U.S. patent application number 12/124590 was filed with the patent office on 2009-09-24 for multitargeting interfering rnas and methods of their use and design.
This patent application is currently assigned to Johnson & Johnson Research PTY. Limited. Invention is credited to Gregory Martin Arndt, Donald John Birkett, Toby Passioura, Michael Poidinger, Laurent Pierre Rivory.
Application Number | 20090239816 12/124590 |
Document ID | / |
Family ID | 38048226 |
Filed Date | 2009-09-24 |
United States Patent
Application |
20090239816 |
Kind Code |
A1 |
Rivory; Laurent Pierre ; et
al. |
September 24, 2009 |
Multitargeting Interfering RNAs And Methods Of Their Use And
Design
Abstract
Interfering RNA molecules are now designed and produced with
specificity for multiple binding sequences present in distinct
genetic contexts in one or more pre-selected target RNA molecules
and are used to modulate expression of the target sequences. Such a
multitargeting interfering RNA approach provides a powerful tool
for gene regulation.
Inventors: |
Rivory; Laurent Pierre; (New
South Wales, AU) ; Poidinger; Michael; (New South
Wales, AU) ; Birkett; Donald John; (New South Wales,
AU) ; Arndt; Gregory Martin; (New South Wales,
AU) ; Passioura; Toby; (New South Wales, AU) |
Correspondence
Address: |
CROWELL & MORING LLP;INTELLECTUAL PROPERTY GROUP
P.O. BOX 14300
WASHINGTON
DC
20044-4300
US
|
Assignee: |
Johnson & Johnson Research PTY.
Limited
New South Wales
AU
|
Family ID: |
38048226 |
Appl. No.: |
12/124590 |
Filed: |
May 21, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/AU2006/001741 |
Nov 21, 2006 |
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12124590 |
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60738441 |
Nov 21, 2005 |
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60738640 |
Nov 21, 2005 |
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Current U.S.
Class: |
514/44R ;
435/235.1; 435/243; 435/320.1; 435/325; 435/455; 435/6.1; 435/6.18;
536/24.5; 800/21; 800/278; 800/298; 800/8 |
Current CPC
Class: |
C12N 15/111 20130101;
C12N 15/1135 20130101; C12N 15/1138 20130101; A61K 31/712 20130101;
A61P 11/00 20180101; A61P 31/18 20180101; A61P 43/00 20180101; C12N
15/1136 20130101; A61P 31/16 20180101; C12N 2310/14 20130101; C12N
15/113 20130101; C12N 2320/50 20130101; A61P 31/14 20180101; C12N
15/1132 20130101; A61K 31/713 20130101; A61K 31/7105 20130101; A61K
31/7115 20130101; A61P 31/12 20180101 |
Class at
Publication: |
514/44 ;
536/24.5; 435/235.1; 435/243; 435/325; 800/298; 800/8; 435/320.1;
800/21; 435/455; 800/278; 435/6 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; C07H 21/02 20060101 C07H021/02; C12N 7/01 20060101
C12N007/01; C12N 1/00 20060101 C12N001/00; C12N 5/10 20060101
C12N005/10; A01H 5/00 20060101 A01H005/00; A01K 67/00 20060101
A01K067/00; C12N 15/63 20060101 C12N015/63; C12N 15/00 20060101
C12N015/00; A01H 1/00 20060101 A01H001/00; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A multitargeting interfering RNA molecule comprising a guide
strand of the Formula (I): 5'-p-XSY-3' wherein p consists of a
terminal phosphate group that is independently present or absent;
wherein S consists of a first nucleotide sequence of a length of
about 5 to about 20 nucleotides that is at least partially
complementary to a first portion of each of at least two binding
sequences present in distinct genetic contexts in one or more
pre-selected target RNA molecules; wherein X is absent or consists
of a second nucleotide sequence; wherein Y is absent or consists of
a third nucleotide sequence, provided that X and Y are not absent
simultaneously; wherein XSY is at least partially complementary to
each of said binding sequences to allow a stable interaction
therewith.
2. The multitargeting interfering RNA molecule of claim 1, wherein
S is completely complementary to the first portion of each of at
least two binding sequences.
3. The multitargeting interfering RNA molecule of claim 1, wherein
the first portion of each of at least two binding sequences is a
seed sequence.
4. The multitargeting interfering RNA molecule of claim 1, wherein
X consists of one or two nucleotides.
5. The multitargeting interfering RNA molecule of claim 1, wherein
Y is at least partially complementary to a second portion of each
of the binding sequences, said second portion is adjacent to and
connected with the 5'-end of said first portion of the binding
sequences.
6. The multitargeting interfering RNA molecule of claim 1, wherein
S is of a length of about 8 to about 15 nucleotides.
7. The multitargeting interfering RNA molecule of claim 1, wherein
XSY is of a length of about 17 to about 25 nucleotides.
8. The multitargeting interfering RNA molecule of any one of claim
1, further comprising a passenger strand that is at least partially
complementary to the guide strand to allow formation of a stable
duplex between the passenger strand and the guide strand.
9. The multitargeting interfering RNA molecule of claim 8, wherein
the passenger strand and the guide strand are completely
complementary to each other.
10. The multitargeting interfering RNA molecule of claim 1
comprising at least one or more terminal overhangs.
11. The multitargeting interfering RNA molecule of claim 10,
wherein the overhang consists of 1 to 5 nucleotides.
12. The multitargeting interfering RNA molecule of claim 1, wherein
the binding sequences are present in distinct genetic contexts in
one pre-selected target RNA molecule.
13. The multitargeting interfering RNA molecule of claim 1, wherein
the binding sequences are present in distinct genetic contexts in
at least two pre-selected target RNA molecules.
14. The multitargeting interfering RNA molecule of claim 1, wherein
at least one of the pre-selected target RNA molecules is a
non-coding RNA molecule.
15. The multitargeting interfering RNA molecule of claim 1, wherein
at least one of the pre-selected target RNA molecules is a
messenger RNA molecule.
16. The multitargeting interfering RNA molecule of claim 1, wherein
at least one of the binding sequences is present in the
3'-untranslated region (3'UTR) of a messenger RNA molecule.
17. The multitargeting interfering RNA molecule of claim 1, wherein
one or more of the pre-selected target RNA molecules are involved
in a disease or disorder.
18. The multitargeting interfering RNA molecule of claim 17,
wherein one or more of the pre-selected target RNA molecules are
involved in a disease or disorder of an animal or a plant.
19. The multitargeting interfering RNA molecule of claim 18,
wherein the animal is selected from the group consisting of a rat,
a mouse, a dog, a cat, a pig, a monkey, and a human.
20. The multitargeting interfering RNA molecule of claim 1, wherein
one or more of the pre-selected target RNA molecules encode a
protein of a class selected from the group consisting of receptors,
cytokines, transcription factors, regulatory proteins, signaling
proteins, cytoskeletal proteins, transporters, enzymes, hormones,
and antigens.
21. The multitargeting interfering RNA molecule of claim 1, wherein
one or more of the pre-selected target RNA molecules encode a
protein selected from the group consisting of ICAM-1, VEGF-A,
MCP-1, IL-8, VEGF-B, IGF-1, Gluc6p, Inppl1, bFGF, PlGF, VEGF-C,
VEGF-D, .beta.-catenin, .kappa.-ras-B, .kappa.-ras-A, EGFR, Bcl-2,
presenilin-1, BACE-1, MALAT-1, BIC, TGF.beta., and TNF alpha.
22. The multitargeting interfering RNA molecule of claim 1 that
decreases expression of any combination of VEGF-A, .quadrature.-ras
and Bcl-2 in an expression system.
23. The multitargeting interfering RNA molecule of claim 1 that
decreases expression of both MALAT-1 and BIC in an expression
system.
24. The multitargeting interfering RNA molecule of claim 1 that
decreases expression of both ICAM-1 and VEGF-A in an expression
system.
25. The multitargeting interfering RNA molecule of claim 1 that
decreases expression of both TGF.beta. and IL-8 in an expression
system.
26. The multitargeting interfering RNA molecule of claim 1 that
decreases expression of both IL-8 and MCP-1 in an expression
system.
27. The multitargeting interfering RNA molecule of claim 1, wherein
one or more of the pre-selected target RNA molecules is viral
RNA.
28. The multitargeting interfering RNA molecule of claim 27,
wherein the virus is selected from the group consisting of a human
immunodeficiency virus (HIV), a hepatitis C virus (HCV), an
influenza virus, a rhinovirus, and a severe acute respiratory
syndrome (SARS) virus.
29. The multitargeting interfering RNA molecule of claim 28,
wherein one or more of the pre-selected target RNA molecules encode
an essential protein for HIV selected from the group consisting of
GAG, POL, VIF, VPR, TAT, NEF, REV, VPU and ENV.
30. The multitargeting interfering RNA molecule of claim 27 wherein
one or more of the preselected RNA molecules comprises Hepatitis C
Virus (HCV) and one or more of the preselected RNA molecules
encodes TNF.alpha..
31. The multitargeting interfering RNA molecule of claim 1
comprising at least one modified ribonucleotide, universal base,
acyclic nucleotide, abasic nucleotide, non-ribonucleotide or
combinations thereof.
32. The multitargeting interfering RNA molecule of claim 1, wherein
S consists essentially of a nucleotide sequence selected from the
group consisting of: TABLE-US-00058 UAUGUGGGUGGG, (SEQ ID NO: 1)
UGUUUUG, (SEQ ID NO: 2) ACCCCGUCUCU, (SEQ ID NO: 5) AGCUGCA, (SEQ
ID NO: 7) AAACAAUGGAAUG, (SEQ ID NO: 8) GGUAGGUGGGUGGG, (SEQ ID NO:
10) CUGCUUGAU, (SEQ ID NO: 12) UCCUUUCCA, (SEQ ID NO: 13)
UUUUUCUUU, (SEQ ID NO: 14) UUCUGAUGUUU, (SEQ ID NO: 15)
UCUUCCUCUAU, (SEQ ID NO: 16) UGGUAGCUGAA, (SEQ ID NO: 17)
CUUUGGUUCCU, (SEQ ID NO: 18) CUACUAAUGCU, (SEQ ID NO: 19)
UCCUGCUUGAU, (SEQ ID NO: 20) AUUCUUUAGUU, (SEQ ID NO: 21)
CCAUCUUCCUG, (SEQ ID NO: 22) CCUCCAAUUCC, (SEQ ID NO: 23)
CUAAUACUGUA, (SEQ ID NO: 24) UUCUGUUAGUG, (SEQ ID NO: 25)
GCUGCUUGAUG, (SEQ ID NO: 26) ACAUUGUACUG, (SEQ ID NO: 27)
UGAUAUUUCUC, (SEQ ID NO: 28) AACAGCAGUUG, (SEQ ID NO: 29)
GUGCUGAUAUU, (SEQ ID NO: 30) CCCAUCUCCAC, (SEQ ID NO: 31)
UAUUGGUAUUA, (SEQ ID NO: 32) CAAAUUGUUCU, (SEQ ID NO: 33)
UACUAUUAAAC, (SEQ ID NO: 34) GCCUAUCAUAU, (SEQ ID NO: 58)
UGGUGCCUGCU, (SEQ ID NO: 59) AAUUAAUAUGGC, (SEQ ID NO: 60)
CCCUCUGGGCU, (SEQ ID NO: 61) UUCUUCCUCAU, (SEQ ID NO: 62)
UAUUUAUACAGA, (SEQ ID NO: 63) CACCAAAAUUC, (SEQ ID NO: 64)
UGAGUNNGAACAUU (SEQ ID NO: 72) where N is any base, CUCCAGG, (SEQ
ID NO: 74) UCAGUGGG, (SEQ ID NO: 76) UCCUCACAGGG, (SEQ ID NO: 78)
GUGCUCAUGGUG, (SEQ ID NO: 79) CCUGGAGCCCUG, (SEQ ID NO: 80)
UCUCAGCUCCAC, (SEQ ID NO: 81) ACCCUCGCACC, (SEQ ID NO: 86)
GUGUUGAAG, (SEQ ID NO: 88) UUCCACAAC, (SEQ ID NO: 90) UCCACUGUC,
(SEQ ID NO: 92) CAGAAUAG, (SEQ ID NO: 93) AACUCUCUA (SEQ ID NO: 94)
and CGUGAAGAC. (SEQ ID NO: 98).
33. The multitargeting interfering RNA molecule of claim 1, wherein
S consists essentially of a nucleotide sequence selected from the
group consisting of: TABLE-US-00059 UAUGUGGGUGGG, (SEQ ID NO: 1)
UCCUCACAGGG, (SEQ ID NO: 78) GUGUUGAAG, (SEQ ID NO: 88) UUCCACAAC,
(SEQ ID NO: 90) AACUCUCUA (SEQ ID NO: 94) and CGUGAAGAC. (SEQ ID
NO: 98)
34. The multitargeting interfering RNA molecule of claim 1, wherein
S consists essentially of a nucleotide sequence of 6 or more
contiguous bases contained within any of the sequences selected
from the group consisting of: TABLE-US-00060 UAUGUGGGUGGG, (SEQ ID
NO: 1) UCCUCACAGGG, (SEQ ID NO: 78) GUGUUGAAG, (SEQ ID NO: 88)
UUCCACAAC, (SEQ ID NO: 90) AACUCUCUA (SEQ ID NO: 94) and CGUGAAGAC.
(SEQ ID NO: 98)
35. The multitargeting interfering RNA molecule of claim 8,
consisting essentially of: TABLE-US-00061 5' UUCCUCACAGGGCAGUGAUUC
3' (SEQ ID NO: 122) 3' UUAAAGAGUGUCCCGUCACUA 5', (SEQ ID NO: 124)
5' UACAAAUCUACUUCAACAUUU 3' (SEQ ID NO: 131) 3 '
GUAUGUUUAGAUGAAGUUGUG 5', (SEQ ID NO: 132) or 5 '
AACAUAUGUUCUUCAACAUUU 3' (SEQ ID NO: 133) 3 ' GUUUGUAUACAAGAAGUUGUG
5',. (SEQ ID NO: 134)
36. The multitargeting interfering RNA molecule of claim 9,
consisting essentially of: TABLE-US-00062 5 ' UAUGUGGGUGGGUGAGUCUAA
3' (SEQ ID NO: 100) 3 ' UUAUACACCCACCCACUCAGA 5', (SEQ ID NO: 101)
5 ' UGUUUUGUUGUUACAUAUGAC 3' (SEQ ID NO: 102) 3 '
UUACAAAACAACAAUGUAUAC 5', (SEQ ID NO: 103) 5 '
UAUGUGGGUGGGGUGUCUCUA 3' (SEQ ID NO: 104) 3 ' UUAUACACCCACCCCACAGAG
5', (SEQ ID NO: 105) 5 ' UAUGUGGGUGGGGUGGUCUAA 3' (SEQ ID NO: 106)
3 ' UUAUACACCCACCCCACCAGA 5', (SEQ ID NO: 107) 5 '
UAUGUGGGUGGGGUGGUGUCU 3' (SEQ ID NO: 108) 3 ' UUAUACACCCACCCCACCACA
5', (SEQ ID NO: 109) 5 ' UAUGUGGGUGGGUGAGUGUCU 3' (SEQ ID NO: 110)
3 ' UUAUACACCCACCCACUCACA 5', (SEQ ID NO: 111) 5 '
CUCACCCACCCACAUACAUUU 3' (SEQ ID NO: 112) 3 ' CUGAGUGGGUGGGUGUAUGUA
5', (SEQ ID NO: 113) 5 ' UCACCCACCCACAUACAUAUU 3' (SEQ ID NO: 114)
3 ' UGAGUGGGUGGGUGUAUGUAU 5', (SEQ ID NO: 115) 5 '
UCACCCACCCACAUACAUUUU 3' (SEQ ID NO: 116) 3 ' UGAGUGGGUGGGUGUAUGUAA
5, (SEQ ID NO: 117) 5 ' UAUGUGGGUGGGUGAGUCUA 3' (SEQ ID NO: 118) 3
' UAUACACCCACCCACUCAGA 5', (SEQ ID NO: 119) 5 '
GGGUUUACCAGGAAGAUGGUU 3' (SEQ ID NO: 120) 3 ' UACCCAAAUGGUCCUUCUACC
5', (SEQ ID NO: 121) 5 ' UUCCUCACAGGGCAGUGAUUC 3' (SEQ ID NO: 122)
3 ' UUAAGGAGUGUCCCGUCACUA 5', (SEQ ID NO: 123) 5 '
UUCCUCACAGGGCAGUGGUUC 3' (SEQ ID NO: 125) 3 ' UUAAGGAGUGUCCCGUCACCA
5', (SEQ ID NO: 126) 5 ' CCCGGACCCUUAGAGAGUUUU 3' (SEQ ID NO: 127)
3 ' ACGGGCCUGGGAAUCUCUCAA 5', (SEQ ID NO: 128) 5 '
UACCCUCGCACCGAUCUCCCAA 3' (SEQ ID NO: 129) 3 '
UUAUGGGAGCGUGGCUAGAGGG 5', (SEQ ID NO: 130) 5 '
UUCCACAACACAAGCUGUGUU 3' (SEQ ID NO: 135) 3 ' UUAAGGUGUUGUGUUCGACAC
5', (SEQ ID NO: 136) 5 ' GGACCCUUAGAGAGUUUCAUU 3' (SEQ ID NO: 137)
3 ' GGCCUGGGAAUCUCUCAAAGU 5', (SEQ ID NO: 138) 5 '
UUCGUGAAGACGGUGGGCCGA 3' (SEQ ID NO: 139) 3 '
dTdTAAGCACUUCUGCCACCCGG 5', (SEQ ID NO: 140) or 5 '
AGACUCACCCACCCAGAUAUU 3' (SEQ ID NO: 141) 3 ' AAUCUGAGUGGGUGGGUCUAU
5' (SEQ ID NO: 142)
37. The multitargeting interfering RNA molecule of claim 9,
consisting essentially of: TABLE-US-00063 5 ' UAUGUGGGUGGGUGAGUCUAA
3' (SEQ ID NO: 100) 3 ' UUAUACACCCACCCACUCAGA 5', (SEQ ID NO: 101)
5 ' GGACCCUUAGAGAGUUUCAUU 3' (SEQ ID NO: 137) 3 '
GGCCUGGGAAUCUCUCAAAGU 5', (SEQ ID NO: 138) or 5 '
UUCGUGAAGACGGUGGGCCGA 3' (SEQ ID NO: 139) 3 '
dTdTAAGCACUUCUGCCACCCGG 5', (SEQ ID NO: 140)
38. The multitargeting interfering RNA molecule of claim 37,
comprising at least one modified ribonucleotide, universal base,
acyclic nucleotide, abasic nucleotide, non-ribonucleotide, overhang
variation or a combination thereof.
39. A biological system comprising a multitargeting interfering RNA
molecule of claim 1.
40. The biological system of claim 39 being a virus, a microbe, a
cell, a plant, or an animal.
41. A vector comprising a nucleotide sequence that encodes the
multitargeting interfering RNA molecule of claim 1.
42. The vector of claim 41 being a viral vector.
43. The vector of claim 42 that is derived from a virus selected
from the group consisting of an adeno-associated virus, a
retrovirus, an adenovirus, a lentivirus, and an alphavirus.
44. A cell comprising the vector of claim 41.
45. The multitargeting interfering RNA molecule of claim 1 produced
from a short hairpin RNA molecule.
46. A vector for the short hairpin RNA molecule of claim 45.
47. A cell comprising the vector of claim 46.
48. A pharmaceutical composition comprising a multitargeting
interfering RNA molecule of claim 1 and an acceptable carrier.
49. A pharmaceutical composition comprising an expression vector of
claim 41 and an acceptable carrier.
50. A pharmaceutical composition comprising an expression vector of
claim 46 and an acceptable carrier.
51. A method of inducing RNA interference in a biological system,
comprising the step of introducing a multitargeting interfering RNA
molecule of claim 1 into the biological system.
52. A method of inducing RNA interference in a biological system,
comprising the steps of: (a) selecting one or more target RNA
molecules; (b) designing a multitargeting interfering RNA molecule
comprising a guide strand that can form stable interactions with at
least two binding sequences present in distinct genetic contexts in
the one or more target RNA molecules; (c) producing the
multitargeting interfering RNA molecule; and (d) administering the
multitargeting interfering RNA molecule into the biological system,
whereby the guide strand of the multitargeting interfering RNA
molecule forms stable interactions with the binding sequences
present in distinct genetic contexts in the target RNA molecules,
and thus induces RNA interference of the target RNA molecules.
53. The method of claim 52, wherein the biological system is a
virus, a microbe, a cell, a plant, or an animal.
54. The method of claim 52, wherein the biological system is an
animal selected from the group consisting of a rat, a mouse, a dog,
a cat, a pig, a monkey, and a human.
55. The method of claim 52, wherein the one or more target RNA
molecules comprises RNA molecules that are involved in a disease or
disorder of the biological system.
56. The method of claim 52, wherein one or more of the target RNA
molecules comprises one or more RNA molecules selected from the
biological system.
57. The method of claim 52, wherein one or more of the target RNA
molecules comprises one or more RNA molecules selected from a
second biological system that is infectious to the biological
system.
58. The method of claim 52, wherein the target RNA molecules
comprise one or more RNA molecules selected from the biological
system and one or more target RNA molecules selected from a second
biological system that is infectious to the biological system.
59. The method of claim 52, wherein the target RNA molecules
comprise one or more RNA molecules selected from an animal or a
plant and one or more RNA molecules selected from a microbe or a
virus that is infectious to the animal or the plant.
60. The method of claim 52, wherein the target RNA molecules
comprise one or more RNA molecules selected from a human and one or
more RNA molecules selected from a virus selected from the group
consisting of a human immunodeficiency virus (HIV), a hepatitis C
virus (HCV), an influenza virus, a rhinovirus, and a severe acute
respiratory syndrome (SARS) virus.
61. The method of claim 52, wherein the one or more target RNA
molecules comprise a RNA molecule encoding a protein of a class
selected from the group consisting of receptors, cytokines,
transcription factors, regulatory proteins, signaling proteins,
cytoskeletal proteins, transporters, enzymes, hormones, and
antigens.
62. The method of claim 52, wherein the one or more target RNA
molecules comprise a RNA molecule encoding a protein selected from
the group consisting of ICAM-1, VEGF-A, MCP-1, IL-8, VEGF-B, IGF-1,
Gluc6p, Inppl1, bFGF, PlGF, VEGF-C, VEGF-D, .beta.-catenin,
.kappa.-ras-B, .kappa.-ras-A, EGFR, Bcl-2, presenilin-1, BACE-1,
MALAT-1, BIC, TGF.beta., and TNF alpha.
63. The method of claim 62, wherein the one or more target RNA
molecules encode ICAM-1 and VEGF-A.
64. The method of claim 52, wherein the one or more target RNA
molecules comprises a viral RNA molecule.
65. The method of claim 52, wherein the one or more target RNA
molecules comprises a virus selected from the group consisting of a
human immunodeficiency virus (HIV), a hepatitis C virus (HCV), an
influenza virus, a rhinovirus, and a severe acute respiratory
syndrome (SARS) virus.
66. The method of claim 65, wherein the one or more target RNA
molecules comprise a RNA molecule encoding an essential protein for
HIV selected from the group consisting of GAG, POL, VIF, VPR, TAT,
NEF, REV, VPU and ENV.
67. The method of claim 59, wherein the target RNA molecules
comprises a RNA molecule encoding a human protein TNFalpha,
LEDGF(p75), BAF, CCR5, CXCR4, furin, NFkB, STAT1.
68. The method of claim 59, wherein one or more of the preselected
RNA molecules comprises Hepatitis C Virus (HCV) and one or more of
the preselected RNA molecules encodes TNF.beta..
69. A method of treating a disease or condition in a subject, the
method comprising the steps of: (a) selecting one or more target
RNA molecules, wherein the modulation in expression of the target
RNA molecules is potentially therapeutic for the treatment of the
disease or condition; (b) designing a multitargeting interfering
RNA molecule comprising a guide strand that can form stable
interactions with at least two binding sequences present in
distinct genetic contexts in the one or more target RNA molecules;
(c) producing the multitargeting interfering RNA molecule; (d)
administering the multitargeting interfering RNA molecule into the
subject, whereby the guide strand of the multitargeting interfering
RNA molecule forms stable interactions with the binding sequences
present in distinct genetic contexts in the one or more target RNA
molecules, and thus induces RNA interference and modulation of
expression of the target RNA molecules.
70. A method for designing a multitargeting interfering RNA
molecule, comprising the steps of: a) selecting one or more target
RNA molecules, wherein the modulation in expression of the target
RNA molecules is desired; b) obtaining at least one nucleotide
sequence for each of the target RNA molecules; c) selecting a seed
sequence of 6 nucleotides or more, said seed sequence occurs in at
least two distinct genetic contexts in nucleotide sequences
obtained in b) for the target RNA molecules; d) selecting at least
two binding sequences, wherein each of binding sequences comprises
the seed sequence, and the binding sequences are present in
distinct genetic contexts in the target molecules; and e) designing
a multitargeting interfering RNA molecule having a guide strand
that shares a substantial degree of complementarity with each of
the at least two binding sequences to allow stable interaction
therewith.
71. The method of claim 70 comprising designing a passenger strand
that is at least partially complementary to the guide strand to
allow formation of a stable duplex between the passenger strand and
the guide strand.
72. A method for designing a multitargeting interfering RNA
molecule, comprising the steps of: a) selecting one or more target
RNA molecules, wherein the modulation in expression of the target
RNA molecules is desired; b) obtaining at least one nucleotide
sequence for each of the target RNA molecules; c) selecting a
length, n, in nucleotides, for a seed sequence; wherein n=about 6
or more; d) generating a collection of candidate seed sequences of
the length n from each nucleotide sequence obtained in step b),
wherein each candidate seed sequence occurs at least once in
nucleotide sequences obtained in step b); e) determining the
genetic context of each of the candidate seed sequences in each
nucleotide sequence obtained in step b), by collecting, for each
occurrence of the candidate seed sequence, a desired amount of the
5' and 3' flanking sequence; f) selecting a seed sequence of the
length n from the candidate seed sequences, wherein the seed
sequence occurs at least in two distinct genetic contexts in
nucleotide sequences obtained in step b); g) selecting a consensus
target sequence, wherein said consensus target sequence comprises
the seed sequence and a desired consensus sequence for the sequence
flanking either one or both of the 5' and 3' ends of the seed; and
h) designing a multitargeting interfering RNA molecule comprising a
guide strand that shares a substantial degree of complementarity
with the consensus target sequence to allow stable interaction
therewith.
73. The method of claim 72 wherein the step of generating a
collection of candidate seed sequences comprises the steps of
beginning at a terminus, sequentially observing the nucleotide
sequence using a window size of n and stepping along the nucleotide
sequence with a step size of 1.
74. The method of claim 72, wherein the step of selecting seed
sequences comprises the step of discarding any sequence of the
length n that: i) is composed of a consecutive string of 5 or more
identical single nucleotides; ii) is composed of only adenosine and
uracil; iii) is predicted to occur with an unacceptably high
frequency in a non-target transcriptome of interest; iv) is
predicted to have a propensity to undesirably modulate the
expression or activity of one or more cellular component; or v) is
any combination of i) to iv).
75. The method of claim 72, wherein the step of selecting a
consensus target sequence comprises the step of discarding any
sequence that is composed of only a single base, is composed only
of A and U, has a consecutive string of 5 or more bases which are
C, is G/C rich at the 3' end, is predicted to occur with
unacceptable frequency in a non-target transcriptome of interest;
or any combination thereof.
76. The method of claim 72 comprising designing a passenger strand
that is at least partially complementary to the guide strand to
allow formation of a stable duplex between the passenger strand and
the guide strand.
77. The method of claim 72, further comprising the step of
modifying the multitargeting interfering RNA molecule, i) to
improve the incorporation of the guide strand of the multitargeting
interfering RNA molecule into the RNA induced silencing complex
(RISC); ii) to increase or decrease the modulation of the
expression of at least one target RNA molecule; iii) to decrease
stress or inflammatory response when the multitargeting interfering
RNA molecule is administered into a subject; iv) to alter half life
in an expression system; or v) any combination of i) to iv).
78. The method of claim 72, further comprising repeating the steps
c) to g) of claim 72 with a new value of n.
79. The method of claim 72, further comprising the steps of making
the designed multitargeting interfering RNA molecule and testing it
in a suitable expression system.
80. A method of designing a full length multitargeting interfering
RNA from a seed sequence, comprising the steps of: a. deducing the
sequence of the complete complement of the seed sequence; b.
generating permutations for the extension of the complete
complement of the seed sequence to the desired length n; c.
creating a collection of putative guide strand sequences, each of
which comprises the sequence of the complete complement of the seed
sequence and one of the permutations generated in step b); d. using
RNAhybrid to determine the binding pattern and the minimum free
energy (mfe) of the putative guide strand sequences created in step
c) against all the target sequences comprising the seed sequence;
e. discarding putative guide strand sequences where i. there is a
contiguous run of 5 or more G residues; and ii. the Load Bias is
<1.2; and f. selecting a guide strand sequence of the length n
for a multitargeting interfering RNA sequence from the list of the
remaining putative guide strand sequences based on their Relative
Activity Score.
81. The method of claim 80, wherein the desired length n is 21
bases.
82. The method of claim 80 further comprising the steps of
producing the multitargeting interfering RNA comprising the guide
strand sequence and testing the multitargeting interfering RNA in
an expression system.
83. A method of making a multitargeting interfering RNA molecule,
comprising the steps of: i) designing a multitargeting interfering
RNA molecule having a guide strand that can form stable
interactions with at least two binding sequences present in
distinct genetic contexts in a set of pre-selected target RNA
molecules; and ii) producing the multitargeting interfering RNA
molecule.
84. A method for making a pharmaceutical composition comprising the
step of mixing a multitargeting interfering RNA molecule of claim 1
and a pharmaceutically acceptable carrier.
85. A multitargeting interfering RNA molecule comprising a guide
strand that forms stable interactions with at least two binding
sequences present in distinct genetic contexts in one or more
pre-selected target RNA molecules.
86. A biological system comprising a multitargeting interfering RNA
molecule comprising Formula (I): 5'-p-XSY-3' wherein p consists of
a terminal phosphate group that is independently present or absent;
wherein S consists of a first nucleotide sequence of a length of
about 5 to about 20 nucleotides that is at least partially
complementary to a first portion of each of at least two binding
sequences present in distinct genetic contexts in one or more
pre-selected target RNA molecules; wherein X is absent or consists
of a second nucleotide sequence; wherein Y is absent or consists of
a third nucleotide sequence, provided that X and Y are not absent
simultaneously; wherein XSY is at least partially complementary to
each of said binding sequences to allow a stable interaction
therewith.
87. A method of introducing a multitargeting interfering RNA
molecule comprising Formula (I) into a cell comprising the steps
of: i) generating a multitargeting interfering RNA molecule
comprising Formula (I) 5'-p-XSY-3' wherein p consists of a terminal
phosphate group that is independently present, or absent; wherein S
consists of a first nucleotide sequence of a length of about 5 to
about 20 nucleotides that is at least partially complementary to a
first portion of each of at least two binding sequences present in
distinct genetic contexts in one or more pre-selected target RNA
molecules; wherein X is absent or consists of a second nucleotide
sequence; wherein Y is absent or consists of a third nucleotide
sequence, provided that X and Y are not absent simultaneously;
wherein XSY is at least partially complementary to each of said
binding sequences to allow a stable interaction therewith; and ii)
contacting the multitargeting interfering RNA molecule with a
cell.
88. The method of claim 87 wherein the multitargeting interfering
RNA is encoded by DNA.
89. The method of claim 87 wherein the RNA is encoded by a DNA or
RNA vector.
90. The method of claim 87 wherein the contacting step further
comprises the step of introducing the RNA molecule or an RNA
molecule encoded by a DNA or RNA vector into the cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT Application No.
PCT/AU2006/001741 filed on Nov. 21, 2006, which claims priority to
Provisional Application No. 60/738,441 filed Nov. 21, 2005 and
60/738,640 filed Nov. 21, 2005.
FIELD OF THE INVENTION
[0002] The present invention concerns methods and reagents useful
in modulating gene expression. Particularly, the invention relates
to modulating gene expression using a multitargeting interfering
RNA molecule that targets multiple target sites on one or more
pre-selected RNA molecules.
BACKGROUND OF THE INVENTION
[0003] RNA interference (RNAi) is a diverse, evolutionarily
conserved mechanism in eukaryotic cells, which inhibits the
transcription and translation of target genes in a
sequence-specific manner. It is now known that single and
double-stranded RNA can modulate expression of or modify processing
of target RNA molecules by a number of mechanisms. Some such
mechanisms tolerate variation in the amount of sequence
complementarity required between the modulatory (or interfering)
RNA and the target RNA. Certain microRNAs can translationally
repress target mRNA having as little as 6 nucleotides of
complementarity with the microRNA. The development of RNA
interfering agents, for example, using double-stranded RNA to
repress expression of disease-related genes is currently an area of
intense research activity.
[0004] Double-stranded RNA of 19-23 bases in length is recognized
by an RNA interference silencing complex (RISC) into which an
effector strand (or "guide strand") of the RNA is loaded. This
guide strand acts as a template for the recognition and destruction
of highly complementary sequences present in the transcriptome.
Alternatively, through the recognition and binding of RNA sequences
of lower complementarity, interfering RNAs may induce translational
repression without mRNA degradation. Such translational repression
appears to be a mechanism of action of endogenous microRNAs, a
group of short non-coding RNAs involved in differentiation and
development.
[0005] Efforts at implementing interfering RNAs therapeutically
thus far have focused on producing specific double stranded RNAs,
each with complete complementarity to a particular target
transcript. Such double-stranded RNAs (dsRNAs) are potentially
effective where a single suitable target can be identified,
however, dsRNAs, particularly those designed against one target,
may have at least two categories of off-target side effects that
need to be avoided or minimized. Undesirable side effects can arise
through the triggering of innate immune response pathways (e.g.
Toll-like Receptor 3, 7, and 8, and the so-called interferon
response) and through inadvertent inhibition of protein expression
from related or unrelated transcripts (either by RNA degradation,
translational repression or other mechanisms). Some bioinformatic
and/or experimental approaches have been developed to try to
minimize off-target effects. Algorithms for in silico hybridization
are known, and others have been developed for predicting target
accessibility and loading bias in an effort to eliminate or
minimize side-effects while maintaining effectiveness.
[0006] Several double-stranded RNA molecules for potentially
treating human diseases of viral and nonviral origin are in various
stages of development. The diseases include Age-related Macular
Degeneration, Amyotrophic Lateral Sclerosis (ALS), and Respiratory
Syncytial Virus (RSV) infection. These RNA molecules, however, are
designed to target only a single site in an RNA sequence. Although
RNA interference may be useful and potent in obtaining knock-down
of specific gene products, many diseases involve complex
interactions between ontologically-unrelated gene products.
Multiple putative targets can be identified for a single disease.
Attempts to confirm that inhibiting these targets one by one is
therapeutically valuable have been disappointing. Indeed, obtaining
therapeutic effectiveness is proving to be extremely challenging,
probably because of multiple levels of redundancy in most signaling
pathways. For example, many disorders, such as cancer, type 2
diabetes, and atherosclerosis, feature multiple biochemical
abnormalities. In addition, some putative targets may be subject to
enhanced mutation rates, thereby negating the effects of
interfering RNAs on any such target.
[0007] For example, therapeutic approaches to viral infections
continue to be major challenges in agriculture, as well as in
animal and human health. The nature of the replication of viruses
makes them highly plastic, "moving targets" therapeutically-capable
of altering structure, infectivity, and host profile. The recent
emergence of viruses such as Severe Acute Respiratory Syndrome
("SARS") and Avian Influenza Virus ("bird flu") exemplify these
challenges. Even well-described viruses such as those involved in
Acquired Immunodeficiency Syndrome or AIDS (e.g. Human
Immunodeficiency Viruses, HIV-1 and HIV-2), continue to defy
efforts at treatment and vaccination because of on-going viral
mutation and evolution.
[0008] Furthermore, although nucleic acid therapeutics such as
interfering RNAs are candidates for viral therapy, in part because
modern rapid gene sequencing techniques allow viral genome
sequences to be determined even before any encoded functions can be
assessed, the error-prone replication of viruses, particularly RNA
viruses, means that substantial genomic diversity can arise rapidly
in an infected population. Thus far, strategies for the development
of nucleic acid therapeutics have largely centered on the targeting
of highly-conserved regions of the viral genome. It is unclear
whether these constructs are efficient at treating viral infection
or preventing emergence of resistant viral clones.
[0009] Therapeutic approaches that involve the design and use of
one interfering RNA for control of several key "drivers" of the
disease are thus desirable. Therefore, there is a need for
interfering RNAs which can target multiple pre-selected target
sequences within one or more target genes to modulate expression of
the targets. Methods for the design and for making such therapeutic
multitargeting interfering RNAs are also needed. Antiviral
interfering RNAs that can be developed rapidly upon the isolation
and identification of new viral pathogens and that can be used to
help slow, or even prevent, the emergence of new, resistant
isotypes are also needed.
SUMMARY OF THE INVENTION
[0010] Interfering RNA molecules are now designed and produced with
specificity for multiple binding sequences present in distinct
genetic contexts in one or more pre-selected target RNA molecules
and are used to decrease expression of the target sequences.
[0011] In a first embodiment, this invention relates to a
multitargeting interfering RNA molecule comprising a guide strand
of the Formula (I):
5'-p-XSY-3'
[0012] wherein p consists of a terminal phosphate group that is
independently present or absent; wherein S consists of a first
nucleotide sequence of a length of about 5 to about 20 nucleotides
that is at least partially complementary to a first portion of each
of at least two binding sequences present in distinct genetic
contexts in one or more pre-selected target RNA molecules; wherein
X is absent or consists of a second nucleotide sequence; wherein Y
is absent or consists of a third nucleotide sequence, provided that
X and Y are not absent simultaneously; wherein XSY is at least
partially complementary to each of said binding sequences to allow
a stable interaction therewith. Preferably S is completely
complementary to the first portion of each of at least two binding
sequences and also preferably, the first portion of each of at
least two binding sequences is a seed sequence. X can consist of
one or two nucleotides and Y can independently be at least
partially complementary to a second portion of each of the binding
sequences, said second portion is adjacent to and connected with
the 5'-end of said first portion of the binding sequences. Also
preferably, S is of a length of about 8 to about 15 nucleotides.
XSY is preferably of a length of about 17 to about 25 nucleotides.
Preferably, the multitargeting interfering RNA molecules of this
invention further comprise a passenger strand that is at least
partially complementary to the guide strand to allow formation of a
stable duplex between the passenger strand and the guide strand and
these RNA molecules preferably include one or more terminal
overhangs and these overhangs preferably are of between 1 to 5
nucleotides. Preferably the passenger strand and the guide strand
are completely complementary to each other. It is possible for the
multitargeting interfering RNA molecules of this invention to
target binding sequences present in distinct genetic contexts in
one or alternatively in at least 2 pre-selected target RNA
molecules. Preferably at least one of the pre-selected target RNA
molecules is a non-coding RNA molecule. Alternatively, at least one
of the pre-selected target RNA molecules can be a messenger RNA
molecule and preferably one or more of the pre-selected target RNA
molecules are involved in a disease or disorder. Preferably, the
disease is a human disease. Also preferably, in the multitargeting
interfering RNA molecules of this invention, at least one of the
binding sequences is present in the 3'-untranslated region (3'UTR)
of a messenger RNA molecule.
[0013] In this embodiment, preferably one or more of the
pre-selected target RNA molecules encode a protein of a class
selected from the group consisting of receptors, cytokines,
transcription factors, regulatory proteins, signaling proteins,
cytoskeletal proteins, transporters, enzymes, hormones, and
antigens. Preferably, the one or more of the pre-selected target
RNA molecules encode a protein selected from the group consisting
of ICAM-1, VEGF-A, MCP-1, IL-8, VEGF-B, IGF-1, Gluc6p, Inppl1,
bFGF, PlGF, VEGF-C, VEGF-D, .beta.-catenin, .kappa.-ras-B,
.kappa.-ras-A, EGFR, Bcl-2, presenilin-1, BACE-1, MALAT-1, BIC,
TGF.beta., and TNF alpha. Also preferably, the multitargeting
interfering RNA molecule decreases expression of any combination of
VEGF-A, .kappa.-ras and Bcl-2 in an expression system or
alternatively decreases expression of both MALAT-1 and BIC in an
expression system or still alternatively decreases expression of
both ICAM-1 and VEGF-A in an expression system. Other
multitargeting interfering RNA molecules decrease expression of
both TGF.beta. and IL-8 in an expression system or alternatively
decrease expression of both IL-8 and MCP-1 in an expression system.
The multitargeting interfering RNA molecules of this invention can
also further be manufactured to comprise a guide strand that forms
stable interactions with at least two binding sequences present in
distinct genetic contexts in one or more pre-selected target RNA
molecules.
[0014] Preferably one or more of the pre-selected target RNA
molecules is viral RNA. The virus is preferably selected from the
group consisting of a human immunodeficiency virus (HIV), a
hepatitis C virus (HCV), an influenza virus, a rhinovirus, and a
severe acute respiratory syndrome (SARS) virus. Where the virus is
HIV, one or more of the pre-selected target RNA molecules
preferably encode an essential protein selected from the group
consisting of GAG, POL, VIF, VPR, TAT, NEF, REV, VPU and ENV. Where
the virus is HCV, one or more of the other preselected RNA
molecules encodes TNF.alpha..
[0015] In the multitargeting interfering RNA molecules of this
invention, the molecules preferably comprise at least one modified
ribonucleotide, universal base, acyclic nucleotide, abasic
nucleotide, non-ribonucleotide or combinations thereof. In other
aspects of this embodiment, S consists essentially of a nucleotide
sequence selected from the group consisting of:
TABLE-US-00001 UAUGUGGGUGGG, (SEQ ID NO: 1) UGUUUUG, (SEQ ID NO: 2)
ACCCCGUCUCU, (SEQ ID NO: 5) AGCUGCA, (SEQ ID NO: 7) AAACAAUGGAAUG,
(SEQ ID NO: 8) GGUAGGUGGGUGGG, (SEQ ID NO: 10) CUGCUUGAU, (SEQ ID
NO: 12) UCCUUUCCA, (SEQ ID NO: 13) UUUUUCUUU, (SEQ ID NO: 14)
UUCUGAUGUUU, (SEQ ID NO: 15) UCUUCCUCUAU, (SEQ ID NO: 16)
UGGUAGCUGAA, (SEQ ID NO: 17) CUUUGGUUCCU, (SEQ ID NO: 18)
CUACUAAUGCU, (SEQ ID NO: 19) UCCUGCUUGAU, (SEQ ID NO: 20)
AUUCUUUAGUU, (SEQ ID NO: 21) CCAUCUUCCUG, (SEQ ID NO: 22)
CCUCCAAUUCC, (SEQ ID NO: 23) CUAAUACUGUA, (SEQ ID NO: 24)
UUCUGUUAGUG, (SEQ ID NO: 25) GCUGCUUGAUG, (SEQ ID NO: 26)
ACAUUGUACUG, (SEQ ID NO: 27) UGAUAUUUCUC, (SEQ ID NO: 28)
AACAGCAGUUG, (SEQ ID NO: 29) GUGCUGAUAUU, (SEQ ID NO: 30)
CCCAUCUCCAC, (SEQ ID NO: 31) UAUUGGUAUUA, (SEQ ID NO: 32)
CAAAUUGUUCU, (SEQ ID NO: 33) UACUAUUAAAC, (SEQ ID NO: 34)
GCCUAUCAUAU, (SEQ ID NO: 58) UGGUGCCUGCU, (SEQ ID NO: 59)
AAUUAAUAUGGC, (SEQ ID NO: 60) CCCUCUGGGCU, (SEQ ID NO: 61)
UUCUUCCUCAU, (SEQ ID NO: 62) UAUUUAUACAGA, (SEQ ID NO: 63)
CACCAAAAUUC, (SEQ ID NO: 64) UGAGUNNGAACAUU (SEQ ID NO: 72) where N
is any base, CUCCAGG, (SEQ ID NO: 74) UCAGUGGG, (SEQ ID NO: 76)
UCCUCACAGGG, (SEQ ID NO: 78) GUGCUCAUGGUG, (SEQ ID NO: 79)
CCUGGAGCCCUG, (SEQ ID NO: 80) UCUCAGCUCCAC, (SEQ ID NO: 81)
ACCCUCGCACC, (SEQ ID NO: 86) GUGUUGAAG, (SEQ ID NO: 88) UUCCACAAC,
(SEQ ID NO: 90) UCCACUGUC, (SEQ ID NO: 92) CAGAAUAG, (SEQ ID NO:
93) AACUCUCUA (SEQ ID NO: 94) and CGUGAAGAC. (SEQ ID NO: 98)
[0016] In other aspects, S consists essentially of a nucleotide
sequence selected from the group consisting of: UAUGUGGGUGGG (SEQ
ID NO: 1), UCCUCACAGGG (SEQ ID NO: 78), GUGUUGAAG (SEQ ID NO: 88),
UUCCACAAC (SEQ ID NO: 90), AACUCUCUA (SEQ ID NO: 94) and CGUGAAGAC
(SEQ ID NO: 98). Preferably, S consists essentially of a nucleotide
sequence of 6 or more contiguous bases contained within any of the
sequences selected from the group consisting of: UAUGUGGGUGGG (SEQ
ID NO: 1), UCCUCACAGGG (SEQ ID NO: 78), GUGUUGAAG (SEQ ID NO: 88),
UUCCACAAC (SEQ ID NO: 90), AACUCUCUA (SEQ ID NO: 94) and CGUGAAGAC
(SEQ ID NO: 98).
[0017] In yet other aspects, the multitargeting interfering RNA
molecule consists essentially of:
TABLE-US-00002 5' UUCCUCACAGGGCAGUGAUUC 3' (SEQ ID NO: 122) 3'
UUAAAGAGUGUCCCGUCACUA 5', (SEQ ID NO: 124) 5' UACAAAUCUACUUCAACAUUU
3' (SEQ ID NO: 131) 3' GUAUGUUUAGAUGAAGUUGUG 5', (SEQ ID NO: 132)
or 5' AACAUAUGUUCUUCAACAUUU 3' (SEQ ID NO: 133) 3'
GUUUGUAUACAAGAAGUUGUG 5', (SEQ ID NO: 134)
Other exemplary multitargeting interfering RNA molecules
include:
TABLE-US-00003 5' UAUGUGGGUGGGUGAGUCUAA 3' (SEQ ID NO: 100) 3'
UUAUACACCCACCCACUCAGA 5', (SEQ ID NO: 101) 5' UGUUUUGUUGUUACAUAUGAC
3' (SEQ ID NO: 102) 3' UUACAAAACAACAAUGUAUAC 5', (SEQ ID NO: 103)
5' UAUGUGGGUGGGGUGUCUCUA 3' (SEQ ID NO: 104) 3'
UUAUACACCCACCCCACAGAG 5', (SEQ ID NO: 105) 5' UAUGUGGGUGGGGUGGUCUAA
3' (SEQ ID NO: 106) 3' UUAUACACCCACCCCACCAGA 5', (SEQ ID NO: 107)
5' UAUGUGGGUGGGGUGGUGUCU 3' (SEQ ID NO: 108) 3'
UUAUACACCCACCCCACCACA 5', (SEQ ID NO: 109) 5' UAUGUGGGUGGGUGAGUGUCU
3' (SEQ ID NO: 110) 3' UUAUACACCCACCCACUCACA 5', (SEQ ID NO: 111)
5' CUCACCCACCCACAUACAUUU 3' (SEQ ID NO: 112) 3'
CUGAGUGGGUGGGUGUAUGUA 5', (SEQ ID NO: 113) 5' UCACCCACCCACAUACAUAUU
3' (SEQ ID NO: 114) 3' UGAGUGGGUGGGUGUAUGUAU 5', (SEQ ID NO: 115)
5' UCACCCACCCACAUACAUUUU 3' (SEQ ID NO: 116) 3'
UGAGUGGGUGGGUGUAUGUAA 5', (SEQ ID NO: 117) 5' UAUGUGGGUGGGUGAGUCUA
3' (SEQ ID NO: 118) 3' UAUACACCCACCCACUCAGA 5', (SEQ ID NO: 119) 5'
GGGUUUACCAGGAAGAUGGUU 3' (SEQ ID NO: 120) 3' UACCCAAAUGGUCCUUCUACC
5', (SEQ ID NO: 121) 5' UUCCUCACAGGGCAGUGAUUC 3' (SEQ ID NO: 122)
3' UUAAGGAGUGUCCCGUCACUA 5', (SEQ ID NO: 123) 5'
UUCCUCACAGGGCAGUGGUUC 3' (SEQ ID NO: 125) 3' UUAAGGAGUGUCCCGUCACCA
5', (SEQ ID NO: 126) 5' CCCGGACCCUUAGAGAGUUUU 3' (SEQ ID NO: .127)
3' ACGGGCCUGGGAAUCUCUCAA 5', (SEQ ID NO: 128) 5'
UACCCUCGCACCGAUCUCCCAA 3' (SEQ ID NO: 129) 3'
UUAUGGGAGCGUGGCUAGAGGG 5', (SEQ ID NO: 130) 5'
UUCCACAACACAAGCUGUGUU 3' (SEQ ID NO: 135) 3' UUAAGGUGUUGUGUUCGACAC
5', (SEQ ID NO: 136) 5' GGACCCUUAGAGAGUUUCAUU 3' (SEQ ID NO: 137)
3' GGCCUGGGAAUCUCUCAAAGU 5', (SEQ ID NO: 138) 5'
UUCGUGAAGACGGUGGGCCGA 3' (SEQ ID NO: 139) 3'
dTdTAAGCACUUCUGCCACCCGG 5', (SEQ ID NO: 140) or 5'
AGACUCACCCACCCAGAUAUU 3' (SEQ ID NO: 141) 3' AAUCUGAGUGGGUGGGUCUAU
5' (SEQ ID NO: 142)
Yet others include:
TABLE-US-00004 5' UAUGUGGGUGGGUGAGUCUAA 3' (SEQ ID NO: 100) 3'
UUAUACACCCACCCACUCAGA 5', (SEQ ID NO: 101) 5' GGACCCUUAGAGAGUUUCAUU
3' (SEQ ID NO: 137) 3' GGCCUGGGAAUCUCUCAAAGU 5', (SEQ ID NO: 138)
or 5' UUCGUGAAGACGGUGGGCCGA 3' (SEQ ID NO: 139) 3'
dTdTAAGCACUUCUGCCACCCGG 5', (SEQ ID NO: 140)
[0018] Preferably the above multitargeting interfering RNA
molecules also include at least one modified ribonucleotide,
universal base, acyclic nucleotide, abasic nucleotide and
non-ribonucleotide, overhang variation or a combination
thereof.
