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Knocking down barriers: advances in siRNA delivery

Knocking down barriers: advances in siRNA delivery REVIEWS Knocking down barriers: advances in siRNA delivery ‡ ‡ Kathryn A. Whitehead*, Robert Langer* and Daniel G. Anderson Abstract | In the 10 years that have passed since the Nobel prize-winning discovery of RNA interference (RNAi), billions of dollars have been invested in the therapeutic application of gene silencing in humans. Today, there are promising data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. Despite these early successes, however, the widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles. Here, we provide an update on the progress of RNAi therapeutics and highlight novel synthetic materials for the encapsulation and intracellular delivery of nucleic acids. RNA interference (RNAi) gained international attention of the siRNA is cleaved . The activated RISC, which RNA interference in 1998 when Fire, Mello and colleagues discovered the contains the antisense strand (or guide strand) of the (RNAi). A fundamental pathway in eukaryotic cells by ability of double-stranded RNA to silence gene expres- siRNA, selectively seeks out and degrades mRNA that is 1 13 which a short piece of RNA is sion in the nematode worm Caenorhabditis elegans . complementary to the antisense strand (FIG. 1). The cleav- able to induce the destruction Three years later, Tuschl and co-workers published their age of mRNA occurs at a position between nucleo tides 10 of mRNA containing a celebrated proof-of-principle experiment demonstrating and 11 on the complementary antisense strand, relative to complementary sequence. that synthetic small interfering RNA (siRNA) could achieve the 5′-end . The activated RISC complex can then move Small interfering RNA sequence-specific gene knockdown in a mammalian cell on to destroy additional mRNA targets, which further (siRNA). RNA fragments 2 15 line . The first successful use of siRNA for gene silencing propagates gene silencing . This extra potency ensures a approximately 21–23 in mice was achieved for a hepatitis C target shortly therapeutic effect for 3–7 days in rapidly dividing cells, nucleotides long that are 3 16 thereafter . Since that time, the biotechnology sector has and for several weeks in non-dividing cells . Eventually, capable of inducing the sequence-specific destruction made considerable efforts in the advancement of siRNA siRNAs are diluted below a certain therapeutic threshold of complementary mRNA. therapeutics for the treatment of various disease targets, or degraded within the cell, and so repeated administra - 4,5 6–8 including viral infections and cancer . tion is necessary to achieve a persistent effect. RNA-induced silencing RNAi is a fundamental pathway in eukaryotic cells Theoretically, when using appropriately designed complex by which sequence-specific siRNA is able to target and siRNA, the RNAi machinery can be exploited to silence (RISC). The protein complex responsible for the binding cleave complementary mRNA . RNAi is triggered by the nearly any gene in the body, giving it a broader thera- and unwinding of the presence of long pieces of double-stranded RNA, which peutic potential than typical small-molecule drugs. double-stranded siRNA are cleaved into the fragments known as siRNA (21–23 Indeed, it has already been reported that synthetic within the cytoplasm, nucleotides long) by the enzyme Dicer . In practice, siRNAs are capable of knocking down targets in various and for the subsequent identification and destruction siRNA can be synthetically produced and then directly diseases in vivo, including hypercholesterolaemia , liver 18 4,19 of the target mRNA. introduced into the cell, thus circumventing Dicer cirr hosis , hepatitis B virus (HBV) , human papilloma- 20 21 22 mechanics (FIG. 1). This shortcut reduces the potential for virus , ovarian cancer and bone cancer . In order for *Department of an innate immune interferon response and the shutdown these advances to be implemented in a clinical setting, safe Chemical Engineering, of cellular protein expression that can occur following the and effective delivery systems must be developed. While The David H. Koch Institute interaction of long pieces (>30 nucleotides) of double- ‘naked’, chemically modified siRNA has shown efficacy for Integrated Cancer 10 23 stranded RNA with intracellular RNA receptors . in certain physiological settings such as the brain and Research, Massachusetts Institute of Technology, Once siRNA is present in the cytoplasm of the cell, the lung , there are many tissues in the body that require Cambridge, Massachusetts it is incorporated into a protein complex called the RNA- an additional delivery system to facilitate transfection. 02142, USA. induced silencing complex (RISC) . Argonaute 2, a multi- This is because naked siRNA is subject to degradation Correspondence to D.G.A. functional protein contained within RISC, unwinds the by endogenous enzymes, and is too large (~13 kDa) e-mail: [email protected] doi:10.1038/nrd2742 siRNA, after which the sense strand (or passenger strand) and too negatively charged to cross cellular membranes. NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 129 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS There are several tissues that are amenable to topical or localized therapy, including the eye, skin, mucus 25–28 membranes, and local tumours (TABLE 1). l ocal siRNA delivery is particularly well-suited for the treat- ment of lung diseases and infections. The direct instillation of siRNA into the lung through intranasal or intra- tracheal routes enables direct contact with lung epithelial cells. These cells play a part in a myriad of lung con- ditions and infections, including cystic fibrosis, asthma, influenza and the common cold . It has been reported that respiratory syncytial virus (RSV) replication can be inhibited by nasally administered siRNA formu- 29,30 lated with or without transfection agents in mice . Progress in the treatment of RSV continues with Phase II clinical trials using an aerosolized siRNA delivery system . Intratracheal administration of siRNA has also been reported to offer prophylactic and therapeu- tic effects in the treatment of severe acute respiratory syndrome . Another example of local delivery is direct intra- tumoral injection of siRNA delivery complexes into various mouse xenograft models. siRNA complexed with the delivery agent polyethyleneimine (PEI) was shown to inhibit tumour growth upon intratumoral injection in mice bearing glioblastoma xenographs . Niu and co-workers have also reported naked siRNA efficacy up on direct injection into a subcutaneous cervical cancer model in mice . Barriers to systemic siRNA delivery in vivo In contrast to the direct accessibility of localized targets, many tissues can only be reached through the systemic administration of delivery agents in the bloodstream. siRNA formulations for systemic application face a series of hurdles in vivo before reaching the cytoplasm of the Figure 1 | The mechanism of r NA interference. Long double-stranded RNA (dsRNA) target cell (FIG. 2). Post-injection, the siRNA complex is introduced into the cytoplasm, where it is cleaved into small interfering RNA (siRNA) by the enzyme Dicer. Alternatively, siRNA can be introduced directly into the cell. The siRNA must navigate the circulatory system of the body while is then incorporated into the RNA-induced silencing complex (RISC), resulting in the avoiding kidney filtration, uptake by phagocytes, aggre- cleavage of the sense strand of RNA by argonaute 2 (AGO2). The activated RISC–siRNA gation with serum proteins, and enzymatic degradation complex seeks out, binds to and degrades complementary mRNA, which leads to the 33 by endogenous nucleases . silencing of the target gene. The activated RISC–siRNA complex can then be recycled for Phagocytosis serves as a significant immunological the destruction of identical mRNA targets. barrier, not only in the bloodstream but also in the extracellular matrix of tissues. Phagocytic cells such as macrophages and monocytes remove foreign material The issue of effective and non-toxic delivery is a key chal- from the body to protect against infection by viruses, lenge and serves as the most significant barrier between bacteria and fungi. u nfortunately, phagocytes are also siRNA technology and its therapeutic application. highly efficient at removing certain therapeutic nano- complexes and macromolecules from the body, and steps Modes of siRNA administration must be taken to avoid opsonization when designing The ease of siRNA delivery is partly dependent on the drug delivery vehicles . accessibility of the target organ or tissue within the Egress from the bloodstream and across the vascu- Antisense strand The strand of the siRNA body. l ocalized siRNA delivery — that is, application lar endothelial barrier poses a significant challenge for molecule that is of siRNA therapy directly to the target tissue — offers delivery of siRNA to many tissues within the body. In complementary to the target several benefits, including the potential for both higher general, molecules larger than 5 nm in diameter do not mRNA, which activates RISC bioavailability given the proximity to the target tissue, readily cross the capillary endothelium, and therefore and has an important role in and reduced adverse effects typically associated with will remain in the circulation until they are cleared from target mRNA identification and destruction. systemic administration. By contrast, systemic delivery, the body. There are certain tissues, however, that allow the meaning the intravenous injection of delivery particles entry of larger molecules, including the liver, spleen, and Transfection that then travel throughout the body to the target organ some tumours. These organs allow the passage of mol- The process of delivering or tissue, requires that particles have the ability to avoid ecules up to 200 nm in diameter, which can accommodate nucleic acid material into the cell. uptake and clearance by non-target tissues (FIG. 2). a typical drug delivery nanocarrier . 130 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS siRNAs can induce nonspecific activation of the immune system through the Toll-like receptor 7 (Tl R7) path- 38,39 way . This effect can be reduced by the incorporation of 2′-O-methyl modifications into the sugar structure of selected nucleotides within both the sense and antisense 38,40 strands (FIG. 3a). 2′-O-methyl modifications have also been shown to confer resistance to endo nuclease activity and to abrogate off-target effects when incorporated into the seed region, which corresponds to nucleo- tides 2–8 on the antisense strand . Other common modification approaches to mitigate enzymatic deg- radation include the introduction of phosphorothioate backbone linkages at the 3′-end of the RNA strands to reduce susceptibility to exonucleases. It is also possible to incorporate alternative 2′ sugar modifications (for example, a fluorine substitution) to increase resistance to endonucleases . Another strategy to improve the therapeutic efficacy of siRNA involves the conjugation of small molecules or peptides to the sense strand of the siRNA. Several small molecules have been reported to increase target-gene knockdown in vitro, including membrane-permeant 44 45 peptides and polyethylene glycol (PEG) . Of particu- lar note are cholesterol-modified siRNAs, which have demonstrated increased binding to serum albumin, resulting in improved biodistribution to certain tar- gets including the liver (FIG. 3b). Cholesterol-modified siRNA were capable of silencing apolipoprotein B (ApoB) targets in mouse liver and jejunum, and of ultimately reducing total cholesterol levels . Another study by DiFiglia and co-workers details the ability of a Figure 2 | Physiological barriers to the systemic delivery of small interfering r NA cholesterol-modified siRNA to knockdown a gene asso- (sir NA) nanoparticles. An injected nanoparticle must avoid filtration, phagocytosis ciated with Huntington’s disease. A single intrastriatal and degradation in the bloodstream (a); be transported across the vascular injection was able to delay the abnormal behavioural endothelial barrier (b); diffuse through the extracellular matrix (c); be taken up into phenotype observed in a rapid-onset mouse model of the cell (d); escape the endosome (e); and unpackage and release the siRNA to the this disease . RNA interference (RNAi) machinery (f). Given the success of cholesterol-modified siRNA in vivo, Wolfrum and co-workers attempted to identify alternative lipid-like molecules to serve as RNA con- After an siRNA complex leaves the bloodstream, it jugates for improved delivery of siRNA . Specifically, must diffuse through the extracellular matrix, which is fatty acids and bile-salt derivatives were conjugated to a dense network of polysaccharides and fibrous proteins siRNA and injected into mice and hamsters in order to that can create resistance to the transport of macro- elucidate how modified siRNA conjugates interact with molecules and nanoparticles . This can slow or even the high-density lipoprotein (HDl ) and low-density halt the drug delivery process and create an additional lipoprotein (l Dl ) receptors that enable delivery to the opportunity for nanoparticles to be taken up by resident liver. It was found that shorter fatty-acid chain lengths macrophages. Having been taken up by the target cell, (<C ) did not induce gene knockdown, whereas bile- particles must then escape the endosome to reach the salt derivatives and fatty-acid conjugates with longer cytoplasm . If the siRNA nanocomplex is unable to exit chain lengths enabled potent silencing in hepatocytes the endosome, it will be trafficked through endomem- via the HDl receptor . brane compartments of decreasing pH and be subject to Another example of the possibility of introducing degradative conditions in the lysosome . Finally, if for- beneficial modifications to nucleic acid therapeutics mulated with delivery agents, siRNA must be released comes from the antisense drug mipomersen. Mipo- from the carrier to the cellular machinery. mersen is a 2′-O-(2-methoxyethyl)-modified single- stranded RNA molecule that is targeted to ApoB , Modified siRNA for improved delivery a protein that has been implicated in cardiovascular Humans have evolved a number of host-defence mecha- disease. Isis Pharmaceuticals has reported promising nisms against siRNA, as it is a feature of certain viral Phase II safety and efficacy results, and a Phase III trial infections. However, chemical modifications can be to assess efficacy in patients with a familial history of introduced into the siRNA molecule to evade immune hypercholesterolaemia is currently underway in a joint defences in vivo. For example, many non-modified venture with Genzyme . NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 131 