Efficient inhibition of HIV-1 expression by LNA modified antisense oligonucleotides and DNAzymes targeted to functionally selected binding sites

Martin R Jakobsen,Joost Haasnoot,Jørgen Kjems,Ben Berkhout,Jesper Wengel

doi:10.1186/1742-4690-4-29

Abstract

BackgroundA primary concern when targeting HIV-1 RNA by means of antisense related technologies is the accessibility of the targets. Using a library selection approach to define the most accessible sites for 20-mer oligonucleotides annealing within the highly structured 5'-UTR of the HIV-1 genome we have shown that there are at least four optimal targets available.ResultsThe biological effect of antisense DNA and LNA oligonucleotides, DNA- and LNAzymes targeted to the four most accessible sites was tested for their abilities to block reverse transcription and dimerization of the HIV-1 RNA template in vitro, and to suppress HIV-1 production in cell culture. The neutralization of HIV-1 expression declined in the following order: antisense LNA > LNAzymes > DNAzymes and antisense DNA. The LNA modifications strongly enhanced the in vivo inhibitory activity of all the antisense constructs and some of the DNAzymes. Notably, two of the LNA modified antisense oligonucleotides inhibited HIV-1 production in cell culture very efficiently at concentration as low as 4 nM.ConclusionLNAs targeted to experimentally selected binding sites can function as very potent inhibitors of HIV-1 expression in cell culture and may potentially be developed as antiviral drug in patients.

Highlights

A primary concern when targeting Human Immunodeficiency Virus (HIV)-1 RNA by means of antisense related technologies is the accessibility of the targets
Construct design and locked nucleic acid (LNA) modification of targeting oligonucleotides Four target sites were selected in the HIV-1 5'-UTR as potential target based on previous accessibility selection studies [22]: 1) a region immediately downstream from the primer binding site (PBSD, 203–222), 2) a region covering the dimerization initiation site (DIS) (DIS, 255–274), 3) a region encompassing the major splice donor site (SD, 278–297) and 4) a region covering the gag initiation site (AUG, 326–345; Fig. 1)
To overcome this barrier we have chosen 4 targets in the HIV-1 genome that were previously selected as optimal annealing sites in vitro, and we tested them as targets for DNA and LNA antisense oligonucleotides, and DNA- and LNA-zymes

Summary

Introduction

A primary concern when targeting HIV-1 RNA by means of antisense related technologies is the accessibility of the targets. In the simplest form, antisense single stranded oligonucleotides (or derivatives hereof) are introduced into the cell to block gene expression by interfering with translation of the mRNA or by degrading the RNA in a DNA/RNA heteroduplex via an RNaseH dependent pathway. Retrovirology 2007, 4:29 http://www.retrovirology.com/content/4/1/29 instability of the single stranded oligonucleotide, chemical toxicity and lack of mRNA target accessibility are possibly obstacles for a lacking antisense effect. The latter problem is mainly due to the formation of stable RNA structures and assembly of the mRNA into nucleoprotein complexes rendering the target site inaccessible to base pairing [5,6]. It has been estimated that only 2–5% of randomly chosen antisense oligonucleotides have any effect on gene expression [5,7] and computer generated structure models are generally not sufficient for rational prediction of effective targets

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Conclusion