Special delivery: targeted therapy with small RNAs

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Keywords: siRNA; drug; liposome; nanoparticle; aptamer. INTRODUCTION .... of treating diseases, such as cancer, that require efficient systemic delivery.
Gene Therapy (2011), 1–7 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt

REVIEW

Special delivery: targeted therapy with small RNAs D Peer1 and J Lieberman2 Harnessing RNA interference using small RNA-based drugs has great potential to develop drugs designed to knock down expression of any disease-causing gene, thereby greatly expanding the universe of possible drug targets. However, delivering small RNAs into specific tissues and cells is still a hurdle. Here, we review recent progress in overcoming systemic, local and cellular barriers to RNA drug delivery, focusing on strategies for targeted uptake. Gene Therapy (2011) 0, 000–000. doi:10.1038/gt.2011.56 Keywords: siRNA; drug; liposome; nanoparticle; aptamer

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animals may have been due to off-target effects.8,9 However, chemical modifications of the RNA can largely eliminate off-target effects without compromising target-gene knockdown. Despite the promise, developing siRNA as therapeutics has proven challenging. Like most drug development, there is no quick fix. Indeed, recently, a large pharmaceutical company that invested considerable sums to develop RNA-based drugs announced it was closing its RNAi subsidiaries. Although many of the hurdles to developing RNAi-based drugs have been easily addressed, the main obstacle is figuring out how to deliver small RNAs into cells in a therapeutically acceptable way. Small RNAs being considered as therapeutic drugs include not only siRNAs designed to knock down one gene at a time, but also mimics of endogenous microRNAs to suppress the expression of many genes but with less efficient suppression of each one. The delivery hurdle that needs to be solved to administer siRNAs and imperfectly paired microRNA mimics is essentially the same (although antagonizing endogenous microRNAs using single-stranded antisense oligonucleotides may be somewhat easier). When injected intravenously, siRNAs are rapidly cleared by renal filtration and are susceptible to degradation by extracellular RNases. The siRNA half-life can be increased—even to days—by chemical modifications to eliminate susceptibility to endogenous exonucleases and endonucleases and by incorporating the RNA into a larger moiety, above the molecular weight cutoff for kidney filtration. However, entering the cell is the biggest obstacle. Because of their large molecular weight (B13 kDa) and net negative charge, naked siRNAs do not cross the plasma membrane.4,8 Although cells can endocytose many types of modified RNAs or RNA-containing particles, another important bottleneck is getting the RNA efficiently out of the endosome into the cytosol where the RNAi machinery resides. Here, we will review recent progress in this emerging field, focusing mostly on in vivo applications with special emphasis on strategies for targeted siRNA delivery.

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INTRODUCTION RNA interference (RNAi) is a natural ubiquitous cellular mechanism for small RNA-guided post-transcriptional suppression of gene expression. The endogenous small RNAs responsible for gene regulation are processed to B19–23 nucleotide imperfectly paired, doublestranded RNAs with 2 unpaired nucleotides at their 5¢-phosphorylated ends and unphosphorylated 3¢ end.1,2 RNAi can be activated exogenously by expressing from viral vectors short hairpin RNAs that are processed intracellularly into small RNAs that mimic the endogenous gene-silencing RNAs or by introducing synthetic small interfering RNAs (siRNAs) directly into the cell cytoplasm.3,4 In the cytoplasm, siRNAs are incorporated into the endogenous machinery responsible for gene silencing, the RNA-induced silencing complex (RISC), which removes one strand. The remaining antisense RNA strand then guides the RISC to bind, cleave and block translation of mRNAs bearing complementary sequences.4 As the target mRNA is destroyed and the antisense strand is protected from degradation within the RISC, the same RNA can be used repeatedly to eliminate many transcripts. In fact, gene knockdown occurs with picomolar concentrations of RNA and can last for up to 7 days in dividing cells (where the therapeutic RNA is diluted with each cell division) and for several weeks in non-dividing cells, even in vivo.5–7 Almost as soon as RNAi was found in mammals just a decade ago,1 synthetic siRNAs were shown to treat disease in mice.6 Small RNAs were quickly heralded as the ‘next new class of drugs’. Enthusiasm ran high because of the potential of small synthetic RNAs to knock down any gene of interest to treat almost any disease by targeting otherwise ‘undruggable’ targets (that is, molecules without ligand-binding domains or enzymatic function). Although, initially, gene knockdown was thought to be impeccably specific for the target gene, it soon became clear that off-target effects were prevalent via suppression of genes harboring non-identical but homologous sequences (as is the case for endogenous microRNAs), and by recognition by innate immune RNA sensors that can initiate interferon and cytokine secretion and activate complement and coagulation cascades. Indeed, some of the early demonstrations of siRNA therapeutic effects in small

