# Patent

## Title
Catalytic molecules

## Abstract

The present invention relates to DNAzymes which are targeted against mRNA molecules encoding EGR-1 (also known as Egr-1 and NGFI-A). The present invention also relates to compositions including these DNAzymes and to methods of treatment involving administration of the DNAzymes.

## Background
FIELD OF THE INVENTION
The present invention relates to DNAzymes which are targeted against mRNA molecules encoding EGR-1 (also known as Egr-1 or NGFI-A). The present invention also relates to compositions including these DNAzymes and to methods of treatment involving administration of the DNAzymes.
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 pathogenesis of these vascular disorders (Jackson & Schwartz, 1992; 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, 1988) 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).
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. Anti-sense DNA molecules also suffer from problems associated with non-specific activity and, at higher concentrations, even toxicity.
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 (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 single-stranded, 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 15 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. 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 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

    
    
        (i) 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-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.
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 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.
In a seventh aspect, the present invention provides a method for 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.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 Sequence of NGFI-ADNAzyme (ED5; SEQ ID NO:21 and SEQ ID NO:22), its scrambled control (ED5SCR; SEQ ID NO:23) and 23 nt synthetic rat substrate. The translational start site is underlined.
FIG. 2 NGFI-A DNAzyme inhibits the induction of NGFI-A mRNA and protein by serum. Northern blot analysis was performed with 25 μg of total RNA. The blot was stripped and reprobed for β-Actin. Autoradiograms were analyzed by scanning densitometry and the ordinate axis is expressed as NGFI-A band intensity as a fraction of β-Actin band intensity. The mean and standard errors of the mean are indicated in the figure. Data is representative of 2 independent experiments. * indicates P<0.05 (Student's paired t-test) as compared to control (FBS alone).
FIG. 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 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: 20). 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 t-test) as compared to control (FBS alone).
FIG. 4 NGFI-A DNAzyme inhibition of neointima formation in the rat carotid artery. Neointimal and medial areas of 5 consecutive sections per rat (5 rats per group) taken at 250 μm 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+ED5SCR groups using the Wilcoxen rank sum test for unpaired data. Lig denotes ligation, Veh denotes vehicle.
FIG. 5 Selective inhibition of human smooth muscle cell proliferation by DzA.
FIG. 6 Specific inhibition of porcine retinal smooth muscle cell proliferation by DzA.



DETAILED DESCRIPTION OF THE INVENTION
Egr-1 (also known as NGFI-A and EGR-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 the equivalent mouse (Egr-1) and rat (NGFI-A) sequences. The present inventors have now developed DNA-based enzymes that cut NGFI-A/Egr-1/EGR-1 RNA with high efficiency and specificity. The NGFI-A “DNAzyme” cleaved synthetic and in vitro transcribed NGFI-A RNA 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, Sp1, or the immediate-early gene product, c-Fos. The DNAzyme blocked serum-inducible 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-1/EGR-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

    
    
        (i) 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-1 mRNA corresponding to nucleotides 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, that strict complementarity may not be required for the DNAzyme to bind to and cleave the EGR-1 mRNA.
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. Pat. No. 5,807,718, the entire contents of which are incorporated herein by reference. In a preferred embodiment, the 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 greater the binding domain length, the more tightly it will bind to its complementary mRNA 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:
(i) 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;
(v) 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:

    (i) 5′-caggggacaGGCTAGCTACAACGAcgttgcggg (SEQ ID NO: 3) targets GU (nt 198, 199); arms hybridise to bp 189-207
    (ii) 5′-tgcaggggaGGCTAGCTACAACGAaccgttgcg (SEQ ID NO: 4) targets GU (nt 200, 201); arms hybridise to bp 191-209
    (iii) 5′-catcctggaGGCTAGCTACAACGAgagcaggct (SEQ ID NO: 5) targets GU (nt 264, 265); arms hybridise to bp 255-273
    (iv) 5′-ccgcggccaGGCTAGCTACAACGAcctggacga (SEQ ID NO: 6) targets AU (nt 271, 272); arms hybridise to bp 262-280
    (v) 5′-ccgctgccaGGCTAGCTACAACGAcccggacgt (SEQ ID NO: 7) targets AU (nt 271, 272); arms hybridise to bp 262-280
    (vi) 5′-gcggggacaGGCTAGCTACAACGAcagctgcat (SEQ ID NO: 8) targets AU (nt 301, 302); arms hybridise to bp 292-310
    (vii) 5′-cagcggggaGGCTAGCTACAACGAatcagctgc (SEQ ID NO: 9) targets GU (nt 303, 304); arms hybridise to bp 294-312
    (viii) 5′-ggtcagagaGGCTAGCTACAACGActgcagcgg (SEQ ID NO: 10) targets AU (nt 316, 317); arms hybridise to bp 307-325.


In a particularly preferred embodiment, the DNAzyme targets the AU site corresponding to nucleotides 271-272 (ie. the translation start codon).
In a further preferred embodiment, the DNAzyme has the sequence: 5′-ccgcggccaGGCTAGCTACAACGAcctggacga (SEQ ID NO: 6).
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 3′-3′ inversion at one or more termini of the DNAzyme. More specifically, a 3′-3′ 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 binding 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.
As will be appreciated by those skilled in the art, given that DNAzymes of the present invention cleave human EGR-1, similar DNAzymes can be produced to cleave the corresponding mRNA in other species, eg. rat (NGFI-A), mouse (Egr-1) etc. In a further aspect, the present invention provides such DNAzymes.
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.
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 proliferation (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).
Within the parameters of the fourth and fifth aspects of the present invention, any suitable mode of administration may be used to administer or deliver the DNAzyme.
In particular, delivery of the nucleic acid agents described may be achieved by one or more of the following methods:

    
    
        (a) Liposomes and liposome-protein conjugates and mixtures.
        (b) Using catheters to deliver intra-luminal formulations of the nucleic acid as a solution or in a complex with a liposome.
        (c) Catheter delivery to adventitial tissue as a solution or in a complex with a liposome.
        (d) Within a polymer formulation such as polyethylenimine (PEI) or pluronic gels or within ethylene vinyl acetate copolymer (EVAc). The polymer is preferably delivered intra-luminally.
        (e) 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.
        (f) Within a viral-liposome complex, such as Sendai virus.
        (g) The nucleic acid may be delivered by a double angioplasty balloon device fixed to catheter.
        (h) 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 excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In the preferred embodiment, the pharmaceutically acceptable carrier is a liposome or a biodegradable polymer. Examples of agents which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI, NII,NIII-tetrapalmitylspermine and dioleoyl phosphatidyl-ethanolamine (DOPE) (GIBCO BRL); (2) Cytofection GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-trimethyl-aimnoniummethylsulfate) (Boehringer Mannheim); (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL); (5) FuGENE6 (Roche Molecular Biochemicals); (6) Superfect (Qiagen); and (7) 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 (1995) 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 1 mg and about 100 mg of the instant DNAzyme. In a further embodiment, the prophylactically effective dose contains between about 10 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 tubular, expanding lattice-type structure appropriate for their function, and can optionally be biodegradable.
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 timely release of the pharmaceutical composition into the surrounding tissue to be treated once the coated stent is administered. Such coating methods, for example, can use the polymer polypyrrole.
In a seventh aspect, the present invention provides a method for 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.







