U.S. patent application number 17/706234 was filed with the patent office on 2022-07-14 for methods and compositions of short small hairpin rnas and micrornas for wound healing.
The applicant listed for this patent is SomaGenics, Inc.. Invention is credited to Anne DALLAS, Heini ILVES, Sumedha JAYASENA, Brian H. JOHNSTON.
Application Number | 20220220484 17/706234 |
Document ID | / |
Family ID | 1000006238984 |
Filed Date | 2022-07-14 |
United States Patent
Application |
20220220484 |
Kind Code |
A1 |
DALLAS; Anne ; et
al. |
July 14, 2022 |
METHODS AND COMPOSITIONS OF SHORT SMALL HAIRPIN RNAS AND MICRORNAS
FOR WOUND HEALING
Abstract
Wound healing is a complex homeostatic process in which several
distinct types coordinate to repair a physical damage. Failure to
close wounds contributes to the pathology of conditions like
diabetes mellitus, particularly in the elderly. Presented herein
are molecules, pharmaceutical compositions, and methods for
applying small RNA oligonucleotide technology to wound healing.
Small RNA oligonucleotide approaches as disclosed herein provide a
therapeutic strategy for improving both basal and pathological
wound healing.
Inventors: |
DALLAS; Anne; (Santa Cruz,
CA) ; ILVES; Heini; (Santa Cruz, CA) ;
JAYASENA; Sumedha; (Santa Cruz, CA) ; JOHNSTON; Brian
H.; (Santa Cruz, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SomaGenics, Inc. |
Santa Cruz |
CA |
US |
|
|
Family ID: |
1000006238984 |
Appl. No.: |
17/706234 |
Filed: |
March 28, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16562118 |
Sep 5, 2019 |
11319538 |
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17706234 |
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15752182 |
Feb 12, 2018 |
10450569 |
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PCT/US16/46884 |
Aug 12, 2016 |
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16562118 |
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62204957 |
Aug 13, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1137 20130101;
C12Y 114/11002 20130101; A61K 45/06 20130101; C07H 21/02 20130101;
A61K 31/713 20130101; C12N 2310/3231 20130101; C12N 15/1131
20130101; A61P 17/02 20180101; C12N 2310/317 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/713 20060101 A61K031/713; C07H 21/02 20060101
C07H021/02; A61K 45/06 20060101 A61K045/06; A61P 17/02 20060101
A61P017/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made in part during work supported by
grant R43GM101725 and R44GM101725 (BHJ) from the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A short small hairpin RNA (sshRNA) for inhibiting prolyl
hydroxylase domain-containing protein 2 (PHD2) comprising: (a) an
antisense sequence that is capable of hybridizing to an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2;
and (b) a sense sequence, wherein the sense and antisense sequences
form a stem having a length of less than or equal to 19 base
pairs.
2. The sshRNA of claim 1, wherein the RNA transcript is encoded by
an Eg1 nine homolog 1 (EGLN1) gene, wherein the EGLN1 gene, minus
introns, is represented by a sequence selected from SEQ ID NO. 1
and SEQ ID NO. 2.
3. The sshRNA of claim 1, comprising a loop region selected from a
direct connection, 1 nucleotide and 2 nucleotides in length.
4. The sshRNA of claim 1, wherein the sshRNA consists of the
antisense sequence, the sense sequence, a loop region, an optional
overhang sequence, and an optional conjugate moiety.
5. The sshRNA of claim 4, wherein the loop region has a length of
zero nucleotides, 1 nucleotide or 2 nucleotides.
6. The sshRNA of claim 5, wherein the loop region consists of zero
nucleotides, 1 nucleotide or 2 nucleotides.
7. The sshRNA of claim 1, wherein the sshRNA consists of the
antisense sequence, the sense sequence, an optional overhang
sequence, and an optional conjugate moiety, wherein the sense
sequence and the antisense sequence are directly connected.
8. The sshRNA of claim 1, wherein the antisense sequence is from
about 80% complementary to 100% complementary to the PHD2
transcript.
9. The sshRNA of claim 1, wherein the sense sequence is from about
10 nucleotides to about 19 nucleotides in length.
10. The sshRNA of claim 1, wherein the sense sequence is from about
11 nucleotides to about 19 nucleotides in length.
11. The sshRNA of claim 1, wherein at least one nucleotide
comprises a chemical modification.
12. The sshRNA of claim 11, wherein the chemical modification
increases the stability of the shRNA in a biological fluid at least
about 20%.
13. The sshRNA of claim 1, wherein the sshRNA is represented by a
sequence selected from SEQ ID NOS: 5-8, 11-31, 49-325.
14. A microRNA (miRNA) antagonist comprising an antisense strand
capable of hybridizing to and inhibiting miR-210.
15. The miRNA antagonist of claim 14, wherein the miR-210 is
represented by SEQ ID NO. 3.
16. The miRNA antagonist of claim 14, wherein the miRNA antagonist
reduces an amount or an activity of the miR-210 from about 10% to
about 99.9%.
17. The miRNA antagonist of claim 14, wherein at least one
ribonucleotide comprises a chemical modification.
18. The miRNA antagonist of claim 14, wherein every ribonucleotide
comprises a chemical modification.
19. The miRNA antagonist of claim 17 or 18, wherein the chemical
modification is a sugar modification.
20. The miRNA antagonist of claim 19, wherein the sugar
modification comprises a 2'-O-methyl modification, a LNA
modification, a DNA modification, or a 2'-F modification.
21. The miRNA antagonist of claim 19, wherein the sugar
modification is a 2'-O-methyl modification.
22. The miRNA antagonist of claim 19, wherein the sugar
modification is a LNA modification.
23. The miRNA antagonist of claim 14, wherein the miRNA antagonist
comprises phosphorothioate internucleotide linkages.
24. The miRNA antagonist of claim 14, wherein the miRNA antagonist
comprises a backbone modification.
25. The miRNA antagonist of claim 24, wherein the backbone
modification is selected from a C3 spacer or ZEN.
26. The miRNA antagonist of claim 14, wherein the miRNA antagonist
comprises a 2'-O-methyl modification at every position and three
phosphorothioate internucleotide linkages at consecutive residues
at both the 5'- and 3'-end.
27. The miRNA antagonist of claim 14, wherein the miRNA antagonist
comprises phosphorothioate linkages at every position and
2'-O-methyl modifications at positions 1, 3, 4, 6, 7, 9, 10, 12,
13, and 15 and LNA modifications at positions 2, 5, 8, 11, and
14.
28. The miRNA antagonist of claim 14, wherein the miRNA antagonist
comprises 2'-O-methyl modifications at every position and ZEN
modifications between position 1 and 2 and also at the 3'-end.
29. The miRNA antagonist of claim 14, wherein the miRNA antagonist
is represented by a sequence selected from SEQ ID NOS: 32-43,
326-328.
30. A pre-miRNA mimic for increasing a steady state level of a
mature miR-21 comprising: (a) a sense sequence; (b) an antisense
sequence; and (c) a loop region, wherein the length of the loop
region is the length of 2 nucleotides or less.
31. The pre-miRNA of claim 30, wherein the mature miR-21 is
represented by SEQ ID NO.4.
32. The pre-miRNA mimic of claim 30, wherein the loop region
consists of a direct connection, or consists of 1-2 nucleotides or
nucleotide moieties.
33. The pre-miRNA mimic of claim 30, wherein the pre-miRNA mimic is
represented by a sequence selected from SEQ ID NOS: 44-48.
34. A pharmaceutical composition comprising a
pharmaceutically-acceptable substrate, carrier or salt, and at
least one oligonucleotide selected from: (a) the sshRNA of claim 1;
(b) the miRNA antagonist of claim 14; and (c) the pre-miRNA mimic
of claim 30.
35. A pharmaceutical composition comprising a
pharmaceutically-acceptable substrate, carrier or salt, and a
combination of oligonucleotides selected from: (a) the sshRNA of
claim 1; (b) the miRNA antagonist of claim 14; and (c) the
pre-miRNA mimic of claim 30.
36. The pharmaceutical composition of claim 34 or 35, wherein the
pharmaceutically-acceptable substrate is a mesh or a dressing.
37. The pharmaceutical composition of claim 36, wherein the mesh is
a layered mesh.
38. The pharmaceutical composition of claim 34 or 35, wherein the
composition is formulated for topical administration.
39. A method for treating a wound in a subject in need thereof
comprising administering a therapeutically effective amount of a
composition selected from: (a) the sshRNA of claim 1; (b) the miRNA
antagonist of claim 14; (c) the pre-miRNA mimic of claim 30; and a
combination thereof.
40. The method of claim 39, wherein the wound is a chronic
wound.
41. The method of claim 39, wherein the wound is a non-healing
wound.
42. The method of claim 39, wherein the subject has diabetes
mellitus.
43. The method of claim 39, wherein the wound is a skin wound.
44. The method of claim 39, comprising administering the
composition topically.
45. The method of claim 39, comprising a dressing, wherein the
dressing comprises the composition.
46. An oligonucleotide according to any one of claim 1, 14, or 30,
and any combination thereof, for use in a method of treating a
wound in an animal or human.
47. An R oligonucleotide NA according to any one of claim 1, 14, or
30, and any combination thereof, for treatment of a wound in a
subject.
48. A kit comprising: (a) the sshRNA of claim 1; (b) the miRNA
antagonist of claim 14; and (c) the pre-miRNA mimic of claim 30.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 16/562,118, filed Sep. 5, 2019, which is a Continuation of U.S.
application Ser. No. 15/752,182, filed Feb. 12, 2018, issued as
U.S. Pat. No. 10,450,569 on Oct. 22, 2019, which is a U.S. National
Stage Entry of PCT/US2016/046884, filed Aug. 12, 2016, which claims
the benefit of U.S. Provisional Application No. 62/204,957, filed
Aug. 13, 2015, each of which are incorporated herein by reference
in their entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 10, 2016, is named 40220_710_302 SL.txt and is 91,878 bytes
in size.
BACKGROUND
[0004] Wound healing is an orchestrated physiological process in
which multiple cell types interact to close a physical insult.
Dysregulation of wound healing contributes to the pathology of
various diseases. New approaches are needed to ameliorate wound
healing in these pathological settings.
SUMMARY OF THE INVENTION
[0005] Disclosed herein, in certain embodiments, is a short small
hairpin RNA (sshRNA) for inhibiting prolyl hydroxylase
domain-containing protein 2 (PHD2) comprising: (a) an antisense
sequence that is capable of hybridizing to an RNA transcript
encoding prolyl hydroxylase domain-containing protein 2; and (b) a
sense sequence, wherein the sense and antisense sequences form a
stem having a length of less than or equal to 19 base pairs. In
some embodiments, the RNA transcript is encoded by an Eg1 nine
homolog 1 (EGLN1) gene, wherein the EGLN1 gene, minus introns, is
represented by a sequence selected from SEQ ID NO. 1 and SEQ ID NO.
2. In some embodiments, RNA transcript is a transcript
corresponding to human PHD2 (SEQ ID NO. 1). In some embodiments,
RNA transcript is a transcript corresponding to mouse Phd2 (SEQ ID
NO. 2). In some embodiments, the sshRNA targets both human PHD2
(SEQ ID NO. 1) and mouse Phd2 (SEQ ID NO. 2). In some embodiments,
the sshRNA consists of the antisense sequence, the sense sequence,
the loop region, an optional overhang sequence, and an optional
conjugate moiety. In some embodiments, loop region is selected from
a direct connection, 1 nucleotide and 2 nucleotides in length. In
some embodiments, the overhang sequence is from about 1 nucleotide
to about 2 nucleotides. In some embodiments, the conjugate moiety
is a detectable label. In some embodiments, the antisense sequence
is from about 15 nucleotides to about 19 nucleotides in length. In
some embodiments, the antisense sequence is from about 16
nucleotides to about 19 nucleotides in length. In some embodiments,
the antisense sequence is from about 60% complementary to 100%
complementary to the PHD2 transcript. In some embodiments, the
antisense sequence is from about 80% complementary to 100%
complementary to the PHD2 transcript. In some embodiments, the
sense sequence is from about 10 nucleotides to about 19 nucleotides
in length. In some embodiments, the sense sequence is from about 11
nucleotides to about 19 nucleotides in length. In some embodiments,
the sense sequence is from about 80% complementary to 100%
complementary to the antisense sequence. In some embodiments, at
least one nucleotide comprises a chemical modification. In some
embodiments, the chemical modification increases the stability of
the shRNA in a biological fluid at least about 20%. In some
embodiments, the chemical modification increases the stability of
the shRNA in a biological fluid about 1% to about 10000%. In some
embodiments, the chemical modification is a sugar modification. In
some embodiments, the sugar modification is chosen from:
2'-O-methyl, 2'-H, and 2'- F. In some embodiments, the sshRNA is
represented by a sequence selected from SEQ ID NOS: 5-8, 11-31,
49-325.
[0006] Disclosed herein, in certain embodiments, is a microRNA
(miRNA) antagonist comprising an antisense strand capable of
hybridizing to and inhibiting miR-210. In some embodiments, the
miR-210 is represented by SEQ ID NO. 3. In some embodiments, the
miRNA antagonist comprises a sequence from about 60% to 100%
complementary to the miR-210 that is represented by SEQ ID NO. 3.
In some embodiments, the miRNA antagonist reduces an amount or an
activity of the miR-210 from about 1% to about 99.9%. In some
embodiments, the miRNA antagonist reduces an amount or an activity
of the miR-210 from about 10% to about 99.9%. In some embodiments,
at least one ribonucleotide comprises a chemical modification. In
some embodiments, every ribonucleotide comprises a chemical
modification. In some embodiments, the chemical modification is a
sugar modification. In some embodiments, the sugar modification
comprises a 2'-O-methyl modification, a LNA modification, a DNA
modification, or a 2'-F modification. In some embodiments, the
sugar modification comprises a 2'-O-methyl modification, a 2'-H, a
LNA modification, a DNA modification, or a 2'-F modification. In
some embodiments, the sugar modification is a 2'-O-methyl
modification. In some embodiments, the sugar modification is a LNA
modification. In some embodiments, the miRNA antagonist comprises
phosphorothioate internucleotide linkages. In some embodiments, the
miRNA antagonist comprises a backbone modification. In some
embodiments, backbone modification is selected from a C3 spacer or
ZEN. In some embodiments, the miRNA antagonist comprises a
2'-O-methyl modification at every position and three
phosphorothioate internucleotide linkages at consecutive residues
at both the 5'- and 3'-end. In some embodiments, the miRNA
antagonist comprises phosphorothioate linkages at every position
and 2'-O-methyl modifications at positions 1, 3, 4, 6, 7, 9, 10,
12, 13, and 15 and LNA modifications at positions 2, 5, 8, 11, and
14. In some embodiments, the miRNA antagonist comprises 2'-O-methyl
modifications at every position and ZEN modifications between
position 1 and 2 and also at the 3'-end. In some embodiments, the
modification reduces miRNA inhibitory activity from about
0.0000000001% to about 50%. In some embodiments, the miRNA
antagonist is represented by a sequence selected from SEQ ID NOS:
32-43, 326-328.
[0007] Disclosed herein, in certain embodiments, is a pre-miRNA
mimic for increasing a steady state level of a mature miR-21
comprising: (a) a sense sequence; (b) an antisense sequence; and
(c) a loop region, wherein the length of the loop region is the
length of 2 nucleotides or less. In some embodiments, the mature
miR-21 is represented by SEQ ID NO.4. In some embodiments, the
sense and antisense sequences are from about 60% to 100%
complementary. In some embodiments, the loop region consists of a
direct connection, or consists of 1-2 nucleotides or nucleotide
moieties. In some embodiments, the pre-miRNA mimic further
comprises an overhang region from about 1 nucleotide to about 2
nucleotides. In some embodiments, the pre-miRNA mimic has at least
one nucleotide modified. In some embodiments, the modification
reduces miRNA-mediated repressive activity from about 0.0000000001%
to about 50%. In some embodiments, the pre-miRNA mimic increases a
steady-state level of the mature miRNA from about 1% to about
10000%. In some embodiments, the pre-miRNA mimic is represented by
a sequence selected from SEQ ID NOS: 44-48.
[0008] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising a pharmaceutically-acceptable
substrate, carrier or salt, and at least one RNA selected from: (a)
a short small hairpin RNA (sshRNA) for inhibiting prolyl
hydroxylase domain-containing protein 2 (PHD2) comprising: (a) an
antisense sequence that is capable of hybridizing to an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2;
and (b) a sense sequence, wherein the sense and antisense sequences
form a stem having a length of less than or equal to 19 base pairs;
(b) a microRNA (miRNA) antagonist comprising an antisense strand
capable of hybridizing to and inhibiting miR-210; and (c) a
pre-miRNA mimic for increasing a steady state level of a mature
miR-21 comprising: (a) a sense sequence; (b) an antisense sequence;
and (c) a loop region, wherein the length of the loop region is the
length of 2 nucleotides or less. Disclosed herein, in certain
embodiments, is a pharmaceutical composition comprising a
pharmaceutically-acceptable substrate, carrier or salt, and a short
small hairpin RNA (sshRNA) for inhibiting prolyl hydroxylase
domain-containing protein 2 (PHD2) comprising: (a) an antisense
sequence that is capable of hybridizing to an RNA transcript
encoding prolyl hydroxylase domain-containing protein 2. Disclosed
herein, in certain embodiments, is a pharmaceutical composition
comprising a pharmaceutically-acceptable substrate, carrier or
salt, and a microRNA (miRNA) antagonist comprising an antisense
strand capable of hybridizing to and inhibiting miR-210. Disclosed
herein, in certain embodiments, is a pharmaceutical composition
comprising a pharmaceutically-acceptable substrate, carrier or
salt, and a pre-miRNA mimic for increasing a steady state level of
a mature miR-21 comprising: (a) a sense sequence; (b) an antisense
sequence; and (c) a loop region, wherein the length of the loop
region is the length of 2 nucleotides or less. In some embodiments,
the short small hairpin RNA (sshRNA) has an antisense sequence that
is from about 60% complementary to 100% complementary to the PHD2
transcript. In some embodiments, the miRNA antagonist comprises a
sequence from about 60% to 100% complementary to the miR-210 that
is represented by SEQ ID NO. 3. In some embodiments, the pre-miRNA
mimic has the sense and antisense sequences that are from about 60%
to 100% complementary. In some embodiments, the
pharmaceutically-acceptable substrate is a mesh or a dressing. In
some embodiments, the mesh is a layered mesh.
[0009] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising a pharmaceutically-acceptable
substrate, carrier or salt, and a combination of RNAs selected
from: (a) a short small hairpin RNA (sshRNA) for inhibiting prolyl
hydroxylase domain-containing protein 2 (PHD2) comprising: (a) an
antisense sequence that is capable of hybridizing to an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2;
and (b) a sense sequence, wherein the sense and antisense sequences
form a stem having a length of less than or equal to 19 base pairs;
(b) a microRNA (miRNA) antagonist comprising an antisense strand
capable of hybridizing to and inhibiting miR-210; and (c) a
pre-miRNA mimic for increasing a steady state level of a mature
miR-21 comprising: (a) a sense sequence; (b) an antisense sequence;
and (c) a loop region, wherein the length of the loop region is the
length of 2 nucleotides or less. Disclosed herein, in certain
embodiments, is a pharmaceutical composition comprising a
pharmaceutically-acceptable substrate, carrier or salt, and a
combination of RNAs comprising: (a) a short small hairpin RNA
(sshRNA) for inhibiting prolyl hydroxylase domain-containing
protein 2 (PHD2) comprising: (a) an antisense sequence that is
capable of hybridizing to an RNA transcript encoding prolyl
hydroxylase domain-containing protein 2; and (b) a sense sequence,
wherein the sense and antisense sequences form a stem having a
length of less than or equal to 19 base pairs; and (b) a microRNA
(miRNA) antagonist comprising an antisense strand capable of
hybridizing to and inhibiting miR-210. Disclosed herein, in certain
embodiments, is a pharmaceutical composition comprising a
pharmaceutically-acceptable substrate, carrier or salt, and a
combination of RNAs comprising: (a) a short small hairpin RNA
(sshRNA) for inhibiting prolyl hydroxylase domain-containing
protein 2 (PHD2) comprising: (a) an antisense sequence that is
capable of hybridizing to an RNA transcript encoding prolyl
hydroxylase domain-containing protein 2; and (b) a sense sequence,
wherein the sense and antisense sequences form a stem having a
length of less than or equal to 19 base pairs; and (c) a pre-miRNA
mimic for increasing a steady state level of a mature miR-21
comprising: (a) a sense sequence; (b) an antisense sequence; and
(c) a loop region, wherein the length of the loop region is the
length of 2 nucleotides or less. Disclosed herein, in certain
embodiments, is a pharmaceutical composition comprising a
pharmaceutically-acceptable substrate, carrier or salt, and a
combination of RNAs comprising: (b) a microRNA (miRNA) antagonist
comprising an antisense strand capable of hybridizing to and
inhibiting miR-210 and (c) a pre-miRNA mimic for increasing a
steady state level of a mature miR-21 comprising: (a) a sense
sequence; (b) an antisense sequence; and (c) a loop region, wherein
the length of the loop region is the length of 2 nucleotides or
less. Disclosed herein, in certain embodiments, is a pharmaceutical
composition comprising a pharmaceutically-acceptable substrate,
carrier or salt, and a combination of RNAs comprising: (a) a short
small hairpin RNA (sshRNA) for inhibiting prolyl hydroxylase
domain-containing protein 2 (PHD2) comprising: (a) an antisense
sequence that is capable of hybridizing to an RNA transcript
encoding prolyl hydroxylase domain-containing protein 2; and (b) a
sense sequence, wherein the sense and antisense sequences form a
stem having a length of less than or equal to 19 base pairs; (b) a
microRNA (miRNA) antagonist comprising an antisense strand capable
of hybridizing to and inhibiting miR-210; and (c) a pre-miRNA mimic
for increasing a steady state level of a mature miR-21 comprising:
(a) a sense sequence; (b) an antisense sequence; and (c) a loop
region, wherein the length of the loop region is the length of 2
nucleotides or less. In some embodiments, the short small hairpin
RNA (sshRNA) has an antisense sequence that is from about 60%
complementary to 100% complementary to the PHD2 transcript. In some
embodiments, the miRNA antagonist comprises a sequence from about
60% to 100% complementary to the miR-210 that is represented by SEQ
ID NO. 3. In some embodiments, the pre-miRNA mimic has the sense
and antisense sequences that are from about 60% to 100%
complementary. In some embodiments, the pharmaceutically-acceptable
substrate is a mesh or a dressing. In some embodiments, the
composition is formulated for topical administration.
[0010] In some embodiments, the mesh is a layered mesh. In some
embodiments, the pharmaceutically-acceptable substrate is formed by
layer-by layer (LbL) fabrication. LbL deposition creates a thin
film, formed by alternating layers of oppositely charged materials.
Formation of layers may comprise a technique selected from
immersion, spin, spray, electromagnetism or fluidics. In some
embodiments, the composition is formulated for topical
administration. In some embodiments, the layered mesh may be
degradable in an aqueous environment. The layered mesh may conform
to a wound. In some embodiments, the layer-by-layer film comprises
a LayerForm.TM. coating. The LayerForm.TM. coating may be chosen
from coatings with various release profiles, to release the
oligonucleotides disclosed herein at an optimal rate for the
subject.
[0011] Disclosed herein, in certain embodiments, is a method for
treating a wound in a subject in need thereof comprising
administering a therapeutically effective amount of a composition
selected from: (a) a short small hairpin RNA (sshRNA) for
inhibiting prolyl hydroxylase domain-containing protein 2 (PHD2)
comprising: (a) an antisense sequence that is capable of
hybridizing to an RNA transcript encoding prolyl hydroxylase
domain-containing protein 2; and (b) a sense sequence, wherein the
sense and antisense sequences form a stem having a length of less
than or equal to 19 base pairs; (b) a microRNA (miRNA) antagonist
comprising an antisense strand capable of hybridizing to and
inhibiting miR-210; (c) a pre-miRNA mimic for increasing a steady
state level of a mature miR-21 comprising: (a) a sense sequence;
(b) an antisense sequence; and (c) a loop region, wherein the
length of the loop region is the length of 2 nucleotides or less;
and a combination thereof. In some embodiments, the wound is a
chronic wound. In some embodiments, the wound is a non-healing
wound. In some embodiments, the subject has diabetes mellitus. In
some embodiments, the wound is a skin wound. In some embodiments,
the method comprises administering the composition topically. In
some embodiments, the method comprises a dressing, wherein the
dressing comprises the composition.
[0012] Disclosed herein, in certain embodiments, is an RNA selected
from: (a) a short small hairpin RNA (sshRNA) for inhibiting prolyl
hydroxylase domain-containing protein 2 (PHD2) comprising: (a) an
antisense sequence that is capable of hybridizing to an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2;
and (b) a sense sequence, wherein the sense and antisense sequences
form a stem having a length of less than or equal to 19 base pairs;
(b) a microRNA (miRNA) antagonist comprising an antisense strand
capable of hybridizing to and inhibiting miR-210; (c) a pre-miRNA
mimic for increasing a steady state level of a mature miR-21
comprising: (a) a sense sequence; (b) an antisense sequence; and
(c) a loop region, wherein the length of the loop region is the
length of 2 nucleotides or less; and any combination thereof, for
use in a method of treating a wound in an animal or human. In some
embodiments, disclosed herein, is a short small hairpin RNA
(sshRNA) for inhibiting prolyl hydroxylase domain-containing
protein 2 (PHD2) comprising: (a) an antisense sequence that is
capable of hybridizing to an RNA transcript encoding prolyl
hydroxylase domain-containing protein 2; and (b) a sense sequence,
wherein the sense and antisense sequences form a stem having a
length of less than or equal to 19 base pairs, for use in a method
of treating a wound in an animal or human. In some embodiments,
disclosed herein, is a microRNA (miRNA) antagonist comprising an
antisense strand capable of hybridizing to and inhibiting miR-210,
for use in a method of treating a wound in an animal or human. In
some embodiments, disclosed herein, is a pre-miRNA mimic for
increasing a steady state level of a mature miR-21 comprising: (a)
a sense sequence; (b) an antisense sequence; and (c) a loop region,
wherein the length of the loop region is the length of 2
nucleotides or less, for use in a method of treating a wound in an
animal or human.
[0013] Disclosed herein, in certain embodiments, is an RNA selected
from: (a) a short small hairpin RNA (sshRNA) for inhibiting prolyl
hydroxylase domain-containing protein 2 (PHD2) comprising: (a) an
antisense sequence that is capable of hybridizing to an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2;
and (b) a sense sequence, wherein the sense and antisense sequences
form a stem having a length of less than or equal to 19 base pairs;
(b) a microRNA (miRNA) antagonist comprising an antisense strand
capable of hybridizing to and inhibiting miR-210; (c) a pre-miRNA
mimic for increasing a steady state level of a mature miR-21
comprising: (a) a sense sequence; (b) an antisense sequence; and
(c) a loop region, wherein the length of the loop region is the
length of 2 nucleotides or less; and any combination thereof, for
treatment of a wound in a subject. In some embodiments, disclosed
herein, is a short small hairpin RNA (sshRNA) for inhibiting prolyl
hydroxylase domain-containing protein 2 (PHD2) comprising: (a) an
antisense sequence that is capable of hybridizing to an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2;
and (b) a sense sequence, wherein the sense and antisense sequences
form a stem having a length of less than or equal to 19 base pairs,
for treatment of a wound in a subject. In some embodiments,
disclosed herein, is a microRNA (miRNA) antagonist comprising an
antisense strand capable of hybridizing to and inhibiting miR-210,
for treatment of a wound in a subject. In some embodiments,
disclosed herein, is a pre-miRNA mimic for increasing a steady
state level of a mature miR-21 comprising: (a) a sense sequence;
(b) an antisense sequence; and (c) a loop region, wherein the
length of the loop region is the length of 2 nucleotides or less,
for treatment of a wound in a subject.
[0014] Disclosed herein, in certain embodiments, is a kit
comprising: (a) a short small hairpin RNA (sshRNA) for inhibiting
prolyl hydroxylase domain-containing protein 2 (PHD2) comprising:
(a) an antisense sequence that is capable of hybridizing to an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2;
and (b) a sense sequence, wherein the sense and antisense sequences
form a stem having a length of less than or equal to 19 base pairs;
(b) a microRNA (miRNA) antagonist comprising an antisense strand
capable of hybridizing to and inhibiting miR-210; and (c) a
pre-miRNA mimic for increasing a steady state level of a mature
miR-21 comprising: (a) a sense sequence; (b) an antisense sequence;
and (c) a loop region, wherein the length of the loop region is the
length of 2 nucleotides or less.
[0015] Disclosed herein, in certain embodiments, is a method for
treating a wound in a subject in need thereof comprising contacting
a cell with a therapeutically effective amount of a composition
selected from: (a) a short small hairpin RNA (sshRNA) for
inhibiting prolyl hydroxylase domain-containing protein 2 (PHD2)
comprising: (a) an antisense sequence that is capable of
hybridizing to an RNA transcript encoding prolyl hydroxylase
domain-containing protein 2; and (b) a sense sequence, wherein the
sense and antisense sequences form a stem having a length of less
than or equal to 19 base pairs; (b) a microRNA (miRNA) antagonist
comprising an antisense strand capable of hybridizing to and
inhibiting miR-210; (c) a pre-miRNA mimic for increasing a steady
state level of a mature miR-21 comprising: (a) a sense sequence;
(b) an antisense sequence; and (c) a loop region, wherein the
length of the loop region is the length of 2 nucleotides or less;
and a combination thereof. Disclosed herein, in certain
embodiments, is a method for treating a wound in a subject in need
thereof comprising contacting a cell with a therapeutically
effective amount of a composition comprising a short small hairpin
RNA (sshRNA) for inhibiting prolyl hydroxylase domain-containing
protein 2 (PHD2) comprising: (a) an antisense sequence that is
capable of hybridizing to an RNA transcript encoding prolyl
hydroxylase domain-containing protein 2. Disclosed herein, in
certain embodiments, is a method for treating a wound in a subject
in need thereof comprising contacting a cell with a therapeutically
effective amount of a composition comprising a microRNA (miRNA)
antagonist comprising an antisense strand capable of hybridizing to
and inhibiting miR-210. Disclosed herein, in certain embodiments,
is a method for treating a wound in a subject in need thereof
comprising contacting a cell with a therapeutically effective
amount of a composition comprising a pre-miRNA mimic for increasing
a steady state level of a mature miR-21 comprising: (a) a sense
sequence; (b) an antisense sequence; and (c) a loop region, wherein
the length of the loop region is the length of 2 nucleotides or
less. Disclosed herein, in certain embodiments, is a method for
treating a wound in a subject in need thereof comprising contacting
a cell with a therapeutically effective amount of a composition
comprising (a) a short small hairpin RNA (sshRNA) for inhibiting
prolyl hydroxylase domain-containing protein 2 (PHD2) comprising:
(a) an antisense sequence that is capable of hybridizing to an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2;
and (b) a sense sequence, wherein the sense and antisense sequences
form a stem having a length of less than or equal to 19 base pairs;
and (b) a microRNA (miRNA) antagonist comprising an antisense
strand capable of hybridizing to and inhibiting miR-210. Disclosed
herein, in certain embodiments, is a method for treating a wound in
a subject in need thereof comprising contacting a cell with a
therapeutically effective amount of a composition comprising (a) a
short small hairpin RNA (sshRNA) for inhibiting prolyl hydroxylase
domain-containing protein 2 (PHD2) comprising: (a) an antisense
sequence that is capable of hybridizing to an RNA transcript
encoding prolyl hydroxylase domain-containing protein 2; and (b) a
sense sequence, wherein the sense and antisense sequences form a
stem having a length of less than or equal to 19 base pairs; and
(c) a pre-miRNA mimic for increasing a steady state level of a
mature miR-21 comprising: (a) a sense sequence; (b) an antisense
sequence; and (c) a loop region, wherein the length of the loop
region is the length of 2 nucleotides or less. Disclosed herein, in
certain embodiments, is a method for treating a wound in a subject
in need thereof comprising contacting a cell with a therapeutically
effective amount of a composition comprising (b) a microRNA (miRNA)
antagonist comprising an antisense strand capable of hybridizing to
and inhibiting miR-210; and (c) a pre-miRNA mimic for increasing a
steady state level of a mature miR-21 comprising: (a) a sense
sequence; (b) an antisense sequence; and (c) a loop region, wherein
the length of the loop region is the length of 2 nucleotides or
less. Disclosed herein, in certain embodiments, is a method for
treating a wound in a subject in need thereof comprising contacting
a cell with a therapeutically effective amount of a composition
comprising: (a) a short small hairpin RNA (sshRNA) for inhibiting
prolyl hydroxylase domain-containing protein 2 (PHD2) comprising:
(a) an antisense sequence that is capable of hybridizing to an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2;
and (b) a sense sequence, wherein the sense and antisense sequences
form a stem having a length of less than or equal to 19 base pairs;
(b) a microRNA (miRNA) antagonist comprising an antisense strand
capable of hybridizing to and inhibiting miR-210; and (c) a
pre-miRNA mimic for increasing a steady state level of a mature
miR-21 comprising: (a) a sense sequence; (b) an antisense sequence;
and (c) a loop region, wherein the length of the loop region is the
length of 2 nucleotides or less. In some embodiments, the short
small hairpin RNA (sshRNA) has an antisense sequence that is from
about 60% complementary to 100% complementary to the PHD2
transcript. In some embodiments, the miRNA antagonist comprises a
sequence from about 60% to 100% complementary to the miR-210 that
is represented by SEQ ID NO. 3. In some embodiments, the pre-miRNA
mimic has the sense and antisense sequences that are from about 60%
to 100% complementary. In some embodiments, the wound is a chronic
wound. In some embodiments, the wound is a non-healing wound. In
some embodiments, the subject has diabetes mellitus. In some
embodiments, the wound is a skin wound. In some embodiments, the
cell is a keratinocyte. In some embodiments, the cell is a
fibroblast. In some embodiments, the method comprises administering
the composition topically. In some embodiments, the method
comprises a dressing, wherein the dressing comprises the
composition.
BRIEF DESCRIPTION OF THE FIGURES
[0016] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which principles of the invention are
utilized, and the accompanying figures of which:
[0017] FIG. 1 diagrams an exemplary HIF-1.alpha. wound healing
network.
[0018] FIG. 2A-FIG. 2B illustrate expression of PHD2 transcript in
response to treatment with an RNA interference (RNAi) molecule.
FIG. 2A illustrates the expression of human PHD2 in human embryonic
kidney 293FT cells transfected with increasing amounts of sshRNA
(SG302) or siRNA (SG303) targeting PHD2 or a control sshRNA
(SG221c). FIG. 2B illustrates the expression of mouse PHD2 in mouse
NIH3T3 fibroblasts transfected with increasing amounts of
PHD2-targeting sshRNAs (SG400 and SG402), PHD2-targeting siRNA
(SG403) or a control sshRNA (SG221c).
[0019] FIG. 3A-FIG. 3C illustrate HIF-1.alpha. pathway measurements
in response to treatment with an RNAi molecule. FIG. 3A illustrates
results of a luciferase reporter assay in human embryonic kidney
293FT cells co-transfected with a plasmid of firefly luciferase
(f-Luc) under the control of a promoter containing HIF-1.alpha.
responsive elements or a plasmid that constitutively expresses
Renilla luciferase (r-Luc) and individual sshRNAs targeting PHD2
(SG300, SG301, or SG302) or with a control sshRNA (NSC sshRNA).
FIG. 3B illustrates Western blotting studies of HIF-1.alpha. and
Lamin protein in Human embryonic kidney 293FT cells. FIG. 3C
illustrates qRT-PCR of VEGF (lanes 1, 4, 7, 10, 13, 16, and 19),
HSP90 (lanes 2, 5, 8, 11, 14, 17, and 20), and HSP70 (lanes 3, 6,
9, 12, 15, 18, and 21) in human embryonic kidney 293FT cells
transfected with increasing amounts of SG302.
[0020] FIG. 4A-FIG. 4C illustrate expression of PHD2 transcript in
response to treatment with an RNAi molecule. FIG. 4A illustrates
qRT-PCR of human PHD2 transcript in human primary normal human
epidermal keratinocytes (NHEK) transfected with increasing amounts
of SG302 or a modified sshRNA targeting PHD2 (SG302m1). FIG. 4B
illustrates qRT-PCR of mouse Phd2 transcript in mouse NIH3T3
fibroblasts transfected with increasing amounts of SG402, a
modified sshRNA targeting PHD2 (SG402m1), or a scrambled control
sshRNA (SG402-scr).
[0021] FIG. 4C illustrates qRT-PCR of mouse PHD2 (mPHD2) transcript
in mouse NIH3T3 fibroblasts transfected with increasing amounts of
an unmodified sshRNA targeting PHD2 (SG404) or SG402m1.
[0022] FIG. 5A-FIG. 5F illustrate cytokine levels in response to
treatment with an RNAi molecule. FIG. 5A illustrates qRT-PCR assays
for IFN-.beta. in cells treated with no inhibitor,
poly-inosine/cytosine (polyI:C), SG302, or SG302m1. FIG. 5B
illustrates qRT-PCR assays for IL-6 in cells treated with no
inhibitor, polyI:C, SG302, or SG302m1. FIG. 5C illustrates qRT-PCR
assays for TNF-.alpha. in cells treated with no inhibitor, polyI:C,
SG302, or SG302m1. FIG. 5D illustrates qRT-PCR for IFN-.beta. in
cells treated with no inhibitor, polyI:C, SG402, SG402m1, or SG404.
FIG. 5E illustrates qRT-PCR assays for IL-6 in cells treated with
no inhibitor, polyI:C, SG402, SG402m1, or SG404. FIG. 5F
illustrates qRT-PCR assays for TNF-.alpha. in cells treated with no
inhibitor, polyI:C, SG402, SG402m1, or SG404.
[0023] FIG. 6 illustrates stability of RNAi molecule in serum.
