U.S. patent application number 10/923451 was filed with the patent office on 2005-11-17 for rna interference mediated inhibition of stearoyl-coa desaturase (scd) gene expression using short interfering nucleic acid (sina).
This patent application is currently assigned to Sirna Therapeutics, Inc.. Invention is credited to Beigelman, Leonid, McSwiggen, James, Thompson, James.
Application Number | 20050256068 10/923451 |
Document ID | / |
Family ID | 46302600 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050256068 |
Kind Code |
A1 |
McSwiggen, James ; et
al. |
November 17, 2005 |
RNA interference mediated inhibition of stearoyl-CoA desaturase
(SCD) gene expression using short interfering nucleic acid
(siNA)
Abstract
This invention relates to compounds, compositions, and methods
useful for modulating Stearoyl-CoA desaturase (SCD) 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 Stearoyl-CoA desaturase (SCD) 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
Stearoyl-CoA desaturase (SCD) genes.
Inventors: |
McSwiggen, James; (Boulder,
CO) ; Beigelman, Leonid; (Longmont, CO) ;
Thompson, James; (Lafayette, CO) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
Sirna Therapeutics, Inc.
Boulder
CO
|
Family ID: |
46302600 |
Appl. No.: |
10/923451 |
Filed: |
August 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10923451 |
Aug 20, 2004 |
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PCT/US03/04317 |
Feb 13, 2003 |
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10923451 |
Aug 20, 2004 |
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PCT/US04/16390 |
May 24, 2004 |
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PCT/US04/16390 |
May 24, 2004 |
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10826966 |
Apr 16, 2004 |
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10826966 |
Apr 16, 2004 |
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10757803 |
Jan 14, 2004 |
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10757803 |
Jan 14, 2004 |
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10720448 |
Nov 24, 2003 |
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10720448 |
Nov 24, 2003 |
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10693059 |
Oct 23, 2003 |
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10693059 |
Oct 23, 2003 |
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10444853 |
May 23, 2003 |
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10444853 |
May 23, 2003 |
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PCT/US03/05346 |
Feb 20, 2003 |
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10444853 |
May 23, 2003 |
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PCT/US03/05028 |
Feb 20, 2003 |
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10923451 |
Aug 20, 2004 |
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PCT/US04/13456 |
Apr 30, 2004 |
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PCT/US04/13456 |
Apr 30, 2004 |
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10780447 |
Feb 13, 2004 |
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10780447 |
Feb 13, 2004 |
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10427160 |
Apr 30, 2003 |
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10427160 |
Apr 30, 2003 |
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PCT/US02/15876 |
May 17, 2002 |
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10923451 |
Aug 20, 2004 |
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10727780 |
Dec 3, 2003 |
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60412304 |
Sep 20, 2002 |
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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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60292217 |
May 18, 2001 |
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60362016 |
Mar 6, 2002 |
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60306883 |
Jul 20, 2001 |
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60311865 |
Aug 13, 2001 |
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60543480 |
Feb 10, 2004 |
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Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
A61K 49/0008 20130101;
A61K 38/00 20130101; C12N 2310/317 20130101; C12N 2310/322
20130101; C12Y 207/11013 20130101; C12N 2310/12 20130101; C12Y
104/03003 20130101; C12Y 207/07049 20130101; C12N 15/113 20130101;
C12N 2310/53 20130101; C12N 15/1137 20130101; C12N 2330/30
20130101; C12Y 604/01002 20130101; C12N 2310/321 20130101; C12Y
301/03048 20130101; C12N 15/1132 20130101; C12N 2310/3521 20130101;
C12N 15/115 20130101; C12N 2310/111 20130101; C12N 15/111 20130101;
C12N 2310/321 20130101; C12Y 114/19001 20130101; C12N 2310/332
20130101; C12Y 103/01022 20130101; C12N 15/1138 20130101; C12Y
207/11001 20130101; C12N 2310/318 20130101; C12N 2310/346 20130101;
C12N 2320/32 20130101; C12N 2310/315 20130101; C12N 2310/121
20130101; C12N 15/87 20130101; C12N 2310/14 20130101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
A61K 048/00; C07H
021/02 |
Claims
What we claim is:
1. A chemically synthesized double stranded short interfering
nucleic acid (siNA) molecule that directs cleavage of a SCD RNA via
RNA interference (RNAi), wherein: a) each strand of said siNA
molecule is about 18 to about 23 nucleotides in length; and b) one
strand of said siNA molecule comprises nucleotide sequence having
sufficient complementarity to said SCD RNA for the siNA molecule to
direct cleavage of the SCD RNA via RNA interference.
2. The siNA molecule of claim 1, wherein said siNA molecule
comprises no ribonucleotides.
3. The siNA molecule of claim 1, wherein said siNA molecule
comprises one or more ribonucleotides.
4. The siNA molecule of claim 1, wherein one strand of said
double-stranded siNA molecule comprises a nucleotide sequence that
is complementary to a nucleotide sequence of a SCD gene or a
portion thereof, and wherein a second strand of said
double-stranded siNA molecule comprises a nucleotide sequence
substantially similar to the nucleotide sequence or a portion
thereof of said SCD RNA.
5. The siNA molecule of claim 4, wherein each strand of the siNA
molecule comprises about 18 to about 23 nucleotides, and wherein
each strand comprises at least about 19 nucleotides that are
complementary to the nucleotides of the other strand.
6. The siNA molecule of claim 1, wherein said siNA molecule
comprises an antisense region comprising a nucleotide sequence that
is complementary to a nucleotide sequence of a SCD gene or a
portion thereof, and wherein said siNA further comprises a sense
region, wherein said sense region comprises a nucleotide sequence
substantially similar to the nucleotide sequence of said SCD gene
or a portion thereof.
7. The siNA molecule of claim 6, wherein said antisense region and
said sense region comprise about 18 to about 23 nucleotides, and
wherein said antisense region comprises at least about 18
nucleotides that are complementary to nucleotides of the sense
region.
8. The siNA molecule of claim 1, wherein said siNA molecule
comprises a sense region and an antisense region, and wherein said
antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence of RNA encoded by a SCD
gene, or a portion thereof, and said sense region comprises a
nucleotide sequence that is complementary to said antisense
region.
9. The siNA molecule of claim 6, wherein said siNA molecule is
assembled from two separate oligonucleotide fragments wherein one
fragment comprises the sense region and a second fragment comprises
the antisense region of said siNA molecule.
10. The siNA molecule of claim 6, wherein said sense region is
connected to the antisense region via a linker molecule.
11. The siNA molecule of claim 10, wherein said linker molecule is
a polynucleotide linker.
12. The siNA molecule of claim 10, wherein said linker molecule is
a non-nucleotide linker.
13. The siNA molecule of claim 6, wherein pyrimidine nucleotides in
the sense region are 2'-O-methyl pyrimidine nucleotides.
14. The siNA molecule of claim 6, wherein purine nucleotides in the
sense region are 2'-deoxy purine nucleotides.
15. The siNA molecule of claim 6, wherein pyrimidine nucleotides
present in the sense region are 2'-deoxy-2'-fluoro pyrimidine
nucleotides.
16. The siNA molecule of claim 9, wherein the fragment comprising
said sense region includes a terminal cap moiety at a 5'-end, a
3'-end, or both of the 5' and 3' ends of the fragment comprising
said sense region.
17. The siNA molecule of claim 16, wherein said terminal cap moiety
is an inverted deoxy abasic moiety.
18. The siNA molecule of claim 6, wherein pyrimidine nucleotides of
said antisense region are 2'-deoxy-2'-fluoro pyrimidine
nucleotides.
19. The siNA molecule of claim 6, wherein purine nucleotides of
said antisense region are 2'-O-methyl purine nucleotides.
20. The siNA molecule of claim 6, wherein purine nucleotides
present in said antisense region comprise 2'-deoxy- purine
nucleotides.
21. The siNA molecule of claim 18, wherein said antisense region
comprises a phosphorothioate internucleotide linkage at the 3' end
of said antisense region.
22. The siNA molecule of claim 6, wherein said antisense region
comprises a glyceryl modification at a 3' end of said antisense
region.
23. The siNA molecule of claim 9, wherein each of the two fragments
of said siNA molecule comprise about 21 nucleotides.
24. The siNA molecule of claim 23, wherein about 19 nucleotides of
each fragment of the siNA molecule are base-paired to the
complementary nucleotides of the other fragment of the siNA
molecule and wherein at least two 3' terminal nucleotides of each
fragment of the siNA molecule are not base-paired to the
nucleotides of the other fragment of the siNA molecule.
25. The siNA molecule of claim 24, wherein each of the two 3'
terminal nucleotides of each fragment of the siNA molecule are
2'-deoxy-pyrimidines.
26. The siNA molecule of claim 25, wherein said 2'-deoxy-pyrimidine
is 2'-deoxy-thymidine.
27. The siNA molecule of claim 23, wherein all of the about 21
nucleotides of each fragment of the siNA molecule are base-paired
to the complementary nucleotides of the other fragment of the siNA
molecule.
28. The siNA molecule of claim 23, wherein about 19 nucleotides of
the antisense region are base-paired to the nucleotide sequence of
the RNA encoded by a SCD gene or a portion thereof.
29. The siNA molecule of claim 23, wherein about 21 nucleotides of
the antisense region are base-paired to the nucleotide sequence of
the RNA encoded by a SCD gene or a portion thereof.
30. The siNA molecule of claim 9, wherein a 5'-end of the fragment
comprising said antisense region optionally includes a phosphate
group.
31. A composition comprising the siNA molecule of claim 1 in an
pharmaceutically acceptable carrier or diluent.
32. A siNA according to claim 1 wherein the SCD RNA comprises
Genbank Accession No. NM.sub.--005063.2.
33. A siNA according to claim 1 wherein said siNA comprises any of
SEQ ID NOs. 1-806.
34. A composition comprising the siNA of claim 32 together with a
pharmaceutically acceptable carrier or diluent.
35. A composition comprising the siNA of claim 33 together with a
pharmaceutically acceptable carrier or diluent.
Description
[0001] This application is a continuation-in-part of International
Patent Application No. PCT/US03/04317, filed Feb. 13, 2003, which
claims the benefit of U.S. Provisional Application No. 60/412,304,
filed Sep. 20, 2002. This application 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, 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, 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. This application is also a
continuation-in-part of International Patent Application No.
PCT/US04/13456, filed Apr. 30, 2004, which is a
continuation-in-part of U.S. patent application Ser. No.
10/780,447, filed Feb. 13, 2004, which is a continuation-in-part of
U.S. patent application Ser. No. 10/427,160, filed Apr. 30, 2003,
which is a continuation-in-part of International Patent Application
No. PCT/US02/15876 filed May 17, 2002, which claims the benefit of
U.S. Provisional Application No. 60/292,217, filed May 18, 2001,
U.S. Provisional Application No. 60/362,016, filed Mar. 6, 2002,
U.S. Provisional Application No. 60/306,883, filed Jul. 20, 2001,
and U.S. Provisional Application No. 60/311,865, filed Aug. 13,
2001. This application is also a continuation-in-part of U.S.
patent application Ser. No. 10/727,780 filed Dec. 3, 2003. This
application also claims the benefit of U.S. Provisional Application
No. 60/543,480, filed Feb. 10, 2004.
[0002] The instant application claims the benefit of all the listed
applications, which are hereby incorporated by reference herein in
their entireties, including the drawings.
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
Stearoyl-CoA desaturase (SCD) 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
Stearoyl-CoA desaturase (SCD) 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
Stearoyl-CoA desaturase (SCD) 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 SCD expression in a subject, such as diabetes
(type I and/or type II), atherosclerosis, cancer, obesity, and
viral infection.
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'-O-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.
[0012] Brownlie et al., International PCT Publication No. WO
01/62954, describe methods and compositions using stearoyl-CoA
desaturase to identify certain triglyceride reducing therapeutic
agents.
SUMMARY OF THE INVENTION
[0013] This invention relates to compounds, compositions, and
methods useful for modulating Stearoyl-CoA desaturase (SCD) 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 SCD 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 SCD genes.
[0014] 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 SCD 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.
[0015] In one embodiment, the invention features one or more siNA
molecules and methods that independently or in combination modulate
the expression of SCD genes encoding proteins, such as proteins
comprising SCD associated with the maintenance and/or development
of diabetes (type I and/or type II), atherosclerosis, cancer,
obesity, and viral infection, such as genes encoding sequences
comprising those sequences referred to by GenBank Accession Nos.
shown in Table I, referred to herein generally as Stearoyl-CoA
desaturase or SCD. The description below of the various aspects and
embodiments of the invention is provided with reference to
exemplary Stearoyl-CoA desaturase gene referred to herein as SCD.
However, the various aspects and embodiments are also directed to
other SCD genes, such as homolog genes and transcript variants, and
polymorphisms (e.g., single nucleotide polymorphism, (SNPs))
associated with certain SCD genes. As such, the various aspects and
embodiments are also directed to other genes that are involved in
SCD 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 SCD 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.
[0016] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a SCD 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 SCD 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 28
nucleotides in length, the first strand of the siNA molecule
comprises nucleotide sequence having sufficient complementarity to
the SCD RNA for the siNA molecule to direct cleavage of the SCD 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 SCD 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 SCD RNA for the siNA molecule to direct cleavage of the SCD 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 SCD 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 SCD RNA for the siNA molecule to direct cleavage of the SCD 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 SCD 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 SCD RNA for the siNA molecule to direct cleavage of the SCD RNA
via RNA interference.
[0021] In one embodiment, the invention features a siNA molecule
that down-regulates expression of a SCD gene, for example, wherein
the SCD gene comprises SCD encoding sequence. In one embodiment,
the invention features a siNA molecule that down-regulates
expression of a SCD gene, for example, wherein the SCD gene
comprises SCD non-coding sequence or regulatory elements involved
in SCD gene expression.
[0022] In one embodiment, a siNA of the invention is used to
inhibit the expression of SCD genes or a SCD 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 SCD 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.
[0023] In one embodiment, the invention features a siNA molecule
having RNAi activity against SCD RNA, wherein the siNA molecule
comprises a sequence complementary to any RNA having SCD 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 SCD RNA, wherein the
siNA molecule comprises a sequence complementary to an RNA having
variant SCD encoding sequence, for example other mutant SCD genes
not shown in Table I but known in the art to be associated with the
maintenance and/or development of diabetes (type I and/or type II),
atherosclerosis, cancer, obesity, and viral infection. 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
SCD gene and thereby mediate silencing of SCD gene expression, for
example, wherein the siNA mediates regulation of SCD gene
expression by cellular processes that modulate the chromatin
structure or methylation patterns of the SCD gene and prevent
transcription of the SCD gene.
[0024] In one embodiment, siNA molecules of the invention are used
to down regulate or inhibit the expression of SCD proteins arising
from SCD haplotype polymorphisms that are associated with a disease
or condition, (e.g., diabetes (type I and/or type II),
atherosclerosis, cancer, obesity, and viral infection). Analysis of
SCD genes, or SCD 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 SCD gene expression. As
such, analysis of SCD protein or RNA levels can be used to
determine treatment type and the course of therapy in treating a
subject. Monitoring of SCD 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 SCD proteins associated with a trait, condition, or
disease.
[0025] 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 SCD protein. The siNA further comprises a sense strand,
wherein said sense strand comprises a nucleotide sequence of a SCD
gene or a portion thereof.
[0026] In another embodiment, a siNA molecule comprises an
antisense region comprising a nucleotide sequence that is
complementary to a nucleotide sequence encoding a SCD protein or a
portion thereof. The siNA molecule further comprises a sense
region, wherein said sense region comprises a nucleotide sequence
of a SCD gene or a portion thereof.
[0027] 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 SCD 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 SCD gene sequence or a portion
thereof.
[0028] In one embodiment, the antisense region of SCD siNA
constructs comprises a sequence complementary to sequence having
any of SEQ ID NOs. 1-290 or 581-596. In one embodiment, the
antisense region of SCD constructs comprises sequence having any of
SEQ ID NOs. 291-580, 605-612, 621-628, 637-644, 653-660, 669-692,
701-708, 717-724, 733-740, 749-756, 765-788, 790, 792, 794, 797,
799, 801, 803, or 806. In another embodiment, the sense region of
SCD constructs comprises sequence having any of SEQ ID NOs. 1-290,
581-604, 613-620, 629-636, 645-652, 661-668, 693-700, 709-716,
725-732, 741-748, 757-764, 789, 791, 793, 795, 796, 798, 800, 802,
804, or 805.
[0029] In one embodiment, a siNA molecule of the invention
comprises any of SEQ ID NOs. 1-806. The sequences shown in SEQ ID
NOs: 1-806 are not limiting. A siNA molecule of the invention can
comprise any contiguous SCD 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 SCD nucleotides).
[0030] 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.
