Note: Descriptions are shown in the official language in which they were submitted.
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INTM/030 PCT
METHODS AND COMPOSITIONS FOR TREATMENT OF CANCER AND
OTHER ANGIOGENESIS-RELATED DISEASES
Cross-Reference to Related Applications
100011 This application claims priority under 35 U.S.C. 119(e) from United
States provisional application 60/958,519, filed July 6, 2007, United States
provisional application 60/966,085, filed August 24, 2007 and United States
provisional application 61/131,876, filed June 12, 2008.
Field of the Invention
[0002] The present invention is in the field of molecular biology and medicine
and relates to short interfering RNA (siRNA) molecules for modulating the
expression of molecules in the angiopoietin/Tie2 signaling pathway.
Background of the Invention
[0003] The angiopoietin/Tie2 signaling pathway has been implicated in several
types of cancer-induced angiogenesis. Several molecules in the Ang-Tie pathway
have been identified (see, e.g., Tables 1 and 13). One of them is the receptor
molecule Tie2 (Tyrosine Kinase with Immunoglobulin and EGF factor homology
domains, also called TIE-2, TEK or epithelial-specific protein receptor
tyrosine
kinase, TEK tyrosine kinase), which is expressed almost exclusively on the
surface
of vascular endothelial cells (ECs) (Sato et al., 1998, Int. Immunol. 10: 1217-
1227). Ligands that bind to Tie2 include angiopoietin-1 and angiopoietin-2
(Yancopoulos et al., 2000, Nature 407: 242-248).
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Table 1. Angiopoietin/Tie2 pathway gene sequence IDs.
UniGene Gene Name Gene
Sequence ID Abbreviation
Hs.89640 H. sapiens receptor protein- Hu Tie2
tyrosine kinase
Mm.14313 M. musculus Tie2 Ms Tie2
Hs.369675 H. sapiens angiopoietin 1 Hu Ang-1
Mm.309336 M. musculus angiopoietin 1 Ms Ang-1
Hs.583870 H. sapiens angiopoietin 2 Hu Ang-2
Mm.435498 M. musculus angiopoietin 2 Ms Ang-2
[0004] Accordingly, there is an urgent need for therapeutic agents targeting
the
Ang-Tie pathway.
Summary of the Invention
[0005] One aspect of the present invention provides a nucleic acid molecule
that
reduces expression of an angiopoietin-1 (Ang-1), an angiopoietin-2 (Ang-2), or
a
tyrosine kinase with immunoglobulin and EGF factor homology domains (Tie2)
gene, wherein the nucleic acid molecule comprises or targets any one of SEQ ID
NOs: 1-648. The present invention also provides a nucleic acid molecule that
reduces expression of an Ang-2 gene, wherein the nucleic acid molecule
comprises
or targets any one of SEQ ID NOs: 487, 489, 525, 526, 553, 554, 639, 640, 643,
and 644. In a particular embodiment, the nucleic acid molecule is a short
interfering RNA (siRNA) molecule. In a preferred embodiment, the invention
provides siRNA of 25 base pairs with blunt ends.
[0006] The present invention also provides a composition comprising a nucleic
acid molecule that comprises or targets any one of SEQ ID NOs: 1-648 and a
pharmaceutically acceptable carrier. In one embodiment, the composition
further
comprises a histidine-lysine copolymer. In a further embodiment, the
composition
further comprises a targeting moiety. The composition may also comprise one or
more additional therapeutic agents.
[0007] The present invention also provides combinations of nucleic acid
molecules that target multiple disease-causing genes or target different
sequences
in the same gene. In one aspect, the invention provides compositions
comprising a
nucleic acid molecule that comprises or targets any one of SEQ ID NOs: 1-648
and
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further comprising one or more additional nucleic acid molecules that induce
RNA
interference and decrease the expression of a gene of interest. In one
embodiment,
the one or more additional nucleic acid molecules decrease the expression of
Ang-
1, Ang-2, or Tie-2.
[0008] The present invention further provides methods for reducing protein
level
expression of Ang- 1, Ang-2, or Tie-2 genes in a cell, comprising introducing
into
the cell any of the nucleic acid molecules or the siRNA molecules of the
invention.
The present invention also provides methods of reducing angiogenesis in a
subject
in need thereof, comprising administering to the subject any of the nucleic
acid
molecules, siRNA molecules, or compositions of the invention. Additionally,
the
present invention provides a method of treating cancer in a subject in need
thereof,
comprising administering to the subject any of the nucleic acid molecules,
siRNA
molecules, or compositions of the invention.
[0009] These and other aspects of the present invention will become apparent
upon references to the following detailed description.
Brief Description of the Drawings
[0010] Figure 1 is a bar graph depicting in vitro inhibition of human Ang-2 by
siRNA molecules in human umbilical vein endothelial (HUVEC) cells at 24 hours
post siRNA transfection.
[00111 Human Ang-2 gene silencing activity of human Ang-2-siRNA sequences
listed in Table 11 was tested in HUVEC cells. Labels #1-#48 on the x-axis
correspond to the siRNA sequences numbered 1-48 in Table 11. The HUVEC
cells were transfected with the Ang-2-siRNAs using a reverse transfection
based
high-through-put (HTP) method with 10 nM of siRNA duplex. A luciferase
specific 25-mer siRNA was used as the negative control (Luc). The effect of
siRNA mediated Ang-2 knockdown was monitored by measuring the concentration
of Ang-2 protein in the medium using a human Ang-2 ELISA kit (R&D).
Significant inhibition of Ang-2 protein level expression in transfected HUVEC
cells was observed at 24 hours post transfection with a majority of the 48 Ang-
2
siRNA candidates tested.
[0012] Figure 2 is a bar graph depicting in vitro inhibition of human Ang-2 by
siRNA molecules in HUVEC cells at 48 hours post siRNA transfection.
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[0013] Human Ang-2 gene silencing activity of human Ang-2-siRNA sequences
listed in Table 11 was tested in HUVEC cells. Labels 1-48 on the x-axis
correspond to the siRNA sequences numbered 1-48 in Table 11. The HUVEC
cells were transfected with the Ang-2-siRNAs using a reverse transfection
based
high-through-put (HTP) method with 10 nM of siRNA duplex. A luciferase
specific 25-mer siRNA was used as the negative control (Luc). The effect of
siRNA mediated Ang-2 knockdown was monitored by measuring the concentration
of Ang-2 protein in the medium using a human Ang-2 ELISA kit (R&D). At 48
hours post siRNA transfection, more than 50% of the transfected HUVEC cells
express less than 20% of Ang-2 protein compared to the mock control.
[0014] Figure 3 is a bar graph depicting the percentage of inhibition of human
Ang-2 by siRNA molecules in HUVEC cells at 48 hours post siRNA transfection.
[0015] Human Ang-2 gene silencing activity of human Ang-2-siRNA sequences
listed in Table 11 was tested in HUVEC cells. Labels 1-48 on the x-axis
correspond to the siRNA sequences numbered 1-48 in Table 11. The HUVEC
cells were transfected with the Ang-2-siRNAs using a reverse transfection
based
high-through-put (HTP) method with 10 nM of siRNA duplex. A luciferase
specific 25-mer siRNA was used as the negative control. The effect of siRNA
mediated Ang-2 knockdown was monitored by measuring the concentration of
Ang-2 protein in the medium using a human Ang-2 ELISA kit (R&D). At 48
hours post transfection, the inhibition effects of Ang-2 siRNA on Ang-2
expression
were more profound, with more than 50% of the Ang-2 siRNA candidates showing
a greater than 80% knockdown of Ang-2 expression compared to the cells
transfected with control Luc-siRNA.
[0016] Figure 4 is a bar graph depicting the cell viability of HUVEC cells
transfected with 10 nM human Ang-2 siRNA molecules at 48 hours post siRNA
transfection.
100171 The HUVEC cells were transfected with the Ang-2-siRNAs using a
reverse transfection based high-through-put (HTP) method with 10 nM of siRNA
duplex. Labels 2-48 on the x-axis correspond to the siRNA sequences numbered
2-48 in Table 11. A luciferase specific 25-mer siRNA was used as the negative
control (Luc). The cell viability of the transfected cells was measured using
a
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WST-1 assay kit (Roche). There was no significant cytotoxicity in the
transfected
HUVEC cells that associated with knockdown of Ang-2 expression.
[0018] Figure 5 is a bar graph depicting in vitro inhibition of human Ang-2 by
siRNA molecules at 2 nM in HUVEC cells at 48 hours post siRNA transfection.
[0019] Human Ang-2 gene silencing activity of human Ang-2-siRNA sequences
listed in Table 11 was further confirmed in HUVEC cells. Labels on the x-axis
correspond to the siRNA sequences numbers in Table 11. The HUVEC cells were
transfected with the Ang-2-siRNAs using a reverse transfection based high-
through-put (HTP) method with 2 nM of siRNA duplex. A control (Ctrl-) siRNA,
which has a 19-nt double-stranded region with dTdT 3'- overhangs on both
strands
and does not has a significant homologous sequence with any known human gene,
was used as the negative control. The effect of siRNA mediated Ang-2
knockdown was monitored by measuring the concentration of Ang-2 protein in the
medium using a human Ang-2 ELISA kit (R&D). At 48 hours post siRNA
transfection, most of the transfected HUVEC cells express less than 16% of Ang-
2
protein compared to mock control.
[0020] Figure 6 is a bar graph depicting the percentage of inhibition of human
Ang-2 by siRNA molecules at 2 nM in HUVEC cells at 48 hours post siRNA
transfection
[0021] The HUVEC cells were transfected with the Ang-2-siRNAs using a
reverse transfection based high-through-put (HTP) method with 2 nM of siRNA
duplex. A control (Ctrl-) siRNA was used as the negative control. The effect
of
siRNA mediated Ang-2 knockdown was monitored by measuring the concentration
of Ang-2 protein in the medium using a human Ang-2 ELISA kit (R&D). At 48
hours post transfection, a majority of the Ang-2 siRNAs demonstrated a greater
than 90% knockdown of Ang-2 expression.
[0022] Figure 7 is a bar graph depicting the cell viability of HUVEC cells
transfected with 2 nM human Ang-2 siRNA molecules at 48 hours post siRNA
transfection.
[0023] The HUVEC cells were transfected with the Ang-2-siRNAs using a
reverse transfection based high-through-put (HTP) method with 2 nM of siRNA
duplex. Labels on the x-axis correspond to the siRNA sequence numbers in Table
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11. A control (Ctrl-) siRNA, which has a 19-nt double-stranded region with
dTdT
3'- overhangs on both strands and does not has a significant homologous
sequence
with any known human gene, was used as the negative control. The cell
viability
of the transfected cells was measured using a WST-1 assay kit (Roche). There
was
no significant cytotoxicity in the transfected HUVEC cells that associated
with
knockdown of Ang-2 expression.
[0024] Figure 8 is a bar graph depicting in vitro inhibition of human Ang-2 by
siRNA molecules at 0.2 nM in HUVEC cells at 48 hours post siRNA transfection.
[0025] Human Ang-2 gene silencing activity of the human Ang-2-siRNA
sequences listed in Table 11 was further confirmed in HUVEC cells. The number
labels on the x-axis correspond to the siRNA sequence numbers in Table 11. The
HUVEC cells were transfected with the Ang-2-siRNAs using a reverse
transfection
based high-through-put (HTP) method with 0.2 nM of siRNA duplex. A control
(Ctrl-) siRNA was used as the negative control. The effect of siRNA mediated
Ang-2 knockdown was monitored by measuring the concentration of Ang-2
protein in the medium using a human Ang-2 ELISA kit (R&D). At 48 hours post
siRNA transfection, some of the transfected HUVEC cells express less than 60%
of Ang-2 protein compared to mock control. siRNA sequence numbers circled
were used for further experiments whose results are shown in Figures 9 and 10.
[0026] Figure 9A-C shows three line graphs depicting the determination of IC50
values of the selected Ang-2 siRNA in HUVEC cells at 48 hours post siRNA
transfection.
[0027] HUVEC cells were transfected with 10 dilutions of each siRNA duplex
(# 10 (Figure 9A), # 14 (Figure 9B), and #31 (Figure 9C) in Table 11). The
dilutions were 0.076 pM, 0.31 pM, 1.2 pM, 4.9 pM, 19.5 pM, 78.1 pM, 312.5 pM,
1.25 nM, 5 nM, and 20nM. The effect of siRNA mediated Ang-2 knockdown was
monitored by measuring the concentration of Ang-2 protein in the medium using
a
human Ang-2 ELISA kit (R&D). The cell viability of the transfected cells was
measured using a WST-1 assay kit (Roche) for normalization of Ang-2
concentration. The IC50 value of each siRNA duplex in HUVEC cells at 48 hours
post siRNA transfection was obtained using the GraphPad Prism program. The
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IC50 of Ang-2-25-10 was 0.363 nM, the IC50 of Ang-2-25-14 was 0.494 nM, and
the IC50 of Ang-2-25-31 was 0.398 nM.
[0028] Figure 1OA-B shows two line graphs depicting the determination of IC50
values of the selected human/mouse Ang-2 siRNA in HUVEC cells at 48 hours
post siRNA transfection.
[0029] HUVEC cells were transfected with 10 dilutions of each siRNA duplex
(#25 (Figure 10A) and #45 (Figure lOB) in Table 11). The dilutions were 0.076
pM, 0.31 pM, 1.2 pM, 4.9 pM, 19.5 pM, 78.1 pM, 312.5 pM, 1.25 nM, 5 nM, and
20nM. The effect of siRNA mediated Ang-2 knockdown was monitored by
measuring the concentration of Ang-2 protein in the medium using a human Ang-2
ELISA kit (R&D). The cell viability of the transfected cells was measured
using a
WST-1 assay kit (Roche) for normalization of Ang-2 concentration. The IC50
value of each siRNA duplex in HUVEC cells at 48 hours post siRNA transfection
was obtained using the GraphPad Prism program. The IC50 of Ang-2-25-25 was
1.634 nM, and the IC50 of Ang-2-25-45 was 0.90 nM.
Detailed Description of the Invention
[0030] The invention provides compositions and methods for treatment of
diseases with unwanted angiogenesis, often an abnormal or excessive
proliferation
and growth of blood vessels. Since angiogenesis also can be a normal
biological
process, inhibition of unwanted angiogenesis is preferably accomplished with
selectivity for a pathological tissue, which preferably requires selective
delivery of
therapeutic molecules to the pathological tissue using targeted nanoparticles.
The
present invention provides compositions and methods to control angiogenesis
through selective inhibition of the Ang-Tie biochemical pathway by nucleic
acid
molecules that induce RNA interference (RNAi), including inhibition of Ang-Tie
pathway gene expression and inhibition localized at pathological angiogenic
tissues. The present invention also provides compositions of and methods for
using a tissue-targeted nanoparticle composition comprising polymer conjugates
and further comprising nucleic acid molecules that induce RNAi.
[0031] The invention is described here in detail, but one skilled in the art
will
appreciate the full extent of the invention.
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Nucleic Acid Molecules for Ang/Tie2Pathway Gene Inhibition
[0032] The present invention provides nucleic acid molecules with a variety of
physicochemical structures for targeting and silencing genes in the Ang/Tie2
pathway by RNAi. In one embodiment, the present invention provides nucleic
acid molecules that result in a reduction in Ang-1, Ang-2, or Tie2 mRNA or
protein levels of at least 50%, 60%, 70%, 80%, 85%, 90%, 95, 96, 97, 98, 99 or
100%. This reduction may result up to 24 hours, up to 36 hours, up to 48
hours, up
to 60 hours, or up to 72 hours post administration of the nucleic acid
molecules.
The nucleic acid molecules that result in this reduction may be administered
at
l OnM siRNA, 5 nM siRNA, 2 nM, 1 nM, 0.5 nM, or 0.2 nM quantities. In one
embodiment, the nucleic acid molecules may have an IC50 for reducing Ang-2
protein levels of 0.75 nM or less, 0.5 nM or less, or 0.4 nM or less.
[0033] The nucleic acid molecules of the invention may be dsRNA or ssRNA. In
one embodiment of the invention, the nucleic acid molecules are siRNA. The
nucleic acid molecules may comprise 15-50, 15-30, 19, 20, 21, 22, 23, 24 or 25
base pairs. The nucleic acid molecules may comprise 5'- or 3'- single-stranded
overhangs. In a certain embodiment, the nucleic acid molecules are blunt-
ended.
In a preferred embodiment, the nucleic acid molecule is a double-stranded
siRNA
of 25 basepairs with blunt ends. Exemplary siRNA sequences of the invention
targeting Ang/Tie2 pathway genes are shown in Tables 2-10. (For all sequences
listed in Tables 2-10, the position is labeled such that the "A" of the ATG
codon is
considered to be position 1.) siRNAs with 25 basepair double-stranded RNA with
blunt ends were previously found to be some of the most potent inhibitors with
the
greatest duration of inhibition (WO 06/1 1 08 1 3). Additionally,
incorporation of
non-naturally occurring chemical analogues may be useful in some embodiments
of the invention. Such analogues include, but are not limited to, 2'-O-Methyl
ribose analogues of RNA, DNA, LNA and RNA chimeric oligonucleotides, and
other chemical analogues of nucleic acid oligonucleotides. In some
embodiments,
the siRNA targets both a human mRNA as well as the homologous or analogous
mRNA in other non-human mammalian species such as primates, mice or rats.
Table 2. siRNA candidates for human TEK (Tie-2) gene.
