U.S. patent application number 13/360442 was filed with the patent office on 2012-08-30 for inhibition of viral gene expression using small interfering rna.
This patent application is currently assigned to SOMAGENICS, INC.. Invention is credited to HEINI ILVES, BRIAN H. JOHNSTON, ROGER L. KASPAR, ATTILA A. SEYHAN, ALEXANDER V. VLASSOV.
Application Number | 20120220033 13/360442 |
Document ID | / |
Family ID | 36060681 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120220033 |
Kind Code |
A1 |
KASPAR; ROGER L. ; et
al. |
August 30, 2012 |
INHIBITION OF VIRAL GENE EXPRESSION USING SMALL INTERFERING RNA
Abstract
The invention provides methods, compositions, and kits
comprising small interfering RNA (shRNA or siRNA) that are useful
for inhibition of viral-mediated gene expression. Small interfering
RNAs as described herein can be used in methods of treatment of HCV
infection. ShRNA and siRNA constructs targetING the internal
ribosome entry site (IRES) sequence of HCV are described.
Inventors: |
KASPAR; ROGER L.; (SANTA
CRUZ, CA) ; ILVES; HEINI; (SANTA CRUZ, CA) ;
SEYHAN; ATTILA A.; (SAN JOSE, CA) ; VLASSOV;
ALEXANDER V.; (SANTA CRUZ, CA) ; JOHNSTON; BRIAN
H.; (SCOTTS VALLEY, CA) |
Assignee: |
SOMAGENICS, INC.
|
Family ID: |
36060681 |
Appl. No.: |
13/360442 |
Filed: |
January 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13039100 |
Mar 2, 2011 |
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13360442 |
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11444901 |
Jun 1, 2006 |
7902351 |
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13039100 |
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PCT/US2005/032768 |
Sep 12, 2005 |
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11444901 |
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60608574 |
Sep 10, 2004 |
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Current U.S.
Class: |
435/375 ;
536/24.5 |
Current CPC
Class: |
C12N 2310/111 20130101;
C12N 2310/53 20130101; C12N 15/1131 20130101; C12N 2770/24211
20130101; A61P 31/14 20180101; A61P 1/16 20180101; C12N 2770/36111
20130101; C12N 2310/14 20130101 |
Class at
Publication: |
435/375 ;
536/24.5 |
International
Class: |
C12N 5/02 20060101
C12N005/02; C12N 15/113 20100101 C12N015/113 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made in part during work supported by
grant no. 5R43AI056611 from the National Institutes of Health. The
government may have certain rights in the invention.
Claims
1. An RNA sequence consisting of a. a first RNA sequence, wherein
the first RNA sequence is SEQ ID NO:34, (SEQ ID NO:35), (SEQ ID
NO:36), (SEQ ID NO:37), (SEQ ID NO:38), (SEQ ID NO:39), (SEQ ID
NO:40), (SEQ ID NO:41), (SEQ ID NO:42), (SEQ ID NO:43), (SEQ ID
NO:44), (SEQ ID NO:45), (SEQ ID NO:46), (SEQ ID NO:47), (SEQ ID
NO:48), (SEQ ID NO:49), (SEQ ID NO:50), (SEQ ID NO:51); (SEQ ID
NO:52), (SEQ ID NO:53), (SEQ ID NO:54), (SEQ ID NO:55), (SEQ ID
NO:56), or a sequence that differs from a foregoing sequence by
one, two, or three nucleotides; b. a second RNA sequence that is a
complement of the first sequence; c. a loop sequence positioned
between the first and second nucleic acid sequence, the loop
sequence consisting of 4-10 nucleotides; and d. optionally, a two
nucleotide overhang.
2.-17. (canceled)
18. A method of inhibiting expression or activity of a hepatitis C
virus, the method comprising a. providing a cell that can express a
hepatitis C virus; and b. contacting the cell with an RNA sequence
of claim 1.
19-61. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of PCT application
PCT/US2005/032768, filed Sep. 12, 2005, which claims priority under
35 U.S.C. .sctn.119 from U.S. Provisional Application No.
60/608,574, filed Sep. 10, 2004, both of which are incorporated
herein by reference in their entirety.
FIELD OF THE INVENTION
[0003] The invention relates to inhibition of viral gene
expression, for example, hepatitis C IRES-mediated gene expression,
with small interfering RNA (shRNA and siRNA).
BACKGROUND OF THE INVENTION
[0004] Treatment and prevention of Hepatitis C virus (HCV)
infections remains a major challenge for controlling this worldwide
health problem; existing therapies are only partially effective and
no vaccine is currently available. Hepatitis C (HCV) virus infects
more than 170 million people worldwide and is the leading cause of
liver transplants. Existing treatments, including ribavirin and
pegylated interferon alpha, are effective only in approximately 50
percent of patients and have substantial side effects. The
development of more effective HCV treatments is hampered by the
lack of a good small animal model, the inability to stably culture
the virus in tissue culture cells, and the high viral mutation rate
[1-3]. The availability of an HCV replicon system has allowed the
study of HCV replication, host-cell interactions and evaluation of
anti-viral agents, and more recently, a transgenic chimeric
humanized mouse liver model was developed that allows full HCV
infection [4-7]. Moreover, the use of in vivo imaging of HCV
IRES-dependent reporter systems has facilitated efficient
evaluation of delivery and inhibition by anti-HCV agents in mouse
liver over multiple time points using the same animals [8].
[0005] RNA interference is an evolutionarily conserved pathway that
leads to down-regulation of gene expression. The discovery that
synthetic short interfering RNAs (siRNAs) of about 19-29 base pairs
can effectively inhibit gene expression in mammalian cells and
animals without activating an immune response has led to a flurry
of activity to develop these inhibitors as therapeutics [9].
Chemical stabilization of siRNAs results in increased serum half
life [10], suggesting that intravenous administration may achieve
positive therapeutic outcomes if delivery issues can be overcome.
Furthermore, small hairpin RNAs (shRNA) have also shown robust
inhibition of target genes in mammalian cells and can be easily
expressed from bacteriophage (e.g. T7, T3 or SP6) or mammalian (pol
III such as U6 or H1 or polII) promoters, making them excellent
candidates for viral delivery [11].
[0006] Efforts have been made to find effective nucleic acid-based
inhibitors against HCV, as existing treatments are not fully
effective (reviewed in [4, 12]). These efforts include traditional
antisense oligonucleotides, phosphorodiamidate morpholino oligomers
[8], ribozymes, and more recently siRNAs. It has been shown that
siRNAs can effectively target HCV in human tissue culture cells
[13-19] and in animal systems [20].
BRIEF SUMMARY OF THE INVENTION
[0007] The invention provides methods, compositions, and kits for
inhibition of IRES-mediated gene expression in a virus, e.g.,
hepatitis C virus (HCV).
[0008] For the inhibitory RNA sequences listed in FIGS. 4A and 10
and Table 1 (e.g., SEQ ID NOs:19-26), a complementary sequence is
implied, as are sequences unrelated to the target that may be
appended one or both ends of each strand, for example the 3' ends,
as will be known to one skilled in the art. The inhibitory
(antisense recognition) sequences shown in FIG. 4A, FIG. 10, and in
Table 1 can be incorporated into either shRNA or siRNA. In the case
of shRNA, the sequence shown is additionally linked to its
complementary sequence by a loop that includes nucleotide residues
usually unrelated to the target. An example of such a loop is shown
in the shRNA sequences depicted in FIG. 1B and FIG. 1C as well as
in FIG. 16A-B. In the case of both siRNAs and shRNAs, the strand
complementary to the target generally is completely complementary,
but in some embodiments, the strand complementary to the target can
contain mismatches (see, for example, SEQ ID NOs:13, 14, and 15).
The sequence can be varied to target one or more genetic variants
or phenotypes of the virus being targeted by altering the targeting
sequence to be complementary to the sequence of the genetic variant
or phenotype. The strand homologous to the target can differ at
about 0 to about 5 sites by having mismatches, insertions, or
deletions of from about 1 to about 5 nucleotides, as is the case,
for example, with naturally occurring microRNAs. In some
embodiments, a sequence can target multiple viral strains, e.g., of
HCV, although the sequence differs from the target of a strain at
least one nucleotide (e.g., one, two, or three nucleotides) of a
targeting sequence
[0009] In one aspect, the invention provides a composition
comprising at least one small interfering RNA that is at least
partially complementary to, and capable of interacting with a
polynucleotide sequence of a virus, such that inhibition of viral
gene expression results from the interaction of the small
interfering RNA with the viral target sequence. In one embodiment,
the composition includes at least one shRNA, for example,
comprising, consisting of, or consisting essentially of a sequence
selected from the group consisting of SEQ ID NO:12, SEQ ID NO:16,
SEQ ID NO:17, and SEQ ID NO:18, or comprising or consisting
essentially of a sequence selected from the group consisting of SEQ
ID NO:27, SEQ ID NO:32, and SEQ ID NO:33. In one embodiment, the
shRNA comprises, consists of, or consists essentially of the
sequence depicted in SEQ ID NO:12. In another embodiment, the
composition includes at least one siRNA. In one embodiment, the
composition includes at least one siRNA or shRNA, for example,
comprising or consisting essentially of a sequence selected from
the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:33. In some
embodiments, the small interfering RNA, e.g., shRNA or siRNA,
interacts with a viral sequence of about 19 to about 30
nucleotides, or about 19 to about 25 nucleotides, for example, any
of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides. In some embodiments, the small interfering RNA binds
to a hepatitis C virus sequence. In one embodiment, the small
interfering RNA binds to a sequence within the internal ribosome
entry site (IRES) sequence of a hepatitis C virus, for example, to
the sequence depicted in SEQ ID NO:26 (residues 344-374 of SEQ ID
NO:11). In one embodiment, the hepatitis C virus is HCV genotype
1a.
[0010] In some embodiments, a composition of the invention
comprises a pharmaceutically acceptable excipient, for example,
water or saline, and optionally, are provided in a therapeutically
effective amount, e.g., for treating HCV infection in a human or in
a non-human primate such as a chimpanzee or new world monkey. In
one embodiment, the composition is a pharmaceutical composition
comprising, consisting of, or consisting essentially of at least
one shRNA or siRNA as described herein and a pharmaceutically
acceptable excipient.
[0011] In another aspect, the invention relates to a kit that
includes any of the compositions described above, and optionally,
further includes instructions for use in a method of inhibiting
gene expression in a virus or treating a viral infection in an
individual as described herein. In one embodiment, the kit is for
use in a method for treating HCV infection in an individual, such
as a human, and comprises an shRNA comprising, consisting of, or
consisting essentially of a sequence selected from the group
consisting of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID
NO:18; or comprising or consisting essentially of a sequence
selected from the group consisting of SEQ ID NO:27, SEQ ID NO:32,
and SEQ ID NO:33, or an siRNA comprising or consisting essentially
of a sequence selected from the group consisting of SEQ ID NO:19,
SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:32, and
SEQ ID NO:33, and optionally further comprises instructions for use
in a method of inhibiting gene expression in a hepatitis C virus,
such as HCV genotype 1a, or instructions for use in a method of
treating a hepatitis C (such as HCV genotype 1a) viral infection in
an individual, such as a human, or a non-human primate such as a
chimpanzee.
[0012] In another aspect, the invention provides a method for
treatment of a viral infection in an individual, such as a mammal,
for example, a human or non-human primate. The method includes
administering to the individual a therapeutically effective amount
of a small interfering RNA, such as shRNA or siRNA, that is at
least partially complementary to and capable of binding to a
polynucleotide sequence of the virus and a pharmaceutically
acceptable excipient, such that binding of the small interfering
RNA to the viral polynucleotide sequence inhibits gene expression
in the virus, e.g., decreases the amount of viral expression in the
individual or decreases the amount of viral expression that would
be expected in an individual that did not receive the small
interfering RNA. In one embodiment, the viral infection comprises a
hepatitis C virus, such as HCV genotype 1a. In some embodiments,
the virus is selected from the group consisting of hepatitis C
genotypes 1a, 1b, 2a, and 2b. In some embodiments, the small
interfering RNA comprises, consists of, or consists essentially of
any of the shRNA or siRNA sequences described herein as well as
sequences located within five nucleotides of one of the siRNA or
shRNA sequences described herein. In some embodiments, the small
interfering RNA is complementary to a viral sequence of about 19 to
about 30 nucleotides, or about 19 to about 25 nucleotides, for
example, any of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30 nucleotides. In one embodiment, the virus is a hepatitis C
virus, such as HCV genotype 1a. In one embodiment, the small
interfering RNA binds to a sequence of about 19 to about 25
nucleotides within the IRES region of HCV 1a depicted in SEQ ID
NO:26. Treatment may include therapy (e.g., amelioration or
decrease in at least one symptom of infection) or cure. In some
embodiments, the shRNA is administered parenterally, for example,
by intravenous injection or infusion.
[0013] In another aspect, the invention provides a method of
inhibiting gene expression in a virus, comprising contacting viral
RNA or viral mRNA with a small interfering RNA or introducing a
small interfering RNA into a virus-containing cell, such that the
small interfering RNA, e.g., shRNA or siRNA, contains a sequence
that is at least partially complementary to a polynucleotide
sequence of the virus and capable of inhibiting viral gene
expression, for example, by inducing cleavage of viral
polynucleotide sequences. In some embodiments, the small
interfering RNA comprises, consists of, or consists essentially of
any one of the shRNA or siRNA sequences described herein. In some
embodiments, the small interfering RNA binds to a viral sequence of
about 19 to about 30 nucleotides, or about 19 to about 25
nucleotides, for example, any of about 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30 nucleotides. In one embodiment, the virus is
a hepatitis C virus, such as HCV 1a. In one embodiment, the small
interfering RNA interacts with a sequence of about 19 to about 30
nucleotides within the IRES region of HCV genotype 1a depicted in
SEQ ID NO:26 as well as sequences located within five nucleotides
of one of the siRNA or shRNA sequences described herein. In yet
other embodiments, at least two small interfering RNAs are
introduced into a cell.