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Biodistribution. The biodistribution of siRNA can be Table 1 | Modes of siRNA delivery and potential targets significantly influenced through formulation with a Mode of Potential organ target Potential disease target r efs delivery vehicle. Systemic administration of synthetic administration delivery nanoparticles often results in accumulation in Topical Eye Macular degeneration 102 the organs of the reticuloendothelial system, including 16,55,56 Skin Atopic dermatitis 25 the liver, spleen, kidneys and lungs . It is no coin- cidence that much of the successful siRNA delivery Vagina Herpes simplex virus 27 seen in recent years has targeted disease within these Rectum Inflammatory bowel disease 105 4,29,30,57,58 organs . Local/direct Lung SARS 32 Excretion through the kidney typically occurs for molecules less than 50 kDa in size . As such, naked Brain Huntington’s disease 23 siRNA experiences rapid renal clearance upon systemic Spinal cord Chronic pain 90 administration . Several studies monitored the biodis- Isolated tumour Glioblastoma multiforme 28 tribution of siRNA in mice after an intravenous injection Systemic Liver Hyper cholestero laemia 57 and observed naked siRNA accumulation in the kidney 60,61 and urine within 5 minutes of administration . By Heart Myocardial infarction 106 complexing siRNA with synthetic materials, the size Kidney Kidney disease 61 of the delivery nanoparticle can be increased to avoid Metastasized tumours Ewing’s sarcoma 97 glomerular filtration through the kidneys and reserve SARS, severe acute respiratory syndrome; siRNA, small interfering RNA. the siRNA for alternative organ targets . Additionally, it has been reported that certain siRNA formulations are capable of accumulation in subcutan- 22,62 Properties of synthetic delivery nanoparticles eous tumours . This phenomenon has been attributed For tissues and cells that are not amenable to the delivery to the enhanced permeability and retention (EPR) effect. of naked or chemically modified siRNA, delivery of It has therefore become a common approach to exploit nanoparticles that incorporate siRNA are used. In gen- the leaky vasculature of tumours for the purposes of 7,62,63 eral, delivery vehicles are designed to both facilitate directed delivery . Others have also reported success uptake into the target tissue of interest and, when used in targeting tumours through conjugation to ligands 64,65 for systemic delivery, to protect siRNA payloads and such as antibodies . inhibit nonspecific delivery. Below, we highlight sev- eral important characteristics of delivery nanoparticles Toxicity. Even the most efficacious siRNA delivery and provide specific examples of their construction agents are rendered useless if they provoke unaccept- and use. able toxicity on either a cellular or systemic level. Viral vectors, which were among the first vehicles to be studied Surface properties. The surface charge of a delivery for siRNA delivery, can induce unacceptable levels of nano particle can significantly influence the way it toxicity through the activation of immune responses . interacts with the target cell and other physiological Therefore, synthetic lipids and polymers have been molecules. In the simplified in vitro setting, a positively developed to offer alternatives to viral vectors for nucleic charged delivery vehicle can facilitate uptake by asso- acid delivery applications, and are carefully formulated 50 39 ciating with the negatively charged cellular membrane . to avoid stimulation of the immune system . Clearance A positive charge also promotes complex formation of larger molecular mass materials typically requires and compression with the polyanionic nucleic acids them to be biodegradable. The use of biodegradable, Enhanced permeability of the siRNA. The situation, however, becomes more high molecular mass polycations and polymers contain- and retention (EPR) effect complicated in vivo as negatively charged serum pro- ing linkages that can be cleaved inside the cell can help The effect by which teins in the bloodstream will often bind to a positively reduce cytotoxicity . macromolecules undergo charged nanocomplex, therefore rendering it ineffective. increased accumulation in The addition of PEG or other hydrophilic conjugates to Synthetic materials for siRNA delivery tumours. This is attributable to the quickly growing the surface of a delivery vehicle can assist in mitigating Synthetic materials have demonstrated potential as effec- tumour vasculature, which 51 this problem . Additionally, PEG conjugation can con- tive non-viral siRNA delivery carriers. Although many is improperly formed and trol particle size and prevent particle aggregation in the types of compound have been investigated as potential subsequently more permeable presence of serum . candidates, this Review focuses on synthetic materials to large molecules. Coating of the nanoparticle with hydrophilic molecules that have successfully delivered nucleic acids in vivo. Liposome such as PEG can also play an important role in the ability An overview of the delivery agents presented here can A type of drug delivery of the siRNA delivery carrier to evade the immune sys- be found in TABLE 2. vehicle made of lipids. tem and associated phagocytes. PEG forms a barrier These nanocomplexes can around nanoparticles that provides steric stabilization Liposomes and lipid-like materials. u nilamellar and be unilamellar (one set of head-groups) or multilamellar and protection from the physiological surroundings . multilamellar liposomes are commonly used as pharma- (two or more sets of 68 The length of the PEG chain can have a significant influ- ceutical delivery vehicles . In an aqueous environment, head-groups). They can be ence on its stabilization and protective properties, and certain materials have the ability to form liposomes, in used to deliver hydrophilic chain length is typically optimized for each individual which a lipid bilayer forms a sphere with an aqueous core. or hydrophobic payloads, 53,54 depending on their structure. delivery system . For example, one set of polar head-groups can create the 132 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Figure 3 | Two common small interfering r NA (sir NA) modifications used for therapeutic applications. a | The 2′-O-methyl sugar modification prevents activation of the Toll-like receptor 7 immune response and confers enzymatic resistance to the siRNA molecule. b | The conjugation of cholesterol to the sense strand of an siRNA duplex improves delivery of naked siRNA to certain cellular targets, including hepatocytes. The cholesterol conjugate is shown in red, and the linker is shown in green. outer surface of the nanocomplex, while another set of in mice have indicated an activation of interferon α and polar head-groups faces the interior hydrophilic core, β, suggesting that stimulation of the immune system 69 72 which houses the nucleic acid payload (FIG. 4). It is also may also have a role in efficacy . possible for liposomes to be amorphous in structure, More recently, Sato and co-workers used vitamin- with the lipids and nucleic acids interspersed through- A-coupled l ipotrust liposomes to deliver anti-gp46 out. l iposomes can be created using single or multiple siRNA to fibrogenic hepatic cells for the treatment of liver types of lipid, which allows for additional flexibility cirrhosis. Five administrations of the siRNA lipocomplexes when optimizing the physical and chemical properties were reported to resolve liver fibrosis and prolong survival of the nanoparticle . in rats that had otherwise lethal liver cirrhosis, in a dose- l iposomes have been used for the delivery of nucleic and duration-dependent manner. It was also shown that acids for over 20 years, originating with studies by Felgner rescue was not related to off-target effects or associated and colleagues detailing the ability of the cationic with recruitment of the innate immune system . lipid DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N- The use of lipid complexes for localized siRNA admin- trimethlyl ammonium chloride) to deliver both DNA istration has also proved beneficial. Vaginal instillation 70,71 and RNA into mouse, rat and human cell lines . More of cationic liposomes comprised of Oligofectamine and recently, stable nucleic acid–lipid particle (SNAl P) for- siRNA targeting herpes simplex virus (HSV)-2 reportedly mulations have demonstrated efficacy in several models led to uptake by the epithelial and lamina propria cells in vivo. A study by Morrissey and co-workers indicated in the vagina in mice and protected against lethal infec- that HBV replication was inhibited through the delivery tion for up to 9 days. The siRNA complexes protected of an siRNA–SNAl P complex that targeted HBV RNA. mice when administered before and/or after (otherwise) Three daily intravenous injections of 3 mg per kg per lethal HSV-2 challenge . In another study, intrathecal day reduced serum HBV levels by at least one order of administration of complexes formulated by i-FECT and magnitude, and the effect was specific, dose-dependent siRNA protected mice from fatal Japanese encephalitis and lasted for up to 7 days after dosing . In addition, virus and West Nile virus after intracranial administra- Zimmerman and colleagues demonstrated the ability tion, and reduced pain receptor expression following of SNAl Ps to enable knockdown of ApoB in the liver of intrathecal administration in rats . cynomolgus monkeys . A single siRNA injection Also of note are several synthetic lipid-based materi- resulted in dose-dependent silencing of ApoB mRNA als that have demonstrated DNA transfection efficiency. expression in the liver 48 hours after administration, Similar materials may also potentially serve as candi- with maximal silencing of more than 90%. Knockdown dates for siRNA delivery, given the similarities involved was confirmed to be caused by ApoB mRNA cleavage at in the two types of nucleic acid delivery . For example, precisely the site predicted for the RNAi mechanism, and Wheeler et al. used liposomes comprising a combination persisted for 11 days at the highest administered dose of of the cationic lipid GAP-Dl RIE (N-(3-aminopropyl)- 2.5 mg per kg . N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium An additional study details the use of SNA l P formu- bromide) and the neutral co-lipid DOPE (dioleoylphos - lations to combat the Zaire strain of Ebola virus, which phatidylethanolamine) to transfect mouse lung with a proves fatal to 90% of its victims via haemorrhagic gene that confers chloramphenicol resistance. After a fever . When contained within the SNAl P formula- single intranasal administration, gene expression was tion, siRNA targeting of the polymerase gene of the virus enhanced by more than 100-fold relative to plasmid DNA completely protected guinea pigs against viraemia and alone, followed by a gradual return to baseline levels death when administered shortly after an Ebola virus by 21 days post-administration . Also of interest are challenge. It is important to note that preliminary studies transactivating transcriptional activator (TAT)-modified NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 133 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Table 2 | Selected synthetic materials for in vivo siRNA delivery Material Model Target r oute Animal r efs Liposomes and lipids i-FECT Japanese encephalitis JEV and WNV Intracranial Mouse 70 virus (JEV) and West envelope Nile virus (WNV) Lipidoids Dyslipidaemia FVII/ApoB Intravenous Mouse, rat, 30 monkey Dyslipidaemia FVII/ApoB Intravenous Mouse, 79 hamster Malaria Haem oxygenase 1 Intravenous Mouse 80 Hypercholesterolaemia PCSK9 Intravenous Mouse, rat 17 LipoTrust Liver cirrhosis gp46 Intravenous Rat 18 Oligofectamine Herpes simplex virus 2 HSV-2-associated Intravaginal Mouse 27 (HSV-2) viral proteins UL27 and UL29 SNALP Hepatitis B virus (HBV) HBV Intravenous Mouse 4 Dyslipidaemia ApoB Intravenous Monkey 56 Ebola (Zaire) Polymerase L Intravenous Guinea pig 69 Cationic polymers Cyclodextrin Ewing’s sarcoma tumour EWS–FLI1 Intravenous Mouse 94 xenograft Healthy monkey model RRM2 Intravenous Monkey 95 Dynamic Dyslipidaemia ApoB/PPARα Intravenous Mouse 98 PolyConjugate Poly- Glioblastoma xenograft PTN Intratumoral Mouse 28 ethyleneimine Formalin-induced pain NMDAR2B Intrathecal Rat 87 Cervical tumour xenograft HPV E6/E7 Intratumoral Mouse 20 Ovarian tumour xenograft HER2 Intraperitoneal Mouse 88 Small interfering RNA (siRNA) conjugates Cholesterol Dyslipidaemia ApoB Intravenous Mouse 46 Huntington’s disease Huntingtin gene Intrastriatal Mouse 23 Fatty acids/ Dyslipidaemia ApoB Intravenous Mouse, 47 bile salts hamster ApoB, apolipoprotein B; EWS–FLI1, Ewing’s sarcoma–friend leukaemia virus integration 1; FVII, factor VII blood protein; gp46, a glycoprotein gene; HER2, human epidermal growth factor receptor 2; HPV E6/E7, human papillomavirus oncogenes; NMDAR2B, N-methyl-d-aspartate receptor type 2B; PCSK9, proprotein convertase subtilisin/kexin type 9; PPARα, peroxisome proliferator- activated receptor α; PTN, pleiotrophin (a secreted growth factor); RRM2, ribonucleoside-diphosphate reductase subunit M2; SNALP, stable nucleic acid–lipid particles. liposomes, which, when complexed with a gene encoding In another study, a combinatorial library of lipid-like green fluorescence protein (GFP) and injected locally, materials was developed for use in siRNA delivery . induced the expression of GFP in l ewis lung carcinoma These ‘lipidoids’ were synthesized through the conju- tumour cells in mice . gate addition of alkyl-acrylates and -amides to primary Although liposomes are among the most popular and secondary amines and then studied in cell culture. nucleic acid delivery agents, some concerns regarding One leading candidate for in vivo gene knockdown was their safety for therapeutic use remain. Toxicity of certain identified as 98N -5(1), which comprises five 12-carbon cationic lipid particles has been reported both in vitro alkyl-acrylamide chains attached to an amine core (FIG. 4d). 76–78 and in vivo , and certain synthetic agents have been Formulations of this material with siRNA were capable of found to induce a gene signature of their own that might achieving potent and persistent silencing of various lung and 79,80 30 increase the off-target effects of siRNA . Despite these liver targets in mice, rats and cynomolgus monkeys . issues, liposomes show promise for future clinical use, as In a more recent primate study, 98N -5(1) was also evidenced by the approval of PEGylated liposomes for used to deliver siRNA against PCSK9, a protein that doxorubicin and amphotericin B delivery by the u S Food regulates l Dl receptor protein levels and function . 