WO2001004290A1

WO2001004290A1 - Triplex-forming oligonucleotides and their use in therapy

Info

Publication number: WO2001004290A1
Application number: PCT/GB2000/002645
Authority: WO
Inventors: You-Xin Jin; Jing Liu
Original assignee: Center for Excellence in Molecular Cell Science of CAS; GENEMEDIX PLC
Current assignee: Center for Excellence in Molecular Cell Science of CAS; GENEMEDIX PLC
Priority date: 1999-07-09
Filing date: 2000-07-10
Publication date: 2001-01-18
Anticipated expiration: 2002-01-09
Also published as: AU5996100A

Abstract

A polynucleotide is capable of selectively binding to the double-stranded promoter region of the c-sis/PDGF-B proto-oncogene to form a triplex, thereby inhibiting the function of the promoter and affecting expression of the proto-oncogene.

Description

TRIPLEX-FORMING OLIGONUCLEOTLDES AND THEIR USE LN THERAPY

Field of the Invention 5 The present invention concerns triplex-forming oligonucleotides and their potential use in gene therapy. Background to the Invention

Platelet-derived growth factor (PDGF) is a ubiquitous, potent mitogen and chemotactic factor for many connective tissue cells that occurs as a three-disulfide-linked o dimer composed of two homologous chains, A and B. Ever since the finding that the v- sis oncogene of the simian sarcoma virus is a retroviral homolog of the cellular gene encoding the B chain of PDGF, in 1983, much attention has been focused on the relationship between PDGF-B expression and malignant transformation. The hypothesis that unscheduled production of PDGF may contribute to the growth of spontaneous 5 tumors is supported by the finding that PDGF is frequently produced by cell lines from human tumors, such as gliblastoma and fibrosarcoma, melanoma, breast carcinoma, lung carcinoma, glioma, esophageal carcinoma, and Kaposi's sarcoma. Gene transfer experiments have shown that overexpression of the normal human PDGF-B gene ( c-sis, proto-oncogene) can cause the generation of fibrosarcoma, vascular connective tissue 0 stroma with no necrosis and tumorigenic and metastatic effects. Furthermore, PDGF has been implicated in the pathogenesis of several non-malignant proliferative diseases including atherosclerosis, fibrosis, restenosis following vascular angioplasty, giant cell arteritis, aseptic loosening and bronchiolitis obliterans syndrome.

The accumulating evidence for the involvement of PDGF in human proliferative 5 disorders has led to a search for specific inhibition of PDGF expression. Currently, antibodies to PDGF and soluble PDGF receptors are the most potent and specific antagonists of PDGF. Other inhibitors of PDGF, such as suramin, neomycin and peptides derived from the PDGF amino acid sequence, have been reported, but, they are either too toxic or lack sufficient specificity or potency to be good drug candidates. Another class o of antagonists of possible clinical utility is tyrphostins that selectively inhibit the PDGF receptor tyrosine kinase.

Oligonucleotides offer enormous potential for manipulating gene function in cells and, as such, constitute a promising new class of pharmaceutical agents. Parallel developments in oligonucleotide technology now offer several alternatives for manipulating gene functions inside cells. Oligonucleotides have been developed that bind site-selectively to DNA (antigene or triplex-forming oligonucleotides ( TFOs ) ), to RNA (antisense and ribozyme oligonucleotides) and to proteins (aptamers). Antisense oligonucleotides, ribozymes and aptamers have been reported to inhibit PDGF expression

Oligonucleotide-directed triple-helix formation involves a pyrimidine-rich (pyrimidine motif, T. A-T and C⁺G-C triplets ) or a purine-rich ( purine motif; T. A-T and G.G-C triplets ) oligonucleotide binding to a homopurine sequence in the major groove of double-helical DNA. This method allows highly sequence-specific DNA recognition. Stabilization of pyrimidine motif triple-helical complexes by cytosine protonation generally requires slightly acidic pH. This requirement does not pertain to the purine motif. Triple-helical complexes are thermodynamically stable near physiological conditions, with half-lives of several hours.

