U.S. patent application number 12/138215 was filed with the patent office on 2009-02-12 for methods and compositions for the specific inhibition of gene expression by double-stranded rna.
This patent application is currently assigned to CITY OF HOPE. Invention is credited to Mark A. BEHLKE, Dongho KIM, John J. ROSSI.
Application Number | 20090043083 12/138215 |
Document ID | / |
Family ID | 34994243 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090043083 |
Kind Code |
A1 |
ROSSI; John J. ; et
al. |
February 12, 2009 |
METHODS AND COMPOSITIONS FOR THE SPECIFIC INHIBITION OF GENE
EXPRESSION BY DOUBLE-STRANDED RNA
Abstract
The invention is directed to compositions and methods for
selectively reducing the expression of a gene product from a
desired target gene in a cell, as well as for treating diseases
caused by the expression of the gene. More particularly, the
invention is directed to compositions that contain double stranded
RNA ("dsRNA"), and methods for preparing them, that are capable of
reducing the expression of target genes in eukaryotic cells. The
dsRNA has a first oligonucleotide sequence that is between 25 and
about 30 nucleotides in length and a second oligonucleotide
sequence that anneals to the first sequence under biological
conditions. In addition, a region of one of the sequences of the
dsRNA having a sequence length of at least 19 nucleotides is
sufficiently complementary to a nucleotide sequence of the RNA
produced from the target gene to trigger the destruction of the
target RNA by the RNAi machinery.
Inventors: |
ROSSI; John J.; (Alta Loma,
CA) ; BEHLKE; Mark A.; (Coralville, IA) ; KIM;
Dongho; (Los Angeles, CA) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W., SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
CITY OF HOPE
Duarte
CA
INTEGRATED DNA TECHNOLOGIES, INC.
Coralville
IA
|
Family ID: |
34994243 |
Appl. No.: |
12/138215 |
Filed: |
June 12, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11079906 |
Mar 15, 2005 |
|
|
|
12138215 |
|
|
|
|
60553487 |
Mar 15, 2004 |
|
|
|
Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 37/06 20180101; A61P 31/14 20180101; A61P 5/14 20180101; A61P
31/08 20180101; C12N 2310/50 20130101; C12N 15/111 20130101; C12N
2320/51 20130101; A61P 1/16 20180101; C12N 2320/50 20130101; A61P
7/04 20180101; A61P 1/00 20180101; A61P 27/16 20180101; A61P 29/00
20180101; A61P 37/08 20180101; A61P 3/10 20180101; A61P 11/00
20180101; C12N 2310/14 20130101; A61P 11/06 20180101; A61P 15/02
20180101; A61P 25/00 20180101; A61P 35/00 20180101; C12N 2330/30
20130101; A61P 1/04 20180101; A61P 21/04 20180101; C12N 2310/33
20130101; A61P 17/00 20180101; A61P 37/00 20180101; A61P 7/06
20180101; A61P 1/02 20180101; C12N 2320/30 20130101; A61P 27/02
20180101; C12N 15/113 20130101; A61P 17/06 20180101; A61P 35/04
20180101; C12N 2310/51 20130101; A61P 37/02 20180101; A61P 25/02
20180101 |
Class at
Publication: |
536/23.1 |
International
Class: |
C07H 21/02 20060101
C07H021/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] The present invention was made in part with Government
support under Grant Numbers A129329 and HL074704 awarded by the
National Institute of Health. The Government has certain rights in
this invention.
Claims
1. An isolated double stranded nucleic acid comprising a first
oligonucleotide strand comprising ribonucleotides and having a 5'
terminus and a 3' terminus and a second oligonucleotide strand
comprising ribonucleotides and having a 5' terminus and a 3'
terminus, wherein starting from the first nucleotide (position 1)
at the 3' terminus of the first oligonucleotide strand, position 1,
2 and/or 3 is substituted with a modified nucleotide, wherein each
of said first and said second strands consists of 25-30
nucleotides, wherein said second strand is 1-4 nucleotides longer
at its 3' terminus than said first strand, and wherein said second
oligonucleotide strand is sufficiently complementary to a target
RNA along at least 19 nucleotides of said second oligonucleotide
strand length to reduce target gene expression when said double
stranded nucleic acid is introduced into a mammalian cell.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a division of U.S. patent
application Ser. No. 11/079,906 filed 15 Mar. 2005, which in turn
is related to and claims priority under 35 U.S.C. .sctn. 119(e) to
U.S. provisional patent application No. 60/553,487 filed 15 Mar.
2004. Each application is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The publications and other materials used herein to
illuminate the background of the invention, and in particular,
cases to provide additional details respecting the practice, are
incorporated by reference, and for convenience are referenced in
the following text by author and date and are listed alphabetically
by author in the appended bibliography.
[0004] The present invention pertains to compositions and methods
for gene-specific inhibition of gene expression by double-stranded
ribonucleic acid (dsRNA) effector molecules. The compositions and
methods are useful in modulating gene expression in a variety of
applications, including therapeutic, diagnostic, target validation,
and genomic discovery.
BACKGROUND OF THE INVENTION
[0005] Suppression of gene expression by double-stranded RNA
(dsRNA) has been demonstrated in a variety of systems including
plants (post-transcriptional gene suppression) (Napoli et al.,
1990), fungi (quelling) (Romano and Marcino, 1992), and nematodes
(RNA interference) (Fire et al., 1998). Early attempts to similarly
suppress gene expression using long dsRNAs in mammalian systems
failed due to activation of interferon pathways that do not exist
in lower organisms. Interferon responses are triggered by dsRNAs
(Stark et al., 1998). In particular, the protein kinase PKR is
activated by dsRNAs of greater than 30 bp long (Manche et al.,
1992) and results in phosphorylation of translation initiation
factor eIF2.alpha. which leads to arrest of protein synthesis and
activation of 2'5'-oligoadenylate synthetase (2'-5'-OAS), which
leads to RNA degradation (Minks et al., 1979).
[0006] In Drosophila cells and cell extracts, dsRNAs of 150 bp
length or greater were seen to induce RNA interference while
shorter dsRNAs were ineffective (Tuschl et al., 1999). Long
double-stranded RNA, however, is not the active effecter molecule;
long dsRNAs are degraded by an RNase III class enzyme called Dicer
(Bernstein et al., 2001) into very short 21-23 bp duplexes that
have 2-base 3'-overhangs (Zamore et al., 2000). These short RNA
duplexes, called siRNAs, direct the RNAi response in vivo and
transfection of short chemically synthesized siRNA duplexes of this
design permits use of RNAi methods to suppress gene expression in
mammalian cells without triggering unwanted interferon responses
(Elbashir et al., 2001a). The antisense strand of the siRNA duplex
serves as a sequence-specific guide that directs activity of an
endoribonuclease function in the RNA induced silencing complex
(RISC) to degrade target mRNA (Martinez et al., 2002).
[0007] In studying the size limits for RNAi in Drosophila embryo
extracts in vitro, a lower threshold of around 38 bp
double-stranded RNA was established for activation of RNA
interference using exogenously supplied double-stranded RNA and
duplexes of 36, 30, and 29 bp length (Elbashir et al., 2001b). The
short 30-base RNAs were not cleaved into active 21-23-base siRNAs
and therefore were deemed inactive for use in RNAi (Elbashir et
al., 2001b). Continuing to work in the Drosophila embryo extract
system, the same group later carefully mapped the structural
features needed for short chemically synthesized RNA duplexes to
function as siRNAs in RNAi pathways. RNA duplexes of 21-bp length
with 2-base 3'-overhangs were most effective, duplexes of 20, 22,
and 23-bp length had slightly decreased potency but did result in
RNAi mediated mRNA degradation, and 24 and 25-bp duplexes were
inactive (Elbashir et al., 2001c).
[0008] Some of the conclusions of these earlier studies may be
specific to the Drosophila system employed. Other investigators
established that longer siRNAs can work in human cells. However,
duplexes in the 21-23-bp range have been shown to be more active
and have become the accepted design (Caplen et al., 2001).
Essentially, chemically synthesized duplex RNAs that mimicked the
natural products that result from Dicer degradation of long duplex
RNAs were identified to be the preferred compound for use in RNAi.
Approaching this problem from the opposite direction, investigators
studying size limits for RNAi in Caenorhabditis elegans found that
although a microinjected 26-bp RNA duplex could function to
suppress gene expression, it required a 250-fold increase in
concentration compared with an 81-bp duplex (Parrish et al.,
2000).
[0009] Despite the attention given to RNAi research recently, the
field is still in the early stages of development. Not all siRNA
molecules are capable of targeting the destruction of their
complementary RNAs in a cell. As a result, complex sets of rules
have been developed for designing RNAi molecules that will be
effective. Those having skill in the art expect to test multiple
siRNA molecules to find functional compositions. (Ji et al., 2003)
Some artisans pool several siRNA preparations together to increase
the chance of obtaining silencing in a single study. (Ji et al.,
2003) Such pools typically contain 20 nM of a mixture of siRNA
oligonucleotide duplexes or more (Ji et al., 2003), despite the
fact that a siRNA molecule can work at concentrations of 1 nM or
less (Holen et al., 2002). This technique can lead to artifacts
caused by interactions of the siRNA sequences with other cellular
RNAs ("off target effects"). (Scherer et al., 2003) Off target
effects can occur when the RNAi oligonucleotides have homology to
unintended targets or when the RISC complex incorporates the
unintended strand from and RNAi duplex. (Scherer et al., 2003)
Generally, these effects tend to be more pronounced when higher
concentrations of RNAi duplexes are used. (Scherer et al.,
2003)
[0010] In addition, the duration of the effect of an effective RNAi
treatment is limited to about 4 days (Holen et al., 2002). Thus,
researchers must carry out siRNA experiments within 2-3 days of
transfection with an siRNA duplex or work with plasmid or viral
expression vectors to obtain longer term silencing.
[0011] The invention provides compositions useful in RNAi for
inhibiting gene expression and provides methods for their use. In
addition, the invention provides RNAi compositions and methods
designed to maximize potency, potentially increase duration of
action and ease site selection criteria, while minimizing "off
target effects." These and other advantages of the invention, as
well as additional inventive features, will be apparent from the
description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
[0012] The present invention is directed to compositions and
methods for selectively reducing the expression of a gene product
from a desired target gene in a eukaryotic cell, as well as for
treating diseases caused by the expression of the gene. More
particularly, the invention is directed to compositions that
contain double stranded RNA ("dsRNA"), and methods for preparing
them, that are capable of reducing the expression of target genes
in eukaryotic cells.
[0013] Thus, in a first aspect, the present invention provides
novel compositions for RNA interference (RNAi). The compositions
comprise dsRNA which is a precursor molecule, i.e., the dsRNA of
the present invention is processed in vivo to produce an active
siRNA. The dsRNA is processed by Dicer to an active siRNA which is
incorporated into the RISC complex for RNA interference of a target
gene. The precursor molecule is also termed a precursor RNAi
molecule herein.
[0014] In one embodiment, the dsRNA, i.e., the precursor RNAi
molecule, has a length sufficient such that it is processed by
Dicer to produce an siRNA. According to this embodiment, the dsRNA
comprises a first oligonucleotide sequence (also termed the sense
strand) that is between 26 and about 30 nucleotides in length and a
second oligonucleotide sequence (also termed the antisense strand)
that anneals to the first sequence under biological conditions,
such as the conditions found in the cytoplasm of a cell. In
addition, a region of one of the sequences, particularly of the
antisense strand, of the dsRNA has a sequence length of at least 19
nucleotides, for example, from about 19 to about 23 nucleotides,
such as 21 nucleotides that are sufficiently complementary to a
nucleotide sequence of the RNA produced from the target gene to
trigger an RNAi response.
[0015] In a second embodiment, the dsRNA, i.e., the precursor RNAi
molecule, has several properties which enhance its processing by
Dicer. According to this embodiment, the dsRNA dsRNA has a length
sufficient such that it is processed by Dicer to produce an siRNA
and at least one of the following properties: (ii) the dsRNA is
asymmetric, e.g., has a 3' overhang on the antisense strand and
(ii) the dsRNA has a modified 3' end on the sense strand to direct
orientation of Dicer binding and processing of the dsRNA to an
active siRNA. According to this embodiment, the sense strand
comprises 22-28 nucleotides and the antisense strand comprises
24-30 nucleotides. In one embodiment, the dsRNA has an overhang on
the 3' end of the antisense strand. In another embodiment, the
sense strand is modified for Dicer binding and processing by
suitable modifiers located at the 3' end of the sense strand.
Suitable modifiers include nucleotides such as
deoxyribonucleotides, acyclonucleotides and the like and sterically
hindered molecules, such as fluorescent molecules and the like.
When nucleotide modifiers are used, they replace ribonucleotides in
the dsRNA such that the length of the dsRNA does not change. In
another embodiment, the dsRNA has an overhang on the 3' end of the
antisense strand and the sense strand is modified for Dicer
processing. In another embodiment, the 5' end of the sense strand
has a phosphate. The sense and antisense strands anneal under
biological conditions, such as the conditions found in the
cytoplasm of a cell. In addition, a region of one of the sequences,
particularly of the antisense strand, of the dsRNA has a sequence
length of at least 19 nucleotides, wherein these nucleotides are in
the 21-nucleotide region adjacent to the 3' end of the antisense
strand and are sufficiently complementary to a nucleotide sequence
of the RNA produced from the target gene. Further in accordance
with this embodiment, the dsRNA, i.e., the precursor RNAi molecule,
may also have one or more of the following additional properties:
(a) the antisense strand has a right shift from the typical 21mer
(i.e., the antisense strand includes nucleotides on the right side
of the molecule when compared to the typical 21mer), (b) the
strands may not be completely complementary, i.e., the strands may
contain simple mismatch pairings and (c) base modifications such as
locked nucleic acid(s) may be included in the 5' end of the sense
strand.
[0016] In a third embodiment, the dsRNA, i.e., the precursor RNAi
molecule, has several properties which enhance its processing by
Dicer. According to this embodiment, the dsRNA has a length
sufficient such that it is processed by Dicer to produce an siRNA
and at least one of the following properties: (i) the dsRNA is
asymmetric, e.g., has a 3' overhang on the sense strand and (ii)
the dsRNA has a modified 3' end on the antisense strand to direct
orientation of Dicer binding and processing of the dsRNA to an
active siRNA. According to this embodiment, the sense strand
comprises 24-30 nucleotides and the antisense strand comprises
22-28 nucleotides. In one embodiment, the dsRNA has an overhang on
the 3' end of the sense strand. In another embodiment, the
antisense strand is modified for Dicer binding and processing by
suitable modifiers located at the 3' end of the antisense strand.
Suitable modifiers include nucleotides such as
deoxyribonucleotides, acyclonucleotides and the like and sterically
hindered molecules, such as fluorescent molecules and the like.
When nucleotide modifiers are used, they replace ribonucleotides in
the dsRNA such that the length of the dsRNA does not change. In
another embodiment, the dsRNA has an overhang on the 3' end of the
sense strand and the antisense strand is modified for Dicer
processing. In one embodiment, the antisense strand has a 5'
phosphate. The sense and antisense strands anneal under biological
conditions, such as the conditions found in the cytoplasm of a
cell. In addition, a region of one of the sequences, particularly
of the antisense strand, of the dsRNA has a sequence length of at
least 19 nucleotides, wherein these nucleotides are adjacent to the
3' end of antisense strand and are sufficiently complementary to a
nucleotide sequence of the RNA produced from the target gene.
Further in accordance with this embodiment, the dsRNA, i.e., the
precursor RNAi molecule, may also have one or more of the following
additional properties: (a) the antisense strand has a left shift
from the typical 21mer (i.e., the antisense strand includes
nucleotides on the left side of the molecule when compared to the
typical 21mer) and (b) the strands may not be completely
complementary, i.e., the strands may contain simple mismatch
pairings.
[0017] In a second aspect, the present invention provides a method
for making a dsRNA, i.e., a precursor RNAi molecule, that has
enhanced processing by Dicer. According to this method an antisense
strand siRNA having a length of at least 19 nucleotides is selected
for a given target gene using conventional techniques and
algorithms. In one embodiment, the antisense siRNA is modified to
include 5-11 ribonucleotides on the 5' end to give a length of
24-30 nucleotides. When the antisense strand has a length of 21
nucleotides, then 3-9 nucleotides, or 4-7 nucleotides or 6
nucleotides are added on the 5' end. Although the added
ribonucleotides may be complementary to the target gene sequence,
full complementarity between the target sequence and the antisense
siRNA is not required. That is, the resultant antisense siRNA is
sufficiently complementary with the target sequence. A sense strand
is then produced that has 22-28 nucleotides. The sense strand is
substantially complementary with the antisense strand to anneal to
the antisense strand under biological conditions. In one
embodiment, the sense strand is synthesized to contain a modified
3' end to direct Dicer processing of the antisense strand. In
another embodiment, the antisense strand of the dsRNA has a 3'
overhang. In a further embodiment, the sense strand is synthesized
to contain a modified 3' end for Dicer binding and processing and
the antisense strand of the dsRNA has a 3' overhang.
[0018] In a second embodiment of this method, the antisense siRNA
is modified to include 1-9 ribonucleotides on the 5' end to give a
length of 22-28 nucleotides. When the antisense strand has a length
of 21 nucleotides, then 1-7 ribonucleotides, or 2-5 ribonucleotides
and or 4 ribonucleotides are added on the 3' end. The added
ribonucleotides may have any sequence. Although the added
ribonucleotides may be complementary to the target gene sequence,
full complementarity between the target sequence and the antisense
siRNA is not required. That is, the resultant antisense siRNA is
sufficiently complementary with the target sequence. A sense strand
is then produced that has 24-30 nucleotides. The sense strand is
substantially complementary with the antisense strand to anneal to
the antisense strand under biological conditions. In one
embodiment, the antisense strand is synthesized to contain a
modified 3' end to direct Dicer processing. In another embodiment,
the sense strand of the dsRNA has a 3' overhang. In a further
embodiment, the antisense strand is synthesized to contain a
modified 3' end for Dicer binding and processing and the sense
strand of the dsRNA has a 3' overhang.
[0019] In a third aspect, the present invention provides
pharmaceutical compositions containing the disclosed dsRNA
compositions.
[0020] In a fourth aspect, the present invention provides methods
for selectively reducing the expression of a gene product from a
desired target gene in a cell, as well as for treating diseases
caused by the expression of the gene. In one embodiment, the method
involves introducing into the environment of a cell an amount of a
dsRNA of the present invention such that a sufficient portion of
the dsRNA can enter the cytoplasm of the cell to cause a reduction
in the expression of the target gene.
[0021] The compositions and methods have an unanticipated level of
potency of the RNAi effect. Although the invention is not intended
to be limited by the underlying theory on which it is believed to
operate, it is thought that this level of potency and duration of
action are caused by the fact the dsRNA serves as a substrate for
Dicer which appears to facilitate incorporation of one sequence
from the dsRNA into the RISC complex that is directly responsible
for destruction of the RNA from the target gene.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A-1F show that 27mer dsRNAs are more potent effectors
of RNAi than a 21+2 siRNA. EGFP expression levels were determined
after cotransfection of HEK293 cells with a fixed amount of EGFP
expression plasmid and varying concentrations of dsRNAs. FIGS.
1A-1C: Transfections were performed using (FIG. 1A) 50 nM, (FIG.
1B) 200 pM and (FIG. 1C) 50 pM of the indicated dsRNAs. FIG. 1D:
Dose-response testing of longer dsRNAs. Transfections were
performed with the indicated concentrations of dsRNA. FIG. 1E:
Depicts in vitro Dicer reactions with the same longer RNAs.
Concentrations and conditions were as described in the Examples.
FIG. 1F: Dose-response curve of dsRNAs transfected into NIH3T3
cells that stably express EGFP. Each graph point represents the
average (with s.d.) of three independent measurements.
[0023] FIGS. 2A-2E show that Dicer processing correlates with RNAi
activity. FIG. 2A: Cleavage of dsRNAs by recombinant Dicer. Each
RNA duplex was incubated in the presence or absence of recombinant
human Dicer for 24 h, separated using nondenaturing PAGE and
visualized (see Examples). FIG. 2B: RNA duplexes used in this test.
Oligos were conjugated with 6FAM at the 5' ends, the 3' ends or
both as shown by the circles. Top and bottom lines indicate sense
and antisense strands in duplex configuration, with the sense in a
5'-to-3' orientation (left to right) and the antisense in a
3'-to-5' orientation (left to right). FIG. 2C: 6FAM
end-modification affects in vitro Dicer activity. RNA duplexes were
incubated with 0.5 units of recombinant human Dicer for 8 h and the
products resolved on a 7.5% nondenaturing polyacrylamide gel. The
RNAs were visualized by ethidium bromide staining. FIG. 2D: 6FAM
modification affects RNAi activity. RNA duplexes at 200 pM were
cotransfected with the EGFP expression plasmid and assayed at 24 h
for EGFP fluorescence as described. Reported values for EGFP
expression represent the average of two independent experiments.