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
67 5'-GCCAUGGACUUGAUCUUGAUCAAUU-3' 40.0 1
3'-CGGUACCUGAACUAGAACUAGUUAA-5' 2
93 5'-CCUACCUCUUGUAUCUGAUGCUGAA-3' 44.0 3
3'-GGAUGGAGAACAUAGACUACGACUU-5' 4
498 5'-CCGGCAUGAAGUACCUGAUAUUCUA-3' 44.0 5
3'-GGCCGUACUUCAUGGACUAUAAGAU-5' 6
744 5'-AAGGACGUGUGAGAAGGCUUGUGAA-3' 48.0 7
3'-UUCCUGCACACUCUUCCGAACACUU-5' 8
1372 5'-CAUAACUUUGCUGUCAUCAACAUCA-3' 36.0 9
3'-GUAUUGAAACGACAGUAGUUGUAGU-5' 10
1784 5'-GCAACUUGACUUCGGUGCUACUUAA-3' 44.0 11
3'-CGUUGAACUGAAGCCACGAUGAAUU-5' 12
1975 5'-UGGACAAUAUUGGAUGGCUAUUCUA-3' 36.0 13
3'-ACCUGUUAUAACCUACCGAUAAGAU-5' 14
2609 5'-CAGGAGAACUGGAAGUUCUUUGUAA-3' 40.0 15
3'-GUCCUCUUGACCUUCAAGAAACAUU-5' 16
2655 5'-CAUCAAUCUCUUAGGAGCAUGUGAA-3' 40.0 17
3'-GUAGUUAGAGAAUCCUCGUACACUU-5' 18
3231 5'-GAAGCCUUAUGAGAGGCCAUCAUUU-3' 44.0 19
3'-CUUCGGAAUACUCUCCGGUAGUAAA-5' 20
204 5'-CCAGGAUCCGCUGGAAGUUACUCAA-3' 52.0 21
3'-GGUCCUAGGCGACCUUCAAUGAGUU-5' 22
319 5'-CGAGGAGAGGCAAUCAGGAUACGAA-3' 52.0 23
3'-GCUCCUCUCCGUUAGUCCUAUGCUU-5' 24
351 5'-GAUGCGUCAACAAGCUUCCUUCCUA-3' 48.0 25
3'-CUACGCAGUUGUUCGAAGGAAGGAU-5' 26
363 5'-AGCUUCCUUCCUACCAGCUACUUUA-3' 44.0 27
3'-UCGAAGGAAGGAUGGUCGAUGAAAU-5' 28
400 5'-GACAAGGGAGAUAACGUGAACAUAU-3' 40.0 29
3'-CUGUUCCCUCUAUUGCACUUGUAUA-5' 30
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
612 5'-CAGGCUGAUAGUCCGGAGAUGUGAA-3' 52.0 31
3'-GUCCGACUAUCAGGCCUCUACACUU-5' 32
660 5'-CAACCAUCUCUGUACUGCUGUAUG-3' 44.0 33
3'-GUUGGUAGAGACAUGACGACAUAC-5' 34
664 5'-CAUCUCUGUACUGCUUGUAUGAACA-3' 40.0 35
3'-GUAGAGACAUGACGAACAUACUUGU-5' 36
771 5'-GCACACGUUUGGCAGAACUUGUAAA-3' 44.0 37
3'-CGUGUGCAAACCGUCUUGAACAUUU-5' 38
805 5'-AGUGGACAAGAGGGAUGCAAGUCUU-3' 48.0 39
3'-UCACCUGUUCUCCCUACGUUCAGAA-5' 40
812 5'-AAGAGGGAUGCAAGUCUUAUGUGUU-3' 40.0 41
3'-UUCUCCCUACGUUCAGAAUACACAA-5' 42
893 5'-GCAAUGAAGCAUGCCACCCUGGUUU-3' 52.0 43
3'-CGUUACUUCGUACGGUGGGACCAAA-5' 44
1049 5'-CAAAGAUAGUGGAUUUGCCAGAUCA-3' 40.0 45
3'-GUUUCUAUCACCUAAACGGUCUAGU-5' 46
1053 5'-GAUAGUGGAUUUGCCAGAUCAUAUA-3' 36.0 47
3'-CUAUCACCUAAACGGUCUAGUAUAU-5' 48
1369 5'-GGACAUAACUUUGCUGUCAUCAACA-3' 40.0 49
3'-CCUGUAUUGAAACGACAGUAGUUGU-5' 50
1455 5'-CGUUAAUCACUAUGAGGCUUGGCAA-3' 44.0 51
3'-GCAAUUAGUGAUACUCCGAACCGUU-5' 52
1463 5'-ACUAUGAGGCUUGGCAACAUAUUCA-3' 40.0 53
3'-UGAUACUCCGAACCGUUGUAUAAGU-5' 54
1636 5'-CCAAGAGGUCUAAAUCUCCUGCCUA-3' 48.0 55
3'-GGUUCUCCAGAUUUAGAGGACGGAU-5' 56
1637 5'-CAAGAGGUCUAAAUCUCCUGCCUAA-3' 44.0 57
3'-GUUCUCCAGAUUUAGAGGACGGAUU-5' 58
1763 5'-AGCAGAAUAUUAAAGUUCCAGGCAA-3' 36.0 59
3'-UCGUCUUAUAAUUUCAAGGUCCGUU-5' 60
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
1781 5'-CAGGCAACUUGACUUCGGUGCUACU-3' 52.0 61
3'-GUCCGUUGAACUGAAGCCACGAUGA-5' 62
1879 5'-GAAGAUCUCACUGCUUGGACCCUUA-3' 48.0 63
3'-CUUCUAGAGUGACGAACCUGGGAAU-5' 64
1898 5'-CCCUUAGUGACAUUCUUCCUCCUCA-3' 48.0 65
3'-GGGAAUCACUGUAAGAAGGAGGAGU-5' 66
1899 5'-CCUUAGUGACAUUCUUCCUCCUCAA-3' 44.0 67
3'-GGAAUCACUGUAAGAAGGAGGAGUU-5' 68
2610 5'-AGGAGAACUGGAAGUUCUUUGUAAA-3' 36.0 69
3'-UCCUCUUGACCUUCAAGAAACAUUU-5' 70
2684 5'-GAGGCUACUUGUACCUGGCCAUUGA-3' 52.0 71
3'-CUCCGAUGAACAUGGACCGGUAACU-5' 72
2723 5'-GAAACCUUCUGGACUUCCUUCGCAA-3' 48.0 73
3'-CUUUGGAAGACCUGAAGGAAGCGUU-5' 74
3020 5'-UCGAGUCACUGAAUUACAGUGUGUA-3' 40.0 75
3'-AGCUCAGUGACUUAAUGUCACACAU-5' 76
3119 5'-GCGGGAUGACUUGUGCAGAACUCUA-3' 52.0 77
3'-CGCCCUACUGAACACGUCUUGAGAU-5' 78
3179 5'-CCCUGAACUGUGAUGAUGAGGUGUA-3' 48.0 79
3'-GGGACUUGACACUACUACUCCACAU-5' 80
3289 5'-GAGGAGCGAAAGACCUACGUGAAUA-3' 48.0 81
3'-CUCCUCGCUUUCUGGAUGCACUUAU-5' 82
72 5'-GGACUUGAUCUUGAUCAAUUCCCUA-3' 40.0 83
3'-CCUGAACUAGAACUAGUUAAGGGAU-5' 84
77 5'-UGAUCUUGAUCAAUUCCCUACCUCU-3' 40.0 85
3'-ACUAGAACUAGUUAAGGGAUGGAGA-5' 86
87 5'-CAAUUCCCUACCUCUUGUAUCUGAU-3' 40.0 87
3'-GUUAAGGGAUGGAGAACAUAGACUA-5' 88
207 5'-GGAUCCGCUGGAAGUUACUCAAGAU-3' 48.0 89
3'-CCUAGGCGACCUUCAAUGAGWCUA-5' 90
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
326 5'-AGGCAAUCAGGAUACGAACCAUGAA-3' 44.0 91
3'-UCCGUUAGUCCUAUGCUUGGUACUU-5' 92
406 5'-GGAGAUAACGUGAACAUAUCUUUCA-3' 36.0 93
3'-CCUCUAUUGCACUUGUAUAGAAAGU-5' 94
571 5'-GCCAGGUAUAUAGGAGGAAACCUCU-3' 48.0 95
3'-CGGUCCAUAUAUCCUCCUUUGGAGA-5' 96
572 5'-CCAGGUAUAUAGGAGGAAACCUCUU-3' 44.0 97
3'-GGUCCAUAUAUCCUCCUUUGGAGAA-5' 98
693 5'-UGUCUGCCAUGAAGAUACUGGAGAA-3' 44.0 99
3'-ACAGACGGUACUUCUAUGACCUCUU-5' 100
774 5'-CACGUUUGGCAGAACUUGUAAAGAA-3' 40.0 101
3'-GUGCAAACCGUCUUGAACAUUUCUU-5' 102
807 5'-UGGACAAGAGGGAUGCAAGUCUUAU-3' 44.0 103
3'-ACCUGUUCUCCCUACGUUCAGAAUA-5' 104
961 5'-GAGAUGUGUGAUCGCUUCCAAGGAU-3' 48.0 105
3'-CUCUACACACUAGCGAAGGUUCCUA-5' 106
970 5'-GAUCGCUUCCAAGGAUGUCUCUGCU-3' 52.0 107
3'-CUAGCGAAGGUUCCUACAGAGACGA-5' 108
1352 5'-CAAACGUGAUUGACACUGGACAUAA-3' 40.0 109
3'-GUUUGCACUAACUGUGACCUGUAUU-5' 110
1364 5'-ACACUGGACAUAACUUUGCUGUCAU-3' 40.0 111
3'-UGUGACCUGUAUUGAAACGACAGUA-5' 112
1385 5'-UCAUCAACAUCAGCUCUGAGCCUUA-3' 44.0 113
3'-AGUAGUUGUAGUCGAGACUCGGAAU-5' 114
1388 5'-UCAACAUCAGCUCUGAGCCUUACUU-3' 44.0 115
3'-AGUUGUAGUCGAGACUCGGAAUGAA-5' 116
1389 5'-CAACAUCAGCUCUGAGCCUUACUUU-3' 44.0 117
3'-GUUGUAGUCGAGACUCGGAAUGAAA-5' 118
1436 5'-AGAAGCUUCUAUACAAACCCGUUAA-3' 36.0 119
3'-UCUUCGAAGAUAUGUUUGGGCAAUU-5' 120
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
1437 5'-GAAGCUUCUAUACAAACCCGUUAAU-3' 36.0 121
3'-CUUCGAAGAUAUGUUUGGGCAAUUA-5' 122
1454 5'-CCGUUAAUCACUAUGAGGCUUGGCA-3' 48.0 123
3'-GGCAAUUAGUGAUACUCCGAACCGU-5' 124
1668 5'-GACCACUCUAAAUUUGACCUGGCAA-3' 44.0 125
3'-CUGGUGAGAUUUAAACUGGACCGUU-5' 126
1791 5'-GACUUCGGUGCUACUUAACAACUUA-3' 40.0 127
3'-CUGAAGCCACGAUGAAUUGUUGAAU-5' 128
1951 5'-ACACACUCCUCGGCUGUGAUUUCUU-3' 48.0 129
3'-UGUGUGAGGAGCCGACACUAAAGAA-5' 130
2050 5'-CACGUUGAUGUGAAGAUAAAGAAUG-3' 36.0 131
3'-GUGCAACUACACUUCUAUUUCUUAC-5' 132
2061 5'-GAAGAUAAAGAAUGCCACCAUCAUU-3' 36.0 133
3'-CUUCUAUUUCUUACGGUGGUAGUAA-5' 134
2141 5'-CAGAGAACAACAUAGGGUCAAGCAA-3' 44.0 135
3'-GUCUCUUGUUGUAUCCCAGUUCGUU-5' 136
2232 5'-GAAGAUGCUGCUUAUAGCCAUCCUU-3' 44.0 137
3'-CUUCUACGACGAAUAUCGGUAGGAA-5' 138
2246 5'-UAGCCAUCCUUGGCUCUGCUGGAAU-3' 52.0 139
3'-AUCGGUAGGAACCGAGACGACCUUA-5' 140
2387 5'-UCAACUCAGGGACUCUGGCCCUAAA-3' 52.0 141
3'-AGUUGAGUCCCUGAGACCGGGAUUU-5' 142
2398 5'-ACUCUGGCCCUAAACAGGAAGGUCA-3' 52.0 143
3'-UGAGACCGGGAUUUGUCCUUCCAGU-5' 144
2603 5'-ACUUUGCAGGAGAACUGGAAGUUCU-3' 44.0 145
3'-UGAAACGUCCUCUUGACCUUCAAGA-5' 146
2608 5'-GCAGGAGAACUGGAAGUUCUUUGUA-3' 44.0 147
3'-CGUCCUCUUGACCUUCAAGAAACAU-5' 148
2618 5'-UGGAAGUUCUUUGUAAACUUGGACA-3' 36.0 149
3'-ACCUUCAAGAAACAUUUGAACCUGU-5' 150
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-14-
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
2722 5'-GGAAACCUUCUGGACUUCCUUCGCA-3' 52.0 151
3'-CCUUUGGAAGACCUGAAGGAAGCGU-5' 152
2767 5'-GACCCAGCAUUUGCCAUUGCCAAUA-3' 48.0 153
3'-CUGGGUCGUAAACGGUAACGGUUAU-5' 154
2958 5'-CCGAGGUCAAGAGGUGUACGUGAAA-3' 52.0 155
3'-GGCUCCAGUUCUCCACAUGCACUUU-5' 156
3072 5'-UGGUGUGUUACUAUGGGAGAUUGUU-3' 40.0 157
3'-ACCACACAAUGAUACCCUCUAACAA-5' 158
3073 5'-GGUGUGUUACUAUGGGAGAUUGUUA-3' 40.0 159
3'-CCACACAAUGAUACCCUCUAACAAU-5' 160
3298 5'-AAGACCUACGUGAAUACCACGCUUU-3' 44.0 161
3'-UUCUGGAUGCACUUAUGGUGCGAAA-5' 162
3300 5'-GACCUACGUGAAUACCACGCUUUAU-3' 44.0 163
3'-CUGGAUGCACUUAUGGUGCGAAAUA-5' 164
3314 5'-CCACGCUUUAUGAGAAGUUUACUUA-3' 36.0 165
3'-GGUGCGAAAUACUCUUCAAAUGAAU-5' 166
Table 3. siRNA candidates for mouse Tie2 gene.
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
612 5'-CAGGCUGAUUGUUCGGAGAUGUGAA-3' 48.0 171
3'-GUCCGACUAACAAGCCUCUACACUU)-5' 172
664 5'-CGUCCUUGUACUACUUGCAAGAACA-3' 44.0 173
3'-GCAGGAACAUGAUGAACGUUCUUGU-5' 174
756 5'-GAAAGCUUGUGAGCCGCACACAUUU-3' 48.0 175
3'-CUUUCGAACACUCGGCGUGUGUAAA-5' 176
812 5'-CAGAAGGAUGCAAGUCUUAUGUGUU-3' 40.0 173
3'-GUCUUCCUACGUUCAGAAUACACAA-5' 174
1032 5'-CAGGCCAAGGAUGACUCCACAGAUA-3' 52.0 175
3'-GUCCGGUUCCUACUGAGGUGUCUAU-5' 176
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
- 15-
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
1049 5'-CACAGAUAGAGGAUUUGCCAGAUCA-3' 44.0 177
3'-GUGUCUAUCUCCUAAACGGUCUAGU-5' 178
1119 5'-UGGGUGGCCACUACCUACUAGUGAA-3' 52.0 179
3'-ACCCACCGGUGAUGGAUGAUCACUU-5' 180
1631 5'-CAAGAGGUCUCAGUCUCCUGCCAAA-3' 52.0 181
3'-GUUCUCCAGAGUCAGAGGACGGUUU-5' 182
1734 5'-GCGAUCCCUGCAAACAACAAGUGAU-3' 48.0 183
3'-CGCUAGGGACGUUUGUUGUUCACUA-5' 184
1760 5'-AGCAGAACAUCAAAGUGCCUGGGAA-3' 48.0 185
3'-UCGUCUUGUAGUUUCACGGACCCUU-5' 186
62 5'-AAGGUGCCAUGGACCUGAUCUUGAU-3' 48.0 187
3"-UUCCACGGUACCUGGACUAGAACUA-5' 188
67 5'-GCCAUGGACCUGAUCUUGAUCAAUU-3' 44.0 189
3'-CGGUACCUGGACUAGAACUAGUUAA-5' 190
93 5'-CCUACCUCUUGUGUCUGAUGCCGAA-3' 52.0 191
3'-GGAUGGAGAACACAGACUACGGCUU-5' 192
162 5'-CAUCACCAUAGGAAGGGACUUUGAA-3' 44.0 193
3'-GUAGUGGUAUCCUUCCCUGAAACUU-5' 194
204 5'-CCAAGAUCCACUGGAGGUUACUCAA-3' 48.0 195
3'-GGUUCUAGGUGACCUCCAAUGAGUU-5' 196
276 5'-GGCCAGUAAGAUUAAUGGUGCUUAU-3' 40.0 197
3'-CCGGUCAUUCUAAUUACCACGAAUA-5' 198
351 5'-GAUGCGUCAACAAGCGUCCUUCCUA-3' 52.0 199
3'-CUACGCAGUUGUUCGCAGGAAGGAU-5' 200
363 5'-AGCGUCCUUCCUACCUGCUACUUUA-3' 48.0 201
3'-UCGCAGGAAGGAUGGACGAUGAAAU-5' 202
572 5'-CCAGGUACAUAGGAGGAAACCUGUU-3' 48.0 203
3'-GGUCCAUGUAUCCUCCUUUGGACAA-5' 204
654 5'-CGACUGUAGCCGUCCUUGUACUACU-3' 52.0 205
3'-GCUGACAUCGGCAGGAACAUGAUGA-5' 206
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-16-
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
744 5'-GAGAACAUGUGAGAAAGCUUGUGAG-3' 44.0 207
3'-CUCUUGUACACUCUUUCGAACACUC-5' 208
756 5'-GAAAGCUUGUGAGCCGCACACAUUU-3' 48.0 209
3'-CUUUCGAACACUCGGCGUGUGUAAA-5' 210
770 5'-CGCACACAUUUGGCAGGACCUGUAA-3' 52.0 211
3'-GCGUGUGUAAACCGUCCUGGACAUU-5' 212
771 5'-GCACACAUUUGGCAGGACCUGUAAA-3' 48.0 213
3'-CGUGUGUAAACCGUCCUGGACAUUU-5' 214
805 5'-AGUGGACCAGAAGGAUGCAAGUCUU-3' 48.0 215
3'-UCACCUGGUCUUCCUACGUUCAGAA-5' 216
928 5'-GACUGUAAGCUCAGGUGCCACUGUA-3' 52.0 217
3'-CUGACAUUCGAGUCCACGGUGACAU-5' 218
1233 5'-CAACCGAGUCUUACCUCCUGACUCA-3' 52.0 219
3'-GUUGGAUCAGAAUGGAGGACUGAGU-5' 220
1453 5'-CCUGUCAAUCAGGCCUGGAAAUACA-3' 48.0 221
3'-GGACAGUUAGUCCGGACCUUUAUGU-5' 222
1458 5'-CAAUCAGGCCUGGAAAUACAUUGAA-3' 40.0 223
3'-GUUAGUCCGGACCUUUAUGUAACUU-5' 224
1956 5'-CACAGCUAUGGUUUCUUGGACAAUA-3' 40.0 225
3'-GUGUCGAUACCAAAGAACCUGUUAU-5' 226
2041 5'-GACCAGCACAUUGAUGUGAAGAUCA-3' 44.0 227
3'-CUGGUCGUGUAACUACACUUCUAGU-5' 228
2047 5'-CACAUUGAUGUGAAGAUCAAGAAUG-3' 36.0 229
3'-GUGUAACUACACUUCUAGUUCUUAC-5' 230
2100 5'-CCUAGAGCCAGAGACUACAUACCAU-3' 48.0 231
3'-GGAUCUCGGUCUCUGAUGUAUGGUA-5' 232
2418 5'-AAACAAUCCGGAUCCCACAAUUUAU-3' 36.0 233
3'-UUUGUUAGGCCUAGGGUGUUAAAUA-5' 234
2456 5'-GGAAUGACAUCAAGUUUCAAGACGU-3' 40.0 235
3'-CCUUACUGUAGUUCAAAGUUCUGCA-5' 236
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-17-
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
2549 5'-CCGCCAUCAAGAGGAUGAAAGAGUA-3' 48.0 237
3'-GGCGGUAGUUCUCCUACUUUCUCAU-5' 238
2559 5'-GAGGAUGAAAGAGUAUGCCUCCAAA-3' 44.0 239
3'-CUAAUACUUUCUCAUACGGAGGUUU-5' 240
2602 5'-GCAGGAGAACUGGAGGUUCUUUGUA-3' 48.0 241
3'-CGUCCUCUUGACCUCCAAGAAACAU-5' 242
2603 5'-CAGGAGAACUGGAGGUUCUUUGUAA-3' 44.0 243
3'-GUCCUCUUGACCUCCAAGAAACAUU-5' 244
2604 5'-AGGAGAACUGGAGGUUCUUUGUAAA-3' 40.0 245
3'-UCCUCUUGACCUCCAAGAAACAUUU-5' 246
2649 5'-CAUCAAUCUCUUGGGAGCAUGUGAA-3' 44.0 247
3'-GUAGUUAGAGAACCCUCGUACACUU-5' 248
2674 5'-CACCGAGGCUAUUUGUACCUAGCUA-3' 48.0 249
3'-GUGGCUCCGAUAAACAUGGAUCGAU-5' 250
2676 5'-CCGAGGCUAUUUGUACCUAGCUAUU-3' 44.0 251
3'-GGCUCCGAUAAACAUGGAUCGAUAA-5' 252
2678 5'-GAGGCUAUUUGUACCUAGCUAUUGA-3' 40.0 253
3'-CUCCGAUAAACAUGGAUCGAUAACU-5' 254
2945 5'-GAUUGUCACGAGGUCAAGAAGUGUA-3' 44.0 255
3'-CUAACAGUGCUCCAGUUCUUCACAU-5' 256
2951 5'-CACGAGGUCAAGAAGUGUAUGUGAA-3' 44.0 257
3'-GUGCUCCAGUUCUUCACAUACACUU-5' 258
2995 5'-CCAGUGCGUUGGAUGGCAAUCGAAU-3' 52.0 259
3'-GGUCACGCAACCUACCGUUAGCUUA-5' 260
3309 5'-CACACUGUAUGAGAAGUUUACCUAU-3' 36.0 261
3'-GUGUGACAUACUCUUCAAAUGGAUA-5' 262
Table 4. siRNA candidates for human/mouse TEK (Tie-2).
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
- 18-
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
77 5'-UGAUCUUGAUCAAUUCCCUACCUCU-3' 40.0 263
3'-ACUAGAACUAGUUAAGGGAUGGAGA-5' 264
161 5'-CCAUCACCAUAGGAAGGGACUUUGA-3' 48.0 265
3'-GGUAGUGGUAUCCUUCCCUGAAACU-5' 266
162 5'-CAUCACCAUAGGAAGGGACUUUGAA-3' 44.0 267
3'-GUAGUGGUAUCCUUCCCUGAAACUU-5' 268
3179 5'-CCCUGAACUGUGAUGAUGAGGUGUA-3' 48.0 269
3'-GGGACUUGACACUACUACUCCACAU-5' 270
Table 5. siRNA candidates for human ANGPT1.