[0014] The invention also relates to an RNA sequence that consists
of (a) a first RNA sequence, such that the first RNA sequence is a
sequence illustrated in FIG. 10 or FIG. 16A-B, e.g., SEQ ID NO:34,
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ
ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,
SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51; SEQ ID NO:52, SEQ ID
NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, or a sequence that
differs from a foregoing sequence by one, two, or three
nucleotides; (b) a second RNA sequence that is a complement of the
first sequence; (c) a loop sequence positioned between the first
and second nucleic acid sequence, the loop sequence consisting of
4-10 nucleotides; and (d) optionally, a two nucleotide overhang. In
some embodiments of the invention, the first RNA sequence is SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ
ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43,
SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID
NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51; SEQ ID NO:52, SEQ
ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56. The RNA
sequence can, in some cases, include at least one modified
nucleotide. The loop sequence of an RNA sequence of the invention
can be, e.g., four nucleotides, five nucleotides, six nucleotides,
seven nucleotides, eight nucleotides, nine nucleotides, ten
nucleotides, or at least ten nucleotides. In some embodiments, the
RNA sequence is an shRNA and includes an HCV target sequence as
described herein and a complementary sequence, linked by a loop
that includes at least one non-nucleotide molecule. In certain
embodiments, the loop of the RNA sequence is 3' to a sense strand
and 5' to the complementary antisense strand of the shRNA. In other
embodiments, the loop of the RNA sequence is 3' to an antisense
strand and 5' to the complementary sense strand of the shRNA. In
some cases, the RNA sequence includes a two nucleotide overhang and
the two nucleotide overhang is a 3'UU. In some cases, the overhang
is one nucleotide, two nucleotides, three nucleotides, or more. In
some cases, the first RNA sequence is any one of SEQ ID NOs:57-79,
SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In some
cases, the RNA sequence is a sequence illustrated in FIG.
16A-B.
[0015] The invention also relates to a DNA sequence that includes a
sequence encoding an RNA sequence disclosed herein (e.g., an RNA
sequence illustrated in FIG. 10 or FIG. 16A-B). The invention also
includes an expression vector comprising such a DNA sequence. Also
included is a retroviral vector that includes such a DNA sequence,
e.g., a retroviral vector that, upon infection of a cell with the
vector, can produce a provirus that can express an RNA sequence of
the invention, for example, without limitation, an shRNA sequence
illustrated in FIG. 16A-B.
[0016] In some aspects, the invention relates to a composition that
includes an RNA sequence as disclosed herein (for example, without
limitation, an shRNA illustrated by FIG. 16A-B) and a
pharmaceutically acceptable excipient. In some embodiments, the
composition comprises a vector as disclosed herein and a
pharmaceutically acceptable excipient. In certain embodiments, a
composition of the invention includes at least two RNA sequences as
disclosed herein.
[0017] In another aspect, the invention includes a method of
inhibiting expression or activity of a hepatitis C virus. The
method includes providing a cell that can express a hepatitis C
virus, and contacting the cell with an RNA sequence as disclosed
herein (non-limiting examples of which are illustrated in FIG.
16A-B). The cell can be in a mammal, e.g., a human or a non-human
primate such as a chimpanzee. In certain embodiments, the cell is
contacted with at least two different RNA sequences.
[0018] In some aspects, the invention relates to a method that
includes identifying a subject infected with or suspected of being
infected with a hepatitis C virus, providing to the subject a
therapeutically effective amount of a composition containing one or
more different RNA sequences disclosed herein. In some embodiments,
the method also includes determining whether the viral load of the
subject is decreased subsequent to providing the composition to the
subject. In some embodiments, the method also includes determining
whether at least one viral protein or viral nucleic acid sequence
is decreased in the subject subsequent to providing the composition
to the subject.
[0019] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0020] Other features and advantages of the invention will be
apparent from the detailed description, drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1A is a representation of the IRES nucleotide sequence
of hepatitis C genotype 1a (see GenBank Accession No. AJ242654).
Nucleotides of a target region, 344-374, are underlined. Various
regions (indicated in bold) have been successfully targeted by
inhibitors, including Heptazyme.TM. ribozyme (siRNA.com; positions
189-207), Chiron 5U5 siRNA [25] (positions 286-304), ISIS 14803
phosphorothioate antisense oligonucleotide [34] (positions
330-349), Mizusawa 331 siRNA [15] (positions 322-340) and a
phosphorodiamidate morpholino oligomer [8, 35] (positions 344-363).
A more complete list of siRNAs that have been tested to
down-regulate the HCV IRES and other HCV elements can be found in
[2, 3].
[0022] FIG. 1B is a representation of RNA sequences of shRNA
HCVa-wt (shRNA1) and mutated variants thereof resulting from pol
III transcription from a U6 promoter of corresponding DNA
templates. Two base pairs (underlined) of HCVa-wt were altered to
create versions of HCVa-wt containing 1 (HCVSNP1 or HCVSNP2) or 2
mismatches (HCVa-mut) shRNAs as shown.
[0023] FIG. 1C is a representation of the sequences of shRNAs
HCVb-wt (sh9), HCVc-wt (sh10), and HCVd-wt (sh11).
[0024] FIG. 1D is a representation of the secondary structure of
the HCV IRES with indicated target sites for shRNA HCVa-wt,
HCVb-wt, HCVc-wt, and HCVd-wt.
[0025] FIG. 1E is a schematic representation of the pCDNA3/HCV IRES
dual luciferase reporter construct used to produce the HCV IRES
target as well as the EMCV IRES control, in which the IRES from
encephalomyocarditis virus replaces the HCV IRES and therefore
lacks any target for the HCV-directed shRNAs. In each case, firefly
luciferase expression is dependent on initiation of translation
from the IRES sequence, whereas Renilla luciferase is expressed in
a cap-dependent manner.
[0026] FIG. 1F is a bar graph depicting the results of a screen of
shRNAs for the ability to inhibit HCV IRES-mediated gene expression
in 293FT cells. 293FT cells were cotransfected with pCDNA3/HCV IRES
dual luciferase reporter construct, pSEAP2 (as a transfection and
specificity control), and an shRNA (at 1 nM) in a well of a 24-well
tissue culture plate. Plasmid pUC18 was added to provide a total of
800 ng nucleic acid per well. 48 hours post-transfection, cells
were lysed and firefly luciferase activity was measured by a
luminometer. All data are the results of individual, independent
experiments performed in triplicate, and normalized to SEAP.
[0027] FIG. 2A is a bar graph depicting the results of experiments
testing inhibition of HCV-IRES driven gene expression in 293FT
cells that were cotransfected with dual luciferase reporter and
SEAP expressing plasmids and 1 pmole of in vitro transcribed
shRNAs. The target plasmid was pCDNA3/HCV IRES dual luciferase
reporter (HCV IRES, as shown in FIG. 1E). Firefly luciferase
activity measured as described in Example 1. Firefly luciferase and
SEAP activities were normalized to 100.
[0028] FIG. 2B is a bar graph depicting the results of experiments
testing HCV versus EMCB inhibition in 293FT cells. The data are
presented as luciferase activity divided by SEAP activity
normalized to 100.
[0029] FIG. 2C is a bar graph depicting the results of experiments
demonstrating the effect of single-base mismatches on potency of
shRNAs. Experimental conditions were as described for FIG. 2A. SNP1
and SNP2 contained mutated base pairs as shown in FIG. 1B.
[0030] FIG. 2D is a line graph depicting the resulting of
experiments testing dose response of inhibition of HCV-IRES-driven
gene expression by HCVa-wt and mutated (HCVa-mut) or control (229)
shRNAs. Experimental conditions were as described for FIG. 2A. The
data are represented as luciferase divided by SEAP normalized to
100. All data are the results of individual, independent
experiments performed in triplicate.
[0031] FIG. 2E is a line graph depicting the resulting of
experiments testing dose response of HCVa-wt, HCVa-mut), and 229
shRNAs on gene expression from a dual-luciferase reporter lacking
shRNA target sites. The procedure was as described for FIG. 2D
except target was firefly luciferase driven by EMCV IRES instead of
HCV IRES.
[0032] FIG. 2F is a reproduction of a Northern blot analysis of
co-transfected 293FT cells treated as follows; 10 .mu.g of total
RNA isolated from cells transfected with no inhibitor (lane 1), 229
(lane 2) HCVa-wt (lane 3), or HCVa-mut (lane 4) were separated by
denaturing gel electrophoresis, transferred to membrane and
hybridized sequentially to .sup.32P-labeled fLuc, SEAP, or
elongation factor 1A (EF1A) cDNA probes. The RNA blot was exposed
to a storage phosphor screen for visualization and quantitation
(BioRad FX Molecular Imager).
[0033] FIG. 3A is a line graph depicting the results of experiments
testing dose response to HCVa-wt and HCVa-mut shRNAs using the
human hepatocyte cell line, Huh7. Procedures were as described for
FIG. 2D, except that Huh7 cells were used.
[0034] FIG. 3B is a line graph depicting the results of experiments
demonstrating that HCVa-wt shRNA does not inhibit a similar target
lacking the HCV IRES. Cells were transfected as in FIG. 3A except
that pCDNA3/EMCV IRES dual luciferase reporter (EMCV IRES) was
added in place of pCDNA3/HCV IRES dual luciferase reporter (HCV
IRES). All data are presented as luciferase activity divided by
SEAP. All data were generated from individual, independent
experiments performed in triplicate.
[0035] FIG. 4A depicts sequences of seven 19 base pair viral
recognition sequences of synthetic siRNAs and shRNAs contained
within the 25 nucleotide target site of HCV genotype 1A (SEQ ID
NO:26) and analysis of their purity on 10% native polyacrylamide
gel stained with ethidium bromide. siRNAs: sense and antisense
strands contained 3'-UU overhangs; shRNAs: loop sequences and
3',5'-end overhangs were identical to those of the 25 base pair
shRNAs.
[0036] FIG. 4B is a bar graph depicting the results of experiments
in which RNA inhibitors (siRNAs and shRNAs) were assayed for
inhibition of HCV IRES-mediated gene expression at an inhibitor
concentration 1 nM in 293 FT cells.
[0037] FIG. 4C is a bar graph depicting the results of experiments
in which RNA inhibitors were assayed for inhibition of HCV
IRES-mediated gene expression at an inhibitor concentration of 0.1
nM in 293 FT cells.
[0038] FIG. 5A is a reproduction of IVIS images of mice in which
dual luciferase HCV IRES reporter plasmid (10 .mu.g) and SEAP
(added to control for injection efficiency and nonspecific
inhibition) were co-injected into the tail veins of mice as
described in Example 1 with 100 .mu.g of the indicated HCV shRNA or
control 229 shRNA) directly or in the form of 100 .mu.g of pol III
expression plasmids expressing shRNA (or pUC18 plasmid as control).
At various time-points (24, 36, 48, 60, 72, 84 and 100 hours)
post-injection, luciferin was administered intraperitoneally, and
the mice were imaged using the IVIS in vivo imaging system. Images
are of representative mice from the 84 hour time point.
[0039] FIG. 5B is a graph depicting the quantitated results of
experiments described for FIG. 5A in which there was direct
delivery of RNA. Quantitation was performed using ImageQuant.TM.
software. Each time-point represents the average of 4-5 mice. At
the 96 hour time point, the mice were bled and the amount of SEAP
activity determined by pNPP assay as described in Example 1. The
quantitated data are presented as luciferase divided by SEAP
activity, normalized to pUC18 control mice (100%, no error bars
shown on pUC18 control for clarity; error bars are similar to the
others shown).
[0040] FIG. 6 is a bar graph depicting the results of experiments
in which shRNA and phosphorodiamidate morpholino oligomer
inhibition of HCV IRES-mediated reporter gene expression in mice
was compared. Mice were co-injected as described in experiments for
FIG. 5 with dual luciferase HCV IRES reporter plasmid and pSEAP
with 100 .mu.g of the indicated HCV shRNA inhibitors or 1 nmole of
a morpholino oligonucleotide previously shown to inhibit HCV IRES
expression construct [8]. The mice were imaged at various times (12
hours, 24 hours, 48 hours, and 144 hours) post-treatment. Data
shown are for the 48 hour time point. The quantitated data are
presented as luciferase and SEAP activities, normalized to pUC18
control (no addition) mice. The results presented are from 3-5 mice
per construct.
[0041] FIG. 7 is a graph depicting the results of experiments in
which BHK-21 cells were transiently transfected with plasmids
expressing an inhibitory shRNA targeting the nsp-1 gene.
Twenty-four hours after transfection, cells were infected with 10
.mu.l of replication-proficient GFP-expressing Semliki Forest virus
(SFV-GFP-VA7; multiplicity of infection (MOI) sufficient for about
100% infection) and assayed for virus-mediated GFP expression by
flow cytometry 24 hours after infection. The level of
siRNA-mediated suppression was about 35%. Labels: Nsp 1. shRNA
targeting Nsp-1 gene (nsp-1#2); empty vector, pU6; naive,
uninfected BHK cells.