[0019] In another aspect of this invention, the invention relates
to a biological system comprising the multitargeting interfering
RNA molecules of this invention and those preferred biological
systems include a virus, a microbe, a cell, a plant, or an animal.
Vectors comprising a nucleotide sequence that encodes the
multitargeting interfering RNA molecules of this invention are also
contemplated. Preferred vectors are viral vectors. Preferred viral
vectors are selected from the group consisting of an
adeno-associated virus, a retrovirus, an adenovirus, a lentivirus,
and an alphavirus. The invention also relates to cells comprising
the vectors of this invention.
[0020] The multitargeting interfering RNA molecules of this
invention can also be short hairpin RNA molecules.
[0021] The invention further relates to pharmaceutical compositions
comprising the multitargeting interfering RNA molecules of this
invention and an acceptable carrier. Alternatively, the composition
can include a vector comprising the RNA molecule and an acceptable
carrier.
[0022] The invention further relates to methods of using the
multitargeting interfering RNA molecules of this invention. In a
preferred method for using the multitargeting interfering RNA
molecules of this invention, the method includes inducing RNA
interference in a biological system, comprising the step of
introducing a multitargeting interfering RNA molecule of this
invention into the biological system. More specifically, the
invention relates to methods of inducing RNA interference in a
biological system, comprising the steps of: (a) selecting one or
more target RNA molecules; (b) designing a multitargeting
interfering RNA molecule comprising a guide strand that can form
stable interactions with at least two binding sequences present in
distinct genetic contexts in the set of one or more target RNA
molecules; (c) producing the multitargeting interfering RNA
molecule; and (d) administering the multitargeting interfering RNA
molecule into the biological system, whereby the guide strand of
the multitargeting interfering RNA molecule forms stable
interactions with the binding sequences present in distinct genetic
contexts in the target RNA molecules, and thus induces RNA
interference of the target RNA molecules. Preferably the biological
system is a virus, a microbe, a cell, a plant, or an animal.
Preferred animals include rats, mice, monkeys, and humans. Also
preferably the target RNA molecules comprise RNA molecules that are
involved in a disease or disorder of the biological system or are
selected from the biological system. Alternatively, the target RNA
molecules comprises one or more RNA molecules selected from a
second biological system that is infectious to the biological
system or where the target RNA molecules are selected from both a
first biological system and a second biological system that is
infectious to the first biological system. For example, the target
RNA molecules can comprise one or more RNA molecules selected from
an animal or a plant and one or more RNA molecules selected from a
microbe or a virus that is infectious to the animal or the plant.
As a more specific example, the target RNA molecules can comprise
one or more RNA molecules selected from a human and one or more RNA
molecules selected from a virus selected from the group consisting
of a human immunodeficiency virus (HIV), a hepatitis C virus (HCV),
an influenza virus, a rhinovirus, and a severe acute respiratory
syndrome (SARS) virus. The target RNA molecules can also be RNA
molecules encoding a protein of a class of proteins, including,
without limitation, receptors, cytokines, transcription factors,
regulatory proteins, signaling proteins, cytoskeletal proteins,
transporters, enzymes, hormones, and antigens. For example groups
of proteins can include ICAM-1, VEGF-A, MCP-1, IL-8, VEGF-B, IGF-1,
Gluc6p, Inppl1, bFGF, PlGF, VEGF-C, VEGF-D, .beta.-catenin,
.kappa.-ras-B, .kappa.-ras-A, EGFR, Bcl-2, presenilin-1, BACE-1,
MALAT-1, BIC, TGF.beta., and TNF alpha. Preferred combinations
include, for example, ICAM-1 and VEGF-A, one or more viral RNA
molecules such as human immunodeficiency virus (HIV), a hepatitis C
virus (HCV), an influenza virus, a rhinovirus, and a severe acute
respiratory syndrome (SARS) virus or an essential protein for HIV
selected from the group consisting of GAG, POL, VIF, VPR, TAT, NEF,
REV, VPU and ENV. The target RNA molecules can also be selected
from human proteins including, for example, TNFalpha, LEDGF(p75),
BAF, CCR5, CXCR4, furin, NFkB, STAT1. Specific combinations include
a virus protein and a human protein associated with the disease
caused by that virus. So, as one example, preselected target RNA
molecules can comprise Hepatitis C Virus (HCV) and one or more of
the other preselected RNA molecules encodes TNF.alpha..
[0023] This invention also relates to methods for treating a
disease or condition in a subject, the method comprising the steps
of: (a) selecting one or more target RNA molecules, wherein the
modulation in expression of the target RNA molecules is potentially
therapeutic for the treatment of the disease or condition; (b)
designing a multitargeting interfering RNA molecule comprising a
guide strand that can form stable interactions with at least two
binding sequences present in distinct genetic contexts in the one
or more target RNA molecules; (c) producing the multitargeting
interfering RNA molecule; and (d) administering the multitargeting
interfering RNA molecule into the subject, whereby the guide strand
of the multitargeting interfering RNA molecule forms stable
interactions with the binding sequences present in distinct genetic
contexts in the one or more target RNA molecules, and thus induces
RNA interference and modulation of expression of the target RNA
molecules.
[0024] The invention also relates to methods for designing a
multitargeting interfering RNA molecule, comprising the steps of:
a) selecting one or more target RNA molecules, wherein modulation
in expression of the target RNA molecules is desired; b) obtaining
at least one nucleotide sequence for each of the target RNA
molecules; c) selecting a seed sequence of 6 nucleotides or more,
wherein said seed sequence occurs in at least two distinct genetic
contexts in nucleotide sequences identified in step b) for the
target RNA molecules; d) selecting at least two binding sequences,
wherein each of binding sequences comprises the seed sequence, and
the binding sequences are present in distinct genetic contexts in
the target molecules; and e) designing a multitargeting interfering
RNA molecule having a guide strand that shares a substantial degree
of complementarity with each of the at least two binding sequences
to allow stable interaction therewith. Preferably the method
further comprises designing a passenger strand that is at least
partially complementary to the guide strand to allow formation of a
stable duplex between the passenger strand and the guide
strand.
[0025] In yet another method for designing a multitargeting
interfering RNA molecule, the method comprises the steps of: a)
selecting one or more target RNA molecules, wherein modulation in
expression of the target RNA molecules is desired; b) identifying
at least one nucleotide sequence for each of the target RNA
molecules; c) selecting a length, n, in nucleotides, for a seed
sequence; wherein n=about 6 or more; d) generating a collection of
candidate seed sequences of the length n from each nucleotide
sequence identified in step b), wherein each candidate seed
sequence occurs at least once in nucleotide sequences obtained in
step b); e) determining the genetic context of each of the
candidate seed sequences in each nucleotide sequence obtained in
step b), by collecting, for each occurrence of the candidate seed
sequence, a desired amount of the 5' and 3' flanking sequence; f)
selecting a seed sequence of the length n from the candidate seed
sequences, wherein the seed sequence occurs at least in two
distinct genetic contexts in nucleotide sequences identified in
step b); g) selecting a consensus target sequence, wherein said
consensus target sequence comprises the seed sequence and a desired
consensus sequence for the sequence flanking either one or both of
the 5' and 3' ends of the seed; and h) designing a multitargeting
interfering RNA molecule comprising a guide strand that shares a
substantial degree of complementarity with the consensus target
sequence to allow stable interaction therewith. Preferably the step
of generating a collection of candidate seed sequences comprises
the steps of beginning at a terminus, sequentially observing the
nucleotide sequence using a window size of n and stepping along the
nucleotide sequence with a step size of 1. Also preferably, the
step of selecting seed sequences comprises the step of discarding
any sequence of the length n that i) is composed of a consecutive
string of 5 or more identical single nucleotides; ii) is composed
of only adenosine and uracil; iii) is predicted to occur with an
unacceptably high frequency in the non-target transcriptome of
interest; iv) is predicted to have a propensity to undesirably
modulate the expression or activity of one or more cellular
component; or v) is any combination of i) to iv). Optionally, steps
c) to g) can be repeated with a new value of n. The multitargeting
interfering RNA molecules, once designed can then be tested in an
expression system.
[0026] In preferred methods for designing a multitargeting
interfering RNA molecule, the step of selecting a consensus target
sequence further comprises the step of discarding any sequence that
is composed of only a single base, is composed only of A and U, has
a consecutive string of 5 or more bases which are C, is G/C rich at
the 3' end, is predicted to occur with unacceptable frequency in
the non-target transcriptome of interest; or any combination
thereof. Also preferably the passenger strand is designed so that
it is at least partially complementary to the guide strand to allow
formation of a stable duplex between the passenger strand and the
guide strand. Once the multitargeting interfering RNA molecule is
designed, it is contemplated that it can be modified. Such
modifying methods are also contemplated within the scope of the
invention and a preferred method comprises modifying the
multitargeting interfering RNA molecule comprising the steps alone
or in combination of i) improving the incorporation of the guide
strand of the multitargeting interfering RNA molecule into the RNA
induced silencing complex (RISC); ii) increasing or decreasing the
modulation of the expression of at least one target RNA molecule;
iii) decreasing stress or inflammatory response when the
multitargeting interfering RNA molecule is administered into a
subject; iv) altering the half life of the multitargeting RNA
molecule in an expression system.
[0027] The invention further relates to a method of designing a
full length multitargeting interfering RNA from a seed sequence,
comprising the steps of: a) deducing the sequence of the complete
complement of the seed sequence; b) generating permutations for the
extension of the complete complement of the seed sequence to the
desired length n; c) creating a collection of putative guide strand
sequences, each of which comprises the sequence of the complete
complement of the seed sequence and one of the permutations
generated in step b); d) using RNAhybrid to determine the binding
pattern and the minimum free energy (mfe) of the putative guide
strand sequences created in step c) against all the target
sequences comprising the seed sequence; e) discarding putative
guide strand sequences where i) there is a contiguous run of 5 or
more G residues; and ii) the Load Bias is <1.2; and f) selecting
a guide strand sequence of the length n for a multitargeting
interfering RNA sequence from the list of the remaining putative
guide strand sequences based on their Relative Activity Score. The
method preferably can additionally comprise the steps of producing
the multitargeting interfering RNA comprising the guide strand
sequence and testing the multitargeting interfering RNA in an
expression system.
[0028] In yet another aspect, the invention relates to a method of
making a multitargeting interfering RNA molecule, comprising the
steps of: i) designing a multitargeting interfering RNA molecule
having a guide strand that can form stable interactions with at
least two binding sequences present in distinct genetic contexts in
a set of pre-selected target RNA molecules; and ii) producing the
multitargeting interfering RNA molecule.
[0029] It is also contemplated within the scope of this invention
that the invention further comprises a pharmaceutical composition
comprising a therapeutically effective amount of one or more
multitargeting interfering RNA molecules together with a
pharmaceutically acceptable carrier.
[0030] Other aspects, features and advantages of the invention will
be apparent from the following disclosure, including the detailed
description of the invention and its preferred embodiments and the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1: A flow-chart highlighting aspects of an exemplary
design process for multitargeting interfering RNAs (CODEMIRs or
VIROMIRs).
[0032] FIG. 2: Graph showing cytotoxicity of control siRNA and
CODEMIRs at a final concentration of 40 nM in ARPE-19 cells in
culture medium at 48 hrs post-transfection. A: mock transfected
cells (Lipofectamine2000 alone); B: irrelevant siRNA control; C:
VEGF-specific siRNA; D: ICAM-specific siRNA; E: CODEMIR-1; F:
CODEMIR-2.
[0033] FIG. 3: Graph showing VEGF (closed bars) and ICAM (open
bars) protein expression as a percent of control (untransfected)
cells in cells treated with various siRNAs and CODEMIRs. A:
Unstimulated cells; B: Untransfected, stimulated cells; C:
Irrelevant control siRNA; D: ICAM-specific siRNA; E: VEGF-specific
siRNA; F: CODEMIR-1; G: CODEMIR-2.
[0034] FIG. 4: Comparison of CODEMIR 1 activity with that of a
naturally-occurring microRNA with some sequence similarity. A:
Untransfected cells; B: Irrelevant siRNA; C: CODEMIR-1; D:
synthetic miR-299 (CODEMIR-84).
[0035] FIG. 5: Example of a VIROMIR targeting two sites in the same
target RNA, in this case, the HIV genome. Top row discloses SEQ ID
NOs: 559 and 560, respectively, in order of appearance. CODEMIR
guide disclosed as SEQ ID NO: 561.
[0036] FIG. 6: Tolerance of CODEMIRs for mismatches at the 5'
extremity for activity against VEGF (closed bars) and ICAM-1 (open
bars) expression. A: Untransfected cells; B: Irrelevant siRNA; C:
ICAM- and VEGF-specific siRNAs; D: CODEMIR-13; E: CODEMIR-14; F:
CODEMIR-15. The presence of a single mismatch at the 5'extremity of
the seed did not significantly reduce the activities of CODEMIRs-14
and -15 relative to the fully matched seed of CODEMIR-13 (see Table
8-2 for sequences).
[0037] FIG. 7: Production of p24 HIV capsid protein in HEK-293
cells co-transfected with pNL4.3 plasmid and 67 ng of either pSIL
vector (control) or pSIL vector encoding sequences for shRNA
approximating VM004, VM006 or VM010. A: Control (empty) vector; B:
VM004 shRNA construct; C: VM006 shRNA construct; D: VM010 shRNA
construct.
[0038] FIG. 8: Survival of HCT116 cells with and without serum
withdrawal following transfection with 40 nM CC014-21 and siRNA
controls. A: Mock-transfected cells; B: Irrelevant control siRNA
(siGC47); C: cytotoxic transfection siRNA control (siTOX); D:
Bcl-2-specific siRNA; E: Kras-specific siRNA; F: VEGF-specific
siRNA (PVE); G: CC014; H: CC015; I: CC016; J: CC017; K: CC018; L:
CC019; M: CC020 and N: CC021.
[0039] FIG. 9: Abundance of K-Ras in HCT116 cells following
transfection with 40 nM CC014-21 as measured by Western and
normalized to beta-actin. A: Mock-transfected cells; B: CC014; C:
CC015; D: CC016; E: CC017; F: CC018; G: CC019; H: CC020; I: CC021;
J: Kras-specific siRNA.
[0040] FIG. 10: Secretion of VEGF by HCT116 cells 48 h
post-transfection with 40 nM CC014-21. VEGF in the cell medium was
measured by ELISA. PVE is a VEGF-specific siRNA active in several
species (human and rodent). A: Mock-transfected cells; B:
Irrelevant control siRNA; C: VEGF-specific siRNA (PVE); D:
Kras-specific siRNA; E: Bcl-2-specific siRNA; F: CC014; G: CC015;
H: CC016; I: CC017; J: CC018; K: CC019; L: CC020; M: CC021.
[0041] FIG. 11: Positivity for Annexin V (early apoptosis) and
Propidium Iodide (necrosis/late apoptosis) staining of HCT116 cells
48 hr following transfection with 40 nM CC014-18 and controls. A:
Untreated cells; B: Mock-transfected cells; C: Irrelevant control
siRNA (siGC47); D: cytotoxic transfection control siRNA (siTOX); E:
VEGF-specific siRNA; F: Bcl-2-specific siRNA; G: Kras-specific
siRNA; H: CC014; I: CC015; J: CC016; K: CC017 and L: CC018.
[0042] FIG. 12: Colony forming ability of HCT116 cells following
transfection with 40 nM siRNAs, CC015 and CC018-21. A: Untreated
cells; B: Irrelevant control siRNA C: VEGF-specific siRNA; D:
Kras-specific siRNA; E: Bcl-2-specific siRNA; F: CC015; G: CC018;
H: CC019; I: CC020 and J: CC021.
[0043] FIG. 13: VEGF Secretion by ARPE-19 cells transfected with 40
nM 2'-F modified CODEMIR-1 analogs. VEGF production was measured by
ELISA of the cell culture supernatant. Each point represents the
mean of triplicate wells; error bars indicate standard deviation of
the mean. A: Untransfected cells; B: Mock-transfected cells; C:
Irrelevant siRNA control; D: CODEMIR-1; E: CODEMIR-33; F:
CODEMIR-87; G: CODEMIR-92; H: CODEMIR-144; I: CODEMIR-145; J:
CODEMIR-165; K: CODEMIR-166 and L: CODEMIR-167.
[0044] FIG. 14: Guide strand terminal modifications of CODEMIR-1
tested in vitro (see FIG. 15 for data), "Oligo" refers to the
active guide strand of CODEMIR-1. A: CODEMIR-146; B: CODEMIR-147;
C: CODEMIR-148; D: CODEMIR-149; E: CODEMIR-150; F: CODEMIR-151; G:
CODEMIR-152; H: CODEMIR-153; I: CODEMIR-154; J: CODEMIR-155 and K:
CODEMIR-156.
[0045] FIG. 15: VEGF secretion by ARPE-19 cells transfected with 10
nM of each terminal conjugated variant of CODEMIR-1. VEGF in cell
culture supernatant was measured by ELISA. Each point represents
the mean of triplicate wells; error bars indicate standard
deviation of the mean. A: Untransfected cells; B: Mock-transfected
cells; C: Irrelevant siRNA control; D: CODEMIR-1; E: CODEMIR-146;
F: CODEMIR-147; G: CODEMIR-148; H: CODEMIR-149; I: CODEMIR-150; J:
CODEMIR-151; K: CODEMIR-152; L: CODEMIR-153; M: CODEMIR-154; N:
CODEMIR-155 and O: CODEMIR-156.
[0046] FIG. 16: Time course of VEGF suppression by CODEMIR-1. Cells
were transfected once on day 0 with 40 nM dsRNA and repeatedly
stimulated with deferroxamine. Supernatant was collected at the
indicated time points and assayed by ELISA. Error bars indicate
standard deviation of the mean. Squares: Untransfected cells;
Triangles: Mock-transfected; Inverted triangles: Irrelevant siRNA
control and Diamonds: CODEMIR-1.
[0047] FIG. 17: Stability of chemically modified variants of
CODEMIR-1 in human serum. 100 nM Duplex RNA was incubated at
37.degree. C. in 10% human AB serum, with RNA concentration
monitored using Oligreen dye. Each point is the mean of triplicate
samples. Error bars (in many cases smaller than symbols) indicate
standard deviation of the mean. Solid squares: CODEMIR-1;
Triangles: CODEMIR-33; Inverted triangles: CODEMIR-87; Diamonds:
CODEMIR-92; Circles: CODEMIR-144 and Open squares: CODEMIR-145.
[0048] FIG. 18: VEGF (closed bars) and ICAM (open bars) secretion
by ARPE-19 cells transfected with chemically modified variants of
CODEMIR-1. ARPE-19 cells were transfected with 40 nM duplex RNA and
VEGF or ICAM secretion was assayed 48 hours post-transfection by
ELISA. Each bar represents the mean of triplicate samples. Error
bars indicate standard deviation of the mean. In 1 (top panel)-A:
Untransfected cells; B: Mock-transfected cells; C: Irrelevant siRNA
control; D: CODEMIR-1; E: CODEMIR-33; F: CODEMIR-87 and G:
CODEMIR-92. In 2 (bottom panel)-A: Untransfected cells; B:
Mock-transfected cells; C: Irrelevant siRNA control; D: CODEMIR-1;
E: CODEMIR-87; F: CODEMIR-144; G: CODEMIR-33 and H:
CODEMIR-145.
[0049] FIG. 19: Effect of mismatches in the seed region of
CODEMIR-1 upon VEGF (open bars) and ICAM (closed bars) suppressive
activity. ARPE-19 cells were transfected with 40 nM duplex RNA and
VEGF (ELISA) or ICAM (FACS) assayed and expressed relative to
results obtained with untransfected cells. Expression was assayed
48 hours post-transfection. Each bar represents the mean of
triplicate samples. Error bars indicate standard deviation of the
mean. A: Untransfected cells; B: Mock-transfected; C: Irrelevant
siRNA control; D: CODEMIR-1; E: CODEMIR-122; F: CODEMIR-123 and G:
CODEMIR-124.
[0050] FIG. 20: Schematic illustration of the design of all 32
variants of CODEMIR-1 that are directly aligned to the VEGF (SEQ ID
NO: 563) and ICAM (SEQ ID NO: 562) mRNAs. The sequence alignment of
two target sequences is shown. A potential guide strand (CODEMIR)
is shown below the sequence alignment (SEQ ID NO: 452). Because of
the lack of total consensus between the two targets, two possible
bases can be used at some positions (alternate bases at mismatch
positions are shown in the row indicated with an arrow). Note the
possible exemplary use of a U to match and wobble-pair the two
target sequences, respectively.
[0051] FIG. 21: Screening 32 variants of CODEMIR-1 for VEGF
suppressive activity. ARPE-19 cells were transfected with 40 nM of
the indicated RNA duplex, and VEGF secretion was measured by ELISA
48 hours post-transfection. The guide strand of each CODEMIR is
shown in the 5' to 3' direction. Each bar represents the mean of
triplicate samples. Error bars indicate standard deviation of the
mean. A: Untransfected; B: Irrelevant siRNA control; C: CODEMIR-1;
D: CODEMIR-52; E: CODEMIR-53; F: CODEMIR-54; G: CODEMIR-55; H:
CODEMIR-56; I: CODEMIR-57; J: CODEMIR-58; K: CODEMIR-59; L:
CODEMIR-60; M: CODEMIR-61; N: CODEMIR-62; O: CODEMIR-63; P:
CODEMIR-64; Q: CODEMIR-65; R: CODEMIR-66; S: CODEMIR-67; T:
CODEMIR-68; U: CODEMIR-69; V: CODEMIR-70; W: CODEMIR-71; X:
CODEMIR-72; Y: CODEMIR-73; Z: CODEMIR-74; AA: CODEMIR-75; BB:
CODEMIR-76; CC: CODEMIR-77; DD: CODEMIR-78; EE: CODEMIR-79; FF:
CODEMIR-80; GG: CODEMIR-81; HH: CODEMIR-82; II: CODEMIR-83 and JJ:
CODEMIR-84.
[0052] FIG. 22: Relationship between target complementarity, length
of 5' complementarity and VEGF suppression for 32 variants of
CODEMIR-1. Open squares: CODEMIR-1 variants with 12 bases of
contiguous complementarity to the VEGF binding sequence; Triangles:
CODEMIR-1 variants with 14 bases of contiguous complementarity;
Inverted triangles: CODEMIR-1 variants with 17 bases of contiguous
complementarity; Open diamonds: CODEMIR-1 variants with 18 bases of
contiguous complementarity; Circles: CODEMIR-1 variants with 19
bases of contiguous complementarity and Solid squares: CODEMIR-1
variant (VAIC) with 21 bases of contiguous complementarity.
[0053] FIG. 23: Screening 31 variants of CODEMIR-1 for suppression
of ICAM production. ARPE-19 cells were transfected with 40 nM of
the indicated RNA duplex, and sICAM secretion was measured by ELISA
48 hours post-transfection. The guide strand of each CODEMIR is
shown in the 5' to 3' direction. Each bar represents the mean of
triplicate samples. Error bars indicate standard deviation of the
mean. A: Untransfected; B: Irrelevant siRNA control; C: CODEMIR-1;
D: CODEMIR-52; E: CODEMIR-53; F: CODEMIR-54; G: CODEMIR-55; H:
CODEMIR-56; I: CODEMIR-57; J: CODEMIR-58; K: CODEMIR-59; L:
CODEMIR-60; M: CODEMIR-61; N: CODEMIR-62; O: CODEMIR-63; P:
CODEMIR-64; Q: CODEMIR-65; R: CODEMIR-66; S: CODEMIR-67; T:
CODEMIR-68; U: CODEMIR-69; V: CODEMIR-70; W: CODEMIR-71; X:
CODEMIR-72; Y: CODEMIR-73; Z: CODEMIR-74; AA: CODEMIR-75; BB:
CODEMIR-76; CC: CODEMIR-77; DD: CODEMIR-78; EE: CODEMIR-79; FF:
CODEMIR-80; GG: CODEMIR-81; HH: CODEMIR-82; II: CODEMIR-83 and JJ:
ICAM-specific siRNA.
[0054] FIG. 24: Comparison of CODEMIR-1 variants with (CODEMIR-56
and CODEMIR-76) and without (CODEMIR-120 and CODEMIR-121) 7 G
motifs. ARPE-19 cells were transfected with 40 nM duplex RNA and
VEGF (open bars) or ICAM (closed bars) expression was assayed 48
hours post-transfection. Each bar represents the mean of triplicate
samples. Error bars indicate standard deviation of the mean. A:
Untransfected; B: Mock-transfected cells; C: Irrelevant siRNA
control; D: VEGF- and ICAM-specific siRNAs; E: CODEMIR-56; F:
CODEMIR-76; G: CODEMIR-120 and H: CODEMIR-121.
[0055] FIG. 25: VEGF and ICAM expression in ARPE-19 cells after
transfection with LNA and inosine containing CODEMIRs. ARPE-19
cells were transfected with 40 nM duplex RNA and VEGF (closed bars)
or ICAM (open bars) expression was assayed 48 hours
post-transfection. Each bar represents the mean of triplicate
samples. Error bars indicate standard deviation of the mean. A:
Untransfected; B: Mock-transfected cells; C: Irrelevant siRNA
control; D: CODEMIR-1; E: CODEMIR-99; F: CODEMIR-100; G:
CODEMIR-101 and H: CODEMIR-102.
[0056] FIG. 26: Comparison of VEGF suppressive activity of CODEMIRs
containing inosine bases or mismatches at positions 13 and/or 15 of
the guide strand. ARPE-19 cells were transfected with 10 nM duplex
RNA and VEGF (ELISA) expression was assayed 48 hours
post-transfection. Each bar represents the mean of triplicate
samples. Error bars indicate standard deviation of the mean. A:
Untransfected; B: Mock-transfected cells; C: Irrelevant siRNA
control; D: CODEMIR-1; E: CODEMIR-68; F: CODEMIR-69; G: CODEMIR-70;
H: CODEMIR-71; I: CODEMIR-100; J: CODEMIR-101 and K:
CODEMIR-102.
[0057] FIG. 27: VEGF production by ARPE-19 cells after transfection
with variants of CODEMIR-1 containing asymmetric bulges. ARPE-19
cells were transfected with 40 nM of the indicated RNA duplex, and
VEGF secretion was measured by ELISA 48 hours post-transfection.
Each bar represents the mean of triplicate samples. Error bars
indicate standard deviation of the mean. A: Untransfected; B:
Mock-transfected cells; C: Irrelevant siRNA control; D: CODEMIR-1;
E: CODEMIR-104; F: CODEMIR-105; G: CODEMIR-106 and H:
CODEMIR-107.
[0058] FIG. 28: Screening multiple seed sites in the VEGF 3' UTR.
ARPE-19 cells were transfected with 40 nM of the indicated RNA
duplex, and VEGF secretion was measured by ELISA 48 hours
post-transfection. Each bar represents the mean of triplicate
samples. Error bars indicate standard deviation of the mean. A:
Untransfected; B: Mock-transfected cells; C: Irrelevant siRNA
control; D: CODEMIR-1; E: CODEMIR-108; F: CODEMIR-109; G:
CODEMIR-110; H: CODEMIR-111; I: CODEMIR-112; J: CODEMIR-113; K:
CODEMIR-114; L: CODEMIR-115; M: CODEMIR-116; N: CODEMIR-117; O:
CODEMIR-118 and P: CODEMIR-119.
[0059] FIG. 29: Design of shRNA based on CODEMIR-1 (SEQ ID NO:
565). Sequence shown is 5' to 3' (upper strand). Sequences in bold
indicate the predicted active CODEMIR-1 duplex.
[0060] FIG. 30: Effect of CODEMIR-1 hairpins on VEGF and ICAM-1
expression. ARPE-19 cells were transfected with 2 .mu.g/ml of each
hairpin plasmid. VEGF (A) and ICAM (B) expression was determined 48
hours post-transfection. The open bars indicate results obtained
with the "empty" vector control and the closed bars are those
obtained with the shRNA constructed to approximate CODEMIR-1. Each
bar represents the mean of triplicate samples. Error bars indicate
standard deviation of the mean. A significant effect of the shRNA
approximating CODEMIR-1 was found for both targets (*=p<0.001;
**=p<0.05).
[0061] FIG. 31: Efficacy of a 1 bp overhang (CODEMIR-24) and
"blunt-ended" variant (CODEMIR-25) of CODEMIR-1 against VEGF
(closed bars) and ICAM-1 (open bars). A: Untransfected; B:
Mock-transfected cells; C: Irrelevant siRNA control; D: ICAM- and
VEGF-specific siRNAs; E: CODEMIR-1; F: CODEMIR-24 and G:
CODEMIR-25.
DETAILED DESCRIPTION OF THE DRAWINGS
[0062] Various publications, articles and patents are cited or
described in the background and throughout the specification; each
of these references is herein incorporated by reference in its
entirety. Discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is for the purpose of providing context for the
present invention. Such discussion is not an admission that any or
all of these matters form part of the prior art with respect to any
inventions disclosed or claimed.
[0063] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention pertains. In this
invention, certain terms are used frequently, which shall have the
meanings set forth as follows. These terms may also be explained in
greater detail later in the specification.
[0064] The following are abbreviations that are at times used in
this specification:
[0065] bp=base pair
[0066] cDNA=complementary DNA
[0067] CODEMIR=COmputationally-DEsigned, Multi-targeting
Interfering RNA
[0068] kb=kilobase; 1000 base pairs
[0069] kDa=kilodalton; 1000 dalton
[0070] mRNA=messenger RNA
[0071] miRNA=microRNA
[0072] ncRNA=non-coding RNA
[0073] nt=nucleotide
[0074] PAGE=polyacrylamide gel electrophoresis
[0075] PCR=polymerase chain reaction
[0076] RISC=RNA interference silencing complex
[0077] RNAi=RNA interference
[0078] SDS=sodium dodecyl sulfate
[0079] siRNA=short interfering RNA
[0080] shRNA=short hairpin RNA
[0081] SNPs=single nucleotide polymorphisms
[0082] UTR=untranslated region
[0083] VIROMIR=multitargeting interfering RNA preferentially
targeted to viral targets
[0084] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a cell" is a reference to one or more
cells and includes equivalents thereof known to those skilled in
the art and so forth.
[0085] An "activity", a "biological activity", or a "functional
activity" of a polypeptide or nucleic acid refers to an activity
exerted by a polypeptide or nucleic acid molecule as determined in
vivo or in vitro, according to standard techniques. Such activities
can be a direct activity, such as an association with or an
enzymatic activity on a second protein, or an indirect activity,
such as a cellular signaling activity mediated by interaction of a
protein with a second protein.
[0086] "Biological system" is meant, material, in a purified or
unpurified form, from biological sources, including but not limited
to human, animal, plant, insect, microbial, viral or other sources,
wherein the system comprises the components required for biologic
activity (e.g., inhibition of gene expression). The term
"biological system" includes, for example, a cell, a virus, a
microbe, an organism, an animal, or a plant.
[0087] A "cell" means an autonomous self-replicating unit that may
constitute an organism (in the case of unicellular organisms) or be
a sub unit of multicellular organisms in which individual cells may
be specialized and/or differentiated for particular functions. A
cell can be prokaryotic or eukaryotic, including bacterial cells
such as E. coli, fungal cells such as yeast, bird cell, mammalian
cells such as cell lines of human, bovine, porcine, monkey, sheep,
apes, swine, dog, cat, and rodent origin, and insect cells such as
Drosophila and silkworm derived cell lines, or plant cells. The
cell can be of somatic or germ line origin, totipotent or hybrid,
dividing or non-dividing. The cell can also be derived from or can
comprise a gamete or embryo, a stem cell, or a fully differentiated
cell. It is further understood that the term "cell" refers not only
to the particular subject cell, but also to the progeny or
potential progeny of such a cell. Because certain modifications can
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0088] The term "complementary" or "complementarity" as used herein
with respect to polynucleotides or oligonucleotides (which terms
are used interchangeably herein) refers to a measure of the ability
of individual strands of such poly- or oligonucleotides to
associate with each other. Two major fundamental interactions in
RNA are stacking and hydrogen bonding. Both contribute to
free-energy changes for associations of oligoribonucleotides. The
RNA-RNA interactions include the standard Watson-Crick pairing (A
opposite U, and G opposite C) and the non-Watson-Crick pairing
(including but not limited to the interaction through the Hoogsteen
edge and/or sugar edge) (see e.g., Leontis et al., 2002, Nucleic
Acids Research, 30: 3497-3531).
[0089] The degree of complementarity between nucleic acid strands
has significant effects on the efficiency and strength of the
association between the nucleic acid strands. "Complementarity"
between two nucleic acid sequences corresponds to free-energy
changes for helix formation. Thus, determination of binding free
energies for nucleic acid molecules is useful for predicting the
three-dimensional structures of RNAs and for interpreting RNA-RNA
associations. e.g., RNAi activity or inhibition of gene expression
or formation of double stranded oligonucleotides. Such
determination can be made using methods known in the art (see,
e.g., Turner et al., 1987, Cold Spring Harb Symp Quant Biol.
52:123-33; Frier et al., 1986, Proc. Nat. Acad. Sci. USA
83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.
109:3783-3785).
[0090] As the skilled artisan will appreciate, complementarity,
where present, can be partial, for example where at least one or
more nucleic acid bases between strands can pair according to the
canonical base pairing rules. For example, the sequences
5'-CTGACAATCG-3' (SEQ ID NO: 546), 5'-CGAAAGTCAG-3' (SEQ ID NO:
547) are partially complementary (also referred to herein as
"incompletely complementary") to each other. "Partial
complementarity" or "partially complementary" as used herein
indicates that only a percentage of the contiguous residues of a
nucleic acid sequence can form Watson-Crick base pairing with the
same number of contiguous residues in a second nucleic acid
sequence in an anti-parallel fashion. For example, 5, 6, 7, 8, 9,
or 10 nucleotides out of a total of 10 nucleotides in the first
oligonucleotide forming Watson-Crick base pairing with a second
nucleic acid sequence having 10 nucleotides represents 50%, 60%,
70%, 80%, 90%, and 100% complementarity respectively.
[0091] Complementarity can also be total where each and every
nucleic acid base of one strand is capable of forming hydrogen
bonds according to the canonical base pairing rules, with a
corresponding base in another, antiparallel strand. For example,
the sequences 5'-CTGACAATCG-3' (SEQ ID NO: 546) and
5'-CGATTGTCAG-3' (SEQ ID NO: 548) are totally complementary (also
referred to herein as "completely complementary") to each other. As
used herein "complete complementarity" or "completely
complementary" indicates that all the contiguous residues of a
nucleic acid sequence can form Watson-Crick base pairing with the
same number of contiguous residues in a second nucleic acid
sequence in an anti-parallel fashion.
[0092] The skilled artisan will appreciate that where there are no
bases that can adequately base pair with corresponding contiguous
residues in an antiparallel strand, the two strands would be
considered to have no complementarity. In certain embodiments
herein, at least portions of two antiparallel strands will have no
complementarity. In certain embodiments such portions may comprise
even a majority of the length of the two strands.
[0093] In addition to the foregoing, the skilled artisan will
appreciate that in strands of equal length that are completely
complementary, all sections of those strands are completely
complementary to each other. Strands which are not of equal length,
i.e. present in a nucleotide duplex having one or both ends not
being blunt, may be considered by those of skill in the art to be
completely complementary, however there will be one or more bases
in the overhanging end or ends ("overhangs") which do not have
corresponding bases in the opposing strand with which to base pair.
In the case of strands that are incompletely or partially
complementary, it is to be understood that there may be portions or
sections of the strands wherein there are several or even many
contiguous bases which are completely complementary to each other,
and other portions of the incompletely complementary strands which
have less than complete complementarity--i.e. those sections are
only partially complementary to each other.
[0094] The percentage of complementarity between a first nucleotide
sequence and a second nucleotide sequence can be evaluated by
sequence identity or similarity between the first nucleotide
sequence and the complement of the second nucleotide sequence. A
nucleotide sequence that is X % complementary to a second
nucleotide sequence is X % identical to the complement of the
second nucleotide sequence. The "complement of a nucleotide
sequence" is completely complementary to the nucleotide sequence,
whose sequence is readily deducible from the nucleotide sequence
using the rules of Watson-Crick base pairing.
[0095] "Sequence identity or similarity", as known in the art, is
the relationship between two or more polypeptide sequences or two
or more polynucleotide sequences, as determined by comparing the
sequences. In the art, identity also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case can be, as determined by the match between strings of such
sequences. To determine the percent identity or similarity of two
amino acid sequences or of two nucleic acids, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid
sequence for optimal alignment with a second amino or nucleic acid
sequence). The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same or
similar amino acid residue or nucleotide as the corresponding
position in the second sequence, then the molecules are identical
or similar at that position. The percent identity or similarity
between the two sequences is a function of the number of identical
or similar positions shared by the sequences (i.e., %
identity=number of identical positions/total number of positions
(e.g., overlapping positions).times.100). In one embodiment, the
two sequences are the same length. Both identity and similarity can
be readily calculated. Methods commonly employed to determine
identity or similarity between sequences include, but are not
limited to those disclosed in Carillo et al, (1988), SIAM J.
Applied Math. 48, 1073. Preferred methods to determine identity are
designed to give the largest match between the sequences tested.
Methods to determine identity and similarity are codified in
computer programs.
[0096] A non-limiting example of a mathematical algorithm utilized
for the comparison of two sequences is the algorithm of Karlin et
al., (1990), Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as
in Karlin et al., (1993), Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul et al., (1990), J Mol. Biol. 215:403-410. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., (1997), Nucleic Acids
Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform
an iterated search which detects distant relationships between
molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. Additionally, the FASTA method
(Atschul et al., (1990), J. Molec. Biol. 215, 403), can also be
used.
[0097] Another non-limiting example of a mathematical algorithm
useful for the comparison of sequences is the algorithm of Myers et
al, (1988), CABIOS 4:11-17. Such an algorithm is incorporated into
the ALIGN program (version 2.0).
[0098] In an embodiment, the percent identity between two sequences
is determined using the Needleman and Wunsch (J. Mol. Biol.
(48):444-453 (1970)) algorithm which has been incorporated into the
GAP program in the GCG software package. The GCG GAP program aligns
two complete sequences to maximize the number of matches and
minimizes the number of gaps.
[0099] In another embodiment, the percent identity between two
sequences is determined using the local homology algorithm of Smith
and Waterman (J Mol. Biol. 1981, 147(1):195-7), which has been
incorporated into the BestFit program in the GCG software package.
The BestFit program makes an optimal alignment of the best segment
of similarity between two sequences. Optimal alignments are found
by inserting gaps to maximize the number of matches.
[0100] Nucleotide sequences that share a substantial degree of
complementarity will form a stable interaction with each other. As
used herein, the term "stable interaction" with respect to two
nucleotide sequences indicates that the two nucleotide sequences
have the natural tendency to interact with each other to form a
double stranded molecule. Two nucleotide sequences can form a
stable interaction with each other within a wide range of sequence
complementarity. In general, the higher the complementarity the
stronger or the more stable the interaction. Different strengths of
interactions may be required for different processes. For example,
the strength of interaction for the purpose of forming a stable
nucleotide sequence duplex in vitro may be different from that for
the purpose of forming a stable interaction between an iRNA and a
binding sequence in vivo. The strength of interaction can be
readily determined experimentally or predicted with appropriate
software by a person skilled in the art.
[0101] Hybridization can be used to test whether two
polynucleotides are substantially complementary to each other and
to measure how stable the interaction is. Polynucleotides that
share a sufficient degree of complementarity will hybridize to each
other under various hybridization conditions. In one embodiment,
polynucleotides that share a high degree of complementarity thus
form a strong stable interaction and will hybridize to each other
under stringent hybridization conditions. "Stringent hybridization
conditions" has the meaning known in the art, as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
(1989). An exemplary stringent hybridization condition comprises
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC and 0.1% SDS at 50-65.degree. C.
[0102] As used herein the term "mismatch" refers to a nucleotide of
either strand of two interacting strands having no corresponding
nucleotide on the corresponding strand or a nucleotide of either
strand of two interacting strands having a corresponding nucleotide
on the corresponding strand that is non-complementary.
[0103] As used herein, a "match" refers to a complementary pairing
of nucleotides.
[0104] As used herein, the term "expression system" refers to any
in vivo or in vitro system that can be used to evaluate the
expression of a target RNA molecule and or the RNAi activity of a
multitargeting RNA molecule of the invention. In particular
embodiments, the "expression system" comprises one or more target
RNA molecules, a multitargeting interfering RNA molecule targeting
the one or more target RNA molecules, and a cell or any type of in
vitro expression system known to a person skilled in the art that
allows expression of the target RNA molecules and RNAi.
[0105] As used herein, the term "RNA" includes any molecule
comprising at least one ribonucleotide residue, including those
possessing one or more natural nucleotides of the following bases:
adenine, cytosine, guanine, and uracil; abbreviated A, C, G, and U,
respectively, modified ribonucleotides, universal base, acyclic
nucleotide, abasic nucleotide and non-ribonucleotides.
"Ribonucleotide" means a nucleotide with a hydroxyl group at the 2'
position of a p-D-ribo-furanose moiety.
[0106] Modified ribonucleotides include, for example 2'deoxy,
2'deoxy-2'-fluoro, 2'O-methyl, 2'O-methoxyethyl, 4'thio or locked
nucleic acid (LNA) ribonucleotides. Also contemplated herein is the
use of various types of ribonucleotide analogues, and RNA with
internucleotide linkage (backbone) modifications. Modified
internucleotide linkages include for example,
phosphorothioate-modified, and even inverted linkages (i.e. 3'-3'
or 5'-5'). Preferred ribonucleotide analogues include
sugar-modified, and nucleobase-modified ribonucleotides, as well as
combinations thereof. In preferred sugar-modified ribonucleotides
the 2'-OH-group is replaced by a substituent selected from H, OR,
R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2 or ON, wherein R is C1-C6
alkyl, alkenyl or alkynyl and halo is F, Cl, Br, or I. In preferred
backbone-modified ribonucleotides the phosphoester group connecting
to adjacent ribonucleotides is replaced by a modified group, e.g. a
phosphorothioate group. Any or all of the above modifications may
be combined. In addition, the 5'termini can be OH, phosphate,
diphosphate or triphosphate. Nucleobase-modified ribonucleotides,
i.e. ribonucleotides wherein the naturally-occurring nucleobase is
replaced with a non-naturally occurring nucleobase instead, for
example, uridines or cytidines modified at the S-position (e.g.
5-(2-amino)propyl uridine, and 5-bromo uridine); adenosines and
guanosines modified at the 8-position (e.g. 8-bromo guanosine);
deaza nucleotides (e.g. 7-deaza-adenosine); O- and N-alkylated
nucleotides (e.g. N6-methyl adenosine) are also contemplated for
use herein.
[0107] The term "universal base" as used herein refers to
nucleotide base analogs that form base pairs with each of the
natural DNA/RNA bases with little discrimination between them.
Non-limiting examples of universal bases include C-phenyl,
C-naphthyl and other aromatic derivatives, inosine, azole
carboxamides, and nitroazole derivatives such as 3-nitropyrrole,
4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art
(see for example Loakes, 2001, Nucleic Acids Research, 29,
2437-2447).
[0108] The term "acyclic nucleotide" as used herein refers to any
nucleotide having an acyclic ribose sugar, for example where any of
the ribose carbons (C1, C2, C3, C4, or C5), are independently or in
combination absent from the nucleotide.
[0109] As used herein with respect to the listing of RNA sequences,
the bases thymidine ("T") and uridine ("U") are frequently
interchangeable depending on the source of the sequence information
(DNA or RNA). Therefore, in disclosure of target sequences, seed
sequences, candidate seeds, consensus target sequences, target RNA
binding sites, and the like, the base "T" is fully interchangeable
with the base "U". However, with respect to specific disclosures of
the interfering RNA molecules of the invention, it is to be
understood that for such sequences the use of the base "U" cannot
be generally substituted with "T" in a functional manner. It is
however known in the art that certain occurrences of the base "U"
in RNA molecules can be substituted with "T" without substantially
deleterious effect on functionality. For example, the substitution
of T for U in overhangs, such as UU overhangs at the 3' end is
known to be silent, or at a minimum, acceptable, and thus is
permissible in the interfering RNA sequences provided herein. Thus,
it is contemplated that the skilled artisan will appreciate how to
vary even the specific interfering RNA sequences disclosed herein
to arrive at other structurally-related and functionally-equivalent
structures that are within the scope of the instant invention and
the appended claims.
[0110] A "target RNA molecule" or a "pre-selected target RNA
molecule" as used herein refers to any RNA molecule whose
expression or activity is desired to be modulated, for example
decreased, by an interfering RNA molecule of the invention in an
expression system. A "target RNA molecule" can be a messenger RNA
(mRNA) molecule that encodes a polypeptide of interest. A messenger
RNA molecule typically includes a coding region and non-coding
regions preceding ("5'UTR") and following ("3'UTR") the coding
region. A "target RNA molecule" can also be a non-coding RNA
(ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA),
small nuclear RNA (snRNA), short interfering RNA (siRNA), small
nucleolar RNA (snoRNA), ribosomal RNA (rRNA), transfer RNA (tRNA)
and precursor RNAs thereof. Such non-coding RNAs can also serve as
target RNA molecules because ncRNA is involved in functional or
regulatory cellular processes. Aberrant ncRNA activity leading to
disease can therefore be modulated by multitargeting interfering
RNA molecules of the invention. The target RNA can further be the
genome of a virus, for example a RNA virus, or a replicative
intermediate of any virus at any stage, as well as any combination
of these.
[0111] The "target RNA molecule" can be a RNA molecule that is
endogenous to a biological system, or a RNA molecule that is
exogenous to the biological system, such as a RNA molecule of a
pathogen, for example a virus, which is present in a cell after
infection thereof. A cell containing the target RNA can be derived
from or contained in any organism, for example a plant, animal,
protozoan, virus, bacterium, or fungus. Non-limiting examples of
plants include nonocots, dicots, or gymnosperms. Non-limiting
examples of animals include vertebrates or invertebrates.
Non-limiting examples of fungi include molds or yeasts.
[0112] A "target RNA molecule" as used herein may include any
variants or polymorphism of a desired RNA molecule. Most genes are
polymorphic in that a low but nevertheless significant rate of
sequence variability occurs in a gene among individuals of the same
species. Thus, a RNA molecule may correlate with multiple sequence
entries, each of which represents a variant or a polymorphism of
the RNA molecule. In designing any gene suppression tool there is
the risk that the selected binding sequence(s) used in the
computer-based design may contain relatively infrequent alleles. As
a result, the active sequence designed might be expected to provide
the required benefit in only a small proportion of individuals. The
frequency, nature and position of most variants (often referred to
as single nucleotide polymorphisms (SNPs)) are easily accessible to
those trained in the art. In this respect, sequences within a
target molecule that are known to be highly polymorphic can be
avoided in the selection of binding sequences during the
bioinformatic screen. Alternatively, a limitless number of
sequences available for any particular target may be used in the
design stages of an interfering RNA of the invention to make sure
that the targeted binding sequence is present in the majority of
allelic variants, with the exception of the situation in which
targeting of the allelic variant is desired (that is, when the
allelic variant itself is implicated in the disease of
interest).
[0113] A "target RNA molecule" comprises at least one targeted
binding sequence that is sufficiently complementary to the guide
sequence of an interfering RNA molecule of the invention to allow a
stable interaction of the binding sequence with the guide sequence.
The targeted binding sequence can be refined to include any part of
the transcript sequence (eg. 5'UTR, ORF, 3'UTR) based on the
desired effect. For example, translational repression is a frequent
mechanism operating in the 3'UTR (eg. as for microRNA). Thus, the
targeted binding sequence can include sequences in the 3' UTR for
effective translational repression.