79,81 and Drug Administration . l iver-specific siRNA silencing of PCSK9 reduced PCSK9 134 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Figure 4 | s ynthetic materials for small interfering r NA (sir NA) delivery. a | A representation of stable nucleic acid–lipid particles (SNALPs), which are liposomes comprising cationic lipids, non-ionic lipids and polyethylene glycol (PEG). siRNA is contained in the hydrophilic interior of the particle. b | Polyethyleneimine is used to fabricate both linear and branched polymeric delivery agents. c | A cyclodextrin-based delivery agent. d | The lipidoid 98N -5(1). mRNA levels by 50–70% in mice and rats, as well as It has also been frequently used for various local siRNA reducing human PCSK9 transcript levels in transgenic delivery applications. Intrathecal administration of PEI– mice by more than 70%. Silencing persisted for up to siRNA complexes was reported to selectively knockdown 3 weeks after a single intravenous dose, indicating that a pain receptor in rats. Maximal effect occurred on day 3 anti-PCSK9–lipidoid complexes may serve as a potent for mRNA levels and day 7 for associated protein levels and effective treatment for hypercholesterolaemia . following an injection of 5 mg of siRNA targeting a Importantly, lipidoid materials were shown to facilitate subunit of the NMDA (N-methyl-d-aspartate) receptor siRNA delivery without disrupting endogenous micro- NR2B . PEI has also shown efficacy in a subcutaneous RNA processing . Finally, Hmox1, a gene expressed in mouse tumour model. The intraperitoneal administra- the liver encoding the protein haem oxygenase 1, has tion of complexed siRNA led to the delivery of the intact also been silenced in mice by siRNA nanoparticles formu- siRNA into the tumours and a marked reduction of lated with lipidoids. Knockdown of Hmox1 may represent tumour growth through siRNA-mediated downregulation a potential approach for the treatment of malaria infection of human epidermal growth factor 2 (HER2; also known 83 91 and disease progression . as ERBB2) .There has been significant concern regarding the toxicity of PEI at higher molecular masses and high 92,93 Polymers. Cationic polymers with a linear or branched doses . However, strategies to modify the structure of structure can serve as efficient transfection agents because PEI to reduce toxicity while retaining its potent ability 94–96 of their ability to bind and condense large nucleic acids to transfect cells are in development . 84,85 into stabilized nanoparticles . Such materials have also Cyclodextrin polymers have also been developed been shown to stimulate nonspecific endocytosis as well as siRNA delivery agents. Tumour growth in a mouse as endosomal escape . A proposed mechanism for this model of metastatic Ewing’s sarcoma was shown to is the ‘proton-sponge’ effect , whereby buffering of the be inhibited by the systemic delivery of nanoparticles endosome leads to an accumulation of ions within this formed by cyclodextrin, the targeting ligand transfer- compartment and an osmotic pressure that eventually rin, and siRNA specific for the EWS–FLI1 fusion gene 86,87 bursts the endosome . commonly associated with the condition. Knockdown PEI is a broadly investigated delivery carrier for the was not observed upon removal of the targeting ligand, administration of a wide range of nucleotide-based ther - nor was there any evidence of immune stimulation or 88,89 97 apies, including DNA, siRNA and oligonucleotides . toxicity . In another study, Heidel and co-workers used NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 135 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS (VEGFR), has shown therapeutic potential in its inhibi- Table 3 | Current clinical trials for siRNA therapeutics tion of the excessive vascularization of the eye that leads c ompany Disease Mode of s tatus to AMD . administration In 2004, the first clinical trial involving siRNA was Allergan Age-related macular Topical Phase II carried out by Acuity Pharmaceuticals for the treatment degeneration of AMD. The completed Phase II trials reported that all Alnylam Respiratory syncytial Local/direct Phase II doses were well tolerated with a lack of adverse systemic virus effects. Testing has now moved into Phase III trials, which Nucleonics Hepatitis B virus Systemic Phase I have been taken over by Opko Health. Allergan is cur- rently conducting a Phase II clinical trial on an siRNA for Quark Pharmaceuticals/ Acute renal failure Systemic Phase I Pfizer AMD, with completed Phase I results indicating minimal side effects and improved vision in some of the patients. Opko Health Age-related macular Topical Phase III Although Silence Therapeutics also had an siRNA product degeneration for AMD in the pipeline, they have now refocused their Silence/Quark/Pfizer Diabetic macular Topical Phase II Phase II clinical study on the treatment of diabetic mac- oedema ular oedema, which is another condition caused by leaky Transderm Pachyonychia Topical Phase Ia/b vasculature within the eye. Importantly, a recent study congenita has reported that anti-VEGF siRNA efficacy in the eye siRNA, small interfering RNA. is not due to specific gene silencing, but is instead caused by nonspecific stimulation of the Tl R3 pathway, which cyclodextrin–transferrin nanoparticles (FIG. 4c) to study can reduce angiogenesis . Although this study calls into the effects of siRNA delivery on the immune system of question the nature of the anti-angiogenic effect reported cynomolgus monkeys . Specifically, an siRNA targeting in AMD clinical trials, it does not explain the therapeutic the M2 subunit of ribonucleotide reductase was delivered effects observed in other applications of siRNA in which intravenously in escalating doses, and it was established appropriate controls have been performed. that multiple, systemic doses of targeted nanoparticles Also of note is Alnylam’s RSV01 formulation, which containing non-chemically modified siRNA can safely targets the nucleocapsid N gene of RSV — a major cause be administered to non-human primates . In addition, of respiratory illness in infants and young children. The cyclodextrin–transferrin nanoparticles demonstrated RSV01 formulation completed Phase I trials and was efficacy in knocking down luciferase and ribonucleotide found to be well tolerated in healthy adults. Since then, 99,100 reductase genes in mice . Phase II trials have demonstrated significant antiviral Polymer–siRNA conjugates have also shown potential efficacy of this formulation in adults . for applications in systemic siRNA delivery. Rozema et al. have developed a polymer-conjugated delivery system Future directions and conclusions called Dynamic PolyConjugates to facilitate delivery of Moving forward, we expect that synthetic nanoparticles siRNA to hepatocytes . Key features of the Dynamic composed of polymers, lipids, lipidoids or conjugates PolyConjugate technology include a membrane-active will have a key role in the systemic application of siRNA cationic polymer, the ability to reversibly mask the activity in the clinic. The incorporation of tissue-specific ligands of this polymer until it reaches the acidic environment into these particles may enable targeting, which will of the endosome, and the ability to target this modified assist with in vivo biodistribution and delivery. We also polymer and its siRNA cargo specifically to hepatocytes anticipate that future developments will require atten- after intravenous injection. Dynamic PolyConjugates tion to nonspecific activation of the immune system by 102,104 were capable of inducing knockdown of two mouse liver siRNA, including Tl R3 and Tl R7 pathways . Given genes. Analyses of serum liver enzyme and cytokine levels the potential for nonspecific effects, it is important that in treated mice indicated that siRNA complexes formed the therapeutic mode of action be validated when possible, with this synthetic polymer were well tolerated . such as through direct measurement of target mRNA levels in vivo. Chemical modification of siRNA — such Clinical trials as 2′-O-methyl substitutions — can minimize nonspecific Today, siRNA therapeutics are progressing in the clinic effects , and we expect that additional improvements (TABLE 3). Many of the most advanced trials rely on forms in both activity and delivery will be mediated by direct of localized delivery, although several ongoing clinical chemical modification of the siRNA sequence. Age-related macular trials involve the use of delivery agents. Not included In summary, the field of RNAi therapeutics has made degeneration in TABLE 3 are many formulations that are in preclinical significant progress since the first demonstration of gene (AMD). This disease of the or animal-study phases at the major pharmaceutical knockdown in mammalian cells. siRNA-based formulations eye is caused by excessive companies developing siRNA therapeutics. offer significant potential as therapeutic agents to induce growth and rupture of blood Several of the most advanced clinical trials focus on the potent, persistent and specific silencing of a broad vessels within the cornea and is a leading cause of the treatment of age-related macular degeneration (AMD), range of genetic targets. Delivery remains the most signifi- blindness. Several early siRNA which is a leading cause of blindness. AMD arises from cant barrier to the widespread use of RNAi therapeutics in clinical trials have targeted excessive blood-vessel growth and rupture within the a clinical setting, and future work focusing on the develop- this disease, primarily due cornea. Naked siRNA targeted to genes for vascular ment of safe and effective delivery materials is needed to to the ease of local delivery to the eye. endothelial growth factor (VEGF) and its receptor ensure the broadest application of RNAi in the clinic. 136 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 1. Fire, A. et al. Potent and specific genetic interference 23. DiFiglia, M. et al. Therapeutic silencing of mutant 46. Soutschek, J. et al. Therapeutic silencing of an by double-stranded RNA in Caenorhabditis elegans. huntingtin with siRNA attenuates striatal and cortical endogenous gene by systemic administration of Nature 391, 806–811 (1998). neuropathology and behavioral deficits. Proc. Natl modified siRNAs. Nature 432, 173–178 (2004). The first study to describe the phenomenon of Acad. Sci. USA 104, 17204–17209 (2007). 47. Wolfrum, C. et al. Mechanisms and optimization of RNAi. 24. de Fougerolles, A. & Novobrantseva, T. siRNA and the in vivo delivery of lipophilic siRNAs. Nature Biotech. 2. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs lung: research tool or therapeutic drug? Curr. Opin. 25, 1149–1157 (2007). mediate RNA interference in cultured mammalian Pharmacol. 8, 280–285 (2008). 48. Merki, E. et al. Antisense oligonucleotide directed to cells. Nature 411, 494–498 (2001). 25. Inoue, T. et al. Modulation of scratching behavior by human apolipoprotein B-100 reduces lipoprotein(a) The first study to demonstrate that exogenous silencing an endogenous cyclooxygenase-1 gene in levels and oxidized phospholipids on human siRNA is capable of sequence-specific knockdown the skin through the administration of siRNA. J. Gene apolipoprotein B-100 particles in lipoprotein(a) in mammalian cells. Med. 9, 994–1001 (2007). transgenic mice. Circulation 118, 743–753 (2008). 3. McCaffrey, A. P. et al. Gene expression: RNA 26. Fattal, E. & Bochot, A. Ocular delivery of nucleic acids: 49. Isis Pharmaceuticals. Genzyme and Isis Begin Second interference in adult mice. Nature 418, 38–39 antisense oligonucleotides, aptamers and siRNA. Phase 3 Trial Of Mipomersen. Isis Pharmaceuticals (2002). Adv. Drug Deliv. Rev. 58, 1203–1223 (2006). web site [online], <http://ir.isispharm.com/ Describes the first observation of sequence-specific 27. Palliser, D. et al. An siRNA-based microbicide protects releasedetail.cfm?ReleaseID=326694> (2008). gene silencing in mice using siRNA. mice from lethal herpes simplex virus 2 infection. 50. Torchilin, V. P. et al. Cell transfection in vitro and in vivo 4. Morrissey, D. V. et al. Potent and persistent in vivo Nature 439, 89–94 (2006). with nontoxic TAT peptide–liposome–DNA complexes. anti-HBV activity of chemically modified siRNAs. 28. Grzelinski, M. et al. RNA interference-mediated gene Proc. Natl Acad. Sci. USA 100, 1972–1977 (2003). Nature Biotech. 23, 1002–1007 (2005). silencing of pleiotrophin through polyethylenimine- 51. Auguste, D. T. et al. Triggered release of siRNA from 5. Okumura, A., Pitha, P. M. & Harty, R. N. complexed small interfering RNAs in vivo exerts poly(ethylene glycol)-protected, pH-dependent ISG15 inhibits Ebola VP40 VLP budding in an antitumoral effects in glioblastoma xenografts. liposomes. J. Control. Release 130, 266–274 (2008). L-domain-dependent manner by blocking Nedd4 Hum. Gene Ther. 17, 751–766 (2006). 52. Martina, M.-S. et al. The in vitro kinetics of the ligase activity. Proc. Natl Acad. Sci. USA 105, 29. Bitko, V., Musiyenko, A., Shulyayeva, O. & Barik, S. interactions between PEG-ylated 3974–3979 (2008). Inhibition of respiratory viruses by nasally magnetic-fluid-loaded liposomes and macrophages. 6. Ptasznik, A., Nakata, Y., Kalota, A., Emerson, S. G. & administered siRNA. Nature Med. 11, 50–55 (2005). Biomaterials 28, 4143–4153 (2007). Gewirtz, A. M. Short interfering RNA (siRNA) targeting 30. Akinc, A. et al. A combinatorial library of lipid-like 53. Mao, S. et al. Influence of polyethylene glycol chain the Lyn kinase induces apoptosis in primary, and drug- materials for delivery of RNAi therapeutics. Nature length on the physicochemical and biological resistant, BCR-ABL1 leukemia cells. Nature Med. 10, Biotech. 26, 561–569 (2008). properties of poly(ethylene imine)-graft-poly(ethylene 1187–1189 (2004). Describes the combinatorial synthesis and glycol) block copolymer/siRNA polyplexes. Bioconjug. 7. Kim, S. H., Jeong, J. H., Lee, S. H., Kim, S. W. & in vivo delivery performance of a large library of Chem. 17, 1209–1218 (2006). Park, T. G. Local and systemic delivery of VEGF siRNA lipid-like materials in multiple animal models and 54. Li, W., Huang, Z., MacKay, J. A., Grube, S. & using polyelectrolyte complex micelles for effective species. Szoka, F. C. Low-pH-sensitive poly(ethylene glycol) treatment of cancer. J. Control. Release 129, 107–116 31. DeVincenzo, J. et al. Evaluation of the safety, (PEG)-stabilized plasmid nanolipoparticles: effects of (2008). tolerability and pharmacokinetics of ALN-RSV01, PEG chain length, lipid composition and assembly 8. Xia, C.-F., Zhang, Y., Zhang, Y., Boado, R. & a novel RNAi antiviral therapeutic directed against conditions on gene delivery. J. Gene Med. 7, 67–79 Pardridge, W. Intravenous siRNA of brain cancer with respiratory syncytial virus (RSV). Antiviral Res. 77, (2005). receptor targeting and avidin–biotin technology. 225–231 (2008). 55. Braasch, D. A. et al. Biodistribution of phosphodiester Pharm. Res. 24, 2309–2316 (2007). 32. Li, B.-J. et al. Using siRNA in prophylactic and and phosphorothioate siRNA. Bioorg. Med. Chem. 9. Bernstein, E., Caudy, A. A., Hammond, S. M. & therapeutic regimens against SARS coronavirus in Lett. 14, 1139–1143 (2004). Hannon, G. J. Role for a bidentate ribonuclease Rhesus macaque. Nature Med. 11, 944–951 (2005). 