21 antisense siNA
UAUUGAGAACCCACUCUCCTT B 726 (5415C) stab22 Uppercase =
ribonucleotide u,c = 2'-deoxy-2'-fluoro U,C T = thymidine B =
inverted deoxy abasic s = phosphorothioate linkage A = deoxy
Adenosine G = deoxy Guanosine G = 2'-O-methyl Guanosine A =
2'-O-methyl Adenosine
TABLE-US-00004 TABLE IV Non-limiting examples of Stabilization
Chemistries for chemically modified siNA constructs Chemistry
pyrimidine Purine cap p = S Strand "Stab 00" Ribo Ribo S/AS "Stab
1" Ribo Ribo -- 5 at 5'-end S/AS 1 at 3'-end "Stab 2" Ribo Ribo --
All Usually AS linkages "Stab 3" 2'-fluoro Ribo -- 4 at 5'-end
Usually S 4 at 3'-end "Stab 4" 2'-fluoro Ribo 5' and 3'- -- Usually
S ends "Stab 5" 2'-fluoro Ribo -- 1 at 3'-end Usually AS "Stab 6"
2'-O- Ribo 5' and 3'- -- Usually S Methyl ends "Stab 7" 2'-fluoro
2'-deoxy 5' and 3'- -- Usually S ends "Stab 8" 2'-fluoro 2'-O- -- 1
at 3'-end S/AS Methyl "Stab 9" Ribo Ribo 5' and 3'- -- Usually S
ends "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'- Usually S ends "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- 5' and 3'- Usually S Methyl ends "Stab
17" 2'-O- 2'-O- 5' and 3'- Usually S Methyl Methyl ends "Stab 18"
2'-fluoro 2'-O- 5' and 3'- Usually S Methyl ends "Stab 19"
2'-fluoro 2'-O- 3'-end S/AS Methyl "Stab 20" 2'-fluoro 2'-deoxy
3'-end Usually AS "Stab 21" 2'-fluoro Ribo 3'-end Usually AS "Stab
22" Ribo Ribo 3'-end Usually AS "Stab 23" 2'-fluoro* 2'-deoxy* 5'
and 3'- Usually S ends "Stab 24" 2'-fluoro* 2'-O- -- 1 at 3'-end
S/AS Methyl* "Stab 25" 2'-fluoro* 2'-O- -- 1 at 3'-end S/AS Methyl*
"Stab 26" 2'-fluoro* 2'-O- -- S/AS Methyl* "Stab 27" 2'-fluoro*
2'-O- 3'-end S/AS Methyl* "Stab 28" 2'-fluoro* 2'-O- 3'-end S/AS
Methyl* "Stab 29" 2'-fluoro* 2'-O- 1 at 3'-end S/AS Methyl* "Stab
30" 2'-fluoro* 2'-O- S/AS Methyl* "Stab 31" 2'-fluoro* 2'-O- 3'-end
S/AS Methyl* "Stab 32" 2'-fluoro 2'-O- S/AS Methyl CAP = any
terminal cap, see for example FIG. 10. All Stab 00-32 chemistries
can comprise 3'-terminal thymidine (TT) residues All Stab 00-32
chemistries typically comprise about 21 nucleotides, but can vary
as described herein. S = sense strand AS = antisense strand *Stab
23 has a single ribonucleotide adjacent to 3'-CAP *Stab 24 and Stab
28 have a single ribonucleotide at 5'-terminus *Stab 25, Stab 26,
and Stab 27 have three ribonucleotides at 5'-terminus *Stab 29,
Stab 30, and Stab 31, any purine at first three nucleotide
positions from 5'-terminus are ribonucleotides p = phosphorothioate
linkage
TABLE-US-00005 TABLE V Reagent Equivalents Amount Wait Time* DNA
Wait Time* 2'-O-methyl Wait Time*RNA A. 2.5 .mu.mol Synthesis Cycle
ABI 394 Instrument 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 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* Reagent
2'-O-methyl/Ribo methyl/Ribo Wait Time* DNA 2'-O-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 1
1
749119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 1ccccucagcc cugcugccc 19219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 2cagcacgagc cugugcucg
19319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 3gcccugccca acgcagaca 19419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 4agccagaccc agggcggcc
19519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 5cccucuggcg gcucugcuc 19619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 6ccucccgaag gaugcuugg
19719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 7gggagugagg cgaagcugg 19819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 8ggcgcuccuc uccccuaca
19919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 9agcagccccc uuccuccau 191019RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 10ucccucuguu cuccugagc
191119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 11ccuucaggag ccugcacca 191219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 12aguccugccu guccuucua
191319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 13acucagcugu uacccacuc 191419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 14cugggaccag cagucuuuc
191519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 15cugauaacug ggagagggc 191619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 16caguaaggag gacuuccug
191719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 17ggagggggug acuguccag 191819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 18gagccuggaa cugugccca
191919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 19acaccagaag ccaucagca 192019RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 20agcaaggaca ccaugcggc
192119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 21cuuccgggug cgaugccag 192219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 22gcucuggccc ucaaaggcg
192319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 23gagcugcugu ugcugucuc 192419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 24cuccuguuac uucuggaac
192519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 25ccacagaucu cucagggcc 192619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 26cuggucguca cacccccgg
192719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 27gggccagagc uuguccuca 192819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 28aaugucucca gcaccuucg
192919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 29guucugaccu gcucggguu 193019RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 30ucagcuccgg ugguguggg
193119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 31gaacggaugu cccaggagc 193219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 32cccccacagg aaauggcca
193319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 33aaggcccagg auggcaccu 193419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 34uucuccagcg ugcucacac
193519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 35cugaccaacc ucacugggc 193619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 36cuagacacgg gagaauacu
193719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 37uuuugcaccc acaaugacu 193819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 38ucccguggac uggagaccg
193919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 39gaugagcgga aacggcucu 194019RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 40uacaucuuug ugccagauc
194119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 41cccaccgugg gcuuccucc 194219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 42ccuaaugaug ccgaggaac
194319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 43cuauucaucu uucucacgg 194419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 44gaaauaacug agaucacca
194519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 45auuccaugcc gaguaacag 194619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 46gacccacagc uggugguga
194719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 47acacugcacg agaagaaag 194819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 48ggggacguug cacugccug
194919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 49guccccuaug aucaccaac 195019RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 50cguggcuuuu cugguaucu
195119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 51uuugaggaca gaagcuaca 195219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 52aucugcaaaa ccaccauug
195319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 53ggggacaggg agguggauu 195419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 54ucugaugccu acuaugucu
195519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 55uacagacucc aggugucau 195619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 56uccaucaacg ucucuguga
195719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 57aacgcagugc agacugugg 195819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 58guccgccagg gugagaaca
195919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 59aucacccuca ugugcauug 196019RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 60gugaucggga augaggugg
196119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 61gucaacuucg aguggacau 196219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 62uacccccgca aagaaagug
196319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 63gggcggcugg uggagccgg 196419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 64gugacugacu uccucuugg
196519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 65gauaugccuu accacaucc 196619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 66cgcuccaucc ugcacaucc
196719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 67cccagugccg aguuagaag 196819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 68gacucgggga ccuacaccu
196919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 69ugcaauguga cggagagug 197019RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 70gugaaugacc aucaggaug
