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Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells

Abstract

RNA interference (RNAi) is the process of sequence-specific, posttranscriptional gene silencing in animals and plants initiated by double-stranded (ds) RNA that is homologous to the silenced gene1,2,3,4,5,6,7. This technology has usually involved injection or transfection of dsRNA in model nonvertebrate organisms. The longer dsRNAs are processed into short (19–25 nucleotides) small interfering RNAs (siRNAs) by a ribonucleotide–protein complex that includes an RNAse III–related nuclease (Dicer)7, a helicase family member8, and possibly a kinase9 and an RNA-dependent RNA polymerase (RdRP)10,11. In mammalian cells it is known that dsRNA 30 base pairs or longer can trigger interferon responses that are intrinsically sequence-nonspecific12, thus limiting the application of RNAi as an experimental and therapeutic agent. Duplexes of 21-nucleotide siRNAs with short 3′ overhangs, however, can mediate RNAi in a sequence-specific manner in cultured mammalian cells12,13. One limitation in the use of siRNA as a therapeutic reagent in vertebrate cells is that short, highly defined RNAs need to be delivered to target cells—a feat thus far only accomplished by the use of synthetic, duplex RNAs delivered exogenously to cells12,13. In this report, we describe a mammalian Pol III promoter system capable of expressing functional double-stranded siRNAs following transfection into human cells. In the case of the 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, we were able to achieve up to 4 logs of inhibition of expression from the HIV-1 DNA.

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Figure 1: Target rev-EGFP and U6 promoter-driven siRNA constructs.
Figure 2: (A) Fluorescence imaging of the effect of siRNA on EGFP expression.
Figure 3: Northern gel analyses.

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Acknowledgements

We thank Aaron Coleman for providing pIND and G. Pavlakis and A. Michienzi for the CMV-rev-EGFP vector. The authors also wish to thank Alessandra Poggi for help with some preliminary experiments. This research was supported by grants from the National Institutes of Health AI 29329, AI42552 and AI 46030.

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Correspondence to John Rossi.

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The authors have filed a patent application covering the technology described in this manuscript. The patent, if issued, would be owned by the Beckman Research Institute of the City of Hope, a not-for-profit organization.

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Lee, N., Dohjima, T., Bauer, G. et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 20, 500–505 (2002). https://doi.org/10.1038/nbt0502-500

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