AU2005242129A1 - Catalytic molecules - Google Patents

Description

039679 3111 Blake Dawson Waldron 16:46:30 06-12-2006 7164 20034680 I

AUSTRALIA

Patents Act 1990 (Cth) Complete Specification (Divisional) UNISEARCH LIMITED, AND JOHNSON JOHNSON RESEARCH PTY LTD Invention Title Catalytic molecules The invention is described in the following statement: Blake Dawson Waldron Pacent Services Level 39, 101 Collins Street Mclbourne VIC 3000 Telephone: t 61 3 9679 3065 Fax: 61 3 9679 3111 6 December 2005 Ref: 03 1393-6432 COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16-46:38 06-12-2006 8 J64 In o 0 CATALYTIC MOLECULES C FIELD OF THE INVENTION Ci The present invention relates to DNAzymes which are targeted against mKRNA molecules encoding EGR-1 (also known as Egr-1 or NGFI-A). The Spresent invention also relates to compositions including these DNAzymes and to methods of treatment involving administrmtion of the DNAzyrnes.

C- BACKGROUND OF THE INVENTION Egr-1 expression in Smooth Muscle Cells Smooth muscle cells (SMCs) are well recognized as a significant cellular component of atherosclerotic and post-angioplasty restenotic lesions (Stary et al, 1995; Holmes et al, 1984). SMC migration and proliferation are key events in the pathoganesis of theso vascular disorders (Jackson Schwartz, 1092: Libby et al, 1995). The promoter regions of many genes that encode mitogenic and migratory factors expressed by SMCs in these lesions (Evanko et al, 1998; Murry et al, 1996; Ueda et al, 1996; Tanizawa et al, 1996; Rekhter Gordon, 1994; Hughes et al, 1993; Brogi et al, 1993; Wilcox et al 1989; Wilcox et al, 1986) contain nucleotide (nt) recognition elements for the nuclear protein and transcription factor, Egr-1 (Khachigian Collins, 1997; Khachigian et al, 1996). Egr-1 is not expressed in the unmanipulated artery wall, but is rapidly activated by mechanical injury (Khachigian et al, 1996; Silverman et al, 1997; Kim et al, 1995). It is also induced in vascular endothelial cells and/or SMCs exposed to fluid biomechanical forces (Khachigian et al, 1997; Sumpio et al, 1998) and multiple other pathophysiologically-relevant agonists (Delbridge Khachigian, 1997).

DNAzvmes 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.

COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16-46:57 06-12-2006 9 164 o

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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 Ci endogenous RNAse H enzyme. This dependence on RNAse H confers t limitations on the design of anti-sense molecules regarding their chemistry o and ability to form stable heteroduplexes with their target mRNA's. Anti- Cl sense DNA molecules also suffer from problems associated with non-specific activity and, at higher concentrations, even toxicity.

S 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 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 regions 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 (1968); 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 teclniques 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)).

Kibozymes, 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)). DNAzy:;Ies are singlestranded, and cleave both RNA (Breaker (1994); Santoro (1997)) and DNA (Carmi (1995)). A general model for the DNAzyme has been proposed, and is known as the "10-23" model. DNAzymes following the "10-23" model, also COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 039679 3111 Blake Dawson Waldron 16:47:20 06-12-2006 10164 o

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referred to simply as "10-23 DNAzymes", have a catalytic domain of dooxyribonucleotides, flanked by two substrate-recognition domains of seven C- to nine deoxyribonucleotides each. In vitro analyses show that this type of SDNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions under physiological conditions (Santoro (1997)).

C DNAzymes show promise as therapeutic agents. However, DNAzyme success against a disease caused by the presence of a known mRNA molecule Sis not predictable. This unpredictability is due, in part, to two factors. First, Ccertain 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. For these reasons, merely knowing of a disease and its causative target mRNA sequence does not alone allow one to reasonably predict the therapeutic success of a DNAzyme against that target is mRNA, absent an inventive step.

SUMMARY OF THE INVENTION Accordingly, in a first aspect the present invention provides a DNAzyme which specifically cleaves EGR-1 mRNA, the DNAzyme including 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 (ii) 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-1 mRNA corresponding to nucleotides 168 to 332 as shown in SEQ ID NO:1, such that the DNAzyme cleaves the EGR-1 mRNA.

In a second aspect the present invention provides a pharmaceutical composition including a DNAzyme according to the first aspect and a pharmaceutically acceptable carrier.

In a third aspect the present invention provides a method of inhibiting EGR-1 activity in cells which includes exposing the cells to a DNAzyme according to the first aspect of the present invention.

COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 0396793111 Blake Dawson Waldron 16:47:37 06-12-2006 11 /64 IO4 0 Lu In a fourth aspect the present invention provides a method of Sinhibiting proliferation or migration of cells in a subject which includes Cl administering to the subject a prophylactically effective dose of a DNAzyme >according to the first aspect of the present invention.

5 In a fifth aspect the present invention provides a method of treating a Scondition associated with cell proliferation or migration in a subject which o ixicludes administering to the subject a prophylacLically effective dose of a O DNAzyme according to the first aspect of the present invention.

C In a sixth aspect the present invention provides an angioplastic stent for inhibiting the onset of restcnouia, which comprises an angioplastic stent operably coated with a prophylactically effective dose of a DNAzyme according to the first aspect.

In a seventh aspect, the present invention provides a method for inhibiting the onset of rusteusis in a subject undergoing angioplasty, which comprises topically administering a stat according to the fifth aspect to the subject at around the time of the angioplasty.

COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 96793111 Blake Dawson Waldron 16:47:61 06-12-2005 12 J64 o

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BRIEF DESCRIPTION OF THE FIGURES 0\ Figurc 1 Sequence of NGFI-A DNAzyme (ED5), its scrambled control and 23 nt synthetic rat substrate. The translational start site is underlined.

Figure 2 NGFI-A DNAzyme inhibits the induction of NGFI-A mRNA and protein by serum. Northern blot analysis was performed with 25 jug of total RNA. The blot was stripped and reprobed foro-Actin. Autoradiograms were to analyzed by scanning densitometry and the ordinate axis is expressed as NGFI-A band intensity as a fraction of P-Actit band intensity. The mean and standard errors of the mean are indicated in the figure. Data is representative of 2 independent experinments. indicates P<0.05 (Student's paired t-test) as compared to control (FBS alone).

Figure 3 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 DNAzynte for 3 days were trypsinized followed by quantitation of the suspension. The sequence of AS2 is GGC CGC TGC CAT-3' (SEQ ID NO: 20). b, Proportion of cells incorporating Trypan Blue after exposure to serum and/or DNAzyme. Cells were stained incubated in 0.2% Trypan Blue at 22 OC for 5 min prior to quantitation by hemocytometer in a blind manner. c, Effect of ED5 on pup SMC proliferation. Growth-arrested VWKY12-22 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 t-test) as compared to control [FBS alone].

Figure 4 NGFI-A DNAzyme inhibition of neointima formation in the rat cifotid artery. Neointimal and medial areas of 5 consecutive sections per rat rats per group) taken at 250 am intervals from the point of ligation wero determined digitally and expressed as a ratio per group. The mean and standard errors of the mean are indicated by the ordinate axis. denotes COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:48:08 06-12-2006 13194

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P<0.05 as compared to the Lig, Lig+Veh or Lig+Veh+EDSSCR groups using O the Wilcoxen rank sum test for unpaired data. Lig denotes ligation, Veh Cl denotes vehicle.

Figure 5 Selective inhibition of human smooth muscle cell proliferation by DzA.

SFigure 6 Specific inhibition of porcine retinal smooth muscle cell cl proliferation by DzA.

COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:48:14 06-12-2006 14164 o o 7 0 DETAILED DESCRIPTION OF THE INVENTION OEr-1 (also known as NFI-A and ECR-1 binds to the promoters of genes whose products influence cell movement and replication in the artery wall. Table 1 shows an alignment of the human EGR-1 cDNA sequence with Clthe equivalent mouse (Egr-1) and rat (NGFI-A) sequences. The present t inventors have now developed DNA-based enzymes that cut NGFI-A/Egro 1/EGR-1 RNA with high efficiency and specificity. The NGFI-A "DNAzyme" Cl cleaved synthetic and in vitro transcribed NGFI-A KNA in a sequence-specific manner and inhibited production of NGFI-A in vascular smooth muscle cells without influencing levels of the related zinc finger protein, Spl, or the immediate-early gene product, c-Fos. The DNAzyme blocked serminducible DNA synthesis in smooth muscle cells and attenuated total cell proliferation. The DNAzyme also inhibited the reparative response to mechanical injury, both in culture and in the rat carotid artery wall. These results indicate a necessary and sufficient role for NGFI-A/Egr-1JEGR-1 in vascular smooth muscle cell growth and provide the first demonstration of a DNAzyme targeted against NGFI-A/Egr-1/EGR-1 transcripts.