197119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 71gaaaaggcca ucaacauca 197219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 72accgugguug agagcggcu
197319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 73uacgugcggc uccugggag 197419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 74gaggugggca cacuacaau
197519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 75uuugcugagc ugcaucgga 197619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 76agccggacac ugcagguag
197719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 77guguucgagg ccuacccac 197819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 78ccgcccacug uccuguggu
197919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 79uucaaagaca accgcaccc 198019RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 80cugggcgacu ccagcgcug
198119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 81ggcgaaaucg cccugucca 198219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 82acgcgcaacg ugucggaga
198319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 83acccgguaug ugucagagc 198419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 84cugacacugg uucgcguga
198519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 85aagguggcag aggcuggcc 198619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 86cacuacacca ugcgggccu
198719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 87uuccaugagg augcugagg 198819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 88guccagcucu ccuuccagc
198919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 89cuacagauca augucccug 199019RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 90guccgagugc uggagcuaa
199119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 91agugagagcc acccugaca 199219RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 92aguggggaac agacagucc
199319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 93cgcugucgug gccggggca 199419RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 94augccccagc cgaacauca
199519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 95aucuggucug ccugcagag 199619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 96gaccucaaaa gguguccac
199719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 97cgugagcugc cgcccacgc 199819RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 98cugcugggga acaguuccg
199919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 99gaagaggaga gccagcugg 1910019RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 100gagacuaacg
ugacguacu 1910119RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 101ugggaggagg agcaggagu
1910219RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 102uuugaggugg ugagcacac 1910319RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 103cugcgucugc
agcacgugg 1910419RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 104gaucggccac ugucggugc
1910519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 105cgcugcacgc ugcgcaacg 1910619RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 106gcugugggcc
aggacacgc 1910719RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 107caggagguca ucguggugc
1910819RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 108ccacacuccu ugcccuuua 1910919RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 109aagguggugg
ugaucucag 1911019RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 110gccauccugg cccuggugg
1911119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 111gugcucacca ucaucuccc 1911219RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 112cuuaucaucc
ucaucaugc 1911319RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 113cuuuggcaga agaagccac
1911419RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 114cguuacgaga uccgaugga 1911519RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 115aaggugauug
agucuguga 1911619RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 116agcucugacg gccaugagu
1911719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 117uacaucuacg uggacccca 1911819RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 118augcagcugc
ccuaugacu 1911919RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 119uccacguggg agcugccgc
1912019RNAArtificial SequenceSynthetic Target Sequence/siNA
sense region 120cgggaccagc uugugcugg 1912119RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 121ggacgcaccc
ucggcucug 1912219RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 122ggggccuuug ggcaggugg
1912319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 123guggaggcca cggcucaug 1912419RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 124ggccugagcc
auucucagg 1912519RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 125gccacgauga aaguggccg
1912619RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 126gucaagaugc uuaaaucca 1912719RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 127acagcccgca
gcagugaga 1912819RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 128aagcaagccc uuaugucgg
1912919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 129gagcugaaga ucaugaguc 1913019RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 130caccuugggc
cccaccuga 1913119RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 131aacgugguca accuguugg
1913219RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 132ggggccugca ccaaaggag 1913319RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 133ggacccaucu
auaucauca 1913419RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 134acugaguacu gccgcuacg
1913519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 135ggagaccugg uggacuacc 1913619RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 136cugcaccgca
acaaacaca 1913719RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 137accuuccugc agcaccacu
1913819RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 138uccgacaagc gccgcccgc 1913919RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 139cccagcgcgg
agcucuaca 1914019RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 140agcaaugcuc ugcccguug
1914119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 141gggcuccccc ugcccagcc 1914219RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 142cauguguccu
ugaccgggg 1914319RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 143gagagcgacg guggcuaca
1914419RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 144auggacauga gcaaggacg 1914519RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 145gagucggugg
acuaugugc 1914619RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 146cccaugcugg acaugaaag
1914719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 147ggagacguca aauaugcag 1914819RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 148gacaucgagu
ccuccaacu 1914919RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 149uacauggccc cuuacgaua
1915019RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 150aacuacguuc ccucugccc 1915119RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 151ccugagagga
ccugccgag 1915219RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 152gcaacuuuga ucaacgagu
1915319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 153ucuccagugc uaagcuaca 1915419RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 154auggaccucg
ugggcuuca 1915519RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 155agcuaccagg uggccaaug
1915619RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 156ggcauggagu uucuggccu 1915719RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 157uccaagaacu
gcguccaca 1915819RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 158agagaccugg cggcuagga
1915919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 159aacgugcuca ucugugaag 1916019RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 160ggcaagcugg
ucaagaucu 1916119RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 161ugugacuuug gccuggcuc
1916219RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 162cgagacauca ugcgggacu 1916319RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 163ucgaauuaca
ucuccaaag 1916419RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 164ggcagcaccu uuuugccuu
1916519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 165uuaaagugga uggcuccgg 1916619RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 166gagagcaucu
ucaacagcc 1916719RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 167cucuacacca cccugagcg
1916819RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 168gacguguggu ccuucggga 1916919RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 169auccugcucu
gggagaucu 1917019RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 170uucaccuugg guggcaccc