Local vs systemic in vivo delivery of siRNA Most of the methods commonly used for in vitro or ex vivo delivery of siRNAs rely on conventional transfection methods. Physical methods,

1Laboratory of Nanomedicine, Department of Cell Research and Immunology, GS Wise Faculty of Life Science and Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, Israel and 2Immune Disease Institute, and Program in Cellular and Molecular Medicine, Children’s Hospital Boston and Department of Pediatrics, Q2 Harvard Medical School, Boston, MA, USA Correspondence: Professor J Lieberman, Department of Pediatrics, Immune Disease Institute, Program in Cellular and Molecular Medicine, Children’s Hospital Boston, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA. E-mail: [email protected] or [email protected] Received 24 January 2011; accepted 16 February 2011

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Systemic siRNA delivery strategies can be divided into passive and active (targeted) delivery. Passive delivery exploits the inherent tendency of nanoparticles to accumulate in filtering organs of the reticuloendothelial system, which contains fenestrated capillaries and phagocytic cells, primarily monocytes and macrophages. These tissues, including lymph nodes, spleen and liver, trap foreign particles to protect us from viruses, bacteria and parasites. As a consequence, many liposomes and nanoparticles concentrate in the liver. Thus, a major focus of RNAi therapeutics research has been to develop siRNA delivery systems for treating liver disease. However, in most phagocytic cells, such as macrophages, although the siRNAs are internalized, they do not efficiently escape from endosomes to the cytoplasm and thus do not knock down gene expression efficiently. Efforts to develop siRNA delivery strategies need to improve and optimize delivery across the cell membrane and then (if the siRNA is endocytosed) across the endosomal membrane. The ultimate success of each delivery strategy will depend upon how much of the therapeutic RNA gets incorporated into the RISC of intended target cells and how long it stays active. High-resolution imaging could be used more fruitfully as a guide to improve siRNA/carrier designs to optimize uptake and trafficking of labeled siRNAs by quantifying the proportion of siRNAs that get internalized, trapped within endosomes, or escape and get incorporated into the RISC.29 Another potentially useful guide for optimizing delivery might be to assess directly how much of the administered siRNA is incorporated into the RISC by argonaute immunoprecipitation of the RISC together with its bound small RNAs. Applying rigorous quantitative methods to follow the siRNA within cells will undoubtedly help optimize design of delivery reagents. At present, it is not clear how many siRNA molecules are needed to knock down a gene or how chemical modifications of the siRNA or properties of the target gene mRNA might affect the durability of gene knockdown.

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such as microinjection and electroporation,10–13 as well as calcium co-precipitation,14 commercially available cationic polymers and lipids,1,15–20 and cell-penetrating peptides21–25 can all be used to knock down genes. Aside from the physical methods (in which siRNAs are directly injected into the cell cytoplasm or enter via membrane pores caused by electrical bursts), all the other methods share a common characteristic complexation with a positive (cationic) charge that enables the siRNAs to interact with the negatively charged plasma membrane. All of the commercial cationic lipids and polymers induce some cytotoxicity (reviewed in Lv et al.26) In addition, recent work also demonstrated that some cationic lipids can agonize Toll-like receptor 4 and induce interferons and proinflammatory cytokines.27 On the contrary, cell-penetrating peptides and proteins are largely non-toxic and have the potential for specific targeting. For some indications, such as prevention or treatment of infectious diseases that enter the body at the mucosa or ocular and skin diseases, the target tissue is readily accessible and direct application of small RNAs should in-principle be possible. However, many of the early studies that claimed RNAi therapeutic benefits in the eye and lung did not rigorously show siRNA intercellular delivery or that the therapeutic benefit was due to gene knockdown. In retrospect, some of the therapeutic benefits were likely due to off-target effects, particularly interferon induction. Epithelial cells in the mucosa and skin can be efficiently transfected using in vitro transfection methods described above. However, the cornified layer of the skin is a significant barrier to transfection and RNA delivery only occurs after abrasion, limiting its usefulness in diseases that affect widespread areas. The female genital mucosa can be very efficiently transfected, providing impressive protection against sexual transmission of herpes virus infection, using either siRNAs lipoplexed with cationic lipids or cholesterolconjugated siRNAs.7,28 Although the lipoplexed siRNA causes mild inflammation, cholesterol-conjugated siRNAs show no evidence of cytotoxicity, inflammation or immune activation. Treatment of diseases that are localized and involve accessible and relatively easily transfected tissues would seem to be the low-hanging fruit for developing RNAi-based therapeutics. Although a few clinical studies have been initiated to treat a congenital skin infection or respiratory infection,4 (http://clinical trials.gov/ct2/show/NCT00716014, 2008) most of the current preclinical and clinical development is focused on the more challenging task of treating diseases, such as cancer, that require efficient systemic delivery. Systemic delivery needs to address not only the need for internalization, release and accumulation of siRNAs in the cytoplasm but also the interaction of the siRNA with blood components, including RNases, entrapment within capillaries, uptake by reticuloendothelial cells, uptake by filtering organs, such as the liver and lung, renal clearance, extravasation from blood vessels to target tissues, and permeation within the tissue. The first successful systemic delivery of siRNAs for therapeutic purposes involved ‘hydrodynamic’ injection (a very large, rapid bolus injection) of naked, unmodified siRNAs in mice to silence the death receptor fas in the liver and prevent autoimmune hepatitis.6 These injections knock down gene expression in highly vascularized central organs, like the liver, lung, pancreas and kidney. Although the precise way these injections work is unknown, the volume overload is hard to control, difficult to scale-up to larger organisms and clinically dangerous. Therefore, this delivery method is not relevant for humans. Because of rapid renal clearance, siRNAs injected under non-hydrodynamic conditions need a carrier to ensure a reasonable circulating half-life, except possibly, if the target tissue is the renal proximal tubule. Those carriers should be made from fully degradable materials to avoid undesired and probably toxic accumulation in the body. Gene Therapy

