Sustained Small Interfering RNA-Mediated HumanImmunodeficiency Virus Type 1 Inhibition in PrimaryMacrophages

Abstract

Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

“…Respectively, we found the intracellular concentration of stabilized siRNA to exceed that of conventional siRNA up to 80 times, even after activation of the T cells. Previous studies including our own unpublished observations did not reveal strong influence of siRNA stabilization on RNAi in long-term cultured cell lines [21,22]. In contrast, we observed a drastic prolongation of target gene silencing by stabilized siRNA in both, murine and human primary T cells.…”

Inhibition of HIV-1 replication with designed miRNAs expressed from RNA polymerase II promoters

“…[7][8][9][10][11] Most studies used polymerase (pol) III promoters to control the expression of the shRNA gene. 5,[12][13][14][15][16][17] The transcription initiation site of a pol III promoter is well defined and transcription termination occurs at a stretch of four consecutive T residues in the DNA template. 18 Well-defined ends of a shRNA are critical for Dicer-mediated processing to generate the mature siRNA.…”

RNA interference and potential therapeutic applications of short interfering RNAs

“…A recent study has demonstrated sustained siRNA-mediated inhibition of HIV type 1 in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. 89 Proof-of-principle experiments have also marked the therapeutic potential of short RNAs against a major class of neurodegenerative disorders, such as Huntington's disease, which are induced by polyglutamine toxicity. 75,90 Other studies have highlighted the potential of RNAi technologies for the treatment of cancer.…”