56. Bogdanov, A. A. Merging molecular imaging and RNA in the initiation step of RNA interference. Nature 409, 33. Alexis, F., Pridgen, E., Molnar, L. K. & Farokhzad, O. C. interference: early experience in live animals. J. Cell 363–366 (2001). Factors affecting the clearance and biodistribution of Biochem. 104, 1113–1123 (2008). 10. Grimm, D. et al. Fatality in mice due to oversaturation polymeric nanoparticles. Mol. Pharm. 5, 505–515 57. Zimmermann, T. S. et al. RNAi-mediated gene silencing of cellular microRNA/short hairpin RNA pathways. (2008). in non-human primates. Nature 441, 111–114 (2006). Nature 441, 537–541 (2006). 34. Scherphof, G. L. in Targeted Drug Delivery The first study to demonstrate sequence-specific 11. Rand, T. A., Ginalski, K., Grishin, N. V. & Wang, X. (ed. Juliano, R. L.) 285–313 (Springer, Berlin, 1991). RNAi in non-human primates. Biochemical identification of Argonaute 2 as the sole 35. Zámecník, J., Vargová, L., Homola, A., Kodet, R. & 58. Yuan, H. et al. Effects of cholesterol-tagged small protein required for RNA-induced silencing complex Syková, E. Extracellular matrix glycoproteins and interfering RNAs targeting 12/15-lipoxygenase on activity. Proc. Natl Acad. Sci. USA 101, 14385–14389 diffusion barriers in human astrocytic tumours. parameters of diabetic nephropathy in a mouse model (2004). Neuropathol. Appl. Neurobiol. 30, 338–350 (2004). of type 1 diabetes. Am. J. Physiol. Renal Physiol. 295, 12. Matranga, C., Tomari, Y., Shin, C., Bartel, D. P. & 36. Oliveira, S., van Rooy, I., Kranenburg, O., Storm, G. & F605–F617 (2008). Zamore, P. D. Passenger-strand cleavage facilitates Schiffelers, R. Fusogenic peptides enhance endosomal 59. Rappaport, J. et al. Transport of phosphorothioate assembly of siRNA into ago2-containing RNAi enzyme escape improving siRNA-induced silencing of oligonucleotides in kidney: implications for molecular complexes. Cell 123, 607–620 (2005). oncogenes. Int. J. Pharm. 331, 211–214 (2007). therapy. Kidney Int. 47, 1462–1469 (1995). 13. Ameres, S. L., Martinez, J. & Schroeder, R. 37. Di Guglielmo, G. M., Le Roy, C., Goodfellow, A. F. & 60. Santel, A. et al. A novel siRNA-lipoplex technology for Molecular basis for target RNA recognition and Wrana, J. L. Distinct endocytic pathways regulate RNA interference in the mouse vascular endothelium. cleavage by human RISC. Cell 130, 101–112 (2007). TGF-β receptor signalling and turnover. Nature Cell Gene Ther. 13, 1222–1234 (2006). 14. Rand, T. A., Petersen, S., Du, F. & Wang, X. Biol. 5, 410–421 (2003). 61. van de Water, F. M. et al. Intravenousely administered Argonaute2 cleaves the anti-guide strand of siRNA 38. Hornung, V. et al. Sequence-specific potent induction short interfering RNA accumulates in the kidney and during RISC activation. Cell 123, 621–629 (2005). of IFN-α by short interfering RNA in plasmacytoid selectively suppresses gene function in renal proximal 15. Hutvagner, G. & Zamore, P. D. A microRNA in a dendritic cells through TLR7. Nature Med 11, tubules. Drug Metab. Dispos. 34, 1393–1397 (2006). multiple-turnover RNAi enzyme complex. Science 263–270 (2005). 62. Takei, Y., Kadomatsu, K., Yuzawa, Y., Matsuo, S. & 297, 2056–2060 (2002). The TLR7 pathway is identified as an important Muramatsu, T. A small interfering RNA targeting 16. Bartlett, D. W. & Davis, M. E. Insights into the kinetics contributor to nonspecific siRNA immune activation. vascular endothelial growth factor as cancer of siRNA-mediated gene silencing from live-cell and 39. Judge, A. D. et al. Sequence-dependent stimulation of therapeutics. Cancer Res. 64, 3365–3370 (2004). live-animal bioluminescent imaging. Nucleic Acids Res. the mammalian innate immune response by synthetic 63. McNamara, J. O. et al. Cell type-specific delivery of 34, 322–333 (2006). siRNA. Nature Biotech. 23, 457–462 (2005). siRNAs with aptamer-siRNA chimeras. Nature Biotech. 17. Frank-Kamenetsky, M. et al. Therapeutic RNAi 40. Judge, A. D., Bola, G., Lee, A. C. H. & MacLachlan, I. 24, 1005–1015 (2006). targeting PCSK9 acutely lowers plasma cholesterol in Design of noninflammatory synthetic siRNA mediating 64. Song, E. et al. Antibody mediated in vivo delivery rodents and LDL cholesterol in nonhuman primates. potent gene silencing in vivo. Mol. Ther. 13, 494–505 of small interfering RNAs via cell-surface receptors. Proc. Natl Acad. Sci. USA 105, 11915–11920 (2006). Nature Biotech. 23, 709–717 (2005). (2008). 41. Vornlocher, H.-P. et al. Nuclease-resistant double- 65. Peer, D., Park, E. J., Morishita, Y., Carman, C. V. & 18. Sato, Y. et al. Resolution of liver cirrhosis using stranded RNA for RNA interference. WO2005115481 Shimaoka, M. Systemic leukocyte-directed siRNA vitamin A-coupled liposomes to deliver siRNA against (2005). delivery revealing Cyclin D1 as an anti-inflammatory a collagen-specific chaperone. Nature Biotech. 26, 42. Jackson, A. L. et al. Position-specific chemical target. Science 319, 627–630 (2008). 431–442 (2008). modification of siRNAs reduces “off-target” transcript 66. Barquinero, J., Eixarch, H. & Perez-Melgosa, M. 19. Song, E. et al. RNA interference targeting Fas silencing. RNA 12, 1197–1205 (2006). Retroviral vectors: new applications for an old tool. protects mice from fulminant hepatitis. Nature Med. 43. Bumcrot, D., Manoharan, M., Koteliansky, V. & Gene Ther. 11, S3–S9 (2004). 9, 347–351 (2003). Sah, D. W. Y. RNAi therapeutics: a potential new class 67. Vandenbroucke, R. E. et al. Prolonged gene silencing 20. Niu, X.-Y., Peng, Z. L., Duan, W. Q., Wang, H. & of pharmaceutical drugs. Nature Chem. Biol. 2, in hepatoma cells and primary hepatocytes after small Wang, P. Inhibition of HPV 16 E6 oncogene expression 711–719 (2006). interfering RNA delivery with biodegradable poly(β- by RNA interference in vitro and in vivo. Int. 44. Muratovska, A. & Eccles, M. R. Conjugate for efficient amino esters). J. Gene Med. 10, 783–794 (2008). J. Gynecol. Cancer 16, 743–751 (2006). delivery of short interfering RNA (siRNA) into 68. Torchilin, V. P. Recent advances with liposomes as 21. Halder, J. et al. Focal adhesion kinase targeting using mammalian cells. FEBS Lett. 558, 63–68 (2004). pharmaceutical carriers. Nature Rev. Drug Discov. 4, in vivo short interfering RNA delivery in neutral 45. Oishi, M., Nagasaki, Y., Itaka, K., Nishiyama, N. & 145–160 (2005). liposomes for ovarian carcinoma therapy. Clin. Cancer Kataoka, K. Lactosylated poly(ethylene glycol)-siRNA 69. MacLachlan, I. in Antisense Drug Technology: Res. 12, 4916–4924 (2006). conjugate through acid-labile β-thiopropionate Principles, Strategies and Applications. 2nd edn Ch. 9 22. Takeshita, F. et al. Efficient delivery of small interfering linkage to construct pH-sensitive polyion complex (ed. Crooke, S. T.) 237–270 (CRC, Boca Raton, 2007). RNA to bone-metastatic tumors by using atelocollagen micelles achieving enhanced gene silencing in 70. Felgner, P. L. et al. Lipofection: a highly efficient, in vivo. Proc. Natl Acad. Sci. USA 102, 12177–12182 hepatoma cells. J. Am. Chem. Soc. 127, 1624–1625 lipid-mediated DNA-transfection procedure. Proc. Natl (2005). (2005). Acad. Sci. USA 84, 7413–7417 (1987). NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 137 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 71. Malone, R. W., Felgner, P. L. & Verma, I. M. Cationic 85. Putnam, D. Polymers for gene delivery across length 98. Heidel, J. D. et al. Administration in non-human liposome-mediated RNA transfection. Proc. Natl scales. Nature Mater. 5, 439–451 (2006). primates of escalating intravenous doses of targeted Acad. Sci. USA 86, 6077–6081 (1989). 86. Akinc, A., Thomas, M., Klibanov, A. M. & Langer, R. nanoparticles containing ribonucleotide reductase 72. Geisbert, T. W. et al. Postexposure protection of Exploring polyethylenimine-mediated DNA subunit M2 siRNA. Proc. Natl Acad. Sci. USA 104, guinea pigs against a lethal ebola virus challenge is transfection and the proton sponge hypothesis. 5715–5721 (2007). conferred by RNA interference. J. Infect. Dis. 193, J. Gene Med. 7, 657–663 (2005). 99. Bartlett, D. W., Su, H., Hildebrandt, I. J., Weber, W. A. 1650–1657 (2006). 87. Wang, Y., Chen, P. & Shen, J. The development and & Davis, M. E. Impact of tumor-specific targeting on 73. Kumar, P., Lee, S. K., Shankar, P. & Manjunath, N. A characterization of a glutathione-sensitive cross-linked the biodistribution and efficacy of siRNA nanoparticles single siRNA suppresses fatal encephalitis induced by polyethylenimine gene vector. Biomaterials 27, measured by multimodality in vivo imaging. Proc. Natl two different Flaviviruses. PLoS Med. 3, e96 (2006). 5292–5298 (2006). Acad. Sci. USA 104, 15549–15554 (2007). 74. Li, W. & Szoka, F. Lipid-based nanoparticles for nucleic 88. Lungwitz, U., Breunig, M., Blunk, T. & Göpferich, A. 100. Bartlett, D. W. & Davis, M. E. Impact of tumor-specific acid delivery. Pharm. Res. 24, 438–449 (2007). Polyethylenimine-based non-viral gene delivery targeting and dosing schedule on tumor growth 75. Wheeler, C. J. et al. A novel cationic lipid greatly systems. Eur. J. Pharm. Biopharm. 60, 247–266 inhibition after intravenous administration of siRNA- enhances plasmid DNA delivery and expression (2005). containing nanoparticles. Biotechnol. Bioeng. 99, in mouse lung. Proc. Natl Acad. Sci. USA 93, 89. Zintchenko, A., Philipp, A., Dehshahri, A. & Wagner, E. 975–985 (2008). 11454–11459 (1996). Simple modifications of branched PEI lead to highly 101. Rozema, D. B. et al. Dynamic PolyConjugates for 76. Lv, H., Zhang, S., Wang, B., Cui, S. & Yan, J. Toxicity of efficient siRNA carriers with low toxicity. Bioconjug. targeted in vivo delivery of siRNA to hepatocytes. cationic lipids and cationic polymers in gene delivery. Chem. 19, 1448–1455 (2008). Proc. Natl Acad. Sci. USA 104, 12982–12987 (2007). J. Control. Release 114, 100–109 (2006). 90. Tan, P. H., Yang, L. C., Shih, H. C., Lan, K. C. & 102. Kleinman, M. E. et al. Sequence- and target- 77. Ma, Z. et al. Cationic lipids enhance siRNA-mediated Cheng, J. T. Gene knockdown with intrathecal siRNA independent angiogenesis suppression by siRNA interferon response in mice. Biochem. Biophys. Res. of NMDA receptor NR2B subunit reduces formalin- via TLR3. Nature 452, 591–597 (2008). Commun. 330, 755–759 (2005). induced nociception in the rat. Gene Ther. 12, 59–66 Demonstration that the anti-angiogenic effects 78. Akhtar, S. & Benter, I. Toxicogenomics of non-viral (2004). seen upon siRNA delivery to the eye can be drug delivery systems for RNAi: potential impact on 91. Urban-Klein, B., Werth, S., Abuharbeid, S., Czubayko, F. nonspecific and caused by TLR3 immune activation. siRNA-mediated gene silencing activity and specificity. & Aigner, A. RNAi-mediated gene-targeting through 103. Alnylam Pharmaceuticals. Alnylam Achieves First Adv. Drug Deliv. Rev. 59, 164–182 (2007). systemic application of polyethylenimine (PEI)- Human Proof of Concept for an RNAi Therapeutic with 79. Akhtar, S. & Benter, I. F. Nonviral delivery of synthetic complexed siRNA in vivo. Gene Ther. 12, 461–466 GEMINI study. Alnylam Pharmaceuticals web site siRNAs in vivo. J. Clin. Invest. 117, 3623–3632 (2004). [online] <http://phx.corporate-ir.net/phoenix. (2007). 92. Kichler, A. Gene transfer with modified zhtml?c=148005&p=irol-newsArticle2&ID=11139 80. Hollins, A. J., Omidi, Y., Benter, I. F. & Akhtar, S. polyethylenimines. J. Gene Med. 6, S3–S10 (2004). 37&highlight=> (2008). Toxicogenomics of drug delivery systems: exploiting 93. Kircheis, R., Wightman, L. & Wagner, E. Design and 104. Robbins, M. et al. Misinterpreting the therapeutic delivery system-induced changes in target gene gene delivery activity of modified polyethylenimines. effects of siRNA caused by immune stimulation. expression to enhance siRNA activity. J. Drug Target. Adv. Drug Deliv. Rev. 53, 341–358 (2001). Hum. Gene Ther. 19 Aug 2008 (doi 10.1089/ 15, 83–88 (2007). 94. Richards Grayson, A., Doody, A. & Putnam, D. hgt.2008.131). 81. Rust, D. M. & Jameson, G. The novel lipid delivery Biophysical and structural characterization of 105. Zhang, Y. et al. Engineering mucosal RNA interference system of amphotericin B: drug profile and relevance polyethylenimine-mediated siRNA delivery in vitro. in vivo. Mol. Ther. 14, 336–342 (2006). to clinical practice. Oncol. Nurs. Forum 25, 35–48 Pharm. Res. 23, 1868–1876 (2006). 106. Arnold, A.-S. et al. Specific β1-adrenergic receptor (1998). 95. Thomas, M. et al. Full deacylation of polyethylenimine silencing with small interfering RNA lowers high blood 82. John, M. et al. Effective RNAi-mediated gene silencing dramatically boosts its gene delivery efficiency and pressure and improves cardiac function in myocardial without interruption of the endogenous microRNA specificity to mouse lung. Proc. Natl Acad. Sci. USA ischemia. J. Hypertens. 25, 197–205 (2007). pathway. Nature 449, 745–747 (2007). 102, 5679–5684 (2005). Demonstration that specific, non-viral RNAi 96. Werth, S. et al. A low molecular weight fraction of Acknowledgements delivery can be achieved without interruption polyethylenimine (PEI) displays increased transfection The authors would like to thank A. de Fougerolles, R. Alvarez of endogenous microRNA processing. efficiency of DNA and siRNA in fresh or lyophilized and E. Deguns for their advice and assistance in the prepara- 83. Epiphanio, S. et al. Heme oxygenase-1 is an anti- complexes. J. Control. Release 112, 257–270 tion of the manuscript. inflammatory host factor that promotes murine (2006). Plasmodium liver infection. Cell Host Microbe 3, 97. Hu-Lieskovan, S., Heidel, J. D., Bartlett, D. W., DATABASES 331–338 (2008). Davis, M. E. & Triche, T. J. Sequence-specific UniProtKB: http://www.uniprot.org 84. Boussif, O. et al. A versatile vector for gene and knockdown of EWS-FLI1 by targeted, nonviral delivery PCSK9 | TLR3 | TLR7 | VEGF | VEGFR oligonucleotide transfer into cells in culture and of small interfering RNA inhibits tumor growth in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA in a murine model of metastatic Ewing’s sarcoma. All li Nks Are Ac Tive i N The o Nli Ne PDf 92, 7297–7301 (1995). Cancer Res. 65, 8984–8992 (2005). 138 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved CoRRigENDuM Kathryn A. Whitehead, Robert Langer & Daniel G. Anderson Nature Reviews Drug Discovery 8, 129–138 (2009) | doi:10.1038/nrd2742 On page 136 in Table 3, the clinical trial for acute renal failure has been attributed to Quark /Pfizer when it should be attributed to Quark alone. In addition, the authors would like to clarify that, following a Phase I/II trial in patients with wet age-related macular degeneration, the siRNA drug candidate PF-4523655 (RTP801i-14) is now being studied in a Phase II trial for diabetic macular oedema conducted by Pfizer in collaboration with Quark. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Reviews. Drug Discovery Pubmed Central

Knocking down barriers: advances in siRNA delivery

Whitehead, Kathryn A.; Langer, Robert; Anderson, Daniel G.
