U.S. patent application number 10/216054 was filed with the patent office on 2003-07-31 for expression system.
Invention is credited to Agami, Reuven, Brummelkamp, Thijn.
Application Number | 20030144232 10/216054 |
Document ID | / |
Family ID | 37663290 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030144232 |
Kind Code |
A1 |
Agami, Reuven ; et
al. |
July 31, 2003 |
Expression system
Abstract
The present invention provides a polynucleotide comprising a RNA
polymerase III promoter, a region encoding a siRNA, and a
transcriptional termination element comprising five consecutive
thymine residues. The invention also provides for vectors, cells
and non-human transgenic animal comprising the polynucleotides of
the invention as well as their use in medicaments for various
conditions.
Inventors: |
Agami, Reuven; (Amsterdam,
NL) ; Brummelkamp, Thijn; (Amsterdam, NL) |
Correspondence
Address: |
GARY CARY WARE & FRIENDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Family ID: |
37663290 |
Appl. No.: |
10/216054 |
Filed: |
August 9, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60377482 |
May 2, 2002 |
|
|
|
Current U.S.
Class: |
514/44A ;
435/320.1; 435/325; 435/456; 435/6.14; 536/23.1; 800/8 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61P 35/00 20180101; C12N 2330/30 20130101; C12N 2310/111 20130101;
C12N 15/111 20130101; C12N 2310/14 20130101; A61P 31/18 20180101;
C12N 15/113 20130101; A61P 35/04 20180101; A61P 1/18 20180101; A61K
49/0008 20130101; A61K 38/00 20130101; A61K 31/711 20130101; A61P
35/02 20180101; C12N 15/1135 20130101; A61P 29/00 20180101; A61P
37/08 20180101; A61P 31/14 20180101; C12N 2310/53 20130101 |
Class at
Publication: |
514/44 ;
536/23.1; 435/456; 435/320.1; 435/6; 435/325; 800/8 |
International
Class: |
A01K 067/00; C12Q
001/68; C07H 021/02; A61K 048/00; C12N 015/861; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 2001 |
GB |
0130955.8 |
Claims
1. A polynucleotide comprising: a RNA polymerase III promoter; a
region encoding a siRNA; and a transcriptional termination element
comprising five consecutive thymine residues in the sense
strand.
2. A polynucleotide according to claim 1, wherein the promoter is
the RNA polymerase III H1-RNA gene promoter or a functional
derivative thereof.
3. A polynucleotide according to claim 1, wherein the promoter is
the RNA polymerase III 5S, U6, adenovirus VA1, Vault, telomerase
RNA, or tRNA gene promoter or a functional derivative thereof.
4. A polynucleotide according to claim 1, 2 or 3, wherein the
region encoding the siRNA comprises: a region complementary to a
target gene and a second region complementary to the first region;
and a spacer separating the two complementary regions.
5. A polynucleotide according to claim 4, wherein: the spacer
region in the polynucleotide comprises two consecutive thymine
residues in the sense strand immediately 3' of the region
complementary to the target gene; and immediately 3' of the region
complementary to the first region are two thymine residues in the
sense strand wherein in the RNA transcript generated from the
polynucleotide the complementary region and spacer are capable of
forming a stem loop structure.
6. A polynucleotide according to claim 4 or 5, wherein the spacer
region is from seven to fifteen nucleotides in length and/or the
region complementary to the target gene is from nineteen to
twenty-one bases in length.
7. A polynucleotide according to claim 5 or claim 6, wherein the
spacer region has the sequence 5' TTCAAGAGA 3'.
8. A polynucleotide according to claim 5, 6 or 7, wherein the stem
loop structure can be cleaved by an enzyme to generate a siRNA
molecule which 3' overhangs at each of its termini each comprising
two uridine residues.
9. A polynucleotide according to any one of claims 4 to 8, wherein
the target gene is Cdh1, p53 or CDC20.
10. A polynucleotide according to any one of the preceding claims,
wherein the siRNA is capable of discriminating between different
alleles of the same gene.
11. A vector comprising a polynucleotide according to any one of
the preceding claims.
12. A cell comprising a polynucleotide according to any one of
claims 1 to 10, or a vector according to claim 9.
13. A cell according to claim 12, wherein the polynucleotide, or
vector is integrated into the host genome.
14. A non-human transgenic animal comprising a polynucleotide
according to any one of claims 1 to 10, a vector according to claim
11, or a cell according to claim 12 or 13.
15. Use of a polynucleotide according to any one of claims 1 to 10
or a vector according to claim 11 to inhibit or reduce the
expression of a gene.
16. A method of identifying an agent capable of modulating the
phenotype of a cell according to claim 12 or 13 or a non-human
transgenic animal according to claim 14, in a desired manner
comprising determining whether a test agent can modulate the
phenotype of the cell or transgenic organism in the desired
manner.
17. A method for identifying: (i) a modulator of transcription
and/or translation of a target gene; and/or (ii) a modulator of the
activity of a target polypeptide, in a cell according to claim 12
or 13 or a non-human transgenic animal according to claim 14 which
method comprises determining whether a test agent can modulate
transcription and/or translation of the target gene or the activity
of the target polypeptide.
18. A method according to claim 16 or 17, wherein the target gene
is a disease gene or the desired modulation in phenotype is the
prevention or treatment of a disease or infection.
19. A method of producing a pharmaceutical composition suitable for
preventing or treating a specific disorder, comprising performing
the method of any one of claims 16 to 18 and formulating the agent
or modulator identified by the method with a pharmaceutically
acceptable carrier or diluent.
20. An agent or modulator identified by a method according to any
one of claims 16 to 18, or a pharmaceutical composition identified
by a method according to claim 19.
21. A kit comprising a polynucleotide according to any one of
claims 1 to 10, a vector according to claim 11, or a cell according
to claim 12 or 13 and a means for detecting and/or quantifying the
expression of the target gene.
22. A pharmaceutical composition comprising a polynucleotide
according to any one of claims 1 to 10, a vector according to claim
11, a cell according to claim 12 or 13, an agent or a modulator
according to claim 20 and a pharmaceutically acceptable
excipient.
23. A polynucleotide according to any one of claims 1 to 10, a
vector according to claim 11, a cell according to claim 12 or 13,
an agent or a modulator according to claim 20 for use in a method
of treatment of the human or animal body by therapy or
diagnosis.
24. Use of a polynucleotide according to any one of claims 1 to 10,
a vector according to claim 11, a cell according to claim 12 or 13,
or an agent or a modulator according to claim 20 in the manufacture
of a medicament for the treatment or prevention of cancer or an
autosomal dominant disorder.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/377,482 dated May 2, 2002 and United Kingdom
Patent Application No. 0130955.8 dated Dec. 24, 2001, both of which
are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to a polynucleotide or vector for
expressing short interfering RNAs (siRNAs) to inhibit the
expression of a target gene. The invention also relates to cells
and non-human transgenic animals comprising the polynucleotide or
vector and their various uses including in target drug validation
and in human therapeutics.
BACKGROUND OF THE INVENTION
[0003] The ability to inhibit or disrupt the function of a specific
gene is highly desirable both from the point of view of studying
gene function and also from a therapeutic perspective.
[0004] Many diseases arise from either the expression of a mutated
gene or from abnormal, and in particular elevated or inappropriate,
expression of a particular gene. Such mutations may be inherited,
such as in the case of autosomal dominant disorders, or occur in
the somatic or germ line tissues of an individual, such as in the
case of cancer. The ability to modulate the expression of a mutated
allele or of an inappropriately expressed wild type allele in
various diseases or disorders may therefore be used to provide
therapies to treat the disorders. In addition, in various
infectious diseases, such as viral infection, the ability to
inhibit the expression of viral genes in the host cell, or of a
gene encoding a host cell protein involved in the life cycle of the
virus, may also lead to possible treatments for infectious
diseases.
[0005] The ability to inhibit gene expression has also been used to
study gene function. Techniques such as classical mutagenesis have
provided great insights into gene function, but such techniques are
labour intensive, expensive and may take long periods of time. Such
techniques simply may not be practical in higher organisms and
require a means to identify the desired mutant. They also do not
offer the possibility of mutating a specific gene of choice.
[0006] Although various methods for targeted gene disruption have
been developed, where a gene of choice can be inhibited or
disrupted, these also suffer from limitations. Techniques such as
gene targeting are highly costly, expensive and time consuming
often taking several years to obtain a homozygous mutant. Gene
targeting also requires detailed knowledge of the structure of the
gene to be disrupted.
[0007] As well as gene targeting antisense technology has also been
developed to try and disrupt a specific gene. However, antisense
RNA is unstable and it is often difficult to achieve high enough
levels of antisense RNA in cells to achieve effective inhibition of
a target gene.
[0008] Recently, it has been found in organisms such as C. elegans,
Drosophilia melanogaster and plants that double stranded RNA
molecules (dsRNA) are capable of inhibiting the expression of a
target gene that they share sequence identity or homology to. The
observed phenomena, sometimes referred to as post transcriptional
gene silencing (PTGS), are thought to represent a possible cellular
defence mechanism against viruses or transposons. Typically, in the
studies carried out in these organisms the dsRNA has been
introduced into cells by techniques such as microinjection or
transfection and the inhibition of a target gene such as a reporter
gene been measured.
[0009] The mechanism by which the dsRNA exerts its inhibitory
effect on the target gene has begun to be elucidated. It is thought
that the dsRNA is processed into duplexes of from 21 to 25
nucleotides in length. These short duplexes have been detected in
plants where PTGS is occurring as well as in extracts of D.
melanogaster schenider-2 (S2) cells transfected with a dsRNA
molecule. It has been found that the processing reaction of a dsRNA
can be carried out in vitro using extracts from these S2 cells.
This provides an in vitro model system in which both the
processing, targeting and transcript cleavage mechanisms involved
in gene silencing can be studied. In the S2 lysate it was observed
that the target mRNA was cleaved at 21 nucleotide intervals and
that synthetic 21 and 22 RNA duplexes added to the lysate were able
to guide efficient sequence specific mRNA degradation. Larger
duplexes of 30 bp dsRNA were found to be active. The 21 nucleotide
RNA products in the system were therefore named small interfering
or silencing RNAs (siRNAs).
[0010] Factors from the target cell are also necessary for gene
silencing. In D. melanogaster a ribonuclease III enzyme, dicer, is
required for processing of the long dsRNAs into siRNA duplexes. It
is thought that genes homologous to dicer exist in other organisms
including mammals and humans as well as homologs or counterparts to
the other host factors necessary. The initial steps in silencing
involve the generation of a siRNA containing endonuclease complex.
The endonuclease may be dicer or a gene homologous to dicer. The
complex then specifically targets the mRNA transcript by a
mechanism involving the exchange of one of the strands of the siRNA
duplex with the region of sequence identity in the target
transcript. Following this strand exchange, cleavage of the mRNA
transcript occurs.
[0011] The cleavage of the target mRNA may occur at the ends of the
duplexed region so, in effect, regenerating the siRNA endonuclease
complex with one of the two strands of the regenerated siRNA coming
from the original siRNA molecule and the other from the target
transcript. Multiple cycles of transcript mRNA cleavage and hence
siRNA regeneration may mean that each initial siRNA molecule can
inactivate multiple copies of the target mRNA. Once the target mRNA
transcript has been cleaved, the cleavage products not in the
regenerated siRNA are rapidly degraded as they either lack the
stabilising cap or pol(A)tail.
[0012] Although experiments investigating gene silencing in lower
organisms have offered promising results it is thought that they
may not be applicable to higher organisms such as mammals. It is
thought that in higher organisms, such as mammals, cellular defence
mechanisms operate which are triggered by dsRNA. It is believed
that dsRNAs activate the interferon response which leads to a
global shut-off in protein synthesis as well as non-specific mRNA
degradation. This can lead to cell death and hence prevent
selective gene inhibition. The presence of such defence mechanisms
means that the applicability of gene silencing employing dsRNA in
higher organisms has been questioned.
[0013] Experiments which have claimed to have demonstrated the
efficacy of dsRNA in inhibiting the expression of a target gene in
higher organisms have either been in non-mammalian systems, such as
zebra fish or chicks, or alternatively in mammalian systems such as
early embryos where the viral defence mechanisms are not thought to
operate.
[0014] Preliminary experiments transfecting and/or microinjecting
synthetic siRNAs, rather than longer dsRNA molecules which can be
processed to give rise to a siRNA, have led to speculation that it
might be possible to overcome the problems of the viral defence
mechanisms in higher organisms. It may be that there is a threshold
for the length of dsRNA necessary to activate the cell's defence
mechanisms. The size of the synthetic siRNAs, and in particular the
double stranded regions in them, introduced into the target cell
may be small enough that they are below this threshold and hence do
not activate the defence mechanisms.
SUMMARY OF THE INVENTION
[0015] It has now been found according to the present invention,
that, by using a RNA polymerase III promoter, and in particular a
type 3 RNA polymerase promoter, in combination with a
transcriptional termination sequence comprising a string of five
consecutive thymine residues in the sense strand that siRNAs can be
efficiently expressed in animal cells and in particular in
mammalian cells.
[0016] By using the RNA polymerase III promoter in conjunction with
the transcriptional terminator this ensures that one of the 3'
overhangs necessary for optimal inhibitory activity is present in
the siRNA generated from the constructs of the invention. The
second 3' overhang may be produced by cleavage of a stem loop
structure in the transcript generated from the construct.
[0017] The fact that the constructs of the invention are DNA
molecules capable of integrating into the genome of the target cell
allows for stable, long term expression of the siRNA and hence long
term inhibition of the target gene.
[0018] Accordingly, the present invention provides a polynucleotide
comprising:
[0019] a RNA polymerase III promoter;
[0020] a region encoding a siRNA; and
[0021] a transcriptional termination element comprising five
consecutive thymine residues.
