U.S. patent application number 16/934864 was filed with the patent office on 2021-05-20 for reduced size self-delivering rnai compounds.
This patent application is currently assigned to Phio Pharmaceuticals Corp.. The applicant listed for this patent is Phio Pharmaceuticals Corp.. Invention is credited to James Cardia, Joanne Kamens, Anastasia Khvorova, William Salomon, Dmitry Samarsky, Tod M. Woolf.
Application Number | 20210147849 16/934864 |
Document ID | / |
Family ID | 1000005370540 |
Filed Date | 2021-05-20 |
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United States Patent
Application |
20210147849 |
Kind Code |
A1 |
Khvorova; Anastasia ; et
al. |
May 20, 2021 |
REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS
Abstract
The present invention relates to RNAi constructs with minimal
double-stranded regions, and their use in gene silencing. RNAi
constructs associated with the invention include a double stranded
region of 8-14 nucleotides and a variety of chemical modifications,
and are highly effective in gene silencing.
Inventors: |
Khvorova; Anastasia;
(Westborough, MA) ; Salomon; William; (Worcester,
MA) ; Kamens; Joanne; (Newton, MA) ; Samarsky;
Dmitry; (Westborough, MA) ; Woolf; Tod M.;
(Sudbury, MA) ; Cardia; James; (Franklin,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Phio Pharmaceuticals Corp. |
Marlborough |
MA |
US |
|
|
Assignee: |
Phio Pharmaceuticals Corp.
Marlborough
MA
|
Family ID: |
1000005370540 |
Appl. No.: |
16/934864 |
Filed: |
July 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14866681 |
Sep 25, 2015 |
10774330 |
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16934864 |
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14278900 |
May 15, 2014 |
9175289 |
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14866681 |
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13120342 |
Oct 7, 2011 |
8796443 |
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PCT/US2009/005247 |
Sep 22, 2009 |
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14278900 |
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61224031 |
Jul 8, 2009 |
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61149946 |
Feb 4, 2009 |
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61192954 |
Sep 22, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/3519 20130101;
C12N 2310/321 20130101; C12N 2310/3521 20130101; C12N 2320/53
20130101; C12N 15/1136 20130101; C12N 2310/3231 20130101; C12N
2310/14 20130101; C12N 2320/51 20130101; C12N 2310/3515 20130101;
C12N 15/111 20130101; C12N 15/113 20130101; C12N 15/1137 20130101;
C12N 2310/3341 20130101; C12N 2310/315 20130101; C12N 2320/32
20130101; C12N 2310/322 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 15/11 20060101 C12N015/11 |
Claims
1.-91. (canceled)
92. An isolated double stranded nucleic acid molecule comprising a
guide strand and a passenger strand, wherein the isolated double
stranded nucleic acid molecule includes a double stranded region
and a single stranded region, wherein the single stranded region is
at the 3' end of the guide strand and is 4-12 nucleotides long,
wherein the single stranded region contains 3, 4, 5, 6, 7, 8, 9,
10, 11 or 12 phosphorothioate modifications, and wherein at least
40% of the nucleotides of the isolated double stranded nucleic acid
molecule are modified.
93. The isolated double stranded nucleic acid molecule of claim 92,
wherein the guide strand has a minimal length of 16
nucleotides.
94. The isolated double stranded nucleic acid molecule of claim 92,
wherein the single stranded region is at least 6 or at least 7
nucleotides long.
95. The isolated double stranded nucleic acid molecule of claim 92,
wherein each nucleotide within the single stranded region has a
phosphorothioate modification.
96. The isolated double stranded nucleic acid molecule of claim 92,
wherein at least one of the nucleotides of the isolated double
stranded molecule that is modified comprises a 2' O-methyl or a
2'-fluoro modification and/or wherein at least one of the
nucleotides of the isolated double stranded nucleic acid molecule
that is modified comprises a hydrophobic modification.
97. The isolated double stranded nucleic acid molecule of claim 92,
wherein a hydrophobic conjugate is attached to the double stranded
nucleic acid molecule.
98. The isolated double stranded nucleic acid molecule of claim 97,
wherein the hydrophobic conjugate is attached at the 3' end of the
double stranded nucleic acid molecule.
99. The isolated double stranded nucleic acid molecule of claim 93,
wherein the guide strand is 16-28 nucleotides long.
100. The isolated double stranded nucleic acid molecule of claim
92, wherein the passenger strand is 8-16 nucleotides long
101. An isolated asymmetric nucleic acid molecule comprising: a
first polynucleotide wherein the first polynucleotide is
complementary to a second polynucleotide and a target gene; and a
second polynucleotide, wherein the second polynucleotide is at
least 6 nucleotides shorter than the first polynucleotide, wherein
the first polynucleotide includes a single stranded region of 6, 7,
8, 9, 10, 11 or 12 nucleotides, wherein the single stranded region
of the first polynucleotide contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or
12 phosphorothioate modifications, wherein the asymmetric nucleic
acid molecule also includes a double stranded region, and wherein
at least 50% of C and U nucleotides in the double stranded region
are 2' O-methyl modified or 2'-fluoro modified.
102. The isolated double stranded nucleic acid molecule of claim
101, wherein the single stranded region is 6 or 7 nucleotides long
and/or wherein each nucleotide within the single stranded region
has a phosphorothioate modification.
103. An isolated double stranded nucleic acid molecule comprising:
a guide strand of 17-21 nucleotides in length that has
complementarity to a target gene, and a passenger strand of 8-16
nucleotides in length, wherein the guide strand and the passenger
strand form the double stranded nucleic acid molecule having a
double stranded region and a single stranded region, wherein the
guide strand has a 3' single stranded region of 4-12 nucleotides in
length, wherein the single stranded region comprises 2-12
phosphorothioate modifications, wherein at least 40% of the
nucleotides of the double stranded nucleic acid are modified,
wherein if the passenger strand is 16 nucleotides long, then one
end of the double-stranded nucleic acid molecule is blunt, and
wherein at least one modification is a hydrophobic base
modification and/or the double stranded nucleic acid molecule is
linked to a hydrophobic conjugate.
104. The isolated double stranded nucleic acid molecule of claim
103, wherein the hydrophobic base modification comprises a
hydrophobic modification of a pyrimidine base, optionally at
position 4 or 5, optionally, wherein the hydrophobic base
modification is selected from the group consisting of a phenyl,
4-pyridyl, 2-pyridyl, indolyl, isobutyl, tryptophanyl
(C.sub.8H.sub.6N)CH.sub.2CH(NH.sub.2)CO), methyl, butyl,
aminobenzyl, and naphthyl modification of a uridine or
cytidine.
105. The isolated double stranded nucleic acid molecule of claim
103, wherein the hydrophobic conjugate is a small molecule,
optionally wherein the small molecule is a sterol-type molecule,
optionally wherein the sterol-type molecule is cholesterol.
106. The isolated double stranded nucleic acid molecule of claim
103, wherein the hydrophobic conjugate is attached to the double
stranded nucleic acid molecule through a linker, optionally wherein
the linker is a TEG linker.
107. A method for inhibiting the expression of a target gene in a
mammalian cell, comprising contacting the mammalian cell with the
isolated double stranded nucleic acid molecule of claim 92.
108. The method of claim 92, wherein one end of the isolated double
stranded nucleic acid molecule is blunt.
109. The isolated double stranded nucleic acid molecule of claim
101, wherein the guide strand has a minimal length of 16
nucleotides.
110. The isolated double stranded nucleic acid molecule of claim
101, wherein one end of the isolated double stranded nucleic acid
molecule is blunt.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/278,900, filed on May 15, 2014, entitled "REDUCED SIZE
SELF-DELIVERING RNAI COMPOUNDS," which is a continuation of U.S.
application Ser. No. 13/120,342, now U.S. Pat. No. 8,796,443, which
issued on Aug. 5, 2014, entitled "REDUCED SIZE SELF-DELIVERING RNAI
COMPOUNDS," which is a national stage filing under 35 U.S.C. .sctn.
371 of international application PCT/US2009/005247, filed Sep. 22,
2009, which was published under PCT Article 21(2) in English, and
claims the benefit under 35 U.S.C. .sctn. 119(e) of U.S.
provisional application serial number U.S. 61/192,954, entitled
"Chemically Modified Polynucleotides and Methods of Using the
Same," filed on Sep. 22, 2008, U.S. 61/149,946, entitled "Minimum
Length Triggers of RNA Interference," filed on Feb. 4, 2009, and
U.S. 61/224,031, entitled "Minimum Length Triggers of RNA
Interference," filed on Jul. 8, 2009, the entire disclosure of each
of which is incorporated by reference herein in its entirety.
FIELD OF INVENTION
[0002] The invention pertains to the field of RNA interference
(RNAi). The invention more specifically relates to nucleic acid
molecules with improved in vivo delivery properties without the use
of a delivering agent and their use in efficient gene
silencing.
BACKGROUND OF INVENTION
[0003] Complementary oligonucleotide sequences are promising
therapeutic agents and useful research tools in elucidating gene
functions. However, prior art oligonucleotide molecules suffer from
several problems that may impede their clinical development, and
frequently make it difficult to achieve intended efficient
inhibition of gene expression (including protein synthesis) using
such compositions in vivo.
[0004] A major problem has been the delivery of these compounds to
cells and tissues. Conventional double-stranded RNAi compounds,
19-29 bases long, form a highly negatively-charged rigid helix of
approximately 1.5 by 10-15 nm in size. This rod type molecule
cannot get through the cell-membrane and as a result has very
limited efficacy both in vitro and in vivo. As a result, all
conventional RNAi compounds require some kind of a delivery vehicle
to promote their tissue distribution and cellular uptake. This is
considered to be a major limitation of the RNAi technology.
[0005] There have been previous attempts to apply chemical
modifications to oligonucleotides to improve their cellular uptake
properties. One such modification was the attachment of a
cholesterol molecule to the oligonucleotide. A first report on this
approach was by Letsinger et al., in 1989. Subsequently, ISIS
Pharmaceuticals, Inc. (Carlsbad, Calif.) reported on more advanced
techniques in attaching the cholesterol molecule to the
oligonucleotide (Manoharan, 1992).
[0006] With the discovery of siRNAs in the late nineties, similar
types of modifications were attempted on these molecules to enhance
their delivery profiles. Cholesterol molecules conjugated to
slightly modified (Soutschek, 2004) and heavily modified (Wolfrum,
2007) siRNAs appeared in the literature. Yamada et al., 2008 also
reported on the use of advanced linker chemistries which further
improved cholesterol mediated uptake of siRNAs. In spite of all
this effort, the uptake of these types of compounds appears to be
inhibited in the presence of biological fluids resulting in highly
limited efficacy in gene silencing in vivo, limiting the
applicability of these compounds in a clinical setting.
[0007] Therefore, it would be of great benefit to improve upon the
prior art oligonucleotides by designing oligonucleotides that have
improved delivery properties in vivo and are clinically
meaningful.
SUMMARY OF INVENTION
[0008] Described herein are asymmetric chemically modified nucleic
acid molecules with minimal double stranded regions, and the use of
such molecules in gene silencing. RNAi molecules associated with
the invention contain single stranded regions and double stranded
regions, and can contain a variety of chemical modifications within
both the single stranded and double stranded regions of the
molecule. Additionally, the RNAi molecules can be attached to a
hydrophobic conjugate such as a conventional and advanced
sterol-type molecule. This new class of RNAi molecules has superior
efficacy both in vitro and in vivo than previously described RNAi
molecules.
[0009] Aspects of the invention relate to asymmetric nucleic acid
molecules including a guide strand, with a minimal length of 16
nucleotides, and a passenger strand forming a double stranded
nucleic acid, having a double stranded region and a single stranded
region, the double stranded region having 8-15 nucleotides in
length, the single stranded region having 5-12 nucleotides in
length, wherein the passenger strand is linked to a lipophilic
group, wherein at least 40% of the nucleotides of the double
stranded nucleic acid are modified, and wherein the single stranded
region has at least 2 phosphorothioate modifications. In some
embodiments position 1 of the guide strand is 5' phosphorylated. In
certain embodiments, position 1 of the guide strand is 2'O-methyl
modified and 5' phosphorylated.
[0010] Aspects of the invention relate to isolated double stranded
nucleic acid molecules including a longer strand of 15-21
nucleotides in length that has complementarily to a miRNA sequence,
a shorter strand of 8-15 nucleotides in length linked at the 3' end
to a lipophilic group, wherein the longer strand and the passenger
strand form the double stranded nucleic acid molecule having a
double stranded region and a single stranded region, wherein the
longer strand has a 3' single stranded region of 2-13 nucleotides
in length, comprising at least two phosphorothioate modification,
and at least 50% nucleotides are modified.
[0011] Further aspects of the invention relate to isolated double
stranded nucleic acid molecules including a guide strand of 17-21
nucleotides in length that has complementarity to a target gene, a
passenger strand of 8-16 nucleotides in length linked at the 3' end
to a lipophilic group, wherein the guide strand and the passenger
strand form the double stranded nucleic acid molecule having a
double stranded region and a single stranded region, wherein the
guide strand has a 3' single stranded region of 2-13 nucleotides in
length, each nucleotide within the single stranded region having a
phosphorothioate modification, wherein the guide strand has a 5'
phosphate modification and wherein at least 50% of C and U
nucleotides in the double stranded region include at least one 2'
O-methyl modification or 2'-fluoro modification.
[0012] In another aspect, the invention is an isolated double
stranded nucleic acid molecule having a guide strand of 17-21
nucleotides in length that has complementarity to a target gene, a
passenger strand of 10-16 nucleotides in length linked at the 3'
end to a lipophilic group, wherein the guide strand and the
passenger strand form the double stranded nucleic acid molecule
having a double stranded region and a single stranded region,
wherein the guide strand has a 3' single stranded region of 5-11
nucleotides in length, at least two nucleotide within the single
stranded region having a phosphorothioate modification, wherein the
guide strand has a 5' phosphate modification and wherein at least
50% of C and U nucleotides in the double stranded region are 2'
O-methyl modification or 2'-fluoro modified.
[0013] The invention in another aspect is an isolated double
stranded nucleic acid molecule having a guide strand of 17-21
nucleotides in length that has complementarity to a target gene, a
passenger strand of 8-16 nucleotides in length linked at the 3' end
to a lipophilic group, wherein the guide strand and the passenger
strand form the double stranded nucleic acid molecule having a
double stranded region and a single stranded region, wherein the
guide strand has a 3' single stranded region of 6-8 nucleotides in
length, each nucleotide within the single stranded region having a
phosphorothioate modification, wherein the guide strand has a 5'
phosphate modification, wherein the passenger strand includes at
least two phosphorothioate modifications, wherein at least 50% of C
and U nucleotides in the double stranded region include a 2'
O-methyl modification or 2'-fluoro modification, and wherein the
double stranded nucleic acid molecule has one end that is blunt or
includes a one-two nucleotide overhang.
[0014] An isolated double stranded nucleic acid molecule having a
guide strand of 17-21 nucleotides in length that has
complementarity to a target gene, a passenger strand of 8-16
nucleotides in length linked at the 3' end to a lipophilic group,
wherein the guide strand and the passenger strand form the double
stranded nucleic acid molecule having a double stranded region and
a single stranded region, wherein the guide strand has a 3' single
stranded region, each nucleotide within the single stranded region
having a phosphorothioate modification, wherein the guide strand
has a 5' phosphate modification, wherein every C and U nucleotide
in position 11-18 of the guide strand has a 2' O-methyl
modification, wherein every nucleotide of the passenger strand is
2' O-methyl modified, and wherein the double stranded nucleic acid
molecule has one end that is blunt or includes a one-two nucleotide
overhang is provided in other aspects of the invention.
[0015] In another aspect the invention is an isolated double
stranded nucleic acid molecule having a guide strand of 17-21
nucleotides in length that has complementarity to a target gene, a
passenger strand of 8-15 nucleotides in length linked at the 3' end
to a lipophilic group, wherein the lipophilic group is selected
from the group consisting of cholesterol and a sterol type molecule
with C17 polycarbon chain of 5-7 or 9-18 carbons in length, wherein
the guide strand and the passenger strand form the double stranded
nucleic acid molecule having a double stranded region and a single
stranded region, wherein the guide strand has a 3' single stranded
region, each nucleotide within the single stranded region having a
phosphorothioate modification, wherein the guide strand has a 5'
phosphate modification, wherein every C and U nucleotide in
position 11-18 of the guide strand has a 2' O-methyl modification,
wherein every C and U nucleotide in position 2-10 of the guide
strand has a 2'F modification, wherein every nucleotide of the
passenger strand is 2' O-methyl modified, and wherein the double
stranded nucleic acid molecule has one end that is blunt or
includes a one-two nucleotide overhang.
[0016] In yet another aspect the invention is an isolated nucleic
acid molecule having a guide sequence that has complementarity to a
target gene, a passenger sequence linked at the 3' end to a
lipophilic group, wherein the guide sequence and the passenger
sequence form a nucleic acid molecule having a double stranded
region and a single stranded region, wherein the guide sequence has
a 3' single stranded region of 2-13 nucleotides in length, each
nucleotide within the single stranded region having a
phosphorothioate modification, wherein the guide sequence has a 5'
phosphate modification, wherein at least 50% of C and U nucleotides
in the double stranded region include at least one 2' O-methyl
modification or 2'-fluoro modification, and wherein the double
stranded nucleic acid molecule has one end that is blunt or
includes a one-two nucleotide overhang.
[0017] An isolated double stranded nucleic acid molecule having a
guide strand and a passenger strand, wherein the region of the
molecule that is double stranded is from 8-14 nucleotides long,
wherein the guide strand contains a single stranded region that is
4-12 nucleotides long, and wherein the single stranded region of
the guide strand contains 2-12 phosphorothioate modifications is
provided in other aspects of the invention.
[0018] In some embodiments the guide strand contains 6-8
phosphorothioate modifications. In other embodiments the single
stranded region of the guide strand is 6 nucleotides long.
[0019] In yet other embodiments the double stranded region is 13
nucleotides long. Optionally the double stranded nucleic acid
molecule has one end that is blunt or includes a one-two nucleotide
overhang.
[0020] In another aspect the invention is an isolated double
stranded nucleic acid molecule having a guide strand, wherein the
guide strand is 16-28 nucleotides long and has complementarity to a
target gene, wherein the 3' terminal 10 nucleotides of the guide
strand include at least two phosphate modifications, and wherein
the guide strand has a 5' phosphate modification and includes at
least one 2' O-methyl modification or 2'-fluoro modification, and a
passenger strand, wherein the passenger strand is 8-14 nucleotides
long and has complementarity to the guide strand, wherein the
passenger strand is linked to a lipophilic group, wherein the guide
strand and the passenger strand form the double stranded nucleic
acid molecule.
[0021] In some embodiments the nucleotide in position one of the
guide strand or sequence has a 2'-O-methyl modification. In other
embodiments at least one C or U nucleotide in positions 2-10 of the
guide strand or sequence has a 2'-fluoro modification. In yet other
embodiments every C and U nucleotide in positions 2-10 of the guide
strand or sequence has a 2'-fluoro modification. At least one C or
U nucleotide in positions 11-18 of the guide strand or sequence may
have a 2'-O-methyl modification. In some embodiments every C and U
nucleotide in positions 11-18 of the guide strand or sequence has a
2'-O-methyl modification.
[0022] In yet other embodiments the 3' terminal 10 nucleotides of
the guide strand include at least four phosphate modifications.
Optionally the 3' terminal 10 nucleotides of the guide strand
include at least eight phosphate modifications. In some embodiments
the guide strand includes 4-14 phosphate modifications. In other
embodiments the guide strand includes 4-10 phosphate modifications.
In yet other embodiments the 3' terminal 6 nucleotides of the guide
strand all include phosphate modifications. The phosphate
modifications may be phosphorothioate modifications.
[0023] In some embodiments every C and U nucleotide on the
passenger strand has a 2'-O-methyl modification. In other
embodiments every nucleotide on the passenger strand has a
2'-O-methyl modification. In an embodiment at least one nucleotide
on the passenger strand is phosphorothioate modified. At least two
nucleotides on the passenger strand are phosphorothioate modified
in other embodiments.
[0024] The lipophilic molecule may be a sterol, such as
cholesterol.
[0025] In some embodiments the guide strand is 18-19 nucleotides
long. In other embodiments the passenger strand is 11-13
nucleotides long.
[0026] The double stranded nucleic acid molecule has one end that
is blunt or includes a one-two nucleotide overhang in other
embodiments.
[0027] In other aspects the invention is an isolated double
stranded nucleic acid molecule comprising a guide strand and a
passenger strand, wherein the guide strand is from 16-29
nucleotides long and is substantially complementary to a target
gene, wherein the passenger strand is from 8-14 nucleotides long
and has complementarity to the guide strand, and wherein the guide
stand has at least two chemical modifications. In some embodiments
the at least two chemical modifications include at least two
phosphorothioate modifications. In some embodiments the double
stranded nucleic acid molecule has one end that is blunt or
includes a one-two nucleotide overhang.
[0028] In some aspects the invention is an isolated double stranded
nucleic acid molecule comprising a guide strand and a passenger
strand, wherein the guide strand is from 16-29 nucleotides long and
is substantially complementary to a target gene, wherein the
passenger strand is from 8-14 nucleotides long and has
complementarity to the guide strand, and wherein the guide stand
has a single stranded 3' region that is 5 nucleotides or longer and
a 5' region that is 1 nucleotide or less. The single stranded
region may contain at least 2 phosphorothioate modifications.
[0029] An isolated double stranded nucleic acid molecule having a
guide strand and a passenger strand, wherein the guide strand is
from 16-29 nucleotides long and is substantially complementary to a
target gene, wherein the passenger strand is from 8-16 nucleotides
long and has complementarity to the guide strand, and wherein the
guide stand has a single stranded 3' region that is 5 nucleotides
or longer and a passenger strand has a sterol type molecule with
C17 attached chain longer than 9 is provided in other aspects of
the invention.
[0030] A duplex polynucleotide is provided in other aspects of the
invention. The polynucleotide has a first polynucleotide wherein
said first polynucleotide is complementary to a second
polynucleotide and a target gene; and a second polynucleotide
wherein said second polynucleotide is at least 6 nucleotides
shorter than said first polynucleotide, wherein said first
polynucleotide includes a single stranded region containing
modifications selected from the group consisting of 40-90%
hydrophobic base modifications, 40-90% phosphorothioates, and
40-90% modifications of the ribose moiety, or any combination
thereof.
[0031] In other aspects the invention is a duplex polynucleotide
having a first polynucleotide wherein said first polynucleotide is
complementary to a second polynucleotide and a target gene; and a
second polynucleotide wherein said second polynucleotide is at
least 6 nucleotides shorter than said first polynucleotide, wherein
the duplex polynucleotide includes a mismatch between nucleotides
9, 11, 12, 13 or 14 on the first polynucleotide and the opposite
nucleotide on the second polynucleotide.
[0032] In other aspects the invention is a method for inhibiting
the expression of a target gene in a mammalian cell, comprising
contacting the mammalian cell with an isolated double stranded
nucleic acid molecule described herein or a duplex polynucleotide
described herein.
[0033] A method of inducing RNAi in a subject is provided in other
aspects of the invention. The method involves administering to a
subject an effective amount for inducing RNAi of an mRNA of a
target gene, an isolated double stranded nucleic acid molecule
described herein or a duplex polynucleotide described herein. In
other embodiment the subject is a human. In other embodiments the
target gene is PPIB, MAP4K4, or SOD1.
[0034] In other aspects an isolated hydrophobic modified
polynucleotide having a polynucleotide, wherein the polynucleotide
is double stranded RNA, attached to a hydrophobic molecule, wherein
the hydrophobic molecule is attached to a base, a ribose or a
backbone of a non-terminal nucleotide and wherein the isolated
double stranded nucleic acid molecule comprises a guide strand and
a passenger strand, wherein the guide strand is from 16-29
nucleotides long and is substantially complementary to a target
gene, wherein the passenger strand is from 8-14 nucleotides long
and has complementarity to the guide strand is provided.
[0035] In one embodiment the hydrophobic molecule is attached to
the guide strand of the double stranded RNA. In another embodiment
the 3' terminal 10 nucleotides of the guide strand include at least
two phosphate modifications, and wherein the guide strand has a 5'
phosphate modification and includes at least one 2' O-methyl
modification or 2'-fluoro modification. In yet another embodiment
the hydrophobic molecule is attached to the passenger strand of the
double stranded RNA.
[0036] The invention provides an isolated hydrophobic modified
polynucleotide having a polynucleotide non-covalently complexed to
a hydrophobic molecule, wherein the hydrophobic molecule is a
polycationic molecule. In some embodiments the polycationic
molecule is selected from the group consisting of protamine,
arginine rich peptides, and spermine.
[0037] In other aspects the invention an isolated hydrophobic
modified polynucleotide having a polynucleotide, wherein the
polynucleotide is double stranded RNA, directly complexed to a
hydrophobic molecule without a linker, wherein the hydrophobic
molecule is not cholesterol.
[0038] A composition having a hydrophobic modified polynucleotide,
wherein the polynucleotide is double stranded RNA, attached to a
hydrophobic molecule, wherein the double stranded nucleic acid
molecule comprises a guide strand and a passenger strand, wherein
the guide strand is from 16-29 nucleotides long and is
substantially complementary to a target gene, wherein the passenger
strand is from 8-14 nucleotides long and has complementarity to the
guide strand, wherein position 1 of the guide strand is 5'
phosphorylated or has a 2' O-methyl modification, wherein at least
40% of the nucleotides of the double stranded nucleic acid are
modified, and wherein the double stranded nucleic acid molecule has
one end that is blunt or includes a one-two nucleotide overhang; a
neutral fatty mixture; and optionally a cargo molecule, wherein the
hydrophobic modified polynucleotide and the neutral fatty mixture
forms a micelle is provided in other aspects of the invention.
[0039] In some embodiments the 3' end of the passenger strand is
linked to the hydrophobic molecule. In other embodiments the
composition is sterile. In yet other embodiments the neutral fatty
mixture comprises a DOPC (dioleoylphosphatidylcholine). In further
embodiments the neutral fatty mixture comprises a DSPC
(distearoylphosphatidylcholine). The neutral fatty mixture further
comprises a sterol such as cholesterol in other embodiments.
[0040] In yet other embodiments the composition includes at least
20% DOPC and at least 20% cholesterol. The hydrophobic portion of
the hydrophobic modified polynucleotide is a sterol in other
embodiments. The sterol may be a cholesterol, a cholesteryl or
modified cholesteryl residue. In other embodiments the hydrophobic
portion of the hydrophobic modified polynucleotide is selected from
the group consisting of bile acids, cholic acid or taurocholic
acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid,
glycolipids, phospholipids, sphingolipids, isoprenoids, vitamins,
saturated fatty acids, unsaturated fatty acids, fatty acid esters,
triglycerides, pyrenes, porphyrines, Texaphyrine, adamantane,
acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red,
digoxygenin, dimethoxytrityl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy5), Hoechst 33258
dye, psoralen, and ibuprofen.
[0041] In yet other embodiments the hydrophobic portion of the
hydrophobic modified polynucleotide is a polycationic molecule,
such as, for instance, protamine, arginine rich peptides, and/or
spermine.
[0042] The composition optionally includes a cargo molecule such as
a lipid, a peptide, vitamin, and/or a small molecule. In some
embodiments the cargo molecule is a commercially available fat
emulsions available for a variety of purposes selected from the
group consisting of parenteral feeding. In some embodiments the
commercially available fat emulsion is an intralipid or a
nutralipid. In other embodiments the cargo molecule is a fatty acid
mixture containing more then 74% of linoleic acid, a fatty acid
mixture containing at least 6% of cardiolipin, or a fatty acid
mixture containing at least 74% of linoleic acid and at least 6% of
cardiolipin. In another embodiment the cargo molecule is a
fusogenic lipid, such as for example, DOPE, and preferably is at
least 10% fusogenic lipid
[0043] In some embodiments the polynucleotide includes chemical
modifications. For instance it may be at least 40% modified.
[0044] A method of inducing RNAi in a subject is provided in
another aspect of the invention. The method involves administering
to a subject an effective amount for inducing RNAi of mRNA of a
target gene, an isolated double stranded nucleic acid molecule or a
duplex polynucleotide or a composition of the invention, wherein
the polynucleotide has at least a region of sequence correspondence
to the target gene, wherein the step of administering is systemic,
intravenous, intraperitoneal, intradermal, topical, intranasal,
inhalation, oral, intramucosal, local injection, subcutaneous, oral
tracheal, or intraocular.
[0045] In other embodiment the subject is a human. In other
embodiments the target gene is PPIB, MAP4K4, or SOD1.
[0046] In some aspects the invention is a single-stranded RNA of
less than 35 nucleotides in length that forms a hairpin structure,
said hairpin includes a double-stranded stem and a single-stranded
loop, said double-stranded stem having a 5'-stem sequence having a
5'-end, and a 3'-stem sequence having a 3'-end; and said 5'-stem
sequence and at least a portion of said loop form a guide sequence
complementary to a transcript of a target gene, wherein said
polynucleotide mediates sequence-dependent gene silencing of
expression of said target gene, wherein each nucleotide within the
single-stranded loop region has a phosphorothioate modification,
and wherein at least 50% of C and U nucleotides in the double
stranded region include a 2' O-methyl modification or 2'-fluoro
modification. In one embodiment every C and U nucleotide in
position 11-18 of the guide sequence has a 2' O-methyl
modification.
[0047] A polynucleotide construct is provided in other aspects, the
polynucleotide having two identical single-stranded
polynucleotides, each of said single-stranded polynucleotide
comprising a 5'-stem sequence having a 5'-end, a 3'-stem sequence
having a 3'-end, and a linker sequence linking the 5'-stem sequence
and the 3'-stem sequence, wherein: (1) the 5'-stem sequence of a
first single-stranded polynucleotide hybridizes with the 3'-stem
sequence of a second single-stranded polynucleotide to form a first
double-stranded stem region; (2) the 5'-stem sequence of the second
single-stranded polynucleotide hybridize with the 3'-stem sequence
of the first single-stranded polynucleotide to form a second
double-stranded stem region; and, (3) the linker sequences of the
first and the second single-stranded polynucleotides form a loop or
bulge connecting said first and said second double-stranded stem
regions, wherein the 5'-stem sequence and at least a portion of the
linker sequence form a guide sequence complementary to a transcript
of a target gene, wherein said polynucleotide construct mediates
sequence-dependent gene silencing of expression of said target
gene, wherein each nucleotide within the single-stranded loop
region has a phosphorothioate modification, and wherein at least
50% of C and U nucleotides in the double stranded regions include a
2' O-methyl modification or 2'-fluoro modification.
[0048] In one embodiment every C and U nucleotide in position 11-18
of the guide sequence has a 2' O-methyl modification.
[0049] In some embodiments, the guide strand is 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides long. In some
embodiments, the passenger strand is 8, 9, 10, 11, 12, 13 or 14
nucleotides long. In some embodiments, the nucleic acid molecule
has a thermodynamic stability (.DELTA.G) of less than -20
kkal/mol.
[0050] Aspects of the invention relate to nucleic acid molecules
that are chemically modified. In some embodiments, the chemical
modification is selected from the group consisting of 5' Phosphate,
2'-O-methyl, 2'-O-ethyl, 2'-fluoro, ribothymidine, C-5 propynyl-dC
(pdC), C-5 propynyl-dU (pdU), C-5 propynyl-C(pC), C-5 propynyl-U
(pU), 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy,
(2,6-diaminopurine), 5'-Dimethoxytrityl-N4-ethyl-2'-deoxyCytidine,
C-5 propynyl-fC (pfC), C-5 propynyl-fU (pfU), 5-methyl fC, 5-methyl
fU, C-5 propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC,
5-methyl mU, LNA (locked nucleic acid), MGB (minor groove binder)
and other base modifications which increase base hydrophobicity.
More than one chemical modification may be present in the same
molecule. In some embodiments, chemical modification increases
stability and/or improves thermodynamic stability (.DELTA.G). In
some embodiments, at least 90% of CU residues on a nucleic acid
molecule are modified.
[0051] In some embodiments, the nucleotide in position one of the
guide strand has a 2'-O-methyl modification and/or a 5' Phosphate
modification. In some embodiments, at least one C or U nucleotide
in positions 2-10 of the guide strand has a 2'-fluoro modification.
In certain embodiments, every C and U nucleotide in positions 2-10
of the guide strand has a 2'-fluoro modification. In some
embodiments, at least one C or U nucleotide in positions 11-18 of
the guide strand has a 2'-O-methyl modification. In certain
embodiments, every C and U nucleotide in positions 11-18 of the
guide strand has a 2'-O-methyl modification. In some embodiments,
every C and U nucleotide on the passenger strand has a 2'-O-methyl
modification. In certain embodiments, every nucleotide on the
passenger strand has a 2'-O-methyl modification.
[0052] In some embodiments, nucleic acid molecules associated with
the invention contain a stretch of at least 4 nucleotides that are
phosphorothioate modified. In certain embodiments, the stretch of
nucleotides that are phosphorothioate modified is at least 12
nucleotides long. In some embodiments, the stretch of nucleotides
that are phosphorothioate modified is not fully single
stranded.
[0053] Nucleic acid molecules associated with the invention may be
attached to a conjugate. In some embodiments, the conjugate is
attached to the guide strand, while in other embodiments the
conjugate is attached to the passenger strand. In some embodiments,
the conjugate is hydrophobic. In some embodiments, the conjugate is
a sterol such as cholesterol. In some embodiments, nucleic acid
molecules associated with the invention are blunt-ended.
[0054] Aspects of the invention relate to double stranded nucleic
acid molecule including a guide strand and a passenger strand,
wherein the region of the molecule that is double stranded is from
8-14 nucleotides long, and wherein the molecule has a thermodynamic
stability (.DELTA.G) of less than -13 kkal/mol.
[0055] In some embodiments, the region of the molecule that is
double stranded is 8, 9, 10, 11, 12, 13, or 14 nucleotides long. In
some embodiments, the molecule has a thermodynamic stability
(.DELTA.G) of less than -20 kkal/mol. The nucleic acid molecules,
in some embodiments are chemically modified. In certain
embodiments, the chemical modification is selected from the group
consisting of 5' Phosphate, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro,
ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU), C-5
propynyl-C(pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U,
5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine),
5'-Dimethoxytrityl-N4-ethyl-2'-deoxyCytidine, C-5 propynyl-fC
(pfC), C-5 propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5
propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC, 5-methyl mU,
LNA (locked nucleic acid), MGB (minor groove binder) and other base
modifications which increase base hydrophobicity. More than one
chemical modification may be present in the same molecule. In some
embodiments, chemical modification increases stability and/or
improves thermodynamic stability (.DELTA.G). In some embodiments,
at least 90% of CU residues on a nucleic acid molecule are
modified.
[0056] In some embodiments, the nucleotide in position one of the
guide strand has a 2'-O-methyl modification and/or a 5' Phosphate
modification. In some embodiments, at least one C or U nucleotide
in positions 2-10 of the guide strand has a 2'-fluoro modification.
In certain embodiments, every C and U nucleotide in positions 2-10
of the guide strand has a 2'-fluoro modification. In some
embodiments, at least one C or U nucleotide in positions 11-18 of
the guide strand has a 2'-O-methyl modification. In certain
embodiments, every C and U nucleotide in positions 11-18 of the
guide strand has a 2'-O-methyl modification. In some embodiments,
every C and U nucleotide on the passenger strand has a 2'-O-methyl
modification. In certain embodiments, every nucleotide on the
passenger strand has a 2'-O-methyl modification.
[0057] The nucleic acid molecules associated with the invention may
contain a stretch of at least 4 nucleotides that are
phosphorothioate modified. In certain embodiments, the stretch of
nucleotides that are phosphorothioate modified is at least 12
nucleotides long. In some embodiments, the stretch of nucleotides
that are phosphorothioate modified is not fully single stranded. In
some embodiments, the nucleic acid molecules are attached to a
conjugate. In some embodiments, the conjugate is attached to the
guide strand, while in other embodiments the conjugate is attached
to the passenger strand. In some embodiments, the conjugate is
hydrophobic. In some embodiments, the conjugate is a sterol such as
cholesterol. In some embodiments, nucleic acid molecules associated
with the invention are blunt-ended. In some embodiments, the
nucleic acid molecules are blunt ended at the 5'end. In certain
embodiments, the nucleic acid molecules are blunt ended at the
5'end where the region of complementarity between the two strands
of the molecule begins.
[0058] Aspects of the invention relate to methods for inhibiting
the expression of a target gene in a mammalian cell. Methods
include contacting the mammalian cell with an isolated double
stranded nucleic acid molecule including a guide strand and a
passenger strand, wherein the guide strand is from 16-29
nucleotides long and has complementarity to a target gene, wherein
the passenger strand is from 8-14 nucleotides long and has
complementarity to the guide strand, and wherein the double
stranded nucleic acid molecule has a thermodynamic stability
(.DELTA.G) of less than -13 kkal/mol.
[0059] The cell may be contacted in vivo or in vitro. In some
embodiments, the guide strand is 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, or 29 nucleotides long. In some embodiments,
the passenger strand is 8, 9, 10, 11, 12, 13 or 14 nucleotides
long. In some embodiments, the nucleic acid molecule has a
thermodynamic stability (.DELTA.G) of less than -20 kkal/mol.
[0060] The nucleic acid molecules associated with methods described
herein may be chemically modified. In some embodiments, the
chemical modification is selected from the group consisting of 5'
Phosphate, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro, ribothymidine, C-5
propynyl-dC (pdC), C-5 propynyl-dU (pdU), C-5 propynyl-C(pC), C-5
propynyl-U (pU), 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU
methoxy, (2,6-diaminopurine),
5'-Dimethoxytrityl-N4-ethyl-2'-deoxyCytidine, C-5 propynyl-fC
(pfC), C-5 propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5
propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC, 5-methyl mU,
LNA (locked nucleic acid), MGB (minor groove binder) and other base
modifications which increase base hydrophobicity. More than one
chemical modification may be present in the same molecule. In some
embodiments, chemical modification increases stability and/or
improves thermodynamic stability (.DELTA.G). In some embodiments,
at least 90% of CU residues on a nucleic acid molecule are
modified.
[0061] In some embodiments, the nucleotide in position one of the
guide strand has a 2'-O-methyl modification and/or a 5' Phosphate
modification. In some embodiments, at least one C or U nucleotide
in positions 2-10 of the guide strand has a 2'-fluoro modification.
In certain embodiments, every C and U nucleotide in positions 2-10
of the guide strand has a 2'-fluoro modification. In some
embodiments, at least one C or U nucleotide in positions 11-18 of
the guide strand has a 2'-O-methyl modification. In certain
embodiments, every C and U nucleotide in positions 11-18 of the
guide strand has a 2'-O-methyl modification. In some embodiments,
every C and U nucleotide on the passenger strand has a 2'-O-methyl
modification. In certain embodiments, every nucleotide on the
passenger strand has a 2'-O-methyl modification.
[0062] In some embodiments, nucleic acid molecules associated with
the invention contain a stretch of at least 4 nucleotides that are
phosphorothioate modified. In certain embodiments, the stretch of
nucleotides that are phosphorothioate modified is at least 12
nucleotides long. In some embodiments, the stretch of nucleotides
that are phosphorothioate modified is not fully single
stranded.
[0063] Nucleic acid molecules associated with the invention may be
attached to a conjugate. In some embodiments, the conjugate is
attached to the guide strand, while in other embodiments the
conjugate is attached to the passenger strand. In some embodiments,
the conjugate is hydrophobic. In some embodiments, the conjugate is
a sterol such as cholesterol. In some embodiments, nucleic acid
molecules associated with the invention are blunt-ended.
[0064] Methods for inhibiting the expression of a target gene in a
mammalian cell described herein include contacting the mammalian
cell with an isolated double stranded nucleic acid molecule
including a guide strand and a passenger strand, wherein the region
of the molecule that is double stranded is from 8-14 nucleotides
long, and wherein the molecule has a thermodynamic stability
(.DELTA.G) of less than -13 kkal/mol.
[0065] In some embodiments, the region of the molecule that is
double stranded is 8, 9, 10, 11, 12, 13, or 14 nucleotides long. In
some embodiments, the molecule has a thermodynamic stability
(.DELTA.G) of less than -20 kkal/mol. The nucleic acid molecules,
in some embodiments are chemically modified. In certain
embodiments, the chemical modification is selected from the group
consisting of 5' Phosphate, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro,
ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU), C-5
propynyl-C(pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U,
5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine),
5'-Dimethoxytrityl-N4-ethyl-2'-deoxyCytidine, C-5 propynyl-fC
(pfC), C-5 propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5
propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC, 5-methyl mU,
LNA (locked nucleic acid), MGB (minor groove binder) and other base
modifications which increase base hydrophobicity. More than one
chemical modification may be present in the same molecule. In some
embodiments, chemical modification increases stability and/or
improves thermodynamic stability (.DELTA.G). In some embodiments,
at least 90% of CU residues on a nucleic acid molecule are
modified.
[0066] In some embodiments, the nucleotide in position one of the
guide strand has a 2'-O-methyl modification and/or a 5' Phosphate
modification. In some embodiments, at least one C or U nucleotide
in positions 2-10 of the guide strand has a 2'-fluoro modification.
In certain embodiments, every C and U nucleotide in positions 2-10
of the guide strand has a 2'-fluoro modification. In some
embodiments, at least one C or U nucleotide in positions 11-18 of
the guide strand has a 2'-O-methyl modification. In certain
embodiments, every C and U nucleotide in positions 11-18 of the
guide strand has a 2'-O-methyl modification. In some embodiments,
every C and U nucleotide on the passenger strand has a 2'-O-methyl
modification. In certain embodiments, every nucleotide on the
passenger strand has a 2'-O-methyl modification.
[0067] The nucleic acid molecules associated with the invention may
contain a stretch of at least 4 nucleotides that are
phosphorothioate modified. In certain embodiments, the stretch of
nucleotides that are phosphorothioate modified is at least 12
nucleotides long. In some embodiments, the stretch of nucleotides
that are phosphorothioate modified is not fully single stranded. In
some embodiments, the nucleic acid molecules are attached to a
conjugate. In some embodiments, the conjugate is attached to the
guide strand, while in other embodiments the conjugate is attached
to the passenger strand. In some embodiments, the conjugate is
hydrophobic. In some embodiments, the conjugate is a sterol such as
cholesterol. In some embodiments, nucleic acid molecules associated
with the invention are blunt-ended.
[0068] In another embodiment, the invention provides a method for
selecting an siRNA for gene silencing by (a) selecting a target
gene, wherein the target gene comprises a target sequence; (b)
selecting a candidate siRNA, wherein said candidate siRNA comprises
a guide strand of 16-29 nucleotide base pairs and a passenger
strand of 8-14 nucleotide base pairs that form a duplex comprised
of an antisense region and a sense region and said antisense region
of said candidate siRNA is at least 80% complementary to a region
of said target sequence; (c) determining a thermodynamic stability
(.DELTA.G) of the candidate siRNA; and (e) selecting said candidate
siRNA as an siRNA for gene silencing, if said thermodynamic
stability is less than -13 kkal/mol.
[0069] Aspects of the invention relate to isolated double stranded
nucleic acid molecules including a guide strand and a passenger
strand, wherein the guide strand is 18-19 nucleotides long and has
complementarity to a target gene, wherein the passenger strand is
11-13 nucleotides long and has complementarity to the guide strand,
and wherein the double stranded nucleic acid molecule has a
thermodynamic stability (.DELTA.G) of less than -13 kkal/mol.
[0070] In some embodiments, the nucleotide in position one of the
guide strand has a 2'-O-methyl modification and/or a 5' Phosphate
modification. In some embodiments, at least one C or U nucleotide
in positions 2-10 of the guide strand has a 2'-fluoro modification.
In certain embodiments, every C and U nucleotide in positions 2-10
of the guide strand has a 2'-fluoro modification. In some
embodiments, at least one C or U nucleotide in positions 11-18 of
the guide strand has a 2'-O-methyl modification. In certain
embodiments, every C and U nucleotide in positions 11-18 of the
guide strand has a 2'-O-methyl modification.
[0071] In some embodiments, the guide strand contains a stretch of
at least 4 nucleotides that are phosphorothioate modified. In
certain embodiments, the guide strand contains a stretch of at
least 8 nucleotides that are phosphorothioate modified. In some
embodiments, every C and U nucleotide on the passenger strand has a
2'-O-methyl modification. In certain embodiments, every nucleotide
on the passenger strand has a 2'-O-methyl modification. In some
embodiments, at least one, or at least two nucleotides on the
passenger strand is phosphorothioate modified. The nucleic acid
molecule can be attached to a conjugate on either the guide or
passenger strand. In some embodiments, the conjugate is a sterol
such as cholesterol.
[0072] Aspects of the invention relate to isolated double stranded
nucleic acid molecules including a guide strand, wherein the guide
strand is 16-28 nucleotides long and has complementarity to a
target gene, wherein the 3' terminal 10 nucleotides of the guide
strand include at least two phosphate modifications, and wherein
the guide strand includes at least one 2' O-methyl modification or
2'-fluoro modification, and a passenger strand, wherein the
passenger strand is 8-28 nucleotides long and has complementarity
to the guide strand, wherein the passenger strand is linked to a
lipophilic group, wherein the guide strand and the passenger strand
form the double stranded nucleic acid molecule.
[0073] In some embodiments, the nucleotide in position one of the
guide strand has a 2'-O-methyl modification and/or a 5' Phosphate
modification. In some embodiments, at least one C or U nucleotide
in positions 2-10 of the guide strand has a 2'-fluoro modification.
In certain embodiments, every C and U nucleotide in positions 2-10
of the guide strand has a 2'-fluoro modification. In some
embodiments, at least one C or U nucleotide in positions 11-18 of
the guide strand has a 2'-O-methyl modification. In certain
embodiments, every C and U nucleotide in positions 11-18 of the
guide strand has a 2'-O-methyl modification.
[0074] In some embodiments, the 3' terminal 10 nucleotides of the
guide strand include at least four, or at least eight phosphate
modifications. In certain embodiments, the guide strand includes
2-14 or 4-10 phosphate modifications. In some embodiments, the 3'
terminal 6 nucleotides of the guide strand all include phosphate
modifications. In certain embodiments, the phosphate modifications
are phosphorothioate modifications.
[0075] In some embodiments, every C and U nucleotide on the
passenger strand has a 2'-O-methyl modification. In certain
embodiments, every nucleotide on the passenger strand has a
2'-O-methyl modification. In some embodiments, at least one, or at
least two nucleotides on the passenger strand is phosphorothioate
modified. In some embodiments, the lipophilic molecule is a sterol
such as cholesterol. In some embodiments, the guide strand is 18-19
nucleotides long and the passenger strand is 11-13 nucleotides
long.
[0076] Aspects of the invention relate to isolated double stranded
nucleic acid molecules including a guide strand and a passenger
strand, wherein the guide strand is from 16-29 nucleotides long and
is substantially complementary to a target gene, wherein the
passenger strand is from 8-14 nucleotides long and has
complementarity to the guide strand, and wherein the guide stand
has at least two chemical modifications. In some embodiments, the
two chemical modifications are phosphorothioate modifications.
[0077] Further aspects of the invention relate to isolated double
stranded nucleic acid molecule comprising a guide strand and a
passenger strand, wherein the guide strand is from 16-29
nucleotides long and is substantially complementary to a target
gene, wherein the passenger strand is from 8-14 nucleotides long
and has complementarity to the guide strand, and wherein the guide
stand has a single stranded 3' region that is 5 nucleotides or
longer. In some embodiments, the single stranded region contains at
least 2 phosphorothioate modifications.
[0078] Further aspects of the invention relate to isolated double
stranded nucleic acid molecules including a guide strand and a
passenger strand, wherein the guide strand is from 18-21
nucleotides long and is substantially complementary to a target
gene, wherein the passenger strand is from 11-14 nucleotides long
and has complementarity to the guide strand, and wherein position
one of the guide stand has 2-OMe and 5' phosphate modifications,
every C and U in positions 2 to 11 of the guide strand are 2-F
modified, every C and U in positions 12-18 of the guide strand are
2'OMe modified, and 80% of Cs and Us on the passenger strand are
2'OMe modified
[0079] Another aspect of the invention relates to isolated double
stranded nucleic acid molecules including a guide strand and a
passenger strand, wherein the guide strand is from 18-21
nucleotides long and is substantially complementary to a target
gene, wherein the passenger strand is from 11-14 nucleotides long
and has complementarity to the guide strand, and wherein the guide
stand has 2-OMe and 5' phosphate modifications at position 1, every
C and U in positions 2 to 11 of the guide strand are 2-F modified,
every C and U in positions 12-18 of the guide strand are 2'OMe
modified, 80% of Cs and Us on the passenger strand are 2'OMe and
the 3' end of the passenger strand is attached to a conjugate. In
some embodiments the conjugate is selected from sterols,
sterol-type molecules, hydrophobic vitamins or fatty acids.
[0080] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways.
BRIEF DESCRIPTION OF DRAWINGS
[0081] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component may be
labeled in every drawing. In the drawings:
[0082] FIG. 1 is a schematic depicting proposed structures of
asymmetric double stranded RNA molecules (adsRNA). Bold lines
represent sequences carrying modification patterns compatible with
RISC loading. Striped lines represent polynucleotides carrying
modifications compatible with passenger strands. Plain lines
represent a single stranded polynucleotide with modification
patterns optimized for cell interaction and uptake. FIG. 1A depicts
adsRNA with extended guide or passenger strands; FIG. 1B depicts
adsRNA with length variations of a cell penetrating polynucleotide;
FIG. 1C depicts adsRNA with 3' and 5' conjugates; FIG. 1D depicts
adsRNAs with mismatches.
[0083] FIG. 2 is a schematic depicting asymmetric dsRNA molecules
with different chemical modification patterns. Several examples of
chemical modifications that might be used to increase
hydrophobicity are shown including 4-pyridyl, 2-pyridyl, isobutyl
and indolyl based position 5 uridine modifications.
[0084] FIG. 3 is a schematic depicting the use of dsRNA binding
domains, protamine (or other Arg rich peptides), spermidine or
similar chemical structures to block duplex charge to facilitate
cellular entry.
[0085] FIG. 4 is a schematic depicting positively charged chemicals
that might be used for polynucleotide charge blockage.
[0086] FIG. 5 is a schematic depicting examples of structural and
chemical compositions of single stranded RISC entering
polynucleotides. The combination of one or more modifications
including 2'd, 2'Ome, 2'F, hydrophobic and phosphorothioate
modifications can be used to optimize single strand entry into the
RISC.
[0087] FIG. 6 is a schematic depicting examples of structural and
chemical composition of RISC substrate inhibitors. Combinations of
one or more chemical modifications can be used to mediate efficient
uptake and efficient binding to preloaded RISC complex.
[0088] FIG. 7 is a schematic depicting structures of
polynucleotides with sterol type molecules attached, where R
represent a polycarbonic tail of 9 carbons or longer. FIG. 7A
depicts an adsRNA molecule; FIG. 7B depicts an siRNA molecule of
approximately 17-30 bp long; FIG. 7C depicts a RISC entering
strand; FIG. 7D depicts a substrate analog strand. Chemical
modification patterns, as depicted in FIG. 7, can be optimized to
promote desired function.
[0089] FIG. 8 is a schematic depicting examples of naturally
occurring phytosterols with a polycarbon chain that is longer than
8, attached at position 17. More than 250 different types of
phytosterols are known.
[0090] FIG. 9 is a schematic depicting examples of sterol-like
structures, with variations in the size of the polycarbon chains
attached at position 17.
[0091] FIG. 10 presents schematics and graphs demonstrating that
the percentage of liver uptake and plasma clearance of lipid
emulsions containing sterol type molecules is directly affected by
the size of the polycarbon chain attached at position 17. This
figure is adapted from Martins et al, Journal of Lipid Research
(1998).
[0092] FIG. 11 is a schematic depicting micelle formation. FIG. 11A
depicts a polynucleotide with a hydrophobic conjugate; FIG. 11B
depicts linoleic acid; FIG. 11C depicts a micelle formed from a
mixture of polynucleotides containing hydrophobic conjugates
combined with fatty acids.
[0093] FIG. 12 is a schematic depicting how alteration in lipid
composition can affect pharmacokinetic behavior and tissue
distribution of hydrophobically modified and/or hydrophobically
conjugated polynucleotides. In particular, use of lipid mixtures
enriched in linoleic acid and cardiolipin results in preferential
uptake by cardiomyocites.
[0094] FIG. 13 is a schematic showing examples of RNAi constructs
and controls used to target MAP4K4 expression. RNAi construct 12083
corresponds to SEQ ID NOs:597 and 598. RNAi construct 12089
corresponds to SEQ ID NO:599.
[0095] FIG. 14 is a graph showing MAP4K4 expression following
transfection with RNAi constructs associated with the invention.
RNAi constructs tested were: 12083 (Nicked), 12085 (13nt Duplex),
12089 (No Stem Pairing) and 12134 (13nt miniRNA). Results of
transfection were compared to an untransfected control sample. RNAi
construct 12083 corresponds to SEQ ID NOs:597 and 598. RNAi
construct 12085 corresponds to SEQ ID NOs:600 and 601. RNAi
construct 12089 corresponds to SEQ ID NO:599. RNAi construct 12134
corresponds to SEQ ID NOs:602 and 603.
[0096] FIG. 15 is a graph showing expression of MAP4K4 24 hours
post-transfection with RNAi constructs associated with the
invention. RNAi constructs tested were: 11546 (MAP4K4 rxRNA), 12083
(MAP4K4 Nicked Construct), 12134 (12 bp soloRNA) and 12241 (14/3/14
soloRNA). Results of transfection were compared to a filler control
sample. RNAi construct 11546 corresponds to SEQ ID NOs:604 and 605.
RNAi construct 12083 corresponds to SEQ ID NOs:597 and 598. RNAi
construct 12134 corresponds to SEQ ID NOs:602 and 603. RNAi
construct 12241 corresponds to SEQ ID NOs:606 and 607.
[0097] FIG. 16 presents a graph and several tables comparing
parameters associated with silencing of MAP4K4 expression following
transfection with RNAi constructs associated with the invention.
The rxRNA construct corresponds to SEQ ID NOs:604 and 605. The
14-3-14 soloRNA construct corresponds to SEQ ID NOs:606 and 607.
The 13/19 duplex (nicked construct) corresponds to SEQ ID NOs:597
and 598. The 12-bp soloRNA construct corresponds to SEQ ID NOs:602
and 603.
[0098] FIG. 17 is a schematic showing examples of RNAi constructs
and controls used to target SOD1 expression. The 12084 RNAi
construct corresponds to SEQ ID NOs:612 and 613.
[0099] FIG. 18 is a graph showing SOD1 expression following
transfection with RNAi constructs associated with the invention.
RNAi constructs tested were: 12084 (Nicked), 12086 (13nt Duplex),
12090 (No Stem Pairing) and 12035 (13nt MiniRNA). Results of
transfection were compared to an untransfected control sample. The
12084 RNAi construct corresponds to SEQ ID NOs:612 and 613. The
12086 RNAi construct corresponds to SEQ ID NOs:608 and 609. The
12035 RNAi construct corresponds to SEQ ID NOs:610 and 611.
[0100] FIG. 19 is a graph showing expression of SOD1 24 hours
post-transfection with RNAi constructs associated with the
invention. RNAi constructs tested were: 10015 (SOD1 rxRNA) and
12084 (SOD1 Nicked Construct). Results of transfection were
compared to a filler control sample. The 10015 RNAi construct
corresponds to SEQ ID NOs:614 and 615. The 12084 RNAi construct
corresponds to SEQ ID NOs:612 and 613.
[0101] FIG. 20 is a schematic indicating that RNA molecules with
double stranded regions that are less than 10 nucleotides are not
cleaved by Dicer.
[0102] FIG. 21 is a schematic revealing a hypothetical RNAi model
for RNA induced gene silencing.
[0103] FIG. 22 is a graph showing chemical optimization of
asymmetric RNAi compounds. The presence of chemical modifications,
in particular 2'F UC, phosphorothioate modifications on the guide
strand, and complete CU 2'OMe modification of the passenger strands
results in development of functional compounds. Silencing of MAP4K4
following lipid-mediated transfection is shown using RNAi molecules
with specific modifications. RNAi molecules tested had sense
strands that were 13 nucleotides long and contained the following
modifications: unmodified; C and U 2'OMe; C and U 2'OMe and 3' Chl;
rxRNA 2'OMe pattern; or full 2'OMe, except base 1. Additionally,
the guide (anti-sense) strands of the RNAi molecules tested
contained the following modifications: unmodified; unmodified with
5'P; C and U 2'F; C and U 2'F with 8 PS 3' end; and unmodified (17
nt length). Results for rxRNA 12/10 Duplex and negative controls
are also shown.
[0104] FIG. 23 demonstrates that the chemical modifications
described herein significantly increase in vitro efficacy in
un-assisted delivery of RNAi molecules in HeLa cells. The structure
and sequence of the compounds were not altered; only the chemical
modification patterns of the molecules were modified. Compounds
lacking 2' F, 2'O-me, phosphorothioate modification, or cholesterol
conjugates were completely inactive in passive uptake. A
combination of all 4 of these types of modifications produced the
highest levels of activity (compound 12386).
[0105] FIG. 24 is a graph showing MAP4K4 expression in Hela cells
following passive uptake transfection of: NT Accell modified siRNA,
MAP4K4 Accell siRNA, Non-Chl nanoRNA (12379) and sd-nanoRNA
(12386).
[0106] FIG. 25 is a graph showing expression of MAP4K4 in HeLa
cells following passive uptake transfection of various
concentrations of RNA molecules containing the following
parameters: Nano Lead with no 3'Chl; Nano Lead; Accell MAP4K4;
21mer GS with 8 PS tail; 21mer GS with 12 PS tail; and 25mer GS
with 12 PS tail.
[0107] FIG. 26 is a graph demonstrating that reduction in
oligonucleotide content increases the efficacy of unassisted
uptake. Similar chemical modifications were applied to assymetric
compounds, traditional siRNA compounds and 25 mer RNAi compounds.
The assymetric small compounds demonstrated the most significant
efficacy.
[0108] FIG. 27 is a graph demonstrating the importance of
phosphorothioate content for un-assisted delivery. FIG. 27A
demonstrates the results of a systematic screen that revealed that
the presence of at least 2-12 phosphorothioates in the guide strand
significantly improves uptake; in some embodiments, 4-8
phosphorothioate modifications were found to be preferred. FIG. 27
B reveals that the presence or absence of phosphorothioate
modifications in the sense strand did not alter efficacy.
[0109] FIG. 28 is a graph showing expression of MAP4K4 in primary
mouse hepatocytes following passive uptake transfection of: Accell
Media-Ctrl-UTC; MM APOB Alnylam; Active APOB Alnylam; nanoRNA
without chl; nanoRNA MAP4K4; Mouse MAP4K4 Accell Smartpool; DY547
Accell Control; Luc Ctrl rxRNA with Dy547; MAP4K4 rxRNA with DY547;
and AS Strand Alone (nano).
[0110] FIG. 29 is a graph showing expression of ApoB in mouse
primary hepatocytes following passive uptake transfection of:
Accell Media-Ctrl-UTC; MM APOB Alnylam; Active APOB Alnylam;
nanoRNA without chl; nanoRNA MAP4K4; Mouse MAP4K4 Accell Smartpool;
DY547 Accell Control; Luc Ctrl rxRNA with Dy547; MAP4K4 rxRNA with
DY547; and AS Strand Alone (nano).