Nature Reviews. Drug Discovery , Volume 8 (2) – May 1, 168
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Abstract

REVIEWS Knocking down barriers: advances in siRNA delivery ‡ ‡ Kathryn A. Whitehead*, Robert Langer* and Daniel G. Anderson Abstract | In the 10 years that have passed since the Nobel prize-winning discovery of RNA interference (RNAi), billions of dollars have been invested in the therapeutic application of gene silencing in humans. Today, there are promising data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. Despite these early successes, however, the widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles. Here, we provide an update on the progress of RNAi therapeutics and highlight novel synthetic materials for the encapsulation and intracellular delivery of nucleic acids. RNA interference (RNAi) gained international attention of the siRNA is cleaved . The activated RISC, which RNA interference in 1998 when Fire, Mello and colleagues discovered the contains the antisense strand (or guide strand) of the (RNAi). A fundamental pathway in eukaryotic cells by ability of double-stranded RNA to silence gene expres- siRNA, selectively seeks out and degrades mRNA that is 1 13 which a short piece of RNA is sion in the nematode worm Caenorhabditis elegans . complementary to the antisense strand (FIG. 1). The cleav- able to induce the destruction Three years later, Tuschl and co-workers published their age of mRNA occurs at a position between nucleo tides 10 of mRNA containing a celebrated proof-of-principle experiment demonstrating and 11 on the complementary antisense strand, relative to complementary sequence. that synthetic small interfering RNA (siRNA) could achieve the 5′-end . The activated RISC complex can then move Small interfering RNA sequence-specific gene knockdown in a mammalian cell on to destroy additional mRNA targets, which further (siRNA). RNA fragments 2 15 line . The first successful use of siRNA for gene silencing propagates gene silencing . This extra potency ensures a approximately 21–23 in mice was achieved for a hepatitis C target shortly therapeutic effect for 3–7 days in rapidly dividing cells, nucleotides long that are 3 16 thereafter . Since that time, the biotechnology sector has and for several weeks in non-dividing cells . Eventually, capable of inducing the sequence-specific destruction made considerable efforts in the advancement of siRNA siRNAs are diluted below a certain therapeutic threshold of complementary mRNA. therapeutics for the treatment of various disease targets, or degraded within the cell, and so repeated administra - 4,5 6–8 including viral infections and cancer . tion is necessary to achieve a persistent effect. RNA-induced silencing RNAi is a fundamental pathway in eukaryotic cells Theoretically, when using appropriately designed complex by which sequence-specific siRNA is able to target and siRNA, the RNAi machinery can be exploited to silence (RISC). The protein complex responsible for the binding cleave complementary mRNA . RNAi is triggered by the nearly any gene in the body, giving it a broader thera- and unwinding of the presence of long pieces of double-stranded RNA, which peutic potential than typical small-molecule drugs. double-stranded siRNA are cleaved into the fragments known as siRNA (21–23 Indeed, it has already been reported that synthetic within the cytoplasm, nucleotides long) by the enzyme Dicer . In practice, siRNAs are capable of knocking down targets in various and for the subsequent identification and destruction siRNA can be synthetically produced and then directly diseases in vivo, including hypercholesterolaemia , liver 18 4,19 of the target mRNA. introduced into the cell, thus circumventing Dicer cirr hosis , hepatitis B virus (HBV) , human papilloma- 20 21 22 mechanics (FIG. 1). This shortcut reduces the potential for virus , ovarian cancer and bone cancer . In order for *Department of an innate immune interferon response and the shutdown these advances to be implemented in a clinical setting, safe Chemical Engineering, of cellular protein expression that can occur following the and effective delivery systems must be developed. While The David H. Koch Institute interaction of long pieces (>30 nucleotides) of double- ‘naked’, chemically modified siRNA has shown efficacy for Integrated Cancer 10 23 stranded RNA with intracellular RNA receptors . in certain physiological settings such as the brain and Research, Massachusetts Institute of Technology, Once siRNA is present in the cytoplasm of the cell, the lung , there are many tissues in the body that require Cambridge, Massachusetts it is incorporated into a protein complex called the RNA- an additional delivery system to facilitate transfection. 02142, USA. induced silencing complex (RISC) . Argonaute 2, a multi- This is because naked siRNA is subject to degradation Correspondence to D.G.A. functional protein contained within RISC, unwinds the by endogenous enzymes, and is too large (~13 kDa) e-mail: [email protected] doi:10.1038/nrd2742 siRNA, after which the sense strand (or passenger strand) and too negatively charged to cross cellular membranes. NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 129 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS There are several tissues that are amenable to topical or localized therapy, including the eye, skin, mucus 25–28 membranes, and local tumours (TABLE 1). l ocal siRNA delivery is particularly well-suited for the treat- ment of lung diseases and infections. The direct instillation of siRNA into the lung through intranasal or intra- tracheal routes enables direct contact with lung epithelial cells. These cells play a part in a myriad of lung con- ditions and infections, including cystic fibrosis, asthma, influenza and the common cold . It has been reported that respiratory syncytial virus (RSV) replication can be inhibited by nasally administered siRNA formu- 29,30 lated with or without transfection agents in mice . Progress in the treatment of RSV continues with Phase II clinical trials using an aerosolized siRNA delivery system . Intratracheal administration of siRNA has also been reported to offer prophylactic and therapeu- tic effects in the treatment of severe acute respiratory syndrome . Another example of local delivery is direct intra- tumoral injection of siRNA delivery complexes into various mouse xenograft models. siRNA complexed with the delivery agent polyethyleneimine (PEI) was shown to inhibit tumour growth upon intratumoral injection in mice bearing glioblastoma xenographs . Niu and co-workers have also reported naked siRNA efficacy up on direct injection into a subcutaneous cervical cancer model in mice . Barriers to systemic siRNA delivery in vivo In contrast to the direct accessibility of localized targets, many tissues can only be reached through the systemic administration of delivery agents in the bloodstream. siRNA formulations for systemic application face a series of hurdles in vivo before reaching the cytoplasm of the Figure 1 | The mechanism of r NA interference. Long double-stranded RNA (dsRNA) target cell (FIG. 2). Post-injection, the siRNA complex is introduced into the cytoplasm, where it is cleaved into small interfering RNA (siRNA) by the enzyme Dicer. Alternatively, siRNA can be introduced directly into the cell. The siRNA must navigate the circulatory system of the body while is then incorporated into the RNA-induced silencing complex (RISC), resulting in the avoiding kidney filtration, uptake by phagocytes, aggre- cleavage of the sense strand of RNA by argonaute 2 (AGO2). The activated RISC–siRNA gation with serum proteins, and enzymatic degradation complex seeks out, binds to and degrades complementary mRNA, which leads to the 33 by endogenous nucleases . silencing of the target gene. The activated RISC–siRNA complex can then be recycled for Phagocytosis serves as a significant immunological the destruction of identical mRNA targets. barrier, not only in the bloodstream but also in the extracellular matrix of tissues. Phagocytic cells such as macrophages and monocytes remove foreign material The issue of effective and non-toxic delivery is a key chal- from the body to protect against infection by viruses, lenge and serves as the most significant barrier between bacteria and fungi. u nfortunately, phagocytes are also siRNA technology and its therapeutic application. highly efficient at removing certain therapeutic nano- complexes and macromolecules from the body, and steps Modes of siRNA administration must be taken to avoid opsonization when designing The ease of siRNA delivery is partly dependent on the drug delivery vehicles . accessibility of the target organ or tissue within the Egress from the bloodstream and across the vascu- Antisense strand The strand of the siRNA body. l ocalized siRNA delivery — that is, application lar endothelial barrier poses a significant challenge for molecule that is of siRNA therapy directly to the target tissue — offers delivery of siRNA to many tissues within the body. In complementary to the target several benefits, including the potential for both higher general, molecules larger than 5 nm in diameter do not mRNA, which activates RISC bioavailability given the proximity to the target tissue, readily cross the capillary endothelium, and therefore and has an important role in and reduced adverse effects typically associated with will remain in the circulation until they are cleared from target mRNA identification and destruction. systemic administration. By contrast, systemic delivery, the body. There are certain tissues, however, that allow the meaning the intravenous injection of delivery particles entry of larger molecules, including the liver, spleen, and Transfection that then travel throughout the body to the target organ some tumours. These organs allow the passage of mol- The process of delivering or tissue, requires that particles have the ability to avoid ecules up to 200 nm in diameter, which can accommodate nucleic acid material into the cell. uptake and clearance by non-target tissues (FIG. 2). a typical drug delivery nanocarrier . 130 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS siRNAs can induce nonspecific activation of the immune system through the Toll-like receptor 7 (Tl R7) path- 38,39 way . This effect can be reduced by the incorporation of 2′-O-methyl modifications into the sugar structure of selected nucleotides within both the sense and antisense 38,40 strands (FIG. 3a). 2′-O-methyl modifications have also been shown to confer resistance to endo nuclease activity and to abrogate off-target effects when incorporated into the seed region, which corresponds to nucleo- tides 2–8 on the antisense strand . Other common modification approaches to mitigate enzymatic deg- radation include the introduction of phosphorothioate backbone linkages at the 3′-end of the RNA strands to reduce susceptibility to exonucleases. It is also possible to incorporate alternative 2′ sugar modifications (for example, a fluorine substitution) to increase resistance to endonucleases . Another strategy to improve the therapeutic efficacy of siRNA involves the conjugation of small molecules or peptides to the sense strand of the siRNA. Several small molecules have been reported to increase target-gene knockdown in vitro, including membrane-permeant 44 45 peptides and polyethylene glycol (PEG) . Of particu- lar note are cholesterol-modified siRNAs, which have demonstrated increased binding to serum albumin, resulting in improved biodistribution to certain tar- gets including the liver (FIG. 3b). Cholesterol-modified siRNA were capable of silencing apolipoprotein B (ApoB) targets in mouse liver and jejunum, and of ultimately reducing total cholesterol levels . Another study by DiFiglia and co-workers details the ability of a Figure 2 | Physiological barriers to the systemic delivery of small interfering r NA cholesterol-modified siRNA to knockdown a gene asso- (sir NA) nanoparticles. An injected nanoparticle must avoid filtration, phagocytosis ciated with Huntington’s disease. A single intrastriatal and degradation in the bloodstream (a); be transported across the vascular injection was able to delay the abnormal behavioural endothelial barrier (b); diffuse through the extracellular matrix (c); be taken up into phenotype observed in a rapid-onset mouse model of the cell (d); escape the endosome (e); and unpackage and release the siRNA to the this disease . RNA interference (RNAi) machinery (f). Given the success of cholesterol-modified siRNA in vivo, Wolfrum and co-workers attempted to identify alternative lipid-like molecules to serve as RNA con- After an siRNA complex leaves the bloodstream, it jugates for improved delivery of siRNA . Specifically, must diffuse through the extracellular matrix, which is fatty acids and bile-salt derivatives were conjugated to a dense network of polysaccharides and fibrous proteins siRNA and injected into mice and hamsters in order to that can create resistance to the transport of macro- elucidate how modified siRNA conjugates interact with molecules and nanoparticles . This can slow or even the high-density lipoprotein (HDl ) and low-density halt the drug delivery process and create an additional lipoprotein (l Dl ) receptors that enable delivery to the opportunity for nanoparticles to be taken up by resident liver. It was found that shorter fatty-acid chain lengths macrophages. Having been taken up by the target cell, (<C ) did not induce gene knockdown, whereas bile- particles must then escape the endosome to reach the salt derivatives and fatty-acid conjugates with longer cytoplasm . If the siRNA nanocomplex is unable to exit chain lengths enabled potent silencing in hepatocytes the endosome, it will be trafficked through endomem- via the HDl receptor . brane compartments of decreasing pH and be subject to Another example of the possibility of introducing degradative conditions in the lysosome . Finally, if for- beneficial modifications to nucleic acid therapeutics mulated with delivery agents, siRNA must be released comes from the antisense drug mipomersen. Mipo- from the carrier to the cellular machinery. mersen is a 2′-O-(2-methoxyethyl)-modified single- stranded RNA molecule that is targeted to ApoB , Modified siRNA for improved delivery a protein that has been implicated in cardiovascular Humans have evolved a number of host-defence mecha- disease. Isis Pharmaceuticals has reported promising nisms against siRNA, as it is a feature of certain viral Phase II safety and efficacy results, and a Phase III trial infections. However, chemical modifications can be to assess efficacy in patients with a familial history of introduced into the siRNA molecule to evade immune hypercholesterolaemia is currently underway in a joint defences in vivo. For example, many non-modified venture with Genzyme . NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 131 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Biodistribution. The