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TARGET HOST FACTORS FOR TREATING VIRAL INFECTION
TECHNICAL FIELD
[0001] The present disclosure relates to methods and compositions for treating
viral infection by
manipulating expression of a target sequence. The methods and compositions
may, for example, be useful in
treating virus infection and/or reducing virus replication and/or virus-
mediated cytotoxicity.
BACKGROUND
[0002] Infections from influenza viruses pose potentially catastrophic global
economic and health
threats. Most current therapeutic strategies are directed at viral entry
prevention, such as vaccination. These
to strategies face challenges because viruses display considerable antigenic
malleability which enable then to
circumvent innate and/or acquired immunity.
[0003] Influenza viruses pose a significant threat to the world population as
causative agents in
seasonal epidemics that claim many lives annually. Furthermore pandemic
outbreaks of influenza can have a
significant health and economic impact. The emergence of highly-pathogenic
avian influenza virus H5N 1
and the pandemic outbreak of swine-origin H IN 1 influenza virus (SOIV)
strongly signals the need for
further investigation of this complex virus and its pathogenicity.
[0004] Consequently, developing strategies for preventing and treating viral
infection is desirable.
New and effective compositions are needed to prevent and treat influenza virus
infection.
SUMMARY
[0005] The present invention relates to methods and compositions for treating
viral infection by
manipulating expression of a target sequence. The methods and compositions may
be useful in treating virus
infection and/or reducing virus replication and/or virus-mediated
cytotoxicity.
[0006] According to the present disclosure there is provided a method of
reducing virus replication
comprising contacting virus-infected cells with an effective amount of at
least one inhibitor of ABI2,
ARRDC3, BAD, BRCA1, C17orf85, Clorf7l, C6orf162, CCNJL, CFLI, GON4L, HCG
1986447,
HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI,
MERTK,
MX2, NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7AI4,
ST8SIA3,
STX3, TMC6, TMTC4, TNFSF12-TNFSFI3, TNFSFI3, TTN, UBXN7, USP47, WNK2, YPEL2,
ZNF251
and/or SCG2.
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[0007] The present disclosure also provides a method of reducing virus-
mediated cytotoxicity comprising
contacting virus-infected cells with an effective amount of at least one
inhibitor of ABI2, ARRDC3, BAD,
BRCA1, C17orf85, Clorf7l, C6orf162, CCNJL, CFLI, GON4L, HCG 1986447,
HISTIH2AB, HPS4,
LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX 1, MERTK, MX2, NRGI,
OR52A1,
PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7AI4, ST8SIA3, STX3, TMC6,
TMTC4,
TNFSFI2-TNFSFI3, TNFSFI3, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2.
[0008] The present disclosure further provides a method for treating a viral
infection in a subject comprising
administering to the subject a composition comprising at least one inhibitor
of ABI2, ARRDC3, BAD,
BRCAI, C17orf85, Clorf7l, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447,
HISTIH2AB, HPS4,
to LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MXI, MERTK, MX2, NRGI,
OR52A1,
PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6,
TMTC4,
TNFSFI2-TNFSFI3, TNFSFI3, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2,
wherein
the composition reduces expression or activity of the ABI2, ARRDC3, BAD,
BRCAI, C 17orf85, C lorf7l,
C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HISTIH2AB, HPS4, LHX8, RPS25,
RPL23, RPL32,
LOC730139, LRRC39, MALTI, MXI, MERTK, MX2, NRGI, OR52A1, PLEKHHI, PTPN13,
PTPRJ,
RLN 1, RNF 19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSF13,
TNFSF13,
TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 when administered to the
subject.
[0009] In the methods as described above the inhibitor may be selected from
siRNA, RNAi, shRNA,
antisense RNA, antisense DNA, decoy molecule, decoy DNA, double stranded DNA,
single-stranded DNA,
complexed DNA, encapsulated DNA, viral DNA, plasmid DNA, naked RNA,
encapsulated RNA, viral
RNA, double stranded RNA, molecules capable of generating RNA interference,
synthetic ligands, peptide
ligands, antagonists, agonists, antibodies, small chemical molecules and
combinations thereof.
[0010] Furthermore in the methods as described above the siRNA may have a
sequence selected from the
group of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:
16, SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ
ID NO: 23, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID 29, SEQ ID NO: 36, SEQ ID 37,
SEQ ID NO: 38,
SEQ ID 39 and combinations thereof and the shRNA may have a sequence selected
from the group of SEQ
ID NO: I to SEQ ID NO: 5, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 25, SEQ
ID NO: 31 to SEQ
ID NO: 35, SEQ ID NO: 40 to SEQ ID NO: 45, SEQ ID NO: 47 to SEQ ID NO: 65, SEQ
ID NO: 67 to SEQ
ID NO: 98, and combinations thereof.
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[0011] Furthermore the ABI2, ARRDC3, BAD, BRCAI, C17orf85, Clorf7l, C6orf162,
CCNJL, CFLI,
GON4L, HCG 1986447, HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139,
LRRC39,
MALTI, MXI, MERTK, MX2,NRGI, OR52Al, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A,
SH3BP4,
SLC7AI4, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSFI3, TNFSFI3, TTN, UBXN7,
USP47,
WNK2, YPEL2, ZNF251 and/or SCG2 inhibitor may act on a polypeptide, DNA, or
RNA.
[0012] In the methods as described above the cells may be contacted by the
inhibitor in vivo and the subject
may be a mammal. More specifically the mammal may be a human. Furthermore the
virus in the methods
described may be an influenza virus.
[0013] The present disclosure further provides a method of screening for an
agent useful for reducing virus
production and/or reducing virus-mediated cytotoxicity in a cell comprising:
i) contacting a cell expressing ABI2, ARRDC3, BAD, BRCA 1, C 17orf85, C 1
orf71, C6orfl 62,
CCNJL, CFL 1, GON4L, HCG 1986447, HISTI H2AB, HPS4, LHX8, RPS25, RPL23, RPL32,
LOC730139,
LRRC39, MALT1, MX1, MERTK, MX2, NRG 1, OR52A1, PLEKHH1, PTPN 13, PTPRJ, RLN 1,
RNFI9A,
SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSFI3, TNFSFI3, TTN,
UBXN7,
USP47, WNK2, YPEL2, ZNF251 and/or SCG2 with a test compound;
ii) comparing the amount of ABI2, ARRDC3, BAD, BRCAI, C 17orf85, C 1 orf7l,
C6orfl 62,
CCNJL, CFLI, GON4L, HCG 1986447, HIST1 H2AB, HPS4, LHX8, RPS25, RPL23, RPL32,
LOC730139,
LRRC39, MALT!, MXI, MERTK, MX2, NRGI, OR52A1, PLEKHHI, PTPN 13, PTPRJ, RLN1,
RNF19A,
SH3BP4, SLC7AI4, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN,
UBXN7,
USP47, WNK2, YPEL2, ZNF251 and/or SCG2 in the cell in the presence and absence
of the test compound;
and
iii) selecting any test compound decreasing the amount of ABI2, ARRDC3, BAD,
BRCA1,
C17orf85, Clorf7l, C6orf162, CCNJL, CFLI, GON4L, HCG 1986447, HISTIH2AB, HPS4,
LHX8,
RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI, MERTK, MX2, NRGI, OR52A1,
PLEKHHI, PTPNI3, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7AI4, ST8SIA3, STX3, TMC6,
TMTC4,
TNFSF 12-TNFSF 13, TNFSF 13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or
SCG2 as useful
for reducing virus production and/or reducing virus-mediated cytotoxicity.
[0014] In addition a siRNA for treating virus infection is provided. The siRNA
may comprise a
sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ
ID NO: 22, SEQ
ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID 29, SEQ ID NO:
36, SEQ ID 37,
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SEQ ID NO: 38, SEQ ID 39. A shRNA for treating virus infection is also
provided. The shRNA may
comprise a sequence represented by SEQ ID NO: I to SEQ ID NO: 5, SEQ ID NO: 11
to SEQ ID NO: 15,
SEQ ID NO: 25, SEQ ID NO: 31 to SEQ ID NO: 35, SEQ ID NO: 40 to SEQ ID NO: 45,
SEQ ID NO: 47 to
SEQ ID NO: 65, SEQ ID NO: 67 to SEQ ID NO: 98.
[0015] The present disclosure also provides a pharmaceutical formulation for
treating a virus
infection comprising at least one inhibitor of ABI2, ARRDC3, BAD, BRCAI,
C17orf85, Clorf7l,
C6orf162, CCNJL, CFLI, GON4L, HCG 1986447, HISTIH2AB, HPS4, LHX8, RPS25,
RPL23, RPL32,
LOC730139, LRRC39, MALTI, MX1, MERTK, MX2, NRGI, OR52A1, PLEKHHI, PTPN13,
PTPRJ,
RLN 1, RNF 19A, SH3BP4, SLC7A 14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF I2-TNFSF
13, TNFSF 13,
TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2. At least one inhibitor
maybe a nucleic acid
and the nucleic acid may be selected from siRNA, RNAi, shRNA, or combinations
thereof or from nucleic
acid molecules capable of encoding siRNA, RNAi, shRNA, or combinations
thereof.
[0016] The siRNA in the pharmaceutical formulation may have a sequence
selected from the group of
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID
NO: 17, SEQ ID
NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:
23, SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID 29, SEQ ID NO: 36, SEQ ID 37, SEQ ID
NO: 38, SEQ ID 39
and combinations thereof and the shRNA may have a sequence selected from the
group SEQ ID NO: 1 to
SEQ ID NO: 5, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 31 to
SEQ ID NO: 35,
SEQ ID NO: 40 to SEQ ID NO: 45, SEQ ID NO: 47 to SEQ ID NO: 65, SEQ ID NO: 67
to SEQ ID NO: 98
and combinations thereof.
[0017] This summary does not necessarily describe all features of the
invention. Other aspects,
features and advantages of the present invention will become apparent to those
of ordinary skill in the art
upon review of the following description of some specific embodiments of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] These and other features of the invention will become more apparent
from the following
description in which reference is made to the appended drawings wherein:
[0019] Figure 1: (A) shows the sequence of SEQ ID NO: 10. (B) shows the
sequence of SEQ ID NO:
103. (C) shows the sequence of SEQ ID NO: 24. (D) shows the sequence of SEQ ID
NO: 30. (E) shows the
sequence of SEQ ID NO: 46. (F) shows the sequence of SEQ ID NO: 66. (G) shows
the sequence of SEQ ID
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NO: 99. (H) shows the sequence of SEQ ID NO: 100. (I) shows the sequence of
SEQ ID NO: 101. (J)
shows the sequence of SEQ ID NO: 102.
[0020] Figure 2: shows a schematic diagram of methods. Details are given in
Methods Summary
(Example 4).
[0021] Figure 3: shows a Venn diagram of the genes (target host factors)
identified in the genome-
wide RNAi screen. Unique number of genes identified in the first and second
biological replicates of the
high-throughput screens. 35 unique genes were identified in both experiments.
[0022] Figure 4 shows PANTHER biological process categorization of the 35
unique genes.
[0023] Figure 5 shows PANTHER molecular functions categorization of the 35
unique genes.
[0024] Figure 6 shows biomolecular interaction network of the identified gene
candidates.
[0025] Figure 7 shows Influenza virus replication in A549 knockdown cells.
Cytopathic effect (CPE)
of cells knocked down with one of indicated shRNA, mock infected (top row), or
infected with NY55 (48
hpi after MOI I - 2nd row), PR8 (72 hpi after MO10.1 - 3rd row), or SOIV (72
hpi after MOI 0.1- bottom
row). Cells were examined with a Nikon Eclipse TE2000-S inverted microscope
and images obtained with a
Canon PowerShot A700 digital camera.
[0026] Figure 8 shows (a) Measurements of cell viability (measured by trypan
blue exclusion) of
normal A549 and A549 knockdown cells at 72hpi after mock infection or
infection with PR8, NY55 and
SOIV virus; (b) Measurements of NY55 (48 hpi), PR8 (72 hpi), and SOIV (72 hpi)
replication in shRNA
knockdown A549 cells at MOI 1, 0.01, and 0.5, respectively. Additional data
presented in Fig. 9; (c) NY55
and SOIV virus replication in siRNA-knockdown A549 cells after 48 h infection
at MOI 0.1. NSi = non-
silencing. Error bars represent standard deviation from 2-3 biological
replicates.
[0027] Figure 9 shows a graph with viability of A549 cells after (a) shRNA or
(b) siRNA treatment
determined by WST-1 assay. NSi = non-silencing. Error bars represent standard
deviation from 2 biological
replicates.
[0028] Figure 10 shows a graph with influenza virus replication in shRNA-
knockdown A549 cells
infected with NY55 at MOI 1. NSi = non-silencing. Error bars represent
standard deviation from 2-3
biological replicates.
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[0029] Figure 11 shows an analysis of critical nodes within the interaction
network of the 31
identified genes, with focus on three unspecified protein complexes.
[0030] Figure 12 shows an analysis of critical nodes within the interaction
network of the 31
identified genes, with focus on BAD. Critical nodes are shown in black circle
and the direct interacting
protein partners of these critical nodes (gene product) are shown in
highlighted boxes.