The relative levels of fluorescence were normalized to those for
luciferase. FIG. 2E: 27mer duplex RNAs are processed to 21mers in
vivo. Total RNA was prepared from cells transfected with duplex 3
and duplex 5 at 10 nM. RNA was hybridized with a 21mer
.sup.32P-labeled oligonucleotide probe. The hybridized samples were
separated by nondenaturing PAGE and visualized by autoradiography.
Size markers are .sup.32P end labeled 21mer and 27mer RNA
duplexes.
[0024] FIG. 3A-3B show RNAi activity of various 21+2 siRNAs. FIG.
3A: Seven possible 21+2 siRNAs predicted from Dicing the 27mer
dsRNA were tested individually or as a pool in co-transfection
assays with the EGFP reporter construct in HEK293 cells. Each graph
depicts the average of duplicate experiments. FIG. 3B: Comparison
of in vitro Diced 27mer dsRNA versus intact 27mer dsRNA for RNAi.
The respective RNAs were co-transfected as in FIG. 4A at the
indicated concentrations of dsRNAs. For the Diced products, a 1
.mu.M 27mer dsRNA was incubated in Dicer reaction buffer without
(column 3) or with Dicer (column 4) at 37.degree. C. for 12 hours.
The mixtures were diluted in water and used directly for
co-transfection with the EGFP reporter. To control for possible
artifacts of residual Dicer in the diluted mixes, the samples in
column 4 were phenol extracted and ethanol precipitated prior to
transfection (column 5).
[0025] FIGS. 4A and 4B show ESI mass spectra of the 27mer duplex
EGFPS1 27+0 before (FIG. 4A) and after (FIG. 4B) incubation with
Dicer. Duplexes separate into single strands and the measured mass
of each strand is indicated. Dicer digestion is performed in the
presence of high salt and some "shadow" peaks represent +1 or +2 Na
species.
[0026] FIGS. 5A-5D show features of 27mer dsRNA in RNAi. FIG. 5A:
Enhanced duration of RNAi by 27mer dsRNAs. Levels of EGFP were
determined after transfection of 5 nM of a 21+2 siRNA or the 27mer
dsRNA into NIH3T3 cells stably expressing EGFP. Graphic
representation of EGFP silencing mediated by a 21+2 siRNA as
compared to the 27mer dsRNA. Duplicate samples were taken on the
indicated days and EGFP expression was determined by fluorometry.
FIG. 5B: 27mer dsRNAs, targeting sites refractory to 21mer siRNAs,
can elicit RNAi. The dsRNAs were transfected along with the EGFP
reporter construct, and EGFP expression was determined (Methods).
Column 1, mock; column 2, 21+2 siRNA targeting EGFPS2; column 3,
27mer dsRNA targeting EGFPS2; column 4, 21+2 siRNA targeting
EGFPS3; column 5, 27mer dsRNA targeting EGFPS3. FIGS. 5C and 5D:
Comparison of 21mer siRNA and 27mer dsRNA in downregulation of
endogenous transcripts. RNAs for a 21+2 siRNA and 27+0 dsRNA were
designed to target sites in the hnRNP H mRNA (FIG. 5C) or La mRNA
(FIG. 5D). HnRNP H knockdown was assayed by western blot and La
knockdown by northern blot analyses. The dsRNAs were used at the
indicated concentrations. .beta.-Actin was used as an internal
specificity and loading standard in both experiments.
[0027] FIG. 6 shows sequence specificity of Dicer substrate 27mer
dsRNAs. The various 27mer dsRNAs were co-transfected at the
indicated concentrations with the EGFP expression plasmid into
HEK93 cells and assayed for EGFP fluorescence.
[0028] FIGS. 7A-7D show that siRNAs and Dicer substrate dsRNAs do
not induce interferons or activate PKR or generate specific "off
target effects." FIGS. 7A and 7B: Interferon alpha (FIG. 7A) and
interferon beta (FIG. 7B) assays: column 1, positive control for
IFN induction (Kim et al., 2004); column 2, no RNA; column 3,
chemically synthesized 21+2 siRNA; column 4, chemically synthesized
27+0 dsRNA. FIG. 7C: PKR activation assay. The lond dsRNA used for
PKR activation (Kim et al., 2004) and the in vitro PKR activation
assay (Manche et al, 1992) have been previously described. Duplex
RNAs were transfected as indicated. FIG. 7D: Summary of microarray
analysis.
[0029] FIGS. 8A-8B show ESI mass spectra of the 27mer duplex EGFPS1
27+0 L before (FIG. 8A) and after (FIG. 8B) incubation with Dicer.
Duplexes separate into single strands and the measured mass of each
strand is indicated.
[0030] FIGS. 8C-8D show ESI mass spectra of the 27mer duplex EGFPS1
27/25 L before (FIG. 8C) and after (FIG. 8D) incubation with Dicer.
Duplexes separate into single strands and the measured mass of each
strand is indicated.
[0031] FIGS. 9A-9B show ESI mass spectra of the 27mer duplex EGFPS1
27+0 R before (FIG. 9A) and after (FIG. 9B) incubation with Dicer.
Duplexes separate into single strands and the measured mass of each
strand is indicated.
[0032] FIGS. 9C-9D show ESI mass spectra of the 27mer duplex EGFPS1
25/27 R before (FIG. 9C) and after (FIG. 9B) incubation with Dicer.
Duplexes separate into single strands and the measured mass of each
strand is indicated.
[0033] FIGS. 10A-10B show that duplexes designed to enhance Dicer
processing are potent effectors of RNAi. EGFP expression levels
were determined after cotransfection of HEK293 cells with a fixed
amount of EGFP expression plasmid and varying concentrations of
dsRNAs. FIG. 10A: Compares the potency of the duplexes EGFPS2-21+2,
EGFPS2-27+0, EGFPS2-27/25 L and EGFPS2-25/27 R. FIG. 10B: Compares
the potency of the duplexes EGFPS1-27/25 L and EGFPS1-25/27 R.
[0034] FIG. 11 is an illustration showing two embodiments of the
present invention with respect to the target sequence and the
relationship between the target sequence and each embodiment.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The present invention is directed to compositions that
contain double stranded RNA ("dsRNA"), and methods for preparing
them, that are capable of reducing the expression of target genes
in eukaryotic cells. One of the strands of the dsRNA contains a
region of nucleotide sequence that has a length that ranges from
about 19 to about 30 nucleotides that can direct the destruction of
the RNA transcribed from the target gene.
[0036] In a first aspect, the present invention provides novel
compositions for RNA interference (RNAi). The compositions comprise
dsRNA which is a precursor molecule, i.e., the dsRNA of the present
invention is processed in vivo to produce an active siRNA. The
dsRNA is processed by Dicer to an active siRNA which is
incorporated into the RISC complex. The precursor molecule is also
termed a precursor RNAi molecule herein. As used herein, the term
active siRNA refers to a double stranded nucleic acid in which each
strand comprises RNA, RNA analog(s) or RNA and DNA. The siRNA
comprises between 19 and 23 nucleotides or comprises 21
nucleotides. The active siRNA has 2 bp overhangs on the 3' ends of
each strand such that the duplex region in the siRNA comprises
17-21 nucleotides, or 19 nucleotides. Typically, the antisense
strand of the siRNA is sufficiently complementary with the target
sequence of the target gene.
[0037] The phrase "duplex region" refers to the region in two
complementary or substantially complementary oligonucleotides that
form base pairs with one another, either by Watson-Crick base
pairing or any other manner that allows for a duplex between
oligonucleotide strands that are complementary or substantially
complementary. For example, an oligonucleotide strand having 21
nucleotide units can base pair with another oligonucleotide of 21
nucleotide units, yet only 19 bases on each strand are
complementary or substantially complementary, such that the "duplex
region" consists of 19 base pairs. The remaining base pairs may,
for example, exist as 5' and 3' overhangs. Further, within the
duplex region, 100% complementarity is not required; substantial
complementarity is allowable within a duplex region. Substantial
complementarity refers to complementarity between the strands such
that they are capable of annealing under biological conditions.
Techniques to empirically determine if two strands are capable of
annealing under biological conditions are well know in the art.
Alternatively, two strands can be synthesized and added together
under biological conditions to determine if they anneal to one
another.
[0038] As used herein, an siRNA having a sequence "sufficiently
complementary" to a target mRNA sequence means that the siRNA has a
sequence sufficient to trigger the destruction of the target mRNA
by the RNAi machinery (e.g., the RISC complex) or process. The
siRNA molecule can be designed such that every residue of the
antisense strand is complementary to a residue in the target
molecule. Alternatively, substitutions can be made within the
molecule to increase stability and/or enhance processing activity
of said molecule. Substitutions can be made within the strand or
can be made to residues at the ends of the strand.
[0039] In one embodiment of the first aspect of the present
invention, the dsRNA, i.e., the precursor RNAi molecule, has a
length sufficient such that it is processed by Dicer to produce an
siRNA. According to this embodiment, a suitable dsRNA contains one
oligonucleotide sequence, a first sequence, that is at least 25
nucleotides in length and no longer than about 30 nucleotides. This
sequence of RNA can be between about 26 and 29 nucleotides in
length. This sequence can be about 27 or 28 nucleotides in length
or 27 nucleotides in length. The second sequence of the dsRNA can
be any sequence that anneals to the first sequence under biological
conditions, such as within the cytoplasm of a eukaryotic cell.
Generally, the second oligonucleotide sequence will have at least
19 complementary base pairs with the first oligonucleotide
sequence, more typically the second oligonucleotides sequence will
have about 21 or more complementary base pairs, or about 25 or more
complementary base pairs with the first oligonucleotide sequence.
In one embodiment, the second sequence is the same length as the
first sequence, and the dsRNA is blunt ended. In another
embodiment, the ends of the dsRNA have overhangs.
[0040] In certain aspects of this first embodiment, the first and
second oligonucleotide sequences of the dsRNA exist on separate
oligonucleotide strands that can be and typically are chemically
synthesized. In some embodiments, both strands are between 26 and
30 nucleotides in length. In other embodiments, both strands are
between 25 and 30 nucleotides in length. In one embodiment, both
strands are 27 nucleotides in length, are completely complementary
and have blunt ends. The dsRNA can be from a single RNA
oligonucleotide that undergoes intramolecular annealing or, more
typically, the first and second sequences exist on separate RNA
oligonucleotides. In one embodiment, one or both oligonucleotide
strands are capable of serving as a substrate for Dicer. In other
embodiments, at least one modification is present that promotes
Dicer to bind to the double-stranded RNA structure in an
orientation that maximizes the double-stranded RNA structure's
effectiveness in inhibiting gene expression. The dsRNA can contain
one or more deoxyribonucleic acid (DNA) base substitutions.
[0041] Suitable dsRNA compositions that contain two separate
oligonucleotides can be chemically linked outside their annealing
region by chemical linking groups. Many suitable chemical linking
groups are known in the art and can be used. Suitable groups will
not block Dicer activity on the dsRNA and will not interfere with
the directed destruction of the RNA transcribed from the target
gene. Alternatively, the two separate oligonucleotides can be
linked by a third oligonucleotide such that a hairpin structure is
produced upon annealing of the two oligonucleotides making up the
dsRNA composition. The hairpin structure will not block Dicer
activity on the dsRNA and will not interfere with the directed
destruction of the RNA transcribed from the target gene.
[0042] The first and second oligonucleotides are not required to be
completely complementary. In fact, in one embodiment, the
3'-terminus of the sense strand contains one or more mismatches. In
one aspect, about two mismatches are incorporated at the 3'
terminus of the sense strand. In another embodiment, the dsRNA of
the invention is a double stranded RNA molecule containing two RNA
oligonucleotides each of which is 27 nucleotides in length and,
when annealed to each other, have blunt ends and a two nucleotide
mismatch on the 3'-terminus of the sense strand (the 5'-terminus of
the antisense strand). The use of mismatches or decreased
thermodynamic stability (specifically at the 3'-sense/5'-antisense
position) has been proposed to facilitate or favor entry of the
antisense strand into RISC (Schwarz et al., 2003; Khvorova et al.,
2003), presumably by affecting some rate-limiting unwinding steps
that occur with entry of the siRNA into RISC. Thus, terminal base
composition has been included in design algorithms for selecting
active 21mer siRNA duplexes (Ui-Tei et al., 2004; Reynolds et al.,
2004). With Dicer cleavage of the dsRNA of this embodiment, the
small end-terminal sequence which contains the mismatches will
either be left unpaired with the antisense strand (become part of a
3'-overhang) or be cleaved entirely off the final 21-mer siRNA.
These "mismatches", therefore, do not persist as mismatches in the
final RNA component of RISC. It was surprising to find that base
mismatches or destabilization of segments at the 3'-end of the
sense strand of Dicer substrate improved the potency of synthetic
duplexes in RNAi, presumably by facilitating processing by
Dicer.
[0043] It has been found empirically that these longer dsRNA
species of from 25 to about 30 nucleotides give unexpectedly
effective results in terms of potency and duration of action.
Without wishing to be bound by the underlying theory of the
invention, it is thought that the longer dsRNA species serve as a
substrate for the enzyme Dicer in the cytoplasm of a cell. In
addition to cleaving the dsRNA of the invention into shorter
segments, Dicer is thought to facilitate the incorporation of a
single-stranded cleavage product derived from the cleaved dsRNA
into the RISC complex that is responsible for the destruction of
the cytoplasmic RNA derived from the target gene. The studies
described herein have shown that the cleavability of a dsRNA
species by Dicer corresponds with increased potency and duration of
action of the dsRNA species.
[0044] In a second embodiment of the first aspect of the present
invention, the dsRNA, i.e., the precursor RNAi molecule, has
several properties which enhance its processing by Dicer. According
to this embodiment, the dsRNA has a length sufficient such that it
is processed by Dicer to produce an active siRNA and at least one
of the following properties: (i) the dsRNA is asymmetric, e.g., has
a 3' overhang on the antisense strand and (ii) the dsRNA has a
modified 3' end on the sense strand to direct orientation of Dicer
binding and processing of the dsRNA to an active siRNA. According
to this embodiment, the longest strand in the dsRNA comprises 24-30
nucleotides. In one embodiment, the dsRNA is asymmetric such that
the sense strand comprises 22-28 nucleotides and the antisense
strand comprises 24-30 nucleotides. Thus, the resulting dsRNA has
an overhang on the 3' end of the antisense strand. The overhang is
1-3 nucleotides, for example 2 nucleotides. The sense strand may
also have a 5' phosphate.
[0045] In another embodiment, the sense strand is modified for
Dicer processing by suitable modifiers located at the 3' end of the
sense strand, i.e., the dsRNA is designed to direct orientation of
Dicer binding and processing. Suitable modifiers include
nucleotides such as deoxyribonucleotides, dideoxyribonucleotides,
acyclonucleotides and the like and sterically hindered molecules,
such as fluorescent molecules and the like. Acyclonucleotides
substitute a 2-hydroxyethoxymethyl group for the
2'-deoxyribofuranosyl sugar normally present in dNMPs. Other
nucleotides modifiers could include 3'-deoxyadenosine (cordycepin),
3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI),
2',3'-dideoxy-3'-thiocytidine (3TC),
2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate
nucleotides of 3'-azido-3'-deoxythymidine (AZT),
2',3'-dideoxy-3'-thiocytidine (3TC) and
2',3'-didehydro-2',3'-dideoxythymidine (d4T). In one embodiment,
deoxynucleotides are used as the modifiers. When nucleotide
modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide
modifiers are substituted for the ribonucleotides on the 3' end of
the sense strand. When sterically hindered molecules are utilized,
they are attached to the ribonucleotide at the 3' end of the
antisense strand. Thus, the length of the strand does not change
with the incorporation of the modifiers. In another embodiment, the
invention contemplates substituting two DNA bases in the dsRNA to
direct the orientation of Dicer processing of the antisense strand.
In a further embodiment of the present invention, two terminal DNA
bases are substituted for two ribonucleotides on the 3'-end of the
sense strand forming a blunt end of the duplex on the 3' end of the
sense strand and the 5' end of the antisense strand, and a
two-nucleotide RNA overhang is located on the 3'-end of the
antisense strand. This is an asymmetric composition with DNA on the
blunt end and RNA bases on the overhanging end.
[0046] The sense and antisense strands anneal under biological
conditions, such as the conditions found in the cytoplasm of a
cell. In addition, a region of one of the sequences, particularly
of the antisense strand, of the dsRNA has a sequence length of at
least 19 nucleotides, wherein these nucleotides are in the
21-nucleotide region adjacent to the 3' end of the antisense strand
and are sufficiently complementary to a nucleotide sequence of the
RNA produced from the target gene.
[0047] Further in accordance with this embodiment, the dsRNA, i.e.,
the precursor RNAi molecule, may also have one or more of the
following additional properties: (a) the antisense strand has a
right shift from the typical 21mer, (b) the strands may not be
completely complementary, i.e., the strands may contain simple
mismatch pairings and (c) base modifications such as locked nucleic
acid(s) may be included in the 5' end of the sense strand. A
"typical" 21mer siRNA is designed using conventional techniques. In
one technique, a variety of sites are commonly tested in parallel
or pools containing several distinct siRNA duplexes specific to the
same target with the hope that one of the reagents will be
effective (Ji et al., 2003). Other techniques use design rules and
algorithms to increase the likelihood of obtaining active RNAi
effector molecules (Schwarz et al., 2003; Khvorova et al., 2003;
Ui-Tei et al., 2004; Reynolds et al., 2004; Krol et al., 2004; Yuan
et al., 2004; Boese et al., 2005). High throughput selection of
siRNA has also been developed (U.S. published patent application
No. 2005/0042641 A1, incorporated herein by reference). Potential
target sites can also be analyzed by secondary structure
predictions (Heale et al., 2005). This 21mer is then used to design
a right shift to include 3-9 additional nucleotides on the 5' end
of the 21mer. The sequence of these additional nucleotides may have
any sequence. In one embodiment, the added ribonucleotides are
based on the sequence of the target gene. Even in this embodiment,
full complementarity between the target sequence and the antisense
siRNA is not required.
[0048] The first and second oligonucleotides are not required to be
completely complementary. They only need to be substantially
complementary to anneal under biological conditions and to provide
a substrate for Dicer that produces an siRNA sufficiently
complementary to the target sequence. Locked nucleic acids, or
LNA's, are well known to a skilled artisan (Elman et al., 2005;
Kurreck et al., 2002; Crinelli et al., 2002; Braasch and Corey,
2001; Bondensgaard et al., 2000; Wahlestedt et al., 2000). In one
embodiment, an LNA is incorporated at the 5' terminus of the sense
strand. In another embodiment, an LNA is incorporated at the 5'
terminus of the sense strand in duplexes designed to include a 3'
overhang on the antisense strand.
[0049] In one embodiment, the dsRNA has an asymmetric structure,
with the sense strand having a 25-base pair length, and the
antisense strand having a 27-base pair length with a 2 base
3'-overhang. In another embodiment, this dsRNA having an asymmetric
structure further contains 2 deoxynucleotides at the 3' end of the
sense strand in place of two of the ribonucleotides.
[0050] Suitable dsRNA compositions that contain two separate
oligonucleotides can be linked by a third structure. The third
structure will not block Dicer activity on the dsRNA and will not
interfere with the directed destruction of the RNA transcribed from
the target gene. In one embodiment, the third structure may be a
chemical linking group. Many suitable chemical linking groups are
known in the art and can be used. Alternatively, the third
structure may be an oligonucleotide that links the two
oligonucleotides of the dsRNA is a manner such that a hairpin
structure is produced upon annealing of the two oligonucleotides
making up the dsRNA composition. The hairpin structure will not
block Dicer activity on the dsRNA and will not interfere with the
directed destruction of the RNA transcribed from the target
gene.
[0051] In a third embodiment of the first aspect of the present
invention, the dsRNA, i.e., the precursor RNAi molecule, has
several properties which enhances its processing by Dicer.
According to this embodiment, the dsRNA has a length sufficient
such that it is processed by Dicer to produce an siRNA and at least
one of the following properties: (i) the dsRNA is asymmetric, e.g.,
has a 3' overhang on the sense strand and (ii) the dsRNA has a
modified 3' end on the antisense strand to direct orientation of
Dicer binding and processing of the dsRNA to an active siRNA.
According to this embodiment, the longest strand in the dsRNA
comprises 24-30 nucleotides. In one embodiment, the sense strand
comprises 24-30 nucleotides and the antisense strand comprises
22-28 nucleotides. Thus, the resulting dsRNA has an overhang on the
3' end of the sense strand. The overhang is 1-3 nucleotides, such
as 2 nucleotides. The antisense strand may also have a 5'
phosphate.
