(19) TZZ__Z_T

(11) EP 1 485 109 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/113 (2010.01) A61K 31/7088 (2006.01) 02.10.2013 Bulletin 2013/40 A61P 9/00 (2006.01) A61P 9/10 (2006.01) A61P 35/00 (2006.01) C12Q 1/68 (2006.01) (21) Application number: 03702205.0 (86) International application number: (22) Date of filing: 27.02.2003 PCT/AU2003/000237

(87) International publication number: WO 2003/072114 (04.09.2003 Gazette 2003/36)

(54) VASCULAR THERAPEUTICS GEFÄSSTHERAPEUTIKA TRAITEMENT VASCULAIRE

(84) Designated Contracting States: • BISWAL S. ET AL.: "Inhibition of cell proliferation AT BE BG CH CY CZ DE DK EE ES FI FR GB GR and AP-1 activity by Acrolein in human A549 lung HU IE IT LI LU MC NL PT SE SI SK TR adenocarcinoma cells due to thiol imbalance and covalent modifications" CHEMICAL RESEARCH (30) Priority: 27.02.2002 AU PS078002 IN TOXICOLOGY, vol. 15, no. 2, February 2002 (2002-02), pages 180-186, XP002531768 (43) Date of publication of application: • SUGGS W.D. ET AL.: "Antisense 15.12.2004 Bulletin 2004/51 oligonucleotides to c- fos and c- jun inhibit intimal thickening in a rat vein graft model" SURGERY, (73) Proprietor: NewSouth Innovations Pty Limited vol. 126, 1999, pages 443-449, XP002531769 Sydney NSW 2052 (AU) • YOSHIDA S. ET AL.: "Involvement of Interleukin- 8, Vascular Endothelial Growth Factor, and Basic (72) Inventor: KHACHIGIAN, Levon, Michael Fibroblast Growth Factor in Tumor Necrosis Ryde, New South Wales 2112 (AU) Factor alpha-dependent angiogenesis" MOLECULAR AND CELLULAR BIOLOGY, vol. 17, (74) Representative: Brasnett, Adrian Hugh et al no. 7, 1 July 1997 (1997-07-01), pages 4015-4023, Mewburn Ellis LLP XP002531770 ISSN: 0270-7306 33 Gutter Lane • BUCHWALD A.B. ET AL.: "Decoy London oligodeoxynucleotide against Activator Protein- EC2V 8AS (GB) 1 reduces neointimal proliferation after coronary angioplasty in hypercholesterolemic minipigs" (56) References cited: JOURNAL OF THE AMERICAN COLLEGE OF WO-A-01/32156 WO-A-95/02051 CARDIOLOGY, vol. 39, no. 4, 20 February 2002 WO-A-95/02051 WO-A-98/46272 (2002-02-20), pages 732-738, XP002531771 WO-A-98/46272 US-A- 5 837 244 • MERCOLA D. AND COHEN J.S.: "Antisense approaches to cancer gene therapy" CANCER • PAN B. ET AL.: "Reversal of Cisplatin resistance GENE THERAPY, vol. 2, no. 1, 1 January 1995 in human ovarian cancer cell lines by a c-jun (1995-01-01), pages 47-59, XP002911890 ISSN: antisense oligodoexynucleotide (ISIS 10582): 0929-1903 evidence for the role of transcription factor • KHACHIGIAN L.M. ET AL.: ’c-Jun regulates overexpression in determining resistant vascular smooth muscle cell growth and phenotype" BIOCHEMICAL PHARMACOLOGY, neointima formation after arterial injury’ vol. 63, no. 9, 1 May 2002 (2002-05-01), pages JOURNALOF BIOLOGICAL CHEMISTRY vol. 277, 1699-1707, XP002531767 no. 25, 21 June 2002, pages 22985 - 22991, XP008101011

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 485 109 B1

Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 1 485 109 B1

• MERCOLA D. ET AL.: ’Antisense approaches to cancer gene therapy’ CANCER GENE THERAPY vol. 2, no. 1, 1995, pages 47 - 59, XP002911890 • PAN B ET AL: "Reversal of Cisplatin resistance in human ovarian cancer cell lines by a c-jun antisense oligodoexynucleotide (ISIS 10582): evidence for the role of transcription factor overexpression in determining resistant phenotype", BIOCHEMICAL PHARMACOLOGY, PERGAMON, OXFORD, GB, vol. 63, no. 9, 1 May 2002 (2002-05-01), pages 1699-1707, XP002531767, ISSN: 0006-2952, DOI: 10.1016/S0006-2952(02)00841-9

2 1 EP 1 485 109 B1 2

Description muscle cell proliferation10. Similarly, YY1 overexpres- sion blocks smooth muscle cell growth without affecting FIELD OF THE INVENTION endothelial cell proliferation11. [0006] c- Jun can repress, as well as activate transcrip- [0001] The present invention relates to methods and 5 tion. c- Jun binds the corepressor TG- interacting factor compositions for reducing or preventing c- Jun mediated (TGIF) to suppress Smad2 transcriptional activity12. c- cellular processes. In particular, the present invention Jun also blocks transforming growth factor beta- medi- relates to methods of reducing or preventing neointima ated transcription by repressing the transcriptional activ- formation, atherosclerosis, restenosis, graft failure or an- ity of Smad313. giogenesis involving the use of DNAzymes. 10 [0007] c- Jun can inhibit, as well as stimulate prolifer- ation. Using antisense oligonucleotides to c- Jun, Kana- BACKGROUND OF THE INVENTION tani and colleagues demonstrated that inhibition of hu- man monocytoid leukemia cell growth by TGF- beta and [0002] The initiating event in the pathogenesis of dexamethasone is mediated by enhanced c- Jun atherosclerosis and restenosis following angioplasty is 15 expression5. injury to cells in the artery wall 1. Injury or stress stimulates [0008] c- Jun, however, has not been directly linked to signalling and transcriptional pathways in vascular the complex process of angiogenesis, which underlies smooth muscle cells, stimulating their migration and pro- many common human diseases including solid tumor liferation and the eventual formation of a neointima. growth and corneal disease. Angiogenesis is a complex Smooth muscle cell proliferation is a key feature of ne- 20 multi- step process involving proteolytic degradation of ointima formation, atherosclerosis, restenosis and graft the basement membrane and surrounding extracellular failure. matrix, microvascular endothelial cell proliferation, mi- [0003] c- Jun, a prototypical member of the basic re- gration, tube formation and structural re- organisation 14. gion- leucine zipper protein family, is transiently induced following arterial injury in animal models 2,3. c- Jun forms 25 DNAzymes both homodimers and heterodimers with other bZIP pro- teins to form the AP- 1 transcription factor. While inves- [0009] In human gene therapy, antisense nucleic acid tigations over the last decade have linked AP- 1 with pro- technology has been one of the major tools of choice to liferation, tumorigenesis and apoptosis, AP- 1 has also inactivate genes whose expression causes disease and been implicated in tumor suppression and 30 cellis thus undesirable. The anti- sense approach employs a differentiation4. Thus, gene- targeting strategies that nucleic acid molecule that is complementary to, and down- regulate c- Jun expression do not necessarily in- thereby hybridizes with, an mRNA molecule encoding an hibit cell proliferation. undesirable gene. Such hybridization leads to the inhibi- [0004] Kanatani et al, (1996)5 have shown that anti- tion of gene expression by mechanisms including nucle- sense oligonucleotides targeting c- Jun dose-dependent- 35 olytic degradation or steric blockade of the translational ly reduce the growth-inhibitory effect of dexamethasone machinery. and TGF.β Recent reports indicate thatJun c- [0010] Anti- sense technology suffers from certain NH2Jerminal kinase / stress activated protein kinase drawbacks. Anti- sense hybridization results in the for- (JNK), an upstream activator of c-Jun and numerous oth- mation of a DNA/ target mRNA heteroduplex. This het- er transcription factors, is expressed by SMCs in human 40 eroduplex serves as a substrate for RNAse H- mediated and rabbit atherosclerotic plaques6,7 and that dominant degradation of the target mRNA component. Here, the negative JNK inhibits neointima formation after balloon DNA anti- sense molecule serves in a passive manner, injury8. c-Jun, however, has not been localised in human in that it merely facilitates the required cleavage by en- atherosclerotic lesions, nor has it been shown to play a dogenous RNAse H enzyme. This dependence on functional role in arterial repair after injury. 45 RNAse H confers limitations on the design of anti- sense [0005] It is clear, however, that the finding that c- Jun, molecules regarding their chemistry and ability to form or any other given gene, is inducibly expressed in the stable heteroduplexes with their target mRNA’s. Anti- artery wall following balloon angioplasty does not neces- sense DNA molecules also suffer from problems asso- sarily translate to it playing a positive regulatory role in ciated with non- specific activity and, at higher concen- transcription, proliferation or neointima formation. For ex- 50 trations, even toxicity. ample, it has been shown that three transcriptional re- [0011] As an alternative to anti-sense molecules, cat- pressors (NAB2, GCF2, and YY1) are activated in vas- alytic nucleic acid molecules have shown promise as cular smooth muscle cells by mechanical injury in vitro, therapeutic agents for suppressing gene expression, and as well as in the rat artery wall. NAB2 directly binds the are widely discussed in the literature15-21. Thus, unlike zinc finger transcription factor Egr-1 and represses Egr- 55 a conventional anti-sense molecule, a catalytic nucleic 1-mediated transcription9. GCF2 is a potent repressor of acid molecule functions by actually cleaving its target the expression of PDGF-A, a well-established mitogen mRNA molecule instead of merely binding to it. Catalytic for vascular smooth muscle cells, and inhibits smooth nucleic acid molecules can only cleave a target nucleic

3 3 EP 1 485 109 B1 4 acid sequence if that target sequence meets certain min- tivity of c-Jun for use in a method of preventing or reduc- imum requirements. The target sequence must be com- ing ocular angiogenesis, or for use in treating or inhibiting plementary to the hybridizing regions of the catalytic nu- melanoma growth, in a subject, wherein the nucleic acid cleic acid, and the target must contain a specific se- is selected from the group consisting of: (a) a DNAzyme quence at the site of cleavage. 5 targeted against c-Jun or a ribozyme targeted against c- [0012] Catalytic RNA molecules ("ribozymes") are well Jun, wherein the cleavage site of the DNAzyme or ri- documented15, 22, 23, and have been shown to be capa- bozyme is within the region of residues U1296 to G1497 ble of cleaving both RNA15 and DNA20 molecules. In- of c_Jun mRNA, or (b) a dsRNA targeted against c-Jun deed, the development of in vitro selection and evolution mRNA, a nucleic acid molecule which results in produc- techniques has made it possible to obtain novel 10 ri- tion of dsRNA targeted against c-Jun mRNA or small bozymes against a known substrate, using either random interfering RNA molecules targeted against c-Jun mR- variants of a known ribozyme or random- sequence RNA NA, wherein the dsRNA or small interfering RNA targets as a starting point16, 24, 25. c-Jun mRNA within the region of residues U1296 to [0013] Ribozymes, however, are highly susceptible to G1497. enzymatic hydrolysis within the cells where they are in- 15 [0019] In another aspect, the invention provides a cat- tended to perform their function. This in turn limits their alytic nucleic acid, wherein the catalytic nucleic acid pharmaceutical applications. cleaves c-Jun mRNA in a GU site corresponding to nu- [0014] Recently, a new class of catalytic molecules cleotides 1311-1312. called "DNAzymes" was created26, 27. DNAzymes are [0020] These and other aspects of the invention are single- stranded, and cleave both RNA 16, 27, and DNA 21. 20 defined in the accompanying claims. A general model for the DNAzyme has been proposed, [0021] Also described herein is a method of screening and is known as the "10- 23" model. DNAzymes following for an agent which inhibits restenosis, neointima forma- the "10- 23" model, also referred to simply as "10- 23 tion, graft failure, atherosclerosis, angiogenesis, and/or DNAzymes", have a catalytic domain of 15 deoxyribonu- solid tumour growth the method comprising testing a pu- cleotides, flanked by two substrate- recognition domains 25 tative agent for the ability to inhibit induction of c-Jun, of variable deoxyribonucleotide arm length. In vitro anal- decrease expression of c-Jun or decrease the nuclear yses show that this type of DNAzyme can effectively accumulation or activity of c-Jun. cleave its substrate RNA at purine: pyrimidine junctions [0022] Also described herein is a catalytic nucleic acid under physiological conditions27. which specifically cleaves c-Jun mRNA in the region of [0015] DNAzymes show promise as therapeutic30 residues A287 to A1501. agents. However, DNAzyme success against a disease [0023] Also described herein is an antisense oligonu- caused by the presence of a known mRNA molecule is cleotide which specifically binds c-Jun mRNA in the re- not predictable. This unpredictability is due, in part, to gion of residues U1296 to G1497. two factors. First, certain mRNA secondary structures [0024] Also described herein is a pharmaceutical com- can impede a DNAzyme’s ability to bind to and cleave 35 position comprising the catalytic nucleic acid of the fifth its target mRNA. Second, the uptake of a DNAzyme by aspect of the invention or the antisense oligonucleotide cells expressing the target mRNA may not be efficient ofthe sixth aspectof theinvention and a pharmaceutically enough to permit therapeutically meaningful results. acceptable carrier. [0016] Investigation of the precise regulatory role of c- [0025] Also described herein is an angioplastic stent Jun in the injured artery wall and indeed, in other disease 40 for inhibiting onset of restenosis comprising an angi- settings such as angiogenesis, has been hampered by oplastic stent operably coated with a prophylactically ef- the lack of a specific pharmacological inhibitor.fective dose of a nucleic acid which decreases the level DNAzymes represent a new class of gene targeting of c-Jun mRNA, c-Jun mRNA translation or nuclear ac- agent with specificity conferred by the sequence of nu- cumulation or activity of c-Jun. cleotides in the two arms flanking a catalytic core 27, with 45 [0026] Also described herein is a method for inhibiting advantages over ribozymes of substrate specificity and the onset of restenosis, neointima formation, graft failure stability27, 28. To date, neither c- Jun nor indeed any other and/or atherosclerosis in a subject undergoing angi- Jun family member has been targeted using catalytic nu- oplasty comprising topically administering a stent ac- cleic acid strategies. cording to the eighth aspect of the invention to the subject [0017] A number of documents relate to c-jun and its 50 at around the time of angioplasty. role in cancer, and some propose targetingc- jun with antisense oligonucleotides 58-66. BRIEF DESCRIPTION OF FIGURES

