U.S. patent application number 09/866557 was filed with the patent office on 2002-10-31 for methods and compositions for rna interference.
Invention is credited to Beach, David, Bernstein, Emily, Caudy, Amy, Hammond, Scott, Hannon, Gregory.
Application Number | 20020162126 09/866557 |
Document ID | / |
Family ID | 26885458 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020162126 |
Kind Code |
A1 |
Beach, David ; et
al. |
October 31, 2002 |
Methods and compositions for RNA interference
Abstract
The present invention provides methods for attenuating gene
expression in a cell using gene-targeted double stranded RNA
(dsRNA). The dsRNA contains a nucleotide sequence that hybridizes
under physiologic conditions of the cell to the nucleotide sequence
of at least a portion of the gene to be inhibited (the "target"
gene).
Inventors: |
Beach, David; (Boston,
MA) ; Bernstein, Emily; (Huntington, NY) ;
Caudy, Amy; (Melville, NY) ; Hammond, Scott;
(Huntington, NY) ; Hannon, Gregory; (Huntington,
NY) |
Correspondence
Address: |
ROPES & GRAY
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
26885458 |
Appl. No.: |
09/866557 |
Filed: |
May 24, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09866557 |
May 24, 2001 |
|
|
|
PCT/US01/08435 |
Mar 16, 2001 |
|
|
|
60189739 |
Mar 16, 2000 |
|
|
|
60243097 |
Oct 24, 2000 |
|
|
|
Current U.S.
Class: |
800/8 ; 435/455;
514/44A |
Current CPC
Class: |
C12N 15/111 20130101;
C12N 2310/53 20130101; C12N 2320/12 20130101; A01K 2217/05
20130101; C12N 2310/14 20130101; C12N 15/1034 20130101; C12N 15/102
20130101 |
Class at
Publication: |
800/8 ; 514/44;
435/455 |
International
Class: |
A01K 067/00; A61K
048/00; C12N 015/87 |
Goverment Interests
[0002] Work described herein was supported by National Institutes
of Health Grant R01-GM62534. The United States Government may have
certain rights in the invention.
Claims
We claim:
1. A method for attenuating expression of a target gene in cultured
cells, comprising introducing double stranded RNA (dsRNA) into the
cells in an amount sufficient to attenuate expression of the target
gene, wherein the dsRNA comprises a nucleotide sequence that
hybridizes under stringent conditions to a nucleotide sequence of
the target gene.
2. A method for attenuating expression of a target gene in a
mammalian cell, comprising (i) activating one or both of a Dicer
activity or an Argonaut activity in the cell, and (ii) introducing
into the cell a double stranded RNA (dsRNA) in an amount sufficient
to attenuate expression of the target gene, wherein the dsRNA
comprises a nucleotide sequence that hybridizes under stringent
conditions to a nucleotide sequence of the target gene.
3. The method of claim 2, wherein the cell is suspended in
culture.
4. The method of claim 2, wherein the cell is in a whole animal,
such as a non-human mammal.
5. The method of claim 1 or 2, wherein is engineered with (i) a
recombinant gene encoding a Dicer activity, (ii) a recombinant gene
encoding an Argonaut activity, or (iii) both.
6. The method of claim 5, wherein the recombinant gene encodes a
protein which includes an amino acid sequence at least 50 percent
identical to SEQ ID No. 2 or 4 or the Argonaut sequence shown in
FIG. 24.
7. The method of claim 5, wherein the recombinant gene includes a
coding sequence hybridizes under wash conditions of 2.times.SSC at
22.degree. C. to SEQ ID No. 1 or 3.
8. The method of claim 1 or 2, wherein an endogenous Dicer gene or
Argonaut gene is activated.
9. The method of claim 1 or 2, wherein the target gene is an
endogenous gene of the cell.
10. The method of claim 1 or 2, wherein the target gene is an
heterologous gene relative to the genome of the cell, such as a
pathogen gene.
11. The method of claim 1 or 2, wherein the cell is treated with an
agent that inhibits protein kinase RNA-activated (PKR) apoptosis,
such as by treatment with agents which inhibit expression of PKR,
cause its destruction, and/or inhibit the kinase activity of
PKF.
12. The method of claim 1 or 2, wherein the cell is a primate cell,
such as a human cell.
13. The method of claim 1 or 2, wherein the dsRNA is at least 20
nucleotides in length.
14. The method of claim 13, wherein the dsRNA is at least 100
nucleotides in length.
15. The method of claim 1 or 2, wherein expression of the target
gene is attenuated by at least 10 fold.
16. An assay for identifying nucleic acid sequences responsible for
conferring a particular phenotype in a cell, comprising (i)
constructing a variegated library of nucleic acid sequences from a
cell in an orientation relative to a promoter to produce double
stranded DNA; (ii) introducing the variegated dsRNA library into a
culture of target cells, which cells have an activated Dicer
activity or Argonaut activity; (iii) identifying members of the
library which confer a particular phenotype on the cell, and
identifying the sequence from a cell which correspond, such as
being identical or homologous, to the library member.
17. A method of conducting a drug discovery business comprising:
(i) identifying, by the assay of claim 16, a target gene which
provides a phenotypically desirable response when inhibited by
RNAi; (ii) identifying agents by their ability to inhibit
expression of the target gene or the activity of an expression
product of the target gene; (iii) conducting therapeutic profiling
of agents identified in step (b), or further analogs thereof, for
efficacy and toxicity in animals; and (iv) formulating a
pharmaceutical preparation including one or more agents identified
in step (iii) as having an acceptable therapeutic profile.
18. The method of claim 17, including an additional step of
establishing a distribution system for distributing the
pharmaceutical preparation for sale, and may optionally include
establishing a sales group for marketing the pharmaceutical
preparation.
19. A method of conducting a target discovery business comprising:
(i) identifying, by the assay of claim 16, a target gene which
provides a phenotypically desirable response when inhibited by
RNAi; (ii) (optionally) conducting therapeutic profiling of the
target gene for efficacy and toxicity in animals; and (iii).
licensing, to a third party, the rights for further drug
development of inhibitors of the target gene.
20. A method for attenuating expression of a target gene in a cell,
comprising introducing into the cell a hairpin nucleic acid in an
amount sufficient to attenuate expression of the target gene,
wherein the hairpin nucleic acid comprises an inverted repeat of a
nucleotide sequence that hybridizes under stringent conditions to a
nucleotide sequence of the target gene.
21. A hairpin nucleic acid for inhibiting expression of a target
gene, comprising a first nucleotide sequence that hybridizes under
stringent conditions to a nucleotide sequence of the target gene,
and a second nucleotide sequence which is an complementary inverted
repeat of said first nucleotide sequence and hybridizes to said
first nucleotide sequence to form a hairpin structure.
22. The method of claim 20 or the hairpin nucleic acid of claim 21,
wherein the hairpin nucleic is RNA.
23. A non-human transgenic mammal having germline and/or somatic
cells comprising a transgene encoding a dsRNA construct.
24. The transgenic animal of claim 23, which is chimeric for said
transgene.
25. The transgenic animal of claim 23, wherein said transgene is
chromosomally incorporated.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of PCT
application PCT/US01/08435, filed Mar. 16, 2001, and claims the
benefit of U.S. Provisional applications U.S. Ser. No. 60/189,739
filed Mar. 16, 2000 and U.S. Ser. No. 60/243,097 filed Oct. 24,
2000. The specifications of such applications are incorporated by
reference herein.
BACKGROUND OF THE INVENTION
[0003] "RNA interference", "post-transcriptional gene silencing",
"quelling"--these different names describe similar effects that
result from the overexpression or misexpression of transgenes, or
from the deliberate introduction of double-stranded RNA into cells
(reviewed in Fire A (1999) Trends Genet 15:358-363; Sharp PA (1999)
Genes Dev 13:139-141; Hunter C (1999) Curr Biol 9:R440-R442;
Baulcombe DC (1999) Curr Biol 9:R599-R601; Vaucheret et al. (1998)
Plant J 16:651-659). The injection of double-stranded RNA into the
nematode Caenorhabditis elegans, for example, acts systemically to
cause the post-transcriptional depletion of the homologous
endogenous RNA (Fire et al. (1998) Nature 391: 806-811; and
Montgomery et al. (1998) PNAS 95:15502-15507). RNA interference,
commonly referred to as RNAi, offers a way of specifically and
potently inactivating a cloned gene, and is proving a powerful tool
for investigating gene function. But the phenomenon is interesting
in its own right; the mechanism has been rather mysterious, but
recent research--the latest reported by Smardon et al. (2000) Curr
Biol 10:169-178--is beginning to shed light on the nature and
evolution of the biological processes that underlie RNAi.
[0004] RNAi was discovered when researchers attempting to use the
antisense RNA approach to inactivate a C. elegans gene found that
injection of sense-strand RNA was actually as effective as the
antisense RNA at inhibiting gene function. Guo et al. (1995) Cell
81:611-620. Further investigation revealed that the active agent
was modest amounts of double-stranded RNA that contaminate in vitro
RNA preparations. Researchers quickly determined the `rules` and
effects of RNAi. Exon sequences are required, whereas introns and
promoter sequences, while ineffective, do not appear to compromise
RNAi (though there may be gene-specific exceptions to this rule).
RNAi acts systemically--injection into one tissue inhibits gene
function in cells throughout the animal. The results of a variety
of experiments, in C. elegans and other organisms, indicate that
RNAi acts to destabilize cellular RNA after RNA processing.
[0005] The potency of RNAi inspired Timmons and Fire (1998 Nature
395: 854) to do a simple experiment that produced an astonishing
result. They fed to nematodes bacteria that had been engineered to
express double-stranded RNA corresponding to the C. elegans unc-22
gene. Amazingly, these nematodes developed a phenotype similar to
that of unc-22 mutants that was dependent on their food source. The
ability to conditionally expose large numbers of nematodes to
gene-specific double-stranded RNA formed the basis for a very
powerful screen to select for RNAi-defective C. elegans mutants and
then to identify the corresponding genes.
[0006] Double-stranded RNAs (dsRNAs) can provoke gene silencing in
numerous in vivo contexts including Drosophila, Caenorhabditis
elegans, planaria, hydra, trypanosomes, fungi and plants. However,
the ability to recapitulate this phenomenon in higher eukaryotes,
particularly mammalian cells, has not be accomplished in the art.
Nor has the prior art demonstrated that this phenomena can be
observe in cultured eukaryotes cells.
SUMMARY OF THE INVENTION
[0007] One aspect of the present invention provides a method for
attenuating expression of a target gene in cultured cells,
comprising introducing double stranded RNA (dsRNA) into the cells
in an amount sufficient to attenuate expression of the target gene,
wherein the dsRNA comprises a nucleotide sequence that hybridizes
under stringent conditions to a nucleotide sequence of the target
gene.
[0008] Another aspect of the present invention provides a method
for attenuating expression of a target gene in a mammalian cell,
comprising
[0009] (i) activating one or both of a Dicer activity or an
Argonaut activity in the cell, and
[0010] (ii) introducing into the cell a double stranded RNA (dsRNA)
in an amount sufficient to attenuate expression of the target gene,
wherein the dsRNA comprises a nucleotide sequence that hybridizes
under stringent conditions to a nucleotide sequence of the target
gene.
[0011] In certain embodiments, the cell is suspended in culture;
while in other embodiments the cell is in a whole animal, such as a
non-human mammal.
[0012] In certain preferred embodiments, the cell is engineered
with (i) a recombinant gene encoding a Dicer activity, (ii) a
recombinant gene encoding an Argonaut activity, or (iii) both. For
instance, the recombinant gene may encode, for a example, a protein
which includes an amino acid sequence at least 50 percent identical
to SEQ ID No. 2 or 4; or be defined by a coding sequence hybridizes
under wash conditions of 2.times.SSC at 22.degree. C. to SEQ ID No.
1 or 3. In certain embodiments, the recombinant gene may encode,
for a example, a protein which includes an amino acid sequence at
least 50 percent identical to the Argonaut sequence shown in FIG.
24.
[0013] In certain embodiments, rather than use a heterologous
expression construct(s), an endogenous Dicer gene or Argonaut gene
can be activated, e.g, by gene activation technology, expression of
activated transcription factors or other signal transduction
protein, which induces expression of the gene, or by treatment with
an endogenous factor which upregualtes the level of expression of
the protein or inhibits the degradation of the protein.
[0014] In certain preferred embodiments, the target gene is an
endogenous gene of the cell. In other embodiments, the target gene
is an heterologous gene relative to the genome of the cell, such as
a pathogen gene, e.g., a viral gene.
[0015] In certain embodiments, the cell is treated with an agent
that inhibits protein kinase RNA-activated (PKR) apoptosis, such as
by treatment with agents which inhibit expression of PKR, cause its
destruction, and/or inhibit the kinase activity of PKF.
[0016] In certain preferred embodiments, the cell is a primate
cell, such as a human cell.
[0017] In certain preferred embodiments, the length of the dsRNA is
at least 20, 21 or 22 nucleotides in length, e.g., corresponding in
size to RNA products produced by Dicer-dependent cleavage. In
certain embodiments, the dsRNA construct is at least 25, 50, 100,
200, 300 or 400 bases. In certain embodiments, the dsRNA construct
is 400-800 bases in length.
[0018] In certain preferred embodiments, expression of the target
gene is attenuated by at least 5 fold, and more preferably at least
10, 20 or even 50 fold, e.g., relative to the untreated cell or a
cell treated with a dsRNA construct which does not correspond to
the target gene.
[0019] Yet another aspect of the present invention provides a
method for attenuating expression of a target gene in cultured
cells, comprising introducing an expression vector having a "coding
sequence" which, when transcribed, produces double stranded RNA
(dsRNA) the cell in an amount sufficient to attenuate expression of
the target gene, wherein the dsRNA comprises a nucleotide sequence
that hybridizes under stringent conditions to a nucleotide sequence
of the target gene. An certain embodiments, the vector includes a
single coding sequence for the dsRNA which is operably linked to
(two) transcriptional regulatory sequences which cause
transcription of in both directions (to form complementary
transcripts of the coding sequence. In other embodiments, the
vector includes two coding sequences which, respectively, give rise
to the two complementary sequences which form the dsRNA when
annealed. In certain embodiments, the vectors are episomal, e.g.,
and transfection is transient. In other embodiments, the vectors
are chromosomally integrated, e.g., to produce a stably transfected
cell line. Preferred vectors for forming such stable cell lines are
the described in U.S. Pat. No. 6,025,192 and PCT publication
WO/9812339, which are incorporated by reference herein.
[0020] Still another aspect of the present invention provides an
assay for identifying nucleic acid sequences responsible for
conferring a particular phenotype in a cell, comprising
[0021] (i) constructing a variegated library of nucleic acid
sequences from a cell in an orientation relative to a promoter to
produce double stranded DNA;
[0022] (ii) introducing the variegated dsRNA library into a culture
of target cells, which cells have an activated Dicer activity or
Argonaut activity;
[0023] (iii) identifying members of the library which confer a
particular phenotype on the cell, and identifying the sequence from
a cell which correspond, such as being identical or homologous, to
the library member.