[0024] FIG. 7A-FIG. 7J illustrate designs for a miRNA antagonist,
expression of PHD2 transcript, expression of miR-210 microRNA
(miRNA), and activity of a miR-210 reporter in response to
treatment with an RNAi molecule or a miRNA antagonist. FIG. 7A
illustrates possible modification patterns for a miR-210 miRNA
antagonist. FIG. 7A discloses SEQ ID NOS 3 and 329, respectively,
in order of appearance. FIG. 7B illustrates qRT-PCR of human PHD2
transcript in human HaCaT keratinocytes transfected with the
following: no inhibitor; an sshRNA targeting human PHD2 (SG302); a
control sshRNA (ssh-NSC); SG302 and a LNA-modified miR-210, miRNA
antagonist (SG302+LNA210); SG302 and a 2'-O-methyl-modified miR-210
miRNA antagonist (SG302+2'-O-methyl 210); ssh-NSC and a
LNA-modified control miRNA antagonist (ssh-NSC+LNA-NSC); LNA210;
2'-O-methyl 210; and LNA-NSC. FIG. 7C illustrates miRNA qRT-PCR
(miR-qRT-PCR) of miR-210 miRNA in human HaCaT keratinocytes
transfected with the following: no inhibitor; SG302; ssh-NSC;
SG302+LNA210; SG302+2'-O-methyl 210; ssh-NSC+LNA-NSC; LNA210;
2'-O-methyl 210; and LNA-NSC. FIG. 7D illustrates a luciferase
assay with a miR-210 reporter in human HaCaT keratinocytes
transfected with no inhibitor or a miR-210 mimic. FIG. 7E
illustrates a luciferase assay with a miR-210 reporter in human
HaCaT keratinocytes treated with the following: no inhibitor
(column 1); CoCl.sub.2 (column 2); CoCl.sub.2 and increasing
concentrations of DNA modified miR-210 miRNA antagonist (DNA210;
columns 3-6); CoCl.sub.2 and increasing concentrations of miR-210
miRNA antagonist (RNA210; columns 7-10); CoCl.sub.2 and increasing
concentrations of 2'-O-methyl modified miR-210 miRNA antagonist
(2'-O-methyl 210; columns 11-14); CoCl.sub.2 and increasing
concentrations of LNA modified miR-210 miRNA antagonist (LNA210;
columns 15-18); and CoCl.sub.2 and increasing concentrations of a
control miRNA antagonist (NSC; columns 19-22). FIG. 7F illustrates
qRT-PCR of human PHD2 transcript in human primary keratinocytes
transfected with increasing amounts of an sshRNA targeting human
PHD2 (SG302) and a control sshRNA (SG221c). FIG. 7G illustrates
qRT-PCR of miR-210 in human primary keratinocytes transfected with
increasing amounts of an sshRNA targeting human PHD2 (SG302) and a
control sshRNA (SG221c). FIG. 7H illustrates qRT-PCR of mouse PHD2
transcript in NIH-3T3 cells transfected with increasing amounts of
an sshRNA targeting mouse PHD2 (SG404). FIG. 7I illustrates qRT-PCR
of miR-210 in NIH-3T3 cells transfected with increasing amounts of
an sshRNA targeting mouse PHD2 (SG404). FIG. 7J illustrates
relative expression of PHD2 and miR-210 both alone and in
combination with PHD2-targeting SG302 sshRNA and antimiR-210
(SG603) in HaCaT cells.
[0025] FIG. 8A-FIG. 8C illustrate cytokine levels in response to
treatment with a miRNA antagonist. FIG. 8A illustrates qRT-PCR for
IFN-.beta. in MRC-5 lung fibroblasts treated with: no inhibitor;
polyI:C; a DNA modified miR-210 miRNA antagonist (210 DNA anti); a
miR-210 miRNA antagonist (210 RNA anti); a 2'-O-methyl modified
miR-210 miRNA antagonist (210 2'-O-methyl anti); a LNA modified
miR-210 miRNA antagonist (210 LNA anti); and a LNA modified control
miRNA antagonist (210 NSC (LNA)). FIG. 8B illustrates qRT-PCR for
IL-6 in cells treated with: no inhibitor; polyI:C; 210 DNA anti;
210 RNA anti; 210 2'-O-methyl anti; 210 LNA anti; and 210 NSC
(LNA). FIG. 8C illustrates qRT-PCR for TNF-.alpha. in cells treated
with: no inhibitor; polyI:C; 210 DNA anti; 210 RNA anti; 210
2'-O-methyl anti; 210 LNA anti; and 210 NSC (LNA).
[0026] FIG. 9 illustrates expression of PHD2 transcript in response
to treatment with an RNAi molecule.
[0027] FIG. 10A-FIG. 10F illustrate wound closure over time in
response to treatment with an RNAi molecule or a miRNA antagonist.
FIG. 10A illustrates the percentage of original wound area over 25
days in the Layer by Layer group (LbL), A6K, HiPerFect, and Control
treatment groups. FIG. 10B illustrates the days until the wound
closed in the LbL, A6K, HiPerFect, and Control treatment groups.
FIG. 10C illustrates the percentage of original wound area over 25
days in the Untreated, SG404, SG603, and SG221c treatment groups.
FIG. 10D illustrates the days until the wound closed in each
treatment group. FIG. 10E illustrates representative images of
fluorescence staining at day 7 for untreated or LbL topical
treatment of control sshRNAs (SG221c) or SG404 sshRNA treatment
group. FIG. 10F illustrates computed values (integrated density) of
vWF staining for each treatment group.
[0028] FIG. 11A-FIG. 11D illustrate expression of PHD2 transcript
in response to treatment with an RNAi molecule. FIG. 11A
illustrates qRT-PCR of human PHD2 transcript in human kidney 293FT
cells transfected with either 1 nM or 10 nM of the following: no
inhibitor; sshRNAs designed to target human PHD2 (SG302,
SG304-SG309); and sshRNAs designed to target mouse PHD2
(SG400-SG402); and a control sshRNA (NSC). FIG. 11B illustrates
qRT-PCR of mouse PHD2 (mPHD2) transcript in mouse NIH3T3
fibroblasts transfected with increasing amounts of sshRNAs designed
to target mouse PHD2 (SG404) or a modified sshRNA designed to
target human PHD2 (SG302m1). FIG. 11C illustrates qRT-PCR of human
PHD2 (hPHD2) transcript in human HaCaT keratinocytes transfected
with increasing amounts of sshRNAs designed to target either human
PHD2 or mouse PHD2 (SG312, and SG314-SG316) or a modified sshRNA
designed to target human PHD2 (SG302m1). FIG. 11D illustrates
qRT-PCR of mouse PHD2 (mPHD2) transcript in mouse NIH3T3
fibroblasts transfected with increasing amounts of sshRNAs designed
to target either human PHD2 or mouse PHD2 (SG312, and SG314-SG316)
or a modified sshRNA designed to target human PHD2 (SG302m1).
[0029] FIG. 12A-FIG. 12B illustrate cell scratch closure over time
in response to treatment with an RNAi molecule and a miRNA
antagonist. FIG. 12A illustrates the percent scratch closure at 24
hours (24 h), 48 h, and 72 h of human HaCaT keratinocytes
transfected with the following: an sshRNA targeting human PHD2
(SG302) and a LNA-modified miR-210 miRNA antagonist (SG302+LNA210);
SG302 and a 2'-O-methyl-modified miR-210 miRNA antagonist
(SG302+2'-O-methyl 210); and a control sshRNA (ssh-NSC) and a
LNA-modified control miRNA antagonist (ssh-NSC+LNA-NSC). FIG. 12B
provides representative images of the scratch wounds at 0 hours (0
h), 24 h, and 48 h of human HaCaT keratinocytes transfected with
SG302+LNA210, SG302+2'-O-methyl 210, and ssh-NSC+LNA-NSC.
[0030] FIG. 13A-FIG. 13F illustrate designs for a pre-miRNA mimic.
FIG. 13A-FIG. 13E illustrate designs for pre-miRNA mimics of miR-21
and disclose SEQ ID NOS 44-48, respectively. FIG. 13F illustrates a
luciferase assay with a miR-21 reporter in human 293FT cells
treated with increasing concentrations of the following: SG701
(FIG. 13A), SG702 (FIG. 13B), SG703 (FIG. 13C), SG703 (replicate 2)
(FIG. 13C), SG704 (FIG. 13D), miRIDIAN miR-21mimic (Dharmacon,
positive control) and cel-67 mimic (Dharmacon, miRIDIAN miRNA mimic
negative control #1).
[0031] FIG. 14A-FIG. 14B illustrate GFP positive cell number in
cells treated with a pre-miRNA mimic. FIG. 14A illustrates the
average (Ave) number of Oct4-GFP-positive colonies in the mock,
miRIDIAN, and Somagenics groups at 10, 12, 14, and 16 days after
transfection. FIG. 14B illustrates a percentage of GFP-positive
cells relative to cells transfected with a control vector over a
time course in the seed mutant, MiRIDIAN, and Somagenics treatment
groups.
[0032] FIG. 15 illustrates uptake of LBL-PHD2 over time in response
to treatment with an RNAi molecule.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Disclosed herein are oligonucleotides for use alone or in
combination in the treatment of wounds in a subject in need
thereof. Oligonucleotides disclosed herein include short small
hairpin RNAs (sshRNAs), micro-RNA (miRNA) antagonists and pre-miRNA
mimics. These oligonucleotides modify the activity and/or
expression of RNAs.
[0034] Wound healing is an orchestrated physiological process in
which multiple cell types interact to close a physical insult.
Dysregulation of wound healing contributes to the pathology of
various diseases. Wound management and healing is currently a great
concern for human patients suffering from diabetes mellitus (DM).
Due to peripheral neuropathy, a complication of DM, wounds, such as
foot ulcers, may often go unnoticed by the diabetic patient. New
approaches are needed to ameliorate wound healing in these
pathological settings.
[0035] Wound healing is a complex and dynamic biological process
with a least three overlapping phases: 1) an inflammatory phase,
characterized by vasoconstriction, platelet aggregation, clot
formation and phagocytosis, which usually occurs in the first 2-5
days of initial insult or injury; 2) a proliferative phase,
characterized by granulation (e.g., fibroblasts lay a collagen
bed), angiogenesis (e.g., new vessel growth), contraction (e.g.,
edges of wounds pull together) and epithelialization, which usually
occurs from 2 days-3 weeks of initial insult or injury; and 3) a
remodeling phase, in which new collagen forms to provide tensile
strength across the wound (3 weeks-2 years from initial insult or
injury). A successful wound management strategy should seek rapid
wound closure through effective inflammatory and proliferative
phases, leading to a productive remodeling phase with minimized
risk of infection.
[0036] There are several biological pathways and mechanisms
involved in wound healing, any of which may be targeted by the
oligonucleotides disclosed herein. However, exemplified herein, is
the pathway involving prolyl hydroxylase domain-containing protein
2 (PHD2), an enzyme encoded by the Eg1 nine homolog 1 (EGLN1) gene.
It is also known as hypoxia-inducible factor prolyl hydroxylase 2
(HIF-PH2). PHD2 is critical for targeting hypoxia-inducible factor
1 alpha (HIF-1.alpha.) for polyubiquitylation and subsequent
degradation. Under normoxia, PHD2 hydroxylates specific proline
residues in cytoplasmic HIF-1.alpha. that are recognized by VHL, a
signal for ubiquitylation to introduce Ubiquitin residues.
Ubiquitylated HIF-1.alpha. is rapidly degraded through the
proteasome pathway. Under hypoxia, PHD2 stays inactive and as a
result, HIF-1.alpha. becomes stabilized and translocates to the
nucleus. It dimerizes with ARNT to bind HRE elements in promoters
of a large number of genes. Upon binding to HRE, HIF-1a/ARNT
complex recruits CBP/p300 to activate gene expression. A knockdown
of PHD2 results in an increase in levels of HIF-1.alpha. under
normoxic conditions. HIF-1.alpha. activates multiple factors that
enhance overall wound healing through stimulating cell survival,
motility and proliferation, angiogenesis, progenitor cell
recruitment and re-epithelialization, including, but not limited
to, VEGF, EPO, PDGF, PLGF, VEGF-rl, ANG-1, ANG-2, iNOS, IGF2,
TGF-beta, PAI-1, SDF-1, TFR, and TF-r.
[0037] Effective wound healing requires that multiple factors play
their individual roles in a time-and tissue-specific manner.
Failure of any element in this process can hamper healing. For
example, under normal wound healing settings, HSPs, PDGF, FGF and
VEGF are induced within 24 h of skin injury and eventually decline
from this elevated level of protein synthesis to a basal level in
7-14 days. However, in chronic wounds, the expression of these
proteins is delayed or inhibited. As such, the induction or
increased activity of these factors may be beneficial. During the
course of wound healing, a variety of HSPs are expressed in a
specific temporal and spatial manner. The biological induction of a
broad spectrum of HSPs may be a better approach to promoting wound
healing than providing just a single HSP. Because wound healing
involves several key molecular pathways and cell types, a
multi-pronged approach is used to modulate a range of biological
molecules and processes to promote an overall increase in
wound-healing activity.
[0038] The present invention is directed to oligonucleotides,
pharmaceutical compositions, polynucleotide vectors, and methods
for performing RNA interference (RNAi), miRNA-mediated inhibition,
and inhibition of miRNAs. Non-limiting examples of oligonucleotides
include short interfering RNAs (siRNAs), small hairpin RNAs
(shRNAs), short small hairpin RNAs (sshRNAs), pre-miRNA mimics,
miRNA antagonists. In some embodiments, the oligonucleotide is an
RNAi agent. Non-limiting examples of RNAi agents include short
interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), short small
hairpin RNAs (sshRNAs), and RNAi duplexes, such as pre-miRNA
mimics.
Oligonucleotides
[0039] Disclosed herein are oligonucleotides, both alone and in
combination, for use in the treatment of wounds in a subject in
need thereof. These oligonucleotides may modify the activity and/or
expression of RNAs by various mechanisms of action.
Oligonucleotides disclosed herein include RNAi agents, such as
short small hairpin RNAs (sshRNAs) and pre-miRNA mimics, as well as
RNAi antagonists, such as micro-RNA (miRNA) antagonists.
[0040] Short small hairpin RNAs (sshRNAs) differ from traditional
shRNAs in that they have a stem sufficiently short, 19 base pairs
or less, to disqualify them as a Dicer substrates. Since sshRNAs
cannot be processed by Dicer in contrast to ordinary shRNAs, they,
therefore, must be processed by a Dicer-independent mechanism.
sshRNAs also differ from ordinary shRNAs in that the loop length
can be much shorter, which confers stability without loss of
activity. sshRNAs are predominantly loaded as intact molecules into
Argonaute (Ago)-containing complexes without prior processing by
Dicer and are activated by Ago2-mediated cleavage of the passenger
arm of the hairpin without prior cleavage of the loop. Advantages
of sshRNAs over traditional shRNAs may include, but are not limited
to, reduced manufacturing cost, fewer off-target effects of
passenger strand, increased stability and resistance to
endonucleases, lower immunostimulatory activity, and increased
potency. sshRNAs disclosed herein may be chemically synthesized.
Chemically synthesized sshRNAs may also be referred to as
"synthetic sshRNAs" or "synthetic shRNAs" or "short synthetic
shRNAs." sshRNAs that are chemically synthesized can contain
modified nucleotides and or backbones in precise modification
patterns that enhance efficacy with diminished immune stimulation
potential.
[0041] While sshRNAs typically inhibit the expression or activity
of messenger RNAs and are duplex RNA, miRNA antagonists typically
inhibit the expression or activity of endogenous miRNA and are
single-stranded.
[0042] Pre-miRNA mimics are synthetic miRNA precursors, meaning
they have not yet been processed into mature miRNA. Pre-miRNA
mimics are hairpin, duplex (double-stranded) RNAs. Pre-miRNA mimics
disclosed herein may have a shorter stem and/or a smaller loop
region than naturally-occurring miRNA precursors. This may make the
pre-miRNA mimics less susceptible to degradation by endonucleases.
For instance, generally, naturally-occurring miRNAs and their
precursors have a loop region of 4 nucleotides or more. Pre-miRNA
mimics disclosed herein may have a loop region of 2 nucleotides or
fewer. Pre-miRNA mimics disclosed herein may have a loop region
consisting of 2 nucleotides or 1 nucleotide. Pre-miRNA mimics
disclosed herein may have a loop region consisting of 2 nucleotides
or 1 nucleotide. Pre-miRNA mimics disclosed herein may have a loop
region consisting of a direct connection. Pre-miRNA mimics
disclosed herein may have a loop region with a length of 2
nucleotides, 1 nucleotide, or zero nucleotides. Pre-miRNA mimics
disclosed herein may have a single stem, wherein the stem is 19 bp
in length or less. Pre-miRNA mimics disclosed herein may have a
single stem, wherein the stem is 10 bp to 19 bp in length.
Pre-miRNA mimics disclosed herein may comprise an overhang region
on either the end of the sense strand or end of the antisense
strand that is not adjacent to the loop region. Pre-miRNA mimics
disclosed herein may comprise only a single overhang region.
[0043] It has been found that introducing pre-miRNA mimics to
cells, rather than the mature miRNAs, is more efficient for
boosting the RNAi activity of endogenous miRNA. Thus, the
oligonucleotides disclosed herein provide means to both inhibit
(via sshRNAs and pre-miRNA mimics) and boost (via miRNA
antagonists) messenger RNA expression and activity.
[0044] While the oligonucleotides disclosed herein mainly comprise
RNA, these oligonucleotides may also comprise some modified
ribonucleotides or substitutions for ribonucleotides, such that
other nucleic acids are incorporated as well. These modified
ribonucleotides may also be referred to as ribonucleotides with
chemical modifications. Modified ribonucleotides include
deoxyribonucleic acids (DNA), locked nucleic acids (LNA), and
peptide nucleic acids (PNA), non-nucleic acids, or any combination
thereof.
[0045] Although the oligonucleotides disclosed herein may comprise
modifications, substitutions, non-nucleotides, nucleotide moieties,
conjugate moieties, etc., the majority of the nucleotides or
nucleotide moieties in the oligonucleotides disclosed herein may be
unmodified ribonucleotides. In some embodiments, at least 30% of
the oligonucleotide consists of unmodified ribonucleotides. In some
embodiments, at least 35% of the oligonucleotide consists of
unmodified ribonucleotides. In some embodiments, at least 40% of
the oligonucleotide consists of unmodified ribonucleotides. In some
embodiments, at least 45% of the oligonucleotide consists of
unmodified ribonucleotides. In some embodiments, at least 50% of
the oligonucleotide consists of unmodified ribonucleotides. In some
embodiments, at least 51% of the oligonucleotide consists of
unmodified ribonucleotides. In some embodiments, at least 55% of
the oligonucleotide consists of unmodified ribonucleotides. In some
embodiments, at least 60% of the oligonucleotide consists of
unmodified ribonucleotides. In some embodiments, at least 65% of
the oligonucleotide consists of unmodified ribonucleotides. In some
embodiments, at least 70% of the oligonucleotide consists of
unmodified ribonucleotides. In some embodiments, at least 75% of
the oligonucleotide consists of unmodified ribonucleotides.
[0046] In some embodiments, the oligonucleotide is at least 55%
RNA. In some embodiments, the oligonucleotide is at least 60% RNA.
In some embodiments, the oligonucleotide is at least 70% RNA. In
some embodiments, the oligonucleotide is at least 80% RNA. In some
embodiments, the oligonucleotide is at least 90% RNA. In some
embodiments, the oligonucleotide is at least 95% RNA.
[0047] shRNAs, sshRNAs, and pre-miRNA mimics are unimolecular
nucleic acid-containing polynucleotides comprising a sense
sequence, a loop region, and an antisense sequence, with the sense
and antisense sequences being at least partially complementary. In
some embodiments, the sense and antisense sequences can be about
60%, about 65%, about 70%, about 75%, about 80%, about 81%, about
82%, about 83%, about 84%, about 85%, about 86%, about 87%, about
88%, about 89%, about 90%, about 91%, about 92%, about 93%, about
94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%
complementary.
[0048] In some embodiments, the sense and antisense sequences are
from at least about 60% to at least about 95% complementary, from
at least about 65% to at least about 95% complementary, from at
least about 70% to at least about 95% complementary, from at least
about 75% to at least about 95% complementary, from at least about
80% to at least about 95% complementary.
[0049] In some embodiments, the antisense sequence or the sense
sequence each independently have a length of about 10 nucleotides,
about 11 nucleotides, about 12 nucleotides, about 13 nucleotides,
about 14 nucleotides, about 15 nucleotides, about 16 nucleotides,
about 17 nucleotides, about 18 nucleotides, about 19 nucleotides,
about 20 nucleotides, about 21 nucleotides, about 22 nucleotides,
about 23 nucleotides, about 24 nucleotides, about 25 nucleotides,
about 26 nucleotides, about 27 nucleotides, about 28 nucleotides,
about 29 nucleotides, or about 30 nucleotides, about 31
nucleotides, about 32 nucleotides, about 33 nucleotides, about 34
nucleotides, about 35 nucleotides, about 36 nucleotides, about 37
nucleotides, about 38 nucleotides, about 39 nucleotides, about 40
nucleotides, about 41 nucleotides, about 42 nucleotides, about 43
nucleotides, about 44 nucleotides, about 45 nucleotides, about 46
nucleotides, about 47 nucleotides, about 48 nucleotides, about 49
nucleotides, or about 50 nucleotides.
[0050] In some embodiments, the antisense sequence or sense
sequence can be from about 10 nucleotides to about 11 nucleotides,
from about 11 nucleotides to about 12 nucleotides, from about 12
nucleotides to about 13 nucleotides, from about 13 nucleotides to
about 14 nucleotides, from about 14 nucleotides to about 15
nucleotides, from about 15 nucleotides to about 16 nucleotides,
from about 16 nucleotides to about 17 nucleotides, from about 17
nucleotides to about 18 nucleotides, from about 18 nucleotides to
about 19 nucleotides, from about 19 nucleotides to about 20
nucleotides, from about 20 nucleotides to about 21 nucleotides,
from about 21 nucleotides to about 22 nucleotides, from about 22
nucleotides to about 23 nucleotides, from about 23 nucleotides to
about 24 nucleotides, from about 24 nucleotides to about 25
nucleotides, from about 25 nucleotides to about 26 nucleotides,
from about 26 nucleotides to about 27 nucleotides, from about 27
nucleotides to about 28 nucleotides, from about 28 nucleotides to
about 29 nucleotides, from about 29 nucleotides to about 30
nucleotides.
[0051] In some embodiments, the shRNA, sshRNA, or pre-miRNA mimic
has a loop region that directly connects the sense and antisense
sequences. The loop region that directly connects the sense and
antisense sequences may also be referred to as a loop having zero
nucleotides. In some embodiments, the shRNA, sshRNA, or pre-miRNA
mimic has a loop region comprising zero nucleotides. In some
embodiments, the loop region has a length of 1 nucleotide, 2
nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6
nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10
nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14
nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18
nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22
nucleotides, 23 nucleotides, or 24 nucleotides.
[0052] In some embodiments, a small loop region confers resistance
to degradation by endonucleases. The small loop region may have a
length of about zero nucleotides, about 1 nucleotide or about 2
nucleotides. In some embodiments, the sshRNAs consist of a sense
strand, a loop region, an antisense strand, and optionally one or
more conjugate moieties, wherein the sense strand has a length of
10-19 nucleotides, the loop region has a length of 2 nucleotides or
less, and the antisense strand has a length of 10-19 nucleotides.
In some embodiments, the sshRNAs consist of a sense strand, a loop
region, an antisense strand, and optionally one or more conjugate
moieties, wherein the sense strand has a length of 10-19
nucleotides, the loop region has a length of less than 2
nucleotides, and the antisense strand has a length of 10-19
nucleotides.
[0053] In some embodiments, the loop region can comprise
non-nucleotide moieties. The loop region may comprise
non-nucleotide moieties from about 1 non-nucleotide moiety to about
2 non-nucleotide moieties, from about 2 non-nucleotide moieties to
about 3 non-nucleotide moieties, from about 3 non-nucleotide
moieties to about 4 non-nucleotide moieties, from about 4
non-nucleotide moieties to about 5 non-nucleotide moieties, from
about 5 non-nucleotide moieties to about 6 non-nucleotide moieties,
from about 6 non-nucleotide moieties to about 7 non-nucleotide
moieties, from about 7 non-nucleotide moieties to about 8
non-nucleotide moieties, from about 8 non-nucleotide moieties to
about 9 non-nucleotide moieties, from about 9 non-nucleotide
moieties to about 10 non-nucleotide moieties, from about 10
non-nucleotide moieties to about 11 non-nucleotide moieties, from
about 11 non-nucleotide moieties to about 12 non-nucleotide
moieties, from about 12 non-nucleotide moieties to about 13
non-nucleotide moieties, from about 13 non-nucleotide moieties to
about 14 non-nucleotide moieties, from about 14 non-nucleotide
moieties to about 15 non-nucleotide moieties, from about 15
non-nucleotide moieties to about 16 non-nucleotide moieties, from
about 16 non-nucleotide moieties to about 17 non-nucleotide
moieties, from about 17 non-nucleotide moieties to about 18
non-nucleotide moieties, from about 18 non-nucleotide moieties to
about 19 non-nucleotide moieties, from about 19 non-nucleotide
moieties to about 20 non-nucleotide moieties, from about 20
non-nucleotide moieties to about 21 non-nucleotide moieties, from
about 21 non-nucleotide moieties to about 22 non-nucleotide
moieties, from about 22 non-nucleotide moieties to about 23
non-nucleotide moieties, or from about 23 non-nucleotide moieties
to about 24 non-nucleotide moieties.
[0054] In some embodiments, the shRNA, sshRNA, or pre-miRNA mimic
has a loop region consisting of: 1 nucleotide or non-nucleotide
moiety; 2 nucleotides, non-nucleotide moieties, or a combination
thereof; 3 nucleotides, non-nucleotide moieties, or a combination
thereof; 4 nucleotides, non-nucleotide moieties, or a combination
thereof; 5 nucleotides, non-nucleotide moieties, or a combination
thereof; 6 nucleotides, non-nucleotide moieties, or a combination
thereof; 7 nucleotides, non-nucleotide moieties, or a combination
thereof; 8 nucleotides, non-nucleotide moieties, or a combination
thereof; 9 nucleotides, non-nucleotide moieties, or a combination
thereof; 10 nucleotides, non-nucleotide moieties, or a combination
thereof 11 nucleotides, non-nucleotide moieties, or a combination
thereof 12 nucleotides, non-nucleotide moieties, or a combination
thereof 13 nucleotides, non-nucleotide moieties, or a combination
thereof 14 nucleotides, non-nucleotide moieties, or a combination
thereof; 15 nucleotides, non-nucleotide moieties, or a combination
thereof 16 nucleotides, non-nucleotide moieties, or a combination
thereof 17 nucleotides, non-nucleotide moieties, or a combination
thereof 18 nucleotides, non-nucleotide moieties, or a combination
thereof 19 nucleotides, non-nucleotide moieties, or a combination
thereof 20 nucleotides, non-nucleotide moieties, or a combination
thereof 21 nucleotides, non-nucleotide moieties, or a combination
thereof 22 nucleotides, non-nucleotide moieties, or a combination
thereof; 23 nucleotides, non-nucleotide moieties, or a combination
thereof; or 24 nucleotides, non-nucleotide moieties, or a
combination thereof. As used herein, the terms nucleotide and
non-nucleotide moiety, may be used interchangeably, unless
otherwise noted.
[0055] In some embodiments, the sequence of the loop region can
include nucleotide residues unrelated to the target. In some
embodiments, the nucleotides of the loop are chosen from: rA, dA,
rC, dC, rG, dG, rU, dU, rT, and dT, wherein r is a nucleotide
comprising a ribose sugar; d is a nucleotide comprising a
deoxyribose sugar; A is a nucleotide comprising an adenine base; C
is a nucleotide comprising a cytosine base; G is a nucleotide
comprising a guanosine base; U is a nucleotide comprising a uracil
base; and T is a nucleotide comprising a thymine base. In some
embodiments, the loop sequence is chosen from: 5'-rUrU-3',
5'-rTrU-3', 5'-rUrT-3', 5'-dUdU-3', 5'-dTdU-3', 5'-dUdT-3',
5'-rTrT-3', and 5'-dTdT-3'.
[0056] In some embodiments, the loop region comprises a
deoxyribonucleotide, a phosphorothioate internucleotide linkage, a
2'-O-alkyl modification, a non-nucleotide monomer or a reversible
linkage, or combinations thereof. In some embodiments, the
reversible linkage is a disulfide bond. In some embodiments, the
2'-O-alkyl modification is a 2'-O-methyl modification.
[0057] An antisense sequence of the oligonucleotides disclosed
herein can be substantially complementary to a messenger RNA
(mRNA), an RNA that is not an mRNA, or a sequence of DNA that is
either coding or non-coding. Non-limiting examples of mRNAs include
5'-methylguanosine capped, 3'-polyadenylated mRNAs;
5'-methylguanosine capped, de-adenylated mRNAs; de-capped,
3'-polyadenylated mRNAs; de-capped, de-adenylated mRNAs; P-body
associated mRNAs; stress granule associated mRNAs; cytosolic mRNAs;
and endoplasmic reticulum associated mRNAs. Non-limiting examples
of non-mRNA RNAs include transfer RNAs (tRNAs), ribosomal RNAs
(rRNAs), heteronuclear RNAs (hnRNAs), Piwi-inducible RNAs (piRNAs),
negative strand viral RNA, and positive stranded viral RNA.
[0058] An RNAi agent is specific for a target transcript when the
antisense sequence is substantially complementary to the target
transcript. Substantially complementary can be from about 80% to
about 85% complementary, from about 85% to about 90% complementary,
from about 90% to about 95% complementary, from about 95% to about
96% complementary, from about 96% to about 97% complementary, from
about 97% to about 98% complementary, from about 98% to about 99%
complementary, or from about 99% to 100% complementary.
[0059] The sense sequence of the pre-miRNA mimic can comprise a
sequence with identity from 5' to 3' to a naturally occurring
miRNA. The sense sequence can comprise a sequence with about 60%,
about 65%, about 70%, about 75%, about 80%, about 81%, about 82%,
about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,
about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,
about 95%, about 96%, about 97%, about 98%, or 100% identity to a
naturally occurring miRNA. The sense sequence can comprise sequence
from about 60% to about 99%, from about 65% to about 99%, from
about 70% to about 99%, from about 75% to about 99%, from about 80%
to about 99%, from about 85% to about 99%, from about 90% to about
99% identity from 5' to 3' to a naturally occurring miRNA.
[0060] The antisense strand of the pre-miRNA mimic has
complementarity from 5' to 3' to the sense strand of the pre-miRNA
mimic. The antisense strand can have about 60%, about 65%, about
70%, about 75%, about 80%, about 81%, about 82%, about 83%, about
84%, about 85%, about 86%, about 87%, about 88%, about 89%, about
90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
96%, about 97%, about 98%, or 100% complementarity from 5' to 3' to
the sense strand of the pre-miRNA mimic. The antisense strand can
have from about 60% to about 65%, from about 65% to about 70%, from
about 70% to about 75%, from about 75% to about 80%, from about 80%
to about 81%, from about 81% to about 82%, from about 82% to about
83%, from about 83% to about 84%, from about 84% to about 85%, from
about 85% to about 86%, from about 86% to about 87%, from about 87%
to about 88%, from about 88% to about 89%, from about 89% to about
90%, from about 90% to about 91%, from about 91% to about 92%, from
about 92% to about 93%, from about 93% to about 94%, from about 94%
to about 95%, from about 95% to about 96%, from about 96% to about
97%, from about 97% to about 98%, or from about 98% to about 100%
complementarity from 5' to 3' to the sense strand of the pre-miRNA
mimic.
[0061] In some embodiments, the siRNA, shRNA, sshRNA, or pre-miRNA
mimic has no overhang or an overhang region of about 1 nucleotide,
about 2 nucleotides, about 3 nucleotides, about 4 nucleotides,
about 5 nucleotides, about 6 nucleotides, about 7 nucleotides,
about 8 nucleotides, about 9 nucleotides, about 10 nucleotides,
about 11 nucleotides, or about 12 nucleotides on the 5' end or the
3' end. In some embodiments, the siRNA, shRNA, sshRNA, or pre-miRNA
mimic can have an overhang region from about 1 nucleotide to about
2 nucleotides, from about 2 nucleotides to about 3 nucleotides,
from about 3 nucleotides to about 4 nucleotides, from about 4
nucleotides to about 5 nucleotides, from about 5 nucleotides to
about 6 nucleotides, from about 6 nucleotides to about 7
nucleotides, from about 7 nucleotides to about 8 nucleotides, from
about 8 nucleotides to about 9 nucleotides, from about 9
nucleotides to about 10 nucleotides, from about 10 nucleotides to
about 11 nucleotides, or from about 11 nucleotides to about 12
nucleotides on the 5' end or the 3' end. Nucleotides of the
overhang region can be unmodified, or can contain one or more
modifications. Non-limiting examples of overhang modifications
include modifications of the 2' position of a sugar, such as
halogen or O-alkyl substitution, cholesterol or alpha-tocopherol,
and modifications of a phosphate group such as phosphorothioate
modification. A nucleotide of the overhang region can be a
ribonucleic acid, a deoxyribonucleic acid, or any combination
thereof.
[0062] In some embodiments, the shRNA, sshRNA, or pre-miRNA mimic
comprises RNAs with base-pair mismatches or bulges. The sense
sequence may be identical to the target transcript or, through
mismatches, insertions, or deletions, have differences at about 1
nucleotide, about 2 nucleotides, about 3 nucleotides, about 4
nucleotides, or about 5 nucleotides from a target transcript.
[0063] A miRNA antagonist is a unimolecular nucleic acid-containing
polynucleotide comprising a sequence complementary to a mature
miRNA. The miRNA antagonist can include RNA, DNA, LNA, PNA, one or
more 2'-O-methyl modifications, phosphorothioate internucleotide
linkage(s), ZEN modification(s), and non-nucleic acids, and any
combination thereof. Non-limiting examples of miRNA antagonists
include antagomiRs, all-DNA anti-miRs, all-RNA anti-miRs, and LNA
anti-miRs. In some embodiments, the miRNA antagonist can have a
length of about 7 nucleotides, about 8 nucleotides, about 9
nucleotides, about 10 nucleotides, about 11 nucleotides, about 12
nucleotides, about 13 nucleotides, about 14 nucleotides, about 15
nucleotides, about 16 nucleotides, about 17 nucleotides, about 18
nucleotides, about 19 nucleotides, about 20 nucleotides, about 21
nucleotides, about 22 nucleotides, about 23 nucleotides, about 24
nucleotides, about 25 nucleotides, about 26 nucleotides, about 27
nucleotides, about 28 nucleotides, about 29 nucleotides, about 30
nucleotides, about 31 nucleotides, about 32 nucleotides, about 33
nucleotides, about 34 nucleotides, about 35 nucleotides, about 36
nucleotides, about 37 nucleotides, about 38 nucleotides, about 39
nucleotides, about 40 nucleotides, about 41 nucleotides, about 42
nucleotides, about 43 nucleotides, about 44 nucleotides, about 45
nucleotides, about 46 nucleotides, about 47 nucleotides, about 48
nucleotides, about 49 nucleotides, or about 50 nucleotides.
[0064] In some embodiments, the miRNA antagonist comprises a
sequence about 60%, about 65%, about 70%, about 75%, about 80%,
about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,
about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99%, or 100% complementary to a mature miRNA.
[0065] In some embodiments, the miRNA antagonist comprises a
sequence from about 60% to about 100%, from about 65% to about
100%, from about 70% to about 100%, from about 75% to about 100%,
from about 80% to about 100%, from about 85% to about 100%, or from
about 90% to about 100%, complementary to a mature miRNA.
[0066] The target of an RNAi agent disclosed herein may be an RNA
transcript encoding prolyl hydroxylase domain-containing protein 2
(PHD2). PHD2, by way of non-limiting example, may be human PHD2,
mouse PHD2, simian PHD2, bovine PHD2, canine PHD2, or PHD2
expressed in any animal in need of a wound healing treatment. The
target of an RNAi agent disclosed herein may be an RNA transcript
encoded by an EGLN1 homolog. The EGLN1 homolog may be at least
about 70%, at least about 75%, at least about 80%, at least about
85%, or at least about 90% homologous to SEQ ID NO. 1 or SEQ ID NO.
2. The terms "homologous," "homology," or "percent homology" when
used herein to describe to an amino acid sequence or a nucleic acid
sequence, relative to a reference sequence, can be determined using
the formula described by Karlin and Altschul (Proc. Natl. Acad.
Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci.
USA 90:5873-5877, 1993). Such a formula is incorporated into the
basic local alignment search tool (BLAST) programs of Altschul et
al. (J. Mol. Biol. 215: 403-410, 1990). Percent homology of
sequences can be determined using the most recent version of BLAST,
as of the filing date of this application. In some embodiments,
EGLN1 is chosen from human EGLN1 (SEQ ID NO. 1) and mouse EGLN1
(SEQ ID NO. 2). In some embodiments, an RNAi agent is designed to
target a transcript of both human EGLN1 (SEQ ID NO. 1) and mouse
EGLN1 (SEQ ID NO. 2). In some embodiments, an sshRNA is designed to
target a transcript of human EGLN1 (SEQ ID NO. 1). In some
embodiments, an sshRNA is designed to target a transcript of mouse
EGLN1 (SEQ ID NO. 2). In some embodiments, an sshRNA is designed to
target transcripts of both human EGLN1 (SEQ ID NO. 1) and mouse
EGLN1 (SEQ ID NO. 2).
[0067] An RNAi agent targeting PHD2 can reduce the amount of PHD2
transcript about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, about 99%, about 99.5%, or about 99.9%.
[0068] An RNAi agent targeting PHD2 can reduce the amount of PHD2
transcript from about 10% to about 99.9%, from about 10% to about
99%, from about 20% to about 99%, from about 30% to about 99%, from
about 40% to about 99%, from about 50% to about 99%, from about 55%
to about 99%, from about 60% to about 99%, from about 65% to about
99%, from about 70% to about 99%, from about 75% to about 99%, from
about 80% to about 99%.
[0069] In some embodiments, RNAi-mediated depletion of PHD2 can
increase the amount of HIF-1.alpha. protein and/or HIF-1.alpha.
target gene expression. Non-limiting examples of HIF-1.alpha.
target genes include VEGF, HSP90, and HSP70. An RNAi agent
targeting PHD2 can increase the amount of HIF-1.alpha. protein
and/or HIF-1.alpha. target gene expression about 1%, about 5%,
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 100%, about 200%, about
300%, about 400%, about 500%, about 600%, about 700%, about 800%,
about 900%, about 1,000%, about 2,000%, about 3,000%, about 4,000%,
about 5,000%, about 6,000%, about 7,000%, about 8,000%, about
9,000%, or about 10,000%.
[0070] An RNAi agent targeting PHD2 may increase the amount of
HIF-1.alpha. protein and/or HIF-1.alpha. target gene expression
from about 10% to about 10,000%, from about 20% to about 10,000%,
from about 30% to about 10,000%, from about 40% to about 10,000%,
from about 50% to about 10,000%, or from about 100% to about
10,000%.
[0071] Disclosed herein are miRNA antagonists and pre-miRNA mimics
designed to antagonize and mimic naturally occurring miRNAs,
respectively. In some embodiments, the pre-miRNA mimics are used to
"boost" the expression of a mature miRNA. In some embodiments, a
naturally occurring miRNA or a mature miRNA can be chosen from
miR-210 (SEQ ID NO. 3) and miR-21 (SEQ ID NO. 4). In some
embodiments, the naturally occurring miRNA or mature miRNA is
miR-210 (SEQ ID NO. 3). In some embodiments, the naturally
occurring miRNA or mature miRNA is miR-21 (SEQ ID NO. 4).
[0072] A pre-miRNA mimic, also referred to herein as an pre-miRNA
mimic, may increase a steady-state level of a mature miRNA about
1%, about 5%, about 10%, about 20%, about 30%, about 40%, about
50%, about 60%, about 70%, about 80%, about 90%, about 100%, about
200%, about 300%, about 400%, about 500%, about 600%, about 700%,
about 800%, about 900%, about 1000%, about 2000%, about 3000%,
about 4000%, about 5000%, about 6000%, about 7000%, about 8000%,
about 9000%, or about 10000%.