SUMMARY OF THE INVENTION [0027] 55 [0018] In one aspect, the present invention provides a Figure 1. c-Jun and Sp1 expression in human nucleic acid which decreases the level of c-Jun mRNA, atherosclerotic lesions. Immunohistochemical stain- c-Jun mRNA translation or nuclear accumulation or ac- ing for c-Jun and Sp1 in 5 mm sections of human ca-

4 5 EP 1 485 109 B1 6 rotid atherosclerotic lesions. The intima and media Figure 4. c-Jun DNAzyme inhibition of smooth mus- are indicated in the figure; L denotes lumen. Staining cle cell repair. Smooth muscle cell regrowth in the is representative of three independent samples. denuded zone three days after scraping and trans- fection with 0.5mM of Dz13 or Dz13scr. The cells Figure 2. Cleavage of in vitro transcribed c- Jun RNA 5 were fixed and stained with hematoxylin and eosin and inhibition of Jun c- induction by Jun c- prior to micrography. DNAzymes. a, Representation of DNAzyme cleav- age sites (arrows) in c- Jun RNA and sizes of expect- Figure 5. Blockade of neointimal thickening in rat ed products. The specific purine hosting the 3’ cleav- common carotid arteries. a, Neointima/media ratios age is indicated for each candidate DNAzyme. Num- 10 for each group (vehicle alone, vehicle containing bering is based on the human c-Jun complete cds Dz13, vehicle containing Dz13scr) 21d after injury. (Accession J04111, NID g186624). The expression * indicates P<0.05 compared with vehicle and vehi- vector used for the T7 RNA polymerase-dependent cle containing Dz13scr groups using Student’s t- test. generation of c-Jun RNA is indicated. b, Integrity The vehicle and vehicle containing Dz13scr groups analysis of DNAzyme (34 nt) (upper panel) and 668 15 were not statistically different.b, Representative nt c-Jun RNA (middle panel) and panning for nucle- cross-sections stained with haematoxylin-eosin. N olytic activity of candidate DNAzymes after 1 h at and single line denotes neointima, M and triple line 37°C (lower panel). DNAzyme integrity was deter- denotes media, arrow denotes preinjured intima. mined by 5’-end labelling with γ32P-dATP and T4 Thrombosis was occasionally observed and not con- RNA polymerase prior to resolution on 12% dena- 20 fined to any particular group. c, Immunoperoxidase turing polyacrylamide gels. Transcript integrity was staining for c-Jun protein six hours after arterial in- determined by random labelling with α32P-UTP and jury. d, Immunoperoxidase staining for Sp1 six hours T7 polynucleotide kinase prior to resolution on 12% after arterial injury. DNAzyme in vehicle (FuGENE6, denaturing polyacrylamide gels. The figure shows MgCl2, PBS, pH 7.4) was applied to the carotid in the 668 nt transcript after the reaction was allowed 25 Pluronicgel (BASF) at thetime of injury. Threeweeks to proceed for the times indicated. Subsequent ex- subsequently the arteries were perfusion-fixed and periments used the 30min run- off. c, Time- and dose- 5mm sections taken for immunohistochemical and dependence of Dz13 cleavage of c-Jun RNA. The morphometric analysis. 474 and 194 nt products are indicated. d, Western blot analysis for c-Jun protein. Extracts of smooth 30 Figure 6. Dz13 inhibits microvascular endothelial muscle cells (10mg) transfected with 0.5mM of microtubule formation on reconstituted basement DNAzyme (Dz13 or Dz13scr) were assessed for c- membranes. HMEC-1 cells, transfected previously Jun immunoreactivity (39 kDa) using rabbit polyclo- with the indicated concentrations of Dz13 and nal anti-peptide antibodies (Santa Cruz Biotechnol- Dz13scr, were plated into 96wps containing matrigel ogy). The Coomassie blue- stain gel shows unbiased 35 and tubule formation was quantitated after 8 h. As- loading. terisk indicates p<0.05 by Student’s t- test relative to control. Western and EMSA revealed that Dz13 in- Figure 3. c-Jun DNAzyme inhibition of smooth mus- hibits c-Jun expression and DNA-binding activity in cle cell proliferation. a, Schematic representation of microvascular endothelial cells (data not shown). c-Jun DNAzyme Dz13 and target site (G 1311T) in hu- 40 (FBS denotes foetal bovine serum) man c-Jun mRNA (upper panel), comparison of Dz13 target site in human, porcine and rat c- Jun mR- Figure 7. A, c-Jun cDNA sequence (Accession NA (middle panel), and comparison of As13 and J04111, NID g186624). B, Location of DNAzyme tar- Dz13 (lower panel). The translational start site of hu- get sites in c-Jun mRNA. man c-Jun mRNA is located at A 1261TG. b, Effect of 45 c-Jun DNAzymes (0.5mM) on serum-inducible pri- Figure 8. Dz13 inhibits microvascular endothelial mary human smooth muscle cell (HASMC) prolifer- cell proliferation. A, Growth- quiescent HMEC- 1 ation inhibited by Dz13. Sequence of Dz13scr is 5’- cells pre- treated with DNAzyme (0.2mM) were ex- GCG ACG TGA GGC TAG CTA CAA CGA GTG posed to serum and total cell counts were deter- GAG GAG X-3’, where X is a 3’- 3’-linked inverted T. 50 mined after 3 days using a Coulter counter. B, Dz13 c, Serum-inducible porcine smooth muscle cell pro- inhibition of microvascular endothelial cell prolifera- liferation (PASMC) inhibited by 0.5mM of Dz13. d, tion is dose- dependent. C, Effect of Dz13 variants Human smooth muscle cell proliferation is inhibited (shorter and longer arm length) on proliferation. Se- by Dz13 and As13 in a dose- dependent manner. The quences of Dz13 (11+11), Dz13 (10+10) and Dz13 concentrations of DNAzyme (0.1-0.4 mM) are indicat- 55 (8+8) are 5’- GA CGG GAG GAA ggc tag cta caa ed in the figure. The sequence of As13scr is 5’- GCG cga GAG GCG TTG AG- Ti- 3’, 5’- A CGG GAG GAA ACG TGA C GTG GAG GAG X-3’, where X is a 3’- ggc tag cta caa cga GAG GCG TTG A- Ti- 3’ and 5’- 3’-linked inverted T. GG GAG GAA ggc tag cta caa cga GAG GCG TT-

5 7 EP 1 485 109 B1 8

Ti- 3’, respectively. Sequences of Dz13 (11+11) scr, CAA CGC CUC G1311| UUC CUC CcG- 3’) are con- Dz13 (10+10) scr and Dz13 (8+8) scr are 5’- GA served in murine c-Jun mRNA (5’-CAA CGC CUC GCG ACG TGA ggc tag cta caa cga GTG GAG GAG G | UUC CUC CaG- 3’). Asterisk indicates p<0.05 by AG- Ti- 3’, 5’- A GCG ACG TGA ggc tag cta caa cga Student’s t-test relative to control. Immunohisto- GTG GAG GAG A- Ti- 3’ and 5’- CG ACG TGA ggc 5 chemical analysis in vascularized human malignant tag cta caa cga GTG GAG GA- Ti- 3’, respectively. cutaneous melanoma tissue revealed that c- Jun and Ti is a 3’- 3’- linked inverted T. Asterisk indicates MMP-2 are expressed in CD31+ endothelium and p<0.05 by Student’s t- test relative to control. surrounding melanoma cells (data not shown). Western blot analysis demonstrated Dz13 inhibition Figure 9. Dz13 inhibits microvascular endothelial re- 10 of c-Jun protein 2h after exposure of the cells to se- growth after scraping in vitro and migration in mod- rum (data not shown). ified Boyden chambers. A, Growth-quiescent HMEC-1 cells pre-treated with DNAzyme (0.2, 0.3 DETAILED DESCRIPTION OF THE INVENTION or 0.4mM) were scraped and the number of cells in the denuded zone was quantitated under microsco- 15 [0028] The present inventors have demonstrated c- py. B, HMEC-1 were plated in modified Boyden Jun expression by smooth muscle cells in the human chambers coated with matrigel or collagen type I atheromatous lesion (Fig. 1). c-Jun is poorly, if at all, ex- (chemoattractant in lower chamber was FGF-2, 20 pressed by smooth muscle cells in the normal media. In ng/ml) and the number of cells on the underside of contrast, the zinc finger transcription factor Sp1 is ex- the membrane was quantitated after 24 h. Asterisk 20 pressed in both the intima and media (Fig. 1). indicates p<0.05 by Student’s t-test relative to con- [0029] Neointima formation is a characteristic feature trol. of common vascular pathologies, such as atherosclero- sis and post- angioplasty restenosis,and involves smooth Figure 10. Dz13 blocks MMP- 2 expression and pro- muscle cell proliferation. teolysis. MMP-2 protein expression was quantitated 25 [0030] In addition to its expression by smooth muscle by enzyme- linked immunosorbent assay. Gelatin zy- cells, the present inventors have also demonstrated that mography demonstrated Dz13 inhibition of MMP-2 c-Jun is linked to the complex process of angiogenesis. proteolysis and rescue by overexpression of c-Jun In particular, expression of c- Jun was found in vascular- cDNA (data not shown). RT- PCR demonstrated that ized primary human melanoma which has not been pre- Dz13 blocked c-Jun mRNA expression (data not 30 viously described. shown). [0031] A gene- specific DNAzyme targeting c- Jun (des- ignated Dz13) was generated. Dz13 cleaves c- Jun RNA Figure 11. Dz13 inhibits VEGF165- induced neovas- and inhibits inducible c-Jun protein expression in vascu- cularization in rat cornea. A, HMEC-1 cells (pre- lar smooth muscle cells with a potency exceeding its ex- transfected with 0.4 mM Dz13, and in medium con- 35 act non-catalytic antisense oligodeoxynucleotide equiv- taining 200 ng/ml of VEGF165) were plated into alent. Moreover, Dz13 abrogated smooth muscle cell re- 96wps containing matrigel and tubule formation was pair after injury in vitro and neointima formation in rat quantitated under microscopy after 8 h. Quantitation carotid arteries in vivo. of B, the number of blood vessels in the rat cornea [0032] Dz13 also blocked endothelial proliferation, mi- and C, the corneal surface area occupied by these 40 grationand microtubule formation with a potency exceed- new vessels. Asterisk indicates p<0.05 by Student’s ing its exact non-catalytic antisense oligodeoxynucle- t-test relative to control. Western blot analysis using otide equivalent. It inhibited neovascularisation in rat cor- polyclonal c-Jun antibodies and extracts of growth- nea and melanoma growth in mice. quiescent HMEC-1 cells pre-treated with DNAzyme [0033] These findings demonstrate the pivotal regula- (0.4mM) 2 h after exposure to VEGF165 (100 mg/ml) 45 tory role of c-Jun in neointima formation in the injured demonstrated inhibition of c-Jun expression with no artery wall, as well as angiogenesis. change in Sp1 expression (data not shown). [0034] Described herein is a method of preventing or reducing angiogenesis and/or neovascularisation in a Figure 12. Dz13 blockade of solid melanoma growth subject, the method comprising administering to the sub- in mice. A, Dz13 inhibition of solid malignant B16 50 ject a prophylactically effective dose of a nucleic acid tumor growth in a sequence-specific manner. Tu- which decreases the level of c- Jun mRNA, c-Jun mRNA mour volumes were evaluated as indicated on the translation or nuclear accumulation or activity of c-Jun. x-axis. B, Mean total body weight in the DNAzyme [0035] Described herein is a method of treating or in- and vehicle-treated cohorts. C, Proliferation 2 days hibiting a condition selected from the group consisting of after exposure of growth- quiescent cultured microv- 55 restenosis, neointima formation, graft failure and athero- ascular endothelial cells pre-treated with DNAzyme sclerosis in a subject, the method comprising adminis- (0.4 mM) to serum. Seventeen of the 18 nucleotides tering to the subject a prophylactically effective dose of in the Dz13 target site in human c-Jun mRNA (5’- a nucleic acid which decreases the level of c- Jun mRNA,