TFO sequence-specific recognition of duplex DNA is a potentially powerful approach to diminishing expression of targeted genes in cells. Oligonucleotide-directed DNA triple-helix formation continues to present an interesting strategy for creating artificial, sequence-specific regulators of DNA function. Summary of the Invention

The present invention is directed to a polynucleotide capable of selectively binding to the double-stranded promoter region of the c-sis/PDGF-B proto-oncogene to form a triplex, thereby inhibiting the function of the promoter and affecting expression of the proto-oncogene The polynucleotide may be useful in therapy, for example, in the preparation of medicaments to inhibit tumour growth and for therapy of atherosclerosis, inflammation, etc. This may result from the ability of the polynucleotide to bind the double-stranded promoter region, thus preventing nuclear factors from binding to the promoter and initiating transcription. Description of the Invention

The present invention is based on the discovery that triplex-forming oligonucleotides (TFOs) can be used to selectively inhibit the c-sis proto-oncogene that codes for human platelet derived growth factor B (PDGF-B).

TFOs are known in the art, as is their ability to selectively bind to double-stranded DNA. The present invention is directed to novel TFOs that are capable of selectively- binding to the double-stranded promoter region of the c-sis proto-oncogene, to form a triplex. It is preferred that the TFO is either a purine-rich or a pyrimidine-rich polynucleotide. More preferably, the TFO is a purine-rich polynucleotide, as pyrimidine- rich polynucleotides typically require acidic conditions in order to protonate the cytosine residue, prior to forming a C⁺G-C motif.

Any TFO that is capable of selectively binding to the c-sis promoter region is understood to be within the scope of the present invention. Such a TFO may substantially consist of purine residues, but may incorporate pyrimidine residues, where appropriate. The skilled person, in attempting to develop additional TFOs according to the present invention, is easily capable of designing and testing TFOs in the light of the methods disclosed herein. The TFO should bind to duplex DNA to form a triplex, and should inhibit the binding of nuclear factors.

By way of example only, a TFO may be complementary to the pyrimidine-rich region (PRR) of the c-sis promoter (SEQ . LD. No 3). Additionally, a TFO may include additional nucleotides that are complementary to part or all of the adjacent SIS proximal element (SPE) region of the promoter (SEQ. LD. No 2). The TFO may include pyrimidine residue complementary to a purine in the SPE, or a complementary purine residue may be used, or another pyrimidine or purine. For example, at position 17 of SEQ. LD. No 2, where the complementary nucleotide should be C, a pyrimidine, the TFO may include C, G, A or T. More preferably, the nucleotide is G or T. Similarly, at position 19 of SEQ. LD. No 2, the nucleotide may be A, T, C or G. More preferably, the nucleotide is A or T.

The invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

Fig. 1. Schematic diagram of the human c-sis PDGF-B gene promoter showing: the locations of the triplex target sequence, SPE (-58 bp to -39 bp) and PRR (-38 to -24); the TATA box (-24 to -20); and the TATA neighboring sequence (TNS) (-18 to -9);

Fig. 2. Electrophoretic mobility shift assays. (A) TFO1. (B) TFO-p triplex formation with the synthetic 15-bp PRR duplex in 20mM Sodium cacodylate-HCl (pH 7.4), 1 OmM MgCl₂. From Lane 1 to 7, 0, 0.1, 0.2, 0.4, 1.6, 6.0 and 12μM TFOl or control TFO-p were added to l .OμM ³²P-labeled 15-bp PRR duplex. (C) Triplex formation by equal amount of TFO2-7 with the synthetic 35-bp-SPE-PRR duplex. Lane 2-7, 12μM TFO2-7 were added to l .OμM ³²P-labeled 35-bp-SPE-PRR duplex relatively. Lane 1, duplex control without TFOs. (D) TFO8. (E) TFO9. (F) TFO10 triplex formation with the synthetic 35-bp-SPE-PRR duplex. From Lane 1 to 6, 0, 0.2, 0.8, 3.0, 6.0, 12μM TFOs were added to l .OμM ³²P-labeled 35-bp-SPE-PRR duplex, t: triplex; d: duplex; Fig. 3. DNase I footprinting analysis demonstrating sequence specific binding of TFO5 to the 262 bp target duplex containing SPE and PRR. Lane 1, no TFOs; Lane 2-6, 1, 5, 10, 15, 20μM TFO8; Lane 7, 20μM TFO2 were incubated with 30nM 262-bp ³²P-labeled fragment followed by limited DNase I digestion;

