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CA 02611528 2007-12-07
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TITLE OF THE INVENTION
TARGETS FOR INHIBITING HCV REPLICATION
RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Application No.
60/692,821,
filed June 22, 2005, and U.S. Provisional Application No. 60/710,006, August
19, 2005, each of which
are hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
The references cited in the present application are not admitted to be prior
art to the
claimed invention.
Exposure to HCV results in an overt acute disease in a small percentage of
cases, while
in most instances the virus establishes a chronic infection causing liver
inflammation and slowly
progresses into liver failure and cirrhosis. (Iwarson, FEMS Microbiol. Rev.,
14:201-204, 1994.)
Epidemiological surveys, indicate HCV plays an important role in
hepatocellular carcinoma pathogenesis.
(Kew, FEMS Microbiol. Rev., 14:211-220, 1994, Alter, Blood, 85:1681-1695,
1995.)
The HCV genome consists of a single strand RNA about 9.5 kb in length,
encoding a
precursor polyprotein about 3000 amino acids. (Choo et al., Science, 244:362-
364, 1989, Choo et al.,
Science, 244:359-362, 1989, Takamizawa et al. J. Virol., 65:1105-1113, 1991.)
The HCV polyprotein
contains the viral proteins in the order: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-
NS5B.
Individual viral proteins are produced by proteolysis of the HCV polyprotein.
Host cell
proteases release the putative structural proteins C, El, E2, and p7, and
create the N-terminus of NS2 at
amino acid 810. (Mizushima et al., J. Virol., 68:2731-2734, 1994, Hijikata et
al., Proc. Natl. Acad. Sci.
USA., 90:10773-10777, 1993.)
The non-structural proteins NS3, NS4A, NS4B, NS5A and NS5B presumably form the
virus replication machinery and are released from the polyprotein. A zinc-
dependent protease associated
with NS2 and the N-terminus of NS3 is responsible for cleavage between NS2 and
NS3. (Grakoui et al.,
J. Virol., 67:1385-1395, 1993, Hijikata et al., Proc. Natl. Acad. Sci. USA,
90:10773-10777, 1993.)
A distinct serine protease located in the N-terminal domain of NS3 is
responsible for
proteolytic cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B
junctions.
(Barthenschlager et al., J. Virol., 67:3835-3844, 1993, Grakoui et al., Proc.
Natl. Acad. Sci. USA,
90:10583-10587, 1993, Tomei et al., J. Virol. 67:4017-4026, 1993.) RNA
stimulated NTPase and
helicase activities are located in the C-terminal domain of NS3.
NS4A provides a cofactor for NS3 protease activity. (Failla et al., J. Virol.,
68:3753-
3760, 1994, De Francesco et al., U.S. Patent No. 5,739,002.)
NSSA is a highly phosphorylated protein conferring interferon resistance.
(Pawlotsky.
J. Viral Hepat. Suppl., 1:47-48, 1999.)
NS5B provides an RNA-dependent RNA polymerase. (De Francesco et al.,
International
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Publication Number WO 96/37619, published November 28, 1996, Behrens et al.,
EMBO 15:12-22,
1996, Lohmann et al., Virology 249:108-118, 1998.)
SUMMARY OF THE INVENTION
Liver expressed proteins involved in HCV replication were identified using a
procedure
measuring the effect of inhibiting expression of host cell proteins on HCV
replicon activity. The
identified proteins and encoding nucleic acid provide targets for inhibiting
HCV replication and for
evaluating the ability of compounds to inhibit HCV replication. Compounds
inhibiting HCV replication
include compounds targeting identified proteins and compounds targeting
nucleic acid encoding the
identified protein. Several of the host genes identified as targets for
inhibiting HCV replication were
also found to be a target for inhibiting HIV replication: The ability to serve
as a target for inhibiting both
HIV and HCV replication indicates that such an identified gene and encoded
protein may be a useful
target for inhibiting replication of different types of viruses and not
limited to inhibiting replication of a
particular virus.
Thus, a first aspect of the present invention describes a method of
identifying a host cell
factor involved in viral (e.g., HCV) replication using a short inhibitory RNA
(siRNA) library. The
method comprises the step of measuring the ability of a siRNA library
targeting different cell factors to
inhibit viral (e.g.,HCV) replication, wherein the siRNA library comprises at
least 10 different siRNA's
targeting a different host factor that was.not previously associated with
viral (e.g.,HCV) replication.
A "library" contains a collection of different siRNA that is screened as part
of an
experiment. The experimental results are obtained at about the same time or
over a limited time period.
In different embodiments, the limited time period is within about a week or
within about a day.
Preferably, the members of the library are tested at the same time.
Reference to the library comprising a certain number of siRNA targeting
different host
cell factors indicates that at least the indicated number of different siRNA
are used. In different
embodiments the library comprises 100, 500 or 1000 members and/or each of the
different members is a
kinase or phosphatase. In another embodiment, the library comprises 5000,
10000, or 20000 members
and is considered to be a genome scale library.
Another aspect of the present invention describes a method of evaluating the
ability of a
compound to inhibit replication of a virus. The method involves:
(a) identifying a compound binding to, or inhibiting the activity or
expression of, a
target protein selected from the group consisting of: AKAP8 (SEQ ID NO: 11),
ALK (SEQ ID NO: 67),
ATM (SEQ ID NO: 68), C140RF24 (SEQ ID NO: 69), DGKD (SEQ ID NO: 15), DGKZ (SEQ
ID NO:
70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ID NO: 71), DUSP6 (SEQ ID NO: 9), DUT
(SEQ ID
NO: 51), DYRK2 (SEQ ID NO: 72), DYRK4 (SEQ ID NO: 53), ENPP5 (SEQ ID NO: 73),
EPHA2
(SEQ ID NO: 13), FGFR2 (SEQ ID NO: 74), FHIT (SEQ ID NO: 75), FRK (SEQ ID NO:
76), GAK
(SEQ ID NO: 55), GCK (SEQ ID NO: 19), MAP2K3 (SEQ ID NO: 77), NME4 (SEQ ID NO:
1),
PANK1 (SEQ ID NO: 78), PCK1 (SEQ ID NO: 3), PFKL (SEQ ID NO: 63), PIK4CA (SEQ
ID NO: 21),
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PRKWNK3 (SEQ ID NO: 5), PRPSILI (SEQ ID NO: 79), PSKHl (SEQ ID NO: 57), PTK9L
(SEQ ID
NO: 80), SOCS5 (SEQ ID NO: 59), SRPK1 (SEQ ]D NO: 17), STK16 (SEQ ID NO: 7),
STK35 (SEQ ID
NO: 81), TAF1 (SEQ ID NO: 82), TBK1 (SEQ ID NO: 83), TJP2 (SEQ ID NO: 84),
TNK1 (SEQ ID
NO: 61), TPKl (SEQ ID NO: 85), TRIB3 (SEQ ID NO: 86), TRPM7 (SEQ ID NO: 87),
VRK (SEQ ID
NO: 88), ALPPL2 (SEQ ID NO: 89), AP4M1 (SEQ ID NO: 23), CAPZAI (SEQ ID NO:
25), DNAH5
(SEQ ID NO: 27), DOM3Z (SEQ ID NO: 90), FTCD (SEQ ID NO: 29), PDIA3 (SEQ ID
NO: 31),
MDM4 (SEQ ID NO: 91), WDR66 (SEQ ID NO: 92), NOP5/NOP58 (SEQ ID NO: 33), NSUN6
(SEQ
ID NO: 93), PAFAHIBI (SEQ ID NO: 35.), PARVB (SEQ ID NO: 37), PHEX (SEQ ID NO:
39), PKN1
(SEQ ID NO: 44), POLR2J2 (SEQ ID NOs: 41 or 65), RAB20 (SEQ ID NO 43), SYNPR
(SEQ ID NO:
45), and TRPM5 (SEQ ID NO: 47); or a protein substantially siznilar to the
target protein, wherein the
substantially similar protein has a sequence identity of at least 95% to the
target protein; and
(b) determining the ability, of the compound identified in step (a) to inhibit
viral
replication.
The initial identification of a compound binding to, or inhibiting the
activity, or
expression of a target protein, can be performed experimentally or based on
known information
concerning the ability of a compound to bind or inhibit one of the identified
targets. Information on the
different targets is available in the scientific literature. Preferably, the
compound is initially identified as
inhibiting activity.,
In preferred embodiments the virus is either H1V or HCV. Determining the
ability of a
compound to inhibit viral replication includes either, or both, (1) the
initial identification of a compound
as able to bind or inhibit viral replication or (2). determining the extent to
which the compound inhibits
viral replication. Inhibition of viral replication can be determined with
quantitative or qualitative
measurements. For example, determining the ability of a compound to inhibit
HCV replication includes
either, or both, (1) the initial identification of a compound as able to bind
or inhibit HCV replication or
(2) determining the extent to which the compound inhibits HCV replication.
Sequence identity to a reference protein sequence is determined by aligning
the protein
sequence with the reference sequence and determining the number of identical
amino acids in the
corresponding regions. This number is divided by the total number of amino
acids in the reference
sequence (e.g., SEQ ID NO: 1) and then multiplied by 100 and rounded to the
nearest whole number.
Another aspect of the present invention describes a method of inhibiting HCV
replication. The method employs an effective amount of a compound inhibiting
the activity or expression
of a target protein selected from the group consisting of: AKAPB (SEQ ID NO:
11), ALK (SEQ ID NO:
67), ATM (SEQ ID NO: 68), C14ORF24 (SEQ ID NO: 69), DGKD (SEQ ID NO: 15), DGKZ
(SEQ ID
NO: 70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ID NO: 71), DUSP6 (SEQ ID NO: 9),
DUT (SEQ
ID NO: 51), DYRK2 (SEQ ID NO: 72), DYRK4 (SEQ ID NO: 53), ENPP5 (SEQ ID NO:
73), EPHA2
(SEQ ID NO: 13), FGFR2 (SEQ ID NO: 74), FHIT (SEQ ID NO: 75, FRK (SEQ ID NO:
76), GAK
(SEQ ID NO: 55), GCK (SEQ ID NO: 19), MAP2K3 (SEQ ID NO: 77), NME4 (SEQ ID NO:
1),
PANKl (SEQ ID NO: 78), PCKI (SEQ ID NO: 3), PFKL (SEQ ID NO: 63), PIK4CA (SEQ
ID NO: 21),
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PRKWNK3 (SEQ ID NO: 5), PRPSiLl (SEQ ID NO: 79), PSKH1 (SEQ ID NO: 57), PTK9L
(SEQ ID
NO: 80), SOCS5 (SEQ ID NO: 59), SRPKl (SEQ ID NO: 17), STK16 (SEQ ID NO: 7),
STK35 (SEQ ID
NO: 81), TAFl (SEQ ID NO: 82), TBK1 (SEQ ID NO: 83), TJP2 (SEQ ID NO: 84),
TNK1 (SEQ ID
NO: 61), TPK1 (SEQ ID NO: 85), TRIB3 (SEQ ID NO: 86), TRPM7 (SEQ ID NO: 87),
VRK (SEQ ID
NO: 88), ALPPL2 (SEQ ID NO: 89), AP4M1 (SEQ ID NO: 23), CAPZAI (SEQ ID NO:
25), DNAH5
(SEQ ID NO: 27), DOM3Z (SEQ ID NO: 90), FTCD (SEQ ID NO: 29), PDIA3 (SEQ ID
NO: 31),
MDM4 (SEQ ID NO: 91), WDR66 (SEQ ID NO: 92), NOP5/NOP58 (SEQ ID NO: 33), NSUN6
(SEQ
ID NO: 93), PAFAHIBI (SEQ ID NO: 35), PARVB (SEQ ID NO: 37), PHEX (SEQ ID NO:
39), PKNl
(SEQ ID NO: 44), POLR2J2 (SEQ ID NOs: 41 or 65), RAB20 (SEQ ID NO 43), SYNPR
(SEQ ID NO:
45), and TRPM5 (SEQ ID NO: 47); or a protein substantially similar to the
target protein, wherein the
substantially similar protein has a sequence identity of at least 95% to the
target protein.