[0073] A pre-miRNA mimic may increase a steady-state level of a
mature miRNA from about 1% to about 5%, from about 5% to about 10%,
from about 10% to about 20%, from about 20% to about 30%, from
about 30% to about 40%, from about 40% to about 50%, from about 50%
to about 60%, from about 60% to about 70%, from about 70% to about
80%, from about 80% to about 90%, from about 90% to about 100%,
from about 100% to about 200%, from about 200% to about 300%, from
about 300% to about 400%, from about 400% to about 500%, from about
500% to about 600%, from about 600% to about 700%, from about 700%
to about 800%, from about 800% to about 900%, from about 900% to
about 1000%, from about 1000% to about 2000%, from about 2000% to
about 3000%, from about 3000% to about 4000%, from about 4000% to
about 5000%, from about 5000% to about 6000%, from about 6000% to
about 7000%, from about 7000% to about 8000%, from about 8000% to
about 9000%, or from about 9000% to about 10000%.
[0074] A miRNA antagonist can reduce the amount or the activity of
a miRNA about 1%, about 5%, about 10%, about 15%, about 20%, about
25%, about 30%, about 35%, about 40%, about 45%, about 50%, about
55%, about 60%, about 65%, about 70%, about 75%, about 80%, about
85%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or
about 99.9%.
[0075] A miRNA antagonist can reduce the amount or the activity of
a miRNA from about 1% to about 5%, from about 5% to about 10%, from
about 10% to about 15%, from about 15% to about 20%, from about 20%
to about 25%, from about 25% to about 30%, from about 30% to about
35%, from about 35% to about 40%, from about 40% to about 45%, from
about 45% to about 50%, from about 50% to about 55%, from about 55%
to about 60%, from about 60% to about 65%, from about 65% to about
70%, from about 70% to about 75%, from about 75% to about 80%, from
about 80% to about 85%, from about 85% to about 90%, from about 90%
to about 91%, from about 91% to about 92%, from about 92% to about
93%, from about 93% to about 94%, from about 94% to about 95%, from
about 95% to about 96%, from about 96% to about 97%, from about 97%
to about 98%, from about 98% to about 99%, from about 99% to about
99.5%, or from about 99.5% to about 99.9%.
[0076] Disclosed herein are oligonucleotide comprising
modifications. Modifications may comprise chemical modifications to
the bases or backbone of the oligonucleotides disclosed herein.
Modifications to oligonucleotides may improve activities of the
oligonucleotide in a cell, in vitro, in vivo, or in a subject.
Non-limiting examples of activities include increased activity,
increased half-life, increased cellular uptake, increased targeting
to a cellular compartment, reduced off-target suppression of
transcripts, and modified immunostimulatory activity. Non-limiting
examples of increased activity include increased RNAi activity,
increased miRNA-mediated repressive activity, and increased miRNA
inhibitory activity. Activities may include increased stability in
a biological fluid. Non-limiting examples of a biological fluid
include plasma, serum, saliva, sputum, stool, breast milk, mucus,
sweat, tears, urine, semen, vaginal secretion, pancreatic juice,
bile, pus, and joint fluid. Characteristics may include modified
immunostimulatory activity. Non-limiting examples of
immunostimulatory activity include expression of interferon-0
(IFN-(3), interleukin-6 (IL-6) secretion, tumor necrosis
factor-.alpha. (TNF-.alpha.) secretion.
[0077] An increase in activities may be about 1%, about 5%, about
10%, about 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about 80%, about 90%, about 100%, about 200%, about 300%,
about 400%, about 500%, about 600%, about 700%, about 800%, about
900%, about 1000%, about 2000%, about 3000%, about 4000%, about
5000%, about 6000%, about 7000%, about 8000%, about 9000%, or about
10000%.
[0078] A reduction in activities may be about 1%, about 5%, about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 91%, about
92%, about 93%, about 94%, about 95%, about 96%, about 97%, about
98%, about 99%, about 99.5%, or about 99.9%.
[0079] In some embodiments, modifications to oligonucleotide reduce
off-target effects of the oligonucleotide. In some embodiments,
modifications to oligonucleotide reduce off-target effects of the
oligonucleotide by about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, about 100%,
about 200%, about 300%, about 400%, about 500%, about 600%, about
700%, about 800%, about 900%, about 1000%, about 2000%, about
3000%, about 4000%, about 5000%, about 6000%, about 7000%, about
8000%, about 9000%, or about 10000%.
[0080] In some embodiments, modifications to oligonucleotides may
cause an increase in RNAi activity, miRNA-mediated repressive
activity, miRNA inhibitory activity, molecular half-life, stability
in a biological fluid, cellular uptake, or targeting to a cellular
compartment. The increase in RNAi activity, miRNA-mediated
repressive activity, miRNA inhibitory activity, molecular
half-life, stability in a biological fluid, cellular uptake, or
targeting to a cellular compartment may be about 10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, about 100%, about 200%, about 300%, about 400%, about
500%, about 600%, about 700%, about 800%, about 900%, about 1000%,
about 2000%, about 3000%, about 4000%, about 5000%, about 6000%,
about 7000%, about 8000%, about 9000%, or about 10000%.
[0081] Non-limiting examples of modifications include modified
nucleotides, modified internucleotide linkages, non-nucleotides,
deoxynucleotides, constrained backbone modifications and nucleotide
analogs. Nucleotide modifications can include modifications to the
base of the nucleotide, to the sugar of the nucleotide, or to the
phosphate group of the nucleotide. Any nucleotide within a sshRNA,
miRNA antagonist or pre-miRNA mimic can be modified. In some
embodiments, the modifications are only to the sense sequence, only
to the antisense sequence, or only to the loop region. In some
embodiments, the modifications are in the sense sequence, antisense
sequence, loop region, or a combination thereof.
[0082] In some embodiments, the oligonucleotide comprises a
conjugate moiety. In some embodiments, the conjugate moiety can be
a detectable label, such as a radioactive label, fluorescent label,
mass label, transferrin, and cholesteryl. In some embodiments, a
small RNA can be targeted to certain organs by conjugating specific
moieties, for example, mannose residues to target to the liver.
[0083] In some embodiments, the conjugate moiety can enhance
delivery, detection, function, specificity, or stability of a
molecule of the present invention. Non-limiting examples of said
conjugate moieties include amino acids, peptides, proteins, sugars,
carbohydrates, lipids, polymers, nucleotides, and polynucleotides,
or any combination thereof. In some embodiments, the conjugate
moiety can be linked to the sense or antisense sequence or loop
region of a shRNA, sshRNA, or pre-miRNA mimic. In some embodiments,
the conjugate moiety can be linked at the 5' or 3' end of the sense
or antisense sequence of a shRNA, sshRNA, or pre-miRNA mimic.
[0084] In some embodiments, a conjugate moiety or a modification of
the oligonucleotide comprises an alkyl group. The alkyl group may
be saturated, unsaturated, substituted, or unsubstituted. In some
embodiments, the alkyl group comprises hydrocarbon chains that are
linear, branched, alkanyl, alkenyl, alkynyl, cyclic, heterocyclic,
aryl, or heteroaryl, any of which is substituted or unsubstituted.
In some embodiments, the alkyl group comprises a functional group
chosen from: ether, ester, amide, amine, carbonyl, ketone, acetic
anhydride, carbamate, thioether, carbonate, sulfone, halide, thiol,
alcohol, thioester, phosphothiorate, phosphate, azo, keto,
aldehyde, carboxyl, nitro, nitroso, nitrile, imidazole, morpholino,
pyrrolidino, hydrazino, hydroxylamino, isocyanate, cyanate,
sulfoxide, sulfide, disulfide, cycloalkyl, cycloalkenyl,
cycloalkynyl, cycloheteroalkyl, cycloheteroalkenyl,
cycloheteroalkynyl, aryl, and heteroaryl, any of which is
substituted or unsubstituted. Non-limiting examples of alkyl groups
include substituted and unsubstituted groups of 1 carbon atom,
about 2 carbon atoms, about 3 carbon atoms, about 4 carbon atoms,
about 5 carbon atoms, about 6 carbon atoms, about 7 carbon atoms,
about 8 carbon atoms, about 9 carbon atoms, about 10 carbon atoms,
11 carbon atoms, about 12 carbon atoms, about 13 carbon atoms,
about 14 carbon atoms, about 15 carbon atoms, 16 carbon atoms,
about 17 carbon atoms, about 18 carbon atoms, about 19 carbon
atoms, about 20 carbon atoms, 21 carbon atoms, about 22 carbon
atoms, about 23 carbon atoms, about 24 carbon atoms, about 25
carbon atoms, 26 carbon atoms, about 27 carbon atoms, about 28
carbon atoms, about 29 carbon atoms, and about 30 carbon atoms.
[0085] A nucleotide of the present invention may comprise a
ribonucleotide, a deoxyribonucleotide, or a modification or analog
thereof. Non-limiting examples of nucleotides include adenine (A),
hypoxanthine (Hy), xanthine (Xt), guanine (G), inosine (I),
cytosine (C), uracil (U), thymine (T), SSICS, NaM, queuosine (Q),
purines, pyrimidines, and derivatives, analogs, or modifications
thereof. Nucleotide analogs comprise nucleotides having
modifications in the chemical structure of the base, sugar or
phosphate.
[0086] In some embodiments, the nucleotide modification is a
modification to the base of the nucleotide. Modified bases refer to
nucleotide bases such as, adenine, guanine, cytosine, thymine, and
uracil, xanthine, inosine, and queuosine that have been modified by
the replacement or addition of one or more atoms or groups.
Non-limiting examples of nucleotide base modifications include
5-position pyrimidine modifications, 8-position purine
modifications, modifications at cytosine exocyclic amines, and
substitution of 5-bromo-uracil. Non-limiting examples of types of
modifications include alkylated, halogenated, thiolated, aminated,
amidated, and acetylated bases, or any combination thereof.
Non-limiting examples of modified bases include, 5-propynyluridine,
5-propynylcytidine, 6-methyladenine, 6-methylguanine,
N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine,
2'-aminoadenine, 1-methylinosine, 3-methyluridine,
5-methylcytidine, 5-methyluridine, 5-methylthymine,
5-hydroxycytidine, 5-formylcytidine, 5-(2-amino)propyluridine,
5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine,
2-methyladenosine, 3-methylcytidine, 6-methyluridine,
2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine,
5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides
such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,
6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as
2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,
pseudouridine, archaeosine, naphthyl- and substituted
naphthyl-conjugated bases, N6-methyladenosine,
5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid,
pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups
such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines
that act as G-clamp nucleotides, 8-substituted adenines and
guanines, 5-substituted uracils and thymines, azapyrimidines,
carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl
nucleotides, and alkylcarbonylalkylated nucleotides.
[0087] In some embodiments, a modification to a nucleotide is a
modification of the sugar or replacement of the sugar with a
non-ribosyl sugar analog. Non-limiting examples of replacement
sugars include mannoses, arabinoses, glucopyranoses,
galactopyranoses, and 4-thioribose. Modifications to the sugar can
occur at the 1' position, 2' position, 3' position, 4' position, 5'
position, or any combination thereof. In some embodiments, the
modification of the sugar is at the 2' position of the sugar.
Non-limiting examples of 2' sugar modifications include replacing
the 2'-OH with H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or
CN, wherein R is an alkyl group. In some embodiments, the 2' sugar
modification is chosen from: 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl,
2'-O-isopropyl, 2'-O-butyl, 2'-O-isobutyl, 2'-O-ethyl-O-methyl
(--OCH2CH2OCH3), and 2'-O-ethyl-OH (--OCH2CH2OH). In some
embodiments, the 2' sugar modification is a methyl group. The 2'
oxygen modification can occur on a nucleotide of the sense
sequence, the antisense sequence, or the loop region.
[0088] In some embodiments, a nucleotide modification can be a
modification of the phosphate group. Non-limiting examples of
phosphate group modifications include methylphosphonates,
phosphorothioates, phosphordithioates, and peptides.
[0089] In some embodiments, a nucleotide modification is a 3' cap
structure, such as an inverted deoxythymidine.
[0090] In some embodiments, a modification is a non-nucleotide
modifier such as
N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN) or a C3
spacer (propanediol). ZEN modification can either be placed at an
internal position between two nucleotides or at a terminus.
[0091] An oligonucleotide disclosed herein may have no
modifications or can have modifications of about 1 nucleotide,
about 2 nucleotides, about 3 nucleotides, about 4 nucleotides,
about 5 nucleotides, about 6 nucleotides, about 7 nucleotides,
about 8 nucleotides, about 9 nucleotides, about 10 nucleotides,
about 11 nucleotides, about 12 nucleotides, about 13 nucleotides,
about 14 nucleotides, about 15 nucleotides, about 16 nucleotides,
about 17 nucleotides, about 18 nucleotides, about 19 nucleotides,
about 20 nucleotides, about 21 nucleotides, about 22 nucleotides,
about 23 nucleotides, about 24 nucleotides, about 25 nucleotides,
about 26 nucleotides, about 27 nucleotides, about 28 nucleotides,
about 29 nucleotides, about 30 nucleotides, about 31 nucleotides,
about 32 nucleotides, about 33 nucleotides, about 34 nucleotides,
about 35 nucleotides, about 36 nucleotides, about 37 nucleotides,
about 38 nucleotides, about 39 nucleotides, about 40 nucleotides,
about 41 nucleotides, about 42 nucleotides, about 43 nucleotides,
about 44 nucleotides, about 45 nucleotides, about 46 nucleotides,
about 47 nucleotides, about 48 nucleotides, about 49 nucleotides,
about 50 nucleotides, about 51 nucleotides, about 52 nucleotides,
about 53 nucleotides, about 54 nucleotides, about 55 nucleotides,
about 56 nucleotides, about 57 nucleotides, about 58 nucleotides,
about 59 nucleotides, about 60 nucleotides, about 61 nucleotides,
about 62 nucleotides, about 63 nucleotides, about 64 nucleotides,
about 65 nucleotides, about 66 nucleotides, about 67 nucleotides,
about 68 nucleotides, about 69 nucleotides, about 70 nucleotides,
about 71 nucleotides, about 72 nucleotides, about 73 nucleotides,
about 74 nucleotides, about 75 nucleotides, or about 76
nucleotides.
[0092] In some embodiments, the oligonucleotide comprises a
plurality of modifications. The plurality of modifications may
occur only on consecutive nucleotides. The plurality of
modifications may occur only on alternating nucleotides. The
plurality of modifications may occur on both consecutive
nucleotides and alternating nucleotides. Alternating nucleotides,
as used herein, refers to two nucleotides separated only by a third
nucleotide.
[0093] A sense or antisense strand of an oligo nucleotide disclosed
herein may have no modifications or can have modifications of about
1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4
nucleotides, about 5 nucleotides, about 6 nucleotides, about 7
nucleotides, about 8 nucleotides, about 9 nucleotides, about 10
nucleotides, about 11 nucleotides, about 12 nucleotides, about 13
nucleotides, about 14 nucleotides, about 15 nucleotides, about 16
nucleotides, about 17 nucleotides, about 18 nucleotides, about 19
nucleotides, about 20 nucleotides, about 21 nucleotides, about 22
nucleotides, about 23 nucleotides, about 24 nucleotides, about 25
nucleotides, about 26 nucleotides, about 27 nucleotides, about 28
nucleotides, about 29 nucleotides, about 30 nucleotides, about 31
nucleotides, about 32 nucleotides, about 33 nucleotides, about 34
nucleotides, about 35 nucleotides, about 36 nucleotides, about 37
nucleotides, about 38 nucleotides, about 39 nucleotides, about 40
nucleotides, about 41 nucleotides, about 42 nucleotides, about 43
nucleotides, about 44 nucleotides, about 45 nucleotides, about 46
nucleotides, about 47 nucleotides, about 48 nucleotides, about 49
nucleotides, or about 50 nucleotides.
[0094] In some embodiments, the oligonucleotides disclosed herein
have modifications at particular positions of the molecule. In some
embodiments, the molecule has a modification at every position of
the oligonucleotide. In some embodiments, the particular modified
positions comprise positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 of the oligonucleotide as
viewed from 5' to 3', or any combination thereof. In some
embodiments, the particular modified positions comprise positions
2, 4, 6, 8, 10, 12, 13, 14, 16, 18, 20, or 22 of the
oligonucleotide as viewed from 5' to 3', or any combination
thereof. In some embodiments, the modification at a particular
position is a sugar modification. Non-limiting examples of sugar
modifications at particular positions include 2'-O-methyl
modifications, LNA modifications, DNA modifications, and 2'-F
modifications. In some embodiments, the sugar modification is at
every position in the oligonucleotide. In some embodiments, the
sugar modifications are at positions 1, 3, 4, 6, 7, 9, 10, 12, 13,
and 15 of the oligonucleotide. In some embodiments, the
modification at a particular position is a phosphorothioate
internucleotide linkage. In some embodiments, the phosphorothioate
linkages are at every position in the oligonucleotide. In some
embodiments, the phosphorothioate linkages are at three consecutive
positions at both the 5' and 3' end in the oligonucleotide. In some
embodiments, the modification at a particular position is a ZEN
modification. In some embodiments, the ZEN modification is between
position 1 and 2 and also at the 3'-end of the oligonucleotide. In
some embodiments, the small RNA to be modified at particular
positions is a miRNA antagonist. In some embodiments, the miRNA
antagonist is a miR-210 antagonist. In some embodiments, the
miR-210 antagonist has 2'-O-methyl modifications. In some
embodiments, the miR-210 antagonist has 2'-O-methyl modifications
at every position. In some embodiments, the miR-210 antagonist has
2'-O-methyl modifications at positions 2, 4, 6, 8, 10, 12, 13, 14,
16, 18, 20, and 22. In some embodiments, the miR-210 antagonist has
2'-O-methyl modifications at positions 1, 3, 4, 6, 7, 9, 10, 12,
13, and 15. In some embodiments, the miR-210 antagonist has
phosphorothioate internucleotide linkage(s). In some embodiments,
the miR-210 antagonist has phosphorothioate linkages are at every
position in the oligonucleotide. In some embodiments, the miR-210
antagonist has phosphorothioate linkages are at three consecutive
positions at both the 5' and 3' end in the oligonucleotide. In some
embodiments, the modification at a particular position is a ZEN
modification. In some embodiments, the miR-210 antagonist has ZEN
modification. In some embodiments, the miR-210 antagonist has ZEN
modification between position 1 and 2 and also at the 3'-end of the
oligonucleotide. In some embodiments, the miR-210 antagonist has
2'-O-methyl modifications at positions 12, 13, and 14, and LNA
modifications at positions 2, 4, 6, 8, 10, 16, 18, 20, and 22. In
some embodiments, the miR-210 antagonist has 2'-O-methyl
modifications at positions 12, 13, and 14, and 2'-F modifications
at positions 2, 4, 6, 8, 10, 16, 18, 20, and 22. In some
embodiments, the miR-210 antagonist has 2'-O-methyl modifications
at positions 12, 13, and 14; 2'-F modifications at positions 4, 8,
18, and 22; and LNA modifications at positions 2, 6, 10, 16, and
20. In some embodiments, the miR-210 antagonist has 2'-O-methyl
modifications at positions 2, 6, 10, 12, 13, 14, 16, and 20, and
2'-F modifications at positions 4, 8, 18, and 22. In some
embodiments, the miRNA antagonist has 2'-O-methyl modifications at
every position and 3 phosphorothioate linkages at consecutive
residues at both the 5'- and 3'-end, (e.g., SG606, SEQ ID No. 326).
In some embodiments, the miRNA antagonist is 15 nucleotides and has
phosphorothioate linkages at every position and 2'-O-methyl
modifications at positions 1, 3, 4, 6, 7, 9, 10, 12, 13, and 15 and
LNA modifications at positions 2, 5, 8, 11, and 14 (e.g., SG607,
SEQ ID No. 327). In some embodiments, the miRNA antagonist has
2'-O-methyl modifications at every position and ZEN modifications
between position 1 and 2 and also at the 3'-end. In some cases this
antagonist is 1 nucleotide shorter than full-length complementarity
to the mature miRNA sequence (e.g., SG615, SEQ ID No. 328).
[0095] In some embodiments, a pre-miRNA mimic has a nucleotide at
its 5' end of the antisense strand in which the phosphate or
hydroxyl group has been replaced with a replacement group.
Non-limiting examples of replacement groups include biotin, alkyl
groups, amine groups, lower alkylamine groups, acetyl groups,
2'-O-alkyls, DMTO (4,4'-dimethoxytrityl with oxygen), fluorosceins,
TexasReds, thiols, and acridines.
Methods of Treatment
[0096] Disclosed herein, are methods for treating a wound in a
subject in need thereof comprising administering a therapeutically
effective amount an oligonucleotide disclosed herein. The
oligonucleotide may be selected from a sshRNA, a miRNA antagonist,
and a pre-miRNA mimic. The method may comprise administering a
therapeutically effective amount of a sshRNA and a therapeutically
effective amount of a miRNA antagonist. The method may comprise
administering a therapeutically effective amount of a sshRNA and a
therapeutically effective amount of a pre-miRNA mimic. The method
may comprise administering a therapeutically effective amount of a
pre-miRNA mimic and a therapeutically effective amount of a miRNA
antagonist. The method may comprise administering a therapeutically
effective amount of a sshRNA, a therapeutically effective amount of
a pre-miRNA mimic, and a therapeutically effective amount of a
miRNA antagonist.
[0097] While the oligonucleotides disclosed herein may be used to
modify gene expression relevant to many biological pathways and
conditions, the experimental examples disclosed herein demonstrate
the usefulness of these oligonucleotides in wound healing. Wound
healing is a complex and dynamic biological process with three
overlapping phases: an inflammatory phase, a proliferative phase,
and a remodeling phase. The oligonucleotides, compositions and
methods disclosed herein especially benefit the inflammatory and
proliferative phases by accelerating the initial healing
process.
[0098] Oligonucleotides and their formulations may be more stable
molecules for wound healing purposes compared to small molecule
inhibitors, recombinant growth factors and other proteins,
facilitating their use in the clinic as well as in resource-limited
environments. Because of the long-lasting pharmacological effects
of the oligonucleotide inhibitors disclosed herein, the effect of a
single application of oligonucleotide inhibitors may be expected to
last from one dressing change to the next.
[0099] The methods disclosed herein may comprise treating a chronic
wound. The chronic wound may be a wound that has existed for more
than 24 hours, more than 3 days, more than a week, more than two
weeks, more than a month, more than two months, more than six
months, or more than a year.
[0100] The methods disclosed herein may comprise treating a
non-healing wound. The non-healing wound may be a wound that still
exists after one or more treatments or therapies have been applied
to the wound, and has not healed in an expected time frame.
[0101] The methods may comprise administering a combination of a
sshRNA, a miRNA antagonist, and a pre-miRNA mimic, wherein the
amount of any one of the sshRNA, a miRNA antagonist, and a
pre-miRNA mimic is administered in an amount that would be less
than therapeutically effective if administered alone. The method
may comprise administering a combination of a sshRNA, a miRNA
antagonist, and a pre-miRNA mimic, wherein at least one of the
sshRNA, a miRNA antagonist, and a pre-miRNA mimic is administered
at a dose that would be subtherapeutic when administered alone. The
combination of two or more of the oligonucleotides disclosed herein
may have an additive therapeutic effect or a synergistic
therapeutic effect.
[0102] Disclosed herein are methods of treating a wound in a
subject comprising administering an sshRNA inhibiting PHD2, a
pre-miRNA mimic enhancing activity of miR-21, and an miRNA
antagonist inhibiting miR-210. The methods may promote wound
healing by stimulating the production of growth factors and
angiogenic effectors, including HSPs, VEGF, PDGF, EPO, ANG 1/2, and
PLGF, and the proliferation of fibroblasts and keratinocytes. HSPs,
VEGF, PDGF, EPO, ANG 1/2, and PLGF may promote wound healing,
especially the initial inflammatory and proliferation phases.
[0103] The reason for inhibiting PHD2 using an sshRNA is to promote
HIF-1.alpha. activity, which is compromised in impaired wound
healing. Under normoxia, PHD2 (prolyl hydroxylase domain-containing
protein 2) hydroxylates prolines on HIF-1a, which triggers its
degradation via the ubiquitin-proteasome pathway. Thus, the
inhibition of PHD2 provides an effective approach to stabilize
levels of HIF-1.alpha. under normoxia. The stabilization of
HIF-1.alpha. in this fashion may induce the expression of factors
like EPO, HSF-1, PDGF, and angiopoietins whose activities promote
the angiogenesis and vascular remodeling that are essential for
wound healing. HIF-1.alpha. may also induce the recruitment of
macrophages and endothelial progenitors from bone marrow to wounded
sites to promote vasculogenesis and microbial resistance. In
addition, it may promote the enhanced expression of heat-shock
proteins (HSPs) by directly up-regulating HSF-1, the transcription
factor that induces the synthesis of other HSPs. Reduced levels of
HSPs are found in chronic human wounds as well as in animal models
of impaired wound healing, suggesting their active role in wound
healing.
[0104] miR-210 is one of the miRNAs that is highly up-regulated by
HIF-1a. Increased levels of miR-210 result in reduced keratinocyte
proliferation and mobility, which are essential activities of the
wound re-epithelialization process. Hence, the stabilization of
HIF-1.alpha. through the inhibition of PHD2 by sshRNA would be
expected to up-regulate miR-210, which is not conducive to healing.
Thus, the rationale for inhibiting miR-210 activity using an
anti-miR-210 is to prevent the repression of keratinocyte growth
and migration that would otherwise be a side-effect of stabilizing
HIF-1a.
[0105] miR-21 plays a role opposite to that of miR-210; it promotes
the migration of fibroblasts, endothelial cells and keratinocytes,
which favor the wound healing process. The migration of fibroblasts
into the wound bed results in recruitment of monocytes and
induction of an inflammatory response, which is important in the
initial phase of wound healing. Another key role of miR-21 is to
promote cell growth and proliferation by regulating the PTEN
pathway and inhibiting apoptotic regulators, both of which are also
important for the initial phases of wound healing. HIF-1.alpha.
up-regulates miR-21, but to a lesser extent than it does miR-210.
Based on these considerations, we hypothesize that further
supplementation of miR-21 activity would be beneficial for
accelerating the healing process.
[0106] miR-210 may inhibit epithelialization by inhibiting
keratinocyte growth and motility, whereas miR-21 may promote
re-epithelialization by enhancing the motility of fibroblasts and
keratinocytes. The inhibition of PHD2 by sshRNAs to stabilize
HIF-1a, the abrogation of miR-210 by an anti-miR, and the
supplementation of miR-21 activity by a pre-miR-21 mimic together
may be an effective therapeutic strategy for wound healing, and may
be superior to the effects of these RNAs alone.
[0107] siRNAs targeting viruses or cancer cells may be well suited
to being combined in cocktails to avoid erosion of their
effectiveness through mutation of single targets. Because the
components of such a cocktail are chemically very similar and what
side-effects they may have are mostly of a class-specific nature,
siRNA cocktails may not have the same level of safety risk as do
combinations of small molecule inhibitors. Based on their different
roles in the wound healing process as summarized above,
simultaneously modulating the activities of PHD2, miR-210 and
miR-21 may profoundly affect the overall wound healing process.
This therapeutic strategy would induce angiogenic factors and
growth factors, and mobilize fibroblasts and keratinocytes within
the wounded tissue to facilitate accelerated wound healing by
regulating multiple biological pathways.
[0108] Because delivery is expected to be topical and the duration
of treatment is only until the wound is healed, administration of
high local doses, if needed to overcome any limitations in
efficiency of tissue uptake, should be relatively safe and
cost-effective. Although there are siRNA- and anti-miR-based drugs
in development, there are currently no approved drug therapies
based on RNAi. Success in this project could represent one of the
first RNAi-based therapies, and the first therapy for synthetic
shRNAs of any design. It would provide a boost to the prospects of
RNAi as a general therapeutic approach for many diseases,
especially skin disorders.
[0109] Described herein are therapeutically effective amounts of
oligonucleotides, pharmaceutical compositions, pharmaceutical
formulations, unit dosage forms, methods and kits to treat wounds
in a subject in need thereof. In some embodiments, a
therapeutically effective amount of a small RNA, pharmaceutical
formulation, unit dosage form, or pharmaceutical composition of the
present invention, either alone or in combination with another
molecule or pharmaceutical composition, can be used for the
treatment of wounds in a subject in need thereof. In some
embodiments, a therapeutically effective amount of an
oligonucleotide, pharmaceutical formulation, unit dosage form, or
pharmaceutical composition of the present invention, either alone
or in combination with another molecule or pharmaceutical
composition, can be used for the manufacture of a medicament for
the treatment of wounds in a subject in need thereof.
[0110] Complications in wound healing are widespread pathologies,
as the inability to complete wound closure and increase the risk of
infection, inflammation, scarring and fibrosis. Non-limiting
examples of wounds include type II diabetic wounds, stab wounds,
gunshot wounds, surgical wounds, thermal wounds, chemical wounds,
electrical wounds, bites, stings, atherosclerotic wounds, deep vein
thrombotic wounds, chronic wounds, acute wounds, ulcerative wounds,
venous ulcerative wounds, pressure ulcerative wounds, infected
wounds, inflammatory wounds, pressure wounds, incision wounds,
superficial wounds, internal organ wounds, foot wounds, bed sore
wounds, non-healing wounds, ischemic wounds, wounds associated with
methicillin-resistant Staphylococcus aureus, pressure ulcerative
wounds, diabetic ulcerative wounds, venous ulcerative wounds,
arterial ulcerative wounds, and wounds of the elderly.
[0111] In some embodiments, a subject in need thereof is diagnosed
with a disease or condition. In some embodiments, the disease or
condition is a disease of pathological wound healing. Non-limiting
examples of diseases or conditions of pathological wound healing
include type II diabetes, diabetes mellitus, ischemia, venous
stasis disease, peripheral vascular disease, an autoimmune disease,
and a disease of dysregulated hypoxia response.
[0112] Non-limiting examples of organs and tissues in which wounds
can occur include the skin, dermis, epidermis, hypodermis,
connective tissue, breast, cervix, gastrointestinal tract, heart,
kidney, large intestine, liver, lung, lymph nodes, ovary, pancreas,
prostate, small intestine, spine or spinal cord, spleen, stomach,
testes, thymus, or uterus. Non-limiting examples of cells in which
a small RNA of the present invention can be contacted include
adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,
neurons, glia, blood cells, megakaryocytes, lymphocytes,
macrophages, neutrophils, eosinophils, basophils, mast cells,
leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts,
osteoclasts, hepatocytes, cells of the endocrine or exocrine
glands, germ cells, nurse cells, epithelial cells, endothelial
cells, hormone secreting cells, contractile cells, skeletal muscle
cells, cardiac muscle cells, blood cells, or cells of the bone,
bone marrow, brain, breast, cervix, gastrointestinal tract, heart,
kidney, large intestine, liver, lung, lymph nodes, ovary, pancreas,
prostate, small intestine, spine or spinal cord, spleen, stomach,
testes, thymus, or uterus.
[0113] Normal wound healing involves a coordinated cascade of
events stimulated in part by the hypoxia resultant from injury to
the vasculature at the wound site. This cascade includes
angiogenesis, vasculogenesis, macrophage recruitment, inhibition of
apoptosis, and the expansion and mobilization of fibroblasts and
keratinocytes for re-epithelialization. In chronic wounds, the
normal response to hypoxia is impaired and many of these cellular
processes are hindered.
[0114] FIG. 1 illustrates the role of the hypoxia network 100 in
this process. In the absence of hypoxia, the regulatory factor PHD2
101 hydroxylates 102 the transcription factor HIF-1.alpha. 103,
leading to HIF-1.alpha. 103 degradation. During hypoxia, PHD2 101
is inactivated, liberating HIF-1.alpha. 103 to engage in
transcriptional activation. In particular, HIF-1.alpha. 103
transcriptionally activates expression 104 of the miRNA miR-210
105. MiR-210 105 then engages in miRNA-mediated repression 106 of
the transcription factor E2F3 107. This blocks the ability of E2F3
107 to transcriptionally activate 108 keratinocytes 109. In
parallel, the miRNA miR-21 110 engages in miRNA-mediated repression
111 to activate keratinocytes 109. MiR-21 110 further engages in
miRNA-mediated repression 112 in recruited fibroblasts 113.
Activated keratinocytes 109 recruit and activate 114 fibroblasts
113. To impact the wound healing process 105, HIF-1.alpha. 103 in
cells at the wound site and recruited fibroblasts 113 elicit gene
expression programs 116 and 117, respectively, including expression
of pro-survival factors, angiogenic factors, progenitor cell
recruiting factors, and chemokines to recruit the cell types
necessary for wound closure.
[0115] In some embodiments, an oligonucleotide or pharmaceutical
composition of the present disclosure can increase wound healing,
wound closure, or re-epithelialization. Non-limiting examples of an
increase in wound healing include an increase in a rate of wound
healing or an increase in an extent of wound healing. An
oligonucleotide or pharmaceutical composition of the present
disclosure can increase wound healing, wound closure, or
re-epithelialization, relative to a control, by about 1%, about 5%,
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 100%, about 200%, about
300%, about 400%, about 500%, about 600%, about 700%, about 800%,
about 900%, about 1000%, about 2000%, about 3000%, about 4000%,
about 5000%, about 6000%, about 7000%, about 8000%, about 9000%, or
about 10000%.
[0116] An oligonucleotide or pharmaceutical composition of the
present disclosure can increase wound healing, wound closure, or
re-epithelialization, relative to a control, from about 1% to about
5%, from about 5% to about 10%, from about 10% to about 20%, from
about 20% to about 30%, from about 30% to about 40%, from about 40%
to about 50%, from about 50% to about 60%, from about 60% to about
70%, from about 70% to about 80%, from about 80% to about 90%, from
about 90% to about 100%, from about 100% to about 200%, from about
200% to about 300%, from about 300% to about 400%, from about 400%
to about 500%, from about 500% to about 600%, from about 600% to
about 700%, from about 700% to about 800%, from about 800% to about
900%, from about 900% to about 1000%, from about 1000% to about
2000%, from about 2000% to about 3000%, from about 3000% to about
4000%, from about 4000% to about 5000%, from about 5000% to about
6000%, from about 6000% to about 7000%, from about 7000% to about
8000%, from about 8000% to about 9000%, or from about 9000% to
about 10000%.
[0117] An oligonucleotide or pharmaceutical composition of the
invention can reduce wound size by about 1%, about 5%, about 10%,
about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,
about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,
about 75%, about 80%, about 85%, about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99%, about 99.5%, about 99.9%, or about 100%.
[0118] An oligonucleotide or pharmaceutical composition of the
invention can reduce wound size from about 1% to about 5%, from
about 5% to about 10%, from about 10% to about 15%, from about 15%
to about 20%, from about 20% to about 25%, from about 25% to about
30%, from about 30% to about 35%, from about 35% to about 40%, from
about 40% to about 45%, from about 45% to about 50%, from about 50%
to about 55%, from about 55% to about 60%, from about 60% to about
65%, from about 65% to about 70%, from about 70% to about 75%, from
about 75% to about 80%, from about 80% to about 85%, from about 85%
to about 90%, from about 90% to about 91%, from about 91% to about
92%, from about 92% to about 93%, from about 93% to about 94%, from
about 94% to about 95%, from about 95% to about 96%, from about 96%
to about 97%, from about 97% to about 98%, from about 98% to about
99%, from about 99% to about 99.5%, from about 99.5% to about
99.9%, or from about 99.9% to about 100%.
[0119] The present invention can be administered intravenously,
intradermally, intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly, intraprostaticaly,
intrapleurally, intratracheally, intranasally, intravitreally,
intravaginally, intrarectally, topically, intratumorally,
intramuscularly, intraperitoneally, subcutaneously,
subconjunctival, transdermally, intravesicularlly, mucosally,
intrapericardially, intraumbilically, intraocularally, orally,
topically, locally, by inhalation, such as aerosol inhalation, by
injection, by infusion, by continuous infusion, by localized
perfusion, via a catheter, via a lavage, in creams, in lipid
compositions, such as liposomes, or a combination thereof. A
therapeutically effective amount of a small RNA of the present
invention administered to a subject in need thereof can be
determined by physical and physiological factors such as body
weight, severity of condition, previous or concurrent therapeutic
interventions, idiopathy of the patient and route of
administration.
[0120] In some embodiments, a therapeutically effective amount of
an oligonucleotide of the invention can comprise about 1 microgram
per kilogram (.mu.g/kg), about 2 .mu.g/kg, about 3 .mu.g/kg, about
4 .mu.g/kg, about 5 .mu.g/kg, about 6 .mu.g/kg, about 7 .mu.g/kg,
about 8 .mu.g/kg, about 9 .mu.g/kg, about 10 .mu.g/kg, about 20
.mu.g/kg, about 30 .mu.g/kg, about 40 .mu.g/kg, about 50 .mu.g/kg,
about 60 .mu.g/kg, about 70 .mu.g/kg, about 80 .mu.g/kg, about 90
.mu.g/kg, about 100 .mu.g/kg, about 200 .mu.g/kg, about 300
.mu.g/kg, about 400 .mu.g/kg, about 500 .mu.g/kg, about 1 milligram
per kilogram (mg/kg), about 2 mg/kg, about 3 mg/kg, about 4 mg/kg,
about 5 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8
mg/kg, about 9 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30
mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70
mg/kg, about 80 mg/kg, about 90 mg/kg, or about 100 mg/kg body
weight.
[0121] In some embodiments, a therapeutically effective amount of
an oligonucleotide can comprise from about 1 .mu.g/kg to about 2
.mu.g/kg, from about 2 .mu.g/kg to about 3 .mu.g/kg, from about 3
.mu.g/kg to about 4 .mu.g/kg, from about 4 .mu.g/kg to about 5
.mu.g/kg, from about 5 .mu.g/kg to about 6 .mu.g/kg, from about 6
.mu.g/kg to about 7 .mu.g/kg, from about 7 .mu.g/kg to about 8
.mu.g/kg, from about 8 .mu.g/kg to about 9 .mu.g/kg, from about 9
.mu.g/kg to about 10 .mu.g/kg, from about 10 .mu.g/kg to about 20
.mu.g/kg, from about 20 .mu.g/kg to about 30 .mu.g/kg, from about
30 .mu.g/kg to about 40 .mu.g/kg, from about 40 .mu.g/kg to about
50 .mu.g/kg, from about 50 .mu.g/kg to about 60 .mu.g/kg, from
about 60 .mu.g/kg to about 70 .mu.g/kg, from about 70 .mu.g/kg to
about 80 .mu.g/kg, from about 80 .mu.g/kg to about 90 .mu.g/kg,
from about 90 .mu.g/kg to about 100 .mu.g/kg, from about 100
.mu.g/kg to about 200 .mu.g/kg, from about 200 .mu.g/kg to about
300 .mu.g/kg, from about 300 .mu.g/kg to about 400 .mu.g/kg, from
about 400 .mu.g/kg to about 500 .mu.g/kg, from about 500 .mu.g/kg
to about 600 .mu.g/kg, from about 600 .mu.g/kg to about 700
.mu.g/kg, from about 700 .mu.g/kg to about 800 .mu.g/kg, from about
800 .mu.g/kg to about 900 .mu.g/kg, from about 900 .mu.g/kg to
about 1 mg/kg, from about 1 mg/kg to about 2 mg/kg, from about 2
mg/kg to about 3 mg/kg, from about 3 mg/kg to about 4 mg/kg, from
about 4 mg/kg to about 5 mg/kg, from about 5 mg/kg to about 6
mg/kg, from about 6 mg/kg to about 7 mg/kg, from about 7 mg/kg to
about 8 mg/kg, from about 8 mg/kg to about 9 mg/kg, from about 9
mg/kg to about 10 mg/kg, from about 10 mg/kg to about 20 mg/kg,
from about 20 mg/kg to about 30 mg/kg, from about 30 mg/kg to about
40 mg/kg, from about 40 mg/kg to about 50 mg/kg, from about 50
mg/kg to about 60 mg/kg, from about 60 mg/kg to about 70 mg/kg,
from about 70 mg/kg to about 80 mg/kg, from about 80 mg/kg to about
90 mg/kg, or from about 90 mg/kg to about 100 mg/kg body
weight.