Accordingly, in a first aspect the present invention provides a DNAzyme which specifically cleaves EGR-1 mRNA, the DNAzyme including 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 catalytcl domain, wherein the binding domains are sufficiently complementary to two regions immediately flanking a purinc:pyrimidine cleavage site within the region of EGR-1 mRNA corresponding to nuclcotides 168 to 332 as shown in SEQ ID NO:1, such that the DNAzyme cleaves the EGR-1 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 of the first aspect of the present invention, the binding domains are complementary to the regions immediately flanking the cleavage site. It will be appreciated by those skilled in the art, however, COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:48:32 06-12-2006 16164 o

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that strict complementarity may not be required for the DNAzyme to bind to and cleave the EGR-1 mRNA.

O0 t The catalytic domain of a DNAzyme of the present invention may be any suitable catalytic domain. Examples of suitable catalytic domains are

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5 described in Santoro andJoyce, 1997 and US 5807718, the entire contents of Swhich are incorporated herein by reference. In a preferred embodiment, the t catalytic domain has the nucleotide sequence GGCTAGCTACAACGA (SEQ ID NO: 2).

Within the parameters of the present invention, 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. It is well established that the gieater the binding domain length, the more tightly it will bind to its complementary mlRNA sequence. Accordingly, in a more preferred embodiment, each domain is nine or more nucleotides in length, Within the context of the present invention, preferred cleavage sites within the region of EGR-1 mRNA corresponding to nucleotides 168 to 332 are as follows: the GU site corresponding to nucleotides 198-199: (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 271-272; 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.

In a further preferred embodiment, the DNAzyme has a sequence selected from: 5'-caggggacaGGCTACCTACAACGAcgttgcggg (SEQ ID NO: 3) targets GU (nt 198, 199); arms hybridise to bp 189-207 (ii) 5'-tgcaggggaGGCTAGCTACACACGAaccgttgcg (SEQ ID NO: 4) targets GU (nt 200, 201); arms hybridise to bp 191-209 COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16-48-61 06-12-2005 16164

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(iii) 5'-catcctggGGCTAGCTACAACGAgagcaggct (SEQ ID NO: N targets GU (nt 284, 265); arms hybridise to bp 255-273 s (iv) 5'-ccgcggccaGGCTAGCTACAACGAcctggacga (SEQ ID NO: 6) targets AU (nt 271, 272); arms hybridise to bp 262-280 0 (tv) 5'-ccgctgccaGGCTAGCTACAACGAcccggacgt (SEQ ID NO: 7) targets AU (nt 271, 272); arms hybridise to bp 282-280 (vi) 5'-cggggacaGGCTAG TACAACGAcagctgeot (SEQ ID NO: 8) targets AU (nt 301, 302); arms hybridise to bp 292-310 (vii) 5'-cag$ggaGGCGCTAGCTACAACAtog c t g c [SEQ ID NO: 9) targets GU (nt 303, 304); arms hybridise to bp 294-312 (viii) 5'-ggtcagaGGCTAGCTACAACGActgcagcgg (SEQ ID NO: targets AU (nt 316, 317); arms hybridise to bp 307-325.

In a particularly preferred embodiment, the TNA2yme targets the AU site corresponding to nucleotides 271-272 (ie. the translation start codon).

In a further preferred embodiment, the DNAzymn has the sequence: (SEQ ID NO: 6).

In applying DNAzyme-based treatments, it is prTcferable 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 terrnini 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 nonmal 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 binding domain contiguous with the 3' end of the catalytic domain. In addition to inversions, the instant DNAzymes maycontain modified nucleotides. Modified nucleotides COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:49:10 06-12-2005 17164 iinverted T at the 3position, o

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include, for example, N3'-P5' phosphoramidate linkages, and peptide-nucleic acid linkages. These are well known in the art.

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

As will be appreciated by those skilled in the art, given that DNAzymes ^c of the present invention cleave human EGR-1, similar DNAzymes can be *I produced to cleave the corresponding mRNA in other species, eg. rat (NGFI- SA), mouse (Egr-1) etc. In a further aspect, the present invention provides CN such DNAsymes.

hI a second aspect the present invention provides a pharmaceutical composition including a DNAzyme according to the first aspect and a pharmaceutically acceptable carrier.

In a third aspect the present invention provides a method of inhibiting EGR-1 activity in cells which includes exposing the cells to a DNAzyme according to the first aspect of the present invention.

In a fourth aspect the present invention provides a method of inhibiting proliferation or migration of cells in a subject which includes administering to the subject a prophylactically effective dose of a DNAzyme according to the first aspect of the present invention.

In a fifth aspect the present invention provides a method of treating a condition associated with cell proliferation or migration in a subject which includes administering to the subject a prophylactically effective dose of a DNAzyme according to the first aspect of the present invention.

In preferred embodiments of the third, fourth and fifth aspects of the present invention, the cells are vascular cells, particularly smooth muscle or endothelial cells. The cells may, however, be cells involved in neoplasia, such as tumour cells and the like.

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

In a preferred embodiment, conditions associated with SMC proliforation(and migration) are selected from post-angioplasty restenosis.

vein graft failure, transplant coronary disease and complications associated with atherosclerosis (cerebrovascular infarction (stroke), myocardial infarction (heart attack), hypertension or peripheral vascular disease (gangrene of the extremities).

COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 96793111 Blake Dawson Waldron 16-49:30 06-12-2005 18 164 o

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^s0 11 Within the parameters of the fourth and fifth aspects of the present invention, any suitable mode of administration may be used to administer or Ci deliver the DNAzyme.

In particular, delivery of the nucleic acid agents described may be achieved by one or more of the following methods: Liposomes and liposome-protein conjugates and mixtures.

tT Using catheters to deliver intra-luminal formulations of the nucleic Sacid as a solution or in a complex with a liposome.

CN Catheter delivery to adventitial tissue as u solution or in a complex with a liposome.

Within a polymer formulation such as polyethyleniminc (PEI) or pluronic gels or within ethylene vinyl acetate copolymer (EVAc). The polymer is preferably delivered intra-luminally.

The nucleic acid may be bound to a delivery agent such as a targetting moiety, or any suitable carrier such as a peptide or fatty acid molecule.

Within a viral-liposomo complex, such as Sendai virus.

The nucleic acid may be delivered by a double angioplasty balloon device fixed to catheter, The nucleic acid could be delivered on a specially prepared stent of the Schatz-Palmaz or derivative type. The stent could be coated with a polymer or agent impregnated with nucleic acid that allows controlled release of the molecules at the vessel wall.

In a preferred embodiment, the mode of administration is topical administration. Topical administration may be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The topical administration can be performed, for example, via catheter and topical injection, and via coated stent as discussed below.

Pharmaceutical carriers for topical administration are well known in the art. as are methods for combining same with active agents to be delivered. 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.

Topical delivery systems include, for example, gels and solutions, and can contain excipicnts such as solubilizers, permeation enhancers fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:49:60 06-12-2005 19164 0 ci IN 12 polymers polycarbophil and polyvinylpyrolidone). In the preferred 0\ embodiment, the pharnmceutically acceptable carrier is a liposome or a C biodegradable polymer. Examples of agents which can be used in this invention include the following: CellFectin, 1:1.5 liposome formulation of the cationic lipid N, N,N,N-tetramethyl-NNI,N,NI- C] tetrapalmitylspermine and dioleoyl phosphatidyl-ethanolamine (DOPE) S(GIBCO BRL): Cytofection GSV, 2:1 liposome formulation of a o catiouic lipid and DOPE (Glen Research); DOTAP (N-[1-(2,3-dioleoyloxy)- N,N,N-trimcthyl-ammoniummethylsulfate) (Boehringer Mannheim); (4) Lipofectamine. 3:1 liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL); FuGENE 0 (Roche Molecular Biochemicals); Superfect (Qiagen); and Lipofectamine 2000 (Gibco-life Technologies).

Examples of suitable methods for topical administration of the DNAzymes of the present invention are described in Autieri et al. (1995).

Simons et al. (1992), Morishita et al. (1993), Bennett and Schwartz (t995) and Frimerman et al. (1999).

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 dose contains between about 0.1 mg and about 1 g of the instant DNAzyme.

In another embodiment, the prophylactically effective dose contains between about I mg and about 100 mg of the instant DNAzyme. In a further embodiment, the prophylactically effective dose contains between about mg and about 50 mg of the instant DNAzyme. In yet a further embodiment, the prophylactically effective does contains about 25 mg of the instant DNAzyme.