[0042] FIG. 8 is a bar graph depicting the results of experiments
in which inhibition of replication-deficient SFV (SFV-PD713P-GFP)
by shRNAs was investigated. BHK-21 cells were transiently
transfected with plasmids expressing inhibitor shRNAs. Forty-six
hours after transfection, cells were infected with SFV-GFP virus at
an MOI of 5 with 8% PEG in serum-free media for one hour. Then
complete media was added and cells were incubated at 37.degree. C.
overnight. Cells were analyzed by flow cytometry at 9, 24, 32, 99,
and 125 hours after infection. For clarity, only three time points
are shown (9, 24 and 32 hours). The amount of inhibition of each
shRNA was normalized to capsid shRNA. Capsid mRNA is not present in
this SFV-GFP replication-deficient virus and therefore capsid shRNA
should have no effect on GFP expression. The transfection
efficiency for the shRNA expression constructs for this experiment
was about 70%, suggesting that actual viral inhibition is
significantly higher than the levels indicated. The fifth set of
bars (Mixed) refers to a mixture of shRNAs targeting nsp 1-4 and
capsid.
[0043] FIG. 9 is a line graph depicting the results of experiments
testing HCV replicon inhibition by shRNAs.
[0044] FIG. 10 is a table depicting sequences and results of a
screen of shRNAs for the ability to inhibit HCV IRES-mediated gene
expression in 293FT cells. Cells were cotransfected (using
Lipofectamine.TM. 2000) with pCDNA3/HCV IRES dual luciferase
reporter construct (40 ng), pSEAP2 (25 ng, as a transfection and
specificity control), and an shRNA (at 1 or 5 nM) in a well of a
48-well tissue culture plate. Plasmid pUC18 was added to provide a
total of 400 ng nucleic acid per well. Forty-eight hours
post-transfection, the supernatants were removed for SEAP analysis,
cells were lysed, and firefly luciferase activity was measured by a
luminometer. All data are the results of at least two independent
experiments performed in triplicate. SEAP levels were uniform in
all samples. Control experiments to assay specificity of shRNAs
were performed on mutated pCDNA3/HCV IRES dual luciferase reporter
construct as well, where C340 (in IRES) was substituted with U.
[0045] FIG. 11 is a diagrammatic representation of 3'-terminal
sequence of the HCV IRES with segments targeted by shRNAs. Mutation
C340.fwdarw.U (used to assay specificity of shRNAs) is
indicated.
[0046] FIG. 12A is a diagrammatic representation of 5'-termini of
HCV IRES and targeting positions for six 19-bp shRNAs.
[0047] FIG. 12B is a bar graph depicting the results of a screen of
shRNAs for the ability to inhibit HCV IRES-mediated gene expression
in 293FT cells. Experiments were conducted as for FIG. 10; shRNA
concentration, 1 nM.
[0048] FIG. 13A is a diagrammatic representation of the sequences
of tested variants of the depicted 25 base pair shRNA, with the
various loop sizes and sequences, as well as 3'-termini that were
tested.
[0049] FIG. 13B is a bar graph depicting the results of a screen of
shRNAs depicted in FIG. 13A for the ability to inhibit HCV
IRES-mediated gene expression in 293FT cells. Experiments were
conducted as for those of FIG. 10. shRNA concentration, 1 nM.
(shRNA sequences are listed in FIG. 16A-B)
[0050] FIG. 14A is a diagrammatic representation of the sequences
of tested variants of the depicted 19-bp shRNA with the various
loop sizes and sequences tested, as well as 3' termini that were
tested.
[0051] FIG. 14B is a bar graph depicting the results of a screen of
shRNAs depicted in FIG. 14A for the ability to inhibit HCV
IRES-mediated gene expression in 293FT cells. Experiments were
conducted as described for FIG. 10. shRNA concentration, 1 nM.
(shRNA sequences are listed in FIG. 16A-B)
[0052] FIG. 15 is a bar graph depicting the results of a screen of
shRNAs (and siRNAs) for the inhibitory activity in the HCV replicon
system. Human hepatocytes (AVA5, a derivative of the Huh7 cell
line) stably expressing HCV subgenomic replicons, were transfected
with RNA inhibitors, and the amount of HCV expression was
determined. A range of concentrations was tested and the
concentration of sh/siRNA that resulted in 50% inhibition (EC50)
was determined. Dark and light bars represent the results of two
independent experiments.
[0053] FIG. 16A-B is a table depicting shRNA sequences targeting
HCV IRES as indicated. ShRNA loops are underlined. Nucleotides
indicated by low-case are non-complementary to the target.
DETAILED DESCRIPTION OF THE INVENTION
[0054] The invention provides compositions, methods, and kits for
inhibiting viral (e.g., hepatitis C) gene expression and/or
treating a viral infection in a mammal.
[0055] RNA interference offers a novel therapeutic approach for
treating viral infections. The present invention provides small
interfering RNAs (e.g., shRNAs and siRNAs) that target a viral
sequence and inhibit (i.e., reduce or eliminate) viral gene
expression, and methods of using such small interfering RNAs for
treatment of a viral infection in a mammal, such as a human. In
some embodiments, the small interfering RNA constructs of the
invention inhibit gene expression of a virus by inducing cleavage
of viral polynucleotide sequences within or near the target
sequence that is recognized by the antisense sequence of the small
interfering RNA.
[0056] As used herein, "small interfering RNA" refers to an RNA
construct that contains one or more short sequences that are at
least partially complementary to and can interact with a
polynucleotide sequence of a virus. Interaction may be in the form
of a direct binding between complementary (antisense) sequences of
the small interfering RNA and polynucleotide sequences of the viral
target, or in the form of an indirect interaction via enzymatic
machinery (e.g., a protein complex) that allows the antisense
sequence of the small interfering RNA to recognize the target
sequence. In some cases, recognition of the target sequence by the
small interfering RNA results in cleavage of viral sequences within
or near the target site that is recognized by the recognition
(antisense) sequence of the small interfering RNA. The small
interfering RNA can exclusively contain ribonucleotide residues, or
the small interfering RNA can contain one or more modified
residues, particularly at the ends of the small interfering RNA or
on the sense strand of the small interfering RNA. The term "small
interfering RNA" as used herein encompasses shRNA and siRNA, both
of which are understood and known to those in the art to refer to
RNA constructs with particular characteristics and types of
configurations.
[0057] As used herein, "shRNA" refers to an RNA sequence comprising
a double-stranded region and a loop region at one end forming a
hairpin loop. The double-stranded region is typically about 19
nucleotides to about 29 nucleotides in length on each side of the
stem, and the loop region is typically about three to about ten
nucleotides in length (and 3'- or 5'-terminal single-stranded
overhanging nucleotides are optional). One example of such an
shRNA, HCVa-wt shRNA, has a 25 base pair double-stranded region
(SEQ ID NO:12), a ten nucleotide loop, a GG extension on the 5'
end, and a UU extension on the 3' end. Additional examples of
suitable shRNAs for use in, e.g., inhibiting HCV expression, are
provided throughout the specification, e.g., FIG. 16A-B.
[0058] As used herein, "siRNA" refers to an RNA molecule comprising
a double-stranded region with a 3' overhang of nonhomologous
residues at each end. The double-stranded region is typically about
18 to about 30 nucleotides in length, and the overhang may be of
any length of nonhomologous residues, for example, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. The siRNA
can also comprise two or more segments of 19-30 base pair separated
by unpaired regions. Without committing to any specific theory, the
unpaired regions may function to prevent activation of innate
immunity pathways. One example of such an siRNA is HCVa-wt siRNA,
which has a 25 base pair double-stranded region (SEQ ID NO:12), and
a UU extension on each 3' end.
[0059] In one embodiment, a small interfering RNA as described
herein comprises a sequence complementary to a sequence of the
internal ribosome entry site (IRES) element of hepatitis C ("HCV").
In one embodiment, the virus is HCV genotype 1a.
[0060] SiRNA gene inhibition has been shown to robustly inhibit
gene expression in a number of mammalian systems. Due to its high
level of secondary structure, the HCV IRES has been suggested to be
a poor target for siRNAs or shRNAs. Mizusawa reported, however,
successful targeting of the HCV IRES in 293 and Huh7 tissue culture
cells, reporting 50 and 74 percent knock-down of gene expression,
respectively. Similarly, Seo and coworkers [25] reported the
ability of 100 nM siRNA to inhibit HCV replication (about 85%
knockdown) in 5-2 Huh7 cells. It has now been demonstrated as
described herein that small interfering RNAs (shRNAs and siRNAs)
directed against the 3' end of the HCV IRES, including and
downstream of the AUG translation start site, can induce 96 percent
knockdown of HCV IRES-dependent luciferase expression at 0.3 nM in
293FT cells and 75 percent knockdown at 0.1 nM in Huh7 cells (see
FIGS. 2D and 3A). Furthermore, direct delivery of shRNA to mouse
liver was shown to potently inhibit HCV IRES-dependent reporter
expression. This is the first demonstration of RNAi-mediated gene
inhibition in an animal model following direct delivery of an RNA
hairpin (not expressed in vivo from a plasmid or viral vector). The
effectiveness of shRNA delivered directly to mouse liver following
hydrodynamic injection was surprising in view of the high levels of
nucleases found in blood. The observation that these shRNAs
effectively knocked down gene expression in liver indicates that
these shRNA inhibitors (1) are very potent and not needed at high
levels in mouse liver to cause gene inhibition, (2) are delivered
sufficiently rapidly to the liver, e.g., before they are cleaved by
nucleases in quantities that prevent an inhibitory effect, or (3)
are inherently stable to nuclease degradation (or a combination of
these characteristics).
[0061] Reports suggest that in vitro-synthesized transcripts from
bacteriophage promoters potently induce interferon (IFN) alpha and
beta due to the presence of an unnatural 5' triphosphate [26].
Furthermore, shRNAs expressed from pol III expression vectors may
also induce IFN [27]. How this interferon induction would affect
use of shRNAs in a clinical setting for HCV infection is unclear.
Current HCV therapy includes treatment with interferon alpha,
suggesting that if interferon is induced by shRNA, it may have a
positive effect. To date, no interferon-related side effects appear
to have been reported in animals following administration of RNAi
[3]. Additional concerns have been raised regarding off-target
effects of siRNA as well as potential cytotoxic effects when siRNAs
or shRNAs are delivered by lentiviral vectors [28].
[0062] The present invention also relates to methods of testing
siRNAs and shRNAs targeting HCV IRES sequences to identify those
sequences having sufficient activity (e.g., the highest activity
among a selected group of such sequences) to be a candidate for use
as a treatment. Testing can also include screening for small
interfering activities having undesirable off-target effects or
general cytotoxic effects. Off-target effects include, without
limitation knockdown of nontargeted genes, inhibition of expression
of non-targeted genes, and competition with natural microRNA
pathways (Birmingham et al., Nat. Methods. 2006 3(3):199-204; Grimm
et al., Nature 2006 441(7092):537-541). Methods of identifying
cytotoxic effects are known in the art (for example, Marques et
al., Nat. Biotechnol. 2006 24(5):559-565; Robbins et al., Nat.
Biotechnol. 2006 24(5):566-571).
[0063] The IRES region in the HCV 5'-UTR is highly conserved
(92-100% identical [15, 29-31]) and has several segments that
appear to be invariant, making the IRES a prime target for nucleic
acid-based inhibitors. The region around the AUG translation
initiation codon is particularly highly conserved, being invariant
at positions +8 to -65 (with the exception of a single nucleotide
variation at position -2) as observed in over 81 isolates from
various geographical locations [32]. Despite the conservation of
sequence in the IRES motif, it is unlikely that targeting a single
sequence, even if highly conserved, will be sufficient to prevent
escape mutants. RNA viruses are known to have high mutation rates
due to the high error rate of the RNA polymerase and the lack of
proofreading activity of that enzyme. On average, each time HCV RNA
is replicated one error is incorporated into the new strand. This
error rate is compounded by the prodigious production of viral
particles in an active infection (approximately a trillion per day
in a chronically infected patient) [33]. Therefore, in some
embodiments of the invention, several conserved sites are targeted
or, alternatively, shRNAs as described herein are used as a
component of a combination treatment, such as with ribaviran and/or
pegylated interferon. As demonstrated herein, a single mismatch
does not completely block shRNA activity (see Example 2; FIG. 2D);
thus each different shRNA may have some activity against a limited
number of mutations. Accordingly, the invention includes methods of
inhibiting HCV expression using an shRNA that may include a
mismatch to the target sequence. The invention also includes
methods of inhibiting HCV expression by administering at least two
different shRNAs targeting an HCV IRES, such that the shRNAs differ
in the targeting sequences.
[0064] McCaffrey and colleagues reported that a phosphorodiamidate
morpholino oligonucleotide directed against a conserved HCV IRES
site at the AUG translation initiation site potently inhibits
reporter gene expression [8]. The same morpholino inhibitor was
used for comparison against the shRNA inhibition described herein.
It was found that both the morpholine and the shRNA targeting the
conserved HCV IRES site potently and robustly inhibited
IRES-dependent gene expression. Four mutations in the morpholino
were required to block activity, whereas two changes in the shRNA
were sufficient, suggesting greater shRNA specificity. This
potential advantage, coupled with the lack of unnatural residues in
the shRNA inhibitor and presumably fewer resultant side effects,
are balanced by the increased stability of the morpholino
oligomer.
[0065] A dual reporter luciferase plasmid was used in which firefly
luciferase (fLuc) expression was dependent on the HCV IRES [24].