6 9 EP 1 485 109 B1 10 c-Jun mRNA translation or nuclear accumulation or ac- GA. tivity of c-Jun. [0046] It is preferred that the DNAzyme cleavage site [0036] Described herein is a method of treating or in- is within the region of residues A287 to A1501, more pref- hibiting solid tumour growth in a subject, the method com- erably U1296 to G1497, of the c-Jun mRNA. It is partic- prising administering to the subject a prophylactically ef- 5 ularly preferred that the cleavage site within the c-Jun fective dose of a nucleic acid which decreases the level mRNA is the GU site corresponding to nucleotides of c-Jun mRNA, c-Jun mRNA translation or nuclear ac- 1311-1312. cumulation or activity of c-Jun. [0047] In a further preferred embodiment, the [0037] In a preferred embodiment the angiogenesis is DNAzyme has the sequencecgggaggaaG- 5’- ocular angiogenesis. 10 GCTAGCTACAACGAgaggcgttg-3’. [0038] In a preferred embodiment the solid tumour is [0048] In applying DNAzyme-based treatments, it is melanoma. preferable that the DNAzymes be as stable as possible [0039] Although the subject may be any animal or hu- against degradation in the intra-cellular milieu. One man, it is preferred that the subject is a human. means of accomplishing this is by incorporating a 3’-3’ [0040] As will be recognised by those skilled in this 15 inversion at one or more termini of the DNAzyme. More field there are a number of means by which the method specifically, a 3’- 3’ inversion (also referred to herein sim- of the present invention may be achieved. ply as an "inversion") means the covalent phosphate [0041] In a preferred embodiment, the method is bonding between the 3’ carbons of the terminal nucle- achieved by cleavage of c-Jun mRNA by a sequence- otide and its adjacent nucleotide. This type of bonding is specific DNAzyme. In a further preferred embodiment, 20 opposed to the normal phosphate bonding between the the DNAzyme comprises 3’ and 5’ carbons of adjacent nucleotides, hence the term "inversion". Accordingly, in a preferred embodiment, the (i) a catalytic domain which cleaves mRNA at a pu- 3’-end nucleotide residue is inverted in the building do- rine:pyrimidine cleavage site; main contiguous with the 3’ end of the catalytic domain. (ii) a first binding domain contiguous with the 5’ end 25 In addition to inversions, the instant DNAzymes may con- of the catalytic domain; and tain modified nucleotides. Modified nucleotides include, (iii) a second binding domain contiguous with the 3’ for example, N3’-P5’ phosphoramidate linkages, and end of the catalytic domain, peptide-nucleic acid linkages. These are well known in the art. wherein the binding domains are sufficiently complemen- 30 [0049] In a particularly preferred embodiment, the tary to two regions immediately flanking a purine:pyrimi- DNAzyme includes an inverted T at the 3’ position. dine cleavage site within the c-Jun mRNA such that the [0050] In order to increase resistance to exonudeolytic DNAzyme cleaves the c-Jun mRNA. degradation and helical thermostability locked nucleic [0042] As used herein, "DNAzyme" means a DNA mol- acid analogues can be produced. Further information re- ecule that specifically recognizes and cleaves a distinct 35 garding these analogues is provided in Vester et al, J. target nucleic acid sequence, which may be either DNA Am. Chem. Soc., 2002, 124, 13682-13683. or RNA. [0051] In another embodiment, the method is achieved [0043] In a preferred embodiment, the binding do- by inhibiting translation of the c-Jun mRNA using syn- mains of the DNAzyme are complementary to the regions thetic antisense DNA molecules that do not act as a sub- immediately flanking the cleavage site. It will be appre- 40 strate for RNase and act by sterically blocking gene ex- ciated by those skilled in the art, however, that strict com- pression. plementarity may not be required for the DNAzyme to [0052] In another embodiment, the method is achieved bind to and cleave the c-Jun mRNA. by inhibiting translation of the c-Jun mRNA by destabil- [0044] The binding domain lengths (also referred to ising the mRNA using synthetic antisense DNA mole- herein as "arm lengths") can be of any permutation, and 45 cules that act by directing the RNase degradation of the can be the same or different. In a preferred embodiment, c-Jun mRNA present in the heteroduplex formed be- the binding domain lengths are at least 6 nucleotides. tween the antisense DNA and mRNA. Preferably, both binding domains have a combined total [0053] In another embodiment, the method is achieved length of at least 14 nucleotides. Various permutations by inhibiting translation of the c-Jun mRNA by cleavage in the length of the two binding domains, such as 7+7, 50 of the mRNA by sequence-specific hammerhead ri- 8+8 and 9+9, are envisioned. Preferably, the length of bozymes and derivatives of the hammerhead ribozyme the two binding domains are 9+9. such as the Minizymes or Mini-ribozymes or where the [0045] The catalytic domain of a DNAzyme of the ribozyme is derived from: present invention may be any suitable catalytic domain. Examples of suitable catalytic domains are described in 55 (i) the hairpin ribozyme, Santoro and Joyce, 199727 and U.S. Patent No. (ii) the Tetrahymena Group I intron, 5,807,718. In a preferred embodiment, the catalytic do- (iii) the Hepatitis Delta Viroid ribozyme or main has the nucleotide sequence GGCTAGCTACAAC- (iv) the Neurospera ribozyme.

7 11 EP 1 485 109 B1 12

[0054] It will be appreciated by those skilled in the art usedpharmaceutically acceptable carriers.Such carriers that the composition of the ribozyme may be; are well known to those skilled in the art. The following delivery systems, which employ a number of routinely (i) made entirely of RNA, used carriers, are only representative of the many em- (ii) made of RNA and DNA bases, or 5 bodiments envisioned for administering the instant com- (iii) made of RNA or DNA and modified bases, sugars position. In one embodiment the delivery vehicle con- and backbones tains Mg2+ or other cation (s) to serve as co- factor (s) for efficient DNAzyme bioactivity. [0055] Within the context of the present invention, the [0064] Transdermal delivery systems include patches, ribozyme may also be either; 10 gels, tapes and creams, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, (i) entirely synthetic or fatty acid esters, fatty alcohols and amino acids), hy- (ii) contained within a transcript from a gene deliv- drophilic polymers (e.g., polycarbophil and polyvinylpy- ered within a virus-derived vector, expression plas- rolidone), and adhesives and tackifiers (e.g., polyisobut- mid, a synthetic gene, homologously or heterolo- 15 ylenes, silicone-based adhesives, acrylates and poly- gously integrated into the patients genome or deliv- butene). ered into cells ex vivo, prior to reintroduction of the [0065] Transmucosal delivery systems include patch- cells of the patient, using one of the above methods. es, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and en- [0056] It is preferred that the ribozyme cleaves the c- 20 hancers (e.g., propylene glycol, bile salts and amino ac- Jun mRNA in the region of residues U1296 to G1497. ids), and other vehicles (e.g., polyethylene glycol, fatty [0057] In another embodiment, the method is achieved acid esters and derivatives, and hydrophilic polymers by inhibition of the ability of the c-Jun gene to bind to its such as hydroxypropylmethylcellulose and hyaluronic target DNA by expression of an antisense c- Jun mRNA. acid). [0058] In a still further embodiment the nucleic acid is 25 [0066] Oral delivery systems include tablets and cap- dsRNA targeted against c- Jun mRNA, a nucleic acid mol- sules. These can contain excipients such as binders ecule which results in production of dsRNA targeted (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilo- against c-Jun mRNA or small interfering RNA molecules done, other cellulosic materials and starch), diluents targeted against c-Jun mRNA. So called "RNA interfer- (e.g., lactose and other sugars, starch, dicalcium phos- ence" or "RNAi" is well known and further information 30 phate and cellulosic materials), disintegrating agents regarding RNAi is provided in Hannon, Nature, Vol 418, (e.g., starch polymers and cellulosic materials) and lu- 2002, 244-251, and McManus et al, Nature Reviews: Ge- bricating agents (e.g., stearates and talc). netics, Vol 3, 2002, 737-747. [0067] Solutions, suspensions and powders for recon- [0059] In one embodiment, the method is achieved by stitutable delivery systems include vehicles such as sus- targeting the c- Jun gene directly using triple helix (triplex) 35 pending agents (e.g., gums, xanthans, cellulosics and methods in which a ssDNA molecule can bind to the dsD- sugars), humectants (e.g., sorbitol), solubilizers (e.g., NA and prevent transcription. ethanol, water, PEG and propylene glycol), surfactants [0060] In another embodiment, the method is achieved (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl by inhibiting transcription of the c- Jun gene using nucleic pyridine), preservatives and antioxidants (e.g., pa- acid transcriptional decoys. Linear sequences can be de- 40 rabens, vitamins E and C, and ascorbic acid), anti- caking signed that form a partial intramolecular duplex which agents, coating agents, and chelating agents (e.g., ED- encodes a binding site for a defined transcriptional factor. TA) . [0061] In another embodiment, the method is achieved [0068] Topical delivery systems include, for example, by inhibition of c- Jun activity as a transcription factor us- gels and solutions, and can contain excipients such as ing transcriptional decoy methods. 45 solubilizers, permeation enhancers (e.g., fatty acids, fatty [0062] In another embodiment, the method is achieved acid esters, fatty alcohols and amino acids), and hy- by inhibition of the ability of the c-Jun gene to bind to its drophilic polymers (e.g., polycarbophil and polyvinylpy- target DNA by drugs that have preference for GC rich rolidone) . In the preferred embodiment, the pharmaceu- sequences. Such drugs include nogalamycin, hedamy- tically acceptable carrier is a liposome or a biodegradable cin and chromomycin A329. 50 polymer. Examples of carriers which can be used in this [0063] Administration of the inhibitory nucleic acid may invention include the following: (1) Fugene6® (Roche) ; be effected or performed using any of the various meth- (2) SUPERFECT® (Qiagen) ; (3) Lipofectamine 2000® ods and delivery systems known to those skilled in the (GIBCO BRL) ; (4) CellFectin, 1: 1.5 (M/M) liposome for- art. The administering can be performed, for example, mulation of the cationic lipid N, NI, NII, NIII- tetramethyl- intravenously, orally, via implant, transmucosally,55 N, NI, NII, NIII- tetrapalmitylspermine and dioleoyl phos- transdermally, topically, intramuscularly, subcutaneous- phatidyl- ethanolamine (DOPE) (GIBCO BRL) ; (5) Cyto- ly or extracorporeally. In addition, the instant pharmaceu- fectin GSV, 2: 1 (M/M) liposome formulation of a cationic tical compositions ideally contain one or more routinely lipid and DOPE (Glen Research) ; (6) DOTAP (N- [1- (2,

8 13 EP 1 485 109 B1 14

3- dioleoyloxy)- N, N, N- trimethyl- ammoniummethylsul- and can contain excipients such as PLGA and polycapr- fate) (Boehringer Mannheim) ; and (7) Lipofectamine, 3: ylactone. 1 (M/M) liposome formulation of the polycationic lipid [0072] It is also envisaged that nucleic acid agents tar- DOSPA and the neutral lipid DOPE (GIBCO BRL) . geting c-Jun may be administered by ex vivo transfection [0069] Delivery of the nucleic acids described may also 5 of cell suspensions, thereby inhibiting angiogenesis. be achieved via one or more, of the following non- limiting [0073] In a fourth aspect, the present invention pro- examples of vehicles: vides a method of screening for an agent which inhibits restenosis, neointima formation, atherosclerosis, graft (a) liposomes and liposome-protein conjugates and failure and/or angiogenesis, the method comprising test- mixtures; 10 ing a putative agent for the ability to inhibit induction of (b) non-liposomal lipid and cationic lipid formula- c-Jun, decrease expression of c- Jun or decrease the nu- tions; clear accumulation or activity of c-Jun. (c) activated dendrimer formulations; [0074] The putative agent may be tested for the ability (d) within a polymer formulation such as polyethyl- to inhibit c-Jun by any suitable means. For example, the enimine (PEI) or pluronic gels or within ethylene vinyl 15 test may involve contacting a cell which expresses c- Jun acetate copolymer (EVAc). The polymer is prefera- with the putative agent and monitoring the production of bly delivered intra-luminally; c-Jun mRNA (by, for example, Northern blot analysis) or (e) within a viral-liposome complex, such as Sendai c-Jun protein (by, for example, immunohistochemical virus; analysis or Western blot analysis or electrophoretic mo- (f) as a peptide-DNA conjugate; 20 bility shift assay). Other suitable tests will be known to (g) using catheters to deliver intra-luminal formula- those skilled in the art. tions of the nucleic acid as a solution or in a complex [0075] In a fifth aspect the present invention consists with a liposome; in a catalytic nucleic acid which specifically cleaves c- (h) catheter delivery to adventitial tissue as a solution Jun mRNA in the region of residues A287 to A1501, pref- or in a complex with a liposome; 25 erably U1296 to G1497. (i) the nucleic acid may be bound to a delivery agent [0076] In a preferred embodiment, the catalytic nucleic such as a targeting moiety, or any suitable carrier acid is a sequence-specific DNAzyme comprising such as a peptide or fatty acid molecule; (j) the nucleic acid may be delivered by a double (i) a catalytic domain which cleaves mRNA at a pu- angioplasty balloon device fixed to catheter; or 30 rine:pyrimidine cleavage site; (k) the nucleic acid could be delivered on a specially (ii) a first binding domain contiguous with the 5’ end prepared stent of the Schatz-Palmaz or derivative of the catalytic domain; and type. The stent could be coated with a polymer or (iii) a second binding domain contiguous with the 3’ agent impregnated with nucleic acid that allows con- end of the catalytic domain, trolled release of the molecules at the vessel wall. 35 wherein the binding domains are sufficiently complemen- [0070] Determining the prophylactically effective dose tary to two regions immediately flanking a purine: pyrimi- of the instant pharmaceutical composition can be done dine cleavage site within the c-Jun mRNA such that the based on animal data using routine computational meth- DNAzyme cleaves the c-Jun mRNA in the region of res- ods. In one embodiment, the prophylactically effective 40 idues U1296 to G1497. does contains between about 0.1 mg and about 1 g of [0077] It is particularly preferred that the cleavage site the instant DNAzyme. In another embodiment, the pro- within the c-Jun mRNA is the GU site corresponding to phylactically effective dose contains between about 1 mg nucleotides 1311-1312. and about 100 mg of the instant DNAzyme. In a further [0078] In a preferred embodiment, the binding do- embodiment, the prophylactically effective does contains 45 mains of the DNAzyme are complementary to the regions between about 10 mg and about 50 mg of the instant immediately flanking the cleavage site. It will be appre- DNAzyme. In yet a further embodiment, the prophylacti- ciated by those skilled in the art, however, that strict com- cally effective does contains about 25 mg of the instant plementarity may not be required for the DNAzyme to DNAzyme. bind to and cleave the c-Jun mRNA. [0071] In the case of the prevention or reduction of an- 50 [0079] The binding domain lengths (also referred to giogenesis or inhibition of solid tumour growth, in a pre- herein as "arm lengths") can be of any permutation, and ferred embodiment, the agent is injected into or proximal can be the same or different. In a preferred embodiment, the tumour. Injectable drug delivery systems include so- the binding domain lengths are at least 6 nucleotides. lutions, suspensions, gels, microspheres and polymeric Preferably, both binding domains have a combined total injectables, and can comprise excipients such as solu- 55 length of at least 14 nucleotides. Various permutations bility-altering agents (e.g., ethanol, propylene glycol and in the length of the two binding domains, such as 7+7, sucrose) and polymers (e.g., polycaprylactones and 8+8 and 9+9, are envisioned. Preferably, the length of PLGA’s). Implantable systems include rods and discs, the two binding domains are 9+9.