biodistribution of siRNA can be Table 1 | Modes of siRNA delivery and potential targets significantly influenced through formulation with a Mode of Potential organ target Potential disease target r efs delivery vehicle. Systemic administration of synthetic administration delivery nanoparticles often results in accumulation in Topical Eye Macular degeneration 102 the organs of the reticuloendothelial system, including 16,55,56 Skin Atopic dermatitis 25 the liver, spleen, kidneys and lungs . It is no coin- cidence that much of the successful siRNA delivery Vagina Herpes simplex virus 27 seen in recent years has targeted disease within these Rectum Inflammatory bowel disease 105 4,29,30,57,58 organs . Local/direct Lung SARS 32 Excretion through the kidney typically occurs for molecules less than 50 kDa in size . As such, naked Brain Huntington’s disease 23 siRNA experiences rapid renal clearance upon systemic Spinal cord Chronic pain 90 administration . Several studies monitored the biodis- Isolated tumour Glioblastoma multiforme 28 tribution of siRNA in mice after an intravenous injection Systemic Liver Hyper cholestero laemia 57 and observed naked siRNA accumulation in the kidney 60,61 and urine within 5 minutes of administration . By Heart Myocardial infarction 106 complexing siRNA with synthetic materials, the size Kidney Kidney disease 61 of the delivery nanoparticle can be increased to avoid Metastasized tumours Ewing’s sarcoma 97 glomerular filtration through the kidneys and reserve SARS, severe acute respiratory syndrome; siRNA, small interfering RNA. the siRNA for alternative organ targets . Additionally, it has been reported that certain siRNA formulations are capable of accumulation in subcutan- 22,62 Properties of synthetic delivery nanoparticles eous tumours . This phenomenon has been attributed For tissues and cells that are not amenable to the delivery to the enhanced permeability and retention (EPR) effect. of naked or chemically modified siRNA, delivery of It has therefore become a common approach to exploit nanoparticles that incorporate siRNA are used. In gen- the leaky vasculature of tumours for the purposes of 7,62,63 eral, delivery vehicles are designed to both facilitate directed delivery . Others have also reported success uptake into the target tissue of interest and, when used in targeting tumours through conjugation to ligands 64,65 for systemic delivery, to protect siRNA payloads and such as antibodies . inhibit nonspecific delivery. Below, we highlight sev- eral important characteristics of delivery nanoparticles Toxicity. Even the most efficacious siRNA delivery and provide specific examples of their construction agents are rendered useless if they provoke unaccept- and use. able toxicity on either a cellular or systemic level. Viral vectors, which were among the first vehicles to be studied Surface properties. The surface charge of a delivery for siRNA delivery, can induce unacceptable levels of nano particle can significantly influence the way it toxicity through the activation of immune responses . interacts with the target cell and other physiological Therefore, synthetic lipids and polymers have been molecules. In the simplified in vitro setting, a positively developed to offer alternatives to viral vectors for nucleic charged delivery vehicle can facilitate uptake by asso- acid delivery applications, and are carefully formulated 50 39 ciating with the negatively charged cellular membrane . to avoid stimulation of the immune system . Clearance A positive charge also promotes complex formation of larger molecular mass materials typically requires and compression with the polyanionic nucleic acids them to be biodegradable. The use of biodegradable, Enhanced permeability of the siRNA. The situation, however, becomes more high molecular mass polycations and polymers contain- and retention (EPR) effect complicated in vivo as negatively charged serum pro- ing linkages that can be cleaved inside the cell can help The effect by which teins in the bloodstream will often bind to a positively reduce cytotoxicity . macromolecules undergo charged nanocomplex, therefore rendering it ineffective. increased accumulation in The addition of PEG or other hydrophilic conjugates to Synthetic materials for siRNA delivery tumours. This is attributable to the quickly growing the surface of a delivery vehicle can assist in mitigating Synthetic materials have demonstrated potential as effec- tumour vasculature, which 51 this problem . Additionally, PEG conjugation can con- tive non-viral siRNA delivery carriers. Although many is improperly formed and trol particle size and prevent particle aggregation in the types of compound have been investigated as potential subsequently more permeable presence of serum . candidates, this Review focuses on synthetic materials to large molecules. Coating of the nanoparticle with hydrophilic molecules that have successfully delivered nucleic acids in vivo. Liposome such as PEG can also play an important role in the ability An overview of the delivery agents presented here can A type of drug delivery of the siRNA delivery carrier to evade the immune sys- be found in TABLE 2. vehicle made of lipids. tem and associated phagocytes. PEG forms a barrier These nanocomplexes can around nanoparticles that provides steric stabilization Liposomes and lipid-like materials. u nilamellar and be unilamellar (one set of head-groups) or multilamellar and protection from the physiological surroundings . multilamellar liposomes are commonly used as pharma- (two or more sets of 68 The length of the PEG chain can have a significant influ- ceutical delivery vehicles . In an aqueous environment, head-groups). They can be ence on its stabilization and protective properties, and certain materials have the ability to form liposomes, in used to deliver hydrophilic chain length is typically optimized for each individual which a lipid bilayer forms a sphere with an aqueous core. or hydrophobic payloads, 53,54 depending on their structure. delivery system . For example, one set of polar head-groups can create the 132 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Figure 3 | Two common small interfering r NA (sir NA) modifications used for therapeutic applications. a | The 2′-O-methyl sugar modification prevents activation of the Toll-like receptor 7 immune response and confers enzymatic resistance to the siRNA molecule. b | The conjugation of cholesterol to the sense strand of an siRNA duplex improves delivery of naked siRNA to certain cellular targets, including hepatocytes. The cholesterol conjugate is shown in red, and the linker is shown in green. outer surface of the nanocomplex, while another set of in mice have indicated an activation of interferon α and polar head-groups faces the interior hydrophilic core, β, suggesting that stimulation of the immune system 69 72 which houses the nucleic acid payload (FIG. 4). It is also may also have a role in efficacy . possible for liposomes to be amorphous in structure, More recently, Sato and co-workers used vitamin- with the lipids and nucleic acids interspersed through- A-coupled l ipotrust liposomes to deliver anti-gp46 out. l iposomes can be created using single or multiple siRNA to fibrogenic hepatic cells for the treatment of liver types of lipid, which allows for additional flexibility cirrhosis. Five administrations of the siRNA lipocomplexes when optimizing the physical and chemical properties were reported to resolve liver fibrosis and prolong survival of the nanoparticle . in rats that had otherwise lethal liver cirrhosis, in a dose- l iposomes have been used for the delivery of nucleic and duration-dependent manner. It was also shown that acids for over 20 years, originating with studies by Felgner rescue was not related to off-target effects or associated and colleagues detailing the ability of the cationic with recruitment of the innate immune system . lipid DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N- The use of lipid complexes for localized siRNA admin- trimethlyl ammonium chloride) to deliver both DNA istration has also proved beneficial. Vaginal instillation 70,71 and RNA into mouse, rat and human cell lines . More of cationic liposomes comprised of Oligofectamine and recently, stable nucleic acid–lipid particle (SNAl P) for- siRNA targeting herpes simplex virus (HSV)-2 reportedly mulations have demonstrated efficacy in several models led to uptake by the epithelial and lamina propria cells in vivo. A study by Morrissey and co-workers indicated in the vagina in mice and protected against lethal infec- that HBV replication was inhibited through the delivery tion for up to 9 days. The siRNA complexes protected of an siRNA–SNAl P complex that targeted HBV RNA. mice when administered before and/or after (otherwise) Three daily intravenous injections of 3 mg per kg per lethal HSV-2 challenge . In another study, intrathecal day reduced serum HBV levels by at least one order of administration of complexes formulated by i-FECT and magnitude, and the effect was specific, dose-dependent siRNA protected mice from fatal Japanese encephalitis and lasted for up to 7 days after dosing . In addition, virus and West Nile virus after intracranial administra- Zimmerman and colleagues demonstrated the ability tion, and reduced pain receptor expression following of SNAl Ps to enable knockdown of ApoB in the liver of intrathecal administration in rats . cynomolgus monkeys . A single siRNA injection Also of note are several synthetic lipid-based materi- resulted in dose-dependent silencing of ApoB mRNA als that have demonstrated DNA transfection efficiency. expression in the liver 48 hours after administration, Similar materials may also potentially serve as candi- with maximal silencing of more than 90%. Knockdown dates for siRNA delivery, given the similarities involved was confirmed to be caused by ApoB mRNA cleavage at in the two types of nucleic acid delivery . For example, precisely the site predicted for the RNAi mechanism, and Wheeler et al. used liposomes comprising a combination persisted for 11 days at the highest administered dose of of the cationic lipid GAP-Dl RIE (N-(3-aminopropyl)- 2.5 mg per kg . N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium An additional study details the use of SNA l P formu- bromide) and the neutral co-lipid DOPE (dioleoylphos - lations to combat the Zaire strain of Ebola virus, which phatidylethanolamine) to transfect mouse lung with a proves fatal to 90% of its victims via haemorrhagic gene that confers chloramphenicol resistance. After a fever . When contained within the SNAl P formula- single intranasal administration, gene expression was tion, siRNA targeting of the polymerase gene of the virus enhanced by more than 100-fold relative to plasmid DNA completely protected guinea pigs against viraemia and alone, followed by a gradual return to baseline levels death when administered shortly after an Ebola virus by 21 days post-administration . Also of interest are challenge. It is important to note that preliminary studies transactivating transcriptional activator (TAT)-modified NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 133 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Table 2 | Selected synthetic materials for in vivo siRNA delivery Material Model Target r oute Animal r efs Liposomes and lipids i-FECT Japanese encephalitis JEV and WNV Intracranial Mouse 70 virus (JEV) and West envelope Nile virus (WNV) Lipidoids Dyslipidaemia FVII/ApoB Intravenous Mouse, rat, 30 monkey Dyslipidaemia FVII/ApoB Intravenous Mouse, 79 hamster Malaria Haem oxygenase 1 Intravenous Mouse 80 Hypercholesterolaemia PCSK9 Intravenous Mouse, rat 17 LipoTrust Liver cirrhosis gp46 Intravenous Rat 18 Oligofectamine Herpes simplex virus 2 HSV-2-associated Intravaginal Mouse 27 (HSV-2) viral proteins UL27 and UL29 SNALP Hepatitis B virus (HBV) HBV Intravenous Mouse 4 Dyslipidaemia ApoB Intravenous Monkey 56 Ebola (Zaire) Polymerase L Intravenous Guinea pig 69 Cationic polymers Cyclodextrin Ewing’s sarcoma tumour EWS–FLI1 Intravenous Mouse 94 xenograft Healthy monkey model RRM2 Intravenous Monkey 95 Dynamic Dyslipidaemia ApoB/PPARα Intravenous Mouse 98 PolyConjugate Poly- Glioblastoma xenograft PTN Intratumoral Mouse 28 ethyleneimine Formalin-induced pain NMDAR2B Intrathecal Rat 87 Cervical tumour xenograft HPV E6/E7 Intratumoral Mouse 20 Ovarian tumour xenograft HER2 Intraperitoneal Mouse 88 Small interfering RNA (siRNA) conjugates Cholesterol Dyslipidaemia ApoB Intravenous Mouse 46 Huntington’s disease Huntingtin gene Intrastriatal Mouse 23 Fatty acids/ Dyslipidaemia ApoB Intravenous Mouse, 47 bile salts hamster ApoB, apolipoprotein B; EWS–FLI1, Ewing’s sarcoma–friend leukaemia virus integration 1; FVII, factor VII blood protein; gp46, a glycoprotein gene; HER2, human epidermal growth factor receptor 2; HPV E6/E7, human papillomavirus oncogenes; NMDAR2B, N-methyl-d-aspartate receptor type 2B; PCSK9, proprotein convertase subtilisin/kexin type 9; PPARα, peroxisome proliferator- activated receptor α; PTN, pleiotrophin (a secreted growth factor); RRM2, ribonucleoside-diphosphate reductase subunit M2; SNALP, stable nucleic acid–lipid particles. liposomes, which, when complexed with a gene encoding In another study, a combinatorial library of lipid-like green fluorescence protein (GFP) and injected locally, materials was developed for use in siRNA delivery . induced the expression of GFP in l ewis lung carcinoma These ‘lipidoids’ were synthesized through the conju- tumour cells in mice . gate addition of alkyl-acrylates and -amides to primary Although liposomes are among the most popular and secondary amines and then studied in cell culture. nucleic acid delivery agents, some concerns regarding One leading candidate for in vivo gene knockdown was their safety for therapeutic use remain. Toxicity of certain identified as 98N -5(1), which comprises five 12-carbon cationic lipid particles has been reported both in vitro alkyl-acrylamide chains attached to an amine core (FIG. 4d). 76–78 and in vivo , and certain synthetic agents have been Formulations of this material with siRNA were capable of found to induce a gene signature of their own that might achieving potent and persistent silencing of various lung and 79,80 30 increase the off-target effects of siRNA . Despite these liver targets in mice, rats and cynomolgus monkeys . issues, liposomes show promise for future clinical use, as In a more recent primate study, 98N -5(1) was also evidenced by the approval of PEGylated liposomes for used to deliver siRNA against PCSK9, a protein that doxorubicin and amphotericin B delivery by the u S Food regulates l Dl receptor protein levels and function . 