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
842 5'-CAUUUAGAGACUGUGCAGAUGUAUA-3' 36.0 271
3'-GUAAAUCUCUGACACGUCUACAUAU-5' 272
978 5'-ACAACAUCGUGAAGAUGGAAGUCUA-3' 40.0 273
3'-UGUUGUAGCACUUCUACCUUCAGAU-5' 274
1003 5'-GAUUUCCAAAGAGGCUGGAAGGAAU-3' 44.0 275
3'-CUAAAGGUUUCUCCGACCUUCCUUA-5' 276
1116 5'-AAGAAUUGAGUUAAUGGACUGGGAA-3' 36.0 277
3'-UUCUUAACUCAAUUACCUGACCCUU-5' 278
1245 5'-CAGCCUGAUCUUACACGGUGCUGAU-3' 52.0 279
3'-GUCGGACUAGAAUGUGCCACGACUA-5' 280
1357 5'-CCCUCCAAUCUAAAUGGAAUGUUCU-3' 40.0 281
3'-GGGAGGUUAGAUUUACCUUACAAGA-5' 282
1358 5'-CCUCCAAUCUAAAUGGAAUGUUCUA-3 36.0 283
3'-GGAGGUUAGAUUUACCUUACAAGAU-5' 284
1443 5'-CAGUUACUCCUUACGUUCCACAACU-3' 44.0 285
3'-GUCAAUGAGGAAUGCAAGGUGUUGA-5' 286
1460 5'-CCACAACUAUGAUGAUUCGACCUUU-3' 40.0 287
3'-GGUGUUGAUACUACUAAGCUGGAAA-5' 288
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-19-
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
1461 5'-CACAACUAUGAUGAUUCGACCUUUA-3' 36.0 289
3'-GUGUUGAUACUACUAAGCUGGAAAU-5' 290
89 5'-GGAGAAGAUAUAACCGGAUUCAACA-3' 40.0 291
3'-CCUCUUCUAUAUUGGCCUAAGUUGU-5' 292
109 5'-CAACAUGGGCAAUGUGCCUACACUU-3' 48.0 293
3'-GUUGUACCCGUUACACGGAUGUGAA-5' 294
112 5'-CAUGGGCAAUGUGCCUACACUUUCA-3' 48.0 295
3'-GUACCCGUUACACGGAUGUGAAAGU-5' 296
125 5'-CCUACACUUUCAUUCUUCCAGAACA-3' 40.0 297
3'-GGAUGUGAAAGUAAGAAGGUCUUGU-5' 298
346 5'-CAGCAGAAUGCAGUUCAGAACCACA-3' 48.0 299
3'-GUCGUCUUACGUCAAGUCUUGGUGU-5' 300
654 5'-CCUUCAAGGCUUGGUUACUCGUCAA-3' 48.0 301
3'-GGAAGUUCCGAACCAAUGAGCAGUU-5' 302
1159 5'-CAGUAUGACAGAUUCCACAUAGGAA-3' 40.0 303
3'-GUCAUACUGUCUAAGGUGUAUCCUU-5' 304
1328 5'-CAGGAGGAUGGUGGUUUGAUGCUUG-3' 52.0 305
3'-GUCCUCCUACCACCAAACUACGAAC-5' 306
95 5'-GAUAUAACCGGAUUCAACAUGGGCA-3' 44.0 307
3'-CUAUAUUGGCCUAAGUUGUACCCGU-5' 308
108 5'-UCAACAUGGGCAAUGUGCCUACACU-3' 48.0 309
3'-AGUUGUACCCGUUACACGGAUGUGA-5' 310
437 5'-CAGAUGUUGAGACCCAGGUACUAAA-3' 44.0 311
3'-GUCUACAACUCUGGGUCCAUGAUUU-5' 312
1168 5'-GACAGAUUCCACAUAGGAAAUGAAA-3' 36.0 313
3'-CUGUCUAAGGUGUAUCCUUUACUUU-5' 314
1412 5'-UGAAUGGGAUAAAGUGGCACUACUU-3' 40.0 315
3'-ACUUACCCUAUUUCACCGUGAUGAA-5' 316
1427 5'-GGCACUACUUCAAAGGGCCCAGUUA-3' 52.0 317
3'-CCGUGAUGAAGUUUCCCGGGUCAAU-5' 318
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-20-
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
163 5'-CGUGAGAGUACGACAGACCAGUACA-3' 52.0 319
3'-GCACUCUCAUGCUGUCUGGUCAUGU-5' 320
166 5'-GAGAGUACGACAGACCAGUACAACA-3' 48.0 321
3'-CUCUCAUGCUGUCUGGUCAUGUUGU-5' 322
176 5'-CAGACCAGUACAACACAAACGCUCU-3' 48.0 323
3'-GUCUGGUCAUGUUGUGUUUGCGAGA-5' 324
213 5'-UCCACACGUGGAACCGGAUUUCUCU-3' 52.0 325
3'-AGGUGUGCACCUUGGCCUAAAGAGA-5' 326
214 5'-CCACACGUGGAACCGGAUUUCUCUU-3' 52.0 327
3'-GGUGUGCACCUUGGCCUAAAGAGAA-5' 328
250 5'-CAACAUCUGGAACAUGUGAUGGAAA-3' 40.0 329
3'-GUUGUAGACCUUGUACACUACCUUU-5' 330
336 5'-GGCCCAGAUACAGCAGAAUGCAGUU-3' 52.0 331
3'-CCGGGUCUAUGUCGUCUUACGUCAA-5' 332
339 5'-CCAGAUACAGCAGAAUGCAGUUCAG-3' 48.0 333
3'-GGUCUAUGUCGUCUUACGUCAAGUC-5' 334
341 5'-AGAUACAGCAGAAUGCAGUUCAGAA-3' 40.0 335
3'-UCUAUGUCGUCUUACGUCAAGUCUU-5' 336
351 5'-GAAUGCAGUUCAGAACCACACGGCU-3' 52.0 337
3'-CUUACGUCAAGUCUUGGUGUGCCGA-5' 338
453 5'-GGUACUAAAUCAAACUUCUCGACUU-3' 36.0 339
3'-CCAUGAUUUAGUUUGAAGAGCUGAA-5' 340
473 5'-GACUUGAGAUACAGCUGCUGGAGAA-3' 48.0 341
3'-CUGAACUCUAUGUCGACGACCUCUU-5' 342
651 5'-GAACCUUCAAGGCUUGGUUACUCGU-3' 48.0 343
3'-CUUGGAAGUUCCGAACCAAUGAGCA-5' 344
653 5'-ACCUUCAAGGCUUGGUUACUCGUCA-3' 48.0 345
3'-UGGAAGUUCCGAACCAAUGAGCAGU-5' 346
658 5'-CAAGGCUUGGUUACUCGUCAAACAU-3' 44.0 347
3'-GUUCCGAACCAAUGAGCAGUUUGUA-5' 348
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-21 -
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
660 5'-AGGCUUGGUUACUCGUCAAACAUAU-3' 40.0 349
3'-UCCGAACCAAUGAGCAGUUUGUAUA-5' 350
662 5'-GCUUGGUUACUCGUCAAACAUAUAU-3' 36.0 351
3'-CGAACCAAUGAGCAGUUUGUAUAUA-5' 352
764 5'-UGGACACAGUCCACAACCUUGUCAA-3' 48.0 353
3'-ACCUGUGUCAGGUGUUGGAACAGUU-5' 354
768 5'-CACAGUCCACAACCUUGUCAAUCUU-3' 44.0 355
3'-GUGUCAGGUGUUGGAACAGUUAGAA-5' 356
770 5'-CAGUCCACAACCUUGUCAAUCUUUG-3' 44.0 357
3'-GUCAGGUGUUGGAACAGUUAGAAAC-5' 358
774 5'-CCACAACCUUGUCAAUCUUUGCACU-3' 44.0 359
3'-GGUGUUGGAACAGUUAGAAACGUGA-5' 360
832 5'-GAAGAGAAACCAUUUAGAGACUGUG-3' 40.0 361
3'-CUUCUCUUUGGUAAAUCUCUGACAC-5' 362
840 5'-ACCAUUUAGAGACUGUGCAGAUGUA-3' 40.0 363
3'-UGGUAAAUCUCUGACACGUCUACAU-5' 364
846 5'-UAGAGACUGUGCAGAUGUAUAUCAA-3' 36.0 365
3'-AUCUCUGACACGUCUACAUAUAGUU-5' 366
991 5'-GAUGGAAGUCUAGAUUUCCAAAGAG-3' 40.0 367
3'-CUACCUUCAGAUCUAAAGGUUUCUC-5' 368
1098 5'-UCAGAGGCAGUACAUGCUAAGAAUU-3' 40.0 369
3'-AGUCUCCGUCAUGUACGAUUCUUAA-5' 370
1147 5'-CGAGCCUAUUCACAGUAUGACAGAU-3' 44.0 371
3'-GCUCGGAUAAGUGUCAUACUGUCUA-5' 372
1164 5'-UGACAGAUUCCACAUAGGAAAUGAA-3' 36.0 373
3'-ACUGUCUAAGGUGUAUCCUUUACUU-5' 374
1257 5'-ACACGGUGCUGAUUUCAGCACUAAA-3' 44.0 375
3'-UGUGCCACGACUAAAGUCGUGAUUU-5' 376
1258 5'-CACGGUGCUGAUUUCAGCACUAAAG-3' 48.0 377
3'-GUGCCACGACUAAAGUCGUGAUUUC-5' 378
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
- 22 -
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
-1260 5'-CGGUGCUGAUUUCAGCACUAAAGAU-3' 44.0 379
3'-GCCACGACUAAAGUCGUGAUUUCUA-5' 380
1282 5'-GAUGCUGAUAAUGACAACUGUAUGU-3' 36.0 381
3'-CUACGACUAUUACUGUUGACAUACA-5' 382
1285 5'-GCUGAUAAUGACAACUGUAUGUGCA-3' 40.0 383
3'-CGACUAUUACUGUUGACAUACACGU-5' 384
1371 5'-UGGAAUGUUCUAUACUGCGGGACAA-3' 44.0 385
3'-ACCUUACAAGAUAUGACGCCCUGUU-5' 386
1409 5'-UGAAUGGGAUAAAGUGGCACUACUU-3' 40.0 387
3'-ACUUACCCUAUUUCACCGUGAUGAA-5' 388
Table 6. siRNA candidates for mouse ANGPT1.
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
706 5'-CAACUUAGUAGAGCUACCAACAACA-3' 40.0 389
3'-GUUGAAUCAUCUCGAUGGUUGUUGU-5' 390
845 5'-CAUUUCGAGACUGUGCAGAUGUAUA-3' 40.0 391
3'-GUAAAGCUCUGACACGUCUACAUAU-5' 392
989 5'-GGGAAGAUGGAAGCCUGGAUUUCCA-3' 52.0 393
3'-CCCUUCUACCUUCGGACCUAAAGGU-5' 394
1052 5'-CCUCUGGUGAAUAUUGGCUCGGGAA-3' 52.0 395
3'-GGAGACCACUUAUAACCGAGCCCUU-5' 396
1119 5'-GAGGAUUGAGCUGAUGGACUGGGAA-3' 52.0 397
3'-CUCCUAACUCGACUACCUGACCCUU-5' 398
1167 5'-CGACAGAUUCCACAUAGGAAAUGAA-3' 40.0 399
3'-GCUGUCUAAGGUGUAUCCUUUACUU-5' 400
1238 5'-GCAAACAGAGCAGCUUGAUCUUACA-3' 44.0 401
3'-CGUUUGUCUCGUCGAACUAGAAUGU-5' 402
1248 5'-CAGCUUGAUCUUACACGGUGCUGAU-3' 48.0 403
3'-GUCGAACUAGAAUGUGCCACGACUA-5' 404
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
- 23 -
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
1360 5'-CCUUCCAAUCUAAAUGGAAUGUUCU-3' 36.0 405
3'-GGAAGGUUAGAUUUACCUUACAAGA-5' 406
1427 5'-GGCACUACUUCAAAGGGCCCAGUUA-3' 52.0 407
3'-CCGUCAUGAAGUUUCCCGGGUCAAU-5' 408
109 5'-CAACAUGGGCAAUGUGCCUACACUU-3' 48.0 409
3'-GUUGUACCCGUUACACGGAUGUGAA-5' 410
112 5'-CAUGGGCAAUGUGCCUACACUUUCA-3' 48.0 411
3'-GUACCCGUUACACGGAUGUGAAAGU-5' 412
125 5'-CCUACACUUUCAUUCUUCCAGAACA-3' 40.0 413
3'-GGAUGUGAAAGUAAGAAGGUCUUGU-5' 414
339 5'-CCAGAUACAACAGAAUGCUGUUCAA-3' 40.0 415
3'-GGUCUAUGUUGUCUUACGACAAGUU-5' 416
437 5'-CAGAUGUUGAGACCCAGGUACUAAA-3' 44.0 417
3'-GUCUACAACUCUGGGUCCAUGAUUU-5' 418
453 5'-GGUACUAAAUCAAACAUCCCGACUU-3' 40.0 416
3'-CCAUGAUUUAGUUUGUAGGGCUGAA-5' 420
467 5'-CAUCCCGACUUGAAAUACAACUGCU-3' 44.0 421
3'-GUAGGGCUGAACUUUAUGUUGACGA-5' 422
473 5'-GACUUGAAAUACAACUGCUAGAGAA-3' 36.0 423
3'-CUGAACUUUAUGUUGACGAUCUCUU-5' 424
509 5'-CAUACAAGCUAGAGAAGCAACUUCU-3' 40.0 425
3'-GUAUGUUCGAUCUCUUCGUUGAAGA-5' 426
525 5'-GCAACUUCUCCAACAGACAAAUGAA-3' 40.0 427
3'-CGUUGAAGAGGUUGUCUGUUUACUU-5' 428
755 5'-UGGAGCUCAUGGACACAGUUCAUAA-3' 44.0 429
3'-ACCUCGAGUACCUGUGUCAAGUAUU-5' 430
1162 5'-CAGUACGACAGAUUCCACAUAGGAA-3' 44.0 431
3'-GUCAUGCUGUCUAAGGUGUAUCCUU-5' 432
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-24-
Table 7. siRNA candidates for human/mouse ANGPT1.
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
109 5'-CAACAUGGGCAAUGUGCCUACACUU-3' 48.0 433
3'-GUUGUACCCGUUACACGGAUGUGAA-5' 434
112 5'-CAUGGGCAAUGUGCCUACACUUUCA-3' 48.0 435
3'-GUACCCGUUACACGGAUGUGAAAGU-5' 436
125 5'-CCUACACUUUCAUUCUUCCAGAACA-3' 40.0 437
3'-GGAUGUGAAAGUAAGAAGGUCUUGU-5' 438
89 5'-GGAGAAGAUAUAACCGGAUUCAACA-3' 40.0 439
3'-CCUCUUCUAUAUUGGCCUAAGUUGU-5' 440
95 5'-GAUAUAACCGGAUUCAACAUGGGCA-3' 44.0 441
3'-CUAUAUUGGCCUAAGUUGUACCCGU-5' 442
108 5'-UCAACAUGGGCAAUGUGCCUACACU-3' 48.0 443
3'-AGUUGUACCCGUUACACGGAUGUGA-5' 444
437 5'-CAGAUGUUGAGACCCAGGUACUAAA-3' 44.0 445
3'-GUCUACAACUCUGGGUCCAUGAUUU-5' 446
1168 5'-GACAGAUUCCACAUAGGAAAUGAAA-3' 36.0 447
3'-CUGUCUAAGGUGUAUCCUUUACUUU-5' 448
1409 5'-UGAAUGGGAUAAAGUGGCACUACUU-3' 40.0 449
3'-ACUUACCCUAUUUCACCGUGAUGAA-5' 450
1412 5'-UGAAUGGGAUAAAGUGGCACUACUU-3' 40.0 451
3'-ACUUACCCUAUUUCACCGUGAUGAA-5' 452
1427 5'-GGCACUACUUCAAAGGGCCCAGUUA-3' 52.0 453
3'-CCGUGAUGAAGUUUCCCGGGUCAAU-5' 454
Table 8. siRNA candidates for human ANGPT2.
