(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.