9 15 EP 1 485 109 B1 16

[0080] The catalytic domain of a DNAzyme of the mid, a synthetic gene, homologously or heterolo- present invention may be any suitable catalytic domain. gously integrated into the patients genome or deliv- Examples of suitable catalytic domains are described in ered into cells ex vivo, prior to reintroduction of the Santoro and Joyce, 199727 and U.S. Patent No. cells of the patient, using one of the above methods. 5,807,718. In a preferred embodiment, the catalytic do- 5 main has the nucleotide sequence GGCTAGCTACAAC- [0087] In a sixth aspect the present invention consists GA. in an antisense oligonucleotide which specifically binds [0081] In a further preferred embodiment, thec-Jun mRNA in the region of residues U1296 to G1497. DNAzyme has the sequencecgggaggaaG- 5’- [0088] It will be understood that the antisense oligonu- GCTAGCTACAACGAgaggcgttg-3’. 10 cleotide need not hybridise to this whole region. It is pre- [0082] In applying DNAzyme-based treatments, it is ferred that the antisense oligonucleotide has the se- preferable that the DNAzymes be as stable as possible quence CGGGAGGAACGAGGCGTTG. against degradation in the intra-cellular milieu. One [0089] In a seventh aspect the present invention con- means of accomplishing this is by incorporating a 3’-3’ sists in a pharmaceutical composition comprising the cat- inversion at one or more termini of the DNAzyme. More 15 alytic nucleic acid of the fifth aspect of the invention or specifically, a 3’-3’ inversion (also referred to herein sim- the antisense oligonucleotide of the sixth aspect of the ply as an "inversion") means the covalent phosphate invention and a pharmaceutically acceptable carrier. bonding between the 3’ carbons of the terminal nucle- [0090] In an eighth aspect the present invention con- otide and its adjacent nucleotide. This type of bonding is sists in an angioplastic stent for inhibiting onset of rest- opposed to the normal phosphate bonding between the 20 enosis comprising an angioplastic stent operably coated 3’ and 5’ carbons of adjacent nucleotides, hence the term with a prophylactically effective dose of a nucleic acid "inversion". Accordingly, in a preferred embodiment, the which decreases the level of c- Jun mRNA, c-Jun mRNA 3’-end nucleotide residue is inverted in the building do- translation or nuclear accumulation or activity of c-Jun. main contiguous with the 3’ end of the catalytic domain. [0091] It is preferred that the agent is the catalytic nu- In addition to inversions, the instant DNAzymes may con- 25 cleic acid of the fifth aspect of the invention or the anti- tain modified nucleotides. Modified nucleotides include, sense oligonucleotide of the sixth aspect of the invention for example, N3’-P5’ phosphoramidate linkages, and [0092] Described herein is a method for inhibiting the peptide-nucleic acid linkages. These are well known in onset of restenosis in a subject undergoing angioplasty the art. comprising topically administering a stent according to [0083] In a particularly preferred embodiment, the30 the eighth aspect of the invention to the subject at around DNAzyme includes an inverted T at the 3’ position. the time of angioplasty. [0084] In another embodiment, the catalytic nucleic ac- [0093] Angioplastic stents, also known by other terms id is a sequence- specific hammerhead ribozyme and de- such as "intravascular stents" or simple "stents", are well rivatives of the hammerhead ribozyme such as the Min- known in the art. They are routinely used to prevent vas- izymes or Mini-ribozymes or where the ribozyme is de- 35 cular closure due to physical anomalies such as unwant- rived from: ed inward growth of vascular tissue due to surgical trau- ma. They often have a tubular, expanding lattice-type (i) the hairpin ribozyme, structure appropriate for their function, and can optionally (ii) the Tetrahymena Group I intron, be biodegradable. (iii) the Hepatitis Delta Viroid ribozyme or 40 [0094] In this invention, the stent can be operably coat- (iv) the Neurospera ribozyme ed with the instant pharmaceutical composition using any suitable means known in the art Here, "operably coating" wherein the ribozyme cleaves the c-Jun mRNA in the a stent means coating it in a way that permits the timely region of residues U1296 to G1497. release of the pharmaceutical composition into the sur- [0085] It will be appreciated by those skilled in the art 45 rounding tissue to be treated once the coated stent is that the composition of the ribozyme may be; administered. Such coating methods, for example, can use the polymer polypyrrole. (i) made entirely of RNA, [0095] As used herein, administration "at around the (ii) made of RNA and DNA bases, or time of angioplasty" can be performed during the proce- (iii) made of RNA or DNA and modified bases, sugars 50 dure, or immediately before or after the procedure. The and backbones. administering can be performed according to known methods such as catheter delivery. [0086] Within the context of the present invention, the [0096] Throughout this specification the word "com- ribozyme may also be either; prise", or variations such as "comprises" or "comprising", 55 will be understood to imply the inclusion of a stated ele- (i) entirely synthetic or ment, integer or step, or group of elements, integers or (ii) contained within a transcript from a gene deliv- steps, but not the exclusion of any other element, integer ered within a virus-derived vector, expression plas- or step, or group of elements, integers or steps.

10 17 EP 1 485 109 B1 18

[0097] Any discussion of documents, acts, materials, a sterile toothpick. Cells were treated with mitomycin C devices, articles or the like which has been included in (Sigma) (20 mM) for 2 h prior to injury to block prolifera- the present specification is solely for the purpose of pro- tion. Seventy- two h after injury, the cells were washed viding a context for the present invention. It is not to be with PBS, pH 7.4, fixed with formaldehyde and stained taken as an admission that any or all of these matters 5 with hematoxylin and eosin. form part of the prior art base or were common general knowledge in the field relevant to the present invention Antibodies as it existed in Australia before the priority date of each claim of this application. [0101] Western immunoblot, and immunohistochemi- [0098] In order that the nature of the present invention 10 cal analysis on human carotid endarterectomy speci- may be more clearly understood, preferred forms thereof mens, were performed using rabbit polyclonal anti-pep- will now be described with reference to the following non- tide antibodies targeting c-Jun and Sp1 (Santa Cruz Bi- limiting and Examples. otechnology) essentially as described11,30.

METHODS 15 Common carotid injury and evaluation of neointima formation DNAzymes, in vitro transcript and cleavage experi- ments [0102] Sprague Dawley rats (450g males) were anaes- thetised using ketamine (60 mg/kg, i.p.) and xylazine (8 [0099] DNAzymes were synthesized by Oligos Etc. or 20 mg/kg, i.p.). The left common and external carotid arter- TriLink with a 3’-3’-linked inverted T and purified by ies were exposed via a midline neck incision, and a lig- HPLC. A 32P-labeled 668 nt c-Jun RNA transcript was ature was applied to the external carotid proximal to the prepared by in vitro transcription (using T7 polymerase) bifurcation. Two hundredm l (at 4°C) containing of pBluescript containing the insert, cut previously with DNAzyme (750 mg), of FuGENE6 (30 ml), MgCl2 (1 mM) XbaI. Reactions were performed in a total volume of 20 25 and P127 Pluronic gel (BASF) was applied around the ml containing 10 mM MgCl2, 5 mM Tris pH 7.5, 150 mM vessel, 6h prior to and again at the time of ligation. The NaCl, 0.5 pmol of in vitro transcribed substrate and 10 solution gelified after contact with the vessel at 37°C. pmol DNAzyme, unless dose-dependent cleavage ex- The incision was sutured and the rats allowed to recover. periments were performed, where stoichiometry is indi- Animals were sacrificed 21 days after injury by lethal in- cated in the figure. Reactions were allowed to proceed 30 jection of ketamine/xylazine, and perfusion fixed with 10 for various times at 37°C and quenched by transferring % (v:v) formaldehyde at 120 mm Hg. Carotids were an aliquot to tubes containing formamide loading buffer. placed in 10 % formaldehyde, embedded in 3 % (w:v) Samples were run on 12% denaturing polyacrylamide agarose, fixed in paraffin and sectioned 1000 mm from gels and autoradiographed overnight at -80°C. the tie. Neointimal and medial areas in 5m m sections 35 stained with hematoxylin and eosin were determined Smooth muscle cell culture, transfection, prolifera- morphometrically and expressed as a mean ratio per tion and wounding assays group of 6 rats.

[0100] Smooth muscle cells derived from human and Endothelial cell culture and transfection porcine coronary arteries were obtained from Cell Appli- 40 cations, Inc (SanDiego, CA), and cultured inWaymouth’s [0103] Human microvascular endothelial 1 cells- medium, pH 7.4, containing 10% fetal bovine serum, 50 (HMEC-1) were grown in MCDB131 medium (GIBCO mg/ml streptomycin and 50 IU/ml penicillin at 37°C in a BRL) containing 10% fetal bovine serum (FBS), 2mM L- humidified atmosphere of 5% CO2. In all in vitro experi- glutamine, 10ng/ml epidermal growth factor, 1 mg/ml hy- ments, smooth muscle cells were not used beyond pas- 45 drocortisone, and 5U/ml penicillin/streptomycin. Murine sage 7. Transfections were performed in smooth muscle brain microvascular endothelial cells (bEND- 3) were cul- cells six h after the change of medium to serum- free, tured in Dulbecco’s modified Eagles medium (DMEM, and again at the time of serum- stimulation 24h after the GIBCO BRL) containing 10% fetal calf serum, 2mM L- start of arrest, using FuGENE6 according to the manu- glutamine and 5 U/ml penicillin/ streptomycin. DNAzyme facturer’s instructions (Roche) . In proliferation assays, 50 transfections were performed with FuGENE6 using sub- growth- arrested smooth muscle cells in 96 well plates confluent cells (60-70%) 6 h after the initiation of growth- (Nunc- InterMed) were transfected with the indicated arrest in serum-free medium. The cells were transfected concentration of DNAzyme or oligonucleotide, then ex- a second time in medium containing serum 18 h after the posed to 5% FBS at 37°C for 72 h. The cells were initial transfection. trypsinized and the suspension quantitated in an auto- 55 mated Coulter counter. In wounding assays, confluent Western Blot Analysis smooth muscle cells in chamber slides (Nunc- InterMed) transfected with DNAzyme were injured by scraping with [0104] Growth- quiescent endothelial cells transfected

11 19 EP 1 485 109 B1 20 twice with DNAzyme were incubated in serum for 2 h der 400x magnification in a blinded manner. prior to the preparation of total cell extracts in 150 mM NaCl, 50 mM Tris- HCl (pH 7.5), 1% sodium deoxycho- HMEC-1 migration and invasion late, 0.1% SDS, 1% Triton X- 100, 5 mM EDTA, 10 mg/ml leupeptin, 1% aprotinin and 2 mM PMSF. These extracts 5 [0108] Polycarbonate membranes (12 mm pore size) were resolved on 12% PAGE gels, transferred onto were coated overnight with matrigel (1mg/ml) (BD Bio- PVDF nylon membranes and probed with the indicated sciences) or collagen type I (1 mg/ml) (Sigma) and air- antibodies (Santa Cruz Biotechnology) . Proteins were dried. A suspension of endothelial cells (43105/ml) pre- detected by chemiluminescence (NEN) . viously transfected with DNAzyme was placed in the up- 10 per chamber of modified Boyden chambers. Media in the Preparation of nuclear extracts and EMSA lower chamber was supplemented with FGF-2 (20 ng/ml). After a 24 h incubation at 37°C, filters were fixed [0105] Cells were scraped into ice-cold phosphate- in methanol, stained with hematoxylin and cells that had buffered saline (PBS), pelleted and resuspended in lysis migrated to the underside of the membrane were quan- 15 buffer containing 10mM Hepes, pH 7.9, 1.5mM MgCl2, titated under 400x magnification. 10mM KCI, 0.5% NP-40, 1mM DTT, 0.5mM PMSF, 4mg/ml aprotinin and 10 mg/ml leupeptin. After incubation RT-PCR on ice for 5 min, the pellets were resuspended in 20mM