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
812 5'-CCACUGUUGCUAAAGAAGAACAAAU-3' 36.0 455
3'-GGUGACAACGAUUUCUUCUUGUUUA-5' 456
837 5'-CAGCUUCAGAGACUGUGCUGAAGUA-3' 48.0 457
3'-GUCGAAGUCUCUGACACGACUUCAU-5' 458
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
871 5'-GGACACACCACAAAUGGCAUCUACA-3' 48.0 459
3'-CCUGUGUGGUGUUUACCGUAGAUGU-5' 460
888 5'-CAUCUACACGUUAACAUUCCCUAAU-3' 36.0 461
3'-GUAGAUGUGCAAUUGUAAGGGAUUA-5' 462
951 5'-UGGAGGAGGCGGGUGGACAAUUAUU-3' 52.0 463
3'-ACCUCCUCCGCCCACCUGUUAAUAA-5' 464
962 5'-GGUGGACAAUUAUUCAGCGACGUGA-3' 48.0 465
3'-CCACCUGUUAAUAAGUCGCUGCACU-5' 466
1082 5'-CGCAACUGACUAAUCAGCAACGCUA-3' 48.0 467
3'-GCGUUGACUGAUUAGUCGUUGCGAU-5' 468
1242 5'-CAGCAUCAGCCAACCAGGAAAUGAU-3' 48.0 469
3'-GUCGUAGUCGGUUGGUCCUUUACUA-5' 470
1354 5'-CCUUCCAACUUGAACGGAAUGUACU-3' 44.0 471
3'-GGAAGGUUGAACUUGCCUUACAUGA-5' 472
1390 5'-CAGAACACAAAUAAGUUCAACGGCA-3' 40.0 473
3'-GUCUUGUGUUUAUUCAAGUUGCCGU-5' 474
34 5'-GAUCUUGUCUUGGCCGCAGCCUAUA-3' 52.0 475
3'-CUAGAACAGAACCGGCGUCGGAUAU-5' 476
47 5'-CCGCAGCCUAUAACAACUUUCGGAA-3' 48.0 477
3'-GGCGUCGGAUAUUGUUGAAAGCCUU-5' 478
241 5'-CAAGUGCUGGAGAACAUCAUGGAAA-3' 44.0 479
3'-GUUCACGACCUCUUGUAGUACCUUU-5' 480
306 5'-GGACAACAUGAAGAAAGAAAUGGUA-3' 36.0 481
3'-CCUGUUGUACUUCUUUCUUUACCAU-5' 482
390 5'-CCUGUUGAACCAAACAGCUGAGCAA-3' 48.0 483
3'-GGACAACUUGGUUUGUCGACUCGUU-5' 484
425 5'-UAACUGAUGUGGAAGCCCAAGUAUU-3' 40.0 485
3'-AUUGACUACACCUUCGGGUUCAUAA-5' 486
458 5'-CCACGAGACUUGAACUUCAGCUCUU-3' 48.0 487
3'-GGUGCUCUGAACUUGAAGUCGAGAA-5' 488
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
877 5'-ACCACAAAUGGCAUCUACACGUUAA-3' 40.0 489
3'-UGGUGUUUACCGUAGAUGUGCAAUU-5' 490
894 5'-CACGUUAACAUUCCCUAAUUCUACA-3' 36.0 491
3'-GUGCAAUUGUAAGGGAUUAAGAUGU-5' 492
1032 5'-GGGAUUUGGUAACCCUUCAGGAGAA-3' 48.0 493
3'-CCCUAAACCAUUGGGAAGUCCUCUU-5' 494
1342 5'-GAUGCAUGUGGUCCUUCCAACUUGA-3' 48.0 495
3'-CUACGUACACCAGGAAGGUUGAACU-5' 496
1410 5'-CGGCAUUAAAUGGUACUACUGGAAA-3' 40.0 497
3'-GCCGUAAUUUACCAUGAUGACCUUU-5' 498
-59 5'-UCUGGACGUGUGUUUGCCCUCAAGU-3' 52.0 499
3'-AGACCUGCACACAAACGGGAGUUCA-5' 500
-57 5'-UGGACGUGUGUUUGCCCUCAAGUUU-3' 48.0 501
3'-ACCUGCACACAAACGGGAGUUCAAA-5' 502
-56 5'-GGACGUGUGUUUGCCCUCAAGUUUG-3' 52.0 503
3'-CCUGUAUAUAAACGGGAGUUCAAAC-5' 504
-13 5'-ACUGAAGAAAGAAUGUGGCAGAUUG-3' 40.0 505
3'-UGACUUCUUUCUUACACCGUCUAAC-5' 506
-10 5'-GAAGAAAGAAUGUGGCAGAUUGUUU-3' 36.0 507
3'-CUUCUUUCUUACACCGUCUAACAAA-5' 508
33 5'-UGAUCUUGUCUUGGCCGCAGCCUAU-3' 52.0 509
3'-ACUAGAACAGAACCGGCGUCGGAUA-5' 510
46 5'-GCCGCAGCCUAUAACAACUUUCGGA-3' 52.0 511
3'-CGGCGUCGGAUAUUGUUGAAAGCCU-5' 512
53 5'-CCUAUAACAACUUUCGGAAGAGCAU-3' 40.0 513
3'-GGAUAUUGUUGAAAGCCUUCUCGUA-5' 514
274 5'-CAGUGGCUAAUGAAGCUUGAGAAUU-3' 40.0 515
3'-GUCACCGAUUACUUCGAACUCUUAA-5' 516
275 5'-AGUGGCUAAUGAAGCUUGAGAAUUA-3' 36.0 517
3'-UCACCGAUUACUUCGAACUCUUAAU-5' 518
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
355 5'-AACCAGACGGCUGUGAUGAUAGAAA-3' 44.0 519
3'-UUGGUCUGCCGACACUACUAUCUUU-5' 520
357 5'-CCAGACGGCUGUGAUGAUAGAAAUA-3' 44.0 521
3'-GGUCUGCCGACACUACUAUCUUUAU-5' 522
403 5'-ACAGCUGAGCAAACGCGGAAGUUAA-3' 48.0 523
3'-UGUCGACUCGUUUGCGCCUUCAAUU-5' 524
414 5'-AACGCGGAAGUUAACUGAUGUGGAA-3' 44.0 525
3'-UUGCGCCUUCAAUUGACUACACCUU-5' 526
419 5'-GGAAGUUAACUGAUGUGGAAGCCCA-3' 48.0 527
3'-CCUUCAAUUGACUACACCUUCGGGU-5' 528
420 5'-GAAGUUAACUGAUGUGGAAGCCCAA-3' 44.0 529
3'-CUUCAAUUGACUACACCUUCGGGUU-5' 530
427 5'-ACUGAUGUGGAAGCCCAAGUAUUAA-3' 40.0 531
3'-UGACUACACCUUCGGGUUCAUAAUU-5' 532
444 5'-AGUAUUAAAUCAGACCACGAGACUU-3' 36.0 533
3'-UCAUAAUUUAGUCUGGUGCUCUGAA-5' 534
483 5'-GGAACACUCCCUCUCGACAAACAAA-3' 48.0 535
3'-CCUUGUGAGGGAGAGCUGUUUGUUU-5' 536
524 5'-UGGACCAGACCAGUGAAAUAAACAA-3' 40.0 537
3'-ACCUGGUCUGGUCACUUUAUUUGUU-5' 538
811 5'-CCCACUGUUGCUAAAGAAGAACAAA-3' 40.0 539
3'-GGGUGACAACGAUUUCUUCUUGUUU-5' 540
820 5'-GCUAAAGAAGAACAAAUCAGCUUCA-3' 36.0 541
3'-CGAUUUCUUCUUGUUUAGUCGAAGU-5' 542
876 5'-CACCACAAAUGGCAUCUACACGUUA-3' 44.0 543
3'-GUGGUGUUUACCGUAGAUGUGCAAU-5' 544
881 5'-CAAAUGGCAUCUACACGUUAACAUU-3' 36.0 545
3'-GUUUACCGUAGAUGUGCAAUUGUAA-5' 546
924 5'-GAUCAAGGCCUACUGUGACAUGGAA-3' 48.0 547
3'-CUAGUUCCGGAUGACACUGUACCUU-5' 548
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
953 5'-GAGGAGGCGGGUGGACAAUUAUUCA-3' 52.0 549
3'-CUCCUCCGCCCACCUGUUAAUAAGU-5' 550
980 5'-GACGUGAGGAUGGCAGCGUUGAUUU-3' 52.0 551
3'-CUGCACUCCUACCGUCGCAACUAAA-5' 552
1066 5'-GGAAAUGAGUUUGUUUCGCAACUGA-3' 40.0 553
3'-CCUUUACUCAAACAAAGCGUUGACU-5' 554
1067 5'-GAAAUGAGUUUGUUUCGCAACUGAC-3' 40.0 555
3'-CUUUACUCAAACAAAGCGUUGACUG-5' 556
1140 5'-GAAUGAGGCUUACUCAUUGUAUGAA-3' 36.0 557
3'-CUUACUCCGAAUGAGUAACAUACUU-5' 558
1144 5'-GAGGCUUACUCAUUGUAUGAACAUU-3' 36.0 559
3'-CUCCGAAUGAGUAACAUACUUGUAA-5' 560
1273 5'-ACAAAGGAUGGAGACAACGACAAAU-3' 40.0 561
3'-UGUUUCCUACCUCUGUUGCUGUUUA-5' 562
1277 5'-AGGAUGGAGACAACGACAAAUGUAU-3' 40.0 563
3'-UCCUACCUCUGUUGCUGUUUACAUA-5' 564
1283 5'-GAGACAACGACAAAUGUAUUUGCAA-3' 36.0 565
3'-CUCUGUUGCUGUUUACAUAAACGUU-5' 566
1359 5'-CAACUUGAACGGAAUGUACUAUCCA-3' 40.0 567
3'-GUUGAACUUGCCUUACAUGAUAGGU-5' 568
1392 5'-GAACACAAAUAAGUUCAACGGCAUU-3' 36.0 589
3'-CUUGUGUUUAUUCAAGUUGCCGUAA-5' 590
1421 5'-GGUACUACUGGAAAGGCUCAGGCUA-3' 52.0 591
3'-CCAUGAUGACCUUUCCGAGUCCGAU-5' 592
1423 5'-UACUACUGGAAAGGCUCAGGCUAUU-3' 44.0 593
3'-AUGAUGACCUUUCCGAGUCCGAUAA-5' 594
1429 5'-UGGAAAGGCUCAGGCUAUUCGCUCA-3' 52.0 595
3'-ACCUUUCCGAGUCCGAUAAGCGAGU-5' 596
1458 5'-CACAACCAUGAUGAUCCGACCAGCA-3' 52.0 597
3'-GUGUUGGUACUACUAGGCUGGUCGU-5' 598
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
1533 5'-AAGACUUAAGCCCAGUGCACUGAAA-3' 44.0 599
3'-UUCUGAAUUCGGGUCACGUGACUUU-5' 600
1620 5'-CCACAUGCUCCAGAUUAGAGCCUGU-3' 52.0 601
3'-GGUGUACGAGGUCUAAUCUCGGACA-5' 602
1621 5'-CACAUGCUCCAGAUUAGAGCCUGUA-3' 48.0 603
3'-GUGUACGAGGUCUAAUCUCGGACAU-5' 604
1623 5'-CAUGCUCCAGAUUAGAGCCUGUAAA-3' 44.0 605
3'-GUACGAGGUCUAAUCUCGGACAUUU-5' 606
1628 5'-UCCAGAUUAGAGCCUGUAAACUUUA-3' 36.0 607
3'-AGGUCUAAUCUCGGACAUUUGAAAU-5' 608
Table 9. siRNA candidates for mouse ANGPT2.
Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
474 5'-GCAGCUUCUCCAACAUUCUAUUUCU-3' 40.0 609
3'-CGUCGAAGAGGUUGUAAGAUAAAGA-5' 610
713 5'-CGGUCAACAACUCGCUCCUUCAGAA-3' 52.0 611
3'-GCCAGUUGUUGAGCGAGGAAGUCUU-5' 612
761 5'-CCGUCAACAGCUUGCUGACCAUGAU-3' 52.0 613
3'- GGCAGUUGUCGAACGACUGGUACUA-5' 614
983 5'-GAGAAGAUGGCAGUGUGGACUUCCA-3' 52.0 615
3'-CUCUUCUACCGUCACACCUGAAGGU-5' 616
1066 5'-GGCAAUGAGUUUGUCUCCCAGCUGA-3' 52.0 617
3'-CCGUUACUCAAACAGAGGGUCGACU-5' 618
1103 5'-GCUACGUGCUUAAGAUCCAGCUGAA-3' 48.0 619
3'-CGAUGCACGAAUUCUAGGUCGACUU-5' 620
1148 3'-GCGUAAGCGACAUACUAGUGAAGAU-5' 44.0 621
5'-CGCAUUCGCUGUAUGAUCACUUCUA-3' 622
1242 5'-UAGCAUCAGCCAACCAGGAAGUGAU-3' 48.0 623
3'-AUCGUAGUCGGUUGGUCCUUCACUA-5' 624
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Start siRNA Sequence GC% SEQ ID
(sense strand/anti-sense strand) NO:
1288 5'-AAUGACAAAUGCAUCUGCAAGUGUU-3' 36.0 625
3'-UUACUGUUUACGUAGACGUUCACAA-5' 626
1354 5'-CCUUCCAACUUGAAUGGACAGUACU-3' 44.0 627
3'-GGAAGGUUGAACUUACCUGUCAUGA-5' 628
475 5'-CAGCUUCUCCAACAUUCUAUUUCUA-3' 36.0 629
31-GUCGAAGAGGUUGUAAGAUAAAGAU-5' 630
742 5'-CAGCAUGACCUAAUGGAGACCGUCA-3' 52.0 631
3'-GUCGUACUGGAUUACCUCUGGCAGU-5' 632
801 5'-CAAGAGCUCGGUUGCUAUCCGUAAA-3' 48.0 633
3'-GUUCUCGAGCCAACGAUAGGCAUUU-5' 634
1342 5'-GACGCAUGUGGUCCUUCCAACUUGA-3' 52.0 635
3'-CUGCGUACACCAGGAAGGUUGAACU-5' 636
Table 10. siRNA candidates for human/mouse ANGPT-2.
Start siRNA Sequence GC% SEQ ID
(sense strand/antisense strand) NO:
922 5'-GAGAUCAAGGCCUACUGUGACAUGG-3' 52.0 637
3'-CUCUAGUUCCGGAUGACACUGUACC-5' 638
923 5'-AGAUCAAGGCCUACUGUGACAUGGA-3' 48.0 639
3'-UCUAGUUCCGGAUGACACUGUACCU-5' 640
1447 5'-UCGCUCAAGGCCACAACCAUGAUGA-3' 52.0 641
3'-AGCGAGUUCCGGUGUUGGUACUACU-5' 642
1448 5'-CGCUCAAGGCCACAACCAUGAUGAU-3' 52.0 643
3'-GCGAGUUCCGGUGUUGGUACUACUA-5' 644
1449 5'-GCUCAAGGCCACAACCAUGAUGAUC-3' 52.0 645
3'-CGAGUUCCGGUGUUGGUACUACUAG-5' 646
1450 5'-CUCAAGGCCACAACCAUGAUGAUCC-3' 52.0 647
3'-GAGUUCCGGUGUUGGUACUACUAGG-5' 648
[0034] The present invention provides methods for inhibition of individual or
combinations of genes active in the Ang-Tie pathway. In some embodiments, the
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present invention provides a method of inhibiting or reducing angiogenesis in
a
tissue associated with undesired angiogenesis comprising administering to the
tissue siRNA molecules that target Tie2 so that expression of Tie2 is
decreased. In
some embodiments, the present invention provides a method of inhibiting or
reducing angiogenesis in a tissue associated with undesired angiogenesis
comprising administering to the tissue siRNA molecules that target Ang-1 so
that
expression of Ang-1 is decreased. In further embodiments, the invention
provides
a method of inhibiting or reducing angiogenesis in a tissue associated with
undesired angiogenesis comprising administering to the tissue siRNA molecules
that target Ang-2 so that expression of Ang-2 is decreased. In one embodiment,
the tissue is a tumor.
Combined Ang/Tie2 Pathway Gene Inhibition
[0035] The compositions and methods of the present invention for inhibition of
angiogenesis are based on several fundamental aspects. First, pathological
angiogenesis is a complex proces and results from interactions of multiple
proteins
which are abnormally expressed or over-expressed in diseased tissues. Second,
nucleic acid agents that activate RNAi are highly selective in a sequence
specific
manner. Third, inhibition of angiogenesis by modulation of protein activity
can be
operative by many methods, including but not limited to an inhibition of
protein
function (antagonists), stimulation of protein function (agonists), reduction
of
protein expression levels, and post transcriptional modification of proteins.
Importantly, it may be desirable in the treatment of disease to effectively
shut
down a particular biological pathway that is critical for disease progression,
by
simultaneously blocking functions of ligands and their receptors,
simultaneously
blocking receptor activity and the activity of down stream signaling proteins,
and/or simultaneously blocking redundant elements of a pathway. Such methods
may be used for treating angiogenesis-related diseases including those that
involve
the Ang/Tie2 pathway.
[0036] Although clinical studies have demonstrated remarkable therapeutic
efficacies, the toxicities of higher dosage and long term safety are major
concerns,
due to the different origins, different manufacturing processes and different
chemistry properties of the components.
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[0037] To overcome these problems, aspects of the present invention provide
compositions of and methods of using nucleic acid molecules, including siRNA
oligonucleotides, to provide a unique advantage, i.e., to achieve
combinatorial
effects with a combination of nucleic acid molecules, including siRNAs, that
target
multiple disease causing genes or target different sequences in the same gene
in the
same treatment. One advantage of the compositions and methods of the present
invention is that all siRNA oligonucleotides are very similar chemically,
pharmacologically, and can be produced from the same source and using the same
manufacturing process. Another advantage provided by the present invention is
that multiple siRNA oligonucleotides can be formulated in a single preparation
such as a nanoparticle preparation.
[0038] Therefore, an aspect of the present invention is to combine nucleic
acid
molecules, including siRNAs, so as to achieve specific and selective silencing
of
multiple genes in the Ang/Tie2 pathway and as a result achieve an inhibition
of
angiogenesis-related disease and a better clinical benefit. The present
invention
provides for combinations of siRNA targets including combinations of two or
more
targets selected from: Tie2, Ang-1 and Ang-2. The present invention also
provides
for combinations of siRNAs targeting one or more sequences within the same
gene
in the Ang/Tie2 pathway. Exemplary siRNA sequences silencing these mRNAs
are listed in Tables 2-10. Such siRNA compositions may also be combined with
siRNA that targets other angiogenic pathways such as the VEGF pathway, PDGF
and EGF and their receptors, downstream signaling factors including RAF and
AKT, and transcription factors including NFxB. Such siRNA compositions may
also be combined with siRNA that target genes downstream of Tie2, Ang-1 and
Ang-2.
[0039] In one embodiment a combination of siRNA inhibiting Tie2 and two of
its ligands Ang-1 and Ang-2 is used. In some embodiments, a combination of
siRNA molecules that target Tie2 and siRNA molecules that target Ang-1 is used
so that expression of both Tie2 and Ang-1 is decreased. In some embodiments, a
combination of siRNA molecules that target Tie2 and siRNA molecules that
target
Ang-2 is used so that expression of both Tie2 and Ang-2 is decreased. In some
embodiments, a combination of siRNA molecules that target Ang-1 and siRNA
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molecules that target Ang-2 is used so that expression of both Ang-1 and Ang-2
is
decreased.
[0040] In some embodiments, the present invention provides a method of
inhibiting or reducing angiogenesis in a tissue associated with undesired
angiogenesis comprising administering to the tissue siRNA molecules that
target
Tie2 and siRNA molecules that target Ang-1 so that expression of Tie2 and Ang-
1
is decreased. In some embodiments, the present invention provides a method of
inhibiting or reducing angiogenesis in a tissue associated with undesired
angiogenesis comprising administering to the tissue siRNA molecules that
target
Tie2 and siRNA molecules that target Ang-2 so that expression of Tie2 and Ang-
2
is decreased. In some embodiments, the present invention provides a method of
inhibiting or reducing angiogenesis in a tissue associated with undesired
angiogenesis comprising administering to the tissue siRNA molecules that
target
Ang-1 and siRNA molecules that target Ang-2 so that expression of Ang-1 and
Ang-2 is decreased. In further embodiments, the present invention provides a
method of inhibiting or reducing angiogenesis in a tissue associated with
undesired
angiogenesis comprising administering to the tissue siRNA molecules that
target
Tie2, siRNA molecules that target Ang-1 and siRNA molecules that target Ang-2
so that expression of Tie2, Ang-1 and Ang-2 is decreased. In one embodiment,
the
tissue is a tumor.
[0041] Another embodiment of the invention is a combination of siRNA
inhibiting Tie2, Ang-1 and Ang-2, PDGF and its receptors, and EGF and its
receptors. Yet another embodiment is a combination of siRNA inhibiting the
Tie2,
Ang-1, and Ang-2 genes and their downstream signaling genes.
[0042] The siRNA oligonucleotides can be combined as a therapeutic for the
treatment of angiogenesis-related disease. In one embodiment of the present
invention they can be mixed together as a cocktail and in another embodiment
they
can be administered sequentially by the same route or by different routes and
formulations and in yet another embodiment some can be administered as a
cocktail and some administered sequentially. Other combinations of siRNA and
methods for their combination will be understood by one skilled in the art to
achieve treatment of angiogenesis-related diseases.
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Therapeutic Methods of Use
[0043] The present invention also provides methods for the treatment of
angiogenesis-related diseases and conditions in a subject. In some
embodiments,
the present invention provides a method of treating a subject afflicted with a
disease or condition associated with undesired angiogenesis comprising
administering to the subject siRNA molecules that target Tie2 so that
expression of
Tie2 is decreased. In some embodiments, the present invention provides a
method
of treating a subject afflicted with a disease or condition associated with
undesired
angiogenesis comprising administering to the subject siRNA molecules that
target
Ang-1 so that expression of Ang-1 is decreased. In further embodiments, the
present invention provides a method of treating a subject afflicted with a
disease or
condition associated with undesired angiogenesis comprising administering to
the
subject siRNA molecules that target Ang-2 so that expression of Ang-2 is
decreased.
[0044] In some embodiments, the present invention provides a method of
treating
a subject afflicted with a disease or condition associated with undesired
angiogenesis comprising administering to the subject siRNA molecules that
target
Tie2 and siRNA molecules that target Ang-1 so that expression of Tie2 and Ang-
1
is decreased. In some embodiments, the present invention provides a method of
treating a subject afflicted with a disease or condition associated with
undesired
angiogenesis comprising administering to the subject siRNA molecules that
target
Tie2 and siRNA molecules that target Ang-2 so that expression of Tie2 and Ang-
2
is decreased. In some embodiments, the present invention provides a method of
treating a subject afflicted with a disease or condition associated with
undesired
angiogenesis comprising administering to the subject siRNA molecules that
target
Ang-1 and siRNA molecules that target Ang-2 so that expression of Ang-I and
Ang-2 is decreased. In further embodiments, the present invention provides a
method of treating a subject afflicted with a disease or condition associated
with
undesired angiogenesis comprising administering to the subject siRNA molecules
that target Tie2, siRNA molecules that target Ang-1 and siRNA molecules that
target Ang-2 so that expression of Tie2, Ang-1 and Ang-2 is decreased.
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[0045] The present invention also provides methods for the treatment of
angiogenesis-related disease in a subject, including cancer, ocular disease,
arthritis,
and inflammatory diseases. The angiogenesis-related diseases include, but are
not
limited to, carcinoma, such as breast, ovarian, stomach, endometrial, salivary
gland, lung, kidney, colon, colorectum, esophageal, thyroid, pancreatic,
prostate
and bladder carcinomas and other neoplastic diseases, such as melanoma, small
cell lung cancer, non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, sarcoma, head and neck cancers, mesothelioma, biliary
(cholangiocarcinoma), small bowel adenocarcinoma, pediatric malignancies and
glioblastoma.
[0046] Antagonizing these molecules is expected to inhibit pathophysiological
processes, and thereby act as a potent therapy for various angiogenesis-
dependent
diseases. Besides solid tumors and their metastases, haematologic
malignancies,
such as leukemias, lymphomas and multiple myeloma, are also angiogenesis-
dependent. Excessive vascular growth contributes to numerous non-neoplastic
disorders. These non-neoplastic angiogenesis-dependent diseases include:
atherosclerosis, haemangioma, haemangioendothelioma, angiofibroma, vascular
malformations (e.g. Hereditary Hemorrhagic Teleangiectasia (HHT), or Osler-
Weber syndrome), warts, pyogenic granulomas, excessive hair growth, Kaposis'
sarcoma, scar keloids, allergic oedema, psoriasis, dysfunctional uterine
bleeding,
follicular cysts, ovarian hyperstimulation, endometriosis, respiratory
distress,
ascites, peritoneal sclerosis in dialysis patients, adhesion formation result
from
abdominal surgery, obesity, rheumatoid arthritis, synovitis, osteomyelitis,
pannus
growth, osteophyte, hemophilic joints, inflammatory and infectious processes
(e.g.
hepatitis, pneumonia, glomerulonephritis), asthma, nasal polyps, liver
regeneration,
pulmonary hypertension, retinopathy of prematurity, diabetic retinopathy, age-
related macular degeneration, leukomalacia, neovascular glaucoma, corneal
graft
neovascularization, trachoma, thyroiditis, thyroid enlargement, and
lymphoproliferative disorders.
[0047] In one embodiment of the invention, the subject treated is a human.
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Compositions and Methods of Administration
[0048] In another aspect, this invention provides compositions comprising the
nucleic acid molecules, including siRNA, of the invention. The siRNA of the
composition may be targeted to mRNA from the Ang-Tie pathway. The
compositions may comprise the nucleic acid molecules and a pharmaceutically
acceptable carrier, for example, a saline solution or a buffered saline
solution.
