Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02534996 2006-02-06
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The l~Jse of siRNA Silencing in the Prevention of Metastasis
The present invention relates to synthesised RNAs, more specifically short
interfering RNAs (siRNAs) that are able to modulate the expression of Tissue
Factor (TF) and the use thereof in the prevention of metastasis and treatment
of
cancer. The present invention also discloses novel siRNA molecules directed
towards murine TF and the use thereof.
Background of the invention
siRNA ihte~;fe~°ence
Mechanisms that silence unwanted gene expression are critical for normal
cellular
function, and RNA silencing is a new field of research that has coalesced
during
the last decade from independent studies on various organisms. It has been
known
for a long time that interactions between homologous DNA and/or RNA sequences
can silence genes and induce DNA W ethylation (Bernstein E, Caudy AA, Hammond
SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA
interference. Nature 409, 363-366 (2001). The discovery of RNA interference
(RNAi) in C. elegans in 1998 focused attention on double-stranded RNA (dsRNA)
as an elicitor of gene silencing, and many gene-silencing effects in plants
are now
known to be mediated by dsRNA (Bernstein E. et al. (2001), "Role for a
bidentate
ribonuclease in the initiation step of RNA interference.",.Nature, 409:363-
366)).
RNAi is usually described as a posttranscriptional gene-silencing (PTGS)
phenomenon in which dsRNA trigger degradation of homologous mRNA in the
cytoplasm (Bernstein E. et al. (2001), supra). However, the potential of
nuclear
dsRNA to enter a pathway leading to epigenetic modifications of homologous
DNA sequences and silencing at the transcriptional level should not be
discounted.
Also, even though the nuclear aspects of RNA silencing have been studied
primarily in plants, there are indications that similar RNA-directed DNA or
chromatin modifications might occur in other organisms as well.
RNAi in animals, and the related phenomena of PTGS in plants, result from the
same highly conserved mechanism, indicating an ancient origin (Bernstein E. et
al.
(2001), supra). The basic process involves a dsRNA that is processed into
shorter
units (called short interfering RNA; siRNA) that guide recognition and
targeted
cleavage of homologous messenger RNA (mRNA). The dsRNAs that (after
processing) trigger RNAi/PTGS can be made in the nucleus or cytoplasm in a
number of ways.
The processing of dsRNA into siRNAs, which in turn degrade mRNA, is a two-
step RNA degradation process. The first step involves a dsRNA endonuclease
(ribonuclease III-like; RNase III-like) activity that processes dsRNA into
sense
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2
and antisense RNAs which are 21 to 2S nucleotides (nt) long, i.e. siRNA. In
Drosophila.this RNase III-type protein is termed Dicer. In the second step the
antisense siRNAs produced combine with, and serve as guides for, a different
ribonuclease complex called RNA-induced silencing complex (RISC), which
S cleaves the homologous single-stranded mRNAs. RISC cuts the mRNA
approximately in the middle of the region paired with the antisense siRNA,
after
which the mRNA is further degraded.
dsRNAs from different sources can enter the processing pathway leading to
RNAi/PTGS. Furthermore, recent work also suggests that there may be more than
one pathway for dsRNA cleavage producing distinct classes of siRNAs that may
not be functionally equivalent.
RNA silencing (which is active at different levels of gene expression in the
cytoplasm and the nucleus) appears to have evolved to counter the
proliferation of
foreign sequences such as transposable elements and viruses (many of which
produce dsRNA during replication). However, as RNAi/PTGS produce a mobile
signal that induces silencing at distant sites, the possibility of injecting
directly
siRNAs to shut down protein synthesis and/or function as a therapeutic tool in
mammalian cells should be considered.
So far, little is known about general effects of mutations or chemical
modifications in a siRNA sequence. Boutla et al. reported that a mutated siRNA
with a single centrally located mismatch relative to the mRNA target sequence
retained substantial activity in Df~osophila (Boutla, A., Delidakis, C.,
Livadaras, L,
Tsagris, M. and Tabler, M. (2001), "Short 5'-phosphorylated double-stranded
RNAs induce RNA interference in Drosophila.", Cus-n. Biol., 11:1776-1780)). In
contrast, Elbashir et al. found that a single mismatch was deleterious to
activity in
an in vitt-o D~°osophila embryo lysate assay (Elbashir, S.M., Martinez,
J.,
Patkaniowska, A., Lendeckel, W. and Tuschl, T. (2001), "Functional anatomy of
siRNAs for mediating effiecient RNAi in Drosophila melanogaster embryo
lysate". EMBO J., 20:6877-6888)). In the present application we have tried to
reconcile these two conflicting results by depicting the RNAi process in vivo
as a
dynamic process where several factors influence the final outcome, among them
siRNA target position, siRNA concentration, mRNA concentration, mRNA
production and siRNA's inherent cleavage activity, an activity that can be
gradually reduced by mismatch mutations.
Some other results have also been reported. For example, Jacque et al (Jacque,
J.M., Triques, K., Stevenson, M. (2002), "Modulation of HIV-1 replication by
RNA interference.", Natune, 418: 435-438)) find that a single mismatch in a
siRNA targeting HIV's LTR did lose only some activity, while another siRNA
targeting HIV's VIF lost almost no activity at all. Four mutations, however,
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WO 2005/040187 PCT/N02004/000238
3
abolished activity completely. Other instances of complete abolishment of
activity
is seen by Gitlin et al (Gitlin, L., Karelsky, S., Andino, R. (2002), "Short
interfering RNA confers intracellular antiviral immunity in human cells.",
Nature,
418:430-434)), Klahre et al (Klahre, U., Crete, P., Leuenberger, S.A.,
Iglesias,
V.A., Meins, F. Jr. (2002), "High molecular weight RNAs and small interfering
RNAs induce systemic posttranscriptional gene silencing in plants.", Proc.
Natl.
Acad. Sci. USA. 99:11981-11986)) and Garrus et al (Garrus, J.E., von
Schwedler,
U.K., Pornillos, O.W., Morham, S.G., Zavitz, K.H., Wang, H.E., Wettstein,
D.A.,
Stray, K.M., Cote, M., Rich, R.L., Myszka, D.G., Sundquist, W.I. (2001),
"Tsg101
and the vacuolar protein sorting pathway are essential for HIV-1 budding.",
Cell,
107:55-65), using 5, 6 and 7 mutations respectively. A central, double
mutations
used by Boutla and our own group (Boutla et al. (2001), "Short 5'-
phosphorylated
double-stranded RNAs induce RNA interference in Drosophila., Curr.Biol.,
11:1776-1780, Elbashir et al. (2001), "Functional anatomy of siRNAs for
mediating efficient RNAi in Drosiphila melanogaster embryo lysate.", EMBO J.,
20:6877-6888), led to severe activity loss also for Yu et al (Yu, J.Y.,
DeRuiter,
S.L., Turner, D.L. (2002), "RNA interference by expression of short-
interfering
RNAs and hairpin RNAs in mammalian cells." PJ°oc. Natl. Acad. Sci.
USA,
99:6047-52)) and Wilda et al. (Wilda, M., Fuchs, U., Wossmann, W., Borkhardt,
A. (2002), "Killing of leukemic cells with a BCR/ABL fusion gene by RNA
interference (RNAi).", Dfzcogerae, 21:5716-24)), the latter using a siRNA with
only 17 basepairs. Interestingly, in view of our very active end-methylated
siRNAs, is Tuschl's report that fully 2'-OH methylated siRNA are inactive.
Further, two published reports of abolishment of activity by a single mutation
exist. One of them, however, the work by Brummelkamp et al (Brummelkamp,
T.R., Bernards, R., Agami, R. (2002), "A system for stable expression of short
interfering RNAs in mammalian cells.", Science, 296:550-3), is using a short
hairpin RNA (shRNA) that is assumed to produce siRNA by action of Dicer .
(Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J., Conklin, D.S.
(2002)
"Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian
cells.", Ge~zes Dev., 16:948-58). This shRNA construct was inactivated either
by a
single mutation in the putative second nucleotide of the shRNA, or by a single
mismatch in the putative ninth nucleotide. Gitlin et al (Gitlin, L., Karelsky,
S.,
Andino, R. (2002), "Short interfering RNA confers intracellular antiviral
immunity in human cells",. Nature, 418:430-434), on the other hand, argued the
case for single mutation inactivation more strongly by isolating siRNA
resistant
polio virus strains containing a single mutation in the target site on the
genomic
RNA, either in the sixth nucleotide of the siRNA or the ninth nucleotide, both
counted from the 5' end of the sense strand. On balance, different siRNA seem
to
be inactivated to different degrees.
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4
Traditionally, chemical modification of nucleic acids has iretes° alia
been used to
protect single stranded nucleic acid sequences against nuclease degradation
and
thus obtaining sequences with longer half life. Fox example, WO 91/15499
discloses 2'O-alkyl oligonucleotides useful as antisense probes. Also, 2-O-
methylation has been used to stabilize hammerhead ribozymes (Amarzguioui M,
Brede G, Babaie E, Grotli M, Sproat B, Prydz H., "Secondary structure
prediction and
in vitro accessibility of mRNA as tools in the selection of target sites for
ribozymes."
Nucleic Acids Res. 2$, 4113-4124 (2000). However, little is known about the
effects
of chemical modifications of siRNAs. Further, the presence of large
substituents in
IO the 2'hydroxyl of the 5'tenninal nucleotide might interfere with the proper
phosphorylation of the siRNA shown to be necessary for the activity of the
siRNA
(Nykanen, A., Haley, B. and Zamore, P.D. (2001), "ATP Requirements and Small
Interfering RNA Structure in the.RNA Interference Pathway.", .Cell, 107:.309-
32I).
Tissue Faetor and metastasis
IS At present, cancer remains a major cause of death and this is often a
consequence
of metastasis. In the process of metastasis, tumour colonies are established
by
malignant cells, which have detached from the original tumour (primary tumour)
and spread throughout the body. The formation of metastasis is a very complex
process and depends on detachment of malignant cells from the primary tmnour,
20 invasion of the extracellular matrix, penetration of the endothelial
basement
membranes to enter the body cavity and vessels, and then after being
transported
by the blood, infiltration of target organs. Finally, the growth of a new
tumour at
the target site depends on angiogenesis. Although one might eliminate the
primary
tumour by surgery, there is always a risk that metastatic deposits already may
exist
25 or may develop due to remnants of the primary tumour after the surgical
intervention. There is therefore a need of anti-metastatic agents to be able
to
prevent metastasis and provide an efficient treatment of cancer patients.
Tissue Factor (TF) is a membrane-bound glycoprotein mainly known as a potent
trigger of blood coagulation (Camerer E, Kolsto AB, Prydz H., "Cell biology of
30 tissue factor, the principal initiator of blood coagulation.", Thno~2b Res.
sl, 1-41
(1996).) and instrumental in causing arterial thrombosis upon rupture of
atherosclerotic plaques. Normally, TF is not found soluble in the circulation
or
accessible to plasma proteins including factor VII/VIIa and the other
coagulation
factors. Expression of TF in the vascular compartment typically results in
35 disseminated intravascular coagulation or localized initiation of clotting.
Several reports suggest that TF may also play a major role in cancer-driven
angiogenesis and metastasis (W.Ruf and B.M. Mueller (1996), "Tissue Factor in
cancer angiogenesis and metastasis.", Cm°~°etat Opi~aion in
Hematology, 3:379-384,
Ohta et al.(2002), "Expression of Tissue Factor in Associated with Clinical
CA 02534996 2006-02-06
WO 2005/040187 PCT/N02004/000238
Features and Angiogenesis in Prostate Cancer", Anticahce~°
Resea~°ch, 22:2991-
2996 (2002)., Bromberg et al. (1995), "Tissue Factor promotes melanoma
metastasis by a pathway independent of blood coagulation",
Pf°oc.Natl.Acad.Sci.
92, 8205-8209, Konigsberg et al. (2001), "The TF:VIIa Complex: Clinical
5 Significance, Structure-function Relationship and its Role in Signaling and
Metastasis", Th~onab, Haemost., 86:757-771).