Hepes, pH7.9, 1.5mM MgCl 2, 420mM NaCl, 0.2mM ED- [0109] Cells were transfected with 0.4 mM of Dz13 or TA, 1mM DTT, 0.5mM PMSF, g/ml 4m aprotinin and 20 Dz13scr 6 h after arrest. Eighteen h later, the cells were 10mg/ml leupeptin, shaking at low speed for 20 min. The incubated with TGF-beta1 (10 ng/ml, Promega) and supernatant was mixed with an equal volume of 20mM transfected again with 0.4 mM Dz13 or Dz13scr. Total Hepes, pH 7.9, 100mM KCI, 0.2mM EDTA, 20% glycerol, RNA was prepared using Trizol (Invitrogen) after 24 h. 1mM DTT, 0.5mM PMSF, 4 mg/ml aprotinin and 10mg/ml Single strand cDNA was synthesized from 4 mg total RNA leupeptin. Double-stranded oligonucleotides were 5’- 25 in a 20-ml volume reaction with 200 U reverse tran- end-labeled with (γ32P)ATP using T4 polynucleotide ki- scriptase (Superscript II), 500 mM dNTPs, and 0.5m g nase (NEB). Reactions were performed in the presence oligo (dT)15 (Life Technologies). PCR was performed in of 10mM Tris-HCl, pH7.5, 50mM NaCl, 0.5mM DTT, a 20 mL volume with 1 U DNA polymerase, 100mM

0.5mM EDTA, 1mM MgCl2, 5% glycerol, 2.5mg poly dI- dNTPs, 30 mM MgCl2 (Invitrogen) and 0.1 mM primers. dC with 150,000cpm of probe for 20min at room temper- 30 MMP-2 PCR was performed at 95°C for 30s, 57°C for ature. Bound complexes were resolved by non- denatur- 30s, and 72°C for 40s over 22 cycles. The predicted ing 8% PAGE in tris- borate-EDTA buffer system. For su- MMP-2 amplification product was 446 bp. cDNA samples pershift studies nuclear extracts were incubated with 2 mg were normalized to GAPDH (452 bp product). Primers of c-Jun antibody 10 min prior to the addition of the 32P- were as follows: MMP-2: Forward 5’- GGG ACA AGA labeled probe. 35 ACC AGA TCA CAT AC-3’, Reverse 5’-CTT CTC AAA GTT GTA GGT GGT GG-3’; GAPDH: Forward 5’-ACC Endothelial proliferation and wounding assays ACA GTC CAT GCC ATC AC-3’, Reverse 5’-TCC ACC ACC CTG TTG CTG TA-3’. [0106] Growth- quiescent endothelial cells treated with DNAzyme were incubated in medium containing serum 40 MMP-2 ELISA for 2 days prior to trypsinization, resuspension in Isoton II (Coulter Electronics) and quantitation using a Coulter [0110] Endothelial cells transfected with DNAzyme counter (Coulter Z series) . Endothelial cells transfected were incubated with 10ng/ml TGF-beta1 for 2 days in with DNAzyme were grown to confluence and injured by medium supplemented with 0.1% FBS. Conditioned me- scraping with a P200 tip. Two days after injury, the cells 45 dia was harvested, centrifuged, normalized for equal pro- were washed twice in PBS, pH 7.4, fixed in 4% parafor- tein, and levels of MMP-2 determined using commercial maldehyde (v/v), and stained in hematoxylin and eosin ELISA (Amersham Biosciences). prior to photomicroscopy. Cell numbers in the denuded zone of each group were determined under 100x mag- SDS-PAGE and gelatin zymography nification in triplicate in a blinded manner. 50 [0111] Bovine type B gelatin (Sigma) was impregnated Microtubule formation assay into a standard 10% PAGE resolving gel mixture (4% stacking) at a final concentration of 1 mg/ml. Where in- [0107] Endothelial cells were grown in 100 mm petri- dicated, endothelial cells transfected with DNAzyme dishes were transfected with DNAzyme then trypsinized 55 were co-transfected with 10 mg of a c-Jun expression and resuspended (30,000 cells per well) into 96 well vector. Equal amounts of protein were loaded and elec- plates coated with 100ml of matrigel (BD Biosciences). trophoresis was performed at 4 °C. Gels were then Microtubule formation was quantified by microscopy un- soaked in 2.5% Triton X-100 (Sigma) and incubated in

12 21 EP 1 485 109 B1 22 substrate buffer (50 mM Tris HCl, pH 7.6, 10mM CaCl 2, (Fig. 2B). One of the active DNAzymes, Dz13, targeting 1311 and 0.02% NaN3) overnight at 37°C. The gels were the G U junction (where the translational start site in stained for 1 h in 0.2% Coomassie Blue R- 250 (Bio-Rad) human c-Jun mRNA is located at A 1261UG), cleaved the in water, methanol and glacial acetic acid as 5: 4:1, then transcript within 15 min in both a time-dependent (Fig. gels were finally destained to reveal gelatinolytic activity 5 2C, upper panel) and dose-dependent (Fig. 2C, lower and photographed. panel) manner, generating 474 and 194 nt products. DNAzyme Dz13scr, in which the hybridizing arms of Immunohistochemistry Dz13 were scrambled without disturbing the integrity of the catalytic domain, failed to cleave the substrate at any [0112] Sections of formalin-fixed, paraffin-embedded 10 time or stoichiometric ratio (Fig. 2C). To demonstrate human cutaneous malignant melanoma in paraffin were Dz13 inhibition of endogenous c-Jun in primary human stained with rabbit anti-peptide polyclonal antibodies to arterial smooth muscle cells, we performed Western blot c-Jun or CD31 or goat anti-peptide antibodies to MMP- analysis on growth-quiescent cells previously transfect- 2, as previously described66. Immunoreactivity was re- ed with 0.5mM Dz13 or Dz13scr and exposed to serum vealed following incubation of the sections with either 15 for 2 h at 37°C. Serum- inducible c-Jun immunoreactivity biotinylated-secondary anti-rabbit or anti-goat antibody, (39 kDa) was strongly inhibited by Dz13, whereas its as appropriate. scrambled counterpart had no effect (Fig. 2D). [0116] All of the c-Jun DNAzymes screened targeted Rat corneal neovascularization model regions in the mRNA likely to be exposed in solution, 20 based on a Zukerian prediction of regions of low free [0113] The corneas of 7 w.o. Sprague Dawley rats energy in the mRNA, and preference for the 5’ end of the were implanted with 0.57 mm diameter nitrocellulose fil- mRNA, where the translational apparatus attaches and ter disks that had previously been soaked for at least 30 moves along the chain. The present study shows that min in 30 mM VEGF165 in 82 mM Tris-HCl (pH 6.9). Zuker analysis does not guarantee the efficacy of any DNAzyme (100 mg) or vehicle alone was subsequently 25 given DNAzyme in intact cells, since only some, but not administered into the conjunctiva adjacent to the disk fol- all the DNAzyme sequences that cleavein vitro tran- lowing implantation. Corneas were carefully removed pri- scribed c-Jun mRNA could actually inhibit cell prolifera- or to quantitation of the (i) area of occupied by neovas- tion. This may be due (although not confined) to differ- cularization (calculated as 0.2 x π x clock hours occupied ences in conformation and site accessibility between in by vascularization x maximum vessel length) and (ii) the 30 vitro transcribed mRNA and endogenous mRNA, number of vessels growing within the cornea 5 d after DNAzyme transfection efficiency, the concentration of implantation. ions and other DNAzyme cofactors in the local cellular millieu, and the possible existence of DNA-binding pro- Tumor xenograft mouse models teins (such as growth factors, signalling molecules, etc) 35 having unintended preference for certain nucleotide se- [0114] B16F10 cells (5x10 4) were injected s.c. into the quences thereby reducing the amount of bioavailable dorsal midback region of C57BL/J6 (6 w.o.) mice with DNAzyme. 750mg of DNAzyme in 200 ml of matrigel. Body weight [0117] The inability of the Zuker analysis to accurately and tumour dimensions were measured digitally at the predict DNAzyme efficiency does not hinder the design times indicated in the figure. Tumor volumes (mm 3) were 40 of effective DNAzyme. Once a particular target is select- determined using the equation length x width x height x ed, eg c- Jun it is a routine task to design and test a range 0.52. of DNAzymes which target the particular mRNA, as shown in Fig. 2. RESULTS 45 Dz13 blocks vascular smooth muscle cell prolifera- Dz13 cleaves c- Jun RNA and blocks inducible c- Jun tion expression in vascular smooth muscle cells [0118] We next determined the influence of Dz13 and [0115] Seven DNAzymes (Fig. 2A), bearing two nine the panel of c-Jun DNAzymes on the growth of primary nucleotide hybridising arms and a single 15 nt catalytic 50 vascular smooth muscle cells derived from human and motif27 targeting various regions of low free energy31 porcine arteries. The Dz13 target site in c-Jun RNA is were evaluated for their capacity to cleave32P-labeled conserved betweenhuman, pigand rat except for a single in vitro transcribed c-Jun RNA. The seven DNAzymes C nt at position 1319 which is an A in pig and rat c-Jun and c-Jun transcript were first resolved by denaturing RNA (Fig. 3A, upper and middle panels). DNAzyme cat- electrophoresis to ensure structural integrity (Fig. 2B). 55 alytic efficiency is largely unaffected by substitution of a The 668 nt c-Jun transcript was cleaved by DNAzymes single pyrimidine nt in the substrate with a purine32, as Dz10, Dz12, Dz13, Dz14 and Dz15, but not by Dz9 and in this case. Dz13 (0.5mM) completely blocked serum- Dz11 within 1 h at 37°C under physiological conditions inducible proliferation in both cell types (Figs. 3B & C)

13 23 EP 1 485 109 B1 24 and was the most potent of the entire DNAzyme panel. centration. c-Jun DNAzymes could serve as new, more Dz13 inhibition was dose-dependent and detectable at potent gene-specific tools to probe the precise function concentrations as low as 100 nM (Fig. 3D). In contrast, (s) of this transcription factor in a wide array of funda- Dz13scr failed to inhibit smooth muscle cell proliferation mental cellular processes. (Fig. 3B), consistent with its inability to affect serum-in- 5 [0121] Since c-Jun has been implicated in the patho- ducible c-Jun protein (Fig. 2D). Surprisingly, some genesis of other fibroproliferative- inflammatory process- DNAzymes (Dz9, Dz11, Dz15) stimulated proliferation es, such as arthritis39, neoplasia40, acute lung injury41, beyond the effect of serum alone (Figs. 3B & C). Addi- scarring42, UV-induced corneal damage43 and tionally, Dz10, which cleaved the c-Jun transcript as ef- osteoperosis44, DNAzymes targeting c-Jun and other fectively as Dz13 (Fig. 2B) failed to modulate smooth 10 key regulatory molecules 33 may, alone or in combination, muscle cell proliferation in either cell type, unlike Dz13 show promise in efforts to inhibit proliferative vascular (Figs. 3B & C). To demonstrate greater potency of the c- disease and other pathological processes. Jun DNAzyme compared to its exact antisense oligonu- cleotide counterpart, we generated As13 which, like Involvement of c-Jun in angiogenesis Dz13, comprises a phosphodiester backbone and a 3’- 15 3’ linked inverted T, but has no catalytic core (Fig. 3A). [0122] Microvascular endothelial cells have become As13 produced dose-dependent inhibition, however, an important target in cancer therapy, since angiogen- Dz13 was twice as potent an inhibitor (Fig. 3D). esis, the formation of new blood vessels, is an absolute requirement for tumor cell growth and metastasis. It is Dz13 inhibits vascular smooth muscle cell repair af- 20 also a key process in the pathogenesis of other common ter injury in vitro and intimal thickening in rat carotid human diseases such as arthritis and diabetic retinopa- arteries thy. Angiogenesis is a complex processes involving en- dothelial cell proliferation, migration, and microtubule for- [0119] Smooth muscle cell regrowth at the wound edge mation. following mechanical scraping in an in vitro model33 was 25 abolished by the presence of 0.5m M Dz13 (Fig. 4), Dz13inhibits c- Jun protein expression, DNA- binding whereas repair in the presence of Dz13scr was not dif- activity, migration, proliferation and tubule forma- ferent from wells without oligonucleotide (Fig. 4). Since tion by microvascular endothelial cells. smooth muscle cell proliferation and repair are processes negatively regulated by Dz13, we next determined30 [0123] c- Jun, unlike the zinc finger transcription factor whether the c-Jun DNAzyme could influence intimal Sp1, is poorly expressed in growth- quiescent human thickening after ligation injury to rat carotid arteries. The microvascular endothelial cells but is induced within 2 h arterial response to injury in rats has provided critical of exposure to serum. Dz13, a DNAzyme targeting the insights on the cellular and molecular events underlying G1311U junction in the coding region of human c- Jun the formation of lesions34. Neointima formation three 35 mRNA, blocked c- Jun protein expression at a concen- weeks after injury, and local administration of Dz13scr tration of 0.4mM. In contrast, c- Jun activation was not was not significantly different from that observed in the affected by the same concentration of Dz13scr, which vehicle alone group (Figs. 5A & B). However, intimal bears the active catalytic domain of Dz13 flanked by thickening was suppressed by Dz13 of the order of 60% scrambled 9+9 nt arms but retaining the 3’- 3’- linked (Figs. 5A & B). Immunohistochemical analysis revealed 40 inverted T that confers stability33. As13, the antisense that Dz13 blocked the induction of c- Jun immunoreactiv- oligonucleotide counterpart of Dz13 (including the 3’ in- ity in the smooth muscle cells of the arterial media, where- verted T) lacking the catalytic domain of the DNAzyme, as Dz13scr had no effect (Fig. 5C). In contrast, neither also inhibited inducible c- Jun protein expression, where- DNAzyme had any influence on levels of Sp1 (Fig. 5C). as the scrambled version, As13scr, had no effect. Sp1 Together, these data demonstrate a crucial role for c- Jun 45 levels were not changed by any of the molecules tested in smooth muscle cell proliferation, wound repair and ne- (data not shown) . ointima formation. [0124] A faint nucleoprotein complex was produced [0120] Arterial neointima formation has previously following electrophoretic mobility shift analysis using been inhibited by phosphorothioate-linked antisense ol- a 32P-labeled oligonucleotide bearing a consensus bind- igonucleotides directed against certain transcription fac- 50 ing element for c- Jun and nuclear extracts from quiescent tors and cell cycle regulatory molecules, including the microvascular endothelial cells. The intensity of this com- p65 subunit of NFκ-B35, c-myb36, c-myc37, and cdc2 ki- plex increased within 2 h of exposure to serum. Inducible nase/proliferating-cell nuclear antigen (PCNA)38. By di- DNA-binding activity was abolished either by Dz13 rectlycomparing aphosphodiester- linked DNAzyme with (0.4mM) and preincubation of the extracts with c- Jun an- an antisense oligonucleotide targeting the same se-55 tibodies (2mg), whereas Dz13scr had no effect (data not quence in c-Jun mRNA, each of identical arm length and shown). bearing a 3’-3’-inverted T, this study demonstrates su- [0125] The preceding findings revealed the capacity of perior inhibition by the former molecule at any given con- c-Jun DNAzymes to block c-Jun protein expression and