[0031] Figure 13 shows an analysis of critical nodes within the interaction
network of the 31
identified genes, with focus on TNFSF I2-13/TNFSF 13. Critical nodes shown in
black circle and the direct
interacting protein partners of these critical nodes (gene product) are shown
in highlighted boxes.
DETAILED DESCRIPTION
[0032] In the description that follows, a number of terms are used, the
following definitions are
provided to facilitate understanding of various aspects of the disclosure. Use
of examples in the
specification, including examples of terms, is for illustrative purposes only
and is not intended to limit the
scope and meaning of the embodiments of the invention herein. Numeric ranges
are inclusive of the numbers
defining the range. In the specification, the word "comprising" is used as an
open-ended term, substantially
equivalent to the phrase "including, but not limited to," and the word
"comprises" has a corresponding
meaning.
[0033] The present disclosure provides, in part, target host factors. By
target host factors is meant a
host polypeptide, DNA or RNA that - when inhibited, decreased or otherwise
interfered with - reduces virus
replication and/or virus-mediated cytotoxicity in a host cell infected with
the virus. Modulation of the
expression level of host factor, or of host factor product activity, prevents
and/or ameliorates disease
progression. For example, virus replication and/or virus-mediated cytotoxicity
and/or apoptosis may be
affected. Thus, compounds that modulate the expression of a target host factor
or the activity of a target host
factor may be used in the diagnosis, treatment, and/or prevention of a viral
infection. In particular, target
host factors in the present disclosure include endogenous genes and gene
products and their variants, as
described herein.
[0034] Viral infections may include for example infections by respiratory
viruses, including but not
limited to, various types of influenza, such as influenza A, influenza B and
numerous other strains of
influenza, including seasonal, avian (e.g., H5N1 strains), and swine (e.g.,
H1NI strains).
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[0035] The present disclosure provides compositions that inhibit target host
factors, for example
nucleic acids, such as polynucleotides. More specifically, it provides siRNAs,
such as shRNAs, that inhibit
target host factors and are therefore useful in treating virus infection
and/or reducing virus replication and/or
virus-mediated cytotoxicity.
[0036] The novel target host factors are, for example, ABI2, ARRDC3, BAD,
BRCA1, C17orf85,
CI orf71, C6orfl62, CCNJL, CFL 1, GON4L, HCG 1986447, HISTI H2AB, HPS4, LHX8,
RPS25, RPL23,
RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHHI,
PTPN13,
PTPRJ, RLNI, RNF19A, SH3BP4, SLC7AI4, ST8SIA3, STX3, TMC6, TMTC4, TNFSF]2-
TNFSFI3,
TNFSF 13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and SCG2 (see Table 1). The
novel target host
factor may be BAD, TNFSF I 2-TNFSF 13, TNFSF 13, MX2 or USP47. The target host
factors may be used
as targets for therapy. The target host factors can also can be used to
identify compounds useful in the
diagnosis, prevention, and/or therapy of virus infection, for example
influenza virus infection.
[0037] By "reduce," "reduction", or "reducing" is meant to destroy, prevent,
control, decrease, slow,
or otherwise interfere with the production, replication, and/or virus-mediated
cytotoxicity of a virus by at
least about 10% to about 100%, at least about 30% to about 100%, at least
about 50% to about 100%, or any
value therebetween for example about 10%, 15%,20%,25%,30%,35%,40%, 45%,
50%,55%,60%,65%,
70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the production or
replication of a virus
in the absence of the inhibitor.
[0038] "Inhibitors" refers to molecules that inhibit and/or block an
identified function. Any molecule
or compound having potential to inhibit and/or block an identified function
can be a "test molecule" or "test
compound", as described herein. For example, referring to anti-viral function
or anti-apoptotic activity by
acting on the ABI2, ARRDC3, BAD, BRCA 1, CI7orf85, Clorf71, C6orfl 62, CCNJL,
CFL1, GON4L, HCG
1986447, HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT],
MX1,
MERTK, MX2, NRG1, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4,
SLC7A14,
ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSFI3, TNFSFI3, TTN, UBXN7, USP47, WNK2,
YPEL2, ZNF251 or SCG2 polypeptide, DNA or RNA such molecules or compounds may
be identified
using in vitro and in vivo assays ofABl2, ARRDC3, BAD, BRCAI, C17orf85,
Clorf71, C6orf162, CCNJL,
CFLI, GON4L, HCG 1986447, HISTI H2AB, HPS4, LHX8, RPS25, RPL23, RPL32,
LOC730139 USP47,
LRRC39, MALT1, MX1, MERTK, MX2,NRG1, OR52AI, PLEKHHI, PTPNI3, PTPRJ, RLNI,
RNF19A,
SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSFI3, TNFSFI3, TTN,
UBXN7,
USP47, WNK2, YPEL2, ZNF251 or SCG2, respectively. Inhibitors are molecules or
compounds that
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partially or totally block ABI2, ARRDC3, BAD, BRCAI, C17orf85, Clorf7l,
C6orfl62, CCNJL, CFLI,
GON4L, HCG 1986447, HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139
USP47,
LRRC39, MALT1, MXI, MERTK, MX2,NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLN1,
RNF19A,
SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSFI3, TNFSF13, TTN,
UBXN7,
USP47, WNK2, YPEL2, ZNF251 and/or SCG2 activity, decrease, prevent, or delay
their activation, or
desensitize the cellular response. This may be accomplished by binding to
ABI2, ARRDC3, BAD, BRCA1,
C17orf85, Clorf7l, C6orfl62, CCNJL, CFL1, GON4L, HCG 1986447, HISTIH2AB, HPS4,
LHX8,
RPS25, RPL23, RPL32, LOC730139 USP47, LRRC39, MALT1, MXI, MERTK, MX2, NRGI,
OR52A1,
PLEKHHI, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6,
TMTC4,
TNFSFI2-TNFSF13, TNFSFI3, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2
factors
directly or via other intermediate molecules. An antagonist or an antibody
that blocks ABI2, ARRDC3,
BAD, BRCAI, C17orf85, Clorf7l, C6orfl62, CCNJL, CFLI, GON4L, HCG 1986447,
HISTIH2AB,
HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI, MERTK, MX2,
NRG1,
OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3,
TMC6,
TMTC4, TNFSF I 2-TNFSF 13, TNFSF 13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251
and/or SCG2
activity, including inhibition of pro-viral function or pro-apoptotic activity
of ABI2, ARRDC3, BAD,
BRCAI, Cl7orf85, Clorf7l, C6orfl62, CCNJL, CFLI, GON4L, HCG 1986447,
HISTIH2AB, HPS4,
LHX8, RPS25, RPL23, RPL32, LOC730139 USP47, LRRC39, MALTI, MXI, MERTK, MX2,
NRGI,
OR52A1, PLEKHHI, PTPNI3, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7AI4, ST8SIA3, STX3,
TMC6,
TMTC4, TNFSFI2-TNFSFI3, TNFSFI3, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or
SCG2,
is considered to be such an inhibitor.
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Table 1. Target host factors
Target host factor Gene ID Examples of Examples of inhibitors of target
(Gene Name) RefSeq Numbers host factor
(mRNA)
ABI2 10152 NM 005759 SEQ ID NO 51
ARRDC3 57561 NM 020801 SEQ ID NO 52
BAD 572 NM_004322 SEQ ID NOs I to 9
NM 032989
BRCAI 672 NM 007294 SEQ ID NOs 53 to 55
NM 007297
NM 007298
NM_007299
NM 007300
C 17orf85 55421 NM001114118 SEQ ID NO 56
NM 018553
Clorf7l (CNST) 163882 NM 001139459 SEQ ID NO 57
NM 152609
C6orfl62 57150 NM 001042493 SEQ ID NO 58
NM 020425
CCNJL 79616 NM 024565 SEQ ID NO 59
CFLI 1072 NM 005507 SEQ ID NO 50
GON4L 54856 NM_001037533 SEQ ID NO 60
NM 032292
HCG 1986447 729324 XR 041499.2 SEQ ID NO 47
HISTIH2AB 8335 NM 003513 SEQ ID NO 61
HPS4 89781 NM 022081 SEQ ID NO 62
NM 152841
LHX8 431707 NM 001001933 SEQ ID NO 63
RPS25 6230 NM 001028 SEQ ID NO 44
RPL23 9349 NM 000978 SEQ ID NO 64
RPL32 6161 NM 000994 SEQ ID NO 65
NM 001007073
LOC730139 730139 XM 001134281.1 SEQ ID NO 67 to 68
LRRC39 127495 NM 144620 SEQ ID NO 69 to70
MALTI 10892 NM 006785 SEQ ID NO 43
NM 173844
MX1 4599 NM 002462 SEQ ID NO 41 to 42
NM 001144925
MERTK 10461 NM 006343 SEQ ID NO 93 to 95
MX2 4600 NM 002463 SEQ ID NOs 25-29
NRGI 3084 NM_001159995 SEQ ID NO 71
NM 001159996
NM_00 1 1 59999
NM 001160001
NM 001160002
NM 001160004
NM 001160005
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Target host factor Gene ID Examples of Examples of inhibitors of target
(Gene Name) RefSeq Numbers host factor
(mRNA)
NM 001160007
NM 001160008
NM_004495
NM 013956
NM 013957
NM 013958
NM_013959
NM_013960
NM 013962
NM 013964
0R52A1 23538 NM 012375 SEQ ID NO 72 to 73
PLEKHHI 57475 NM 020715 SEQ ID NO 74
PTPN 13 5783 NM 006264 SEQ ID NO 75
NM 080683
NM_080684
NM 080685
PTPRJ 5795 NM_001098503 SEQ ID NO 76 to 77
NM 002843
RLNI 6013 NM 006911 SEQ ID NO 78
RNF19A 25897 NM 015435 SEQ ID NO 79
NM 183419
SH3BP4 23677 NM 014521 SEQ ID NO 45
SLC7AI4 57709 NM 020949 SEQ ID NO 80
ST8SIA3 51046 NM 015879 SEQ ID NO 81 to 82
STX3 6809 NM 004177 SEQ ID NO 83
TMC6 11322 NM_001127198 SEQ ID NO 49
NM 007267
TMTC4 84899 NM_001079669 SEQ ID NO 84 to 85
NM 032813
TNFSFI2-TNFSFI3, 407977 NM 172089 SEQ ID NOs 11 to 19
TNFSFI3 8741 NM 003808 SEQ ID NOs 11 to 23
NM_172087
NM 172088
TTN 7273 NM 003319 SEQ ID NO 86 to 87
NM_133378
NM_133379
NM 133432
NM 133437
UBXN7 26043 NM_015562 SEQ ID NO 88
USP47 55031 NM 017944.3 SEQ ID NOs 31 to 40
WNK2 65268 NM 006648 SEQ ID NO 48
YPEL2 388403 NM 001005404 SEQ ID NO 89 to 90
ZNF251 90987 NM 138367 SEQ ID NO 91 to 92
SCG2 7857 NM 003469 SEQ ID NO 96 to 98
CA 02709132 2010-07-07
[0039] Inhibitors may be for example siRNA, RNAi, shRNA, antisense RNA,
antisense DNA, decoy
molecules, decoy DNA, double stranded DNA, single-stranded DNA, complexed DNA,
encapsulated DNA,
viral DNA, plasmid DNA, naked RNA, encapsulated RNA, viral RNA, double
stranded RNA, molecules
capable of generating RNA interference, or combinations thereof. The group of
inhibitors also includes
genetically modified versions of AB12, ARRDC3, BAD, BRCA 1, C 17orf85, C 1
orf71, C6orf162, CCNJL,
CFLI, GON4L, HCG 1986447, HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32,
LOC730139,
LRRC39, MALTI, MXI, MERTK, MX2,NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI,
RNF19A,
SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSF13, TNFSFI3, TTN,
UBXN7,
USP47, WNK2, YPEL2, ZNF251 and SCG2, for example, versions with altered
activity. The group thus is
inclusive of the naturally occurring protein with altered activity, as well as
synthetic ligands, peptide ligands,
antagonists, agonists, antibodies, small chemical molecules and the like.
Screening for Inhibitors of Target Host Factors
[0040] A "test substance" or "test compound" is a compound or mixture of
compounds, whose ability
to modulate AB12, ARRDC3, BAD, BRCA 1, CI7orf85, CIorf7I, C6orfl62, CCNJL, CFL
1, GON4L, HCG
1986447, HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI,
MXI,
MERTK, MX2, NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4,
SLC7AI4,
ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSFI3, TNFSFI3, TTN, UBXN7, USP47, WNK2,
YPEL2, ZNF251 and/or SCG2 activity may be defined by various assays. A "test
substance" is also referred
to as a "candidate drug" or "candidate compound" in the present description.
[0041 ] Test substances may be screened from large libraries of synthetic or
natural compounds. Numerous
means are currently used for random and directed synthesis of saccharide,
peptide, and nucleic acid based
compounds. Synthetic compound libraries are commercially available from
Maybridge Chemical Co.
(Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates
(Merrimack, N.H.), and
Microsource (New Milford, Conn.). A rare chemical library is available from
Aldrich (Milwaukee, Wis.).
Alternatively, libraries of natural compounds in the form of bacterial,
fungal, plant and animal extracts are
available from, e.g., Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or
are readily producible.