[0052] In another embodiment, the antisense strand is modified for
Dicer processing by suitable modifiers located at the 3' end of the
antisense strand, i.e., the dsRNA is designed to direct orientation
of Dicer binding and processing. Suitable modifiers include
nucleotides such as deoxyribonucleotides, dideoxyribonucleotides,
acyclonucleotides and the like and sterically hindered molecules,
such as fluorescent molecules and the like. Acyclonucleotides
substitute a 2-hydroxyethoxymethyl group for the
2'-deoxyribofuranosyl sugar normally present in dNMPs. Other
nucleotide modifiers could include 3'-deoxyadenosine (cordycepin),
3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI),
2',3'-dideoxy-3'-thiocytidine (3TC),
2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate
nucleotides of 3'-azido-3'-deoxythymidine (AZT),
2',3'-dideoxy-3'-thiocytidine (3TC) and
2',3'-didehydro-2',3'-dideoxythymidine (d4T). In one embodiment,
deoxynucleotides are used as the modifiers. When nucleotide
modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide
modifiers are substituted for the ribonucleotides on the 3' end of
the antisense strand. When sterically hindered molecules are
utilized, they are attached to the ribonucleotide at the 3' end of
the antisense strand. Thus, the length of the strand does not
change with the incorporation of the modifiers. In another
embodiment, the invention contemplates substituting two DNA bases
in the dsRNA to direct the orientation of Dicer processing. In a
further invention, two terminal DNA bases are located on the 3' end
of the antisense strand in place of two ribonucleotides forming a
blunt end of the duplex on the 5' end of the sense strand and the
3' end of the antisense strand, and a two-nucleotide RNA overhang
is located on the 3'-end of the sense strand. This is an asymmetric
composition with DNA on the blunt end and RNA bases on the
overhanging end.
[0053] The sense and antisense strands anneal under biological
conditions, such as the conditions found in the cytoplasm of a
cell. In addition, a region of one of the sequences, particularly
of the antisense strand, of the dsRNA has a sequence length of at
least 19 nucleotides, wherein these nucleotides are adjacent to the
3' end of antisense strand and are sufficiently complementary to a
nucleotide sequence of the RNA produced from the target gene.
[0054] Further in accordance with this embodiment, the dsRNA, i.e.,
the precursor RNAi molecule, may also have one or more of the
following additional properties: (a) the antisense strand has a
right shift from the typical 21mer and (b) the strands may not be
completely complementary, i.e., the strands may contain simple
mismatch pairings. A "typical" 21mer siRNA is designed using
conventional techniques, such as described above. This 21mer is
then used to design a right shift to include 1-7 additional
nucleotides on the 5' end of the 21mer. The sequence of these
additional nucleotides may have any sequence. Although the added
ribonucleotides may be complementary to the target gene sequence,
full complementarity between the target sequence and the antisense
siRNA is not required. That is, the resultant antisense siRNA is
sufficiently complementary with the target sequence. The first and
second oligonucleotides are not required to be completely
complementary. They only need to be substantially complementary to
anneal under biological conditions and to provide a substrate for
Dicer that produces an siRNA sufficiently complementary to the
target sequence.
[0055] In one embodiment, the dsRNA has an asymmetric structure,
with the antisense strand having a 25-base pair length, and the
sense strand having a 27-base pair length with a 2 base
3'-overhang. In another embodiment, this dsRNA having an asymmetric
structure further contains 2 deoxynucleotides at the 3' end of the
antisense strand.
[0056] Suitable dsRNA compositions that contain two separate
oligonucleotides can be linked by a third structure. The third
structure will not block Dicer activity on the dsRNA and will not
interfere with the directed destruction of the RNA transcribed from
the target gene. In one embodiment, the third structure may be a
chemical linking group. Many suitable chemical linking groups are
known in the art and can be used. Alternatively, the third
structure may be an oligonucleotide that links the two
oligonucleotides of the dsRNA is a manner such that a hairpin
structure is produced upon annealing of the two oligonucleotides
making up the dsRNA composition. The hairpin structure will not
block Dicer activity on the dsRNA and will not interfere with the
directed destruction of the RNA transcribed from the target
gene.
[0057] One feature of the dsRNA compositions of the present
invention is that they can serve as a substrate for Dicer.
Typically, the dsRNA compositions of this invention will not have
been treated with Dicer, other RNases, or extracts that contain
them. In the current invention this type of pretreatment can
prevent Dicer annealing. Several methods are known and can be used
for determining whether a dsRNA composition serves as a substrate
for Dicer. For example, Dicer activity can be measured in vitro
using the Recombinant Dicer Enzyme Kit (GTS, San Diego, Calif.)
according to the manufacturer's instructions. Dicer activity can be
measured in vivo by treating cells with dsRNA and maintaining them
for 24 h before harvesting them and isolating their RNA. RNA can be
isolated using standard methods, such as with the RNeaSy.TM. Kit
(Qiagen) according to the manufacturer's instructions. The isolated
RNA can be separated on a 10% PAGE gel which is used to prepare a
standard RNA blot that can be probed with a suitable labeled
deoxyoligonucleotide, such as an oligonucleotide labeled with the
Starfire.RTM. Oligo Labeling System (Integrated DNA Technologies,
Inc., Coralville, Iowa).
[0058] The effect that a dsRNA has on a cell can depend upon the
cell itself. In some circumstances a dsRNA could induce apoptosis
or gene silencing in one cell type and not another. Thus, it is
possible that a dsRNA could be suitable for use in one cell and not
another. To be considered "suitable" a dsRNA composition need not
be suitable under all possible circumstances in which it might be
used, rather it need only be suitable under a particular set of
circumstances.
[0059] Modifications can be included in the dsRNA, i.e., the
precursor RNAi molecule, of the present invention so long as the
modification does not prevent the dsRNA composition from serving as
a substrate for Dicer. In one embodiment, one or more modifications
are made that enhance Dicer processing of the dsRNA. In a second
embodiment, one or more modifications are made that result in more
effective RNAi generation. In a third embodiment, one or more
modifications are made that support a greater RNAi effect. In a
fourth embodiment, one or more modifications are made that result
in greater potency per each dsRNA molecule to be delivered to the
cell. Modifications can be incorporated in the 3'-terminal region,
the 5'-terminal region, in both the 3'-terminal and 5'-terminal
region or in some instances in various positions within the
sequence. With the restrictions noted above in mind any number and
combination of modifications can be incorporated into the dsRNA.
Where multiple modifications are present, they may be the same or
different. Modifications to bases, sugar moieties, the phosphate
backbone, and their combinations are contemplated. Either
5'-terminus can be phosphorylated.
[0060] Examples of modifications contemplated for the phosphate
backbone include phosphonates, including methylphosphonate,
phosphorothioate, and phosphotriester modifications such as
alkylphosphotriesters, and the like. Examples of modifications
contemplated for the sugar moiety include 2'-alkyl pyrimidine, such
as 2'-O-methyl, 2'-fluoro, amino, and deoxy modifications and the
like (see, e.g., Amarzguioui et al., 2003). Examples of
modifications contemplated for the base groups include abasic
sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil,
5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the
like. Locked nucleic acids, or LNA's, could also be incorporated.
Many other modifications are known and can be used so long as the
above criteria are satisfied. Examples of modifications are also
disclosed in U.S. Pat. Nos. 5,684,143, 5,858,988 and 6,291,438 and
in U.S. published patent application No. 2004/0203145 A1, each
incorporated herein by reference. Other modifications are disclosed
in Herdewijn (2000), Eckstein (2000), Rusckowski et al. (2000),
Stein et al. (2001) and Vorobjev et al. (2001).
[0061] Additionally, the dsRNA structure can be optimized to ensure
that the oligonucleotide segment generated from Dicer's cleavage
will be the portion of the oligonucleotide that is most effective
in inhibiting gene expression. For example, in one embodiment of
the invention a 27-bp oligonucleotide of the dsRNA structure is
synthesized wherein the anticipated 21 to 22-bp segment that will
inhibit gene expression is located on the 3'-end of the antisense
strand. The remaining bases located on the 5'-end of the antisense
strand will be cleaved by Dicer and will be discarded. This cleaved
portion can be homologous (i.e., based on the sequence of the
target sequence) or non-homologous and added to extend the nucleic
acid strand.
[0062] RNA may be produced enzymatically or by partial/total
organic synthesis, and modified ribonucleotides can be introduced
by in vitro enzymatic or organic synthesis. In one embodiment, each
strand is prepared chemically. Methods of synthesizing RNA
molecules are known in the art, in particular, the chemical
synthesis methods as described in Verma and Eckstein (1998) or as
described herein.
[0063] As is known, RNAi methods are applicable to a wide variety
of genes in a wide variety of organisms and the disclosed
compositions and methods can be utilized in each of these contexts.
Examples of genes which can be targeted by the disclosed
compositions and methods include endogenous genes which are genes
that are native to the cell or to genes that are not normally
native to the cell. Without limitation these genes include
oncogenes, cytokine genes, idiotype (Id) protein genes, prion
genes, genes that expresses molecules that induce angiogenesis,
genes for adhesion molecules, cell surface receptors, proteins
involved in metastasis, proteases, apoptosis genes, cell cycle
control genes, genes that express EGF and the EGF receptor,
multi-drug resistance genes, such as the MDR1 gene.
[0064] More specifically, the target mRNA of the invention
specifies the amino acid sequence of a cellular protein (e.g., a
nuclear, cytoplasmic, transmembrane, or membrane-associated
protein). In another embodiment, the target mRNA of the invention
specifies the amino acid sequence of an extracellular protein
(e.g., an extracellular matrix protein or secreted protein). As
used herein, the phrase "specifies the amino acid sequence" of a
protein means that the mRNA sequence is translated into the amino
acid sequence according to the rules of the genetic code. The
following classes of proteins are listed for illustrative purposes:
developmental proteins (e.g., adhesion molecules, cyclin kinase
inhibitors, Wnt family members, Pax family members, Winged helix
family members, Hox family members, cytokines/lymphokines and their
receptors, growth/differentiation factors and their receptors,
neurotransmitters and their receptors); oncogene-encoded proteins
(e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB,
EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK,
LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML, RET, SRC,
TALI, TCL3, and YES); tumor suppressor proteins (e.g., APC, BRCA1,
BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53, and WTI); and enzymes
(e.g., ACC synthases and oxidases, ACP desaturases and
hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol
dehydrogenases, amylases, amyloglucosidases, catalases, cellulases,
chalcone synthases, chitinases, cyclooxygenases, decarboxylases,
dextriinases, DNA and RNA polymerases, galactosidases, glucanases,
glucose oxidases, granule-bound starch synthases, GTPases,
helicases, hernicellulases, integrases, inulinases, invertases,
isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes,
nopaline synthases, octopine synthases, pectinesterases,
peroxidases, phosphatases, phospholipases, phosphorylases,
phytases, plant growth regulator synthases, polygalacturonases,
proteinases and peptidases, pullanases, recombinases, reverse
transcriptases, RUBISCOs, topoisomerases, and xylanases).
[0065] In one aspect, the target mRNA molecule of the invention
specifies the amino acid sequence of a protein associated with a
pathological condition. For example, the protein may be a
pathogen-associated protein (e.g., a viral protein involved in
immunosuppression of the host, replication of the pathogen,
transmission of the pathogen, or maintenance of the infection), or
a host protein which facilitates entry of the pathogen into the
host, drug metabolism by the pathogen or host, replication or
integration of the pathogen's genome, establishment or spread of
infection in the host, or assembly of the next generation of
pathogen. Pathogens include RNA viruses such as flaviviruses,
picornaviruses, rhabdoviruses, filoviruses, retroviruses, including
lentiviruses, or DNA viruses such as adenoviruses, poxviruses,
herpes viruses, cytomegaloviruses, hepadnaviruses or others.
Additional pathogens include bacteria, fungi, helminths,
schistosomes and trypanosomes. Other kinds of pathogens can include
mammalian transposable elements. Alternatively, the protein may be
a tumor-associated protein or an autoimmune disease-associated
protein.
[0066] The target gene may be derived from or contained in any
organism. The organism may be a plant, animal, protozoa, bacterium,
virus or fungus. See e.g., U.S. Pat. No. 6,506,559, incorporated
herein by reference.
[0067] In another aspect, the present invention provides for a
pharmaceutical composition comprising the dsRNA of the present
invention. The dsRNA sample can be suitably formulated and
introduced into the environment of the cell by any means that
allows for a sufficient portion of the sample to enter the cell to
induce gene silencing, if it is to occur. Many formulations for
dsRNA are known in the art and can be used so long as dsRNA gains
entry to the target cells so that it can act. See, e.g., U.S.
published patent application Nos. 2004/0203145 A1 and 2005/0054598
A1, each incorporated herein by reference. For example, dsRNA can
be formulated in buffer solutions such as phosphate buffered saline
solutions, liposomes, micellar structures, and capsids.
Formulations of dsRNA with cationic lipids can be used to
facilitate transfection of the dsRNA into cells. For example,
cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188,
incorporated herein by reference), cationic glycerol derivatives,
and polycationic molecules, such as polylysine (published PCT
International Application WO 97/30731, incorporated herein by
reference), can be used. Suitable lipids include Oligofectamine,
Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals,
Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used
according to the manufacturer's instructions.
[0068] It can be appreciated that the method of introducing dsRNA
into the environment of the cell will depend on the type of cell
and the make up of its environment. For example, when the cells are
found within a liquid, one preferable formulation is with a lipid
formulation such as in lipofectamine and the dsRNA can be added
directly to the liquid environment of the cells. Lipid formulations
can also be administered to animals such as by intravenous,
intramuscular, or intraperitoneal injection, or orally or by
inhalation or other methods as are known in the art. When the
formulation is suitable for administration into animals such as
mammals and more specifically humans, the formulation is also
pharmaceutically acceptable. Pharmaceutically acceptable
formulations for administering oligonucleotides are known and can
be used. In some instances, it may be preferable to formulate dsRNA
in a buffer or saline solution and directly inject the formulated
dsRNA into cells, as in studies with oocytes. The direct injection
of dsRNA duplexes may also be done. For suitable methods of
introducing dsRNA see U.S. published patent application No.
2004/0203145 A1, incorporated herein by reference.
[0069] Suitable amounts of dsRNA must be introduced and these
amounts can be empirically determined using standard methods.
Typically, effective concentrations of individual dsRNA species in
the environment of a cell will be about 50 nanomolar or less 10
nanomolar or less, or compositions in which concentrations of about
1 nanomolar or less can be used. In other embodiment, methods
utilize a concentration of about 200 picomolar or less and even a
concentration of about 50 picomolar or less can be used in many
circumstances.
[0070] The method can be carried out by addition of the dsRNA
compositions to any extracellular matrix in which cells can live
provided that the dsRNA composition is formulated so that a
sufficient amount of the dsRNA can enter the cell to exert its
effect. For example, the method is amenable for use with cells
present in a liquid such as a liquid culture or cell growth media,
in tissue explants, or in whole organisms, including animals, such
as mammals and especially humans.
[0071] Expression of a target gene can be determined by any
suitable method now known in the art or that is later developed. It
can be appreciated that the method used to measure the expression
of a target gene will depend upon the nature of the target gene.
For example, when the target gene encodes a protein the term
"expression" can refer to a protein or transcript derived from the
gene. In such instances the expression of a target gene can be
determined by measuring the amount of mRNA corresponding to the
target gene or by measuring the amount of that protein. Protein can
be measured in protein assays such as by staining or immunoblotting
or, if the protein catalyzes a reaction that can be measured, by
measuring reaction rates. All such methods are known in the art and
can be used. Where the gene product is an RNA species expression
can be measured by determining the amount of RNA corresponding to
the gene product. Several specific methods for detecting gene
expression are described in Example 1. The measurements can be made
on cells, cell extracts, tissues, tissue extracts or any other
suitable source material.
[0072] The determination of whether the expression of a target gene
has been reduced can be by any suitable method that can reliably
detect changes in gene expression. Typically, the determination is
made by introducing into the environment of a cell undigested dsRNA
such that at least a portion of that dsRNA enters the cytoplasm and
then measuring the expression of the target gene. The same
measurement is made on identical untreated cells and the results
obtained from each measurement are compared.
[0073] The dsRNA can be formulated as a pharmaceutical composition
which comprises a pharmacologically effective amount of a dsRNA and
pharmaceutically acceptable carrier. A pharmacologically or
therapeutically effective amount refers to that amount of a dsRNA
effective to produce the intended pharmacological, therapeutic or
preventive result. The phrases "pharmacologically effective amount"
and "therapeutically effective amount" or simply "effective amount"
refer to that amount of an RNA effective to produce the intended
pharmacological, therapeutic or preventive result. For example, if
a given clinical treatment is considered effective when there is at
least a 20% reduction in a measurable parameter associated with a
disease or disorder, a therapeutically effective amount of a drug
for the treatment of that disease or disorder is the amount
necessary to effect at least a 20% reduction in that parameter.
[0074] The phrase "pharmaceutically acceptable carrier" refers to a
carrier for the administration of a therapeutic agent. Exemplary
carriers include saline, buffered saline, dextrose, water,
glycerol, ethanol, and combinations thereof. For drugs administered
orally, pharmaceutically acceptable carriers include, but are not
limited to pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract. The pharmaceutically acceptable
carrier of the disclosed dsRNA composition may be micellar
structures, such as a liposomes, capsids, capsoids, polymeric
nanocapsules, or polymeric microcapsules.
[0075] Polymeric nanocapsules or microcapsules facilitate transport
and release of the encapsulated or bound dsRNA into the cell. They
include polymeric and monomeric materials, especially including
polybutylcyanoacrylate. A summary of materials and fabrication
methods has been published (see Kreuter, 1991). The polymeric
materials which are formed from monomeric and/or oligomeric
precursors in the polymerization/nanoparticle generation step, are
per se known from the prior art, as are the molecular weights and
molecular weight distribution of the polymeric material which a
person skilled in the field of manufacturing nanoparticles may
suitably select in accordance with the usual skill.
[0076] Suitably formulated pharmaceutical compositions of this
invention can be administered by any means known in the art such as
by parenteral routes, including intravenous, intramuscular,
intraperitoneal, subcutaneous, transdermal, airway (aerosol),
rectal, vaginal and topical (including buccal and sublingual)
administration. In some embodiments, the pharmaceutical
compositions are administered by intravenous or intraparenteral
infusion or injection.
[0077] In general a suitable dosage unit of dsRNA will be in the
range of 0.001 to 0.25 milligrams per kilogram body weight of the
recipient per day, or in the range of 0.01 to 20 micrograms per
kilogram body weight per day, or in the range of 0.01 to 10
micrograms per kilogram body weight per day, or in the range of
0.10 to 5 micrograms per kilogram body weight per day, or in the
range of 0.1 to 2.5 micrograms per kilogram body weight per day.
Pharmaceutical composition comprising the dsRNA can be administered
once daily. However, the therapeutic agent may also be dosed in
dosage units containing two, three, four, five, six or more
sub-doses administered at appropriate intervals throughout the day.
In that case, the dsRNA contained in each sub-dose must be
correspondingly smaller in order to achieve the total daily dosage
unit. The dosage unit can also be compounded for a single dose over
several days, e.g., using a conventional sustained release
formulation which provides sustained and consistent release of the
dsRNA over a several day period. Sustained release formulations are
well known in the art. In this embodiment, the dosage unit contains
a corresponding multiple of the daily dose. Regardless of the
formulation, the pharmaceutical composition must contain dsRNA in a
quantity sufficient to inhibit expression of the target gene in the
animal or human being treated. The composition can be compounded in
such a way that the sum of the multiple units of dsRNA together
contain a sufficient dose.
[0078] Data can be obtained from cell culture assays and animal
studies to formulate a suitable dosage range for humans. The dosage
of compositions of the invention lies within a range of circulating
concentrations that include the ED.sub.50 (as determined by known
methods) 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
of the compound that includes the IC.sub.50 (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 of dsRNA in plasma may be measured by standard
methods, for example, by high performance liquid
chromatography.
[0079] In a further aspect, the present invention relates to a
method for treating a subject having a disease or at risk of
developing a disease caused by the expression of a target gene. In
this embodiment, the dsRNA can act as novel therapeutic agents for
controlling one or more of cellular proliferative and/or
differentiative disorders, disorders associated with bone
metabolism, immune disorders, hematopoietic disorders,
cardiovascular disorders, liver disorders, viral diseases, or
metabolic disorders. The method comprises administering a
pharmaceutical composition of the invention to the patient (e.g.,
human), such that expression of the target gene is silenced.
Because of their high specificity, the dsRNAs of the present
invention specifically target mRNAs of target genes of diseased
cells and tissues.
[0080] In the prevention of disease, the target gene may be one
which is required for initiation or maintenance of the disease, or
which has been identified as being associated with a higher risk of
contracting the disease. In the treatment of disease, the dsRNA can
be brought into contact with the cells or tissue exhibiting the
disease. For example, dsRNA substantially identical to all or part
of a mutated gene associated with cancer, or one expressed at high
levels in tumor cells, e.g. aurora kinase, may be brought into
contact with or introduced into a cancerous cell or tumor gene.