Expression of the upstream renilla luciferase is not HCV
IRES-dependent and is translated in a Cap-dependent process. Direct
transfection of HCV IRES shRNAs, or alternatively shRNAs expressed
from polIII-promoter vectors, efficiently blocked HCV IRES-mediated
fLuc expression in human 293FT and Huh7 cells. Control shRNAs
containing a double mutation had little or no effect on fLuc
expression, and shRNAs containing only a single mutation showed
partial inhibition. These shRNAs were also evaluated in a mouse
model where DNA constructs were delivered to cells in the liver by
hydrodynamic transfection via the tail vein. The dual luciferase
expression plasmid, the shRNAs, and secreted alkaline phosphatase
plasmid were used to transfect cells in the liver, and the animals
were imaged at time points over 12 to 96 hours. In vivo imaging
revealed that HCV IRES shRNA directly, or alternatively expressed
from a polIII-plasmid vector, inhibited HCV IRES-dependent reporter
gene expression; mutant or irrelevant shRNAs had little or no
effect. These results indicate that shRNAs, delivered as RNA or
expressed from viral or nonviral vectors, are useful as effective
antivirals for the control of HCV and related viruses.
[0066] Assay of three additional shRNAs targeting different sites
on HCV IRES domain IV revealed another potent shRNA, HCVd-wt, whose
target position is shifted six nucleotides from that of HCVa-wt.
HCVb-wt and HCVc-wt were much less efficient inhibitors.
[0067] To further investigate local sequence effects on potency,
seven in vitro-transcribed shRNA constructs comprising a 19 base
pair sequence complementary to a sequence of the HCV IRES and the
corresponding synthetic siRNA comprising the same 19 base pair
sequences, targeting all possible positions within the 25 base pair
site of HCVa-wt (344-368), were assayed for inhibitory activity. A
25 base pair synthetic siRNA corresponding to HCVa-wt shRNA was
also tested. All of the tested constructs exhibited a high level of
activity. In general, 19 base pair siRNAs were more potent than 19
base pair shRNAs. The most potent, siHCV19-3 was effective at 1 nM
(>90% inhibition), 0.1 nM (about 90% inhibition) and even at a
concentration of 0.01 nM (about 40% inhibition). Thus, 19-25 base
pair shRNAs and siRNAs designed to target the region 344-374 on the
HCV IRES are generally potent inhibitors of HCV expression, with
some local differences.
[0068] Small hairpin RNAs of the invention can, optionally, include
structures resulting in strong noncovalent bonds between the sense
and antisense strands of the shRNA. Examples of such noncovalent
bonds include cross-links mediated by metal ions. Such cross-links
can be formed between natural or modified nucleotide residues,
including, for example, modified bases, sugars, and terminal
groups, as described in Kazakov and Hecht 2005, Nucleic Acid-Metal
Ion Interactions. In: King, R. B. (ed.), Encyclopedia of Inorganic
Chemistry. 2nd ed., Wiley, Chichester, vol. VI, pp. 3690-3724,
e.g., section 5.4.3. Additional non-limiting examples of variants
of such bonds are found patent application WO
99/09045(US2006074041; e.g., FIG. 10. In general the location of
cross-linkable nucleotide residues is at the ends of the
complementary RNA strands that lie in close proximity upon duplex
formation. The addition of certain metal ions (or metal ion
coordination compounds) can result in the cross-linking of
functional groups that have strong affinity for these metal ions,
such as --SH, --SCH3, phosphorothioates, imidazolides,
o-phenanthrolines, and others. These modified nucleotides are
introduced during chemical synthesis of the sense and antisense RNA
strands. The modified nucleotides in sense and antisense strands
may either form base pairs or be part of 1-3 nucleotide
overhangs.
Targeting Sequences
[0069] Examples of targeting sequences are provided throughout the
specification. Non-limiting examples of targeting sequences are
provided in, for example, Table 1 and FIG. 10. Non-limiting
examples of shRNAs and siRNAs incorporating targeting sequences are
found throughout the specification, e.g., in FIG. 1 and FIG.
16A-B.
Loops
[0070] Effects of size and sequence of loop region of the shRNA
were also investigated. The loop region of the shRNA stem-loop can
be as small as two to three nucleotides and does not have a clear
upper limit on size; generally, a loop is between four and nine
nucleotides, and is generally a sequence that does not cause
unintended effects, e.g., by being complementary to a non-target
gene. Highly structured loop sequences such as a GNRA tetraloop can
be used in the loop region (e.g., as the loop) in an shRNA. The
loop can be at either end of the molecule; that is, the sense
strand can be either 5' or 3' relative to the loop. Also, a
noncomplementary duplex region (approximately one to six base
pairs, for example, four CG base pairs) can be placed between the
targeting duplex and the loop, for example to serve as a "CG clamp"
to strengthen duplex formation. At least 19 base pairs of
target-complementary duplex are needed if a noncomplementary duplex
is used.
[0071] A loop structure can also include reversible linkages such
as S--S bonds, which can be formed by oxidation of --SH groups
introduced into nucleotide residues, e.g., as described in
(Earnshaw et al., J. Mol. Biol., 1997, 274: 197-212; Sigurdsson et
al. (Thiol-Containing RNA for the Study of Structure and Function
of Ribozymes. METHODS: A Companion to Methods in Enzymology, 1999,
18: 71-77). A non-limiting example of the location for nucleotide
residues with SH groups is at the ends of the complementary RNA
strands that lie in close proximity upon duplex formation. Such
modified nucleotides are introduced during chemical synthesis of
the sense and antisense RNA strands of the small interfering RNA.
The modified nucleotides in sense and antisense strands may either
form base pairs or form non-complementary overhangs of one to three
nucleotides.
[0072] Additional non-limiting examples of loops and their
applications, e.g., in shRNA and siRNA targeting HCV, can be found
in the Examples.
Termini
[0073] The 3' terminus of an shRNA as described herein can have a
non-target-complementary overhang of two or more nucleotides, for
example, UU or dTdT, however, the overhangs can be any nucleotide
including chemically modified nucleotides that, for example,
promote enhanced nuclease resistance. In other embodiments, there
are one or zero nucleotides overhanging on the 3' end.
[0074] The 5' end can have a noncomplementary extension, e.g., two
Gs (as shown in FIG. 1B), a GAAAAAA sequence, or only one or zero
nucleotides extending beyond the target-complementary duplex
region. In the sequence shown in FIG. 1B, the two 5' G's are
included primarily for ease of transcription from a T7
promoter.
Additional Features
[0075] Additional features that can optionally be included in
shRNAs used to inhibit HCV expression and that are encompassed by
the invention are length variations between about 19 base pairs and
about 30 base pairs for the target complementary duplex region,
small shifts in the sequence targeted (generally zero to about two
nucleotides, and shifts as large as about ten nucleotides in either
direction along the target may lie within the targetable region).
Similarly, mismatches are also tolerated: about one to about two in
the antisense strand and about one to about nine in the sense
strand (the latter destabilizing the hairpin duplex but not
affecting the strength of binding of the antisense strand to the
target; the number tolerated depends partly on the length of the
target-complementary duplex. As described herein, an shRNA having
at least seven G-U mismatches within a 29 base pair
target-complementary duplex region can be used successfully for
inhibiting HCV expression, e.g., using sequence targeting the HCV
IRES. Note that the two mutations shown in FIG. 1B largely
abrogated inhibition, but other mutants having mutations in other
positions, particularly if they are closely spaced and/or near the
end, can be better tolerated. Certain variations are known in the
art or demonstrated in the instant application.
Vectors
[0076] Suitable vectors for producing shRNAs and siRNAs are
described herein and are known in the art. In non-limiting
examples, shRNAs can be expressed using Pol III promoters such as
U6 or H1, in the context of vectors derived from adeno-associated
virus or lentiviruses. The human U6 nuclear RNA promoter and human
H1 promoter are among the pol III promoters for expressing
shRNAs.
[0077] One feature that is generally desirable in a vector is
relatively prolonged transgene expression. Lentiviral vectors are
able to transduce nondividing cells and maintain sustained
long-term expression of transgene. Adeno-associated virus serotype
8 is considered safe and is characterized by prolonged transgene
expression.
Candidate shRNA and siRNA
[0078] In some cases, one or more small interfering RNAs are
identified as having activity for inhibiting a targeted virus such
as HCV. Additional tests can be carried out to further characterize
the suitability of such RNAs for use, e.g., for inhibiting HCV
expression in an animal. Animal models can be used for such
testing. One non-limiting examples includes a mouse model, e.g., as
illustrated in Example 3 (infra). Other animal models suitable for
testing an treatment for HCV are known in the art, for example,
using chimpanzees.
Methods
[0079] The invention relates to methods of inhibiting gene
expression in a virus, comprising contacting the virus with a small
interfering RNA, such as a shRNA or siRNA as described herein that
comprises a sequence that is at least partially complementary to,
and is capable of interacting with a polynucleotide sequence of the
virus. In some embodiments, contacting the virus comprises
introducing the small interfering RNA into a cell that contains the
virus, i.e., a virus infected cell. "Inhibiting gene expression" as
used herein refers to a reduction (i.e., decrease in level) or
elimination of expression of at least one gene of a virus. For
example, reduction in expression compared to corresponding cell or
animal infected with the virus. In some embodiments, inhibition of
gene expression is accomplished by cleavage of the viral target
sequence to which the small interfering RNA binds. Gene expression
can be assayed by assaying viral RNA or viral protein. In some
cases, efficacy of a method (for example, a treatment using a
composition described herein) is assayed by evaluating an infected
animal for a decrease in symptoms or a change (e.g., decrease) in
the expression or activity of a protein associated with viral
infection, e.g., a viral protein such as p24, or a host protein
such as an interferon.
[0080] The invention also relates to methods for treating a viral
infection or for treating a subject suspected of being infected
(including a subject exposed to virus for prophylactic treatment)
in a mammal, comprising administering to the mammal a composition
comprising a therapeutically effective amount of a small
interfering RNA, such as a shRNA or siRNA as described herein that
includes a sequence that is at least partially complementary to,
and capable of interacting with (e.g., hybridizing to under
physiological conditions, or effecting RNAi activity), a
polynucleotide sequence of the virus, e.g., the IRES sequence of
HCV. In some embodiments, the mammal is human. In one embodiment,
the mammal is a human and the viral infection is a HCV infection,
such as an infection with HCV genotype 1a, and the small
interfering RNA comprises a sequence that is at least complementary
to a sequence of the IRES of the HCV.
[0081] As used herein, a "therapeutically effective amount" is an
amount of a small interfering RNA that can render a desired
therapeutic outcome (e.g., reduction or elimination of a viral
infection). A therapeutically effective amount may be administered
in one or more doses. Non-limiting examples of doses are about 0.1
mg/kg to about 50 mg/kg, e.g., about 1 to about 5 mg/kg. Suitable
methods of delivery are known in the art and include, for example,
intravenous administration (e.g., via a peripheral vein of via a
catheter). Non-limiting examples include delivery via the hepatic
artery or the portal vein.
[0082] Generally, in methods for treating a viral infection in a
mammal, the small interfering RNA is administered with a
pharmaceutically acceptable carrier. As used herein, a
"pharmaceutically acceptable carrier" (also interchangeably termed
"pharmaceutically acceptable excipient" herein) is a relatively
inert substance that facilitates administration of the small
interfering RNA or RNAs. For example, a carrier can give form or
consistency to the composition or can act as a diluent. A
pharmaceutically acceptable carrier is biocompatible (i.e., not
toxic to the host) and suitable for a particular route of
administration for a pharmacologically effective substance.
Suitable pharmaceutically acceptable carriers include but are not
limited to stabilizing agents, wetting and emulsifying agents,
salts for varying osmolarity, encapsulating agents, buffers, and
skin penetration enhancers. In some embodiments, the
pharmaceutically acceptable carrier is water or saline. Examples of
pharmaceutically acceptable carriers are described in Remington's
Pharmaceutical Sciences (Alfonso R. Gennaro, ed., 18th edition,
1990).
[0083] In methods for treating a viral infection, small interfering
RNAs as described herein are generally administered parenterally,
e.g., subcutaneously, intravenously, or intramuscularly.
Compositions
[0084] The invention provides compositions for inhibiting viral
gene expression and/or treating a viral infection in a mammal
comprising at least one small interfering RNA as described herein.
Compositions of the invention may comprise two or more small
interfering RNAs as described herein. In accordance with the
invention, a small interfering RNA, e.g., shRNA or siRNA, comprises
a sequence that is substantially complementary to a viral
polynucleotide sequence of about 19 to about 30 nucleotides,
wherein interaction of the substantially complementary sequence of
the small interfering RNA with the polynucleotide sequence of the
virus inhibits viral gene expression, for example, by cleavage of
viral polynucleotide sequences.
[0085] In some embodiments, the composition comprises an shRNA that
includes a sequence selected from the group consisting of SEQ ID
NOs: 12, 17, 18, 19, 20, 21, 22, 23, 24, and 25. In some
embodiments, the composition comprises an shRNA that includes one
of the following: SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID
NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ
ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,
SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID
NO:51; SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or
SEQ ID NO:56 (Table 10). In some embodiments, the composition
comprises one or more shRNAs of SEQ ID NO:57-110. In some
embodiments, the composition comprises a siRNA comprising a
sequence selected from SEQ ID NOs:19, 20, 21, 22, 23, 24, and 25.