Additionally, natural and synthetically produced libraries and compounds are
readily modified through
conventional chemical, physical, and biochemical means (Blondelle et al.,
TIBTech, 1996; 14:60).
[0042] A modulatory effect may be determined by an in vitro method using a
recombinant ABI2,
3o ARRDC3, BAD, BRCAI, C17orf85, Clorf7l, C6orf162, CCNJL, CFLI, GON4L, HCG
1986447,
HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MX1,
MERTK,
11
CA 02709132 2010-07-07
MX2, NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7AI4,
ST8SIA3,
STX3, TMC6, TMTC4, TNFSF 12-TNFSF 13, TNFSF 13, TTN, UBXN7, USP47, WNK2,
YPEL2, ZNF251
or SCG2 reporter gene promoter activity system. Reporter genes encode
detectable proteins, including, but
not limited to, chloramphenicol transferase (CAT), beta-galactosidase (beta-
gal), luciferase, green
fluorescent protein (GFP) and derivatives thereof, yellow fluorescent protein
and derivatives thereof,
alkaline phosphatase, other enzymes that can be adapted to produce a
detectable product, and other gene
products that can be detected, e.g., immunologically (by immunoassay).
[0043] A screen involves detecting a change in the expression of the reporter
gene by the host cell
when contacted with a test substance. If there is no change in expression of
the reporter gene, the test
substance may not be an effective modulator. If reporter gene expression is
modified, in particular reduced
or eliminated, the test substance has modulated, e.g., inhibited, ABI2,
ARRDC3, BAD, BRCAI, C 17orf85,
C I orf7l, C6orfl62, CCNJL, CFLI, GON4L, HCG 1986447, HISTI H2AB, HPS4, LHX8,
RPS25, RPL23,
RPL32, LOC730139, LRRC39, MALTI, MX!, MERTK, MX2, NRGI, OR52A1, PLEKHHI,
PTPN13,
PTPRJ, RLNI, RNF19A, SH3BP4, SLC7A]4, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-
TNFSFI3,
TNFSFI3, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 -mediated gene
expression, and is
thus a candidate for development as a ABI2, ARRDC3, BAD, BRCAI, C17orf85,
CIorf71, C6orfl62,
CCNJL, CFLI, GON4L, HCG 1986447, HISTI H2AB, HPS4, LHX8, RPS25, RPL23, RPL32,
LOC730139,
LRRC39, MALT1, MX1, MERTK, MX2,NRG1, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLN1,
RNFI9A,
SH3BP4, SLC7AI4, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSFI3, TNFSF13, TTN,
UBXN7,
USP47, WNK2, YPEL2, ZNF251 or SCG2 modulator, for use as inhibitor of virus
replication and virus-
mediated cytotoxicity. The reporter gene assay system described herein may be
used in a high-throughput
primary screen for antagonists, or it may be used as a secondary functional
screen for candidate compounds
identified by a different primary screen, e.g., a binding assay screen that
identifies compounds that modulate
ABI2, ARRDC3, BAD, BRCAI, Cl7orf85, CIorf71, C6orfl62, CCNJL, CFL1, GON4L, HCG
1986447,
HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT!, MXI,
MERTK,
MX2, NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7A14,
ST8SIA3,
STX3, TMC6, TMTC4, TNFSF 12-TNFSF 13, TNFSF 13, TTN, UBXN7, USP47, WNK2,
YPEL2, ZNF251
or SCG2 transcription activity.
[0044] Potential drugs may be identified by screening in high-throughput
assays, including without
limitation cell-based or cell-free assays. It will be appreciated by those
skilled in the art that different types
of assays can be used to detect different types of agents. Several methods of
automated assays have been
developed in recent years so as to permit screening of tens of thousands of
compounds in a short period of
12
CA 02709132 2010-07-07
time. Such high-throughput screening methods are particularly useful when
screening for candidates for
further testing.
[0045] Examples of target host factor modulators include the polynucleotides
such as siRNAs.
"siRNA" refers to small interfering RNAs, which includes short hairpin RNA
("shRNA") (Paddison et al.,
Genes & Dev. 16: 948-958, 2002), that are capable of causing interference (as
described herein for RNAi)
and can cause post-transcriptional silencing of specific genes in cells, for
example, mammalian cells
(including human cells) and in the body, for example, mammalian bodies
(including humans). The
phenomenon of RNA interference (RNAi) is described and discussed in Bass,
Nature, 411:428-29, 2001;
Elbashir et al., Nature, 411:494-98, 2001; and Fire et al., Nature, 391:806-
11, 1998, wherein methods of
making interfering RNA also are discussed. Exemplary siRNAs could have up to
29 bps, 25 bps, 22 bps, 21
bps, 20 bps, 15 bps, 10 bps, 5 bps or any integer thereabout or therebetween.
[0046] Expression of selected genes can be suppressed in human cells by
transfecting with
exogenous, short RNA duplexes (siRNA) where one strand corresponds to a target
region of the mRNA of
interest (Elbashir et al., Nature, 2001; 411:494-498). Upon entry into the
cell, siRNA causes the degradation
of single-stranded (ssRNAs) RNAs of with identical or near identical
sequences, including endogenous
mRNAs. siRNA is more potent than standard anti-sense technology since it acts
through a catalytic
mechanism. Effective strategies to deliver siRNAs to target cells, for
example, include physical or chemical
transfection. An alternative strategy uses the endogenous expression of siRNAs
by various Pol III promoter
expression cassettes that allow transcription of functional siRNAs or their
precursors (Scherr et al., Curr.
Med. Chem., 2003; 10(3):245-56). Recently, the RNA-polymerase III dependent
promoter (H1-RNA
promoter) was inserted in the lentiviral genome to drive the expression of a
small hairpin RNA (shRNA)
against enhanced green fluorescent protein (Abbas-Turki et al., Hum. Gene
Ther., 2002; 13(18):2197-201).
siRNA can also be delivered in a viral vector derived, e.g., from a lentivirus
(Tiscornia et al., Proc. Natl.
Acad. Sci. U.S.A., 2003; 100:1844-8).
13
CA 02709132 2010-07-07
Target host factors and siRNAs
BAD
[0047] According to an aspect of the present disclosure, it has been
determined that BAD may be a
suitable target when looking at inhibition of virus replication or virus-
mediated cytotoxicity for example
influenza virus.
[0048] The present disclosure provides polynucleotides that inhibit expression
of a polypeptide
encoded by a BAD coding region. As used herein a BAD coding region refers to
the genomic nucleotide
sequence disclosed under GenelD: 572. Examples of target mRNA encoding a BAD
polypeptide are the
sequences available at Genbank accession numbers NM004322 (SEQ IDNO:10) or NM
032989 (SEQ ID
NO: 103).
[0049] Polynucleotides of the present disclosure that will act to inhibit
expression of a BAD
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of SEQ ID NO: 10 or SEQ ID NO: 103. Examples of such polynucleotides that will
act to inhibit expression
of a polypeptide encoded by a BAD coding region include SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3,
SEQ ID NO:4. SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID
NO:9.
Table 2: Inhibitors of BAD
BAD shRNA species SEQ ID NO SEQUENCE
V2HS 15289 1 CTCACTACCAAATGTTAAT
V2HS 243025 2 CAGTGACCTTCGCTCCACA
V2HS 201511 3 GAGTTTGTGGACTCCTTTA
V2HS 202976 4 GTGCTCACTACCAAATGTT
V2HS 262043 5 GACTTGGACTTGGATGTAA
shRNA species SEQ ID NO SEQUENCE
J-003870-09 6 GAUCGGAACUUGGGCAGGG
J-003870-10 7 CAGAGUUUGAGCCGAGUGA
J-003870-11 8 GAGCUCCGGAGGAUGAGUG
J-003870-12 9 UUGUGGACUCCUUUAAGAA
TNFSFI2-13 (TWE-PRIL)
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[0050] The present disclosure provides polynucleotides that inhibit expression
ofa fusion polypeptide
encoded by a TNFSF12-13 coding region. As used herein a TNFSFI2-13 coding
region refers to the
genomic nucleotide sequence disclosed under Gene1D: 407977. An example of a
target mRNA encoding a
TNFSF 12-13 fusion polypeptide is the sequence available at Genbank accession
number NM_! 72089 (SEQ
ID NO: 99).
[00511 Polynucleotides ofthe present disclosure that will act to inhibit
expression of a TNFSF 12-13
fusion polypeptide, include polynucleotides with a sense strand that is
substantially identical or identical to a
region of SEQ ID NO: 99. Examples of such polynucleotides that will act to
inhibit expression of a
polypeptide encoded by a TNFSFI2-13 coding region include SEQ ID NO: 11, SEQ
ID NO: 12. SEQ ID
Io NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID
NO: 18 and SEQ ID
NO: 19.
Table 3: Inhibitors of TNFSFI2-TNFSF13
TNFSF12-TNFSFI3 shRNA species SEQ ID NO SEQUENCE
TW E-PRIL
V2HS 17313 11 GCCGCCCTCTGCTAGGGAA
V2HS 17314 12 GATATTCTGAGTGTCATAA
V2HS 17316 13 GGTGCCTTCGCAGTCAAAT
V2HS 17317 14 GAGACTCTATTCCGATGTA
V2HS 17318 15 CTCCAGAGATGTAGCTATT
siRNA species SEQ ID NO SEQUENCE
J-032530-05 16 GGGCAAGGGCGAAACUUAA
J-032530-06 17 GCAGGUGUCUUCCAUUUAC
J-032530-07 18 UGACAGAGGUGAUGUGGCA
J-032530-08 19 GGAGUUUAUCUGCUGUAUA
TNFSFI3 (APRIL)
[0052] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a TNFSF 13 coding region. As used herein a TNFSF 13
coding region refers to the
genomic nucleotide sequence disclosed for example under GenelD: 8741. Several
splice variants of the
TNFSF 13 coding region are expressed and encode polypeptides including a TNFSF
13 alpha polypeptide, a
TNFSF 13 beta polypeptide, and a TNFSF13 gamma polypeptide.
CA 02709132 2010-07-07
[0053] An example of a target mRNA encoding a TNFSF 13 alpha polypeptide is
the sequence
available at Genbank accession number NM003808 (SEQ ID NO: 100). An example of
a target mRNA
encoding a TNFSF 13 beta polypeptide is the sequence available at Genbank
accession number NM_172087
(SEQ ID NO: 101). An example of a target mRNA encoding a TNFSF 13 gamma
polypeptide is the sequence
available at Genbank accession number NM_172088 (SEQ ID NO: 102).
[0054] A preferred target mRNA includes a sequence that is present in all
three splice variants.
Polynucleotides of the present disclosure that will act to inhibit expression
of a TNFSF 13 alpha polypeptide,
a TNFSF 13 beta polypeptide, and a TNFSF 13 gamma polypeptide include
polynucleotides with a sense
strand that is substantially identical or identical to a region of SEQ ID NO:
100.
[0055] Examples of such polynucleotides that will act to inhibit expression of
a polypeptide encoded
by a TNFSFI3 coding region include SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:
13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ
ID NO: 20, SEQ
ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.
Table 4: Inhibitors of TNFSFI3 (APRIL)
TNFSF13 (APRIL) shRNA species SEQ ID NO SEQUENCE
V2HS 17313 11 GCCGCCCTCTGCTAGGGAA
V2HS 17314 12 GATATTCTGAGTGTCATAA
V2HS 17316 13 GGTGCCTTCGCAGTCAAAT
V2HS 17317 14 GAGACTCTATTCCGATGTA
V2HS 17318 15 CTCCAGAGATGTAGCTATT
siRNA species SEQ ID NO SEQUENCE
J-032530-05 16 GGGCAAGGGCGAAACUUAA
J-032530-06 17 GCAGGUGUCUUCCAUUUAC
J-032530-07 18 UGACAGAGGUGAUGUGGCA
J-032530-08 19 GGAGUUUAUCUGCUGUAUA
J-011523-05 20 GGGCAAGGGCGAAACUUAA
J-011523-06 21 GCAGGUGUCUUCCAUUUAC
J-011523-07 22 UGACAGAGGUGAUGUGGCA
J-01 1523-08 23 GGAGUUUAUCUGCUGUAUA
MX2
16
CA 02709132 2010-07-07
[0056] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of
a polypeptide encoded by a MX2 coding region. As used herein a MX2 coding
region refers to the
genomic nucleotide sequence disclosed under GeneID: 4600. An example of a
target mRNA encoding a
MX2 polypeptide is the sequence available at Genbank accession number
NM_002463 (SEQ ID NO:
24).