Zhang et al. (1994) (Zhang et al (1994), J. Clin.hZVest., 94:1320-1327)
suggested
that TF influenced tumour angiogenesis based on experiments utilizing sense
and
anti-sense TF cDNA constructs in Meth-A sarcoma cells. On the other side,
Toomey et al (1997) concluded that there is no relationship between tumour
growth and the~presence or absence of tumour-derived TF (Toomey et al. (1997),
"Effect of tissue factor deficiency on mouse and tumor development.",
P~°oc.Natl.Acad.Sci., 94: 6922-6926). .However, others have reported
that various
tissue factor inhibitors have metastasis reducing abilities (Hu and Garen
(2001),
"Targeting tissue factor on tumor vascular endothelial cells and tumor cells
for
immunotherapy in mouse models of prostatic cancer.", P~oc.Natl.Acad.Sci, 98
(21): 10180-12185, A. Amirkhosravi et al. (2002), "Tissue Factor Pathway
Inhibitor Reduces Experimental Lung Metastasis of B 16 Melanomas.",
TIm°omb.
Haemost., 87: 930-936). Although the mechanism of the metastatic capability of
TF still remains unknown, Bromberg et al (1999) have found that
phosphorylation
of the extracellular domain of TF and complex forming with VIIa is required
for
the metastatic effect of TF (Bromberg et al. (1999), "Role of Tissue Factor in
Metastasis: Functions of the Cytoplasmic and Extracellular Domains of the
Molecule", Th~omb Haeniost., 82:88-92).
Hitherto, no anti-metastatic agents are available that are based on the
inhibition of
TF in spite of the various reports about the correlation between TF and
metastasis.
Thus, there is clearly a need for methods to modulate or silence TF to prevent
metastasis in cancer patients. The present inventors have now found that siRNA
molecules directed towards TF are surprisingly efficient in preventing
metastasis
as will be apparent from the detailed description and examples below.
The use of siRNA in silenciiZg TF.
Patent application WO 01/75164 (A2) discloses a Drosophila if2 vita°o
system
which is used to demonstrate that dsRNA is processed to RNA segments 21-23
nucleotides (nt) in length, wherein these 21-23 nt fragments are specific
mediators
of RNA degradation. Caplen et al. reports that synthetic siRNA directed
towards
the CAT gene and C. elega~2s unc-22 gene reduced the expression in vertebrate
and
inveutebrate systems respectively (Caplen, N.J. et al. (2001 ), "Specific
inhibition
of gene expression by small double-stranded RNAs in invertebrate and
vertebrate
systems.", Ps°oc. Natl. Acad. Sci. USA, 98:1.7, 9742-47). However,
neither WO
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6
01/75164 nor~Caplen et al. (2001), sups°a disclose anything regarding
siRNAs
which are able to directly modulate the expression of TF in mammals. Janowsky
and Schwenzer and Schwenzer (1998) reports that the activation of a hammerhead
ribozyme by oligonucleotide facilitators exampled inte~° alia with a
hammerhead
ribozyme construct and oligonucleotide facilitators directed towards hTF
(Janowsky, E., and Schwenzer, B. (1998), "Oligonucleotide facilitators enable
a
hammerhead ribozyme to cleave long RNA substrates with multiple-turnover
activity.", Euf°. J. Biochem., 254, 129-134). However, the mechanism to
inhibit
gene expression with hammerhead ribozymes and oligonucleotide facilitators as
utilized by Janowsky and Schwenzer (Janowsky, E., and Schwenzer, B. (1998),
supra) are clearly different from the mechanism by which siRNAs inhibit the
expression of any gene such as the TF coding gene.
Apart from preliminary studies on antibodies, no clinically useful direct
inhibitor
of TF is available, nor can it be usefully regulated at the level of gene
expression.
Studies on silencing of transgenes in plants has led to a rather general
opportunity
for suppressing gene expression, and dsRNA is already established as a routine
tool for gene silencing in e.g. plants, C. elegans and Drosophila (Clemens, J.
C. et
al., "Use of double-stranded RNA interference in Drosophila cell lines to
dissect
signal transduction pathways.", P~oc. Natl Acael. Sci. USA 97, 6499-6503
(2000).
However, dsRNA cannot be used in mammalian cells because of unspecified
effects. Furtheumore, even though all gene expression can, in principle, be
suppressed by use of e.g. oligonucleotide.(synthetic chains), ribozymes or
siRNA
molecules, it is extremely hard to find exactly what part of an mRNA sequence
that should be used in order to synthesise siRNA(s) which are active in
suppressing a specific gene as siRNAs are heavily position-dependent. This
aspect
is further supported by the results reported by Harborth et al. (Harborth et
al.
(2001), "Identification of essential genes in cultured mammalian cells using
small
interfering RNAs", J. Cell Science, 114, 4557-4565), which experienced that
without revealing any unusual features, siRNA-sequences directed towards
different sequences of the same gene exerted quite dissimilar efficiency. In
addition, as sites on the mRNA target can also be differentially accessible to
ribozymes (Amarzguioui M., Brede G. Babaie E., Grotli M., Sproat B., Prydz H.,
"Secondary structure prediction and in vitro accessibility of mRNA as tools in
the
selection of target sites for ribozymes", Nucleic Acid Res., 28, 4113-4124
(2000)),
efforts to identify really efficient ribozymes towards TF with little or no
toxicity,
have not yet succeeded.
Recently, it was demonstrated that the siRNA molecules directed towards TF
modulate the activity of TF and that the TF reducing activity is highly
sequence
specific (PCT/N003/00045, Holen, T. et al. (2000), "Positional effects of
short
interfering RNAs targeting the human coagulation trigger Tissue Factor",
Nucleic
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7
~4cid Res., 30, 1757-1766). The use of siRNA to inhibit TF and thus prevent
metastasis would constitute a promising step forward in the cancer therapy.
Summary of the invention
It is therefore an object of the present invention to provide siRNA that,
together
with RISC, are able to directly modulate the expression of TF in mammals and
thus prevent the formation, of metastasis. These objects have been obtained by
the
present invention, characterised by the enclosed claims. Generally, the
present
invention relates to short interfering RNA molecules which are double or
single
stranded and comprise at least 19 nucleotides, and wherein said siRNAs are
able to
modulate the gene expression of TF.
siRNAs are dsRNAs of ~ 21-25 nucleotides that have been shown to function as
key intermediates in triggering sequence-specific RNA degradation. Recently it
was demonstrated that siRNAs towards TF can bypass the RNAse III-like RNAi
initiator Dicer and directly charge the effector nuclease RISC so that TF mRNA
is
degraded (PCT/N003/00045). It was also demonstrated that different siRNAs
against the same target vary in efficiency, and thus, siRNAs may be
synthesised
against different parts of TF mRNA, after which they combine with RISC which
is
then guided for specific degradation/silencing of TF mRNA.
The present inventors have found that siRNA molecules directed towards TF
reduce malignant cells abilities to settle and form new tumours irz vivo in a
mouse
model. The metastasis reducing effect of siRNA targeting TF is also
demonstrated
after systemic injection of siRNA. Thus, the siRNA molecules directed towards
TF may be useful in the prevention of metastasis and treatment of cancer in
vertebrates, preferably mammals, more preferably humans.
More specific, the present inventions relates to the use of short interfering
RNA
molecules (siRNAs) directed towards TF fox the preparation of a pharmaceutical
composition for preventing metastasis.
Furthermore, the present invention relates to the use of double or single
stranded
siRNA directed towards. a tissue factor (TF) coding nucleic acid sequence or
fragments thereof, and wherein the siRNA molecule is selected from the group
consisting of
(a) a siRNA molecule having the nucleic acid.sequence depicted in
SEQ ID NO 1 to SEQ ID NO 8 or SEQ ID NO 32 to SEQ ID NO
37, the complement of which is SEQ ID NO 48 - SEQ ID 53;
(b) a siRNA molecule having a sequence which is about 90
homologous to a siRNA molecule of (a);
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8
(c) a siRNA molecule which compromise a sequence having a target
site which is shifted up to 7 nucleotides in either the 5' or 3'
tenninal direction of the SEQ ID NO 1 to SEQ ID NO 8 or SEQ ID
NO 32 to SEQ ID NO 37, the complement of which is SEQ ID NO
48 - SEQ ID 53;
(d) a siRNA molecule having a sequence which is about 90
homologous to a siRNA molecule of (c); and
(e) a siRNA having the nucleic acid sequence in (a) - (d) wherein the
sequences are modified by the introduction of a CI-C3-alkyl, Ci-C3-
alkenyl or C1-C3-alkylyl .group in one or more of the 2' OH
hydroxyl group in the sequence and/or by replacing the
phosphodiester bond with a phosphorothioate bond.
The use of siRNA to prevent metastasis according to the present invention may
provide a better and more efficient treatment of cancer and preferably lead to
increased survival rate. Preferably the said siRNAs are double stranded.
It is further preferably that said siRNAs induces cleavage of TF mRNA, more
preferably identified by SEQ ID NO 1 or SEQ ID NO 2.
According to another aspect of the present invention, said composition
comprise
siRNAs which are 21-25 nucleotides long, more preferably 21 nucleotides long
and even more preferably identified by SEQ ID NO 1 to SEQ ID NO 8.
According to still another aspect of the invention, the siRNAs are directed to
TF
or fragments thereof, which are of vertebrate origin, preferably mammalian
origin,
more preferably human origin.
Moreover, according to another aspect, it is preferred that the siRNA
molecules
comprises a sequence which is about 90 % homologous to a siRNA molecule
depicted in SEQ ID NO 1 to SEQ ID NO 8 or that the siRNA comprises a
sequence as depicted in SEQ 'ID NO 1 to SEQ ID NO 8 wherein a C~-C3-alkyl, Cl-
C3-alkenyl or Ci-C3-alkylyl group is introduced in one or more of the 2' OH
hydroxyl group. Preferably, the siRNA molecule has the sequence as depicted in
SEQ ID NO 9 to SEQ ID NO 11).
Further, according to another aspect it is preferred that said siRNA molecules
comprise a sequence as depicted SEQ ID NO 1 to SEQ ID NO 8, wherein the
phosphodiester bond has been replaced by a thiophosphodiester bond.
Preferably,
the modified sequence is the sequence SEQ ID NO 24, the complement of which
is SEQ ID NO 40, SEQ ID NO 28, the complement of which is SEQ ID NO 44 or
SEQ ID NO 29, the complement of which is SEQ ID NO 45.
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9
Furthermore, the present invention also provides novel murine siRNA sequences
useful as e.g. a research tool for studying the mechanism of metastasis and
the role
of TF. According to a preferred aspect of the present invention, the siRNA's
have
the nucleic acid sequences depicted in SEQ ID NO 32 to SEQ ID NO 37, the
complement of which is SEQ ID NO 48 to SEQ ID NO 53, respectively.
The composition prepared according to the present use may furthermore comprise
e.g. diluents, lubricants, binders, carriers, disintegration means, absorption
means,
colourings, sweeteners andlor flavourings. It is also favourable that the
composition comprises adjuvants and/or other therapeutically principles.
Further, in still another aspect, the said composition may be administered
e.g.
parenterally (e.g. by subcutaneous, intravenous, intramuscular or
intraperitoneal
injection or infusion}, orally, nasally, buccally, rectally, vaginally and/or
by
inhalation or insufflation. More preferably, the composition may be formulated
as
e.g. infusion solutions or suspensions, an aerosol, capsules, tablets, pills,
spray,
suppositories etc., in dosage formulations containing conventional non-toxic
pharmaceutically-acceptable carriers, adjuvants and/or vehicles. The
composition
may be administered in one dose, in divided doses or by way of sustained
release
devices, preferably alone or together with other pharmaceuticals.
The administration rout according to the method of the present invention is
e.g.
parenterally (e.g. by subcutaneous, intravenous, intramuscular or
intraperitoneal
injection or infusion), orally, nasally, buccally, rectally, vaginally andlor
by
inhalation or insuffiatians.