Another aspect of the present inventioii describes a method of inhibiting
replication of a
virus in a host. The host is provided with an effective amount of a compound
able to inhibit the activity
or expression of a target protein selected from the group consisting of: AKAP8
(SEQ ID NO: 11), ALK
(SEQ ID:NO: 67), ATM(SEQ ID NO: 68), C140RF24 (SEQ ID NO: 69), DGKD (SEQ ID
NO: 15),
DGKZ (SEQ ID NO: 70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ID NO: 71), DUSP6
(SEQ ID NO:
9), DUT (SEQ ID NO: 51), DYRK2 (SEQ ID NO: 72), DYRK4 (SEQ ID NO: 53), ENPP5
(SEQ ID
NO: 73), EPHA2 (SEQ ID NO: 13), FGFR2 (SEQ ID NO: 74), FHTT (SEQ ID NO: 75),
FRK (SEQ ID
NO: 76), GAK (SEQ ID NO: 55), GCK (SEQ ID NO: 19), MAP2K3 (SEQ ID NO: 77),
NME4 (SEQ ID
NO: 1), PANK1 (SEQ ID NO: 78), PCKl (SEQ ID NO: 3), PFKL (SEQ ID NO: 63),
PIK4CA (SEQ ID
NO: 21), PRKWNK3 (SEQ ID NO: 5), PRPSILI (SEQ ID NO: 79), PSKHl (SEQ ID NO:
57), PTK9L
(SEQ ID NO: 80), SOCS5 (SEQ ID NO: 59), SRPK1 (SEQ ID NO: 17), STK16 (SEQ ID
NO: 7), STK35
(SEQ ID NO: 81), TAFl (SEQ ID NO: 82), TBKl (SEQ ID NO: 83), TJP2 (SEQ ID NO:
84), TNKl
(SEQ ID NO: 61), TPKl(SEQ ID NO: 85), TRIB3 (SEQ ID NO: 86), TRPM7 (SEQ ID NO:
87), VRK
(SEQ ID NO: 88), ALPPL2 (SEQ ID NO: 89), AP4M1 (SEQ ID NO: 23), CAPZAl (SEQ ID
NO: 25),
DNAH5 (SEQ ID NO: 27), DOM3Z (SEQ ID NO: 90), FTCD (SEQ ID NO: 29), PDIA3 (SEQ
ID NO:
31), MDM4 (SEQ ID NO: 91), WDR66 (SEQ ID NO: 92), NOP5/NOP58 (SEQ ID NO: 33),
NSUN6
(SEQ ID NO: 93), PAFAHIB 1(SEQ ID NO: 35), PARVB (SEQ ID NO: 37), PHEX (SEQ ID
NO: 39),
PKN1 (SEQ ID NO: 44), POLR2J2 (SEQ ID NOs: 41 or 65), RAB20 (SEQ ID NO 43),
SYNPR (SEQ ID
NO: 45), and TRPM5 (SEQ ID NO: 47); or a protein substantially similar to said
target protein, wherein
said substantially similar protein has a sequence identity of at least 95% to
said target protein.
Reference to "host" indicates a cell, animal, or human that is, or can be,
infected with the
virus. The compound can be administered prior to viral infection or to a host
infected with the virus.
Reference to open-ended terms such as "comprises" allows for additional
elements or
steps. Occasionally phrases such as "one or more" are used with or without
open-ended terms to
highlight the possibility of additional elements or steps.
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Unless explicitly stated reference to terms such as "a" or "an" is not limited
to one. For
example, "a cell" does not exclude "cells". Occasionally phrases such as one
or more are used to
highlight the possible presence of a plurality.
Other features and advantages of the present invention are apparent from the
additional
descriptions provided herein including the different examples. The provided
examples illustrate different
components and methodology useful in practicing the present invention. The
examples do not lirrut the
claimed invention. Based on the present disclosure the skilled artisan can
identify and employ other
components and methodology useful for practicing the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides results illustrating the time course for knockdown of PIK4CA
mRNA
levels and HCV RNA levels after transfection of siRNA targeting PIK4CA.
Figure 2A provides results illustrating the production of a rabbit polyclonal
antisera that
recognizes PIK4CA protein. Figure 2B provides results showing inhibition of
HCV replication after
transfection of two separate siRNAs targeting PIK4CA, or an siRNA directly
targeting HCV but not after
transfection of a non-silencing siRNA (NS1). Figure 2C provides results
showing that the'siRNAs
targeting PIK4CA used in figure 2B also knock down PIK4CA protein levels, but
the siRNA targeting
HCV and the nonsilencing siRNA (NS 1) do not affect PIK4CA protein levels.
Figure 3 provides resulting illustrating the ability of siRNA targeting
different host genes
to inhibit HIV replication. siRNA results for the following genes are provided
in Figure 1: 1- CycTl; 2-
PFKL; 3- PIK4CA; 4- DYRK4; 5- SYNPR; 6- GAK; 7- DUSP19; 8- DUT; 9- PSKH1; 10-
LUC; 11-
SOCS5; 12- PARVB; and 13- TRPM5.
DETAILED DESCRIPTION OF THE INVENTION
Different host cell protein targets for inhibiting HCV replication were
identified using a
procedure measuring the effect of inhibiting expression of host cell protein
on HCV replicon activity and
taking into account liver expression of targeted proteins. The proteins
identified as involved in HCV
replication and the encoding nucleic acid provide targets for inhibiting HCV
replication and for
evaluating the ability of compounds to inhibit HCV replication.
Several of the host genes identified as targets for inhibiting HCV replication
were also
found to inhibit HIV replication. The ability to serve as a target for both
HIV and HCV indicates that
such a target may be a useful target for inhibiting replication different
types of viruses and not limited to
a inhibiting replication of particular virus.
Inhibiting viral replication, such as HIV and HCV, has research and
therapeutic
implications. Research applications include providing tools to study viral
replication and expression, for
example, HCV or HIV replication and expression. Therapeutic applications
include using those
compounds having appropriate pharmacological properties such as efficacy and
lack of unacceptable
toxicity to treat or inhibit onset of viral infection in a patient (e.g., HCV
or HIV infection).
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Identified Targets
The targets for inhibiting viral replication, such as HCV replication,
identified herein are
host cell factors. Nucleic acid sequences encoding the identified host cell
factors, host cell factors, and
substantially similar nucleic acid or protein can be used as a target for
inhibiting viral replication, such as
HCV replication. Table 1 provides information on the identified host cell
factors for inhibiting HCV
replication, and in some cases HIV replication.
The targets provide in Table 1 may also be useful for inhibiting viral
replication beyond
HCV. A subset of the HCV targets were tested for inhibiting,HIV replication.
As described in Example
13, infra inhibition of PIK4CA, SYNPR, DYRK4, PFKL, GAK, DUSP19, and DUT also
inhibited HIV
replication. The ability to inhibit both HCV and HIV replication indicates a
role for such target in the
replication of different types of viruses.
TABLE 1
Gene Acc. No. Amino acid Sequence Additional Information
AKAP8 NM_005858 SEQ ID NO: 11 SEQ ID NO: 12, ORF = 62-2140
ALK NM_004304 SEQ ID NO: 67
ATM NM_000051 SEQ ID NO: 68
C140RF24 NM_173607 SEQ ID NO: 69
CSNK2A1 NM_001895
CSNK2B NM_001320
DDR1 NM_001954
DGKD NM_003648 SEQ ID NO: 15 SEQ ID NO: 16, ORF = 80-3592
DGKZ NM_003646 SEQ ID NO: 70
DUSP19 NM_080876 SEQ ID NO: 49 SEQ ID NO: 50, ORF=183-836
DUSP22 NM_020185 SEQ ID NO: 71
DUSP6 NM_001946 SEQ ID NO: 9 SEQ ID NO: 10, ORF = 481-1626
DUT NM_001948 SEQ ID NO: 51 SEQ ID NO: 52, ORF=85-579
DYRK2 NM_006482 SEQ ID NO: 72
DYRK4 NM_003845 SEQ ID NO: 53 SEQ ID NO: 54, ORF=161-1723
ENPP5 NM_021572 SEQ ID NO: 73
EPHA2 NM_004431 SEQ ID NO: 13 SEQ ID NO: 14, ORF = 138-3068
FGFR2 NM000141 SEQ ID NO: 74
FHIT NM_002012 SEQ ID NO: 75
FRK NM_002031 SEQ ID NO: 76
GAK NM_005255 SEQ ID NO: 55 SEQ ID NO: 56, ORF= 1-3936
GCK NM_000162 SEQ ID NO: 19 SEQ ID NO: 20, ORF = 487-1884
MAP2K3 NM_002756 SEQ ID NO: 77
MAP2K6 NM_002758
NME4 NM_005009 SEQ ID NO: 1 SEQ ID NO: 2, ORF = 32-595
PANKl NM_148977 SEQ ID NO: 78
PCK1 NM_002591 SEQ ID NO: 3 SEQ ID NO: 4
PFKL NM_002626 SEQ ID NO: 63 SEQ ID NO: 64, ORF = 60-2402
PIK4CA NM_058004 SEQ ID NO: 21 SEQ ID NO: 22, ORF = 1-6135
PRKCL2 NM_006256
PRKWNK3 NM_020922 SEQ ID NO: 5 SEQ ID NO: 6, ORF = 440-5842
PRPS1L1 NM_175886 SEQ ID NO: 79
PSKH1 NM_006742 SEQ ID NO: 57 SEQ ID NO: 58, ORF= 131-1405
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TABLE 1
Gene Acc. No. Amino acid Sequence Additional Information
PTK9L NM_007284 SEQ ID NO: 80
SOCS5 NM_014011 SEQ ID NO: 59 SEQ ID NO:60, ORF=165-1775
SRPKl NM_003137 SEQ ID NO: 17 SEQ ID NO: 18, ORF = 124-2091
STK16 NM_003691 SEQ ID NO: 7 SEQ ID NO: 8, ORF = 400-1317
STK35 NM080836 SEQ ID NO: 81 SEQ ID NO: 28, ORF = 1-1206
TAF1 NM004606 SEQ ID NO: 82
TBKl NM_013254 SEQ ID NO: 83
TJP2 NM_004817 SEQ ID NO: 84
TNK1 NM_003985 SEQ ID NO: 61 SEQ ID NO: 62, ORF=117-2117
TPK1 NM_022445 SEQ ID NO: 85
TRIB3 NM_021158 SEQ ID NO: 86
TRPM7 NM_017672 SEQ ID NO: 87
VRK1 NM003384 SEQ ID NO: 88
ALPPL2 NM031313 SEQ ID NO: 89 SEQ ID NO: 24
AP4MI NM_004722 SEQ ID NO: 23
CAPZAI NM_006135 SEQ ID NO: 25
DNAH5 NM_001369 SEQ ID NO: 27
DOM3Z NM_005510 SEQ ID NO: 90
FTCD NM_006657 SEQ ID NO: 29 NM_206965 is provided by SEQ ID NO 30, ORF=45-
1670
NM206965
NM 206965 is the predominant isoform, both isoforms
are transcribed and make the same protein. The tested
siRNAs should affect both isoforms. The difference
between the two isoforms is in the 3'UTR
PDIA3 NM005313 SEQ ID NO: 31 SEQ ID NO: 32, ORF=149-1666
MDM4 NM_002393 SEQ ID NO: 91 SEQ ID NO: 26, ORF 163-1635
WDR66 NM_144668 SEQ ID NO: 92
NOP5/ NM_015934 SEQ ID NO: 33 SEQ ID NO: 34, ORF=151-1740
NOP58
NSUN6 NM_182543 SEQ ID NO: 93
PAFAHIB 1 NM_000430 SEQ ID NO: 35 SEQ ID NO: 36, ORF=566-1788
PARVB NM_013327 SEQ ID NO: 94
ISOFORM B
PARVB NM001003828 SEQ ID NO: 37 SEQ ID NO: 38, ORF=53-1147
ISOFORM A
PHEX NM_000444 SEQ ID NO: 39 SEQ ID NO: 40, ORF = 604-2853
PKN1 NM002741 SEQ ID NO: 44 The siRNAs tested targeted both isoforms
NM_213560
POLR2J2 NM_145325 ISOFORMI: ISOFORM 1(NM_145325) ORF=129-605: (SEQ ID
NM_032959 SEQ ID NO: 41 NO: 42)
ISOFORM 3 (NM_032959) ORF=129-476:
ISOFORM3: SEQ ID NO: 66
SEQ ID NO: 65 siRNAs were identified that targeted both NM_145325
and NM 032959.