Fig. 4. Synthesis of the 255-bp c-sis/PDGF-B promoter by the technique of oligonucleotide overlap extension and PCR amplification. The sequences of oligonucleotides A, B, C, D, E, F, primer 1, primer2 were as follows: A: 5' C CCATG GTCAC TGTGC TGAGG GGCGG GACGG TGGGT CACCC CTAGT TCTTT TTTCC CCAGG GCCAG ATTCA TGGAC TGAAG GGTTG 3'; B: 5' C CTTCA GTCCA TGAAT CTGGC CCTGG GGAAA AAAGA ACTAG GGGTG ACCCA CCGTC CCGCC CCTCA GCACA GTGAC CATGG GAGCT 3'; C: 5' CTCGG CTCTC AGAGA CCCCC TAAGC GCCCC GCCCT GGCCC CAAGC CCTCC CCCAG CTCCC GCGTC CCCCC CCTCC TGGCG CTGAC 3'; D: 5' G CGCCA GGAGG GGGGG GACGC GGGAG CTGGG GGAGG GCTTG GGGCC AGGGC GGGGC GCTTA GGGGG TCTCT GAGAG CCGAG CAAC 3'; E: 5' TCCGG GCCAG AAGAG GAAAG GCTGT CTCCA CCCAC CTCTC GCACT CTCCC TTCTC CTTTA TAAAG GCCGG AACAG CTGAA AGGGT CCC 3'; F: 5' GGG ACCCT TTCAG CTGTT CCGGC CTTTA TAAAG GAGAA GGGAG AGTGC

GAGAG GTGGG TGGAG ACAGC CTTTC CTCTT CTGGC CCGGA GTCA 3'; primerl: 5' ATTCG AGCTC CCATG GTCAC TGTGC 3'; primer2: 3' TTGTC GACTT TCCCA GGGCC CCTAG 5'. Oligonucleotides A-F were 5' phosphorylated with ATP by T4 kinase and A-B, C-D and E-F were annealed separately before ligation with T4 DNA ligase. The resulting 255-bp fragment was amplified by PCR using primer

1 and primer 2; Fig. 5. Electrophoretic mobility shift analysis of the effect of TFOs on the binding of nuclear factors present in PMA-treated K562 cells to the target region of the c- sis/PDGF-B promoter. Radiolabeled duplex target, the 255bp promoter fragment isolated from the puclδpromoter, (0.1 μM) was preincubated 50μM TFO2 (lane 1), no TFOs ( lane2-4) increasing concentration (1, 10, 50μM ) of single-stranded TFO 1 ( lanes 5-7), TFO8 ( lanes 8-10) and TFO9 ( lanes 11-13), Samples were then incubated with the nuclear extracts from PMA-treated K562 cells except in lane 2, which containing labeled duplex only. In lane 3, 20-fold more unlabeled duplex target was added, a, b: protein-DNA complexes; c: free duplex probe; and Fig. 6. Luciferase assays demonstrating specific inhibition of c-sis/PDGF-B transcription. (A) Transient transfection. (B) Stable transfection. Oligonucleotides: All oligonucleotides used with or without modification were synthesized on a Beckman-OligolOOO DNA synthesizer and purified by preparative 15- 20% polyacrylamide gel electrophoresis. Double-stranded oligonucleotides were prepared by mixing equal amounts of complementary single strands in the presence of 0.25M NaCl. The mixtures were annealed and gel-purified on a 12% polyacrylamide gel, eluted, and concentrated by ethanol precipitation.