In a sixth aspect the present invention provides an angioplastic stent for inhibiting the onset of restenosis, which comprises an angioplastic stent operably coated with a prophylactically effective dose of a DNAzyme according to the first aspect Angioplastic stents, also known by other terms such as "intravascular stents" or simple "stents", are well known in the art. They are routinely used to prevent vascular closure due to physical anomalies such as unwanted inward growth of vascular tissue due to surgical trauma. They often have a COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 0396793111 Blake Dawson Waldron 166:1 06-12-2006 20164 o O 13

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tubular, expanding lattice-type structure appropriate for their function, and can optionally be biodegradable.

Cl In this invention, the stent can be operably coated with the instant pharmaceutical composition using any suitable means known in the art.

Here, "operably coating" a stent means coating it in a way that permits the Cl timely release of the pharmaceutical composition into the surrounding tissue I to be treated once the coated stent is administered. Such coating methods, Sfor example, can use the polymer polypyrrole.

Cl In a seventh aspect, the present invention provides a method for o0 inhibiting the onset of restenosis in a subject undergoing angioplasty, which comprises topically administering a stent according to the fifth aspect to the subject at around the time of the angioplasty.

As used herein, administration "at around the time of angioplasty" can be performed during the procedure, or immediately before or after the procedure. The administering can be performed according to known methods such as catheter delivery.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting Figures and Examples.

COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 31 11 Blake Dawson Waldron 16:50:26 06-12-2006 21164 14 Table 1 Symbol vQmpariso table: GenRunDate:pileupdna.amp CapWeight: 5.000 GepLengthweight: 0.300 ISGRlan.mf MSF: 4388 Type! N April 7, 199a Name: mouseEGR1 Len: 4388 Check: 8340 Weight: Name: ratEGRl Len: 4388 Chek: 8587 Weight; Nane humanEGAI Ten: 4388 Check: 8180 Weight: CompCheck: 6876 12:07 Checkt $107 1.0(SEQ ID NO:11) 1.O(SEQ ID N012) 1.00 (SEQ ID NOzI) N. THIS ZS RT Ng-A numberin5 3. mouseEgr reINGETA CGCGGAGCC TCAGCTCTAC GEGCCTGGCG, CCCTCCCTAC CCCGCCCCTCC hvmanEGRI 51 100 nous eEGRI ratEGRI CCGACTCWCG CGCCGTTCA GGCCCGSGT TGCOAACCAA AGGACGGGGAG huranEGRI 101 150 MMU-50eEGR1 ratEGPl GGrGGCrGCC CCSACCCCGA AACACCATM AAGGAGCAGG AAGGATCCCC humv-EGRI 51 mC'uoEGRl ratEGRI CGCCGGAAVA hurranEGRi 201 mOuSeEGRI ratM;KI TOOCCCTGCQ hlnianEGRI GACCTTA'rIC GGGCAGCGOC TTMAGPG GCTTCCCCCT CTCGGALmAG GGGCGAACGG 251 MouseFR1 ratEGRI GGGGGCAAGC TGMAACTCC AQGAGCCTAG CCCGGiAGGC huannEGRI mouzeEGRI ratEGRI 'uThIanEGRI 301 GTTCCAATAC TAGCCTTTCC AGGAGCCTGA GCCCTCAGGG 200

TGGCCCANT,

250

GGGTTGGGGC

300 ZACTGCCGC7 350 Tc;CCGGaGC 400

TCTTCACGTC

CCTGCTTCCC

500 351 mouseEGRI rattGR1 GGTCCCAGC, VGGAAGCGCC u..a u.

.R.

402 mouseEGRi ratEGRI ACTCCGGGTC CTCCGGTCG buvt nEG.

CACCGCTCTT OGATGGGAGO GTCCTTCCAT ATTAuGnCTTIl COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron160:1 6-206214 16:50:41 06-12-2006 22164 Mis aESRI ratEf3Rl human SCRI mrOIuseEGfll ratEG.A1 humarnEGRI mouseEGkl rEGRI humanEGRi mous aSGRI ratEGRi hurnanEcRI2 rnOUrseGRl ntEURl humanEGRI MOUSeEGRI rat]ZGFL humanEGrIF YRQuseEGRi -atEGRI humanEGftl incus eEG R ratEGRI huinanEGRi.

racuseEGRI ratEGRi hUinanEGRI Iuo.us.EiGRi raLEGRi hunanEGR1 lnouacEGRI 53 ratEri~k buinanEGRI inauseEGRI 00 ratEGRI ATATATCC A7TgACCTCA O(GOGGArOC CGGGCCCGTGC; TGrrCGAC CCTT7GAAAkTA GAGGCCGATT CGGB3GAQTC;G CGAGPLGATCC: CAGOGGCAG

CCCAG

551 600 GGGGA GttGcccccc CGATTCGCCG CCGCUiSCCAG CTTCCGCCGC AACTTGGGGA GCCGCCGCCG CGATTCGCCG CCGCCGCCAG CTTCCGCCGC AACTTGGGGA GCCGCCGCCG CCATCCGCCG CCGCAGCCAQ CTTCCGCCC 601 650 CCAGATCG GCC CTGCCC CACCTOCGO GGCOGCCCTG CGTCCACCAC CGCAAGATCG CeeC;!toCCo CAGCCTCCGC GCCAMCCCTrG CGTCCACCAC CGCAGraACC-G GCCCCTGCCC CAGG;CTCCGjC AGCCGCGGCG CGTCCACGCC 651 GGGCZ-GCGG7C TACCGCCAGC CTGGGGGCCC ACCTACACTC CCCGCAt3TGT GGGjCZCCGGC CA=t;CCAGa CTGGGGGCCC ACCTACACTC ccCGCAGTGT CGCC:;1CGCC CAGGG'CGACT CUGGGTCGCC GCCTCACGC TTCTCAG;TGT 701. 750 GCCCCTGCAC CCCGCATGTA ACCCCCCCAA CCCOGGCGA GT1GTGCCCTz GCCCT-TCCAC: CCCGCATG'rA ACCCGGCCAA CATCOGGCGA C-Z'TGCCCTZ TCC.CGCGC: CCCSCATGTA ACCCaCCC CCCCCGCAA CGC-TGTCCCC iS: 5800l AGTASCTTCG GCCCCGGGCT GCCCACC. .ACCCAAr-AT CAGTTrTrCA AG!tAGCTTCS. CCCCCG47GC' GCGCCCACC. ACC CAALCAT CASC-TCTCCA TGCAGCTCCA GCCCCGGGCT GCPZCCCCCC GCCCCGACAC CAGCTCCA 801. 050 GCTCGCTGGT CCGGGA'IGGC AGCSGCCAAG GCCACATjOC MT7TCATGTC GCCGCACGT CCGGGATGZ AGrc^GcCAAG SCCGAGATOGC AATTGAn7SW CCCTCXCG2' CCAGCATGCC CGCG*CAAG- GCCGAGATGC ACGCTGATtC WtS (rat) arms hybridise 'to bp 001-825 in rat seqtt hZDS(hum) ants hybridise to bp 242-280 in hum, seqn 851 900 TCCGCTGCAG ATCTCTGACC CGTTCC-GCTC CTTTCCTCAC TOACCCACCA TCCGCTGC-AW ATCTCTGACC CGTTCCGCTC CTTTCCTCAC TCACCCACCA CCCGGTGCAC ATCTCTGACC CGTTCGC.ATC CTTTCCTCAC TCCGCCCACCA 901 950 TGGACAACA CCCCAAACTC CACACATGA TGcTGCTGAG CACSCGCT TGGACAACTA CCCZ:AAACTG GAGGACATGA TGCTGCPGAG CkC4GOGGCT 'rGGACAACTAP CCCTAAGQCTG GAGGAGATGA TGCTCIGAG ChACc-GGGCT 931 1000 CCCCAGTTC- *cci3-,Torc CGGAACCCCA GAGGGCAGCG GcGGTAAT..

CCWCAGTTCC TCGGTG ,CRC _CCGAMCCCCA a'.ZC;CCACC CCGGCAATIA CCCCA$oTTCC TCGOCGCCGC CGGCGCCCA GAcGGCAGCG GCAGC-AACAS i0o: 10D50 AGO A.GCAGC-AGCA CC-AGCACC GGG.CGGTGGT GGGS:CGGCA CASCACCASOC ASCAGCACC-A GCAGCAGC!GG GGGCrGTGC;T GGrnCG.,_GC:A COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 1:10 61220 36 16:51:02 06-12-2005 23164 bumanEoRI ITIOUSPECGR1 ratEcri flume nEC-R zzous cECRi ZatEGA1 hhArtEG;Rl IncUS EQR1 ZetEGRJ.

humanEc.R1 Mous eEGR2.