[0057] Polynucleotides of the present disclosure that will act to inhibit
expression of a MX2
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a
region of SEQ ID NO: 24. Examples of such polynucleotides that will act to
inhibit expression of a
polypeptide encoded by a MX2 coding region include SEQ ID NO: 25, SEQ ID NO:
26, SEQ ID NO:
27. SEQ ID NO: 28 and SEQ ID NO: 29.
Table 5: Inhibitors of MX2
MX2 shRNA species SEQ ID NO SEQUENCE
V2HS 152031 25 GACAAGATGTTCTTTCTAA
siRNA species SEQ ID NO SEQUENCE
J-0 11736-05 26 GAGCACGAUUGAAGACAUA
J-011736-06 27 GGAGAAUGAGACCCGUUUA
J-011736-07 28 GAAUUUACCGGCUCACUCA
J-01 1736-08 29 GGGACGCCUUCACAGAAUA
USP47
[0058] The present disclosure includes polynucleotides that inhibit expression
of a polypeptide
encoded by a USP47 coding region. As used herein a USP47 coding region refers
to the genomic nucleotide
sequence disclosed under GeneID: 55031. An example of a target mRNA encoding a
USP47 polypeptide is
the sequence available at Genbank accession number NM_017944.3 (SEQ ID NO:
30).
[0059] Polynucleotides of the present disclosure that will act to inhibit
expression of a USP47
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of SEQ ID NO:30. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a USP47 coding region include SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID
NO: 33. SEQ ID NO:
34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39
and SEQ ID NO:
40.
17
CA 02709132 2010-07-07
Table 6: Inhibitor of USP 47
USP47 shRNA species SEQ ID NO SEQUENCE
V2HS 174637 31 GAATCTGTCTTGAAACCAA
V2HS 174639 32 CGCAATACATGCAAGATAA
V2HS 174641 33 GGATTCCTTTGGATGATAT
V2HS 174642 34 GATTTAGACTGGAATCCTA
V2HS 21.8228 35 CAATGACTTGCTATTTGAA
V2HS-174640 40 CTTATAAGATGATGGATTT
siRNA species SEQ ID NO SEQUENCE
J-006093-05 36 GCAACGAUUUCUCCAAUGA
J-006093-06 37 CAACAUGUCAGCAGGAUAA
J-006093-07 38 GCUGUCGCCUUGUUAAAUA
J-006093-08 39 CGCAATACAUGCAAGAUAA
MX1
[0060] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a MXI coding region. As used herein a MX] coding region
refers to the genomic
nucleotide sequence disclosed under GenelD: 4599. An example of a target mRNA
encoding a MX1
polypeptide is the sequence available at Genbank accession numbers NM 002462
and NM_001144925.
[0061] Polynucleotides of the present disclosure that will act to inhibit
expression of a MX1 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM_002462 or NM_001144925. Examples of such polynucleotides that will act to
inhibit expression of a
1o polypeptide encoded by a MX1 coding region include SEQ ID NO: 41, SEQ ID
NO: 42.
MALT1
[0062] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a MALTI coding region. As used herein a MALT1 coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 10892. An example of a
target mRNA encoding a
MALT] polypeptide is the sequence available at Genbank accession numbers
NM_006785 or NMI 73844.
[0063] Polynucleotides of the present disclosure that will act to inhibit
expression of a MALT I polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
18
CA 02709132 2010-07-07
NM_006785 or NM_173844. Examples of such polynucleotides that will act to
inhibit expression of a
polypeptide encoded by a MALT1 coding region include SEQ ID NO: 43.
RPS25
[0064] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a RPS25 coding region. As used herein a RPS25 coding
region refers to the genomic
nucleotide sequence disclosed under GeneID: 6230. An example of a target mRNA
encoding a RPS25
polypeptide is the sequence available at Genbank accession number NM-00 1028.
[0065] Polynucleotides of the present disclosure that will act to inhibit
expression of a RPS25 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
1o NM_001028. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide encoded
by a RPS25 coding region include SEQ ID NO: 44.
SH3BP4
[0066] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a SH3BP4 coding region. As used herein a SH3BP4 coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 23677. An example of a
target mRNA encoding a
SH3BP4 polypeptide is the sequence available at Genbank accession number NM-0
1452 1.
[0067] Polynucleotides ofthe present disclosure that will act to inhibit
expression of a SH3BP4 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM_014521. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide encoded
by a SH3BP4 coding region include SEQ ID NO: 45.
HCG 1986447
[0068] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a HCG 1986447 coding region. As used herein a HCG
1986447 coding region refers
to the genomic nucleotide sequence disclosed under GenelD: 729324. An example
of a target mRNA
encoding a HCG 1986447 polypeptide is the sequence available at SEQ ID NO: 46.
[0069] Polynucleotides of the present disclosure that will act to inhibit
expression of a HCG1986447
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
19
CA 02709132 2010-07-07
of SEQ ID NO: 46. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a HCG 1986447 coding region include SEQ ID NO: 47.
WNK2
[0070] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a WNK2 coding region. As used herein a WNK2 coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 65268. An example of a
target mRNA encoding a
WNK2 polypeptide is the sequence available at Genbank accession number
NM_006648.
[0071 ] Polynucleotides of the present disclosure that will act to inhibit
expression of a WNK2 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
io NM_006648. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide encoded
by a WNK2 coding region include SEQ ID NO: 48.
TMC6
[0072] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a TMC6 coding region. As used herein a TMC6 coding
region refers to the genomic
nucleotide sequence disclosed under GenelD: 11322. An example of a target mRNA
encoding a TMC6
polypeptide are the sequences available at Genbank accession numbers
NM_001127198 and NM_007267.
[0073] Polynucleotides of the present disclosure that will act to inhibit
expression of a TMC6 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM_001127198 or NM_007267. Examples of such polynucleotides that will act to
inhibit expression of a
polypeptide encoded by a TMC6 coding region include SEQ ID NO: 49.
CFLI
[0074] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a CFL I coding region. As used herein a CFLI coding
region refers to the genomic
nucleotide sequence disclosed under GenelD: 1072. An example of a target mRNA
encoding a CFLI
polypeptide is the sequence available at Genbank accession number NM_005507.
[0075] Polynucleotides of the present disclosure that will act to inhibit
expression of a CFLI polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
CA 02709132 2010-07-07
NM_005507. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide encoded
by a CFLI coding region include SEQ ID NO: 50.
ABI2
[0076] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a ABI2 coding region. As used herein a AB12 coding
region refers to the genomic
nucleotide sequence disclosed under GenelD: 10152. An example of a target mRNA
encoding a ABI2
polypeptide is the sequence available at Genbank accession number NM_005759.
[0077] Polynucleotides of the present disclosure that will act to inhibit
expression of a ABI2 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM_005759. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide encoded
by a AB12 coding region include SEQ ID NO: 51.
ARRDC3
[0078] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a ARRDC3 coding region. As used herein a ARRDC3 coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 57561. An example of a
target mRNA encoding a
ARRDC3 polypeptide is the sequence available at Genbank accession number
NM_020801.
[0079] Polynucleotides of the present disclosure that will act to inhibit
expression of a ARRDC3
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_020801. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a ARRDC3 coding region include SEQ ID NO: 52.
BRCA1
[0080] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a BRCA I coding region. As used herein a BRCA I coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 672. An example of a
target mRNA encoding a
BRCA I polypeptide are the sequence available at Genbank accession numbers
NM007294, NM_007297,
NM_007298, NM_007299 and NM_007300.
[0081 ] Polynucleotides of the present disclosure that will act to inhibit
expression of a BRCA 1 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
21
CA 02709132 2010-07-07
NM007294, NM_007297, NM_007298, NM007299 or NM_007300. Examples of such
polynucleotides
that will act to inhibit expression of a polypeptide encoded by a BRCA I
coding region include SEQ ID NO:
53, SEQ ID NO: 54 and SEQ ID NO: 55.
C17orJ85
[0082] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a C 17orf85 coding region. As used herein a C l 7orf85
coding region refers to the
genomic nucleotide sequence disclosed under GenelD: 55421. An example of a
target mRNA encoding a
C17orf85 polypeptide are the sequences available at Genbank accession numbers
NM_001114118 and
NM_018553.
[0083] Polynucleotides of the present disclosure that will act to inhibit
expression of a C17orf85
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_001114118 or NM_018553. Examples of such polynucleotides that will act
to inhibit expression of a
polypeptide encoded by a C 17orf85 coding region include SEQ ID NO: 56.
Clorf7l (CNST)
[0084] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a C 1 orf71 coding region. As used herein a C 1 orf7l
coding region refers to the
genomic nucleotide sequence disclosed under GenelD: 163882. An example of a
target mRNA encoding a
C I orf71 polypeptide are the sequences available at Genbank accession numbers
NM_001139459 and
NM_152609.
[0085] Polynucleotides of the present disclosure that will act to inhibit
expression of a C I orf71 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM_00 1 1 39459 or NM-I52609. Examples of such polynucleotides that will act
to inhibit expression of a
polypeptide encoded by a CIorf7l coding region include SEQ ID NO: 57.
C6orfl62
[0086] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a C6orf162 coding region. As used herein a C6orf162
coding region refers to the
genomic nucleotide sequence disclosed under GenelD: 57150. An example of a
target mRNA encoding a
C6orf162 polypeptide are the sequences available at Genbank accession numbers
NM_001042493 and
NM_020425.
22
CA 02709132 2010-07-07
[0087] Polynucleotides of the present disclosure that will act to inhibit
expression of a C6orfl62
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
ofNM_001042493 or NM_020425. Examples of such polynucleotides that will act to
inhibit expression of a
polypeptide encoded by a C6orfl62 coding region include SEQ ID NO: 58.
CCNJL
[0088] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a CCNJL coding region. As used herein a CCNJL coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 79616. An example of a
target mRNA encoding a
CCNJL polypeptide is the sequence available at Genbank accession number
NM_024565.
[0089] Polynucleotides of the present disclosure that will act to inhibit
expression of a CCNJL polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM_024565. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide encoded
by a CCNJL coding region include SEQ ID NO: 59.
GON4L
[0090] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a GON4L coding region. As used herein a GON4L coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 54856. An example of a
target mRNA encoding a
GON4L polypeptide are the sequences available at Genbank accession numbers
NM_001037533 and
NM_032292.
[0091] Polynucleotides of the present disclosure that will act to inhibit
expression of a GON4L polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM_001037533 or NM_032292. Examples of such polynucleotides that will act to
inhibit expression of a
polypeptide encoded by a GON4L coding region include SEQ ID NO: 60.
HISTI H2AB
[0092] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a HIST I H2AB coding region. As used herein a HIST I
H2AB coding region refers to
the genomic nucleotide sequence disclosed under GeneID: 8335. An example of a
target mRNA encoding a
HISTI H2AB polypeptide is the sequence available at Genbank accession number
NM_003513.
23
CA 02709132 2010-07-07
[0093] Polynucleotides of the present disclosure that will act to inhibit
expression of a HISTIH2AB
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_003513. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a HISTIH2AB coding region include SEQ ID NO: 61.
HPS4
[0094] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a HPS4 coding region. As used herein a HPS4 coding
region refers to the genomic
nucleotide sequence disclosed under GenelD: 89781. An example of a target mRNA
encoding a HPS4
polypeptide are the sequences available at Genbank accession numbers NM_022081
and NM_152841.
[0095] Polynucleotides of the present disclosure that will act to inhibit
expression of a HPS4 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM_022081 or NM_152841. Examples of such polynucleotides that will act to
inhibit expression of a
polypeptide encoded by a HPS4 coding region include SEQ ID NO: 62.
LHX8
[0096] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a LHX8 coding region. As used herein a LHX8 coding
region refers to the genomic
nucleotide sequence disclosed under GeneID: 431707. An example of a target
mRNA encoding a LHX8
polypeptide is the sequence available at Genbank accession number
NM_001001933.
[0097] Polynucleotides of the present disclosure that will act to inhibit
expression of a LHX8 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM001001933. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a LHX8 coding region include SEQ ID NO: 63.
RPL23
[0098] In another aspect, the present disclosure includes polynucleotides that
inhibit expression of a
polypeptide encoded by a RPL23 coding region. As used herein a RPL23 coding
region refers to the
genomic nucleotide sequence disclosed under GeneID: 9349. An example of a
target mRNA encoding a
RPL23 polypeptide is the sequence available at Genbank accession number NM
000978.
24
CA 02709132 2010-07-07
[0099] Polynucleotides of the present disclosure that will act to inhibit
expression of a RPL23 polypeptide,
include polynucleotides with a sense strand that is substantially identical or
identical to a region of
NM_000978. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide encoded
by a RPL23 coding region include SEQ ID NO: 64.
RPL32
[00100] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a RPL32 coding region. As used herein a RPL32 coding
region refers to the
genomic nucleotide sequence disclosed under GeneID: 6161. An example of a
target mRNA encoding a
RPL32 polypeptide are the sequences available at Genbank accession numbers
NM000994 and
NM_001007073.
[00101] Polynucleotides of the present disclosure that will act to inhibit
expression of a RPL32
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_000994 or NM_001007073. Examples of such polynucleotides that will act
to inhibit expression of
a polypeptide encoded by a RPL32 coding region include SEQ ID NO: 65.
LOC730139
[00102] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a LOC730139 coding region. As used herein a LOC730139
coding region refers to
the genomic nucleotide sequence disclosed under GenelD: 730139. An example of
a target mRNA encoding
a LOC730139 polypeptide is the sequence of SEQ ID NO: 66.
[00103] Polynucleotides of the present disclosure that will act to inhibit
expression of a LOC730139
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of SEQ ID NO: 66. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a LOC730139 coding region include SEQ ID NO: 67 and SEQ ID NO: 68.