The term "double-stranded" as used herein means a nucleic acid molecule having
both a sense and an anti-sense strand. The sense strand and the antisense
strand
can be from the same nucleic acid molecule or assembled from two nucleic acid
molecules and covalently connected via a linker molecule (e:g., a
polynucleotide
linker or a non-nucleotide linker).
The term "nucleic acid molecule," "oligonucleotide," or "nucleobase oligomer"
as
used herein means any chain of nucleotides or nucleic acid mimetics. Included
in
this definition are natural and non-natural oligonucleotides, both modified
and
unmodified.
Furthermore, by "pharmaceutically acceptable carrier" is meant a carrier that
is
physiologically acceptable to the treated mammal while retaining the
therapeutic
properties of the compound with which it is administered. One exemplary
pharmaceutically acceptable carrier substance is physiological saline. Other
physiologically acceptable carriers and their formulations are known to one
skilled
in the art and described, for example, in RernirZgtorz 's Plaarrnaaceutical
Sciences,
(20tli edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins,
Philadelphia, PA.
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By "reduce or inhibit" as used herein means the ability to cause an overall
decrease, preferably of 20% or greater, more preferably of 50% or greater, and
most preferably of 75% or greater, in the level of protein or oligonucleotide
as
compared to a reference sample (e.g., a sample not treated with siRNA). This
5 reduction or inhibition of RNA or protein expression can occur through
'targeted
mRNA cleavage or degradation. Assays for protein expression or nucleic acid
expression are known in the art and include, for example, ELISA, western blot
analysis for protein expression, Southern blotting or PCR for DNA analysis,
and
northern blotting or RNase protection assays for RNA. By "reduce or inhibit"
is
10 also meant an overall decrease preferably of 20% or greater, more
preferably of
50% or greater, and most preferably of 75% or greater, in the biological
activity of
TF. Assays for TF activity are known in the art and include in vitr~o
coagulation
assays, one-stage clotting assyas, two-stage clotting assays, TF clotting
time.
assays, and prothrombin time assays.
By "small interfering RNA" or "siRNA" as used herein is meant an isolated RNA
molecule, preferably greater than 10 nucleotides in length, more preferably
greater
than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24,
25,
26, 27, 28, 29, or 30 nucleotides in length that is used to identify a target
gene or
mRNA to be degraded. A range of 19-25 nucleotides is the most preferred size
for
siRNAs. siRNAs can also include short hairpin RNAs (shRNA) in which both
strands of an siRNA duplex are included within a single RNA molecule. Double-
stranded siRNAs generally consist of a sense and anti-sense strand. Single-
stranded siRNAs generally consist of only the anti-sense strand that is
complementary to the target gene. siRNA includes any form of RNA, preferably
dsRNA (proteolytically cleaved products of larger dsRNA, partially purified
RNA,
essentially pure RNA, synthetic RNA, recombinantly produced RNA) as well as
altered RNA 'that differs from naturally occurring RNA by the addition,
deletion,
substitution, and/or alteration of one or more nucleotides. Such alterations
can
include the addition of non-nucleotide material, such as to the ends) of the
21 to
23 nucleotide RNA or internally (at one or mare nucleotides of the RNA). In a
preferred embodiment, the RNA molecule contains a 3'hydroxyl group.
Nucleotides in the RNA molecules of the present invention can also comprise
non-
standard nucleotides, including non-naturally occurring nucleotides or
deoxyribonucleotides. The double-stranded oligonucleotide may contain a
modified backbone, for example, phosphorathioate, phosphorodithioate, or other
modified backbones known in the art, or may contain non-natural
internucleoside
linkages. Collectively, all such altered RNAs axe referred to as modified
siRNAs.
siRNAs of the present invention need only be sufficiently similar to natural
RNA
such that it has the ability to mediate RNAi. As used herein "mediate RNAi"
refers to the ability to distinguish or identify which RNAs are to be
degraded.
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11
Preferably, RNAi is capable of decreasing the expression of TF in a cell by at
least
10%, 20%, 30%, or 40%, more preferably by at least 50%, 60%, or 70%, and most
preferably by at least 75%, 80%, 90%, 95% or more. In one preferred
embodiment, short 21, 22, 23, 24, or 25 nucleotide double stranded RNAs are
used
to down regulate TF expression. Such RNAs are effective at down-regulating
gene expression in mammalian tissue culture cell lines (Elbashir et al.,
Natuf~e
411:494-498, 2001, hereby incorporated by reference).
By "shRNA" as used herein is meant an RNA comprising a duplex region
complementary to an mRNA. For example, a short hairpin RNA (shRNA) may
comprise a duplex region containing nucleotides, where the duplex is between
19
and 29 bases in length, and the strands are separated by a single-stranded 3,
4, 5,
6, 7, 8, 9, or 10 base linker region. Optimally, the linker region is 6 bases
in
length.
By "tissue factor protein" as used herein is meant any chain of amino acids,
regardless of length or post-translational modification (for example,
glycosylation
or phosphorylation) that is substantially identical to any mammalian TF or TF
precursor molecule. See, for example, GenBank accession numbers AAH11029
(human), NP001984 (human), P20352 (mouse), AAH24886 (mouse), AAH16397
(mouse), P42533 (rat), P30931 (bovine), Q9JLU8 (guinea pig). TF is an integral
membrane glycoprotein that can trigger blood coagulation via the extrinsic
pathway (Back et al., J..Biol Chem. 256, 8324-8331 (1981)). TF consists of a
protein component (previously referred to as TF apoprotein-III) and a
phospholipid (Osterud and Rapaport, Pf°oc. Natl. Acad. Sci. 74, 5260-
5264
(1977)). TF from various organs and species has been reported to have a
relative
molecular mass of 42,000 to 53,000. Purification of TF has been reported from
various tissues such as human brain (Guha et al. Pf~oc. Natl. Acad. Sci: 83,
299-
302 (1986) and Broze et al., J. Biol. ClZenz. 260, 10917-10920 (1985)); bovine
brain (Bach et al., J. Biol. Chenz. 256, 8324-8331 (1981)); human placenta
(Bom et
al., Thr°onzbosis Res. 42:635-643 (1986); and, Andoh et al.,
Thnonzbosis Res.
43:275-286 (1986)); ovine brain (Carlsen et al., Tlzromb. Haetzzostas. 48, 315-
319
(1982)); and lung (Glas, and Astrup Arn. J. Physiol. 219, 1140-1146 (1970)).
It
has been shown that bovine and hmnan tissue thromboplastin is identical in
size
and' function (see fox example Broze et al., J. Biol. them. 260, 10917-10920
(1985,)). It is widely accepted that while there are differences in structure
of TF
protein between species there are no functional differences as measured by irz
vitf°o
coagulation assays. As used herein, TF includes TF protein from any of the
species or tissues described herein having TF biological activity. TF
biological
activity can be measured by any of several assays known in the art. Non-
limiting
examples include i~z vita°o coagulation assays, one-stage clotting
assays, two-stage
clotting assays (Pitlick and Nemerson, Methods E~azynzol., 45: 37-48 (1976)),
TF
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12
clotting time assay (Santucci et al., Tlaf°omb. Haemost. 83:445-454,
2000), and
prothrombin time assays.
By "tissue factor nucleic acid" is meant a nucleic acid molecule (e.g., DNA,
cDNA, genomic, mRNA, RNA, dsRNA, antisense RNA, shRNA) substantially
.identical to any mammalian TF or TF precursor nucleic acid molecule or any
nucleic acid molecule that encodes any of the TF proteins described above.
See,
for example, GenBank accession numbers M16553 (human), BC011029 (human)
NM01993 (human), AF540377 (human); U07619 (rat), M57896 (mouse), .and
M55390 (rabbit).
Furthermore, the terms "treating" or "treatment" as used herein means
administering a compound or a pharmaceutical composition for prophylactic
and/or therapeutic purposes. To "treat a disease" or use for "therapeutic
treatment" refers to administering treatment to a subject already suffering
from a
disease to improve the subject's condition. Preferably, the subject is
diagnosed as
suffering from a coagulation disorder or a tumour with metastatic potential.
To
"prevent disease" refers to prophylactic treatment of a subject who is not yet
ill,
but who is susceptible to, or otherwise at risk of, developing a particular
disease.
In one example, a subject is determined to be at risk of developing a
coagulation
disorder based on a family history of coagulation disorders or prior cardiac
events.
In another example, a subject is determined to be at risk of developing a
tumour
metastasis if the subject has been diagnosed with a malignant tumour. Thus, in
the
claims and embodiments, treating is the administration to a mammal either for
therapeutic or prophylactic purposes.
By "tumour" is meant an abnormal group of cells or tissue that grows by a
rapid,
uncontrolled cellular proliferation and continues to grow after the stimuli
that
initiated the new growth cease. Tumours show partial or complete lack of
structural organization and functional coordination with the normal tissue,
and
usually form a distinct mass of tissue, which may be either benign or
malignant.
Non-limiting examples of tumours include bladder, blood, bone, brain, breast,
cartilage, colon, kidney,. liver, lung, lymph node, nervous tissue, ovarian,
pancreatic, prostate, skeletal muscle, skin, spinal cord,, spleen, stomach,
testicular,
thymus, thyroid, trachea, urogenital tract, ureter, urethrea, uterine, and
vaginal
tumours.
By "metastasis" is meant the spread of cancer cells from its original site to
another
part of the body. The formation of metastasis is a very complex process and
depends on detachment of malignant cells from the primary tumour, invasion of
the extracellular matrix, penetration of the endothelial basement membranes to
enter the body cavity and vessels, and then, after being transported by the
blood,
infiltration of target organs. Finally, the growth of a new tumour at the
target site
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13
depends on angiogenesis. Tumour metastasis often occurs even after the removal
of the primary tumour because tumour cells or components may remain and
develop metastatic potential.
Other features and advantages of the invention will be apparent from the
following
description of the preferred embodiments thereof, and from the claims.
The present invention will now be described in more detail, with reference to
figures and examples.
Brief description of the figures
Figure 1 siRNAs, reporter construct and RNAi of transgene expression; a) The
sense (top) and antisense (bottom) strands of siRNA species targeting eight
sites
within human TF (Genbank entry Acc. No. M16553) mRNA are shown, b)
Luciferase reporter construct of human TF and c) RNAi by siRNA in
cotransfection assays (averages of three or more independent experiments each
in
triplicate, ~ s.d. are shown).
Figure 2 Efficacy of the siRNAs in standard cotransfection assays in HaCaT
cells.
Different synthetic batches of the hTFl67i siRNA showed similar efficacy.
Results are averages of at least three experiments, each in triplicate.
Figure 3 siRNA mediated reduction of endogenous TF expression; a) hTFl67i and
hTF372i induced cleavage of mRNA in transfected cells. The Northern analysis
of
TF mRNA was performed after transfection of HaCaT cells with siRNA (100 nM)
with GADPH as control. Arrowhead indicates cleavage fragments resulting from
siRNA action, b) Measurements of the effect of siRNAs on steady state mRNA
levels (filled bars), procoagulant activity (dotted bars) and TF protein
(antigen)
expression (hatched bars) show that siRNA reduces mRNA, TF antigen levels and
procoagulant activity. For measurement of procoagulant activity and antigen,
cells
were harvested 48 h after si transfection to accommodate the 7-8 h half life
of TF
protein. Data are from a representative experiment in triplicate.
Figure 4 Dose-response curve for hTF 167i.
Figure 5 Time-dependence of siRNA-mediated RNAi; a) Inhibitory activity is
reduced when mutations (M1 and M2 refer to one and two mutations,
respectively)
are introduced into the siRNAs. Cells were transfected with 100 nM siRNA and
harvested for mRNA isolation 4, 8, 24 and 48 h (filled bars, lined bars, white
bars
with black dots and hatched bars, respectively). Expression levels were
normalised
to GADPH and standardised to mock-transfected cells at all time-points, b)
Time-
course of decay of inhibitory effect for mRNA levels (closed diamonds),
reporter
gene activity (open triangles) and procoagulant activity (filled bars).