[0031] 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 SCD 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.
[0032] 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 SCD 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.
[0033] In one embodiment, a siNA molecule of the invention has RNAi
activity that modulates expression of RNA encoded by a SCD gene.
Because SCD genes can share some degree of sequence homology with
each other, siNA molecules can be designed to target a class of SCD
genes or alternately specific SCD genes (e.g., polymorphic
variants) by selecting sequences that are either shared amongst
different SCD targets or alternatively that are unique for a
specific SCD target. Therefore, in one embodiment, the siNA
molecule can be designed to target conserved regions of SCD RNA
sequences having homology among several SCD gene variants so as to
target a class of SCD genes with one siNA molecule. Accordingly, in
one embodiment, the siNA molecule of the invention modulates the
expression of one or both SCD alleles in a subject. In another
embodiment, the siNA molecule can be designed to target a sequence
that is unique to a specific SCD RNA sequence (e.g., a single SCD
allele or SCD single nucleotide polymorphism (SNP)) due to the high
degree of specificity that the siNA molecule requires to mediate
RNAi activity.
[0034] 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.
[0035] In one embodiment, the invention features one or more
chemically-modified siNA constructs having specificity for SCD
expressing nucleic acid molecules, such as RNA encoding a SCD
protein. In one embodiment, the invention features a RNA based siNA
molecule (e.g., a siNA comprising 2'-OH nucleotides) having
specificity for SCD 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.
[0036] 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.
[0037] One aspect of the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that down-regulates
expression of a SCD 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 SCD gene, and the second strand of the
double-stranded siNA molecule comprises a nucleotide sequence
substantially similar to the nucleotide sequence of the SCD gene or
a portion thereof.
[0038] In another embodiment, the invention features a
double-stranded short interfering nucleic acid (siNA) molecule that
down-regulates expression of a SCD gene comprising an antisense
region, wherein the antisense region comprises a nucleotide
sequence that is complementary to a nucleotide sequence of the SCD
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 SCD 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.
[0039] In another embodiment, the invention features a
double-stranded short interfering nucleic acid (siNA) molecule that
down-regulates expression of a SCD 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 SCD gene or a portion thereof and the sense
region comprises a nucleotide sequence that is complementary to the
antisense region.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a SCD 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.
[0044] In one embodiment, the invention features double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a SCD 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 SCD 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 SCD 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 SCD 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 SCD 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 SCD gene can comprise, for
example, sequences referred to in Table I.
[0045] In one embodiment, a siNA molecule of the invention
comprises no ribonucleotides. In another embodiment, a siNA
molecule of the invention comprises ribonucleotides.
[0046] 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 SCD 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 SCD 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 SCD 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 SCD gene or a portion thereof.
[0047] 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 SCD
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 SCD gene can comprise, for example, sequences referred
in to Table I.
[0048] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a SCD 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 SCD 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-methyl
pyrimidine 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.
[0049] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a SCD 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.
[0050] 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.
[0051] 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.
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.
[0052] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a SCD 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 SCD 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.
[0053] In one embodiment, the antisense region of a siNA molecule
of the invention comprises sequence complementary to a portion of a
SCD transcript having sequence unique to a particular SCD 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.
[0054] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a SCD 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 SCD 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
SCD gene. In any of the above embodiments, the 5'-end of the
fragment comprising said antisense region can optionally include a
phosphate group.
[0055] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits the
expression of a SCD RNA sequence (e.g., wherein said target RNA
sequence is encoded by a SCD gene involved in the SCD 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).
[0056] In one embodiment, the invention features a chemically
synthesized double stranded RNA molecule that directs cleavage of a
SCD 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 SCD RNA for the RNA molecule to direct
cleavage of the SCD 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.
[0057] In one embodiment, the invention features a medicament
comprising a siNA molecule of the invention.
[0058] In one embodiment, the invention features an active
ingredient comprising a siNA molecule of the invention.
[0059] 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 SCD 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 SCD 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
SCD 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 SCD gene. In any of the
above embodiments, the 5'-end of the fragment comprising said
antisense region can optionally include a phosphate group.
[0060] 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 SCD 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 SCD 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.
[0061] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits,
down-regulates, or reduces expression of a SCD 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 SCD 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.
[0062] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits,
down-regulates, or reduces expression of a SCD 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 SCD 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.
[0063] In any of the above-described embodiments of a
double-stranded short interfering nucleic acid (siNA) molecule that
inhibits expression of a SCD 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 SCD 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 SCD RNA or a portion thereof.
[0064] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits
expression of a SCD 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 SCD 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.
[0065] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits
expression of a SCD 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 SCD 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 SCD RNA.
[0066] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits
expression of a SCD 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 SCD 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 SCD RNA or a portion
thereof that is present in the SCD RNA.
[0067] In one embodiment, the invention features a composition
comprising a siNA molecule of the invention in a pharmaceutically
acceptable carrier or diluent.
[0068] 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.
[0069] 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.
[0070] 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 SCD 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.
[0071] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against SCD 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: 1
[0072] 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).
[0073] 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.
[0074] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against SCD 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: 2
[0075] 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-OSH, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-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 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.
[0076] 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.
[0077] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) against SCD 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: 3
[0078] 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-OSH, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-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 SCD inside a
cell or reconstituted in vitro system, wherein the chemical
modification comprises a 5'-terminal phosphate group having Formula
IV: 4
[0082] wherein each X and Y is independently O, S, N, alkyl,
substituted alkyl, or alkylhalo; wherein each Z and W is
independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl,
alkaryl, aralkyl, 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 SCD 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 I 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 antisense 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 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 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:
5
[0098] 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-OSH, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-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.
[0099] 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: 6
[0100] 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-OSH, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-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.
[0101] 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: 7
[0102] 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-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,
S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,
aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,
O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, 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.
[0103] In another embodiment, the invention features a compound
having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups,
n=1, and R3 comprises O 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).
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[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), 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).
[0109] 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.
[0110] 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).
[0111] 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.
[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 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.
[0114] 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).
[0115] 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).
[0116] 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 SCD 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).
[0117] 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.
[0118] 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 deoxyabaisc moiety,
at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense
strand.
[0119] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid molecule (siNA)
capable of mediating RNA interference (RNAi) against SCD 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.
[0120] 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-nucleotid- e 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.)
[0121] 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 C l position of the sugar.
[0122] 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 oligonculeotide 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 oligonculeotide 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 presense 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.
[0123] 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.
[0124] 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.
[0125] 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 l-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.
[0126] In one embodiment, the invention features a method for
modulating the expression of a SCD 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 SCD gene; and (b) introducing
the siNA molecule into a cell under conditions suitable to modulate
the expression of the SCD gene in the cell.
[0127] In one embodiment, the invention features a method for
modulating the expression of a SCD 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 SCD 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 SCD gene in the cell.
[0128] In another embodiment, the invention features a method for
modulating the expression of more than one SCD 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 SCD genes; and (b)
introducing the siNA molecules into a cell under conditions
suitable to modulate the expression of the SCD genes in the
cell.
[0129] In another embodiment, the invention features a method for
modulating the expression of two or more SCD 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 SCD 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 SCD
genes in the cell.
[0130] In another embodiment, the invention features a method for
modulating the expression of more than one SCD 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 SCD 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 SCD genes in
the cell.
[0131] 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 SCD 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 SCD 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 SCD 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 SCD gene in that organism.
[0132] In one embodiment, the invention features a method of
modulating the expression of a SCD 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 SCD 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 SCD 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 SCD gene in that organism.
[0133] In another embodiment, the invention features a method of
modulating the expression of more than one SCD 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 SCD
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 SCD 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 SCD genes in that organism.
[0134] In one embodiment, the invention features a method of
modulating the expression of a SCD 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 SCD gene; and (b)
introducing the siNA molecule into the subject or organism under
conditions suitable to modulate the expression of the SCD gene in
the subject or organism. The level of SCD protein or RNA can be
determined using various methods well-known in the art.
[0135] In another embodiment, the invention features a method of
modulating the expression of more than one SCD 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 SCD
genes; and (b) introducing the siNA molecules into the subject or
organism under conditions suitable to modulate the expression of
the SCD genes in the subject or organism. The level of SCD protein
or RNA can be determined as is known in the art.
[0136] In one embodiment, the invention features a method for
modulating the expression of a SCD 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 SCD gene; and (b)
introducing the siNA molecule into a cell under conditions suitable
to modulate the expression of the SCD gene in the cell.
[0137] In another embodiment, the invention features a method for
modulating the expression of more than one SCD 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 SCD gene;
and (b) contacting the cell in vitro or in vivo with the siNA
molecule under conditions suitable to modulate the expression of
the SCD genes in the cell.
[0138] In one embodiment, the invention features a method of
modulating the expression of a SCD 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 SCD
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 SCD 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 SCD gene in that subject or organism.
[0139] In another embodiment, the invention features a method of
modulating the expression of more than one SCD 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 SCD 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 SCD
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 SCD genes in that subject or organism.
[0140] In one embodiment, the invention features a method of
modulating the expression of a SCD 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 SCD
gene; and (b) introducing the siNA molecule into the subject or
organism under conditions suitable to modulate the expression of
the SCD gene in the subject or organism.
[0141] In another embodiment, the invention features a method of
modulating the expression of more than one SCD 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 SCD gene; and (b) introducing the siNA molecules into the
subject or organism under conditions suitable to modulate the
expression of the SCD genes in the subject or organism.
[0142] In one embodiment, the invention features a method of
modulating the expression of a SCD 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 SCD gene in the subject or organism.
[0143] In one embodiment, the invention features a method for
treating or preventing diabetes (type I and/or type II) 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 SCD gene in the subject or
organism.
[0144] In one embodiment, the invention features a method for
treating or preventing atherosclerosis 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 SCD gene in the subject or organism.
[0145] 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 SCD gene in the subject or organism.
[0146] In one embodiment, the invention features a method for
treating or preventing obesity 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 SCD gene in the subject or organism.
[0147] In one embodiment, the invention features a method for
inducing weight loss 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 SCD gene in
the subject or organism.
[0148] In one embodiment, the invention features a method for
treating or preventing a viral infection 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 SCD gene in the subject or organism.
[0149] In another embodiment, the invention features a method of
modulating the expression of more than one SCD 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 SCD genes in the subject or
organism.
[0150] The siNA molecules of the invention can be designed to down
regulate or inhibit target (e.g., SCD) 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).
[0151] 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 SCD family genes. As such, siNA
molecules targeting multiple SCD 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 diabetes (type I and/or type II),
atherosclerosis, cancer, obesity, and viral infection.
[0152] 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, SCD genes
encoding RNA sequence(s) referred to herein by Genbank Accession
number, for example, Genbank Accession Nos. shown in Table I.
[0153] 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.
[0154] In one embodiment, the invention features a method
comprising: (a) generating a randomized library of siNA constructs
having a predetermined complexity, such as of 4N, where N
represents the number of base paired nucleotides in each of the
siNA construct strands (eg. for a siNA construct having 21
nucleotide sense and antisense strands with 19 base pairs, the
complexity would be 419); and (b) assaying the siNA constructs of
(a) above, under conditions suitable to determine RNAi target sites
within the target SCD 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 SCD
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 SCD RNA sequence. The target SCD 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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 diabetes
(type I and/or type II), atherosclerosis, cancer, obesity, and
viral infection in a subject or organism comprising administering
to the subject a composition of the invention under conditions
suitable for the treatment or prevention of diabetes (type I and/or
type II), atherosclerosis, cancer, obesity, and viral infection in
the subject or organism.
[0159] In another embodiment, the invention features a method for
validating a SCD 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 SCD target gene; (b) introducing the siNA molecule into
a cell, tissue, subject, or organism under conditions suitable for
modulating expression of the SCD 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.
[0160] In another embodiment, the invention features a method for
validating a SCD 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 SCD target gene; (b) introducing the siNA molecule into
a biological system under conditions suitable for modulating
expression of the SCD target gene in the biological system; and (c)
determining the function of the gene by assaying for any phenotypic
change in the biological system.
[0161] 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.
[0162] 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.
[0163] 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 SCD 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 SCD target gene in a biological system,
including, for example, in a cell, tissue, subject, or
organism.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] In one embodiment, the invention features siNA constructs
that mediate RNAi against SCD, 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.
[0172] 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.
[0173] In another embodiment, the invention features a method for
generating siNA molecules with improved toxicologic profiles (e.g.,
have attenuated or no immunstimulatory 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.
[0174] 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.
[0175] 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).
[0176] In one embodiment, the invention features siNA constructs
that mediate RNAi against SCD, 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.
[0177] 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.
[0178] In one embodiment, the invention features siNA constructs
that mediate RNAi against SCD, 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.
[0179] In one embodiment, the invention features siNA constructs
that mediate RNAi against SCD, 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.
[0180] 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.
[0181] 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.
[0182] In one embodiment, the invention features siNA constructs
that mediate RNAi against SCD, 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.
[0183] 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.
[0184] In one embodiment, the invention features
chemically-modified siNA constructs that mediate RNAi against SCD
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.
[0185] In another embodiment, the invention features a method for
generating siNA molecules with improved RNAi activity against SCD
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.
[0186] In yet another embodiment, the invention features a method
for generating siNA molecules with improved RNAi activity against
SCD 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.
[0187] In yet another embodiment, the invention features a method
for generating siNA molecules with improved RNAi activity against
SCD 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.
[0188] In one embodiment, the invention features siNA constructs
that mediate RNAi against SCD, wherein the siNA construct comprises
one or more chemical modifications described herein that modulates
the cellular uptake of the siNA construct.
[0189] In another embodiment, the invention features a method for
generating siNA molecules against SCD 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.
[0190] In one embodiment, the invention features siNA constructs
that mediate RNAi against SCD, 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.
[0191] 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.
[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 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.
[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 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.
[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 is incapable of
acting as a guide sequence for mediating RNA interference.
[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 does not have a
terminal 5'-hydroxyl (5'-OH) or 5'-phosphate group.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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). 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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).
[0206] 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.
[0207] 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 or the
pre-transcriptional level. In a non-limiting example, epigenetic
regulation of gene expression by siNA molecules of the invention
can result from siNA mediated modification of chromatin structure
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).
[0208] 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).
[0209] 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 SCD RNA (see for example
target sequences in Tables II and III).
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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
nucleolar 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. Abberant 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.
[0215] 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)-N3-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.
[0216] By "SCD" as used herein is meant, any stearoyl-CoA
desaturase protein, peptide, or polypeptide having any stearoyl-CoA
desaturase activity, such as encoded by SCD Genbank Accession Nos.
shown in Table I. The term SCD also refers to nucleic acid
sequences encoding any stearoyl-CoA desaturase protein, peptide, or
polypeptide having SCD activity. The term "SCD" is also meant to
include other SCD encoding sequence, such as other SCD isoforms,
mutant SCD genes, splice variants of SCD genes, and SCD 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 SCD gene expression are used for preventing or
treating diabetes (type I and/or type II), atherosclerosis, cancer,
obesity, and viral infection in a subject or organism.
[0224] In one embodiment, the siNA molecules of the invention are
used to treat diabetes (type I and/or type II), atherosclerosis,
cancer, obesity, and viral infection in a subject or organism.
[0225] In one embodiment, the siNA molecules of the invention are
used to induce or promote weight loss in a subject or organism. In
one embodiment, the siNA molecules of the invention are used to
lower the amount of body fat in a subject or organism.
[0226] By "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 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 any other cancer that can respond to the modulation of
disease related gene expression in a cell or tissue, alone or in
combination with other therapies.
[0227] 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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] By "RNA" is meant a molecule comprising at least one
ribonucleotide residue. By "ribonucleotide" is meant a nucleotide
with a hydroxyl group at the 2' position of a .beta.-D-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.
[0232] 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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).
[0237] 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.
[0238] 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 diabetes (type I and/or
type II), atherosclerosis, cancer, obesity, and viral infection in
a subject or organism.
[0239] 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.
[0240] In a further embodiment, the siNA molecules can be used in
combination with other known treatments to prevent or treat
diabetes (type I and/or type II), atherosclerosis, cancer, obesity,
and viral infection 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
diabetes (type I and/or type II), atherosclerosis, cancer, obesity,
and viral infection in a subject or organism as are known in the
art.
[0241] 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.
[0242] In another embodiment, the invention features a mammalian
cell, for example, a human cell, including an expression vector of
the invention.
[0243] 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.
[0244] 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.
[0245] 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.
[0246] By "vectors" is meant any nucleic acid- and/or viral-based
technique used to deliver a desired nucleic acid.
[0247] 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
[0248] 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.
[0249] 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. 4A-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 SCD siNA
sequence. Such chemical modifications can be applied to any SCD
sequence and/or SCD 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 SCD 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 SCD 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 SCD 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 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. 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
bifuctional 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 bifuctional 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 complex 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 complex 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.