[0114] The "targeted binding sequence", "binding sequence", or
"target sequence" shall all mean a portion of a target RNA molecule
sequence comprising a seed sequence and the sequence flanking
either one or both ends of the seed, said binding sequence is
predicted to form a stable interaction with the guide strand of a
multitargeting interfering RNA of the invention based on the
complementarity between the guide strand and the binding
sequence.
[0115] As used herein, the term "non-target transcriptome" or
"non-targeted transcriptome" indicates the transcriptome aside from
the targeted RNA molecules. For example, when a multitargeting
interfering RNA is designed to target a viral RNA, the non-targeted
transcriptome is that of the host. When a multitargeting
interfering RNA is designed to target a given RNA in a biological
system, the non-targeted transcriptome is the transcriptome of the
biological system aside from the targeted given RNA.
[0116] As used herein the term "seed" or "seed sequence" or "seed
region sequence" refers to a sequence of at least about 6
contiguous nucleotides present in a target RNA that is completely
complementary to a portion of the guide strand of an interfering
RNA. Although 6 or more contiguous bases are preferred, the
expression "about 6" refers to the fact that windows of at least 5
or more contiguous bases or more can provide useful candidates in
some cases and can ultimately lead to the design of useful
interfering RNAs. Thus, all such seed sequences are contemplated
within the scope of the instant invention.
[0117] "Conservation or conserved" indicates the extent to which a
specific sequence, such as the seed sequence, is found to be
represented in a group of related target sequences, regardless of
the genetic context of the specific sequence.
[0118] "Genetic context" refers to the flanking sequences that
surround a specific identified sequence and that are sufficiently
long to enable one of average skill in the art to determine its
position within a genome or RNA molecule relative to sequence
annotations or other markers in common use.
[0119] As used herein, the term "interfering RNA" is used to
indicate single or double stranded RNA molecules that modulate the
presence, processing, transcription, translation, or half-life of a
target RNA molecule, for example by mediating RNA interference
("RNAi"), in a sequence-specific manner. As used herein, the term
"RNA interference" or "RNAi" is meant to be equivalent to other
terms used to describe sequence specific RNA interference, such as
post-transcriptional gene silencing, translational inhibition, or
epigenetics. This includes, for example, RISC-mediated degradation
or translational repression, as well as transcriptional silencing,
altered RNA editing, competition for binding to regulatory
proteins, and alterations of mRNA splicing. It also encompasses
degradation and/or inactivation of the target RNA by other
processes known in the art, including but not limited to
nonsense-mediated decay, and translocation to P bodies. Thus, the
interfering RNAs provided herein (e.g. CODEMIRs and VIROMIRs) may
exert their functional effect via any of the foregoing mechanisms
alone, or in combination with one or more other means of RNA
modulation known in the art. The interfering RNAs provided herein
can be used to manipulate or alter the genotype or phenotype of an
organism or cell, by intervening in cellular processes such as
genetic imprinting, transcription, translation, or nucleic acid
processing (e.g., transamination, methylation, etc.).
[0120] The term "interfering RNA" is meant to be equivalent to
other terms used to describe nucleic acid molecules that are
capable of mediating sequence specific RNAi, for example short
interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA
(miRNA), short hairpin RNA (shRNA), short interfering
oligonucleotide, short interfering nucleic acid, short interfering
modified oligonucleotide, chemically-modified siRNA,
post-transcriptional gene silencing RNA (ptgsRNA), and others.
[0121] The "interfering RNA" can be, for example, a double-stranded
polynucleotide molecule comprising self-complementary sense and
antisense strand. The "sense" also named "passenger" strand is
required for presentation of the "antisense" also named "guide",
"guiding", or "target-complementary" strand to the RISC. The guide
strand is retained in the active RISC complex and guides the RISC
to the target nucleotide sequence by means of complementary
base-pairing, which in turn results in RNAi. The relative
thermodynamic characteristics of the 5' termini of the two strands
of a double-stranded interfering RNA determine which strand will
serve the function of a passenger or a guide strand during RNAi.
Indeed, the asymmetric RISC formation can be defined by the
relative thermodynamic strength of the first four nucleotide-pairs
of the 5' termini of an interfering RNA calculated by the
nearest-neighbor methods. Hutvagner (2005), FEBS Letters 579:
5850-5857. Thus, in designing an interfering RNA of the invention,
the guide strand can be pre-determined by the 5' termini
thermodynamic characteristics.
[0122] In an interfering RNA of the invention, the guide strand can
have a sequence completely complementary to one or more but not all
binding sequences present in the one or more target RNA molecules.
It can also be partially complementary to a binding sequence
present in a target RNA molecule, so long as the complementarity is
sufficient for the formation of a stable interaction between the
guide strand and the binding sequence on the target molecule. The
"passenger strand" can be completely or partially complementary to
the guide strand, so long as the complementarity is sufficient for
the formation of a stable interaction between the guide strand and
the passenger strand. Thus, the passenger strand can be completely
or partially identical to the binding sequence on a target
molecule. Both the passenger strand and the guide strand can be
modified and refined to enhance some aspect of the function of the
interfering RNA molecule of the invention. For example, various
pharmacophores, dyes, markers, ligands, conjugates, antibodies,
antigens, polymers, peptides and other molecules can be
conveniently linked to the molecules of the invention. The
interfering RNA can further comprise a terminal phosphate group,
such as a 5'-phosphate or 5',3'-diphosphate. These may be of use to
improve cell uptake, stability, tissue targeting or any combination
thereof.
[0123] The "interfering RNA" can be assembled from two separate
oligonucleotides, where one oligonucleotide is the sense strand and
the other is the antisense strand. The "interfering RNA" can also
be assembled from a single oligonucleotide, where the
self-complementary sense and antisense regions of the interfering
RNA are linked by means of a nucleic acid based or non-nucleic
acid-based linker(s). The "interfering RNA" can be a polynucleotide
with a duplex, asymmetric duplex, hairpin or asymmetric hairpin
secondary structure, having self-complementary sense and antisense
regions. The "interfering RNA" can also be a single-stranded
polynucleotide having one or more loop structures and a stem
comprising self-complementary regions (e.g. short hairpin RNA,
shRNA), wherein the polynucleotide can be processed either in vivo
or in vitro to generate one or more double stranded interfering RNA
molecules capable of mediating RNA inactivation. The cleavage of
the self-paired region or regions of the single strand RNA to
generate double-stranded RNA can occur in vitro or in vivo, both of
which are contemplated for use herein.
[0124] The "interfering RNA" can also be a single stranded
polynucleotide having nucleotide sequence complementary to a
nucleotide sequence in a target nucleic acid molecule or a portion
thereof (i.e., the guide strand), for example, where such
interfering RNA molecule does not require the presence within the
molecule of nucleotide sequence corresponding to the target nucleic
acid sequence or a portion thereof (i.e., the passenger
strand).
[0125] As used herein, the term "interfering RNA" need not be
limited to those molecules containing only RNA, but further
encompasses those possessing one or more modified ribonucleotides
and non-nucleotides, such as those described supra.
[0126] The term "interfering RNA" includes double-stranded RNA,
single-stranded RNA, isolated RNA such as partially purified RNA,
essentially pure RNA, synthetic RNA, recombinantly produced RNA, as
well as altered RNA that differs from naturally occurring RNA by
the addition, deletion, substitution and/or alteration of one or
more nucleotides. Such alterations can include addition of
non-nucleotide material, such as to the end(s) of the
multitargeting interfering RNA or internally, for example at one or
more nucleotides of the RNA. Nucleotides in the RNA molecules of
the instant invention can also comprise non-standard nucleotides,
such as non-naturally occurring nucleotides or chemically
synthesized nucleotides or deoxynucleotides. These altered RNAs can
be referred to as analogs or analogs of naturally-occurring
RNA.
[0127] The interfering RNA of the invention, also termed
"multitargeting interfering RNA" is an interfering RNA having a
guide strand that can form stable interactions with at least two
binding sites present in distinct genetic contexts on one or more
target RNA molecules. Examples of the multitargeting interfering
RNA include CODEMIRs, COmputationally-DEsigned, Multi-targeting
Interfering RNAs, and VIROMIRs, where these multitargeting
interfering RNA molecules are preferentially targeted to viral
targets.
[0128] "Sequence" means the linear order in which monomers occur in
a polymer, for example, the order of amino acids in a polypeptide
or the order of nucleotides in a polynucleotide.
[0129] A "subject" as used herein, refers to an organism to which
the nucleic acid molecules of the invention can be administered. A
subject can be an animal or a plant, preferably a mammal, most
preferably a human, who has been the object of treatment,
observation or experiment, or any cell thereof.
[0130] The term "therapeutically effective amount" as used herein,
means that amount of active compound or pharmaceutical agent that
elicits the biological or medicinal response in a subject that is
being sought by a researcher, veterinarian, medical doctor or other
clinician, which includes preventing, ameliorating or alleviating
the symptoms of the disease or disorder being treated. Methods are
known in the art for determining therapeutically effective doses
for the instant pharmaceutical composition.
[0131] A "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One
type of vector is a "plasmid", which refers to a circular double
stranded DNA loop into which additional DNA segments can be
inserted. Another type of vector is a viral vector, wherein
additional DNA segments can be inserted. Certain vectors are
capable of autonomous replication in a host cell into which they
are introduced (e.g., bacterial vectors having a bacterial origin
of replication and episomal mammalian vectors). Other vectors
(e.g., non-episomal mammalian vectors) are integrated into the
genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome. Moreover,
certain vectors, expression vectors, are capable of directing the
expression of genes to which they are operably linked.
[0132] As used herein, "modulate (or modulation of) the expression
of an RNA molecule" means any RNA interference mediated regulation
of the level and/or biological activity of the RNA molecule. It
includes any RNAi-related transcriptional or post-transcriptional
gene silencing, such as by cleaving, destabilizing the target RNA
molecule or preventing RNA translation. In one embodiment, the term
"modulate" can mean "inhibit," but the use of the word "modulate"
is not limited to this definition. The modulation of the target RNA
molecule is determined in a suitable expression system, for example
in vivo, in one or more suitable cells, or in an acellular or in
vitro expression system such as are known in the art. Routine
methods for measuring parameters of the transcription, translation,
or other aspects of expression relating to RNA molecules are known
in the art, and any such measurements are suitable for use
herein.
[0133] By "inhibit", "down-regulate", "reduce", or "decrease" as
with respect to a target RNA or its expression it is meant that the
expression of the gene or level and/or biological activity of
target RNA molecules is reduced below that observed in the absence
of the nucleic acid molecules (e.g., multitargeting interfering
RNA) of the invention. In one embodiment, inhibition,
down-regulation or reduction with a multitargeting interfering RNA
molecule is greater than that observed in the presence of an
inactive or attenuated molecule. In another embodiment, inhibition,
down-regulation, or reduction with a multitargeting interfering RNA
molecule is greater than that observed in the presence of, for
example, multitargeting interfering RNA molecule with scrambled
sequence or with mismatches. In another embodiment, inhibition,
down-regulation, or reduction of gene expression with a nucleic
acid molecule of the instant invention is greater in the presence
of the nucleic acid molecule than in its absence.
[0134] "Inhibit", "down-regulate", "reduce", or "decrease" as with
respect to a target RNA or its expression encompasses, for example,
reduction of the amount or rate of transcription or translation of
a target RNA, reduction of the amount or rate of activity of the
target RNA, and/or a combination of the foregoing in a selected
expression system. The skilled artisan will appreciate that a
decrease in the total amount of transcription, the rate of
transcription, the total amount of translation, or the rate of
translation, or even the activity of an encoded gene product are
indicative of such a decrease. The "activity" of an RNA refers to
any detectable effect the RNA may have in a cell or expression
system, including for example, any effect on transcription, such as
enhancing or suppressing transcription of itself or another RNA
molecule. The measurement of a "decrease" in expression or the
determination of the activity of a given RNA can be performed in
vitro or in vivo, in any system known or developed for such
purposes, or adaptable thereto. Preferably the measurement of a
"decrease" in expression by a particular interfering RNA is made
relative to a control, for example, in which no interfering RNA is
used. In some comparative embodiments such measurement is made
relative to a control in which some other interfering RNA or
combination of interfering RNAs is used. Most preferably a change,
such as the decrease is statistically significant based on a
generally accepted test of statistical significance. However,
because of the large number of possible measures and the need for
the ability to rapidly screen candidate interfering RNAs, it is
contemplated herein that a given RNA need only show an arithmetic
decrease in one such in vitro or in vivo assay to be considered to
show a "decrease in expression" as used herein.
[0135] More particularly, the biological modulating activity of the
multitargeting interfering RNA is not limited to, or necessarily
reliant on, degradation or translational repression by conventional
RISC protein complexes involved in siRNA and microRNA
gene-silencing, respectively. Indeed, short double-stranded and
single-stranded RNA have been shown to have other possible
sequence-specific roles via alternative mechanisms. For example,
short double-stranded RNA (dsRNA) species may act as modulatory
effectors of differentiation/cell activity, possibly through
binding to regulatory proteins (Kuwabara, T., et al., (2004), Cell,
116: 779-93). Alternatively, dsRNA may lead to the degradation of
mRNA through the involvement of AU-rich element (ARE)-binding
proteins (Jing, Q., et al., (2005), Cell, 120: 623-34). Further,
dsRNA may also induce epigenetic transcriptional silencing (Morris,
K. V., et al., (2004) Science, 305: 1289-89). Processing of mRNA
can also be altered through A to I editing and modified
splicing.
[0136] As used herein, "palindrome" or "palindromic sequence" means
a nucleic acid sequence that is completely complementary to a
second nucleotide sequence that is identical to the nucleic acid
sequence, e.g., UGGCCA. The term also includes a nucleic acid
molecule comprising two nucleotide sequences that are palindromic
sequences.
[0137] "Phenotypic change" as used herein refers to any detectable
change to a cell or an organism that occurs in response to contact
or treatment with a nucleic acid molecule of the invention. Such
detectable changes include, but are not limited to, changes in
shape, size, proliferation, motility, protein expression or RNA
expression or other physical or chemical changes as can be assayed
by methods known in the art. The detectable change can also include
expression of reporter genes/molecules such as Green Fluorescent
Protein (GFP) or various tags that are used to identify an
expressed protein or any other cellular component that can be
assayed.
[0138] The present invention provides a multitargeting interfering
RNA molecule comprising a guide strand that forms stable
interactions with at least two binding sequences present in
distinct genetic contexts in one or more pre-selected target RNA
molecules. In one general aspect, the present invention provides a
multitargeting interfering RNA molecule comprising a guide strand
of the Formula (I):
5'-p-XSY-3' (Formula I)
[0139] In Formula (I), p consists of a terminal phosphate group
that can be present or absent from the 5'-end of the guide strand.
Any terminal phosphate group known to a person skilled in the art
can be used. Such phosphate groups include, but are not limited to,
monophosphate, diphosphate, triphosphate, cyclic phosphate or to a
chemical derivative of phosphate such as a phosphate ester
linkage.
[0140] In Formula (I), S consists of a first nucleotide sequence of
a length of about 5 to about 20 nucleotides that is at least
partially, preferably completely, complementary to a first portion
of each of at least two binding sequences present in distinct
genetic contexts in one or more pre-selected target RNA molecules.
In particular embodiments, S has a length of about 6 to about 15
nucleotides, such as a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15 nucleotides that are at least partially, preferably completely,
complementary to the first portion of the at least two binding
sequences. In one embodiment, S is completely complementary to a
seed sequence of each of one, two, three, four, five, or more
distinct binding sequences present in distinct genetic contexts in
one or more pre-selected target RNA molecules. The skilled artisan
will appreciate that the at least two distinct binding sequences
may be on the same target RNA molecule, or they can be on different
RNA molecules.
[0141] In certain embodiments, S consists essentially of a
nucleotide sequence selected from the group consisting of:
TABLE-US-00005 UAUGUGGGUGGG, (SEQ ID NO; 1) UGUUUUG, (SEQ ID NO: 2)
ACCCCGUCUCU, (SEQ ID NO: 5) AGCUGCA, (SEQ ID NO: 7) AAACAAUGGAAUG,
(SEQ ID NO: 8) GGUAGGUGGGUGGG, (SEQ ID NO: 10) CUGCUUGAU, (SEQ ID
NO: 12) UCCUUUCCA, (SEQ ID NO: 13) UUUUUCUUU, (SEQ ID NO: 14)
UUCUGAUGUUU, (SEQ ID NO: 15) UCUUCCUCUAU, (SEQ ID NO: 16)
UGGUAGCUGAA, (SEQ ID NO: 17) CUUUGGUUCCU, (SEQ ID NO: 18)
CUACUAAUGCU, (SEQ ID NO: 19) UCCUGCUUGAU, (SEQ ID NO: 20)
AUUCUUUAGUU, (SEQ ID NO: 21) CCAUCUUCCUG, (SEQ ID NO: 22)
CCUCCAAUUCC, (SEQ ID NO: 23) CUAAUACUGUA, (SEQ ID NO: 24)
UUCUGUUAGUG, (SEQ ID NO: 25) GCUGCUUGAUG, (SEQ ID NO: 26)
ACAUUGUACUG, (SEQ ID NO: 27) UGAUAUUUCUC, (SEQ ID NO: 28)
AACAGCAGUUG, (SEQ ID NO: 29) GUGCUGAUAUU, (SEQ ID NO: 30)
CCCAUCUCCAC, (SEQ ID NO: 31) UAUUGGUAUUA, (SEQ ID NO: 32)
CAAAUUGUUCU, (SEQ ID NO: 33) UACUAUUAAAC, (SEQ ID NO: 34)
GCCUAUCAUAU, (SEQ ID NO: 58) UGGUGCCUGCU, (SEQ ID NO: 59)
AAUUAAUAUGGC, (SEQ ID NO: 60) CCCUCUGGGCU, (SEQ ID NO: 61)
UUCUUCCUCAU, (SEQ ID NO: 62) UAUUUAUACAGA, (SEQ ID NO: 63)
CACCAAAAUUC, (SEQ ID NO: 64) UGAGUNNGAACAUU (SEQ ID NO: 72) where N
is any base, CUCCAGG, (SEQ ID NO: 74) UCAGUGGG, (SEQ ID NO: 76)
UCCUCACAGGG, (SEQ ID NO: 78) GUGCUCAUGGUG, (SEQ ID NO: 79)
CCUGGAGCCCUG, (SEQ ID NO: 80) UCUCAGCUCCAC, (SEQ ID NO: 81)
ACCCUCGCACC, (SEQ ID NO: 86) GUGUUGAAG, (SEQ ID NO: 88) UUCCACAAC,
(SEQ ID NO: 90) UCCACUGUC, (SEQ ID NO: 92) CAGAAUAG, (SEQ ID NO:
93) AACUCUCUA (SEQ ID NO: 94) and CGUGAAGAC. (SEQ ID NO: 98)
In certain preferred embodiments, S consists essentially of a
nucleotide sequence selected from the group consisting of:
UAUGUGGGUGGG (SEQ ID NO: 1), UCCUCACAGGG (SEQ ID NO: 78), GUGUUGAAG
(SEQ ID NO: 88), UUCCACAAC (SEQ ID NO: 90), AACUCUCUA (SEQ ID NO:
94) and CGUGAAGAC (SEQ ID NO: 98).
[0142] In other embodiments, S consists essentially of a nucleotide
sequence of 6 or more contiguous bases contained within any of the
sequences selected from the group consisting of: UAUGUGGGUGGG (SEQ
ID NO: 1), UCCUCACAGGG (SEQ ID NO: 78), GUGUUGAAG (SEQ ID NO: 88),
UUCCACAAC (SEQ ID NO: 90), AACUCUCUA (SEQ ID NO: 94) and CGUGAAGAC
(SEQ ID NO: 98).
[0143] In certain embodiments, S is partially complementary to a
first portion of at least two distinct binding sequences present in
distinct genetic contexts in one or more pre-selected target RNA
molecules, such as 6 of 7, 7 of 8, 8 of 9, 9 of 10, 10 of 11, 11 of
12, 12 of 13, 13 of 14, 14 of 15, or 15 of 16 consecutive
nucleotides of S are completely complementary to the first portion
of at least two target RNA binding sequences. In other embodiments,
S and the first portion of the distinct binding sequences have
lesser overall complementarity such as 10 of 12, 11 of 13, 12 of
14, 13 of 15, or 14 of 16 nucleotides of complete
complementarity.
[0144] In Formula (I), X is absent or consists of a second
nucleotide sequence. In particular embodiments, X consists of one
or two nucleotides.
[0145] In Formula (I), Y is absent or consists of a third
nucleotide sequence, provided that X and Y are not absent
simultaneously. Y has complementarity that ranges from complete to
nonexistent with respect to a second portion of each of the at
least two distinct binding sequences, where the second portion is
adjacent to and connected with the 5'-end of the first portion of
the binding sequences. In one embodiment, Y is at least partially
complementary to a second portion of at least one binding sequence,
thus allowing the guide strand to have improved interaction with
the at least one binding sequence. Preferably, Y provides optimal
or desired binding to each of the second portions of the distinct
binding sequences by comprising a consensus-like sequence to which
these second portions can bind. This aspect of having a region of
less than complete complementarity in the guide strand is
particularly useful in certain embodiments, for example, by
providing an area of some consensus between distinct binding
sequences.
[0146] In particular embodiments of the invention, by combining in
the guide strand, S with complete complementarity to a seed portion
of each binding sequence, and Y, that is incompletely complementary
to a second portion of each binding sequence, the overall
nucleotide sequence of XSY is such that it is at least partially
complementary to each of the distinct binding sequences to allow a
stable interaction with each of the binding sequences, thus
providing multitargeting interfering RNA of any target molecules
comprising the binding sequences. In some embodiments XSY may be
fully complementary to at least one of the distinct binding
sequences. In other embodiments, XSY is partially complementary to
both distinct binding sequences.
[0147] The multitargeting interfering RNA can comprise both a guide
strand of formula (I) described supra and a passenger strand that
is at least partially complementary to the guide strand to allow
formation of stable duplexes between the passenger strand and the
guide strand. The passenger strand and the guide strand can be
completely complementary to each other. The passenger strand and
the guide strand can have the same or different length. In an
embodiment of the present invention, each strand of a
multitargeting interfering RNA molecule of the invention is
independently about 17 to about 25 nucleotides in length, in
specific embodiments about 17, 18, 19, 20, 21, 22, 23, 24, and 25
nucleotides in length. Using shorter length interfering RNA
molecules without the need for the generation of multiple active
sequences through processing of RNA by enzymes such as Dicer and
RNaseIII, provides advantages, for example, in reduction of cost,
manufacturing, and chance of off-target effects.
[0148] The interaction between the passenger strand and the guide
strand can be adjusted to improve loading of the guide strand into
the cellular RISC complex (Khvorova et al. (2003) Cell, 115:
209-16; Schwarz et al. (2003) Cell, 115: 199-208), or to otherwise
improve the functional aspects of the multitargeting interfering
RNA. The skilled artisan will appreciate that there are routine
methods for altering the strength and other properties of the base
paired strands through the addition, deletion, or substitution of
one or more bases in either strand of the synthetic duplex. In
particular as one example, these strategies can be applied to the
design of the extremities of the duplex to ensure that the
predicted thermodynamics of the duplex are conducive to the loading
of the desired strand. These strategies are well known to persons
skilled in the art.
[0149] It is also contemplated herein that a substantially
double-stranded RNA molecule comprises a single-stranded RNA
molecule with, for example, a hairpin loop or similar secondary
structure that allows the molecule to self-pair to form at least a
region of double-stranded nucleic acid comprising the guide strand
of Formula (I).
[0150] The skilled artisan will appreciate that the double-stranded
RNA molecules provide certain advantages for use in therapeutic
applications. Although blunt-ended molecules are disclosed herein
for certain embodiments, in various other embodiments, overhangs,
for example of 1-5 nucleotides, are present at either or both
termini. In some embodiments, the overhangs are 2 or 3 bases in
length. Presently preferred overhangs include 3'-UU overhangs in
certain embodiments. Other overhangs exemplified for use herein
include, but are not limited to, 3'-AA, 3'-CA, 3'-AU, 3'-UC, 3'-CU,
3'-UG, 3'-CC, 3'-UA, 3'-U, and 3'-A. Still other either 5'-, or
more preferably 3'-, overhangs of various lengths and compositions
are contemplated for use herein on the RNA molecules provided.
[0151] In certain embodiments at least one target RNA molecule is
an mRNA. More specifically, in some embodiments at least one target
encodes a receptor, cytokine, transcription factor, regulatory
protein, signaling protein, cytoskeletal protein, transporter,
enzyme, hormone, or antigen. As such, the potential range of
protein targets in the cell is not limited, however the skilled
artisan will appreciate that certain targets are more likely to be
of value in a particular disease state or process. In addition, the
skilled artisan will appreciate that target RNA molecules, whether
coding or regulatory, originating from a pathogen (e.g. a virus)
are useful with the multitargeting RNAs and methods provided
herein.
[0152] In one embodiment, at least one of the binding sequences is
in the 3' UTR of an mRNA. In embodiments featuring multitargeting
of different RNA molecules, preferably the target RNAs are not
solely splice variants of a single gene, nor solely isoforms of
each other. In other embodiments where it is vital or preferred to
modulate some or all such splice variants or isoforms, the multiple
targets may encompass such sequences.
[0153] The inclusion of one target or more targets does not
preclude the use of, or intention for, a particular interfering RNA
to target another selected target. Such targeting of any additional
RNA target molecules may result in less, equal, or greater effect
in an expression system. Notwithstanding the foregoing, the
multitargeting interfering RNAs of the instant invention are
preferably screened for off-target effects, especially those that
are likely. For example, reviewing the potential binding to the
entire transcriptome, or as much as of it as is known at the time
provides a useful approach to such screening. For example, where a
genome has been completely sequenced, the skilled artisan will
appreciate that the entire transcriptome can be conveniently
screened for likely off-target effects. In cases for which local
delivery of multitargeting interfering RNA is anticipated,
specialized tissue-specific transcriptomes (eg retina for ocular
applications) may be more relevant because non-target transcripts
that are identified through bioinformatic approaches from the
complete transcriptome may actually not be present in the tissue
into which the multitargeting interfering RNA is applied.
[0154] In one embodiment, the guide strand of a multitargeting
interfering RNA of the invention forms stable interactions with at
least two targeted binding sequences present in distinct genetic
contexts on a single target RNA molecule, thus modulating the
expression or activity of the RNA molecule. Targeting multiple
binding sites on a single target RNA molecule with a single guide
strand can provide more effective RNAi of the target RNA molecule.
This approach is particularly useful for the modulation of virus
gene expression where the mutation rate is high.
[0155] In another embodiment, the guide strand of a multitargeting
interfering RNA of the invention forms stable interactions with at
least two distinct binding sequences present in distinct genetic
contexts on multiple pre-selected target RNA molecules, thus
modulating the expression or activity of multiple pre-selected
target RNA molecules. Targeting multiple target RNA molecules with
a single guide strand represents an alternative to the prototypical
one-drug, one-target approach. In considering the complexity of
biological systems, designing a drug selective for multiple targets
will lead to new and more effective medications for a variety of
diseases and disorders.
[0156] In specific embodiments, RNA molecules that are involved in
a disease or disorder of a biological system are pre-selected and
targeted by a multitargeting interfering RNA molecule of the
invention. The biological system can be, for example, a plant, or
an animal such as a rat, a mouse, a dog, a pig, a monkey, and a
human. The pre-selected target RNA molecules can, for example,
encode a protein of a class selected from the group consisting of
receptors, cytokines, transcription factors, regulatory proteins,
signaling proteins, cytoskeletal proteins, transporters, enzymes,
hormones, and antigens. The pre-selected target RNA molecules can,
for example, encode a protein selected from the group consisting of
ICAM-1, VEGF-A, MCP-1, IL-8, VEGF-B, IGF-1, Gluc6p, Inppl1, bFGF,
PlGF, VEGF-C, VEGF-D, .beta.-catenin, .kappa.-ras-B, .kappa.-ras-A,
EGFR, Bcl-2, presenilin-1, BACE-1, MALAT-1, BIC, TGF.beta. and TNF
alpha. Therefore, the multitargeting interfering RNA molecule of
the invention can, for example, decrease expression of any
combination of ICAM-1, VEGF-B, VEGF-C, VEGF-D, IL-8, bFGF, PIGF,
MCP-1 and IGF-1, any combination of ICAM-1, VEGF-A and IGF-1, any
combination of .beta.-catenin, .kappa.-ras, and EGFR, both ICAM-1
and VEGF-A, both presenilin-1 and one or more isoforms of BACE-1,
both VEGF-A and bFGF, any combination of VEGF-A, ICAM-1, PlGF, and
IGF-1, any combination of VEGF-A, .kappa.-ras, EGFR and Bcl-2, both
MALAT-1 and BIC, both TGF.beta., and IL-8, both IL-8 and MCP-1, any
combination of VEGF-A, Bcl-2 and .kappa.-ras, or both Gluc6p and
Inppl1, in a biological system, such as an animal.
[0157] The pre-selected target RNA molecule can also encode a
protein, including an essential protein, for a virus. Such
essential proteins can be a protein, for example, that is involved
in the replication, transcription, translation, or packaging
activity of the virus. Exemplary essential proteins for a HIV virus
are GAG, POL, VIF, VPR, TAT, NEF, REV, VPU and ENV, all of which
can be a pre-selected target molecule of the invention. The
multitargeting interfering RNA of the invention can be used to
modulate RNA expression in viruses, including but not limited to, a
human immunodeficiency virus (HIV), a hepatitis C virus (HCV), an
influenza virus, a rhinovirus, and a severe acute respiratory
syndrome (SARS) virus or a combination thereof.
[0158] In some embodiments, the multitargeting interfering RNA of
the invention are designed to target one or more target RNA
molecules in a first biological system and one or more target
molecules in a second biological system that is infectious to the
first biological system. In particular embodiments, the
multitargeting interfering RNA of the invention are designed to
target one or more host RNA molecules and one or more RNA molecules
of a virus or a pathogen for the host. Therefore, the
multitargeting interfering RNA molecule of the invention can, for
example, decrease expression of TNF alpha and modulate expression
of a hepatitis C virus (HCV).
[0159] In particular embodiments of the invention, specific
multitargeting interfering RNA molecules are provided which are
functional against specific targets. These multitargeting
interfering RNA molecules are useful for modulating expression of
RNA, for example, their intended target RNA molecules, and data
supporting the activity are also provided herein in the working
examples.
[0160] In one embodiment, synthetic multitargeting interfering RNA
molecules comprising the sequence UAUGUGGGUGGG (SEQ ID NO: 1), or
UGUUUUG (SEQ ID NO: 2), are provided. In one embodiment the
molecules are double stranded in at least the region comprising the
recited sequence. Preferably the molecules decrease expression of
both VEGF-A and ICAM-1 in an expression system. In another
embodiment, the multitargeting interfering RNA molecules decrease
expression of at least one target RNA molecule comprising the
sequence CCCACCCACAUA (SEQ ID NO: 3), or CAAAACA (SEQ ID NO: 4).
More preferably they target multiple sites on two or more RNAs.
[0161] Multitargeting interfering RNA molecules comprising the
sequence ACCCCGUCUCU (SEQ ID NO: 5) are also provided herein. In
one embodiment the molecules are double stranded in at least the
region comprising the recited sequence. Preferably, the
multitargeting interfering RNAs decrease expression of any
combination of ICAM-1, VEGF-A and IGF-1 in an expression system. In
another embodiment, in an expression system, the multitargeting
interfering RNA molecules decrease expression of at least one
target RNA molecule comprising the sequence AGAGACGGGGU (SEQ ID NO:
6). More preferably they target multiple sites on two or more
RNAs.
[0162] Also provided herein are multitargeting interfering RNA
molecules comprising the sequence AGCUGCA (SEQ ID NO: 7). In one
embodiment the molecules are double stranded in at least the region
comprising the recited sequence. The multitargeting interfering
RNAs preferably decrease expression of any combination of ICAM-1,
VEGF-B, VEGF-C, VEGF-D, IL-8, bFGF, PlGF, MCP-1 and IGF-1 in an
expression system. In one embodiment, in an expression system the
multitargeting interfering RNA molecules modulate expression of at
least one target RNA molecule comprising the sequence UGCAGCU. More
preferably they target multiple sites on two or more RNAs.
[0163] In another embodiment, multitargeting interfering RNA
molecules comprising the sequence AAACAAUGGAAUG (SEQ ID NO: 8) are
provided. In one embodiment the molecules are double stranded in at
least the region comprising the recited sequence. The
multitargeting interfering RNAs preferably modulate expression of
any combination of .kappa.-ras, EGFR and .beta. catenin in an
expression system. Preferably in an expression system, the
multitargeting interfering RNA molecules modulate expression of at
least one target RNA molecule comprising the sequence CAUUCCAUUGUUU
(SEQ ID NO: 9). More preferably they target multiple sites on two
or more RNAs.
[0164] Also provided herein are multitargeting interfering RNA
molecules comprising the sequence GGUAGGUGGGUGGG (SEQ ID NO: 10).
In one embodiment the molecules are double stranded in at least the
region comprising the recited sequence. The multitargeting
interfering RNAs preferably modulate expression of gluc6p and
Inppl1 in an expression system. In an expression system, the
multitargeting interfering molecules preferably modulate expression
of at least one target RNA molecule comprising the sequence
CCCACCCACCUACC (SEQ ID NO: 11). More preferably they target either
multiple sites on a single RNA or multiple sites on two or more
RNAs.
[0165] Also provided herein are multitargeting interfering RNA
molecules comprising the sequence CUGCUUGAU (SEQ ID NO: 12),
UCCUUUCCA (SEQ ID NO: 13), UUUUUCUUU (SEQ ID NO: 14), UUCUGAUGUUU
(SEQ ID NO: 15), UCUUCCUCUAU (SEQ ID NO: 16), UGGUAGCUGAA (SEQ ID
NO: 17), CUUUGGUUCCU (SEQ ID NO: 18), CUACUAAUGCU (SEQ ID NO: 19),
UCCUGCUUGAU (SEQ ID NO: 20), AUUCUUUAGUU (SEQ ID NO: 21),
CCAUCWUCCUG (SEQ ID NO: 22), CCUCCAAUUCC (SEQ ID NO: 23),
CUAAUACUGUA (SEQ ID NO: 24), UUCUGUUAGUG (SEQ ID NO: 25),
GCUGCUUGAUG (SEQ ID NO: 26), ACAUUGUACUG (SEQ ID NO: 27),
UGAUAUUUCUC (SEQ ID NO: 28), AACAGCAGUUG (SEQ ID NO: 29),
GUGCUGAUAUU (SEQ ID NO: 30), CCCAUCUCCAC (SEQ ID NO: 31),
UAUUGGUAUUA (SEQ ID NO: 32), CAAAUUGUUCU (SEQ ID NO: 33), or
UACUAUUAAAC (SEQ ID NO: 34). In one embodiment the molecules are
double stranded in at least the region comprising the recited
sequence. In an expression system, the multitargeting interfering
RNAs preferably modulate expression of at least one RNA encoded by
an HIV genome. More preferably the multitargeting interfering
molecules modulate expression of at least two RNAs so encoded. In
another embodiment the multitargeting interfering RNA molecules
modulate expression of at least three, or even four, HIV RNAs.
Preferably, the multitargeting interfering RNA molecules decrease
expression of at least one target RNA molecule comprising the
sequence AUCAAGCAG (SEQ ID NO: 35), UGGAAAGGA (SEQ ID NO: 36),
AAAGAAAAA (SEQ ID NO: 37), AAACAUCAGAA (SEQ ID NO: 38), AUAGAGGAAGA
(SEQ ID NO: 39), UUCAGCUACCA (SEQ ID NO: 40), AGGAACCAAAG (SEQ ID
NO: 41), AGCAUUAGUAG (SEQ ID NO: 42), AUCAAGCAGGA (SEQ ID NO: 43),
AACUAAAGAAU (SEQ ID NO: 44), CAGGAAGAUGG (SEQ ID NO: 45),
GGAAUUGGAGG (SEQ ID NO: 46), UACAGUAUUAG (SEQ ID NO: 47),
CACUAACAGAA (SEQ ID NO: 48), CAUCAAGCAGC (SEQ ID NO: 49),
CAGUACAAUGU (SEQ ID NO: 50), GAGAAAUAUCA (SEQ ID NO: 51),
CAACUGCUGUU (SEQ ID NO: 52), AAUAUCAGCAC (SEQ ID NO: 53),
GUGGAGAUGGG (SEQ ID NO: 54), UAAUACCAAUA (SEQ ID NO: 0.55),
AGAACAAUUUG (SEQ ID NO: 56) or GUUUAAUAGUA (SEQ ID NO: 57), when
measured in a selected expression system. More preferably they
target multiple sites on two or more RNAs.
[0166] In another embodiment, multitargeting interfering RNA
molecules comprising the sequence GCCUAUCAUAU (SEQ ID NO: 58),
UGGUGCCUGCU (SEQ ID NO: 59), AAUUAAUAUGGC (SEQ ID NO: 60),
CCCUCUGGGCU (SEQ ID NO: 61), UUCUUCCUCAU (SEQ ID NO: 62),
UAUUUAUACAGA (SEQ ID NO: 63), or CACCAAAAUUC (SEQ ID NO: 64) are
provided. In one embodiment the molecules are double stranded in at
least the region comprising the recited sequence. Preferably the
multitargeting interfering RNAs modulate expression of presenilin-1
and one or more isoforms of BACE-1 in an expression system. Also
preferred are such multitargeting interfering RNA molecules that in
an expression system modulate expression of at least one target RNA
molecule comprising the sequence AUAUGAUAGGC (SEQ ID NO: 65),
AGCAGGCACCA (SEQ ID NO: 66), GCCAUAUUAAUU (SEQ ID NO: 67),
AGCCCAGAGGG (SEQ ID NO: 68), AUGAGGAAGAA (SEQ ID NO: 69),
UCUGUAUAAAUA (SEQ ID NO: 70), or GAAUUUUGGUG (SEQ ID NO: 71). More
preferably they target multiple sites on two or more RNAs.
[0167] Also provided herein are multitargeting interfering RNA
molecules comprising the sequence UGAGUNNGAACAUU (SEQ ID NO: 72),
where N is any base. In one embodiment the molecules are double
stranded in at least the region comprising the recited sequence. In
a preferred embodiment, the multitargeting interfering RNAs
modulate expression of VEGF-A and bFGF in an expression system. The
multitargeting interfering RNA molecules preferably modulate
expression in an expression system of at least one target RNA
molecule comprising the sequence AAUGUUCVVACUCA (SEQ ID NO: 73)
where VV is CC or AG. More preferably they target multiple sites on
two or more RNAs.
[0168] Also provided herein are multitargeting interfering RNA
molecules comprising the sequence CUCCAGG (SEQ ID NO: 74). In one
embodiment the molecules are double stranded in at least the region
comprising the recited sequence. These multitargeting interfering
RNAs, in one embodiment, modulate expression of any combination of
VEGF-A, ICAM-1, PlGF and IGF-1 in an expression system. The
multitargeting interfering RNA molecules preferably modulate
expression, in an expression system, of at least one target RNA
molecule comprising the sequence CCUGGAG (SEQ ID NO: 75). More
preferably they target multiple sites on two or more RNAs.
[0169] In another embodiment, multitargeting interfering RNA
molecules comprising the sequence UCAGUGGG (SEQ ID NO: 76) are
provided herein. In one embodiment the molecules are double
stranded in at least the region comprising the recited sequence.
Preferably, the multitargeting interfering RNAs modulate expression
of any combination of VEGF-A, .kappa.-ras, EGFR and bcl 2 in an
expression system. Also preferred are those multitargeting
interfering RNA molecules that modulate expression of at least one
target RNA molecule comprising the sequence CCCACUGA (SEQ ID NO:
77) in such an expression system. More preferably they target
multiple sites on two or more RNAs.
[0170] In yet another embodiment, provided are multitargeting
interfering RNA molecules comprising the sequence UCCUCACAGGG (SEQ
ID NO: 78), GUGCUCAUGGUG (SEQ ID NO: 79), CCUGGAGCCCUG (SEQ ID NO:
80), or UCUCAGCUCCAC (SEQ ID NO: 81). In one embodiment the
molecules are double stranded in at least the region comprising the
recited sequence. The multitargeting interfering RNAs preferably
decrease expression of TNF.alpha. and at least one RNA encoded by
the HCV genome in an expression system. The multitargeting
interfering RNA molecules preferably decrease expression of at
least one target RNA molecule comprising the sequence CCCUGUGAGGA
(SEQ ID NO: 82), CACCAUGAGCAC (SEQ ID NO: 83), CAGGGCUCCAGG (SEQ ID
NO: 84), or GUGGAGCUGAGA (SEQ ID NO: 85) in an expression system.
More preferably they target multiple sites on two or more RNAs.
[0171] Also provided herein are multitargeting interfering RNA
molecules comprising the sequence ACCCUCGCACC (SEQ ID NO: 86). In
one embodiment the molecules are double stranded in at least the
region comprising the recited sequence. The multitargeting
interfering RNAs preferably modulate expression of MALAT-1 and BIC
in an expression system. In an expression system, the
multitargeting interfering molecules preferably modulate expression
of at least one target RNA molecule comprising the sequence
GGUGCGAGGGU (SEQ ID NO: 87). More preferably they target either
multiple sites on a single RNA or multiple sites on two or more
RNAs.
[0172] In yet another embodiment, provided are multitargeting
interfering RNA molecules comprising the sequence GUGUUGAAG (SEQ ID
NO: 88). In one embodiment the molecules are double stranded in at
least the region comprising the recited sequence. The
multitargeting interfering RNAs preferably modulate expression of
TGF.beta. and IL-8 in an expression system. In an expression
system, the multitargeting interfering molecules preferably
modulate expression of at least one target RNA molecule comprising
the sequence CUUCAACAC (SEQ ID NO: 89). More preferably they target
either multiple sites on a single RNA or multiple sites on two or
more RNAs.
[0173] In another embodiment, provided are multitargeting
interfering RNA molecules comprising the sequence UUCCACAAC (SEQ ID
NO: 90). In one embodiment the molecules are double stranded in at
least the region comprising the recited sequence. The
multitargeting interfering RNAs preferably modulate expression of
IL-8 and MCP-1 in an expression system. In an expression system,
the multitargeting interfering molecules preferably modulate
expression of at least one target RNA molecule comprising the
sequence GUUGUGGAA (SEQ ID NO: 91). More preferably they target
either multiple sites on a single RNA or multiple sites on two or
more RNAs.
[0174] Also provided herein are multitargeting interfering RNA
molecules comprising the sequences UCCACUGUC (SEQ ID NO: 92),
CAGAAUAG (SEQ ID NO: 93) or AACUCUCUA (SEQ ID NO: 94). In one
embodiment the molecules are double stranded in at least the region
comprising the recited sequence. These multitargeting interfering
RNAs, in one embodiment, modulate expression of any combination of
VEGF-A, Bcl-2 and .kappa.-Ras in an expression system. The
multitargeting interfering RNA molecules preferably modulate
expression, in an expression system, of at least one target RNA
molecule comprising the sequences GACAGUGGA (SEQ ID NO: 95),
CUAUUCUG (SEQ ID NO: 96) or UAGAGAGUU (SEQ ID NO: 97). More
preferably they target multiple sites on two or more RNAs.
[0175] In another embodiment, provided are multitargeting
interfering RNA molecules comprising the sequence CGUGAAGAC (SEQ ID
NO: 98). In one embodiment the molecules are double stranded in at
least the region comprising the recited sequence. The
multitargeting interfering RNAs preferably modulate expression of
HCV in an expression system. In an expression system, the
multitargeting interfering molecules preferably modulate expression
of at least one target RNA molecule comprising the sequence
GUCUUCACG (SEQ ID NO: 99). More preferably they target either
multiple sites on a single RNA or multiple sites on two or more
RNAs.
[0176] It will be understood by one skilled in the art that these
exemplary seeds, and their complete complements, also subsume any
number of shorter seeds and their complete complements,
respectively, and that these are envisaged as part of the
invention. For example, the 12-base seed: CCCACCCACAUA (SEQ ID NO:
3) comprises further two 11-base, three 10-base, four 9-base, five
8-base, six 7-base and seven 6-base seeds, all of which could be
used in the design of useful multitargeting interfering RNA.
[0177] Also provided herein are multitargeting RNA duplexes
consisting essentially of:
TABLE-US-00006 5' UAUGUGGGUGGGUGAGUCUAA 3' (SEQ ID NO: 100) 3'
UUAUACACCCACCCACUCAGA 5', (SEQ ID NO: 101) 5' UGUUUUGUUGUUACAUAUGAC
3' (SEQ ID NO: 102) 3' UUACAAAACAACAAUGUAUAC 5', (SEQ ID NO: 103)
5' UAUGUGGGUGGGGUGUCUCUA 3' (SEQ ID NO: 104) 3'
UUAUACACCCACCCCACAGAG 5', (SEQ ID NO: 105) 5' UAUGUGGGUGGGGUGGUCUAA
3' (SEQ ID NO: 106) 3' UUAUACACCCACCCCACCAGA 5', (SEQ ID NO: 107)
5' UAUGUGGGUGGGGUGGUGUCU 3' (SEQ ID NO: 108) 3'
UUAUACACCCACCCCACCACA 5', (SEQ ID NO: 109) 5' UAUGUGGGUGGGUGAGUGUCU
3' (SEQ ID NO: 110) 3' UUAUACACCCACCCACUCACA 5', (SEQ ID NO: 111)
5' CUCACCCACCCACAUACAUUU 3' (SEQ ID NO: 112) 3'
CUGAGUGGGUGGGUGUAUGUA 5', (SEQ ID NO: 113) 5' UCACCCACCCACAUACAUAUU
3' (SEQ ID NO: 114) 3' UGAGUGGGUGGGUGUAUGUAU 5', (SEQ ID NO: 115)
5' UCACCCACCCACAUACAUUUU 3' (SEQ ED NO: 116) 3'
UGAGUGGGUGGGUGUAUGUAA 5', (SEQ ID NO: 117) 5' UAUGUGGGUGGGUGAGUCUA
3' (SEQ ID NO: 118) 3' UAUACACCCACCCACUCAGA 5', (SEQ ID NO: 119) 5'
GGGUUUACCAGGAAGAUGGUU 3' (SEQ ID NO: 120) 3' UACCCAAAUGGUCCUUCUACC
5', (SEQ ID NO: 121) 5' UUCCUCACAGGGCAGUGAUUC 3' (SEQ ID NO: 122)
3' UUAAGGAGUGUCCCGUCACUA 5', (SEQ ID NO: 123) 5'
UUCCUCACAGGGCAGUGAUUC 3' (SEQ ID NO: 122) 3' UUAAAGAGUGUCCCGUCACUA
5', (SEQ ID NO: 124) 5' UUCCUCACAGGGCAGUGGUUC 3' (SEQ ID NO: 125)
3' UUAAGGAGUGUCCCGUCACCA 5', (SEQ ID NO: 126) 5'
CCCGGACCCUUAGAGAGUUUU 3' (SEQ ID NO: 127) 3' ACGGGCCUGGGAAUCUCUCAA
5', (SEQ ID NO: 128) 5' UACCCUCGCACCGAUCUCCCAA 3' (SEQ ID NO: 129)
3' UUAUGGGAGCGUGGCUAGAGGG 5', (SEQ ID NO: 130) 5'
UACAAAUCUACUUCAACAUUU 3' (SEQ ID NO: 131) 3' GUAUGUUUAGAUGAAGUUGUG
5', (SEQ ID NO: 132) 5' AACAUAUGUUCUUCAACAUUU 3' (SEQ ID NO: 133)
3' GUUUGUAUACAAGAAGUUGUG 5', (SEQ ID NO: 134) 5'
UUCCACAACACAAGCUGUGUU 3' (SEQ ID NO: 135) 3' UUAAGGUGUUGUGUUCGACAC
5', (SEQ ID NO: 136) 5' GGACCCUUAGAGAGUUUCAUU 3' (SEQ ID NO: 137)
3' GGCCUGGGAAUCUCUCAAAGU 5', (SEQ ID NO: 138) 5'
UUCGUGAAGACGGUGGGCCGA 3' (SEQ ID NO: 139) 3'
dTdTAAGCACUUCUGCCACCCGG 5', (SEQ ID NO: 140) or 5'
AGACUCACCCACCCAGAUAUU 3' (SEQ ID NO: 141) 3' AAUCUGAGUGGGUGGGUCUAU
5' (SEQ ID NO: 142)
[0178] Such molecules, the skilled artisan will appreciate, can
target multiple sites on a single RNA or multiple sites on two or
more RNAs and are useful to decrease the expression of such one or
preferably two or more such targeted RNAs in an expression
system.
[0179] In some embodiments, a given multitargeting interfering RNA
will be more effective at modulating expression of one of several
target RNAs than another. In other cases, the multitargeting
interfering RNA will similarly affect all targets in one or more
expression systems. Various factors can be responsible for causing
variations in silencing or RNAi efficiency: (i) asymmetry of
assembly of the RISC causing the passenger strand to enter more
efficiently into the RISC than the guide strand; (ii)
inaccessibility of the targeted segment on the target RNA molecule;
(iii) a high degree of off-target activity by the interfering RNA;
(iv) sequence-dependent variations for natural processing of RNA,
and (v) the balance of the structural and kinetic effects described
in (i) to (iv). See Hossbach et al. (2006), RNA Biology 3: 82-89.
