U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells

Miyagishi, Makoto; Taira, Kazunari

doi:10.1038/nbt0502-497

Cited by 672 publications

(392 citation statements)

References 19 publications

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“…Numerous additional reports indicate that certain individuals or groups are immune to HIV-1 altogether (Baur et al 1989, Bryson et al 1995, Deacon et al 1995, Roques et al 1995, Paxton et al 1996, Kaul et al 2000, Kulkarni et al 2003, while others remain AIDS free for extensive periods of time . In order to develop sufficient human protection against HIV-1 variants, a number of homologous sequences will be required in the endogenous repertoire (Hiroshika et al 2000, Donze & Picard 2002, Hohjoh 2002, Jeong et al 2002, Leirdal & Sioud 2002, Miyagishi & Taira 2002, Paddison et al 2002, Paul et al 2002, Sui et al 2002, Yu et al 2002. Through genetic technology, however, a rapid HIV-1 inhibitory siRNAs specific repertoire can perhaps be designed.…”

Section: Role Of Sirnas In Development Of Hiv-1 Vaccinementioning

confidence: 99%

“…Through genetic technology, however, a rapid HIV-1 inhibitory siRNAs specific repertoire can perhaps be designed. These siRNAs could be encoded and delivered by appropriate vectors and then utilized in preventative or therapeutic vaccines (Hiroshika et al 2000, Donze & Picard 2002, Hohjoh 2002, Jeong et al 2002, Leirdal & Sioud 2002, Miyagishi & Taira 2002, Paddison et al 2002, Paul et al 2002, Sui et al 2002, Yu et al 2002.…”

Section: Role Of Sirnas In Development Of Hiv-1 Vaccinementioning

confidence: 99%

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RNA interference: The molecular immune system

Bagasra

Prilliman²

2004

Histochem J

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SummaryIntroduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS). The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer. siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo-and endo-nucleases as well as other proteins. Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner. RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals. In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression. In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21-to 23-nt-long siRNAs. However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi. siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells. Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements. Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described. Recent data in C. elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed. However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined. Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells. RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins. RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla. We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements. The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed.

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Section: Role Of Sirnas In Development Of Hiv-1 Vaccinementioning

confidence: 99%

Section: Role Of Sirnas In Development Of Hiv-1 Vaccinementioning

confidence: 99%

RNA interference: The molecular immune system

Bagasra

Prilliman²

2004

Histochem J

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“…19,20 Of note, though, this amplification step has not been demonstrated in mammals, and recent initial data suggest that it may not take place in either mammals or Drosophila. 21 siRNAs, which seem to always require short 3 0 overhangs, 3,22 were first shown to be useful in altering gene product expression by transfection into invertebrates. These siRNAs can be chemically synthesized or created via in vitro transcription systems, and maintain their activity after introduction into mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of HIV-1 fusion with small interfering RNAs targeting the chemokine coreceptor CXCR4

et al. 2004

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RNA interference (RNAi) is an evolutionarily conserved process by which plants and animals protect their genomes utilizing small, double-stranded RNAs to degrade target RNAs in a sequence-specific manner. Post-transcriptional gene silencing by these moieties can lead to degradation of both cellular and viral RNAs. It has recently been shown that double-stranded, small interfering RNAs (siRNAs) of 21-25 nucleotides can be transfected into relevant cells to target specific RNAs. This approach was utilized to inhibit human immunodeficiency virus type I (HIV-1) infection in human cells. siRNAs with homology to a motif in the mRNA that encodes for the HIV-1 chemokine coreceptor CXCR4 was utilized. Complementary studies via immunofluorescence microscopy and fluorescence-activated cell sorting demonstrated downregulation of CXCR4 from the surface of cells transfected with the specific siRNAs. As well, siRNAs without sequence homology to CXCR4 were used as controls and demonstrated no downregulation of CXCR4. siRNAs targeted to another chemokine coreceptor, APJ, showed specificity for downregulation of APJ but had no effects on CXCR4.Transfections with siRNAs targeting CXCR4 mRNA were shown to inhibit HIV-1 envelope fusion, which is relatively resistant to most viral inhibitors targeting chemokine coreceptors. The specificity of this effect was demonstrated by the inhibition of fusion by CXCR4-tropic and dual-tropic (CXCR4 and CCR5) envelope glycoproteins from HIV-1 on CXCR4+ indicator cells, but the lack of effects by siRNAs targeting CXCR4 mRNA on dual-tropic HIV-1 envelopes in CCR5+ indicator cells utilizing these fusion assays. Interestingly, siRNAs targeting CXCR4 selectively inhibited CXCR4-tropic cell-free virus infection of human cells but at only modest levels as compared to cell:cell fusion. siRNA may be a potential molecular therapeutic approach to alter a cellular cofactor critical for infection of human cells by relevant strains of HIV-1. The targeting of a cellular cofactor, rather than the HIV-1-specific mRNAs or genomic RNA, holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi directly against this virus.

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“…27,28 Upon expression in the cell, the complementary strands anneal to form siRNA molecules with overhanging 3 0 ends. Probably due to the lower efficiency of requiring two separate RNA strands to find and anneal to each other, these dual expression cassette designs have been found to be generally less effective than hairpin designs, though they can achieve substantial gene inhibition.…”

Section: Codon Optimization and Codon Pairing Will Affect Transcriptimentioning

confidence: 99%

Gene Therapy Progress and Prospects: Recent progress in transgene and RNAi expression cassettes

Ill¹,

Chiou²

2005

Gene Ther

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Plasmid expression cassette design must include a thoughtful analysis of potentially every nucleotide comprising a covalently closed circular or end-protected linear DNA. This review will discuss recent studies in unraveling the mechanisms of postdelivery gene silencing, codon optimization and promoter identification. The recent discovery of potent RNA interference (RNAi) mechanisms for sequence-specific gene silencing has also invoked a great deal of interest in development of expression cassettes that can produce double-stranded RNA molecules for RNAi. Expression cassettes based on both RNA polymerase II and polymerase III transcription units that generate double-stranded RNA molecules for RNAi will also be discussed. Gene Therapy (2005) Whether a mammalian cell expression cassette is produced by bacterial amplification, minicircle recombination, or by purely synthetic means, the primary DNA

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U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells

Cited by 672 publications

References 19 publications

RNA interference: The molecular immune system

RNA interference: The molecular immune system

Inhibition of HIV-1 fusion with small interfering RNAs targeting the chemokine coreceptor CXCR4

Gene Therapy Progress and Prospects: Recent progress in transgene and RNAi expression cassettes

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