Therapeutic application of RNAi: is mRNA targeting finally ready for prime time?

Grimm, Dirk; Kay, Mark A.

doi:10.1172/jci34129

Cited by 138 publications

(79 citation statements)

References 78 publications

(113 reference statements)

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“…[1][2][3][4] RNAi technology is commonly used in reverse genetics approaches to study gene function and to show targets of therapeutic potential in cancer. [5][6][7] Several synthetic methods of silencing gene expression integral to disease phenotype have been developed. 8,9 Short hairpin RNA (shRNA) transcribed from an expression vector intrinsically differs from synthetic double-stranded small interfering RNA (siR-NA) with respect to intracellular trafficking and nucleotide preference 10 and can result in enhanced gene knockdown effects.…”

Section: Introductionmentioning

confidence: 99%

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

et al. 2010

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RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel 'bifunctional' RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations.

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Section: Introductionmentioning

confidence: 99%

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

et al. 2010

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“…The lethality did not appear to be target or sequence specific but was dose dependent. 8 This effect was later found to be caused by overexpression of shRNA saturating the endogenous miRNA pathway, particularly the exportin-5-mediated nuclear export and the downstream RNA-induced silencing complex component, Argonaute-2, 9 resulting in excess and toxic concentrations of shRNA competing with endogenous miRNAs. This lethal effect was alleviated by the use of an RNA polymerase II promoter-driven shRNA expression cassette that does not overwhelm the miRNA pathway and exportin-5-mediated nuclear export and allows for greater regulation.…”

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confidence: 99%

Comparative assessment of siRNA and shRNA off target effects: what is slowing clinical development

et al. 2009

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This review considers comparisons of the off-target effects of siRNA to shRNA and their potential impact on the efficacy and toxicity of RNAi based therapeutics. Cancer Gene Therapy (2009) 16, 807-809; doi:10.1038/cgt.2009 published online 28 August 2009 Keywords: RNAi; siRNA; shRNA; miRNA; cancer therapyThe discovery of RNA interference (RNAi), 1 an innate biological process through which the expression of specifically targeted genes can be modulated and/or silenced, ushered in a new world of research and potential therapeutic applications thereby setting the stage for a paradigm shift in oncology. Targeted gene knockdown can be used as a preclinical tool for reverse genetics to determine the function of a given gene as well as delineating its functional connectivity. In the therapeutic arena, the potential of targeted therapy with high specificity can finally be considered a realizable goal. The effect of RNAi and associated technical advances on the scientific community is attested to by more than 20 000 related publications in the PubMed database. A number of in vivo animal studies have shown the activity of RNAi technology implying likely potential for therapeutic efficacy in humans; in fact, a few RNAibased therapeutic strategies are currently being tested through clinical trials. 2 RNA interference holds strong promise for cancer gene therapy where differentially expressed genes, designated as malignancy process-dependent (drivers 3,4 ) by virtue of high connectivity and, most likely, 'in-betweenness', can be targeted as a presumptive, individual tumor's 'Achilles' heel'.5 RNAi can be effected through two types of molecules; a synthetic small interfering RNA (siRNA) and a vector-based short hairpin RNA (shRNA). siRNAs are 21-23 nucleotide (nt), double-stranded RNAs with 2-nt overhangs on the 3 0 ends capable of being incorporated into an RNA-induced silencing complex, whereas shRNAs are composed of RNA folded into stem-loop structures similar to pre-miRNA hairpins requiring processing by DICER before incorporation into the RNA-induced silencing complex. Although both siRNA and shRNA are reported to be able to achieve target-specific silencing, insofar as they are mechanistically different, one could postulate varying levels of therapeutic effectiveness based on considerations of both silencing efficiency and off-target effects (as a safety parameter).Soon after the discovery, in fact, the dream of target specificity was somewhat dampened by the finding of offtarget effects associated with the applications of RNAi. Induction of interferon in cells transfected with siRNA was the first reported off-target effect of RNAi. siRNA duplexes longer than 29-30 bp can readily induce a cellular interferon response similar to double-stranded RNA. However, structural modification or shortening the length of the siRNA in large part abrogates interferon induction.6 There are also recent reports of siRNA-or shRNA-induced stimulation of Toll-like receptors (TLRs) including the membrane-bound TLR4 and endosomal TLR3 (dsR...

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“…It can also be obtained by in vivo synthesis through siRNA expression using plasmid or viral vectors, or through a siRNA expression cassette prepared by PCR. 12 Among these cassettes, the adenoviral vector for intracellular siRNA expression is convenient and inexpensive, making it one of the most widely used biological viral vectors. It has many advantages, including a wide host range, low pathogenicity for humans and effective proliferation.…”

Section: Discussionmentioning

confidence: 99%

Screening siRNAs targeting a novel gene (HA117) and the development of a derivative recombinant adenovirus delivery system

et al. 2011

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A novel gene, HA117, was discovered in our previous work. Using the pSOS-HUS vector method which we designed at previous study, we screened for small interfering RNAs (siRNAs) that targeted HA117. The pSOS-HUS siRNA screening results were verified and a delivery system was developed that contained a recombinant adenovirus carrying DNA templates for the transcription of the HA117 siRNAs. Of five pairs of DNA templates, siRNA transcribed from HAi5 produced the strongest effect against HA117. A recombinant adenovirus containing HAi5 (Ad-HAi5) was successfully constructed and evaluated. This work has laid the foundation for further study of HA117 gene function using RNA interference technology and has showed the pSOS-HUS vector method was successfully utilized as a rapid and effective screen of siRNAs for a target gene.

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Therapeutic application of RNAi: is mRNA targeting finally ready for prime time?

Cited by 138 publications

References 78 publications

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

Comparative assessment of siRNA and shRNA off target effects: what is slowing clinical development

Screening siRNAs targeting a novel gene (HA117) and the development of a derivative recombinant adenovirus delivery system

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