RAB20 NM_017817 SEQ ID NO: 43
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TABLE 1
Gene Acc. No. Amino acid Sequence Additional Information
SYNPR NM_144642 SEQ ID NO: 45 SEQ ID NO: 46, ORF=99-896:
TRPM5 NM014555 SEQ ID NO: 47 SEQ ID NO: 48, ORF=10-3507
Nucleic acid and protein substantially similar to a particular identified
sequence provide
sequences with a small number of changes to the particular identified
sequence. Substantially similar
sequences include sequences containing one or more naturally occurring
polymorphisms or artificial
changes.
A substantially similar protein sequence is at least 95% identical to a
reference sequence.
The substantially similar protein sequence should also not have significantly
less activity than the
reference sequence. Significantly less activity is less than about 80%
activity of the identified protein.
In different embodiments, the substantially similar protein sequence differs
from the
reference sequence by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16,
17, 18, 19, or 20 amino acid
alterations. Each-amino acid alteration is independently an addition, deletion
or substitution. Preferred
substantially similar sequences are naturally occurring variants.
A substantially similar nucleic acid is at least 95% identical to a reference
sequence. The
substantially similar nucleic acid sequence should encode a protein that does
not have significantly less
activity than the protein encoded by the reference sequence. Significantly
less activity is less than about
80% activity of the identified protein.
Sequence identity to a reference nucleic acid sequence is determined by
aligning the
nucleic acid sequence with the reference sequence and determining the number
of identical nucleotides in
the corresponding regions. This number is divided by the total number of
nucleotides in the reference
sequence (e.g., SEQ ID NO: 2) and then multiplied by 100 and rounded to the
nearest whole number.
In different embodiments, the substantially similar nucleic acid sequence
differs from the
reference sequence by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 nucleotide
alterations. Each nucleic acid alteration is independently an addition,
deletion or substitution. Preferred
substantially similar sequences are naturally occurring variants.
Protein Production
Proteins can be produced using techniques well known in the art including
those
involving chemical synthesis and those involving purification from a cell
producing the protein.
Techniques for chemical synthesis of proteins are well known in the art. (See
e.g., Vincent, Peptide arad
Protein Drug Delivery, New York, N.Y., Decker, 1990.) Techniques for
recombinant protein production
and purification are also well known in the art. (See for example, Ausubel,
Current Protocols in
Molecular Biology, John Wiley, 1987-2002.)
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Obtaining a protein from a cell is facilitated using recombinant nucleic acid
techniques
to produce the protein. Recombinant nucleic acid techniques for producing a
protein involve
introducing, or producing, a recombinant gene encoding the protein in a cell
and expressing the protein.
A recombinant gene contains nucleic acid encoding a protein along with
regulatory
elements for protein expression. The recombinant gene can be present in a
cellular genome or can be
part of an expression vector.
The regulatory elements that may be present as part of a recombinant gene
include those
naturally associated with the protein encoding sequence and exogenous
regulatory elements not naturally
associated with the protein encoding sequence. Exogenous regulatory elements
such as an exogenous
promoter can be useful for expressing a recombinant gene in a particular host
or increasing the level of
expression. Generally, the regulatory elements that are present in a
recombinant gene include a
transcriptional promoter, a ribosome binding site, a terminator, and an
optionally present operator. A
preferred element for processing in eukaryotic cells is a polyadenylation
signal.
Expression of a recombinant gene in a cell is facilitated through the use of
an expression
vector. Preferably, an expression vector in addition to a recombinant gene
also contains an origin of
replication for autonomous replication in a host cell, a selectable marker, a
limited number of useful
restriction enzyme sites, and a potential for high copy number. Examples of
expression vectors are
cloning vectors, modified cloning vectors, specifically designed plasmids and
viruses.
Due to the degeneracy of the genetic code, a large number of different
encoding nucleic
acid sequences can be used to code for a particular protein. The degeneracy of
the genetic code arises
because almost all amino acids are encoded by different combinations of
nucleotide triplets or "codons".
Amino acids are encoded by codons as follows:
A=Ala=Alanine: codons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codons UGC, UGU
D=Asp=Aspartic acid: codons GAC, GAU
E=Glu=Glutamic acid: codons GAA, GAG
F=Phe=Phenylalanine: codons UUC, UUU
G=Gly=Glycine: codons GGA, GGC, GGG, GGU
H=His=Histidine: codons CAC, CAU
I=Ile=lsoleucine: codons AUA, AUC, AUU
K=Lys=Lysine: codons AAA, AAG
L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU
M=Met=Methionine: codon AUG
N=Asn=Asparagine: codons AAC, AAU
P=Pro=Proline: codons CCA, CCC, CCG, CCU
Q=Gln=Glutamine: codons CAA, CAG
R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU
S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU
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T=Thr=Threonine: codons ACA, ACC, ACG, ACU
V=Va1=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG
Y=Tyr=Tyrosine: codons UAC, UAU
Techniques for recombinant gene production, introduction into a cell, and
recombinant
gene expression are well known in the art. Examples of such techniques are
provided in references such
as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002, and
Sambrook et al.,
Molecular Cloning, A Laboratory Manual, 2d Edition, Cold Spring Harbor
Laboratory Press, 1989.
If desired, expression in a particular host can be enhanced through codon
optimization.
Codon optimization includes use of more preferred codons. Techniques for codon
optimization in
different hosts are well known in the art.
Identifying a Compound Inhibiting Binding to, or Activity or Expression of, a
Target Protein
The initial identification of a compound inhibiting binding to, or activity or
expression
of, a target protein can be determined experimental or based on available
information concerning a target. ;
Compounds binding to, or inhibiting protein activity, are directed at the
protein. Compounds inhibiting
protein expression are directed at nucleic acid encoding the protein or having
a regulatory function.
The ability of a compound to bind to a protein can be determined using
techniques such
as competitive and non-competitive binding assays. Such assays can be
performed, for example, using a
labeled compound to directly measure binding or measuring binding using a
detectable reagent that binds
to compound.
Techniques for assaying kinase ancl phosphatase activity are well known in the
art. For
example, references describing techniques that can be used for measuring
activity of NME4, PCK1,
STK16, DUSP6, PIK4CA, PKWNK3, AKAP8, GCK, SRPK1, DGKD, DUT, GAK, PFKL, PSKHl,
SOCS5, TNKl, DUSP19, and EPHA2 include: Kowluru et al., Arch. Biocheni.
Bioplzys., 398(2):160-169,
2002 (NME4); Mahoud et al., Biochemica Biophysica Acta, 1336:549-556, 1997
(PCK1); Ohta et al.,
Biochern J., 350 (Pt 2):395-404, 2000 (STK16); Kim et al., Biochemistry,
42(51):15197-207, 2003
(DUSP6); Varsanyi et al., Eur. J. Biochem., 179: 473-479, 1989 (PIK4CA); Xu et
al., J. Biol. Clzem.
275:16795-16801, 2000, (PRKWNK3); Akileswaran et al., J. Biol. Chern.,
18:276(20):17448-54, 2001
(AKAP8); Van Schaftingen et al., Eur. J. Bioch.ern., 179:179 -184, 1989 (GCK);
Aubol et al., Proc. Natl.
Acad. Sci., U S A., 100(22):12601-6, 2003 (SRPK1); Imai et al., J. Biol.
Chenz. 277(38):35323-32, 2002
(DGKD); Cliniie et al., Protein Express Purif. 5(3):252-258 (1993) (DUT);
Greener et al., J. Biol. Clzem.
275(2):1365-1370, 2000 and Kimura et al., Genonzics 44:179-187 (1997) (GAK);
Furuya and Uyeda, J.
Biol. Chem. 256(14):7109-7112, 1981, Hirada et al., Biosci. Biotechnol.
Biochem. 64(10):2047-2052,
2000 (PFKL); Brede et al., Genoinics 70(1): 82-92. 2000, Brede et al., Nucl.
Acid Res. 30(23):5301-
5309, 2002 (PSKHl); Kario et al., J. Biol Clzein., 280 (8):7038-7048, 2005
(SOCS5); Felschow et al.,
Biochezn. Biophys. Res. Comm. 273:294-301, 2000 (TNK1); Zama et al., J. Biol.