[0111] FIG. 30 is a graph showing expression of MAP4K4 in primary
human hepatocytes following passive uptake transfection of: 11550
MAP4K4 rxRNA; 12544 MM MAP4K4 nanoRNA; 12539 Active MAP4K4 nanoRNA;
Accell Media; and UTC.
[0112] FIG. 31 is a graph showing ApoB expression in primary human
hepatoctyes following passive uptake transfection of: 12505 Active
ApoB chol-siRNA; 12506 MM ApoB chol-siRNA; Accell Media; and
UTC.
[0113] FIG. 32 is an image depicting localization of
sd-rxRNA.sup.nano localization.
[0114] FIG. 33 is an image depicting localization of Chol-siRNA
(Alnylam).
[0115] FIG. 34 is a schematic of 1.sup.st generation (G1)
sd-rxRNA.sup.nano molecules associated with the invention
indicating regions that are targeted for modification, and
functions associated with different regions of the molecules.
[0116] FIG. 35 depicts modification patterns that were screened for
optimization of sd-rxRNA.sup.nano (G1). The modifications that were
screened included, on the guide strand, lengths of 19, 21 and 25
nucleotides, phosphorothioate modifications of 0-18 nucleotides,
and replacement of 2'F modifications with 2'OMe, 5 Methyl C and/or
ribo Thymidine modifications. Modifications on the sense strand
that were screened included nucleotide lengths of 11, 13 and 19
nucleotides, phosphorothiote modifications of 0-4 nucleotides and
2'OMe modifications.
[0117] FIG. 36 is a schematic depicting modifications of
sd-rxRNA.sup.nano that were screened for optimization.
[0118] FIG. 37 is a graph showing percent MAP4K4 expression in
Hek293 cells following transfection of: Risc Free siRNA; rxRNA;
Nano (unmodified); GS alone; Nano Lead (no Chl); Nano (GS: (3)
2'OMe at positions 1, 18, and 19, 8 PS, 19 nt); Nano (GS: (3) 2'OMe
at positions 1, 18, and 19, 8 PS, 21 nt); Nano (GS: (3) 2'OMe at
positions 1, 18, and 19, 12 PS, 21 nt); and Nano (GS: (3) 2'OMe at
positions 1, 18, and 19, 12 PS, 25 nt);
[0119] FIG. 38 is a graph showing percent MAP4K4 expression in HeLa
cells following passive uptake transfection of: GS alone; Nano
Lead; Nano (GS: (3) 2'OMe at positions 1, 18, and 19, 8 PS, 19 nt);
Nano (GS: (3) 2'OMe at positions 1, 18, and 19, 8 PS, 21 nt); Nano
(GS: (3) 2'OMe at positions 1, 18, and 19, 12 PS, 21 nt); Nano (GS:
(3) 2'OMe at positions 1, 18, and 19, 12 PS, 25 nt).
[0120] FIG. 39 is a graph showing percent MAP4K4 expression in
Hek293 cells following lipid mediated transfection of: Guide Strand
alone (GS: 8PS, 19 nt); Guide Strand alone (GS: 18PS, 19 nt); Nano
(GS: no PS, 19 nt); Nano (GS: 2 PS, 19 nt); Nano (GS: 4 PS, 19 nt);
Nano (GS: 6 PS, 19 nt); Nano Lead (GS: 8 PS, 19 nt); Nano (GS: 10
PS, 19 nt); Nano (GS: 12 PS, 19 nt); and Nano (GS: 18 PS, 19
nt).
[0121] FIG. 40 is a graph showing percent MAP4K4 expression in
Hek293 cells following lipid mediated transfection of: Guide Strand
alone (GS: 8PS, 19 nt); Guide Strand alone (GS: 18PS, 19 nt); Nano
(GS: no PS, 19 nt); Nano (GS: 2 PS, 19 nt); Nano (GS: 4 PS, 19 nt);
Nano (GS: 6 PS, 19 nt); Nano Lead (GS: 8 PS, 19 nt); Nano (GS: 10
PS, 19 nt); Nano (GS: 12 PS, 19 nt); and Nano (GS: 18 PS, 19
nt).
[0122] FIG. 41 is a graph showing percent MAP4K4 expression in HeLa
cells following passive uptake transfection of: Nano Lead (no Chl);
Guide Strand alone (18 PS); Nano (GS: 0 PS, 19 nt); Nano (GS: 2 PS,
19 nt); Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt); Nano Lead
(GS: 8 PS, 19 nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS, 19
nt); and Nano (GS: 18 PS, 19 nt).
[0123] FIG. 42 is a graph showing percent MAP4K4 expression in HeLa
cells following passive uptake transfection of: Nano Lead (no Chl);
Guide Strand alone (18 PS); Nano (GS: 0 PS, 19 nt); Nano (GS: 2 PS,
19 nt); Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt); Nano Lead
(GS: 8 PS, 19 nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS, 19
nt); and Nano (GS: 18 PS, 19 nt).
[0124] FIG. 43 is a schematic depicting guide strand chemical
modifications that were screened for optimization.
[0125] FIG. 44 is a graph showing percent MAP4K4 expression in
Hek293 cells following reverse transfection of: RISC free siRNA; GS
only (2'F C and Us); GS only (2'OMe C and Us); Nano Lead (2'F C and
Us); nano (GS: (3) 2'OMe, positions 16-18); nano (GS: (3) 2'OMe,
positions 16, 17 and 19); nano (GS: (4) 2'OMe, positions 11,
16-18); nano (GS: (10) 2'OMe, C and Us); nano (GS: (6) 2'OMe,
positions 1 and 5-9); nano (GS: (3) 2'OMe, positions 1, 18 and 19);
and nano (GS: (5) 2'OMe Cs).
[0126] FIG. 45 is a graph demonstrating efficacy of various
chemical modification patterns. In particular, 2-OMe modification
in positions 1 and 11-18 was well tolerated. 2'OMe modifications in
the seed area resulted in a slight reduction of efficacy (but were
still highly efficient). Ribo-modifications in the seed were well
tolerated. This data enabled the generation of self delivering
compounds with reduced or no 2'F modifications. This is significant
because 2'F modifications may be associated with toxicity in
vivo.
[0127] FIG. 46 is a schematic depicting sense strand
modifications.
[0128] FIG. 47 is a graph demonstrating sense strand length
optimization. A sense strand length between 10-15 bases was found
to be optimal in this assay. Increasing sense strand length
resulted in a reduction of passive uptake of these compounds but
may be tolerated for other compounds. Sense strands containing LNA
modification demonstrated similar efficacy to non-LNA containing
compounds. In some embodiments, the addition of LNA or other
thermodynamically stabilizing compounds can be beneficial,
resulting in converting non-functional sequences into functional
sequences.
[0129] FIG. 48 is a graph showing percent MAP4K4 expression in HeLa
cells following passive uptake transfection of: Guide Strand Alone
(2'F C and U); Nano Lead; Nano Lead (No Chl); Nano (SS: 11 nt 2'OMe
C and Us, Chl); Nano (SS: lint, complete 2'OMe, Chl); Nano (SS: 19
nt, 2'OMe C and Us, Chl); Nano (SS: 19 nt, 2'OMe C and Us, no
Chl).
[0130] FIG. 49 is a graph showing percent MAP4K4 expression in HeLa
cells following passive uptake transfection of: Nano Lead (No Chl);
Nano (SS no PS); Nano Lead (SS:2 PS); Nano (SS:4 PS).
[0131] FIG. 50 is a schematic depicting a sd-rxRNA.sup.nano second
generation (GII) lead molecule.
[0132] FIG. 51 presents a graph indicating EC50 values for MAP4K4
silencing in the presence of sd-rxRNA, and images depicting
localization of DY547-labeled rxRNA.sup.ori and DY547-labeled
sd-rxRNA.
[0133] FIG. 52 is a graph showing percent MAP4K4 expression in HeLa
cells in the presence of optimized sd-rxRNA molecules.
[0134] FIG. 53 is a graph depicting the relevance of chemistry
content in optimization of sd-rxRNA efficacy.
[0135] FIG. 54 presents schematics of sterol-type molecules and a
graph revealing that sd-rxRNA compounds are fully functional with a
variety of linker chemistries. GII asymmetric compounds were
synthesized with steroltype molecules attached through TEG and
amino caproic acid linkers. Both linkers showed identical potency.
This functionality independent of linker chemistry indicates a
significant difference between the molecules described herein and
previously described molecules, and offers significant advantages
for the molecules described herein in terms of scale up and
synthesis.
[0136] FIG. 55 demonstrates the stability of chemically modified
sd-rxRNA compounds in human serum in comparison to non modified
RNA. The oligonucleotides were incubated in 75% serum at 37.degree.
C. for the number of hours indicated. The level of degradation was
determined by running the samples on non-denaturing gels and
staining with SYBGR.
[0137] FIG. 56 is a graph depicting optimization of cellular uptake
of sd-rxRNA through minimizing oligonucleotide content.
[0138] FIG. 57 is a graph showing percent MAP4K4 expression after
spontaneous cellular uptake of sd-rxRNA in mouse PEC-derived
macrophages, and phase and fluorescent images showing localization
of sd-rxRNA.
[0139] FIG. 58 is a graph showing percent MAP4K4 expression after
spontaneous cellular uptake of sd-rxRNA (targeting) and sd-rxRNA
(mismatch) in mouse primary hepatocytes, and phase and fluorescent
images showing localization of sd-rxRNA.
[0140] FIG. 59 presents images depicting localization of
DY547-labeled sd-rxRNA delivered to RPE cells with no
formulation.
[0141] FIG. 60 is a graph showing silencing of MAP4K4 expression in
RPE cells treated with sd-rxRNA.sup.nano without formulation.
[0142] FIG. 61 presents a graph and schematics of RNAi compounds
showing the chemical/structural composition of highly effective
sd-rxRNA compounds. Highly effective compounds were found to have
the following characteristics: antisense strands of 17-21
nucleotides, sense strands of 10-15 nucleotides, single-stranded
regions that contained 2-12 phosphorothioate modifications,
preferentially 6-8 phosphorothioate modifications, and sense
strands in which the majority of nucleotides were 2'OMe modified,
with or without phosphorothioate modification. Any linker chemistry
can be used to attach these molecules to hydrophobic moieties such
as cholesterol at the 3' end of the sense strand. Version GIIa-b of
these RNA compounds demonstrate that elimination of 2'F content has
no impact on efficacy.
[0143] FIG. 62 presents a graph and schematics of RNAi compounds
demonstrating the superior performance of sd-rxRNA compounds
compared to compounds published by Wolfrum et. al. Nature Biotech,
2007. Both generation I and II compounds (GI and GIIa) developed
herein show great efficacy. By contrast, when the chemistry
described in Wolfrum et al. (all oligos contain cholesterol
conjugated to the 3' end of the sense strand) was applied to the
same sequence in a context of conventional siRNA (19 bp duplex with
two overhang) the compound was practically inactive. These data
emphasize the significance of the combination of chemical
modifications and assymetrical molecules described herein,
producing highly effective RNA compounds.
[0144] FIG. 63 presents images showing that sd-rxRNA accumulates
inside cells while other less effective conjugate RNAs accumulate
on the surface of cells.
[0145] FIG. 64 presents images showing that sd-rxRNA molecules, but
not other molecules, are internalized into cells within
minutes.
[0146] FIG. 65 presents images demonstrating that sd-rxRNA
compounds have drastically better cellular and tissue uptake
characteristics when compared to conventional cholesterol
conjugated siRNAs (such as those published by Soucheck et al). FIG.
65A,B compare uptake in RPE cells, FIG. 65C,D compare uptake upon
local administration to skin and FIG. 65E,F compare uptake by the
liver upon systemic administration. The level of uptake is at least
an order of magnitude higher for the sd-rxRNA compounds relative to
the regular siRNA-cholesterol compounds.
[0147] FIG. 66 presents images depicting localization of
rxRNA.sup.ori and sd-rxRNA following local delivery.
[0148] FIG. 67 presents images depicting localization of sd-rxRNA
and other conjugate RNAs following local delivery.
[0149] FIG. 68 presents a graph revealing the results of a screen
performed with sd-rxRNAGII chemistry to identify functional
compounds targeting the SPP1 gene. Multiple effective compounds
were identified, with 14131 being the most effective. The compounds
were added to A-549 cells and the level of the ratio of SPP1/PPIB
was determined by B-DNA after 48 hours.
[0150] FIG. 69 presents a graph and several images demonstrating
efficient cellular uptake of sd-rxRNA within minutes of exposure.
This is a unique characteristics of the sd-rxRNA compounds
described herein, not observed with any other RNAi compounds. The
Soutschek et al. compound was used as a negative control.
[0151] FIG. 70 presents a graph and several images demonstrating
efficient uptake and silencing of sd-rxRNA compounds in multiple
cell types with multiple sequences. In each case silencing was
confirmed by looking at target gene expression using a Branched DNA
assay.
[0152] FIG. 71 presents a graph revealing that sd-rxRNA is active
in the presence and absence of serum. A slight reduction in
efficacy (2-5 fold) was observed in the presence of serum. This
minimal reduction in efficacy in the presence of serum
differentiates the sd-rxRNA compounds described herein from
previously described RNAi compounds, which had a greater reduction
in efficacy, and thus creates a foundation for in vivo efficacy of
the sd-rxRNA molecules described herein.
[0153] FIG. 72 presents images demonstrating efficient tissue
penetration and cellular uptake upon single intradermal injection
of sd-rxRNA compounds described herein. This represents a model for
local delivery of sd-rxRNA compounds as well as an effective
demonstration of delivery of sd-rxRNA compounds and silencing of
genes in dermatological applications.
[0154] FIG. 73 presents images and a graph demonstrating efficient
cellular uptake and in vivo silencing with sd-rxRNA following
intradermal injection.
[0155] FIG. 74 presents graphs demonstrating that sd-rxRNA
compounds have improved blood clearance and induce effective gene
silencing in vivo in the liver upon systemic administration.
[0156] FIG. 75 presents a graph demonstrating that the presence of
5-Methyl C in an RNAi compound resulted in an increase in potency
of lipid mediated transfection, demonstrating that hydrophobic
modification of Cs and Us in the content of RNAi compounds can be
beneficial. In some embodiments, these types of modifications can
be used in the context of 2' ribose modified bases to insure
optimal stability and efficacy.
[0157] FIG. 76 presents a graph showing percent MAP4K4 expression
in HeLa cells following passive uptake transfection of: Guide
strand alone; Nano Lead; Nano Lead (No cholesterol); Guide Strand
w/5MeC and 2'F Us Alone; Nano Lead w/GS 5MeC and 2'F Us; Nano Lead
w/GS riboT and 5 Methyl Cs; and Nano Lead w/Guide dT and 5 Methyl
Cs.
[0158] FIG. 77 presents images comparing localization of sd-rxRNA
and other RNA conjugates following systemic delivery to the
liver.
[0159] FIG. 78 presents schematics demonstrating 5-uridyl
modifications with improved hydrophobicity characteristics (FIG.
78A). Incorporation of such modifications into sd-rxRNA compounds
can increase cellular and tissue uptake properties. FIG. 78B
presents a new type of RNAi compound modification which can be
applied to compounds to improve cellular uptake and pharmacokinetic
behavior. This type of modification, when applied to sd-rxRNA
compounds, may contribute to making such compounds orally
available.
[0160] FIG. 79 presents schematics revealing the structures of
synthesized modified sterol type molecules, where the length and
structure of the C17 attached tail is modified. Without wishing to
be bound by any theory, the length of the C17 attached tail may
contribute to improving in vitro and in vivo efficacy of sd-rxRNA
compounds.
[0161] FIG. 80 presents a schematic demonstrating the lithocholic
acid route to long side chain cholesterols.
[0162] FIG. 81 presents a schematic demonstrating a route to
5-uridyl phosphoramidite synthesis.
[0163] FIG. 82 presents a schematic demonstrating synthesis of
tri-functional hydroxyprolinol linker for 3'-cholesterol
attachment.
[0164] FIG. 83 presents a schematic demonstrating synthesis of
solid support for the manufacture of a shorter asymmetric RNAi
compound strand.
[0165] FIG. 84 demonstrates SPPI sd-rxRNA compound selection.
Sd-rxRNA compounds targeting SPP1 were added to A549 cells (using
passive transfection) and the level of SPP1 expression was
evaluated after 48 hours. Several novel compounds effective in SPP1
silencing were identified, the most potent of which was compound
14131.
[0166] FIG. 85 demonstrates independent validation of sd-rxRNA
compounds 14116, 14121, 14131, 14134, 14139, 14149, and 14152
efficacy in SPP1 silencing.
[0167] FIG. 86 demonstrates results of sd-rxRNA compound screens to
identify sd-rxRNA compounds functional in CTGF knockdown.
[0168] FIG. 87 demonstrates results of sd-rxRNA compound screens to
identify sd-rxRNA functional in CTGF knockdown.
[0169] FIG. 88 demonstrates a systematic screen identifying the
minimal length of the asymmetric compounds. The passenger strand of
10-19 bases was hybridized to a guide strand of 17-25 bases. In
this assay, compounds with duplex regions as short as 10 bases were
found to be effective in inducing.
[0170] FIG. 89 demonstrates that positioning of the sense strand
relative to the guide strand is critical for RNAi Activity. In this
assay, a blunt end was found to be optimal, a 3' overhang was
tolerated, and a 5' overhang resulted in complete loss of
functionality.
[0171] FIG. 90 demonstrates that the guide strand, which has
homology to the target only at nucleotides 2-17, resulted in
effective RNAi when hybridized with sense strands of different
lengths. The compounds were introduced into HeLa cells via lipid
mediated transfection.
[0172] FIG. 91 is a schematic depicting a panel of sterol-type
molecules which can be used as a hydrophobic entity in place of
cholesterol. In some instances, the use of sterol-type molecules
comprising longer chains results in generation of sd-rxRNA
compounds with significantly better cellular uptake and tissue
distribution properties.
[0173] FIG. 92 presents schematics depicting a panel of hydrophobic
molecules which might be used as a hydrophobic entity in place of
cholesterol. These list just provides representative examples; any
small molecule with substantial hydrophobicity can be used.
DETAILED DESCRIPTION
[0174] Aspects of the invention relate to methods and compositions
involved in gene silencing. The invention is based at least in part
on the surprising discovery that asymmetric nucleic acid molecules
with a double stranded region of a minimal length such as 8-14
nucleotides, are effective in silencing gene expression. Molecules
with such a short double stranded region have not previously been
demonstrated to be effective in mediating RNA interference. It had
previously been assumed that that there must be a double stranded
region of 19 nucleotides or greater. The molecules described herein
are optimized through chemical modification, and in some instances
through attachment of hydrophobic conjugates.
[0175] The invention is based at least in part on another
surprising discovery that asymmetric nucleic acid molecules with
reduced double stranded regions are much more effectively taken up
by cells compared to conventional siRNAs. These molecules are
highly efficient in silencing of target gene expression and offer
significant advantages over previously described RNAi molecules
including high activity in the presence of serum, efficient self
delivery, compatibility with a wide variety of linkers, and reduced
presence or complete absence of chemical modifications that are
associated with toxicity.
[0176] In contrast to single-stranded polynucleotides, duplex
polynucleotides have been difficult to deliver to a cell as they
have rigid structures and a large number of negative charges which
makes membrane transfer difficult. Unexpectedly, it was found that
the polynucleotides of the present invention, although partially
double-stranded, are recognized in vivo as single-stranded and, as
such, are capable of efficiently being delivered across cell
membranes. As a result the polynucleotides of the invention are
capable in many instances of self delivery. Thus, the
polynucleotides of the invention may be formulated in a manner
similar to conventional RNAi agents or they may be delivered to the
cell or subject alone (or with non-delivery type carriers) and
allowed to self deliver. In one embodiment of the present
invention, self delivering asymmetric double-stranded RNA molecules
are provided in which one portion of the molecule resembles a
conventional RNA duplex and a second portion of the molecule is
single stranded.
[0177] The polynucleotides of the invention are referred to herein
as isolated double stranded or duplex nucleic acids,
oligonucleotides or polynucleotides, nano molecules, nano RNA,
sd-rxRNA.sup.nano, sd-rxRNA or RNA molecules of the invention.
[0178] The oligonucleotides of the invention in some aspects have a
combination of asymmetric structures including a double stranded
region and a single stranded region of 5 nucleotides or longer,
specific chemical modification patterns and are conjugated to
lipophilic or hydrophobic molecules. This new class of RNAi like
compounds have superior efficacy in vitro and in vivo. Based on the
data described herein it is believed that the reduction in the size
of the rigid duplex region in combination with phosphorothioate
modifications applied to a single stranded region are new and
important for achieving the observed superior efficacy. Thus, the
RNA molecules described herein are different in both structure and
composition as well as in vitro and in vivo activity.
[0179] In a preferred embodiment the RNAi compounds of the
invention comprise an asymmetric compound comprising a duplex
region (required for efficient RISC entry of 10-15 bases long) and
single stranded region of 4-12 nucleotides long; with a 13
nucleotide duplex. A 6 nucleotide single stranded region is
preferred in some embodiments. The single stranded region of the
new RNAi compounds also comprises 2-12 phosphorothioate
internucleotide linkages (referred to as phosphorothioate
modifications). 6-8 phosphorothioate internucleotide linkages are
preferred in some embodiments. Additionally, the RNAi compounds of
the invention also include a unique chemical modification pattern,
which provides stability and is compatible with RISC entry. The
combination of these elements has resulted in unexpected properties
which are highly useful for delivery of RNAi reagents in vitro and
in vivo.
[0180] The chemically modification pattern, which provides
stability and is compatible with RISC entry includes modifications
to the sense, or passenger, strand as well as the antisense, or
guide, strand. For instance the passenger strand can be modified
with any chemical entities which confirm stability and do not
interfere with activity. Such modifications include 2' ribo
modifications (O-methyl, 2' F, 2 deoxy and others) and backbone
modification like phosphorothioate modifications. A preferred
chemical modification pattern in the passenger strand includes
Omethyl modification of C and U nucleotides within the passenger
strand or alternatively the passenger strand may be completely
Omethyl modified.
[0181] The guide strand, for example, may also be modified by any
chemical modification which confirms stability without interfering
with RISC entry. A preferred chemical modification pattern in the
guide strand includes the majority of C and U nucleotides being 2'
F modified and the 5' end being phosphorylated. Another preferred
chemical modification pattern in the guide strand includes 2'
Omethyl modification of position 1 and C/U in positions 11-18 and
5' end chemical phosphorylation. Yet another preferred chemical
modification pattern in the guide strand includes 2'Omethyl
modification of position 1 and C/U in positions 11-18 and 5' end
chemical phosphorylation and and 2'F modification of C/U in
positions 2-10.
[0182] It was surprisingly discovered according to the invention
that the above-described chemical modification patterns of the
oligonucleotides of the invention are well tolerated and actually
improved efficacy of asymmetric RNAi compounds. See, for instance,
FIG. 22.
[0183] It was also demonstrated experimentally herein that the
combination of modifications to RNAi when used together in a
polynucleotide results in the achievement of optimal efficacy in
passive uptake of the RNAi. Elimination of any of the described
components (Guide strand stabilization, phosphorothioate stretch,
sense strand stabilization and hydrophobic conjugate) or increase
in size results in sub-optimal efficacy and in some instances
complete lost of efficacy. The combination of elements results in
development of compound, which is fully active following passive
delivery to cells such as HeLa cells. (FIG. 23). The degree to
which the combination of elements results in efficient self
delivery of RNAi molecules was completely unexpected.
[0184] The data shown in FIGS. 26, 27 and 43 demonstrated the
importance of the various modifications to the RNAi in achieving
stabilization and activity. For instance, FIG. 26 demonstrates that
use off asymmetric configuration is important in getting efficacy
in passive uptake. When the same chemical composition is applied to
compounds of traditional configurations (19-21 bases duplex and 25
mer duplex) the efficacy was drastically decreased in a length
dependent manner. FIG. 27 demonstrated a systematic screen of the
impact of phosphorothioate chemical modifications on activity. The
sequence, structure, stabilization chemical modifications,
hydrophobic conjugate were kept constant and compound
phosphorothioate content was varied (from 0 to 18 PS bond). Both
compounds having no phosphorothioate linkages and having 18
phosphorothioate linkages were completely inactive in passive
uptake. Compounds having 2-16 phosphorothioate linkages were
active, with compounds having 4-10 phosphorothioate being the most
active compounds.
[0185] The data in the Examples presented below demonstrates high
efficacy of the oligonucleotides of the invention both in vitro in
variety of cell types (supporting data) and in vivo upon local and
systemic administration. For instance, the data compares the
ability of several competitive RNAi molecules having different
chemistries to silence a gene. Comparison of sd-rxRNA
(oligonucleotides of the invention) with RNAs described in Soucheck
et al. and Wolfrum at al., as applied to the same targeting region,
demonstrated that only sd-rxRNA chemistry showed a significant
functionality in passive uptake. The composition of the invention
achieved EC50 values of 10-50 pM. This level of efficacy is
un-attainable with conventional chemistries like those described in
Sauthceck at al and Accell. Similar comparisons were made in other
systems, such as in vitro (RPE cell line), in vivo upon local
administration (wounded skin) and systemic (50 mg/kg) as well as
other genes (FIGS. 65 and 68). In each case the oligonucleotides of
the invention achieved better results. FIG. 64 includes data
demonstrating efficient cellular uptake and resulting silencing by
sd-rxRNA compounds only after 1 minute of exposure. Such an
efficacy is unique to this composition and have not been seen with
other types of molecules in this class. FIG. 70 demonstrates
efficient uptake and silencing of sd-rxRNA compounds in multiple
cell types with multiple sequences. The sd-rxRNA compounds are also
active in cells in presence and absence of serum and other
biological liquids. FIG. 71 demonstrates only a slight reduction in
activity in the presence of serum. This ability to function in
biologically aggressive environment effectively further
differentiates sd-rxRNA compounds from other compounds described
previously in this group, like Accell and Soucheck et al, in which
uptake is drastically inhibited in a presence of serum.
[0186] Significant amounts of data also demonstrate the in vivo
efficacy of the compounds of the invention. For instance FIGS.
72-74 involve multiple routes of in vivo delivery of the compounds
of the invention resulting in significant activity. FIG. 72, for
example, demonstrates efficient tissue penetration and cellular
uptake upon single intradermal injection. This is a model for local
delivery of sd-rxRNA compounds as well as an effective delivery
mode for sd-rxRNA compounds and silencing genes in any dermatology
applications. FIG. 73 demonstrated efficient tissue penetration,
cellular uptake and silencing upon local in vivo intradermal
injection of sd-rxRNA compounds. The data of FIG. 74 demonstrate
that sd-rxRNA compounds result in highly effective liver uptake
upon IV administration. Comparison to Souicheck at al molecule
showed that the level of liver uptake at identical dose level was
quite surprisingly, at least 50 fold higher with the sd-rxRNA
compound than the Souicheck at al molecule.
[0187] The sd-rxRNA can be further improved in some instances by
improving the hydrophobicity of compounds using of novel types of
chemistries. For example one chemistry is related to use of
hydrophobic base modifications. Any base in any position might be
modified, as long as modification results in an increase of the
partition coefficient of the base. The preferred locations for
modification chemistries are positions 4 and 5 of the pyrimidines.
The major advantage of these positions is (a) ease of synthesis and
(b) lack of interference with base-pairing and A form helix
formation, which are essential for RISC complex loading and target
recognition. Examples of these chemistries is shown in FIGS. 75-83.
A version of sd-rxRNA compounds where multiple deoxy Uridines are
present without interfering with overall compound efficacy was
used. In addition major improvement in tissue distribution and
cellular uptake might be obtained by optimizing the structure of
the hydrophobic conjugate. In some of the preferred embodiment the
structure of sterol is modified to alter (increase/decrease) C17
attached chain. This type of modification results in significant
increase in cellular uptake and improvement of tissue uptake
prosperities in vivo.
[0188] This invention is not limited in its application to the
details of construction and the arrangement of components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments and of being practiced or
of being carried out in various ways. Also, the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of "including,"
"comprising," or "having," "containing," "involving," and
variations thereof herein, is meant to encompass the items listed
thereafter and equivalents thereof as well as additional items.
[0189] Thus, aspects of the invention relate to isolated double
stranded nucleic acid molecules comprising a guide (antisense)
strand and a passenger (sense) strand. As used herein, the term
"double-stranded" refers to one or more nucleic acid molecules in
which at least a portion of the nucleomonomers are complementary
and hydrogen bond to form a double-stranded region. In some
embodiments, the length of the guide strand ranges from 16-29
nucleotides long. In certain embodiments, the guide strand is 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides
long. The guide strand has complementarity to a target gene.
Complementarity between the guide strand and the target gene may
exist over any portion of the guide strand. Complementarity as used
herein may be perfect complementarity or less than perfect
complementarity as long as the guide strand is sufficiently
complementary to the target that it mediates RNAi. In some
embodiments complementarity refers to less than 25%, 20%, 15%, 10%,
5%, 4%, 3%, 2%, or 1% mismatch between the guide strand and the
target. Perfect complementarity refers to 100% complementarity.
Thus the invention has the advantage of being able to tolerate
sequence variations that might be expected due to genetic mutation,
strain polymorphism, or evolutionary divergence. For example, siRNA
sequences with insertions, deletions, and single point mutations
relative to the target sequence have also been found to be
effective for inhibition. Moreover, not all positions of a siRNA
contribute equally to target recognition. Mismatches in the center
of the siRNA are most critical and essentially abolish target RNA
cleavage. Mismatches upstream of the center or upstream of the
cleavage site referencing the antisense strand are tolerated but
significantly reduce target RNA cleavage. Mismatches downstream of
the center or cleavage site referencing the antisense strand,
preferably located near the 3' end of the antisense strand, e.g. 1,
2, 3, 4, 5 or 6 nucleotides from the 3' end of the antisense
strand, are tolerated and reduce target RNA cleavage only
slightly.
[0190] While not wishing to be bound by any particular theory, in
some embodiments, the guide strand is at least 16 nucleotides in
length and anchors the Argonaute protein in RISC. In some
embodiments, when the guide strand loads into RISC it has a defined
seed region and target mRNA cleavage takes place across from
position 10-11 of the guide strand. In some embodiments, the 5' end
of the guide strand is or is able to be phosphorylated. The nucleic
acid molecules described herein may be referred to as minimum
trigger RNA.
[0191] In some embodiments, the length of the passenger strand
ranges from 8-14 nucleotides long. In certain embodiments, the
passenger strand is 8, 9, 10, 11, 12, 13 or 14 nucleotides long.
The passenger strand has complementarity to the guide strand.
Complementarity between the passenger strand and the guide strand
can exist over any portion of the passenger or guide strand. In
some embodiments, there is 100% complementarity between the guide
and passenger strands within the double stranded region of the
molecule.
[0192] Aspects of the invention relate to double stranded nucleic
acid molecules with minimal double stranded regions. In some
embodiments the region of the molecule that is double stranded
ranges from 8-14 nucleotides long. In certain embodiments, the
region of the molecule that is double stranded is 8, 9, 10, 11, 12,
13 or 14 nucleotides long. In certain embodiments the double
stranded region is 13 nucleotides long. There can be 100%
complementarity between the guide and passenger strands, or there
may be one or more mismatches between the guide and passenger
strands. In some embodiments, on one end of the double stranded
molecule, the molecule is either blunt-ended or has a
one-nucleotide overhang. The single stranded region of the molecule
is in some embodiments between 4-12 nucleotides long. For example
the single stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12
nucleotides long. However, in certain embodiments, the single
stranded region can also be less than 4 or greater than 12
nucleotides long. In certain embodiments, the single stranded
region is 6 nucleotides long.
[0193] RNAi constructs associated with the invention can have a
thermodynamic stability (.DELTA.G) of less than -13 kkal/mol. In
some embodiments, the thermodynamic stability (.DELTA.G) is less
than -20 kkal/mol. In some embodiments there is a loss of efficacy
when (.DELTA.G) goes below -21 kkal/mol. In some embodiments a
(.DELTA.G) value higher than -13 kkal/mol is compatible with
aspects of the invention. Without wishing to be bound by any
theory, in some embodiments a molecule with a relatively higher
(.DELTA.G) value may become active at a relatively higher
concentration, while a molecule with a relatively lower (.DELTA.G)
value may become active at a relatively lower concentration. In
some embodiments, the (.DELTA.G) value may be higher than -9
kkcal/mol. The gene silencing effects mediated by the RNAi
constructs associated with the invention, containing minimal double
stranded regions, are unexpected because molecules of almost
identical design but lower thermodynamic stability have been
demonstrated to be inactive (Rana et al 2004).
[0194] Without wishing to be bound by any theory, results described
herein suggest that a stretch of 8-10 bp of dsRNA or dsDNA will be
structurally recognized by protein components of RISC or co-factors
of RISC. Additionally, there is a free energy requirement for the
triggering compound that it may be either sensed by the protein
components and/or stable enough to interact with such components so
that it may be loaded into the Argonaute protein. If optimal
thermodynamics are present and there is a double stranded portion
that is preferably at least 8 nucleotides then the duplex will be
recognized and loaded into the RNAi machinery.
[0195] In some embodiments, thermodynamic stability is increased
through the use of LNA bases. In some embodiments, additional
chemical modifications are introduced. Several non-limiting
examples of chemical modifications include: 5' Phosphate,
2'-O-methyl, 2'-O-ethyl, 2'-fluoro, ribothymidine, C-5 propynyl-dC
(pdC) and C-5 propynyl-dU (pdU); C-5 propynyl-C(pC) and C-5
propynyl-U (pU); 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU
methoxy, (2,6-diaminopurine),
5'-Dimethoxytrityl-N4-ethyl-2'-deoxyCytidine and MGB (minor groove
binder). It should be appreciated that more than one chemical
modification can be combined within the same molecule.
[0196] Molecules associated with the invention are optimized for
increased potency and/or reduced toxicity. For example, nucleotide
length of the guide and/or passenger strand, and/or the number of
phosphorothioate modifications in the guide and/or passenger
strand, can in some aspects influence potency of the RNA molecule,
while replacing 2'-fluoro (2'F) modifications with 2'-O-methyl
(2'OMe) modifications can in some aspects influence toxicity of the
molecule. Specifically, reduction in 2'F content of a molecule is
predicted to reduce toxicity of the molecule. The Examples section
presents molecules in which 2'F modifications have been eliminated,
offering an advantage over previously described RNAi compounds due
to a predicted reduction in toxicity. Furthermore, the number of
phosphorothioate modifications in an RNA molecule can influence the
uptake of the molecule into a cell, for example the efficiency of
passive uptake of the molecule into a cell. Preferred embodiments
of molecules described herein have no 2'F modification and yet are
characterized by equal efficacy in cellular uptake and tissue
penetration. Such molecules represent a significant improvement
over prior art, such as molecules described by Accell and Wolfrum,
which are heavily modified with extensive use of 2'F.
[0197] In some embodiments, a guide strand is approximately 18-19
nucleotides in length and has approximately 2-14 phosphate
modifications. For example, a guide strand can contain 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14 or more than 14 nucleotides that are
phosphate-modified. The guide strand may contain one or more
modifications that confer increased stability without interfering
with RISC entry. The phosphate modified nucleotides, such as
phosphorothioate modified nucleotides, can be at the 3' end, 5' end
or spread throughout the guide strand. In some embodiments, the 3'
terminal 10 nucleotides of the guide strand contains 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10 phosphorothioate modified nucleotides. The guide
strand can also contain 2'F and/or 2'OMe modifications, which can
be located throughout the molecule. In some embodiments, the
nucleotide in position one of the guide strand (the nucleotide in
the most 5' position of the guide strand) is 2'OMe modified and/or
phosphorylated. C and U nucleotides within the guide strand can be
2'F modified. For example, C and U nucleotides in positions 2-10 of
a 19 nt guide strand (or corresponding positions in a guide strand
of a different length) can be 2'F modified. C and U nucleotides
within the guide strand can also be 2'OMe modified. For example, C
and U nucleotides in positions 11-18 of a 19 nt guide strand (or
corresponding positions in a guide strand of a different length)
can be 2'OMe modified. In some embodiments, the nucleotide at the
most 3' end of the guide strand is unmodified. In certain
embodiments, the majority of Cs and Us within the guide strand are
2'F modified and the 5' end of the guide strand is phosphorylated.
In other embodiments, position 1 and the Cs or Us in positions
11-18 are 2'OMe modified and the 5' end of the guide strand is
phosphorylated. In other embodiments, position 1 and the Cs or Us
in positions 11-18 are 2'OMe modified, the 5' end of the guide
strand is phosphorylated, and the Cs or Us in position 2-10 are 2'F
modified.
[0198] In some aspects, an optimal passenger strand is
approximately 11-14 nucleotides in length. The passenger strand may
contain modifications that confer increased stability. One or more
nucleotides in the passenger strand can be 2'OMe modified. In some
embodiments, one or more of the C and/or U nucleotides in the
passenger strand is 2'OMe modified, or all of the C and U
nucleotides in the passenger strand are 2'OMe modified. In certain
embodiments, all of the nucleotides in the passenger strand are
2'OMe modified. One or more of the nucleotides on the passenger
strand can also be phosphate-modified such as phosphorothioate
modified. The passenger strand can also contain 2' ribo, 2'F and 2
deoxy modifications or any combination of the above. As
demonstrated in the Examples, chemical modification patterns on
both the guide and passenger strand are well tolerated and a
combination of chemical modifications is shown herein to lead to
increased efficacy and self-delivery of RNA molecules.
[0199] Aspects of the invention relate to RNAi constructs that have
extended single-stranded regions relative to double stranded
regions, as compared to molecules that have been used previously
for RNAi. The single stranded region of the molecules may be
modified to promote cellular uptake or gene silencing. In some
embodiments, phosphorothioate modification of the single stranded
region influences cellular uptake and/or gene silencing. The region
of the guide strand that is phosphorothioate modified can include
nucleotides within both the single stranded and double stranded
regions of the molecule. In some embodiments, the single stranded
region includes 2-12 phosphorothioate modifications. For example,
the single stranded region can include 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, or 12 phosphorothioate modifications. In some instances, the
single stranded region contains 6-8 phosphorothioate
modifications.
[0200] Molecules associated with the invention are also optimized
for cellular uptake. In RNA molecules described herein, the guide
and/or passenger strands can be attached to a conjugate. In certain
embodiments the conjugate is hydrophobic. The hydrophobic conjugate
can be a small molecule with a partition coefficient that is higher
than 10. The conjugate can be a sterol-type molecule such as
cholesterol, or a molecule with an increased length polycarbon
chain attached to C17, and the presence of a conjugate can
influence the ability of an RNA molecule to be taken into a cell
with or without a lipid transfection reagent. The conjugate can be
attached to the passenger or guide strand through a hydrophobic
linker. In some embodiments, a hydrophobic linker is 5-12C in
length, and/or is hydroxypyrrolidine-based. In some embodiments, a
hydrophobic conjugate is attached to the passenger strand and the
CU residues of either the passenger and/or guide strand are
modified. In some embodiments, at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90% or 95% of the CU residues on the passenger
strand and/or the guide strand are modified. In some aspects,
molecules associated with the invention are self-delivering (sd).
As used herein, "self-delivery" refers to the ability of a molecule
to be delivered into a cell without the need for an additional
delivery vehicle such as a transfection reagent.
[0201] Aspects of the invention relate to selecting molecules for
use in RNAi. Based on the data described herein, molecules that
have a double stranded region of 8-14 nucleotides can be selected
for use in RNAi. In some embodiments, molecules are selected based
on their thermodynamic stability (.DELTA.G). In some embodiments,
molecules will be selected that have a (.DELTA.G) of less than -13
kkal/mol. For example, the (.DELTA.G) value may be -13, -14, -15,
-16, -17, -18, -19, -21, -22 or less than -22 kkal/mol. In other
embodiments, the (.DELTA.G) value may be higher than -13 kkal/mol.
For example, the (.DELTA.G) value may be -12, -11, -10, -9, -8, -7
or more than -7 kkal/mol. It should be appreciated that .DELTA.G
can be calculated using any method known in the art. In some
embodiments .DELTA.G is calculated using Mfold, available through
the Mfold internet site
(http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cfi). Methods for
calculating .DELTA.G are described in, and are incorporated by
reference from, the following references: Zuker, M. (2003) Nucleic
Acids Res., 31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M.
and Turner, D. H. (1999) J. Mol. Biol. 288:911-940; Mathews, D. H.,
Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and
Turner, D. H. (2004) Proc. Natl. Acad. Sci. 101:7287-7292; Duan,
S., Mathews, D. H., and Turner, D. H. (2006) Biochemistry
45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I. L., and
Schuster, P. (1999) Biopolymers 49:145-165.
[0202] Aspects of the invention relate to using nucleic acid
molecules described herein, with minimal double stranded regions
and/or with a (.DELTA.G) of less than -13 kkal/mol, for gene
silencing. RNAi molecules can be administered in vivo or in vitro,
and gene silencing effects can be achieved in vivo or in vitro.
[0203] In certain embodiments, the polynucleotide contains 5'-
and/or 3'-end overhangs. The number and/or sequence of nucleotides
overhang on one end of the polynucleotide may be the same or
different from the other end of the polynucleotide. In certain
embodiments, one or more of the overhang nucleotides may contain
chemical modification(s), such as phosphorothioate or 2'-OMe
modification.
[0204] In certain embodiments, the polynucleotide is unmodified. In
other embodiments, at least one nucleotide is modified. In further
embodiments, the modification includes a 2'-H or 2'-modified ribose
sugar at the 2nd nucleotide from the 5'-end of the guide sequence.
The "2nd nucleotide" is defined as the second nucleotide from the
5'-end of the polynucleotide.
[0205] As used herein, "2'-modified ribose sugar" includes those
ribose sugars that do not have a 2'-OH group. "2'-modified ribose
sugar" does not include 2'-deoxyribose (found in unmodified
canonical DNA nucleotides). For example, the 2'-modified ribose
sugar may be 2'-O-alkyl nucleotides, 2'-deoxy-2'-fluoro
nucleotides, 2'-deoxy nucleotides, or combination thereof.
[0206] In certain embodiments, the 2'-modified nucleotides are
pyrimidine nucleotides (e.g., C/U). Examples of 2'-O-alkyl
nucleotides include 2'-O-methyl nucleotides, or 2'-O-allyl
nucleotides.
[0207] In certain embodiments, the miniRNA polynucleotide of the
invention with the above-referenced 5'-end modification exhibits
significantly (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) less "off-target"
gene silencing when compared to similar constructs without the
specified 5'-end modification, thus greatly improving the overall
specificity of the RNAi reagent or therapeutics.
[0208] As used herein, "off-target" gene silencing refers to
unintended gene silencing due to, for example, spurious sequence
homology between the antisense (guide) sequence and the unintended
target mRNA sequence.
[0209] According to this aspect of the invention, certain guide
strand modifications further increase nuclease stability, and/or
lower interferon induction, without significantly decreasing RNAi
activity (or no decrease in RNAi activity at all).
[0210] In some embodiments, the 5'-stem sequence may comprise a
2'-modified ribose sugar, such as 2'-O-methyl modified nucleotide,
at the 2.sup.nd nucleotide on the 5'-end of the polynucleotide and,
in some embodiments, no other modified nucleotides. The hairpin
structure having such modification may have enhanced target
specificity or reduced off-target silencing compared to a similar
construct without the 2'-O-methyl modification at said
position.
[0211] Certain combinations of specific 5'-stem sequence and
3'-stem sequence modifications may result in further unexpected
advantages, as partly manifested by enhanced ability to inhibit
target gene expression, enhanced serum stability, and/or increased
target specificity, etc.
[0212] In certain embodiments, the guide strand comprises a
2'-O-methyl modified nucleotide at the 2.sup.nd nucleotide on the
5'-end of the guide strand and no other modified nucleotides.
[0213] In other aspects, the miniRNA structures of the present
invention mediates sequence-dependent gene silencing by a microRNA
mechanism. As used herein, the term "microRNA" ("miRNA"), also
referred to in the art as "small temporal RNAs" ("stRNAs"), refers
to a small (10-50 nucleotide) RNA which are genetically encoded
(e.g., by viral, mammalian, or plant genomes) and are capable of
directing or mediating RNA silencing. An "miRNA disorder" shall
refer to a disease or disorder characterized by an aberrant
expression or activity of an miRNA.
[0214] microRNAs are involved in down-regulating target genes in
critical pathways, such as development and cancer, in mice, worms
and mammals. Gene silencing through a microRNA mechanism is
achieved by specific yet imperfect base-pairing of the miRNA and
its target messenger RNA (mRNA). Various mechanisms may be used in
microRNA-mediated down-regulation of target mRNA expression.
[0215] miRNAs are noncoding RNAs of approximately 22 nucleotides
which can regulate gene expression at the post transcriptional or
translational level during plant and animal development. One common
feature of miRNAs is that they are all excised from an
approximately 70 nucleotide precursor RNA stem-loop termed
pre-miRNA, probably by Dicer, an RNase III-type enzyme, or a
homolog thereof. Naturally-occurring miRNAs are expressed by
endogenous genes in vivo and are processed from a hairpin or
stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or other
RNAses. miRNAs can exist transiently in vivo as a double-stranded
duplex but only one strand is taken up by the RISC complex to
direct gene silencing.
[0216] In some embodiments a version of sd-rxRNA compounds, which
are effective in cellular uptake and inhibiting of miRNA activity
are described. Essentially the compounds are similar to RISC
entering version but large strand chemical modification patterns
are optimized in the way to block cleavage and act as an effective
inhibitor of the RISC action. For example, the compound might be
completely or mostly Omethyl modified with the PS content described
previously. For these types of compounds the 5' phosphorilation is
not necessary. The presence of double stranded region is preferred
as it is promotes cellular uptake and efficient RISC loading.
[0217] Another pathway that uses small RNAs as sequence-specific
regulators is the RNA interference (RNAi) pathway, which is an
evolutionarily conserved response to the presence of
double-stranded RNA (dsRNA) in the cell. The dsRNAs are cleaved
into .about.20-base pair (bp) duplexes of small-interfering RNAs
(siRNAs) by Dicer. These small RNAs get assembled into multiprotein
effector complexes called RNA-induced silencing complexes (RISCs).
The siRNAs then guide the cleavage of target mRNAs with perfect
complementarity.
[0218] Some aspects of biogenesis, protein complexes, and function
are shared between the siRNA pathway and the miRNA pathway. The
subject single-stranded polynucleotides may mimic the dsRNA in the
siRNA mechanism, or the microRNA in the miRNA mechanism.
[0219] In certain embodiments, the modified RNAi constructs may
have improved stability in serum and/or cerebral spinal fluid
compared to an unmodified RNAi constructs having the same
sequence.
[0220] In certain embodiments, the structure of the RNAi construct
does not induce interferon response in primary cells, such as
mammalian primary cells, including primary cells from human, mouse
and other rodents, and other non-human mammals. In certain
embodiments, the RNAi construct may also be used to inhibit
expression of a target gene in an invertebrate organism.
[0221] To further increase the stability of the subject constructs
in vivo, the 3'-end of the hairpin structure may be blocked by
protective group(s). For example, protective groups such as
inverted nucleotides, inverted abasic moieties, or amino-end
modified nucleotides may be used. Inverted nucleotides may comprise
an inverted deoxynucleotide. Inverted abasic moieties may comprise
an inverted deoxyabasic moiety, such as a 3',3'-linked or
5',5'-linked deoxyabasic moiety.
[0222] The RNAi constructs of the invention are capable of
inhibiting the synthesis of any target protein encoded by target
gene(s). The invention includes methods to inhibit expression of a
target gene either in a cell in vitro, or in vivo. As such, the
RNAi constructs of the invention are useful for treating a patient
with a disease characterized by the overexpression of a target
gene.
[0223] The target gene can be endogenous or exogenous (e.g.,
introduced into a cell by a virus or using recombinant DNA
technology) to a cell. Such methods may include introduction of RNA
into a cell in an amount sufficient to inhibit expression of the
target gene. By way of example, such an RNA molecule may have a
guide strand that is complementary to the nucleotide sequence of
the target gene, such that the composition inhibits expression of
the target gene.
[0224] The invention also relates to vectors expressing the nucleic
acids of the invention, and cells comprising such vectors or the
nucleic acids. The cell may be a mammalian cell in vivo or in
culture, such as a human cell.
[0225] The invention further relates to compositions comprising the
subject RNAi constructs, and a pharmaceutically acceptable carrier
or diluent.
[0226] Another aspect of the invention provides a method for
inhibiting the expression of a target gene in a mammalian cell,
comprising contacting the mammalian cell with any of the subject
RNAi constructs.
[0227] The method may be carried out in vitro, ex vivo, or in vivo,
in, for example, mammalian cells in culture, such as a human cell
in culture.
[0228] The target cells (e.g., mammalian cell) may be contacted in
the presence of a delivery reagent, such as a lipid (e.g., a
cationic lipid) or a liposome.
[0229] Another aspect of the invention provides a method for
inhibiting the expression of a target gene in a mammalian cell,
comprising contacting the mammalian cell with a vector expressing
the subject RNAi constructs.
[0230] In one aspect of the invention, a longer duplex
polynucleotide is provided, including a first polynucleotide that
ranges in size from about 16 to about 30 nucleotides; a second
polynucleotide that ranges in size from about 26 to about 46
nucleotides, wherein the first polynucleotide (the antisense
strand) is complementary to both the second polynucleotide (the
sense strand) and a target gene, and wherein both polynucleotides
form a duplex and wherein the first polynucleotide contains a
single stranded region longer than 6 bases in length and is
modified with alternative chemical modification pattern, and/or
includes a conjugate moiety that facilitates cellular delivery. In
this embodiment, between about 40% to about 90% of the nucleotides
of the passenger strand between about 40% to about 90% of the
nucleotides of the guide strand, and between about 40% to about 90%
of the nucleotides of the single stranded region of the first
polynucleotide are chemically modified nucleotides.
[0231] In an embodiment, the chemically modified nucleotide in the
polynucleotide duplex may be any chemically modified nucleotide
known in the art, such as those discussed in detail above. In a
particular embodiment, the chemically modified nucleotide is
selected from the group consisting of 2' F modified nucleotides,
2'-O-methyl modified and 2'deoxy nucleotides. In another particular
embodiment, the chemically modified nucleotides results from
"hydrophobic modifications" of the nucleotide base. In another
particular embodiment, the chemically modified nucleotides are
phosphorothioates. In an additional particular embodiment,
chemically modified nucleotides are combination of
phosphorothioates, 2'-O-methyl, 2'deoxy, hydrophobic modifications
and phosphorothioates. As these groups of modifications refer to
modification of the ribose ring, back bone and nucleotide, it is
feasible that some modified nucleotides will carry a combination of
all three modification types.
[0232] In another embodiment, the chemical modification is not the
same across the various regions of the duplex. In a particular
embodiment, the first polynucleotide (the passenger strand), has a
large number of diverse chemical modifications in various
positions. For this polynucleotide up to 90% of nucleotides might
be chemically modified and/or have mismatches introduced.
[0233] In another embodiment, chemical modifications of the first
or second polynucleotide include, but not limited to, 5' position
modification of Uridine and Cytosine (4-pyridyl, 2-pyridyl,
indolyl, phenyl (C.sub.6H.sub.5OH); tryptophanyl
(C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl;
naphthyl, etc), where the chemical modification might alter base
pairing capabilities of a nucleotide. For the guide strand an
important feature of this aspect of the invention is the position
of the chemical modification relative to the 5' end of the
antisense and sequence. For example, chemical phosphorylation of
the 5' end of the guide strand is usually beneficial for efficacy.
O-methyl modifications in the seed region of the sense strand
(position 2-7 relative to the 5' end) are not generally well
tolerated, whereas 2'F and deoxy are well tolerated. The mid part
of the guide strand and the 3' end of the guide strand are more
permissive in a type of chemical modifications applied. Deoxy
modifications are not tolerated at the 3' end of the guide
strand.
[0234] A unique feature of this aspect of the invention involves
the use of hydrophobic modification on the bases. In one
embodiment, the hydrophobic modifications are preferably positioned
near the 5' end of the guide strand, in other embodiments, they
localized in the middle of the guides strand, in other embodiment
they localized at the 3' end of the guide strand and yet in another
embodiment they are distributed thought the whole length of the
polynucleotide. The same type of patterns is applicable to the
passenger strand of the duplex.
[0235] The other part of the molecule is a single stranded region.
The single stranded region is expected to range from 7 to 40
nucleotides.
[0236] In one embodiment, the single stranded region of the first
polynucleotide contains modifications selected from the group
consisting of between 40% and 90% hydrophobic base modifications,
between 40%-90% phosphorothioates, between 40%-90% modification of
the ribose moiety, and any combination of the preceding.
[0237] Efficiency of guide strand (first polynucleotide) loading
into the RISC complex might be altered for heavily modified
polynucleotides, so in one embodiment, the duplex polynucleotide
includes a mismatch between nucleotide 9, 11, 12, 13, or 14 on the
guide strand (first polynucleotide) and the opposite nucleotide on
the sense strand (second polynucleotide) to promote efficient guide
strand loading.
[0238] More detailed aspects of the invention are described in the
sections below.
Duplex Characteristics
[0239] Double-stranded oligonucleotides of the invention may be
formed by two separate complementary nucleic acid strands. Duplex
formation can occur either inside or outside the cell containing
the target gene.
[0240] As used herein, the term "duplex" includes the region of the
double-stranded nucleic acid molecule(s) that is (are) hydrogen
bonded to a complementary sequence. Double-stranded
oligonucleotides of the invention may comprise a nucleotide
sequence that is sense to a target gene and a complementary
sequence that is antisense to the target gene. The sense and
antisense nucleotide sequences correspond to the target gene
sequence, e.g., are identical or are sufficiently identical to
effect target gene inhibition (e.g., are about at least about 98%
identical, 96% identical, 94%, 90% identical, 85% identical, or 80%
identical) to the target gene sequence.
[0241] In certain embodiments, the double-stranded oligonucleotide
of the invention is double-stranded over its entire length, i.e.,
with no overhanging single-stranded sequence at either end of the
molecule, i.e., is blunt-ended. In other embodiments, the
individual nucleic acid molecules can be of different lengths. In
other words, a double-stranded oligonucleotide of the invention is
not double-stranded over its entire length. For instance, when two
separate nucleic acid molecules are used, one of the molecules,
e.g., the first molecule comprising an antisense sequence, can be
longer than the second molecule hybridizing thereto (leaving a
portion of the molecule single-stranded). Likewise, when a single
nucleic acid molecule is used a portion of the molecule at either
end can remain single-stranded.
[0242] In one embodiment, a double-stranded oligonucleotide of the
invention contains mismatches and/or loops or bulges, but is
double-stranded over at least about 70% of the length of the
oligonucleotide. In another embodiment, a double-stranded
oligonucleotide of the invention is double-stranded over at least
about 80% of the length of the oligonucleotide. In another
embodiment, a double-stranded oligonucleotide of the invention is
double-stranded over at least about 90%-95% of the length of the
oligonucleotide. In another embodiment, a double-stranded
oligonucleotide of the invention is double-stranded over at least
about 96%-98% of the length of the oligonucleotide. In certain
embodiments, the double-stranded oligonucleotide of the invention
contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, or 15 mismatches.
Modifications
[0243] The nucleotides of the invention may be modified at various
locations, including the sugar moiety, the phosphodiester linkage,
and/or the base.
[0244] Sugar moieties include natural, unmodified sugars, e.g.,
monosaccharide (such as pentose, e.g., ribose, deoxyribose),
modified sugars and sugar analogs. In general, possible
modifications of nucleomonomers, particularly of a sugar moiety,
include, for example, replacement of one or more of the hydroxyl
groups with a halogen, a heteroatom, an aliphatic group, or the
functionalization of the hydroxyl group as an ether, an amine, a
thiol, or the like.
[0245] One particularly useful group of modified nucleomonomers are
2'-O-methyl nucleotides. Such 2'-O-methyl nucleotides may be
referred to as "methylated," and the corresponding nucleotides may
be made from unmethylated nucleotides followed by alkylation or
directly from methylated nucleotide reagents. Modified
nucleomonomers may be used in combination with unmodified
nucleomonomers. For example, an oligonucleotide of the invention
may contain both methylated and unmethylated nucleomonomers.
[0246] Some exemplary modified nucleomonomers include sugar- or
backbone-modified ribonucleotides. Modified ribonucleotides may
contain a non-naturally occurring base (instead of a naturally
occurring base), such as uridines or cytidines modified at the
5'-position, e.g., 5'-(2-amino)propyl uridine and 5'-bromo uridine;
adenosines and guanosines modified at the 8-position, e.g., 8-bromo
guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; and
N-alkylated nucleotides, e.g., N6-methyl adenosine. Also,
sugar-modified ribonucleotides may have the 2'-OH group replaced by
a H, alxoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as
NH2, NHR, NR.sub.2,), or CN group, wherein R is lower alkyl,
alkenyl, or alkynyl.
[0247] Modified ribonucleotides may also have the phosphodiester
group connecting to adjacent ribonucleotides replaced by a modified
group, e.g., of phosphorothioate group. More generally, the various
nucleotide modifications may be combined.
[0248] Although the antisense (guide) strand may be substantially
identical to at least a portion of the target gene (or genes), at
least with respect to the base pairing properties, the sequence
need not be perfectly identical to be useful, e.g., to inhibit
expression of a target gene's phenotype. Generally, higher homology
can be used to compensate for the use of a shorter antisense gene.
In some cases, the antisense strand generally will be substantially
identical (although in antisense orientation) to the target
gene.
[0249] The use of 2'-O-methyl modified RNA may also be beneficial
in circumstances in which it is desirable to minimize cellular
stress responses. RNA having 2'-O-methyl nucleomonomers may not be
recognized by cellular machinery that is thought to recognize
unmodified RNA. The use of 2'-O-methylated or partially
2'-O-methylated RNA may avoid the interferon response to
double-stranded nucleic acids, while maintaining target RNA
inhibition. This may be useful, for example, for avoiding the
interferon or other cellular stress responses, both in short RNAi
(e.g., siRNA) sequences that induce the interferon response, and in
longer RNAi sequences that may induce the interferon response.
[0250] Overall, modified sugars may include D-ribose, 2'-O-alkyl
(including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino,
2'-S-alkyl, 2'-halo (including 2'-fluoro), 2'-methoxyethoxy,
2'-allyloxy (--OCH.sub.2CH.dbd.CH.sub.2), 2'-propargyl, 2'-propyl,
ethynyl, ethenyl, propenyl, and cyano and the like. In one
embodiment, the sugar moiety can be a hexose and incorporated into
an oligonucleotide as described (Augustyns, K., et al., Nucl.
Acids. Res. 18:4711 (1992)). Exemplary nucleomonomers can be found,
e.g., in U.S. Pat. No. 5,849,902, incorporated by reference
herein.
[0251] The term "alkyl" includes saturated aliphatic groups,
including straight-chain alkyl groups (e.g., methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.),
branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl,
etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl
groups, and cycloalkyl substituted alkyl groups. In certain
embodiments, a straight chain or branched chain alkyl has 6 or
fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.6 for
straight chain, C.sub.3-C.sub.6 for branched chain), and more
preferably 4 or fewer. Likewise, preferred cycloalkyls have from
3-8 carbon atoms in their ring structure, and more preferably have
5 or 6 carbons in the ring structure. The term C.sub.1-C.sub.6
includes alkyl groups containing 1 to 6 carbon atoms.
