Circular dumbbell decoy oligodeoxynucleotides (cdodn) containing dna bindings sites of transcription

Abstract

The present invention provides a circular dumbbell oligodeoxynucleotide (CDODN) comprising two loop structures and a stem structure, wherein the stem structure comprises a nucleotide sequence capable of binding the DNA-binding domain of a transcriptional factor. The present invention further provides a pharmaceutical composition comprising said CDODN. The pharmaceutical composition can be used for treating and / or preventing a disease or disorder related to such a transcriptional factor. The present invention also provides a method for treating and / or preventing a disease or disorder related to such a transcriptional factor, comprising administering to the subject a therapeutically effective amount of a CDODN comprising two loop structures and a stem structure, wherein the stem structure comprises a nucleotide sequence capable of binding the DNA-binding domain of the transcriptional factor.

Description

TECHNICAL FIELD [0001] This invention is in the field of gene therapy. In particular, it is directed to novel decoy oligodeoxynucleotides and uses thereof. BACKGROUND ART [0002] Double stranded oligodeoxynucleotides (ODN or “decoys”) for reducing trans-activity of transcription factors are an innovative and attractive strategy for gene therapy and for the functional study of gene products. Several different double-stranded DNA structures, including unmodified oligonucleotide duplexes, ab-anomeric oligonucleotides, phosphorothioate oligonucleotide duplexes, and dumbbell oligonucleotides, have been introduced as decoys for transcription factors (see Scholer H R and Gruss P., Cell 1984; 36: 403-411; Cereghini Set al., Genes Dev 1988; 2: 957-974; Berkowitz L A et al., Mol Cell Biol 1989; 9: 4272-4281; Tanaka H et al., Nucleic Acids Res 1994; 22: 3069-3074; Bielinska A et al., Science 1990; 250:997-1000; Clusel C et al., Nucleic Acids Res 1993; 21: 3405-3411; Lim C S et al., Nucleic Acid...

AI Technical Summary

Benefits of technology

[0026] We developed a novel AP-1 decoy ODN with a circular dumbbell structure (CDODN) to avoid destruction by exonucleases. This new form of AP-1 decoy ODN was more stable, largely preserving its structural integrity after incubation in the presence of either exonuclease III or serum, than phosphorothioate linear decoy ODN (PSODN). Transfection of AP-1 decoy ODN strongly inhibited both proliferation and migration of vascular smooth muscle cells. AP-1 decoy ODN also inhibited high glucose- and serum-induced transcriptional expression of PCNA and cyclin A genes. Consistent with in vitro data, administration of AP-1 decoy ODN in vivo using the Hemagglutinating virus of Japan (HVJ)-liposome method almost completely inhibited neointima formation after balloon injury of rat carotid artery. As compared to conventional PSODN, CDODN was more effective in the inhibition of proliferation of smooth muscle cells in vitro and neointima formation in vivo. Approximately half the dose of CDODN, as compared to PSODN, was enough to obtain similar effects on growth inhibition of vascular smooth muscle cells in vitro as well as in vivo. Moreover, the sequence specificity of the CDODN of AP-1 binding was unexpectedly more 10 times greater than conventional PSODN.

[0027] This invention thus shows that employment of a more stable CDODN against AP-1 with the highly effective HVJ-liposome delivery method provides a new therapeutic strategy for the prevention of restenosis after angioplasty in humans.

[0028] Further, the present invention is concerned with the transcription factor E2F which plays a critical role in the trans-activation of several genes involved in cell-cycle regulation. Previous studies have shown that the transfection of cis element double stranded oligodeoxynucleotides (decoys), corresponding to E2F binding domains, could inhibit vascular smooth muscle cell (VSMC) proliferation and neointimal hyperplasia in injured vessels. In the present study, we developed a novel E2F decoy with a circular dumbbell structure (CD-E2F) and compared the effect of this CD-E2F with conventional phosphorothioated E2F decoy (PS-E2F). We found that the CD-E2F was more stable, largely preserving its structural integrity after incubation in the presence of either nucleases or serum, than PS-E2F. The CD-E2F more strongly inhibited high glucose- and serum-induced transcriptional expression of cell-cycle regulatory genes as compared with the PS-E2F. Transfection of CD-E2F was more effective in the inhibition of VSMC proliferation as well as neointima formation in vivo, as compared with PS-E2F. CD-E2F in a 40-50% reduced dose compared with PS-E2F was enough to obtain a similar effect on VSMC growth inhibition in vitro as well as neointima formation in vivo. Moreover, CD-E2F unexpectedly showed a 10 times greater sequence specificity against E2F than PS-E2F.

[0038] In one embodiment of the invention, the compound may competitively inhibit binding of the AP-1 to the promoters.

[0045] In one embodiment of the invention, the compound may competitively inhibit binding of said AP-1 to said promoters.

Problems solved by technology

The main limitation of unmodified oligonucleotide ODNs is that they are easily degraded by nucleases present in serum and in cells.

However, these modified ODNs exhibit problems such as insensitivity to RNase H, the possibility of recycling of hydrolyzed modified nucleotides into cellular DNA, lack of sequence-specific binding effects of ODN-based gene therapy, and immune activation (see Moon I J, et al., J Biol Chem.

There have been a number of trials with pharmacological agents to reduce the incidence and rate of restenosis after PTCA, but the results have not been satisfactory.

However, it is not known whether inhibition of AP-1 binding would prevent neointima formation.

Although there have been successful application of decoys to some diseases and disorders related to transcriptional factors, the above-mentioned main limitation of unmodified oligonucleotide ODNs where they are easily degraded by nucleases present in serum and in cells, significantly reduces the efficacy of decoys in treatment and prevention of such diseases and disorders.

However, there have been no reports showing that such covalently closed ODNs or circular dumbbell oligonucleotides are effective in treating or preventing diseases or disorders.

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