1917119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 171ccuuacccag agcugccca 1917219RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 172augaacgagc
aguucuaca 1917319RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 173aaugccauca aacgggguu
1917419RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 174uaccgcaugg cccagccug 1917519RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 175gcccaugccu
ccgacgaga 1917619RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 176aucuaugaga ucaugcaga
1917719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 177aagugcuggg aagagaagu 1917819RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 178uuugagauuc
ggccccccu 1917919RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 179uucucccagc uggugcugc
1918019RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 180cuucucgaga gacuguugg 1918119RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 181ggcgaagguu
acaaaaaga 1918219RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 182aaguaccagc agguggaug
1918319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 183gaggaguuuc ugaggagug 1918419RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 184gaccacccag
ccauccuuc 1918519RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 185cggucccagg cccgcuugc
1918619RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 186ccuggguucc auggccucc 1918719RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 187cgaucucccc
uggacacca 1918819RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 188agcuccgucc ucuauacug
1918919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 189gccgugcagc ccaaugagg 1919019RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 190ggugacaacg
acuauauca 1919119RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 191aucccccugc cugacccca
1919219RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 192aaacccgagg uugcugacg 1919319RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 193gagggcccac
uggaggguu 1919419RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 194ucccccagcc uagccagcu
1919519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 195uccacccuga augaaguca 1919619RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 196aacaccuccu
caaccaucu 1919719RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 197uccugugaca gcccccugg
1919819RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 198gagccccagg acgaaccag 1919919RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 199gagccagagc
cccagcuug 1920019RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 200gagcuccagg uggagccgg
1920119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 201gagccagagc uggaacagu 1920219RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 202uugccggauu
cggggugcc 1920319RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 203ccugcgccuc gggcggaag
1920419RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 204gcagaggaua gcuuccugu 1920519RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 205uagggggcug
gccccuacc 1920619RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 206ccugcccugc cugaagcuc
1920719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 207cccccccugc cagcaccca 1920819RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 208agcaucuccu
ggccuggcc 1920919RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 209cugaccgggc uuccuguca
1921019RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 210agccaggcug cccuuauca 1921119RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 211agcugucccc
uucuggaag 1921219RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 212gcuuucugcu ccugacgug
1921319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 213guugugcccc aaacccugg 1921419RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 214gggcuggcuu
aggaggcaa 1921519RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 215agaaaacugc aggggccgu
1921619RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 216ugaccagccc ucugccucc 1921719RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 217cagggaggcc
aacugacuc 1921819RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 218cugagccagg guuccccca
1921919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 219agggaacuca guuuuccca 1922019RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 220auauguaaga
ugggaaagu 1922119RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 221uuaggcuuga ugacccaga
1922219RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 222aaucuaggau ucucucccu 1922319RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 223uggcugacag
guggggaga 1922419RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 224accgaauccc ucccuggga
1922519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 225aagauucuug gaguuacug 1922619RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 226gaggugguaa
auuaacuuu 1922719RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 227uuuucuguuc agccagcua
1922819RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 228accccucaag gaaucauag 1922919RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 229gcucucuccu
cgcacuuuu 1923019RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 230uuauccaccc aggagcuag
1923119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 231gggaagagac ccuagccuc 1923219RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 232cccuggcugc
uggcugagc 1923319RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 233cuagggccua gccuugagc
1923419RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 234caguguugcc ucauccaga 1923519RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 235aagaaagcca
gucuccucc 1923619RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 236ccuaugaugc cagucccug
1923719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 237gcguucccug gcccgagcu 1923819RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 238uggucugggg
ccauuaggc 1923919RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 239cagccuaauu aaugcugga
1924019RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 240aggcugagcc aaguacagg 1924119RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 241gacaccccca
gccugcagc 1924219RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 242cccuugccca gggcacuug
1924319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 243ggagcacacg cagccauag 1924419RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 244gcaagugccu
gugucccug 1924519RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 245guccuucagg cccaucagu
1924619RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 246uccuggggcu uuuucuuua 1924719RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 247aucacccuca
gucuuaauc 1924819RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 248ccauccacca gagucuaga
1924919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 249aaggccagac gggccccgc 1925019RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 250caucugugau
gagaaugua 1925119RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 251aaaugugcca guguggagu
1925219RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 252uggccacgug ugugugcca 1925319RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 253aguauauggc
ccuggcucu 1925419RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 254ugcauuggac cugcuauga
1925519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 255aggcuuugga ggaaucccu 1925619RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 256ucacccucuc
ugggccuca 1925719RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 257aguuuccccu ucaaaaaau
1925819RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 258ugaauaaguc ggacuuauu 1925919RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 259uaacucugag
ugccuugcc 1926019RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 260cagcacuaac auucuagag
1926119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 261guauuccagg ugguugcac 1926219RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 262cauuugucca
gaugaagca 1926319RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 263aaggccauau acccuaaac
1926419RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 264cuuccauccu gggggucag 1926519RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 265gcugggcucc
ugggagauu 1926619RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 266uccagaucac acaucacac
1926719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 267cucuggggac ucaggaacc 1926819RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 268caugccccuu
ccccaggcc 1926919RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 269ccccagcaag ucucaagaa
1927019RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 270acacagcugc acaggccuu 1927119RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 271ugacuuagag