-ra-tEGAi humnanEGRl IiiouseEGRl retECRi hUmanEGRI inougtezos ratEGjRi hlnan.EGRI incus eEGR1 raLECRI &-'rnaLECRl IflouseCGRI retEORi hurnnEGRl Incus eErftl EaLEGQI buUmaIIEiRZ 2nouseESGRj.

s0 ratEGRI h=manEGRi inouseEGRI aldEGRI humanzGR1 inOUs eECRI ratEGRI CA.GCACCAQC AGCACGCuc CEGGTEOACC CCCCGGOGGC; 1051 CCAACAGCGG. CAGCACGCFC TTCAATCCTC AAGUCCACC CC?%ACAGCGG C-AGCAGCGCT TT CMTCQTC AAGGGAGCC GCACCAGACAG C-A(CAGCACC TTCAACCCTC AGGCGG.ACAC

GGCAGCAACA

1100

GAGCGAACAA

GAGCGAACI

GGGCGAGCAG

1101 CCCTATGAGC ACCTCAcCAc AG A&TC CCCTACGAGC ACGTGACCALC ASGTAAGCSG CCCTAGGAGC ACCTGACCGC AG..*.AGTCT 1151 TAATG.AGAA.G CGATGGTrCC AGACG3AGTTA TCCCC-CTTCG 'rCACTA-CCT AACGTCCA~rT CAACGAGAAG GTGCTCTGQt AGACCAGTA, 1201 TGCCTCCCAT CACCTALTACT GOCC;CTTCZ ATCTAGATCT TAGGG ACGG ATGUGATTT TGCCCCCCAT CACCTATACT GGCCGCTrTTT 1251 ASP GGCAACA CTTTGTGGCC TGAACCCCT TCCAGGGACT TGTGTTrAAG GGATGTCTGC AGTGGCAACA CCrTGTGGCC CGAGCCCCTC 1301 CGTGAGCATG ACCAATCCTC CGACCTCTTC CTTGCGGGTG CGCCCACCGtC AGACCGTTTC AGTGAGCATG ACCAACCCAC CGCCcac 1.351 CTCfcT7AT GTCTTCCTCT GCCTCCCACA TTC-CGTGGGT GCT GGAGT CCCC'rCC CG. CTC0 OCTCCCAGA 101C

G

9 IGCZGTZ-CA ACG;A:AGCAG TCCCATCTAC GAGCAkGGGTT GC CCCC TCCCCCGCC GIGC:ATCCAN AGGaACCAG TCCATTTAC 145i.

ThCTCCCAACL ACTGACATTT VFCCTG AUCC TCCA CrrGT TCCCAACGA COGCTCAAAT GAG-GCAAC ACTGAOATTT TCCCTQAGcc 1501 GV2000-CAG CACAGGOTTG CJLGTACCCG5C CCGOCCAGGG TAGGGGCOCO CA'ZTAOCTGT GCTCCACO CAEAGCGCTC CAGTACCCC 1551 AAAOOTOGTT TCCAOCTTCC CA'PGATCCC.;T GGATTCCCTC ACCCCGCACG CCTGCTGCGG PAGZTGOCT TCCAGGTTCC CAGATCCCC 1601 ACAGZ3GAGAC CTGAGCUTGG CCACCCCAGA AGAGSOOCPLT TCI'CTGTrFG CGTCAGCrGT 115D TTTTCTGACA TCGCTCTGAA TGGTCTGCGC CGAGGCTGAA TTTCCTGACA TCZ'CTCTG;AA 1200 TCCCAGCAJ ACGACTCGGT CGCTTTOCACC ACCCACCTGC CCCCAWGCGAA ACCACTCGAC 1250 CCCTGSA&7CC COCACCOC CCCTCTATVc cAcAcAGC CCT-Gt;AGCC rGrCACCCAAc 1303) T'TCAGCCPAG TCACTOGCT GGACCCCCA ACCCTCCATC TTCACTTII TCTC-GCCT 330 ATCCTCGGCG CCTTCTCCAC TTT7GUATG( AGAAC;tC-AAG GW7CCTCACCA CCATC-TCC.6G 1400 CGCCC*CCCCT CACCTCTGCCC GGUGi GAGGGT TTGTTTTCAT GCCCACCCCT GAGCTGCGCA 1450 TCGGCTCCGC CCACCTTTCC GCC-CfCGC GAGCCTTGTT TCAGCOOCAC CCACCTTCCC 1500 CCAAAS;CCAO GCCTTTCCTG CTOTCACCrAG GGATGTCCCG ACAAASCCAO GCCTTCCCGG 1550 CIOCTOCCTA CCCTGCCACC GGZC ACTAS GGTGCTOOCG CTCCVCTA CCCTOCCGCC 1500 GACTAOYCTCT TTCCACAACA AGCGCTCTCA CAOCTOCAGT CACTACCTc2T TICCAC-'AGCA 1650 CCAGMIUGCCC TTCCASGGTC CGAAATqGCT CT CC COMS ID No: SBMI-02132168 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 1:12 61220 46 16:61:22 06-12-2005 24164 hunianTA&1 inouaCEGR1 6 ratEGRI humanEGlI mou&EtGrl raLE(GR1 bUnianEGa 2 mousesopl ratECfli huisanEGRI rnouaeEGRl ral;DG31 hUinanEGR2 inouseEGR1 tEGR1 huleanEGRI incus tECR1 ratGH1 htunantGR2.

rnouseeGRi raLEGfll buzponMnGRI incus eEGRI ratEGRl huznanEGRl ncu-s eEGHI ratEGRl, hunaknzopj IEOuzeEGkl XALZGRl huinanTGRI inOUSeEGRi 5,3 rat;SGR1 huant~nGRl inoUSeESRl ratEG;Ri oCAGGOOGAT CTrGGccrGG ~CAccccGAGA CCAGAAkGCCC nTCCASGGCC 1651 1700 TGGAGPACCG TACCCAG-CAG CCTTCGaZCA CTOCACTATC CACTATTAAA CACTGG&GCA GGTCCAGGAA CATTGC:AATC TO.CTOCTATC AATTATTrAAC TGCAGAGCCG CACCOAGGAG CCTTCGCTAA CCCCTCTGTC TAcTATAAG 17?01 1750 GCCTTCGCCA CTCACTCGCrg CCCCAGGA C TTAAAS GCTOTTA CAtCATCGACA G;TCAGTGGTA CCGGCGAGC TOTTOGCCTG occGCTTC; GCCTTTGCCA CPCAGTCGGG CTCCCAiGAC CTCAAG GCZCTCA 1751 1000 ATACCACCTA CCAATCCCAG CTC-ATCA. .A ACCCAG7CCGC ATGCGCAAGT CTCTCATCG-T CCAGTGATTG CTCI'CCAG.TA ACCAGGCCTC TCtGTTCTCZ.

ATACCAGCTA CCAGTCCCAC CTCATCA-.A A&CCOAGOCOGC ATGCGCAAGT' 1601 1850 ACCCCAACCG GCCCAGCMAG ACACOCCOC ATGAA~vCGCCC A'TATG.CTTGC T CCTGCCAG AjTCCTTTTC TiCaAA1CGCT CTGAATAACS ALGAXO rC ATcCC-AACCC GCCCAGCAAG ACQC=CCCCC ACCAACCCCC TTACc-CTTG^C 1851 1S00 CCTGTCGAGT CCTGCGATCO CCCCTFDTTCT COOT OGGATO ACTTACOCC CTGGTGGAGA CAAGTTATCC CAGCQAAACT ACOCGGrToC CTCCCATCA.C CC-AGI'GW$GT CCTCTGATCG COOTTCTCC CCCTCCGACG AGcTUAC~crG 19111 1950 CCATAPrCCGC ATOCACACAG GCCAGAACCC CTTCCAGTGT CGAATCZ'CA CIATACTGGC COCTrTTCC TGIGAGCCTGC ACCCAACACT GG7CAACACTT COACATCCGC ATCCACAG-AG GCCAGAAGcc C'rlFCCAGTGC CGCATCTGCA 195i 2000 7GCGTAACTT CAGTCGTAG? GACCACOTTA CCACCCACAT CCCACCCAC TGTG^GC-CGA ACCCCTTTTC AGCCTAGTC-A CTCGCCTTGT GAGCATGACO TIGCGCAACTr CAGGOGO:AGO GACC ACCICA CCACCCACAT CCCACCCAC 2001. 20$0 -ACGGAGA AGOCCTYTTGC CTCTCACATT TGTGGGA-CA ACTTTCCCAG AZACCCTCCAA CCTCTTCATC CTC-ACCGCCT TCTCCAGCTG CTTOATOOTOT ACAGGCCAAA AGCCCTTCGC CTGCGACATC TGTG4GAAGAA AGTTTGCCAG.

