EP1660658A2 - The use of sirna silencing in the prevention of metastasis - Google Patents

Abstract

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.

Description

The Use 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 interference

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 methylation (Bernstein E, Caudy AA, Hammond SM, Harmon 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 Ill-like; RNase Ill-like) activity that processes dsRNA into sense and antisense RNAs which are 21 to 25 nucleotides (nt) long, i.e. siRNA. In Drosophila.this RNase Ill-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 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 Drosophila (Boutla, A., Delidakis, C, Livadaras, I., Tsagri.s, M. and Tabler, M. (2001), "Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila.", Curr. Biol, 11:1776-1780)). In contrast, Elbashir et al. found that a single mismatch was deleterious to activity in an in vitro Drosophila embryo lysate assay (Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W. and Tuschl, T. (2001), "Functional anatomy of siRNAs for mediating effϊecient 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.", Nature, 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, 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), "TsglOl 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." Proc. 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).", Oncogene, 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., Harmon, G.J., Conklin, D.S. (2002) "Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.", Genes 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. Traditionally, chemical modification of nucleic acids has inter alia been used to protect single stranded nucleic acid sequences against nuclease degradation and thus obtaining sequences with longer half life. For 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. 28, 4113-4124 (2000). However, little is known about the effects of chemical modifications of siRNAs. Further, the presence of large substituents in the 2'hydroxyl of the 5'terminal 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, 107:309-321).

Tissue Factor and metastasis 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 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 depends on angiogenesis. Although one might eliminate the primary tumour by surgery, there is always a risk that metastatic deposits already may exist 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 tissue factor, the principal initiator of blood coagulation.", Thromb Res. 81, 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 Vll/VIIa and the other coagulation factors. Expression of TF in the vascular compartment typically results in 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.", Current Opinion in Hematology, 3:379-384, Ohta et al.(2002), "Expression of Tissue Factor in Associated with Clinical Features and Angiogenesis in Prostate Cancer", Anticancer Research, 22:2991- 2996 (2002)., Bromberg et al. (1995), "Tissue Factor promotes melanoma metastasis by a pathway independent of blood coagulation", Proc.Natl.Acad.Sci. 92, 8205-8209, Konigsberg et al. (2001), "The TF:VIIa Complex: Clinical Significance, Structure-function Relationship and its Role in Signaling and Metastasis", Thromb, Haemost., 86:757-771).

Zhang et al. (1994) (Zhang et al (1994), J. Clin. Invest, 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.", Proc.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.", Proc.Natl.Acad.Sci, 98 (21): 10180-12185, A. Amirkhosravi et al. (2002), "Tissue Factor Pathway Inhibitor Reduces Experimental Lung Metastasis of B16 Melanomas.", Thromb. 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 Vila 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", Thromb Haemost, 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 silencing TF.

Patent application WO 01/75164 (A2) discloses a Drosophila in vitro 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. elegans unc-22 gene reduced the expression in vertebrate and invertebrate systems respectively (Caplen, N.J. et al. (2001), "Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.", Proc. Natl. Acad. Sci. USA, 98: 17, 9742-47). However, neither WO 01/75164 nor¹ Caplen et al. (2001), supra 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 inter 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.", Eur. 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.", Proc. Natl Acad. Sci. USA 97, 6499-6503 (2000). However, dsRNA cannot be used in mammalian cells because of unspecified effects. Furthermore, 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/NO03/00045, Holen, T. et al. (2000), "Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor", Nucleic Acid 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 Ill-like RNAi initiator Dicer and directly charge the effector nuclease RISC so that TF mRNA is degraded (PCT/NO03/00045). 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 in 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 for 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); (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 ' terminal 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 C]-C₃-alkyl, alkenyl or Cι-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, C C₃-alkenyl or Ci-C 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. Furtheπnore, 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 and/or 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 and/or by inhalation or insufflations.