[0081] Examples of cellular proliferative and/or differentiative
disorders include cancer, e.g., carcinoma, sarcoma, metastatic
disorders or hematopoietic neoplastic disorders, e.g., leukemias. A
metastatic tumor can arise from a multitude of primary tumor types,
including but not limited to those of prostate, colon, lung, breast
and liver origin. As used herein, the terms "cancer,"
"hyperproliferative," and "neoplastic" refer to cells having the
capacity for autonomous growth, i.e., an abnormal state of
condition characterized by rapidly proliferating cell growth. These
terms are meant to include all types of cancerous growths or
oncogenic processes, metastatic tissues or malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness. Proliferative disorders also include
hematopoietic neoplastic disorders, including diseases involving
hyperplastic/neoplastic cells of hematopoietic origin, e.g.,
arising from myeloid, lymphoid or erythroid lineages, or precursor
cells thereof.
[0082] The present invention can also be used to treat a variety of
immune disorders, in particular those associated with
overexpression of a gene or expression of a mutant gene. Examples
of hematopoietic disorders or diseases include, without limitation,
autoimmune diseases (including, for example, diabetes mellitus,
arthritis (including rheumatoid arthritis, juvenile rheumatoid
arthritis, osteoarthritis, psoriatic arthritis), multiple
sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus
erythematosis, autoimmune thyroiditis, dermatitis (including atopic
dermatitis and eczematous dermatitis), psoriasis, Sjogren's
Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing, loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Graves' disease, sarcoidosis,
primary biliary cirrhosis, uveitis posterior, and interstitial lung
fibrosis), graft-versus-host disease, cases of transplantation, and
allergy.
[0083] In another embodiment, the invention relates to a method for
treating viral diseases, including but not limited to human
papilloma virus, hepatitis C, hepatitis B, herpes simplex virus
(HSV), HIV-AIDS, poliovirus, and smallpox virus. dsRNAs of the
invention are prepared as described herein to target expressed
sequences of a virus, thus ameliorating viral activity and
replication. The molecules can be used in the treatment and/or
diagnosis of viral infected tissue, both animal and plant. Also,
such molecules can be used in the treatment of virus-associated
carcinoma, such as hepatocellular cancer.
[0084] The dsRNA of the present invention can also be used to
inhibit the expression of the multi-drug resistance 1 gene
("MDR1"). "Multi-drug resistance" (MDR) broadly refers to a pattern
of resistance to a variety of chemotherapeutic drugs with unrelated
chemical structures and different mechanisms of action. Although
the etiology of MDR is multifactoral, the overexpression of
P-glycoprotein (Pgp), a membrane protein that mediates the
transport of MDR drugs, remains the most common alteration
underlying MDR in laboratory models (Childs and Ling, 1994).
Moreover, expression of Pgp has been linked to the development of
MDR in human cancer, particularly in the leukemias, lymphomas,
multiple myeloma, neuroblastoma, and soft tissue sarcoma (Fan et
al.). Recent studies showed that tumor cells expressing
MDR-associated protein (MRP) (Cole et al., 1992), lung resistance
protein (LRP) (Scheffer et al., 1995) and mutation of DNA
topoisomerase II (Beck, 1989) also may render MDR.
[0085] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA, genetics,
immunology, cell biology, cell culture and transgenic biology,
which are within the skill of the art. See, e.g., Maniatis et al.,
1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd
Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel
et al., 1992), Current Protocols in Molecular Biology (John Wiley
& Sons, including periodic updates); Glover, 1985, DNA Cloning
(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow
and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology,
6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan
et al., Manipulating the Mouse Embryo, (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M.,
The zebrafish book. A guide for the laboratory use of zebrafish
(Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000).
EXAMPLES
[0086] The present invention is described by reference to the
following Examples, which are offered by way of illustration and
are not intended to limit the invention in any manner. Standard
techniques well known in the art or the techniques specifically
described below were utilized.
Example 1
Preparation of Double-Stranded RNA Oligonucleotides
[0087] Oligonucleotide Synthesis and Purification
[0088] RNA oligonucleotides were synthesized using solid phase
phosphoramidite chemistry, deprotected and desalted on NAP-5
columns (Amersham Pharmacia Biotech, Piscataway, N.J.) using
standard techniques (Damha and Olgivie, 1993; Wincott et al.,
1995). The oligomers were purified using ion-exchange high
performance liquid chromatography (IE-HPLC) on an Amersham Source
15Q column (1.0 cm.times.25 cm) (Amersham Pharmacia Biotech,
Piscataway, N.J.) using a 15 min step-linear gradient. The gradient
varied from 90:10 Buffers A:B to 52:48 Buffers A:B, where Buffer A
was 100 mM Tris pH 8.5 and Buffer B was 100 mM Tris pH 8.5, 1 M
NaCl. Samples were monitored at 260 nm and peaks corresponding to
the full-length oligonucleotide species were collected, pooled,
desalted on NAP-5 columns, and lyophilized.
[0089] The purity of each oligomer was determined by capillary
electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.,
Fullerton, Calif.). The CE capillaries had a 100 .mu.m inner
diameter and contained ssDNA 100R Gel (Beckman-Coulter). Typically,
about 0.6 nmole of oligonucleotide was injected into a capillary,
ran in an electric field of 444 V/cm and detected by UV absorbance
at 260 nm. Denaturing Tris-Borate-7 M-urea running buffer was
purchased from Beckman-Coulter. Oligoribonucleotides were at least
90% pure as assessed by CE for use in experiments described below.
Compound identity was verified by matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectroscopy on a
Voyager DE.TM. Biospectometry Work Station (Applied Biosystems,
Foster City, Calif.) following the manufacturer's recommended
protocol. Relative molecular masses of all oligomers were within
0.2% of expected molecular mass.
[0090] Preparation of Duplexes
[0091] Single-stranded RNA (ssRNA) oligomers were resuspended at
100 .mu.M concentration in duplex buffer consisting of 100 mM
potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and
antisense strands were mixed in equal molar amounts to yield a
final solution of 50 .mu.M duplex. Samples were heated to
95.degree. C. for 5' and allowed to cool to room temperature before
use. Double-stranded RNA (dsRNA) oligomers were stored at
-20.degree. C. Single-stranded RNA oligomers were stored
lyophilized or in nuclease-free water at -80.degree. C.
[0092] Nomenclature
[0093] For consistency, the following nomenclature has been
employed throughout the specification and Examples. Names given to
duplexes indicate the length of the oligomers and the presence or
absence of overhangs. A "21+2" duplex contains two RNA strands both
of which are 21 nucleotides in length, also termed a 21mer siRNA
duplex, and having a 2 base 3'-overhang. A "21-2" design is a 21mer
siRNA duplex with a 2 base 5'-overhang. A 21-0 design is a 21mer
siRNA duplex with no overhangs (blunt). A "21+2UU" is a 21mer
duplex with 2-base 3'-overhang and the terminal 2 bases at the
3'-ends are both U residues (which may result in mismatch with
target sequence). A "25/27" is an asymmetric duplex having a 25
base sense strand and a 27 base antisense strand with a 2-base
3'-overhang. A "27/25" is an asymmetric duplex having a 27 base
sense strand and a 25 base antisense strand.
Example 2
Increased Potency of 25mers
[0094] This example demonstrates that dsRNAs having strands that
are 25 nucleotides in length or longer have surprisingly increased
potency in mammalian systems than known 21mer to 23mer siRNAs.
[0095] During investigations of the effects of different 5' and 3'
end structures of dsRNAs made through bacteriophage T7 in vitro
transcription (Kim et al., 2004), we observed that some seemed to
have greater potency than synthetic 21mer siRNAs directed to the
same target site, and that this property seemed to correlate with
length. To further explore this phenomenon, we systematically
studied the silencing properties of chemically synthesized duplex
RNAs of different lengths and designs.
[0096] Cell Culture, Transfection, and EGFP Assays
[0097] HEK 293 cells were split in 24-well plates to 60% confluency
in DMEM medium 1 d before transfection. After adding the aliquot of
each RNA, 50 .mu.l of Opti Media containing the reporter vectors
was added. Next, 50 .mu.l of Opti Media containing 1.5 .mu.l of
Lipofectamine 2000 (Invitrogen) was mixed and incubated for 15 min.
The cells were then added in 0.4 ml of DMEM medium. To normalize
for transfection efficiency, each assay included cotransfection of
the target and/or duplex RNAs with either firefly luciferase or a
red fluorescent protein (RFP) reporter plasmid (all other assays).
For the luciferase assay, the Steady Glo Luciferase assay kit was
used according to manufacturer's instructions (Promega). For RFP
cotransfection, the indicated amount of EGFP reporter plasmid
(pLEGFP-C1 vector, Clontech) was transfected with 20 ng of RFP
reporter plasmid (pDsRed2-C1, BD Sciences). After 24 h, RFP
expression was monitored by fluorescence microscopy. Only
experiments where transfection efficiency was >90% (as assessed
by RFP expression) were evaluated. EGFP expression was measured 24
h later. EGFP expression was determined either from the median
number of EGFP-fluorescent cells determined by FACS (live cells) or
by fluorometer readings (cell extracts).
[0098] For EGFP assays using NIH3T3 cells stably expressing EGFP,
measurements were determined using a VersaFluor Fluorometer
(Bio-Rad) using excitation filter D490 and emission filter D520.
Before transfections, cells were seeded to approximately 30%
confluency in a 24-well plate. On day 0, cells were transfected as
described above and the medium was changed on day 1 after
transfection. The first EGFP assay was carried out on day 3. For
extract preparation 1.times.10.sup.5 cells were taken and
1.times.10.sup.4 cells were further propagated for the day 6 EGFP
assays. On days 6 and 9 the same procedure was repeated.
[0099] Extract measurements of EGFP were obtained as follows:
1.times.10.sup.5 cells were suspended in 300 .mu.l of PBS and
sonicated for 10 s followed by a 2-min microcentrifugation. The
supernatants were used for fluorescence measurements. Percentages
of EGFP expression were determined relative to extracts prepared
from untreated NIH3T3 cells.
[0100] Nucleic Acid Reagents
[0101] The reporter system employed EGFP either as a transfection
plasmid vector pEGFP-C1 (Clontech, Palo Alto, Calif.) or as a
stable transformant in an NIH 3T3 cell line. The coding sequence of
EGFP is shown in Table 1, from Genbank accession #U55763. The ATG
start codon and TAA stop codons are highlighted in bold font and
sites target by siRNA reagents in this Example and other Examples
are underscored.
TABLE-US-00001 TABLE 1 Nucleotide Sequence of EGFP
atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggt
cgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagg
gcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcacc
accggcaagctgcccgtgccctggcccaccctcgtgaccaccctgaccta
cggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgact
atcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatctt
cttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagg
gcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggag
gacggcaacatcctggggcacaagctggagtacaactacaacagccacaa
cgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttca
agatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactac
cagcagaacacccccatcggcgacggccccgtgctgctgcccgacaacca
ctacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcg
atcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggc
atggacgagctgtacaagtaa (SEQ ID NO:1)
[0102] Site-1 used for siRNA targeting in EGFP for this example
was:
TABLE-US-00002 SITE 1: (SEQ ID NO:2) 5'
GCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGC 3'.
[0103] RNA duplexes were synthesized and prepared as described in
Example 1. RNA duplexes targeting EGFP Site-1 are summarized in
Table 2. Some sequences had the dinucleotide sequence "UU" placed
at the 3'-end of the sense strand (Elbashir et al., 2001c; Hohjoh,
2002). Mismatches that resulted from including 3'-terminal "UU" or
where a mismatch was intentionally positioned are highlighted in
bold and underscored.
TABLE-US-00003 TABLE 2 Summary of Oligonucleotide Reagents, EGFP
Site-1 Sequence Name SEQ ID NO. 5'
GCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGC 3' EGFP Site-1 SEQ ID NO:2
5' GCAAGCUGACCCUGAAGUUCA EGFPS1-21 - 2 SEQ ID No:3 3'
UUCGACUGGGACUUCAAGUAG SEQ ID NO:4 5' AAGCUGACCCUGAAGUUCAUC
EGFPS1-21 + 0 SEQ ID No:5 3' UUCGACUGGGACUUCAAGUAG SEQ ID No:6 5'
CCUGAAGUUCAUCUGCACCAC EGFPS1-21 + 2 (1) SEQ ID No:7 3'
UGGGACUUCAAGUAGACGUGG SEQ ID No:8 5' CCCUGAAGUUCAUCUGCACCA
EGFPS1-21 + 2 (2) SEQ ID No:9 3' CUGGGACUUCAAGUAGACGUG SEQ ID No:10
5' ACCCUGAAGUUCAUCUGCACC EGFPS1-21 + 2 (3) SEQ ID No:11 3'
ACUGGGACUUCAAGUAGACGU SEQ ID No:12 5' GACCCUGAAGUUCAUCUGCAC
EGFPS1-21 + 2 (4) SEQ ID No:13 3' GACUGGGACUUCAAGUAGACG SEQ ID
No:14 5' UGACCCUGAAGUUCAUCUGCA EGFPS1-21 + 2 (5) SEQ ID No:15 3'
CGACUGGGACUUCAAGUAGAC SEQ ID No:16 5' CUGACCCUGAAGUUCAUCUGC
EGFPS1-21 + 2 (6) SEQ ID No:17 3' UCGACUGGGACUUCAAGUAGA SEQ ID
No:18 5' GCUGACCCUGAAGUUCAUCUG EGFPS1-21 + 2 (7) SEQ ID No:19 3'
UUCGACUGGGACUUCAAGUAG SEQ ID No:20 5' GCAAGCUGACCCUGAAGUUCAUU
EGFPS1-23 - 2UU SEQ ID No:21 3' UUCGACUGGGACUUCAAGUAGAC SEQ ID
No:22 5' GCUGACCCUGAAGUUCAUCUGUU EGFPS1-23 + 2UU SEQ ID No:23 3'
UUCGACUGGGACUUCAAGUAGAC SEQ ID No:24 5' GCAAGCUGACCCUGAAGUUCAUUU
EGFPS1-24 - 2UU SEQ ID No:25 3' UUCGACUGGGACUUCAAGUAGACG SEQ ID
No:26 5' GCUGACCCUGAAGUUCAUCUGCUU EGFPS1-24 + 2UU SEQ ID No:27 3'
UUCGACUGGGACUUCAAGUAGACG SEQ ID No:28 5' GCAAGCUGACCCUGAAGUUCAUCUU
EGFPS1-25 - 2UU SEQ ID No:29 3' UUCGACUGGGACUUCAAGUAGACGU SEQ ID
No:30 5' GCUGACCCUGAAGUUCAUCUGCAUU EGFPS1-25 + 2UU SEQ ID No:31 3'
UUCGACUGGGACUUCAAGUAGACGU SEQ ID No:32 5'
AAGCUGACCCUGAAGUUCAUCUGCAC EGFPS1-26 + 0 SEQ ID No:33 3'
UUCGACUGGGACUUCAAGUAGACGUG SEQ ID No:34 5'
AAGCUGACCCUGAAGUUCAUCUGCUU EGFPS1-26 + 0UU SEQ ID No:35 3'
UUCGACUGGGACUUCAAGUAGACGUG SEQ ID No:36 5'
GCAAGCUGACCCUGAAGUUCAUCUUU EGFPS1-26 - 2UU SEQ ID No:37 3'
UUCGACUGGGACUUCAAGUAGACGUG SEQ ID No:38 5'
GCUGACCCUGAAGUUCAUCUGCACUU EGFPS1-26 + 2UU SEQ ID No:39 3'
UUCGACUGGGACUUCAAGUAGACGUG SEQ ID No:40 5'
AAGCUGACCCUGAAGUUCAUCUGCACC EGFPS1-27 + 0 SEQ ID No:41 3'
UUCGACUGGGACUUCAAGUAGACGUGG SEQ ID No:42 5'
F-AAGCUGACCCUGAAGUUCAUCUGCACC EGFPS1-27 + 0 SEQ ID No:43 3'
UUCGACUGGGACUUCAAGUAGACGUGG-F FAM #1 SEQ ID No:44 5'
AAGCUGACCCUGAAGUUCAUCUGCACC-F EGFPS1-27 + 0 SEQ ID No:45 3'
UUCGACUGGGACUUCAAGUAGACGUGG-F FAM #2 SEQ ID No:44 5'
F-AAGCUGACCCUGAAGUUCAUCUGCACC EGFPS1-27 + 0 SEQ ID No:43 3'
F-UUCGACUGGGACUUCAAGUAGACGUGG FAM #4 SEQ ID No:46 5'
AAGCUGACCCUGAAGUUCAUCUGCACC-F EGFPS1-27 + 0 SEQ ID No:45 3'
F-UUCGACUGGGACUUCAAGUAGACGUGG FAM #5 SEQ ID No:46 5'
AAGCUGACCCUGAAGUUCAUCUGCAUU EGFPS1-27 + 0UU SEQ ID No:47 3'
UUCGACUGGGACUUCAAGUAGACGUGG SEQ ID No:48 5'
GCAAGCUGACCCUGAAGUUCAUCUGUU EGFPS1-27 - 2UU SEQ ID No:49 3'
UUCGACUGGGACUUCAAGUAGACGUGG SEQ ID No:50 5'
GCUGACCCUGAAGUUCAUCUGCACAUU EGFPS1-27 + 2UU/ SEQ ID No:51 3'
UUCGACUGGGACUUCAAGUAGACGUGG 25 SEQ ID No:52 5'
AAGCUGACCCUGAAGAUCAUCUGCAUU EGFPS1-27 + 0UU/ SEQ ID No:53 3'
UUCGACUGGGACUUCUAGUAGACGUGG 16 SEQ ID No:54 5'
AAGCUGACCCUGAAGAACAUCUGCAUU EGFPS1-27 + 0UU/ SEQ ID No:55 3'
UUCGACUGGGACUUCUUGUAGACGUGG 16, 17 SEQ ID No:56 5'
AAGCUGACCCUGAACAACAUCUGCAUU EGFPS1-27 + 0UU/ SEQ ID No:57 3'
UUCGACUGGGACUUGUUGUAGACGUGG 15, 16, 17 SEQ ID No:58 5'
AAGCUGACCCUGUUCAUCAUCUGCACC EGFPS1-27 + 0-mut SEQ ID No:59 3'
UUCGACUGGGACAAGUAGUAGACGUGG SEQ ID No:60 5'
AAGCUGACCCUGAAGUUCAUCUGCACCA EGFPS1-28 + 0 SEQ ID No:61 3'
UUCGACUGGGACUUCAAGUAGACGUGGU SEQ ID No:62 5'
AAGCUGACCCUGAAGUUCAUCUGCACCAC EGFPS1-29 + 0 SEQ ID No:63 3'
UUCGACUGGGACUUCAAGUAGACGUGGUG SEQ ID No:64 5'
AAGCUGACCCUGAAGUUCAUCUGCACCACC EGFPS1-30 + 0 SEQ ID No:65 3'
UUCGACUGGGACUUCAAGUAGACGUGGUGG SEQ ID No:66 5'
AAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAA EGFPS1-35 + 0 SEQ ID NO:67 3'
UUCGACUGGGACUUCAAGUAGACGUGGUGGCCGUU SEQ ID NO:68 5'
AAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGC EGFPS1-40 + 0 SEQ ID NO:69
3' UUCGACUGGGACUUCAAGUAGACGUGGUGGCCGUUCGACG SEQ ID NO:70 5'
AAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUG EGFPS1-45 + 0 SEQ ID
NO:71 3' UUCGACUGGGACUUCAAGUAGACGUGGUGGCCGUUCGACGGGCAC SEQ ID
NO:72
[0104] Results
[0105] An expanded set of synthetic RNA duplexes of varying length
containing 3' overhangs, 5' overhangs or blunt ends were tested for
their relative potency in a model system targeting `site 1` in EGFP
(Kim et al., 2003). We carried out our initial transfections using
50 nM of the various RNA duplexes (FIG. 1A). The real potency of
the longer duplexes was shown only when transfections were done at
subnanomolar concentrations. Using duplex RNA concentrations of 200
pM (FIG. 1B) and 50 pM (FIG. 1C), we observed that potency
increased with length. Blunt, 5'-overhanging and 3'-overhanging
ends were of similar potency. Increased potencies of the longer
duplex RNAs were observed even in the NIH3T3 cells stably
expressing EGFP (FIG. 1F). Duplexes longer than 27 nt were
synthesized and tested for RNAi efficacy (FIG. 1D). Maximal
inhibitory activity was seen at a duplex length of 27 bp (FIG. 1D).
Longer duplexes showed progressive loss of functional RNAi activity
and by 40-45 bp were wholly inactive at the tested concentrations,
which also correlated with poor in vitro cleavage of these duplexes
by Dicer (FIG. 1E).
