Nature Biotechnology
20, 500 - 505 (2002)
doi:10.1038/nbt0502-500
Expression of small interfering RNAs targeted against HIV-1 rev
transcripts in human cellsNan Sook Lee1, Taikoh Dohjima1, Gerhard Bauer1, Haitang Li1, Ming-Jie Li1, Ali Ehsani1, 3, Paul Salvaterra2, 3
& John Rossi1, 31
Division of Molecular Biology, Graduate School of
Biological Sciences, City of Hope, Duarte, CA
91010. 2
Division of Neuroscience, Graduate School of
Biological Sciences, City of Hope, Duarte, CA
91010. 3
Division of Beckman Research Institute of the City
of Hope, Graduate School of Biological Sciences, City of Hope,
Duarte, CA 91010.
Correspondence should be addressed to John Rossi jrossi@bricoh.eduRNA 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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