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14
Figure 6 siRNA modifications. (A) Mutated and wild type versions of the siRNA
hTFl67i. The sequence of the sense strand of wild type (wt) siRNA corresponds
to
position 167-187 in human Tissue Factor (Ass. No. M16553). Single (sl, s2, s3,
s4, s7, s10, sl l, s13, sl6) and double mutants (ds7/10, dsl0/11, dsl0/13,
dsl0/16)
are all named according to the position of the mutation, counted from the
5'end of
the sense strand. All mutations (in bold) are GC inversions relative to the
wild
type. (B) Chemically modified versions of the siRNA hTF167i. Non-modified
ribonucleotides are in lower case. Phosphorothioate linkages are indicated by
.
asteriscs (*), while 2'-O-methylated and 2'-O-allylated ribonucleotides are in
normal and underlined bold upper case, respectively.
Figure 7 Activity of mutants against endogenous hTF mRNA. HaCaT cells were
harvested for mRNA isolation 24h post-transfection. TF expression was
normalised to that of GAPDH. Normalised expression in mock-transfected cells
was set as 100%. Data are averages + s.d. of at least three independent
experiments.
Figure 8, Activity of chemically modified siRNA against endogenous TF mRNA.
Experiments were performed and analysed as described in figure 7.
Figure 9 Persistence of TF silencing by chemically modified siRNAs. A)
Specific
TF expression 5 days post-transfection of 100nM siRNA. B) Time-course of TF
mRNA silencing. Cells harvested 1-3-5 days after single transfection of 100nM
siRNA. Medium was replaced every second day.
Figure 10 shows the effect of i.v. injection of TF siRNA-transfected B 16
cells in
lungs of C57 BL/6 mice.
Figure 11 shows the effect of systemic application of siRNA. Mice in the
control group
received one i.v. inj ection with B 16 melanoma cells. Mice in the test group
received
additionally three i.p. injections of siRNA targeting TF. These injections
were done 1 day
before, and 3 and 6 days after injection of the cells.
Detailed description of the invention
Despite the suggested role of TF in tumour metastasis, no clinically useful
direct
inhibitors of TF have yet been identified. There are neither any clear
evidence that
TF may be successfully regulated at the level of gene expression and thus to
prevent metastasis.
The present invention provides compositions comprising siRNA directed towards
TF, which can be used for the treatment and prevention of tumour metastasis.
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RNAi is a form of post-transcriptional gene silencing initiated by the
introduction
of siRNAs. Short 21 to 25 nucleotide double-stranded RNAs are effective at
down-regulating gene expression in nematodes (Zamore et al., Cell 101:25-33,
2000) and in mammalian tissue culture cell lines (Elbashir et al.,
Natm°e 411:494-
5 498, 2001). The further therapeutic effectiveness of this approach in
mammals
was demonstrated i~z vivo by McCaffrey et al. (Nature 418:38-39, 2002). The
nucleic acid sequence of a mammalian gene, such as TF, can be used to design
small.interfering RNAs (siRNAs) that will inactivate TF target genes that have
the
specific 21 to 25 nucleotide RNA sequences used. siRNAs that target TF may be
10 used, for example, as therapeutics to treat or prevent a coagulation
disorder or a
metastatic tumour.
Provided with the sequence of a mammalian gene, siRNAs may be designed to
inactivate target genes of interest and screened for effective gene silencing,
as
described herein. In addition to the siRNAs disclosed herein, additional
siRNAs
15 may be designed using standard methods. Short hairpin RNAs (shRNAs) can
also
be used for RNAi as described in Paddison et al. (Proc. Natl. Aeael. Sci USA,
99:6047-6052, 2002; Genes & Dev, 16:948-958, 2002).
While various parameters are used to identify promising RNAi targets, the most
effective siRNA and shRNA candidate'sequences are identified by empirical
testing. One strategy for such testing is to construct a large library of non-
overlapping synthetic siRNAs or shRNA encoding vectors that give good coverage
of a tissue factor gene of interest, according to its largest sequenced cDNA,
which
includes partial 5' and 3'UTR sequences. Provided with knowledge of the intron-
exon structure of tissue factor and with sensitive means of measuring target
knock-down, such as Taqman quantitative RT-PCR and ELISA assays, the process
of siRNA or shRNA selection is relatively straightforward once conditions have
been optimized for transfection and target measurements.
As is known in the art, a nucleoside is a nucleobase-sugar combination. The
base
portion of the nucleoside is normally a heterocyclic base. The two most common
classes of such heterocyclic bases are the purines and the pyrimidines..
Nucleotides are nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those nucleosides that
include a
pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3'
or 5'
hydroxyl moiety of the sugar. In forning oligonucleotides, the phosphate
groups
covalently link adjacent nucleosides to one another to form a linear polymeric
compound. In turn, the respective ends of this linear polymeric structure can
be
further joined to form a circular structure; open linear structures are
generally
preferred. Within the oligonucleotide structure, the phosphate groups are
commonly referred to as forming the backbone of the oligonucleotide. The
normal
linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
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16
siRNA molecules used according to the present invention preferably include
oligonucleotides containing modified backbones or non-natural internucleoside
linkages. As defined in this specification, oligonucleotides having modified
backbones include those that retain a phosphorus atom in the backbone and
those
that do not have a phosphorus atom in the backbone. For the purposes of this
specification, modified nucleobase oligomers that do not have a phosphorus
atom
in their internucleoside backbone are also considered to be nucleobase
oligomers.
Non-limited examples of nucleobase oligomers having modified oligonucleotide
backbones include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and
other alkyl phosphonates including 3'-alkylene phosphonates and chiral
phosphonates, phosphinates, phosphoramidates including 3'-amino
phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates
- having normal 3'-5' linkages, 2'-5' linked analogs o.f these, and those
having
inverted polarity, wherein the adjacent pairs of nucleoside units are linked
3'-5' to
5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are
also
included. Methods for the preparation of such phosphorus-containing linkages
are
well known to the skilled person such as those disclosed in U.S. Patent Nos.
3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;
5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;
5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein
incorporated by reference.
Nucleobase oligomers having modified backbones that do not include a
phosphorus atom therein have backbones that are formed by short chain alkyl or
cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl
internucleoside.linkages, or one or more short chain heteroatomic or
heterocyclic
interriucleoside linkages. These include those having morpholino linkages
(formed in part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; fonnacetyl and thioformacetyl
backbones; methylene formacetyl and thiofonnacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide
backbones; and others having mixed N, O, S and CH2 component parts. Methods
for the preparation of the above oligonucleotides are disclosed in e.g. U.S.
Patent
Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;
5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;
5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;
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17
5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439,
each of which is herein incorporated by reference.
In other types of oligonucleotides, both the sugar and the internucleoside
linkage,
i.e., the backbone, are replaced with novel groups. One such class of
molecules is
referred to as Peptide Nucleic Acids (PNA). PNA compounds contain an amide
backbone, more specifically an aminoethylglycine backbone. The nucleobases are
retained and are bound directly or indirectly to aza nitrogen atoms of the
amide
portion of the backbone. Methods for making and using these nucleobase
oligomers are described, for example, in "Peptide Nucleic Acids: Protocols and
Applications" Ed. P.E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999.
Methods for the preparation of PNAs are disclosed e.g in U.S. Patent Nos.:
5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by
reference.
In particular embodiments of the invention, the nucleobase oligomers have
phosphorothioate backbones and nucleosides with heteroatom backbones, and in
particular -CHZ-NH-O-CH2-, -CHZ-N(CH3)-O-CH2- (known as a methylene
(methylimino) or MMI backbone), -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-
CHZ-, and -O-N(CH3)-CH2-CHa-. In other embodiments, the oligonucleotides
have morpholino backbone structures as described in U.S. Patent No. 5,034,506.
Nucleobase oligomers may also contain one or more substituted sugar moieties.
Nucleobase oligomers comprise one of the following at the 2' position: OH; F;
O-,
S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N--alkynyl; or O-alkyl-O-
alkyl,
wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1
to
,Clo alkyl or C2 to Clo alkenyl and alkynyl. Particularly preferred are
O~(CH2)n0],nCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, arid
O(CHZ)nON[(CH2)nCH3))2, where n and m are from 1 to about 10. Other preferred
nucleobase oligomers include one of the following at the 2' position: C1 to
Clo
lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-
aralkyl, SH,
SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, S02CH3, ONOZ, NOZ, NHZ,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,
substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a
group
for improving the phannacokinetic properties of a nucleobase oligomer, or a
group
for improving the phannacodynamic properties of an nucleobase oligomer, and
other substituents having similar properties. Preferred modifications are 2'-O-
methyl and 2'-methoxyethoxy (2'-O-CHZCHZOCH3, also known as 2'-O-(2-
methoxyethyl) or 2'-MOE). Another desirable modification is 2'-
dimethylaminooxyethoxy (i.e., O(CHZ)ZON(CH3)2), also known as 2'-DMAOE.
Other modifications include, 2'-aminopropoxy (2'-OCH2CHZCHaNHz) and 2'-
fluoro (2'-F). Similar modifications may also be made at other positions on an
oligonucleotide, particularly the 3' position of the sugar on the 3' terminal
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18
nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5'
terminal
nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl
moieties in place of the pentofuranosyl sugar. Methods for the preparation of
such
modified sugar structures are disclosed in e.g. U.S. Patent Nos.: 4,981,957;
5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is
herein incorporated by reference in its entirety.
Oligonucleotides may also include nucleobase modifications or substitutions.
As
used herein, "unmodified" or '.'natural" nucleobases include the purine bases
adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine
(C)
and uracil (U). Modified nucleobases include other synthetic and natural
nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,
xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of
adenine and guanine; 2-propyl and other alkyl derivatives of adenine and
guanine;
2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5-
propynyl uracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil
(pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-
hydroxyl and
other 8-substituted adenines and guanines; 5-halo (e.g., 5-bromo), 5-
trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine
and
7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-
deazaadenine; and 3-deazaguanine and 3-deazaadenine. Further nucleobases
include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The
Concise
Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz,
J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte
Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi,
Y.
S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.
T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are
particularly useful for increasing the binding affinity of an antisense
oligonucleotide of the invention. These include 5-substituted pyrimidines, 6-
azapyrimidines, and N-2, N-6 and O-6 substituted purines, including 2-
aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex stability by 0.6-
1.2
degrees Celsius per base pair. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B.,
eds.,
Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278)
and are desirable base substitutions, even more particularly when combined
with
2'-O-methoxyethyl or 2'-O-methyl sugar modifications. Methods for the
preparation of certain of the above noted modified nucleobases as well as
other
modified nucleobases are well known to the person skilled in the art, e.g. as
disclosed in U.S. Patent Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273;
5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;
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19
5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and
5,750,692,
each of which is herein incorporated by reference.
Another modification of a nucleobase oligomer of the invention involves
chemically linking to the nucleobase oligomer one or more moieties or
conjugates
that enhance the activity, cellular distribution, or cellular uptake of the
oligonucleotide. Such moieties include but are not limited to lipid moieties
such
as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553-
6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let, 4:1053-
1060,
1994), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.
Acad.
Sci., 660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-
2770, 1993), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 20:533-
538:
1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison- .
Behmoaras et al., EMBO J., 10:1111-1118, 1991; Kabanov et al., FEBS Lett.,
259:327-330, 1990; Svinarchuk et al., Biochimie, 75:49-54, 1993), a
phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-
glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 36:3651-3654,
1995; Shea et al., Nucl. Acids Res., 18:3777-3783, 1990), a polyamine or a
polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 14:969-
973, 1995), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett.,
36:3651-3654, 1995), a pahnityl moiety (Mishra et al., Biochim. Biophys. Acta,
1264:229-237, 1995), or an octadecylamine or hexylamino-carbonyl-
oxycholesterol moiety (Crooke et al., J. Phannacol. Exp. Ther., 277:923-937,
1996. Methods for the preparation of nucleobase oligomer conjugates as
mentioned above is disclosed im U.S. Patent Nos.: 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 4,835,263; 4,876,335;
4,904,582; 4,948,882; 4,958,013; 5,082,830; 5,109,124; 5,112,963; 5,118,802;
5,138,045; 5,214,136; 5,218,105; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,414,077; 5,416,203,
5,451,463; 5,486,603; 5,510,475; 5,512,439; 5,512,667; 5,514,785; 5,525,465;
5,541,313; 5,545,730; 5,552,538; 5,565,552; 5,567,810; 5,574,142; 5,578,717;
5,578,718; 5,580,731; 5,585,481; 5,587,371; 5,591,584; 5,595,726; 5,597,696;
5,599,923; 5,599,928; 5,608,046; and 5,688,941, each of which is herein
incorporated by reference.