79,81 and Drug Administration . l iver-specific siRNA silencing of PCSK9 reduced PCSK9 134 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Figure 4 | s ynthetic materials for small interfering r NA (sir NA) delivery. a | A representation of stable nucleic acid–lipid particles (SNALPs), which are liposomes comprising cationic lipids, non-ionic lipids and polyethylene glycol (PEG). siRNA is contained in the hydrophilic interior of the particle. b | Polyethyleneimine is used to fabricate both linear and branched polymeric delivery agents. c | A cyclodextrin-based delivery agent. d | The lipidoid 98N -5(1). mRNA levels by 50–70% in mice and rats, as well as It has also been frequently used for various local siRNA reducing human PCSK9 transcript levels in transgenic delivery applications. Intrathecal administration of PEI– mice by more than 70%. Silencing persisted for up to siRNA complexes was reported to selectively knockdown 3 weeks after a single intravenous dose, indicating that a pain receptor in rats. Maximal effect occurred on day 3 anti-PCSK9–lipidoid complexes may serve as a potent for mRNA levels and day 7 for associated protein levels and effective treatment for hypercholesterolaemia . following an injection of 5 mg of siRNA targeting a Importantly, lipidoid materials were shown to facilitate subunit of the NMDA (N-methyl-d-aspartate) receptor siRNA delivery without disrupting endogenous micro- NR2B . PEI has also shown efficacy in a subcutaneous RNA processing . Finally, Hmox1, a gene expressed in mouse tumour model. The intraperitoneal administra- the liver encoding the protein haem oxygenase 1, has tion of complexed siRNA led to the delivery of the intact also been silenced in mice by siRNA nanoparticles formu- siRNA into the tumours and a marked reduction of lated with lipidoids. Knockdown of Hmox1 may represent tumour growth through siRNA-mediated downregulation a potential approach for the treatment of malaria infection of human epidermal growth factor 2 (HER2; also known 83 91 and disease progression . as ERBB2) .There has been significant concern regarding the toxicity of PEI at higher molecular masses and high 92,93 Polymers. Cationic polymers with a linear or branched doses . However, strategies to modify the structure of structure can serve as efficient transfection agents because PEI to reduce toxicity while retaining its potent ability 94–96 of their ability to bind and condense large nucleic acids to transfect cells are in development . 84,85 into stabilized nanoparticles . Such materials have also Cyclodextrin polymers have also been developed been shown to stimulate nonspecific endocytosis as well as siRNA delivery agents. Tumour growth in a mouse as endosomal escape . A proposed mechanism for this model of metastatic Ewing’s sarcoma was shown to is the ‘proton-sponge’ effect , whereby buffering of the be inhibited by the systemic delivery of nanoparticles endosome leads to an accumulation of ions within this formed by cyclodextrin, the targeting ligand transfer- compartment and an osmotic pressure that eventually rin, and siRNA specific for the EWS–FLI1 fusion gene 86,87 bursts the endosome . commonly associated with the condition. Knockdown PEI is a broadly investigated delivery carrier for the was not observed upon removal of the targeting ligand, administration of a wide range of nucleotide-based ther - nor was there any evidence of immune stimulation or 88,89 97 apies, including DNA, siRNA and oligonucleotides . toxicity . In another study, Heidel and co-workers used NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 135 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS (VEGFR), has shown therapeutic potential in its inhibi- Table 3 | Current clinical trials for siRNA therapeutics tion of the excessive vascularization of the eye that leads c ompany Disease Mode of s tatus to AMD . administration In 2004, the first clinical trial involving siRNA was Allergan Age-related macular Topical Phase II carried out by Acuity Pharmaceuticals for the treatment degeneration of AMD. The completed Phase II trials reported that all Alnylam Respiratory syncytial Local/direct Phase II doses were well tolerated with a lack of adverse systemic virus effects. Testing has now moved into Phase III trials, which Nucleonics Hepatitis B virus Systemic Phase I have been taken over by Opko Health. Allergan is cur- rently conducting a Phase II clinical trial on an siRNA for Quark Pharmaceuticals/ Acute renal failure Systemic Phase I Pfizer AMD, with completed Phase I results indicating minimal side effects and improved vision in some of the patients. Opko Health Age-related macular Topical Phase III Although Silence Therapeutics also had an siRNA product degeneration for AMD in the pipeline, they have now refocused their Silence/Quark/Pfizer Diabetic macular Topical Phase II Phase II clinical study on the treatment of diabetic mac- oedema ular oedema, which is another condition caused by leaky Transderm Pachyonychia Topical Phase Ia/b vasculature within the eye. Importantly, a recent study congenita has reported that anti-VEGF siRNA efficacy in the eye siRNA, small interfering RNA. is not due to specific gene silencing, but is instead caused by nonspecific stimulation of the Tl R3 pathway, which cyclodextrin–transferrin nanoparticles (FIG. 4c) to study can reduce angiogenesis . Although this study calls into the effects of siRNA delivery on the immune system of question the nature of the anti-angiogenic effect reported cynomolgus monkeys . Specifically, an siRNA targeting in AMD clinical trials, it does not explain the therapeutic the M2 subunit of ribonucleotide reductase was delivered effects observed in other applications of siRNA in which intravenously in escalating doses, and it was established appropriate controls have been performed. that multiple, systemic doses of targeted nanoparticles Also of note is Alnylam’s RSV01 formulation, which containing non-chemically modified siRNA can safely targets the nucleocapsid N gene of RSV — a major cause be administered to non-human primates . In addition, of respiratory illness in infants and young children. The cyclodextrin–transferrin nanoparticles demonstrated RSV01 formulation completed Phase I trials and was efficacy in knocking down luciferase and ribonucleotide found to be well tolerated in healthy adults. Since then, 99,100 reductase genes in mice . Phase II trials have demonstrated significant antiviral Polymer–siRNA conjugates have also shown potential efficacy of this formulation in adults . for applications in systemic siRNA delivery. Rozema et al. have developed a polymer-conjugated delivery system Future directions and conclusions called Dynamic PolyConjugates to facilitate delivery of Moving forward, we expect that synthetic nanoparticles siRNA to hepatocytes . Key features of the Dynamic composed of polymers, lipids, lipidoids or conjugates PolyConjugate technology include a membrane-active will have a key role in the systemic application of siRNA cationic polymer, the ability to reversibly mask the activity in the clinic. The incorporation of tissue-specific ligands of this polymer until it reaches the acidic environment into these particles may enable targeting, which will of the endosome, and the ability to target this modified assist with in vivo biodistribution and delivery. We also polymer and its siRNA cargo specifically to hepatocytes anticipate that future developments will require atten- after intravenous injection. Dynamic PolyConjugates tion to nonspecific activation of the immune system by 102,104 were capable of inducing knockdown of two mouse liver siRNA, including Tl R3 and Tl R7 pathways . Given genes. Analyses of serum liver enzyme and cytokine levels the potential for nonspecific effects, it is important that in treated mice indicated that siRNA complexes formed the therapeutic mode of action be validated when possible, with this synthetic polymer were well tolerated . such as through direct measurement of target mRNA levels in vivo. Chemical modification of siRNA — such Clinical trials as 2′-O-methyl substitutions — can minimize nonspecific Today, siRNA therapeutics are progressing in the clinic effects , and we expect that additional improvements (TABLE 3). Many of the most advanced trials rely on forms in both activity and delivery will be mediated by direct of localized delivery, although several ongoing clinical chemical modification of the siRNA sequence. Age-related macular trials involve the use of delivery agents. Not included In summary, the field of RNAi therapeutics has made degeneration in TABLE 3 are many formulations that are in preclinical significant progress since the first demonstration of gene (AMD). This disease of the or animal-study phases at the major pharmaceutical knockdown in mammalian cells. siRNA-based formulations eye is caused by excessive companies developing siRNA therapeutics. offer significant potential as therapeutic agents to induce growth and rupture of blood Several of the most advanced clinical trials focus on the potent, persistent and specific silencing of a broad vessels within the cornea and is a leading cause of the treatment of age-related macular degeneration (AMD), range of genetic targets. Delivery remains the most signifi- blindness. Several early siRNA which is a leading cause of blindness. AMD arises from cant barrier to the widespread use of RNAi therapeutics in clinical trials have targeted excessive blood-vessel growth and rupture within the a clinical setting, and future work focusing on the develop- this disease, primarily due cornea. Naked siRNA targeted to genes for vascular ment of safe and effective delivery materials is needed to to the ease of local delivery to the eye. endothelial growth factor (VEGF) and its receptor ensure the broadest application of RNAi in the clinic. 136 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 1. Fire, A. et al. Potent and specific genetic interference 23. DiFiglia, M. et al. Therapeutic silencing of mutant 46. Soutschek, J. et al. Therapeutic silencing of an by double-stranded RNA in Caenorhabditis elegans. huntingtin with siRNA attenuates striatal and cortical endogenous gene by systemic administration of Nature 391, 806–811 (1998). neuropathology and behavioral deficits. Proc. Natl modified siRNAs. Nature 432, 173–178 (2004). The first study to describe the phenomenon of Acad. Sci. USA 104, 17204–17209 (2007). 47. Wolfrum, C. et al. Mechanisms and optimization of RNAi. 24. de Fougerolles, A. & Novobrantseva, T. siRNA and the in vivo delivery of lipophilic siRNAs. Nature Biotech. 2. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs lung: research tool or therapeutic drug? Curr. Opin. 25, 1149–1157 (2007). mediate RNA interference in cultured mammalian Pharmacol. 8, 280–285 (2008). 48. Merki, E. et al. Antisense oligonucleotide directed to cells. Nature 411, 494–498 (2001). 25. Inoue, T. et al. Modulation of scratching behavior by human apolipoprotein B-100 reduces lipoprotein(a) The first study to demonstrate that exogenous silencing an endogenous cyclooxygenase-1 gene in levels and oxidized phospholipids on human siRNA is capable of sequence-specific knockdown the skin through the administration of siRNA. J. Gene apolipoprotein B-100 particles in lipoprotein(a) in mammalian cells. Med. 9, 994–1001 (2007). transgenic mice. Circulation 118, 743–753 (2008). 3. McCaffrey, A. P. et al. Gene expression: RNA 26. Fattal, E. & Bochot, A. Ocular delivery of nucleic acids: 49. Isis Pharmaceuticals. Genzyme and Isis Begin Second interference in adult mice. Nature 418, 38–39 antisense oligonucleotides, aptamers and siRNA. Phase 3 Trial Of Mipomersen. Isis Pharmaceuticals (2002). Adv. Drug Deliv. Rev. 58, 1203–1223 (2006). web site [online], <http://ir.isispharm.com/ Describes the first observation of sequence-specific 27. Palliser, D. et al. An siRNA-based microbicide protects releasedetail.cfm?ReleaseID=326694> (2008). gene silencing in mice using siRNA. mice from lethal herpes simplex virus 2 infection. 50. Torchilin, V. P. et al. Cell transfection in vitro and in vivo 4. Morrissey, D. V. et al. Potent and persistent in vivo Nature 439, 89–94 (2006). with nontoxic TAT peptide–liposome–DNA complexes. anti-HBV activity of chemically modified siRNAs. 28. Grzelinski, M. et al. RNA interference-mediated gene Proc. Natl Acad. Sci. USA 100, 1972–1977 (2003). Nature Biotech. 23, 1002–1007 (2005). silencing of pleiotrophin through polyethylenimine- 51. Auguste, D. T. et al. Triggered release of siRNA from 5. Okumura, A., Pitha, P. M. & Harty, R. N. complexed small interfering RNAs in vivo exerts poly(ethylene glycol)-protected, pH-dependent ISG15 inhibits Ebola VP40 VLP budding in an antitumoral effects in glioblastoma xenografts. liposomes. J. Control. Release 130, 266–274 (2008). L-domain-dependent manner by blocking Nedd4 Hum. Gene Ther. 17, 751–766 (2006). 52. Martina, M.-S. et al. The in vitro kinetics of the ligase activity. Proc. Natl Acad. Sci. USA 105, 29. Bitko, V., Musiyenko, A., Shulyayeva, O. & Barik, S. interactions between PEG-ylated 3974–3979 (2008). Inhibition of respiratory viruses by nasally magnetic-fluid-loaded liposomes and macrophages. 6. Ptasznik, A., Nakata, Y., Kalota, A., Emerson, S. G. & administered siRNA. Nature Med. 11, 50–55 (2005). Biomaterials 28, 4143–4153 (2007). Gewirtz, A. M. Short interfering RNA (siRNA) targeting 30. Akinc, A. et al. A combinatorial library of lipid-like 53. Mao, S. et al. Influence of polyethylene glycol chain the Lyn kinase induces apoptosis in primary, and drug- materials for delivery of RNAi therapeutics. Nature length on the physicochemical and biological resistant, BCR-ABL1 leukemia cells. Nature Med. 10, Biotech. 26, 561–569 (2008). properties of poly(ethylene imine)-graft-poly(ethylene 1187–1189 (2004). Describes the combinatorial synthesis and glycol) block copolymer/siRNA polyplexes. Bioconjug. 7. Kim, S. H., Jeong, J. H., Lee, S. H., Kim, S. W. & in vivo delivery performance of a large library of Chem. 17, 1209–1218 (2006). Park, T. G. Local and systemic delivery of VEGF siRNA lipid-like materials in multiple animal models and 54. Li, W., Huang, Z., MacKay, J. A., Grube, S. & using polyelectrolyte complex micelles for effective species. Szoka, F. C. Low-pH-sensitive poly(ethylene glycol) treatment of cancer. J. Control. Release 129, 107–116 31. DeVincenzo, J. et al. Evaluation of the safety, (PEG)-stabilized plasmid nanolipoparticles: effects of (2008). tolerability and pharmacokinetics of ALN-RSV01, PEG chain length, lipid composition and assembly 8. Xia, C.-F., Zhang, Y., Zhang, Y., Boado, R. & a novel RNAi antiviral therapeutic directed against conditions on gene delivery. J. Gene Med. 7, 67–79 Pardridge, W. Intravenous siRNA of brain cancer with respiratory syncytial virus (RSV). Antiviral Res. 77, (2005). receptor targeting and avidin–biotin technology. 225–231 (2008). 55. Braasch, D. A. et al. Biodistribution of phosphodiester Pharm. Res. 24, 2309–2316 (2007). 32. Li, B.-J. et al. Using siRNA in prophylactic and and phosphorothioate siRNA. Bioorg. Med. Chem. 9. Bernstein, E., Caudy, A. A., Hammond, S. M. & therapeutic regimens against SARS coronavirus in Lett. 14, 1139–1143 (2004). Hannon, G. J. Role for a bidentate ribonuclease Rhesus macaque. Nature Med. 11, 944–951 (2005). 