TABLE 1







Symbol comparison table: GenRunData:pileupdna.cmp CompCheck: 6876


      GapWeight: 5.000


GapLengthWeight: 0.300


EGR1align.msf MSF: 4388 Type: N Apr. 7, 1998 12:07 Check: 5107


Name: mouseEGR1 Len: 4388 Check: 8340 Weight: 1.0 (SEQ ID NO:11)


Name: ratEGR1  Len: 4388 Check: 8587 Weight: 1.0 (SEQ ID NO:12)


Name: humanEGR1 Len: 4388 Check: 8180 Weight: 1.00 (SEQ ID NO:1)


NB. THIS IS RAT NGFI-A numbering









   1                                                50


mouseEgr1
.......... .......... .......... .......... ..........


  ratNGFIA
CCGCGGAGCC TCAGCTCTAC GCGCCTGGCG CCCTCCCTAC GCGGGCGTCC


humanEGR1
.......... .......... .......... .......... ..........


 



  51                                               100


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
CCGACTCCCG CGCGCGTTCA GGCTCCGGGT TGGGAACCAA GGAGGGGGAG


humanEGR1
.......... .......... .......... .......... ..........


 



 101                                               150


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
GGTGGGTGCG CCGACCCGGA AACACCATAT AAGGAGCAGG AAGGATCCCC


humanEGR1
.......... .......... .......... .......... ..........


 



 151                                               200


mouseEGR1
.......... .......... .......... .......... ..........


ratEGR1
CGCCGGAACA GACCTTATTT GGGCAGCGCC TTATATGGAG TGGCCCAATA


humanEGR1
.......... .......... .......... .......... ..........


 



 201                                               250


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
TGGCCCTGCC GCTTCCGGCT CTGGGAGGAG GGGCGAACGG GGGTTGGGGC


humanEGR1
.......... .......... .......... .......... ..........


 



 251                                               300


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
GGGGGCAAGC TGGGAACTCC AGGAGCCTAG CCCGGGAGGC CACTGCCGCT


humanEGR1
.......... .......... .......... .......... ..........


 



 301                                               350


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
GTTCCAATAC TAGGCTTTCC AGGAGCCTGA GCGCTCAGGG TGCCGGAGCC


humanEGR1
.......... .......... .......... .......... ..........


 



 351                                               400


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
GGTCGCAGGG TGGAAGCGCC CACCGCTCTT GGATGGGAGG TCTTCACGTC


humanEGR1
.......... .......... .......... .......... ..........


 



 401                                               450


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
ACTCCGGGTC CTCCCGGTCG GTCCTTCCAT ATTAGGGCTT CCTGCTTCCC


humanEGR1
.......... .......... .......... .......... ..........


 



 451                                               500


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
ATATATGGCC ATGTACGTCA CGGCGGAGGC GGGCCCGTGC TGTTTCAGAC


humanEGR1
.......... .......... .......... .......... ..........


 



 501                                               550


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
CCTTGAAATA GAGGCCGATT CGGGGAGTCG CGAGAGATCC CAGCGCGCAG


humanEGR1
.......... .......... .......... .......... ....CCGCAG


 



 551                                               600


mouseEGR1
.....GGGGA GCCGCCGCCG CGATTCGCCG CCGCCGCCAG CTTCCGCCGC


  ratEGR1
AACTTGGGGA GCCGCCGCCG CGATTCGCCG CCGCCGCCAG CTTCCGCCGC


humanEGR1
AACTTGGGGA GCCGCCGCCG CCATCCGCCG CCGCAGCCAG CTTCCGCCGC


 



 601                                               650


mouseEGR1
CGCAAGATCG GCCCCTGCCC CAGCCTCCGC GGCAGCCCTG CGTCCACCAC


  ratEGR1
CGCAAGATCG GCCCCTGCCC CAGCCTCCGC GGCAGCCCTG CGTCCACCAC


humanEGR1
CGCAGGACCG GCCCCTGCCC CAGCCTCCGC AGCCGCGGCG CGTCCACGCC


 



 651                                               700


mouseEGR1
GGGCCGCGGC TACCGCCAGC CTGGGGGCCC ACCTACACTC CCCGCAGTGT


  ratEGR1
GGGCCGCGGC CACCGCCAGC CTGGGGGCCC ACCTACACTC CCCGCAGTGT


humanEGR1
CGCCCGCGCC CAGGGCGAGT CGGGGTCGCC GCCTGCACGC TTCTCAGTGT


 



 701                                               750


mouseEGR1
GCCCCTGCAC CCCGCATGTA ACCCGGCCAA CCCCCGGCGA GTGTGCCCTC


  ratEGR1
GCCCCTGCAC CCCGCATGTA ACCCGGCCAA CATCCGGCGA GTGTGCCCTC


humanEGR1
TCCCC.GCGC CCCGCATGTA ACCCGGCCAG GCCCCCGCAA CGGTGTCCCC


 



 751                                               800


mouseEGR1
AGTAGCTTCG GCCCCGGGCT GCGCCCACC. .ACCCAACAT CAGTTCTCCA


  ratEGR1
AGTAGCTTCG GCCCCGGGCT GCGCCCACC. .ACCCAACAT CAGCTCTCCA


humanEGR1
TGCAGCTCCA GCCCCGGGCT GCACCCCCCC GCCCCGACAC CAGCTCTCCA


 



 801                                               850


mouseEGR1
GCTCGCTGGT CCGGGATGGC AGCGGCCAAG GCCGAGATGC AATTGATGTC


  ratEGR1
GCTCGCACGT CCGGGATGGC AGCGGCCAAG GCCGAGATGC AATTGATGTC


humanEGR1
GCCTGCTCGT CCAGGATGGC CGCGGCCAAG GCCGAGATGC AGCTGATGTC


 



ED5  (rat) arms hybridise to bp 807-825 in rat sequ



hED5(hum) arms hybridise to bp 262-280 in hum sequ


 