[0024] Yet another aspect of the present invention provides a
method of conducting a drug discovery business comprising:
[0025] (i) identifying, by the subject assay, a target gene which
provides a phenotypically desirable response when inhibited by
RNAi;
[0026] (ii) identifying agents by their ability to inhibit
expression of the target gene or the activity of an expression
product of the target gene;
[0027] (iii) conducting therapeutic profiling of agents identified
in step (b), or further analogs thereof, for efficacy and toxicity
in animals; and
[0028] (iv) formulating a pharmaceutical preparation including one
or more agents identified in step (iii) as having an acceptable
therapeutic profile.
[0029] The method may include an additional step of establishing a
distribution system for distributing the pharmaceutical preparation
for sale, and may optionally include establishing a sales group for
marketing the pharmaceutical preparation.
[0030] Another aspect of the present invention provides a method of
conducting a target discovery business comprising:
[0031] (i) identifying, by the subject assay, a target gene which
provides a phenotypically desirable response when inhibited by
RNAi;
[0032] (ii) (optionally) conducting therapeutic profiling of the
target gene for efficacy and toxicity in animals; and
[0033] (iii). licensing, to a third party, the rights for further
drug development of inhibitors of the target gene.
[0034] Another aspect of the invention provides a method for
inhibiting RNAi by inhibiting the expression or activity of an RNAi
enzyme. Thus, the subject method may include inhibiting the
acitivity of Dicer and/or the 22-mer RNA.
[0035] Still another aspect relates to the a method for altering
the specificity of an RNAi by modifying the sequence of the RNA
component of the RNAi enzyme.
[0036] Another aspect of the invention relates to purified or
semi-purified preparations of the RNAi enzyme or components
thereof. In certain embodiments, the preparations are used for
identifying compounds, especially small organic molecules, which
inhibit or potentiate the RNAi activity. Small molecule inhibitors,
for example, can be used to inhibit dsRNA responses in cells which
are purposefully being transfected with a virus which produces
double stranded RNA.
[0037] The dsRNA construct may comprise one or more strands of
polymerized ribonucleotide. It may include modifications to either
the phosphate-sugar backbone or the nucleoside. The double-stranded
structure may be formed by a single self-complementary RNA strand
or two complementary RNA strands. RNA duplex formation may be
initiated either inside or outside the cell. The dsRNA construct
may be introduced in an amount which allows delivery of at least
one copy per cell. Higher doses of double-stranded material may
yield more effective inhibition. Inhibition is sequence-specific in
that nucleotide sequences corresponding to the duplex region of the
RNA are targeted for genetic inhibition. dsRNA constructs
containing a nucleotide sequences identical to a portion of the
target gene is preferred for inhibition. RNA sequences with
insertions, deletions, and single point mutations relative to the
target sequence have also been found to be effective for
inhibition. Thus, sequence identity may optimized by alignment
algorithms known in the art and calculating the percent difference
between the nucleotide sequences. Alternatively, the duplex region
of the RNA may be defined functionally as a nucleotide sequence
that is capable of hybridizing with a portion of the target gene
transcript.
[0038] Yet another aspect of the invention pertains to transgenic
non-human mammals which include a transgene encoding a dsRNA
construct, preferably which is stably integrated into the genome of
cells in which it occurs. The animals can be derived by oocyte
microinjection, for example, in which case all of the nucleated
cells of the animal will include the transgene, or can be derived
using embryonic stem (ES) cells which have been transfected with
the transgene, in which case the animal is a chimera and only a
portion of its nucleated cells will include the transgene. In
certain instances, the sequence-independent dsRNA response, e.g.,
the PKR response, is also inhibited in those cells including the
transgene.
[0039] In still other embodiments, dsRNA itself can be introduced
into an ES cell in order to effect gene silencing, and that
phenotype will be carried for at least several rounds of division,
e.g., into the progeny of that cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1: RNAi in S2 cells. a, Drosophila S2 cells were
transfected with a plasmid that directs lacZ expression from the
copia promoter in combination with dsRNAs corresponding to either
human CD8 or lacZ, or with no dsRNA, as indicated. b, S2 cells were
co-transfected with a plasmid that directs expression of a GFP-US9
fusion protein (12) and dsRNAs of either lacZ or cyclin E, as
indicated. Upper panels show FACS profiles of the bulk population.
Lower panels show FACS profiles from GFP-positive cells. c, Total
RNA was extracted from cells transfected with lacZ, cyclin E, fizzy
or cyclin A dsRNAs, as indicated. Northern blots were hybridized
with sequences not present in the transfected dsRNAs.
[0041] FIG. 2: RNAi in vitro. a, Transcripts corresponding to
either the first 600 nucleotides of Drosophila cyclin E (E600) or
the first 800 nucleotides of lacZ (Z800) were incubated in lysates
derived from cells that had been transfected with either lacZ or
cyclin E (cycE) dsRNAs, as indicated. Time points were 0, 10, 20,
30, 40 and 60 min for cyclin E and 0, 10, 20, 30 and 60 min for
lacZ. b, Transcripts were incubated in an extract of S2 cells that
had been transfected with cyclin E dsRNA (cross-hatched box,
below). Transcripts corresponded to the first 800 nucleotides of
lacZ or the first 600, 300, 220 or 100 nucleotides of cyclin E, as
indicated. Eout is a transcript derived from the portion of the
cyclin E cDNA not contained within the transfected dsRNA. E-ds is
identical to the dsRNA that had been transfected into S2 cells.
Time points were 0 and 30 min. c, Synthetic transcripts
complementary to the complete cyclin E cDNA (Eas) or the final 600
nucleotides (Eas600) or 300 nucleotides (Eas300) were incubated in
extract for 0 or 30 min.
[0042] FIG. 3: Substrate requirements of the RISC. Extracts were
prepared from cells transfected with cyclin E dsRNA. Aliquots were
incubated for 30 min at 30.degree. C. before the addition of either
the cyclin E (E600) or lacZ (Z800) substrate. Individual 20-.mu.l
aliquots, as indicated, were pre-incubated with 1 mM CaCl.sub.2 and
5 mM EGTA, 1 mM CaCl.sub.2, 5 mM EGTA and 60 U of micrococcal
nuclease, 1 mM CaCl.sub.2 and 60 U of micrococcal nuclease or 10 U
of DNase I (Promega) and 5 mM EGTA. After the 30-min
pre-incubation, EGTA was added to those samples that lacked it.
Yeast tRNA (1 .mu.g) was added to all samples. Time points were at
0 and 30 min.
[0043] FIG. 4: The RISC contains a potential guide RNA. a, Northern
blots of RNA from either a crude lysate or the S100 fraction
(containing the soluble nuclease activity, see Methods) were
hybridized to a riboprobe derived from the sense strand of the
cyclin E mRNA. b, Soluble cyclin-E-specific nuclease activity was
fractionated as described in Methods. Fractions from the
anion-exchange resin were incubated with the lacZ, control
substrate (upper panel) or the cyclin E substrate (centre panel).
Lower panel, RNA from each fraction was analysed by northern
blotting with a uniformly labelled transcript derived from sense
strand of the cyclin E cDNA. DNA oligonucleotides were used as size
markers.
[0044] FIG. 5: Generation of 22 mers and degradation of mRNA are
carried out by distinct enzymatic complexes. A. Extracts prepared
either from 0-12 hour Drosophila embryos or Drosophila S2 cells
(see Methods) were incubated 0, 15, 30, or 60 minutes (left to
right) with a uniformly-labeled double-stranded RNA corresponding
to the first 500 nucleotides of the Drosophila cyclin E coding
region. M indicates a marker prepared by in vitro transcription of
a synthetic template. The template was designed to yield a 22
nucleotide transcript. The doublet most probably results from
improper initiation at the +1 position. B. Whole-cell extracts were
prepared from S2 cells that had been transfected with a dsRNA
corresponding to the first 500 nt. of the luciferase coding region.
S10 extracts were spun at 30,000.times.g for 20 minutes which
represents our standard RISC extract.sup.6. S100 extracts were
prepared by further centrifugation of S10 extracts for 60 minutes
at 100,000.times.g. Assays for mRNA degradation were carried out as
described previously.sup.6 for 0, 30 or 60 minutes (left to right
in each set) with either a single-stranded luciferase mRNA or a
single-stranded cyclin E mRNA, as indicated. C. S10 or S100
extracts were incubated with cyclin E dsRNAs for 0, 60 or 120
minutes (L to R).
[0045] FIG. 6: Production of 22 mers by recombinant CG4792/Dicer.
A. Drosophila S2 cells were transfected with plasmids that direct
the expression of T7-epitope tagged versions of Drosha,
CG4792/Dicer-1 and Homeless. Tagged proteins were purified from
cell lysates by immunoprecipitation and were incubated with cyclin
E dsRNA. For comparison, reactions were also performed in
Drosophila embryo and S2 cell extracts. As a negative control,
immunoprecipitates were prepared from cells transfected with a
.beta.-galactosidase expression vector. Pairs of lanes show
reactions performed for 0 or 60 minutes. The synthetic marker (M)
is as described in the legend to FIG. 1. B. Diagrammatic
representations of the domain structures of CG4792/Dicer-1, Drosha
and Homeless are shown. C. Immunoprecipitates were prepared from
detergent lysates of S2 cells using an antiserum raised against the
C-terminal 8 amino acids of Drosophila Dicer-1 (CG4792). As
controls, similar preparations were made with a pre-immune serum
and with an immune serum that had been pre-incubated with an excess
of antigenic peptide. Cleavage reactions in which each of these
precipitates was incubated with an .about.500 nt. fragment of
Drosophila cyclin E are shown. For comparsion, an incubation of the
substrate in Drosophila embryo extract was electrophoresed in
parallel. D. Dicer immunoprecipitates were incubated with dsRNA
substrates in the presence or absence of ATP. For comparison, the
same substrate was incubated with S2 extracts that either contained
added ATP or that were depleted of ATP using glucose and hexokinase
(see methods). E. Drosophila S2 cells were transfected with
uniformly, 32P-labelled dsRNA corresponding to the first 500 nt. of
GFP. RISC complex was affinity purified using a histidine-tagged
version of D.m. Ago-2, a recently identified component of the RISC
complex (Hammond et al., in prep). RISC was isolated either under
conditions in which it remains ribosome associated (ls, low salt)
or under conditions that extract it from the ribosome in a soluble
form (hs, high salt).sup.6. For comparison, the spectrum of
labelled RNAs in the total lysate is shown. F. Guide RNAs produced
by incubation of dsRNA with a Dicer immunoprecipitate are compared
to guide RNAs present in a affinity-purified RISC complex. These
precisely comigrate on a gel that has single-nucleotide resolution.
The lane labelled control is an affinity selection for RISC from
cell that had been transfected with labeled dsRNA but not with the
epitope-tagged D.m. Ago-2.
[0046] FIG. 7: Dicer participates in RNAi. A. Drosophila S2 cells
were transfected with dsRNAs corresponding to the two Drosophila
Dicers (CG4792 and CG6493) or with a control dsRNA corresponding to
murine caspase 9. Cytoplasmic extracts of these cells were tested
for Dicer activity. Transfection with Dicer dsRNA reduced activity
in lysates by 7.4-fold. B. The Dicer-1 antiserum (CG4792) was used
to prepare immunoprecipitates from S2 cells that had been treated
as described above. Dicer dsRNA reduced the activity of Dicer-1 in
this assay by 6.2-fold. C. Cells that had been transfected two days
previously with either mouse caspase 9 dsRNA or with Dicer dsRNA
were cotransfected with a GFP expression plasmid and either
control, luciferase dsRNA or GFP dsRNA. Three independent
experiments were quantified by FACS. A comparison of the relative
percentage of GFP-positive cells is shown for control (GFP plasmid
plus luciferase dsRNA) or silenced (GFP plamsid plus GFP dsRNA)
populations in cells that had previously been transfected with
either control (caspase 9) or Dicer dsRNAs.
[0047] FIG. 8: Dicer is an evolutionarily conserved ribonuclease.
A. A model for production of 22 mers by Dicer. Based upon the
proposed mechanism of action of Ribonuclease III, we propose that
Dicer acts on its substrate as a dimer. The positioning of the two
ribonuclease domains (RIIIa and RIIIb) within the enzyme would thus
determine the size of the cleavage product. An equally plausible
alternative model could be derived in which the RIIIa and RIIIb
domains of each Dicer enzyme would cleave in concert at a single
position. In this model, the size of the cleavage product would be
determined by interaction between two neighboring Dicer enzymes. B.
Comparison of the domain structures of potential Dicer homologs in
various organisms (Drosophila--CG4792, CG6493, C. elegans--K12H4.8,
Arabidopsis--CARPEL FACTORY.sup.24, T25K16.4, AC012328.sub.--1,
human Helicase-MOI.sup.25 and S. pombe--YC9A_SCHPO). The ZAP
domains were identified both by analysis of individual sequences
with Pfam.sup.27 and by Psi-blast.sup.28 searches. The ZAP domain
in the putative S. pombe Dicer is not detected by PFAM but is
identified by Psi-Blast and is thus shown in a different color. For
comparison, a domain structure of the RDE1/QDE2/ARGONAUTE family is
shown. It should be noted that the ZAP domains are more similar
within each of the Dicer and ARGONAUTE families than they are
between the two groups. C. An alignment of the ZAP domains in
selected Dicer and Argonaute family members is shown. The alignment
was produced using ClustalW.
[0048] FIG. 9: Purification strategy for RISC. (second step in RNAi
model).
[0049] FIG. 10: Fractionation of RISC activity over sizing column.
Activity fractionates as 500 KD complex. Also, antibody to dm
argonaute 2 cofractionates with activity.
[0050] FIGS. 11-13: Fractionation of RISC over monoS, monoQ,
Hydroxyapatite columns. Dm argonaute 2 protein also
cofactionates.
[0051] FIG. 14: Alignment of dm argonaute 2 with other family
members.
[0052] FIG. 15: Confirmation of dm argonaute 2. S2 cells were
transfected with labeled dsRNA and His tagged argonaute. Argonaute
was isolated on nickel agarose and RNA component was identified on
15% acrylamide gel.
[0053] FIG. 16: S2 cell and embryo extracts were assayed for 22 mer
generating activity.
[0054] FIG. 17: RISC can be separated from 22 mer generating
activity (dicer). Spinning extracts (S100) can clear RISC activity
from supernatant (left panel) however, S100 spins still contain
dicer activity (right panel).
[0055] FIG. 18: Dicer is specific for dsRNA and prefers longer
substrates.
[0056] FIG. 19: Dicer was fractionated over several columns.
[0057] FIG. 20: Identification of dicer as enzyme which can process
dsRNA into 22 mers. Various RNaseIII family members were expressed
with n terminal tags, immunoprecipitated, and assayed for 22 mer
generating activity (left panel). In right panel, antibodies to
dicer could also precipitate 22 mer generating activity.
[0058] FIG. 21: Dicer requires ATP.
[0059] FIG. 22: Dicer produces RNAs that are the same size as RNAs
present in RISC.
[0060] FIG. 23: Human dicer homolog when expressed and
immunoprecipitated has 22 mer generating activity.
[0061] FIG. 24: Sequence of dm argonaute 2. Peptides identified by
microsequencing are shown in underline.