Vectors: pucl δSPE was constructed by inserting the synthetic 38bp double- stranded oligonucleotides (containing both SPE and PRR): C TCTCC ACCCA CCTCT CGCACTCTCC CTTCTC CTTTCCC

TCGAGAGAGGTGGGTGGAGAGCGTGAGAGGGAAGAGGAAAGGG between Sad and Smal site of pucl δ. Plasmid pGL3promoter was constructed as follows: A synthetic PDGF-B promoter (-252-+3) was constructed by a technique of oligonucleotide overlap extension and amplification by PCR (Fig. 4). The resulting 255bp fragment was cloned between a Sad and Smal site of pucl8 to give puclδpromoter, sequenced, then subcloned into the Sacl-Hindlll site of the pGL3 Basic vector (Promega). PBluecat was a gift from Dr. Kan Liao. Digestion with Hindlll released a 1.5 kb neo^rgene fragment, which was purified through agarose gel electrophoresis and ligated into Hindlll-digested pBSK(+) to give pneo^rBSK(+). To create neo^rpGL3 promoter and neo^rpGL3 control, pneo^rBSK(+) was digested with Kpnl and Sad to release the neo^r gene fragment, which was in turn subcloned into the Kpnl-Sacl site of pGL3 promoter and pGL3 control ( Promega ).

Cells: Human K562 erythroleukemia cells were obtained from American Type Culture Collection (CCL243, Rockville, MD) and maintained in complete RPMI (RPMI 1640 supplemented with 10% fetal bovine serum, ImM L-glutamine, lOmM hepes, lOOu/ml penicillin and 50μg/ml of streptomycin) in a 37°C /5 % CO₂ incubator. Selection of Target sites and Design of Triplex-forming Oligonucleotides: The human c-sis/PDGF-B promoter contains a 20-bp purine-pyrimidine rich sequence (SPE) located at -58 to -39 from the transcription start site. This region is the binding site for the Sp family of transcription factors. The sequence of this region has a run of 16 pyrimidines interrupted by three T-As and one C-G, while the 15bp sequence downstream (PRR) is strictly homopurine/homopyrimidine run (Fig. 1). A series of purine motif TFOs of different sequence or length was designed (SEQ ID NOS. 4-15) A 16-mer TFO1 was designed to target the perfect 15-bp PRR. Guanine residues were placed in the oligonucleotide in apposition to each G-C pair in the target (G:GC triplet) and adenine residues in apposition to each A-T pair ( A:AT triplet). Eight 25-mers TFO2-9 were designed to target part of the SPE and the entire PRR, while 35-mer TFO 10 was designed to span the entire SPE and PRR. At each interrupt site in TFO2-9, adenine or thymine or guanine or cytosine was aligned with the C-G (A: CG or T: CG or G: CG or C: CG triplets) and adenine or thymine was aligned with the T-A (A:TA or T:TA triplets). TFOl-10 were anti-parallel in orientation to the target purine strand, while another TFO, TFO-p, with the same sequence as TFO1, was in parallel orientation. Triplex Formation with the c-sis/PDGF-B promoter: Triplex formation was demonstrated by gel mobility shift analysis and DNase I footprinting. Gel Mobility Shift Analysis of Triplex Formation: the synthetic coding strand of PRR

(15bp) or SPE-PRR (35bp) triplex target sequence was ³²P-end-labeled with (γ-³²P)ATP by T4 kinase and annealed to its oligonucleotide complement. The resulting double- stranded oligonucleotides were gel-purified on a 12% polyacrylamide gel, eluted, and concentrated by ethanol precipitation. Potential TFOs were heated at 95 °C for 3 min to avoid self-aggregation of the G-rich oligonucleotides, then cooled on ice before adding to the labeled 15bp or 35bp targets in 20mM Sodium cacodylate-HCl (pH 7.4), lOmM MgCl₂ and incubated at 4°C overnight Samples were analyzed by electrophoresis on 10- 15% native polyacrylamide gels at 4°C at 100 V for 4h Gels were dried and autoradiographed at -70°C Both gel and running buffer contained 90mM Tris borate (pH 8 0) , 10mM MgCl₂ Gel mobility shift analysis Triplex DNA migrates more slowly than duplex because of its decreased charge density First, the 16mer TFOs in antiparallel (TFO1) and parallel (TFO-p) orientation were used to bind PRR (15bp duplex target). The results are shown in Fig 2 A and B respectively As expected, increasing concentrations of TFO 1 shifted the 15-bp-duplex target (d) to a distinct, more slowly migrating band (t), indicating the complete formation of triplex DNA at 12 OμM concentration (12-fold molar excess relative to target) More than 30% of the duplex was converted to triplex at 0 8μM and 50% at 1 6 μM (by darkness scanning) In contrast, the control, TFO-p, with the identical sequence but in a parallel orientation to the target purine strand, did not show any triplex formation under the same conditions Then, eight 25-mer TFO2-9 were screened for the best choice for triplex forming