Passive systemic siRNA delivery Stable nucleic acid–lipid particles (SNALPs) are B100 nm nontargeted liposomes with low cationic lipid content that incorporate siRNAs and are coated with a diffusible polyethylene glycol–lipid (PEG–lipid) conjugate.30,31 The PEG–lipid coat stabilizes the particle during formation and provides a neutral and hydrophilic exterior that prevents rapid systemic clearance. The lipid bilayer contains a mixture of cationic and fusogenic lipids that facilitate internalization of the SNALP and endosomal escape of its siRNA payload. However, it is uncertain how much of the internalized siRNAs actually get delivered to the cytosol. About 28% of the siRNA payload in intravenously injected SNALPs accumulates in the liver and only 0.3% in the lungs. SNALPs encapsulating ApoB siRNAs significantly reduce ApoB mRNA levels in mice and non-human primates at reasonable doses (B2.5 mg kg 1). Although cationic lipids are known to cause toxicity,26 the only reported adverse effects in animals are transient liver enzyme release. A Phase I clinical study that involved a single injection of ApoB siRNA-containing SNALPs showed target gene knockdown and serum cholesterol reduction and acceptable safety, although flu-like symptoms, indicative of stimulation of innate immune receptors, occurred at the highest dose. On the basis of these results, a clinical trial is in progress to test the ability of SNALPs to deliver siRNAs targeting a gene required for mitotic spindle formation (PLK1) for liver cancer treatment. SNALPs encapsulating siRNAs directed against the polymerase gene of the Zaire strain of Ebola virus also protect guinea pigs from lethal viral challenge.32 Other formulations of cationic liposomes, with higher cationic lipid content than the SNALPs, have not only induced effective gene

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polyconjugates’.43 In this intelligently designed delivery system, siRNAs are conjugated to membrane-penetrating polymers whose activity is unmasked only after reaching the acidic environment of the endosome. Therefore, in principle, cell membrane toxicity by the polymer is limited. The polymers are conjugated to PEG to reduce nonspecific uptake by phagocytic cells en route to the liver and with N-acetylgalactosamine, which binds to the asialoglycoprotein receptor expressed on hepatocytes, but not other resident cells in the liver. These hepatocyte-targeted particles, when conjugated to ApoB–siRNAs, specifically decrease ApoB mRNA levels in the liver for a week more efficiently than cholesterol-conjugated siRNAs and with comparable efficiency as SNALPs. Although potential toxicity was not evaluated thoroughly, there was some suggestion that dynamic polyconjugates cause some immune stimulation and hepatic toxicity, but these side effects might be clinically acceptable. The potential application of other small molecule–siRNA conjugates to deliver siRNAs and induce gene silencing to tissues outside the liver has not been extensively explored and might be a fruitful avenue for further research. One clever example takes advantage of native nucleic acid receptors on immune cells as a way to both activate innate immunity and induce gene silencing to improve the endogenous immune response to xenografted tumors.44 Intratumoral or intravenous injection of siRNAs conjugated to a CpG oligonucleotide agonist of Toll-like receptor 9 targeted myeloid cells and B cells that express Toll-like receptor 9. These conjugated siRNAs simultaneously knock down Stat3 and activate Toll-like receptor 9 activation to induce antitumor immunity by reducing immunosuppressive regulatory T cells and enhancing tumor infiltration with antitumor killer T cells. Thereby, CpG–Stat3 siRNAs inhibit tumor outgrowth. However, gene silencing does not occur with an antagonistic oligonucleotide, suggesting that it might be difficult to get siRNA delivery via innate immune nucleic acid receptors without simultaneously triggering innate immunity. Although in some circumstances, such as in the setting of tumor therapy, this may provide synergistic therapeutic benefit, it may be difficult to control the inflammatory side effects caused by systemic immune activation of dendritic cells and macrophages outside the tumor.