[0062] FIG. 25: Molecular charaterization of dm argonaute 2. The
presence of an intron in coding sequence was determined by northern
blotting using intron probe. This results in a different 5' reading
frame that that published genome seqeunce. Number of polyglutaine
repeats was determined by genomic PCR.
[0063] FIG. 26: Dicer activity can be created in human cells by
expression of human dicer gene. Host cell was 293. Crude extracts
had dicer activity, while activity was absent from untransfected
cells. Activity is not dissimilar to that seen in drosophila embryo
extracts.
[0064] FIG. 27: An .about.500 nt. fragment of the gene that is to
be silenced (X) is inserted into the modified vector as a stable
direct repeat using standard cloning procedures. Treatment with
commercially available cre recombinase reverses sequences within
the loxP sites (L) to create an inverted repeat. This can be stably
maintained and amplified in an sbc mutant bacterial strain (DL759).
Transcription in vivo from the promoter of choice (P) yields a
hairpin RNA that causes silencing. A zeocin resistance marker is
included to insure maintenance of the direct and inverted repeat
structures; however this is non-essential in vivo and could be
removed by pre-mRNA splicing if desired. Smith, N. A. et al. Total
silencing by intron-spliced hairpin RNAs. Nature 407, 319-20
(2000).
[0065] FIG. 28: Hela, Chinese hamster ovary, and P19 (pluripotent,
mouse embryonic carcinoma) cell lines transfected with plasmids
expressing Photinus pyralis (firefly) Renilla reniformis (sea
pansy) luciferases and with dsRNA 500 mers (400 ng), either
homologous to firefly luciferase mRNA (dsLUC) or non-homologous
(dsGFP). Dual luciferase assays were carried out using an
Analytical Scientific Instruments model 3010 Luminometer. In this
assay Renilla luciferase serves as an internal control for
dsRNA-specific suppression of firefly luciferase activity. These
data demonstrate that 500 mer dsRNA can specifically suppress
cognate gene expression in vivo.
[0066] FIG. 29: P19 (a pluripontent, mouse embryonic cell line)
cells transfected with plasmids expressing Photinus pyralis
(firefly) Renilla reniformis (sea pansy) luciferases and with dsRNA
500 mers (500ng), either homologous to firefly luciferase mRNA
(dsLUC) or non-homologous (dsGFP). Dual luciferase assays were
carried out using an Analytical Scientific Instruments model 3010
Luminometer. In this assay Renilla luciferase serves as an internal
control for dsRNA-specific suppression of firefly luciferase
activity. These data further demonstrate that 500 mer dsRNA can
specifically suppress cognate gene expression in vivo and that the
effect is stable over time .
[0067] FIG. 30: S10 fractions from P19 cell lysates were used for
in vitro translations of mRNA coding for Photinus pyralis (firefly)
Renilla reniformis (sea pansy) luciferases. Translation reactions
were programmed with various amounts of dsRNA 500 mers, either
homologous to firefly luciferase mRNA (dsLUC) or non-homologous
(dsGFP). Reactions were carried out at 30 degrees for 1 hour, after
which dual luciferase assays were carried out using an Analytical
Scientific Instruments model 3010 Luminometer. In this assay
Renilla luciferase serves as an internal control for dsRNA-specific
suppression of firefly luciferase activity. These data demonstrate
that 500 mer dsRNA can specifically suppress cognate gene
expression in vitro in a manner consistent with
post-transcriptional gene silencing. Anti-sense firefly RNA did not
differ significantly from dsGFP control (approximately 10%) (data
not shown).
[0068] FIG. 31: S10 fractions from P19 cell lysates were used for
in vitro translations of mRNA coding for Photinus pyralis (firefly)
Renilla reniformis (sea pansy) luciferases. Translation reactions
were programmed with dsRNA or asRNA 500 mers, either complementary
to firefly luciferase mRNA (asLUC and dsLUC) or non-complementary
(dsGFP). Reactions were carried out at 30 degrees for 1 hour, after
a 30 min preincubation with dsRNA or asRNA. Dual luciferase assays
were carried out using an Analytical Scientific Instruments model
3010 Luminometer. In this assay Renilla luciferase serves as an
internal control for dsRNA-specific suppression of firefly
luciferase activity. These data demonstrate that 500 mer
double-stranded RNA (dsRNA) but not anti-sense RNA (asRNA)
suppresses cognate gene expression in vitro in a manner consistent
with post-transcriptional gene silencing.
[0069] FIG. 32: P19 cells were grown in 6-well tissue culture
plates to approximately 60% confluence. Various amounts of dsRNA,
either homologous to firefly luciferase mRNA (dsLUC) or
non-homologous (dsGFP), were added to each well and incubated for
12 hrs under normal tissue culture conditions. Cells were then
transfected with plasmids expressing Photinus pyralis (firefly)
Renilla reniformis (sea pansy) luciferases and with dsRNA 500 mers
(500 ng). Dual luciferase assays were carried out 12 hrs
post-transfection using an Analytical Scientific Instruments model
3010 Luminometer. In this assay Renilla luciferase serves as an
internal control for dsRNA-specific suppression of firefly
luciferase activity. These data show that 500 mer dsRNA can
specifically suppress cognate gene expression in vivo without
transfection under normal tissue culture conditions.
[0070] FIG. 33: Is a graph illustrating the relative rate of
expression luciferase in cells which are treated with various
antisense and dsRNA constructs.
DETAILED DESCRIPTION OF THE CERTAIN PREFERRED EMBODIMENTS
I. Overview
[0071] The present invention provides methods for attenuating gene
expression in a cell using gene-targeted double stranded RNA
(dsRNA). The dsRNA contains a nucleotide sequence that hybridizes
under physiologic conditions of the cell to the nucleotide sequence
of at least a portion of the gene to be inhibited (the "target"
gene).
[0072] A significant aspect to certain embodiments of the present
invention relates to the demonstration in the present application
that RNAi can in fact be accomplished in cultured cells, rather
than whole organisms as described in the art.
[0073] Another salient feature of the present invention concerns
the ability to carry out RNAi in higher eukaryotes, particularly in
non-oocytic cells of mammals, e.g., cells from adult mammals as an
example.
[0074] As described in further detail below, the present
invention(s) are based on the discovery that the RNAi phenomenum is
mediated by a set of enzyme activities, including an essential RNA
component, that are evolutionarily conserved in eukaryotes ranging
from plants to mammals.
[0075] One enzyme contains an essential RNA component. After
partial purification, a multi-component nuclease (herein "RISC
nuclease") co-fractionates with a discrete, 22-nucleotide RNA
species which may confer specificity to the nuclease through
homology to the substrate mRNAs. The short RNA molecules are
generated by a processing reaction from the longer input dsRNA.
Without wishing to be bound by any particular theory, these 22 mer
guide RNAs may serve as guide sequences that instruct the RISC
nuclease to destroy specific mRNAs corresponding to the dsRNA
sequences.
[0076] As illustrated in FIG. 33, double stranded forms of the
22-mer guide RNA can be sufficient in length to induce
sequence-dependent dsRNA inhibition of gene expression. In the
illustrated example, dsRNA contructs are administered to cells
having a recombinant luciferase reporter gene. The control cell,
e.g., no exogeneously added RNA, the level of expression of the
luciferase reporter is normalized to be the value of "1". As
illustrated, both long (500-mer) and short (22-mer) dsRNA
constructs complementary to the luciferase gene could inhibit
expression of that gene product relative to the control cell. On
the other hand, similarly sized dsRNA complementary to the coding
sequence for another protein, green fluorescence protein (GFP), did
not significantly effect the expression of luciferase--indicating
that the inhibitory phenomena was in each case sequence-dependent.
Likewise, single stranded 22-mers of luciferase did not inhibit
expression of that gene--indicating that the inhibitory phenomena
is double stranded-dependent.
[0077] The appended examples also identify an enzyme, Dicer, that
can produce the putative guide RNAs. Dicer is a member of the RNAse
III family of nucleases that specifically cleave dsRNA and is
evolutionarily conserved in worms, flies, plants, fungi and, as
described herein, mammals. The enzyme has a distinctive structure
which includes a helicase domain and dual RNAse III motifs. Dicer
also contains a region of homology to the RDE1/QDE2/ARGONAUTE
family, which have been genetically linked to RNAi in lower
eukaryotes. Indeed, activation of, or overexpression of Dicer may
be sufficient in many cases to permit RNA interference in otherwise
non-receptive cells, such as cultured eukaryotic cells, or
mammalian (non-oocytic) cells in culture or in whole organisms.
[0078] In certain embodiments, the cells can be treated with an
agent(s) that inhibits the general double-stranded RNA response(s)
by the host cells, such as may give rise to sequence-independent
apoptosis. For instance, the cells can be treated with agents that
inhibit the dsRNA-dependent protein kinase known as PKR (protein
kinase RNA-activated). Double stranded RNAs in mammalian cells
typically activate protein kinase PKR and leads to apoptosis. The
mechanism of action of PKR includes phosphorylation and
inactivation eIF2a (Fire (1999) Trends Genet 15:358). It has also
been reported that induction of NF-.kappa.B by PKR is involved in
apoptosis commitment and this process is mediated through
activation of the IKK complex. This sequence-independent response
may reflect a form of primitive immune response, since the presence
of dsRNA is a common feature of many viral lifecycles.
[0079] As described herein, Applicants have demonstrated that the
PKR response can be overcome in favor of the sequence-specific RNAi
response. However, in certain instances, it can be desirable to
treat the cells with agents which inhibit expression of PKR, cause
its destruction, and/or inhibit the kinase activity of PKF are
specifically contemplated for use in the present method. Likewise,
overexpression of or agents which ectopic activate IF2.alpha.a. can
be used. Other agents which can be used to suppress the PKR
response include inhibitors of IKK phosphorylation of I.kappa.B,
inhibitors of I.kappa.B ubiquitination, inhibitors of I.kappa.B
degradation, inhibitors of NF-.kappa.B nuclear translocation, and
inhibitors of NF-.kappa.B interaction with .kappa.B response
elements.
[0080] Other inhibitors of sequence-independent dsRNA response in
cells include the gene product of the vaccinia virus E3L. The E3L
gene product contains two distinct domains. A conserved
carboxy-terminal domain has been shown to bind double-stranded RNA
(dsRNA) and inhibit the antiviral dsRNA response by cells.
Expression of at least that portion of the E3L gene in the host
cell, or the use of polypeptide or peptidomimetics thereof, can be
used to suppress the general dsRNA response. Caspase inhibitors
sensitized cells to killing by double-stranded RNA. Accordingly,
ectopic expression or activated of caspases in the host cell can be
used to suppress the general dsRNA response.
[0081] In other embodiments, the subject method is carried out in
cells which have little or no general response to double stranded
RNA, e.g., have no PKR-dependent dsRNA response, at least under the
culture conditions. As illustrated in FIGS. 28-32, CHO and P19
cells can be used without having to inhibit PKR or other general
dsRNA responses.
[0082] Thus, the present invention provides a process and
compositions for inhibiting expression of a target gene in a cell,
expecially a mammalian cell. In certain embodiments, the process
comprises introduction of RNA (the "dsRNA construct") with partial
or fully double-stranded character into the cell or into the
extracellular environment. Inhibition is specific in that a
nucleotide sequence from a portion of the target gene is chosen to
produce the dsRNA construct. In preferred embodiments, the method
utilizes a cell in which Dicer and/or Argonaute activities are
recombinantly expressed or otherwise ectopically activated. This
process can be (1) effective in attenuating gene expression, (2)
specific to the targeted gene, and (3) general in allowing
inhibition of many different types of target gene.
II. Definitions
[0083] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0084] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to that it
has been linked. One type of vector is a genomic integrated vector,
or "integrated vector", which can become integrated into the
chromsomal DNA of the host cell. Another type of vector is an
episomal vector, i.e., a nucleic acid capable of extra-chromosomal
replication. Vectors capable of directing the expression of genes
to that they are operatively linked are referred to herein as
"expression vectors". In the present specification, "plasmid" and
"vector" are used interchangeably unless otherwise clear from the
context.
[0085] As used herein, the term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA). The term should also be
understood to include, as applicable to the embodiment being
described, single-stranded (such as sense or antisense) and
double-stranded polynucleotides.
[0086] As used herein, the term "gene" or "recombinant gene" refers
to a nucleic acid comprising an open reading frame encoding a
polypeptide of the present invention, including both exon and
(optionally) intron sequences. A "recombinant gene" refers to
nucleic acid encoding such regulatory polypeptides, that may
optionally include intron sequences that are derived from
chromosomal DNA. The term "intron" refers to a DNA sequence present
in a given gene that is not translated into protein and is
generally found between exons. As used herein, the term
"transfection" means the introduction of a nucleic acid, e.g., an
expression vector, into a recipient cell by nucleic acid-mediated
gene transfer.
[0087] A "protein coding sequence" or a sequence that "encodes" a
particular polypeptide or peptide, is a nucleic acid sequence that
is transcribed (in the case of DNA) and is translated (in the case
of mRNA) into a polypeptide in vitro or in vivo when placed under
the control of appropriate regulatory sequences. The boundaries of
the coding sequence are determined by a start codon at the 5'
(amino) terminus and a translation stop codon at the 3' (carboxy)
terminus. A coding sequence can include, but is not limited to,
cDNA from procaryotic or eukaryotic mRNA, genomic DNA sequences
from procaryotic or eukaryotic DNA, and even synthetic DNA
sequences. A transcription termination sequence will usually be
located 3' to the coding sequence.
[0088] Likewise, "encodes", unless evident from its context, will
be meant to include DNA sequences that encode a polypeptide, as the
term is typically used, as well as DNA sequences that are
transcribed into inhibitory antisense molecules.
[0089] The term "loss-of-function", as it refers to genes inhibited
by the subject RNAi method, refers a diminishment in the level of
expression of a gene when compared to the level in the absense of
dsRNA constructs.
[0090] The term "expression" with respect to a gene sequence refers
to transcription of the gene and, as appropriate, translation of
the resulting mRNA transcript to a protein. Thus, as will be clear
from the context, expression of a protein coding sequence results
from transcription and translation of the coding sequence.
[0091] "Cells," "host cells" or "recombinant host cells" are terms
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0092] The term "cultured cells" refers to cells suspended in
culture, e.g., dispersed in culture or in the form tissue. It does
not, however, include oocytes or whole embryos (including
blastocysts and the like) which may be provided in culture. In
certain embodiments, the cultured cells are adults cells, e.g.,
non-embryonic.
[0093] By "recombinant virus" is meant a virus that has been
genetically altered, e.g., by the addition or insertion of a
heterologous nucleic acid construct into the particle.
[0094] As used herein, the terms "transduction" and "transfection"
are art recognized and mean the introduction of a nucleic acid,
e.g., an expression vector, into a recipient cell by nucleic
acid-mediated gene transfer. "Transformation", as used herein,
refers to a process in which a cell's genotype is changed as a
result of the cellular uptake of exogenous DNA or RNA, and, for
example, the transformed cell expresses a dsRNA contruct.
[0095] "Transient transfection" refers to cases where exogenous DNA
does not integrate into the genome of a transfected cell, e.g.,
where episomal DNA is transcribed into mRNA and translated into
protein.