LRRC39
[00104] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a LRRC39 coding region. As used herein a LRRC39 coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 127495. An example of a
target mRNA encoding a
LRRC39 polypeptide is the sequence available at Genbank accession number
NM_144620.
CA 02709132 2010-07-07
[00105] Polynucleotides of the present disclosure that will act to inhibit
expression of a LRRC39
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM144620. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a LRRC39 coding region include SEQ ID NO: 69 and SEQ ID NO: 70.
NRGI
[00106] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a NRG I coding region. As used herein a NRG 1 coding
region refers to the genomic
nucleotide sequence disclosed under GenelD: 3084. An example of a target mRNA
encoding a NRG 1
polypeptide is the sequence available at Genbank accession number
NM_001159995, NM_001159996,
NM-00 1 1 59999, NM_001160001, NM_001160002, NM_001160004, NM_001160005,
NM_001160007,
NM_00 1 1 60008, NM_004495, NM_013956, NM_013957, NM_013958, NM_013959, NM
013960,
NM_013962 and NM_013964.
[00107] Polynucleotides of the present disclosure that will act to inhibit
expression of a NRGI
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM 001159995, NM_001159996, NM_001159999, NM-001 160001, NM-001 160002, NM-
001 160004,
NM-00 1 1 60005, NM_001160007, NM_00 1 1 60008, NM004495, NM_013956,
NM_013957, NM_013958,
NM_013959, NM_013960, NM_013962 or NM_013964. Examples of such polynucleotides
that will act to
inhibit expression of a polypeptide encoded by a NRGI coding region include
SEQ ID NO: 71.
OR52A1
[00108] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a OR52AI coding region. As used herein a OR52A1 coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 23538. An example of a
target mRNA encoding a
OR52A1 polypeptide is the sequence available at Genbank accession number
NM_012375.
[00109] Polynucleotides of the present disclosure that will act to inhibit
expression of a OR52A 1
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_012375. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a OR52A1 coding region include SEQ ID NO: 72 and SEQ ID NO: 73.
PLEKHHI
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CA 02709132 2010-07-07
[00110] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a PLEKHH I coding region. As used herein a PLEKHH I
coding region refers to the
genomic nucleotide sequence disclosed under GenelD: 57475. An example of a
target mRNA encoding a
PLEKHH I polypeptide is the sequence available at Genbank accession number
NM_020715.
[00111] Polynucleotides of the present disclosure that will act to inhibit
expression of a PLEKHH I
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM020715. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a PLEKHHI coding region include SEQ ID NO: 74.
PTPN13
[00112] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a PTPN13 coding region. As used herein a PTPN13 coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 5783. An example of a
target mRNA encoding a
PTPN 13 polypeptide is the sequence available at Genbank accession number NM
006264, NM080683,
NM080684 and NM080685.
[00113] Polynucleotides of the present disclosure that will act to inhibit
expression of a PTPN 13
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
ofNM_006264, NM_080683, NM_080684 or NM080685. Examples of such
polynucleotides that will act
to inhibit expression of a polypeptide encoded by a PTPN 13 coding region
include SEQ ID NO: 75.
PTPRJ
[00114] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a PTPRJ coding region. As used herein a PTPRJ coding
region refers to the
genomic nucleotide sequence disclosed under GenelD: 5795. An example of a
target mRNA encoding a
PTPRJ polypeptide is the sequence available at Genbank accession number
NM_001098503 and
NM_002843.
[00115] Polynucleotides of the present disclosure that will act to inhibit
expression of a PTPRJ
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_001098503 or NM_002843. Examples of such polynucleotides that will act
to inhibit expression of a
polypeptide encoded by a PTPN13 coding region include SEQ ID NO: 76 and SEQ ID
NO: 77.
RLNJ
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CA 02709132 2010-07-07
[00116] In another aspect, the present disclosure includes polynucleotides
that inhibit expression ofa
polypeptide encoded by a RLN I coding region. As used herein a RLN 1 coding
region refers to the genomic
nucleotide sequence disclosed under GeneID: 6013. An example of a target mRNA
encoding a RLN I
polypeptide is the sequence available at Genbank accession number NM_006911.
[00117] Polynucleotides of the present disclosure that will act to inhibit
expression of a RLNI
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_006911. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a RLNI coding region include SEQ ID NO: 78.
RNF19A
[00118] In another aspect, the present disclosure includes polynucleotides
that inhibit expression ofa
polypeptide encoded by a RNF 19A coding region. As used herein a RNF I 9A
coding region refers to the
genomic nucleotide sequence disclosed under GenelD: 25897. An example of a
target mRNA encoding a
RNF19A polypeptide are the sequence available at Genbank accession numbers
NM_015435 and
NM_183419.
[00119] Polynucleotides of the present disclosure that will act to inhibit
expression of a RNF19A
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_015435 or NM_183419. Examples of such polynucleotides that will act to
inhibit expression of a
polypeptide encoded by a RLN I coding region include SEQ ID NO: 79.
SLC7A14
[00120] In another aspect, the present disclosure includes polynucleotides
that inhibit expression ofa
polypeptide encoded by a SLC7A 14 coding region. As used herein a SLC7A 14
coding region refers to the
genomic nucleotide sequence disclosed under GenelD: 57709. An example of a
target mRNA encoding a
SLC7A 14 polypeptide is the sequence available at Genbank accession number
NM_020949.
[00121] Polynucleotides of the present disclosure that will act to inhibit
expression of a SLC7A 14
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_020949. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a SLC7A14 coding region include SEQ ID NO: 80.
ST8SIA3
28
CA 02709132 2010-07-07
[00122] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a ST8SIA3 coding region. As used herein a ST8SIA3
coding region refers to the
genomic nucleotide sequence disclosed under GeneID: 51046. An example of a
target mRNA encoding a
ST8SIA3 polypeptide is the sequence available at Genbank accession number
NM_015879.
[00123] Polynucleotides of the present disclosure that will act to inhibit
expression of a ST8SIA3
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_015879. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a SLC7A14 coding region include SEQ ID NO: 81 and SEQ ID NO: 82.
TMTC4
[00124] In another aspect, the present disclosure includes polynucleotides
that inhibit expression ofa
polypeptide encoded by a TMTC4 coding region. As used herein a TMTC4 coding
region refers to the
genomic nucleotide sequence disclosed under GeneID: 84899. An example of a
target mRNA encoding a
TMTC4 polypeptide are the sequences available at Genbank accession numbers NM
001079669 and
NM032813.
[00125] Polynucleotides of the present disclosure that will act to inhibit
expression of a TMTC4
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
ofNM_001079669 orNM_032813. Examples of such polynucleotides that will act to
inhibit expression of a
polypeptide encoded by a TMTC4 coding region include SEQ ID NO: 84 and SEQ ID
NO: 85.
7TN
[00126] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a TTN coding region. As used herein a TTN coding region
refers to the genomic
nucleotide sequence disclosed under GeneID: 7273. An example of a target mRNA
encoding a TTN
polypeptide is the sequence available at Genbank accession numbers NM003319,
NM_133378,
NM133379, NM_133432 and NM_133437.
[00127] Polynucleotides of the present disclosure that will act to inhibit
expression of a TTN
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_003319, NM_133378, NM_133379, NM_133432 or NM_133437. Examples of such
polynucleotides that will act to inhibit expression of a polypeptide encoded
by a TTN coding region include
SEQ ID NO: 86 and SEQ ID NO: 87.
29
CA 02709132 2010-07-07
UBXN7
[00128] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a UBXN7 coding region. As used herein a UBXN7 coding
region refers to the
genomic nucleotide sequence disclosed under GeneID: 26043. An example of a
target mRNA encoding a
UBXN7 polypeptide is the sequence available at Genbank accession number
NM_015562.
[00129] Polynucleotides of the present disclosure that will act to inhibit
expression of a UBXN7
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_015562. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a UBXN7 coding region include SEQ ID NO: 88.
1o YPEL2
[00130] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a YPEL2 coding region. As used herein a YPEL2 coding
region refers to the
genomic nucleotide sequence disclosed under GeneID: 388403. An example of a
target mRNA encoding a
YPEL2 polypeptide is the sequence available at Genbank accession number
NM_001005404.
[00131] Polynucleotides of the present disclosure that will act to inhibit
expression of a YPEL2
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM_001005404. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a YPEL2 coding region include SEQ ID NO: 89 and SEQ ID NO: 90.
ZNF251
[00132] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a ZNF251 coding region. As used herein a ZNF251 coding
region refers to the
genomic nucleotide sequence disclosed under GeneID: 90987. An example of a
target mRNA encoding a
ZNF251 polypeptide is the sequence available at Genbank accession number
NM_138367.
[00133] Polynucleotides of the present disclosure that will act to inhibit
expression of a ZNF251
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM-I38367. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a ZNF251 coding region include SEQ ID NO: 91 and SEQ ID NO: 92.
MERTK
CA 02709132 2010-07-07
[00134] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a MERTK coding region. As used herein a MERTK coding
region refers to the
genom ic nucleotide sequence disclosed under GenelD: 10461. An example of a
target mRNA encoding a
MERTK polypeptide is the sequence available at Genbank accession number
NM_006343.
[00135] Polynucleotides of the present disclosure that will act to inhibit
expression of a MERTK
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM006343. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a MERTK coding region include SEQ ID NO: 93, SEQ ID NO: 94 and SEQ
ID NO: 95.
SCG2
to [00136] In another aspect, the present disclosure includes polynucleotides
that inhibit expression of a
polypeptide encoded by a SCG2 coding region. As used herein a SCG2 coding
region refers to the genomic
nucleotide sequence disclosed under GeneID: 7857. An example of a target mRNA
encoding a SCG2
polypeptide is the sequence available at Genbank accession number NM_003469.
[00137] Polynucleotides of the present disclosure that will act to inhibit
expression of a SCG2
polypeptide, include polynucleotides with a sense strand that is substantially
identical or identical to a region
of NM003469. Examples of such polynucleotides that will act to inhibit
expression of a polypeptide
encoded by a SCG2 coding region include SEQ ID NO: 96, SEQ ID NO: 97 and SEQ
ID NO: 98.
[00138] A person skilled in the art will appreciate that other polynucleotides
can be designed to inhibit
the target host factor polypeptides. Methods for designing such molecules are
known in the art. For instance,
polynucleotides that inhibit the expression of one of the polypeptides
described herein may be identified by
the use of cell lines including, but not limited to, HT29 and KM20. A
candidate polynucleotide is the
polynucleotide that is being tested to determine if it decreases expression of
one of the polypeptides
described herein. The candidate polynucleotide can be identical to nucleotides
located in the region encoding
the polypeptide, or located in the 5' or 3' untranslated regions of the mRNA.
Other methods are known in the
art and used for designing and selecting candidate polynucleotides. Candidate
polynucleotides are typically
screened using publicly available algorithms (e.g., BLAST) to compare the
candidate polynucleotide
sequences with coding sequences. Those that are likely to forma duplex with an
mRNA expressed by a non-
target coding region are typically eliminated from further consideration. The
remaining candidate
polynucleotides may then be tested to determine if they inhibit expression of
one of the polypeptides
described herein.
31
CA 02709132 2010-07-07
[00139] In general, candidate polynucleotides are individually tested by
introducing a candidate
polynucleotide into a cell that expresses the appropriate polypeptide. The
candidate polynucleotides may be
prepared in vitro and then introduced into a cell. Methods for in vitro
synthesis include, for instance,
chemical synthesis with a conventional DNA/RNA synthesizer. Commercial
suppliers of synthetic
polynucleotides and reagents for such synthesis are well known. Methods for in
vitro synthesis also include,
for instance, in vitro transcription using a circular or linear vector in a
cell free system.
[00140] When evaluating whether a candidate polynucleotide functions to
inhibit expression of one of
the polypeptides described herein, the amount of target mRNA in a cell
containing a candidate
polynucleotide can be measured and compared to the same type of cell that does
not contain the candidate
polynucleotide. Methods for measuring mRNA levels in a cell are known in the
art. Such methods include
quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Primers
and specific conditions for
amplification of an mRNA vary depending upon the mRNA, and can be readily
determined by the skilled
person. Other methods include, for instance, Northern blotting, and array
analysis.
[00141] Other methods for evaluating whether a candidate polynucleotide
functions to inhibit
expression of one of the polypeptides described herein include monitoring the
polypeptide. For instance,
assays can be used to measure a decrease in the amount of polypeptide encoded
by the mRNA, or to measure
a decrease in the activity of the polypeptide encoded by the mRNA. Methods for
measuring a decrease in the
amount of a polypeptide include assaying for the polypeptide present in cells
containing a candidate
polynucleotide and comparing to the same type of cell that does not contain
the candidate polynucleotide.
For instance, antibody to one of the polypeptides described herein can be used
in Western immunoblot,
immunoprecipitation, or immunohistochemistry.
[00142] Methods for measuring a decrease in the activity of one of the
polypeptides, e.g., ABI2,
ARRDC3, BAD, BRCAI, C17orf85, Clorf7l, C6orf162, CCNJL, CFLI, GON4L, HCG
1986447,
HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI,
MERTK,
MX2, NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7AI4,
ST8SIA3,
STX3, TMC6, TMTC4, TNFSFI2-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2,
ZNF251
or SCG2, vary depending upon the polypeptide. In general, methods for
measuring a decrease in the activity
of a polypeptide include assaying the appropriate activity present in a cell
containing a candidate
polynucleotide and comparing to the same type of cell that does not contain
the candidate polynucleotide.