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silencing but also elevated cytokine levels and toxicity and thus cannot be used clinically. Considering the significant toxicity that has been associated with cationic liposomes, neutral liposomes are promising carriers for systemic delivery of siRNAs. 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine non-pegylated liposomes encapsulating siRNA against genes expressed in melanoma and ovarian cancer inhibit tumor growth in human cancer xenograft models in mice.33,34 These liposomes accumulate in the cancers. This is thought to be due to the enhanced permeability and retention effect—increased permeability of blood vessels in tumors caused by rapid and defective angiogenesis and defective lymphatic drainage that retains the accumulated liposomes. Cationic synthetic lipid-like molecules, termed lipidoids, can also be used to construct siRNA-containing liposomes to induce effective gene silencing in the liver. At doses between 1 and 10 mg kg 1, ApoB and/or Factor VII mRNA levels decrease in the liver in mice, rats and cynomolgus monkeys. A single intravenous injection of cationic lipidoid-containing liposomes encapsulating ApoB–siRNA reduces protein levels by 50% for 2 weeks. Although no immune response was reported, small increases in serum levels of two liver enzymes and a reduction in platelet count suggests a potential concern about liver toxicity.35,36 Further design modifications of liposomes may improve the therapeutic index by enhancing intracellular delivery and/or further reducing cytotoxicity and immune stimulation. Some positively charged peptides can be used to introduce RNA into cells. One example is atelocollagen, produced from pepsindigested type I collagen from calf dermis, which is rich in positively charged residues (lysine and hydroxylysine), and thereby complexes with negatively charged siRNAs and interacts with the plasma membrane. Although these particles have not been modified to target tumors, passive targeting due to the enhanced permeability and retention effect, causes the selective accumulation within the cancerous tissues as shown in several studies with tumor xenografts.23,37–39 Initial studies indicated that atelocollagen particles could be administered safely without induction of cytokines or observed toxicity to the tissues.

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Active (targeted) systemic siRNA delivery The ability to target siRNAs only into specific cell types in vivo should provide the dual benefit of reducing the dose required for a therapeutic benefit and minimizing toxicity from uptake into bystander cells. Targeting has been achieved by directly conjugating the siRNA with a small molecule that specifically binds to the cell of interest, by attaching a targeting molecule or antibody to liposomes or nanoparticles that encapsulate the RNA, or by complexing the siRNA with a protein that contains both a targeting peptide and an RNA-binding peptide (Figure 1).

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Conjugated siRNAs. siRNAs can be conjugated to small molecules to direct binding to cell-surface receptors. The first demonstration of this used cholesterol, which can be joined to either siRNA strand. The cholesterol moiety enhances retention of the conjugated siRNA in the circulation by binding to albumin, low-density lipoprotein and highdensity lipoprotein particles, as well as uptake in hepatocytes by binding to low-density lipoprotein receptors and uptake in the liver, gut, kidney and steroidogenic organs by binding to the scavenger receptor class B, type I receptors, which take up high-density lipoprotein.40 Conjugation of cholesterol, as well as a-tocopherol,41 lithocholic acid or lauric acid42 to ApoB–siRNAs reduces serum cholesterol and ApoB mRNA levels in the liver after intravenous injection. Another example of this approach is ‘siRNA dynamic

Aptamer–siRNA chimeric RNAs. A variation of conjugated siRNAs are aptamer–siRNA chimeras that fuse an siRNA with a structured piece of RNA, called an aptamer, that can be selected to bind with high affinity and specificity to a cell-surface ligand. Aptamer–siRNA chimeras that utilize an aptamer that recognizes prostate surface membrane antigen specifically deliver siRNAs and inhibit tumor outgrowth in a xenograft model of human prostate cancer.45,46 Similarly, a chimeric RNA containing an aptamer to HIV env protein specifically delivers siRNAs to HIV-infected CD4+ cells, which are otherwise refractory to transfection, and when joined with siRNAs that target viral genes can be used to inhibit HIV replication in vitro.47,48 An siRNA–aptamer encoding a CD4 aptamer knocks down gene expression specifically in human CD4+ T cells, macrophages and dendritic cells.49 Intravaginal administration of CD4 aptamer–siRNA chimeras designed to knock down CCR5 and/or viral genes can even prevent sexual transmission of HIV in humanized mice. Because these chimeric RNAs only contain RNA, they are not expected to elicit antibody responses, but this has not been examined. Moreover, they are not cytotoxic and do not trigger interferon or inflammatory cytokines. Because of their small size compared with antibodies, nanoparticles or liposomes (discussed below), aptamer–siRNA chimeras (and conjugated siRNAs in general) are likely to have superior tissue penetration. An additional potential advantage of Gene Therapy