In designing a multitargeting interfering RNA molecule of the
invention, special attention can be given to each of the listed
factors to increase or decrease the RNAi efficiency on a given
target RNA molecule.
[0180] Another general aspect of the invention is a method for
designing a multitargeting interfering RNA. The method of the
invention includes various means leading to a multitargeting
interfering RNA that effectively targets distinct binding sequences
present in distinct genetic contexts in one or more pre-selected
target RNA molecules. In one embodiment, a multitargeting
interfering RNA can be designed by visual or computational
inspection of the sequences of the target molecules, for example,
by comparing target sequences and identifying sequences of length n
which occur in the one or more target sequences. Alternatively, all
possible sequences of a pre-selected length n can be generated by
virtue of each permutation possible for each nucleotide position to
a given length (4.sup.n) and then examining for their occurrence in
the target sequences.
[0181] In preferred embodiments, the methods provided herein design
interfering RNA molecules, either single or duplex in nature, which
have at least one region (S) of complete complementarity to a first
portion (a seed) of the distinct binding sequences, and in certain
embodiments, further comprises a second region (Y) of at least
partial complementarity to a second portion of one or more distinct
binding sequences. In other embodiments, the region Y can have
complete complementarity to a second portion of one or more
distinct binding sequences, and in yet others there may be no
complementarity between Y and a second portion of one or more
distinct binding sequences.
[0182] In one embodiment, a method of designing a multitargeting
interfering RNA comprises the steps of a) selecting one or more
target RNA molecules, wherein the modulation in expression of the
one or more target RNA molecules is desired; b) obtaining at least
one nucleotide sequence for each of the one or more target RNA
molecules; c) selecting a seed sequence of 6 nucleotides or more,
said seed sequence occurs in distinct genetic contexts in the
target RNA molecules; d) selecting at least two distinct binding
sequences, each of which comprises the seed sequence and is present
in distinct genetic contexts in the target molecules; e) designing
a multitargeting interfering RNA molecule having a guide strand
that shares a substantial degree of complementarity with each of
the at least two binding sequences to allow stable interaction
therewith.
[0183] In one embodiment, the method comprises designing a
passenger strand that is at least partially complementary to the
guide strand to allow formation of a stable duplex between the
passenger strand and the guide strand.
[0184] In another embodiment, the method of the invention comprises
the steps of: a) selecting one or more target RNA molecules,
wherein the modulation in expression of the target RNA molecules is
desired; b) obtaining at least one nucleotide sequence for each of
the target RNA molecules; c) selecting a length, n, in nucleotides,
for a seed sequence; wherein n=about 6 or more; d) generating a
collection of candidate seed sequences of the length n from each of
the nucleotide sequences obtained in step b), wherein each
candidate seed sequence occurs at least once in nucleotide
sequences obtained in step b); e) determining the genetic context
of each of the candidate seed sequences in nucleotide sequences
obtained in step b), by collecting, for each occurrence of the
candidate seed sequence, a desired amount of the 5' and 3' flanking
sequence; f) selecting a seed sequence of the length n from the
candidate seed sequences, wherein the seed sequence occurs in at
least two distinct genetic contexts in nucleotide sequences
obtained in step b); g) selecting a consensus target sequence,
wherein said consensus target sequence comprises the seed sequence
and a desired consensus sequence for the sequence flanking either
one or both of the 5' and 3' ends of the seed; and h) designing a
multitargeting interfering RNA molecule that comprises a guide
strand that shares a substantial degree of complementarity with the
consensus target sequence to allow stable interaction
therewith.
[0185] In one embodiment, the method comprises designing a
passenger strand that is at least partially complementary to the
guide strand to allow formation of a stable duplex between the
passenger strand and the guide strand.
[0186] One of skill in the art would recognise that several
iterations of various steps of the methods can be performed. For
example, in some embodiments, the method further comprises
repeating steps c) to h) with a different value of n. In other
embodiments, the method may comprise repeating steps f) to h) for a
different seed sequence. In yet other embodiments, the method may
comprise selecting a different consensus target sequence based
around a particular seed and flanking sequence. The skilled person
will recognise that various combinations of the above are also
envisaged.
[0187] In other embodiments, the method of the invention further
comprises the steps of making the multitargeting interfering RNA
molecule and testing it in an expression system.
[0188] Preferred target RNA molecules are strategically-selected
molecules, for example viral or host RNAs involved in disease
processes, viral genomes, particularly those of clinical
significance, and the like. A detailed discussion of target RNA is
provided above and applies equally to this and other aspects of the
invention, as if set out in its entirety here. The basis for the
selection of a target RNA molecule will be appreciated by those of
skill in the art. Preferred target RNAs are those involved in
diseases or disorders one wishes to control by the administration
of the multitargeting interfering RNA
[0189] The step of obtaining the sequences for the selected target
is conducted by obtaining sequences from publicly available
sources, such as the databases provided by the National Center For
Biotechnology Information (NCBI) (through the National Institutes
of Health (NIH) in the United States), the European Molecular
Biology Laboratories (through the European Bioinformatics Institute
throughout Europe) available on the World-Wide Web, or proprietary
sources such as fee-based databases and the like. Sequences can
also be obtained by direct determination. This may be desirable
where a clinical isolate or an unknown gene is involved or of
interest, for example, in a disease process. Either complete or
incomplete sequences of a target RNA molecule can be used for the
design of multitargeting interfering RNA of the invention.
[0190] Also provided herein are methods wherein a plurality of
independent target nucleotide sequences are obtained in step b) for
each of one or more target RNA molecules selected in step a). The
databases described above frequently have multiple sequences
available for particular targets. This is especially true where
genetic variation is naturally higher, for example with viral
sequences. In various embodiments, the plurality of target
nucleotide sequences represents strain variation, allelic
variation, mutation, or multiple species. The number of such a
plurality of sequences may range from several or a low multiple, to
numerous--for example dozens or even hundreds or thousands of
sequences for a given target. It is especially possible to have
such numbers of sequences when working with viral sequences.
[0191] The sequences chosen can be further limited based on
additional desirable or undesirable features such as areas of low
sequence complexity, poor sequence quality, or those that contain
artifacts relating to cloning or sequencing such as inclusion of
vector-related sequences. Furthermore, regions with extensive
inaccessible secondary structure could be filtered out at this
stage. Indeed, Luo and Chang have demonstrated that siRNA targeting
accessible regions of mRNA structure such as loops were more likely
to be effective than those aligned with stems (Luo & Chang,
(2604), Biochem. Biophys. Res. Commun., 318: 303-10). The sequences
chosen, however, need not be limited to 3'UTR sequences or regions
of low secondary structure (as illustrated in some of the specific
examples.)
[0192] The step of selecting a length of n nucleotide bases for a
seed sequence is preferably an iterative process that does not
require any particular basis or logic at first glance--i.e. the
starting seed length may be any number of bases above about 5. The
longer the length that is chosen for a seed, the less likely it
will appear in a portion of the target RNA, e.g. in a target RNA
binding sequence. The shorter the seed sequence length, the more
frequently it will occur as would be expected. Preferably, an
iterative process is used to find the preferred sequences for
candidate seeds as described below. Thus, after a particular value
for n is used to identify candidate seeds of length n, another
value (e.g. n+1, n-1) will be used and the process can be repeated
to identify candidate seed sequences of length n+1, n-1 and so on.
In one embodiment, the first scan through target sequences will
begin with any seed length (e.g. n=9) and subsequent rounds of
searching will either increase or decrease the seed length (e.g.
based on the number of seeds returned in previous scans). A person
of ordinary skill in the art will recognize that the number of
candidate seeds will increase as the length of the seed is
decreased.
[0193] In a situation where a plurality of sequences are available
for particular target RNA molecule (eg viral isolates), it will be
appreciated by one of skill in the art that the totality of the
sequences can be searched for candidate seeds. However, candidate
seeds may in some cases be found only in a proportion of the
sequences for the RNA molecule. In these situations, it may be
desirable to prioritise those seeds which occur in a larger
proportion of the sequences.
[0194] The seeds can be selected from a pool of "candidate seeds,"
also referred to herein as "seed candidates." Seed candidates
include sequences of a particularly desired or selected length
present at least once in each of the target molecules. The
candidate seeds are preferably generated by computer, for example
by moving stepwise along a target sequence with a "window"
(expressed in terms of a fixed number of contiguous nucleotides) of
the desired or selected seed length. Preferably each step is a
single base progression, thus generating a "moving window" of
selected length through which the target sequence is sequentially
viewed. Other step distances are contemplated, however, the skilled
artisan will appreciate that only a step of one nucleotide will
allow the generation of all possible seed sequences.
[0195] When there is only one target RNA molecule, the pool of
candidate seeds includes any sequences of the selected length from
the "moving window" scan of the target RNA molecule. When there are
multiple target RNA molecules, the pool of candidate seeds includes
those sequences of the selected length from the "moving window"
scan of the target RNA molecules, which occur at least once in each
of the target RNA molecules. Therefore when there are multiple
target RNA molecules, step (d) above comprises two substeps: (i)
generating a collection of nucleotide sequences of the selected
length n from each nucleotide sequence obtained in step b); and
(ii) selecting candidate seed sequences of the length n from the
collections of nucleotide sequences, wherein each candidate seed
sequence occurs at least once in the nucleotide sequences obtained
in step b).
[0196] As used herein, the term "distribution" in the context of a
seed or candidate seed means the overall average frequency, or
number of occurrences, of a seed or candidate seed within a
nucleotide sequence of interest. For example, Example 13 (below)
has the following pattern of occurrence for a 9 base seed in HCV
isolates of genotype 1a/1b:
[0197] 4 times in genome: 5/155 isolates
[0198] 3 times in genome: 68/155 isolates
[0199] 2 times in genome: 50/155 isolates
[0200] 1 time in genome: 31/155 isolates
[0201] 0 times in genome: 1/155 isolates
[0202] This equates to a "distribution" of 2.29. In comparison, the
"conservation" (see below) would be 154/155 (=99%).
[0203] In one embodiment, the method further comprises the step of
discarding those candidate seed sequences that do not occur with at
least a predetermined minimum average rate of occurrences within
the sequences obtained for a target RNA molecule. This step allows
the method to take into account the average distribution of the
candidate seed sequences within sequences for the same target
RNA.
[0204] In another embodiment, the method further comprises the step
of discarding those candidate seed sequences that do not occur
within at least a predetermined minimum percentage of the target
nucleotide sequences obtained for a target nucleotide molecule
(that is, the extent of conservation). This step allows the method
to more fully take into account the frequency with which a
particular candidate seed occurs across multiple target sequences
obtained for a target RNA. The multitargeting function of preferred
RNAs made by the methods provided herein is enhanced by the higher
representation of a candidate seed sequence both within and across
such sets of target sequences.
[0205] Preferably the method ultimately chosen will include one or
more of these steps, or all of them as needed. For example, in one
embodiment, the method further comprises the step of discarding any
candidate seed sequence that: is composed of only a single base, is
composed only of A and U, has a consecutive string of 5 or more
bases which are C, is predicted to have a propensity to undesirably
modulate the expression or activity of one or more cellular
components, is predicted to occur with unacceptable frequency in
the non-target transcriptome of interest; or any combination
thereof. In the situation in which a double stranded multitargeting
interfering RNA is to be designed, candidate seed sequences may
also be discarded if they contain sequences predicted to have a
propensity to activate a cellular sensor of foreign nucleic acid,
have a consecutive string of 5 or more bases which are G, or a
combination thereof.
[0206] Seeds then are selected from the pool of candidate sequences
as the ones that are present in two different genetic contexts in
the one or more target sequences. Genetic contexts are determined
by collecting, for each occurrence of the candidate seed sequence,
a desired amount of the 5' and 3' flanking sequence.
[0207] In an exemplary process of making a multitargeting
interfering RNA of the invention, a seed sequence is used to
generate one or more "consensus target sequences", which comprises
the seed sequence and a desired consensus sequence for the sequence
flanking either one or both of the 5' and 3' ends of the seed. The
term "consensus target sequence" does not suggest that there is
only one best sequence approximating multiple binding sequences on
target molecule(s), rather a population of one or more alternative
sequences may all be consensus target sequences. The "consensus
sequence for the sequence flanking either one or both of the 5' and
3' ends of the seed" is readily derived from the examination of the
genetic context of seed sequence in each of the target molecules by
visual inspection, or through the use of bioinformatic tools or
calculations. While the seed sequence portion of a consensus target
sequence will usually have complete identity to a corresponding
portion in each of targeted binding sites, the consensus sequence
for the sequence flanking either one or both of the 5' and 3' ends
of the seed need not be completely identical to the sequence
flanking the 5' and 3' ends of the seed of some but not all of the
sequences of the target molecules. However, it may be identical to
some, all or none of the sequences.
[0208] Preferably, when a double stranded molecule is to be
designed, the consensus target sequence does not include any
sequence that is predicted to have a propensity to undesirably
modulate the expression or activity of one or more cellular
components.
[0209] Consensus target sequences may be determined by eye or by
algorithm. For example, a computer algorithm can be used to score
all possible permutations of paired nucleotides in the positions in
which the sequences are different between the at least two targets.
This is particularly useful when the targets have some identity
beyond the seed, but for which an alignment by eye proves
difficult. This method can be used to determine the consensus
target sequence, or alternatively, the antisense strand of the
candidate multitargeting interfering RNA directly. When designing
the antisense strand directly, the algorithm scores wobbles (G:U,
U:G), other non-canonical pairs (eg A:C, C:A) and remaining
mismatches with increasingly large negative penalty terms,
respectively. These penalty terms are adjusted for their position
within the multitargeting interfering RNA, with those placed at the
3' extremity incurring lower fractional penalties. Penalties are
also adjusted in the presence of multiple contiguous mismatches or
wobbles. Those proposed multitargeting interfering RNAs with the
lowest aggregate penalty scores for all targets are prioritized for
further evaluation.
[0210] A further alternative approach that is particularly useful
when there is little identity between the target sequences outside
of the seed sequence or when a large number of target sequences
need to be considered (eg when large numbers of nucleotide
sequences for viral isolates are screened) is to generate all
possible permutations of the extension from the complement of the
seed in the 3' direction to a required length, thereby generating
the putative SY of Formula (I) and hybridising each putative SY
against the target sequences of interest in silico to determine
those which demonstrate the most favourable properties in terms of
hybridisation to the target and preferential strand loading.
[0211] In most cases, when a duplex molecule is being designed, the
overhangs, if required, are considered as part of the hybridization
process. Hybridization is typically examined from a thermodynamic
perspective using RNAhybrid software (Rehmsmeier et al., 2004, RNA,
10: 1507-17) or similar algorithm.
[0212] In one embodiment, the invention provides an algorithm for
determination of the guide strand of a length n for a
multitargeting interfering RNA comprising a seed sequence. [0213]
1) Definition of terms [0214] a. Load Bias: Scoring A or U as "1"
and G or C as "2" in each candidate guide strand: [0215] i.
Multiply the A-U/G-C score by the relevant scaling factor in the
Table below [0216] ii. sum the products for the residues at
positions 1-5. [0217] iii. sum the products for the residues at
positions n to n-4. [0218] iv. divide the total at (iii) by the
total at (ii)
TABLE-US-00007 [0218] 3' end of the guide 5' end of the guide
strand (excepting strand overhangs) Residue Scaling Residue Scaling
position factor position factor 1 5 n - 4 1 2 4 n - 3 2 3 3 n - 2 3
4 2 n - 1 4 5 1 N 5
[0219] b. Activity Score: For each target T.sub.1 . . . T.sub.n,
calculate the product of the minimum free energy (mfe) of the
binding of the guide strand and the number of contiguous bases
complementary to the target at the 5' end of the guide strand.
(r-5'). The product of these scores is the Activity Score.
[0219] T.sub.1(mfe*r-5')*T.sub.2(mfe*r-5')* . . . T.sub.n(mfe*r-5')
[0220] c. Maximum Activity Score: For each target T.sub.1 . . .
T.sub.n calculate the product of the mfe of the binding of that
guide strand which is completely complementary to the target and
the length of the guide strand (for example: 21 bases as shown
below). The product of these scores is the Maximum Activity
Score.
[0220] T.sub.1(mfe*21)*T.sub.2(mfe*21)* . . . T.sub.n(mfe*21)
[0221] d. Relative Activity Score: The Activity Score for an
interfering RNA divided by the Maximum Activity Score for the
targets. [0222] 2) Starting with the complement of the seed
sequence, generate all possible permutations for the extension of
the complement of the seed to a length of n, for example 21 bases,
thus creating a complete list of putative guide strands [0223] 3)
Use RNAhybrid to determine the binding pattern and minimum free
energy of all the putative guide strands against all the target
sequences which contain the seed sequence; [0224] 4) Discard all
putative guide strands where [0225] a. There is a contiguous run of
5 or more G residues [0226] b. The Load Bias is <1.2 [0227] 5)
Rank the remaining putative guide strands by their Relative
Activity Score and select a guide strand sequence of the length n
for a multitargeting interfering RNA sequence from the list of the
remaining putative guide strand sequences based on their Relative
Activity Score.
[0228] Consider a certain number or fraction (eg the top 1%) of the
putative guide strands for additional steps, such as overhangs,
chemical modifications etc, synthesis and determination of
biological activity.
[0229] In a particular embodiment, a simple algorithm that may be
useful in the case of a fully complementary duplex is the
following:
[0230] Scoring A or U as "1" and G or C as "2" in the guide strand:
[0231] i. multiply the A-U/G-C score by the relevant scaling factor
in the Table A below; [0232] ii. sum the products for the residues
at positions 1-5; [0233] iii. sum the products for the residues at
positions n-4 to n (excepting overhangs if present); [0234] iv.
divide the total at (iii) by the total at (ii); and [0235] v.
scores greater than 1.2 for the guide strand indicate likely
favourable loading of that strand.
TABLE-US-00008 [0235] TABLE A 5' end of guide 3' end of guide
strand strand Residue Scaling Residue Scaling position factor
position factor 1 5 n - 4 1 2 4 n - 3 2 3 3 n - 2 3 4 2 n - 1 4 5 1
n 5 n = number of nucleotides involved in forming the duplex
(excluding overhangs if present).
[0236] It will be appreciated by one skilled in the art that in the
case of a double stranded multitargeting interfering RNA, ensuring
maximum strand loading of the desired guide strand is beneficial
not only with respect to the potency of the molecule but also will
greatly reduce inadvertent off-target effects resulting from
incorporation of the passenger strand into RISC.
[0237] Sequences demonstrating strong binding (typically having
mean free energies of <-20 kcal/mol) are of particular interest
for the multitargeting interfering RNA. As before, one skilled in
the art will appreciate that this method, used to design the active
multitargeting interfering RNA can, with slight modification, be
used to design suitable consensus target sequences, which are then
converted to putative SY sequences. Regardless of the flow path of
design, the candidate SY sequences are then prioritized for testing
not only on this basis but also taking into account other features
that may be important for the functionality of the multitargeting
interfering RNA (by, for example, use of appropriate penalty
terms). This may involve discarding those putative SY sequences
which are composed of only a single base, are composed only of A
and U, are predicted to be involved in substantial intramolecular
base pairing, have a consecutive string of 5 or more bases which
are G, are predicted to occur with unacceptable frequency in the
antiparallel orientation in the non-target transcriptome of
interest; are predicted to have a propensity to activate a cellular
sensor of foreign nucleic acid, or any combination thereof. In some
cases, the addition of one or two nucleotides to the 5' end of the
putative SY (ie X) is considered. This is particularly relevant
when an otherwise useful SY sequence is G/C rich at the 5' end and
this is predicted to disfavor loading in the case of a double
stranded multitargeting interfering RNA. The addition of one or two
A/U nucleotides to the 5' extremity of the putative SY sequence,
will most likely improve loading. Another situation that
necessitates this addition includes, but is not limited to, when
there is a need for the double stranded multitargeting interfering
RNA to be able to support target cleavage. In most cases, cleavage
requires the active strand to be complementary to the target
spanning the site bounded by the 10 and 11.sup.th nucleotides. When
the seed is of insufficient length (eg n=9), the addition of two
additional nucleotides to the 5' end of SY will shift S along
sufficiently to fulfill this requirement. Because multitargeting
interfering RNA in most cases tolerate mismatches at positions 1
and 2, the addition of this additional region X, which need not be
complementary to the at least two target sequences, further
increases the flexibility of design.
[0238] In the case of a double stranded multitargeting interfering
RNA of the invention, a further embodiment comprises the step of
discarding seeds that are extremely G/C rich at their 3' extremity.
A G/C rich 3' extremity of the seed may be undesirable as the
resulting matching guide strand could be disadvantaged from a
loading point of view, depending on the G/C content of the 3' end
of the guide strand (excluding the overhangs). Whilst this may not
be preferable, strategies can be used to overcome the predicted
poor loading bias when seeds are G/C rich at their 3' end. These
include, but are not limited to, the use of an A/U-rich extension
in the 5' end of the guide strand, i.e. "X" in Formula (I), or the
selection of G/C rich termini in the design of the 3' end of the
guide strand i.e. `Y` in Formula (I). Also, the substitution of U
for C in the corresponding passenger strand by virtue of wobble
base pairing will at least partially rectify strand loading when
there is a G near the 5' terminus of the guide strand. Finally, one
skilled in the art will appreciate that modifications that disfavor
strand loading could be used on the passenger strand to further
enhance the loading of the desired strand. Such modifications also
include manipulation of the length and composition of the
overhangs. Alternatively, chemical modifications that increase the
binding energy of RNA duplexes such as LNA, 2'-O-methyl and
2'-methoxyethyl can be used for bases positioned at the 5' end of
the passenger strand so as to favor loading of the guide
strand.
[0239] Various steps can optionally be added individually or in
combination. Such steps are used to further the rational process of
designing the multitargeting interfering RNAs--such as to reduce
the number of sequences unlikely to work for the intended purpose,
to increase the effectiveness of the RNAs, to reduce off target
effects and the like. Many of these steps can be automated, or
require only a limited amount of input from an operator, though the
use of bioinformatic computer systems, which as the skilled artisan
will appreciate, will facilitate the methods.
[0240] Similar to the situation with antisense, for which it is now
recognized that there are specific sequences that have a high
propensity to activate cellular sensors of foreign DNA, other
receptors may detect particular RNA sequences and produce stress
responses (for example, see Sioud, M. (2005), J Mol Biol 348,
1079-1090. Specific "motifs" associated with increased inflammatory
responses (Hornung, V. et al. (2005) Nat Med 11, 263-270; Judge, A.
D. et al. (2005) Nat Med 11, 263-270) could be easily excluded.
[0241] DNA sequences with stretches of contiguous guanosines are
known to produce additional effects not related to targeting of
mRNA. Although the situation in the case of RNA is less clear, most
manufacturers recommend not selecting dsRNA duplexes containing
long runs of guanosines (G) for their experiments. It was found in
this invention that greater than 4 consecutive G greatly reduced
the activity of the corresponding CODEMIR (see Example 17).
Therefore, many seeds could be eliminated if they contain 5 or more
contiguous C. One skilled in the art will recognise that the
presence of 5 or more Cs in a seed will correspond to 5 or more Gs
in the active portion of the RNA molecule of the invention.
However, we also consider that it is desirable to further exclude
the occurrence of 5 or more Gs in the passenger strand when the
molecule is double stranded, therefore the occurrence of 5 or more
contiguous Gs in the seed in combination with the consensus target
sequence is also considered to be undesirable when a duplex
molecule is to be constructed.
[0242] A further embodiment to excluding seeds specifically related
to duplex molecules of the invention applies to the presence of a
G/C rich region at the 3' end and to the presence of 5 or more
contiguous Gs in the seed or consensus target sequence (which
includes the seed).
[0243] In another embodiment, the method further comprises the step
of discarding any consensus target sequence that: is composed of
only a single base, is composed only of A and U, has a consecutive
string of 5 or more bases which are C, is G/C rich at the 3' end,
is predicted to occur with unacceptable frequency in the non-target
transcriptome of interest; or any combination thereof. In the
situation in which a double stranded multitargeting interfering RNA
is to be designed, consensus target sequences may also be discarded
if they contain sequences predicted to have a propensity to
activate a cellular sensor of foreign nucleic acid, if they are
predicted to be involved in intramolecular base pairing, have a
consecutive string of 5 or more bases which are G, or a combination
thereof.
[0244] Like candidate seeds and seeds, consensus target sequences
can be used as intermediates in the design of a multitargeting
interfering RNA of the invention. In particular, the consensus
target sequences are used to design a strand of the corresponding
multitargeting interfering RNA The consensus target sequences are
at least substantially the complement of a "guide strand" of a
candidate multitargeting interfering RNA. Preferably they are the
complete complement of a "guide strand" of a candidate
multitargeting interfering RNA. The consensus target sequence is at
least possibly, but not necessarily, identical to the "passenger
strand" of the corresponding candidate multitargeting interfering
RNA, when these are double stranded. They may be non-identical
because "passenger strands" are frequently further refined to
optimize RISC loading and other functionality through the use of
sequence modifications, for example, end modifications, such as
inclusion of overhangs (non-blunt ends, e.g. 3'-UU), and the
incorporation of mismatches and wobble bases. Such modifications
will be understood by those of skill in the art.
[0245] Scanning the consensus target sequences against a non-target
transcriptome of interest for prediction of off-target effects, and
eliminating any sequence predicted to have unacceptable off-target
effects on a non-target transcriptome of interest are also useful
ways of reducing the number of consensus target sequences, and any
of the foregoing may be added as a step in the process. This is
performed by searching for similar sequences using, for example,
BLAST software. An alternative, but not necessarily equivalent
procedure includes the in silico hybridization of the complement of
the consensus target sequence against the transcriptome using, for
example, RNAhybrid or equivalent software. In practice, it is
prudent to routinely screen specific designed multitargeting
interfering RNAs, e.g. CODEMIRs, VIROMIRs and the like, for
cytotoxicity, due to unforeseen, but problematic, off-target
effects.
[0246] Any undesirable properties for such a therapeutic RNA, as
would be understood by those of skill in the art, can be used as a
basis on which to discard candidate seed sequences, consensus
binding sites or proposed multitargeting interfering RNA.
[0247] The RNA molecules obtained from the basic method outlined
above can be modified and often will be to improve their
properties. The methods then can further comprise the step of
modifying a multitargeting interfering RNA duplex designed in step
h) to improve actual or predicted loading into a RISC complex, to
improve activity against at least one target RNA molecule, to
decrease stress or inflammatory response when administered in a
host cell; to alter half-life in an expression system, or any
combination thereof.
[0248] In certain embodiments, the designed multitargeting
interfering RNA molecule can be modified, for example, i) to
improve the incorporation of the guide strand of the multitargeting
interfering RNA molecule into the RNA induced silencing complex
(RISC); ii) to increase or decrease the modulation of the
expression of at least one target RNA molecule; iii) to decrease
stress or inflammatory response when the multitargeting interfering
RNA molecule is administered into a subject; or iv) any combination
of i) to iii).
[0249] The skilled artisan will understand how to modify the RNA
molecules either in the laboratory, or preferably in silico. In
preferred embodiments the modifying step comprises one or more of
altering, deleting, or introducing one or more nucleotide bases to
create at least one mismatched base pair, wobble base pair, or
terminal overhang, or to increase RISC mediated processing.
Techniques for doing so are known in the art. Preferably the
modifications are at least initially performed in silico, and the
effects of such modifications can be readily tested against
experimental parameters to determine which offer improved
properties of the interfering RNA products.
[0250] In a presently preferred embodiment, the methods, through to
the step of actually making an RNA, are conducted entirely in
silico, or by visual inspection and determination. In one
embodiment the method further comprises the step of choosing a new
value for the seed length, n, and repeating each of the remaining
steps. It is clear that the method can be iterative and the
benefits of computers for such purposes are well known. Also
provided herein are methods that further comprise the step of
actually making and testing at least one designed interfering RNA
in a suitable cellular expression system. This will be necessary so
as to identify those interfering RNA that have the required or
sufficient activity against the target RNA molecules or that
produce the required phenotype in the model system (eg death of
cancer cell, inhibition of angiogenesis, suppression of lesion
formation, accelerated wound healing etc).
[0251] As will be appreciated, large numbers of seeds and thereby
potential multitargeting interfering RNAs can be generated using
the above methodology. While the rules above can be used to filter
potential candidates based on undesirable properties, one skilled
in the art will appreciate that with access to high throughput
screening methodologies as well as recent improvements in fidelity,
cost and access to RNA synthesis that testing of hundreds to
thousands of candidates can be easily performed to further assist
in the development of active multitargeting interfering RNAs. Thus,
it is occasionally preferable to screen significant numbers of
candidates as opposed to prioritising a few candidates solely on
the basis of algorithmic design. A combination of careful in silico
design along with biological testing of candidates can be used to
identify candidates with superior activity in an efficient
manner.
[0252] Screens that can be considered for the high throughput
assessment of candidates include reporter assays, multiplexed
ELISAs, viral replicon systems, dot-blot assays, RT-PCR etc.
[0253] Candidate multitargeting interfering RNA are routinely
synthesized as double-stranded RNA molecules with 19 bp of
complementarity and 3' two nucleotide overhangs. For the guide
strand (the strand with complementarity to the target RNAs and
which is predicted to be incorporated into RISC), the two
nucleotide overhangs are routinely designed to be complementary to
the target RNAs, although dTdT or UU overhangs may also suit. The
passenger strand (complementary to the guide strand) can be usually
designed to include a 3' two nucleotide UU overhang. However, other
types and lengths of overhangs could be considered, as could
"blunt-ended" duplexes. Candidate multitargeting interfering RNA
can also be single-stranded molecules.
[0254] When produced by an expression system such as a vector or
plasmid, it is possible to assemble multiple multitargeting
interfering RNAs into a single therapeutic product. Skilled
artisans will realize that multiple multitargeting interfering RNAs
can be co-expressed by several strategies, including but not
limited to, expression of individual multitargeting interfering
RNAs from multiple expression vectors (plasmid or viral),
expression from multiple expression cassettes contained within a
single vector, with each expression cassette containing a promoter,
a single multitargeting interfering RNA and terminator. Multiple
multitargeting interfering RNAs can also be generated through a
single polycistronic transcript, which contains a series of
multitargeting interfering RNAs.
[0255] The multiple multitargeting interfering RNAs can be
expressed sequentially (sense/intervening loop/antisense) or
expressed with the sense sequence of each multitargeting
interfering RNA sequentially linked 5' to 3', joined directly or
with intervening loop/spacer sequence, followed by the antisense
sequence of each multitargeting interfering RNAs sequentially
linked 5' to 3'.
[0256] In the first instance, multitargeting interfering RNA are
typically tested in cell culture using an appropriate cell line
representative of the targeted tissue. Some non-limiting specific
conditions used are outlined in the specific examples.
Multitargeting interfering RNA that modulate target RNA expression
or activity can then be studied further. Specifically,
semi-quantitative RT-PCR for the target RNA may be performed to
establish whether repression is likely to be mediated by RNA
degradation. In general, cells are transfected with the
multitargeting interfering RNA at a concentration of 5-40 nM in the
culture medium and after 48 hours, are washed, trypsinized and
harvested for total RNA using a RNeasy kit (Qiagen). RT-PCR is then
performed using primer sets specific for the target RNAs.
[0257] Proteomic and microarray experiments may be used to assess
off-target effects. Likewise, to select active multitargeting
interfering RNA with little propensity for activation of innate
immune response, analysis of markers of IFN-response (eg STAT1,
IFNb, IL-8, phosphoEIF etc) can be performed on treated cells.
[0258] Preferably, the candidate multitargeting interfering RNA are
tested for non-specific toxic effects by, for example, direct
assays of cell toxicity. Alternatively, in some cases such as
cancer, cytotoxicity is the desired outcome and may reflect the
successful repression of key oncogenic signaling pathways.
Multitargeting interfering RNA are additionally assessed for their
ability to repress the production of specific target proteins.
Multitargeting interfering RNA demonstrating efficacy in this
respect are then assessed for additional evidence of off-target
effects, including arrest of non-target protein production and
activation of Protein Kinase R mediated responses.
[0259] The RNA molecule may be expressed from transcription units
inserted into vectors. The vector may be a recombinant DNA or RNA
vector, and includes DNA plasmids or viral vectors. The viral
vectors expressing the multitargeting interfering RNA molecules can
be constructed based on, but not limited to, adeno-associated
virus, retrovirus, adenovirus, lentivirus or alphavirus.
[0260] Preferably the vector is an expression vector suitable for
expression in a mammalian cell.
[0261] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing a sequence which
encodes the multi target RNA molecule. These methods include in
vitro recombinant DNA techniques, synthetic techniques and in vivo
recombination or genetic recombination. Such techniques are
described in Sambrook et al (1989) Molecular Cloning, A laboratory
manual, Cold Spring Harbor Press, Plainview N.Y. and Asubel F M et
al (1989) Current Protocols in Molecular Biology, John Wiley &
Sons, New York N.Y. Suitable routes of administration of the
pharmaceutical composition of the present invention may, for
example, include oral, rectal, transmucosal, or intestinal
administration; parenteral delivery, including intramuscular,
intravenous and subcutaneous injections.
[0262] Alternatively, the pharmaceutical composition may be
administered in a local rather than systemic manner, for example,
via injection of the pharmaceutical composition directly into a
target organ or tissue, such as intramedullary, intrathecal, direct
intraventricular, intraperitoneal, or intraocular injections, often
in a depot or sustained release formulation. Intravesicular
instillation and intranasal/inhalation delivery are other possible
means of local administration as is direct application to the skin
or affected area. Ex vivo applications are also envisaged.
[0263] Furthermore, the pharmaceutical composition of the present
invention may be delivered in a targeted delivery system, for
example, in a liposome coated with target cell-specific antibody.
The liposomes will be targeted to and taken up selectively by the
target cell. Other delivery strategies include, but are not limited
to, dendrimers, polymers, nanoparticles and ligand conjugates of
the RNA.
[0264] The multitargeting interfering RNA molecules of the
invention can be added directly, or can be complexed with cationic
lipids, packaged within liposomes, or otherwise delivered to target
cells or tissues. The nucleic acid or nucleic acid complexes can be
locally administered to relevant tissues ex vivo, or in vivo
through injection, infusion pump or stent, with or without their
incorporation in biopolymers.
[0265] In another aspect, the invention provides biological systems
containing one or more multitargeting interfering RNA molecule of
this invention. The biological system can be, for example, a virus,
a microbe, a plant, an animal, or a cell. The invention also
provides a vector comprising a nucleotide sequence that encodes the
multitargeting interfering RNA molecule of the invention. In
particular embodiment, the vector is viral, from example, derived
from a virus selected from the group consisting of an
adeno-associated virus, a retrovirus, an adenovirus, a lentivirus,
and an alphavirus. The multitargeting interfering RNA can be short
a hairpin RNA molecule, which can be expressed from a vector of the
invention. The invention further provides a pharmaceutical
composition comprising a multitargeting interfering RNA molecule of
the invention and an acceptable carrier. In particular embodiments,
the pharmaceutical composition comprises a vector for a
multitargeting interfering RNA molecule of the invention.
[0266] In another general aspect, the present invention provides a
method of inducing RNA interference in a biological system, which
comprises the step of introducing a multitargeting interfering RNA
molecule of the invention into the biological system.
[0267] In a particular embodiment, the present invention provides a
method of inducing RNA interference in a biological system,
comprising the steps of: (a) selecting a set of target RNA
molecules; (b) designing a multitargeting interfering RNA molecule
comprising a guide strand that can form stable interactions with at
least two binding sequences present in distinct genetic contexts in
the set of target RNA molecules; (c) producing the multitargeting
interfering RNA molecule; and (d) administering the multitargeting
interfering RNA molecule into the biological system, whereby the
guide strand of the multitargeting interfering RNA molecule forms
stable interactions with the binding sequences present in distinct
genetic contexts in the set of target RNA molecules, and thus
induces RNA interference of the target RNA molecules.
[0268] In another particular embodiment, the present invention
provides a method of treating a disease or condition in a subject,
the method comprising the steps of: (a) selecting a set of target
RNA molecules, wherein the modulation in expression of the target
RNA molecules is potentially therapeutic for the treatment of the
disease or condition; (b) designing a multitargeting interfering
RNA molecule comprising a guide strand that can form stable
interactions with at least two binding sequences present in
distinct genetic contexts in the set of target RNA molecules; (c)
producing the multitargeting interfering RNA molecule; (d)
administering the multitargeting interfering RNA molecule into the
subject, whereby the guide strand of the multitargeting interfering
RNA molecule forms stable interactions with the binding sequences
present in distinct genetic contexts in the set of target RNA
molecules, and thus induces modulation of expression of the target
RNA molecules.
[0269] One skilled in the art will recognize that these design
steps can be performed in a different order to produce an
equivalent final product. Also, one skilled in the art will
recognize that some steps can be substituted with alternative
procedures that are broadly equivalent.
[0270] The target RNA molecule or molecules can be selected, for
example, from any RNA in a cell or virus. A viral genome, or even
multiple viral genomes, for example two or more related or
unrelated viruses, can also be conveniently targeted. The useful or
desirable targets for any disease or related process may be
identified by any of one or more means including, for example,
projected or validated drug targets from the literature, including
the patent literature, or from target discovery processes. The
skilled artisan will understand and appreciate how to select useful
or desirable targets. The target genes are then prioritized based
on evidence supporting a key role for their products in the disease
process of interest.
[0271] In some cases, specific attention may need to be paid to the
accuracy and/or relevance of the sequence to the disease of
interest. For example, in targeting cancer, it is advisable to
avoid mutational "hot-spots". It is also to be noted that the
sequence used need not be the complete sequence. Also, selective
targeting of a specific splice variant or isoform may be desired
and the target sequence used in the design of multitargeting
interfering RNAs may need to be restricted to that predominantly
present only in the diseased tissue that is targeted by the
multitargeting interfering RNA of the invention.
[0272] The use of single- or double-stranded RNA compounds that can
target multiple sites within a viral genome, for example, viral RNA
targets, is also provided herein. The multitargeting interfering
RNA molecules that target multiple sites in the genome of one or
multiple isolates of a virus are sometimes referred herein as
"VIROMIRs". Targeting repeated sequence elements in viral genomes
is an attractive approach for viral therapy. Such multitargeting is
calculated to create a formidable hurdle to the emergence of
resistant clones, which would require multiple, simultaneous,
mutations. Also, multiple sites can be chosen to maximize coverage
of sequence variations across a range of viral isolates. Elements
can be identified computationally that are present in a
pre-selected percentage of isolates, such as a majority or even the
totality of known isolates, thereby ensuring maximal therapeutic
benefit. Alternatively, isolates of greatest actual or projected
clinical significance can be preferentially targeted. The design
process can also facilitate development, manufacture, and
ultimately administration of the therapeutic compounds. The
additional targeting of one or more host proteins or other
intermediates of the pathway involved in the pathogenesis of the
viral disease can also be designed. Bacterial infection and any
other disease caused by a pathogen can be targeted by a similar
approach.
[0273] The RNA compounds of the present invention can be used to
treat or prevent diseases in plants, animals and in particular
humans. The RNA compounds can be either cell-expressed into the
relevant plant, animal or human cell to derive the required effect
or be administered as a chemically synthesized compound directly or
indirectly by means of a delivery molecule or device. The RNA
compounds of the present invention can be used to treat or minimise
pest attack on plants and animals. The pests may be vertebrate or
invertebrate.
[0274] The present invention may be used for treatment or
prevention of a disease state resulting from expression of the
target genes. Disease states include, but are not limited to,
autoimmune diseases, inherited diseases, cancer, or infection by a
pathogen. Treatment would include prevention or amelioration of any
symptom or clinical indication associated with the disease.
[0275] In a preferred embodiment, the disease state is cancer (eg
colorectal adenocarcinoma), diabetes, diabetic retinopathy, age
related macular degeneration (AMD), psoriasis, HIV infection, HCV
infection or Alzheimer's disease. The current invention therefore
encompasses, but is not limited to, the concept of treating these
diseases with multitargeting interfering RNAs. As can be readily
appreciated, the same principles can be applied to the treatment of
all other complex diseases.
[0276] The selection of target sequences for the multitargeting
interfering RNAs will be determined by the disease(s) against which
the therapeutic is desired. The target RNA sequence may include
mRNA, and noncoding RNA and may be encoded either by the host or
infectious agent or both. The target RNA set for any one
multitargeting interfering RNA can be selected from any of these
types and may represent any combination of gene or gene products
including but not limited to: receptors, cytokines, transcription
factors, viral genes, bacterial genes, plant genes, insect genes,
yeast and fungal genes, regulatory and/or signalling proteins,
non-coding RNA, cytoskeletal proteins, transporters, enzymes,
hormones, antigens, hypothetical proteins and proteins of unknown
function.
[0277] Additionally, targeting of different isolates of a
pathogenic agent is envisaged. The multiple target sites can then
be chosen to maximise coverage of sequence variations across a
range of isolates.
[0278] The modulation of the target RNA molecule is determined in a
suitable expression system, for example in vivo, in one or more
suitable cells, or in an acellular or in vitro expression system
such as are known in the art. Routine methods for measuring
parameters of the transcription, translation, or other aspects of
expression relating to RNA molecules are known in the art, and any
such measurements are suitable for use herein.
[0279] The multitargeting RNAs in accordance with various aspects
of the invention are useful to modulate the expression of one or
more target RNA in an expression system. More preferably, they are
used to reduce expression of one or more target RNA. Such decrease
can occur directly or indirectly by any mechanism known in the art,
or which is yet to be discovered, for the decrease of RNA
expression as defined herein by an RNA. In some embodiments, they
may completely eliminate expression of the one or more RNA targets.
In some embodiments, a given RNA will be more effective at
modulating expression of one of several target RNAs than another.
In other cases, the RNA may similarly affect all targets in one or
more expression systems.
[0280] The multitargeting RNAs provided herein are of particular
value in the treatment of complex multigenic diseases in which
single gene-specific therapeutics may be at a disadvantage because
of the multiple redundancies in pathophysiologic pathways. The
current invention enables a conscious and calculated approach in
which multiple or key proteins or pathways, such as signaling
pathways or molecules, can be targeted with a single agent to
generate greatly increased therapeutic potential.
[0281] In some cases, the targets of interest may be at least
partially controlled by a common "master regulator," for example,
an upstream pleiotropic factor. Such common regulators are often
transcription factors. For example, IL-8 and MCP-1 could
theoretically be down-regulated by targeting the nuclear factor,
NFkappaB. However, by way of example, NFkappaB is also a factor
involved in the survival of Retinal Pigmented Epithelial cells
(RPE), particularly in times of stress. Thus, the indiscriminate
down-regulation of such a cell-survival factor would likely lead to
the undesirable consequence of increased death of RPE cells in
diseased eyes. Rather than identifying upstream pleiotropic
controllers as potential targets with the concomitant risk of
negatively impacting a desirable pathway or process, the novel
approach disclosed herein is amenable to the modulation of multiple
specific targets of interest without having to indiscriminately
modulate common upstream factors.
[0282] An additional aspect of the multitargeting interfering RNAs
provided herein is applicable to the treatment of diseases
characterized by cellular heterogeneity. For example, in solid
tumours, the presence of mutated genes and activated pathways may
vary widely within the same tumour, between tumours in the same
patient, as well as between tumours of a similar histology in
different patients. The development of an RNA molecule active
against several key pathways may derive synergistic activity
against cells reliant on several of these targeted pathways.
However, activity against a greater proportion of the tumour cells
will also be likely because of the "multi-targeted" nature of the
RNA molecule. Furthermore, targeting of several key pathways will
"cover" or allow treatment of more of the patient population.
Hence, improved clinical outcomes are likely with treatment with
the RNA molecules provided herein.
[0283] In certain embodiments, for example where RISC is involved
in the mechanism of action, the targeting of multiple
disease-related transcripts with a single multitargeting
interfering RNA makes optimal use of available RISC, in contrast to
the administration of multiple siRNA molecules, which could
saturate the available intracellular machinery.
[0284] Targeting multiple sites within the same RNA target sequence
is also envisioned for the interfering RNAs provided herein, i.e.
the multitargeting aspect is not limited to multiple targets within
multiple target RNA molecules. Many human diseases, including
cancer and viral infections, are characterized by RNA targets
exhibiting high mutation rates. This increases the likelihood of
resistance to nucleic acid therapeutics arising in these diseases,
due to mutation of the target RNA. Targeting multiple sites within
the target RNA decreases the likelihood of such resistance arising,
since at least two simultaneous mutations would be required for
resistance. Therefore in certain embodiments, the multi-targeting
approach used with multitargeting interfering RNAs can be directed
to the generation of multiple hits against a single target RNA
molecule, for example, to prevent escape mutants. Targeting of
multiple sites within the same transcript (for example, with RNA
viruses) may also produce synergistic effects on the inhibition of
viral growth. Further, employing a mechanism or mechanisms
requiring only partial complementarity with the target RNA molecule
can decrease the possibility of developing resistance through
single point mutation.
[0285] This invention will be better understood by reference to the
examples that follow. Those skilled in the art will readily
appreciate that these examples are only illustrative of the
invention and not limiting.
Example 1
Selection of CODEMIRs for Diabetic Retinopathy (DR)
[0286] CODEMIRs suitable for therapy for DR were sought. In the
disease state, VEGF-A and ICAM-1 are likely drivers of the loss of
integrity of the blood-retinal barrier, which loss leads for
example to diabetic macular edema, a prelude to angiogenesis and
DR. Therefore, VEGF-A and ICAM-1 were selected as targets for the
design of CODEMIRs. Such CODEMIRs were also of therapeutic interest
for treatment of psoriasis and other conditions characterized by
mononuclear cell infiltration and angiogenesis.
[0287] Transcript sequences corresponding to the 3' untranslated
regions (3' UTRs) of VEGF-A and ICAM-1 were used to search for a
suitable seed of at least 6 contiguous bases using a searching
algorithm combined with a database. Publicly available sequences
related to VEGF-A and ICAM-1 obtained from the Ensembl database
were used to perform the initial analysis (see Table 1-1). (Ensembl
is a joint project between the European Molecular Biology
Laboratory (EMBL)/European Bioinformatics Institute (EBI) and the
Wellcome Trust Sanger Institute (WTSI)). The database and related
tools are publicly available on the World Wide Web at the Ensembl
website.
TABLE-US-00009 TABLE 1-1 Ensembl Transcript IDs of selected human
target sequences (Ensembl database, release 33) BFGF
ENST00000264498 IGF-1 ENST00000337514 MCP-1 ENST00000225831 VEGF-A
ENST00000356655 VEGF-B ENST00000309422 VEGF-C ENST00000280193
VEGF-D ENST00000297904 ICAM-1 ENST00000264832 IL-8 ENST00000307407
PlGF ENST00000256315
[0288] A pool of all possible candidate seeds of length 6 or
greater was generated using the specified length as window and
moving sequentially along the sequence in a stepwise fashion by
advancing the window one base at a time. Low complexity seeds were
eliminated. The pool of candidate seed sequences was further
restricted to those for which at least three contiguous bases were
predicted to bind to an unpaired region in at least 50% of optimal
and suboptimal folded structures. Optimal and suboptimal (within -1
kcal/mol of the optimal fold energy) folded structures were
determined using the Vienna RNA package (Hofacker, (2003), Nucleic
Acids Res., 31: 3429-31).
[0289] Two different seeds were selected that optimally fulfilled
the selection criteria. The first seed was selected on the basis of
its length of 12 nucleotides. There were also several smaller seeds
recorded. One 7 nt seed was in a genetic context that favored the
design of the consensus target sequence by virtue of additional,
albeit discontinuous, regions of further identity between the two
target sequences near the seed. The two seeds were used to generate
two sets of consensus target sequences. The consensus target
sequences for the first seed were determined by optical alignment
of the two target sequences and determining the likely effect
various base changes would have on the binding of the complementary
sequence to both target RNA. In the case of the second seed, a
permutation analysis was favoured. In this case, the two target
sequences differ at only seven positions over the 14 bases that are
5' to the seed. Because the extreme tail of the multitargeting
interfering RNA has less requirement for complementarity, only two
possible terminal triplets were considered for the 5'end of the
consensus target sequence; namely TAT and GTC. This reduced by four
the number of possible permutations. Systematic generation of all
remaining 32 possible consensus target sequences (32=2.sup.5) was
followed by in silico hybridization of the 21 base complementary
sequences to the two targets. The sequence providing the best
overall hybridization to the two targets was designated CODEMIR-2.
The consensus target sequences identified for each of the two seeds
are listed in Table 1-2.