[0022] The invention also provides for
[0023] a vector comprising a polynucleotide of the invention;
[0024] a cell comprising a polynucleotide or vector of the
invention;
[0025] a non-human transgenic animal comprising a polynucleotide or
vector of the invention;
[0026] the use of a polynucleotide or vector of the invention to
inhibit or reduce the expression of a target gene.
[0027] The invention also provides for a method of identifying an
agent capable of modulating the phenotype of a cell or non-human
transgenic animal of the invention, in a desired manner comprising
determining whether a test agent can modulate the phenotype of the
cell or transgenic organism in the desired manner.
[0028] The invention further provides for a method for
identifying:
[0029] (i) a modulator of transcription and/or translation of a
target gene; and/or
[0030] (ii) a modulator of the activity of a target polypeptide, in
a cell or a non-human transgenic animal of the invention, which
method comprises determining whether a test agent can modulate
transcription and/or translation of the target gene or the activity
of the target polypeptide.
[0031] The invention also provides:
[0032] an agent or modulator identified by a method of the
invention;
[0033] a pharmaceutical composition identified by a method of the
invention.
[0034] a kit comprising a polynucleotide, vector, or cell of the
invention and a means for detecting and/or quantifying the
expression of the target gene;
[0035] a pharmaceutical composition comprising a polynucleotide,
vector, cell, agent or modulator of the invention and a
pharmaceutically acceptable excipient;
[0036] a polynucleotide, vector, cell, agent or modulator of the
invention for use in a method of treatment of the human or animal
body by therapy or diagnosis; and
[0037] the use of a polynucleotide, vector, cell, agent or
modulator of the invention in the manufacture of a medicament for
the treatment or prevention of cancer or an autosomal dominant
disorder.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 shows suppression of gene expression in mammalian
cells by a vector of the invention. FIG. 1(a) shows a schematic
drawing of the basic pSUPER vector. FIG. 1(b) depicts the synthetic
siRNA used to target CDH1 and the predicted secondary structures of
the three pSUPER-CDH1 transcripts (A, B and C) from each of the
three pSUPER-Cdh1 constructs evaluated. FIG. 1(c) shows a western
blot for Cdh1. The cell extracts on the blot are from cells
transfected with (from left to right) a control plasmid expressing
GFP, Cdh1-siRNA, the empty pSUPER construct, the three pSUPER
constructs capable of expressing the transcripts A, B and C
indicated in FIG. 1(b) and finally empty pSUPER. FIG. 1(d) shows a
northern blot of RNA extracted from cells transfected with the
various constructs indicated. The position of the stem loop and
siRNA are indicated on the blot. The 5S-RNA band was also measured
with Ethidium Bromide staining as a control for loading.
[0039] FIG. 2(a) shows a western blot of cells transfected with
increasing amounts of the pSUPER-p53 vector, that is predicted to
produce the transcript depicted above the blot. Cells were either
irradiated (+IR, 20 Gy) or left untreated, harvested, blotted and
then probed with anti-p53 antibody as indicated and also probed for
a control protein to show equal loading. FIG. 2(b) shows flow
cytometric analysis of cells transfected either with empty pSUPER
or with pSUPER expressing the siRNA against p53. The cells have
either been irradiated (+IR, 10Gy) or are unirradiated controls
(-). Cells with a G1-phase DNA content are indicated with an arrow.
FIG. 2(c) shows cells transfected with 1 .mu.g pSUPER vectors and
0.1 .mu.g pBabe-puro plasmid which were selected with 1 .mu.g/ml
puromycin 48 hours later for 12 days. Plates were irradiated (20
Gy) and after 4 hours fixed and stained to detect p53. Shown also
are the phase contrast images of the same colonies. The left and
right images are of two different colonies.
[0040] FIG. 3(a) depicts the intact target recognition sequence
required to suppress CDH1 by the pSUPER-CDH1 vector. The CDH1 19
nucleotide target-recognition sequence was mutated to give one
basepair substitution at position 9 or 2 of the stem. The predicted
secondary structures of the transcripts are shown (mutations are in
bold and underlined). FIG. 3(b) shows an immunoblot against CDH1 of
cells transfected with the constructs displayed in FIG. 3(a) probed
with anti-CDH1 antibody. Cyclin D1 protein was used to demonstrate
equal loading.
[0041] FIG. 4 shows suppression of CDC20 expression by both
synthetic SiRNA and pSUPER-CDC20. Shown are the sequences of the
SiRNA and the predicted transcript of pSUPER-CDC20 utilized to
ihibit CDC20 expression. The indicated SiRNAs and plasmids were
transfected into cells, cell extracts immunoblotted and probed to
detect Cdc2O and Cyclin D1 proteins.
[0042] FIG. 5 shows the use of a vector of the invention to
interfere with p53 mRNA expression. FIG. 5A shows a northern blot
of RNA from MCF-7 cells transfected with pSUPER or the pSUPER-p53
vector. MCF-7 cells were electroporated with pSUPER-p53 or vector
and total RNA was extracted 48 hours later. Thirty .mu.g of RNA was
separated on agarose gel, blotted and probed with a p53 specific
.sup.32P labeled probe. The rRNAs controls for loading were
visualized by Ethidium Bromide staining of the blot. FIG. 5B shows
siRNA interference mediated by the same stem-loop transcript can be
expressed from retro viral vectors. Self-inactivating retro viral
vectors (pRETRO-SUPER) expressing the puromycin marker gene were
cloned to harbor either an empty pol-III promoter or one that
targets p53 (FIG. 5D) as depicted. U2-OS cells containing the
Ecotropic-receptor were infected three times with these vectors and
one day later cells were selected for 4 days with 1 .mu.g/ml
puromycin and plated on glass slides. One day later, slides were
irradiated (20Gy), fixed four hours later and stained with anti-p53
antibody. Immuno-florescence with a FITC-conjugated secondary
antibody is shown together with the phase contrast of the same
field. Both pictures were taken using the same settings of the
camera and microscope. FIG. 5(C) shows a schematic for pRETRO-SUPER
with long terminal repeats (LTRs) at either end, a puromycin
selectable marker with the HI RNA gene promoter, target sequence
and terminator also inserted.
[0043] FIG. 6 shows a schematic representation of the various
elements typically present in the construct of the invention. These
are three consecutive cytosine residues, immediately after which
transcription begins, the region encoding the siRNA, and the
transcriptional terminator comprising 5 consecutive thymidine
residues.
[0044] FIG. 7 shows the use of retroviral vectors to mediate RNA
interference. FIG. 7A is a schematic drawing of retroviral
pRETRO-SUPER RNA interference vector (pRS). DNA fragments
containing the H1-RNA promoter with no insert or with an insert to
target human p53 (as described in Example 2) were digested
(EcoRI-XhoI) from corresponding pSUPER constructs and cloned into a
self inactivating MSCV to generate pRS and pRS-p53, respectively.
FIG. 7B shows immuno-stained cells. Human U2-OS cells that stably
express the murine ecotropic receptor (to allow retorviral entry
into cells) were infected with pRS and pRS-p53 retrovirus and
selected for one week with puromycin. Polyclonal populations of
puromycin-resistant cells were immuno-stained for p53 (in green)
and for actin (in red). FIG. 7C shows a Western blot in which whole
cell extracts were made from the same infected polyclonal
populations of U2-OS cels as in FIG. 7B, separated by
SDS-polyacrylamide gel electrophoresis (PAGE), and immuno-blotted
to detect p53 protein. FIG. 7D shows Northern blot analysis, in
which 30 .quadrature.g of total RNA from the same infected cell
population described in FIG. 7B was preformed and probed with the
sense 19 nucleotide targeting p53 sequence, as described in Example
2.
[0045] FIG. 8 shows the selective suppression of oncogenic
K-RAS.sup.V12. FIG. 8A shows the sequences of the wild type and V12
mutant alleles of human K-RAS and the predicted mutant-specific
short hairpin transcript encoded by pSUPER-K-RAS.sup.V2. FIG. 8B
shows an immunoblot. The 19 nt sequence spanning the V12 mutation
of K-RAS.sup.V12 was used to generate a pSUPER-K-RAS.sup.V12
(pS-K-RAS.sup.V12) construct. This construct, an empty pSUPER (pS)
and H2B-GFP plasmids were electroporated into CAPAN-1 cells and
whole cell extracts were prepared as described in Agami, et al,
Cell 102, 55-66 (2000)). Immunoblot analysis was preformed using a
specific anti K-RAS antibody (sc-30, Santa Cruz) and anti cyclin-D1
as control. FIG. 8C shows a Western blot produced as follows. The
pSUPER cassette, containing the K-RAS.sup.V12 targeting sequence,
was cloned into pRS as described in FIG. 7, and virus stock was
produced. A stable polyclonal pool of CAPAN-1 cells that expresses
the murine ecotropic receptor was infected with the indicated viral
stocks. Cells were selected with puromycin for three days and whole
cell extracts were used for immunoblot analysis to detect K-RAS
protein and the controls p53 and actin. FIG. 8D shows a Western
blot produced as follows. Stable polyclonal pools of CAPAN-1 and EJ
cells that express the ecotropic receptor were infected with the
indicated virus stocks, drug selected and immunoblotted to detect
K-RAS, p53 and actin proteins.
[0046] FIG. 9 shows stable and selective loss of tumorigenicity by
a retroviral vector that targets the K-RAS.sup.V12 oncogene. The
same CAPAN-1 (harbor mutant K-RAS.sup.V12) and EJ (harbor wild type
K-RAS) cell populations as in FIG. 8 were infected with the
indicated RETRO-SUPER viruses and selected for three days using 3
.quadrature.g/ml puromycin. FIG. 9A is one representative of three
independent experiments in which 2.times.10 selected cells from the
indicated infections were plated in duplicates in 2.5 cm diameter
plates containing soft agar. FIG. 9B shows athymic mice into which
1.times.10.sup.6 selected cells from pRS and pRS-K-RAS.sup.V12
infections were injected subcutaneously as indicated. Four weeks
later, mice were inspected for the presence of tumors at the site
of injection.
BRIEF DESCRIPTION OF THE SEQUENCES
[0047] SEQ ID NO:1 provides the sequence for the human H1 RNA gene
as available from GenBank under accession number X16612.
[0048] SEQ ID NO:2 provides the sequence for the preferred H1 RNA
gene promoter and corresponds to from nucleotide 146 to nucleotide
374 of the sequence of SEQ ID NO:1.
[0049] SEQ ID NO:3 provides the sequence of the sense strand of the
synthetic siRNA against Cdh1 depicted in FIG. 1(b).
[0050] SEQ ID NO:4 provides the sequence of the antisense strand of
the synthetic siRNA against Cdh1 depicted in FIG. 1(b).
[0051] SEQ ID NO:5 provides the sequence of the predicted stem loop
transcript generated from pSUPER-Cdh11-A depicted in FIG. 1(b).
[0052] SEQ ID NO:6 provides the sequence of the predicted stem loop
transcript generated from pSUPER-Cdh11-B depicted in FIG. 1(b).
[0053] SEQ ID NO:7 provides the sequence of the predicted stem loop
transcript generated from pSUPER-Cdh11-C depicted in FIG. 1(b).
[0054] SEQ ID NO:8 provides the sequence of the predicted stem loop
transcript generated from pSUPER-p53 which is also depicted in FIG.
2(a).
[0055] SEQ ID NO:9 provides the sequence of the predicted stem loop
transcript generated from the pSUPER-Cdh11-B vector as depicted in
FIG. 3(a).
[0056] SEQ ID NO:10 provides the sequence of the predicted stem
loop transcript generated from the pSUPER-Cdh11-B(mut-9) vector as
depicted in FIG. 3(a).
[0057] SEQ ID NO:11 provides the sequence of the predicted stem
loop transcript generated from the pSUPER-Cdh11-B(mut-2) vector as
depicted in FIG. 3(a).
[0058] SEQ ID NO:12 provides the sequence of the sense strand of
the synthetic siRNA against CDC20 depicted in FIG. 4.
[0059] SEQ ID NO:13 provides the sequence of the antisense strand
of the synthetic siRNA against CDC20 depicted in FIG. 4.
[0060] SEQ ID NO:14 provides the sequence of the predicted stem
loop transcript generated from the pSUPER-CDC20 vector as depicted
in FIG. 4.
[0061] SEQ ID NO:15 provides the sequence of an oligonucleotide
used to generate pS-K-RAS.sup.V12.
[0062] SEQ ID NO:16 provides the sequence of an oligonucleotide
used to generate pS-K-RAS.sup.V12.
[0063] SEQ ID NO:17 provides the sequence of a region of wild type
K-RAS spanning residue 12.
[0064] SEQ ID NO:18 provides the sequence of a region of mutant
K-RAS spanning residue 12.
[0065] SEQ ID NO:19 provides the sequence of the predicted stem
loop transcript generated from the pSUPER-K-RAS.sup.V12 vector as
depicted in FIG. 8A.
[0066] SEQ ID NO:20 provides the sequence of a preferred spacer
region.
DETAILED DESCRIPTION OF THE INVENTION
[0067] Throughout the present specification and the accompanying
claims the words "comprise" and "include" and variations such as
"comprises", "comprising", "includes" and "including" are to be
interpreted inclusively. That is, these words are intended to
convey the possible inclusion of other elements or integers not
specifically recited, where the context allows. Where the word
"comprising" is used the invention encompasses embodiments which
consist essentially of the elements specified.
[0068] The present invention provides various polynucleotides,
vectors and constructs capable of producing siRNAs. By construct it
is meant either a polynucleotide or vector of the invention. The
polynucleotides of the invention comprises:
[0069] a RNA polymerase III promoter;
[0070] a region encoding a siRNA; and
[0071] a transcriptional termination element comprising five
consecutive
[0072] thymidine residues.
