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.

Abstract

Small interfering RNAs (siRNAs) are widely used to study gene function owing to the ease with which they silence target genes, and there is considerable interest in their potential for therapeutic applications. In a remarkably short time since their discovery, siRNAs have entered human clinical trials in various disease areas. However, rapid acceptance of the use of siRNAs has been accompanied by recognition of several hurdles for the technology, including a lack of specificity. Off-target activity can complicate the interpretation of phenotypic effects in gene-silencing experiments and can potentially lead to unwanted toxicities. Here, we describe the types of off-target effects of siRNAs and methods to mitigate them, to help enable effective application of this exciting technology.

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Figure 1: Types of off-target effects observed with small interfering RNAs.
Figure 2: Immune responses to small interfering RNAs.

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FURTHER INFORMATION

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Glossary

RNA interference

(RNAi). A fundamental pathway in eukaryotic cells by which a short piece of RNA induces the destruction of mRNA(s) with sequence complementarity.

Small interfering RNA

(siRNA). RNA fragments (typically exogenous), 21–23 nt long, that induce the sequence-dependent destruction of complementary mRNA.

microRNA

Genomically-encoded non-coding RNAs that regulate gene expression through a mechanism that involves the inhibition of translation and transcript degradation.

Guide strand

The strand of the siRNA or microRNA that is complementary to the target mRNA sequence(s).

RNA-induced silencing complex (RISC)

A multiprotein complex that incorporates one strand of an siRNA or microRNA as a template for targeting a complementary mRNA and is responsible for the destruction of the target mRNA.

Seed region

The portion of the guide strand implicated in target recognition; it encompasses positions 1–8 at the 5′ end of the guide strand.

Lipid nanoparticle

A drug delivery vehicle made of lipids that can encapsulate the siRNA or small molecule for delivery across the cell membrane.

Immunostimulatory

Capable of activating the innate immune response for host defense against invading pathogens.

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Jackson, A., Linsley, P. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov 9, 57–67 (2010). https://doi.org/10.1038/nrd3010

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