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 ␣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.

DNA triple helices: Biological consequences and therapeutic potential

“…A number of chemical modifications have been incorporated into TFOs to facilitate their uptake. These include, but are not limited to: polyamine analogs [88] cholesterol derivatives [89] nuclear targeting peptide conjugates [90]; 6-phosphate-bovine serum albumin [91]; and polypropylenimine dendrimers [92].…”

Bioconjugation of Oligonucleotides for Treating Liver Fibrosis

“…We have also conjugated cholesterol to a TFO molecule against α1(I) collagen gene promoter at 3' end via disulfide bond formation (428). Around 4-fold increase in the cellular uptake and enhanced bioactivity in inhibition of α1(I) collagen gene transcription were observed in HSC-T6 cell culture.…”

“…The wide variation in nuclear accumulation was also observed in different cell types ranging from 7% to 30% of the total cellular uptake. We have also studied the cellular uptake and subcellular distribution of a 25mer purine-rich TFO targeting to rat α1(I) collagen promoter (428). Around 12% of the dose was found to be associated with cells in HSC-T6 cell line, in which 30% was associated with nuclear structures.…”

“…Alefelder et al (477) reported modification of postsynthetic PS-ODNs with either α-(bromoacetamido)-3-cholesterol or 2-(5'-nitropyridyl)-3-cholesterol disulfide. We conjugated thiocholesterol to TFOs containing sulfhydryl group at 3'-end via activation by bis-(5-nitro-2-pyridyl)-disulfide ( Figure 11A) (428). The disulfide bond formation between cholesterol and TFO has advantage over other non-cleavable bonds in releasing TFO molecules within target cells.…”

Small interfering RNAs: heralding a new era in gene therapy

“…Conjugation of siRNAs with lipids and polymers increases thermodynamic stability, protects siRNAs' strands from nucleases and improves the biodistribution and 77 Small Interfering RNAs: Heralding a New Era in Gene Therapy pharmacokinetic profiles of siRNAs along with their targeting to specific cell types (Cheng 2006, Lorenz 2004, Soutschek 2004, Wolfrum 2007, DiFiglia 2007, Mahat 1999Schepers 2005). Therefore, conjugation of siRNA with lipids either enhances the uptake via receptormediated endocytosis, or increases penetration across the cell membrane, as demonstrated by the studies where cholesterol-conjugated siRNAs were effectively delivered to cells in cell culture, liver and other organs (Cheng 2006).…”