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Figure 1 siRNA delivery strategies under development include siRNAs that are directly conjugated to cholesterol (a) or other small targeting molecules (b), joined to an aptamer that binds to a cell-surface receptor (c), conjugated to membrane-penetrating polymers linked to targeting small molecules (d), complexed with fusion proteins composed of an antibody fragment or targeting peptide linked to an RNA-binding domain that is either protamine (e) or polyarginine (f), or encapsulated within nanoparticles (g) or liposomes (h) bearing targeting moieties.

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this single all-RNA molecule is the potential for large-scale chemical synthesis at relatively low cost. Regulatory hurdles are also likely more tractable than for complex particle delivery systems.

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siRNA-fusion protein complexes. Dual-function proteins, composed of a targeting peptide, such as an antibody fragment that recognizes a cell-surface receptor expressed on the target cells or a peptidic receptor ligand, linked to an RNA-binding peptide can be complexed to siRNAs for systemic, targeted siRNA delivery. The two functional domains can be expressed in a single fusion protein or can be chemically conjugated. In an initial study, antibody fragments, either an Fab fragment or a single-chain antibody (scFv), were fused to a protamine peptide. Protamines are relatively small (5–8 kDa) highly basic proteins that condense DNA in sperm. They bind siRNAs and other nucleic acids by a charge interaction. Each fusion protein binds about six siRNAs and forms a highly condensed nanoparticle. Fusion Gene Therapy

proteins targeting HIV envelope specifically transfect cells bearing HIV envelope, whereas fusion proteins containing Her2–scFv target Her2+ breast cancer cells specifically. Intravenous injection of these fusion proteins complexed with siRNAs directed against oncogenes or PLK1, required for formation of the mitotic spindle, inhibits outgrowth of tumors bearing the antibody ligands.50,51 Hematopoietic cells are a special challenge for nucleic acid delivery, as they are disseminated through the body and are resistant to all conventional in vitro transfection methods other than electroporation or infection with viral vectors. Having a way to induce gene silencing in leukocytes could be the basis for therapies for autoimmune and inflammatory diseases, transplant rejection, leukemia and lymphoma, and some viral infections. All hematopoietic cells express integrins that mediate cell–cell and cell–matrix interactions and are important for directing white blood cell trafficking within the body.52 Antibody– protamine fusion proteins constructed using scFvs that bind to the

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Targeted nanoparticles. Nanoparticles carrying siRNAs have also been used to induce functional silencing in subcutaneously transplanted tumors in nude mice. In one early study, polyethyleneimine polymers, linked to PEG to enhance circulating half-life and to an RGD (Arg– Gly–Asp) peptide that binds to integrins, can be complexed with siRNAs to form polyplexes that reportedly deliver siRNAs to tumor vasculature and tumor cells that overexpress av-integrins.56 However, this study did not convincingly show either siRNA delivery or target gene knockdown and did not look at immune stimulation or toxicity (polyethyleneimine is known to have significant cytotoxicity). Another polymer on the basis of a cyclodextrin-containing polycation (CDP) also delivers siRNAs into subcutaneous tumor xenografts in mice.57 CDP is a polymer with a cyclic oligomeric glucose backbone that assembles into a colloidal 50–70 nm particle when complexed with siRNAs. To achieve targeting, transferin (Tf)-coupled PEG was attached to the surface of the particles to exploit the upregulation of Tf receptors on cancer cells. However, despite the fact that CDP is less toxic than other cationic polymers (such as polyethyleneimine), injection of CDP nanoparticles into non-human primates at the high concentration tested led to elevated blood urea (which might indicate kidney toxicity) and a mild increase in serum liver enzymes and IL-6. Multiple injections of the particles induced antibodies to human Tf. Despite these potential problems, Tf-coupled CDP nanoparticles that contain siRNAs targeting a ribonucleotide reductase subunit gene are being tested in Phase I studies in patients with refractory solid tumors.58 In a few patients, gene knockdown and siRNA delivery to biopsied melanoma tumors was demonstrated, but no clinical effects have yet been reported.59