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 Remington 's Pharmaceutical Sciences, (20^th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA. 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 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 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 vitro 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 end(s) of the 21 to 23 nucleotide RNA or internally (at one or more 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, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages. Collectively, all such altered RNAs are 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. 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., Nature 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 (Bach 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, Proc. 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. Proc. Natl. Acad. Sci-. 83, 299- 302 (1986) and Broze et al., J. Biol. Chem. 260, 10917-10920 (1985)); bovine brain (Bach et al, J. Biol. Chem. 256, 8324-8331 (1981)); human placenta (Bom et al., Thrombosis Res. 42:635-643 (1986); and, Andoh et al., Thrombosis Res. 43:275-286 (1986)); ovine brain (Carlsen et al., Thromb. Haemostas. 48, 315-319 (1982)); and lung (Glas, and Astrup Am. J. Ph siol. 219, 1 140-1146 (1970)). It has been shown that bovine and human tissue thromboplastin is identical in size and function (see for example Broze et al., J. Biol. Chem. 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 in vitro 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 in vitro coagulation assays, one-stage clotting assays, two-stage clotting assays (Pitlick and Nemerson, Methods En∑ymol, 45: 37-48 (1976)), TF clotting time assay (Santucci et al., Thromb. 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 Ml 6553 (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, uro genital 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 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 Ace. 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 hTF167i 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) hTF167i 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 hTF167i.

Figure 5 Time-dependence of siRNA-mediated RNAi; a) Inhibitory activity is reduced when mutations (Ml 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 noπnalised 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). Figure 6 siRNA modifications. (A) Mutated and wild type versions of the siRNA hTF167i. The sequence of the sense strand of wild type (wt) siRNA corresponds to position 167-187 in human Tissue Factor (Ass. No. M16553). Single (si, s2, s3, s4, s7, slO, sl l, sl3, sl6) and double mutants (ds 7/10, ds 10/11, dslO/13, dslO/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'-<9-mefhylated and 2'-( -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 lOOnM siRNA. B) Time-course of TF mRNA silencing. Cells harvested 1-3-5 days after single transfection of lOOnM 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. injection with B16 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. 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., Nature 411 :494- 498, 2001). The further therapeutic effectiveness of this approach in mammals was demonstrated in 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 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 may be designed using standard methods. Short hairpin RNAs (shRNAs) can also be used for RNAi as described in Paddison et al. (Proc. Natl. Acad. 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 forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to foπn a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to foπn a circular structure; open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as fonning the backbone of the oligonucleotide. The nonnal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage. 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 phosphor amidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having noπnal 3'-5' linkages, 2'-5' linked analogs of 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 fonns 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; 5,550,1 11; 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 internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; foπnacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyl eneimino 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; 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 -CH₂-NH-O-CH₂-, -CH₂-N(CH₃)-O-CH₂- (known as a methylene (methylimino) or MMI backbone), -CH₂-O-N(CH₃)-CH₂-, -CH₂-N(CH₃)-N(CH₃)- CH₂-, and -O-N(CH₃)-CH₂-CH₂-. 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 Ci to Cj_.o alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_nO]_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_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: Cj_. to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, NH₂, heterocycloalkyl, heterocycloalkaryl, amino alkyl amino, 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 pharmacodynamic properties of an nucleobase oligomer, and other substituents having similar properties. Preferred modifications are 2'-O- methyl and 2'-methoxyethoxy (2'-O-CH₂CH₂OCH₃, also known as 2'-O-(2- methoxyethyl) or 2'-MOE). Another desirable modification is 2'- dimethylaminooxyethoxy (i.e., O(CH₂)₂ON(CH₃)₂), also known as 2'-DMAOE. Other modifications include, 2'-aminopropoxy (2'-OCH₂CH₂CH₂NH₂) 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 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- trifluorom ethyl 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. I., 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 ; 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 palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1264:229-237, 1995), or an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 277:923-937, 1996. Methods for the preparation of nucleobase oligomer conjugates as mentioned above is disclosed in 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, 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. 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. 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; 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 synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Bio systems (Foster City, Calif). 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 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 term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active 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, calcium₅ 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. Pharma 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 foπn 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 "phaπnaceutical addition salt" includes a pharmaceutically acceptable salt of an acid foπn 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, maleic 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, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-l,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the foπnation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Phannaceutically acceptable salts of compounds may also be prepared with a phannaceutically acceptable cation.

Suitable phannaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations.

For oligonucleotides and other nucleobase oligomers, suitable phannaceutically acceptable salts include (i) salts fonned 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, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic 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, bromine, and iodine.

The present invention also includes phannaceutical compositions and fonnulations that include the nucleobase oligomers of the invention. The phaπnaceutical 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-pair 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 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 promoters U6 or HI, which are able to generate a transcript of defined sequence. An example of a suitable polymerase II promoter is the 763-base-ρair 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 pUCl 18, pBR322, or other known plasmid 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). The plasmid vector may also include a selectable marker such as the β lactamase gene for ampicillin 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. WO95/22618.