Example 3
Dicer Substrates
[0106] This example demonstrates a method for determining whether a
dsRNA serves as a substrate for Dicer.
[0107] In Vitro Dicer Cleavage Assays
[0108] RNA duplexes (100 pmol) were incubated in 20 .mu.l of 20 mM
Tris pH 8.0, 200 mM NaCl, 2.5 mM MgCl.sub.2 with 1 unit of
recombinant human Dicer (Stratagene) for 24 h. A 3 .mu.l aliquot of
each reaction (15 pmol RNA) was separated in a 15% nondenaturing
polyacrylamide gel, stained with GelStar (Ambrex) and visualized
using UV excitation.
[0109] Results
[0110] Incubation of 21-bp to 27-bp RNA duplexes with recombinant
human Dicer resulted in cleavage of the 23mer, 25mer and 27mer
duplexes, but not the 21mer duplex (FIG. 2A). Determinations of
relative efficiencies of Dicer cleavage were not possible under the
in vitro conditions recommended by the supplier owing to the slow
kinetics of this reaction. Aside from the possibility that the
dsRNAs longer than 30 bp may need to be preprocessed by Drosha, a
micro RNA processing enzyme, to be good substrates for Dicer, we do
not have an explanation for why these longer dsRNAs are both poor
substrates for Dicer and poor triggers for RNAi.
Example 4
Effect of End-Modification on Dicer Activity
[0111] The effect of end-modification of dsRNA was tested using the
in vitro Dicer cleavage assay described in Example 3.
[0112] For the 27mer duplexes, we tested the effects of fluorescein
end-modification on Dicer activity and the ability to trigger RNAi
silencing. RNA oligonucleotides were synthesized for the EGFPS1
site with 6-carboxyfluorescein (6FAM) attached to the 5' end of the
sense (S) strand, 3' end of the S-strand, 5' end of the antisense
(AS) strand and 3' end of the AS strand. Pairwise combinations were
used to make duplex RNAs (FIG. 2B). Duplex 3 was the unmodified
wild-type EGFPS1 27+0 duplex. The structure of the 6FAM modified
duplexes are shown in Table 2. RNA duplexes were incubated for 24 h
with recombinant human Dicer, separated by nondenaturing
polyacrylamide gel electrophoresis (PAGE), stained and visualized
by UV excitation (FIG. 2C). Unlike in earlier experiments, in which
RNA duplexes were fully cleaved during a 24-h incubation (FIG. 2A),
all of the modified duplexes showed some degree of resistance to
cleavage by Dicer. Only the unmodified wild-type sequence (duplex
3) was fully cleaved in the in vitro Dicer reaction. The duplex
bearing a 3'-6FAM on the S strand and a 3'-6FAM on the AS strand
(duplex 5) was totally resistant to cleavage under these
conditions. Functional potencies of these five duplexes were
compared in EGFP cotransfection assays (FIG. 2D) using 200 pM RNA
concentrations. Parallel to the patterns seen for in vitro Dicer
cleavage, all of the 27mer duplexes with 6FAM-modified ends were
less potent than the unmodified duplexes in reducing EGFP
expression. Duplexes 1, 2 and 4, which showed partial cleavage with
recombinant Dicer, had three- to six-fold reduced RNAi activity.
Duplex 5, which showed no cleavage with recombinant Dicer, had
minimal RNAi activity, establishing a direct correlation between
the relative effectiveness of in vitro cleavage by Dicer and RNAi
in cell culture.
Example 5
In Vivo Processing by Dicer
[0113] This example confirms that the 27mer dsRNA are processed in
vivo by Dicer.
[0114] Assay for Intracellular Processing of 27mer RNAs
[0115] RNA duplexes were transfected as described above into HEK293
cells in a six-well plate at 10 nM. After 14 h, total RNA was
prepared as described below. First, 20 .mu.g of total RNA was
heated for 10 min at 75.degree. C. and mixed with .sup.32P
5'-end-labeled oligonucleotide probe (5'-ACCCTGAAGTTCATCTGCACC-3';
SEQ ID NO:73) and hybridized in 150 mM NaCl, 50 mM Tris-HCl, pH.
7.4, 1 mM EDTA) at 24.degree. C. Samples were loaded on 7.5%
nondenaturing polyacrylamide gel and separated at 200 V for 3 h at
4.degree. C., and the gel was exposed to X-ray film.
.sup.32P-end-labeled 27mer and 21mer duplex RNA oligos were used as
size standards.
[0116] Results
[0117] To confirm that the 27mer dsRNAs are processed by Dicer in
vivo, we transfected HEK293 cells with 10 nM of the duplex 3
(unmodified) or duplex 5 (both 3' ends modified with 6FAM). After
14 h, total RNA was isolated and hybridized with a .sup.32P end
labeled 21mer probe oligo (S strand). RNA was separated by
nondenaturing PAGE and visualized by autoradiography (FIG. 2E).
Similar to the results seen with in vitro Dicer cleavage, in RNA
prepared from cells transfected with duplex 3 (unmodified 27mer), a
smaller species was observed migrating with a 21mer duplex marker,
consistent with Dicer cleavage product. This 21mer species was not
detected in RNA from cells transfected with duplex 5 (3'
end-modified 27mer).
Example 6
Potency of dsRNAs
[0118] This example examines the potency of potential cleavage
products of the 27mer by Dicer.
[0119] Cleavage of a 27mer by Dicer could result in a variety of
distinct 21mers depending on where cleavage occurs; it is possible
that one or a mix of these possible 21mers is significantly more
potent than the specific 21mer that we used as our standard for
comparison. To test this possibility we synthesized seven different
21mers that could be derived from the EGFPS1 27+0 duplex, walking
in single-base steps along the antisense strand, using the
traditional 21+2 design. These seven duplexes were tested for RNAi
activity in the HEK293 cell cotransfection assay individually and
as a pool (FIG. 3A). At concentrations of 50 or 200 pM, neither the
individual 21mer duplexes nor the pooled set of seven 21mer
duplexes showed activity comparable to the 27mer duplex. In vitro
Dicer cleavage of the 27mers before transfection did not
significantly enhance efficacy (FIG. 3B). As an additional control,
we transfected a mutated EGFP 27mer duplex (Table 2),
EGFPS1-27+0/mut) harboring four consecutive, centrally placed
mismatched bases. The mismatches virtually eliminated any RNAi
activity (FIG. 3B).
Example 7
Analysis of Dicer Cleavage Products
[0120] This example analyzed the in vitro Dicer cleavage products
by mass spectroscopy.
[0121] Electrospray-Ionization Liquid Chromatography Mass
Spectroscopy
[0122] Electrospray-ionization liquid chromatography mass
spectroscopy (ESI-LCMS) of duplex RNAs before and after treatment
with Dicer were done using an Oligo HTCS system (Novatia), which
consisted of ThermoFinnigan TSQ7000, Xcalibur data system, ProMass
data processing software and Paradigm MS4 HPLC (Michrom
BioResources). The liquid chromatography step used before injection
into the mass spectrometer (LC-MS) removes most of the cations
complexed with the nucleic acids; some sodium ions can remain bound
to the RNA and are visualized as minor +22 or +44 species,
reflecting the net mass gain seen with substitution of sodium for
hydrogen.
[0123] Results
[0124] The species that are actually produced by incubation of the
EGFPS1 27+0 duplex with recombinant Dicer in vitro were identified
using electrospray ionization mass spectrometry (ESI MS) (FIGS. 4A
and 4B). Calculated masses for each possible digestion product that
could result from in vitro Dicer cleavage are shown in Table 3. The
ESI MS analyses of the in vitro cleavage products are consistent
with the known activity of this enzyme.
TABLE-US-00004 TABLE 3 Molecular Weights of Possible 21mer Duplexes
Derived from the 27mer Duplex by Dicer Processing Sequence (SEO ID
NO:) Name Mol Wt* 5' AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1-27 + 0
8552 3' UUCGACUGGGACUUCAAGUAGACGUGG (42) 8689 5'
ACCCUGAAGUUCAUCUGCACC (11) EGFPS1-21 + 2 (3) 6672** 3'
ACUGGGACUUCAAGUAGACGU (12) 6816** 5' GACCCUGAAGUUCAUCUGCAC (13)
EGFPS1-21 + 2 (4) 6712** 3' GACUGGGACUUCAAGUAGACG (14) 6855 5'
UGACCCUGAAGUUCAUCUGCA (15) EGFPS1-21 + 2 (5) 6713** 3'
CGACUGGGACUUCAAGUAGAC (16) 6815** 5' CUGACCCUGAAGUUCAUCUGC (17)
EGFPS1-21 + 2 (6) 6689 3' UCGACUGGGACUUCAAGUAGA (18) 6816** 5'
GCUGACCCUGAAGUUCAUCUG (19) EGFPS1-21 + 2 (7) 6729 3'
UUCGACUGGGACUUCAAGUAG (20) 6793 * Molecular weight of 27mer is the
original chemically synthesized duplex with hydroxyl ends.
Calculated weights of 21mers assume 5'phosphate on each strand
after Dicer Processing. ** Indicates masses that were consistent
with visualized peaks in FIG. 4B.
Example 8
Further Characterization of Inhibitory Properties of 27mer
dsRNA
[0125] To further characterize the inhibitory properties of the
27mer dsRNA in cells stably expressing the EGFP target, stably
transfected NIH3T3 cells expressing EGFP were transfected with 21+2
and 27+0 dsRNA duplexes (both at 5 nM). To obtain a quantitative
estimate of the duration of gene suppression, we carried out a
time-course experiment, observing EGFP expression on days 2, 4, 6,
8 and 10 after transfection. Cell extracts were prepared and
measured for EGFP fluorescence using a fluorometer (FIG. 5A). EGFP
suppression lasted approximately 4 d using the 21+2 siRNA,
consistent with previous observations (Persengiev et al., 2004),
whereas inhibition obtained with the 27+0 dsRNA persisted up to 10
d. A class of `hyperfunctional` 21+2 siRNAs has been reported
showing a similar extended duration of silencing (Reynolds et al.,
2004); however, these sequences are rare and difficult to find or
predict. Use of the 27mer dsRNA design may permit longer, more
potent RNAi to be achieved at a greater variety of target
sites.
Example 9
Effect of 27mer dsRNA on Site Selection
[0126] A frequent problem in using RNAi as a tool to systematically
inhibit the expression of any gene is that not all target sites are
equally susceptible to suppression by siRNAs (Sherer and Rossi,
2004), necessitating complex design algorithms to predict effective
sites (Reynolds et al., 2004; Ui-Tei et al., 2004; Amarzguioui and
Prydz, 2004). We therefore asked whether the increased potency of
the 27mer dsRNA permits effective targeting at sites that are not
active using traditional 21mer siRNAs. Duplex RNAs were made having
21+2 and 27+0 designs to two sites in EGFP (`EGFP-S2` and
`EGFP-S3`) both previously shown to be refractory to RNAi using
standard siRNAs (Kime and Rossi, 2003).
[0127] Nucleic Acid Reagents
[0128] The reporter system employed EGFP as in SEQ ID NO: 1 above.
Site-2 (also termed bad site 1) and Site-3 (also termed bad site 2)
in EGFP were targeted. RNA duplexes were synthesized and prepared
as described in Example 1. Site-2 and Site 3 used for siRNA
targeting in EGFP for this example were:
TABLE-US-00005 (SEQ ID NO:74) SITE 2: 5'
UGAAGCAGCACGACUUCUUCAAGUCCGCCAUG 3' and (SEQ ID NO:75) SITE 3: 5'
UGAAGUUCGAGGGCGACACCCUGGUGAACCGCAU 3'.
[0129] RNA duplexes targeting EGFP Site-2 and EGFP Site-3 are
summarized in Table 4.
TABLE-US-00006 TABLE 4 Summary of Oligonucleotide Reagents, EGFP
Site-2 Sequence Name SEQ ID NO: 5' UGAAGCAGCACGACUUCUUCAAGUCCGCCAUG
3' EGFP Site-2 SEQ ID NO:74 5' GCAGCACGACUUCUUCAAGUU EGFPS2-21 + 2
SEQ ID NO:76 3' UUCGUCGUGCUGAAGAAGUUC SEQ ID NO:77 5'
AAGCAGCACGACUUCUUCAAGUCCGCC EGFPS2-27 + 0 SEQ ID NO:78 3'
UUCGUCGUGCUGAAGAAGUUCAGGCGG SEQ ID NO:79 5'
UGAAGUUCGAGGGCGACACCCUGGUGAACCGCAU 3' EGFP Site-3 SEQ ID NO:75 5'
GUUCGAGGGCGACACCCUGUU EGFPS3-21 + 2 SEQ ID NO:80 3'
UUCAAGCUCCCGCUCUGGGAC SEQ ID NO:81 5' GUUCGAGGGCGACACCCUGGUGAACUU
EGFPS3-27 + 0UU SEQ ID NO:82 3' UUCAAGCUCCCGCUCUGGGACCACUUGGC SEQ
ID NO:83
[0130] Results
[0131] The duplexes were transfected into HEK293 cells using the
cotransfection assay format (FIG. 5B) at 1 nM and 10 nM. At these
doses, standard 21+2 siRNAs were ineffective at both sites, whereas
the 27mer dsRNAs reduced EGFP expression by 80-90% at EGFP-S2 and
by 50% (1 nM) and 80% (10 nM) at EGFP-S3. Despite the increased
potency of Dicer substrate dsRNAs, empirical testing of target
sites is still useful to find the most sensitive targets for RNAi.
In this regard, it is important that Dicer products of some 27mers
generated poorly functional siRNAs. By better understanding Dicer
substrate preferences, it should be possible to design substrate
RNAs that will generate the desired 21mers. We have observed that a
two-base 3' overhang on only one end of the 27mer will
preferentially guide Dicer to cleave 21-23 nt upstream of the
two-base 3' overhang.
[0132] This example demonstrates that dsRNAs of the invention can
efficiently target sites within the EGFP gene that were previously
considered poor targets by previously known methods. Use of the
method of the invention will therefore simplify site selection and
design criteria for RNAi. This example also shows that the
intentional placement of mismatches at the 3'-terminus of the sense
strand increases the potency of the 27mer duplex.
Example 10
Effect of 27mer dsRNA on Other Genes
[0133] To ensure that the increased potency of the 27mer dsRNAs was
not an artifact of targeting a reporter construct, we targeted two
endogenous transcripts, human hnRNP H mRNA (Markovtsov et al.,
2000) and mRNA encoding the La protein (Wolin and Cedervall,
2002).
[0134] RNAi Assays Against hnRNP H and La
[0135] HEK293 cells were plated to 30% confluency in a six-well
plate. The next day, the cells were transfected with the indicated
amount of dsRNA, and the medium was changed on the following day.
The cells were harvested in 300 .mu.l PBS 72 h after transfection.
Extracts were prepared as described above for the EGFP assays. For
western blots, 2 .mu.l of cell extract was loaded on a 10% SDS-PAGE
gel. Endogenous hnRNP H was detected using a rabbit polyclonal
anti-hnRNP H antibody (Markovtsov et al., 2000) and anti-rabbit
antibody conjugated to alkaline phosphatase (Sigma). .beta.-Actin
was detected with a mouse-derived anti-actin antibody (Sigma) and
anti-mouse antibody conjugated to alkaline phosphatase (Sigma). For
northern blot analyses, harvested cells were mixed with RNA STAT-60
(Tel-Test B) and total RNA was extracted according to the
manufacturer's protocol. RNA was electrophoresed in a 6% denaturing
polyacrylamide gel, transferred to a nylon membrane and probed with
.sup.32P-end-labeled oligos (La, 5'-CCAAAGGTACCCAGCCTTCATCCAGTT-3'
(SEQ ID NO:84); .beta.-actin, 5'-GTGAGGATGCCTCTCTTGCTCTGGGCCTCG-3'
(SEQ ID NO:85)). Hybridizations were carried out in 10 ml of
hybridization solution (1 ml 50.times.Denhardt's, 3 ml
20.times.SSPE, 0.5 ml 10% SDS) for 3 h at 37.degree. C. After
hybridization, the blot was washed three times with 2.times.SSPE at
37.degree. C.
[0136] Nucleic Acid Reagents
[0137] The coding sequence of Homo sapiens heterogeneous nuclear
ribonucleoprotein H (hnRPH) mRNA (Genbank accession No.
NM.sub.--005520) is shown in Table 5. The ATG start codon and TAA
stop codons are highlighted in bold font and site target by siRNA
reagents is underscored.
TABLE-US-00007 TABLE 5 Nucleotide Sequence of HNRPH
ttttttttttcgtcttagccacgcagaagtcgcg (SEQ ID NO: 86)
tgtctagtttgtttcgacgccggaccgcgtaaga
gacgatgatgttgggcacggaaggtggagaggga
ttcgtggtgaaggtccggggcttgccctggtctt
gctcggccgatgaagtgcagaggtttttttctga
ctgcaaaattcaaaatggggctcaaggtattcgt
ttcatctacaccagagaaggcagaccaagtggcg
aggcttttgttgaacttgaatcagaagatgaagt
caaattggccctgaaaaaagacagagaaactatg
ggacacagatatgttgaagtattcaagtcaaaca
acgttgaaatggattgggtgttgaagcatactgg
tccaaatagtcctgacacggccaatgatggcttt
gtacggcttagaggacttccctttggatgtagca
aggaagaaattgttcagttcttctcagggttgga
aatcgtgccaaatgggataacattgccggtggac
ttccaggggaggagtacgggggaggccttcgtgc
agtttgcttcacaggaaatagctgaaaaggctct
aaagaaacacaaggaaagaatagggcacaggtat
attgaaatctttaagagcagtagagctgaagtta
gaactcattatgatccaccacgaaagcttatggc
catgcagcggccaggtccttatgacagacctggg
gctggtagagggtataacagcattggcagaggag
ctggctttgagaggatgaggcgtggtgcttatgg
tggaggctatggaggctatgatgattacaatggc
tataatgatggctatggatttgggtcagatagat
ttggaagagacctcaattactgtttttcaggaat
gtctgatcacagatacggggatggtggctctact
ttccagagcacaacaggacactgtgtacacatgc
ggggattaccttacagagctactgagaatgacat
ttataattttttttcaccgctcaaccctgtgaga
gtacacattgaaattggtcctgatggcagagtaa
ctggtgaagcagatgtcgagttcgcaactcatga
agatgctgtggcagctatgtcaaaagacaaagca
aatatgcaacacagatatgtagaactcttcttga
attctacagcaggagcaagcggtggtgcttacga
acacagatatgtagaactcttcttgaattctaca
gcaggagcaagcggtggtgcttatggtagccaaa
tgatgggaggcatgggcttgtcaaaccagtccag
ctacgggggcccagccagccagcagctgagtggg
ggttacggaggcggctacggtggccagagcagca
tgagtggatacgaccaagttttacaggaaaactc
cagtgattttcaatcaaacattgcataggtaacc
aaggagcagtgaacagcagctactacagtagtgg
aagccgtgcatctatgggcgtgaacggaatggga
gggttgtctagcatgtccagtatgagtggtggat
ggggaatgtaattgatcgatcctgatcactgact
cttggtcaacctttttttttttttttttttcttt
aagaaaacttcagtttaacagtttctgcaataca
agcttgtgatttatgcttactctaagtggaaatc
aggattgttatgaagacttaaggcccagtatttt
tgaatacaatactcatctaggatgtaacagtgaa
gctgagtaaactataactgttaaacttaagttcc
agcttttctcaagttagttataggatgtacttaa
gcagtaagcgtatttaggtaaaagcagttgaatt
atgttaaatgttgccctttgccacgttaaattga
acactgttttggatgcatgttgaaagacatgctt
ttattttttttgtaaaacaatataggagctgtgt
ctactattaaaagtgaaacattttggcatgtttg
ttaattctagtttcatttaataacctgtaaggca
cgtaagtttaagctttttttttttttaagttaat
gggaaaaatttgagacgcaataccaatacttagg
attttggtcttggtgtttgtatgaaattctgagg
ccttgatttaaatctttcattgtattgtgatttc
cttttaggtatattgcgctaagtgaaacttgtca aataaatcctccttttaaaaactgc
[0138] The coding sequence of the La protein mRNA (Genbank
accession No. NM.sub.--005520) is shown in Table 6. The ATG start
codon and TAA stop codons are highlighted in bold font and site
target by siRNA reagents is underscored.