2051 2100 &ACTCATCAA CGCANGAGOC ATACCAAAAT CCATTTAAGA CACAA'CCACA TTCCCT(300 TCCCASAGCC CACOrCGAG CTGIrGCCC-TG CCGTCCAACG SAGCGATGM CGC-AAGAGGC; ATACCAAGMT CCACTTGCCG CACAAGCACA 2101 2150 AGAAAGCAGA CAAAAGT'GTG GTGGCCTCCC CG.GCTG;C. CTCTTCACT ACAGCAS'TCC CATTTACTCA GCTGCACCP CCTTTCCTAC TCCCAACACT AGAAASCAGA CAAAAGTGOOT GT-GGCCTCTT COOCCACCTC cTCtaeTCT 2151 2200 TCTTCTTAC CCATCCCCAG ?G(GCTACCTC GAC-ATTTTTC CTO-ACCCCA MAGCCAGGCC TCT"ACCCTr CCCCGGTZGC TAhOOTCTTAC CCGTCCCCGS TTACTACC'rC 2201 2250 CTACCCNTCC CCTGCCACCA CCTCATTCCC ATCCCCTGTG CCACTTCCT TTTCCTGGCT CTGCAGGCAC AG3CCVrGCAG~ rACCGCCTC CT-C-CTACCC COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 0396793111 Blake Dawson Waldron1:141 0-2062/4 16:51:41 06-12-2006 25164 humanEGP.2 teouseEGRI atEGRI hunianEGI ratEGRI hunt EGRI mOq**5

EGRI

is zrctEGRi hunflJtIEGR2.

ziusreEGRi ratEGRi human grRi rpoueEGRi ratEGR1 mouseEGRl rAttGRl humanSGRI rnOUSeEGR1 ratEsRi humanS;J9J rnojseSGi ratzGR1 hwmanzEGRl mnouseEGRI r at-t&co.-I bureanEuRl mouseEGRI ratGA1 hurnanEGRI mougseEGRI ratEGRi bunnanEGRi jTvouselEGR1 SO rateGR.

TTATCCATZC CCGGCCAC:A CC-TCATACCG ATCCCTGrG ccCACCTCCT 2251 2300 ACTCCTCTCC TGGCTCCTCC ACCTACCCAT CrTCGCA CAGTGGCTTC TGCL2ACCAAG (GTGG-T?1 t AGQ.TTCCCAT GATCCCVGzAC 27ACTCrFTC TCTCCTCTCC CGGCTGCT:G ACCTACCCAT CCCCTGTGC-A CAGiTGG=TC 2301 CCGTCG7CCQT CAGTGGCCAC CLCCTTTQCC TCCGTTCC CACAACAACA r5GGAGACCTG AGCCTGGGCA COCCAGACCA GAAGCCCTTC CCCTCCCCC7 CGGTGSOCAC cCGrTACTCC TCTGTTCCC 2351 2400 ACCTGC TTTCCCCACC CAGGTCAGCA UCflCCCSTC 'rSCGCGCGTC CAGGGTCTGG AGAACCGTAC CC-AGCGCCT TCGCTC:ACTC ICACTATCCAC CCIGC TTTCCGCCC CAG.GTCAGCA GC'flCcCTTC CTCAGCTCTC 2401 2450 AGC:AGCTCCT TCAGCAC CTC: AACTGCTCTT TCAGAGATGA cAGCGACCrT 2A'I-AAAkGCC TTCGCCA6CTC AGTCGCTC- CC-AGGACTTA AAGGCTCTTA ALCCAACTCT TCAGCGCCTC CACACCTT T CGCGACATQCA CAS.CAACCTT 2451 '2500 TTCTCCC-AGG ACAATTGAAA TTTCCTMAAG GGA AAAAAC. ATAACACCTA CC-AGTCCCAA CTCATCAAAC GCAGCCGCMT GCSCAAGT..

T7CTCCCAGU ACPATTGAAA TTTGCTAAA.G GGAAAGGGGA AAGAAAGGGA 2501 2550 AAGCRAAG GGAGAGGCAG GAAAGACATA AARGCA. C AGGAGGGAAG .ACC:CAACC GGCCCAGCAA GACACCOCCOC CATGAACGCC CGTATGCTTG AAAGGGAGAA AAAGAAACAC AAGAGACTTA AGACAGGA CGAGGAGATGj 2S'. 2600 AGATGCCCGC AAGAGGFGGC!C ACCTCTTAGG TCAGATqGAAL GATCT1CACAG cCCCrrGAG TCCTCCCATC GCCGC'1TTTTC TCGCTCCGATI CAGcT1'ACAC GCCATAGW C& ACMMGGGTT. .CTCTTAC;G TCAGATGGAG G;TTCTCAGAG; 2601 265C CCAAGTCCTT CTACTCACcA GA. .GCA'G ACCGTTGGCC AAkCAGCCCTT GCCACATCCG. cAI'CATACA GGC. CAGAR .CCCTTCC-AG TG~cGAATCT CCAAG3TCCTC CCTCTCTACT GGAG'TGQAAG rTCATr'IGC( CALACAATCCT 2651 27 0 C TCACTTACCA TCCCTGCCTC CCCCGTCCTG TTCCCTTGA CTTCAGCTGC GCAkTc3TAA rrTCAGTCGT AGTGACCACC TTACCACCCA CATCCCCACC TTCTGCCCAC TTCCCCTICC CCPATTACTA TTCCCTTTGA CTTCAGCTGC 270D. 2750 CTGAAkAAGC CATC.TC CAAC TTCflCACt Ci'ATrCCAAAG G;ACTTGATTT C. -ACACAOG CGAGAAGCCT TTTGCCTCTC ACArrTGTGG GACXAGTT CTGAAACABC CATCTCCAAkG TTCTlCACcr CTATCCAAAd AACTTGATTT 2751 2800 GCATOGG. .TATTOGIAT AAAITCArrTC CCCAGGAGTG ATQAACGCAA CAGGCAkTACC ARRATrCACT TAAGACACAR.

GOATGA. TTTTGMAT AAATCATfl'C 2B01 2850 CCATC ACATCCCTGC CCCTTGCTCC CTTCAOCGCT AGACC.ATCAAt GSACAAGAAA GCAGACWAA G.TGTCGTSGC( CTCCTCAGCT GCCTCT1'CCC COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 0396793111 Blake Dawison Waldron162:3 6--26264 16:62:03 06-12-2006 26164 bcmantsRl ISouseEGRI z atCGR1 buh.anS-fti rnou.s etGRI ratCR1 huwmatEGRI mouSSEGRal ratEGRi bumanEGal MoonseEr.R1 ratEcRI humanEGR1 InaureeEGAl zatEGR1 hu~manEGIL1 oouS eECR1 so rtatEGal hUinanECRi MouseEGRI ratE(R1 humanEGR) mau U eEGRi tatEGRi huma.2cEup incus sEGRI 459 ratEGRi huluanEGRI InouseEGRI ZatEGRi humeiEaR 1 inouaeFGRl raLEGPi htuaanLEoRl rnouselSGRl ratEGRI CCACA TATGCCTAC CCCTT3CTCC CTTCAATGCT AGAATOON 2851: GTTGGCATAA AGAAAAAAAA AVIGrGTTTCG GCCCTCACAA CCCrGCCCTG TC7CTTCCTA CCCATCCCCA GTGGCTACCT CCTACCCALTC CCGGEACC GZTGGC AMA? GGGQGTTTGGG CCCCTCNGAG CCCTGCCCTG 2350 CATCTTrGTA CAGCATr-tGT GCQCATGGATT TTGTTTTCCT TGGGTATTC ACCTCALTITC CATCCCCAkGT CCCCACC CT TACTCCTrCTC CCGGCTCCTC- CACCCTTGTA CAGTGTCTGT SCCATGGATT TCGTTT2TCT Tr(GGTAC'rC 2951 3000 TTGATCTGAA CATAATTTCC ATACT. CTATTGTAT TACCTACCCG; TCTCCTGC:AC ACACTOOCT'] CCCATCGCCC TCGG;TCCCCA TTGAT GTGAA GATAALTTTGC ATAT CTATTSTAV rAT??GGAGT 3001 3050 ThAATCCTCA CTTTGGCG;., GAGOGGCAG CAPAGCCAASG CAAACC-AATc; CCA4CCTATCC CTVCISTCC.. CALCCTr3C'LTT CCCTGCCCA(.i UTCAUCACCT TACQTCCTCA CITTGGGGAA AAAAAAAAAA MAAAGC:CAAQ; CAAACCAATG 305i 3100 ATGATCCTCT ATTTTGTGAT CACTCrGCTG TCCAGTeCGC AGGGCAGC AA.CTCCTCA GCACC-TCAAC- QGGTZ:TTTCA GTrGATCC-TCT ATflTGTCAT GA.TGCTGTCA sic: 3150 GGTTTCAA( CATT 'TTTTT'r TTCAACCAGC ACTCCTflGGT ATTJ'ACTCCA GACATGACAG CAACCTTTTC TCC2AGiGACA ?J-TGAAAT'F? ,ACrrTC-A ACC'rTTTTTT ?TGMAACACC AGTCCCAG. TATTCTCA 315~.3200 *.-GCATGTGT CACAGTGTTG TTCCGTTAAT ?'1-GTAAATA CTGGcTCGAC ATGAAAGAGA SCARACOC~AG G(Q3AGCGCG.A G.AGACAA 4 TAA AGGACAGGAG GAOATGTGT CASAGTGTTG flCCGTT/tac CTTTTTGTAA ATACTGCTG7 3201 3250 TGTAACTCT CACATGTGAC AAAGTATCCT TTCTTGrGTT CGTTTTCTT GCGAAGAAAT GGCCCGCAAG AGGGCTGCC TCTTAGCTCAL CATCC-AAGAT ACCGTACTCT CACATGTG;GC AAAATATGGT TTGGTTTTTC ITTTT~rT-T 3251 3300 rFTGAGAAT*T TTTTTCCCCri- TCCCTTTCCrT TTCAAPAGT? TCACGTCTTC CTCAGAkCCCA AGTCCTTCTA GTCAGTAGAA GGCCCGTTGS CCALCCAI3CCC 7rGAAALGTCT TT1TTTCTTCC TCCT-TTGGT TTAAAAAGTT TCACC;TCTTG, 330:- 3350 GTGCCTTTTG TGI'GACACCC CTT.CCGATG GCTI'CACATC COCA..