In other embodiments, the composition comprises a siRNA that
includes a sequence of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51; SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, or SEQ ID NO:56 (FIG. 10). In some embodiments, the
composition comprises a shRNA or siRNA that binds to, i.e.,
comprises a sequence substantially complementary to, a sequence of
about 19 to about 30 nucleotides within the IRES element of HCV,
for example, HCV genotype 1a. A composition can include more than
one different shRNA, e.g., shRNAs targeting different sequences of
an IRES or different alleles or mutations of a target sequence. An
shRNA or siRNA as described herein can include more than one of the
identified sequences. Certain compositions contain more than one
different shRNA or siRNA sequences.
[0086] In some embodiments, the invention provides a pharmaceutical
composition comprising a small interfering RNA as described herein
and a pharmaceutically acceptable carrier. In some embodiments, the
pharmaceutical composition is formulated for parenteral
administration to a mammal, for example, a human.
[0087] A pharmaceutical composition that includes a short
interfering RNA (e.g., an siRNA or an shRNA) is formulated to be
compatible with its intended route of administration. Examples of
routes of administration include parenteral, e.g., intravenous,
intradermal, subcutaneous, inhalation, transdermal (topical),
transmucosal, and rectal administration; or oral. Solutions or
suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerin, propylene glycol or other synthetic
solvents; antibacterial agents such as benzyl alcohol or methyl
parabens; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as ethylenediaminetetraacetic acid; buffers
such as acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or dextrose. pH can
be adjusted with acids or bases, such as hydrochloric acid or
sodium hydroxide. A parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0088] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the selected particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In some cases, isotonic
agents are included, for example, sugars, or polyalcohols such as
manitol, sorbitol, or sodium chloride. Prolonged absorption of an
injectable composition can be effected by including in the
composition an agent which delays absorption, for example, aluminum
monostearate or gelatin.
[0089] Sterile injectable solutions can be prepared by
incorporating the active compound in the specified amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as needed, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and other ingredients selected from those enumerated above
or others known in the art. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0090] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Pharmaceutically compatible binding agents can be included as part
of the composition. The tablets, pills, capsules, troches and the
like can contain any of the following ingredients, or compounds of
a similar nature: a binder such as microcrystalline cellulose, gum
tragacanth or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0091] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser that contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0092] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0093] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0094] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0095] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the selected pharmaceutical carrier.
[0096] Toxicity and therapeutic efficacy of compounds disclosed
herein can be determined by pharmaceutical procedures known in the
art, for example, in cell cultures or experimental animals, e.g.,
for determining the LD50 (the dose lethal to 50% of the population)
and the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Compounds that exhibit high therapeutic indices are
preferred. While compounds that exhibit toxic side effects may be
used, care should be taken to design a delivery system that targets
such compounds to the site of affected tissue to minimize potential
damage to uninfected cells and, thereby, reduce side effects.
[0097] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0098] The invention also relates to a method of making a
medicament for use in treating a subject, e.g., for HCV infection.
Such medicaments can also be used for prophylactic treatment of a
subject at risk for or suspected of having an HCV infection.
Kits
[0099] The invention provides kits comprising a small interfering
RNA as described herein. In some embodiments, the kits also include
instructions for use in the methods for inhibiting viral gene
expression and/or methods for treatment of a viral infection in a
mammal described herein. Instructions may be provided in printed
form or in the form of an electronic medium such as a floppy disc,
CD, or DVD, or in the form of a website address where such
instructions may be obtained.
[0100] In some embodiments, the kits include a pharmaceutical
composition of the invention, for example including at least one
unit dose of at least one small interfering RNA such as a shRNA or
a siRNA, and instructions providing information to a health care
provider regarding usage for treating or preventing a viral
infection. The small interfering RNA is often included as a sterile
aqueous pharmaceutical composition or dry powder (e.g.,
lyophilized) composition.
[0101] Suitable packaging is provided. As used herein, "packaging"
refers to a solid matrix or material customarily used in a system
and capable of holding within fixed limits a composition of the
invention suitable for administration to an individual. Such
materials include glass and plastic (e.g., polyethylene,
polypropylene, and polycarbonate) bottles, vials, paper, plastic,
and plastic-foil laminated envelopes and the like. If e-beam
sterilization techniques are employed, the packaging should have
sufficiently low density to permit sterilization of the
contents.
[0102] Kits may also optionally include equipment for
administration of a pharmaceutical composition of the invention,
such as, for example, syringes or equipment for intravenous
administration, and/or a sterile solution, e.g., a diluent such as
water, saline, or a dextrose solution, for preparing a dry powder
(e.g., lyophilized) composition for administration.
TABLE-US-00001 TABLE 1 Listing of Targeting Sequences Disclosed in
the Application that can be Incorporated into shRNA or siRNA and
Examples of such shRNAs and siRNAs Target Examples of Sequence
Antisense sequence Position on shRNA or ID # (5'-3') HCV IRES siRNA
SEQ ID NO: 27 UCUUUGAGGUUUAGGAUUCGUGCUC 344-368 HCVa-wt shRNA SEQ
ID NO: 28 UCUUUGAGGUUUAGGAUUGGUGCUC 344-368 HCVa-SNP1 shRNA SEQ ID
NO: 29 UCUUUGAGCUUUAGGAUUCGUGCUC 344-368 HCVa-SNP2 shRNA SEQ ID NO:
30 UCUUUGAGCUUUAGGAUUGGUGCUC 344-368 HCVa-mut shRNA SEQ ID NO: 31
CCUCCCGGGGCACUCGCAAGCACCC 299-323 HCVb-wt shRNA SEQ ID NO: 32
UGGUGCACGGUCUACGAGACCUCCC 318-342 HCVc-wt shRNA SEQ ID NO: 33
GGUUUUUCUUUGAGGUUUAGGAUUC 350-374 HCVd-wt shRNA SEQ ID NO: 19
AGGUUUAGGAUUCGUGCUC 344-362 siRNA#1, shRNA#1 SEQ ID NO: 20
GAGGUUUAGGAUUCGUGCU 345-363 siRNA#2, shRNA#2 SEQ ID NO: 21
UGAGGUUUAGGAUUCGUGC 346-364 siRNA#3, shRNA#3 SEQ ID NO: 22
UUGAGGUUUAGGAUUCGUG 347-365 siRNA#4, shRNA#4 SEQ ID NO: 23
UUUGAGGUUUAGGAUUCGU 348-366 siRNA#5, shRNA#5 SEQ ID NO: 24
CUUUGAGGUUUAGGAUUCG 349-367 siRNA#6, shRNA#6 SEQ ID NO: 25
UCUUUGAGGUUUAGGAUUC 350-368 siRNA#7, shRNA#7
[0103] FIG. 16A-B illustrates examples of shRNAs containing
sequence targeting HCV IRES, and tested using methods described
herein.
EXAMPLES
[0104] The invention is further illustrated by the following
examples. The examples are provided for illustrative purposes only.
They are not to be construed as limiting the scope or content of
the invention in any way.
Example 1
Design and Construction of shRNA Expression Cassettes, T7
Transcription Reactions, and Reporter Gene Assays
[0105] Chemically synthesized oligonucleotides were obtained from
IDT (Coralville, Iowa), resuspended in RNase- and pyrogen-free
water (Biowhittaker), and annealed as described below. The
following oligonucleotide pairs, for making shRNA, contain a T7
promoter element (doubly underlined), sense and antisense HCV IRES
sequence and a miR-23 microRNA loop structure (reported to
facilitate cytoplasmic localization [21, 22]).
TABLE-US-00002 T7-HCVa-wt fw: (SEQ ID NO: 1)
5'-taatacgactcactatagggagcacgaatcctaaacctca
aagaCTTCCTGTCAtctttgaggtttaggattcgtgctcTT-3'; T7-HCVa-wt rev: (SEQ
ID NO: 2) 5'-AAgagcacgaatcctaaacctcaaagaTGACAGGAA
Gtctttgaggtttaggattcgtgct ccctatagtgagtcgtatta-3'
[0106] (T7 promoter sequence doubly underlined). T7 transcripts for
HCVa-mut shRNA were identical with the exception that nucleotide
changes (G.fwdarw.C and C.fwdarw.G) were incorporated into the
synthesized oligonucleotides at the singly underlined residues.
[0107] HCVa-wt shRNA (FIG. 1B) was designed to target the region
344-374 on the HCV IRES; HCVb-wt was designed to target the region
299-323 (FIG. 1C); HCVc-wt was designed to target the region
318-342 (FIG. 1C); and HCVd-wt was designed to target the region
350-374 (FIG. 1C).
[0108] ShRNAs #1-7 (targeting positions 344-362, 345-363, 346-364,
347-365, 348-366, 349-367, 350-368 on the HCV IRES; See FIG. 4A,
which depicts the 19 base pair viral recognition sequences) were in
vitro transcribed using the MEGAscript.RTM. kit (Ambion) and
contained the same loop sequences and 5',3'-overhangs as HCVa-wt
shRNA. SiRNAs #1-7 (see FIG. 4A, which depicts the 19 base pair
viral recognition sequences) were chemically synthesized at
Dharmacon (Lafayette, Colo.) and contained 3'-UU overhangs on both
sense and antisense strands.
[0109] The oligonucleotide pair used to prepare the control shRNA
229 (which targets tumor necrosis factor alpha) is
229-5'-TAATACGACTCACTATAGGGGCG
GTGCCTATGTCTCAGCCTCTTCTCACTTCCTGTCATGAGAAGAGGCTGAGACA TAGGCACCGCC
TT-3' (SEQ ID NO:3) and 229-3'-AAGGCG GTGCCTATGTC TCAGCC TCT TCTCA
TGACAGGAAG TGAGA AGAGGCTGA GACATAGGCACCCCTATAGTGAGTCGTATTA-5' (SEQ
ID NO:4).
Pol III U6 shRNA Expression Vector Construction--Design of Small
Hairpin shRNA Expression Vectors
[0110] Oligonucleotide pairs were incubated together at 95.degree.
C. for two minutes in RNA polymerase buffer (e.g., 120 .mu.l of
each 100 .mu.M oligonucleotide in 60 .mu.l 5.times. annealing
buffer (Promega; 1.times.=10 mM Tris-HCl (pH 7.5), 50 mM NaCl) and
slowly cooled (annealed) over 1 hour to less than 40.degree. C. The
oligonucleotides were designed to provide 4-base overhangs for
rapid cloning into Bbs1/BamH1-digested pCRII-U6 plasmid (Bbs1 and
BamH1 recognition sites or overhangs are underlined in the
oligonucleotide sequences). The pCRII-U6 pol III expression plasmid
was prepared by subcloning the PCR product obtained from human
HT-1080 genomic DNA using primers and huU6-5'
ATCGATCCCCAGTGGAAAGACGCGCAG (SEQ ID NO:5) and
huU6-3'-GGATCCGAATTCGAAGACCACGGTGTTTCGTCCTTTCCACAA-5'
[0111] (SEQ ID NO:6) [23] into the pCRII vector (Invitrogen) using
the TA cloning kit (Invitrogen). The cassette consisting of the
annealed oligonucleotides (encoding the HCV IRES shRNA) was ligated
into the Bbs1/BamH1-digested pCRII-U6 plasmid. The expressed shRNA
contains a miR-23 microRNA loop structure to facilitate cytoplasmic
localization [21, 22]. The final pCRII-U6 constructs were confirmed
by sequencing. The primers pairs used were: pHCVa-wt 5'-ACCG
GAGCACGAATCCTAAACCTCAAAGA CTTCCTGTCA TCTTTGAGGTTTAGGATTCGTGCTC
TTTTTTG-3' (SEQ ID NO:7) and 5'-GATCCAAAAAA
GAGCACGAATCCTAAACCTCAAAGA TGACAGGAAG TCTTTGAGGTTTAGGATTCGTGCTC-3'
(SEQ ID NO:8). Oligonucleotides containing the appropriate sequence
changes at the underlined residues (see above) were used to
generate the pCRII-U6/HCVa-mut (double mutation), HCVsnp1 (single
change at 5' side) and HCVsnp2 (single change at 3' end) as
depicted in FIG. 1B and described above. The control pCRII-U6/229
was prepared is similar fashion using the oligonucleotides
TABLE-US-00003 (SEQ ID NO: 9) 5'-ACCGGGCG
GTGCCTATGTCTCAGCCTCTTCTCACTTCCTGTCATGA
GAAGAGGCTGAGACATAGGCACCGCCTTTTTT-3' and (SEQ ID NO: 10) 3'-
GATCAAAAAAGGCGGTGCCTATGTCTCAGCCTCTTCTCATGACAGGAAGT
GAGAAGAGGCTGAGACATAGGCACCGCC-5'.
T7 Transcription Reactions
[0112] Oligonucleotide pairs were incubated at 95.degree. C. for
two minutes in RNA polymerase buffer (e.g., 120 .mu.l of each 100
.mu.M oligonucleotide in 60 .mu.l 5.times. transcription buffer
(Promega)) and slowly cooled (annealed) over 1 hour to less than
40.degree. C. ShRNA was transcribed at 42.degree. C. for four hours
from 5 .mu.M of the resulting annealed double-stranded DNA template
using the AmpliScribe.TM. T7 Flash transcription kit (Epicentre
Technologies) followed by purification on a gel filtration spin
column (Microspin.TM. G-50, Amersham Biosciences) that had been
thoroughly washed three times with phosphate buffered saline (PBS)
to remove preservative.
siRNAs
[0113] siRNAs were prepared by annealing chemically synthesized
(Dharmacon) complementary strands of RNA, each containing the
appropriate recognition sequence plus an (overhanging) UU extension
on the 3'end.