TFO2-7, nearly have no effect on the target at the same concentration (Fig. 2C), while increasing concentration of TFO8 and TFO9, respectively, shifted the 35-bp-duplex to triplex, at 12μM concentration, TFO8 shifted 100% and TFO9 shifted 50% duplex to triplex At higher concentration (more that 5 OμM) TFO2-7 can bind part of the target(data not shown) According to SEQ LD NOS 12 and 13 , at the C-G interrupt site,

A or C or G was placed in TFO2-7, while T was placed in TFO 8, 9 TFO8 had an A while TFO9 had a T against the TA interrupt site These results indicate that single base change in TFO sequence at the interrupting site will dramatically reduce its ability to bind it's the duplex target, which further shows the sequence specificity of TFOs Therefore, another 35-mer TFO 10 was designed with a T at the C-G conversion site and A at the three T-A conversion sites As shown in Fig 2F, increasing concentration of TFO 10 can shift the target duplex to triplex and at 12μM concentration, TFO 10 shifted 100% duplex to triplex DNase I Footprinήng: a 262bp fragment with HindIII(399) and PvuII(628) sites containing the SPE ( 439-444 ) was isolated from puclδSPE This fragment was end- labeled at the Hindlll site with (α-³²P)dATP using the Klenow fragment of Escherichia coli DNA polymerase I. After incubated at 55°C for 5 min then cooling on ice, the labeled 262bp fragment was incubated with TFO2 and increasing concentrations of TFO8, which have been heated at 95 °C for 3 min and cooled on immediately, in 20mM Sodium cacodylate-HCl (pH 7.4), lOmM MgCl₂ at 4°C overnight. Samples were precooled on ice, then digested with DNase I for 40s on ice. Digestion was terminated by the addition of 20mM EDTA in 98% formamide followed by heating at 95°C for 5 min to inactivate DNase I. Samples were quickly cooled on ice, then analyzed by electrophoresis on a 8M urea, 9% polyacrylamide sequencing gel at 50 watts. The gel was dried and autoradiographed at -70 °C. DNase I footprinting experiments support the results obtained from gel mobility shift analysis and confirm that triplex formation occurs in a sequence specific manner. As shown in Fig. 3, protection of the target sequence by TFO8 is concentration dependent in a manner consistent with the gel mobility shift analysis. At oligonucleotide concentration of 20μM (-700 fold molar excess with respect of the 262-bp target duplex), the antiparallel TFO8 yields DNase I protection patterns documenting complete protection of the 25-bp target sequence. TFO2 was used as negative control since it has been proven in the gel mobility shift analysis to bind the duplex poorly. As shown in lane7, 20μM TFO2 has no effect on the protection of target sequence. These data further suggest that triplex formation is completely sequence-specific. Protein Binding Assays: Nuclear extracts from PMA-treated K562 cells were prepared according to the method of Dignam et al. [55 ] . A 255-bp fragment of the human PDGF-B gene promoter (-252-+3) with EcoRI and Smal ends isolated from pucl δpromoter was labeled with (α-³²P)dATP at EcoRI site using the Klenow fragment of Escherichia coli DNA polymerase I. 0.1 μM (approximately 20,00 cpm) was incubated with increasing concentrations of TFOs ( l-50μM) in 3μl 20mM Sodium cacodylate-HCl (pH 7.4), lOmM MgCl₂. After preincubation at 4°C overnight, 6μg PMA-treated K562 cells nuclear extracts were added, resulting in a final volume of lOμl containing 20 mM Tris.HCl(pH8.0), 5mM MgCl₂, 5mM CaCl₂, O. lmM EDTA, O. lmM DTT, 0.5 μg BSA, lOOmMKCl, 3% glycerol, and lμg Poly(dl-dC). The DNA-protein mixtures were kept at room temperature for 15min, Followed by electrophoresis in a 5% polyacrylamide gel in 0.25'TBE (22.5mM Tris-borate, 0.5mM EDTA) at lOV/cm 2 h, gels were dried and autoradiographed at -70°C.