'PTTCALCTTAG CG7SCCC-TGCC CTC. CCCmi-r (CCCGGTCCTT TTGACTTCAG CTCCCTTflG TCTCATCrC CTTCCTCATC G.CTTGACATC, TGCAAT....

3351 3400 CGATCTCA SC'GACACCCT ZACOTTALCCC .000 CCTACCAGTG CTG-CCTGAAA CA(3n;ACG'SC :;MGI7'CTTC ACCT... MCIA TCCAAACGAPC TGTGA GGACA2GCT ZACCTCTAGC CTTAAGGGc.G CCAGGOACTG 3401 3450 ATCTGTTG~c SCAGCCA GAC-CAAC GAGGAAGAGG3 GCTGAGC'SGA TTCATTTGCA TGT-ATTGGA TANACCATTT CACCATCA7C TCCACCACAT C0MG ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 96793111 Blake Dawson Waldron 1:22 61-062 6 16:52:25 06-12-2006 27164 humanEGAi incus tEGR1 humanEc-Pi inogs cEGRi ratEGRi.

hiimanEcAl ratBGRI huranSGRl ioufseSGR] ravEGR3.

hunaniEGRIf razECIRL huxnanEGR 1 nouse]EGRI ratEGRI moUsIeERIC ratFGI bUriEnGRl incuseZRI s0 ratEGGR.

humanEGal ratEGRI huinanEcRI mousetGRl ratEGRl

ATGATTTGGG

3451 GCTTCGOCc

GCC:GGCCOT

GCTTCGCTTC

3503.

CTCTCAAAA3

AATGGGTC

C-TCTCAAAc3 3551

TCAGGAGTTG

GCATCTGTGC

TCAGGACTTG

3601 ATGrILATAA TAATT'G tAT

ATGTTATGMA

3651 flGTGTTTGC 0A3 rGrGAGC

TTGTGTTTGC

2101

GCGCTCTATT

ATCCT OCT 1? cICGCI STATT

TTAAAACCAA

GaTCCTAflGTA

,TTAMCGAA

GTAALATACrTG 3851 T(CrTTGGGTT 2901 CCTrTCGTTT 395L.

GSC CGA7GOCT 4001

TTAAGCGGCT

GGAa(GCrTTTG

TCCCAGAATGT

TGCTCCCTTC

TCCAWATGr CCAOCAAMAT AAOGAAGAGCC GcCAGCTGA 3500 AAGAkGAAAA ATtTZAAACA AAAACTG;A AGCACTAGAA CATCAAGTTG GCGAAAAAA AAGAkMCAA AATCTAAAAC pAAATCT(AA TCTATTTTTrC TAAACT-GAAA TC7GCCCTCA CAACCCTGCC TCTATTTTTT TAA.CTSAAA CAC TGTGrG OTTACCTACT CATGGXDTTT GflTTCCTTG GAAT GTTGTA GTTACCTACT CATGNXGTTc ATTATTTTGT ACTPCTATTG.T ACTATTTOGA CATGCAGTTC ATThrrrGT 1'TAARCAAAG TAACa-GTTT AA.AFCCAAGC AAACCAATGG TTAAA.CAAAG TG?.CTCTT1' GCCCATOG.. QATATOTO SACAT'IAGGT TTGAAACTTT GCCCATGG.. ATATOTG TTAACTGCAS CATGTGTCM, AA!TAA)GYAz CTGCGATTG; 3550 AkTG'rAAATTT ATACATOTAT CTGTA.TCTTT OTACA-- ATGTAAAITT ATAAATATAT 3600 GA'TAGGCTG CPLGI'TTTI GT CCTAI-rCTl GATa~rCAAGA GAGTAGCCG C&.ATTTTTG? 3650 GGTTTTATTT TACTTTGTAC GTTAAATTCT, CACTTlGG;G GGTTarATr?: TACTTST AC.

3700 GGCTT49rAFAA CALCATTGAAT TGATCCTCTA ?TTQTCTGAjTS CCCTTATAAA CT.CATTGAAT 375;0 CTGTTATCC ?TChGMAA TTTTTTITTTT TGAAGCAGCA GTGTPATAIC;C T*.CCAAA.MA 3600 3850 GTGACAAAPAT ACGOI rZGTI' 3900 AAATrTTTTT TrOOCCCTC GCCTTTST.'1 GCACACCTT 4000 CCACACCEC ALCCTCTACCC 4050 GGCTTTAGAG CACQATOAGG CTCGACT STA ACTCTCACAT TT?'rGTTa:;T TTGAA CAAAAGTTTC ACGTC=TGG? OCACATGTGC AATCSTGAGG ACrcIGrATG TTTcAc.GCCA COMS I D No: S BMI1-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawso~n Waldron 16:62:42 06-12-206 28 /64 21 humanEGRi =uuscEGR1 rattr.1 hunanSQRl 4051 AAncCCG#Ct AGCTGAGCIT TGGTTCTCCA GAATGTAAGA 4100

AAAAAATT

4150 4101 moue eECRI ratSGRl AAACAAAAA humanEGR 4151 motuneNGR1 ratEGR2 GATTTATCCA humariEGR TC'GAACTC CAAAAGTCTA TTTTTTTAAC tGAAAA'gG-TA 4200 TGTTCGGGAG TTGGATGCT GCGGTTACCT ACCiGTAG G 4201 4250 rauseEGR LaLEGRI CGSTGACTTT rGTATGCTAT QAACATGAAG TTCATTATTT TGTGGTWTTA humanER..

mouoEEGR ratEGRI TTTTACTTCG TACTTGTGT TGCTTAAACA huranEG 4300 AAGTGACTTG TrTGGCTTAI 4301 4350 moua oEGR2 zatEGRI humanEGRI AAAOACATIG AATGCGCTTT ACTGCGA'IG (MIATTvrflG TGTGTATCCT 4351 4388 mouseEGR retEGR] TCAGAAAAA? TAAMAGCNM ATAAAGPW TVkCT;GT humanEGR COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:62:64 06-12-2OC6 29164

I

Example 1 Characterisation of DNAzymes ED5 and Materials and Methods C 5 ODN synthesis. DNAzymes were synthesized commercially (Oligos SEtc., Inc.) with an inverted T at the 3' position unless otherwise indicated.

tf Substrates in cleavage reactions were synthesized with no such modification.

O Where indicated ODNs were 5'-nd labeled withy2P-dATP and T4 0 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 pl containing 10 mM MgClz, 5 mM Tris pH 7.5, 150 mM NaCl. 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 autoradiographcd overnight at -60 °C.

Culture conditions and DNAzyme transfection. Primary rat aortic SMCs wore obtained from Cell Applications. Inc.. and grown in Waymouth's medium, pH 7.4, containing 10% fetal bovine serutu (FBS), streptomycin and 50 IU/ml penicillin at 37 "C in a humidified atmosphere of COz. SMCs were used in experiments between passages 3-7. Pup rat SMCs (WKY12-22 (as described in Lemire at 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 (SPM) fur 8t h prior to DNAzyme (or antisease ODN, where indicated) transfection (0.1 pM) using Superfect in accordance with manufacturer's instructions (Qiagen). After 18 h, the cells were washed with phosphate-buffered saline [PBS), pH 7.4 prior to trensfection a second time in

FBS.

COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:53:12 06-12-2006 30164 o

O

VO 23 Northern blot analysis. Total RNA was isolated using the TRIzol Sreagent (Life Technologies) and 25 Mg was resolved by electrophoresis prior Clto transfer to IIybond-N+ membranes (NEN-DuPont). Prehybridization, C hybridization with a 32 P-dCTP-labeled Egr-l or P-Actin cDNA, and washing was performed essentially as previously described (Khachigian et al, 1995).

Cl Western blot analysis. Growth-quiescent SMCs in 100 mm plates (Nunc-InterMed) were transfected with ED5 or ED5SCR as above, and o incubated with 5% FBS for 1 h. The cells were washed in cold PBS, pH 7.4, Cl and extracted in 150 mM NaCi, 50 mM Tris-HCl, plH 7.3, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 5 mM EIDTA, 1% trasylol, MAg/ml leupeptin, 1% aprotinin and 2 mM PMSF. Twenty four Ag protein samples were loaded onto 10% denaturing SDS-polyscrylamide 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 chcmiluminescent detection (NEN-DuPont).

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

Assessment ufDNAzyme stability. DNAzymes were 5'-end labeled with y3P-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 autoradiogmaphed overnight at -80 UC.

SMC wounding assay. Confluent growth-quiescent SMCs irn chamber slides (Nunc-InterMed) were exposed to ED5 or ED5SCR for 18 h prior to a single scrape with a sterile toothpick. Cells were treated with mitomycin C (Sigma) (20 gM) for 2 h prior to injury (Pitsch et al, 1996; Horodyski COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 96793111 Blake Dawson Waldron 16:53-31 06-1 2-2OC6 31 164 o

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24 Powell, 1996). Seventy-two h after injury, the cells were washed with PBS, p pH 7,4, fixed with formaldehyde then stained with hematoxylin-eosin.

Rat arterial ligation model and analysis. Adult male Sprague Dawley C, rats weighing 300-350 g were anaesthetised using ketanine (60 rg/kg, i.p.) and xylazine (8 mg/kg, The right common carotid artery was exposed Clup to the carotid bifurcation via a midline neck incision. Size 6/0 nonabsorbable suture was tied around the common carotid proximal to the Sbifurcation, ensuring cessation of blood flow distally. A 200 il solution at Cl 4'C containing 500 Ag of DNAzyme (in DEPC-treated H2O), 30 pl of transfecting agent and Pluronic gal P127 (BASF) was applied around the vessel in each group of 5 rats, extending proximally from the ligature for 12mm. These agents did not inhibit the solidification of the gel at 37 C.

After 3 days. vehicle with or without 500 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 nmm 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 irm sections were prepared at 250 Mm intervals along the vessel from the point of ligation and stained with hcmatoxylin and eosin. The neoinLimal and medial areas of 5 consecutive sections per rat were determined digitally using a customized software package (Magellan) (Halasz Martin, 1884) 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 7 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 exonuclase digestion. The sequence in both arms of EDS was scrambled (SCR) without altering the catalytic domain to produce DNAzyme EDSSCR (Figure 1).

A synthetic RNA substrate comprised of 23 nts, matching nts 805 to 827 of NGFI-A mRNA (Figure 1) was used to determine whether ED5 had the capacity to cleave target RNA. EDS cleaved the 'P-5'-end labeled 23-mer within 10 min, The 12-ixer product corresponds to the length between the COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:63:60 06-12-2006 32164

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o O A(816)-U(8t7) junction and the 5' end of the substrate (Figure In contrast, SEDSSCR had no demonstrable effect on this synthetic substrate. Specific C EDS catalysis was further demonstrated by the inability of the human equivalent of this DNAzymne (hED5) to cleave the rat substrate over a wide range of stoichiometric ratios. Similar results were obtained using E.llSCR S(data not shown), hED5 differs from the rat ED5 sequence by 3 of 18 nts in t its hybridizing arms (Table The catalytic effect of ED5 on a 2 P-labeled 206 nt fragment of native NGFI-A mRNA prepared byin vitro transcription Cl was then determined. The cleavage reaction produced two radiolabeled species of 163 and 43 nt length consistent with DNAzyma cleavage at the A(816)-U[817) junction. In other experiments. ED5 also cleaved a 2 P-labeled NGFI-A transcript of 1960 nt length in a specific and time-dependent manner (data not shown).

COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:54:00 06-12-2006 33164 o

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o 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 c- percentage. The target sequence of ED5 in NGFI-A mRNA is 5'-807-A tn CGU CCG GGA UGG CAG CGG-825-3' (SEQ ID NO: 13) (rat NGFI-A o sequence), and that of hED5 in EGR-1 is 5'-262-U CGU CCA GGA UGG CCG NC CGG-280-3' (SEQ ID NO: 14) (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 64.2 100 MurineSpl AF022363 66.7 66.7 Human c-Fos KU0650 66.7 66.7 Murine c-Fos XU 760 61.1 66.7 Humal Sp3 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 IM]) inhibited serum-inducible steady-state NGFI-A mRNA levels by 55% (Figure 2a), whereas EDSSGR had no effect (Figure 2a). 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 EDSSCR nor EDC. a DNAzyme bearing an identical catalytic domain as ED5 and EDSSCR but flanked by nonsense arms had any influence on the induction of NGFI-A (data not COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 0396793111 Blake Dawson Waldron 16:54:14 06-12-2006 34164 0

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Is0 27 0 shown). EDS failed to affect levels of the constitutively expressed, structurally -related zinc-finger protein, Spl. It was also unable to block Sserum-induction of the immediate-early gene product, c-Fos whose induction, like NGFI-A, is dependent upon serum response elements in its promoter and phosphorylation mediated by extracellular-signal regulated C' kinase (Treisman, 1990, 1994 and 1995; Gashler Sukhatme, 1995). These t findings, taken together, demonstrate the capacity of ED5 to inhibit o production of NGFI-A mRNA and protein in a gene-specific and sequencespecific 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 then determined. Growthquiescent SMCs were incubated with DNAzyme prior to exposure to serum and the assessment of cell numbers after 3 days. ED5 (0.1 IM) inhibited SMC proliferation stimulated by serumn by 70% (Figure 3a). In contrast, ED5SCR failed to influence SMC growth (Figure 3a). ASZ, an antisense NGFI-A ODN able to inhibit SMC growth at 1 M failed to inhibit proliferation at the lower concentration (Figure 3a). Additional experiments revealed that ED5 also blocked serum-inducible aH-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 3b).

Cultured SMCs derived from the aortae of 2 week-old rats (WKYZ1-22) 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 WrKY12-22 cells and neointimal cells contraats 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 Klhachigian, 1998], consistent with a "synthetic" phenotype (Majesky at al, 1992; Campbell Campbell. 1985). ED35 attenuated serum-inducible WKY12- 22 proiiferation by approximately 75% (Figure 3c). ED5SCR had no inhibitory effect: surprisingly, it appeared to stimulate growth (Figure 3c].

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

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To ensure that differences in the biological effects of EDS and were not the consequence of dissimilar intracellular localization, both DNAzymes were 3'-end labeled with fluorescein isothiocyanate (FITC) and Iincubated with SMCs. Fluorescence microscopy revealed that both FITCc 5 ED5 and FITC-EDSSCR localized mainly within the nuclei. Punctate fluorescence in this cellular compartment was independent of DNAzyme t sequence. Fluorescence was also observed in the cytoplasm, albeit with less Sintensity. Cultures not exposed to DNAzyme showed no evidence of 0 autofluorescence.

Both molecules were 5'-end labeled with y"P-dATP and incubated in culture medium to ascertain whether cellular responsiveness to ED5 and was a consequence of differences in DNAzymo stability. Both 32

P-

and 3 2 P-EDSSCR remained intact even after 48 h. In contrast to 32 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. 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 Powel, 1006) 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 0 days (data not shown).

That ED5SCR had no effect in this system further demonstrates sequencespecific inhibition by The effect of EDS on neoiatima 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 4) nor the vehicle control (Figure 4) had any effect on neointima formation. These findings demonstrate the capacity of COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 31112 Blake Dawson Waldron 16:64:88 08-12-2006 36164 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 at al, 1998; Tharlow et al, 1996).

Further experiments revealed the capacity of hED5 tc cleave (human) EGR-iRNA. hED5 cleaved its substrate in a dose-dependent manner over a wide range of stoichiomnetric ratios. hEDb also cleaved in a time-dependent manner, whereas hEDSSCR its scrambled counterpart. had nu such catalytic property (data not shown).