[0049] In certain embodiments, this invention provides "naked" nucleic acid
molecules or nucleic acid molecules in a vehicle which can be a naturally
occurring or synthetic vector, such as a viral vector, a liposome, polylysine,
or a
cationic polymer. In one embodiment, the composition may comprise the siRNA
of the invention and a complex-forming agent, such as a cationic polymer. The
cationic polymer may be a histidine-lysine (HK) copolymer or a
polyethyleneimine.
[0050] In certain embodiments, the cationic polymer is an HK copolymer. This
HK copolymer is a copolymer of histidine and lysine. In certain embodiments,
the
HK copolymer is synthesized from any appropriate combination of polyhistidine,
polylysine, histidine and/or lysine. In certain embodiments, the HK copolymer
is
linear. In certain preferred embodiments, the HK copolymer is branched.
[0051] In certain preferred embodiments, the branched HK copolymer comprises
a polypeptide backbone. Preferably, the polypeptide backbone comprises 1-10
amino acid residues, and more preferably 2-5 amino acid residues.
[0052] In certain preferred embodiments, the polypeptide backbone consists of
lysine amino acid residues.
[0053] In certain preferred embodiments, the number of branches on the
branched HK copolymer is one greater than the number of backbone amino acid
residues. In certain preferred embodiments, the branched HK copolymer contains
1-11 branches. In certain more preferred embodiments, the branched HK
copolymer contains 2-5 branches. In certain even more preferred embodiments,
the branched HK copolymer contains 4 branches.
[0054] In some embodiments, the branch of the branched HK copolymer
comprises 10-100 amino acid residues. In certain preferred embodiments, the
branch comprises 10-50 amino acid residues. In certain more preferred
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embodiments, the branch comprises 15-25 amino acid residues. In certain
embodiments, the branch of the branched HK copolymer comprises at least 3
histidine amino acid residues in every subsegment of 5 amino acid residues. In
certain other embodiments, the branch comprises at least 3 histidine amino
acid
residues in every subsegment of 4 amino acid residues. In certain other
embodiments, the branch comprises at least 2 histidine amino acid residues in
every subsegment of 3 amino acid residues. In certain other embodiments, the
branch comprises at least 1 histidine amino acid residues in every subsegment
of 2
amino acid residues.
[0055] In certain embodiments, at least 50% of the branch of the HK copolymer
comprises units of the sequence KHHH. In certain preferred embodiments, at
least
75% of the branch comprises units of the sequence KHHH.
[0056] In certain embodiments, the HK copolymer branch comprises an amino
acid residue other than histidine or lysine. In certain preferred embodiments,
the
branch comprises a cysteine amino acid residue, wherein the cysteine is a N-
terminal amino acid residue.
[0057] In certain embodiments, the HK copolymer has the structure
(KHHHKHHHKHHHHKHHHK)4-KKK. In certain other embodiments, the HK
copolymer has the structure (CKHHHKHHHKHHHHKHHHK)4-KKK.
[0058] Some suitable examples of HK copolymers can be found, for example, in
U.S. Patent Nos. 6,692,911 and 7,163,695, which are both incorporated herein
by
reference.
[0059] In one embodiment, the compositions of the invention may comprise the
siRNA of the invention and a complex-forming agent that is used to make a
nanoparticle. The nanoparticle may optionally comprise a steric polymer and/or
a
targeting moiety. The targeting moiety may be a peptide, an antibody, or an
antigen-binding portion. The targeting moiety may serve as a means for
targeting
vascular endothelial cells, such as a peptide comprising the sequence Arg-Gly-
Asp (RGD). Such a peptide may be cyclic or linear. In one embodiment, this
peptide is RGDFK. In a certain embodiment, this peptide is cyclo (RGD-D-FK).
[0060] The nucleic acid molecules, compositions, and therapeutic methods of
the
invention can be used alone or in combination with other therapeutic agents
and
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modalities including targeted therapeutics and including Ang-Tie pathway
antagonists, such as monoclonal antibodies and small molecule inhibitors, and
targeted therapeutics inhibiting EGF and its receptor, PDGF and its receptors,
or
MEK or Bcr-Abl, and other immunotherapeutic and chemotherapeutic agents, such
as EGFR inhibitors VECTIBIX (panitumumab) and TARCEVA (erlotinib),
Her-2-targeted therapy HERCEPTIN (trastuzumab), or anti-angiogenesis drugs
such as AVASTIN (bevacizumab) and SUTENT (sunitinib malate). The
nucleic acid molecules, compositions, and methods also may be combined
therapeutically with other treatment modalities including radiation, laser
therapy,
surgery and the like.
[0061] Methods of administration for the nucleic acids and compositions of the
invention are known to those of ordinary skill in the art. Administration may
be
intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous,
cutaneous,
or transdermal. In one embodiment, administration may be systemic. In a
further
embodiment, administration may be local. For example, the nucleic acid
molecules of the invention may be delivered via direct injections into tumor
tissue
and directly into or near angiogenic tissue or tissue with undesirable
neovasculature. For certain applications, the nucleic acid molecules and
compositions may be administered with application of an electric field. In
certain
embodiments, this invention provides for administration of "naked" siRNA.
Preparation of nanoparticles containing nucleic acid molecules modulating
expression of Ang/Tie2 pathway genes
[0062] One embodiment of the present invention provides compositions and
methods for nanoparticle preparations of anti-Ang/Tie2 pathway nucleic acid
molecules, including siRNAs. The nanoparticles may comprise one or more of a
histidine-lysine copolymer, polyethylene glycol, or polyethyleneimine. In one
embodiment of the invention, RGD-mediated ligand-directed nanoparticles may be
prepared. In one method for the manufacture of RGD-mediated tissue-targeted
nanoparticles containing siRNA, the targeting ligand, an RGD-containing
peptide,
is conjugated to a steric polymer such as polyethylene glycol, or other
polymers
with similar properties. This ligand-steric polymer conjugate is further
conjugated
to a polycation such as polyethyleneimine or other effective material such as
a
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histidine-lysine copolymer. The conjugation can be by covalent or non-covalent
bonds and the covalent bonds can be non-cleavable or they can be cleavable
such
as by hydrolysis or by reducing agents. A solution comprising the polymer
conjugate, or comprising a mixture of a polymer conjugate with other polymer,
lipid, or micelle such as materials comprising a ligand or a steric polymer or
fusogen, is mixed with a solution comprising the nucleic acid, in one
embodiment
an siRNA targeted against specific mRNA of interest, in desirable ratios to
obtain
nanoparticles that contain siRNA. Such ratios may produce nanoparticles of a
desired size, stability, or other characteristics.
[0063] In one embodiment, nanoparticles are formed by layered nanoparticle
self-assembly comprising mixing the polymer conjugate with excess polycation
and the nucleic acid. Non-covalent electrostatic interactions between the
negatively charged nucleic acid and the positively charged segment of the
polymer
conjugate drive the self-assembly process that leads to formation of
nanoparticles.
This process involves simple mixing of the solutions where one of the
solutions
containing the nucleic acid is added to another solution containing the
polymer
conjugate and excess polycation followed by or concurrently with stirring. In
one
embodiment, the ratio between the positively charged components and the
negatively charged components in the mixture is determined by appropriately
adjusting the concentrations of each solution or by adjusting the volume of
solution
added. In another embodiment, the two solutions are mixed under continuous
flow
conditions using mixing apparatus such as static mixer. In this embodiment,
two
or more solutions are introduced into a static mixer at rates and pressures
giving a
ratio of the solutions, where the streams of solutions get mixed within the
static
mixer. Arrangements are possible for mixers to be arranged in parallel or in
series.
[0064] The present invention, thus generally described, will be understood
more
readily by reference to the following examples, which are provided by way of
illustration and are not intended to be limiting of the present invention.
The invention is illustrated by the following examples but one skilled in the
art will
appreciate that the invention is not limited.
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Examples:
Example 1: Selection of 48 human Ang-2 siRNA candidates for potency screeniny,
[0065] To select potent human Ang-2 siRNA, 48 siRNA candidates were
selected from Table 8 and Table 10 (Table 11). These siRNA were synthesized in
plate-format at 20 nmol scale and used for in vitro potency screening.
Table 11. Human Ang-2 siRNA candidates for in vitro screening
No. Start siRNA Sequence GC% SEQ
(sense strand/antisense strand) ID
NO:
1 -56 5'-GGACGUGUGUUUGCCCUCAAGUUUG-3' 52.0 503
3'-CCUGUAUAUAAACGGGAGUUCAAAC-5' 504
2 34 5'-GAUCUUGUCUUGGCCGCAGCCUAUA-3' 52.0 475
3'-CUAGAACAGAACCGGCGUCGGAUAU-5' 476
3 47 5'-CCGCAGCCUAUAACAACUUUCGGAA-3' 48.0 477
3'-GGCGUCGGAUAUUGUUGAAAGCCUU-5' 478
4 241 5'-CAAGUGCUGGAGAACAUCAUGGAAA-3' 44.0 479
31-GUUCACGACCUCUUGUAGUACCUUU-5' 480
5 274 5'-CAGUGGCUAAUGAAGCUUGAGAAUU-3' 40.0 515
3'-GUCACCGAUUACUUCGAACUCUUAA-5' 516
6 306 5'-GGACAACAUGAAGAAAGAAAUGGUA-3' 36.0 481
3'-CCUGUUGUACUUCUUUCUUUACCAU-5' 482
7 357 5'-CCAGACGGCUGUGAUGAUAGAAAUA-3' 44.0 521
3'-GGUCUGCCGACACUACUAUCUUUAU-5' 522
8 390 5'-CCUGUUGAACCAAACAGCUGAGCAA-3' 48.0 483
3'-GGACAACUUGGUUUGUCGACUCGUU-5' 484
9 403 5'-ACAGCUGAGCAAACGCGGAAGUUAA-3' 48.0 523
3'-UGUCGACUCGUUUGCGCCUUCAAUU-5' 524
414 5'-AACGCGGAAGUUAACUGAUGUGGAA-3' 44.0 525
3'-UUGCGCCUUCAAUUGACUACACCUU-5' 526
11 420 5'-GAAGUUAACUGAUGUGGAAGCCCAA-3' 44.0 529
3'-CUUCAAUUGACUACACCUUCGGGUU-5' 530
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No. Start siRNA Sequence GC% SEQ
(sense strand/antisense strand) ID
NO:
12 425 5'-UAACUGAUGUGGAAGCCCAAGUAUU-3' 40.0 485
3'-AUUGACUACACCUUCGGGUUCAUAA-5' 486
13 427 5'-ACUGAUGUGGAAGCCCAAGUAUUAA-3' 40.0 531
3'-UGACUACACCUUCGGGUUCAUAAUU-5' 532
14 458 5'-CCACGAGACUUGAACUUCAGCUCUU-3' 48.0 487
3'-GGUGCUCUGAACUUGAAGUCGAGAA-5' 488
15 483 5'-GGAACACUCCCUCUCGACAAACAAA-3' 48.0 535
3'-CCUUGUGAGGGAGAGCUGUUUGUUU-5' 536
16 524 5'-UGGACCAGACCAGUGAAAUAAACAA-3' 40.0 537
3'-ACCUGGUCUGGUCACUUUAUUUGUU-5' 538
17 812 5'-CCACUGUUGCUAAAGAAGAACAAAU-3' 36.0 455
3'-GGUGACAACGAUUUCUUCUUGUUUA-5' 456
18 820 5'-GCUAAAGAAGAACAAAUCAGCUUCA-3' 36.0 541
3'-CGAUUUCUUCUUGUUUAGUCGAAGU-5' 542
19 837 5'-CAGCUUCAGAGACUGUGCUGAAGUA-3' 48.0 457
3'-GUCGAAGUCUCUGACACGACUUCAU-5' 458
20 871 5'-GGACACACCACAAAUGGCAUCUACA-3' 48.0 459
3'-CCUGUGUGGUGUUUACCGUAGAUGU-5' 460
21 877 5'-ACCACAAAUGGCAUCUACACGUUAA-3' 40.0 489
3'-UGGUGUUUACCGUAGAUGUGCAAUU-5' 490
22 888 5'-CAUCUACACGUUAACAUUCCCUAAU-3' 36.0 461
3'-GUAGAUGUGCAAUUGUAAGGGAUUA-5' 462
23 894 5'-CACGUUAACAUUCCCUAAUUCUACA-3' 36.0 491
3'-GUGCAAUUGUAAGGGAUUAAGAUGU-5' 492
24 922 5'-GAGAUCAAGGCCUACUGUGACAUGG-3' 52.0 637
3'-CUCUAGUUCCGGAUGACACUGUACC-5' h/m 638
25 923 5'-AGAUCAAGGCCUACUGUGACAUGGA-3' 48.0 639
3'-UCUAGUUCCGGAUGACACUGUACCU-5' h/m 640
26 924 5'-GAUCAAGGCCUACUGUGACAUGGAA-3' 48.0 547
3'-CUAGUUCCGGAUGACACUGUACCUU-5' 548
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No. Start siRNA Sequence GC% SEQ
(sense strand/antisense strand) ID
NO:
27 951 5'-UGGAGGAGGCGGGUGGACAAUUAUU-3' 52.0 463
3'-ACCUCCUCCGCCCACCUGUUAAUAA-5' 464
28 962 5'-GGUGGACAAUUAUUCAGCGACGUGA-3' 48.0 465
3'-CCACCUGUUAAUAAGUCGCUGCACU-5' 466
29 980 5'-GACGUGAGGAUGGCAGCGUUGAUUU-3' 52.0 551
3'-CUGCACUCCUACCGUCGCAACUAAA-5' 552
30 1032 5'-GGGAUUUGGUAACCCUUCAGGAGAA-3' 48.0 493
3'-CCCUAAACCAUUGGGAAGUCCUCUU-5' 494
31 1066 5'-GGAAAUGAGUUUGUUUCGCAACUGA-3' 40.0 553
3'-CCUUUACUCAAACAAAGCGUUGACU-5' 554
32 1082 5'-CGCAACUGACUAAUCAGCAACGCUA-3' 48.0 467
3'-GCGUUGACUGAUUAGUCGUUGCGAU-5' 468
33 1140 5'-GAAUGAGGCUUACUCAUUGUAUGAA-3' 36.0 557
3'-CUUACUCCGAAUGAGUAACAUACUU-5' 558
34 1144 5'-GAGGCUUACUCAUUGUAUGAACAUU-3' 36.0 559
3'-CUCCGAAUGAGUAACAUACUUGUAA-5' 560
35 1242 5'-CAGCAUCAGCCAACCAGGAAAUGAU-3' 48.0 469
3'-GUCGUAGUCGGUUGGUCCUUUACUA-5' 470
36 1277 5'-AGGAUGGAGACAACGACAAAUGUAU-3' 40.0 563
3'-UCCUACCUCUGUUGCUGUUUACAUA-5' 564
37 1283 5'-GAGACAACGACAAAUGUAUUUGCAA-3' 36.0 565
3'-CUCUGUUGCUGUUUACAUAAACGUU-5' 566
38 1342 5'-GAUGCAUGUGGUCCUUCCAACUUGA-3' 48.0 495
3'-CUACGUACACCAGGAAGGUUGAACU-5' 496
39 1354 5'-CCUUCCAACUUGAACGGAAUGUACU-3' 44.0 471
3'-GGAAGGUUGAACUUGCCUUACAUGA-5' 472
40 1359 5'-CAACUUGAACGGAAUGUACUAUCCA-3' 40.0 567
3'-GUUGAACUUGCCUUACAUGAUAGGU-5' 568
41 1390 5'-CAGAACACAAAUAAGUUCAACGGCA-3' 40.0 473
3'-GUCUUGUGUUUAUUCAAGUUGCCGU-5' 474
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No. Start siRNA Sequence GC% SEQ
(sense strand/antisense strand) ID
NO:
42 1410 5'-CGGCAWAAAUGGUACUACUGGAAA-3' 40.0 497
3'-GCCGUAAUUUACCAUGAUGACCUUU-5' 498
43 1421 5'-GGUACUACUGGAAAGGCUCAGGCUA-3' 52.0 571
3'-CCAUGAUGACCUUUCCGAGUCCGAU-5' 572
44 1447 5'-UCGCUCAAGGCCACAACCAUGAUGA-3' 52.0 641
3'-AGCGAGUUCCGGUGUUGGUACUACU-5' h/m 642
45 1448 5'-CGCUCAAGGCCACAACCAUGAUGAU-3' 52.0 643
3'-GCGAGUUCCGGUGUUGGUACUACUA-5' h/m 644
46 1449 5'-GCUCAAGGCCACAACCAUGAUGAUC-3' 52.0 645
3'-CGAGWCCGGUGUUGGUACUACUAG-5' h/m 646
47 1450 5'-CUCAAGGCCACAACCAUGAUGAUCC-3' 52.0 647
3'-GAGUUCCGGUGWGGUACUACUAGG-5' h/m 648
48 1623 5'-CAUGCUCCAGAUUAGAGCCUGUAAA-3' 44.0 605
3'-GUACGAGGUCUAAUCUCGGACAUUU-5' 606
Example 2: High-through-put screening of human Ang-2 siRNA for their
potency in inhibiting An -g 2 expression in HUVEC cells
100661 A reverse transfection based high-through-put (HTP) method was used to
screen 48 human Ang-2 siRNAs (Table 11) for their potency in inhibiting Ang-2
expression in HUVEC cells. Briefly, 10 nM of siRNA duplex was spotted onto the
bottom of a 96-well plate followed by addition of 0.25 1 of LipofectamineTM
RNAiMAX (Invitrogen). A luciferase specific 25-mer siRNA was used as the
negative control. The plate was incubated at room temperature for 10-20
minutes,
and 7,500 HUVEC cells in 100 ul growth medium was added to each wells. The
plate was mixed gently by rocking the plate back and forth, and then incubated
for
24-48 hours at 37 C in a CO2 incubator. The effect of siRNA mediated Ang-2
knockdown was monitored by measuring the concentration of Ang-2 protein in the
medium using a human Ang-2 ELISA kit (R&D). The cell viability of the
transfected cells was measured using a WST-1 assay kit (Roche) for
normalization
of Ang-2 concentration.
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[0067] Significant inhibition of Ang-2 protein level expression in transfected
HUVEC cells was observed at 24 hours post transfection with a majority of the
48
Ang-2 siRNA candidates tested (Figure 1). At 48 hours post transfection, the
inhibition effects were more profound (Figure 2), with about 50% of the Ang-2
siRNA candidates showing a greater than 80% inhibition of Ang-2 expression
compared to cells transfected with control Luc-siRNA (Figure 3). There was no
cytotoxicity in the transfected HUVEC cells that associated with knockdown of
Ang-2 expression (Figure 4).
Example 3: Confirmation of Ang-2 epression knockdown in HUVEC cells
transfected with 2 nM Ang-2 siRNA
[0068] In a separate experiment, 38 Ang-2 siRNA candidates that demonstrated a
high percentage of Ang-2 knockdown in previous HTP screening (Figure 1-3) were
further examined for their potency in inhibiting Ang-2 expression in HUVEC
cells
using a reverse transfection method. Briefly, 2 nM of siRNA duplex was spotted
onto the bottom of a 96-well plate followed by addition of 0.25 l of
LipofectamineTM RNAiMAX (Invitrogen). A negative control (Ctrl-) siRNA,
which has a 19-nt double-stranded region with dTdT 3'- overhangs on both
strands
and does not has a significant homologous sequence with any known human gene,
was used as the negative control. The plate was incubated at room temperature
for
10-20 minutes, and 7,500 HUVEC cells in 100 l growth medium was added to
each well. The plate was mixed gently by rocking the plate back and forth, and
then incubated for 48 hours at 37 C in a CO2 incubator. The effect of siRNA
mediated Ang-2 knockdown was monitored by measuring the concentration of
Ang-2 protein in the medium using a human Ang-2 ELISA kit (R&D). The cell
viability of the transfected cells was measured using a WST-1 assay kit
(Roche) for
normalization of Ang-2 concentration.
[0069] Significant inhibition (>90%) of Ang-2 protein level expression in
transfected HUVEC cells was observed at 48 hours post transfection with a
majority of the 38 Ang-2 siRNA candidates tested (Figure 5), including many
siRNA candidates with a greater than 90% knockdown of Ang-2 protein level
expression (Figure 6). In addition, 3 siRNA that target both human and mouse
Ang-2 also demonstrated high potency in knocking down human Ang-2 expression
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(Figures 5 and 6). Finally, there was no cytotoxicity in the transfected HUVEC
cells that associated with knockdown of Ang-2 expression (Figure 7).