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 transfened 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, 6:682-690, 1988), Feigner and Holm, (Bethesda Res. Lab. Focus, 11 :21 , 1989) and Maurer (Bethesda Res. Lab. Focus, 1 1 :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 rule, 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., Proc. Natl. Acad. Sci. USA, 89:2581-2584, 1992; Stratford-Pereicadet et al, J. Clin. Invest, 90:626-630, 1992; and Rosenfeld et al., Cell, 68: 143-155, 1992). 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 HI, U6, BL, SMK, 7SK, tRNA polIII, fRNA(met)-derived, and T7 promoters, a cloning site for the stem-looped RNA coding insert, and a 4-5- thymidine transcription tennination 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- thymidine 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 for transfection, or introduction, of dsRNA into mammalian cells. For example, there are several commercially available transfection reagents including but not limited to: TransIT-TKO3 (Minis, Cat. # MIR 2150), Transmessenger3 (Qiagen, Cat. # 301525), and Oligofectamine3 (Invitrogen, Cat. # MIR 12252-011). 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 nM to 100 nM, and most preferably 1 nM to 50 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 noraially encode the small nuclear RNA U6 or the human RNAse P RNA HI. 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 (Miyagishi M. and Taira K., Nature Biotech., 20:497-500, 2002; Brummelkamp T.R. et al., Science, 296:550-553, 2002). If desired, stable cell lines for RNAi of TF can be generated using the above techniques. Assays for 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 β-gal reporter assays, western blots, and immunological methods such as ELISAs.

Therapeutic 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 present invention can be used to treat or prevent tumour metastasis in a wann- 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- 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. 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.

An siRNA molecule of the invention may be administered together with a phannaceutically acceptable diluent, carrier, or excipient, in unit dosage fonn. Conventional phannaceutical 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, intracistemal, 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 fonnulations, in the fonn 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. Methods well known in the art for making fonnulations 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 lactide 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 ethyl ene- 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 fonnulations 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 foraiulation 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 phaπnaceutical 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. Preparation of siRNA directed towards 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/NO03/00045. 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. Thromb 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/NO03/00045.

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 (Ace. No. AF416989). Numbering of the fusion construct refers to that of the genbank entry for TF and to the pGL3-enhancer plasmid (Promega) for LUC. The plasmid pcDNA3-Rluc (Ace. 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 epideπnal growth factor and 25 μg/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. Lipofectamine-mediated transient co-transfections were perfoπned in triplicate in 12-well plates with 0,40 μg/ml plasmid (0,38 μg/ml reporter and 20 ng/ l control) and typically 30 nM siRNA (0,43 μg/ml) essentially as described (Amarzguioui M. et al. (2000), supra). Luciferase activity levels were measured on 25 μl 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. Lipofectamine2000™ was used for higher transfection efficiency. Poly(A) mRNA was isolated 24 h after transfection using Dynabeads oligo(dT)₂₅ (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 NaCl) and scraped into BBS. Immediately after harvesting and homogenisation the activity was measured in a one-stage clotting assay using noπnal citrated platelet poor plasma mixed from two donors and 10 mM CaCl₂. 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. Thromb 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), supra, and Camerer E. et al., (1996), supra). 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 Diagnostica, Greenwich, CT, USA). This ELISA recognises TF apoprotein, TF and TF: Coagulation Factor VII (FVII) complexes. The samples were left to thaw at 37°C and homogenised. An aliquot of each homogenate (100 μl) was diluted in phosphate-buffered saline containing 1% BSA and 0,1% Triton X-100. This sample was then added to the ELIS A-well and the procedure from the manufacturer followed. The antigen levels were noπnalised to the total protein content in the cell homogenates. 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 phannaceutical 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, earners disintegration and/or absorption means, colourings, sweeteners flavourings etc., all known in the art. Furthennore, 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 fonn of solutions/spray), buccally, rectally (e.g. in the form of suppositories), vaginally (e.g. in the fonn 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 fonnulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and/or vehicles. The phaπnaceutical 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/NO03/00045. 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 contrary to data in lower organisms, as only a few sites on the TF mRNA were highly susceptible to the conesponding siRNAs. As can be seen from Example 2 the depletion of TF mRNA results in marked reduction of TF protein and procoagulant activity. All the mTF siRNA sequences target site lie within a 200 bp region corresponding to the region harbouring the best siRNA targets in hTF (cf. PCT/NO03/00045). Specifically, three different target sites, shifted 3 bp 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 possible match with the human sequence. All siRNAs are synthesized with 2 bp 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.