The specific, growth-inhibitory properties of EDS reported herein suggest that DNAzymes may be useful as therapeutic tools in the treatment of vascular disorders involving inappropriate SMC growth.

Example 2 Cleavage of human EGR-1 RNA by panel of candidate DNAzymes To evaluate which specific DNAzymes targeting human EGR-1 (other than hED5) efficiently cleave ECR-1 RNA, we prepared in vitro transcribed 351-labeled EGR-1 RNA and incubated this substrate with candidate DNAzymes for various times. The EGR-1 plasmid template (hs164) was prepared by subcloning bps 108-332 of human EGR-1 into pGEM-T-easy. A 388 nt 35S-labeled substrate was prepared byin vitro transcription using SPa polymerase. Time-dependent cleavage of the substrate was tested using the following DNzymes: DzA: 5'-CAGGGGACAGGCGCTCTACAACGACGTI'GCGGG-X-3' (SEQ ID NO: 15) tzB: 5'-TGCAGGGGAGGCTAGCTACAACGAACCGTTGCG-X-3' (SEQ ID NO: 101 DzC: 5'-CATCCTGGAGGCTaAGCTACAACGAGAGCAGGC'r-X-3' (SEQ ID NO: 17) I lzE: 5'-TCAG AG GGCPAGCTCACAACGACICGGCCTT-X-S' (SEQ ID NO; Jn 18)n; and DzF: 5'-GCGGGGACAGGCCTACTACAACGACAGCTGCT-X-3' (SEQ ID NO: 19)35 where X deotes a -3-linked T.

where X deotes a 3'-3-linked COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:66-16 06-12-2006 37164 o

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The DNAzyme that cleaved most effectively of this group was DzA, Sthen DzB, then DzC. In contrast, DzE was inactive.

c Example 3 inhibition of induction of EGR-1 in human SMC by DzA V' To determine whether DzA could block the induction of endogenous 0 human EGR-1, we incubated growth-quiescent human aortic smooth muscle Scells with 5% fetal bovine serum and observed the production of EGR-1 protein by Western blot analysis, This band representing the EGR-1 protein was blocked by 0.5 gM DzA, delivered using FuGNEB (Roche Molecular Biochemicals] and unaffected by DzE. The blot was then stripped and reprobed with antibodies to the transcription factor Spl. Results obtained showed that neither serum nor DzA affected induction of Spl. A Coomassie Blue gel indicated that equal protein had been loaded.

The data demonstrate that DzA cleaves EGR-1 mRNA and blocks the induction of EGR-I protein.

Example 4 Inhibition of human SMC proliferation by TzA To ascertain whether proliferation of human SMCs could be inhibited by DzA, a population of SMCs was quantitated with and without exposure to DzA or DzE. SMC proliferation stimulated by 5% fetal bovine serum was significantly inhibited by 0.5 jiM DzA (Figure In contrast, neither DzE nor had any effect (Figure These data demonstrate that DzA inhibits human SMC proliferation.

Example Inhibition of porcine SMC proliferation by DzA The porcine and human EGR-1 sequences are remarkably well conserved Porcine retinal SMCs were used to determine whether DzA could block the growth of porcine SMCs. Our studies indicate that DzA uM) could inhibit the proliferation of these cells (Figure In contrast, DzE had no effect (Figure 6).

COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:66:31 06-12-2006 38164 o N Vs 31 Example 6 Delivery of DNAzvme into the orcine coronary artery wall C 5 Porcine angioplasty and stenting are accepted models of human in- C stent restenosis (Karas et al. 1992). The porcine coronary anatomy, 1t) dimensions and histological response to steanting are similar to the human o (Muller et al. 1992). The Transport Catheter has previously been used to C deliver antisense DNA targeting c-myc in humans (Serrys at al.

io 1998) and the pig (Gunn Cumberland, 1990) via the intraluminal route.

Using this catheter, FITC-labeled DNAzyme was applied to the inner wall of a porcine coronary artery, ex vivo, from a newly explanted pig heart.

DNAzymo (1000 ~l8) was delivered via the catheter in 2ml MilliQ containing 300td FuGENE6 and ImM MgCJ2. The FITC-labeled DNAzyme localised into the intimal cells of the vessel wall. These studies demonstrate that DNAzyne can be delivered to cells within the artery wall via an intraluminal catheter.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

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, In addition.

various documents are cited throughout this application. The disclosures of these documents are hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.

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COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06

Claims (16)

1. A DNAzyme which specifically cleaves EGR-1 mENA, the DNAzyme comprising C a catalytic domain which cleaves mRNA at a purine:pyrimidine t£n cleavage site; 0 (ii) a first binding domain contiguous with the 5' end of the catalytic 0, domain; and (iii) a second binding domain contiguous with the 3' end of the catalytic domain, wherein the binding domains are sufficiently complementary to the two regions immediately flanking a purine:pyrimidine cleavage site within the region of EGR-1 mRNA corresponding to nucleotides 168 to 332 as shown in SEQ ID NO:1, such that the DNAzyn cleaves the EGR-1 mRNA.

2. ADNAzyme as claimed in claim 1 wherein each binding domain is nine or more nucleotides in length.

3. A DNAzyme as claimed in claim 1 or claim 2 in which the catalytic domain has the nucleotide sequence GGCTAGCTACAAGGA (SEQ ID NO: 2).

4. A DNAzyme as claimed in any one of claims I to 3 in which the cleavage site is selected from the group consisting of the GU site corresponding to nucleotides 198-199; (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 271-272; 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. A DNAzyme as claimed in claim 4 in which the cleavage site is the AU site corresponding to nucleotides 271-272. COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 0396793111 Blake Dawson Waldron 16:7:48 06-12-2006 47164 0 0 ci IND 0. ADNAzyme as claimed in claim i which has a sequence selected from the group consisting of: 5'-caggggacaGGCTAGCTACAACGAcgttgcggg (SEQ ID NO: P;-A (ii) 5'-tgcaggggaGGCTAGCTACAACGAaccgttgcg (SEQ ID NO: D L (iii) 5'-catcctggaGGCTAGCTACAACAgagcaggct (SEQ ID NO: Cot (iv) 5'-ccgcggccaGGCTAGCTACAACGAcctggacga (SEQ ID NO: 6); C 5'-ccgctgccaGGCTAGCTACAACGAcccggacg t (SEQ ID NO: 7); (vi) 5'-gcggggacaGGCTAGCTACAACGAcagctgoat (SEQ ID NO: D 2 (vii) 5'-cagcggggaGGCTAGCTACAACGAatcagctc (SEQ ID NO: and (viii) 5'-ggtcagagaCGCTACCTACAACGActgcagcgg (SEQ ID NO:

7. A DNAzyme as claimed in claim 6 which has the sequence: (SEQ ID NO: 8).

8. A DNAzyme as claimed in any one of claims 1 to 7, wherein the '-end nucicotide residue is inverted in the binding domain contiguous with the 3' end of the catalytic domain.

9. A pharmaceutical composition comprising a DNAzyme according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. A method of inhibiting EGR-1 activity in cells which comprises exposing the cells to a DNAzyme according to any one of claims 1 to 8.

11. A method of inhibiting proliferation or migration of cells in a subject which comprises administering to the sulbject a prophylactically effective dose of the pharmaceutical composition according to claim 9.

12. A method of treating a condition associated with cell proliferation or migration in a subject which comprises administering to the subject a therapeutically effective dose of the pharmaceutical composition according to claim 9. COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06 03 9679 3111 Blake Dawson Waldron 16:88:03 06-12-2005 48164 0 0 are cells involved in neoplasia. ci 41

13. A method as claimed in any one of claims 10 to 12 wherein the cells are vascular cells. Cl 0 14. A moethod as claimed in any one of claims 10 to 12 wherein the cells care cells involved in neoplasia. O

15. A method as claimed in claim 12 wherein the condition associated Swith coll proliferation or migration is selected from the group consisting of post-angioplasty restenosis, vein graft failure, hypertension, traasplant coronary disease and complications associated with atherosclerosis or peripheral vascular disease.

16. An angioplastic stent for inhibiting the onset of restenosis, which comprises an angioplastic stent operably coated with a prophylactically effective dose of a DNAsyme according to any one of claims 1 to 8.

17. A method for inhibiting the onset of restsnosis in a subject undergoing angioplasty, which comprises topically administering a prophylactically effective dose of a pharmaceutical composition according to claim 9 to the subject at around the time of the angioplasty.

18. A method according to claim 17 in which the pharmaceutical composition is administered by catheter.

19. A method for inhibiting the onset of restenosis in a subject undergoing angioplasty, which comprises topically administering a stent according to claim 15 to the subject at around the time of the angioplasty. COMS ID No: SBMI-02132166 Received by IP Australia: Time 17:01 Date 2005-12-06