The present invention also includes nucleobase oligomers that are chimeric
compounds. "Chimeric" nucleobase oligomers are nucleobase oligomers,
particularly oligonucleotides, that contain two or more chemically distinct
regions,
each made up of at least one monomer unit, i.e., a nucleotide in the case of
an
oligonucleotide. These nucleobase oligomers typically contain at least one
region
where the nucleobase oligomer.is modified to confer, upon the nucleobase
oligomer, increased resistance to nuclease degradation, increased cellular
uptake,
CA 02534996 2006-02-06
WO 2005/040187 PCT/N02004/000238
and/or increased binding affinity for the target nucleic acid. An additional
region
of the nucleobase oligomer may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a
cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
5 Activation of RNase H, therefore, results in cleavage of the RNA target,
thereby
greatly enhancing the efficiency of nucleobase oligomer inhibition of gene .
expression. Consequently, comparable results can often be obtained with
shorter
nucleobase~ oligomers when chimeric nucleobase oligomers are used, compared to
phosphorothioate oligodeoxynucleotides hybridizing to the same target region.
10 Chimeric nucleobase oligomers of the invention may be formed as composite
structures of two or more nucleobase oligomers as described above. Such
nucleobase oligomers, when oligonucleotides, have also been referred to in the
art
as hybrids or gapmers. Representative United States.patents that teach the
preparation of such hybrid structures include U.S. Patent Nos.: 5,013,830;
15 5,149,,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;
5,565,350;
5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein
incorporated by reference in its entirety.
The nucleobase oligomers used in accordance with this invention may be
conveniently and routinely made through the well-known technique of solid
phase
20 synthesis. Equipment for such synthesis is sold by several vendors
including, for
example, Applied Biosystems (Foster City, Cali~). Any other means for such
synthesis known in the art may additionally or alternatively be employed. It
is
well known to use similar techniques to prepare oligonucleotides such as the
phosphorothioates and alkylated derivatives.
The nucleobase oligomers of the invention may also be admixed, encapsulated,
conjugated or otherwise associated with other molecules, molecule structures
or
mixtures of compounds, as for example, liposomes, receptor targeted molecules,
oral; rectal, topical or other formulations, for assisting in uptake,
distribution
and/or absorption. The following patents represent various non-limited
examples
of publications disclosing the preparation of suitable formulations provided
for
assisting uptake, distribution and/or absorption assisting formulations: U.S.
Patent
Nos.: 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158;
5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921;
5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978;
5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by reference.
The nucleobase oligomers of the invention encompass any pharmaceutically
acceptable salts, esters, or salts of such esters, or any other compound that,
upon
administration to an animal, is capable of providing (directly or indirectly)
the
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21
biologically active metabolite or residue thereof. Accordingly, for example,
the
disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of
the
compounds of the invention, pharmaceutically acceptable salts of such
prodrugs,
and other bioequivalents.
The teen "prodrug" indicates a therapeutic agent that is prepared in an
inactive
form that is converted to awactive form (i.e., drug) within the body or cells
thereof
by the action of endogenous enzymes or other chemicals and/or conditions.
The term "pharmaceutically acceptable salts", refers to salts that retain the
desired
biological activity of the parent compound and do not impart undesired
toxicological effects thereto. Pharmaceutically acceptable base addition salts
are
formed with metals or amines, such as alkali and alkaline earth metals or
organic
amines. Examples of metals used as cations are sodium, potassium, magnesium,
calcium; and the like. Examples of suitable amines are N,N'-dibenzylethylene-
diamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylene-
diamine, N-methylglucamine, and procaine (see, for example, Berge et al., J.
Pharna Sci., 66:1-19, 1977). The base addition salts of acidic compounds are
prepared by contacting the free acid form with a sufficient amount of the
desired
base to produce the salt in the conventional manner. The free acid form may be
regenerated by contacting the salt form with an acid and isolating the free
acid in.
the conventional manner. The free acid forms differ from their respective salt
forms somewhat in certain physical properties such as solubility in polar
solvents,
but otherwise the salts are equivalent to their respective free acid for
purposes of
the present invention. As used herein, a "pharmaceutical addition salt"
includes a
pharmaceutically acceptable salt of an acid form of one of the components of
the
compositions of the invention. These include organic or inorganic acid salts
of the
amines. Preferred acid salts are the hydrochlorides, acetates, salicylates,
nitrates
and phosphates. Other suitable pharmaceutically acceptable salts are well
known
to the person skilled in the art and include basic salts of a variety of
inorganic and
organic acids, such as, for example, with inorganic acids, such as for example
hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with
organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic
acids, for example acetic acid, propionic acid, glycolic acid, succinic acid,
malefic
acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid,
tartaric
acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid,
citric
acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-
aminosalicylic
acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic
acid
or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids
involved in the synthesis of proteins in nature, for example glutamic acid or
aspartic acid, and also with phenylacetic acid, methanesulfonic acid,
ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,
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22
benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic
acid,
naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-
phosphate,
N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other
acid
organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of
compounds may also be prepared with a pharmaceutically acceptable cation.
Suitable pharmaceutically acceptable cations are well known to those skilled
in the
art and include alkaline, alkaline earth, ammonium.and quaternary ammonium
Cat10x1S.
For oligonucleotides and other nucleobase oligomers, suitable pharmaceutically
acceptable salts include (i) salts formed with cations such as sodium,
potassium,
ammonium, magnesium, calcium, polyamines such as spermine and spermidine,
etc.; (ii) acid addition salts formed with inorganic acids, for example
hydrochloric
acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the
like; (iii)
salts formed with organic acids such as, for example, acetic acid, oxalic
acid,
tartaric acid, succinic acid, malefic acid, fumaric acid, gluconic acid,
citric acid,
malic acid, ascorbic acid, benzoic acid, tannic acid, palnitic acid, alginic
acid,
polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-
toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and
the
like; and (iv) salts formed from elemental anions such as chlorine, brouine,
and
iodine.
The present invention also includes pharmaceutical compositions and
formulations
that include the nucleobase oligomers of the invention. The pharmaceutical
compositions of the present invention may be administered in a number of ways
depending upon whether local or systemic treatment is desired and upon the
area
to be treated. Administration may be topical (including ophthalmic and to
mucous
membranes including vaginal and rectal delivery), pulmonary, e.g., by
inhalation
or insufflation of powders or aerosols, including by nebulizer; intratracheal,
intranasal, epidermal and transdermal), oral, or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal, or
intramuscular injection or infusion; or intracranial, e.g., intrathecal or
intraventricular, administration.
In addition to the modifications described above, mutations of the siRNA
molecules directed towards TF are also included in the present invention.
Preferred mutations include single base-pair mutations, including but not
limited
to those described in Example 5, and double base-p~.ir mutations, also
including
but not limited to those described in Example 5.
Introduction of siRNA into cells
One way to simplify the manipulation and handling of the siRNA molecules is to
place a cDNA cassette encoding the siRNA molecule under the control of a
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23
suitable promoter. The promoter must be capable of driving expression of the
siRNA in the desired target host cell. The selection of appropriate promoters
can
readily be accomplished. Preferably, one would use a high expression promoter.
Examples of suitable promoters include the self contained polymerase III
S promoters U6 or H1, which are able to generate a transcript of defined
sequence.
An example of a suitable polymerase II promoter is the 763-base-pair
cytomegalovirus (CMV) promoter.
Other elements that enhance the expression may also be included, e.g.,
enhancers
or a system that results in high levels of expression such as a tat gene and
tar
element. The recombinant vector can be a plasmid vector such as pUC118,
pBR322, or other known plasznid vectors, that includes, for example, an E.
coli
origin of replication (see, Sambrook, et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory press, 1989). v The plasmid vector may
also include a selectable marker such as the (3 lactamase gene for ampicillin
I S resistance, provided that the marker polypeptide does not adversely affect
the
metabolism of the organism being treated. The cassette can also be bound to a
nucleic acid binding moiety in a synthetic delivery system, such as the system
disclosed in PCT Publication No. W095/22618.
The nucleic acid can be introduced into the cells by any means appropriate for
the
vector employed. Many such methods are well known in the art (Sambrook et al.,
supra, and Watson et al., "Recombinant DNA", Chapter 12, 2d edition,
Scientific
American Books, 1992). Recombinant vectors can be transferred by methods such.
as calcium phosphate precipitation, electroporation, liposome-mediated
transfection, gene gun, microinjection; viral capsid-mediated transfer,
polybrene-
mediated transfer, or protoplast fusion. For a review of the procedures for
liposome preparation; targeting and delivery of contents, see Mannino and
Gould-
Fogerite, (Bio Techniques, 6:682-69.0, 1988), Felgner and Holm, (Bethesda Res.
Lab. Focus, I I:21, 1989) and Maurer (Betl2esda Res. Lab. Focus, 11:25, 1989).
Transfer of the recombinant vector (either plasmid vector or viral vectors)
can be
accomplished through direct injection into the amniotic fluid or intravenous
delivery. Gene delivery using adenoviral vectors or adeno-associated vectors
(AAV) can also be used. Adenoviruses are present in a large number of animal
species, are not very pathogenic, and can replicate equally well in dividing
and
quiescent cells. As a general z-ule, adenoviruses used for gene delivery are
lacking
one or more genes required for viral replication. Replication-defective
recombinant adenoviral vectors can be produced in accordance with art-known
techniques (see Quantin et al., P~°oc. Natl. Acad. Sci. USA, 89:2581-
2584, 1992;
Stratford-Perricadet et al., J. Clin. Invest., 90:626-630, 1992; and Rosenfeld
et al.,
Ce~l, 68:143-I55, 1992).
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24
For expression of siRNAs or shRNAs within cells, plasmid or viral vectors may
contain, for example, a promoter, including, but not limited to the polymerase
I, II,
and III H1, U6, BL, SMK, 7SK, tRNA poIIII, tRNA(met)-derived, and T7
promoters, a cloning site for the stern-looped RNA coding insert, and a 4-5-
thynidine transcription termination signal. The Polymerase III promoters
generally have well-defined initiation and stop sites and their transcripts
lack
poly(A) tails. The termination signal,for these promoters is defined by the
poly-
thyrnidine tract, and the transcript is typically cleaved after the second
uridine.
Cleavage at this position generates a 3' UU overhang in the expressed shRNA,
which is similar to the 3' overhangs of synthetic siRNAs.
A variety of methods is available fox transfection, or introduction, of dsRNA
into
mammalian cells. For example, there are several commercially available
transfection reagents including but not limited to: TransIT-TK03 (Minus, Cat.
#
MIR 2150), Transznessenger3 (Qiagen, Cat. # 301525), and Oligofectamine3
(Invitrogen, Cat. # MIR 12252-Ol 1). Protocols for each transfection reagent
are
available from the manufacturer. Additional formulations that aid in the
delivery
of oligonucleotides or other nucleobase oligomers to cells are described in
(see,
e.g., U.S. Patents 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959,
6,346,613, and 6,353,055).
The concentration of siRNA used for each target and each cell line varies but
in
general ranges from 0.05 nM to 500 nM, more preferably 0.1 nlvl to 100 nM, and
most preferably 1 nM to SO nM. If desired, cells can be transfected multiple
times, using multiple siRNAs to optimize the gene-silencing effect.