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 perfonned in HeLa cells transiently cotransfected with hTF-LUC (Fig. lb) and hTF siRNA (Fig. la) using the Dual Luciferase system (Promega). Ratios of LUC to Rluc expression were normalised to levels in cells transfected with a representative irrelevant siRNA, Protein Serine Kinase 314i (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 luciferase activity in HeLa cells (Fig. lc). Furthermore, also a positional effect was found, as hTF562i showed only intennediate 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 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. J., 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). 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 hTF176i) were synthesized which targeted sites shifted at both sides of hTF167i in increments of 3 nts, wherein each of them shared 18 out of 21 nts with its neighbours (see Fig. lc). 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 hTF167i that was more pronounced for the upstream siRNAs. The two siRNAs hTF158i and hTF161i were shifted only nine and six nucleotides away, respectively, from hTF167i, yet their activity was severely diminished. These results suggest that local factor(s) 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 difficult 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 nonnalised 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. Furtheπnore, 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.5b). The effect of siRNAs on total cellular TF protein was also measured (Fig. 3b), and demonstrated an inhibitory effect that was generally greater than the observed effect on pro coagulation 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 target 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. Nature 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 silencing after 24 hours (Fig. 5a). There seemed to be a difference in the apparent depletion rate, as hTF167i reduced the mRNA level more than hTF173i at each time-point. Similar observations were made for modified versions of hTF 167i, in which the induced mutations (Ml and M2) resulted in reduced inhibitory activity. Mutations in the anti-sense strand had a more pronounced effect than the coreesponding mutations in the sense strand. The fact that siRNA-induced target degradation was incomplete (a level of approximately 10% of the target mRNA 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 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. Curr Biol. 9, 59-66 (1999) and nematodes (Fire, A. RNA-triggered gene silencing. Trends Genet. 15, 358-363 (1999), Grishok A, Tabara H, Mello CC. Genetic requirements for inheritance of

RNAi in C. elegans. Science 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 low cell density was measured. In an experiment based on serial transfection of reporter constructs there was a gradual recovery of expression between 3 and 5 days post-transfection, and the time-dependence of the siRNA effect on endogenous TF mRNA was similar (Fig. 5b). 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. Interestingly, the procoagulant activity showed little indication of recovering to control levels in transfected cells (Fig. 5b, 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 (si, s2, s3, s4, s7, sl l, sl3, sl6, i.e. SEQ ID NO 9- 17). The previously described central single-mutant Ml (example 4) was included in this analysis and renamed slO. 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 LIPOFECTAMLNE2000-mediated transfection, as previously described. A summary of the data is shown in figure 7. The wild type siRNA, and the mutant slO, 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, slO, sl l), 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 (slO) was mutated in conjunction with one additional position (s7, sl l, si 3, si 6), 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). 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 (Pl+1, Ml+1, Al+1, 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 chemically synthesized 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, M0+2 and AO+2, i.e. SEQ ID NO 23, 27 and 31, respectively, cf. 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. Genes 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' tenninal 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 Small 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 hTF167.

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 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 1 1% in cells 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 silencing by analysing TF mRNA expression 5 days after a single transfection of lOOnM siRNA. At this point, TF expression in cells transfected with wild type siRNA had recovered almost completely (80% residual expression compared to mock-trans feet ed 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 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%) 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).

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 foπn lung tumors in mice. The' cells were transfected 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 of cells before injection was with siRNA against PSKH1, 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 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/NO04/00007.

Table 1: Number of pulmonary metastases 10 days after injection of TF siRNA.transfected B16 cells in C57 mice.

Table 2: Number of pulmonary metastases 15 days after injection of TF siRNA.transfected B16 cells in C57 mice.

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 bp conesponding to the region harbouring the best siRNA targets in hTF (Holen, T. et al. (2002), supra). Lipofectamine2000-mediated transfection of B16 cells with 100 nM siRNA demonstrated a highly variable efficiency of the different target sequences, consistent with previous observations (Holen, T. et al. (2002), supra). 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 canied out to investigate if knockdown of Tissue Factor (TF) in B16 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), supra), the control siRNA hTF167i contained substantial mismatches against mTF, and had no effect at all on mTF expression in cultured B16 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 from the 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 B16 cells with active mTF siRNA in vitro 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 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 B16 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/NO04/00007.