[0286] FIG. 22 shows a non-limiting example of reduction of SCD
mRNA in A549 cells mediated by chemically modified siNAs that
target SCD mRNA. A549 cells were transfected with 0.25 ug/well of
lipid complexed with 25 nM siNA. Active siNA constructs comprising
various stabilization chemistries (see Tables III and IV) were
compared to untreated cells, a matched chemistry irrelevant siNA
control construct (IC; 32072/32075), and cells transfected with
lipid alone (transfection control). As shown in the figure, the
siNA constructs significantly reduce SCD RNA expression.
DETAILED DESCRIPTION OF THE INVENTION
[0287] Mechanism of Action of Nucleic Acid Molecules of the
Invention
[0288] 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.
[0289] 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.
[0290] 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.
[0291] 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.
[0292] 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.
[0293] Synthesis of Nucleic Acid Molecules
[0294] 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.
[0295] 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 calorimetric 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 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.
[0296] 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.
[0297] 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-dioxide0.05 M in acetonitrile) is
used.
[0298] 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.cndot.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.
[0299] 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.cndot.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.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] 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.
[0304] 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.
[0305] 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.
[0306] 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.
[0307] Optimizing Activity of the Nucleic Acid Molecule of the
Invention.
[0308] 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.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] 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).
[0313] 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.
[0314] 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.
[0315] The term "biodegradable" as used herein, refers to
degradation in a biological system, for example, enzymatic
degradation or chemical degradation.
[0316] 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.
[0317] 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.
[0318] 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.
[0319] 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.
[0320] 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.
[0321] 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.
[0322] 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.
[0323] 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).
[0324] 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.
[0325] 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 I 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.
[0326] 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.
[0327] 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.
[0328] 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.
[0329] 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.
[0330] By "unmodified nucleoside" is meant one of the bases
adenine, cytosine, guanine, thymine, or uracil joined to the 1'
carbon of P-D-ribo-furanose.
[0331] 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.
[0332] In connection with 2'-modified nucleotides as described for
the present invention, by "amino" is meant 2'-NH2 or 2'-O- NH2,
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.
[0333] 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.
[0334] Administration of Nucleic Acid Molecules
[0335] A siNA molecule of the invention can be adapted for use to
prevent or treat diabetes (type I and/or type II), atherosclerosis,
cancer, obesity, and viral infection, and/or any other trait,
disease, disorder or condition that is related to or will respond
to the levels of SCD 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 U.S. patent application
Publication No. U.S. 2002130430), biodegradable nanocapsules, and
bioadhesive microspheres, or by proteinaceous vectors (O'Hare and
Normand, International PCT Publication No. WO 00/53722). 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 U.S.
patent application Publication No. 20030077829, incorporated by
reference herein in its entirety.
[0336] 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.
[0337] 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.
[0338] 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.
[0339] 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,
dichlorotetrafluoroethan- e 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 U.S. patent application Ser. No.
20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728;
6,565,885.
[0340] 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).
[0341] 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).
[0342] 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, Phramaceutical 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.
[0343] 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.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] 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.
[0348] 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, DF 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.
[0349] 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.
[0350] 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.
[0351] 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.
[0352] 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.
[0353] 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.
[0354] 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.
[0355] 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.
[0356] 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 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.
[0357] 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.
[0358] 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.
[0359] 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.
[0360] 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.
[0361] 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.
[0362] 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.
[0363] 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.
[0364] 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.
[0365] 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.
[0366] 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.
[0367] 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. Pat. 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 intra-muscular 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).
[0368] 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).
[0369] 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).
[0370] 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 III 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).
[0371] 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.
[0372] 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.
[0373] 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.
[0374] SCD Biology and Biochemistry
[0375] Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in
the biosynthesis of monounsaturated fatty acids, including oleate
(C18:1) and palmitoleate (C16:1). SCD catalyzes the introduction of
the cis double bond in the delta-9 position of fatty acyl-CoA
substrates, such as palmitoyl-CoA and stearoyl-CoA, which are
converted to palmitoleoyl-CoA (16:1) and oleoyl-CoA (18:1),
respectively. These fatty acids are essential components of
membrane phospholipids, triglycerides, cholesterol esters, and wax
esters. Modulation of phospholipid composition ultimately
determines physical properties, such as membrane fluidity. Further,
effects on the composition of cholesterol esters and triglycerides
can substantially affect lipoprotein metabolism and adiposity. SCD
expression is sensitive to dietary factors, including
polyunsaturated fatty acids, cholesterol and vitamin A, hormonal
changes (i.e., insulin leptin, and glucagon), developmental
processes, temperature changes, thiazolinediones, metals, alcohol,
peroxisomal proliferators, and certain phenolic compounds. High SCD
activity has been implicated in a wide range of diseases and
disorders including diabetes, atherosclerosis, cancer, obesity, and
viral infection (Ntambi et al., 2002, PNAS USA, 99,
11482-11486).
[0376] Several SCD isoforms (SCD1-3) have been identified in the
mouse. Mice having defective SCD expression provide useful
information in determining the relevance of SCD in models of
obesity, diabetes, and insulin resistance. For example, new
insights into the physiological role of the SCD1 gene and its
endogenous products have been reported from recent studies of the
asebia mouse strains (ab.sup.j and ab.sup.2j) that have naturally
occurring mutations in SCD1 (Zheng et al., 1999, Nature Genetics,
23, 268-270), as well as a laboratory mouse model with a targeted
disruption (SCD1 -/-) (Miyazaki et al., 2001, J. Biol. Chem., 276,
39455-39461). Mice having a targeted disruption of the SCD1 isoform
have reduced body adiposity, increased insulin sensitivity, and are
resistant to diet-induced weight gain. The protection from obesity
in these mice involves increased energy expenditure and increased
oxygen consumption. Compared with the wild-type mice, SCD1-/- mice
have increased levels of plasma ketone bodies, but also have
reduced levels of plasma insulin and leptin. The expression of
several genes controlling lipid oxidation are up-regulated in
SCD1-/- mice, whereas lipid synthesis genes are down-regulated in
SCD1-/- mice. These observations suggest that a consequence of SCD1
deficiency is an activation of lipid oxidation in addition to
reduced triglyceride synthesis and storage. Aside from the dramatic
alterations in triglyceride and cholesterol metabolism, the SCD1-/-
mice are considerably leaner than their wild-type counterparts.
(Ntambi et al., 2002, PNAS USA, 99, 11482-11486).
[0377] The use of small interfering nucleic acid molecules
targeting SCD, therefore provides a class of novel therapeutic
agents that can be used in the treatment, alleviation, or
prevention of diabetes (type I and/or type II), atherosclerosis,
cancer, obesity, and viral infection, alone or in combination with
other therapies.
EXAMPLES
[0378] 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
[0379] 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.
[0380] 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.
[0381] 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
Bromotripyrrolidinophosphoniumhexaflurorophosphate (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
1.5 M NH.sub.4H.sub.2CO.sub.3.
[0382] Purification of the siNA duplex can be readily accomplished
using solid phase extraction, for example, using a Waters C18
SepPak 1 g 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 H2O followed by 1 CV 1M NaCl and additional
H2O. The siNA duplex product is then eluted, for example, using 1
CV 20% aqueous CAN.
[0383] 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
[0384] 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
[0385] The following non-limiting steps can be used to carry out
the selection of siNAs targeting a given gene sequence or
transcript.
[0386] 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.
[0387] 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.
[0388] 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.
[0389] 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.
[0390] 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.
[0391] 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.
[0392] 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' WU on the antisense sequence).
These sequences allow one to design siNA molecules with terminal TT
thymidine dinucleotides.
[0393] 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.
[0394] 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.
[0395] 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.
[0396] In an alternate approach, a pool of siNA constructs specific
to a SCD target sequence is used to screen for target sites in
cells expressing SCD RNA, such as such HepG2, MDA-MB-231 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 1-806. Cells expressing SCD are
transfected with the pool of siNA constructs and cells that
demonstrate a phenotype associated with SCD 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 SCD
mRNA levels or decreased SCD protein expression), are sequenced to
determine the most suitable target site(s) within the target SCD
RNA sequence.
Example 4
SCD Targeted siNA Design
[0397] siNA target sites were chosen by analyzing sequences of the
SCD 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.
[0398] 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
[0399] siNA molecules can be designed to interact with various
sites in the RNA message, for example, target sequences within the
RNA sequences described herein. The sequence of one strand of the
siNA molecule(s) is complementary to the target site sequences
described above. The siNA molecules can be chemically synthesized
using methods described herein. Inactive siNA molecules that are
used as control sequences can be synthesized by scrambling the
sequence of the siNA molecules such that it is not complementary to
the target sequence. Generally, siNA constructs can by synthesized
using solid phase oligonucleotide synthesis methods as described
herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683;
5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117;
6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400;
6,111,086 all incorporated by reference herein in their
entirety).
[0400] 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).
[0401] 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.
[0402] 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
[0403] An in vitro assay that recapitulates RNAi in a cell-free
system is used to evaluate siNA constructs targeting SCD 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 SCD 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 SCD 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 pM
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.
[0404] 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.
[0405] In one embodiment, this assay is used to determine target
sites in the SCD RNA target for siNA mediated RNAi cleavage,
wherein a plurality of siNA constructs are screened for RNAi
mediated cleavage of the SCD 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 SCD Target RNA
[0406] siNA molecules targeted to the human SCD 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 SCD RNA are given in Tables II and III.
[0407] Two formats are used to test the efficacy of siNAs targeting
SCD. First, the reagents are tested in cell culture using, for
example, HepG2, MDA-MB-231 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 SCD target as described herein.
RNA inhibition is measured after delivery of these reagents by a
suitable transfection agent to, for example, HepG2, MDA-MB-231 or
A549 cells. Relative amounts of target RNA are measured versus
actin using real-time PCR monitoring of amplification (eg., ABI
7700 TAQMAN.RTM.). A comparison is made to a mixture of
oligonucleotide sequences made to unrelated targets or to a
randomized siNA control with the same overall length and chemistry,
but randomly substituted at each position. Primary and secondary
lead reagents are chosen for the target and optimization performed.
After an optimal transfection agent concentration is chosen, a RNA
time-course of inhibition is performed with the lead siNA molecule.
In addition, a cell-plating format can be used to determine RNA
inhibition.
[0408] Delivery of siNA to Cells
[0409] Cells such as HepG2, MDA-MB-231 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.
[0410] TAQMAN.RTM. (Real-Time PCR Monitoring of Amplification) and
Lightcycler Quantification of mRNA
[0411] 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, 133 TaqMan PCR reaction buffer
(PE-Applied Biosystems), 5.5 mM MgCl.sub.2, 300 .mu.M each DATP,
dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ
GOLD.RTM. (DNA polymerase) (PE-Applied Biosystems) and 10U 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 lightcyler. A standard curve is generated for each
primer pair using control cRNA. Values are represented as relative
expression to GAPDH in each sample.
[0412] Western Blotting
[0413] 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 SCD Gene
Expression
[0414] Cell Culture
[0415] There are numerous cell culture systems that can be used to
analyze reduction of SCD levels either directly or indirectly by
measuring downstream effects. For example, cultured HepG2,
MDA-MB-231 or A549 cells can be used in cell culture experiments to
assess the efficacy of nucleic acid molecules of the invention. As
such, cells treated with nucleic acid molecules of the invention
(e.g., siNA) targeting SCD RNA would be expected to have decreased
SCD expression capacity compared to matched control nucleic acid
molecules having a scrambled or inactive sequence. In a
non-limiting example, cells are cultured and SCD expression is
quantified, for example, by time-resolved immuno fluorometric
assay. SCD 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 SCD protein and RNA
levels are quantitated. Dose response assays are then performed to
establish dose dependent inhibition of SCD expression.
[0416] Liver stearoyl-CoA desaturase (SCD) activity has been
implicated in excessive adiposity in chickens. Studies suggest that
the difference in SCD activity between fat and lean chickens is
explained by a difference in SCD I gene expression. (Lefevre et
al., 1999, Archives of Biochemistry and Biophysics, 368, 329-337).
Lefevre et al. describe a study using primary cultures of
6-week-old chicken hepatocytes, in which SCD1 gene expression was
analyzed as the result of insulin and glucagon action. The studies
showed that insulin increased SCD1 activity and mRNA levels,
whereas glucagon dramatically decreased both SCD1 enzyme activity
and SCD1 mRNA levels. Nuclear run-on transcription assays and mRNA
stability investigations demonstrated that insulin and glucagon
effects on SCD1 gene expression were primarily transcriptional in
nature. Furthermore, the results indicated that the
glucagon-mediated inhibition of SCD1 gene transcription was more
potent than simply counteracting the insulin-mediated effect.
Moreover, among hepatic genes involved in lipid metabolism in
chickens, SCD1 was the first gene shown to be regulated at the
transcriptional level by insulin, in the absence of
triiodothyronine. These studies demonstrate that the growing
chicken hepatocyte culture model is a useful model in studying the
effect of SCD inhibitors, including siNA molecules of the present
invention.
[0417] 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.
[0418] Evaluating the efficacy of anti-SCD agents in animal models
is an important prerequisite to human clinical trials. Stearoyl-CoA
desaturase (SCD) mRNA is overexpressed in various animal models of
obesity and type 2 diabetes, see for example Ntambi et al., 2000,
Biomedical and Health Research, 37, 69-78 and Miyazaki et al.,
2001, J. Biol. Chem., 276, 39455-39461. SCD expression is known to
dramatically increase during the differentiation of preadipocytes
into adipocytes. Thiazolidinediones (TZDs), ligands for the
adipocyte-specific nuclear peroxisome proliferator-activated
receptor gamma 2 (PPAR-gamma-2), enhance the conversion of
preadipocytes into mature adipocytes in vivo and in vitro, and can
exert potent antidiabetic effects by enhancing sensitivity to
insulin in target tissues. TZDs have been shown to repress the
expression of the SCD1 gene isoform in differentiating
preadipocytes and mature adipocytes (Ntambi et al., 2000,
Biomedical and Health Research, 37, 69-78). The repression of
several other adipogenic genes, including the SCD2 isoform, were
not affected by TZDs. In differentiating preadipocytes and mature
adipocytes, the downregulation of the SCD1 gene expression was
accompanied by a decrease in SCD protein and enzyme activity, as
well as a dramatic decrease in palmitoleate (C16:1n-7) composition.
The adipocytes were smaller in size and contained smaller fat
droplets as observed by Oil Red O staining. These data indicate
that the TZDs specifically target SCD1 gene expression in both
differentiating and mature adipocytes, which consequently decreases
the C16:1n-7 composition of triglycerides. The models described by
Ntambi et al., 2000, Biomedical and Health Research, 37, 69-78;
Miyazaki et al., 2001, J. Biol. Chem., 276, 39455-39461; and Zheng
et al., 1999, Nature Genetics, 23, 268-270, such as asebia mouse
strains (ab.sup.j and ab.sup.2j) and mouse models with a targeted
disruption (SCD1 -/-) can be used to study the effect of SCD
inhibition using siNA molecules of the invention.
Example 9
RNAi Mediated Inhibition of SCD Expression
[0419] siNA constructs (Table III) are tested for efficacy in
reducing SCD RNA expression in, for example, HepG2, MDA-MB-231 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.
[0420] In a non-limiting example, chemically modified siNA
constructs (Table III) were tested for efficacy as described above
in reducing SCD RNA expression in A549 cells. Active siNAs were
evaluated compared to untreated cells, a matched chemistry
irrelevant control (IC, 32072/32075), and a transfection control.
Results are summarized in FIG. 22. FIG. 22 shows results for
chemically modified siNA constructs targeting various sites in SCD
mRNA. As shown in FIG. 22, the active siNA constructs provide
significant inhibition of SCD gene expression in cell culture
experiments as determined by levels of SCD mRNA when compared to
appropriate controls.
Example 10
Indications
[0421] The present body of knowledge in SCD research indicates the
need for methods to assay SCD activity and for compounds that can
regulate SCD expression for research, diagnostic, and therapeutic
use. As described herein, the nucleic acid molecules of the present
invention can be used in assays to diagnose disease state related
of SCD levels. In addition, the nucleic acid molecules can be used
to treat disease state related to SCD levels.
[0422] Particular conditions and disease states that can be
associated with SCD expression modulation include, but are not
limited to diabetes (type I and/or type II), atherosclerosis,
cancer, obesity, and viral infection, and any other diseases,
conditions or disorders that are related to or will respond to the
levels of SCD in a cell or tissue, alone or in combination with
other therapies.