ugacagccg 1927219RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 272gguguccugg aaagcccca
1927319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 273aagcagcugc cccagggac 1927419RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 274caugggaaga
ccacgggac 1927519RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 275ccucuuucac uacccacga
1927619RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 276augaccuccg gggguaucc 1927719RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 277cugggcaaaa
gggacaaag 1927819RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 278gagggcaaau gagaucacc
1927919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 279cuccugcagc ccaccacuc 1928019RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 280ccagcaccug
ugccgaggu 1928119RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 281ucugcgucga agacagaau
1928219RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 282uggacaguga ggacaguua 1928319RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 283augucuugua
aaagacaag 1928419RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 284gaagcuucag augguaccc
1928519RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 285ccaagaagga ugugagagg 1928619RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 286guggccgcuu
ggaguuugc 1928719RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 287ccccucaccc accagcugc
1928819RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 288ccccaucccu gaggcagcg 1928919RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 289gcuccauggg
gguaugguu 1929019RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 290uuugucacug cccagaccu
1929119RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 291uagcagugac aucucauug 1929219RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 292guccccagcc
cagugggca 1929319RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 293auuggaggug ccaggggag
1929419RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 294gucaggguug uagccaaga 1929519RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 295acgcccccgc
acggggagg 1929619RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 296gguugggaag ggggugcag
1929719RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 297ggaagcucaa ccccucugg 1929819RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 298ggcaccaacc
cugcauugc 1929919RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 299cagguuggca ccuuacuuc
1930019RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 300cccugggauc cccagaguu 1930119RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 301ugguccaagg
agggagagu 1930219RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 302uggguucuca auacgguac
1930319RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 303ccaaagauau aaucaccua 1930419RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 304agguuuacaa
auauuuuua 1930519RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 305aggacucacg uuaacucac
1930619RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 306cauuuauaca gcagaaaug 1930719RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 307gcuauuuugu
augcuguua 1930819RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 308aaguuuuucu aucugugua
1930919RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 309acuuuuuuuu aagggaaag 1931019RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 310gauuuuaaua
uuaaaccug 1931119RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 311aaccuggugc uucucacuc
1931219RNAArtificial SequencesiNA antisense region 312gggcagcagg
gcugagggg 1931319RNAArtificial SequencesiNA antisense region
313cgagcacagg cucgugcug 1931419RNAArtificial SequencesiNA antisense
region 314ugucugcguu gggcagggc 1931519RNAArtificial SequencesiNA
antisense region 315ggccgcccug ggucuggcu 1931619RNAArtificial
SequencesiNA antisense region 316gagcagagcc gccagaggg
1931719RNAArtificial SequencesiNA antisense region 317ccaagcaucc
uucgggagg 1931819RNAArtificial SequencesiNA antisense region
318ccagcuucgc cucacuccc 1931919RNAArtificial SequencesiNA antisense
region 319uguaggggag aggagcgcc 1932019RNAArtificial SequencesiNA
antisense region 320auggaggaag ggggcugcu 1932119RNAArtificial
SequencesiNA antisense region 321gcucaggaga acagaggga
1932219RNAArtificial SequencesiNA antisense region 322uggugcaggc
uccugaagg 1932319RNAArtificial SequencesiNA antisense region
323uagaaggaca ggcaggacu 1932419RNAArtificial SequencesiNA antisense
region 324gaguggguaa cagcugagu 1932519RNAArtificial SequencesiNA
antisense region 325gaaagacugc uggucccag 1932619RNAArtificial
SequencesiNA antisense region 326gcccucuccc aguuaucag
1932719RNAArtificial SequencesiNA antisense region 327caggaagucc
uccuuacug 1932819RNAArtificial SequencesiNA antisense region
328cuggacaguc acccccucc 1932919RNAArtificial SequencesiNA antisense
region 329ugggcacagu uccaggcuc 1933019RNAArtificial SequencesiNA
antisense region 330ugcugauggc uucuggugu 1933119RNAArtificial
SequencesiNA antisense region 331gccgcauggu guccuugcu
1933219RNAArtificial SequencesiNA antisense region 332cuggcaucgc
acccggaag 1933319RNAArtificial SequencesiNA antisense region
333cgccuuugag ggccagagc 1933419RNAArtificial SequencesiNA antisense
region 334gagacagcaa cagcagcuc 1933519RNAArtificial SequencesiNA
antisense region 335guuccagaag uaacaggag 1933619RNAArtificial
SequencesiNA antisense region 336ggcccugaga gaucugugg
1933719RNAArtificial SequencesiNA antisense region 337ccgggggugu
gacgaccag 1933819RNAArtificial SequencesiNA antisense region
338ugaggacaag cucuggccc 1933919RNAArtificial SequencesiNA antisense
region 339cgaaggugcu ggagacauu 1934019RNAArtificial SequencesiNA
antisense region 340aacccgagca ggucagaac 1934119RNAArtificial
SequencesiNA antisense region 341cccacaccac cggagcuga
1934219RNAArtificial SequencesiNA antisense region 342gcuccuggga
cauccguuc 1934319RNAArtificial SequencesiNA antisense region
343uggccauuuc cuguggggg 1934419RNAArtificial SequencesiNA antisense
region 344aggugccauc cugggccuu 1934519RNAArtificial SequencesiNA
antisense region 345gugugagcac gcuggagaa 1934619RNAArtificial
SequencesiNA antisense region 346gcccagugag guuggucag
1934719RNAArtificial SequencesiNA antisense region 347aguauucucc
cgugucuag 1934819RNAArtificial SequencesiNA antisense region
348agucauugug ggugcaaaa 1934919RNAArtificial SequencesiNA antisense
region 349cggucuccag uccacggga 1935019RNAArtificial SequencesiNA
antisense region 350agagccguuu ccgcucauc 1935119RNAArtificial
SequencesiNA antisense region 351gaucuggcac aaagaugua
1935219RNAArtificial SequencesiNA antisense region 352ggaggaagcc
cacgguggg 1935319RNAArtificial SequencesiNA antisense region
353guuccucggc aucauuagg 1935419RNAArtificial SequencesiNA antisense
region 354ccgugagaaa gaugaauag 1935519RNAArtificial SequencesiNA
antisense region 355uggugaucuc
aguuauuuc 1935619RNAArtificial SequencesiNA antisense region
356cuguuacucg gcauggaau 1935719RNAArtificial SequencesiNA antisense
region 357ucaccaccag cuguggguc 1935819RNAArtificial SequencesiNA
antisense region 358cuuucuucuc gugcagugu 1935919RNAArtificial
SequencesiNA antisense region 359caggcagugc aacgucccc
1936019RNAArtificial SequencesiNA antisense region 360guuggugauc
auaggggac 1936119RNAArtificial SequencesiNA antisense region
361agauaccaga aaagccacg 1936219RNAArtificial SequencesiNA antisense
region 362uguagcuucu guccucaaa 1936319RNAArtificial SequencesiNA
antisense region 363caaugguggu uuugcagau 1936419RNAArtificial
SequencesiNA antisense region 364aauccaccuc ccugucccc
1936519RNAArtificial SequencesiNA antisense region 365agacauagua
ggcaucaga 1936619RNAArtificial SequencesiNA antisense region
366augacaccug gagucugua 1936719RNAArtificial SequencesiNA antisense
region 367ucacagagac guugaugga 1936819RNAArtificial SequencesiNA
antisense region 368ccacagucug cacugcguu 1936919RNAArtificial
SequencesiNA antisense region 369uguucucacc cuggcggac
1937019RNAArtificial SequencesiNA antisense region 370caaugcacau
gagggugau 1937119RNAArtificial SequencesiNA antisense region
371ccaccucauu cccgaucac 1937219RNAArtificial SequencesiNA antisense
region 372auguccacuc gaaguugac 1937319RNAArtificial SequencesiNA
antisense region 373cacuuucuuu gcgggggua 1937419RNAArtificial
SequencesiNA antisense region 374ccggcuccac cagccgccc
1937519RNAArtificial SequencesiNA antisense region 375ccaagaggaa
gucagucac 1937619RNAArtificial SequencesiNA antisense region
376ggauguggua aggcauauc 1937719RNAArtificial SequencesiNA antisense
region 377ggaugugcag gauggagcg 1937819RNAArtificial SequencesiNA
antisense region 378cuucuaacuc ggcacuggg 1937919RNAArtificial
SequencesiNA antisense region 379agguguaggu ccccgaguc
1938019RNAArtificial SequencesiNA antisense region 380cacucuccgu
cacauugca 1938119RNAArtificial SequencesiNA antisense region
