Báo cáo khoa học: Staying on message: design principles for controlling nonspecific responses to

MINIREVIEW

Staying on message: design principles for controlling

nonspecific responses to siRNA

Shirley Samuel-Abraham1 and Joshua N. Leonard1,2

1 Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA

2 Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA

Introduction

In the decade since RNA interference (RNAi) was

initially discovered in Caenorhabditis elegans [1] and

shown to be inducible in mammalian cells [2,3],

technologies for harnessing this mechanism to induce

targeted gene silencing have become routine laboratory

tools and, increasingly, are making their way into

clinical trials (reviewed in Castanotto & Rossi [4]).

Over this same period, however, it has become clear

that the short interfering RNA (siRNA) commonly

delivered to induce RNAi can also induce multiple

nonspecific effects. A poignant example comes from the

first system for which clinical trials of RNAi were

Keywords

innate immunity; OAS1; off-target; RIG-I;

RNA interference; RNAi; short interfering

RNA; siRNA; TLR; Toll-like receptors

Correspondence

J. N. Leonard, Department of Chemical and

Biological Engineering, Northwestern

University, 2145 Sheridan Rd, Room E-136,

Evanston, IL 60208 USA

Fax: +1 847 491 3728

Tel: +1 847 491 7455

E-mail: [email protected]

(Received 7 July 2010, accepted 26 August

2010)

doi:10.1111/j.1742-4658.2010.07905.x

Short interfering RNAs (siRNA) are routinely used in the laboratory to

induce targeted gene silencing by RNA interference, and increasingly, this

technology is being translated to the clinic. However, there are multiple

mechanisms by which siRNA may be recognized by receptors of the innate

immune system, including both endosomal Toll-like receptors and cytoplas￾mic receptors. Signaling through these receptors may induce multiple non￾specific effects, including general reductions in gene expression and the

production of type I interferons and inflammatory cytokines, which can

lead to systemic inflammation in vivo. The pattern of immune activation

varies depending upon the types of cells and receptors that are stimulated

by a particular siRNA. Although we are still discovering the mechanisms

by which these recognition events occur, our current understanding pro￾vides useful guidelines for avoiding immune activation. In this minireview,

we present a design-based approach for developing siRNA-based experi￾ments and therapies that evade innate immune recognition and control

nonspecific effects. We describe strategies and trade-offs related to siRNA

design considerations including the choice of siRNA target sequence, chem￾ical modifications to the RNA backbone and the influence of the delivery

method on immune activation. Finally, we provide suggestions for conduct￾ing appropriate controls for siRNA experiments, because some commonly

employed strategies do not adequately account for known nonspecific

effects and can lead to misinterpretation of the data. By incorporating

these principles into siRNA design, it is generally possible to control

nonspecific effects, and doing so will help to best utilize this powerful

technology for both basic science and therapeutics.

Abbreviations

dsRNA, double-stranded RNA; GFP, greem fluorescent protein; IFN, interferon; IL, interleukin; OAS1, 2¢-5¢-oligoadenylate synthetase;

PKR, protein kinase R; RIG-I, retinoic acid-inducible gene I; RISC, RNA-induced silencing complex; RNAi, RNA interference; siRNA,

short interfering RNA; ssRNA, single-stranded RNA; TLR, Toll-like receptor.

4828 FEBS Journal 277 (2010) 4828–4836 ª 2010 The Authors Journal compilation ª 2010 FEBS