[0423] Thiazolidinediones (TZDs), insulin, and PTP-1B inhibitors
(see for example McSwiggen, U.S. Ser. No. 10/206,705) are
non-limiting examples of pharmaceutical agents that can be combined
with or used in conjunction with the nucleic acid molecules (e.g.
siRNA molecules) of the instant invention. Those skilled in the art
will recognize that other drugs, such as anti-diabetes and
anti-obesity compounds and therapies, can similarly be readily
combined with the nucleic acid molecules of the instant invention
(e.g. siRNA molecules) and are hence within the scope of the
instant invention.
Example 11
Diagnostic Uses
[0424] 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).
[0425] In a specific example, siNA molecules that cleave only
wild-type or mutant forms of the target RNA are used for the assay.
The first siNA molecules (i.e., those that cleave only wild-type
forms of target RNA) are used to identify wild-type RNA present in
the sample and the second siNA molecules (i.e., those that cleave
only mutant forms of target RNA) are used to identify mutant RNA in
the sample. As reaction controls, synthetic substrates of both
wild-type and mutant RNA are cleaved by both siNA molecules to
demonstrate the relative siNA efficiencies in the reactions and the
absence of cleavage of the "non-targeted" RNA species. The cleavage
products from the synthetic substrates also serve to generate size
markers for the analysis of wild-type and mutant RNAs in the sample
population. Thus, each analysis requires two siNA molecules, two
substrates and one unknown sample, which is combined into six
reactions. The presence of cleavage products is determined using an
RNase protection assay so that full-length and cleavage fragments
of each RNA can be analyzed in one lane of a polyacrylamide gel. It
is not absolutely required to quantify the results to gain insight
into the expression of mutant RNAs and putative risk of the desired
phenotypic changes in target cells. The expression of mRNA whose
protein product is implicated in the development of the phenotype
(i.e., disease related or infection related) is adequate to
establish risk. If probes of comparable specific activity are used
for both transcripts, then a qualitative comparison of RNA levels
is adequate and decreases the cost of the initial diagnosis. Higher
mutant form to wild-type ratios are correlated with higher risk
whether RNA levels are compared qualitatively or
quantitatively.
[0426] 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.
[0427] 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.
[0428] 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.
[0429] 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. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of", and "consisting of" may be replaced
with either of the other two terms. 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.
[0430] 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.
1TABLE I SCD Accession Numbers XM_208174 Homo sapiens similar to
stearoyl-CoA desaturase (delta-9-desaturase) [Homo sapiens]
(LOC284202), mRNA
gi.vertline.27500564.vertline.ref.vertline.XM_208174.1.vertline.[27500564-
] NM_005063 Homo sapiens stearoyl-CoA desaturase (delta-9-
desaturase) (SCD), mRNA gi.vertline.19923295.vertline.ref-
.vertline.NM_005063.2.vertline.[19923295] Y13647 Homo sapiens mRNA
for stearoyl-CoA desaturase gi.vertline.2190403.vert-
line.emb.vertline.Y13647.1.vertline.HSSTEACOA[2190403] AF320307
Homo sapiens stearoyl-CoA desaturase (SCD) gene, exon 1 and intron
1 and partial cds gi.vertline.14150490.vertline.gb.vertlin-
e.AF320307.1.vertline.AF320307[14150490] AU098830 AU098830 Sugano
Homo sapiens cDNA library Homo sapiens cDNA clone HEP02242 similar
to Homo sapiens stearoyl-CoA desaturase (SCD) mRNA, MRNA sequence
gi.vertline.13549959.vertline.dbj.vertline.AU-
098830.1.vertline.[13549959] BG319495 Tmdh05 Human Keratinocyte
Subtraction Library- Downregulated Transcripts Homo sapiens cDNA
similar to stearoyl-CoA desaturase (delta-9-desaturase) (SCD), MRNA
sequence gi.vertline.13129361.ve-
rtline.gb.vertline.BG319495.1.vertline.[13129361] BG319433 4dh85
Human Keratinocyte Subtraction Library- Downregulated Transcripts
Homo sapiens cDNA similar to stearoyl-CoA desaturase (SCD), MRNA
sequence gi.vertline.13129299.vertline.gb.ver-
tline.BG319433.1.vertline.[13129299] AU076658 AU076658 Sugano cDNA
library Homo sapiens cDNA clone HEP02242 similar to 5'-end region
of Homo sapiens mRNA for stearoyl-CoA desaturase, MRNA sequence
gi.vertline.7439136.vertline.dbj.vertli-
ne.AU076658.1.vertline.[7439136] AB032261 Homo sapiens Scd mRNA for
stearoyl-CoA desaturase, complete cds
gi.vertline.7415720.vertline.dbj.vertline.AB032261.1.vertline.[7415720]
AF097514 Homo sapiens stearoyl-CoA desaturase (SCD) mRNA, complete
cds gi.vertline.4808600.vertline.gb.vertline.AF097-
514.1.vertline.AF097514[4808600] S70284 stearoyl-CoA desaturase
[human, adipose tissue, mRNA Partial, 712 nt]
gi.vertline.546517.vertline.bbm.vertline.339737.vertline.bbs.vertline.148-
167.vertline.gb.vertline.S70284.1.vertline.S70284[546517]
[0431]
2TABLE II SCD siNA AND TARGET SEQUENCES SCD NM_005063.2 Pos Seq Seq
ID UPos Upper seq Seq ID LPos Lower seq Seq ID 3
AAAAGGGGGCUGAGGAAAU 1 3 AAAAGGGGGCUGAGGAAAU 1 21
AUUUCCUCAGCCCCCUUUU 291 21 UACCGGACACGGUCACCCG 2 21
UACCGGACACGGUCACCCG 2 39 CGGGUGACCGUGUCCGGUA 292 39
GUUGCCAGCUCUAGCCUUU 3 39 GUUGCCAGCUCUAGCCUUU 3 57
AAAGGCUAGAGCUGGCAAC 293 57 UAAAUUCCCGGCUCGGGGA 4 57
UAAAUUCCCGGCUCGGGGA 4 75 UCCCCGAGCCGGGAAUUUA 294 75
ACCUCCACGCACCGCGGCU 5 75 ACCUCCACGCACCGCGGCU 5 93
AGCCGCGGUGCGUGGAGGU 295 93 UAGCGCCGACAACCAGCUA 6 93
UAGCGCCGACAACCAGCUA 6 111 UAGCUGGUUGUCGGCGCUA 296 111
AGCGUGCAAGGCGCCGCGG 7 111 AGCGUGCAAGGCGCCGCGG 7 129
CCGCGGCGCCUUGCACGCU 297 129 GCUCAGCGCGUACCGGCGG 8 129
GCUCAGCGCGUACCGGCGG 8 147 CCGCCGGUACGCGCUGAGC 298 147
GGCUUCGAAACCGCAGUCC 9 147 GGCUUCGAAACCGCAGUCC 9 165
GGACUGCGGUUUCGAAGCC 299 165 CUCCGGCGACCCCGAACUC 10 165
CUCCGGCGACCCCGAACUC 10 183 GAGUUCGGGGUCGCCGGAG 300 183
CCGCUCCGGAGCCUCAGCC 11 183 CCGCUCCGGAGCCUCAGCC 11 201
GGCUGAGGCUCCGGAGCGG 301 201 CCCCUGGAAAGUGAUCCCG 12 201
CCCCUGGAAAGUGAUCCCG 12 219 CGGGAUCACUUUCCAGGGG 302 219
GGCAUCCGAGAGCCAAGAU 13 219 GGCAUCCGAGAGCCAAGAU 13 237
AUCUUGGCUCUCGGAUGCC 303 237 UGCCGGCCCACUUGCUGCA 14 237
UGCCGGCCCACUUGCUGCA 14 255 UGCAGCAAGUGGGCCGGCA 304 255
AGGACGAUAUCUCUAGCUC 15 255 AGGACGAUAUCUCUAGCUC 15 273
GAGCUAGAGAUAUCGUCCU 305 273 CCUAUACCACCACCACCAC 16 273
CCUAUACCACCACCACCAC 16 291 GUGGUGGUGGUGGUAUAGG 306 291
CCAUUACAGCGCCUCCCUC 17 291 CCAUUACAGCGCCUCCCUC 17 309
GAGGGAGGCGCUGUAAUGG 307 309 CCAGGGUCCUGCAGAAUGG 18 309
CCAGGGUCCUGCAGAAUGG 18 327 CCAUUCUGCAGGACCCUGG 308 327
GAGGAGAUAAGUUGGAGAC 19 327 GAGGAGAUAAGUUGGAGAC 19 345
GUCUCCAACUUAUCUCCUC 309 345 CGAUGCCCCUCUACUUGGA 20 345
CGAUGCCCCUCUACUUGGA 20 363 UCCAAGUAGAGGGGCAUCG 310 363
AAGACGACAUUCGCCCUGA 21 363 AAGACGACAUUCGCCCUGA 21 381
UCAGGGCGAAUGUCGUCUU 311 381 AUAUAAAAGAUGAUAUAUA 22 381
AUAUAAAAGAUGAUAUAUA 22 399 UAUAUAUCAUCUUUUAUAU 312 399
AUGACCCCACCUACAAGGA 23 399 AUGACCCCACCUACAAGGA 23 417
UCCUUGUAGGUGGGGUCAU 313 417 AUAAGGAAGGCCCAAGCCC 24 417
AUAAGGAAGGCCCAAGCCC 24 435 GGGCUUGGGCCUUCCUUAU 314 435
CCAAGGUUGAAUAUGUCUG 25 435 CCAAGGUUGAAUAUGUCUG 25 453
CAGACAUAUUCAACCUUGG 315 453 GGAGAAACAUCAUCCUUAU 26 453
GGAGAAACAUCAUCCUUAU 26 471 AUAAGGAUGAUGUUUCUCC 316 471
UGUCUCUGCUACACUUGGG 27 471 UGUCUCUGCUACACUUGGG 27 489
CCCAAGUGUAGCAGAGACA 317 489 GAGCCCUGUAUGGGAUCAC 28 489
GAGCCCUGUAUGGGAUCAC 28 507 GUGAUCCCAUACAGGGCUC 318 507
CUUUGAUUCCUACCUGCAA 29 507 CUUUGAUUCCUACCUGCAA 29 525
UUGCAGGUAGGAAUCAAAG 319 525 AGUUCUACACCUGGCUUUG 30 525
AGUUCUACACCUGGCUUUG 30 543 CAAAGCCAGGUGUAGAACU 320 543
GGGGGGUAUUCUACUAUUU 31 543 GGGGGGUAUUCUACUAUUU 31 561
AAAUAGUAGAAUACCCCCC 321 561 UUGUCAGUGCCCUGGGCAU 32 561
UUGUCAGUGCCCUGGGCAU 32 579 AUGCCCAGGGCACUGACAA 322 579
UAACAGCAGGAGCUCAUCG 33 579 UAACAGCAGGAGCUCAUCG 33 597
CGAUGAGCUCCUGCUGUUA 323 597 GUCUGUGGAGCCACCGCUC 34 597
GUCUGUGGAGCCACCGCUC 34 615 GAGCGGUGGCUCCACAGAC 324 615
CUUACAAAGCUCGGCUGCC 35 615 CUUACAAAGCUCGGCUGCC 35 633
GGCAGCCGAGCUUUGUAAG 325 633 CCCUACGGCUCUUUCUGAU 36 633
CCCUACGGCUCUUUCUGAU 36 651 AUCAGAAAGAGCCGUAGGG 326 651
UCAUUGCCAACACAAUGGC 37 651 UCAUUGCCAACACAAUGGC 37 669
GCCAUUGUGUUGGCAAUGA 327 669 CAUUCCAGAAUGAUGUCUA 38 669
CAUUCCAGAAUGAUGUCUA 38 687 UAGACAUCAUUCUGGAAUG 328 687
AUGAAUGGGCUCGUGACCA 39 687 AUGAAUGGGCUCGUGACCA 39 705
UGGUCACGAGCCCAUUCAU 329 705 ACCGUGCCCACCACAAGUU 40 705
ACCGUGCCCACCACAAGUU 40 723 AACUUGUGGUGGGCACGGU 330 723
UUUCAGAAACACAUGCUGA 41 723 UUUCAGAAACACAUGCUGA 41 741
UCAGCAUGUGUUUCUGAAA 331 741 AUCCUCAUAAUUCCCGACG 42 741
AUCCUCAUAAUUCCCGACG 42 759 CGUCGGGAAUUAUGAGGAU 332 759
GUGGCUUUUUCUUCUCUCA 43 759 GUGGCUUUUUCUUCUCUCA 43 777
UGAGAGAAGAAAAAGCCAC 333 777 ACGUGGGUUGGCUGCUUGU 44 777
ACGUGGGUUGGCUGCUUGU 44 795 ACAAGCAGCCAACCCACGU 334 795
UGCGCAAACACCCAGCUGU 45 795 UGCGCAAACACCCAGCUGU 45 813
ACAGCUGGGUGUUUGCGCA 335 813 UCAAAGAGAAGGGGAGUAC 46 813
UCAAAGAGAAGGGGAGUAC 46 831 GUACUCCCCUUCUCUUUGA 336 831
CGCUAGACUUGUCUGACCU 47 831 CGCUAGACUUGUCUGACCU 47 849
AGGUCAGACAAGUCUAGCG 337 849 UAGAAGCUGAGAAACUGGU 48 849
UAGAAGCUGAGAAACUGGU 48 867 ACCAGUUUCUCAGCUUCUA 338 867
UGAUGUUCCAGAGGAGGUA 49 867 UGAUGUUCCAGAGGAGGUA 49 885
UACCUCCUCUGGAACAUCA 339 885 ACUACAAACCUGGCUUGCU 50 885
ACUACAAACCUGGCUUGCU 50 903 AGCAAGCCAGGUUUGUAGU 340 903
UGCUGAUGUGCUUCAUCCU 51 903 UGCUGAUGUGCUUCAUCCU 51 921
AGGAUGAAGCACAUCAGCA 341 921 UGCCCACGCUUGUGCCCUG 52 921
UGCCCACGCUUGUGCCCUG 52 939 CAGGGCACAAGCGUGGGCA 342 939
GGUAUUUCUGGGGUGAAAC 53 939 GGUAUUUCUGGGGUGAAAC 53 957
GUUUCACCCCAGAAAUACC 343 957 CUUUUCAAAACAGUGUGUU 54 957
CUUUUCAAAACAGUGUGUU 54 975 AACACACUGUUUUGAAAAG 344 975
UCGUUGCCACUUUCUUGCG 55 975 UCGUUGCCACUUUCUUGCG 55 993
CGCAAGAAAGUGGCAACGA 345 993 GAUAUGCUGUGGUGCUUAA 56 993
GAUAUGCUGUGGUGCUUAA 56 1011 UUAAGCACCACAGCAUAUC 346 1011
AUGCCACCUGGCUGGUGAA 57 1011 AUGCCACCUGGCUGGUGAA 57 1029
UUCACCAGCCAGGUGGCAU 347 1029 ACAGUGCUGCCCACCUCUU 58 1029
ACAGUGCUGCCCACCUCUU 58 1047 AAGAGGUGGGCAGCACUGU 348 1047
UCGGAUAUCGUCCUUAUGA 59 1047 UCGGAUAUCGUCCUUAUGA 59 1065
UCAUAAGGACGAUAUCCGA 349 1065 ACAAGAACAUUAGCCCCCG 60 1065
ACAAGAACAUUAGCCCCCG 60 1083 CGGGGGCUAAUGUUCUUGU 350 1083