 851                                               900


mouseEGR1
TCCGCTGCAG ATCTCTGACC CGTTCGGCTC CTTTCCTCAC TCACCCACCA


  ratEGR1
TCCGCTGCAG ATCTCTGACC CGTTCGGCTC CTTTCCTCAC TCACCCACCA


humanEGR1
CCCGCTGCAG ATCTCTGACC CGTTCGGATC CTTTCCTCAC TCGCCCACCA


 



 901                                               950


mouseEGR1
TGGACAACTA CCCCAAACTG GAGGAGATGA TGCTGCTGAG CAACGGGGCT


  ratEGR1
TGGACAACTA CCCCAAACTG GAGGAGATGA TGCTGCTGAG CAACGGGGCT


humanEGR1
TGGACAACTA CCCTAAGCTG GAGGAGATGA TGCTGCTGAG CAACGGGGCT


 



 951                                              1000


mouseEGR1
CCCCAGTTCC TCGGTGCTGC CGGAACCCCA GAGGGCAGCG GCGGTAAT..


  ratEGR1
CCCCAGTTCC TCGGTGCTGC CGGAACCCCA GAGGGCAGCG GCGGCAATAA


humanEGR1
CCCCAGTTCC TCGGCGCCGC CGGGGCCCCA GAGGGCAGCG GCAGCAACAG


 



1001                                              1050


mouseEGR1
.......AGC AGCAGCAGCA CCAGCAGCGG GGGCGGTGGT GGGGGCGGCA


  ratEGR1
CAGCAGCAGC AGCAGCAGCA GCAGCAGCGG GGGCGGTGGT GGGGGCGGCA


humanEGR1
CAGCAGCAGC AGCAGCGGGG GCGGTGGAGG CGGCGGGGGC GGCAGCAACA


 



1051                                              1100


mouseEGR1
GCAACAGCGG CAGCAGCGCC TTCAATCCTC AAGGGGAGCC GAGCGAACAA


  ratEGR1
GCAACAGCGG CAGCAGCGCT TTCAATCCTC AAGGGGAGCC GAGCGAACAA


humanEGR1
GCAGCAGCAG CAGCAGCACC TTCAACCCTC AGGCGGACAC GGGCGAGCAG


 



1101                                              1150


mouseEGR1
CCCTATGAGC ACCTGACCAC AG...AGTCC TTTTCTGACA TCGCTCTGAA


  ratEGR1
CCCTACGAGC ACCTGACCAC AGGTAAGCGG TGGTCTGCGC CGAGGCTGAA


humanEGR1
CCCTACGAGC ACCTGACCGC AG...AGTCT TTTCCTGACA TCTCTCTGAA


 



1151                                              1200


mouseEGR1
TAATGAGAAG GCGATGGTGG AGACGAGTTA TCCCAGCCAA ACGACTCGGT


  ratEGR1
TCCCCCTTCG TGACTACCCT AACGTCCAGT CCTTTGCAGC ACGGACCTGC


humanEGR1
CAACGAGAAG GTGCTGGTGG AGACCAGTTA CCCCAGCCAA ACCACTCGAC


 



1201                                              1250


mouseEGR1
TGCCTCCCAT CACCTATACT GGCCGCTTCT CCCTGGAGCC CGCACCCAAC


  ratEGR1
ATCTAGATCT TAGGGACGGG ATTGGGATTT CCCTCTATTC ..CACACAGC


humanEGR1
TGCCCCCCAT CACCTATACT GGCCGCTTTT CCCTGGAGCC TGCACCCAAC


 



1251                                              1300


mouseEGR1
AGTGGCAACA CTTTGTGGCC TGAACCCCTT TTCAGCCTAG TCAGTGGCCT


  ratEGR1
TCCAGGGACT TGTGTTAGAG GGATGTCTGG GGACCCCCCA ACCCTCCATC


humanEGR1
AGTGGCAACA CCTTGTGGCC CGAGCCCCTC TTCAGCTTGG TCAGTGGCCT


 



1301                                              1350


mouseEGR1
CGTGAGCATG ACCAATCCTC CGACCTCTTC ATCCTCGGCG CCTTCTCCAG


  ratEGR1
CTTGCGGGTG CGCGGAGGGC AGACCGTTTG TTTTGGATGG AGAACTCAAG


humanEGR1
AGTGAGCATG ACCAACCCAC CGGCCTCCTC GTCCTCAGCA CCATCTCCAG


 



1351                                              1400


mouseEGR1
CTGCTTCATC GTCTTCCTCT GCCTCCCAGA GCCCGCCCCT GAGCTGTGCC


  ratEGR1
TTGCGTGGGT GGCT...... .....GGAGT GGGGGAGGGT TTGTTTTGAT


humanEGR1
CGGCCTCCTC CGC...CTCC GCCTCCCAGA GCCCACCCCT GAGCTGCGCA


 



1401                                              1450


mouseEGR1
GTGCCGTCCA ACGACAGCAG TCCCATCTAC TCGGCTGCGC CCACCTTTCC


  ratEGR1
GAGCAGGGTT GC....CCCC TCCCCCGCGC GCGTTGTCGC GAGCCTTGTT


humanEGR1
GTGCCATCCA ACGACAGCAG TCCCATTTAC TCAGCGGCAC CCACCTTCCC


 



1451                                              1500


mouseEGR1
TACTCCCAAC ACTGACATTT TTCCTGAGCC CCAAAGCCAG GCCTTTCCTG


  ratEGR1
TGCAGCTTGT TCCCAAGGAA GGGCTGAAAT CTGTCACCAG GGATGTCCCG


humanEGR1
CACGCCGAAC ACTGACATTT TCCCTGAGCC ACAAAGCCAG GCCTTCCCGG


 



1501                                              1550


mouseEGR1
GCTCGGCAGG CACAGCCTTG CAGTACCCGC CTCCTGCCTA CCCTGCCACC


  ratEGR1
CCGCCCAGGG TAGGGGCGCG CATTAGCTGT GGCC.ACTAG GGTGCTGGCG


humanEGR1
GCTCGGCAGG GACAGCGCTC CAGTACCCGC CTCCTGCCTA CCCTGCCGCC


 



1551                                              1600


mouseEGR1
AAAGGTGGTT TCCAGGTTCC CATGATCCCT GACTATCTGT TTCCACAACA


  ratEGR1
GGATTCCCTC ACCCCGGACG CCTGCTGCGG AGCGCTCTCA GAGCTGCAGT


humanEGR1
AAGGGTGGCT TCCAGGTTCC CATGATCCCC GACTACCTGT TTCCACAGCA


 



1601                                              1650


mouseEGR1
ACAGGGAGAC CTGAGCCTGG GCACCCCAGA CCAGAAGCCC TTCCAGGGTC


  ratEGR1
AGAGGGGGAT TCTCTGTTTG CGTCAGCTGT CGAAATGGCT CT......GC


humanEGR1
GCAGGGGGAT CTGGGCCTGG GCACCCCAGA CCAGAAGCCC TTCCAGGGCC


 



