key: cord-0997995-9663hfi4
authors: Behlke, Mark A.
title: Progress towards in Vivo Use of siRNAs
date: 2016-12-14
journal: Mol Ther
DOI: 10.1016/j.ymthe.2006.01.001
sha: 5d44f1076d94bf7a07973c9c7c53336b3a11e5ab
doc_id: 997995
cord_uid: 9663hfi4

RNA interference (RNAi) has become the method of choice to suppress gene expression in vitro. It is also emerging as a powerful tool for in vivo research with over 90 studies published using synthetic small interfering RNAs in mammals. These reports demonstrate the potential for use of synthetic small interfering RNAs (siRNAs) as therapeutic agents, especially in the areas of cancer and viral infection. The number of reports using siRNAs for functional genomics applications, for validation of targets for small-molecule drug development programs, and to address questions of basic biology will rapidly grow as methods and protocols for use in animals become more established. This review will first discuss aspects of RNAi biochemistry and biology that impact in vivo use, especially as relates to experimental design, and will then provide an overview of published work with a focus on methodology.

Compounds that can selectively alter expression levels of any gene have been sought by basic scientists and clinicians for many years. Nucleic acid hybridization is very specific, and theoretically the sequence of any desired gene could be uniquely targeted by an oligonucleotide of complementary sequence. The first bantisenseQ experiments were performed over 25 years ago and employed DNA oligonucleotides to block translation of Rous sarcoma virus RNA [1, 2] . Experiments demonstrating the use of antisense RNA to inhibit gene expression soon followed [3, 4] . Most antisense methods that use single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) oligonucleotides rely upon either RNase H-mediated degradation of the target mRNA or steric blockade of translation for their mechanism of action [5] . However, other pathways have been exploited with favorable results. For example, 2V,5V-oligoadenylate (2-5A) triggers action of RNase L; conjugation of 2-5L to an antisense oligonucleotide sequence enables sequence-specific targeting of this otherwise nonspecific ribonuclease [6] .

Extensive medicinal chemistry has been done on antisense nucleic acids and hundreds of compounds have been tested in a search for modifications that improve nuclease stability, increase binding affinity (melting temperature, or T m ), and improve the in vivo pharmacodynamic properties of oligonucleotides as drugs [7, 8] . Pharmaceutical companies have been working to develop antisense drugs for more than 15 years and extensive knowledge of the pharmacology and toxicology of synthetic nucleic acids in vivo has been compiled [9] . As of 2005, one antisense drug is on the market. Formivirsen (Vitravene), a product of Isis Pharmaceuticals and Novartis Ophthalmics, is a 21-mer phosphorothioate DNA oligonucleotide that is complementary to the immediate early region 2 of cytomegalovirus (CMV). This compound has been shown to be effective in the treatment of CMV retinitis and was approved by the FDA for use by direct intravitreal injection in 1998 [10, 11] . Currently, over 20 antisense drugs are in various stages of clinical testing [12] . Although antisense methods have been successfully used for years to study gene function, antisense technology has failed to gain widespread acceptance as a gene knockdown tool, largely due to the extensive experimental testing that often needs to be done to find effective target sites within the gene of interest.

In 1998 Fire and Mello described a process in Caenorhabditis elegans in which long dsRNA (several hundred bases in length) caused suppression of complementary genes and called this process RNA interference (RNAi) [13] . Although the mechanism was not understood at the time, RNAi had actually been seen as early as 1990 in plants as the phenomenon of cosuppression [14] . In plants, it is suspected that the degradative pathways of RNAi evolved as an antiviral defense mechanism. In higher organisms, antiviral defense mechanisms are more complex and long dsRNAs induce interferon secretion, activating a cascade of interferon-stimulated genes (ISGs) as well as RNAi. Therefore, long dsRNAs should not be used as an experimental tool to trigger RNAi in mammalian cells. Fortunately, long dsRNAs do not mediate suppression directly. Instead, long dsRNAs are processed by the endoribonuclease Dicer into short 21-bp duplexes with 2-base 3V overhangs called small interfering (si) RNAs, which are the actual effector species [15] [16] [17] . Synthetic siRNAs can be transfected into mammalian cells and specifically suppress expression of complementary genes [18] , usually without triggering interferon responses. The siRNA enters a multimember protein complex called RISC (RNA induced silencing complex) by which one strand of the duplex (the bpassenger strandQ) is cleaved and discarded [19, 20] . The other strand is retained and serves as the bguide strand,Q which directs the sequence specificity of RISC and therefore of RNAi silencing. Some of the components of human RISC have been identified and have assigned functions. Argonaute 2 (Ago2) functions as bslicer,Q the RNA endonuclease that cleaves a target RNA species as directed by the siRNA guide strand [21] [22] [23] . Dicer and TRBP form a heterodimer, which processes long dsRNA into siRNAs [24, 25] , and together complex with the siRNA and Ago2 to form a functional silencing complex [26] . The biochemistry of degradative RNAi has been extensively studied and has been well summarized in recent reviews [27] [28] [29] [30] [31] .

RNAi exploits a complex, natural pathway that regulates gene expression and includes machinery for sequence-specific mRNA degradation. Specific siRNA design rules have been developed from studies that compared effective vs ineffective duplexes and it is now possible to obtain potent RNAi reagents without the extensive testing needed when using traditional antisense approaches [32] [33] [34] [35] [36] [37] . SiRNAs can be surprisingly potent. Transfections done using subnanomolar concentrations of RNA sometimes achieve N90% reduction in mRNA levels [38] . RNAi has rapidly been adopted by the research community and a keyword search for bRNAi or siRNA or RNA interferenceQ finds that almost 9000 citations already exist in the PubMed database. RNAi has largely replaced antisense as the gene knockdown tool of choice for most applications in vitro and is showing similar promise for in vivo applications.

Two different general approaches can be used to trigger RNA interference in vivo. First, synthetic RNA duplexes can be introduced directly into animals; the RNAi effect will be transient. In this case, the dsRNA is usually chemically synthesized and the problems surrounding its use parallel those encountered in small-molecule or antisense drug development. Alternatively, dsRNA can be expressed inside cells using DNA templates that direct synthesis of RNA duplexes or short hairpin RNAs; in this case the RNAi effect can be sustained and could theoretically be permanent, depending upon the vector employed and whether expression levels are adequately maintained long term. DNA-directed RNAi (ddRNAi) expression cassettes can be introduced into animals using plasmid or viral vectors. Problems surrounding this approach parallel those encountered with other forms of gene therapy. The present review is restricted to the use of chemically synthesized siRNA duplexes in vivo with a focus on methodology. Other recent reviews consider in vivo use of siRNA from different perspectives [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] .

A number of important issues need to be considered before embarking on in vivo studies using RNAi. These include site selection, compound design and chemistry, controls, route of administration, and use of a delivery vehicle.

Site selection and optimization do not appear to be as difficult for siRNA as would be expected from historical experience with antisense oligonucleotide site selection. Effective site selection algorithms are available from several commercial and academic sources and key elements of design criteria have been published [32] [33] [34] [35] [36] [37] . Before undertaking an in vivo experiment, however, actual validation of duplexes in vitro should be performed; it is usually necessary to test only a small number of duplexes, possibly as few as three or four, to find suitable candidates. It is preferable to have more than one effective siRNA available for each target to help control for off-target effects (see below). For siRNA therapeutic applications, more extensive optimization will of course be expected.

