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

Abstract

Background: A 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.

Results: The 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.

Conclusion: LNAs 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.

Figure 1

Oligonucleotides and their respective targets in the 5' end of the HIV-1 RNA genome. (A) Secondary structural model of the HIV-1 leader RNA. The stem-loops are named according to assigned function (see text for details) and the sequence is numbered from the 5' end of the RNA transcript. (B) The targets for the various oligonucleotide constructs. The annealing sites for the oligonucleotides are indicated by a solid line and the cleavage sites of the DNA/LNAzymes are marked by arrows. (C) Sequences of the antisense oligonucleotides and DNAzymes containing the 10–23 catalytic motif [10] named according to their target sites shown in panel B. The selected target sequences for the antisense constructs include sequences downstream of the primer binding site (PBSD), the dimerization initiation site (DIS), the splice donor site (SD) and the Gag initiation codon (AUG). The nucleotides that are substituted with LNA residues in the LNA antisense gap-mers and LNAzymes constructs are circled. The target sequences of the "10–23" DNAzymes are indicated with grey letters.

Figure 2

The effect of antisense LNA on reverse transcription of HIV-1 RNA. An equimolar amount (1pmol) of HIV RNA and LNA oligo was mixed and incubated prior to primer extension using a primer complementary to position 384–401. Antisense LNA oligonucleotides, included LNA_AUG (lane 1), LNA_SD (lane 2), LNA_DIS (lane 3), LNA_PBSD (lane 4), and the major sites of transcriptional termination are indicated to the right. Read through to the 5' end of the HIV-1 RNA is denoted by +1. A sequence latter obtained by dideoxysequencing of the HIV-1 RNA is included in lanes 6–9. The level of read through reverse transcription is calculated as read-through/(read through + paused) × 100% and indicated below.

Figure 3

The effect of antisense LNA versus RNA and DNA on dimerization of HIV-1 RNA. The ability of (A) antisense LNA oligonucleotides directed towards different targets or (B) antisense LNA, RNA or DNA oligonucleotide directed towards the DIS target, to inhibit the formation of the DIS dimer-complex during 30 min incubation were investigated. Monomeric and dimer bands are indicated to the left. (C) A similar experiment, but where the indicated antisense oligonucleotide was added after the dimers were allowed to pre-form for 30 mins and subsequently incubated for the indicated time, hence evaluating the efficiency of breaking a stable DIS dimer-complex as a function of time time. Diamonds = LNA_DIS; Bullet = RNA_DIS; Triangle = DNA_DIS.

Figure 4

In vitro cleavage of HIV-1 RNA by DNA- and LNAzymes. One hundred nmol 5' end labeled leader RNA (+1–355) was incubated with 5 nmol, 100 nmol or 2 pmol DNAzymes or LNAzymes for the indicated time. The DNAzymes targeted to the PBSD and DIS regions cleaved primarily at the expected site, yielding a 5'-end labeled fragment of 205 and 261 nucleotides, respectively (product; panel A and C). The same bands were obtained using the LNAzyme (panel B and C). The experiment was made in duplicates yielding essentially the same result and the cleavage efficiencies indicated below each autoradiogram were calculated as (cleaved RNA/cleaved RNA and uncleaved RNA) × 100% averaged over both experiments.

Figure 5

Inhibition of intracellular HIV-1 production in the presence of antisense LNA and LNAzymes. One hundred nanograms of HIV-1 genomic LAI plasmid was co-transfected with a renilla luciferase expression construct and the indicated amounts of antisense LNA or LNAzyme, and the HIV-1 production was measured by CA-p24 ELISA 72 hours later. (A) HIV-1 production in the presence 20 nM of the four different HIV-1 specific LNAs and 2 LNA controls (Mock 1 and Mock 2). (B) Measuring HIV-1 production in the presence of low range concentration of LNA_PBSD (0.16–20 nM). The inhibition is calculated as the average value of two independent experiments where the relative CA-p24 expression is normalized for unspecific inhibition of renilla expression. (C) Comparing the inhibitory capacity of DNA versus LNA containing antisense or 10–23 enzymes targeted to the DIS and PBSD regions. The identity and the concentration of the oligonucleotide are indicated below. The assay was performed in duplicates.