[0073] RNA Polymerase III Promoters
[0074] The expression of the siRNA in the constructs of the
invention is driven by a RNA polymerase III promoter. Such
promoters are typically capable of producing a high level of
expression of a particular gene and often well in excess of the
levels achievable with RNA polymerase II promoters. This high level
of expression can help ensure that a high level of inhibition of
the target gene is achievable.
[0075] Typically, the level of inhibition of the target gene is at
least 20%, preferably is at least 30%, preferably at least 40%,
even more preferably is at least 50%, still more preferably is at
least 60% of the normal level of expression of the allele or of the
elevated level of expression of the targeted where the target gene
is abnormally expressed. The level of inhibition may be in excess
of 60%, preferably in excess of 75%, more preferably in excess of
90%, even more preferably in excess of 95% of the normal level of
expression of the allele or of the elevated level of expression of
the targeted where the target gene is abnormally expressed. The
fact that the level of inhibition may be specifically chosen is one
advantage over gene targeting and other conventional mutagensis
methods, where a gene is rendered completely inactive, without the
option for gradations of gene inhibition. Thus for a particular
situation any of the levels of inhibition specified herein may be
used or a level of inhibition as appropriate.
[0076] The particular level of inhibition may be chosen, because of
the use the methods of the invention are being put to. For example,
in cases where a disease is being modelled that involves reduced
expression of a gene, but not total elimination of the expression
of that gene the level of inhibition may be chosen to match the
reduction seen in the disease condition. Alternatively, in some
therapeutic methods, where a specific gene is expressed at an
elevated level, it may be desired to return the level of expression
of that gene to the normal level expression rather than to
completely inhibit expression of that target gene. For target
validation and drug screening less than a 100% inhibition may be
required such as from 20 to 30%, more preferably from 30 to 40% or
still more preferably from 40 to 50%.
[0077] In a preferred embodiment of the invention the level of
inhibition is, or almost is, 100%, and hence the cell or organism
will in effect have the phenotype equivalent to a so called "knock
out" of a gene. However, in some embodiments it may be preferred to
achieve only partial inhibition so that the phenotype is equivalent
to a so called "knock down" of the gene.
[0078] The RNA polymerase III (pol III) promoters are responsible
for the expression of a variety of genes including H1 RNA gene, 5S,
U6, adenovirus VA1, Vault, telomerase RNA, and tRNA genes. There
are three major types of pol III promoters: types 1, 2 and 3. In
addition to type 1 to 3 promoters, several other pol III promoter
elements have been reported including those responsible for the
expression of Epstein-Barr-virus-encoded RNAs (EBER), and human 7SL
RNA. Any of these RNA polymerase III promoters, or functional
derivatives thereof, may be used in the present invention to drive
expression of the siRNA, the promoter may typically be a type 3 RNA
promoter and in particular most preferably the promoter is a type 3
HI RNA gene promoter. Preferably the RNA polymerase III promoter
responsible for the expression of the H1I RNA may be employed. The
HI RNA is the RNA component of the human RNAse P. Type 3 promoters
are preferred as they are "external" promoters in other words they
are self contained, in that they do no require specific elements to
be present downstream of the transcriptional start site for
transcription to occur such as in the case of type 1 or 2
promoters. In an especially preferred embodiment of the invention
the promoter employed is an external promoter.
[0079] As well as various known RNA polymerase III promoter various
functional derivatives of such promoters may be employed and in
particular a functional derivative of the Hi RNA gene promoter may
be employed. Such derivatives will be capable of being recognised
by RNA polymerase III resulting in a transcript being generated.
Such functional derivatives may comprise combinations of the
various elements known to be important in RNA polymerase III
promoters.
[0080] The promoter will be operably linked to the region encoding
the siRNA. Typically, the sequences encoding the siRNA will be
immediately downstream of the transcriptional start site or be
separated by a minimal distance such less than twenty base pairs,
preferably less than ten base pairs, even more preferably less than
five base pairs and still more preferably by two or less base
pairs.
[0081] Typically the RNA polymerase III promoter employed will
comprise three consecutive cytosines i.e. CCC, these will normally
be the last three nucleotides of the promoter and transcription
will start immediately downstream of this CCC sequence. This is
especially the case where the promoter is a HI RNA gene promoter or
a functional derivative thereof.
[0082] In addition, to the RNA polymerase III promoter the
constructs of the invention may comprise various elements to allow
for tissue specific, or temporally (time) specific expression.
Methods to achieve such tissue or temporally controlled expression
are known in the art and any of these may be employed to achieve
such expression. By using such mechanisms this may allow the
inhibition of the target gene to occur in a specific cell type or
stage of development. This may have applications in both therapy
and developmental biology for example, where the aberrant
expression or mutated allele is only being expressed in a
particular cell type or it is not wished to disrupt expression in
other cell types or where a gene is only expressed during a
particular stage of embryonic development or maturation of the
adult organism. It may also allow for the study of essential
embryonic genes in mature adults.
[0083] By disrupting or inhibiting genes in a tissue or temporally
controlled manner insights into gene function can be gained as well
as into the function of specific cell types. In gene knockouts
often a phenotype is severe or affects multiple cell types so that
it is hard to tell the role of a gene in a given cell type which
may be important in developing therapies. As well as in
developmental biology such methods may also be important in the
study of the immune system as it involves multiple lineages and
cell types. It may also be possible to eliminate a specific lineage
or cell type by disrupting an essential gene only in that cell type
or lineage. Again this may be important in animal models, screening
and target validation as well as studying the function of the
lineage or cell type eliminated.
[0084] Transcriptional Termination
[0085] A transcriptional termination element is included in the
polynucleotide of the invention. The transcriptional terminator is
downstream of the region encoding the siRNA and is preferably
immediately downstream of the encoding region or separated by a
minimal distance.
[0086] Typically the termination element will comprise a series of
consecutive thymidines and in particular five consecutive thymidine
residues in the sense strand of the vector. The advantage of such a
transcriptional terminator is that the transcript initiated by the
preferred promoter of the invention is normally cleaved after the
second uricil to give rise to a transcript ending with two
consecutive uridines. These uridines can form one of the 3'
overhangs in the siRNA necessary for optimal activity. The cleavage
site and hence the overhang generated may vary depending on the
precise nature of the type 3 RNA polymerase promoter used, some
will lead to the generation of overhangs of two, three, four or
five uridines and the particular system will be chosen to give rise
to the overhang of choice, which will typically be two uridine
residues.
[0087] Region Encoding the siRNA
[0088] The polynucleotides of the invention comprise a region
encoding a siRNA. By siRNA it is meant a short double stranded RNA
molecule which comprises a double stranded region which is
identical in sequence to a target gene. The siRNA is capable of
silencing or inhibiting the specific target gene.
[0089] The inhibitory effect of the siRNA of the invention is
mediated by the double stranded region of the molecule. It is the
double stranded region which is responsible for the specificity of
the inhibition and the mechanism by which the siRNA acts. The
formation of a complex with a nuclease and subsequent strand
exchange of one of the strands of the siRNA with the target RNA
transcript all subsequent cleavage of the transcript all involve a
double stranded region.
[0090] Typically, the dsRNA region of the siRNA has overhangs at
one or preferably both of its 3' termini, these overhangs are
preferably only a few nucleotides in length and in particular are
one or two nucleotides in length and preferably are two nucleotides
in length. Although less preferred, the siRNA may be blunt ended or
have single nucleotide 5' overhangs at one or both 5' termini.
[0091] In situations where the 3'overhangs are dinucleotides, then
the preferred 3' overhangs are derived from the first two
nucleotides of the loop, being preferably UU or UG, and from the
last two nucleotides in the transcript which are invariably UU. In
a particularly preferred embodiment of the invention, one or
preferably both of the overhangs are UU.
[0092] Typically, the double stranded region which is identical in
sequence to the target is generated from a stem looped single
stranded precursor. The precursor comprises a region identical to a
region of the sense strand of the target gene and a second region
which is the complement of the first and hence which corresponds to
the antisense strand of the target gene. The two complementary
regions are usually separated by a short spacer region such that
when the two complementary regions hybridise a stem loop or hairpin
structure is formed with the spacer forming the loop.
[0093] Typically, the region immediately 3' of the first
complementary region comprise two consecutive uridine residues and
the loop structure can be cleaved. The cleavage typically occurs 3'
to the two uridine residues and just before the region
complementary to the first. This results in the generation of a
siRNA with a dsRNA region identical to the target and with the
dinucleotide 3' overhangs necessary for activity. The two
nucleotides which give rise to the 3'overhang may be any of the
preferred dinucleotides mentioned above. Typically, the cleavage is
carried out by an endogenous enzyme and in particular by a homolog
of dicer. Alternatively, the construct may also encode such an
enzyme.
[0094] Typically, the region of sequence identity to the target
gene is from eighteen to thirty nucleotides in length, preferably
from nineteen to twenty-three nucleotides in length, even more
preferably is 21 or 22 nucleotides in length, and still more
preferably the region is 21 nucleotides in length. Preferably the
region of sequence identity does not exceed 30 bases. The loop of
the stem structure may be any size above 6 nucleotides. Typically,
the loop may be from 6 to 100 nucleotides in length, preferably it
is from 7 to 50 nucleotides in length, more preferably is from 9 to
20 nucleotides in length. In an especially preferred embodiment of
the invention the loop is 9 nucleotides in length. The loop, and
hence the region encoding, may include various elements such as a
regulatory elements which influence transcription or elements which
influence RNA stability.
[0095] As the polynucleotide of the invention generates a siRNA
from a single RNA precursor with a stem loop structure this is
preferable to many methods in the art for generating siRNAs where
complementary single stranded RNAs are annealed and then the double
stranded siRNA has to be purified from unannealed single stranded
RNA to ensure optimal performance. It is also more efficient than
the use of plasmids comprising opposing promoters transcribing
through the same region to produce sense and antisense transcripts
which again have to be annealed.
[0096] As the invention uses a polynucleotide molecule to express
the siRNA rather than transfecting or microinjecting the siRNA
itself, this also ensures longer term expression of the siRNA and
hence inhibition of the target gene. In addition, the delivery of a
DNA molecule such as polynucleotide to a target cell is
considerably easier and less time-consuming than the generation of
a siRNA and its introduction to the target cell.
[0097] Whilst not being wished to be constrained to a particular
mechanism it is believed that the siRNA effectively acts as a guide
RNA in a sequence specific RNA degradation process. The siRNA is
thought to form a complex with a nuclease followed by exchange of
one of the strands in the siRNA by the equivalent strand of the
transcript of the endogenous gene to be targeted. This means that
one of the strands of the siRNA is released and replaced by the
region of sequence identity in the target RNA. The strand exchange
is followed by cleavage of the transcript, probably at each end of
the duplex region.
[0098] The cleavage products which are separate from the duplex
region are rapidly degraded as they lack either a stabilising cap
or poly (A) tail. This cleavage therefore prevents expression of
the targeted transcript, but also regenerates the initial complex
of a siRNA and nuclease. This means that the regenerated complex
can again inactivate another target transcript and so on. The
mechanism of action means there does not necessarily have to be an
excess in the initial amount of siRNA to be expressed in comparison
to the target transcript.
[0099] Preferably the region of the target gene which is also
present in the siRNA is an exonic region. Typically the region is
towards the 5' end of the targeted transcript. In some embodiments
of the invention several siRNAs are expressed targeting different
regions of the same gene to help ensure maximal inhibition. The
different siRNAs will preferably be expressed as separate
transcripts, but may be encoded on the same construct. Constructs
are also provided which are capable of inhibiting multiple genes by
expressing siRNAs specific for each gene. Alternatively, multiple
constructs may be used, each of which expresses one or more siRNA
specific for a particular gene.
[0100] Embodiments of the invention allowing for the inhibition of
multiple genes may be used for inhibiting several genes in the same
pathway or redundant family members. This may be important in
disease models, target validation, drug discovery and the other
applications of the invention. The inhibition of multiple genes may
allow multifactorial disorders to be modelled.
[0101] Often when one gene is inhibited a second gene is able to
compensate for the first either totally or at least to some extent.
By inhibiting the compensatory gene or genes as well this can be
used to produce cells or organisms totally lacking a particular
property or function. For example, all of the kinases capable of
phosphorylating a particular substrate or class of substrate may be
eliminated or embryonic development can be altered.
[0102] In situations where several genes in a pathway are
inhibited, this may ensure total elimination of the pathway or
allow the pathway to be engineered to produce a particular
phenotype such as to produce a particular substance such as a
desirable metabolite, in excess. Pathways often have feedback
mechanisms controlling them and some of these may be eliminated
using the methods of the invention.
[0103] In some cases, the same siRNA produced may be able to target
several genes. Such siRNAs will typically be specific for a
sequence present in two or more genes such as an evolutionary
conserved sequence in a gene family. Again, this means that two or
more genes capable of functionally compensating for each other may
be inhibited, but also that a particular gene class may be
inhibited. In some embodiments, the siRNA produced may chosen to be
able to inhibit homologous genes in different species because of
sequence identity or homology between the genes in the two species.
Such embodiments may, for example, be useful where the siRNA
inhibits a gene of a pathogen such as a viral gene and is capable
of inhibiting that gene in several species or strains of viruses
because of sequence conservation.
[0104] In some embodiments it may be desired to inhibit two or more
transgenes, for example in a tissue specific manner, so that they
are only active when chosen. In such embodiments, the transgenes
may be tagged with a specific sequence present in all of them
allowing for them all to be inhibited with a single siRNA.
