REVIEWS Strategies for silencing human disease using RNA interference Daniel H Kim and John J Rossi Abstract Since the first description of RNA interference RNAi in animals less than a decade ago there has been rapid progress towards its use as a therapeutic modality against human diseases Advances in our understanding of the mechanisms of RNAi and studies of RNAi in vivo indicate that RNAi based therapies might soon provide a powerful new arsenal against pathogens and diseases for which treatment options are currently limited Recent findings have highlighted both promise and challenges in using RNAi for therapeutic applications Design and delivery strategies for RNAi effector molecules must be carefully considered to address safety concerns and to ensure effective successful treatment of human diseases The realm of RNA interference RNAi has expanded at a remarkable rate since the initial characterization of RNAi in the nematode Caenorhabditis elegans1 Soon after this RNAi was shown to occur in mammalian cells in response to double stranded small interfering RNAs siRNAs of 21 nt in length2 that serve as the effector molecules of sequence specific gene silencing Mechanistic insights followed rapidly during the ensuing years and with them came the increasing hope that RNAi pathways could be harnessed for the therapeutic interven tion of human diseases3 The key therapeutic advantage of using RNAi lies in its ability to specifically and potently knock down the expression of disease causing genes of known sequence Furthermore the relatively short turnaround time for efficacy testing of potential therapeutic RNAi molecules and the fact that even newly discovered pathogens are theoretically amen able to rapid targeting has caused great excitement about the potential of RNAi for treating a wide range of diseases Recent findings have highlighted the effectiveness of RNAi in therapeutically relevant settings the results of which have spurred cautious optimism about the promise of RNAi based therapies The first clinical applications of RNAi have been directed at the treatment of wet age related macular degeneration AMD 4 5 and respiratory syncytial virus RSV infection6 Therapies based on RNAi are also in preclinical development for other viral diseases7 8 neurodegenerative disorders9 and cancers10 although a number of challenges need to be addressed and improvements made for RNAi based ther apies to realize their full potential A progressively more detailed understanding of the basic mechanisms of RNAi has been important in developing diverse RNAi effector molecules with improved levels of potency and efficacy For example synthetic siRNAs and expressed short hairpin RNAs shRNAs 11 both have specific advantages and disadvantages which are important considerations when designing RNAi based therapies for a particular disease In addition although many in vivo stud ies have shown the potential effectiveness of various RNAi based strategies other studies have highlighted challenges that arise as a result of using an endogenous cellular mechanism for therapeutic benefit Unwanted side effects have included induction of type 1 interferon IFN responses12 and saturation of endogenous RNAi pathway components13 indicating that caution is neces sary when designing effector molecules for delivery into target cells The issue of cell specific or tissue specific delivery is another key challenge in developing RNAi based therapies Various strategies for non viral and viral delivery of RNAi triggers have recently been shown to be effective in disease models raising the hope that clinical studies of RNAi based therapies will be extended to an increasing list of diseases in the near future14 Here we provide an overview of the current mecha nistic understanding of RNAi leading into a discussion of how potency can be maximized and off target effects OTEs minimized or avoided in therapeutic applications We then discuss strategies that have been investigated for effective delivery in vivo Finally we conclude with an overview of the therapeutic applications of RNAi cur rently in development and the outlook for the wider use of RNAi based therapies in the future Short hairpin RNAs shRNAs A class of small RNAs with a stem of 19 29 base pairs and a loop of 4 10 nucleotides that are processed by Dicer into small interfering RNAs shRNAs are expressed from vectors to induce RNAi Interferons A class of glycoproteins that are upregulated in response to exogenous ssRNA or dsRNA as a cellular defence mechanism against RNA viral infection Division of Molecular Biology Graduate School of Biological Sciences Beckman Research Institute of the City of Hope 1450 East Duarte Road Duarte California 91010 USA Correspondence to J J R e mail jrossi bricoh edu doi 10 1038 nrg2006 NATURE REVIEWS GENETICS VOLUME 8 MARCH 2007 173 2007 Nature Publishing Group R E V I E W S Mechanisms of RNAi mediated gene silencing RNAi pathways are guided by small RNAs that include siRNAs and microRNAs miRNAs which derive from imperfectly paired hairpin RNA structures naturally encoded in the genome15 RNAi effector molecules induce gene silencing in several ways they direct sequence specific cleavage of perfectly complementary mRNAs and translational repression and transcript degradation for imperfectly complementary targets RNAi pathways can also direct transcriptional gene silencing TGS in the nucleus16 17 although mechanistic details of TGS are not yet well established in mammalian systems FIG 1 siRNA DNA Pol II Methylation of H3K9 and H3K27 Pri miRNA m7G Drosha DGCR8 AAAA Transcriptional gene silencing Pre miRNA Exportin 5 Nucleus Cytoplasm dsRNA Dicer TRBP PACT miRNA RISC AGO2 Translational repression TRBP PACT Dicer m7G m7G siRNA RISC AGO2 ORF mRNA AAAA m7G mRNA 3 UTR AAAA mRNA cleavage mRNA degradation AAAA P body DCP1 DCP2 Figure 1 Mechanisms of RNA interference in mammalian cells As shown in the pathway at the bottom left cytoplasmic double stranded RNAs dsRNAs are processed by a complex consisting of Dicer TAR RNA binding protein TRBP and protein activator of protein kinase PKR PACT into small interfering RNAs siRNAs which are loaded into Argonaute 2 AGO2 and the RNA induced silencing complex RISC The siRNA guide strand recognizes target sites to direct mRNA cleavage which is carried out by the catalytic domain of AGO2 siRNAs complementary to promoter regions direct transcriptional gene silencing in the nucleus through chromatin changes involving