Example 4: Final selection of Ang-2 siRNA based on Ang-2 gene expression
knockdown in HUVEC cells transfected with 0.2 nM
[0070] In another experiment, 18 Ang-2 siRNA candidates that demonstrated a
higher than 94% knockdown of Ang-2 expression in a previous experiment (Figure
6) and 3 human/mouse Ang-2 siRNA were further examined for their potency in
inhibiting Ang-2 expression in HUVEC cells using a reverse transfection method
with a lower dose of siRNA. Briefly, 0.2 nM of siRNA duplex was spotted onto
the bottom of a 96-well plate followed by addition of 0.25 l of
LipofectamineTM
RNAiMAX (Invitrogen). A negative control (Ctrl-) siRNA, which has a 19-nt
double-stranded region with dTdT 3'- overhangs on both strands and does not
has
a significant homologous sequence with any known human gene, was used as the
negative control. The plate was incubated at room temperature for 10-20
minutes,
and 7,500 HUVEC cells in 100 l growth medium was added to each well. The
plate was mixed gently by rocking the plate back and forth, and then incubated
for
48 hours at 37 C in a CO2 incubator. The effect of siRNA mediated Ang-2
knockdown was monitored by measuring the concentration of Ang-2 protein in the
medium using a human Ang-2 ELISA kit (R&D). The cell viability of the
transfected cells was measured using a WST-1 assay kit (Roche) for
normalization
of Ang-2 concentration.
[0071] When transfected with only 0.2 nM of siRNA, significant inhibition (30-
50%) of Ang-2 protein level expression in transfected HUVEC cells was observed
at 48 hours post transfection with a majority of the 38 Ang-2 siRNA candidates
tested (Figure 8), including one siRNA which targets both human and mouse Ang-
2.
[0072] Three Ang-2 siRNA, #10 (Ang-2-25-10), #14 (Ang-2-25-14), and #31
(Ang-2-25-3 1) were selected for further experiments as Ang-2 siRNA. In
addition,
#25 (Ang-2-25-25) and #45 (Ang-2-25-45) were selected for further experiments
as human/mouse Ang-2 siRNA.
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Example 5: Determination of IC50 values of AnR-2 siRNA
100731 Upon the confirmation of Ang-2 siRNA candidates, experiments were
conducted to determine the IC50 value of Ang-2 siRNA (Ang-2-25-10, Ang-2-25-
14, and Ang-2-25-31) in HUVEC cells. Briefly, 10 dilutions of each siRNA
duplex were spotted onto the bottom of a 96-well plate followed by addition of
0.25 l of LipofectamineTM RNAiMAX (Invitrogen). The siRNA dilutions were
0.076 pM, 0.31 pM, 1.2 pM, 4.9 pM, 19.5 pM, 78.1 pM, 312.5 pM, 1.25 nM, 5
nM, and 20nM. The plate was incubated at room temperature for 10-20 minutes,
and 7,500 HUVEC cells in 100 l growth medium was added to each well. The
plate was mixed gently by rocking the plate back and forth, and then incubated
for
48 hours at 37 C in a CO2 incubator. The effect of siRNA-mediated Ang-2
knockdown was monitored by measuring the concentration of Ang-2 protein in the
medium using a human Ang-2 ELISA kit (R&D). The cell viability of the
transfected cells was measured using a WST-1 assay kit (Roche) for
normalization
of Ang-2 concentration.
[0074] The IC50 value of each siRNA duplex in HUVEC cells at 48 hours post
siRNA transfection was obtained using the GraphPad Prism program (Figure 9).
The IC50 of Ang-2-25-10 was 0.363 nM, the IC50 of Ang-2-25-14 was 0.494 nM,
and the IC50 of Ang-2-25-31 was 0.398 nM (Figure 9 and Table 12).
Example 6: Determination of IC50 values of human/mouse Ang-2 siRNA
[0075] Upon the confirmation of human/mouse Ang-2 siRNA candidates that
target both human and mouse Ang-2 mRNA, experiments were conducted to
determine the IC50 value of human/mouse Ang-2 siRNA (Ang-2-25-25 and Ang-
2-25-45) in HUVEC cells. Briefly, 10 dilutions of each siRNA duplex were
spotted onto the bottom of a 96-well plate followed by addition of 0.25 l of
LipofectamineTM RNAiMAX (Invitrogen). The siRNA dilutions were 0.076 pM,
0.31 pM, 1.2 pM, 4.9 pM, 19.5 pM, 78.1 pM, 312.5 pM, 1.25 nM, 5 nM, and
20nM. The plate was incubated at room temperature for 10-20 minutes, and 7,500
HUVEC cells in 100 l growth medium was added to each well. The plate was
mixed gently by rocking the plate back and forth, and then incubated for 48
hours
at 37 C in a CO2 incubator. The effect of siRNA-mediated Ang-2 knockdown was
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monitored by measuring the concentration of Ang-2 protein in the medium using
a
human Ang-2 ELISA kit (R&D). The cell viability of the transfected cells was
measured using a WST-1 assay kit (Roche) for normalization of Ang-2
concentration.
[0076] The IC50 value of each siRNA duplex in HUVEC cells at 48 hours post
siRNA transfection was obtained using the GraphPad Prism program (Figure 10).
The IC50 of Ang-2-25-25 was 1.634 nM, and the IC50 of Ang-2-25-45 was 0.90
nM (Figure 10 and Table 12).
Table 12. IC50 of selected Ang-2-siRNA in transfected HUVEC cells
siRNA IC50 (nM)
48 hours post-transfection_
human Ang-2-25mer-siRNA# 10 0.363
human Ang-2-25mer-siRNA# 14 0.494
human Ang-2-25mer-siRNA#31 0.398
human&mouse Ang-2-25mer-siRNA#25 1.634
human&mouse Ang-2-25mer-siRNA#45 0.9
Table 13. Ang-1, Ang-2, and Tie2 mRNA sequence table
Gene: TEK (Tie2)
Species: human
NCBI Accession No.: NM000459
SEQ ID NO: 649
Sequence:
AGTTTCCCGCCTATGAGAGGATACCCCTATTGTTTCTGAAAATGCTGAC
CGGGACCCACACTTCCAACAAAAATTCCTCTGCCCCTACAGCAGCAGC
AAAAGCAGCAGCAGAAGCAACAGCAACAGATAAGTGTTTTGATGAATT
GCGAGATGGATAGGGCTTGAGTGCCCCCAGCCCTGCTGATACCAAATG
CCTTTAAGATACAGCCTTTCCCATCCTAATCTACAAAGGAAACAGGAA
AAAGGAACTTAAAACTCCCTGTGCTCAGACAGAAATGAGACTGTTACA
GCCTGCTTCTGTGCTGTTCCTTCTTGCCTCTAACTTGTAAACAAGACGTA
GTAGGACGATGCTAATGGAAAGTCACAAACCGCTGGGTTTTTGAAAGG
ATCCTTGGGACCTCATGCACATTTGTGGAAACTGGATGGAGAGATTTGG
GGAAGCATGGACTCTTTAGCCAGCTTAGTTCTCTGTGGAGTCAGCTTGC
TCCTTTCTGGAACTGTGGAAGGTGCCATGGACTTGATCTTGATCAATTC
CCTACCTCTTGTATCTGATGCTGAAACATCTCTCACCTGCATTGCCTCTG
GGTGGCGCCCCCATGAGCCCATCACCATAGGAAGGGACTTTGAAGCCT
TAATGAACCAGCACCAGGATCCGCTGGAAGTTACTCAAGATGTGACCA
GAGAATGGGCTAAAAAAGTTGTTTGGAAGAGAGAAAAGGCTAGTAAG
ATCAATGGTGCTTATTTCTGTGAAGGGCGAGTTCGAGGAGAGGCAATC
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AGGATACGAACCATGAAGATGCGTCAACAAGCTTCCTTCCTACCAGCT
ACTTTAACTATGACTGTGGACAAGGGAGATAACGTGAACATATCTTTCA
AAAAGGTATTGATTAAAGAAGAAGATGCAGTGATTTACAAAAATGGTT
CCTTCATCCATTCAGTGCCCCGGCATGAAGTACCTGATATTCTAGAAGT
ACACCTGCCTCATGCTCAGCCCCAGGATGCTGGAGTGTACTCGGCCAG
GTATATAGGAGGAAACCTCTTCACCTCGGCCTTCACCAGGCTGATAGTC
CGGAGATGTGAAGCCCAGAAGTGGGGACCTGAATGCAACCATCTCTGT
ACTGCTTGTATGAACAATGGTGTCTGCCATGAAGATACTGGAGAATGC
ATTTGCCCTCCTGGGTTTATGGGAAGGACGTGTGAGAAGGCTTGTGAAC
TGCACACGTTTGGCAGAACTTGTAAAGAAAGGTGCAGTGGACAAGAGG
GATGCAAGTCTTATGTGTTCTGTCTCCCTGACCCCTATGGGTGTTCCTGT
GCCACAGGCTGGAAGGGTCTGCAGTGCAATGAAGCATGCCACCCTGGT
TTTTACGGGCCAGATTGTAAGCTTAGGTGCAGCTGCAACAATGGGGAG
ATGTGTGATCGCTTCCAAGGATGTCTCTGCTCTCCAGGATGGCAGGGGC
TCCAGTGTGAGAGAGAAGGCATACCGAGGATGACCCCAAAGATAGTGG
ATTTGCCAGATCATATAGAAGTAAACAGTGGTAAATTTAATCCCATTTG
CAAAGCTTCTGGCTGGCCGCTACCTACTAATGAAGAAATGACCCTGGT
GAAGCCGGATGGGACAGTGCTCCATCCAAAAGACTTTAACCATACGGA
TCATTTCTCAGTAGCCATATTCACCATCCACCGGATCCTCCCCCCTGACT
CAGGAGTTTGGGTCTGCAGTGTGAACACAGTGGCTGGGATGGTGGAAA
AGCCCTTCAACATTTCTGTTAAAGTTCTTCCAAAGCCCCTGAATGCCCC
AAACGTGATTGACACTGGACATAACTTTGCTGTCATCAACATCAGCTCT
GAGCCTTACTTTGGGGATGGACCAATCAAATCCAAGAAGCTTCTATAC
AAACCCGTTAATCACTATGAGGCTTGGCAACATATTCAAGTGACAAAT
GAGATTGTTACACTCAACTATTTGGAACCTCGGACAGAATATGAACTCT
GTGTGCAACTGGTCCGTCGTGGAGAGGGTGGGGAAGGGCATCCTGGAC
CTGTGAGACGCTTCACAACAGCTTCTATCGGACTCCCTCCTCCAAGAGG
TCTAAATCTCCTGCCTAAAAGTCAGACCACTCTAAATTTGACCTGGCAA
CCAATATTTCCAAGCTCGGAAGATGACTTTTATGTTGAAGTGGAGAGA
AGGTCTGTGCAAAAAAGTGATCAGCAGAATATTAAAGTTCCAGGCAAC
TTGACTTCGGTGCTACTTAACAACTTACATCCCAGGGAGCAGTACGTGG
TCCGAGCTAGAGTCAACACCAAGGCCCAGGGGGAATGGAGTGAAGATC
TCACTGCTTGGACCCTTAGTGACATTCTTCCTCCTCAACCAGAAAACAT
CAAGATTTCCAACATTACACACTCCTCAGCTGTGATTTCTTGGACAATA
TTGGATGGCTATTCTATTTCTTCTATTACTATCCGTTACAAGGTTCAAGG
CAAGAATGAAGACCAGCACGTTGATGTGAAGATAAAGAATGCCACCAT
CACTCAGTATCAGCTCAAGGGCCTAGAGCCTGAAACAGCATACCAGGT
GGACATTTTTGCAGAGAACAACATAGGGTCAAGCAACCCAGCCTTTTCT
CATGAACTGGTGACCCTCCCAGAATCTCAAGCACCAGCGGACCTCGGA
GGGGGGAAGATGCTGCTTATAGCCATCCTTGGCTCTGCTGGAATGACCT
GCCTGACTGTGCTGTTGGCCTTTCTGATCATATTGCAATTGAAGAGGGC
AAATGTGCAAAGGAGAATGGCCCAAGCCTTCCAAAACGTGAGGGAAG
AACCAGCTGTGCAGTTCAACTCAGGGACTCTGGCCCTAAACAGGAAGG
TCAAAAACAACCCAGATCCTACAATTTATCCAGTGCTTGACTGGAATGA
CATCAAATTTCAAGATGTGATTGGGGAGGGCAATTTTGGCCAAGTTCTT
AAGGCGCGCATCAAGAAGGATGGGTTACGGATGGATGCTGCCATCAAA
AGAATGAAAGAATATGCCTCCAAAGATGATCACAGGGACTTTGCAGGA
GAACTGGAAGTTCTTTGTAAACTTGGACACCATCCAAACATCATCAATC
CA 02692632 2010-01-05
WO 2009/008990 -49 PCT/US2008/008232
-
TCTTAGGAGCATGTGAACATCGAGGCTACTTGTACCTGGCCATTGAGTA
CGCGCCCCATGGAAACCTTCTGGACTTCCTTCGCAAGAGCCGTGTGCTG
GAGACGGACCCAGCATTTGCCATTGCCAATAGCACCGCGTCCACACTG
TCCTCCCAGCAGCTCCTTCACTTCGCTGCCGACGTGGCCCGGGGCATGG
ACTACTTGAGCCAAAAACAGTTTATCCACAGGGATCTGGCTGCCAGAA
ACATTTTAGTTGGTGAAAACTATGTGGCAAAAATAGCAGATTTTGGATT
GTCCCGAGGTCAAGAGGTGTATGTGAAAAAGACAATGGGAAGGCTCCC
AGTGCGCTGGATGGCCATCGAGTCACTGAATTACAGTGTGTACACAAC
CAACAGTGATGTATGGTCCTATGGTGTGTTACTATGGGAGATTGTTAGC
TTAGGAGGCACACCCTACTGCGGGATGACTTGTGCAGAACTCTACGAG
AAGCTGCCCCAGGGCTACAGACTGGAGAAGCCCCTGAACTGTGATGAT
GAGGTGTATGATCTAATGAGACAATGCTGGCGGGAGAAGCCTTATGAG
AGGCCATCATTTGCCCAGATATTGGTGTCCTTAAACAGAATGTTAGAGG
AGCGAAAGACCTACGTGAATACCACGCTTTATGAGAAGTTTACTTATGC
AGGAATTGACTGTTCTGCTGAAGAAGCGGCCTAGGACAGAACATCTGT
ATACCCTCTGTTTCCCTTTCACTGGCATGGGAGACCCTTGACACCTGCT
GAGAAAACATGCCTCTGCCAAAGGATGTGATATATAAGTGTACATATG
TGCTGTACACCTGGGACCTTCACCACTGTAGATCCCATGCATGGATCTA
TGTAGTATGCTCTGACTCTAATAGGACTGTATATACTGTTTTAAGAATG
GGCTGAAATCAGAATGCCTGTTTGTGGTTTCATATGCAATAATATATTT
TTTTAAAAATGTGGACTTCATAGGAAGGCGTGAGTACAATTAGTATAAT
GCATAACTCATTGTTGTCCTAGATATTTTGATATTTACCTTTATGTTGAA
TGCTATTAAATGTTTTCCTGTGTCAAAGTAAAATATTGTTAATAAACCT
AACAATGACCCTGATAGTACAGGTTAAGTGAGAGAACTATATGAATTC
TAACAAGTCATAGGTTAATATTTAAGACACTGAAAAATCTAAGTGATA
TAAATCAGATTCTTCTCTCTCAATTTTATCCCTCACCTGTAGCAGCCAGT
CCCGTTTCATTTAGTCATGTGACCACTCTGTCTTGTGTTTCCACAGCCTG
CAAGTCAGTCCAGGATGCTAACATCTAAAAATAGACTTAAATCTCATTG
CTTACAAGCCTAAGAATCTTTAGAGAAGTATACATAAGTTTAGGATAA
AATAATGGGATTTTCTTTTCTTTTCTCTGGTAATATTGACTTGTATATTT
TAAGAAATAACAGAAAGCCTGGGTGACATTTGGGAGACATGTGACATT
TATATATTGAATTAATATCCCTACATGTATTGCACATTGTAAAAAGTTT
TAGTTTTGATGAGTTGTGAGTTTACCTTGTATACTGTAGGCACACTTTGC
ACTGATATATCATGAGTGAATAAATGTCTTGCCTACTCACGTCTCAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAA
Gene: TEK (Tie2)
Species: mouse
NCBI Accession No.: NM_013690
SEQ ID NO: 650
Sequence:
GAGCAGGAGCCGGAGCAGGAGCAGAAGATAAGCCTTGGATGAAGGGC
AAGATGGATAGGGCTCGCTCTGCCCCAAGCCCTGCTGATACCAAGTGC
CTTTAAGATACAGCCTTTCCCATCCTAATCTGCAAAGGAAACAGGAAA
AAGGAACTTAACCCTCCCTGTGCTCAGACAGAAATGAGACTGTTACCG
CCTGCTTCTGTGGTGTTTCTCCTTGCCGCCAACTTGTAAACAAGAGCGA
GTGGACCATGCGAGCGGGAAGTCGCAAAGTTGTGAGTTGTTGAAAGCT
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-50-
TCCCAGGGACTCATGCTCATCTGTGGACGCTGGATGGGGAGATCTGGG
GAAGTATGGACTCTTTAGCCGGCTTAGTTCTCTGTGGAGTCAGCTTGCT
CCTTTATGGAGTAGTAGAAGGCGCCATGGACCTGATCTTGATCAATTCC