[0096] A cell has been "stably transfected" with a nucleic acid
construct when the nucleic acid construct is capable of being
inherited by daughter cells.
[0097] As used herein, a "reporter gene construct" is a nucleic
acid that includes a "reporter gene" operatively linked to at least
one transcriptional regulatory sequence. Transcription of the
reporter gene is controlled by these sequences to which they are
linked. The activity of at least one or more of these control
sequences can be directly or indirectly regulated by the target
receptor protein. Exemplary transcriptional control sequences are
promoter sequences. A reporter gene is meant to include a
promoter-reporter gene construct that is heterologously expressed
in a cell.
[0098] As used herein, "transformed cells" refers to cells that
have spontaneously converted to a state of unrestrained growth,
i.e., they have acquired the ability to grow through an indefinite
number of divisions in culture. Transformed cells may be
characterized by such terms as neoplastic, anaplastic and/or
hyperplastic, with respect to their loss of growth control. For
purposes of this invention, the terms "transformed phenotype of
malignant mammalian cells" and "transformed phenotype " are
intended to encompass, but not be limited to, any of the following
phenotypic traits associated with cellular transformation of
mammalian cells: immortalization, morphological or growth
transformation, and tumorigenicity, as detected by prolonged growth
in cell culture, growth in semi-solid media, or tumorigenic growth
in immuno-incompetent or syngeneic animals.
[0099] As used herein, "proliferating" and "proliferation" refer to
cells undergoing mitosis.
[0100] As used herein, "immortalized cells" refers to cells that
have been altered via chemical, genetic, and/or recombinant means
such that the cells have the ability to grow through an indefinite
number of divisions in culture.
[0101] The "growth state" of a cell refers to the rate of
proliferation of the cell and the state of differentiation of the
cell.
III. Exemplary Embodiments of Isolation Method
[0102] One aspect of the invention provides a method for
potentiating RNAi by induction or ectopic activation of an RNAi
enzyme in a cell (in vivo or in vitro) or cell-free mixtures. In
preferred embodiments, the RNAi activity is activated or added to a
mammalian cell, e.g., a human cell, which cell may be provided in
vitro or as part of a whole organism. In other embodiments, the
subject method is carried out using eukaryotic cells generally
(except for oocytes) in culture. For instance, the Dicer enzyme may
be activated by virtue of being recombinantly expressed or it may
be activated by use of an agent which (i) induces expression of the
endogenous gene, (ii) stabilizes the protein from degradation,
and/or (iii) allosterically modies the enzyme to increase its
activity (by altering its Kcat, Km or both).
[0103] A. Dicer and Argonaut Activities
[0104] In certain embodiment, at least one of the activated RNAi
enzymes is Dicer, or a homolog thereof. In certain preferred
embodiments, the present method provides for ectopic activation of
Dicer. As used herein, the term "Dicer" refers to a protein which
(a) mediates an RNAi response and (b) has an amino acid sequence at
least 50 percent identical, and more preferablty at least 75, 85,
90 or 95 percent identical to SEQ ID No. 2 or 4, and/or which can
be encoded by a nucleic acid which hybridizes under wash conditions
of 2.times.SSC at 22.degree. C., and more preferably 0.2.times.SSC
at 65.degree. C., to a nucleotide represented by SEQ ID No. 1 or 3.
Accordingly, the method may comprise introducing a dsRNA contruct
into a cell in which Dicer has been recombinantly expressed or
otherwise ectopically activated.
[0105] In certain embodiment, at least one of the activated RNAi
enzymes is Argonaut, or a homolog thereof. In certain preferred
embodiments, the present method provides for ectopic activation of
Argonaut. As used herein, the term "Argonaut" refers to a protein
which (a) mediates an RNAi response and (b) has an amino acid
sequence at least 50 percent identical, and more preferablty at
least 75, 85, 90 or 95 percent identical to the amino acid sequence
shown in FIG. 24. Accordingly, the method may comprise introducing
a dsRNA contruct into a cell in which Argonaut has been
recombinantly expressed or otherwise ectopically activated.
[0106] This invention also provides expression vectors containing a
nucleic acid encoding a Dicer or Argonaut polypeptides, operably
linked to at least one transcriptional regulatory sequence.
Operably linked is intended to mean that the nucleotide sequence is
linked to a regulatory sequence in a manner which allows expression
of the nucleotide sequence. Regulatory sequences are art-recognized
and are selected to direct expression of the subject Dicer or
Argonaut proteins. Accordingly, the term transcriptional regulatory
sequence includes promoters, enhancers and other expression control
elements. Such regulatory sequences are described in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). For instance, any of a wide variety of
expression control sequences, sequences that control the expression
of a DNA sequence when operatively linked to it, may be used in
these vectors to express DNA sequences encoding Dicer or Argonaut
polypeptides of this invention. Such useful expression control
sequences, include, for example, a viral LTR, such as the LTR of
the Moloney murine leukemia virus, the early and late promoters of
SV40, adenovirus or cytomegalovirus immediate early promoter, the
lac system, the trp system, the TAC or TRC system, T7 promoter
whose expression is directed by T7 RNA polymerase, the major
operator and promoter regions of phage .lambda., the control
regions for fd coat protein, the promoter for 3-phosphoglycerate
kinase or other glycolytic enzymes, the promoters of acid
phosphatase, e.g., Pho5, the promoters of the yeast .alpha.-mating
factors, the polyhedron promoter of the baculovirus system and
other sequences known to control the expression of genes of
prokaryotic or eukaryotic cells or their viruses, and various
combinations thereof. It should be understood that the design of
the expression vector may depend on such factors as the choice of
the host cell to be transformed and/or the type of protein desired
to be expressed.
[0107] Moreover, the vector's copy number, the ability to control
that copy number and the expression of any other proteins encoded
by the vector, such as antibiotic markers, should also be
considered.
[0108] The recombinant Dicer or Argonaut genes can be produced by
ligating nucleic acid encoding a Dicer or Argonaut polypeptide into
a vector suitable for expression in either prokaryotic cells,
eukaryotic cells, or both. Expression vectors for production of
recombinant forms of the subject Dicer or Argonaut polypeptides
include plasmids and other vectors. For instance, suitable vectors
for the expression of a Dicer or Argonaut polypeptide include
plasmids of the types: pBR322-derived plasmids, pEMBL-derived
plasmids, pEX-derived plasmids, pBTac-derived plasmids and
pUC-derived plasmids for expression in prokaryotic cells, such as
E. coli.
[0109] A number of vectors exist for the expression of recombinant
proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2,
and YRP17 are cloning and expression vehicles useful in the
introduction of genetic constructs into S. cerevisiae (see, for
example, Broach et al. (1983) in Experimental Manipulation of Gene
Expression, ed. M. Inouye Academic Press, p. 83, incorporated by
reference herein). These vectors can replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the
replication determinant of the yeast 2 micron plasmid. In addition,
drug resistance markers such as ampicillin can be used. In an
illustrative embodiment, a Dicer or Argonaut polypeptide is
produced recombinantly utilizing an expression vector generated by
sub-cloning the coding sequence of a Dicer or Argonaut gene.
[0110] The preferred mammalian expression vectors contain both
prokaryotic sequences, to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine
papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived
and p205) can be used for transient expression of proteins in
eukaryotic cells. The various methods employed in the preparation
of the plasmids and transformation of host organisms are well known
in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells, as well as general recombinant
procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989) Chapters 16 and 17.
[0111] In yet another embodiment, the subject invention provides a
"gene activation" construct which, by homologous recombination with
a genomic DNA, alters the transcriptional regulatory sequences of
an endogenous Dicer or Argonaut gene. For instance, the gene
activation construct can replace the endogenous promoter of a Dicer
or Argonaut gene with a heterologous promoter, e.g., one which
causes constitutive expression of the Dicer or Argonaut gene or
which causes inducible expression of the gene under conditions
different from the normal expression pattern of Dicer or Argonaut.
A variety of different formats for the gene activation constructs
are available. See, for example, the Transkaryotic Therapies, Inc
PCT publications WO93/09222, WO95/31560, WO96/29411, WO95/31560 and
WO94/12650.
[0112] In preferred embodiments, the nucleotide sequence used as
the gene activation construct can be comprised of (1) DNA from some
portion of the endogenous Dicer or Argonaut gene (exon sequence,
intron sequence, promoter sequences, etc.) which direct
recombination and (2) heterologous transcriptional regulatory
sequence(s) which is to be operably linked to the coding sequence
for the genomic Dicer or Argonaut gene upon recombination of the
gene activation construct. For use in generating cultures of Dicer
or Argonaut producing cells, the construct may further include a
reporter gene to detect the presence of the knockout construct in
the cell.
[0113] The gene activation construct is inserted into a cell, and
integrates with the genomic DNA of the cell in such a position so
as to provide the heterologous regulatory sequences in operative
association with the native Dicer or Argonaut gene. Such insertion
occurs by homologous recombination, i.e., recombination regions of
the activation construct that are homologous to the endogenous
Dicer or Argonaut gene sequence hybridize to the genomic DNA and
recombine with the genomic sequences so that the construct is
incorporated into the corresponding position of the genomic
DNA.
[0114] The terms "recombination region" or "targeting sequence"
refer to a segment (i.e., a portion) of a gene activation construct
having a sequence that is substantially identical to or
substantially complementary to a genomic gene sequence, e.g.,
including 5' flanking sequences of the genomic gene, and can
facilitate homologous recombination between the genomic sequence
and the targeting transgene construct.
[0115] As used herein, the term "replacement region" refers to a
portion of a activation construct which becomes integrated into an
endogenous chromosomal location following homologous recombination
between a recombination region and a genomic sequence.
[0116] The heterologous regulatory sequences, e.g., which are
provided in the replacement region, can include one or more of a
variety elements, including: promoters (such as constitutive or
inducible promoters), enhancers, negative regulatory elements,
locus control regions, transcription factor binding sites, or
combinations thereof.
[0117] Promoters/enhancers which may be used to control expression
of the targeted gene in vivo include, but are not limited to, the
cytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989,
J. Exp. Med., 169:13), the human .beta.-actin promoter (Gunning et
al. (1987) PNAS 84:4831-4835), the glucocorticoid-inducible
promoter present in the mouse mammary tumor virus long terminal
repeat (MMTV LTR) (Klessig et al. (1984) Mol. Cell Biol.
4:1354-1362), the long terminal repeat sequences of Moloney murine
leukemia virus (MuLV LTR) (Weiss et al. (1985) RNA Tumor Viruses,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), the SV40
early or late region promoter (Bernoist et al. (1981) Nature
290:304-310; Templeton et al. (1984) Mol. Cell Biol., 4:817; and
Sprague et al. (1983) J. Virol., 45:773), the promoter contained in
the 3' long terminal repeat of Rous sarcoma virus (RSV) (Yamamoto
et al., 1980, Cell, 22:787-797), the herpes simplex virus (HSV)
thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS
82:3567-71), and the herpes simplex virus LAT promoter (Wolfe et
al. (1992) Nature Genetics, 1:379-384).
[0118] In still other embodiments, the replacement region merely
deletes a negative transcriptional control element of the native
gene, e.g., to activate expression, or ablates a positive control
element, e.g., to inhibit expression of the targeted gene.
[0119] B. Cell/Organism
[0120] The cell with the target gene may be derived from or
contained in any organism (e.g., plant, animal, protozoan, virus,
bacterium, or fungus). The dsRNA construct may be synthesized
either in vivo or in vitro. Endogenous RNA polymerase of the cell
may mediate transcription in vivo, or cloned RNA polymerase can be
used for transcription in vivo or in vitro. For generating double
stranded transcripts from a transgene in vivo, a regulatory region
may be used to transcribe the RNA strand (or strands).
[0121] Furthermore, genetic manipulation becomes possible in
organisms that are not classical genetic models. Breeding and
screening programs may be accelerated by the ability to rapidly
assay the consequences of a specific, targeted gene disruption.
Gene disruptions may be used to discover the function of the target
gene, to produce disease models in which the target gene are
involved in causing or preventing a pathological condition, and to
produce organisms with improved economic properties.
[0122] The cell with the target gene may be derived from or
contained in any organism. The organism may a plant, animal,
protozoan, bacterium, virus, or fungus. The plant may be a monocot,
dicot or gymnosperm; the animal may be a vertebrate or
invertebrate. Preferred microbes are those used in agriculture or
by industry, and those that are pathogenic for plants or animals.
Fungi include organisms in both the mold and yeast
morphologies.
[0123] Plants include arabidopsis; field crops (e.g., alfalfa,
barley, bean, com, cotton, flax, pea, rape, rice, rye, safflower,
sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops
(e.g., asparagus, beet, broccoli, cabbage, carrot, cauliflower,
celery, cucumber, eggplant, lettuce, onion, pepper, potato,
pumpkin, radish, spinach, squash, taro, tomato, and zucchini);
fruit and nut crops (e.g., almond, apple, apricot, banana,
blackberry, blueberry, cacao, cherry, coconut, cranberry, date,
faJoa, filbert, grape, grapefruit, guava, kiwi, lemon, lime, mango,
melon, nectarine, orange, papaya, passion fruit, peach, peanut,
pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine,
walnut, and watermelon); and ornamentals (e.g., alder, ash, aspen,
azalea, birch, boxwood, camellia, carnation, chrysanthemum, elm,
fir, ivy, jasmine, juniper, oak, palm, poplar, pine, redwood,
rhododendron, rose, and rubber).
[0124] Examples of vertebrate animals include fish, mammal, cattle,
goat, pig, sheep, rodent, hamster, mouse, rat, primate, and
human.
[0125] Invertebrate animals include nematodes, other worms,
drosophila, and other insects. Representative generae of nematodes
include those that infect animals (e.g., Ancylostoma, Ascaridia,
Ascaris, Bunostomum, Caenorhabditis, Capillaria, Chabertia,
Cooperia, Dictyocaulus, Haernonchus, Heterakis, Nematodirus,
Oesophagostomum, Ostertagia, Oxyuris, Parascaris, Strongylus,
Toxascaris, Trichuris, Trichostrongylus, Tflichonema, Toxocara,
Uncinaria) and those that infect plants (e.g., B ursaphalenchus,
Criconerriella, Diiylenchus, Ditylenchus, Globodera,
Helicotylenchus, Heterodera, Longidorus, Melodoigyne, Nacobbus,
Paratylenchus, Pratylenchus, Radopholus, Rotelynchus, Tylenchus,
and Xiphinerna). Representative orders of insects include
Coleoptera, Diptera, Lepidoptera, and Homoptera.
[0126] The cell having the target gene may be from the germ line or
somatic, totipotent or pluripotent, dividing or non-dividing,
parenchyma or epithelium, immortalized or transformed, or the like.
The cell may be a stem cell or a differentiated cell. Cell types
that are differentiated include adipocytes, fibroblasts, myocytes,
cardiomyocytes, endothelium, neurons, glia, blood cells,
megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils,
basophils, mast cells, leukocytes, granulocytes, keratinocytes,
chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of
the endocrine or exocrine glands.
