siSPOTR: a tool for designing highly specific and potent siRNAs for human and mouse

Abstract

RNA interference (RNAi) serves as a powerful and widely used gene silencing tool for basic biological research and is being developed as a therapeutic avenue to suppress disease-causing genes. However, the specificity and safety of RNAi strategies remains under scrutiny because small inhibitory RNAs (siRNAs) induce off-target silencing. Currently, the tools available for designing siRNAs are biased toward efficacy as opposed to specificity. Prior work from our laboratory and others’ supports the potential to design highly specific siRNAs by limiting the promiscuity of their seed sequences (positions 2–8 of the small RNA), the primary determinant of off-targeting. Here, a bioinformatic approach to predict off-targeting potentials was established using publically available siRNA data from more than 50 microarray experiments. With this, we developed a specificity-focused siRNA design algorithm and accompanying online tool which, upon validation, identifies candidate sequences with minimal off-targeting potentials and potent silencing capacities. This tool offers researchers unique functionality and output compared with currently available siRNA design programs. Furthermore, this approach can greatly improve genome-wide RNAi libraries and, most notably, provides the only broadly applicable means to limit off-targeting from RNAi expression vectors.

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

“…Although there has been much progress in siRNA design to avoid immunogenicity, toxicity, and off-target effects (4,8,11,17,19,23,36,50,62), the use of loss of function through RNAi has been limited in polarized, pseudostratified ALI epithelial cultures because of inefficiencies in delivery and the presence of barriers for entry of RNAi reagents (4,8,11,17,19,23,36,50,62). To overcome this limitation we developed and applied a simple approach that exploits the susceptibility of cells to transfection at the time of plating.…”

RNAi or overexpression: Alternative therapies for Spinocerebellar Ataxia Type 1

“…While earlier work in our group demonstrated short term efficacy using an RNAi approach in the B05 model (Xia et al, 2004), we have substantially improved the safety of vector-based platforms by moving from shRNA systems to those based on endogenous miRNA backbones (Boudreau and Davidson, 2012; Boudreau et al, 2008, 2009a, 2012; McBride et al, 2008). For this work, we also took advantage of recent progress in minimizing off-sequence silencing to design an RNAi trigger that would target human ataxin-1 but have low off-target potential (Boudreau et al, 2009a, 2011).…”

Gene Therapy for Dominantly Inherited Retinal Degeneration

“…With regard to potency and longevity, synthesized RNAi can be chemically modifi ed to increase longevity and to optimize cell transfection or viral and nonviral vectors can provide a sustained supply of RNAibased suppression (Burnett and Rossi 2012 ;Bhavsar et al 2012 ;Bramsen and Kjems 2013 ). In parallel, methodologies to minimize risk of toxicity associated with off-target effects of RNAi and/or recruitment and saturation of the endogenous cellular machinery have been developed (Birmingham et al 2007 ;Jackson and Linsley 2010 ;Boudreau et al 2013 ). Recently, singlestranded RNAi (ssRNAi) has been used to recruit the endogenous RNAi machinery and in principle should eliminate the risk of the sense strand of dsRNA entering RISC thereby minimizing potential off-targets (Lima et al 2012 ;Liu et al 2013 ).…”