Effect of Triplex Formation on Nuclear Factors Binding: The effect of oligonucleotide- directed triplex formation on nuclear factors binding to the 255-bp promoter fragment was determined by protein binding assays (Fig. 5). This fragment, lying immediately 5' to the SIS/PDGF-B mRNA initiation site (+1), contains SPE between -58 to -39, which is the binding site of the Sp family of transcription factors including at least Spl and Sp3. As shown in Fig. 5, Lane 2 is the labeled 255-bp c-sis/PDGF-B promoter alone, while lane 4 shows that the labeled 255-bp target is bound by the nuclear factors as evidence by retardation of labeled duplex target following incubation with nuclear extracts of PMA-treated K562 cells. In lane 3, 20-fold unlabeled 255-bp target competed with the labeled target and bound most of the nuclear factors. Increasing amounts of TFO (1, 10, 5 OμM) eliminated the formation of protein DNA complexes partly (TFOl, lanes 5-7) and completely (TFO8, lanes 8-10; TFO10, lanes 11-13). In contrast, lane 1 shows that 5 OμM TFO2 has little effect on the binding between nuclear factors and duplex target. Transient Transfection Experiments: 40μg pGL3promoter was incubated with 0-30 nmol TFOs in 20mM sodium cacodylate-HCl (pH 7.4), lOmM MgCl₂ at 4°C overnight before transfection. Exponentially growing K562 cells were counted, harvested by centrifugation, and resuspended in RPMI 1640 to a concentration of 1.25x10⁷ cells/ml. For each transfection, 6.25x 10⁶ cells (0.5ml) were mixed on ice with pGL3promoter-TFO mixtures and 0.5ng pRLSV40 plasmid (Promega). The cell DNA mixtures were pulsed at 300V/975μF capacitance using a Bio-Rad Gene Pulser and immediately resuspended in complete RPMI. Approximately 24 h after electroporation, the cell culture volumes were split equally into two dishes and treated with either PMA ( Sigma Chemical Co.; final concentration, 2ng/ml) or solvent (ethanol). Cultures were harvested approximately 48 h after electroporation by centrifugation, washed three times with cold PBS, and consulted to 500μl lysis buffer ( Luciferase Assay System, Promega ). After 1 h at room temperature, the lysate were collected. A volume of 30μl of the lysate was added to 30μl of luciferase assay Reagent II (Promega) in a clear polystyrene 12^χ75mm tube, which was immediately placed in a luminometer (Lumat LB 9507, EG&G Berthold) and light production was measured for 10 s. The first measurement was the firefly luciferase activity from pGL3 promoter plasmid, then 30μl of Stop & Glu reagent ( Promega ) was added immediately to measure the Re lla Luciferase activity from pPRLS V40 plasmid Stable Transfection Experiments: To establish the K562 luciferase or control cell line, 20μg neo^rpGL3 promoter or neo^rpGL3 control was mixed with 6 25 ^χ l0⁶ cells in 0 5ml RPMI 1640 on ice and electroporated as described above Approximately 48hr after transfection, 0 5mg neomycin analogue G418 (Gibco BRL) was added to culture medium per ml The transfectants were replenished with fresh selection medium every 2-3 days Resistant colonies were pooled into a 96-well plate after 2 weeks of selection and propagated in the presence of 0 5mg/ml of G418 K562 luciferase and control cells were incubated with 50, 10, 1, 0 1 and 0 01 μM TFOs for 72 hours TFO attrition was compensated for by the addition of fresh TFO-containing medium every 24 hours After, the cultures were washed twice with PBS prior to firefly luciferase assay as described above

