AU784305B2

AU784305B2 - Treatment of cancer

Info

Publication number: AU784305B2
Application number: AU11169/01A
Authority: AU
Inventors: Levon Michael Khachigian
Original assignee: Unisearch Ltd
Current assignee: Unisearch Ltd
Priority date: 1999-10-26
Filing date: 2000-10-26
Publication date: 2006-03-09
Anticipated expiration: 2020-10-26
Also published as: AU1116901A

Description

WO 01/30394 PCT/AU00/01315 1 Treatment of cancer FIELD OF THE INVENTION The present invention relates to compositions and methods for the treatment of cancer.

BACKGROUND OF THE INVENTION Cancer Cancer accounted for over half a million deaths in the United States in 1998 alone, or approximately 23 of all deaths (Landis et al., 1998). Only cardiovascular disease consistently claims more lives (Cotran et al., 1999).

There is growing evidence that the cellular and molecular mechanisms underlying tumour growth involves more than just tumour cell proliferation and migration. Importantly, tumour growth and metastasis are critically dependent upon ongoing angiogenesis, the process of new blood vessel formation (Crystal, 1999). Angiogenesis (also known as neovascularisation) is mediated by the migration and proliferation of vascular endothelial cells that sprout from existing blood vessels to form a growing network of microvessels that supply growing tumours with vital nutrients. Primary solid tumours cannot grow beyond 1-2 mm diameter without active angiogenesis (Harris, 1998).

Human HepG2 hepatocellular carcinoma cells have been used as a model cancer cell line for the assessment of anti-neoplastic drugs (Yang et al., 1997). These cells basally and inducibly express the immediately-early gene and transcriptional regulator, early growth response factor-1 (EGR-1) (Kosaki et al., 1995).

Early Growth Response Protein (EGR-1) Early growth response factor-1 (EGR-1, also known as Egr-1, NGFI-A, zif268, krox24 and TIS8) is the product of an immediate early gene and a prototypical member of the zinc finger family of transcriptional regulators (Gashler et al., 1995). Egr-1 binds to the promoters of a spectrum of genes implicated in the pathogenesis of atherosclerosis and restenosis. These WO 01/30394 PCT/AU00/01315 2 include the platelet-derived growth factor (PDGF) A-chain (Khachigian et al., 1995), PDGF-B (Khachigian et al., 1996), transforming growth factor-P1 (Liu et al, 1996,1998), fibroblast growth factor-2 (FGF-2) (Hu et al., 1994; Biesiada et al., 1996), membrane type 1 matrix metalloproteinase (Haas et al., 1999), tissue factor (Cui et al., 1996) and intercellular adhesion molecule-1 (Malzman et al., 1996). EGR-1 has also been localised to endothelial cells and smooth muscle cells in human atherosclerotic plaques (McCaffrey et al., 2000). Suppression of Egr-1 gene induction using sequence-specific catalytic DNA inhibits intimal thickening in the rat carotid artery following balloon angioplasty (Santiago et al., 1999a).

DNAzymes In human gene therapy, antisense nucleic acid technology has been one of the major tools of choice to inactivate genes whose expression causes disease and is thus undesirable. The anti-sense approach employs a nucleic acid molecule that is complementary to, and thereby hybridizes with, an mRNA molecule encoding an undesirable gene. Such hybridization leads to the inhibition of gene expression.

Anti-sense technology suffers from certain drawbacks. Anti-sense hybridization results in the formation of a DNA/target mRNA heteroduplex.

This heteroduplex serves as a substrate for RNAse H-mediated degradation of the target mRNA component. Here, the DNA anti-sense molecule serves in a passive manner, in that it merely facilitates the required cleavage by endogenous RNAse H enzyme. This dependence on RNAse H confers limitations on the design of anti-sense molecules regarding their chemistry and ability to form stable heteroduplexes with their target mRNA's. Antisense DNA molecules also suffer from problems associated with non-specific activity and, at higher concentrations, even toxicity. An example of an alternative mechanism of antisense inhibition of target mRNA expression is steric inhibition of movement of the translational apparatus along the mRNA.

As an alternative to anti-sense molecules, catalytic nucleic acid molecules have shown promise as therapeutic agents for suppressing gene expression, and are widely discussed in the literature (Haseloff (1988); Breaker (1994); Koizumi (1989); Otsuka; Kashani-Sabet (1992); Raillard (1996); and Carmi (1996)). Thus, unlike a conventional anti-sense molecule, a catalytic nucleic acid molecule functions by actually cleaving its target WO 01/30394 PCT/AU00/01315 3 mRNA molecule instead of merely binding to it. Catalytic nucleic acid molecules can only cleave a target nucleic acid sequence if that target sequence meets certain minimum requirements. The target sequence must be complementary to the hybridizing arms of the catalytic nucleic acid, and the target must contain a specific sequence at the site of cleavage.

Catalytic RNA molecules ("ribozymes") are well documented (Haseloff (1988); Symonds (1992); and Sun (1997)), and have been shown to be capable of cleaving both RNA (Haseloff (1988)) and DNA (Raillard (1996)) molecules.

Indeed, the development of in vitro selection and evolution techniques has made it possible to obtain novel ribozymes against a known substrate, using either random variants of a known ribozyme or random-sequence RNA as a starting point (Pan (1992); Tsang (1994); and Breaker (1994)).

Ribozymes, however, are highly susceptible to enzymatic hydrolysis within the cells where they are intended to perform their function. This in turn limits their pharmaceutical applications.

Recently, a new class of catalytic molecules called "DNAzymes" was created (Breaker and Joyce (1995); Santoro (1997)). DNAzymes are singlestranded, and cleave both RNA (Breaker (1994); Santoro (1997)) and DNA (Carmi (1996)). A general model for the DNAzyme has been proposed, and is known as the "10-23" model. DNAzymes following the "10-23" model, also referred to simply as "10-23 DNAzymes", have a catalytic domain of deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. In vitro analyses show that this type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions under physiological conditions (Santoro (1997)).

DNAzymes show promise as therapeutic agents. However, DNAzyme success against a disease caused by the presence of a known mRNA molecule is not predictable. This unpredictability is due, in part, to two factors. First, certain mRNA secondary structures can impede a DNAzyme's ability to bind to and cleave its target mRNA. Second, the uptake of a DNAzyme by cells expressing the target mRNA may not be efficient enough to permit therapeutically meaningful results.

WO 01/30394 PCT/AU00/01315 4 SUMMARY OF THE INVENTION The present inventors have established that EGR-1 is critical in vascular endothelial cell replication and migration and that DNA-based, sequencespecific catalytic molecules targeting EGR-1 inhibit the growth of malignant cells in culture. These findings show that inhibitors of EGR or related EGR family members are useful in the treatment of tumours and that two separate mechanisms of action may involved. Specifically, inhibitors of EGR family members may inhibit tumour growth indirectly by inhibiting angiogenesis and/or directly by blocking the EGR family member in tumour cells.

When used herein the term "EGR" refers to a member of the EGR family.

Members of the EGR family are described in Gashler et al., 1995 and include EGR-1 to EGR-4. It is currently preferred that the EGR family member is EGR-1.

Accordingly, in a first aspect the present invention provides a method for the treatment of a tumour, the method comprising administering to a subject in need thereof an agent which inhibits induction of EGR, an agent which decreases expression of EGR or an agent which decreases the nuclear accumulation or activity of EGR.

In a second aspect, the present invention provides a method for inhibiting the growth or proliferation of a tumour cell, the method comprising contacting a tumour cell with an agent which inhibits induction of EGR, an agent which decreases expression of EGR or an agent which decreases the nuclear accumulation or activity of EGR.

In a third aspect, the present invention provides a tumour cell which has been transformed by introducing into the cell a nucleic acid molecule, the nucleic acid molecule comprising or encoding an agent which inhibits induction of EGR, (ii) an agent which decreases expression of EGR, or (iii) an agent which decreases the nuclear accumulation or activity of EGR.

In a fourth aspect, the present invention provides a method of screening for an agent which inhibits angiogenesis, the method comprising testing a putative agent for the ability to inhibit induction of EGR, decrease expression of EGR or decrease the nuclear accumulation or activity of EGR.

In a preferred embodiment of the present invention the agent is selected from the group consisting of an EGR antisense oligonucleotide, a ribozyme targeted against EGR, a ssDNA targeted against EGR dsDNA such that the ssDNA forms a triplex with the EGR-1 ds DNA, and a DNAzyme targeted against EGR.

WO 01/30394 PCT/AU00/01315 BRIEF DESCRIPTION OF THE FIGURES Figure 1. Insulin stimulates Egr-1-dependent gene expression in vascular endothelial cells. Growth-arrested bovine aortic endothelial cells previously transfected with pEBSl'foscat using FuGENE6 were incubated with D-glucose (5-30 mM), insulin (100 nM) or FGF-2 (25 ng/ml) as indicated for 24 h prior to preparation of cell lysates. CAT activity was normalized to the concentration of protein in the lysates.

Figure 2. Insulin-induced DNA synthesis in aortic endothelial cells is blocked by antisense oligonucleotides targeting Egr-1. A, Insulin stimulates DNA synthesis. Growth-arrested endothelial cells were incubated with insulin (100 nM or 500 nM) or FBS for 18 h prior to 'H-thymidine pulse for a further 6 h. B, Antisense Egr-1 oligonucleotides inhibit insulin-inducible DNA synthesis.

Endothelial cells were incubated with 0.8 pM of either AS2, AS2C or E3 prior to exposure to insulin (500 nM or 1000 nM) for 18 h and 'H-thymidine pulse for 6 h. C, Dose-dependent inhibition of insulin-inducible DNA synthesis. DNA synthesis stimulated by insulin (500 nM) was assessed in endothelial cells incubated with 0.4 pM or 0.8 p.M of AS2 or AS2C. TCA-precipitable 'Hthymidine incorporation into DNA was assessed using a p-scintillation counter.

Figure 3. Insulin-inducible DNA synthesis in cultured aortic endothelial cells is MEK/ERK-dependent. Growth quiescent endothelial cells were preincubated for 2 h with either PD98059 (10 p/M or 30 SB202190 (100 nM or 500 nM) or wortmannin (300 nM or 1000 nM) prior to the addition of insulin (500 nM) for 18 h and 3 H-thymidine pulse. TCA-precipitable 3 H-thymidine incorporation into DNA was assessed using a p-scintillation counter.

Figure 4. Wound repair after endothelial injury is potentiated by insulin in an Egr-1-dependent manner. The population of cells in the denuded zone 3 d after injury in the various groups was quantitated and presented histodiagrammatically.

Figure 5. Human microvascular endothelial cell proliferation is inhibited by DNA enzymes targeting human EGR-1. SV40-transformed HMEC-1 cells were grown in MCDB 131 medium with EGF (10 ng/ml) and hydrocortisone (1 Ag/ml) WO 01/30394 PCT/AU00/01315 6 supplements and 10% FBS. Forty-eight hours after incubation in serum-free medium without supplements, the cells were transfected with the indicated DNA enzyme (0.4 pjM) and transfected again 72 h after the change of medium, when 10% serum was added. The cells were quantitated by Coulter counter, 24 h after the addition of serum.

Figure 6. Sequence of NGFI-A DNAzyme (ED5), its scrambled control and 23 nt synthetic rat substrate. The translational start site is underlined.

Figure 7. NGFI-A DNAzyme inhibits the induction of NGFI-A protein by serum (FBS). Western blot analysis was performed using antibodies to NGFI-A, Spl or c-Fos. The Coomassie Blue stained gel demonstrates that uniform amounts of protein were loaded per lane. The sequence of EDC is 5'-CGC CAT TAG GCT AGC TAC AAC GAC CTA GTG AT-3' (SEQ ID NO:1); 3' T is inverted.

SFM denotes serum-free medium.

Figure 8. SMC proliferation is inhibited by NGFI-A DNAzyme. a, Assessment of total cell numbers by Coulter counter. Growth-arrested SMCs that had been exposed to serum and/or DNAzyme for 3 days were trypsinized followed by quantitation of the suspension. The sequence of AS2 is 5'-CTT GGC CGC TGC CAT-3' (SEQ ID NO:2) b, Proportion of cells incorporating Trypan Blue after exposure to serum and/or DNAzyme. Cells were stained incubated in 0.2% (w:v) Trypan Blue at 22 °C for 5 min prior to quantitation by hemocytometer in a blind manner. c, Effect of ED5 on pup SMC proliferation. Growth-arrested WKY12-22 cells exposed to serum and/or DNAzyme for 3 days were resuspended and numbers were quantitated by Coulter counter. Data is representative of 2 independent experiments performed in triplicate. The mean and standard errors of the mean are indicated in the figure. indicates P<0.05 (Student's paired ttest) as compared to control (FBS alone).

Figure 9. NGFI-A DNAzyme inhibition of neointima formation in the rat carotid artery. A neointima was achieved 18 days after permanent ligation of the right common carotid artery. DNAzyme (500 tpg) or vehicle alone was applied adventitially at the time of ligation and again after 3 days. Sequence-specific inhibition of neointima formation. Neointimal and medial areas of 5 consecutive WO 01/30394 PCT/AU00/01315 7 sections per rat (5 rats per group) taken at 250 I/m intervals from the point of ligation were determined digitally and expressed as a ratio per group. The mean and standard errors of the mean are indicated by the ordinate axis. denotes P<0.05 as compared to the Lig, Lig+Veh or Lig+Veh+EDSSCR groups using the Wilcoxen rank sum test for unpaired data. Lig denotes ligation, Veh denotes vehicle.

Figure 10. HepG2 cell proliferation is inhibited by 0.75piM of DNAzyme DzA.

Assessment of total cell numbers by Coulter counter. Growth-arrested cells that had been exposed to serum and/or DNAzyme for 3 days were trypsinized followed by quantitation of the suspension. The sequence of DzA is caggggacaGGCTAGCTACAACGAcgttgcggg (SEQ ID NO:3).

WO 01/30394 PCT/AU00/01315 8 DETAILED DESCRIPTION OF THE INVENTION In a first aspect the present invention provides a method for the treatment of a tumour, the method comprising administering to a subject in need thereof an agent which inhibits induction of an EGR, an agent which decreases expression of an EGR or an agent which decreases the nuclear accumulation or activity of an EGR.

The method of the first aspect may involve indiract inhibition of tumour growth by inhibiting angiogenesis and/or direct inhibition by blocking EGR in tumour cells.

In a preferred embodiment of the first aspect, the tumour is a solid tumour. The tumour may be selected from, without being limited to, a prostate tumour, a hepatocellular carcinoma, a skin carcinoma or a breast tumour.

As will be recognised by those skilled in this field there are a number means by which the method of the present invention may be achieved.

In a preferred embodiment of the present invention, the EGR is EGR-1.

In one embodiment, the method is achieved by targeting the EGR gene directly using triple helix (triplex) methods in which a ssDNA molecule can bind to the dsDNA and prevent transcription.

In another embodiment, the method is achieved by inhibiting transcription of the EGR gene using nucleic acid transcriptional decoys.

Linear sequences can be designed that form a partial intramolecular duplex which encodes a binding site for a defined transcriptional factor. Evidence suggests that EGR transcription is dependent upon the binding of Spl, AP1 or serum response factors to the promoter region. It is envisaged that inhibition of this binding of one or more of these transcription factors would inhibit transcription of the EGR gene.