Chein. 277(26):23909-
23918, 2002; and Pratt et al.,Oncogezze 21(50):7690-9, 2002 (EPHA2).
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Inhibitors of different kinases and phosphatases are well known in art.
Examples of such
inhibitors include: 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933),
which inhibits ATM
(Hickson et al., Cancer Res., 64(24):9152-9, 2004); (5R,6S)-5-Azido-6-
benzoyloxy-2-cyclohexen-l-one,
which inhibits PIK4CA (Pelyvas et al., J. Med. Chein., 44: 627-632, 2001); 1-
allyl-3-butyl-8-
methylxanthine, which inhibits PCK1 (Foley et al., Bioorg. Med. Chem. Lett.,
13(20):3607-10, 2003); 5-
mercuri-dUTP and other UTP analogues that inhibit DUT (Climie et al., Protein
Express Purif 5(3):252-
258, 1993, Beck et al., Adv. Exp. Med. Biol., 195 Pt B (97-104), 1986; and N-
bromoaceylethanolamine
phosphate, which inhibits PFKL (Hirada et al., Biosci. Biotechzzol. Biochem.
64(10):2047-2052, 2000).
Techniques that can be used for assaying the enzymatic activity and/or
compound
binding for the non-kinase target proteins described in Table 1 are also
known. Examples of references
describing techniques for activity assays that can be used for measuring
TRPM5, PHEX, MDM4, PDIA3,
APPL2, and PARVB include: Prawitt et al., Proc Natl Acad Sci USA, 100:15166-
15171, 2003 (TRPM5);
Boileau et al., Biochem J. 355:707-713, 2001 (PHEX); Badciong and Haas, J.
Biol. Chem., 277:49668-
49775, 2002 (MDM4); Frickel et al., J. Biol. Chem., 279:18277-18287, 2004,
(PDIA3); Hoylaerts et al.,
Biochem. J. 277:49808-49814, 1992 (ALPPL2); Olski et al., J. Cell Sci. 114:525-
538 (2000) (PARVB).
Although PAFAHIB 1 is the catalytically inactive subunit of platelet
activating factor acetylhydrolase,
assay conditions for the full acetylhydrolase enzyme complex can be found in
Hattori and Inoue, J. Biol.
Chem. 268:18748-18753, 1993 (PAFAHIBI).
The encoding nucleic acid sequence of an identified protein provides a target
for
compounds able to hybridize to the nucleic acid. Examples of compounds able to
hybridize to a nucleic
acid sequence include siRNA, ribozymes, and antisense nucleic acid. The
mechanism of inhibition
varies depending upon the type of compound. Techniques for producing and using
sRNAi, ribozymes,
and antisense nucleic acid are well known in the art. (E.g., Probst, Methods
22:271-281, 2000; Zhang et
al., Methods in Molecular Medicine Vol. 106: Azztisense Tlzerapeutics 2"d
Edition, p. 11-34, Edited by I.
Philips, Humana Press Inc., Totowa, NJ, 2005.) In addition, the examples
provided below illustrate the
use of siRNA.
Vector for delivering nucleic acid based compounds include plasmid and viral
based
vectors. Preferred vectors for therapeutic applications are retroviral and
adenovirus based vectors.
(Devroe et al., Expert Opin. Biol. Ther. 4(3):319-327, 2004, Zhang et al.,
Virology 320:135-143, 2004.)
Measurinp; HCV Inhibitory Activity
The ability of a compound to inhibit HCV replication can be measured in vitro
or using
animal models. (Pietschmann et al., Clitz Liver Dis. 7(1):23-43, 2003.) In
vitro techniques for
measuring the ability of a compound to inhibit HCV replication involve using
HCV or an HCV replicon.
Because HCV is difficult to grow in culture, preferred in vitro techniques
employ an HCV replicon.
An HCV replicon is an RNA molecule able to autonomously replicate in a
cultured cell,
such as Huh7. The HCV replicon expresses the HCV derived components of the
replication machinery
and contains cis-elements required for replication in a cultured cell.
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The production and use of HCV replicons are described in different references.
(See, for
example, Lohmann et al., Science, 285:110-113, 1999; Blight et al., Science,
290:1972-1974, 2000;
Lohmann et al., Journal of Virology, 75:1437-1449, 2001; Pietschmann et al.,
Journal of Virology,
75:1252-1264, 2001; Grobler et al., J. Biol. Chetn., 278:16741-16746, 2003;
Murray et al., J. Virol.,
77(5):2928-2935, 2003; Zuck et al., Anal. Briochenz.334(2):344-355, 2004;
Ludmerer et al., Arztimicrob.
Agents Chemother,. 49(5):2059-69, 2005; Rice et al., International Publication
Number WO 01/89364,
published November 29, 2001; Bichko, International Publication Number WO
02/238793, published
May 16, 2002; Kukolj et al., International Publication Number WO 02/052015,
published July 4, 2002;
De Francesco et al., International Publication Number WO 02/059321, published
August 1, 2002; Glober
et al., International Publication Number WO 04/074507, published September 2,
2004; and
Bartenschlager U.S. Patent No. 6,630,343.)
The ability of a compound to inhibit HCV replication can be measured in
naturally
occurring or artificial animal models susceptible to HCV infection. Only a few
animals such as humans
and chimpanzees are susceptible to HCV infection. Chimpanzees have been used
as animal models for
determining the effect of a compound on HCV infection.
Artificial animal models susceptible to HCV infection have been produced by
transplanting human livers cells into a mouse. (Pietschmann et al., Clin Liver
Dis., 7(1):23-43, 2003.)
The use of transgenic mice with chimeric mouse-human livers provides for a
small animal model.
Measuring HIV Inhibitory Activity
The ability of a compound to inhibit HN replication can be measured in vitro
or using
animal models. In vitro techniques for measuring the ability of a compound to
inhibit HIV replication
include, for example, techniques measuring early steps in the viral life cycle
(entry through integration),
or involve using H1V infection of T-cell lines, or peripheral blood
mononuclear cells to follow a
spreading viral infection. (E.g., Joyce et al., J. Biol. Chem. 277(48):45811-
20, 2002; Nunberg et al., J.
Virol 65(9): 4887-4892, 1991; Goldman et al., Antitnicrob Agents Chenzother.
36(5):1019-1023, 1992.)
The ability of a compound to inhibit HIV replication can be measured in non-
human
primate models susceptible to infection with either HIV; or a chimeric virus
created by combining
fragments of the HIV and SN (simian immunodeficiency virus) genome, termed a
SHIV (simian-human
immunodeficiency virus). Only a few animals such as humans and chimpanzees are
susceptible to HIV
infection. Chimpanzees have been used as animal models for determining the
effect of a compound on
HIV infection. (Grob et al., Nat Med. 3(6):665-670, 1997.) More frequently,
determining the effect of a
compound on HIV infection in non-human primates is carried out in rhesus
macaques infected with
SHIV. (Hazuda et al., Sci.erzce 305(5683):528-32, 2004.)
Administration
Guidelines for pharmaceutical administration of a therapeutic compound in
general are
provided in, for example, Reinington's Pharrnaceutical Sciences 20'h Editiorz,
Ed. Gennaro, Mack
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Publishing, 2000; and Modern Pharnzaceutics 2d Edition, Eds. Banker and
Rhodes, Marcel Dekker, Inc.,
1990.
Compounds active at a therapeutic target having appropriate functional groups
can be
prepared as acidic or base salts. Pharmaceutically acceptable salts (in the
form of water- or oil-soluble or
dispersible products) include conventional non-toxic salts or the quaternary
ammonium salts that are
formed, e.g., from inorganic or organic acids or bases. Examples of such salts
include acid addition salts
such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate,
camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate,
fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,
hexanoate, hydrochloride,
hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-
naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-
phenylpropionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and
undecanoate; and base salts such as
ammonium salts, alkali metal salts such as sodium and potassium salts,
alkaline earth metal salts such as
calcium and magnesium salts, salts with organic bases such as
dicyclohexylamine salts, N-methyl-D-
glucamine, and salts with amino acids such as arginine and lysine.
Compounds can be administered using different routes including oral, nasal,
by, injection,
transdermal, and transmucosally. Active ingredients to be administered orally
as a suspension can be
prepared according to techniques well known in the art of pharmaceutical
formulation and may contain
microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate
as a suspending agent,
methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents. As
immediate release tablets,
these compositions may contain microcrystalline cellulose, dicalcium
phosphate, starch, magnesium
stearate and lactose and/or other excipients, binders, extenders,
disintegrants, diluents and lubricants.
When administered by nasal aerosol, or inhalation, compositions can be
prepared
according to techniques well known in the art of pharmaceutical formulation
and may be prepared as
solutions in saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to
enhance bioavailability, fluorocarbons, and/or other solubilizing or
dispersing agents.
The compounds may also be administered in intravenous (both bolus and
infusion),
intraperitoneal, subcutaneous, topical with or without occlusion, or
intramuscular form, all using forms
well known to those of ordinary skill in the pharmaceutical arts. When
administered by injection, the
injectable solutions or suspensions may be formulated using suitable non-
toxic, parenterally-acceptable
diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's
solution or isotonic sodium
chloride solution, or suitable dispersing or wetting and suspending agents,
such as sterile, bland, fixed
oils, including synthetic mono- or diglycerides, and fatty acids, including
oleic acid.
When rectally administered in the form of suppositories, compositions may be
prepared
by mixing the drug with a suitable non-irritating excipient, such as cocoa
butter, synthetic glyceride
esters or polyethylene glycols, which are solid at ordinary temperatures, but
liquidify and/or dissolve in
the rectal cavity to release the drug.
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Suitable dosing regimens for the therapeutic applications of the present
invention are
selected taking into account factors well known in the art including age,
weight, sex and medical
condition of the patient; the severity of the condition to be treated; the
route of administration; the renal
and hepatic function of the patient; and the particular compound employed.
Guidelines for
pharmaceutical administration and pharmaceutical compositions are provided in,
for example,
Remington's Plaarmaceutical Sciences 20'hEdition, supra. and Modern
Pharmaceutics 2"d Edition, supra.
Optimal precision in achieving concentrations of drug within the range that
yields
efficacy without toxicity requires a regimen based on the kinetics of the
drug's availability to target sites.
This involves. a consideration of the distribution, equilibrium, and
elimination of a drug. The daily dose
for a patient is expected to be between 0.01 and 1,000 mg per adult patient
per day.
Examples
Examples are provided below further illustrating different features of the
present
invention. The examples also illustrate useful methodology, for practicing the
invention. The examples
do not limit the claimed invention.