TABLE-US-00008 TABLE 6 Nucleotide Sequence of La Protein
ccggcggcgctgggaggtggagtcgttgctgttg (SEQ ID NO:87)
ctgtttgtgagcctgtggcgcggcttctgtgggc
cggaaccttaaagatagccgtaatggctgaaaat
ggtgataatgaaaagatggctgccctggaggcca
aaatctgtcatcaaattgagtattattttggcga
cttcaatttgccacgggacaagtttctaaaggaa
cagataaaactggatgaaggctgggtacctttgg
agataatgataaaattcaacaggttgaaccgtct
aacaacagactttaatgtaattgtggaagcattg
agcaaatccaaggcagaactcatggaaatcagtg
aagataaaactaaaatcagaaggtctccaagcaa
acccctacctgaagtgactgatgagtataaaaat
gatgtaaaaaacagatctgtttatattaaaggct
tcccaactgatgcaactcttgatgacataaaaga
atggttagaagataaaggtcaagtactaaatatt
cagatgagaagaacattgcataaagcatttaagg
gatcaatttttgttgtgtttgatagcattgaatc
tgctaagaaatttgtagagacccctggccagaag
tacaaagaaacagacctgctaatacttttcaagg
acgattactttgccaaaaaaaatgaagaaagaaa
acaaaataaagtggaagctaaattaagagctaaa
caggagcaagaagcaaaacaaaagttagaagaag
atgctgaaatgaaatctctagaagaaaagattgg
atgcttgctgaaattttcgggtgatttagatgat
cagacctgtagagaagatttacacatacttttct
caaatcatggtgaaataaaatggatagacttcgt
cagaggagcaaaagaggggataattctatttaaa
gaaaaagccaaggaagcattgggtaaagccaaag
atgcaaataatggtaacctacaattaaggaacaa
agaagtgacttgggaagtactagaaggagaggtg
gaaaaagaagcactgaagaaaataatagaagacc
aacaagaatccctaaacaaatggaagtcaaaagg
tcgtagatttaaaggaaaaggaaagggtaataaa
gctgcccagcctgggtctggtaaaggaaaagtac
agtttcagggcaagaaaacgaaatttgctagtga
tgatgaacatgatgaacatgatgaaaatggtgca
actggacctgtgaaaagagcaagagaagaaacag
acaaagaagaacctgcatccaaacaacagaaaac
agaaaatggtgctggagaccagtagtttagtaaa
ccaattttttattcattttaaataggttttaaac
gacttttgtttgcggggcttttaaaaggaaaacc
gaattaggtccacttcaatgtccacctgtgagaa
aggaaaaatttttttgttgtttaacttgtctttt
tgttatgcaaatgagatttctttgaatgtattgt
tctgtttgtgttatttcagatgattcaaatatca
aaaggaagattcttccattaaattgcctttgtaa
tatgagaatgtattagtacaaactaactaataaa atatatactatatgaaaagagc
[0139] RNA duplexes were synthesized and prepared as described in
Example 1. RNA duplexes targeting HNRPH1 are summarized in Table
7.
TABLE-US-00009 TABLE 7 Summary of Oligonucleotide Reagents Sequence
Name SEQ ID NO: 5' GUUGAACUUGAAUCAGAAGAUGAAGUCAAAUUGGC 3' HNRPH1
Site-1 SEQ ID No:88 5' CUUGAAUCAGAAGAUGAAGUU HNRPH1-21 + 2 SEQ ID
No:89 3' UUGAACUUAGUCUUCUACUUC SEQ ID No:90 5'
AACUUGAAUCAGAAGAUGAAGUCAAAU HNRPH1-27 + 0 SEQ ID No:91 3'
UUGAACUUAGUCUUCUACUUCAGUUUA SEQ ID No:92 5'
AUAAAACUGGAUGAAGGCUGGGUACCUUUGGAGAU 3' La Site-1 SEQ ID NO:93 5'
CUGGAUGAAGGCUGGGUACUU La-21 + 2 SEQ ID NO:94 3'
UUGACCUACUUCCGACCCAUG SEQ ID NO:95 5' AACUGGAUGAAGGCUGGGUACCUUUUU
La-21 + 2 SEQ ID NO:96 3' UUGACCUACUUCCGACCCAUGGAAACC SEQ ID
NO:97
[0140] Results
[0141] RNA duplexes were synthesized to target randomly chosen
sites in the human hnRNP H mRNA (analyzed by western blotting) and
the mRNA encoding the La protein (analyzed by northern blotting
(FIGS. 5C and 5D). For both targets the 27mer duplex was more
potent than the 21mer siRNAs targeting these messages.
Example 111
Sequence Specificity of 27mer
[0142] As a test for the sequence specificity of the 27mer dsRNA, a
series of 27+0 dsRNAs with one, two or three mismatches to the EGFP
target mRNA were synthesized and tested at concentrations of 0 nM,
1 nM and 200 pM in the cotransfection assay (FIG. 6). The sequences
of the mutated 27+0 dsRNAs are shown in Table 2. At 200 pM, each of
the mismatched sequences was less potent than the wild-type 27mer
dsRNA; the triple mismatch 27mer dsRNA was completely ineffective
at triggering RNAi at all concentrations tested. Similar results
were obtained using a 27mer dsRNA targeted to `site 2` of EGFP.
Example 12
Lack of Interferon Response
[0143] This example demonstrates that the dsRNA duplexes of the
invention do not activate the interferon response.
[0144] Interferon and PKR Assays
[0145] After transfection of 293 cells with 20 nM of each RNA as
described previously, medium was collected after 24 h and used for
ELISA assays of interferon .alpha. and .beta. as previously
described (Kim et al., 2004). The PKR activation assay was done as
previously described (Gunnery and Mathews, 1998). PKR in the lysate
was first activated by co-incubation of the indicated RNAs and
radiolabeled by its auto-kinase reaction. The radiolabeled PKR was
then immunoprecipitated for analysis. To determine the activity of
PKR in the cell lysate without prior activation, dsRNA was omitted.
The reaction was incubated at 30.degree. C. for 20 min. PKR from
the reaction was immunoprecipitated using polyclonal antibody. The
polyclonal antibody was added to the reaction, which was then
placed on ice for 1 h, followed by addition of 50 .mu.l of 10%
protein A-Sepharose in IPP500 (10 mM Tris, pH 8, 500 mM NaCl, 0.1%
Nonidet P-40). This mixture was rocked for 30 min at 4.degree. C.
The protein A-Sepharose beads were washed with 1 ml IPP100 buffer
(10 mM Tris, pH 8, 100 mM NaCl, 0.1% Nonidet P-40) five times.
After the last wash, the beads were boiled in protein sample buffer
and loaded onto an SDS-polyacrylamide gel followed by
autoradiography.
[0146] Results
[0147] A potential problem in the use of the longer dsRNAs is
activation of PKR and induction of interferons (Manche et al.,
1992). We therefore assessed whether a transfected 27mer dsRNA
activates interferon-.alpha. (FIG. 7A) or interferon-.beta. (FIG.
7B). As a positive control for interferon induction, we transfected
a triphosphate-containing single-stranded RNA which potently
activated interferon-.alpha. and -.beta., as reported previously
(Kim et al., 2004). Neither cytokine was detected when either the
21+2 siRNA or 27+0 dsRNA was used. We have extended this
observation to two other 27mer sequences specific for the EGFP-S2
and EGFP-S3 sites. PKR activation in cell lysates was also assayed
as described previously (Gunnery and Mathews, 1998). The lysate was
treated with the indicated RNAs, followed by immunoprecipitation.
The positive control long dsRNA elicited PKR activation, but none
of the shorter RNAs activated PKR (FIG. 7C).
Example 13
Asymmetric 27mer Duplex Design and Base Modifications can Influence
Dicing Patterns and Allow Intelligent Design of 27mer
[0148] This examples demonstrates that multiple species are
produced by Dicer action on the 27mer and that design of the 27mer
and/or inclusion of base modifications can be employed to direct
these degradation patterns, limit heterogeneity, and predict end
products.
[0149] It was demonstrated in Example 6, FIGS. 3A and 3B that all
of the individual 21mers that could be produced by Dicer action on
the EGFPS1 27mer duplex are less potent in suppressing EGFP than
the 27mer duplex. Nevertheless, which 21mers are produced can
influence ultimate potency. An electrospray mass spectrometry assay
was used to determine which 21mers are the actual products that
result from enzymatic digestion of a 27mer substrate RNA by Dicer.
More than one 21mer can result from Dicer digestion of the 27mer.
Dicing patterns can be controlled, permitting intelligent
design.
[0150] Electrospray Mass Spectrometry Assay of In Vitro Dicing
Reactions
[0151] RNA duplexes (100 pmoles) were incubated in 20 .mu.l of 20
mM Tris pH 8.0, 200 mM NaCl, 2.5 mM MgCl.sub.2 with 1 unit of
recombinant human Dicer (Stratagene, La Jolla, Calif.) for 12-24
hours. Electrospray-ionization liquid chromatography mass
spectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment with
Dicer were done using an Oligo HTCS system (Novatia, Princeton,
N.J.), which consisted of ThermoFinnigan TSQ7000, Xcalibur data
system, ProMass data processing software and Paradigm MS4.TM. HPLC
(Michrom BioResources, Auburn, Calif.). The liquid chromatography
step employed before injection into the mass spectrometer (LC-MS)
removes most of the cations complexed with the nucleic acids; some
sodium ion can remain bound to the RNA and are visualized as minor
+22 or +44 species, which is the net mass gain seen with
substitution of sodium for hydrogen. Accuracy of this assay is
around +/-2 Daltons for oligonucleotides of this size.
[0152] Results
[0153] The EGFPS1-27+0 duplex was digested with Dicer and mass
spectra were obtained pre-digestion (FIG. 8A) and post-digestion
(FIG. 8B). In general, Dicer will cleave a 27mer duplex into 21mer
length fragments with a 5'-phosphate. Lesser amounts of 22mers are
also usually generated. Small amounts of 20mer and 23mers can also
sometimes be observed. By comparing observed masses with the
starting sequence, it can be deduced that 4 duplexes with 2-base
3'-overhangs and 5'-phosphate were produced in this dicing
reaction. These species represent two major cleavage products, both
of which resulted in 21mer and 22mer duplexes. Lower case "p"
represents a phosphate group. Calculated masses for each possible
digestion product that could result from in vitro Dicer cleavage
are shown in Table 8.
TABLE-US-00010 TABLE 8 Molecular Weights of Possible Duplexes
Derived from the 27 mer Duplex by Dicer Processing Sequence (SEQ ID
NO:) Name Mol Wt 5' AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1-27 + 0
L 8552 3' UUCGACUGGGACUUCAAGUAGACGUGG (42) 8689 5'
pACCCUGAAGUUCAUCUGCACC (11) EGFPS1-21 + 2 (3) 6672 3'
ACUGGGACUUCAAGUAGACGUp (12) 6816 5' pGACCCUGAAGUUCAUCUGCACC (98)
EGFPS1-22 + 2 (3) 7017 3' GACUGGGACUUCAAGUAGACGUp (99) 7161 5'
pUGACCCUGAAGUUCAUCUGCA (15) EGFPS1-21 + 2 (5) 6713 3'
CGACUGGGACUUCAAGUAGACp (16) 6815 5' pCUGACCCUGAAGUUCAUCUGCA (100)
EGFPS1-22 + 2 (5) 7018 3' UCGACUGGGACUUCAAGUAGACp (101) 7121
[0154] It was demonstrated in Example 2, FIGS. 1A-1C that blunt
duplexes or duplexes with 5'-overhangs can show similar or better
potency than duplexes with 3'-overhangs. In these studies, both
ends of the duplex were symmetric (i.e., both ends were blunt, both
ends were 3'-overhang, or both ends were 5'-overhang). In similar
studies, it was found that a blunt 27mer duplex with two bases
mismatch at one end had higher potency than the symmetric blunt,
3'-overhang, or 5'-overhang species tested. The blunt duplex with
2-base mismatch on one end might mimic the behavior of an
asymmetric duplex with a 2-base 3'-overhang on one end and blunt on
the other end. Asymmetric 27mer duplexes of this kind were tested
and found to have increased potency and fewer diced products (less
heterogeneity) and resulted in a more predictable pattern.
[0155] The double-stranded RNA binding domains of many enzymes
often specifically recognize RNA and not DNA or some modified RNAs.
Insertion of DNA residues into the ends of blunt 27mer duplexes was
studied. Asymmetric designs with DNA residues in the 3'-end of one
strand resulted in fewer diced products (less heterogeneity) and
generally produced a predicable pattern which was opposite that of
the asymmetric 3'-overhang designs.
[0156] The EGFPS1-27+0 duplex was modified to have an asymmetric
3'-overhang on one side and 2 DNA bases at the 3'-end of the other
side. A 5-phosphate was placed on the recessed strand at the
3-overhang to mimic dicer processing. The final duplex of this
design is show below aligned with the original blunt 27mer. Lower
case "t" represents DNA dT base and lower case "p" represents a
phosphate group. Calculated masses are shown in Table 9.
TABLE-US-00011 TABLE 9 Molecular Weights of Duplexes Sequence (SEQ
ID NO:) Name Mol Wt 5' AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1-27 +
0 L 8552 3' UUCGACUGGGACUUCAAGUAGACGUGG (42) 8689 5'
AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1-27/25 L 8552 3'
ttCGACUGGGACUUCAAGUAGACGUp (102) 8075
[0157] Mass spectra were obtained pre-digestion (FIG. 8C) and
post-digestion (FIG. 8D). Analysis of the dicing products made from
the modified asymmetric duplex was much simpler than for the
symmetric duplex (compare FIGS. 8B with 8D). A single cleavage
product was observed which was mostly 21mer duplex with small
amounts of 22mer detectable. Lower case "t" represents DNA dT base
and lower case "p" represents a phosphate group. Calculated masses
are shown in Table 10.
TABLE-US-00012 TABLE 10 Molecular Weights of Possible Duplexes
Derived from the 27 mer/25 mer Duplex by Dicer Processing Sequence
(SEQ ID NO:) Name Mol Wt 5' AAGCUGACCCUGAAGUUCAUCUGCACC (41)
EGFPS1-27/25 L 8552 3' ttCGACUGGGACUUCAAGUAGACGUp (102) 8075 5'
pACCCUGAAGUUCAUCUGCACC (11) EGFPS1-21 + 2 (3) 6672 3'
ACUGGGACUUCAAGUAGACGUp (12) 6816 3' GACUGGGACUUCAAGUAGACGUp (99)
7161
[0158] Use of asymmetric design with a single 2-base 3'-overhang
and selective incorporation of DNA residues simplifies the dicing
reaction to yield a single major cleavage product. This design
additionally permits prediction of the dicing pattern and allows
for intelligent design of 27mers such that specific, desired 21mers
can be produced from dicing. As demonstration, a second 27mer
duplex, EGFPS1-27-R was studied which overlaps the EGFPS1-27-L
sequence. Calculated masses are shown in Table 11.
TABLE-US-00013 TABLE 11 Molecular Weights of Duplexes Sequence (SEQ
ID NO:) Name Mol Wt 5' AAGCUGACCCUGAAGUUCAUCUGCACC (41) EGFPS1-27 +
0 L 8552 3' UUCGACUGGGACUUCAAGUAGACGUGG (42) 8689 5'
UGACCCUGAAGUUCAUCUGCACCACCG (103) EGFPS1-27 + 0 R 8528 3'
ACUGGGACUUCAAGUAGACGUGGUGGC (104) 8728
[0159] Mass spectra were obtained pre-digestion (FIG. 9A) and
post-digestion (FIG. 9B). Analysis of the dicing products made from
the EGFPS1-27+0 R duplex showed a complex pattern similar to that
seen with the EGFPS1-27+0 L duplex. Two major cleavage products
were observed and both 21mer and 22mer species were present. Very
minor 20mer species were also seen. Lower case "p" represents a
phosphate group. Calculated masses for each possible digestion
product that could result from in vitro Dicer cleavage are shown in
Table 12.
TABLE-US-00014 TABLE 12 Molecular Weights of Possible Duplexes
Derived from the 27 mer Duplex by Dicer Processing Sequence (SEQ ID
NO:) Name Mol Wt 5' UGACCCUGAAGUUCAUCUGCACCACCG (103) EGFPS1-27 + 0
R 8528 3' ACUGGGACUUCAAGUAGACGUGGUGGC (104) 8728 5'
pUGAAGUUCAUCUGCACCACCG (105) EGFPS1-21 (1) R 6712 5'
pCCUGAAGUUCAUCUGCACCACC (106) EGFPS1-22 (3)R 6977 3'
UGGGACUUCAAGUAGACGUGGUp (107) 7178 5' pACCCUGAAGUUCAUCUGCACCACC
(108) EGFPS1-27 + 0 R 6672 3' ACUGGGACUUCAAGUAGACGUGGUp (109) 6816
3' ACUGGGACUUCAAGUAGACGUGp (110) EGFPS1-22 (5)R 7161
[0160] The EGFPS1-27+0R duplex was converted to a DNA-modified,
asymmetric 25/27mer duplex as shown below and this duplex was
studied in the in vitro dicing assay. Lower case "cg" represents
DNA dCdG base and lower case "p" represents a phosphate group.
[0161] Calculated masses are shown in Table 13.
TABLE-US-00015 TABLE 13 Molecular Weights of Duplexes Sequence (SEQ
ID NO:) Name Mol Wt 5' UGACCCUGAAGUUCAUCUGCACCACCG (103) EGFPS1-27
+ 0 R 8528 3' ACUGGGACUUCAAGUAGACGUGGUGGC (104) 8728 5'
pACCCUGAAGUUCAUCUGCACCACcg (111) EGFPS1-25/27 R 7925 3'
ACUGGGACUUCAAGUAGACGUGGUGGC (112) 8728
[0162] Mass spectra were obtained pre-digestion (FIG. 9C) and
post-digestion (FIG. 9D). The DNA-modified asymmetric EGFPS1-25/25
R duplex showed a clean, single diced 21mer species, as summarized
in Table 14. Lower case "p" represents a phosphate group.
TABLE-US-00016 TABLE 14 Molecular Weight of Possible Duplex Derived
from the 25 mer/27 mer Duplex by Dicer Processing Sequence (SEQ ID
NO:) Name Mol Wt 5' pACCCUGAAGUUCAUCUGCACCACcg (111) EGFPS1-25/27 R
7925 3' ACUGGGACUUCAAGUAGACGUGGUGGC (112) 8728 5'
pACCCUGAAGUUCAUCUGCACC (11) EGFPS1-21 + 2 (3) 6672 3'
ACUGGGACUUCAAGUAGACGUp (12) 6816
[0163] If the results of FIGS. 8D and 9D are compared, it can be
seen that digestion of the two different asymmetric duplexes
EGFPS1-27/25 L and EGFPS1-25/27R result in formation of the same
21mer species, EGFPS1-21(3). Lower case "t" or "cg" represents DNA
bases and lower case "p" represents a phosphate group. Calculated
masses are shown in Table 15.
TABLE-US-00017 TABLE 15 Molecular Weights of Duplexes Sequence (SEQ
ID NO:) Name Mol Wt 5' AAGCUGACCCUGAAGUUCAUCUGCACC (41)
EGFPS1-27/25 L 8552 3' ttCGACUGGGACUUCAAGUAGACGUp (102) 8075 5'
pACCCUGAAGUUCAUCUGCACCACcg (111) EGFPS1-25/27 R 7925 3'
ACUGGGACUUCAAGUAGACGUGGUGGC (112) 8728 5' pACCCUGAAGUUCAUCUGCACC
(11) EGFPS1-21 + 2 (3) 6672 3' ACUGGGACUUCAAGUAGACGUp (12) 6816
[0164] Therefore use of the DNA-modified asymmetric duplex design
as taught by the invention reduces complexity of the dicing
reaction for 27mer RNA species and permits intelligent design of
27mers for use in RNAi such that it is possible to specifically
target a desired location. Cleavage of the substrate 27mer by Dicer
results in a unique, predictable 21mer wherein one end of the 21mer
coincides with the 3'-overhang end of the substrate 27mer.
Example 14
Asymmetric 27mer Duplex Designs with Base Modifications can Improve
Potency Over Blunt 27Mers
[0165] This examples demonstrates that the new asymmetric RNA
duplexes as taught by the invention, having a 2-base 3'-overhang on
one side and blunt with 3-base 3'-DNA modification on the other
side, can improve potency over blunt 27mer duplexes.
[0166] It was demonstrated in Example 13, FIGS. 8A-8D and 9A-9D,
that use of asymmetric duplexes can direct dicing and result in a
single major 21mer cleavage product. Further, the 27/25 L and 25/27
R asymmetric duplexes both result in the same 21mer duplex after
dicing. Since the same 21mer duplex is produced from each of the
two asymmetric 27mers, it would be anticipated by one skilled in
the art that these compounds should functionally have similar
potency. It is shown in this example that this is not the case and
that the 25/27 R design unexpectedly has increased potency relative
to both the 27/25 L duplex and the blunt 27+0 duplex.
[0167] RNA duplexes targeting EGFPS2 were co-transfected into
HEK293 cells with an EGFP expression plasmid and assayed for EGFP
activity after 24 hours incubation according to methods described
above. Transfected duplexes are shown in Table 16. Lower case "p"
represents a phosphate group and lower case bases "cc" and "gt"
represent DNA bases while uppercase bases represent RNA.