In another embodiment, the method is achieved by inhibiting translation of the EGR mRNA using synthetic antisense DNA molecules that do not act as a substrate for RNase H and act by sterically blocking gene expression.

In another embodiment, the method is achieved by inhibiting translation of the EGR mRNA by destabilising the mRNA using synthetic antisense DNA molecules that act by directing the RNase H-mediated degradation of the EGR mRNA present in the heteroduplex formed between the antisense DNA and mRNA.

WO 01/30394 PCT/AU00/01315 9 In one preferrede embodiment of the present invention, the antisense oligonucleotide has a sequence selected from the group consisting of ACA CTT TTG TCT GCT (SEQ ID NO:4), and (ii) CTT GGC CGC TGC CAT (SEQ ID NO:2).

In another embodiment, the method is achieved by inhibiting translation of the EGR mRNA by cleavage of the mRNA by sequence-specific hammerhead ribozymes and derivatives of the hammerhead ribozyme such as the Minizymes or Mini-ribozymes or where the ribozyme is derived from: the hairpin ribozyme, (ii) the Tetrahymena Group I intron, (iii) the Hepatitis Delta Viroid ribozyme or (iv) the Neurospera ribozyme.

It will be appreciated by those skilled in the art that the composition of the ribozyme may be; made entirely of RNA, (ii) made of RNA and DNA bases, or (iii) made of RNA or DNA and modified bases, sugars and backbones Within the context of the present invention, the ribozyme may also be either; entirely synthetic or (ii) contained within a transcript from a gene delivered within a virusderived vector, expression plasmid, a synthetic gene, homologously or heterologously integrated into the patients genome or delivered into cells ex vivo, prior to reintroduction of the cells of the patient, using one of the above methods.

In another embodiment, the method is achieved by inhibition of the ability of the EGR gene to bind to its target DNA by expression of an antisense EGR-1 mRNA.

In another embodiment, the method is achieved by inhibition of EGR activity as a transcription factor using transcriptional decoy methods.

In another embodiment, the method is achieved by inhibition of the ability of the EGR gene to bind to its target DNA by drugs that have preference for GC rich sequences. Such drugs include nogalamycin, hedamycin and chromomycin A3 (Chiang et al J. Biol. Chem 1996; 271:23999).

WO 01/30394 PCT/AU00/01315 In a preferred embodiment, the method is achieved by cleavage of EGR mRNA by a sequence-specific DNAzyme. In a further preferred embodiment, the DNAzyme comprises a catalytic domain which cleaves mRNA at a purine:pyrimidine cleavage site; (ii) a first binding domain contiguous with the 5' end of the catalytic domain; and (iii) a second binding domain contiguous with the 3' end of the catalytic domain, wherein the binding domains are sufficiently complementary to two regions immediately flanking a purine:pyrimidine cleavage site within the region of EGR mRNA corresponding to nucleotides 168 to 332 as shown in SEQ ID NO:15, such that the DNAzyme cleaves the EGR mRNA.

As used herein, "DNAzyme" means a DNA molecule that specifically recognizes and cleaves a distinct target nucleic acid sequence, which may be either DNA or RNA.

In a preferred embodiment, the binding domains of the DNAzyme are complementary to the regions immediately flanking the cleavage site. It will be appreciated by those skilled in the art, however, that strict complementarity may not be required for the DNAzyme to bind to and cleave the EGR mRNA.

The binding domain lengths (also referred to herein as "arm lengths") can be of any permutation, and can be the same or different. In a preferred embodiment, the binding domain lengths are at least 6 nucleotides.

Preferably, both binding domains have a combined total length of at least 14 nucleotides. Various permutations in the length of the two binding domains, such as 7+7, 8+8 and 9+9, are envisioned.

The catalytic domain of a DNAzyme of the present invention may be any suitable catalytic domain. Examples of suitable catalytic domains are described in Santoro and Joyce, 1997 and U.S. Patent No. 5,807,718. In a preferred embodiment, the catalytic domain has the nucleotide sequence GGCTAGCTACAACGA (SEQ ID Within the context of the present invention, preferred cleavage sites within the region of EGR mRNA corresponding to nucleotides 168 to 332 are as follows: the GU site corresponding to nucleotides 198-199; WO 01/30394 WO 0130394PCT/AUOO/01315 11 (ii) the CU site corresponding to nucleotides 200-201; (iii) the CU site corresponding to nucleotides 264-265; (iv) the AU site corresponding to nucleotides 271-272; the AU site corresponding to nucleotides 292-293; (vi) the AU site corresponding to nucleotides 301-302; (vii) the GU site corresponding to nucleotides 303-304; and (viii) the AU site corresponding to nucleotides 316-3 17.

In a further preferred embodiment, the DNAzyme has a sequence selected from: 5'-caggggacaGGGTAGCTACAAGGAcgttgcggg (SEQ ID NO:3) targets CU (bp 198, 199); arms hybildise to bp 189-20 7 (ii) 5'-tgcaggggaGGCTAGGT'ACAACGAaccgttgcg (SEQ lID NO:6) targets CU (bp 200, 201); arms hybridise to bp 191-209 (iii) 5'-catcctggaGGGTAGGTACAACCAgagcaggct (SEQ ID NO:7) targets CU (bp 264, 265); arnis hybridise to bp 255-2 73 (iv) 5'-ccgcggccaGGCTAGCTAGAACGAcctggacga (SEQ ID NO:8) targets AU (bp 271, 272); arms hybridise to bp 262-280 5'-ccgctgccaGCCTAC C'fACAACGAcccggacgt (SEQ DD NO:9) targets AU (bp 271, 272); arms hybridise to bp 262-280 (vi) 5'-gcggggacaCGCTAGCTACAACGAcagctgcat (SEQ ID targets AU (bp 301, 302); arms hybridise to bp 292-310 (vii) 5'-cagcggggaCCCTrACTAGAACCAatcagctgc (SEQ ID NO:11) targets GU (bp 303, 304); arms hybridise to bp 294-3 12 (viii) 5'-ggtcagagaGCCTAGCTACAAGGActgcagcgg (SEQ ID NO:12) targets AU (bp 316, 317); arms hybridise to bp 307-325.

WO 01/30394 PCT/AU00/01315 12 In a particularly preferred embodiment, the DNAzyme targets the the GU site corresponding to nucleotides 198-199, the AU site corresponding to nucleotides 271-272 or the AU site corresponding to nucleotides 301-302.

In a further preferred embodiment, the DNAzyme has the sequence: 5'-caggggacaGGCTAGCTACAACGAcgttgcggg (SEQ ID NO:3), (SEQ ID (SEQ ID NO:8) or (SEQ ID NO:9).

In applying DNAzyme-based treatments, it is preferable that the DNAzymes be as stable as possible against degradation in the intra-cellular milieu. One means of accomplishing this is by incorporating a inversion at one or more termini of the DNAzyme. More specifically, a inversion (also referred to herein simply as an "inversion") means the covalent phosphate bonding between the 3' carbons of the terminal nucleotide and its adjacent nucleotide. This type of bonding is opposed to the normal phosphate bonding between the 3' and 5' carbons of adjacent nucleotides, hence the term "inversion". Accordingly, in a preferred embodiment, the 3'end nucleotide residue is inverted in the building domain contiguous with the 3' end of the catalytic domain. In addition to inversions, the instant DNAzymes may contain modified nucleotides. Modified nucleotides include, for example, N3'-P5' phosphoramidate linkages, and peptide-nucleic acid linkages. These are well known in the art.

In a particularly preferred embodiment, the DNAzyme includes an inverted T at the 3' position.

Although the subject may be any animal or human, it is preferred that the subject is a human.

Within the context of the present invention, the EGR inhibitory agents may be administered either alone or in combination with one or more additional anti-cancer agents which will be known to a person skilled in the art.

Administration of the inhibitory agents may be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, via implant, transmucosally, transdermally, topically, intramuscularly, subcutaneously or extracorporeally. In addition, the instant pharmaceutical compositions ideally contain one or more routinely used WO 01/30394 PCT/AU00/01315 13 pharmaceutically acceptable carriers. Such carriers are well known to those skilled in the art. The following delivery systems, which employ a number of routinely used carriers, are only representative of the many embodiments envisioned for administering the instant composition. In one embodiment the delivery vehicle contains Mg" or other cation(s) to serve as co-factor(s) for efficient DNAzyme bioactivity.

Transdermal delivery systems include patches, gels, tapes and creams, and can contain excipients such as solubilizers, permeation enhancers fatty acids, fatty acid esters, fatty alcohols and amino acids), hydrophilic polymers polycarbophil and polyvinylpyrolidone), and adhesives and tackifiers polyisobutylenes, silicone-based adhesives, acrylates and polybutene).

Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers propylene glycol, bile salts and amino acids), and other vehicles polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

Oral delivery systems include tablets and capsules. These can contain excipients such as binders hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents starch polymers and cellulosic materials) and lubricating agents stearates and talc).

Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents gums, zanthans, cellulosics and sugars), humectants sorbitol), solubilizers ethanol, water, PEG and propylene glycol), surfactants sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents EDTA).

Topical delivery systems include, for example, gels and solutions, and can contain excipients such as solubilizers, permeation enhancers fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers polycarbophil and polyvinylpyrolidone). In the preferred embodiment, the pharmaceutically acceptable carrier is a liposome or a WO 01/30394 PCT/AU00/01315 14 biodegradable polymer. Examples of carriers which can be used in this invention include the following: Fugene6® (Roche); (2) SUPERFECT'(Qiagen); Lipofectamine 2000®(GIBCO BRL); CellFectin, 1:1.5 liposome formulation of the cationic lipid N,NI,NII,NIIItetramethyl-N,NI,N,NIIII-tetrapalmitylspermine and dioleoyl phosphatidylethanolainine (DOPE)(GIBCO BRL); Cytofectin GSV, 2:1 liposome formulation of a cationic lipid and DOPE (Glen Research); DOTAP (2,3-dioleoyloxy)-N,N,N-trimethyl-ammoniummethylsulfate) (Boehringer Manheim); and Lipofectamine, 3:1 liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).

In a preferred embodiment, the agent is injected into or proximal the solid tumour. Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents ethanol, propylene glycol and sucrose) and polymers polycaprylactones and PLGA's).

Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone.

Delivery of the nucleic acid agents described may also be achieved via one or more, of the following non-limiting examples of vehicles: liposomes and liposome-protein conjugates and mixtures; non-liposomal lipid and cationic lipid formulations; activated dendrimer formulations; within polymer formulations such pluronic gels or within ethylene vinyl acetate coploymer (EVAc). The polymer may be delivered intra-luminally; within a viral-liposome complex, such as Sendai virus; or as a peptide-DNA conjugate.

Determining the prophylactically effective dose of the instant pharmaceutical composition can be done based on animal data using routine computational methods. In one embodiment, the prophylactically effective does contains between about 0.1 mg and about 1 g of the instant DNAzyme.

In another embodiment, the prophylactically effective dose contains between about 1 mg and about 100 mg of the instant DNAzyme. In a further embodiment, the prophylactically effective does contains between about mg and about 50 mg of the instant DNAzyme. In yet a further embodiment, WO 01/30394 PCT/AU00/01315 the prophylactically effective does contains about 25 mg of the instant DNAzyme.

It is also envisaged that nucleic acid agents targeting EGR may be administered by ex vivo transfection of cell suspensions, thereby inhibiting tumour growth, differentiation and/or metastasis.

In a preferred embodiment of the third and fourth aspects, the agent is selected from the group consisting of an EGR antisense oligonucleotide or mRNA, a sequence-specific ribozyme targeted against EGR, a ssDNA targeted against EGR dsDNA and a sequence specific DNAzyme targeted against EGR.

The putative agent may be tested for the ability to inhibit EGR by any suitable means. For example, the test may involve contacting a cell which expresses EGR with the putative agent and monitoring the production of EGR nRNA (by, for example, Northern blot analysis) or EGR protein (by, for example, immunohistochemical analysis or Western blot analysis). Other suitable tests will be known to those skilled in the art.

WO 01/30394 WO 0130394PCT/AUOO/01315 16 For reference, Table 1 below sets forth a comparison between the DNA sequences of mouse, rat and human EGR-1.

Table I MousP. Rat and HumnmarxER-1 Symbol comparison table: GenRunData:pileupdna.cmp GapWeight: 5.000 GapLengthWeight: 0.300 EGRliign.msf MSF: 4388 Type: N April 7, 1998 Name: mouseEGRI Len: 4388 Check: 8340 Weight: Name: ratEGRi Len: 4388 Check: 8587 Weight: Name: humanEGRi Len: 4388 Check: 8180 Weight: CompCheck: 6876 12:07 Check: 5107 1 00 (SEQ ID NO:13) 1. 00 (SEQ ID NO:14) 1. 00 (SEQ ID NB. THIS IS RAT NGFI-A numbering mou seEgr 1 ratNGFIA humanEGR1 mouseEGRi ratEGRi hurnanEGRi mouseEGR1 ratEGRl humanEGRI mouseEGR1 ratEGRi hunianEGRI iouseEGRi ratEGRI huxnanEGRl mouseEGR1 ratEGRi humanEGR1 inouseEGR1 ratEGR1 humanEGRl mouseEGR1 ratEGR1 humanEGRI

CCGCGGAGCC

51

CCGACTCCCG

101

GGTGGGTGCG

TCAGCTCTAC GCGCCTGGCG CCCTCCCTAC GCGGGCGTCC CGCGCGTTCA GGCTCCGGGT TGGGAACCAA CCGACCCGGA AACACCATAT AAGGAGCAGG 100

GGAGGGGGAG

150 AAGGAT CCC C 200

TGGCCCAATA

250

GGGTTGGGGC

300

CACTGCCGCT

CGCCGGAACA GACCTTATTT GGGCAGCGCC TTATATGGAG 201 TGGCCCTGCC GCTTCCGGCT CTGGGAGGAG GGGCGAACGG 251 GGGGGCAAGC TGGGAACTCC AGGAGCCTAG CCCGGGAGGC GTTCCAATAC TAGGCTTTCC AGGAGCCTGA GCGCTCAGGG TGCCGGAGCC 351

GGTCGCAGGG

400 TGGAAGCGCC CACCGCTCTT GGATGGGAGG TCTTCACGTC mouseEGRl WO 01/30394 WO 0130394PCT/AUOO/01315 ratEGRI humariEGRI ACTCCGGGTC CTCCCGGTCG GTCCTTCCAT ATTAGGGCTT CCTGCTTCCC mouseEGRi ratEGRI hwnmanEGRl mouseEGRi ratEGR1 humanEGR1 mouseEGRi ratEGRi huwnanEGRI mouseEGRi ratEGR1 humanEGRI mouseEGRi ratEGRi humanEGR1 mouseEGRi ratEGRi liumanEGRI iouseEGRi ratEGR1 humanEGRI mouseEGRi ratEGRi humanEGR1 ATATATGGCC ATGTACGTCA CGGCGGAGGC GGGCCCGTGC TGTTTCAGAC CCTTGAAATA GAGGCCGATT CGGGGAGTCG CGAGAGATCC 551

GGGGA

AACTTGGGGA

601

CGCAAGATCG

CGCAGGACCG

651

GGGCCGCGGC

CGCCCGCGCC

701

GCCCCTGCAC

TCCCC. GCGC 751

AGTAGCTTCG

TGCAGCTCCA

801

GCTCGCTGGT

GCTCGCACGT

GCCTGC2'CGT

GCCGCCGCCG

GCCCCTGCCC

TACCGCCAGC

CACCGCCAGC

CAGGGCGAGT

CCCGCATGTA

CCCGCATGrfA

CCCGCATGTA

GCCCCGGGCT

CCGGGATGGC

CCAGGA TGGC

CGATTCGCCG

CCATCCGCCG

CAGCCTCCGC

CTGGGGGCCC

CGGGGTCGCC

ACCCGGCCAA

ACCCGGCCAG

GCGCCCACC.