GGGAGAAUAUCCUGGUUUC 61 1083 GGGAGAAUAUCCUGGUUUC 61 1101
GAAACCAGGAUAUUCUCCC 351 1101 CACUUGGAGCUGUGGGUGA 62 1101
CACUUGGAGCUGUGGGUGA 62 1119 UCACCCACAGCUCCAAGUG 352 1119
AGGGCUUCCACAACUACCA 63 1119 AGGGCUUCCACAACUACCA 63 1137
UGGUAGUUGUGGAAGCCCU 353 1137 ACCACUCCUUUCCCUAUGA 64 1137
ACCACUCCUUUCCCUAUGA 64 1155 UCAUAGGGAAAGGAGUGGU 354 1155
ACUACUCUGCCAGUGAGUA 65 1155 ACUACUCUGCCAGUGAGUA 65 1173
UACUCACUGGCAGAGUAGU 355 1173 ACCGCUGGCACAUCAACUU 66 1173
ACCGCUGGCACAUCAACUU 66 1191 AAGUUGAUGUGCCAGCGGU 356 1191
UCACCACAUUCUUCAUUGA 67 1191 UCACCACAUUCUUCAUUGA 67 1209
UCAAUGAAGAAUGUGGUGA 357 1209 AUUGCAUGGCCGCCCUCGG 68 1209
AUUGCAUGGCCGCCCUCGG 68 1227 CCGAGGGCGGCCAUGCAAU 358 1227
GUCUGGCCUAUGACCGGAA 69 1227 GUCUGGCCUAUGACCGGAA 69 1245
UUCCGGUCAUAGGCCAGAC 359 1245 AGAAAGUCUCCAAGGCCGC 70 1245
AGAAAGUCUCCAAGGCCGC 70 1263 GCGGCCUUGGAGACUUUCU 360 1263
CCAUCUUGGCCAGGAUUAA 71 1263 CCAUCUUGGCCAGGAUUAA 71 1281
UUAAUCCUGGCCAAGAUGG 361 1281 AAAGAACCGGAGAUGGAAA 72 1281
AAAGAACCGGAGAUGGAAA 72 1299 UUUCCAUCUCCGGUUCUUU 362 1299
ACUACAAGAGUGGCUGAGU 73 1299 ACUACAAGAGUGGCUGAGU 73 1317
ACUCAGCCACUCUUGUAGU 363 1317 UUUGGGGUCCCUCAGGUUU 74 1317
UUUGGGGUCCCUCAGGUUU 74 1335 AAACCUGAGGGACCCCAAA 364 1335
UCCUUUUUCAAAAACCAGC 75 1335 UCCUUUUUCAAAAACCAGC 75 1353
GCUGGUUUUUGAAAAAGGA 365 1353 CCAGGCAGAGGUUUUAAUG 76 1353
CCAGGCAGAGGUUUUAAUG 76 1371 CAUUAAAACCUCUGCCUGG 366 1371
GUCUGUUUAUUAACUACUG 77 1371 GUCUGUUUAUUAACUACUG 77 1389
CAGUAGUUAAUAAACAGAC 367 1389 GAAUAAUGCUACCAGGAUG 78 1389
GAAUAAUGCUACCAGGAUG 78 1407 CAUCCUGGUAGCAUUAUUC 368 1407
GCUAAAGAUGAUGAUGUUA 79 1407 GCUAAAGAUGAUGAUGUUA 79 1425
UAACAUCAUCAUCUUUAGC 369 1425 AACCCAUUCCAGUACAGUA 80 1425
AACCCAUUCCAGUACAGUA 80 1443 UACUGUACUGGAAUGGGUU 370 1443
AUUCUUUUAAAAUUCAAAA 81 1443 AUUCUUUUAAAAUUCAAAA 81 1461
UUUUGAAUUUUAAAAGAAU 371 1461 AGUAUUGAAAGCCAACAAC 82 1461
AGUAUUGAAAGCCAACAAC 82 1479 GUUGUUGGCUUUCAAUACU 372 1479
CUCUGCCUUUAUGAUGCUA 83 1479 CUCUGCCUUUAUGAUGCUA 83 1497
UAGCAUCAUAAAGGCAGAG 373 1497 AAGCUGAUAUUAUUUCUUC 84 1497
AAGCUGAUAUUAUUUCUUC 84 1515 GAAGAAAUAAUAUCAGCUU 374 1515
CUCUUAUCCUCUCUCUCUU 85 1515 CUCUUAUCCUCUCUCUCUU 85 1533
AAGAGAGAGAGGAUAAGAG 375 1533 UCUAGGCCCAUUGUCCUCC 86 1533
UCUAGGCCCAUUGUCCUCC 86 1551 GGAGGACAAUGGGCCUAGA 376 1551
CUUUUCACUUUAUUGCUAU 87 1551 CUUUUCACUUUAUUGCUAU 87 1569
AUAGCAAUAAAGUGAAAAG 377 1569 UCGCCCUCCUUUCCCUUAU 88 1569
UCGCCCUCCUUUCCCUUAU 88 1587 AUAAGGGAAAGGAGGGCGA 378 1587
UUGCCUCCCAGGCAAGCAG 89 1587 UUGCCUCCCAGGCAAGCAG 89 1605
CUGCUUGCCUGGGAGGCAA 379 1605 GCUGGUCAGUCUUUGCUCA 90 1605
GCUGGUCAGUCUUUGCUCA 90 1623 UGAGCAAAGACUGACCAGC 380 1623
AGUGUCCAGCUUCCAAAGC 91 1623 AGUGUCCAGCUUCCAAAGC 91 1641
GCUUUGGAAGCUGGACACU 381 1641 CCUAGACAACCUUUCUGUA 92 1641
CCUAGACAACCUUUCUGUA 92 1659 UACAGAAAGGUUGUCUAGG 382 1659
AGCCUAAAACGAAUGGUCU 93 1659 AGCCUAAAACGAAUGGUCU 93 1677
AGACCAUUCGUUUUAGGCU 383 1677 UUUGCUCCAGAUAACUCUC 94 1677
UUUGCUCCAGAUAACUCUC 94 1695 GAGAGUUAUCUGGAGCAAA 384 1695
CUUUCCUUGAGCUGUUGUG 95 1695 CUUUCCUUGAGCUGUUGUG 95 1713
CACAACAGCUCAAGGAAAG 385 1713 GAGCUUUGAAGUAGGUGGC 96 1713
GAGCUUUGAAGUAGGUGGC 96 1731 GCCACCUACUUCAAAGCUC 386 1731
CUUGAGCUAGAGAUAAAAC 97 1731 CUUGAGCUAGAGAUAAAAC 97 1749
GUUUUAUCUCUAGCUCAAG 387 1749 CAGAAUCUUCUGGGUAGUC 98 1749
CAGAAUCUUCUGGGUAGUC 98 1767 GACUACCCAGAAGAUUCUG 388 1767
CCCCUGUUGAUUAUCUUCA 99 1767 CCCCUGUUGAUUAUCUUCA 99 1785
UGAAGAUAAUCAACAGGGG 389 1785 AGCCCAGGCUUUUGCUAGA 100 1785
AGCCCAGGCUUUUGCUAGA 100 1803 UCUAGCAAAAGCCUGGGCU 390 1803
AUGGAAUGGAAAAGCAACU 101 1803 AUGGAAUGGAAAAGCAACU 101 1821
AGUUGCUUUUCCAUUCCAU 391 1821 UUCAUUUGACACAAAGCUU 102 1821
UUCAUUUGACACAAAGCUU 102 1839 AAGCUUUGUGUCAAAUGAA 392 1839
UCUAAAGCAGGUAAAUUGU 103 1839 UCUAAAGCAGGUAAAUUGU 103 1857
ACAAUUUACCUGCUUUAGA 393 1857 UCGGGGGAGAGAGUUAGCA 104 1857
UCGGGGGAGAGAGUUAGCA 104 1875 UGCUAACUCUCUCCCCCGA 394 1875
AUGUAUGAAUGUAAGGAUG 105 1875 AUGUAUGAAUGUAAGGAUG 105 1893
CAUCCUUACAUUCAUACAU 395 1893 GAGGGAAGCGAAGCAAGAG 106 1893
GAGGGAAGCGAAGCAAGAG 106 1911 CUCUUGCUUCGCUUCCCUC 396 1911
GGAACCUCUCGCCAUGAUC 107 1911 GGAACCUCUCGCCAUGAUC 107 1929
GAUCAUGGCGAGAGGUUCC 397 1929 CAGACAUACAGCUGCCUAC 108 1929
CAGACAUACAGCUGCCUAC 108 1947 GUAGGCAGCUGUAUGUCUG 398 1947
CCUAAUGAGGACUUCAAGC 109 1947 CCUAAUGAGGACUUCAAGC 109 1965
GCUUGAAGUCCUCAUUAGG 399 1965 CCCCACCACAUAGCAUGCU 110 1965
CCCCACCACAUAGCAUGCU 110 1983 AGCAUGCUAUGUGGUGGGG 400 1983
UUCCUUUCUCUCCUGGCUC 111 1983 UUCCUUUCUCUCCUGGCUC 111 2001
GAGCCAGGAGAGAAAGGAA 401 2001 CGGGGUAAAAAGUGGCUGC 112 2001
CGGGGUAAAAAGUGGCUGC 112 2019 GCAGCCACUUUUUACCCCG 402 2019
CGGUGUUUGGCAAUGCUAA 113 2019 CGGUGUUUGGCAAUGCUAA 113 2037
UUAGCAUUGCCAAACACCG 403 2037 AUUCAAUGCCGCAACAUAU 114 2037
AUUCAAUGCCGCAACAUAU 114 2055 AUAUGUUGCGGCAUUGAAU 404 2055
UAGUUGAGGCCGAGGAUAA 115 2055 UAGUUGAGGCCGAGGAUAA 115 2073
UUAUCCUCGGCCUCAACUA 405 2073 AAGAAAAGACAUUUUAAGU 116 2073
AAGAAAAGACAUUUUAAGU 116 2091 ACUUAAAAUGUCUUUUCUU 406 2091
UUUGUAGUAAAAGUGGUCU 117 2091 UUUGUAGUAAAAGUGGUCU 117 2109
AGACCACUUUUACUACAAA 407 2109 UCUGCUGGGGAAGGGUUUU 118 2109
UCUGCUGGGGAAGGGUUUU 118 2127 AAAACCCUUCCCCAGCAGA 408 2127
UCUUUUCUUUUUUUCUUUA 119 2127 UCUUUUCUUUUUUUCUUUA 119 2145
UAAAGAAAAAAAGAAAAGA 409 2145 AAUAACAAGGAGAUUUCUU 120 2145
AAUAACAAGGAGAUUUCUU 120 2163 AAGAAAUCUCCUUGUUAUU 410 2163
UAGUUCAUAUAUCAAGAAG 121 2163 UAGUUCAUAUAUCAAGAAG 121 2181
CUUCUUGAUAUAUGAACUA 411 2181 GUCUUGAAGUUGGGUGUUU 122 2181
GUCUUGAAGUUGGGUGUUU 122 2199 AAACACCCAACUUCAAGAC 412 2199
UCCAGAAUUGGUAAAAACA 123 2199 UCCAGAAUUGGUAAAAACA 123 2217
UGUUUUUACCAAUUCUGGA 413 2217 AGCAGCUCAUGGAAUUUUG 124 2217
AGCAGCUCAUGGAAUUUUG 124 2235 CAAAAUUCCAUGAGCUGCU 414 2235
GAGUAUUCCAUGAGCUGCU 125 2235 GAGUAUUCCAUGAGCUGCU 125 2253
AGCAGCUCAUGGAAUACUC 415 2253 UCAUUACAGUUCUUUCCUC 126 2253
UCAUUACAGUUCUUUCCUC 126 2271 GAGGAAAGAACUGUAAUGA 416 2271
CUUUCUGCUCUGCCAUCUU 127 2271 CUUUCUGCUCUGCCAUCUU 127 2289
AAGAUGGCAGAGCAGAAAG 417 2289 UCAGGAUAUUGGUUCUUCC 128 2289
UCAGGAUAUUGGUUCUUCC 128 2307 GGAAGAACCAAUAUCCUGA 418 2307
CCCUCAUAGUAAUAAGAUG 129 2307 CCCUCAUAGUAAUAAGAUG 129 2325
CAUCUUAUUACUAUGAGGG 419 2325 GGCUGUGGCAUUUCCAAAC 130 2325
GGCUGUGGCAUUUCCAAAC 130 2343 GUUUGGAAAUGCCACAGCC 420 2343
CAUCCAAAAAAAGGGAAGG 131 2343 CAUCCAAAAAAAGGGAAGG 131 2361
CCUUCCCUUUUUUUGGAUG 421 2361 GAUUUAAGGAGGUGAAGUC 132 2361
GAUUUAAGGAGGUGAAGUC 132 2379 GACUUCACCUCCUUAAAUC 422 2379
CGGGUCAAAAAUAAAAUAU 133 2379 CGGGUCAAAAAUAAAAUAU 133 2397
AUAUUUUAUUUUUGACCCG 423 2397 UAUAUACAUAUAUACAUUG 134 2397
UAUAUACAUAUAUACAUUG 134 2415 CAAUGUAUAUAUGUAUAUA 424 2415
GCUUAGAACGUUAAACUAU 135 2415 GCUUAGAACGUUAAACUAU 135 2433
AUAGUUUAACGUUCUAAGC 425 2433 UUAGAGUAUUUCCCUUCCA 136 2433
UUAGAGUAUUUCCCUUCCA 136 2451 UGGAAGGGAAAUACUCUAA 426 2451
AAAGAGGGAUGUUUGGAAA 137 2451 AAAGAGGGAUGUUUGGAAA 137 2469
UUUCCAAACAUCCCUCUUU 427 2469 AAAACUCUGAAGGAGAGGA 138 2469
AAAACUCUGAAGGAGAGGA 138 2487 UCCUCUCCUUCAGAGUUUU 428 2487
AGGAAUUAGUUGGGAUGCC 139 2487 AGGAAUUAGUUGGGAUGCC 139 2505
GGCAUCCCAACUAAUUCCU 429 2505 CAAUUUCCUCUCCACUGCU 140 2505
CAAUUUCCUCUCCACUGCU 140 2523 AGCAGUGGAGAGGAAAUUG 430 2523
UGGACAUGAGAUGGAGAGG 141 2523 UGGACAUGAGAUGGAGAGG 141 2541
CCUCUCCAUCUCAUGUCCA 431 2541 GCUGAGGGACAGGAUCUAU 142 2541
GCUGAGGGACAGGAUCUAU 142 2559 AUAGAUCCUGUCCCUCAGC 432 2559
UAGGCAGCUUCUAAGAGCG 143 2559 UAGGCAGCUUCUAAGAGCG 143 2577
CGCUCUUAGAAGCUGCCUA 433 2577 GAACUUCACAUAGGAAGGG 144 2577
GAACUUCACAUAGGAAGGG 144 2595 CCCUUCCUAUGUGAAGUUC 434 2595
GAUCUGAGAACACGUUGCC 145 2595 GAUCUGAGAACACGUUGCC 145 2613
GGCAACGUGUUCUCAGAUC 435 2613 CAGGGGCUUGAGAAGGUUA 146 2613
CAGGGGCUUGAGAAGGUUA 146 2631 UAACCUUCUCAAGCCCCUG 436 2631
ACUGAGUGAGUUAUUGGGA 147 2631 ACUGAGUGAGUUAUUGGGA 147 2649
UCCCAAUAACUCACUCAGU 437 2649 AGUCUUAAUAAAAUAAACU 148 2649
AGUCUUAAUAAAAUAAACU 148 2667 AGUUUAUUUUAUUAAGACU 438 2667
UAGAUAUUAGGUCCAUUCA 149 2667 UAGAUAUUAGGUCCAUUCA 149 2685
UGAAUGGACCUAAUAUCUA 439 2685 AUUAAUUAGUUCCAGUUUC 150 2685
AUUAAUUAGUUCCAGUUUC 150 2703 GAAACUGGAACUAAUUAAU 440 2703
CUCCUUGAAAUGAGUAAAA 151 2703 CUCCUUGAAAUGAGUAAAA 151 2721
UUUUACUCAUUUCAAGGAG 441 2721 AACUAGAAGGCUUCUCUCC 152 2721
AACUAGAAGGCUUCUCUCC 152 2739 GGAGAGAAGCCUUCUAGUU 442 2739
CACAGUGUUGUGCCCCUUC 153 2739 CACAGUGUUGUGCCCCUUC 153 2757
GAAGGGGCACAACACUGUG 443 2757 CACUCAUUUUUUUUUGAGG 154 2757
CACUCAUUUUUUUUUGAGG 154 2775 CCUCAAAAAAAAAUGAGUG 444 2775
GAGAAGGGGGUCUCUGUUA 155 2775 GAGAAGGGGGUCUCUGUUA 155 2793
UAACAGAGACCCCCUUCUC 445 2793 AACAUCUAGCCUAAAGUAU 156 2793
AACAUCUAGCCUAAAGUAU 156 2811 AUACUUUAGGCUAGAUGUU 446 2811
UACAACUGCCUGGGGGGCA 157 2811 UACAACUGCCUGGGGGGCA 157 2829
UGCCCCCCAGGCAGUUGUA 447 2829 AGGGUUAGGAAUCUCUUCA 158 2829
AGGGUUAGGAAUCUCUUCA 158 2847 UGAAGAGAUUCCUAACCCU 448 2847
ACUACCCUGAUUCUUGAUU 159 2847 ACUACCCUGAUUCUUGAUU 159 2865
AAUCAAGAAUCAGGGUAGU 449 2865 UCCUGGCUCUACCCUGUCU 160 2865
UCCUGGCUCUACCCUGUCU 160 2883 AGACAGGGUAGAGCCAGGA 450 2883
UGUCCCUUUUCUUUGACCA 161 2883 UGUCCCUUUUCUUUGACCA 161 2901
UGGUCAAAGAAAAGGGACA 451 2901 AGAUCUUUCUCUUCCCUGA 162 2901
AGAUCUUUCUCUUCCCUGA 162 2919 UCAGGGAAGAGAAAGAUCU 452 2919
AACGUUUUCUUCUUUCCCU 163 2919 AACGUUUUCUUCUUUCCCU 163 2937
AGGGAAAGAAGAAAACGUU 453 2937 UGGACAGGCAGCCUCCUUU 164 2937
UGGACAGGCAGCCUCCUUU 164 2955 AAAGGAGGCUGCCUGUCCA 454 2955
UGUGUGUAUUCAGAGGCAG 165 2955 UGUGUGUAUUCAGAGGCAG 165 2973
CUGCCUCUGAAUACACACA 455 2973 GUGAUGACUUGCUGUCCAG 166 2973
GUGAUGACUUGCUGUCCAG 166 2991 CUGGACAGCAAGUCAUCAC 456 2991
GGCAGCUCCCUCCUGCACA 167 2991 GGCAGCUCCCUCCUGCACA 167 3009
UGUGCAGGAGGGAGCUGCC 457 3009 ACAGAAUGCUCAGGGUCAC 168 3009
ACAGAAUGCUCAGGGUCAC 168 3027 GUGACCCUGAGCAUUCUGU 458 3027
CUGAACCACUGCUUCUCUU 169 3027 CUGAACCACUGCUUCUCUU 169 3045
AAGAGAAGCAGUGGUUCAG 459 3045 UUUGAAAGUAGAGCUAGCU 170 3045
UUUGAAAGUAGAGCUAGCU 170 3063 AGCUAGCUCUACUUUCAAA 460 3063
UGCCACUUUCACGUGGCCU 171 3063 UGCCACUUUCACGUGGCCU 171 3081
AGGCCACGUGAAAGUGGCA 461 3081 UCCGCAGUGUCUCCACCUA 172 3081
UCCGCAGUGUCUCCACCUA 172 3099 UAGGUGGAGACACUGCGGA 462 3099
ACACCCCUGUGCUCCCCUG 173 3099 ACACCCCUGUGCUCCCCUG 173 3117
CAGGGGAGCACAGGGGUGU 463 3117 GCCACACUGAUGGCUCAAG 174 3117
GCCACACUGAUGGCUCAAG 174 3135 CUUGAGCCAUCAGUGUGGC 464 3135
GACAAGGCUGGCAAACCCU 175 3135 GACAAGGCUGGCAAACCCU 175 3153
AGGGUUUGCCAGCCUUGUC 465 3153 UCCCAGAAACAUCUCUGGC 176 3153
UCCCAGAAACAUCUCUGGC 176 3171 GCCAGAGAUGUUUCUGGGA 466 3171
CCCAGAAAGCCUCUCUCUC 177 3171 CCCAGAAAGCCUCUCUCUC 177 3189
GAGAGAGAGGCUUUCUGGG 467 3189 CCCUCCCUCUCUCAUGAGG 178 3189