CTACCTCTTGTGTCTGATGCCGAAACATCCCTCACCTGCATTGCCTCTG
GGTGGCACCCCCATGAGCCCATCACCATAGGAAGGGACTTTGAAGCCT
TAATGAACCAGCACCAAGATCCACTGGAGGTTACTCAAGATGTGACCA
GAGAATGGGCGAAAAAAGTTGTTTGGAAGAGAGAAAAGGCCAGTAAG
ATTAATGGTGCTTATTTCTGTGAAGGTCGAGTTCGAGGACAGGCTATAA
GGATACGGACCATGAAGATGCGTCAACAAGCATCCTTCCTACCTGCTA
CTTTAACTATGACCGTGGACAGGGGAGATAATGTGAACATATCTTTCAA
AAAGGTGTTAATTAAAGAAGAAGATGCAGTGATTTACAAAAATGGCTC
CTTCATCCACTCAGTGCCCCGGCATGAAGTACCTGATATTTTAGAAGTT
CACTTGCCGCATGCTCAGCCCCAGGATGCTGGTGTGTACTCGGCCAGGT
ACATAGGAGGAAACCTGTTCACCTCAGCCTTCACCAGGCTGATTGTTCG
GAGATGTGAAGCTCAGAAGTGGGGGCCCGACTGTAGCCGTCCTTGTAC
TACTTGCAAGAACAATGGAGTCTGCCATGAAGATACCGGGGAATGCAT
TTGCCCTCCTGGGTTTATGGGGAGAACATGTGAGAAAGCTTGTGAGCC
GCACACATTTGGCAGGACCTGTAAAGAAAGGTGTAGTGGACCAGAAGG
ATGCAAGTCTTATGTGTTCTGTCTCCCAGACCCTTACGGGTGTTCCTGTG
CCACAGGCTGGAGGGGGTTGCAGTGCAATGAAGCATGCCCATCTGGTT
ACTACGGACCAGACTGTAAGCTCAGGTGCCACTGTACCAATGAAGAGA
TATGTGATCGGTTCCAAGGATGCCTCTGCTCTCAAGGATGGCAAGGGCT
GCAGTGTGAGAAAGAAGGCAGGCCAAGGATGACTCCACAGATAGAGG
ATTTGCCAGATCACATTGAAGTAAACAGTGGAAAATTTAACCCCATCTG
CAAAGCCTCTGGGTGGCCACTACCTACTAGTGAAGAAATGACCCTAGT
GAAGCCAGATGGGACAGTGCTCCAACCAAATGACTTCAACTATACAGA
TCGTTTCTCAGTGGCCATATTCACTGTCAACCGAGTCTTACCTCCTGACT
CAGGAGTCTGGGTCTGCAGTGTGAACACAGTGGCTGGGATGGTGGAAA
AGCCTTTCAACATTTCCGTCAAAGTTCTTCCAGAGCCCCTGCACGCCCC
AAATGTGATTGACACTGGACATAACTTTGCTATCATCAATATCAGCTCT
GAGCCTTACTTTGGGGATGGACCCATCAAATCCAAGAAGCTTTTCTATA
AACCTGTCAATCAGGCCTGGAAATACATTGAAGTGACGAATGAGATTT
TCACTCTCAACTACTTGGAGCCGCGGACTGACTACGAGCTGTGTGTGCA
GCTGGCCCGTCCTGGAGAGGGTGGAGAAGGGCATCCTGGGCCTGTGAG
ACGATTTACAACAGCGTCTATCGGACTCCCTCCTCCAAGAGGTCTCAGT
CTCCTGCCAAAAAGCCAGACAGCTCTAAATTTGACTTGGCAACCGATAT
TTACAAACTCAGAAGATGAATTTTATGTGGAAGTCGAGAGGCGATCCC
TGCAAACAACAAGTGATCAGCAGAACATCAAAGTGCCTGGGAACCTGA
CCTCGGTGCTACTGAGCAACTTAGTCCCCAGGGAGCAGTACACAGTCC
GAGCTAGAGTCAACACCAAGGCGCAGGGGGAGTGGAGTGAAGAACTC
AGGGCCTGGACCCTTAGTGACATTCTCCCTCCTCAACCAGAAAACATCA
AGATCTCCAACATCACTGACTCCACAGCTATGGTTTCTTGGACAATAGT
GGATGGCTATTCGATTTCTTCCATCATCATCCGGTATAAGGTTCAGGGC
AAAAATGAAGACCAGCACATTGATGTGAAGATCAAGAATGCTACCGTT
ACTCAGTACCAGCTCAAGGGCCTAGAGCCAGAGACTACATACCATGTG
GATATTTTTGCTGAGAACAACATAGGATCAAGCAACCCAGCCTTTTCTC
ATGAACTGAGGACGCTTCCACATTCCCCAGCCTCTGCAGACCTCGGAG
GGGGAAAGATGCTACTCATAGCCATCCTTGGGTCGGCTGGAATGACTT
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-51-
GCATCACCGTGCTGTTGGCGTTTCTGATTATGTTGCAACTGAAGAGAGC
AAATGTCCAAAGGAGAATGGCTCAGGCATTCCAGAACGTGAGAGAAG
AACCAGCTGTGCAGTTTAACTCAGGAACTCTGGCCCTTAACAGGAAGG
CCAAAAACAATCCGGATCCCACAATTTATCCTGTGCTTGACTGGAATGA
CATCAAGTTTCAAGACGTGATCGGAGAGGGCAACTTTGGCCAGGTTCT
GAAGGCACGCATCAAGAAGGATGGGTTACGGATGGATGCCGCCATCAA
GAGGATGAAAGAGTATGCCTCCAAAGATGATCACAGGGACTTCGCAGG
AGAACTGGAGGTTCTTTGTAAACTTGGACACCATCCAAACATCATTAAT
CTCTTGGGAGCATGTGAACACCGAGGCTATTTGTACCTAGCTATTGAGT
ATGCCCCGCATGGAAACCTCCTGGACTTCCTGCGTAAGAGCAGAGTGC
TAGAGACAGACCCTGCTTTTGCCATCGCCAACAGTACAGCTTCCACACT
GTCCTCCCAACAGCTTCTTCATTTTGCTGCAGATGTGGCCCGGGGGATG
GACTACTTGAGCCAGAAACAGTTTATCCACAGGGACCTGGCTGCCAGA
AACATTTTAGTTGGTGAAAACTACATAGCCAAAATAGCAGATTTTGGA
TTGTCACGAGGTCAAGAAGTGTATGTGAAAAAGACAATGGGAAGGCTC
CCAGTGCGTTGGATGGCAATCGAATCACTGAACTATAGTGTCTATACAA
CCAACAGTGATGTCTGGTCCTATGGTGTATTGCTCTGGGAGATTGTTAG
CTTAGGAGGCACCCCCTACTGCGGCATGACGTGCGCGGAGCTCTATGA
GAAGCTACCCCAGGGCTACAGGCTGGAGAAGCCCCTGAACTGTGATGA
TGAGGTGTATGATCTAATGAGACAGTGCTGGAGGGAGAAGCCTTATGA
GAGACCATCATTTGCCCAGATATTGGTGTCCTTAAACAGGATGCTGGAA
GAACGGAAGACATACGTGAACACCACACTGTATGAGAAGTTTACCTAT
GCAGGAATTGACTGCTCTGCGGAAGAAGCAGCCTAGAGCAGAACTCTT
CATGTACAACGGCCATTTCTCCTCACTGGCGCGAGAGCGCCTTGACACC
TGTACCAAGCAAGCCACCCACTGCCAAGAGATGTGATATATAAGTGTA
TATATTGTGCTGTGTTTGGGACCCTCCTCATACAGCTCGTGCGGATCTG
CAGTGTGTTCTGACTCTAATGTGACTGTATATACTGCTCGGAGTAAGAA
TGTGCTAAGATCAGAATGCCTGTTCGTGGTTTCATATAATATATTTTTCT
AAAAGCATAGATTGCACAGGAAGGTATGAGTACAAATACTGTAATGCA
TAACTTGTTATTGTCCTAGATGTGTTTGATATTTTTCCTTTACAACTGAA
TGCTATAAAAGTGTTTTGCTGTGTACACATAAGATACTGTTCGTTAAAA
TAAGCATTCCCTTGACAGCACAGGAAGAAAAGCGAGGGAAATGTATGG
ATTATATTAAATGTGGGTTACTACACAAGAGGCCGAACATTCCAAGTA
GCAGAAGAGAGGGTCTCTCAACTCTGCTCCTCACCTGCAGAAGCCAGT
TTGTTTGGCCATGTGACAATTGTCCTGTGTTTTTATAGCACCCAAATCAT
TCTAAAATATGAACATCTAAAAACTTTGCTAGGAGACTAAGAACCTTTG
GAGAGATAGATATAAGTACGGTCAAAAAACAAAACTGTGGGACTTACA
TTTATTTTCTATAGTAATCTGTTGTACATTTTAAGAAGTAAAACTAGGA
ATTTAGGAGTGATGTGTGACATTTCTGACATGGAGTTACCATCCCCACA
TGTATCACATACTGTCATATTCCCACATGTATCACACATGTATTGTAAA
ATTTTGTAGTTTTGATCACTTGTGAATTTACTGTTGATGTGGTAGCCACC
TGCTGCAATGGTTCCTCTTGTAGGTGAATAAATGTCTTGTCTACCCACA
Gene: ANGPTI (Ang-1)
Species: human
NCBI Accession No.: NM_001146
SEQ ID NO: 651
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
- 52 -
Sequence:
GGGGCACACTCATGCATTCCTGTCAAGTCATCTTGTGAAAGGCTGCCTG
CTTCCAGCTTGGCTTGGATGTGCAACCTTAATAAAACTCACTGAGGTCT
GGGAGAAAATAGCAGATCTGCAGCAGATAGGGTAGAGGAAAGGGTCT
AGAATATGTACACGCAGCTGACTCAGGCAGGCTCCATGCTGAACGGTC
ACACAGAGAGGAAACAATAAATCTCAGCTACTATGCAATAAATATCTC
AAGTTTTAACGAAGAAAAACATCATTGCAGTGAAATAAAAAATTTTAA
AATTTTAGAACAAAGCTAACAAATGGCTAGTTTTCTATGATTCTTCTTC
AAACGCTTTCTTTGAGGGGGAAAGAGTCAAACAAACAAGCAGTTTTAC
CTGAAATAAAGAACTAGTTTTAGAGGTCAGAAGAAAGGAGCAAGTTTT
GCGAGAGGCACGGAAGGAGTGTGCTGGCAGTACAATGACAGTTTTCCT
TTCCTTTGCTTTCCTCGCTGCCATTCTGACTCACATAGGGTGCAGCAATC
AGCGCCGAAGTCCAGAAAACAGTGGGAGAAGATATAACCGGATTCAA
CATGGGCAATGTGCCTACACTTTCATTCTTCCAGAACACGATGGCAACT
GTCGTGAGAGTACGACAGACCAGTACAACACAAACGCTCTGCAGAGAG
ATGCTCCACACGTGGAACCGGATTTCTCTTCCCAGAAACTTCAACATCT
GGAACATGTGATGGAAAATTATACTCAGTGGCTGCAAAAACTTGAGAA
TTACATTGTGGAAAACATGAAGTCGGAGATGGCCCAGATACAGCAGAA
TGCAGTTCAGAACCACACGGCTACCATGCTGGAGATAGGAACCAGCCT
CCTCTCTCAGACTGCAGAGCAGACCAGAAAGCTGACAGATGTTGAGAC
CCAGGTACTAAATCAAACTTCTCGACTTGAGATACAGCTGCTGGAGAA
TTCATTATCCACCTACAAGCTAGAGAAGCAACTTCTTCAACAGACAAAT
GAAATCTTGAAGATCCATGAAAAAAACAGTTTATTAGAACATAAAATC
TTAGAAATGGAAGGAAAACACAAGGAAGAGTTGGACACCTTAAAGGA
AGAGAAAGAGAACCTTCAAGGCTTGGTTACTCGTCAAACATATATAAT
CCAGGAGCTGGAAAAGCAATTAAACAGAGCTACCACCAACAACAGTGT
CCTTCAGAAGCAGCAACTGGAGCTGATGGACACAGTCCACAACCTTGT
CAATCTTTGCACTAAAGAAGGTGTTTTACTAAAGGGAGGAAAAAGAGA
GGAAGAGAAACCATTTAGAGACTGTGCAGATGTATATCAAGCTGGTTT
TAATAAAAGTGGAATCTACACTATTTATATTAATAATATGCCAGAACCC
AAAAAGGTGTTTTGCAATATGGATGTCAATGGGGGAGGTTGGACTGTA
ATACAACATCGTGAAGATGGAAGTCTAGATTTCCAAAGAGGCTGGAAG
GAATATAAAATGGGTTTTGGAAATCCCTCCGGTGAATATTGGCTGGGG
AATGAGTTTATTTTTGCCATTACCAGTCAGAGGCAGTACATGCTAAGAA
TTGAGTTAATGGACTGGGAAGGGAACCGAGCCTATTCACAGTATGACA
GATTCCACATAGGAAATGAAAAGCAAAACTATAGGTTGTATTTAAAAG
GTCACACTGGGACAGCAGGAAAACAGAGCAGCCTGATCTTACACGGTG
CTGATTTCAGCACTAAAGATGCTGATAATGACAACTGTATGTGCAAATG
TGCCCTCATGTTAACAGGAGGATGGTGGTTTGATGCTTGTGGCCCCTCC
AATCTAAATGGAATGTTCTATACTGCGGGACAAAACCATGGAAAACTG
AATGGGATAAAGTGGCACTACTTCAAAGGGCCCAGTTACTCCTTACGTT
CCACAACTATGATGATTCGACCTTTAGATTTTTGAAAGCGCAATGTCAG
AAGCGATTATGAAAGCAACAAAGAAATCCGGAGAAGCTGCCAGGTGA
GAAACTGTTTGAAAACTTCAGAAGCAAACAATATTGTCTCCCTTCCAGC
AATAAGTGGTAGTTATGTGAAGTCACCAAGGTTCTTGACCGTGAATCTG
GAGCCGTTTGAGTTCACAAGAGTCTCTACTTGGGGTGACAGTGCTCACG
TGGCTCGACTATAGAAAACTCCACTGACTGTCGGGCTTTAAAAAGGGA
AGAAACTGCTGAGCTTGCTGTGCTTCAAACTACTACTGGACCTTATTTT
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
- 53 -
GGAACTATGGTAGCCAGATGATAAATATGGTTAATTTCATGTAAAACA
GAAAAAAAGAGTGAAAAAGAGAATATACATGAAGAATAGAAACAAGC
CTGCCATAATCCTTTGGAAAAGATGTATTATACCAGTGAAAAGGTGTTA
TATCTATGCAAACCTACTAACAAATTATACTGTTGCACAATTTTGATAA
AAATTTAGAACAGCATTGTCCTCTGAGTTGGTTAAATGTTAATGGATTT
CAGAAGCCTAATTCCAGTATCATACTTACTAGTTGATTTCTGCTTACCC
ATCTTCAAATGAAAATTCCATTTTTGTAAGCCATAATGAACTGTAGTAC
ATGGACAATAAGTGTGTGGTAGAAACAAACTCCATTACTCTGATTTTTG
ATACAGTTTTCAGAAAAAGAAATGAACATAATCAAGTAAGGATGTATG
TGGTGAAAACTTACCACCCCCATACTATGGTTTTCATTTACTCTAAAAA
CTGATTGAATGATATATAAATATATTTATAGCCTGAGTAAAGTTAAAAG
AATGTAAAATATATCATCAAGTTCTTAAAATAATATACATGCATTTAAT
ATTTCCTTTGATATTATACAGGAAAGCAATATTTTGGAGTATGTTAAGT
TGAAGTAAAAGCAAGTACTCTGGAGCAGTTCATTTTACAGTATCTACTT
GCATGTGTATACATACATGTAACTTCATTATTTTAAAAATATTTTTAGA
ACTCCAATACTCACCCTGTTATGTCTTGCTAATTTAAATTTTGCTAATTA
ACTGAAACATGCTTACCAGATTCACACTGTTCCAGTGTCTATAAAAGAA
ACACTTTGAAGTCTATAAAAAATAAAATAATTATAAATATCATTGTACA
TAGCATGTTTATATCTGCAAAAAACCTAATAGCTAATTAATCTGGAATA
TGCAACATTGTCCTTAATTGATGCAAATAACACAAATGCTCAAAGAAA
TCTACTATATCCCTTAATGAAATACATCATTCTTCATATATTTCTCCTTC
AGTCCATTCCCTTAGGCAATTTTTAATTTTTAAAAATTATTATCAGGGG
AGAAAAATTGGCAAAACTATTATATGTAAGGGAAATATATACAAAAAG
AAAATTAATCATAGTCACCTGACTAAGAAATTCTGACTGCTAGTTGCCA
TAAATAACTCAATGGAAATATTCCTATGGGATAATGTATTTTAAGTGAA
TTTTTGGGGTGCTTGAAGTTACTGCATTATTTTATCAAGAAGTCTTCTCT
GCCTGTAAGTGTCCAAGGTTATGACAGTAAACAGTTTTTATTAAAACAT
GAGTCACTATGGGATGAGAAAATTGAAATAAAGCTACTGGGCCTCCTC
TCATAAAAGAGACAGTTGTTGGCAAGGTAGCAATACCAGTTTCAAACT
TGGTGACTTGATCCACTATGCCTTAATGGTTTCCTCCATTTGAGAAAAT
AAAGCTATTCACATTGTTAAGAAAAATACTTTTTAAAGTTTACCATCAA
GTCTTTTTTATATTTATGTGTCTGTATTCTACCCCTTTTTGCCTTACAAGT
GATATTTGCAGGTATTATACCATTTTTCTATTCTTGGTGGCTTCTTCATA
GCAGGTAAGCCTCTCCTTCTAAAAACTTCTCAACTGTTTTCATTTAAGG
GAAAGAAAATGAGTATTTTGTCCTTTTGTGTTCCTACAGACACTTTCTT
AAACCAGTTTTTGGATAAAGAATACTATTTCCAAACTCATATTACAAAA
ACAAAATAAAATAATAAAAAAAGAAAGCATGATATTTACTGTTTTGTT
GTCTGGGTTTGAGAAATGAAATATTGTTTCCAATTATTTATAATAAATC
AGTATAAAATGTTTTATGATTGTTATGTGTATTATGTAATACGTACATG
TTTATGGCAATTTAACATGTGTATTCTTTTAATTGTTTCAGAATAGGATA
ATTAGGTATTCGAATTTTGTCTTTAAAATTCATGTGGTTTCTATGCAAAG
TTCTTCATATCATCACAACATTATTTGATTTAAATAAAATTGAAAGTAA
TATTTGTGCAA
Gene: Angpt 1(Ang-1)
Species: mouse
NCBI Accession No.: NM_009640
SEQ ID NO: 652
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-54-
Sequence:
GGAAAGGGGCTAGAATATGTACTCGCAGCTGACGCGGGCAGGCTCCAC
GCTGAACGGTTACACAGAGAGGAAACAATAAATCTAAGCTACTATTGC
AATAAATATCTCAAGTTTTAACGAAGGAAACTATCATTACAGTTAAAAT
TTTTTAAAGTAACGCTTTTTTAGAACAAAGCTAACAAATGGCTAGTTTT
CTGTGGATCTTCTTCAAACGCTTTCTTTAACGGGGAAAGAGTCAAACAA
GCAGTTTTACCTGAAATAAAGAACTAGTTTAAAGGTCAGAAGAGAAGA
GCAAGCTTTGCAGGAGGCACGGAAGGCAAGCGCTGGCAGTACAATGAC
AGTTTTCCTTTCCTTTGCATTCTTCGCTGCCATTCTGACTCACATAGGGT
GCAGCAACCAGCGCCGAAATCCAGAAAACGGAGGGAGAAGATATAAC
CGGATTCAACATGGGCAATGTGCCTACACTTTCATTCTTCCAGAACACG
ACGGGAACTGCCGTGAGAGTGCGACAGAGCAGTACAACACCAACGCTC
TGCAAAGGGATGCTCCACACGTGGAGCCGGATTTCTCTTCCCAGAAACT
TCAGCATCTGGAGCATGTGATGGAAAATTATACTCAGTGGCTGCAAAA
ACTTGAGAATTACATTGTGGAAAATATGAAGTCGGAGATGGCCCAGAT
ACAACAGAATGCTGTTCAAAACCACACGGCCACCATGCTTGAGATAGG
AACCAGTCTCTTATCTCAGACTGCAGAGCAGACCCGAAAGCTGACAGA
TGTTGAGACCCAGGTACTAAATCAAACATCCCGACTTGAAATACAACT
GCTAGAGAATTCATTATCAACATACAAGCTAGAGAAGCAACTTCTCCA
ACAGACAAATGAAATTCTGAAGATTCACGAAAAAAACAGTTTACTAGA
GCACAAAATCTTAGAAATGGAGGGAAAACACAAAGAAGAATTGGACA
CCTTGAAGGAGGAGAAAGAAAACCTTCAAGGCTTGGTTTCTCGTCAGA
CATTCATCATCCAGGAGTTGGAGAAGCAACTTAGTAGAGCTACCAACA
ACAACAGCATCCTGCAGAAGCAACAACTGGAGCTCATGGACACAGTTC
ATAACCTTATCAGCCTTTGCACTAAAGAAGGTGTTTTGCTAAAGGGAGG
AAAAAGAGAAGAAGAGAAACCATTTCGAGACTGTGCAGATGTATATCA
AGCTGGTTTTAATAAAAGTGGAATCTACACTATTTATTTTAATAATATG
CCAGAACCCAAAAAGGTATTTTGCAATATGGATGTGAATGGGGGAGGT
TGGACAGTAATACAACACCGGGAAGATGGAAGCCTGGATTTCCAGAGG
GGCTGGAAGGAGTATAAAATGGGTTTTGGGAATCCCTCTGGTGAATAT
TGGCTTGGGAACGAGTTCATTTTTGCAATAACCAGTCAGAGGCAGTAC
ATGCTGAGGATTGAGCTGATGGACTGGGAAGGGAACCGAGCCTACTCA
CAGTACGACAGATTCCACATAGGAAATGAAAAGCAGAACTATAGGTTA
TATTTAAAAGGTCACACAGGGACAGCAGGCAAACAGAGCAGCTTGATC
TTACACGGTGCCGATTTCAGCACGAAGGATGCTGATAACGACAACTGT
