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

Inactive Publication Date: 2007-01-18
LEE IN KYU DR +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018] Further, an object of the invention is to provide means such as a decoy ODN containing a transcriptional factor such as AP-1 binding site for transfection of VSMC, which w

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 wheth

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

example 1

Inhibitory Effects of Novel AP-1 Decoy Oligodeoxynucleotides on Proliferation of Vascular Smooth Muscle Cells In Vitro and Neointima Formation In Vivo

Materials and Methods

Animals

[0130] Nine- to ten-week old male Sprague-Dawley rats (Hyochang, Taegu, Korea) weighing 280 to 320 g were used. All procedures were in accordance with institutional guidelines for animal research.

Cell Culture

[0131] Human VSMC were harvested as described in Ahn et al 2001 supra, and rat aortic smooth muscle cells w re harvested from the thoracic aorta of adult male Sprague-Dawley rats (200-250 g). VSMC were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Grand Island, N.Y., USA) containing 20% fetal bovine serum (Gibco BRL). VSMC purity was characterized by positive staining with smooth muscle specific α-actin monoclonal antibodies (Sigma, St. Louis, Mo., USA).

Construction of CDODN

[0132] The sequences of dumbbell type and phosphorothioate double-stranded ODN derived from the AP-1 b...

example 2

Effects of Novel E2F Decoy Oligodeoxynucleotides

Materials and Methods

Animals

[0155] Nine- to ten-week old male Sprague-Dawley (SD) rats weighing 280 to 320 g were used. All procedures were in accordance with institutional guidelines for animal research.

Cell Culture

[0156] Human VSMCs were isolated from thoracic aortas of heart transplant donors. The collection of this tissue was approved by the Ethics Committee of the institution. Rat VSMCs were harvested from thoracic aortas of adult male SD rats. VSMCs were cultured in DMEM (Gibco BRL, Grand Island, N.Y., USA) containing 20% FBS (Gibco BRL). VSMC purity was characterized by positive staining with smooth muscle specific α-actin monoclonal antibodies (Sigma, St. Louis, Mich., USA).

[0157] After reaching 80-90% confluence in 100-mm dishes, human VSMC were serum-starved for 24 h in serum free medium, and were subjected to either control normal glucose medium (DMEM containing 5.5 mmol / l D-glucose) or conditioned medium (DMEM cont...

example 3

Effects of Novel NF-κB Decoy Oligodeoxynucleotides

Construction of Dumbbell Type Decoy ODN

[0178] The sequences of dumbbell type and phosphorothioated double-stranded ODN against NFκB binding site and mutated ODN used in this invention are as follows: CD-NF (note; consensus sequences are underlined), 5′-GGATCCGGGGATTTCTATTGCAAAAGCAATAGCGCGAAAC-3′ (SEQ ID NO: 15); phosphorothioate NFκB decoy (PS-NF), 5′-ATsTTAAGGGGATTTCCCTTTCTCAsAs-3′ (SEQ ID NO: 16); mutated E2F decoy (M-NF), 5′-GGATCCGGGGATATTTATTGCAAAAGCAATAAATCGAAAC-3′ (SEQ ID NO: 17). CD-NF was anticipated to form a stem-loop structure. The stem is formed by complementary sequences at both ends of each oligo. The 5′ terminus of the stem has 6 bases of a single-stranded sequence of 5′-GGATCC-3′ as enzyme site of BamHI. Two oligo molecules were joined by the complementary 6 base sequences at both 5′ ends. ODN were annealed for 2 h, while the temperature descended from 80° C. to 25° C. One unit of T4 DNA ligase was added and incub...

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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...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K9/127C07H21/04C12N15/11A61K31/70A61K31/7088A61K38/00A61P1/04A61P3/06A61P5/14A61P9/00A61P9/04A61P9/10A61P11/00A61P13/12A61P25/00A61P29/00A61P35/00A61P35/04A61P37/02A61P43/00C12N15/09C12N15/10C12N15/113
CPCA61K38/00C12N2310/53C12N2310/13C12N15/113A61P1/04A61P11/00A61P13/12A61P25/00A61P29/00A61P35/00A61P3/06A61P35/04A61P37/02A61P43/00A61P5/14A61P9/00A61P9/04A61P9/10C12N15/11C12N15/10A61K48/00
Inventor LEE, IN-KYUMORISHITA, RYUICHI
Owner LEE IN KYU DR
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