CCCUCCCUCUCUCAUGAGG 178 3207 CCUCAUGAGAGAGGGAGGG 468 3207
GCACAGCCAAGCCAAGCGC 179 3207 GCACAGCCAAGCCAAGCGC 179 3225
GCGCUUGGCUUGGCUGUGC 469 3225 CUCAUGUUGAGCCAGUGGG 180 3225
CUCAUGUUGAGCCAGUGGG 180 3243 CCCACUGGCUCAACAUGAG 470 3243
GCCAGCCACAGAGCAAAAG 181 3243 GCCAGCCACAGAGCAAAAG 181 3261
CUUUUGCUCUGUGGCUGGC 471 3261 GAGGGUUUAUUUUCAGUCC 182 3261
GAGGGUUUAUUUUCAGUCC 182 3279 GGACUGAAAAUAAACCCUC 472 3279
CCCUCUCUCUGGGUCAGAA 183 3279 CCCUCUCUCUGGGUCAGAA 183 3297
UUCUGACCCAGAGAGAGGG 473 3297 ACCAGAGGGCAUGCUGAAU 184 3297
ACCAGAGGGCAUGCUGAAU 184 3315 AUUCAGCAUGCCCUCUGGU 474 3315
UGCCCCCUGCUUACUUGGU 185 3315 UGCCCCCUGCUUACUUGGU 185 3333
ACCAAGUAAGCAGGGGGCA 475 3333 UGAGGGUGCCCCGCCUGAG 186 3333
UGAGGGUGCCCCGCCUGAG 186 3351 CUCAGGCGGGGCACCCUCA 476 3351
GUCAGUGCUCUCAGCUGGC 187 3351 GUCAGUGCUCUCAGCUGGC 187 3369
GCCAGCUGAGAGCACUGAC 477 3369 CAGUGCAAUGCUUGUAGAA 188 3369
CAGUGCAAUGCUUGUAGAA 188 3387 UUCUACAAGCAUUGCACUG 478 3387
AGUAGGAGGAAACAGUUCU 189 3387 AGUAGGAGGAAACAGUUCU 189 3405
AGAACUGUUUCCUCCUACU 479 3405 UCACUGGGAAGAAGCAAGG 190 3405
UCACUGGGAAGAAGCAAGG 190 3423 CCUUGCUUCUUCCCAGUGA 480 3423
GGCAAGAACCCAAGUGCCU 191 3423 GGCAAGAACCCAAGUGCCU 191 3441
AGGCACUUGGGUUCUUGCC 481 3441 UCACCUCGAAAGGAGGCCC 192 3441
UCACCUCGAAAGGAGGCCC 192 3459 GGGCCUCCUUUCGAGGUGA 482 3459
CUGUUCCCUGGAGUCAGGG 193 3459 CUGUUCCCUGGAGUCAGGG 193 3477
CCCUGACUCCAGGGAACAG 483 3477 GUGAACUGCAAAGCUUUGG 194 3477
GUGAACUGCAAAGCUUUGG 194 3495 CCAAAGCUUUGCAGUUCAC 484 3495
GCUGAGACCUGGGAUUUGA 195 3495 GCUGAGACCUGGGAUUUGA 195 3513
UCAAAUCCCAGGUCUCAGC 485 3513 AGAUACCACAAACCCUGCU 196 3513
AGAUACCACAAACCCUGCU 196 3531 AGCAGGGUUUGUGGUAUCU 486 3531
UGAACACAGUGUCUGUUCA 197 3531 UGAACACAGUGUCUGUUCA 197 3549
UGAACAGACACUGUGUUCA 487 3549 AGCAAACUAACCAGCAUUC 198 3549
AGCAAACUAACCAGCAUUC 198 3567 GAAUGCUGGUUAGUUUGCU 488 3567
CCCUACAGCCUAGGGCAGA 199 3567 CCCUACAGCCUAGGGCAGA 199 3585
UCUGCCCUAGGCUGUAGGG 489 3585 ACAAUAGUAUAGAAGUCUG 200 3585
ACAAUAGUAUAGAAGUCUG 200 3603 CAGACUUCUAUACUAUUGU 490 3603
GGAAAAAAACAAAAACAGA 201 3603 GGAAAAAAACAAAAACAGA 201 3621
UCUGUUUUUGUUUUUUUCC 491 3621 AAUUUGAGAACCUUGGACC 202 3621
AAUUUGAGAACCUUGGACC 202 3639 GGUCCAAGGUUCUCAAAUU 492 3639
CACUCCUGUCCCUGUAGCU 203 3639 CACUCCUGUCCCUGUAGCU 203 3657
AGCUACAGGGACAGGAGUG 493 3657 UCAGUCAUCAAAGCAGAAG 204 3657
UCAGUCAUCAAAGCAGAAG 204 3675 CUUCUGCUUUGAUGACUGA 494 3675
GUCUGGCUUUGCUCUAUUA 205 3675 GUCUGGCUUUGCUCUAUUA 205 3693
UAAUAGAGCAAAGCCAGAC 495 3693 AAGAUUGGAAAUGUACACU 206 3693
AAGAUUGGAAAUGUACACU 206 3711 AGUGUACAUUUCCAAUCUU 496 3711
UACCAAACACUCAGUCCAC 207 3711 UACCAAACACUCAGUCCAC 207 3729
GUGGACUGAGUGUUUGGUA 497 3729 CUGUUGAGCCCCAGUGCUG 208 3729
CUGUUGAGCCCCAGUGCUG 208 3747 CAGCACUGGGGCUCAACAG 498 3747
GGAAGGGAGGAAGGCCUUU 209 3747 GGAAGGGAGGAAGGCCUUU 209 3765
AAAGGCCUUCCUCCCUUCC 499 3765 UCUUCUGUGUUAAUUGCGU 210 3765
UCUUCUGUGUUAAUUGCGU 210 3783 ACGCAAUUAACACAGAAGA 500 3783
UAGAGGCUACAGGGGUUAG 211 3783 UAGAGGCUACAGGGGUUAG 211 3801
CUAACCCCUGUAGCCUCUA 501 3801 GCCUGGACUAAAGGCAUCC 212 3801
GCCUGGACUAAAGGCAUCC 212 3819 GGAUGCCUUUAGUCCAGGC 502 3819
CUUGUCUUUUGAGCUAUUC 213 3819 CUUGUCUUUUGAGCUAUUC 213 3837
GAAUAGCUCAAAAGACAAG 503 3837 CACCUCAGUAGAAAAGGAU 214 3837
CACCUCAGUAGAAAAGGAU 214 3855 AUCCUUUUCUACUGAGGUG 504 3855
UCUAAGGGAAGAUCACUGU 215 3855 UCUAAGGGAAGAUCACUGU 215 3873
ACAGUGAUCUUCCCUUAGA 505 3873 UAGUUUAGUUCUGUUGACC 216 3873
UAGUUUAGUUCUGUUGACC 216 3891 GGUCAACAGAACUAAACUA 506 3891
CUGUGCACCUACCCCUUGG 217 3891 CUGUGCACCUACCCCUUGG 217 3909
CCAAGGGGUAGGUGCACAG 507 3909 GAAAUGUCUGCUGGUAUUU 218 3909
GAAAUGUCUGCUGGUAUUU 218 3927 AAAUACCAGCAGACAUUUC 508 3927
UCUAAUUCCACAGGUCAUC 219 3927 UCUAAUUCCACAGGUCAUC 219 3945
GAUGACCUGUGGAAUUAGA 509 3945 CAGAUGCCUGCUUGAUAAU 220 3945
CAGAUGCCUGCUUGAUAAU 220 3963 AUUAUCAAGCAGGCAUCUG 510 3963
UAUAUAAACAAUAAAAACA 221 3963 UAUAUAAACAAUAAAAACA 221 3981
UGUUUUUAUUGUUUAUAUA 511 3981 AACUUUCACUUCUUCCUAU 222 3981
AACUUUCACUUCUUCCUAU 222 3999 AUAGGAAGAAGUGAAAGUU 512 3999
UUGUAAUCGUGUGCCAUGG 223 3999 UUGUAAUCGUGUGCCAUGG 223 4017
CCAUGGCACACGAUUACAA 513 4017 GAUCUGAUCUGUACCAUGA 224 4017
GAUCUGAUCUGUACCAUGA 224 4035 UCAUGGUACAGAUCAGAUC 514 4035
ACCCUACAUAAGGCUGGAU 225 4035 ACCCUACAUAAGGCUGGAU 225 4053
AUCCAGCCUUAUGUAGGGU 515 4053 UGGCACCUCAGGCUGAGGG 226 4053
UGGCACCUCAGGCUGAGGG 226 4071 CCCUCAGCCUGAGGUGCCA 516 4071
GCCCCAAUGUAUGUGUGGC 227 4071 GCCCCAAUGUAUGUGUGGC 227 4089
GCCACACAUACAUUGGGGC 517 4089 CUGUGGGUGUGGGUGGGAG 228 4089
CUGUGGGUGUGGGUGGGAG 228 4107 CUCCCACCCACACCCACAG 518 4107
GUGUGUCUGCUGAGUAAGG 229 4107 GUGUGUCUGCUGAGUAAGG 229 4125
CCUUACUCAGCAGACACAC 519 4125 GAACACGAUUUUCAAGAUU 230 4125
GAACACGAUUUUCAAGAUU 230 4143 AAUCUUGAAAAUCGUGUUC 520 4143
UCUAAAGCUCAAUUCAAGU 231 4143 UCUAAAGCUCAAUUCAAGU 231 4161
ACUUGAAUUGAGCUUUAGA 521 4161 UGACACAUUAAUGAUAAAC 232 4161
UGACACAUUAAUGAUAAAC 232 4179 GUUUAUCAUUAAUGUGUCA 522 4179
CUCAGAUCUGAUCAAGAGU 233 4179 CUCAGAUCUGAUCAAGAGU 233 4197
ACUCUUGAUCAGAUCUGAG 523 4197 UCCGGAUUUCUAACAGUCC 234 4197
UCCGGAUUUCUAACAGUCC 234 4215 GGACUGUUAGAAAUCCGGA 524 4215
CCUGCUUUGGGGGGUGUGC 235 4215 CCUGCUUUGGGGGGUGUGC 235 4233
GCACACCCCCCAAAGCAGG 525 4233 CUGACAACUUAGCUCAGGU 236 4233
CUGACAACUUAGCUCAGGU 236 4251 ACCUGAGCUAAGUUGUCAG 526 4251
UGCCUUACAUCUUUUCUAA 237 4251 UGCCUUACAUCUUUUCUAA 237 4269
UUAGAAAAGAUGUAAGGCA 527 4269 AUCACAGUGUUGCAUAUGA 238 4269
AUCACAGUGUUGCAUAUGA 238 4287 UCAUAUGCAACACUGUGAU 528 4287
AGCCUGCCCUCACUCCCUC 239 4287 AGCCUGCCCUCACUCCCUC 239 4305
GAGGGAGUGAGGGCAGGCU 529 4305 CUGCAGAAUCCCUUUGCAC 240 4305
CUGCAGAAUCCCUUUGCAC 240 4323 GUGCAAAGGGAUUCUGCAG 530 4323
CCUGAGACCCUACUGAAGU 241 4323 CCUGAGACCCUACUGAAGU 241 4341
ACUUCAGUAGGGUCUCAGG 531 4341 UGGCUGGUAGAAAAAGGGG 242 4341
UGGCUGGUAGAAAAAGGGG 242 4359 CCCCUUUUUCUACCAGCCA 532 4359
GCCUGAGUGGAGGAUUAUC 243 4359 GCCUGAGUGGAGGAUUAUC 243 4377
GAUAAUCCUCCACUCAGGC 533 4377 CAGUAUCACGAUUUGCAGG 244 4377
CAGUAUCACGAUUUGCAGG 244 4395 CCUGCAAAUCGUGAUACUG 534 4395
GAUUCCCUUCUGGGCUUCA 245 4395 GAUUCCCUUCUGGGCUUCA 245 4413
UGAAGCCCAGAAGGGAAUC 535 4413 AUUCUGGAAACUUUUGUUA 246 4413
AUUCUGGAAACUUUUGUUA 246 4431 UAACAAAAGUUUCCAGAAU 536 4431
AGGGCUGCUUUUCUUAAGU 247 4431 AGGGCUGCUUUUCUUAAGU 247 4449
ACUUAAGAAAAGCAGCCCU 537 4449 UGCCCACAUUUGAUGGAGG 248 4449
UGCCCACAUUUGAUGGAGG 248 4467 CCUCCAUCAAAUGUGGGCA 538 4467
GGUGGAAAUAAUUUGAAUG 249 4467 GGUGGAAAUAAUUUGAAUG 249 4485
CAUUCAAAUUAUUUCCACC 539 4485 GUAUUUGAUUUAUAAGUUU 250 4485
GUAUUUGAUUUAUAAGUUU 250 4503 AAACUUAUAAAUCAAAUAC 540 4503
UUUUUUUUUUUUUGGGUUA 251 4503 UUUUUUUUUUUUUGGGUUA 251 4521
UAACCCAAAAAAAAAAAAA 541 4521 AAAAGAUGGUUGUAGCAUU 252 4521
AAAAGAUGGUUGUAGCAUU 252 4539 AAUGCUACAACCAUCUUUU 542 4539
UUAAAAUGGAAAAUUUUCU 253 4539 UUAAAAUGGAAAAUUUUCU 253 4557
AGAAAAUUUUCCAUUUUAA 543 4557 UCCUUGGUUUGCUAGUAUC 254 4557
UCCUUGGUUUGCUAGUAUC 254 4575 GAUACUAGCAAACCAAGGA 544 4575
CUUGGGUGUAUUCUCUGUA 255 4575 CUUGGGUGUAUUCUCUGUA 255 4593
UACAGAGAAUACACCCAAG 545 4593 AAGUGUAGCUCAAAUAGGU 256 4593
AAGUGUAGCUCAAAUAGGU 256 4611 ACCUAUUUGAGCUACACUU 546 4611
UCAUCAUGAAAGGUUAAAA 257 4611 UCAUCAUGAAAGGUUAAAA 257 4629
UUUUAACCUUUCAUGAUGA 547 4629 AAAGCGAGGUGGCCAUGUU 258 4629
AAAGCGAGGUGGCCAUGUU 258 4647 AACAUGGCCACCUCGCUUU 548 4647
UAUGCUGGUGGUUAAGGCC 259 4647 UAUGCUGGUGGUUAAGGCC 259 4665
GGCCUUAACCACCAGCAUA 549 4665 CAGGGCCUCUCCAACCACU 260 4665
CAGGGCCUCUCCAACCACU 260 4683 AGUGGUUGGAGAGGCCCUG 550 4683
UGUGCCACUGACUUGCUGU 261 4683 UGUGCCACUGACUUGCUGU 261 4701
ACAGCAAGUCAGUGGCACA 551 4701 UGUGACCCUGGGCAAGUCA 262 4701
UGUGACCCUGGGCAAGUCA 262 4719 UGACUUGCCCAGGGUCACA 552 4719
ACUUAACUAUAAGGUGCCU 263 4719 ACUUAACUAUAAGGUGCCU 263 4737
AGGCACCUUAUAGUUAAGU 553 4737 UCAGUUUUCCUUCUGUUAA 264 4737
UCAGUUUUCCUUCUGUUAA 264 4755 UUAACAGAAGGAAAACUGA 554 4755
AAAUGGGGAUAAUAAUACU 265 4755 AAAUGGGGAUAAUAAUACU 265 4773
AGUAUUAUUAUCCCCAUUU 555 4773 UGACCUACCUCAAAGGGCA 266 4773
UGACCUACCUCAAAGGGCA 266 4791 UGCCCUUUGAGGUAGGUCA 556 4791
AGUUUUGAGGCAUGACUAA 267 4791 AGUUUUGAGGCAUGACUAA 267 4809
UUAGUCAUGCCUCAAAACU 557 4809 AUGCUUUUUAGAAAGCAUU 268 4809
AUGCUUUUUAGAAAGCAUU 268 4827 AAUGCUUUCUAAAAAGCAU 558 4827
UUUGGGAUCCUUCAGCACA 269 4827 UUUGGGAUCCUUCAGCACA 269 4845
UGUGCUGAAGGAUCCCAAA 559 4845 AGGAAUUCUCAAGACCUGA 270 4845
AGGAAUUCUCAAGACCUGA 270 4863 UCAGGUCUUGAGAAUUCCU 560 4863
AGUAUUUUUUAUAAUAGGA 271 4863 AGUAUUUUUUAUAAUAGGA 271 4881
UCCUAUUAUAAAAAAUACU 561 4881 AAUGUCCACCAUGAACUUG 272 4881
AAUGUCCACCAUGAACUUG 272 4899 CAAGUUCAUGGUGGACAUU 562 4899
GAUACGUCCGUGUGUCCCA 273 4899 GAUACGUCCGUGUGUCCCA 273 4917
UGGGACACACGGACGUAUC 563 4917 AGAUGCUGUCAUUAGUCUA 274 4917
AGAUGCUGUCAUUAGUCUA 274 4935 UAGACUAAUGACAGCAUCU 564 4935
AUAUGGUUCUCCAAGAAAC 275 4935 AUAUGGUUCUCCAAGAAAC 275 4953
GUUUCUUGGAGAACCAUAU 565 4953 CUGAAUGAAUCCAUUGGAG 276 4953
CUGAAUGAAUCCAUUGGAG 276 4971 CUCCAAUGGAUUCAUUCAG 566 4971
GAAGCGGUGGAUAACUAGC 277 4971 GAAGCGGUGGAUAACUAGC 277 4989
GCUAGUUAUCCACCGCUUC 567 4989 CCAGACAAAAUUUGAGAAU 278 4989
CCAGACAAAAUUUGAGAAU 278 5007 AUUCUCAAAUUUUGUCUGG 568 5007
UACAUAAACAACGCAUUGC 279 5007 UACAUAAACAACGCAUUGC 279 5025
GCAAUGCGUUGUUUAUGUA 569 5025 CCACGGAAACAUACAGAGG 280 5025
CCACGGAAACAUACAGAGG 280 5043 CCUCUGUAUGUUUCCGUGG 570 5043
GAUGCCUUUUCUGUGAUUG 281 5043 GAUGCCUUUUCUGUGAUUG 281 5061
CAAUCACAGAAAAGGCAUC 571 5061 GGGUGGGAUUUUUUCCCUU 282 5061
GGGUGGGAUUUUUUCCCUU 282 5079 AAGGGAAAAAAUCCCACCC 572 5079
UUUUAUGUGGGAUAUAGUA 283 5079 UUUUAUGUGGGAUAUAGUA 283 5097
UACUAUAUCCCACAUAAAA 573 5097 AGUUACUUGUGACAAAAAU 284 5097
AGUUACUUGUGACAAAAAU 284 5115 AUUUUUGUCACAAGUAACU 574 5115
UAAUUUUGGAAUAAUUUCU 285 5115 UAAUUUUGGAAUAAUUUCU 285 5133
AGAAAUUAUUCCAAAAUUA 575 5133 UAUUAAUAUCAACUCUGAA 286 5133
UAUUAAUAUCAACUCUGAA 286 5151 UUCAGAGUUGAUAUUAAUA 576 5151
AGCUAAUUGUACUAAUCUG 287 5151 AGCUAAUUGUACUAAUCUG 287 5169
CAGAUUAGUACAAUUAGCU 577 5169 GAGAUUGUGUUUGUUCAUA 288 5169
GAGAUUGUGUUUGUUCAUA 288 5187 UAUGAACAAACACAAUCUC 578 5187
AAUAAAAGUGAAGUGAAUC 289 5187 AAUAAAAGUGAAGUGAAUC 289 5205
GAUUCACUUCACUUUUAUU 579 5201 GAAUCUAAAAAAAAAAAAA 290 5201
GAAUCUAAAAAAAAAAAAA 290 5219 UUUUUUUUUUUUUAGAUUC 580 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 Figures 4 and 5, or having modifications
described in Table IV or any combination thereof.
[0432]
3TABLE III SCD Synthetic Modified siNA Constructs Target Seq Seq
Pos Target ID Cmpd# Aliases Sequence ID 993 GAUAUGCUGUGGUGCUUAAUGCC
581 31021 SCD:995U21 sense siNA UAUGCUGUGGUGCUUAAUGTT 597 2518