[0252] Moreover, unless otherwise specified, the term alkyl
includes both "unsubstituted alkyls" and "substituted alkyls," the
latter of which refers to alkyl moieties having independently
selected substituents replacing a hydrogen on one or more carbons
of the hydrocarbon backbone. Such substituents can include, for
example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. Cycloalkyls can be further
substituted, e.g., with the substituents described above. An
"alkylaryl" or an "arylalkyl" moiety is an alkyl substituted with
an aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also
includes the side chains of natural and unnatural amino acids. The
term "n-alkyl" means a straight chain (i.e., unbranched)
unsubstituted alkyl group.
[0253] The term "alkenyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but that contain at least one double bond. For
example, the term "alkenyl" includes straight-chain alkenyl groups
(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,
octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,
cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,
cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl
substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl
substituted alkenyl groups. In certain embodiments, a straight
chain or branched chain alkenyl group has 6 or fewer carbon atoms
in its backbone (e.g., C.sub.2-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain). Likewise, cycloalkenyl groups
may have from 3-8 carbon atoms in their ring structure, and more
preferably have 5 or 6 carbons in the ring structure. The term
C.sub.2-C.sub.6 includes alkenyl groups containing 2 to 6 carbon
atoms.
[0254] Moreover, unless otherwise specified, the term alkenyl
includes both "unsubstituted alkenyls" and "substituted alkenyls,"
the latter of which refers to alkenyl moieties having independently
selected substituents replacing a hydrogen on one or more carbons
of the hydrocarbon backbone. Such substituents can include, for
example, alkyl groups, alkynyl groups, halogens, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0255] The term "alkynyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but which contain at least one triple bond. For
example, the term "alkynyl" includes straight-chain alkynyl groups
(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl,
octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups,
and cycloalkyl or cycloalkenyl substituted alkynyl groups. In
certain embodiments, a straight chain or branched chain alkynyl
group has 6 or fewer carbon atoms in its backbone (e.g.,
C.sub.2-C.sub.6 for straight chain, C.sub.3-C.sub.6 for branched
chain). The term C.sub.2-C.sub.6 includes alkynyl groups containing
2 to 6 carbon atoms.
[0256] Moreover, unless otherwise specified, the term alkynyl
includes both "unsubstituted alkynyls" and "substituted alkynyls,"
the latter of which refers to alkynyl moieties having independently
selected substituents replacing a hydrogen on one or more carbons
of the hydrocarbon backbone. Such substituents can include, for
example, alkyl groups, alkynyl groups, halogens, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0257] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to five carbon atoms in its backbone structure.
"Lower alkenyl" and "lower alkynyl" have chain lengths of, for
example, 2-5 carbon atoms.
[0258] The term "alkoxy" includes substituted and unsubstituted
alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen
atom. Examples of alkoxy groups include methoxy, ethoxy,
isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of
substituted alkoxy groups include halogenated alkoxy groups. The
alkoxy groups can be substituted with independently selected groups
such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulffiydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfmyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moieties. Examples of halogen
substituted alkoxy groups include, but are not limited to,
fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,
dichloromethoxy, trichloromethoxy, etc.
[0259] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.
[0260] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.- (with an appropriate counterion).
[0261] The term "halogen" includes fluorine, bromine, chlorine,
iodine, etc. The term "perhalogenated" generally refers to a moiety
wherein all hydrogens are replaced by halogen atoms.
[0262] The term "substituted" includes independently selected
substituents which can be placed on the moiety and which allow the
molecule to perform its intended function. Examples of substituents
include alkyl, alkenyl, alkynyl, aryl, (CR'R'').sub.0-3 NR'R'',
(CR'R'').sub.0-3 CN, NO.sub.2, halogen, (CR'R'').sub.0-3
C(halogen).sub.3, (CR'R'').sub.0-3 CH(halogen).sub.2,
(CR'R'').sub.0-3CH.sub.2(halogen), (CR'R'').sub.0-3 CONR'R'',
(CR'R'').sub.0-3 S(O).sub.1-2 NR'R'', (CR'R'').sub.0-3 CHO,
(CR'R'').sub.0-3O(CR'R'').sub.0-3 H, (CR'R'').sub.0-3
S(O).sub.0-2R', (CR'R'').sub.0-3O(CR'R'').sub.0-3 H,
(CR'R'').sub.0-3 COR', (CR'R'').sub.0-3 CO.sub.2R', or
(CR'R'').sub.0-3OR' groups; wherein each R' and R'' are each
independently hydrogen, a C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5
alkenyl, C.sub.2-C.sub.5 alkynyl, or aryl group, or R' and R''
taken together are a benzylidene group or a
--(CH.sub.2).sub.2O(CH.sub.2).sub.2-- group.
[0263] The term "amine" or "amino" includes compounds or moieties
in which a nitrogen atom is covalently bonded to at least one
carbon or heteroatom. The term "alkyl amino" includes groups and
compounds wherein the nitrogen is bound to at least one additional
alkyl group. The term "dialkyl amino" includes groups wherein the
nitrogen atom is bound to at least two additional alkyl groups.
[0264] The term "ether" includes compounds or moieties which
contain an oxygen bonded to two different carbon atoms or
heteroatoms. For example, the term includes "alkoxyalkyl," which
refers to an alkyl, alkenyl, or alkynyl group covalently bonded to
an oxygen atom which is covalently bonded to another alkyl
group.
[0265] The term "base" includes the known purine and pyrimidine
heterocyclic bases, deazapurines, and analogs (including
heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine),
derivatives (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic- and
1-alkynyl derivatives) and tautomers thereof. Examples of purines
include adenine, guanine, inosine, diaminopurine, and xanthine and
analogs (e.g., 8-oxo-N6-methyladenine or 7-diazaxanthine) and
derivatives thereof. Pyrimidines include, for example, thymine,
uracil, and cytosine, and their analogs (e.g., 5-methylcytosine,
5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and
4,4-ethanocytosine). Other examples of suitable bases include
non-purinyl and non-pyrimidinyl bases such as 2-aminopyridine and
triazines.
[0266] In a preferred embodiment, the nucleomonomers of an
oligonucleotide of the invention are RNA nucleotides. In another
preferred embodiment, the nucleomonomers of an oligonucleotide of
the invention are modified RNA nucleotides. Thus, the
oligunucleotides contain modified RNA nucleotides.
[0267] The term "nucleoside" includes bases which are covalently
attached to a sugar moiety, preferably ribose or deoxyribose.
Examples of preferred nucleosides include ribonucleosides and
deoxyribonucleosides. Nucleosides also include bases linked to
amino acids or amino acid analogs which may comprise free carboxyl
groups, free amino groups, or protecting groups. Suitable
protecting groups are well known in the art (see P. G. M. Wuts and
T. W. Greene, "Protective Groups in Organic Synthesis", 2.sup.nd
Ed., Wiley-Interscience, New York, 1999).
[0268] The term "nucleotide" includes nucleosides which further
comprise a phosphate group or a phosphate analog.
[0269] As used herein, the term "linkage" includes a naturally
occurring, unmodified phosphodiester moiety (--O--(PO.sup.2)--O--)
that covalently couples adjacent nucleomonomers. As used herein,
the term "substitute linkage" includes any analog or derivative of
the native phosphodiester group that covalently couples adjacent
nucleomonomers. Substitute linkages include phosphodiester analogs,
e.g., phosphorothioate, phosphorodithioate, and
P-ethyoxyphosphodiester, P-ethoxyphosphodiester,
P-alkyloxyphosphotriester, methylphosphonate, and nonphosphorus
containing linkages, e.g., acetals and amides. Such substitute
linkages are known in the art (e.g., Bjergarde et al. 1991. Nucleic
Acids Res. 19:5843; Caruthers et al. 1991. Nucleosides Nucleotides.
10:47). In certain embodiments, non-hydrolizable linkages are
preferred, such as phosphorothiate linkages.
[0270] In certain embodiments, oligonucleotides of the invention
comprise hydrophobicly modified nucleotides or "hydrophobic
modifications." As used herein "hydrophobic modifications" refers
to bases that are modified such that (1) overall hydrophobicity of
the base is significantly increased, and/or (2) the base is still
capable of forming close to regular Watson-Crick interaction.
Several non-limiting examples of base modifications include
5-position uridine and cytidine modifications such as phenyl,
4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C6H5OH);
tryptophanyl (C8H6N)CH2CH(NH2)CO), Isobutyl, butyl, aminobenzyl;
phenyl; and naphthyl.
[0271] In certain embodiments, oligonucleotides of the invention
comprise 3' and 5' termini (except for circular oligonucleotides).
In one embodiment, the 3' and 5' termini of an oligonucleotide can
be substantially protected from nucleases e.g., by modifying the 3'
or 5' linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For
example, oligonucleotides can be made resistant by the inclusion of
a "blocking group." The term "blocking group" as used herein refers
to substituents (e.g., other than OH groups) that can be attached
to oligonucleotides or nucleomonomers, either as protecting groups
or coupling groups for synthesis (e.g., FITC, propyl
(CH.sub.2--CH.sub.2--CH.sub.3), glycol
(--O--CH.sub.2--CH.sub.2--O--) phosphate (PO.sub.3.sup.2-),
hydrogen phosphonate, or phosphoramidite). "Blocking groups" also
include "end blocking groups" or "exonuclease blocking groups"
which protect the 5' and 3' termini of the oligonucleotide,
including modified nucleotides and non-nucleotide exonuclease
resistant structures.
[0272] Exemplary end-blocking groups include cap structures (e.g.,
a 7-methylguanosine cap), inverted nucleomonomers, e.g., with 3'-3'
or 5'-5' end inversions (see, e.g., Ortiagao et al. 1992. Antisense
Res. Dev. 2:129), methylphosphonate, phosphoramidite,
non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers,
conjugates) and the like. The 3' terminal nucleomonomer can
comprise a modified sugar moiety. The 3' terminal nucleomonomer
comprises a 3'-0 that can optionally be substituted by a blocking
group that prevents 3'-exonuclease degradation of the
oligonucleotide. For example, the 3'-hydroxyl can be esterified to
a nucleotide through a 3'.fwdarw.3' internucleotide linkage. For
example, the alkyloxy radical can be methoxy, ethoxy, or
isopropoxy, and preferably, ethoxy. Optionally, the
3'.fwdarw.3'linked nucleotide at the 3' terminus can be linked by a
substitute linkage. To reduce nuclease degradation, the 5' most
3'.fwdarw.5' linkage can be a modified linkage, e.g., a
phosphorothioate or a P-alkyloxyphosphotriester linkage.
Preferably, the two 5' most 3'.fwdarw.5' linkages are modified
linkages. Optionally, the 5' terminal hydroxy moiety can be
esterified with a phosphorus containing moiety, e.g., phosphate,
phosphorothioate, or P-ethoxyphosphate.
[0273] Another type of conjugates that can be attached to the end
(3' or 5' end), the loop region, or any other parts of the miniRNA
might include a sterol, sterol type molecule, peptide, small
molecule, protein, etc. In some embodiments, a miniRNA may contain
more than one conjugates (same or different chemical nature). In
some embodiments, the conjugate is cholesterol.
[0274] Another way to increase target gene specificity, or to
reduce off-target silencing effect, is to introduce a
2'-modification (such as the 2'-O methyl modification) at a
position corresponding to the second 5'-end nucleotide of the guide
sequence. This allows the positioning of this 2'-modification in
the Dicer-resistant hairpin structure, thus enabling one to design
better RNAi constructs with less or no off-target silencing.
[0275] In one embodiment, a hairpin polynucleotide of the invention
can comprise one nucleic acid portion which is DNA and one nucleic
acid portion which is RNA. Antisense (guide) sequences of the
invention can be "chimeric oligonucleotides" which comprise an
RNA-like and a DNA-like region.
[0276] The language "RNase H activating region" includes a region
of an oligonucleotide, e.g., a chimeric oligonucleotide, that is
capable of recruiting RNase H to cleave the target RNA strand to
which the oligonucleotide binds. Typically, the RNase activating
region contains a minimal core (of at least about 3-5, typically
between about 3-12, more typically, between about 5-12, and more
preferably between about 5-10 contiguous nucleomonomers) of DNA or
DNA-like nucleomonomers. (See, e.g., U.S. Pat. No. 5,849,902).
Preferably, the RNase H activating region comprises about nine
contiguous deoxyribose containing nucleomonomers.
[0277] The language "non-activating region" includes a region of an
antisense sequence, e.g., a chimeric oligonucleotide, that does not
recruit or activate RNase H. Preferably, a non-activating region
does not comprise phosphorothioate DNA. The oligonucleotides of the
invention comprise at least one non-activating region. In one
embodiment, the non-activating region can be stabilized against
nucleases or can provide specificity for the target by being
complementary to the target and forming hydrogen bonds with the
target nucleic acid molecule, which is to be bound by the
oligonucleotide.
[0278] In one embodiment, at least a portion of the contiguous
polynucleotides are linked by a substitute linkage, e.g., a
phosphorothioate linkage.
[0279] In certain embodiments, most or all of the nucleotides
beyond the guide sequence (2'-modified or not) are linked by
phosphorothioate linkages. Such constructs tend to have improved
pharmacokinetics due to their higher affinity for serum proteins.
The phosphorothioate linkages in the non-guide sequence portion of
the polynucleotide generally do not interfere with guide strand
activity, once the latter is loaded into RISC.
[0280] Antisense (guide) sequences of the present invention may
include "morpholino oligonucleotides." Morpholino oligonucleotides
are non-ionic and function by an RNase H-independent mechanism.
Each of the 4 genetic bases (Adenine, Cytosine, Guanine, and
Thymine/Uracil) of the morpholino oligonucleotides is linked to a
6-membered morpholine ring. Morpholino oligonucleotides are made by
joining the 4 different subunit types by, e.g., non-ionic
phosphorodiamidate inter-subunit linkages. Morpholino
oligonucleotides have many advantages including: complete
resistance to nucleases (Antisense & Nucl. Acid Drug Dev. 1996.
6:267); predictable targeting (Biochemica Biophysica Acta. 1999.
1489:141); reliable activity in cells (Antisense & Nucl. Acid
Drug Dev. 1997. 7:63); excellent sequence specificity (Antisense
& Nucl. Acid Drug Dev. 1997. 7:151); minimal non-antisense
activity (Biochemica Biophysica Acta. 1999. 1489:141); and simple
osmotic or scrape delivery (Antisense & Nucl. Acid Drug Dev.
1997. 7:291). Morpholino oligonucleotides are also preferred
because of their non-toxicity at high doses. A discussion of the
preparation of morpholino oligonucleotides can be found in
Antisense & Nucl. Acid Drug Dev. 1997. 7:187.
[0281] The chemical modifications described herein are believed,
based on the data described herein, to promote single stranded
polynucleotide loading into the RISC. Single stranded
polynucleotides have been shown to be active in loading into RISC
and inducing gene silencing. However, the level of activity for
single stranded polynucleotides appears to be 2 to 4 orders of
magnitude lower when compared to a duplex polynucleotide.
[0282] The present invention provides a description of the chemical
modification patterns, which may (a) significantly increase
stability of the single stranded polynucleotide (b) promote
efficient loading of the polynucleotide into the RISC complex and
(c) improve uptake of the single stranded nucleotide by the cell.
FIG. 5 provides some non-limiting examples of the chemical
modification patterns which may be beneficial for achieving single
stranded polynucleotide efficacy inside the cell. The chemical
modification patterns may include combination of ribose, backbone,
hydrophobic nucleoside and conjugate type of modifications. In
addition, in some of the embodiments, the 5' end of the single
polynucleotide may be chemically phosphorylated.
[0283] In yet another embodiment, the present invention provides a
description of the chemical modifications patterns, which improve
functionality of RISC inhibiting polynucleotides. Single stranded
polynucleotides have been shown to inhibit activity of a preloaded
RISC complex through the substrate competition mechanism. For these
types of molecules, conventionally called antagomers, the activity
usually requires high concentration and in vivo delivery is not
very effective. The present invention provides a description of the
chemical modification patterns, which may (a) significantly
increase stability of the single stranded polynucleotide (b)
promote efficient recognition of the polynucleotide by the RISC as
a substrate and/or (c) improve uptake of the single stranded
nucleotide by the cell. FIG. 6 provides some non-limiting examples
of the chemical modification patterns that may be beneficial for
achieving single stranded polynucleotide efficacy inside the cell.
The chemical modification patterns may include combination of
ribose, backbone, hydrophobic nucleoside and conjugate type of
modifications.
[0284] The modifications provided by the present invention are
applicable to all polynucleotides. This includes single stranded
RISC entering polynucleotides, single stranded RISC inhibiting
polynucleotides, conventional duplexed polynucleotides of variable
length (15-40 bp), asymmetric duplexed polynucleotides, and the
like. Polynucleotides may be modified with wide variety of chemical
modification patterns, including 5' end, ribose, backbone and
hydrophobic nucleoside modifications.
Synthesis
[0285] Oligonucleotides of the invention can be synthesized by any
method known in the art, e.g., using enzymatic synthesis and/or
chemical synthesis. The oligonucleotides can be synthesized in
vitro (e.g., using enzymatic synthesis and chemical synthesis) or
in vivo (using recombinant DNA technology well known in the
art).
[0286] In a preferred embodiment, chemical synthesis is used for
modified polynucleotides. Chemical synthesis of linear
oligonucleotides is well known in the art and can be achieved by
solution or solid phase techniques. Preferably, synthesis is by
solid phase methods. Oligonucleotides can be made by any of several
different synthetic procedures including the phosphoramidite,
phosphite triester, H-phosphonate, and phosphotriester methods,
typically by automated synthesis methods.
[0287] Oligonucleotide synthesis protocols are well known in the
art and can be found, e.g., in U.S. Pat. No. 5,830,653; WO
98/13526; Stec et al. 1984. J. Am. Chem. Soc. 106:6077; Stec et al.
1985. J. Org. Chem. 50:3908; Stec et al. J. Chromatog. 1985.
326:263; LaPlanche et al. 1986. Nucl. Acid. Res. 1986. 14:9081;
Fasman G. D., 1989. Practical Handbook of Biochemistry and
Molecular Biology. 1989. CRC Press, Boca Raton, Fla.; Lamone. 1993.
Biochem. Soc. Trans. 21:1; U.S. Pat. Nos. 5,013,830; 5,214,135;
5,525,719; Kawasaki et al. 1993. J. Med. Chem. 36:831; WO 92/03568;
U.S. Pat. Nos. 5,276,019; and 5,264,423.
[0288] The synthesis method selected can depend on the length of
the desired oligonucleotide and such choice is within the skill of
the ordinary artisan. For example, the phosphoramidite and
phosphite triester method can produce oligonucleotides having 175
or more nucleotides, while the H-phosphonate method works well for
oligonucleotides of less than 100 nucleotides. If modified bases
are incorporated into the oligonucleotide, and particularly if
modified phosphodiester linkages are used, then the synthetic
procedures are altered as needed according to known procedures. In
this regard, Uhlmann et al. (1990, Chemical Reviews 90:543-584)
provide references and outline procedures for making
oligonucleotides with modified bases and modified phosphodiester
linkages. Other exemplary methods for making oligonucleotides are
taught in Sonveaux. 1994. "Protecting Groups in Oligonucleotide
Synthesis"; Agrawal. Methods in Molecular Biology 26:1. Exemplary
synthesis methods are also taught in "Oligonucleotide Synthesis--A
Practical Approach" (Gait, M. J. IRL Press at Oxford University
Press. 1984). Moreover, linear oligonucleotides of defined
sequence, including some sequences with modified nucleotides, are
readily available from several commercial sources.
[0289] The oligonucleotides may be purified by polyacrylamide gel
electrophoresis, or by any of a number of chromatographic methods,
including gel chromatography and high pressure liquid
chromatography. To confirm a nucleotide sequence, especially
unmodified nucleotide sequences, oligonucleotides may be subjected
to DNA sequencing by any of the known procedures, including Maxam
and Gilbert sequencing, Sanger sequencing, capillary
electrophoresis sequencing, the wandering spot sequencing procedure
or by using selective chemical degradation of oligonucleotides
bound to Hybond paper. Sequences of short oligonucleotides can also
be analyzed by laser desorption mass spectroscopy or by fast atom
bombardment (McNeal, et al., 1982, J. Am. Chem. Soc. 104:976;
Viari, et al., 1987, Biomed. Environ. Mass Spectrom. 14:83;
Grotjahn et al., 1982, Nuc. Acid Res. 10:4671). Sequencing methods
are also available for RNA oligonucleotides.
[0290] The quality of oligonucleotides synthesized can be verified
by testing the oligonucleotide by capillary electrophoresis and
denaturing strong anion HPLC (SAX-HPLC) using, e.g., the method of
Bergot and Egan. 1992. J. Chrom. 599:35.
[0291] Other exemplary synthesis techniques are well known in the
art (see, e.g., Sambrook et al., Molecular Cloning: a Laboratory
Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D N
Glover Ed. 1985); Oligonucleotide Synthesis (M J Gait Ed, 1984;
Nucleic Acid Hybridisation (B D Hames and S J Higgins eds. 1984); A
Practical Guide to Molecular Cloning (1984); or the series, Methods
in Enzymology (Academic Press, Inc.)).
[0292] In certain embodiments, the subject RNAi constructs or at
least portions thereof are transcribed from expression vectors
encoding the subject constructs. Any art recognized vectors may be
use for this purpose. The transcribed RNAi constructs may be
isolated and purified, before desired modifications (such as
replacing an unmodified sense strand with a modified one, etc.) are
carried out.
Delivery/Carrier
Uptake of Oligonucleotides by Cells
[0293] Oligonucleotides and oligonucleotide compositions are
contacted with (i.e., brought into contact with, also referred to
herein as administered or delivered to) and taken up by one or more
cells or a cell lysate. The term "cells" includes prokaryotic and
eukaryotic cells, preferably vertebrate cells, and, more
preferably, mammalian cells. In a preferred embodiment, the
oligonucleotide compositions of the invention are contacted with
human cells.
[0294] Oligonucleotide compositions of the invention can be
contacted with cells in vitro, e.g., in a test tube or culture
dish, (and may or may not be introduced into a subject) or in vivo,
e.g., in a subject such as a mammalian subject. Oligonucleotides
are taken up by cells at a slow rate by endocytosis, but
endocytosed oligonucleotides are generally sequestered and not
available, e.g., for hybridization to a target nucleic acid
molecule. In one embodiment, cellular uptake can be facilitated by
electroporation or calcium phosphate precipitation. However, these
procedures are only useful for in vitro or ex vivo embodiments, are
not convenient and, in some cases, are associated with cell
toxicity.
[0295] In another embodiment, delivery of oligonucleotides into
cells can be enhanced by suitable art recognized methods including
calcium phosphate, DMSO, glycerol or dextran, electroporation, or
by transfection, e.g., using cationic, anionic, or neutral lipid
compositions or liposomes using methods known in the art (see e.g.,
WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No. 4,897,355;
Bergan et al. 1993. Nucleic Acids Research. 21:3567). Enhanced
delivery of oligonucleotides can also be mediated by the use of
vectors (See e.g., Shi, Y. 2003. Trends Genet 2003 Jan. 19:9;
Reichhart J M et al. Genesis. 2002. 34(1-2):1604, Yu et al. 2002.
Proc. Natl. Acad Sci. USA 99:6047; Sui et al. 2002. Proc. Natl.
Acad Sci. USA 99:5515) viruses, polyamine or polycation conjugates
using compounds such as polylysine, protamine, or Ni, N12-bis
(ethyl) spermine (see, e.g., Bartzatt, R. et al. 1989. Biotechnol.
Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl. Acad.
Sci. 88:4255).
[0296] In certain embodiments, the miniRNA of the invention may be
delivered by using various beta-glucan containing particles, such
as those described in US 2005/0281781 A1, WO 2006/007372, and WO
2007/050643 (all incorporated herein by reference). In certain
embodiments, the beta-glucan particle is derived from yeast. In
certain embodiments, the payload trapping molecule is a polymer,
such as those with a molecular weight of at least about 1000 Da,
10,000 Da, 50,000 Da, 100 kDa, 500 kDa, etc. Preferred polymers
include (without limitation) cationic polymers, chitosans, or PEI
(polyethylenimine), etc.
[0297] Such beta-glucan based delivery system may be formulated for
oral delivery, where the orally delivered beta-glucan/miniRNA
constructs may be engulfed by macrophages or other related
phagocytic cells, which may in turn release the miniRNA constructs
in selected in vivo sites. Alternatively or in addition, the
miniRNA may changes the expression of certain macrophage target
genes.
[0298] The optimal protocol for uptake of oligonucleotides will
depend upon a number of factors, the most crucial being the type of
cells that are being used. Other factors that are important in
uptake include, but are not limited to, the nature and
concentration of the oligonucleotide, the confluence of the cells,
the type of culture the cells are in (e.g., a suspension culture or
plated) and the type of media in which the cells are grown.
Encapsulating Agents
[0299] Encapsulating agents entrap oligonucleotides within
vesicles. In another embodiment of the invention, an
oligonucleotide may be associated with a carrier or vehicle, e.g.,
liposomes or micelles, although other carriers could be used, as
would be appreciated by one skilled in the art. Liposomes are
vesicles made of a lipid bilayer having a structure similar to
biological membranes. Such carriers are used to facilitate the
cellular uptake or targeting of the oligonucleotide, or improve the
oligonucleotide's pharmacokinetic or toxicologic properties.
[0300] For example, the oligonucleotides of the present invention
may also be administered encapsulated in liposomes, pharmaceutical
compositions wherein the active ingredient is contained either
dispersed or variously present in corpuscles consisting of aqueous
concentric layers adherent to lipidic layers. The oligonucleotides,
depending upon solubility, may be present both in the aqueous layer
and in the lipidic layer, or in what is generally termed a
liposomic suspension. The hydrophobic layer, generally but not
exclusively, comprises phopholipids such as lecithin and
sphingomyelin, steroids such as cholesterol, more or less ionic
surfactants such as diacetylphosphate, stearylamine, or
phosphatidic acid, or other materials of a hydrophobic nature. The
diameters of the liposomes generally range from about 15 nm to
about 5 microns.
[0301] The use of liposomes as drug delivery vehicles offers
several advantages. Liposomes increase intracellular stability,
increase uptake efficiency and improve biological activity.
Liposomes are hollow spherical vesicles composed of lipids arranged
in a similar fashion as those lipids which make up the cell
membrane. They have an internal aqueous space for entrapping water
soluble compounds and range in size from 0.05 to several microns in
diameter. Several studies have shown that liposomes can deliver
nucleic acids to cells and that the nucleic acids remain
biologically active. For example, a lipid delivery vehicle
originally designed as a research tool, such as Lipofectin or
LIPOFECTAMINE.TM. 2000, can deliver intact nucleic acid molecules
to cells.
[0302] Specific advantages of using liposomes include the
following: they are non-toxic and biodegradable in composition;
they display long circulation half-lives; and recognition molecules
can be readily attached to their surface for targeting to tissues.
Finally, cost-effective manufacture of liposome-based
pharmaceuticals, either in a liquid suspension or lyophilized
product, has demonstrated the viability of this technology as an
acceptable drug delivery system.
[0303] In some aspects, formulations associated with the invention
might be selected for a class of naturally occurring or chemically
synthesized or modified saturated and unsaturated fatty acid
residues. Fatty acids might exist in a form of triglycerides,
diglycerides or individual fatty acids. In another embodiment, the
use of well-validated mixtures of fatty acids and/or fat emulsions
currently used in pharmacology for parenteral nutrition may be
utilized.
[0304] Liposome based formulations are widely used for
oligonucleotide delivery. However, most of commercially available
lipid or liposome formulations contain at least one positively
charged lipid (cationic lipids). The presence of this positively
charged lipid is believed to be essential for obtaining a high
degree of oligonucleotide loading and for enhancing liposome
fusogenic properties. Several methods have been performed and
published to identify optimal positively charged lipid chemistries.
However, the commercially available liposome formulations
containing cationic lipids are characterized by a high level of
toxicity. In vivo limited therapeutic indexes have revealed that
liposome formulations containing positive charged lipids are
associated with toxicity (i.e. elevation in liver enzymes) at
concentrations only slightly higher than concentration required to
achieve RNA silencing.
[0305] New liposome formulations, lacking the toxicity of the prior
art liposomes have been developed according to the invention. These
new liposome formulations are neutral fat-based formulations for
the efficient delivery of oligonucleotides, and in particular for
the delivery of the RNA molecules of the invention. The
compositions are referred to as neutral nanotransporters because
they enable quantitative oligonucleotide incorporation into
non-charged lipids mixtures. The lack of toxic levels of cationic
lipids in the neutral nanotransporter compositions of the invention
is an important feature.
[0306] The neutral nanotransporters compositions enable efficient
loading of oligonucleotide into neutral fat formulation. The
composition includes an oligonucleotide that is modified in a
manner such that the hydrophobicity of the molecule is increased
(for example a hydrophobic molecule is attached (covalently or
no-covalently) to a hydrophobic molecule on the oligonucleotide
terminus or a non-terminal nucleotide, base, sugar, or backbone),
the modified oligonucleotide being mixed with a neutral fat
formulation (for example containing at least 25% of cholesterol and
25% of DOPC or analogs thereof). A cargo molecule, such as another
lipid can also be included in the composition. This composition,
where part of the formulation is build into the oligonucleotide
itself, enables efficient encapsulation of oligonucleotide in
neutral lipid particles.
[0307] One of several unexpected observations associated with the
invention was that the oligonucleotides of the invention could
effectively be incorporated in a lipid mixture that was free of
cationic lipids and that such a composition could effectively
deliver the therapeutic oligonucleotide to a cell in a manner that
it is functional. Another unexpected observation was the high level
of activity observed when the fatty mixture is composed of a
phosphatidylcholine base fatty acid and a sterol such as a
cholesterol. For instance, one preferred formulation of neutral
fatty mixture is composed of at least 20% of DOPC or DSPC and at
least 20% of sterol such as cholesterol. Even as low as 1:5 lipid
to oligonucleotide ratio was shown to be sufficient to get complete
encapsulation of the oligonucleotide in a non charged formulation.
The prior art demonstrated only a 1-5% oligonucleotide
encapsulation with non-charged formulations, which is not
sufficient to get to a desired amount of in vivo efficacy. Compared
to the prior art using neutral lipids the level of oligonucleotide
delivery to a cell was quite unexpected.
[0308] Stable particles ranging in size from 50 to 140 nm were
formed upon complexing of hydrophobic oligonucleotides with
preferred formulations. It is interesting to mention that the
formulation by itself typically does not form small particles, but
rather, forms agglomerates, which are transformed into stable
50-120 nm particles upon addition of the hydrophobic modified
oligonucleotide.
[0309] The neutral nanotransporter compositions of the invention
include a hydrophobic modified polynucleotide, a neutral fatty
mixture, and optionally a cargo molecule. A "hydrophobic modified
polynucleotide" as used herein is a polynucleotide of the invention
(i.e. sd-rxRNA) that has at least one modification that renders the
polynucleotide more hydrophobic than the polynucleotide was prior
to modification. The modification may be achieved by attaching
(covalently or non-covalently) a hydrophobic molecule to the
polynucleotide. In some instances the hydrophobic molecule is or
includes a lipophilic group.
[0310] The term "lipophilic group" means a group that has a higher
affinity for lipids than its affinity for water. Examples of
lipophilic groups include, but are not limited to, cholesterol, a
cholesteryl or modified cholesteryl residue, adamantine,
dihydrotesterone, long chain alkyl, long chain alkenyl, long chain
alkynyl, olely-lithocholic, cholenic, oleoyl-cholenic, palmityl,
heptadecyl, myrisityl, bile acids, cholic acid or taurocholic acid,
deoxycholate, oleyl litocholic acid, oleoyl cholenic acid,
glycolipids, phospholipids, sphingolipids, isoprenoids, such as
steroids, vitamins, such as vitamin E, fatty acids either saturated
or unsaturated, fatty acid esters, such as triglycerides, pyrenes,
porphyrines, Texaphyrine, adamantane, acridines, biotin, coumarin,
fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl,
t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3
or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. The cholesterol
moiety may be reduced (e.g. as in cholestan) or may be substituted
(e.g. by halogen). A combination of different lipophilic groups in
one molecule is also possible.
[0311] The hydrophobic molecule may be attached at various
positions of the polynucleotide. As described above, the
hydrophobic molecule may be linked to the terminal residue of the
polynucleotide such as the 3' of 5'-end of the polynucleotide.
Alternatively, it may be linked to an internal nucleotide or a
nucleotide on a branch of the polynucleotide. The hydrophobic
molecule may be attached, for instance to a 2'-position of the
nucleotide. The hydrophobic molecule may also be linked to the
heterocyclic base, the sugar or the backbone of a nucleotide of the
polynucleotide.
[0312] The hydrophobic molecule may be connected to the
polynucleotide by a linker moiety. Optionally the linker moiety is
a non-nucleotidic linker moiety. Non-nucleotidic linkers are e.g.
abasic residues (dSpacer), oligoethyleneglycol, such as
triethyleneglycol (spacer 9) or hexaethylenegylcol (spacer 18), or
alkane-diol, such as butanediol. The spacer units are preferably
linked by phosphodiester or phosphorothioate bonds. The linker
units may appear just once in the molecule or may be incorporated
several times, e.g. via phosphodiester, phosphorothioate,
methylphosphonate, or amide linkages.
[0313] Typical conjugation protocols involve the synthesis of
polynucleotides bearing an aminolinker at one or more positions of
the sequence, however, a linker is not required. The amino group is
then reacted with the molecule being conjugated using appropriate
coupling or activating reagents. The conjugation reaction may be
performed either with the polynucleotide still bound to a solid
support or following cleavage of the polynucleotide in solution
phase. Purification of the modified polynucleotide by HPLC
typically results in a pure material.
[0314] In some embodiments the hydrophobic molecule is a sterol
type conjugate, a PhytoSterol conjugate, cholesterol conjugate,
sterol type conjugate with altered side chain length, fatty acid
conjugate, any other hydrophobic group conjugate, and/or
hydrophobic modifications of the internal nucleoside, which provide
sufficient hydrophobicity to be incorporated into micelles.
[0315] For purposes of the present invention, the term "sterols",
refers or steroid alcohols are a subgroup of steroids with a
hydroxyl group at the 3-position of the A-ring. They are
amphipathic lipids synthesized from acetyl-coenzyme A via the
HMG-CoA reductase pathway. The overall molecule is quite flat. The
hydroxyl group on the A ring is polar. The rest of the aliphatic
chain is non-polar. Usually sterols are considered to have an 8
carbon chain at position 17.
[0316] For purposes of the present invention, the term "sterol type
molecules", refers to steroid alcohols, which are similar in
structure to sterols. The main difference is the structure of the
ring and number of carbons in a position 21 attached side
chain.
[0317] For purposes of the present invention, the term
"PhytoSterols" (also called plant sterols) are a group of steroid
alcohols, phytochemicals naturally occurring in plants. There are
more then 200 different known PhytoSterols
[0318] For purposes of the present invention, the term "Sterol side
chain" refers to a chemical composition of a side chain attached at
the position 17 of sterol-type molecule. In a standard definition
sterols are limited to a 4 ring structure carrying a 8 carbon chain
at position 17. In this invention, the sterol type molecules with
side chain longer and shorter than conventional are described. The
side chain may branched or contain double back bones.
[0319] Thus, sterols useful in the invention, for example, include
cholesterols, as well as unique sterols in which position 17 has
attached side chain of 2-7 or longer then 9 carbons. In a
particular embodiment, the length of the polycarbon tail is varied
between 5 and 9 carbons. FIG. 9 demonstrates that there is a
correlation between plasma clearance, liver uptake and the length
of the polycarbon chain. Such conjugates may have significantly
better in vivo efficacy, in particular delivery to liver. These
types of molecules are expected to work at concentrations 5 to 9
fold lower then oligonucleotides conjugated to conventional
cholesterols.
[0320] Alternatively the polynucleotide may be bound to a protein,
peptide or positively charged chemical that functions as the
hydrophobic molecule. The proteins may be selected from the group
consisting of protamine, dsRNA binding domain, and arginine rich
peptides. Exemplary positively charged chemicals include spermine,
spermidine, cadaverine, and putrescine.
[0321] In another embodiment hydrophobic molecule conjugates may
demonstrate even higher efficacy when it is combined with optimal
chemical modification patterns of the polynucleotide (as described
herein in detail), containing but not limited to hydrophobic
modifications, phosphorothioate modifications, and 2' ribo
modifications.
[0322] In another embodiment the sterol type molecule may be a
naturally occurring PhytoSterols such as those shown in FIG. 8. The
polycarbon chain may be longer than 9 and may be linear, branched
and/or contain double bonds. Some PhytoSterol containing
polynucleotide conjugates may be significantly more potent and
active in delivery of polynucleotides to various tissues. Some
PhytoSterols may demonstrate tissue preference and thus be used as
a way to delivery RNAi specifically to particular tissues.
[0323] The hydrophobic modified polynucleotide is mixed with a
neutral fatty mixture to form a micelle. The neutral fatty acid
mixture is a mixture of fats that has a net neutral or slightly net
negative charge at or around physiological pH that can form a
micelle with the hydrophobic modified polynucleotide. For purposes
of the present invention, the term "micelle" refers to a small
nanoparticle formed by a mixture of non charged fatty acids and
phospholipids. The neutral fatty mixture may include cationic
lipids as long as they are present in an amount that does not cause
toxicity. In preferred embodiments the neutral fatty mixture is
free of cationic lipids. A mixture that is free of cationic lipids
is one that has less than 1% and preferably 0% of the total lipid
being cationic lipid. The term "cationic lipid" includes lipids and
synthetic lipids having a net positive charge at or around
physiological pH. The term "anionic lipid" includes lipids and
synthetic lipids having a net negative charge at or around
physiological pH.
[0324] The neutral fats bind to the oligonucleotides of the
invention by a strong but non-covalent attraction (e.g., an
electrostatic, van der Waals, pi-stacking, etc. interaction).
[0325] The neutral fat mixture may include formulations selected
from a class of naturally occurring or chemically synthesized or
modified saturated and unsaturated fatty acid residues. Fatty acids
might exist in a form of triglycerides, diglycerides or individual
fatty acids. In another embodiment the use of well-validated
mixtures of fatty acids and/or fat emulsions currently used in
pharmacology for parenteral nutrition may be utilized.
[0326] The neutral fatty mixture is preferably a mixture of a
choline based fatty acid and a sterol. Choline based fatty acids
include for instance, synthetic phosphocholine derivatives such as
DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, and DEPC. DOPC (chemical
registry number 4235-95-4) is dioleoylphosphatidylcholine (also
known as dielaidoylphosphatidylcholine, dioleoyl-PC,
dioleoylphosphocholine, dioleoyl-sn-glycero-3-phosphocholine,
dioleylphosphatidylcholine). DSPC (chemical registry number
816-94-4) is distearoylphosphatidylcholine (also known as
1,2-Distearoyl-sn-Glycero-3-phosphocholine).
[0327] The sterol in the neutral fatty mixture may be for instance
cholesterol. The neutral fatty mixture may be made up completely of
a choline based fatty acid and a sterol or it may optionally
include a cargo molecule. For instance, the neutral fatty mixture
may have at least 20% or 25% fatty acid and 20% or 25% sterol.
[0328] For purposes of the present invention, the term "Fatty
acids" relates to conventional description of fatty acid. They may
exist as individual entities or in a form of two- and
triglycerides. For purposes of the present invention, the term "fat
emulsions" refers to safe fat formulations given intravenously to
subjects who are unable to get enough fat in their diet. It is an
emulsion of soy bean oil (or other naturally occurring oils) and
egg phospholipids. Fat emulsions are being used for formulation of
some insoluble anesthetics. In this disclosure, fat emulsions might
be part of commercially available preparations like Intralipid,
Liposyn, Nutrilipid, modified commercial preparations, where they
are enriched with particular fatty acids or fully de
novo-formulated combinations of fatty acids and phospholipids.
[0329] In one embodiment, the cells to be contacted with an
oligonucleotide composition of the invention are contacted with a
mixture comprising the oligonucleotide and a mixture comprising a
lipid, e.g., one of the lipids or lipid compositions described
supra for between about 12 hours to about 24 hours. In another
embodiment, the cells to be contacted with an oligonucleotide
composition are contacted with a mixture comprising the
oligonucleotide and a mixture comprising a lipid, e.g., one of the
lipids or lipid compositions described supra for between about 1
and about five days. In one embodiment, the cells are contacted
with a mixture comprising a lipid and the oligonucleotide for
between about three days to as long as about 30 days. In another
embodiment, a mixture comprising a lipid is left in contact with
the cells for at least about five to about 20 days. In another
embodiment, a mixture comprising a lipid is left in contact with
the cells for at least about seven to about 15 days. 50%-60% of the
formulation can optionally be any other lipid or molecule. Such a
lipid or molecule is referred to herein as a cargo lipid or cargo
molecule. Cargo molecules include but are not limited to
intralipid, small molecules, fusogenic peptides or lipids or other
small molecules might be added to alter cellular uptake, endosomal
release or tissue distribution properties. The ability to tolerate
cargo molecules is important for modulation of properties of these
particles, if such properties are desirable. For instance the
presence of some tissue specific metabolites might drastically
alter tissue distribution profiles. For example use of Intralipid
type formulation enriched in shorter or longer fatty chains with
various degrees of saturation affects tissue distribution profiles
of these type of formulations (and their loads).
[0330] An example of a cargo lipid useful according to the
invention is a fusogenic lipid. For instance, the zwiterionic lipid
DOPE (chemical registry number 4004-5-1,
1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine) is a preferred cargo
lipid.
[0331] Intralipid may be comprised of the following composition: 1
000 mL contain: purified soybean oil 90 g, purified egg
phospholipids 12 g, glycerol anhydrous 22 g, water for injection
q.s. ad 1 000 mL. pH is adjusted with sodium hydroxide to pH
approximately 8. Energy content/L: 4.6 MJ (190 kcal). Osmolality
(approx.): 300 mOsm/kg water. In another embodiment fat emulsion is
Liposyn that contains 5% safflower oil, 5% soybean oil, up to 1.2%
egg phosphatides added as an emulsifier and 2.5% glycerin in water
for injection. It may also contain sodium hydroxide for pH
adjustment. pH 8.0 (6.0-9.0). Liposyn has an osmolarity of 276 m
Osmol/liter (actual).
[0332] Variation in the identity, amounts and ratios of cargo
lipids affects the cellular uptake and tissue distribution
characteristics of these compounds. For example, the length of
lipid tails and level of saturability will affect differential
uptake to liver, lung, fat and cardiomyocytes. Addition of special
hydrophobic molecules like vitamins or different forms of sterols
can favor distribution to special tissues which are involved in the
metabolism of particular compounds. Complexes are formed at
different oligonucleotide concentrations, with higher
concentrations favoring more efficient complex formation (FIGS.
21-22).
[0333] In another embodiment, the fat emulsion is based on a
mixture of lipids. Such lipids may include natural compounds,
chemically synthesized compounds, purified fatty acids or any other
lipids. In yet another embodiment the composition of fat emulsion
is entirely artificial. In a particular embodiment, the fat
emulsion is more then 70% linoleic acid. In yet another particular
embodiment the fat emulsion is at least 1% of cardiolipin. Linoleic
acid (LA) is an unsaturated omega-6 fatty acid. It is a colorless
liquid made of a carboxylic acid with an 18-carbon chain and two
cis double bonds.
[0334] In yet another embodiment of the present invention, the
alteration of the composition of the fat emulsion is used as a way
to alter tissue distribution of hydrophobicly modified
polynucleotides. This methodology provides for the specific
delivery of the polynucleotides to particular tissues (FIG.
12).
[0335] In another embodiment the fat emulsions of the cargo
molecule contain more then 70% of Linoleic acid (C18H32O2) and/or
cardiolipin are used for specifically delivering RNAi to heart
muscle.
[0336] Fat emulsions, like intralipid have been used before as a
delivery formulation for some non-water soluble drugs (such as
Propofol, re-formulated as Diprivan). Unique features of the
present invention include (a) the concept of combining modified
polynucleotides with the hydrophobic compound(s), so it can be
incorporated in the fat micelles and (b) mixing it with the fat
emulsions to provide a reversible carrier. After injection into a
blood stream, micelles usually bind to serum proteins, including
albumin, HDL, LDL and other. This binding is reversible and
eventually the fat is absorbed by cells. The polynucleotide,
incorporated as a part of the micelle will then be delivered
closely to the surface of the cells. After that cellular uptake
might be happening though variable mechanisms, including but not
limited to sterol type delivery.
Complexing Agents
[0337] Complexing agents bind to the oligonucleotides of the
invention by a strong but non-covalent attraction (e.g., an
electrostatic, van der Waals, pi-stacking, etc. interaction). In
one embodiment, oligonucleotides of the invention can be complexed
with a complexing agent to increase cellular uptake of
oligonucleotides. An example of a complexing agent includes
cationic lipids. Cationic lipids can be used to deliver
oligonucleotides to cells. However, as discussed above,
formulations free in cationic lipids are preferred in some
embodiments.
[0338] The term "cationic lipid" includes lipids and synthetic
lipids having both polar and non-polar domains and which are
capable of being positively charged at or around physiological pH
and which bind to polyanions, such as nucleic acids, and facilitate
the delivery of nucleic acids into cells. In general cationic
lipids include saturated and unsaturated alkyl and alicyclic ethers
and esters of amines, amides, or derivatives thereof.
Straight-chain and branched alkyl and alkenyl groups of cationic
lipids can contain, e.g., from 1 to about 25 carbon atoms.
Preferred straight chain or branched alkyl or alkene groups have
six or more carbon atoms. Alicyclic groups include cholesterol and
other steroid groups. Cationic lipids can be prepared with a
variety of counterions (anions) including, e.g., Cl.sup.-,
Br.sup.-, I.sup.-, F.sup.-, acetate, trifluoroacetate, sulfate,
nitrite, and nitrate.
[0339] Examples of cationic lipids include polyethylenimine,
polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a
combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE.TM.
(e.g., LIPOFECTAMINE.TM. 2000), DOPE, Cytofectin (Gilead Sciences,
Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.).
Exemplary cationic liposomes can be made from
N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride
(DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium
methylsulfate (DOTAP),
3.beta.-[N--(N',N'-dimethylaminoethane)carbamoyl]cholesterol
(DC-Chol),
2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-
ium trifluoroacetate (DOSPA),
1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide;
and dimethyldioctadecylammonium bromide (DDAB). The cationic lipid
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTMA), for example, was found to increase 1000-fold the antisense
effect of a phosphorothioate oligonucleotide. (Vlassov et al.,
1994, Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides
can also be complexed with, e.g., poly (L-lysine) or avidin and
lipids may, or may not, be included in this mixture, e.g.,
steryl-poly (L-lysine).
[0340] Cationic lipids have been used in the art to deliver
oligonucleotides to cells (see, e.g., U.S. Pat. Nos. 5,855,910;
5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996.
Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998. Molecular
Membrane Biology 15:1). Other lipid compositions which can be used
to facilitate uptake of the instant oligonucleotides can be used in
connection with the claimed methods. In addition to those listed
supra, other lipid compositions are also known in the art and
include, e.g., those taught in U.S. Pat. Nos. 4,235,871; 4,501,728;
4,837,028; 4,737,323.
[0341] In one embodiment lipid compositions can further comprise
agents, e.g., viral proteins to enhance lipid-mediated
transfections of oligonucleotides (Kamata, et al., 1994. Nucl.
Acids. Res. 22:536). In another embodiment, oligonucleotides are
contacted with cells as part of a composition comprising an
oligonucleotide, a peptide, and a lipid as taught, e.g., in U.S.
Pat. No. 5,736,392. Improved lipids have also been described which
are serum resistant (Lewis, et al., 1996. Proc. Natl. Acad. Sci.
93:3176). Cationic lipids and other complexing agents act to
increase the number of oligonucleotides carried into the cell
through endocytosis.
[0342] In another embodiment N-substituted glycine oligonucleotides
(peptoids) can be used to optimize uptake of oligonucleotides.
Peptoids have been used to create cationic lipid-like compounds for
transfection (Murphy, et al., 1998. Proc. Natl. Acad. Sci.
95:1517). Peptoids can be synthesized using standard methods (e.g.,
Zuckermann, R. N., et al. 1992. J. Am. Chem. Soc. 114:10646;
Zuckermann, R. N., et al. 1992. Int. J. Peptide Protein Res.
40:497). Combinations of cationic lipids and peptoids, liptoids,
can also be used to optimize uptake of the subject oligonucleotides
(Hunag, et al., 1998. Chemistry and Biology. 5:345). Liptoids can
be synthesized by elaborating peptoid oligonucleotides and coupling
the amino terminal submonomer to a lipid via its amino group
(Hunag, et al., 1998. Chemistry and Biology. 5:345).
[0343] It is known in the art that positively charged amino acids
can be used for creating highly active cationic lipids (Lewis et
al. 1996. Proc. Natl. Acad. Sci. U.S.A. 93:3176). In one
embodiment, a composition for delivering oligonucleotides of the
invention comprises a number of arginine, lysine, histidine or
ornithine residues linked to a lipophilic moiety (see e.g., U.S.
Pat. No. 5,777,153).
[0344] In another embodiment, a composition for delivering
oligonucleotides of the invention comprises a peptide having from
between about one to about four basic residues. These basic
residues can be located, e.g., on the amino terminal, C-terminal,
or internal region of the peptide. Families of amino acid residues
having similar side chains have been defined in the art. These
families include amino acids with basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine (can
also be considered non-polar), asparagine, glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine). Apart from the
basic amino acids, a majority or all of the other residues of the
peptide can be selected from the non-basic amino acids, e.g., amino
acids other than lysine, arginine, or histidine. Preferably a
preponderance of neutral amino acids with long neutral side chains
are used.
[0345] In one embodiment, a composition for delivering
oligonucleotides of the invention comprises a natural or synthetic
polypeptide having one or more gamma carboxyglutamic acid residues,
or .gamma.-Gla residues. These gamma carboxyglutamic acid residues
may enable the polypeptide to bind to each other and to membrane
surfaces. In other words, a polypeptide having a series of
.gamma.-Gla may be used as a general delivery modality that helps
an RNAi construct to stick to whatever membrane to which it comes
in contact. This may at least slow RNAi constructs from being
cleared from the blood stream and enhance their chance of homing to
the target.
[0346] The gamma carboxyglutamic acid residues may exist in natural
proteins (for example, prothrombin has 10 .gamma.-Gla residues).
Alternatively, they can be introduced into the purified,
recombinantly produced, or chemically synthesized polypeptides by
carboxylation using, for example, a vitamin K-dependent
carboxylase. The gamma carboxyglutamic acid residues may be
consecutive or non-consecutive, and the total number and location
of such gamma carboxyglutamic acid residues in the polypeptide can
be regulated/fine tuned to achieve different levels of "stickiness"
of the polypeptide.
[0347] In one embodiment, the cells to be contacted with an
oligonucleotide composition of the invention are contacted with a
mixture comprising the oligonucleotide and a mixture comprising a
lipid, e.g., one of the lipids or lipid compositions described
supra for between about 12 hours to about 24 hours. In another
embodiment, the cells to be contacted with an oligonucleotide
composition are contacted with a mixture comprising the
oligonucleotide and a mixture comprising a lipid, e.g., one of the
lipids or lipid compositions described supra for between about 1
and about five days. In one embodiment, the cells are contacted
with a mixture comprising a lipid and the oligonucleotide for
between about three days to as long as about 30 days. In another
embodiment, a mixture comprising a lipid is left in contact with
the cells for at least about five to about 20 days. In another
embodiment, a mixture comprising a lipid is left in contact with
the cells for at least about seven to about 15 days.
[0348] For example, in one embodiment, an oligonucleotide
composition can be contacted with cells in the presence of a lipid
such as cytofectin CS or GSV (available from Glen Research;
Sterling, Va.), GS3815, GS2888 for prolonged incubation periods as
described herein.
[0349] In one embodiment, the incubation of the cells with the
mixture comprising a lipid and an oligonucleotide composition does
not reduce the viability of the cells. Preferably, after the
transfection period the cells are substantially viable. In one
embodiment, after transfection, the cells are between at least
about 70% and at least about 100% viable. In another embodiment,
the cells are between at least about 80% and at least about 95%
viable. In yet another embodiment, the cells are between at least
about 85% and at least about 90% viable.
[0350] In one embodiment, oligonucleotides are modified by
attaching a peptide sequence that transports the oligonucleotide
into a cell, referred to herein as a "transporting peptide." In one
embodiment, the composition includes an oligonucleotide which is
complementary to a target nucleic acid molecule encoding the
protein, and a covalently attached transporting peptide.
[0351] The language "transporting peptide" includes an amino acid
sequence that facilitates the transport of an oligonucleotide into
a cell. Exemplary peptides which facilitate the transport of the
moieties to which they are linked into cells are known in the art,
and include, e.g., HIV TAT transcription factor, lactoferrin,
Herpes VP22 protein, and fibroblast growth factor 2 (Pooga et al.
1998. Nature Biotechnology. 16:857; and Derossi et al. 1998. Trends
in Cell Biology. 8:84; Elliott and O'Hare. 1997. Cell 88:223).
[0352] Oligonucleotides can be attached to the transporting peptide
using known techniques, e.g., (Prochiantz, A. 1996. Curr. Opin.
Neurobiol. 6:629; Derossi et al. 1998. Trends Cell Biol. 8:84; Troy
et al. 1996. J. Neurosci. 16:253), Vives et al. 1997. J. Biol.
Chem. 272:16010). For example, in one embodiment, oligonucleotides
bearing an activated thiol group are linked via that thiol group to
a cysteine present in a transport peptide (e.g., to the cysteine
present in the .beta. turn between the second and the third helix
of the antennapedia homeodomain as taught, e.g., in Derossi et al.
1998. Trends Cell Biol. 8:84; Prochiantz. 1996. Current Opinion in
Neurobiol. 6:629; Allinquant et al. 1995. J Cell Biol. 128:919). In
another embodiment, a Boc-Cys-(Npys)OH group can be coupled to the
transport peptide as the last (N-terminal) amino acid and an
oligonucleotide bearing an SH group can be coupled to the peptide
(Troy et al. 1996. J. Neurosci. 16:253).
[0353] In one embodiment, a linking group can be attached to a
nucleomonomer and the transporting peptide can be covalently
attached to the linker. In one embodiment, a linker can function as
both an attachment site for a transporting peptide and can provide
stability against nucleases. Examples of suitable linkers include
substituted or unsubstituted C.sub.1-C.sub.20 alkyl chains,
C.sub.2-C.sub.20 alkenyl chains, C.sub.2-C.sub.20 alkynyl chains,
peptides, and heteroatoms (e.g., S, O, NH, etc.). Other exemplary
linkers include bifinctional crosslinking agents such as
sulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g.,
Smith et al. Biochem J 1991.276: 417-2).
[0354] In one embodiment, oligonucleotides of the invention are
synthesized as molecular conjugates which utilize receptor-mediated
endocytotic mechanisms for delivering genes into cells (see, e.g.,
Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559,
and the references cited therein).
Targeting Agents
[0355] The delivery of oligonucleotides can also be improved by
targeting the oligonucleotides to a cellular receptor. The
targeting moieties can be conjugated to the oligonucleotides or
attached to a carrier group (i.e., poly(L-lysine) or liposomes)
linked to the oligonucleotides. This method is well suited to cells
that display specific receptor-mediated endocytosis.
[0356] For instance, oligonucleotide conjugates to
6-phosphomannosylated proteins are internalized 20-fold more
efficiently by cells expressing mannose 6-phosphate specific
receptors than free oligonucleotides. The oligonucleotides may also
be coupled to a ligand for a cellular receptor using a
biodegradable linker. In another example, the delivery construct is
mannosylated streptavidin which forms a tight complex with
biotinylated oligonucleotides. Mannosylated streptavidin was found
to increase 20-fold the internalization of biotinylated
oligonucleotides. (Vlassov et al. 1994. Biochimica et Biophysica
Acta 1197:95-108).
[0357] In addition specific ligands can be conjugated to the
polylysine component of polylysine-based delivery systems. For
example, transferrin-polylysine, adenovirus-polylysine, and
influenza virus hemagglutinin HA-2 N-terminal fusogenic
peptides-polylysine conjugates greatly enhance receptor-mediated
DNA delivery in eucaryotic cells. Mannosylated glycoprotein
conjugated to poly(L-lysine) in aveolar macrophages has been
employed to enhance the cellular uptake of oligonucleotides. Liang
et al. 1999. Pharmazie 54:559-566.
[0358] Because malignant cells have an increased need for essential
nutrients such as folic acid and transferrin, these nutrients can
be used to target oligonucleotides to cancerous cells. For example,
when folic acid is linked to poly(L-lysine) enhanced
oligonucleotide uptake is seen in promyelocytic leukaemia (HL-60)
cells and human melanoma (M-14) cells. Ginobbi et al. 1997.
Anticancer Res. 17:29. In another example, liposomes coated with
maleylated bovine serum albumin, folic acid, or ferric
protoporphyrin IX, show enhanced cellular uptake of
oligonucleotides in murine macrophages, KB cells, and 2.2.15 human
hepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.
[0359] Liposomes naturally accumulate in the liver, spleen, and
reticuloendothelial system (so-called, passive targeting). By
coupling liposomes to various ligands such as antibodies are
protein A, they can be actively targeted to specific cell
populations. For example, protein A-bearing liposomes may be
pretreated with H-2K specific antibodies which are targeted to the
mouse major histocompatibility complex-encoded H-2K protein
expressed on L cells. (Vlassov et al. 1994. Biochimica et
Biophysica Acta 1197:95-108).
[0360] Other in vitro and/or in vivo delivery of RNAi reagents are
known in the art, and can be used to deliver the subject RNAi
constructs. See, for example, U.S. patent application publications
20080152661, 20080112916, 20080107694, 20080038296, 20070231392,
20060240093, 20060178327, 20060008910, 20050265957, 20050064595,
20050042227, 20050037496, 20050026286, 20040162235, 20040072785,
20040063654, 20030157030, WO 2008/036825, WO04/065601, and
AU2004206255B2, just to name a few (all incorporated by
reference).
Administration
[0361] The optimal course of administration or delivery of the
oligonucleotides may vary depending upon the desired result and/or
on the subject to be treated. As used herein "administration"
refers to contacting cells with oligonucleotides and can be
performed in vitro or in vivo. The dosage of oligonucleotides may
be adjusted to optimally reduce expression of a protein translated
from a target nucleic acid molecule, e.g., as measured by a readout
of RNA stability or by a therapeutic response, without undue
experimentation.
[0362] For example, expression of the protein encoded by the
nucleic acid target can be measured to determine whether or not the
dosage regimen needs to be adjusted accordingly. In addition, an
increase or decrease in RNA or protein levels in a cell or produced
by a cell can be measured using any art recognized technique. By
determining whether transcription has been decreased, the
effectiveness of the oligonucleotide in inducing the cleavage of a
target RNA can be determined.