[0105] In some embodiments of the invention, such as to ensure a
particular secondary structure in the transcript or siRNA, it may
be that the construct or transcript does not have any dinucleotide,
trinucleotide, tetranucleotide, or hexanucleotide repeats with more
than a certain number of repeats of the dinucleotide,
trinucleotide, tetranucleotide or hexanucleotide, such as having
less than five, preferably less than ten, more preferably less than
fifteen, even more preferably less than twenty such repeats, still
more preferably less than twenty five repeats of the dinucleotide,
trinucleotide, tetranucleotide or hexnucleotide. In some
embodiments of the invention, the limitation on the number of
repeats may apply specifically to the number of repeats in the loop
of the stem loop and any of the limits mentioned above may apply
specifically to the loop although the limitation may also be on the
number of repeats in the stem or alternatively on any regions
outside the hairpin such as at single stranded regions outside the
stem loop. It may also be desired in some cases that these
limitations apply to a specific dinucleotide such as GC or a
specific tetranucleotide such as AGCT or a specific hexanucleotide
such as GAATTC.
[0106] Polynucleotides & Vectors
[0107] The polynucleotides of the invention may be provided as
simple polynucleotides or alternatively in the form of vectors.
Preferably, they are provided in the form of vectors such as a
plasmid. Such vectors may be shuttle vectors such that they are
capable of being reproduced in large amounts in prokaryotic or
eukaryotic bacterial systems and then introduced into the target
cells.
[0108] Many suitable vectors are known in the art. These include
without limitation plasmid vectors, such as pBSK, pBR322, pUC
vectors, vectors that contain markers that can be selected in
mammalian cells, such as pcDNA3.1, episomally replicating vectors,
such as the pREP series of vectors, retroviral vectors, such as the
pBABE vector series, adenovirus-associated vectors or adenoviral
vectors. In particular, the preferred vector is pBSK
(Bluescript).
[0109] Such vectors may include various selection markers and/or
reporter genes. These may be used for selection in the bacterial
system the plasmid are grown in, but also for selection of
transfected and in particular stably transfected cell lines.
Examples of reporter genes which may be employed to identify
transfected cell lines include alkaline phosphatase (AP), beta
galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol
acetyltransferase (CAT), green fluorescent protein (GFP),
horseradish peroxidase (HRP), and luciferase (Luc). Possible
antibiotic selectable markers include those that confer resistance
to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,
kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin,
and tetracyclin. The construct of the invention transcribed to
generate the siRNA may be double or single stranded nucleic acid,
especially preferred is the situation where the construct is double
stranded.
[0110] The vector of the invention may be one which is capable of
integrating into the genome of the cell. Possible viruses which may
typically be used to integrate the constructs include retroviral
vectors, such as the pBABE vectors, lentiviral vectors,
Adeno-associated virus (AAV) vectors. However, most plasmids can
integrate at some frequency and hence may be used to generate
integrants. Alternatively the vector may be one which is capable of
replicating as an extrachromosomal element such as an artificial
chromosome or an Epstein Barr based virus.
[0111] The vector used to introduce the polynucleotide of the
invention into target cells may be a viral vector such as an
adenoviral vector, retroviral viral vector, reovirus vector or
lentivirus vector. Retroviral vectors are particularly useful for
embodiments where it is desired to integrate the vector into the
host genome. Various viral vectors, and in particular retroviral
and adenoviral vectors are known in the art and any of these may be
employed.
[0112] The constructs of the invention may be introduced into the
target cell or organism using a variety of methods. Where the
polynucleotide is introduced into a cell in vitro conventional
techniques such as by transfection, liposomes or viruses may be
employed. Typically electroporation may be employed.
[0113] Electroporation may also be used to introduce the constructs
into embryos.
[0114] In the case of organisms any conventional method of
introducing nucleic acids may be employed. A viral construct
packaged into a viral particle may be employed. For example viruses
such as adeno associated virus, lentivirus, reovirus or a
retrovirus may be used. Lipid-mediated carrier transport such as
liposomes may be used. Physical means such as bombardment with
particles comprising the nucleic acid may be used. The method of
delivery may mean that the construct is delivered to a particular
location such as an organ or a diseased or inflamed sited. In some
situations, the construct will be delivered into the blood, lymph,
or cerobrospinal fluid.
[0115] The polynucleotides of the invention also includes
transcripts and derivatives generated by transcription of the
constructs of the invention. In particular, the molecules will
comprise the stem loop structure prior to cleavage. The transcript
will include the double stranded region responsible for the
specificity of the resulting siRNA. Preferably, this region will be
specific for a human or viral gene, more preferably the region will
be specific for a target gene present in the genome of the target
cell, even more preferably the target gene will be an endogenous
gene present in the host cell genome, but may be a transgene or
viral gene integrated into the host genome. Therefore the target
gene of the siRNA molecule will be present in a host chromosome,
but may be on an episome or even a plasmid or extrachromosomal
element or a viral genome. The transcripts and derivatives may have
any of the characteristics or properties specified herein such as
size of stem loop, or overhangs etc. Although not a preferred
embodiment of the invention, also envisaged are situations where
the constructs of the invention are used to generate siRNAs in one
system, such as any of the cells mentioned herein, and then
transferred into another system to inhibit or modulate a target
system.
[0116] Target Genes
[0117] The target gene may be any gene of which it is desired to
inhibit or modulate the function of. The purpose of the inhibition
may be therapeutic or to study the function of a particular gene.
The inhibition of the gene may be to alter the phenotype of a cell
or organism in some desired way such as to improve the
characteristics of a commercially reared animal. Typically, the
target gene will be a eukaryotic gene, but alternatively the target
gene may be prokaryotic such as a viral gene being expressed in a
eukaryotic host cell. The target gene may encode a polypeptide or
alternatively a structural or enzymatic RNA. However, preferably
the target gene encodes a polypeptide.
[0118] The target gene may be a developmentally important gene, it
may encode a cytokine, lymphokine, a growth or differentiation
factor, a neurotransmitter, an oncogene, a tumour suppressor gene,
a membrane channel or component thereof. The gene may encode a
receptor and in particular one for the gene products of any of the
genes mentioned herein. The target gene may be one involved in
apoptosis. Typically, the target gene will be one associated with a
disease or disorder and the methods of the invention may be used to
treat, prevent, or ameliorate that disease or disorder.
[0119] The system may be used to treat, prevent or ameliorate
cancers. For example, the target gene may be an oncogene, tumour
suppressor gene, or gene involved in the control of the cell cycle.
Cancers which may be treated include solid tumors and leukemias
(for example B cell, mixed cell, null cell, T cell, T-cell chronic,
HTLV-II-associated, lymphocytic acute, lymphocytic chronic, mast
cell, and myeloid leukemias, melanoma, fibrosarcoma, osteosarcoma,
neuroblastoma, neurofibroma, sarcoma (for example Ewing,
experimental, Kaposi, and mast cell sarcomas). The cancer may be
one of the bone, breast, digestive system, colorectal, liver,
pancreatic, pituitary, testicular, central nervous system, lung,
urogenital system or prostate. The tumour may be benign or
malignant, typically it will be malignant. The tumour may be a
primary or secondary tumour and may be metastatic. The medicaments
of the invention may be administered on their own or in combination
with other anti-cancer treatments such as in conjunction with
chemotherapy or radiotherapy. The target gene may be one of a
pathogen or host gene responsible for entry of the pathogen into
its host, its subsequent replication or other functions such as
integration of the pathogen's genome into the hosts, establishment
or spread of an infection in the host, or assembly of the next
generation of pathogen. The inhibition of the gene may be used
prophylactically (i.e., prevention) or to decrease risk of
infection, as well as to reduce the frequency or severity of
symptoms associated with infection.
[0120] In some situations disorders are caused by the elevated or
inappropriate expression of a particular gene. For example in
inflammatory disorders or autoimmune disorders inappropriate
expression of a particular gene may play a part in the pathogenesis
of the disorder. In conditions such as arthritis, emphysema, adult
respiratory distress syndrome and the like expression of
inflammatory mediators, receptors for such mediators, adhesion
molecules, and bactericidal activities such as proteases or the
respiratory burst may play an important part in the tissue damage
occurring. By employing the methods and constructs of the present
invention to inhibit genes such as these, and in particular at the
particular site of inflammation, these conditions may be prevented,
treated or ameliorated. In such embodiments, methods which allow
the inhibition to be confined to a particular cell type are
particularly preferred.
[0121] The target gene may be present in a host cell chromosome or
may alternatively be an episomal element or present associated with
a pathogenic structure present in the cell such as a viral protein.
The target gene may be an endogenous gene or a transgene.
Typically, the target gene is a mammalian gene or alternatively a
viral gene. In embodiments where the target gene is a viral gene it
may be integrated into the host chromosome or present as a non
integrated element. The target gene may be a gene on a viral
construct or some other vector introduced into a cell.
[0122] In many embodiments the target gene is not a reporter gene
or a selectable marker although such target genes are also
envisioned as possible target genes. Examples of such reporter and
selectable markers include any of those mentioned herein and in
particular beta galactosidase (LacZ), beta glucoronidase (GUS),
chloramphenicol acetyltransferase (CAT), green fluorescent protein
(GFP), horseradish peroxidase (HRP), or luciferase (Luc).
[0123] Allele Specific Inhibition
[0124] The polynucleotides of the invention may be used to inhibit
expression of a specific allele, whilst allowing normal expression
of the other allele. In such embodiments the two alleles of the
gene will have some difference in sequence which will allow them to
be discriminated between by the siRNA.
[0125] Many disorders result from the mutation of one allele whilst
the other allele is normal. These include autosomal dominant
conditions as well as some cancers such as where mutation of one of
the two copies of a proto-oncogene results in the generation of an
oncogene and hence cancer or puts the individual one step closer to
developing cancer. By specifically blocking expression of the
mutated allele this may allow treatment of the disorder as the
remaining wild type allele in the cell may be able to render the
cell normal or at least regress the cancer, or ameliorate the
condition.
[0126] In such embodiments the region of the polynucleotide of the
invention identical to the target sequence will be identical to the
target allele, but different in sequence to the other allele. Thus,
for example, the polynucleotide may include a nucleotide
substitution, deletion, insertion or duplication which allows the
siRNA generated to discriminate between the two alleles. The siRNA
may target an allele generated by chromosomal translocation such as
in the case of Burkitt's Lymphoma or Philadelphia chromosome but
neither of the wild type alleles of the genes involved in the
fusion.
[0127] Typically, the sequence difference will be the mutation
responsible for the disorder in question. The mutation may be one
which is responsible for converting a proto oncogene into an
oncogene. For example the mutation may be one in an oncogene such
as ras, jun, myc, src, sis, fos, bcl-1 or 2, or abl. However, in
some cases the sequence difference may not be the mutation
responsible for the disorder, but instead may be a polymorphism
allowing the two alleles to be discriminated between. This may mean
that the specific mutation associated with the disease may not have
to be identified in each individual to be treated as a polymorphism
may be more convenient to genotype for. In some conditions, such as
those associated with the expansion of a trinucleotide repeat, it
may be difficult to generate a siRNA capable of specifically
recognising the mutation as the only difference is a duplication or
expansion of a repeat in one allele. It may be easier to generate a
siRNA specific for a polymorphism within the gene rather than the
mutation in question.
[0128] Typically, in the siRNA the sequence variation which allows
discrimination between two alleles might be located at or near the
centre of the double stranded region, such as from five to ten
bases into the double stranded region, preferably from seven to ten
bases and even more preferably will be nine or ten nucleotides into
the duplexed region. In situations where the mutation is not at the
centre of the duplex region, it will preferably be located between
the 3' end and the middle of the antisense strand of the siRNA. In
some embodiments the mutation be close to the end of the double
stranded region such as two, three or four nucleotides away.
[0129] In some situations allele specific siRNAs of the invention
may be used where it is desired to inhibit both endogenous alleles
of a gene whilst allowing expression of a transgenic allele. For
example, in many cases where a knockout is generated transgenic
alleles of the mutated gene are introduced to determine whether the
transgene can rescue the phenotype associated with the knock out.
This can allow functional analysis of a gene. Therefore, a
polynucleotide of the invention may be employed which generates a
siRNA capable of inhibiting the expression of both copies of a gene
but not of a transgenic allele. The discrimination may be on the
basis of a specific polymorphism introduced into the transgenic
allele. Preferably, such a polymorphism does not involve an amino
acid change or only results in a conservative amino acid
substitution. Such methods may also be employed in therapies where
it is desired to inhibit the expression of both alleles of a target
gene and then express a particular transgenic allele. In some
situations, where it is desired to inhibit both copies of an
endogenous gene, the two alleles will each have a specific mutation
or polymorphism so that a separate siRNA can be employed to inhibit
each allele.
[0130] The system of the invention may be used to selectively
inhibit the expression of particular splice variants. For example,
the polynucleotide of the invention may produce a siRNA which
targets a particular splice variant which contains an exon it has
sequence identity to, but leave intact splice variants lacking that
exon.
[0131] Target Cells & Organisms
[0132] The system of the invention may be employed to inhibit gene
expression in a variety of cells and organisms. The system may also
be used to inhibit the expression or viral genes in their host
cells. Typically, the system is used to inhibit expression in
eukaryotic cells and organisms and in particular in mammalian cells
or organisms.
[0133] The target cell or organism may any organism in which an RNA
polymerase m promoter is capable of being expressed in. The
organism will usually be eukaryotic, and may be inverterbrate or
verterbrate, but is preferably a verterbrate. Preferably, the
target cell or organism is mammalian in origin such as of rat,
mouse, cow, pig, sheep, or primate origin. In a particularly
preferred embodiment of the invention the cell or organism is
human. The target may be a virus and in particular a virus when it
is present in a host cell.
[0134] The cell in which the polynucleotide or vector of the
invention may be introduced into or the target gene is expressed in
may be from the germ line or somatic cells, totipotent or
pluripotent, dividing or non-dividing, immortalized or transformed.
The cell may be a multipotent cell or a differentiated cell.