Example 1: Screening the Protein Kinase and Phosphatase siRNA library
Potential host cell factor targets for inhibiting HCV replicon replication
were identified
using an HCV Conl-lb (.3-lactamase-expressing, replicon expressed in Huh-7
cells and a siRNA library
targeting host siRNA. The procedure was performed as follows:
Day 1: Plate HuH-7'cells expressing a tissue culture-adapted HCV Conl-lb (3-
lactamase-expressing
replicon (Zuck et al., Anal. Biochem., 334(2):344-355, 2004), at 2000
cells/well in black, clear-bottom
96-well plates (Costar, cat # 3603). Plate cells in 100 L DMEM (Invitrogen,
21063-029), 10% Fetal
bovine serum (Invitrogen 16140-071), 1X Non-essential amino acids (Invitrogen,
11140-050), 1X
Glutamax (Invitrogen, 35050-061). Allow cells to attach and grow overnight at
37 C, 5% C02.
Day 2: Transfect plated HuH-7/replicon cells as follows:
1. Resuspend siRNA by adding 200 L of 1X buffer (Dharmacon) to each well,
producing a final
siRNA concentration of 10 M.
Control siRNAs:
(+ control) HCV siRNA (Randall et al., Virus Res. 102(1):19-25, 2004)
AACCUCAAAGAAAAACCAACdTdT (SEQ ID NO: 95)
(+ control) HVAP33 siRNA (TGI) CUGUUCCACUGAAUGCAUCdTdT (SEQ ID NO: 96)
(TGI) AUCGGCACCUGAGAGAUGAdTdT (SEQ ID NO: 96)
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(Zhang et al., Virology. 320(1):135-143, 2004) GAUGGACCUAUGCCAAAACdTdT (SEQ ID
NO: 97)
(- control) NS 1 siRNA (Dharmacon)
Use controls at 10 M as well. The final concentration of siRNA added to the
cells will be 50
nM.
2. Dispense 33 L of Optimem/well into a sterile 96-well plate, leaving the
12"' column empty.
3. Transfer 1 L of siRNA from each well of the siRNA stock plate into the
Optimem-containing
plates such that the siRNA from well A3 of the mother plate is transferred
into well A2 of the
daughter plate (1 I. of siRNA from each well is transferred into the
corresponding plate into the
same row position and the N-1 colun-in position).
4. Mix by pipetting up and down.
5. In a tube, add 100 L Oligofectamine (Invitrogen, 12252-011), 1210 L
Optimem Reduced
Serum Media (Invitrogen, 11058-021). Incubate 5 minutes at room temperature.
6. Dispense 6 L of the Oligofectamine to each well and mix by pipetting up
and down. Incubate
the plate at room temperature for 15 minutes.
7. Remove media from the cells and replace with 80 L (DMEM (phenol red free),
1X Non-
essential amino acids, 1X Glutamax). Add 20 L of the siRNA/Oligofectamine
mixture to each
corresponding well.
8. Incubate the plate at 37 C, 5% CO2 for 4 hours.
9. Add 50 L of DMEM (phenol red free) + 30% Fetal bovine serum, 1X Non-
essential amino
acids, 1X Glutamax. Incubate the cells at 37 C, 5% C02 for 48 hours.
10. Remove media from cells and replace with 100 gL/ well DMEM (phenol red
free), 10% FBS,
1X non-essential amino acids, 1X Glutamax and 0.5 ttM Clavulanic acid.
Incubate cells at 37 C,
5% CO2 for 24 hours.
11. Remove media from cells and replace with 50 L of the following solution:
12 mL DMEM
(phenol red-free), 24 pL CCF4 (1 mM solution in DMSO), 2 mL Solution C
(Aurora), 120 ,l.
Solution B (Pluronic Acid, Aurora).
12. Incubate cells in the dark at room temperature for 1.5 hours.
13. Read the plates at 460 nm and 530 nm using a plate reader capable of
reading fluorescence
intensity.
14. Data analysis: Determine an average background for both A460 and A538
(average of counts
from column 12). Subtract background from each well. Divide A460 by A538 after
background
subtraction to determine blue/green ratio for each cell. Calculate average and
standard deviation
for each transfection.
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This analysis was carried out in duplicate for each plate of the Dharmacon
Kinase and
Phosphatase siRNA libraries. Hits were considered to be those siRNA that
suppressed replication of the
replicon by 40% or more, as measured by (3-lactamase activity (A460/A530 ratio
compared with NS 1
siRNA-transfected control) and did not inhibit cell viability by more than 20%
as measured by the A530
reading compared with NS1 siRNA-transfected control.
Results:
Eighty-nine hits were identified in the primary screen: AKAPB, ALK, ASP, ATM,
C140RF24, CSNKIE, CSNK2A1, CSNK2B, CXCL10, DDRl, DGKD, DGKG, DGKZ, DLG2,
DUSP18, DUSP19, DUSP22, DUSP5, DUSP6, DUT, DYRK2, DYRK4, EGFR, ENPP5, EPHA2,
EPHA3, FBP2, FGFR2, FGFR4, FHIT, FLJ20442, FLJ35107, FN3K, FRK, GAK, GCK,
HIPK3, HK3; I-
4, IGBPl, IMPK, LOC339221, LPPR4, MAP2K3, MAP2K6, MAP3Kl, MAP3K7IP1, MGC1136,
MTMRl, MTMR7, NME4, PANKl, PCKl, PCK2, PCTK1, PFKL, PIK4CA, PHKA2, PHKG2,
PPAP2C, PPP1R11, PPP1R2, PRKCL2, PRKD2, PRKWNK3, PRKY, PRPS1, PRPS1Ll,
PRPSAPI,
PSKHl, PTK2B, PTK9L, PTP4A3, PTPN2, RIPK2, SOCS5, SRPKl, STK16, STK3, STK33,
STK35,
STK38, TAF1, TBK1, TJP2, TNKl, TPKl, TRPM7, TXK, ULK2, and VRKl.
Exapple 2: Counter Screening the Hits for Liver Expression using the Body
Atlas
Since HCV replicates in the liver, one requirement for host HCV targets is
that the gene
is expressed in the liver. The gene expression for the 89 genes described in
Example 1 in different
tissues were evaluated using a previously generated Body Atlas. The Body Atlas
provides the relative
gene expression for different genes in different tissues compared to
expression of an mRNA pool from
multiple tissues.
Genes were considered to have liver high expression if the relative expression
level was
greater than 1.5 relative intensity, medium expression if the gene was between
0.5 and 1.5 relative
intensity, and low expression if the gene was between 0.05 and 0.5 relative
intensity. Genes with less
than 0.05 relative intensity were not considered to be expressed in the liver.
Genes eliminated from further consideration as targets included those not
expressed in
liver and those with low level liver expression that were only identified as
hits in one of the two replicate
screens. The genes that were not expressed in liver included: ASP, FBP2,1-4,
LPPR4, MTMR7,
PRP4A3, and STK33. These genes were considered to be false positives or genes
that were essential for
maintenance of the replicon in cultured cells only.
Genes with moderate to high levels of expression within the liver were
selected for
further analysis. Genes with low level liver expression were only selected for
further analysis if they
inhibited HCV replication in each of the two times the library was screened.
Genes with low level liver
expression that were not chosen for further analysis included CSNKIE, DGKG,
DLG2, DUSP5,
MAP3K1, MAP3K7IP1, PCTKl, PTK2B, RIPK2, and STK38.
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Table 2 provides a list of the targeted genes chosen for further analysis and
an indication
of their relative level of expression in liver:
TABLE 2
Liver Expression Gene Name
Level
Hi h IMPK, FGFR4, FRK, PANKl, PCK1, PCK2, PFKL
Moderate C140RF24, CXCL10, DUSP6, EGFR, EPHA3, FGFR2, FHRT, DUSP23,
FLJ35107, FN3K, GAK, GCK, ENPP7, MAP2K3, MAP2K6, NME4, PHKA2,
PHKG2, PPAP2C, PPP1R11, PRKD2, PRKWNK3, PRKY, PRPS1, PRPSILI,
PRPSAPl, PSKHl, STK16, TAFl, TBKl, TJP2, TNKl, TRPM7, TXK
Low AKAP8, ALK, ATM, CSNK2A1, CSNK2B, DDR1, DGKD, DGKZ, DUSP18,
DUSP19, DUSP22, DUT, DYRK2, DYRK4, ENPP5, EPHA2, HIPK3, HK3,
IGBPI, MGC1136, MTMR1, PIK4CA, PPP1R2, PRKCL2, PTK9L, PTPN2,
SOCS5, SRPKl, STK3, STK35, TPK1, ULK2, VRK1
Example 3: Confirmation of siRNA Hits
From the primary, screen of the kinase and phosphatase library, 80 siRNA pools
were
selected that both suppressed the HCV replicon and also were expressed in
liver. siRNA pools were
designed to have four siRNAs directed against the same gene in each well of
the pool plate. To confirm
the hits, each of the four siRNAs in the pool were tested individually in an
i;-point, 2-fold titration.
Confirmed hits were expected to demonstrate titrateable inhibition with at
least two of the individual
siRNAs present in the pool (since it is highly unlikely that two distinct
siRNA sequences would have the
same off-target effect).
DAY1:
Reagents and Consumables:
CM.10 Cells
Trypsin (Invitrogen)
DMEM Plating Media: DMEM (Invitrogen), 10% FBS (Invitrogen), 1X GlutaMax
(Invitrogen), 1X
Non-Essential Amino Acids (Invitrogen)
Trypan Blue (Invitrogen)
96-well Cell Plate (Corning)
Procedure:
1. Trypsinize a flask of CM.10 cells.
2. Inactivate the Trypsin by adding DMEM Plating Media to the flask.
3. Remove a 10 ul aliquot of the cell suspension and dilute it 1:2 in Trypan
Blue for viability
staining.
4. Count the cells using a hemocytometer.
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5. Dilute the cells in DMEM Plating Media to a final concentration of 20,000
cells/ml.
6. Plate the cells at a density of 2000 cells/well by adding 100 ul of the
cell suspension [20,000
cells/ml] to columns 1-11 of a 96-well Cell Plate.
7. Incubate the 96-well Cell Plate under normal growth conditions (37 C, 5%
C02) for 18-20
hours.
DAY 2
Reagents and Consumables:
siRNA Master Plate
Oligofectamine (Invitrogen)
Optimem-I (Invitrogen)
DMEM Transfection Media: DMEM (Invitrogen), 1X GlutaMax (Invitrogen), 1X Non-
Essential Amino
Acids (Invitrogen)
96-well Titration Plate (Corning)
Procedure:
1. Add Optimem-I to the 96-well Titration Plate (Duplicate eight times). The
Titration plate can be
set up in a grid with colunms numbered 1-12 and rows A-H. Colum.n 1, rows A-H
gets 33 ul:
Columns 2-11 row A gets 66 ul; columns 2-11, rows B-H gets 34 ul; and column
12 is kept
empty.
2. A 96-well Master Plate is set up in a grid with columns numbered 1-12 and
rows A-H. Column
1, rows A-H is a control; Columns 2-11 rows A-H are sample; and column 11 is
empty.
3. Transfer 2 uL of siRNA [10 uM] (Samples) from the wells in Row A, Columns 2-
11 of the
Master Plate to the wells in Row A, Columns 2-11 of the 96-well Titration
Plate.
