The Multiple Inhibitory Mechanisms of GEM 91®, agagAntisense Phosphorothioate Oligonucleotide, for Human Immunodeficiency Virus Type 1

Abstract

GEM 91 (gene expression modulator) is a 25-mer oligonucleotide phosphorothioate complementary to the gag initiation site of HIV-1. GEM 91 has been studied in various in vitro cell culture models to examine inhibitory effects on different stages of HIV-1 replication. Experiments were focused on the binding of virions to the cell surface, inhibition of virus entry, reverse transcription (HIV DNA production), inhibition of steady state viral mRNA levels, inhibition of virus production from chronically infected cells, and inhibition of HIV genome packaging within virions. Experiments were also performed in vitro in an attempt to generate strains of HIV with reduced sensitivity to GEM 91. We observed sequence-dependent inhibition of virus entry/reverse transcription and a reduction in steady state viral RNA levels. We also observed sequence-independent inhibition of virion binding to cells and inhibition of virus production by chronically infected cells. Using in vitro methods that were successful in generating HIV strains with reduced sensitivity to AZT, we were unable to generate strains with reduced sensitivity to GEM 91.

Is antisense an appropriate nomenclature or design for oligodeoxynucleotides aimed at the inhibition of HIV-1 replication?

“…PS-ODNs have been shown to inhibit the replication of HIV-1 in vitro by both sequencespecific 19,29,37,38 and non-sequence-specific [29][30][31][32] processes depending on the cell culture model employed. In acute-infection models, non-antisense inhibition of PS-ODNs has been well documented 1 and might occur, because of interference with virus adsorption, by binding to the CD4 receptor or the V3 loop of viral gp120, [39][40][41] or with reverse transcription. 42 In chronic-infection models, sequence-specific inhibition of HIV production has been reported, 19,32 but the precise mechanism of action of PS-ODNs still remains to be elucidated.…”

“…4,28 In a recent study, inhibition of virus production in chronically infected H9 cells was evaluated at about 60% (as measured by p24 determination) with either the antisense molecule GEM 91 or the mismatched oligo control at 1 μM, suggesting that inhibition of HIV-1 production in this chronic-infection model was also a non-sequencespecific phenomenon. 41 However, these authors found that treatment of the chronically infected H9 cells with GEM 91 caused a significant decrease in gag mRNA expression (60%-70% inhibition), while mismatched oligo treatment resulted in gag mRNA expression similar to the control, suggesting that GEM 91 had a sequencespecific effect at this stage of the viral-replicative cycle. Nevertheless, a reduction in mRNA level does not necessarily result in biological activity since a lower mRNA level might be sufficient for protein function.…”

Phosphorothioate Oligonucleotides Inhibit Human Immunodeficiency Virus Type 1 Fusion by Blocking gp41 Core Formation

“…Several studies have suggested that the antiviral activity of phosphorothioated oligonucleotides (PS-ONs) with different sequences or different intended mechanisms of action (i.e., antisense or sequence-specific aptamers) all work as inhibitors of human immunodeficiency virus (HIV) viral entry by binding to the V3 loop of gp120 and preventing CD4 interaction and attachment to the host cell (13,43). The general conservation of this mechanism of action with different oligonucleotides having different sequences suggested that the antiviral activity of oligonucleotides against HIV was sequence independent.…”

Identification of a Key Target Sequence To Block Human Immunodeficiency Virus Type 1 Replication within the gag-pol Transframe Domain

“…Targeting various regulatory genes of the HIV-1 genome has not always been effective against diverse HIV-1 strains (2, 64, 66, 83-85, 92, 141). The structural gag gene has also been targeted by antisense PsODN at a gag translation initiation site or various regions of capsid-encoding domain (4,5,66,86,141,148). Such an anti-gag antisense strategy, however, exhibited only a modest, at most, antiviral effect in tissue culture.…”

“…Antisense PsODN targeting the same sequence as PNA PR2 (PsODN PR2 ) did not show a substantial antiviral effect in chronically infected H9 LAI or H9 RF cells in our laboratory (data not shown). The antiviral effects of antisense PsODN are exhibited through several mechanisms, including sequence-specific suppression of transcription or translation and a sequence-independent inhibition of viral adsorption (83,85,148). Moreover, the translational suppression induced by antisense PsODN is predominantly mediated by degradation of target RNA by RNase H (9, 14, 139), unlike PNA, which blocks ribosomal elongation in an RNase H-independent manner (45).…”