381cauccugaug gucauucac 1938219RNAArtificial SequencesiNA antisense
region 382ugauguugau ggccuuuuc 1938319RNAArtificial SequencesiNA
antisense region 383agccgcucuc aaccacggu 1938419RNAArtificial
SequencesiNA antisense region 384cucccaggag ccgcacgua
1938519RNAArtificial SequencesiNA antisense region 385auuguagugu
gcccaccuc 1938619RNAArtificial SequencesiNA antisense region
386uccgaugcag cucagcaaa 1938719RNAArtificial SequencesiNA antisense
region 387cuaccugcag uguccggcu 1938819RNAArtificial SequencesiNA
antisense region 388guggguaggc cucgaacac 1938919RNAArtificial
SequencesiNA antisense region 389accacaggac agugggcgg
1939019RNAArtificial SequencesiNA antisense region 390gggugcgguu
gucuuugaa 1939119RNAArtificial SequencesiNA antisense region
391cagcgcugga gucgcccag 1939219RNAArtificial SequencesiNA antisense
region 392uggacagggc gauuucgcc 1939319RNAArtificial SequencesiNA
antisense region 393ucuccgacac guugcgcgu 1939419RNAArtificial
SequencesiNA antisense region 394gcucugacac auaccgggu
1939519RNAArtificial SequencesiNA antisense region 395ucacgcgaac
cagugucag 1939619RNAArtificial SequencesiNA antisense region
396ggccagccuc ugccaccuu 1939719RNAArtificial SequencesiNA antisense
region 397aggcccgcau gguguagug 1939819RNAArtificial SequencesiNA
antisense region 398ccucagcauc cucauggaa 1939919RNAArtificial
SequencesiNA antisense region 399gcuggaagga gagcuggac
1940019RNAArtificial SequencesiNA antisense region 400cagggacauu
gaucuguag 1940119RNAArtificial SequencesiNA antisense region
401uuagcuccag cacucggac 1940219RNAArtificial SequencesiNA antisense
region 402ugucagggug gcucucacu 1940319RNAArtificial SequencesiNA
antisense region 403ggacugucug uuccccacu 1940419RNAArtificial
SequencesiNA antisense region 404ugccccggcc acgacagcg
1940519RNAArtificial SequencesiNA antisense region 405ugauguucgg
cuggggcau 1940619RNAArtificial SequencesiNA antisense region
406cucugcaggc agaccagau 1940719RNAArtificial SequencesiNA antisense
region 407guggacaccu uuugagguc 1940819RNAArtificial SequencesiNA
antisense region 408gcgugggcgg cagcucacg 1940919RNAArtificial
SequencesiNA antisense region 409cggaacuguu ccccagcag
1941019RNAArtificial SequencesiNA antisense region 410ccagcuggcu
cuccucuuc 1941119RNAArtificial SequencesiNA antisense region
411aguacgucac guuagucuc 1941219RNAArtificial SequencesiNA antisense
region 412acuccugcuc cuccuccca 1941319RNAArtificial SequencesiNA
antisense region 413gugugcucac caccucaaa 1941419RNAArtificial
SequencesiNA antisense region 414ccacgugcug cagacgcag
1941519RNAArtificial SequencesiNA antisense region 415gcaccgacag
uggccgauc 1941619RNAArtificial SequencesiNA antisense region
416cguugcgcag cgugcagcg 1941719RNAArtificial SequencesiNA antisense
region 417gcguguccug gcccacagc 1941819RNAArtificial SequencesiNA
antisense region 418gcaccacgau gaccuccug 1941919RNAArtificial
SequencesiNA antisense region 419uaaagggcaa ggagugugg
1942019RNAArtificial SequencesiNA antisense region 420cugagaucac
caccaccuu 1942119RNAArtificial SequencesiNA antisense region
421ccaccagggc caggauggc 1942219RNAArtificial SequencesiNA antisense
region 422gggagaugau ggugagcac 1942319RNAArtificial SequencesiNA
antisense region 423gcaugaugag gaugauaag 1942419RNAArtificial
SequencesiNA antisense region 424guggcuucuu cugccaaag
1942519RNAArtificial SequencesiNA antisense region 425uccaucggau
cucguaacg 1942619RNAArtificial SequencesiNA antisense region
426ucacagacuc aaucaccuu 1942719RNAArtificial SequencesiNA antisense
region 427acucauggcc gucagagcu 1942819RNAArtificial SequencesiNA
antisense region 428ugggguccac guagaugua 1942919RNAArtificial
SequencesiNA antisense region 429agucauaggg cagcugcau
1943019RNAArtificial SequencesiNA antisense region 430gcggcagcuc
ccacgugga 1943119RNAArtificial SequencesiNA antisense region
431ccagcacaag cuggucccg 1943219RNAArtificial SequencesiNA antisense
region 432cagagccgag ggugcgucc 1943319RNAArtificial SequencesiNA
antisense region 433ccaccugccc aaaggcccc 1943419RNAArtificial
SequencesiNA antisense region 434caugagccgu ggccuccac
1943519RNAArtificial SequencesiNA antisense region 435ccugagaaug
gcucaggcc 1943619RNAArtificial SequencesiNA antisense region
436cggccacuuu caucguggc 1943719RNAArtificial SequencesiNA antisense
region 437uggauuuaag caucuugac 1943819RNAArtificial SequencesiNA
antisense region 438ucucacugcu gcgggcugu 1943919RNAArtificial
SequencesiNA antisense region 439ccgacauaag ggcuugcuu
1944019RNAArtificial SequencesiNA antisense region 440gacucaugau
cuucagcuc 1944119RNAArtificial SequencesiNA antisense region
441ucaggugggg cccaaggug 1944219RNAArtificial SequencesiNA antisense
region 442ccaacagguu gaccacguu 1944319RNAArtificial SequencesiNA
antisense region 443cuccuuuggu gcaggcccc 1944419RNAArtificial
SequencesiNA antisense region 444ugaugauaua gaugggucc
1944519RNAArtificial SequencesiNA antisense region 445cguagcggca
guacucagu 1944619RNAArtificial SequencesiNA antisense region
446gguaguccac caggucucc 1944719RNAArtificial SequencesiNA antisense
region 447uguguuuguu gcggugcag 1944819RNAArtificial SequencesiNA
antisense region 448aguggugcug caggaaggu 1944919RNAArtificial
SequencesiNA antisense region 449gcgggcggcg cuugucgga
1945019RNAArtificial SequencesiNA antisense region 450uguagagcuc
cgcgcuggg 1945119RNAArtificial SequencesiNA antisense region
451caacgggcag agcauugcu 1945219RNAArtificial SequencesiNA antisense
region 452ggcugggcag ggggagccc 1945319RNAArtificial SequencesiNA
antisense region 453ccccggucaa ggacacaug 1945419RNAArtificial
SequencesiNA antisense region 454uguagccacc gucgcucuc
1945519RNAArtificial SequencesiNA antisense region 455cguccuugcu
cauguccau 1945619RNAArtificial SequencesiNA antisense region
456gcacauaguc caccgacuc 1945719RNAArtificial SequencesiNA antisense
region 457cuuucauguc cagcauggg 1945819RNAArtificial SequencesiNA
antisense region 458cugcauauuu gacgucucc 1945919RNAArtificial
SequencesiNA antisense region 459aguuggagga cucgauguc
1946019RNAArtificial SequencesiNA antisense region 460uaucguaagg
ggccaugua 1946119RNAArtificial SequencesiNA antisense region
461gggcagaggg aacguaguu 1946219RNAArtificial SequencesiNA antisense
region 462cucggcaggu ccucucagg 1946319RNAArtificial SequencesiNA
antisense region 463acucguugau caaaguugc 1946419RNAArtificial
SequencesiNA antisense region 464uguagcuuag cacuggaga
1946519RNAArtificial SequencesiNA antisense region 465ugaagcccac
gagguccau 1946619RNAArtificial SequencesiNA antisense region
466cauuggccac cugguagcu 1946719RNAArtificial SequencesiNA antisense
region 467aggccagaaa cuccaugcc 1946819RNAArtificial SequencesiNA
antisense region 468uguggacgca guucuugga 1946919RNAArtificial
SequencesiNA antisense region 469uccuagccgc caggucucu
1947019RNAArtificial SequencesiNA antisense region 470cuucacagau
gagcacguu 1947119RNAArtificial SequencesiNA antisense region
471agaucuugac cagcuugcc 1947219RNAArtificial SequencesiNA antisense
region 472gagccaggcc aaagucaca 1947319RNAArtificial SequencesiNA
antisense region 473agucccgcau gaugucucg 1947419RNAArtificial
SequencesiNA antisense region 474cuuuggagau guaauucga
1947519RNAArtificial SequencesiNA antisense region 475aaggcaaaaa
ggugcugcc 1947619RNAArtificial SequencesiNA antisense region
476ccggagccau ccacuuuaa 1947719RNAArtificial SequencesiNA antisense
region 477ggcuguugaa gaugcucuc 1947819RNAArtificial SequencesiNA
antisense region 478cgcucagggu gguguagag 1947919RNAArtificial
SequencesiNA antisense region 479ucccgaagga ccacacguc
1948019RNAArtificial SequencesiNA antisense region 480agaucuccca
gagcaggau
1948119RNAArtificial SequencesiNA antisense region 481gggugccacc
caaggugaa 1948219RNAArtificial SequencesiNA antisense region
482ugggcagcuc uggguaagg 1948319RNAArtificial SequencesiNA antisense
region 483uguagaacug cucguucau 1948419RNAArtificial SequencesiNA
antisense region 484aaccccguuu gauggcauu 1948519RNAArtificial
SequencesiNA antisense region 485caggcugggc caugcggua
1948619RNAArtificial SequencesiNA antisense region 486ucucgucgga
ggcaugggc 1948719RNAArtificial SequencesiNA antisense region
487ucugcaugau cucauagau 1948819RNAArtificial SequencesiNA antisense
region 488acuucucuuc ccagcacuu 1948919RNAArtificial SequencesiNA
antisense region 489aggggggccg aaucucaaa 1949019RNAArtificial
SequencesiNA antisense region 490gcagcaccag cugggagaa
1949119RNAArtificial SequencesiNA antisense region 491ccaacagucu
cucgagaag 1949219RNAArtificial SequencesiNA antisense region
492ucuuuuugua accuucgcc 1949319RNAArtificial SequencesiNA antisense
region 493cauccaccug cugguacuu 1949419RNAArtificial SequencesiNA
antisense region 494cacuccucag aaacuccuc 1949519RNAArtificial
SequencesiNA antisense region 495gaaggauggc ugggugguc