Transfections and Reporter Gene Assays
[0114] Human 293FT (Invitrogen) and Huh7 cells (American Type
Culture Collection (ATCC), Manassas, Va.) were maintained in DMEM
(Biowhittaker.RTM.) with 10% fetal bovine serum (HyClone),
supplemented with 2 mM L-glutamine and 1 mM sodium pyruvate. The
day prior to transfection, cells were seeded at 1.7.times.10.sup.5
cells/well in a 24-well plate, resulting in about 80% cell
confluency at the time of transfection. Cells were transfected with
Lipofectamine.TM. 2000 (Invitrogen, Carlsbad, Calif.) following the
manufacturer's instructions. For the inhibition experiments, 293FT
or Huh7 cells were cotransfected (in triplicate) with 40 ng
pCDNA3/HCV IRES dual luciferase (renilla and firefly) reporter
construct, 50 ng pSEAP2-control plasmid (BD Biosciences Clontech,
as transfection controls) and the indicated amounts of T7-generated
shRNA (typical amount 1 pmole) or pCRII-U6 shRNA expression
construct (710 ng). Compensatory pUC18 plasmid was added to the
transfection mix to give a final concentration of 800 ng total
nucleic acid per transfection. Forty-eight hours later, supernatant
was removed, heated at 65.degree. C. for 15-30 minutes, and 5-10
.mu.l of the supernatant was added to 150 .mu.l p-nitrophenyl
phosphate liquid substrate system (pNPP, Sigma). After a 30-60
minute incubation at room temperature, samples were read (405 nm)
on a Molecular Devices Thermomax microplate reader and quantitated
using SOFTmax software (Molecular Devices). The remaining cells
were lysed and luciferase activity measured using the
Dual-Luciferase Reporter assay system (Promega) and MicroLumat LB
96 P luminometer (Berthold).
Mice
[0115] Six-week old female Balb/c mice were obtained from the
animal facility of Stanford University. Animals were treated
according to the NIH Guidelines for Animal Care and the Guidelines
of Stanford University.
Mouse Hydrodynamic Injections and In Vivo Imaging
[0116] Hydrodynamic tail vein injections were performed as
described by McCaffrey and colleagues with minor modifications
including omission of RNasin [24]. A total volume of 1.8 ml of
phosphate-buffered saline containing inhibitor (RNA or plasmid), 10
.mu.g of pHCV Dual Luc plasmid, and 2 .mu.g pSEAP2-control plasmid
(BD Biosciences Clontech, contains the SV40 early promoter), was
steadily injected into the mouse tail vein over about five seconds
(N=4-6 animals per group). At the indicated times, 100 of 30 mg/ml
luciferin was injected intraperitoneally. Ten minutes following the
injection, live anesthetized mice were analyzed using the IVIS7
imaging system (Xenogen Corp., Alameda, Calif.) and the resulting
light emission data quantitated using LivingImage software
(Xenogen). Raw values are reported as relative detected light per
minute and standard errors of the mean for each group (N=4-5
animals) are shown.
Secreted Alkaline Phosphatase (SEAP) Assay
[0117] At day 5, mice were bled through the retro-orbital vein of
the eye. The serum was separated from blood cells by
microcentrifugation, heated at 65.degree. C. for 30 minutes to
inactivate endogenous alkaline phosphates, and 5-10 .mu.l of the
serum was added to 150 .mu.l pNPP liquid substrate system (see
above). After a 30-60 minute incubation at room temperature,
samples were read (405 nm) and quantitated as described above.
Example 2
shRNA Inhibition of HCV IRES-Mediated Gene Expression in Human
Tissue Culture Cells
[0118] In this study, short interfering RNAs (shRNAs and siRNAs)
designed and constructed as in Example 1 to target a conserved
region of the hepatitis C IRES were tested for their ability to
inhibit HCV IRES-mediated reporter expression in human tissue
culture cells.
[0119] FIG. 1A shows the HCV IRES target site (panel A) as well as
the HCV shRNA resulting from T7 transcription of a template
prepared from hybridized oligonucleotides containing a T7 promoter
sequence and HCV IRES target (FIG. 1B). The underlined residues are
those that were changed to generate the mutant HCV shRNAs. The
shRNAs contain a mir-23 microRNA loop structure that was previously
suggested to facilitate cytoplasmic localization [21, 22] and a 25
base pair RNA stem with two nucleotides at the 5' (two guanines)
and 3' (two uridines) ends that may also hybridize though non
Watson-Crick G:U base pairings. For vector-delivered shRNAs,
overlapping oligonucleotides were subcloned into a poIII expression
vector (pCRII-U6, see Example 1).
[0120] Three other shRNAs were also designed with the same stem
length and loop sequence that target nearby positions in Domain IV
of the HCV IRES (FIG. 1C). HCVb-wt shRNA targets a highly
structured region (used as negative control, to compare
efficiency), while HCVc-wt and HCVd-wt shRNA target regions that
are more `accessible` according to biochemical footprinting studies
(FIG. 1D; Brown et al., Nucleic Acids Res., 1992, 20:5041-5). All
RNAs were in vitro transcribed from dsDNA templates containing a T7
promoter, similar to the HCVa-wt shRNA.
[0121] To test the effectiveness of the HCV shRNAs to inhibit HCV
IRES-mediated gene expression, human 293FT or hepatocyte Huh7 cells
were co-transfected with pCDNA3/HCV IRES dual luciferase expression
plasmid, secreted alkaline phosphatase expression plasmid (pSEAP2,
to control for efficiency of transfection) as well as in vitro
synthesized shRNAs or alternatively, pol III expression vectors
containing the corresponding shRNA cassettes.
[0122] As seen in FIG. 1F, both HCVa-wt and HCVd-wt shRNAs, which
target the region of the IRES immediately downstream of the AUG
translation start site (positions 344-368 and 350-374,
respectively), strongly inhibit HCV IRES-mediated fLuc expression
in human 293FT cells. HCVc-wt (targeting 318-342) showed moderate
inhibition and HCVb-wt (299-323) displayed little if any activity,
as expected. Thus, preliminary screening revealed a potent shRNA,
HCVa-wt, that was chosen for further detailed studies.
Specificity and Potency of Inhibition of HCV IRES-Mediated Gene
Expression by shRNAs in 293FT Cells
[0123] To further test inhibition of HCV-IRES driven gene
expression, 293FT cells were cotransfected with dual luciferase
reporter and SEAP expressing plasmids as well as 1 pmole of in
vitro transcribed shRNAs. The target plasmid was pCDNA3/HCV IRES
dual luciferase reporter (HCV IRES, as shown in FIG. 1E). pUC18
plasmid was added to the transfection mix to give a final total
nucleic acid concentration of 800 ng per transfection per well
(24-well tissue culture plates). Forty-eight hours later,
supernatant was removed for SEAP analysis, then cells were lysed
and firefly and renilla (not shown) luciferase activity measured as
described in Example 1. Firefly luciferase and SEAP activities were
normalized to 100. Results are shown in FIG. 2A.
[0124] HCVa-wt shRNA targeting the region of the IRES immediately
downstream of the AUG translation start site strongly inhibited HCV
IRES-mediated fLuc expression in both human 293FT (FIG. 2) and
hepatocyte Huh7 (FIG. 3B) cell lines. Little or no inhibition was
observed using either a mutant shRNA (HCVa-mut) containing two
changes in the pairing of the RNA hairpin (for mismatch location,
see FIG. 1B) or an unrelated TNF (229) shRNA. The 229 TNF shRNA is
highly effective at inhibiting TNF expression (Seyhan et al., RNA,
2005, 11:837-846), suggesting that this shRNA is utilized
effectively by the RNAi apparatus. Single nucleotide changes in the
hairpin region, at either the upstream or downstream position (SNP1
and SNP2 respectively; see FIG. 2C), had a partial effect.
[0125] Little or no inhibition was observed when the HCV shRNA was
targeted to a similar dual luciferase construct in which the HCV
IRES was replaced by the encephalomyocarditis virus (EMCV) IRES
(FIGS. 2B and 3B). Thus, the data of FIG. 2B illustrate that
HCVa-wt shRNA does not inhibit a similar target lacking the HCV
IRES. In this experiment, cells were transfected as for FIG. 2A
except that pCDNA3/EMCV dual luciferase reporter (EMCV IRES) was
used as target in place of pCDNA3/HCV. These data are presented in
FIG. 2B as luciferase activity divided by SEAP activity normalized
to 100.
[0126] To confirm that the shRNAs were acting by degrading their
target mRNA, a Northern blot analysis was performed (FIG. 2F).
Equal amounts of total RNA, isolated from cells transfected with no
inhibitor or HCVa-wt, HCVmut1/2, or 229 shRNAs, were separated by
gel electrophoresis. The separated RNA was transferred to a
membrane and hybridized to radiolabeled cDNA probes specific for
fLuc, SEAP and elongation factor 1A (EF1A). HCVa-wt shRNA (lane 3)
specifically inhibited fLuc mRNA accumulation (63% inhibition
compared to 229 shRNA (lane 2) when corrected for SEAP and EF1A
mRNA levels; no inhibition was observed for HCVa-mut1/2) (compare
lanes 3 and 4) following quantitation by phosphorimager. These data
demonstrate that the shRNAs were degrading target mRNA.
[0127] Dose response experiments showed that the HCVa-wt shRNA
effectively inhibited HCV IRES-dependent gene expression at 0.3 nM
in 293FT cells (96 percent inhibition, see FIG. 2D) and 0.1 nM in
Huh7 cells (75 percent inhibition, see FIG. 3A).
[0128] To further investigate local sequence effects on potency,
seven in vitro-transcribed 19 bp shRNA and the corresponding
synthetic 19 base pair siRNA, targeting all possible positions
within the 31-base pair site of HCVa (344-374; FIG. 4A), were
assayed for inhibitory activity. A 25base pair synthetic siRNA
corresponding to HCVa-wt shRNA was also tested. All of them
exhibited a high level of activity (FIG. 4B). The most potent were
siRNA and shRNA versions of HCVa as well as siRNA #3, which was
effective at 1 nM (>90% inhibition, FIG. 4B) and 0.1 nM (about
90% inhibition, FIG. 4C). Thus, 19-25 base pair shRNAs and siRNAs
designed to target the region 344-374 on the HCV IRES are potent,
with some local differences.
Example 3
shRNA Inhibition of HCV IRES-Mediated Gene Expression in a Mouse
Model System
[0129] The ability of the HCV shRNA and HCV shRNA expression
plasmid to inhibit target gene expression was extended to a mouse
model system using hydrodynamic injection to deliver the nucleic
acids to mouse liver. FIG. 5 shows the results of injecting a large
volume of PBS (1.8 ml) containing pHCV dual Luc, pSEAP2, and shRNAs
(10 fold excess over the target on a mass basis of either shRNA or
pol III expression vectors expressing the shRNAs) into the tail
veins of mice (n=4-5 mice). At the time points shown in FIG. 5B,
luciferin was injected intraperitoneally and the mice were imaged
with a high sensitivity, cooled CCD camera. (FIG. 5A shows
representative mice chosen from each set (4-5 mice per set) at the
84 hour time point.) At all time points tested, HCV shRNA robustly
inhibited luciferase expression ranging from 98% (84 hour time
point) to 94% (48-hour time point) inhibition compared to mice
injected with pUC18 in place of shRNA inhibitor. Mutant (mut) or
control (229) shRNAs had little or no effect. It should be noted
that luciferase activity decreases with time, possibly due to loss
of DNA or promoter silencing [8] and that the data are normalized
within each time point (see description of FIG. 5 above).
[0130] FIG. 6 shows a comparison of HCVa-wt shRNA inhibitory
activity with a phosphoramidite morpholino oligomer that was
previously shown to effectively target this same site [8]. Both the
HCVa-wt shRNA and morpholino oligomers effectively blocked
luciferase expression at all time-points tested. Data are shown for
the 48-hour time-point, where inhibition was 99.95 and 99.88
percent, respectively for the HCVa-wt shRNA and morpholino
inhibitors.
Example 4
Inhibition of Semliki Forest Virus (SFV) Using shRNAs
[0131] SFV has been used as a model system for more virulent
positive-strand RNA viruses. To examine the inhibitory effect of
RNAi on SFV growth, shRNAs targeting four SFV genes (nsp-1, nsp-2
and nsp-4, and capsid) and one mismatched control for the nsp-4
site were generated and expressed from a U6 promoter. Their ability
to tested their ability to inhibit the proliferation of
SFV-A7-EGFP, a version of the replication-proficient SFV strain
SFV-A7 that expresses a eGFP reporter gene [49]. A modest reduction
(about 35%) of SFV-GFP replication was seen with shRNAs targeting
the nsp-1 (FIG. 7) but not nsp-2, nsp-4 or capsid coding regions,
nor with the mismatched siRNA (not shown).
[0132] A site within the capsid coding region that was previously
shown to be effective on Sindbis virus [50] was not effective on
SFV. The Sindbis-SFV sequence homology at this site is only 77%.
SFV is a very rapidly growing virus, producing up to 200,000
cytoplasmic RNAs during its infectious cycle. To see if cells could
better protected from a slower-growing virus, the effects of these
siRNAs on a replication-deficient strain of SFV-GFP were tested in
two separate experiments. FIG. 8 shows that U6-expressed shRNAs
targeting this SFV strain can reduce viral expression by
.gtoreq.70% over a time period of up to five days. This effect was
seen with siRNAs targeting the nonstructural genes nsp-1, nsp-2,
and nsp-4 as well as an siRNA with one mismatch to nsp-4, but not
for the capsid gene (which is lacking in this crippled virus) or
other controls (FIG. 8). Note that the length of the sequence
targeted by the shRNAs is 29 nucleotides and the single mismatch
used in the nsp-4 mismatch shRNA is apparently not disruptive for
the RNAi effect. The wide variation in effectiveness of the various
shRNAs underscores the importance of a library approach for finding
the best siRNAs and shRNAs when dealing with rapidly replicating
and highly mutagenic viruses such as SFV.