GCACCCCCCC

AGCGGCCAAG

CGCGGCCAAG

CCGCCGCCAG

CCGCAGCCAG

GGCAGCCCTG

AGCCGCGGCG

ACCTACACTC

GCCTGCACGC

CCCCCGGCGA

CATCCGGCGA

GCCCCCGCAA

ACCCAACAT

GCCCCGACAC

GCCGAGATGC

CAGCGCGCAG

CCGCAG

600

CTTCCGCCGC

650

CGTCCACCAC

CGTCCACGCC

700

CCCGCAGTGT

TTCTCAGTGT

750

GTGTGCCCTC

CGGTGTCCCC

800

CAGTTCTCCA

CAGCTCTCCA

850

AATTGATGTC

AGCTGATGTC

(rat) arms hybridise to bp 807-825 in rat sequ hEDS(hum) arms hybridise to bp 262-280 in hum sequ mouseEGRI ra tEGRi humanEGRI mouseEGRi ratEGRi hurnanEGRi mouseEGR1 ratEGR1 huxuanEGR1 851

TCCGCTGCAG

CCCGCTGCAG

901

TGGACAACTA

951

CCCCAGTTCC

ATCTCTGACC

CCCCAAACTG

CCCTAAGCTG

TCGGTGCTGC

TCGGCGCCGC

CGTTCGGCTC

CGTTCGGATC

GAGGAGATGA

CGGAACCCCA

CGGGGCCCCA

CTTTCCTCAC

TGCTGCTGAG

GAGGGCAGCG

900

TCACCCACCA

TCGCCCACCA

950

CAACGGGGCT

CAACGGGGCT*

1000

GCGGTAAT..

GCGGCAATAA

GCAGCAACAG

WO 01/30394 WO 0130394PCT/AUOO/01315 mouse EGR1 ratEGRi humanEGRi mouseEGRi rat EGRI huiuanEGRl mouseEGRI ratEGRi humanEGRI mouseEGRi ratEGR1 hunanEGRl rouseEGRl ratEGRi human EGRi mouse EGRi ratEGRI humanEGRi inous eEGRl ratEGR1 humanEGRI mouse EGR I ratEGRi huinanEGRi mouseEGRI ratEGRI humanEGR1 mouseEGRi ratEGRi hurnanEGRi mouseEGRI ratEGRi humanEGRi mouseEGRI ratEGRi humanEGRI 1001

.AGC

CAGCAGCAGC

1051

GCAACAGCGG

GCAGCAGCAG

1101

CCCTATGAGC

CCCTACGAGC

1151

TAATGAGAAG

TCCCCCTTCG

CAACGAGAAG

1201

TGCCTCCCAT

ATCTAGATCT

TGCCCCCCAT

1251

AGTGGCAACA

TCCAGGGACT

AGTGGCAACA

1301

CGTGAGCATG

CTTGCGGGTG

AGTGAGCATG

1351

CTGCTTCATC

TTGCGTGGGT

CGGCCTCCTC

1401

GTGCCGTCCA

GAGCAGGGTT

GTGCCATCCA

1451

TACTCCCAAC

TGCAGCTTGT

CACGCCGAAC

1501

GCTCGGCAGG

CCGCCCAGGG

GCTCGGCAGG

1551

AAAGGTGGTT

GGATTCCCTC

AAGGGTGGCT

AGCAGCAGCA

AGCAGCGGGG

CAGCAGCGCC

CAGCAGCGCT

CAGCAGCACC

ACCTGACCAC

ACCTGACCGC

GCGATGGTGG

TGACTACCCT

GTGCTGGTGG

CACCTATACT

TAGGGACGGG

CACCTATACT

CTTTGTGGCC

TGTGTTAGAG

CCTTGTGGCC

ACCAATCCTC

CGCGGAGGGC

ACCAACCCAC

GTCTTCCTCT

GGCT..

CGC... CTCC

ACGACAGCAG

GC. CCCC

ACGACAGCAG

ACTGACATTT

TCCCAAGGAA

ACTGACATTT

CACAGCCTTG

TAGGGGCGCG

GACAGCGCTC

TCCAGGTTCC

ACCCCGGACG

TCCAGGTTCC

CCAGCAGCGG

GCAGCAGCGG

GCGGTGGAGG

TTCAATCCTC

TTCAACCCTC

AG.. .AGTCC

AGGTAAGCGG

AG. AGTCT

AGACGAGTTA

AACGTCCAGT

AGACCAGTTA

GGCCGCTTCT

ATTGGGATTT

GGCCGCTTTT

TGAACCCCTT

GGATGTCTGG

CGAGCCCCTC

CGACCTCTTC

AGACCGTTTG

CGGCCTCC'rC

GCCTCCCAGA

GGAGT

GCCTCCCAGA

TCCCATCTAC

TCCCCCGCGC

TCCCATTTAC

TTCC'rGAGCC

GGGCTGAAAT

TCCCTGAGCC

CAGTACCCGC

CATTAGCTGT

CAGTACCCGC

CATGATCCCT

CCTGCTGCGG

CATGATCCCC

GGGCGGTGGT

CGGCGGGGGC

AAGGGGAGCC

AGGCGGACAC

TTTTCTGACA

TGGTCTGCGC

TTTCCTGACA

TCCCAGCCAA

CCTTTGCAGC

CCCCAGCC.AA

CCCTGGAGCC

CCCTCTATTC

CCCTGGAGCC

TTCAGCCTAG

GGACCCCCCA

TTCAGCTTGG

AT CCT CGGC C

TTTTGGATGG

GTCCTCAGCA

GCCCGCCCCT

GGGGGAGGGT

GCCCACCCCT

TCGGCTGCGC

GCGTTGTCGC

TCAGCGGCAC

CCAAAGCCAG

CTGTCACCAG

ACAAAGCCAG

CTCCTGCCTA

GGCC .ACTAG

CTCCTGCCTA

GACTATCTGT

AGCGCTCTCA

GACTACCTGT

1050

GGGGGCGGC-A

GGGGGCGGCA

GGCAGCAACA

1100

GAGCGAACAA

GGGCGAGCAG

1150

TCGCTCTGAA

CGAGGCTGAA

TCTCTCTGAA

1200

ACGACTCGGT

ACGGACCTGC

ACCACTCGAC

1250

CGCACCCAAC

.CACACAGC

TGCACCCAAC

1300

TCAGTGGCCT

ACCCTCCATC

TCAGTGGCCT

1350

CCTTCTCCAG

AGAACTCAAG

CCATCTCCAG

1400

GAGCTGTGCC

TTGTTTTGAT

GAGCTGCGCA

1450

CCACCTTTCC

GAGCCTTGTT

CCACCTTCCC

1500

GCCTTTCCTG

GGATGTCCCG

GCCTTCCCGG

1550

CCCTGCCACC

GGTGCTGGCG

CCCTGCCGCC

1600

TTCCACAACA

GAGCTGCAGT

TTCCACAGCA

WO 01/30394 WO 0130394PCT/AUOO/01315 mouseEGR1 rat EGRi humanEGRi mouse EGRI rat EGRI humanEGRI mouseEGRI ratEGRi humanEGRI mouseEGR1 ratEGRi humanEGRi mouseEGRi rat EGRI humnanEGRi mouse EGRi ra tEGRi humanEGRI mouse EGRi rat EGR1 humanEGRi mouseEGR1 ratEGR1 huinanEGRI mouseEGRi ratEGRi humanEGRi mou seEGR1 ratEGRI humanEGRI mouseEGR1 ratEGR1 humanEGRi mouseEGRi ratEGRI humanEGR1 1601

ACAGGGAGAC

AGAGGGGGAT

GCAGGGGGAT

1651

TGGAGAACCG

CACTGGAGCA

TGGAGAGCCG

1701

GCCTTCGCCA

CACATCGAGA

GCCTTTGCCA

1751

ATACCACCTA

CT CT CAT CGT

ATACCAGCTA

1801

ACCCCAACCG

TTCCTGCCAG

ATCCCAACCG

1851

CCTGTCGAGT

CTGGTGGAGA

CCAGTGGAGT

1901

CCATATCCGC

CTATACTGGC

CCACATCCGC

1951

TGCGTAACTT

TGTGGCCTGA

TGCGCAACTT

2001

ACAGGCGAGA

AACCCTCCAA

ACAGGCGAAA

2051

GAGTGATGAA

TTCCTCTGCC

GAGCGATGAA

2101

AGAAAGCAGA

ACAGCAGTCC

AGAAAGCAGA

CTGAGCCTGG

TCTCTGTTTG

CTGGGCCTGG

TACCCAGCAG

GGTCCAGGAA

CACCCAGCAG

CTCAGTCGGG

GTCAGTGGTA

CTCAGTCGGG

CCAATCCCAG

CCAGTGATTG

CCAGTCCCAG

GCCCAGCAAG

AGTCCTrTTTC

GCCCAGCAAG

CCTGCGATCG

CAAGTTATCC

CCTGTGATCG

ATCCACACAG

CGCTTCTCCC

ATCCACACAG

CAGTCGTAGT

ACCCCTTTTC

CAGCCGCAGC

AGCCTTTTGC

CCTCTTCATC

AGCCCTTCGC

CGCAAGAGGC

TCCCAGAGCC

CGCAAGAGGC

CAAAAGTGTG

CATTTACTCA

CAAAAGTGTT

GCACCCCAGA

CGTCAGCTGT

GCACCCCAGA

CCTTCGCTCA

CATTGCAATC

CCTTCGCTAA

CTCCCAGGAC

GCCGGGCGAC

CTCCCAGGAC

CTCATCA.. A

CTCTCCAGTA

CTCATCA. .A

ACACCCCCCC

TGACATCGCT

ACGCCCCCCC

CCGCTTTTCT

CAGCCAAACT

CCGCTTCTCC

GCCAGAAGCC

TGGAGCCTGC

GCCAGAAGCC

GACCACCTTA

AGCCTAGTCA

GACCACCTCA

CTGTGACATT

CTCAGCGCCT

CTGCGACATC

ATACCAAAAT

CACCCCTGAG

ATACCAAGAT

GTGGCCTCCC

GCTGCACCCA

GTGGCCTCTT

CTCTTCTTAC

GACATTTTTC

TACCTCTTAC

CCAGAAGCCC

CGAAATGGCT

CCAGAAGCCC

CTCCACTATC

TGCTGCTATC

CCCCTCTGTC

TTAAAG...

CTCTTGCCTG

CTGAAG...

ACCCAGCCGC

ACCAGGCCTC

ACCCAGCCGC

ATGAACGCCC

CTGAATAACG

ACGAACGCCC

CGCTCGGATG

ACCCGGTTGC

CGCTCCGACG

CTTCCAGTGT

ACCCAACAGT

CTTCCAGTGC

CCACCCACAT

GTGGCCTTGT

CCACCCACAT

TGTGGGAGGA

TCTCCAGCTG

TGTGGAAGAA

CCATTTAAGA

CTGTGCCGTG

CCACTTGCGG

CGGCTG

CCTTTCCTAC

CGGCCACCTC

CCATCCCCAG

CTGAGCCCCA

CCGTCCCCGG

1650

TTCCAGGGTC

CT GC

TTCCAGGGCC

1700

CACTATTAAA

AATTATTAAC

TACTATTAAG

1750

GCTCTTA

GCCGCTTCGG

GCCCTCA

1800

ATGCGCAAGT

TCTGTTCTCT

ATGCGCAAGT

1850

ATATGCTTGC

AGAAG. .GCG

TTACGCTTGC

1900

AGCTTACCCG

CTCCCATCAC

AGCTCACCCG

1950

CGAATCTGCA

GGCAACACTT

CGCATCTGCA

2000

CCGCACCCAC

GAGCATGACC

CCGCACCCAC

2050

AGTTTGCCAG

CTTCATCGTC

AGTTTGCCAG

2100

CAGAAGGACA

CCGTCCAACG

CAGAAGGACA

2150

CTCTTCACT

TCCCAACACT

CTCTCTCTCT

2200

TGGCTACCTC

AAGCCAGGCC

TTACTACCTC

2151 TCCTACCCGT CCCCGGTTGC WO 01/30394 WO 0130394PCT/AUOO/01315 mouseEGR1 ratEGR1 humanEGRi mouseEGRI ratEGRi huxnanEGRl mous eEGR1 ratEGR1 hutnanEGRi mouseEGRI ratEGRI humanEGRi mouseEGR1 ratEGRI humanEGRi inouseEGRI ratEGR1 huinanEGRi mouse EGRi ratEGRi humanEGR1 mouseEGR1 ratEGR1 humanEGRi mouseEGRi rat EGR1 humanEGRi mouseEGRi ratEGRi humanEGR1 mouseEGRI ratEGRi liumanEGRi mouseEGRi ratEGR1 humanEGR1 2201

CTACCCATCC

TTTCCTGGCT

TTAfC CAT CC 2251

ACTCCTCTCC

TGCCACCAAG

TCTCCTCTCC

2301

CCGTCGCCGT

CACAACAACA

CCCTCCCCGT

2351

ACCTGC

CAGGGTCTGG

CCTGC

2401

AGCAGCTCCT

TATCAAAGCC

ACCAACTCCT

2451

TTCTCCCAGG

ATAACACCTA

TTCTCCCAGG

2501

AAAGCAAAG

ACCCCAACC

AAAGGGAGAA

2551

AGATGGCCGC

CCCTGTTGAG

GCCATAGGAG

2601

CCAAGTCCTT

GCCACATCCG

CCAAGTCCTC

2651

TCACTTACCA

GCATGCGTAA

TTCTGCCCAC

2701

CTGAAACAGC

C. .ACACAGG

CTGAAACAGC

2751

GCATGG...

GCCAGGAGTG

GCATGGA...

CCTGCCACCA

CTGCAGGCAC

CCGGCCACCA

TGGCTCCTCC

GGTGGTTTCC

CGGCTCCTCG

CAGTGGCCAC

GGGAGACCTG

CGGTGGCCAC

TTTCCCCACC

AGAACCGTAC

TTTCCCGGCC

TCAGCACCTC

TTCGCCACTC

TCAGCGCCTC

ACAATTGAAA

CCAGTCCCAA

ACAATTGAAA

GGAGAGGCAG

GGCCCAGCAA

AAAGAAACAC

AAGAGGGGCC

TCCTGCGATC

AGGAGGGTT.