1949619RNAArtificial SequencesiNA antisense region 496gcaagcgggc
cugggaccg 1949719RNAArtificial SequencesiNA antisense region
497ggaggccaug gaacccagg 1949819RNAArtificial SequencesiNA antisense
region 498ugguguccag gggagaucg 1949919RNAArtificial SequencesiNA
antisense region 499caguauagag gacggagcu 1950019RNAArtificial
SequencesiNA antisense region 500ccucauuggg cugcacggc
1950119RNAArtificial SequencesiNA antisense region 501ugauauaguc
guugucacc 1950219RNAArtificial SequencesiNA antisense region
502uggggucagg cagggggau 1950319RNAArtificial SequencesiNA antisense
region 503cgucagcaac cucggguuu 1950419RNAArtificial SequencesiNA
antisense region 504aacccuccag ugggcccuc 1950519RNAArtificial
SequencesiNA antisense region 505agcuggcuag gcuggggga
1950619RNAArtificial SequencesiNA antisense region 506ugacuucauu
cagggugga 1950719RNAArtificial SequencesiNA antisense region
507agaugguuga ggagguguu 1950819RNAArtificial SequencesiNA antisense
region 508ccagggggcu gucacagga 1950919RNAArtificial SequencesiNA
antisense region 509cugguucguc cuggggcuc 1951019RNAArtificial
SequencesiNA antisense region 510caagcugggg cucuggcuc
1951119RNAArtificial SequencesiNA antisense region 511ccggcuccac
cuggagcuc 1951219RNAArtificial SequencesiNA antisense region
512acuguuccag cucuggcuc 1951319RNAArtificial SequencesiNA antisense
region 513ggcaccccga auccggcaa 1951419RNAArtificial SequencesiNA
antisense region 514cuuccgcccg aggcgcagg 1951519RNAArtificial
SequencesiNA antisense region 515acaggaagcu auccucugc
1951619RNAArtificial SequencesiNA antisense region 516gguaggggcc
agcccccua 1951719RNAArtificial SequencesiNA antisense region
517gagcuucagg cagggcagg 1951819RNAArtificial SequencesiNA antisense
region 518ugggugcugg caggggggg 1951919RNAArtificial SequencesiNA
antisense region 519ggccaggcca ggagaugcu 1952019RNAArtificial
SequencesiNA antisense region 520ugacaggaag cccggucag
1952119RNAArtificial SequencesiNA antisense region 521ugauaagggc
agccuggcu 1952219RNAArtificial SequencesiNA antisense region
522cuuccagaag gggacagcu 1952319RNAArtificial SequencesiNA antisense
region 523cacgucagga gcagaaagc 1952419RNAArtificial SequencesiNA
antisense region 524ccaggguuug gggcacaac 1952519RNAArtificial
SequencesiNA antisense region 525uugccuccua agccagccc
1952619RNAArtificial SequencesiNA antisense region 526acggccccug
caguuuucu 1952719RNAArtificial SequencesiNA antisense region
527ggaggcagag ggcugguca 1952819RNAArtificial SequencesiNA antisense
region 528gagucaguug gccucccug 1952919RNAArtificial SequencesiNA
antisense region 529ugggggaacc cuggcucag 1953019RNAArtificial
SequencesiNA antisense region 530ugggaaaacu gaguucccu
1953119RNAArtificial SequencesiNA antisense region 531acuuucccau
cuuacauau 1953219RNAArtificial SequencesiNA antisense region
532ucugggucau caagccuaa 1953319RNAArtificial SequencesiNA antisense
region 533agggagagaa uccuagauu 1953419RNAArtificial SequencesiNA
antisense region 534ucuccccacc ugucagcca 1953519RNAArtificial
SequencesiNA antisense region 535ucccagggag ggauucggu
1953619RNAArtificial SequencesiNA antisense region 536caguaacucc
aagaaucuu 1953719RNAArtificial SequencesiNA antisense region
537aaaguuaauu uaccaccuc 1953819RNAArtificial SequencesiNA antisense
region 538uagcuggcug aacagaaaa 1953919RNAArtificial SequencesiNA
antisense region 539cuaugauucc uugaggggu 1954019RNAArtificial
SequencesiNA antisense region 540aaaagugcga ggagagagc
1954119RNAArtificial SequencesiNA antisense region 541cuagcuccug
gguggauaa 1954219RNAArtificial SequencesiNA antisense region
542gaggcuaggg ucucuuccc 1954319RNAArtificial SequencesiNA antisense
region 543gcucagccag cagccaggg 1954419RNAArtificial SequencesiNA
antisense region 544gcucaaggcu aggcccuag 1954519RNAArtificial
SequencesiNA antisense region 545ucuggaugag gcaacacug
1954619RNAArtificial SequencesiNA antisense region 546ggaggagacu
ggcuuucuu 1954719RNAArtificial SequencesiNA antisense region
547cagggacugg caucauagg 1954819RNAArtificial SequencesiNA antisense
region 548agcucgggcc agggaacgc 1954919RNAArtificial SequencesiNA
antisense region 549gccuaauggc cccagacca 1955019RNAArtificial
SequencesiNA antisense region 550uccagcauua auuaggcug
1955119RNAArtificial SequencesiNA antisense region 551ccuguacuug
gcucagccu 1955219RNAArtificial SequencesiNA antisense region
552gcugcaggcu ggggguguc 1955319RNAArtificial SequencesiNA antisense
region 553caagugcccu gggcaaggg 1955419RNAArtificial SequencesiNA
antisense region 554cuauggcugc gugugcucc 1955519RNAArtificial
SequencesiNA antisense region 555cagggacaca ggcacuugc
1955619RNAArtificial SequencesiNA antisense region 556acugaugggc
cugaaggac 1955719RNAArtificial SequencesiNA antisense region
557uaaagaaaaa gccccagga 1955819RNAArtificial SequencesiNA antisense
region 558gauuaagacu gagggugau 1955919RNAArtificial SequencesiNA
antisense region 559ucuagacucu gguggaugg 1956019RNAArtificial
SequencesiNA antisense region 560gcggggcccg ucuggccuu
1956119RNAArtificial SequencesiNA antisense region 561uacauucuca
ucacagaug 1956219RNAArtificial SequencesiNA antisense region
562acuccacacu ggcacauuu 1956319RNAArtificial SequencesiNA antisense
region 563uggcacacac acguggcca 1956419RNAArtificial SequencesiNA
antisense region 564agagccaggg ccauauacu 1956519RNAArtificial
SequencesiNA antisense region 565ucauagcagg uccaaugca
1956619RNAArtificial SequencesiNA antisense region 566agggauuccu
ccaaagccu 1956719RNAArtificial SequencesiNA antisense region
567ugaggcccag agaggguga 1956819RNAArtificial SequencesiNA antisense
region 568auuuuuugaa ggggaaacu 1956919RNAArtificial SequencesiNA
antisense region 569aauaaguccg acuuauuca 1957019RNAArtificial
SequencesiNA antisense region 570ggcaaggcac ucagaguua
1957119RNAArtificial SequencesiNA antisense region 571cucuagaaug
uuagugcug 1957219RNAArtificial SequencesiNA antisense region
572gugcaaccac cuggaauac 1957319RNAArtificial SequencesiNA antisense
region 573ugcuucaucu ggacaaaug 1957419RNAArtificial SequencesiNA
antisense region 574guuuagggua uauggccuu 1957519RNAArtificial
SequencesiNA antisense region 575cugaccccca ggauggaag
1957619RNAArtificial SequencesiNA antisense region 576aaucucccag
gagcccagc 1957719RNAArtificial SequencesiNA antisense region
577gugugaugug ugaucugga 1957819RNAArtificial SequencesiNA antisense
region 578gguuccugag uccccagag 1957919RNAArtificial SequencesiNA
antisense region 579ggccugggga aggggcaug 1958019RNAArtificial
SequencesiNA antisense region 580uucuugagac uugcugggg
1958119RNAArtificial SequencesiNA antisense region 581aaggccugug
cagcugugu 1958219RNAArtificial SequencesiNA antisense region
582cggcugucac ucuaaguca 1958319RNAArtificial SequencesiNA antisense
region 583uggggcuuuc caggacacc 1958419RNAArtificial SequencesiNA
antisense region 584gucccugggg cagcugcuu 1958519RNAArtificial
SequencesiNA antisense region 585gucccguggu cuucccaug
1958619RNAArtificial SequencesiNA antisense region 586ucguggguag
ugaaagagg 1958719RNAArtificial SequencesiNA antisense region
587ggauaccccc ggaggucau 1958819RNAArtificial SequencesiNA antisense
region 588cuuugucccu uuugcccag 1958919RNAArtificial SequencesiNA
antisense region 589ggugaucuca uuugcccuc 1959019RNAArtificial
SequencesiNA antisense region 590gagugguggg cugcaggag
1959119RNAArtificial SequencesiNA antisense region 591accucggcac
aggugcugg 1959219RNAArtificial SequencesiNA antisense region
592auucugucuu cgacgcaga 1959319RNAArtificial SequencesiNA antisense
region 593uaacuguccu cacugucca 1959419RNAArtificial SequencesiNA
antisense region 594cuugucuuuu acaagacau 1959519RNAArtificial
SequencesiNA antisense region 595ggguaccauc ugaagcuuc
1959619RNAArtificial SequencesiNA antisense region 596ccucucacau
ccuucuugg 1959719RNAArtificial SequencesiNA antisense region
597gcaaacucca agcggccac 1959819RNAArtificial SequencesiNA antisense
region 598gcagcuggug ggugagggg 1959919RNAArtificial SequencesiNA
antisense region 599cgcugccuca gggaugggg 1960019RNAArtificial
SequencesiNA antisense region 600aaccauaccc ccauggagc
1960119RNAArtificial SequencesiNA antisense region 601aggucugggc
agugacaaa 1960219RNAArtificial SequencesiNA antisense region
602caaugagaug ucacugcua 1960319RNAArtificial SequencesiNA antisense
region 603ugcccacugg gcuggggac 1960419RNAArtificial SequencesiNA
antisense region 604cuccccuggc accuccaau 1960519RNAArtificial
SequencesiNA antisense region 605ucuuggcuac aacccugac
1960619RNAArtificial SequencesiNA antisense region
606ccuccccgug
cgggggcgu 1960719RNAArtificial SequencesiNA antisense region
607cugcaccccc uucccaacc 1960819RNAArtificial SequencesiNA antisense
region 608ccagaggggu ugagcuucc 1960919RNAArtificial SequencesiNA
antisense region 609gcaaugcagg guuggugcc 1961019RNAArtificial
SequencesiNA antisense region 610gaaguaaggu gccaaccug