inflammation to inhibit colitis in a mouse model.60 I-tsNPs are B80 nm neutral liposomes loaded with siRNAs precondensed with protamine. Each liposome carries a high payload (B4000 siRNAs per particle), allowing therapeutic efficacy at a low dose (B2.5 mg kg 1). The particles are coated with hyaluronan, a naturally occurring glycosaminoglycan that stabilizes siRNA entrapment, inhibits nonspecific reticuloendothelial system uptake in vivo, and serves as the attachment site for an integrin monoclonal antibody. For reducing gut inflammation, a monoclonal antibody against b7-integrin, which is highly expressed in gut mononuclear leukocytes, was used. As I-tsNP are made from natural biomaterials, they offer a safe platform for siRNA delivery, avoiding cytokine induction and liver damage. The I-tsNP platform carrying an lymphocyte function-associated antigen1 antibody was also used to deliver CCR5 siRNAs to human lymphocytes and monocytes and protect mice from HIV challenge.61 Lymphocyte function-associated antigen-1 I-tsNPs loaded with CCR5–siRNAs also do not induce an interferon response or inflammatory cytokine secretion. Another example of targeted liposomes takes advantage of the high expression of the vitamin A-binding receptor, retinol-binding protein, on perisinusoidal hepatic stellate cells to deliver siRNAs specifically to this specialized cell type in the rat liver.62 Stellate cells have a central role in organismal vitamin A uptake and storage, but are also responsible for liver fibrosis in response to liver injury. They express a heat shock protein (gp46) that acts as a chaperone for collagen and is needed to secrete and deposit extracellular collagen in fibrotic liver. In this study, commercially available lyophilized liposomes composed of cholesterol, dioleoylphosphatidylethanolamine and the cationic lipid O,O¢-ditetradecanoyl-N-(a-trimethylammonio-acetyl) diethanolamine chloride were reconstituted in water containing retinol and an siRNA-targeting gp46. After intravenous injection of these liposomes to rats with fibrotic livers, the incorporated retinol delivers the siRNA payload almost exclusively to hepatic stellate cells and not to other resident liver cells (hepatocytes and Kupffer cells). Rats treated with 0.75 mg kg 1 gp46 siRNA-containing vitamin A liposomes two or three times a week show impressive protection from fibrotic liver damage-related death caused by dimethylnitrosamine. Knocking down gp46 reduces collagen secretion and causes stellate cell apoptosis. Importantly, the siRNA treatment also reverses already established fibrosis in this model. In rats without fibrosis, there was some uptake in macrophages. It might be worth investigating whether direct conjugation of vitamin A to the siRNA would also work and circumvent the need for liposomes. Another liposome strategy has been used to target liposomes to tumors. Liposomes encapsulating Her2 siRNAs with histadine–lysine peptides that facilitate endosomal release were targeted to prostate cancer xenografts by incorporating an scFv to the Tf receptor, whose expression is increased on the membranes of tumor cells.63

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lymphocyte function-associated antigen-1 integrin, which is expressed exclusively and universally on all leukocytes, can be used for selective targeting of leukocytes both in vitro and in vivo.53 A fusion protein constructed with an scFv that recognizes all conformations of lymphocyte function-associated antigen-1 delivers siRNAs to both resting and activated leukocytes, whereas an scFv that only binds to the activated conformation selectively targets only activated leukocytes, potentially providing a way to manipulate unwanted immune activation without causing global immunosuppression. Importantly, these fusion protein–siRNA complexes do not activate the cells they transfect or induce innate immunity. Other methods to transfect immune cells substitute a 9 amino acid poly-(D)-arginine peptide for protamine and conjugate it to a CD7 scFv designed to bind to CD7 on human T lymphocytes or to a peptide (DC3) that binds to an unknown receptor on dendritic cells. In all, 2–10 polyarginine fusion protein molecules bind one siRNA, suggesting that the binding capacity is at least 10-fold less than with protamine fusion proteins. Systemic injection of the CD7 scFv–9 amino acid poly-(D)-arginine peptide, complexed with siRNAs directed against CCR5 (a chemokine receptor that is an HIV coreceptor) and HIV vif and tat, suppresses HIV infection in humanized mice without inducing toxicity.54 A similar approach can be used to suppress dengue virus infection in vitro in monocyte-derived dendritic cells using DC3–9 amino acid poly-(D)-arginine peptide for delivering siRNAs to suppress expression of dengue genes or TNF-a, which has a major role in dengue pathogenesis. These complexes also suppress TNF-a production induced by innate immune stimulation of dendritic cells in vivo.55

Targeted liposomes. Integrin antibodies have also been used to construct liposomes that efficiently knock down gene expression in specific subsets of immune cells in vivo. Stabilized liposomes (termed I-tsNP) can deliver siRNAs into leukocytes involved in gut