Stable expression of siRNA
Recently, a DNA template method has been used to create and deliver siRNA
molecules (reviewed in T. Tuschl, Nature Biotechnology, 20:446-448, 2002). The
siRNA template is cloned into RNA polymerase III transcription units, which
normally encode the small nuclear RNA U6 or the human RNAse P RNA H1.
These expression cassettes allow for the expression of both sense and anti-
sense
RNA. The endogenous expression of siRNA from introduced DNA templates is
thought to overcome some limitations of exogenous siRNA delivery, in
particular
the transient loss of phenotype. In fact, stable cell lines have been obtained
using
these siRNA expression cassettes allowing for a stable loss of function
phenotype
(Mi~yagishi M. and Taira K., Natm°e Biotech., 20:497-500, 2002;
Brummelkamp
T.R. et al., Scief~ce, 296:550-553, 2002). If desired, stable cell lines for
RNAi of
TF can be generated using the above techniques.
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Assays fo~~ evaluating gene silencing effect
mRNA and protein expression can be analyzed using any of a variety of art
known
methods including but not limited to northern blot analysis, RNAse protection
assays, luciferase or 13-gal reporter assays, western blots, and immunological
methods such as ELISAs.
TlaeT~apeutic Application
The siRNAs according to the present invention can be used to down-regulate the
expression or biological activity of mammalian TF. Thus, the siRNAs of the
10 present invention can be used to treat or prevent tumour metastasis in a
wane-
blooded animal including, but not limited to, a human, cow, horse, pig, sheep,
bird,
mouse, rat, dog, cat, monkey, baboon, or the like. Treatment generally begins
at a
hospital so that the doctor can observe the therapy's effects closely and make
any
adjustments that are needed. The duration of the therapy depends on the tumour-
15 metastasis being treated, the age and condition of the patient, the stage
and type of
the patient's disease, and how the patient's body responds to the treatment.
Therapy may be performed at different intervals (e.g., daily, weekly, or
monthly).
Therapy may be given in on-and-off cycles that include rest periods so that
the
patient's body has a chance to build healthy new cells and regain its
strength.
20 Therapeutic treatments for metastatic tumours can be used to prevent tumour
metastasis, slow the metastasis, slow the tumour-driven angiogenesis, to slow
the
tumour's growth, to kill or arrest tumour cells that may have spread to other
parts of
the body from the original tumour, or to relieve symptoms caused by the
cancer.
25 An siRNA molecule of the invention may be administered together with a
pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage
form.
Conventional pharmaceutical practice may be employed to provide suitable
formulations or compositions to administer the compounds to patients suffering
from a disease that is caused by excessive cell proliferation. Administration
may
begin before the patient is symptomatic. Any appropriate route of
administration
may be employed, for example, administration may be parenteral, intravenous,
intraarterial, subcutaneous, intratumoral, intramuscular, intracranial,
intraorbital,
ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal,
intracisternal,
intraperitoneal, intranasal, aerosol, suppository, or oral administration. For
example, therapeutic formulations may be in the form of liquid solutions or
suspensions; for oral administration, formulations may be in the form of
tablets or
capsules; and for intranasal formulations, in the form of powders, nasal
drops, or
aerosols. In one example, intravenous administration can be used to inject
siRNAs
directly into the blood stream to treat a coagulation disorder. In another
example,
direct injection of siRNA into tumours can be used to treat metastatic
tumours.
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26
Methods well known in the art for making formulations are found, for example,
in
"Remington: The Science and Practice of Pharmacy" Ed. A.R. Gennaro,
Lippincourt Williams & Wilkins, Philadelphia, PA, 2000. Fonnulations for
parenteral administration may, for example, contain excipients, sterile water,
or
saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lacti~de polymer,
lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers
may be used to control the release of the compounds. -Other potentially useful
parenteral delivery systems for tissue factor modulatory compounds include
ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable
infusion
systems, and liposomes. Formulations for inhalation may contain excipients,
for
example, lactose, or may be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily
solutions for administration in the form of nasal drops, or as a gel.
The formulations can be administered to human patients in therapeutically
effective
amounts (e.g., amounts which prevent, eliminate, or reduce a pathological
condition) to provide therapy for a disease or condition. The preferred dosage
of an
siRNA molecule of the invention is likely to depend on such variables as the
type
and extent of the disorder, the overall health status of the particular
patient, the
formulation of the compound excipients, and its route of administration.
In providing a mammal with the siRNA molecules of the present invention the
dosage of administered siRNAs will vary depending upon such factors as the
mammal's age, weight, height, sex, general medical condition, previous medical
history, disease progression, tumour burden', and the like. The dose is
administered
as indicated. Other therapeutic drugs may be administered in conjunction with
the
siRNA molecules. The pharmaceutical composition used for the treatment of
tumours may optionally contain other chemotherapeutic agents, antibodies,
antivirals, exogenous immunomodulators or the like. The pharmaceutical
composition used for the treatment of coagulation disorders may optionally
contain
additional thrombolytic agents or anticoagulants such as heparin.
The efficacy of treatment using the siRNAs described herein may be assessed by
determination of alterations in the expression, concentration, or biological
activity
of the DNA, RNA or gene product of TF; clot dissolution; clot prevention;
tumor
regression; metastasis regression; metastasis prevention; or a reduction of
the
pathology or symptoms associated with the tumour.
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27
Pr°epar°atioh of siRNA dir°ected towar°ds TF
In order to provide the siRNAs to obtain silencing of human TF (hTF), 21-
nucleotide RNAs were chemically synthesised using phoshoramidites (Pharmacia
and ABI) as described in PCT/N003/00045. Thirteen siRNAs against hTF mRNA
(Wiiger MT, Pringle S, Pettersen KS, Narahara N, Prydz H. Effects of binding
of
ligand (FVIIa) to induced tissue factor in human endothelial cells. Thr~ornb
Res. 98,
311-321 (2000) were synthesised (Fig.la). Eight of these siRNAs are termed SEQ
ID NO 1 to SEQ ID NO 8, respectively. The present invention relates inter alia
to
the use of the synthesised siRNAs according to SEQ ID NO 1 to SEQ ID NO 31
disclosed in PCT/N003/00045.
Furthermore, the invention relates to novel murin siRNA molecules and the use
thereof, specifically siRNA sequences having the nucleic acid sequence
depicted
in SEQ ID NO 32 to SEQ ID NO 37, the complement of which is SEQ ID NO 48
to SEQ ID NO 53, respectively.
Various siRNAs directed toward TF have also been mapped more systematically.
To avoid affecting the duplex stability of the siRNA, only GC pairs were
targeted
for mutation, by inversion of the pairs as described in example 5 below.
The reporter constructs of human TF to be used in the Dual Luciferase system
(Promega) were designed using the coding region of TF which were cloned in-
frame with the Firefly luciferase (LUC) gene, producing the fusion construct
TF-
LUC (Acc. No. AF416989). Numbering of the fusion construct refers to that of
the
genbank entry for TF and to the pGL3-enhancer plasmid (Promega) for LUG. The
plasmid pcDNA3-Rluc (Acc. No. AF416990), encoding Renilla luciferase (Rluc;
not shown) was used as internal control. Regions of TF and LUC cDNA contained
within the construct are indicated in Figure lb. The Dual. Luciferase system
is a
reporter system which is used to detect how much TF mRNA that is degraded by
siRNA(s).
HeLa, Cos-1 and 293 cells were maintained in Dulbecco's Minimal Essential
Medium (DMEM) supplemented with 10% fetal calf serum (Gibco BRL). The
human keratinocyte cell line HaCaT was cultured in serum free keratinocyte
medium supplemented with 2,5 ng/ml epidermal growth factor and 25 ~g/ml
bovine pituitary extract. All cell lines were regularly passaged at sub-
confluence.
The day before the experiment cells cultured in DMEM were trypsinized and
resuspended in full medium before plating. HaCaT cells were trypsinized until
detachment. Trypsin inhibitor was then added and the cells centrifuged for 5
min
at 400x g before resuspension in supplemented medium and plating. Cells were
transfected one or two days later.
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28
Lipofectamine-mediated transient co-transfections were performed in triplicate
in
12-well plates with 0,40 ~,g/ml plasmid (0,38 ~,g/ml reporter and 20 nghnl
control)
and typically 30 nM siRNA (0,43 ~g/ml) essentially as described (Amarzguioui
M. et al. (2000), sups°a). Luciferase activity levels were measured on
25 ~,l cell
lysate 24 h after transfection using the Dual Luciferase assay (Promega).
Serial
transfections were performed by transfecting initially with 100 nM siRNA,
followed by transfection with reporter and internal control plasmids before
harvest
time points.
For Northern analyses, HaCaT cells in 6-well plates were transfected with 100
nM
siRNA in serum-free medium. Lipofectamine2000TM was used for higher
transfection efficiency. Poly(A) mRNA was isolated 24 h after transfection
using
Dynabeads oligo(dT)ZS (Dynal). Isolated mRNA was fractionated for 16-18 h on
1,3% agarose/formaldehyde (0,8 M) gels and blotted on to nylon membranes
(MagnaCharge, Micron Separations Inc.). Membranes were hybridised with
random-primed TF (position 61-1217 in cDNA) and GAPDH (1,2 kb) cDNA
probes in PerfectHyb hybridisation buffer (Sigma) as recommended by the
manufacturer.
For TF activity measurements HaCaT cell monolayers were washed thrice with
ice-cold barbital buffered saline (BBS) pH 7,4 (BBS, 3 mM sodium barbital, 140
mM NaCI) and scraped into BBS. Immediately after harvesting and
homogenisation the activity was measured in a one-stage clotting assay using
nonnal citrated platelet poor plasma mixed from two donors and 10 mM CaClz.
The activity was related to a standard (Wiiger MT, Pringle S, Pettersen KS,
Narahara N, Prydz H. Effects of binding of ligand (FVIIa) to induced tissue
factor in
human endothelial cells. The°on2b Res. 98, 311-321 (2000), Camerer E,
Pringle S,
Skartlien AH, Wiiger M, Prydz K, Kolsto AB, Prydz H. Opposite sorting of
tissue
factor in human umbilical vein endothelial cells and Madin-Darby canine kidney
epithelial cells. Blood. 88, 1339-1349 (1996). One unit (U) TF corresponds to
1,5 ng
TF as detennined in the TF ELISA (Wiiger MT et al., (2000), sups°a, and
Camerer
E. et al., (1996), sups°a). The activity was normalised to the protein
content in the
cell homogenates, as measured by the BioRad DC assay.
TF antigen was quantified using the Imubind Tissue Factor ELISA kit (American
Diagnostics, Greenwich, CT, USA). This ELISA recognises TF apoprotein, TF
and TF:Coagulation Factor VII (FVII) complexes. The samples were left to thaav
at 37°C and homogenised. An aliquot of each homogenate (100 ~1) was
diluted in
phosphate-buffered saline containing 1% BSA and 0,1% Triton X-100. This
sample was then added to the ELISA-well and the procedure from the
manufacturer followed. The antigen levels were normalised to the total protein
content in the cell homogenates.
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WO 2005/040187 PCT/N02004/000238
29
All the various mutant siRNAs were analysed for depletion of endogenous TF
mRNA in HaCaT cells, 24h after LIPOFECTAMINE2000-mediated transfection,
as described previously for the wild type siRNA sequences.
The preparation of a pharmaceutical composition according to the present use
of
the invention may be provided by using techniques well-known to the person
skilled in the art. The composition may comprise one or more of said siRNAs,
and
optionally diluents, lubricants, binders, carriers disintegration and/or
absorption
means, colourings, sweeteners flavourings etc., all known in the art.
Furthermore,
the said composition may also comprise adjuvants and/or other therapeutical
principles, and may be administered alone or together with other
pharmaceuticals.
Said composition may be used before, simultaneous or after conventional cancer
treatment regimes, e.g. cytostatica treatments, radiation etc. Said
composition may
also be used before, simultaneous or after surgical intervention, e.g. to
prevent
metastasis from remnants of the primary tumour.