ATGTGCAAATGCGCTCTCATGCTAACAGGAGGTTGGTGGTTCGATGCCT
GTGGCCCTTCCAATCTAAATGGAATGTTCTACACTGCGGGACAAAATCA
TGGAAAACTGAATGGGATAAAGTGGCACTACTTCAAAGGGCCCAGTTA
CTCCTTACGTTCCACCACCATGATGATCCGGCCCTTGGACTTTTGAAGG
TGCTCTGCCAGTATTAGAAAGCTGCAAAGAAAGCTGGGCATGTTCCCA
GATGAGAAGCTAGTCAGAGGCTTCAGAAACAACCAACATTGTCTCCAT
TCCAGCAGCAAGTGGTTATGTCATGTCACCTGGGTTTGGAGCCTTCTGA
GGTCAACAGAATCGCCACTTGGGTCCAGAGAATGCCACTCACAATCAT
GTTTAAAAGGGAAGAAACTTCTCAGCTTGCTGCACTTCAAAGTGCTACT
GGATCACATTCTGAACTTATAACATCCTGATGCTGAATGCAACTTGTTT
CATGTAAAAGCAAAAGAAGAAGAAACAGCAAATGGGAACAGGCTTTC
CAGAATCTGTTGAAGATGGATTGTGGAGGTGACCTGGTATCACTGTAG
GAAATCCTGCTAACAATACATCACTGCCCAAAAGAGACATAAAGAAAA
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-55-
GTTTTGTCTACTGAGTTGGCTAAAAGTTAGTGGAGTTCACCTGCCCATT
TCCAGTATCATATTTACTAGCTGATTTCAGGTTTCCTGTGTTCAAATGTA
AACTCTGTTCTTGTAAGCCATGATACAATATAGTACATGGAGGATAAG
AGTTGGGGGTAGAAGGTGCCTAAAGACTCTTGAGTTTCTGGGGATTCA
GTTTTCAAAAGATATAAAATATAATCAAGAATGGATAAAACAGGTGAA
AATCACACTCATGCTACAGTGTTCCTTTACATGAAATTTGATTAACTGA
TCCACAAGAATGTTTAGAGCCTGAGTATATATAAAGACTGGAAGTGTT
ATCACCCAGTTCTCAAAACAATAAGCAGGCAGTTAACATTCTCATTGAC
AGTATGTAGGAGAGCAATATGTGGAGTACTTGAGTTGGAACAGCCCAT
TGTACAGATCTTGCATGTATTTGCATATGTATGGCATTATTATTTTTAAA
GTGTTCGTAGGCCTTCAATTCTTCATACAGATTTTTCATGCTAATTTAAT
TTTTGTTAATTAACTGCAATGTACTTACTAAATATATCCTACTCCAGTTT
TTTATGAGTTATACTTTAAAGTCTACAAATAATAGAAGAATTTTAAATA
TCATTGTACATAATATCTTATACCTGTCCATGCTAAACTCAATAATTGTT
TAGTCTGGAATATATGATGCTGTCCACAACTGATGACTATAAATATGAT
TGTTTAAAGACAGTTACCATACTATTGATTAAATATATTACTCTGCATA
GTTTTTCTCCTCCAGGATCTGTTTCTTCAAGCAATTTCTACCTTGTAAAA
TAATGGTAGTAGAGAAAATTGACATAACTCCTTGTACAAAAGAATTAT
AGAAAAAATTACAGTCATTTGACTAGGAAGTTTCTGATTGTTAGCTGCT
ATAAGTGCCTTAGTTAAGATGCCCCTGTGTTATAATATGTAGTAAATGA
AGTTTTGGACACAGGATTCTGTGATAACCTGATGTGACTGCAGTATTCT
ATCAAGTTCTCTTTGTTGTTAAATGTTCAAGGTTATAGTAGAAAAAAAA
CATTCAATCAAACACAATTTGCCATGAAAGGAGAGAACTAAATGTAGG
CACCAGTTCTGTTTTCTCAGAGAAGGAGAAGACTTTCTGGGACGTACAT
GTACCAAAATATAAATCTTGATAACCGCAGCCACAAAGCCTTAGTGAC
TTTCCTCTACCTGGTAAGACAGAGCTCTTCATGCTTTTAAGAAAAGATT
CTGAATGCTTCCCACCACATCTTTCTTATATTTATATGTGTTCATAAAGT
ACTATTTTGCCTTACAAGAGGTATGTGCCGACATTACAGGATTTTTCTA
CTATAGTGACTCCTTCACAGCTTTCTTAAGCCTAGCCCTCTAAAAGCTT
CCTTCTCATTTAGATGAAAGAAAATGAGTATTTTTGTGATTCTGGTGAT
TGTGGTGGTTGTTGTTGTTGTTGTTGTTGTTCCCACAGATGTTCGAAAAC
TCATCTTGGGTAAATTGTTTTTCAATCCACATTACAAAAATAAAGCGAA
ACAAGGAGAAAAAAAAGCATGGAATTTACTGATTTGTTATGTGGGTTT
GAAAAATAAGATATTGTTTTCAGTTATTTATAATAAAGCAGTATAATGT
GTACATTGTATAATGCCAACATGTGTGTAGCAATTTGATACGCATAGCT
TTTTGCATTTAATTAATGCAGGGCAGAAAAATTAGATAACTCGAACTTT
GTCTTGAAGTTTCTATTTCAATAAAAGCTGTGTCATTTCTATGAAAA
Gene: ANGPT2 (Ang-2)
Species: mouse
NCBI Accession No.: NM_009640
SEQ ID NO: 653
Sequence:
AAAGTGATTGATTCGGATACTGACACTGTAGGATCTGGGGAGAGAGGA
ACAAAGGACCGTGAAAGCTGCTCTGTAAAAGCTGACACAGCCCTCCCA
AGTGAGCAGGACTGTTCTTCCCACTGCAATCTGACAGTTTACTGCATGC
CTGGAGAGAACACAGCAGTAAAAACCAGGTTTGCTACTGGAAAAAGA
GGAAAGAGAAGACTTTCATTGACGGACCCAGCCATGGCAGCGTAGCAG
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
- 56 -
CCCTGCGTTTTAGACGGCAGCAGCTCGGGACTCTGGACGTGTGTTTGCC
CTCAAGTTTGCTAAGCTGCTGGTTTATTACTGAAGAAAGAATGTGGCAG
ATTGTTTTCTTTACTCTGAGCTGTGATCTTGTCTTGGCCGCAGCCTATAA
CAACTTTCGGAAGAGCATGGACAGCATAGGAAAGAAGCAATATCAGGT
CCAGCATGGGTCCTGCAGCTACACTTTCCTCCTGCCAGAGATGGACAAC
TGCCGCTCTTCCTCCAGCCCCTACGTGTCCAATGCTGTGCAGAGGGACG
CGCCGCTCGAATACGATGACTCGGTGCAGAGGCTGCAAGTGCTGGAGA
ACATCATGGAAAACAACACTCAGTGGCTAATGAAGCTTGAGAATTATA
TCCAGGACAACATGAAGAAAGAAATGGTAGAGATACAGCAGAATGCA
GTACAGAACCAGACGGCTGTGATGATAGAAATAGGGACAAACCTGTTG
AACCAAACAGCGGAGCAAACGCGGAAGTTAACTGATGTGGAAGCCCA
AGTATTAAATCAGACCACGAGACTTGAACTTCAGCTCTTGGAACACTCC
CTCTCGACAAACAAATTGGAAAAACAGATTTTGGACCAGACCAGTGAA
ATAAACAAATTGCAAGATAAGAACAGTTTCCTAGAAAAGAAGGTGCTA
GCTATGGAAGACAAGCACATCATCCAACTACAGTCAATAAAAGAAGAG
AAAGATCAGCTACAGGTGTTAGTATCCAAGCAAAATTCCATCATTGAA
GAACTAGAAAAAAAAATAGTGACTGCCACGGTGAATAATTCAGTTCTT
CAGAAGCAGCAACATGATCTCATGGAGACAGTTAATAACTTACTGACT
ATGATGTCCACATCAAACTCAGCTAAGGACCCCACTGTTGCTAAAGAA
GAACAAATCAGCTTCAGAGACTGTGCTGAAGTATTCAAATCAGGACAC
ACCACGAATGGCATCTACACGTTAACATTCCCTAATTCTACAGAAGAG
ATCAAGGCCTACTGTGACATGGAAGCTGGAGGAGGCGGGTGGACAATT
ATTCAGCGACGTGAGGATGGCAGCGTTGATTTTCAGAGGACTTGGAAA
GAATATAAAGTGGGATTTGGTAACCCTTCAGGAGAATATTGGCTGGGA
AATGAGTTTGTTTCGCAACTGACTAATCAGCAACGCTATGTGCTTAAAA
TACACCTTAAAGACTGGGAAGGGAATGAGGCTTACTCATTGTATGAAC
ATTTCTATCTCTCAAGTGAAGAACTCAATTATAGGATTCACCTTAAAGG
ACTTACAGGGACAGCCGGCAAAATAAGCAGCATCAGCCAACCAGGAA
ATGATTTTAGCACAAAGGATGGAGACAACGACAAATGTATTTGCAAAT
GTTCACAAATGCTAACAGGAGGCTGGTGGTTTGATGCATGTGGTCCTTC
CAACTTGAACGGAATGTACTATCCACAGAGGCAGAACACAAATAAGTT
CAACGGCATTAAATGGTACTACTGGAAAGGCTCAGGCTATTCGCTCAA
GGCCACAACCATGATGATCCGACCAGCAGATTTCTAAACATCCCAGTC
CACCTGAGGAACTGTCTCGAACTATTTTCAAAGACTTAAGCCCAGTGCA
CTGAAAGTCACGGCTGCGCACTGTGTCCTCTTCCACCACAGAGGGCGTG
TGCTCGGTGCTGACGGGACCCACATGCTCCAGATTAGAGCCTGTAAACT
TTATCACTTAAACTTGCATCACTTAACGGACCAAAGCAAGACCCTAAAC
ATCCATAATTGTGATTAGACAGAACACCTATGCAAAGATGAACCCGAG
GCTGAGAATCAGACTGACAGTTTACAGACGCTGCTGTCACAACCAAGA
ATGTTATGTGCAAGTTTATCAGTAAATAACTGGAAAACAGAACACTTAT
GTTATACAATACAGATCATCTTGGAACTGCATTCTTCTGAGCACTGTTT
ATACACTGTGTAAATACCCATATGTCCTGAATTCACCATCACTATCACA
ATTAAAAGGAAGAAAAAAACTCTCTAAGCCATAAAAAGACATATTCAG
GGATATTCTGAGAAGGGGTTACTAGAAGTTTAATATTTGGAAAAACAG
TTAGTGCATTTTTACTCCATCTCTTAGGTGCTTTAAATTTTTATTTCAAA
AACAGCGTATTTACATTTATGTTGACAGCTTAGTTATAAGTTAATGCTC
AAATACGTATTTCAAATTTATATGGTAGAAACTTCCAGAATCTCTGAAA
TTATCAACAGAAACGTGCCATTTTAGTTTATATGCAGACCGTACTATTT
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-57-
TTTTCTGCCTGATTGTTAAATATGAAGGTATTTTTAGTAATTAAATATAA
CTTATTAGGGGATATGCCTATGTTTAACTTTTATGATAATATTTACAATT
TTATAATTTGTTTCCAAAAGACCTAATTGTGCCTTGTGATAAGGAAACT
TCTTACTTTTAATGATGAGGAAAATTATACATTTCATTCTATGACAAAG
AAACTTTACTATCTTCTCACTATTCTAAAACAGAGGTCTGTTTTCTTTCC
TAGTAAGATATATTTTTATAGAACTAGACTACAATTTAATTTCTGGTTG
AGAAAAGCCTTCTATTTAAGAAATTTACAAAGCTATATGTCTCAAGATT
CACCCTTAAATTTACTTAAGGAAAAAAATAATTGACACTAGTAAGTTTT
TTTATGTCAATCAGCAAACTGAAAAAAAAAAAAGGGTTTCAAAGTGCA
AAAACAAAATCTGATGTTCATAATATATTTAAATATTTACCAAAAATTT
GAGAACACAGGGCTGGGCGCAGTGGCTCACACCTATAATCCCAGTACA
TTGGTAGGCAAGGTGGGCAGATCACCTGAGGTCAGGAGTTCAAGACCA
GCCTGGACAACATGGTGAAACCCTGTCTCTACTAAATAATACAAAAAT
TAGCCAGGCGTGCTGGCGGGCACCTGTAATCCCAGCTACTCGGGAGGC
TGAGGCAGGGAGAATTGCTTGCACCAGGGAGGTAGAGGTTGCAGTGAG
CCAAGATCGCACCACTGCACTCCAGCCGGGGCAACAGAGCAAGACTCC
ATCTCAAAAAAAAAAAAAAAAAAAGAAAGAAAAGAAAATTTGAGAAC
ACAGCTTTATACTCGGGACTACAAAACCATAAACTCCTGGAGTTTTAAC
TCCTTTTGAAATTTTCATAGTACAATTAATACTAATGAACATTTGTGTA
AAGCTTTATAATTTAAAGGCAATTTCTCATATATTCTTTTCTGAATCATT
TGCAAGGAAGTTCAGAGTCCAGTCTGTAACTAGCATCTACTATATGTCT
GTCTTCACCTTACAGTGTTCTACCATTATTTTTTCTTTATTCCATTTCAAA
ATCTAATTTATTTTACCCCAACTTCTCCCCACCACTTGACGTAGTTTTAG
AACACACAGGTGTTGCTACATATTTGGAGTCAATGATGGACTCTGGCA
AAGTCAAGGCTCTGTTTTATTTCCACCAAGGTGCACTTTTCCAACAACT
ATTTAACTAGTTAAGAACCTCCCTATCTTAGAACTGTATCTACTTTATAT
TTAAGAAGGTTTTATGAATTCAACAACGGTATCATGGCCTTGTATCAAG
TTGAAAAACAACTGAAAATAAGAAAATTTCACAGCCTCGAAAGACAAC
AACAAGTTTCTAGGATATCTCAATGACAAGAGTGATGGATACTTAGGT
AGGGAAACGCTAATGCAGGAAAAACTGGCAACAACACAATTTATATCA
ATTCTCTTTGTAGGCAGGTGATAAAAAATTCAAGGACAAATCTCATTAT
GTCATTGTGCATCATATATAATCTCTTATGAGCGAGAATGGGGGGAATT
TGTGTTTTTACTTTACACTTCAATTCCTTACACGGTATTTCAAACAAACA
GTTTTGCTGAGAGGAGCTTTTGTCTCTCCTTAAGAAAATGTTTATAAAG
CTGAAAGGAAATCAAACAGTAATCTTAAAAATGAAAACAAAACAACCC
AACAACCTAGATAACTACAGTGATCAGGGAGCACAGTTCAACTCCTTG
TTATGTTTTAGTCATATGGCCTACTCAAACAGCTAAATAACAACACCAG
TGGCAGATAAAAATCACCATTTATCTTTCAGCTATTAATCTTTTGAATG
AATAAACTGTGACAAACAAATTAACATTTTTGAACATGAAAGGCAACT
TCTGCACAATCCTGTATCCAAGCAAACTTTAAATTATCCACTTAATTAT
TACTTAATCTTAAAAAAAATTAGAACCCAGAACTTTTCAATGAAGCATT
TGAAAGTTGAAGTGGAATTTAGGAAAGCCATAAAAATATAAATACTGT
TATCACAGCACCAGCAAGCCATAATCTTTATACCTATCAGTTCTATTTC
TATTAACAGTAAAAACATTAAGCAAGATATAAGACTACCTGCCCAAGA
ATTCAGTCTTTTTTCATTTTTGTTTTTCTCAGTTCTGAGGATGTTAATCGT
CAAATTTTCTTTGGACTGCATTCCTCACTACTTTTTGCACAATGGTCTCA
CGTTCTCACATTTGTTCTCGCGAATAAATTGATAAAAGGTGTTAAGTTC
TGTGAATGTCTTTTTAATTATGGGCATAATTGTGCTTGACTGGATAAAA
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-58-
ACTTAAGTCCACCCTTATGTTTATAATAATTTCTTGAGAACAGCAAACT
GCATTTACCATCGTAAAACAACATCTGACTTACGGGAGCTGCAGGGAA
GTGGTGAGACAGTTCGAACGGCTCCTCAGAAATCCAGTGACCCAATTC
TAAAGACCATAGCACCTGCAAGTGACACAACAAGCAGATTTATTATAC
ATTTATTAGCCTTAGCAGGCAATAAACCAAGAATCACTTTGAAGACAC
AGCAAAAAGTGATACACTCCGCAGATCTGAAATAGATGTGTTCTCAGA
CAACAAAGTCCCTTCAGAATCTTCATGTTGCATAAATGTTATGAATATT
AATAAAAAGTTGATTGAGAAAAA
Gene: Angpt2 (Ang-2)
Species: mouse
NCBI Accession No.: NM_007426
SEQ ID NO: 654
Sequence:
GATACTGACACTGTAGACTCAGGGGAGAAACAAAGAGTCCGTGCAGAC
CTCTGGAGTGAGCAGGGCTGCTCCTTCCTCTCAGGACAGCTCCGAGTGT
GCCGGGGAGAAGAGAAGAGAAGAGACAGGCACTGGGAAAGAGCCTGC
TGCGGGACGGAGAAGGCTCTCACTGATGGACTTATTCACACGGCACAG
CCCTGTGCCTTAGACAGCAGCTGAGAGCTCAGGACGCAAGTTTGCTGA
ACTCACAGTTTAGAACCCAAAAAGAGAGAGAGAATGTGGCAGATCATT
TTCCTAACTTTTGGCTGGGATCTTGTCTTGGCCTCAGCCTACAGTAACTT
TAGGAAGAGCGTGGACAGCACAGGCAGAAGGCAGTACCAGGTCCAGA
ACGGACCCTGCAGCTACACGTTCCTGCTGCCGGAGACCGACAGCTGCC
GATCTTCCTCCAGCCCCTACATGTCCAATGCCGTGCAGAGGGATGCACC
CCTCGACTACGACGACTCAGTGCAAAGGCTGCAGGTGCTGGAGAACAT
TCTAGAGAACAACACACAGTGGCTGATGAAGCTGGAGAATTACATTCA
GGACAACATGAAGAAGGAGATGGTGGAGATCCAACAGAATGTGGTGC
AGAACCAGACAGCTGTGATGATAGAGATTGGAACCAGCTTGCTGAACC
AGACAGCAGCACAAACTCGGAAACTGACTGATGTGGAAGCCCAAGTAC
TAAACCAGACGACAAGACTCGAGCTGCAGCTTCTCCAACATTCTATTTC
TACCAACAAATTGGAAAAGCAGATTTTGGATCAGACCAGTGAAATAAA
CAAGCTACAAAATAAGAACAGCTTCCTAGAACAGAAAGTTCTGGACAT
GGAGGGCAAGCACAGCGAGCAGCTACAGTCCATGAAGGAGCAGAAGG
ACGAGCTCCAGGTGCTGGTGTCCAAGCAGAGCTCTGTCATTGACGAGC
TGGAGAAGAAGCTGGTGACAGCCACGGTCAACAACTCGCTCCTTCAGA
AGCAGCAGCATGACCTAATGGAGACCGTCAACAGCTTGCTGACCATGA
TGTCATCACCCAACTCCAAGAGCTCGGTTGCTATCCGTAAAGAAGAGC
AAACCACCTTCAGAGACTGTGCGGAAATCTTCAAGTCAGGACTCACCA
CCAGTGGCATCTACACACTGACCTTCCCCAACTCCACAGAGGAGATCA
AGGCCTACTGTGACATGGACGTGGGTGGAGGAGGGTGGACAGTCATCC
AACACCGAGAAGATGGCAGTGTGGACTTCCAGAGGACGTGGAAAGAA
TACAAAGAGGGCTTCGGGAGCCCTCTGGGAGAGTACTGGCTGGGCAAT
GAGTTTGTCTCCCAGCTGACCGGTCAGCACCGCTACGTGCTTAAGATCC
AGCTGAAGGACTGGGAAGGCAACGAGGCGCATTCGCTGTATGATCACT
TCTACCTCGCTGGTGAAGAGTCCAACTACAGGATTCACCTTACAGGACT
CACGGGGACCGCGGGCAAAATAAGTAGCATCAGCCAACCAGGAAGTG
ATTTTAGCACAAAGGATTCGGACAATGACAAATGCATCTGCAAGTGTT
CCCAGATGCTCTCAGGAGGCTGGTGGTTTGACGCATGTGGTCCTTCCAA
CA 02692632 2010-01-05
WO 2009/008990 PCT/US2008/008232
-59-
CTTGAATGGACAGTACTACCCACAAAAACAGAATACAAATAAGTTTAA
CGGTATCAAGTGGTACTACTGGAAGGGGTCCGGCTACTCGCTCAAGGC
CACAACCATGATGATCCGGCCAGCAGATTTCTAAATGCCTGCCTACACT
ACCAGAAGAACTTGCTGCATCCAAAGATTAACTCCAAGGCACTGAGAG
ACACCAATGCATAGCAGCCCCTTTCCACATCAGGAAGTGCTCCTGGGG
GTGGGGAGGGTCTGTGTGTACCAGACTGAAGCGCATCACTTAAGCCTG
CACCGCTAACCAACCAAAGGCACTGCAGTCTGGAGAAACACTTCTGGG
AAGGTTGTGGCTGAGGATCAGAAGGACAGCGTGCAGACTCTGTCACAG
GGAAGAATGTTCCGTGGGAGTTCAGCAGTAAATAACTGGAAAACAGAA
CACTTAGATGGTGCAGATAAATCTTGGGACCACATTCCTCTAAGCACGG
TTTCTAGAGTGAATACATTCACAGCTCGGCTGTCACAATGACAAGGCCG
TGTCCTCGCACTGTGGCAGCCAGTATCCAGGGACTTCTAAGTGGTGGGC
ACAGGTTATCATCTGGAGAAGCACACATTCATTGTTTTCCTCTTGGGTG
CTTTACATGTTCATTTGAAAACAACACATTTACCTATCTTGATGGCTTA
GTTTTTAATGGCTGGCTACTATTTACTATATGGCAAAAATGCCCACATC
TCTGGAATAACCACCAAATAAGCGCCATGTTGGTGAATGCGGAGACTG
TACTATTTTGTTTTCTTCCTGGCTGTTAAATATGAAGGTATTTTTAGTAA
TTAAATATAAGTTATT
