Enhanced hepatic uptake and bioactivity of type α1(I) collagen gene promoter-specific triplex-forming oligonucleotides after conjugation with cholesterol

Kun Cheng, Zhaoyang Ye, Ramareddy Guntaka, Ram I. Mahato

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Abstract

A triplex-forming oligonucleotide (TFO) specific for type α1(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 3′ 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 α1(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 TFO-Chol was higher than TFO in cell culture system and in vivo studies. In conclusion, TFO-Chol is a potential antifibrotic agent.

Original languageEnglish (US)
Pages (from-to)797-805
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Volume317
Issue number2
DOIs
StatePublished - May 1 2006

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Collagen Type I
Oligonucleotides
Cholesterol
Liver
Genes
Transfection
Hepatic Stellate Cells
Reverse Transcriptase Polymerase Chain Reaction
Disulfides
Liver Cirrhosis
Real-Time Polymerase Chain Reaction
Hepatocytes
Pharmacokinetics
Cell Culture Techniques

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

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title = "Enhanced hepatic uptake and bioactivity of type α1(I) collagen gene promoter-specific triplex-forming oligonucleotides after conjugation with cholesterol",
abstract = "A triplex-forming oligonucleotide (TFO) specific for type α1(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 3′ 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 α1(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 TFO-Chol was higher than TFO in cell culture system and in vivo studies. In conclusion, TFO-Chol is a potential antifibrotic agent.",
author = "Kun Cheng and Zhaoyang Ye and Ramareddy Guntaka and Mahato, {Ram I.}",
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T1 - Enhanced hepatic uptake and bioactivity of type α1(I) collagen gene promoter-specific triplex-forming oligonucleotides after conjugation with cholesterol

AU - Cheng, Kun

AU - Ye, Zhaoyang

AU - Guntaka, Ramareddy

AU - Mahato, Ram I.

PY - 2006/5/1

Y1 - 2006/5/1

N2 - A triplex-forming oligonucleotide (TFO) specific for type α1(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 3′ 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 α1(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 TFO-Chol was higher than TFO in cell culture system and in vivo studies. In conclusion, TFO-Chol is a potential antifibrotic agent.

AB - A triplex-forming oligonucleotide (TFO) specific for type α1(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 3′ 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 α1(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 TFO-Chol 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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