Most in vivo siRNA experiments reported to date used 21-mer duplexes that have a 19-base central doublestranded domain with terminal 2-base 3V overhangs. These reagents mimic the products naturally produced by Dicer processing in vivo. Other designs are available that may be worth consideration. Blunt 19-mer duplexes with extensive 2V-O-methyl modifications have been shown to have favorable properties [49] ; blunt 25-mer duplexes, with sense-strand 2V-O-methyl modifications, have also been used with good results [50] . Asymmetric 27-mer duplexes that are substrates for Dicer have been shown to have increased potency in vitro compared with 21-mers [38, 51] and may have similar benefit in vivo.

Although duplex RNA is more resistant to nuclease attack than single-stranded DNA, unmodified siRNAs are nevertheless rapidly degraded when administered intravenously in mammals; degradation can be delayed or avoided by chemical modification of the oligonucleotide and/or by complexation with a carrier/delivery particle [52] . A wide variety of chemical modifications that confer nuclease resistance have been successfully used in single-stranded antisense oligonucleotides and ribozymes; many of these modifications can be imported directly into siRNA use and will provide similar benefit.

Phosphorothioate or boranophosphate modification of the internucleoside linkage improves nuclease stability. Boranophosphate-modified RNAs have substantial nuclease resistance; unfortunately, boranophosphate-modified RNAs cannot simply be manufactured using standard chemical synthesis methods. Instead, borano-modified bases are incorporated in RNA made by enzymatic synthesis using in vitro transcription [53] , which makes siteselective placement of this modification difficult. Phosphorothioate (PS) modifications can be easily placed in the RNA duplex at any desired position and will prolong the life of the duplex when exposed to serum or other nuclease sources. Limited PS modification preserves potency of the siRNA; however, extensively modified duplexes show reduced potency and/or toxicity [54] [55] [56] [57] . Extensive PS modification can also lead to artifacts due to nonspecific protein binding [58] , so it may be preferable to limit the use of this modification to terminal positions (or 3V overhangs) where added nuclease resistance is probably most important. Other nuclease-resistant modifications that do not have these kinds of problems are available.

Modification of the 2V position of the ribose can increase duplex stability (T m ) and confers varying degrees of nuclease resistance. 2V-O-methyl RNA is a naturally occurring base that is present in mammalian ribosomal RNA. A number of reports have described patterns of 2V-O-methyl incorporation that retain full potency of the siRNA compared with unmodified RNA and are stable in serum [49, 55, 57] . The precise pattern of incorporation is important and RNAs that are entirely substituted with 2V-O-methyl are inactive as triggers of RNAi. Alternating the 2V-O-methyl bases retains potency and confers substantial nuclease resistance [49] . The 2V-fluoro (2V-F) modification has also been used with good results [54] [55] [56] . This modification is usually incorporated selectively at pyrimidine bases due to reagent issues. Their use either alone or in combination with 2V-O-methyl purines will stabilize RNA duplexes. In fact, duplexes can be fully modified such that no unmodified RNA remains [59] . Locked nucleic acids (LNAs) are a different class of 2V modification in which a methylene bridge connects the 2V-O with the 4V-C of the ribose. This modification blocksQ the ribose in the 3V-endo conformation and improves both duplex stability and nuclease resistance. LNAs can be incorporated into siRNAs; however, placement to achieve best effect is more restricted than for 2V-O-methyl or 2V-F bases [56, 60, 61] .

Several studies have already been conducted in mice that compared the functional performance of modified vs unmodified siRNA duplexes. Layzer and colleagues modified an anti-luciferase siRNA with 2V-F pyrimidines and compared it with unmodified siRNA in vivo using hydrodynamic tail vein injection. SiRNAs were co-injected with a luciferase expression plasmid and luciferase activity in the liver was assessed by in vivo bioluminescence imaging 2-7 days after injection [62] . Despite improved nuclease stability, no functional difference in luciferase knockdown was observed between modified and unmodified siRNAs. Using hydrodynamic delivery, injected nucleic acids are rapidly taken up by hepatocytes, thus nuclease resistance may not be as important for siRNA administered using this approach than when using more traditional low-pressure intravenous administration. Unfortunately, only a single 10-Ag siRNA dose was used, making it difficult to assess potency accurately without the additional data obtained from dose-response curves. Other groups have reported substantial benefit from the use of highly modified siRNA duplexes. Morrissey and colleagues compared activity of an unmodified siRNA with that of a highly modified duplex that included DNA bases, 2V-O-methyl purines, 2V-F pyrimidines, terminal inverted-dT bases, and PS linkage modification at select positions (leaving no unmodified RNA). A hepatitis B virus (HBV) expression plasmid was co-injected with modified or unmodified duplexes into the tail vein of mice using the same hydrodynamic delivery method, and production of HBV DNA and hepatitis B surface antigen was assayed 72 h postinjection [63] . A 2-log dose-response curve was performed and the modified duplexes were significantly more potent. Unmodified RNA may be suitable for use in some in vivo research applications, especially if a delivery vehicle that offers protection from serum nucleases is employed. However, there may be reasons beyond nuclease resistance to include modified bases in siRNAs used in vivo (see discussion of innate immunity below).

Various chemical groups can be placed at the ends of oligonucleotides that facilitate entry into cells or improve biodistribution. Experience with modifications in this category has been gained from work done to improve properties of antisense oligonucleotides and can be applied directly to siRNAs [8] . For example, conjugation of cholesterol to a siRNA can improve serum protein binding, improve pharmacokinetics, and increase delivery to hepatocytes [64, 65] . Other lipid conjugates can be used, although synthesis is currently more complex [64] . A dinitrophenol end modification has been shown to improve transfection and increase intracellular stability in tissue culture [66] . Perhaps of greatest interest, peptide-oligonucleotide conjugates can have improved cell permeation properties as well as nuclear targeting, if desired [67] . Peptide delivery will be considered in greater detail below.

It seems unlikely that major adverse effects will be observed from the use of siRNAs in vivo that could not be predicted from prior experience with the toxicology of antisense oligonucleotides and ribozymes. Early results suggest that the biodistribution of siRNAs will be grossly similar to that seen using single-stranded DNA oligonucleotides [68] . Phase I clinical trials are already ongoing and more information will soon be available about the pharmacology of siRNAs in humans. Some specific problems have already been identified, which need to be considered in experimental design and inclusion of appropriate controls, including stimulation of the innate immune system (leading to interferon pathway cascade) and off-target effects (OTEs).

Perhaps the most significant unexpected bside effectQ encountered in the use of antisense DNA oligonucleotides was a sequence-specific ability to trigger the innate immune system. This property was traced to the recognition of motifs that contained nonmethylated dC bases in the context of a bCpGQ dinucleotide by Toll-like receptor 9 (TLR9). Although originally viewed as a nuisance side effect, this observation opened an entirely new therapeutic use for DNA oligonucleotides as modulators of the immune system [69] [70] [71] [72] . The innate immune system relies upon a series of receptor molecules that recognize bpathogen-associated molecular patternsQ and that initiate a variety of cellular responses when these motifs are encountered [73, 74] . One class of these receptors, the Toll-like receptor family, can recognize compounds that range from bacterial lipopolysaccharide (LPS) to DNA.