TABLE-US-00010 TABLE 1-2 Exemplary Seed Sequences and Consensus
Target Sequences for the design of multitargeting interfering RNAs,
e.g. CODEMIRs targeting VEGF-A and ICAM.1 (all 5' to 3'). Seed
Sequence Consensus Target Sequences CCCACCCACATA
TAGAGACACCCCACCCACATA (SEQ ID NO: 144) (SEQ ID NO: 143)
TTAGACCACCCCACCCACATA (SEQ ID NO: 145) AGACACCACCCCACCCACATA (SEQ
ID NO: 146) TTAGACTCACCCACCCACATA *.sup.1 (SEQ ID NO: 147)
AGACACTCACCCACCCACATA (SEQ ID NO: 148) CAAAACA
TATATGTGTAGCATCAAAACA (SEQ ID NO: 149) (SEQ ID NO: 4)
GTCATGTGTAGCATCAAAACA (SEQ ID NO: 150) TATATGTGTAGCAACAAAACA (SEQ
ID NO: 151) GTCATGTGTAGCAACAAAACA (SEQ ID NO: 152)
TATATGTGTAGAATCAAAACA (SEQ ID NO: 153) GTCATGTGTAGAATCAAAACA (SEQ
ID NO: 154) TATATGTGTAGAAACAAAACA (SEQ ID NO: 155)
GTCATGTGTAGAAACAAAACA (SEQ ID NO: 156) TATATGTGTAACATCAAAACA (SEQ
ID NO: 157) GTCATGTGTAACATCAAAACA (SEQ ID NO: 158)
TATATGTGTAACAACAAAACA (SEQ ID NO: 159) GTCATGTGTAACAACAAAACA (SEQ
ID NO: 160) TATATGTGTAAAATCAAAACA (SEQ ID NO: 161)
GTCATGTGTAAAATCAAAACA (SEQ ID NO: 162) TATATGTGTAAAAACAAAACA (SEQ
ID NO: 163) GTCATGTGTAAAAACAAAACA (SEQ ID NO: 164)
TATATATGTAGCATCAAAACA (SEQ ID NO: 165) GTCATATGTAGCATCAAAACA (SEQ
ID NO: 166) TATATATGTAGCAACAAAACA (SEQ ID NO: 167)
GTCATATGTAGCAACAAAACA (SEQ ID NO: 168) TATATATGTAGAATCAAAACA (SEQ
ID NO: 169) GTCATATGTAGAATCAAAACA (SEQ ID NO: 170)
TATATATGTAGAAACAAAACA (SEQ ID NO: 171) GTCATATGTAGAAACAAAACA (SEQ
ID NO: 172) TATATATGTAACATCAAAACA (SEQ ID NO: 173)
GTCATATGTAACATCAAAACA (SEQ ID NO: 174) TATATATGTAACAACAAAACA (SEQ
ID NO: 175) GTCATATGTAACAACAAAACA *.sup.2 (SEQ ID NO: 176)
TATATATGTAAAATCAAAACA (SEQ ID NO: 177) GTCATATGTAAAATCAAAACA (SEQ
ID NO: 178) TATATATGTAAAAACAAAACA (SEQ ID NO: 179)
GTCATATGTAAAAACAAAACA (SEQ ID NO: 180) *.sup.1,2 These consensus
target sequences were used to design CODEMIR1 and CODEMIR2,
respectively.
[0290] Only several exemplary sequences are listed in Table 1-2
because, as mentioned above, additional consensus target sequences
with partial identity to VEGF-A and ICAM-1 could have been readily
generated, particularly for the second, shorter seed. In FIG. 1,
five consensus target sequences for the 12 nt seed are shown. The
predicted hybridization of the CODEMIR guide strands to the target
VEGF-A and ICAM-1 transcripts is also shown. CODEMIRs targeting
VEGF-A and ICAM-1 were designed using the methods provided.
Consensus target sequences (VAIC1-01 to -05) were derived by
aligning the target mRNA sequences 5' of the seed region to
generate sites of 21 nucleotides in length. Candidate CODEMIR guide
strand sequences complementary to these consensus target sequences
were determined, and hybridization between the CODEMIR guide
sequences and the targets was predicted by in silico modeling.
Based on the results, the consensus target sequence, VAIC1-04, was
used to generate CODEMIR 1 (FIGS. 2 and 3).
[0291] Candidate CODEMIRs were synthesized as double-stranded RNA
molecules with 19 bp of complementarity and 3' overhangs of two
nucleotides in length. The two nucleotide overhangs for the guide
strands were designed to be complementary to the consensus target
sequences. The complementary passenger strand was designed to
include a two nucleotide 3'-UU overhang.
[0292] Two CODEMIRs were selected for more extensive testing, one
each designed from the 12 nt seed and the 7 nt seed. The sequences
of the guide strands and their predicted hybridization to the
targeted VEGF-A and ICAM-1 3' UTRs are shown in Table 1-3. It can
be seen that even with a seed sequence only 7 nt in length, using
the approach provided herein, CODEMIRs (for example CODEMIR2, Table
1-3) can be designed to achieve significant hybridization with at
least two different targets. The degree of 3' identity that the
CODEMIRs have with the intended targets is evident.
TABLE-US-00011 TABLE 1-3 Exemplary CODEMIRs multitargeting both
VEGF-A and ICAM-1. CODEMIR-1 CODEMIR-2 Guide sequence
UAUGUGGGUGGGUGAGUCUAA (SEQ ID NO: 100) UGUUUUGUUGUUACAUAUGAC (SEQ
ID NO: 102) (5' to 3') CODEMIR 5' UAUGUGGGUGGGUGAGUCUAA 3' (SEQ ID
NO: 100) 5' UGUUUUGUUGUUACAUAUGAC 3' 3' UUAUACACCCACCCACUCAGA 5'
(SEQ ID NO: 101) 3' UUACAAAACAACAAUGUAUAC 5' top strand (SEQ ID NO:
102) bottom strand (SEQ ID NO: 103) VEGF-A binding G A C U A A
(VEGF-A = upper UAGAC CACCCACCCACAUA (SEQ ID NO: 181) UUAUAUGUAA
AACAAAACA (SEQ ID NO: 182) strand) AUCUG GUGGGUGGGUGUAU (SEQ ID NO:
100) AGUAUACAUU UUGUUUUGU (SEQ ID NO: 102) Top strand 5' to 3' A A
C G Bottom 3' to 5' ICAM-1 binding G CCAC C A U C (ICAM-1 upper
UUAG CUC CCCACCCACAUA (SEQ ID NO: 183) CAUGUGUAGCA CAAAACA (SEQ ID
NO: 184) strand) AAUC GAG GGGUGGGUGUAU (SEQ ID NO: 100) GUAUACAUUGU
GUUUUGU (SEQ ID NO: 102) Top strand 5' to 3' U U CA U Bottom 3' to
5' Representations of the predicted binding of CODEMIR-1 and
CODEMIR-2 to each of the targets. The likely mismatches and bulges
are shown. The seeds in the consensus target sites are shown in
bold font.
Testing CODEMIRs in a Human Cell Culture System Producing VEGF-A
and ICAM-1
[0293] Cells and Culturing: RPE (retinal pigmented epithelium)
cells in culture were used to screen the anti-angiogenic CODEMIRs
designed, as described above. The human cell line, ARPE-19, was
used. ARPE-19 cells were grown in Dulbecco's Modified Eagle's
Medium (DMEM) supplemented with 10% fetal bovine serum and 10 mM
glutamine. For ELISA detection of secreted proteins of interest, or
in situ cell surface antigen immunostaining, ARPE cells were seeded
at 4.times.10.sup.3 cells per well in a 96 well tissue culture
plate. For FACS analysis, ARPE-19 cells were seeded at
2.5.times.10.sup.4 cells per well in a 24 well tissue culture
plate. Cells were transfected 24 hours after seeding using
lipofectamine (InVitrogen) at a ratio of 1 microL lipofectamine per
20 pmol of CODEMIR RNA duplex or control siRNA. In most studies,
medium was replaced 24 hours after transfection at which time
deferroxamine (130 .mu.M) or IL-1.beta. (1 ng/mL) was added for the
VEGF-A and ICAM-1 experiments, respectively. Experiments were
performed in triplicate and repeated at least twice.
[0294] Assays for VEGF-A and ICAM-1: The ARPE-19 cells were assayed
to confirm production of both VEGF-A and ICAM-1. VEGF-A was assayed
in the supernatant using a commercially available ELISA assay
(R&D Systems) according to the manufacturer's instructions.
Cell surface ICAM-1 was assayed either by immunostaining followed
by fluorescence activated cell sorting (FACS), by in situ
immunostaining of cell-surface ICAM-1 in 96 well plates using
colorimetric detection, or alternatively by ELISA of cell lysates
using a commercial ICAM ELISA kit (R&D systems).
[0295] Cytotoxity and Off-Target Effects: Apart from being a source
of proangiogenic factors, RPE cells are critical to the survival of
the neurosensory photoreceptor cells. Hence, the CODEMIRs were
screened for cytotoxic effects in ARPE-19 cells to enable selection
of CODEMIRs without significant cytotoxicity. CODEMIRs were
transfected into ARPE-19 cells, typically at final concentrations
of 1-100 nM in the culture medium. Cell survival was measured 48
hours after transfection using the Cell Titer Blue Assay, which
measures cellular respiration.
[0296] Activity of the CODEMIRs against multiple RNA targets: The
CODEMIRs were assessed for their ability to repress the production
of each of the target proteins. Specific siRNAs individually
targeting either VEGF-A or ICAM-1 were used as single target
comparative controls. (See e.g. Table 1-4).
[0297] An siRNA which targeted neither VEGF nor ICAM (siRNA
CONTROL) was used as a non-targeting control (Table 1-4):
TABLE-US-00012 TABLE 1-4 Sequences of comparative and non-targeting
control siRNA (guide strand). Control guide strand sequence (5' to
3') siRNA CONTROL GUCUGCGAUCGCAUACAAU dTdT (SEQ ID NO: 185)
siICAM-1 UAGAGGUACGUGCUGAGGC dTdT (SEQ ID NO: 186) siVEGF
GUGCUGGCCUUGGUGAGGU dTdT (SEQ ID NO: 187)
[0298] Results: CODEMIR-1 and CODEMIR-2, as well as comparative and
non-targeting control siRNAs were evaluated for cytotoxicity in
ARPE-19 cells. All were found to have negligible toxicity over the
concentration range of 0-40 nM (FIG. 2) when transfected over a
period of 48-72 hours. None of the CODEMIRs tested showed any
appreciable toxicity at concentrations less than about 50 nM.
Individual designed CODEMIRs were routinely screened for potential
cytotoxicity due to off-target effects.
[0299] The supernatants from the cell culture 48 hours after
changing of medium (plus deferroxamine in the case of VEGF-A and
IL-1.beta. for ICAM-1) were assayed for VEGF-A and ICAM-1 and cells
harvested. FIG. 3 shows protein suppression by CODEMIRs
multitargeting both VEGF-A and ICAM-1. ARPE-19 cells were treated
with a siRNA not targeted to either VEGF or ICAM (siRNA Control),
or RNAs specific for VEGF-A (siVEGF), ICAM-1 (siICAM), or CODEMIR-1
or -2, each at a concentration of 40 nM. VEGF-A (as determined by
ELISA) and cell-surface ICAM-1 (as determined by FACS) proteins
were assayed 48 hrs after treatment and expressed as a percentage
of those from untransfected control cells. In experiments measuring
VEGF-A secretion, cells were stimulated with the hypoxia mimic
deferroxamine (130 .mu.M) 24 hrs post-transfection, whereas
IL-1.beta. was added at 1 ng/mL in the corresponding experiment for
ICAM-1. Data from unstimulated and stimulated but untransfected
cells are shown for comparison. Depicted results are composite data
from several experiments. The comparative controls each
significantly reduced the expression of their respective targets
(ANOVA and Dunnett's multiple comparison test versus control siRNA,
p<0.01), without reducing the other, non-targeted protein. In
fact, increased ICAM-1 expression was observed with siVEGF (ANOVA
and Dunnett's multiple comparison test versus control siRNA,
p<0.01).
[0300] CODEMIR-1 profoundly repressed VEGF-A secretion to an extent
that exceeded the comparative siRNA control at the same
concentration. The same CODEMIR also suppressed ICAM-1 expression
in a comparable manner to the ICAM-1 comparative control. CODEMIR-2
also repressed both ICAM-1 and VEGF-A to a lesser, but nevertheless
significant extent (ANOVA and Dunnett's multiple comparison test
versus control siRNA, p<0.01). An siRNA duplex targeting the
exact CODEMIR1 site in VEGF-A produced only slightly greater
knock-down of VEGF-A secretion (data not shown). Thus, a lack of
total complementarity in the interfering RNA molecules provided
herein is not significantly deleterious, and the methods provided
herein of designing individual CODEMIRs each of which target
multiple RNAs, can be used to generate such interfering
molecules.
[0301] The activity of CODEMIR-1 and CODEMIR-2 was assessed, in
terms of VEGF mRNA expression, by RT-PCR. Comparative controls were
analysed alongside; siVEGF (a comparative control siRNA targeting a
different site in VEGF) and siCONTROL (a non-targeting control
siRNA) and, finally, a sample not containing DNA (water control).
VAIC, a multitargeting interfering RNA designed to be fully
complementary to the target binding sequence of CODEMIR-1 was also
examined. Two RT-PCR reactions were run on samples of RNA prepared
from ARPE-19 cells. Specifically, ARPE-19 cells were transfected
with Lipofectamine 2000 and 40 nM of the respective RNA treatment,
incubated for 24 hrs, then treated with 130 microM deferroxamine
for an additional 24 hrs before harvest and extraction of RNA. In
one reaction, the PCR product was a 266 nt PCR amplicon from part
of the Open Reading Frame (ORF) of VEGF. In the second reaction,
the amplified amplicon (243 nt) is located in the 3'UTR of VEGF. To
control for loading, PCR amplification of a GAPDH "house-keeping"
transcript was also determined. The relative intensities of the
GAPDH bands were equal in all treatment lanes. Both VEGF amplicons
were significantly reduced in intensity for the ARPE-19 cells
transfected with either CODEMIR-1 or siVAIC. The comparative siVEGF
control had visibly reduced intensity for the ORF amplicon but not
the 3'UTR amplicon. Overall, these results indicate that CODEMIR-1
induced substantial mRNA degradation, whereas CODEMIR-2 resulted in
less VEGF mRNA degradation. Without limiting aspects of the
invention to any particular theory of operation, it is at least
noteworthy that CODEMIR-1 has a relatively high degree of
complementarity to the VEGF mRNA target, including the central base
pairs adjacent to the RISC cleavage site, while CODEMIR2 has a
central mismatch with the VEGF target mRNA. Thus, CODEMIRs with
less than complete complementarity to a given target RNA sequence,
may induce mRNA degradation, for example by RISC processing, as an
additional means of modulation, or as an alternative to other
mechanisms, such as repressing translation of the target
[0302] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules (CODEMIRs) will comprise
the sequence corresponding to the complement of the seed. In this
example of RNA molecules for altering expression of VEGF-A and
ICAM-1, the complementary sequences are UAUGUGGGUGGG (SEQ ID NO: 1)
and UGUUUUG (SEQ ID NO: 2) for CODEMIRs 1 and 2 respectively.
[0303] Additional CODEMIRs targeting both VEGF and ICAM were
designed based on each of the consensus target sequences shown in
FIG. 1. Each of the CODEMIRS so designed was also significantly
active in modulating the target RNAs (CODEMIR1 previously
described, other data not shown). The active CODEMIRs are as
follows:
TABLE-US-00013 5' UAUGUGGGUGGGGUGUCUCUA 3' (SEQ ID NO: 104) 3'
UUAUACACCCACCCCACAGAG 5' (SEQ ID NO: 105) 5' UAUGUGGGUGGGGUGGUCUAA
3' (SEQ ID NO: 106) 3' UUAUACACCCACCCCACCAGA 5' (SEQ ID NO: 107) 5'
UAUGUGGGUGGGGUGGUGUCU 3' (SEQ ID NO: 108) 3' UUAUACACCCACCCCACCACA
5' (SEQ ID NO: 109) 5' UAUGUGGGUGGGUGAGUGUCU 3' (SEQ ID NO: 110) 3'
UUAUACACCCACCCACUCACA 5' (SEQ ID NO: 111)
[0304] Variants of CODEMIR-1 with altered overhang lengths were
also tested in the ARPE system and found to have significant
activity, although CODEMIR-25 had negligible activity on ICAM-1
(data not shown). The sequences of these two CODEMIRs are as
follows:
TABLE-US-00014 CODEMIR-24 5' UAUGUGGGUGGGUGAGUCUA 3' (SEQ ID NO:
118) 3' UAUACACCCACCCACUCAGA 5' (SEQ ID NO: 119) CODEMIR-25 5'
UAUGUGGGUGGGUGAGUCU 3' (SEQ ID NO: 188) 3' AUACACCCACCCACUCAGA 5'
(SEQ ID NO: 189)
Example 2
Comparison of CODEMIR-1 Activity with that of a Naturally Occurring
MicroRNA with Some Homology
[0305] After CODEMIR-1 was designed for multitargeting of both
VEGF-A and ICAM-1 targets, according to the methods provided herein
(see e.g., Example 1), it was noted that CODEMIR-1 has some
homology with the naturally occurring human microRNA miR-299.
[0306] Processing of miR-299 is predicted to produce two active
strands, miR-299-5p and miR-299-3p. As can be seen below,
miR-299-3p has a region with 12 of 15 of the same bases as the
guide strand of CODEMIR-1, and the first 8 of those bases are
identical. That region of CODEMIR-1 corresponds to the complement
of the seed sequence used in its design. Although the two sequences
share this homology, they have different central and 3' tail
regions (see below).
##STR00001##
[0307] Little is known about the activity of miR-299. No targets
for miR-299-3p or miR-299-5p are reported in the Tarbase database,
a central repository of validated interactions between mRNAs and
miRNAs available on the World Wide Web at the University of
Pennsylvania's DNA & Protein Analysis Lab (DIANA Lab) world
wide website site at diana.pcbi.upenn.edu/tarbase.html. After
further investigation, no published studies of the activity of
miR-299-3p could be located. Thus, a comparison was made of the
activity of CODEMIR-1 and miR-299 against each of the targets,
VEGF-A and ICAM-1, in an expression system.
[0308] As can be seen in FIG. 4, when compared to an RNA duplex
negative control not specific for the targets tested, a RNA duplex
comprising the predicted mature strands of miR-299 demonstrated
some VEGF suppressive activity, although less than that obtained
with CODEMIR-1. The miR-299 had no significant activity against
ICAM-1 (data not shown). CODEMIR-1, therefore, has a markedly
different activity profile from the naturally occurring miR-299.
Despite some similarities in structure to CODEMIR-1, miR-299 did
not have any demonstrable activity with respect to the second RNA
(ICAM-1) which CODEMIR-1 was specifically designed to target. This
shows the effectiveness of using a rational design process to
obtain a functional multitargeting interfering RNA, and the
advantages that can be gained, even over naturally occurring
sequences that have homology to the seed region used in the design
process.
Example 3
Further Exemplification of Multitargeting of Angiogenic Factors
[0309] More complex angiogenic phenotypes than diabetic retinopathy
are found in advanced cancer and in Age-related Macular
Degeneration. With Age-related Macular Degeneration (AMD), it is
currently thought that accumulation of partially phagocytosed
remnants in and below the Retinal Pigmented Epithelial (RPE) layer
causes cellular stresses that lead to the production of angiogenic
cytokines or chemokines by the RPE cells (eg VEGF-A, IL-8, MCP-1).
Additional proteins expressed on the membrane surfaces of RPE cells
may further drive this process (e.g. ICAM-1). Overall, the
angiogenic factors involved in AMD include: VEGF-A, VEGF-B, IGF-1,
MCP-1, IL-8, ICAM-1, bFGF, and PlGF. Indeed, this multitude of
pro-angiogenic cytokines acting in combination is analogous to the
angiogenesis seen in advanced cancer. In order to increase the
number of angiogenic factors that could be covered by a CODEMIR,
the full mRNA transcripts derived from the Ensembl database (see
Table 1-1) for VEGF-A, ICAM-1 and IGF-1 were used in the search of
suitable seeds as previously outlined. A seed consisting of 11 bp
present in all 3 transcripts was identified (Table 3-1). Consensus
target sequences were derived as described above (Table 3-1). Guide
strand sequences for CODEMIRs targeting these sites were
determined, and the predicted binding of these CODEMIRs to the 3
target sequences was assessed using RNAhybrid software. Because the
3' seed region contains 4 contiguous G bases, the loading bias is
unlikely to be favorable. As shown in Table 3-2, the high duplex
binding energy at the 5' end of the guide strand can be reduced by
the judicious introduction of mismatched base pairings by
modification of the passenger strand.
TABLE-US-00015 TABLE 3-1 Target sequences aligned with candidate
CODEMIR consensus target sequences for targeting VEGF-A, ICAM-1 and
IGF-1 Target Site Sequences (5' to 3') VEGF-A
AAGTCGAGGAAGAGAGAGACGGGGTCAGAG (SEQ ID NO: 191) ICAM-1
TTTTTTTTTTTTCCAGAGACGGGGTCTCGC (SEQ ID NO: 192) IGF-1
TTTGGATTTTTAATAGAGACGGGGTTTTAC (SEQ ID NO: 193) Consensus target
Sequences TGGTTAACAGAGACGGGGTCA (SEQ ID NO: 542)
TGGTTAACAGAGACGGGGTCT (SEQ ID NO: 194) ATGGTTAACAGAGACGGGGTA (SEQ
ID NO: 195) GATGGTTAACAGAGACGGGGT (SEQ ID NO: 196)
GGTTAACAGAGACGGGGTCTA (SEQ ID NO: 197) GGTTAACAGAGACGGGGTCTT (SEQ
ID NO: 198) GGGTAACAGAGACGGGGTCTA (SEQ ID NO: 199)
GGGTAACAGAGACGGGGTCTT (SEQ ID NO: 200)
TABLE-US-00016 TABLE 3-2 Alteration of guide strand 5' terminal
stability by introduction of mismatched base pairings through
modification of the passenger strand. CODEMIR duplex (lower strand
= guide, 3' to 5') mfe* UGGUUAACAGAGACGGGGUUU (SEQ ID NO: 201)
-12.2 CUACCAAUUGUCUCUGCCCCA (SEQ ID NO: 202) UGGUUAACAGAGACGGGAUUU
(SEQ ID NO: 203) -9.7 CUACCAAUUGUCUCUGCCCCA (SEQ ID NO: 202)
UGGUUAACAGAGACGGAGUUU (SEQ ID NO: 204) -6.4 CUACCAAUUGUCUCUGCCCCA
(SEQ ID NO: 202) UGGUUAACAGAGACGAGGUUU (SEQ ID NO: 205) -6.1
CUACCAAUUGUCUCUGCCCCA (SEQ ID NO: 202) UGGUUAACAGAGACAGGGUUU (SEQ
ID NO: 206) -9.4 CUACCAAUUGUCUCUGCCCCA (SEQ ID NO: 202)
UGGUUAACAGAGACGGAAUUU (SEQ ID NO: 207) -6.4 CUACCAAUUGUCUCUGCCCCA
(SEQ ID NO: 202) UGGUUAACAGAGACGAAGUUU (SEQ ID NO: 208) -3.1
CUACCAAUUGUCUCUGCCCCA (SEQ ID NO: 202) UGGUUAACAGAGACAAGGUUU (SEQ
ID NO: 209) -6.1 CUACCAAUUGUCUCUGCCCCA (SEQ ID NO: 202) NOTE:
Underlining in this table indicates the seed, which in some
instances has been modified (bolding). *Minimum free energy of
terminal (5' of theguide) pentamer (kcal/mol)
[0310] Alternatively, a broader coverage of the angiogenic factors
can be derived. For example, the 7 nt seed TGCAGCT (SEQ ID NO: 210)
can be used to construct CODEMIRs targeting simultaneously: VEGF-B,
-C, -D, IL-8, bFGF, PlGF, MCP-1, ICAM-1 and IGF-1.
[0311] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules (CODEMIRs) will comprise
the sequence corresponding to the complement of the seed.
Accordingly, in the example of an RNA molecule for altering
expression of any combination of ICAM-1, VEGF-A and IGF-1, the
complementary sequence to the seed in Table 3-1
TABLE-US-00017 (AGAGACGGGGT) (SEQ ID NO: 211) is ACCCCGUCUCU. (SEQ
ID NO: 5)
[0312] In the example of an RNA molecule for altering expression of
any combination of ICAM-1, VEGF-B, VEGF-C, VEGF-D, IL-8, bFGF,
PlGF, MCP-1 and IGF-1, the complementary sequence to the seed above
(TGCAGCT) (SEQ ID NO: 210) is AGCUGCA (SEQ ID NO: 7).
Example 4
CODEMIRs for Metabolic Disorders
[0313] CODEMIRs may also be suitable for the treatment of complex
metabolic diseases such as type 2 diabetes. Two potential gene
targets for the treatment of this disease are glucose-6-phosphatase
and Inppl1. Full transcript sequences were examined for the
presence of common candidate seeds. In this case, a seed of 14 nt
identity (CCCACCCACCTACC) (SEQ ID NO: 212) was identified (top of
Table 4-1). Candidate CODEMIRs were then designed, via the
intermediate of consensus target sites that are shown in Table
4-1.
[0314] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules (CODEMIRs) will comprise
the sequence corresponding to the complement of the seed. In this
example, these complementary sequences will comprise GGUAGGUGGGUGGG
(SEQ ID NO: 10).
TABLE-US-00018 TABLE 4-1 Target sequences aligned with CODEMIR
consensus target sites for targeting Gluc6p and Inppl1. Sequences
(5' to 3') Gluc6p CAGAGTATTTCTGCCCCACCCACCTACCCCCCAAAAA (SEQ ID NO:
213) Inppl1 GAGGAGATCTCCTTCCCACCCACCTACCGCTATGAGC (SEQ ID NO: 214)
Candidate "consensus target sites" CTCCGCCCCACCCACCTACCA (SEQ ID
NO: 215) CTCCTCCCCACCCACCTACCA (SEQ ID NO: 216)
TCTCCGCCCCACCCACCTACC (SEQ ID NO: 217) TCTCCTCCCCACCCACCTACC (SEQ
ID NO: 218)
Example 5
Multitargeting of Cancer-Related Gene Products
[0315] CODEMIRs can also be applied to target multiple unrelated
cancer genes in order to more competently control the tumour
phenotype. By way of example, inappropriate activation of
.quadrature.-catenin, K-Ras and EGFR is found in many advanced
colorectal adenocarcinomas. Simultaneous targeting of these three
genes was sought with a CODEMIR. The full-length transcripts for
all three genes were used in the search for candidate seeds. In the
case of K-ras, both alternative transcripts (a and b) were
included. A 13 bp seed (CAUUCCAUUGUUU) (SEQ ID NO: 9) was found and
appropriate candidate CODEMIRs identified. Some alternative CODEMIR
consensus target sequences are listed in Table 5-1.
[0316] CODEMIRs targeting this seed and which comprise a complement
to this seed may be of interest for the treatment of colorectal and
other cancer, especially those in which altered
.quadrature.-catenin, .quadrature.-ras and/or EGFR signaling
contributes to the malignant phenotype.
[0317] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules (CODEMIRs) will comprise
the sequence corresponding to the complement of the seed. In this
example, the complementary sequence is AAACAAUGGAAUG (SEQ ID NO:
8).
TABLE-US-00019 TABLE 5-1 Target site sequences aligned with
candidate CODEMIR consensus sequences for the targeting of
.quadrature.-catenin, K-ras-B, K-ras-A and EGFR. Target Site
Sequences (5' to 3') beta- CAGAGGACUAAAUACCAUUCCAUUGUUUGUGCAG (SEQ
ID NO: 219) Catenin K-ras-A CUGGUAACAGUAAUACAUUCCAUUGUUUUAGUAA (SEQ
ID NO: 220) K-ras-B CUGGUAACAGUAAUACAUUCCAUUGUUUUAGUAA (SEQ ID NO:
220) EGFR GACUUGUUUGUCUUCCAUUCCAUUGUUUUGAAAC (SEQ ID NO: 221)
Candidate consensus target sequences AUAAUCCAUUCCAUUGUUUUA (SEQ ID
NO: 222) AUAAUACAUUCCAUUGUUUUA (SEQ ID NO: 223)
AUAUUCCAUUCCAUUGUUUUA (SEQ ID NO: 224) AUAUUACAUUCCAUUGUUUUA (SEQ
ID NO: 225) GUAAUCCAUUCCAUUGUUUUA (SEQ ID NO: 226)
GUAAUACAUUCCAUUGUUUUA (SEQ ID NO: 227) GUAUUCCAUUCCAUUGUUUUA (SEQ
ID NO: 228) GUAUUACAUUCCAUUGUUUUA (SEQ ID NO: 229)
CUAUAAUCCAUUCCAUUGUUU (SEQ ID NO: 230) CUAUAAUACAUUCCAUUGUUU (SEQ
ID NO: 231) CUAUAUUCCAUUCCAUUGUUU (SEQ ID NO: 232)
CUAUAUUACAUUCCAUUGUUU (SEQ ID NO: 233) CUGUAAUCCAUUCCAUUGUUU (SEQ
ID NO: 234) CUGUAAUACAUUCCAUUGUUU (SEQ ID NO: 235)
CUGUAUUCCAUUCCAUUGUUU (SEQ ID NO: 236) CUGUAUUACAUUCCAUUGUUU (SEQ
ID NO: 237) CAAUAAUCCAUUCCAUUGUUU (SEQ ID NO: 238)
CAAUAAUACAUUCCAUUGUUU (SEQ ID NO: 239) CAAUAUUCCAUUCCAUUGUUU (SEQ
ID NO: 240) CAAUAUUACAUUCCAUUGUUU (SEQ ID NO: 241)
CAGUAAUCCAUUCCAUUGUUU (SEQ ID NO: 242) CAGUAAUACAUUCCAUUGUUU (SEQ
ID NO: 243) CAGUAUUCCAUUCCAUUGUUU (SEQ ID NO: 244)
CAGUAUUACAUUCCAUUGUUU (SEQ ID NO: 245)
Example 6
Targeting of Multiple Sites within the HIV Genome
[0318] The CODEMIR approach can be used to target proteins of
interest that are likely to be mutated in chronic forms of disease.
Mutations may be particularly prevalent in cancer and viral disease
in which drug-resistant forms often evolve. In this example,
VIROMIRs were designed to target multiple sites in the Human
Immunodeficiency Virus (HIV). The requirement for simultaneous
mutation at several sites, in order to overcome the effects of such
a VIROMIR, is likely to provide a high genetic hurdle to the
emergence of resistant viral clones or quasispecies. The genome of
the HXB2 strain of HIV I serotype B (GenBank Accession K03455) was
used as the principal sequence of interest and was examined with
bioinformatics methods detailed elsewhere in this application to
find seeds occurring at more than one location. All HIV I clade B
isolates in the LANL database as of 1 Aug. 2005 which contain full
sequences for any of the GAG, ENV, POL, TAT, VIF, VPR, VPU and NEF
genes were used in these analyses.
[0319] Three 9-base seeds were found to occur in 4 genetic contexts
in this reference sequence. These were: ATCAAGCAG (SEQ ID NO: 246),
TGGAAAGGA (SEQ ID NO: 247) and AAAGAAAAA (SEQ ID NO: 37).
[0320] In the population of Clade B isolates described above, the
first seed (ATCAAGCAG) (SEQ ID NO: 246), which is of greater
interest because of its greater base complexity, was found to be
present in 91%, 76%, 78% and 74% of the isolates with respect to
the GAG, POL, VIF and ENV genetic contexts of this seed,
respectively. This is shown below as the seed flanked by the
genetic context at each site.
TABLE-US-00020 (SEQ ID NO: 248)
AAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAG GAG 91% (SEQ ID NO:
249) CCTGTTGGTGGGCGGGAATCAAGCAGGAATTTGGAATTCCCTAC POL 76% (SEQ ID
NO: 250) TAGCCCTAGGTGTGAATATCAAGCAGGACATAACAAGGTAGGAT VIF 78% (SEQ
ID NO: 251) AACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTG ENV
7.4%
[0321] The bolded regions correspond to independent occurrences of
the seed. These regions have been annotated to the genes GAG, POL,
VIF, and ENV.
[0322] An additional twenty 11-base seeds occurred at two sites
within the reference HIV genome. There were no seeds of a length of
10 bases that were not subsumed in the 11 base seeds. These seeds
are listed below, each with its genetic context, the HIV gene in
which it occurs, and its rate of occurrence in that genetic
context.
TABLE-US-00021 1 (SEQ ID NO: 252)
AGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGA
AGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTT GAG 52% (SEQ ID NO: 253)
ACAACATCTGTTGAGGTGGGGACTTACCACACCAGACAAAAAACATCAGA
AAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATC POL 84% 2 (SEQ ID NO: 254)
AAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAG
AGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGC GAG 81% (SEQ ID NO: 255)
AACAGGGAGACTAAATTAGGAAAAGCAGGATATGTTACTAATAGAGGAAG
ACAAAAAGTTGTCACCCTAACTGACACAACAAATCAGAAG POL 23% 3 (SEQ ID NO: 256)
AGGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATTCAGCTACC
ATAATGATGCAGAGAGGCAATTTTAGGAACCAAAGAAAGA GAG 31% (SEQ ID NO: 257)
CTTGGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACC
ACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGT REV 42% 4 (SEQ ID NO: 258)
AACAAATTCAGCTACCATAATGATGCAGAGAGGCAATTTTAGGAACCAAA
GAAAGATTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCA GAG 58% (SEQ ID NO: 259)
ACCCAGGGATTAAAGTAAGGCAATTATGTAAACTCCTTAGAGGAACCAAA
GCACTAACAGAAGTAATACCACTAACAGAAGAAGCAGAGC POL 12% 5 (SEQ ID NO: 260)
CAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTA
GAAATTTGTACAGAGATGGAAAAGGAAGGGAAAATTTCAA POL 68% (SEQ ID NO: 261)
TAGTACATGTAACGCAACCTATACCAATAGTAGCAATAGTAGCATTAGTA
GTAGCAATAATAATAGCAATAGTTGTGTGGTCCATAGTAA VPU 72% 6 (SEQ ID NO: 262)
CACCGGTGCTACGGTTAGGGCCGCCTGTTGGTGGGCGGGAATCAAGCAGG
AATTTGGAATTCCCTACAATCCCCAAAGTCAAGGAGTAGT POL 75% (SEQ ID NO: 263)
AAGGCCTTATTAGGACACATAGTTAGCCCTAGGTGTGAATATCAAGCAGG
ACATAACAAGGTAGGATCTCTACAATACTTGGCACTAGCA VIF 78% 7 (SEQ ID NO: 264)
CAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAA
TTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTT POL 52% (SEQ ID NO: 265)
CTCAAATATTGGTGGAATCTCCTACAGTATTGGAGTCAGGAACTAAAGAA
TAGTGCTGTTAGCTTGCTCAATGCCACAGCCATAGCAGTA REV 68% 8 (SEQ ID NO: 266)
ACAGGAGCAGATGATACAGTATTAGAAGAAATGAGTTTGCCAGGAAGATG
GAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAA POL 95% (SEQ ID NO: 267)
GAAACAGGGCAGGAAACAGCATATTTTCTTTTAAAATTAGCAGGAAGATG
GCCAGTAAAAACAATACATACTGACAATGGCAGCAATTTC POL 95% 9 (SEQ ID NO: 268)
AATGAGTTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAG
GTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGA POL 98% (SEQ ID NO: 269)
AAAGGAAAAGGTCTATCTGGCATGGGTACCAGCACACAAAGGAATTGGAG
GAAATGAACAAGTAGATAAATTAGTCAGTGCTGGAATCAG POL 92% 10 (SEQ ID NO:
270) GGCAACTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTA
GAAGAAATGAGTTTGCCAGGAAGATGGAAACCAAAAATGA POL 80% (SEQ ID NO: 271)
ATCAGATACTCATAGAAATCTGTGGACATAAAGCTATAGGTACAGTATTA
GTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGT POL 92% 11 (SEQ ID NO:
272) AAAGTAAGGCAATTATGTAAACTCCTTAGAGGAACCAAAGCACTAACAGA
AGTAATACCACTAACAGAAGAAGCAGAGCTAGAACTGGC POL 85% (SEQ ID NO: 273)
AAACTCCTTAGAGGAACCAAAGCACTAACAGAAGTAATACCACTAACAGA
AGAAGCAGAGCTAGAACTGGCAGAAAACAGAGAGATTCT POL 85% 12 (SEQ ID NO: 274)
CCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAG
CCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGC GAG 89% (SEQ ID NO: 275)
TTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAG
CTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGG ENV 74% 13 (SEQ ID NO:
276) CATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATG
TGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCA POL 86% (SEQ ID NO: 277)
AAGACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAATG
TACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTG ENV 84% 14 (SEQ ID NO:
278) ACTATTTTTAGATGGAATAGATAAGGCCCAAGATGAACATGAGAAATATC
ACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTGCC POL 61% (SEQ ID NO: 279)
CTAATAGAAAGAGCAGAAGACAGTGGCAATGAGAGTGAAGGAGAAATATC
AGCACTTGTGGAGATGGGGGTGGAGATGGGGCACCATGCT ENV 3% 15 (SEQ ID NO: 280)
TACTTGGGCAGGAGTGGAAGCCATAATAAGAATTCTGCAACAACTGCTGT
TTATCCATTTTCAGAATTGGGTGTCGACATAGCAGAATAG VPR 77% (SEQ ID NO: 281)
AGTACAATGTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGT
TAAATGGCAGTCTAGCAGAAGAAGAGGTAGTAATTAGATC ENV 61% 16 (SEQ ID NO:
282) TAGAAAGAGCAGAAGACAGTGGCAATGAGAGTGAAGGAGAAATATCAGCA
CTTGTGGAGATGGGGGTGGAGATGGGGCACCATGCTCCTTG ENV 2% (SEQ ID NO: 283)
AATGATAATGGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCA
CAAGCATAAGAGGTAAGGTGCAGAAAGAATATGCATTTTTT ENV 3% 17 (SEQ ID NO:
284) AGACAGTGGCAATGAGAGTGAAGGAGAAATATCAGCACTTGTGGAGATGG
GGGTGGAGATGGGGCACCATGCTCCTTGGGATGTTGATGA ENV 57% (SEQ ID NO: 285)
TGAGAGTGAAGGAGAAATATCAGCACTTGTGGAGATGGGGGTGGAGATGG
GGCACCATGCTCCTTGGGATGTTGATGATCTGTAGTGCTA ENV 10% 18 (SEQ ID NO:
286) TCTGTGTTAGTTTAAAGTGCACTGATTTGAAGAATGATACTAATACCAAT
AGTAGTAGCGGGAGAATGATAATGGAGAAAGGAGAGATAA ENV 16% (SEQ ID NO: 287)
AAGGTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATACCAAT
AGATAATGATACTACCAGCTATAAGTTGACAAGTTGTAAC ENV 1% 19 (SEQ ID NO: 288)
AATGGAATAACACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAATTT
GGAAATAATAAAACAATAATCTTTAAGCAATCCTCAGGAG ENV 63% (SEQ ID NO: 289)
GTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTT
GCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTC ENV 20% 20 (SEQ ID NO:
290) ATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGT
ACTTGGTTTAATAGTACTTGGAGTACTGAAGGGTCAAATA ENV 79% (SEQ ID NO: 291)
TTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGTTTAATAGT
ACTTGGAGTACTGAAGGGTCAAATAACACTGAAGGAAGTG ENV 20%
[0323] Ultimately, an effective RNA therapeutic of the invention
should provide broad coverage of the affected population and it is
obviously desirable to target sequences that are highly represented
in this patient population. Therefore, of the seeds presented
above, those with undesirably low rates of occurrence in their
specific genetic contexts in the Clade B isolates available from
the LANL Database as defined previously were removed from present
consideration. For the purposes of this example, undesirably low
rates of occurrence in their specific genetic contexts was defined
as <50% of the Clade B isolates.
[0324] In order to prioritize and test candidate VIROMIRs, it is
important to have screening methods that are compatible with the
intended target sequence. The pNL4.3 assay is widely used in the
field of HIV research as a valuable, validated screen for drugs
active in HIV and was used by us to test candidate VIROMIRs.
However, there are some differences between the sequences of the
HIV component of the pNL4.3 plasmid and that of the reference HIV
strain (K03455) used in the design of the VIROMIRs. Therefore,
comparison of the sequences of the reference strain and the
sequence of the pNL4.3 plasmid was carried out and only the
designed VIROMIRs from the above-detailed steps with conserved seed
sites present in the plasmid were selected. VIROMIRs corresponding
to seeds #1 and #12 from the previous list of 11-base seeds were
excluded on this basis. However, one skilled in the art will
realize that these 2 VIROMIRs may be of use therapeutically and
that the decision here was simply based on compatibility with the
testing system chosen. Other testing systems such as viral
challenge assays, fusion reporters, viral pseudoparticles among
others, each representing any multitude of therapeutically relevant
or irrelevant sequences could equally be considered.
[0325] There were then nine 11-base seeds for consideration and, of
these, Seed 6 (POL/VIF; see above) was chosen as an example of the
design of a VIROMIR to target multiple sites in a viral genome. A
number of consensus target sequences for the two seed 6 occurrences
were derived (Table 6-1), candidate VIROMIR guide sequences were
identified, and the predicted hybridization of these sequences to
the HIV target sites was assessed using RNAhybrid software. The
predicted VIROMIR RNA duplexes were further analyzed with respect
to factors likely to generate the desired strand loading bias. An
example of a CODEMIR guide strand targeting seed site 6 in the HIV
genome is shown in FIG. 5.
TABLE-US-00022 TABLE 6-1 Target site sequences and aligned
candidate CODEMIR consensus target sequences for targeting two
sites in the HIV genome. Target Site Sequences (5' to 3') Target
site 1 TGGTGGGCGGGAATCAAGCAGGAATTTG (SEQ ID NO: 292) Target site 2
CTAGGTGTGAATATCAAGCAGGACATAA (SEQ ID NO: 293) Candidate consensus
target sequences TTGTGCGAAAATCAAGCAGGA (SEQ ID NO: 294)
TTGTGCGAATATCAAGCAGGA (SEQ ID NO: 295) TTGGGCGAAAATCAAGCAGGA (SEQ
ID NO: 296) TTGGGCGAATATCAAGCAGGA (SEQ ID NO: 297)
TGGTGCGAAAATCAAGCAGGA (SEQ ID NO: 298) TGGTGCGAATATCAAGCAGGA (SEQ
ID NO: 299) TGGGGCGAAAATCAAGCAGGA (SEQ ID NO: 300)
TGGGGCGAATATCAAGCAGGA (SEQ ID NO: 301) GTGTGCGAAAATCAAGCAGGA (SEQ
ID NO: 302) GTGTGCGAATATCAAGCAGGA (SEQ ID NO: 303)
GTGGGCGAAAATCAAGCAGGA (SEQ ID NO: 304) GTGGGCGAATATCAAGCAGGA (SEQ
ID NO: 305) GGGTGCGAAAATCAAGCAGGA (SEQ ID NO: 306)
GGGTGCGAATATCAAGCAGGA (SEQ ID NO: 307) GGGGGCGAAAATCAAGCAGGA (SEQ
ID NO: 308) GGGGGCGAATATCAAGCAGGA (SEQ ID NO: 309)
ATGGGCGAAAATCAAGCAGGA (SEQ ID NO: 310)
[0326] Consensus target sequences were similarly designed for the
one selected 9-base seed and the other eight 11-base seeds. As
appreciated by one skilled in the art and as outlined in other
examples of the invention and Table 6-1, there are many possible
consensus target sequences, although only 1 such sequence in each
case was used here. The guide strands were generated as the
complements of these consensus target sequences as indicated above.
The corresponding passenger strands were designed to be the
complement of the guide strand, minus the first 2 bases at the
5'-extremity and with a 3'-extremity extension of UU, thereby
generating dual 2-base overhangs at each 3' extremity.
[0327] The 10 VIROMIR candidates were thus:
TABLE-US-00023 VM001 5' GUGGGCGAACAUCAAGCAGUU 3' (SEQ ID NO: 311)
3' GGCACCCGCUUGUAGUUCGUC 5' (SEQ ID NO: 312) VM002 5'
GCAAUAAAAGCAUUAGUAGUU 3' (SEQ ID NO: 313) 3' AUCGUUAUUUUCGUAAUCAUC
5' (SEQ ID NO: 314) VM003 5' GGGCGAAAAUCAAGCAGGAUU 3' (SEQ ID NO:
315) 3' UACCCGCUUUUAGUUCGUCCU 5' (SEQ ID NO: 316) VM004 5'
GAGUCAGAAACUAAAGAAUUU 3' (SEQ ID NO: 317) 3' GUCUCAGUCUUUGAUUUCUUA
5' (SEQ ID NO: 318) VH005 5' GCAAUAGAUACAGUAUUAGUU 3' (SEQ ID NO:
319) 3' UUCGUUAUCUAUGUCAUAAUC 5' (SEQ ID NO: 320) VM006 5'
GGGUUUACCAGGAAGAUGGUU 3' (SEQ ID NO: 120) 3' UACCCAAAUGGUCCUUCUACC
5' (SEQ ID NO: 121) VM007 5' CGCUAAAAGGAAUUGGAGGUU 3' (SEQ ID NO:
321) 3' UUGCGAUUUUCCUUAACCUCC 5' (SEQ ID NO: 322) VM008 5'
CGUGAUAACAGUACAAUGUUU 3' (SEQ ID NO: 323) 3' UUGCACUAUUGUCAUGUUACA
5' (SEQ ID NO: 324) VM009 5' AGCCAUAGCACUAACAGAAUU 3' (SEQ ID NO:
325) 3' CUUCGGUAUCGUGAUUGUCUU 5' (SEQ ID NO: 326) VM010 5'
UUCCACCUCAACUGCUGUUUU 3' (SEQ ID NO: 327) 3' UUAAGGUGGAGUUGACGACAA
5' (SEQ ID NO: 328)
[0328] As an added precaution, the predicted hybridization of the
ten guide strand sequences of these VIROMIRs to the pNL4.3 target
sequence was assessed using RNAhybrid to ensure appropriate binding
as predicted based on the presence of the seed. In some cases, this
binding analysis identified other possible seed-based binding
interactions between the candidate guide strands and other sites on
the HIV genome.
[0329] HIV generally causes chronic infection with in vivo viral
reservoirs. Consequently, VIROMIRs targeting HIV are most likely to
be therapeutically effective as cell-expressed short hairpin RNAs
(shRNAs) rather than as synthetic RNA duplexes because of a need
for continued therapeutic cover to prevent re-emergence from latent
sites.
[0330] Three representative VIROMIRs were selected (VM004, VM006
and VM010) for expression as shRNA. Contiguous DNA sequences
corresponding to: BamHI restriction site, G initiator, VIROMIR
passenger, Xho loop sequence (ACTCGAGA), VIROMIR guide strand,
polIII terminator and HindIII restriction site were assembled and
prepared as dsDNA. They were then cloned into a pSIL vector under
the control of a H1 promoter. By way of example, the
double-stranded DNA insert designed to encode an shRNA VIROMIR
approximating VM006 is shown below (loop sequence in parentheses
and terminator italicized):
TABLE-US-00024 (SEQ ID NO: 329) 5'GATCCGATGGGTTTACCAGGAAGATGG
(ACTCGAGA) CCATCTTCC TGGTAAACCCATTTTTTTGGAA-3' (SEQ ID NO: 330)
3'GCTACCCAAATGGTCCTTCTACC (TGAGCTCT) GGTAGAAGGACCA
TTTGGGTAAAAAAACCTTTCGA-5'
[0331] One skilled in the art will appreciate that when
transcribed, the encoded RNA folds into a hairpin structure, which
is modified by the cellular Drosha and Dicer proteins to generate
active VIROMIR RNA duplex(es). The skilled artisan will also
recognize that a number of variations of the design of the shRNA
construct could be considered. These include but are not limited
to: length, sequence and orientation of the shRNA duplex components
(guide strand, passenger strand, precursors), length and sequence
of the loop, choice of promoter, initiator and terminator sequences
as well as the cloning strategies used to assemble the final
construct.
[0332] Each of these shRNA contructs was tested in HEK-293 cells by
co-transfecting with the pNL4.3 plasmid. Specifically, HEK-293
cells were seeded at density of 2.times.10 5 cells in 1 ml Optimem
medium/well in a 12-well plate. Cells were transfected 24 hr later
with 200 .mu.L DNA: Lipofectamine mix (200 ng pNL4.3 plasmid, 67 ng
VIROMIR pSIL construct in 100 .mu.L complexed with 2.7 .mu.L
Lipofectamine 2000 in Optimem). After changing the medium 24 hours
later, the production of p24 was assayed by collection of the
supernatant after a further 24 hours of incubation.
[0333] The production of p24 was expressed as a percentage of the
production from cells transfected with the empty control plasmid.
As shown in FIG. 7, HIV was suppressed by 60% in the case of the
shRNA form of VM006, whereas the other two constructs had no
detectable activity.
