Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application

Key Points Small interfering RNAs (siRNAs) are widely used to study gene function and there is general excitement about their potential for therapeutic applications. However, several hurdles, including a lack of specificity, will have to be overcome before it is possible to use siRNAs therapeutically. siRNAs have multiple types of off-target effects, but there are several methods to help mitigate them. microRNA-like off-target effects refer to siRNA-induced sequence-dependent regulation of unintended transcripts, through partial sequence complementarity to their 3′ UTRs, to produce false-positive phenotypes. Such off-target effects follow the same dose response as on-target effects and therefore cannot be selectively eliminated by reducing siRNA concentration. Off-target siRNA effects are also species-specific. microRNA-like off-target effects can be mitigated by siRNA redundancy, siRNA pooling or chemical modification such as 2′- O -methyl modification. A significant obstacle to the therapeutic application of RNAi is the ability of siRNAs and/or their delivery vehicles (such as cationic lipids) to stimulate the innate immune system and trigger an inflammatory response through activation of Toll-like receptors. Immunostimulatory siRNAs can also produce unwanted toxicities, including elevated levels of serum alanine and aspartate aminotransaminases, and reduced numbers of lymphocytes and platelets. Immunostimulation by siRNAs can be reduced by sequence selection or chemical modification such as substitution of the 2′ position of ribose with 2′- O -methyl, 2′-fluoro, 2′-deoxy or locked nucleic acid. A novel non-specific effect of RNAi expression relates to saturation of the RNAi machinery; exogenous siRNAs can saturate the endogenous RNAi machinery causing widespread effects on microRNA processing and function. Understanding the sources of siRNA off-target activity is yielding insights into siRNA design and chemical modification. Much progress has been made in the design of effective siRNAs with reduced off-target liabilities and continued research in this area will undoubtedly lead to further improvements in the development of siRNAs for therapeutic applications. The therapeutic potential of gene silencing by RNA interference has generated considerable interest and investment, with clinical trials now in progress in various disease areas. However, achieving target specificity remains a key challenge. Here, Jackson and Linsley discuss the off-target effects