Preferred cells include stem cells such as haematopoietic stem
cells. Differentiated cell types which may be targeted include
without limitation adipocytes, fibroblasts, myocytes,
cardiomyocytes, endothelium, neurons, glia, blood cells,
megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils,
basophils, mast cells, leukocytes, natural killer cells, dendritic
cells, keratinocytes, chondrocytes, osteoblasts, osteoclasts,
hepatocytes, and cells of the endocrine or exocrine glands. The
cells may be those of an established cell or freshly isolated
cells. The target cells may be transformed, in particular they may
be cancerous and especially malignant cells or cell lines. The
cancer may be any of those mentioned herein. Alternatively the
target cells may be those infected with a particular pathogen. The
target gene may be specifically inhibited in the target cell as
opposed to other lineages.
[0135] The nucleotide or vector may be delivered ex vivo with the
target cell being recovered from the subject, the polynucleotide
introduced, and the cells then returned to the subject. Optionally,
various selection stages or assessments may be carried out to
select and identify clones or cells where the vector has integrated
and the target gene is inhibited. Alternatively, the polynucleotide
may be introduced into multipotent cells, the cells differentiated
into the desired cell type and then introduced into the subject to
be treated. Again, optional stages of selection and
characterisation may be carried out. Such embodiments are
especially preferred for disorders and situations where it is not
necessary to inhibit the target gene in all of the particular cells
type, and inhibiting expression in a proportion will suffice. Such
embodiments may also be used in target validation and drug
identification.
[0136] In some situations it may be desired to introduce the vector
into a multipotent cell and then differentiate it into a number of
different cell types to allow screening in several different cell
types. In embodiments where the construct is a multipotent cell
this may also be used to study the differentiation and
differentiation potential of that cell when the target gene is
inhibited. This may elucidation of whether a gene plays a role in
the differentiation process and if so what role it plays. It may
also be used to identify agents or treatments which are capable of
influencing the differentiation in a preferable way when the target
gene is inhibited.
[0137] The polynucleotides or constructs of the invention may be
introduced into the target cell or organism via a variety of
mechanisms. Where the polynucleotide is introduced into a cell in
vitro conventional techniques such as by transfection, liposomes or
viruses may be employed. Typically electroporation may be
employed.
[0138] In the case of organisms any conventional method of
introducing nucleic acids may be employed. A viral construct
packaged into a viral particle may be employed. For example viruses
such as adeno associated virus or a retrovirus may be used.
Lipid-mediated carrier transport such as liposomes may be used.
Physical means such as bombardment with particles comprising the
nucleic acid may be used. The polynucleotide may be introduced into
the vascular or extravascular circulation, the blood or lymph
system, or the cerebrospinal fluid.
[0139] Measurement of Gene Inhibition
[0140] In many embodiments of the invention it will be desired to
check the efficacy of the siRNAs in blocking the expression of the
target gene. The inhibition of the gene may be measured in a
variety ways, typically at the RNA, protein or phenotypic
level.
[0141] Inhibition may be confirmed using biochemical techniques
such as Northern blotting, nuclease protection, 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).
[0142] Where the target gene is a mutated allele and the object of
the inhibition is to specifically inhibit the mutated allele whilst
allowing normal expression of the wild type allele methods may be
used to assess inhibition which can discriminate between expression
of the wild type allele and the mutated allele such as by single
stranded conformational polymorphism, denaturing gel
electrophoresis, allele specific PCR or antibodies capable of
discriminating between the wild type and mutated proteins.
[0143] Inhibition in a cell line or whole organism, may be measured
by using a reporter or drug resistance gene whose protein product
is easily assayed. Such reporter genes and selection markers
include any of those mentioned herein. Inhibition may also be
measured at the phenotypic level. For example, the appearance of a
phenotype similar to that associated with disruption of the
targeted gene may be looked for. Where the purpose of the siRNA is
to block expression of a gene associated with a disease whether or
not the disease is prevented, ameliorated or treatable using the
siRNA may be measured. Where the purpose of the siRNA is to treat
an infectious disease any reduction in viral or bacterial load may
be assessed or alternatively the presence, absence or severity of
symptoms associated with the disorder may be measured.
[0144] Integration
[0145] In a preferred embodiment of the invention the
polynucleotide or vector of the invention is integrated into the
genome of the target cell. This helps ensure that the expression of
the siRNA is permanent rather than the transient expression
associated with non-integrating vectors.
[0146] Typically, the polynucleotide or vector of the invention
will be integrated into a chromosome of the host cell, although
alternatively it may introduced in the form of an artificial
chromosome such as a human artificial chromosome or some other
episomal element capable of self replication and maintenance in the
host cell. Preferably, however the vector or polynucleotide is
integrated into a host chromosome.
[0147] Particular vectors are known in the art which integrate into
the genome of a cell more frequently. For example, the vector used
to introduce the polynucleotide of the invention may be a
retrovirus or retrovirus based vector capable of integrating into
the host genome. Preferred vectors for such embodiments include
retroviral vectors, such as the pBABE vectors, lentiviral vectors,
Adeno-associated virus (AAV) vectors, retroviral, lentiviral,
adeno-associated and adenoviral vectors. Plasmid vectors such as
pcDNA 3.1 integrate as well, albeit at lower frequency.
[0148] Although episomal vectors may not integrate into the genome
at a high level integrants may still be obtained as a low level of
integration normally occurs when such vectors are employed. Almost
all vectors will integrate into host chromosomes at some level,
even if they do so infrequently, as such integrants can probably be
generated for any vector. Various methods are known in the art for
promoting integration such as irradiation and such methods may be
employed.
[0149] Preferably, the polynucleotide is integrated into the host
genome by random integration. Alternatively, the vector or
polynucleotide may be targeted to a specific location in the host
cell by methods known in the art such as a site specific
recombinase or integrase to integrate the polynucleotide into a
specific site. This may allow the vector to be targeted into a
known region with particular characteristics such as being
permissive for expression or to avoid integration in a gene of the
host cell.
[0150] After introduction of the target cell of the polynucleotide
into the target cell various selection and/or screening techniques
may be employed to identify clones in which the vector has
integrated and to further characterise them. By employing a
selectable marker this may allow selection of the clones in which
the vector has integrated such as by looking for expression of a
reporter gene such as green flourescent protein (GFP) or by
antibiotic selection such as with G418. FACS sorting may be
employed to collect cells expressing a particular marker gene such
as GFP.
[0151] Typically, after transfection the cells will be grown for a
sufficient period of time such that transient expression will not
be the reason for drug resistance or reporter gene expression. For
example, the cells may be grown for more than a week, preferably
for ten days and more preferably for two weeks before selection and
characterisation.
[0152] The vector or polynucleotide may also include means by which
the selectable marker or reporter gene can be removed leaving the
region capable of expressing the siRNA present in the cell. For
example, the selectable marker may be flanked by recognition sites
for a site specific recombinase. The selected clone may be
transiently transfection with a plasmid capable of expressing the
recombinase and then the transfected cells plated and clones from
which the selectable marker has been excised selected or
identified.
[0153] Clones which have integrated the vector or polynucleotide of
the invention may be further characterised. For example, Southern
blotting or PCR may be carried out to check the plasmid has
integrated, determine the site of integration and copy number of
the integrated plasmid. The site of integration may be
characterised to ensure that it is not an endogenous gene or other
important element. Northern blotting or other such techniques may
be carried out to determine whether the siRNA is being expressed
and to check whether the target gene is being inhibited. Any of the
techniques mentioned herein for measuring the inhibition of the
target gene may be employed and checks may be made to ensure that
the inhibition is specific.
[0154] Transgenic Organisms
[0155] The polynucleotides of the invention may be used to generate
non-human transgenic organisms in which the expression of a target
gene is inhibited or reduced. The transgenic animals will
preferably have a polynucleotide or vector of the invention
integrated into its genome and hence can transmit the integrated
polynucleotide or vector to its progeny. However, the invention
also encompasses normal animals into which cells comprising the
polynucleotide or vector of the invention are transplanted or
transferred into. Such animals may provide a model for a particular
therapies involving ex vivo treatments.
[0156] The transgenic animals may be generated by any of the
techniques known in the art for introducing transgenes into animal
and in particular by pronuclear injection where the vector or
polynucleotide is microinjected into the pronucleus of an oocyte.
Transgenic organisms can also be generated by introducing nucleic
acid constructs into early embryos such as by electroporation and
such methods may be employed to generate the transgenic organisms
of the invention. The non-human transgenic animal may be a
transgenic rodent, such as a mouse or rat, a primate, or a
commercially important animal such as a sheep, cow, or pig.
Preferably the organism is a mouse or rat.
[0157] The transgenic organisms of the invention may be used as
animal models. Alternatively, the transgenic organism may be a
commercially raised animal and the introduction of a polynucleotide
or vector of the invention means the transgenic organism has a
desirable phenotype such as disease or pathogen resistance.
[0158] In addition, to comprising a polynucleotide or vector of the
invention the transgenic animal may also comprise additional
transgenes. For example, the transgenic animal may comprise a
modified allele of the target gene and the siRNA be specific for
the endogenous alleles of the gene. This may allow an animal model
to be developed to assess the functionality of the modified allele
introduced as a transgene.
[0159] Disease Models
[0160] The methods of the invention allow the generation of models
of various disease conditions and disorders. For example, they may
be used to generate a cell line or an organism in which a specific
gene is inhibited. They may also be used to generate models in
which both copies of a chosen endogenous gene are inhibited and a
mutated allele of the endogenous gene is expressed so modeling
conditions such as an autosomal dominant condition or cancer.
[0161] Models produced using the methods of the invention may be
used to assess the therapeutic efficacy of test agents. The
prevention, relief or amelioration of the conditions or symptoms
associated with a disorder may be measured. The model may be an a
model of an infectious disease such as viral infection and the
assay may be used to assess whether infection can be prevented, the
load of the pathogen can be reduced, viral integration can be
prevented or other symptoms can be treated or ameliorated.
[0162] The model may be of the entire disease condition or may be
of part of, or a stage in, the condition such as, a step involved
in the underlying pathogenesis of the disorder. The model may be of
a particular cellular function thought important in the disorder
such as, for example, migration, chemotaxis, apoptosis,
degranulation, adhesion, phagocytosis or any of the cellular
functions mentioned herein.
[0163] A large number of genes have been implicated in, or are
known to cause, specific disorders and by modulating the expression
of these genes using the methods of the invention the same
disorders can be modeled in cells or organisms. Various knockout
and classically generated mutant models exist and equivalent models
may be generated by inhibiting the expression of the gene in
question. This may be particularly useful where the existing model
is only available for one species, strain or cell and it is desired
to rapidly generate a model in a different species, strain or cell
line by inhibiting the same gene or its homolog. The methods of the
invention may also allow multiple genes to be disrupted in the same
organism without having to undergo laborious and lengthy breeding
programs. This means that multifactorial disorders can be simply
and rapidly modeled.
[0164] One of the preferred uses of the model systems of the
invention is in screening and target validation. Thus the model
may, for example, be used to screen agents to identify those agents
which may be useful in treating or preventing the condition being
modeled. Promising agents from initial screens may be assessed and
characterised further, such as by studying them in more detail in
the same or other model systems of the invention. For example, the
initial screen may be cell based and may then be followed by
characterisation of promising candidate agents from the initial
screen in a transgenic organism of the invention.
[0165] As in the case of conventional knockouts, using the model
systems of the invention means that therapies can be tested and
evaluated before they are applied to the actual disease sufferers
and also provide the possibility of high throughput screening so
that a varying large number of candidate agents can be screened to
identify promising candidates for therapeutic use and further
assessment. The model systems of the invention, and in particular
the transgenic organisms, may also be used to develop, improve or
assess methodology in treating conditions such as improved surgical
methods.
[0166] The model system may be cell based and the particular cell
type important in the condition or affected in the condition may
typically be used. For example, immune cells may be used in models
of inflammatory disorders or for cancers the particular cell type
involved in the type of cancer may be used. Alternatively, other
cell types known to be suitable for the particular assay methods
being employed may be used, rather than those cell types affected
in the specific disorder being modeled. Any of the cell types
mentioned herein may be used in disease modeling. The cell type may
be a multipotent cell and be differentiated into different types of
cell to allow screening, target validation and the other
applications of the invention to be carried out on multiple
lineages including cells in the process of differentiation. The
model system may involve multiple different cell types. The
interactions of the cell types may, for example, be monitored.
[0167] The cells may be assessed in a variety of ways such as at
the biochemical, molecular level or functional level. This is
discussed further below. The cells may be treated with various
agents, or be exposed to specific conditions, which facilitate the
modeling of the disease condition. In essence, any of the factors
involved in a disorder such as those thought to be important in
triggering its onset or involved in its subsequent development may
be administered. Such agents may also be used in the animal models
of the invention. The substance may be, for example, the actual
substance involved in the condition or another substance capable of
having an equivalent effect. For example, the cells may be exposed
to agents that cause apoptosis, cell death, cell activation,
degranulation, transformation or other cellular functions such as
any of those mentioned herein. The cells may be exposed to a
particular allergens, immunogenic substances, or inflammatory
mediators such as those involved in a disorder.
[0168] The model system may be a non-human transgenic animal of the
invention. Alternatively, cells of the invention may be introduced
into normal animals or mutant animals. In many conditions, a
specific cell type or lineage may be implicated in the pathogenesis
and these may be introduced into an organism. For example, cells of
the immune system are implicated in various inflammatory disorders
and immune cells or their progenitors, in which a target gene has
been modulated using the method of the invention, may be introduced
into an animal. The recipient animal may lack the cell types being
transferred into it, for example it may have been irradiated in the
case of immune cells or may be an animal suffering from SCID or
some other immunodeficiency meaning that it lacks specific cell
types. The cells being introduced from the animal may originate
from that animal.
[0169] The generation of the model may also involve various stages
such as physical or chemical insult or surgical methods to
replicate or induce the disorder being modeled. For example, spinal
injury may be induced or liver damage induced using agents such as,
for example, carbon tetrachloride. Immune disorders may be induced
by, for example, exposure to specific antigens. In many cases the
agents known to lead to a disorder, or ones having an equivalent
effect, will be administered to induce or model the desired
condition. The models may involve infection with pathogens such as
viruses. Such methods apply to both cell based and animal models of
the invention.