Methods for measuring the activity of a ABI2, ARRDC3, BAD, BRCA 1, C 17orf85,
C 1 orf71, C6orf162,
CCNJL, CFL1, GON4L, HCG 1986447, HISTI H2AB, HPS4, LHX8, RPS25, RPL23, RPL32,
LOC730139,
32
CA 02709132 2010-07-07
LRRC39, MALTI, MXI, MERTK, MX2, NRGI, OR52A1, PLEKHH 1, PTPN 13, PTPRJ, RLN I,
RNF19A,
SH3BP4, SLC7AI4, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSFI3, TNFSF13, TTN,
UBXN7,
USP47, WNK2, YPEL2, ZNF251 or SCG2 polypeptide are known in the art.
[00143] A candidate polynucleotide that is able to decrease the expression ofa
polypeptide encoded by
a ABI2, ARRDC3, BAD, BRCA 1, C l 7orf85, Cl orf7 1, C6orfl62, CCNJL, CFL 1,
GON4L, HCG 1986447,
HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI,
MERTK,
MX2, NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7AI4,
ST8SIA3,
STX3, TMC6, TMTC4, TNFSFI2-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2,
ZNF251
or SCG2 coding region, a polypeptide encoded by a ABI2, ARRDC3, BAD, BRCA 1, C
17orf85, Cl orf7 1,
1o C6orfl62, CCNJL, CFL1, GON4L, HCG 1986447, HISTIH2AB, HPS4, LHX8, RPS25,
RPL23, RPL32,
LOC730139, LRRC39, MALTI, MXI, MERTK, MX2, NRGI, OR52A1, PLEKHHI, PTPN13,
PTPRJ,
RLN 1, RNF 19A, SH3BP4, SLC7A 14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF 12-TNFSF
13, TNFSF 13,
TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 coding region, or a target mRNA
by at least
80%, or at least 90%, or up to 100%, is considered to be a polynucleotide of
the present disclosure.
[00144] An inhibiting polynucleotide of the present disclosure can be present
in a vector. A vector is a
replicating polynucleotide, such as a plasmid, phage, or cosmid, to which
another polynucleotide may be
attached so as to bring about the replication of the attached polynucleotide.
Construction of vectors
containing a polynucleotide employs standard ligation techniques known in the
art. See, e.g., Sambrook et al,
Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press
(1989). A vector can
provide for further cloning (amplification of the polynucleotide), i.e., a
cloning vector, or for expression of
the polynucleotide, i.e., an expression vector. The term vector includes, but
is not limited to, plasmid
vectors, viral vectors, cosmid vectors, or artificial chromosome vectors.
Examples of viral vectors include,
for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral
vectors, retroviral vectors, and
herpes virus vectors. A vector may result in integration into a cell's genomic
DNA. Typically, a vector is
capable of replication in a bacterial host, for instance E. coli. Preferably
the vector is a plasmid. A
polynucleotide of the present disclosure can be present in a vector as two
separate complementary
polynucleotides, each of which can be expressed to yield a sense and an
antisense strand of a dsRNA, or as a
single polynucleotide containing a sense strand, a loop region, and an
antisense strand, which can be
expressed to yield an RNA polynucleotide having a sense and an antisense
strand of the dsRNA.
[00145] Selection of a vector depends upon a variety of desired
characteristics in the resulting
construct, such as a selection marker, vector replication rate, and the like.
Suitable host cells for cloning or
33
CA 02709132 2010-07-07
expressing the vectors herein are prokaryotic or eukaryotic cells. Suitable
eukaryotic cells include
mammalian cells, such as murine cells and human cells. Suitable prokaryotic
cells include eubacteria, such
as gram-negative organisms, for example, E. COIL
[0034] An expression vector optionally includes regulatory sequences operably
linked to the
polynucleotide of the present disclosure. Typically, the promoter results in
the production of an RNA
polynucleotide. Examples of such promoters include, but are not limited to,
those that cause binding of an
RNA polymerase III complex to initiate transcription of an operably linked
polynucleotide of the present
disclosure. Examples of such promoters include U6 and H I promoters. Vectors
may also include inducible
or regulatable promoters for expression of a polynucleotide of the present
disclosure in a particular tissue or
intracellular environment. The polynucleotide of the present disclosure also
typically includes a transcription
terminator. Suitable transcription terminators are known in the art and
include, for instance, a stretch of 5
consecutive thymidine nucleotides.
[0035] Polynucleotides of the present disclosure can be produced in vitro or
in vivo. For instance,
methods for in vitro synthesis include, but are not limited to, chemical
synthesis with a conventional
DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and
reagents for such synthesis
are well known. Methods for in vitro synthesis also include, for instance, in
vitro transcription using a
circular or linear expression vector in a cell free system. Expression vectors
can also be used to produce a
polynucleotide of the present disclosure in a cell, and the polynucleotide
then isolated from the cell.
[00146] The present disclosure is further directed to methods of treating
viral infection and virus-
mediated cytotoxicity by inhibiting the target host factors of the present
disclosure.
[00147] RNAi, antisense, ribozyme and other nucleic acid therapeutics can be
used to inhibit
expression of ABI2, ARRDC3, BAD, BRCAI, C17orf85, Clorf7l, C6orfl62, CCNJL,
CFLI, GON4L,
HCG 1986447, HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39,
MALTI,
MXI, MERTK, MX2, NRGI, OR52A1, PLEKHHI, PTPNI3, PTPRJ, RLNI, RNF19A, SH3BP4,
SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF I 2-TNFSF 13, TNFSF13, TTN, UBXN7,
USP47,
WNK2, YPEL2, ZNF251 or SCG2 or a combination thereof in patients suffering
from virus infection. For
example, a ABI2, ARRDC3, BAD, BRCAI, CI 7orf85, C l orf7l, C6orf162, CCNJL,
CFLI, GON4L, HCG
1986447, HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1,
MX1,
MERTK, MX2, NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4,
SLC7AI4,
ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2,
34
CA 02709132 2010-07-07
YPEL2, ZNF251 and/or SCG2 antisense strand (either RNA or DNA) may be directly
introduced into the
cells in a form that is capable of binding to the mRNA transcripts.
Alternatively, a vector containing a
sequence which once within the target cells, is transcribed into the
appropriate antisense mRNA, may be
administered. Antisense nucleic acids which hybridize to target mRNA decrease
or inhibit production of the
polypeptide product encoded by a gene by associating with the normally single-
stranded mRNA transcript,
thereby interfering with translation and thus, expression of the protein. For
example, DNA containing a
promoter, e.g., a tissue-specific is operably linked to a DNA sequence (an
antisense template), which is
transcribed into an antisense RNA. By "operably linked" is meant that a coding
sequence and a regulatory
sequence(s) (i.e., a promoter) are connected in such a way as to permit gene
expression when the appropriate
1o molecules (e.g., transcriptional activator proteins) are bound to the
regulatory sequence(s).
[00148] Oligonucleotides complementary to various portions of ABI2, ARRDC3,
BAD, BRCAI,
C17orf85, Clorf7l, C6orf162, CCNJL, CFLI, GON4L, HCG 1986447, HISTIH2AB, HPS4,
LHX8,
RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI, MERTK, MX2, NRGI, OR52A1,
PLEKHHI, PTPNl3, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6,
TMTC4,
TNFSFI2-TNFSF13, TNFSFI3, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 can
be
determined in vitro for their ability to decrease production of ABI2, ARRDC3,
BAD, BRCA 1, C 17orf85,
CIorf71, C6orf162, CCNJL, CFLI, GON4L, HCG 1986447, HISTIH2AB, HPS4, LHX8,
RPS25, RPL23,
RPL32, LOC730139, LRRC39, MALT1, MXI, MERTK, MX2, NRGI, OR52A1, PLEKHHI,
PTPN13,
PTPRJ, RLN1, RNF19A, SH3BP4, SLC7AI4, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-
TNFSFI3,
TNFSFI3, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 in human cells
according to standard
methods. A reduction in ABI2, ARRDC3, BAD, BRCA1, CI7orf85, CIorf71, C6orf162,
CCNJL, CFLI,
GON4L, HCG 1986447, HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139,
LRRC39,
MALT1, MXI, MERTK, MX2, NRG1, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNFI9A,
SH3BP4,
SLC7AI4, ST8SIA3, STX3, TMC6, TMTC4, TNFSFI2-TNFSFI3, TNFSF13, TTN, UBXN7,
USP47,
WNK2, YPEL2, ZNF251 or SCG2 gene product in cells contacted with the candidate
antisense composition
compared to cells cultured in the absence of the candidate composition is
detected using ABI2, ARRDC3,
BAD, BRCA1, Cl7orf85, Clorf7l, C6orf162, CCNJL, CFLI, GON4L, HCG 1986447,
HISTIH2AB,
HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT], MX1, MERTK, MX2,
NRGI,
OR52A1, PLEKHHI, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3,
TMC6,
3o TMTC4, TNFSF 12-TNFSF 13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251
or SCG2-
specific antibodies or other detection strategies. Sequences which decrease
production of ABI2, ARRDC3,
BAD, BRCAI, C17orf85, Clorf7l, C6orfl62, CCNJL, CFLI, GON4L, HCG 1986447,
HISTIH2AB,
HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI, MERTK, MX2,
NRGI,
CA 02709132 2010-07-07
OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3,
TMC6,
TMTC4, TNFSFI2-TNFSFI3, TNFSFI3, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or
SCG2 in
vitro cell-based or cell-free assays are then be tested in vivo in rats or
mice to confirm decreased ABI2,
ARRDC3, BAD, BRCAI, C17orf85, Clorf7l, C6orf162, CCNJL, CFLI, GON4L, HCG
1986447,
HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI,
MERTK,
MX2, NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7AI4,
ST8SIA3,
STX3, TMC6, TMTC4, TNFSF 12-TNFSF 13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2,
ZNF251
or SCG2 production in animals with virus infection.
[00149] Antisense therapy may be carried out by administering to a patient an
antisense nucleic acid by
standard vectors and/or gene delivery systems. Suitable gene delivery systems
may include liposomes,
polymers, receptor-mediated delivery systems, naked DNA, and viral vectors
such as herpes viruses,
retroviruses, adenoviruses and adeno-associated viruses, among others. A
therapeutic nucleic acid
composition is formulated in a pharmaceutically acceptable carrier. The
therapeutic composition may also
include a gene delivery system as described above.
[00150] The present disclosure is also directed to compositions including one
or more inhibitors ofthe
target host factor polypeptide of the present disclosure. Such compositions
typically include a
pharmaceutically acceptable carrier. As used herein "pharmaceutically
acceptable carrier" includes saline,
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying
agents, and the like, compatible with pharmaceutical administration.
Additional active compounds can also
be incorporated into the compositions.
[00151] Pharmaceutically acceptable carriers are biologically compatible
vehicles which are suitable
for administration to an animal: e.g., physiological saline. A therapeutically
effective amount of a compound
is an amount which is capable of producing a medically desirable result such
as reduced production of a
ABI2, ARRDC3, BAD, BRCA1, C17orf85, CIorf7l, C6orf162, CCNJL, CFL1, GON4L, HCG
1986447,
HISTIH2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI,
MERTK,
MX2, NRGI, OR52A1, PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7AI4,
ST8SIA3,
STX3, TMC6, TMTC4, TNFSFI2-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2,
ZNF251
or SCG2 gene product in a treated animal.
[00152] Parenteral administration, such as intravenous, subcutaneous,
intramuscular, and
intraperitoneal delivery routes, may be used to deliver nucleic acids or ABI2,
ARRDC3, BAD, BRCAI,
C17orf85, Clorf7l, C6orfl62, CCNJL, CFLI, GON4L, HCG 1986447, HISTIH2AB, HPS4,
LHX8,
36
CA 02709132 2010-07-07
RPS25, RPL23, RPL32, LOC730139, LRRC39, MALTI, MXI, MERTK, MX2, NRGI, OR52A1,
PLEKHHI, PTPN13, PTPRJ, RLNI, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6,
TMTC4,
TNFSFI2-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2-
inhibitory
peptides on non-peptide compounds. Liposome formulations of therapeutic
compounds may also facilitate
activity.
[00153] Dosages for any one patient depends upon many factors, including the
patient's size, body
surface area, age, the particular nucleic acid to be administered, sex, time
and route of administration,
general health, and other drugs being administered concurrently.
[00154] A list of sequence identification numbers of the present disclosure is
given in Table 7.
to Table 7. List of Sequence Identification numbers.