Conclusions Although no siRNA delivery system has yet progressed beyond Phase II studies, a number of delivery strategies have shown promise for harnessing the potential of siRNAs as drugs to treat a multitude of human diseases. However, there is still a lot of work to be done. Each approach we have described has its own merits and shortcomings. The optimal delivery strategy may vary depending on the target tissue and cell type and the disease being treated. At this stage there is certainly room for multiple approaches for specific indications and for further improvement of each to optimize delivery, not only to the target cell but also within the cell to the RISC, while minimizing cytotoxicity from membrane damage and unintended immune activation. Gene Therapy

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This work was supported, in part, by grants from the Alon Foundation, the Marie Curie IRG-FP7 of the European Union, Levy Family Trust and ISF (181/10) to DP, the Breast Cancer Research Program of the US Department of Defense to JL, and by a joint BSF grant (2009107) to DP and JL.

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ACKNOWLEDGEMENTS

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CONFLICT OF INTEREST D Peer declares financial interest in Quiet Therapeutics; J Lieberman is on the Scientific Advisory Board of Alnylam Pharmaceuticals.

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A recombinant H1 histone-based system for efficient delivery of nucleic acids. J Biotechnol 2003; 105: 215–226. 26 Lv H, Zhang S, Wang B, Cui S, Yan J. Toxicity of cationic lipids and cationic polymers in gene delivery. J Control Release 2006; 114: 100–109. 27 Kedmi R, Ben-Arie N, Peer D. The systemic toxicity of positively charged lipid nanoparticles and the role of Toll-like receptor 4 in immune activation. Biomaterials 2010; 31: 6867–6875. 28 Wu Y, Navarro F, Lal A, Basar E, Pandey RK, Manoharan M et al. Durable protection from Herpes Simplex Virus-2 transmission following intravaginal application of siRNAs targeting both a viral and host gene. Cell Host Microbe 2009; 5: 84–94. 29 Lee YS, Pressman S, Andress AP, Kim K, White JL, Cassidy JJ et al. Silencing by small RNAs is linked to endosomal trafficking. Nat Cell Biol 2009; 11: 1150–1156. 30 Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN et al. RNAi-mediated gene silencing in non-human primates. Nature 2006; 441: 111–114. 31 Morrissey DV, Lockridge JA, Shaw L, Blanchard K, Jensen K, Breen W et al. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat Biotechnol 2005; 23: 1002–1007. 32 Geisbert TW, Hensley LE, Kagan E, Yu EZ, Geisbert JB, Daddario-DiCaprio K et al. Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference. J Infect Dis 2006; 193: 1650–1657. 33 Villares GJ, Zigler M, Wang H, Melnikova VO, Wu H, Friedman R et al. Targeting melanoma growth and metastasis with systemic delivery of liposome-incorporated protease-activated receptor-1 small interfering RNA. Cancer Res 2008; 68: 9078–9086. 34 Landen Jr CN, Chavez-Reyes A, Bucana C, Schmandt R, Deavers MT, Lopez-Berestein G et al. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res 2005; 65: 6910–6918. 35 Anderson DG, Akinc A, Hossain N, Langer R. Structure/property studies of polymeric gene delivery using a library of poly(beta-amino esters). Mol Ther 2005; 11: 426–434. 36 Akinc A, Zumbuehl A, Goldberg M, Leshchiner ES, Busini V, Hossain N et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol 2008; 26: 561–569. 37 Honma K, Iwao-Koizumi K, Takeshita F, Yamamoto Y, Yoshida T, Nishio K et al. RPN2 gene confers docetaxel resistance in breast cancer. Nat Med 2008; 14: 939–948. 38 Fujii T, Saito M, Iwasaki E, Ochiya T, Takei Y, Hayashi S et al. Intratumor injection of small interfering RNA-targeting human papillomavirus 18 E6 and E7 successfully inhibits the growth of cervical cancer. Int J Oncol 2006; 29: 541–548. 39 Mu P, Nagahara S, Makita N, Tarumi Y, Kadomatsu K, Takei Y. Systemic delivery of siRNA specific to tumor mediated by atelocollagen: combined therapy using siRNA targeting Bcl-xL and cisplatin against prostate cancer. Int J Cancer 2009; 125: 2978–2990. 40 Wolfrum C, Shi S, Jayaprakash KN, Jayaraman M, Wang G, Pandey RK et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat Biotechnol 2007; 25: 1149–1157. 41 Nishina K, Unno T, Uno Y, Kubodera T, Kanouchi T, Mizusawa H et al. Efficient in vivo delivery of siRNA to the liver by conjugation of alpha-tocopherol. Mol Ther 2008; 16: 734–740. 42 Lorenz C, Hadwiger P, John M, Vornlocher HP, Unverzagt C. Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells. Bioorg Med Chem Lett 2004; 14: 4975–4977. 43 Rozema DB, Lewis DL, Wakefield DH, Wong SC, Klein JJ, Roesch PL et al. Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc Natl Acad Sci USA 2007; 104: 12982–12987. 44 Kortylewski M, Swiderski P, Herrmann A, Wang L, Kowolik C, Kujawski M et al. In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses. Nat Biotechnol 2009; 27: 925–932. 45 McNamara II JO, Andrechek ER, Wang Y, Viles KD, Rempel RE, Gilboa E et al. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat Biotechnol 2006; 24: 1005–1015. 46 Dassie JP, Liu XY, Thomas GS, Whitaker RM, Thiel KW, Stockdale KR et al. Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMAexpressing tumors. Nat Biotechnol 2009; 27: 839–849. 47 Zhou J, Li H, Li S, Zaia J, Rossi JJ. Novel dual inhibitory function aptamer-siRNA delivery system for HIV-1 therapy. Mol Ther 2008; 16: 1481–1489. 48 Zhou J, Swiderski P, Li H, Zhang J, Neff CP, Akkina R et al. Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells. Nucleic Acids Res 2009; 37: 3094–3109. 49 Wheeler L, Trifonova R, Vrbanac V, Basar E, McKernan S, Xu Z et al. CD4 aptamersiRNA chimeras inhibit HIV infection in primary CD4+ cells in vitro and in polarized human cervicovaginal explants and prevent vaginal transmission in humanized mice. J Clin Invest 2011; accepted for publication.