[0122] In some embodiments, a therapeutically effective amount of
an oligonucleotide can comprise about 50 .mu.g, about 100 .mu.g,
about 150 .mu.g, about 200 .mu.g, about 250 .mu.g, about 300 .mu.g,
about 350 .mu.g, about 400 .mu.g, about 450 .mu.g, about 500 .mu.g,
about 600 .mu.g, about 700 .mu.g, about 800 .mu.g, about 900 .mu.g,
about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg,
about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg,
about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg,
about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 11 mg,
about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg,
about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 25 mg,
about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg,
about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg,
about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150
mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about
200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg,
about 700 mg, about 800 mg, about 900 mg, or about 1 g.
[0123] In some embodiments, a therapeutically effective amount of
an oligonucleotide can comprise from about 50 .mu.g to about 100
.mu.g, from about 100 .mu.g to about 150 .mu.g, from about 150
.mu.g to about 200 .mu.g, from about 200 .mu.g to about 250 .mu.g,
from about 250 .mu.g to about 300 .mu.g, from about 300 .mu.g to
about 350 .mu.g, from about 350 .mu.g to about 400 .mu.g, from
about 400 .mu.g to about 450 .mu.g, from about 450 .mu.g to about
500 .mu.g, from about 500 .mu.g to about 600 .mu.g, from about 600
.mu.g to about 700 .mu.g, from about 700 .mu.g to about 800 .mu.g,
from about 800 .mu.g to about 900 .mu.g, from about 900 .mu.g to
about 1 mg, from about 1 mg to about 1.5 mg, from about 1.5 mg to
about 2 mg, from about 1 mg to about 1.5 mg, from about 1.5 mg to
about 2 mg, from about 2 mg to about 2.5 mg, from about 2.5 mg to
about 3 mg, from about 3 mg to about 3.5 mg, from about 3.5 mg to
about 4 mg, from about 4 mg to about 4.5 mg, from about 4.5 mg to
about 5 mg, from about 5 mg to about 5.5 mg, from about 5.5 mg to
about 6 mg, from about 6 mg to about 6.5 mg, from about 6.5 mg to
about 7 mg, from about 7 mg to about 7.5 mg, from about 7.5 mg to
about 8 mg, from about 8 mg to about 8.5 mg, from about 8.5 mg to
about 9 mg, from about 9 mg to about 9.5 mg, from about 9.5 mg to
about 10 mg, from about 10 mg to about 11 mg, from about 11 mg to
about 12 mg, from about 12 mg to about 13 mg, from about 13 mg to
about 14 mg, from about 14 mg to about 15 mg, from about 15 mg to
about 16 mg, from about 16 mg to about 17 mg, from about 17 mg to
about 18 mg, from about 18 mg to about 19 mg, from about 19 mg to
about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to
about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to
about 40 mg, from about 40 mg to about 45 mg, from about 45 mg to
about 50 mg, from about 50 mg to about 60 mg, from about 60 mg to
about 70 mg, from about 70 mg to about 80 mg, from about 80 mg to
about 90 mg, from about 90 mg to about 100 mg, from about 100 mg to
about 110 mg, from about 110 mg to about 120 mg, from about 120 mg
to about 130 mg, from about 130 mg to about 140 mg, from about 140
mg to about 150 mg, from about 150 mg to about 160 mg, from about
160 mg to about 170 mg, from about 170 mg to about 180 mg, from
about 180 mg to about 190 mg, from about 190 mg to about 200 mg,
from about 200 mg to about 300 mg, from about 300 mg to about 400
mg, from about 400 mg to about 500 mg, from about 500 mg to about
600 mg, from about 600 mg to about 700 mg, from about 700 mg to
about 800 mg, from about 800 mg to about 900 mg, or from about 900
mg to about 1 g.
[0124] Polynucleotide Vectors
[0125] The present disclosure provides nucleic acids encoding the
oligonucleotides disclosed herein, delivered by a suitable method,
for example, a polynucleotide vector, to a subject in need
thereof.
[0126] Delivery of a nucleic acid to a cell with a polynucleotide
vector can be accomplished by a number of methods. Viral nucleic
acid vector delivery methods use recombinant viruses for nucleic
acid vector transfer. Non-viral nucleic acid vector delivery can
comprise injecting naked DNA or RNA, use of carriers including
lipid carriers, polymer carriers, chemical carriers and biological
carriers such as biologic membranes, bacteria, and virus-like
particles, and physical or mechanical approaches. A combination of
viral and non-viral nucleic acid vector delivery methods can be
used for efficient gene therapy.
[0127] Non-viral nucleic acid vector transfer can include injection
of naked nucleic acid vector, for example, nucleic acid that is not
protected or devoid of a carrier. In vivo, naked nucleic acid
vectors can be subject to rapid degradation, low transfection
levels, and poor tissue-targeting ability. Hydrodynamic injection
methods can increase the targeting ability of naked nucleic acid
vectors.
[0128] Non-viral nucleic acid vector delivery systems can include
chemical carriers. These systems can include lipoplexes,
polyplexes, dendrimers, and inorganic nanoparticles. A lipoplex is
a complex of a lipid and a nucleic-acid that protects the nucleic
acid from degradation and facilitates entry into cells. Lipoplexes
can be prepared from neutral, anionic, or cationic lipids.
Preparation of lipoplexes with cationic lipids can facilitate
encapsulation of negatively charged nucleic acids. Lipoplexes with
a net positive charge can interact more efficiently with a
negatively charged cell membrane. Preparation of lipoplexes with a
slight excess of positive charges can confer higher transfection
efficiency. Lipoplexes can enter cells by endocytosis. Once inside
the cell, lipoplexes can release the nucleic acid contents into the
cytoplasm. A polyplex is a complex of a polymer and a nucleic acid.
Most polyplexes are prepared from cationic polymers that facilitate
assembly by ionic interactions between nucleic acids and polymers.
Uptake of polyplexes into cells can occur by endocytosis. Inside
the cells, polyplexes require co-transfected endosomal rupture
agents such as inactivated adenovirus, for the release of the
polyplex particle from the endocytic vesicle. Examples of polymeric
carriers include polyethyleneimine, chitosan, poly(beta-amino
esters) and polyphosphoramidate. Polyplexes show low toxicity, high
loading capacity, and ease of fabrication. A dendrimer is a highly
branched molecule. Dendrimers can be constructed to have a
positively-charged surface or carry functional groups that aid
temporary association of the dendrimer with nucleic acids. These
dendrimer-nucleic acid complexes can be used for gene therapy. The
dendrimer-nucleic acid complex can enter the cell by endocytosis.
Nanoparticles prepared from inorganic material can be used for
nucleic acid vector delivery. Examples of inorganic material can
include gold, silica/silicate, silver, iron oxide, and calcium
phosphate. Inorganic nanoparticles with a size of less than 100 nm
can be used to encapsulate nucleic acids efficiently. The
nanoparticles can be taken up by the cell via endocytosis. Inside
the cell, the nucleic acid can be released from the endosome
without degradation. Nanoparticles based on quantum dots can be
prepared and offers the use of a stable fluorescence marker coupled
with gene therapy. Organically modified silica or silicate can be
used to target nucleic acids to specific cells in an organism.
[0129] Non-viral nucleic acid vector delivery systems can include
biological methods including bactofection, biological liposomes,
and virus-like particles (VLPs). Bactofection method comprises
using attenuated bacteria to deliver nucleic acids to a cell.
Biological liposomes, such as erythrocyte ghosts and secretion
exosomes, are derived from the subject receiving gene therapy to
avoid an immune response. Virus-like particles (VLP) or empty viral
particles are produced by transfecting cells with only the
structural genes of a virus and harvesting the empty particles. The
empty particles are loaded with nucleic acids to be transfected for
gene therapy.
[0130] Delivery of nucleic acid vectors can be enhanced by physical
methods. Examples of physical methods include electroporation, gene
gun, sonoporation, and magnetofection. The electroporation method
uses short high-voltage pulses to transfer nucleic acid across the
cell membrane. These pulses can lead to formation of temporary
pores in the cell membrane, thereby allowing nucleic acid to enter
the cell. Electroporation can be efficient for a broad range of
cells. Electron-avalanche transfection is a type of electroporation
method that uses very short, for example, microsecond, pulses of
high-voltage plasma discharge for increasing efficiency of nucleic
acid vector delivery. The gene gun method utilizes nucleic
acid-coated gold particles that are shot into the cell using
high-pressure gas. Force generated by the gene gun allows
penetration of nucleic acid into the cells, while the gold is left
behind on a stopping disk. The sonoporation method uses ultrasonic
frequencies to modify permeability of cell membrane. Change in
permeability allows uptake of nucleic acid into cells. The
magnetofection method uses a magnetic field to enhance nucleic acid
uptake. In this method, nucleic acid is complexed with magnetic
particles. A magnetic field is used to concentrate the nucleic acid
complex and bring them in contact with cells.
[0131] Viral nucleic acid vector delivery systems use recombinant
viruses to deliver nucleic acids for gene therapy. Non-limiting
examples of viruses that can be used to deliver nucleic acids
include retrovirus, adenovirus, herpes simplex virus,
adeno-associated virus, vesicular stomatitis virus, reovirus,
vaccinia, pox virus, and measles virus.
[0132] Retroviral vectors can be used in the disclosure. Retrovirus
is an enveloped virus that contains a single-stranded RNA genome.
Retroviruses can integrate inside a host cell via reverse
transcription. Retroviruses can enter a host cell by binding to
specific membrane-bound receptors. Inside the host cell cytoplasm,
retroviral reverse transcriptase generates double-stranded DNA from
the viral RNA genome template. Retroviral enzyme integrase
incorporates the new viral DNA into host cell genome, where the
viral DNA is transcribed and translated along with host cell genes.
Retroviral gene therapy vectors can be used for chromosomal
integration of the transferred vector genomes, thereby leading to
stable genetic modification of treated cells. Non-limiting examples
of retroviral vectors include Moloney murine leukemia viral (MMLV)
vectors, HIV-based viral vectors, gammaretroviral vectors, C-type
retroviral vectors, and lentiviral vectors. Lentivirus is a
subclass of retrovirus. While some retroviruses can infect only
dividing cells, lentiviruses can infect and integrate into the
genome of actively dividing cells and non-dividing cells.
[0133] Adenovirus-based vectors can be used in the disclosure.
Adenovirus is a non-enveloped virus with a linear double-stranded
genome. Adenoviruses can enter host cells using interactions
between viral surface proteins and host cell receptors that lead to
endocytosis of the adenovirus particle. Once inside the host cell
cytoplasm, the adenovirus particle is released by the degradation
of the endosome. Using cellular microtubules, the adenovirus
particle gains entry into the host cell nucleus, where adenoviral
DNA is released. Inside the host cell nucleus, the adenoviral DNA
is transcribed and translated. Adenoviral DNA is not integrated
into the host cell genome. Adenoviral DNA is not replicated during
host cell division. Gene therapy using adenoviral vectors can
require multiple administrations if the host cell population is
replicating.
[0134] Herpes simplex virus (HSV)-based vectors can be used in the
disclosure. HSV is an enveloped virus with a linear double-stranded
DNA genome. Interactions between surface proteins on the host cell
and HSV lead to pore formation in the host cell membrane. These
pores allow HSV to enter the host cell cytoplasm. Inside the host
cell, HSV uses the nuclear entry pore to enter the host cell
nucleus where HSV DNA is released. HSV can persist in host cells in
a state of latency. Herpes simplex virus 1 and 2 (HSV-1 and HSV-2),
also known as human herpes virus 1 and 2 (HHV-1 and HHV-2), are
members of the herpes virus family.
[0135] Alphavirus-based vectors can be used to deliver nucleic
acids. Examples of alphavirus-based vectors include vectors derived
from semliki forest virus and sindbis virus. Alphavirus-based
vectors can provide high transgene expression and the ability to
transduce a wide variety of cells. Alphavirus vectors can be
modified to target specific tissues. Alphaviruses can persist in a
latent state in host cells, thereby offering the advantage of
long-term nucleic acid expression in the cell.
[0136] Pox/vaccinia-based vectors such as orthopox or avipox
vectors can be used in the disclosure. Pox virus is a double
stranded DNA virus that can infect diving and non-dividing cells.
Pox viral genome can accommodate up to 25 kilobase transgenic
sequence. Multiple genes can be delivered using a single vaccinia
viral vector.
[0137] In one aspect, the present disclosure provides a recombinant
virus, such as an adeno-associated virus (AAV), as a vector to
deliver a nucleic acid encoding a shRNA, sshRNA, or pre-miRNA mimic
to a subject in need thereof.
[0138] Adeno-associated virus (AAV) is a small, nonenveloped virus
that belongs to the Parvoviridae family. AAV genome is a linear
single-stranded DNA molecule of about 4,800 nucleotides. The AAV
DNA comprises two inverted terminal repeats (ITRs) at both ends of
the genome and two sets of open reading frames. The ITRs serve as
origins of replication for the viral DNA and as integration
elements. The open reading frames encode for the Rep
(non-structural replication) and Cap (structural capsid) proteins.
AAV can infect dividing cells and quiescent cells. AAV is common in
the general population and can persist naturally in the host.
[0139] AAV can be engineered for use as a gene therapy vector by
substituting the coding sequence for both AAV genes with a
transgene (transferred nucleic acid) to be delivered to a cell. The
substitution eliminates immunologic or toxic side effects due to
expression of viral genes. The transgene can be placed between the
two ITRs (145 basepair (bp)) on the AAV DNA molecule. AAV-based
vectors can transencapsidate the genome allowing large variations
in vector biology and tropism.
[0140] When producing recombinant AAV (rAAV), the viral genes or
adenovirus genes providing helper functions to AAV can be supplied
in trans to allow for production of the rAAV particles. In this
way, rAAV can be produced through a three-plasmid system,
decreasing the probability of production of wild-type virus.
[0141] AAV vector of the present disclosure can be generated using
any AAV serotype. Non-limiting examples of serotypes include AAV1,
AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,
AAV11, AAV12, rh10, and hybrids thereof.
[0142] AAV vectors can be modified for immune evasion or to enhance
therapeutic output.
[0143] The modifications can include genetic manipulation of the
viral capsid. Proteins in the viral capsid can be rationally
designed. The viral capsid can be modified by introducing exogenous
agents such as antibodies, copolymers, and cationic lipids to evade
the immune system. AAV vectors can be engineered to enhance the
targeting ability. Targeting peptides or ligands can be inserted
onto the capsid surface to enhance transduction into specific
tissue. Capsid proteins from more than one serotype of AAV can be
combined to produce a mosaic AAV vector comprising a capsid
particle with enhanced targeting ability of the AAV vector.
Tissue-specific promoters can be added to the viral vector to
express the transgene in desired tissue types.
[0144] AAV vector can be modified to be self-complementary. A
self-complementary AAV vector can comprise both strands of the
viral DNA, thereby alleviating the requirement for host-cell
second-strand DNA synthesis. The use of self-complementary AAV
vectors can promote efficient transfer of nucleic acids into host
genome.
[0145] A pseudotyped virus can be used for the Delivery of nucleic
acid vectors. Psuedotyping involves substitution of endogenous
envelope proteins of the virus by envelope proteins from other
viruses or chimeric proteins. The foreign envelope proteins can
confer a change in host tropism or alter stability of the virus. An
example of a pseudotyped virus useful for gene therapy includes
vesicular stomatitis virus G-pseudotyped lentivirus (VSV
G-pseudotyped lentivirus) that is produced by coating the
lentivirus with the envelope G-protein from Vesicular stomatitis
virus. VSV G-pseudotyped lentivirus can transduce almost all
mammalian cell types.
[0146] A hybrid vector having properties of two or more vectors can
be used for nucleic acid vector delivery to a host cell. Hybrid
vectors can be engineered to reduce toxicity or improve therapeutic
transgene expression in target cells. Non-limiting examples of
hybrid vectors include AAV/adenovirus hybrid vectors, AAV/phage
hybrid vectors, and retrovirus/adenovirus hybrid vectors.
[0147] A viral vector can be replication-competent. A
replication-competent vector contains all the genes necessary for
replication, making the genome lengthier than replication-defective
viral vectors. A viral vector can be replication-defective, wherein
the coding region for the genes essential for replication and
packaging are deleted or replaced with other genes.
Replication-defective viruses can transduce host cells and transfer
the genetic material, but do not replicate. A helper virus can be
supplied to help a replication-defective virus replicate.
[0148] A viral vector can be derived from any source, for example,
humans, non-human primates, dogs, fowl, mouse, cat, sheep, and
pig.
[0149] The composition and methods of the disclosure provide for
the delivery of a nucleic acid that encodes for a shRNA, sshRNA, or
pre-miRNA mimic to a subject in need thereof. The nucleic acid can
be delivered by a viral vector, for example, an adeno-associated
virus (AAV), adenovirus, retrovirus, herpes simplex virus,
lentivirus, poxvirus, hemagglutinating virus of Japan-liposome
(HVJ) complex, Moloney murine leukemia virus, or HIV-based virus.
The nucleic acid can be delivered by a suitable non-viral method,
for example, injection of naked nucleic acid, use of carriers such
as lipid, polymer, biological or chemical carriers, or
physical/mechanical approaches. The nucleic acid can be delivered
by a combination of viral and non-viral methods.
[0150] The nucleic acid of the disclosure can be generated using
any method. The nucleic acid can be synthetic, recombinant,
isolated, or purified. The nucleic acid can comprise, for example,
a nucleic acid sequence that encodes a shRNA, sshRNA, or pre-miRNA
mimic.
[0151] A vector of the present disclosure can comprise one or more
types of nucleic acids. The nucleic acids can include DNA or RNA.
RNA nucleic acids can include a transcript of a gene of interest,
for example, a shRNA, sshRNA, pre-miRNA mimic, introns,
untranslated regions, and termination sequences. DNA nucleic acids
can include the gene of interest, promoter sequences, untranslated
regions, and termination sequences. A combination of DNA and RNA
can be used. The nucleic acids can be double-stranded or
single-stranded. The nucleic acid can include non-natural or
altered nucleotides.
[0152] A vector of the disclosure can comprise additional nucleic
acid sequences including promoters, enhancers, repressors,
insulators, polyadenylation signals (polyA), untranslated regions
(UTRs), termination sequences, transcription terminators, internal
ribosome entry sites (IRES), introns, origins of replication
sequence, primer binding sites, att sites, encapsidation sites,
polypurine tracts, Long Terminal Repeats (LTRs), and linker
sequences. The vector can be modified to target specific cells, for
example, cancer cells, or to a tissue, for example, retina.
[0153] Expression of a shRNA, sshRNA, pre-miRNA mimic can be under
the control of a regulatory sequence. The regulatory sequence can
comprise a promoter. Promoters from any suitable source including
virus, mammal, human, insect, plant, yeast, and bacteria, can be
used. Tissue-specific promoters can be used. Promoters can be
constitutive, inducible, or repressible. Promoters can be
unidirectional (initiating transcription in one direction) or
bi-directional (initiating transcription in either a 3' or 5'
direction). Non-limiting examples of promoters include the T7
bacterial expression system, pBAD (araA) bacterial expression
system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the
Rous sarcoma virus promoter, MMT promoter, EF-1 alpha promoter, UB6
promoter, chicken beta-actin promoter, CAG promoter, RPE65
promoter, opsin promoter, HIV-1 promoter, HIV-2 promoter, AAV
promoter, adenovirus promoters such as from the E1A, E2A, or MLP
region, cauliflower mosaic virus promoter, HSV-TK promoter, avian
sarcoma virus promoter, MLV promoter, MMTV promoter, and rat
insulin promoter. Inducible promoters can include, for example, the
Tet system, the ecdysone inducible system, the T-REX.TM. system,
LACSWITCH.TM. System, and the Cre-ERT tamoxifen inducible
recombinase system.
[0154] A promoter sequence, intron sequence, polyA sequence,
untranslated region, or linker sequence can be about 1 base or base
pair, about 10 bases or base pairs, about 20 bases or base pairs,
about 50 bases or base pairs, about 100 bases or base pairs, about
500 bases or base pairs, about 1000 bases or base pairs, about 2000
bases or base pairs, about 3000 bases or base pairs, about 4000
bases or base pairs, about 5000 bases or base pairs, about 6000
bases or base pairs, about 7000 bases or base pairs, about 8000
bases or base pairs, about 9000 bases or base pairs, or about 10000
bases or base pairs. A promoter sequence, intron sequence, polyA
sequence, untranslated region, or linker sequence can be a length
from about 1 to about 10 bases or base pairs, from about 10 to
about 20 bases or base pairs, from about 20 to about 50 bases or
base pairs, from about 50 to about 100 bases or base pairs, from
about 100 to about 500 bases or base pairs, from about 500 to about
1000 bases or base pairs, from about 1000 to about 2000 bases or
base pairs, from about 2000 to about 3000 bases or base pairs, from
about 3000 to about 4000 bases or base pairs, from about 4000 to
about 5000 bases or base pairs, from about 5000 to about 6000 bases
or base pairs, from about 6000 to about 7000 bases or base pairs,
from about 7000 to about 8000 bases or base pairs, from about 8000
to about 9000 bases or base pairs, or from about 9000 to about
10000 bases or base pairs in length.
[0155] A vector of the disclosure can comprise nucleic acids
encoding a selectable marker. The selectable marker can be
positive, negative or bifunctional. The selectable marker can be an
antibiotic-resistance gene. Examples of antibiotic resistance genes
include markers conferring resistance to kanamycin, gentamicin,
ampicillin, chloramphenicol, tetracycline, doxycycline, hygromycin,
puromycin, zeomycin, or blasticidin. The selectable marker can
allow imaging of the host cells, for example, a fluorescent
protein. Examples of imaging marker genes include GFP, eGFP, RFP,
CFP, YFP, dsRed, Venus, mCherry, mTomato, and mOrange.
[0156] A vector of the disclosure can comprise fusion proteins. The
fusion partner can comprise a signal polypeptide that targets the
protein to the desired site. The fusion partner can comprise a
polypeptide tag, for example, a poly-His or a Flag peptide, that
facilitates purification of the protein. The fusion partner can
comprise an imaging tag, for example, a fluorescent protein, for
imaging the cells. A vector of the disclosure can comprise chemical
conjugates.
[0157] A vector of the disclosure can comprise components to confer
additional properties to the vector. These properties can include
targeting of the vector to a specific tissue, uptake of vector into
a host cell, entry of nucleic acid into nucleus, incorporation of
nucleic acid into host cell genome, transgene expression in host
cell, immune evasion, and vector stability.
[0158] A vector of the disclosure can be generated by any suitable
methods. The method can include use of transgenic cells including
for example, mammalian cells such as HEK293, insect cells such as
Sf9, animal cells or fungal cells.
[0159] A viral vector of the disclosure can be measured as plaque
forming units (pfu). The pfu of a viral vector can be, for example,
from about 10.sup.1 to about 10.sup.18 pfu. A viral vector of the
disclosure can be, for example, at least 10.sup.1, at least
10.sup.2, at least 10.sup.3, at least 10.sup.4, at least 10.sup.5,
at least 10.sup.6, at least 10.sup.7, at least 10.sup.8, at least
10.sup.9, at least 10.sup.10, at least 10.sup.11, at least
10.sup.12, at least 10.sup.13, at least 10.sup.14, at least
10.sup.15, at least 10.sup.16, at least 10.sup.17, or at least
10.sup.18 pfu. A viral vector of the disclosure can be, for
example, at most 10.sup.1, at most 10.sup.2, at most 10.sup.3, at
most 10.sup.4, at most 10.sup.5, at most 10.sup.6, at most
10.sup.7, at most 10.sup.8, at most 10.sup.9, at most 10.sup.10, at
most 10.sup.11, at most 10.sup.12, at most 10.sup.13, at most
10.sup.14, at most 10.sup.15, at most 10.sup.16, at most 10.sup.17,
or at most 10.sup.18 pfu.
[0160] A viral vector of the disclosure can be measured as vector
genomes. A viral vector of the disclosure can be, for example, from
about 10.sup.1 to about 10.sup.18 vector genomes. A viral vector of
the disclosure can be, for example, at least 10.sup.1, at least
10.sup.2, at least 10.sup.3, at least 10.sup.4, at least 10.sup.5,
at least 10.sup.6, at least 10.sup.7, at least 10.sup.8, at least
10.sup.9, at least 10.sup.10, at least 10.sup.11, at least
10.sup.12, at least 10.sup.13, at least 10.sup.14, at least
10.sup.15, at least 10.sup.16, at least 10.sup.17, or at least
10.sup.18 vector genomes. A viral vector of the disclosure can be,
for example, at most 10.sup.1, at most 10.sup.2, at most 10.sup.3,
at most 10.sup.4, at most 10.sup.5, at most 10.sup.6, at most
10.sup.7, at most 10.sup.8, at most 10.sup.9, at most 10.sup.10, at
most 10.sup.11, at most 10.sup.12, at most 10.sup.13, at most
10.sup.14, at most 10.sup.15, at most 10.sup.16, at most 10.sup.17,
or at most 10.sup.18 vector genomes.
[0161] A viral vector of the disclosure can be measured using
multiplicity of infection (MOI). MOI can be, for example, the
ratio, or multiple of vector or viral genomes to the cells to which
the nucleic acid can be delivered. A viral vector of the disclosure
can be, for example, from about 10.sup.1 to about 10.sup.18MOI. A
viral vector of the disclosure can be, for example, at least about
10.sup.1, at least 10.sup.2, at least 10.sup.3, at least 10.sup.4,
at least 10.sup.5, at least 10.sup.6, at least 10.sup.7, at least
10.sup.8, at least 10.sup.9, at least 10.sup.10, at least
10.sup.11, at least 10.sup.12, at least 10.sup.13, at least
10.sup.14, at least 10.sup.15, at least 10.sup.16, at least
10.sup.17, or at least 10.sup.18 MOI. A viral vector of the
disclosure can be, for example, at most 10.sup.1, at most 10.sup.2,
at most 10.sup.3, at most 10.sup.4, at most 10.sup.5, at most
10.sup.6, at most 10.sup.7, at most 10.sup.8, at most 10.sup.9, at
most 10.sup.10, at most 10.sup.11, at most 10.sup.12, at most
10.sup.13, at most 10.sup.14, at most 10.sup.15, at most 10.sup.16,
at most 10.sup.17, or at most 10.sup.18MOI.
[0162] The amount of nucleic acid vector can be, for example, from
about 1 .mu.g to about 1 ng. The amount of nucleic acid vector can
be, for example, from about 1 ng to about 1 .mu.g. The amount of
nucleic acid vector can be, for example, from about 1 .mu.g to
about 1 mg. The amount of nucleic acid vector can be, for example,
from about 1 mg to about 1 g. The amount of nucleic acid vector can
be, for example, from about 1 g to about 5 g. The amount of nucleic
acid vector can be, for example, about 1 pg, about 10 pg, about 100
pg, about 200 pg, about 300 pg, about 400 pg, about 500 pg, about
600 pg, about 700 pg, about 800 pg, about 900 pg, about 1 ng, about
10 ng, about 100 ng, about 200 ng, about 300 ng, about 400 ng,
about 500 ng, about 600 ng, about 700 ng, about 800 ng, about 900
ng, about 1 .mu.g, about 10 .mu.g, about 100 .mu.g, about 200
.mu.g, about 300 .mu.g, about 400 .mu.g, about 500 .mu.g, about 600
.mu.g, about 700 .mu.g, about 800 .mu.g, about 900 .mu.g, about 1
mg, about 10 mg, about 100 mg, about 200 mg, about 300 mg, about
400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg,
about 900 mg, about 1 g, about 2 g, about 3 g, about 4 g, or about
5 g.
[0163] The amount of nucleic acid vector can be from about 1 .mu.g
to about 10 pg, from about 10 pg to about 100 pg, from about 100 pg
to about 200 pg, from about 200 pg to about 300 pg, from about 300
pg to about 400 pg, from about 400 pg to about 500 pg, from about
500 pg to about 600 pg, from about 600 pg to about 700 pg, from
about 700 pg to about 800 pg, from about 800 pg to about 900 pg,
from about 900 pg to about 1 ng, from about 1 ng to about 10 ng,
from about 10 ng to about 100 ng, from about 100 ng to about 200
ng, from about 200 ng to about 300 ng, from about 300 ng to about
400 ng, from about 400 ng to about 500 ng, from about 500 ng to
about 600 ng, from about 600 ng to about 700 ng, from about 700 ng
to about 800 ng, from about 800 ng to about 900 ng, from about 900
ng to about from about 1 .mu.g to about 10 .mu.m, from about 10
.mu.g to about 100 .mu.m, from about 100 .mu.g to about 200 .mu.g,
from about 200 .mu.g to about 300 .mu.g, from about 300 .mu.g to
about 400 .mu.g, from about 400 .mu.g to about 500 .mu.g, from
about 500 .mu.g to about 600 .mu.g, from about 600 .mu.g to about
700 .mu.g, from about 700 .mu.g to about 800 .mu.g, from about 800
.mu.g to about 900 .mu.g, from about 900 .mu.g to about 1 mg, from
about 1 mg to about 10 mg, from about 10 mg to about 100 mg, from
about 100 mg to about 200 mg, from about 200 mg to about 300 mg,
from about 300 mg to about 400 mg, from about 400 mg to about 500
mg, from about 500 mg to about 600 mg, from about 600 mg to about
700 mg, from about 700 mg to about 800 mg, from about 800 mg to
about 900 mg, from about 900 mg to about 1 g, from about 1 g to
about 2 g, from about 2 g to about 3 g, from about 3 g to about 4
g, or from about 4 g to about 5 g.
[0164] A viral vector of the disclosure can be measured as
recombinant viral particles. A viral vector of the disclosure can
be, for example, from about 10.sup.1 to about 10.sup.18 recombinant
viral particles. A viral vector of the disclosure can be, for
example, at least about 10.sup.1, at least about 10.sup.2, at least
about 10.sup.3, at least about 10.sup.4, at least about 10.sup.5,
at least about 10.sup.6, at least about 10.sup.7, at least about
10.sup.8, at least about 10.sup.9, at least about 10.sup.10, at
least about 10.sup.11, at least about 10.sup.12, at least about
10.sup.13, at least about 10.sup.14, at least about 10.sup.15, at
least about 10.sup.16, at least about 10.sup.17, or at least about
10.sup.18 recombinant viral particles. A viral vector of the
disclosure can be, for example, at most about 10.sup.1, at most
about 10.sup.2, at most about 10.sup.3, at most about 10.sup.4, at
most about 10.sup.5, at most about 10.sup.6, at most about
10.sup.7, at most about 10.sup.8, at most about 10.sup.9, at most
about 10.sup.10, at most about 10.sup.11, at most about 10.sup.12,
at most about 10.sup.13, at most about 10.sup.14, at most about
10.sup.15, at most about 10.sup.16, at most about 10.sup.17, or at
most about 10.sup.18 recombinant viral particles.
Pharmaceutical Formulations
[0165] The oligonucleotides of the invention can be administered to
a subject alone or in the form of a pharmaceutical composition.
Pharmaceutical compositions can be formulated using one or more
pharmaceutically-acceptable carriers, diluents, substrates,
excipients or auxiliaries which facilitate processing of the
oligonucleotides into preparations which can be used
pharmaceutically. Formulation is dependent upon the route of
administration chosen.
[0166] For topical administration, the oligonucleotides of the
invention can be formulated as solutions, gels, ointments, creams,
or suspensions, onto transdermal patches, meshes, or films.
Systemic formulations include those designed for administration by
injection, such as subcutaneous, intravenous, intramuscular,
intrathecal or intraperitoneal injection, as well as those designed
for transdermal, transmucosal, inhalation, oral or pulmonary
administration. For injection, the nucleic acids of the invention
can be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiological saline buffer. The solution can
contain formulatory agents such as suspending, stabilizing or
dispersing agents. Alternatively, the oligonucleotides can be in
powder form for constitution with a suitable vehicle, such as
sterile pyrogen-free water, before use. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation.
[0167] In some embodiments, the oligonucleotides of the invention
can be formulated onto transdermal patches, meshes, or films.
Non-limiting examples of transdermal patches, meshes, or films
include dressings, medical dressings, occlusive dressings,
transparent dressings, and waterproof dressings. Oligonucleotides
of the invention can be applied together or sequentially with an
alternating charged material to form a layer on a
pharmaceutically-acceptable substrate, such as a transdermal patch,
mesh or film. Non-limiting examples of the layer include a printed
layer and a cationic layer. The thin layer biodegrades to allow the
oligonucleotide to undergo sustained release. In some embodiments,
the sustained release can occur over about 1 day, about 2 days,
about 3 days, about 4 days, about 5 days, about 6 days, about 7
days, about 8 days, about 9 days, about 10 days, about 11 days,
about 12 days, about 13 days, about 14 days, or about 15 days. In
some embodiments, the sustained release can occur from about 1 day
to about 2 days, from about 2 days to about 3 days, from about 3
days to about 4 days, from about 4 days to about 5 days, from about
5 days to about 6 days, from about 6 days to about 7 days, from
about 7 days to about 8 days, from about 8 days to about 9 days,
from about 9 days to about 10 days, from about 10 days to about 11
days, from about 11 days to about 12 days, from about 12 days to
about 13 days, from about 13 days to about 14 days, or from about
14 days to about 15 days.
[0168] For oral administration, the nucleic acids can be readily
formulated by combining the molecules with
pharmaceutically-acceptable carriers. Oligonucleotides of the
present invention can be formulated with a
pharmaceutically-acceptable carrier to generate tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, and suspensions
for oral ingestion by a subject in need thereof. For oral solid
formulations, such as powders, capsules and tablets, suitable
excipients include fillers chosen from: sugars, cellulose
preparations, granulating agents, binding agents, and
disintegrating agents. Non-limiting examples of sugars include
lactose, sucrose, mannitol and sorbitol. Non-limiting examples of
cellulose preparations include maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and
polyvinylpyrrolidone (PVP). Non-limiting examples of disintegrating
agents include cross-linked polyvinylpyrrolidone, agar, and alginic
acid or a salt thereof such as sodium alginate. If desired, solid
dosage forms can be sugar-coated or enteric-coated. For oral liquid
preparations, such as suspensions, elixirs and solutions, suitable
carriers, excipients or diluents include water, glycols, oils, and
alcohols. Flavoring agents, preservatives, and coloring agents can
further be added. For buccal administration, the molecules can take
the form of tablets or lozenges. For administration by inhalation,
the oligonucleotides can be delivered in the form of an aerosol
spray from pressurized packs or a nebulizer, with the use of a
suitable propellant, such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, and carbon
dioxide. In the case of a pressurized aerosol, the dosage unit can
be determined by providing a valve to deliver a metered amount.
Capsules and cartridges of gelatin for use in an inhaler or
insufflator can be formulated containing a powder mix of the
nucleic acids and a suitable powder base such as lactose or starch.
The oligonucleotides can also be formulated in rectal or vaginal
compositions such as suppositories and retention enemas, for
example containing conventional suppository bases such as cocoa
butter and other glycerides.
[0169] In addition to the formulations described previously, the
oligonucleotides can also be formulated as a depot preparation.
Such long acting formulations can be administered by implantation
subcutaneously or intramuscularly or by intramuscular injection.
For depot formulation, oligonucleotides can be formulated with:
suitable polymeric or hydrophobic materials, for example, as an
emulsion in an acceptable oil; ion exchange resins; or sparingly
soluble derivatives, for example, a sparingly soluble salt.
[0170] In some embodiments, liposomes and emulsions can be
formulated as delivery vehicles to deliver nucleic acids of the
present invention. A nucleic acid of the invention can be
administered in combination with a carrier or lipid to increase
cellular uptake. For example, the small RNA can be administered in
combination with a cationic lipid. Non-limiting examples of
cationic lipids include lipofectin, DOTMA, DOPE, DOTAP,
DOTAP:cholesterol, and cholesterol derivative formulations. In some
embodiments, lipid or liposomal formulations include
nanoparticles.
[0171] In some embodiments, oligonucleotides can be administered in
combination with a cationic amine such as poly(L-lysine).
[0172] Oligonucleotides can be delivered using a sustained-release
system, such as semipermeable matrices of solid polymers containing
the small RNA. Sustained-release capsules can, depending on their
chemical nature, release the small RNA for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks,
about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about
11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15
weeks, or about 16 weeks.
[0173] Nucleic acids can be included in any of the above-described
formulations as the free acids, free bases, or as
pharmaceutically-acceptable salts. Pharmaceutically-acceptable
salts include, for example, acid-addition salts and base-addition
salts. The acid that is added to the compound to form an
acid-addition salt can be an organic acid or an inorganic acid. A
base that is added to the compound to form a base-addition salt can
be an organic base or an inorganic base. In some embodiments, a
pharmaceutically-acceptable salt is a metal salt. In some
embodiments, a pharmaceutically-acceptable salt is an ammonium
salt.
[0174] Acid addition salts can arise from the addition of an acid
to a compound of the invention. In some embodiments, the acid is
organic. In some embodiments, the acid is inorganic. In some
embodiments, the acid is hydrochloric acid, hydrobromic acid,
hydroiodic acid, nitric acid, nitrous acid, sulfuric acid,
sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid,
salicylic acid, tartaric acid, ascorbic acid, gentisinic acid,
gluconic acid, glucaronic acid, saccaric acid, formic acid, benzoic
acid, glutamic acid, pantothenic acid, acetic acid, propionic acid,
butyric acid, fumaric acid, succinic acid, methanesulfonic acid,
ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
citric acid, oxalic acid, or maleic acid. Acid addition salts can
be, for example, monosalts, disalts, trisalts, tetrasalts, or
higher salts. Such forms can have counterions that are all the same
anion, or encompass a plurality of chemically-distinct anions.
[0175] In some embodiments, the salt is a hydrochloride salt, a
hydrobromide salt, a hydroiodide salt, a nitrate salt, a nitrite
salt, a sulfate salt, a sulfite salt, a phosphate salt,
isonicotinate salt, a lactate salt, a salicylate salt, a tartrate
salt, an ascorbate salt, a gentisinate salt, a gluconate salt, a
glucaronate salt, a saccarate salt, a formate salt, a benzoate
salt, a glutamate salt, a pantothenate salt, an acetate salt, a
propionate salt, a butyrate salt, a fumarate salt, a succinate
salt, a methanesulfonate (mesylate) salt, an ethanesulfonate salt,
a benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt,
an oxalate salt, or a maleate salt.
