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Asymmetric siRNA: new strategy to improve specificity and reduce off-target gene expression.

ABSTRACT: Small interfering RNAs (siRNAs) have been used extensively in reverse genetic research, and many have made their way into clinical trials. The most widely used siRNA structure consists of double-stranded RNA with 19 base pairs and 2-nucleotide overhangs at the 3'-end of both strands (19+2). Although widely used, this symmetric structure bears inherent disadvantages in both research and clinical applications. One of the most common caveats is the off-target effect leading to adverse effects in clinical application. In the current study, using C-C chemokine receptor (CCR5) as a target, we have shown that 19+2 siRNA could still cause considerable global off-target effects regardless of rational design based on its thermodynamic asymmetry. However, we demonstrated that structurally asymmetric siRNA targeting CCR5 could be adopted to improve the strand specificity and greatly reduce the off-target effects without significantly compromising its on-target effects. Data from microarray analysis suggest that an unidentified mechanism resulting in global gene down-regulation might be avoided through strand shortening. Taken together, our work suggested a promising and simple way to improve strand specificity and overcome the off-target gene-expression effects without introducing more complications while retaining the efficacy of siRNA.

SUBMITTER: Yuan Z

PROVIDER: S-EPMC3360511 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Asymmetric siRNA: new strategy to improve specificity and reduce off-target gene expression.

Yuan Zhongping Z Wu Xilin X Liu Chao C Xu Genxing G Wu Zhiwei Z

Human gene therapy 20120309 5

Small interfering RNAs (siRNAs) have been used extensively in reverse genetic research, and many have made their way into clinical trials. The most widely used siRNA structure consists of double-stranded RNA with 19 base pairs and 2-nucleotide overhangs at the 3'-end of both strands (19+2). Although widely used, this symmetric structure bears inherent disadvantages in both research and clinical applications. One of the most common caveats is the off-target effect leading to adverse effects in cl ...[more]

PMID: 22229698

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Project description:BackgroundA direct and independent role of inflammation in atherothrombosis was recently highlighted by the Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) trial, showing the benefit of inhibiting signaling molecules, eg, interleukins. Accordingly, we sought to devise a flexible platform for preventing the inflammatory drivers at their source to preserve plaque endothelium and mitigate procoagulant risk.Methodsp5RHH-siRNA nanoparticles were formulated through self-assembly processes. The therapeutic efficacy of p5RHH-JNK2 siRNA nanoparticles was evaluated both in vitro and in vivo.ResultsBecause JNK2 is critical to macrophage uptake of oxidized lipids through scavenger receptors that engender expression of myriad inflammatory molecules, we designed an RNA-silencing approach based on peptide-siRNA nanoparticles (p5RHH-siRNA) that localize to atherosclerotic plaques exhibiting disrupted endothelial barriers to achieve control of JNK2 expression by macrophages. After seven doses of p5RHH-JNK2 siRNA nanoparticles over 3.5 weeks in ApoE-/- mice on a Western diet, both JNK2 mRNA and protein levels were significantly decreased by 26% (P=0.044) and 42% (P=0.042), respectively. Plaque-macrophage populations were markedly depleted and NF?B and STAT3-signaling pathways inhibited by 47% (P<0.001) and 46% (P=0.004), respectively. Endothelial barrier integrity was restored (2.6-fold reduced permeability to circulating 200 nm nanoparticles in vivo, P=0.003) and thrombotic risk attenuated (200% increased clotting times to carotid artery injury, P=0.02), despite blood-cholesterol levels persistently exceeding 1,000 mg/dL. No adaptive or innate immunoresponses toward the nanoparticles were observed, and blood tests after the completion of treatment confirmed the largely nontoxic nature of this approach.ConclusionThe ability to formulate these nanostructures rapidly and easily interchange or multiplex their oligonucleotide content represents a promising approach for controlling deleterious signaling events locally in advanced atherosclerosis.

Project description:The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.

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Asymmetric siRNA: new strategy to improve specificity and reduce off-target gene expression.

Publications

Asymmetric siRNA: new strategy to improve specificity and reduce off-target gene expression.

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