[0170] As well as use in target validation, screening and the
development of various treatments the models of the invention may
be used to gain an insight into the pathogenesis of disorders and
into the genes involved. The models may be studied to determine how
the disease develops. They may be used to confirm the role of a
candidate gene in a disorder. The models may allow a better
understanding of the disease to be gained at the biochemical,
molecular, genetic, or cellular levels and hence may allow the
rational design of new therapies. Various mutated alleles of the
gene involved in the disorder may be tested to see if they can be
used to rescue or prevent a disease phenotype to analyse what are
the essential regions of a particular portion of a gene and what
the function of a particular region of a gene or its protein
product is. They may be used to demonstrate that a particular
portion of a gene has a given function such as enzymatic
activity.
[0171] The ability to model a human disease in an animal or a cell
means that various tests and assays not possible on samples from
human patients can be carried out helping to generate further
understanding of, and treatments for, the disorders. This may also
help save on the inconvenience for patients of having to repeatedly
provide samples and be important in cases where a condition is rare
in incidence and hence patient samples are not readily
available.
[0172] Screening & Target Validation
[0173] The invention provides for the use of a cell or animal model
of the invention to be used to screen candidate agents and identify
those that can prevent, treat or ameliorate the condition in
question. The model may also be used in target validation to
further characterise candidate agents thought to have potential
therapeutic value in a condition or to confirm that a candidate
gene is involved in a disorder.
[0174] Preferably, the assays will be high throughput assays.
Assays which can screen large numbers of test agents may typically
be employed such as various multiwell plate based assays. These may
involve all, or the majority, of the stages of the invention being
carried out in the multi-well plate or may involve individual
stages of the assay being carried out in the multiwell plate.
[0175] The assay may involve growing or culturing cells of the
invention in a multi-well plate, contacting them with a test agent,
and then looking for such particular phenotype. In some cases the
phenotype may be assessed from observing the cells or by employing
an assay systems that uses the same plate. The assay may involve
growing cells in one multiwell plate, and then removing culture
supernatant or cells to be analysed, typically in another multiwell
plate. The assay may involve analysing multiple test samples from
animals of the invention in a multiwell plate. Preferably, the
screening methods employed may be partially or totally automated.
Multiwell plate formats are particularly well suited to automation.
Various ways to streamline or screen multiple samples are known in
the art and these may be employed.
[0176] Stages such as the analysis of phenotype may also be
automated or performed by an operative. The results obtained may be
analysed by computer. Techniques, employing various labels and
colour changes may be employed and are often suitable to
automation. The label may, for example be enzymatic, radioactive,
or fluorescent. Techniques such as PCR, antibody based assays and
ELISA may be used as again these may allow multiple samples to be
screened and give the option of automation. Where the change being
monitored is at the genetic level, such as expression of a
transcript or a protein, various assays such as microarrays, chips
and membrane based assays may be used. FACS may also be used.
[0177] Test agents may be used in an initial screen of, for
example, 10 agents per reaction, and the agents of these batches
which show the desired phenotype tested individually. Test agents
may, for example, be used at a concentration of from 1 nM to 1000
.mu.M, preferably from 1 .mu.M to 100 .mu.M, more preferably from 1
.mu.M to 10 .mu.M. The activity of a test agent may be compared to
the activity shown by a molecule used to treat the condition in
question
[0178] The assay may be such that the desired agent gives rise to
the expression of a reporter gene or of a selectable marker. This
may also facilitate the screening of large numbers of test agents
and make it easier to identify the desired clones. Any of the
selectable markers and reported genes mentioned herein may be used
in such embodiments.
[0179] Suitable test agents which can be tested include
combinatorial libraries, defined chemical entities and compounds,
peptide and peptide mimetics, oligonucleotides and natural product
libraries, such as display (e.g. phage display libraries) and
antibody products. Typically, organic molecules will be screened,
preferably small organic molecules which have a molecular weight of
from 50 to 2500 daltons. Candidate products can be biomolecules
including, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof. Candidate agents are obtained from a wide variety of
sources including libraries of synthetic or natural compounds.
Known pharmacological agents may be subjected to directed or random
chemical modifications, such as acylation, alkylation,
esterification, amidification, etc. to produce structural
analogs.
[0180] The agent may be a polynucleotide (single or double
stranded) typically with a length of at least 10 nucleotides, for
example at least 15, 20, 30 or more polynucleotides. The agent may
be molecule which is structurally related to polynucleotides that
comprises units (such as purines or pyrimidines) able to
participate in Watson-Crick base pairing. The agent may be a
polypeptide, typically with a length of at least 10 amino acids,
such as at least 20, 30, 50, 100 or more amino acids.
[0181] A number of mutated alleles of the gene being inhibited by
the siRNA may be introduced into a cell or animal of the invention
and the phenotype of the cell or organism. Alternatively, the
introduced nucleic acids may be different genes from that inhibited
by the siRNA. Various nucleic acid libraries may be screened to
identify a nucleic acid capable of producing the desired phenotype.
Various mutagenesis techniques may be used to generate the
libraries being screened such as to generate mutants from a given
sequence either in a directed or random way. A test gene may be a
candidate nucleic acid for gene therapy and various variants
assessed to identify the optimal sequences. Various delivery
methods for delivering a given nucleic acid to a cell may be
assessed. Target validation, gene therapy, and other therapeutic
applications may well require the administration of multiple genes
or nucleic acids. The expression of multiple genes may be
advantageous for the treatment of a variety of conditions and the
models can be generated where multiple nucleic acids are
delivered.
[0182] Knowledge about the condition being modeled in the screen
may be used to help select what agents are to be screened. For
example, the candidate agents for screening may be chosen by
rational design. Rational drug design (RDD) methods accelerate the
discovery process for useful pharmaceutical agents. RDD typically
involves the design and optimization of small, organic therapeutics
from the ideal case, where a protein structure is available. RDD
may employ techniques such as molecular graphics and simulation
technology. RDD may employ three dimensional searching of large
databases to identify small molecule fragments which can interact
with specific sites in a target molecule, bridging fragments with
the correct size and geometry, or framework structures which can
support functional groups at favorable orientations. A three
dimensional pharmacophore hypothesis or a quantitative
structure-activity model (QSAR) may be developed, that is converted
into a search query or a predictive formula to search a three
dimensional database for structures that fit the hypothesis within
a certain tolerance, or the QSAR model may be used to predict
activities on novel compounds. Cluster analysis and two dimensional
and three dimensional similarity search techniques to identify
potential new leads may be employed.
[0183] The ability to monitor the phenotype of a cell or organism
of the invention is important in the various screening and target
validation methods of the invention. Thus the ability of a test
agent to modulate the phenotype of a cell or organism, such as in
preventing a specific phenotype from developing, causing it to
develop, or causing it to regress to a more normal phenotype, may
be monitored to identify desirable agents or methods such as, for
example, for therapeutic or diagnostic use.
[0184] As used herein the term phenotype refers to the
characteristics of a cell or organism resulting from the
interaction between its genetic makeup and the environment. The
phenotype in question will typically be any manifestation of a
specific disorder or infection including any of those mentioned
herein. Alternatively, the particular phenotype may be some
desirable non-disease associated phenotype which it is wished to
obtain, such as an increase in the yield of a desirable product in
a particular cell type or organism.
[0185] The term phenotype is intended to include characteristics
such as ones at the biochemical, molecular, cellular, tissue,
organ, developmental, cognitive, or behavioural level. The
phenotype being assessed may be one resulting from injury, trauma,
or chemical or physical insult. The assessment may be at the
genetic level such as to see whether the expression of a particular
gene, other than that targeted by the siRNA, is modulated by
candidate agents. The activity of a receptor, signal transduction
protein, membrane channel or enzyme may be monitored. Particular
cellular functions such as, for example, migration, adhesion,
degranulation, phagocytosis, apoptosis, differentiation, and
chemotaxis may be monitored and any change observed. The
transformation of a cell or the acquisition of characteristics
associated with a cancer may be monitored as a possible phenotype.
For many genes the symptoms and associated phenotype of a
particular disorder or infectious disease are known as may be the
underlying pathogenesis of the disorder. This means that the
particular characteristic being studied may be chosen on the basis
of such knowledge. The phenotype may be one associated with any of
the diseases, disorders, infections, conditions or states mentioned
herein.
[0186] The assessment of phenotype may be performed on an animal
model. This may be done after an initial screen to identify
promising candidate agents in cell based assays or may be the
primary screen. The animal model may be one of an infectious
disease and characteristics such as viral load, infectivity,
prevention, amelioration or treatment of the infection may be
measured. Preferably the characteristic being measured will be one
of central importance to the disease and one whose prevention may
improve the condition of sufferers of the disease.
[0187] The sensitivity of an animal model of the invention to
developing tumours may be monitored. These may have arisen in the
animal or have been transplanted into it. The metastasis of tumours
from one site to another may be monitored. The early stages, before
a tumour is actually malignant or metastatic may be monitored.
Developmental disorders and in particular those of the embryo may
be monitored. In such cases embryos may be harvested from an animal
at various stages of development such as is appropriate. Techniques
such as embryo transfer may be used to return the embryos to a
pseudopregnant female may be carried out to monitor their
subsequent development.
[0188] Pain may be monitored using any suitable assay for
monitoring the behavioral response of an animal to pain stimuli.
Control responses may be determined by testing an animal prior to
administration of a candidate agent. Learning or cognitive ability
may be assessed using such methods as mazes. Aggression may be
monitored. The ability of a model organism to raise and care for
its young successfully may be measured.
[0189] In the screening and target validation methods of the
invention various controls such as cells or animals without
inhibition of the target gene, to which no agent has been
administered or a placebo has been given. Positive controls may
include existing modulators which it is desired to improve on.
[0190] Generally a test agent may be considered to influence a
phenotype if it inhibits or enhances the phenotype, for example
expression of a phenotype may be increased or decreased by at least
5%, for example by at least 10%, at least 15%, at least 20% or at
least 25%, preferably by at least 30%, for example at least 40% or
at least 50%, more preferably by at least 70%, for example, at
least 80% or at least 90% compared to controls. In a preferred
embodiment of the invention the test agent will be able to turn an
abnormal phenotype into a normal one or prevent the development of
an abnormal phenotype. The agent may reduce or eliminate a specific
symptom associated with a disease.
[0191] The methods of the invention may be used to confirm that a
candidate gene is actually involved in a particular condition,
phenotype or function. For example, the candidate gene may have
been identified on the basis of gene mapping to a particular area
containing several genes, due to its homology to a known gene (such
as a known disease gene) or using a functional based gene cloning
strategy. The gene may have been identified as a candidate as it is
one of those whose expression changes in a disorder.
[0192] Whether or not the candidate gene is actually involved in
the function in question may then be confirmed by inhibiting the
gene using the methods of the invention. The phenotype of the cell
or organism produced may then be studied such as, for example, by
any of the methods described herein or employing assays known in
the art for assessing such functions. The characteristic may, for
example, be one at the biochemical, molecular, genetic, cellular,
or organism level, it may be any of those mentioned herein. In the
case of cells the characteristic being studied may typically be at
the biochemical, molecular, genetic, or cellular levels.
[0193] In the case of organisms the characteristic may be at the
biochemical, molecular, genetic, or cellular levels or may be, for
example, at the organ or system level. The characteristic may be
behavioural or cognitive or it may be a symptom associated with a
disease. Whether or not the model generated mirrors the disease in
question will typically be studied.
[0194] In some cases the candidate gene may not be matched to a
particular condition. For example, the candidate gene may have
homology to a known disease, but whether it is actually implicated
in a disorder, and if so what disorder, may not be known. By using
the methods of the invention what function the gene plays and what,
if any, disorder it may play a part in may be elucidated. This may
the identification of specific genes playing a role in a condition.
In some cases the genes may be known, but not have been previously
been associated with such a disorder and this may provide new
therapeutic targets for that condition.
[0195] Libraries
[0196] The vectors of the invention can also be used to generate
large collections of siRNAs to perform genome-wide screens for
genes that act in biologically relevant pathways. Therefore
libraries of siRNAs can be generated using the invention. Genetic
"loss-of-function phenotype" screens using such libraries may yield
novel therapeutic targets that are candidates for drug development
or may be used to evaluate the contribution of a limited number of
candidate genes to a biological response.
[0197] Phenotypic genetic screens using cDNA expression libraries
have been very successful for selection of genes that act in a
dominant fashion to modulate cell behavior. The siRNA gene
libraries allow, for the first time, a genome-wide evaluation for
loss-of-function phenotypes in mammalian systems. This means that
the equivalent of a homozygote for a recessive mutation may be
generated.
[0198] Sequences to be inserted in the siRNA vector of the
invention can be selected in silico by screening the appropriate
databases for unique short nucleotide sequences, of the lengths
specified herein for the double stranded region of the siRNA of the
invention, such as typically 19mers, for every known gene and every
EST or a substantial proportion of these. Collections of unique
short nucleotide sequences may be synthesized as part of a longer
oligonucleotides, such that it will form the characteristic
stem-loop structure described herein, then expressed in the siRNA
vector, and will be inserted in the siRNA vector.
[0199] Such libraries may be based on human gene sequences for use
in human cell systems or of species such any of those mentioned
herein and in particular those of mammalian origin, or
alternatively pathogenic origin such as viral origin.
[0200] Libraries of siRNAs can be introduced into the appropriate
cell system and a response of the cells can be monitored. Any of
the assays mentioned herein may be used to monitor the cells. The
cells that show an altered response can be identified in various
ways, depending on the nature of the biological system, and the
siRNA that is expressed in the identified cell type can be
recovered by several strategies, including PCR-based amplification
of the specific siRNA insert using vector-specific primers.