56. Bogdanov, A. A. Merging molecular imaging and RNA in the initiation step of RNA interference. Nature 409, 33. Alexis, F., Pridgen, E., Molnar, L. K. & Farokhzad, O. C. interference: early experience in live animals. J. Cell 363–366 (2001). Factors affecting the clearance and biodistribution of Biochem. 104, 1113–1123 (2008). 10. Grimm, D. et al. Fatality in mice due to oversaturation polymeric nanoparticles. Mol. Pharm. 5, 505–515 57. Zimmermann, T. S. et al. RNAi-mediated gene silencing of cellular microRNA/short hairpin RNA pathways. (2008). in non-human primates. Nature 441, 111–114 (2006). Nature 441, 537–541 (2006). 34. Scherphof, G. L. in Targeted Drug Delivery The first study to demonstrate sequence-specific 11. Rand, T. A., Ginalski, K., Grishin, N. V. & Wang, X. (ed. Juliano, R. L.) 285–313 (Springer, Berlin, 1991). RNAi in non-human primates. Biochemical identification of Argonaute 2 as the sole 35. Zámecník, J., Vargová, L., Homola, A., Kodet, R. & 58. Yuan, H. et al. Effects of cholesterol-tagged small protein required for RNA-induced silencing complex Syková, E. Extracellular matrix glycoproteins and interfering RNAs targeting 12/15-lipoxygenase on activity. Proc. Natl Acad. Sci. USA 101, 14385–14389 diffusion barriers in human astrocytic tumours. parameters of diabetic nephropathy in a mouse model (2004). Neuropathol. Appl. Neurobiol. 30, 338–350 (2004). of type 1 diabetes. Am. J. Physiol. Renal Physiol. 295, 12. Matranga, C., Tomari, Y., Shin, C., Bartel, D. P. & 36. Oliveira, S., van Rooy, I., Kranenburg, O., Storm, G. & F605–F617 (2008). Zamore, P. D. Passenger-strand cleavage facilitates Schiffelers, R. Fusogenic peptides enhance endosomal 59. Rappaport, J. et al. Transport of phosphorothioate assembly of siRNA into ago2-containing RNAi enzyme escape improving siRNA-induced silencing of oligonucleotides in kidney: implications for molecular complexes. Cell 123, 607–620 (2005). oncogenes. Int. J. Pharm. 331, 211–214 (2007). therapy. Kidney Int. 47, 1462–1469 (1995). 13. Ameres, S. L., Martinez, J. & Schroeder, R. 37. Di Guglielmo, G. M., Le Roy, C., Goodfellow, A. F. & 60. Santel, A. et al. A novel siRNA-lipoplex technology for Molecular basis for target RNA recognition and Wrana, J. L. Distinct endocytic pathways regulate RNA interference in the mouse vascular endothelium. cleavage by human RISC. Cell 130, 101–112 (2007). TGF-β receptor signalling and turnover. Nature Cell Gene Ther. 13, 1222–1234 (2006). 14. Rand, T. A., Petersen, S., Du, F. & Wang, X. Biol. 5, 410–421 (2003). 61. van de Water, F. M. et al. Intravenousely administered Argonaute2 cleaves the anti-guide strand of siRNA 38. Hornung, V. et al. Sequence-specific potent induction short interfering RNA accumulates in the kidney and during RISC activation. Cell 123, 621–629 (2005). of IFN-α by short interfering RNA in plasmacytoid selectively suppresses gene function in renal proximal 15. Hutvagner, G. & Zamore, P. D. A microRNA in a dendritic cells through TLR7. Nature Med 11, tubules. Drug Metab. Dispos. 34, 1393–1397 (2006). multiple-turnover RNAi enzyme complex. Science 263–270 (2005). 62. Takei, Y., Kadomatsu, K., Yuzawa, Y., Matsuo, S. & 297, 2056–2060 (2002). The TLR7 pathway is identified as an important Muramatsu, T. A small interfering RNA targeting 16. Bartlett, D. W. & Davis, M. E. Insights into the kinetics contributor to nonspecific siRNA immune activation. vascular endothelial growth factor as cancer of siRNA-mediated gene silencing from live-cell and 39. Judge, A. D. et al. Sequence-dependent stimulation of therapeutics. Cancer Res. 64, 3365–3370 (2004). live-animal bioluminescent imaging. Nucleic Acids Res. the mammalian innate immune response by synthetic 63. McNamara, J. O. et al. Cell type-specific delivery of 34, 322–333 (2006). siRNA. Nature Biotech. 23, 457–462 (2005). siRNAs with aptamer-siRNA chimeras. Nature Biotech. 17. Frank-Kamenetsky, M. et al. Therapeutic RNAi 40. Judge, A. D., Bola, G., Lee, A. C. H. & MacLachlan, I. 24, 1005–1015 (2006). targeting PCSK9 acutely lowers plasma cholesterol in Design of noninflammatory synthetic siRNA mediating 64. Song, E. et al. Antibody mediated in vivo delivery rodents and LDL cholesterol in nonhuman primates. potent gene silencing in vivo. Mol. Ther. 13, 494–505 of small interfering RNAs via cell-surface receptors. Proc. Natl Acad. Sci. USA 105, 11915–11920 (2006). Nature Biotech. 23, 709–717 (2005). (2008). 41. Vornlocher, H.-P. et al. Nuclease-resistant double- 65. Peer, D., Park, E. J., Morishita, Y., Carman, C. V. & 18. Sato, Y. et al. Resolution of liver cirrhosis using stranded RNA for RNA interference. WO2005115481 Shimaoka, M. Systemic leukocyte-directed siRNA vitamin A-coupled liposomes to deliver siRNA against (2005). delivery revealing Cyclin D1 as an anti-inflammatory a collagen-specific chaperone. Nature Biotech. 26, 42. Jackson, A. L. et al. Position-specific chemical target. Science 319, 627–630 (2008). 431–442 (2008). modification of siRNAs reduces “off-target” transcript 66. Barquinero, J., Eixarch, H. & Perez-Melgosa, M. 19. Song, E. et al. RNA interference targeting Fas silencing. RNA 12, 1197–1205 (2006). Retroviral vectors: new applications for an old tool. protects mice from fulminant hepatitis. Nature Med. 43. Bumcrot, D., Manoharan, M., Koteliansky, V. & Gene Ther. 11, S3–S9 (2004). 9, 347–351 (2003). Sah, D. W. Y. RNAi therapeutics: a potential new class 67. Vandenbroucke, R. E. et al. Prolonged gene silencing 20. Niu, X.-Y., Peng, Z. L., Duan, W. Q., Wang, H. & of pharmaceutical drugs. Nature Chem. Biol. 2, in hepatoma cells and primary hepatocytes after small Wang, P. Inhibition of HPV 16 E6 oncogene expression 711–719 (2006). interfering RNA delivery with biodegradable poly(β- by RNA interference in vitro and in vivo. Int. 44. Muratovska, A. & Eccles, M. R. Conjugate for efficient amino esters). J. Gene Med. 10, 783–794 (2008). J. Gynecol. Cancer 16, 743–751 (2006). delivery of short interfering RNA (siRNA) into 68. Torchilin, V. P. Recent advances with liposomes as 21. Halder, J. et al. Focal adhesion kinase targeting using mammalian cells. FEBS Lett. 558, 63–68 (2004). pharmaceutical carriers. Nature Rev. Drug Discov. 4, in vivo short interfering RNA delivery in neutral 45. Oishi, M., Nagasaki, Y., Itaka, K., Nishiyama, N. & 145–160 (2005). liposomes for ovarian carcinoma therapy. Clin. Cancer Kataoka, K. Lactosylated poly(ethylene glycol)-siRNA 69. MacLachlan, I. in Antisense Drug Technology: Res. 12, 4916–4924 (2006). conjugate through acid-labile β-thiopropionate Principles, Strategies and Applications. 2nd edn Ch. 9 22. Takeshita, F. et al. Efficient delivery of small interfering linkage to construct pH-sensitive polyion complex (ed. Crooke, S. T.) 237–270 (CRC, Boca Raton, 2007). RNA to bone-metastatic tumors by using atelocollagen micelles achieving enhanced gene silencing in 70. Felgner, P. L. et al. Lipofection: a highly efficient, in vivo. Proc. Natl Acad. Sci. USA 102, 12177–12182 hepatoma cells. J. Am. Chem. Soc. 127, 1624–1625 lipid-mediated DNA-transfection procedure. Proc. Natl (2005). (2005). Acad. Sci. USA 84, 7413–7417 (1987). NATu RE REVIEWS | Drug Discovery VOlu M E 8 | FEB Ru ARy 2009 | 137 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 71. Malone, R. W., Felgner, P. L. & Verma, I. M. Cationic 85. Putnam, D. Polymers for gene delivery across length 98. Heidel, J. D. et al. Administration in non-human liposome-mediated RNA transfection. Proc. Natl scales. Nature Mater. 5, 439–451 (2006). primates of escalating intravenous doses of targeted Acad. Sci. USA 86, 6077–6081 (1989). 86. Akinc, A., Thomas, M., Klibanov, A. M. & Langer, R. nanoparticles containing ribonucleotide reductase 72. Geisbert, T. W. et al. Postexposure protection of Exploring polyethylenimine-mediated DNA subunit M2 siRNA. Proc. Natl Acad. Sci. USA 104, guinea pigs against a lethal ebola virus challenge is transfection and the proton sponge hypothesis. 5715–5721 (2007). conferred by RNA interference. J. Infect. Dis. 193, J. Gene Med. 7, 657–663 (2005). 99. Bartlett, D. W., Su, H., Hildebrandt, I. J., Weber, W. A. 1650–1657 (2006). 87. Wang, Y., Chen, P. & Shen, J. The development and & Davis, M. E. Impact of tumor-specific targeting on 73. Kumar, P., Lee, S. K., Shankar, P. & Manjunath, N. A characterization of a glutathione-sensitive cross-linked the biodistribution and efficacy of siRNA nanoparticles single siRNA suppresses fatal encephalitis induced by polyethylenimine gene vector. Biomaterials 27, measured by multimodality in vivo imaging. Proc. Natl two different Flaviviruses. PLoS Med. 3, e96 (2006). 5292–5298 (2006). Acad. Sci. USA 104, 15549–15554 (2007). 74. Li, W. & Szoka, F. Lipid-based nanoparticles for nucleic 88. Lungwitz, U., Breunig, M., Blunk, T. & Göpferich, A. 100. Bartlett, D. W. & Davis, M. E. Impact of tumor-specific acid delivery. Pharm. Res. 24, 438–449 (2007). Polyethylenimine-based non-viral gene delivery targeting and dosing schedule on tumor growth 75. Wheeler, C. J. et al. A novel cationic lipid greatly systems. Eur. J. Pharm. Biopharm. 60, 247–266 inhibition after intravenous administration of siRNA- enhances plasmid DNA delivery and expression (2005). containing nanoparticles. Biotechnol. Bioeng. 99, in mouse lung. Proc. Natl Acad. Sci. USA 93, 89. Zintchenko, A., Philipp, A., Dehshahri, A. & Wagner, E. 975–985 (2008). 11454–11459 (1996). Simple modifications of branched PEI lead to highly 101. Rozema, D. B. et al. Dynamic PolyConjugates for 76. Lv, H., Zhang, S., Wang, B., Cui, S. & Yan, J. Toxicity of efficient siRNA carriers with low toxicity. Bioconjug. targeted in vivo delivery of siRNA to hepatocytes. cationic lipids and cationic polymers in gene delivery. Chem. 19, 1448–1455 (2008). Proc. Natl Acad. Sci. USA 104, 12982–12987 (2007). J. Control. Release 114, 100–109 (2006). 90. Tan, P. H., Yang, L. C., Shih, H. C., Lan, K. C. & 102. Kleinman, M. E. et al. Sequence- and target- 77. Ma, Z. et al. Cationic lipids enhance siRNA-mediated Cheng, J. T. Gene knockdown with intrathecal siRNA independent angiogenesis suppression by siRNA interferon response in mice. Biochem. Biophys. Res. of NMDA receptor NR2B subunit reduces formalin- via TLR3. Nature 452, 591–597 (2008). Commun. 330, 755–759 (2005). induced nociception in the rat. Gene Ther. 12, 59–66 Demonstration that the anti-angiogenic effects 78. Akhtar, S. & Benter, I. Toxicogenomics of non-viral (2004). seen upon siRNA delivery to the eye can be drug delivery systems for RNAi: potential impact on 91. Urban-Klein, B., Werth, S., Abuharbeid, S., Czubayko, F. nonspecific and caused by TLR3 immune activation. siRNA-mediated gene silencing activity and specificity. & Aigner, A. RNAi-mediated gene-targeting through 103. Alnylam Pharmaceuticals. Alnylam Achieves First Adv. Drug Deliv. Rev. 59, 164–182 (2007). systemic application of polyethylenimine (PEI)- Human Proof of Concept for an RNAi Therapeutic with 79. Akhtar, S. & Benter, I. F. Nonviral delivery of synthetic complexed siRNA in vivo. Gene Ther. 12, 461–466 GEMINI study. Alnylam Pharmaceuticals web site siRNAs in vivo. J. Clin. Invest. 117, 3623–3632 (2004). [online] <http://phx.corporate-ir.net/phoenix. (2007). 92. Kichler, A. Gene transfer with modified zhtml?c=148005&p=irol-newsArticle2&ID=11139 80. Hollins, A. J., Omidi, Y., Benter, I. F. & Akhtar, S. polyethylenimines. J. Gene Med. 6, S3–S10 (2004). 37&highlight=> (2008). Toxicogenomics of drug delivery systems: exploiting 93. Kircheis, R., Wightman, L. & Wagner, E. Design and 104. Robbins, M. et al. Misinterpreting the therapeutic delivery system-induced changes in target gene gene delivery activity of modified polyethylenimines. effects of siRNA caused by immune stimulation. expression to enhance siRNA activity. J. Drug Target. Adv. Drug Deliv. Rev. 53, 341–358 (2001). Hum. Gene Ther. 19 Aug 2008 (doi 10.1089/ 15, 83–88 (2007). 94. Richards Grayson, A., Doody, A. & Putnam, D. hgt.2008.131). 81. Rust, D. M. & Jameson, G. The novel lipid delivery Biophysical and structural characterization of 105. Zhang, Y. et al. Engineering mucosal RNA interference system of amphotericin B: drug profile and relevance polyethylenimine-mediated siRNA delivery in vitro. in vivo. Mol. Ther. 14, 336–342 (2006). to clinical practice. Oncol. Nurs. Forum 25, 35–48 Pharm. Res. 23, 1868–1876 (2006). 106. Arnold, A.-S. et al. Specific β1-adrenergic receptor (1998). 95. Thomas, M. et al. Full deacylation of polyethylenimine silencing with small interfering RNA lowers high blood 82. John, M. et al. Effective RNAi-mediated gene silencing dramatically boosts its gene delivery efficiency and pressure and improves cardiac function in myocardial without interruption of the endogenous microRNA specificity to mouse lung. Proc. Natl Acad. Sci. USA ischemia. J. Hypertens. 25, 197–205 (2007). pathway. Nature 449, 745–747 (2007). 102, 5679–5684 (2005). Demonstration that specific, non-viral RNAi 96. Werth, S. et al. A low molecular weight fraction of Acknowledgements delivery can be achieved without interruption polyethylenimine (PEI) displays increased transfection The authors would like to thank A. de Fougerolles, R. Alvarez of endogenous microRNA processing. efficiency of DNA and siRNA in fresh or lyophilized and E. Deguns for their advice and assistance in the prepara- 83. Epiphanio, S. et al. Heme oxygenase-1 is an anti- complexes. J. Control. Release 112, 257–270 tion of the manuscript. inflammatory host factor that promotes murine (2006). Plasmodium liver infection. Cell Host Microbe 3, 97. Hu-Lieskovan, S., Heidel, J. D., Bartlett, D. W., DATABASES 331–338 (2008). Davis, M. E. & Triche, T. J. Sequence-specific UniProtKB: http://www.uniprot.org 84. Boussif, O. et al. A versatile vector for gene and knockdown of EWS-FLI1 by targeted, nonviral delivery PCSK9 | TLR3 | TLR7 | VEGF | VEGFR oligonucleotide transfer into cells in culture and of small interfering RNA inhibits tumor growth in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA in a murine model of metastatic Ewing’s sarcoma. All li Nks Are Ac Tive i N The o Nli Ne PDf 92, 7297–7301 (1995). Cancer Res. 65, 8984–8992 (2005). 138 | FEB Ru ARy 2009 | VOlu M E 8 w w w.nature.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. All rights reserved CoRRigENDuM Kathryn A. Whitehead, Robert Langer & Daniel G. Anderson Nature Reviews Drug Discovery 8, 129–138 (2009) | doi:10.1038/nrd2742 On page 136 in Table 3, the clinical trial for acute renal failure has been attributed to Quark /Pfizer when it should be attributed to Quark alone. In addition, the authors would like to clarify that, following a Phase I/II trial in patients with wet age-related macular degeneration, the siRNA drug candidate PF-4523655 (RTP801i-14) is now being studied in a Phase II trial for diabetic macular oedema conducted by Pfizer in collaboration with Quark.

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Nature Reviews. Drug Discovery – Pubmed Central

Published: May 1, 168

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