While long dsRNA is a well-known trigger of type-1 interferon (IFN) responses in mammals, it was originally hoped that short dsRNAs, like siRNAs, would be bsafeQ to use. However, siRNAs are also capable of triggering IFN responses. These effects can be both sequence specific and cell-type specific and therefore are not always encountered. Members of the Toll-like receptor family that recognize nucleic acids reside intracellularly within endosomal or lysosomal compartments (other TLRs reside on the cell surface). Of the 11 known TLRs, 3 that specifically interact with RNA have been identified, including TLR3, 7, and 8 [75] [76] [77] . Other molecules that recognize dsRNA and can trigger IFN pathway responses include dsRNA-dependent protein kinase (PKR), 2V,5V-oligoadenylate synthetase (OAS), and retinoic acid-inducible gene I (RIG-I) [78, 79] .

Sledz and colleagues reported that 21-mer siRNAs could cause interferon-mediated activation of the JAK-STAT pathway and a dose-dependent induction of ISGs in T98G cells that was attributed to PKR activation [80] . This effect appeared to be sequence independent as two different siRNAs that did not share any obvious sequence motifs triggered the same effect. In contrast, stimulation of IFN-a, IFN-h, or phospho-PKR was not detected after administration of either 21-mer or 27-mer dsRNAs in HEK293 cells [38] . Activation of 2V,5V-OAS by dsRNA requires a minimum RNA length of 30 bp and maximal activation is not achieved until length reaches 60-80 bp [81] , so direct triggering of 2V,5V-OAS by siRNAs is unlikely to be a problem. Requirements for RIG-I activation are under investigation, but it appears that it can recognize short siRNAs and may be a major pathway for IFN production and activation of ISGs, at least in vitro (J. T. Marques and B. R. G. Williams, personal communication). RNA synthesized by in vitro transcription retains a triphosphate group at the 5V end, which can trigger a robust IFN response [82] ; this is easily avoided by use of chemically synthesized RNA of known structure and purity.

Toll-like receptor 3 recognizes dsRNA [83] and certain siRNAs have been shown to trigger release of IFN-a, IFNh, and interleukin 8 (IL-8) in HEK293 cells; this effect is dose dependent and is magnified by overexpression of TLR3 [84, 85] . Toll-like receptors 7 and 8 recognize ssRNA [86] and can be triggered by siRNAs in a sequence-specific fashion. Single-stranded RNA is more effective than double-stranded RNA in triggering TLR7 and 8 responses [87] . The sequences UGUGU [88, 89] and GUCCUUCAA [90] have been demonstrated to stimulate secretion of a variety of cytokines from TLR7-or 8-bearing cell types. TLR7 and 8 are expressed in a limited number of cell types including myeloid dendritic cells, plasmacytoid dendritic cells, neutrophils, B cells, and monocytes. However, these cells are found throughout the body; siRNAs can trigger IFN secretion from human peripheral blood mononuclear cells (PBMCs), so stimulation of the innate immune system must be considered when using any kind of systemic administration [89] . TLR3, 7, and 8 are localized intracellularly in endosomes and delivery to this compartment must take place for activation to occur. In cell culture, cationic lipid transfection agents deliver nucleic acids to endosomal compartments, while electroporation does not, and siRNA delivery done using electroporation is less likely to stimulate IFN secretion [87] . These same principles appear to apply in vivo. Intravenous administration to mice of unmodified siR-NAs complexed in lipid particles is more likely to cause IFN pathway activation [52] than intravenous administration of unmodified bnakedQ siRNAs using high-volume hydrodynamic delivery [91] . Cationic DOTAP liposomes significantly enhance the ability of siRNA to trigger IFN responses in PBMCs and in mice [92, 93] . More information about siRNA activation of innate immunity via TLR signaling can be found in a recent review by Marques and Williams [94] .

As more and more sequences that are capable of activating IFN via TLR7/8 binding are being identified [87] , a better strategy than simply avoiding bbadQ motifs may be needed to generalize use of siRNA in vivo, where all cell types are present. Use of chemically modified RNAs may overcome many of these problems. A recent study by Kariko and colleagues compared the capacity of RNA from prokaryotic and eukaryotic sources for their ability to trigger IFN responses. They observed that prokaryotic RNAs were a much more potent trigger of signaling through TLR3, 7, and 8 pathways than eukaryotic RNAs. Weaker or absent receptor signaling from eukaryotic RNAs was not sequence dependent but rather was due to the base and/or sugar modifications that were present in these nucleic acids. Much like 5-methyl-dC, which can reduce or prevent activation of TLR9 in CpG motif DNA, the presence of 5-methyl-C, N 6 -methyl-A, pseudouridine, or 2V-O-methyl bases prevents recognition of RNA by TLR3, 7, and 8 [95] . Importantly, it has been shown that extensive 2V modification of a sequence that is otherwise strongly immunostimulatory as unmodified RNA can prevent an immune response when that same sequence is administered intravenously (iv) in mice using a lipid-based particle delivery system [52] . However, it might not be necessary to modify an RNA duplex this extensively to block immune responses. Judge and colleagues reported that substitution of as few as two 2V-O-methyl bases in one strand of a siRNA duplex can be sufficient to block TLR activation both in vitro (human PBMCs) and in vivo (iv delivery in mice using stabilized nucleic acid lipid particle (SNALP)-facilitated delivery). Modified U and G bases were most effective in achieving this affect [96] . Since selective incorporation of 2V-O-methyl bases is inexpensive and does not compromise siRNA functional activity, use of this modification in all duplexes intended for in vivo applications should be considered.

Just as certain sequences are active in stimulating innate immune responses via TLR pathways, some sequences can inhibit or block these responses. Barrat and colleagues described several sequence motifs that prevent TLR7 and/or 9 activation when present in ssDNA oligonucleotides [97] . It would also be interesting to see if endosomal inhibitors such as chloroquine might blunt innate immune responses to siRNAs.

Given the sequence-specific variation in the ability of a duplex to trigger innate immune responses, it may be impossible to ensure that any single bnegative control duplexQ is really a valid control. It might therefore be judicious to measure serum cytokine levels routinely to verify whether IFN induction has actually occurred to assist with interpretation of in vivo results, even if modified duplexes are employed.

Nucleic acid base pairing is highly specific and mismatches at one or a small number of positions in a short sequence are often sufficient to prevent hybridization completely under physiological conditions. Based upon antisense oligonucleotide experience, it was originally hoped that performing a genome-wide homology search to eliminate sites with significant cross-hybridization potential would ensure specificity of siRNA gene targeting. Indeed, even a single mismatch can disrupt siRNA action against an intended target [57, 98] . Systematic analysis of the functional impact caused by base changes at all positions in a target site has been reported and can be used as a guide to assist with siRNA design [99] . Unfortunately, this kind of traditional homology and cross-hybridization analysis is insufficient and siRNAs can cause widespread changes in expression levels in seemingly unrelated genes [100] [101] [102] .