[0201] Therapeutics
[0202] The various polynucleotides, vectors, cell lines and agents
identified using the screening methods of the invention may be used
in methods of treatment of the human or animal body by therapy or
diagnosis. They may be used to prevent, treat, ameliorate or
diagnose specific disease conditions or infections condition.
[0203] The disease conditions may be any of those associated with
the possible target genes mentioned herein. They may be any
condition involving a dominant mutation which is either inherited
or which results from a dominant mutation in the somatic or germ
line tissue of an organism. They may also be conditions which
result from the aberrant or inappropriate expression of a target
gene.
[0204] The condition may be a cancer, and in particular a malignant
cancer and especially one which is metastatic. The cancer may be
any of those mentioned herein. The condition may be an inflammatory
disorder or an autoimmune disorder. The condition may be a
developmental disorder. It may be an inherited autosomal dominant
condition. Infectious diseases may also be treated or prevented and
in particular viral diseases such as retroviral diseases and
especially HIV.
[0205] The polynucleotide, vector, cell, or agent of the invention
may be formulated with standard pharmaceutically acceptable
carriers and/or excipients as is routine in the pharmaceutical art.
For example, a suitable agent may be dissolved in physiological
saline or water for injections. The exact nature of a formulation
ill depend upon several factors including the particular agent of
the invention to be administered and the desired route of
administration. Suitable types of formulation are fully described
in Remington's Pharmaceutical Sciences, Mack Publishing Company,
Eastern Pennsylvania, 17.sup.th Ed. 1985, the disclosure of which
is included herein of its entirety by way of reference.
[0206] The therapeutic entity may be administered by enteral or
parenteral routes such as via oral, buccal, anal, pulmonary,
intravenous, intra-arterial, intramuscular, intraperitoneal,
topical or other appropriate administration routes.
[0207] A therapeutically effective dose of the therapeutic molecule
or agent of the invention is administered to a patient. The dose
may be determined according to various parameters, especially
according to the agent used; the age, weight and condition of the
patient to be treated; the route of administration; and the
required regimen. A physician will be able to determine the
required route of administration and dosage for any particular
patient. A typical daily dose is from about 0.1 to 50 mg per kg of
body weight, according to the activity of the specific modulator,
the age, weight and conditions of the subject to be treated, the
type and severity of the degeneration and the frequency and route
of administration. Preferably, daily dosage levels are from 5 mg to
2 g.
[0208] In the case of the nucleic acids of the invention these may
be administered by any available technique. For example, the
nucleic acid may be introduced by needle injection, preferably
intradermally, subcutaneously or intramuscularly. Alternatively,
the nucleic acid may be delivered directly across the skin using a
nucleic acid delivery device such as particle-mediated gene
delivery. The nucleic acid may be administered topically to the
skin, or to mucosal surfaces for example by intranasal, oral,
intravaginal or intrarectal administration.
[0209] Uptake of nucleic acid constructs may be enhanced by several
known transfection techniques, for example those including the use
of transfection agents. Examples of these agents includes cationic
agents, for example, calcium phosphate and DEAE-Dextran and
lipofectants, for example, lipofectam and transfectam. The dosage
of the nucleic acid to be administered can be altered. Typically
the nucleic acid is administered in the range of 1 pg to 1 mg,
preferably to 1 pg to 10 .mu.g nucleic acid for particle mediated
gene delivery and 10 .mu.g to 1 mg for other routes.
EXAMPLES
[0210] The following Examples further illustrate the present
invention.
Example 1
[0211] Introduction of synthetic short interfering RNAs (siRNAs)
into mammalian cells can significantly suppress expression of
specific genes. However, this reduction in gene expression is
transient. To overcome this limitation, an expression vector,
termed pSUPER was generated which directs the synthesis of
siRNA-like transcripts (pSUPER, suppression of endogenous RNA). The
pSUPER vector was made by digestion of the pBSKII+(Bluescript)
plasmid with EcoRI and BgLII and ligating to it the PCR product of
H1-RNA gene promoter.
[0212] The human HI RNA gene sequence available on the NCBI
database, accession number X16612, was used. The HI RNA gene
promoter sequences from nucleotide 146 of the genbank sequence (an
Eco RI restriction enzyme cleavage site) up to nucleotide 374 were
cloned to generate the pSUPER vector. The last three nucleotides of
the H1 RNA gene promoter in the vector are CCC, transcription
starts immediately downstream of this CCC sequence in the H1 RNA
gene. As such, the CCC sequence is a relevant part of the promoter
construct. The termination sequence is a stretch of 5 consecutive T
residues and was added by PCR downstream of the promoter in the
vector.
[0213] A schematic drawing of the basic pSUPER vector is depicted
in FIG. 1(a) The H1-RNA promoter is cloned in front of the gene
specific targeting sequence (typically 19 nucleotide of sequence
from the target transcript separated by a short spacer from the
reverse complement of the same sequence) and five thymidines (T5)
in the sense strand of the vector as termination signal. The basic
pSUPER construct was then modified to express a variety of stem
loop structures capable of giving rise to siRNAs.
[0214] Three pSUPER constructs, pSUPER-Cdh1 A, B and C were
generated which contain a 19 nucleotide region identical in
sequence to a portion of the Cdh1 gene. FIG. 1(b) depicts the
synthetic SiRNA used to target CDH1 generated from the constructs
and the predicted secondary structures of the three pSUPER-CDH1
transcripts from the tree constructs A, B and C. The constructs
were then transfected into MCF-7 cells using the protocol described
in Agami, & Bernards. Cell 102, 55-66 (2000) which gives a
transfection efficiency of more than 90%. 1 .mu.g from the
indicated DNA constructs and 1.5 .mu.g of SiRNA were transfected
into the cells. Sixty hours later whole cell extracts were
prepared, separated on 10% SDS-PAGE and immunoblotted to detect
CDH1 protein. An immunoblot with anti-Cyclin D1 antibody was used
as a control for protein loading. FIG. 1(c) shows the resulting
western blot. From left to right the lanes are loaded with cell
extracts from cells transfected with a control plasmid expressing
GFP, Cdh1-siRNA, the empty pSUPER construct, the three pSUPER
constructs capable of expressing the transcripts A, B and C and
finally empty pSUPER.
[0215] The results show that the pSUPER-Cdh1B construct capable of
expressing transcript B which has a stem loop structure where the
loop has is 9 nucleotides in length is capable of eliminating up to
90% of Cdh1 expression and achieves an equivalent level of
inhibition to the transfection of the synthetic siRNA itself. The
pSUPER-Cdh1A construct, where the resulting transcript has a loop
of seven nucleotides result in some inhibition of Cdh1 expression
whereas the pSUPER-Cdh1C construct where the loop is five
nucleotides is inactive. This emphasises the importance of the size
of the loop of the stem loop structure in generating siRNA.
[0216] Importantly neither the transfection of the synthetic CDH1
SiRNA, nor introduction of the SiRNA expression vectors, had any
detrimental effect on cell survival or cell cycle profile (data not
shown).
[0217] U2OS cells were also transfected as described above with
either the synthetic siRNA, empty pSUPER vector, the pSUPER-Cdh1B
construct. Total RNA was extracted 60 hours later. Thirty 1 g of
RNA was loaded on an 11% denaturing polyacrylamide gel, separated
and blotted as described in Lee et al., Cell 75, 843-54 (1993) with
a .sup.32P-labeled anti-sense 19 nt Cdh1 target oligonucleotide and
visualized by PhophorImager (4 hours exposure). The blots were also
probed with a sense strand. The control 5S-RNA band was detected
with EtBr staining as a control for RNA loading. The resulting blot
is shown in FIG. 1(d). The blot shows that the pSUPER-Cdh1B
construct results in the generation of an RNA molecule similar in
size to the siRNA molecule itself implying that the hairpin loop is
cleave to give rise to siRNA molecules.
Example 2
[0218] The ability of the methods of the invention to inhibit p53
was assessed. The tumour suppressor p53 is a transcription factor
that is stabilized following ionizing radiation (IR) and plays a
crucial role in the maintenance of cell cycle arrest in G1
following DNA damage (Agami & Bernard, supra and Pluquet, &
Hainaut, Cancer Lett 2001, 174, 1-15).
[0219] A pSUPER construct, pSUPER-p53, was generated capable of
giving rise to the transcript depicted in FIG. 2(a) was generated.
Again the vector include a 19 nucleotide region of sequence
identity to a region of the p53 gene and a complement of that
region.
[0220] MCF-7 cells were transfected with increasing amounts
pSUPER-p53. Sixty hours after transfection cells were either
irradiated (+IR, 20 Gy) or left untreated, harvested 2 hours later
and separated on 10% SDS-PAGE. Immunoblot with anti-p53 antibody
was preformed as well as a blot to act as a control for protein
loading. The results obtained are depicted in FIG. 2(a). The bands
corresponding to p53 protein and a loading control are indicated.
Cells transfected with the pSUPER-p53 construct or empty pSUPER
were analysed by flow cytometry. MCF-7 cells were transfected,
irradiated (+IR, 10Gy) after 60 hours and analyzed 24 hours later
for DNA content as described in Agami & Bernards (supra). The
results obtained are depicted in FIG. 2(b) and cells with a
GI-phase DNA content are indicated with an arrow.
[0221] The results obtained show that transfection of as little as
0.5 .mu.g of pSUPER-p53 reduced p53 protein to very low levels and
prevented entirely its induction following IR. When
vector-transfected cells were irradiated, they arrested within 24
hours in either G1 or G2 with very few cells remaining in S phase.
In contrast, cells transfected with the pSUPER-p53 almost
completely lost their p53-dependent arrest in G1, but were able to
establish a p53-independent G2/M arrest (FIG. 2b). These results
indicate that the pSUPER-p53 vector can suppress the endogenous p53
to the extent that it abrogates the function of p53 in the DNA
damage response.
[0222] The transfected cells were also studied by microscopy. FIG.
2(c) FIG. 2(c) shows cells transfected with 1 .mu.g pSUPER vectors
and 0.1 .mu.g pBabe-puro plasmid which were selected with 1
.mu.g/ml puromycin 48 hours later for 12 days. Plates were
irradiated (20 Gy) and after 4 hours fixed and stained to detect
p53. Shown also are the phase contrast images of the same colonies.
The left and right images are of two different colonies.
Example 3
[0223] The ability of the methods of the invention to suppress the
expression of a specific allele was assessed.
[0224] The CDH1 19 nt target-recognition sequence was mutated to
give one basepair substitution at position 9 or 2 of the stem.
Constructs capable of expressing each of the transcripts depicted
in FIG. 3(a) were generated with the mutations highlighted in
bold.
[0225] U2OS cells were transfected exactly as previously. Whole
cell lysates were prepared after 60 hours, separated on 10%
SDS-PAGE and analyzed by immunoblotting with anti-CDH1 antibody.
Cyclin D1 protein was used to demonstrate equal loading. The
results obtained are shown in FIG. 3(b). Empty pSUPER was
constructed as a control as well as a construct capable of
expressing GFP to determine transfection efficiency. The results
obatined show that whilst the construct capable of generating a
siRNA with complete sequence identity to the 19 nucleotide region
of the Cdh1 gene could inhibit expression of Cdh1 as effectively as
siRNA neither of the constructs with the point mutations were
capable of inhibiting expression. This means that the constructs of
the invention can discriminate between two alleles of the same gene
inhibiting expression of one allele whilst allowing normal
expression of the other.
Example 4
[0226] The ability of a the methods of the invention to inhibit the
expression of a further gene, CDC20 was assessed.
[0227] FIG. 4 shows the sequences of the SiRNA and the predicted
transcript of pSUPER-CDC20 utilized to inhibit CDC20 expression.
The indicated SiRNAs and plasmids were transfected into MCF-7 cells
as described above. Whole cell extracts were separated on 10%
SDS-PAGE and immunoblotted to detect Cdc2O and Cyclin D1 proteins.
The results show that the construct against CDC20 inhibited the
desired gene and also that this inhibition is specific and not
merely a non-specific response to dsRNA as transfection with the
pSUPER-CDH1-B construct had no effect on CDC20 expression.
Example 5
[0228] The effect of pSUPER-p53 vector on p53 mRNA stability was
examined. MCF-7 cells were electroporated with pSUPER-p53 or vector
and total RNA was extracted 48 hours later. Thirty .mu.g of RNA was
separated on agarose gel, blotted and probed with a p53 specific
.sup.32P labeled probe. The rRNAs controls were visualized by
Ethidium Bromide staining of the blot as a control for loading. The
Northern blot obtained and control gel for rRNA loading are shown
in FIG. 5(A). The cells transfected with the pSUPER-p53 vector have
a substantially decreased level of p53 mRNA in comparison to cells
transfected with the empty vector pSUPER.
[0229] siRNA interference mediated by the same stem-loop transcript
can be expressed from retro viral vectors. Self-inactivating retro
viral vectors (pRETRO-SUPER) expressing the puromycin marker gene
were cloned to harbor either an empty pol-III promoter or one that
targets p53 (see FIG. 2A) as depicted. The vector pRETRO-SUPER was
constructed by restriction enzyme digestion of the self
inactivating-retro viral vector (MSCVpuro) with EcoRI and XhoI and
ligating to it the insert from the appropriate pSUPER plasmid
digested with the same enzymes.
[0230] U2-OS cells containing the Ecotropic-receptor were infected
three times with these vectors and one day later cells were
selected for 4 days with 1 .mu.g/ml puromycin and plated on glass
slides. One day later, slides were irradiated (20Gy), fixed four
hours later and stained with anti-p53 antibody. Immuno-florescence
with a FITC-conjugated secondary antibody is shown together with
the phase contrast of the same field. Both pictures were taken
using the same settings of the camera and microscope. The resulting
pictures are shown in FIG. 5(B).
[0231] A schematic drawing of the pRETRO-SUPER is given in FIG.
5(c) indicating the various elements present in the vector.
