Reversible covalent attachment of cholesterol to oligodeoxyribonucleotides for studies of the mechanisms of their penetration into eucaryotic cells

Boutorine, Alexandre S.; Kostina, E. V.

doi:10.1016/0300-9084(93)90022-k

Cited by 60 publications

(39 citation statements)

References 14 publications

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“…It occupies the space between the saturated hydrocarbon chains of sphingolipids in the cell membrane and makes the fluid membrane rigid (Simons and Ikonen, 2000). Cholesterol attached to the TFO may facilitate the anchoring of the conjugate to lipophilic cellular structure (MacKellar et al, 1992) and prevent translocation to the nucleus where TFO needs to form triple helix with genomic DNA to function as an antigene (Boutorine and Kostina, 1993). In the present study, we conjugated cholesterol to the TFO via a disulfide bond that is known to be cleaved inside the cells with intracellular thiols or by the action of redox enzyme.…”

Section: Discussionmentioning

confidence: 99%

Enhanced Hepatic Uptake and Bioactivity of Type α1(I) Collagen Gene Promoter-Specific Triplex-Forming Oligonucleotides after Conjugation with Cholesterol

Cheng

Guntaka

et al. 2006

J Pharmacol Exp Ther

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A triplex-forming oligonucleotide (TFO) specific for type ␣1(I) collagen promoter is a promising candidate for treating liver fibrosis. Earlier, we determined the pharmacokinetics and biodistribution of TFO after systemic administration into normal and fibrotic rats. In this study, we conjugated cholesterol to the 3Ј end of the TFO via a disulfide bond and determined its cellular and nuclear uptake and bioactivity using HSC-T6 cell lines in vitro, followed by biodistribution at whole-body, organ (liver), and subcellular levels. Conjugation with cholesterol had little effect on the triplex-forming ability of the TFO with target duplex DNA, and the cellular uptake of 33 P-TFO-cholesterol (Chol) increased by 2-to approximately 4-fold. Real-time reverse transcriptase-polymerase chain reaction analysis after transfection of HSC-T6 cells with TFO-Chol or TFO indicated that TFO-Chol had higher inhibition on type ␣1(I) collagen primary transcript than naked TFO at low concentration (200 nM) but showed similar inhibition at higher concentration (500 and 1000 nM). There was increase in the inhibition on primary transcript with transfection time. The hepatic uptake of 33 P-TFO-Chol after systemic administration was 72.22% of the dose compared with 45.8% of 33 P-TFO. There was significant increase in the uptake of 33 P-TFO-Chol by hepatic stellate cells and hepatocytes. More importantly, the nuclear uptake of TFOChol was higher than TFO in cell culture system and in vivo studies. In conclusion, TFO-Chol is a potential antifibrotic agent.

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Section: Discussionmentioning

confidence: 99%

Enhanced Hepatic Uptake and Bioactivity of Type α1(I) Collagen Gene Promoter-Specific Triplex-Forming Oligonucleotides after Conjugation with Cholesterol

Cheng

Guntaka

et al. 2006

J Pharmacol Exp Ther

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“…Cholesterol modification has been shown to increase both the cell association and activity of phosphodiester and phosphorothioate oligonucleotides (Boutorine et al, 1989;Letsinger et al, 1989;Boutorine et al, 1993;Krieg et al, 1993). These compounds have been shown to be taken up, at least in part, by the LDL receptor (de Smidt et al, 1991;Krieg et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

“…The mechanism of action of cholesteryl-phosphodiester oligonucleotides is unclear since sequence non-specific effects were found in an HIV model (Letsinger et al, 1989), in which the non-coupled oligonucleotide was reported to act in a sequence-specific manner (Zamecnik et al, 1986;Goodchild et al, 1988). Lack of specificity is presumably caused by the increased hydrophobicity associated with coupling to cholesterol or other hydrophobic residues in several viral and cellular models (Shea et al, 1990;Boutorine et al, 1993). Other authors, however, have reported that cholesteryl-modified oligonucleotides act in a sequence specific manner (Krieg et al, 1993;Svinarchuk et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes

1994

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Encapsulation of oligonucleotides in antibody-targeted liposomes (immunoliposomes) which bind to target cells permits intracellular delivery of the oligonucleotides. This approach circumvents problems of extracellular degradation by nucleases and poor membrane permeability which free phosphodiester oligonucleotides are subject to, but leaves unresolved the inefficiency of encapsulation of oligonucleotides in liposomes. We have coupled oligonucleotides to cholesterol via a reversible disulfide bond. This modification of oligonucleotides improved their association with immunoliposomes by a factor of about 10 in comparison to unmodified oligonucleotides. The presence of cholesteryl-modified oligonucleotides incorporated in the bilayer of liposomes did not interfere with the coupling of the targeting protein to the liposome surface. Free or cholesterol coupled oligonucleotides associated with liposomes and directed against the tat gene of HIV-1 were tested for inhibition of HIV-1 proliferation in acutely infected cells. We demonstrate that the cholesteryl-modified as well as unmodified oligonucleotides acquire the target specificity of the antibody on the liposome. Their antiviral activity when delivered into cells is sequence-specific. The activity of these modified or unmodified oligonucleotides to inhibit the replication of HIV was the same on an equimolar basis (ECs0 around 0.1 /~M). Cholesterol coupled oligonucleotides thus offer increased liposome association without loss of antiviral activity.

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“…It is known that the oligonucleotides are incorporated into the cell by endocytosis, in a concentration dependent manner: below the concentration of 1 µM predominantly via a receptor-like mechanism (3) and at higher concentrations, mainly by fluid-phase endocytosis (2). To be most effective, the ASOs should be also delivered to the nuclei (4) and therefore several uptake improvement strategies have been developed such as the use of streptolysin O (5), chemical modifications of the oligonucleotides (6), attachment of the oligonucleotides to cholesterol (7), or the use of nanosized cationic hydrogels (8). Perhaps one of the most successful strategies is the use of cationic lipids, which are known to increase the oligonucleotide uptake up to 250-fold as well as its cytoplasmic bioavailability (1,9).…”

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confidence: 99%

Evidence for a Specific Intracellular Localization of an Antisense Oligonucleotide in K562 Cells

et al. 2005

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Abstract. In order to be active, antisense oligonucleotides (ASOs) should be delivered to the nuclei of cells. The lack of effect of some ASOs might be explained by poor distribution inside the cell. Here we describe the study of the intracellular distribution of an ASO in a leukemic cell line in which the ASO was not showing an effect. We used fluorescein isothiocyanate-labeled ASO and fluorescent or confocal microscopy. The internalised ASO was localized in a specific intracellular juxtanuclear region, showing no cytoplasmic or nuclear diffusion. Transfection of the ASO improved cellular distribution to the cytoplasm and nuclei and improved the ASO effect.

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Reversible covalent attachment of cholesterol to oligodeoxyribonucleotides for studies of the mechanisms of their penetration into eucaryotic cells

Cited by 60 publications

References 14 publications

Enhanced Hepatic Uptake and Bioactivity of Type α1(I) Collagen Gene Promoter-Specific Triplex-Forming Oligonucleotides after Conjugation with Cholesterol

Enhanced Hepatic Uptake and Bioactivity of Type α1(I) Collagen Gene Promoter-Specific Triplex-Forming Oligonucleotides after Conjugation with Cholesterol

Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes

Evidence for a Specific Intracellular Localization of an Antisense Oligonucleotide in K562 Cells

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