[0334] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules will comprise the sequence
corresponding to the complement of the seed. In this example, these
complementary sequences will
TABLE-US-00025 CUGCUUGAU, (SEQ ID NO: 12) UCCUUUCCA, (SEQ ID NO:
13) UUUUUCUUU, (SEQ ID NO: 14) UUCUGAUGUUU, (SEQ ID NO: 15)
UCUUCCUCUAU, (SEQ ID NO: 16) UGGUAGCUGAA, (SEQ ID NO: 17)
CUUUGGUUCCU, (SEQ ID NO: 18) CUACUAAUGCU, (SEQ ID NO: 19)
UCCUGCUUGAU, (SEQ ID NO: 20) AUUCUUUAGUU, (SEQ ID NO: 21)
CCAUCUUCCUG, (SEQ ID NO: 22) CCUCCAAUUCC, (SEQ ID NO: 23)
CUAAUACUGUA, (SEQ ID NO: 24) UUCUGUUAGUG, (SEQ ID NO: 25)
GCUGCUUGAUG, (SEQ ID NO: 26) ACAUUGUACUG, (SEQ ID NO: 27)
UGAUAUUUCUC, (SEQ ID NO: 28) AACAGCAGUUG, (SEQ ID NO: 29)
GUGCUGAUAUU, (SEQ ID NO: 30) CCCAUCUCCAC, (SEQ ID NO: 31)
UAUUGGUAUUA, (SEQ ID NO: 32) CAAAUUGUUCU, (SEQ ID NO: 33)
UACUAUUAAAC (SEQ ID NO: 34)
Example 7
Multitargeting of Gene Products Implicated in Alzheimer's
Disease
[0335] In some cases it may be beneficial to include multiple
transcript variants corresponding to at least one of the targets of
interest. For example, down-regulation of presenilin-1 and the four
variant isoforms of BACE-1 could be therapeutically advantageous
for the treatment of Alzheimer's disease. In this case, examination
of the five corresponding sequences yields several candidate seeds
of 11 and 12 bp identity including: ATATGATAGGC (SEQ ID NO: 331),
AGCAGGCACCA (SEQ ID NO: 66), GCCATATTAATT (SEQ ID NO: 332),
AGCCCAGAGGG (SEQ ID NO: 68), ATGAGGAAGAA (SEQ ID NO: 333),
TCTGTATAAATA (SEQ ID NO: 334) and GAATTTTGGTG (SEQ ID NO: 335).
From these, those that have appropriate secondary characteristics
(strand loading bias, continued partial identity, sequence
complexity etc) may be of specific interest for the design of
CODEMIRs useful in the treatment of Alzheimer's disease.
[0336] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules (CODEMIRs) will comprise
the sequence corresponding to the complement of the seed. In this
example, these complementary sequences are GCCUAUCAUAU (SEQ ID NO:
58), UGGUGCCUGCU (SEQ ID NO: 59), AAUUAAUAUGGC (SEQ ID NO: 60),
CCCUCUGGGCU (SEQ ID NO: 61), UUCUUCCUCAU (SEQ ID NO: 62),
UAUUUAUACAGA (SEQ ID NO: 63), and CACCAAAAUUC (SEQ ID NO: 64).
Example 8
Use of Wobbles and Mismatches in the Design of CODEMIRs
[0337] The length of the seed may, in some cases be reduced to
enable the requisite multi-targeting to occur. However, in these
cases, it is advisable to limit the selection of these short seeds
to those that have regions of further identity juxtaposed near the
identified seed. For example, VEGF-A and bFGF targeting could be
achieved through the design of a CODEMIR guide strand that would be
predicted to bind to AATGTTCCCACTCA (VEGF-A) (SEQ ID NO: 336) and
AATGTTCAGACTCA (bFGF) (SEQ ID NO: 337). In this instance, this
further region of identity may compensate for the short ACTCA (SEQ
ID NO: 338) seed.
[0338] Alternatively, the seed could include mismatches that would
correspond to wobble base-pairing between the target and the
CODEMIR guide strand. In this situation, G:U wobble base pairing
can be utilized to design the 5' region of a CODEMIR guide strand
with predicted binding to several target transcripts. In Table 8-1,
the 5'region of a CODEMIR targeting VEGF-A, ICAM-1, PlGF and IGF-1
is derived from a suitable seed (CCTGGAG) (SEQ ID NO: 339) in the
corresponding target mRNA.
TABLE-US-00026 TABLE 8-1 Use of wobble-base pairing compatible
mis-matches Gene of interest Target sequence (5' - 3') VEGF-A
AGTCCTGGAG (SEQ ID NO: 340) ICAM-1 AACCCTGGAG (SEQ ID NO: 341) P1GF
GGCCCTGGAG (SEQ ID NO: 342) IGF-1 GGTCCTGGAG (SEQ ID NO: 343)
Consensus CODEMIR 5'region RRYCCTGGAG* 5' CUCCAGGGUU 3' (SEQ ID NO:
344) (SEQ ID NO: 345) *Y indicates pyrimidine and R purine
[0339] Further cases of tolerated mismatches in CODEMIRs have been
examined in the context of the first base of the seed. Tolerability
of a mismatch in this position would greatly enhance the
multi-targeting nature of CODEMIRs. For example, in a situation in
which two targets share the same seed, but a third target has a
mismatch in the first seed position, maintaining activity in spite
of this mismatch against the third target enhances the repertoire
of CODEMIR activity. Flexibility in this position also enables
modification of the 5'terminus of the CODEMIR guide strand with the
goal of modulating strand-loading bias, which may also impact on
activity. The efficacy of CODEMIRs with a 5' mismatch of the guide
strand with the target was investigated with CODEMIRs 13 to 15
(Table 8-2). These results, shown in FIG. 6, indicate that CODEMIRs
with a single mismatch retain activity against the target.
TABLE-US-00027 TABLE 8-2 Examples of CODEMIRs targeting VEGF and
ICAM-1 containing CODEMIR-target mismatches (bold face) at the 5'
end of the guide strand CODEMIR (Guide strand on bottom 3' to 5')
CODEMIR-13 5'CUCACCCACCCACAUACAUUU 3' (SEQ ID NO: 112)
3'CUGAGUGGGUGGGUGUAUGUA 5' (SEQ ID NO: 113) CODEMIR-14
5'UCACCCACCCACAUACAUAUU 3' (SEQ ID NO: 114) 3'UGAGUGGGUGGGUGUAUGUAU
5' (SEQ ID NO: 115) CODEMIR-15 5'UCACCCACCCACAUACAUUUU 3' (SEQ ID
NO: 116) 3'UGAGUGGGUGGGUGUAUGUAA 5' (SEQ ID NO: 117) VEGF binding*
5'A A A 3' GAC CACCCACCCACAUACAU (SEQ ID NO: 346) CUG
GUGGGUGGGUGUAUGUA (SEQ ID NO: 113) 3' A 5' 5' A C 3'
CACCCACCCACAUACAUA (SEQ ID NO: 348) GUGGGUGGGUGUAUGUAU (SEQ ID NO:
115) 3'UGA 5' 5'A A 3' CACCCACCCACAUACAU (SEQ ID NO: 349)
GUGGGUGGGUGUAUGUA (SEQ ID NO: 117) 3'UGA A 5' ICAM-1 binding* 5' C
U 3' CUC CCCACCCACAUACAU (SEQ ID NO: 347) GAG GGGUGGGUGUAUGUA (SEQ
ID NO: 113) 3'CU U 5' 5'C U 3' CUC CCCACCCACAUACAU (SEQ ID NO: 347)
GAG GGGUGGGUGUAUGUA (SEQ ID NO: 115) 3'U U U 5' 5'C U 3' CUC
CCCACCCACAUACAUU (SEQ ID NO: 350) GAG GGGUGGGUGUAUGUAA (SEQ ID NO:
117) 3'U U 5' *Upper strand =ztarget mRNA, lower strand CODEMIR
guide. Predicted mismatches are shown in bold font
[0340] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules (CODEMIRs) will comprise
the sequence corresponding to the complement of the seed. In the
example of an RNA molecule for altering expression of VEGF-A and
bFGF, a guide strand could comprise UGAGUNNGAACAUU (SEQ ID NO: 72)
where N is any base. In the example of an RNA molecule for altering
expression of any combination of VEGF-A, ICAM-1, PlGF and IGF-1,
the complementary sequence to the seed CCUGGAG (SEQ ID NO: 75)
comprises CUCCAGG (SEQ ID NO: 74).
Example 9
Co-Suppression of Virus and Disease-Implicated Host Protein
[0341] In the case of infectious diseases, CODEMIRs can also be
utilized to target both the genome of the infectious agent and one
or more key host "drivers" of the disease. For example, TNF-alpha
is considered a major disease-associated factor in Hepatitis C
Virus infection and its sequelae. Analysis of the genome of HCV and
the TNF-alpha mRNA sequence was used to identify seeds consisting
of: CCCTGTGAGGA (SEQ ID NO: 351), CACCATGAGCAC (SEQ ID NO: 352),
CAGGGCTCCAGG (SEQ ID NO: 353) and, GTGGAGCTGAGA (SEQ ID NO:
354).
1. The first seed, CCCTGTGAGGA (SEQ ID NO: 351) was found to be
highly conserved (92%) in the 155 available sequences for genotype
1a/1b isolates and, therefore, an attractive target sequence from a
therapeutic point of view. In terms of genetic context, the seed is
in the 5'NTR of HCV and the ORF of TNFalpha
TABLE-US-00028 (SEQ ID NO: 355)
GGGCGACACTCCACCATGAATCACTCCCCTGTGAGGAACTACTGTCTTCA CGCAGAAAGC HCV
43 (SEQ ID NO: 356)
TGGGCAGGTCTACTTTGGGATCATTGCCCTGTGAGGAGGACGAACATCCA ACCTTCCCAA TNFa
864
A consensus target site is, possibly:
TABLE-US-00029 5'-GAATCACTGCCCTGTGAGGAA-3' (SEQ ID NO: 357)
and duplex:
TABLE-US-00030 5'-UUCCUCACAGGGCAGUGAUUC-3' (SEQ ID NO: 122)
3'-UUAAGGAGUGUCCCGUCACUA-5' (SEQ ID NO: 123)
with predicted binding:
TABLE-US-00031 HCV 5' U C C 3' GAAUCACU CCCUGUGAGGAA (SEQ ID NO:
358) CUUAGUGA GGGACACUCCUU 3' C 5' (SEQ ID NO: 122) guide mfe:
-38.8 kcal/mol TNFa 5' G G 3' GGAUCAUUGCCCUGUGAGGAG (SEQ ID NO:
359) CUUAGUGACGGGACACUCCUU 3' 5' (SEQ ID NO: 122) guide mfe: -42.6
kcal/mol
[0342] One skilled in the art would recognize that this interfering
RNA could be further modified to improve its strand loading bias
and stability. This could be achieved through the use of modified
bases (eg LNA, 2'-F or 2'-O-methyl) in the 3' duplex section of the
guide strand (would also likely improve stability), for
example:
TABLE-US-00032 5'-UUCCUCACAGGGCAGUGAUUC-3' (SEQ ID NO: 539)
3'-UUAAGGAGUGUCCCGUCACUA-5' (SEQ ID NO: 123)
in which the bolding indicates LNA-modified bases. Alternatively,
judicious introduction of mismatches such as, for example:
TABLE-US-00033 5'-UUCCUCACAGGGCAGUGAUUC-3' (SEQ ID NO: 122)
3'-UUAAAGAGUGUCCCGUCACUA-5' (SEQ ID NO: 124)
in which the bolding indicates a mismatched base in the non-active
passenger strand could be used. Alternatively, one could consider
substitution of bases in the 3' duplex part of the guide strand and
corresponding changes in the passenger strand such as:
TABLE-US-00034 5'-UUCCUCACAGGGCAGUGGUUC-3' (SEQ ID NO: 125)
3'-UUAAGGAGUGUCCCGUCACCA-5' (SEQ ID NO: 126)
in which the bolding indicates the changed bases. The change in the
3' end of the guide strand is possibly associated with minimal
change in activity or hybridization given the G:U wobble that would
occur with the target binding sequences as shown below:
TABLE-US-00035 HCV 5' U C C 3' GAAUCACU CCCUGUGAGGAA (SEQ ID NO:
358) CUUGGUGA GGGACACUCCUU (SEQ ID NO: 125) 3' C 5' guide TNFa 5' G
G 3' GGAUCAUUGCCCUGUGAGGAG (SEQ ID NO: 359) CUUGGUGACGGGACACUCCUU
(SEQ ID NO: 125) 3' 5' guide
[0343] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules (CODEMIRs) will comprise
the sequence corresponding to the complement of the seed. In this
example, these complementary sequences are UCCUCACAGGG (SEQ ID NO:
78), GUGCUCAUGGUG (SEQ ID NO: 79), CCUGGAGCCCUG (SEQ ID NO: 80) and
UCUCAGCUCCAC (SEQ ID NO: 81).
Example 10
Simultaneous Multitargeting of Tumour and Stromal Factors Important
in Cancer Progression
[0344] An extension of this concept can be considered in the case
of some diseases in which several tissue "compartments" act
together in augmenting the deleterious effects of the disease.
Examples of such a situation would encompass, for example, the role
of the stromal cells in cancer. In this situation, blocking the
secretion of paracrine factors (eg growth factors such as VEGF) by
the stroma would be advantageous. Indeed, targeting the
neovasculature simultaneously with cytotoxic chemotherapy has
significant clinical benefit in the case of colorectal cancer. Some
of these factors also function in an autocrine fashion and cancer
cells produce, for example, VEGF and other growth factors. Also,
the simultaneous down-regulation of an anti-apoptotic protein (eg
bcl-2, bcl-X.sub.L) might permit greater anti-cancer activity.
Therefore, a CODEMIR targeting VEGF-A, K-ras, EGFR and bcl-2 would
be of considerable interest. An example of a seed common to all
these targets was identified as: CCCACTGA. In this example, the
complementary sequence is UCAGUGGG (SEQ ID NO: 76).
[0345] Seeds common to the mRNA sequences of the more limited
sub-set of VEGF, Bcl-2 and K-Ras were identified as: GACAGTGGA,
CTATTCTG and TAGAGAGTT. The sequences complementary to these seeds
are: UCCACUGUC (SEQ ID NO: 92), CAGAAUAG (SEQ ID NO: 93) and
AACUCUCUA (SEQ ID NO: 94).
[0346] A set of 8 CODEMIRs (CC014-CC021) were designed from the
latter three seeds. In this early and preliminary experiment, the
design of the CODEMIRs did not necessarily follow the guidelines
eventually developed. For example, the positioning of the
seed-binding region was not necessarily constrained to the region
proximal to the 5' end of the guide strand. The predicted binding
to all three targets is shown in Table 10-1.
[0347] According to eventual design guidelines, one of these
CODEMIRs (CC014) would be predicted to be sub-optimal based on
reduced likelihood of loading because of its G/C rich 5' terminus
of the guide strand relative to the passenger strand. CC015 was of
particular interest for the targeting of K-ras, because of the
presence of 2 binding sites featuring the seed. All of these
CODEMIRs were synthesized and tested against all three targets as
part of the evaluation.
[0348] The two colon cancer cell lines HCT116 and SW480 were used
for the evaluation of anticancer CODEMIRs. Cells were plated at
4000 cells per well of a 96-well plate. Transfection was performed
24 hours later using 0.2-0.3 .mu.L Lipofectamine2000 per well and
sufficient dsRNA to yield a final concentration of 40 nM.
Transfection efficiency was evaluated by measuring the effect of
siTOX (a cytotoxic siRNA from Dharmacon) on cellular survival
relative to mock and inactive controls using the Cell Titer Blue
assay (CTB).
[0349] All cancer CODEMIRs were then screened for effects on
cellular survival using this assay. Briefly, 8 h post-transfection,
the serum-containing medium was removed and replaced with OptiMEM
(serum-free) as we found this to improve the dynamic window of the
assay in separating specific (siTOX) versus non-specific (siGC47)
cell death. Different times for serum withdrawal and recovery were
tested. The optimum protocol appeared to be withdrawal of serum for
16 h followed by a 48 h recovery period in 10% FCS (fetal calf
serum). Survival was measured 72 hr post-transfection. This assay
was used to measure the effects of all cancer CODEMIRs on the
survival of HCT116 cells (FIG. 8). Transfection with siRNA
specifically designed to target K-Ras or Bcl-2 had greater effects
on the survival of serum-starved cells as compared to non-starved
cells. Transfection with a VEGF-specific siRNA with activity in
several species (human, rodent), PVE, did not affect the survival
of HCT116 cells under either of these conditions, relative to a
non-targeting siRNA, siGC47. Of the cancer CODEMIRs, CC015, in
particular, reduced cellular survival under both conditions.
TABLE-US-00036 TABLE 10-1 Cancer CODEMIRs and predicted
hybridization to target mRNA Duplex (top strand 5' to 3', bottom
strand Name 3' to 5') (a) Predicted target hybridization CC014
Passenger KRAS 5' U A 3' CAAAAAUGACAGUGGACGAUU AAAAUGACAGUGGA (SEQ
ID NO: 362) GGGUUUUUACUGUCACCUGCU UUUUACUGUCACCU (SEQ ID NO: 361)
Guide guide 3' GGGU GCU 5' Top strand BCL-2 5' G U 3' (SEQ ID NO:
360) CAAGAGUGACAGUGGAU (SEQ ID NO: 363) Bottom strand
GUUUUUACUGUCACCUG (SEQ ID NO: 361) (SEQ ID NO: 361) guide 3' GG CU
5' VEGF 5' C CCGGC CG C 3' CCCGG GG GACAGUGGACG (SEQ ID NO: 364)
GGGUU UU CUGUCACCUGC (SEQ ID NO: 361) guide 3' UA U CC015 Passenger
KRAS 5' G A GUGUGAACCUUUGAGCUUUCA C 3' GGACCCUUAGAGAGUUUCAUU #1 CC
GGCCCU UAGAGAGUUUCA (SEQ ID NO: 365) GGCCUGGGAAUCUCUCAAAGU GG
CUGGGA AUCUCUCAAAGU (SEQ ID NO: 138) Guide guide 3' C 5' Top strand
KRAS 5' GA CU A C 3' (SEQ ID NO: 137) #2 G UUC UAGAGACUUUCA (SEQ ID
NO: 366) Bottom strand C GGG AUCUCUCAAAGU (SEQ ID NO: 138) (SEQ ID
NO: 138) guide 3' GG CU A 5' BCL2 5' U G 3' GACCUUUAGAGAGUU (SEQ ID
NO: 367) CUGGGAAUCUCUCAA (SEQ ID NO: 138) guide 3' GGC AGU 5' VEGF
5' C AGA U3' CCGGA UUAGAGAGUUUUA (SEQ ID NO: 368) GGCCU
AAUCUCUCAAAGU (SEQ ID NO: 138) guide 3' GGG 5' CC016 Passenger
ACCCUUAGAGAGUUUCACAUU KRAS 5' A UCAGCUUUCA G 3'
CCUGGGAAUCUCUCAAAGUGU ACCUU UAGAGAGUUUCACA (SEQ ID NO: 371) Guide
UGGGA AUCUCUCAAAGUGU (SEQ ID NO: 370) Top strand guide 3' CC 5'
(SEQ ID NO: 369) BCL-2 5' U GCU U 3' Bottom strand GACCUUUAGAGAGUU
UUACG (SEQ ID NO: 372) (SEQ ID NO: 370) CUGGGAAUCUCUCAA AGUGU (SEQ
ID NO: 370) 3' C 5' VEGF 5' A GGAAGA U 3' GCCC UUAGAGAGUUUUAU (SEQ
ID NO: 373) UGGG AAUCUCUCAAAGUG (SEQ ID NO: 370) guide 3' CC U 5'
CC017 Passenger KRAS 5' U G U 3' AUCCCUAUUCUGUUCUUUAUU
CAUCCCUAUUCUGU UUUUA (SEQ ID NO: 376) CGUAGGGAUAAGACAAGAAAU
GUAGGGAUAAGACA GAAAU (SEQ ID NO: 375) Guide guide 3' C A 5' Top
strand BCL2 5' A A A 3' (SEQ ID NO: 374) UUCUAUUCUG UCUU (SEQ ID
NO: 377) Bottom strand GGGAUAAGAC AGAA (SEQ ID NO: 375) (SEQ ID NO:
375) guide 3' CGUA A AU 5' VEGF 5' A G U 3' CAUU CUAUUCUGUUUUUUA
(SEQ ID NO: 378) GUAG GAUAAGACAAGAAAU (SEQ ID NO: 375) guide 3' C G
5' CC018 Passenger KRAS 5' U G U 3' CAUCCCUAUUCUGUUCUUAUU
CAUCCCUAUUCUGU UUUUA (SEQ ID NO: 376) CUGUAGGGAUAAGACAAGAAU
GUAGGGAUAAGACA AGAAU (SEQ ID NO: 380) Guide guide 3' CU 5' Top
Strand BCL-2 5' A A U 3' (SEQ ID NO: 379) UUCUAUUCUG UCUUA (SEQ ID
NO: 381) Bottom strand GGGAUAAGAC AGAAU (SEQ ID NO: 380) (SEQ ID
NO: 380) guide 3' CUGUA A 5' VEGF 5' A G U 3' GACAUU CUAUUCUGUUUUU
(SEQ ID NO: 382) CUGUAG GAUAAGACAAGAA (SEQ ID NO: 380) guide 3' G U
5' CC019 Passenger KRAS 5' G U UGUGA UGAGCUUUCA U 3'
CCCGGACCCUUAGAGAGUUUU GCCC G ACCUU UAGAGAGUU (SEQ ID NO: 383)
ACGGGCCUGGGAAUCUCUCAA CGGG C UGGGA AUCUCUCAA (SEQ ID NO: 128) Guide
guide 3' A C 5' Top strand BCL-2 5' G UU G 3' (SEQ ID NO: 127) GU G
ACCUUUAGAGAGUU (SEQ ID NO: 384) Bottom strand CG C UGGGAAUCUCUCAA
(SEQ ID NO: 128) (SEQ ID NO: 128) guide 3' A GG C 5' VEGF 5' A AGA
U 3' GCCCGGA UUAGAGAGUU (SEQ ID NO: 385) CGGGCCU AAUCUCUCAA (SEQ ID
NO: 128) guide 3' A GGG 5' CC020 Passenger KRAS 5' G U UGUGA
UGAGCUUUCA C 3' CCGGACCCUUAGAGAGUUUUU GCCC G ACCUU UAGAGAGUUU (SEQ
ID NO: 388) CGGGCCUGGGAAUCUCUCAAA CGGG C UGGGA AUCUCUCAAA (SEQ ID
NO: 387) Guide guide 3' C 5' Top strand BCL-2 5' G UU G 3' (SEQ ID
NO: 386) GU G ACCUUUAGAGAGUU (SEQ ID NO: 384) Bottom strand CG C
UGGGAAUCUCUCAA (SEQ ID NO: 387) (SEQ ID NO: 387) guide 3' GG C A 5'
VEGF 5' A AGA U 3' GCCCGGA UUAGAGAGUUU (SEQ ID NO: 564) CGGGCCU
AAUCUCUCAAA (SEQ ID NO: 387) guide 3' GGG 5' CC021 Passenger KRAS
5' C U UGA UGACCUUUCA A 3' CGGACCCUUAGAGAGUUUCUU CCUG G ACCUU
UAGAGAGUUUC (SEQ ID NO: 390) GGGCCUGGGAAUCUCUCAAAG GGGC C UGGGA
AUCUCUCAAAG (SEQ ID NO: 389) Guide guide 3' 5' Top strand BCL-2 5'
U G 3' (SEQ ID NO: 543) GACCUUUAGAGAGUU (SEQ ID NO: 391) Bottom
strand CUGGGAAUCUCUCAA (SEQ ID NO: 389) (SEQ ID NO: 389) guide 3'
GGCC AG 5' VEGF 5' G AGA A 3' CCCGGA UUAGAGAGUUUU (SEQ ID NO: 392)
GGGCCU AAUCUCUCAAAG (SEQ ID NO: 389) guide 3' GGG 5'
[0350] The abundance of Bcl-2 in HCT116 cells was measured by ELISA
(R&D systems) and by Western. The signal for this protein was
relatively weak and appreciable knock-down could only be detected
for HCT-116 cells transfected with a Bcl-2-specific siRNA (siBcl2)
when measured by Western blotting. No impact of any of the CODEMIRs
or even siBcl2 was detected using the ELISA assay.
[0351] The abundance of K-Ras following transfection with CODEMIRs
CC014-CC021 was measured in HCT116 cell extracts by Western (FIG.
9). CC015 reduced K-Ras to a similar level as a K-ras-specific
siRNA (siKRas) while CC020 and CC021 also had some effect (FIG.
9).
[0352] VEGF production by HCT-116 cells was measured using the same
VEGF ELISA (R&D systems) used to measure the effects of
CODEMIR-1 (AM001) in ARPE cells. HCT116 cells produce moderate
levels of VEGF (.about.200 pg/mL in 48 hrs when seeded at 4000
cells/well) as measured by ELISA. CODEMIRs CC015, CC019, CC020 and
CC021 all resulted in a decrease in the production of VEGF by
HCT116 cells. The most marked effect was caused by CC019 which
resulted in a .about.55% reduction as compared to untransfected
cells (FIG. 10). CC015, which also had suppressive activity against
K-ras, reduced VEGF by .about.20%.
[0353] Annexin V and PI staining followed by FACS analysis was used
to analyze apoptosis in HCT116 cells following transfection with
various siRNAs and CODEMIRs. Cells that are positive for Annexin V
and negative for Propidium Iodide (PI) are considered to be cells
undergoing early apoptosis. This assay was used to determine the
number of dead (PI positive) and apoptotic cells following
transfection with several cancer CODEMIRs into HCT116 (FIG. 11) and
SW480 (data not shown) cells. SiTOX, siBcl2 and siKRas each caused
a marked increase in the Annexin V binding in HCT116 cells while
another VEGF-specific siRNA only caused a slight increase (FIG.
11). Transfection of HCT116 cells with CC015 resulted in Annexin V
binding similar to that for the KRas and Bcl2-specific siRNAs.
CC015 also increased Annexin V binding in SW480 cells, as did
transfection with siKRas (data not shown).
[0354] A Caspase 3/7 activation assay (FACS staining with caspase
substrate) was performed to gain more insight into the mode of
apoptosis in HCT116 cells following transfection with CC015. There
was no noticeable increase in caspase activation following
transfection with siKRas, siTOX or CC015 (data not shown).
[0355] Soft agar-based anchorage independent growth assays are of
interest because it has been demonstrated that the ability of a
cell to form a colony in agar is linked to its ability to form a
tumour in vivo. A conventional soft-agar assay was performed in
which the cells were transfected and harvested 24 h later, at which
point cells from each treatment were counted and re-plated in a
6-well plate in 0.4% low-melting agarose. Seven days
post-transfection the colonies that formed were counted under a
microscope. With HCT116 cells, CC015 was very effective at reducing
colony formation to an even greater extent than siKRas and siBcl-2
while CC018, CC019, CC020 and CC021 had no apparent effect on
colony formation relative to a non-targeting siRNA, siGC47 (FIG.
12). In SW480 cells, transfection with CC015 also markedly reduced
the ability of the cells to form colonies while CC019, CC020 and
CC021 had no effect (data not shown).
[0356] Overall, we found that CC015, which was designed using a
seed found in the mRNA of Bcl-2, K-ras and VEGF, was able to
suppress K-ras and to a lesser extent VEGF. In keeping with our
experiments performed with CODEMIR-1, however, it was clear that
having the seed-binding region of the guide strand upstream of 3
mismatched bases, could not be expected to lead to suppression of
Bcl-2. Likewise, the positioning of the seed-binding region of
CC016-18 in relation to this target was sub-optimal. However,
technical issues with measuring Bcl-2 mean that we cannot rule out
a possible effect on Bcl-2. The cancer CODEMIR CC015 was found to
induce cell death in HCT-116 and SW480 cells but not the non-cancer
cell line ARPE-19 (data not shown). CC015 also markedly inhibited
colony formation in soft-agar. Table 10-2 summarizes the effects of
CC015 in HCT116 and SW480 cells.
[0357] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules (CODEMIRs) will comprise
the sequence corresponding to the complement of the seed. In this
example, these complementary sequences are UCCACUGUC (SEQ ID NO:
92), CAGAAUAG (SEQ ID NO: 93) and AACUCUCUA (SEQ ID NO: 94).
TABLE-US-00037 TABLE 10-2 Summary of the effects of CC015 on cancer
cells. HCT116 SW480 Survival (in serum) Decreased Decreased
Survival (serum withdrawal) Decreased survival Not tested Annexin V
staining Increased Increased PI staining Increased Increased Colony
forming assay Decreased Decreased VEGF secretion Reduced (15-20%)
Not determined K-Ras abundance Reduced (40%) Not determined Bcl-2
abundance No change Not determined
Example 11
Multitargeting when at Least One Target is a Non-Coding RNA
[0358] Although we present above many examples relating to
targeting mRNA encoding protein targets, the concept is equally
applicable to non-coding RNAs. In cancer and other diseases,
non-coding RNA such as MALAT-1, BIC, PCGEM1, DD3 and BC200 have
been shown to be associated with more aggressive progression and
worse patient outcomes. Targeting seeds common to some or all of
these could be considered in the design of CODEMIRs. Other
non-coding RNA (eg. such as microRNA, pre- and pri-microRNA, snoRNA
etc) can equally be considered.
[0359] In the case of a CODEMIR designed to target two
cancer-related non-coding RNA molecules, MALAT-1 and BIC, an
exemplary seed sequence is: GGTGCGAGGGT (SEQ ID NO: 393
[0360] When viewed in the relationship to the genetic context of
this seed in the two target RNA sequences:
TABLE-US-00038 (SEQ ID NO: 394) AACGTGGCAGGGACGCCGGGGGACTTC
GGTGCGAGGGT CACCGCCGGG TTAACTGGC (MALAT-1) (SEQ ID NO: 395)
CAAGTAGGGTACGGACTTTGGGGGATT GGTGCGAGGGT AGTGGGTGAG TGGCCTACT
(BIC)
it is apparent that a suitable consensus target site can easily be
generated with the methods provided elsewhere in the invention.
Because of the G/C-rich region near the 3' end of the seed, the
5'end of the active (guide) strand of the duplex multitargeting
interfering RNA would need to be shifted away from this G/C region,
although the presence of a G/C-region in the 5' extension of the
seed (with the exception of the desired overhangs) would assist in
mitigating these effects. A candidate consensus sequence could be
(seed underlined and bolded):
TABLE-US-00039 5'-TTGGGAGATCGGTGCGAGGGTA-3' (SEQ ID NO: 396)
with the resulting duplex being, for example:
TABLE-US-00040 (SEQ ID NO: 129) 5'-UACCCUCGCACCGAUCUCCCAA-3'
(guide) (SEQ ID NO: 130) 3'-UUAUGGGAGCGUGGCUAGAGGG-5'
(passenger)
[0361] The guide strand of this CODEMIR has predicted binding to
the two targets (RNAhybrid) as follows:
TABLE-US-00041 MALAT-1 5' C CU C 3' GGGGGA UCGGUGCGAGGGU (SEQ ID
NO: 397) CCCUCU AGCCACGCUCCCA (SEQ ID NO: 129) 3' AA U 5' guide
mfe: -43.6 kcaL/mol BIC 5'U G 3' UUGGGGGAUUGGUGCGAGGGUA (SEQ ID NO:
398) AACCCUCUAGCCACGCUCCCAU (SEQ ID NO: 129) 3' 5' guide mfe: -48.4
kcal/mol
[0362] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules will comprise the sequence
corresponding to the complement of the seed. In this example, this
complementary sequence is:
TABLE-US-00042 ACCCUCGCACC. (SEQ ID NO: 86)
Example 12
CODEMIRs for the Treatment or Prophylaxis of Pulmonary Fibrosis
[0363] Pulmonary fibrosis is frequently observed as a sequelae of
lung injury (smoke inhalation, pneumonia, trauma etc). It also
occurs without a known causative factor in idiopathic pulmonary
fibrosis, which is progressive and almost universally fatal.
Regardless of the etiology, it appears that the transforming growth
factor .quadrature. (TGF.quadrature.) axis is a major signaling
pathway in pulmonary fibrosis, although other factors such as
Connective Tissue Growth Factor (CTGF), IL-8 and MCP-1 are likely
to play a role. CODEMIRs targeting TGFb, IL-8 and MCP-1 were
sought. The 3'UTR of these mRNA were used in this search as
described in other examples. A seed common to IL-8 and TGFb was
identified as CUUCAACAC (SEQ ID NO: 89). Two consensus target
sequences were designed by eye from the aligned mRNA sequences for
these two targets. As shown in Table 12-1, the bias of
complementarity for the two targets was reversed for PF007 and
PF008. That is, the consensus target sequence was in one case made
more similar to IL-8 (PF007) and in the second was more similar to
TGF-.quadrature. (PF008). This aspect of design of the
multitargeting interfering RNA of the invention will be seen by one
skilled in the art to enable the titration of the activity of the
multitargeting interfering RNA against the one or more target RNA.
In this instance, PF007 and PF008 had similar activity against TGFb
with .about.50% reduction of TGFb secretion by A549 cells when
assayed by ELISA 48 hours post transfection with 40 nM dsRNA and
lipofectamine. In contrast, IL-8 secretion was suppressed by 80%
and 35% with PF008 and PF007, respectively. Thus these two CODEMIRs
would be expected to be of potential utility for the treatment of
pulmonary fibrosis. They could also be further improved to increase
strand loading. Given that the 5' terminal base of the guide strand
is a G, the corresponding base used in the passenger strand was a U
to provide weaker wobble-base pairing. However, one skilled in the
art would realize that additional modification could be envisaged
which is exemplified but not limited to the inclusion of an
additional C or G to the 3'end of the guide strand, an additional A
or U to the 5'end of the guide strand, or both. Matching these in
the corresponding passenger strand would improve further the
loading bias without necessarily any deleterious impact on their
functional activity.
TABLE-US-00043 TABLE 12-1 CODEMIRs targeting pulmonary fibrosis
targets (TGF.beta., and IL-8). Duplex Target Binding (RNA hybrid)
PF007 Passenger TGFB1 5' G U G U A 3' UACAAAUCUACUUCAACAUUU (SEQ ID
NO: 131) CAUAUA AU U CUUCAACAC (SEQ ID NO: 399)
GUAUGUUUAGAUGAAGUUGUG (SEQ ID NO: 132) GUAUGU UA A GAAGUUGUG (SEQ
ID NO: 132) Guide PF007 3' U G U 5' IL8 5' A U 3'
GCAAAUCUACUUCAACAC (SEQ ID NO: 400) UGUUUAGAUGAAGUUGUG (SEQ ID NO:
132) PF007 3' GUA 5' PF008 Passenger TGFB1 5' G U A 3'
AACAUAUGUUCUUCAACAUUU (SEQ ID NO: 133) CA AUAUAUGUUCUUCAACAC (SEQ
ID NO: 399) GUUUGUAUACAAGAAGUUGUG (SEQ ID NO: 134) GU
UGUAUACAAGAAGUUGUG (SEQ ID NO: 134) Guide FF008 3' U 5' IL-B 5' U A
CUA U 3' CAAGCA AU CUUCAACAC (SEQ ID NO: 544) GUUUGU UA GAAGUUGUG
(SEQ ID NO: 134) FF008 3' A CAA 5'
[0364] Other active CODEMIRs were found including PF018 which
targets the seed of GUUGUGGAA in IL-8 and MCP-1. This CODEMIR, with
the guide sequence UUCCACAACACAAGCUGUGUU (SEQ ID NO: 135),
suppressed IL-8 and MCP-1 secretion by 45% and 60%, respectively.
The CODEMIR PF018 duplex is as follows:
TABLE-US-00044 (SEQ ID NO: 135) 5'-UUCCACAACACAAGCUGUGUU-3' (guide
strand) (SEQ ID NO: 136) 3'-UUAAGGUGUUGUGUUCGACAC-5' (passenger
strand)
[0365] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules will comprise the sequence
corresponding to the complement of the seed. In this example, these
complementary sequences are GUGUUGAAG (SEQ ID NO: 88) and UUCCACAAC
(SEQ ID NO: 90).
Example 13
CODEMIRs for the Treatment of HCV
[0366] All possible 6, 7, 8, 9, 10, 11 and 12mer seeds present at
least twice in at least one of the 155 clade 1a and 1b HCV genome
sequences available from the LANL database have been generated.
[0367] These break down as:
seed length number of seeds
[0368] 6 4012
[0369] 7 10683
[0370] 8 11352
[0371] 9 5942
[0372] 10 2267
[0373] 11 725
[0374] 12 236
This is a total of 35217 sequences.
[0375] One of the seeds from the set identified was:
5'-GTCTTCACG-3' (SEQ ID NO: 401). This seed was selected on the
basis of its high conservation in HCV 1a/1b genotype sequences.
Indeed it was found at least once in the sequences of 154 of 155
1a/1b isolates (conservation of 99%). When examined against other
genotypes (1a-6a), it was found at least once in 97% of isolates.
The other important feature of this seed is its high distribution,
occurring more than once in most sequences. This distribution,
relative to 155 isolates was as follows:
4 times in genome: 5/155 isolates 3 times in genome: 68/155
isolates 2 times in genome: 50/155 isolates 1 time in genome:
31/155 isolates
[0376] A further selection criterion was that this seed occurs only
rarely in the 3'UTR portion of the human transcriptome, indicating
that this seed would be unlikely to generate broad non-specific
effects to the host tissue.
[0377] This seed, however, is G/C rich at the 3' extremity, such
that a complementary guide sequence would, in the context of a
double stranded multitargeting interfering RNA, be unlikely to load
if the guide strand were to be completely complementary to this
sequence and the 3' extremity of the passenger strand was not
similarly G/C rich.
[0378] The genetic contexts of the seed in a representative isolate
shown below were used to devise a strategy to adjust the loading
bias.
TABLE-US-00045 1 (SEQ ID NO: 402) ATCACTCCCCTGTGAGGAACTACT
GTCTTCACG CAGAAAGCGTCTAGC 2 (SEQ ID NO: 403)
ATGGAGACCACTATGCGGTCTCCG GTCTTCACG GACAACTCATCTCCC 3 (SEQ ID NO:
404) GATCACCTGGAGTTCTGGGAGAGC GTCTTCACG GGCCTCACCCACATA 4 (SEQ ID
NO: 405) CAGGAGGATGCGGCGAGCCTACGA GTCTTCACG GAGGCTATGACTAGG
[0379] Through examination of the genetic context, it is clear that
the complementary sequence (S according to claim definition) could
be extended in the 5'direction with the addition of UU, thereby
generating a candidate XS sequence: 5'-UUCGUGAAGAC-3' (SEQ ID NO:
406).
[0380] All possible 21 bp sequences that contain this candidate XS
sequence were generated. Each putative full length XSY sequence was
then tested for its load bias, to favour those XSY sequences that
would, in the context of a double stranded multitargeting
interfering RNA, be likely to have the guide strand loaded into the
RISC complex. This was accomplished by examining the base
composition for the 5 bases at the 5' terminus and the 5 bases at
the 3' terminus of each XSY sequence and scoring them according to
the following table:
TABLE-US-00046 Position from the 5' or 3' terminus Base 1.sup.st
2.sup.nd 3.sup.rd 4.sup.th 5.sup.th G/C 10 8 6 4 2 A/U 5 4 3 2
1
[0381] All putative full-length XSY sequences for which the ratio
of the sum of the scores of the 5 bases at the 3' terminus relative
to the 5 bases at the 5' terminus was less than 1.2 were discarded.
In addition, any XSY sequence that contained a contiguous run of 5
or more G nucleotides was also discarded. The resulting initial set
of XSY sequences contained 422848 sequences. The RNAhybrid program
[Hofacker, (2003), Nucleic Acids Res., 31: 3429-31] was then used
to determine the binding pattern for each of these 422848 sequences
against each of the 4 genetic contexts in a representative HCV
strain (Genbank accession AB049092). The RNAhybrid analysis
required an exact binding of the XS sequence (UUCGUGAAGAC) (SEQ ID
NO: 406) to each genetic context for positions 3 to 11. For each of
the binding patterns, the minimum free energy (mfe) and maximum
length of contiguous completely complementary sequence were used to
generate a relative binding score according to the following
algorithm:
Score=(mfe of XSY sequence.times.length of contiguous
complementarity.times.100)(mfe of the completely complementary
sequence at that genetic context.times.21).
[0382] The average score for each XSY sequence across the 4 genetic
contexts was then determined. From the 422848 sequences, the 183
potential XSY sequences that satisfied the criteria of being in the
top 100 average scores or having a score of >40 in at least 3 of
the four genetic contexts were considered for further analysis. The
RNAhybrid and scoring analysis described above was used to analyse
the binding patterns of the 183 selected XSY sequences against the
sequences of the 4 genetic contexts of all 155 clade 1a/1b isolates
(620 sequences). In this analysis an exact binding of the XS
sequence (UUCGUGAAGAC) (SEQ ID NO: 406) was not required.
[0383] Using the criterion of a score>=50, XSY sequences were
selected which gave the highest number of scores>=50 across the
620 genetic contexts. In this case, there were 9 XSY sequences
which each gave a score of >=50 in 147 of the 620 genetic
contexts. These 9 XSY sequences are:
TABLE-US-00047 UUCGUGAAGACGGUGGGCCGA (SEQ ID NO: 139)
UUCGUGAAGACGGUGGGCCGG (SEQ ID NO: 407) UUCGUGAAGACGGUGGGCCGC (SEQ
ID NO: 408) UUCGUGAAGACGGUGGGCCGU (SEQ ID NO: 409)
UUCGUGAAGACGGUAGGCCGA (SEQ ID NO: 410) UUCGUGAAGACGGUAGGCCGG (SEQ
ID NO: 411) UUCGUGAAGACGGUAGGCCGC (SEQ ID NO: 412)
UUCGUGAAGACGGUAGGCCGU (SEQ ID NO: 413) UUCGUGAAGACAGUGGGCCGC (SEQ
ID NO: 414) UUCGUGAAGACAGUAGGCCGC (SEQ ID NO: 415)
[0384] All 9 XSY sequences had very similar binding patterns at
each genetic context. Columns 2 and 3 shown below in Table 13-2
represent the six possible binding patterns for the 9 XSY sequences
to the four genetic contexts.
TABLE-US-00048 TABLE 13-2 Possible binding patterns to four genetic
contexts of HCV seed Genetic Context Binding patterns (top strand
is target 5'-3', guide strand at bottom 3'-5') 1 5' A AA C-3' GG
CUACUGUCUUCACG (SEQ ID NO: 416) CC GRUGRCAGAAGUGC (SEQ ID NO: 417)
3'NG G UU-5' 2 U C G C UG C G C GCGGUCU C GUCUUCACGGA (SEQ ID NO:
418) CGGUCU C GUCUUCACGGA (SEQ ID NO: 418) YGCCGGR G CAGAAGUGCUU
(SEQ ID NO: 419) GCCGGR G CAGAAGUGCUU (SEQ ID NO: 420) U R R U R 3
G AGA G GG GC GUCUUCACGG (SEQ ID NO: 421) CC UG CAGAAGUGCU (SEQ ID
NO: 417) NG GGR R U 4 G A GA G GG A GA G GCG GCCUAC GUCUUCACGGA
(SEQ ID NO: 422) CG GCCUAC GUCUUCACGGA (SEQ ID NO: 422) YGC CGGRUG
CAGAAGUGCUU (SEQ ID NO: 419) GC CGGRUG CAGAAGUGCUU (SEQ ID NO: 420)
R R R (N = any of A, G, C, U. R = either of A, G. Y = either of C,
T.)
[0385] The further construction of a VIROMIR is shown below using
the first of the nine sequences shown above as an example: 5'-
TABLE-US-00049 UUCGUGAAGACGGUGGGCCGA-3'. (SEQ ID NO: 139)
[0386] As a fully complementary duplex, the above strand would be
predicted to have a loading bias in its favor relative to the
passenger strand. With the addition of exemplary overhangs, the
final candidate HCV VIROMIR could have the following formula:
TABLE-US-00050 5'-UUCGUGAAGACGGUGGGCCGA-3' (SEQ ID NO: 139)
3'-dTdTAAGCACUUCUGCCACCCGG-5' (SEQ ID NO: 140)
[0387] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules will comprise the sequence
corresponding to the complement of the seed. In this example, this
complementary sequence is CGUGAAGAC (SEQ ID NO: 98).
Example 14
Chemically-Modified Codemirs
[0388] Because dsRNA has limited stability in vivo, it is well
understood by one skilled in the art that it may be desirable to
chemically modify the multitargeting interfering RNA in order to
improve stability and activity. Any chemical modification to the
multitargeting interfering RNA is within the scope of the
invention. As a non-limiting example, we considered the use of 2'-F
modified nucleotides within the context of CODEMIR-1 (also known as
AM001). To investigate the stability of the modified CODEMIRs,
either the Oligreen or Sybr Green I fluorescent dye was used to
assess CODEMIR degradation in 10% human serum. These, in particular
Sybr Green I, bind avidly to dsRNA to produce enhanced
fluorescence. Thus, monitoring of fluorescence during the
incubation of dsRNA in 10% human serum, or other biological
solution, produces a facile method for monitoring stability. To
further clarify the presence of the products of degradation
produced, CODEMIR-1 and chemically-modified analogs (with one or
both strands 2'-F modified at every C and U position) were
incubated in 10% serum for 30 minutes and separated by PAGE.
Unmodified duplex RNA exhibited substantial degradation, and was
presumably completely inactive. By contrast, duplex RNA in which
both strands were 2'-Fluoro modified, exhibited no degradation at
all, which corresponds well to the results observed in the
fluorescence assay (data not shown). The two duplexes in which only
a single strand was modified appeared to be incompletely degraded
to a duplex presumably a hybrid between the full-length modified
strand and a partially degraded unmodified strand. Because this
degradation product would be likely to have greatly decreased
activity, it seems reasonable to infer that only constructs which
demonstrate no degradation in the fluorescence assay would retain
maximal activity.
[0389] However, replacement of all pyrimidine nucleotides with
their corresponding 2'-F analogs was somewhat deleterious to
activity when assessed by the suppression of VEGF secretion by
ARPE-19 cells. In contrast, similar modification of the guide
strand did not significantly alter activity. Because efficient
loading of the guide strand into RISC requires (in some cases) the
cleavage of the passenger strand, we hypothesized that 2'-F
modification of the passenger strand inhibited strand loading. To
test this, variants of CODEMIR-1 with 2'-F modification at each C
and U position in the guide and/or passenger strand, were designed
(Table 14-1). In particular, the influence of modification of
position 9 (pos9) of the passenger strand (the position critical
for passenger strand cleavage during RISC loading) was examined.
When tested for their ability to suppress VEGF expression in
ARPE-19 cells, a modest difference was observed between the
pos9-Fluoro and pos9-ribo variants in which only the passenger
strand was modified (ie CODEMIR-144 and 87) although this was not
significant. By contrast, the pos9-ribo variant in which both
strands were otherwise modified (CODEMIR-145) was significantly
(p<0.01) more active than the comparable pos9-Fluoro variant
(CODEMIR-33). As compared to unmodified CODEMIR-1, the activity of
the pos9-Fluoro variant in which only the passenger strand was
modified (CODEMIR-87), but not the pos9-ribo variant (CODEMIR-144),
was significantly decreased (p<0.01).
[0390] The stability of the pos9-ribo and pos9-F variants of
CODEMIR-1 was assessed in 10% human serum using Sybr Green I. No
difference between the pos9-ribo and pos9-Fluoro variants was
observed. However, as discussed above, the end product of
degradation in the case where only a single strand is modified is
unlikely to possess activity. The RNase activity of serum is due to
RNase-A like endonuclease(s) (Haupenthal, J. (2006) Biochem
Pharmacol 71, 702-710). These RNases are single-strand specific,
only cleave 3' of C and U bases and are blocked by 2'-F
modification (Kelemen, B. R. (2000) Biochemistry 39, 14487-14494).
Thus, a likely model of short RNA duplex degradation in serum is
that the "breathing" ends of the duplex are the most vulnerable to
RNase degradation whereas the central section of the duplex is
protected by its duplex nature. To assess this model, variants of
CODEMIR-1 in which only the ends of each strand were 2'-F modified
were designed (Table 14-1). Modification of 3 bases from the end of
the duplex region (CODEMIR-165) produced a modest improvement in
stability, but this was abolished if only the 3' termini were
modified (CODEMIR-167). Interestingly, modification of 5 bases from
the end of the duplex region (CODEMIR-166) also produced an
increase in stability. However, when tested for VEGF suppressive
activity, all of the terminus-modified CODEMIRs displayed
significantly impaired activity relative to CODEMIR-1; comparable
in activity to the other 2'-F modified CODEMIRs containing a
passenger pos9-ribo base (FIG. 13).
[0391] Overall, therefore, 2'Fluoro modification represents a
viable strategy for the chemical modification of the multitargeting
interfering RNA of the invention, although extensive modification,
particularly that of the pos-9 nucleotide of the passenger strand
may reduce activity in cell-based assays. Although the trade-off
for stability may in some cases not appear worthwhile, the use of
transfection reagents (in this case Lipofectamine) may mask some of
the benefits of chemical modification because lipid-based
complexation protects nucleic acid from degradation by nucleases.
Thus while CODEMIR-1 was amongst the most potent in the cell screen
assay (with lipofection), it had minimal stability in serum.
Ultimately, whereas the differential activity of multitargeting
interfering RNA can be tested in a predictive manner in cell-based
assays, the impact of chemical modification would need testing in
the context intended for the therapeutic molecule.