TABLE-US-00018 TABLE 16 Transfected Duplexes Sequence Name SEQ ID
NO: 5' GCAGCACGACUUCUUCAAGUU EGFPS2-21 + 2 SEQ ID NO:76 3'
UUCGUCGUGCUGAAGAAGUUC SEQ ID NO:77 5' AAGCAGCACGACUUCUUCAAGUCCGCC
EGFPS2-27 + 0 SEQ ID NO:78 3' UUCGUCGUGCUGAAGAAGUUCAGGCGG SEQ ID
NO:79 5' CAUGAAGCAGCACGACUUCUUCAAGUC EGFPS2-27/25 L SEQ ID NO:113
3' gtACUUCGUCGUGCUGAAGAAGUUCp SEQ ID NO:114 5'
pGCAGCACGACUUCUUCAAGUCCGcc EGFPS2-25/27 R SEQ ID NO:115 3'
UUCGUCGUGCUGAAGAAGUUCAGGCGG SEQ ID NO:79
[0168] The EGFPS2-21+2 and EGFPS2-27+0 RNA duplexes were employed
previously in Example 9. The EGFPS2-27/25 L and EGFPS2-25/27 R
duplexes are new asymmetric dsRNAs designed according to the
present invention and target the same site, site II of EGFP. In the
in vitro dicing electrospray mass spectrometry assay as described
in Example 13, both the EGFPS2-27/25 L and EGFPS2-25/27 R duplexes
yield the same 21mer product after digestion by Dicer, similar to
the EGFPS2-21+2 duplex.
[0169] Transfection results are shown in FIG. 10A. As previously
shown, the EGFPS2-21+2 duplexes had minimal activity in suppressing
EGFP expression while the 27+0 duplex showed significant
inhibition. The 27/25 L duplex was slightly less potent than the
27+0 duplex and the 25/27 R duplex was most potent. Based upon the
teaching of prior art, this finding is unexpected, since both of
the asymmetric duplexes produce the same 21mer species following
dicing.
[0170] Similar transfections were done using the EGFPS1 duplexes
27/25 L and 25/27 R. These duplexes produce the same 21mer product,
the EGFPS1-21(3) duplex, after dicing (Example 13). Transfected
duplexes are shown in Table 17. Lower case "p" represents a
phosphate group and lower case bases "tt" represent DNA bases while
uppercase bases represent RNA.
TABLE-US-00019 TABLE 17 Transfected Duplexes Sequence Name SEQ ID
NO: 5' AAGCUGACCCUGAAGUUCAUCUGCACC EGFPS1-27/25 L SEQ ID NO:41 3'
ttCGACUGGGACUUCAAGUAGACGUp SEQ ID NO:102 5'
pACCCUGAAGUUCAUCUGCACCACcg EGFPS1-25/27 R SEQ ID NO:111 3'
ACUGGGACUUCAAGUAGACGUGGUGGC SEQ ID NO:112
[0171] EGFP expression after transfection is shown in FIG. 10B. As
before, the 25/27 R duplex was significantly more potent than the
27/25 L duplex in reducing EGFP expression. In a similar experiment
in which the blunt ended 27mer was compared with 25/27 R duplex and
27/25 L duplex, it was found that the dsRNAs had the following
potencies: [0172] 25/27 R duplex>27/25 L duplex>27mer.
[0173] 27mer duplex RNAs can show significantly higher potency than
21mer duplexes in suppressing targeted gene expression. The blunt
27mers can result in a variety of 21mer species after dicing, so
precise performance of a blunt 27mer duplex cannot always be
predicted. The novel asymmetric duplexes of the present invention
wherein one side of the duplex has a 2-base 3'-overhang and the
other side is blunt and has base modifications, such as DNA, at the
3'-end, force dicing to occur in a predictable way so that precise
21mers result. These asymmetric duplexes, i.e., 27/25 L and 25/27
R, are each also more potent than the 21mers. The asymmetric 25/27
R design is the most potent embodiment of the present
invention.
[0174] FIG. 11 is an illustration comparing the embodiments of the
present invention. The target gene sequence is illustrated by SEQ
ID NO:116. The "typical" parent 21mer used as an siRNA molecule is
shown aligned with the target gene sequence. Aligned with the
target gene and parent 21mer sequences is the L 27mer v2.1
containing a 3' overhang on the sense strand and two DNA bases at
the 3' end of the antisense strand. The diced cleavage product is
also shown. This alignment illustrates the left shift in designing
these precursor RNAi molecules. Also aligned with the target gene
and parent 21mer sequences is the R 27mer v2.1 containing a 3'
overhang on the antisense strand and two DNA bases at the 3' end of
the sense strand. The diced cleavage product is also shown. This
alignment illustrates the right shift in designing these precursor
RNAi molecules.
Example 15
Determination of Effective Dose
[0175] This example demonstrates a method for determining an
effective dose of the dsRNA of the invention in a mammal. A
therapeutically effective amount of a composition containing a
sequence that encodes a dsRNA, (i.e., an effective dosage), is an
amount that inhibits expression of the product of the target gene
by at least 10 percent. Higher percentages of inhibition, e.g., 20,
50, 90 95, 99 percent or higher may be desirable in certain
circumstances. Exemplary doses include milligram or microgram
amounts of the molecule per kilogram of subject or sample weight
(e.g., about 1 microgram per kilogram to about 5 milligrams per
kilogram, about 100 micrograms per kilogram to about 0.5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram). The compositions can be administered one
or more times per week for between about 1 to 10 weeks, e.g.,
between 2 to 8 weeks, or between about 3 to 7 weeks, or for about
4, 5, or 6 weeks, as deemed necessary by the attending physician.
Treatment of a subject with a therapeutically effective amount of a
composition can include a single treatment or a series of
treatments.
[0176] Appropriate doses of a particular dsRNA composition depend
upon the potency of the molecule with respect to the expression or
activity to be modulated. One or more of these molecules can be
administered to an animal, particularly a mammal, and especially
humans, to modulate expression or activity of one or more target
genes. A physician may, for example, prescribe a relatively low
dose at first, subsequently increasing the dose until an
appropriate response is obtained. In addition, it is understood
that the specific dose level for any particular subject will depend
upon a variety of other factors including the severity of the
disease, previous treatment regimen, other diseases present,
off-target effects of the active agent, age, body weight, general
health, gender, and diet of the patient, the time of
administration, the route of administration, the rate of excretion,
any drug combination, and the degree of expression or activity to
be modulated.
[0177] The efficacy of treatment can be monitored by measuring the
amount of the target gene mRNA (e.g. using real time PCR) or the
amount of product encoded by the target gene such as by Western
blot analysis. In addition, the attending physician can monitor the
symptoms associated with the disease or disorder afflicting the
patient and compare with those symptoms recorded prior to the
initiation of treatment.
[0178] It is clear from recent studies that the effects of RNAi are
not entirely specific and that undesired `off-target` effects can
occur of a magnitude dependent on the concentration of siRNA
(Persengiev et al., 2004). The new Dicer substrate dsRNA approach
may facilitate use of lower concentrations of duplex RNA than are
needed with traditional 21mer siRNAs. It is clear from published
data that `off-target` effects can occur in certain cell lines
using 21mer siRNAs (Persengiev et al., 2004; Jackson et al., 2003),
but these also can be minimized by using reagents that have
efficacy in the low to subnanomolar range (Persengiev et al.,
2004). To examine the potential for `off-target` effects using
Dicer substrate dsRNAs, we carried out microarray analyses
comparing an siRNA 21mer with the 27mer, each targeting EGFP site
1. NIH3T3 cells that stably express EGFP were transfected with
concentrations of siRNA that give effective target knockdowns (FIG.
2A, FIG. 7D). Total cellular RNAs were prepared from cells 24 and
48 h after transfection and analyzed by hybridization to an
oligonucleotide microarray as described in FIG. 7D. Among the
16,282 mouse genes analyzed, only a small fraction showed
upregulation or downregulation more than twofold above or below
control values (FIG. 7D). The 27mer and 21mer gave comparable
results at their effective RNAi concentrations. There was an
increase in the number of transcripts upregulated when the 27mer
was used at the higher 25 nM concentration, but comparisons of the
targets affected at 24 versus 48 h and at 5 nM versus 25 nM showed
no overlap. Rather than specific `off-target` effects, these
changes are more consistent with statistical scatter among the
16,282 genes examined. The same assay was repeated using the
EGFP-S2 27+0 duplex RNA with comparable results.
[0179] Given the increase in potency of the 27mer dsRNAs relative
to 21+2 siRNAs, it is of interest that this observation has not
been previously reported. Although others have used dsRNAs of up to
27 bp for RNAi studies in mammalian cells (Bohula et al., 2003;
Caplen et al., 2001), no differences in efficacy were reported as
compared with traditional 21+2 duplexes. This discrepancy between
previous studies and our own may simply be due to differences in
the concentration of dsRNAs tested. "Good" sites for 21mer siRNAs
can have potencies in the nanomolar range (Reynolds et al., 2004).
When `good` sites are tested at high concentrations of transfected
RNA, differences between 21mer siRNAs and 27mer dsRNAs will most
likely be small and easily overlooked. Marked differences in
potency are best shown by testing at low nanomolar or picomolar
concentrations, something that is not routinely done in most
laboratories.
[0180] Thus far, the 27mer dsRNA design has shown increased RNAi
potency relative to 21+2 siRNAs at every site examined. Within the
set of 27mers studied here, however, a range of potencies is
nevertheless seen between different target sites within the same
gene (FIG. 3B). We have shown that, even in the absence of fully
optimized design rules, use of Dicer substrate dsRNA approach can
increase RNAi potency relative to traditional 21+2 siRNAs.
Additionally, the use of 27mer dsRNAs allows targeting of some
sites within a given sequence that are refractory to suppression
with traditional 21mer siRNAs. Use of Dicer substrate dsRNAs to
trigger RNAi should result in enhanced efficacy and longer duration
of RNAi at lower concentrations of RNA than are required for 21+2
applications. Consistent with our results linking Dicer cleavage to
enhanced RNAi efficacy, it has recently been shown that chemically
synthesized hairpin RNAs that are substrates for Dicer are more
potent inducers of RNAi than conventional siRNAs and, moreover,
that a two-base 3' overhang directs Dicer cleavage (Siolas et al.,
2005).
[0181] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0182] Embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those embodiments may become apparent to
those of ordinary skill in the art upon reading the foregoing
description. The inventors expect skilled artisans to employ such
variations as appropriate, and the inventors intend for the
invention to be practiced otherwise than as specifically described
herein. Accordingly, this invention includes all modifications and
equivalents of the subject matter recited in the claims appended
hereto as permitted by applicable law. Moreover, any combination of
the above-described elements in all possible variations thereof is
encompassed by the invention unless otherwise indicated herein or
otherwise clearly contradicted by context.
BIBLIOGRAPHY
[0183] Amarzguioui, M. and Prydz (2004). An algorithm for selection
of functional siRNA sequences. Biochem Biophys Res Commun
316:1050-1058. [0184] Amarzguioui, M. et al. (2003). Tolerance for
Mutation and Chemical Modifications in a siRNA. Nucleic Acids
Research 31:589-595. [0185] Beale, S. E. et al. (2005). siRNA
target site secondary structure predictions using local stable
substructures. Nucl Acids Res 33:e30 (pp 1-10). [0186] Beck, W. T.
(1989). Unknotting the complexities of multidrug resistance: the
involvement of DNA topoisomerases in drug action and resistance. J
Natl Cancer Inst 81:1683-1685. [0187] Bernstein, E. et al. (2001)
Role for a bidentate ribonuclease in the initiation step of RNA
interference. Nature 409:363-366. [0188] Boese, Q. et al. (2005).
Mechanistic insights aid computational short intervening RNA
design. Methods Enzymol 392:73-96. [0189] Bondensgaard, K. et al.
(2000). Structural studies of LNA:RNA duplexes by NMR:
conformations and implications for RNase H activity. Chemistry
6:2687-2695. [0190] Braasch, D. A. and Corey, D. R. (2001). Locked
nucleic acid (LNA): fine-tuning the recognition of DNA and RNA.
Chem Biol 8:1-7. [0191] Braasch et al. (2003). RNA Interference in
Mammalian Cells by Chemically-Modified RNA. Biochemistry
42:7967-7975. [0192] Bohula, E. A. et al. (2003). The efficacy of
small interfering RNAs targeted to the type 1 insulin-like growth
factor receptor (IGF1R) is influenced by secondary structure in the
IGF1R transcript. J Biol Chem 278:15991-15997. [0193] Bridge et al.
(2003). Induction of an Interferon Response by RNAi Vectors in
Mammalian Cells. Nature Genetics 34:263-264. [0194] Caplen, N. J.
et al. (2001). Specific inhibition of gene expression by small
double-stranded RNAs in invertebrate and vertebrate systems. Proc
Natl Acad Sci USA 98:9742-9747. [0195] Chang et al. (1985). Gene
Expression from Both Intronless and Intron-Containing Rous Sarcoma
Virus Clones is Specifically Inhibited by Anti-Sense RNA. Molecular
and Cellular Biology 5:2341-2348. [0196] Check (2003). RNA to the
Rescue? Nature 425:10-12. [0197] Childs, S. and V. Ling (1994). The
MDR superfamily of genes and its biological implications. Important
Adv Oncol, pp. 21-36. [0198] Chiu et al. (2003). siRNA Function in
RNAi: A Chemical Modification Analysis. RNA 9:1034-1048. [0199]
Chiu, Y. L., and Rana, T. R. (2002). RNAi in human cells: basic
structural and functional features of small interfering RNA. Mol
Cell 10:549-561. [0200] Cole, S. P. et al. (1992). Overexpression
of a transporter gene in a multidrug-resistant human lung cancer
cell line. Science 258:1650-1654. [0201] Crinelli, R. et al.
(2002). Design and characterization of decoy oligonucleotides
containing locked nucleic acids. Nucleic Acids Res 30:2435-2443.
[0202] Czauderna et al. (2003). Structural Variations and
Stabilising Modifications of Synthetic siRNAs in Mammalian Cells.
Nucleic Acids Research 31:2705-2716. [0203] Damha, M. J., and
Ogilvie, K. K. (1993). Oligoribonucleotide synthesis. The
silyl-phosphoramidite method. Methods Mol Biol 20:81-114. [0204]
Eckstein, F. (2000). Phosphorothioate oligodeoxynucleotides: what
is their origin and what is unique about them? Antisense Nucleic
Acid Drug Dev 10:117-21. [0205] Elbashir, S. M. et al. (2001a).
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured
mammalian cells. Nature 411:494-498. [0206] Elbashir, S. M. et al.
(2001b). RNA interference is mediated by 21- and 22-nucleotide
RNAs. Genes & Dev 15:188-200. [0207] Elbashir, S. M. et al.
(2001c). Functional Anatomy of siRNAs for Mediating Efficient RNAi
in Drosophila melanogaster Embryo Lysate. EMBO J 20:6877-6888.
[0208] Elman, J. et al. (2005). Locked nucleic acid (LNA) mediated
improvements in siRNA stability and functionality. Nucleic Acids
Res 33:439-447. [0209] Fan., D. et al., (1994). Reversal of
multidrug resistance In Reversal of Multidrug Resistance in Cancer,
Kellen, J. A., ed., CRC Press, Boca Raton, Fla., pp. 93-125. [0210]
Fire, A. et al. (1998). Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811.
[0211] Graessmann, M. et al. (1991). Inhibition of SV40 gene
expression by microinjected small antisense RNA and DNA molecules.
Nucleic Acids Res 19:53-59. [0212] Gunnery, S., and Mathews, M. B.
(1998) RNA binding and modulation of PKR activity. Methods
15:189-98. [0213] Hamada et al. (2002). Effects on RNA Interference
in Gene Expression (RNAi) in Cultured Mammalian Cells of Mismatches
and the Introduction of Chemical Modifications at the 3'-Ends of
siRNAs. Antisense and Nucleic Acid Drug Development 12:301-309.
[0214] Hannon (2002). RNA Interference. Nature 418:244-251. [0215]
Harborth, J. et al. (2003). Sequence, chemical, and structural
variation of small interfering RNAs and short hairpin RNAs and the
effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev
13:83-105. [0216] Herdewijn, P. (2000). Heterocyclic modifications
of oligonucleotides and antisense technology. Antisense Nucleic
Acid Drug Dev 10:297-310. [0217] Hohjoh, J. (2002) RNA interference
(RNAi) induction with various types of synthetic oligonucleotide
duplexes in cultured human cells. FEBS Lett 521:195-199. [0218]
Holen, T. (2002). Positional effects of short interfering RNAs
targeting the human coagulation trigger tissue factor. Nucleic
Acids Res 30:1757-1766. [0219] Jackson, A. L. et al. (2003).
Expression profiling reveals off-target gene regulation by RNAi.
Nat. Biotechno. 21:635-637. [0220] Ji, J. et al. (2003). Enhanced
gene silencing by the application of multiple specific small
interfering RNAs. FEBS Lett 552:247-252. [0221] Khvorova, A. et al.
(2003). Functional siRNAs and miRNAs exhibit strand bias. Cell
115:209-216. [0222] Kim, D. H., and J. J. Rossi (2003). Coupling of
RNAi-mediated target downregulation with gene replacement.
Antisense Nucleic Acid Drug Dev 13:151-155. [0223] Kim, D. H. et
al. (2004). Interferon induction by siRNAs and ssRNAs synthesized
by phage polymerase. Nat Biotechnol 22:321-325. [0224]
Kretshmer-Kazemi Far et al. (2003). The Activity of siRNA in
Mammalian Cells is Related to Structural Target Accessibility: A
Comparison with Antisense Oligonucleotides. Nucleic Acids Research
31: 4417-4424. [0225] Krol, J. et al. (2004). Structural features
of microRNA (miRNA) precursors and their relevance to miRNA
biogenesis and small interfering RNA/short hairpin RNA design. J
Biol Chem 279:42230-42239. [0226] Kreuter, J. (1991)
Nanoparticles-preparation and applications. In: Microcapsules and
nanoparticles in medicine and pharmacy, Donbrow M., ed, CRC Press,
Boca Raton, Fla., pp. 125-14. [0227] Kurreck, J. et al. (2002).
Design of antisense oligonucleotides stabilized by locked nucleic
acids. Nucleic Acids Res 30:1911-1918. [0228] Liu, et al. (2003).
R2D2, a Bridge Between the Initiator and Effector Steps of the
Drosophila RNAi Pathway. Science 301:1921-1925. [0229] Manche, L.
et al. (1992). Interactions between double-stranded RNA regulators
and the protein kinase DAI. Mol Cell Biol 12:5238-5248. [0230]
Markovtsov, V. et al. (2000). Cooperative assembly of an hnRNP
complex induced by a tissue specific homolog of polypyrimidine
tract binding protein. Mol Cell Biol 20:7463-79. [0231] Martinez,
J. et al. (2002). Single-stranded antisense siRNAs guide target RNA
cleavage in RNAi. Cell 110:563-574. [0232] McManus et al. (2002).
Gene Silencing in Mammals by Small Interfering RNAs. Nature Reviews
Genetics 3:737-747. [0233] Melton, D. A. (1985). Injected
anti-sense RNAs specifically block messenger RNA translation in
vivo. Proc Natl Acad Sci USA 82:144-148. [0234] Minks, M. A. et al.
(1979). Structural requirements of double-stranded RNA for the
activation of the 2'-5'-oligo(A) polymerase and protein kinase of
interferon-treated HeLa cells. Biol Chem 254:10180-10183. [0235]
Napoli, C. et al. (1990). Introduction of a chimeric chalcone
synthase gene into petunia results in reversible co-suppression of
homologous genes in trans. Plant Cell 2:279-289. [0236] Ngo et al.
(1998). Double-Stranded RNA Induces mRNA Degradation in Trypanosoma
brucei. Proc Natl Acad Sci USA 95:14687-14692. [0237] Parrish, S.
et al. (2000). Functional anatomy of a dsRNA trigger: differential
requirements for the two trigger strands in RNA interference. Mol
Cell 6: 1077-1087. [0238] Pellino et al. (2003). R2D2 Leads the
Silencing Trigger to mRNA's Death Star. Cell 115:132-133. [0239]
Persengiev, S. P. et al. (2004). Nonspecific,
concentration-dependent stimulation and repression of mammalian
gene expression by small interfering RNAs (siRNAs). RNA 10:12-18.
[0240] Reynolds, A. et al. (2004). Rational siRNA design for RNA
interference. Nat Biotechnol 22:326-330. [0241] Romano, N. and G.
Macino (1992). Quelling: transient inactivation of gene expression
in Neurospora crassa by transformation with homologous sequences.
Mol Microbiol 6:3343-53. [0242] Rusckowski, M. et al. (2000).