[0176] Metal salts can arise from the addition of an inorganic base
to a compound of the invention. The inorganic base consists of a
metal cation paired with a basic counterion, such as, hydroxide,
carbonate, bicarbonate, or phosphate. The metal can be an alkali
metal, alkaline earth metal, transition metal, or main group metal.
In some embodiments, the metal is lithium, sodium, potassium,
cesium, cerium, magnesium, manganese, iron, calcium, strontium,
cobalt, titanium, aluminum, copper, or zinc. In some embodiments, a
metal salt is a lithium salt, a sodium salt, a potassium salt, a
cesium salt, a cerium salt, a magnesium salt, a manganese salt, an
iron salt, a calcium salt, a strontium salt, a cobalt salt, a
titanium salt, an aluminum salt, a copper salt, a cadmium salt, or
a zinc salt.
[0177] Ammonium salts can arise from the addition of ammonia or an
organic amine to a compound of the invention. In some embodiments,
the organic amine is triethyl amine, diisopropyl amine, ethanol
amine, diethanol amine, triethanol amine, morpholine,
N-methylmorpholine, piperidine, N-methylpiperidine,
N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrrazole,
pipyrrazole, imidazole, pyrazine, or pipyrazine. In some
embodiments, an ammonium salt is a triethyl amine salt, a
diisopropyl amine salt, an ethanol amine salt, a diethanol amine
salt, a triethanol amine salt, a morpholine salt, an
N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine
salt, an N-ethylpiperidine salt, a dibenzylamine salt, a piperazine
salt, a pyridine salt, a pyrrazole salt, a pipyrrazole salt, an
imidazole salt, a pyrazine salt, or a pipyrazine salt.
[0178] Pharmaceutical compositions of the present invention
comprise an effective amount of one or more oligonucleotides
dissolved or dispersed in a pharmaceutically-acceptable carrier.
Pharmaceutically-acceptable carriers include any solvents,
dispersion media, coatings, surfactants, antioxidants,
preservatives, antibacterial agents, antifungal agents, isotonic
agents, absorption delaying agents, salts, preservatives, drugs,
drug stabilizers, gels, binders, excipients, disintegration agents,
lubricants, sweetening agents, flavoring agents, dyes, or a
combination thereof (see, for example, Remington's Pharmaceutical
Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329,
incorporated herein by reference). Except insofar as any
conventional carrier is incompatible with the active ingredient,
its use in the therapeutic or pharmaceutical compositions is
contemplated.
[0179] The oligonucleotides can comprise different types of
carriers depending on whether it is to be administered in solid,
liquid or aerosol form, and whether it need to be sterile for such
routes of administration as injection.
[0180] In some embodiments, pharmaceutical compositions can
comprise about 0.1%, about 0.5%, about 1%, about 5%, about 10%,
about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,
about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,
about 75%, about 80%, about 85%, about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99%, about 99.5%, or about 99.9% small RNA by mass. In some
embodiments, pharmaceutical compositions can comprise from about
0.1% to about 0.5%, from about 0.5% to about 1%, from about 1% to
about 5%, from about 5% to about 10%, from about 10% to about 15%,
from about 15% to about 20%, from about 20% to about 25%, from
about 25% to about 30%, from about 30% to about 35%, from about 35%
to about 40%, from about 40% to about 45%, from about 45% to about
50%, from about 50% to about 55%, from about 55% to about 60%, from
about 60% to about 65%, from about 65% to about 70%, from about 70%
to about 75%, from about 75% to about 80%, from about 80% to about
85%, from about 85% to about 90%, from about 90% to about 95%, from
about 95% to about 96%, from about 96% to about 97%, from about 97%
to about 98%, from about 98% to about 99%, from about 99% to about
99.5%, or from about 99.5% to about 99.9% small RNA by mass.
[0181] In some embodiments, a composition of the present invention
can have a concentration of the small RNA of about 0.01 milligrams
per milliliter (mg/mL), about 0.05 mg/mL, about 0.1 mg/mL, about
0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about
0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about
1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 2.5 mg/mL, about 3
mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, about 5
mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL,
about 10 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL,
about 13 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL,
about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, about 20 mg/mL,
about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL,
about 45 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL,
about 80 mg/mL, about 90 mg/mL, or about 100 mg/mL.
[0182] In some embodiments, a composition of the present invention
can have a concentration of the small RNA from about 0.01 mg/mL to
about 0.05 mg/mL, from about 0.05 mg/mL to about 0.1 mg/mL, from
about 0.1 mg/mL to about 0.2 mg/mL, from about 0.2 mg/mL to about
0.3 mg/mL, from about 0.3 mg/mL to about 0.4 mg/mL, from about 0.4
mg/mL to about 0.5 mg/mL, from about 0.5 mg/mL to about 0.6 mg/mL,
from about 0.6 mg/mL to about 0.7 mg/mL, from about 0.7 mg/mL to
about 0.8 mg/mL, from about 0.8 mg/mL to about 0.9 mg/mL, from
about 0.9 mg/mL to about 1 mg/mL, from about 1 mg/mL to about 1.5
mg/mL, from about 1.5 mg/mL to about 2 mg/mL, from about 2 mg/mL to
about 2.5 mg/mL, from about 2.5 mg/mL to about 3 mg/mL, from about
3 mg/mL to about 3.5 mg/mL, from about 3.5 mg/mL to about 4 mg/mL,
from about 4 mg/mL to about 4.5 mg/mL, from about 4.5 mg/mL to
about 5 mg/mL, from about 5 mg/mL to about 6 mg/mL, from about 6
mg/mL to about 7 mg/mL, from about 7 mg/mL to about 8 mg/mL, from
about 8 mg/mL to about 9 mg/mL, from about 9 mg/mL to about 10
mg/mL, from about 10 mg/mL to about 11 mg/mL, from about 11 mg/mL
to about 12 mg/mL, from about 12 mg/mL to about 13 mg/mL, from
about 13 mg/mL to about 14 mg/mL, from about 14 mg/mL to about 15
mg/mL, from about 15 mg/mL to about 16 mg/mL, from about 16 mg/mL
to about 17 mg/mL, from about 17 mg/mL to about 18 mg/mL, from
about 18 mg/mL to about 19 mg/mL, from about 19 mg/mL to about 20
mg/mL, from about 20 mg/mL to about 25 mg/mL, from about 25 mg/mL
to about 30 mg/mL, from about 30 mg/mL to about 35 mg/mL, from
about 35 mg/mL to about 40 mg/mL, from about 40 mg/mL to about 45
mg/mL, from about 45 mg/mL to about 50 mg/mL, from about 50 mg/mL
to about 60 mg/mL, from about 60 mg/mL to about 70 mg/mL, from
about 70 mg/mL to about 80 mg/mL, from about 80 mg/mL to about 90
mg/mL, or from about 90 mg/mL to about 100 mg/mL.
[0183] In some embodiments, the composition can comprise an
antioxidant to retard oxidation of one or more component.
Additionally, compositions can comprise preservatives such as
various antibacterial and antifungal agents. Non-limiting examples
of preservatives include parabens, such as methylparabens and
propylparabens, chlorobutanol, phenol, sorbic acid, thimerosal, or
a combination thereof.
[0184] In some embodiments, the composition is in a liquid form in
which a carrier can be a solvent or dispersion medium comprising
water, ethanol, a polyol such as glycerol, propylene glycol, and
liquid polyethylene glycol, a lipid such as triglycerides,
vegetable oils, and liposomes, or a combination thereof. Fluidity
can be maintained by the use of a coating, such as lecithin; by the
maintenance of the required particle size by dispersion in carriers
such as liquid polyol or lipids; by the use of surfactants, such as
hydroxypropylcellulose; or a combination thereof. In some
embodiments, the composition will comprise isotonic agents, such as
sugars, sodium chloride or a combination thereof. In some
embodiments, the compositions of the present invention are stored
in a lyophilized form for solubilization with a liquid carrier as
described herein. In some embodiments, the lyophilized form is
stored in an airtight container with a rubber stopper seal.
[0185] In some embodiments, the composition can be administered as
eye drops, nasal solutions, nasal sprays, aerosols or inhalants. In
some embodiments, the drops, sprays, solutions, aerosols or
inhalants are isotonic with a pH from about 7.0 to about 7.4 or
slightly buffered to maintain a pH of about 5.5 to about 6.5. In
some embodiments, the composition can further comprise
antimicrobial preservatives as described above. In some
embodiments, the composition can further comprise drugs such as
antibiotics or antihistamines.
[0186] In some embodiments, the oligonucleotides are formulated for
oral administration as a solid or liquid composition. In some
embodiments, the oral composition can comprise solutions,
suspensions, emulsions, tablets, pills, capsules such as hard or
soft shelled gelatin capsules, sustained release formulations,
buccal compositions, troches, elixirs, suspensions, syrups, wafers,
or a combination thereof. Oral compositions can be incorporated
directly with the food of the diet of the subject. Non-limiting
examples of carriers for oral administration include inert
diluents, assimilable edible carriers and a combination thereof. In
some embodiments, the syrup or elixir can comprise a sweetening
agent, a preservative, a flavoring agent, a dye, a preservative, or
a combination thereof.
[0187] In some embodiments, the composition can comprise one or
more of the following: a binder such as gum tragacanth, acacia,
cornstarch, gelatin or a combination thereof; an excipient such as
dicalcium phosphate, mannitol, lactose, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate or a combination
thereof; a disintegrating agent such as corn starch, potato starch,
alginic acid or a combination thereof; a lubricant such as
magnesium stearate; a sweetening agent such as sucrose, lactose,
saccharin or a combination thereof; a flavoring agent such as
peppermint, oil of wintergreen, cherry flavoring, orange flavoring;
or a combination thereof. A capsule can further comprise carriers
such as a liquid carrier. In some embodiments, the dosage unit,
such as tablets, capsules, or pills, can comprise a coating, such
as shellac, sugar or a combination thereof.
[0188] In some embodiments, prolonged absorption of an injectable
composition can be brought about by the use in the compositions of
agents delaying absorption, such as aluminum monostearate, gelatin,
biodegradable matrices or gels, time-release matrices or gels, or a
combination thereof. Non-limiting examples of time-release matrices
or gels include agarose, agarose colloid matrix, such 0.4% (w/v),
12 kDa, hydrogels, sodium carboxymethylcellulose, and
polyethyleneimine. An agent delaying absorption can release a
composition over about 1 minute, about 5 minutes, about 10 minutes,
about 20 minutes, about 30 minutes, about 40 minutes, about 50
minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 12 hours, about 1 day, about 2
days, about 3 days, about 4 days, about 5 days, about 6 days, about
1 week, about 8 days, about 9 days, about 10 days, about 11 days,
about 12 days, about 13 days, about 2 weeks, about 15 days, about
16 days, about 17 days, about 18 days, about 19 days, about 20
days, about 3 weeks, or about 1 month. An agent delaying absorption
can release a composition over from about 1 minute to about 5
minutes, from about 5 minutes to about 10 minutes, from about 10
minutes to about 20 minutes, from about 20 minutes to about 30
minutes, from about 30 minutes to about 40 minutes, from about 40
minutes to about 50 minutes, from about 50 minutes to about 1 hour,
from about 1 hour to about 2 hours, from about 2 hours to about 3
hours, from about 3 hours to about 4 hours, from about 4 hours to
about 5 hours, from about 5 hours to about 6 hours, from about 6
hours to about 12 hours, from about 12 hours to about 1 day, from
about 1 day to about 2 days, from about 2 days to about 3 days,
from about 3 days to about 4 days, from about 4 days to about 5
days, from about 5 days to about 6 days, from about 6 days to about
1 week, from about 1 week to about 8 days, from about 8 days to
about 9 days, from about 9 days to about 10 days, from about 10
days to about 11 days, from about 11 days to about 12 days, from
about 12 days to about 13 days, from about 13 days to about 2
weeks, from about 2 weeks to about 15 days, from about 15 days to
about 16 days, from about 16 days to about 17 days, from about 17
days to about 18 days, from about 18 days to about 19 days, from
about 19 days to about 20 days, from about 20 days to about 3
weeks, or from about 3 weeks to about 1 month,
[0189] In some embodiments, the composition is formulated to be
sterile. In some embodiments, endotoxin contamination is less than
about 0.5 nanograms per milligram (ng/mg) protein.
Kits
[0190] The present invention can include kits for regulating
expression of a target transcript in a cell, the kits comprising
oligonucleotides as described herein. The kits may comprise any one
of the sshRNAs, miRNA antagonists or pre-miRNA mimics, as disclosed
herein, or any combination thereof. The kits may comprise an sshRNA
targeting PHD2, an miRNA antagonist targeting miR-210, a pre-miRNA
mimic targeting miR-21, and any combination thereof. In certain
embodiments, the kits include systems that allow for the storage,
transport, or delivery of reaction reagents, for example, a small
RNA and a culture medium, and supporting materials, for example,
buffers, written instructions for performing the assay, from one
location to another. In some embodiments, the kits can include one
or more control reagents, such as a non-chemically modified small
RNA or a non-targeting small RNA. In some embodiments, kits
comprise a container, such as a box, comprising the reaction
reagents and supporting materials. Such contents can be delivered
to the intended recipient together or separately.
[0191] In some embodiments, the kits can further include
instructions for using the components of the kit to practice the
methods of the present invention. The instructions can be recorded
on a suitable recording medium, for example, paper, plastic,
cardboard, or a digital format. In some embodiments, the
instructions can be present as a package insert, on the labeling of
the container of the kit or components thereof, such as the
packaging or sub-packaging of the kit. In some embodiments, the
instructions are present as an electronic storage data file present
on a suitable computer readable storage medium, for example, a
CD-ROM or flash drive. In yet other embodiments, the actual
instructions are not present in the kit, but means for obtaining
the instructions from a remote source, such as the Internet, are
provided. In some examples, the Internet instructions can be
accessed from a web address where the instructions can be viewed or
downloaded.
[0192] A listing of relevant sequences disclosed herein is provided
in TABLE 1. Nucleotides preceded by a "d" correspond to 2'-H
modified nucleotides. Nucleotides preceded by a "1" correspond to
LNA modified nucleotides. Nucleotides preceded by a "f" correspond
to 2'-F modified nucleotides. Underlined nucleotides correspond to
2'-O-methyl modified nucleotides. Phosphorothioate linkages are
indicated by an asterisk. Nucleotides preceded by an +-sign
correspond to nucleotides to which a C6-amine-TexasRed conjugate
moiety is attached (dT-C6-NH (Alexa594). /ZEN/corresponds to an
N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine chemical
modifier.
[0193] SEQ ID NOS: 1 and 2 correspond to DNA sequences encoding
transcripts for human and mouse PHD2 transcripts, respectively. SEQ
ID NOS: 3 and 4 correspond to sequences for naturally occurring
miR-210 and miR-21, respectively. SEQ ID NOS: 44-48 correspond to
sequences representing pre-miRNA mimics disclosed herein. SEQ ID
NOS: 5-8, 11-27, 30, 31, 49-325 correspond to sequences
representing sshRNAs targeting PHD2, disclosed herein. SEQ ID NOS:
32-43, 326-328 correspond to sequences representing miRNA
antagonists disclosed herein. SEQ ID NOS: 9, 10, 28, 29 correspond
to sequences (guide (g) or passenger (p) strands) that make up
siRNA targeting PHD2. For example, SEQ ID NOS: 28 and 29 are the
individual strands that make up an siRNA-SG403g is guide
(antisense) and SG403p is passenger (sense).
TABLE-US-00001 TABLE 1 SEQ ID NO Name Sequence 1 human
TTAGGGGCAGAAAAACATTTGTAATAATTAATGGCTTTGAGAGA PHD2
CACAAGGCTTTGTTTGCCCCAGAGTATTAGTTAACCCACCTAGTG
CTCCTAATCATACAATATTAAGGATTGGGAGGGACATTCATTGCC
TCACTCTCTATTTGTTTCACCTTCTGTAAAATTGGTAGAATAATA
GTACCCACTTCATAGCATTGTATGATGATTAAATTGGTTAATATT
TTTAAAATGCTTAGAACACAGATTGGGCACATAACAGCAAGCAC
CACATGTGTTTATAAGATAAATTCCTTTGTGTTGCCTTCCGTTAA
AGTTTAAATAAGTAAATAAATAAATAAATACTTGCATGACATTTT
GAAGTCTCTCTATAACATCTGAGTAAGTGGCGGCTGCGACAATG
CTACTGGAGTTCCAGAATCGTGTTGGTGACAAGATTGTTCACCAG
CATATGGTGTGGTGAAAACTCACTAATTTGGAATTAGTTCAGATT
ATTAAGCCTGAATAGGTGAAAATCCTGAAATCAAGGATCTTTGG
AACTATTTGAAATCAGTATTTTATATTTTCCTGTTGTATTCATTAA
AGTGTTGCAAGTGTTCTATTTGATGGATTAAGTATATTTAGGATA
TACATGTTCAATTTGTGATTTTGTATACTTAATTGGAACAAGAAA
GCTAATAAAGGTTTTGATATGGACATCTATTCTTTTAAGTAAACT
TCAATGAAAATATATGAGTAGAGCATATAGAGATGTAAATAATT
TGTGGACACACCACAGACTGAAATAGCAAATTTAAAAGAAATTG
TTGGAAGAATCAAGTGTTTGTGGAATGAGTCCTCCTAGTAAAGTT
CCTGCTCTTGTGAATAATTAAGCCTCATGTATAATTACTATAGCA
AAAGGAAGCCTAAGAAGTATTAGACTCTACTTGTATTTAAATTAC
ATTTTACATAATTTATGTGTATGAAAAATGTTTTAAATGCTTATTT
TCGTAAGCCATGAGATAGCTCCTTTATATTTTAAGAATTTCTGAA
TTAATTTGCTTGGATTTTATTAGTGCAAATGGCAGAGCTAGCAAT
TCCTTTTTCTGTGTTCCCATTCCATCCTATTCATCCCTCTTTTAGG
AAACTCTGAACTCTGGATTGTCCTTGTTTACATACCTGCCTCCTG
CATTGGACTATGTGTCTCTGAGTGTAGTATGACTAATTCATTTGT
TTGTCAAGGACTCTCAATGCATTTGTTGAACAGCCTAATTAGTAA
TGTCTGCAACAATGACATTTTACTGTATTTAATAAAGCTCTGGGA
AAGTAGGATACACATAAGACAGGTCTAGGTCTAAATTCTTTACA
GAAACTTGGATTTTTAGTTCGGTTTGAAATTTGAAGATGTGAGTA
TATTTATCTCAGTTTCCCAAAGGACAAGCTAATTGGAATTATCAT
CCTCTTTCACTTGATTGGATCCCCAGAATGCCATTTACGCATGCA
GCAGGATTTTATAACAGTTTTAAATTCTGTATATTTGATGAAGAG
GTTTTATATTTTTGGATTCAAGCCTCTTTTTAAACTTCTACAATAT
GGTTTACAATAATTCCTTATATCCTGCTTTTGAAATACATATTAC
AACTTTTTAAGTTTGGAAGGCTATATTTCAAGGACTGAAGTTACA
GTATACTCAAGTGATACACAAGCCTAGCACCCCACTTTCCACATA
GTGTTCGATAAAGATTGATAAACTCGAAATCACAGACCTTTTAAT
TCTTAAGACAAATAGCAGCAGAAAGAAACATCTTTGGCTTATTTC
TGGTAAGGTTTTTATGCTCTGTAAAACAAAGAATTGTATTCATCC
GCGCAGCACAGATTCTATTAAAAATAAATGTGAGAGTCGTTAAT
GTAGTACTGCTCATTTACCATCAAAATTCACTTTTCAGGAATAAT
CCCATCAGTTTAAATTGGATATTGGAATGAGCATTGATTACATTT
AACTTGGTAGCCCAAAATTTCTTCATGGGGTTTTGAACTCGGCGG
GATTTCAAAGGTTTTAAAAATGAGTTTTTGATTTTTTTTAAAACC
CTCAAATTTCATTACCTTTAAACTAGGTCGAAACGGGGCGCAAG
AGATTGGATTAACACCATAGTAATACTTATTTTGTTCTTAACCAT
TTCAGGGCTTCTTGAAATAGAGGCTGTATGGTGTAATGGAAAAA
ACAGCCTTGGAATCTGGGAGCCTGATTCCTGGATTCAGTCCCAGT
TTTGCGTGACCTTGGGCAAGTTACTTTACTTCTCTGAATTTCCGTT
TCCTCCTCTGCAAAATGAGGATCGCAATAGCCACCTTGCAACCTT
GACTGGAGCGAGCCTCGCACACCCCGCGCCGGCCTGGAGGAAGA
GCAGCCATGATTACGCCGCCTTCGCTCCGCTACCCGCTTGCGGCT
GGCGCCCTCCTCCAGCAGGTGTAGGCGCTGCCGCGCTGCCCCAC
GCCTTTCCGCCGCTCGCGGGCCTGCGCCTCGGCGTCCCCGAGGAG
GCCGCTGCGGGCTGAGGTAGCGCACCGGCCTCTCGGCGTCCCAG
TCCGGTCCCGGGCGGAGGGAAAGCGGGCGACCCACCTCCGAGGC
AGAAGCCGAGGCCCGGCCCCGCCGAGTGCGGAGGAGCGCAGGC
AGCCCCCGCCCCTCGGCCCTCCCCCCGGCCCTCCCGGCCCTCCCT
CCGCCCCCTCCGCCCTCGCGCGCCGCCCGCCCGGGTCGCCGCGG
GGCCGTGGTGTACGTGCAGAGCGCGCAGAGCGAGTGGCGCCCGT
ATGCCCTGCGCTCCTCCACAGCCTGGGCCGGGCCGCCCGGGACG
CTGAGGCGGCGGCGGCGGCCGAGGGGGCCGGTCTTGCGCTCCCC
AGGCCCGCGCGCCTGAGCCCAGGTTGCCATTCGCCGCACAGGCC
CTATTCTCTCAGCCCTCGGCGGCGATGAGGCGCTGAGGCGGCTG
CCGGCGCTGCGCCGGAGCTTAGGACTCGGAAGCGGCCGGGCCGA
GGGCGTGGGGTGCCGGCCTCCCTGAGGCGAGGGTAGCGGGTGCA
TGGCGCAGTAACGGCCCCTATCTCTCTCCCCGCTCCCCAGCCTCG
GGCGAGGCCGTCCGGCCGCTACCCCTCCTGCTCGGCCGCCGCAG
TCGCCGTCGCCGCCGCCGCCGCCGCCATGGCCAATGACAGCGGC
GGGCCCGGCGGGCCGAGCCCGAGCGAGCGAGACCGGCAGTACT
GCGAGCTGTGCGGGAAGATGGAGAACCTGCTGCGCTGCAGCCGC
TGCCGCAGCTCCTTCTACTGCTGCAAGGAGCACCAGCGTCAGGA
CTGGAAGAAGCACAAGCTCGTGTGCCAGGGCAGCGAGGGCGCCC
TCGGCCACGGAGTGGGCCCACACCAGCATTCCGGCCCCGCGCCG
CCGGCTGCAGTGCCGCCGCCCAGGGCCGGGGCCCGGGAGCCCAG
GAAGGCAGCGGCGCGCCGGGACAACGCCTCCGGGGACGCGGCC
AAGGGAAAAGTAAAGGCCAAGCCCCCGGCCGACCCAGCGGCGG
CCGCGTCGCCGTGTCGTGCGGCCGCCGGCGGCCAGGGCTCGGCG
GTGGCTGCCGAAGCCGAGCCCGGCAAGGAGGAGCCGCCGGCCC
GCTCATCGCTGTTCCAGGAGAAGGCGAACCTGTACCCCCCAAGC
AACACGCCCGGGGATGCGCTGAGCCCCGGCGGCGGCCTGCGGCC
CAACGGGCAGACGAAGCCCCTGCCGGCGCTGAAGCTGGCGCTCG
AGTACATCGTGCCGTGCATGAACAAGCACGGCATCTGTGTGGTG
GACGACTTCCTCGGCAAGGAGACCGGACAGCAGATCGGCGACGA
GGTGCGCGCCCTGCACGACACCGGGAAGTTCACGGACGGGCAGC
TGGTCAGCCAGAAGAGTGACTCGTCCAAGGACATCCGAGGCGAT
AAGATCACCTGGATCGAGGGCAAGGAGCCCGGCTGCGAAACCAT
TGGGCTGCTCATGAGCAGCATGGACGACCTGATACGCCACTGTA
ACGGGAAGCTGGGCAGCTACAAAATCAATGGCCGGACGAAAGC
CATGGTTGCTTGTTATCCGGGCAATGGAACGGGTTATGTACGTCA
TGTTGATAATCCAAATGGAGATGGAAGATGTGTGACATGTATAT
ATTATCTTAATAAAGACTGGGATGCCAAGGTAAGTGGAGGTATA
CTTCGAATTTTTCCAGAAGGCAAAGCCCAGTTTGCTGACATTGAA
CCCAAATTTGATAGACTGCTGTTTTTCTGGTCTGACCGTCGCAAC
CCTCATGAAGTACAACCAGCATATGCTACAAGGTACGCAATAAC
TGTTTGGTATTTTGATGCAGATGAGAGAGCACGAGCTAAAGTAA
AATATCTAACAGGTGAAAAAGGTGTGAGGGTTGAACTCAATAAA
CCTTCAGATTCGGTCGGTAAAGACGTCTTCTAGAGCCTTTGATCC
AGCAATACCCCACTTCACCTACAATATTGTTAACTATTTGTTAAC
TTGTGAATACGAATAAATGGGATAAAGAAAAATAGACAACCAGT
TCGCATTTTAATAAGGAAACAGAAACAACTTTTTGTGTTGCATCA
AACAGAAGATTTTGACTGCTGTGACTTTGTACTGCATGATCAACT
TCAAATCTGTGATTGCTTACAGGAGGAAGATAAGCTACTAATTG
AAAATGGTTTTTACATCTGGATATGAAATAAGTGCCCTGTGTAGA
ATTTTTTTCATTCTTATATTTTGCCAGATCTGTTATCTAGCTGAGT
TCATTTCATCTCTCCCTTTTTTATATCAAGTTTGAATTTGGGATAA
TTTTTCTATATTAGGTACAATTTATCTAAACTGAATTGAGAAAAA
ATTACAGTATTATTCCTCAAAATAACATCAATCTATTTTTGTAAA
CCTGTTCATACTATTAAATTTTGCCCTAAAAGACCTCTTAATAAT
GATTGTTGCCAGTGACTGATGATTAATTTTATTTTACTTAAAATA
AGAAAAGGAGCACTTTAATTACAACTGAAAAATCAGATTGTTTT
GTAGTCCTTCCTTACACTAATTTGAACTGTTAAAGATTGCTGCTTT
TTTTTTGACATTGTCAATAACGAAACCTAATTGTAAAACAGTCAC
CATTTACTACCAATAACTTTTAGTTAATGTTTTACAAGGAAAAAG
ACACAAGAAGAGTTTAAATTTTTTTGTTTTGTTTTGTTTTTTTGAG
ACAGTCTTGCTCTGTTACCCAGGCTGGAGGGGAGTGGTGCATTCT
TGGCTCACTGCAACCTCCGCCTCCCAGGTTCAAGCAATCCTCCCA
CCTCAGCCTCCCAACTAGCTGGGACTGCAGGCACACACCACCAT
GCCTGACTAATTTTTGTATGTTTAGTAGAGACGGGGTTTTGCCAT
GTTGCCTAGGCTGGGGTTTAAGTTAAATTTTTTAAAAAACTAAAG
TGACTGGCACTAAGTGAACTTGAGATTATCCTCAGCTTCAAGTTC
CTAAGATAAGGGCTTTCTTAAGCTTTCAGGTGTATGTATCCTCTA
GATGTAGACAATAATGTCCCATTTCTAAGTCTTTTCCTTTTGCTTC
TCCTTAAATTGATTGTACTTCCAAATTTGCTGTTATGTTTTTTTCC
TAATACTGTGATCTATCTGATCTGCAGACAAGAACCTTGTCTCTG
TTGAAGAGCATCAAGGGGAGATTATGTACACATTGAAACTGAAG
TGTGGTGTTACTGACGGAATGTGCAGTAACTCCTCAGATATCTGT
TAAGGCATTTCCCAGATGTGATGCCAGCCTTCTTACCTGTACTGA
AAGATGCTTAGCTTAGAAAAAAACAAAACAGATGCAAAATCAG
ATAATTTTATTTTGTTTCATGGGTTTTCTTATTTACTTTTTAAACA
AGGAAGGAATATTAGAAAATCACACAAGGCCTCACATACATGTT
ATTTAAAGAATGAATTGGGACGGATGTCTTAGACTTCACTTTCCT
AGGCTTTTTAGCAAAACCTAAAGGGTGGTATCCATATTTTGCGTG
AATTATGGGTGTAAGACCTTGCCCACTTAGGTTTTCTATCTCTGT
CCTTGATCTTCTTTGCCAAAATGTGAGTATACAGAAATTTTCTGT
ATATTTCAACTTAAGACATTTTTAGCATCTGTATAGTTTGTATTCA
ATTTGAGACCTTTTCTATGGGAAGCTCAGTAATTTTTATTAAAAG