[0363] Any of the above-described oligonucleotide compositions can
be used alone or in conjunction with a pharmaceutically acceptable
carrier. As used herein, "pharmaceutically acceptable carrier"
includes appropriate solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, it can be used in the therapeutic compositions.
Supplementary active ingredients can also be incorporated into the
compositions.
[0364] Oligonucleotides may be incorporated into liposomes or
liposomes modified with polyethylene glycol or admixed with
cationic lipids for parenteral administration. Incorporation of
additional substances into the liposome, for example, antibodies
reactive against membrane proteins found on specific target cells,
can help target the oligonucleotides to specific cell types.
[0365] Moreover, the present invention provides for administering
the subject oligonucleotides with an osmotic pump providing
continuous infusion of such oligonucleotides, for example, as
described in Rataiczak et al. (1992 Proc. Natl. Acad. Sci. USA
89:11823-11827). Such osmotic pumps are commercially available,
e.g., from Alzet Inc. (Palo Alto, Calif.). Topical administration
and parenteral administration in a cationic lipid carrier are
preferred.
[0366] With respect to in vivo applications, the formulations of
the present invention can be administered to a patient in a variety
of forms adapted to the chosen route of administration, e.g.,
parenterally, orally, or intraperitoneally. Parenteral
administration, which is preferred, includes administration by the
following routes: intravenous; intramuscular; interstitially;
intraarterially; subcutaneous; intra ocular; intrasynovial; trans
epithelial, including transdermal; pulmonary via inhalation;
ophthalmic; sublingual and buccal; topically, including ophthalmic;
dermal; ocular; rectal; and nasal inhalation via insufflation.
[0367] Pharmaceutical preparations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
or water-dispersible form. In addition, suspensions of the active
compounds as appropriate oily injection suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, or dextran, optionally, the
suspension may also contain stabilizers. The oligonucleotides of
the invention can be formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution. In addition, the oligonucleotides may be
formulated in solid form and redissolved or suspended immediately
prior to use. Lyophilized forms are also included in the
invention.
[0368] Pharmaceutical preparations for topical administration
include transdermal patches, ointments, lotions, creams, gels,
drops, sprays, suppositories, liquids and powders. In addition,
conventional pharmaceutical carriers, aqueous, powder or oily
bases, or thickeners may be used in pharmaceutical preparations for
topical administration.
[0369] Pharmaceutical preparations for oral administration include
powders or granules, suspensions or solutions in water or
non-aqueous media, capsules, sachets or tablets. In addition,
thickeners, flavoring agents, diluents, emulsifiers, dispersing
aids, or binders may be used in pharmaceutical preparations for
oral administration.
[0370] For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are known in the art, and include, for
example, for transmucosal administration bile salts and fusidic
acid derivatives, and detergents. Transmucosal administration may
be through nasal sprays or using suppositories. For oral
administration, the oligonucleotides are formulated into
conventional oral administration forms such as capsules, tablets,
and tonics. For topical administration, the oligonucleotides of the
invention are formulated into ointments, salves, gels, or creams as
known in the art.
[0371] Drug delivery vehicles can be chosen e.g., for in vitro, for
systemic, or for topical administration. These vehicles can be
designed to serve as a slow release reservoir or to deliver their
contents directly to the target cell. An advantage of using some
direct delivery drug vehicles is that multiple molecules are
delivered per uptake. Such vehicles have been shown to increase the
circulation half-life of drugs that would otherwise be rapidly
cleared from the blood stream. Some examples of such specialized
drug delivery vehicles which fall into this category are liposomes,
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres.
[0372] The described oligonucleotides may be administered
systemically to a subject. Systemic absorption refers to the entry
of drugs into the blood stream followed by distribution throughout
the entire body. Administration routes which lead to systemic
absorption include: intravenous, subcutaneous, intraperitoneal, and
intranasal. Each of these administration routes delivers the
oligonucleotide to accessible diseased cells. Following
subcutaneous administration, the therapeutic agent drains into
local lymph nodes and proceeds through the lymphatic network into
the circulation. The rate of entry into the circulation has been
shown to be a function of molecular weight or size. The use of a
liposome or other drug carrier localizes the oligonucleotide at the
lymph node. The oligonucleotide can be modified to diffuse into the
cell, or the liposome can directly participate in the delivery of
either the unmodified or modified oligonucleotide into the
cell.
[0373] The chosen method of delivery will result in entry into
cells. Preferred delivery methods include liposomes (10-400 nm),
hydrogels, controlled-release polymers, and other pharmaceutically
applicable vehicles, and microinjection or electroporation (for ex
vivo treatments).
[0374] The pharmaceutical preparations of the present invention may
be prepared and formulated as emulsions. Emulsions are usually
heterogeneous systems of one liquid dispersed in another in the
form of droplets usually exceeding 0.1 .mu.m in diameter. The
emulsions of the present invention may contain excipients such as
emulsifiers, stabilizers, dyes, fats, oils, waxes, fatty acids,
fatty alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives, and anti-oxidants may also be present in emulsions
as needed. These excipients may be present as a solution in either
the aqueous phase, oily phase or itself as a separate phase.
[0375] Examples of naturally occurring emulsifiers that may be used
in emulsion formulations of the present invention include lanolin,
beeswax, phosphatides, lecithin and acacia. Finely divided solids
have also been used as good emulsifiers especially in combination
with surfactants and in viscous preparations. Examples of finely
divided solids that may be used as emulsifiers include polar
inorganic solids, such as heavy metal hydroxides, nonswelling clays
such as bentonite, attapulgite, hectorite, kaolin,
montrnorillonite, colloidal aluminum silicate and colloidal
magnesium aluminum silicate, pigments and nonpolar solids such as
carbon or glyceryl tristearate.
[0376] Examples of preservatives that may be included in the
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Examples of antioxidants
that may be included in the emulsion formulations include free
radical scavengers such as tocopherols, alkyl gallates, butylated
hydroxyanisole, butylated hydroxytoluene, or reducing agents such
as ascorbic acid and sodium metabisulfite, and antioxidant
synergists such as citric acid, tartaric acid, and lecithin.
[0377] In one embodiment, the compositions of oligonucleotides are
formulated as microemulsions. A microemulsion is a system of water,
oil and amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution. Typically microemulsions
are prepared by first dispersing an oil in an aqueous surfactant
solution and then adding a sufficient amount of a 4th component,
generally an intermediate chain-length alcohol to form a
transparent system.
[0378] Surfactants that may be used in the preparation of
microemulsions include, but are not limited to, ionic surfactants,
non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers,
polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310),
tetraglycerol monooleate (M0310), hexaglycerol monooleate (P0310),
hexaglycerol pentaoleate (P0500), decaglycerol monocaprate
(MCA750), decaglycerol monooleate (M0750), decaglycerol sequioleate
(S0750), decaglycerol decaoleate (DA0750), alone or in combination
with cosurfactants. The cosurfactant, usually a short-chain alcohol
such as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules.
[0379] Microemulsions may, however, be prepared without the use of
cosurfactants and alcohol-free self-emulsifying microemulsion
systems are known in the art. The aqueous phase may typically be,
but is not limited to, water, an aqueous solution of the drug,
glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and
derivatives of ethylene glycol. The oil phase may include, but is
not limited to, materials such as Captex 300, Captex 355, Capmul
MCM, fatty acid esters, medium chain (C.sub.8-C.sub.12) mono, di,
and tri-glycerides, polyoxyethylated glyceryl fatty acid esters,
fatty alcohols, polyglycolized glycerides, saturated polyglycolized
C.sub.8-C.sub.10 glycerides, vegetable oils and silicone oil.
[0380] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both oil/water and water/oil)
have been proposed to enhance the oral bioavailability of
drugs.
[0381] Microemulsions offer improved drug solubilization,
protection of drug from enzymatic hydrolysis, possible enhancement
of drug absorption due to surfactant-induced alterations in
membrane fluidity and permeability, ease of preparation, ease of
oral administration over solid dosage forms, improved clinical
potency, and decreased toxicity (Constantinides et al.,
Pharmaceutical Research, 1994, 11:1385; Ho et al., J. Pharm. Sci.,
1996, 85:138-143). Microemulsions have also been effective in the
transdermal delivery of active components in both cosmetic and
pharmaceutical applications. It is expected that the microemulsion
compositions and formulations of the present invention will
facilitate the increased systemic absorption of oligonucleotides
from the gastrointestinal tract, as well as improve the local
cellular uptake of oligonucleotides within the gastrointestinal
tract, vagina, buccal cavity and other areas of administration.
[0382] In an embodiment, the present invention employs various
penetration enhancers to affect the efficient delivery of nucleic
acids, particularly oligonucleotides, to the skin of animals. Even
non-lipophilic drugs may cross cell membranes if the membrane to be
crossed is treated with a penetration enhancer. In addition to
increasing the diffusion of non-lipophilic drugs across cell
membranes, penetration enhancers also act to enhance the
permeability of lipophilic drugs.
[0383] Five categories of penetration enhancers that may be used in
the present invention include: surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants. Other
agents may be utilized to enhance the penetration of the
administered oligonucleotides include: glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-15 pyrrol, azones,
and terpenes such as limonene, and menthone.
[0384] The oligonucleotides, especially in lipid formulations, can
also be administered by coating a medical device, for example, a
catheter, such as an angioplasty balloon catheter, with a cationic
lipid formulation. Coating may be achieved, for example, by dipping
the medical device into a lipid formulation or a mixture of a lipid
formulation and a suitable solvent, for example, an aqueous-based
buffer, an aqueous solvent, ethanol, methylene chloride, chloroform
and the like. An amount of the formulation will naturally adhere to
the surface of the device which is subsequently administered to a
patient, as appropriate. Alternatively, a lyophilized mixture of a
lipid formulation may be specifically bound to the surface of the
device. Such binding techniques are described, for example, in K.
Ishihara et al., Journal of Biomedical Materials Research, Vol. 27,
pp. 1309-1314 (1993), the disclosures of which are incorporated
herein by reference in their entirety.
[0385] The useful dosage to be administered and the particular mode
of administration will vary depending upon such factors as the cell
type, or for in vivo use, the age, weight and the particular animal
and region thereof to be treated, the particular oligonucleotide
and delivery method used, the therapeutic or diagnostic use
contemplated, and the form of the formulation, for example,
suspension, emulsion, micelle or liposome, as will be readily
apparent to those skilled in the art. Typically, dosage is
administered at lower levels and increased until the desired effect
is achieved. When lipids are used to deliver the oligonucleotides,
the amount of lipid compound that is administered can vary and
generally depends upon the amount of oligonucleotide agent being
administered. For example, the weight ratio of lipid compound to
oligonucleotide agent is preferably from about 1:1 to about 15:1,
with a weight ratio of about 5:1 to about 10:1 being more
preferred. Generally, the amount of cationic lipid compound which
is administered will vary from between about 0.1 milligram (mg) to
about 1 gram (g). By way of general guidance, typically between
about 0.1 mg and about 10 mg of the particular oligonucleotide
agent, and about 1 mg to about 100 mg of the lipid compositions,
each per kilogram of patient body weight, is administered, although
higher and lower amounts can be used.
[0386] The agents of the invention are administered to subjects or
contacted with cells in a biologically compatible form suitable for
pharmaceutical administration. By "biologically compatible form
suitable for administration" is meant that the oligonucleotide is
administered in a form in which any toxic effects are outweighed by
the therapeutic effects of the oligonucleotide. In one embodiment,
oligonucleotides can be administered to subjects. Examples of
subjects include mammals, e.g., humans and other primates; cows,
pigs, horses, and farming (agricultural) animals; dogs, cats, and
other domesticated pets; mice, rats, and transgenic non-human
animals.
[0387] Administration of an active amount of an oligonucleotide of
the present invention is defined as an amount effective, at dosages
and for periods of time necessary to achieve the desired result.
For example, an active amount of an oligonucleotide may vary
according to factors such as the type of cell, the oligonucleotide
used, and for in vivo uses the disease state, age, sex, and weight
of the individual, and the ability of the oligonucleotide to elicit
a desired response in the individual. Establishment of therapeutic
levels of oligonucleotides within the cell is dependent upon the
rates of uptake and efflux or degradation. Decreasing the degree of
degradation prolongs the intracellular half-life of the
oligonucleotide. Thus, chemically-modified oligonucleotides, e.g.,
with modification of the phosphate backbone, may require different
dosing.
[0388] The exact dosage of an oligonucleotide and number of doses
administered will depend upon the data generated experimentally and
in clinical trials. Several factors such as the desired effect, the
delivery vehicle, disease indication, and the route of
administration, will affect the dosage. Dosages can be readily
determined by one of ordinary skill in the art and formulated into
the subject pharmaceutical compositions. Preferably, the duration
of treatment will extend at least through the course of the disease
symptoms.
[0389] Dosage region may be adjusted to provide the optimum
therapeutic response. For example, the oligonucleotide may be
repeatedly administered, e.g., several doses may be administered
daily or the dose may be proportionally reduced as indicated by the
exigencies of the therapeutic situation. One of ordinary skill in
the art will readily be able to determine appropriate doses and
schedules of administration of the subject oligonucleotides,
whether the oligonucleotides are to be administered to cells or to
subjects.
[0390] Physical methods of introducing nucleic acids include
injection of a solution containing the nucleic acid, bombardment by
particles covered by the nucleic acid, soaking the cell or organism
in a solution of the nucleic acid, or electroporation of cell
membranes in the presence of the nucleic acid. A viral construct
packaged into a viral particle would accomplish both efficient
introduction of an expression construct into the cell and
transcription of nucleic acid encoded by the expression construct.
Other methods known in the art for introducing nucleic acids to
cells may be used, such as lipid-mediated carrier transport,
chemical-mediated transport, such as calcium phosphate, and the
like. Thus the nucleic acid may be introduced along with components
that perform one or more of the following activities: enhance
nucleic acid uptake by the cell, inhibit annealing of single
strands, stabilize the single strands, or other-wise increase
inhibition of the target gene.
[0391] Nucleic acid may be directly introduced into the cell (i.e.,
intracellularly); or introduced extracellularly into a cavity,
interstitial space, into the circulation of an organism, introduced
orally or by inhalation, or may be introduced by bathing a cell or
organism in a solution containing the nucleic acid. Vascular or
extravascular circulation, the blood or lymph system, and the
cerebrospinal fluid are sites where the nucleic acid may be
introduced.
[0392] The cell with the target gene may be derived from or
contained in any organism. The organism may a plant, animal,
protozoan, bacterium, virus, or fungus. The plant may be a monocot,
dicot or gymnosperm; the animal may be a vertebrate or
invertebrate. Preferred microbes are those used in agriculture or
by industry, and those that are pathogenic for plants or
animals.
[0393] Alternatively, vectors, e.g., transgenes encoding a siRNA of
the invention can be engineered into a host cell or transgenic
animal using art recognized techniques.
[0394] Another use for the nucleic acids of the present invention
(or vectors or transgenes encoding same) is a functional analysis
to be carried out in eukaryotic cells, or eukaryotic non-human
organisms, preferably mammalian cells or organisms and most
preferably human cells, e.g. cell lines such as HeLa or 293 or
rodents, e.g. rats and mice. By administering a suitable nucleic
acid of the invention which is sufficiently complementary to a
target mRNA sequence to direct target-specific RNA interference, a
specific knockout or knockdown phenotype can be obtained in a
target cell, e.g. in cell culture or in a target organism.
[0395] Thus, a further subject matter of the invention is a
eukaryotic cell or a eukaryotic non-human organism exhibiting a
target gene-specific knockout or knockdown phenotype comprising a
fully or at least partially deficient expression of at least one
endogenous target gene wherein said cell or organism is transfected
with at least one vector comprising DNA encoding an RNAi agent
capable of inhibiting the expression of the target gene. It should
be noted that the present invention allows a target-specific
knockout or knockdown of several different endogenous genes due to
the specificity of the RNAi agent.
[0396] Gene-specific knockout or knockdown phenotypes of cells or
non-human organisms, particularly of human cells or non-human
mammals may be used in analytic to procedures, e.g. in the
functional and/or phenotypical analysis of complex physiological
processes such as analysis of gene expression profiles and/or
proteomes. Preferably the analysis is carried out by high
throughput methods using oligonucleotide based chips.
Assays of Oligonucleotide Stability
[0397] In some embodiments, the oligonucleotides of the invention
are stabilized, i.e., substantially resistant to endonuclease and
exonuclease degradation. An oligonucleotide is defined as being
substantially resistant to nucleases when it is at least about
3-fold more resistant to attack by an endogenous cellular nuclease,
and is highly nuclease resistant when it is at least about 6-fold
more resistant than a corresponding oligonucleotide. This can be
demonstrated by showing that the oligonucleotides of the invention
are substantially resistant to nucleases using techniques which are
known in the art.
[0398] One way in which substantial stability can be demonstrated
is by showing that the oligonucleotides of the invention function
when delivered to a cell, e.g., that they reduce transcription or
translation of target nucleic acid molecules, e.g., by measuring
protein levels or by measuring cleavage of mRNA. Assays which
measure the stability of target RNA can be performed at about 24
hours post-transfection (e.g., using Northern blot techniques,
RNase Protection Assays, or QC-PCR assays as known in the art).
Alternatively, levels of the target protein can be measured.
Preferably, in addition to testing the RNA or protein levels of
interest, the RNA or protein levels of a control, non-targeted gene
will be measured (e.g., actin, or preferably a control with
sequence similarity to the target) as a specificity control. RNA or
protein measurements can be made using any art-recognized
technique. Preferably, measurements will be made beginning at about
16-24 hours post transfection. (M. Y. Chiang, et al. 1991. J Biol
Chem. 266:18162-71; T. Fisher, et al. 1993. Nucleic Acids Research.
21 3857).
[0399] The ability of an oligonucleotide composition of the
invention to inhibit protein synthesis can be measured using
techniques which are known in the art, for example, by detecting an
inhibition in gene transcription or protein synthesis. For example,
Nuclease S1 mapping can be performed. In another example, Northern
blot analysis can be used to measure the presence of RNA encoding a
particular protein. For example, total RNA can be prepared over a
cesium chloride cushion (see, e.g., Ausebel et al., 1987. Current
Protocols in Molecular Biology (Greene & Wiley, New York)).
Northern blots can then be made using the RNA and probed (see,
e.g., Id.). In another example, the level of the specific mRNA
produced by the target protein can be measured, e.g., using PCR. In
yet another example, Western blots can be used to measure the
amount of target protein present. In still another embodiment, a
phenotype influenced by the amount of the protein can be detected.
Techniques for performing Western blots are well known in the art,
see, e.g., Chen et al. J. Biol. Chem. 271:28259.
[0400] In another example, the promoter sequence of a target gene
can be linked to a reporter gene and reporter gene transcription
(e.g., as described in more detail below) can be monitored.
Alternatively, oligonucleotide compositions that do not target a
promoter can be identified by fusing a portion of the target
nucleic acid molecule with a reporter gene so that the reporter
gene is transcribed. By monitoring a change in the expression of
the reporter gene in the presence of the oligonucleotide
composition, it is possible to determine the effectiveness of the
oligonucleotide composition in inhibiting the expression of the
reporter gene. For example, in one embodiment, an effective
oligonucleotide composition will reduce the expression of the
reporter gene.
[0401] A "reporter gene" is a nucleic acid that expresses a
detectable gene product, which may be RNA or protein. Detection of
mRNA expression may be accomplished by Northern blotting and
detection of protein may be accomplished by staining with
antibodies specific to the protein. Preferred reporter genes
produce a readily detectable product. A reporter gene may be
operably linked with a regulatory DNA sequence such that detection
of the reporter gene product provides a measure of the
transcriptional activity of the regulatory sequence. In preferred
embodiments, the gene product of the reporter gene is detected by
an intrinsic activity associated with that product. For instance,
the reporter gene may encode a gene product that, by enzymatic
activity, gives rise to a detectable signal based on color,
fluorescence, or luminescence. Examples of reporter genes include,
but are not limited to, those coding for chloramphenicol acetyl
transferase (CAT), luciferase, beta-galactosidase, and alkaline
phosphatase.
[0402] One skilled in the art would readily recognize numerous
reporter genes suitable for use in the present invention. These
include, but are not limited to, chloramphenicol acetyltransferase
(CAT), luciferase, human growth hormone (hGH), and
beta-galactosidase. Examples of such reporter genes can be found in
F. A. Ausubel et al., Eds., Current Protocols in Molecular Biology,
John Wiley & Sons, New York, (1989). Any gene that encodes a
detectable product, e.g., any product having detectable enzymatic
activity or against which a specific antibody can be raised, can be
used as a reporter gene in the present methods.
[0403] One reporter gene system is the firefly luciferase reporter
system. (Gould, S. J., and Subramani, S. 1988. Anal. Biochem.,
7:404-408 incorporated herein by reference). The luciferase assay
is fast and sensitive. In this assay, a lysate of the test cell is
prepared and combined with ATP and the substrate luciferin. The
encoded enzyme luciferase catalyzes a rapid, ATP dependent
oxidation of the substrate to generate a light-emitting product.
The total light output is measured and is proportional to the
amount of luciferase present over a wide range of enzyme
concentrations.
[0404] CAT is another frequently used reporter gene system; a major
advantage of this system is that it has been an extensively
validated and is widely accepted as a measure of promoter activity.
(Gorman C. M., Moffat, L. F., and Howard, B. H. 1982. Mol. Cell.
Biol., 2:1044-1051). In this system, test cells are transfected
with CAT expression vectors and incubated with the candidate
substance within 2-3 days of the initial transfection. Thereafter,
cell extracts are prepared. The extracts are incubated with acetyl
CoA and radioactive chloramphenicol. Following the incubation,
acetylated chloramphenicol is separated from nonacetylated form by
thin layer chromatography. In this assay, the degree of acetylation
reflects the CAT gene activity with the particular promoter.
[0405] Another suitable reporter gene system is based on
immunologic detection of hGH. This system is also quick and easy to
use. (Selden, R., Burke-Howie, K. Rowe, M. E., Goodman, H. M., and
Moore, D. D. (1986), Mol. Cell, Biol., 6:3173-3179 incorporated
herein by reference). The hGH system is advantageous in that the
expressed hGH polypeptide is assayed in the media, rather than in a
cell extract. Thus, this system does not require the destruction of
the test cells. It will be appreciated that the principle of this
reporter gene system is not limited to hGH but rather adapted for
use with any polypeptide for which an antibody of acceptable
specificity is available or can be prepared.
[0406] In one embodiment, nuclease stability of a double-stranded
oligonucleotide of the invention is measured and compared to a
control, e.g., an RNAi molecule typically used in the art (e.g., a
duplex oligonucleotide of less than 25 nucleotides in length and
comprising 2 nucleotide base overhangs) or an unmodified RNA duplex
with blunt ends.
[0407] The target RNA cleavage reaction achieved using the siRNAs
of the invention is highly sequence specific. Sequence identity may
determined by sequence comparison and alignment algorithms known in
the art. To determine the percent identity of two nucleic acid
sequences (or of two amino acid sequences), the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the first sequence or second sequence for optimal
alignment). A preferred, non-limiting example of a local alignment
algorithm utilized for the comparison of sequences is the algorithm
of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA
87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into
the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. Additionally, numerous commercial entities, such
as Dharmacon, and Invitrogen provide access to algorithms on their
website. The Whitehead Institute also offers a free siRNA Selection
Program. Greater than 90% sequence identity, e.g., 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity,
between the siRNA and the portion of the target gene is preferred.
Alternatively, the siRNA may be defined functionally as a
nucleotide sequence (or oligonucleotide sequence) that is capable
of hybridizing with a portion of the target gene transcript.
Examples of stringency conditions for polynucleotide hybridization
are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and
Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,
eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4,
incorporated herein by reference.
Therapeutic Use
[0408] By inhibiting the expression of a gene, the oligonucleotide
compositions of the present invention can be used to treat any
disease involving the expression of a protein. Examples of diseases
that can be treated by oligonucleotide compositions, just to
illustrate, include: cancer, retinopathies, autoimmune diseases,
inflammatory diseases (i.e., ICAM-1 related disorders, Psoriasis,
Ulcerative Colitus, Crohn's disease), viral diseases (i.e., HIV,
Hepatitis C), miRNA disorders, and cardiovascular diseases.
[0409] In one embodiment, in vitro treatment of cells with
oligonucleotides can be used for ex vivo therapy of cells removed
from a subject (e.g., for treatment of leukemia or viral infection)
or for treatment of cells which did not originate in the subject,
but are to be administered to the subject (e.g., to eliminate
transplantation antigen expression on cells to be transplanted into
a subject). In addition, in vitro treatment of cells can be used in
non-therapeutic settings, e.g., to evaluate gene function, to study
gene regulation and protein synthesis or to evaluate improvements
made to oligonucleotides designed to modulate gene expression or
protein synthesis. In vivo treatment of cells can be useful in
certain clinical settings where it is desirable to inhibit the
expression of a protein. There are numerous medical conditions for
which antisense therapy is reported to be suitable (see, e.g., U.S.
Pat. No. 5,830,653) as well as respiratory syncytial virus
infection (WO 95/22,553) influenza virus (WO 94/23,028), and
malignancies (WO 94/08,003). Other examples of clinical uses of
antisense sequences are reviewed, e.g., in Glaser. 1996. Genetic
Engineering News 16:1. Exemplary targets for cleavage by
oligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf
kinase, p53, c-myb, and the bcr/abl fusion gene found in chronic
myelogenous leukemia.
[0410] The subject nucleic acids can be used in RNAi-based therapy
in any animal having RNAi pathway, such as human, non-human
primate, non-human mammal, non-human vertebrates, rodents (mice,
rats, hamsters, rabbits, etc.), domestic livestock animals, pets
(cats, dogs, etc.), Xenopus, fish, insects (Drosophila, etc.), and
worms (C. elegans), etc.
[0411] The invention provides methods for inhibiting or preventing
in a subject, a disease or condition associated with an aberrant or
unwanted target gene expression or activity, by administering to
the subject a nucleic acid of the invention. If appropriate,
subjects are first treated with a priming agent so as to be more
responsive to the subsequent RNAi therapy. Subjects at risk for a
disease which is caused or contributed to by aberrant or unwanted
target gene expression or activity can be identified by, for
example, any or a combination of diagnostic or prognostic assays
known in the art. Administration of a prophylactic agent can occur
prior to the manifestation of symptoms characteristic of the target
gene aberrancy, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
target gene aberrancy, for example, a target gene, target gene
agonist or target gene antagonist agent can be used for treating
the subject.
[0412] In another aspect, the invention pertains to methods of
modulating target gene expression, protein expression or activity
for therapeutic purposes. Accordingly, in an exemplary embodiment,
the methods of the invention involve contacting a cell capable of
expressing target gene with a nucleic acid of the invention that is
specific for the target gene or protein (e.g., is specific for the
mRNA encoded by said gene or specifying the amino acid sequence of
said protein) such that expression or one or more of the activities
of target protein is modulated. These methods can be performed in
vitro (e.g., by culturing the cell with the agent), in vivo (e.g.,
by administering the agent to a subject), or ex vivo. The subjects
may be first treated with a priming agent so as to be more
responsive to the subsequent RNAi therapy if desired. As such, the
present invention provides methods of treating a subject afflicted
with a disease or disorder characterized by aberrant or unwanted
expression or activity of a target gene polypeptide or nucleic acid
molecule. Inhibition of target gene activity is desirable in
situations in which target gene is abnormally unregulated and/or in
which decreased target gene activity is likely to have a beneficial
effect.
[0413] Thus the therapeutic agents of the invention can be
administered to subjects to treat (prophylactically or
therapeutically) disorders associated with aberrant or unwanted
target gene activity. In conjunction with such treatment,
pharmacogenomics (i.e., the study of the relationship between an
individual's genotype and that individual's response to a foreign
compound or drug) may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, a physician or clinician may
consider applying knowledge obtained in relevant pharmacogenomics
studies in determining whether to administer a therapeutic agent as
well as tailoring the dosage and/or therapeutic regimen of
treatment with a therapeutic agent. Pharmacogenomics deals with
clinically significant hereditary variations in the response to
drugs due to altered drug disposition and abnormal action in
affected persons.
[0414] For the purposes of the invention, ranges may be expressed
herein as from "about" one particular value, and/or to "about"
another particular value. When such a range is expressed, another
embodiment includes from the one particular value and/or to the
other particular value. Similarly, when values are expressed as
approximations, by use of the antecedent "about," it will be
understood that the particular value forms another embodiment. It
will be further understood that the endpoints of each of the ranges
are significant both in relation to the other endpoint, and
independently of the other endpoint.
[0415] Moreover, for the purposes of the present invention, the
term "a" or "an" entity refers to one or more of that entity; for
example, "a protein" or "a nucleic acid molecule" refers to one or
more of those compounds or at least one compound. As such, the
terms "a" (or "an"), "one or more" and "at least one" can be used
interchangeably herein. It is also to be noted that the terms
"comprising", "including", and "having" can be used
interchangeably. Furthermore, a compound "selected from the group
consisting of" refers to one or more of the compounds in the list
that follows, including mixtures (i.e., combinations) of two or
more of the compounds. According to the present invention, an
isolated, or biologically pure, protein or nucleic acid molecule is
a compound that has been removed from its natural milieu. As such,
"isolated" and "biologically pure" do not necessarily reflect the
extent to which the compound has been purified. An isolated
compound of the present invention can be obtained from its natural
source, can be produced using molecular biology techniques or can
be produced by chemical synthesis.
[0416] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Example 1: Inhibition of Gene Expression Using Minimum Length
Trigger RNAs
[0417] Transfection of Minimum Length Trigger (mlt) RNA
[0418] mltRNA constructs were chemically synthesized (Integrated
DNA Technologies, Coralville, Iowa) and transfected into HEK293
cells (ATCC, Manassas, Va.) using the Lipofectamine RNAiMAX
(Invitrogen, Carlsbad, Calif.) reagent according to manufacturer's
instructions. In brief, RNA was diluted to a 12.times.
concentration and then combined with a 12.times. concentration of
Lipofectamine RNAiMAX to complex. The RNA and transfection reagent
were allowed to complex at room temperature for 20 minutes and make
a 6.times. concentration. While complexing, HEK293 cells were
washed, trypsinized and counted. The cells were diluted to a
concentration recommended by the manufacturer and previously
described conditions which was at 1.times.10.sup.5 cells/ml. When
RNA had completed complexing with the RNAiMAX transfection reagent,
20 ul of the complexes were added to the appropriate well of the
96-well plate in triplicate. Cells were added to each well (100 ul
volume) to make the final cell count per well at 1.times.10.sup.4
cells/well. The volume of cells diluted the 6.times. concentration
of complex to 1.times. which was equal to a concentration noted
(between 10-0.05 nM). Cells were incubated for 24 or 48 hours under
normal growth conditions.
[0419] After 24 or 48 hour incubation cells were lysed and gene
silencing activity was measured using the QuantiGene assay
(Panomics, Freemont, Calif.) which employs bDNA hybridization
technology. The assay was carried out according to manufacturer's
instructions.
.DELTA.G Calculation
[0420] .DELTA.G was calculated using Mfold, available through the
Mfold internet site
(http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cfi). Methods for
calculating .DELTA.G are described in, and are incorporated by
reference from, the following references: Zuker, M. (2003) Nucleic
Acids Res., 31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M.
and Turner, D. H. (1999) J. Mol. Biol. 288:911-940; Mathews, D. H.,
Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and
Turner, D. H. (2004) Proc. Natl. Acad. Sci. 101:7287-7292; Duan,
S., Mathews, D. H., and Turner, D. H. (2006) Biochemistry
45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I. L., and
Schuster, P. (1999) Biopolymers 49:145-165.
Example 2: Optimization of sd-rxRNA.sup.nano Molecules for Gene
Silencing
[0421] Asymmetric double stranded RNAi molecules, with minimal
double stranded regions, were developed herein and are highly
effective at gene silencing. These molecules can contain a variety
of chemical modifications on the sense and/or anti-sense strands,
and can be conjugated to sterol-like compounds such as
cholesterol.
[0422] FIGS. 1-3 present schematics of RNAi molecules associated
with the invention. In the asymmetric molecules, which contain a
sense and anti-sense strand, either of the strands can be the
longer strand. Either strand can also contain a single-stranded
region. There can also be mismatches between the sense and
anti-sense strand, as indicated in FIG. 1D. Preferably, one end of
the double-stranded molecule is either blunt-ended or contains a
short overhang such as an overhang of one nucleotide. FIG. 2
indicates types of chemical modifications applied to the sense and
anti-sense strands including 2'F, 2'OMe, hydrophobic modifications
and phosphorothioate modifications. Preferably, the single stranded
region of the molecule contains multiple phosphorothioate
modifications. Hydrophobicity of molecules can be increased using
such compounds as 4-pyridyl at 5-U, 2-pyridyl at 5-U, isobutyl at
5-U and indolyl at 5-U (FIG. 2). Proteins or peptides such as
protamine (or other Arg rich peptides), spermidine or other similar
chemical structures can also be used to block duplex charge and
facilitate cellular entry (FIG. 3). Increased hydrophobicity can be
achieved through either covalent or non-covalent modifications.
Several positively charged chemicals, which might be used for
polynucleotide charge blockage are depicted in FIG. 4.
[0423] Chemical modifications of polynucleotides, such as the guide
strand in a duplex molecule, can facilitate RISC entry. FIG. 5
depicts single stranded polynucleotides, representing a guide
strand in a duplex molecule, with a variety of chemical
modifications including 2'd, 2'OMe, 2'F, hydrophobic modifications,
phosphorothioate modifications, and attachment of conjugates such
as "X" in FIG. 5, where X can be a small molecule with high
affinity to a PAZ domain, or sterol-type entity. Similarly, FIG. 6
depicts single stranded polynucleotides, representing a passenger
strand in a duplex molecule, with proposed structural and chemical
compositions of RISC substrate inhibitors. Combinations of chemical
modifications can ensure efficient uptake and efficient binding to
preloaded RISC complexes.
[0424] FIG. 7 depicts structures of polynucleotides with
sterol-type molecules attached, where R represents a polycarbonic
tail of 9 carbons or longer. FIG. 8 presents examples of naturally
occurring phytosterols with a polycarbon chain longer than 8
attached at position 17. More than 250 different types of
phytosterols are known. FIG. 9 presents examples of sterol-like
structures with variations in the sizes of the polycarbon chains
attached at position 17. FIG. 91 presents further examples of
sterol-type molecules that can be used as a hydrophobic entity in
place of cholesterol. FIG. 92 presents further examples of
hydrophobic molecules that might be used as hydrophobic entities in
place of cholestesterol. Optimization of such characteristics can
improve uptake properties of the RNAi molecules. FIG. 10 presents
data adapted from Martins et al. (J Lipid Research), showing that
the percentage of liver uptake and plasma clearance of lipid
emulsions containing sterol-type molecules is directly affected by
the size of the attached polycarbon chain at position 17. FIG. 11
depicts a micelle formed from a mixture of polynucleotides attached
to hydrophobic conjugates and fatty acids. FIG. 12 describes how
alteration in lipid composition can affect pharmacokinetic behavior
and tissue distribution of hydrophobically modified and/or
hydrophobically conjugated polynucleotides. In particular, the use
of lipid mixtures that are enriched in linoleic acid and
cardiolipin results in preferential uptake by cardiomyocites.
[0425] FIG. 13 depicts examples of RNAi constructs and controls
designed to target MAP4K4 expression. FIGS. 14 and 15 reveal that
RNAi constructs with minimal duplex regions (such as duplex regions
of approximately 13 nucleotides) are effective in mediating RNA
silencing in cell culture. Parameters associated with these RNA
molecules are shown in FIG. 16. FIG. 17 depicts examples of RNAi
constructs and controls designed to target SOD1 expression. FIGS.
18 and 19 reveal the results of gene silencing experiments using
these RNAi molecules to target SOD1 in cells. FIG. 20 presents a
schematic indicating that RNA molecules with double stranded
regions that are less than 10 nucleotides are not cleaved by Dicer,
and FIG. 21 presents a schematic of a hypothetical RNAi model for
RNA induced gene silencing.
[0426] The RNA molecules described herein were subject to a variety
of chemical modifications on the sense and antisense strands, and
the effects of such modifications were observed. RNAi molecules
were synthesized and optimized through testing of a variety of
modifications. In first generation optimization, the sense
(passenger) and anti-sense (guide) strands of the sd-rxRNA'
molecules were modified for example through incorporation of C and
U 2'OMe modifications, 2'F modifications, phosphorothioate
modifications, phosphorylation, and conjugation of cholesterol.
Molecules were tested for inhibition of MAP4K4 expression in cells
including HeLa, primary mouse hepatocytes and primary human
hepatocytes through both lipid-mediated and passive uptake
transfection.
[0427] FIG. 22 reveals that chemical modifications can enhance gene
silencing. In particular, modifying the guide strand with 2'F UC
modifications, and with a stretch of phosphorothioate
modifications, combined with complete CU O'Me modification of the
passenger strands, resulted in molecules that were highly effective
in gene silencing. The effect of chemical modification on in vitro
efficacy in un-assisted delivery in HeLa cells was also examined.
FIG. 23 reveals that compounds lacking any of 2'F, 2'OMe, a stretch
of phosphorothioate modifications, or cholesterol conjugates, were
completely inactive in passive uptake. A combination of all 4 types
of chemical modifications, for example in compound 12386, was found
to be highly effective in gene silencing. FIG. 24 also shows the
effectiveness of compound 12386 in gene silencing.
[0428] Optimization of the length of the oligonucleotide was also
investigated. FIGS. 25 and 26 reveal that oligonucleotides with a
length of 21 nucleotides were more effective than oligonucletides
with a length of 25 nucleotides, indicating that reduction in the
size of an RNA molecule can improve efficiency, potentially by
assisting in its uptake. Screening was also conducted to optimize
the size of the duplex region of double stranded RNA molecules.
FIG. 88 reveals that compounds with duplexes of 10 nucleotides were
effective in inducing gene silencing. Positioning of the sense
strand relative to the guide strand can also be critical for
silencing gene expression (FIG. 89). In this assay, a blunt end was
found to be most effective. 3' overhangs were tolerated, but 5'
overhangs resulted in a complete loss of functionality. The guide
strand can be effective in gene silencing when hybiridized to a
sense strand of varying lengths (FIG. 90). In this assay presented
in FIG. 90, the compounds were introduced into HeLa cells via lipid
mediated transfection.
[0429] The importance of phosphorothioate content of the RNA
molecule for unassisted delivery was also investigated. FIG. 27
presents the results of a systematic screen that identified that
the presence of at least 2-12 phosphorothioates in the guide strand
as being highly advantageous for achieving uptake, with 4-8 being
the preferred number. FIG. 27 also shows that presence or absence
of phosphorothioate modifications in the sense strand did not alter
efficacy.
[0430] FIGS. 28-29 reveal the effects of passive uptake of RNA
compounds on gene silencing in primary mouse hepatocytes. nanoRNA
molecules were found to be highly effective, especially at a
concentration of 1 .mu.M (FIG. 28). FIGS. 30 and 31 reveal that the
RNA compounds associated with the invention were also effective in
gene silencing following passive uptake in primary human
hepatocytes. The cellular localization of the RNA molecules
associated with the invention was examined and compared to the
localization of Chol-siRNA (Alnylam) molecules, as shown in FIGS.
32 and 33.
[0431] A summary of 1.sup.st generation sd-rxRNA molecules is
presented in FIG. 21. Chemical modifications were introduced into
the RNA molecules, at least in part, to increase potency, such as
through optimization of nucleotide length and phosphorothioate
content, to reduce toxicity, such as through replacing 2'F
modifications on the guide strand with other modifications, to
improve delivery such as by adding or conjugating the RNA molecules
to linker and sterol modalities, and to improve the ease of
manufacturing the RNA molecules. FIG. 35 presents schematic
depictions of some of the chemical modifications that were screened
in 1.sup.st generation molecules. Parameters that were optimized
for the guide strand included nucleotide length (e.g., 19, 21 and
25 nucleotides), phosphorothioate content (e.g., 0-18
phosphorothioate linkages) and replacement of 2'F groups with 2'OMe
and 5 Me C or riboThymidine. Parameters that were optimized for the
sense strand included nucleotide length (e.g., 11, 13 and 19
nucleotides), phosphorothioate content (e.g., 0-4 phosphorothioate
linkages), and 2'OMe modifications. FIG. 36 summarizes parameters
that were screened. For example, the nucleotide length and the
phosphorothioate tail length were modified and screened for
optimization, as were the additions of 2'OMe C and U modifications.
Guide strand length and the length of the phosphorothioate modified
stretch of nucleotides were found to influence efficacy (FIGS.
37-38). Phosphorothioate modifications were tolerated in the guide
strand and were found to influence passive uptake (FIGS.
39-42).
[0432] FIG. 43 presents a schematic revealing guide strand chemical
modifications that were screened. FIGS. 44 and 45 reveal that 2'OMe
modifications were tolerated in the 3' end of the guide strand. In
particular, 2'OMe modifications in positions 1 and 11-18 were well
tolerated. The 2'OMe modifications in the seed area were tolerated
but resulted in slight reduction of efficacy. Ribo-modifications in
the seed were also well tolerated. These data indicate that the
molecules associated with the invention offer the significant
advantage of having reduced or no 2'F modification content. This is
advantageous because 2'F modifications are thought to generate
toxicity in vivo. In some instances, a complete substitution of 2'F
modifications with 2'OMe was found to lead to some reduction in
potency. However, the 2' OMe substituted molecules were still very
active. A molecule with 50% reduction in 2'F content (including at
positions 11, 16-18 which were changed to 2'OMe modifications), was
found to have comparable efficacy to a compound with complete 2'F C
and U modification. 2'OMe modification in position was found in
some instances to reduce efficacy, although this can be at least
partially compensated by 2'OMe modification in position 1 (with
chemical phosphate). In some instances, 5 Me C and/or ribothymidine
substitution for 2'F modifications led to a reduction in passive
uptake efficacy, but increased potency in lipid mediated
transfections compared to 2'F modifications. Optimization results
for lipid mediated transfection were not necessarily the same as
for passive uptake.
[0433] Modifications to the sense strand were also developed and
tested, as depicted in FIG. 46. FIG. 47 reveals that in some
instances, a sense strand length between 10-15 bases was found to
be optimal. For the molecules tested in FIG. 47, an increase in the
sense strand length resulted in reduction of passive uptake,
however an increase in sense strand length may be tolerated for
some compounds. FIG. 47 also reveals that LNA modification of the
sense strand demonstrated similar efficacy to non-LNA containing
compounds. In general, the addition of LNA or other
thermodynamically stabilizing compounds has been found to be
beneficial, in some instances resulting in converting
non-functional sequences to functional sequences. FIG. 48 also
presents data on sense strand length optimization, while FIG. 49
shows that phosphorothioate modification of the sense strand is not
required for passive uptake.
[0434] Based on the above-described optimization experiments,
2.sup.nd generation RNA molecules were developed. As shown in FIG.
50, these molecules contained reduced phosphorothioate modification
content and reduced 2'F modification content, relative to 1.sup.st
generation RNA molecules. Significantly, these RNA molecules
exhibit spontaneous cellular uptake and efficacy without a delivery
vehicle (FIG. 51). These molecules can achieve self-delivery (i.e.,
with no transfection reagent) and following self-delivery can
exhibit nanomolar activity in cell culture. These molecules can
also be delivered using lipid-mediated transfection, and exhibit
picomolar activity levels following transfection. Significantly,
these molecules exhibit highly efficient uptake, 95% by most cells
in cell culture, and are stable for more than three days in the
presence of 100% human serum. These molecules are also highly
specific and exhibit little or no immune induction. FIGS. 52 and 53
reveal the significance of chemical modifications and the
configurations of such modifications in influencing the properties
of the RNA molecules associated with the invention.
[0435] Linker chemistry was also tested in conjunction with the RNA
molecules associated with the invention. As depicted in FIG. 54,
2.sup.nd generation RNA molecules were synthesized with sterol-type
molecules attached through TEG and amino caproic acid linkers. Both
linkers showed identical potency. This functionality of the RNA
molecules, independent of linker chemistry offers additional
advantages in terms of scale up and synthesis and demonstrates that
the mechanism of function of these RNA molecules is very different
from other previously described RNA molecules.
[0436] Stability of the chemically modified sd-rxRNA molecules
described herein in human serum is shown in FIG. 55 in comparison
to unmodified RNA. The duplex molecules were incubated in 75% serum
at 37.degree. C. for the indicated periods of time. The level of
degradation was determined by running the samples on non-denaturing
gels and staining with SYBGR.
[0437] FIGS. 56 and 57 present data on cellular uptake of the
sd-rxRNA molecules. FIG. 56 shows that minimizing the length of the
RNA molecule is importance for cellular uptake, while FIG. 57
presents data showing target gene silencing after spontaneous
cellular uptake in mouse PEC-derived macrophages. FIG. 58
demonstrates spontaneous uptake and target gene silencing in
primary cells. FIG. 59 shows the results of delivery of sd-rxRNA
molecules associated with the invention to RPE cells with no
formulation. Imaging with Hoechst and DY547 reveals the clear
presence of a signal representing the RNA molecule in the sd-rxRNA
sample, while no signal is detectable in the other samples
including the samples competing a competing conjugate, an rxRNA,
and an untransfected control. FIG. 60 reveals silencing of target
gene expression in RPE cells treated with sd-rxRNA molecules
associated with the invention following 24-48 hours without any
transfection formulation.
[0438] FIG. 61 shows further optimization of the
chemical/structural composition of sd-rxRNA compounds. In some
instances, preferred properties included an antisense strand that
was 17-21 nucleotides long, a sense strand that was 10-15
nucleotides long, phosphorothioate modification of 2-12 nucleotides
within the single stranded region of the molecule, preferentially
phosphorothioate modification of 6-8 nucleotides within the single
stranded region, and 2'OMe modification at the majority of
positions within the sense strand, with or without phosphorothioate
modification. Any linker chemistry can be used to attach the
hydrophobic moiety, such as cholesterol, to the 3' end of the sense
strand. Version GM molecules, as shown in FIG. 61, have no 2'F
modifications. Significantly, there is was no impact on efficacy in
these molecules.
[0439] FIG. 62 demonstrates the superior performance of sd-rxRNA
compounds compared to compounds published by Wolfrum et. al. Nature
Biotech, 2007. Both generation I and II compounds (GI and GIIa)
developed herein show great efficacy in reducing target gene
expression. By contrast, when the chemistry described in Wolfrum et
al. (all oligos contain cholesterol conjugated to the 3' end of the
sense strand) was applied to the same sequence in a context of
conventional siRNA (19 bp duplex with two overhang) the compound
was practically inactive. These data emphasize the significance of
the combination of chemical modifications and assymetrical
molecules described herein, producing highly effective RNA
compounds.
[0440] FIG. 63 shows localization of sd-rxRNA molecules developed
herein compared to localization of other RNA molecules such as
those described in Soutschek et al. (2004) Nature, 432:173.
sd-rxRNA molecules accumulate inside the cells whereas competing
conjugate RNAs accumulate on the surface of cells. Significantly,
FIG. 64 shows that sd-rxRNA molecules, but not competitor molecules
such as those described in Soutschek et al. are internalized within
minutes. FIG. 65 compares localization of sd-rxRNA molecules
compared to regular siRNA-cholesterol, as described in Soutschek et
al. A signal representing the RNA molecule is clearly detected for
the sd-rxRNA molecule in tissue culture RPE cells, following local
delivery to compromised skin, and following systemic delivery where
uptake to the liver is seen. In each case, no signal is detected
for the regular siRNA-cholesterol molecule. The sd-rxRNA molecule
thus has drastically better cellular and tissue uptake
characteristics when compared to conventional cholesterol
conjugated siRNAs such as those described in Soutschek et al. The
level of uptake is at least order of magnitude higher and is due at
least in part to the unique combination of chemistries and
conjugated structure. Superior delivery of sd-rxRNA relative to
previously described RNA molecules is also demonstrated in FIGS. 66
and 67.
[0441] Based on the analysis of 2.sup.nd generation RNA molecules
associated with the invention, a screen was performed to identify
functional molecules for targeting the SPP1/PPIB gene. As revealed
in FIG. 68, several effective molecules were identified, with 14131
being the most effective. The compounds were added to A-549 cells
and then the level of SPP1/PPIB ratio was determined by B-DNA after
48 hours.
[0442] FIG. 69 reveals efficient cellular uptake of sd-rxRNA within
minutes of exposure. This is a unique characteristics of these
molecules, not observed with any other RNAi compounds. Compounds
described in Soutschek et al. were used as negative controls. FIG.
70 reveals that the uptake and gene silencing of the sd-rxRNA is
effective in multiple different cell types including SH-SY5Y
neuroblastoma derived cells, ARPE-19 (retinal pigment epithelium)
cells, primary hepatocytes, and primary macrophages. In each case
silencing was confirmed by looking at target gene expression by a
Branched DNA assay.
[0443] FIG. 70 reveals that sd-rxRNA is active in the presence or
absence of serum. While a slight reduction in efficacy (2-5 fold)
was observed in the presence of serum, this small reduction in
efficacy in the presence of serum differentiate the sd-rxRNA
molecules from previously described molecules which exhibited a
larger reduction in efficacy in the presence of serum. This
demonstrated level of efficacy in the presence of serum creates a
foundation for in vivo efficacy.
[0444] FIG. 72 reveals efficient tissue penetration and cellular
uptake upon single intradermal injection. This data indicates the
potential of the sd-rxRNA compounds described herein for silencing
genes in any dermatology applications, and also represents a model
for local delivery of sd-rxRNA compounds. FIG. 73 also demonstrates
efficient cellular uptake and in vivo silencing with sd-rxRNA
following intradermal injection. Silencing is determined as the
level of MAP4K4 knockdown in several individual biopsies taken from
the site of injection as compared to biopsies taken from a site
injected with a negative control. FIG. 74 reveals that sd-rxRNA
compounds has improved blood clearance and induced effective gene
silencing in vivo in the liver upon systemic administration. In
comparison to the RNA molecules described by Soutschek et al., the
level of liver uptake at identical dose level is at least 50 fold
higher with the sd-rxRNA molecules. The uptake results in
productive silencing. sd-rxRNA compounds are also characterized by
improved blood clearance kinetics.
[0445] The effect of 5-Methly C modifications was also examined.
FIG. 75 demonstrates that the presence of 5-Methyl C in an RNAi
molecule resulted in increased potency in lipid mediated
transfection. This suggests that hydrophobic modification of Cs and
Us in an RNAi molecule can be beneficial. These types of
modifications can also be used in the context 2' ribose modified
bases to ensure optimal stability and efficacy. FIG. 76 presents
data showing that incorporation of 5-Methyl C and/or ribothymidine
in the guide strand can in some instances reduce efficacy.
[0446] FIG. 77 reveals that sd-rxRNA molecules are more effective
than competitor molecules such as molecules described in Soutschek
et al., in systemic delivery to the liver. A signal representing
the RNA molecule is clearly visible in the sample containing
sd-rxRNA, while no signal representing the RNA molecule is visible
in the sample containing the competitor RNA molecule.
[0447] The addition of hydrophobic conjugates to the sd-rxRNA
molecules was also explored (FIGS. 78-83). FIG. 78 presents
schematics demonstrating 5-uridyl modifications with improved
hydrophobicity characteristics. Incorporation of such modifications
into sd-rxRNA compounds can increase cellular and tissue uptake
properties. FIG. 78B presents a new type of RNAi compound
modification which can be applied to compounds to improve cellular
uptake and pharmacokinetic behavior. Significantly, this type of
modification, when applied to sd-rxRNA compounds, may contribute to
making such compounds orally available. FIG. 79 presents schematics
revealing the structures of synthesized modified sterol-type
molecules, where the length and structure of the C17 attached tail
is modified. Without wishing to be bound by any theory, the length
of the C17 attached tail may contribute to improving in vitro and
in vivo efficacy of sd-rxRNA compounds.
[0448] FIG. 80 presents a schematic demonstrating the lithocholic
acid route to long side chain cholesterols. FIG. 81 presents a
schematic demonstrating a route to 5-uridyl phosphoramidite
synthesis. FIG. 82 presents a schematic demonstrating synthesis of
tri-functional hydroxyprolinol linker for 3'-cholesterol
attachment. FIG. 83 presents a schematic demonstrating synthesis of
solid support for the manufacture of a shorter asymmetric RNAi
compound strand.
[0449] A screen was conducted to identify compounds that could
effectively silence expression of SPP1 (Osteopontin). Compounds
targeting SPP1 were added to A549 cells (using passive
transfection), and the level of SPP1 expression was evaluated at 48
hours. Several novel compounds effective in SPP1 silencing were
identified. Compounds that were effective in silencing of SPP1
included 14116, 14121, 14131, 14134, 14139, 14149, and 14152 (FIGS.
84-86). The most potent compound in this assay was 14131 (FIG. 84).
The efficacy of these sd-rxRNA compounds in silencing SPP1
expression was independently validated (FIG. 85).
[0450] A similar screen was conducted to identify compounds that
could effectively silence expression of CTGF (FIGS. 86-87).
Compounds that were effective in silencing of CTGF included 14017,
14013, 14016, 14022, 14025, 14027.
Methods
Transfection of Sd-rxRNA.sup.nano
Lipid Mediated Transfection
[0451] sd-rxRNA.sup.nano constructs were chemically synthesized
(Dharmacon, Lafayette, Colo.) and transfected into HEK293 cells
(ATCC, Manassas, Va.) using Lipofectamine RNAiMAX (Invitrogen,
Carlsbad, Calif.) according to the manufacturer's instructions. In
brief, RNA was diluted to a 12.times. concentration in
Opti-MEM.RTM. Reduced Serum Media (Invitrogen, Carlsbad, Calif.)
and then combined with a 12.times. concentration of Lipofectamine
RNAiMAX. The RNA and transfection reagent were allowed to complex
at room temperature for 20 minutes and make a 6.times.
concentration. While complexing, HEK293 cells were washed,
trypsinized and counted. The cells were diluted to a concentration
recommended by the manufacturer and previously described of
1.times.10.sup.5 cells/ml. When RNA had completed complexing with
the RNAiMAX transfection reagent, 20 ul of the complexes were added
to the appropriate well of the 96-well plate in triplicate. Cells
were added to each well (100 ul volume) to make the final cell
count per well 1.times.10.sup.4 cells/well. The volume of cells
diluted the 6.times. concentration of complex to 1.times. (between
10-0.05 nM). Cells were incubated for 24 or 48 hours under normal
growth conditions. After 24 or 48 hour incubation, cells were lysed
and gene silencing activity was measured using the QuantiGene assay
(Panomics, Freemont, Calif.) which employs bDNA hybridization
technology. The assay was carried out according to manufacturer's
instructions.
Passive Uptake Transfection
[0452] sd-rxRNA.sup.nano constructs were chemically synthesized
(Dharmacon, Lafayette, Colo.). 24 hours prior to transfection, HeLa
cells (ATCC, Manassas, Va.) were plated at 1.times.10.sup.4
cells/well in a 96 well plate under normal growth conditions (DMEM,
10% FBS and 1% Penicillin and Streptomycin). Prior to transfection
of HeLa cells, sd-rxRNA.sup.nano were diluted to a final
concentration of 0.01 uM to 1 uM in Accell siRNA Delivery Media
(Dharmacon, Lafayette, Colo.). Normal growth media was aspirated
off cells and 100 uL of Accell Delivery media containing the
appropriate concentration of sd-rxRNAnano was applied to the cells.
48 hours post transfection, delivery media was aspirated off the
cells and normal growth media was applied to cells for an
additional 24 hours.
[0453] After 48 or 72 hour incubation, cells were lysed and gene
silencing activity was measured using the QuantiGene assay
(Panomics, Freemont, Calif.) according to manufacturer's
instructions.