CTACTCACGA

CATCCATACA

CCTCTCTACT

TCCCTGCCTC

TTTCAGTCGT

TTCCCCTTCC

CATGTCCAAG

CGAGAAGCCT

CATGTCCAAG

*..TATTGGAT

ATGAACGCAA

*..TTTTGGAT

CCTCATTCCC

AGCCTTGCAG

CCTCATACCC

ACCTACCCAT

AGGTTCCCAT

ACCTACCCAT

CACCTTTGCC

AGCCTGGGCA

CACGTACTCC

CAGGTCAGCA

CCAGCAGCCT

CAGGTCAGCA

AACTGGTCTT

AGTCGGGCTC

CACAGGGCTT

TTTGCTAAAG

CTCATCAAAC

TTTGCTAAAG

GAAAGACATA

GACACCCCCC

AAGAGACTTA

ACCTCTTAGG

GCCGCTTTTC

CCTCTTAGG

GTA. .GAAGG

GGC..CAGAA

GGAGTGGAAG

CCCCGTCCTG

AGTGACCACC

CCAATTACTA

TTCTTCACCT

TTTGCCTGTG

TTCTTCACCT

AAATCATTTC

GAGGCATACC

AAATCATTTC

ATCCCCTGTG

TACCCGCCTC

ATCCCCTGTG

CTCCTGCGCA

GATCCCTGAC

CCCCTGTGCA

TCCGTTCC..

CCCCAGACCA

TCTGTTC CC.

GCTTCCCGTC

TCGCTCACTC

GCTTCCCTTC

TCAGACATGA

CCAGGACrTA

TCGGACATGA

2250

CCCACTTCCT

CTGCCTACCC

CCCACCTCCT

2300

CAGTGGCTTC

TATCTGTTTC

CAGTGGCTTC

2350

GAAGCCCTTC

2400

TGCGGGCGTC

CACTATCCAC

CTCAGCTGTC

2450

CAGCGACCTT

AAGGCTCTTA

CAGCAACCTT

2500 GGA ATAAAAG..

CCAGCCGCAT GCGCAAGT..

GGAAAGGGGA AAGAAAGGGA 2550 GC.. C AGGAGGGAAG CATGAACGCC CGTATGC'rTG AAGGACAGGA GGAGGAGATG 2600 TCAGATGGAA GATCTCAGAG TCGCTCGGAT GAGCTTACAC TCAGATGGAG GTTCTCAGAG

ACCGTTGGCC

GCCCTTCCAG

GTCTATTGGC

TTCCCTTTGA

TTACCACCCA

TTCCCTTTGA

CTATCCAAAG

ACATTTGTGG

CTATCCAAAG

AGTATCCTCT

AAAATCCACT

AGTATCATCT

2650

AACAGCCCTT

TGTCGAATCT

CAACAATCCT

2700

CTTCAGCTGC

CATCCGCACC

CTTCAGCTGC

2750

GACTTGATTT

GAGAAAGTTT

AACTTGATTT

2800

TAAGACAGAA

WO 01/30394 WO 0130394PCT/AUOO/01315 mouseEGRI rat EGRI hurnanEGRi rnous eEGRI ratEGRI humnanEGRi mouseEGRi ratEGRi humanEGR1 mous eEGR1 rat EGR1 humanEGRi mouseEGRi rat EGRi humanEGRi mouseEGRi ratEGRi hurnanEGRI mouseEGRi rat EGR 1 humanEGRi mouseEGRi ratEGR1 humanEGR1 mouseEGR1 rat EGRi humanEGRi mouseEGRi rat EGR1 huinanEGRI mouseEGR1 ratEGR1 humanEGRI mouseEGR1 ratEGRI humanEGR1 2801

CCATC

GGACAAGAAA

CCATCA

2851

GTTGGCATAA

TCTCTTCCTA

GTTGG 2901

CATCTTTGTA

ACCTCATTTC

CACCCTTGTA

2951

TTGATGTGAA

TACCTACCCG

TTGATGTGAA

3001

TAAATCCTCA

CCACCTATGC

TAGGTCCTCA

3051

ATGATCCTCT

TCCAGTCTGC

GTGATCCTCT

3101 GGTTrTGAAG

GACATGACAG

AGTTTGA

3151

.GCATGTGT

ATGAAAGAGA

GAGCATGTGT

3201

TGTAACTCT

GGAAGAAAT

ACCGTACTCT

3251 T'rTGAGAATT

CTCAGAGCCA

TTGAAAGTGT

3301

GTGCCTTTTG

TTTCACTTAG

GTGCCTTTTG

3351

GATGTGA

CTGCCTGAAA

TGTGA

ACATGCCTGG

GCAGACAAAA

TATGCCTGAC

AGAAAAAAAA

CCCATCCCCA

PAAA

CAGCATCTGT

CATCCCC*AGT

CAGTGTCTGT

GATAATTTGC

TCTCCTGCAC

GATAATTTGC

CTTTGGGG..

CTCCGTCC..

CTTGGGGGAA

ATTTTGTGAT

AGGGGTCAGC

ATTTTGTGAT

CATTTTTTTT

CAACCTTTTC

ACCTTTTTTT

CAGAGTGTTG

GCAAAGGGAG

CAGAGTGTTG

CAC:ATGTGAC

GGCCCGCAAG

CACATGTGGC

TTTTTGCCCG

AGTCCTTCTA

TTTT'rCTTCG

TGTGACACGC

CGTCCCTGCC

TGTGATGCCC

GGGACACGCT

CAGCCACGTC

GGGACATGCT

CCCTTGCTCC

GTGTCGTGGC

CCCTTGCTCC

ATGGGTTTGG

GTGGCTACCT

GGGGTTTGGG

GCCATGGATT

GCCCACCTCT

GCCATGGATT

ATACT

ACAGTGGCTT

ATATT

GAGGGGGGAG

CACCTGCTTT

AAAAAAAAAA

GACTCTGCTG

AACTCCTTCA

GATGCTGTGA

TTCAAGCAGC

TCCTAGGACA

TTGAAACAGC

TTCCGTTAAT

GGGAGCGCGA

TTCCGTTAAC

AAAGTATGGT

AGGGGCTGCC

AAAATATGGT

TCCCTTTGGT

GTCAGTAGAA

TCCTI'TTGGT

CTT.CCGATG

CTC .CCCAGT

CTTGCTGATG

CACCTTAGCC

CAAGTTCTTC

CACCTCTAGC

CTTCAGCGCT

CTCCTCAGCT

CTTCAATGCT

GCCCTCAGAA

CCTACCCATC

CCCCTCAGAG

TTGTTTTCCT

TACTCCTCTC

TCGTTTTTCT

CTATTGTAT

CCCATCGCCC

CTATTGTAT

CAAAGCCAAG

CCCTGCCCAG

AAAAGCCAAG

TGACATTA..

GCACCTCAAC

CAATA..

AGTCCTAGGT

ATTGAAATTT

AGTCCCAG..

TTTGTAAATA

GAGACAATAA

CTTTTTGTAA

TTGTTTGGTT

TCTTAGGTCA

TTGGTTTTTC

TTCAAAAGTT

GGCCCGTTGG

TTAAAAAGTT

GCTTGACATG

CCCGGTCCTT

GCTTGACATG

TTA... GGG ACCT. .CTA

CTTAAGGGGG

2850

AGACCATCAA

GCCTCTTCCC

AGAAAATCGA

2900

CCCTGCCCTG

CCCCGCCACC

CCCTGCCCTG

2950

TGGGGTATTC

CGGGCTCCTC

TGGGGTACTC

3000

TATTTGGAGT

TCGGTGGCCA

TATTTGGAGT

3050

CAAACCAATG

GTCAGCACCT

CAAACCAATG

3100

GGGTCTTTCA

3150

ATTAACTGGA

GCTAAAGGGA

.TATTCTCA

3200

CTGGCTCGAC

AGGACAGGAG

ATACTGCTTG

3250

GGGTTTTGTT

GATGGAAGAT

TTTTTTTTTT

3300

TCACGTCTTG

CCACCAGCCC

TCACGTCTTG

3350

CGCA..

TTGACTTCAG

TGCAAT....

3400

GGTAGGAGTG

TCCAAAGGAC

GCAGGGAGTG

WO 01/30394 WO 0130394PCT/AUOO/01315 mou seEGR1 ratEGRi humanEGRI mouseEGRI ratEGRI humanEGRI mouseEGR1 ra tEGRi humanEGRi mous eEGRI ratEGRI humanEGRi mous eEGR1 ra tEGRi humanEGR1 mouseEGRi ratEGRi hurnanEGR1 inouseEGR1 ratEGRI humanEGR1 inouseEGRI ratEGR1 humanEGRi ma us eEGR 1 rat EGRI huinanEGRI inouseEGRI ra tEGRI humanEGRi mouseEGR1 ratEGRi humanEGRI mouseEGRI ratEGRi hunanEGR1 3401

ATGTGTTGGG

TTGATTTGCA

ATGATTTGGG

3451

GCTTTCGGTC

GCCTGGCCCT

GCTTCGGTTC

3501

CTCTCAAAAG

AAAATGGGTC

CTCTCAAAAG

3551

TCAGGAGTTG

GCATCTGTGC

TCAGGAGTTG

3601

ATGTTATGAA

TAATTTGCAT

ATGTTATGAA

3651

TTGTGTTTGC

GAGGGGGAGC

TTGTGTTTGC

3701

GCGCTCTATT

ATCCTGCTGT

GCGCTTTATT

3751

TTAAAAGGAA

GTCCTAGGTA

TTAAAACGAA

GGAGGCTTGA

TGGTATTGGA

GGAGGCTTTG

TCCAGAATGT

TGCTCCCTTC

TCCAGAATGT

TCTATTTTTC

TGGGCCCTCA

TCTATTTTTT

GAGTGTTGTG

CATGGATTTT

GAATGTTGTA

CATGAAGTTC

ACTCTATTGT

CATGCAGTTC

TTAAACAAAG

AAAGCCAAGC

TTAAACAAAG

GCCCATGG..

GACATTAGGT

GCCCATGG..

AAAT..

TTAACTGGAG

AATAAAGTAG

GAGCAAAAAC

TAAACCATTT

GGAGCAAAAT

AAGAAGAAAA

AGCACTAGAA

AAGAAAACAA

TAAACTGAAA

GAACCCTGCC

TAA. CTGAAA

GTTACCTACT

GTTTTCCTTG

GTTACCTACT

ATTATTTTGT

ACTATTTGGA

ATTATTTTGT

TAACCTGTTT

AAACCAATGG

TGA. CTGTTT

*..GATATGTG

TTGAAACTTT

*..GATATGTG

3450 GAGGAAGAGG GCTGAGCTGA CAGCATCATC TCCACCACAT AAGGAAGAGG GCTGAGCTGA 3500 AATTfTAAACA AAAATCTGAA CATCAAGTTG GCTGAAAAAA AATCTAAAAC AAANFCTGAA

ATGTAAATTT

CTGTATCTTT

ATGTAAATTT

GAGTAGGCTG

GGGTATTCTT

GAGTAGGCGG

GGTTTTATTT

GTTAAATTCT

GGTTCTATTT

GGCTTATAAA

TGATCCTCTA

GGCTTATAAA

GTGTGTATCC

TTTTTTTTTT

GTGTATATCC

3550

ATACATCTAT

GTACA

ATAAATATAT

3600

CAGTTTTTGT

GATGTGAAGA

CGATTTTTGT

3650

TACTTTGTAC

CACTTTGGGG

TACTTTGTAC

3700

CACATTGAAT

TTTTGTGATG

CACATTGAAT

3750

TTCAGAAAAA

TGAAGCAGCA

TTCCAAAAAA

3800 CATGTGTCAG AGTGTTGTTC CGTTAATTTT CTGCGATTGG 3801 3850 GTAAATACTG CTCGACTGTA ACTCTCAC.AT GTGACAAAAT ACGGTTTGTT 3851 3900 TGGTTGGGTT TTTTGTTGTT TTTGAAAAAA AAATTTTTTT TTTGCCCGTC 3901 3950 CCTTTGGTTT CAAAAGTTTC ACGTCTTGGT GCCTTTGTGT GACACACCTT 3951 GCCGATGGCT GGACATGTGC 4000 AATCGTGAGG GGACACGCTC ACCTCTAGCC WO 01/30394 PCT/AU00/01315 mouseEGR1 ratEGR1 humanEGR1 mouseEGR1 ratEGR1 humanEGR1 mouseEGR1 ratEGRI humanEGRI mouseEGRI ratEGR1 humanEGR1 mouseEGR1 ratEGR1 huinanEGR1 mouseEGR1 ratEGR1 humanEGRI mouseEGR1 ratEGRI humanEGRI mouseEGR1 ratEGRI humanEGRI 4001 4050 I TTAAGGGGGT AGGAGTGATG TTTCAGGGGA GGCTTTAGAG CACGATGAGG 4051 4100 AAGAGGGCTG AGCTGAGCTT TGGTTCTCCA GAATGTAAGA AGAAAAATTT 4101 4150 AAAACAAAAA TCTGAACTCT CAAAAGTCTA TTTTTTTAAC TGAAAATGTA 4151 4200 GATTTATCCA TGTTCGGGAG TTGGAATGCT GCGGTTACCT ACTGAGTAGG 4201 4250 CGGTGACTTT TGTATGCTAT GAACATGAAG TTCATTATTT TGTGGTTTTA 4251 4300 TTTTACTTCG TACTTGTGTT TGCTTAAACA AAGTGACTTG TTTGGCTTAT 4301 4350 AAACACATTG AATGCGCTTT ACTGCCCATG GGATATGTGG TGTGTATCCT 4351 4388 TCAGAAAAAT ATAAAGAAAC TAACTGGT........

TCAGAAAAAT TAAAAGGAAA ATAAAGAAAC TAACTGGT WO 01/30394 PCT/AU00/01315 24 EXPERIMENTAL DETAILS EXAMPLE 1 Role of EGR-1 in endothelial cell proliferation and migration Materials and Methods Oligonucleotides and chemicals. Phosphorothioate-linked antisense oligonucleotides directed against the region comprising the translational start site of Egr-1 mRNA were synthesized commercially (Genset Pacific) and purified by high performance liquid chromatography. The target sequence of AS2 (5'-CsTsTsGsGsCsCsGsCsTsGsCsCsAsT-3') (SEQ ID NO:16) is conserved in mouse, rat and human Egr-1 mRNA. For control purposes, we used AS2C (5'-GsCsAsCsTsTsCsTsGsCsTsGsTsCsC-3') (SEQ ID NO:17), a size-matched phosphorothioate-linked counterpart of AS2 with similar base composition.

Phorbol-12-myristrate 13-acetate (PMA) and fibroblast growth factor-2 were purchased from Sigma-Aldrich.

Cell culture. Bovine aortic endothelial cells were obtained from Cell Applications, Inc. and used between passages 5-9. The endothelial cells were grown in Dulbecco's modified Eagles' medium (Life Technologies), pH 7.4, containing 10% fetal bovine serum supplemented with 50 /g/mL streptomycin and 50 IU/mL penicillin. The cells were routinely passaged with trypsin/EDTA and maintained at 37 0 C in a humidified atmosphere of C02/95% air.

Transient transfection analysis and.CATassay. The endothelial cells were grown to 60-70% confluence in 100mm dishes and transiently transfected with 10 g of the indicated chloramphenicol acetyl transferase (CAT)-based promoter reporter construct using FuGENE6 (Roche). The cells were rendered growth-quiescent by incubation 48 h in 0.25% FBS, and stimulated with various agonists for 24 h prior to harvest and assessment of CAT activity. CAT activity was measured and normalized to the concentration of protein in the lysates (determined by Biorad Protein Assay) as previously described (Khachigian et al., 1999).