ATTGCCATTGCTATTCATGTAAAACATGGAAAAAAATTGTGTAGT
GAAGCCAACAGTGGACTTAGGATGGGATTGAATGTTCAGTATAG
TGATCTCACTTAGGAGAATTTGCAGGAGAAAGTGATAGTTTATTG
TTTTTTCCTCGCCCATATTCAGTTTTGTTCTACTTCCTCCCCTTCC
TTCCAGATGATAACATCACATCTCTACAGTAAGTGCCTCTGCCAGC
CCAACCCAGGAGCGCAAGTTGTCTTTGCCATCTGGTCTATAGTAC
AGTGCGCGGCGTTAGGCCACAACTCAAAAGCATTATCTTTTTTAG
GGTTAGTAGAAATTGTTTTATGTTGATGGGAGGTTTGTTTGATTG
TCAAAATGTACAGCCACAGCCTTTTAATTTGGGAGCCCCTGTTGT
CATTCAAATGTGTACCTCTACAGTTGTAAAAAGTATTAGATTCTA
CTATCTGTGGGTTGTGCTTGCCAGACAGGTCTTAAATTGTATATT
TTTTGGAAAAGTTTATATACTCTCTTAGGAATCATTGTGAAAAGA
TCAAGAAATCAGGATGGCCATTTATTTAATATCCATTCATTTCAT
GTTAGTGGGACTATTAACTTGTCACCAAGCAGGACTCTATTTCAA
ACAAAATTTAAAACTGTTTGTGGCCTATATGTGTTTAATCCTGGT
TAAAGATAAAGCTTCATAATGCTGTTTTTATTCAACACATTAACC
AGCTGTAAAACACAGACCTTTATCAAGAGTAGGCAAAGATTTTC
AGGATTCATATACAGATAGACTATAAAGTCATGTAATTTGAAAA
GCAGTGTTTCATTATGAAAGAGCTCTCAAGTTGCTTGTAAAGCTA
ATCTAATTAAAAAGATGTATAAATGTTGTTGAAACATTAAAAAA 2 mouse
GGCTGGGCCCGCCCGCCCAGGGCGCTGTGCGCCGCGCAGGCCGC PHD2
GCTCTCTCCGGCGCGATGCGGCGCTAGGCGGCCCCGGGCAAGGC
AGGCGAGGCCAGGGCGCGCGCGGCCTCCCGCAGCGGGCGGCGG
CCCCGGGCGGGCGCCCCGACGGCCCCGCCGCCGCCCCGCTCCCG
GCCCGCGGCCCGCCCTGCCGCGGCCATGGCCAGTGACAGCGGCG
GGCCCGGCGTGCTGAGCGCCAGCGAGCGCGACCGGCAGTACTGC
GAGCTGTGCGGGAAGATGGAGAACCTGCTGCGCTGCGGCCGCTG
CCGCAGCTCCTTCTACTGCTGCAAAGAGCACCAGCGCCAGGACT
GGAAGAAGCACAAGCTGGTGTGCCAGGGCGGCGAGGCCCCCCG
CGCGCAGCCCGCGCCGGCGCAGCCCCGCGTCGCGCCCCCGCCCG
GTGGGGCCCCCGGAGCCGCGCGCGCCGGCGGGGCGGCCCGGCGC
GGGGACAGCGCGGCGGCCTCGCGCGTACCGGGCCCGGAGGACG
CGGCGCAGGCCCGGAGCGGCCCCGGCCCAGCAGAGCCCGGCTCC
GAGGATCCTCCGCTTAGCCGGTCTCCGGGCCCCGAGCGCGCCAG
CCTGTGCCCAGCGGGTGGCGGCCCCGGGGAGGCGCTGAGTCCCG
GTGGAGGGCTGCGGCCCAACGGGCAGACCAAGCCGTTGCCCGCG
TTGAAGCTGGCTCTGGAGTACATCGTGCCGTGCATGAACAAGCA
CGGCATCTGCGTGGTGGACGACTTCCTGGGCAGGGAGACCGGGC
AGCAGATCGGCGATGAGGTGCGCGCCCTGCACGACACCGGCAAG
TTCACGGACGGGCAGCTGGTCAGCCAGAAGAGTGACTCTTCCAA
GGACATCCGGGGGGACCAGATCACCTGGATCGAGGGCAAAGAG
CCCGGCTGCGAAACCATCGGCCTGCTCATGAGCAGCATGGACGA
CCTGATCCGCCACTGCAGCGGGAAGCTGGGCAACTACAGGATAA
ACGGCCGAACGAAAGCCATGGTTGCTTGTTACCCAGGCAACGGA
ACAGGCTATGTCCGTCACGTTGATAACCCAAATGGAGATGGAAG
ATGCGTGACATGTATATATTATCTAAATAAAGACTGGGACGCCA
AGGTAAGTGGAGGTATTCTTCGAATTTTTCCAGAAGGCAAAGCC
CAGTTTGCTGACATTGAACCCAAATTTGATAGACTGCTGTTTTTC
TGGTCTGACCGGCGTAACCCTCATGAAGTACAGCCAGCATACGC
CACAAGGTACGCAATAACTGTTTGGTATTTTGATGCAGATGAGC
GAGCGAGAGCTAAAGTAAAATATCTAACAGGTGAGAAAGGTGT
GAGGGTTGAACTCAAGCCCAATTCAGTCAGCAAAGACGTCTAGT
GGGGCCTTGGGTCCGGCAGTACCCACGTCACCTACAGCCTCTCA
GTTGCCTTCTGTGGACTCGTGGACAGGATGGACAGAGAGACACC
TGCCTGGTATTTCAGCTGGGAGCCAGGCGACTTCGCCGGGTGTCA
TCCAACAGAGGGCTCCATCTGCTGGGACTGTACTGTGGGGTCAG
CTCCAGATCTGTGACTGCTCTTGGCTGCTGACCCAAGAGGAGAC
GCTGTCGGAGGAGAGTAGCTTTTCCATCTGGACACGAAACAAGG
GCCCTTTGTAGGAATTTCTTCAGTCTTCTATTTTGCCAGACCTGTC
ACCTAACTGAGTTCATTTCATCTCTTTTTTATATCAAGTTTTGAAT
TCGGGGAATTTTTGTATTAGGTACAATTTATCAAAACTGAATTAA
GAAAAAAAAATTTACAGTATTATTCTCAAAATAACATCAATCTAT
TTTTGTAAACCTCTTCATGCTATTAAATTTTGCCCTCAAGGCCTCC
TGCGATGATTGTTGCCAGTGAGTGACGACGTGTTGCTTCTGCCTG
AACGTAAAGGACGGGCGGGCGCTGTGTCCCAGCCCGAGTGCACG
AGGTTTTTCTTGGCCCGTCTCTCAGTGATTCCAACCTGTAAAGGT
CACTGCTCTCGCGCTTCGACCGACCTAACAGTAGATGGTTGCCAC
TGGCACTCAACTAACTCAACATAGTTACAAGAGGAAACAAGCCA
CAGGAGAGGGTTTGTCTCTTCAGTTAATTTTTTTAAAGCGAAGTG
ACGGGCACTAAATGAACTCGGGGCTCTCCCTCAGCTTCGGGTTCC
TGAGACAAAGGGCTTTCTTCTGCGGCAGGTCTAGCCTGCCTACAG
CCGTGTCCCACTGCCGCAGGTTTCCTTGTGGCTTCTCCGTAGTTTT
GACTGTGCTTCCAGACCCTTCCAGGTCAGGGCTGTGTTCTTGTGG
CAGGGCACCTGGTGGACCCAGGCACGTGAATGTGGTATGTGGTT
GTAGCCTCAATCGTGGCCATCGGCTCCTTGGACAGCCACGAGCC
ATTTTCATACCCAATAATGAAAGCTGTGTGCTAGCTTAGAAATCA
AAGGGGGTGTAAAAGCACACATTCTTTGTTTTATGGGTTTTTCTC
TTTTTAGAGGACAGAGGGACAACCACACGAGGCTGCCAGACTCC
TGTCACCTCTACAGTCCCCTTAGAAAGCCAGAGTTTGCACAGATT
GTGGGTATAACTCCTGTCCCCTTAGGTGTTCTATCTCCGACCTTG
ATCTTTGCCAAAATGTGTGTATGCAGAACTATTTCTGTGTATTTTC
CTTGACACCCGTCTTAGCACCTGTGTAGTTTGTATCCGGTTAGAA
ACCTTTTCTATGGAAAGCTCAGTAATTCTTATTAAGAGATTGCTA
TTGTTCATGTAAAACATGAAAACAACCAAGTAGAGCCGTGTGTG
GATGAGGGCCCACTCAGCACTGTGCTTGCTTGAGGGGCTCTCGG
CAGGAAGTCTCCTTCTGACCCATATCCGCTGACCACACCTCTCCA
GCAAGTGCCTCTGCCGCTGGCCAGCTCAAGGTTTGCCCACCTGGC
CCCGAAGCACCGTGTTTCGGAGTTGGGAGGAACTGTTTGGCATT
GTTGGCAGAAGGTGTGATTGCCTGGAGCAGCAGCCTTTTAAATTC
TGGAGACCCTGTAGTCCTTTGTATCTCAGACCTTTACTGATGTAC
CAGGTCCCAGATTCTGTGGCAGGGGATGGGGTGGGGTGTGCTTG
CCAGACGAAATTTAAATTATCTATCTTTTGGGAAGTGTGTGCTTT
CCTGGAGGTCACTGTGAAAACAAACAAACAAATCAGGACCGTTA
ACCCCTTAATGCCCACTTAAACTCAATTTCATGTTAGGACTCTTG
TTTAAAACCATTTGTGGCCTGTATGTGTTCATCCTGGTTAGAGAG
AAAGCTTTATGACGCTGTTTCTGTTCAACACATTAACCAGCTGTG
GAACAGCCCTTTTTGCACGACAGGCAGGGCACTTCAGGATTCGC
AGAGAGACTCGTGTGGTTTGGAAGTGGTATTTCCTATGAAAGCCT
CTCACGTTGCTTGTAAAGCTAATCTAATTAAAAAGATGTATAAAT GTTCTTGAAAAAAATC 3
miR- CUGUGCGUGUGACAGCGGCUGA 210 4 miR- UAGCUUAUCAGACUGAUGUUGA
21 5 SG300 CUUCCUUUUGCUAUAGUAAUUUUACUAUAGCAAAAGGAAGUU 6 SG301
UGUUGCAGACAUUACUAAUUUAUUAGUAAUGUCUGCAACAUU 7 SG302
UAUACCUCCACUUACCUUGUUCAAGGUAAGUGGAGGUAUAUU 8 SG302m1
UAUACCUCCACUUACCUUGUUCAAGGUAAGUGGAGGUAUATT 9 SG303g
UAUACCUCCACUUACCUUGUU 10 SG303p CAAGGUAAGUGGAGGUAUAUU 11 SG304
AUUCGAAGUAUACCUCCACUUGUGGAGGUAUACUUCGAAUUU 12 SG305
CGAAGUAUACCUCCACUUAUUUAAGUGGAGGUAUACUUCGUU 13 SG306
AGUAUACCUCCACUUACCUUUAGGUAAGUGGAGGUAUACUUU 14 SG307
UACCUCCACUUACCUUGGCUUGCCAAGGUAAGUGGAGGUAUU 15 SG308
CUCCACUUACCUUGGCAUCUUGAUGCCAAGGUAAGUGGAGUU 16 SG309
CACUUACCUUGGCAUCCCAUUUGGGAUGCCAAGGUAAGUGUU 17 SG310
AGGUUCUCCAUCUUCCCGCUUGCGGGAAGAUGGAGAACCUUU 18 SG311
AUGCCGUGCUUGUUCAUGCUUGCAUGAACAAGCACGGCAUUU 19 SG312
UCACUCUUCUGGCUGACCAUUUGGUCAGCCAGAAGAGUGAUU 20 SG313
AUCUUCCAUCUCCAUUUGGUUCCAAAUGGAGAUGGAAGAUUU 21 SG314
UGUGCUUCUUCCAGUCCUGUUCAGGACUGGAAGAAGCACAUU 22 SG315
AUCAGGUCGUCCAUGCUGCUUGCAGCAUGGACGACCUGAUUU 23 SG316
AUACCUCCACUUACCUUGgUUcCAAGGUAAGUGGAGGUAUUU 24 SG400
UGGCGUAUGCUGGCUGUACUUGUACAGCCAGCAUACGCCAUU 25 SG401
CUCACACCUUUCUCACCUGUUCAGGUGAGAAAGGUGUGAGUU 26 SG402
CUGAAUUGGGCUUGAGUUCUUGAACUCAAGCCCAAUUCAGUU 27 SG402m1
CUGAAUUGGGCUUGAGUUCUUGAACUCAAGCCCAAUUCAGTT 28 SG403p
GAACUCAAGCCCAAUUCAGUU 29 SG403g CUGAAUUGGGCUUGAGUUCUU 30 SG404
CUGAAUUGGGCUUGAGUUCUUGAACUCAAGCCCAAUUCAG 31 SG405
CUGAAUUGGGCUUGAGUUC+TUGAACUCAAGCCCAAUUCAG 32 SG601
UCAGCCGCUGUCACACGCACAG 33 SG602
dTdCdAdGdCdCdGdCdTdGdTdCdAdCdAdCdGdCdAdCdAdG 34 SG603
UCAGCCGCUGUCACACGCACAG 35 SG604 GCCGCTGTCACACGCACA 36 SG605
AGAGCTCCCTTCAATCCAAA 37 SG613 UlCAlGClCGlCUlGUCACAlCGlCAlCAlG 38
SG614 UlCAfGClCGfCUlGUCACAlCGfCAlCAfG 39 SG608
UfCAfGCfCGfCUfGUCACAfCGfCAfCAfG 40 SG609 UCAfGCCGfCUGUCACACGfCACAfG
41 SG610 UCAGCCGCUGUCACACGCACAG 42 SG611
UlCAlGClCGlCUlGUlClAlCAlCGlCAlCAlG 43 SG612
UfCAfGCfCGfCUfGUfCfAfCAfCGfCAfCAfG 44 SG701
UAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACA CCAGUCGAUGGGCUUU 45
SG702 UAGCUUAUCAGACUGAUGUUGACAUCAGUCUGAUAAGCUC 46 SG703
UAGCUUAUCAGACUGAUGUUGACAUCAGUCUGAUAAGCUAUU 47 SG704
UAGCUUAUCAGACUGAUGUUGAUUUCAACAUCAGUCUGAUAA GCUAUU 48 SG705
UAGCUUAUCAGACUGAUGUUGAUUUCAACAUCAGUCUGAUAA GCUAUU 49 PHD2-
CCGGGCCCGCCGCUGUCAUUUAUGACAGCGGCGGGCCCGGUU 17 50 PHD2-
GCCGGGCCCGCCGCUGUCAUUUGACAGCGGCGGGCCCGGCUU 18 51 PHD2-
CGCCGGGCCCGCCGCUGUCUUGACAGCGGCGGGCCCGGCGUU 19 52 PHD2-
CCGCCGGGCCCGCCGCUGUUUACAGCGGCGGGCCCGGCGGUU 20 53 PHD2-
CUCGCAGUACUGCCGGUCUUUAGACCGGCAGUACUGCGAGUU 21 54 PHD2-
GCUCGCAGUACUGCCGGUCUUGACCGGCAGUACUGCGAGCUU 22 55 PHD2-
AGCUCGCAGUACUGCCGGUUUACCGGCAGUACUGCGAGCUUU 23 56 PHD2-
CAGCUCGCAGUACUGCCGGUUCCGGCAGUACUGCGAGCUGUU 24 57 PHD2-
ACAGCUCGCAGUACUGCCGUUCGGCAGUACUGCGAGCUGUUU 25 58 PHD2-
CACAGCUCGCAGUACUGCCUUGGCAGUACUGCGAGCUGUGUU 26 59 PHD2-
GCACAGCUCGCAGUACUGCUUGCAGUACUGCGAGCUGUGCUU 27 60 PHD2-
CGCACAGCUCGCAGUACUGUUCAGUACUGCGAGCUGUGCGUU 28 61 PHD2-
CCGCACAGCUCGCAGUACUUUAGUACUGCGAGCUGUGCGGUU 29 62 PHD2-
CCCGCACAGCUCGCAGUACUUGUACUGCGAGCUGUGCGGGUU 30 63 PHD2-
UCCCGCACAGCUCGCAGUAUUUACUGCGAGCUGUGCGGGAUU 31 64 PHD2-
UUCCCGCACAGCUCGCAGUUUACUGCGAGCUGUGCGGGAAUU 32 65 PHD2-
CUUCCCGCACAGCUCGCAGUUCUGCGAGCUGUGCGGGAAGUU 33 66 PHD2-
UCUUCCCGCACAGCUCGCAUUUGCGAGCUGUGCGGGAAGAUU 34 67 PHD2-
AUCUUCCCGCACAGCUCGCUUGCGAGCUGUGCGGGAAGAUUU 35 68 PHD2-
CAUCUUCCCGCACAGCUCGUUCGAGCUGUGCGGGAAGAUGUU 36 69 PHD2-
CCAUCUUCCCGCACAGCUCUUGAGCUGUGCGGGAAGAUGGUU 37 70 PHD2-
UCCAUCUUCCCGCACAGCUUUAGCUGUGCGGGAAGAUGGAUU 38 71 PHD2-
CUCCAUCUUCCCGCACAGCUUGCUGUGCGGGAAGAUGGAGUU 39 72 PHD2-
UCUCCAUCUUCCCGCACAGUUCUGUGCGGGAAGAUGGAGAUU 40 73 PHD2-
UUCUCCAUCUUCCCGCACAUUUGUGCGGGAAGAUGGAGAAUU 41 74 PHD2-
GUUCUCCAUCUUCCCGCACUUGUGCGGGAAGAUGGAGAACUU 42 75 PHD2-
GGUUCUCCAUCUUCCCGCAUUUGCGGGAAGAUGGAGAACCUU 43 76 PHD2-
AGGUUCUCCAUCUUCCCGCUUGCGGGAAGAUGGAGAACCUUU 44 77 PHD2-
CAGGUUCUCCAUCUUCCCGUUCGGGAAGAUGGAGAACCUGUU 45 78 PHD2-
GCAGGUUCUCCAUCUUCCCUUGGGAAGAUGGAGAACCUGCUU 46 79 PHD2-
AGCAGGUUCUCCAUCUUCCUUGGAAGAUGGAGAACCUGCUUU 47 80 PHD2-
CAGCAGGUUCUCCAUCUUCUUGAAGAUGGAGAACCUGCUGUU 48 81 PHD2-
GCAGCAGGUUCUCCAUCUUUUAAGAUGGAGAACCUGCUGCUU 49 82 PHD2-
CGCAGCAGGUUCUCCAUCUUUAGAUGGAGAACCUGCUGCGUU 50 83 PHD2-
GCGCAGCAGGUUCUCCAUCUUGAUGGAGAACCUGCUGCGCUU 51 84 PHD2-
AGCGCAGCAGGUUCUCCAUUUAUGGAGAACCUGCUGCGCUUU 52 85 PHD2-
CAGCGCAGCAGGUUCUCCAUUUGGAGAACCUGCUGCGCUGUU 53 86 PHD2-
GCAGCGCAGCAGGUUCUCCUUGGAGAACCUGCUGCGCUGCUU 54 87 PHD2-
UGCAGCGCAGCAGGUUCUCUUGAGAACCUGCUGCGCUGCAUU 55 88 PHD2-
AGGAGCUGCGGCAGCGGCUUUAGCCGCUGCCGCAGCUCCUUU 56 89 PHD2-
AAGGAGCUGCGGCAGCGGCUUGCCGCUGCCGCAGCUCCUUUU 57 90 PHD2-
GAAGGAGCUGCGGCAGCGGUUCCGCUGCCGCAGCUCCUUCUU 58 91 PHD2-
AGAAGGAGCUGCGGCAGCGUUCGCUGCCGCAGCUCCUUCUUU 59 92 PHD2-
UAGAAGGAGCUGCGGCAGCUUGCUGCCGCAGCUCCUUCUAUU 60 93 PHD2-
GUAGAAGGAGCUGCGGCAGUUCUGCCGCAGCUCCUUCUACUU 61 94 PHD2-
AGUAGAAGGAGCUGCGGCAUUUGCCGCAGCUCCUUCUACUUU 62 95 PHD2-
CAGUAGAAGGAGCUGCGGCUUGCCGCAGCUCCUUCUACUGUU 63 96 PHD2-
GCAGUAGAAGGAGCUGCGGUUCCGCAGCUCCUUCUACUGCUU 64 97 PHD2-
AGCAGUAGAAGGAGCUGCGUUCGCAGCUCCUUCUACUGCUUU 65 98 PHD2-
CAGCAGUAGAAGGAGCUGCUUGCAGCUCCUUCUACUGCUGUU 66 99 PHD2-
GCAGCAGUAGAAGGAGCUGUUCAGCUCCUUCUACUGCUGCUU 67 100 PHD2-
UGCAGCAGUAGAAGGAGCUUUAGCUCCUUCUACUGCUGCAUU 68 101 PHD2-
UUGCAGCAGUAGAAGGAGCUUGCUCCUUCUACUGCUGCAAUU 69
102 PHD2- CUUGCAGCAGUAGAAGGAGUUCUCCUUCUACUGCUGCAAGUU 70 103 PHD2-
GUGCUUCUUCCAGUCCUGAUUUCAGGACUGGAAGAAGCACUU 71 104 PHD2-
UGUGCUUCUUCCAGUCCUGUUCAGGACUGGAAGAAGCACAUU 72 105 PHD2-
UUGUGCUUCUUCCAGUCCUUUAGGACUGGAAGAAGCACAAUU 73 106 PHD2-
CUUGUGCUUCUUCCAGUCCUUGGACUGGAAGAAGCACAAGUU 74 107 PHD2-
GCUUGUGCUUCUUCCAGUCUUGACUGGAAGAAGCACAAGCUU 75 108 PHD2-
AGCUUGUGCUUCUUCCAGUUUACUGGAAGAAGCACAAGCUUU 76 109 PHD2-
GAGCUUGUGCUUCUUCCAGUUCUGGAAGAAGCACAAGCUCUU 77 110 PHD2-
CUGCCCGUUGGGCCGCAGGUUCCUGCGGCCCAACGGGCAGUU 78 111 PHD2-
UCUGCCCGUUGGGCCGCAGUUCUGCGGCCCAACGGGCAGAUU 79 112 PHD2-
GUCUGCCCGUUGGGCCGCAUUUGCGGCCCAACGGGCAGACUU 80 113 PHD2-
CGUCUGCCCGUUGGGCCGCUUGCGGCCCAACGGGCAGACGUU 81 114 PHD2-
GCACGGCACGAUGUACUCGUUCGAGUACAUCGUGCCGUGCUU 82 115 PHD2-
UGCACGGCACGAUGUACUCUUGAGUACAUCGUGCCGUGCAUU 83 116 PHD2-
AUGCACGGCACGAUGUACUUUAGUACAUCGUGCCGUGCAUUU 84 117 PHD2-
CAUGCACGGCACGAUGUACUUGUACAUCGUGCCGUGCAUGUU 85 118 PHD2-
UCAUGCACGGCACGAUGUAUUUACAUCGUGCCGUGCAUGAUU 86 119 PHD2-
UUCAUGCACGGCACGAUGUUUACAUCGUGCCGUGCAUGAAUU 87 120 PHD2-
GUUCAUGCACGGCACGAUGUUCAUCGUGCCGUGCAUGAACUU 88 121 PHD2-
UGUUCAUGCACGGCACGAUUUAUCGUGCCGUGCAUGAACAUU 89 122 PHD2-
UUGUUCAUGCACGGCACGAUUUCGUGCCGUGCAUGAACAAUU 90 123 PHD2-
CUUGUUCAUGCACGGCACGUUCGUGCCGUGCAUGAACAAGUU 91 124 PHD2-
GCUUGUUCAUGCACGGCACUUGUGCCGUGCAUGAACAAGCUU 92 125 PHD2-
UGCUUGUUCAUGCACGGCAUUUGCCGUGCAUGAACAAGCAUU 93 126 PHD2-
GUGCUUGUUCAUGCACGGCUUGCCGUGCAUGAACAAGCACUU 94 127 PHD2-
CGUGCUUGUUCAUGCACGGUUCCGUGCAUGAACAAGCACGUU 95 128 PHD2-
CCGUGCUUGUUCAUGCACGUUCGUGCAUGAACAAGCACGGUU 96 129 PHD2-
GCCGUGCUUGUUCAUGCACUUGUGCAUGAACAAGCACGGCUU 97 130 PHD2-
UGCCGUGCUUGUUCAUGCAUUUGCAUGAACAAGCACGGCAUU 98 131 PHD2-
AUGCCGUGCUUGUUCAUGCUUGCAUGAACAAGCACGGCAUUU 99 132 PHD2-
GAUGCCGUGCUUGUUCAUGUUCAUGAACAAGCACGGCAUCUU 100 133 PHD2-
AGAUGCCGUGCUUGUUCAUUUAUGAACAAGCACGGCAUCUUU 101 134 PHD2-
CAGAUGCCGUGCUUGUUCAUUUGAACAAGCACGGCAUCUGUU 102 135 PHD2-
ACAGAUGCCGUGCUUGUUCUUGAACAAGCACGGCAUCUGUUU 103 136 PHD2-
GUGCAGGGCGCGCACCUCGUUCGAGGUGCGCGCCCUGCACUU 104 137 PHD2-
CGUGCAGGGCGCGCACCUCUUGAGGUGCGCGCCCUGCACGUU 105 138 PHD2-
UCGUGCAGGGCGCGCACCUUUAGGUGCGCGCCCUGCACGAUU 106 139 PHD2-
GUCGUGCAGGGCGCGCACCUUGGUGCGCGCCCUGCACGACUU 107 140 PHD2-
UGUCGUGCAGGGCGCGCACUUGUGCGCGCCCUGCACGACAUU 108 141 PHD2-
GUGUCGUGCAGGGCGCGCAUUUGCGCGCCCUGCACGACACUU 109 142 PHD2-
GGUGUCGUGCAGGGCGCGCUUGCGCGCCCUGCACGACACCUU 110 143 PHD2-
CGGUGUCGUGCAGGGCGCGUUCGCGCCCUGCACGACACCGUU 111 144 PHD2-
CCGGUGUCGUGCAGGGCGCUUGCGCCCUGCACGACACCGGUU 112 145 PHD2-
CCCGGUGUCGUGCAGGGCGUUCGCCCUGCACGACACCGGGUU 113 146 PHD2-
CUGCCCGUCCGUGAACUUCUUGAAGUUCACGGACGGGCAGUU 114 147 PHD2-
GCUGCCCGUCCGUGAACUUUUAAGUUCACGGACGGGCAGCUU 115 148 PHD2-
AGCUGCCCGUCCGUGAACUUUAGUUCACGGACGGGCAGCUUU 116 149 PHD2-
CAGCUGCCCGUCCGUGAACUUGUUCACGGACGGGCAGCUGUU 117 150 PHD2-
CCAGCUGCCCGUCCGUGAAUUUUCACGGACGGGCAGCUGGUU 118 151 PHD2-
ACCAGCUGCCCGUCCGUGAUUUCACGGACGGGCAGCUGGUUU 119 152 PHD2-
GACCAGCUGCCCGUCCGUGUUCACGGACGGGCAGCUGGUCUU 120 153 PHD2-
UGACCAGCUGCCCGUCCGUUUACGGACGGGCAGCUGGUCAUU 121 154 PHD2-
CUGACCAGCUGCCCGUCCGUUCGGACGGGCAGCUGGUCAGUU 122 155 PHD2-
GCUGACCAGCUGCCCGUCCUUGGACGGGCAGCUGGUCAGCUU 123 156 PHD2-
GGCUGACCAGCUGCCCGUCUUGACGGGCAGCUGGUCAGCCUU 124 157 PHD2-
UGGCUGACCAGCUGCCCGUUUACGGGCAGCUGGUCAGCCAUU 125 158 PHD2-
CUGGCUGACCAGCUGCCCGUUCGGGCAGCUGGUCAGCCAGUU 126 159 PHD2-
UCUGGCUGACCAGCUGCCCUUGGGCAGCUGGUCAGCCAGAUU 127 160 PHD2-
UUCUGGCUGACCAGCUGCCUUGGCAGCUGGUCAGCCAGAAUU 128 161 PHD2-
CUUCUGGCUGACCAGCUGCUUGCAGCUGGUCAGCCAGAAGUU 129 162 PHD2-
UCUUCUGGCUGACCAGCUGUUCAGCUGGUCAGCCAGAAGAUU 130 163 PHD2-
CUCUUCUGGCUGACCAGCUUUAGCUGGUCAGCCAGAAGAGUU 131 164 PHD2-
ACUCUUCUGGCUGACCAGCUUGCUGGUCAGCCAGAAGAGUUU 132 165 PHD2-
CACUCUUCUGGCUGACCAGUUCUGGUCAGCCAGAAGAGUGUU 133 166 PHD2-
UCACUCUUCUGGCUGACCAUUUGGUCAGCCAGAAGAGUGAUU 134 167 PHD2-
GUCACUCUUCUGGCUGACCUUGGUCAGCCAGAAGAGUGACUU 135 168 PHD2-
AGUCACUCUUCUGGCUGACUUGUCAGCCAGAAGAGUGACUUU 136 169 PHD2-
GAGUCACUCUUCUGGCUGAUUUCAGCCAGAAGAGUGACUCUU 137 170 PHD2-
CGAGUCACUCUUCUGGCUGUUCAGCCAGAAGAGUGACUCGUU 138 171 PHD2-
CCUCGAUCCAGGUGAUCUUUUAAGAUCACCUGGAUCGAGGUU 139 172 PHD2-
CCCUCGAUCCAGGUGAUCUUUAGAUCACCUGGAUCGAGGGUU 140 173 PHD2-
GCCCUCGAUCCAGGUGAUCUUGAUCACCUGGAUCGAGGGCUU 141 174 PHD2-
UGCCCUCGAUCCAGGUGAUUUAUCACCUGGAUCGAGGGCAUU 142 175 PHD2-
UUGCCCUCGAUCCAGGUGAUUUCACCUGGAUCGAGGGCAAUU 143 176 PHD2-
CUUGCCCUCGAUCCAGGUGUUCACCUGGAUCGAGGGCAAGUU 144 177 PHD2-
GGUUUCGCAGCCGGGCUCCUUGGAGCCCGGCUGCGAAACCUU 145 178 PHD2-
UGGUUUCGCAGCCGGGCUCUUGAGCCCGGCUGCGAAACCAUU 146 179 PHD2-
AUGGUUUCGCAGCCGGGCUUUAGCCCGGCUGCGAAACCAUUU 147 180 PHD2-
AAUGGUUUCGCAGCCGGGCUUGCCCGGCUGCGAAACCAUUUU 148 181 PHD2-
CAUGCUGCUCAUGAGCAGCUUGCUGCUCAUGAGCAGCAUGUU 149 182 PHD2-
CCAUGCUGCUCAUGAGCAGUUCUGCUCAUGAGCAGCAUGGUU 150 183 PHD2-
UCCAUGCUGCUCAUGAGCAUUUGCUCAUGAGCAGCAUGGAUU 151 184 PHD2-
GUCCAUGCUGCUCAUGAGCUUGCUCAUGAGCAGCAUGGACUU 152 185 PHD2-
CGUCCAUGCUGCUCAUGAGUUCUCAUGAGCAGCAUGGACGUU
153 186 PHD2- UCGUCCAUGCUGCUCAUGAUUUCAUGAGCAGCAUGGACGAUU 154 187
PHD2- GUCGUCCAUGCUGCUCAUGUUCAUGAGCAGCAUGGACGACUU 155 188 PHD2-
GGUCGUCCAUGCUGCUCAUUUAUGAGCAGCAUGGACGACCUU 156 189 PHD2-
AGGUCGUCCAUGCUGCUCAUUUGAGCAGCAUGGACGACCUUU 157 190 PHD2-
CAGGUCGUCCAUGCUGCUCUUGAGCAGCAUGGACGACCUGUU 158 191 PHD2-
UCAGGUCGUCCAUGCUGCUUUAGCAGCAUGGACGACCUGAUU 159 192 PHD2-
AUCAGGUCGUCCAUGCUGCUUGCAGCAUGGACGACCUGAUUU 160 193 PHD2-
UAUCAGGUCGUCCAUGCUGUUCAGCAUGGACGACCUGAUAUU 161 194 PHD2-
AGCAACCAUGGCUUUCGUCUUGACGAAAGCCAUGGUUGCUUU 162 195 PHD2-
AAGCAACCAUGGCUUUCGUUUACGAAAGCCAUGGUUGCUUUU 163 196 PHD2-
CAAGCAACCAUGGCUUUCGUUCGAAAGCCAUGGUUGCUUGUU 164 197 PHD2-
ACAAGCAACCAUGGCUUUCUUGAAAGCCAUGGUUGCUUGUUU 165 198 PHD2-
AACAAGCAACCAUGGCUUUUUAAAGCCAUGGUUGCUUGUUUU 166 199 PHD2-
UAACAAGCAACCAUGGCUUUUAAGCCAUGGUUGCUUGUUAUU 167 200 PHD2-
AUAACAAGCAACCAUGGCUUUAGCCAUGGUUGCUUGUUAUUU 168 201 PHD2-
UCUUCCAUCUCCAUUUGGAUUUCCAAAUGGAGAUGGAAGAUU 169 202 PHD2-
AUCUUCCAUCUCCAUUUGGUUCCAAAUGGAGAUGGAAGAUUU 170 203 PHD2-
CAUCUUCCAUCUCCAUUUGUUCAAAUGGAGAUGGAAGAUGUU 171 204 PHD2-
ACAUCUUCCAUCUCCAUUUUUAAAUGGAGAUGGAAGAUGUUU 172 205 PHD2-
AUAAUAUAUACAUGUCACAUUUGUGACAUGUAUAUAUUAUUU 173 206 PHD2-
GAUAAUAUAUACAUGUCACUUGUGACAUGUAUAUAUUAUCUU 174 207 PHD2-
AGAUAAUAUAUACAUGUCAUUUGACAUGUAUAUAUUAUCUUU 175 208 PHD2-
AAGAUAAUAUAUACAUGUCUUGACAUGUAUAUAUUAUCUUUU 176 209 PHD2-
ACCUCCACUUACCUUGGCAUUUGCCAAGGUAAGUGGAGGUUU 177 210 PHD2-
UACCUCCACUUACCUUGGCUUGCCAAGGUAAGUGGAGGUAUU 178 211 PHD2-
AUACCUCCACUUACCUUGGUUCCAAGGUAAGUGGAGGUAUUU 179 212 PHD2-
UAUACCUCCACUUACCUUGUUCAAGGUAAGUGGAGGUAUAUU 180 213 PHD2-
UUCUGGAAAAAUUCGAAGUUUACUUCGAAUUUUUCCAGAAUU 181 214 PHD2-
CUUCUGGAAAAAUUCGAAGUUCUUCGAAUUUUUCCAGAAGUU 182 215 PHD2-
CCUUCUGGAAAAAUUCGAAUUUUCGAAUUUUUCCAGAAGGUU 183 216 PHD2-
GCCUUCUGGAAAAAUUCGAUUUCGAAUUUUUCCAGAAGGCUU 184 217 PHD2-
UGCCUUCUGGAAAAAUUCGUUCGAAUUUUUCCAGAAGGCAUU 185 218 PHD2-
UUGCCUUCUGGAAAAAUUCUUGAAUUUUUCCAGAAGGCAAUU 186 219 PHD2-
UUUGCCUUCUGGAAAAAUUUUAAUUUUUCCAGAAGGCAAAUU 187 220 PHD2-
CUUUGCCUUCUGGAAAAAUUUAUUUUUCCAGAAGGCAAAGUU 188 221 PHD2-
GCUUUGCCUUCUGGAAAAACCAGAAGGCAAAGCUU 189 222 PHD2-
GGCUUUGCCUUCUGGAAAAUUUUUUCCAGAAGGCAAAGCCUU 190 223 PHD2-
GGGCUUUGCCUUCUGGAAAUUUUUCCAGAAGGCAAAGCCCUU 191 224 PHD2-
UGGGCUUUGCCUUCUGGAAUUUUCCAGAAGGCAAAGCCCAUU 192 225 PHD2-
CUGGGCUUUGCCUUCUGGAUUUCCAGAAGGCAAAGCCCAGUU 193 226 PHD2-
ACUGGGCUUUGCCUUCUGGUUCCAGAAGGCAAAGCCCAGUUU 194 227 PHD2-
AACUGGGCUUUGCCUUCUGUUCAGAAGGCAAAGCCCAGUUUU 195 228 PHD2-
AAACUGGGCUUUGCCUUCUUUAGAAGGCAAAGCCCAGUUUUU 196 229 PHD2-
CAAACUGGGCUUUGCCUUCUUGAAGGCAAAGCCCAGUUUGUU 197 230 PHD2-
GCAAACUGGGCUUUGCCUUUUAAGGCAAAGCCCAGUUUGCUU 198 231 PHD2-
AGCAAACUGGGCUUUGCCUUUAGGCAAAGCCCAGUUUGCUUU 199 232 PHD2-
CAGCAAACUGGGCUUUGCCUUGGCAAAGCCCAGUUUGCUGUU 200 233 PHD2-
UCAGCAAACUGGGCUUUGCUUGCAAAGCCCAGUUUGCUGAUU 201 234 PHD2-
GUCAGCAAACUGGGCUUUGUUCAAAGCCCAGUUUGCUGACUU 202 235 PHD2-
UGUCAGCAAACUGGGCUUUUUAAAGCCCAGUUUGCUGACAUU 203 236 PHD2-
AUGUCAGCAAACUGGGCUUUUAAGCCCAGUUUGCUGACAUUU 204 237 PHD2-
AAUGUCAGCAAACUGGGCUUUAGCCCAGUUUGCUGACAUUUU 205 238 PHD2-
CAAUGUCAGCAAACUGGGCUUGCCCAGUUUGCUGACAUUGUU 206 239 PHD2-
UCAAUGUCAGCAAACUGGGUUCCCAGUUUGCUGACAUUGAUU 207 240 PHD2-
UUCAAUGUCAGCAAACUGGUUCCAGUUUGCUGACAUUGAAUU 208 241 PHD2-
GUUCAAUGUCAGCAAACUGUUCAGUUUGCUGACAUUGAACUU 209 242 PHD2-
GGUUCAAUGUCAGCAAACUUUAGUUUGCUGACAUUGAACCUU 210 243 PHD2-
GGGUUCAAUGUCAGCAAACUUGUUUGCUGACAUUGAACCCUU 211 244 PHD2-
UGGGUUCAAUGUCAGCAAAUUUUUGCUGACAUUGAACCCAUU 212 245 PHD2-
UUGGGUUCAAUGUCAGCAAUUUUGCUGACAUUGAACCCAAUU 213 246 PHD2-
UUUGGGUUCAAUGUCAGCAUUUGCUGACAUUGAACCCAAAUU 214 247 PHD2-
AUUUGGGUUCAAUGUCAGCUUGCUGACAUUGAACCCAAAUUU 215 248 PHD2-
AAUUUGGGUUCAAUGUCAGUUCUGACAUUGAACCCAAAUUUU 216 249 PHD2-
AAAUUUGGGUUCAAUGUCAUUUGACAUUGAACCCAAAUUUUU 217 250 PHD2-
CAAAUUUGGGUUCAAUGUCUUGACAUUGAACCCAAAUUUGUU 218 251 PHD2-
UCAAAUUUGGGUUCAAUGUUUACAUUGAACCCAAAUUUGAUU 219 252 PHD2-
AUCAAAUUUGGGUUCAAUGUUCAUUGAACCCAAAUUUGAUUU 220 253 PHD2-
UAUCAAAUUUGGGUUCAAUUUAUUGAACCCAAAUUUGAUAUU 221 254 PHD2-
CUAUCAAAUUUGGGUUCAAUUUUGAACCCAAAUUUGAUAGUU 222 255 PHD2-
UCUAUCAAAUUUGGGUUCAUUUGAACCCAAAUUUGAUAGAUU 223 256 PHD2-
GUCUAUCAAAUUUGGGUUCUUGAACCCAAAUUUGAUAGACUU 224 257 PHD2-
AGUCUAUCAAAUUUGGGUUUUAACCCAAAUUUGAUAGACUUU 225 258 PHD2-
CAGUCUAUCAAAUUUGGGUUUACCCAAAUUUGAUAGACUGUU 226 259 PHD2-
GCAGUCUAUCAAAUUUGGGUUCCCAAAUUUGAUAGACUGCUU 227 260 PHD2-
AGCAGUCUAUCAAAUUUGGUUCCAAAUUUGAUAGACUGCUUU 228 261 PHD2-
CAGCAGUCUAUCAAAUUUGUUCAAAUUUGAUAGACUGCUGUU 229 262 PHD2-
ACAGCAGUCUAUCAAAUUUUUAAAUUUGAUAGACUGCUGUUU 230 263 PHD2-
AACAGCAGUCUAUCAAAUUUUAAUUUGAUAGACUGCUGUUUU 231 264 PHD2-
AAACAGCAGUCUAUCAAAUUUAUUUGAUAGACUGCUGUUUUU 232 265 PHD2-
AAAACAGCAGUCUAUCAAAUUUUUGAUAGACUGCUGUUUUUU 233 266 PHD2-
AAAAACAGCAGUCUAUCAAUUUUGAUAGACUGCUGUUUUUUU 234 267 PHD2-
GAAAAACAGCAGUCUAUCAUUUGAUAGACUGCUGUUUUUCUU 235 268 PHD2-
AGAAAAACAGCAGUCUAUCUUGAUAGACUGCUGUUUUUCUUU 236
269 PHD2- CAGAAAAACAGCAGUCUAUUUAUAGACUGCUGUUUUUCUGUU 237 270 PHD2-
CCAGAAAAACAGCAGUCUAUUUAGACUGCUGUUUUUCUGGUU 238 271 PHD2-
ACCAGAAAAACAGCAGUCUUUAGACUGCUGUUUUUCUGGUUU 239 272 PHD2-
GACCAGAAAAACAGCAGUCUUGACUGCUGUUUUUCUGGUCUU 240 273 PHD2-
AGACCAGAAAAACAGCAGUUUACUGCUGUUUUUCUGGUCUUU 241 274 PHD2-
CAGACCAGAAAAACAGCAGUUCUGCUGUUUUUCUGGUCUGUU 242 275 PHD2-
UCAGACCAGAAAAACAGCAUUUGCUGUUUUUCUGGUCUGAUU 243 276 PHD2-
GUCAGACCAGAAAAACAGCUUGCUGUUUUUCUGGUCUGACUU 244 277 PHD2-
GGUCAGACCAGAAAAACAGUUCUGUUUUUCUGGUCUGACCUU 245 278 PHD2-
CGGUCAGACCAGAAAAACAUUUGUUUUUCUGGUCUGACCGUU 246 279 PHD2-
ACGGUCAGACCAGAAAAACUUGUUUUUCUGGUCUGACCGUUU 247 280 PHD2-
UUGUACUUCAUGAGGGUUGUUCAACCCUCAUGAAGUACAAUU 248 281 PHD2-
AGUUAUUGCGUACCUUGUAUUUACAAGGUACGCAAUAACUUU 249 282 PHD2-
CAGUUAUUGCGUACCUUGUUUACAAGGUACGCAAUAACUGUU 250 283 PHD2-
ACAGUUAUUGCGUACCUUGUUCAAGGUACGCAAUAACUGUUU 251 284 PHD2-
AACAGUUAUUGCGUACCUUUUAAGGUACGCAAUAACUGUUUU 252 285 PHD2-
AAACAGUUAUUGCGUACCUUUAGGUACGCAAUAACUGUUUUU 253 286 PHD2-
CAAACAGUUAUUGCGUACCUUGGUACGCAAUAACUGUUUGUU 254 287 PHD2-
CCAAACAGUUAUUGCGUACUUGUACGCAAUAACUGUUUGGUU 255 288 PHD2-
ACCAAACAGUUAUUGCGUAUUUACGCAAUAACUGUUUGGUUU 256 289 PHD2-
UACCAAACAGUUAUUGCGUUUACGCAAUAACUGUUUGGUAUU 257 290 PHD2-
AUACCAAACAGUUAUUGCGUUCGCAAUAACUGUUUGGUAUUU 258 291 PHD2-
AAUACCAAACAGUUAUUGCUUGCAAUAACUGUUUGGUAUUUU 259 292 PHD2-
AAAUACCAAACAGUUAUUGUUCAAUAACUGUUUGGUAUUUUU 260 293 PHD2-
CAAACAGUUAUUUUAAUAACUGUUUGGUAUUUUUU 261 294 PHD2-
CAAAAUACCAAACAGUUAUUUAUAACUGUUUGGUAUUUUGUU 262 295 PHD2-
UCAAAAUACCAAACAGUUAUUUAACUGUUUGGUAUUUUGAUU 263 296 PHD2-
AUCAAAAUACCAAACAGUUUUAACUGUUUGGUAUUUUGAUUU 264 297 PHD2-
CAUCAAAAUACCAAACAGUUUACUGUUUGGUAUUUUGAUGUU 265 298 PHD2-
GCAUCAAAAUACCAAACAGUUCUGUUUGGUAUUUUGAUGCUU 266 299 PHD2-
UGCAUCAAAAUACCAAACAUUUGUUUGGUAUUUUGAUGCAUU 267 300 PHD2-
CUGCAUCAAAAUACCAAACUUGUUUGGUAUUUUGAUGCAGUU 268 301 PHD2-
UCUGCAUCAAAAUACCAAAUUUUUGGUAUUUUGAUGCAGAUU 269 302 PHD2-
AUCUGCAUCAAAAUACCAAUUUUGGUAUUUUGAUGCAGAUUU 270 303 PHD2-
CAUCUGCAUCAAAAUACCAUUUGGUAUUUUGAUGCAGAUGUU 271 304 PHD2-
UCAUCUGCAUCAAAAUACCUUGGUAUUUUGAUGCAGAUGAUU 272 305 PHD2-
CUCAUCUGCAUCAAAAUACUUGUAUUUUGAUGCAGAUGAGUU 273 306 PHD2-
UCUCAUCUGCAUCAAAAUAUUUAUUUUGAUGCAGAUGAGAUU 274 307 PHD2-
GAUAUUUUACUUUAGCUCGUUCGAGCUAAAGUAAAAUAUCUU 275 308 PHD2-
AGAUAUUUUACUUUAGCUCUUGAGCUAAAGUAAAAUAUCUUU 276 309 PHD2-
UAGAUAUUUUACUUUAGCUUUAGCUAAAGUAAAAUAUCUAUU 277 310 PHD2-
UUAGAUAUUUUACUUUAGCUUGCUAAAGUAAAAUAUCUAAUU 278 311 PHD2-
GUUAGAUAUUUUACUUUAGUUCUAAAGUAAAAUAUCUAACUU 279 312 PHD2-
UGUUAGAUAUUUUACUUUAUUUAAAGUAAAAUAUCUAACAUU 280 313 PHD2-
CUGUUAGAUAUUUUACUUUUUAAAGUAAAAUAUCUAACAGUU 281 314 PHD2-
CCUGUUAGAUAUUUUACUUUUAAGUAAAAUAUCUAACAGGUU 282 315 PHD2-
ACCUGUUAGAUAUUUUACUUUAGUAAAAUAUCUAACAGGUUU 283 316 PHD2-
CACCUGUUAGAUAUUUUACUUGUAAAAUAUCUAACAGGUGUU 284 317 PHD2-
UCACCUGUUAGAUAUUUUAUUUAAAAUAUCUAACAGGUGAUU 285 318 PHD2-
UUCACCUGUUAGAUAUUUUUUAAAAUAUCUAACAGGUGAAUU 286 319 PHD2-
UUCAACCCUCACACCUUUUUUAAAAGGUGUGAGGGUUGAAUU 287 320 PHD2-
GUUCAACCCUCACACCUUUUUAAAGGUGUGAGGGUUGAACUU 288 321 PHD2-
AGUUCAACCCUCACACCUUUUAAGGUGUGAGGGUUGAACUUU 289 322 PHD2-
GAGUUCAACCCUCACACCUUUAGGUGUGAGGGUUGAACUCUU 290 323 PHD2-
UGAGUUCAACCCUCACACCUUGGUGUGAGGGUUGAACUCAUU 291 324 PHD2-
UUGAGUUCAACCCUCACACUUGUGUGAGGGUUGAACUCAAUU 292 325 PHD2-
AUUGAGUUCAACCCUCACAUUUGUGAGGGUUGAACUCAAUUU 293 326 SG606
U*C*A*GCCGCUGUCACACGCA*C*A*G 327 SG607
G*1C*U*G*1T*C*A*C*A*C*1G*C*A*1C*A 328 SG615
U/ZEN/CAGCCGCUGUCACACGCACA/ZEN/Q1 329 miR- UCAGCCGCUGUCACACGCACAG
210 antisense
EXAMPLES
Example 1. Targeting of PHD2 with sshRNA
[0194] FIG. 2A shows quantitative reverse transcription PCR
(qRT-PCR) of human PHD2 (SEQ ID NO. 1) transcript in human
embryonic kidney 293FT cells transfected with increasing amounts of
a sshRNA (SG302, SEQ ID NO. 7) or siRNA (SG303, SEQ ID NO. 9 and
SEQ ID NO. 10) targeting PHD2 or a control sshRNA (SG221c).