4. Transfer 1 uL of siRNA [ 10 uM] (Controls) from the wells in Column 1 of
the Master Plate to
the wells in Column 1 of the 96-well Titration Plate.
5. Mix the samples by pipetting up and down gently.
6. Perform an 8-point, 2-fold serial dilution of the siRNA (Sample) by
transferring and mixing 34
uL of sample from the wells in Row A, Columns 2-11 to the wells in Row B,
Columns 2-11.
Continue the titration by transferring and mixing 34 uL of sample from Row C
to D, D to E, E to
F, etc. The final concentration of siRNA at each titration point will be 50
nM, 25 nM, 12.5 nM,
6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM and 0.390625 nM. Repeat the plate
setup and
titration setup eight times to account for all eight rows of samples from the
Master plate.
7. In a 15 mL conical tube, add 5.50 mL of Optimem-I and 0.50 rnL of
Oligofectamine. Mix gently.
8. Incubate the mixture for 5 minutes at room temperature.
9. Add 6 uL of the Oligofectamine mixture to each well of the 96-well
Titration Plate, containing
siRNA. Mix gently.
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10. Incubate the siRNA:Oligofectamine mixtures for 15 minutes at room
temperature to form the
transfection complexes.
11. During the siRNA:Oligofectamine incubation, remove the media from the
cells in the 96-well
Cell Plate with a multi-channel pipettor.
12. Replace the media with 50 uL of DMEM Transfection Media.
13. After the 15 minutes needed for the siRNA:Oligofectamine complex formation
has expired,
transfer 20 uL of complex from the 96-well Titration Plate to the matching
wells on the 96-well
Cell Plate. The final concentration of siRNA at each titration point will be
50 nM, 25 nM, 12.5
nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM and 0.390625 nM.
14. Incubate the 96-well Cell Plate under normal growth conditions (37 C, 5%
C02) for 4 hours.
15. After the 4-hour incubation time has expired, add 30 uL of DMEM FBS Media
to each of the
wells containing cells and transfection mixes on the 96-well Cell Plate.
16. Incubate the 96-well Cell Plate under normal growth conditions (37 C, 5%
C02) for 48 hours.
DAY 4
Reagents and Consumables:
Clavulanic Acid
DMEM Plating Media: DMEM (Invitrogen), 10% FBS (Invitrogen), 1X GlutaMax
(Invitrogen), 1X Non-
Essential Amino Acids (Invitrogen)
Procedure:
1. Remove the media from the wells on the 96-well Cell Plates.
2. Add 100 uL of DMEM Plating Media; 0.5 uM Clavulanic Acid.
3. Incubate the 96-well Cell Plate under normal growth conditions (37 C, 5%
C02) for 24 hours.
DAY 5
Reagents and Consumables:
DMEM Staining Media: DMEM (Invitrogen)
1 M HEPES
CCF4
Solution B (Aurora, CET338P41),
Solution C (Aurora, CET338P39),
Procedure:
1. Prepare the staining solution as follows (sufficient amount for eight
plates):
a. 72 mL of DMEM Staining Media; 25 mM HEPES
b. 0.144 mL of CCF4 [ 1 mMl
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c. 0.720 mL of Solution B
d. 12 ml of Solution C
2. Remove the media from the wells on the 96-well Cell Plates.
3. Add 50 uL of staining solution to all of the wells on the 96-well Cell
Plates, including the wells
in Column 12.
4. Incubate the plates at room temperature in the dark for 1.5 hours.
5. Read fluorescence intensity at A460 and A530
Data Analysis:
Subtract background (no cell wells) from each sample reading. Calculate the
A460/A530 ratio. Normalize as a percentage of Nonsilencing control siRNA.
Graph the data for each
siRNA as % of control A460/A530 vs. Concentration of siRNA transfected.
For an siRNA to be considered validated, at least two of the four siRNAs
tested inhibited
HCV replication by at least 40% to 60% of control levels and the inhibition
should titrate with
decreasing amounts of transfected siRNA.
Genes that were validated as essential for maintenance of the HCV replicon
using the
above criteria were then categorized into four groups, with priority 1 having
the most desirable criteria
for a HCV target. Gene classification was determined as follows:
Priority 1: The most potent siRNA resulted in _> 70% inhibition of HCV
replicon replication. Moderate
to high expression in Liver. These genes and encoded proteins are the
preferred targets for
inhibiting HCV infection.
Priority 2: The most potent siRNA resulted in >_ 60% inhibition of HCV
replicon replication. Any level
liver expression.
Priority 3: The most potent siRNA resulted in between 60 and 50% inhibition of
HCV replicon
replication.
Priority 4: The most potent siRNA resulted in between 50 and 40% inhibition of
HCV replicon
replication.
Genes essential for HCV replicon replication and their designated categories
are shown
in Table 3.
TABLE 3
Priority Gene
1 NME4, PCK1, PRKWNK3, STK16, DUSP6
2 SOCS5, SRPK1, DGKD, DGKZ, ENPP5, EPHA2, GAK, GCK, PANK1, PFKL, PIK4CA,
PTK9L, STK35, TAF1, FHIT, AKAP8
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3 CSNK2B, DYRK2, DYRK4, MAP2K3, MAP2K6, PRPSILI, PSKHl, TBKl, TJP2,
TRPM7, VRK1, DUSP19
4 ALK, ATM, CSNK2A1, DDRl, FGFR2, FRK, PRKCL2, TNK1, TPK1, TRIB3,
C140RF24, DUSP22, DUT
Example 4: Screening of a Genome-Scale siRNA library
Screening of a library of over 22,000 siRNA pools was carried out essentially
using the
methods described in example 1. Briefly, HuH-7 cells containing the HCV conl-
1b replicon expressing
beta-lactamase (CM. 10) were plated onto 384-well plates. The following day
the cells were transfected
with an array of siRNA pools including control siRNAs using Oligofectamine
according to the
manufacturer's directions. 72 hours after transfection, the cells were stained
for beta-lactamase
expression using CCF4, according to the manufacturer's instructions. Unlike in
example 1, clavulanic
acid was not added to the cells.
640 siRNA pools that affected beta-lactamase expression were then assayed an
additional three times to confirm their effect on HCV replication.
39 siRNA pools were then selected for further confirmation. Six individual
siRNAs
targeting each of the genes targeted by the 39 siRNA pools were transfected
separately into CM. 10 cells
and assayed as above. Effective inhibition of HCV replication by a minimum of
two siRNAs targeting
one of the tested genes was considered to be confirmation of the importance of
the host factor for HCV
conl lb replication.
Results:
Nineteen genes were confirmed in this screen, including: ALPPL2, AP4M1,
CAPZAl,
DNAH5, DOM3Z, FTCD, PDIA3, MDM4, WDR66, NOP5/NOP58, NSUN6, PAFAHIB 1, PARVB,
PHEX, PKN, POLR2J2, RAB20, SYNPR, and TRPM5. All of the confirmed siRNA pools
targeted genes
that had at least low levels of liver expression.
Example 5: Screening of Hits Using a Chimeric BK:2b(NS5b) Replicon
An experiment was performed to determine whether a chimeric BK:2b(NS5b)
replicon is
sensitive to knock-down of the same genes as the Conl lb replicon. The
employed procedure is
described in Examples 1 and 4, except a BK replicon containing a genotype 2b
NS5b sequence was used.
The BK:2b(NS5b) replicon is described by Grobler et al., J. Biol. Chein.,
278:16741-16746, 2003.
Data analysis was carried for each confirmed siRNA hit. The effects of siRNA
transfection on the BK:2b(NS5b) replicon are shown in Table 4:
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TABLE 4
Activity in BK:2b(NS5b)
Gene
Replicon
>39% inhibition SRPKl, EPHA1, TNKl, STK16, DYRK4, EPHA2, MAP2K6, PRPSILI,
NME4, DGKD, TPK1, PRKWNK3, GCK, PIK4CA, FTCD
TRPM7, PFKL, ALK, CSNK2B, FHIT, PTK9L, FGFR2, DUSP19, DUT,
20-39% inhibition DGKZ, TJP2, PRKCL2, DDR1, DUSP22, SOCS5, SYNPR, POLR2J2,
ALPPL2, NOP5/NOP58,
AKAP8, Cl4orf20, TBK1, MAP2K3, GAK, ATM, FRK, PANK1,
No effect PSKH1, DYRK2, CSNK2Al, TAFl, RAB20, TRPM5, WDR66,
NSUN6, DNAH5, PARVB, AP4M1, PAFAHIBI, DOM3Z, PHEX,
CAPZAl
Increased BK lb STK35
replication
Example 6: Activity against HCV Genotype la, Con-lb and 2a Replicons
A quantitative, multiplex TaqMan assay was established to measure the relative
quantities of HCV Replicon RNA in three HCV Replicon cell lines, Con-lb, la
and 2a. Briefly, total
RNA was isolated from replicon cells transfected with siRNAs using the RNeasy
96-well Kit (Qiagen,
#74182). TaqMan reactions utilized the TaqMan EZ RT-PCR Kit (Applied
Biosystems, #403028),
TaqMan PDAR Control Reagent Human Cyclophilin A (Applied Biosystems,
#4310883E) as well as a
probe and primer set targeting the neomycin resistance gene of the HCV
Replicon genome (Neo fwd:
SEQ ID NO: 98; Neo rev: SEQ ID NO: 99; and Neo probe 5' FAM-SEQ ID NO: 100-
TAMRA 3' ). The
final concentration of each component in the reaction mixture was as follow:
1X TaqMan EZ Buffer, 3
mM Mn(Oac)2, 0.3 mM dATP, 0.3 mM dCTP, 0.3 mM dGTP, 0.6 mM dUTP, 0.2 mM
Forward Primer,
0.2 mM Reverse Primer, 0.1 mM Probe, 1X PDAR Cyclophilin A Mix, 0.1 Unit/ l
rTth DNA
Polymerase, 0.01 Unit/ l AmpErase UNG, 10 l of total RNA and H2O to 50 l.
The 96-tube optical
plate (Applied Biosystems #N801-0560) was covered with an optical adhesive
cover (Applied
Biosystems, #4311971) and mixed by inverting several times. The samples were
placed in an ABI 7700
(Applied Biosystems) for multiplex TaqMan analysis by setting the entire plate
to the FAM dye layer for
"unknowns" (HCV) and to the VIC dye layer for the Endogenous control,
Cyclophilin A. The cycling
parameters were set to 50 C, 2 min.; 60 C, 30 min.; 95 C, 5 min.; (94 C, 20
sec.; 55 C, 1 min.) 40
cycles, utilizing spectral compensation and an exposure time of 10
milliseconds.
