Comparative Assessment of Sirna and Shrna Off Target Effects

Mini Review Comparative assessment of siRNA and shRNA off target effects: what is slowing clinical development DD Rao1, N Senzer1,2, MA Cleary3 and J Nemunaitis1,2 1Gradalis Inc., Dallas, TX, USA; 2Mary Crowley Cancer Research Centers, Dallas, TX, USA and 3Rosetta Inpharmatics LLC, a wholly owned subsidiary of Merck & Co. Inc., Seattle, WA, USA

This review considers comparisons of the off-target effects of siRNA to shRNA and their potential impact on the efficacy and toxicity of RNAi based therapeutics. Cancer Gene Therapy (2009) 16, 807–809; doi:10.1038/cgt.2009.53; published online 28 August 2009 Keywords: RNAi; siRNA; shRNA; miRNA; cancer therapy

The discovery of RNA interference (RNAi),1 an innate processing by DICER before incorporation into the biological process through which the expression of RNA-induced silencing complex. Although both specifically targeted genes can be modulated and/or siRNA and shRNA are reported to be able to silenced, ushered in a new world of research and potential achieve target-specific silencing, insofar as they are therapeutic applications thereby setting the stage for a mechanistically different, one could postulate varying paradigm shift in oncology. Targeted gene knockdown levels of therapeutic effectiveness based on considerations can be used as a preclinical tool for reverse genetics to of both silencing efficiency and off-target effects (as a determine the function of a given gene as well as safety parameter). delineating its functional connectivity. In the therapeutic Soon after the discovery, in fact, the dream of target arena, the potential of targeted therapy with high specificity was somewhat dampened by the finding of off- specificity can finally be considered a realizable goal. target effects associated with the applications of RNAi. The effect of RNAi and associated technical advances on Induction of interferon in cells transfected with siRNA the scientific community is attested to by more than was the first reported off-target effect of RNAi. siRNA 20 000 related publications in the PubMed database. A duplexes longer than 29–30 bp can readily induce a number of in vivo animal studies have shown the activity cellular interferon response similar to double-stranded of RNAi technology implying likely potential for RNA. However, structural modification or shortening the therapeutic efficacy in humans; in fact, a few RNAi- length of the siRNA in large part abrogates interferon based therapeutic strategies are currently being tested induction.6 There are also recent reports of siRNA- or through clinical trials.2 shRNA-induced stimulation of Toll-like receptors (TLRs) RNA interference holds strong promise for cancer gene including the membrane-bound TLR4 and endosomal therapy where differentially expressed genes, designated TLR3 (dsRNA), TLR7 and 8 (ssRNA) and TLR9 as malignancy process-dependent (drivers3,4) by virtue of (unmethylated CpG), and downstream immune (includ- high connectivity and, most likely, ‘in-betweenness’, can ing interferon) responses.7 The use of unmethylated CpG- be targeted as a presumptive, individual tumor’s ‘Achilles’ free plasmid or minimal plasmid vector attenuates this heel’.5 RNAi can be effected through two types of potentially toxic effect. molecules; a synthetic small interfering RNA (siRNA) A recent murine study has shown that shRNA and a vector-based short hairpin RNA (shRNA). siRNAs expression driven by highly active RNA polymerase III are 21–23 nucleotide (nt), double-stranded RNAs with type promoters (U6 or H1) delivered through liver- 2-nt overhangs on the 30 ends capable of being incor- directed adeno-associated virus can result in significant porated into an RNA-induced silencing complex, whereas lethality. The lethality did not appear to be target or shRNAs are composed of RNA folded into stem-loop sequence specific but was dose dependent.8 This effect was structures similar to pre-miRNA hairpins requiring later found to be caused by overexpression of shRNA saturating the endogenous miRNA pathway, particularly the exportin-5-mediated nuclear export and the down- Correspondence: Dr J Nemunaitis, Executive Medical Director, stream RNA-induced silencing complex component, Mary Crowley Cancer Research Centers, 1700 Pacific Ave., Suite 9 1100, Dallas, TX 75201, USA. Argonaute-2, resulting in excess and toxic concentrations E-mail: [email protected] of shRNA competing with endogenous miRNAs. This Received 9 March 2009; revised 27 June 2009; accepted 8 July 2009; lethal effect was alleviated by the use of an RNA poly- published online 28 August 2009 merase II promoter-driven shRNA expression cassette Efficacy and toxicity of siRNA vs shRNA therapy DD Rao et al 808 that does not overwhelm the miRNA pathway and related to mechanistic differences in the manner in which exportin-5-mediated nuclear export and allows for greater these molecules enter the RNAi pathway.14 Using RNA regulation.10 polymerase II promoter, the primary transcript of shRNA All RNAi-based therapeutics have off-target effects is polyadenylated, similar to pri-miRNA hairpins and that impact transcript expression and translation resulting initially processed in the nucleus by complex with in off-target phenotypes.11 siRNAs can act similarly to Drosha/DGCR8 and, therefore, subject to normal cellular miRNAs as a result of complementarity of their seed regulatory processes of nuclear export and RNA-induced region sequence (positions 2–7) to unintended mRNA silencing complex loading in the cytoplasm.15 In contrast, sites producing off-target effect in a dose–responsive siRNA, which is not integrated into nuclear and early manner.12 This ‘miRNA type’ of off-target effect is cytoplasmic processing and requires higher concentra- elicited by both the passenger strand and the guide tions and more frequent dosing to achieve similar levels strand. In addition, short mismatches between siRNA of knockdown, is more likely to result in off-target effects. sequence and unintended mRNA sequences can result In addition, unprotected siRNA in the cytoplasm may in off-target knockdown. Chemical modification of the be more susceptible to degradation and metabolic seed region sequence of the guide strand (particularly processes, which may further contribute to nonspecific position 2) significantly improves target specificity.13 target recognition. Some researchers have taken a further Comparison of the off-target effects of siRNA to shRNA step to alleviate toxicity and enhance specificity by has uncovered differences in gene expression profiles embedding shRNA sequences into miRNA contexts, such produced by siRNA and shRNA effectors with identical as the miR-30 stem loop precursor, to create artificial pri- nucleotide targeting sequences. In general, when induced miRNA transcripts.16 It remains to be seen whether such from an integrated vector, shRNA produces less up- and an approach has an impact on the overall off-target downregulation of off-target transcripts than a corres- regulations caused by shRNA. ponding siRNA dosed to achieve a comparable level of As our understanding of the RNAi process expands target knockdown (M Mehaffey, T Ward and M Cleary, and criteria for higher affinity and target-specific effectors in preparation, Figure 1). This difference in off-target broaden, the potential of a paradigm shift in oncologi- activity of shRNA in comparison with siRNA is likely cal therapeutics becomes a more foreseeable reality.