1651                                              1700


mouseEGR1
TGGAGAACCG TACCCAGCAG CCTTCGCTCA CTCCACTATC CACTATTAAA


  ratEGR1
CACTGGAGCA GGTCCAGGAA CATTGCAATC TGCTGCTATC AATTATTAAC


humanEGR1
TGGAGAGCCG CACCCAGCAG CCTTCGCTAA CCCCTCTGTC TACTATTAAG


 



1701                                              1750


mouseEGR1
GCCTTCGCCA CTCAGTCGGG CTCCCAGGAC TTAAAG.... ...GCTCTTA


  ratEGR1
CACATCGAGA GTCAGTGGTA GCCGGGCGAC CTCTTGCCTG GCCGCTTCGG


humanEGR1
GCCTTTGCCA CTCAGTCGGG CTCCCAGGAC CTGAAG.... ...GCCCTCA


 



1751                                              1800


mouseEGR1
ATACCACCTA CCAATCCCAG CTCATCA..A ACCCAGCCGC ATGCGCAAGT


  ratEGR1
CTCTCATCGT CCAGTGATTG CTCTCCAGTA ACCAGGCCTC TCTGTTCTCT


humanEGR1
ATACCAGCTA CCAGTCCCAG CTCATCA..A ACCCAGCCGC ATGCGCAAGT


 



1801                                              1850


mouseEGR1
ACCCCAACCG GCCCAGCAAG ACACCCCCCC ATGAACGCCC ATATGCTTGC


  ratEGR1
TTCCTGCCAG AGTCCTTTTC TGACATCGCT CTGAATAACG AGAAG..GCG


humanEGR1
ATCCCAACCG GCCCAGCAAG ACGCCCCCCC ACGAACGCCC TTACGCTTGC


 



1851                                              1900


mouseEGR1
CCTGTCGAGT CCTGCGATCG CCGCTTTTCT CGCTCGGATG AGCTTACCCG


  ratEGR1
CTGGTGGAGA CAAGTTATCC CAGCCAAACT ACCCGGTTGC CTCCCATCAC


humanEGR1
CCAGTGGAGT CCTGTGATCG CCGCTTCTCC CGCTCCGACG AGCTCACCCG


 



1901                                              1950


mouseEGR1
CCATATCCGC ATCCACACAG GCCAGAAGCC CTTCCAGTGT CGAATCTGCA


  ratEGR1
CTATACTGGC CGCTTCTCCC TGGAGCCTGC ACCCAACAGT GGCAACACTT


humanEGR1
CCACATCCGC ATCCACACAG GCCAGAAGCC CTTCCAGTGC CGCATCTGCA


 



1951                                              2000


mouseEGR1
TGCGTAACTT CAGTCGTAGT GACCACCTTA CCACCCACAT CCGCACCCAC


  ratEGR1
TGTGGCCTGA ACCCCTTTTC AGCCTAGTCA GTGGCCTTGT GAGCATGACC


humanEGR1
TGCGCAACTT CAGCCGCAGC GACCACCTCA CCACCCACAT CCGCACCCAC


 



2001                                              2050


mouseEGR1
ACAGGCGAGA AGCCTTTTGC CTGTGACATT TGTGGGAGGA AGTTTGCCAG


  ratEGR1
AACCCTCCAA CCTCTTCATC CTCAGCGCCT TCTCCAGCTG CTTCATCGTC


humanEGR1
ACAGGCGAAA AGCCCTTCGC CTGCGACATC TGTGGAAGAA AGTTTGCCAG


 



2051                                              2100


mouseEGR1
GAGTGATGAA CGCAAGAGGC ATACCAAAAT CCATTTAAGA CAGAAGGACA


  ratEGR1
TTCCTCTGCC TCCCAGAGCC CACCCCTGAG CTGTGCCGTG CCGTCGAACG


humanEGR1
GAGCGATGAA CGCAAGAGGC ATACCAAGAT CCACTTGCGG CAGAAGGACA


 



2101                                              2150


mouseEGR1
AGAAAGCAGA CAAAAGTGTG GTGGCCTCCC CGGCTGC... .CTCTTCACT


  ratEGR1
ACAGCAGTCC CATTTACTCA GCTGCACCCA CCTTTCCTAC TCCCAACACT


humanEGR1
AGAAAGCAGA CAAAAGTGTT GTGGCCTCTT CGGCCACCTC CTCTCTCTCT


 



2151                                              2200


mouseEGR1
.......... .......... CTCTTCTTAC CCATCCCCAG TGGCTACCTC


  ratEGR1
.......... .......... GACATTTTTC CTGAGCCCCA AAGCCAGGCC


humanEGR1
TCCTACCCGT CCCCGGTTGC TACCTCTTAC CCGTCCCCGG TTACTACCTC


 



2201                                              2250


mouseEGR1
CTACCCATCC CCTGCCACCA CCTCATTCCC ATCCCCTGTG CCCACTTCCT


  ratEGR1
TTTCCTGGCT CTGCAGGCAC AGCCTTGCAG TACCCGCCTC CTGCCTACCC


humanEGR1
TTATCCATCC CCGGCCACCA CCTCATACCC ATCCCCTGTG CCCACCTCCT


 



2251                                              2300


mouseEGR1
ACTCCTCTCC TGGCTCCTCC ACCTACCCAT CTCCTGCGCA CAGTGGCTTC


  ratEGR1
TGCCACCAAG GGTGGTTTCC AGGTTCCCAT GATCCCTGAC TATCTGTTTC


humanEGR1
TCTCCTCTCC CGGCTCCTCG ACCTACCCAT CCCCTGTGCA CAGTGGCTTC


 



2301                                              2350


mouseEGR1
CCGTCGCCGT CAGTGGCCAC CACCTTTGCC TCCGTTCC.. ..........


  ratEGR1
CACAACAACA GGGAGACCTG AGCCTGGGCA CCCCAGACCA GAAGCCCTTC


humanEGR1
CCCTCCCCGT CGGTGGCCAC CACGTACTCC TCTGTTCCC. ..........