RNAi is mediated by multimember protein complexes that mechanistically repress gene expression in different ways when using perfect vs imperfect duplexes. Degradative RNAi leads to sequence-specific cleavage of a target mRNA as directed by RISC using Ago2 [21, 22, 103] and initiation of this event requires perfect or near-perfect base pairing with the siRNA-derived guide strand. Conversely, the microRNA (miRNA) pathway results in translational repression and is directed by imperfect base pairing between target and guide strand [104, 105] . This pathway is theorized to be mediated by a variant of RISC using Ago1. Functionally, the siRNA degradative pathway and miRNA translational repression pathway overlap. Exogenously supplied siRNA duplexes can function as miRNAs [106, 107] and miRNAs can direct target cleavage events [108, 109] . It seems likely that many of the OTEs attributed to siRNAs derive from miRNA-like target interactions, which can be triggered by surprisingly limited sequence homology. Target specificity for miR-NAs is largely directed by a 7-nucleotide bseed regionQ at the 5V end of the antisense strand of the miRNA [110] . Unfortunately, even this small amount of homology can give rise to OTEs. Lin and colleagues reported results from a large-scale siRNA library screen in which their top three bhitsQ all derived from off-target events and, further, that two of these events resulted from only a 7-bp complementation between the siRNA and the boff targetQ [111] .

Given the expected frequency of finding 7-base matches between a siRNA and nontargeted genes within the entire transcriptome of a mammal, simple homology screening alone is insufficient to predict or eliminate offtarget events. It is possible that some pattern of chemical modification will decrease the ability of a siRNA to participate in this class of OTE. When using siRNAs as research tools, a simple strategy to help ensure that results are not misinterpreted because of unsuspected OTEs is to require that two independent siRNAs against the same target produce the same biological results. For development of siRNAs as therapeutic agents, extensive testing may be needed to ensure that a potential drug candidate has an acceptable degree of OTEs. It is not clear what metrics should be employed to assess OTEs in vivo. While microarrays have been employed to document changes in gene expression profiles that result from siRNA treatment on a genome-wide scale in vitro, no studies have yet reported this kind of experiment in vivo. Further, up or down regulation of bunrelatedQ genes might or might not result in biologically significant adverse events in vivo. Traditional badverse eventQ assessment, as is routinely performed in small-molecule drug development programs, may be the most relevant method to consider unwanted affects from siRNA drugs without the need for additional studies to catalog the presence or absence of bunintended gene regulation events.Q

The cellular machinery responsible for degradative RNA interference also participates in processing miRNAs. MicroRNAs are important in cellular gene regulation networks and anything that disrupts or alters miRNA processing could potentially have broad affects on gene expression in a cell. It is theoretically possible that high doses of exogenous siRNAs administered as therapeutics or investigational tools could bsaturateQ the RNAi machinery and thereby alter miRNA levels, further complicating the spectrum of OTEs. Hong and colleagues described that high doses of a siRNA can induce expression of some genes involved in siRNA degradation and processing (meri-1 and adar-1) [112] . Stimulation of IFN pathways, OTEs, and desired gene knockdown are all dose dependent. The precise dose needed to trigger each response can be different and it may be possible to influence the relative magnitude of undesired side effects by careful attention to dosing. Dose-response profiles should be performed in vivo and the siRNA regents subsequently administered at the lowest levels that achieve the desired degree of target knockdown or therapeutic effect.

The route of siRNA administration will affect the total dose needed, the effective tissue distribution, potential side effects, and the need for chemical modification or use of an agent that facilitates delivery. Although iv administration is often considered for broadest tissue distribution, it is important to consider alternative injection sites such as intraperitoneal (ip) and subcutaneous (sc) routes. Local delivery can be used to achieve high concentration at the intended target site while using a low dose and minimizing risk of systemic side effects. Local delivery can be achieved by direct injection (such as into a tumor or joint or intraocularly) or by topical application to a surface (such as transepithelial absorption through oral, rectal, or vaginal mucosa). Direct administration into the central nervous system (CNS) can be achieved by intrathecal or intraventricular injection, bypassing issues with blood-brain barrier penetration. Inhaled, intranasal, or intratracheal administration can be employed for pulmonary delivery.

Although reports about the life span of unmodified siRNA in serum vary from minutes to days, it is obvious that chemically modified duplexes will survive longer in nuclease-rich environments than unmodified RNA [49, 56, 59] . Even when chemical modifications are employed to stabilize a duplex from degradation, plasma clearance may still be rapid and use of a delivery vehicle can improve pharmacokinetics. Morrissey and colleagues reported that the plasma half-life of an unmodified siRNA duplex in mice was 0.03 h, the half-life of a modified duplex was 0.8 h, and the half-life of a modified duplex packaged in a SNALP was 6.5 h [52] . Similarly, Soutschek and colleagues reported studies using modified siRNAs in rats in which an unconjugated duplex had an elimination half-life of 0.1 h, while a cholesterol-conjugated duplex had a half-life of 1.5 h. While it may be reasonable to employ unmodified duplexes for basic research applications in vivo, a more sustained effect and potential therapeutic benefit will be achieved using chemically modified duplexes together with some kind of delivery system.

A wide variety of methods have been used to facilitate delivery of nucleic acids. Nucleic acids have a high negative charge density and cationic polymers are usually employed to bind the nucleic acid, neutralize charge, and assist with transport across the cell membrane. Cationic lipids are routinely used with great success as transfection agents in tissue culture; however, their in vivo use by iv administration presents significant problems as these reagents can be quite toxic. Despite problems with iv use, cationic lipids can sometimes be employed for ip injection [113] [114] [115] , for CNS injection [116, 117] , or in topical epithelial surface application [118, 119] . Toxicity varies with the precise chemical composition of the lipids employed, dose, and a variety of other factors. Variations in chemical composition can have a large impact on the functional properties of cationic lipid mixtures [120] and lipoplex/liposomal preparations have been devised with decreased toxicity that are more compatible with iv administration. Liposomal formulations have been FDA approved for smallmolecule drug delivery since the late 1990s [121] . Liposomes can be modified with ligands such as folate or small peptides, which assist with delivery and help target specific cell types or tissues [122, 123] . Through the use of neutral polyethylene glycol-substituted surfaces and other approaches, liposomes can be stabilized and made more bstealthy,Q showing reduced clearance and improved pharmacokinetics [124, 125] . These kinds of lipid nanoparticles have been successfully used to deliver antisense oligonucleotides and siRNAs in vivo [52, [126] [127] [128] .

Polyethylenimine (PEI) has been used for many years to facilitate nucleic acid delivery [129] ; however, due to toxicity and variable performance it has not found generalized acceptance as a delivery tool for either antisense oligonucleotides or siRNAs. Nevertheless, PEI can be used as a bbaseQ for formulation of more complex particles with improved properties. For example, a PEGylated PEI particle with a vasculature-targeting RDG peptide has been shown to deliver siRNAs successfully in a mouse xenograft tumor model [130] . Deacylation of PEI lowers toxicity and improves pulmonary delivery of siRNAs in mice [131] and folate-modified PEGylated PEI is being tested for siRNA delivery in tumor cells [132] .