Northern blot analysis. Total RNA (12 pg/well) of growth-arrested endothelial cells (prepared using TRIzol Reagent (Life Technologies) in WO 01/30394 PCT/AU00/01315 accordance with the manufacturer's instructions) previously exposed to various agonists for 1 h was resolved by electrophoresis on denaturing 1% agarose-formaldehyde gels. Following transfer overnight to Hybond- N+ nylon membranes (Amersham), the blots were hybridized with 3 2 P-labeled Egr-1 cDNA prepared using the Nick Translation Kit overnight (Roche). The membranes were washed and radioactivity visualized by autoradiography as previously described (Khachigian et al., 1995).

RT-PCR. Reverse transcription was performed with 8 pg of total RNA using M-MLV reverse transcriptase. Egr-1 cDNA was amplified (334 bp product (Delbridge et al., 1997)) using Taq polymerase by heating for 1 min at 94"C, and cycling through 94°C for 1 min, 94°C for 1 min, 55"C for 1 min, and 72"C for 1 min. Following thirty cycles, a 5 min extension at 72°C was carried out. Samples were electrophoresed on 1.5% agarose gel containing ethidium bromide and photographed under ultraviolet illumination. P-actin amplification (690 bp product) was performed essentially as above. The sequences of the primers were: Egr-1 forward primer (5'-GCA CCC AAC AGT GGC AAC-3') (SEQ ID NO:18), Egr-1 reverse primer (5'-GGG ATC ATG GGA ACC TGG-3') (SEQ ID NO:19), p-actin forward primer (5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA (SEQ ID NO:20), and P-actin reverse primer (5'-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3') (SEQ ID NO:21).

Antisense oligonucleotide delivery and Western blot analysis. Growtharrested cells in 100 mm dishes were incubated with the indicated oligonucleotides 24 h and 48 h after the initial change of medium. When oligonucleotide was added a second time, the cells were incubated with various concentrations of insulin and harvest 1 h subsequently. The cells were washed in cold phosphate-buffered saline (PBS), pH 7.4, and solubilized in RIPA buffer (150 mM NaCI, 50 mM Tris-HCl, pH 7.5, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, 1% trasylol, pg/ml leupeptin, 1% aprotinin, 2 pM PMSF). Lysates were resolved by electrophoresis on 8% denaturing SDS-polyacrylamide gels, transferred to PDVF nylon membranes (NEN-DuPont), blocked with skim milk powder, then incubated with polyclonal antibodies to Egr-1 (Santa Cruz Biotechnology, Inc) and monoclonal horseradish peroxidase-linked mouse anti-rabbit Ig secondary antibodies followed by chemiluminescent detection (NEN-DuPont).

WO 01/30394 PCT/AU00/01315 26 'H-Thymidine incorporation into DNA. Growth-arrested endothelial cells at 90% confluence in 96 well plates were incubated twice with the oligonucleotides prior to the addition of insulin. When signaling inhibitors (PD98059, SB202190, wortmannin) were used in experiments, these agents were added 2 h before the addition of insulin. After 18 h of exposure to insulin, the cells were pulsed with 200,000 cpm/well of methyl- 3 H thymidine (NEN-DuPont) for 6 h. Lysates were prepared by washing first in cold PBS, pH7.4, then fixing with cold 10% trichloroacetic acid, washing with cold ethanol and solubilizing in 0.1 M NaOH. 3 H-Thymidine in the lysates was quantitated with ACSII scintillant using P-scintillation counter (Packard).

In vitro injury. Growth-arrested cells at 90% confluence were incubated with antisense oligonucleotides and insulin at various concentrations as described above, then were scraped by drawing a sterile wooden toothpick across the monolayer (Khachigian et al., 1996). Following 48-72 h, the cells were fixed in 4% formalin, stained with hematoxylin/eosin then photographed.

HMEC-1 culture and proliferation assay. SV40-transformed HMEC-1 cells were grown in MCDB 131 medium with EGF (10 ng/ml) and hydrocortisone (1 g/ml) supplements and 10% FBS. Forty-eight h after incubation in serum-free medium without supplements, the cells were transfected with the indicated DNA enzyme (0.4 AM) and transfected again 72 h after the change of medium, when 10% serum was added. The cells were quantitated by Coulter counter, 24 h after the addition of serum.

Antisense Egr-1 mRNA overexpression. Bovine aortic endothelial cells or rat vascular smooth muscle cells were grown to 60% confluence in 96-well plates then transfected with 3k g of construct pcDNA3-A/SEgr-1 (in which a 137bp fragment of Egr-1 cDNA (732-869) was cloned in antisense orientation into the BamHI/EcoRI site of pcDNA3), or pcDNA3 alone, using Fugene6 in accordance with the manufacturer's instructions. Growth arrested cells were incubated with 5% FBS in Waymouth's medium (SMC) or DMEM (EC) and trypisinised after 3 days prior to quantitation of the cell populations by Coulter counting.

Results and Discussion Insulin, but not Glucose, Stimulates Egr-1 Activity in Vascular Endotlielial Cells. High glucose may activate normally-quiescent vascular WO 01/30394 PCT/AU00/01315 27 endothelium by stimulating mitogen-activated protein (MAP) kinase activity and the expression of immediate-early genes (Frodin et al., 1995; Kang et al., 1999). These signaling and transcriptional events may, in turn, induce the expression of other genes whose products then alter endothelial phenotype and facilitate the development of lesions. To determine the effect of glucose on Egr-1 activity in vascular endothelial cells, we performed transient transfection analysis in endothelial cells transfected with pEBS1'foscat, a chloramphenical acetyltransferase (CAT)-based reporter vector driven by three high-affinity Egr-1 binding sites placed upstream of the c-fos TATA box (Gashler et al., 1993). Exposure of growth-arrested endothelial cells to various concentrations of glucose (5 to 30 nM) over 24 h did not increase Egr-1 binding activity (Figure However, Egr-1 binding activity did increase in cells exposed to insulin (100 nM) (Figure Reporter activity also increased upon incubation with FGF-2, a known inducer of Egr-1 transcription and binding activity in vascular endothelial cells (Santiago et al., 1999b) (Figure 1).

Insulin and FGF-2 Induce Egr-1 mRNA Expression in Vascular Endothelial Cells. The preceding findings using reporter gene analysis provided evidence for increased Egr-1 expression in endothelial cells exposed to insulin. We next used reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis to demonstrate directly the capacity of insulin to increase levels of Egr-1 mRNA. RT-PCR revealed that Egr-1 is weakly expressed in growth-quiescent endothelial cells (data not shown).

Insulin, like FGF-2, increased Egr-1 expression within 1 h of exposure to the agonist. In contrast, levels of P-actin mRNA were unchanged. Northern blot analysis confirmed these qualitative data by demonstrating that insulin, FGF- 2, and phorbol 12-myristate 13-acetate (PMA), a second potent inducer of Egr-1 expression (Khachigian et al., 1995) elevated steady-state Egr-1 mRNA levels within 1 h without increasing levels of ribosomal 28S and 18S mRNA (data not shown).

Insulin-Stimulated Egr-1 Protein Synthesis in Endothelial Cells is Inhibited by Antisense Oligonucleotides Targeting Egr-1 mRNA. To reconcile our demonstration of insulin-induced Egr-1 mRNA expression with the binding activity of the transcription factor (Figure we performed Western immunoblot analysis using polyclonal antibodies directed against Egr-1 protein. Insulin (at 100 nM and 500 nM) induced Egr-1 protein synthesis in WO 01/30394 PCT/AU00/01315 28 growth-arrested endothelial cells within 1 h (data not shown). These findings, taken together, demonstrate that insulin elevates Egr-1 mRNA, protein and binding activity in vascular endothelial cells.

We recently developed phosphorothioate-based antisense oligonucleotides targeting the translational start site in Egr-1 mRNA (Santiago et al., 1999c). These oligonucleotides lack phosphorothioate Gquartet sequences that have been associated with non-specific biological activity (Stein, 1997). Western blot analysis revealed that prior incubation of growth-arrested endothelial cells with 0.8 AM antisense Egr-1 oligonucleotides (AS2) inhibited insulin-inducible Egr-1 protein synthesis, despite equal loading of protein. The lack of attenuation in insulin-inducible Egr-1 protein following exposure of the cells to an identical concentration of AS2C demonstrates the sequence-specific inhibitory effect of the antisense Egr-1 oligonucleotides.

Insulin Stimulates Endothelial Cell DNA Synthesis which is Inhibited by Antisense Oligonucleotides Targeting Egr-1 mRNA. These oligonucleotides, which attenuate the induction of Egr-1 protein, were used in 'H-thymidine incorporation assays to determine the involvement of Egr-1 in insulininducible DNA synthesis. This assay evaluates 'H-thymidine uptake into DNA precipitable with trichloroacetic acetic (TCA) (Khachigian et al., 1992).

In initial experiments, growth-arrested endothelial cells exposed to insulin (100 nM) increased the extent of DNA synthesis by 100%, whereas 500 nM insulin caused a 200% increase in DNA synthesis (Figure 2A).

We next determined the effect of AS2 and AS2C on insulin-inducible endothelial DNA synthesis. In the absence of added insulin, AS2 (0.8 PM) inhibited basal endothelial DNA synthesis facilitated by low concentrations of serum (0.25% v:v) (Figure 2B). In contrast, the scrambled control (0.8 AM) or a third oligonucleotide, E3 (0.8 AM), a size-matched phosphorothioate directed toward another region of Egr-1 mRNA (Santiago et al., 1999c) had little effect on basal DNA synthesis (Figure 2B). Furthermore, unlike AS2 and E3, AS2 significantly inhibited DNA synthesis inducible by insulin (500 nM and 1000 nM) (Figure 2B). To demonstrate concentration-dependent inhibition of DNA synthesis, we incubated the endothelial cells with 0.4 UM as well as 0.8 PM of Egr-1 oligonucleotide. Since this lower concentration of AS2 inhibited 3H-thymidine incorporation less effectively (compare to AS2C) indicates dose-dependent and sequence-specific inhibition by the antisense WO 01/30394 PCT/AU00/01315 29 Egr-1 oligonucleotide (Figure 2C). These findings thus demonstrate the requirement for Egr-1 protein in endothelial cell DNA synthesis inducible by insulin.

Insulin-Stimulated DNA Synthesis in Endothelial Cells is Inhibited by PD98059 and Woltmannin, But Not by SB202190. Inducible Egr-1 transcription is governed by the activity of extracellular signal-regulated kinase (ERK) (Santiago et al., 1999b) which phosphorylates factors at serum response elements in the Egr-1 promoter (Gashler et al, 1995). Since there is little known about signaling pathways mediating insulin-inducible proliferation of vascular endothelial cells, we determined the relevance of MEK/ERK in this process using the specific MEK/ERK inhibitor, PD98059.

This compound (at 10 and 30 pM) inhibited insulin-inducible DNA synthesis in a dose-dependent manner (Figure Likewise, wortmannin (0.3 and 1 pM), the phosphatidylinositol 3-kinase inhibitor which also inhibits c-Jun Nterminal kinase UNK) (Ishizuka et al, 1999; Day et al., 1999; Kumahara et al., 1999), ERK (Barry et al., 1999) and p38 kinase (Barry et al., 1999) inhibited DNA synthesis in a dose-dependent manner (Figure In contrast, SB202190 (100 and 500 nM), a specific p38 kinase inhibitor failed to affect DNA synthesis (Figure These findings demonstrate the critical role for MEK/ERK, and possibly JNK, in insulin-inducible endothelial cell proliferation, and the lack of p38 kinase involvement in this process.

Insulin Stimulates Endothelial Cell Regrowth After Mechanical Injury In Vitro in an Egr-1-Dependent Manner. Mechanically wounding vascular endothelial (and smooth muscle) cells in culture results in migration and proliferation at the wound edge and the eventual recoverage of the denuded area. We hypothesized that insulin would accelerate this cellular response to mechanical injury. Acutely scraping the growth-quiescent (rendered by 48 h incubation in 0.25% serum) endothelial monolayer resulted in a distinct wound edge (data not shown). Continued incubation of the cultures in medium containing low serum for a further 3 days resulted in weak regrowth in the denuded zone but aggressive regrowth in the presence of optimal amounts of serum When insulin (500 nM) was added to growthquiescent cultures at the time of injury the population of cells in the denuded zone significantly increased, albeit as expected, less efficiently than the 10% serum control.

WO 01/30394 PCT/AU00/01315 To investigate the involvement of Egr-1 in endothelial regrowth potentiated by insulin after injury we incubated the cultures with antisense Egr-1 oligonucleotides prior to scraping and again at the time of injury and the addition of insulin. AS2 (0.8 /LM) significantly inhibited endothelial regrowth stimulated by insulin. In contrast, regrowth in the presence of AS2C (0.8 M) was not significantly different from cultures in which oligonucleotide was omitted. Similar findings were observed when higher concentrations (1.2 AM) of AS2 and AS2C were used. Thus, endothelial regrowth after injury stimulated by insulin proceeds in an Egr-1-dependent manner. These observations are quantitated in Figure 4.

These results show that insulin-induced proliferation and regrowth after injury are processes critically dependent upon the activation of Egr-1.

Northern blot, RT-PCR and Western inmunoblot analysis reveal that insulin induces Egr-1 mRNA and protein expression. Antisense oligonucleotides which block insulin-induced synthesis of Egr-1 protein in a sequence-specific and dose-dependent manner, also inhibit proliferation and regrowth after mechanical injury. These findings using nucleic acids specifically targeting Egr-1 demonstrate the functional involvement of this transcription factor in endothelial growth.

Insulin signaling involves the activation of a growing number of immediate-early genes and transcription factors. These include c-fos (Mohn et al., 1990; Jhun et al, 1995; Harada et al., 1996), c-jun (Mohn et al., 1990), nuclear factor-KB (Bertrand et al., 1998), SOCS3 (Emanuelli et al., 2000) and the forkhead transcription factor FKHR (Nakae et al., 1999). Insulin also induces the expression of Egr-1 in mesangial cells (Solow et al., 1999), fibroblasts (Jhun et al., 1995), adipocytes (Alexander-Bridges et al., 1992) and Chinese hamster ovary cells (Harada et al., 1996). This study is the first to describe the induction of Egr-1 by insulin in vascular endothelial cells.

Insulin activates several subclasses within the MAP kinase superfamily, including ERK, JNK and p38 kinase (Guo et al., 1998). Our findings indicate that the specific ERK inhibitor PD98059, which binds to MEK and prevents phosphorylation by Raf, inhibits insulin-inducible endothelial cell proliferation. Egr-1 transcription is itself dependent upon the phosphorylation activity of ERK via its activation of ternary complex factors (such as Elk-1) at serum response elements (SRE) in the Egr-1 promoter. Six SREs appear in the Egr-1 promoter whereas only one is present WO 01/30394 PCT/AU00/01315 31 in the c-fos promoter (Gashler et al., 1995). PD98059 blocks insulininducible Elk-1 transcriptional activity at the c-fos SRE in vascular cells (Xi et al., 1997). These published findings are consistent with the present demonstration of the involvement of Egr-1 in insulin-inducible proliferation.

To provide evidence, independent of insulin, that endothelial proliferation is an Egr-1-dependent process, we incubated human microvascular endothelial cells (HMEC-1) separately with two DNA enzymes (DzA and DzF) each targeting different sites in human EGR-1 mRNA, at a final concentration of 0.4 pM. DzA and DzF both inhibited HMEC-1 replication (total cell counts) in the presence of 5% serum (Figure In contrast, DzFscr, was unable to modulate proliferation at the same concentration (Figure DzFscr bears the same active 15nt catalytic domain as DzF and has the same net charge but has scrambled hybridizing arms.