 



2351                                              2400


mouseEGR1
....ACCTGC TTTCCCCACC CAGGTCAGCA GCTTCCCGTC TGCGGGCGTC


  ratEGR1
CAGGGTCTGG AGAACCGTAC CCAGCAGCCT TCGCTCACTC CACTATCCAC


humanEGR1
.....CCTGC TTTCCCGGCC CAGGTCAGCA GCTTCCCTTC CTCAGCTGTC


 



2401                                              2450


mouseEGR1
AGCAGCTCCT TCAGCACCTC AACTGGTCTT TCAGACATGA CAGCGACCTT


  ratEGR1
TATCAAAGCC TTCGCCACTC AGTCGGGCTC CCAGGACTTA AAGGCTCTTA


humanEGR1
ACCAACTCCT TCAGCGCCTC CACAGGGCTT TCGGACATGA CAGCAACCTT


 



2451                                              2500


mouseEGR1
TTCTCCCAGG ACAATTGAAA TTTGCTAAAG GGA....... .ATAAAAG..


  ratEGR1
ATAACACCTA CCAGTCCCAA CTCATCAAAC CCAGCCGCAT GCGCAAGT..


humanEGR1
TTCTCCCAGG ACAATTGAAA TTTGCTAAAG GGAAAGGGGA AAGAAAGGGA


 



2501                                              2550


mouseEGR1
.AAAGCAAAG GGAGAGGCAG GAAAGACATA AAAGCA...C AGGAGGGAAG


  ratEGR1
.ACCCCAACC GGCCCAGCAA GACACCCCCC CATGAACGCC CGTATGCTTG


humanEGR1
AAAGGGAGAA AAAGAAACAC AAGAGACTTA AAGGACAGGA GGAGGAGATG


 



2551                                              2600


mouseEGR1
AGATGGCCGC AAGAGGGGCC ACCTCTTAGG TCAGATGGAA GATCTCAGAG


  ratEGR1
CCCTGTTGAG TCCTGCGATC GCCGCTTTTC TCGCTCGGAT GAGCTTACAC


humanEGR1
GCCATAGGAG AGGAGGGTT. .CCTCTTAGG TCAGATGGAG GTTCTCAGAG


 



2601                                              2650


mouseEGR1
CCAAGTCCTT CTACTCACGA GTA..GAAGG ACCGTTGGCC AACAGCCCTT


  ratEGR1
GCCACATCCG CATCCATACA GGC..CAGAA GCCCTTCCAG TGTCGAATCT


humanEGR1
CCAAGTCCTC CCTCTCTACT GGAGTGGAAG GTCTATTGGC CAACAATCCT


 



2651                                              2700


mouseEGR1
TCACTTACCA TCCCTGCCTC CCCCGTCCTG TTCCCTTTGA CTTCAGCTGC


  ratEGR1
GCATGCGTAA TTTCAGTCGT AGTGACCACC TTACCACCCA CATCCGCACC


humanEGR1
TTCTGCCCAC TTCCCCTTCC CCAATTACTA TTCCCTTTGA CTTCAGCTGC


 



2701                                              2750


mouseEGR1
CTGAAACAGC CATGTCCAAG TTCTTCACCT CTATCCAAAG GACTTGATTT


  ratEGR1
C..ACACAGG CGAGAAGCCT TTTGCCTGTG ACATTTGTGG GAGAAAGTTT


humanEGR1
CTGAAACAGC CATGTCCAAG TTCTTCACCT CTATCCAAAG AACTTGATTT


 



2751                                              2800


mouseEGR1
GCATGG.... ..TATTGGAT AAATCATTTC AGTATCCTCT ..........


  ratEGR1
GCCAGGAGTG ATGAACGCAA GAGGCATACC AAAATCCACT TAAGACAGAA


humanEGR1
GCATGGA... ..TTTTGGAT AAATCATTTC AGTATCATCT ..........


 



2801                                              2850


mouseEGR1
.....CCATC ACATGCCTGG CCCTTGCTCC CTTCAGCGCT AGACCATCAA


  ratEGR1
GGACAAGAAA GCAGACAAAA GTGTCGTGGC CTCCTCAGCT GCCTCTTCCC


humanEGR1
.....CCATCA TATGCCTGAC CCCTTGCTCC CTTCAATGCT AGAAAATCGA


 



2851                                              2900


mouseEGR1
GTTGGCATAA AGAAAAAAAA ATGGGTTTGG GCCCTCAGAA CCCTGCCCTG


  ratEGR1
TCTCTTCCTA CCCATCCCCA GTGGCTACCT CCTACCCATC CCCCGCCACC


humanEGR1
GTTGGC.... .....AAAAT GGGGTTTGGG CCCCTCAGAG CCCTGCCCTG


 



2901                                              2950


mouseEGR1
CATCTTTGTA CAGCATCTGT GCCATGGATT TTGTTTTCCT TGGGGTATTC


  ratEGR1
ACCTCATTTC CATCCCCAGT GCCCACCTCT TACTCCTCTC CGGGCTCCTC


humanEGR1
CACCCTTGTA CAGTGTCTGT GCCATGGATT TCGTTTTTCT TGGGGTACTC


 



2951                                              3000


mouseEGR1
TTGATGTGAA GATAATTTGC ATACT..... .CTATTGTAT TATTTGGAGT


  ratEGR1
TACCTACCCG TCTCCTGCAC ACAGTGGCTT CCCATCGCCC TCGGTGGCCA


humanEGR1
TTGATGTGAA GATAATTTGC ATATT..... .CTATTGTAT TATTTGGAGT


 



3001                                              3050


mouseEGR1
TAAATCCTCA CTTTGGGG.. GAGGGGGGAG CAAAGCCAAG CAAACCAATG


  ratEGR1
CCACCTATGC CTCCGTCC.. CACCTGCTTT CCCTGCCCAG GTCAGCACCT


humanEGR1
TAGGTCCTCA CTTGGGGGAA AAAAAAAAAA AAAAGCCAAG CAAACCAATG


 



3051                                              3100


mouseEGR1
ATGATCCTCT ATTTTGTGAT GACTCTGCTG TGACATTA.. ..........


  ratEGR1
TCCAGTCTGC AGGGGTCAGC AACTCCTTCA GCACCTCAAC GGGTCTTTCA


humanEGR1
GTGATCCTCT ATTTTGTGAT GATGCTGTGA CAATA..... ..........