TABLE-US-00001 TABLE 1 Oligo Accession Gene ID Number Number number
Gene Name Symbol APOB-10167-20-12138 12138 NM_000384 Apolipoprotein
B (including APOB Ag(x) antigen) APOB-10167-20-12139 12139
NM_000384 Apolipoprotein B (including APOB Ag(x) antigen)
MAP4K4-2931-13-12266 12266 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
MAP4K4-2931-16-12293 12293 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
MAP4K4-2931-16-12383 12383 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
MAP4K4-2931-16-12384 12384 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
MAP4K4-2931-16-12385 12385 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
MAP4K4-2931-16-12386 12386 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
MAP4K4-2931-16-12387 12387 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
MAP4K4-2931-15-12388 12388 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
MAP4K4-2931-13-12432 12432 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
MAP4K4-2931-13-12266.2 12266.2 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
APOB--21-12434 12434 NM_000384 Apolipoprotein B (including APOB
Ag(x) antigen) APOB--21-12435 12435 NM_000384 Apolipoprotein B
(including APOB Ag(x) antigen) MAP4K4-2931-16-12451 12451 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4-2931-16-12452 12452 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4-2931-16-12453 12453 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4-2931-17-12454 12454 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4-2931-17-12455 12455 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4-2931-19-12456 12456 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 --27-12480 12480 --27-12481 12481
APOB-10167-21-12505 12505 NM_000384 Apolipoprotein B (including
APOB Ag(x) antigen) APOB-10167-21-12506 12506 NM_000384
Apolipoprotein B (including APOB Ag(x) antigen)
MAP4K4-2931-16-12539 12539 NM_004834 Mitogen-Activated Protein
Kinase MAP4K4 Kinase Kinase Kinase 4 (MAP4K4), transcript variant 1
APOB-10167-21-12505.2 12505.2 NM_000384 Apolipoprotein B (including
APOB Ag(x) antigen) APOB-10167-21-12506.2 12506.2 NM_000384
Apolipoprotein B (including APOB Ag(x) antigen) MAP4K4--13-12565
12565 MAP4K4 MAP4K4-2931-16-12386.2 12386.2 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4-2931-13-12815 12815 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 APOB--13-12957 12957 NM_000384
Apolipoprotein B (including APOB Ag(x) antigen) MAP4K4--16-12983
12983 Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase
4 (MAP4K4), transcript variant 1 MAP4K4--16-12984 12984
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12985 12985
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12986 12986
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12987 12987
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12988 12988
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12989 12989
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12990 12990
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12991 12991
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12992 12992
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12993 12993
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12994 12994
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4--16-12995 12995
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4-2931-19-13012 13012 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 MAP4K4-2931-19-13016 13016 NM_004834
Mitogen-Activated Protein Kinase MAP4K4 Kinase Kinase Kinase 4
(MAP4K4), transcript variant 1 PPIB--13-13021 13021 NM_000942
Peptidylprolyl Isomerase B PPIB (cyclophilin B) pGL3-1172-13-13038
13038 U47296 Cloning vector pGL3-Control pGL3 pGL3-1172-13-13040
13040 U47296 Cloning vector pGL3-Control pGL3 --16-13047 13047
SOD1-530-13-13090 13090 NM_000454 Superoxide Dismutase 1, soluble
SOD1 (amyotrophic lateral sclerosis 1 (adult)) SOD1-523-13-13091
13091 NM_000454 Superoxide Dismutase 1, soluble SOD1 (amyotrophic
lateral sclerosis 1 (adult)) SOD1-535-13-13092 13092 NM_000454
Superoxide Dismutase 1, soluble SOD1 (amyotrophic lateral sclerosis
1 (adult)) SOD1-536-13-13093 13093 NM_000454 Superoxide Dismutase
1, soluble SOD1 (amyotrophic lateral sclerosis 1 (adult))
SOD1-396-13-13094 13094 NM_000454 Superoxide Dismutase 1, soluble
SOD1 (amyotrophic lateral sclerosis 1 (adult)) SOD1-385-13-13095
13095 NM_000454 Superoxide Dismutase 1, soluble SOD1 (amyotrophic
lateral sclerosis 1 (adult)) SOD1-195-13-13096 13096 NM_000454
Superoxide Dismutase 1, soluble SOD1 (amyotrophic lateral sclerosis
1 (adult)) APOB-4314-13-13115 13115 NM_000384 Apolipoprotein B
(including APOB Ag(x) antigen) APOB-3384-13-13116 13116 NM_000384
Apolipoprotein B (including APOB Ag(x) antigen) APOB-3547-13-13117
13117 NM_000384 Apolipoprotein B (including APOB Ag(x) antigen)
APOB-4318-13-13118 13118 NM_000384 Apolipoprotein B (including APOB
Ag(x) antigen) APOB-3741-13-13119 13119 NM_000384 Apolipoprotein B
(including APOB Ag(x) antigen) PPIB--16-13136 13136 NM_000942
Peptidylprolyl Isomerase B PPIB (cyclophilin B) APOB-4314-15-13154
13154 NM_000384 Apolipoprotein B (including APOB Ag(x) antigen)
APOB-3547-15-13155 13155 NM_000384 Apolipoprotein B (including APOB
Ag(x) antigen) APOB-4318-15-13157 13157 NM_000384 Apolipoprotein B
(including APOB Ag(x) antigen) APOB-3741-15-13158 13158 NM_000384
Apolipoprotein B (including APOB Ag(x) antigen) APOB--13-13159
13159 NM_000384 Apolipoprotein B (including APOB Ag(x) antigen)
APOB--15-13160 13160 NM_000384 Apolipoprotein B (including APOB
Ag(x) antigen) SOD1-530-16-13163 13163 NM_000454 Superoxide
Dismutase 1, soluble SOD1 (amyotrophic lateral sclerosis 1 (adult))
SOD1-523-16-13164 13164 NM_000454 Superoxide Dismutase 1, soluble
SOD1 (amyotrophic lateral sclerosis 1 (adult)) SOD1-535-16-13165
13165 NM_000454 Superoxide Dismutase 1, soluble SOD1 (amyotrophic
lateral sclerosis 1 (adult)) SOD1-536-16-13166 13166 NM_000454
Superoxide Dismutase 1, soluble SOD1 (amyotrophic lateral sclerosis
1 (adult)) SOD1-396-16-13167 13167 NM_000454 Superoxide Dismutase
1, soluble SOD1 (amyotrophic lateral sclerosis 1 (adult))
SOD1-385-16-13168 13168 NM_000454 Superoxide Dismutase 1, soluble
SOD1 (amyotrophic lateral sclerosis 1 (adult)) SOD1-195-16-13169
13169 NM_000454 Superoxide Dismutase 1, soluble SOD1 (amyotrophic
lateral sclerosis 1 (adult)) pGL3-1172-16-13170 13170 U47296
Cloning vector pGL3-Control pGL3 pGL3-1172-16-13171 13171 U47296
Cloning vector pGL3-Control pGL3 MAP4k4-2931-19-13189 13189
NM_004834 Mitogen-Activated Protein Kinase MAP4k4 Kinase Kinase
Kinase 4 (MAP4K4), transcript variant 1 CTGF-1222-13-13190 13190
NM_001901.2 connective tissue growth factor CTGF CTGF-813-13-13192
13192 NM_001901.2 connective tissue growth factor CTGF
CTGF-747-13-13194 13194 NM_001901.2 connective tissue growth factor
CTGF CTGF-817-13-13196 13196 NM_001901.2 connective tissue growth
factor CTGF CTGF-1174-13-13198 13198 NM_001901.2 connective tissue
growth factor CTGF CTGF-1005-13-13200 13200 NM_001901.2 connective
tissue growth factor CTGF CTGF-814-13-13202 13202 NM_001901.2
connective tissue growth factor CTGF CTGF-816-13-13204 13204
NM_001901.2 connective tissue growth factor CTGF CTGF-1001-13-13206
13206 NM_001901.2 connective tissue growth factor CTGF
CTGF-1173-13-13208 13208 NM_001901.2 connective tissue growth
factor CTGF CTGF-749-13-13210 13210 NM_001901.2 connective tissue
growth factor CTGF CTGF-792-13-13212 13212 NM_001901.2 connective
tissue growth factor CTGF CTGF-1162-13-13214 13214 NM_001901.2
connective tissue growth factor CTGF CTGF-811-13-13216 13216
NM_001901.2 connective tissue growth factor CTGF CTGF-797-13-13218
13218 NM_001901.2 connective tissue growth factor CTGF
CTGF-1175-13-13220 13220 NM_001901.2 connective tissue growth
factor CTGF CTGF-1172-13-13222 13222 NM_001901.2 connective tissue
growth factor CTGF CTGF-1177-13-13224 13224 NM_001901.2 connective
tissue growth factor CTGF CTGF-1176-13-13226 13226 NM_001901.2
connective tissue growth factor CTGF CTGF-812-13-13228 13228
NM_001901.2 connective tissue growth factor CTGF CTGF-745-13-13230
13230 NM_001901.2 connective tissue growth factor CTGF
CTGF-1230-13-13232 13232 NM_001901.2 connective tissue growth
factor CTGF CTGF-920-13-13234 13234 NM_001901.2 connective tissue
growth factor CTGF
CTGF-679-13-13236 13236 NM_001901.2 connective tissue growth factor
CTGF CTGF-992-13-13238 13238 NM_001901.2 connective tissue growth
factor CTGF CTGF-1045-13-13240 13240 NM_001901.2 connective tissue
growth factor CTGF CTGF-1231-13-13242 13242 NM_001901.2 connective
tissue growth factor CTGF CTGF-991-13-13244 13244 NM_001901.2
connective tissue growth factor CTGF CTGF-998-13-13246 13246
NM_001901.2 connective tissue growth factor CTGF CTGF-1049-13-13248
13248 NM_001901.2 connective tissue growth factor CTGF
CTGF-1044-13-13250 13250 NM_001901.2 connective tissue growth
factor CTGF CTGF-1327-13-13252 13252 NM_001901.2 connective tissue
growth factor CTGF CTGF-1196-13-13254 13254 NM_001901.2 connective
tissue growth factor CTGF CTGF-562-13-13256 13256 NM_001901.2
connective tissue growth factor CTGF CTGF-752-13-13258 13258
NM_001901.2 connective tissue growth factor CTGF CTGF-994-13-13260
13260 NM_001901.2 connective tissue growth factor CTGF
CTGF-1040-13-13262 13262 NM_001901.2 connective tissue growth
factor CTGF CTGF-1984-13-13264 13264 NM_001901.2 connective tissue
growth factor CTGF CTGF-2195-13-13266 13266 NM_001901.2 connective
tissue growth factor CTGF CTGF-2043-13-13268 13268 NM_001901.2
connective tissue growth factor CTGF CTGF-1892-13-13270 13270
NM_001901.2 connective tissue growth factor CTGF CTGF-1567-13-13272
13272 NM_001901.2 connective tissue growth factor CTGF
CTGF-1780-13-13274 13274 NM_001901.2 connective tissue growth
factor CTGF CTGF-2162-13-13276 13276 NM_001901.2 connective tissue
growth factor CTGF CTGF-1034-13-13278 13278 NM_001901.2 connective
tissue growth factor CTGF CTGF-2264-13-13280 13280 NM_001901.2
connective tissue growth factor CTGF CTGF-1032-13-13282 13282
NM_001901.2 connective tissue growth factor CTGF CTGF-1535-13-13284
13284 NM_001901.2 connective tissue growth factor CTGF
CTGF-1694-13-13286 13286 NM_001901.2 connective tissue growth
factor CTGF CTGF-1588-13-13288 13288 NM_001901.2 connective tissue
growth factor CTGF CTGF-928-13-13290 13290 NM_001901.2 connective
tissue growth factor CTGF CTGF-1133-13-13292 13292 NM_001901.2
connective tissue growth factor CTGF CTGF-912-13-13294 13294
NM_001901.2 connective tissue growth factor CTGF CTGF-753-13-13296
13296 NM_001901.2 connective tissue growth factor CTGF
CTGF-918-13-13298 13298 NM_001901.2 connective tissue growth factor
CTGF CTGF-744-13-13300 13300 NM_001901.2 connective tissue growth
factor CTGF CTGF-466-13-13302 13302 NM_001901.2 connective tissue
growth factor CTGF CTGF-917-13-13304 13304 NM_001901.2 connective
tissue growth factor CTGF CTGF-1038-13-13306 13306 NM_001901.2
connective tissue growth factor CTGF CTGF-1048-13-13308 13308
NM_001901.2 connective tissue growth factor CTGF CTGF-1235-13-13310
13310 NM_001901.2 connective tissue growth factor CTGF
CTGF-868-13-13312 13312 NM_001901.2 connective tissue growth factor
CTGF CTGF-1131-13-13314 13314 NM_001901.2 connective tissue growth
factor CTGF CTGF-1043-13-13316 13316 NM_001901.2 connective tissue
growth factor CTGF CTGF-751-13-13318 13318 NM_001901.2 connective
tissue growth factor CTGF CTGF-1227-13-13320 13320 NM_001901.2
connective tissue growth factor CTGF CTGF-867-13-13322 13322
NM_001901.2 connective tissue growth factor CTGF CTGF-1128-13-13324
13324 NM_001901.2 connective tissue growth factor CTGF
CTGF-756-13-13326 13326 NM_001901.2 connective tissue growth factor
CTGF CTGF-1234-13-13328 13328 NM_001901.2 connective tissue growth
factor CTGF CTGF-916-13-13330 13330 NM_001901.2 connective tissue
growth factor CTGF CTGF-925-13-13332 13332 NM_001901.2 connective
tissue growth factor CTGF CTGF-1225-13-13334 13334 NM_001901.2
connective tissue growth factor CTGF CTGF-445-13-13336 13336
NM_001901.2 connective tissue growth factor CTGF CTGF-446-13-13338
13338 NM_001901.2 connective tissue growth factor CTGF
CTGF-913-13-13340 13340 NM_001901.2 connective tissue growth factor
CTGF CTGF-997-13-13342 13342 NM_001901.2 connective tissue growth
factor CTGF CTGF-277-13-13344 13344 NM_001901.2 connective tissue
growth factor CTGF CTGF-1052-13-13346 13346 NM_001901.2 connective
tissue growth factor CTGF CTGF-887-13-13348 13348 NM_001901.2
connective tissue growth factor CTGF CTGF-914-13-13350 13350
NM_001901.2 connective tissue growth factor CTGF CTGF-1039-13-13352
13352 NM_001901.2 connective tissue growth factor CTGF
CTGF-754-13-13354 13354 NM_001901.2 connective tissue growth factor
CTGF CTGF-1130-13-13356 13356 NM_001901.2 connective tissue growth
factor CTGF CTGF-919-13-13358 13358 NM_001901.2 connective tissue
growth factor CTGF CTGF-922-13-13360 13360 NM_001901.2 connective
tissue growth factor CTGF CTGF-746-13-13362 13362 NM_001901.2
connective tissue growth factor CTGF CTGF-993-13-13364 13364
NM_001901.2 connective tissue growth factor CTGF CTGF-825-13-13366
13366 NM_001901.2 connective tissue growth factor CTGF
CTGF-926-13-13368 13368 NM_001901.2 connective tissue growth factor
CTGF CTGF-923-13-13370 13370 NM_001901.2 connective tissue growth
factor CTGF CTGF-866-13-13372 13372 NM_001901.2 connective tissue
growth factor CTGF CTGF-563-13-13374 13374 NM_001901.2 connective
tissue growth factor CTGF CTGF-823-13-13376 13376 NM_001901.2
connective tissue growth factor CTGF CTGF-1233-13-13378 13378
NM_001901.2 connective tissue growth factor CTGF CTGF-924-13-13380
13380 NM_001901.2 connective tissue growth factor CTGF
CTGF-921-13-13382 13382 NM_001901.2 connective tissue growth factor
CTGF CTGF-443-13-13384 13384 NM_001901.2 connective tissue growth
factor CTGF CTGF-1041-13-13386 13386 NM_001901.2 connective tissue
growth factor CTGF CTGF-1042-13-13388 13388 NM_001901.2 connective
tissue growth factor CTGF CTGF-755-13-13390 13390 NM_001901.2
connective tissue growth factor CTGF CTGF-467-13-13392 13392
NM_001901.2 connective tissue growth factor CTGF CTGF-995-13-13394
13394 NM_001901.2 connective tissue growth factor CTGF
CTGF-927-13-13396 13396 NM_001901.2 connective tissue growth factor
CTGF SPP1-1025-13-13398 13398 NM_000582.2 Osteopontin SPP1
SPP1-1049-13-13400 13400 NM_000582.2 Osteopontin SPP1
SPP1-1051-13-13402 13402 NM_000582.2 Osteopontin SPP1
SPP1-1048-13-13404 13404 NM_000582.2 Osteopontin SPP1
SPP1-1050-13-13406 13406 NM_000582.2 Osteopontin SPP1
SPP1-1047-13-13408 13408 NM_000582.2 Osteopontin SPP1
SPP1-800-13-13410 13410 NM_000582.2 Osteopontin SPP1
SPP1-492-13-13412 13412 NM_000582.2 Osteopontin SPP1
SPP1-612-13-13414 13414 NM_000582.2 Osteopontin SPP1
SPP1-481-13-13416 13416 NM_000582.2 Osteopontin SPP1
SPP1-614-13-13418 13418 NM_000582.2 Osteopontin SPP1
SPP1-951-13-13420 13420 NM_000582.2 Osteopontin SPP1
SPP1-482-13-13422 13422 NM_000582.2 Osteopontin SPP1
SPP1-856-13-13424 13424 NM_000582.2 Osteopontin SPP1
SPP1-857-13-13426 13426 NM_000582.2 Osteopontin SPP1
SPP1-365-13-13428 13428 NM_000582.2 Osteopontin SPP1
SPP1-359-13-13430 13430 NM_000582.2 Osteopontin SPP1
SPP1-357-13-13432 13432 NM_000582.2 Osteopontin SPP1
SPP1-858-13-13434 13434 NM_000582.2 Osteopontin SPP1
SPP1-1012-13-13436 13436 NM_000582.2 Osteopontin SPP1
SPP1-1014-13-13438 13438 NM_000582.2 Osteopontin SPP1
SPP1-356-13-13440 13440 NM_000582.2 Osteopontin SPP1
SPP1-368-13-13442 13442 NM_000582.2 Osteopontin SPP1
SPP1-1011-13-13444 13444 NM_000582.2 Osteopontin SPP1
SPP1-754-13-13446 13446 NM_000582.2 Osteopontin SPP1
SPP1-1021-13-13448 13448 NM_000582.2 Osteopontin SPP1
SPP1-1330-13-13450 13450 NM_000582.2 Osteopontin SPP1
SPP1-346-13-13452 13452 NM_000582.2 Osteopontin SPP1
SPP1-869-13-13454 13454 NM_000582.2 Osteopontin SPP1
SPP1-701-13-13456 13456 NM_000582.2 Osteopontin SPP1
SPP1-896-13-13458 13458 NM_000582.2 Osteopontin SPP1
SPP1-1035-13-13460 13460 NM_000582.2 Osteopontin SPP1
SPP1-1170-13-13462 13462 NM_000582.2 Osteopontin SPP1
SPP1-1282-13-13464 13464 NM_000582.2 Osteopontin SPP1
SPP1-1537-13-13466 13466 NM_000582.2 Osteopontin SPP1
SPP1-692-13-13468 13468 NM_000582.2 Osteopontin SPP1
SPP1-840-13-13470 13470 NM_000582.2 Osteopontin SPP1
SPP1-1163-13-13472 13472 NM_000582.2 Osteopontin SPP1
SPP1-789-13-13474 13474 NM_000582.2 Osteopontin SPP1
SPP1-841-13-13476 13476 NM_000582.2 Osteopontin SPP1
SPP1-852-13-13478 13478 NM_000582.2 Osteopontin SPP1
SPP1-209-13-13480 13480 NM_000582.2 Osteopontin SPP1
SPP1-1276-13-13482 13482 NM_000582.2 Osteopontin SPP1
SPP1-137-13-13484 13484 NM_000582.2 Osteopontin SPP1
SPP1-711-13-13486 13486 NM_000582.2 Osteopontin SPP1
SPP1-582-13-13488 13488 NM_000582.2 Osteopontin SPP1
SPP1-839-13-13490 13490 NM_000582.2 Osteopontin SPP1
SPP1-1091-13-13492 13492 NM_000582.2 Osteopontin SPP1
SPP1-884-13-13494 13494 NM_000582.2 Osteopontin SPP1
SPP1-903-13-13496 13496 NM_000582.2 Osteopontin SPP1
SPP1-1090-13-13498 13498 NM_000582.2 Osteopontin SPP1
SPP1-474-13-13500 13500 NM_000582.2 Osteopontin SPP1
SPP1-575-13-13502 13502 NM_000582.2 Osteopontin SPP1
SPP1-671-13-13504 13504 NM_000582.2 Osteopontin SPP1
SPP1-924-13-13506 13506 NM_000582.2 Osteopontin SPP1
SPP1-1185-13-13508 13508 NM_000582.2 Osteopontin SPP1
SPP1-1221-13-13510 13510 NM_000582.2 Osteopontin SPP1
SPP1-347-13-13512 13512 NM_000582.2 Osteopontin SPP1
SPP1-634-13-13514 13514 NM_000582.2 Osteopontin SPP1
SPP1-877-13-13516 13516 NM_000582.2 Osteopontin SPP1
SPP1-1033-13-13518 13518 NM_000582.2 Osteopontin SPP1
SPP1-714-13-13520 13520 NM_000582.2 Osteopontin SPP1
SPP1-791-13-13522 13522 NM_000582.2 Osteopontin SPP1
SPP1-813-13-13524 13524 NM_000582.2 Osteopontin SPP1
SPP1-939-13-13526 13526 NM_000582.2 Osteopontin SPP1
SPP1-1161-13-13528 13528 NM_000582.2 Osteopontin SPP1
SPP1-1164-13-13530 13530 NM_000582.2 Osteopontin SPP1
SPP1-1190-13-13532 13532 NM_000582.2 Osteopontin SPP1
SPP1-1333-13-13534 13534 NM_000582.2 Osteopontin SPP1
SPP1-537-13-13536 13536 NM_000582.2 Osteopontin SPP1
SPP1-684-13-13538 13538 NM_000582.2 Osteopontin SPP1
SPP1-707-13-13540 13540 NM_000582.2 Osteopontin SPP1
SPP1-799-13-13542 13542 NM_000582.2 Osteopontin SPP1
SPP1-853-13-13544 13544 NM_000582.2 Osteopontin SPP1
SPP1-888-13-13546 13546 NM_000582.2 Osteopontin SPP1
SPP1-1194-13-13548 13548 NM_000582.2 Osteopontin SPP1
SPP1-1279-13-13550 13550 NM_000582.2 Osteopontin SPP1
SPP1-1300-13-13552 13552 NM_000582.2 Osteopontin SPP1
SPP1-1510-13-13554 13554 NM_000582.2 Osteopontin SPP1
SPP1-1543-13-13556 13556 NM_000582.2 Osteopontin SPP1
SPP1-434-13-13558 13558 NM_000582.2 Osteopontin SPP1
SPP1-600-13-13560 13560 NM_000582.2 Osteopontin SPP1
SPP1-863-13-13562 13562 NM_000582.2 Osteopontin SPP1
SPP1-902-13-13564 13564 NM_000582.2 Osteopontin SPP1
SPP1-921-13-13566 13566 NM_000582.2 Osteopontin SPP1
SPP1-154-13-13568 13568 NM_000582.2 Osteopontin SPP1
SPP1-217-13-13570 13570 NM_000582.2 Osteopontin SPP1
SPP1-816-13-13572 13572 NM_000582.2 Osteopontin SPP1
SPP1-882-13-13574 13574 NM_000582.2 Osteopontin SPP1
SPP1-932-13-13576 13576 NM_000582.2 Osteopontin SPP1
SPP1-1509-13-13578 13578 NM_000582.2 Osteopontin SPP1
SPP1-157-13-13580 13580 NM_000582.2 Osteopontin SPP1
SPP1-350-13-13582 13582 NM_000582.2 Osteopontin SPP1
SPP1-511-13-13584 13584 NM_000582.2 Osteopontin SPP1
SPP1-605-13-13586 13586 NM_000582.2 Osteopontin SPP1
SPP1-811-13-13588 13588 NM_000582.2 Osteopontin SPP1
SPP1-892-13-13590 13590 NM_000582.2 Osteopontin SPP1
SPP1-922-13-13592 13592 NM_000582.2 Osteopontin SPP1
SPP1-1169-13-13594 13594 NM_000582.2 Osteopontin SPP1
SPP1-1182-13-13596 13596 NM_000582.2 Osteopontin SPP1
SPP1-1539-13-13598 13598 NM_000582.2 Osteopontin SPP1
SPP1-1541-13-13600 13600 NM_000582.2 Osteopontin SPP1
SPP1-427-13-13602 13602 NM_000582.2 Osteopontin SPP1
SPP1-533-13-13604 13604 NM_000582.2 Osteopontin SPP1 APOB--13-13763
13763 NM_000384 Apolipoprotein B (including APOB Ag(x) antigen)
APOB--13-13764 13764 NM_000384 Apolipoprotein B (including APOB
Ag(x) antigen) MAP4K4--16-13766 13766 MAP4K4 PPIB--13-13767 13767
NM_000942 peptidylprolyl isomerase B PPIB (cyclophilin B)
PPIB--15-13768 13768 NM_000942 peptidylprolyl isomerase B PPIB
(cyclophilin B) PPIB--17-13769 13769 NM_000942 peptidylprolyl
isomerase B PPIB (cyclophilin B) MAP4K4--16-13939 13939 MAP4K4
APOB-4314-16-13940 13940 NM_000384 Apolipoprotein B (including APOB
Ag(x) antigen) APOB-4314-17-13941 13941 NM_000384 Apolipoprotein B
(including APOB Ag(x) antigen) APOB--16-13942 13942 NM_000384
Apolipoprotein B (including APOB Ag(x) antigen) APOB--18-13943
13943 NM_000384 Apolipoprotein B (including APOB Ag(x) antigen)
APOB--17-13944 13944 NM_000384 Apolipoprotein B (including APOB
Ag(x) antigen) APOB--19-13945 13945 NM_000384 Apolipoprotein B
(including APOB Ag(x) antigen) APOB-4314-16-13946 13946 NM_000384
Apolipoprotein B (including APOB Ag(x) antigen) APOB-4314-17-13947
13947 NM_000384 Apolipoprotein B (including APOB Ag(x) antigen)
APOB--16-13948 13948 NM_000384 Apolipoprotein B (including APOB
Ag(x) antigen) APOB--17-13949 13949 NM_000384 Apolipoprotein B
(including APOB Ag(x) antigen) APOB--16-13950 13950 NM_000384
Apolipoprotein B (including APOB Ag(x) antigen) APOB--18-13951
13951 NM_000384 Apolipoprotein B (including APOB Ag(x) antigen)
APOB--17-13952 13952 NM_000384 Apolipoprotein B (including APOB
Ag(x) antigen) APOB--19-13953 13953 NM_000384 Apolipoprotein B
(including APOB Ag(x) antigen) MAP4K4--16-13766.2 13766.2 MAP4K4
CTGF-1222-16-13980 13980 NM_001901.2 connective tissue growth
factor CTGF CTGF-813-16-13981 13981 NM_001901.2 connective tissue
growth factor CTGF CTGF-747-16-13982 13982 NM_001901.2 connective
tissue growth factor CTGF CTGF-817-16-13983 13983 NM_001901.2
connective tissue growth factor CTGF CTGF-1174-16-13984 13984
NM_001901.2 connective tissue growth factor CTGF CTGF-1005-16-13985
13985 NM_001901.2 connective tissue growth factor CTGF
CTGF-814-16-13986 13986 NM_001901.2 connective tissue growth factor
CTGF CTGF-816-16-13987 13987 NM_001901.2 connective tissue growth
factor CTGF CTGF-1001-16-13988 13988 NM_001901.2 connective tissue
growth factor CTGF CTGF-1173-16-13989 13989 NM_001901.2 connective
tissue growth factor CTGF CTGF-749-16-13990 13990 NM_001901.2
connective tissue growth factor CTGF CTGF-792-16-13991 13991
NM_001901.2 connective tissue growth factor CTGF CTGF-1162-16-13992
13992 NM_001901.2 connective tissue growth factor CTGF
CTGF-811-16-13993 13993 NM_001901.2 connective tissue growth factor
CTGF CTGF-797-16-13994 13994 NM_001901.2 connective tissue growth
factor CTGF CTGF-1175-16-13995 13995 NM_001901.2 connective tissue
growth factor CTGF CTGF-1172-16-13996 13996 NM_001901.2 connective
tissue growth factor CTGF CTGF-1177-16-13997 13997 NM_001901.2
connective tissue growth factor CTGF CTGF-1176-16-13998 13998
NM_001901.2 connective tissue growth factor CTGF CTGF-812-16-13999
13999 NM_001901.2 connective tissue growth factor CTGF
CTGF-745-16-14000 14000 NM_001901.2 connective tissue growth factor
CTGF CTGF-1230-16-14001 14001 NM_001901.2 connective tissue growth
factor CTGF CTGF-920-16-14002 14002 NM_001901.2 connective tissue
growth factor CTGF CTGF-679-16-14003 14003 NM_001901.2 connective
tissue growth factor CTGF CTGF-992-16-14004 14004 NM_001901.2
connective tissue growth factor CTGF
CTGF-1045-16-14005 14005 NM_001901.2 connective tissue growth
factor CTGF CTGF-1231-16-14006 14006 NM_001901.2 connective tissue
growth factor CTGF CTGF-991-16-14007 14007 NM_001901.2 connective
tissue growth factor CTGF CTGF-998-16-14008 14008 NM_001901.2
connective tissue growth factor CTGF CTGF-1049-16-14009 14009
NM_001901.2 connective tissue growth factor CTGF CTGF-1044-16-14010
14010 NM_001901.2 connective tissue growth factor CTGF
CTGF-1327-16-14011 14011 NM_001901.2 connective tissue growth
factor CTGF CTGF-1196-16-14012 14012 NM_001901.2 connective tissue
growth factor CTGF CTGF-562-16-14013 14013 NM_001901.2 connective
tissue growth factor CTGF CTGF-752-16-14014 14014 NM_001901.2
connective tissue growth factor CTGF CTGF-994-16-14015 14015
NM_001901.2 connective tissue growth factor CTGF CTGF-1040-16-14016
14016 NM_001901.2 connective tissue growth factor CTGF
CTGF-1984-16-14017 14017 NM_001901.2 connective tissue growth
factor CTGF CTGF-2195-16-14018 14018 NM_001901.2 connective tissue
growth factor CTGF CTGF-2043-16-14019 14019 NM_001901.2 connective
tissue growth factor CTGF CTGF-1892-16-14020 14020 NM_001901.2
connective tissue growth factor CTGF CTGF-1567-16-14021 14021
NM_001901.2 connective tissue growth factor CTGF CTGF-1780-16-14022
14022 NM_001901.2 connective tissue growth factor CTGF
CTGF-2162-16-14023 14023 NM_001901.2 connective tissue growth
factor CTGF CTGF-1034-16-14024 14024 NM_001901.2 connective tissue
growth factor CTGF CTGF-2264-16-14025 14025 NM_001901.2 connective
tissue growth factor CTGF CTGF-1032-16-14026 14026 NM_001901.2
connective tissue growth factor CTGF CTGF-1535-16-14027 14027
NM_001901.2 connective tissue growth factor CTGF CTGF-1694-16-14028
14028 NM_001901.2 connective tissue growth factor CTGF
CTGF-1588-16-14029 14029 NM_001901.2 connective tissue growth
factor CTGF CTGF-928-16-14030 14030 NM_001901.2 connective tissue
growth factor CTGF CTGF-1133-16-14031 14031 NM_001901.2 connective
tissue growth factor CTGF CTGF-912-16-14032 14032 NM_001901.2
connective tissue growth factor CTGF CTGF-753-16-14033 14033
NM_001901.2 connective tissue growth factor CTGF CTGF-918-16-14034
14034 NM_001901.2 connective tissue growth factor CTGF
CTGF-744-16-14035 14035 NM_001901.2 connective tissue growth factor
CTGF CTGF-466-16-14036 14036 NM_001901.2 connective tissue growth
factor CTGF CTGF-917-16-14037 14037 NM_001901.2 connective tissue
growth factor CTGF CTGF-1038-16-14038 14038 NM_001901.2 connective
tissue growth factor CTGF CTGF-1048-16-14039 14039 NM_001901.2
connective tissue growth factor CTGF CTGF-1235-16-14040 14040
NM_001901.2 connective tissue growth factor CTGF CTGF-868-16-14041
14041 NM_001901.2 connective tissue growth factor CTGF
CTGF-1131-16-14042 14042 NM_001901.2 connective tissue growth
factor CTGF CTGF-1043-16-14043 14043 NM_001901.2 connective tissue
growth factor CTGF CTGF-751-16-14044 14044 NM_001901.2 connective
tissue growth factor CTGF CTGF-1227-16-14045 14045 NM_001901.2
connective tissue growth factor CTGF CTGF-867-16-14046 14046
NM_001901.2 connective tissue growth factor CTGF CTGF-1128-16-14047
14047 NM_001901.2 connective tissue growth factor CTGF
CTGF-756-16-14048 14048 NM_001901.2 connective tissue growth factor
CTGF CTGF-1234-16-14049 14049 NM_001901.2 connective tissue growth
factor CTGF CTGF-916-16-14050 14050 NM_001901.2 connective tissue
growth factor CTGF CTGF-925-16-14051 14051 NM_001901.2 connective
tissue growth factor CTGF CTGF-1225-16-14052 14052 NM_001901.2
connective tissue growth factor CTGF CTGF-445-16-14053 14053
NM_001901.2 connective tissue growth factor CTGF CTGF-446-16-14054
14054 NM_001901.2 connective tissue growth factor CTGF
CTGF-913-16-14055 14055 NM_001901.2 connective tissue growth factor
CTGF CTGF-997-16-14056 14056 NM_001901.2 connective tissue growth
factor CTGF CTGF-277-16-14057 14057 NM_001901.2 connective tissue
growth factor CTGF CTGF-1052-16-14058 14058 NM_001901.2 connective
tissue growth factor CTGF CTGF-887-16-14059 14059 NM_001901.2
connective tissue growth factor CTGF CTGF-914-16-14060 14060
NM_001901.2 connective tissue growth factor CTGF CTGF-1039-16-14061
14061 NM_001901.2 connective tissue growth factor CTGF
CTGF-754-16-14062 14062 NM_001901.2 connective tissue growth factor
CTGF CTGF-1130-16-14063 14063 NM_001901.2 connective tissue growth
factor CTGF CTGF-919-16-14064 14064 NM_001901.2 connective tissue
growth factor CTGF CTGF-922-16-14065 14065 NM_001901.2 connective
tissue growth factor CTGF CTGF-746-16-14066 14066 NM_001901.2
connective tissue growth factor CTGF CTGF-993-16-14067 14067
NM_001901.2 connective tissue growth factor CTGF CTGF-825-16-14068
14068 NM_001901.2 connective tissue growth factor CTGF
CTGF-926-16-14069 14069 NM_001901.2 connective tissue growth factor
CTGF CTGF-923-16-14070 14070 NM_001901.2 connective tissue growth
factor CTGF CTGF-866-16-14071 14071 NM_001901.2 connective tissue
growth factor CTGF CTGF-563-16-14072 14072 NM_001901.2 connective
tissue growth factor CTGF CTGF-823-16-14073 14073 NM_001901.2
connective tissue growth factor CTGF CTGF-1233-16-14074 14074
NM_001901.2 connective tissue growth factor CTGF CTGF-924-16-14075
14075 NM_001901.2 connective tissue growth factor CTGF
CTGF-921-16-14076 14076 NM_001901.2 connective tissue growth factor
CTGF CTGF-443-16-14077 14077 NM_001901.2 connective tissue growth
factor CTGF CTGF-1041-16-14078 14078 NM_001901.2 connective tissue
growth factor CTGF CTGF-1042-16-14079 14079 NM_001901.2 connective
tissue growth factor CTGF CTGF-755-16-14080 14080 NM_001901.2
connective tissue growth factor CTGF CTGF-467-16-14081 14081
NM_001901.2 connective tissue growth factor CTGF CTGF-995-16-14082
14082 NM_001901.2 connective tissue growth factor CTGF
CTGF-927-16-14083 14083 NM_001901.2 connective tissue growth factor
CTGF SPP1-1091-16-14131 14131 NM_000582.2 Osteopontin SPP1
PPIB--16-14188 14188 NM_000942 peptidylprolyl isomerase B PPIB
(cyclophilin B) PPIB--17-14189 14189 NM_000942 peptidylprolyl
isomerase B PPIB (cyclophilin B) PPIB--18-14190 14190 NM_000942
peptidylprolyl isomerase B PPIB (cyclophilin B) pGL3-1172-16-14386
14386 U47296 Cloning vector pGL3-Control pGL3 pGL3-1172-16-14387
14387 U47296 Cloning vector pGL3-Control pGL3 MAP4K4-2931-25-14390
14390 NM_004834 Mitogen-Activated Protein Kinase MAP4K4 Kinase
Kinase Kinase 4 (MAP4K4), transcript variant 1 miR-122--23-14391
14391 miR-122 14084 NM_000582.2 Osteopontin SPP1 14085 NM_000582.2
Osteopontin SPP1 14086 NM_000582.2 Osteopontin SPP1 14087
NM_000582.2 Osteopontin SPP1 14088 NM_000582.2 Osteopontin SPP1
14089 NM_000582.2 Osteopontin SPP1 14090 NM_000582.2 Osteopontin
SPP1 14091 NM_000582.2 Osteopontin SPP1 14092 NM_000582.2
Osteopontin SPP1 14093 NM_000582.2 Osteopontin SPP1 14094
NM_000582.2 Osteopontin SPP1 14095 NM_000582.2 Osteopontin SPP1
14096 NM_000582.2 Osteopontin SPP1 14097 NM_000582.2 Osteopontin
SPP1 14098 NM_000582.2 Osteopontin SPP1 14099 NM_000582.2
Osteopontin SPP1 14100 NM_000582.2 Osteopontin SPP1 14101
NM_000582.2 Osteopontin SPP1 14102 NM_000582.2 Osteopontin SPP1
14103 NM_000582.2 Osteopontin SPP1 14104 NM_000582.2 Osteopontin
SPP1 14105 NM_000582.2 Osteopontin SPP1 14106 NM_000582.2
Osteopontin SPP1 14107 NM_000582.2 Osteopontin SPP1 14108
NM_000582.2 Osteopontin SPP1 14109 NM_000582.2 Osteopontin SPP1
14110 NM_000582.2 Osteopontin SPP1 14111 NM_000582.2 Osteopontin
SPP1 14112 NM_000582.2 Osteopontin SPP1 14113 NM_000582.2
Osteopontin SPP1 14114 NM_000582.2 Osteopontin SPP1 14115
NM_000582.2 Osteopontin SPP1 14116 NM_000582.2 Osteopontin SPP1
14117 NM_000582.2 Osteopontin SPP1 14118 NM_000582.2 Osteopontin
SPP1 14119 NM_000582.2 Osteopontin SPP1 14120 NM_000582.2
Osteopontin SPP1 14121 NM_000582.2 Osteopontin SPP1 14122
NM_000582.2 Osteopontin SPP1 14123 NM_000582.2 Osteopontin SPP1
14124 NM_000582.2 Osteopontin SPP1 14125 NM_000582.2 Osteopontin
SPP1 14126 NM_000582.2 Osteopontin SPP1 14127 NM_000582.2
Osteopontin SPP1 14128 NM_000582.2 Osteopontin SPP1 14129
NM_000582.2 Osteopontin SPP1 14130 NM_000582.2 Osteopontin SPP1
14132 NM_000582.2 Osteopontin SPP1 14133 NM_000582.2 Osteopontin
SPP1 14134 NM_000582.2 Osteopontin SPP1 14135 NM_000582.2
Osteopontin SPP1 14136 NM_000582.2 Osteopontin SPP1 14137
NM_000582.2 Osteopontin SPP1 14138 NM_000582.2 Osteopontin SPP1
14139 NM_000582.2 Osteopontin SPP1 14140 NM_000582.2 Osteopontin
SPP1 14141 NM_000582.2 Osteopontin SPP1 14142 NM_000582.2
Osteopontin SPP1 14143 NM_000582.2 Osteopontin SPP1 14144
NM_000582.2 Osteopontin SPP1 14145 NM_000582.2 Osteopontin SPP1
14146 NM_000582.2 Osteopontin SPP1 14147 NM_000582.2 Osteopontin
SPP1 14148 NM_000582.2 Osteopontin SPP1 14149 NM_000582.2
Osteopontin SPP1 14150 NM_000582.2 Osteopontin SPP1 14151
NM_000582.2 Osteopontin SPP1 14152 NM_000582.2 Osteopontin SPP1
14153 NM_000582.2 Osteopontin SPP1 14154 NM_000582.2 Osteopontin
SPP1 14155 NM_000582.2 Osteopontin SPP1 14156 NM_000582.2
Osteopontin SPP1 14157 NM_000582.2 Osteopontin SPP1 14158
NM_000582.2 Osteopontin SPP1 14159 NM_000582.2 Osteopontin SPP1
14160 NM_000582.2 Osteopontin SPP1 14161 NM_000582.2 Osteopontin
SPP1 14162 NM_000582.2 Osteopontin SPP1 14163 NM_000582.2
Osteopontin SPP1 14164 NM_000582.2 Osteopontin SPP1 14165
NM_000582.2 Osteopontin SPP1 14166 NM_000582.2 Osteopontin SPP1
14167 NM_000582.2 Osteopontin SPP1 14168 NM_000582.2 Osteopontin
SPP1 14169 NM_000582.2 Osteopontin SPP1 14170 NM_000582.2
Osteopontin SPP1 14171 NM_000582.2 Osteopontin SPP1 14172
NM_000582.2 Osteopontin SPP1 14173 NM_000582.2 Osteopontin SPP1
14174 NM_000582.2 Osteopontin SPP1 14175 NM_000582.2 Osteopontin
SPP1 14176 NM_000582.2 Osteopontin SPP1 14177 NM_000582.2
Osteopontin SPP1 14178 NM_000582.2 Osteopontin SPP1 14179
NM_000582.2 Osteopontin SPP1 14180 NM_000582.2 Osteopontin SPP1
14181 NM_000582.2 Osteopontin SPP1 14182 NM_000582.2 Osteopontin
SPP1 14183 NM_000582.2 Osteopontin SPP1 14184 NM_000582.2
Osteopontin SPP1 14185 NM_000582.2 Osteopontin SPP1 14186
NM_000582.2 Osteopontin SPP1 14187 NM_000582.2 Osteopontin SPP1
TABLE-US-00002 TABLE 2 Antisense backbone, chemistry, and sequence
information. ID Oligo AntiSense AntiSense AntiSense SEQ ID Number
Number Backbone Chemistry Sequence NO: APOB-10167-20-12138 12138
ooooooooooooo 00000000000000 AUUGGUAUUCAGUGUGA 1 oooooo 000000m UG
APOB-10167-20-12139 12139 ooooooooooooo 00000000000000
AUUCGUAUUGAGUCUGA 2 oooooo 000000m UC MAP4K4-2931-13-12266 12266
MAP4K4-2931-16-12293 12293 ooooooooooooo Pf000fffff0f00
UAGACUUCCACAGAACU 3 oooooo 00fff0 CU MAP4K4-2931-16-12383 12383
ooooooooooooo 00000000000000 UAGACUUCCACAGAACU 4 oooooo 00000 CU
MAP4K4-2931-16-12384 12384 ooooooooooooo P0000000000000
UAGACUUCCACAGAACU 5 oooooo 000000 CU MAP4K4-2931-16-12385 12385
ooooooooooooo Pf000fffff0f00 UAGACUUCCACAGAACU 6 oooooo 00fff0 CU
MAP4K4-2931-16-12386 12386 oooooooooosss Pf000fffff0f00
UAGACUUCCACAGAACU 7 ssssso 00fff0 CU MAP4K4-2931-16-12387 12387
oooooooooosss P0000000000000 UAGACUUCCACAGAACU 8 ssssso 000000 CU
MAP4K4-2931-15-12388 12388 ooooooooooooo 00000000000000
UAGACUUCCACAGAACU 9 oooo 000 MAP4K4-2931-13-12432 12432
MAP4K4-2931-13-12266.2 12266.2 APOB--21-12434 12434 ooooooooooooo
00000000000000 AUUGGUAUUCAGUGUGA 10 oooooooo 000000m UGAC
APOB--21-12435 12435 ooooooooooooo 00000000000000 AUUCGUAUUGAGUCUGA
11 oooooooo 000000m UCAC MAP4K4-2931-16-12451 12451 oooooooooosss
Pf000fffff0f00 UAGACUUCCACAGAACU 12 ssssso 00ffmm CU
MAP4K4-2931-16-12452 12452 oooooooooosss Pm000fffff0f00
UAGACUUCCACAGAACU 13 ssssso 00ffmm CU MAP4K4-2931-16-12453 12453
oooooosssssss Pm000fffff0f00 UAGACUUCCACAGAACU 14 ssssso 00ffmm CU
MAP4K4-2931-17-12454 12454 oooooooooooos Pm000fffff0f00
UAGACUUCCACAGAACU 15 ssssssso 00ffffmm CUUC MAP4K4-2931-17-12455
12455 oooooooosssss Pm000fffff0f00 UAGACUUCCACAGAACU 16 ssssssso
00ffffmm CUUC MAP4K4-2931-19-12456 12456 oooooooooooos
Pm000fffff0f00 UAGACUUCCACAGAACU 17 ssssssssssso 00ffffff00mm
CUUCAAAG --27-12480 12480 --27-12481 12481 APOB-10167-21-12505
12505 ooooooooooooo 00000000000000 AUUGGUAUUCAGUGUGA 18 ooooooos
000000m UGAC APOB-10167-21-12506 12506 ooooooooooooo 00000000000000
AUUCGUAUUGAGUCUGA 19 ooooooos 000000m UCAC MAP4K4-2931-16-12539
12539 oooooooooooss Pf000fffff0f00 UAGACUUCCACAGAACU 20 ssssss
00fff0 CU APOB-10167-21-12505.2 12505.2 ooooooooooooo
00000000000000 AUUGGUAUUCAGUGUGA 21 oooooooo 000000m UGAC
APOB-10167-21-12506.2 12506.2 ooooooooooooo 00000000000000
AUUCGUAUUGAGUCUGA 22 oooooooo 000000m UCAC MAP4K4--13-12565 12565
MAP4K4-2931-16-12386.2 12386.2 oooooooooosss Pf000fffff0f00
UAGACUUCCACAGAACU 23 ssssso 00fff0 CU MAP4K4-2931-13-12815 12815
APOB--13-12957 12957 MAP4K4--16-12983 12983 oooooooooooos
Pm000fffff0m00 uagacuuccacagaacu 24 ssssso 00mmm0 cu
MAP4K4--16-12984 12984 oooooooooooos Pm000fffff0m00
uagacuuccacagaacu 25 sssss 00mmm0 cu MAP4K4--16-12985 12985
oooooooooooos Pm000fffff0m00 uagacuuccacagaacu 26 ssssso 00mmm0 cu
MAP4K4--16-12986 12986 oooooooooosss Pf000fffff0f00
UAGACUUCCACAGAACU 27 ssssso 00fff0 CU MAP4K4--16-12987 12987
ooooooooooooo P0000f00ff0m00 UagacUUccacagaacU 28 ssssss 00m0m0 cU
MAP4K4--16-12988 12988 ooooooooooooo P0000f00ff0m00
UagacUUccacagaacU 29 ssssss 00m0m0 cu MAP4K4--16-12989 12989
ooooooooooooo P0000ff0ff0m00 UagacuUccacagaacU 30 ssssss 00m0m0 cu
MAP4K4--16-12990 12990 ooooooooooooo Pf0000ff000000
uagaCuuCCaCagaaCu 31 ssssss 000m00 Cu MAP4K4--16-12991 12991
ooooooooooooo Pf0000fff00m00 uagaCuucCacagaaCu 32 ssssss 000mm0 cu
MAP4K4--16-12992 12992 ooooooooooooo Pf000fffff0000
uagacuuccaCagaaCu 33 ssssss 000m00 Cu MAP4K4--16-12993 12993
ooooooooooooo P0000000000000 UagaCUUCCaCagaaCU 34 ssssss 000000 CU
MAP4K4--16-12994 12994 ooooooooooooo P0000f0f0f0000
UagacUuCcaCagaaCu 35 ssssss 000m00 Cu MAP4K4--16-12995 12995
oooooooooooos Pf000fffff0000 uagacuuccaCagaaCu 36 ssssso 000000 CU
MAP4K4-2931-19-13012 13012 MAP4K4-2931-19-13016 13016
PPIB--13-13021 13021 pGL3-1172-13-13038 13038 pGL3-1172-13-13040
13040 --16-13047 13047 oooooooooooos Pm000000000m00
UAGACUUCCACAGAACU 37 sssss 00mmm0 CU SOD1-530-13-13090 13090
SOD1-523-13-13091 13091 SOD1-535-13-13092 13092 SOD1-536-13-13093
13093 SOD1-396-13-13094 13094 SOD1-385-13-13095 13095
SOD1-195-13-13096 13096 APOB-4314-13-13115 13115 APOB-3384-13-13116
13116 APOB-3547-13-13117 13117 APOB-4318-13-13118 13118
APOB-3741-13-13119 13119 PPIB--16-13136 13136 oooooooooooos
Pm0fffff0f00mm UGUUUUUGUAGCCAAAU 38 sssss 000mm0 CC
APOB-4314-15-13154 13154 APOB-3547-15-13155 13155
APOB-4318-15-13157 13157 APOB-3741-15-13158 13158 APOB--13-13159
13159 APOB--15-13160 13160 SOD1-530-16-13163 13163 oooooooooooos
Pm0ffffffff0mm UACUUUCUUCAUUUCCA 39 ssssso mmm0m0 CC
SOD1-523-16-13164 13164 oooooooooooos Pmff0fffff0fmm
UUCAUUUCCACCUUUGC 40 ssssso mm0mm0 CC SOD1-535-16-13165 13165
oooooooooooos Pmfff0f0ffffmm CUUUGUACUUUCUUCAU 41 ssssso mm0mm0 UU
SOD1-536-16-13166 13166 oooooooooooos Pmffff0f0fffmm
UCUUUGUACUUUCUUCA 42 ssssso mmm0m0 UU SOD1-396-16-13167 13167
oooooooooooos Pmf00f00ff0f0m UCAGCAGUCACAUUGCC 43 ssssso m0mmm0 CA
SOD1-385-16-13168 13168 oooooooooooos Pmff0fff000fmm
AUUGCCCAAGUCUCCAA 44 ssssso mm00m0 CA SOD1-195-16-13169 13169
oooooooooooos Pmfff0fff0000m UUCUGCUCGAAAUUGAU 45 ssssso m00m00 GA
pGL3-1172-16-13170 13170 oooooooooooos Pm00ff0f0ffm0f
AAAUCGUAUUUGUCAAU 46 ssssso f00mm0 CA pGL3-l172-16-13171 13171
ooooooooooooo Pm00ff0f0ffm0f AAAUCGUAUUUGUCAAU 47 ssssss f00mm0 CA
MAP4k4-2931-19-13189 13189 ooooooooooooo 00000000000000
UAGACUUCCACAGAACU 48 oooooo 00000 CU CTGF-1222-13-13190 13190
CTGF-813-13-13192 13192 CTGF-747-13-13194 13194 CTGF-817-13-13196
13196
CTGF-1174-13-13198 13198 CTGF-1005-13-13200 13200 CTGF-814-13-13202
13202 CTGF-816-13-13204 13204 CTGF-1001-13-13206 13206
CTGF-1173-13-13208 13208 CTGF-749-13-13210 13210 CTGF-792-13-13212
13212 CTGF-1162-13-13214 13214 CTGF-811-13-13216 13216
CTGF-797-13-13218 13218 CTGF-1175-13-13220 13220 CTGF-1172-13-13222
13222 CTGF-1177-13-13224 13224 CTGF-1176-13-13226 13226
CTGF-812-13-13228 13228 CTGF-745-13-13230 13230 CTGF-1230-13-13232
13232 CTGF-920-13-13234 13234 CTGF-679-13-13236 13236
CTGF-992-13-13238 13238 CTGF-1045-13-13240 13240 CTGF-1231-13-13242
13242 CTGF-991-13-13244 13244 CTGF-998-13-13246 13246
CTGF-1049-13-13248 13248 CTGF-1044-13-13250 13250
CTGF-1327-13-13252 13252 CTGF-1196-13-13254 13254 CTGF-562-13-13256
13256 CTGF-752-13-13258 13258 CTGF-994-13-13260 13260
CTGF-1040-13-13262 13262 CTGF-1984-13-13264 13264
CTGF-2195-13-13266 13266 CTGF-2043-13-13268 13268
CTGF-1892-13-13270 13270 CTGF-1567-13-13272 13272
CTGF-1780-13-13274 13274 CTGF-2162-13-13276 13276
CTGF-1034-13-13278 13278 CTGF-2264-13-13280 13280
CTGF-1032-13-13282 13282 CTGF-1535-13-13284 13284
CTGF-1694-13-13286 13286 CTGF-1588-13-13288 13288 CTGF-928-13-13290
13290 CTGF-1133-13-13292 13292 CTGF-912-13-13294 13294
CTGF-753-13-13296 13296 CTGF-918-13-13298 13298 CTGF-744-13-13300
13300 CTGF-466-13-13302 13302 CTGF-917-13-13304 13304
CTGF-1038-13-13306 13306 CTGF-1048-13-13308 13308
CTGF-1235-13-13310 13310 CTGF-868-13-13312 13312 CTGF-1131-13-13314
13314 CTGF-1043-13-13316 13316 CTGF-751-13-13318 13318
CTGF-1227-13-13320 13320 CTGF-867-13-13322 13322 CTGF-1128-13-13324
13324 CTGF-756-13-13326 13326 CTGF-1234-13-13328 13328
CTGF-916-13-13330 13330 CTGF-925-13-13332 13332 CTGF-1225-13-13334
13334 CTGF-445-13-13336 13336 CTGF-446-13-13338 13338
CTGF-913-13-13340 13340 CTGF-997-13-13342 13342 CTGF-277-13-13344
13344 CTGF-1052-13-13346 13346 CTGF-887-13-13348 13348
CTGF-914-13-13350 13350 CTGF-1039-13-13352 13352 CTGF-754-13-13354
13354 CTGF-1130-13-13356 13356 CTGF-919-13-13358 13358
CTGF-922-13-13360 13360 CTGF-746-13-13362 13362 CTGF-993-13-13364
13364 CTGF-825-13-13366 13366 CTGF-926-13-13368 13368
CTGF-923-13-13370 13370 CTGF-866-13-13372 13372 CTGF-563-13-13374
13374 CTGF-823-13-13376 13376 CTGF-1233-13-13378 13378
CTGF-924-13-13380 13380 CTGF-921-13-13382 13382 CTGF-443-13-13384
13384 CTGF-1041 13-13386 13386 CTGF-1042-13-13388 13388
CTGF-755-13-13390 13390 CTGF-467-13-13392 13392 CTGF-995-13-13394
13394 CTGF-927-13-13396 13396 SPP1-1025-13-13398 13398
SPP1-1049-13-13400 13400 SPP1-1051-13-13402 13402
SPP1-1048-13-13404 13404 SPP1-1050-13-13406 13406
SPP1-1047-13-13408 13408 SPP1-800-13-13410 13410 SPP1-492-13-13412
13412 SPP1-612-13-13414 13414 SPP1-481-13-13416 13416