Cyclodextrin-modified PEI has favorable properties for nucleic acid delivery. Compared with unmodified PEI, the cyclodextrin polymer (CDP) has relatively low toxicity and with the addition of adamantine-PEG, will spontaneously form stable particles with the nucleic acid cargo under physiological conditions. PEI-based CDPs have been shown to function for in vivo delivery of nucleic acids [133] . CDPs have been synthesized using other polycations (than PEI) and optionally can include terminal imidazole groups to improve endosomal trafficking [183] . Conjugation of transferrin to a linear cyclodextrincontaining polymer/adamantine/PEG complex improves tumor targeting in vivo and this kind of polyplex has been shown to effectively deliver siRNAs in mice [134] . As a general strategy, transferrin-conjugated polycations were shown to facilitate transfection of DNA 15 years ago [135] . A variety of different conjugates that facilitate delivery of nucleic acids have been characterized [8] and novel smallmolecule libraries are under development that assist targeting of different cell types and may eventually offer a bmenuQ that enables selective delivery of therapeutic compounds wherever desired [136] .

Other polycations can be complexed with siRNAs to facilitate delivery. Protamine has been employed as a carrier for nucleic acids and can be conjugated to a variety of molecules that improve its performance, including lipids [137] . Protamine can be conjugated to antibody Fab fragments to target delivery selectively to specific cell types and this approach has been successfully used to deliver siRNAs in mice [138] . Cationic peptides have also been used to facilitate nucleic acid entry into cells by exploiting the capability of certain sequences to enter cells directly (cell-penetrating peptides) or to enter specific cell types selectively (cell-targeting peptides) [67, 139] . The peptides MPG, penetratin, and transportin have all been shown to promote delivery of siRNAs in tissue culture [140] [141] [142] and may have promise for in vivo use. General peptide carrier effects can also be exploited, and atelocollagen, a purified pepsin digest of collagen, has been shown to function as a delivery vehicle for siRNAs in mice [143] . The DNA-packaging motor of bacteriophage f29 has been conjugated to siRNAs and shown to facilitate cell entry [144] .

Modified virus envelopes can be adapted to deliver nucleic acids to cells. Fusogenic influenza virus envelopes were used to deliver fluorescent dye-labeled siRNAs by ip injection into mice [145] , hemagglutinating virus of Japan envelopes were used to deliver siRNAs via direct intratumor injection in mice [146] , and SV40 pseudovirions are being tested as a delivery vehicle [147] . The potential antigenicity of these delivery tools will need to be addressed for consideration in therapeutic applications.

Physical and mechanical delivery methods should also be considered. In fact, the single most commonly employed strategy for in vivo siRNA delivery to date has been the bhydrodynamic deliveryQ (HD) method [62, 63, 91, 112, [148] [149] [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] [160] [161] [162] [163] [164] [165] . In this approach, a large volume of a nucleic acid solution is rapidly injected into the tail vein of a mouse, which transiently disrupts vascular and tissue integrity and achieves delivery [166] . Morbidity can be significant and success using this method is technique dependent. Nevertheless, HD is useful as a research tool and is especially effective in delivering nucleic acids to the liver. Recent reports have also demonstrated success using electroporation [167] [168] [169] [170] [171] [172] , ultrasound [173] , and particle-based bgene gunQ [174] delivery systems.

Although a large number of studies have reported successful in vivo delivery of nucleic acids using liposome or polyplex reagents, issues of toxicity remain and must be considered. Chien and colleagues compared the toxicity of iv injection of cationic DOTAP liposomes directly with the cardiolipin analogue CCLA:DOPE liposomes in mice as part of an evaluation of the use of this delivery system with siRNAs [128] . Liposomes were administered by tail vein injection and mice were followed for 2 weeks. Two of three mice given a single dose of 100 mg/kg DOTAP liposomes died compared with 0/3 mice given CCLA liposomes. The CCLA liposomes were also studied with a multiple dose regimen and 1/3 mice that received 100 mg/kg/day for 3 consecutive days died. Animals may survive yet still suffer delivery-related morbidity, which can affect experimental results and must be considered.

Much of the toxicity of cationic lipids relates to electrostatic effects, and precise complexation ratios of the positively charged lipid with the negatively charged cargo nucleic acid are important. Interaction with negatively charged serum proteins can inactivate the complex [175] and particle aggregation with clumping in capillary beds can be problematic; interaction with complement proteins can lead to inflammation. When loaded with a nucleic acid cargo, cationic lipids can trigger the release of a variety of cytokines (see discussion of TLR activation above). Lymphopenia, thrombocytopenia, and hepatic necrosis can occur with higher doses of lipid [176] . Newer classes of lipid/nucleic acids delivery systems fully encapsulate (not just complex with) the nucleic acid cargo and present a stabilized, more neutral surface to the intravascular environment that lowers toxicity, improves stability, and prolongs serum circulation time [177] . Recent reviews by Dass [178] , Audouy [179] , and Simberg [180] provide greater detail on cationic liposome delivery methods and in vivo toxicity.

Polyplex delivery systems have similar toxicity problems. The LD 50 of linear PEI is around 4 mg/kg in mice [181] , significantly limiting in vivo utility. Cyclodextrin polymers are much less toxic, having an LD 50 of~200 mg/kg in mice [182] . Grafting of h-cyclodextrin onto PEI reduces toxicity but also can decrease efficiency of nucleic acid transfection, so careful optimization is needed. The addition of adamantine-PEG forms inclusion complexes that coat the complex in a way that further stabilizes the polymer/nucleic acid particle under physiological salt conditions without increasing size and helps further lower toxicity [133] . Transferrin or other ligands can be added to the polymer to help with celltype targeting. For an overview of issues involved with polyplex design, see the recent review by Davis and colleagues [183] .

It is also possible to perform in vivo gene knockdown experiments using ex vivo delivery methods, which avoids potential complications and/or toxicity associated with each of the various in vivo delivery methods. If a cell line can be transfected with siRNAs in tissue culture using standard methods, it can subsequently be implanted into recipient animals in which changes in phenotype and behavior in vivo can be studied, avoiding the challenges associated with traditional in vivo delivery. For example, in one study that employed ex vivo methods, human melanoma tumor cells were transfected in culture using electroporation with either control siRNAs or siRNAs specific for a mutant V599E B-Raf gene. In this study, bstealthQ siRNAs were employed, which are chemically modified blunt 25-mer duplexes with selective incorporation of 2V-O-methyl RNA in the sense strand. Transfected cells were implanted in nude mice. Cells transfected with control siRNAs took~5 days to develop 10-mm tumor foci, whereas cells transfected with anti--V599E B-Raf siRNAs took 14-16 days to develop 10 mm tumor foci [184] .

Over 90 reports that involve in vivo administration of siRNAs in mammals have been published. The following survey of these studies will focus on the methodologies employed, such as route of administration, use of delivery vehicles, use of chemically modified duplexes, and controls (if any) for OTE or IFN induction. Table 1 includes a listing of studies organized by bsystemQ studied. Selected studies are examined in greater detail below, organized by route of administration.