 



3101                                              3150


mouseEGR1
.GGTTTGAAG CATTTTTTTT TTCAAGCAGC AGTCCTAGGT ATTAACTGGA


  ratEGR1
GACATGACAG CAACCTTTTC TCCTAGGACA ATTGAAATTT GCTAAAGGGA


humanEGR1
...AGTTTGA ACCTTTTTTT TTGAAACAGC AGTCCCAG.. ..TATTCTCA


 



3151                                              3200


mouseEGR1
..GCATGTGT CAGAGTGTTG TTCCGTTAAT TTTGTAAATA CTGGCTCGAC


  ratEGR1
ATGAAAGAGA GCAAAGGGAG GGGAGCGCGA GAGACAATAA AGGACAGGAG


humanEGR1
GAGCATGTGT GAGAGTGTTG TTCCGTTAAC CTTTTTGTAA ATACTGCTTG


 



3201                                              3250


mouseEGR1
.TGTAACTCT CACATGTGAC AAAGTATGGT TTGTTTGGTT GGGTTTTGTT


  ratEGR1
.GGAAGAAAT GGCCCGCAAG AGGGGCTGCC TCTTAGGTCA GATGGAAGAT


humanEGR1
ACCGTACTCT CACATGTGGC AAAATATGGT TTGGTTTTTC TTTTTTTTTT


 



3251                                              3300


mouseEGR1
TTTGAGAATT TTTTTGCCCG TCCCTTTGGT TTCAAAAGTT TCACGTCTTG


  ratEGR1
CTCAGAGCCA AGTCCTTCTA GTCAGTAGAA GGCCCGTTGG CCACCAGCCC


humanEGR1
TTGAAAGTGT TTTTTCTTCG TCCTTTTGGT TTAAAAAGTT TCACGTCTTG


 



3301                                              3350


mouseEGR1
GTGCCTTTTG TGTGACACGC CTT.CCGATG GCTTGACATG CGCA......


  ratEGR1
TTTCACTTAG CGTCCCTGCC CTC.CCCAGT CCCGGTCCTT TTGACTTCAG


humanEGR1
GTGCCTTTTG TGTGATGCCC CTTGCTGATG GCTTGACATG TGCAAT....


 



3351                                              3400


mouseEGR1
...GATGTGA GGGACACGCT CACCTTAGCC TTAA...GGG GGTAGGAGTG


  ratEGR1
CTGCCTGAAA CAGCCACGTC CAAGTTCTTC ACCT...CTA TCCAAAGGAC


humanEGR1
.....TGTGA GGGACATGCT CACCTCTAGC CTTAAGGGGG GCAGGGAGTG


 



3401                                              3450


mouseEGR1
ATGTGTTGGG GGAGGCTTGA GAGCAAAAAC GAGGAAGAGG GCTGAGCTGA


  ratEGR1
TTGATTTGCA TGGTATTGGA TAAACCATTT CAGCATCATC TCCACCACAT


humanEGR1
ATGATTTGGG GGAGGCTTTG GGAGCAAAAT AAGGAAGAGG GCTGAGCTGA


 



3451                                              3500


mouseEGR1
GCTTTCGGTC TCCAGAATGT AAGAAGAAAA AATTTAAACA AAAATCTGAA


  ratEGR1
GCCTGGCCCT TGCTCCCTTC AGCACTAGAA CATCAAGTTG GCTGAAAAAA


humanEGR1
GCTTCGGTTC TCCAGAATGT AAGAAAACAA AATCTAAAAC AAAATCTGAA


 



3501                                              3550


mouseEGR1
CTCTCAAAAG TCTATTTTTC TAAACTGAAA ATGTAAATTT ATACATCTAT


  ratEGR1
AAAATGGGTC TGGGCCCTCA GAACCCTGCC CTGTATCTTT GTACA.....


humanEGR1
CTCTCAAAAG TCTATTTTTT TAA.CTGAAA ATGTAAATTT ATAAATATAT


 



3551                                              3600


mouseEGR1
TCAGGAGTTG GAGTGTTGTG GTTACCTACT GAGTAGGCTG CAGTTTTTGT


  ratEGR1
GCATCTGTGC CATGGATTTT GTTTTCCTTG GGGTATTCTT GATGTGAAGA


humanEGR1
TCAGGAGTTG GAATGTTGTA GTTACCTACT GAGTAGGCGG CGATTTTTGT


 



3601                                              3650


mouseEGR1
ATGTTATGAA CATGAAGTTC ATTATTTTGT GGTTTTATTT TACTTTGTAC


  ratEGR1
TAATTTGCAT ACTCTATTGT ACTATTTGGA GTTAAATTCT CACTTTGGGG


humanEGR1
ATGTTATGAA CATGCAGTTC ATTATTTTGT GGTTCTATTT TACTTTGTAC


 



3651                                              3700


mouseEGR1
TTGTGTTTGC TTAAACAAAG TAACCTGTTT GGCTTATAAA CACATTGAAT


  ratEGR1
GAGGGGGAGC AAAGCCAAGC AAACCAATGG TGATCCTCTA TTTTGTGATG


humanEGR1
TTGTGTTTGC TTAAACAAAG TGA.CTGTTT GGCTTATAAA CACATTGAAT


 



3701                                              3750


mouseEGR1
GCGCTCTATT GCCCATGG.. ..GATATGTG GTGTGTATCC TTCAGAAAAA


  ratEGR1
ATCCTGCTGT GACATTAGGT TTGAAACTTT TTTTTTTTTT TGAAGCAGCA


humanEGR1
GCGCTTTATT GCCCATGG.. ..GATATGTG GTGTATATCC TTCCAAAAAA


 



3751                                              3800


mouseEGR1
TTAAAAGGAA AAAT...... .......... .......... ..........


  ratEGR1
GTCCTAGGTA TTAACTGGAG CATGTGTCAG AGTGTTGTTC CGTTAATTTT


humanEGR1
TTAAAACGAA AATAAAGTAG CTGCGATTGG G......... ..........


 



3801                                              3850


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
GTAAATACTG CTCGACTGTA ACTCTCACAT GTGACAAAAT ACGGTTTGTT


humanEGR1
.......... .......... .......... .......... ..........


 



3851                                              3900


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
TGGTTGGGTT TTTTGTTGTT TTTGAAAAAA AAATTTTTTT TTTGCCCGTC


humanEGR1
.......... .......... .......... .......... ..........


 



3901                                              3950


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
CCTTTGGTTT CAAAAGTTTC ACGTCTTGGT GCCTTTGTGT GACACACCTT


humanEGR1
.......... .......... .......... .......... ..........


 



3951                                              4000


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
GCCGATGGCT GGACATGTGC AATCGTGAGG GGACACGCTC ACCTCTAGCC


humanEGR1
.......... .......... .......... .......... ..........


 



4001                                              4050


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
TTAAGGGGGT AGGAGTGATG TTTCAGGGGA GGCTTTAGAG CACGATGAGG


humanEGR1
.......... .......... .......... .......... ..........


 



4051                                              4100


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
AAGAGGGCTG AGCTGAGCTT TGGTTCTCCA GAATGTAAGA AGAAAAATTT


humanEGR1
.......... .......... .......... .......... ..........


 



4101                                              4150


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
AAAACAAAAA TCTGAACTCT CAAAAGTCTA TTTTTTTAAC TGAAAATGTA


humanEGR1
.......... .......... .......... .......... ..........