14 25 EP 1 485 109 B1 26

DNA-binding activity in a sequence- specific manner. We membrane was blocked 50% by Dz13 but not Dz13scr next determined the effect of these molecules on en- (Fig. 9B). Cell migration through filters coated with col- dothelial tubule formation, proliferation and migration. lagen type I was greater than with matrigel and also in- [0126] Endothelial cells spontaneously form a three- hibited by 50% by Dz13, but not Dz13scr (Fig. 9B). dimensional microtubular capillary-like network on 5 matrigel. Endothelial cells align on the matrigel and form Dz13 inhibits microvascular endothelial cell MMP-2 cords within hours of plating. Dz13 blocked tubulogene- mRNA, protein expression and proteolytic activity. sis in a dose-dependent manner (Fig. 6). This process was unchanged by the presence of Dz13scr (Fig. 6). [0131] Matrix metalloproteinases (MMPs), proteinas- These findings demonstrate that c- Jun is required for en- 10 es that cleave basement membrane and extracellular dothelial network formation. The target site of Dz13 and matrix molecules, are key to the process of other DNAzymes are shown in Fig. 7B. angiogenesis45. For example, mice deficient in MMP-2 [0127] Endothelial cell growth in the presence of serum (also known as gelatinase A) have compromised tumor- was inhibited by Dz13 (Fig. 8A) but not by Dz13scr (Fig. inducible angiogenesis and progression 46. We hypothe- 8A). Dz13 inhibition was sequence-specific and dose- 15 sised that Dz13 activity is mediated by its capacity to dependent maximal at 0.3m M (Fig. 8B). As13 also atten- inhibit the expression of MMP-2. Assessment of MMP-2 uated endothelial proliferation (Fig. 8A), although with mRNA and protein expression by semi-quanitative RT- less potency than the c- Jun DNAzyme at the same con- PCR and enzyme-linked immunosorbent assay, respec- centration (Fig. 8A), consistent with the effect of these tively, demonstrated reduced MMP-2 expression upon agents on the expression of c-Jun. 20 treatment with Dz13 but no change using Dz13scr (Fig. [0128] To determine effect of arm length on the biolog- 10). Analysis of MMP-2 activity secreted into the culture ical activity of Dz13, bearing 9+9 nt arms, we synthesized medium by gelatin zymography revealed significantly re- a nested series of DNAzymes with 10+10, 11+11 and duced MMP-2 proteolysis of gelatin by Dz13, which was 8+8 nt arms (each with an 3’ inverted T), together with rescued by overexpression of Jun. c- There was no their scrambled arm counterparts. At a concentration of 25 change in MMP-2 activity in the presence of Dz13scr. 0.4 mM, Dz13 (11+11) and Dz13 (8+8) inhibited endothe- lial proliferation as effectively as native Dz13 (Fig. 8C) . Dz13 inhibits VEGF165-induced neovascularization However, when these DNAzymes were used at a lower in rat cornea. concentration (0.2mM), it became apparent that Dz13 (11+11) and Dz13 (8+8) were less potent inhibitors of 30 [0132] Corneal neovascularization is a sight- threaten- endothelial proliferation than Dz13 (Fig. 8C) . Dz13 ing condition usually associated with inflammatory or in- (10+10), in comparison to Dz13, Dz13 (11+11) or Dz13 fectious disorders47. A hallmark process in corneal dis- (8+8), was a poor inhibitor. ease is the invasion of blood vessels into what is normally [0129] Western blot analysis revealed that Dz13 avascular tissue48. We evaluated the capacity of Dz13 (11+11) and Dz13 (8+8), like Dz13, inhibited serum- in- 35 to inhibit angiogenesis in rat model of corneal neovascu- ducible c- Jun expression, whereas Dz13 (10+10), Dz13 larization, a process involving MMP- 2 expression 49. Im- (11+11) scr and Dz13 (8+8) scr had little effect. Reprob- plantation of vascular endothelial growth factor ing the stripped blot with antibodies to Sp1 revealed un- (VEGF)165- soaked disks in the normally avascular rat altered levels of this nuclear protein. In support of these cornea stimulates new blood vessel growth from the lim- data, Dz13 (11+11) and Dz13 (8+8), like Dz13, cleaved 40 bus toward the implant within 5 days. This growth factor their 40 nt 32P- labeled synthetic RNA substrate in a time- is also strongly implicated in the pathogenesis of human dependent manner, whereas Dz13 (10+10), Dz13 corneal neovascularization50. Western blot analysis

(11+11 ) scr and Dz13 (8+8) scr failed to cleave (data demonstrates that VEGF165 can induce c- Jun expres- not shown) . sion and that this is blocked by Dz13 but not by Dz13scr [0130] To demonstrate a role for c- Jun in microvascu- 45 (data not shown) . Sp1 levels were unchanged. Dz13 lar endothelial cell migration, we scraped an endothelial also inhibited VTGF165- inducible microvascular tubule monolayer in vitro and quantitated the population of cells formation in vitro. Slit lamp biomicroscopic visualization in the denuded zone after 2 days, in the absence and demonstrated that Dz13 blocked the corneal angiogenic presence of DNAzyme. Dz13 inhibited this reparative re- response to VEGF165 following its conjunctival adminis- sponse to injury in a dose- and sequence-dependent 50 tration in a sequence- specific manner. Quantitative de- manner. Modest inhibition of regrowth was apparent in termination of neovascularization revealed 81% inhibi- the presence of 0.2mM Dz13 (Fig. 9A) with almost com- tion in the number of blood vessels (Fig. 11A) . Dz13 plete inhibition observed at 0.4 mM (Fig. 9A). Dz13scr did inhibited the corneal surface area occupied by these new not interfere with endothelial regrowth in this concentra- vessels by 74% (Fig. 11B) . tion range (Fig. 9A). These findings were confirmed using 55 modified Boyden chambers coated with a reconstituted Dz13 inhibits solid melanoma growth in mice. basement membrane (matrigel). Microvascular endothe- lial cell invasion through matrigel to the underside of the [0133] Aggressive melanoma lesions are associated

15 27 EP 1 485 109 B1 28 with a significant increase in blood vessel density 51. Pre- 3. Watson, L. et al. JNK and c- Jun but not ERK and vious studies have demonstrated that the in vivo growth c-Fos are associated with sustained neointima-for- of solid B16 melanoma is blocked by administration of mation after balloon injury. Eur. J. Clin. Invest. 30, anti-Flk-1 monoclonal antibodies52 and MMP-2 11-17 (2000). inhibitors53,54 indicating the dependence of tumor growth 5 on angiogenesis and matrix degradation. Immunohisto- 4. Wagner, E.F. AP-1. Oncogene 20, 2334-2335 chemical analysis of primary human cutaneous malig- (2001). nant melanoma demonstrates that c-Jun is strongly ex- pressed in endothelial cell-specific CD31+blood vessels 5. Kanatani et al., Transforming growth factor beta and in surrounding melanoma cells (data not shown). 10 and dexamethasone cooperatively enhance c-Jun Intense cytoplasmic staining in both cell types was also gene expression and inhibit the growth of human apparent using antibodies to MMP-2 (data not shown). monocytoid leukemia cells. Cell Growth Differ 1996; c-Jun expression in primary melanoma has hitherto not 7:187-196. been described. [0134] Dz13 blocked solid B16 growth in C57BL/J6 15 6. Nishio, H., Matsui, K., Tsuji, H. & Suzuki, K. Im- mice in both a time-dependent and sequence-specific munohistochemical study of the phosphoylated and manner (Fig.12A). The c-Jun DNAzyme inhibited tumor activated form of c-Jun NH2-terminal kinase in hu- growth by approximately 70% within 14 days, whereas man aorta. Histochem. J. 33, 167-171 (2001). Dz13scr-treated tumors were indistinguishable from the vehicle group (Fig. 12A). Dz13 efficacy was not associ- 20 7. Metzler, B., Hu, Y., Dietrich, H. & Xu, Q. Increased ated with any difference in body weight relative to the expression and activation of stress- activated protein other treatment groups (Fig. 12B). There was also no ev- kinases/c-Jun NH2-terminal protein kinases in idence of lethargy, ruffled fur, skin erythema, and soft atherosclerotic lesions coincide with p53. Am. J. faeces, consistent with the lack of a toxic effect. Dz13 Pathol. 156,1875-1886 (2000). blocked c-Jun protein expression (data not shown) and 25 proliferation (Fig. 12C) of murine microvascular endothe- 8. Izumi, Y. et al. Gene transfer of dominant- negative lial cells, whereas Dz13scr failed to inhibit either process. mutants of extracellular signal- regulated kinase and [0135] Strategies that target specific genes in complex c-Jun NH2-terminal kinase prevents neointimal for- biological milieu may be achieved with synthetic agents mation in balloon-injured rat artery. Circ. Res. 88 including ribozymes, minizymes, antisense oligonucle- 30 (2001). otides, RNA interference and DNAzymes. DNAzymes have been used versatile tools that tease out the precise 9. Silverman et al., Vascular smooth muscle cells functions of the targeted gene in a variety of cellular express the transcriptional corepressor NAB2 in re- processes55. These molecules have also been used as sponse to injury. Am J Pathol 1999; 155:1311-1317. inhibitors of restenosis and in- stent restenosis, process- 35 es involving vascular smooth muscle cell10. Khachigian et al., GC factor 2 represses platelet- hyperplasia33,56,57. This study has shown that derived growth factor A- chain gene transcription and DNAzymes targetingc- Jun can serve as potent inhibitors is itself induced by arterial injury. Circ Res. 1999; 11; of microvascular endothelial cell mitogenesis, migration, 84:1258-1267. corneal neovascularization and solid tumor growth. Ac- 40 cordingly, we provide here the first direct evidence for 11. Santiago et al., Induction of the transcriptional the key role of c- Jun in angiogenesis. repressor Yin Yang-1 by vascular cell injury. Auto- crine/paracrine role of endogenous fibroblast growth References factor-2. J Biol Chem. 2001;276:41143-1149. 45 [0136] 12. Pessah et al., c- Jun interacts with the corepres- sor TG-interacting factor (TGIF) to suppress Smad2 1. Ross, R., Glomset, J.A., Kariya, B. & Harker, L. A transcriptional activity. Proc Natl Acad Sci USA platelet-dependent serum factor that stimulates the 2001;98:6198-6203. proliferation of arterial smooth muscle cells in vitro. 50 Proc. Natl. Acad. Sci. USA 71, 1207-1210 (1974). 13. Dennler et al., c- Jun inhibits transforming growth factor beta-mediated transcription by repressing 2. Miano, J.M., Tota, R.R., Vlasic, N., Danishefsky, Smad3 transcriptional activity. J Biol Chem. 2000; K.J. & Stemerman, M.B. Early proto-oncogene ex- 275:28858-28865. pression in rat aortic smooth muscle cells following 55 endothelial removal. Am. J. Pathol. 137, 761-765 14. Hanahan, D., and Folkman, J. (1996). Patterns (1990). and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353-364.