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Once developed, each of these platforms can be applied to deliver any siRNA sequence. For some of the active delivery systems described, alternate cell types might also be readily targeted by substituting alternate targeting ligands or antibodies. This flexibility, which is inherent to RNAi, distinguishes the development of siRNA-based drugs from the development of other small molecule drugs where extensive development work is required to improve or adapt an individual candidate drug. If the delivery hurdle can be overcome, the same platform could be used to treat a diversity of diseases, customizing the treatment to address the unique molecular abnormalities of individual patients.

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tumor growth in a murine model of metastatic Ewing¢s sarcoma. Cancer Res 2005; 65: 8984–8992. Davis ME. The first targeted delivery of siRNA in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to clinic. Mol Pharm 2009; 6: 659–668. Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010. Peer D, Park EJ, Morishita Y, Carman CV, Shimaoka M. Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target. Science 2008; 319: 627–630. Kim SS, Peer D, Kumar P, Subramanya S, Wu H, Asthana D et al. RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT mice. Mol Ther 2010; 18: 370–376. Klier M, Anastasov N, Hermann A, Meindl T, Angermeier D, Raffeld M et al. Specific lentiviral shRNA-mediated knockdown of cyclin D1 in mantle cell lymphoma has minimal effects on cell survival and reveals a regulatory circuit with cyclin D2. Leukemia 2008; 22: 2097–2105. Pirollo KF, Rait A, Zhou Q, Hwang SH, Dagata JA, Zon G et al. Materializing the potential of small interfering RNA via a tumor-targeting nanodelivery system. Cancer Res 2007; 67: 2938–2943.

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50 Song E, Zhu P, Lee SK, Chowdhury D, Kussman S, Dykxhoorn DM et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol 2005; 23: 709–717. 51 Yao Y, Sun T, Huang S, Dou S, Lin L, Mao C et al. Targeted Delivery of PLK1-siRNA by Single-Chain Antibody Suppresses Her2+ Breast Cancer Growth and Metastasis. 2011. Submitted. 52 Shimaoka M, Takagi J, Springer TA. Conformational regulation of integrin structure and function. Annu Rev Biophys Biomol Struct 2002; 31: 485–516. 53 Peer D, Zhu P, Carman CV, Lieberman J, Shimaoka M. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1. Proc Natl Acad Sci USA 2007; 104: 4095–4100. 54 Kumar P, Ban HS, Kim SS, Wu H, Pearson T, Greiner DL et al. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 2008; 134: 577–586. 55 Subramanya S, Kim SS, Abraham S, Yao J, Kumar M, Kumar P et al. Targeted delivery of small interfering RNA to human dendritic cells to suppress dengue virus infection and associated proinflammatory cytokine production. J Virol 2010; 84: 2490–2501. 56 Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res 2004; 32: e149. 57 Hu-Lieskovan S, Heidel JD, Bartlett DW, Davis ME, Triche TJ. Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits

Gene Therapy