The first published reports of the in vivo use of siRNAs in mammals appeared in mid-2002. McCaffrey and colleagues used HD delivery to introduce 2 Ag of a luciferase expression plasmid with 40 Ag of unmodified control or anti-luciferase siRNAs into mice [148] . A human a-1 antitrypsin (hAAT) expression plasmid was co-injected and serum levels of hAAT were used for normalization between animals. Luciferase activity in the liver was assessed by in vivo bioluminescence imaging 3 days after injection and the anti-luciferase siRNA was found to specifically reduce light emission by 81%. IFN levels (or other cytokines) were not measured; however, use of the hAAT marker served as a control for nonspecific effects. Similar results using HD delivery to suppress luciferase in mice were reported by Lewis and colleagues [149] . In this case RNAi activity was demonstrated to occur in multiple tissue types, including liver, kidney, spleen, lung, and pancreas. Duration of silencing was investigated using an expression plasmid encoding secreted human placental alkaline phosphatase (SEAP). Ten micrograms of the pSEAP plasmid and 5 Ag of an anti-SEAP siRNA were administered by HD injection. SEAP levels were suppressed by 83% on day 1 postinjection and by 32% on day 4 and were at control levels on day 14 (possibly sooner, but intermediate time points were not reported). Song and colleagues reported using siRNAs to protect mice from liver damage in two model systems involving Fas-mediated apoptosis [150] . Anti-Fas siRNAs or control anti-EGFP siRNAs (50 Ag in 1 ml PBS) were administered using HD delivery for three doses. Both Fas mRNA and Fas protein levels were shown to be specifically reduced about 10-fold (Fas/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio) in recipient mouse hepatocytes relative to controls. Suppression lasted~10 days. Mice treated with the anti-Fas siRNA did not develop fulminant hepatitis or hepatic fibrosis after treatment with concanavalin A (which induces hepatitis). Treatment with the anti-Fas siRNA was also protective against treatment with an agonist, an anti-Fas antibody (Jo2), which is normally fatal within 2-3 days of injection. Similar beneficial effects were reported using the same anti-Fas siRNA in protecting mice from renal ischemia-reperfusion injury [156] . SiR-NAs were administered by HD delivery via the tail vein (50 Ag in 1 ml PBS) or by low-pressure injection directly into the renal vein. In one series of experiments, renal ischemia was induced by clamping one renal pedicle for 35 min, and the other kidney was removed. Survival was significantly higher (8/10) for mice treated with anti-Fas siRNA than with control siRNA or saline (2/10). Caspase 8 is a critical enzyme in the Fas apoptosis pathway. Zender and colleagues reported use of siRNAs targeting caspase 8 to block hepatic necrosis following challenge with the agonist anti-Fas antibody (Jo2) or recombinant adenovirus encoding Fas ligand [152] . SiR-NAs were administered using HD tail vein injection at a dose of 0.6 nmol/g (8 mg/kg, or 200 Ag per dose per 25-g mouse) or a 0.45 nmol infusion directly into the portal vein. The caspase 8 mRNA levels were reduced about fourfold (caspase 8/GAPDH ratio) following siRNA treatment in mouse hepatocytes. Following administration of the Jo2 Fas-agonist antibody, 5/11 mice treated with the anti-caspase 8 siRNA survived, while 0/17 control animals survived. Using a strategy similar to that discussed previously for protection of Fas-induced renal ischemiareperfusion injury, Contreras and colleagues reported success in using anti-caspase 8 and anti-caspase 3 siRNAs in protecting mice from hepatic ischemia-reperfusion injury [154] . A variant of HD delivery was employed in which low dose 0.45 nmol (6 Ag) siRNA was administered in 1 ml volume directly into the portal vein. SiRNAs were administered 1 h before injury, hepatic blood supply was clamped for 90 min, and hepatic caspase 3 and 8 levels were assayed 1 h after reperfusion. Both caspase enzymes are strongly induced by ischemia; however, the relative level of induction was reduced approximately twofold by siRNA treatment. In one arm of the study, mice were observed for 30 days after a 75-min ischemic injury to the liver, with or without siRNA pretreatment. Mice treated with anti-caspase 3 showed 50% survival (5/10), mice treated with anti-caspase 8 showed 30% survival (3/10), while control mice showed 10% survival (1/10, control siRNA) or no survival (0/10, vehicle).

Hydrodynamic delivery has also been used to study the antiviral potential of siRNAs in vivo. Giladi and colleagues demonstrated that a plasmid encoding the hepatitis B virus (HBV) genome can be codelivered with siRNAs to the liver of mice using the HD method to study the effects of anti-HBV siRNAs on viral replication [151] . Five HBV siRNAs were tested, which, not surprisingly, showed different functional potency. The most potent reagent was capable of reducing HBV titers produced in transfected livers by 10 5 on day 2 and 10 2 on day 3 at the dose studied (15 Ag HBV plasmid with 1 nmol, or 13 Ag siRNA). Morrissey and colleagues employed a similar HBV plasmid model system to study the use of anti-HBV siRNAs in mice, also using HD delivery [63] , and compared performance of modified vs unmodified RNAs. The modified duplexes (called bsmall interfering nucleic acids, or siNAs) performed better, showing a 1.5-log greater reduction in HBV DNA and surface antigen levels 72 h postinjection than when the same sequence was administered as an unmodified siRNA. Importantly, only the modified duplexes suppressed HBV when treatment was started 3 days after binfectionQ (via delivery of the HBV plasmid by HD injection); however, a significantly higher dose was needed in this setting (30 mg/kg). Although HD has largely been viewed as a research tool, Hagstrom and colleagues reported success using this method to deliver siRNAs and other nucleic acids to skeletal muscle in rats, dogs, and rhesus macaques [155] . In this variant of the HD method, a limb is functionally isolated by application of a blood pressure cuff or tourniquet and a large-volume bolus of nucleic acid solution is administered intravenously. Therapeutic delivery of plasmids or siRNAs in humans using this approach was proposed; if care is taken in technique, only moderate, reversible tissue damage was observed [185] .

Despite the relative susceptibility of unmodified siRNA duplexes to degradation in serum, several groups have reported positive results using unmodified siRNAs administered by low-volume intravenous injection in mice without the use of any facilitated delivery system [186] [187] [188] [189] [190] . Filleur and colleagues administered unmodified siRNAs targeting vascular endothelial growth factor (VEGF) to nude mice bearing subcutaneous implants of rat fibrosarcoma cJ4 cells that express luciferase [187] . SiRNAs were injected iv, ip, or sc at a dose of 3 Ag per injection (125 Ag/kg/day) in PBS. Animals given a single anti-VEGF siRNA by all three of these routes showed 40-50% suppression of luciferase activity in the tumor implants at day 3 postinjection compared with animals receiving a control siRNA. Other animals were given daily ip injections of siRNAs in PBS and after 16 days those receiving anti-VEGF treatment showed a 66% reduction in tumor volume and a 70% reduction in tumorassociated VEGF levels compared with controls.