To calculate the relative quantities of each target RNA in the samples, a
standard curve
was generated for the HCV Neo primer and probe sets as well as the endogenous
control, Cyclophilin A
Pre-Developed Assay Reagent (PDAR) in each of the three HCV Replicon cell
lines. Total RNA was
isolated from the Replicons using the RNeasy Mini Kit (Qiagen, # 74104)
following the manufacturer's
protocol. The TaqMan Cycle Threshold (Ct) values was determined for a 7-point,
five-fold titration of
the total RNA. The natural log of the mass (ng) of total RNA was determined
and plotted over the Ct
values. The equation of the line for each probe and primer set in each of the
HCV replicon cell lines was
calculated to derive the relative quantity of the HCV RNA and the endogenous
control, Cyclophilin A
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mRNA, from their respective Ct values. HCV RNA levels were normalized to the
endogenous control,
Cyclophilin A, mRNA levels.
Results
Priority 1 targets: siRNAs inhibited replication of HCV genotype la, Con-lb ,
and 2a
replicons by greater than 30%: DUSP19, DUT, DYRK4, GAK, PARVB, PFKL, PIK4CA,
PSKHl,
SOCS5, SYNPR, and TRPM5.
Priority 2 targets: siRNAs inhibited replication of two HCV genotypes by at
least 50%:
PAFAH 1 B 1, STK16, and TNK 1.
Priority 3 targets: siRNAs inhibited replication of two HCV genotypes by at
least 30%:
AKAP8, ALK, AP4M1, CAPZAI, DGKD, DNAH5, DYRK2, EPHA2, FGFR2, FRK, FTCD, GCK,
NOP5/NOP58, PDIA3, PHEX, POLR2J2, PRKWNK3, RAB20, STK35, TJP2, TPK1, TRIB3,
and
TRPM7.
Priority. 4 targets: siRNAs inhibited replication of one HCV genotype by at
least 30%:
ALPPL2, CSNK2A1, CSNK2B, DDR1, DGKZ, DOM3Z, DUSP22, DUSP6, MAP2K6, NME4, PCKl,
PRPSILI, PTK9L, SRPKI, TAF1, TBK1, VRKl, and WDR66.
Example 7: Inhibition of HCV lb Subgenomic Replication in HeLa Cells
The ability of Priority 1 targets to inhibit HCV lb subgenomic replication in
a replicon
clone engineered to replicate in HeLa cells, was tested. Inhibition of
subgenomic replication in HeLa
cells serves as evidence that the requirement for the target gene is not an
artifact of the HuH-7 cell line.
The HCV HeLa replication system was as described in Zhu et al., J. Virol,
77(17):9204-9210, 2003.
siRNA transfection and quantification of subgenomic replication were carried
out as described in
Example 6.
Results:
The priority 1 targets DUT, GAK, PFKL, PIK4CA, PSKHl, and SYNPR inhibited HCV
con lb subgenomic replication in HeLa cells by at least 30%. PARVB, SOCS5, and
TRPM5 siRNAs
were toxic in these cells and effects on HCV replication could not be
assessed. DUSP19 and DYRK 4
siRNAs were ineffective against HCV subgenomic replication in HeLa cells.
Example 8: Inhibition of HCV Replication with Wortmannin
PIK4CA has two reported isoforms. Isoform 1 (mRNA: NM_002650, protein:
NP_002641) is shorter and lacks much of the N-terminal portion of the Isoform
2 (mRNA: NM_058004,
protein: NP477352) protein. PIK4CA isoform 1 has been characterized as a type
.II
phosphatidylinositol 4-kinase, which is sensitive to adenosine and insensitive
to wortmannin (Wong and
Cantley, J. Biol. Clzena., 269:28878-28884, 1994), while PIK4CA isoform 2 has
been characterized as a
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type III phosphatidylinositol 4-kinase, which is sensitive to wortmannin and
insensitive to adenosine
(Gehrmann et al., Biochim. Biophys. Acta, 1437:341-356, 1999).
To ascertain whether isoform 2 was the relevant isoform for HCV replication,
we tested
the effect of the kinase inhibitor, wortmannin, which inhibits type III
phosphatidylinositol 4-kinases in
cells at micromolar levels, on HCV replication. Briefly, the beta-lactamase
expressing, HCV Conl-b
CM10 cell line was plated at 7500 cells/well in 50 ul of cell Complete Media
(DMEM, 10% FBS, lx
NEAA, lx Glutamax, (-) Pen/Strep) on 96-well Black Tissue Culture Treated
Plate. A 10 mM DMSO
stock of wortmannin (Sigma, #W1628) was diluted to 100 M in 200 l of
Complete Media and titrated
over a seven point, 2.5-fold dilution series. Fifty. (50) l from each of the
dilution points was transferred
to the assay plate containing the HCV Conl-b CM10 cells to produce the
following concentrations of
wortmannin [ M]: 50, 20, 8, 3.2, 1.28, 0.512 and 0.2048. The cells were
incubated at 37 C, 5% CO2 for
24 hours.
To inhibit endogenous 13-lactamase activity in the HCV Conl-b CM10 cell line,
a 1 mM
DMSO stock of Clavulanic acid (US Pharmocopeia, #1134426) was diluted to 5.5
M in Complete
Media of which 10 l was added to the cells to produce a final concentration
of 0.5 M. The cells were
incubated at 37 C, 5%CO2 for 24 hours.
To measure the I3-lactamase activity in the HCV Con1-b CM10 cell line, the
GeneBlazer
13-lactamase (Invitrogen, #K1085) stain mixture was prepared based on the
manufacturer's protocol. The
cell culture/compound media was removed from the cells and.replaced with 50 l
of the GeneBlazer B-
lactamase stain mixture. The cells were incubated in the dark at room
temperature for 1.5-2.0 hours.
The A460 and A530 fluorescence intensities at were measured on an LJL Analyst.
Results:
Wortmannin inhibited HCV replication with an IC50 of 7.1 M. The data is
consistent
with HCV replication having a requirement for PIK4CA function.
Example 9: siRNAs Specifically Tar etin PIK4CA Isoform 2 Disrupt HCV
Replication
To demonstrate the requirement for PIK4CA isoform 2 in HCV replication, siRNAs
targeting isoform 2 mRNA only (NM_058004) were tested for inhibition of HCV
replication. The
following siRNAs were transfected into CM. 10 cells and tested for inhibition
of HCV subgenomic
replication as described in Example 1:
PIK.4CA2-l: sense 5' UCAACGGUUCACAUAUAAdTdT 3' (SEQ ID NO: 101)
Antisense 5' UUAUAUGUGACACCGUUGAdTdT 3' (SEQ ID NO: 102)
PIK4CA2-2: sense 5' GGUCCGUCCUCCAGUAUAAdTdT 3' (SEQ ID NO: 103)
Antisense 5' UUAUACUGGAGGACGGACCdTdT 3' (SEQ ID NO: 104)
PIK4CA2-3 sense 5' CAGACCGGAUCCACAAUGAdTdT 3' (SEQ ID NO: 105)
Antisense 5' UCAUUGUGGAUCCGGUCUGdTdT 3' (SEQ ID NO: 106)
PIK4CA2-4 sense 5' GGAGUACUCAUUCCUGUAAdTdT 3' (SEQ ID NO: 107)
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Antisense 5' UUACAGGAAUGAGUACUCCdTdT 3' (SEQ ID NO: 108)
PIK4CA2-5 sense 5' UGAUUGCAGUCGCGGACAAdTdT 3' (SEQ ID NO: 109)
Antisense 5' UUGUCCGCGACUGCAAUCAdTdT 3' (SEQ ID NO: 110)
PIK4CA2-6 sense 5' AAAGACUACUCCAACUUCAdTdT 3' (SEQ ID NO: 111)
Antisense 5' UGAAGUUGGAGUAGUCUUUdTdT 3' (SEQ ID NO: 112)
Result:
All six siRNAs targeting PIK4CA inhibited HCV subgenomic replication,
confirming
that isoform 2 of PIK4CA is the relevant isoform for HCV replication.
Example 10: siRNAs Targeting PIK4CA Knock Down PIK4CA mRNA Levels Prior to
Disrupting HCV'
Replication
If HCV replication is dependent upon PIK4CA expression or activity, the
decrease in
HCV replication occurring after transfection of PIK4CA-targeting siRNA should
occur subsequent to
loss of PIK4CA expression. To verify this, HuH-7 cells containing the HCV Conl-
lb replicon were
transfected with siRNA targeting PIK4CA. At 0, 12, 24, 36, 48, 60 and 72 h
following transfection, total
RNA was isolated from the cells and PIK4CA and HCV RNA levels were determined
as described in
Example 6.
Results
As shown in Figure 1, siRNAs targeting PIK4CA lead to maximal inhibition of
PIK4CA
mRNA levels at between 12 and 24 h post-transfection. In contrast, HCV RNA
levels begin to decrease
between 36 and 48 h post-transfection and continue to decrease through the 72
h time point.
Example 11: Validation of knock down of PIK4CA Protein Levels Using a
Polyclonal PIK4CA antibody
Antibodies were raised against antigens IP1240 SEQ ID NO: 113 (aa 893-904) and
IP1241 SEQ ID NO: 114 (aa 696-707}. Antisera from two of the rabbits (D3792
and D3793) was
sensitive enough to detect endogenous levels of PIK4CA when assayed by western
blot. V5-tagged
PIK4CA or empty pCDNA 3.1 vectors were over-expressed in HCV HB1 Con1b cells.
The cell lysates
were harvested in RIPA buffer and loaded on a 4% Tris-Glycine SDS-PAGE Gel.
The custom antibodies
(lmg/ml) were diluted 1:1000 for western blot analysis using the Licor Odyssey
Imaging system.
To determine the gel migration characteristics of PIK4CA, we performed a dual-
probe western blot using
the V5 and D3972 antibodies. The dual color western blot demonstrates that the
less intense, lower band
seen in the D3972 blot migrates at the same size as the V5-tagged protein
(Figure 2A).
To determine if the PIK4CA protein is reduced in the presence of PIK4CA siRNA,
we
transfected HCV HB1 Conlb cells with control siRNAs as well as PIK4CA siRNAs
targeting the ORF or
the 3'UTR of the PIK4CA mRNA. The results demonstrate that HCV RNA levels are
reduced by
PIK4CA siRNAs as well as the positive control, HCV siRNA (Figure 2B). In
addition, the PIK4CA
siRNAs targeting both the ORF and 3'UTR of the PIK4CA mRNA reduce PIK4CA
protein to
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undetectable levels by western blot (as noted above, the lower, less intense
band represents PIK4CA,
Figure 2C). These data both validate the knockdown of PIK4CA protein levels by
the siRNAs used and
also confirm the identity of the PIK4CA band recognized by the D3972 antibody.
Example 12: Additional Information on Some Targets
Additional information on some of the targets is provided below:
DUSP19 (Dual Specificity Phosphatase 19)
DUSP19 is a member of a family of dual specificity mitogen-activated protein
kinase
phosphatases. The protein sequence and encoding cDNA sequence are provided by
SEQ ID NOs: 49 and
50. DUSP19 polymorphisms are shown in Table 5.
TABLE 5
mRNA position NM_080876 se , Polymo hism NP543152 seq Amino Acid chan e
830 T G Ser Arg
DUT (dUTP Pyrophosphatase)
DUT maintains dUTP at low levels to prevent misincorporation into DNA during
replication, mediates resistance to 5-fluorouracil, and may regulate
peroxisome proliferation. The
protein sequence and encoding cDNA sequences are provided by SEQ ID NOs: 51
and 52. DUT
polymorphisms are shown in Table 6.