SPP1-614-13-13418 13418 SPP1-951-13-13420 13420 SPP1-482-13-13422
13422 SPP1-856-13-13424 13424 SPP1-857-13-13426 13426
SPP1-365-13-13428 13428 SPP1-359-13-13430 13430 SPP1-357-13-13432
13432 SPP1-858-13-13434 13434 SPP1-1012-13-13436 13436
SPP1-1014-13-13438 13438 SPP1-356-13-13440 13440 SPP1-368-13-13442
13442 SPP1-1011-13-13444 13444 SPP1-754-13-13446 13446
SPP1-1021-13-13448 13448 SPP1-1330-13-13450 13450 SPP1-346-13-13452
13452 SPP1-869-13-13454 13454 SPP1-701-13-13456 13456
SPP1-896-13-13458 13458 SPP1-1035-13-13460 13460 SPP1-1170-13-13462
13462 SPP1-1282-13-13464 13464 SPP1-1537-13-13466 13466
SPP1-692-13-13468 13468 SPP1-840-13-13470 13470 SPP1-1163-13-13472
13472 SPP1-789-13-13474 13474 SPP1-841-13-13476 13476
SPP1-852-13-13478 13478 SPP1-209-13-13480 13480 SPP1-1276-13-13482
13482 SPP1-137-13-13484 13484 SPP1-711-13-13486 13486
SPP1-582-13-13488 13488 SPP1-839-13-13490 13490 SPP1-1091-13-13492
13492 SPP1-884-13-13494 13494 SPP1-903-13-13496 13496
SPP1-1090-13-13498 13498 SPP1-474-13-13500 13500 SPP1-575-13-13502
13502 SPP1-671-13-13504 13504 SPP1-924-13-13506 13506
SPP1-1185-13-13508 13508 SPP1-1221-13-13510 13510 SPP1-347-13-13512
13512 SPP1-634-13-13514 13514 SPP1-877-13-13516 13516
SPP1-1033-13-13518 13518 SPP1-714-13-13520 13520 SPP1-791-13-13522
13522 SPP1-813-13-13524 13524 SPP1-939-13-13526 13526
SPP1-1161-13-13528 13528 SPP1-1164-13-13530 13530
SPP1-1190-13-13532 13532 SPP1-1333-13-13534 13534 SPP1-537-13-13536
13536 SPP1-684-13-13538 13538 SPP1-707-13-13540 13540
SPP1-799-13-13542 13542 SPP1-853-13-13544 13544 SPP1-888-13-13546
13546 SPP1-1194-13-13548 13548 SPP1-1279-13-13550 13550
SPP1-1300-13-13552 13552 SPP1-1510-13-13554 13554
SPP1-1543-13-13556 13556 SPP1-434-13-13558 13558 SPP1-600-13-13560
13560 SPP1-863-13-13562 13562 SPP1-902-13-13564 13564
SPP1-921-13-13566 13566 SPP1-154-13-13568 13568 SPP1-217-13-13570
13570 SPP1-816-13-13572 13572 SPP1-882-13-13574 13574
SPP1-932-13-13576 13576 SPP1-1509-13-13578 13578 SPP1-157-13-13580
13580 SPP1-350-13-13582 13582 SPP1-511-13-13584 13584
SPP1-605-13-13586 13586 SPP1-811-13-13588 13588 SPP1-892-13-13590
13590 SPP1-922-13-13592 13592 SPP1-1169-13-13594 13594
SPP1-1182-13-13596 13596 SPP1-1539-13-13598 13598
SPP1-1541-13-13600 13600 SPP1-427-13-13602 13602 SPP1-533-13-13604
13604 APOB--13-13763 13763 APOB--13-13764 13764 MAP4K4--16-13766
13766 oooooooooooos Pm000fffff0m00 UAGACUUCCACAGAACU 49 ssssso
00mmm0 CU PPIB--13-13767 13767 PPIB--15-13768 13768 PPIB--17-13769
13769 MAP4K4--16-13939 13939 oooooooooooos m000f0ffff0m0m
UAGACAUCCUACACAGC 50 ssssso 00m0m AC APOB-4314-16-13940 13940
oooooooooooos Pm0fffffff000m UGUUUCUCCAGAUCCUU 51 ssssso mmmm00 GC
APOB-4314-17-13941 13941 oooooooooooos Pm0fffffff000m
UGUUUCUCCAGAUCCUU 52 ssssso mmmm00 GC APOB--16-13942 13942
oooooooooooos Pm00f000f000mm UAGCAGAUGAGUCCAUU 53 ssssso m0mmm0 UG
APOB--18-13943 13943 ooooooooooooo Pm00f000f000mm UAGCAGAUGAGUCCAUU
54 ooosssssso m0mmm00000 UGGAGA APOB--17-13944 13944 oooooooooooos
Pm00f000f000mm UAGCAGAUGAGUCCAUU 55 ssssso m0mmm0 UG APOB--19-13945
13945 ooooooooooooo Pm00f000f000mm UAGCAGAUGAGUCCAUU 56 ooosssssso
m0mmm00000 UGGAGA APOB-4314-16-13946 13946 oooooooooooos
Pmf0ff0ffffmmm AUGUUGUUUCUCCAGAU 57 ssssso 000mm0 CC
APOB-4314-17-13947 13947 oooooooooooos Pmf0ff0ffffmmm
AUGUUGUUUCUCCAGAU 58 ssssso 000mm0 CC APOB--16-13948 13948
oooooooooooos Pm0fff000000mm UGUUUGAGGGACUCUGU 59 ssssso mm0m00 GA
APOB--17- 13949 oooooooooooos Pm0fff000000mm UGUUUGAGGGACUCUGU 60
13949 ssssso mm0m00 GA APOB--16- 13950 oooooooooooos Pmff00f0fff00m
AUUGGUAUUCAGUGUGA 61 13950 ssssso 0m00m0 UG APOB--18- 13951
ooooooooooooo Pmff00f0fff00m AUUGGUAUUCAGUGUGA 62 13951 ooosssssso
0m00m00m00 UGACAC APOB--17- 13952 oooooooooooos Pmff00f0fff00m
AUUGGUAUUCAGUGUGA 63 13952 ssssso 0m00m0 UG APOB--19- 13953
ooooooooooooo Pmff00f0fff00m AUUGGUAUUCAGUGUGA 64 13953 ooosssssso
0m00m00m00 UGACAC MAP4K4-- 13766.2 oooooooooooos Pm000fffff0m00
UAGACUUCCACAGAACU 65 16-13766.2 ssssso 00mmm0 CU CTGF-1222- 13980
oooooooooooos Pm0f0ffffffm0m UACAUCUUCCUGUAGUA 66 16-13980 ssssso
00m0m0 CA CTGF-813- 13981 oooooooooooos Pm0f0ffff0mmmm
AGGCGCUCCACUCUGUG 67 16-13981 ssssso 0m000 GU CTGF-747- 13982
oooooooooooos Pm0ffffff00mm0 UGUCUUCCAGUCGGUAA 68 16-13982 ssssso
m0000 GC CTGF-817- 13983 oooooooooooos Pm00f000f0fmmm
GAACAGGCGCUCCACUC 69 16-13983 ssssso 0mmmm0 UG CTGF-1174- 13984
oooooooooooos Pm00ff0f00f00m CAGUUGUAAUGGCAGGC 70 16-13984 ssssso
000m00 AC CTGF-1005- 13985 oooooooooooos Pmff000000mmm0
AGCCAGAAAGCUCAAAC 71 16-13985 ssssso 00mm0 UU CTGF-814- 13986
oooooooooooos Pm000f0ffff0mm CAGGCGCUCCACUCUGU 72 16-13986 ssssso
mm0m00 GG CTGF-816- 13987 oooooooooooos Pm0f000f0ffmm0
AACAGGCGCUCCACUCU 73 16-13987 ssssso mmmm00 GU CTGF-1001- 13988
oooooooooooos Pm0000fff000mm AGAAAGCUCAAACUUGA 74 16-13988 ssssso
m00m0 UA CTGF-1173- 13989 oooooooooooos Pmff0f00f00m00
AGUUGUAAUGGCAGGCA 75
16-13989 ssssso 0m0m0 CA CTGF-749- 13990 oooooooooooos
Pmf0ffffff00mm CGUGUCUUCCAGUCGGU 76 16-13990 ssssso 00m00 AA
CTGF-792- 13991 oooooooooooos Pm00ff000f00mm GGACCAGGCAGUUGGCU 77
16-13991 ssssso 00mmm0 CU CTGF-1162- 13992 oooooooooooos
Pm000f0f000mmm CAGGCACAGGUCUUGAU 78 16-13992 ssssso m00m00 GA
CTGF-811- 13993 oooooooooooos Pmf0ffff0ffmm0 GCGCUCCACUCUGUGGU 79
16-13993 ssssso m00mm0 CU CTGF-797- 13994 oooooooooooos
Pm0fff000ff000 GGUCUGGACCAGGCAGU 80 16-13994 ssssso m00mm0 UG
CTGF-1175- 13995 oooooooooooos Pmf00ff0f00m00 ACAGUUGUAAUGGCAGG 81
16-13995 ssssso m000m0 CA CTGF-1172- 13996 oooooooooooos
Pmff0f00f00m00 GUUGUAAUGGCAGGCAC 82 16-13996 ssssso 0m0m00 AG
CTGF-1177- 13997 oooooooooooos Pm00f00ff0f00m GGACAGUUGUAAUGGCA 83
16-13997 ssssso 00m000 GG CTGF-1176- 13998 oooooooooooos
Pm0f00ff0f00m0 GACAGUUGUAAUGGCAG 84 16-13998 ssssso 0m0000 GC
CTGF-812- 13999 oooooooooooos Pm0f0ffff0fmmm GGCGCUCCACUCUGUGG 85
16-13999 ssssso 0m00m0 UC CTGF-745- 14000 oooooooooooos
Pmfffff00ff00m UCUUCCAGUCGGUAAGC 86 16-14000 ssssso 000mm0 CG
CTGF-1230- 14001 oooooooooooos Pm0fffff0f0m0m UGUCUCCGUACAUCUUC 87
16-14001 ssssso mmmmm0 CU CTGF-920- 14002 oooooooooooos
Pmffff0f0000mm AGCUUCGCAAGGCCUGA 88 16-14002 ssssso m00m0 CC
CTGF-679- 14003 oooooooooooos Pm0ffffff0f00m CACUCCUCGCAGCAUUU 89
16-14003 ssssso 0mmmm0 CC CTGF-992- 14004 oooooooooooos
Pm00fff00f000m AAACUUGAUAGGCUUGG 90 16-14004 ssssso mm0000 AG
CTGF-1045- 14005 oooooooooooos Pmffff0f0000mm ACUCCACAGAAUUUAGC 91
16-14005 ssssso m00mm0 UC CTGF-1231- 14006 oooooooooooos
Pmf0fffff0f0m0 AUGUCUCCGUACAUCUU 92 16-14006 ssssso mmmmm0 CC
CTGF-991- 14007 oooooooooooos Pm0fff00f000mm AACUUGAUAGGCUUGGA 93
16-14007 ssssso m00000 GA CTGF-998- 14008 oooooooooooos
Pm00fff000fmm0 AAGCUCAAACUUGAUAG 94 16-14008 ssssso 0m0000 GC
CTGF-1049- 14009 oooooooooooos Pmf0f0ffff0m00 ACAUACUCCACAGAAUU 95
16-14009 ssssso 00mmm0 UA CTGF-1044- 14010 oooooooooooos
Pmfff0f0000mmm CUCCACAGAAUUUAGCU 96 16-14010 ssssso 00mmm0 CG
CTGF-1327- 14011 oooooooooooos Pm0f0ff0ff0000 UGUGCUACUGAAAUCAU 97
16-14011 ssssso mm0mm0 UU CTGF-1196- 14012 oooooooooooos
Pm0000f0ff0mm0 AAAGAUGUCAUUGUCUC 98 16-14012 ssssso mmmmm0 CG
CTGF-562- 14013 oooooooooooos Pmf0f0ff00f0mm GUGCACUGGUACUUGCA 99
16-14013 ssssso m0m000 GC CTGF-752- 14014 oooooooooooos
Pm00f0f0fffmmm AAACGUGUCUUCCAGUC 100 16-14014 ssssso 00mm00 GG
CTGF-994- 14015 oooooooooooos Pmf000fff00m00 UCAAACUUGAUAGGCUU 101
16-14015 ssssso 0mmm00 GG CTGF-1040- 14016 oooooooooooos
Pmf0000fff00mm ACAGAAUUUAGCUCGGU 102 16-14016 ssssso m00m00 AU
CTGF-1984- 14017 oooooooooooos Pmf0f0ffff0mmm UUACAUUCUACCUAUGG 103
16-14017 ssssso 0m00m0 UG CTGF-2195- 14018 oooooooooooos
Pm00ff00ff00mm AAACUGAUCAGCUAUAU 104 16-14018 ssssso 0m0m00 AG
CTGF-2043- 14019 oooooooooooos Pm0fff000f0000 UAUCUGAGCAGAAUUUC 105
16-14019 ssssso mmmmm0 CA CTGF-1892- 14020 oooooooooooos
Pmf00fff000m00 UUAACUUAGAUAACUGU 106 16-14020 ssssso mm0m00 AC
CTGF-1567- 14021 oooooooooooos Pm0ff0fff0f0m0 UAUUACUCGUAUAAGAU 107
16-14021 ssssso 000m00 GC CTGF-1780- 14022 oooooooooooos
Pm00ff0fff00mm AAGCUGUCCAGUCUAAU 108 16-14022 ssssso m00mm0 CG
CTGF-2162- 14023 oooooooooooos Pm00f00000fm0m UAAUAAAGGCCAUUUGU 109
16-14023 ssssso mm0mm0 UC CTGF-1034- 14024 oooooooooooos
Pmff00fff00m0m UUUAGCUCGGUAUGUCU 110 16-14024 ssssso 0mmmm0 UC
CTGF-2264- 14025 oooooooooooos Pmf0fffff00m00 ACACUCUCAACAAAUAA 111
16-14025 ssssso 0m0000 AC CTGF-1032- 14026 oooooooooooos
Pm00fff00f0m0m UAGCUCGGUAUGUCUUC 112 16-14026 ssssso mmmm00 AU
CTGF-1535- 14027 oooooooooooos Pm00fffffff0mm UAACCUUUCUGCUGGUA 113
16-14027 ssssso 00m0m0 CC CTGF-1694- 14028 oooooooooooos
Pmf000000f00mm UUAAGGAACAACUUGAC 114 16-14028 ssssso m00mm0 UC
CTGF-1588- 14029 oooooooooooos Pmf0f0ffff000m UUACACUUCAAAUAGCA 115
16-14029 ssssso 00m000 GG CTGF-928- 14030 oooooooooooos
Pmff000ff00mmm UCCAGGUCAGCUUCGCA 116 16-14030 ssssso m0m000 AG
CTGF-1133- 14031 oooooooooooos Pmffffff0f00mm CUUCUUCAUGACCUCGC 117
16-14031 ssssso mm0mm0 CG CTGF-912- 14032 oooooooooooos
Pm000fff00fm0m AAGGCCUGACCAUGCAC 118 16-14032 ssssso 0m0m00 AG
CTGF-753- 14033 oooooooooooos Pm000f0f0ffmmm CAAACGUGUCUUCCAGU 119
16-14033 ssssso m00mm0 CG CTGF-918- 14034 oooooooooooos
Pmfff0f0000mmm CUUCGCAAGGCCUGACC 120 16-14034 ssssso 00mm00 AU
CTGF-744- 14035 oooooooooooos Pmffff00ff00m0 CUUCCAGUCGGUAAGCC 121
16-14035 ssssso 00mm00 GC CTGF-466- 14036 oooooooooooos
Pmf00ffff0f00m CCGAUCUUGCGGUUGGC 122 16-14036 ssssso m00mm0 CG
CTGF-917- 14037 oooooooooooos Pmff0f0000fmm0 UUCGCAAGGCCUGACCA 123
16-14037 ssssso 0mm0m0 UG CTGF-1038- 14038 oooooooooooos
Pm00fff00fmm0m AGAAUUUAGCUCGGUAU 124 16-14038 ssssso 0m00 GU
CTGF-1048- 14039 oooooooooooos Pm0f0ffff0f000 CAUACUCCACAGAAUUU 125
16-14039 ssssso 0mmm00 AG CTGF-1235- 14040 oooooooooooos
Pm0ff0f0fffmmm UGCCAUGUCUCCGUACA 126 16-14040 ssssso 0m0m0 UC
CTGF-868- 14041 oooooooooooos Pm000f0ff0fm0m GAGGCGUUGUCAUUGGU 127
16-14041 ssssso m00m00 AA CTGF-1131- 14042 oooooooooooos
Pmffff0f00fmmm UCUUCAUGACCUCGCCG 128 16-14042 ssssso 0mm0m0 UC
CTGF-1043- 14043 oooooooooooos Pmff0f0000fmm0 UCCACAGAAUUUAGCUC 129
16-14043 ssssso 0mmm00 GG CTGF-751- 14044 oooooooooooos
Pm0f0f0ffffmm0 AACGUGUCUUCCAGUCG 130 16-14044 ssssso 0mm000 GU
CTGF-1227- 14045 oooooooooooos Pmfff0f0f0fmmm CUCCGUACAUCUUCCUG 131
16-14045 ssssso mmm0m0 UA CTGF-867- 14046 oooooooooooos
Pm0f0ff0ff0mm0 AGGCGUUGUCAUUGGUA 132 16-14046 ssssso 0m000 AC
CTGF-1128- 14047 oooooooooooos PmfCf00ffff0mm UCAUGACCUCGCCGUCA 133
16-14047 ssssso 0mm000 GG CTGF-756- 14048 oooooooooooos
Pm0ff000f0f0mm GGCCAAACGUGUCUUCC 134 16-14048 ssssso mmmm00 AG
CTGF-1234- 14049 oooooooooooos Pmff0f0ffffmm0 GCCAUGUCUCCGUACAU 135
16-14049 ssssso m0mm0 CU CTGF-916- 14050 oooooooooooos
Pmf0f0000ffm00 UCGCAAGGCCUGACCAU 136 16-14050 ssssso mm0m00 GC
CTGF-925- 14051 oooooooooooos Pm0ff00fffmm00 AGGUCAGCUUCGCAAGG 137
16-14051 ssssso 00m0 CC CTGF-1225- 14052 oooooooooooos
Pmf0f0f0fffmmm CCGUACAUCUUCCUGUA 138 16-14052 ssssso m0m000 GU
CTGF-445- 14053 oooooooooooos Pm00ff0000fm0m GAGCCGAAGUCACAGAA 139
16-14053 ssssso 000000 GA CTGF-446- 14054 oooooooooooos
Pm000ff0000mm0 GGAGCCGAAGUCACAGA 140 16-14054 ssssso m00000 AG
CTGF-913- 14055 oooooooooooos Pm0000fff00mm0 CAAGGCCUGACCAUGCA 141
16-14055 ssssso m0m0m0 CA CTGF-997- 14056 oooooooooooos
Pmfff000ffm00m AGCUCAAACUUGAUAGG 142 16-14056 ssssso 000m0 CU
CTGF-277- 14057 oooooooooooos Pmf0f00ffff00m CUGCAGUUCUGGCCGAC 143
16-14057 ssssso m00m00 GG CTGF-1052- 14058 oooooooooooos
Pm0f0f0f0ffmm0 GGUACAUACUCCACAGA 144 16-14058 ssssso m00000 AU
CTGF-887- 14059 oooooooooooos Pmf0fffffff00m CUGCUUCUCUAGCCUGC 145
16-14059 ssssso mm0m00 AG CTGF-914- 14060 oooooooooooos
Pmf0000fff00mm GCAAGGCCUGACCAUGC 146 16-14060 ssssso 0m0m00 AC
CTGF-1039- 14061 oooooooooooos Pm0000fff00mmm CAGAAUUUAGCUCGGUA 147
16-14061 ssssso 00m0m0 UG CTGF-754- 14062 oooooooooooos
Pmf000f0f0fmmm CCAAACGUGUCUUCCAG 148 16-14062 ssssso mm00m0 UC
CTGF-1130- 14063 oooooooooooos Pmfff0f00ffmmm CUUCAUGACCUCGCCGU 149
16-14063 ssssso m0mm0 CA CTGF-919- 14064 oooooooooooos
Pmffff0f0000mm GCUUCGCAAGGCCUGAC 150 16-14064 ssssso m00mm0 CA
CTGF-922- 14065 oooooooooooos Pmf00ffff0f000 UCAGCUUCGCAAGGCCU 151
16-14065 ssssso 0mmm00 GA CTGF-746- 14066 oooooooooooos
Pmffffff00fm0m GUCUUCCAGUCGGUAAG 152 16-14066 ssssso 000m0 CC
CTGF-993- 14067 oooooooooooos Pm000fff00f000 CAAACUUGAUAGGCUUG 153
16-14067 ssssso mmm000 GA CTGF-825- 14068 oooooooooooos
Pm0ffff0000m00 AGGUCUUGGAACAGGCG 154 16-14068 ssssso 0m0m0 CU
CTGF-926- 14069 oooooooooooos Pm000ff00ffmmm CAGGUCAGCUUCGCAAG 155
16-14069 ssssso 00000 GC CTGF-923- 14070 oooooooooooos
Pmff00ffff0m00 GUCAGCUUCGCAAGGCC 156 16-14070 ssssso 00mmm0 UG
CTGF-866- 14071 oooooooooooos Pm0f0ff0ff0mm0 GGCGUUGUCAUUGGUAA 157
16-14071 ssssso 0m00m0 CC CTGF-563- 14072 oooooooooooos
Pmf0f0ff00m0mm CGUGCACUGGUACUUGC 158 16-14072 ssssso m0m00 AG
CTGF-823- 14073 oooooooooooos Pmffff0000f000 GUCUUGGAACAGGCGCU 159
16-14073 ssssso m0mmm0 CC CTGF-1233- 14074 oooooooooooos
Pmf0f0fffff0m0 CCAUGUCUCCGUACAUC 160 16-14074 ssssso m0mmm0 UU
CTGF-924- 14075 oooooooooooos Pm0ff00ffff0m0 GGUCAGCUUCGCAAGGC 161
16-14075 ssssso 000mm0 CU CTGF-921- 14076 oooooooooooos
Pm00ffff0f0000 CAGCUUCGCAAGGCCUG 162 16-14076 ssssso mmm000 AC
CTGF-443- 14077 oooooooooooos Pmff0000ff0m00 GCCGAAGUCACAGAAGA 163
16-14077 ssssso 000000 GG CTGF-1041- 14078 oooooooooooos
Pm0f0000fff00m CACAGAAUUUAGCUCGG 164 16-14078 ssssso mm00m0 UA
CTGF-1042- 14079 oooooooooooos Pmf0f0000ffm00 CCACAGAAUUUAGCUCG 165
16-14079 ssssso mmm000 GU CTGF-755- 14080 oooooooooooos
Pmff000f0f0mmm GCCAAACGUGUCUUCCA 166 16-14080 ssssso mmm000 GU
CTGF-467- 14081 oooooooooooos Pmf0f00ffff0m0 GCCGAUCUUGCGGUUGG 167
16-14081 ssssso mm00m0 CC CTGF-995- 14082 oooooooooooos
Pmff000fff00m0 CUCAAACUUGAUAGGCU 168 16-14082 ssssso 00mmm0 UG
CTGF-927- 14083 oooooooooooos Pmf000ff00fmmm CCAGGUCAGCUUCGCAA 169
16-14083 ssssso 0m0000 GG SPP1-1091- 14131 oooooooooooos
Pmff00ff000m0m UUUGACUAAAUGCAAAG 170 16-14131 ssssso 0000m0 UG
PPIB--16-14188 14188 ooooooooooooo Pm0fffff0f00mm UGUUUUUGUAGCCAAAU
171 ssssss 000mm0 CC PPIB--17-14189 14189 ooooooooooooo
Pm0fffff0f00mm UGUUUUUGUAGCCAAAU 172 ssssss 000mm0 CC
PPIB--18-14190 14190 ooooooooooooo Pm0fffff0f00mm UGUUUUUGUAGCCAAAU
173 ssssss 000mm0 CC pGL3-l172- 14386 oooooooooooos Pm00ff0f0ffm0m
AAAUCGUAUUUGUCAAU 174 16-14386 ssssso m00mm0 CA pGL3-l172- 14387
oooooooooooos Pm00ff0f0ffm0m AAAUCGUAUUUGUCAAU 175 16-14387 ssssso
m00mm0 CA MAP4K4-2931- 14390 25-14390 miR-122--23-14391 14391 14084
oooooooooooos Pmff00fff0f000 UCUAAUUCAUGAGAAAU 616 ssssso 000m00 AC
14085 oooooooooooos Pm00ff00fffm00 UAAUUGACCUCAGAAGA 617 ssssso
0000m0 UG 14086 oooooooooooos Pmff00ff00fmmm UUUAAUUGACCUCAGAA 618
ssssso 000000 GA 14087 oooooooooooos Pm0ff00ffff000
AAUUGACCUCAGAAGAU 619 ssssso 000m00 GC 14088 oooooooooooos
Pmf00ff00ffmm0 UUAAUUGACCUCAGAAG 620 ssssso 000000 AU 14089
oooooooooooos Pmff00ffff0000 AUUGACCUCAGAAGAUG 621 ssssso 00m0m0 CA
14090 oooooooooooos Pmf0fff00ff00m UCAUCCAGCUGACUCGU 622 ssssso
mm0mm0 UU 14091 oooooooooooos Pm0fff0ff0000m AGAUUCAUCAGAAUGGU 623
ssssso 00m00 GA 14092 oooooooooooos Pm0Cffff00fmm0
UGACCUCAGUCCAUAAA 624 ssssso m000m0 CC 14093 oooooooooooos
Pm0f00f0000mmm AAUGGUGAGACUCAUCA 625 ssssso 0mm000 GA 14094
oooooooooooos Pmff00ffff00mm UUUGACCUCAGUCCAUA 626 ssssso m0m000 AA
14095 oooooooooooos Pmff0f00ff0m00 UUCAUGGCUGUGAAAUU 627 ssssso
00mmm0 CA 14096 oooooooooooos Pm00f00f0000mm GAAUGGUGAGACUCAUC 628
ssssso m0mm00 AG 14097 oooooooooooos Pm0Cffffff0mmm
UGGCUUUCCGCUUAUAU 629 ssssso 0m0m00 AA 14098 oooooooooooos
Pmf00ffffff0mm UUGGCUUUCCGCUUAUA 630 ssssso m0m0m0 UA 14099
oooooooooooos Pmf0fff0f0f00m UCAUCCAUGUGGUCAUG 631 ssssso m0m000 GC
14100 oooooooooooos Pmf0f00ff0f00m AUGUGGUCAUGGCUUUC 632 ssssso
mmmm00 GU 14101 oooooooooooos Pmf00ff0f00mmm GUGGUCAUGGCUUUCGU 633
ssssso mm0mm0 UG 14102 oooooooooooos Pmff00fffffmmm
AUUGGCUUUCCGCUUAU 634 ssssso m0m00 AU 14103 oooooooooooos
Pm00f0f0000mmm AAAUACGAAAUUUCAGG 635 ssssso m000m0 UG 14104
oooooooooooos Pm000f0f0000mm AGAAAUACGAAAUUUCA 636 ssssso mm000 GG
14105 oooooooooooos Pm00ff0f00fmmm UGGUCAUGGCUUUCGUU 637 ssssso
m0mm00 GG 14106 oooooooooooos PmfCff0fff0m0m AUAUCAUCCAUGUGGUC 638
ssssso 00mm00 AU 14107 oooooooooooos Pm0f0f0000fmmm
AAUACGAAAUUUCAGGU 639 ssssso 000m00 GU 14108 oooooooooooos
Pm0ff000000mm0 AAUCAGAAGGCGCGUUC 640 ssssso mmm00 AG 14109
oooooooooooos Pmfff0f000000m AUUCAUGAGAAAUACGA 641 ssssso 0m0000 AA
14110 oooooooooooos Pmf0fff0f00000 CUAUUCAUGAGAGAAUA 642 ssssso
00m000 AC 14111 oooooooooooos Pmfff0ff000mmm UUUCGUUGGACUUACUU 643
ssssso 0mmm00 GG 14112 oooooooooooos Pmf0fffff0fm0m
UUGCUCUCAUCAUUGGC 644 ssssso m00mm0 UU 14113 oooooooooooos
Pmff00fffffmmm UUCAACUCCUCGCUUUC 645 ssssso mmmm0 CA 14114
oooooooooooos Pm00ff0ff00mm0 UGACUAUCAAUCACAUC 646 ssssso m0mm00 GG
14115 oooooooooooos Pm0f0f0ff0mmm0 AGAUGCACUAUCUAAUU 647 ssssso
0mmm0 CA 14116 oooooooooooos Pm0f000f0f0m0m AAUAGAUACACAUUCAA 648
ssssso mm00m0 CC 14117 oooooooooooos Pmffffff0f0000
UUCUUCUAUAGAAUGAA 649 ssssso m000m0 CA 14118 oooooooooooos
Pm0ff0ff000m00 AAUUGCUGGACAACCGU 650 ssssso mm0m00 GG 14119
oooooooooooos Pmf0ffffff0m0m UCGCUUUCCAUGUGUGA 651 ssssso 0m0000 GG
14120 oooooooooooos Pm0Cfff000fm0m UAAUCUGGACUGCUUGU 652 ssssso
mm0m00 GG 14121 oooooooooooos Pmf0f0fff00mm0 ACACAUUCAACCAAUAA 653
ssssso 0m0000 AC 14122 oooooooooooos Pmfff0ffff0m00
ACUCGUUUCAUAACUGU 654 ssssso mm0mm0 CC 14123 oooooooooooos
Pmf00fff000mm0 AUAAUCUGGACUGCUUG 655 ssssso mmm0m0 UG 14124
oooooooooooos Pmffff0fff0m0m UUUCCGCUUAUAUAAUC 656 ssssso 00mmm0 UG
14125 oooooooooooos Pm0fff00ff00m0 UGUUUAACUGGUAUGGC 657 ssssso
m00m00 AC 14126 oooooooooooos Pm0f0000f000m0 UAUAGAAUGAACAUAGA 658
ssssso m000m0 CA 14127 oooooooooooos Pmffffff00fm0m
UUUCCUUGGUCGGCGUU 659 ssssso 0mmm0 UG 14128 oooooooooooos
Pmf0f0f0ff0mmm GUAUGCACCAUUCAACU 660 ssssso 00mmm0 CC 14129
oooooooooooos Pmf00ff0ff0m0m UCGGCCAUCAUAUGUGU 661 ssssso 0m0mm0 CU
14130 oooooooooooos Pm0fff000ff0mm AAUCUGGACUGCUUGUG 662 ssssso
m0m000 GC 14132 oooooooooooos Pmf0ff0000f0mm ACAUCGGAAUGCUCAUU 663
ssssso m0mm00 GC 14133 oooooooooooos Pm00fffff00mm0
AAGUUCCUGACUAUCAA 664 ssssso mm00m0 UC 14134 oooooooooooos
Pmf00ff000f0m0 UUGACUAAAUGCAAAGU 665 ssssso 000m00 GA 14135
oooooooooooos Pm0fff0ff000mm AGACUCAUCAGACUGGU 666 ssssso 00m00 GA
14136 oooooooooooos Pmf0f0f0f0fmm0 UCAUAUGUGUCUACUGU 667 ssssso
mm0mC0 GG 14137 oooooooooooos Pmf0fffff0fmm0 AUGUCCUCGUCUGUAGC 668
ssssso m00m00 AU 14138 oooooooooooos Pm00fff0f00mm0
GAAUUCACGGCUGACUU 669 ssssso 0mmmm0 UG 14139 oooooooooooos
Pmf0fffff000mm UUAUUUCCAGACUCAAA 670 ssssso m000m0 UA 14140
oooooooooooos Pm000ff0f000mm GAAGCCACAAACUAAAC 671 ssssso 000mm0 UA
14141 oooooooooooos Pmffff0ff000mm CUUUCGUUGGACUUACU 672 ssssso
m0mmm0 UG 14142 oooooooooooos Pmfff0f0000mmm GUCUGCGAAACUUCUUA 673
ssssso mmm000 GA 14143 oooooooooooos Pm0f0fff0ff0mm
AAUGCUCAUUGCUCUCA 674 ssssso mmm0m0 UC 14144 oooooooooooos
Pmf0f0ff0ffm00 AUGCACUAUCUAAUUCA 675 ssssso mmm0m0 UG 14145
oooooooooooos Pmff0f0f0f0mm0 CUUGUAUGCACCAUUCA 676 ssssso mmm000 AC
14146 oooooooooooos Pm00fff0fffm0m UGACUCGUUUCAUAACU 677 ssssso
00mm00 GU 14147 oooooooooooos Pmff00f0fffm00 UUCAGCACUCUGGUCAU 678
ssssso mm0mm0 CC 14148 oooooooooooos Pm00fff0f00mm0
AAAUUCAUGGCUGUGGA 679 ssssso m00000 AU 14149 oooooooooooos
Pmf0fff00ff00m ACAUUCAACCAAUAAAC 680 ssssso 000mm0 UG
14150 oooooooooooos Pm0f0f0fff00mm UACACAUUCAACCAAUA 681 ssssso
00m000 AA 14151 oooooooooooos Pmff00ff0ffmmm AUUAGUUAUUUCCAGAC 682
ssssso 000mm0 UC 14152 oooooooooooos Pmffff0fff0m00
UUUCUAUUCAUGAGAGA 683 ssssso 000000 AU 14153 oooooooooooos
Pmff00ff0ff00m UUCGGUUGCUGGCAGGU 684 ssssso 000mm0 CC 14154
oooooooooooos Pm0f0f0f0000m0 CAUGUGUGAGGUGAUGU 685 ssssso 0m0mm0 CC
14155 oooooooooooos Pmf0ff0fff00mm GCACCAUUCAACUCCUC 686 ssssso
mmmm00 GC 14156 oooooooooooos Pm0fff00ff00mm CAUCCAGCUGACUCGUU 687
ssssso m0mmm0 UC 14157 oooooooooooos Pmfffff0fff0m0
CUUUCCGCUUAUAUAAU 688 ssssso m00mm0 CU 14158 oooooooooooos
Pm0ff0f0ff0000 AAUCACAUCGGAAUGCU 689 ssssso m0mmm0 CA 14159
oooooooooooos Pmf0f0ff00fm0m ACACAUUAGUUAUUUCC 690 ssssso mmmm00 AG
14160 oooooooooooos Pmfff0f0000m00 UUCUAUAGAAUGAACAU 691 ssssso
0m0m00 AG 14161 oooooooooooos Pm0f00f00f00mm UACAGUGAUAGUUUGCA 692
ssssso m0m0m0 UU 14162 oooooooooooos Pmf000f00ff00m
AUAAGCAAUUGACACCA 693 ssssso 0mm0m0 CC 14163 oooooooooooos
Pmff0ff00ff0mm UUUAUUAAUUGCUGGAC 694 ssssso 000m00 AA 14164
oooooooooooos Pmf0ff0000fmmm UCAUCAGAGUCGUUCGA 695 ssssso m0000 GU
14165 oooooooooooos Pmf000ff0f0mm0 AUAAACCACACUAUCAC 696 ssssso
mm0mm0 CU 14166 oooooooooooos Pmf0ff0ff00mmm UCAUCAUUGGCUUUCCG 697
ssssso mmm0m0 CU 14167 oooooooooooos Pmfffff00fm0mm
AGUUCCUGACUAUCAAU 698 ssssso 00mm0 CA 14168 oooooooooooos
Pmff0f00ff00mm UUCACGGCUGACUUUGG 699 ssssso mm0000 AA 14169
oooooooooooos Pmffff0f00f00m UUCUCAUGGUAGUGAGU 700 ssssso 000mm0 UU
14170 oooooooooooos Pm0ff00fff0mmm AAUCAGCCUGUUUAACU 701 ssssso
00mm00 GG 14171 oooooooooooos Pm0ffff00f0mmm GGUUUCAGCACUCUGGU 702
ssssso m00mm0 CA 14172 oooooooooooos Pmff0000f0fmm0
AUCGGAAUGCUCAUUGC 703 ssssso mm0mm0 UC 14173 oooooooooooos
Pm00ff0f0000mm UGGCUGUGGAAUUCACG 704 ssssso m0m000 GC 14174
oooooooooooos Pm000f00ff00m0 UAAGCAAUUGACACCAC 705 ssssso mm0mm0 CA
14175 oooooooooooos Pm00fffff0f00m CAAUUCUCAUGGUAGUG 706 ssssso
00m000 AG 14176 oooooooooooos Pm00fffff0fm00 UGGCUUUCGUUGGACUU 707
ssssso 0mmm00 AC 14177 oooooooooooos Pm0ff00f00fm00
AAUCAGUGACCAGUUCA 708 ssssso mmm0m0 UC 14178 oooooooooooos
Pmfff0f000mm0m AGUCCAUAAACCACACU 709 ssssso 0mm0C AU 14179
oooooooooooos Pm00f0ffff00mm CAGCACUCUGGUCAUCC 710 ssssso 0mmm00 AG
14180 oooooooooooos Pm0ff00ff0f0mm UAUCAAUCACAUCGGAA 711 ssssso
0000m0 UG 14181 oooooooooooos Pmfff0f00ff00m AUUCACGGCUGACUUUG 712
ssssso mmm000 GA 14182 oooooooooooos Pmf000f0f0f0mm
AUAGAUACACAUUCAAC 713 ssssso m00mm0 CA 14183 oooooooooooos
Pmffff000ffm00 UUUCCAGACUCAAAUAG 714 ssssso 0m0000 AU 14184
oooooooooooos Pmf00ff0ff000m UUAAUUGCUGGACAACC 715 ssssso 00mm00 GU
14185 oooooooooooos Pm0ff00ff0fm00 UAUUAAUUGCUGGACAA 716 ssssso
0m00m0 CC 14186 oooooooooooos Pmff0fff000mm0 AGUCGUUCGAGUCAAUG 717
ssssso 0m000 GA 14187 oooooooooooos Pmff0ff00f000m
GUUGCUGGCAGGUCCGU 718 ssssso mm0m00 GG o: phosphodiester; s:
phosphorothioate; P: 5' phosphorylation; 0: 2'-OH; F: 2'-fluoro; m:
2' O-methyl; +: LNA modification. Capital letters in the sequence
signify riobonucleotides, lower case letters signify
deoxyribonucleotides.
TABLE-US-00003 TABLE 3 Sense backbone, chemistry, and sequence
information. OHang SEQ Oligo Sense Sense Sense Sense ID ID Number
Number Chem. Backbone Chemistry Sequence NO: APOB-10167- 12138 chl
oooooooooooo 0000000000000 GUCAUCACACUGA 176 20-12138 oooooooso
0000000 AUACCAAU APOB-10167- 12139 chl oooooooooooo 0000000000000
GUGAUCAGACUCA 177 20-12139 oooooooso 0000000 AUACGAAU MAP4K4- 12266
chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 178 2931-13- o 12266
MAP4K4- 12293 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 179
2931-16- o 12293 MAP4K4- 12383 chl oooooooooooo mm0m00000mmm0
CUGUGGAAGUCUA 180 2931-16- o 12383 MAP4K4- 12384 chl oooooooooooo
mm0m00000mmm0 CUGUGGAAGUCUA 181 2931-16- o 12384 MAP4K4- 12385 chl
oooooooooooo mm0m00000mmm0 CUGUGGAAGUCUA 182 2931-16- o 12385
MAP4K4- 12386 chl ooooooooooss 0mm0m00000mmm CUGUGGAAGUCUA 183
2931-16- o 0 12386 MAP4K4- 12387 chl oooooooooooo mm0m00000mmm0
CUGUGGAAGUCUA 184 2931-16- o 12387 MAP4K4- 12388 chl oooooooooooo
mm0m00000mmm0 CUGUGGAAGUCUA 185 2931-15- o 12388 MAP4K4- 12432 chl
oooooooooooo DY547mm0m0000 CUGUGGAAGUCUA 186 2931-13- o 0mmm0 12432
MAP4K4- 12266.2 chl ooooooooooos mm0m00000mmm0 CUGUGGAAGUCUA 187
2931-13- s 12266.2 APOB--21- 12434 chl oooooooooooo 0000000000000
GUCAUCACACUGA 188 12434 oooooooso 0000000 AUACCAAU APOB--21- 12435
chl oooooooooooo DY54700000000 GUGAUCAGACUCA 189 12435 oooooooso
000000000000 AUACGAAU MAP4K4- 12451 chl ooooooooooos 0mm0m00000mmm
CUGUGGAAGUCUA 190 2931-16- s 0 12451 MAP4K4- 12452 chl ooooooooooos
mm0m00000mmm0 CUGUGGAAGUCUA 191 2931-16- s 12452 MAP4K4- 12453 chl
ooooooooooos mm0m00000mmm0 CUGUGGAAGUCUA 192 2931-16- s 12453
MAP4K4- 12454 chl ooooooooooos 0mm0m00000mmm CUGUGGAAGUCUA 193
2931-17- s 0 12454 MAP4K4- 12455 chl ooooooooooos mm0m00000mmm0
CUGUGGAAGUCUA 194 2931-17- s 12455 MAP4K4- 12456 chl ooooooooooos
mm0m00000mmm0 CUGUGGAAGUCUA 195 2931-19- s 12456 --27-12480 12480
chl oooooooooooo DY547mm0f000f UCAUAGGUAACCU 196 oooooooooooo
0055f5f00mm00 CUGGUUGAAAGUG sso 000m000 A --27-12481 12481 chl
oooooooooooo DY547mm05f050 CGGCUACAGGUGC 197 oooooooooooo
00f05ff0m0000 UUAUGAAGAAAGU sso 0000m00 A APOB-10167- 12505 chl
oooooooooooo 0000000000000 GUCAUCACACUGA 198 21-12505 oooooooos
00000000 AUACCAAU APOB-10167- 12506 chl oooooooooooo 0000000000000
GUGAUCAGACUCA 199 21-12506 oooooooos 00000000 AUACGAAU MAP4K4-
12539 chl ooooooooooos DY547mm0m0000 CUGUGGAAGUCUA 200 2931-16- s
0mmm0 12539 APOB-10167- 12505.2 chl oooooooooooo 0000000000000
GUCAUCACACUGA 201 21-12505.2 oooooooso 0000000 AUACCAAU APOB-10167-
12506.2 chl oooooooooooo 0000000000000 GUGAUCAGACUCA 202 21-12506.2
oooooooso 0000000 AUACGAAU MAP4K4--13- 12565 Chl oooooooooooo
m0m0000m0mmm0 UGUAGGAUGUCUA 203 12565 o MAP4K4- 12386.2 chl
oooooooooooo 0mm0m00000mmm CUGUGGAAGUCUA 204 2931-16- o 0 12386.2
MAP4K4- 12815 chl oooooooooooo m0m0m0m0m0m0m CUGUGGAAGUCUA 205
2931-13- o 0m0m0m0m0m0m0 12815 APOB--13- 12957 Chl ooooooooooos
0mmmmmmmmmmmm ACUGAAUACCAAU 206 12957 TEG s m MAP4K4--16- 12983 chl
ooooooooooos mm0m00000mmm0 CUGUGGAAGUCUA 207 12983 s MAP4K4--16-
12984 Chl oooooooooooo mm0m00000mmm0 CUGUGGAAGUCUA 208 12984 oo
MAP4K4--16- 12985 chl ooooooooooss mmmmmmmmmmmmm CUGUGGAAGUCUA 209
12985 o MAP4K4--16- 12986 chl ooooooooooss mmmmmmmmmmmmm
CUGUGGAAGUCUA 210 12986 o MAP4K4--16- 12987 chl ooooooooooss
mm0m00000mmm0 CUGUGGAAGUCUA 211 12987 o MAP4K4--16- 12988 chl
ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 212 12988 o MAP4K4--16-
12989 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 213 12989 o
MAP4K4--16- 12990 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 214
12990 o MAP4K4--16- 12991 chl ooooooooooss mm0m00000mmm0
CUGUGGAAGUCUA 215 12991 o MAP4K4--16- 12992 chl ooooooooooss
mm0m00000mmm0 CUGUGGAAGUCUA 216 12992 o MAP4K4--16- 12993 chl
ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 217 12993 o MAP4K4--16-
12994 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 218 12994 o
MAP4K4--16- 12995 chl ooooooooooss mm0m00000mmm0 CUGUGGAAGUCUA 219
12995 o MAP4K4- 13012 chl oooooooooooo 0000000000000 AGAGUUCUGUGGA
220 2931-19- ooooooo 00000000 AGUCUA 13012 MAP4K4- 13016 chl
oooooooooooo DY54700000000 AGAGUUCUGUGGA 221 2931-19- ooooooo
0000000000000 AGUCUA 13016 PPIB--13- 13021 Chl oooooooooooo
0mmm00mm0m000 AUUUGGCUACAAA 222 13021 o pGL3-1172- 13038 chl
oooooooooooo 00m000m0m00mm ACAAAUACGAUUU 223 13-13038 o m
pGL3-1172- 13040 chl oooooooooooo DY5470m000m0m ACAAAUACGAUUU 224
13-13040 00mm --16-13047 13047 Chl oooooooooooo mm0m00000mmm0
CUGUGGAAGUCUA 225 oo SOD1-530- 13090 chl oooooooooooo 00m00000000m0
AAUGAAGAAAGUA 226 13-13090 o SOD1-523- 13091 chl oooooooooooo
000m00000m000 AGGUGGAAAUGAA 227 13-13091 o SOD1-535- 13092 chl
oooooooooooo 000000m0m0000 AGAAAGUACAAAG 228 13-13092 o SOD1-536-
13093 chl oooooooooooo 00000m0m00000 GAAAGUACAAAGA 229 13-13093 o
SOD1-396- 13094 chl oooooooooooo 0m0m00mm0mm00 AUGUGACUGCUGA 230
13-13094 o SOD1-385- 13095 chl oooooooooooo 000mmm000m00m
AGACUUGGGCAAU 231 13-13095 o SOD1-195- 13096 chl oooooooooooo
0mmmm000m0000 AUUUCGAGCAGAA 232 13-13096 o APOB-4314- 13115 Chl
oooooooooooo 0mmm0000000m0 AUCUGGAGAAACA 233 13-13115 0 APOB-3384-
13116 Chl oooooooooooo mm0000m000000 UCAGAACAAGAAA 234 13-13116 o
APOB-3547- 13117 Chl oooooooooooo 00mmm0mmm0mm0 GACUCAUCUGCUA 235
13-13117 o APOB-4318- 13118 Chl oooooooooooo 0000000m00m0m
GGAGAAACAACAU 236 13-13118 o APOB-3741- 13119 Chl oooooooooooo
00mmmmmm000m0 AGUCCCUCAAACA 237 13-13119 o PPIB--16- 13136 Chl
oooooooooooo 00mm0m00000m0 GGCUACAAAAACA 238 13136 oo APOB-4314-
13154 chl oooooooooooo 000mmm0000000 AGAUCUGGAGAAA 239 15-13154 oo
m0 CA APOB-3547- 13155 chl oooooooooooo m000mmm0mmm0m UGGACUCAUCUGC
240 15-13155 oo m0 UA APOB-4318- 13157 chl oooooooooooo
mm0000000m00m CUGGAGAAACAAC 241 15-13157 oo 0m AU APOB-3741- 13158
chl oooooooooooo 0000mmmmmm000 AGAGUCCCUCAAA 242 15-13158 oo m0 CA
APOB--13- 13159 chl oooooooooooo 0mm000m0mm00m ACUGAAUACCAAU 243
13159 APOB--15- 13160 chl oooooooooooo 0m0mm000m0mm0 ACACUGAAUACCA
244 13160 oo 0m AU SOD1-530- 13163 chl oooooooooooo 00m00000000m0
AAUGAAGAAAGUA 245 16-13163 o SOD1-523- 13164 chl oooooooooooo
000m00000m000 AGGUGGAAAUGAA 246 16-13164 o SOD1-535- 13165 chl
oooooooooooo 000000m0m0000 AGAAAGUACAAAG 247 16-13165 o SOD1-536-
13166 chl oooooooooooo 00000m0m00000 GAAAGUACAAAGA 248 16-13166 o
SOD1-396- 13167 chl oooooooooooo 0m0m00mm0mm00 AUGUGACUGCUGA
249
16-13167 o SOD1-385- 13168 chl oooooooooooo 000mmm000m00m
AGACUUGGGCAAU 250 16-13168 o SOD1-195- 13169 chl oooooooooooo
0mmmm000m0000 AUUUCGAGCAGAA 251 16-13169 o pGL3-1172- 13170 chl
oooooooooooo 0m000m0m00mmm ACAAAUACGAUUU 252 16-13170 o pGL3-1172-
13171 chl oooooooooooo DY5470m000m0m ACAAAUACGAUUU 253 16-13171 o
00mmm MAP4k4- 13189 chl oooooooooooo 0000000000000 AGAGUUCUGUGGA
254 2931-19- ooooooo 00000000 AGUCUA 13189 CTGF-1222- 13190 Chl
oooooooooooo 0m0000000m0m0 ACAGGAAGAUGUA 255 13-13190 o CTGF-813-
13192 Chl oooooooooooo 000m0000m0mmm GAGUGGAGCGCCU 256 13-13192 o
CTGF-747- 13194 Chl oooooooooooo m00mm000000m0 CGACUGGAAGACA 257
13-13194 o CTGF-817- 13196 Chl oooooooooooo 0000m0mmm0mmm
GGAGCGCCUGUUC 258 13-13196 o CTGF-1174- 13198 Chl oooooooooooo
0mm0mm0m00mm0 GCCAUUACAACUG 259 13-13198 o CTGF-1005- 13200 Chl
oooooooooooo 000mmmmmm00mm GAGCUUUCUGGCU 260 13-13200 o CTGF-814-
13202 Chl oooooooooooo 00m0000m0mmm0 AGUGGAGCGCCUG 261 13-13202 o
CTGF-816- 13204 Chl oooooooooooo m0000m0mmm0mm UGGAGCGCCUGUU 262
13-13204 o CTGF-1001- 13206 Chl oooooooooooo 0mmm000mmmmmm
GUUUGAGCUUUCU 263 13-13206 o CTGF-1173- 13208 Chl oooooooooooo
m0mm0mm0m00mm UGCCAUUACAACU 264 13-13208 o CTGF-749- 13210 Chl
oooooooooooo 0mm000000m0m0 ACUGGAAGACACG 265 13-13210 o CTGF-792-
13212 Chl oooooooooooo 00mm0mmm00mmm AACUGCCUGGUCC 266 13-13212 o
CTGF-1162- 13214 Chl oooooooooooo 000mmm0m0mmm0 AGACCUGUGCCUG 267
13-13214 o CTGF-811- 13216 Chl oooooooooooo m0000m0000m0m
CAGAGUGGAGCGC 268 13-13216 o CTGF-797- 13218 Chl oooooooooooo
mmm00mmm000mm CCUGGUCCAGACC 269 13-13218 o CTGF-1175- 13220 Chl
oooooooooooo mm0mm0m00mm0m CCAUUACAACUGU 270 13-13220 o CTGF-1172-
13222 Chl oooooooooooo mm0mm0mm0m00m CUGCCAUUACAAC 271 13-13222 o
CTGF-1177- 13224 Chl oooooooooooo 0mm0m00mm0mmm AUUACAACUGUCC 272
13-13224 o CTGF-1176- 13226 Chl oooooooooooo m0mm0m00mm0mm
CAUUACAACUGUC 273 13-13226 o CTGF-812- 13228 Chl oooooooooooo
0000m0000m0mm AGAGUGGAGCGCC 274 13-13228 o CTGF-745- 13230 Chl
oooooooooooo 0mm00mm000000 ACCGACUGGAAGA 275 13-13230 o CTGF-1230-
13232 Chl oooooooooooo 0m0m0m00000m0 AUGUACGGAGACA 276 13-13232 o
CTGF-920- 13234 Chl oooooooooooo 0mmmm0m0000mm GCCUUGCGAAGCU 277
13-13234 o CTGF-679- 13236 Chl oooooooooooo 0mm0m000000m0
GCUGCGAGGAGUG 278 13-13236 o CTGF-992- 13238 Chl oooooooooooo
0mmm0mm000mmm GCCUAUCAAGUUU 279 13-13238 o CTGF-1045- 13240 Chl
oooooooooooo 00mmmm0m0000m AAUUCUGUGGAGU 280 13-13240 o CTGF-1231-
13242 Chl oooooooooooo m0m0m00000m0m UGUACGGAGACAU 281 13-13242 o
CTGF-991- 13244 Chl oooooooooooo 00mmm0mm000mm AGCCUAUCAAGUU 282
13-13244 o CTGF-998- 13246 Chl oooooooooooo m000mmm000mmm
CAAGUUUGAGCUU 283 13-13246 o CTGF-1049- 13248 Chl oooooooooooo
mm0m0000m0m0m CUGUGGAGUAUGU 284 13-13248 o CTGF-1044- 13250 Chl
oooooooooooo 000mmmm0m0000 AAAUUCUGUGGAG 285 13-13250 o CTGF-1327-
13252 Chl oooooooooooo mmmm00m00m0m0 UUUCAGUAGCACA 286 13-13252 o
CTGF-1196- 13254 Chl oooooooooooo m00m00m0mmmmm CAAUGACAUCUUU 287
13-13254 o CTGF-562- 13256 Chl oooooooooooo 00m0mm00m0m0m
AGUACCAGUGCAC 288 13-13256 o CTGF-752- 13258 Chl oooooooooooo
000000m0m0mmm GGAAGACACGUUU 289 13-13258 o CTGF-994- 13260 Chl
oooooooooooo mm0mm000mmm00 CUAUCAAGUUUGA 290 13-13260 o CTGF-1040-
13262 Chl oooooooooooo 00mm000mmmm0m AGCUAAAUUCUGU 291 13-13262 o
CTGF-1984- 13264 Chl oooooooooooo 000m0000m0m00 AGGUAGAAUGUAA 292
13-13264 o CTGF-2195- 13266 Chl oooooooooooo 00mm00mm00mmm
AGCUGAUCAGUUU 293 13-13266 o CTGF-2043- 13268 Chl oooooooooooo
mmmm0mmm000m0 UUCUGCUCAGAUA 294 13-13268 o CTGF-1892- 13270 Chl
oooooooooooo mm0mmm000mm00 UUAUCUAAGUUAA 295 13-13270 o CTGF-1567-
13272 Chl oooooooooooo m0m0m000m00m0 UAUACGAGUAAUA 296 13-13272 o
CTGF-1780- 13274 Chl oooooooooooo 00mm000m00mmm GACUGGACAGCUU 297
13-13274 o CTGF-2162- 13276 Chl oooooooooooo 0m00mmmmm0mm0
AUGGCCUUUAUUA 298 13-13276 o CTGF-1034- 13278 Chl oooooooooooo
0m0mm000mm000 AUACCGAGCUAAA 299 13-13278 o CTGF-2264- 13280 Chl
oooooooooooo mm0mm00000m0m UUGUUGAGAGUGU 300 13-13280 o CTGF-1032-
13282 Chl oooooooooooo 0m0m0mm000mm0 ACAUACCGAGCUA 301 13-13282 o
CTGF-1535- 13284 Chl oooooooooooo 00m0000000mm0 AGCAGAAAGGUUA 302
13-13284 o CTGF-1694- 13286 Chl oooooooooooo 00mm0mmmmmm00
AGUUGUUCCUUAA 303 13-13286 o CTGF-1588- 13288 Chl oooooooooooo
0mmm0000m0m00 AUUUGAAGUGUAA 304 13-13288 o CTGF-928- 13290 Chl
oooooooooooo 000mm00mmm000 AAGCUGACCUGGA 305 13-13290 o CTGF-1133-
13292 Chl oooooooooooo 00mm0m0000000 GGUCAUGAAGAAG 306 13-13292 o
CTGF-912- 13294 Chl oooooooooooo 0m00mm000mmmm AUGGUCAGGCCUU 307
13-13294 o CTGF-753- 13296 Chl oooooooooooo 00000m0m0mmm0
GAAGACACGUUUG 308 13-13296 o CTGF-918- 13298 Chl oooooooooooo
000mmmm0m0000 AGGCCUUGCGAAG 309 13-13298 o CTGF-744- 13300 Chl
oooooooooooo m0mm0mm00000 UACCGACUGGAAG 310 13-13300 o CTGF-466-
13302 Chl oooooooooooo 0mm0m0000mm0 ACCGCAAGAUCGG 311 13-13302 o
CTGF-917- 13304 Chl oooooooooooo m000mmmm0m000 CAGGCCUUGCGAA 312
13-13304 o CTGF-1038- 13306 Chl oooooooooooo m000mm000mmmm
CGAGCUAAAUUCU 313 13-13306 o CTGF-1048- 13308 Chl oooooooooooo
mmm0m0000m0m0 UCUGUGGAGUAUG 314 13-13308 o CTGF-1235- 13310 Chl
oooooooooooo m00000m0m00m0 CGGAGACAUGGCA 315 13-13310 o CTGF-868-
13312 Chl oooooooooooo 0m00m00m0mmmm AUGACAACGCCUC 316 13-13312 o
CTGF-1131- 13314 Chl oooooooooooo 0000mm0m00000 GAGGUCAUGAAGA 317
13-13314 o CTGF-1043- 13316 Chl oooooooooooo m000mmmm0m000
UAAAUUCUGUGGA 318 13-13316 o CTGF-751- 13318 Chl oooooooooooo
m000000m0m0mm UGGAAGACACGUU 319 13-13318 o CTGF-1227- 13320 Chl
oooooooooooo 0000m0m0m0000 AAGAUGUACGGAG 320 13-13320 0 CTGF-867-
13322 Chl oooooooooooo 00m00m00m0mmm AAUGACAACGCCU 321 13-13322 o
CTGF-1128- 13324 Chl oooooooooooo 00m0000mm0m00 GGCGAGGUCAUGA 322
13-13324 o CTGF-756- 13326 Chl oooooooooooo 00m0m0mmm00mm
GACACGUUUGGCC 323 13-13326 o CTGF-1234- 13328 Chl oooooooooooo
0m00000m0m00m ACGGAGACAUGGC 324 13-13328 o CTGF-916- 13330 Chl
oooooooooooo mm000mmmm0m00 UCAGGCCUUGCGA 325 13-13330 o CTGF-925-
13332 Chl oooooooooooo 0m0000mm00mmm GCGAAGCUGACCU 326 13-13332 o
CTGF-1225- 13334 Chl oooooooooooo 000000m0m0m00 GGAAGAUGUACGG 327
13-13334 o CTGF-445- 13336 Chl oooooooooooo 0m00mmmm00mmm
GUGACUUCGGCUC 328 13-13336 o CTGF-446- 13338 Chl oooooooooooo
m00mmmm00mmmm UGACUUCGGCUCC 329 13-13338 o CTGF-913- 13340 Chl
oooooooooooo m00mm000mmmm0 UGGUCAGGCCUUG 330 13-13340 o CTGF-997-
13342 Chl oooooooooooo mm000mmm000mm UCAAGUUUGAGCU 331 13-13342 o
CTGF-277- 13344 Chl oooooooooooo 0mm0000mm0m00 GCCAGAACUGCAG 332
13-13344 o
CTGF-1052- 13346 Chl oooooooooooo m0000m0m0m0mm UGGAGUAUGUACC 333
13-13346 o CTGF-887- 13348 Chl oooooooooooo 0mm0000000m00
GCUAGAGAAGCAG 334 13-13348 o CTGF-914- 13350 Chl oooooooooooo
00mm000mmmm0m GGUCAGGCCUUGC 335 13-13350 o CTGF-1039- 13352 Chl
oooooooooooo 000mm000mmmm0 GAGCUAAAUUCUG 336 13-13352 o CTGF-754-
13354 Chl oooooooooooo 0000m0m0mmm00 AAGACACGUUUGG 337 13-13354 o
CTGF-1130- 13356 Chl oooooooooooo m0000mm0m0000 CGAGGUCAUGAAG 338
13-13356 o CTGF-919- 13358 Chl oooooooooooo 00mmmm0m0000m
GGCCUUGCGAAGC 339 13-13358 o CTGF-922- 13360 Chl oooooooooooo
mmm0m0000mm00 CUUGCGAAGCUGA 340 13-13360 o CTGF-746- 13362 Chl
oooooooooooo mm00mm000000m CCGACUGGAAGAC 341 13-13362 o CTGF-993-
13364 Chl oooooooooooo mmm0mm000mmm0 CCUAUCAAGUUUG 342 13-13364 o
CTGF-825- 13366 Chl oooooooooooo m0mmmm0000mmm UGUUCCAAGACCU 343
13-13366 o CTGF-926- 13368 Chl oooooooooooo m0000mm00mmm0
CGAAGCUGACCUG 344 13-13368 o CTGF-923- 13370 Chl oooooooooooo
mm0m0000mm00m UUGCGAAGCUGAC 345 13-13370 o CTGF-866- 13372 Chl
oooooooooooo m00m00m00m0mm CAAUGACAACGCC 346 13-13372 o CTGF-563-
13374 Chl oooooooooooo 0m0mm00m0m0m0 GUACCAGUGCACG 347 13-13374 o
CTGF-823- 13376 Chl oooooooooooo mmm0mmmm0000m CCUGUUCCAAGAC 348
13-13376 o CTGF-1233- 13378 Chl oooooooooooo m0m00000m0m00
UACGGAGACAUGG 349 13-13378 o CTGF-924- 13380 Chl oooooooooooo
m0m0000mm00mm UGCGAAGCUGACC 350 13-13380 o CTGF-921- 13382 Chl
oooooooooooo mmmm0m0000mm0 CCUUGCGAAGCUG 351 13-13382 o CTGF-443-
13384 Chl oooooooooooo mm0m00mmmm00m CUGUGACUUCGGC 352 13-13384 o
CTGF-1041- 13386 Chl oooooooooooo 0mm000mmmm0m0 GCUAAAUUCUGUG 353
13-13386 o CTGF-1042- 13388 Chl oooooooooooo mm000mmmm0m00
CUAAAUUCUGUGG 354 13-13388 o CTGF-755- 13390 Chl oooooooooooo
000m0m0mmm00m AGACACGUUUGGC 355 13-13390 o CTGF-467- 13392 Chl
oooooooooooo mm0m0000mm00m CCGCAAGAUCGGC 356 13-13392 o CTGF-995-
13394 Chl oooooooooooo m0mm000mmm000 UAUCAAGUUUGAG 357 13-13394 o
CTGF-927- 13396 Chl oooooooooooo 0000mm00mmm00 GAAGCUGACCUGG 358
13-13396 o SPP1-1025- 13398 Chl oooooooooooo mmm0m000mm000
CUCAUGAAUUAGA 359 13-13398 o SPP1-1049- 13400 Chl oooooooooooo
mm0000mm00mm0 CUGAGGUCAAUUA 360 13-13400 o SPP1-1051- 13402 Chl
oooooooooooo 0000mm00mm000 GAGGUCAAUUAAA 361 13-13402 o SPP1-1048-
13404 Chl oooooooooooo mmm0000mm00mm UCUGAGGUCAAUU 362 13-13404 o
SPP1-1050- 13406 Chl oooooooooooo m0000mm00mm00 UGAGGUCAAUUAA 363
13-13406 o SPP1-1047- 13408 Chl oooooooooooo mmmm0000mm00m
UUCUGAGGUCAAU 364 13-13408 o SPP1-800- 13410 Chl oooooooooooo
0mm00mm000m00 GUCAGCUGGAUGA 365 13-13410 o SPP1-492- 13412 Chl
oooooooooooo mmmm00m000mmm UUCUGAUGAAUCU 366 13-13412 o SPP1-612-
13414 Chl oooooooooooo m000mm0000mm0 UGGACUGAGGUCA 367 13-13414 o
SPP1-481- 13416 Chl oooooooooooo 000mmmm0mm0mm GAGUCUCACCAUU 368
13-13416 o SPP1-614- 13418 Chl oooooooooooo 00mm0000mm000
GACUGAGGUCAAA 369 13-13418 o SPP1-951- 13420 Chl oooooooooooo
mm0m00mm0m000 UCACAGCCAUGAA 370 13-13420 o SPP1-482- 13422 Chl
oooooooooooo 00mmmm0mm0mmm AGUCUCACCAUUC 371 13-13422 o SPP1-856-
13424 Chl oooooooooooo 000m000000mm0 AAGCGGAAAGCCA 372 13-13424 o
SPP1-857- 13426 Chl oooooooooooo 00m000000mm00 AGCGGAAAGCCAA 373
13-13426 o SPP1-365- 13428 Chl oooooooooooo 0mm0m0m000m00
ACCACAUGGAUGA 374 13-13428 o SPP1-359- 13430 Chl oooooooooooo
0mm0m00mm0m0m GCCAUGACCACAU 375 13-13430 o SPP1-357- 13432 Chl
oooooooooooo 000mm0m00mm0m AAGCCAUGACCAC 376 13-13432 o SPP1-858-
13434 Chl oooooooooooo 0m000000mm00m GCGGAAAGCCAAU 377 13-13434 o
SPP1-1012- 13436 Chl oooooooooooo 000mmmm0m0mmm AAAUUUCGUAUUU 378
13-13436 o SPP1-1014- 13438 Chl oooooooooooo 0mmmm0m0mmmmm
AUUUCGUAUUUCU 379 13-13438 o SPP1-356- 13440 Chl oooooooooooo
0000mm0m00mm0 AAAGCCAUGACCA 380 13-13440 o SPP1-368- 13442 Chl
oooooooooooo 0m0m000m00m0m ACAUGGAUGAUAU 381 13-13442 o SPP1-1011-
13444 Chl oooooooooooo 0000mmmm0m0mm GAAAUUUCGUAUU 382 13-13444 o
SPP1-754- 13446 Chl oooooooooooo 0m0mmmmmm00mm GCGCCUUCUGAUU 383
13-13446 o SPP1-1021- 13448 Chl oooooooooooo 0mmmmmm0m000m
AUUUCUCAUGAAU 384 13-13448 o SPP1-1330- 13450 Chl oooooooooooo
mmmmm0m000m00 CUCUCAUGAAUAG 385 13-13450 o SPP1-346- 13452 Chl
oooooooooooo 000mmm00m0000 AAGUCCAACGAAA 386 13-13452 o SPP1-869-
13454 Chl oooooooooooo 0m00m00000m00 AUGAUGAGAGCAA 387 13-13454 o
SPP1-701- 13456 Chl oooooooooooo 0m000000mm000 GCGAGGAGUUGAA 388
13-13456 o SPP1-896- 13458 Chl oooooooooooo m00mm00m00mm0
UGAUUGAUAGUCA 389 13-13458 o SPP1-1035- 13460 Chl oooooooooooo
000m00m0m0mmm AGAUAGUGCAUCU 390 13-13460 o SPP1-1170- 13462 Chl
oooooooooooo 0m0m0m0mmm0mm AUGUGUAUCUAUU 391 13-13462 o SPP1-1282-
13464 Chl oooooooooooo mmmm0m0000000 UUCUAUAGAAGAA 392 13-13464 o
SPP1-1537- 13466 Chl oooooooooooo mm0mmm00m00mm UUGUCCAGCAAUU 393
13-13466 o SPP1-692- 13468 Chl oooooooooooo 0m0m000000m00
ACAUGGAAAGCGA 394 13-13468 o SPP1-840- 13470 Chl oooooooooooo
0m00mmm000mm0 GCAGUCCAGAUUA 395 13-13470 o SPP1-1163- 13472 Chl
oooooooooooo m00mm000m0m0m UGGUUGAAUGUGU 396 13-13472 o SPP1-789-
13474 Chl oooooooooooo mm0m0000m000m UUAUGAAACGAGU 397 13-13474 o
SPP1-841- 13476 Chl oooooooooooo m00mmm000mm0m CAGUCCAGAUUAU 398
13-13476 o SPP1-852- 13478 Chl oooooooooooo 0m0m000m00000
AUAUAAGCGGAAA 399 13-13478 o SPP1-209- 13480 Chl oooooooooooo
m0mm00mm000m0 UACCAGUUAAACA 400 13-13480 o SPP1-1276- 13482 Chl
oooooooooooo m0mmm0mmmm0m0 UGUUCAUUCUAUA 401 13-13482 o SPP1-137-
13484 Chl oooooooooooo mm00mm0000000 CCGACCAAGGAAA 402 13-13484 o
SPP1-711- 13486 Chl oooooooooooo 000m00m0m0m0m GAAUGGUGCAUAC 403
13-13486 o SPP1-582- 13488 Chl oooooooooooo 0m0m00m00mm00
AUAUGAUGGCCGA 404 13-13488 o SPP1-839- 13490 Chl oooooooooooo
00m00mmm000mm AGCAGUCCAGAUU 405 13-13490 o SPP1-1091- 13492 Chl
oooooooooooo 0m0mmm00mm000 GCAUUUAGUCAAA 406 13-13492 o SPP1-884-
13494 Chl oooooooooooo 00m0mmmm00m0m AGCAUUCCGAUGU 407 13-13494 o
SPP1-903- 13496 Chl oooooooooooo m00mm00000mmm UAGUCAGGAACUU 408
13-13496 o SPP1-1090- 13498 Chl oooooooooooo m0m0mmm00mm00
UGCAUUUAGUCAA 409 13-13498 o SPP1-474- 13500 Chl oooooooooooo
0mmm00m000mmm GUCUGAUGAGUCU 410 13-13500 o SPP1-575- 13502 Chl
oooooooooooo m000m0m0m0m00 UAGACACAUAUGA 411 13-13502 o SPP1-671-
13504 Chl oooooooooooo m000m00000m0m CAGACGAGGACAU 412 13-13504 o
SPP1-924- 13506 Chl oooooooooooo m00mm0m000mmm CAGCCGUGAAUUC 413
13-13506 o SPP1-1185- 13508 Chl oooooooooooo 00mmm00000m00
AGUCUGGAAAUAA 414 13-13508 o SPP1-1221- 13510 Chl oooooooooooo
00mmm0m00mmmm AGUUUGUGGCUUC 415 13-13510 o SPP1-347- 13512 Chl
oooooooooooo 00mmm00m00000 AGUCCAACGAAAG 416
13-13512 o SPP1-634- 13514 Chl oooooooooooo 000mmmm0m000m
AAGUUUCGCAGAC 417 13-13514 o SPP1-877- 13516 Chl oooooooooooo