ACUGCUGGACAUGAGAUGGAGAG 582 31022 SCD:2520U21 sense siNA
UGCUGGACAUGAGAUGGAGTT 598 3783 UAGAGGCUACAGGGGUUAGCCUG 583 31023
SCD:3785U21 sense siNA GAGGCUACAGGGGUUAGCCTT 599 4772
CUGACCUACCUCAAAGGGCAGUU 584 31024 SCD:4774U21 sense siNA
GACCUACCUCAAAGGGCAGTT 600 660 ACACAAUGGCAUUCCAGAAUGAU 585 31635
SCD:662U21 sense siNA ACAAUGGCAUUCCAGAAUGTT 601 2879
UGUCUGUCCCUUUUCUUUGACCA 586 31636 SCD:2881U21 sense siNA
UCUGUCCCUUUUCUUUGACTT 602 3557 AACCAGCAUUCCCUACAGCCUAG 587 31637
SCD:3559U21 sense siNA CCAGCAUUCCCUACAGCCUTT 603 4970
AGAAGCGGUGGAUAACUAGCCAG 588 31638 SCD:4972U21 sense siNA
AAGCGGUGGAUAACUAGCCTT 604 993 GAUAUGCUGUGGUGCUUAAUGCC 581 31097
SCD:1013L21 antisense siNA CAUUAAGCACCACAGCAUATT 605 (995C) 2518
ACUGCUGGACAUGAGAUGGAGAG 582 31098 SCD:2538L21 antisense siNA
CUCCAUCUCAUGUCCAGCATT 606 (2520C) 3783 UAGAGGCUACAGGGGUUAGCCUG 583
31099 SCD:3803L21 antisense siNA GGCUAACCCCUGUAGCCUCTT 607 (3785C)
4772 CUGACCUACCUCAAAGGGCAGUU 584 31100 SCD:4792L21 antisense siNA
CUGCCCUUUGAGGUAGGUCTT 608 (4774C) 660 ACACAAUGGCAUUCCAGAAUGAU 585
31639 SCD:680L21 antisense siNA CAUUCUGGAAUGCCAUUGUTT 609 (662C)
2879 UGUCUGUCCCUUUUCUUUGACCA 586 31640 SCD:2899L21 antisense siNA
GUCAAAGAAAAGGGACAGATT 610 (2881C) 3557 AACCAGCAUUCCCUACAGCCUAG 587
31641 SCD:3577L21 antisense siNA AGGCUGUAGGGAAUGCUGGTT 611 (3559C)
4970 AGAAGCGGUGGAUAACUAGCCAG 588 31642 SCD:4990L21 antisense siNA
GGCUAGUUAUCCACCGCUUTT 612 (4972C) 993 GAUAUGCUGUGGUGCUUAAUGCC 581
30873 SCD:995U21 sense siNA B uAuGcuGuGGuGcuuAAuGTT B 613 stab04
2518 ACUGCUGGACAUGAGAUGGAGAG 582 30874 SCD:2520U21 sense siNA B
uGcuGGAcAuGAGAuGGAGTT B 614 stab04 3783 UAGAGGCUACAGGGGUUAGCCUG 583
30875 SCD:3785U21 sense siNA B GAGGcuAcAGGGGuuAGccTT B 615 stab04
4772 CUGACCUACCUCAAAGGGCAGUU 584 30876 SCD:4774U21 sense siNA B
GAccuAccucAAAGGGcAGTT B 616 stab04 660 ACACAAUGGCAUUCCAGAAUGAU 585
SCD:662U21 sense siNA B AcAAuGGcAuuccAGAAuGTT B 617 stab04 2879
UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2881U21 sense siNA B
ucuGucccuuuucuuuGAcTT B 618 stab04 3557 AACCAGCAUUCCCUACAGCCUAG 587
SCD:3559U21 sense siNA stab04 B ccAGcAuucccuAcAGccuTT B 619 4970
AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4972U21 sense siNA stab04 B
AAGcGGuGGAuAAcuAGccTT B 620 993 GAUAUGCUGUGGUGCUUAAUGCC 581 30877
SCD:1013L21 antisense siNA cAuuAAGcAccAcAGcAuATsT 621 (995C) stab05
2518 ACUGCUGGACAUGAGAUGGAGAG 582 30878 SCD:2538L21 antisense siNA
cuccAucucAuGuccAGcATsT 622 (2520C) stab05 3783
UAGAGGCUACAGGGGUUAGCCUG 583 30879 SCD:3803L21 antisense siNA
GGcuAAccccuGuAGccucTsT 623 (3785C) stab05 4772
CUGACCUACCUCAAAGGGCAGUU 584 30880 SCD:4792L21 antisense siNA
cuGcccuuuGAGGuAGGucTsT 624 (4774C) stab05 660
ACACAAUGGCAUUCCAGAAUGAU 585 SCD:680L21 antisense siNA
cAuucuGGAAuGccAuuGuTsT 625 (662C) stab05 2879
UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNA
GucAAAGAAAAGGGAcAGATsT 626 (2881C) stab05 3557
AACCAGCAUUCCCUACAGCCUAG 587 SCD:3577L21 antisense siNA
AGGcuGuAGGGAAuGcuGGTsT 627 (3559C) stab05 4970
AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNA
GGcuAGuuAuccAccGcuuTsT 628 (4972C) stab05 993
GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:995U21 sense siNA stab07 B
uAuGcuGuGGuGcuuAAuGTT B 629 2518 ACUGCUGGACAUGAGAUGGAGAG 582
SCD:2520U21 sense siNA stab07 B uGcuGGAcAuGAGAuGGAGTT B 630 3783
UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3785U21 sense siNA stab07 B
GAGGcuAcAGGGGuuAGccTT B 631 4772 CUGACCUACCUCAAAGGGCAGUU 584
SCD:4774U21 sense siNA stab07 B GAccuAccucAAAGGGcAGTT B 632 660
ACACAAUGGCAUUCCAGAAUGAU 585 SCD:662U21 sense siNA stab07 B
AcAAuGGcAuuccAGAAuGTT B 633 2879 UGUCUGUCCCUUUUCUUUGACCA 586
SCD:2881U21 sense siNA stab07 B ucuGucccuuuucuuuGAcTT B 634 3557
AACCAGCAUUCCCUACAGCCUAG 587 31871 SCD:3559U21 sense siNA stab07 B
ccAGcAuucccuAcAGccuTT B 635 4970 AGAAGCGGUGGAUAACUAGCCAG 588
SCD:4972U21 sense siNA stab07 B AAGcGGuGGAuAAcuAGccTT B 636 993
GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:1013L21 antisense siNA
cAuuAAGcAccAcAGcAuATsT 637 (995C) stab11 2518
ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNA
cuccAucucAuGuccAGcATsT 638 (2520C) stab11 3783
UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3803L21 antisense siNA
GGcuAAccccuGuAGccucTsT 639 (3785C) stab11 4772
CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNA
cuGcccuuuGAGGuAGGucTsT 640 (4774C) stab11 660
ACACAAUGGCAUUCCAGAAUGAU 585 SCD:680L21 antisense siNA
cAuucuGGAAuGccAuuGuTsT 641 (662C) stab11 2879
UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNA
GucAAAGAAAAGGGAcAGATsT 642 (2881C) stab11 3557
AACCAGCAUUCCCUACAGCCUAG 587 SCD:3577L21 antisense siNA
AGGcuGuAGGGAAuGcuGGTsT 643 (3559C) stab11 4970
AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNA
GGcuAGuuAuccAccGcuuTsT 644 (4972C) stab11 993
GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:995U21 sense siNA stab18 B
uAuGcuGuGGuGcuuAAuGTT B 645 2518 ACUGCUGGACAUGAGAUGGAGAG 582
SCD:2520U21 sense siNA stab18 B uGcuGGAcAuGAGAuGGAGTT B 646 3783
UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3785U21 sense siNA stab18 B
GAGGcuAcAGGGGuuAGccTT B 647 4772 CUGACCUACCUCAAAGGGCAGUU 584
SCD:4774U21 sense siNA stab18 B GAccuAccucAAAGGGcAGTT B 648 660
ACACAAUGGCAUUCCAGAAUGAU 585 SCD:662U21 sense siNA stab18 B
AcAAuGGcAuuccAGAAuGTT B 649 2879 UGUCUGUCCCUUUUCUUUGACCA 586
SCD:2881U21 sense siNA stab18 B ucuGucccuuuucuuuGAcTT B 650 3557
AACCAGCAUUCCCUACAGCCUAG 587 SCD:3559U21 sense siNA stab18 B
ccAGcAuucccuAcAGccuTT B 651 4970 AGAAGCGGUGGAUAACUAGCCAG 588
SCD:4972U21 sense siNA stab18 B AAGcGGuGGAuAAcuAGccTT B 652 993
GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:1013L21 antisense siNA
cAuuAAGcAccAcAGcAuATsT 653 (995C) stab08 2518
ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNA
cuccAucucAuGuccAGcATsT 654 (2520C) stab08 3783
UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3803L21 antisense siNA
GGcuAAccccuGuAGccucTsT 655 (3785C) stab08 4772
CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNA
cuGcccuuuGAGGuAGGucTsT 656 (4774C) stab08 660
ACACAAUGGCAUUCCAGAAUGAU 585 SCD:680L21 antisense siNA
cAuucuGGAAuGccAuuGuTsT 657 (662C) stab08 2879
UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNA
GucAAAGAAAAGGGAcAGATsT 658 (2881C) stab08 3557
AACCAGCAUUCCCUACAGCCUAG 587 31877 SCD:3577L21 antisense siNA
AGGcuGuAGGGAAuGcuGGTsT 659 (3559C) stab08 4970
AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNA
GGcuAGuuAuccAccGcuuTsT 660 (4972C) stab08 993
GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:995U21 sense siNA stab09 B
UAUGCUGUGGUGCUUAAUGTT B 661 2518 ACUGCUGGACAUGAGAUGGAGAG 582
SCD:2520U21 sense siNA stab09 B UGCUGGACAUGAGAUGGAGTT B 662 3783
UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3785U21 sense siNA stab09 B
GAGGCUACAGGGGUUAGCCTT B 663 4772 CUGACCUACCUCAAAGGGCAGUU 584
SCD:4774U21 sense siNA stab09 B GACCUACCUCAAAGGGCAGTT B 664 660
ACACAAUGGCAUUCCAGAAUGAU 585 SCD:662U21 sense siNA stab09 B
ACAAUGGCAUUCCAGAAUGTT B 665 2879 UGUCUGUCCCUUUUCUUUGACCA 586
SCD:2881U21 sense siNA stab09 B UCUGUCCCUUUUCUUUGACTT B 666 3557
AACCAGCAUUCCCUACAGCCUAG 587 SCD:3559U21 sense siNA stab09 B
CCAGCAUUCCCUACAGCCUTT B 667 4970 AGAAGCGGUGGAUAACUAGCCAG 588
SCD:4972U21 sense siNA stab09 B AAGCGGUGGAUAACUAGCCTT B 668 993
GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:1013L21 antisense siNA
CAUUAAGCACCACAGCAUATsT 669 (995C) stab10 2518
ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNA
CUCCAUCUCAUGUCCAGCATsT 670 (2520C) stab10 3783
UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3803L21 antisense siNA
GGCUAACCCCUGUAGCCUCTsT 671 (3785C) stab10 4772
CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNA
CUGCCCUUUGAGGUAGGUCTsT 672 (4774C) stab10 660
ACACAAUGGCAUUCCAGAAUGAU 585 SCD:680L21 antisense siNA
CAUUCUGGAAUGCCAUUGUTsT 673 (662C) stab10 2879
UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNA
GUCAAAGAAAAGGGACAGATsT 674 (2881C) stab10 3557
AACCAGCAUUCCCUACAGCCUAG 587 SCD:3577L21 antisense siNA
AGGCUGUAGGGAAUGCUGGTsT 675 (3559C) stab10 4970
AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNA
GGCUAGUUAUCCACCGCUUTsT 676 (4972C) stab10 993
GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:1013L21 antisense siNA
cAuuAAGcAccAcAGcAuATT B 677 (995C) stab19 2518
ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNA
cuccAucucAuGuccAGcATT B 678 (2520C) stab19 3783
UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3803L21 antisense siNA
GGcuAAccccuGuAGccucTT B 679 (37850) stab19 4772
CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNA
cuGcccuuuGAGGuAGGucTT B 680 (4774C) stab19 660
ACACAAUGGCAUUCCAGAAUGAU 585 SCD:680L21 antisense siNA
cAuucuGGAAuGccAuuGuTT B 681 (662C) stab19 2879
UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNA
GucAAAGAAAAGGGAcAGATT B 682 (2881C) stab19 3557
AACCAGCAUUCCCUACAGCCUAG 587 SCD:3577L21 antisense siNA
AGGcuGuAGGGAAuGcuGGTT B 683 (3559C) stab19 4970
AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNA
GGcuAGuuAuccAccGcuuTT B 684 (4972C) stab19 993
GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:1013L21 antisense siNA
CAUUAAGCACCACAGCAUATT B 685 (9950) stab22 2518
ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNA
CUCCAUCUCAUGUCCAGCATT B 686 (2520C) stab22 3783
UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3803L21 antisense siNA
GGCUAACCCCUGUAGCCUCTT B 687 (3785C) stab22 4772
CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNA
CUGCCCUUUGAGGUAGGUCU B 688 (4774C) stab22 660
ACACAAUGGCAUUCCAGAAUGAU 585 SCD:680L21 antisense siNA
CAUUCUGGAAUGCCAUUGUTT B 689 (662C) stab22 2879
UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNA
GUCAAAGAAAAGGGACAGATT B 690 (2881C) stab22 3557
AACCAGCAUUCCCUACAGCCUAG 587 SCD:3577L21 antisense siNA
AGGCUGUAGGGAAUGCUGGTT B 691 (3559C) stab22 4970
AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNA
GGCUAGUUAUCCACCGCUUTT B 692 (4972C) stab22 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:647U21 sense siNA
CUGAUCAUUGCCAACACAATT 693 1192 CACCACAUUCUUCAUUGAUUGCA 590
SCD:1194U21 sense siNA CCACAUUCUUCAUUGAUUGTT 694 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2025U21 sense siNA
UUGGCAAUGCUAAUUCAAUTT 695 2728 AGGCUUCUCUCCACAGUGUUGUG 592
SCD:2730U21 sense siNA GCUUCUCUCCACAGUGUUGTT 696 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3556U21 sense siNA