TABLE 6
mRNA position NM_001948 se Pol mo hism NP_001939 se Amino Acid change
545 G T Gl Val.
565 A C Thr Pro
DYRK4 (Dual Specificity Tyrosine-Regulated Kinase 4)
DYRK4 is a member of the DYRK family of protein tyrosine kinases. The protein
sequence and encoding cDNA sequences are provided by SEQ ID NOs: 53 and 54.
DYRK4
polymorphisms are shown in Table 7.
TABLE 7
mRNA position NM_003845 se Pol mo hism NP_003836 se Amino Acid chan e
284 C T Leu42 No Change
341 G A A1a61 Thr
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445 C T Va195 No change
726 A G Asn189 Ser
1135 C T A1a325 No Change
1433 G C G1u425 Gln
1521 A T Asp454 Val
GAK (Cyclin G Associated Kinase)
GAK is a putative serine/threonine protein kinase that shares homology with
tensin and
auxilin, and may play a role in cell cycle regulation. The protein sequence
and encoding cDNA sequence
for GAK are provided by SEQ ID NOs: 55 and 56. GAK nucleotide polymorphisms
are shown in Table
8
TABLE 8
mRNA position NM_005255 se Pol mo hism NP_005246 se Amino Acid change
3891 C T As 1297 No Change
3889 G A Asp1297 Asn
3819 T C A1a1273 No change
3794 A G Lys1265 Arg
3135 A G A1a1045 No change
3003 C T Ser1001 No chan e
2618 A C G1u873 Ala
2330 A G Asp779 Gly
1457 A G Tyr486 Cys
1237 A G Ee413 Val
1197 C T Ser399 No chan e
PARVB (Parvin beta) Isofor z A:
PARVB is a focal adhesion protein containing two calponin homology domains
that
binds integrin-linked kinase and is likely involved in integrin-ILK signaling
to establish cell-substrate
adhesion. The protein sequence and encoding cDNA sequence for PARVB are
provided by SEQ ID NOs:
37 and 38. PARVB nucleotide polymorphisms are shown in Table 9.
TABLE 9
mRNA position NM_013327 se Pol mo hism NP_037459 se Amino Acid change
225 T C Va158 Ala
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253 T C Leu67 No Change
790 C T Phe246 No change
1015 C T Phe321 No change
PFKL (Phosphofructokinase, liver)
Liver phosphofructokinase catalyses the phosphorylation of fructose-6-
phosphate to
fructose-1,6-bisphosphate in glycolysis. Deficiency is linked to glycogenosis
type VII while
overexpression may lead to the cognitive disabilities of Down's syndrome. The
protein and encoding
cDNA sequence for PFKL are provided by SEQ ID NOs: 63 and 64.
PIK4CA
PIK4CA is a type III phosphatidylinositol-4 kinase. It catalyzes the first
step in the
formation of phosphatidylinosito14,5-bisphosphate and its activity is
inhibited by high concentrations of
wortmannin. The protein and encoding cDNA sequence for PIK4CA are provided by
SEQ ID NOs: 21
and 22. PIK4CA nucleotide polymorphisms are shown in Table 10.
TABLE 10
mRNA position NM_058004 se Polymorphism NP_477352 se Amino Acid change
6119 A T Asn2040 11e2040
5734 T C Gl 1911 No change
5613 T C Thr1871 No change
5505 C T C s 1835 No change
5377 G C Va11793 Leu1793
5376 G A L s 1792 No change
5305 G T Asp1769 T r1769
4869 G A G1n1623 No change
3903 C T Leu1301 No change
3854 C T His1284 No change
3826 G A G1 1276 Ar 1276
3603 G A Thr1201 No change
PSKH1 (Protein Serine Kinase H1)
PSKHl is a protein serine kinase that undergoes calcium-dependent
autophosphorylation. Overexpression of PSKH1 leads to nuclear reorganization
of splicing factors
SFRS 1 and SFRS2 and stimulates RNA splicing. The protein and encoding cDNA
sequence for PSKH1
are provided by SEQ ID NOs: 57 and 58.
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SOCS5 (Suppressor of Cytokine Signaling 5)
SOCS5 is a cytokine-inducible protein containing an SH2 domain and a SOCS box.
It
negatively regulates cytokine signaling via the JAK-STAT pathways. The protein
and encoding cDNA
sequence for SOCS5 are provided by SEQ ID NOs: 59 and 60. Nucleotide
polymorphisms identified for
SOCS5 are shown in Table 11.
TABLE 11
mRNA position NM_014011 se Pol mo hism NP054730 se Amino Acid change
911 A G Thr249 No change
1296 G A Gly378 Arg
1634 G A A1a490 No change
SYNPR (Synaptoporin)
Synaptoporin is a protein with high homology to rat synaptophysin, an integral-
membrane synaptic vesicle protein involved in targeting of synaptic vesicles.
It contains a membrane-
associating domain, often found in lipid-associating proteins. The protein and
encoding cDNA sequence
for SYNPR are provided by SEQ ID NOs: 45 and 46.
TRPM5 (Transient Receptor Poteiztial Cation Charznel, Subfamily M, Mernber 5)
TRPM5 is related to the transient receptor potential family of cation
channels. It has six
predicted transmembrane domains. The protein and encoding cDNA sequence for
TRPM5 are provided
by SEQ ID NOs: 47 and 48. Nucleotide polymorphisms identified for TRPM5 are
presented in Table 12.
TABLE 12
mRNA position NM_014555 se Pol mo hism NP055370 se Amino Acid change
3291 G T Gl 1094 No change
2517 G A Thr836 No change
1752 T C Leu581 No change
1742 G A Arg578 G1n578
1270 C T Ser421 No change
770 T C Va1254 A1a254
756 G A Pro249 No change
713 A G Asn235 Ser235
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PAFAHI B1 (Platelet-Activatitzg Factor Acetylhydrolase (Isofortiz lb) Alpha
Subunit (45kD)
PAFAHIB 1 is the noncatalytic subunit of a heterotrimeric enzyme that
inactivates
platelet-activating factor. The protein and encoding cDNA sequence for PAFAHIB
1 are provided by
SEQ ID NOs: 35 and 36. Nucleotide polymorphisnls for PAFAHIB 1 are shown in
Table 13.
TABLE 13
mRNA position NM 000430 se Pol mo hism NP 000421 se Amino Acid change
1248 A T Tlu 231 No change
1614 T C Ile 353 No change
STKJ6 (Serine/Threonizze Kinase 16)
Serine/threonine kinase 16 is a myristoylated and palmitoylated protein kinase
that may
regulate transcription in response to signaling by transforming growth factor
beta. The protein and
encodingcDNA sequence for STK16 are provided by SEQ ID NOs: 7 and 8.
TNKI (Tyrosine Kinase,Non-receptor 1)
TNK1 is a kinase that interacts with phospholipase C gamma 1(PLCGl). It may
regulate
phospholipid signaling pathways during fetal development and in adult cells of
the lymphohematopoietic
system. The protein and encoding cDNA sequence for TNKl are provided by SEQ ID
NOs: 61 and 62.
Example 13: siRNA hits that also block H1V Infection
High priority siRNA pools were tested for their ability to disrupt HIV
infection in HeLa
cells. The procedure was performed as follows:
Day 1: Plate HeLa (P4/R5) cells at 2000 cells per well in 4x96-well plates.
Day 2: Transfect HeLa (P4/R5) cells with siRNA pools as follows:
1. siRNAs were transfected at a final concentration of 100 nM using
OligofectamineTM reagent
(Invitrogen) at a final concentration of 0.5%. Positive and negative control
siRNAs are included
as follows:
Cyclin Tl (positive control): purchased from Santa Cruz Biotechnology (Cat.
No. sc-35144)
Luciferase (negative control): CGUACGCGGAAUACLTUCGAdTdT (SEQ ID NO: 115)
siRNAs tested in duplicate included a pool of 3 siRNAs targeting: DUSP19, DUT,
DYRK4
GAK, PARVB, PFKL, PIK4CA, PSKH1, SOCS5, SYNPR and TRPM5
2. Dispensed 66 pL of OptiMEM/ well into a sterile 96-well plate, leaving the
12th column
empty.
3. Transferred 2 pL of each siRNA (resuspended at 10 M) into the OptiMEM-
containing
plate.
4. Mixed by pipetting up and down.
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WO 2007/001928 PCT/US2006/023646
5. In a tube, added 240 .L Oligofectamine, 1210 gL OptiMEM. Incubated 5
minutes at room
temperature.
6. Dispensed 12 pL of the Oligofectamine to each well and mixed by pipetting
up and down.
Incubated the plate at room temperature for 15 minutes.
7. Added 20 pL of the siRNA-Oligofectamine complex to each well of the HeLa
(P4/R5) cells.
Day 3: Transfected HeLa (P4/R5) cells were infected with HXB2 HIV as follows:
1. Media was removed from the cells.
2. 80 L fresh media was added to each well.
3. HXB2 HIV was diluted with media. 40 E.iL of diluted HXB2 was added to each
well.
4. Viral infection was allowed to proceed for 96 hours.
Day 5: Beta-galactosidase activity, an indication of viral infection, was
measured as follows:
1. Media was removed from the cells.
2. Cells were washed with 200 pL PBS per well.
3. 20 L lysis buffer (Galacto-Light Plus, Tropix, Cat. No. BL100P) containing
DTT was
added to each well, and the plates were shaken for 10 nlinutes.
4. 80 pL of substrate was then added to each well and the plates were
incubated at room
temperature in the dark for 1 hour.
5. 100 L of enhancing solution was added to each well and the plates were
read using a Dynex
luminometer.
Data was analyzed in the following manner. Readings for each plate were
normalized to
the reading for the luciferase negative control and expressed as "percent of
Luciferase Control".
Preferred targets were considered to be those siRNA pools that suppressed beta-
galactosidase activity by
an average of 40% or more.
Results:
siRNAs targeting PIK4CA, SYNPR, DYRK4, and PFKL inhibited HIV replication by
greater than 40% (see Figure 3). Thus, these genes are essential for the
replication of both HIV and
HCV in human cell lines. Between 30 and 40% inhibition of HIV replication was
also observed with
siRNAs targeting GAK, DUSP19, and DUT, indicating these genes may also be
targets for HIV
infection.
As in Example 7, toxicity associated with SOCS5, PARVB, and TRPM5 siRNAs in
HeLa cells precludes any understanding of the role these genes play in viral
infection in these cells. No
effect on H1V replication was observed with PSKH1 siRNAs.
Other embodiments are within the following claims. While several embodiments
have
been shown and described, various modifications may be made without departing
from the spirit and
scope of the present invention.
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