[0127] C. Targeted Genes
[0128] The target gene may be a gene derived from the cell, an
endogenous gene, a transgene, or a gene of a pathogen which is
present in the cell after infection thereof. Depending on the
particular target gene and the dose of double stranded RNA material
delivered, the procedure may provide partial or complete loss of
function for the target gene. Lower doses of injected material and
longer times after administration of dsRNA may result in inhibition
in a smaller fraction of cells. Quantitation of gene expression in
a cell may show similar amounts of inhibition at the level of
accumulation of target mRNA or translation of target protein.
[0129] "Inhibition of gene expression" refers to the absence (or
observable decrease) in the level of protein and/or mRNA product
from a target gene. "Specificity" refers to the ability to inhibit
the target gene without manifest effects on other genes of the
cell. The consequences of inhibition can be confirmed by
examination of the outward properties of the cell or organism (as
presented below in the examples) or by biochemical techniques such
as RNA solution hybridization, nuclease protection, Northern
hybridization, reverse transcription, gene expression monitoring
with a microarray, antibody binding, enzyme linked immunosorbent
assay (ELISA), Western blotting, radioImmunoassay (RIA), other
immunoassays, and fluorescence activated cell analysis (FACS). For
RNA-mediated inhibition in a cell line or whole organism, gene
expression is conveniently assayed by use of a reporter or drug
resistance gene whose protein product is easily assayed. Such
reporter genes include acetohydroxyacid synthase (AHAS), alkaline
phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase
(GUS), chloramphenicol acetyltransferase (CAT), green fluorescent
protein (GFP), horseradish peroxidase (HRP), luciferase (Luc),
nopaline synthase (NOS), octopine synthase (OCS), and derivatives
thereof multiple selectable markers are available that confer
resistance to ampicillin, bleomycin, chloramphenicol, gentamycin,
hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,
puromycin, and tetracyclin.
[0130] Depending on the assay, quantitation of the amount of gene
expression allows one to determine a degree of inhibition which is
greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell
not treated according to the present invention. Lower doses of
injected material and longer times after administration of dsRNA
may result in inhibition in a smaller fraction of cells (e.g., at
least 10%, 20%, 50%, 75%,90%, or 95% of targeted cells).
Quantitation of gene expression in a cell may show similar amounts
of inhibition at the level of accumulation of target mRNA or
translation of target protein. As an example, the efficiency of
inhibition may be determined by assessing the amount of gene
product in the cell: mRNA may be detected with a hybridization
probe having a nucleotide sequence outside the region used for the
inhibitory double-stranded RNA, or translated polypeptide may be
detected with an antibody raised against the polypeptide sequence
of that region.
[0131] As disclosed herein, the present invention may is not
limited to any type of target gene or nucleotide sequence. But the
following classes of possible target genes are listed for
illustrative purposes: developmental genes (e.g., adhesion
molecules, cyclin kinase inhibitors, Writ 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); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL,
CSFIR, ERBA, ERBB, EBRB2, ETSI, ETS1, ETV6, FGR, FOS, FYN, HCR,
HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS,
PIM 1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor genes
(e.g., APC, BRCA 1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, 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, dextrinases, DNA and RNA polymerases,
galactosidases, glucanases, glucose oxidases, granule-bound starch
synthases, GTPases, helicases, hemicellulases, 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).
[0132] D. dsRNA constructs
[0133] The dsRNA construct may comprise one or more strands of
polymerized ribonucleotide. It may include modifications to either
the phosphate-sugar backbone or the nucleoside. For example, the
phosphodiester linkages of natural RNA may be modified to include
at least one of a nitrogen or sulfur heteroatom. Modifications in
RNA structure may be tailored to allow specific genetic inhibition
while avoiding a general panic response in some organisms which is
generated by dsRNA. Likewise, bases may be modified to block the
activity of adenosine deaminase. The dsRNA construct may be
produced enzymatically or by partial/total organic synthesis, any
modified ribonucleotide can be introduced by in vitro enzymatic or
organic synthesis.
[0134] The dsRNA construct may be directly introduced into the cell
(i.e., intracellularly); or introduced extracellularly into a
cavity, interstitial space, into the circulation of an organism,
introduced orally, or may be introduced by bathing an organism in a
solution containing RNA. Methods for oral introduction include
direct mixing of RNA with food of the organism, as well as
engineered approaches in which a species that is used as food is
engineered to express an RNA, then fed to the organism to be
affected. Physical methods of introducing nucleic, acids include
injection directly into the cell or extracellular injection into
the organism of an RNA solution.
[0135] The double-stranded structure may be formed by a single
self-complementary RNA strand or two complementary RNA strands. RNA
duplex formation may be initiated either inside or outside the
cell. The RNA may be introduced in an amount which allows delivery
of at least one copy per cell. Higher doses (e.g., at least 5, 10,
100, 500 or 1000 copies per cell) of double-stranded material may
yield more effective inhibition; lower doses may also be useful for
specific applications. Inhibition is sequence-specific in that
nucleotide sequences corresponding to the duplex region of the RNA
are targeted for genetic inhibition.
[0136] dsRNA constructs containing a nucleotide sequences identical
to a portion of the target gene are preferred for inhibition. RNA
sequences with insertions, deletions, and single point mutations
relative to the target sequence have also been found to be
effective for inhibition. Thus, sequence identity may optimized by
sequence comparison and alignment algorithms known in the art (see
Gribskov and Devereux, Sequence Analysis Primer, Stockton Press,
1991, and references cited therein) and calculating the percent
difference between the nucleotide sequences by, for example, the
Smith-Waterman algorithm as implemented in the BESTFIT software
program using default parameters (e.g., University of Wisconsin
Genetic Computing Group). Greater than 90% sequence identity, or
even 100% sequence identity, between the inhibitory RNA and the
portion of the target gene is preferred. Alternatively, the duplex
region of the RNA may be defined functionally as a nucleotide
sequence that is capable of hybridizing with a portion of the
target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM
EDTA, 50.degree. C. or 70.degree. C. hybridization for 12-16 hours;
followed by washing). In certain preferred embodiments, the length
of the dsRNA is at least 20, 21 or 22 nucleotides in length, e.g.,
corresponding in size to RNA products produced by Dicer-dependent
cleavage. In certain embodiments, the dsRNA construct is at least
25, 50, 100, 200, 300 or 400 bases. In certain embodiments, the
dsRNA construct is 400-800 bases in length.
[0137] 100% sequence identity between the RNA and the target gene
is not required to practice the present invention. Thus the
invention has the advantage of being able to tolerate sequence
variations that might be expected due to genetic mutation, strain
polymorphism, or evolutionary divergence.
[0138] The dsRNA construct may be synthesized either in vivo or in
vitro. Endogenous RNA polymerase of the cell may mediate
transcription in vivo, or cloned RNA polymerase can be used for
transcription in vivo or in vitro. For transcription from a
transgene in vivo or an expression construct, a regulatory region
(e.g., promoter, enhancer, silencer, splice donor and acceptor,
polyadenylation) may be used to transcribe the dsRNA strand (or
strands). Inhibition may be targeted by specific transcription in
an organ, tissue, or cell type; stimulation of an environmental
condition (e.g., infection, stress, temperature, chemical
inducers); and/or engineering transcription at a developmental
stage or age. The RNA strands may or may not be polyadenylated; the
RNA strands may or may not be capable of being translated into a
polypeptide by a cell's translational apparatus. The dsRNA
construct may be chemically or enzymatically synthesized by manual
or automated reactions. The dsRNA construct may be synthesized by a
cellular RNA polymerase or a bacteriophage RNA polymerase (e.g.,
T3, T7, SP6). The use and production of an expression construct are
known in the art 32,33,34 (see also WO 97/32016; U.S. Pat. Nos.
5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693; and the
references cited therein). If synthesized chemically or by in vitro
enzymatic synthesis, the RNA may be purified prior to introduction
into the cell. For example, RNA can be punified from a mixture by
extraction with a solvent or resin, precipitation, electrophoresis,
chromatography or a combination thereof. Alternatively, the dsRNA
construct may be used with no or a minimum of purification to avoid
losses due to sample processing. The dsRNA construct may be dried
for storage or dissolved in an aqueous solution. The solution may
contain buffers or salts to promote annealing, and/or stabilization
of the duplex strands.
[0139] Physical methods of introducing nucleic acids include
injection of a solution containing the dsRNA construct, bombardment
by particles covered by the dsRNA construct, soaking the cell or
organism in a solution of the RNA, or electroporation of cell
membranes in the presence of the dsRNA construct. A viral construct
packaged into a viral particle would accomplish both efficient
introduction of an expression construct into the cell and
transcription of dsRNA construct encoded by the expression
construct. Other methods known in the art for introducing nucleic
acids to cells may be used, such as lipid-mediated carrier
transport, chemicalmediated transport, such as calcium phosphate,
and the like. Thus the dsRNA construct may be introduced along with
components that perform one or more of the following activities:
enhance RNA uptake by the cell, promote annealing of the duplex
strands, stabilize the annealed strands, or other-wise increase
inhibition of the target gene.
[0140] E. Illustrative Uses
[0141] One utility of the present invention is as a method of
identifying gene function in an organism, especially higher
eukaryotes comprising the use of double-stranded RNA to inhibit the
activity of a target gene of previously unknown function. Instead
of the time consuming and laborious isolation of mutants by
traditional genetic screening, functional genomics would envision
determining the function of uncharacterized genes by employing the
invention to reduce the amount and/or alter the timing of target
gene activity. The invention could be used in determining potential
targets for pharmaceutics, understanding normal and pathological
events associated with development, determining signaling pathways
responsible for postnatal development/aging, and the like. The
increasing speed of acquiring nucleotide sequence information from
genomic and expressed gene sources, including total sequences for
mammalian genomes, can be coupled with the invention to determine
gene function in a cell or in a whole organism. The preference of
different organisms to use particular codons, searching sequence
databases for related gene products, correlating the linkage map of
genetic traits with the physical map from which the nucleotide
sequences are derived, and artificial intelligence methods may be
used to define putative open reading frames from the nucleotide
sequences acquired in such sequencing projects.
[0142] A simple assay would be to inhibit gene expression according
to the partial sequence available from an expressed sequence tag
(EST). Functional alterations in growth, development, metabolism,
disease resistance, or other biological processes would be
indicative of the normal role of the EST's gene product.
[0143] The ease with which the dsRNA construct can be introduced
into an intact cell/organism containing the target gene allows the
present invention to be used in high throughput screening (HTS).
For example, duplex RNA can be produced by an amplification
reaction using primers flanking the inserts of any gene library
derived from the target cell or organism. Inserts may be derived
from genomic DNA or mRNA (e.g., cDNA and cRNA). Individual clones
from the library can be replicated and then isolated in separate
reactions, but preferably the library is maintained in individual
reaction vessels (e.g., a 96 well microtiter plate) to minimize the
number of steps required to practice the invention and to allow
automation of the process.
[0144] In an exemplary embodiment, the subject invention provides
an arrayed library of RNAi constructs. The array may in the form of
solutions, such as multi-well plates, or may be "printed" on solid
substrates upon which cells can be grown. To illustrate, solutions
containing duplex RNAs that are capable of inhibiting the different
expressed genes can be placed into individual wells positioned on a
microtiter plate as an ordered array, and intact cells/organisms in
each well can be assayed for any changes or modifications in
behavior or development due to inhibition of target gene
activity.
[0145] In one embodiment, the subject method uses an arrayed
library of RNAi constructs to screen for combinations of RNAi that
is lethal to host cells. Synthetic lethality is a bedrock principle
of experimental genetics. A synthetic lethality describes the
properties of two mutations which, individually, are tolerated by
the organism but which, in combination, are lethal. The subject
arrays can be used to identify loss-of-function mutations that are
lethal in combination with alterations in other genes, such as
activated oncogenes or loss-of-function mutations to tumor
suppressors. To achieve this, one can create "phenotype arrays"
using cultured cells. Expression of each of a set of genes, such as
the host cell's genome, can be individually systematically
disrupted using RNA interference. Combination with alterations in
oncogene and tumor suppressor pathways can be used to identify
synthetic lethal interactions that may identify novel therapeutic
targets.
[0146] In certain embodiments, the RNAi constructs can be fed
directly to, injected into, the cell/organism containing the target
gene. Alternatively, the duplex RNA can be produced by in vivo or
in vitro transcription from an expression construct used to produce
the library. The construct can be replicated as individual clones
of the library and transcribed to produce the RNA; each clone can
then be fed to, or injected into, the cell/organism containing the
target gene. The function of the target gene can be assayed from
the effects it has on the cell/organism when gene activity is
inhibited. This screening could be amenable to small subjects that
can be processed in large number, for example, tissue culture cells
derived from mammals, especially primates, and most preferably
humans.
[0147] If a characteristic of an organism is determined to be
genetically linked to a polymorphism through RFLP or QTL analysis,
the present invention can be used to gain insight regarding whether
that genetic polymorphism might be directly responsible for the
characteristic. For example, a fragment defining the genetic
polymorphism or sequences in the vicinity of such a genetic
polymorphism can be amplified to produce an RNA, the duplex RNA can
be introduced to the organism or cell, and whether an alteration in
the charactenstic is correlated with inhibition can be determined.
Of course, there may be trivial explanations for negative results
with this type of assay, for example: inhibition of the target gene
causes lethality, inhibition of the target gene may not result in
any observable alteration, the fragment contains nucleotide
sequences that are not capable of inhibiting the target gene, or
the target gene's activity is redundant.
[0148] The present invention may be useful in allowing the
inhibition of essential genes. Such genes may be required for cell
or organism viability at only particular stages of development or
cellular compartments. The functional equivalent of conditional
mutations may be produced by inhibiting activity of the target gene
when or where it is not required for viability. The invention
allows addition of RNA at specific times of development and
locations in the organism without introducing permanent mutations
into the target genome.
[0149] If alternative splicing produced a family of transcripts
that were distinguished by usage of characteristic exons, the
present invention can target inhibition through the appropriate
exons to specifically inhibit or to distinguish among the functions
of family members. For example, a hormone that contained an
alternatively spliced transmembrane domain may be expressed in both
membrane bound and secreted forms. Instead of isolating a nonsense
mutation that terminates translation before the transmembrane
domain, the functional consequences of having only secreted hormone
can be determined according to the invention by targeting the exon
containing the transmembrane domain and thereby inhibiting
expression of membrane-bound hormone.
[0150] The present invention may be used alone or as a component of
a kit having at least one of the reagents necessary to carry out
the in vitro or in vivo introduction of RNA to test samples or
subjects. Preferred components are the dsRNA and a vehicle that
promotes introduction of the dsRNA. Such a kit may also include
instructions to allow a user of the kit to practice the
invention.
[0151] Alternatively, an organism may be engineered to produce
dsRNA which produces commercially or medically beneficial results,
for example, resistance to a pathogen or its pathogenic effects,
improved growth, or novel developmental patterns.
IV. Exemplification
[0152] The invention, now being generally described, will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention and are not intended to
limit the invention.