Figure 1 HCT-116 colon carcinoma cells were treated with either a small interfering RNA (siRNA) duplex or an inducible short hairpin RNA (shRNA) of the same core sequence targeting TP53. RNA was collected from cells 24 h post-treatment, and gene expression changes were

analyzed by microarray hybridization. Microarray data are shown plotted as the log10 expression ratio of treated to untreated cells (y axis) versus the log10 fluorescence intensity of the individual probes on the array (x axis). A substantially higher proportion of upregulated or downregulated mRNA transcripts are observed following siRNA knockdown than following shRNA knockdown of TP53.

Cancer Gene Therapy Efficacy and toxicity of siRNA vs shRNA therapy DD Rao et al 809 Identification and elucidation of direct (sequence-related) 4 Wood LD, Parsons DW, Jones S, Lin J, Sjoblom T, and indirect (immune-mediated) off-target effects has Leary RJ et al. The genomic landscapes of human breast lead to modifications in product design and targeting and colorectal cancers. Science 2007; 318: 1108–1113. strategy. Indeed, improvements in the targeted delivery 5 Nemunaitis J, Senzer N, Khalil I, Shen Y, Kumar P, Tong A of stable RNAi effectors will further augment the et al. Proof concept for clinical justification of network specificity and selectivity of the process and extend the mapping for personalized cancer therapeutics. Cancer Gene Ther 2007; 14: 686–695. application of RNAi-based therapeutics from localized to 6 Bumcrot D, Manoharan M, Koteliansky V, Sah DW. RNAi systemic diseases. Despite the highly publicized in vivo therapeutics: a potential new class of pharmaceutical drugs. miRNA-related toxicity of shRNA (discussed above), Nat Chem Biol 2006; 2: 711–719. which was initially perceived as a strong deterrent to its 7 Yew NS, Scheule RK. Toxicity of cationic lipid-DNA clinical application, shRNA has several advantages over complexes. Adv Genet 2005; 53: 189–214. siRNA: fewer off-target effects, multiple target silencing 8 Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, capacity without a corresponding increase in dose, Davis CR et al. Fatality in mice due to oversaturation of durability of effect and inducible application. When cellular microRNA/short hairpin RNA pathways. Nature integrated with an effective delivery system, there are 2006; 441: 537–541. compelling data to support implementation of shRNA as 9 Grimm D, Kay MA. Therapeutic application of RNAi: is mRNA targeting finally ready for prime time? J Clin Invest a next generation therapeutic strategy for cancer. 2007; 117: 3633–3641. 10 Giering JC, Grimm D, Storm TA, Kay MA. Expression of shRNA from a tissue-specific pol II promoter is an effective Conflict of interest and safe RNAi therapeutic. Mol Ther 2008; 16: 1630–1636. 11 Fedorov Y, Anderson EM, Birmingham A, Reynolds A, DD Rao, N Senzer and J Nemunaitis have direct Karpilow J, Robinson K et al. Off-target effects by siRNA can induce toxic phenotype. RNA (New York, NY) 2006; 12: affiliation with Gradalis, Inc., in which they own common 1188–1196. stock. The authors declare there are no other competing 12 Jackson AL, Burchard J, Schelter J, Chau BN, Cleary M, financial interest in relation to the work described herein. Lim L et al. Widespread siRNA ‘off-target’ transcript silencing mediated by seed region sequence complementarity. RNA (New York, NY) 2006; 12: 1179–1187. References 13 Hoerter JA, Walter NG. Chemical modification resolves the asymmetry of siRNA strand degradation in human 1 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, blood serum. RNA (New York, NY) 2007; 13: 1887–1893. Mello CC. Potent and specific genetic interference by double- 14 Rao DD, Vorhies JS, Senzer N, Nemunaitis J. siRNA vs. stranded RNA in Caenorhabditis elegans. Nature 1998; 391: shRNA: similarities and differences. Adv Drug Deliv Rev 806–811. 2009; 61: 746–759. 2 Macron D. Breakdown of RNAi-based drugs in the clinic. 15 Zeng Y, Cai X, Cullen BR. Use of RNA polymerase II to RNAi News 2008 (online publication at www.rnainews.com transcribe artificial microRNAs. Methods Enzymol 2005; October 16, 2008). 392: 371–380. 3 Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, 16 Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Barber TD et al. The consensus coding sequences of Hannon GJ et al. Probing tumor phenotypes using stable human breast and colorectal cancers. Science 2006; 314: and regulated synthetic microRNA precursors. Nat Genet 268–274. 2005; 37: 1289–1295.

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