A pharmaceutical composition prepared according to the present use may be
administered e.g. parenterally (e.g. by subcutaneous, intravenous,
intramuscular or
intraperitoneal injection or infusion of sterile solutions or suspensions),
orally
(e.g. in the form of capsules, tablets, pills, suspensions or solutions),
nasally (e.g.
in form of solutions/spray), buccally, rectally (e.g. in the form of
suppositories),
vaginally (e.g. in the form of suppositories), by inhalation or insufflation
(e.g. in
the form of an aerosol or solution/spray), via an implanted reservoir, or by
any
other suitable route of administration, in dosage formulations containing
conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and/or
vehicles. The pharmaceutical composition may further be administered in one
dose, in divided doses or by way of sustained release devices.
EXAMPLES
The invention will now be described by way of examples. Although the examples
represent preferred embodiments of the present inventions, they are not to be
contemplated as restrictive to the scope of the present invention.
In order to obtain siRNAs that provide silencing of human TF, siRNAs according
to SEQ ID NO 1 to SEQ ID NO 31 and the novel murine TF (mTF) siRNA
sequences according to SEQ ID NO 32 to SEQ ID NO 37 were chemically
synthesised according to the method described in PCT/N003/00045.
Double-stranded siRNA complementary to a certain partial sequence on the
targeted TF mRNA sequence induces degradation of this specific mRNA in
mammalian cells (see Example 1). This effect was highly sequence-dependent,
and
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WO 2005/040187 PCT/N02004/000238
contrary to data in lower organisms, as only a few sites on the TF mRNA were
highly susceptible to the corresponding siRNAs. As can be seen from Example 2
the depletion of TF .mRNA results in marked reduction of TF protein and
procoagulant activity.
5 All the mTF siRNA sequences target site lie within a 200 by region
corresponding
to the region harbouring the best siRNA targets in hTF (cf. PCT/N003/00045).
Specifically, three different target sites, shifted 3 by relative to each
other, were
designed against the locus corresponding to the hTF167i. Where possible, the
positioning of the siRNA within each locus has been chosen to achieve the
highest
10 possible match with the human sequence. All siRNAs are synthesized with 2
by
target specific ribonucleotide overhangs (i.e. no DNA in the ends). SEQ ID NO
32-37 showed significantly metastasis reducing activity in a mouse model as
described in Example 7.
15 Example 1 Analysis of hTF siRNA efficacy in cells transiently cotransfected
with hTF-LUC and hTF siRNA (i.e. analysis of RNAi by siRNA(s) in
cotransfection assays)
The initial analysis of TF siRNA efficacy was performed in HeLa cells
transiently
cotransfected with hTF-LUC (Fig.lb) and hTF siRNA (Fig.la) using the Dual
20 Luciferase system (Promega). Ratios of LUC to Rluc expression were
normalised
to levels in cells transfected with a representative irrelevant siRNA, Protein
Serine
Kinase 3141 (PSK314i).
The siRNAs had potent and specific effects in the cotransfection assays, with
the
best candidates, hTF167i and hTF372i, resulting in only 10-15 % residual
25 luciferase activity in HeLa cells (Fig.lc). Furthermore, also a positional
effect was
found, as hTF562i .showed only intermediate effect, and hTF478i had very low
activity. This pattern was also found in 293, COS-1 and HaCaT cells (Fig.lc),
and
with siRNAs from different synthetic batches and at various concentrations
(the
siRNAs caused the same degree of inhibition over a concentration range of 1-
100
30 nM in cotransfection assays; data not shown).
Coculturing siRNA transfected cells with reporter plasmid transfected cells,
both
in HeLa cells and in the contact-inhibited growth of HaCaT cells, gave no
indication of siRNA transfer between cells (data not shown), despite the
medium-
mediated transfer previously reported by other investigators (Caplen, N. 3.,
Fleenor,
J., Fire, A. & Morgan, R. A. dsRNA-mediated gene silencing in cultured
Drosophila
cells: a tissue culture model for the analysis of RNA interference. Gene 252,
95-105
(2000).
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Example 2 Investigation of siRNA position-dependence at codon-level
resolution
The accessibility of the region surrounding the target site of the best siRNA
(i.e.
hTF167i) at a higher resolution was investigated. siRNAs (hTF158i, hTF161i,
hTF164i, hTF170i, hTF173i and hTFl76i) were synthesized which targeted sites
shifted at both sides of hTF 167i in increments of 3 nts, wherein each of them
shared 18 out of 21 nts with its neighbours (see Fig. 1 c). Surprisingly it
was found
that despite the minimal sequence and position-differences between these
siRNAs,
they displayed a wide range of activities (Figure 2). There was a gradual
change
away from the full activity of hTFl67i that was more pronounced for the
upstream
siRNAs. The two siRNAs hTFl58i and hTF161i were shifted only nine and six
nucleotides away, respectively, from hTFl67i, yet their activity was severely
diminished. These results suggest that local factors) caused the positional
effect.
Example 3 Analysis of hTF siRNA efficacy on endogenous mRNA
The results of cotransfection assays involving the use of forced expression of
reporter genes as substrates may be difficulf to interpret. The effect of
siRNA was
therefore also measured on endogenous mRNA targets in HaCaT cells (Fig. 3a)
which express TF constitutively. The two best TF siRNAs, hTF167i and hTF372i,
showed strong activity also in this assay, as normalised TF mRNA was reduced
to
10% and 26%, respectively (Fig. 3a). Interestingly, cleavage products, whose
sizes
were consistent with primary cleavages at the target sequences, were clearly
visible below the depleted main band, though cleavage assays of mRNA based on
RNAi have so far failed in mammalian systems (Tuschl T, Zamore PD, Lehmann R,
Bartel DP, Sharp PA. Targeted mRNA degradation by double-stranded RNA in
vitro.
,Genes Dev. 13, 3191-3197 (1999)). Thus,~the,present invention also relates to
siRNA which is able to cleave mRNA in mammalian cells. Furthermore, the
observed effect suggests that RISC may be active also in mammals. The third
best:
siRNA in cotransfection assays, hTF256i, also resulted in significant
depletion of
TF mRNA levels (57% residual expression, data not shown). The remaining TF
siRNA did not show any activity as measured by Northern assays (Fig. 3b), nor
did they stimulate TF expression, a point of some interest, as transfection
with
chemically modified ribozymes can induce TF mRNA three-fold (data not shown).
Thus, this relative inertness of irrelevant siRNAs (i.e. siRNAs with «non-
specific»
effects) further enhances the promise of siRNA-based drugs.
The coagulation activity in the HaCaT cells was reduced 5-fold and 2-fold,
respectively, in cells transfected with hTF167i and hTF372i, compared to mock-
transfected cells (Fig.3b~and Fig.Sb). The effect of siRNAs on total cellular
TF
protein was also measured (Fig. 3b), and demonstrated an inhibitory effect
that
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was generally greater than the observed effect on procoagulation activity. We
therefore conclude that the siRNAs hTF167i and hTF372i display specificity and
potency in a complex physiological system, and that we have demonstrated
positional effects, as other siRNA molecules against the same taxget mRNA are
basically inactive. Data from a new series of TF siRNA are in support of this
conclusion (data not shown), and this inactivity of certain siRNAs might be
due to
mRNA .folding structure or blockage of cleavage sites by impenetrable protein
coverage (Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T.
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian
cells. Natuf~e 411, 494-498 (2001)).
Example 4 Analysis of the time-course and persistence of siRNA silencing
The time-course of mRNA silencing was measured, and Northern analysis of cells
harvested 4, 8, 24 and 48 hours after start of transfection showed maximum
siRNA
1S silencing after 24 hours (Fig. Sa). There seemed to be a difference in the
apparent
depletion rate, as hTFl67i reduced the mRNA level more than hTFl73i at each
time-point. Similar observations were made for modified versions of hTF 167i,
in
which the induced mutations (M1 and M2) resulted in reduced inhibitory
activity.
Mutations in the anti-sense strand had a more pronounced effect than the
corresponding mutations in the sense strand. The fact that siRNA-induced
target
degradation was incomplete (a level of approximately 10% of the target znRNA
remained even with the most effective, siRNAs), may be due to the presence of
a
fraction of mRNA in a protected compartment, e.g. in spliceosomes or in other
nuclear locations. However, in view of the above data and data from
competition
2S experiments, a more likely possibility may be a kinetically determined
balance
between transcription and degradation, the latter being a time-consuming
process.
Experiments in plants (Palauqui JC, Balzergue S. Activation of systemic
acquired
silencing by localised introduction of DNA. Cure Biol. 9, S9-66 (1999) and
nematodes (Fire, A. RNA-triggered gene silencing. Ti~e~zds Gefzet. 15, 358-363
(1999), Grishok A, Tabara H, Mello CC. Genetic requirements for inheritance of
RNAi in C. elegans. Seie~zce 287, 2494-2497 (2000)) have suggested the
existence of
a system whereby certain siRNA genes are involved in the heritability of
induced
phenotypes. To investigate the existence of such propagators in mammalian cell
lines, the persistence of the siRNA silencing in HaCaT cells transfected at a
very
3S low cell density was measured. In an experiment based on serial
transfection of
reporter constructs thexe was a gradual recovery of expression between 3 and S
days post-transfection, and the time-dependence of the siRNA effect on
endogenous TF mRNA was similar (Fig. Sb). The level of TF mRNA in mock-
transfected control cells declined gradually during the experiment, in what
appeared to be cell expansion-dependent down-regulation of expression.
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Interestingly, the procoagulant activity showed little indication of
recovering to
control levels in transfected cells (Fig. Sb, columns). Similar observations
were
made with hTF372i and with a combination of hTF167i, hTF372i and hTF562i
(data not shown).
Example 5 Analysis of the effect of introducing base-pairing mutations in the
siRNA sequences.
As mentioned previously, siRNA were mapped more systematically in order to
detennine whether mutations were equally tolerated within the whole siRNA. A
total of 8 different new single-mutant siRNA were designed and named according
to the position (starting from the 5' of the sense strand) of the mutation
(sl, s2, s3,
s4, s7, sl l, s13, s16, i.e. SEQ ID NO 9- 17). The previously described
central
single-mutant Ml (example 4) was included in this analysis and renamed s10.
All
siRNAs were analysed for productive annealing by denaturing PAGE (15%).
All the various mutant siRNAs were analysed for depletion of endogenous TF
mRNA in HaCaT cells, 24h after LIPOFECTAMINE2000-mediated transfection,
as previously described. A summary of the data is shown in figure 7. The wild
type siRNA, and the mutant s 10, included as positive controls, depleted TF
mRNA
to ca 10% and 20% residual levels, as expected and previously reported. The
activities of the other mutants fall in three different groups depending on
their
position along the siRNA. Mutations in the extreme 5' end of the siRNA (sl-s3)
were very well tolerated, exhibiting essentially the same activity as the wild
type.
Mutations located further in, up to the approximate midpoint of the siRNA (s4,
s7,
s 10, s 11 ), were slightly impaired in their activity, resulting in depletion
of mRNA
to 25-30% residual levels. Both the mutations in the 3' half of the siRNA,
however, exhibited severely impaired activity. This suggested to us a bias in
the
tolerance for mutations in the siRNA. The activities of several double
mutants, in
which the central position (s10) was mutated in conjunction with one
additional
position (s7, sl 1, s13, sl6), were also analysed. The bias in mutation
tolerance
was also evident for these double mutants, as the rank order of their activity
mirrored that of the activity of the single mutants of the variant position.
This
observation strengthens the proposition that the differential activity of
mutants is
due to an intrinsic bias in the tolerance for target mismatches along the
sequence
of the siRNA. The reason for such a bias might be linked to the proposed
existence
of a ruler region in the siRNA which is primarily used by the RISC complex to
"measure up" the target mRNA for cleavage (15).
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Example 6 Effects of chemical modification of the siRNA sequences.