These data obtained using a second endothelial cell type demonstrate inhibition of endothelial proliferation using sequence-specific strategies targeting human EGR-1.

Finally, we found that CMV-mediated overexpression of antisense Egr- 1 mRNA inhibited proliferation of both endothelial cells and smooth muscle cells. Replication of both endothelial and smooth muscle cell pcDNA3- A/SEgr-1 transfectants was significantly lower than those transfected with the backbone vector alone, pcDNA3 (data not shown). These findings demonstrate that antisense EGR mRNA strategies can inhibit proliferation of arterial endothelial cells and at least one other vascular cell type.

Despite the availability and clinical use of a large number of chemotherapeutic agents for the clinical management of neoplasia, solid tumours remain a major cause of mortality in the Western world. Drugs currently used to treat such tumours are generally non-specific poisons that can be toxic to non-cancerous tissue and require high doses for efficacy.

There is growing evidence that the cellular and molecular mechanisms underlying tumour growth involves more than just tumour cell proliferation and migration. Importantly, tumour growth and metastasis are critically dependent upon ongoing angiogenesis, the process new blood vessel formation (Crystal et al., 1999). The present findings, which demonstrate that Egr-1 is critical in vascular endothelial cell replication and migration, strongly implicate this transcription factor as a key regulator in angiogenesis and tumorigenesis.

WO 01/30394 PCT/AU00/01315 32 Example 2 Characterisation of DNAzyme targeting rat Egr-1 (NGFI-A) Materials and Methods ODN synthesis. DNAzyines were synthesized commercially (Oligos Etc., Inc.) with an inverted T at the 3' position unless otherwise indicated.

Substrates in cleavage reactions were synthesized with no such modification.

Where indicated ODNs were 5'-end labeled with y 3 P-dATP and T4 polynucleotide kinase (New England Biolabs). Unincorporated label was separated from radiolabeled species by centrifugation on columns (Clontech).

In vitro transcript and cleavage experiments. A "P-labelled 206 nt NGFI-A RNA transcript was prepared by in vitro transcription (T3 polymerase) of plasmid construct pJDM8 (as described in Milbrandt, 1987, the entire contents of which are incorporated herein by reference) previously cut with Bgl II. Reactions were performed in a total volume of 20 pj containing 10 mM MgCl 2 5 mM Tris pH 7.5, 150 mM NaCI, 4.8 pmol of in vitro transcribed or synthetic RNA substrate and 60 pmol DNAzyme (1:12.5 substrate to DNAzyme ratio), unless otherwise indicated. Reactions were allowed to proceed at 37 "C for the times indicated and quenched by transferring an aliquot to tubes containing formamide loading buffer (Sambrook et al, 1989). Samples were run on 12% denaturing polyacrylamide gels and autoradiographed overnight at -80 "C.

Culture conditions and DNAzyme transfection. Primary rat aortic SMCs were obtained from Cell Applications, Inc., and grown in Waymouth's medium, pH 7.4, containing 10% fetal bovine serum (FBS), 50 g/ml streptomycin and 50 IU/ml penicillin at 37 "C in a humidified atmosphere of CO,. SMCs were used in experiments between passages 3-7. Pup rat SMCs (WKY12-22 (as described in Lemire et al, 1994, the entire contents of which are incorporated herein by reference)) were grown under similar conditions. Subconfluent (60-70%) SMCs were incubated in serum-free medium (SFM) for 6 h prior to DNAzyme (or antisense ODN, where indicated) transfection (0.1 pM) using Superfect in accordance with manufacturer's instructions (Qiagen). After 18 h, the cells were washed with WO 01/30394 PCT/AU00/01315 33 phosphate-buffered saline (PBS), pH 7.4 prior to transfection a second time in

FBS.

Northern blot analysis. Total RNA was isolated using the TRIzol reagent (Life Technologies) and 25 kg was resolved by electrophoresis prior to transfer to Hybond-N+ membranes (NEN-DuPont). Prehybridization, hybridization with a2P-dCTP-labeled Egr-1 or P-Actin cDNA, and washing was performed essentially as previously described (Khachigian et al, 1995).

Western blot analysis. Growth-quiescent SMCs in 100 mm plates (Nunc-InterMed) were transfected with ED5 or ED5SCR as above, and incubated with 5% FBS for 1 h. The cells were washed in cold PBS, pH 7.4, and extracted in 150 mM NaCI, 50 mM Tris-HCl, pH 7.5, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, 1% trasylol, /g/ml leupeptin, 1% aprotinin and 2 mM PMSF. Twenty four /g protein samples were loaded onto 10% denaturing SDS-polyacrylamide gels and electroblotted onto PVDF nylon membranes (NEN-DuPont). Membranes were air dried prior to blocking with non-fat skim milk powder in PBS containing 0.05% Tween 20. Membranes were incubated with rabbit antibodies to Egr-1 or Spl (Santa Cruz Biotechnology, Inc.) (1:1000) then with HRP-linked mouse anti-rabbit Ig secondary antiserum (1:2000). Where mouse monoclonal c-Fos (Santa Cruz Biotechnology, Inc.) was used, detection was achieved with HRP-linked rabbit anti-mouse Ig. Proteins were visualized by chemiluminescent detection (NEN-DuPont).

Assays of cell proliferation. Growth-quiescent SMCs in 96-well titer plates (Nunc-InterMed) were transfected with ED5 or ED5SCR as above, then exposed to 5% FBS at 37 OC for 72 h. The cells were rinsed with PBS, pH 7.4, trypsinized and the suspension was quantitated using an automated Coulter counter.

Assessment of DNAzyme stability. DNAzymes were 5'-end labeled with y"P-dATP and separated from free label by centrifugation. Radiolabeled DNAzymes were incubated in 5% FBS or serum-free medium at 37 °C for the times indicated. Aliquots of the reaction were quenched by transfer to tubes containing formamide loading buffer (Sambrook et al, 1989). Samples were applied to 12% denaturing polyacrylamide gels and autoradiographed overnight at -80 °C.

SMC wounding assay. Confluent growth-quiescent SMCs in chamber slides (Nunc-InterMed) were exposed to ED5 or ED5SCR for 18 h prior to a WO 01/30394 PCT/AU00/01315 34 single scrape with a sterile toothpick. Cells were treated with mitomycin C (Sigma) (20 jM) for 2 h prior to injury (Pitsch et al, 1996; Horodyski Powell, 1996). Seventy-two h after injury, the cells were washed with PBS, pH 7.4, fixed with formaldehyde then stained with hematoxylin-eosin.

Rat arterial ligation model and analysis. Adult male Sprague Dawley rats weighing 300-350 g were anaesthetised using ketamine (60 mg/kg, i.p.) and xylazine (8 mg/kg, The right common carotid artery was exposed up to the carotid bifurcation via a midline neck incision. Size 6/0 nonabsorbable suture was tied around the common carotid proximal to the bifurcation, ensuring cessation of blood flow distally. A 200 pl solution at 4°C containing 500 jg of DNAzyme (in DEPC-treated H 2 1mM MgCIl, 30 /l of transfecting agent (Fugene 6) and Pluronic gel P127 (BASF) was applied around the vessel in each group of 5 rats, extending proximally from the ligature for 12-15 mm. These agents did not inhibit the solidification of the gel at 37 OC. After 3 days, vehicle with or without 500 Aig of DNAzyme was administered a second time. Animals were sacrificed 18 days after ligation by lethal injection of phenobarbitone, and perfusion fixed using 10% (v:v) formaldehyde perfused at 120 mm Hg. Both carotids were then dissected free and placed in 10% formaldehyde, cut in 2 mm lengths and embedded in 3% agarose prior to fixation in paraffin. Five Am sections were prepared at 250 im intervals along the vessel from the point of ligation and stained with hernatoxylin and eosin. The neointimal and medial areas of 5 consecutive sections per rat were determined digitally using a customized software package (Magellan) (Halasz Martin, 1984) and expressed as a mean ratio per group of 5 rats.

Results and Discussion The 7x7 nt arms flanking the 15 nt DNAzyme catalytic domain in the original DNAzyme design (Santoro and Joyce, 1997) were extended by 2 nts per arm for improved specificity Sun, data not shown) (Figure The 3' terminus of the molecule was capped with an inverted 3'-3'-linked thymidine to confer resistance to exonuclease digestion. The sequence in both arms of ED5 was scrambled (SCR) without altering the catalytic domain to produce DNAzyme ED5SCR (Figure 6).

A synthetic RNA substrate comprised of 23 nts, matching nts 805 to 827 of NGFI-A mRNA (Figure 6) was used to determine whether ED5 had the WO 01/30394 PCT/AU00/01315 capacity to cleave target RNA. ED5 cleaved the "P-5'-end labeled 23-mer within 10 min (data not shown). The 12-mer product corresponds to the length between the A(816)-U(817) junction and the 5' end of the substrate (Figure In contrast, ED5SCR had no demonstrable effect on this synthetic substrate. Specific ED5 catalysis was further demonstrated by the inability of the human equivalent of this DNAzyme (hED5) to cleave the rat substrate over a wide range of stoichiometric ratios (data not shown). Similar results were obtained using ED5SCR (data not shown). hED5 differs from the rat sequence by 3 of 18 nts in its hybridizing arms (Table The catalytic effect of ED5 on a "P-labeled 206 nt fragment of native NGFI-A mRNA prepared by in vitro transcription was then determined. The cleavage reaction produced two radiolabeled species of 163 and 43 nt length consistent with DNAzyme cleavage at the A(816)-U(817) junction. In other experiments, ED5 also cleaved a "P-labeled NGFI-A transcript of 1960 nt length in a specific and time-dependent manner (data not shown).

WO 01/30394 PCT/AU00/01315 36 Table 2. DNAzyme target sites in mRNA.

Similarity between the 18 nt arms of ED5 or hED5 and the mRNA of rat NGFI-A or human EGR-1 (among other transcription factors) is expressed as a percentage. The target sequence of ED5 in NGFI-A mRNA is 5'-807-A CGU CCG GGA UGG CAG CGG-825-3' (SEQ ID NO: 22) (rat NGFI-A sequence), and that of hED5 in EGR-1 is 5'-262-U CGU CCA GGA UGG CCG CGG-280-3' (SEQ ID NO: 23) (Human EGR-1 sequence). Nucleotides in bold indicate mismatches between rat and human sequences. Data obtained by a gap best fit search in ANGIS using sequences derived from Genbank and EMBL. Rat sequences for Spl and c-Fos have not been reported.

Gene Accession Best homology over 18 nts number Rat NGFI-A M18416 100 84.2 Human EGR-1 X52541 84.2 100 Murine Spl AF022363 66.7 66.7 Human c-Fos K00650 66.7 66.7 Murine c-Fos X06769 61.1 66.7 Human Spl AF044026 38.9 28.9 To determine the effect of the DNAzymes on endogenous levels of NGFI-A mRNA, growth-quiescent SMCs were exposed to ED5 prior to stimulation with serum. Northern blot and densitometric analysis revealed that ED5 (0.1 inhibited serum-inducible steady-state NGFI-A mRNA levels by 55% (data not shown), whereas ED5SCR had no effect (data not shown). The capacity of ED5 to inhibit NGFI-A synthesis at the level of protein was assessed by Western blot analysis. Serum-induction of NGFI-A protein was suppressed by ED5. In contrast, neither ED5SCR nor EDC, a DNAzyme bearing an identical catalytic domain as ED5 and ED5SCR but flanked by nonsense arms had any influence on the induction of NGFI-A WO 01/30394 PCT/AU00/01315 37 (Figure ED5 failed to affect levels of the constitutively expressed, structurally-related zinc-finger protein, Spl (Figure It was also unable to block serum-induction of the immediate-early gene product, c-Fos (Figure 7) whose induction, like NGFI-A, is dependent upon serum response elements in its promoter and phosphorylation mediated by extracellular-signal regulated kinase (Treisman, 1990, 1994 and 1995; Gashler Sukhatme, 1995). These findings, taken together, demonstrate the capacity of ED5 to inhibit production of NGFI-A mRNA and protein in a gene-specific and sequence-specific manner, consistent with the lack of significant homology between its target site in NGFI-A mRNA and other mRNA (Table 2).

The effect of ED5 on SMC replication was next determined. Growthquiescent SMCs were incubated with DNAzyme prior to exposure to serum and the assessment of cell numbers after 3 days. ED5 (0.1 tM) inhibited SMC proliferation stimulated by serum by 70% (Figure 8a). In contrast, ED5SCR failed to influence SMC growth (Figure 8a). AS2, an antisense NGFI-A ODN able to inhibit SMC growth at 1 pM failed to inhibit proliferation at the lower concentration (Figure 8a). Additional experiments revealed that ED5 also blocked serum-inducible 'H-thymidine incorporation into DNA (data not shown). ED5 inhibition was not a consequence of cell death since no change in morphology was observed, and the proportion of cells incorporating Trypan Blue in the presence of serum was not influenced by either DNAzyme (Figure 8b).

Cultured SMCs derived from the aortae of 2 week-old rats (WKY12-22) are morphologically and phenotypically similar to SMCs derived from the neointima of balloon-injured rat arteries (Seifert et al, 1984; Majesky et al, 1992). The epitheloid appearance of both WKY12-22 cells and neointimal cells contrasts with the elongated, bipolar nature of SMCs derived from normal quiescent media (Majesky et al, 1988). WKY12-22 cells grow more rapidly than medial SMCs and overexpress a large number of growthregulatory molecules (Lemire et al, 1994), such as NGFI-A (Rafty Khachigian, 1998), consistent with a "synthetic" phenotype (Majesky et al, 1992; Campbell Campbell, 1985). ED5 attenuated serum-inducible WKY12- 22 proliferation by approximately 75% (Figure 8c). ED5SCR had no inhibitory effect; surprisingly, it appeared to stimulate growth (Figure 8c).

Trypan Blue exclusion revealed that DNAzyme inhibition was not a consequence of cytotoxicity (data not shown).

WO 01/30394 PCT/AU00/01315 38 To ensure that differences in the biological effects of ED5 and were not the consequence of dissimilar intracellular localization, both DNAzymes were 5'-end labeled with fluorescein isothiocyanate (FITC) and incubated with SMCs. Fluorescence microscopy revealed that both FITC- ED5 and FITC-ED5SCR localized mainly within the nuclei. Punctate fluorescence in this cellular compartment was independent of DNAzyme sequence. Fluorescence was also observed in the cytoplasm, albeit with less intensity. Cultures not exposed to DNAzyme showed no evidence of autofluorescence.

Both molecules were 5'-end labeled with y 3 P-dATP and incubated in culture medium to ascertain whether cellular responsiveness to ED5 and was a consequence of differences in DNAzyme stability. Both 3 2

P-

and 3 2 P-ED5SCR remained intact even after 48 h (data not shown). In contrast to 32 P-ED5 bearing the 3' inverted T, degradation of 3 P-ED5 bearing its 3' T in the correct orientation was observed as early as 1 h. Exposure to serum-free medium did not result in degradation of the molecule even after 48 h (data not shown). These findings indicate that inverse orientation of the 3' base in the DNAzyme protects the molecule from nucleolytic cleavage by components in serum.