[0133] Dose-response experiments were performed to examine
inhibition of an HCV replicon system in Huh7 cells by HCVa-wt shRNA
and HCVa-mut shRNA as well as a non-specific control shRNA (229).
The antiviral activity of test compounds was assayed in the stably
HCV RNA-replicating cell line, AVA5, derived by transfection of the
human hepatoblastoma cell line, Huh7 (Blight, et al., Science,
2000, 290:1972). RNA-based inhibitors were co-transfected with
DsRed expression plasmid into cultures that were about 80 percent
confluent. HCV RNA levels were assessed 48 hours after transfection
using dot blot hybridization. Assays were conducted in triplicate
cultures. A total of 4-6 untreated control cultures, and triplicate
cultures treated with 10, 3, and 1 IU/ml .alpha.-interferon (active
antiviral with no cytotoxicity), and 100, 10, and 1 uM ribavirin
(no antiviral activity and cytotoxic) served as positive antiviral
and toxicity controls. The transfection efficiency was estimated by
fluorescence microscopy (DsRed expression). Both HCV and b-actin
RNA levels in triplicate treated cultures were determined as a
percentage of the mean levels of RNA detected in untreated cultures
(6 total). Beta-actin RNA levels are used both as a measure of
toxicity, and to normalize the amount of cellular RNA in each
sample. A level of 30% or less HCV RNA (relative to control
cultures) is considered to be a positive antiviral effect, and a
level of 50% or less b-actin RNA (relative to control cultures) is
considered to be a cytotoxic effect. Cytotoxicity is measured using
an established neutral red dye uptake assay (Korba, B. E. and J. L.
Gerin (1992). Use of a standardized cell culture assay to determine
activities of nucleoside analogs against hepatitis B virus
replication (Antivir. Res. 19:55-70).
[0134] ]Inhibition of an HCV replicon system in Huh7 cells by
HCVa-wt shRNA and HCVa-mut shRNA as well as an irrelevant control
shRNA (229); dose response. The antiviral activity of test
compounds was assayed in the stably HCV RNA-replicating cell line,
AVA5, derived by transfection of the human hepatoblastoma cell
line, Huh7 (Blight et al. Science, 2000, 290:1972). RNA-based
inhibitors were co-transfected with DsRed expression plasmid into
.about.80 percent confluent cultures and HCV RNA levels were
assessed 48 hours after transfection using dot blot hybridization.
Assays were conducted in triplicate cultures. A total of 4-6
untreated control cultures, and triplicate cultures treated with
10, 3, and 1 IU/ml a-interferon (active antiviral with no
cytotoxicity), and 100, 10, and 1 uM ribavirin (no antiviral
activity and cytotoxic) served as positive antiviral and toxicity
controls. The transfection efficiency was estimated by fluorescence
microscopy (DsRed expression). Both HCV and beta-actin RNA levels
in triplicate treated cultures were determined as a percentage of
the mean levels of RNA detected in untreated cultures (6 total).
Beta actin RNA levels were used both as a measure of toxicity, and
to normalize the amount of cellular RNA in each sample. A level of
30% or less HCV RNA (relative to control cultures) was considered
to be a positive antiviral effect, and a level of 50% or less
beta-actin RNA (relative to control cultures) was considered to be
a cytotoxic effect. Cytotoxicity was measured using an established
neutral red dye uptake assay (Korba et al., Antiviral Res., 1992,
19:55-70). Use of a standardized cell culture assay to determine
activities of nucleoside analogs against hepatitis B virus
replication (Korba et al., 1992 supra).
Example 5
Identification of shRNAs that Inhibit HCV IRES-Dependent Gene
Expression in Tissue Culture Cells
[0135] The ability of in vitro-transcribed small hairpin RNAs
(shRNAs) to inhibit hepatitis C virus internal ribosome entry site
(HCV IRES)-dependent gene expression in cultured cells was
investigated. As disclosed supra, a 25 base pair shRNA HCVa-wt that
targets the 3' end of the HCV IRES, near the AUG translation start
site (Table 2) was found to be effective for disrupting expression
of HCV. To assess the ability of co-transfected shRNA constructs to
interfere with the function of the IRES, a reporter construct (pHCV
Dual Luciferase plasmid) in which firefly luciferase (fLuc)
expression is dependent on the HCV IRES was used (FIG. 1; Wang et
al., Mol. Ther., 2005, 12:562-568. In these experiments, 293FT
cells were cultured and transfected with a reporter construct and
HCVa-wt or one of the other test sequences as described in Wang et
al., 2005, supra.
[0136] It was found that at a concentration of 1 nM, HCVa-wt caused
90% inhibition of HCV IRES-dependent luciferase expression in 293FT
cells (Wang et al., 2005, supra). In subsequent experiments, 26
additional shRNAs targeting various regions of the HCV IRES were
designed and tested (FIG. 10, FIG. 16A-B); 3 of the 26 were
duplicates of those described above (HCVb, HCVc, HCVd-wt); 23 were
new sequences) to identify additional inhibitors of HCV. The goal
was to identify shRNAs that can be used either in combination with
HCVa-wt), making it harder for the virus to develop resistance by
mutating the HCVa-wt target site, or as alternatives to HCVa-wt.
The shRNAs to be tested were chosen to avoid regions that vary
among different HCV genotypes. Some test sequences were selected
using the algorithm available at (e.g.,
jura.wi.mit.edu/bioc/siRNAext/, and other test sequences
intentionally targeted HCV-IRES sequences that, due to their CG
content and other characteristics, would not be recommended by most
algorithms would rule out, such as GC-rich or highly structured
regions. The shRNAs were generated by in vitro transcription from
dsDNA templates using T7 RNA polymerase and, to promote
transcription efficiency, began with the sequence 5'-pppGGG. This
5' sequence formed an overhang of two to three nucleotides, the
exact length depending on whether the target site contains one or
more guanosine residues at its 5' end (see FIG. 16A-B). If the last
nucleotide of the RNA sense strand matching a target sequence was
`G,` only two more Gs had to be added for efficient transcription,
and those Gs are single-stranded on the 5'-end of the shRNA, not
complimentary to the target. If the last nucleotide of the shRNA
sense strand matching the target, was not a G, then for efficient
transcription in the test systems, three Gs had to be added that
were not complimentary to the target. All shRNAs tested in this set
of experiments had a duplex stem length of 21-25 base pairs and a
10 nucleotide loop derived from microRNA-23, as described for
HCVa-wt.
[0137] All of the shRNAs (27 total, including HCVa-wt were assayed
for activity as described in Wang, 2005. Briefly, human 293FT cells
were co-transfected with pHCV Dual Luciferase.RTM. Reporter
expression plasmid (Promega, Madison, Wis.), and a secreted
alkaline phosphatase expression plasmid (pSEAP2, Clontech, Mountain
View, Calif.) to control for efficiency of transfection and
possible off-target effects), and shRNA. Results are shown in FIG.
10. SEAP levels were uniform in all samples, indicating efficient
transfection and the absence of nonspecific inhibitory or toxic
effects, at shRNA concentrations of 1 nM to 5 nM. Most of the
shRNAs displayed only moderate activity (less than 60% inhibition
at 1 nM). Without committing to any particular theory, this effect
is likely because the targeted areas on IRES are highly structured.
The exceptions were HCVd-wt, sh37, sh39, hcv17, which target the
IRES positions near the HCVa-wt site. These shRNAs caused 85-90%
inhibition of HCV IRES dependent gene expression at 1 nM
concentration. The low shRNA concentration of 1 nM was chosen to
allow easy identification of hyper-functional shRNAs. If the
screening were performed at 10 nM shRNA, more shRNAs would display
high activity; however, significant nonspecific inhibition was seen
at that concentration in some cases. Thus, the screening revealed a
44 nucleotide region (positions 331-374 on the HCV IRES) where five
overlapping shRNAs display high activity.
Example 6
Effect of Single Base Mismatches on shRNA Activity
[0138] It is desirable that a treatment for HCV be effective
against mutated HCV. To determine the performance of the RNAs
described herein in this regard (e.g., shRNAs targeting HCV IRES),
and to address whether off-target effects are problematic, the
sensitivity of selected shRNA directed against HCV IRES to point
mutations in the target sequence was tested. For these experiments,
a C340U mutation was introduced in the HCV IRES using the
QuikChange.RTM. II Site-Directed Mutagenesis Kit (Stratagene, La
Jolla, Calif.). Of the 27 shRNAs that were assayed, nine targeted
the mutated region (FIG. 11), therefore their activity could
theoretically be affected by this mutation. All of these shRNAs
were assayed with the mutated version of pHCV, along with selected
shRNAs targeting other sites as controls. For all tested shRNAs,
activity was found to be unaffected or slightly decreased compared
to the activity of original, perfectly matched target (FIG.
10).
[0139] However, in the replicon system, shRNAs were surprisingly
found to be SNP-sensitive (see below).
Example 7
Fine Mapping of Target Sites
[0140] Six short 19 base pair shRNAs were designed to target a 44
nucleotide site near the 3'-terminus of the HCV IRES: three
targeting nucleotides 331-353 and three targeting nucleotides
354-374. These molecules contained 10 nucleotide loops and 5'-GG
and 3'-UU overhangs. Screening was performed to identify of
non-overlapping candidates that were most effective among those
sequences tested for inhibition of HCV expression. All six of the
shRNAs tested were able to inhibit activity in the assay system.
Three of the six shRNAs (sh52, sh53, and sh54) were identified as
the most effective (FIG. 12). This does not preclude the use of
those shRNAs that were less effective in a composition, e.g., for
treating HCV, for example as part of a composition that includes
more than one shRNA and/or siRNA.
Example 8
shRNA Design: Effects of Stem Length, Loop Length and Sequence, and
3'-Terminus
[0141] Additional experiments were performed to test how shRNA
design affects gene silencing activity. HCVa-wt contained a 25 base
pair stem with 5'-GG and 3'-UU overhangs (which may form
non-canonical base pairs) and a ten nucleotide miR-23 loop. To test
the importance of these parameters in the effectiveness for
inhibition of expression, each of these parameters was separately
varied (FIG. 13A). The microRNA-23 loop sequence was initially
selected because it is a naturally occurring sequence
(Lagos-Quintana et al., Science, 2001, 293:854-258) and was
therefore unlikely to be toxic. Two alternative ten nucleotide
loops were tested, along with loops of six nucleotides, five
nucleotides, and four nucleotides, each in two versions of a
sequence. Neither loop size nor sequence was found to affect the
activity of these 25 base pair shRNAs (FIG. 13B; see FIG. 16A-B for
sequences).
[0142] Small hairpin RNAs lacking the 3'-UU terminal sequence
(single-stranded overhang) had the same efficacy as the parental
shRNA containing this feature. Control shRNA with full-length (25
nucleotide) sense but short (13 nucleotide) antisense regions had
no activity, confirming the importance of duplex structure in the
targeting sequence. shRNAs having a 3'-CC instead of 3'-UU terminus
(allowing formation of 2 additional Watson-Crick base pairs) were
more effective than HCVa-wt for decreasing HCV expression, but also
affected SEAP levels. This nonspecific inhibition could be a
consequence of the longer stem (27 base pairs), which can induce
genes of the interferon responsive pathway and activate protein
kinase R (PKR). Surprisingly, moving the loop to the other end of
the shRNA resulted in a dramatic reduction of activity (15%
inhibition at 1 nM instead of 90%). Possible explanations for this
effect include a shift in the position of Dicer processing (and
therefore the sequence targeted) as well as a different GC content
at the 5'-end.
[0143] Because 19 base pair shRNAs were shown to display potency
similar to 25 base pair shRNA, the effects of loop variations for
19 base pair shRNAs were examined. The results are shown in FIG.
14; see FIG. 16A-B for sequences. Loop sizes of 10, 6, 5, and 4
nucleotides were tested, each in two sequence versions. Sequences
containing all loop sizes demonstrated the ability to inhibit gene
expression. However, in contrast to the results with 25base pair
shRNAs, reduction of loop size, especially below 5 nucleotides,
resulted in reduced activity for the 19 base pair shRNAs for both
loop sequences tested. Loops of at least 5-6 nucleotides
demonstrated the most activity.
[0144] Removal of the 3'-UU also resulted in dramatic reduction of
activity for 19 base pair as well as 20 base pair (but not 25 base
pair) shRNAs. Without committing to any particular theory, the
3'-UU and 5'-GG may form non-canonical base pairs and the overall
size of shRNA duplex is important such that the duplex cannot be
less than 21 base pairs for efficient processing. Thus, for 25 base
pair shRNAs, neither the size of the loop nor the presence of a
3'-UU matters, whereas these parameters are important for potency
of short, e.g., 19 base pair shRNAs. Without committing to any
particular theory, it may be that Dicer binds at the termini prior
to processing and does not "sense" the loop in the case of longer
shRNAs, but for 19 base pair shRNAs the loop is "felt" as Dicer
"measures" 19-21 nucleotides from the ends.
[0145] Accordingly, it was found that 19 base pair shRNAs can be as
potent as 25 base pair shRNAs and 19 base pair siRNA. It was also
found that some shRNA molecules were active at low concentrations
of 0.1-1 nM ("hyper-potent shRNAs." Other groups typically use
10-25-50-100 nM siRNA).