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17 31 EP 1 485 109 B1 32 otides. Br. J. Ophthalmol. 79, 277-281 (1995). tanabe, F., Kawada, K., Toda, K., Ohtani, M., Sugita, K., and Yoshioka, T. (1999). Correlation of antiang- 44. Shevde, N.K., Bendixen, A.C., Dienger, K.M. & iogenic and antitumor efficacy of N-biphenyl sulfo- Pike, J.W. Estrogens suppress RANK ligand-in- nyl-phenylalanine hydroxiamic acid (BPHA), an oral- duced osteoclast differentiation via a stromal cell in- 5 ly-active, selective matrix metalloproteinase inhibi- dependent mechanism involving c-Jun repression. tor. Cancer Res 59, 1231-1235. Proc. Natl. Acad. Sci. USA 97, 7829-7834 (2000). 54. Shalinsky, D.R., Brekken, J., Zou, H., McDer- 45. Haas, T.L., and Madri, J.A. (1999). Extracellular mott, C.D., Forsyth, P., Edwards, D., Margosiak, S., matrix-driven matrix metalloproteinase production in 10 Bender, S., Truitt, G., Wood, A., Varki, N.M., and endothelial cells: implications for angiogenesis. Appelt, K. (1999). Broad antitumor and antiang- Trends Cardiovasc. Med. 9, 70-77. iogenic activities of AG3340, a potent and selective MMP inhibitor undergoing advanced oncology clini- 46. Itoh, T., Tanioka, M., Yoshida, H., Yoshioka, T., cal trials. Ann N Y Acad Sci 878, 236-270. Nishimoto, H., and Itohara, S. (1998). Reduced an- 15 giogenesis and tumor progression in gelatinase A- 55. Khachigian, L.M. (2002). DNAzymes: cutting a deficient mice. Cancer Res. 58, 1048-1051. path to a new class of therapeutics. Curr. Opin. Mol. Therap. 4,119-121. 47. Chang, J.H., Gabison, E.E., Kato, T., and Azar, D.T. (2001). Corneal neovascularization. Curr. Opin. 20 56. Lowe, H.C., Fahmy, R.G., Kavurma, M.M., Bak- Ophthalmol. 12, 242-249. er, A., Chesterman, C.N., and Khachigian, L.M. (2001). Catalytic oligodeoxynucleotides define a key 48. Klintworth, G.K. (1977). The contribution of mor- regulatory role for early growth response factor- 1 in phology to our understanding of the pathogenesis of the porcine model of coronary in-stent restenosis. experimentally produced corneal vascularization. In- 25 Circulation Research 89, 670-677. vest. Ophthalmol. Vis. Sci. 16, 281-285. 57. Lowe, H.C, Chesterman, CN., and Khachigian, 49. Kvanta, A., Sarman, S., Fagerholm, P., Sere- L.M. (2002). Catalytic antisense DNA molecules tar- gard, S., and Steen, B. (2000). Expression of matrix geting Egr-1 inhibit neointima formation following metalloproteinase-2 (MMP-2) and vascular en-30 permanent ligation of rat common cartoid arteries. dothelial growth factor (VEGF) in inflammation-as- Thromb. Haemost. 87, 134-140. sociated corneal neovascularization. Exp Eye Res 70, 419-428. 58: WO 95/02051 A (Biognostik Gesellschaft Fur Bi- omolekulare Diagnostik Mbh[DE]) 19 January 1995 50. Lai, C.M., Spilsbury, K., Brankov, M., Zaknich, 35 T., and Rakoczy, P.E. (2002). Inhibition of corneal 59: WO 98/46272 A (ISIS Pharmaceuticals, Inc. neovascularization by recombinant adenovirus me- [US]) 22 October 1998 diated antisense VEGF RNA. Exp Eye Res. 75, 625-634. 60: Pan B. et al.: "Reversal of Cisplatin resistance 40 in human ovarian cancer cell lines by a cjun anti- 51. de Waal, R.M., van Altena, M.C., Erhard, H., Wei- sense oligodoexynucleotide (ISIS 10582): evidence dle, U.H., Nooijen, P.T., and Ruiter, D.J. (1997). Lack for the role of transcription factor overexpression in of lymphangiogenesis in human primary cutaneous determining resistant phenotype" Biochemical Phar- melanoma. Consequences for the mechanism of macology, vol. 63, no. 9, 1 May 2002, pages lymphatic dissemination. Am. J. Pathol. 150,45 1699-1707 1951-1957. 61: Biswal S. et al.: "Inhibition of cell proliferation and 52. Prewett, M., Huber, J., Li, Y., Santiago, A., AP-1 activity by Acrolein in human A549 lung aden- O’Connor, W., King, K., Overholser, J., Hooper, A., ocarcinoma cells due to thiol imbalance and covalent Pytowski, B., Witte, L., Bohlen, P., and Hicklin, D.J. 50 modifications" Chemical Research In Toxicology, (1999). Antivascular endothelial growth factor recep- vol. 15, no. 2, February 2002, pages 180-186; tor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse 62: WO 01/32156 A (DALHOUSIE UNIVERSITY and human tumors. Cancer Res. 59, 5209-5218. [CA]) 10 May 2001; 55 53. Maekawa, R., Maki, H., Yoshida, H., Hojo, K., 63: Suggs W.D. et al.: "Antisense oligonucleotides Tanaka, H., Wada, T., Uchida, N., Takeda, Y., Kasai, to c-fos and c-jun inhibit intimal thickening in a rat H., Okamoto, H., Tsuzuki, H., Kambayashi, Y., Wa- vein graft model" Surgery, vol. 126,1999, pages

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443-449 ing domains are sufficiently complementary to two regions immediately flanking a purine:pyri- 64: Yoshida S. et al.: "Involvement of Interleukin-8. midine cleavage site within the c-Jun mRNA. Vascular Endothelial Growth Factor, and Basic Fi- broblast Growth Factor in Tumor Necrosis Factor al- 5 4. A nucleic acid according to claim 3, wherein the bind- pha-dependent angiogenesis" Molecular And Cellu- ing domain lengths of the DNAzyme are at least 6 lar Biology, vol. 17, no. 7, 1 July 1997, pages nucleotides. 4015-4023 5. A nucleic acid according to claim 3 or 4, wherein the 65: Buchwald A.B. et al.: "Decoy oligodeoxynucle- 10 binding domain lengths of the DNAzyme have a com- otide against Activator Protein- 1 reduces neointimal bined total length of 14 nucleotides. proliferation after coronary angioplasty in hypercho- lesterolemic minipigs" Journal Of The American Col- 6. A nucleic acid according to claim 3 or 4, wherein the lege Of Cardiology, vol. 39, no. 4, 20 February 2002, length of each binding domain of the DNAzyme is 9 pages 732-738 15 nucleotides.

66: Mercola D. and Cohen J.S.: "Antisense ap- 7. A nucleic acid according to any one of claims 3 to 6, proaches to cancer gene therapy "Cancer Gene wherein the cleavage site of the DNAzyme is a GU Therapy, vol. 2, no. 1, 1 January 1995, pages 47-59. site corresponding to nucleotides 1311-1312 of the 20 c-Jun mRNA.

Claims 8. A nucleic acid according to any one of claims 1 to 7, wherein the DNAzyme has the sequence 5’- cgggag- 1. A nucleic acid which decreases the level of c-Jun gaaGGCTAGCTACAACGAgaggcgttg-3’. mRNA, c-Jun mRNA translation or nuclear accumu- 25 lation or activity of c-Jun for use in a method of pre- 9. A nucleic acid according to any one of claims 1 to 8, venting or reducing ocular angiogenesis, or for use wherein the DNAzyme incorporates a 3’- 3’ inversion in treating or inhibiting melanoma growth, in a sub- at one or more termini. ject, wherein the nucleic acid is selected from the group consisting of: 30 10. A DNAzyme targeted against c-Jun mRNA for use in a method of treating or inhibiting atherosclerosis, (a) a DNAzyme targeted against c-Jun or a ri- restenosis, graft failure, neointima formation, or solid bozyme targeted against c-Jun, wherein the tumour growth in a subject, or preventing or reducing cleavage site of the DNAzyme or ribozyme is angiogenesis and/or neovascularisation, in a sub- within the region of residues U1296 to G1497 of 35 ject, wherein the DNAzyme comprises: c_Jun mRNA, or (b) a dsRNA targeted against c-Jun mRNA, a a. a catalytic domain which cleaves mRNA at a nucleic acid molecule which results in produc- purine:pyrimidine cleavage site; tion of dsRNA targeted against c-Jun mRNA or b. first binding domain contiguous with the 5’ end small interfering RNA molecules targeted40 of the catalytic domain; and against c-Jun mRNA, wherein the dsRNA or c. a second binding domain contiguous with the small interfering RNA targets c- Jun mRNA with- 3’ end of the catalytic domain; in the region of residues U1296 to G1497. wherein the binding domains are sufficiently com- 2. A nucleic acid according to claim 1, wherein the nu- 45 plementary to two regions immediately flanking a pu- cleicacid is a DNAzyme having a cleavage site within rine:pyrimidinecleavage sitewithin thec- JunmRNA, the region of residues U1296 to G1497 of the c- Jun and wherein the cleavage site is a GU site corre- mRNA. sponding to nucleotides 1311 to 1312 of the c-Jun mRNA. 3. A nucleic acid according to claim 2, wherein the50 DNAzyme comprises: 11. A DNAzyme according to claim 10, wherein the an- giogenesis is ocular angiogenesis. a. a catalytic domain which cleaves mRNA at a purine:pyrimidine cleavage site; 12. A DNAzyme according to claim 10, wherein the solid b. first binding domain contiguous with the 5’ end 55 tumour growth is melanoma growth. of the catalytic domain; and c. a second binding domain contiguous with the 13. A DNAzyme according to any one of claims 10 to 3’ end of the catalytic domain; wherein the bind- 12, wherein the binding domain lengths are at least

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6 nucleotides. 25. An angioplastic stent for inhibiting onset of resteno- sis comprising an angiogenic stent operably coated 14. A DNAzyme according to any one of claims 10 to with a prophylactically effective dose of a catalytic 13, wherein the binding domains have a combined nucleic acid according to any one of claims 18 to 23. length of at least 14 nucleotides. 5

15. A DNAzyme according to any one of claims 10 to Patentansprüche 13, wherein the length of each binding domain is 9 nucleotides. 1. Nucleinsäure, die das Ausmaß an c- Jun- mRNA, c- 10 Jun- mRNA- Translation oder Akkumulation oder Ak- 16. A DNAzyme according to any one of claims 10 to tivität von c- Jun im Kern senkt, zur Verwendung in 15, wherein the DNAzyme has the sequence 5’-cg- einem Verfahren zur Prävention oder Reduktion von ggaggaaGGCTAGCTACAACGAgaggcgttg-3’. Augenangiogenese oder zur Verwendung bei der Behandlung oder Hemmung von Melanomwachs- 17. A DNAzyme according to any one of claims 10 to 15 tum bei einem Individuum, worin die Nucleinsäure 16, wherein the DNAzyme incorporates a 3’- 3’ inver- aus der aus Folgendem bestehenden Gruppe aus- sion at one or more termini. gewählt ist:

18. A catalytic nucleic acid, wherein the catalytic nucleic (a) einem gegen c- Jun gerichteten DNAzym acidcleaves c- Jun mRNA in a GU site corresponding 20 oder einem gegen c- Jun gerichteten Ribozym, to nucleotides 1311-1312. worin die Spaltstelle des DNAzyms oder des Ri- bozyms innerhalb der Region der Reste U1296 19. A catalytic nucleic acid according to claim 18, where- bis G1497 von c- Jun- mRNA liegt, und in the catalytic nucleic acid is a sequence-specific (b) einer gegen c- Jun- mRNA gerichteten dsR- DNAzyme comprising: 25 NA, einem Nucleinsäuremolekül, das die Pro- duktion von gegen c- Jun- mRNA gerichteter a. a catalytic domain which cleaves mRNA at a dsRNA herbeiführt, oder gegen c- Jun- mRNA purine:pyrimidine cleavage site; gerichteten small- interfering- RNA- Molekülen, b. first binding domain contiguous with the 5’ end worin die dsRNA oder die small- interfering- of the catalytic domain; and 30 RNA innerhalb der Region der Reste U1296 bis c. a second binding domain contiguous with the G1497 gegen c- Jun- mRNA gerichtet ist. 3’ end of the catalytic domain; 2. Nucleinsäure nach Anspruch 1, worin die Nuclein- wherein the binding domains are sufficiently com- säure ein DNAzym mit einer Spaltstelle innerhalb plementary to two regions immediately flanking a pu- 35 der Region der Reste U1296 bis G1497 der c-Jun- rine:pyrimidine cleavage site within the c- Jun mRNA mRNA ist. such that the DNAzyme cleaves c- Jun mRNA in the GU site corresponding to nucleotides 1311-1312. 3. Nucleinsäure nach Anspruch 2, worin das DNAzym Folgendes umfasst: 20. A catalytic nucleic acid according to claim 19, where- 40 in the binding domain lengths are at least 6 nucle- a.eine katalytische Domäne,die mRNAan einer otides. Purin-Pyrimidin-Spaltstelle spaltet; b. eine erste Bindedomäne, die mit dem 5’- Ende 21. A catalytic nucleic acid according to claim 20, where- der katalytischen Domäne zusammenhängt; in the length of each binding domain is 9 nucleotides. 45 und c. eine zweite Bindedomäne, die mit dem 3’- En- 22. A catalytic nucleic acid accoding to any one of claims de der katalytischen Domäne zusammenhängt; 18 to 21, having the sequence cgggaggaaG- 5’- GCTAGCTACAACGAgaggcgttg-3’. worin die Bindedomänen ausreichend komplemen- 50 tär zu zwei eine Purin-Pyrimidin-Spaltstelle inner- 23. A catalytic nucleic acid according to any one of halb der c- Jun-mRNA unmittelbar flankierenden Re- claims 18 to 22, wherein the DNAzyme incorporates gionen sind. a 3’-3’ inversion at one or more termini. 4. Nucleinsäure nach Anspruch 3, worin die Länge der 24. A pharmaceutical composition comprising the cata- 55 Bindedomänen des DNAzyms zumindest 6 Nucleo- lytic nucleic acid according to any one of claims 18 tide beträgt. to 23 and a pharmaceutically acceptable carrier. 5. Nucleinsäure nachAnspruch 3 oder4, worin die Län-

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ge der Bindedomänen des DNAzyms eine kombi- 15. DNAzym nach einem der Ansprüche 10 bis 13, worin nierte Gesamtlänge von 14 Nucleotiden ausmacht. die Länge jeder Bindedomäne 9 Nucleotide beträgt.