[0232] To accomplish more efficient delivery of short interfering
RNAs, whether retroviruses that carry the pSUPER cassette can
mediate gene silencing was tested. The entire pSUPER expression
cassette from the p53 knockdown vector described in Example 2 was
cloned into a self-inactivating pMSCV-puro retroviral vector. The
3' LTR of the murine stem cell virus (MSCV) was inactivated by an
internal (NheI-XbaI) deletion to generate a self-inactivating virus
(.DELTA.LTR). Upon integration to the genome of the virus generated
from this vector, the 3' .DELTA.LTR is duplicated to the 5' LTR to
generate a provirus that lacks all LTR's enhancer-promoter
activities. The resulting vector, pRETRO-SUPER-p53 (pRS-p53), is
shown in FIG. 7A.
[0233] Viral stocks were generated from this vector, and control
pRETRO-SUPER vector, and used to infect U2-OS cells that express
the murine ecotropic receptor to allow infection by ecotropic
virus. After infection, cells were drug-selected and immuno-stained
for p53 protein. FIG. 7B shows that the vast majority of the cells
which were infected with the pRS-p53 virus stained only weakly for
p53, whereas all of the pRS-control infected cells showed a clear
nuclear p53 staining. As expected, the red staining of the control
actin protein was similar in both polyclonal populations. Western
blot analysis of these cells confirmed clear suppression of p53
expression mediated by pRS-p53 virus infection (FIG. 7C).
Consistent with this, Northern blot analysis with the sense-19 nt
p53 target sequence as a probe detected 21-22 nt siRNAs generated
only by the pRS-p53 construct (FIG. 7D).
[0234] It was shown recently that RNA viruses are sensitive to RNA
interference (Gitlin, et al, Nature 26, 26 (2002); Novina et al.,
Nat Med 8, 681-6. (2002); Jacque, et al, Nature 26, 26 (2002)).
Nevertheless, high titer retroviral supernatants of pRS-p53
(10.sup.6/ml) were obtained in spite of the fact that the
full-length retroviral transcript produced by pRS-p53 also contains
the p53 sequence that is targeted by the virally-encoded siRNAs.
Apparently, the full-length retroviral transcript does not fall
victim to self-inflicted RNA interference. One possible explanation
could be that the intra-molecular base pairing of the p53 target
sequence with its complementary sequence within the retroviral
transcript precludes siRNA recognition. Alternatively, rapid
packaging of retroviral transcript in a viral coat may render the
full-length transcript relatively resistant to RNA interference.
Whatever the explanation, these results indicate that retroviral
vectors can be used to mediate efficient integration of pSUPER
cassettes in human cells and direct the synthesis of siRNAs to
suppress gene expression.
Example 6
[0235] To study the effects of inhibition of oncogenic RAS
expression on the tumorigenic phenotype of human cancer cells, the
expression of the endogenous mutant K-RAS.sup.V12 allele was
targeted with the pSUPER vector in the human pancreatic cell line
CAPAN-1 (FIG. 8A). To target specifically the mutant K-RAS.sup.V12
allele, a 19 nt targeting sequence spanning the region encoding
valine 12 of mutant K-RAS was cloned into the pSUPER vector,
yielding pSUPER-K-RAS.sup.V12. The two oligos used to generate the
pS-K-RAS.sup.V12 are:
1
5'gatccccgttggagctgTtggcgtagttcaagagaCTACGCCAACAGCTCCAACtttttggaa-
a3' 5'agcttttccaaaaaGTTGGAGCTGTTGGCGTAGtctcttgaaCTACGCCAA-
CAGCTCCAACggg3'
[0236] where the 19 nt K-RAS.sup.V12 target sequences are in
capital letters and the G-T mutation that generates the Gly-Val
substitution in amino acid 12 of K-RAS is in bold. These oligos
were annealed to generate an insert with compatible ends to a BglII
and HindIII digested pSUPER vector. FIG. 8B shows that CAPAN-1
human pancreatic carcinoma cells transiently transfected with
pSUPER-K-RAS.sup.V12 had significant suppression of endogenous
K-RAS.sup.V12 expression, whereas control cyclin-D1 protein levels
were unaffected (FIG. 8B).
[0237] The pSUPER-K-RAS.sup.V12 cassette was then cloned into the
pRETRO-SUPER retroviral vector of Example 5. pRS-K-RAS.sup.V12
virus was then used to infect CAPAN-1 cells stably expressing the
murine-ecotropic receptor (to allow retroviral infection). Parental
pRS and pRS-p53 viral stocks were used for control infections.
Following drug selection, a Western blot analysis with anti-K-RAS
specific antibodies revealed that the K-RAS.sup.V12 expression in
the PRS-K-RAS.sup.V12-infected CAPAN-1 cells was markedly
suppressed compared to control infections (FIG. 8C).
[0238] Next, the specificity of the targeting construct was tested
by examining the expression of wt K-RAS. EJ cells, which
endogenously express two wild type K-RAS alleles, but harbor
oncogenic H-RAS.sup.V12 were used. Western blot analysis revealed
that comparable levels of wt K-RAS protein were expressed in EJ
cells, irrespective of whether they were infected with the same
pRS-K-RAS.sup.V12, pRS-p53 or pRS retroviral stocks used for the
CAPAN-1 cells (FIG. 8D, lanes 1,3,4,6). In contrast, p53 expression
was suppressed equally by pRS-p53 in both EJ and CAPAN-1 cell
types, ruling out the possibilities that the EJ cells were not
infected or lacked components necessary for RNA interference (lanes
2 and 5). Thus, the RNA interference response provoked by the
PRS-K-RAS.sup.V12 retrovirus is powerful and sufficiently selective
to distinguish between the wild type and K-RAS.sup.V12 alleles,
which differ by one base pair only.
[0239] The presence of oncogenic K-RAS alleles is frequent in human
tumors, but almost invariably associated with multiple other
genetic events. To address the question whether the oncogenic
phenotype of late stage human tumors still depends on the
expression of oncogenic mutant K-RAS, CAPAN-1 cells were again
used. One phenotype that is associated with tumorigenicity is the
ability to grow independent of anchorage when plated in a
semi-solid media (soft agar assay). CAPAN-1 and EJ cells were
infected with either pRS-K-RAS.sup.V12 or with control pRS-p53 and
pRS virus. After drug selection, 2.times.10.sup.4 cells were plated
in soft agar and allowed to grow for three weeks. As expected from
transformed human tumor cell lines, both CAPAN-1 and EJ cell lines
were able to grow and form colonies when infected with pRS and
pRS-p53 control viruses (FIG. 8A and Table 1A). In contrast,
infection of pRS-K-RAS.sup.V12 abolished almost completely the
colony growth of CAPAN-1 cells in this assay. Importantly, the
effect of pRS-K-RAS.sup.V12 was specific as soft agar growth of EJ
cells (which contain the H-RAS.sup.V12 oncogene) was unaffected
(FIG. 9A and Table 1).
2 TABLE 1 pRS- pRS- Cell line PRS K-RAS.sup.V12 p53 CAPAN-1 150-200
0-2 150-200 EJ 300-400 300-400 300-400 Growth in soft agar. The
averge number of soft agar colonies from three independent
experiments are represented.
[0240] Finally, we tested if down-regulation of K-RAS.sup.V12
expression in CAPAN-1 cells affected their ability to form tumors
in nude mice. CAPAN-1 cells were infected with either a
pRS-K-RAS.sup.V12 virus or pRS control virus and drug selected for
three days to eliminate uninfected cells. After this,
1.times.10.sup.6 infected cells were injected subcutaneously into
athymic nude mice. As shown in FIG. 9B and Table 2, control pRS
infected CAPAN-1 cells gave rise to tumors within 4 weeks in all
mice, whereas none of the six animals infected with the
pRS-K-RAS.sup.V12 virus developed tumors.
3TABLE 2 Cell line: PRS pRS-K-RAS CAPAN-1 6/6 0/6 Tumorigenicity in
athymic nude mice of cells infected with K-RAS.sup.V12 or control
knockdown vector.
[0241] These results demonstrate for the first time that viral
vectors can be used to integrate expression cassettes in the
genomes of human cells, which mediate RNA interference to induce
persistent loss-of function phenotypes. Vectors like these have at
least two potential applications. In gene therapy, the selective
down-regulation of only the mutant version of a gene allows for
highly specific effects on tumor cells, while leaving the normal
cells alone. This feature greatly reduces the need to design viral
vectors with tumor-specific infection and/or expression. By
designing target sequences that span chromosomal translocation
breakpoints found in cancer, these vectors may also be used to
specifically inhibit the chimeric transcripts of these translocated
chromosomes. In addition, these vectors can be used to efficiently
identify the genetic events that are required for cancer cells to
manifest a tumorigenic phenotype. Through use of this technology,
out of the many genetic alterations present in most human cancer
cells, the most effective targets for drug development can be
rapidly identified.
Sequence CWU 1
1
21 1 1057 DNA Homo sapiens 1 ttatagggag ctgaagggaa gggggtcaca
gtaggtggca tcgttccttt ctgactgccc 60 gccccccgca tgccgtcccg
cgatattgag ctccgaacct ctcgccctgc cgccgccggt 120 gctccgtcgc
cgccgcgccg ccatggaatt cgaacgctga cgtcatcaac ccgctccaag 180
gaatcgcggg cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa
240 gatggctgtg agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc
tatgtgttct 300 gggaaatcac cataaacgtg aaatgtcttt ggatttggga
atcttataag ttctgtatga 360 gaccactctt tcccataggg cggagggaag
ctcatcagtg gggccacgag ctgagtgcgt 420 cctgtcactc cactcccatg
tcccttggga aggtctgaga ctagggccag aggcggccct 480 aacagggctc
tccctgagct tcggggaggt gagttcccag agaacggggc tccgcgcgag 540
gtcagactgg gcaggagatg ccgtggaccc cgcccttcgg ggaggggccc ggcggatgcc
600 tcctttgccg gagcttggaa cagactcacg gccagcgaag tgagttcaat
ggctgaggtg 660 aggtaccccg caggggacct cataacccaa ttcagactac
tctcctccgc ccatttttgg 720 aaaaaaaaaa aaaaaaaaaa aacaaaacga
aaccgggccg ggcgcggtgg ttcacgccta 780 taatcccagc actttgggag
gccgaggcgg gcggatcaca aggtcaggag gtcgagacca 840 tccaggctaa
cacggtgaaa ccccccccca tctctactaa aaaaaaaaaa tacaaaaaat 900
tagccattag ccgggcgtgg tggcgggcgc ctataatccc agctacttgg gaggctgaag
960 cagaatggcg tgaacccggg aggcggacgt tgcagtgagc cgagatcgcg
ccgactgcat 1020 tccagcctgg gcgacagagc gagtctcaaa aaaaaaa 1057 2 229
DNA Homo sapiens 2 gaattcgaac gctgacgtca tcaacccgct ccaaggaatc
gcgggcccag tgtcactagg 60 cgggaacacc cagcgcgcgt gcgccctggc
aggaagatgg ctgtgaggga caggggagtg 120 gcgccctgca atatttgcat
gtcgctatgt gttctgggaa atcaccataa acgtgaaatg 180 tctttggatt
tgggaatctt ataagttctg tatgagacca ctctttccc 229 3 21 DNA Artificial
sequence Sense sequence 3 ugagaagucu cccagucagn n 21 4 21 DNA
Artificial sequence Antisense sequence 4 cugacuggga gacuucucan n 21
5 47 RNA Artificial sequence Predicted stem loop transcript 5
ugagaagucu cccagucagc agagcucuga cugggagacu ucucauu 47 6 49 RNA
Artificial sequence Predicted stem loop transcript 6 ugagaagucu
cccagucagu ucaagagacu gacugggaga cuucucauu 49 7 45 RNA Artificial
sequence Predicted stem loop transcript 7 ugagaagucu cccagucagu
ucgacugacu gggagacuuc ucauu 45 8 49 RNA Artificial sequence
Predicted stem loop transcript 8 gacuccagug guaaucuacu ucaagagagu
agauuaccac uggagucuu 49 9 49 RNA Artificial sequence Predicted stem
loop transcript 9 ugagaagucu cccagucagu ucaagagacu gacugggaga
cuucucauu 49 10 49 RNA Artificial sequence Predicted stem loop
transcript 10 ugagaaguau cccagucagu ucaagagacu gacugggaua cuucucauu
49 11 49 RNA Artificial sequence Predicted stem loop transcript 11
uaagaagucu cccagucagu ucaagagacu gacugggaga cuucuuauu 49 12 21 DNA
Artificial sequence Sense sequence 12 cggcaggacu ccgggccgan n 21 13
21 DNA Artificial sequence Antisense sequence 13 ucggcccgga
guccugccgn n 21 14 49 RNA Artificial sequence Predicted stem loop
transcript 14 cggcaggacu ccgggccgau ucaagagauc ggcccggagu ccugccguu
49 15 64 DNA Artificial sequence Mutant K-RAS target sequence 15
gatccccgtt ggagctgttg gcgtagttca agagactacg ccaacagctc caactttttg
60 gaaa 64 16 64 DNA Artificial sequence Mutant K-RAS target
sequence 16 agcttttcca aaaagttgga gctgttggcg tagtctcttg aactacgcca
acagctccaa 60 cggg 64 17 19 DNA Homo sapiens 17 gttggagctg
gtggcgtag 19 18 19 DNA Homo sapiens 18 gttggagctg ttggcgtag 19 19
49 RNA Artificial sequence Predicted stem loop transcript 19
guuggagcug uuggcguagu ucaagagacu acgccaacag cuccaacuu 49 20 9 DNA
Artificial sequence Preferred spacer region 20 ttcaagaga 9 21 49
DNA Artificial sequence Region encoding a siRNA 21 gttggagctg
ttggcgtagt tcaagagact acgccaacag ctccaactt 49
* * * * *