00m00m000m0mm AGCAAUGAGCAUU 418 13-13516 o SPP1-1033- 13518 Chl
oooooooooooo mm000m00m0m0m UUAGAUAGUGCAU 419 13-13518 o SPP1-714-
13520 Chl oooooooooooo m00m0m0m0m000 UGGUGCAUACAAG 420 13-13520 o
SPP1-791- 13522 Chl oooooooooooo 0m0000m000mm0 AUGAAACGAGUCA 421
13-13522 o SPP1-813- 13524 Chl oooooooooooo mm0000m0mm000
CCAGAGUGCUGAA 422 13-13524 o SPP1-939- 13526 Chl oooooooooooo
m00mm0m000mmm CAGCCAUGAAUUU 423 13-13526 o SPP1-1161- 13528 Chl
oooooooooooo 0mm00mm000m0m AUUGGUUGAAUGU 424 13-13528 o SPP1-1164-
13530 Chl oooooooooooo 00mm000m0m0m0 GGUUGAAUGUGUA 425 13-13530 o
SPP1-1190- 13532 Chl oooooooooooo 00000m00mm00m GGAAAUAACUAAU 426
13-13532 o SPP1-1333- 13534 Chl oooooooooooo mm0m000m00000
UCAUGAAUAGAAA 427 13-13534 o SPP1-537- 13536 Chl oooooooooooo
0mm00m00mm000 GCCAGCAACCGAA 428 13-13536 o SPP1-684- 13538 Chl
oooooooooooo m0mmmm0m0m0m0 CACCUCACACAUG 429 13-13538 o SPP1-707-
13540 Chl oooooooooooo 00mm000m00m0m AGUUGAAUGGUGC 430 13-13540 o
SPP1-799- 13542 Chl oooooooooooo 00mm00mm000m0 AGUCAGCUGGAUG 431
13-13542 o SPP1-853- 13544 Chl oooooooooooo m0m000m000000
UAUAAGCGGAAAG 432 13-13544 o SPP1-888- 13546 Chl oooooooooooo
mmmm00m0m00mm UUCCGAUGUGAUU 433 13-13546 o SPP1-1194- 13548 Chl
oooooooooooo 0m00mm00m0m0m AUAACUAAUGUGU 434 13-13548 o SPP1-1279-
13550 Chl oooooooooooo mm0mmmm0m0000 UCAUUCUAUAGAA 435 13-13550 o
SPP1-1300- 13552 Chl oooooooooooo 00mm0mm0mm0m0 AACUAUCACUGUA 436
13-13552 o SPP1-1510- 13554 Chl oooooooooooo 0mm00mm0mmm0m
GUCAAUUGCUUAU 437 13-13554 o SPP1-1543- 13556 Chl oooooooooooo
00m00mm00m000 AGCAAUUAAUAAA 438 13-13556 o SPP1-434- 13558 Chl
oooooooooooo 0m00mmmm00m00 ACGACUCUGAUGA 439 13-13558 o SPP1-600-
13560 Chl oooooooooooo m00m0m00mmm0m UAGUGUGGUUUAU 440 13-13560 o
SPP1-863- 13562 Chl oooooooooooo 000mm00m00m00 AAGCCAAUGAUGA 441
13-13562 o SPP1-902- 13564 Chl oooooooooooo 0m00mm00000mm
AUAGUCAGGAACU 442 13-13564 o SPP1-921- 13566 Chl oooooooooooo
00mm00mm0m000 AGUCAGCCGUGAA 443 13-13566 o SPP1-154- 13568 Chl
oooooooooooo 0mm0mm0m00000 ACUACCAUGAGAA 444 13-13568 o SPP1-217-
13570 Chl oooooooooooo 000m000mm00mm AAACAGGCUGAUU 445 13-13570 o
SPP1-816- 13572 Chl oooooooooooo 000m0mm0000mm GAGUGCUGAAACC 446
13-13572 o SPP1-882- 13574 Chl oooooooooooo m000m0mmmm00m
UGAGCAUUCCGAU 447 13-13574 o SPP1-932- 13576 Chl oooooooooooo
00mmmm0m00mm0 AAUUCCACAGCCA 448 13-13576 o SPP1-1509- 13578 Chl
oooooooooooo m0mm00mm0mmm0 UGUCAAUUGCUUA 449 13-13578 o SPP1-157-
13580 Chl oooooooooooo 0mm0m00000mm0 ACCAUGAGAAUUG 450 13-13580 o
SPP1-350- 13582 Chl oooooooooooo mm00m00000mm0 CCAACGAAAGCCA 451
13-13582 o SPP1-511- 13584 Chl oooooooooooo mm00mm0mm00mm
CUGGUCACUGAUU 452 13-13584 o SPP1-605- 13586 Chl oooooooooooo
m00mmm0m000mm UGGUUUAUGGACU 453 13-13586 o SPP1-811- 13588 Chl
oooooooooooo 00mm0000m0mm0 GACCAGAGUGCUG 454 13-13588 o SPP1-892-
13590 Chl oooooooooooo 00m0m00mm00m0 GAUGUGAUUGAUA 455 13-13590 o
SPP1-922- 13592 Chl oooooooooooo 0mm00mm0m000m GUCAGCCGUGAAU 456
13-13592 o SPP1-1169- 13594 Chl oooooooooooo 00m0m0m0mmm0m
AAUGUGUAUCUAU 457 13-13594 o SPP1-1182- 13596 Chl oooooooooooo
mm000mmm00000 UUGAGUCUGGAAA 458 13-13596 o SPP1-1539- 13598 Chl
oooooooooooo 0mmm00m00mm00 GUCCAGCAAUUAA 459 13-13598 o SPP1-1541-
13600 Chl oooooooooooo mm00m00mm00m0 CCAGCAAUUAAUA 460 13-13600 o
SPP1-427- 13602 Chl oooooooooooo 00mmm000m00mm GACUCGAACGACU 461
13-13602 o SPP1-533- 13604 Chl oooooooooooo 0mmm0mm00m00m
ACCUGCCAGCAAC 462 13-13604 o APOB--13- 13763 Chl oooooooooooo 0m +
00 + m0 + m0 + m ACtGAaUAcCAaU 463 13763 TEG o APOB--13- 13764 Chl
oooooooooooo 0mm000m0mm00m ACUGAAUACCAAU 464 13764 TEG o
MAP4K4--16- 13766 Chl oooooooooooo DY547mm0m0000 CUGUGGAAGUCUA 465
13766 o 0mmm0 PPIB--13- 13767 Chl oooooooooooo mmmmmmmmmmmmm
GGCUACAAAAACA 466 13767 o PPIB--15- 13768 Chl oooooooooooo
mm00mm0m00000 UUGGCUACAAAAA 467 13768 ooo m0 CA PPIB--17- 13769 Chl
oooooooooooo 0mmm00mm0m000 AUUUGGCUACAAA 468 13769 ooooo 00m0 AACA
MAP4K4--16- 13939 Chl oooooooooooo m0m0000m0mmm0 UGUAGGAUGUCUA 469
13939 o APOB-4314- 13940 Chl oooooooooooo 0mmm0000000m0
AUCUGGAGAAACA 470 16-13940 o APOB-4314- 13941 Chl oooooooooooo
000mmm0000000 AGAUCUGGAGAAA 471 17-13941 ooo m0 CA APOB--16- 13942
Chl oooooooooooo 00mmm0mmm0mm0 GACUCAUCUGCUA 472 13942 o APOB--18-
13943 Chl oooooooooooo 00mmm0mmm0mm0 GACUCAUCUGCUA 473 13943 o
APOB--17- 13944 Chl oooooooooooo m000mmm0mmm0m UGGACUCAUCUGC 474
13944 ooo m0 UA APOB--19- 13945 Chl oooooooooooo m000mmm0mmm0m
UGGACUCAUCUGC 475 13945 ooo m0 UA APOB-4314- 13946 Chl oooooooooooo
0000000m00m0m GGAGAAACAACAU 476 16-13946 o APOB-4314- 13947 Chl
oooooooooooo mm0000000m00m CUGGAGAAACAAC 477 17-13947 ooo 0m AU
APOB--16- 13948 Chl oooooooooooo 00mmmmmm000m0 AGUCCCUCAAACA 478
13948 o APOB--17- 13949 Chl oooooooooooo 0000mmmmmm000
AGAGUCCCUCAAA 479 13949 ooo m0 CA APOB--16- 13950 Chl oooooooooooo
0mm000m0mm00m ACUGAAUACCAAU 480 13950 o APOB--18- 13951 Chl
oooooooooooo 0mm000m0mm00m ACUGAAUACCAAU 481 13951 o APOB--17-
13952 Chl oooooooooooo 0m0mm000m0mm0 ACACUGAAUACCA 482 13952 ooo 0m
AU APOB--19- 13953 Chl oooooooooooo 0m0mm000m0mm0 ACACUGAAUACCA 483
13953 ooo 0m AU MAP4K4--16- 13766.2 Chl oooooooooooo DY547mm0m0000
CUGUGGAAGUCUA 484 13766.2 o 0mmm0 CTGF-1222- 13980 Chl oooooooooooo
0m0000000m0m0 ACAGGAAGAUGUA 485 16-13980 o CTGF-813- 13981 Chl
oooooooooooo 000m0000mmmm GAGUGGAGCGCCU 486 16-13981 o CTGF-747-
13982 Chl oooooooooooo m0mm000000m0 CGACUGGAAGACA 487 16-13982 o
CTGF-817- 13983 Chl oooooooooooo 0000mmmm0mmm GGAGCGCCUGUUC 488
16-13983 o CTGF-1174- 13984 Chl oooooooooooo 0mm0mm0m00mm0
GCCAUUACAACUG 489 16-13984 o CTGF-1005- 13985 Chl oooooooooooo
000mmmmmm00mm GAGCUUUCUGGCU 490 16-13985 o CTGF-814- 13986 Chl
oooooooooooo 00m0000mmmm0 AGUGGAGCGCCUG 491 16-13986 o CTGF-816-
13987 Chl oooooooooooo m0000mmmm0mm UGGAGCGCCUGUU 492 16-13987 o
CTGF-1001- 13988 Chl oooooooooooo 0mmm000mmmmmm GUUUGAGCUUUCU 493
16-13988 o CTGF-1173- 13989 Chl oooooooooooo m0mm0mm0m00mm
UGCCAUUACAACU 494 16-13989 o CTGF-749- 13990 Chl oooooooooooo
0mm000000m0m ACUGGAAGACACG 495 16-13990 o CTGF-792- 13991 Chl
oooooooooooo 00mm0mmm00mmm AACUGCCUGGUCC 496 16-13991 o CTGF-1162-
13992 Chl oooooooooooo 000mmm0m0mmm0 AGACCUGUGCCUG 497 16-13992 o
CTGF-811- 13993 Chl oooooooooooo m0000m0000mm CAGAGUGGAGCGC 498
16-13993 o CTGF-797- 13994 Chl oooooooooooo mmm00mmm000mm
CCUGGUCCAGACC 499 16-13994 o
CTGF-1175- 13995 Chl oooooooooooo mm0mm0m00mm0m CCAUUACAACUGU 500
16-13995 o CTGF-1172- 13996 Chl oooooooooooo mm0mm0mm0m00m
CUGCCAUUACAAC 501 16-13996 o CTGF-1177- 13997 Chl oooooooooooo
0mm0m00mm0mmm AUUACAACUGUCC 502 16-13997 o CTGF-1176- 13998 Chl
oooooooooooo m0mm0m00mm0mm CAUUACAACUGUC 503 16-13998 o CTGF-812-
13999 Chl oooooooooooo 0000m0000mmm AGAGUGGAGCGCC 504 16-13999 o
CTGF-745- 14000 Chl oooooooooooo 0mm0mm000000 ACCGACUGGAAGA 505
16-14000 o CTGF-1230- 14001 Chl oooooooooooo 0m0m0m0000m0
AUGUACGGAGACA 506 16-14001 o CTGF-920- 14002 Chl oooooooooooo
0mmmm0m000mm GCCUUGCGAAGCU 507 16-14002 o CTGF-679- 14003 Chl
oooooooooooo 0mm0m00000m0 GCUGCGAGGAGUG 508 16-14003 o CTGF-992-
14004 Chl oooooooooooo 0mmm0mm000mmm GCCUAUCAAGUUU 509 16-14004 o
CTGF-1045- 14005 Chl oooooooooooo 00mmmm0m0000m AAUUCUGUGGAGU 510
16-14005 o CTGF-1231- 14006 Chl oooooooooooo m0m0m0000m0m
UGUACGGAGACAU 511 16-14006 o CTGF-991- 14007 Chl oooooooooooo
00mmm0mm000mm AGCCUAUCAAGUU 512 16-14007 o CTGF-998- 14008 Chl
oooooooooooo m000mmm000mmm CAAGUUUGAGCUU 513 16-14008 o CTGF-1049-
14009 Chl oooooooooooo mm0m0000m0m0m CUGUGGAGUAUGU 514 16-14009 o
CTGF-1044- 14010 Chl oooooooooooo 000mmmm0m0000 AAAUUCUGUGGAG 515
16-14010 o CTGF-1327- 14011 Chl oooooooooooo mmmm00m00m0m0
UUUCAGUAGCACA 516 16-14011 o CTGF-1196- 14012 Chl oooooooooooo
m00m00m0mmmmm CAAUGACAUCUUU 517 16-14012 o CTGF-562- 14013 Chl
oooooooooooo 00m0mm00m0m0m AGUACCAGUGCAC 518 16-14013 o CTGF-752-
14014 Chl oooooooooooo 000000m0mmmm GGAAGACACGUUU 519 16-14014 o
CTGF-994- 14015 Chl oooooooooooo mm0mm000mmm00 CUAUCAAGUUUGA 520
16-14015 o CTGF-1040- 14016 Chl oooooooooooo 00mm000mmmm0m
AGCUAAAUUCUGU 521 16-14016 o CTGF-1984- 14017 Chl oooooooooooo
000m0000m0m00 AGGUAGAAUGUAA 522 16-14017 o CTGF-2195- 14018 Chl
oooooooooooo 00mm00mm00mmm AGCUGAUCAGUUU 523 16-14018 o CTGF-2043-
14019 Chl oooooooooooo mmmm0mmm000m0 UUCUGCUCAGAUA 524 16-14019 o
CTGF-1892- 14020 Chl oooooooooooo mm0mmm000mm00 UUAUCUAAGUUAA 525
16-14020 o CTGF-1567- 14021 Chl oooooooooooo m0m0m00m00m0
UAUACGAGUAAUA 526 16-14021 o CTGF-1780- 14022 Chl oooooooooooo
00mm000m00mmm GACUGGACAGCUU 527 16-14022 o CTGF-2162- 14023 Chl
oooooooooooo 0m00mmmmm0mm0 AUGGCCUUUAUUA 528 16-14023 o CTGF-1034-
14024 Chl oooooooooooo 0m0mm00mm000 AUACCGAGCUAAA 529 16-14024 o
CTGF-2264- 14025 Chl oooooooooooo mm0mm00000m0m UUGUUGAGAGUGU 530
16-14025 o CTGF-1032- 14026 Chl oooooooooooo 0m0m0mm00mm0
ACAUACCGAGCUA 531 16-14026 o CTGF-1535- 14027 Chl oooooooooooo
00m0000000mm0 AGCAGAAAGGUUA 532 16-14027 o CTGF-1694- 14028 Chl
oooooooooooo 00mm0mmmmmm00 AGUUGUUCCUUAA 533 16-14028 o CTGF-1588-
14029 Chl oooooooooooo 0mmm0000m0m00 AUUUGAAGUGUAA 534 16-14029 o
CTGF-928- 14030 Chl oooooooooooo 000mm00mmm000 AAGCUGACCUGGA 535
16-14030 o CTGF-1133- 14031 Chl oooooooooooo 00mm0m0000000
GGUCAUGAAGAAG 536 16-14031 o CTGF-912- 14032 Chl oooooooooooo
0m00mm000mmmm AUGGUCAGGCCUU 537 16-14032 o CTGF-753- 14033 Chl
oooooooooooo 00000m0mmmm0 GAAGACACGUUUG 538 16-14033 o CTGF-918-
14034 Chl oooooooooooo 000mmmm0m000 AGGCCUUGCGAAG 539 16-14034 o
CTGF-744- 14035 Chl oooooooooooo m0mm0mm00000 UACCGACUGGAAG 540
16-14035 o CTGF-466- 14036 Chl oooooooooooo 0mmm0000mm0
ACCGCAAGAUCGG 541 16-14036 o CTGF-917- 14037 Chl oooooooooooo
m000mmmm0m00 CAGGCCUUGCGAA 542 16-14037 o CTGF-1038- 14038 Chl
oooooooooooo m00mm000mmmm CGAGCUAAAUUCU 543 16-14038 o CTGF-1048-
14039 Chl oooooooooooo mmm0m0000m0m0 UCUGUGGAGUAUG 544 16-14039 o
CTGF-1235- 14040 Chl oooooooooooo m0000m0m00m0 CGGAGACAUGGCA 545
16-14040 o CTGF-868- 14041 Chl oooooooooooo 0m00m00mmmmm
AUGACAACGCCUC 546 16-14041 o CTGF-1131- 14042 Chl oooooooooooo
0000mm0m00000 GAGGUCAUGAAGA 547 16-14042 o CTGF-1043- 14043 Chl
oooooooooooo m000mmmm0m000 UAAAUUCUGUGGA 548 16-14043 o CTGF-751-
14044 Chl oooooooooooo m000000m0mmm UGGAAGACACGUU 549 16-14044 o
CTGF-1227- 14045 Chl oooooooooooo 0000m0m0m000 AAGAUGUACGGAG 550
16-14045 o CTGF-867- 14046 Chl oooooooooooo 00m00m00mmmm
AAUGACAACGCCU 551 16-14046 o CTGF-1128- 14047 Chl oooooooooooo
00m000mm0m00 GGCGAGGUCAUGA 552 16-14047 o CTGF-756- 14048 Chl
oooooooooooo 00m0m0mmm00mm GACACGUUUGGCC 553 16-14048 o CTGF-1234-
14049 Chl oooooooooooo 0m00000m0m00m ACGGAGACAUGGC 554 16-14049 o
CTGF-916- 14050 Chl oooooooooooo mm000mmmm0m00 UCAGGCCUUGCGA 555
16-14050 o CTGF-925- 14051 Chl oooooooooooo 0m0000mm00mmm
GCGAAGCUGACCU 556 16-14051 o CTGF-1225- 14052 Chl oooooooooooo
000000m0m0m00 GGAAGAUGUACGG 557 16-14052 o CTGF-445- 14053 Chl
oooooooooooo 0m00mmmm00mmm GUGACUUCGGCUC 558 16-14053 o CTGF-446-
14054 Chl oooooooooooo m00mmmm00mmmm UGACUUCGGCUCC 559 16-14054 o
CTGF-913- 14055 Chl oooooooooooo m00mm000mmmm0 UGGUCAGGCCUUG 560
16-14055 o CTGF-997- 14056 Chl oooooooooooo mm000mmm000mm
UCAAGUUUGAGCU 561 16-14056 o CTGF-277- 14057 Chl oooooooooooo
0mm0000mm0m00 GCCAGAACUGCAG 562 16-14057 o CTGF-1052- 14058 Chl
oooooooooooo m0000m0m0m0mm UGGAGUAUGUACC 563 16-14058 o CTGF-887-
14059 Chl oooooooooooo 0mm0000000m00 GCUAGAGAAGCAG 564 16-14059 o
CTGF-914- 14060 Chl oooooooooooo 00mm000mmmm0m GGUCAGGCCUUGC 565
16-14060 O CTGF-1039- 14061 Chl oooooooooooo 000mm000mmmm0
GAGCUAAAUUCUG 566 16-14061 o CTGF-754- 14062 Chl oooooooooooo
0000m0m0mmm00 AAGACACGUUUGG 567 16-14062 o CTGF-1130- 14063 Chl
oooooooooooo m0000mm0m0000 CGAGGUCAUGAAG 568 16-14063 o CTGF-919-
14064 Chl oooooooooooo 00mmmm0m0000m GGCCUUGCGAAGC 569 16-14064 o
CTGF-922- 14065 Chl oooooooooooo mmm0m0000mm00 CUUGCGAAGCUGA 570
16-14065 o CTGF-746- 14066 Chl oooooooooooo mm00mm000000m
CCGACUGGAAGAC 571 16-14066 o CTGF-993- 14067 Chl oooooooooooo
mmm0mm000mmm0 CCUAUCAAGUUUG 572 16-14067 o CTGF-825- 14068 Chl
oooooooooooo m0mmmm0000mmm UGUUCCAAGACCU 573 16-14068 o CTGF-926-
14069 Chl oooooooooooo m0000mm00mmm0 CGAAGCUGACCUG 574 16-14069 o
CTGF-923- 14070 Chl oooooooooooo mm0m0000mm00m UUGCGAAGCUGAC 575
16-14070 o CTGF-866- 14071 Chl oooooooooooo m00m00m00m0mm
CAAUGACAACGCC 576 16-14071 o CTGF-563- 14072 Chl oooooooooooo
0m0mm00m0m0m0 GUACCAGUGCACG 577 16-14072 o CTGF-823- 14073 Chl
oooooooooooo mmm0mmmm0000m CCUGUUCCAAGAC 578 16-14073 o CTGF-1233-
14074 Chl oooooooooooo m0m00000m0m00 UACGGAGACAUGG 579 16-14074 o
CTGF-924- 14075 Chl oooooooooooo m0m0000mm00mm UGCGAAGCUGACC 580
16-14075 o CTGF-921- 14076 Chl oooooooooooo mmmm0m0000mm0
CCUUGCGAAGCUG 581 16-14076 o CTGF-443- 14077 Chl oooooooooooo
mm0m00mmmm00m CUGUGACUUCGGC 582 16-14077 o CTGF-1041- 14078 Chl
oooooooooooo 0mm000mmmm0m0 GCUAAAUUCUGUG 583 16-14078 o
CTGF-1042- 14079 Chl oooooooooooo mm000mmmm0m00 CUAAAUUCUGUGG 584
16-14079 o CTGF-755- 14080 Chl oooooooooooo 000m0m0mmm00m
AGACACGUUUGGC 585 16-14080 o CTGF-467- 14081 Chl oooooooooooo
mm0m0000mm00m CCGCAAGAUCGGC 586 16-14081 o CTGF-995- 14082 Chl
oooooooooooo m0mm000mmm000 UAUCAAGUUUGAG 587 16-14082 o CTGF-927-
14083 Chl oooooooooooo 0000mm00mmm00 GAAGCUGACCUGG 588 16-14083 o
SPP1-1091- 14131 Chl oooooooooooo 0m0mmm00mm000 GCAUUUAGUCAAA 589
16-14131 o PPIB--16- 14188 Chl oooooooooooo mmmmmmmmmmmmm
GGCUACAAAAACA 590 14188 o PPIB--17- 14189 Chl oooooooooooo
mm00mm0m00000 UUGGCUACAAAAA 591 14189 ooo m0 CA PPIB--18- 14190 Chl
oooooooooooo 0mmm00mm0m000 AUUUGGCUACAAA 592 14190 ooooo 00m0 AACA
pGL3-1172- 14386 chl oooooooooooo 0m000m0m00mmm ACAAAUACGAUUU 593
16-14386 o pGL3-1172- 14387 chl oooooooooooo DY5470m000m0m
ACAAAUACGAUUU 594 16-14387 o 00mmm MAP4K4- 14390 Chl oooooooooooo
Pmmmmmmmmmmmm CUUUGAAGAGUUC 595 2931-25- oooooooooooo 000mmmmmmmmmm
UGUGGAAGUCUA 14390 o miR-122-- 14391 Chl ssoooooooooo mmmmmmmmmmmmm
ACAAACACCAUUG 596 23-14391 ooooooossss mmmmmmmmmm UCACACUCCA 14084
Chl oooooooooooo mmm0m000mm000 CUCAUGAAUUAGA 719 o 14085 Chl
oooooooooooo mm0000mm00mm0 CUGAGGUCAAUUA 720 o 14086 Chl
oooooooooooo 0000mm00mm000 GAGGUCAAUUAAA 721 o 14087 Chl
oooooooooooo mmm0000mm00mm UCUGAGGUCAAUU 722 o 14088 Chl
oooooooooooo m0000mm00mm00 UGAGGUCAAUUAA 723 o 14089 Chl
oooooooooooo mmmm0000mm00m UUCUGAGGUCAAU 724 o 14090 Chl
oooooooooooo 0mm00mm000m00 GUCAGCUGGAUGA 725 o 14091 Chl
oooooooooooo mmmm00m000mmm UUCUGAUGAAUCU 726 o 14092 Chl
oooooooooooo m000mm0000mm0 UGGACUGAGGUCA 727 o 14093 Chl
oooooooooooo 000mmmm0mm0mm GAGUCUCACCAUU 728 o 14094 Chl
oooooooooooo 00mm0000mm000 GACUGAGGUCAAA 729 o 14095 Chl
oooooooooooo mm0m00mm0m000 UCACAGCCAUGAA 730 o 14096 Chl
oooooooooooo 00mmmm0mm0mmm AGUCUCACCAUUC 731 o 14097 Chl
oooooooooooo 000m00000mm0 AAGCGGAAAGCCA 732 o 14098 Chl
oooooooooooo 00m00000mm00 AGCGGAAAGCCAA 733 o 14099 Chl
oooooooooooo 0mm0m0m000m00 ACCACAUGGAUGA 734 o 14100 Chl
oooooooooooo 0mm0m00mm0m0m GCCAUGACCACAU 735 o 14101 Chl
oooooooooooo 000mm0m00mm0m AAGCCAUGACCAC 736 o 14102 Chl
oooooooooooo 0m00000mm00m GCGGAAAGCCAAU 737 o 14103 Chl
oooooooooooo 000mmmmm0mmm AAAUUUCGUAUUU 738 o 14104 Chl
oooooooooooo 0mmmmm0mmmmm AUUUCGUAUUUCU 739 o 14105 Chl
oooooooooooo 0000mm0m00mm0 AAAGCCAUGACCA 740 o 14106 Chl
oooooooooooo 0m0m000m00m0m ACAUGGAUGAUAU 741 o 14107 Chl
oooooooooooo 0000mmmmm0mm GAAAUUUCGUAUU 742 o 14108 Chl
oooooooooooo 0mmmmmmm00mm GCGCCUUCUGAUU 743 o 14109 Chl
oooooooooooo 0mmmmmm0m000m AUUUCUCAUGAAU 744 o 14110 Chl
oooooooooooo mmmmm0m000m00 CUCUCAUGAAUAG 745 o 14111 Chl
oooooooooooo 000mmm00m000 AAGUCCAACGAAA 746 o 14112 Chl
oooooooooooo 0m00m00000m00 AUGAUGAGAGCAA 747 o 14113 Chl
oooooooooooo 0m00000mm000 GCGAGGAGUUGAA 748 o 14114 Chl
oooooooooooo m00mm00m00mm0 UGAUUGAUAGUCA 749 o 14115 Chl
oooooooooooo 000m00m0m0mmm AGAUAGUGCAUCU 750 o 14116 Chl
oooooooooooo 0m0m0m0mmm0mm AUGUGUAUCUAUU 751 o 14117 Chl
oooooooooooo mmmm0m0000000 UUCUAUAGAAGAA 752 o 14118 Chl
oooooooooooo mm0mmm00m00mm UUGUCCAGCAAUU 753 o 14119 Chl
oooooooooooo 0m0m000000m0 ACAUGGAAAGCGA 754 o 14120 Chl
oooooooooooo 0m00mmm000mm0 GCAGUCCAGAUUA 755 o 14121 Chl
oooooooooooo m00mm000m0m0m UGGUUGAAUGUGU 756 o 14122 Chl
oooooooooooo mm0m0000m00m UUAUGAAACGAGU 757 o 14123 Chl
oooooooooooo m00mmm000mm0m CAGUCCAGAUUAU 758 o 14124 Chl
oooooooooooo 0m0m000m0000 AUAUAAGCGGAAA 759 o 14125 Chl
oooooooooooo m0mm00mm000m0 UACCAGUUAAACA 760 o 14126 Chl
oooooooooooo m0mmm0mmmm0m0 UGUUCAUUCUAUA 761 o 14127 Chl
oooooooooooo mm0mm0000000 CCGACCAAGGAAA 762 o 14128 Chl
oooooooooooo 000m00m0m0m0m GAAUGGUGCAUAC 763 o 14129 Chl
oooooooooooo 0m0m00m00mm0 AUAUGAUGGCCGA 764 o 14130 Chl
oooooooooooo 00m00mmm000mm AGCAGUCCAGAUU 765 o 14132 Chl
oooooooooooo 00m0mmmm0m0m AGCAUUCCGAUGU 766 o 14133 Chl
oooooooooooo m00mm00000mmm UAGUCAGGAACUU 767 o 14134 Chl
oooooooooooo m0m0mmm00mm00 UGCAUUUAGUCAA 768 o 14135 Chl
oooooooooooo 0mmm00m000mmm GUCUGAUGAGUCU 769 o 14136 Chl
oooooooooooo m000m0m0m0m00 UAGACACAUAUGA 770 o 14137 Chl
oooooooooooo m000m0000m0m CAGACGAGGACAU 771 o 14138 Chl
oooooooooooo m00mmm000mmm CAGCCGUGAAUUC 772 o 14139 Chl
oooooooooooo 00mmm00000m00 AGUCUGGAAAUAA 773 o 14140 Chl
oooooooooooo 00mmm0m00mmmm AGUUUGUGGCUUC 774 o 14141 Chl
oooooooooooo 00mmm00m0000 AGUCCAACGAAAG 775 o 14142 Chl
oooooooooooo 000mmmmm000m AAGUUUCGCAGAC 776 o 14143 Chl
oooooooooooo 00m00m000m0mm AGCAAUGAGCAUU 777 o 14144 Chl
oooooooooooo mm000m00m0m0m UUAGAUAGUGCAU 778 o 14145 Chl
oooooooooooo m00m0m0m0m000 UGGUGCAUACAAG 779 o 14146 Chl
oooooooooooo 0m0000m00mm0 AUGAAACGAGUCA 780 o 14147 Chl
oooooooooooo mm0000m0mm000 CCAGAGUGCUGAA 781 o 14148 Chl
oooooooooooo m00mm0m000mmm CAGCCAUGAAUUU 782 o 14149 Chl
oooooooooooo 0mm00mm000m0m AUUGGUUGAAUGU 783 o 14150 Chl
oooooooooooo 00mm000m0m0m0 GGUUGAAUGUGUA 784 o 14151 Chl
oooooooooooo 00000m00mm00m GGAAAUAACUAAU 785 o 14152 Chl
oooooooooooo mm0m000m00000 UCAUGAAUAGAAA 786 o 14153 Chl
oooooooooooo 0mm00m00mm00 GCCAGCAACCGAA 787 o 14154 Chl
oooooooooooo m0mmmm0m0m0m0 CACCUCACACAUG 788 o
14155 Chl oooooooooooo 00mm000m00m0m AGUUGAAUGGUGC 789 o 14156 Chl
oooooooooooo 00mm00mm000m0 AGUCAGCUGGAUG 790 o 14157 Chl
oooooooooooo m0m000m00000 UAUAAGCGGAAAG 791 o 14158 Chl
oooooooooooo mmmm0m0m00mm UUCCGAUGUGAUU 792 o 14159 Chl
oooooooooooo 0m00mm00m0m0m AUAACUAAUGUGU 793 o 14160 Chl
oooooooooooo mm0mmmm0m0000 UCAUUCUAUAGAA 794 o 14161 Chl
oooooooooooo 00mm0mm0mm0m0 AACUAUCACUGUA 795 o 14162 Chl
oooooooooooo 0mm00mm0mmm0m GUCAAUUGCUUAU 796 o 14163 Chl
oooooooooooo 00m00mm00m000 AGCAAUUAAUAAA 797 o 14164 Chl
oooooooooooo 0m0mmmm00m00 ACGACUCUGAUGA 798 o 14165 Chl
oooooooooooo m00m0m00mmm0m UAGUGUGGUUUAU 799 o 14166 Chl
oooooooooooo 000mm00m00m00 AAGCCAAUGAUGA 800 o 14167 Chl
oooooooooooo 0m00mm00000mm AUAGUCAGGAACU 801 o 14168 Chl
oooooooooooo 00mm00mmm000 AGUCAGCCGUGAA 802 o 14169 Chl
oooooooooooo 0mm0mm0m00000 ACUACCAUGAGAA 803 o 14170 Chl
oooooooooooo 000m000mm00mm AAACAGGCUGAUU 804 o 14171 Chl
oooooooooooo 000m0mm0000mm GAGUGCUGAAACC 805 o 14172 Chl
oooooooooooo m000m0mmmm0m UGAGCAUUCCGAU 806 o 14173 Chl
oooooooooooo 00mmmm0m00mm0 AAUUCCACAGCCA 807 o 14174 Chl
oooooooooooo m0mm00mm0mmm0 UGUCAAUUGCUUA 808 o 14175 Chl
oooooooooooo 0mm0m00000mm0 ACCAUGAGAAUUG 809 o 14176 Chl
oooooooooooo mm00m0000mm0 CCAACGAAAGCCA 810 o 14177 Chl
oooooooooooo mm00mm0mm00mm CUGGUCACUGAUU 811 o 14178 Chl
oooooooooooo m00mmm0m000mm UGGUUUAUGGACU 812 o 14179 Chl
oooooooooooo 00mm0000m0mm0 GACCAGAGUGCUG 813 o 14180 Chl
oooooooooooo 00m0m00mm00m0 GAUGUGAUUGAUA 814 o 14181 Chl
oooooooooooo 0mm00mmm000m GUCAGCCGUGAAU 815 o 14182 Chl
oooooooooooo 00m0m0m0mmm0m AAUGUGUAUCUAU 816 o 14183 Chl
oooooooooooo mm000mmm00000 UUGAGUCUGGAAA 817 o 14184 Chl
oooooooooooo 0mmm00m00mm00 GUCCAGCAAUUAA 818 o 14185 Chl
oooooooooooo mm00m00mm00m0 CCAGCAAUUAAUA 819 o 14186 Chl
oooooooooooo 00mmm00m0mm GACUCGAACGACU 820 o 14187 Chl oooooooooooo
0mmm0mm00m00m ACCUGCCAGCAAC 821 o o: phosphodiester; s:
phosphorothioate; P: 5' phosphorylation; 0: 2'-OH; F: 2'-fluoro; m:
2' O-methyl; +: LNA modification. Capital letters in the sequence
signify ribonucleotides, lower case letters signify
deoxyribonucleotides.
[0454] Having thus described several aspects of at least one
embodiment of this invention, it is to be appreciated various
alterations, modifications, and improvements will readily occur to
those skilled in the art. Such alterations, modifications, and
improvements are intended to be part of this disclosure, and are
intended to be within the spirit and scope of the invention.
Accordingly, the foregoing description and drawings are by way of
example only.
EQUIVALENTS
[0455] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0456] All references, including patent documents, disclosed herein
are incorporated by reference in their entirety. This application
incorporates by reference the entire contents, including all the
drawings and all parts of the specification (including sequence
listing or amino acid/polynucleotide sequences) of the co-pending
U.S. Provisional Application No. 61/135,855, filed on Jul. 24,
2008, entitled "SHORT HAIRPIN RNAI CONSTRUCTS AND USES THEREOF,"
and U.S. Provisional Application No. 61/197,768, filed on Oct. 30,
2008, entitled "MINIRNA CONSTRUCTS AND USES THEREOF."
Sequence CWU 1
1
821119RNAArtificial Sequencesynthetic oligonucleotide 1auugguauuc
agugugaug 19219RNAArtificial Sequencesynthetic oligonucleotide
2auucguauug agucugauc 19319RNAArtificial Sequencesynthetic
oligonucleotide 3uagacuucca cagaacucu 19419RNAArtificial
Sequencesynthetic oligonucleotide 4uagacuucca cagaacucu
19519RNAArtificial Sequencesynthetic oligonucleotide 5uagacuucca
cagaacucu 19619RNAArtificial Sequencesynthetic oligonucleotide
6uagacuucca cagaacucu 19719RNAArtificial Sequencesynthetic
oligonucleotide 7uagacuucca cagaacucu 19819RNAArtificial
Sequencesynthetic oligonucleotide 8uagacuucca cagaacucu
19917RNAArtificial Sequencesynthetic oligonucleotide 9uagacuucca
cagaacu 171021RNAArtificial Sequencesynthetic oligonucleotide
10auugguauuc agugugauga c 211121RNAArtificial Sequencesynthetic
oligonucleotide 11auucguauug agucugauca c 211219RNAArtificial
Sequencesynthetic oligonucleotide 12uagacuucca cagaacucu
191319RNAArtificial Sequencesynthetic oligonucleotide 13uagacuucca
cagaacucu 191419RNAArtificial Sequencesynthetic oligonucleotide
14uagacuucca cagaacucu 191521RNAArtificial Sequencesynthetic
oligonucleotide 15uagacuucca cagaacucuu c 211621RNAArtificial
Sequencesynthetic oligonucleotide 16uagacuucca cagaacucuu c
211725RNAArtificial Sequencesynthetic oligonucleotide 17uagacuucca
cagaacucuu caaag 251821RNAArtificial Sequencesynthetic
oligonucleotide 18auugguauuc agugugauga c 211921RNAArtificial
Sequencesynthetic oligonucleotide 19auucguauug agucugauca c
212019RNAArtificial Sequencesynthetic oligonucleotide 20uagacuucca
cagaacucu 192121RNAArtificial Sequencesynthetic oligonucleotide
21auugguauuc agugugauga c 212221RNAArtificial Sequencesynthetic
oligonucleotide 22auucguauug agucugauca c 212319RNAArtificial
Sequencesynthetic oligonucleotide 23uagacuucca cagaacucu
192419RNAArtificial Sequencesynthetic oligonucleotide 24uagacuucca
cagaacucu 192519RNAArtificial Sequencesynthetic oligonucleotide
25uagacuucca cagaacucu 192619RNAArtificial Sequencesynthetic
oligonucleotide 26uagacuucca cagaacucu 192719RNAArtificial
Sequencesynthetic oligonucleotide 27uagacuucca cagaacucu
192819RNAArtificial Sequencesynthetic oligonucleotide 28uagacuucca
cagaacucu 192919RNAArtificial Sequencesynthetic oligonucleotide
29uagacuucca cagaacucu 193019RNAArtificial Sequencesynthetic
oligonucleotide 30uagacuucca cagaacucu 193119RNAArtificial
Sequencesynthetic oligonucleotide 31uagacuucca cagaacucu
193219RNAArtificial Sequencesynthetic oligonucleotide 32uagacuucca
cagaacucu 193319RNAArtificial Sequencesynthetic oligonucleotide
33uagacuucca cagaacucu 193419RNAArtificial Sequencesynthetic
oligonucleotide 34uagacuucca cagaacucu 193519RNAArtificial
Sequencesynthetic oligonucleotide 35uagacuucca cagaacucu
193619RNAArtificial Sequencesynthetic oligonucleotide 36uagacuucca
cagaacucu 193719RNAArtificial Sequencesynthetic oligonucleotide
37uagacuucca cagaacucu 193819RNAArtificial Sequencesynthetic
oligonucleotide 38uguuuuugua gccaaaucc 193919RNAArtificial
Sequencesynthetic oligonucleotide 39uacuuucuuc auuuccacc
194019RNAArtificial Sequencesynthetic oligonucleotide 40uucauuucca
ccuuugccc 194119RNAArtificial Sequencesynthetic oligonucleotide
41cuuuguacuu ucuucauuu 194219RNAArtificial Sequencesynthetic
oligonucleotide 42ucuuuguacu uucuucauu 194319RNAArtificial
Sequencesynthetic oligonucleotide 43ucagcaguca cauugccca
194419RNAArtificial Sequencesynthetic oligonucleotide 44auugcccaag
ucuccaaca 194519RNAArtificial Sequencesynthetic oligonucleotide
45uucugcucga aauugauga 194619RNAArtificial Sequencesynthetic
oligonucleotide 46aaaucguauu ugucaauca 194719RNAArtificial
Sequencesynthetic oligonucleotide 47aaaucguauu ugucaauca
194819RNAArtificial Sequencesynthetic oligonucleotide 48uagacuucca
cagaacucu 194919RNAArtificial Sequencesynthetic oligonucleotide
49uagacuucca cagaacucu 195019RNAArtificial Sequencesynthetic
oligonucleotide 50uagacauccu acacagcac 195119RNAArtificial
Sequencesynthetic oligonucleotide 51uguuucucca gauccuugc
195219RNAArtificial Sequencesynthetic oligonucleotide 52uguuucucca
gauccuugc 195319RNAArtificial Sequencesynthetic oligonucleotide
53uagcagauga guccauuug 195423RNAArtificial Sequencesynthetic
oligonucleotide 54uagcagauga guccauuugg aga 235519RNAArtificial
Sequencesynthetic oligonucleotide 55uagcagauga guccauuug
195623RNAArtificial Sequencesynthetic oligonucleotide 56uagcagauga
guccauuugg aga 235719RNAArtificial Sequencesynthetic
oligonucleotide 57auguuguuuc uccagaucc 195819RNAArtificial
Sequencesynthetic oligonucleotide 58auguuguuuc uccagaucc
195919RNAArtificial Sequencesynthetic oligonucleotide 59uguuugaggg
acucuguga 196019RNAArtificial Sequencesynthetic oligonucleotide
60uguuugaggg acucuguga 196119RNAArtificial Sequencesynthetic
oligonucleotide 61auugguauuc agugugaug 196223RNAArtificial
Sequencesynthetic oligonucleotide 62auugguauuc agugugauga cac
236319RNAArtificial Sequencesynthetic oligonucleotide 63auugguauuc
agugugaug 196423RNAArtificial Sequencesynthetic oligonucleotide
64auugguauuc agugugauga cac 236519RNAArtificial Sequencesynthetic
oligonucleotide 65uagacuucca cagaacucu 196619RNAArtificial
Sequencesynthetic oligonucleotide 66uacaucuucc uguaguaca
196719RNAArtificial Sequencesynthetic oligonucleotide 67aggcgcucca
cucuguggu 196819RNAArtificial Sequencesynthetic oligonucleotide
68ugucuuccag ucgguaagc 196919RNAArtificial Sequencesynthetic
oligonucleotide 69gaacaggcgc uccacucug 197019RNAArtificial
Sequencesynthetic oligonucleotide 70caguuguaau ggcaggcac
197119RNAArtificial Sequencesynthetic oligonucleotide 71agccagaaag
cucaaacuu 197219RNAArtificial Sequencesynthetic oligonucleotide
72caggcgcucc acucugugg 197319RNAArtificial Sequencesynthetic
oligonucleotide 73aacaggcgcu ccacucugu 197419RNAArtificial
Sequencesynthetic oligonucleotide 74agaaagcuca aacuugaua
197519RNAArtificial Sequencesynthetic oligonucleotide 75aguuguaaug
gcaggcaca 197619RNAArtificial Sequencesynthetic oligonucleotide
76cgugucuucc agucgguaa 197719RNAArtificial Sequencesynthetic
oligonucleotide 77ggaccaggca guuggcucu 197819RNAArtificial
Sequencesynthetic oligonucleotide 78caggcacagg ucuugauga
197919RNAArtificial Sequencesynthetic oligonucleotide 79gcgcuccacu
cuguggucu 198019RNAArtificial Sequencesynthetic oligonucleotide
80ggucuggacc aggcaguug 198119RNAArtificial Sequencesynthetic
oligonucleotide 81acaguuguaa uggcaggca 198219RNAArtificial
Sequencesynthetic oligonucleotide 82guuguaaugg caggcacag
198319RNAArtificial Sequencesynthetic oligonucleotide 83ggacaguugu
aauggcagg 198419RNAArtificial Sequencesynthetic oligonucleotide
84gacaguugua auggcaggc 198519RNAArtificial Sequencesynthetic
oligonucleotide 85ggcgcuccac ucugugguc 198619RNAArtificial
Sequencesynthetic oligonucleotide 86ucuuccaguc gguaagccg
198719RNAArtificial Sequencesynthetic oligonucleotide 87ugucuccgua
caucuuccu 198819RNAArtificial Sequencesynthetic oligonucleotide
88agcuucgcaa ggccugacc 198919RNAArtificial Sequencesynthetic
oligonucleotide 89cacuccucgc agcauuucc 199019RNAArtificial
Sequencesynthetic oligonucleotide 90aaacuugaua ggcuuggag
199119RNAArtificial Sequencesynthetic oligonucleotide 91acuccacaga
auuuagcuc 199219RNAArtificial Sequencesynthetic oligonucleotide
92augucuccgu acaucuucc 199319RNAArtificial Sequencesynthetic
oligonucleotide 93aacuugauag gcuuggaga 199419RNAArtificial
Sequencesynthetic oligonucleotide 94aagcucaaac uugauaggc
199519RNAArtificial Sequencesynthetic oligonucleotide 95acauacucca
cagaauuua 199619RNAArtificial Sequencesynthetic oligonucleotide
96cuccacagaa uuuagcucg 199719RNAArtificial Sequencesynthetic
oligonucleotide 97ugugcuacug aaaucauuu 199819RNAArtificial
Sequencesynthetic oligonucleotide 98aaagauguca uugucuccg
199919RNAArtificial Sequencesynthetic oligonucleotide 99gugcacuggu
acuugcagc 1910019RNAArtificial Sequencesynthetic oligonucleotide
100aaacgugucu uccagucgg 1910119RNAArtificial Sequencesynthetic
oligonucleotide 101ucaaacuuga uaggcuugg 1910219RNAArtificial
Sequencesynthetic oligonucleotide 102acagaauuua gcucgguau
1910319RNAArtificial Sequencesynthetic oligonucleotide
103uuacauucua ccuauggug 1910419RNAArtificial Sequencesynthetic
oligonucleotide 104aaacugauca gcuauauag 1910519RNAArtificial
Sequencesynthetic oligonucleotide 105uaucugagca gaauuucca
1910619RNAArtificial Sequencesynthetic oligonucleotide
106uuaacuuaga uaacuguac 1910719RNAArtificial Sequencesynthetic
oligonucleotide 107uauuacucgu auaagaugc 1910819RNAArtificial
Sequencesynthetic oligonucleotide 108aagcugucca gucuaaucg
1910919RNAArtificial Sequencesynthetic oligonucleotide
109uaauaaaggc cauuuguuc 1911019RNAArtificial Sequencesynthetic
oligonucleotide 110uuuagcucgg uaugucuuc 1911119RNAArtificial
Sequencesynthetic oligonucleotide 111acacucucaa caaauaaac
1911219RNAArtificial Sequencesynthetic oligonucleotide
112uagcucggua ugucuucau 1911319RNAArtificial Sequencesynthetic
oligonucleotide 113uaaccuuucu gcugguacc 1911419RNAArtificial
Sequencesynthetic oligonucleotide 114uuaaggaaca acuugacuc
1911519RNAArtificial Sequencesynthetic oligonucleotide
115uuacacuuca aauagcagg 1911619RNAArtificial Sequencesynthetic
oligonucleotide 116uccaggucag cuucgcaag 1911719RNAArtificial
Sequencesynthetic oligonucleotide 117cuucuucaug accucgccg
1911819RNAArtificial Sequencesynthetic oligonucleotide
118aaggccugac caugcacag 1911919RNAArtificial Sequencesynthetic
oligonucleotide 119caaacguguc uuccagucg 1912019RNAArtificial
Sequencesynthetic oligonucleotide 120cuucgcaagg ccugaccau
1912119RNAArtificial Sequencesynthetic oligonucleotide
121cuuccagucg guaagccgc 1912219RNAArtificial Sequencesynthetic
oligonucleotide 122ccgaucuugc gguuggccg 1912319RNAArtificial
Sequencesynthetic oligonucleotide 123uucgcaaggc cugaccaug
1912419RNAArtificial Sequencesynthetic oligonucleotide
124agaauuuagc ucgguaugu 1912519RNAArtificial Sequencesynthetic
oligonucleotide 125cauacuccac agaauuuag 1912619RNAArtificial
Sequencesynthetic oligonucleotide 126ugccaugucu ccguacauc
1912719RNAArtificial Sequencesynthetic oligonucleotide
127gaggcguugu cauugguaa 1912819RNAArtificial Sequencesynthetic
oligonucleotide 128ucuucaugac cucgccguc 1912919RNAArtificial
Sequencesynthetic oligonucleotide 129uccacagaau uuagcucgg
1913019RNAArtificial Sequencesynthetic oligonucleotide
130aacgugucuu ccagucggu 1913119RNAArtificial Sequencesynthetic
oligonucleotide 131cuccguacau cuuccugua 1913219RNAArtificial
Sequencesynthetic oligonucleotide 132aggcguuguc auugguaac
1913319RNAArtificial Sequencesynthetic oligonucleotide
133ucaugaccuc gccgucagg 1913419RNAArtificial Sequencesynthetic
oligonucleotide 134ggccaaacgu gucuuccag 1913519RNAArtificial
Sequencesynthetic oligonucleotide 135gccaugucuc cguacaucu
1913619RNAArtificial Sequencesynthetic oligonucleotide
136ucgcaaggcc ugaccaugc 1913719RNAArtificial Sequencesynthetic
oligonucleotide 137aggucagcuu cgcaaggcc 1913819RNAArtificial
Sequencesynthetic oligonucleotide 138ccguacaucu uccuguagu
1913919RNAArtificial Sequencesynthetic oligonucleotide
139gagccgaagu cacagaaga 1914019RNAArtificial Sequencesynthetic
oligonucleotide 140ggagccgaag ucacagaag 1914119RNAArtificial
Sequencesynthetic oligonucleotide 141caaggccuga ccaugcaca
1914219RNAArtificial Sequencesynthetic oligonucleotide
142agcucaaacu ugauaggcu 1914319RNAArtificial Sequencesynthetic
oligonucleotide 143cugcaguucu ggccgacgg 1914419RNAArtificial
Sequencesynthetic oligonucleotide 144gguacauacu ccacagaau
1914519RNAArtificial Sequencesynthetic oligonucleotide
145cugcuucucu agccugcag 1914619RNAArtificial Sequencesynthetic
oligonucleotide 146gcaaggccug accaugcac 1914719RNAArtificial
Sequencesynthetic oligonucleotide 147cagaauuuag cucgguaug
1914819RNAArtificial Sequencesynthetic oligonucleotide
148ccaaacgugu cuuccaguc 1914919RNAArtificial Sequencesynthetic
oligonucleotide 149cuucaugacc ucgccguca 1915019RNAArtificial
Sequencesynthetic oligonucleotide 150gcuucgcaag gccugacca
1915119RNAArtificial Sequencesynthetic oligonucleotide
151ucagcuucgc aaggccuga 1915219RNAArtificial Sequencesynthetic
oligonucleotide 152gucuuccagu cgguaagcc 1915319RNAArtificial
Sequencesynthetic oligonucleotide 153caaacuugau aggcuugga
1915419RNAArtificial Sequencesynthetic oligonucleotide
154aggucuugga acaggcgcu 1915519RNAArtificial Sequencesynthetic
oligonucleotide 155caggucagcu ucgcaaggc 1915619RNAArtificial
Sequencesynthetic oligonucleotide 156gucagcuucg caaggccug
1915719RNAArtificial Sequencesynthetic oligonucleotide
157ggcguuguca uugguaacc 1915819RNAArtificial Sequencesynthetic
oligonucleotide 158cgugcacugg uacuugcag 1915919RNAArtificial
Sequencesynthetic oligonucleotide 159gucuuggaac aggcgcucc
1916019RNAArtificial Sequencesynthetic oligonucleotide
160ccaugucucc guacaucuu 1916119RNAArtificial Sequencesynthetic
oligonucleotide 161ggucagcuuc gcaaggccu 1916219RNAArtificial
Sequencesynthetic oligonucleotide 162cagcuucgca aggccugac
1916319RNAArtificial Sequencesynthetic oligonucleotide
163gccgaaguca cagaagagg 1916419RNAArtificial Sequencesynthetic
oligonucleotide 164cacagaauuu agcucggua 1916519RNAArtificial
Sequencesynthetic oligonucleotide 165ccacagaauu uagcucggu
1916619RNAArtificial Sequencesynthetic oligonucleotide
166gccaaacgug ucuuccagu 1916719RNAArtificial Sequencesynthetic
oligonucleotide 167gccgaucuug cgguuggcc 1916819RNAArtificial
Sequencesynthetic oligonucleotide 168cucaaacuug auaggcuug
1916919RNAArtificial Sequencesynthetic oligonucleotide
169ccaggucagc uucgcaagg 1917019RNAArtificial Sequencesynthetic
oligonucleotide 170uuugacuaaa ugcaaagug 1917119RNAArtificial
Sequencesynthetic oligonucleotide 171uguuuuugua gccaaaucc
1917219RNAArtificial Sequencesynthetic oligonucleotide
172uguuuuugua gccaaaucc 1917319RNAArtificial Sequencesynthetic
oligonucleotide 173uguuuuugua gccaaaucc 1917419RNAArtificial
Sequencesynthetic oligonucleotide 174aaaucguauu ugucaauca
1917519RNAArtificial Sequencesynthetic oligonucleotide
175aaaucguauu ugucaauca 1917621RNAArtificial Sequencesynthetic
oligonucleotide 176gucaucacac ugaauaccaa u 2117721RNAArtificial
Sequencesynthetic oligonucleotide 177gugaucagac ucaauacgaa u
2117813RNAArtificial Sequencesynthetic oligonucleotide
178cuguggaagu cua 1317913RNAArtificial Sequencesynthetic
oligonucleotide 179cuguggaagu cua 1318013RNAArtificial
Sequencesynthetic oligonucleotide 180cuguggaagu cua
1318113RNAArtificial Sequencesynthetic oligonucleotide
181cuguggaagu cua 1318213RNAArtificial Sequencesynthetic
oligonucleotide 182cuguggaagu cua 1318313RNAArtificial
Sequencesynthetic oligonucleotide 183cuguggaagu cua
1318413RNAArtificial Sequencesynthetic oligonucleotide
184cuguggaagu cua 1318513RNAArtificial Sequencesynthetic
oligonucleotide 185cuguggaagu cua 1318613RNAArtificial
Sequencesynthetic oligonucleotide 186cuguggaagu cua
1318713RNAArtificial Sequencesynthetic oligonucleotide
187cuguggaagu cua 1318821RNAArtificial Sequencesynthetic
oligonucleotide 188gucaucacac ugaauaccaa u 2118921RNAArtificial
Sequencesynthetic oligonucleotide 189gugaucagac ucaauacgaa u
2119013RNAArtificial Sequencesynthetic oligonucleotide
190cuguggaagu cua 1319113RNAArtificial Sequencesynthetic
oligonucleotide 191cuguggaagu cua 1319213RNAArtificial
Sequencesynthetic oligonucleotide 192cuguggaagu cua
1319313RNAArtificial Sequencesynthetic oligonucleotide
193cuguggaagu cua 1319413RNAArtificial Sequencesynthetic
oligonucleotide 194cuguggaagu cua 1319513RNAArtificial
Sequencesynthetic oligonucleotide 195cuguggaagu cua
1319627RNAArtificial Sequencesynthetic oligonucleotide
196ucauagguaa ccucugguug aaaguga 2719727RNAArtificial
Sequencesynthetic oligonucleotide 197cggcuacagg ugcuuaugaa gaaagua
2719821RNAArtificial Sequencesynthetic oligonucleotide
198gucaucacac ugaauaccaa u 2119921RNAArtificial Sequencesynthetic
oligonucleotide 199gugaucagac ucaauacgaa u 2120013RNAArtificial
Sequencesynthetic oligonucleotide 200cuguggaagu cua
1320121RNAArtificial Sequencesynthetic oligonucleotide
201gucaucacac ugaauaccaa u 2120221RNAArtificial Sequencesynthetic
oligonucleotide 202gugaucagac ucaauacgaa u 2120313RNAArtificial
Sequencesynthetic oligonucleotide 203uguaggaugu cua
1320413RNAArtificial Sequencesynthetic oligonucleotide
204cuguggaagu cua 1320513RNAArtificial Sequencesynthetic
oligonucleotide 205cuguggaagu cua 1320613RNAArtificial
Sequencesynthetic oligonucleotide 206acugaauacc aau
1320713RNAArtificial Sequencesynthetic oligonucleotide
207cuguggaagu cua 1320813RNAArtificial Sequencesynthetic
oligonucleotide 208cuguggaagu cua 1320913RNAArtificial
Sequencesynthetic oligonucleotide 209cuguggaagu cua
1321013RNAArtificial Sequencesynthetic oligonucleotide
210cuguggaagu cua 1321113RNAArtificial Sequencesynthetic
oligonucleotide 211cuguggaagu cua 1321213RNAArtificial
Sequencesynthetic oligonucleotide 212cuguggaagu cua
1321313RNAArtificial Sequencesynthetic oligonucleotide
213cuguggaagu cua 1321413RNAArtificial Sequencesynthetic
oligonucleotide 214cuguggaagu cua 1321513RNAArtificial
Sequencesynthetic oligonucleotide 215cuguggaagu cua
1321613RNAArtificial Sequencesynthetic oligonucleotide
216cuguggaagu cua 1321713RNAArtificial Sequencesynthetic
oligonucleotide 217cuguggaagu cua 1321813RNAArtificial
Sequencesynthetic oligonucleotide 218cuguggaagu cua
1321913RNAArtificial Sequencesynthetic oligonucleotide
219cuguggaagu cua 1322019RNAArtificial Sequencesynthetic
oligonucleotide 220agaguucugu ggaagucua 1922119RNAArtificial
Sequencesynthetic oligonucleotide 221agaguucugu ggaagucua
1922213RNAArtificial Sequencesynthetic oligonucleotide
222auuuggcuac aaa 1322313RNAArtificial Sequencesynthetic
oligonucleotide 223acaaauacga uuu 1322413RNAArtificial
Sequencesynthetic oligonucleotide 224acaaauacga uuu
1322513RNAArtificial Sequencesynthetic oligonucleotide
225cuguggaagu cua 1322613RNAArtificial Sequencesynthetic
oligonucleotide 226aaugaagaaa gua 1322713RNAArtificial
Sequencesynthetic oligonucleotide 227agguggaaau gaa
1322813RNAArtificial Sequencesynthetic oligonucleotide
228agaaaguaca aag 1322913RNAArtificial Sequencesynthetic
oligonucleotide 229gaaaguacaa aga 1323013RNAArtificial
Sequencesynthetic oligonucleotide 230augugacugc uga
1323113RNAArtificial Sequencesynthetic oligonucleotide
231agacuugggc aau 1323213RNAArtificial Sequencesynthetic
oligonucleotide 232auuucgagca gaa 1323313RNAArtificial
Sequencesynthetic oligonucleotide 233aucuggagaa aca
1323413RNAArtificial Sequencesynthetic oligonucleotide
234ucagaacaag aaa 1323513RNAArtificial Sequencesynthetic
oligonucleotide 235gacucaucug cua 1323613RNAArtificial
Sequencesynthetic oligonucleotide 236ggagaaacaa cau
1323713RNAArtificial Sequencesynthetic oligonucleotide
237agucccucaa aca 1323813RNAArtificial Sequencesynthetic
oligonucleotide 238ggcuacaaaa aca 1323915RNAArtificial
Sequencesynthetic oligonucleotide 239agaucuggag aaaca
1524015RNAArtificial Sequencesynthetic oligonucleotide
240uggacucauc ugcua 1524115RNAArtificial Sequencesynthetic
oligonucleotide 241cuggagaaac aacau 1524215RNAArtificial
Sequencesynthetic oligonucleotide 242agagucccuc aaaca
1524313RNAArtificial Sequencesynthetic oligonucleotide
243acugaauacc aau 1324415RNAArtificial Sequencesynthetic
oligonucleotide 244acacugaaua ccaau 1524513RNAArtificial
Sequencesynthetic oligonucleotide 245aaugaagaaa gua
1324613RNAArtificial Sequencesynthetic oligonucleotide
246agguggaaau gaa 1324713RNAArtificial Sequencesynthetic
oligonucleotide 247agaaaguaca aag 1324813RNAArtificial
Sequencesynthetic oligonucleotide 248gaaaguacaa aga
1324913RNAArtificial Sequencesynthetic oligonucleotide
249augugacugc uga 1325013RNAArtificial Sequencesynthetic
oligonucleotide 250agacuugggc aau 1325113RNAArtificial
Sequencesynthetic oligonucleotide 251auuucgagca gaa
1325213RNAArtificial Sequencesynthetic oligonucleotide
252acaaauacga uuu
1325313RNAArtificial Sequencesynthetic oligonucleotide
253acaaauacga uuu 1325419RNAArtificial Sequencesynthetic
oligonucleotide 254agaguucugu ggaagucua 1925513RNAArtificial
Sequencesynthetic oligonucleotide 255acaggaagau gua
1325613RNAArtificial Sequencesynthetic oligonucleotide
256gaguggagcg ccu 1325713RNAArtificial Sequencesynthetic
oligonucleotide 257cgacuggaag aca 1325813RNAArtificial
Sequencesynthetic oligonucleotide 258ggagcgccug uuc
1325913RNAArtificial Sequencesynthetic oligonucleotide
259gccauuacaa cug 1326013RNAArtificial Sequencesynthetic
oligonucleotide 260gagcuuucug gcu 1326113RNAArtificial
Sequencesynthetic oligonucleotide 261aguggagcgc cug
1326213RNAArtificial Sequencesynthetic oligonucleotide
262uggagcgccu guu 1326313RNAArtificial Sequencesynthetic
oligonucleotide 263guuugagcuu ucu 1326413RNAArtificial
Sequencesynthetic oligonucleotide 264ugccauuaca acu
1326513RNAArtificial Sequencesynthetic oligonucleotide
265acuggaagac acg 1326613RNAArtificial Sequencesynthetic
oligonucleotide 266aacugccugg ucc 1326713RNAArtificial