6. Nucleinsäure nach Anspruch 3 oder 4, worin die Län- 16. DNAzym nach einem der Ansprüche 10 bis 15, worin ge jeder Bindedomäne des DNAzyms 9 Nucleotide 5 das DNAzym die Sequenz cgggaggaaGGC- 5’- beträgt. TAGCTACAACGAgaggcgttg-3’ aufweist.

7. Nucleinsäure nach einem der Ansprüche 3 bis 6, 17. DNAzym nach einem der Ansprüche 10 bis 16, worin worin die Spaltstelle des DNAzyms eine GU-Stelle das DNAzym eine 3’-3’-Inversion an einem oder ist, die den Nucleotiden 1311 bis 1312 der c-Jun- 10 mehreren Termini inkorporiert. mRNA entspricht. 18. Katalytische Nucleinsäure, worin die katalytische 8. Nucleinsäure nach einem der Ansprüche 1 bis 7, Nucleinsäure c-Jun-mRNA an einer GU-Stelle spal- worin das DNAzym die Sequenzcgggag- 5’- tet, die den Nucleotiden 1311 bis 1312 entspricht. gaaGGCTAGCTACAACGAgaggcgttg-3’ aufweist. 15 19. Katalytische Nucleinsäure nach Anspruch 18, worin 9. Nucleinsäure nach einem der Ansprüche 1 bis 8, die katalytische Nucleinsäure ein sequenzspezifi- worin das DNAzym eine 3’-3’-Inversion an einem sches DNAzym ist, das Folgendes umfasst: oder mehreren Termini inkorporiert. 20 a.eine katalytische Domäne,die mRNAan einer 10. DNAzym, gerichtet gegen c- Jun- mRNA, zur Ver- Purin-Pyrimidin-Spaltstelle spaltet; wendung in einem Verfahren zur Behandlung oder b. eine erste Bindedomäne, die mit dem 5’- Ende Hemmung von Atherosklerose, Restenose, Trans- der katalytischen Domäne zusammenhängt; plantatversagen, Neointima- Bildung oder dem und Wachstum eines soliden Tumors bei einem Indivi- 25 c. eine zweite Bindedomäne, die mit dem 3’- En- duum oder zur Prävention oder Reduktion von An- de der katalytischen Domäne zusammenhängt; giogenese und/ oder Gefäßneubildung bei einem In- dividuum, worin das DNAzym Folgendes umfasst: worin die Bindedomänen ausreichend komplemen- tär zu zwei eine Purin-Pyrimidin-Spaltstelle inner- a. eine katalytische Domäne, die mRNA an einer 30 halb der c- Jun-mRNA unmittelbar flankierenden Re- Purin- Pyrimidin- Spaltstelle spaltet; gionen sind, sodass das DNAzym c-Jun-mRNA an b. eine erste Bindedomäne, die mit dem 5’- Ende der GU-Stelle spaltet, die den Nucleotiden 1311 bis der katalytischen Domäne zusammenhängt; 1312 entspricht. und c. eine zweite Bindedomäne, die mit dem 3’- En- 35 20. Katalytische Nucleinsäure nach Anspruch 19, worin de der katalytischen Domäne zusammenhängt; die Länge der Bindedomänen zumindest 6 Nucleo- tide beträgt. worin die Bindedomänen ausreichend komplemen- tär zu zwei eine Purin-Pyrimidin-Spaltstelle inner- 21. Katalytische Nucleinsäure nach Anspruch 20, worin halb der c- Jun-mRNA unmittelbar flankierenden Re- 40 die Länge jeder Bindedomäne 9 Nucleotide beträgt. gionen sind und worin die Spaltstelle eine GU- Stelle ist, die den Nucleotiden 1311 bis 1312 der c-Jun- 22. Katalytische Nucleinsäure nach einem der Ansprü- mRNA entspricht. che 18 bis 21 mit der Sequenz 5’-cgggaggaaGGC- TAGCTACAACGAgaggcgttg-3’. 11. DNAzym nach Anspruch 10, worin die Angiogenese 45 Augenangiogenese ist. 23. Katalytische Nucleinsäure nach einem der Ansprü- che 18 bis 22, worin das DNAzym eine 3’-3’-Inver- 12. DNAzym nach Anspruch 10, worin das Wachstum sion an einem oder mehreren Termini inkorporiert. eines soliden Tumors Melanomwachstum ist. 50 24. Pharmazeutische Zusammensetzung, die eine ka- 13. DNAzym nach einem der Ansprüche 10 bis 12, worin talytische Nucleinsäure nach einem der Ansprüche die Länge der Bindedomänen zumindest 6 Nucleo- 18 bis 23 und einen pharmazeutisch annehmbaren tide beträgt. Träger umfasst.

14. DNAzym nach einem der Ansprüche 10 bis 13, worin 55 25. Angioplastischer Stent zur Hemmung des Aus- die Bindedomänen eine kombinierte Länge von zu- bruchs von Restenose, der einen angioplastischen mindest 14 Nucleotiden aufweisen. Stent umfasst, der operabel mit einer prophylaktisch wirksamen Dosis einer katalytischen Nucleinsäure

21 39 EP 1 485 109 B1 40

nach einem der Ansprüche 18 bis 23 überzogen ist. 7. Acide nucléique selon l’une quelconque des reven- dications 3 à 6, où le site de clivage de l’ADNzyme est un site GU correspondant aux nucléotides Revendications 1311-1312 de l’ARNm de c-Jun. 5 1. Acide nucléique qui réduit le taux de l’ARNm de c- 8. Acide nucléique selon l’une quelconque des reven- Jun, la traduction de l’ARNm de c-Jun ou l’accumu- dications 1 à 7, où l’ADNzyme possède la séquence lation nucléaire ou l’activité de c-Jun, destiné à être 5’-cgggaggaaGGCTAGCTACAACGAgaggcgttg-3’. utilisé dans un procédé de prévention ou de réduc- tion de l’angiogenèse oculaire, ou destiné à être uti- 10 9. Acide nucléique selon l’une quelconque des reven- lisé dans le traitement ou l’inhibition de la croissance dications 1 à 8, où l’ADNzyme incorpore une inver- d’un mélanome, chez un sujet, où l’acide nucléique sion 3’-3’ à une ou plusieurs terminaisons. est sélectionné dans le groupe consistant en : 10. ADNzyme ciblée contre l’ARNm de c-Jun destinée (a) une ADNzyme ciblée contre c-Jun ou un ri- 15 à être utilisée dans un procédé de traitement ou d’in- bozyme ciblé contre c-Jun, où le site de clivage hibition de l’athérosclérose, la resténose, l’échec de de l’ADNzyme ou du ribozyme est dans la région greffe, la formation de la néo- intima, ou la croissance des résidus U1296 à G1497 de l’ARNm de c- d’une tumeur solide chez un sujet, ou de prévention Jun, ou ou de réduction de l’angiogenèse et/ou de la néo- (b)un ARNdb ciblé contre l’ARNm de c- Jun, une 20 vascularisation, chez un sujet, où l’ADNzyme molécule d’acide nucléique qui entraîne la pro- comprend : duction d’un ARNdb ciblé contre l’ARNm de c- Jun ou des molécules de petit ARN interférent a. un domaine catalytique qui clive l’ARNm au ciblées contre l’ARNm de c- Jun, où l’ARNdb ou niveau d’un site de clivage purine:pyrimidine ; le petit ARN interférent cible l’ARNm de c-Jun 25 b. un premier domaine de liaison contigu à l’ex- dans la région des résidus U1296 à G1497. trémité 5’ du domaine catalytique ; et c. un second domaine de liaison contigu à l’ex- 2. Acide nucléique selon la revendication 1, où l’acide trémité 3’ du domaine catalytique ; nucléique est une ADNzyme comportant un site de clivage dans la région des résidus U1296 à G1497 30 où les domaines de liaison sont suffisamment com- de l’ARNm de c-Jun. plémentaires à deux régions flanquant immédiate- ment un site de clivage purine: pyrimidine au sein de 3. Acide nucléique selon la revendication 2, où l’ADN- l’ARNm de c-Jun, et où le site de clivage est un site zyme comprend : GU correspondant aux nucléotides 1311-1312 de 35 l’ARNm de c-Jun. a. un domaine catalytique qui clive l’ARNm au niveau d’un site de clivage purine:pyrimidine ; 11. ADNzyme selon la revendication 10, où l’angioge- b. un premier domaine de liaison contigu à l’ex- nèse est l’angiogenèse oculaire. trémité 5’ du domaine catalytique ; et c. un second domaine de liaison contigu à l’ex- 40 12. ADNzyme selon la revendication 10, où la croissan- trémité 3’ du domaine catalytique ; ce d’une tumeur solide est la croissance d’un méla- nome. où les domaines de liaison sont suffisamment com- plémentaires à deux régions flanquant immédiate- 13. ADNzyme selon l’une quelconque des revendica- ment un site de clivage purine: pyrimidine au sein de 45 tions 10 à 12, où les longueurs des domaines de l’ARNm de c-Jun. liaison sont d’au moins 6 nucléotides.

4. Acide nucléique selon la revendication 3, où les lon- 14. ADNzyme selon l’une quelconque des revendica- gueurs des domaines de liaison de l’ADNzyme sont tions 10 à 13, où les domaines de liaison ont une d’au moins 6 nucléotides. 50 longueur combinée d’au moins 14 nucléotides.

5. Acide nucléique selon la revendication 3 ou 4, où les 15. ADNzyme selon l’une quelconque des revendica- longueurs des domaines de liaison de l’ADNzyme tions 10 à 13, où la longueur de chaque domaine de ont une longueur totale combinée de 14 nucléotides. liaison est de 9 nucléotides. 55 6. Acide nucléique selon la revendication 3 ou 4, où la 16. ADNzyme selon l’une quelconque des revendica- longueur de chaque domaine de liaison de l’ADNzy- tions 10 à 15, où l’ADNzyme possède la séquence me est de 9 nucléotides. 5’-cgggaggaaGGCTAGCTACAACGAgaggcgttg-3’.

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17. ADNzyme selon l’une quelconque des revendica- tions 10 à 16, où l’ADNzyme incorpore une inversion 3’-3’ à une ou plusieurs terminaisons.

18. Acide nucléique catalytique, où l’acide nucléique ca- 5 talytique clive l’ARNm de c- Jun dans un site GU cor- respondant aux nucléotides 1311-1312.

19. Acide nucléique catalytique selon la revendication 18, où l’acide nucléique catalytique est une ADNzy- 10 me spécifique d’une séquence comprenant :

a. un domaine catalytique qui clive l’ARNm au niveau d’un site de clivage purine:pyrimidine ; b. un premier domaine de liaison contigu à l’ex- 15 trémité 5’ du domaine catalytique ; et c. un second domaine de liaison contigu à l’ex- trémité 3’ du domaine catalytique ;

où les domaines de liaison sont suffisamment com- 20 plémentaires à deux régions flanquant immédiate- ment un site de clivage purine: pyrimidine au sein de l’ARNm de c-Jun, de sorte que l’ADNzyme clive l’ARNm de c- Jun dans le site GU correspondant aux nucléotides 1311-1312. 25

20. Acide nucléique catalytique selon la revendication 19, où les longueurs des domaines de liaison sont d’au moins 6 nucléotides. 30 21. Acide nucléique catalytique selon la revendication 20, où la longueur de chaque domaine de liaison est de 9 nucléotides.

22. Acide nucléique catalytique selon l’une quelconque 35 des revendications 18 à 21, ayant la séquence 5’- cgggaggaaGGCTAGCTACAACGAgaggcgttg-3’.

23. Acide nucléique catalytique selon l’une quelconque des revendications 18 à 22, où l’ADNzyme incorpore 40 une inversion 3’-3’ à une ou plusieurs terminaisons.

24. Composition pharmaceutique comprenant l’acide nucléique catalytique selon l’une quelconque des re- vendications 18 à 23 et un véhicule pharmaceuti- 45 quement acceptable.

25. Stent d’angioplastie destiné à inhiber la survenue d’une resténose, comprenant un stent angiogénique revêtu de manière fonctionnelle avec une dose pro- 50 phylactiquement efficace d’un acide nucléique cata- lytique selon l’une quelconque des revendications 18 à 23.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

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