1961119RNAArtificial SequencesiNA antisense region 611aacucugggg
aucccaggg 1961219RNAArtificial SequencesiNA antisense region
612acucucccuc cuuggacca 1961319RNAArtificial SequencesiNA antisense
region 613guaccguauu gagaaccca 1961419RNAArtificial SequencesiNA
antisense region 614uaggugauua uaucuuugg 1961519RNAArtificial
SequencesiNA antisense region 615uaaaaauauu uguaaaccu
1961619RNAArtificial SequencesiNA antisense region 616gugaguuaac
gugaguccu 1961719RNAArtificial SequencesiNA antisense region
617cauuucugcu guauaaaug 1961819RNAArtificial SequencesiNA antisense
region 618uaacagcaua caaaauagc 1961919RNAArtificial SequencesiNA
antisense region 619uacacagaua gaaaaacuu 1962019RNAArtificial
SequencesiNA antisense region 620cuuucccuua aaaaaaagu
1962119RNAArtificial SequencesiNA antisense region 621cagguuuaau
auuaaaauc 1962219RNAArtificial SequencesiNA antisense region
622gagugagaag caccagguu 1962323RNAArtificial SequenceSynthetic
Target Sequence/siNA sense region 623ugccuguccu ucuacucagc ugu
2362423RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 624ggagguggau ucugaugccu acu 2362523RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 625ggcacacuac
aauuugcuga gcu 2362623RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 626cgagugcugg agcuaaguga gag
2362723RNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 627cucgaauuac aucuccaaag gca 2362823RNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 628cugcuaugag
gcuuuggagg aau 2362923RNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 629gacaaagagg gcaaaugaga uca
2363023DNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 630agggagagug gguucucaau acg 2363121DNAArtificial
SequencesiNA sense region 631ccuguccuuc uacucagcut t
2163221DNAArtificial SequencesiNA sense region 632agguggauuc
ugaugccuat t 2163321DNAArtificial SequencesiNA sense region
633cacacuacaa uuugcugagt t 2163421DNAArtificial SequencesiNA sense
region 634agugcuggag cuaagugagt t 2163521DNAArtificial SequencesiNA
sense region 635cgaauuacau cuccaaaggt t 2163621DNAArtificial
SequencesiNA sense region 636gcuaugaggc uuuggaggat t
2163721DNAArtificial SequencesiNA sense region 637caaagagggc
aaaugagaut t 2163821DNAArtificial SequencesiNA sense region
638ggagaguggg uucucaauat t 2163921DNAArtificial SequencesiNA
antisense region 639agcugaguag aaggacaggt t 2164021DNAArtificial
SequencesiNA antisense region 640uaggcaucag aauccaccut t
2164121DNAArtificial SequencesiNA antisense region 641cucagcaaau
uguagugugt t 2164221DNAArtificial SequencesiNA antisense region
642cucacuuagc uccagcacut t 2164321DNAArtificial SequencesiNA
antisense region 643ccuuuggaga uguaauucgt t 2164421DNAArtificial
SequencesiNA antisense region 644uccuccaaag ccucauagct t
2164521DNAArtificial SequencesiNA antisense region 645aucucauuug
cccucuuugt t 2164621DNAArtificial SequencesiNA antisense region
646uauugagaac ccacucucct t 2164721DNAArtificial SequencesiNA sense
region 647ccuguccuuc uacucagcut t 2164821DNAArtificial SequencesiNA
sense region 648agguggauuc ugaugccuat t 2164921DNAArtificial
SequencesiNA sense region 649cacacuacaa uuugcugagt t
2165021DNAArtificial SequencesiNA sense region 650agugcuggag
cuaagugagt t 2165121DNAArtificial SequencesiNA sense region
651cgaauuacau cuccaaaggt t 2165221DNAArtificial SequencesiNA sense
region 652gcuaugaggc uuuggaggat t 2165321DNAArtificial SequencesiNA
sense region 653caaagagggc aaaugagaut t 2165421DNAArtificial
SequencesiNA sense region 654ggagaguggg uucucaauat t
2165521DNAArtificial SequencesiNA antisense region 655agcugaguag
aaggacaggt t 2165621DNAArtificial SequencesiNA antisense region
656uaggcaucag aauccaccut t 2165721DNAArtificial SequencesiNA
antisense region 657cucagcaaau uguagugugt t 2165821DNAArtificial
SequencesiNA antisense region 658cucacuuagc uccagcacut t
2165921DNAArtificial SequencesiNA antisense region 659ccuuuggaga
uguaauucgt t 2166021DNAArtificial SequencesiNA antisense region
660uccuccaaag ccucauagct t 2166121DNAArtificial SequencesiNA
antisense region 661aucucauuug cccucuuugt t 2166221DNAArtificial
SequencesiNA antisense region 662uauugagaac ccacucucct t
2166321DNAArtificial SequencesiNA sense region 663ccuguccuuc
uacucagcut t 2166421DNAArtificial SequencesiNA sense region
664agguggauuc ugaugccuat t 2166521DNAArtificial SequencesiNA sense
region 665cacacuacaa uuugcugagt t 2166621DNAArtificial SequencesiNA
sense region 666agugcuggag cuaagugagt t 2166721DNAArtificial
SequencesiNA sense region 667cgaauuacau cuccaaaggt t
2166821DNAArtificial SequencesiNA sense region 668gcuaugaggc
uuuggaggat t 2166921DNAArtificial SequencesiNA sense region
669caaagagggc aaaugagaut t 2167021DNAArtificial SequencesiNA sense
region 670ggagaguggg uucucaauat t 2167121DNAArtificial SequencesiNA
antisense region 671agcugaguag aaggacaggt t 2167221DNAArtificial
SequencesiNA antisense region 672uaggcaucag aauccaccut t
2167321DNAArtificial SequencesiNA antisense region 673cucagcaaau
uguagugugt t 2167421DNAArtificial SequencesiNA antisense region
674cucacuuagc uccagcacut t 2167521DNAArtificial SequencesiNA
antisense region 675ccuuuggaga uguaauucgt t 2167621DNAArtificial
SequencesiNA antisense region 676uccuccaaag ccucauagct t
2167721DNAArtificial SequencesiNA antisense region 677aucucauuug
cccucuuugt t 2167821DNAArtificial SequencesiNA antisense region
678uauugagaac ccacucucct t 2167921DNAArtificial SequencesiNA sense
region 679ccuguccuuc uacucagcut t 2168021DNAArtificial SequencesiNA
sense region 680agguggauuc ugaugccuat t 2168121DNAArtificial
SequencesiNA sense region 681cacacuacaa uuugcugagt t
2168221DNAArtificial SequencesiNA sense region 682agugcuggag
cuaagugagt t 2168321DNAArtificial SequencesiNA sense region
683cgaauuacau cuccaaaggt t 2168421DNAArtificial SequencesiNA sense
region 684gcuaugaggc uuuggaggat t 2168521DNAArtificial SequencesiNA
sense region 685caaagagggc aaaugagaut t 2168621DNAArtificial
SequencesiNA sense region 686ggagaguggg uucucaauat t
2168721DNAArtificial SequencesiNA antisense region 687agcugaguag
aaggacaggt t 2168821DNAArtificial SequencesiNA antisense region
688uaggcaucag aauccaccut t 2168921DNAArtificial SequencesiNA
antisense region 689cucagcaaau uguagugugt t 2169021DNAArtificial
SequencesiNA antisense region 690cucacuuagc uccagcacut t
2169121DNAArtificial SequencesiNA antisense region 691ccuuuggaga
uguaauucgt t 2169221DNAArtificial SequencesiNA antisense region
692uccuccaaag ccucauagct t 2169321DNAArtificial SequencesiNA
antisense region 693aucucauuug cccucuuugt t 2169421DNAArtificial
SequencesiNA antisense region 694uauugagaac ccacucucct t
2169521DNAArtificial SequencesiNA sense region 695ccuguccuuc
uacucagcut t 2169621DNAArtificial SequencesiNA sense region
696agguggauuc ugaugccuat t 2169721DNAArtificial SequencesiNA sense
region 697cacacuacaa uuugcugagt t 2169821DNAArtificial SequencesiNA
sense region 698agugcuggag cuaagugagt t 2169921DNAArtificial
SequencesiNA sense region 699cgaauuacau cuccaaaggt t
2170021DNAArtificial SequencesiNA sense region 700gcuaugaggc
uuuggaggat t 2170121DNAArtificial SequencesiNA sense region
701caaagagggc aaaugagaut t 2170221DNAArtificial SequencesiNA sense
region 702ggagaguggg uucucaauat t 2170321DNAArtificial SequencesiNA
antisense region 703agcugaguag aaggacaggt t 2170421DNAArtificial
SequencesiNA antisense region 704uaggcaucag aauccaccut t
2170521DNAArtificial SequencesiNA antisense region 705cucagcaaau
uguagugugt t 2170621DNAArtificial SequencesiNA antisense region
706cucacuuagc uccagcacut t 2170721DNAArtificial SequencesiNA
antisense region 707ccuuuggaga uguaauucgt t 2170821DNAArtificial
SequencesiNA antisense region 708uccuccaaag ccucauagct t
2170921DNAArtificial SequencesiNA antisense region 709aucucauuug
cccucuuugt t 2171021DNAArtificial SequencesiNA antisense region
710uauugagaac ccacucucct t 2171121DNAArtificial SequencesiNA
antisense region 711agcugaguag aaggacaggt t 2171221DNAArtificial
SequencesiNA antisense region 712uaggcaucag aauccaccut t
2171321DNAArtificial SequencesiNA antisense region 713cucagcaaau
uguagugugt t 2171421DNAArtificial SequencesiNA antisense region
714cucacuuagc uccagcacut t 2171521DNAArtificial SequencesiNA
antisense region 715ccuuuggaga uguaauucgt t 2171621DNAArtificial
SequencesiNA antisense region 716uccuccaaag ccucauagct t
2171721DNAArtificial SequencesiNA antisense region 717aucucauuug
cccucuuugt t 2171821DNAArtificial SequencesiNA antisense region
718uauugagaac ccacucucct t 2171921DNAArtificial SequencesiNA
antisense region 719agcugaguag aaggacaggt t 2172021DNAArtificial
SequencesiNA antisense region 720uaggcaucag aauccaccut t
2172121DNAArtificial SequencesiNA antisense region 721cucagcaaau
uguagugugt t 2172221DNAArtificial SequencesiNA antisense region