SEQ ID NO: Description Table/Figure
1 BAD shRNA V2HS_15289 Table 2 and 8A
2 BAD shRNA V2HS243025 Table 2 and 8A
3 BAD shRNA V2HS_20151 l Table 2 and 8A
4 BAD shRNA V2HS_202976 Table 2 and 8A
5 BAD shRNA V2HS_262043 Table 2 and 8A
6 BAD siRNA J-003870-09 Table 2
7 BAD siRNA J-003870-10 Table 2
8 BAD siRNA J-003870-11 Table 2
9 BAD siRNA J-003870-12 Table 2
BAD transcript variant I (NM_004322.3) Figure I A
11 TNFSF12-TNFSF13/TNFSF13 shRNA V2HS 17313 Table 3 and 8A
12 TNFSFI2-TNFSF]3/TNFSFI3 shRNA V2HS 17314 Table 3 and 8A
13 TNFSF12-TNFSFI3/TNFSF13 shRNA V2HS 17316 Table 3 and 8A
14 TNFSFI2-TNFSFI3/TNFSFI3 shRNA V2HS 17317 Table 3 and 8A
TNFSFI2-TNFSFI3/TNFSFI3 shRNA V2HS 17318 Table 3 and 8A
16 TNFSFI2-TNFSF13/TNFSFI3 siRNA J-032530-05 Table 3
17 TNFSFI2-TNFSFI3/TNFSFI3 siRNA J-032530-06 Table 3
18 TNFSFI2-TNFSFI3/TNFSF13 siRNA J-032530-07 Table 3
37
CA 02709132 2010-07-07
19 TNFSF12-TNFSFI3/TNFSFI3 siRNA J-032530-08 Table 3
20 TNFSFI2-TNFSF13 siRNA J-011523-05 Table 4
21 TNFSFI2-TNFSFI3 siRNA J-011523-06 Table 4
22 TNFSFI2-TNFSFI3 siRNA J-011523-07 Table 4
23 TNFSFI2-TNFSFI3 siRNA J-011523-08 Table 4
24 MX2 transcript Figure I C
25 MX2 shRNA V2HS_l52031 Table 5 and 8A
26 MX2 siRNA J-01 1736-05 Table 5
27 MX2 siRNA J-0 1 1 736-06 Table 5
28 MX2 siRNA J-0 1 1 736-07 Table 5
29 MX2 siRNA J-01 1736-08 Table 5
30 USP 47 transcript Figure I D
31 USP47 shRNA V2HS174637 Table 6 and 8A
32 USP47 shRNA V2HS174639 Table 6 and 8A
33 USP47 shRNAV2HS_174641 Table 6 and 8A
34 USP47 shRNA V2HS174642 Table 6 and 8A
35 USP47 shRNA V2HS218228 Table 6 and 8A
36 USP47 siRNA J-006093-05 Table 6 and 8A
37 USP47 siRNA J-006093-06 Table 6 and 8A
38 USP47 siRNA J-006093-07 Table 6 and 8A
39 USP47 siRNA J-006093-08 Table 6 and 8A
40 USP47 shRNA V2HS-174640 Table 6 and 8A
41 MXI shRNA V2HS_152026 Table 8 A
42 MXI shRNA V2HS_152028 Table 8 A
43 MALT1 shRNA V2HS84222 Table 8 A
44 RPS25 shRNA V2HS 93855 Table 8 A
45 SH3BP4 shRNA V2HS 260346 Table 8 A
46 HCG 1986447 transcript Figure I E
47 HCG 1986447 shRNA V2HS29650 Table 8 A
48 WNK2 shRNA V2HS 238923 Table 8 A
49 TMC6 shRNA V2HS_198493 Table 8 A
50 CFLI shRNA V2HS 64314 Table 8 A
38
CA 02709132 2010-07-07
51 ABI2 shRNA V2HS_196634 Table 8 A
52 ARRDC3 shRNA V2HS_217697 Table 8 A
53 BRCAI shRNA V2HS254648 Table 8 A
54 BRCAI shRNA V2HS 280394 Table 8 A
55 BRCAI shRNA V2HS_255064 Table 8 A
56 C17orf85 shRNA V2HS_176062 Table 8 A
57 C lorf 71 shRNA V2HS 44617 Table 8 A
58 C6orfl62 shRNA V2HS35766 Table 8 A
59 CCNJL shRNA V2HS_136349 Table 8 A
60 GON4L shRNA V2HS 138350 Table 8 A
61 HISTIH2AB shRNA V2HS33954 Table 8 A
62 HPS4 shRNA V2HS_70495 Table 8 A
63 LHX8 shRNA V2HS_75780 Table 8 A
64 RPL23 shRNA V2HS_23046 Table 8 A
65 RPL32 shRNA V2HS 165267 Table 8 A
66 LOC730139 transcript Figure 1 F
67 LOC730139 shRNA V2HS_25169 Table 8 A
68 LOC730139 shRNA V2HS 25168 Table 8 A
69 LRRC39 shRNA V2HS_18852 Table 8 A
70 LRRC39 shRNA V2HS 18851 Table 8 A
71 NRG1 shRNA V2HS 84939 Table 8 A
72 OR52A 1 shRNA V2HS 244561 Table 8 A
73 OR52AI shRNA V2HS 49243 Table 8 A
74 PLEKHHI shRNA V2HS_46786 Table 8 A
75 PTPN 13 shRNA V2HS 57273 Table 8 A
76 PTPRJ shRNA V2HS 91546 Table 8 A
77 PTPRJ shRNA V2HS 171000 Table 8 A
78 RLN I shRNA V2HS 94799 Table 8 A
79 RNF 19A shRNA V2HS 96523 Table 8 A
80 SLC7A14 shRNA V2HS 57109 Table 8 A
81 ST8SIA3 shRNA V2HS_l 14878 Table 8 A
82 ST8SIA3 shRNA V2HS 114879 Table 8 A
39
CA 02709132 2010-07-07
83 STX3 shRNA V2HS 33937 Table 8 A
84 TMTC4 shRNA V2HS 177667 Table 8 A
85 TMTC4 shRNA V2HS 275500 Table 8 A
86 TTN shRNA V2HS 171633 Table 8 A
87 TTN shRNA V2HS 171637 Table 8 A
88 UBXN7 shRNA V2HS 130208 Table 8 A
89 YPEL2 shRNA V2HS 77698 Table 8 A
90 YPEL2 shRNA V2HS 77701 Table 8 A
91 ZNF251 shRNA V2HS 250202 Table 8 A
92 ZNF251 shRNA V2HS 215547 Table 8 A
93 MERTK shRNA V2HS 1643 Table 8 B
94 MERTK shRNA V2HS 168768 Table 8 B
95 MERTK shRNA V2HS 197158 Table 8 B
96 SCG2 shRNA V2HS 172404 Table 8 B
97 SCG2 shRNA V2HS 172401 Table 8 B
98 SCG2 shRNA V2HS 172400 Table 8 B
99 TNFSFI2 -TNFSFI3 transcript Fig IG
100 TNFSF 13 transcript variant alpha Fig I H
101 TNFSFI3 transcript variant beta Fig 1 I
102 TNFSFI3 transcript variant gamma Fig I J
103 BAD transcript variant 2 (NM_032989) Fig. lB
[00155] The present invention will be further illustrated in the following
examples. However it is to be
understood that these examples are for illustrative purposes only, and should
not be used to limit the scope
of the present invention in any manner.
Example 1
[00156] Drosophila-based screens5 have been used to identify genes involved in
influenza replication,
and two very recent studies employed genome-wide shRNA arrays to identify
mammalian host proteins
involved in various stages of the influenza virus lifecycle.6'' Severe
influenza pathology has been seen with
pandemic 1918 virus" and SOIV-infected patient lung pathology showed alveolar
damage and hemorrhage
CA 02709132 2010-07-07
suggestive of atypical immune responses., Studies in pigtailed macaques
reported that the activation of
apoptotic pathways may contribute to tissue damage during infection10.
[00157] Human genes (target host factors) required for viral replication and
cytotoxicity were
identified. Interference with these proteins provided a basis for inhibiting
viral production while protecting
infected cells from virus-induced death.
[00158] Multiple genome-wide lentiviral-based shRNAmir screens consistently
identified 35 annotated
candidate genes associated with cell survival despite infection (see Table 1).
Initial analysis of 4 candidate
genes (BAD, TNFSFI2-13/TNFSF13, MX2, and USP47), using lentiviral-based and
synthetic RNAi
silencing confirmed protective roles with dramatic inhibition of virus
replication after challenge with
influenza viruses of multiple H and N types, including the contemporary
pandemic swine-origin H IN I virus.
High throughput genorne-wide screens
[00159] The availability of human genome-wide lentiviral-based shRNAi (short
hairpin RNA
interference) libraries provides the ability to identify host genes important
in influenza pathology. RNAi
regulates gene expression through sequence-specific mRNA targeting, and genome-
wide RNAi screens
produce genome-wide loss-of-function phenotypes.,) This allows for a system-
level understanding ofthe host
cellular processes and comprehensive identification of molecular components
underlying influenza
pathogenicity. The lentiviral-based Decode- RNAi library (Open Biosystems),
consisting of 7 pools, each
with - 10,000 shRNAmir constructs, was used to establish stably transduced
genome-wide knockdown of
every host gene in human lung adenocarcinoma A549 cells (Fig. 2). Cells were
transduced at an MOI of 0.3,
which allowed 100-fold coverage of each clone and reduced the risk of any
single cell being multiply
transduced to less than 4% of the cell population. The transduced cells were
then infected with influenza
virus strain A/New York/55/2004(H3N2) (NY55) at an MOI of 7 PFU/cell to ensure
>99% of cells were
initially infected (Fig. 2). Illumina -mediated high throughput sequencing of
all 7 pools was then
individually performed on two independent biological replicate screens of
surviving transduced, infected
cells. The combined high throughput genome-wide screens identified more than
1000 potential targets (Fig.
3). 35 genes were identified in both trials (Fig. 3), knockdown of which
provided protection against
influenza A/NY/55/2005 infection. Many of these genes' ontological functions
are shown in Fig. 4-6 and
discussed below. Of the 35 genes found in both high-throughput screens (Fig.
3), 4 genes-BAD, TNFSFI2-
TNFSFI3/TNFSFI3, MX2, and USP47- appeared at 100- to 1000-fold higher
frequencies during Illumina
high-throughput sequencing than did most of the other genes (Table 8). BAD,
TNFSFI2-
TNFSFI3/TNFSFI3, MX2, and USP47 genes were also identified in a preliminary
manual screen of 2 library
41
CA 02709132 2010-07-07
pools in which transduced cell clones that survived NY55 infection were
individually isolated, amplified
(which demonstrated each survived long-term transduction and infection), and
sequenced to identify the
shRNA insert.
42
CA 02709132 2010-07-07
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b vi LL N v g p C d C 7 y A O M
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Example 2
Target host factor Validation
[00160] As an initial confirmation that BAD, TNFSFI2-TNFSFI3/TNFSFI3, MX2, and
USP47
knockdown eliminate influenza virus-mediated cytotoxicity, sets of A549 cells
were individually transduced
with shRNA-lentiviruses specifically targeting each transcript. The shRNA
constructs contained puromycin
markers for positive selection of transduced cells. Stably transduced cells
were passaged in puromycin at
least twice to remove non-transduced cells, followed by infection with various
influenza A viruses. Non-
transduced cells, as well as cells transduced with an irrelevant non-silencing
shRNA, were killed after being
infected with NY55 at an MOI of 1. In contrast, there was no observable
cytopathic effect (CPE) in influenza
lo virus infected cells that had been transduced with BAD-, TNFSF 12-TNFSF
13/TNFSF 13-, MX2-, or USP47-
specific shRNAs (Fig. 7). Knockdown of these genes also resulted in
significant reductions in virus titer
compared to non-transduced and non-silencing transduction, with virus titers
being generally reduced to
about 20% of control levels (Fig. 8b). The shRNA constructs that target
TNFSFI3 mRNA also inhibits
TNFSFI2-13 mRNA. WST-1 cell viability assay (Roche) showed no significant
reduction in knockdown
cell viability compared to controls (Fig. 9a).
[00161] As further validation, to ensure that lack of cytotoxicity and reduced
virus replication were not
caused by off-target effects or other artifacts, protein knockdowns were
repeated with siRNA duplexes
(Dharmacon ). Sets of A549 cells were treated with each of four distinct
siRNAs that target each of the
four proteins (plus an irrelevant non-silencing control) twice, 24 h apart,
and after a further 24 h, were
infected with influenza virus. Influenza virus replication was dramatically
reduced by each ofthe four BAD-,
TNFSF I 2-TNFSF I 3/TNFSF 13-, MX2-, and USP47-specific siRNAs (Fig. 8c; and
Fig 10) and no detectable
CPE was observed.
[00162] Since NY55 is an H3N2 virus (like the 1968 pandemic "Hong Kong"
virus), it was then
determined whether these observations could be extended more broadly to other
influenza virus subtypes.
Therefore, the effects of knocking down these genes were examined on
cytopathology and replication ofthe
A/Puerto Rico/8/1934 (H IN 1) (PR8) and contemporary pandemic SOIV H 1N1
isolates. Similar to effects
observed with NY55, knockdown of BAD, TNFSFI2- TNFSFI3/TNFSFI3, MX2, and USP47
also
protected the host cell from virus-induced cytopathology during PR8 (HIN1) and
SOIV (HINI) infections
(Fig. 7a) and resulted in reduced virus replication (Fig. 8b,c). These data
strongly indicate that MX2, BAD,
TNFSFI2-13, TNFSFI3, and USP47 knockdown protect human lung epithelial A549
cells from virus-
mediated cytotoxicity during infection by viruses of multiple H and N types.
