Expression profiling reveals off-target gene regulation by RNAi

Jackson, Aimee L.; Bartz, Steven R.; Schelter, Janell M.; Kobayashi, Sumire; Burchard, Julja; Mao, Mao; Li, Bin; Cavet, Guy; Linsley, Peter S.

doi:10.1038/nbt831

Cited by 2,081 publications

(1,642 citation statements)

References 8 publications

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“…A primary source of sequence-dependent off-target effects, at least in mammalian systems, appears to be the relatively high tolerance for mismatches between the siRNA guide strand, the ultimate targeting molecule, and the complementary target mRNA sequence, outside of the short 'core' targeting region, that is, bases 2-8, known as the guide strand's 'seed region' 3,4 . Indeed, this type of partial complementarity has been shown to underlie the mechanism of target silencing exhibited by microRNAs (miRNAs; for example, see ref.…”

Section: Specificity Of Rnai Phenotypes: Experimental Controls Lead Tmentioning

confidence: 99%

Minimizing the risk of reporting false positives in large-scale RNAi screens

et al. 2006

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Section: Specificity Of Rnai Phenotypes: Experimental Controls Lead Tmentioning

confidence: 99%

Minimizing the risk of reporting false positives in large-scale RNAi screens

et al. 2006

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“…However, off-target translational repression in the case of RNAi led to the observation that shorter sequences, comprising as few as 7 contiguous complementary base pairs, may be sufficient for direct RNAi-mediated silencing, which seems particularly relevant in the case of microRNA (miRNA) action. [61][62][63][64] MicroRNAs miRNAs are endogenous non-coding small RNAs (19-23 nt) found in both animal and plant genomes. [65][66][67] In plants, miRNAs seem to bind to mRNA targets by perfect or nearly perfect complementarity, and cleave the target molecules.…”

Section: Rnai Mechanism Of Actionmentioning

confidence: 99%

“…Moreover, their results suggested that the most effective siRNA overhangs have a UU or TT sequence. Since then, most of the reported siRNA sequences have been 21 bp in length (although there have also been reports of effective 23 bp [123][124][125][126][127] The action of siRNA depends on many cellular proteins; therefore, various organisms may respond to siRNA in slightly different manners. As cellular antiviral systems are intertwined with the siRNA system, the innate immune system (specifically the interferon response and the NF-kB-modulated inflammation response through Toll-like receptors) may be stimulated by siRNA.…”

Section: Rnai and Functional Genomicsmentioning

confidence: 99%

siRNA-based approaches in cancer therapy

2006

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The availability of the human genome sequence has revolutionized the strategy of employing nucleic acids with sequences complementary to specific target genes to improve drug discovery and target validation. Development of sequence-specific DNA or RNA analogs that can block the activity of selected single-stranded genetic sequences offers the possibility of rational design with high specificity, lacking in many current drug treatments for various diseases including cancer, at relatively inexpensive costs. Antisense technology is one such example that has shown promising results and boasts of yielding the only approved drug to date in the genomics field. However, in vivo delivery issues have yet to be completely overcome for widespread clinical applications. In contrast to antisense oligonucleotides, the mechanism of silencing an endogenous gene by the introduction of a homologous double-stranded RNA (dsRNA), transgene or virus is called post-transcriptional gene silencing (PTGS) or RNA interference. PTGS is a natural mechanism whereby metazoan cells suppress expansion of genes when they come across dsRNA molecules with the same sequence. Short interfering RNA is currently the fastest growing sector of this antigene field for target validation and therapeutic applications. Although, in theory, the development of genomics-based agents to inhibit gene expression is simple and straightforward, the fundamental concern relies upon the capacity of the oligonucleotide to gain access to the target RNA. This paper summarizes the advances in the last decade in the field of PTGS using RNA interference approaches and provides relevant comparisons with other oligonucleotide-based approaches with a specific focus on oncology applications. Cancer Gene Therapy (2006) Genomic-based strategiesThe American Cancer Society estimates that, in 2005, a total of 1 372 910 new cancer cases and 570 280 deaths are expected in the United States. 1 These morbid statistics demonstrate the necessity of newer therapeutic modalities for achieving successful cancer treatment and cure. Downregulation of genes that contribute to cancer progression has been the goal of targeted genomics-based strategies, with the expectation that such an approach may lead to selective and specific inhibition of tumor growth with minimal untoward side effects on normal cells. Identification of target genes involved in neoplastic transformation and tumor progression has triggered the idea that nucleotide sequences of cancer-relevant genes could lead to the development of tailored anticancer agents that lack many of the toxic side effects of traditional cytotoxic drugs. This has led to a recent acceleration in the development and optimization of genomic-based strategies for cancer therapy. This idea dates back to the 1960s when RNA sequences were shown to serve as endogenous inhibitors of gene expression in procaryotes. The antigene approaches include the following:(1) Ribozymes: 2,3 These were discovered in the 1970s, and the elucidation of the transactivating hammerh...

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“…Thus, for clinically significant OI caused by structural defects in collagen, suppression of the Mut allele could in principle convert severe types III and IV to the minimally symptomatic OI type I, a situation particularly favorable to therapeutic strategies based on gene silencing. The promising preliminary data on stem cell transplantation in both murine models [18][19][20] and human OI 21,22 contributes to an appealing possibility for correcting the patient's own stem cells in vitro, and transplanting them back to the patient, after checking chromosomal rearrangements 23 or off-target effects 24 in vitro.…”

Section: Introductionmentioning

confidence: 99%

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

et al. 2013

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Gene silencing approaches have the potential to become a powerful curative tool for a variety of monogenic diseases caused by gain-of-function mutations. Classical osteogenesis imperfecta (OI), a dominantly inherited bone dysplasia, is characterized in its more severe forms by synthesis of structurally abnormal type I collagen, which exerts a negative effect on extracellular matrix. Specific suppression of the mutant (Mut) allele would convert severe OI forms to the mild type caused by a quantitative defect in normal collagen. Here, we describe the in vitro and ex vivo investigation of a small interfering RNA (siRNA) approach to allele-specific gene silencing using Mut Col1a1 from the Brtl mouse, a well-characterized model for classical human OI. A human embryonic kidney cell line, which expresses the firefly luciferase gene, combined with either wild-type or Mut Brtl Col1a1 exon 23 sequences, was used for the first screening. The siRNAs selected based on their specificity and the corresponding short hairpin RNAs (shRNAs) subcloned in a lentiviral vector were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction of the Mut allele of up to 52% was associated with about 40% decrease of the Mut protein, with no alteration of cell proliferation. Interestingly, a downregulation of HSP47, a specific collagen chaperone known to be upregulated in some OI cases, was detected. Our data support further testing of shRNAs and their delivery by lentivirus as a strategy to specifically suppress the Mut allele in mesenchymal stem cells of OI patients for autologous transplantation.

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Expression profiling reveals off-target gene regulation by RNAi

Cited by 2,081 publications

References 8 publications

Minimizing the risk of reporting false positives in large-scale RNAi screens

Minimizing the risk of reporting false positives in large-scale RNAi screens

siRNA-based approaches in cancer therapy

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

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