722cucacuuagc uccagcacut t 2172321DNAArtificial SequencesiNA
antisense region 723ccuuuggaga uguaauucgt t 2172421DNAArtificial
SequencesiNA antisense region 724uccuccaaag ccucauagct t
2172521DNAArtificial SequencesiNA antisense region 725aucucauuug
cccucuuugt t 2172621DNAArtificial SequencesiNA antisense region
726uauugagaac ccacucucct t 2172721DNAArtificial SequencesiNA sense
region 727nnnnnnnnnn nnnnnnnnnn 2172821DNAArtificial SequencesiNA
antisense region 728nnnnnnnnnn nnnnnnnnnn 2172921DNAArtificial
SequencesiNA sense region 729nnnnnnnnnn nnnnnnnnnn
2173021DNAArtificial SequencesiNA antisense region 730nnnnnnnnnn
nnnnnnnnnn 2173121DNAArtificial SequencesiNA sense region
731nnnnnnnnnn nnnnnnnnnn 2173221DNAArtificial SequencesiNA
antisense region 732nnnnnnnnnn nnnnnnnnnn 2173321DNAArtificial
SequencesiNA sense region 733nnnnnnnnnn nnnnnnnnnn
2173421DNAArtificial SequencesiNA sense region 734nnnnnnnnnn
nnnnnnnnnn 2173521DNAArtificial SequencesiNA antisense region
735nnnnnnnnnn nnnnnnnnnn 2173621DNAArtificial SequencesiNA sense
region 736ucugaugccu acuaugucut t 2173721DNAArtificial SequencesiNA
antisense region 737agacauagua ggcaucagat 2173821DNAArtificial
SequencesiNA sense region 738ucugaugccu acuaugucut t
2173921DNAArtificial SequencesiNA antisense region 739agacauagua
ggcaucagat 2174021DNAArtificial SequencesiNA sense region
740ucugaugccu acuaugucut t 2174121DNAArtificial
SequencesiNA antisense region 741agacauagua ggcaucagat
2174221DNAArtificial SequencesiNA sense region 742ucugaugccu
acuaugucut 2174321DNAArtificial SequencesiNA sense region
743ucugaugccu acuaugucut 2174421DNAArtificial SequencesiNA
antisense region 744agacauagua ggcaucagat 2174514RNAArtificial
SequenceSynthetic Target Sequence 745auauaucuau uucg
1474614RNAArtificial SequenceSynthetic Complement to Synthetic
Target Sequence 746cgaaauagua uaua 1474722RNAArtificial
SequenceSynthetic appended target/complement 747cgaaauagau
auaucuauuu cg 2274824DNAArtificial SequenceSynthetic duplex forming
oligonucleotide 748cgaaauagau auaucuauuu cgtt 247495598RNAHomo
sapiens 749ggccccucag cccugcugcc cagcacgagc cugugcucgc ccugcccaac
gcagacagcc 60agacccaggg cggccccucu ggcggcucug cuccucccga aggaugcuug
gggagugagg 120cgaagcuggg cgcuccucuc cccuacagca gcccccuucc
uccaucccuc uguucuccug 180agccuucagg agccugcacc aguccugccu
guccuucuac ucagcuguua cccacucugg 240gaccagcagu cuuucugaua
acugggagag ggcaguaagg aggacuuccu ggagggggug 300acuguccaga
gccuggaacu gugcccacac cagaagccau cagcagcaag gacaccaugc
360ggcuuccggg ugcgaugcca gcucuggccc ucaaaggcga gcugcuguug
cugucucucc 420uguuacuucu ggaaccacag aucucucagg gccuggucgu
cacacccccg gggccagagc 480uuguccucaa ugucuccagc accuucguuc
ugaccugcuc ggguucagcu ccgguggugu 540gggaacggau gucccaggag
cccccacagg aaauggccaa ggcccaggau ggcaccuucu 600ccagcgugcu
cacacugacc aaccucacug ggcuagacac gggagaauac uuuugcaccc
660acaaugacuc ccguggacug gagaccgaug agcggaaacg gcucuacauc
uuugugccag 720aucccaccgu gggcuuccuc ccuaaugaug ccgaggaacu
auucaucuuu cucacggaaa 780uaacugagau caccauucca ugccgaguaa
cagacccaca gcugguggug acacugcacg 840agaagaaagg ggacguugca
cugccugucc ccuaugauca ccaacguggc uuuucuggua 900ucuuugagga
cagaagcuac aucugcaaaa ccaccauugg ggacagggag guggauucug
960augccuacua ugucuacaga cuccaggugu cauccaucaa cgucucugug
aacgcagugc 1020agacuguggu ccgccagggu gagaacauca cccucaugug
cauugugauc gggaaugagg 1080uggucaacuu cgaguggaca uacccccgca
aagaaagugg gcggcuggug gagccgguga 1140cugacuuccu cuuggauaug
ccuuaccaca uccgcuccau ccugcacauc cccagugccg 1200aguuagaaga
cucggggacc uacaccugca augugacgga gagugugaau gaccaucagg
1260augaaaaggc caucaacauc accgugguug agagcggcua cgugcggcuc
cugggagagg 1320ugggcacacu acaauuugcu gagcugcauc ggagccggac
acugcaggua guguucgagg 1380ccuacccacc gcccacuguc cugugguuca
aagacaaccg cacccugggc gacuccagcg 1440cuggcgaaau cgcccugucc
acgcgcaacg ugucggagac ccgguaugug ucagagcuga 1500cacugguucg
cgugaaggug gcagaggcug gccacuacac caugcgggcc uuccaugagg
1560augcugaggu ccagcucucc uuccagcuac agaucaaugu cccuguccga
gugcuggagc 1620uaagugagag ccacccugac aguggggaac agacaguccg
cugucguggc cggggcaugc 1680cccagccgaa caucaucugg ucugccugca
gagaccucaa aaggugucca cgugagcugc 1740cgcccacgcu gcuggggaac
aguuccgaag aggagagcca gcuggagacu aacgugacgu 1800acugggagga
ggagcaggag uuugaggugg ugagcacacu gcgucugcag cacguggauc
1860ggccacuguc ggugcgcugc acgcugcgca acgcuguggg ccaggacacg
caggagguca 1920ucguggugcc acacuccuug cccuuuaagg ugguggugau
cucagccauc cuggcccugg 1980uggugcucac caucaucucc cuuaucaucc
ucaucaugcu uuggcagaag aagccacguu 2040acgagauccg auggaaggug
auugagucug ugagcucuga cggccaugag uacaucuacg 2100uggaccccau
gcagcugccc uaugacucca cgugggagcu gccgcgggac cagcuugugc
2160ugggacgcac ccucggcucu ggggccuuug ggcagguggu ggaggccacg
gcucauggcc 2220ugagccauuc ucaggccacg augaaagugg ccgucaagau
gcuuaaaucc acagcccgca 2280gcagugagaa gcaagcccuu augucggagc
ugaagaucau gagucaccuu gggccccacc 2340ugaacguggu caaccuguug
ggggccugca ccaaaggagg acccaucuau aucaucacug 2400aguacugccg
cuacggagac cugguggacu accugcaccg caacaaacac accuuccugc
2460agcaccacuc cgacaagcgc cgcccgccca gcgcggagcu cuacagcaau
gcucugcccg 2520uugggcuccc ccugcccagc cauguguccu ugaccgggga
gagcgacggu ggcuacaugg 2580acaugagcaa ggacgagucg guggacuaug
ugcccaugcu ggacaugaaa ggagacguca 2640aauaugcaga caucgagucc
uccaacuaca uggccccuua cgauaacuac guucccucug 2700ccccugagag
gaccugccga gcaacuuuga ucaacgaguc uccagugcua agcuacaugg
2760accucguggg cuucagcuac cagguggcca auggcaugga guuucuggcc
uccaagaacu 2820gcguccacag agaccuggcg gcuaggaacg ugcucaucug
ugaaggcaag cuggucaaga 2880ucugugacuu uggccuggcu cgagacauca
ugcgggacuc gaauuacauc uccaaaggca 2940gcaccuuuuu gccuuuaaag
uggauggcuc cggagagcau cuucaacagc cucuacacca 3000cccugagcga
cguguggucc uucgggaucc ugcucuggga gaucuucacc uuggguggca
3060ccccuuaccc agagcugccc augaacgagc aguucuacaa ugccaucaaa
cgggguuacc 3120gcauggccca gccugcccau gccuccgacg agaucuauga
gaucaugcag aagugcuggg 3180aagagaaguu ugagauucgg ccccccuucu
cccagcuggu gcugcuucuc gagagacugu 3240ugggcgaagg uuacaaaaag
aaguaccagc agguggauga ggaguuucug aggagugacc 3300acccagccau
ccuucggucc caggcccgcu ugccuggguu ccauggccuc cgaucucccc
3360uggacaccag cuccguccuc uauacugccg ugcagcccaa ugagggugac
aacgacuaua 3420ucaucccccu gccugacccc aaacccgagg uugcugacga
gggcccacug gaggguuccc 3480ccagccuagc cagcuccacc cugaaugaag
ucaacaccuc cucaaccauc uccugugaca 3540gcccccugga gccccaggac
gaaccagagc cagagcccca gcuugagcuc cagguggagc 3600cggagccaga
gcuggaacag uugccggauu cggggugccc ugcgccucgg gcggaagcag
3660aggauagcuu ccuguagggg gcuggccccu acccugcccu gccugaagcu
cccccccugc 3720cagcacccag caucuccugg ccuggccuga ccgggcuucc
ugucagccag gcugcccuua 3780ucagcugucc ccuucuggaa gcuuucugcu
ccugacgugu ugugccccaa acccuggggc 3840uggcuuagga ggcaagaaaa
cugcaggggc cgugaccagc ccucugccuc cagggaggcc 3900aacugacucu
gagccagggu ucccccaggg aacucaguuu ucccauaugu aagaugggaa
3960aguuaggcuu gaugacccag aaucuaggau ucucucccug gcugacaggu
ggggagaccg 4020aaucccuccc ugggaagauu cuuggaguua cugagguggu
aaauuaacuu uuuucuguuc 4080agccagcuac cccucaagga aucauagcuc
ucuccucgca cuuuuuaucc acccaggagc 4140uagggaagag acccuagccu
cccuggcugc uggcugagcu agggccuagc cuugagcagu 4200guugccucau
ccagaagaaa gccagucucc ucccuaugau gccagucccu gcguucccug
4260gcccgagcug gucuggggcc auuaggcagc cuaauuaaug cuggaggcug
agccaaguac 4320aggacacccc cagccugcag cccuugccca gggcacuugg
agcacacgca gccauagcaa 4380gugccugugu cccuguccuu caggcccauc
aguccugggg cuuuuucuuu aucacccuca 4440gucuuaaucc auccaccaga
gucuagaagg ccagacgggc cccgcaucug ugaugagaau 4500guaaaugugc
caguguggag uggccacgug ugugugccag uauauggccc uggcucugca
4560uuggaccugc uaugaggcuu uggaggaauc ccucacccuc ucugggccuc
aguuuccccu 4620ucaaaaaaug aauaagucgg acuuauuaac ucugagugcc
uugccagcac uaacauucua 4680gaguauucca ggugguugca cauuugucca
gaugaagcaa ggccauauac ccuaaacuuc 4740cauccugggg gucagcuggg
cuccugggag auuccagauc acacaucaca cucuggggac 4800ucaggaacca
ugccccuucc ccaggccccc agcaagucuc aagaacacag cugcacaggc
4860cuugacuuag agugacagcc gguguccugg aaagccccaa gcagcugccc
cagggacaug 4920ggaagaccac gggaccucuu ucacuaccca cgaugaccuc
cggggguauc cugggcaaaa 4980gggacaaaga gggcaaauga gaucaccucc
ugcagcccac cacuccagca ccugugccga 5040ggucugcguc gaagacagaa
uggacaguga ggacaguuau gucuuguaaa agacaagaag 5100cuucagaugg
uaccccaaga aggaugugag agguggccgc uuggaguuug ccccucaccc
5160accagcugcc ccaucccuga ggcagcgcuc caugggggua ugguuuuguc
acugcccaga 5220ccuagcagug acaucucauu guccccagcc cagugggcau
uggaggugcc aggggaguca 5280ggguuguagc caagacgccc ccgcacgggg
aggguuggga agggggugca ggaagcucaa 5340ccccucuggg caccaacccu
gcauugcagg uuggcaccuu acuucccugg gauccccaga 5400guugguccaa
ggagggagag uggguucuca auacgguacc aaagauauaa ucaccuaggu
5460uuacaaauau uuuuaggacu cacguuaacu cacauuuaua cagcagaaau
gcuauuuugu 5520augcuguuaa guuuuucuau cuguguacuu uuuuuuaagg
gaaagauuuu aauauuaaac 5580cuggugcuuc ucacucac 5598
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