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

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 gene(1-7). 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 member(8), and possibly a kinase(9) 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-nonspecific(12), 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 cells(12,13). 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 cells(12,13). 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.