Thus, different influenza virus
46
CA 02709132 2010-07-07
subtypes require certain common cellular pathways for replication and the
roles played by BAD, TNFSFI2-
TNFSF 13/TNFSF 13, MX2, and USP47 are virus-type independent. Each ofthese 4
genes (BAD, TNFSFI2-
TNFSFI3/TNFSFI3, MX2, and USP47), and many of the other 31 consistently
identified genes, participate
in integrated cellular pathways with broad biological impact, including host
defence, autoimmunity, cancer
and cell cycle, and inflammation. For example, BAD is a pro-apoptotic protein
that belongs to the BH3-only
subfamily. In light of the pro-apoptotic nature of BAD, a reduction in viral
replication seen here suggests a
synergistic role of BAD in the virus' lifecycle by sequestering Bcl-2 and
promoting apoptosis.
[00163] TNFSFI3 and the fusion protein TNFSFI2-13-also known as APRIL and TWE-
PRIL,
respectively-belong to the tumour necrosis factor (TNF) ligand superfamily.1-
1t Influenza virus infection up-
regulates TNFSFI3 expression in lung tissues and affects recruitment of
macrophages to the site of infection
in TNFSFI3 deficient mice.14 Human MX2 protein has no anti-influenza virus
activity described to date;
however, it localizes to the nuclear membrane where it may play a role in
nucleocytoplasmic
trafficking. 15.16.17.19 USP47 is a deubiquitinase, whose specific role in
cellular processes remains relatively
unknown.,, Although there are previous reports indicating the importance of
the ubiquitin-proteasome
pathway in other viruses, the involvement of deubiquitylation in influenza
remains relatively unknown.
More studies on the cellular functions and their involvement in influenza
infection are warranted to delineate
the specific molecular mechanisms to which TNFSFI2-13, TNFSFI3 and USP47
proteins contribute.
[00164] In order to delineate host biomolecular interactions that may function
in conferring protection
against viral infection, network analysis= was performed. The genes identified
in this study participated in
175 binary interactions and three regulatory protein complexes (Fig. 6, Table
9 and Fig.I 1-13). Network
analysis highlighted the role of BAD and TNFSFI2-13/TNFSF]3 as being critical
nodes within the
pathways (Fig. 12), and predicted the involvement ofNF-KB, MAPK and AKT
pathways in host processes
that confer host cell survival.
[00165] Knockdown of host MX2, BAD, TNFSFI2-13, TNFSFI3, or USP47 genes
confers broad, non-
lethal and long-lasting virus type-independent protection against influenza
virus-mediated cytotoxicity and
promotes host cell survival.
Example 3
Target Host Factors
47
CA 02709132 2010-07-07
[00166] Cytopathology induced during influenza infection is a contributing
factor to tissue damage and
has been suggested to be a catalyst for aberrant host immune response during
disease progression, but the
underlying molecular mechanisms remain obscure.
[00167] BAD is a pro-apoptotic protein that belongs to the BH3-only subfamily.
Its interaction with
Bcl-2 and Bcl-XL inhibits the function of these two anti-apoptotic molecules
to promote apoptosis.1,2 In West
Nile virus (WNV)'a, the human immunodeficiency virus (HIV),,,, and Heptitis B
and C viruses, viral-
induction of apoptosis are significant contributing factors to disease
pathology. It also has been recently
reported that influenza viruses initiate and require cellular apoptosis for
efficient virus replication, and that
the anti-apoptotic Bcl-2 protein negatively effects influenza virus
replication.-' Similarly, a study with the
1918 pandemic virus in macaques indicate up-regulation of cell death and
inflammatory related genes''.
Without whishing to be bound by theory the results suggest a synergistic role
of BAD in the virus' lifecycle
by sequestering Bcl-2 and promoting apoptosis.
[00168] TNFSFI3 and the fusion protein TNFSFI2-13 belong to the tumour
necrosis factor (TNF)
ligand superfamily.'Z" Intergenic splicing between exon 6 of TNFSFI2, another
member of the TNF
superfamily, and exon 2 of TNFSFI3 leads to the production ofa fusion protein
called TNFSF12-13, which
displays the same receptor specificity as TNFSFI3.1' TNFSFl3 and TNFSFI2-13
are reported to induce
cellular proliferation and be involved in innate and adaptive immune
responses.', TNFSF 13 is a proliferation
inducing ligand located in the cytosol and reportedly involved in class switch
recombination in B cell
responses.] 5 Influenza virus infection has been shown to upregulate TNFSFI3
expression in lung tissues and
affect recruitment of macrophages to the site of infection in TNFSFI3
deficient mice . 16
[00169] Human MX2 protein has no anti-influenza virus activity described to
date; however, it
localizes to the nuclear membrane where it may playa role in nucleocytoplasmic
trafficking. 17,18,19 Without
whishing to be bound by theory the results suggest that MX2 has a role in
trafficking specific viral proteins
or protein complexes between the cytoplasm and the nucleus.2 21
[00170] USP47 is a deubiquitinase, whose specific role in cellular processes
remains relatively
unknown.22 Recently, USP47 has been reported to play a function in scattering
responses in epithelial cells.=1
Although there are previous reports indicating the importance of the ubiquitin-
proteasome pathway in other
viruses, the involvement of deubiquitylation in influenza remains relatively
unknown.
[00171] 31 of the 35 gene candidates identified in the Illumina screen (4
genes had no known
functions) were used for bioinformatics interrogation in order to identify
host biomolecular interactions that
48
CA 02709132 2010-07-07
these candidates participated in employing a open source interaction database
analysis platform24-25.
Computational network analysis demonstrated that several members of the two
regulatory pathways Nuclear
factor (NF)-KB and the mitogen activated protein kinase (MAPK) pathways were
direct interactors of the
identified gene candidates. For example, (i) the gene product of MALT-I
interacts with IKBKG (NF-KB
essential modulator, NEMO), TRAF-6 (regulator of TLRNF-KB pathway), MAP3K and
MAKP9, and
TNFAIP3 (a negative regulator of NF-KB), (ii) RPL23 and PTPNI3 directly
interacts with NF-KBIA (a
regulator ofNF-KB), (iii) RPL23 participated in direct interactions with
members of the MAPK pathway and
TRAF-2 (TRAF-2 is required for the activation of both MAPK/JNK and NF-KB
pathways), (iv) Members of
MAPK family and AKT, known to phosphorylate NF-icB subunits26 were
demonstrated to be direct
io interacting protein partner with BAD, (v) 14-3-3 family of proteins, known
to be involved in protection
against viral infections27,including in AKT signalling pathwaysx, were found
to be direct interacting partners
for four of the 31 candidates, BAD, SH3BP4, CFLI and RPL23 (Table 9). (vi)
TNFSF-12-13 (TWE-PRIL) a
critical node in the analyses was previously demonstrated to be involved in
stimulating lymphocyte
proliferation. Further, the 31 identified gene candidates were analyzed for
the overrepresentation of
transcription factor binding sites. Over represented transcription factors
(TFs) were defined if the binding
sites for the factors were predicted in the promoter region of at least three
of the candidate genes. This
analysis revealed 36 transcription factor binding sites to be enriched within
the promoter regions of the
submitted genes, among which several TFs were known to be active in host
responses downstream of the
NF-KB and MAPK pathways. Over represented TFs such as MEF2A, AIRE, SRF,
CREBIand IRF 1, all are
known to be involved in host responses against pathogenic infections,
including viral infections=-',." 32-'4. This
analysis was consistent with the interaction network analysis which also
demonstrated the involvement of
both NF-KB and MAPK pathways in the activity of the identified candidates. The
human genome-wide
screen identified genes that play significant roles in protecting host cells
from virus-induced cytopathic
effect, and also are important in influenza propagation.
49
CA 02709132 2010-07-07
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CA 02709132 2010-07-07
Example 4
Methods Summary
[00172] Human whole-genome screen: A549 cells were transduced at MOI 0.3 with
each of 7
Decode RNA GIPZ Lentiviral Positive Screening Library pools according to
manufacturer's protocol (Open
Biosystems). After 72 h, cells were washed twice with I x phosphate-buffered
saline (PBS) and infected with
NY55 at an MOI of 7 PFU per cell. At 72 hpi, cells were washed twice with I x
PBS and harvested.
Genomic DNA was isolated by phenol/chloroform extraction followed by ethanol
precipitation.
[00173] PCR: PCR was carried out on isolated genomic DNA using Expand High
Fidelity polymerase
mix (Roche) and product was purified from polyacrylamide gels. Pooled cDNA was
sequenced by high-
throughput Illumina sequencing technology at Canada's Michael Smith Genome
Sciences Centre
(Vancouver, British Columbia).
[00174] Lentivirus packaging and transduction: Individual human shRNAmir
lentiviral clones
(Thermo Scientific Open Biosystems; 3-4 for each gene target of interest) were
prepared and isolated
according to manufacturer's protocol. Individual shRNAs were packaged into
lentivirus particles by co-
transfection of each shRNAmir with pMD2.G and psPAX2 (Addgene plasmid 12260)
in individual sets of
HEK-293T cells according to Open Biosystems' Trans-lentiviral Packaging
protocol. A549 cells were
transduced with lentivirus at an MOI of 0.5. At 72 h post transduction, 3
g/ml puromycin (Sigma) was
added to the media. Cells were passaged twice in puromycin-supplemented
completed media to select
transductants before they were infected with virus (described below).
[00175] siRNA Transfection: Sets of A549 cells were treated with 25 nM of each
of 4 ON-Targetplus
siRNA (Dharmacon) targeting each of the USP47, TNFSF 12-13, TNFSF 13, and BAD
genes. siRNAs were
introduced into cells with Lipofectamine RNAiMAX (Invitrogen). Each cell set
was re-treated with the same
siRNA 24 h later. After a further 24 h, cells were infected with virus at an
MOI 0.1 and harvested for
analysis 48 hpi.
[00176] Influenza virus infections: Sets of transduced or transfected A549
cells were infected with
influenza virus strains A/NY/55/2004(H3N2) at an MOI of 1 PFU/cell, or with
A/PR/8/34(H I N 1) at an MOI
of 0.01, or with SOIV at an MOI of 0.1 and harvested at 48 hpi for virus
titration by plaque assay.
[00177] Influenza plaque assay: Influenza plaque assay was carried out on MDCK
cells as previously
described22.
CA 02709132 2010-07-07
[00178] Bioinformatics Analysis: Sequences were analysed by an in-house
computer algorithm.
Genes were functionally categorized using PANTHER ontology system2?='. Network
analysis were done with
InnateDBZ database and visualization employing Cerebral2l.
Table 10: Replication of A/NY/55/2004(H3N2) in shRNA knockdown A549 cells.
Cells were transduced
with shRNA---packaged lentivirus at MOI 0.5, and infected 72 h later at MOI 1.
Standard Deviation
represents 3 biological replicates.
Gene shRNA 46 of r c n-silencing Standard Deviation
i+l -Soendec 100.00 OAO
(MxB) 152C31 1S 91 _C 54
ITNFSFI 17313 3038 15.24
1-314 0746 12,54
11316 22.73 15.40
1731735 1509
17318 16.53 2025
(BAD) 15289 23 29 14 97
201511 1193 2252
277976 29 91 36 77
(USP47) 174637 13.23 20.92
174641 2545 3242
174642 176.72 3388
174639 59 96 113 71
218228 42 49 90 22
Table 11: Replication of A/NY/55/2004(H3N2) in A549 siRNA transfected cells.
Cells were treated with 25
pmol siRNA twice, 24 h apart, and after a further 24 h were infected at MOI
0.1. Standard Deviation
1o Represents 2 biological replicates.
71
CA 02709132 2010-07-07
Gene siRNA t of non.54enc.ng Standard Devation
Non-sendng MOD 0.00
USiP47 05 4.48 - 42
s06 9.95 12.22
s i07 1S.92 1.07
Sias 18.91 30.15
BAD 5i09 22 39 9.4C
silo 16.42 17.47
=111 19 91 10 1
si12 23.38 19.09
Mx$ s105 9 42 11 11
si06 1.49 0.09
iu7 0 2.21
si08 3.48 5.43
TPJFSFI2-13 sio5 5.47 1.72
si06 5.97 3.45
-i07 2.49 2.72
si08 15.42 3.16
TP4FSFI3 si05 16.42 8.23
Sio6 12.44 0.26
siO7 1.99 2.38
si08 3.98 335
[00179] All citations are herein incorporated by reference, as if each
individual publication was
specifically and individually indicated to be incorporated by reference herein
and as though it were fully set
forth herein. Citation of references herein is not to be construed nor
considered as an admission that such
references are prior art to the present invention.
[00180] One or more currently preferred embodiments of the invention have been
described by way of
example. The invention includes all embodiments, modifications and variations
substantially as
hereinbefore described and with reference to the examples and figures. It will
be apparent to persons skilled
in the art that a number of variations and modifications can be made without
departing from the scope of the
invention as defined in the claims. Examples of such modifications include the
substitution of known
equivalents for any aspect of the invention in order to achieve the same
result in substantially the same way.
72
CA 02709132 2010-07-07
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