UAACCAGCAUUCCCUACAGTT 697 4316 CUUUGCACCUGAGACCCUACUGA 594
SCD:4318U21 sense siNA UUGCACCUGAGACCCUACUTT 698 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4320U21 sense siNA
GCACCUGAGACCCUACUGATT 699 4775 ACCUACCUCAAAGGGCAGUUUUG 596
SCD:4777U21 sense siNA CUACCUCAAAGGGCAGUUUTT 700 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21 antisense siNA
UUGUGUUGGCAAUGAUCAGTT 701 (647C) 1192 CACCACAUUCUUCAUUGAUUGCA 590
SCD:1212L21 antisense siNA CAAUCAAUGAAGAAUGUGGTT 702 (1194C) 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2043L21 antisense siNA
AUUGAAUUAGCAUUGCCAATT 703 (2025C) 2728 AGGCUUCUCUCCACAGUGUUGUG 592
SCD:2748L21 antisense siNA CAACACUGUGGAGAGAAGCTT 704 (2730C) 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3574L21 antisense siNA
CUGUAGGGAAUGCUGGUUATT 705 (3556C) 4316 CUUUGCACCUGAGACCCUACUGA 594
SCD:4336L21 antisense siNA AGUAGGGUCUCAGGUGCAATT 706 (4318C) 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4338L21 antisense siNA
UCAGUAGGGUCUCAGGUGCTT 707 (4320C) 4775 ACCUACCUCAAAGGGCAGUUUUG 596
SCD:4795L21 antisense siNA AAACUGCCCUUUGAGGUAGTT 708 (4777C) 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:647U21 sense siNA stab04 B
cuGAucAuuGccAAcAcAATT B 709 1192 CACCACAUUCUUCAUUGAUUGCA 590
SCD:1194U21 sense siNA stab04 B ccAcAuucuucAuuGAuuGTT B 710 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2025U21 sense siNA stab04 B
uuGGcAAuGcuAAuucAAuTT B 711 2728 AGGCUUCUCUCCACAGUGUUGUG 592
SCD:2730U21 sense siNA stab04 B GcuucucuccAcAGuGuuGTT B 712 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3556U21 sense siNA stab04 B
uAAccAGcAuucccuAcAGTT B 713 4316 CUUUGCACCUGAGACCCUACUGA 594
SCD:4318U21 sense siNA stab04 B uuGcAccuGAGAcccuAcuTT B 714 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4320U21 sense siNA stab04 B
GCAccuGAGAcccuAcuGATT B 715 4775 ACCUACCUCAAAGGGCAGUUUUG 596
SCD:4777U21 sense siNA stab04 B cuAccucAAAGGGcAGuuuTT B 716 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21 antisense siNA
uuGuGuuGGcAAuGAucAGTsT 717 (647C) stab05 1192
CACCACAUUCUUCAUUGAUUGCA 590 SCD:1212L21 antisense siNA
cAAucAAuGAAGAAuGuGGTsT 718 (1194C) stab05 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2043L21 antisense siNA
AuuGAAuuAGcAuuGccAATsT 719 (2025C) stab05 2728
AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNA
cAAcAcuGuGGAGAGAAGcTsT 720 (2730C) stab05 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3574L21 antisense siNA
cuGuAGGGAAuGcuGGuuATsT 721 (3556C) stab05 4316
CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNA
AGuAGGGucucAGGuGcAATsT 722 (4318C) stab05 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4338L21 antisense siNA
ucAGuAGGGucucAGGuGcTsT 723 (4320C) stab05 4775
ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNA
AAAcuGcccuuuGAGGuAGTsT 724 (4777C) stab05 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:647U21 sense siNA stab07 B
cuGAucAuuGccAAcAcAATT B 725 1192 CACCACAUUCUUCAUUGAUUGCA 590
SCD:1194U21 sense siNA stab07 B ccAcAuucuucAuuGAuuGTT B 726 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2025U21 sense siNA stab07 B
uuGGcAAuGcuAAuucAAuTT B 727 2728 AGGCUUCUCUCCACAGUGUUGUG 592
SCD:2730U21 sense siNA stab07 B GcuucucuccAcAGuGuuGTT B 728 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3556U21 sense siNA stab07 B
uAAccAGcAuucccuAcAGTT B 729 4316 CUUUGCACCUGAGACCCUACUGA 594
SCD:4318U21 sense siNA stab07 B uuGcAccuGAGAcccuAcuTT B 730 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4320U21 sense siNA stab07 B
GcAccuGAGAcccuAcuGATT B 731 4775 ACCUACCUCAAAGGGCAGUUUUG 596
SCD:4777U21 sense siNA stab07 B cuAccucAAAGGGcAGuuuTT B 732 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21 antisense siNA
uuGuGuuGGcAAuGAucAGTsT 733 (647C) stab11 1192
CACCACAUUCUUCAUUGAUUGCA 590 SCD:1212L21 antisense siNA
cAAucAAuGAAGAAuGuGGTsT 734 (1194C) stab11 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2043L21 antisense siNA
AuuGAAuuAGcAuuGccAATsT 735 (2025C) stab11 2728
AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNA
cAAcAcuGuGGAGAGAAGcTsT 736 (2730C) stab11 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3574L21 antisense siNA
cuGuAGGGAAuGcuGGuuATsT 737 (3556C) stab11 4316
CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNA
AGuAGGGucucAGGuGcAATsT 738 (4318C) stab11 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4338L21 antisense siNA
ucAGuAGGGucucAGGuGcTsT 739 (4320C) stab11 4775
ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNA
AAAcuGcccuuuGAGGuAGTsT 740 (4777C) stab11 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:647U21 sense siNA B
cuGAucAuuGccAAcAcAATT B 741 stab18 1192 CACCACAUUCUUCAUUGAUUGCA 590
SCD:1194U21 sense siNA stab18 B ccAcAuucuucAuuGAuuGTT B 742 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2025U21 sense siNA stab18 B
uuGGcAAuGcuAAuucAAuTT B 743 2728 AGGCUUCUCUCCACAGUGUUGUG 592
SCD:2730U21 sense siNA stab18 B GcuucucuccAcAGuGuuGTT B 744 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3556U21 sense siNA stab18 B
uAAccAGcAuucccuAcAGTT B 745 4316 CUUUGCACCUGAGACCCUACUGA 594
SCD:4318U21 sense siNA stab18 B uuGcAccuGAGAcccuAcuTT B 746 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4320U21 sense siNA stab18 B
GcAccuGAGAcccuAcuGATT B 747 4775 ACCUACCUCAAAGGGCAGUUUUG 596
SCD:4777U21 sense siNA stab18 B cuAccucAAAGGGcAGuuuTT B 748 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21 antisense siNA
uuGuGuuGGcAAuGAucAGTsT 749 (647C) stab08 1192
CACCACAUUCUUCAUUGAUUGCA 590 SCD:1212L21 antisense siNA
cAAucAAuGAAGAAuGuGGTsT 750 (1194C) stab08 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2043L21 antisense siNA
AuuGAAuuAGcAuuGccAATsT 751 (2025C) stab08 2728
AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNA
cAAcAcuGuGGAGAGAAGcTsT 752 (2730C) stab08 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3574L21 antisense siNA
cuGuAGGGAAuGcuGGuuATsT 753 (3556C) stab08 4316
CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNA
AGuAGGGucucAGGuGcAATsT 754 (4318C) stab08 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4338L21 antisense siNA
ucAGuAGGGucucAGGuGcTsT 755 (4320C) stab08 4775
ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNA
AAAcuGcccuuuGAGGuAGTsT 756 (4777C) stab08 645
UUCUGAUCAUUGCCAACACAAUG 589 37108 SCD:647U21 sense siNA stab09 B
CUGAUCAUUGCCAACACAATT B 757 1192 CACCACAUUCUUCAUUGAUUGCA 590 37109
SCD:1194U21 sense siNA stab09 B CCACAUUCUUCAUUGAUUGTT B 758 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 37110 SCD:2025U21 sense siNA stab09 B
UUGGCAAUGCUAAUUCAAUTT B 759 2728 AGGCUUCUCUCCACAGUGUUGUG 592 37111
SCD:2730U21 sense siNA stab09 B GCUUCUCUCCACAGUGUUGTT B 760 3554
ACUAACCAGCAUUCCCUACAGCC 593 37112 SCD:3556U21 sense siNA stab09 B
UAACCAGCAUUCCCUACAGTT B 761 4316 CUUUGCACCUGAGACCCUACUGA 594 37113
SCD:4318U21 sense siNA stab09 B UUGCACCUGAGACCCUACUTT B 762 4318
UUGCACCUGAGACCCUACUGAAG 595 37114 SCO:4320U21 sense siNA stab09 B
GCACCUGAGACCCUACUGATT B 763 4775 ACCUACCUCAAAGGGCAGUUUUG 596 37115
SCD:4777U21 sense siNA stab09 B CUACCUCAAAGGGCAGUUUTT B 764 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21 antisense siNA
UUGUGUUGGCAAUGAUCAGTsT 765 (647C) stab10 1192
CACCACAUUCUU0AUUGAUUGCA 590 SCD:1212L21 antisense siNA
CAAUCAAUGAAGAAUGUGGTsT 766 (1194C) stab10 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2043L21 antisense siNA
AUUGAAUUAGOAUUGOCAATsT 767 (2025C) stab10 2728
AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNA
CAACACUGUGGAGAGAAGCTsT 768 (2730C) stab10 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3574L21 antisense siNA
CUGUAGGGAAUGCUGGUUATsT 769 (3556C) stab10 4316
CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNA
AGUAGGGUCUCAGGUGCAATsT 770 (4318C) stab10 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4338L21 antisense siNA
UCAGUAGGGUCUCAGGUGCTsT 771 (4320C) stab10 4775
ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNA
AAACUGCCCUUUGAGGUAGTsT 772 (4777C) stab10 645
UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21 antisense siNA
uuGuGuuGGcAAuGAucAGTT B 773 (647C) stab19 1192
CACCACAUUCUUCAUUGAUUGCA 590 SCD:1212L21 antisense siNA
cAAucAAuGAAGAAuGuGGTT B 774 (1194C) stab19 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2043L21 antisense siNA
AuuGAAuuAGcAuuGccAATT B 775 (2025C) stab19 2728
AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNA
cAAcAcuGuGGAGAGAAGcTT B 776 (2730C) stab19 3554
ACUAACCAGCAUUCCCUACAGCC 593 SCD:3574L21 antisense siNA
cuGuAGGGAAuGcuGGuuATT B 777 (3556C) stab19 4316
CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNA
AGuAGGGucucAGGuGcAATT B 778 (4318C) stab19 4318
UUGCACCUGAGACCCUACUGAAG 595 SCD:4338L21 antisense siNA
ucAGuAGGGucucAGGuGcTT B 779 (4320C) stab19 4775
ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNA
AAAcuGcccuuuGAGGuAGTT B 780 (4777C) stab19 645
UUCUGAUCAUUGCCAACACAAUG 589 37116 SCD:665L21 antisense siNA
UUGUGUUGGCAAUGAUCAGTT B 781 (647C) stab22 1192
CACCACAUUCUUCAUUGAUUGCA 590 37117 SCD:1212L21 antisense siNA
CAAUCAAUGAAGAAUGUGGTT B 782 (1194C) stab22 2023
GUUUGGCAAUGCUAAUUCAAUGC 591 37118 SCD:2043L21 antisense siNA
AUUGAAUUAGCAUUGCCAATT B 783 (2025C) 2728 AGGCUUCUCUCCACAGUGUUGUG
592 37119 SCD:2748L21 antisense siNA CAACACUGUGGAGAGAAGCTT B 784
(2730C) stab22 3554 ACUAACCAGCAUUCCCUACAGCC 593 37120 SCD:3574L21
antisense siNA CUGUAGGGAAUGCUGGUUATT B 785 (3556C) stab22 4316
CUUUGCACCUGAGACCCUACUGA 594 37121 SCD:4336L21 antisense siNA
AGUAGGGUCUCAGGUGCAATT B 786 (4318C) stab22 4318
UUGCACCUGAGACCCUACUGAAG 595 37122 SCD:4338L21 antisense siNA
UCAGUAGGGUCUCAGGUGCTT B 787 (4320C) stab22 4775
ACCUACCUCAAAGGGCAGUUUUG 596 37123 SCD:4795L21 antisense siNA
AAACUGCCCUUUGAGGUAGTT B 788 (4777C) 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
[0433]
4TABLE IV Non-limiting examples of Stabilization Chemistries for
chemically modified siNA constructs pyri- Chemistry midine Purine
cap p = S Strand "Stab 00" Ribo Ribo TT at 3'- S/AS ends "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
[0434]
5TABLE 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 Amount: Wait
Time* Equivalents: DNA/ DNA/2'-O- Wait Time* 2'-O- Wait Time*
Reagent 2'-O-methyl/Ribo methyl/Ribo DNA methyl 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
* * * * *