Example 1
An RNA-directed Nuclease Mediates RNAi Gene Silencing
[0153] In a diverse group of organisms that includes Caenorhabditis
elegans, Drosophila, planaria, hydra, trypanosomes, fungi and
plants, the introduction of double-stranded RNAs inhibits gene
expression in a sequence-specific manner.sup.1-7. These responses,
called RNA interference or post-transcriptional gene silencing, may
provide anti-viral defence, modulate transposition or regulate gene
expression.sup.1, 6, 8-10. We have taken a biochemical approach
towards elucidating the mechanisms underlying this genetic
phenomenon. Here we show that `loss-of-function` phenotypes can be
created in cultured Drosophila cells by transfection with specific
double-stranded RNAs. This coincides with a marked reduction in the
level of cognate cellular messenger RNAs. Extracts of transfected
cells contain a nuclease activity that specifically degrades
exogenous transcripts homologous to transfected double-stranded
RNA. This enzyme contains an essential RNA component. After partial
purification, the sequence-specific nuclease co-fractionates with a
discrete, .about.25-nucleotide RNA species which may confer
specificity to the enzyme through homology to the substrate
mRNAs.
[0154] Although double-stranded RNAs (dsRNAs) can provoke gene
silencing in numerous biological contexts including
Drosophila.sup.11, 12, the mechanisms underlying this phenomenon
have remained mostly unknown. We therefore wanted to establish a
biochemically tractable model in which such mechanisms could be
investigated.
[0155] Transient transfection of cultured, Drosophila S2 cells with
a lacZ expression vector resulted in .beta.-galactosidase activity
that was easily detectable by an in situ assay (FIG. 1a). This
activity was greatly reduced by co-transfection with a dsRNA
corresponding to the first 300 nucleotides of the lacZ sequence,
whereas co-transfection with a control dsRNA (CD8) (FIG. 1a) or
with single-stranded RNAs of either sense or antisense orientation
(data not shown) had little or no effect. This indicated that
dsRNAs could interfere, in a sequence-specific fashion, with gene
expression in cultured cells.
[0156] To determine whether RNA interference (RNAi) could be used
to target endogenous genes, we transfected S2 cells with a dsRNA
corresponding to the first 540 nucleotides of Drosophila cyclin E,
a gene that is essential for progression into S phase of the cell
cycle. During log-phase growth, untreated S2 cells reside primarily
in G2/M (FIG. 1b). Transfection with lacZ dsRNA had no effect on
cell-cycle distribution, but transfection with the cyclin E dsRNA
caused a G1-phase cell-cycle arrest (FIG. 1b). The ability of
cyclin E dsRNA to provoke this response was length-dependent.
Double-stranded RNAs of 540 and 400 nucleotides were quite
effective, whereas dsRNAs of 200 and 300 nucleotides were less
potent. Double-stranded cyclin E RNAs of 50 or 100 nucleotides were
inert in our assay, and transfection with a single-stranded,
antisense cyclin E RNA had virtually no effect.
[0157] One hallmark of RNAi is a reduction in the level of mRNAs
that are homologous to the dsRNA. Cells transfected with the cyclin
E dsRNA (bulk population) showed diminished endogenous cyclin E
mRNA as compared with control cells (FIG. 1c). Similarly,
transfection of cells with dsRNAs homologous to fizzy, a component
of the anaphase-promoting complex (APC) or cyclin A, a cyclin that
acts in S, G2 and M, also caused reduction of their cognate mRNAs
(FIG. 1c). The modest reduction in fizzy mRNA levels in cells
transfected with cyclin A dsRNA probably resulted from arrest at a
point in the division cycle at which fizzy transcription is
low.sup.14, 15. These results indicate that RNAi may be a generally
applicable method for probing gene function in cultured Drosophila
cells.
[0158] The decrease in mRNA levels observed upon transfection of
specific dsRNAs into Drosophila cells could be explained by effects
at transcriptional or post-transcriptional levels. Data from other
systems have indicated that some elements of the dsRNA response may
affect mRNA directly (reviewed in refs 1 and 6). We therefore
sought to develop a cell-free assay that reflected, at least in
part, RNAi.
[0159] S2 cells were transfected with dsRNAs corresponding to
either cyclin E or lacZ. Cellular extracts were incubated with
synthetic mRNAs of lacZ or cyclin E. Extracts prepared from cells
transfected with the 540-nucleotide cyclin E dsRNA efficiently
degraded the cyclin E transcript; however, the lacZ transcript was
stable in these lysates (FIG. 2a). Conversely, lysates from cells
transfected with the lacZ dsRNA degraded the lacZ transcript but
left the cyclin E mRNA intact. These results indicate that RNAi
ablates target mRNAs through the generation of a sequence-specific
nuclease activity. We have termed this enzyme RISC (RNA-induced
silencing complex). Although we occasionally observed possible
intermediates in the degradation process (see FIG. 2), the absence
of stable cleavage end-products indicates an exonuclease (perhaps
coupled to an endonuclease). However, it is possible that the RNAi
nuclease makes an initial endonucleolytic cut and that non-specific
exonucleases in the extract complete the degradation
process.sup.16. In addition, our ability to create an extract that
targets lacZ in vitro indicates that the presence of an endogenous
gene is not required for the RNAi response.
[0160] To examine the substrate requirements for the dsRNA-induced,
sequence-specific nuclease activity, we incubated a variety of
cyclin-E-derived transcripts with an extract derived from cells
that had been transfected with the 540-nucleotide cyclin E dsRNA
(FIG. 2b, c). Just as a length requirement was observed for the
transfected dsRNA, the RNAi nuclease activity showed a dependence
on the size of the RNA substrate. Both a 600-nucleotide transcript
that extends slightly beyond the targeted region (FIG. 2b) and an
.about.1-kilobase (kb) transcript that contains the entire coding
sequence (data not shown) were completely destroyed by the extract.
Surprisingly, shorter substrates were not degraded as efficiently.
Reduced activity was observed against either a 300- or a
220-nucleotide transcript, and a 100-nucleotide transcript was
resistant to nuclease in our assay. This was not due solely to
position effects because .about.100-nucleotide transcripts derived
from other portions of the transfected dsRNA behaved similarly
(data not shown). As expected, the nuclease activity (or
activities) present in the extract could also recognize the
antisense strand of the cyclin E mRNA. Again, substrates that
contained a substantial portion of the targeted region were
degraded efficiently whereas those that contained a shorter stretch
of homologous sequence (.about.130 nucleotides) were recognized
inefficiently (FIG. 2c, as600). For both the sense and antisense
strands, transcripts that had no homology with the transfected
dsRNA ( FIG. 2b, Eout; FIG. 2c, as300) were not degraded. Although
we cannot exclude the possibility that nuclease specificity could
have migrated beyond the targeted region, the resistance of
transcripts that do not contain homology to the dsRNA is consistent
with data from C. elegans. Double-stranded RNAs homologous to an
upstream cistron have little or no effect on a linked downstream
cistron, despite the fact that unprocessed, polycistronic mRNAs can
be readily detected.sup.17, 18. Furthermore, the nuclease was
inactive against a dsRNA identical to that used to provoke the RNAi
response in vivo (FIG. 2b). In the in vitro system, neither a 5'
cap nor a poly(A) tail was required, as such transcripts were
degraded as efficiently as uncapped and non-polyadenylated
RNAs.
[0161] Gene silencing provoked by dsRNA is sequence specific. A
plausible mechanism for determining specificity would be
incorporation of nucleic-acid guide sequences into the complexes
that accomplish silencing.sup.19. In accord with this idea,
pre-treatment of extracts with a Ca.sup.2+-dependent nuclease
(micrococcal nuclease) abolished the ability of these extracts to
degrade cognate mRNAs (FIG. 3). Activity could not be rescued by
addition of non-specific RNAs such as yeast transfer RNA. Although
micrococcal nuclease can degrade both DNA and RNA, treatment of the
extract with DNAse I had no effect (FIG. 3). Sequence-specific
nuclease activity, however, did require protein (data not shown).
Together, our results support the possibility that the RNAi
nuclease is a ribonucleoprotein, requiring both RNA and protein
components. Biochemical fractionation (see below) is consistent
with these components being associated in extract rather than being
assembled on the target mRNA after its addition.
[0162] In plants, the phenomenon of co-suppression has been
associated with the existence of small (.about.25-nucleotide) RNAs
that correspond to the gene that is being silenced.sup.19. To
address the possibility that a similar RNA might exist in
Drosophila and guide the sequence-specific nuclease in the choice
of substrate, we partially purified our activity through several
fractionation steps. Crude extracts contained both
sequence-specific nuclease activity and abundant, heterogeneous
RNAs homologous to the transfected dsRNA (FIGS. 2 and 4a). The RNAi
nuclease fractionated with ribosomes in a high-speed centrifugation
step. Activity could be extracted by treatment with high salt, and
ribosomes could be removed by an additional centrifugation step.
Chromatography of soluble nuclease over an anion-exchange column
resulted in a discrete peak of activity (FIG. 4b, cyclin E). This
retained specificity as it was inactive against a heterologous mRNA
(FIG. 4b, lacZ). Active fractions also contained an RNA species of
25 nucleotides that is homologous to the cyclin E target (FIG. 4b,
northern). The band observed on northern blots may represent a
family of discrete RNAs because it could be detected with probes
specific for both the sense and antisense cyclin E sequences and
with probes derived from distinct segments of the dsRNA (data not
shown). At present, we cannot determine whether the 25-nucleotide
RNA is present in the nuclease complex in a double-stranded or
single-stranded form.
[0163] RNA interference allows an adaptive defence against both
exogenous and endogenous dsRNAs, providing something akin to a
dsRNA immune response. Our data, and that of others.sup.19, is
consistent with a model in which dsRNAs present in a cell are
converted, either through processing or replication, into small
specificity determinants of discrete size in a manner analogous to
antigen processing. Our results suggest that the
post-transcriptional component of dsRNA-dependent gene silencing is
accomplished by a sequence-specific nuclease that incorporates
these small RNAs as guides that target specific messages based upon
sequence recognition. The identical size of putative specificity
determinants in plants.sup.19 and animals predicts a conservation
of both the mechanisms and the components of dsRNA-induced,
post-transcriptional gene silencing in diverse organisms. In
plants, dsRNAs provoke not only post-transcriptional gene silencing
but also chromatin remodelling and transcriptional
repression.sup.20, 21. It is now critical to determine whether
conservation of gene-silencing mechanisms also exists at the
transcriptional level and whether chromatin remodelling can be
directed in a sequence-specific fashion by these same dsRNA-derived
guide sequences.
[0164] Methods
[0165] Cell Culture and RNA Methods
[0166] S2 (ref. 22) cells were cultured at 27.degree. C. in 90%
Schneider's insect media (Sigma), 10% heat inactivated fetal bovine
serum (FBS). Cells were transfected with dsRNA and plasmid DNA by
calcium phosphate co-precipitation.sup.23. Identical results were
observed when cells were transfected using lipid reagents (for
example, Superfect, Qiagen). For FACS analysis, cells were
additionally transfected with a vector that directs expression of a
green fluorescent protein (GFP)-US9 fusion protein.sup.13. These
cells were fixed in 90% ice-cold ethanol and stained with propidium
iodide at 25 .mu.g ml.sup.-1. FACS was performed on an Elite flow
cytometer (Coulter). For northern blotting, equal loading was
ensured by over-probing blots with a control complementary DNA
(RP49). For the production of dsRNA, transcription templates were
generated by polymerase chain reaction such that they contained T7
promoter sequences on each end of the template. RNA was prepared
using the RiboMax kit (Promega). Confirmation that RNAs were double
stranded came from their complete sensitivity to RNAse III (a gift
from A. Nicholson). Target mRNA transcripts were synthesized using
the Riboprobe kit (Promega) and were gel purified before use.
[0167] Extract Preparation
[0168] Log-phase S2 cells were plated on 15-cm tissue culture
dishes and transfected with 30 .mu.g dsRNA and 30 .mu.g carrier
plasmid DNA. Seventy-two hours after transfection, cells were
harvested in PBS containing 5 mM EGTA washed twice in PBS and once
in hypotonic buffer (10 mM HEPES pH 7.3, 6 mM
.beta.-mercaptoethanol). Cells were suspended in 0.7 packed-cell
volumes of hypotonic buffer containing Complete protease inhibitors
(Boehringer) and 0.5 units ml.sup.-1 of RNasin (Promega). Cells
were disrupted in a dounce homogenizer with a type B pestle, and
lysates were centrifuged at 30,000 g for 20 min. Supernatants were
used in an in vitro assay containing 20 mM HEPES pH 7.3, 110 mM
KOAc, 1 mM Mg(OAc).sub.2, 3 mM EGTA, 2 mM CaCl.sub.2, 1 mM DTT.
Typically, 5 .mu.l extract was used in a 10 .mu.l assay that
contained also 10,000 c.p.m. synthetic mRNA substrate.
[0169] Extract Fractionation
[0170] Extracts were centrifuged at 200,000 g for 3 h and the
resulting pellet (containing ribosomes) was extracted in hypotonic
buffer containing also 1 mM MgCl.sub.2 and 300 mM KOAc. The
extracted material was spun at 100,000 g for 1 h and the resulting
supernatant was fractionated on Source 15Q column (Pharmacia) using
a KCl gradient in buffer A (20 mM HEPES pH 7.0, 1 mM
dithiothreitol, 1 mM MgCl.sub.2). Fractions were assayed for
nuclease activity as described above. For northern blotting,
fractions were proteinase K/SDS treated, phenol extracted, and
resolved on 15% acrylamide 8M urea gels. RNA was electroblotted
onto Hybond N+ and probed with strand-specific riboprobes derived
from cyclin E mRNA. Hybridization was carried out in 500 mM
NaPO.sub.4 pH 7.0, 15% formamide, 7% SDS, 1% BSA. Blots were washed
in 1 SSC at 37-45.degree. C.
References Cited in Example 1
[0171] 1. Sharp, P. A. RNAi and double-strand RNA. Genes Dev. 13,
139-141 (1999).
[0172] 2. Sanchez-Alvarado, A. & Newmark, P. A. Double-stranded
RNA specifically disrupts gene expression during planarian
regeneration. Proc. Natl Acad. Sci. USA 96, 5049-5054 (1999).
[0173] 3. Lohmann, J. U., Endl, I. & Bosch, T. C. Silencing of
developmental genes in Hydra. Dev. Biol. 214, 211-214 (1999).
[0174] 4. Cogoni, C. & Macino, G. Gene silencing in Neurospora
crassa requires a protein homologous to RNA-dependent RNA
polymerase. Nature 399, 166-169 (1999).
[0175] 5. Waterhouse, P. M., Graham, M. W. & Wang, M. B. Virus
resistance and gene silencing in plants can be induced by
simultaneous expression of sense and antisense RNA. Proc. Natl
Acad. Sci. USA 95, 13959-13964 (1998).
[0176] 6. Montgomery, M. K. & Fire, A. Double-stranded RNA as a
mediator in sequence-specific genetic silencing and co-suppression.
Trends Genet. 14, 225-228 (1998).
[0177] 7. Ngo, H., Tschudi, C., Gull, K. & Ullu, E.
Double-stranded RNA induces mRNA degradation in Trypanosoma brucei.
Proc. Natl Acad. Sci. USA 95, 14687-14692 (1998).
