Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA

Abstract

ISIS 2922 is a phosphorothioate oligonucleotide that is complementary to human cytomegalovirus (CMV) immediate-early (IE) RNA and that exhibits potent and specific antiviral activity against CMV in cell culture assays. Specific assay systems were developed to separately characterize the antisense and nonantisense components of the antiviral activity mediated by ISIS 2922. In U373 cells transformed with cDNA encoding the CMV IE 55-kDa (IE55) protein, expression was inhibited at nanomolar concentrations comparable to effective concentrations in antiviral assays. The specificity of inhibition was demonstrated by using control oligonucleotides incorporating progressive base changes to destabilize oligonucleotide-RNA base pairing and by showing a lack of inhibition of the CMV IE72 product expressed from the same promoter. Inhibition of IE55 protein expression correlated with a reduction in mRNA levels consistent with an RNase H-mediated termination event. Studies with virus-infected cells demonstrated that antisense and nonantisense mechanisms contribute to the antiviral activity of ISIS 2922. Base complementarity to target RNA was important for optimal activity in antiviral assays, but base changes affecting parameters other than hybridization affinity also influenced antiviral activity. Sequence-independent inhibition of virus adsorption to host cells by phosphorothioate oligonucleotides was also observed at high concentrations. Therefore, at least three different mechanisms may contribute to the antiviral activity of ISIS 2922 in cell culture: antisense-mediated inhibition of target gene expression; nonantisense, sequence-dependent inhibition of virus replication; and sequence-independent inhibition of virus adsorption to host cells.

Cytomegalovirus Infection Decreases Expression of Thrombospondin‐1 and ‐2 in Cultured Human Retinal Glial Cells: Effects of Antiviral Agents

“…The antiviral effects of ganciclovir, ISIS 2922, ISIS 26062, SN50, and SN50M were tested in confluent cultures of retinal glial cells infected with the AD169 strain at an MOI of 2. Treatment with ISIS 2922 or ISIS 26062 was performed according to established protocols [30][31][32]. Briefly, cell monolayers were pretreated with oligonucleotides at different concentrations overnight in MEM containing 2% FBS and then were washed 3 times with PBS before virus infection.…”

Topical antisense-oligonucleotides targeting IFN-gamma mRNA improve incidence and severity of herpetic stromal keratitis by cytokine specific and sequence unspecific effects

“…A recent report described that repetitive motifs in telomeric DNA of mammals can suppress Th1 responses of the immune response by interference with the IFN-γ signalling [30]. Indeed, it was shown that the efficacy of ASON is often a mixture of diverse effects [1]. Unwanted side effects of ASON are known to be concentration-dependent [18], but for this model already very small amounts were effective to improve HSK.…”

Carbon nanotubes in drug delivery: focus on infectious diseases

“…Antisense oligonucleotides research is considered a very promising technology for use in the development of drugs with both high target specificity and reduced side effects. Studies have demonstrated antisense inhibition of viral gene expression in biochemical assays, in cultured cells and in animal models [85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102]. Currently, there is ongoing antisense oligonucleotides research targeting human cytomegalovirus and human immunodeficiency virus, which is being evaluated in clinical trials.…”

“…A targeted novel approach of delivering antisense therapy and other entities which are typically too problematic to deliver may be instrumental. Therefore achieving a combination of targeted delivery of oligonucleotides, other genetic disrupters and other viral targets not normally feasible with current systems may be advantageous [31,32,[85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102].…”

“…Current treatments have not demonstrated promising results, thus demanding innovative treatments [85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102]. One promising antiviral strategy implemented against HCV is the use of RNA interference (RNAi), a specific gene silencing process mediated by small interfering RNA (siRNA) duplexes.…”