Transcript levels were quantified using the 2.sup.-.DELTA..DELTA.Ct
method relative to cells not transfected with inhibitor and
normalized to GAPDH transcript. FIG. 2B shows qRT-PCR of mouse PHD2
(SEQ ID NO. 2) transcript in mouse NIH3T3 fibroblasts transfected
with increasing amounts of PHD2-targeting sshRNAs (SG400, SEQ ID
NO. 24 and SG402, SEQ ID NO. 26), PHD2-targeting siRNA (SG403, SEQ
ID NO. 28 and SEQ ID NO. 29) or a control sshRNA (SG221c).
Transcript levels were quantified using the 2.sup.-.DELTA..DELTA.Ct
method relative to cells not transfected with inhibitor and
normalized to GAPDH transcript.
Example 2. Targeting of the HIF-1.alpha. Network with PHD2
sshRNA
[0195] FIG. 3A shows the results of a luciferase reporter assay in
which a plasmid of firefly luciferase (f-Luc) is under the control
of a promoter containing HIF-1.alpha. responsive elements. A
plasmid that constitutively expresses Renilla luciferase (r-Luc)
was used as a transfection normalization control. Human embryonic
kidney 293FT cells were co-transfected with the above plasmids and
individual sshRNAs targeting PHD2 (SG300 (SEQ ID NO. 5), SG301 (SEQ
ID NO. 6), or SG302 (SEQ ID NO. 7)) or with a control sshRNA (NSC
sshRNA). 48 hours after transfection, the f-Luc signal normalized
to r-Luc was determined. Values are normalized to a NSC sshRNAs
control. FIG. 3B shows Western blotting studies of HIF-1.alpha. and
Lamin protein in Human embryonic kidney 293FT cells treated with no
inhibitor (lane 1), two concentrations of control sshRNA (Neg
control sshRNA; lanes 2 and 3), two concentrations each of SG303
(SEQ ID NO. 9 and SEQ ID NO. 10) and SG302 (SEQ ID NO. 7) (lanes 4
and 5 and 6 and 7, respectively), and untransfected cells treated
with cobalt chloride (CoCl.sub.2; lane 8). Values of HIF-1.alpha.
are calculated relative to no inhibitor treatment and are
normalized to Lamin. FIG. 3C shows qRT-PCR of VEGF (lanes 1, 4, 7,
10, 13, 16, and 19), HSP90 (lanes 2, 5, 8, 11, 14, 17, and 20), and
HSP70 (lanes 3, 6, 9, 12, 15, 18, and 21) in human embryonic kidney
293FT cells transfected with increasing amounts of SG302 (SEQ ID
NO. 7). Total RNA was isolated 48 hours after transfection.
Quantification is expressed as fold-induction relative to
untransfected cells and normalized to GAPDH using the
2.sup.-.DELTA..DELTA.Ct method.
Example 3. Effect of sshRNA Modification on RNAi
[0196] FIG. 4A shows qRT-PCR of human PHD2 transcript in human
primary normal human epidermal keratinocytes (NHEK) transfected
with increasing amounts of SG302 (SEQ ID NO. 7) or a modified
sshRNA targeting PHD2 (SG302m1, SEQ ID NO. 8). Total RNA was
isolated 48 h after transfection. PHD2 transcripts were quantified
by the 2.sup.-.DELTA..DELTA.Ct method, normalizing to GAPDH.
Quantification is expressed as fold-inhibition relative to
untransfected cells. FIG. 4B shows qRT-PCR of mouse PHD2 transcript
in mouse NIH3T3 fibroblasts transfected with increasing amounts of
SG402 (SEQ ID NO. 26), a modified sshRNA targeting PHD2 (SG402m1,
SEQ ID NO. 27), or a scrambled control sshRNA (SG402-scr). Values
are normalized relative to a control transcript (GAPDH) and to
untransfected mouse cells and quantified using the
2.sup.-.DELTA..DELTA.Ct method. FIG. 4C shows qRT-PCR of mouse PHD2
(mPHD2) transcript in mouse NIH3T3 fibroblasts transfected with
increasing amounts of an unmodified sshRNA targeting PHD2 (SG404,
SEQ ID NO. 30) or SG402m1 (SEQ ID NO. 27). Data were analyzed as
described in FIG. 4B.
Example 4. Effect of sshRNAs on Immunostimulatory Pathways
[0197] MRC-5 human lung fibroblasts were seeded in 24-well plates
at 6.times.10.sup.4 cells per well with Minimum Essential Medium
containing 10% fetal calf serum. Transfections were performed using
Lipofectamine 2000. 20 nanomolar (nM) sshRNAs or an equivalent
amount of poly-inosine/cytosine (poly I:C) were transfected in
triplicate. Untransfected cells and cells receiving Lipofectamine
2000 alone were used as negative controls. 24 hours later, the
cells were lysed in TRIzol.RTM. (Invitrogen.TM. Carlsbad, Calif.)
and total RNA was extracted according to the manufacturer's
instructions. QRT-PCR was performed using a High-Capacity cDNA
Reverse Transcription Kit with the TaqMan.RTM. Universal PCR Master
Mix, IFN-.beta., IL-6, TNF-.alpha., and GAPDH TaqMan.RTM. probes,
and a 7500 Fast real-time PCR system (Applied Biosystems, Foster
City, Calif.) following the manufacturer's protocol. IFN-.beta.,
IL-6, and TNF-.alpha. transcripts were quantified by the
2.sup.-.DELTA..DELTA.Ct method, normalizing to GAPDH. FIG. 5A shows
qRT-PCR assays for IFN-.beta. in cells treated with no inhibitor,
poly-inosine/cytosine (polyI:C), SG302 (SEQ ID NO. 7), or SG302m1
(SEQ ID NO. 8). FIG. 5B shows qRT-PCR assays for IL-6 in cells
treated with no inhibitor, polyI:C, SG302 (SEQ ID NO. 7), or
SG302m1 (SEQ ID NO. 8). FIG. 5C shows qRT-PCR assays for
TNF-.alpha. in cells treated with no inhibitor, polyI:C, SG302 (SEQ
ID NO. 7), or SG302m1 (SEQ ID NO. 8). FIG. 5D shows qRT-PCR for
IFN-.beta. in cells treated with no inhibitor, polyI:C, SG402 (SEQ
ID NO. 26), SG402m1 (SEQ ID NO. 27), or SG404 (SEQ ID NO. 30). FIG.
5E shows qRT-PCR assays for IL-6 in cells treated with no
inhibitor, polyI:C, SG402 (SEQ ID NO. 26), SG402m1 (SEQ ID NO. 27),
or SG404 (SEQ ID NO. 30). FIG. 5F shows qRT-PCR assays for
TNF-.alpha. in cells treated with no inhibitor, polyI:C, SG402 (SEQ
ID NO. 26), SG402m1 (SEQ ID NO. 27), or SG404 (SEQ ID NO. 30).
Example 5. Effect of sshRNA Modification on Serum Stability
[0198] FIG. 6 shows serum stability assays for unmodified sshRNAs
(SG402, SEQ ID NO. 26) and modified shRNAs (SG404, SEQ ID NO. 30).
3.35 micrograms (.mu.g) of SG404 (SEQ ID NO. 30) and SG402 (SEQ ID
NO. 26) were incubated with 10% human serum (Sigma-Aldrich, St
Louis, Mo.) in phosphate-buffered saline at 37.degree. C. for
various times (from 0 minutes (0') to 24 hours (24 h)). At each
time point, an aliquot was taken out, mixed with 2.times. gel
loading buffer (Ambion.RTM., Austin, Tex.), and immediately stored
in -80.degree. C. The samples were analyzed by 12% denaturing
polyacrylamide gel electrophoresis (12% polyacrylamide, 20%
formamide, and 8M urea) and were stained with SYBR Gold
(Invitrogen.TM., Carlsbad, Calif.). Bands at about 40 nucleotides
correspond to the full-length sshRNA.
Example 6. Effect of Combining sshRNA with miRNA Antagonist
[0199] FIG. 7A shows possible modification patterns for a miR-210
miRNA antagonist. FIG. 7B shows qRT-PCR of human PHD2 transcript in
human HaCaT keratinocytes transfected with the following: no
inhibitor; an sshRNA targeting human PHD2 (SG302, SEQ ID NO. 7); a
control sshRNA (ssh-NSC); SG302 (SEQ ID NO. 7) and a LNA-modified
miR-210 (SEQ ID NO. 3) miRNA antagonist (SG302+LNA210); SG302 (SEQ
ID NO. 7) and a 2'-O-methyl-modified miR-210 miRNA antagonist
(SG302+2'-O-methyl 210); ssh-NSC and a LNA-modified control miRNA
antagonist (ssh-NSC+LNA-NSC); LNA210; 2'-O-methyl 210; and LNA-NSC.
Values are normalized relative to a control transcript (GAPDH) and
to untransfected human HaCaT keratinocytes. FIG. 7C shows miRNA
qRT-PCR (miR-qRT-PCR) of miR-210 miRNA in human HaCaT keratinocytes
transfected with the following: no inhibitor; SG302; ssh-NSC;
SG302+LNA210; SG302+2'-O-methyl 210; ssh-NSC+LNA-NSC; LNA210;
2'-O-methyl 210; and LNA-NSC. Values are normalized relative to a
control small nucleolar RNA (RNU44) and to untransfected human
HaCaT keratinocytes. FIG. 7D shows a luciferase assay with a
miR-210 reporter in human HaCaT keratinocytes transfected with no
inhibitor or a miR-210 mimic. The miR-210 reporter was generated by
sub-cloning four tandem miR-210 binding sites derived from the
target found in the 3' untranslated region (3' UTR) of E2F3), each
separated by a 8 basepair (bp) sequence, into the 3' UTR of Renilla
luciferase (r-Luc) of the psiCheck-2 dual luciferase reporter
vector (Promega). This vector also contains constitutively
expressed firefly luciferase (f-Luc) for transfection
normalization. FIG. 7E shows a luciferase assay with a miR-210
reporter in human HaCaT keratinocytes treated with the following:
no inhibitor (column 1); CoCl.sub.2 (column 2); CoCl.sub.2 and
increasing concentrations of DNA modified miR-210 miRNA antagonist
(DNA210; columns 3-6); CoCl.sub.2 and increasing concentrations of
miR-210 miRNA antagonist (RNA210; columns 7-10); CoCl.sub.2 and
increasing concentrations of 2'-O-methyl modified miR-210 miRNA
antagonist (2'-O-methyl 210; columns 11-14); CoCl.sub.2 and
increasing concentrations of LNA modified miR-210 miRNA antagonist
(LNA210; columns 15-18); and CoCl.sub.2 and increasing
concentrations of a control miRNA antagonist (NSC; columns 19-22).
Values are normalized as in FIG. 7D. FIG. 7F shows qRT-PCR of human
PHD2 transcript in human primary keratinocytes transfected with
increasing amounts of an sshRNA targeting human PHD2 (SG302, SEQ ID
NO. 7) and a control sshRNA (SG221c). Transcript levels were
quantified using the 2.sup.-.DELTA..DELTA.Ct method relative to
cells not transfected with inhibitor and normalized to GAPDH
transcript. FIG. 7G shows qRT-PCR of miR-210 in human primary
keratinocytes transfected with increasing amounts of an sshRNA
targeting human PHD2 (SG302, SEQ ID NO. 7) and a control sshRNA
(SG221c). miRNA levels were quantified using the
2.sup.-.DELTA..DELTA.Ct method relative to cells not transfected
with inhibitor and normalized to RNU44 small nucleolar RNA. FIG.
711 shows qRT-PCR of mouse PHD2 transcript in NIH-3T3 cells
transfected with increasing amounts of an sshRNA targeting mouse
PHD2 (SG404, SEQ ID NO. 30). Transcript levels were quantified
using the 2.sup.-.DELTA..DELTA.Ct method relative to cells not
transfected with inhibitor and normalized to GAPDH transcript. FIG.
7I shows qRT-PCR of miR-.sub.210 in NIH-3T3 cells transfected with
increasing amounts of an sshRNA targeting mouse PHD2 (SG404, SEQ ID
NO. 30). miRNA levels were quantified using the
2.sup.-.DELTA..DELTA.Ct method relative to cells not transfected
with inhibitor and normalized to sno-234 small nucleolar RNA. FIG.
7J shows relative expression of PHD2 and miR-210 both alone and in
combination with PHD2-targeting SG302 sshRNA (SEQ ID NO. 7) and
antimiR-210 (SG603, SEQ ID NO. 34) in HaCaT cells.
Example 7. Effect of miRNA Antagonists on Immunostimulatory
Pathways
[0200] FIG. 8A shows qRT-PCR for IFN-.beta. in MRC-5 lung
fibroblasts treated with: no inhibitor; polyI:C; a DNA modified
miR-210 miRNA antagonist (210 DNA anti (SG602, SEQ ID NO. 33)); a
miR-210 miRNA antagonist (210 RNA anti (SG601, SEQ ID NO. 32)); a
2'-O-methyl modified miR-210 miRNA antagonist (210 2'-O-methyl anti
(SG603, SEQ ID NO. 34)); a LNA modified miR-210 miRNA antagonist
(210 LNA anti (SG604, SEQ ID NO. 35)); and a LNA modified control
miRNA antagonist (210 NSC (LNA) (SG605, SEQ ID NO. 36)). FIG. 8B
shows qRT-PCR for IL-6 in cells treated with: no inhibitor;
polyI:C; 210 DNA anti; 210 RNA anti; 210 2'-O-methyl anti; 210 LNA
anti; and 210 NSC (LNA). FIG. 8C shows qRT-PCR for TNF-.alpha. in
cells treated with: no inhibitor; polyI:C; 210 DNA anti; 210 RNA
anti; 210 2'-O-methyl anti; 210 LNA anti; and 210 NSC (LNA).
Example 8. Effect of Fluorescent Conjugate Moiety on sshRNA
Activity
[0201] FIG. 9 shows qRT-PCR of mouse PHD2 (mPHD2) transcript in
mouse cells transfected with increasing amounts of an unmodified
sshRNA targeting PHD2 (SG404, SEQ ID NO. 30) or a PHD2-targeting
sshRNA modified to have a C6-amine-TexasRed (AlexaFluor594)
conjugated to the first nucleotide of the loop (SG405, SEQ ID NO.
31). Values are normalized relative to a control transcript (GAPDH)
and to untransfected mouse cells by the 2.sup.-.DELTA..DELTA.Ct
method.
Example 9. Effect of sshRNAs on Diabetic Wound Healing
[0202] The leptin receptor deficient db/db type II diabetes mouse
model was used as a model of diabetic wound healing. Males aged 8
to 12 weeks were used for the study. Isoflurane was administered as
an anesthetic and full-thickness skin punch biopsies (6-mm in
diameter) were created in pairs on the dorsa of db/db mice,
equidistant from the midline. The skin and underlying panniculus
was removed from the biopsy area. Following the punch biopsy,
rubber donuts with a hole about 6 mm in diameter in their center
was sutured in place around the wound to splint the wound open,
preventing wound contracture and allowing healing to proceed
through re-epithelialization The animals were returned to the
housing facility after recovering from anesthesia. All procedures
were performed using aseptic techniques. Sterile instruments were
used, and animals were clipped and prepped with Betadine. The
wounds were covered with a dressing to limit infection.
[0203] Oligonucleotide formulations were then applied to the
animals the following day. Each treatment group had 4 animals with
two wounds per animal (number of wounds=8/group). The Layer by
Layer group (LbL) received topical treatment with the dressing on
which a mesh comprising an sshRNA targeting mouse PHD2 (SG404) was
printed. The A6K peptide group (A6K) was injected with 300
picomoles (pmol) of SG404 with the A6K peptide. The HiPerFect group
(HiPerFect) was injected with 300 pmol SG404 with the HiPerFect
reagent. The control group was injected with PBS as done for the
A6K and HiPerFect groups. For the A6K, HiPerFect, and PBS groups,
injections were intradermal, with four injections administered
locally around the wound. Images of the wound were captured every
other day to monitor progress of wound closure. The wound area was
calculated digitally, and a time to closure curve was generated.
FIG. 10A shows the percentage of original wound area over 25 days
in the LbL, A6K, HiPerFect, and Control treatment groups. * over
time points on the graph in FIG. 10A indicate p-values<0.05
between the SG404-LbL group (diamond marker) vs the control group
(square marker). Treatment with an sshRNA targeting PHD2 (SG404)
improves diabetic wound healing. FIG. 10B shows the days until the
wound closed in the LbL, A6K, HiPerFect, and Control treatment
groups. Average time to wound closure in SG404-LbL group was
17.+-.1.05 days vs control group (20.6.+-.0.45 days). * indicates
p-value=0.01 between SG404 LbL group and control group with respect
to days to wound closure. PHD2-targeting sshRNAs shows a
therapeutically significant increase in rate of wound closure and
reduction in time to wound closure in db/db mice.
[0204] A further study was performed in which db/db mice were
wounded as described above. Animals were then untreated or given a
LbL topical treatment of a control sshRNA (SG221c), SG404, or a
miR-210 antagonist (SG603). FIG. 10C shows the percentage of
original wound area over 25 days in the Untreated, SG404, SG603,
and SG221c treatment groups. FIG. 10D shows the days until the
wound closed in each treatment group. Average days to wound
closure.+-.SEM were as follows: SG404 (14.00.+-.0.68 days), SG603
(15.33.+-.0.61 days), SG221c control (16.33.+-.0.07 days),
untreated control (18.33.+-.1.42 days). P-values for SG404 vs
untreated and SG404 vs SG221c control sshRNAs were 0.021 and 0.034,
respectively. P-value for SG603 vs untreated control was 0.08.
[0205] In a further study in which db/db mice were wounded as
described above, histological analysis was performed to examine the
effect of sshRNA treatment on neovascularization in the wound area.
Animals were untreated or given a LbL topical treatment of control
sshRNAs (SG221c) or SG404 sshRNA for a total of 6 wounds per
treatment group. Animals were sacrificed at day 7 after wounding,
and histology slides were prepared and stained with an antibody to
von Willebrand Factor (vWF) (EMD Millipore). 3 images were captured
per slide. A blinded analysis was performed with fluorescence
intensity measured by Image J software. FIG. 10E shows
representative images of fluorescence staining at day 7 for each
treatment group. In FIG. 10F, computed values (integrated density)
of vWF staining for each treatment group are plotted. Staining for
vWF showed a significant increase in neovascularization in the
wound area at Day 7 post wounding for SG404-treated wounds. * on
the graph between treatment arms represent p-values<0.05.
Example 10. Development of sshRNAs Targeting Both Mouse PHD2 and
Human PHD2
[0206] FIG. 11A shows qRT-PCR of human PHD2 transcript in human
kidney 293FT cells transfected with either 1 nM or 10 nM of the
following: no inhibitor; sshRNAs designed to target human PHD2
(SG302 (SEQ ID NO. 7), SG304-SG309 (SEQ ID NO. 11-SEQ ID NO. 16));
and sshRNAs designed to target mouse PHD2 (SG400-SG402, SEQ ID NO.
24-SEQ ID NO. 26); and a control sshRNA (NSC). Values are
normalized relative to a control transcript (GAPDH) and to
untransfected human HaCaT keratinocytes by the
2.sup.-.DELTA..DELTA.Ct method. FIG. 11B shows qRT-PCR of mouse
PHD2 (mPHD2) transcript in mouse NIH3T3 fibroblasts transfected
with increasing amounts of sshRNAs designed to target mouse PHD2
(SG404, SEQ ID NO. 30) or a modified sshRNA designed to target
human PHD2 (SG302m1, SEQ ID NO. 8). Values are normalized relative
to a control transcript (GAPDH) and to untransfected mouse NIH3T3
fibroblasts by the 2.sup.-.DELTA..DELTA.Ct method. FIG. 11C shows
qRT-PCR of human PHD2 (hPHD2) transcript in human HaCaT
keratinocytes transfected with increasing amounts of sshRNAs
designed to target either human PHD2 or mouse PHD2 (SG312 (SEQ ID
NO. 19), and SG314-SG316 (SEQ ID NO. 21-SEQ ID NO. 23)) or a
modified sshRNA designed to target human PHD2 (SG302m1, SEQ ID NO.
8). Values are normalized relative to a control transcript (GAPDH)
and to untransfected human HaCaT keratinocytes by the
2.sup.-.DELTA..DELTA.Ct method. FIG. 11D shows qRT-PCR of mouse
Phd2 (mPHD2) transcript in mouse NIH3T3 fibroblasts transfected
with increasing amounts of sshRNAs designed to target either human
PHD2 or mouse Phd2 (SG312 (SEQ ID NO. 19), and SG314-SG316 (SEQ ID
NO. 21-SEQ ID NO. 23)) or a modified sshRNA designed to target
human PHD2 (SG302m1, SEQ ID NO. 8). Values are normalized relative
to a control transcript (Gapdh) and to untransfected NIH3T3
fibroblasts.
Example 11. Effect of sshRNAs and miRNA Antagonists on Scratch
Wound Closure
[0207] To measure cell migration, human HaCaT keratinocytes were
plated at about 60% to about 70% confluence in 12-well plates and
were transfected with sshRNAs and microRNA antagonists. When cells
reached .about.100% confluence, the medium was replaced with
Dulbecco's Modified Eagle Medium containing 0.5% fetal bovine serum
two hours before scratching with a 200 .mu.L pipet tip. Photographs
and measurements across the cell-free scratch were taken
immediately after scratching and at the indicated time points at
six marked places per transfection condition. The percent closure
at each time point was calculated relative to the initial
scratching width. FIG. 12A shows the percent scratch closure at 24
hours (24 h), 48 h, and 72 h of human HaCaT keratinocytes
transfected with the following: an sshRNA targeting human PHD2
(SG302, SEQ ID NO. 7) and a LNA-modified miR-210 miRNA antagonist
(SG302 (SEQ ID NO. 7)+LNA210); SG302 (SEQ ID NO. 7) and a
2'-O-methyl-modified miR-210 miRNA antagonist (SG302+2'-O-methyl
210); and a control sshRNA (ssh-NSC) and a LNA-modified control
miRNA antagonist (ssh-NSC+LNA-NSC).
[0208] FIG. 12B provides representative images of the scratch
wounds at 0 hours (0 h), 24 h, and 48 h of human HaCaT
keratinocytes transfected with SG302+LNA210, SG302+2'-O-methyl 210,
and ssh-NSC+LNA-NSC. In bold is the scratch wound distance in
microns (.mu.all). The scale bar for all images is 100 .mu.m.
[0209] Similar scratch wound studies were performed with normal
human epidermal keratinocytes (NHEK) as described above. The
results are shown in TABLE 2.
TABLE-US-00002 TABLE 2 Inhibitors Percent wound closure (10 h)
SG302 (SEQ ID NO. 7) + LNA210 33 .+-. 6% SG302 (SEQ ID NO. 7) +
2'-O-methyl 41 .+-. 8% 210 NSC sshRNA + NSC 210 20 .+-. 5%
Example 12. Designs and Efficacy of Pre-miRNA Mimics
[0210] FIG. 13A-E show designs for pre-miRNA mimics of miR-21.
Mature miR-21 (SEQ ID NO. 4) is the sequence within the black
boxes. Lines between nucleotides correspond to base-pairing
interactions. Dots between nucleotides correspond to spaces
opposite a bulged nucleotide. Modified nucleotides are underlined.
FIG. 13F shows a luciferase assay with a miR-21 reporter in human
293FT cells transfected with no mimic or a miR-21 mimic. The miR-21
reporter was generated by sub-cloning four tandem miR-21
complementary binding sites, each separated by a 8 basepair (bp)
sequence, into the 3' UTR of Renilla luciferase (r-Luc) of the
psiCheck-2 dual luciferase reporter vector (Promega). This vector
also contains constitutively expressed firefly luciferase (f-Luc)
for transfection normalization. FIG. 13F shows a luciferase assay
with a miR-21 reporter in human 293FT cells treated with increasing
concentrations of the following: SG701, SEQ ID NO. 44 (FIG. 13A),
SG702, SEQ ID NO. 45 (FIG. 13B), SG703, SEQ ID NO. 46 (FIG. 13C),
SG703, SEQ ID NO. 46 (replicate 2) (FIG. 13C), SG704, SEQ ID NO. 47
(FIG. 13D), miRIDIAN miR-21 mimic (Dharmacon, positive control) and
cel-67 mimic (Dharmacon, miRIDIAN miRNA mimic negative control #1).
Values are computed relative to cells that were not transfected
with a mimic as in FIG. 7D.
Example 13. Effect of Pre-miRNA Mimic Design
[0211] Mouse embryonic fibroblasts (MEFs) that express green
fluorescent protein under the control of the Oct4 promoter
(Oct4-GFP) were mock transfected, transfected with a standard
miR-302b miRNA (miRIDIAN), with a miR-302b seed mutant, or a
sshRNA-pre-miR-302b (Somagenics) comparable to the pre-miR-21
design in FIG. 13E. Two days later, these cells were transduced
with a cocktail of retroviral vectors expressing Oct4, Klf4 and
Sox2. FIG. 14A shows the average (Ave) number of Oct4-GFP-positive
colonies in the mock, miRIDIAN, and Somagenics groups at 10, 12,
14, and 16 days after transfection. FIG. 14B shows a percentage of
GFP-positive cells relative to cells transfected with a control
vector over a time course in the seed mutant, MiRIDIAN, and
Somagenics treatment groups.
Example 14. Combination Effect of a sshRNA, miRNA Antagonist, and
miRNA Mimic in Diabetic Wound Healing
[0212] Skin wounding of leptin receptor the deficient db/db type II
diabetes mouse model is performed as described in Example 9.
[0213] Oligonucleotide formulations are then applied to the animals
the following day. All formulations are given topically as an LbL
mesh on a dressing on which the oligonucleotides are printed. In
group 1, the oligonucleotide is SG404 (SEQ ID NO. 30). In group 2,
the oligonucleotide is SG603 (SEQ ID NO. 34). In group 3, the
oligonucleotide is a chemically-modified miR-21 pre-miRNA mimic
presented in FIG. 13C (pre-miR-21). In group 4, the
oligonucleotides are SG404 (SEQ ID NO. 30) and SG603 (SEQ ID NO.
34). In group 5, the oligonucleotides are SG404 (SEQ ID NO. 30) and
pre-miR-21. In group 6, the oligonucleotides are SG603 (SEQ ID NO.
34) and pre-miR-21. In group 7, the oligonucleotides are SG404 (SEQ
ID NO. 30), SG603 (SEQ ID NO. 34), and pre-miR-21. In group 8, the
oligonucleotides are the sshRNA control SG221c, the miRNA
antagonist control NSC, and a control scrambled version of
pre-miR-21 (pre-miR-21-sc). Images of the wound are captured, the
wound area is calculated, and the time to closure curve is
generated as in Example 9. Comparisons are made between: each of
groups 1-7 and group 8; group 4 and groups 1 and 2; group 5 and
groups 1 and 3; group 6 and groups 2 and 3; and group 7 and groups
4, 5, and 6.
Example 15. Therapeutic Administration of Oligonucleotides to a
Subject with Chronic Diabetic Wounds
[0214] A subject in need thereof diagnosed with diabetes mellitus
experiences chronic, non-healing, diabetic foot wounds. To treat
these wounds, a dressing is applied to a foot wound of the subject.
On the dressing is an LbL mesh on which is printed as layers an
sshRNA targeting human PHD2, an LNA, 2'-O-methyl-modified miR-210
miRNA antagonist, and a 2'-O-methyl-modified pre-miR-21 pre-miRNA
mimic. After cleaning the wound, the dressing is applied such that
the mesh is in close physical contact with the wound. The dressing
is maintained and, if necessary, exchanged for a fresh dressing
with the LbL mesh containing the oligonucleotides. Treatment with
the dressing is complete upon complete wound closure.
Example 16. Therapeutic Administration of a Polynucleotide Vector
of a sshRNA to a Subject with Chronic Diabetic Wounds
[0215] A subject in need thereof diagnosed with diabetes mellitus
experiences chronic, non-healing, diabetic foot wounds. To treat
these wounds, a dressing is applied to a foot wound of the subject.
On the dressing is an LbL mesh on which is printed as layers a
recombinant adeno-associated virus (rAAV) of a sshRNA targeting
human PHD2 expressed from a EF1.alpha. promoter. The rAAV is of the
AAV1 serotype and is generated to 10.sup.11pfu. In producing the
virus, the adenoviral genes that provide helper functions to AAV
are supplied in trans to allow for production of the rAAV
particles, so that the rAAV is generated through a three-plasmid
system to decrease the probability of production of wild-type AAV.
After cleaning the wound, the dressing is applied such that the
mesh is in close physical contact with the wound. The dressing is
maintained and, if necessary, exchanged for a fresh dressing with
the LbL mesh containing the virus. Treatment with the dressing is
complete upon complete wound closure.
Example 17. Uptake of LBL-PHD2 sshRNA in db/db Mice Measured by
Fluorescence
[0216] Skin wounding of leptin receptor the deficient db/db type II
diabetes mouse model was performed as described in Example 9.
Oligonucleotide formulations LbL-SG405 (SEQ ID NO. 31) containing a
Alexa Fluor 594 label was then applied to the animals the following
day. Wounds were harvested at day 2, day 4, and day 6.
Cryopreserved sections were prepared and imaged by fluorescence
microscopy. FIG. 15 shows representative images at each of the time
points showing that LbL-SG405 (SEQ ID NO. 31) has been taken up
into cells in the wound area. 6 representative images of each time
point were obtained and the area of fluorescence was quantified
using Image J software. Results are plotted in FIG. 15. Uptake of
SG405 (SEQ ID NO. 31) was observed at all time points.