[0178] 8. Tabara, H. et al. The rde-1 gene, RNA interference, and
transposon silencing in C. elegans. Cell 99, 123-132 (1999).
[0179] 9. Ketting, R. F., Haverkamp, T. H. A., van Luenen, H. G. A.
M. & Plasterk, R. H. A. mut-7 of C. elegans, required for
transposon silencing and RNA interference, is a homolog of Werner
Syndrome helicase and RnaseD. Cell 99, 133-141 (1999).
[0180] 10. Ratcliff, F., Harrison, B. D. & Baulcombe, D. C. A
similarity between viral defense and gene silencing in plants.
Science 276, 1558-1560 (1997).
[0181] 11. Kennerdell, J. R. & Carthew, R. W. Use of
dsRNA-mediated genetic interference to demonstrate that frizzled
and frizzled 2 act in the wingless pathway. Cell 95, 1017-1026
(1998).
[0182] 12. Misquitta, L. & Paterson, B. M. Targeted disruption
of gene function in Drosophila by RNA interference: a role for
nautilus in embryonic somatic muscle formation. Proc. Natl Acad.
Sci. USA 96, 1451-1456 (1999).
[0183] 13. Kalejta, R. F., Brideau, A. D., Banfield, B. W. &
Beavis, A. J. An integral membrane green fluorescent protein
marker, Us9-GFP, is quantitatively retained in cells during
propidium iodine-based cell cycle analysis by flow cytometry. Exp.
Cell. Res. 248, 322-328 (1999).
[0184] 14. Wolf, D. A. & Jackson, P. K. Cell cycle: oiling the
gears of anaphase. Curr. Biol. 8, R637-R639 (1998).
[0185] 15. Kramer, E. R., Gieffers, C., Holz, G., Hengstschlager,
M. & Peters, J. M. Activation of the human anaphase-promoting
complex by proteins of the CDC20/fizzy family. Curr. Biol. 8,
1207-1210 (1998).
[0186] 16. Shuttleworth, J. & Colman, A. Antisense
oligonucleotide-directe- d cleavage of mRNA in Xenopus oocytes and
eggs. EMBO J. 7, 427-434 (1988).
[0187] 17. Tabara, H., Grishok, A. & Mello, C. C. RNAi in C.
elegans: soaking in the genome sequence. Science 282, 430-432
(1998).
[0188] 18. Bosher, J. M., Dufourcq, P., Sookhareea, S. &
Labouesse, M. RNA interference can target pre-mRNA. Consequences
for gene expression in a Caenorhabditis elegans operon. Genetics
153, 1245-1256 (1999).
[0189] 19. Hamilton, J. A. & Baulcombe, D. C. A species of
small antisense RNA in posttranscriptional gene silencing in
plants. Science 286, 950-952 (1999).
[0190] 20. Jones, L. A., Thomas, C. L. & Maule, A. J. De novo
methylation and co-suppression induced by a cytoplasmically
replicating plant RNA virus. EMBO J. 17, 6385-6393 (1998).
[0191] 21. Jones, L. A. et al. RNA-DNA interactions and DNA
methylation in post-transcriptional gene silencing. Plant Cell 11,
2291-2301 (1999).
[0192] 22. Schneider, I. Cell lines derived from late embryonic
stages of Drosophila melanogaster. J. Embryol. Exp. Morpho. 27,
353-365 (1972).
[0193] 23. Di Nocera, P. P. & Dawid, I. B. Transient expression
of genes introduced into cultured cells of Drosophila. Proc. Natl
Acad. Sci. USA 80, 7095-7098 (1983).
Example 2
Role for a Bidentate Ribonuclease in the Initiation Step of RNA
Interference
[0194] Genetic approaches in worms, fungi and plants have
identified a group of proteins that are essential for
double-stranded RNA-induced gene silencing. Among these are
ARGONAUTE family members (e.g. RDE1, QDE2).sup.9,10,30, recQ-family
helicases (MUT-7, QDE3).sup.11,12, and RNA-dependent RNA
polymerases (e.g. EGO-1, QDE1, SGS2/SDE1).sup.13-16. While
potential roles have been proposed, none of these genes has been
assigned a definitive function in the silencing process.
Biochemical studies have suggested that PTGS is accomplished by a
multicomponent nuclease that targets mRNAs for
degradation.sup.6,8,17. We have shown that the specificity of this
complex may derive from the incorporation of a small guide sequence
that is homologous to the mRNA substrate.sup.6. Originally
identified in plants that were actively silencing transgenes.sup.7,
these .about.22 nt. RNAs have been produced during RNAi in vitro
using an extract prepared from Drosophila embryos.sup.8. Putative
guide RNAs can also be produced in extracts from Drosophila S2
cells (FIG. 5a). With the goal of understanding the mechanism of
post-transcriptional gene silencing, we have undertaken both
biochemical fractionation and candidate gene approaches to identify
the enzymes that execute each step of RNAi.
[0195] Our previous studies resulted in the partial purification of
a nuclease, RISC, that is an effector of RNA interference. See
Example 1. This enzyme was isolated from Drosophila S2 cells in
which RNAi had been initiated in vivo by transfection with dsRNA.
We first sought to determine whether the RISC enzyme and the enzyme
that initiates RNAi via processing of dsRNA into 22 mers are
distinct activities. RISC activity could be largely cleared from
extracts by high-speed centrifugation (100,000.times.g for 60 min.)
while the activity that produces 22 mers remained in the
supernatant (FIG. 5b,c). This simple fractionation indicated that
RISC and the 22 mer-generating activity are separable and thus
distinct enzymes. However, it seems likely that they might interact
at some point during the silencing process.
[0196] RNAse III family members are among the few nucleases that
show specificity for double-stranded RNA.sup.18. Analysis of the
Drosophila and C. elegans genomes reveals several types of RNAse
III enzymes. First is the canonical RNAse III which contains a
single RNAse III signature motif and a double-stranded RNA binding
domain (dsRBD; e.g. RNC_CAEEL). Second is a class represented by
Drosha.sup.19, a Drosophila enzyme that contains two RNAse III
motifs and a dsRBD (CeDrosha in C. elegans). A third class contains
two RNAse III signatures and an amino terminal helicase domain
(e.g. Drosophila CG4792, CG6493, C. elegans K12H4.8), and these had
previously been proposed by Bass as candidate RNAi
nucleases.sup.20. Representatives of all three classes were tested
for the ability to produce discrete, .about.22 nt. RNAs from dsRNA
substrates.
[0197] Partial digestion of a 500 nt. cyclin E dsRNA with purified,
bacterial RNAse III produced a smear of products while nearly
complete digestion produced a heterogeneous group of .about.11-17
nucleotide RNAs (not shown). In order to test the dual-RNAse III
enzymes, we prepared T7 epitope-tagged versions of Drosha and
CG4792. These were expressed in transfected S2 cells and isolated
by immunoprecipitation using antibody-agarose conjugates. Treatment
of the dsRNA with the CG4792 immunoprecipitate yielded .about.22
nt. fragments similar to those produced in either S2 or embryo
extracts (FIG. 6a). Neither activity in extract nor activity in
immunoprecipitates depended on the sequence of the RNA substrate
since dsRNAs derived from several genes were processed equivalently
(see Supplement 1). Negative results were obtained with Drosha and
with immunoprecipitates of a DExH box helicase (Homeless.sup.21;
see FIGS. 6a,b). Western blotting confirmed that each of the tagged
proteins was expressed and immunoprecipitated similarly (see
Supplement 2). Thus, we conclude that CG4792 may carry out the
initiation step of RNA interference by producing .about.22 nt.
guide sequences from dsRNAs. Because of its ability to digest dsRNA
into uniformly sized, small RNAs, we have named this enzyme Dicer
(Dcr). Dicer mRNA is expressed in embryos, in S2 cells, and in
adult flies, consistent with the presence of functional RNAi
machinery in all of these contexts (see Supplement 3).
[0198] The possibility that Dicer might be the nuclease responsible
for the production of guide RNAs from dsRNAs prompted us to raise
an antiserum directed against the carboxy-terminus of the Dicer
protein (Dicer-1, CG4792). This antiserum could immunoprecipitate a
nuclease activity from either Drosophila embryo extracts or from S2
cell lysates that produced .about.22 nt. RNAs from dsRNA substrates
(FIG. 6C). The putative guide RNAs that are produced by the Dicer-1
enzyme precisely comigrate with 22 mers that are produced in
extract and with 22 mers that are associated with the RISC enzyme
(FIGS. 6 D,F). It had previously been shown that the enzyme that
produced guide RNAs in Drosophila embryo extracts was
ATP-dependent.sup.8. Depletion of this cofactor resulted in an
.about.6-fold lower rate of dsRNA cleavage and in the production of
RNAs with a slightly lower mobility. Of interest was the fact that
both Dicer-1 immunoprecipitates and extracts from S2 cells require
ATP for the production of 22 mers (FIG. 6D). We do not observe the
accumulation of lower mobility products in these cases, although we
do routinely observe these in ATP-depleted embryo extracts. The
requirement of this nuclease for ATP is a quite unusual property.
We hypothesize that this requirement could indicate that the enzyme
may act processively on the dsRNA, with the helicase domain
harnessing the energy of ATP hydrolysis both for unwinding guide
RNAs and for translocation along the substrate.
[0199] Efficient induction of RNA interference in C. elegans and in
Drosophila has several requirements. For example, the initiating
RNA must be double-stranded, and it must be several hundred
nucleotides in length. To determine whether these requirements are
dictated by Dicer, we characterized the ability of extracts and of
immunoprecipitated enzyme to digest various RNA substrates. Dicer
was inactive against single stranded RNAs regardless of length (see
Supplement 4). The enzyme could digest both 200 and 500 nucleotide
dsRNAs but was significantly less active with shorter substrates
(see Supplement 4). Double-stranded RNAs as short as 35 nucleotides
could be cut by the enzyme, albeit very inefficiently (data not
shown). In contrast, E. coli RNAse III could digest to completion
dsRNAs of 35 or 22 nucleotides (not shown). This suggests that the
substrate preferences of the Dicer enzyme may contribute to but not
wholly determine the size dependence of RNAi.
[0200] To determine whether the Dicer enzyme indeed played a role
in RNAi in vivo, we sought to deplete Dicer activity from S2 cells
and test the effect on dsRNA-induced gene silencing. Transfection
of S2 cells with a mixture of dsRNAs homologous to the two
Drosophila Dicer genes (CG4792 and CG6493) resulted in an
.about.6-7 fold reduction of Dicer activity either in whole cell
lysates or in Dicer-1 immunoprecipitates (FIGS. 7A,B). Transfection
with a control dsRNA (murine caspase 9) had no effect.
Qualitatively similar results were seen if Dicer was examined by
Northern blotting (not shown). Depletion of Dicer in this manner
substantially compromised the ability of cells to silence
subsequently an exogenous, GFP transgene by RNAi (FIG. 7C). These
results indicate that Dicer is involved in RNAi in vivo. The lack
of complete inhibition of silencing could result from an incomplete
suppression of Dicer (which is itself required for RNAi) or could
indicate that in vivo, guide RNAs can be produced by more than one
mechanism (e.g. through the action of RNA-dependent RNA
polymerases).
[0201] Our results indicate that the process of RNA interference
can be divided into at least two distinct steps. According to this
model, initiation of PTGS would occur upon processing of a
double-stranded RNA by Dicer into .about.22 nucleotide guide
sequences, although we cannot formally exclude the possibility that
another, Dicer-associated nuclease may participate in this process.
These guide RNAs would be incorporated into a distinct nuclease
complex (RISC) that targets single-stranded mRNAs for degradation.
An implication of this model is that guide sequences are themselves
derived directly from the dsRNA that triggers the response. In
accord with this model, we have demonstrated that .sup.32P-labeled,
exogenous dsRNAs that have been introduced into S2 cells by
transfection are incorporated into the RISC enzyme as 22 mers (FIG.
7E). However, we cannot exclude the possibility that RNA-dependent
RNA polymerases might amplify 22 mers once they have been generated
or provide an alternative method for producing guide RNAs.
[0202] The structure of the Dicer enzyme provokes speculation on
the mechanism by which the enzyme might produce discretely sized
fragments irrespective of the sequence of the dsRNA (see Supplement
1, FIG. 8a). It has been established that bacterial RNAse III acts
on its substrate as a dimer.sup.18,22,23. Similarly, a dimer of
Dicer enzymes may be required for cleavage of dsRNAs into .about.22
nt. pieces. According to one model, the cleavage interval would be
determined by the physical arrangement of the two RNAse III domains
within Dicer enzyme (FIG. 8a). A plausible alternative model would
dictate that cleavage was directed at a single position by the two
RIII domains in a single Dicer protein. The 22 nucleotide interval
could be dictated by interaction of neighboring Dicer enzymes or by
translocation along the mRNA substrate. The presence of an integral
helicase domain suggests that the products of Dicer cleavage might
be single-stranded 22 mers that are incorporated into the RISC
enzyme as such.
[0203] A notable feature of the Dicer family is its evolutionary
conservation. Homologs are found in C. elegans (K12H4.8),
Arabidopsis (e.g., CARPEL FACTORY.sup.24, T25K16.4,
AC012328.sub.--1), mammals (Helicase-MOI.sup.25) and S. pombe
(YC9A_SCHPO) (FIG. 8b, see Supplements 6,7 for sequence
comparisons). In fact, the human Dicer family member is capable of
generating .about.22 nt. RNAs from dsRNA substrates (Supplement 5)
suggesting that these structurally similar proteins may all share
similar biochemical functions. It has been demonstrated that
exogenous dsRNAs can affect gene function in early mouse
embryos.sup.29, and our results suggest that this regulation may be
accomplished by an evolutionarily conserved RNAi machinery.
[0204] In addition to RNAseIII and helicase motifs, searches of the
PFAM database indicate that each Dicer family member also contains
a ZAP domain (FIG. 8c).sup.27. This sequence was defined based
solely upon its conservation in the Zwille/ARGONAUTE/Piwi family
that has been implicated in RNAi by mutations in C. elegans
(Rde-1).sup.9 and Neurospora (Qde-2).sup.10. Although the function
of this domain is unknown, it is intriguing that this region of
homology is restricted to two gene families that participate in
dsRNA-dependent silencing. Both the ARGONAUTE and Dicer families
have also been implicated in common biological processes, namely
the determination of stem-cell fates. A hypomorphic allele of
carpel factory, a member of the Dicer family in Arabidopsis, is
characterized by increased proliferation in floral
meristems.sup.24. This phenotype and a number of other
characteristic features are also shared by Arabidopsis ARGONAUTE
(ago1-1) mutants.sup.26 (C. Kidner and R. Martiennsen, pers.
comm.). These genetic analyses begin to provide evidence that RNAi
may be more than a defensive response to unusual RNAs but may also
play important roles in the regulation of endogenous genes.
[0205] With the identification of Dicer as a catalyst of the
initiation step of RNAi, we have begun to unravel the biochemical
basis of this unusual mechanism of gene regulation. It will be of
critical importance to determine whether the conserved family
members from other organisms, particularly mammals, also play a
role in dsRNA-mediated gene regulation.