[0146] These data demonstrate that sequences that do not include a
3'-UU that are at least 22 base pairs, e.g., 23 base pairs, 24 base
pairs, or 25 base pairs, can be suitable for inhibition of HCV
expression. Similarly, loop size is not critical for shRNAs that
are at least 22 base pairs in length.
Example 9
HCV Replicon System
[0147] A number of shRNA and siRNA inhibitors along with negative
controls were used to transfect human hepatocytes (AVA5, a
derivative of the Huh7 cell line) stably expressing HCV subgenomic
replicons (Blight et al., Science, 2000, 290:5498), and the amount
of HCV expression was determined. A range of concentrations was
tested and the concentration of RNA resulting in 50% inhibition
(IC50 or EC50) was determined. IC50s from two independent
experiments are shown side-by-side in FIG. 15. The results
generally correlated with the data obtained using the fLuc/IRES
system in 293 FT cells, with the following differences: (1) 19 base
pair shRNAs are more potent than 19 base pair siRNAs in the
replicon system, whereas with the reporter system, 19 base pair
siRNAs were more potent than shRNAs; (2) shRNA HCVa-wt with point
mutations did not demonstrate activity in the replicon system,
while it was effective in fLuc/IRES reporter system; (3) in
general, 25 base pair siRNA and 25 base pair shRNA had less
activity than other 19 base pair shRNAs and siRNAs tested. In
general, the IRES and replicon systems are useful for
identification of candidate sequences. Methods of confirming the
efficacy (e.g., for inhibiting expression of HCV in a subject) of a
selected shRNA or siRNA can be further tested using methods
described herein and methods known in the art.
Other Embodiments
[0148] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
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Sequence CWU 1
1
113181DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1taatacgact cactataggg agcacgaatc
ctaaacctca aagacttcct gtcatctttg 60aggtttagga ttcgtgctct t
81281DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2aagagcacga atcctaaacc tcaaagatga
caggaagtct ttgaggttta ggattcgtgc 60tccctatagt gagtcgtatt a
81389DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 3taatacgact cactataggg gcggtgccta
tgtctcagcc tcttctcact tcctgtcatg 60agaagaggct gagacatagg caccgcctt
89486DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 4attatgctga gtgatatccc cacggataca
gagtcggaga agagtgaagg acagtactct 60tctccgactc tgtatccgtg gcggaa
86527DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 5atcgatcccc agtggaaaga cgcgcag
27642DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 6aacacctttc ctgctttgtg gcaccagaag
cttaagccta gg 42771DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 7accggagcac gaatcctaaa
cctcaaagac ttcctgtcat ctttgaggtt taggattcgt 60gctctttttt g
71871DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 8gatccaaaaa agagcacgaa tcctaaacct
caaagatgac aggaagtctt tgaggtttag 60gattcgtgct c 71978DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9accgggcggt gcctatgtct cagcctcttc tcacttcctg
tcatgagaag aggctgagac 60ataggcaccg cctttttt 781078DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10ccgccacgga tacagagtcg gagaagagtg aaggacagta
ctcttctccg actctgtatc 60cgtggcggaa aaaactag 7811393RNAHepatitis C
Virus 11gccagccccc gauugggggc gacacuccac cauagaucac uccccuguga
ggaacuacug 60ucuucacgca gaaagcgucu agccauggcg uuaguaugag ugucgugcag
ccuccaggac 120ccccccuccc gggagagcca uaguggucug cggaaccggu
gaguacaccg gaauugccag 180gacgaccggg uccuuucuug gaucaacccg
cucaaugccu ggagauuugg gcgugccccc 240gcgagacugc uagccgagua
guguuggguc gcgaaaggcc uugugguacu gccugauagg 300gugcuugcga
gugccccggg aggucucgua gaccgugcac caugagcacg aauccuaaac
360cucaaagaaa aaccaaacgu aacaccaacc gcc 3931264RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 12gggagcacga auccuaaacc ucaaagacuu ccugucaucu
uugagguuua ggauucgugc 60ucuu 641364RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13gggagcacca auccuaaacc ucaaagacuu ccugucaucu
uugagguuua ggauuggugc 60ucuu 641464RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14gggagcacga auccuaaagc ucaaagacuu ccugucaucu
uugagcuuua ggauucgugc 60ucuu 641564RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 15gggagcacca auccuaaagc ucaaagacuu ccugucaucu
uugagcuuua ggauuggugc 60ucuu 641660RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 16gggugcuugc gagugccccg ggaggcuucc ugucaccucc
cggggcacuc gcaagcaccc 601760RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 17gggaggucuc
guagaccgug caccacuucc ugucauggug cacggucuac gagaccuccc
601862RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 18gggaaugcua aaccucaaag aaaaacccuu
ccugucaggu uuuucuuuga gguuuacgau 60uc 621919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 19cucgugcuua ggauuugga 192019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20ucgugcuuag gauuuggag 192119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 21cgugcuuagg auuuggagu 192219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22gugcuuagga uuuggaguu 192319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 23ugcuuaggau uuggaguuu 192419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 24gcuuaggauu uggaguuuc 192519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 25cuuaggauuu ggaguuucu 192631RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26gagcacgaau ccuaaaccuc aaagaaaaac c
312725RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 27ucuuugaggu uuaggauucg ugcuc
252825RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 28ucuuugaggu uuaggauugg ugcuc
252925RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 29ucuuugagcu uuaggauucg ugcuc
253025RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 30ucuuugagcu uuaggauugg ugcuc
253125RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 31ccucccgggg cacucgcaag caccc
253225RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 32uggugcacgg ucuacgagac cuccc
253325RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 33gguuuuucuu ugagguuuag gauuc
253423RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 34cucacagggg agugaucuau ggu
233525RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 35aguaguuccu cacaggggag ugauc
253625RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 36cuuucugcgu gaagacagua guucc
253722RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 37auggcuagac gcuuucugcg ug
223822RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 38cuaacgccau ggcuagacgc uu
223925RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 39ucauacuaac gccauggcua gacgc
254025RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 40acgacacuca uacuaacgcc auggc
254125RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 41ccgguuccgc agaccacuau ggcuc
254225RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 42caauuccggu guacucaccg guucc
254322RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 43cauugagcgg guugauccaa ga
224421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 44cuccaggcau ugagcggguu g
214525RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 45agucucgcgg gggcacgccc aaauc
254625RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 46cuuucgcgac ccaacacuac ucggc
254725RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 47acccuaucag gcaguaccac aaggc
254822RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 48cgcaagcacc cuaucaggca gu
224923RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 49uggugcacgg ucuacgagac cuc
235021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 50uggugcacgg ucuacgagac c
215124RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 51cucauggugc acggucuacg agac
245221RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 52cucauggugc acggucuacg a
215325RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 53uucgugcuca uggugcacgg ucuac
255425RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 54ggauucgugc ucauggugca cgguc
255525RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 55uuuaggauuc gugcucaugg ugcac
255623RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 56uuuaggauuc gugcucaugg ugc
235759RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 57gggaccauag aucacucccc ugugagcuuc
cugucacuca caggggagug aucuauggu 595862RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 58gggaucacuc cccugugagg aacuacucuu ccugucaagu
aguuccucac aggggaguga 60uc 625961RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 59gggaacuacu
gucuucacgc agaaagcuuc cugucacuuu cugcgugaag acaguaguuc 60c
616057RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 60gggcacgcag aaagcgucua gccaucuucc
ugucaauggc uagacgcuuu cugcgug 576157RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 61gggaagcguc uagccauggc guuagcuucc ugucacuaac
gccauggcua gacgcuu 576262RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 62gggcgucuag
ccauggcguu aguaugacuu ccugucauca uacuaacgcc auggcuagac 60gc
626362RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 63gggccauggc guuaguauga gugucgucuu
ccugucaacg acacucauac uaacgccaug 60gc 626462RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 64gggagccaua guggucugcg gaaccggcuu ccugucaccg
guuccgcaga ccacuauggc 60uc 626561RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 65gggaaccggu
gaguacaccg gaauugcuuc cugucacaau uccgguguac ucaccgguuc 60c
616657RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 66gggucuugga ucaacccgcu caaugcuucc
ugucacauug agcggguuga uccaaga 576755RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 67gggcaacccg cucaaugccu ggagcuuccu gucacuccag
gcauugagcg gguug 556862RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 68gggauuuggg
cgugcccccg cgagacucuu ccugucaagu cucgcggggg cacgcccaaa 60uc
626962RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 69gggccgagua guguuggguc gcgaaagcuu
ccugucacuu ucgcgaccca acacuacucg 60gc 627062RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 70gggccuugug guacugccug auagggucuu ccugucaacc
cuaucaggca guaccacaag 60gc 627157RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 71gggacugccu
gauagggugc uugcgcuucc ugucacgcaa gcacccuauc aggcagu
577258RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 72gggaggucuc guagaccgug caccacuucc
ugucauggug cacggucuac gagaccuc 587353RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 73gggucucgua gaccgugcac cacuuccugu cauggugcac
ggucuacgag acc 537460RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 74gggucucgua
gaccgugcac caugagcuuc cugucacuca uggugcacgg ucuacgagac
607555RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 75gggucguaga ccgugcacca ugagcuuccu
gucacucaug gugcacgguc uacga 557662RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 76ggguagaccg
ugcaccauga gcacgaacuu ccugucauuc gugcucaugg ugcacggucu 60ac
627762RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 77gggaccgugc accaugagca cgaaucccuu
ccugucagga uucgugcuca uggugcacgg 60uc 627862RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 78gggugcacca ugagcacgaa uccuaaacuu ccugucauuu
aggauucgug cucauggugc 60ac 627958RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 79gggcaccaug
agcacgaauc cuaaacuucc ugucauuuag gauucgugcu cauggugc
588052RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 80gggaccgugc accaugagca ccuuccuguc
agugcucaug gugcacgguc uu 528153RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 81gggcgugcac
caugagcacg aacuuccugu cauucgugcu cauggugcac guu 538252RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 82gggugcacca ugagcacgaa ucuuccuguc aauucgugcu
cauggugcac uu 528353RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 83gggccuaaac cucaaagaaa
aacuuccugu cauuuuucuu ugagguuuag guu 538453RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 84gggcuaaacc ucaaagaaaa accuuccugu caguuuuucu
uugagguuua guu 538553RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 85ggguaaaccu
caaagaaaaa cccuuccugu cagguuuuuc uuugagguuu auu 538652RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 86gggcacgaau ccuaaaccuc acuuccuguc augagguuua
ggauucgugc uu 528752RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 87gggcacgaau ccuaaaccuc
acaacaacaa cugagguuua ggauucgugc uu 528848RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 88gggcacgaau ccuaaaccuc acuuccuuga gguuuaggau
ucgugcuu 488948RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 89gggcacgaau ccuaaaccuc
aaacaacuga gguuuaggau ucgugcuu 489047RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 90gggcacgaau ccuaaaccuc auucuuugag guuuaggauu
cgugcuu 479147RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 91gggcacgaau ccuaaaccuc
acaauaugag guuuaggauu cgugcuu 479246RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 92gggcacgaau ccuaaaccuc
agaaaugagg uuuaggauuc gugcuu 469346RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 93gggcacgaau ccuaaaccuc acucuugagg uuuaggauuc
gugcuu 469450RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 94gggcacgaau ccuaaaccuc
acuuccuguc augagguuua ggauucgugc 509552RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 95gggcacgaau ccuaaaccuc aacuuccugu cauugagguu
uaggauucgu gc 529664RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 96gggagcacga auccuaaacc
ucaaagacaa caacaacucu uugagguuua ggauucgugc 60ucuu
649760RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 97gggagcacga auccuaaacc ucaaagaaac
aacucuuuga gguuuaggau ucgugcucuu 609860RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 98gggagcacga auccuaaacc ucaaagacuu ccuucuuuga
gguuuaggau ucgugcucuu 609959RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 99gggagcacga
auccuaaacc ucaaagacaa uaucuuugag guuuaggauu cgugcucuu
5910059RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 100gggagcacga auccuaaacc ucaaagauuc
uuucuuugag guuuaggauu cgugcucuu 5910158RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 101gggagcacga auccuaaacc ucaaagacuc uucuuugagg
uuuaggauuc gugcucuu 5810258RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 102gggagcacga
auccuaaacc ucaaagagaa aucuuugagg uuuaggauuc gugcucuu
5810362RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 103gggagcacga auccuaaacc ucaaagacuu
ccugucaucu uugagguuua ggauucgugc 60uc 6210450RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 104gggagcacga auccuaaacc ucaaagacuu ccugucaucu
uugagguuua 5010565RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 105gggagaaacu ccaaauccua
agcacgagcu uccugucacu cgugcuuagg auuuggaguu 60ucuuu
6510652RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 106gggagcacga auccuaaacc ucuuccuguc
aagguuuagg auucgugcuc uu 5210753RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 107gggagcacga
auccuaaacc uccuuccugu cagagguuua ggauucgugc uuu
5310852RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 108gggcacgaau ccuaaaccuc acuuccuguc
augagguuua ggauucgugc uu 5210953RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 109gggcacgaau
ccuaaaccuc aacuuccugu cauugagguu uaggauucgu guu
5311053RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 110gggacgaauc cuaaaccuca aacuuccugu
cauuugaggu uuaggauucg uuu 5311110RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 111cuuccuguca
1011210RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 112caacaacaac 1011352RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 113gggcacgaau ccuaaaccuc acuuccuguc augagguuua
ggauucgugc uu 52
* * * * *