Biodistribution and metabolism of a mixed backbone oligonucleotide
(GEM 231) following single and multiple dose administration in
mice. Antisense Nucleic Acid Drug Dev 10:333-345. [0243] Scheffer,
G. L. et al. (1995). The drug resistance-related protein LRP is the
human major vault protein. Nat Med 1:578-582. [0244] Scherer, L.
and J. J. Rossi (2004). RNAi applications in mammalian cells.
Biotechniques 36:557-561. [0245] Scherer et al. (2003). Approaches
for the Sequence-Specific Knockdown of mRNA, Nature Biotechnology
21:1457-1465. [0246] Schwarz, D. S. et al. (2003). Asymmetry in the
assembly of the RNAi enzyme complex. Cell 115:199-208. [0247]
Siolas, D. et al. (2005). Synthetic shRNAs as potent RNAi triggers.
Nat Biotechnol 23:227-231. [0248] Skipper, (2003). Elegant Tour de
Force. Nature Reviews Genetics 4: 79-80. [0249] Sledz et al.
(2003). Activation of the Interferon System by Short-Interfering
RNAs. Nature Cell Biology 5:834-839. [0250] Stark, G. R. et al.
(1998). How cells respond to interferons. Annu Rev Biochem
67:227-264. [0251] Stein, D. A. et al. (2001). Inhibition of
Vesivirus infections in mammalian tissue culture with antisense
morpholino oligomers. Antisense Nucleic Acid Drug Dev 11:317-25.
[0252] Tuschl, T. et al. (1999). Targeted mRNA degradation by
double-stranded RNA in vitro. Genes & Dev 13:3191-3197. [0253]
Ui-Tei, K. et al. (2004). Guidelines for the selection of highly
effective siRNA sequences for mammalian and chick RNA interference.
Nucleic Acids Res 32:936-948. [0254] Verma, S, and F. Eckstein
(1998). Modified oligonucleotides: synthesis and strategy for
users. Annu Rev Biochem 67:99-134. [0255] Vorobjev, P. E. et al.
(2001). Nuclease resistance and RNase H sensitivity of
oligonucleotides bridged by oligomethylenediol and oligoethylene
glycol linkers. Antisense Nucleic Acid Drug Dev 11:77-85. [0256]
Wahlestedt, C. et al. (2000). Potent and nontoxic antisense
oligonucleotides containing locked nucleic acids. Proc Natl Acad
Sci USA 97:5633-5638. [0257] Waterhouse et al. (2003). Exploring
Plant Genomes by RNA-Induced Gene Silencing. Nature Reviews
Genetics 4: 29-38. [0258] Wincott, F. et al. (1995). Synthesis,
deprotection, analysis and purification of RNA and ribozymes.
Nucleic Acids Res 23:2677-84. [0259] Wolin, S. L. and T. Cedervall
(2002). The La protein. Annu Rev Biochem 71:375-403. [0260] Xu et
al. (2003). Effective Small Interfering RNAs and Phosphorothioate
Antisense DNAs Have Different Preferences for Target Sites in the
Luciferase mRNAs. Biochemical and Biophysical Research
Communications 306:712-717. [0261] Yuan, B. et al. (2004). siRNA
Selection Server: an automated siRNA oligonucleotide prediction
server. Nucl Acids Res 32(Webserver issue):W130-134. [0262] Zamore,
P. D. et al. (2000). RNAi: double-stranded RNA directs the
ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.
Cell 101:25-33.
Sequence CWU 1
1
1161720DNAArtificialEGFP from cloning vector pEGFP-C1 1atggtgagca
agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60ggcgacgtaa
acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
ctggcccacc 180ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
gctaccccga ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc
gaaggctacg tccaggagcg caccatcttc 300ttcaaggacg acggcaacta
caagacccgc gccgaggtga agttcgaggg cgacaccctg 360gtgaaccgca
tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
420aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
gcagaagaac 480ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
acggcagcgt gcagctcgcc 540gaccactacc agcagaacac ccccatcggc
gacggccccg tgctgctgcc cgacaaccac 600tacctgagca cccagtccgc
cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660ctgctggagt
tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
720239RNAArtificialmRNA from EGFP from cloning vector pEGFP-C1
2gcaagcugac ccugaaguuc aucugcacca ccggcaagc 39321RNAArtificialsiRNA
3gcaagcugac ccugaaguuc a 21421RNAArtificialsiRNA 4gaugaacuuc
agggucagcu u 21521RNAArtificialsiRNA 5aagcugaccc ugaaguucau c
21621RNAArtificialsiRNA 6gaugaacuuc agggucagcu u
21721RNAArtificialsiRNA 7ccugaaguuc aucugcacca c
21821RNAArtificialsiRNA 8ggugcagaug aacuucaggg u
21921RNAArtificialsiRNA 9cccugaaguu caucugcacc a
211021RNAArtificialsiRNA 10gugcagauga acuucagggu c
211121RNAArtificialsiRNA 11acccugaagu ucaucugcac c
211221RNAArtificialsiRNA 12ugcagaugaa cuucaggguc a
211321RNAArtificialsiRNA 13gacccugaag uucaucugca c
211421RNAArtificialsiRNA 14gcagaugaac uucaggguca g
211521RNAArtificialsiRNA 15ugacccugaa guucaucugc a
211621RNAArtificialsiRNA 16cagaugaacu ucagggucag c
211721RNAArtificialsiRNA 17cugacccuga aguucaucug c
211821RNAArtificialsiRNA 18agaugaacuu cagggucagc u
211921RNAArtificialsiRNA 19gcugacccug aaguucaucu g
212021RNAArtificialsiRNA 20gaugaacuuc agggucagcu u
212123RNAArtificialsiRNA 21gcaagcugac ccugaaguuc auu
232223RNAArtificialsiRNA 22cagaugaacu ucagggucag cuu
232323RNAArtificialsiRNA 23gcugacccug aaguucaucu guu
232422RNAArtificialsiRNA 24cagugaacuu cagggucagc uu
222524RNAArtificialsiRNA 25gcaagcugac ccugaaguuc auuu
242624RNAArtificialsiRNA 26gcagaugaac uucaggguca gcuu
242724RNAArtificialsiRNA 27gcugacccug aaguucaucu gcuu
242824RNAArtificialsiRNA 28gcagaugaac uucaggguca gcuu
242925RNAArtificialsiRNA 29gcaagcugac ccugaaguuc aucuu
253025RNAArtificialsiRNA 30ugcagaugaa cuucaggguc agcuu
253125RNAArtificialsiRNA 31gcugacccug aaguucaucu gcauu
253225RNAArtificialsiRNA 32ugcagaugaa cuucaggguc agcuu
253326RNAArtificialsiRNA 33aagcugaccc ugaaguucau cugcac
263426RNAArtificialsiRNA 34gugcagauga acuucagggu cagcuu
263526RNAArtificialsiRNA 35aagcugaccc ugaaguucau cugcuu
263626RNAArtificialsiRNA 36gugcagauga acuucagggu cagcuu
263726RNAArtificialsiRNA 37gcaagcugac ccugaaguuc aucuuu
263826RNAArtificialsiRNA 38gugcagauga acuucagggu cagcuu
263926RNAArtificialsiRNA 39gcugacccug aaguucaucu gcacuu
264026RNAArtificialsiRNA 40gugcagauga acuucagggu cagcuu
264127RNAArtificialsiRNA 41aagcugaccc ugaaguucau cugcacc
274227RNAArtificialsiRNA 42ggugcagaug aacuucaggg ucagcuu
274327RNAArtificialsiRNA 43aagcugaccc ugaaguucau cugcacc
274427RNAArtificialsiRNA 44ggugcagaug aacuucaggg ucagcuu
274527RNAArtificialsiRNA 45aagcugaccc ugaaguucau cugcacc
274627RNAArtificialsiRNA 46ggugcagaug aacuucaggg ucagcuu
274727RNAArtificialsiRNA 47aagcugaccc ugaaguucau cugcauu
274827RNAArtificialsiRNA 48ggugcagaug aacuucaggg ucagcuu
274927RNAArtificialsiRNA 49gcaagcugac ccugaaguuc aucuguu
275027RNAArtificialsiRNA 50ggugcagaug aacuucaggg ucagcuu
275127RNAArtificialsiRNA 51gcugacccug aaguucaucu gcacauu
275227RNAArtificialsiRNA 52ggugcagaug aacuucaggg ucagcuu
275327RNAArtificialsiRNA 53aagcugaccc ugaagaucau cugcauu
275427RNAArtificialsiRNA 54ggugcagaug aucuucaggg ucagcuu
275527RNAArtificialsiRNA 55aagcugaccc ugaagaacau cugcauu
275627RNAArtificialsiRNA 56ggugcagaug uucuucaggg ucagcuu
275727RNAArtificialsiRNA 57aagcugaccc ugaacaacau cugcauu
275827RNAArtificialsiRNA 58ggugcagaug uuguucaggg ucagcuu
275927RNAArtificialsiRNA 59aagcugaccc uguucaucau cugcacc
276027RNAArtificialsiRNA 60ggugcagaug augaacaggg ucagcuu
276128RNAArtificialsiRNA 61aagcugaccc ugaaguucau cugcacca
286228RNAArtificialsiRNA 62uggugcagau gaacuucagg gucagcuu
286329RNAArtificialsiRNA 63aagcugaccc ugaaguucau cugcaccac
296429RNAArtificialsiRNA 64guggugcaga ugaacuucag ggucagcuu
296530RNAArtificialsiRNA 65aagcugaccc ugaaguucau cugcaccacc
306630RNAArtificialsiRNA 66gguggugcag augaacuuca gggucagcuu
306735RNAArtificialsiRNA 67aagcugaccc ugaaguucau cugcaccacc ggcaa
356835RNAArtificialsiRNA 68uugccggugg ugcagaugaa cuucaggguc agcuu
356940RNAArtificialsiRNA 69aagcugaccc ugaaguucau cugcaccacc
ggcaagcugc 407040RNAArtificialsiRNA 70gcagcuugcc gguggugcag
augaacuuca gggucagcuu 407145RNAArtificialsiRNA 71aagcugaccc
ugaaguucau cugcaccacc ggcaagcugc ccgug 457244RNAArtificialsiRNA
72cacgggcagc uugccguggu gcagaugaac uucaggguca gcuu
447321DNAArtificialprobe 73accctgaagt tcatctgcac c
217432RNAArtificialmRNA from EGFP from cloning vector pEGFP-C1
74ugaagcagca cgacuucuuc aaguccgcca ug 327534RNAArtificialmRNA from
EGFP from cloning vector pEGFP-C1 75ugaaguucga gggcgacacc
cuggugaacc gcau 347621RNAArtificialsiRNA 76gcagcacgac uucuucaagu u
217721RNAArtificialsiRNA 77cuugaagaag ucgugcugcu u
217827RNAArtificialsiRNA 78aagcagcacg acuucuucaa guccgcc
277927RNAArtificialsiRNA 79ggcggacuug aagaagucgu gcugcuu
278021RNAArtificialsiRNA 80guucgagggc gacacccugu u
218121RNAArtificialsiRNA 81cagggucucg cccucgaacu u
218227RNAArtificialsiRNA 82guucgagggc gacacccugg ugaacuu
278329RNAArtificialsiRNA 83cgguucacca gggucucgcc cucgaacuu
298427DNAArtificialprobe 84ccaaaggtac ccagccttca tccagtt
278530DNAArtificialprobe 85gtgaggatgc ctctcttgct ctgggcctcg
30862201DNAHomo sapiens 86tttttttttt cgtcttagcc acgcagaagt
cgcgtgtcta gtttgtttcg acgccggacc 60gcgtaagaga cgatgatgtt gggcacggaa
ggtggagagg gattcgtggt gaaggtccgg 120ggcttgccct ggtcttgctc
ggccgatgaa gtgcagaggt ttttttctga ctgcaaaatt 180caaaatgggg
ctcaaggtat tcgtttcatc tacaccagag aaggcagacc aagtggcgag
240gcttttgttg aacttgaatc agaagatgaa gtcaaattgg ccctgaaaaa
agacagagaa 300actatgggac acagatatgt tgaagtattc aagtcaaaca
acgttgaaat ggattgggtg 360ttgaagcata ctggtccaaa tagtcctgac
acggccaatg atggctttgt acggcttaga 420ggacttccct ttggatgtag
caaggaagaa attgttcagt tcttctcagg gttggaaatc 480gtgccaaatg
ggataacatt gccggtggac ttccagggga ggagtacggg ggaggccttc
540gtgcagtttg cttcacagga aatagctgaa aaggctctaa agaaacacaa
ggaaagaata 600gggcacaggt atattgaaat ctttaagagc agtagagctg
aagttagaac tcattatgat 660ccaccacgaa agcttatggc catgcagcgg
ccaggtcctt atgacagacc tggggctggt 720agagggtata acagcattgg
cagaggagct ggctttgaga ggatgaggcg tggtgcttat 780ggtggaggct
atggaggcta tgatgattac aatggctata atgatggcta tggatttggg
840tcagatagat ttggaagaga cctcaattac tgtttttcag gaatgtctga
tcacagatac 900ggggatggtg gctctacttt ccagagcaca acaggacact
gtgtacacat gcggggatta 960ccttacagag ctactgagaa tgacatttat
aatttttttt caccgctcaa ccctgtgaga 1020gtacacattg aaattggtcc
tgatggcaga gtaactggtg aagcagatgt cgagttcgca 1080actcatgaag
atgctgtggc agctatgtca aaagacaaag caaatatgca acacagatat
1140gtagaactct tcttgaattc tacagcagga gcaagcggtg gtgcttacga
acacagatat 1200gtagaactct tcttgaattc tacagcagga gcaagcggtg
gtgcttatgg tagccaaatg 1260atgggaggca tgggcttgtc aaaccagtcc
agctacgggg gcccagccag ccagcagctg 1320agtgggggtt acggaggcgg
ctacggtggc cagagcagca tgagtggata cgaccaagtt 1380ttacaggaaa
actccagtga ttttcaatca aacattgcat aggtaaccaa ggagcagtga
1440acagcagcta ctacagtagt ggaagccgtg catctatggg cgtgaacgga
atgggagggt 1500tgtctagcat gtccagtatg agtggtggat ggggaatgta
attgatcgat cctgatcact 1560gactcttggt caaccttttt tttttttttt
ttttctttaa gaaaacttca gtttaacagt 1620ttctgcaata caagcttgtg
atttatgctt actctaagtg gaaatcagga ttgttatgaa 1680gacttaaggc
ccagtatttt tgaatacaat actcatctag gatgtaacag tgaagctgag
1740taaactataa ctgttaaact taagttccag cttttctcaa gttagttata
ggatgtactt 1800aagcagtaag cgtatttagg taaaagcagt tgaattatgt
taaatgttgc cctttgccac 1860gttaaattga acactgtttt ggatgcatgt
tgaaagacat gcttttattt tttttgtaaa 1920acaatatagg agctgtgtct
actattaaaa gtgaaacatt ttggcatgtt tgttaattct 1980agtttcattt
aataacctgt aaggcacgta agtttaagct tttttttttt ttaagttaat
2040gggaaaaatt tgagacgcaa taccaatact taggattttg gtcttggtgt
ttgtatgaaa 2100ttctgaggcc ttgatttaaa tctttcattg tattgtgatt
tccttttagg tatattgcgc 2160taagtgaaac ttgtcaaata aatcctcctt
ttaaaaactg c 2201871620DNAHomo sapiens 87ccggcggcgc tgggaggtgg
agtcgttgct gttgctgttt gtgagcctgt ggcgcggctt 60ctgtgggccg gaaccttaaa
gatagccgta atggctgaaa atggtgataa tgaaaagatg 120gctgccctgg
aggccaaaat ctgtcatcaa attgagtatt attttggcga cttcaatttg
180ccacgggaca agtttctaaa ggaacagata aaactggatg aaggctgggt
acctttggag 240ataatgataa aattcaacag gttgaaccgt ctaacaacag
actttaatgt aattgtggaa 300gcattgagca aatccaaggc agaactcatg
gaaatcagtg aagataaaac taaaatcaga 360aggtctccaa gcaaacccct
acctgaagtg actgatgagt ataaaaatga tgtaaaaaac 420agatctgttt
atattaaagg cttcccaact gatgcaactc ttgatgacat aaaagaatgg
480ttagaagata aaggtcaagt actaaatatt cagatgagaa gaacattgca
taaagcattt 540aagggatcaa tttttgttgt gtttgatagc attgaatctg
ctaagaaatt tgtagagacc 600cctggccaga agtacaaaga aacagacctg
ctaatacttt tcaaggacga ttactttgcc 660aaaaaaaatg aagaaagaaa
acaaaataaa gtggaagcta aattaagagc taaacaggag 720caagaagcaa
aacaaaagtt agaagaagat gctgaaatga aatctctaga agaaaagatt
780ggatgcttgc tgaaattttc gggtgattta gatgatcaga cctgtagaga
agatttacac 840atacttttct caaatcatgg tgaaataaaa tggatagact
tcgtcagagg agcaaaagag 900gggataattc tatttaaaga aaaagccaag
gaagcattgg gtaaagccaa agatgcaaat 960aatggtaacc tacaattaag
gaacaaagaa gtgacttggg aagtactaga aggagaggtg 1020gaaaaagaag
cactgaagaa aataatagaa gaccaacaag aatccctaaa caaatggaag
1080tcaaaaggtc gtagatttaa aggaaaagga aagggtaata aagctgccca
gcctgggtct 1140ggtaaaggaa aagtacagtt tcagggcaag aaaacgaaat
ttgctagtga tgatgaacat 1200gatgaacatg atgaaaatgg tgcaactgga
cctgtgaaaa gagcaagaga agaaacagac 1260aaagaagaac ctgcatccaa
acaacagaaa acagaaaatg gtgctggaga ccagtagttt 1320agtaaaccaa
ttttttattc attttaaata ggttttaaac gacttttgtt tgcggggctt
1380ttaaaaggaa aaccgaatta ggtccacttc aatgtccacc tgtgagaaag
gaaaaatttt 1440tttgttgttt aacttgtctt tttgttatgc aaatgagatt
tctttgaatg tattgttctg 1500tttgtgttat ttcagatgat tcaaatatca
aaaggaagat tcttccatta aattgccttt 1560gtaatatgag aatgtattag
tacaaactaa ctaataaaat atatactata tgaaaagagc 16208835RNAHomo sapiens
88guugaacuug aaucagaaga ugaagucaaa uuggc 358921RNAArtificialsiRNA
89cuugaaucag aagaugaagu u 219021RNAArtificialsiRNA 90cuucaucuuc
ugauucaagu u 219127RNAArtificialsiRNA 91aacuugaauc agaagaugaa
gucaaau 279227RNAArtificialsiRNA 92auuugacuuc aucuucugau ucaaguu
279335RNAHomo sapiens 93auaaaacugg augaaggcug gguaccuuug gagau
359421RNAArtificialsiRNA 94cuggaugaag gcuggguacu u
219521RNAArtificialsiRNA 95guacccagcc uucauccagu u
219627RNAArtificialsiRNA 96aacuggauga aggcugggua ccuuuuu
279727RNAArtificialsiRNA 97ccaaagguac ccagccuuca uccaguu
279822RNAArtificialsiRNA 98gacccugaag uucaucugca cc
229922RNAArtificialsiRNA 99ugcagaugaa cuucaggguc ag
2210022RNAArtificialsiRNA 100cugacccuga aguucaucug ca
2210122RNAArtificialsiRNA 101cagaugaacu ucagggucag cu
2210225DNAArtificialsiRNA 102ugcagaugaa cuucaggguc agctt
2510327RNAArtificialsiRNA 103ugacccugaa guucaucugc accaccg
2710427RNAArtificialsiRNA 104cgguggugca gaugaacuuc aggguca
2710521RNAArtificialsiRNA 105ugaaguucau cugcaccacc g
2110622RNAArtificialsiRNA 106ccugaaguuc aucugcacca cc
2210722RNAArtificialsiRNA 107uggugcagau gaacuucagg gu
2210824RNAArtificialsiRNA 108acccugaagu ucaucugcac cacc
2410924RNAArtificialsiRNA 109uggugcagau gaacuucagg guca
2411022RNAArtificialsiRNA 110gugcagauga acuucagggu ca
2211125DNAArtificialsiRNA 111acccugaagu ucaucugcac caccg
2511227RNAArtificialsiRNA 112cgguggugca gaugaacuuc aggguca
2711327RNAArtificialsiRNA 113caugaagcag cacgacuucu ucaaguc
2711425DNAArtificialsiRNA 114cuugaagaag ucgugcugcu ucatg
2511525DNAArtificialsiRNA 115gcagcacgac uucuucaagu ccgcc
2511648DNAArtificialEGFP from cloning vector pEGFP-C1 116acggcaagct
gaccctgaag ttcatctgca ccaccggcaa gctgcccg 48
* * * * *