A series of siRNAs with one modification each in the extreme 5' and 3' ends of
the siRNA strands (P1+l, Ml+1, Al+l, i.e. SEQ ID NO 22, the compliment of
which is. SEQ ID NO 38, SEQ ID NO 26, the compliment of which is SEQ ID NO
42 and SEQ ID NO 30, the compliment of which is SEQ ID NO 46, respectively)
was initially synthesized. The 5' end of the chemicallysynthesized siRNAs
might
be more sensitive to modification since it has to be phosphorylated in vivo to
be
active (Nykanen, A., Haley, B. and Zamore, P.D. (2001) ATP Requirements and
Small Interfering RNA Structure in the RNA Interference Pathway. Cell, 107:309-
321). We therefore also included siRNAs with two modifications only in the non-
base pairing 3' overhangs (siRNAs PO+2, MO+2 and AO+2, i.e. SEQ ID NO 23, 27
and 31, respectively, c~ figure 6),_which were known to be tolerant for
various
types of modifications (Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber
K,
Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured
mammalian cells. Nature 411, 494-498 (2001), Elbashir, S.M., Martinez,. J.,
Patkaniowska, A., Lendeckel, W. and Tuschl, T. (2001) Functional anatomy of
siRNAs for mediating effiecient RNAi in Drosophila melanogaster embryo lysate.
EMBO J.,-20:6877-6888, Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001)
RNA interference is mediated by 21 and 22 nt RNAs. Genres Dev., 15:188-200).
Northern analysis of transfected HaCaT cells demonstrated essentially
undiminished activity of all the modified siRNAs, with the exception of the
siRNA
with allylation at both ends (figure 8). Allyl-modification in the 3' end only
had
no effect on activity. The presence of a large substituent in 2'-hydroxyl of
5'
terminal nucleotide might interfere with the proper phosphorylation of the
siRNA
shown to be necessary by Nykanen et al (Nykanen, A., Haley, B. and Zamore,
P.D. (2001) ATP Requirements~and SW all Interfering RNA Structure in the RNA
Interference Pathway. Cell, 107:309-321).
We next wanted to know if any of these modifications were sufficient to
increase
the persistence of siRNA-mediated silencing. Endogenous TF mRNA recovers
gradually 3-5 days after transfection with wild type siRNA targeting hTFl67.
Transfecting HaCaT cells with active and chemically modified siRNA in
parallel,
we could not demonstrate any significant difference in the silencing
activities 3
and 5 days post-transfection (data not shown). The moderate modifications we
had
introduced, although exhibiting full initial activity, were therefore clearly
not
sufficient to substantially stabilize the siRNAs in vivo.
With the activity of the siRNA still intact after our initial moderate
modifications,
the degree of modifications was extended to include either two on both sides
or
two on the 5' end in combination with four in the 3' end. Due to the less
promising results with the allylated versions from the first series, and the
higher
cost associated with these modifications, we decided to restrict ourselves to
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phophorothioate modifications and methylations for the next series. The new
set of
siRNAs were likewise analysed for initial activity 24h following transfection
into
HaCaT cells. Normalized expression levels in cells transfected with modified
siRNAs~were slightly elevated, at 16-18% residual levels compared to 11% in
cells
5 transfected with wild type. The most extensively phosphorothioated siRNA
proved
to be toxic to cells, resulting in approximately 70% cell death compared to
mock-
transfected cells (measured as the expression level of the control mRNA
GAPDH).
Due to these complications, this siRNA species was not included in further
analysis. The remaining siRNA species were evaluated for increased persistence
of
10 silencing by analysing TF mRNA expression 5 days after a single
transfection of
100nM siRNA. At this point, TF expression in cells transfected with wild type
siRNA had recovered almost completely (80% residual expression compared to
mock-transfected cells) (figure 9a). In cells transfected with the most
extensively
modified siRNA (M2+4; SEQ ID NO 29, the compliment of which is SEQ ID NO
15 45), however, strong silencing was still evident (32% residual expression).
The
less extensively modified siRNA species (P2+2, M2+2; SEQ ID NO 24, the
compliment of which is SEG ID NO 40 and SEQ ID NO 28, the, compliment of
which is SEG ID NO 44, respectively), although less effective than Me2+4,
consistently resulted in lower TF expression 5 days post-transfection (55-60%)
20 than the wild type. Time-course experiments demonstrated that the wild type
siRNA was still the most effective 3 days post-transfection, when silencing
was
relatively unimpaired, but that silencing drops off at a much higher rate
thereafter
(figure 9b).
25 Example 7 siRNA toward murin TF reduces circulating malignant cells ability
to form pulmonary tumours.
The experiments were designed and carried out to investigate if knockdown of
_ Tissue Factor (TF) reduced the ability of tail vein - injected cells to
settle in the
pulmonary circulation and fonn lung tumors in mice. The cells were transfected
30 with siRNA against murine TF before injection of the knockdown cells, which
had
a reduction in their TF level down to 10 - 20 percent of control mouse before
injection. The mice were sacrificed 6 - 25 days after injection of the cells.
In each
mouse had 0.2 to 1.0 million cells in 0.2 ml medium been injected. All mice
used
.were C57B1. Three groups were tested in each experiment: Group 1:
pretreatment
35 of cells before injection was with siRNA against PSKHl, a serine kinase of
unknown function. Group 2: pretreatment of the cells with siRNA against murine
TF. Group: 3 pretreatment of the cells with siRNA against human TF. Number of
macroscopically visible tumours in the lungs was counted after autopsy of the
mice. Examples of the results are given in Table 1 and 2. Mice receiving cells
retreated with siRNA against murine TF (Group 2) develop a low number of
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tumours. Group 1 mice develop around 10 times more tumours than group 2, and
mice of group 3 have more than 500 tumours in their lungs. Since the effect of
siRNA on its target mRNA is highly specific (if the siRNA sequence has been
properly selected to not bind other nucleotide sequences), the group 3 mice
may
have up to 200 - 250 - fold increase in their lung tumours when compared to
mice
in group 2. The experiments show that the level of TF in the injected cells
shows a
highly specific effect i.e. that of increasing the ability to form tumours in
the
lungs.
Thus, siRNA directed towards TF are useful in the preventions of metastasis
and
may be used in the treatment of cancer in mammals. Highly specific TF siRNA
molecules adapted to the TF sequence of a specific species may be found by the
screening method disclosed in PCT/N004/00007.
Table 1: Number of pulmonary metastases 10 days after injection of TF
siRNA.transfected B16 cells in C57 mice.
Group Mouse Mouse Mouse Mouse Mouse Mean
1 2 3 4 5
1 82 11 5 79 36 43
2 12 0 37 64 4 23
3 188 127 13' 404 263 199
Table 2: Number of pulmonary metastases 15 days after injection of TF
siRNA.transfected B 16 cells in C57 mice.
Group Mouse Mouse Mouse Mouse Mouse Mouse Mean
1 2 3 4 5 6
1 239 174 56 79 66 342 159
2 17 12 2 13 11 10 11
3 > 500 > 500 > 500 > 500 > 500 > 500 > 500
ConfluentConfluentConfluentConfluentConfluentConfluentConfluent
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Example 8 siRNA toward murin TF reduces circulating malignant cells ability
to form pulmonary tumours.
This example represents a follow-up of example 7. We designed eight siRNA
specific for murine TF (mTF), targeting sites located within 200 by
corresponding
to the region harbouring the best siRNA targets in hTF (Holen, T. et al.
(2002),
supra). Lipofectamine2000-mediated transfection of B 16 cells with 100 nM
siRNA demonstrated a highly variable efficiency of the different target
sequences,
consistent with previous observations (Holen, T. et al. (2002),
sups°a). The two
most effective siRNA, mTF223i and mTF321i, consistently depleted mTF mRNA
by 70-80% in cultured B 16 cells.
Tail vein injections have been assumed to represent a model for tumor take of
blood-borne metastases. We used a well established model in which tail-vein
injection of B16-F10 (B16) murine melanoma cells into C57BL/6 mice results in
pulmonary colonization within 10-14 days. Experiments were designed and
carried
out to investigate if knockdown of Tissue Factor (TF) in B 16 cells in vitro
reduced
the tendency for pulmonary metastasis following intra tail-vein injection. The
day
before injection, cells were transfected with a control siRNA against hTF
(hTF167i) or with either of two different siRNA targeting mTF (mTF223i,
mTF321i). Although highly active against its intended target (Holen, T. et al.
(2002), sups°a), the control siRNA hTFl67i contained substantial
mismatches
against mTF, and had no effect at all on mTF expression in cultured B 16
cells.
A total of three independent blinded experiments were performed, with at least
five mice in each experimental group and harvesting time point. Mice were
harvested on day 10 in the first experiment, on days 10 and ~15 in the second
experiment, and on days 15 and 20 in the third experiment. The data from all
experiments are summarized in Table 1. A picture of representative lungs from
mice treated fromythe test and control groups harvested at days 10 and 15 is
shown
in Figure 10. Both groups of mice that were treated with cells transiently
transfected with active (mTF) siRNA, and therefore exhibiting reduced
expression
of TF, developed significantly less tumors than the control group of mice at
all
time-points investigated. Thus, our experiments demonstrate that a single
liposome-mediated transfection of B 16 cells with active mTF siRNA in
vita°o
results in a target-sequence specific delay in development of pulmonary tumors
of
intravenously injected cells. This is directly attributable to the transient
knockdown of TF expression.
The window of protection achieved by the single administration of siRNA in
vitro
was estimated by observing the survival of mice injected with control- and
test-
transfected cells. Five or 6 mice in each group were inspected several times
daily
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and sacrificed at the first.indication of tumor-associated stress. The average
survival of the mice increased significantly (P=0.01), from 22 days for the
control
group to 27 days for mice injected with active mTF223i siRNA.
In conclusion, our results clearly demonstrate that TF has a crucial function
in
promoting lung tumor metastasis of B 16 melanoma cells in the C57BL/6 mice.
Thus, siRNA directed towards TF is useful in the prevention of metastasis and
may be used in the treatment of cancer in mammals. Highly specific TF siRNA
molecules adapted to the TF sequence of a specific species may be found by the
screening method disclosed in PCT/N004/00007.
Table 1. Tumor incidence from all experiW ents. The average number of tumors
and the total number of mice included in the analysis (parentheses) are given
for
each experimental group and harvest time point. The level of significance of
the
differences in tumors for test (mTF223i, mTF321i) and control (hTF167i) groups
are indicated by asterisks (*: P=0.01, **: P<0.001). n.d.: not determined.
siRNA Day 10 Day I S Day 20
hTF167i 107 (n=12) >500 (n=l1) >500 (n=5)
mTF223i 10* (n=12) 33** (n=11) 74** (n=8)
mTF321i n.d. 16** (n=5) 41** (n=5)
Example 9. Effect of systemic application of siRNA
In example 7 and 8, it is demonstrated that knockdown of TF prevents
colonization of the lungs by B 16 melanoma cells. The inventors have
furthermore
demonstrated that that the effect of siRNA targeting TF is evident also after
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systemic injection of siRNA, thus allowing potential therapeutic use of the
siRNA
(cf figure 11). hTF167i was used as a negative control.
An injection mixture was prepared for intraperitonal (i.p.) injection in a
total
volume of l5ml for each mixture of mTF siRNA and hTF siRNA:
~ 1050 ~,l annealed siRNA (SO~.M, 50 pg/ml) + 6500 p.l OptiMEM
~ 1500 ~.l Lipofectamine200 + 6000 ~,l OptiMEM
The two solutions were combined after 5 minutes and then incubated for 20,
minutes before injection. In the injection, mixture for the controls siRNA was
replaced by 10 mM Tris pH 7.5.
In the in vivo animal experiments, mice were divided into groups of ten
animals
each. The groups received one i.v. injection with B16 melanoma cells. The test
group additionally received i.p injections of the injection mixture comprising
the
TF targeting siRNA molecules to be tested. One injection were administered one
day previous to the administration of the B16 melanoma cells, and additionally
one inj ection the third and sixth day after said melanoma cell inj ection,
respectively.
The results shown figure 11 demonstrate that siRNA directed towards TF inhibit
colonization of the lungs by B 16 melanoma cells and thus that siRNA silencing
is
useful in prevention of metastasis.