TABLE-US-00051 TABLE 14-1 2'-Fluoro-modified variants of CODEMIR-1
CODEMIR Duplex Sequence (pass/guide) CODEMIR-1 5'
AGACUCACCCACCCACAUAUU-3' (SEQ ID NO: 101) 3'
AAUCUGAGUGGGUGGGUGUAU-5' (SEQ ID NO: 100) CODEMIR-92
AGACUCACCCACCCACAUAUU (SEQ ID NO: 101) AAUCUGAGUGGGUGGGUGUAU (SEQ
ID NO: 423) CODEMIR-144 AGACUCACCCACCCACAUAb UU (SEQ ID NO: 424)
AAUCUGAGUGGGUGGGUGUAU (SEQ ID NO: 100) CODEMIR-87
AGACUCACCCACCCACAUAUU (SEQ ID NO: 425) AAUCUGAGUGGGUGGGUGUAU (SEQ
ID NO: 100) CODEMIR-145 AGACUCACCCACCCACAUAb UU (SEQ ID NO: 424)
AAUCUGAGUGGGUGGGUGUAU (SEQ ID NO: 423) CODEMIR-33
AGACUCACCCACCCACAUAb UU (SEQ ID NO: 425) AAUCUGAGUGGGUGGGUGUAU (SEQ
ID NO: 423) CODEMIR-165 AGACUCACCCACCCACAUAUU (SEQ ID NO: 426)
AAUCUGAGUGGGUGGGUGUAU (SEQ ID NO: 541) CODEMIR-166
AGACUCACCCACCCACAUAUU (SEQ ID NO: 427) AAUCUGAGUGGGUGGGUGUAU (SEQ
ID NO: 423) CODEMIR-167 AGACUCACCCACCCACAUAUU (SEQ ID NO: 426)
AAUCUGAGUGGGUGGGUGUAU (SEQ ID NO: 428) (2'-F-modified nucleotides
are bolded and underlined).
[0392] It is well recognized by those skilled in the art that the
terminal conjugation of nucleic acid therapeutics to various dyes,
pharmacophores, ligands, peptides, linkers, conjugates, polymers,
lipids, peptides and other molecules can be used to improve or
monitor the uptake, distribution, tissue targeting, stability or
biological potency of the said nucleic acid. In most cases, the
required conjugation reactions are performed through the formation
of a phosphoester linkage by means of an aliphatic chain. To
investigate the compatibility of such a strategy in relation to the
multitargeting interfering RNA of the invention, we investigated
the biological activity of analogs of CODEMIR-1 in which the active
guide strand was linked at either the 5' or 3' oligonucleotide
terminal. These include phosphate linked aliphatic chains with
hydroxyl or amino moieties, polyethylene glycols and abasic sugars
(CODEMIRs 146-156; FIG. 14). All of these CODEMIRs demonstrated
high VEGF suppressive activity when transfected into ARPE-19 cells
at 10 nM (FIG. 15), indicating the compatibility of these
modifications with biological activity of the molecules of the
invention. The stability (FIG. 17) and further RNAi activity (FIG.
18) of the chemically modified variants of CODEMIR-1 were also
analyzed. It is possible and indeed likely that cell
phosphoesterases caused the release of the linker once delivered
within the cell and that this would account for the high and
uniform activity of these analogs. This indicates that at least
some of the linkers could be used in a prodrug approach in which
the targeting or protective ligand is shed once the multitargeting
interfering RNA has penetrated into cells.
Example 15
Use of ARPE-19 Cells for the Evaluation of Anti-Angiogenic
CODEMIRs
[0393] CODEMIR-1 has been the prototype sequence in which the
influence of chemical and sequence modifications have been tested.
This CODEMIR may be particularly useful for the treatment of the
wet forms of AMD as well as macular edema and diabetic retinopathy.
This is because secreted VEGF-A plays a major role in all of these
diseases (Witmer et al (2003) Prog Retin Eye Res, 22, 1-29),
although ICAM-1 overexpression may be an early initiating event,
particularly for diabetic retinopathy and macular edema (Funatsu et
al., (2005) Ophthalmology, 112, 806-16; Joussen et al. (2002) Am J
Pathol, 160, 501-9; Lu et al. (1999) Invest Ophthalmol Vis Sci, 40,
1808-12). We have shown that CODEMIR-1 has demonstrated the ability
to suppress both VEGF-A and ICAM-1 production by human retinal
epithelium cells (ARPE-19 cell line). Retinal pigmented epithelial
cells are a major contributor to the production of these proteins
in these ocular angiogenic diseases (Matsuoka et al., (2004) Br J
Ophthalmol, 88, 809-15, Yeh et al. (2004), Invest Ophthalmol Vis
Sci, 45, 2368-73). RPE cells are also the primary site of uptake of
foreign nucleic acids in the eye and, for these two reasons, are
the appropriate cell model for evaluation of anti-angiogenic
CODEMIRs. The in vivo activities of two oligonucleotide drugs
correlated with their activity against RPE cells in culture
(Garrett et al. (2001) J Gene Med, 3, 373-83; Rakoczy et al.
(1996), Antisense Nucleic Acid Drug Dev, 6, 207-13) demonstrating
the value of this cell culture model. An advantage of this cell
line is that it forms polarized monolayers that mimic the RPE layer
of the eye (Dunn et al., (1996), Exp Eye Res, 62, 155-69), and
which can be studied for protracted periods of time. This property
was used to evaluate VEGF secretion by repeated sampling of the
supernatant of ARPE-19 monolayers (ICAM-1 cannot be studied in this
same way because it requires harvesting of the cells). VEGF
secretion was suppressed for at least 9 days following a single
dose of CODEMIR-1 (FIG. 16). This indicates that CODEMIR-1 is
expected to produce a durable inhibition of VEGF production in the
eye.
Example 16
Mismatches in the Seed Region Impair CODEMIR-1 Activity
[0394] In order to validate that seed binding was essential for
CODEMIR activity, a number of RNA duplexes based upon CODEMIR-1,
but with mismatches to the targets in the seed region, were
designed and tested. These were aligned with the human
transcriptome using BLAST and three sequences with the lowest
predicted off-target siRNA activity were chosen (CODEMIRs 122-124).
These feature mismatches at positions 4, 4+6 and 4+6+8,
respectively (Table 16-1). Each of these CODEMIRs was assessed for
activity against VEGF and ICAM in ARPE-19 cells (FIG. 19). A
mismatch at position 4 exhibited slightly impaired activity against
both VEGF and ICAM-1, whereas mismatches at 4 and 6 or at 4+6+8
greatly reduced VEGF suppression and abolished ICAM-1 suppression,
demonstrating that seed binding is important for CODEMIR
activity.
TABLE-US-00052 TABLE 16-1 Variants of CODEMIR-1 with seed
mismatches (mismatches are bold). Duplex mRNA binding (RNA hybrid)
CODEMIR-1 Passenger VEGF 5' G A C 3' AGACUCACCCACCCACAUAUU (SEQ ID
NO: 101) UAGAC CACCCACCCACAUA (SEQ ID NO: 181)
AAUCUGAGUGGGUGGGUGUAU (SEQ ID NO: 100) AUCUG GUGGGUGGGUGUAU (SEQ ID
NO: 100) Guide 3' A A 5' ICAM 5' G CCAC C 3' UUAG CUC CCCACCCACAUA
(SEQ ID NO: 183) AAUC GAG GGGUGGGUGUAU (SEQ ID NO: 100) 3' U U 5'
CODEMIR-122 Passenger VEGF 5' G A C C 3' AGACUCACCCACCCAGAUAUU (SEQ
ID NO: 141) UAGAC CACCCACCCA AUA (SEQ ID NO: 181)
AAUCUGAGUGGGUGGGUCUAU (SEQ ID NO: 142) AUCUG GUGGGUGGGU UAU (SEQ ID
NO: 142) Guide 3' A A C 5' ICAM 5' G CCAC C C 3' UUAG CUC CCCACCCA
AUA (SEQ ID NO: 183) AAUC GAG GGGUGGGU UAU (SEQ ID NO: 142) 3' U U
C 5' CODEMIR-123 Passenger VEGF 5' G A C 3' AGACUCACCCACCGAGAUAUU
(SEQ ID NO: 429) UAGAC CACCCACC (SEQ ID NO: 431)
AAUCUGAGUGGGUGGCUCUAU (SEQ ID NO: 430) AUCUG GUGGGUGG (SEQ ID NO:
430) Guide 3' A A CUCUAU 5' ICAM 5' G CCAC CACAUA 3' UUAG CUC
CCCACC (SEQ ID NO: 549) AAUC GAG GGGUGG (SEQ ID NO: 430) 3' U U
CUCUAU 5' CODEMIR-124 Passenger VEGF 5' G A C 3'
AGACUCACCCAGCGAGAUAUU (SEQ ID NO: 550) UAGAC CACCCA (SEQ ID NO:
433) AAUCUGAGUGGGUCGCUCUAU (SEQ ID NO: 432) AUCUG GUGGGU (SEQ ID
NO: 432) Guide 3' A A CGCUCUAU 5' ICAM 5' G CCAC CCCACAUA 3' UUAG
CUC CCCA (SEQ ID NO: 549) AAUC GAG GGGU (SEQ ID NO: 432) 3' U U
CGCUCUAU 5'
[0395] The results demonstrated the more microRNA-like, less
siRNA-like, qualities of CODEMIRs with respect to mismatches in the
seed.
Example 17
Screening of 32 Variants of CODEMIR-1 for VEGF and ICAM-1 RNAi
Activity
[0396] The RNAi efficacy of an additional 32 variants of CODEMIR-1,
differing in the composition of the 3' tail of the guide strand was
analyzed. A consensus sequence of the VEGF and ICAM-1 target sites
was generated (allowing wobble base pairing between guide strand
and the target site--i.e. allowing G to be equivalent to A and U to
be equivalent to C; FIG. 20). CODEMIRs representing the 32
(2.sup.5) possible 3' tails targeting both transcripts were
designed (Table 17-1). ARPE-19 cells treated with 40 nM of each of
these CODEMIRs demonstrated VEGF suppression ranging from
.about.50% to .about.90% (FIG. 21). CODEMIRs with complementarity
to the VEGF mRNA at position 13 of the guide strand (i.e. having 14
contiguous bases of complementarity to the target were
substantially more effective that those with a mismatch at position
13 (12 bases of contiguous complementarity to the target);
presumably because a mismatch at position 13 impairs RISC mediated
cleavage of the VEGF mRNA.
[0397] In keeping with the effects of central mismatches on the
activity of siRNAs targeting the CODEMIR-1 binding site (above), no
CODEMIR demonstrated less than 40% suppression of VEGF. Indeed,
transfection of ARPE-19 cells with a synthetic microRNA duplex
(hsa-mir-299 herein named CODEMIR-84), which shares part of the
CODEMIR-1 seed binding region but has little complementarity to the
VEGF mRNA in the 3' tail (Table 17-1), also inhibited VEGF
expression by .about.40%. It appears that this level of VEGF
suppression represents the translational repression induced by
nearly any short RNA that binds with perfect complementarity to the
CODEMIR-1 seed site of the VEGF mRNA. Of those CODEMIRs that
displayed greater than 40% VEGF suppression, there was a strong
correlation between activity and both the degree of complementarity
(an inverse correlation with the number of mismatches) and the
length of the complementary seed region (FIG. 22). CODEMIRs with a
12 base complementary seed produced only .about.70% suppression,
even with only a single mismatch (at position 13). By contrast,
CODEMIRs with a 14 base complementary seed produced strong
suppression (.about.85%), but activity of these CODEMIRs was
impaired by mismatches in the remainder of the 3' tail. All of the
CODEMIRs with 17 base complementary seed regions were highly
active, with mismatches in the 3' tail having little effect upon
activity. The CODEMIRs with a complementary seed longer than 17
bases had activity that appeared to more closely correlate with
strand loading than with any other factor.
[0398] In contrast to the VEGF suppression data, when the 32
CODEMIR-1 variants were assayed for ICAM-1 suppressive activity, no
clear trend was discernable with respect to the length of 5'
complementarity or the number of mismatches to the target (FIG.
23). In part, this may have been because those CODEMIRs with a high
degree of complementarity to the ICAM-1 mRNA also contained long
sequences of contiguous guanosines (CODEMIR-52, 56, 60, 64, 68, 72,
76 and 80), which may be detrimental to activity. To test this,
variants of CODEMIR-56 and 76 were designed in which the guanosine
at position 14 of the guide strand (which forms a G:U pairing with
the ICAM-1 mRNA) was replaced with an A (to generate an A:U pair
with the ICAM-1 mRNA) in order to break the contiguous Gs. These
variants (CODEMIRs 120 and 121-Table 17-2) demonstrated a marked
increase in both VEGF and ICAM-1 suppressive activity compared to
their respective analogues containing a guanosine at position 14
(FIG. 24). The increase in ICAM-1 suppressive activity is
potentially attributable to the introduction of an A:U pair in
place of a G:U pair in the guide/target interaction (rather than
being a direct effect of removing the 7 G motif). However, the fact
that activity of these CODEMIRs against VEGF was also improved
(despite the fact that the predicted binding of these CODEMIRs to
the VEGF mRNA is unchanged compared with CODEMIRs 56 and 76-Table
17-2) shows that the 7 G motif was detrimental to the activity of
CODEMIRs 56 and 76.
[0399] One possible strategy to increase the activity of CODEMIRs
that have substantial complementarity to the target mRNA is to
include inosine bases at crucial points (sites that cannot be
matched to all transcripts and are near to the RISC cleavage site).
To test the tolerance for inosine bases by the RNAi machinery,
three variants of CODEMIR-1 were designed which included inosine
bases at positions 13, 15 or 13 and 15 of the guide strand
(CODEMIR-100, 101 and 102, respectively; Table 17-3). These
CODEMIRs showed comparable ICAM-1 suppressive activity to CODEMIR-1
(which contains a mismatch at position 13), but reduced VEGF
suppression relative to CODEMIR-1 in the case of CODEMIR-100 and
102 (FIG. 25). The comparable activity against ICAM-1 may result
from translational repression that is largely dependent upon the
seed binding alone, and so is not affected by alterations in the 3'
tail. The VEGF suppressive activity of inosine containing variants
of CODEMIR-1 was also compared to similar variants of CODEMIR-1
with mismatches to the VEGF mRNA at positions 13, 15 or 13 and 15
(CODEMIRs 68-71). For this assay, ARPE-19 cells were transfected
with 10 nM RNA duplex in order to increase the dynamic range of the
assay. None of the inosine containing variants demonstrated
substantially improved activity compared to its respective
mismatched variant, although, the variant with an inosine at
position 13 was slightly more active than the mismatched variant
(compare CODEMIR-70 with CODEMIR-100 in FIG. 26). Nevertheless,
these experiments indicate that inosine substitution can be
considered in the design of CODEMIRs.
[0400] An alternative to inosine containing CODEMIRs as a strategy
for increasing the length of the 5' complementary region is to
introduce a single base pair loop (either on the target or the
guide strand) so as to increase the length of complementarity on
either the target or the guide strand. Two such CODEMIRs (one with
a predicted target loop (CODEMIR 105) and one with a predicted
guide strand loop (CODEMIR 106) after position 14 of the guide
strand-Table 17-3) were tested for the ability to suppress VEGF and
ICAM-1 expression (FIG. 27). Interestingly, each of these displayed
substantially reduced activity compared to CODEMIR-1 (which has an
A:A mismatch at position 15). This suggests that symmetrical
mismatches (loops) may be tolerated better than asymmetrical
mismatches, and is a potentially useful design principle.
[0401] Another strategy for increasing the activity of a CODEMIR
against multiple targets is to increase the hybridisation strength
of the 3' tail by inclusion of chemically modified bases that have
increased hybridization potential in RNA:RNA duplexes. Such
modified bases include LNA (locked nucleic acid), ENA (ethylene
bridge nucleic acid), 2'Fluoro, 2'O-methyl and 2'O-alkyl-ribose
among others. Strengthening of hybridisation in the seed region
could also be envisaged, however, this may impact negatively on
strand-loading bias. Modification of the G at position 16 of the
guide strand of CODEMIR-1 was chosen in an effort to increase
hybridization and therefore stability of the binding of the tail of
the guide strand of CODEMIR-1 to both targets. This modified
CODEMIR (CODEMIR-99) has the ribo-base at position 16 replaced with
a LNA base. When tested for the ability to suppress VEGF and ICAM-1
expression in ARPE-19 cells, this CODEMIR exhibited comparable
activity to CODEMIR-1 (FIG. 25). As with the inosine containing
variants of CODEMIR-1 (and most variants of CODEMIR-1 generally),
the comparable ICAM-1 suppression may reflect a near maximal
translational suppression that cannot be improved upon without
induction of RISC mediated cleavage. Again, however, this
experiment indicates that such chemical modifications are tolerated
and can be considered in the design of CODEMIRs. Modifications such
as the LNA modified base could be used in the 3'tail of a candidate
multitargeting interfering RNA so as to strengthen the interaction
of this portion of the guide strand with the corresponding part of
the passenger strand, thereby improving loading bias. Therefore,
there are a number of uses for modified bases that are envisaged in
this invention.
[0402] The data primarily relates to principles of CODEMIR design
and in particular supports the approach of obtaining maximum
hybridisation of the active RNA to each of its targets. However,
these data also support the fact that complete complementarity is
not necessary for maximal activity and that significant activity
can be obtained with the guide strand even when the guide strand
has complete complementarity to just the seed region.
TABLE-US-00053 TABLE 17-1 Sequence listing of 32 systematically
designed variants of CODEMIR-1 Guide strand VEGF binding (5' to 3')
(upper = VEGF mRNA) CODEMIR-1 UAUGUGGGUGGGUGAGUCUAA 5' G A C 3'
(SEQ ID NO: 100) UAGAC CACCCACCCACAUA AUCUG GUGGGUGGGUGUAU 3' A A
5' Top strand (SEQ ID NO: 181) Bottom strand (SEQ ID NO: 100)
CODEMIR-52 UAUGUGGGUGGGGGGGUCUCU 5' U UGUAGACACA C 3' (SEQ ID NO:
434) GGGAUUCC CCCACCCACAUA CUCUGGGG GGGUGGGUGUAU 3' U 5' Top strand
(SEQ ID NO: 435) Bottom strand (SEQ ID NO: 434) CODEMIR-53
UAUGUGGGUGGGUGGGUCUCU 5' U A C 3' (SEQ ID NO: 437) AGAC
CACCCACCCACAUA UCUG GUGGGUGGGUGUAU 3' UC G 5' Top strand (SEQ ID
NO: 438) Bottom strand (SEQ ID NO: 437) CODEMIR-54
UAUGUGGGUGGGGGUGUCUCU 5' U A C 3' (SEQ ID NO: 439) AGACAC
CCCACCCACAUA UCUGUG GGGUGGGUGUAU 3'UC G 5' Top strand (SEQ ID NO:
438) Bottom strand (SEQ ID NO: 439) CODEMIR-55
UAUGUGGGUGGGUGUGUCUCU 5' U C 3' (SEQ ID NO: 441)
AGACACACCCACCCACAUA UCUGUGUGGGUGGGUGUAU 3' UC 5' Top strand (SEQ ID
NO: 438) Bottom strand (SEQ ID NO: 441) CODEMIR-56
UAUGUGGGUGGGGGGGUGUCU 5' U A C 3' (SEQ ID NO: 442) AGACAC
CCCACCCACAUA UCUGUG GGGUGGGUGUAU 3' GGG 5' Top strand (SEQ ID NO:
438) Bottom strand (SEQ ID NO: 442) CODEMIR-57
UAUGUGGGUGGGUGGGUGUCU 5' U C 3' (SEQ ID NO: 443) AGACAC
ACCCACCCACAUA UCUGUG UGGGUGGGUGUAU 3' GG 5' Top strand (SEQ ID NO:
438) Bottom strand (SEQ ID NO: 443) CODEMIR-58
UAUGUGGGUGGGGGUGUGUCU 5' U C 3' (SEQ ID NO: 444) AGACACA
CCCACCCACAUA UCUGUGU GGGUGGGUGUAU 3' GG 5' Top strand (SEQ ID NO:
438) Bottom strand (SEQ ID NO: 444) CODEMIR-59
UAUGUGGGUGGGUGUGUGUCU 5' G UCCUGUAG C 3' (SEQ ID NO: 445) GGAU
ACACACCCACCCACAUA UCUG UGUGUGGGUGGGUGUAU 3' 5' Top strand (SEQ ID
NO: 446) Bottom strand (SEQ ID NO: 445) CODEMIR-60
UAUGUGGGUGGGGGGGUCGCU 5' U G UGUAGACACA C 3' (SEQ ID NO: 447) G
GAUUCC CCCACCCACAUA C CUGGGG GGGUGGGUGUAU 3' U G 5' Top strand (SEQ
ID NO: 435) Bottom strand (SEQ ID NO: 447) CODEMIR-61
UAUGUGGGUGGGUGGGUCGCU 5' A A C 3' (SEQ ID NO: 448) GAC
CACCCACCCACAUA CUG GUGGGUGGGUGUAU 3' UCG G 5' Top strand (SEQ ID
NO: 449) Bottom strand (SEQ ID NO: 448) CODEMIR-62
UAUGUGGGUGGGGGUGUCGCU 5' A A C 3' (SEQ ID NO: 450) GACAC
CCCACCCACAUA CUGUG GGGUGGGUGUAU 3' UCG G 5' Top strand (SEQ ID NO:
449) Bottom strand (SEQ ID NO: 450) CODEMIR-63
UAUGUGGGUGGGUGUGUCGCU 5' A C 3' (SEQ ID NO: 451) GACACACCCACCCACAUA
CUGUGUGGGUGGGUGUAU 3' UCG 5' Top strand (SEQ ID NO: 449) Bottom
strand (SEQ ID NO: 451) CODEMIR-64 UAUGUGGGUGGGGGGGUGGCU 5' U A A C
3' (SEQ ID NO: 452) AG CAC CCCACCCACAUA UC GUG GGGUGGGUGUAU 3' G
GGG 5' Top strand (SEQ ID NO: 438) Bottom strand (SEQ ID NO: 452)
CODEMIR-65 UAUGUGGGUGGGUGGGUGGCU 5' U UG AGACA C 3' (SEQ ID NO:
453) CC U CACCCACCCACAUA GG G GUGGGUGGGUGUAU 3' UC UG 5' Top strand
(SEQ ID NO: 454) Bottom strand (SEQ ID NO: 453) CODEMIR-66
UAUGUGGGUGGGGGUGUGGCU 5' U AG A C 3' (SEQ ID NO: 551) GU ACAC
CCCACCCACAUA CG UGUG GGGUGGGUGUAU 3' U G G 5' Top strand (SEQ ID
NO: 545) Bottom strand (SEQ ID NO: 551) CODEMIR-67
UAUGUGGGUGGGUGUGUGGCU 5' U AG C 3' (SEQ ID NO: 455) GU
ACACACCCACCCACAUA CG UGUGUGGGUGGGUGUAU 3' U G 5' Top strand (SEQ ID
NO: 545) Bottom strand (SEQ ID NO: 455) CODEMIR-68
UAUGUGGGUGGGGCGGUCUAU 5' U ACA C 3' (SEQ ID NO: 456) GUAGAC
CCCACCCACAUA UAUCUG GGGUGGGUGUAU 3' GGG 5' Top strand (SEQ ID NO:
545) Bottom strand (SEQ ID NO: 456) CODEMIR-69
UAUGUGGGUGGGUGGGUCUAU 5' U A C 3' (SEQ ID NO: 458) GUAGAC
CACCCACCCACAUA UAUCUG GUGGGUGGGUGUAU 3' G 5' Top strand (SEQ ID NO:
545) Bottom strand (SEQ ID NO: 458) CODEMIR-70
UAUGUGGGUGGGGGUGUCUAU 5' U A C 3' (SEQ ID NO: 459) GUAGACAC
CCCACCCACAUA UAUCUGUG GGGUGGGUGUAU 3' G 5' Top strand (SEQ ID NO:
545) Bottom strand (SEQ ID NO: 459) CODEMIR-71
UAUGUGGGUGGGUGUGUCUAU 5' U C 3' (SEQ ID NO: 460)
GUAGACACACCCACCCACAUA UAUCUGUGUGGGUGGGUGUAU 3' 5' Top strand (SEQ
ID NO: 545) Bottom strand (SEQ ID NO: 460) CODEMIR-72
UAUGUGGGUGGGGGGGUGUAU 5' G A C 3' (SEQ ID NO: 461) ACAC
CCCACCCACAUA UGUG GGGUGGGUGUAU 3' UA GGG 5' Top strand (SEQ ID NO:
462) Bottom strand (SEQ ID NO: 461) CODEMIR-73
UAUGUGGGUGGGUGGGUGUAU 5' U G A C 3' (SEQ ID NO: 464) GUA AC
CACCCACCCACAUA UAU UG GUGGGUGGGUGUAU 3' G G 5' Top strand (SEQ ID
NO: 545) Bottom strand (SEQ ID NO: 464) CODEMIR-74
UAUGUGGGUGGGGGUGUGUAU 5' U G A C 3' (SEQ ID NO: 465) GUA ACAC
CCCACCCACAUA UAU UGUG GGGUGGGUGUAU 3' C G 5' Top strand (SEQ ID NO:
545) Bottom strand (SEQ ID NO: 465) CODEMIR-75
UAUGUCCGUGGGUGUGUCUAU 5' U G C 3' (SEQ ID NO: 466) GUA
ACACACCCACCCACAUA UAU UGUGUGCCUGGGUGUAU 3' G 5' Top strand (SEQ ID
NO: 545) Bottom strand (SEQ ID NO: 466) CODEMIR-76
UAUGUGGGUGGGCCCGUCGAU 5' A ACA C 3' (SEQ ID NO: 468) GAC
CCCACCCACAUA CUG GGGUGGGUGUAU 3' UAG GGG 5' Top strand (SEQ ID NO:
449) Bottom strand (SEQ ID NO: 468) CODEMIR-77
UAUGUGGGUGGGUGGGUCGAU 5' A A C 3' (SEQ ID NO: 470) GAC
CACCCACCCACAUA CUG GUGGCUGGGUGUAU 3' UAG C 5' Top strand (SEQ ID
NO: 449) Bottom strand (SEQ ID NO: 470) CODEMIR-78
UAUGUGGGUGGGGGUGUCGAU 5' A A C 3' (SEQ ID NO: 471) GACAC
CCCACCCACAUA CUGUG GGGUGGGUGUAU 3' UAG G 5' Top strand (SEQ ID NO:
449) Bottom strand (SEQ ID NO: 471) CODEMIR-79
UAUGUGCGUGGGUGUCUCGAU 5' A C 3' (SEQ ID NO: 473) GACACACCCACCCACAUA
CUCUCUGGGUGGGUGUAU 3' UAG 5' Top strand (SEQ ID NO: 449) Bottom
strand (SEQ ID NO: 473) CODEMIR-80 UAUGUGGGUGGGGGGGUGGAU 5' U UG
AGACA A C 3' (SEQ ID NO: 474) UCC U C CCCACCCACAUA AGG G G
GGCUGGGUGUAU 3' U UG G 5' Top strand (SEQ ID NO: 475) Bottom strand
(SEQ ID NO: 474) CODEMIR-81 UAUGUGGGUGGGUGGGUGGAU 5' U UG AGACA C
3' (SEQ ID NO: 477) UCC U CACCCACCCACAUA AGG G GUGGGUGGGUGUAU 3' U
UG 5' Top strand (SEQ ID NO: 475) Bottom strand (SEQ ID NO: 477)
CODEMIR-82 UAUGUGGGUGGGGGUGUGGAU 5' U UGUAG A C 3' (SEQ ID NO: 478)
UCC ACAC CCCACCCACAUA AGG UGUG GGGUGGGUGUAU 3' U G 5' Top strand
(SEQ ID NO: 475) Bottom strand (SEQ ID NO: 478) CODEMIR-83
UAUGUGGGUGGGUGUGUGGAU 5' U UGUAG C 3' (SEQ ID NO: 479) UCC
ACACACCCACCCACAUA AGG UGUGUGGGUGGGUGUAU 3' U 5' Top strand (SEQ 10
NO: 475) Bottom strand (SEQ ID NO: 479) CODEMIR-84
UAUGUGGGAUGGUAAACCGCUU 5' C C 3' (hsa-mir-299) (SEQ ID NO: 190) CCA
CCCACAUA GGU GGGUGUAU 3'UUCGCCAAAU A 5' Top strand (SEQ ID NO: 480)
Bottom strand (SEQ ID NO: 190) ICAM-1 binding (upper = ICAM-1 mRNA)
CODEMIR-1 5' G CCAC C 3'
UUAG CUC CCCACCCACAUA AAUC GAG GGGUGGGUGUAU 3' U U 5' Top strand
(SEQ ID NO: 183) Bottom strand (SEQ ID NO: 100) CODEMIR-52 5' U CC
C 3' AG ACCUCCCCACCCACAUA UC UGGGGGGGUGGGUGUAU 3' UC 5' Top strand
(SEQ ID NO: 436) Bottom strand (SEQ ID NO: 434) CODEMIR-53 5' U CC
C C 3' AG ACCU CCCACCCACAUA UC UGGG GGGUGGGUGUAU 3' UC U 5' Top
strand (SEQ ID NO: 436) Bottom strand (SEQ ID NO: 437) CODEMIR-54
5' U CUUUGUUAGCCACCU C 3' GACAC CCCCACCCACAUA CUGUG GGGGUGGGUGUAU
3' UCU 5' Top strand (SEQ ID NO: 440) Bottom strand (SEQ ID NO:
439) CODEMIR-55 5' U C CUC C 3' AG CAC CCCACCCACAUA UC GUG
GGGUGGGUGUAU 3' UC U U 5' Top strand (SEQ ID NO: 436) Bottom strand
(SEQ ID NO: 441) CODEMIR-56 5' U C C 3' AG CACCUCCCCACCCACAUA UC
GUGGGGGGGUGGGUGUAU 3' U 5' Top strand (SEQ ID NO: 436) Bottom
strand (SEQ ID NO: 442) CODEMIR-57 5' U C C C 3' AG CACCU
CCCACCCACAUA UC GUGGG GGGUGGGUGUAU 3' U U 5' Top strand (SEQ ID NO:
436) Bottom strand (SEQ ID NO: 443) CODEMIR-58 5' U C C C 3' AG CAC
UCCCCACCCACAUA UC GUG GGGGGUGGGUGUAU 3' U U 5' Top strand (SEQ ID
NO: 436) Bottom strand (SEQ ID NO: 444) CODEMIR-59 5' U CUUUGUUAGCC
CUC C 3' GACAC AC CCCACCCACAUA CUGUG UG GGGUGGGUGUAU 3' U U 5' Top
strand (SEQ ID NO: 440) Bottom strand (SEQ ID NO: 445) CODEMIR-60
5' U C C 3' AGC ACCUCCCCACCCACAUA UCG UGGGGGGGUGGGUGUAU 3' C 5' Top
strand (SEQ ID NO: 436) Bottom strand (SEQ ID NO: 447) CODEMIR-61
5' U C C C 3' AGC ACCU CCCACCCACAUA UCG UGGG GGGUGGGUGUAU 3' C U 5'
Top strand (SEQ ID NO: 346) Bottom strand (SEQ ID NO: 448)
CODEMIR-62 5' U C C C 3' AGC AC UCCCCACCCACAUA UCG UG
GGGGGUGGGUGUAU 3' C U 5' Top strand (SEQ ID NO: 436) Bottom strand
(SEQ ID NO: 450) CODEMIR-63 5' U C UC C 3' AGC AC C CCCACCCACAUA
UCG UG G GGGUGGGUGUAU 3' C U U 5' Top strand (SEQ ID NO: 436)
Bottom strand (SEQ ID NO: 451) CODEMIR-64 5' U C 3'
AGCCACCUCCCCACCCACAUA UCGGUGGGGGGGUGGGUGUAU 3' 5' Top strand (SEQ
ID NO: 436) Bottom strand (SEQ ID NO: 452) CODEMIR-65 5' U C C 3'
AGCCACCU CCCACCCACAUA UCGGUGGG GGGUGGGUGUAU 3' U 5' Top strand (SEQ
ID NO: 436) Bottom strand (SEQ ID NO: 453) CODEMIR-66 5' U C C 3'
AGCCAC UCCCCACCCACAUA UCGGUG GGGGGUGGGUGUAU 3' U 5' Top strand (SEQ
ID NO: 436) Bottom strand (SEQ ID NO: 551) CODEMIR-67 5' U UC C 3'
AGCCAC C CCCACCCACAUA UCGGUG G GGGUGGGUGUAU 3' U U 5' Top strand
(SEQ ID NO: 436) Bottom strand (SEQ ID NO: 455) CODEMIR-68 5' U CC
C 3' UAG ACCUCCCCACCCACAUA AUC UGGGGGGGUGGGUGUAU 3' U 5' Top strand
(SEQ ID NO: 457) Bottom strand (SEQ ID NO: 456) CODEMIR-69 5' U CC
C C 3' UAG ACCU CCCACCCACAUA AUC UGGG GGGUGGGUGUAU 3' U U 5' Top
strand (SEQ ID NO: 457) Bottom strand (SEQ ID NO: 458) CODEMIR-70
5' U C CU C 3' UAG CAC CCCCACCCACAUA AUC GUG GGGGUGGGUGUAU 3' U U
5' Top strand (SEQ ID NO: 457) Bottom strand (SEQ ID NO: 459)
CODEMIR-71 5' U C CUC C 3' UAG CAC CCCACCCACAUA AUC GUG
GGGUGGGUGUAU 3' U U U 5' Top strand (SEQ ID NO: 457) Bottom strand
(SEQ ID NO: 460) CODEMIR-72 5' C C 3' CACCUCCCCACCCACAUA
GUGGGGGGGUGGGUGUAU 3' UAU 5' Top strand (SEQ ID NO: 463) Bottom
strand (SEQ ID NO: 461) CODEMIR-73 5' C C C 3' CACCU CCCACCCACAUA
GUGGG GGGUGGGUGUAU 3' UAU U 5' Top strand (SEQ ID NO: 463) Bottom
strand (SEQ ID NO: 464) CODEMIR-74 5' C C C 3' CAC UCCCCACCCACAUA
GUG GGGGGUGGGUGUAU 3' UAU U 5' Top strand (SEQ ID NO: 463) Bottom
strand (SEQ ID NO: 465) CODEMIR-75 5' A CUC C 3' GC CAC
CCCACCCACAUA UC GUG GGGUGGGUGUAU 3' UA U U 5' Top strand (SEQ ID
NO: 467) Bottom strand (SEQ ID NO: 466) CODEMIR-76 5' C C 3'
ACCUCCCCACCCACAUA UGGGGGGGUGGGUGUAU 3' UAGC 5' Top strand (SEQ ID
NO: 469) Bottom strand (SEQ ID NO: 468) CODEMIR-77 5' C C C 3' ACCU
CCCACCCACAUA UGGG GGGUGGGUGUAU 3' UAGC U 5' Top strand (SEQ ID NO:
469) Bottom strand (SEQ ID NO: 470) CODEMIR-78 5' C CUUUGUUAGCCACCU
C 3' UCACAC CCCCACCCACAUA GCUGUG GCGGUGGCUGUAU 3' UA 5' Top strand
(SEQ ID NO: 472) Bottom strand (SEQ ID NO: 471) CODEMIR-79 5' A C
CUC C 3' G CAC CCCACCCACAUA C GUG GGGUCCGUGUAU 3 UAG U U 5' Top
strand (SEQ ID NO: 467) Bottom strand (SEQ ID NO: 473) CODEMIR-80
5' G C 3' CCACCUCCCCACCCACAUA GGUGGGGGGGUGGGUGUAU 3' UA 5' Top
strand (SEQ ID NO: 476) Bottom strand (SEQ ID NO: 474) CODEMIR-81
5' G C C 3' CCACCU CCCACCCACAUA GGUGGG GGGUGGGUGUAU 3' UA U 5' Top
strand (SEQ ID NO: 476) Bottom strand (SEQ ID NO: 477) CODEMIR-82
5' G C C 3' CCAC UCCCCACCCACAUA GGUG GGGGGUGGGUGUAU 3' UA U 5' Top
strand (SEQ ID NO: 476) Bottom strand (SEQ ID NO: 478) CODEMIR-83
5' G UC C 3' CCAC C CCCACCCACAUA GGUG G GGGUGGGUGUAU 3' UA UU 5'
Top strand (SEQ ID NO: 476) Bottom strand (SEQ ID NO: 479)
CODEMIR-84 5' U CACCUCC C 3' (hsa-mir-299) AGC CCA CCCACAUA UCG GGU
GGGUGUAU 3' U CCAAAU A 5' Top strand (SEQ ID NO: 436) Bottom strand
(SEQ ID NO: 190)
TABLE-US-00054 TABLE 17-2 Variants of CODEMIR-56 and 76 without 7 G
motifs. Duplex mRNA binding (RNA hybrid) CODEMIR-56 Passenger VEGF
5' U A C 3' ACACCCCCCCACCCACAUAUU (SEQ ID NO: 481) AGACAC
CCCACCCACAUA (SEQ ID NO: 438) UCUGUGGGGGGGUGGGUGUAU (SEQ ID NO:
482) UCUGUG GGGUGGGUGUAU Guide 3' GGG 5' (SEQ ID NO: 482) ICAM-1 5'
U C C 3' AG CACCUCCCCACCCACAUA (SEQ ID NO: 436) UC
GUGGGGGGGUGGGUGUAU 3' U 5' (SEQ ID NO: 482) CODEMIR-76 Passenger
VEGF 5' A ACA C 3' CGACCCCCCCACCCACAUAUU (SEQ ID NO: 483) GAC
CCCACCCACAUA (SEQ ID NO: 449) UAGCUGGGGGGGUGGGUGUAU (SEQ ID NO:
484) CUG GGGUGGGUGUAU Guide 3' UAG GGG 5' (SEQ ID NO: 484) ICAM-1
5' C C 3' ACCUCCCCACCCACAUA (SEQ ID NO: 485) UGGGGGGGUGGGUGUAU 3'
UAGC 5' (SEQ ID NO: 484) CODEMIR-120 Passenger VEGF 5' U A C 3'
ACACCUCCCCACCCACAUAUU (SEQ ID NO: 486) AGACAC CCCACCCACAUA (SEQ ID
NO: 438) UCUGUGGAGGGGUGGGUGUAU (SEQ ID NO: 487) UCUGUG GGGUGGGUGUAU
Guide 3' GAG 5' (SEQ ID NO: 487) ICAM-1 5' U C C 3' AG
CACCUCCCCACCCACAUA (SEQ ID NO: 436) UC GUGGAGGGGUGGGUGUAU 3' U 5'
(SEQ ID NO: 487) CODEMIR-121 Passenger VEGF 5' A ACA C 3'
CGACCUCCCCACCCACAUAUU (SEQ ID NO: 488) GAC CCCACCCACAUA (SEQ ID NO:
449) UAGCUGGAGGGGUGGGUGUAU (SEQ ID NO: 489) CUG GGGUGGGUGUAU Guide
3' UAG GAG 5' (SEQ ID NO: 489) ICAM-1 5' C C 3' ACCUCCCCACCCACAUA
(SEQ ID NO: 469) UGGAGGGGUGGGUGUAU 3' UAGC 5' (SEQ ID NO: 489)
TABLE-US-00055 TABLE 17-3 Variants of CODEMIR-1 incorporating LNA
or inosine bases, or asymmetric loops. Duplex mRNA binding (RNA
hybrid) CODEMIR-1 Passenger VEGF 5' G A C 3' AGACUCACCCACCCACAUAUU
(SEQ ID NO: 101) UAGAC CACCCACCCACAUA (SEQ ID NO: 181)
AAUCUGAGUGGGUGGGUGUAU (SEQ ID NO: 100) AUCUG GUGGGUGGGUGUAU (SEQ ID
NO: 100) Guide 3' A A 5' ICAM 5' G CCAC C 3' UUAG CUC CCCACCCACAUA
(SEQ ID NO: 183) AAUC GAG GGGUGGGUGUAU (SEQ ID NO: 100) 3' U U 5'
CODEMIR- Passenger VEGF 5' G A C 3' 99 AGACUCACCCACCCACAUAUU (SEQ
ID NO: 101) UAGAC CACCCACCCACAUA (SEQ ID NO: 181)
AAUCUGAGUGGGUGGGUGUAU (SEQ ID NO: 490) AUCUG GUGGGUGGGUGUAU (SEQ ID
NO: 490) Guide 3' A A 5' ICAM 5' G CCAC C 3' UUAG CUC CCCACCCACAUA
(SEQ ID NO: 183) AAUC GAG GGGUGGGUGUAU (SEQ ID NO: 490) 3' U U 5'
CODEMIR- Passenger VEGF 5' G A C 3' 100 AGACUCACCCACCCACAUAUU (SEQ
ID NO: 101) UAGAC CACCCACCCACAUA (SEQ ID NO: 181)
AAUCUGAGIGGGUGGGUGUAU (SEQ ID NO: 491) AUCUG GIGGGUGGGUGUAU (SEQ ID
NO: 491) Guide 3' A A 5' ICAM 5' G CC C C 3' UUAG AC UCCCCACCCACAUA
(SEQ ID NO: 183) AAUC UG GIGGGUGGGUGUAU (SEQ ID NO: 491) 3' A 5'
CODEMIR- Passenger VEGF 5' G C 3' 101 AGACUCACCCACCCACAUAUU (SEQ ID
NO: 101) UAGACACACCCACCCACAUA (SEQ ID NO: 181)
AAUCUGIGUGGGUGGGUGUAU (SEQ ID NO: 492) AUCUGIGUGGGUGGGUGUAU (SEQ ID
NO: 492) Guide 3' A 5' ICAM 5' G CC C C 3' UUAG ACCU CCCACCCACAUA
(SEQ ID NO: 183) AAUC UGIG GGGUGGGUGUAU (SEQ ID NO: 492) 3' U 5'
CODEMIR- Passenger VEGF 5' G C 3' 102 AGACUCACCCACCCACAUAUU (SEQ ID
NO: 101) UAGACACACCCACCCACAUA (SEQ ID NO: 181)
AAUCUGIGIGGGUGGGUGUAU (SEQ ID NO: 493) AUCUGIGIGGGUGGGUGUAU (SEQ ID
NO: 493) Guide 3' A 5' ICAM 5' G CC C 3' UUAG ACCUCCCCACCCACAUA
(SEQ ID NO: 183) AAUC UGIGIGGGUGGGUGUAU (SEQ ID NO: 493) 3' 5'
CODEMIR- Passenger VEGF 5' CU A C 3' 104 AGACUCACCCACCCACAUAUU (SEQ
ID NO: 101) GUAGAC CACCCACCCACAUA (SEQ ID NO: 495)
CAUCUGAGUGGGUGGGUGUAU (SEQ ID NO: 494) CAUCUG GUGGGUGGGUGUAU (SEQ
ID NO: 494) Guide 3' A 5' CODEMIR- Passenger VEGF 5' C A C 3' 105
UAGACCACCCACCCACAUAUU (SEQ ID NO: 496) UGUAGAC CACCCACCCACAUA (SEQ
ID NO: 495) ACAUCUGGUGGGUGGGUGUAU (SEQ ID NO: 497) ACAUCUG
GUGGGUGGGUGUAU (SEQ ID NO: 497) Guide 3' 5' CODEMIR- Passenger VEGF
5' UG C 3' 106 GACAUCACCCACCCACAUAUU (SEQ ID NO: 498) UAGACA
CACCCACCCACAUA (SEQ ID NO: 545) AUCUGUAGUGGGUGGGUGUAU (SEQ ID NO:
499) AUCUGU GUGGGUGGGUGUAU (SEQ ID NO: 499) Guide 3' A 5' CODEMIR-
Passenger VEGF 5' CU C 3' 107 AGACACACCCACCCACAUAUU (SEQ ID NO:
500) GUAGACACACCCACCCACAUA (SEQ ID NO: 495) CAUCUGUGUGGGUGGGUGUAU
(SEQ ID NO: 501) CAUCUGUGUGGGUGGGUGUAU (SEQ ID NO: 501) Guide 3' 5'
Bold face indicates LNA modified nucleotide; I indicates inosine
base
Example 18
Screening Multiple Seeds for VEGF Activity
[0403] The data acquired from the systematic analysis of the VEGF
suppressive activity of 32 variants of CODEMIR-1 (above)
demonstrated that the length of 5' complementarity to the target as
well as the total complementarity to the target are critical
determinants of CODEMIR activity, particularly when hybridization
of the guide strand supports RISC-mediated cleavage. These factors,
as well as those factors related to loading of the active strand in
RISC, are the most important CODEMIR design criteria elucidated to
date. However, these factors do not allow discrimination between
active and inactive seed sites, which is critical for in silico
identification of active CODEMIRs. Some of the factors that may be
expected to influence the activity of CODEMIR activity at a given
seed site are: i) secondary structure at the target site of the
mRNA, ii) an adenosine at the final position of the target site
(i.e. a uracil at the first position of the guide strand--naturally
occurring microRNAs have a preference for this pairing) and iii)
the number of seed sites in the target mRNA (i.e. the number of
guide strand binding sites in the target). To assess these factors,
12 CODEMIRs were designed (Table 18-1) with seed sites that had the
characteristics: i) free target secondary structure, no initial A
and only a single site (CODEMIRs 108-110), ii) free target
secondary structure, an initial A and only a single site (CODEMIRs
111-113), iii) not free target secondary structure, an initial A
and only a single site (CODEMIRs 114-116), and iv) not free target
secondary structure, an initial A and two sites (CODEMIRs 117-119).
To exclude complications arising from differing 3' tails on the
guide strands of these CODEMIRs a uniform 3' tail was incorporated,
using the sequence from CODEMIR-84 (miR-299), which has favourable
strand loading characteristics and supports translational
suppression of VEGF (see above and FIG. 4). Each of these CODEMIRs
was assayed for VEGF suppressive activity in ARPE-19 cells (FIG.
28). However, no discernable trend was observed with respect to the
seed characteristics under investigation, despite the fact that
some of these CODEMIRs had significant activity relative to the
irrelevant control.
TABLE-US-00056 TABLE 18-1 CODEMIRs targeting different seed sites
in the VEGF 3'UTR. CODEMIR Features Duplex VEGF binding 108 Free
2.degree. Passenger 5' A A 3' No A GCGGUUUACCCCUGAAAUGUU (SEQ ID
NO: 502) CCUGAAAUG (SEQ ID NO: 504) 1 site UUCGCCAAAUGGGGACUUUAC
(SEQ ID NO: 503) GGACUUUAC (SEQ ID NO: 503) Guide 3' UUCGCCAAAUGG
5' 109 Passenger 5' C AAG A 3' GCGGUUUACCAGAAGAGACUU (SEQ ID NO:
505) GGCG AGAAGAGAC (SEQ ID NO: 507) UUCGCCAAAUGGUCUUCUCUG (SEQ ID
NO: 506) UCGC UCUUCUCUG (SEQ ID NO: 506) Guide 3' U CAAAUGG 5' 110
Passenger 5' U AAC UA A 3' GCGGUUUACCUUUAUAUGUUU (SEQ ID NO: 508)
GGC UUG UUUGUGUGU (SEQ ID NO: 510) UUCGCCAAAUGGAAAUAUACA (SEQ ID
NO: 509) UCG AAU AAAUAUACA (SEQ ID NO: 509) Guide 3' U CCA GG 5'
111 Free 2.degree. Passenger 5' U GGAAAAG U 3' A
GCGGUUUACCAUAUUAACAUU (SEQ ID NO: 511) GUGG AUAUUAACA (SEQ ID NO:
513) 1 site UUCGCCAAAUGGUAUAAUUGU (SEQ ID NO: 512) CGCC UAUAAUUGU
(SEQ ID NO: 512) Guide 3' UU AAAUGG 5' 112 Passenger 5' C A 3'
GCGGUUUACCGUAUAUAAAUU (SEQ ID NO: 514) CGGUUU UUGUAUAUAAA (SEQ ID
NO: 516) UUCGCCAAAUGGCAUAUAUUU (SEQ ID NO: 515) GCCAAA GGCAUAUAUUU
(SEQ ID NO: 515) Guide 3' UUC U 5' 113 Passenger 5' G CAG CCUCUCCCC
CAGG C 3' GCGGUUUACCAAUGUGCAAUU (SEQ ID NO: 517) GGGC GGU UGCC
AAUGUGCAA UUCGCCAAAUGGUUACACGUU (SEQ ID NO: 518) UUCG CCA AUGG
UUACACGUU Guide 3' A 5' Top strand (SEQ ID NO: 519) Bottom strand
(SEQ ID NO: 518) 114 Not Free Passenger 5' A AG U 3' 2.degree.