Table 1. Tumor incidence from all experiments. 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 15 Day 20

hTF167i 107 (n=12) >500 (n=l l) >500 (n=5)

mTF223i 10* (n=12) 33** (n=l l) 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 B16 melanoma cells. The inventors have furthennore demonstrated that that the effect of siRNA targeting TF is evident also after 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 15ml for each mixture of mTF siRNA and hTF siRNA: • 1050 μl annealed siRNA (50μM, 50 μg/ml) + 6500 μl OptiMEM • 1500 μl Lipofectamine200 + 6000 μl OptiMEM

The two solutions were combined after 5 minutes and then incubated for 2 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 injection the third and sixth day after said melanoma cell injection, respectively.

The results shown figure 11 demonstrate that siRNA directed towards TF inhibit colonization of the lungs by B16 melanoma cells and thus that siRNA silencing is useful in prevention of metastasis.

Claims

1. The use of one or more short interfering RNA molecule (siRNA) directed towards tissue factor (TF) for the preparation of a phannaceutical composition useful for the prevention of metastasis.

2. The use according to claim 1, wherein the TF or fragments thereof is of vertebrate origin, preferably mammalian origin, more preferably human origin.

3. The use according to any of the claims 1-2, wherein the siRNA is a single or double stranded siRNA comprising at least 19 nucleotides and which is 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% homologue to a siRNA molecule of (a); (c) a siRNA molecule which comprise a sequence having a target site which is shifted up to 7 nucleotides in either the 5' or 3' terminal 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 Cι-C₃-alkyl, Cι-C₃- alkenyl or Cι-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 .

4. The use according to claim 1, wherein said siRNA is double stranded.

5. The use according to claims 1, wherein said siRNA is 21-25 nucleotides long, preferably 21 nucleotides long.

6. The use according to any of the claims 1-5, wherein said siRNA is identified by SEQ ID NO 1 to SEQ ID NO 8.

7. The use according to any of the claims 1-6, wherein the siRNA induces cleavage of mRNA.

8. The use according to claim 7, wherein the siRNA is identified by SEQ ID NO 1 or SEQ ID NO 2.

9. The use according to claim 8, wherein the siRNA are the sequences as depicted in SEQ ID NO 10 to 31.

10. The use according to claim 9, wherein the sequences are modified by the introduction of a Cι-C -alkyl, Cι-C₃- alkenyl or Cι-C₃-alkylyl group in one or more of the 2' OH hydroxyl group in the sequence.

1 1. The use according to claim 10, wherein the siRNA the sequences depicted in SEQ ID NO 9, 10 OR 11.

12. The use according to claim 11, wherein the sequences are modified by replacing the phosphodiester bond with a thiophosphodiester bond.

13. RNA molecules (siRNA) according to claim 12, wherein the siRNA the sequences depicted in 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.

14. The use according to claim 13, wherein the siRNA the sequences depicted in SEQ ID NO 29, the complement of which is SEQ ID NO 45.

15. The use according to any of the claims 1-14, wherein the pharmaceutical composition optionally comprises e.g. diluents, lubricants, binders, carriers disintegration means, absorption means, colourings, sweeteners and/or flavourings.

16. The use according to any of the claims 1-15, wherein it comprises adjuvants and/or other therapeutically principles.

17. The use according to any of the claims 1-16 wherein the pharmaceutical composition is foπnulated for parenteral (subcutaneous, intravenous, intramuscular or intraperitoneal injection or infusion), oral, nasal, buccal, rectal, vaginal administration.

18. The use according to any of the claims 1-17, wherein said phaπnaceutical composition is formulated as e.g. infusion solutions or suspensions, an aerosol, capsules, tablets, pills, spray, suppositories etc., in dosage fonnulations containing conventional non-toxic phannaceutically-acceptable carriers, adjuvants and/or vehicles.

19. The use according to any of the claims 1-18, wherein said pharmaceutical composition is administered in one dose, in single or multiple doses or by sustained release formulations.

20. The use according to any of the claims 1-19, wherein said phannaceutical composition is administered alone or together with other pharmaceuticals .

21. The use according to any of the claims 1-5, wherein the siRNA is identified by SEQ ID NO 32 to SEQ ID NO 37, the complement of which is SEQ ID NO 48 to SEQ ID NO 53, respectively.

22. siRNA molecules characterized by 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.