Duxbury and colleagues studied the effects that changes in levels of ribonucleotide reductase (RR) had on the efficacy of gemcitabine treatment of pancreatic tumor cells implants in nude mice [188] . A high level of RR is believed to be one cause of resistance to gemcitabine chemotherapy. RR levels can be reduced using siRNAs targeting the M2 subunit of RR (RRM2) and this treatment increases tumor cell susceptibility to gemcitabine in vitro. MIAPaCa2 pancreatic tumor cells were implanted in nude mice and were given control treatments, an anti-RRM2 siRNA (150 Ag/kg iv), and/or gemcitabine (150 mg ip) twice weekly for 6 weeks. Mice treated with the anti-RRM2 siRNA showed reduced RRM2 levels in tumor extracts by Western blot compared with control siRNA-treated mice. Treatment with gemcitabine or the anti-RRM2 siRNA alone resulted in a modest reduction in tumor volume, while treatment with the combination of gemcitabine with anti-RRM2 siRNA showed a synergistic effect with 87% reduction in tumor size. Mice receiving combination treatment had no detectable liver metastasis, whereas 80% of control and 40% of gemcitabine or siRNA monotherapy animals had significant tumor burden in their livers. This group continued to investigate using siRNAs as a therapy for pancreatic adenocarcinoma using a different target, CEA-CAM6, a cell-surface adhesion molecule thought to play a role in tumor progression and metastasis. As before, unmodified siRNAs were administered at a dose of 150 Ag/kg in PBS iv via the tail vein twice weekly. BxPC3 cells were implanted in the pancreas of nude mice and animals were given PBS alone, a control siRNA, or an anti-CEACAM6 siRNA for 6 weeks. Mice treated with targetspecific siRNAs had smaller tumors, 46% lower CEACAM6 levels, reduced evidence of angiogenesis (CD34 + structures using immunohistochemical staining), and reduced hepatic metastasis compared to control mice. Survival after 6 weeks of treatment was 0/10 for PBS-treated mice, 0/10 for control siRNA-treated mice, and 9/10 for anti-CEACAM6treated mice [189] .

A variety of delivery systems have been successfully used to improve siRNA uptake following iv injection, including liposomes, polymer-based nanoparticles, peptides, antibodies, and small molecule ligands. Soutschek and colleagues described the use of cholesterol-conjugated siRNAs to reduce expression of apolipoprotein B (apoB) in mice and assessed the impact that this had on serum cholesterol and lipoprotein particle levels [65] . Duplexes were chemically modified by selective incorporation of 2V-O-methyl RNA bases and phosphorothioate linkages. Cholesterol modification increased serum half-life 15fold compared with unconjugated siRNAs. Following iv injection, siRNA levels present in various tissues were measured using a sensitive RNase protection assay (RPA) and only the modified, conjugated RNA duplexes were detectable; these siRNAs were seen in liver, heart, kidney, adipose, and lung tissue. Extensive sequence optimization was performed; 84 siRNAs were screened in vitro and the 2 best candidates were used in vivo. Mice received the anti-apoB siRNAs or a control siRNA at a dose of 50 mg/kg iv daily for 3 days and cholesterol levels were measured 24 h after the last dose. ApoB-100 protein levels were reduced by 68% (siRNA apoB-1) and by 31% (siRNA apoB-2); the more potent siRNA showed a 37% reduction in total serum cholesterol. Although IFN or other cytokines were not measured directly to assess activation of the innate immune system, mRNA levels for three unrelated control genes were found to be unaffected by siRNA treatment. Mechanism of action was demonstrated in vivo using a 5V-RACE procedure. ApoB species were identified that had a cleavage point exactly at the site predicted using the apoB-1 siRNA (10 bp from the 5V end of the antisense strand of the siRNA).

It is informative to compare what dose is needed to achieve the same physiological effects if the same siRNA sequence was delivered using a liposomal or polymerbased delivery system. Judge and colleagues administered a siRNA having the same sequence as apoB-1 discussed above in mice intravenously using a lipid-based delivery vehicle (SNALP). The siRNA was not cholesterol conjugated and had a different modification pattern (compared to that employed by Soutschek) and was limited to five 2V-O-methyl-U residue substitutions in the sense strand [96] . A 50% reduction in serum cholesterol was achieved using a dose of 5 mg/kg/day for 3 days, 1/10 the dose used previously for the cholesterol-modified anti-apoB siRNA. Using a traditional antisense approach, Crooke and colleagues reported that ip administration of a 2V-O-methoxyethyl/DNA bgapmerQ phosphorothioate oligonucleotide specific for apoB reduced total cholesterol to a similar degree when mice were given a dose of 25 mg/kg twice weekly for 8 weeks and by 55% when dose was increased to 50 mg/kg [191] .

A synthetic cardiolipin analogue (CCLA) has been used to make a new family of cationic liposomes that can be used for iv administration of siRNAs. Chien and colleagues described the use of the CCLA liposomes to deliver siRNAs by tail vein injection in mice [128] . Delivery efficiency was estimated to be approximately sevenfold better than with traditional DOTAP liposomes and had lower toxicity. The Ras/Raf/MAPK signaling pathway is thought to be important in around 30% of human cancers. An unmodified siRNA specific for c-raf was administered via the tail vein at a dose of 7.5 mg/kg twice a day for 5 days to SCID mice bearing sc implants of the breast cancer tumor MDA-MB-231. Tumors in mice treated with naked siRNAs grew at the same rate as controls, while tumors in mice treated with liposomal-siRNAs were 73% smaller at day 8, suggesting that the delivery vehicle had a significant impact on the therapeutic efficacy of the anti-c-Raf siRNA treatment. A similar series of experiments studied the effect of an anti-Raf-1 siRNA with or without the chemotherapy agent Taxotere in suppressing the growth of sc implants of the prostate cancer cell line PC-3 in SCID mice [127] . Mice were administered unmodified siRNAs at a dose of 7.5 mg/kg twice a day for 5 days, either naked or complexed in CCLA liposomes, with or without the coadministration of Taxotere (10 mg/kg on day 2 and 5 mg/kg on day 5). Tumors were excised and Western blots were performed to examine Raf-1 protein levels. Relative to the control siRNA, administration of free Raf-1 siRNAs reduced Raf-1 protein levels by 37% (Raf-1/GAPDH ratio), liposomal Raf-1 siRNAs reduced Raf-1 levels by 89%, and the combination of liposomal siRNAs + Taxotere reduced Raf-1 levels by 98%. Naked siRNAs had no effect on tumor growth, while the liposomal anti-Raf-1 siRNA inhibited tumor growth by 49%, and, when combined with Taxotere, inhibited tumor growth by 89%.

Use of neutral liposomes made of 1,2-dioleoyl-snglycero-3-phosphatidylcholine (DOPC) to deliver siRNAs iv in mice was described by Landen and colleagues [192] . Dye-labeled RNA duplexes were injected into mice iv and the use of naked, DOTAP liposome, and DOPC liposome delivery was compared using fluorescence microscopy. The DOPC liposomes gave an estimated 10-fold improved delivery compared with DOTAP liposomes and 30-fold improved delivery compared with naked RNA; the greatest uptake of siRNA was seen in liver, kidney, and lung. EphA2 is a tyrosine kinase receptor that is overexpressed in some human cancers and is associated with poor outcome in ovarian cancer. Nude mice with ip implants of the ovarian cancer cell line HeyA8 or SKOV3ip1 were given control or anti-EphA2 siRNA (150 Ag/kg iv twice weekly in DOPC liposomes) for 4 weeks, with or without the chemotherapeutic agent Paclitaxel (100 Ag ip weekly). Compared with mice receiving the control siRNA, average tumor size was reduced by 35-50% using the anti-EphA2 siRNA, 45-68% using Paclitaxel, and 86-91% with combination therapy. Several studies have reported similar observations in which the combination of siRNA with a cytotoxic chemotherapeutic agent worked additively or synergistically in reducing tumor size. It is also worth noting the very low dose of siRNA employed in this study, presumably due to improved delivery using the neutral liposome carrier.