Physical trauma imparted to SMCs in culture results in outward migration from the wound edge and proliferation in the denuded zone. We determined whether ED5 could modulate this response to injury by exposing growth-quiescent SMCs to either DNazyme and Mitomycin C, an inhibitor of proliferation (Pitsch et al, 1996; Horodyski Powell, 1996) prior to scraping.

Cultures in which DNAzyme was absent repopulated the entire denuded zone within 3 days. ED5 inhibited this reparative response to injury and prevented additional growth in this area even after 6 days (data not shown).

That ED5SCR had no effect in this system further demonstrates sequencespecific inhibition by EDS.

The effect of ED5 on neointima formation was investigated in a rat model. Complete ligation of the right common carotid artery proximal to the bifurcation results in migration of SMCs from the media to the intima where proliferation eventually leads to the formation of a neointima (Kumar Lindner, 1997; Bhawan et al, 1977; Buck, 1961). Intimal thickening 18 days after ligation was inhibited 50% by ED5 (Figure In contrast, neither its scrambled counterpart (Figure 9) nor the vehicle control (Figure 9) had any WO 01/30394 PCT/AU00/01315 39 effect on neointima formation. These findings demonstrate the capacity of to suppress SMC accumulation in the vascular lumen in a specific manner, and argue against inhibition as a mere consequence of a "mass effect" (Kitze et al, 1998; Tharlow et al, 1996). Sequence specific inhibition of inducible NGFI-A protein expression and intimal thickening by ED5 was also observed in the rat carotid balloon injury model (Santiago et al., 1999a).

Further experiments revealed the capacity of hED5 to cleave (human) EGR-1 RNA. hED5 cleaved its substrate in a dose-dependent manner over a wide range of stoichiometric ratios. hED5 also cleaved in a time-dependent manner, whereas hED5SCR, its scrambled counterpart, had no such catalytic property (data not shown).

The specific, growth-inhibitory properties of antisense EGR-1 strategies reported herein suggest that EGR-1 inhibitors may be useful as therapeutic tools in the treatment of vascular disorders involving inappropriate SMC growth, endothelial growth and tumour growth.

EXAMPLE 3 Use of DNAzymes to inhibit growth of malignant cells Materials and Methods HepG2 cells were routinely grown in DMEM, pH 7.4, containing 10 fetal calf serum supplemented with antibiotics. The cells were trypsinized, resuspended in growth medium (to 10,000 cells/200 Al) and 200 p 1 transferred into sterile 96 well titre plates. Two days subsequently, 180 Al of the culture supernatant was removed, the cells were washed with PBS, pH 7.4, and refed with 180 A 1 of serum free media. After 6 h, the first transfection of DNAzyme (2 pg/200li wall, 0.75 iM final) was performed in tubes containing serum free media using FuGENE6 at a ratio of 1:3 After 15 min incubation at room temperature, 180 1l of the culture supernantant was replaced with 180 Al of the transfection mix. After 24 h, 180.l of the supernatant was replaced with 180 Al of new transfection mix, but this time in 5% FBS media. After 3 days, the cells were washed in PBS, pH 7.4, and resuspended by trypsinization in 100 pil trypsin-EDTA. The cells were shaken for approximately 5 min to ensure the cells were in suspension. The entire suspension was placed into 10 ml of Isoton I. That all the cells were transferred was ensured by pipetting Isoton II solution from tubes back into wells several times. Using Isoton I only, background cell WO 01/30394 PCT/AU00/01315 number was determined. Each sample was counted three times and used to calculate mean counts and standard errors of each mean.

Results and Discussion Our results indicate that serum stimulated HepG2 cell proliferation after 3 days (Figure 10). Proliferation was almost completely suppressed by 0.75 pM of DzA (5'-caggggacaGGCTAGCTACAACGAcgttgcggg (SEQ ID NO:3), catalytic moiety in capitals), a DNAzyme targeting human EGR-1 mRNA (arms hybridize to nts 189-207) (Figure 10). In contrast, HepG2 cell growth was not inhibited by ED5SCR (Figure 10). Western blot analysis revealed that DzA strongly inhibited EGR-1 expression in HepG2 cells, whereas a size matched DNAzyme with different sequence (5'-tcagctgcaGGCTAGCTACAACGActcggcctt) (SEQ ID NO:24) had no effect (data not shown). These data indicate that inducible proliferation of this model human malignant cell line can be blocked by the EGR-1 DNAzyme. These findings suggest that EGR inhibitors may be clinically useful in therapeutic strategies targeting human cancer.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which the invention pertains.

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EDITORIAL NOTE APPLICATION NUMBER 11169/01 The following Sequence Listing pages 1/9 to 9/9 are part of the description. The claims pages follow on pages "49" to "52".

WO 01/30394 PCT/AU00/01315 1/9 SEQUENCE LISTING <110> Unisearch Limited <120> Treatment of cancer <160> 24 <170> PatentIn Ver. 2.1 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNAzyme <400> 1 cgccattagg ctagctacaa cgacctagtg at 32 <210> 2 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: antisense oligonucleotide <400> 2 cttggccgct gccat <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNAzyme <400> 3 caggggacag gctagctaca acgacgttgc ggg 33 <210> 4 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:antisense oligonucleotide WO 01/30394 PCT/AU00/01315 2/9 <400> 4 acacttttgt ctgct <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: catalytic domain of DNAzyme <400> ggctagctac aacga <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNAzyme <400> 6 tgcaggggag gctagctaca acgaaccgtt gcg 33 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNAzyme <400> 7 catcctggag gctagctaca acgagagcag get 33 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNAzyme <400> 8 ccgcggccag gctagctaca acgacctgga cga 33 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence WO 01/30394 WO 0130394PCT/AUOO/01315 3/9 <220> <223> Description of Artificial Sequence: DNAzyme <400> 9 ccgctgccag gctagctaca acgacccgga cgt <210> <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNAzyme <400> gcggqgacag gctagctaca acgacagctg cat <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNAzyne <400> 11 cagcggggag gctagctaca acgaatcagc tgc <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNAzyme <400> 12 ggtcagagag gctagctaca acgactgcag cgg <210> 13 <211> 3068 <212> DNA <213> Mus inusculus <400> 13 gggga gccgc tgccccagcc ggcccaccta ggcgagtgtg ccagctcgct cagatctctg ctggaggaga cgccgcgatt tccgcggcag cactccccgc ccctcag tag ggtccgggat acccgttcgg tgatgctgct cgccgccgcc ccctgcgtcc agtgtgcccc cttcggcccc ggcagcggcc ctcctttcct gagcaacggg gccagcttcc accacgggcc tgcaccccgc gggctgcgcc aaggccgaga cactcaccca gctccccagt gccgccgcaa gcggctaccg atgtaacccg caccacccaa tgcaattgat ccatggacaa tcctcggtgC gatcggcccc ccagcctggg gccaa ccccc catcagttct gtctccgctg ctaccccaaa tgccggaacc WO 01/30394 WO 0130394PCT/AUO/01315 ccagagggca ggcagcaaca gagcacctga gagacgagtt tccctggagc gtcagtggcc gctgcttcat aacgacagca tttcctgagc cctcctgcct tttccacaac ctggagaacc actcagtcgg aaacccagcc ccatatgctt cgccatatcc ttcagtcgta gcctgtgaca atccatttaa tcttcactct tcattcccat cctgcgcaca gctttcccca tcaactggtc agggaataaa atggccgcaa actcacqagt gtcctgttcc ccaaaggact cctggccctt tttgggccct ttccttgggg aaatcctcac ttgtgatgac aggtattaac actgtaactc ttttttgccc ccttccgatg taggagtgat tttcggtctc taittttcta tacctactga ttttatttta cat tgaa tgc ggaaaaai gcggcgg taa gcggcagcag ccacagaqtc atcccagcca ccgcacccaa tcgtgagcat cgtcttcctc gtcccatcta cccaaagcca accctgccac aacagggaga gtacccagca gctcccagga gcatgcgcaa gccctgtcga gcatccacac gtgaccacct tttgtgggag gacagaagga cttcttaccc cccctgtgcc gtggcttccc cccaggtcag t ttcagacat agaaagcaaa gaggggccac agaaggaccg ctttgacttc tgatttgcat gctcccttca cagaaccctg tattcttgat tttgggggag tctgctgtga tggagcatgt tcaca tgtga gtccctttgg gcttgacatg gtgttggggg cagaatgtaa aactgaaaat gtaggctgca ctttgtactt gctctattgc tagcagcagc cgccttcaat cttttctgac aacgactcgg cagtggcaac gaccaatcct tgcctcccag ctcggctgcg ggcctttcct caaaggtggt cctgagcctg gccttcgctc cttaaaggct gtaccccaac gtcctgcgat aggccagaag taccacccac gaagtttgcc caagaaagca atccccagtg cacttcctac gtcgccgtca cagcttcccg gacagcgacc gggagaggca ctcttaggtc ttggccaaca agctgcctga ggtattggat gcgctagacc ccctgcatct gtgaagataa gggggagcaa cat taggttt gtcagagtlgt caaagtatgg tttcaaaagt cgcagatgtg aggcttgaga gaagaaaaaa gtaaatttat gtttttgtat gtgtttgctt ccatgggata agcaccagca cctcaagggg atcgctctqa t tgcctccca actttgtggc ccgacctctt agcccgcccc cccacctttc ggctcggcag ttccaggttc ggcaccccag actccactat cttaatacca cggcccagca cgccgctttt cccttccagt atccgcaccc aggagtgatg gacaaaagtg gctacctcct tcctctcctg gtggccacca tctgcgggcg ttttctccca ggaaagacat agatggaaga gccctttcac aacagccatg aaatcatttc atcaagttgg ttgtacagca tttgcatact agccaagcaa gaagcatttt tgttccgtta tttgtttggt ttcacgtctt agggacacgc gcaaaaacga tttaaacaaa acatctattc gttatgaaca aaacaaagta tgtggtgtgt gcgggggcgg agccgagcga ataatgagaa tcacctatac ctgaacccct catcctcggc tgagctgtgc ctactcccaa gcacagcctt ccatgatccc accagaagcc ccactattaa cctaccaatc agacaccccc ctcgctcgga gtcgaatctg acacaggcga aacgcaagag tggtggcctc acccatcccc gctcctccac cctttgcctc tcagcagctc ggacaattga aaaagcacag tctcagagcc ttaccatccc tccaagttct agtatcctct cataaagaaa tctgtgccat ctattgtatt accaatgatg ttttttcaag attttgtaaa tgggttttgt ggtgcctttt tcaccttagc ggaagagggc aatctgaact aggagttgga tgaagttcat acctgtttgg atccttcaga tggtgggggc acaaccctat ggcgatggtg tggccgcttc tttcaqccta gccttctcca cgtgccgtcc cactgacatt gcagtacccg tgactatctg cttccagggt agccttcgcc ccagctcatc c catg a acg c tgagcttacc catgcgtaac gaagcctttt gcataccaaa cccggctgcc tgccaccacc ctacccatct cgttccacct cttcagcacc aatttgctaa gagggaagag aagtccttct tgcctccccc tcacctctat ccatcacatg aaaaaatgqg ggattttgtt atttggagtt atcctctatt cagcagtcct tactggctcg ttttgagaat gtgtgacacg cttaagggg tgagctgagc ctcaaaagtc gtgttgtggt tattttgtgg cttataaaca aaaattaaaa 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3068 <210> 14 <211> 4321 <212> DNA <213> Rattus rattus <400> 14 ccgcggagcc cgcgcgttca aacaccatat ttatatggag tcagctctac gcgcctggcg ccctccctac gcgggcgtcc ccgactcccg ggctccgggt tgggaaccaa ggagggggag ggtgggtgcg ccgacccgga 120 aaggagcagg aaggatcccc cgccggaaca gaccttattt gggcagcgcc 180 tggcccaata tggccctgcc gcttccggct ctgggaggag gggcgaacgg 240 WO 01/30394 WO 0130394PCT/AUOO/01315 gggttggggc gttccaatac tggaagcgcc gtccttccat gggcccgtgc cagcgcgcag cgcaagatcg caccgccagc acccggccaa cccaacatca ttgatgtctc gacaactacc ggtgctgccg agcagcgggg ggggagccga aggctgaatc ctagatctta ttagagggat cgtttgtttt ttgatgagca ttcccaagga gcattagctg agcgctctca ctgccactgg gagagtcagt attgctctcc cgctctgaat gcctcccatc tttgtggcct aacctcttca cccacccctg cacctttcct ctctgcaggc ccaggttccc caccccagac tccactatcc taataacacc gcccagcaag ccgctt tt ct cttccagtgt ccgcacccac qagtgatgaa caaaagtgtc tacctcctac ctctccgggc ggccaccacc tgcaggggtc ttctcctagg cgagagacaa cagatggaag cctttcactt acagccacgt aaccatttca tcaagttggc acagcatctg catactctat agcaaaccaa gggggcaagc taggctttcc caccgctctt attagggctt tgtttcagac aacttgggga gcccctgccc ctgggggccc catccggcga gctctccagc cgctgcagat ccaaactgga gaaccccaga gcggtggtgg gcgaacaacc ccccttcgtg gggacgggat gtctggggac ggatggagaa gggttgcccc agggctgaaa tggccactag gagctgcagt agcaggtcca ggtagccggg agtaaccagg aacgagaagg acctatactg gaaccccttt tcctcagcgc agctgtgccg actcccaaca acagccttgc atgatccctg cagaagccct actatcaaag taccagtccc acaccccccc cgctcggatg cgaatctgca acaggcgaga cgcaagaggc gtggcct cct ccatcccccg tcctctacct tatgcctccg agcaactcct acaattgaaa taaaggacag atctcaqagc agcgtccctg ccaagttctt gcatcatctc Lgaaaaaaaa tgccatggat tgtactattt tggtgatcct tgggaactcc aggagcctga gga tgggagg cctgcttccc ccttgaaata gccgccgccg cagcctccgc acctacactc gtgtgccctc Lcgcacg tcc ctctgacccg ggagatgatg gggcagcggc gggcggcagc ctacgagcac actaccctaa tgggatttcc cccccaaccc ctcaagttgc ctcccccgcg tctgtcacca ggtgctggcg agagggggat ggaacattgc cgacctcttg cctctctgtt cgctggtgga gccgcttctc tcagcctagt cttctccagc tgccgtccaa ctgacatttt agtacccgcc actatctgtt Lccagggtct ccttcgccac aactcatcaa atgaacgccc agcttacacg tgcgtaattt agccttttgc ataccaaaat cagctgcctc ccaccacctc acccgtctcc tcccacctgc tcagcacct c tttqctaaag gagggaagaa caagtccttc ccctccccag cacctctatc caccacatgc aatgggtctg tttgttttcc ggagttaaat ctattttgtg aggagcctag gcgctcaggg tcttcacgtc atatatggcc gaggccgatt cgattcgccg ggcagccctg cccgcagtgt agtagcttcg gggatggcag ttcggctcct ctgctgagca ggcaataaca aacagcggca ctgaccacag cgtccagtcc ctctattcca t ccat cc ttg gtgggtggct cgcgttgtcg gggatgtccc ggattccctc tctctgtttg aatctgctgc cctggccgct ctctttcctg gacaagttat cctggagcct cagtggcctt tgcttcatcg cgacagcagt tcctgagccc tcctgcctac tccacaacaa ggagaaccgt tcagtcgggc acccagccgc gtatgcttgc ccacatccgc cagtcgtagt ctgtgacatt ccacttaaga Ltccctctct atttccatcc tgcacacagt tttccctgcc aacgggtctt ggaatgaaag a Lggcccgca tagtcagtag Lcccggtcct caaaggactt ctggcccttg ggccctcaga ttggggtatt tctcactttg atgatcctgc cccgggaggc tgccggagcc actccgggtc atgtacgtca cggggagtcg ccgccgccag cgtccaccac gcccctgcac gccccgggct cggccaaggc ttcctcactc acggggctcc cjcagcagcag gcagcgcttt gtaagcggtg tttgcagcac cacagctcca cgggtgcgcg ggagtggggg cgagccttgt gccgcccagg accccggacg cgtcagctgt tatcaattat tcggctctca ccagagtcct cccagccaaa gcacccaaca gtgagcatga tcttcctctg cccatttact caaagccagg cctgccacca cagggagacc acccagcagc tcccaggact atgcgcaag L cctgttgagt atccatacag gaccacctta tgtgggagaa cagaaggaca tcctacccat ccagtgccca ggcttcccat caggtcagca tcagacatga agagcaaagg agaggggctg aaggcccgtt tttgacttca gatttgcatg ctcccttcag accctgccct cttgatgtga ggggaggggg tgtgacatta cactgccgct ggtcgcaggg ctcccggtcg cggcggaggc cgagagatcc cttccgccgc gggccgcggc cccgcatgta gcgcccacca cgagatgcaa accc accatg ccagttcctc cagcagcagc caatcctcaa gtctgcgccg ggacctgcat gggacttgtg gagggcagac agggtttgtt ttgcagcttg gtaggggcgc cctgctgcgg cgaaatggct taaccacatc tcgtccagtg tttctgacat ctacccggtt gtggcaacac ccaaccctcc cctcccagag cagctgcacc cctttcctgg agggtggttt tgagcctggg cttcgctcac taaaggctct accccaaccg cctgcgatcg gccagaagcc ccacccacat agtttgccag agaaagcaga ccccag Lggc cctcttactc cgccctcggt ccttccagtc cagcaacctt gaggggagcg cctcttaggt ggccaccagc gctgcctgaa gtattggata cactagaaca gtatctttgt agataatttg agcaaagcca ggtttgaaac 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 WO 01/30394 WO 0130394PCT/AUOO/01315 tttttttttt t t ccg t taat gtttggttgg tttcaaaagt tgcaatcgtg ggaggcttta agaagaaaaa gtagatttat ttttgtatgc gtttgcttaa atgggatatg t ttttgaagca tttgtaaata gttttttgtt ttcacgtctt aggggacacg gagcacgatg tttaaaacaa ccatgttcgg tatgaacatg a ca aa g tga c tggtgtgtat gcagtcctag ctgctcgact gtttttgaaa ggtgcctttg ctcacctcta aggaagaggg aaatctgaac gagttggaat aagttcatta ttgtttggct ccttcagaaa gtattaactg gtaactctca aaaaaatttt tgtgacacac gccttaaggg ctgagctgag tctcaaaagt gctgcggtta ttttgtggtt tataaacaca aattaaaagg gagcatgtgt catgtgacaa ttttttgccc cttgccgatg ggtaggagtg ctttggttct ctattttttt cctactgagt ttattttact ttgaatgcgc aaaataaaga cagagtgttg aatacggttt gtccctttgg gctggacatg atgtttcagg ccagaatgta aactgaaaat aggcggtgac tcgtacttgt tttactgccc aactaactgg 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4321 <210> <211> 3132 <212> DNA <213> Homo sapiens <400> ccgcagaact ggaccqgccc gcgagtcggg gccaggcccc gacaccagct atgtccccgc aactacccta gccgccgggg ggaggcggcg gacacgggcg aacaacgaga atcacctata cccgagcccc tcgtcctcag agctgcgcag acgccgaaca acagcgctcc atgatccccg caqaagccct actattaagg taccagtccc acgccccccc cgctccgacg cgcatctgca acaggcgaaa cgcaagaggc gtggcctctt ccgtccccgg cccacctcct ccctccccgt agcagcttcc atgacagcaa gggaaaaggg ggagaggagg ctggagtgga tattcccttt agaacttgat tggggagccg ctgccccagc gtcgccgcct cgcaacggtg ctccagcctg tgcagatctc agctggagga ccccagaggg ggggcggcag agcagcccta aggtgctggt ctggccgctt tcttcagctt caccatctcc tgccatccaa ctgacatttt agtacccgcc actacctgtt tccagggcct cctttgccac agctcatcaa acgaacgccc agctcacccg tgc Jcaactt agcccttcgc ataccaagat cggccacctc ttactacctc tctcctct cc cggtggccac cttcctcagc ccttttctcc agaaaaagaa gttcctctta aggtctattg gacttcagct ttgcatggat ccgccgccat ctccgcagcc gcacgcttct tcccctgcag ctcgtccagg tgacccgttc gatgatgctg cagcggcagc caacagcagc cgagcacctg ggagaccagt ttccctggag ggtcagtggc agcggcctcc cgacagcagt ccctgagcca tcctgcctac tccacagcag ggagagccgc tcagtcgggc accagccgc ttacgcttgc cca cat ccgc cagcccjcagc ctgcgacatc ccacttgcgg ctctctctct ttatccatcc cggctcctcg cacgtactcc tgtcaccaac caggacaatt acacaagaga ggtcagatgg gccaacaatc gcctgaaaca tttggataaa ccgccgccgc gcggcgcgtc cagtgttccc ctccagcccc atggccgcgg ggatcctttc ctgagcaacg aacaqcagca agcagcagca accgcagagt taccccagcc cctgcaccca ctagtgagca tccgcctccg cccatttact caaagccagg cctgccgcca cagggggatc acccagcagc tcccaggacc atgcgcaagt ccagtggagt atccacacag gaccacctca tgtggaagaa cagaaggaca tcctacccgt ccggccacca acctacccat tctgttcccc tccttcagcg gaaatttgct cttaaaggac aggttctcag ctttctgccc gccatgtcca tcatttcagt agccagcttc ca cgcccgcc cgcgccccgc gggctgcacc ccaaggccga ctcactcgcc gggctcccca gcagcagcag gcaccttcaa cttttcctga aaaccactcg acaqtggcaa tgaccaaccc cctcccagag cagcggcacc ccttcccggg agggtggctt tgggcctggg cttcgctaac tgaaggccct atcccaaccg cctgtgatcg gccagaagcc ccacccacat agtttgccag agaaagcaga ccccggttgc cctcataccc cccctgtgca ctgctttccc cctccacagg aaagggaaag aggaggagga agccaagtcc acttcccctt agttcttcac atcatctcca cgccgccgca cgcgcccagg atgtaacccg cccccgcccc gatgcagctg caccatggac gttcctcggc cgggggcggt ccctcaggcg catctctctg actgcccccc caccttgtgg accggcctcc cccacccctg caccttcccc ctcggcaggg ccaggttcc caccccagac ccctctgtct caataccagc gcccagcaag ccgcttctcc cttccagtgc ccgcacccac gag cga tgaa caaaagtgtt tacctcttac atcccctgtg cagtggcttc ggcccaggtc gctttcggac gggaaagaaa gatggccata tccctctcta ccccaattac ctctatccaa tcatatgcct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 WO 01/30394 WO 0130394PCT/AUOO/01315 gaccccttgc agagccctgc actcttgatg tgggggaaaa tgctgtgaca atgtgtcaga tgtggcaaaa tttggtttaa yacatgtgca atttggggga agaatgtaag ctgaaaatgt aggcggcgat ttgtacttgt ttattgccca gctgcgattg tcccttcaat cctgcaccct tgaagataat aaaaaaaaaa ataagtttga gtgttgttcc tatggtttgg aaagtttcac attgtgaggg ggctttggga aaaacaaaat aaatttataa ttttgtatgt gtttgcttaa tgggatatgt gg gctagaaaat tgtacagtgt ttgcatattc aagccaagca accttttttt gttaaccttt tttttctttt gtcttggtgc acatgctcac gcaaaataag ctaaaacaaa atatattcag tatgaacatg acaaagtgac ggtgtatatc cgagttggca ctgtgccatg tat tg tat ta aaccaatgg t ttgaaacagc ttgtaaatac ttttttttga cttttgtgtg ctctagcctt gaagagggct atctgaactc gacjttggaat cagttcatta tgtttggctt cttccaaaaa aaatggggtt gatttcgttt tttggagtta gatcctctat agtcccagta tgcttgaccg aagtgttttt atgccccttg aaggggggca gagctgagct tcaaaagtct gttgtagtta ttttgtggtt ataaacacat attaaaacga tgggcccctc ttcttggggt ggtcctcact tttgtgatga ttctcagagc tactctcaca tcttcgtcct ctgatggctt gggagtgatg tcggttctcc atttttttaa cctactgagt ctattttact tgaatgcgct aaataaagta 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3132 <210> 16 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: phosphorothioate-linked antisense oligonucleotide <400> 16 cttggccgct gccat <210> 17 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: phosphorothioate-linked antisense oligonucleotide <400) 17 gcacttctgc tgtcc (210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 2CR primers <400> 18 gcacccaaca gtggcaac <210> 19 WO 01/30394 PCT/AU00/01315 8/9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primers <400> 19 gggatcatgg gaacctgg 18 <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primers <400> tgacggggtc acccacactg tgcccatcta <210> 21 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primers <400> 21 ctagaagcat ttgcggtgga cgatggaggg <210> 22 <211> 19 <212> RNA <213> Rattus rattus <400> 22 acguccggga uggcagcgg 19 <210> 23 <211> 19 <212> RNA <213> Homo sapiens <400> 23 ucguccagga uggccgcgg 19 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence WO 01/30394 PCT/AUOO/01315 9/9 <220> <223> Description of Artificial Sequence: DNAzyne <400> 24 tcagctgcag gctagctaca acgactcggc ctt