 



4151                                              4200


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
GATTTATCCA TGTTCGGGAG TTGGAATGCT GCGGTTACCT ACTGAGTAGG


humanEGR1
.......... .......... .......... .......... ..........


 



4201                                              4250


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
CGGTGACTTT TGTATGCTAT GAACATGAAG TTCATTATTT TGTGGTTTTA


humanEGR1
.......... .......... .......... .......... ..........


 



4251                                              4300


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
TTTTACTTCG TACTTGTGTT TGCTTAAACA AAGTGACTTG TTTGGCTTAT


humanEGR1
.......... .......... .......... .......... ..........


 



4301                                              4350


mouseEGR1
.......... .......... .......... .......... ..........


  ratEGR1
AAACACATTG AATGCGCTTT ACTGCCCATG GGATATGTGG TGTGTATCCT


humanEGR1
.......... .......... .......... .......... ..........


 



4351                                 4388


mouseEGR1
.......... .......... .......... ........


  ratEGR1
TCAGAAAAAT TAAAAGGAAA ATAAAGAAAC TAACTGGT


humanEGR1
.......... .......... .......... ........









EXAMPLE 1
Characterisation of DNAzymes ED5 and hED5
Materials and Methods
ODN synthesis. DNAzymes 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 γ32P-dATP and T4 polynucleotide kinase (New England Biolabs). Unincorporated label was separated from radiolabeled species by centrifugation on Chromaspin-10 columns (Clontech).
In vitro transcript and cleavage experiments. A 32P-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 μl containing 10 mM MgCl2, 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 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 μg/ml streptomycin and 50 IU/ml penicillin at 37° C. in a humidified atmosphere of 5% CO2. 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 μM) using Superfect iii accordance with manufacturers instructions (Qiagen). After 18 h, the cells were washed with phosphate-buffered saline (PBS). pH 7.4 prior to transfection a second time in 5% FBS.
Northern blot analysis. Total RNA was isolated using the TRIzol reagent (Life Technologies) and 25 μg was resolved by electrophoresis prior to transfer to Hybond-N+ membranes (NEN-DuPont). Prehybridization, hybridization with α32P-dCTP-labeled Egr-1 or β-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 NaCl, 50 mM Tris-HCl, pH 7.5, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, 1% trasylol, 10 μg/ml leupeptin, 1% aprotinin and 2 mM PMSF. Twenty four μg 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% (w:v) Tween 20. Membranes were incubated with rabbit antibodies to Egr-1 or Sp1 (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° C. 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 γ32P-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 single scrape with a sterile toothpick. Cells were treated with mitomycin C (Sigma) (20 μM) 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, i.p.). The right common carotid artery was exposed up to the carotid bifurcation via a midline neck incision. Size 6/0 non-absorbable suture was tied around the common carotid proximal to the bifurcation, ensuring cessation of blood flow distally. A 200 μl solution at 4° C. containing 500 μg of DNAzyme (in DEPC-treated H2O), 30 μl of transfecting agent 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° C. After 3 days, vehicle with or without 500 μg 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% (w:v) agarose prior to fixation in paraffin. Five μm sections were prepared at 250 μm intervals along the vessel from the point of ligation and stained with hematoxylin 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 7×7 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 (L.-Q. Sun, data not shown) (FIG. 1). The 3′ terminus of the molecule was capped with an inverted 3′-3′-linked thymidine (T) to confer resistance to 3′->5′ exonuclease digestion. The sequence in both arms of ED5 was scrambled (SCR) without altering the catalytic domain to produce DNAzyme ED5SCR (FIG. 1).
A synthetic RNA substrate comprised of 23 nts, matching nts 805 to 827 of NGFI-A mRNA (FIG. 1) was used to determine whether ED5 had the capacity to cleave target RNA. ED5 cleaved the 32P-5′-end labeled 23-mer within 10 min. The 12-mer product corresponds to the length between the A(816)-U(817) junction and the 5′ end of the substrate (FIG. 1). 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. Similar results were obtained using ED5SCR (data not shown) hED5 differs from the rat ED5 sequence by 3 of 18 nts in its hybridizing arms (Table 2). The catalytic effect of ED5 on a 32P-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 32P-labeled NGFI-A transcript of 1960 nt length in a specific and time-dependent manner (data not shown).
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: 13) (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: 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 Sp1 and c-Fos have not been reported.


















Best homology over 18 nts





Accession
(%)