GCGGUUUACCACUGGCAGAUU (SEQ ID NO: 520) GG GCCACUGGCAGA (SEQ ID NO:
522) A UUCGCCAAAUGGUGACCGUCU (SEQ ID NO: 521) CC UGGUGACCGUCU (SEQ
ID NO: 521) 1 site Guide 3' UUCG AAA 5' 115 Passenger 5' A A 3'
GCGGUUUACCAGAAAUUAAUU (SEQ ID NO: 523) AUUAGAAAUUAA (SEQ ID NO:
525) UUCGCCAAAUGGUCUUUAAUU (SEQ ID NO: 524) UGGUCUUUAAUU (SEQ ID
NO: 524) Guide 3' UUCGCCAAA 5' 116 Passenger 5' U A AUGU G 3'
GCGGUUUACCCGGCGAAGAUU (SEQ ID NO: 526) GGC G CCCGGCGAAGA (SEQ ID
NO: 528) UUCGCCAAAUGGGCCGCUUCU (SEQ ID NO: 527) UCG C GGGCCGCUUCU
(SEQ ID NO: 527) Guide 3' U C AAAU 5' 117 Not Free Passenger 5' A
AAAG C 3' 2.degree. GCGGUUUACCGAUACAGAAUU (SEQ ID NO: 529) GG
ACUGAUACAGAA (SEQ ID NO: 531) A UUCGCCAAAUGGCUAUGUCUU (SEQ ID NO:
530) CC UGGCUAUGUCUU (SEQ ID NO: 530) 2 sites Guide 3' UUCG AAA 5'
118 Passenger 5' U ACUUGA UU A 3' GCGGUUUACCGGAAGAGGAUU (SEQ ID NO:
532) GUGG G GGGAGGGGA (SEQ ID NO: 534) UUCGCCAAAUGGCCUUCUCCU (SEQ
ID NO: 533) CGCC U CCUUCUCCU (SEQ ID NO: 533) Guide 3' UU AAA GG 5'
119 Passenger 5' G CAG CCUCUCC A 3' GCGGUUUACCUGCCCAGGAUU (SEQ ID
NO: 535) GGGC GGU CCUGCCCAGGA UUCGCCAAAUGGACGGGUCCU (SEQ ID NO:
536) UUCG CCA GGACGGGUCCU Guide 3' AAU 5' Top strand (SEQ ID NO:
537) Bottom strand (SEQ ID NO: 536)
Example 19
Expression of CODEMIRs as shRNAs
[0404] To further confirm, as shown in the HIV VIROMIR example,
that CODEMIRs may be expressed as short hairpin RNAs (shRNA), a
shRNA CODEMIR was designed based on CODEMIR-1. This hairpin was
designed to include the 21 nucleotide core of CODEMIR-1 at the free
(non-loop) terminus of the hairpin (FIG. 29). The hairpin was
cloned into a plasmid vector and expressed from an H1 promoter.
ARPE-19 cells were seeded into 96-well plated (4000 cells/well) and
transfected with 200 ng plasmid DNA 24 hours later. VEGF and ICAM-1
was evaluated 48 hours later by ELISA and FACS, respectively. The
hairpin demonstrated VEGF and ICAM-1 suppressive activity relative
to a length matched hairpin control (FIG. 30); demonstrating the
applicability of expressed short-hairpin CODEMIRs
[0405] Results from the study demonstrated that CODEMIRs can be
expressed as hairpins. Expression of interfering RNAs as hairpins
is well documented and these findings demonstrate that the concept
of CODEMIRs is applicable to hairpins as well as to synthetic
duplexes.
[0406] One skilled in the art will appreciate that the construction
of a suitable expression system for shRNA requires consideration of
many factors which, for example, influence the amount of RNA
produced (promoter, length and stability of transcript etc). Hence,
it is evident that further optimisation of an expression system for
a CODEMIR-1 shRNA precursor could be considered and would likely
lead to higher suppressive capacity.
Example 20
Blunt-Ended CODEMIRs
[0407] Experiments using cell lysates from Drosophila have
identified that 2 nucleotide 3' overhangs at the extremities of the
duplex are optimal for RNA silencing. However, in some experiments
with mammalian cells, "blunt-ended" duplexes were also found to be
considerably active and had increased stability in culture medium
containing FBS (Czauderna et al., (2003), Nucleic Acids Res, 31,
2705-16).
[0408] To test the effect of blunt ends on CODEMIR activity,
variants of CODEMIR-1 were designed with either 1 or 0 base
overhangs (Table 20-1). When transfected in ARPE-19 cells at 40 nM,
these CODEMIRs demonstrated decreased efficacy against VEGF and
ICAM-1 as compared to CODEMIR-1 (FIG. 31). In contrast to the
results of Czauderna et al, it was found that blunt-ended duplexes
were not appreciably more stable (data not shown). Furthermore,
data have been generated indicating that the presence of a blunt
end reduces the loading of the shorter strand (data not shown),
thereby explaining the negative result observed in the above
experiment. Therefore, whilst blunt-ended duplexes may not be of
utility in enhancing the stability of the molecule, this strategy
can be used to enhance the loading of an active strand by designing
the passenger strand to be shorter than the guide strand and having
its 5' end located at the blunt end of the duplex.
TABLE-US-00057 TABLE 20-1 Variants of CODEMIR-1 with truncated 3'
termini CODEMIR duplex Predicted binding (RNA Hybrid) CODEMIR-
Passenger VEGF 5' G A C 3' 1 AGACUCACCCACCCACAUAUU (SEQ ID NO: 101)
UAGAC CACCCACCCACAUA (SEQ ID NO: 181) (CO-1) AAUCUGAGUGGGUGGGUGUAU
(SEQ ID NO: 100) AUCUG GUGGGUGGGUGUAU (SEQ ID NO: 100) Guide CO-1
3' A A 5' ICAM 5' G CCAC C 3' UUAG CUC CCCACCCACAUA (SEQ ID NO:
183) AAUC GAG GGGUGGGUGUAU (SEQ ID NO: 100) CO-1 3' U U 5' CODEMIR-
Passenger VEGF 5' G A C 3' 24 AGACUCACCCACCCACAUAU (SEQ ID NO: 119)
UAGAC CACCCACCCACAUA (SEQ ID NO: 181) AUCUGAGUGGGUGGGUGUAU (SEQ ID
NO: 538) AUCUG GUGGGUGGGUGUAU (SEQ ID NO: 538) Guide AM024 3' A 5'
ICAM 5' U CCAC C 3' UAG CUC CCCACCCACAUA (SEQ ID NO: 457) AUC GAG
GGGUGGGUGUAU (SEQ ID NO: 538) AM024 3' U U 5' CODEMIR- Passenger
VEGF 5' U A C 3' 25 AGACUCACCCACCCACAUA (SEQ ID NO: 189) AGAC
CACCCACCCACAUA (SEQ ID NO: 438) UCUGAGUGGGUGGGUGUAU (SEQ ID NO:
188) UCUG GUGGGUGGGUGUAU (SEQ ID NO: 188) Guide AM025 3' A 5' ICAM
5' C C 3' CUC CCCACCCACAUA (SEQ ID NO: 540) GAG GGGUGGGUGUAU (SEQ
ID NO: 188) AM025 3' UCU U 5'
Example 21
In Vivo Studies with CODEMIR-1
[0409] The activity of CODEMIR-1 and other multitargeting RNA of
the invention could be tested in various preclinical models known
to those skilled in the art. As a non-limiting example, CODEMIR-1
could be tested in a retinopathy of prematurity model. This model
is well known to those working in the field of ocular angiogenesis
and is used extensively as one of several models for the
development of drugs active against the diseases of interest (AMD,
diabetic retinopathy etc). The study could comprise of a suitable
number of mouse or rat neonate pups equally divided into treatment
groups. The treatment groups could include negative controls such
as vehicle, irrelevant or scrambled sequence controls plus a number
of multitargeting RNA including CODEMIR-1. One could also consider
including siRNA to VEGF or other validated angiogenic targets as
known comparators.
[0410] In this model, beginning on Day 1 of life, litters are
exposed to cycles of hyperoxia followed by several days of room
air. The injections could be performed on the last day of cycling,
prior to the 4 day normoxia period. Several days later, animals
could be injected with FITC-dextran and sacrificed. Fluorescence
images of the retinal flat mounts could used to estimate the extent
of neoangiogenesis in each animal. In addition, measurement of the
production of the target RNA molecules or their encoded proteins
(in this case, VEGF and ICAM) could be measured by analysis of
homogenized samples or, alternatively, with in situ
hybridization.
[0411] As a further non-limiting example, CODEMIR-1 could
alternatively be evaluated in vivo for inhibition of
disease-related angiogenesis using the laser-induced Choroidal
Neovascularization (CNV) model in rats or primates. In this model,
animals under general anaesthesia have their pupils dilated and
retina photographed. Choroidal neovascularisation (CNV) is induced
by krypton laser photocoagulation. This is performed using laser
irradiation to either the left or alternatively, the right eye of
each animal from all treatment groups through a slit lamp. A total
of 6-11 laser burns are generally applied to each eye surrounding
the optic nerve at the posterior pole.
[0412] At a suitable time following laser injury, the
multitargeting RNA are injected into the affected eyes. The
suitable time can be the day following laser induction, or for an
assessment against established CNV, the injections can be performed
several days or weeks following injury. Intravitreal injections of
the oligonucleotides are performed by inserting a 30- or 32-gauge
needle into the vitreous. Insertion and infusion can be performed
and directly viewed through an operating microscope. Care is taken
not to injure the lens or the retina. Ideally, the test compounds
are placed in the superior and peripheral vitreous cavity.
Periodically after treatment, the neoangiogenesis is evaluated by
either imaging and/or direct sampling (eg histology,
immunohistochemistry). In all cases, the assessment of CNV is best
performed by a skilled operator blinded to the actual treatment to
ensure a lack of bias in the recording of the information.
[0413] An example of a direct imaging method is Colour Fundus
Photography (CFP). Again, under anaesthesia as described above, the
pupils are dilated. The fundus is then photographed with a camera
using the appropriate film.
[0414] Alternatively, or preferably in addition to CFP, fluorescein
angiography is used to image the vessels and areas of vascular
leakage in the retina. This is performed on all of the animals
following the intraperitoneal or intravenous injection of sodium
fluorescein. The retinal vasculature is then photographed using the
same camera as used for CFP but with a barrier filter for
fluorescein angiography added. Single photographs can be taken at
0.5-1 minute intervals immediately after the administration of
sodium fluorescein. The extent of fluorescein leakage is scored by
a trained operator. The mean severity scores from each of the time
points are compared by a suitable statistical analysis and
differences considered significant at p<0.05. In addition, the
frequency of each lesion score is counted, tabulated and
represented graphically.
[0415] Alternatively, or in addition, rats can be euthanised at
selected time points following treatment (for example 7, 14 and 28
days post injection) and eyes examined by conventional
histopathology. A reduction in the number and severity of lesions
is expected to be seen with samples treated by active
oligonucleotides of the invention.
[0416] Other non-limiting examples including testing the
multitargeting RNA of the invention in other preclinical models
such as those that are well known to those skilled in the art. A
non-exhaustive list includes pulmonary fibrosis (bleomycin
induced), paw inflammation (carrageen), joint arthritis, diabetes,
viral infection, tumour xenografts etc.
[0417] While the foregoing specification teaches the principles of
the present invention, with examples provided for the purpose of
illustration, it will be understood that the practice of the
invention encompasses all of the usual variations, adaptations
and/or modifications as come within the scope of the following
claims and their equivalents. All references are hereby
incorporated into this application in their entirety.
Sequence CWU 1
1
565112RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1uaugugggug gg 1227RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2uguuuug 7312RNAHomo sapiens 3cccacccaca ua
1247RNAHomo sapiens 4caaaaca 7511RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 5accccgucuc u
11611RNAHomo sapiens 6agagacgggg u 1177RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7agcugca 7813RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 8aaacaaugga aug
13913RNAHomo sapiens 9cauuccauug uuu 131014RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10gguagguggg uggg 141114RNAHomo sapiens
11cccacccacc uacc 14129RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 12cugcuugau
9139RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 13uccuuucca 9149RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14uuuuucuuu 91511RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 15uucugauguu u
111611RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 16ucuuccucua u 111711RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 17ugguagcuga a 111811RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 18cuuugguucc u 111911RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 19cuacuaaugc u 112011RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20uccugcuuga u 112111RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 21auucuuuagu u 112211RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22ccaucuuccu g 112311RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 23ccuccaauuc c 112411RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 24cuaauacugu a 112511RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 25uucuguuagu g 112611RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26gcugcuugau g 112711RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 27acauuguacu g 112811RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 28ugauauuucu c 112911RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29aacagcaguu g 113011RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30gugcugauau u 113111RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 31cccaucucca c 113211RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 32uauugguauu a 113311RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 33caaauuguuc u 113411RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 34uacuauuaaa c 11359RNAHuman immunodeficiency virus
type 1 35aucaagcag 9369RNAHuman immunodeficiency virus type 1
36uggaaagga 9379RNAHuman immunodeficiency virus type 1 37aaagaaaaa
93811RNAHuman immunodeficiency virus type 1 38aaacaucaga a
113911RNAHuman immunodeficiency virus type 1 39auagaggaag a
114011RNAHuman immunodeficiency virus type 1 40uucagcuacc a
114111RNAHuman immunodeficiency virus type 1 41aggaaccaaa g
114211RNAHuman immunodeficiency virus type 1 42agcauuagua g
114311RNAHuman immunodeficiency virus type 1 43aucaagcagg a
114411RNAHuman immunodeficiency virus type 1 44aacuaaagaa u
114511RNAHuman immunodeficiency virus type 1 45caggaagaug g
114611RNAHuman immunodeficiency virus type 1 46ggaauuggag g
114711RNAHuman immunodeficiency virus type 1 47uacaguauua g
114811RNAHuman immunodeficiency virus type 1 48cacuaacaga a
114911RNAHuman immunodeficiency virus type 1 49caucaagcag c
115011RNAHuman immunodeficiency virus type 1 50caguacaaug u
115111RNAHuman immunodeficiency virus type 1 51gagaaauauc a
115211RNAHuman immunodeficiency virus type 1 52caacugcugu u
115311RNAHuman immunodeficiency virus type 1 53aauaucagca c
115411RNAHuman immunodeficiency virus type 1 54guggagaugg g
115511RNAHuman immunodeficiency virus type 1 55uaauaccaau a
115611RNAHuman immunodeficiency virus type 1 56agaacaauuu g
115711RNAHuman immunodeficiency virus type 1 57guuuaauagu a
115811RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 58gccuaucaua u 115911RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 59uggugccugc u 116012RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 60aauuaauaug gc 126111RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 61cccucugggc u 116211RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 62uucuuccuca u 116312RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 63uauuuauaca ga 126411RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 64caccaaaauu c 116511RNAHomo sapiens 65auaugauagg c
116611RNAHomo sapiens 66agcaggcacc a 116712RNAHomo sapiens
67gccauauuaa uu 126811RNAHomo sapiens 68agcccagagg g 116911RNAHomo
sapiens 69augaggaaga a 117012RNAHomo sapiens 70ucuguauaaa ua
127111RNAHomo sapiens 71gaauuuuggu g 117214RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 72ugagunngaa cauu 147314RNAHomo
sapiensmodified_base(8)..(9)This region may encompass "CC" or "AG"
73aauguucnna cuca 14747RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 74cuccagg 7757RNAHomo
sapiens 75ccuggag 7768RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 76ucaguggg
8778RNAHomo sapiens 77cccacuga 87811RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 78uccucacagg g 117912RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 79gugcucaugg ug 128012RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 80ccuggagccc ug 128112RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 81ucucagcucc ac 128211RNAHomo sapiens 82cccugugagg
a 118312RNAHomo sapiens 83caccaugagc ac 128412RNAHomo sapiens
84cagggcucca gg 128512RNAHomo sapiens 85guggagcuga ga
128611RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 86acccucgcac c 118711RNAHomo sapiens
87ggugcgaggg u 11889RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 88guguugaag 9899RNAHomo sapiens
89cuucaacac 9909RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 90uuccacaac 9919RNAHomo sapiens
91guuguggaa 9929RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 92uccacuguc 9938RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 93cagaauag 8949RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 94aacucucua
9959RNAHomo sapiens 95gacagugga 9968RNAHomo sapiens 96cuauucug
8979RNAHomo sapiens 97uagagaguu 9989RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 98cgugaagac 9999RNAHepatitis C virus 99gucuucacg
910021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 100uaugugggug ggugagucua a
2110121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 101agacucaccc acccacauau u
2110221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 102uguuuuguug uuacauauga c
2110321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 103cauauguaac aacaaaacau u
2110421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 104uaugugggug gggugucucu a
2110521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 105gagacacccc acccacauau u
2110621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 106uaugugggug ggguggucua a
2110721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 107agaccacccc acccacauau u
2110821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 108uaugugggug gggugguguc u
2110921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 109acaccacccc acccacauau u
2111021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 110uaugugggug ggugaguguc u
2111121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 111acacucaccc acccacauau u
2111221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 112cucacccacc cacauacauu u
2111321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 113auguaugugg gugggugagu c
2111421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 114ucacccaccc acauacauau u
2111521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 115uauguaugug ggugggugag u
2111621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 116ucacccaccc acauacauuu u
2111721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 117aauguaugug ggugggugag u
2111820RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 118uaugugggug ggugagucua
2011920RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 119agacucaccc acccacauau
2012021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 120ggguuuacca ggaagauggu u
2112121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 121ccaucuuccu gguaaaccca u
2112221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 122uuccucacag ggcagugauu c
2112321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 123aucacugccc ugugaggaau u
2112421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 124aucacugccc ugugagaaau u
2112521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 125uuccucacag ggcagugguu c
2112621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 126accacugccc ugugaggaau u
2112721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 127cccggacccu uagagaguuu u
2112821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 128aacucucuaa ggguccgggc a
2112922RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 129uacccucgca ccgaucuccc aa
2213022RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 130gggagaucgg ugcgagggua uu
2213121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 131uacaaaucua cuucaacauu u
2113221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 132guguugaagu agauuuguau g
2113321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 133aacauauguu cuucaacauu u
2113421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 134guguugaaga acauauguuu g
2113521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 135uuccacaaca caagcugugu u
2113621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 136cacagcuugu guuguggaau u
2113721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 137ggacccuuag agaguuucau u
2113821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 138ugaaacucuc uaaggguccg g
2113921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 139uucgugaaga cggugggccg a
2114021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 140ggcccaccgu cuucacgaat t
2114121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 141agacucaccc acccagauau u
2114221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 142uaucugggug ggugagucua a 2114312DNAHomo
sapiens 143cccacccaca ta 1214421DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 144tagagacacc
ccacccacat a 2114521DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 145ttagaccacc ccacccacat a
2114621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 146agacaccacc ccacccacat a
2114721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 147ttagactcac ccacccacat a
2114821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 148agacactcac ccacccacat a
2114921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 149tatatgtgta gcatcaaaac a
2115021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 150gtcatgtgta gcatcaaaac a
2115121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 151tatatgtgta gcaacaaaac a
2115221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 152gtcatgtgta gcaacaaaac a
2115321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 153tatatgtgta gaatcaaaac a
2115421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 154gtcatgtgta gaatcaaaac a
2115521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 155tatatgtgta gaaacaaaac a
2115621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 156gtcatgtgta gaaacaaaac a
2115721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 157tatatgtgta acatcaaaac a
2115821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 158gtcatgtgta acatcaaaac a
2115921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 159tatatgtgta acaacaaaac a
2116021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 160gtcatgtgta acaacaaaac a
2116121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 161tatatgtgta aaatcaaaac a
2116221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 162gtcatgtgta aaatcaaaac a
2116321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 163tatatgtgta aaaacaaaac a
2116421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 164gtcatgtgta aaaacaaaac a
2116521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 165tatatatgta gcatcaaaac a
2116621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 166gtcatatgta gcatcaaaac a
2116721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 167tatatatgta gcaacaaaac a
2116821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 168gtcatatgta gcaacaaaac a
2116921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 169tatatatgta gaatcaaaac a
2117021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 170gtcatatgta gaatcaaaac a
2117121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 171tatatatgta gaaacaaaac a
2117221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 172gtcatatgta gaaacaaaac a
2117321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 173tatatatgta acatcaaaac a
2117421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 174gtcatatgta acatcaaaac a
2117521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 175tatatatgta acaacaaaac a
2117621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 176gtcatatgta acaacaaaac a
2117721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 177tatatatgta aaatcaaaac a
2117821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 178gtcatatgta aaatcaaaac a
2117921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 179tatatatgta aaaacaaaac a
2118021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 180gtcatatgta aaaacaaaac a 2118122RNAHomo
sapiens 181guagacacac ccacccacau ac 2218222RNAHomo sapiens
182uuuauaugua aaaacaaaac aa 2218325RNAHomo sapiens 183guuagccacc
uccccaccca cauac 2518421RNAHomo sapiens 184acauguguag caucaaaaca c
2118521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 185gucugcgauc gcauacaaut t
2118621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 186uagagguacg ugcugaggct t
2118721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 187gugcuggccu uggugaggut t
2118819RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 188uaugugggug ggugagucu
1918919RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 189agacucaccc acccacaua 1919022RNAHomo
sapiens 190uaugugggau gguaaaccgc uu 2219130DNAHomo sapiens
191aagtcgagga agagagagac ggggtcagag 3019230DNAHomo sapiens
192tttttttttt ttccagagac ggggtctcgc 3019330DNAHomo sapiens
193tttggatttt taatagagac ggggttttac 3019421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 194tggttaacag agacggggtc t 2119521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 195atggttaaca gagacggggt a 2119621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 196gatggttaac agagacgggg t 2119721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 197ggttaacaga gacggggtct a 2119821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 198ggttaacaga gacggggtct t 2119921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 199gggtaacaga gacggggtct a 2120021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 200gggtaacaga gacggggtct t 2120121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 201ugguuaacag agacgggguu u 2120221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 202accccgucuc uguuaaccau c 2120321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 203ugguuaacag agacgggauu u 2120421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 204ugguuaacag agacggaguu u 2120521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 205ugguuaacag agacgagguu u 2120621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 206ugguuaacag agacaggguu u 2120721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 207ugguuaacag agacggaauu u 2120821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 208ugguuaacag agacgaaguu u 2120921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 209ugguuaacag agacaagguu u 212107DNAHomo sapiens
210tgcagct 721111DNAHomo sapiens 211agagacgggg t 1121214DNAHomo
sapiens 212cccacccacc tacc 1421337DNAHomo sapiens 213cagagtattt
ctgccccacc cacctacccc ccaaaaa 3721437DNAHomo sapiens 214gaggagatct
ccttcccacc cacctaccgc tatgagc 3721521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 215ctccgcccca cccacctacc a 2121621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 216ctcctcccca cccacctacc a 2121721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 217tctccgcccc acccacctac c 2121821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 218tctcctcccc acccacctac c 2121934RNAHomo sapiens
219cagaggacua aauaccauuc cauuguuugu gcag 3422034RNAHomo sapiens
220cugguaacag uaauacauuc cauuguuuua guaa 3422134RNAHomo sapiens
221gacuuguuug ucuuccauuc cauuguuuug aaac 3422221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 222auaauccauu ccauuguuuu a 2122321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 223auaauacauu ccauuguuuu a 2122421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 224auauuccauu ccauuguuuu a 2122521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 225auauuacauu ccauuguuuu a 2122621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 226guaauccauu ccauuguuuu a 2122721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 227guaauacauu ccauuguuuu a 2122821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 228guauuccauu ccauuguuuu a 2122921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 229guauuacauu ccauuguuuu a 2123021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 230cuauaaucca uuccauuguu u 2123121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 231cuauaauaca uuccauuguu u 2123221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 232cuauauucca uuccauuguu u 2123321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 233cuauauuaca uuccauuguu u
2123421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 234cuguaaucca uuccauuguu u
2123521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 235cuguaauaca uuccauuguu u
2123621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 236cuguauucca uuccauuguu u
2123721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 237cuguauuaca uuccauuguu u
2123821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 238caauaaucca uuccauuguu u
2123921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 239caauaauaca uuccauuguu u
2124021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 240caauauucca uuccauuguu u
2124121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 241caauauuaca uuccauuguu u
2124221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 242caguaaucca uuccauuguu u
2124321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 243caguaauaca uuccauuguu u
2124421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 244caguauucca uuccauuguu u
2124521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 245caguauuaca uuccauuguu u 212469DNAHuman
immunodeficiency virus type 1 246atcaagcag 92479DNAHuman
immunodeficiency virus type 1 247tggaaagga 924844DNAHuman
immunodeficiency virus type 1 248aaacacagtg gggggacatc aagcagccat
gcaaatgtta aaag 4424944DNAHuman immunodeficiency virus type 1
249cctgttggtg ggcgggaatc aagcaggaat ttggaattcc ctac 4425044DNAHuman
immunodeficiency virus type 1 250tagccctagg tgtgaatatc aagcaggaca
taacaaggta ggat 4425144DNAHuman immunodeficiency virus type 1
251aactcacagt ctggggcatc aagcagctcc aggcaagaat cctg 4425289DNAHuman
immunodeficiency virus type 1 252agggagctag aacgattcgc agttaatcct
ggcctgttag aaacatcaga agctgtagac 60aaatactggg acagctacaa ccatccctt
8925389DNAHuman immunodeficiency virus type 1 253acaacatctg
ttgaggtggg gacttaccac accagacaaa aaacatcaga aagaacctcc 60attcctttgg
atgggttatg aactccatc 8925490DNAHuman immunodeficiency virus type 1
254aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
agcaaaacaa 60aagtaagaaa aaagcacagc aagcagcagc 9025590DNAHuman
immunodeficiency virus type 1 255aacagggaga ctaaattagg aaaagcagga
tatgttacta atagaggaag acaaaaagtt 60gtcaccctaa ctgacacaac aaatcagaag
9025690DNAHuman immunodeficiency virus type 1 256aggcaagagt
tttggctgaa gcaatgagcc aagtaacaaa ttcagctacc ataatgatgc 60agagaggcaa
ttttaggaac caaagaaaga 9025790DNAHuman immunodeficiency virus type 1
257cttggcactt atctgggacg atctgcggag cctgtgcctc ttcagctacc
accgcttgag 60agacttactc ttgattgtaa cgaggattgt 9025890DNAHuman
immunodeficiency virus type 1 258aacaaattca gctaccataa tgatgcagag
aggcaatttt aggaaccaaa gaaagattgt 60taagtgtttc aattgtggca aagaagggca
9025990DNAHuman immunodeficiency virus type 1 259acccagggat
taaagtaagg caattatgta aactccttag aggaaccaaa gcactaacag 60aagtaatacc
actaacagaa gaagcagagc 9026090DNAHuman immunodeficiency virus type 1
260caaaagttaa acaatggcca ttgacagaag aaaaaataaa agcattagta
gaaatttgta 60cagagatgga aaaggaaggg aaaatttcaa 9026190DNAHuman
immunodeficiency virus type 1 261tagtacatgt aacgcaacct ataccaatag
tagcaatagt agcattagta gtagcaataa 60taatagcaat agttgtgtgg tccatagtaa
9026290DNAHuman immunodeficiency virus type 1 262caccggtgct
acggttaggg ccgcctgttg gtgggcggga atcaagcagg aatttggaat 60tccctacaat
ccccaaagtc aaggagtagt 9026390DNAHuman immunodeficiency virus type 1
263aaggccttat taggacacat agttagccct aggtgtgaat atcaagcagg
acataacaag 60gtaggatctc tacaatactt ggcactagca 9026490DNAHuman
immunodeficiency virus type 1 264caggggaaag aatagtagac ataatagcaa
cagacataca aactaaagaa ttacaaaaac 60aaattacaaa aattcaaaat tttcgggttt
9026590DNAHuman immunodeficiency virus type 1 265ctcaaatatt
ggtggaatct cctacagtat tggagtcagg aactaaagaa tagtgctgtt 60agcttgctca
atgccacagc catagcagta 9026690DNAHuman immunodeficiency virus type 1
266acaggagcag atgatacagt attagaagaa atgagtttgc caggaagatg
gaaaccaaaa 60atgatagggg gaattggagg ttttatcaaa 9026790DNAHuman
immunodeficiency virus type 1 267gaaacagggc aggaaacagc atattttctt
ttaaaattag caggaagatg gccagtaaaa 60acaatacata ctgacaatgg cagcaatttc
9026890DNAHuman immunodeficiency virus type 1 268aatgagtttg
ccaggaagat ggaaaccaaa aatgataggg ggaattggag gttttatcaa 60agtaagacag
tatgatcaga tactcataga 9026990DNAHuman immunodeficiency virus type 1
269aaaggaaaag gtctatctgg catgggtacc agcacacaaa ggaattggag
gaaatgaaca 60agtagataaa ttagtcagtg ctggaatcag 9027090DNAHuman
immunodeficiency virus type 1 270ggcaactaaa ggaagctcta ttagatacag
gagcagatga tacagtatta gaagaaatga 60gtttgccagg aagatggaaa ccaaaaatga
9027190DNAHuman immunodeficiency virus type 1 271atcagatact
catagaaatc tgtggacata aagctatagg tacagtatta gtaggaccta 60cacctgtcaa
cataattgga agaaatctgt 9027289DNAHuman immunodeficiency virus type 1
272aaagtaaggc aattatgtaa actccttaga ggaaccaaag cactaacaga
agtaatacca 60ctaacagaag aagcagagct agaactggc 8927389DNAHuman
immunodeficiency virus type 1 273aaactcctta gaggaaccaa agcactaaca
gaagtaatac cactaacaga agaagcagag 60ctagaactgg cagaaaacag agagattct
8927490DNAHuman immunodeficiency virus type 1 274cccacaagat
ttaaacacca tgctaaacac agtgggggga catcaagcag ccatgcaaat 60gttaaaagag
accatcaatg aggaagctgc 9027590DNAHuman immunodeficiency virus type 1
275ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
ctccaggcaa 60gaatcctggc tgtggaaaga tacctaaagg 9027690DNAHuman
immunodeficiency virus type 1 276catacctagt ataaacaatg agacaccagg
gattagatat cagtacaatg tgcttccaca 60gggatggaaa ggatcaccag caatattcca
9027790DNAHuman immunodeficiency virus type 1 277aagacgttca
atggaacagg accatgtaca aatgtcagca cagtacaatg tacacatgga 60attaggccag
tagtatcaac tcaactgctg 9027890DNAHuman immunodeficiency virus type 1
278actattttta gatggaatag ataaggccca agatgaacat gagaaatatc
acagtaattg 60gagagcaatg gctagtgatt ttaacctgcc 9027990DNAHuman
immunodeficiency virus type 1 279ctaatagaaa gagcagaaga cagtggcaat
gagagtgaag gagaaatatc agcacttgtg 60gagatggggg tggagatggg gcaccatgct
9028090DNAHuman immunodeficiency virus type 1 280tacttgggca
ggagtggaag ccataataag aattctgcaa caactgctgt ttatccattt 60tcagaattgg
gtgtcgacat agcagaatag 9028190DNAHuman immunodeficiency virus type 1
281agtacaatgt acacatggaa ttaggccagt agtatcaact caactgctgt
taaatggcag 60tctagcagaa gaagaggtag taattagatc 9028291DNAHuman
immunodeficiency virus type 1 282tagaaagagc agaagacagt ggcaatgaga
gtgaaggaga aatatcagca cttgtggaga 60tgggggtgga gatggggcac catgctcctt
g 9128391DNAHuman immunodeficiency virus type 1 283aatgataatg
gagaaaggag agataaaaaa ctgctctttc aatatcagca caagcataag 60aggtaaggtg
cagaaagaat atgcattttt t 9128490DNAHuman immunodeficiency virus type
1 284agacagtggc aatgagagtg aaggagaaat atcagcactt gtggagatgg
gggtggagat 60ggggcaccat gctccttggg atgttgatga 9028590DNAHuman
immunodeficiency virus type 1 285tgagagtgaa ggagaaatat cagcacttgt
ggagatgggg gtggagatgg ggcaccatgc 60tccttgggat gttgatgatc tgtagtgcta
9028690DNAHuman immunodeficiency virus type 1 286tctgtgttag
tttaaagtgc actgatttga agaatgatac taataccaat agtagtagcg 60ggagaatgat
aatggagaaa ggagagataa 9028790DNAHuman immunodeficiency virus type 1
287aaggtgcaga aagaatatgc atttttttat aaacttgata taataccaat
agataatgat 60actaccagct ataagttgac aagttgtaac 9028890DNAHuman
immunodeficiency virus type 1 288aatggaataa cactttaaaa cagatagcta
gcaaattaag agaacaattt ggaaataata 60aaacaataat ctttaagcaa tcctcaggag
9028990DNAHuman immunodeficiency virus type 1 289gtacaggcca
gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct 60attgaggcgc
aacagcatct gttgcaactc 9029090DNAHuman immunodeficiency virus type 1
290attgtggagg ggaatttttc tactgtaatt caacacaact gtttaatagt
acttggttta 60atagtacttg gagtactgaa gggtcaaata 9029190DNAHuman
immunodeficiency virus type 1 291ttttctactg taattcaaca caactgttta
atagtacttg gtttaatagt acttggagta 60ctgaagggtc aaataacact gaaggaagtg
9029228DNAHuman immunodeficiency virus type 1 292tggtgggcgg
gaatcaagca ggaatttg 2829328DNAHuman immunodeficiency virus type 1
293ctaggtgtga atatcaagca ggacataa 2829421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 294ttgtgcgaaa atcaagcagg a 2129521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 295ttgtgcgaat atcaagcagg a 2129621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 296ttgggcgaaa atcaagcagg a 2129721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 297ttgggcgaat atcaagcagg a 2129821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 298tggtgcgaaa atcaagcagg a 2129921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 299tggtgcgaat atcaagcagg a 2130021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 300tggggcgaaa atcaagcagg a 2130121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 301tggggcgaat atcaagcagg a 2130221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 302gtgtgcgaaa atcaagcagg a 2130321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 303gtgtgcgaat atcaagcagg a 2130421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 304gtgggcgaaa atcaagcagg a 2130521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 305gtgggcgaat atcaagcagg a 2130621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 306gggtgcgaaa atcaagcagg a 2130721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 307gggtgcgaat atcaagcagg a 2130821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 308gggggcgaaa atcaagcagg a 2130921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 309gggggcgaat atcaagcagg a 2131021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 310atgggcgaaa atcaagcagg a 2131121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 311gugggcgaac aucaagcagu u 2131221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 312cugcuugaug uucgcccacg g 2131321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 313gcaauaaaag cauuaguagu u 2131421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 314cuacuaaugc uuuuauugcu a 2131521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 315gggcgaaaau caagcaggau u 2131621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 316uccugcuuga uuuucgccca u 2131721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 317gagucagaaa cuaaagaauu u 2131821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 318auucuuuagu uucugacucu g 2131921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 319gcaauagaua caguauuagu u 2132021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 320cuaauacugu aucuauugcu u 2132121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 321cgcuaaaagg aauuggaggu u 2132221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 322ccuccaauuc cuuuuagcgu u 2132321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 323cgugauaaca guacaauguu u 2132421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 324acauuguacu guuaucacgu u 2132521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 325agccauagca cuaacagaau u 2132621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 326uucuguuagu gcuauggcuu c 2132721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 327uuccaccuca acugcuguuu u 2132821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 328aacagcaguu gagguggaau u 2132966DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 329gatccgatgg gtttaccagg aagatggact cgagaccatc
ttcctggtaa acccattttt 60ttggaa 6633066DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 330agctttccaa aaaaatgggt ttaccaggaa gatggtctcg
agtccatctt cctggtaaac 60ccatcg 6633111DNAHomo sapiens 331atatgatagg
c 1133212DNAHomo sapiens 332gccatattaa tt 1233311DNAHomo sapiens
333atgaggaaga a
1133412DNAHomo sapiens 334tctgtataaa ta 1233511DNAHomo sapiens
335gaattttggt g 1133614DNAHomo sapiens 336aatgttccca ctca
1433714DNAHomo sapiens 337aatgttcaga ctca 143385DNAHomo sapiens
338actca 53397DNAHomo sapiens 339cctggag 734010DNAHomo sapiens
340agtcctggag 1034110DNAHomo sapiens 341aaccctggag 1034210DNAHomo
sapiens 342ggccctggag 1034310DNAHomo sapiens 343ggtcctggag
1034410DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 344rrycctggag 1034510RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 345cuccaggguu 1034623RNAHomo sapiens 346agacacaccc
acccacauac aua 2334720RNAHomo sapiens 347ccuccccacc cacauacauu
2034820RNAHomo sapiens 348acacccaccc acauacauac 2034919RNAHomo
sapiens 349acacccaccc acauacaua 1935021RNAHomo sapiens
350ccuccccacc cacauacauu u 2135111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 351ccctgtgagg a
1135212DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 352caccatgagc ac 1235312DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 353cagggctcca gg 1235412DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 354gtggagctga ga 1235560DNAHepatitis C virus
355gggcgacact ccaccatgaa tcactcccct gtgaggaact actgtcttca
cgcagaaagc 6035660DNAHomo sapiens 356tgggcaggtc tactttggga
tcattgccct gtgaggagga cgaacatcca accttcccaa 6035721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 357gaatcactgc cctgtgagga a 2135823RNAHepatitis C
virus 358ugaaucacuc cccugugagg aac 2335923RNAHomo sapiens
359gggaucauug cccugugagg agg 2336021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 360caaaaaugac aguggacgau u 2136121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 361ucguccacug ucauuuuugg g 2136216RNAHomo sapiens
362uaaaaugaca guggaa 1636319RNAHomo sapiens 363gcaagaguga caguggauu
1936427RNAHomo sapiens 364ccccggccgg cggcggacag uggacgc
2736544RNAHomo sapiens 365gccaggcccu gugugaaccu uugagcuuuc
auagagaguu ucac 4436622RNAHomo sapiens 366gagcuuucau agagaguuuc ac
2236717RNAHomo sapiens 367ugaccuuuag agaguug 1736823RNAHomo sapiens
368cccggaagau uagagaguuu uau 2336921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 369acccuuagag aguuucacau u 2137021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 370ugugaaacuc ucuaaggguc c 2137131RNAHomo sapiens
371aaccuuugag cuuucauaga gaguuucaca g 3137225RNAHomo sapiens
372ugaccuuuag agaguugcuu uacgu 2537326RNAHomo sapiens 373agcccggaag
auuagagagu uuuauu 2637421RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 374aucccuauuc
uguucuuuau u 2137521RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 375uaaagaacag aauagggaug c
2137622RNAHomo sapiens 376ucaucccuau ucuguguuuu au 2237717RNAHomo
sapiens 377auucuauucu gaucuua 1737822RNAHomo sapiens 378acauugcuau
ucuguuuuuu au 2237921RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 379caucccuauu
cuguucuuau u 2138021RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 380uaagaacaga auagggaugu c
2138118RNAHomo sapiens 381auucuauucu gaucuuau 1838222RNAHomo
sapiens 382agacauugcu auucuguuuu uu 2238337RNAHomo sapiens
383ggcccugugu gaaccuuuga gcuuucauag agaguuu 3738421RNAHomo sapiens
384gguuugaccu uuagagaguu g 2138522RNAHomo sapiens 385agcccggaag
auuagagagu uu 2238621RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 386ccggacccuu
agagaguuuu u 2138721RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 387aaacucucua aggguccggg c
2138838RNAHomo sapiens 388ggcccugugu gaaccuuuga gcuuucauag agaguuuc
3838921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 389gaaacucucu aaggguccgg g 2139037RNAHomo
sapiens 390cccuguguga accuuugagc uuucauagag aguuuca 3739117RNAHomo
sapiens 391ugaccuuuag agaguug 1739223RNAHomo sapiens 392gcccggaaga
uuagagaguu uua 2339311DNAHomo sapiens 393ggtgcgaggg t
1139457DNAHomo sapiens 394aacgtggcag ggacgccggg ggacttcggt
gcgagggtca ccgccgggtt aactggc 5739557DNAHomo sapiens 395caagtagggt
acggactttg ggggattggt gcgagggtag tgggtgagtg gcctact
5739622DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 396ttgggagatc ggtgcgaggg ta
2239723RNAHomo sapiens 397cgggggacuu cggugcgagg guc 2339824RNAHomo
sapiens 398uuugggggau uggugcgagg guag 2439923RNAHomo sapiens
399gcauauauau guucuucaac aca 2340020RNAHomo sapiens 400agcaaaucua
cuucaacacu 204019DNAHepatitis C virus 401gtcttcacg
940248DNAHepatitis C virus 402atcactcccc tgtgaggaac tactgtcttc
acgcagaaag cgtctagc 4840348DNAHepatitis C virus 403atggagacca
ctatgcggtc tccggtcttc acggacaact catctccc 4840448DNAHepatitis C
virus 404gatcacctgg agttctggga gagcgtcttc acgggcctca cccacata
4840548DNAHepatitis C virus 405caggaggatg cggcgagcct acgagtcttc
acggaggcta tgactagg 4840611RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 406uucgugaaga c
1140721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 407uucgugaaga cggugggccg g
2140821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 408uucgugaaga cggugggccg c
2140921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 409uucgugaaga cggugggccg u
2141021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 410uucgugaaga cgguaggccg a
2141121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 411uucgugaaga cgguaggccg g
2141221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 412uucgugaaga cgguaggccg c
2141321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 413uucgugaaga cgguaggccg u
2141421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 414uucgugaaga cagugggccg c
2141521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 415uucgugaaga caguaggccg c
2141620RNAHepatitis C virus 416aggaacuacu gucuucacgc
2041721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 417uucgugaaga crgurggccg n
2141823RNAHepatitis C virus 418ugcggucucc ggucuucacg gac
2341921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 419uucgugaaga crgurggccg y
2142021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 420uucgugaaga crgurggccg r
2142119RNAHepatitis C virus 421gggagagcgu cuucacggg
1942225RNAHepatitis C virus 422ggcgagccua cgagucuuca cggag
2542321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 423uaugugggug ggugagucua a
2142421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 424agacucaccc acccacauau u
2142521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 425agacucaccc acccacauau u
2142621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 426agacucaccc acccacauau u
2142721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 427agacucaccc acccacauau u
2142821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 428uaugugggug ggugagucua a
2142921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 429agacucaccc accgagauau u
2143021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 430uaucucggug ggugagucua a
2143116RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 431guagacacac ccaccc 1643221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 432uaucucgcug ggugagucua a 2143314RNAHomo sapiens
433guagacacac ccac 1443421RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 434uaugugggug
ggggggucuc u 2143532RNAHomo sapiens 435ugggauuccu guagacacac
ccacccacau ac 3243623RNAHomo sapiens 436uagccaccuc cccacccaca uac
2343721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 437uaugugggug ggugggucuc u 2143821RNAHomo
sapiens 438uagacacacc cacccacaua c 2143921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 439uaugugggug ggggugucuc u 2144035RNAHomo sapiens
440ugacaccuuu guuagccacc uccccaccca cauac 3544121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 441uaugugggug ggugugucuc u 2144221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 442uaugugggug gggggguguc u 2144321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 443uaugugggug gguggguguc u 2144421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 444uaugugggug gggguguguc u 2144521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 445uaugugggug gguguguguc u 2144631RNAHomo sapiens
446gggauuccug uagacacacc cacccacaua c 3144721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 447uaugugggug ggggggucgc u 2144821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 448uaugugggug ggugggucgc u 2144920RNAHomo sapiens
449agacacaccc acccacauac 2045021RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 450uaugugggug
ggggugucgc u 2145121RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 451uaugugggug ggugugucgc u
2145221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 452uaugugggug gggggguggc u
2145321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 453uaugugggug gguggguggc u 2145426RNAHomo
sapiens 454uccuguagac acacccaccc acauac 2645521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 455uaugugggug gguguguggc u 2145621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 456uaugugggug ggggggucua u 2145724RNAHomo sapiens
457uuagccaccu ccccacccac auac 2445821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 458uaugugggug ggugggucua u 2145921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 459uaugugggug ggggugucua u 2146021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 460uaugugggug ggugugucua u 2146121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 461uaugugggug ggggggugua u 2146219RNAHomo sapiens
462gacacaccca cccacauac 1946320RNAHomo sapiens 463ccaccucccc
acccacauac 2046421RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 464uaugugggug ggugggugua u
2146521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 465uaugugggug ggggugugua u
2146621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 466uaugugggug ggugugugua u
2146722RNAHomo
sapiens 467agccaccucc ccacccacau ac 2246821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 468uaugugggug ggggggucga u 2146919RNAHomo sapiens
469caccucccca cccacauac 1947021RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 470uaugugggug
ggugggucga u 2147121RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 471uaugugggug ggggugucga u
2147236RNAHomo sapiens 472cugacaccuu uguuagccac cuccccaccc acauac
3647321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 473uaugugggug ggugugucga u
2147421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 474uaugugggug ggggggugga u 2147527RNAHomo
sapiens 475uuccuguaga cacacccacc cacauac 2747621RNAHomo sapiens
476gccaccuccc cacccacaua c 2147721RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 477uaugugggug
ggugggugga u 2147821RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 478uaugugggug ggggugugga u
2147921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 479uaugugggug ggugugugga u 2148013RNAHomo
sapiens 480cccacccaca uac 1348121RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 481acaccccccc
acccacauau u 2148221RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 482uaugugggug gggggguguc u
2148321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 483cgaccccccc acccacauau u
2148421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 484uaugugggug ggggggucga u 2148519RNAHomo
sapiens 485caccucccca cccacauac 1948621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 486acaccucccc acccacauau u 2148721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 487uaugugggug gggagguguc u 2148821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 488cgaccucccc acccacauau u 2148921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 489uaugugggug gggaggucga u 2149021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 490uaugugggug ggugagucua a 2149121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 491uaugugggug ggngagucua a 2149221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 492uaugugggug ggugngucua a 2149321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 493uaugugggug ggngngucua a 2149421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 494uaugugggug ggugagucua c 2149524RNAHomo sapiens
495cuguagacac acccacccac auac 2449621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 496uagaccaccc acccacauau u 2149721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 497uaugugggug gguggucuac a 2149821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 498gacaucaccc acccacauau u 2149921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 499uaugugggug ggugaugucu a 2150021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 500agacacaccc acccacauau u 2150121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 501uaugugggug ggugugucua c 2150221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 502gcgguuuacc ccugaaaugu u 2150321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 503cauuucaggg guaaaccgcu u 2150411RNAHomo sapiens
504accugaaaug a 1150521RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 505gcgguuuacc
agaagagacu u 2150621RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 506gucucuucug guaaaccgcu u
2150718RNAHomo sapiens 507cggcgaagag aagagaca 1850821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 508gcgguuuacc uuuauauguu u 2150921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 509acauauaaag guaaaccgcu u 2151022RNAHomo sapiens
510uggcaacuug uauuugugug ua 2251121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 511gcgguuuacc auauuaacau u 2151221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 512uguuaauaug guaaaccgcu u 2151322RNAHomo sapiens
513uguggggaaa agauauuaac au 2251421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 514gcgguuuacc guauauaaau u 2151521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 515uuuauauacg guaaaccgcu u 2151619RNAHomo sapiens
516ccgguuuuug uauauaaaa 1951721RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 517gcgguuuacc
aaugugcaau u 2151821RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 518uugcacauug guaaaccgcu u
2151938RNAHomo sapiens 519ggggccaggg uccucucccc ugcccaggaa ugugcaag
3852021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 520gcgguuuacc acuggcagau u
2152121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 521ucugccagug guaaaccgcu u 2152218RNAHomo
sapiens 522aggaggccac uggcagau 1852321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 523gcgguuuacc agaaauuaau u 2152421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 524uuaauuucug guaaaccgcu u 2152514RNAHomo sapiens
525aauuagaaau uaaa 1452621RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 526gcgguuuacc
cggcgaagau u 2152721RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 527ucuucgccgg guaaaccgcu u
2152822RNAHomo sapiens 528uggcagaugu cccggcgaag ag
2252921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 529gcgguuuacc gauacagaau u
2153021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 530uucuguaucg guaaaccgcu u 2153120RNAHomo
sapiens 531aggaaagacu gauacagaac 2053221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 532gcgguuuacc ggaagaggau u 2153321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 533uccucuuccg guaaaccgcu u 2153424RNAHomo sapiens
534uguggacuug aguugggagg ggaa 2453521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 535gcgguuuacc ugcccaggau u 2153621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 536uccugggcag guaaaccgcu u 2153730RNAHomo sapiens
537ggggccaggg uccucucccc ugcccaggaa 3053820RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 538uaugugggug ggugagucua 2053921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 539uuccucacag ggcagugauu c 2154017RNAHomo sapiens
540ccuccccacc cacauac 1754121RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 541uaugugggug
ggugagucua a 2154221DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 542tggttaacag agacggggtc a
2154321RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 543cggacccuua gagaguuucu u 2154423RNAHomo
sapiens 544ucaagcaaau cuacuucaac acu 2354523RNAHomo sapiens
545uguagacaca cccacccaca uac 2354610DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 546ctgacaatcg 1054710DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 547cgaaagtcag 1054810DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 548cgattgtcag 1054924RNAHomo sapiens 549guuagccacc
uccccaccca caua 2455021RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 550agacucaccc
agcgagauau u 2155121RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 551uaugugggug gggguguggc u
2155230RNAHomo sapiens 552gggauuccug uagacacacc cacccacaua
3055330RNAHomo sapiens 553accuuuguua gccaccuccc cacccacaua
3055421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 554uagagacacc ccacccacau a
2155521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 555uuagaccacc ccacccacau a
2155621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 556agacaccacc ccacccacau a
2155721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 557uuagacucac ccacccacau a
2155821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 558agacacucac ccacccacau a
2155923RNAHuman immunodeficiency virus type 1 559ggugggcggg
aaucaagcag gaa 2356022RNAHuman immunodeficiency virus type 1
560aggugugaau aucaagcagg ac 2256121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 561uccugcuuga uuuucgcccc a 2156221RNAHomo sapiens
562agccaccucc ccacccacau a 2156321RNAHomo sapiens 563guagacacac
ccacccacau a 2156423RNAHomo sapiens 564agcccggaag auuagagagu uuu
2356548RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 565agacucaccc acccacauaa cucgagauau
gugggugggu gagucuaa 48
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