[0206] Methods
[0207] Plasmid constructs. A full-length cDNA encoding Drosha was
obtained by PCR from an EST sequenced by the Berkeley Drosophila
genome project. The Homeless clone was a gift from Gillespie and
Berg (Univ. Washington). The T7 epitope-tag was added to the amino
terminus of each by PCR, and the tagged cDNAs were cloned into
pRIP, a retroviral vector designed specifically for expression in
insect cells (E. Bernstein, unpublished). In this vector,
expression is driven by the Orgyia pseudotsugata IE2 promoter
(Invitrogen). Since no cDNA was available for CG4792/Dicer, a
genomic clone was amplified from a bacmid (BACR23F10; obtained from
the BACPAC Resource Center in the Dept. of Human Genetics at the
Roswell Park Cancer Institute). Again, during amplification, a T7
epitope tag was added at the amino terminus of the coding sequence.
The human Dicer gene was isolated from a cDNA library prepared from
HaCaT cells (GJH, unpublished). A T7-tagged version of the complete
coding sequence was cloned into pCDNA3 (Invitrogen) for expression
in human cells (LinX-A).
[0208] Cell culture and extract preparation. S2 and embryo culture.
S2 cells were cultured at 27.degree. C. in 5% CO.sub.2 in
Schneider's insect media supplemented with 10% heat inactivated
fetal bovine serum (Gemini) and 1% antibiotic-antimycotic solution
(Gibco BRL). Cells were harvested for extract preparation at
10.times.10.sup.6 cells/ml. The cells were washed 1.times. in PBS
and were resuspended in a hypotonic buffer (10 mM Hepes pH 7.0, 2
mM MgCl2, 6 mM .beta.ME) and dounced. Cell lysates were spun
20,000.times.g for 20 minutes. Extracts were stored at -80.degree.
C. Drosophila embryos were reared in fly cages by standard
methodologies and were collected every 12 hours. The embryos were
dechorionated in 50% chlorox bleach and washed thoroughly with
distilled water. Lysis buffer (10 mM Hepes, 10 mM KCl, 1.5 mM
MgCl.sub.2, 0.5 mM EGTA, 10 mM .beta.-glycerophosphate, 1 mM DTT,
0.2 mM PMSF) was added to the embryos, and extracts were prepared
by homogenization in a tissue grinder. Lysates were spun for two
hours at 200,000.times.g and were frozen at -80.degree. C. LinX-A
cells, a highly-transfectable derivative of human 293 cells, (Lin
Xie and GJH, unpublished) were maintained in DMEM/10%FCS.
[0209] Transfections and immunoprecipitations. S2 cells were
transfected using a calcium phosphate procedure essentially as
previously described.sup.6. Transfection rates were .about.90% as
monitored in controls using an in situ .beta.-galactosidase assay.
LinX-A cells were also transfected by calcium phosphate
co-precipitation. For immunoprecipitations, cells
(.about.5.times.10.sup.6 per IP) were transfected with various
clones and lysed three days later in IP buffer (125 mM KOAc, 1 mM
MgOAc, 1 mM CaCl.sub.2, 5 mM EGTA, 20 mM Hepes pH 7.0, 1 mM DTT, 1%
NP-40 plus Complete protease inhibitors (Roche)). Lysates were spun
for 10 minutes at 14,000.times.g and supernatants were added to T7
antibody-agarose beads (Novagen). Antibody binding proceeded for 4
hours at 4.degree. C. Beads were centrifuged and washed in lysis
buffer three times, and once in reaction buffer. The Dicer
antiserum was raised in rabbits using a KLH-conjugated peptide
corresponding to the C-terminal 8 amino acids of Drosophila Dicer-1
(CG4792).
[0210] Cleavage reactions. RNA preparation. Templates to be
transcribed into dsRNA were generated by PCR with forward and
reverse primers, each containing a T7 promoter sequence. RNAs were
produced using Riboprobe (Promega) kits and were uniformly labeling
during the transcription reaction with .sup.32P-UTP.
Single-stranded RNAs were purified from 1% agarose gels. dsRNA
cleavage. Five microliters of embryo or S2 extracts were incubated
for one hour at 30.degree. C. with dsRNA in a reaction containing
20 mM Hepes pH 7.0, 2 mM MgOAc, 2 mM DTT, 1 mM ATP and 5% Superasin
(Ambion). Immunoprecipitates were treated similarly except that a
minimal volume of reaction buffer (including ATP and Superasin) and
dsRNA were added to beads that had been washed in reaction buffer
(see above). For ATP depletion, Drosophila embryo extracts were
incubated for 20 minutes at 30.degree. C. with 2 mM glucose and
0.375 U of hexokinase (Roche) prior to the addition of dsRNA.
[0211] Northern and Western analysis. Total RNA was prepared from
Drosophila embryos (0-12 hour), from adult flies, and from S2 cells
using Trizol (Lifetech). Messenger RNA was isolated by affinity
selection using magnetic oligo-dT beads (Dynal). RNAs were
electrophoresed on denaturing formaldehyde/agarose gels, blotted
and probed with randomly primed DNAs corresponding to Dicer. For
Western analysis, T7-tagged proteins were immunoprecipitated from
whole cell lysates in IP buffer using anti-T7-antibody-agarose
conjugates. Proteins were released from the beads by boiling in
Laemmli buffer and were separated by electrophoresis on 8% SDS
PAGE. Following transfer to nitrocellulose, proteins were
visualized using an HRP-conjugated anti-T7 antibody (Novagen) and
chemiluminescent detection (Supersignal, Pierce).
[0212] RNAi of Dicer. Drosophila S2 cells were transfected either
with a dsRNA corresponding to mouse caspase 9 or with a mixture of
two dsRNAs corresponding to Drosophila Dicer-1 and Dicer-2 (CG4792
and CG6493). Two days after the initial transfection, cells were
again transfected with a mixture containing a GFP expression
plasmid and either luciferase dsRNA or GFP dsRNA as previously
described.sup.6. Cells were assayed for Dicer activity or
fluorescence three days after the second transfection.
Quantification of fluorescent cells was done on a Coulter EPICS
cell sorter after fixation. Control transfections indicated that
Dicer activity was not affected by the introduction of caspase 9
dsRNA.
References Cited Example 2
[0213] 1. Baulcombe, D. C. RNA as a target and an initiator of
post-transcriptional gene silencing in transgenic plants. Plant Mol
Biol 32, 79-88 (1996).
[0214] 2. Wassenegger, M. & Pelissier, T. A model for
RNA-mediated gene silencing in higher plants. Plant Mol Biol 37,
349-62 (1998).
[0215] 3. Montgomery, M. K. & Fire, A. Double-stranded RNA as a
mediator in sequence-specific genetic silencing and co-suppression
[see comments]. Trends Genet 14, 255-8 (1998).
[0216] 4. Sharp, P. A. RNAi and double-strand RNA. Genes Dev 13,
139-41 (1999).
[0217] 5. Sijen, T. & Kooter, J. M. Post-transcriptional
gene-silencing: RNAs on the attack or on the defense? [In Process
Citation]. Bioessays 22, 520-31 (2000).
[0218] 6. Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G.
J. An RNA-directed nuclease mediates post-transcriptional gene
silencing in Drosophila cells. Nature 404, 293-6 (2000).
[0219] 7. Hamilton, A. J. & Baulcombe, D. C. A species of small
antisense RNA in posttranscriptional gene silencing in plants [see
comments]. Science 286, 950-2 (1999).
[0220] 8. Zamore, P. D., Tuschl, T., Sharp, P. A. & Bartel, D.
P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of
mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33 (2000).
[0221] 9. Tabara, H. et al. The rde-1 gene, RNA interference, and
transposon silencing in C. elegans. Cell 99, 123-32 (1999).
[0222] 10. Catalanotto, C., Azzalin, G., Macino, G. & Cogoni,
C. Gene silencing in worms and fungi. Nature 404, 245 (2000).
[0223] 11. Ketting, R. F., Haverkamp, T. H., van Luenen, H. G.
& Plasterk, R. H. Mut-7 of C. elegans, required for transposon
silencing and RNA interference, is a homolog of Werner syndrome
helicase and RNaseD. Cell 99, 133-41 (1999).
[0224] 12. Cogoni, C. & Macino, G. Posttranscriptional gene
silencing in Neurospora by a RecQ DNA helicase. Science 286, 2342-4
(1999).
[0225] 13. Cogoni, C. & Macino, G. Gene silencing in Neurospora
crassa requires a protein homologous to RNA-dependent RNA
polymerase. Nature 399, 166-9 (1999).
[0226] 14. Smardon, A. et al. EGO-1 is related to RNA-directed RNA
polymerase and functions in germ-line development and RNA
interference in C. elegans [published erratum appears in Curr Biol
May 18, 2000;10(10):R393-4]. Curr Biol 10, 169-78 (2000).
[0227] 15. Mourrain, P. et al. Arabidopsis SGS2 and SGS3 genes are
required for posttranscriptional gene silencing and natural virus
resistance. Cell 101, 533-42 (2000).
[0228] 16. Dalmay, T., Hamilton, A., Rudd, S., Angell, S. &
Baulcombe, D. C. An RNA-dependent RNA polymerase gene in
Arabidopsis is required for posttranscriptional gene silencing
mediated by a transgene but not by a virus. Cell 101, 543-53
(2000).
[0229] 17. Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P.
& Sharp, P. A. Targeted mRNA degradation by double-stranded RNA
in vitro. Genes Dev 13, 3191-7 (1999).
[0230] 18. Nicholson, A. W. Function, mechanism and regulation of
bacterial ribonucleases. FEMS Microbiol Rev 23, 371-90 (1999).
[0231] 19. Filippov, V., Solovyev, V., Filippova, M. & Gill, S.
S. A novel type of RNase III family proteins in eukaryotes. Gene
245, 213-21 (2000).
[0232] 20. Bass, B. L. Double-stranded RNA as a template for gene
silencing. Cell 101, 235-8 (2000).
[0233] 21. Gillespie, D. E. & Berg, C. A. Homeless is required
for RNA localization in Drosophila oogenesis and encodes a new
member of the DE-H family of RNA-dependent ATPases. Genes Dev 9,
2495-508 (1995).
[0234] 22. Robertson, H. D., Webster, R. E. & Zinder, N. D.
Purification and properties of ribonuclease III from Escherichia
coli. J Biol Chem 243, 82-91 (1968).
[0235] 23. Dunn, J. J. RNase III cleavage of single-stranded RNA.
Effect of ionic strength on the fideltiy of cleavage. J Biol Chem
251, 3807-14 (1976).
[0236] 24. Jacobsen, S. E., Running, M. P. & Meyerowitz, E. M.
Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes
unregulated cell division in floral meristems. Development 126,
5231-43 (1999).
[0237] 25. Matsuda, S. et al. Molecular cloning and
characterization of a novel human gene (HERNA) which encodes a
putative RNA-helicase. Biochim Biophys Acta 1490, 163-9 (2000).
[0238] 26. Bohmert, K. et al AGO1 defines a novel locus of
Arabidopsis controlling leaf development. Embo J 17, 170-80
(1998).
[0239] 27. Sonnhammer, E. L., Eddy, S. R. & Durbin, R. Pfam: a
comprehensive database of protein domain families based on seed
alignments. Proteins 28, 405-20 (1997).
[0240] 28. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res
25, 3389-402 (1997).
[0241] 29. Wianny, F. and Zernicka-Goetz, M. Specific interference
with gene function by double-stranded RNA in early mouse
development. Nature Cell Biol. 2, 70-75 (2000).
[0242] 30. Fagard, M., Boutet, S., Morel, J.-B., Bellini, C. and
Vaucheret, H. Ago-1, Qde-2 and Rde-1 are related proteins required
for post-transcriptional gene silencing in plants, quelling in
fungi, and RNA interference in animals. Proc. Natl. Acad. Sci. USA
97, 11650-11654 (2000).
Example 3
A simplified Method for the Creation of Hairpin Constructs for RNA
Interference
[0243] In numerous model organisms, double stranded RNAs have been
shown to cause effective and specific suppression of gene function
(ref. 1). This response, termed RNA interference or
post-transcriptional gene silencing, has evolved into a highly
effective reverse genetic tool in C. elegans, Drosophila, plants
and numerous other systems. In these cases, double-stranded RNAs
can be introduced by injection, transfection or feeding; however,
in all cases, the response is both transient and systemic.
Recently, stable interference with gene expression has been
achieved by expression of RNAs that form snap-back or hairpin
structures (refs 2-7). This has the potential not only to allow
stable silencing of gene expression but also inducible silencing as
has been observed in trypanosomes and adult Drosophila (refs
2,4,5). The utility of this approach is somewhat hampered by the
difficulties that arise in the construction of bacterial plasmids
containing the long inverted repeats that are necessary to provoke
silencing. In a recent report, it was stated that more than 1,000
putative clones were screed to identify the desired construct (ref
7).
[0244] The presence of hairpin structures often induces plasmid
rearrangement, in part due to the E. coli sbc proteins that
recognize and cleave cruciform DNA structures (ref 8). We have
developed a method for the construction of hairpins that does not
require cloning of inverted repeats, per se. Instead, the fragment
of the gene that is to be silenced is cloned as a direct repeat,
and the inversion is accomplished by treatment with a site-specific
recombinase, either in vitro (or potentially in vivo) (see FIG.
27). Following recombination, the inverted repeat structure is
stable in a bacterial strain that lacks an intact SBC system
(DL759). We have successfully used this strategy to construct
numerous hairpin expression constructs that have been successfully
used to provoke gene silencing in Drosophila cells.
Literature Cited in Example 3
[0245] 1. Bosher, J. M. & Labouesse, M. RNA interference:
genetic wand and genetic watchdog. Nat Cell Biol 2, E31-6
(2000).
[0246] 2. Fortier, E. & Belote, J. M. Temperature-dependent
gene silencing by an expressed inverted repeat in Drosophila
[published erratum appears in Genesis; May 27, 2000; (1):47].
Genesis 26, 240-4 (2000).
[0247] 3. Kennerdell, J. R. & Carthew, R. W. Heritable gene
silencing in Drosophila using double-stranded RNA. Nat Biotechnol
18, 896-8 (2000).
[0248] 4. Lam, G. & Thummel, C. S. Inducible expression of
double-stranded RNA directs specific genetic interference in
Drosophila [In Process Citation]. Curr Biol 10, 957-63 (2000).
[0249] 5. Shi, H. et al. Genetic interference in Trypanosoma brucei
by heritable and inducible double-stranded RNA. Rna 6, 1069-76
(2000).
[0250] 6. Smith, N. A. et al Total silencing by intron-spliced
hairpin RNAs. Nature 407, 319-20 (2000).
[0251] 7. Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A.
& Driscoll, M. Heritable and inducible genetic interference by
double-stranded RNA encoded by transgenes. Nat Genet 24, 180-3
(2000).
[0252] 8. Connelly, J. C. & Leach, D. R. The sbcC and sbcD
genes of Escherichia coli encode a nuclease involved in palindrome
inviability and genetic recombination. Genes Cells 1, 285-91
(1996).
V. EQUIVALENTS
[0253] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0254] All of the above-cited references and publications are
hereby incorporated by reference.
* * * * *