Effect of Triplex Formation on c-sis/PDGF-B transcription: A reporter plasmid pGL3promoter carrying a firefly luciferase gene driven by the 255-bp c-sis/PDGF promoter was constructed to measure the in vivo effects of TFOs on c-sis/PDGF-B transcription Triplex was formed in vitro by incubating supercoil pGL3promoter plasmid with TFOs, and then the entire DNA complex was transfected into K562 cells. Incubation of TFO 1 results in a 56 2% luciferase activity while TFO6 causes 85.3% and TFO8 76.3% decrease in luciferase activity As a non-triplex forming control, TFO2 had little effect on the expression of the firefly luciferase (Fig 6A) Two new K562 cell lines were created for the stable transfection experiments One was the K562 luciferase cell line, which was stably transfected with neo^rpGL3 promoter, a luciferase reporter gene under the control of the 255-bp c-sis/PDGF promoter The other was K562 control cell line, which was stably transfected with neo^rpGL3 control, a luciferase reporter gene under the control of the SV40 promoter After incubating both cell lines with the S-TFOs of increasing concentration for 72 hours, a concentration-dependent decrease of luciferase activity was observed At 50μM, the inhibition of luciferase activity was as high as 50 7% by TFO1, 65 8 % by TFO8 and 84% by TFO 10, but TFO2 had no effect at the same concentration (Fig 6B) The effect of the TFOs on transcription from a promoter unrelated to that of PDGF-B was examined by treating stably transfected K562 cells expressing a luciferase reporter gene driven by the SV40 promoter with either TFO or control oligonucleotides. As shown in Fig. 6B, at the same concentration which inhibits PDGF-B transcription, the TFOs had little effect on transcription from the SV40 promoter under the same conditions. These results suggest that inhibition of PDGF-B transcription by TFOs is due to sequence specific triplex formation within the PDGF-B promoter.

In short, the results from gel mobility shift analysis, DNase I footprinting, protein binding assays, transient transfection experiments and stable transfection experiments demonstrate that the TFOs can form sequence-specific stable triplex with the target, and can effectively inhibit transcription factors binding and thereby suppress the activity of PDGF-B promoter. They can be used for preparation of drugs to inhibit tumor growth and for the therapy of atherosclerosis, inflammation, etc, which have the advantage of being affordable, specific and stable.

Oligonucleotides are especially attractive as potential therapeutic agents for several reasons: (1) they can be synthesized using automated procedures; (2) oligonucleotides with improved properties can be selected in vitro using "directed- evolution' methods; and (3) modified bases, backbone components and reactively chemical moieties can be incorporated to confer stability, increase binding or improve functional characteristics. Compared with antisense oligonucleotides or ribozymes, The triplex strategy has a potential stoichiometric advantage, because there are generally one to two targets per cell as compared with the hundreds to thousands of mRNA targets for antisense oligonucleotides, thus offering the potential for low dose long-acting therapeutics.

A product of the invention may be formulated in known manner, together with a conventional carrier or diluent. It may be administered to a patient in any form or by any route compatible with its intended use, as will be readily apparent to one of ordinary skill in the art. The amount of the active agent to be administered will be chosen according to the usual factors, and this again is within the practice of one of ordinary skill in the art.

Claims

1 A polynucleotide capable of selectively binding to the double-stranded promoter region of the c-sis/PDGF-B proto-oncogene to form a triplex, thereby inhibiting the function of the promoter and affecting expression of the proto-oncogene 2 A polynucleotide according to claim 1 , which is capable of binding to a strand of the promoter having SEQ LD No 3

3 A polynucleotide according to claim 2, comprising SEQ LD No 5

4 A polynucleotide according to claim 1 , which is capable of binding to a strand of the promoter having SEQ ID No 2 5 A polynucleotide according to claim 4, comprising SEQ LD No 12

6 A polynucleotide according to claim 1, comprising SEQ LD No 13

7 A polynucleotide according to any previous claim, for therapeutic use

8 Use of a polynucleotide according to any of claims 1 to 6, for the manufacture of a medicament for the treatment of a condition associated with expression of c- sis/PDGF-B

9 Use according to claim 8, wherein the condition is a tumour, atherosclerosis or inflamation