Sequence CWU 1
1
32917102DNAHomo sapiens 1ttaggggcag aaaaacattt gtaataatta
atggctttga gagacacaag gctttgtttg 60ccccagagta ttagttaacc cacctagtgc
tcctaatcat acaatattaa ggattgggag 120ggacattcat tgcctcactc
tctatttgtt tcaccttctg taaaattggt agaataatag 180tacccacttc
atagcattgt atgatgatta aattggttaa tatttttaaa atgcttagaa
240cacagattgg gcacataaca gcaagcacca catgtgttta taagataaat
tcctttgtgt 300tgccttccgt taaagtttaa ataagtaaat aaataaataa
atacttgcat gacattttga 360agtctctcta taacatctga gtaagtggcg
gctgcgacaa tgctactgga gttccagaat 420cgtgttggtg acaagattgt
tcaccagcat atggtgtggt gaaaactcac taatttggaa 480ttagttcaga
ttattaagcc tgaataggtg aaaatcctga aatcaaggat ctttggaact
540atttgaaatc agtattttat attttcctgt tgtattcatt aaagtgttgc
aagtgttcta 600tttgatggat taagtatatt taggatatac atgttcaatt
tgtgattttg tatacttaat 660tggaacaaga aagctaataa aggttttgat
atggacatct attcttttaa gtaaacttca 720atgaaaatat atgagtagag
catatagaga tgtaaataat ttgtggacac accacagact 780gaaatagcaa
atttaaaaga aattgttgga agaatcaagt gtttgtggaa tgagtcctcc
840tagtaaagtt cctgctcttg tgaataatta agcctcatgt ataattacta
tagcaaaagg 900aagcctaaga agtattagac tctacttgta tttaaattac
attttacata atttatgtgt 960atgaaaaatg ttttaaatgc ttattttcgt
aagccatgag atagctcctt tatattttaa 1020gaatttctga attaatttgc
ttggatttta ttagtgcaaa tggcagagct agcaattcct 1080ttttctgtgt
tcccattcca tcctattcat ccctctttta ggaaactctg aactctggat
1140tgtccttgtt tacatacctg cctcctgcat tggactatgt gtctctgagt
gtagtatgac 1200taattcattt gtttgtcaag gactctcaat gcatttgttg
aacagcctaa ttagtaatgt 1260ctgcaacaat gacattttac tgtatttaat
aaagctctgg gaaagtagga tacacataag 1320acaggtctag gtctaaattc
tttacagaaa cttggatttt tagttcggtt tgaaatttga 1380agatgtgagt
atatttatct cagtttccca aaggacaagc taattggaat tatcatcctc
1440tttcacttga ttggatcccc agaatgccat ttacgcatgc agcaggattt
tataacagtt 1500ttaaattctg tatatttgat gaagaggttt tatatttttg
gattcaagcc tctttttaaa 1560cttctacaat atggtttaca ataattcctt
atatcctgct tttgaaatac atattacaac 1620tttttaagtt tggaaggcta
tatttcaagg actgaagtta cagtatactc aagtgataca 1680caagcctagc
accccacttt ccacatagtg ttcgataaag attgataaac tcgaaatcac
1740agacctttta attcttaaga caaatagcag cagaaagaaa catctttggc
ttatttctgg 1800taaggttttt atgctctgta aaacaaagaa ttgtattcat
ccgcgcagca cagattctat 1860taaaaataaa tgtgagagtc gttaatgtag
tactgctcat ttaccatcaa aattcacttt 1920tcaggaataa tcccatcagt
ttaaattgga tattggaatg agcattgatt acatttaact 1980tggtagccca
aaatttcttc atggggtttt gaactcggcg ggatttcaaa ggttttaaaa
2040atgagttttt gatttttttt aaaaccctca aatttcatta cctttaaact
aggtcgaaac 2100ggggcgcaag agattggatt aacaccatag taatacttat
tttgttctta accatttcag 2160ggcttcttga aatagaggct gtatggtgta
atggaaaaaa cagccttgga atctgggagc 2220ctgattcctg gattcagtcc
cagttttgcg tgaccttggg caagttactt tacttctctg 2280aatttccgtt
tcctcctctg caaaatgagg atcgcaatag ccaccttgca accttgactg
2340gagcgagcct cgcacacccc gcgccggcct ggaggaagag cagccatgat
tacgccgcct 2400tcgctccgct acccgcttgc ggctggcgcc ctcctccagc
aggtgtaggc gctgccgcgc 2460tgccccacgc ctttccgccg ctcgcgggcc
tgcgcctcgg cgtccccgag gaggccgctg 2520cgggctgagg tagcgcaccg
gcctctcggc gtcccagtcc ggtcccgggc ggagggaaag 2580cgggcgaccc
acctccgagg cagaagccga ggcccggccc cgccgagtgc ggaggagcgc
2640aggcagcccc cgcccctcgg ccctcccccc ggccctcccg gccctccctc
cgccccctcc 2700gccctcgcgc gccgcccgcc cgggtcgccg cggggccgtg
gtgtacgtgc agagcgcgca 2760gagcgagtgg cgcccgtatg ccctgcgctc
ctccacagcc tgggccgggc cgcccgggac 2820gctgaggcgg cggcggcggc
cgagggggcc ggtcttgcgc tccccaggcc cgcgcgcctg 2880agcccaggtt
gccattcgcc gcacaggccc tattctctca gccctcggcg gcgatgaggc
2940gctgaggcgg ctgccggcgc tgcgccggag cttaggactc ggaagcggcc
gggccgaggg 3000cgtggggtgc cggcctccct gaggcgaggg tagcgggtgc
atggcgcagt aacggcccct 3060atctctctcc ccgctcccca gcctcgggcg
aggccgtccg gccgctaccc ctcctgctcg 3120gccgccgcag tcgccgtcgc
cgccgccgcc gccgccatgg ccaatgacag cggcgggccc 3180ggcgggccga
gcccgagcga gcgagaccgg cagtactgcg agctgtgcgg gaagatggag
3240aacctgctgc gctgcagccg ctgccgcagc tccttctact gctgcaagga
gcaccagcgt 3300caggactgga agaagcacaa gctcgtgtgc cagggcagcg
agggcgccct cggccacgga 3360gtgggcccac accagcattc cggccccgcg
ccgccggctg cagtgccgcc gcccagggcc 3420ggggcccggg agcccaggaa
ggcagcggcg cgccgggaca acgcctccgg ggacgcggcc 3480aagggaaaag
taaaggccaa gcccccggcc gacccagcgg cggccgcgtc gccgtgtcgt
3540gcggccgccg gcggccaggg ctcggcggtg gctgccgaag ccgagcccgg
caaggaggag 3600ccgccggccc gctcatcgct gttccaggag aaggcgaacc
tgtacccccc aagcaacacg 3660cccggggatg cgctgagccc cggcggcggc
ctgcggccca acgggcagac gaagcccctg 3720ccggcgctga agctggcgct
cgagtacatc gtgccgtgca tgaacaagca cggcatctgt 3780gtggtggacg
acttcctcgg caaggagacc ggacagcaga tcggcgacga ggtgcgcgcc
3840ctgcacgaca ccgggaagtt cacggacggg cagctggtca gccagaagag
tgactcgtcc 3900aaggacatcc gaggcgataa gatcacctgg atcgagggca
aggagcccgg ctgcgaaacc 3960attgggctgc tcatgagcag catggacgac
ctgatacgcc actgtaacgg gaagctgggc 4020agctacaaaa tcaatggccg
gacgaaagcc atggttgctt gttatccggg caatggaacg 4080ggttatgtac
gtcatgttga taatccaaat ggagatggaa gatgtgtgac atgtatatat
4140tatcttaata aagactggga tgccaaggta agtggaggta tacttcgaat
ttttccagaa 4200ggcaaagccc agtttgctga cattgaaccc aaatttgata
gactgctgtt tttctggtct 4260gaccgtcgca accctcatga agtacaacca
gcatatgcta caaggtacgc aataactgtt 4320tggtattttg atgcagatga
gagagcacga gctaaagtaa aatatctaac aggtgaaaaa 4380ggtgtgaggg
ttgaactcaa taaaccttca gattcggtcg gtaaagacgt cttctagagc
4440ctttgatcca gcaatacccc acttcaccta caatattgtt aactatttgt
taacttgtga 4500atacgaataa atgggataaa gaaaaataga caaccagttc
gcattttaat aaggaaacag 4560aaacaacttt ttgtgttgca tcaaacagaa
gattttgact gctgtgactt tgtactgcat 4620gatcaacttc aaatctgtga
ttgcttacag gaggaagata agctactaat tgaaaatggt 4680ttttacatct
ggatatgaaa taagtgccct gtgtagaatt tttttcattc ttatattttg
4740ccagatctgt tatctagctg agttcatttc atctctccct tttttatatc
aagtttgaat 4800ttgggataat ttttctatat taggtacaat ttatctaaac
tgaattgaga aaaaattaca 4860gtattattcc tcaaaataac atcaatctat
ttttgtaaac ctgttcatac tattaaattt 4920tgccctaaaa gacctcttaa
taatgattgt tgccagtgac tgatgattaa ttttatttta 4980cttaaaataa
gaaaaggagc actttaatta caactgaaaa atcagattgt tttgtagtcc
5040ttccttacac taatttgaac tgttaaagat tgctgctttt tttttgacat
tgtcaataac 5100gaaacctaat tgtaaaacag tcaccattta ctaccaataa
cttttagtta atgttttaca 5160aggaaaaaga cacaagaaga gtttaaattt
ttttgttttg ttttgttttt ttgagacagt 5220cttgctctgt tacccaggct
ggaggggagt ggtgcattct tggctcactg caacctccgc 5280ctcccaggtt
caagcaatcc tcccacctca gcctcccaac tagctgggac tgcaggcaca
5340caccaccatg cctgactaat ttttgtatgt ttagtagaga cggggttttg
ccatgttgcc 5400taggctgggg tttaagttaa attttttaaa aaactaaagt
gactggcact aagtgaactt 5460gagattatcc tcagcttcaa gttcctaaga
taagggcttt cttaagcttt caggtgtatg 5520tatcctctag atgtagacaa
taatgtccca tttctaagtc ttttcctttt gcttctcctt 5580aaattgattg
tacttccaaa tttgctgtta tgtttttttc ctaatactgt gatctatctg
5640atctgcagac aagaaccttg tctctgttga agagcatcaa ggggagatta
tgtacacatt 5700gaaactgaag tgtggtgtta ctgacggaat gtgcagtaac
tcctcagata tctgttaagg 5760catttcccag atgtgatgcc agccttctta
cctgtactga aagatgctta gcttagaaaa 5820aaacaaaaca gatgcaaaat
cagataattt tattttgttt catgggtttt cttatttact 5880ttttaaacaa
ggaaggaata ttagaaaatc acacaaggcc tcacatacat gttatttaaa
5940gaatgaattg ggacggatgt cttagacttc actttcctag gctttttagc
aaaacctaaa 6000gggtggtatc catattttgc gtgaattatg ggtgtaagac
cttgcccact taggttttct 6060atctctgtcc ttgatcttct ttgccaaaat
gtgagtatac agaaattttc tgtatatttc 6120aacttaagac atttttagca
tctgtatagt ttgtattcaa tttgagacct tttctatggg 6180aagctcagta
atttttatta aaagattgcc attgctattc atgtaaaaca tggaaaaaaa
6240ttgtgtagtg aagccaacag tggacttagg atgggattga atgttcagta
tagtgatctc 6300acttaggaga atttgcagga gaaagtgata gtttattgtt
ttttcctcgc ccatattcag 6360ttttgttcta cttcctcccc ttccttccag
atgataacat cacatctcta cagtaagtgc 6420ctctgccagc ccaacccagg
agcgcaagtt gtctttgcca tctggtctat agtacagtgc 6480gcggcgttag
gccacaactc aaaagcatta tcttttttag ggttagtaga aattgtttta
6540tgttgatggg aggtttgttt gattgtcaaa atgtacagcc acagcctttt
aatttgggag 6600cccctgttgt cattcaaatg tgtacctcta cagttgtaaa
aagtattaga ttctactatc 6660tgtgggttgt gcttgccaga caggtcttaa
attgtatatt ttttggaaaa gtttatatac 6720tctcttagga atcattgtga
aaagatcaag aaatcaggat ggccatttat ttaatatcca 6780ttcatttcat
gttagtggga ctattaactt gtcaccaagc aggactctat ttcaaacaaa
6840atttaaaact gtttgtggcc tatatgtgtt taatcctggt taaagataaa
gcttcataat 6900gctgttttta ttcaacacat taaccagctg taaaacacag
acctttatca agagtaggca 6960aagattttca ggattcatat acagatagac
tataaagtca tgtaatttga aaagcagtgt 7020ttcattatga aagagctctc
aagttgcttg taaagctaat ctaattaaaa agatgtataa 7080atgttgttga
aacattaaaa aa 710223524DNAMus sp. 2ggctgggccc gcccgcccag ggcgctgtgc
gccgcgcagg ccgcgctctc tccggcgcga 60tgcggcgcta ggcggccccg ggcaaggcag
gcgaggccag ggcgcgcgcg gcctcccgca 120gcgggcggcg gccccgggcg
ggcgccccga cggccccgcc gccgccccgc tcccggcccg 180cggcccgccc
tgccgcggcc atggccagtg acagcggcgg gcccggcgtg ctgagcgcca
240gcgagcgcga ccggcagtac tgcgagctgt gcgggaagat ggagaacctg
ctgcgctgcg 300gccgctgccg cagctccttc tactgctgca aagagcacca
gcgccaggac tggaagaagc 360acaagctggt gtgccagggc ggcgaggccc
cccgcgcgca gcccgcgccg gcgcagcccc 420gcgtcgcgcc cccgcccggt
ggggcccccg gagccgcgcg cgccggcggg gcggcccggc 480gcggggacag
cgcggcggcc tcgcgcgtac cgggcccgga ggacgcggcg caggcccgga
540gcggccccgg cccagcagag cccggctccg aggatcctcc gcttagccgg
tctccgggcc 600ccgagcgcgc cagcctgtgc ccagcgggtg gcggccccgg
ggaggcgctg agtcccggtg 660gagggctgcg gcccaacggg cagaccaagc
cgttgcccgc gttgaagctg gctctggagt 720acatcgtgcc gtgcatgaac
aagcacggca tctgcgtggt ggacgacttc ctgggcaggg 780agaccgggca
gcagatcggc gatgaggtgc gcgccctgca cgacaccggc aagttcacgg
840acgggcagct ggtcagccag aagagtgact cttccaagga catccggggg
gaccagatca 900cctggatcga gggcaaagag cccggctgcg aaaccatcgg
cctgctcatg agcagcatgg 960acgacctgat ccgccactgc agcgggaagc
tgggcaacta caggataaac ggccgaacga 1020aagccatggt tgcttgttac
ccaggcaacg gaacaggcta tgtccgtcac gttgataacc 1080caaatggaga
tggaagatgc gtgacatgta tatattatct aaataaagac tgggacgcca
1140aggtaagtgg aggtattctt cgaatttttc cagaaggcaa agcccagttt
gctgacattg 1200aacccaaatt tgatagactg ctgtttttct ggtctgaccg
gcgtaaccct catgaagtac 1260agccagcata cgccacaagg tacgcaataa
ctgtttggta ttttgatgca gatgagcgag 1320cgagagctaa agtaaaatat
ctaacaggtg agaaaggtgt gagggttgaa ctcaagccca 1380attcagtcag
caaagacgtc tagtggggcc ttgggtccgg cagtacccac gtcacctaca
1440gcctctcagt tgccttctgt ggactcgtgg acaggatgga cagagagaca
cctgcctggt 1500atttcagctg ggagccaggc gacttcgccg ggtgtcatcc
aacagagggc tccatctgct 1560gggactgtac tgtggggtca gctccagatc
tgtgactgct cttggctgct gacccaagag 1620gagacgctgt cggaggagag
tagcttttcc atctggacac gaaacaaggg ccctttgtag 1680gaatttcttc
agtcttctat tttgccagac ctgtcaccta actgagttca tttcatctct
1740tttttatatc aagttttgaa ttcggggaat ttttgtatta ggtacaattt
atcaaaactg 1800aattaagaaa aaaaaattta cagtattatt ctcaaaataa
catcaatcta tttttgtaaa 1860cctcttcatg ctattaaatt ttgccctcaa
ggcctcctgc gatgattgtt gccagtgagt 1920gacgacgtgt tgcttctgcc
tgaacgtaaa ggacgggcgg gcgctgtgtc ccagcccgag 1980tgcacgaggt
ttttcttggc ccgtctctca gtgattccaa cctgtaaagg tcactgctct
2040cgcgcttcga ccgacctaac agtagatggt tgccactggc actcaactaa
ctcaacatag 2100ttacaagagg aaacaagcca caggagaggg tttgtctctt
cagttaattt ttttaaagcg 2160aagtgacggg cactaaatga actcggggct
ctccctcagc ttcgggttcc tgagacaaag 2220ggctttcttc tgcggcaggt
ctagcctgcc tacagccgtg tcccactgcc gcaggtttcc 2280ttgtggcttc
tccgtagttt tgactgtgct tccagaccct tccaggtcag ggctgtgttc
2340ttgtggcagg gcacctggtg gacccaggca cgtgaatgtg gtatgtggtt
gtagcctcaa 2400tcgtggccat cggctccttg gacagccacg agccattttc
atacccaata atgaaagctg 2460tgtgctagct tagaaatcaa agggggtgta
aaagcacaca ttctttgttt tatgggtttt 2520tctcttttta gaggacagag
ggacaaccac acgaggctgc cagactcctg tcacctctac 2580agtcccctta
gaaagccaga gtttgcacag attgtgggta taactcctgt ccccttaggt
2640gttctatctc cgaccttgat ctttgccaaa atgtgtgtat gcagaactat
ttctgtgtat 2700tttccttgac acccgtctta gcacctgtgt agtttgtatc
cggttagaaa ccttttctat 2760ggaaagctca gtaattctta ttaagagatt
gctattgttc atgtaaaaca tgaaaacaac 2820caagtagagc cgtgtgtgga
tgagggccca ctcagcactg tgcttgcttg aggggctctc 2880ggcaggaagt
ctccttctga cccatatccg ctgaccacac ctctccagca agtgcctctg
2940ccgctggcca gctcaaggtt tgcccacctg gccccgaagc accgtgtttc
ggagttggga 3000ggaactgttt ggcattgttg gcagaaggtg tgattgcctg
gagcagcagc cttttaaatt 3060ctggagaccc tgtagtcctt tgtatctcag
acctttactg atgtaccagg tcccagattc 3120tgtggcaggg gatggggtgg
ggtgtgcttg ccagacgaaa tttaaattat ctatcttttg 3180ggaagtgtgt
gctttcctgg aggtcactgt gaaaacaaac aaacaaatca ggaccgttaa
3240ccccttaatg cccacttaaa ctcaatttca tgttaggact cttgtttaaa
accatttgtg 3300gcctgtatgt gttcatcctg gttagagaga aagctttatg
acgctgtttc tgttcaacac 3360attaaccagc tgtggaacag ccctttttgc
acgacaggca gggcacttca ggattcgcag 3420agagactcgt gtggtttgga
agtggtattt cctatgaaag cctctcacgt tgcttgtaaa 3480gctaatctaa
ttaaaaagat gtataaatgt tcttgaaaaa aatc 3524322RNAUnknownDescription
of Unknown miR-210 oligonucleotide 3cugugcgugu gacagcggcu ga
22422RNAUnknownDescription of Unknown miR-21 oligonucleotide
4uagcuuauca gacugauguu ga 22542RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 5cuuccuuuug
cuauaguaau uuuacuauag caaaaggaag uu 42642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6uguugcagac auuacuaauu uauuaguaau gucugcaaca uu
42742RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 7uauaccucca cuuaccuugu ucaagguaag
uggagguaua uu 42842RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 8uauaccucca cuuaccuugu
ucaagguaag uggagguaua uu 42921RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 9uauaccucca
cuuaccuugu u 211021RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 10caagguaagu ggagguauau u
211142RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 11auucgaagua uaccuccacu uguggaggua
uacuucgaau uu 421242RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 12cgaaguauac cuccacuuau
uuaaguggag guauacuucg uu 421342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 13aguauaccuc
cacuuaccuu uagguaagug gagguauacu uu 421442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14uaccuccacu uaccuuggcu ugccaaggua aguggaggua uu
421542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 15cuccacuuac cuuggcaucu ugaugccaag
guaaguggag uu 421642RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 16cacuuaccuu ggcaucccau
uugggaugcc aagguaagug uu 421742RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 17agguucucca
ucuucccgcu ugcgggaaga uggagaaccu uu 421842RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 18augccgugcu uguucaugcu ugcaugaaca agcacggcau uu
421942RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 19ucacucuucu ggcugaccau uuggucagcc
agaagaguga uu 422042RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 20aucuuccauc uccauuuggu
uccaaaugga gauggaagau uu 422142RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 21ugugcuucuu
ccaguccugu ucaggacugg aagaagcaca uu 422242RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22aucaggucgu ccaugcugcu ugcagcaugg acgaccugau uu
422342RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 23auaccuccac uuaccuuggu uccaagguaa
guggagguau uu 422442RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 24uggcguaugc uggcuguacu
uguacagcca gcauacgcca uu 422542RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 25cucacaccuu
ucucaccugu ucaggugaga aaggugugag uu 422642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26cugaauuggg cuugaguucu ugaacucaag cccaauucag uu
422742DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 27cugaauuggg cuugaguucu ugaacucaag
cccaauucag tt 422821RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 28gaacucaagc ccaauucagu u
212921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 29cugaauuggg cuugaguucu u
213040RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 30cugaauuggg cuugaguucu ugaacucaag
cccaauucag 403139RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 31cugaauuggg cuugaguucu
gaacucaagc ccaauucag 393222RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 32ucagccgcug
ucacacgcac ag 223322DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 33tcagccgctg tcacacgcac ag
223422RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 34ucagccgcug ucacacgcac ag
223518DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 35gccgctgtca cacgcaca 183620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 36agagctccct tcaatccaaa 203722RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 37ucagccgcug ucacacgcac ag 223822RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 38ucagccgcug ucacacgcac ag 223922RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 39ucagccgcug ucacacgcac ag 224022RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 40ucagccgcug ucacacgcac ag 224122RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 41ucagccgcug ucacacgcac ag 224222RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 42ucagccgcug ucacacgcac ag 224322RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 43ucagccgcug ucacacgcac ag 224459RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 44uagcuuauca gacugauguu gacuguugaa ucucauggca
acaccagucg augggcuuu 594540RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 45uagcuuauca
gacugauguu gacaucaguc ugauaagcuc 404642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 46uagcuuauca gacugauguu gacaucaguc ugauaagcua uu
424748RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 47uagcuuauca gacugauguu gauuucaaca
ucagucugau aagcuauu 484848RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 48uagcuuauca
gacugauguu gauuucaaca ucagucugau aagcuauu 484942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 49ccgggcccgc cgcugucauu uaugacagcg gcgggcccgg uu
425042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 50gccgggcccg ccgcugucau uugacagcgg
cgggcccggc uu 425142RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 51cgccgggccc gccgcugucu
ugacagcggc gggcccggcg uu 425242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 52ccgccgggcc
cgccgcuguu uacagcggcg ggcccggcgg uu 425342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 53cucgcaguac ugccggucuu uagaccggca guacugcgag uu
425442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 54gcucgcagua cugccggucu ugaccggcag
uacugcgagc uu 425542RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 55agcucgcagu acugccgguu
uaccggcagu acugcgagcu uu 425642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 56cagcucgcag
uacugccggu uccggcagua cugcgagcug uu 425742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 57acagcucgca guacugccgu ucggcaguac ugcgagcugu uu
425842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 58cacagcucgc aguacugccu uggcaguacu
gcgagcugug uu 425942RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 59gcacagcucg caguacugcu
ugcaguacug cgagcugugc uu 426042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 60cgcacagcuc
gcaguacugu ucaguacugc gagcugugcg uu 426142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 61ccgcacagcu cgcaguacuu uaguacugcg agcugugcgg uu
426242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 62cccgcacagc ucgcaguacu uguacugcga
gcugugcggg uu 426342RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 63ucccgcacag cucgcaguau
uuacugcgag cugugcggga uu 426442RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 64uucccgcaca
gcucgcaguu uacugcgagc ugugcgggaa uu 426542RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 65cuucccgcac agcucgcagu ucugcgagcu gugcgggaag uu
426642RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 66ucuucccgca cagcucgcau uugcgagcug
ugcgggaaga uu 426742RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 67aucuucccgc acagcucgcu
ugcgagcugu gcgggaagau uu 426842RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 68caucuucccg
cacagcucgu ucgagcugug cgggaagaug uu 426942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 69ccaucuuccc gcacagcucu ugagcugugc gggaagaugg uu
427042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 70uccaucuucc cgcacagcuu uagcugugcg
ggaagaugga uu 427142RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 71cuccaucuuc ccgcacagcu
ugcugugcgg gaagauggag uu 427242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 72ucuccaucuu
cccgcacagu ucugugcggg aagauggaga uu 427342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 73uucuccaucu ucccgcacau uugugcggga agauggagaa uu
427442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 74guucuccauc uucccgcacu ugugcgggaa
gauggagaac uu 427542RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 75gguucuccau cuucccgcau
uugcgggaag auggagaacc uu 427642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 76agguucucca
ucuucccgcu ugcgggaaga uggagaaccu uu 427742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 77cagguucucc aucuucccgu ucgggaagau ggagaaccug uu
427842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 78gcagguucuc caucuucccu ugggaagaug
gagaaccugc uu 427942RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 79agcagguucu ccaucuuccu
uggaagaugg agaaccugcu uu 428042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 80cagcagguuc
uccaucuucu ugaagaugga gaaccugcug uu 428142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 81gcagcagguu cuccaucuuu uaagauggag aaccugcugc uu
428242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 82cgcagcaggu ucuccaucuu uagauggaga
accugcugcg uu 428342RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 83gcgcagcagg uucuccaucu
ugauggagaa ccugcugcgc uu 428442RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 84agcgcagcag
guucuccauu uauggagaac cugcugcgcu uu 428542RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 85cagcgcagca gguucuccau uuggagaacc ugcugcgcug uu
428642RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 86gcagcgcagc agguucuccu uggagaaccu
gcugcgcugc uu 428742RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 87ugcagcgcag cagguucucu
ugagaaccug cugcgcugca uu 428842RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 88aggagcugcg
gcagcggcuu uagccgcugc cgcagcuccu uu 428942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 89aaggagcugc ggcagcggcu ugccgcugcc gcagcuccuu uu
429042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 90gaaggagcug cggcagcggu uccgcugccg
cagcuccuuc uu 429142RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 91agaaggagcu gcggcagcgu
ucgcugccgc agcuccuucu uu 429242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 92uagaaggagc
ugcggcagcu ugcugccgca gcuccuucua uu 429342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 93guagaaggag cugcggcagu ucugccgcag cuccuucuac uu
429442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 94aguagaagga gcugcggcau uugccgcagc
uccuucuacu uu 429542RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 95caguagaagg agcugcggcu
ugccgcagcu ccuucuacug uu 429642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 96gcaguagaag
gagcugcggu uccgcagcuc cuucuacugc uu 429742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 97agcaguagaa ggagcugcgu ucgcagcucc uucuacugcu uu
429842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 98cagcaguaga aggagcugcu ugcagcuccu
ucuacugcug uu 429942RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 99gcagcaguag aaggagcugu
ucagcuccuu cuacugcugc uu 4210042RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 100ugcagcagua
gaaggagcuu uagcuccuuc uacugcugca uu 4210142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 101uugcagcagu agaaggagcu ugcuccuucu acugcugcaa uu
4210242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 102cuugcagcag uagaaggagu ucuccuucua
cugcugcaag uu 4210342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 103gugcuucuuc
caguccugau uucaggacug gaagaagcac uu 4210442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 104ugugcuucuu ccaguccugu ucaggacugg aagaagcaca uu
4210542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 105uugugcuucu uccaguccuu uaggacugga
agaagcacaa uu 4210642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 106cuugugcuuc
uuccaguccu uggacuggaa gaagcacaag uu 4210742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 107gcuugugcuu cuuccagucu ugacuggaag aagcacaagc uu
4210842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 108agcuugugcu ucuuccaguu uacuggaaga
agcacaagcu uu 4210942RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 109gagcuugugc
uucuuccagu ucuggaagaa gcacaagcuc uu 4211042RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 110cugcccguug ggccgcaggu uccugcggcc caacgggcag uu
4211142RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 111ucugcccguu gggccgcagu ucugcggccc
aacgggcaga uu 4211242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 112gucugcccgu
ugggccgcau uugcggccca acgggcagac uu 4211342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 113cgucugcccg uugggccgcu ugcggcccaa cgggcagacg uu
4211442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 114gcacggcacg auguacucgu ucgaguacau
cgugccgugc uu 4211542RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 115ugcacggcac
gauguacucu ugaguacauc gugccgugca uu 4211642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 116augcacggca cgauguacuu uaguacaucg ugccgugcau uu
4211742RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 117caugcacggc acgauguacu uguacaucgu
gccgugcaug uu 4211842RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 118ucaugcacgg
cacgauguau uuacaucgug ccgugcauga uu 4211942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 119uucaugcacg gcacgauguu uacaucgugc cgugcaugaa uu
4212042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 120guucaugcac ggcacgaugu ucaucgugcc
gugcaugaac uu 4212142RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 121uguucaugca
cggcacgauu uaucgugccg ugcaugaaca uu 4212242RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 122uuguucaugc acggcacgau uucgugccgu gcaugaacaa uu
4212342RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 123cuuguucaug cacggcacgu ucgugccgug
caugaacaag uu 4212442RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 124gcuuguucau
gcacggcacu ugugccgugc augaacaagc uu 4212542RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 125ugcuuguuca ugcacggcau uugccgugca ugaacaagca uu
4212642RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 126gugcuuguuc augcacggcu ugccgugcau
gaacaagcac uu 4212742RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 127cgugcuuguu
caugcacggu uccgugcaug aacaagcacg uu 4212842RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 128ccgugcuugu ucaugcacgu ucgugcauga acaagcacgg uu
4212942RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 129gccgugcuug uucaugcacu ugugcaugaa
caagcacggc uu 4213042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 130ugccgugcuu
guucaugcau uugcaugaac aagcacggca uu 4213142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 131augccgugcu uguucaugcu ugcaugaaca agcacggcau uu
4213242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 132gaugccgugc uuguucaugu ucaugaacaa
gcacggcauc uu 4213342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 133agaugccgug
cuuguucauu uaugaacaag cacggcaucu uu 4213442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 134cagaugccgu gcuuguucau uugaacaagc acggcaucug uu
4213542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 135acagaugccg ugcuuguucu ugaacaagca
cggcaucugu uu 4213642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 136gugcagggcg
cgcaccucgu ucgaggugcg cgcccugcac uu 4213742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 137cgugcagggc gcgcaccucu ugaggugcgc gcccugcacg uu
4213842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic
oligonucleotide 138ucgugcaggg cgcgcaccuu uaggugcgcg cccugcacga uu
4213942RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 139gucgugcagg gcgcgcaccu uggugcgcgc
ccugcacgac uu 4214042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 140ugucgugcag
ggcgcgcacu ugugcgcgcc cugcacgaca uu 4214142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 141gugucgugca gggcgcgcau uugcgcgccc ugcacgacac uu
4214242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 142ggugucgugc agggcgcgcu ugcgcgcccu
gcacgacacc uu 4214342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 143cggugucgug
cagggcgcgu ucgcgcccug cacgacaccg uu 4214442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 144ccggugucgu gcagggcgcu ugcgcccugc acgacaccgg uu
4214542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 145cccggugucg ugcagggcgu ucgcccugca
cgacaccggg uu 4214642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 146cugcccgucc
gugaacuucu ugaaguucac ggacgggcag uu 4214742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 147gcugcccguc cgugaacuuu uaaguucacg gacgggcagc uu
4214842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 148agcugcccgu ccgugaacuu uaguucacgg
acgggcagcu uu 4214942RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 149cagcugcccg
uccgugaacu uguucacgga cgggcagcug uu 4215042RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 150ccagcugccc guccgugaau uuucacggac gggcagcugg uu
4215142RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 151accagcugcc cguccgugau uucacggacg
ggcagcuggu uu 4215242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 152gaccagcugc
ccguccgugu ucacggacgg gcagcugguc uu 4215342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 153ugaccagcug cccguccguu uacggacggg cagcugguca uu
4215442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 154cugaccagcu gcccguccgu ucggacgggc
agcuggucag uu 4215542RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 155gcugaccagc
ugcccguccu uggacgggca gcuggucagc uu 4215642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 156ggcugaccag cugcccgucu ugacgggcag cuggucagcc uu
4215742RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 157uggcugacca gcugcccguu uacgggcagc
uggucagcca uu 4215842RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 158cuggcugacc
agcugcccgu ucgggcagcu ggucagccag uu 4215942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 159ucuggcugac cagcugcccu ugggcagcug gucagccaga uu
4216042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 160uucuggcuga ccagcugccu uggcagcugg
ucagccagaa uu 4216142RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 161cuucuggcug
accagcugcu ugcagcuggu cagccagaag uu 4216242RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 162ucuucuggcu gaccagcugu ucagcugguc agccagaaga uu
4216342RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 163cucuucuggc ugaccagcuu uagcugguca
gccagaagag uu 4216442RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 164acucuucugg
cugaccagcu ugcuggucag ccagaagagu uu 4216542RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 165cacucuucug gcugaccagu ucuggucagc cagaagagug uu
4216642RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 166ucacucuucu ggcugaccau uuggucagcc
agaagaguga uu 4216742RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 167gucacucuuc
uggcugaccu uggucagcca gaagagugac uu 4216842RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 168agucacucuu cuggcugacu ugucagccag aagagugacu uu
4216942RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 169gagucacucu ucuggcugau uucagccaga
agagugacuc uu 4217042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 170cgagucacuc
uucuggcugu ucagccagaa gagugacucg uu 4217142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 171ccucgaucca ggugaucuuu uaagaucacc uggaucgagg uu
4217242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 172cccucgaucc aggugaucuu uagaucaccu
ggaucgaggg uu 4217342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 173gcccucgauc
caggugaucu ugaucaccug gaucgagggc uu 4217442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 174ugcccucgau ccaggugauu uaucaccugg aucgagggca uu
4217542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 175uugcccucga uccaggugau uucaccugga
ucgagggcaa uu 4217642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 176cuugcccucg
auccaggugu ucaccuggau cgagggcaag uu 4217742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 177gguuucgcag ccgggcuccu uggagcccgg cugcgaaacc uu
4217842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 178ugguuucgca gccgggcucu ugagcccggc
ugcgaaacca uu 4217942RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 179augguuucgc
agccgggcuu uagcccggcu gcgaaaccau uu 4218042RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 180aaugguuucg cagccgggcu ugcccggcug cgaaaccauu uu
4218142RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 181caugcugcuc augagcagcu ugcugcucau
gagcagcaug uu 4218242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 182ccaugcugcu
caugagcagu ucugcucaug agcagcaugg uu 4218342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 183uccaugcugc ucaugagcau uugcucauga gcagcaugga uu
4218442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 184guccaugcug cucaugagcu ugcucaugag
cagcauggac uu 4218542RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 185cguccaugcu
gcucaugagu ucucaugagc agcauggacg uu 4218642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 186ucguccaugc ugcucaugau uucaugagca gcauggacga uu
4218742RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 187gucguccaug cugcucaugu ucaugagcag
cauggacgac uu 4218842RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 188ggucguccau
gcugcucauu uaugagcagc auggacgacc uu 4218942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 189aggucgucca ugcugcucau uugagcagca uggacgaccu uu
4219042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 190caggucgucc augcugcucu ugagcagcau
ggacgaccug uu 4219142RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 191ucaggucguc
caugcugcuu uagcagcaug gacgaccuga uu 4219242RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 192aucaggucgu ccaugcugcu ugcagcaugg acgaccugau uu
4219342RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 193uaucaggucg uccaugcugu ucagcaugga
cgaccugaua uu 4219442RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 194agcaaccaug
gcuuucgucu ugacgaaagc caugguugcu uu 4219542RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 195aagcaaccau ggcuuucguu uacgaaagcc augguugcuu uu
4219642RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 196caagcaacca uggcuuucgu ucgaaagcca
ugguugcuug uu 4219742RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 197acaagcaacc
auggcuuucu ugaaagccau gguugcuugu uu 4219842RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 198aacaagcaac cauggcuuuu uaaagccaug guugcuuguu uu
4219942RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 199uaacaagcaa ccauggcuuu uaagccaugg
uugcuuguua uu 4220042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 200auaacaagca
accauggcuu uagccauggu ugcuuguuau uu 4220142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 201ucuuccaucu ccauuuggau uuccaaaugg agauggaaga uu
4220242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 202aucuuccauc uccauuuggu uccaaaugga
gauggaagau uu 4220342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 203caucuuccau
cuccauuugu ucaaauggag auggaagaug uu 4220442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 204acaucuucca ucuccauuuu uaaauggaga uggaagaugu uu
4220542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 205auaauauaua caugucacau uugugacaug
uauauauuau uu 4220642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 206gauaauauau
acaugucacu ugugacaugu auauauuauc uu 4220742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 207agauaauaua uacaugucau uugacaugua uauauuaucu uu
4220842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 208aagauaauau auacaugucu ugacauguau
auauuaucuu uu 4220942RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 209accuccacuu
accuuggcau uugccaaggu aaguggaggu uu 4221042RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 210uaccuccacu uaccuuggcu ugccaaggua aguggaggua uu
4221142RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 211auaccuccac uuaccuuggu uccaagguaa
guggagguau uu 4221242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 212uauaccucca
cuuaccuugu ucaagguaag uggagguaua uu 4221342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 213uucuggaaaa auucgaaguu uacuucgaau uuuuccagaa uu
4221442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 214cuucuggaaa aauucgaagu ucuucgaauu
uuuccagaag uu 4221542RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 215ccuucuggaa
aaauucgaau uuucgaauuu uuccagaagg uu 4221642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 216gccuucugga aaaauucgau uucgaauuuu uccagaaggc uu
4221742RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 217ugccuucugg aaaaauucgu ucgaauuuuu
ccagaaggca uu 4221842RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 218uugccuucug
gaaaaauucu ugaauuuuuc cagaaggcaa uu 4221942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 219uuugccuucu ggaaaaauuu uaauuuuucc agaaggcaaa uu
4222042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 220cuuugccuuc uggaaaaauu uauuuuucca
gaaggcaaag uu 4222142RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 221gcuuugccuu
cuggaaaaau uuuuuuccag aaggcaaagc uu 4222242RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 222ggcuuugccu ucuggaaaau uuuuuccaga aggcaaagcc uu
4222342RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 223gggcuuugcc uucuggaaau uuuuccagaa
ggcaaagccc uu 4222442RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 224ugggcuuugc
cuucuggaau uuuccagaag gcaaagccca uu 4222542RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 225cugggcuuug ccuucuggau uuccagaagg caaagcccag uu
4222642RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 226acugggcuuu gccuucuggu uccagaaggc
aaagcccagu uu 4222742RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 227aacugggcuu
ugccuucugu ucagaaggca aagcccaguu uu 4222842RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 228aaacugggcu uugccuucuu uagaaggcaa agcccaguuu uu
4222942RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 229caaacugggc uuugccuucu ugaaggcaaa
gcccaguuug uu 4223042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 230gcaaacuggg
cuuugccuuu uaaggcaaag cccaguuugc uu 4223142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 231agcaaacugg gcuuugccuu uaggcaaagc ccaguuugcu uu
4223242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 232cagcaaacug ggcuuugccu uggcaaagcc
caguuugcug uu 4223342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 233ucagcaaacu
gggcuuugcu ugcaaagccc aguuugcuga uu 4223442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 234gucagcaaac ugggcuuugu ucaaagccca guuugcugac uu
4223542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 235ugucagcaaa cugggcuuuu uaaagcccag
uuugcugaca uu 4223642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 236augucagcaa
acugggcuuu uaagcccagu uugcugacau uu 4223742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 237aaugucagca aacugggcuu uagcccaguu ugcugacauu uu
4223842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 238caaugucagc aaacugggcu ugcccaguuu
gcugacauug uu
4223942RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 239ucaaugucag caaacugggu ucccaguuug
cugacauuga uu 4224042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 240uucaauguca
gcaaacuggu uccaguuugc ugacauugaa uu 4224142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 241guucaauguc agcaaacugu ucaguuugcu gacauugaac uu
4224242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 242gguucaaugu cagcaaacuu uaguuugcug
acauugaacc uu 4224342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 243ggguucaaug
ucagcaaacu uguuugcuga cauugaaccc uu 4224442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 244uggguucaau gucagcaaau uuuugcugac auugaaccca uu
4224542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 245uuggguucaa ugucagcaau uuugcugaca
uugaacccaa uu 4224642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 246uuuggguuca
augucagcau uugcugacau ugaacccaaa uu 4224742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 247auuuggguuc aaugucagcu ugcugacauu gaacccaaau uu
4224842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 248aauuuggguu caaugucagu ucugacauug
aacccaaauu uu 4224942RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 249aaauuugggu
ucaaugucau uugacauuga acccaaauuu uu 4225042RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 250caaauuuggg uucaaugucu ugacauugaa cccaaauuug uu
4225142RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 251ucaaauuugg guucaauguu uacauugaac
ccaaauuuga uu 4225242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 252aucaaauuug
gguucaaugu ucauugaacc caaauuugau uu 4225342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 253uaucaaauuu ggguucaauu uauugaaccc aaauuugaua uu
4225442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 254cuaucaaauu uggguucaau uuugaaccca
aauuugauag uu 4225542RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 255ucuaucaaau
uuggguucau uugaacccaa auuugauaga uu 4225642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 256gucuaucaaa uuuggguucu ugaacccaaa uuugauagac uu
4225742RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 257agucuaucaa auuuggguuu uaacccaaau
uugauagacu uu 4225842RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 258cagucuauca
aauuuggguu uacccaaauu ugauagacug uu 4225942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 259gcagucuauc aaauuugggu ucccaaauuu gauagacugc uu
4226042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 260agcagucuau caaauuuggu uccaaauuug
auagacugcu uu 4226142RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 261cagcagucua
ucaaauuugu ucaaauuuga uagacugcug uu 4226242RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 262acagcagucu aucaaauuuu uaaauuugau agacugcugu uu
4226342RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 263aacagcaguc uaucaaauuu uaauuugaua
gacugcuguu uu 4226442RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 264aaacagcagu
cuaucaaauu uauuugauag acugcuguuu uu 4226542RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 265aaaacagcag ucuaucaaau uuuugauaga cugcuguuuu uu
4226642RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 266aaaaacagca gucuaucaau uuugauagac
ugcuguuuuu uu 4226742RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 267gaaaaacagc
agucuaucau uugauagacu gcuguuuuuc uu 4226842RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 268agaaaaacag cagucuaucu ugauagacug cuguuuuucu uu
4226942RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 269cagaaaaaca gcagucuauu uauagacugc
uguuuuucug uu 4227042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 270ccagaaaaac
agcagucuau uuagacugcu guuuuucugg uu 4227142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 271accagaaaaa cagcagucuu uagacugcug uuuuucuggu uu
4227242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 272gaccagaaaa acagcagucu ugacugcugu
uuuucugguc uu 4227342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 273agaccagaaa
aacagcaguu uacugcuguu uuucuggucu uu 4227442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 274cagaccagaa aaacagcagu ucugcuguuu uucuggucug uu
4227542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 275ucagaccaga aaaacagcau uugcuguuuu
ucuggucuga uu 4227642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 276gucagaccag
aaaaacagcu ugcuguuuuu cuggucugac uu 4227742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 277ggucagacca gaaaaacagu ucuguuuuuc uggucugacc uu
4227842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 278cggucagacc agaaaaacau uuguuuuucu
ggucugaccg uu 4227942RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 279acggucagac
cagaaaaacu uguuuuucug gucugaccgu uu 4228042RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 280uuguacuuca ugaggguugu ucaacccuca ugaaguacaa uu
4228142RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 281aguuauugcg uaccuuguau uuacaaggua
cgcaauaacu uu 4228242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 282caguuauugc
guaccuuguu uacaagguac gcaauaacug uu 4228342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 283acaguuauug cguaccuugu ucaagguacg caauaacugu uu
4228442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 284aacaguuauu gcguaccuuu uaagguacgc
aauaacuguu uu 4228542RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 285aaacaguuau
ugcguaccuu uagguacgca auaacuguuu uu 4228642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 286caaacaguua uugcguaccu ugguacgcaa uaacuguuug uu
4228742RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 287ccaaacaguu auugcguacu uguacgcaau
aacuguuugg uu 4228842RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 288accaaacagu
uauugcguau uuacgcaaua acuguuuggu uu 4228942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 289uaccaaacag uuauugcguu uacgcaauaa cuguuuggua uu
4229042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 290auaccaaaca guuauugcgu ucgcaauaac
uguuugguau uu 4229142RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 291aauaccaaac
aguuauugcu ugcaauaacu guuugguauu uu 4229242RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 292aaauaccaaa caguuauugu ucaauaacug uuugguauuu uu
4229342RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 293aaaauaccaa acaguuauuu uaauaacugu
uugguauuuu uu 4229442RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 294caaaauacca
aacaguuauu uauaacuguu ugguauuuug uu 4229542RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 295ucaaaauacc aaacaguuau uuaacuguuu gguauuuuga uu
4229642RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 296aucaaaauac caaacaguuu uaacuguuug
guauuuugau uu 4229742RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 297caucaaaaua
ccaaacaguu uacuguuugg uauuuugaug uu 4229842RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 298gcaucaaaau accaaacagu ucuguuuggu auuuugaugc uu
4229942RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 299ugcaucaaaa uaccaaacau uuguuuggua
uuuugaugca uu 4230042RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 300cugcaucaaa
auaccaaacu uguuugguau uuugaugcag uu 4230142RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 301ucugcaucaa aauaccaaau uuuugguauu uugaugcaga uu
4230242RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 302aucugcauca aaauaccaau uuugguauuu
ugaugcagau uu 4230342RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 303caucugcauc
aaaauaccau uugguauuuu gaugcagaug uu 4230442RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 304ucaucugcau caaaauaccu ugguauuuug augcagauga uu
4230542RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 305cucaucugca ucaaaauacu uguauuuuga
ugcagaugag uu 4230642RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 306ucucaucugc
aucaaaauau uuauuuugau gcagaugaga uu 4230742RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 307gauauuuuac uuuagcucgu ucgagcuaaa guaaaauauc uu
4230842RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 308agauauuuua cuuuagcucu ugagcuaaag
uaaaauaucu uu 4230942RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 309uagauauuuu
acuuuagcuu uagcuaaagu aaaauaucua uu 4231042RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 310uuagauauuu uacuuuagcu ugcuaaagua aaauaucuaa uu
4231142RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 311guuagauauu uuacuuuagu ucuaaaguaa
aauaucuaac uu 4231242RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 312uguuagauau
uuuacuuuau uuaaaguaaa auaucuaaca uu 4231342RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 313cuguuagaua uuuuacuuuu uaaaguaaaa uaucuaacag uu
4231442RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 314ccuguuagau auuuuacuuu uaaguaaaau
aucuaacagg uu 4231542RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 315accuguuaga
uauuuuacuu uaguaaaaua ucuaacaggu uu 4231642RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 316caccuguuag auauuuuacu uguaaaauau cuaacaggug uu
4231742RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 317ucaccuguua gauauuuuau uuaaaauauc
uaacagguga uu 4231842RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 318uucaccuguu
agauauuuuu uaaaauaucu aacaggugaa uu 4231942RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 319uucaacccuc acaccuuuuu uaaaaggugu gaggguugaa uu
4232042RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 320guucaacccu cacaccuuuu uaaaggugug
aggguugaac uu 4232142RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 321aguucaaccc
ucacaccuuu uaagguguga ggguugaacu uu 4232242RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 322gaguucaacc cucacaccuu uaggugugag gguugaacuc uu
4232342RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 323ugaguucaac ccucacaccu uggugugagg
guugaacuca uu 4232442RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 324uugaguucaa
cccucacacu ugugugaggg uugaacucaa uu 4232542RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 325auugaguuca acccucacau uugugagggu ugaacucaau uu
4232622RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 326ucagccgcug ucacacgcac ag
2232715DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 327gcugtcacac gcaca 1532820RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 328cagccgcugu cacacgcaca 2032922RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 329ucagccgcug ucacacgcac ag 22
* * * * *