Gene
number
ED5
hED5
















Rat NGFI-A
M18416
100
84.2



Human EGR-1
X52541
84.2
100



Murine Sp1
AF022363
66.7
66.7



Human c-Fos
K00650
66.7
66.7



Murine c-Fos
X06769
61.1
66.7



Human Sp1
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 μM) inhibited serum-inducible steady-state NGFI-A mRNA levels by 55% (FIG. 2a), whereas ED5SCR had no effect (FIG. 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 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 (data not shown). ED5 failed to affect levels of the constitutively expressed, structurally-related zinc-finger protein, Sp1. It was also unable to block serum-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 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 then determined. Growth-quiescent SMCs were incubated with DNAzyme prior to exposure to serum and the assessment of cell numbers after 3 days. ED5 (0.1 μM) inhibited SMC proliferation stimulated by serum by 70% (FIG. 3a). In contrast, ED5SCR failed to influence SMC growth (FIG. 3a). AS2, an antisense NGH-A ODN able to inhibit SMC growth at 1 μM failed to inhibit proliferation at the lower concentration (FIG. 3a). Additional experiments revealed that ED5 also blocked serum-inducible 3H-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 (FIG. 3b).
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 growth-regulatory 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% (FIG. 3c). ED5SCR had no inhibitory effect; surprisingly, it appeared to stimulate growth (FIG. 3c). Trypan Blue exclusion revealed that DNAzyme inhibition was not a consequence of cytotoxicity (data not shown).
To ensure that differences in the biological effects of ED5 and ED5SCR 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 γ32P-dATP and incubated in culture medium to ascertain whether cellular responsiveness to ED5 and ED5SCR was a consequence of differences in DNAzyme stability. Both 32P-ED5 and 32P-ED5SCR remained intact even after 48 h. In contrast to 32P-ED5 bearing the 3′ inverted T, degradation of 32P-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 & 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 sequence-specific inhibition by ED5.
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 (FIG. 4). In contrast, neither its scrambled counterpart (FIG. 4) nor the vehicle control (FIG. 4) had any effect on neointima formation. These findings demonstrate the capacity of ED5 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).
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 ED5 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 EGR-1 RNA, we prepared in vitro transcribed 35S-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 168-332 of human EGR-1 into pGEM-T-easy. A 388 nt 35S-labeled substrate was prepared by in vitro transcription using SP6 polymerase. Time-dependent cleavage of the substrate was tested using the following DNZzymes:
DzA: 5′-CAGGGGACAGGCTAGCTACAACGACGTTGCGGG-X-3′ (SEQ ID NO: 15);
DzB: 5′-TGCAGGGGAGGCTAGCTACAACGAACCGTTGCG-X-3′ (SEQ ID NO: 16);
DzC: 5′-CATCCTGGAGGCTAGCTACAACGAGAGCAGGCT-X-3′ (SEQ ID NO: 17);
DzE: 5′-TCAGCTGCAGGCTAGCTACAACGACTCGGCCTT-X-3′ (SEQ ID NO: 18); and
DzF: 5′-GCGGGGACAGGCTAGCTACAACGACAGCTGCAT-X-3′ (SEQ ID NO: 19)
where X denotes a 3′-3-linked T.
The DNAzyme that cleaved most effectively of this group was DzA, then DzB, then DzC. In contrast, DzE was inactive.
EXAMPLE 3
Inhibition of Induction of EGR-1 in Human SMC by DzA
To determine whether DzA could block the induction of endogenous human EGR-1, we incubated growth-quiescent human aortic smooth muscle cells 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 μM DzA, delivered using FuGENE6 (Roche Molecular Biochemicals) and unaffected by DzE. The blot was then stripped and reprobed with antibodies to the transcription factor Sp1. Results obtained showed that neither serum nor DzA affected induction of Sp1. 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-1 protein.
EXAMPLE 4
Inhibition of Human SMC Proliferation by DzA
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 μM DzA (FIG. 5). In contrast, neither DzE nor ED5SCR had any effect (FIG. 5). These data demonstrate that DzA inhibits human SMC proliferation.
EXAMPLE 5
Inhibition of Porcine SMC Proliferation by DzA
The porcine and human EGR-1 sequences are remarkably well conserved (91%). Porcine retinal SMCs were used to determine whether DzA could block the growth of porcine SMCs. Our studies indicate that DzA (0.5 μM) could inhibit the proliferation of these cells (FIG. 6). In contrast, DzE had no effect (FIG. 6).
EXAMPLE 6
Delivery of DNAzyme into the Porcine Coronary Artery Wall
Porcine angioplasty and stenting are accepted models of human in-stent restenosis (Karas et al. 1992). The porcine coronary anatomy, dimensions and histological response to stenting are similar to the human (Muller et al. 1992). The Transport Catheter has previously been used to deliver antisense DNA targeting c-myc in humans (Serrys et al. 1998) and the pig (Gunn & Cumberland, 1996) 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. DNAzyme (1000 μg) was delivered via the catheter in 2 ml MilliQ H2O containing 300 μl FuGENE6 and 1 mM MgCl2. The FITC-labeled DNAzyme localised into the intimal cells of the vessel wall. These studies demonstrate that DNAzyme 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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## Claims

1. A DNAzyme which specifically cleaves EGR-1 mRNA, the DNAzyme comprising
(i) a catalytic domain which cleaves mRNA at a purine:pyrimidine cleavage site;
(ii) a first binding domain continuous with the 5′ end of the catalytic domain; and
(iii) a second binding domain continuous 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 168-332 as shown in SEQ ID No: 1, such that the DNAzyme cleaves the EGR-1 mRNA.

2. A DNAzyme as claimed in claim 1 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

3. A DNAzyme as claimed in claim 1 in which the cleavage site is selected from the group consisting of
(i) 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;
(v) 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.

4. A DNAzyme as claimed in claim 3 in which the cleavage site is the AU site corresponding to nucleotides 271-272.

5. A DNAzyme as claimed in claim 3 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

6. A DNAzyme as claimed in claim 4 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 31-end of the catalytic domain.

7. A DNAzyme as claimed in claim 1 in which the catalytic domain has the nucleotide sequence GGCTAGCTACAACGA [SEQ. ID. NO:2].

8. A DNAzyme as claimed in claim 7 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

9. A DNAzyme as claimed in claim 7 in which the cleavage site is selected from the group consisting of
(i) 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;
(v) 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.

10. A DNAzyme as claimed in claim 9 in which the cleavage site is the AU site corresponding to nucleotides 271-272.

11. A DNAzyme as claimed in claim 9 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

12. A DNAzyme as claimed in claim 9 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

13. A DNAzyme as claimed in claim 1 wherein each binding domain is nine or more nucleotides in length.

14. A DNAzyme as claimed in claim 13 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

15. A DNAzyme as claimed in claim 13 in which the cleavage site is selected from the group consisting of
(i) 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;
(v) 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.

16. A DNAzyme as claimed in claim 15 in which the cleavage site is the AU site corresponding to nucleotides 271-272.

17. A DNAzyme as claimed in claim 15 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

18. A DNAzyme as claimed in claim 16 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

19. A DNAzyme as claimed in claim 13 in which the catalytic domain has the nucleotide sequence GGCTAGCTACAACGA [SEQ ID NO: 2].

20. A DNAzyme as claimed in claim 19 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

21. A DNAzyme as claimed in claim 19 in which the cleavage site is selected from the group consisting of
(i) 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;
(v) 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.

22. A DNAzyme as claimed in claim 21 in which the cleavage site is the AU site corresponding to nucleotides 271-272.

23. A DNAzyme as claimed in claim 21 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

24. A DNAzyme as claimed in claim 22 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

25. A DNAzyme as claimed in claim 1 which has a sequence selected from the group consisting of:
(i) 5′-caggggacaGGCTAGCTACAACGAcgttgcggg (SEQ ID NO: 3);
(ii) 5′-tgcaggggaGGCTAGCTACAACGAaccgttgcg (SEQ ID NO: 4);
(iii) 5′-catcctggaGGCTAGCTACAACGAgagcaggct (SEQ ID NO: 5);
(iv) 5′-ccgcggccaGGCTAGCTACAACGAcctggacga (SEQ ID NO: 6);
(v) 5′-ccgctgccaGGCTAGCTACAACGAcccggacgt (SEQ ID NO: 7);
(vi) 5′-gcggggacaGGCTAGCTACAACGAcagctgcat (SEQ ID NO: 8);
(vii) 5′-cagcggggaGGCTAGCTACAACGAatcagctgc (SEQ ID NO: 9); and
(viii) 5′-ggtcagagaGGCTAGCTACAACGActgcagcgg (SEQ ID NO: 10).

26. A DNAzyme as claimed in claim 25 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

27. A DNAzyme as claimed in claim 25 which has the sequence:
5′-ccgcggccaGGCTAGCTACAACCAcctggacga (SEQ ID NO: 6).

28. A DNAzyme as claimed in claim 27 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

29. A pharmaceutical composition comprising a DNAzyme according to claim 1 and a pharmaceutically acceptable carrier.