Claims

1. A method for the treatment of a tumour, the method comprising administering to a subject in need thereof an agent which inhibits induction of an EGR, an agent which decreases expression of an EGR or an agent which decreases the nuclear accumulation or activity of an EGR; wherein said agent is a sequence specific DNAzyme.

3. A method as claimed in claim 1 wherein the DNAzyme directly inhibits proliferation of the tumour cells.

4. A method as claimed in claims 1 or 2 in which the tumour is a solid tumour.

5. A method as claimed in any one of claims 1 to 4 in which the EGR is EGR-1.

6. A method as claimed in any one of claims 1 to 5 in which the DNAzyme comprises a catalytic domain which cleaves mRNA at a purine:pyrimidine cleavage site; (ii) a first binding domain contiguous with the 5' end of the catalytic domain; and :15 (iii) a second binding domain contiguous with the 3' end of the catalytic domain, wherein the binding domains are sufficiently complementary to two regions immediately flanking a purine:pyrimidine cleavage site within the region of EGR mRNA corresponding to nucleotides 168 to 332 as shown in SEQ ID NO:15, such that the DNAzyme cleaves the EGR mRNA.

7. A method as claimed in claim 6 in which the catalytic domain has the nucleotide sequence GGCTAGCTACAACGA.

8. A method as claimed in claim 6 or 7 in which the cleavage site is selected from the group consisting of the GU site corresponding to nucleotides 198-199; 141792670_3 (ii) the GU site corresponding to nucleotides 200-201; (iii) the GU site corresponding to nucleotides 264-265; (iv) the AU site corresponding to nucleotides 27 1-272; the AU site corresponding to nucleotides 301-302; (vi) the GU site corresponding to nucleotides 303-304; and (vii) the AU site corresponding to nucleotides 316-317.

9. A method as claimed in claim 8 in which the cleavage site is the GU site corresponding to nucleotidles 198-199, the AU site corresponding to nucleotides 271-272 or the AU site corresponding to nucleotides 301-302.

10. A method as claimed in any one of claims 6 to 9 in which the DNAzyme has a sequence selected from the group consisting of: (1 5'cggaaGTGTCAGctggg(E (fi) 5'-tcaggggaGGCTAGCTACAACGAacgttgcg (SEQ ID NO:3); (vi) Y-gcagggacaGGCTAGCTACAACGAacgtgcgt (SEQ ID NO:61); (iii) 5'-catctgagaGGCTAGCTACAACGAcagcagg (SEQ ID NO:71). cg ggc GGCAGCCAC cgtt ctgg g (SEQ ID NO: 8) 'gcggggacaGGCTAGCTACAACGAcagctgcat (SEQ ID NO: 14 179267Q3

12. A method as claimed in claim 10 in which the DNAzyme has the sequence: ccgcggccaGGCTAGCTACAACGAcctggacga (SEQ ID NO:8) or ccgctgccaGGCTAGCTACAACGAcccggacgt (SEQ ID NO:9).

13. A method as claimed in any one of claims 6 to 12, wherein the 3'-end nucleotide residue of the DNAzyme is inverted in the binding domain contiguous with the 3' end of the catalytic domain.

14. A method as claimed in any one of claims 1 to 13 which further comprises administering one or more additional anti-cancer agents. A method for inhibiting the growth or proliferation of a tumour cell, the method comprising contacting a tumour cell with an agent which inhibits induction of EGR, an agent which decreases S 10 expression of EGR or an agent which decreases the nuclear accumulation or activity of EGR; wherein said agent is a sequence specific DNAzyme.

16. A tumour cell which has been transformed by introducing into the cell a nucleic acid molecule, the nucleic acid molecule comprising or encoding an agent which inhibits induction of EGR, (ii) an agent which decreases expression of EGR, or (iii) an agent which decreases the S: 15 nuclear accumulation or activity of EGR; wherein said agent is a sequence specific DNAzyme.

17. A method for inhibiting angiogenesis, the method comprising administering to a subject in need thereof an agent which inhibits induction of an EGR, an agent which decreases expression of an EGR or an agent which decreases the nuclear accumulation or activity of an EGR.

18. A method as claimed in claim 1 in which the EGR is EGR-1.

19. A method as claimed in claim 1 or 2 in which the expression of EGR is decreased by the use of EGR antisense oligonucleotide. A method as claimed in claim 19 in which the antisense oligonucleotide has a sequence selected from the group consisting of ACA CTT TTG TCT GCT (SEQ ID NO:4), and (ii) CTT GGC CGC TGC CAT (SEQ ID NO:2). 141792670 3 52

21. A method as claimed in claim 17 in which the expression of EGR is decreased by the cleavage of EGR mRNA by a sequence specific ribozyme.

22. A method as claimed in claim 17 in which the expression of EGR is decreased by the use of a ssDNA targeted against EGR dsDNA the ssDNA molecule being selected so as to form a triple helix with the dsDNA.

23. A method as claimed claim 17 in which the expression of EGR is decreased by inhibiting transcription of the EGR gene using a nucleic acid transcriptional decoy.

24. A method as claimed in claim 17 in which the expression of EGR is decreased by the expression of antisense EGR mRNA. 10 25. A method as claimed in any one of claims 17 to 24 which further comprises administering *ee one or more additional anti-cancer agents.

26. A method of screening for an agent which inhibits angiogenesis, the method comprising testing a putative agent for the ability to inhibit induction of EGR, decrease expression of EGR or decrease the nuclear accumulation or activity of EGR. o 4 15 Dated 22 December 2005 Unisearch Limited S* Patent Attorneys for the Applicant: Blake Dawson Waldron Patent Services 141792670_3