Sequencesynthetic oligonucleotide 267agaccugugc cug
1326813RNAArtificial Sequencesynthetic oligonucleotide
268cagaguggag cgc 1326913RNAArtificial Sequencesynthetic
oligonucleotide 269ccugguccag acc 1327013RNAArtificial
Sequencesynthetic oligonucleotide 270ccauuacaac ugu
1327113RNAArtificial Sequencesynthetic oligonucleotide
271cugccauuac aac 1327213RNAArtificial Sequencesynthetic
oligonucleotide 272auuacaacug ucc 1327313RNAArtificial
Sequencesynthetic oligonucleotide 273cauuacaacu guc
1327413RNAArtificial Sequencesynthetic oligonucleotide
274agaguggagc gcc 1327513RNAArtificial Sequencesynthetic
oligonucleotide 275accgacugga aga 1327613RNAArtificial
Sequencesynthetic oligonucleotide 276auguacggag aca
1327713RNAArtificial Sequencesynthetic oligonucleotide
277gccuugcgaa gcu 1327813RNAArtificial Sequencesynthetic
oligonucleotide 278gcugcgagga gug 1327913RNAArtificial
Sequencesynthetic oligonucleotide 279gccuaucaag uuu
1328013RNAArtificial Sequencesynthetic oligonucleotide
280aauucugugg agu 1328113RNAArtificial Sequencesynthetic
oligonucleotide 281uguacggaga cau 1328213RNAArtificial
Sequencesynthetic oligonucleotide 282agccuaucaa guu
1328313RNAArtificial Sequencesynthetic oligonucleotide
283caaguuugag cuu 1328413RNAArtificial Sequencesynthetic
oligonucleotide 284cuguggagua ugu 1328513RNAArtificial
Sequencesynthetic oligonucleotide 285aaauucugug gag
1328613RNAArtificial Sequencesynthetic oligonucleotide
286uuucaguagc aca 1328713RNAArtificial Sequencesynthetic
oligonucleotide 287caaugacauc uuu 1328813RNAArtificial
Sequencesynthetic oligonucleotide 288aguaccagug cac
1328913RNAArtificial Sequencesynthetic oligonucleotide
289ggaagacacg uuu 1329013RNAArtificial Sequencesynthetic
oligonucleotide 290cuaucaaguu uga 1329113RNAArtificial
Sequencesynthetic oligonucleotide 291agcuaaauuc ugu
1329213RNAArtificial Sequencesynthetic oligonucleotide
292agguagaaug uaa 1329313RNAArtificial Sequencesynthetic
oligonucleotide 293agcugaucag uuu 1329413RNAArtificial
Sequencesynthetic oligonucleotide 294uucugcucag aua
1329513RNAArtificial Sequencesynthetic oligonucleotide
295uuaucuaagu uaa 1329613RNAArtificial Sequencesynthetic
oligonucleotide 296uauacgagua aua 1329713RNAArtificial
Sequencesynthetic oligonucleotide 297gacuggacag cuu
1329813RNAArtificial Sequencesynthetic oligonucleotide
298auggccuuua uua 1329913RNAArtificial Sequencesynthetic
oligonucleotide 299auaccgagcu aaa 1330013RNAArtificial
Sequencesynthetic oligonucleotide 300uuguugagag ugu
1330113RNAArtificial Sequencesynthetic oligonucleotide
301acauaccgag cua 1330213RNAArtificial Sequencesynthetic
oligonucleotide 302agcagaaagg uua 1330313RNAArtificial
Sequencesynthetic oligonucleotide 303aguuguuccu uaa
1330413RNAArtificial Sequencesynthetic oligonucleotide
304auuugaagug uaa 1330513RNAArtificial Sequencesynthetic
oligonucleotide 305aagcugaccu gga 1330613RNAArtificial
Sequencesynthetic oligonucleotide 306ggucaugaag aag
1330713RNAArtificial Sequencesynthetic oligonucleotide
307auggucaggc cuu 1330813RNAArtificial Sequencesynthetic
oligonucleotide 308gaagacacgu uug 1330913RNAArtificial
Sequencesynthetic oligonucleotide 309aggccuugcg aag
1331013RNAArtificial Sequencesynthetic oligonucleotide
310uaccgacugg aag 1331113RNAArtificial Sequencesynthetic
oligonucleotide 311accgcaagau cgg 1331213RNAArtificial
Sequencesynthetic oligonucleotide 312caggccuugc gaa
1331313RNAArtificial Sequencesynthetic oligonucleotide
313cgagcuaaau ucu 1331413RNAArtificial Sequencesynthetic
oligonucleotide 314ucuguggagu aug 1331513RNAArtificial
Sequencesynthetic oligonucleotide 315cggagacaug gca
1331613RNAArtificial Sequencesynthetic oligonucleotide
316augacaacgc cuc 1331713RNAArtificial Sequencesynthetic
oligonucleotide 317gaggucauga aga 1331813RNAArtificial
Sequencesynthetic oligonucleotide 318uaaauucugu gga
1331913RNAArtificial Sequencesynthetic oligonucleotide
319uggaagacac guu 1332013RNAArtificial Sequencesynthetic
oligonucleotide 320aagauguacg gag 1332113RNAArtificial
Sequencesynthetic oligonucleotide 321aaugacaacg ccu
1332213RNAArtificial Sequencesynthetic oligonucleotide
322ggcgagguca uga 1332313RNAArtificial Sequencesynthetic
oligonucleotide 323gacacguuug gcc 1332413RNAArtificial
Sequencesynthetic oligonucleotide 324acggagacau ggc
1332513RNAArtificial Sequencesynthetic oligonucleotide
325ucaggccuug cga 1332613RNAArtificial Sequencesynthetic
oligonucleotide 326gcgaagcuga ccu 1332713RNAArtificial
Sequencesynthetic oligonucleotide 327ggaagaugua cgg
1332813RNAArtificial Sequencesynthetic oligonucleotide
328gugacuucgg cuc 1332913RNAArtificial Sequencesynthetic
oligonucleotide 329ugacuucggc ucc 1333013RNAArtificial
Sequencesynthetic oligonucleotide 330uggucaggcc uug
1333113RNAArtificial Sequencesynthetic oligonucleotide
331ucaaguuuga gcu 1333213RNAArtificial Sequencesynthetic
oligonucleotide 332gccagaacug cag 1333313RNAArtificial
Sequencesynthetic oligonucleotide 333uggaguaugu acc
1333413RNAArtificial Sequencesynthetic oligonucleotide
334gcuagagaag cag 1333513RNAArtificial Sequencesynthetic
oligonucleotide 335ggucaggccu ugc 1333613RNAArtificial
Sequencesynthetic oligonucleotide 336gagcuaaauu cug
1333713RNAArtificial Sequencesynthetic oligonucleotide
337aagacacguu ugg 1333813RNAArtificial Sequencesynthetic
oligonucleotide 338cgaggucaug aag 1333913RNAArtificial
Sequencesynthetic oligonucleotide 339ggccuugcga agc
1334013RNAArtificial Sequencesynthetic oligonucleotide
340cuugcgaagc uga 1334113RNAArtificial Sequencesynthetic
oligonucleotide 341ccgacuggaa gac 1334213RNAArtificial
Sequencesynthetic oligonucleotide 342ccuaucaagu uug
1334313RNAArtificial Sequencesynthetic oligonucleotide
343uguuccaaga ccu 1334413RNAArtificial Sequencesynthetic
oligonucleotide 344cgaagcugac cug 1334513RNAArtificial
Sequencesynthetic oligonucleotide 345uugcgaagcu gac
1334613RNAArtificial Sequencesynthetic oligonucleotide
346caaugacaac gcc 1334713RNAArtificial Sequencesynthetic
oligonucleotide 347guaccagugc acg 1334813RNAArtificial
Sequencesynthetic oligonucleotide 348ccuguuccaa gac
1334913RNAArtificial Sequencesynthetic oligonucleotide
349uacggagaca ugg 1335013RNAArtificial Sequencesynthetic
oligonucleotide 350ugcgaagcug acc 1335113RNAArtificial
Sequencesynthetic oligonucleotide 351ccuugcgaag cug
1335213RNAArtificial Sequencesynthetic oligonucleotide
352cugugacuuc ggc 1335313RNAArtificial Sequencesynthetic
oligonucleotide 353gcuaaauucu gug 1335413RNAArtificial
Sequencesynthetic oligonucleotide 354cuaaauucug ugg
1335513RNAArtificial Sequencesynthetic oligonucleotide
355agacacguuu ggc 1335613RNAArtificial Sequencesynthetic
oligonucleotide 356ccgcaagauc ggc 1335713RNAArtificial
Sequencesynthetic oligonucleotide 357uaucaaguuu gag
1335813RNAArtificial Sequencesynthetic oligonucleotide
358gaagcugacc ugg 1335913RNAArtificial Sequencesynthetic
oligonucleotide 359cucaugaauu aga 1336013RNAArtificial
Sequencesynthetic oligonucleotide 360cugaggucaa uua
1336113RNAArtificial Sequencesynthetic oligonucleotide
361gaggucaauu aaa 1336213RNAArtificial Sequencesynthetic
oligonucleotide 362ucugagguca auu 1336313RNAArtificial
Sequencesynthetic oligonucleotide 363ugaggucaau uaa
1336413RNAArtificial Sequencesynthetic oligonucleotide
364uucugagguc aau 1336513RNAArtificial Sequencesynthetic
oligonucleotide 365gucagcugga uga 1336613RNAArtificial
Sequencesynthetic oligonucleotide 366uucugaugaa ucu
1336713RNAArtificial Sequencesynthetic oligonucleotide
367uggacugagg uca 1336813RNAArtificial Sequencesynthetic
oligonucleotide 368gagucucacc auu 1336913RNAArtificial
Sequencesynthetic oligonucleotide 369gacugagguc aaa
1337013RNAArtificial Sequencesynthetic oligonucleotide
370ucacagccau gaa 1337113RNAArtificial Sequencesynthetic
oligonucleotide 371agucucacca uuc 1337213RNAArtificial
Sequencesynthetic oligonucleotide 372aagcggaaag cca
1337313RNAArtificial Sequencesynthetic oligonucleotide
373agcggaaagc caa 1337413RNAArtificial Sequencesynthetic
oligonucleotide 374accacaugga uga 1337513RNAArtificial
Sequencesynthetic oligonucleotide 375gccaugacca cau
1337613RNAArtificial Sequencesynthetic oligonucleotide
376aagccaugac cac 1337713RNAArtificial Sequencesynthetic
oligonucleotide 377gcggaaagcc aau
1337813RNAArtificial Sequencesynthetic oligonucleotide
378aaauuucgua uuu 1337913RNAArtificial Sequencesynthetic
oligonucleotide 379auuucguauu ucu 1338013RNAArtificial
Sequencesynthetic oligonucleotide 380aaagccauga cca
1338113RNAArtificial Sequencesynthetic oligonucleotide
381acauggauga uau 1338213RNAArtificial Sequencesynthetic
oligonucleotide 382gaaauuucgu auu 1338313RNAArtificial
Sequencesynthetic oligonucleotide 383gcgccuucug auu
1338413RNAArtificial Sequencesynthetic oligonucleotide
384auuucucaug aau 1338513RNAArtificial Sequencesynthetic
oligonucleotide 385cucucaugaa uag 1338613RNAArtificial
Sequencesynthetic oligonucleotide 386aaguccaacg aaa
1338713RNAArtificial Sequencesynthetic oligonucleotide
387augaugagag caa 1338813RNAArtificial Sequencesynthetic
oligonucleotide 388gcgaggaguu gaa 1338913RNAArtificial
Sequencesynthetic oligonucleotide 389ugauugauag uca
1339013RNAArtificial Sequencesynthetic oligonucleotide
390agauagugca ucu 1339113RNAArtificial Sequencesynthetic
oligonucleotide 391auguguaucu auu 1339213RNAArtificial
Sequencesynthetic oligonucleotide 392uucuauagaa gaa
1339313RNAArtificial Sequencesynthetic oligonucleotide
393uuguccagca auu 1339413RNAArtificial Sequencesynthetic
oligonucleotide 394acauggaaag cga 1339513RNAArtificial
Sequencesynthetic oligonucleotide 395gcaguccaga uua
1339613RNAArtificial Sequencesynthetic oligonucleotide
396ugguugaaug ugu 1339713RNAArtificial Sequencesynthetic
oligonucleotide 397uuaugaaacg agu 1339813RNAArtificial
Sequencesynthetic oligonucleotide 398caguccagau uau
1339913RNAArtificial Sequencesynthetic oligonucleotide
399auauaagcgg aaa 1340013RNAArtificial Sequencesynthetic
oligonucleotide 400uaccaguuaa aca 1340113RNAArtificial
Sequencesynthetic oligonucleotide 401uguucauucu aua
1340213RNAArtificial Sequencesynthetic oligonucleotide
402ccgaccaagg aaa 1340313RNAArtificial Sequencesynthetic
oligonucleotide 403gaauggugca uac 1340413RNAArtificial
Sequencesynthetic oligonucleotide 404auaugauggc cga
1340513RNAArtificial Sequencesynthetic oligonucleotide
405agcaguccag auu 1340613RNAArtificial Sequencesynthetic
oligonucleotide 406gcauuuaguc aaa 1340713RNAArtificial
Sequencesynthetic oligonucleotide 407agcauuccga ugu
1340813RNAArtificial Sequencesynthetic oligonucleotide
408uagucaggaa cuu 1340913RNAArtificial Sequencesynthetic
oligonucleotide 409ugcauuuagu caa 1341013RNAArtificial
Sequencesynthetic oligonucleotide 410gucugaugag ucu
1341113RNAArtificial Sequencesynthetic oligonucleotide
411uagacacaua uga 1341213RNAArtificial Sequencesynthetic
oligonucleotide 412cagacgagga cau 1341313RNAArtificial
Sequencesynthetic oligonucleotide 413cagccgugaa uuc
1341413RNAArtificial Sequencesynthetic oligonucleotide
414agucuggaaa uaa 1341513RNAArtificial Sequencesynthetic
oligonucleotide 415aguuuguggc uuc 1341613RNAArtificial
Sequencesynthetic oligonucleotide 416aguccaacga aag
1341713RNAArtificial Sequencesynthetic oligonucleotide
417aaguuucgca gac 1341813RNAArtificial Sequencesynthetic
oligonucleotide 418agcaaugagc auu 1341913RNAArtificial
Sequencesynthetic oligonucleotide 419uuagauagug cau
1342013RNAArtificial Sequencesynthetic oligonucleotide
420uggugcauac aag 1342113RNAArtificial Sequencesynthetic
oligonucleotide 421augaaacgag uca 1342213RNAArtificial
Sequencesynthetic oligonucleotide 422ccagagugcu gaa
1342313RNAArtificial Sequencesynthetic oligonucleotide
423cagccaugaa uuu 1342413RNAArtificial Sequencesynthetic
oligonucleotide 424auugguugaa ugu 1342513RNAArtificial
Sequencesynthetic oligonucleotide 425gguugaaugu gua
1342613RNAArtificial Sequencesynthetic oligonucleotide
426ggaaauaacu aau 1342713RNAArtificial Sequencesynthetic
oligonucleotide 427ucaugaauag aaa 1342813RNAArtificial
Sequencesynthetic oligonucleotide 428gccagcaacc gaa
1342913RNAArtificial Sequencesynthetic oligonucleotide
429caccucacac aug 1343013RNAArtificial Sequencesynthetic
oligonucleotide 430aguugaaugg ugc 1343113RNAArtificial
Sequencesynthetic oligonucleotide 431agucagcugg aug
1343213RNAArtificial Sequencesynthetic oligonucleotide
432uauaagcgga aag 1343313RNAArtificial Sequencesynthetic
oligonucleotide 433uuccgaugug auu 1343413RNAArtificial
Sequencesynthetic oligonucleotide 434auaacuaaug ugu
1343513RNAArtificial Sequencesynthetic oligonucleotide
435ucauucuaua gaa 1343613RNAArtificial Sequencesynthetic
oligonucleotide 436aacuaucacu gua 1343713RNAArtificial
Sequencesynthetic oligonucleotide 437gucaauugcu uau
1343813RNAArtificial Sequencesynthetic oligonucleotide
438agcaauuaau aaa 1343913RNAArtificial Sequencesynthetic
oligonucleotide 439acgacucuga uga 1344013RNAArtificial
Sequencesynthetic oligonucleotide 440uagugugguu uau
1344113RNAArtificial Sequencesynthetic oligonucleotide
441aagccaauga uga 1344213RNAArtificial Sequencesynthetic
oligonucleotide 442auagucagga acu 1344313RNAArtificial
Sequencesynthetic oligonucleotide 443agucagccgu gaa
1344413RNAArtificial Sequencesynthetic oligonucleotide
444acuaccauga gaa 1344513RNAArtificial Sequencesynthetic
oligonucleotide 445aaacaggcug auu 1344613RNAArtificial
Sequencesynthetic oligonucleotide 446gagugcugaa acc
1344713RNAArtificial Sequencesynthetic oligonucleotide
447ugagcauucc gau 1344813RNAArtificial Sequencesynthetic
oligonucleotide 448aauuccacag cca 1344913RNAArtificial
Sequencesynthetic oligonucleotide 449ugucaauugc uua
1345013RNAArtificial Sequencesynthetic oligonucleotide
450accaugagaa uug 1345113RNAArtificial Sequencesynthetic
oligonucleotide 451ccaacgaaag cca 1345213RNAArtificial
Sequencesynthetic oligonucleotide 452cuggucacug auu
1345313RNAArtificial Sequencesynthetic oligonucleotide
453ugguuuaugg acu 1345413RNAArtificial Sequencesynthetic
oligonucleotide 454gaccagagug cug 1345513RNAArtificial
Sequencesynthetic oligonucleotide 455gaugugauug aua
1345613RNAArtificial Sequencesynthetic oligonucleotide
456gucagccgug aau 1345713RNAArtificial Sequencesynthetic
oligonucleotide 457aauguguauc uau 1345813RNAArtificial
Sequencesynthetic oligonucleotide 458uugagucugg aaa
1345913RNAArtificial Sequencesynthetic oligonucleotide
459guccagcaau uaa 1346013RNAArtificial Sequencesynthetic
oligonucleotide 460ccagcaauua aua 1346113RNAArtificial
Sequencesynthetic oligonucleotide 461gacucgaacg acu
1346213RNAArtificial Sequencesynthetic oligonucleotide
462accugccagc aac 1346313DNAArtificial Sequencesynthetic
oligonucleotide 463actgaauacc aau 1346413RNAArtificial
Sequencesynthetic oligonucleotide 464acugaauacc aau
1346513RNAArtificial Sequencesynthetic oligonucleotide
465cuguggaagu cua 1346613RNAArtificial Sequencesynthetic
oligonucleotide 466ggcuacaaaa aca 1346715RNAArtificial
Sequencesynthetic oligonucleotide 467uuggcuacaa aaaca
1546817RNAArtificial Sequencesynthetic oligonucleotide
468auuuggcuac aaaaaca 1746913RNAArtificial Sequencesynthetic
oligonucleotide 469uguaggaugu cua 1347013RNAArtificial
Sequencesynthetic oligonucleotide 470aucuggagaa aca
1347115RNAArtificial Sequencesynthetic oligonucleotide
471agaucuggag aaaca 1547213RNAArtificial Sequencesynthetic
oligonucleotide 472gacucaucug cua 1347313RNAArtificial
Sequencesynthetic oligonucleotide 473gacucaucug cua
1347415RNAArtificial Sequencesynthetic oligonucleotide
474uggacucauc ugcua 1547515RNAArtificial Sequencesynthetic
oligonucleotide 475uggacucauc ugcua 1547613RNAArtificial
Sequencesynthetic oligonucleotide 476ggagaaacaa cau
1347715RNAArtificial Sequencesynthetic oligonucleotide
477cuggagaaac aacau 1547813RNAArtificial Sequencesynthetic
oligonucleotide 478agucccucaa aca 1347915RNAArtificial
Sequencesynthetic oligonucleotide 479agagucccuc aaaca
1548013RNAArtificial Sequencesynthetic oligonucleotide
480acugaauacc aau 1348113RNAArtificial Sequencesynthetic
oligonucleotide 481acugaauacc aau 1348215RNAArtificial
Sequencesynthetic oligonucleotide 482acacugaaua ccaau
1548315RNAArtificial Sequencesynthetic oligonucleotide
483acacugaaua ccaau 1548413RNAArtificial Sequencesynthetic
oligonucleotide 484cuguggaagu cua 1348513RNAArtificial
Sequencesynthetic oligonucleotide 485acaggaagau gua
1348613RNAArtificial Sequencesynthetic oligonucleotide
486gaguggagcg ccu 1348713RNAArtificial Sequencesynthetic
oligonucleotide 487cgacuggaag aca 1348813RNAArtificial
Sequencesynthetic oligonucleotide 488ggagcgccug uuc
1348913RNAArtificial Sequencesynthetic oligonucleotide
489gccauuacaa cug 1349013RNAArtificial Sequencesynthetic
oligonucleotide 490gagcuuucug gcu 1349113RNAArtificial
Sequencesynthetic oligonucleotide 491aguggagcgc cug
1349213RNAArtificial Sequencesynthetic oligonucleotide
492uggagcgccu guu 1349313RNAArtificial Sequencesynthetic
oligonucleotide 493guuugagcuu ucu 1349413RNAArtificial
Sequencesynthetic oligonucleotide 494ugccauuaca acu
1349513RNAArtificial Sequencesynthetic oligonucleotide
495acuggaagac acg 1349613RNAArtificial Sequencesynthetic
oligonucleotide 496aacugccugg ucc 1349713RNAArtificial
Sequencesynthetic oligonucleotide 497agaccugugc cug
1349813RNAArtificial Sequencesynthetic oligonucleotide
498cagaguggag cgc 1349913RNAArtificial Sequencesynthetic
oligonucleotide 499ccugguccag acc 1350013RNAArtificial
Sequencesynthetic oligonucleotide 500ccauuacaac ugu
1350113RNAArtificial Sequencesynthetic oligonucleotide
501cugccauuac aac 1350213RNAArtificial Sequencesynthetic
oligonucleotide 502auuacaacug ucc 1350313RNAArtificial
Sequencesynthetic oligonucleotide 503cauuacaacu guc
1350413RNAArtificial Sequencesynthetic oligonucleotide
504agaguggagc gcc 1350513RNAArtificial Sequencesynthetic
oligonucleotide 505accgacugga aga 1350613RNAArtificial
Sequencesynthetic oligonucleotide 506auguacggag aca
1350713RNAArtificial Sequencesynthetic oligonucleotide
507gccuugcgaa gcu 1350813RNAArtificial Sequencesynthetic
oligonucleotide 508gcugcgagga gug 1350913RNAArtificial
Sequencesynthetic oligonucleotide 509gccuaucaag uuu
1351013RNAArtificial Sequencesynthetic oligonucleotide
510aauucugugg agu 1351113RNAArtificial Sequencesynthetic
oligonucleotide 511uguacggaga cau 1351213RNAArtificial
Sequencesynthetic oligonucleotide 512agccuaucaa guu
1351313RNAArtificial Sequencesynthetic oligonucleotide
513caaguuugag cuu 1351413RNAArtificial Sequencesynthetic
oligonucleotide 514cuguggagua ugu 1351513RNAArtificial
Sequencesynthetic oligonucleotide 515aaauucugug gag
1351613RNAArtificial Sequencesynthetic oligonucleotide
516uuucaguagc aca 1351713RNAArtificial Sequencesynthetic
oligonucleotide 517caaugacauc uuu 1351813RNAArtificial
Sequencesynthetic oligonucleotide 518aguaccagug cac
1351913RNAArtificial Sequencesynthetic oligonucleotide
519ggaagacacg uuu 1352013RNAArtificial Sequencesynthetic
oligonucleotide 520cuaucaaguu uga 1352113RNAArtificial
Sequencesynthetic oligonucleotide 521agcuaaauuc ugu
1352213RNAArtificial Sequencesynthetic oligonucleotide
522agguagaaug uaa 1352313RNAArtificial Sequencesynthetic
oligonucleotide 523agcugaucag uuu 1352413RNAArtificial
Sequencesynthetic oligonucleotide 524uucugcucag aua
1352513RNAArtificial Sequencesynthetic oligonucleotide
525uuaucuaagu uaa 1352613RNAArtificial Sequencesynthetic
oligonucleotide 526uauacgagua aua 1352713RNAArtificial
Sequencesynthetic oligonucleotide 527gacuggacag cuu
1352813RNAArtificial Sequencesynthetic oligonucleotide
528auggccuuua uua 1352913RNAArtificial Sequencesynthetic
oligonucleotide 529auaccgagcu aaa 1353013RNAArtificial
Sequencesynthetic oligonucleotide 530uuguugagag ugu
1353113RNAArtificial Sequencesynthetic oligonucleotide
531acauaccgag cua 1353213RNAArtificial Sequencesynthetic
oligonucleotide 532agcagaaagg uua 1353313RNAArtificial
Sequencesynthetic oligonucleotide 533aguuguuccu uaa
1353413RNAArtificial Sequencesynthetic oligonucleotide
534auuugaagug uaa 1353513RNAArtificial Sequencesynthetic
oligonucleotide 535aagcugaccu gga 1353613RNAArtificial
Sequencesynthetic oligonucleotide 536ggucaugaag aag
1353713RNAArtificial Sequencesynthetic oligonucleotide
537auggucaggc cuu 1353813RNAArtificial Sequencesynthetic
oligonucleotide 538gaagacacgu uug 1353913RNAArtificial
Sequencesynthetic oligonucleotide 539aggccuugcg aag
1354013RNAArtificial Sequencesynthetic oligonucleotide
540uaccgacugg aag 1354113RNAArtificial Sequencesynthetic
oligonucleotide 541accgcaagau cgg 1354213RNAArtificial
Sequencesynthetic oligonucleotide 542caggccuugc gaa
1354313RNAArtificial Sequencesynthetic oligonucleotide
543cgagcuaaau ucu 1354413RNAArtificial Sequencesynthetic
oligonucleotide 544ucuguggagu aug 1354513RNAArtificial
Sequencesynthetic oligonucleotide 545cggagacaug gca
1354613RNAArtificial Sequencesynthetic oligonucleotide
546augacaacgc cuc 1354713RNAArtificial Sequencesynthetic
oligonucleotide 547gaggucauga aga 1354813RNAArtificial
Sequencesynthetic oligonucleotide 548uaaauucugu gga
1354913RNAArtificial Sequencesynthetic oligonucleotide
549uggaagacac guu 1355013RNAArtificial Sequencesynthetic
oligonucleotide 550aagauguacg gag 1355113RNAArtificial
Sequencesynthetic oligonucleotide 551aaugacaacg ccu
1355213RNAArtificial Sequencesynthetic oligonucleotide
552ggcgagguca uga 1355313RNAArtificial Sequencesynthetic
oligonucleotide 553gacacguuug gcc 1355413RNAArtificial
Sequencesynthetic oligonucleotide 554acggagacau ggc
1355513RNAArtificial Sequencesynthetic oligonucleotide
555ucaggccuug cga 1355613RNAArtificial Sequencesynthetic
oligonucleotide 556gcgaagcuga ccu 1355713RNAArtificial
Sequencesynthetic oligonucleotide 557ggaagaugua cgg
1355813RNAArtificial Sequencesynthetic oligonucleotide
558gugacuucgg cuc 1355913RNAArtificial Sequencesynthetic
oligonucleotide 559ugacuucggc ucc 1356013RNAArtificial
Sequencesynthetic oligonucleotide 560uggucaggcc uug
1356113RNAArtificial Sequencesynthetic oligonucleotide
561ucaaguuuga gcu 1356213RNAArtificial Sequencesynthetic
oligonucleotide 562gccagaacug cag 1356313RNAArtificial
Sequencesynthetic oligonucleotide 563uggaguaugu acc
1356413RNAArtificial Sequencesynthetic oligonucleotide
564gcuagagaag cag 1356513RNAArtificial Sequencesynthetic
oligonucleotide 565ggucaggccu ugc 1356613RNAArtificial
Sequencesynthetic oligonucleotide 566gagcuaaauu cug
1356713RNAArtificial Sequencesynthetic oligonucleotide
567aagacacguu ugg 1356813RNAArtificial Sequencesynthetic
oligonucleotide 568cgaggucaug aag 1356913RNAArtificial
Sequencesynthetic oligonucleotide 569ggccuugcga agc
1357013RNAArtificial Sequencesynthetic oligonucleotide
570cuugcgaagc uga 1357113RNAArtificial Sequencesynthetic
oligonucleotide 571ccgacuggaa gac 1357213RNAArtificial
Sequencesynthetic oligonucleotide 572ccuaucaagu uug
1357313RNAArtificial Sequencesynthetic oligonucleotide
573uguuccaaga ccu 1357413RNAArtificial Sequencesynthetic
oligonucleotide 574cgaagcugac cug 1357513RNAArtificial
Sequencesynthetic oligonucleotide 575uugcgaagcu gac
1357613RNAArtificial Sequencesynthetic oligonucleotide
576caaugacaac gcc 1357713RNAArtificial Sequencesynthetic
oligonucleotide 577guaccagugc acg 1357813RNAArtificial
Sequencesynthetic oligonucleotide 578ccuguuccaa gac
1357913RNAArtificial Sequencesynthetic oligonucleotide
579uacggagaca ugg 1358013RNAArtificial Sequencesynthetic
oligonucleotide 580ugcgaagcug acc 1358113RNAArtificial
Sequencesynthetic oligonucleotide 581ccuugcgaag cug
1358213RNAArtificial Sequencesynthetic oligonucleotide
582cugugacuuc ggc 1358313RNAArtificial Sequencesynthetic
oligonucleotide 583gcuaaauucu gug 1358413RNAArtificial
Sequencesynthetic oligonucleotide 584cuaaauucug ugg
1358513RNAArtificial Sequencesynthetic oligonucleotide
585agacacguuu ggc 1358613RNAArtificial Sequencesynthetic
oligonucleotide 586ccgcaagauc ggc 1358713RNAArtificial
Sequencesynthetic oligonucleotide 587uaucaaguuu gag
1358813RNAArtificial Sequencesynthetic oligonucleotide
588gaagcugacc ugg 1358913RNAArtificial Sequencesynthetic
oligonucleotide 589gcauuuaguc aaa 1359013RNAArtificial
Sequencesynthetic oligonucleotide 590ggcuacaaaa aca
1359115RNAArtificial Sequencesynthetic oligonucleotide
591uuggcuacaa aaaca 1559217RNAArtificial Sequencesynthetic
oligonucleotide 592auuuggcuac aaaaaca 1759313RNAArtificial
Sequencesynthetic oligonucleotide 593acaaauacga uuu
1359413RNAArtificial Sequencesynthetic oligonucleotide
594acaaauacga uuu 1359525RNAArtificial Sequencesynthetic
oligonucleotide 595cuuugaagag uucuguggaa gucua 2559623RNAArtificial
Sequencesynthetic oligonucleotide 596acaaacacca uugucacacu cca
2359719RNAArtificial sequencesynthetic oligonucleotide
597uagacuucca cagaacucu 1959813RNAArtificial sequencesynthetic
oligonucleotide 598cuguggaagu cua 1359932RNAArtificial
sequencesynthetic oligonuclotide 599uagacuucca cagaacucug
acaccuucag au 3260013RNAArtificial sequencesynthetic
oligonucleotide 600uagacuucca cag 1360113RNAArtificial
sequencesynthetic oligonucleotide 601cuguggaagu cua
1360231RNAArtificial sequencesynthetic oligonucleotide
602uagacuucca cagaacucuu guggaagucu a 3160331RNAArtificial
sequencesynthetic oligonucleotide 603uagacuucca cagaacucuu
guggaagucu a 3160425RNAArtificial sequencesynthetic oligonucleotide
604cuuugaagag uucuguggaa gucua 2560525RNAArtificial
sequencesynthetic oligonucleotide 605uagacuucca cagaacucuu caaag
2560631RNAArtificial sequencesynthetic oligonucleotide
606uagacuucca cagaacuucu guggaagucu a 3160731RNAArtificial
sequencesynthetic oligonucleotide 607uagacuucca cagaacuucu
guggaagucu a 3160813RNAArtificial sequencesynthetoc oligonucleotide
608uacuuucuuc auu 1360913RNAArtificial sequencesynthetic
oligonucleotide 609aaugaagaaa gua 1361032RNAArtificial
sequencesynthetic oligonucleotide 610uacuuucuuc auuuccacca
augaagaaag ua 3261132RNAArtificial sequencesynthetic
oligonucleotide 611uacuuucuuc auuuccacca augaagaaag ua
3261219RNAArtificial sequencesynthetic oligonucleotide
612uacuuucuuc auuuccacc 1961313RNAArtificial sequencesynthetic
oligonucleotide 613aaugaagaaa gua 1361425RNAArtificial
sequencesynthetic oligonucleotide 614uacuuucuuc auuuccaccu uugcc
2561525RNAArtificial sequencesynthetic oligonucleotide
615ggcaaaggug gaaaugaaga aagua 2561619RNAArtificial
Sequencesynthetic oligonucleotide 616ucuaauucau gagaaauac
1961719RNAArtificial Sequencesynthetic oligonucleotide
617uaauugaccu cagaagaug 1961819RNAArtificial Sequencesynthetic
oligonucleotide 618uuuaauugac cucagaaga 1961919RNAArtificial
Sequencesynthetic oligonucleotide 619aauugaccuc agaagaugc
1962019RNAArtificial Sequencesynthetic oligonucleotide
620uuaauugacc ucagaagau 1962119RNAArtificial Sequencesynthetic
oligonucleotide 621auugaccuca gaagaugca 1962219RNAArtificial
Sequencesynthetic oligonucleotide 622ucauccagcu gacucguuu
1962319RNAArtificial Sequencesynthetic oligonucleotide
623agauucauca gaaugguga 1962419RNAArtificial Sequencesynthetic
oligonucleotide 624ugaccucagu ccauaaacc 1962519RNAArtificial
Sequencesynthetic oligonucleotide 625aauggugaga cucaucaga
1962619RNAArtificial Sequencesynthetic oligonucleotide
626uuugaccuca guccauaaa 1962719RNAArtificial Sequencesynthetic
oligonucleotide 627uucauggcug ugaaauuca 1962819RNAArtificial
Sequencesynthetic oligonucleotide 628gaauggugag acucaucag
1962919RNAArtificial Sequencesynthetic oligonucleotide
629uggcuuuccg cuuauauaa 1963019RNAArtificial Sequencesynthetic
oligonucleotide 630uuggcuuucc gcuuauaua 1963119RNAArtificial
Sequencesynthetic oligonucleotide 631ucauccaugu ggucauggc
1963219RNAArtificial Sequencesynthetic oligonucleotide
632auguggucau ggcuuucgu 1963319RNAArtificial Sequencesynthetic
oligonucleotide 633guggucaugg cuuucguug 1963419RNAArtificial
Sequencesynthetic oligonucleotide 634auuggcuuuc cgcuuauau
1963519RNAArtificial Sequencesynthetic oligonucleotide
635aaauacgaaa uuucaggug 1963619RNAArtificial Sequencesynthetic
oligonucleotide 636agaaauacga aauuucagg 1963719RNAArtificial
Sequencesynthetic oligonucleotide 637uggucauggc uuucguugg
1963819RNAArtificial Sequencesynthetic oligonucleotide
638auaucaucca uguggucau 1963919RNAArtificial Sequencesynthetic
oligonucleotide 639aauacgaaau uucaggugu 1964019RNAArtificial
Sequencesynthetic oligonucleotide 640aaucagaagg cgcguucag
1964119RNAArtificial Sequencesynthetic oligonucleotide
641auucaugaga aauacgaaa 1964219RNAArtificial Sequencesynthetic
oligonucleotide 642cuauucauga gagaauaac 1964319RNAArtificial
Sequencesynthetic oligonucleotide 643uuucguugga cuuacuugg
1964419RNAArtificial Sequencesynthetic oligonucleotide
644uugcucucau cauuggcuu 1964519RNAArtificial Sequencesynthetic
oligonucleotide 645uucaacuccu cgcuuucca 1964619RNAArtificial
Sequencesynthetic oligonucleotide 646ugacuaucaa ucacaucgg
1964719RNAArtificial Sequencesynthetic oligonucleotide
647agaugcacua ucuaauuca 1964819RNAArtificial Sequencesynthetic
oligonucleotide 648aauagauaca cauucaacc 1964919RNAArtificial
Sequencesynthetic oligonucleotide 649uucuucuaua gaaugaaca
1965019RNAArtificial Sequencesynthetic oligonucleotide
650aauugcugga caaccgugg 1965119RNAArtificial Sequencesynthetic
oligonucleotide 651ucgcuuucca ugugugagg 1965219RNAArtificial
Sequencesynthetic oligonucleotide 652uaaucuggac ugcuugugg
1965319RNAArtificial Sequencesynthetic oligonucleotide
653acacauucaa ccaauaaac 1965419RNAArtificial Sequencesynthetic
oligonucleotide 654acucguuuca uaacugucc 1965519RNAArtificial
Sequencesynthetic oligonucleotide 655auaaucugga cugcuugug
1965619RNAArtificial Sequencesynthetic oligonucleotide
656uuuccgcuua uauaaucug 1965719RNAArtificial Sequencesynthetic
oligonucleotide 657uguuuaacug guauggcac 1965819RNAArtificial
Sequencesynthetic oligonucleotide 658uauagaauga acauagaca
1965919RNAArtificial Sequencesynthetic oligonucleotide
659uuuccuuggu cggcguuug 1966019RNAArtificial Sequencesynthetic
oligonucleotide 660guaugcacca uucaacucc 1966119RNAArtificial
Sequencesynthetic oligonucleotide 661ucggccauca uaugugucu
1966219RNAArtificial Sequencesynthetic oligonucleotide
662aaucuggacu gcuuguggc 1966319RNAArtificial Sequencesynthetic
oligonucleotide 663acaucggaau gcucauugc 1966419RNAArtificial
Sequencesynthetic oligonucleotide 664aaguuccuga cuaucaauc
1966519RNAArtificial Sequencesynthetic oligonucleotide
665uugacuaaau gcaaaguga 1966619RNAArtificial Sequencesynthetic
oligonucleotide 666agacucauca gacugguga 1966719RNAArtificial
Sequencesynthetic oligonucleotide 667ucauaugugu cuacugugg
1966819RNAArtificial Sequencesynthetic oligonucleotide
668auguccucgu cuguagcau 1966919RNAArtificial Sequencesynthetic
oligonucleotide 669gaauucacgg cugacuuug 1967019RNAArtificial
Sequencesynthetic oligonucleotide 670uuauuuccag acucaaaua
1967119RNAArtificial Sequencesynthetic oligonucleotide
671gaagccacaa acuaaacua 1967219RNAArtificial Sequencesynthetic
oligonucleotide 672cuuucguugg acuuacuug 1967319RNAArtificial
Sequencesynthetic oligonucleotide 673gucugcgaaa cuucuuaga
1967419RNAArtificial Sequencesynthetic oligonucleotide
674aaugcucauu gcucucauc 1967519RNAArtificial Sequencesynthetic
oligonucleotide 675augcacuauc uaauucaug 1967619RNAArtificial
Sequencesynthetic oligonucleotide 676cuuguaugca ccauucaac
1967719RNAArtificial Sequencesynthetic oligonucleotide
677ugacucguuu cauaacugu 1967819RNAArtificial Sequencesynthetic
oligonucleotide 678uucagcacuc uggucaucc 1967919RNAArtificial
Sequencesynthetic oligonucleotide 679aaauucaugg cuguggaau
1968019RNAArtificial Sequencesynthetic oligonucleotide
680acauucaacc aauaaacug 1968119RNAArtificial Sequencesynthetic
oligonucleotide 681uacacauuca accaauaaa 1968219RNAArtificial
Sequencesynthetic oligonucleotide 682auuaguuauu uccagacuc
1968319RNAArtificial Sequencesynthetic oligonucleotide
683uuucuauuca ugagagaau 1968419RNAArtificial Sequencesynthetic
oligonucleotide 684uucgguugcu ggcaggucc 1968519RNAArtificial
Sequencesynthetic oligonucleotide 685caugugugag gugaugucc
1968619RNAArtificial Sequencesynthetic oligonucleotide
686gcaccauuca acuccucgc 1968719RNAArtificial Sequencesynthetic
oligonucleotide 687cauccagcug acucguuuc 1968819RNAArtificial
Sequencesynthetic oligonucleotide 688cuuuccgcuu auauaaucu
1968919RNAArtificial Sequencesynthetic oligonucleotide
689aaucacaucg gaaugcuca 1969019RNAArtificial Sequencesynthetic
oligonucleotide 690acacauuagu uauuuccag 1969119RNAArtificial
Sequencesynthetic oligonucleotide 691uucuauagaa ugaacauag
1969219RNAArtificial Sequencesynthetic oligonucleotide
692uacagugaua guuugcauu 1969319RNAArtificial Sequencesynthetic
oligonucleotide 693auaagcaauu gacaccacc 1969419RNAArtificial
Sequencesynthetic oligonucleotide 694uuuauuaauu gcuggacaa
1969519RNAArtificial Sequencesynthetic oligonucleotide
695ucaucagagu cguucgagu 1969619RNAArtificial Sequencesynthetic
oligonucleotide 696auaaaccaca cuaucaccu 1969719RNAArtificial
Sequencesynthetic oligonucleotide 697ucaucauugg cuuuccgcu
1969819RNAArtificial Sequencesynthetic oligonucleotide
698aguuccugac uaucaauca 1969919RNAArtificial Sequencesynthetic
oligonucleotide 699uucacggcug acuuuggaa 1970019RNAArtificial
Sequencesynthetic oligonucleotide 700uucucauggu agugaguuu
1970119RNAArtificial Sequencesynthetic oligonucleotide
701aaucagccug uuuaacugg 1970219RNAArtificial Sequencesynthetic
oligonucleotide 702gguuucagca cucugguca 1970319RNAArtificial
Sequencesynthetic oligonucleotide 703aucggaaugc ucauugcuc
1970419RNAArtificial Sequencesynthetic oligonucleotide
704uggcugugga auucacggc 1970519RNAArtificial Sequencesynthetic
oligonucleotide 705uaagcaauug acaccacca 1970619RNAArtificial
Sequencesynthetic oligonucleotide 706caauucucau gguagugag
1970719RNAArtificial Sequencesynthetic oligonucleotide
707uggcuuucgu uggacuuac 1970819RNAArtificial Sequencesynthetic
oligonucleotide 708aaucagugac caguucauc 1970919RNAArtificial
Sequencesynthetic oligonucleotide 709aguccauaaa ccacacuau
1971019RNAArtificial Sequencesynthetic oligonucleotide
710cagcacucug gucauccag 1971119RNAArtificial Sequencesynthetic
oligonucleotide 711uaucaaucac aucggaaug 1971219RNAArtificial
Sequencesynthetic oligonucleotide 712auucacggcu gacuuugga
1971319RNAArtificial Sequencesynthetic oligonucleotide
713auagauacac auucaacca 1971419RNAArtificial Sequencesynthetic
oligonucleotide 714uuuccagacu caaauagau 1971519RNAArtificial
Sequencesynthetic oligonucleotide 715uuaauugcug gacaaccgu
1971619RNAArtificial Sequencesynthetic oligonucleotide
716uauuaauugc uggacaacc 1971719RNAArtificial Sequencesynthetic
oligonucleotide 717agucguucga gucaaugga 1971819RNAArtificial
Sequencesynthetic oligonucleotide 718guugcuggca gguccgugg
1971913RNAArtificial Sequencesynthetic oligonucleotide
719cucaugaauu aga 1372013RNAArtificial Sequencesynthetic
oligonucleotide 720cugaggucaa uua 1372113RNAArtificial
Sequencesynthetic oligonucleotide 721gaggucaauu aaa
1372213RNAArtificial Sequencesynthetic oligonucleotide
722ucugagguca auu 1372313RNAArtificial Sequencesynthetic
oligonucleotide 723ugaggucaau uaa 1372413RNAArtificial
Sequencesynthetic oligonucleotide 724uucugagguc aau
1372513RNAArtificial Sequencesynthetic oligonucleotide
725gucagcugga uga 1372613RNAArtificial Sequencesynthetic
oligonucleotide 726uucugaugaa ucu 1372713RNAArtificial
Sequencesynthetic oligonucleotide 727uggacugagg uca
1372813RNAArtificial Sequencesynthetic oligonucleotide
728gagucucacc auu 1372913RNAArtificial Sequencesynthetic
oligonucleotide 729gacugagguc aaa 1373013RNAArtificial
Sequencesynthetic oligonucleotide 730ucacagccau gaa
1373113RNAArtificial Sequencesynthetic oligonucleotide
731agucucacca uuc 1373213RNAArtificial Sequencesynthetic
oligonucleotide 732aagcggaaag cca 1373313RNAArtificial
Sequencesynthetic oligonucleotide 733agcggaaagc caa
1373413RNAArtificial Sequencesynthetic oligonucleotide
734accacaugga uga 1373513RNAArtificial Sequencesynthetic
oligonucleotide 735gccaugacca cau 1373613RNAArtificial
Sequencesynthetic oligonucleotide 736aagccaugac cac
1373713RNAArtificial Sequencesynthetic oligonucleotide
737gcggaaagcc aau 1373813RNAArtificial Sequencesynthetic
oligonucleotide 738aaauuucgua uuu 1373913RNAArtificial
Sequencesynthetic oligonucleotide 739auuucguauu ucu
1374013RNAArtificial Sequencesynthetic oligonucleotide
740aaagccauga cca 1374113RNAArtificial Sequencesynthetic
oligonucleotide 741acauggauga uau 1374213RNAArtificial
Sequencesynthetic oligonucleotide 742gaaauuucgu auu
1374313RNAArtificial Sequencesynthetic oligonucleotide
743gcgccuucug auu 1374413RNAArtificial Sequencesynthetic
oligonucleotide 744auuucucaug aau 1374513RNAArtificial
Sequencesynthetic oligonucleotide 745cucucaugaa uag
1374613RNAArtificial Sequencesynthetic oligonucleotide
746aaguccaacg aaa 1374713RNAArtificial Sequencesynthetic
oligonucleotide 747augaugagag caa 1374813RNAArtificial
Sequencesynthetic oligonucleotide 748gcgaggaguu gaa
1374913RNAArtificial Sequencesynthetic oligonucleotide
749ugauugauag uca 1375013RNAArtificial Sequencesynthetic
oligonucleotide 750agauagugca ucu 1375113RNAArtificial
Sequencesynthetic oligonucleotide 751auguguaucu auu
1375213RNAArtificial Sequencesynthetic oligonucleotide
752uucuauagaa gaa 1375313RNAArtificial Sequencesynthetic
oligonucleotide 753uuguccagca auu 1375413RNAArtificial
Sequencesynthetic oligonucleotide 754acauggaaag cga
1375513RNAArtificial Sequencesynthetic oligonucleotide
755gcaguccaga uua 1375613RNAArtificial Sequencesynthetic
oligonucleotide 756ugguugaaug ugu 1375713RNAArtificial
Sequencesynthetic oligonucleotide 757uuaugaaacg agu
1375813RNAArtificial Sequencesynthetic oligonucleotide
758caguccagau uau 1375913RNAArtificial Sequencesynthetic
oligonucleotide 759auauaagcgg aaa 1376013RNAArtificial
Sequencesynthetic oligonucleotide 760uaccaguuaa aca
1376113RNAArtificial Sequencesynthetic oligonucleotide
761uguucauucu aua 1376213RNAArtificial Sequencesynthetic
oligonucleotide 762ccgaccaagg aaa 1376313RNAArtificial
Sequencesynthetic oligonucleotide 763gaauggugca uac
1376413RNAArtificial Sequencesynthetic oligonucleotide
764auaugauggc cga 1376513RNAArtificial Sequencesynthetic
oligonucleotide 765agcaguccag auu 1376613RNAArtificial
Sequencesynthetic oligonucleotide 766agcauuccga ugu
1376713RNAArtificial Sequencesynthetic oligonucleotide
767uagucaggaa cuu 1376813RNAArtificial Sequencesynthetic
oligonucleotide 768ugcauuuagu caa 1376913RNAArtificial
Sequencesynthetic oligonucleotide 769gucugaugag ucu
1377013RNAArtificial Sequencesynthetic oligonucleotide
770uagacacaua uga 1377113RNAArtificial Sequencesynthetic
oligonucleotide 771cagacgagga cau 1377213RNAArtificial
Sequencesynthetic oligonucleotide 772cagccgugaa uuc
1377313RNAArtificial Sequencesynthetic oligonucleotide
773agucuggaaa uaa 1377413RNAArtificial Sequencesynthetic
oligonucleotide 774aguuuguggc uuc 1377513RNAArtificial
Sequencesynthetic oligonucleotide 775aguccaacga aag
1377613RNAArtificial Sequencesynthetic oligonucleotide
776aaguuucgca gac 1377713RNAArtificial Sequencesynthetic
oligonucleotide 777agcaaugagc auu 1377813RNAArtificial
Sequencesynthetic oligonucleotide 778uuagauagug cau
1377913RNAArtificial Sequencesynthetic oligonucleotide
779uggugcauac aag 1378013RNAArtificial Sequencesynthetic
oligonucleotide 780augaaacgag uca 1378113RNAArtificial
Sequencesynthetic oligonucleotide 781ccagagugcu gaa
1378213RNAArtificial Sequencesynthetic oligonucleotide
782cagccaugaa uuu 1378313RNAArtificial Sequencesynthetic
oligonucleotide 783auugguugaa ugu 1378413RNAArtificial
Sequencesynthetic oligonucleotide 784gguugaaugu gua
1378513RNAArtificial Sequencesynthetic oligonucleotide
785ggaaauaacu aau 1378613RNAArtificial Sequencesynthetic
oligonucleotide 786ucaugaauag aaa 1378713RNAArtificial
Sequencesynthetic oligonucleotide 787gccagcaacc gaa
1378813RNAArtificial Sequencesynthetic oligonucleotide
788caccucacac aug 1378913RNAArtificial Sequencesynthetic
oligonucleotide 789aguugaaugg ugc 1379013RNAArtificial
Sequencesynthetic oligonucleotide 790agucagcugg aug
1379113RNAArtificial Sequencesynthetic oligonucleotide
791uauaagcgga aag 1379213RNAArtificial Sequencesynthetic
oligonucleotide 792uuccgaugug auu 1379313RNAArtificial
Sequencesynthetic oligonucleotide 793auaacuaaug ugu
1379413RNAArtificial Sequencesynthetic oligonucleotide
794ucauucuaua gaa 1379513RNAArtificial Sequencesynthetic
oligonucleotide 795aacuaucacu gua 1379613RNAArtificial
Sequencesynthetic oligonucleotide 796gucaauugcu uau
1379713RNAArtificial Sequencesynthetic oligonucleotide
797agcaauuaau aaa 1379813RNAArtificial Sequencesynthetic
oligonucleotide 798acgacucuga uga 1379913RNAArtificial
Sequencesynthetic oligonucleotide 799uagugugguu uau
1380013RNAArtificial Sequencesynthetic oligonucleotide
800aagccaauga uga 1380113RNAArtificial Sequencesynthetic
oligonucleotide 801auagucagga acu 1380213RNAArtificial
Sequencesynthetic oligonucleotide 802agucagccgu gaa
1380313RNAArtificial Sequencesynthetic oligonucleotide
803acuaccauga gaa 1380413RNAArtificial Sequencesynthetic
oligonucleotide 804aaacaggcug auu 1380513RNAArtificial
Sequencesynthetic oligonucleotide 805gagugcugaa acc
1380613RNAArtificial Sequencesynthetic oligonucleotide
806ugagcauucc gau 1380713RNAArtificial Sequencesynthetic
oligonucleotide 807aauuccacag cca 1380813RNAArtificial
Sequencesynthetic oligonucleotide 808ugucaauugc uua
1380913RNAArtificial Sequencesynthetic oligonucleotide
809accaugagaa uug 1381013RNAArtificial Sequencesynthetic
oligonucleotide 810ccaacgaaag cca 1381113RNAArtificial
Sequencesynthetic oligonucleotide 811cuggucacug auu
1381213RNAArtificial Sequencesynthetic oligonucleotide
812ugguuuaugg acu 1381313RNAArtificial Sequencesynthetic
oligonucleotide 813gaccagagug cug 1381413RNAArtificial
Sequencesynthetic oligonucleotide 814gaugugauug aua
1381513RNAArtificial Sequencesynthetic oligonucleotide
815gucagccgug aau 1381613RNAArtificial Sequencesynthetic
oligonucleotide 816aauguguauc uau 1381713RNAArtificial
Sequencesynthetic oligonucleotide 817uugagucugg aaa
1381813RNAArtificial Sequencesynthetic oligonucleotide
818guccagcaau uaa 1381913RNAArtificial Sequencesynthetic
oligonucleotide 819ccagcaauua aua 1382013RNAArtificial
Sequencesynthetic oligonucleotide 820gacucgaacg acu
1382113RNAArtificial Sequencesynthetic oligonucleotide
821accugccagc aac 13
* * * * *
References