A different type of lipid particle was employed by Morrissey and colleagues to deliver anti-HBV siRNAs in a mouse hepatitis B model system [52] . The particle, called a SNALP, contained cationic, neutral, and fusogenic lipids that fully encapsulate the nucleic acid cargo; an earlier version of this particle was reported to improve delivery of phosphorothioate antisense oligonucleotides in mice [193] . Both unmodified and highly modified siRNAs (including 2V-O-methyl, 2V-F, DNA, and PS bonds) were tested by Morrissey. Unmodified siRNAs administered using this delivery method strongly stimulated innate immune responses while the highly modified duplexes did not. Delivery of a fluorescent siRNA was examined and use of SNALPs resulted in highest levels of RNA in liver and spleen but showed little accumulation in lung. Plasma half-life of modified siNAs in SNALPs was 6.5 h compared with 2 min for unmodified naked siRNAs. Hepatitis binfectionQ was initiated with HD delivery of 0.3 Ag of pWTD HBV plasmid and siRNA therapy was started 6 days later. Mice received control or anti-HBV siRNAs in SNALPs at doses of 0.3 or 3 mg/kg/day for 3 days. Serum HBV DNA levels dropped by 1.5 log 10 for the higher dose, and reduction in HBV DNA levels persisted for 6 weeks after the last siNA dose. Effective reduction in HBV DNA was achieved using 3 mg/kg dosing with SNALPs compared with 30 mg/kg for the same sequences administered using HD delivery [63] .

Nonliposomal compounds are being developed to deliver nucleic acids in vivo that form a bnanoparticle drug delivery complex.Q Typically, this kind of compound includes a cationic polymer that binds the DNA or RNA cargo (such as PEI), a neutral bstealthQ coating (such as PEG), and a targeting ligand (such as transferrin). Schiffelers and colleagues described the use of a bsterically stabilized nanoparticleQ to deliver unmodified siRNAs to neuroblastoma N2A tumors in nude mice [130] . The particles contained PEI (bPQ nanoparticles), PEI + PEG (bPPQ nanoparticles) or PEI + PEG plus an Arg-Gly-Asp (RGD) peptide that promotes vascular targeting (bRPPQ nanoparticles). Complex formation of siRNAs in the particles (nanoplexes) protected unmodified siRNAs from degradation in serum and altered in vivo delivery patterns. A fluorescence-labeled naked siRNA did not accumulate in tumors, lung, or liver and was mostly degraded and/or excreted in urine. The fluorescence-labeled P nanoplex accumulated more in lung than in liver or tumor, while the labeled RPP nanoplex improved uptake more selectively in the N2A tumor masses (distribution of PP nanoplexes was not reported). SiRNA duplexes specific for the murine vascular endothelial growth factor receptor-2 (VEGFR2) were administered to mice bearing N2A tumors at a dose of 40 Ag iv every 3 days. After three cycles of treatment, mice receiving the anti-VEGFR2 siRNA as an RPP nanoplex showed N90% inhibition of tumor growth compared with vehicle-only or control siRNA-treated mice. Newer formulations of this particle that may have even greater potential in siRNA drug development have been devised [194] .

Another novel particle employs a linear cyclodextrincontaining polymer stabilized with adamantine-PEG and has been shown to deliver both small molecule drugs as well as nucleic acids in vivo [183] . This cyclodextrinpolycation (bpolyplexQ) was conjugated to transferrin to improve uptake into tumor cells and used to deliver unmodified siRNAs in a mouse model of Ewing sarcoma [134] . Many Ewing sarcoma tumors contain a translocation, t (11;22) , that results in production of an aberrant transcript, EWS-FL11, which is thought to function as an oncogenic transcription factor. An anti-EWS-F11 siRNA (siEFB2) and a control siRNA were administered to SCID mice bearing tumors formed from TC71 cells that stably express luciferase (enabling bioluminescence imaging of tumor implants). Administration of siEFB2 for 3 days using the transferrin polyplex at a dose of 50 Ag (2.5 mg/kg/day) resulted in transient suppression of the luciferase signal and 60% reduction in EWS-F11 mRNA levels (normalized to h-actin) in estab-lished tumors. In another series of experiments, siRNA administration was started concurrent with injection of the TC71-Luc tumor cells. Mice were followed for up to 8 weeks with twice weekly dosing (2.5 mg/kg) of siRNAs. Mice receiving control siRNAs (in any form) and those receiving naked siEFB2 had rapid tumor development. Mice receiving siEFB2 with nontargeting polyplexes had delayed onset of tumors and those receiving siEGB2 delivered with transferrin polyplexes had minimal to no tumor formation. Ogris and colleagues described the use of transferrin and epidermal growth factor as ligands to improve targeting of DNA polyplexes (PEGylated PEI) to tumor cells in mice [195] . Employing some kind of specific cell-surface marker or receptor targeting technique seems generally to improve in vivo delivery of nucleic acid polyplexes.

Antibodies have high specificity and a variety of wellcharacterized reagents are available boff the shelfQ that recognize many different mouse or human cell surface markers. Song and colleagues described a method in which protamine was conjugated to the C-terminus of an antibody Fab fragment and used as a cell-marker-specific reagent to deliver unmodified siRNAs in mice [138] . In one series of experiments, a Fab fragment (F105) specific for the HIV envelope glycoprotein gp160 was coupled to protamine (F105-P). Six siRNAs formed a delivery complex with one F105-P carrier. This complex was effective in delivering siRNAs to all cell types tested that expressed the HIV env gene, even T cells (which are notoriously difficult to transfect). Administration of siRNAs using the F105-P carrier did not elicit an immune response in B16 melanoma cells in vitro (h-IFN, STAT1, and OAS1 mRNA levels were assayed using qRT-PCR). While B16 cells are competent for some TLR responses and express TLR3 [196] , it may be worthwhile to determine if this method of delivery triggered any innate immune response in myeloid or dendritic cell lines or in vivo. Murine B16 cells were implanted in C57BL/6 mice and different siRNAs were administered iv with or without the F105-P carrier. A fluorescent dye-labeled control siRNA was used to assess specificity of delivery. Naked siRNA did not enter tumor cells. Using the F105-P carrier, gp160-B16 tumors showed high uptake of the dye-labeled siRNA, whereas native B16 tumors did not, demonstrating the specificity of this delivery method. Tumor gp160-B16 cells were injected into the flanks of recipient mice and a cocktail of three siRNAs specific for c-myc, MDM2, and VEGF was administered either iv or by direct tumor injection with or without the F105-P carrier on days 0, 1, and 3 at a dose of 80 Ag (~4 mg/kg); tumors were removed at day 9. Both intratumor and iv administration of the siRNA cocktail with F105-P delivery significantly reduced tumor size, whereas naked siRNAs or control treatments had no effect.

Although complicated polyplex delivery systems can offer significant advantages in administration of siRNAs in vivo, some very simple cationic polymers have also been used with success. Ge and colleagues reported the use of PEI to deliver siRNAs targeting influenza virus via retroorbital iv injection in mice [197] . Unmodified siRNAs specific for genes encoding nucleocapsid protein (NP) or viral transcriptase (PA and PB1) were administered at doses of 30, 60, or 120 Ag iv complexed to PEI in 200 Al PBS (using a nitrogen to phosphorus ratio of 5). This treatment did not trigger an obvious innate immune response and IFN-a was not detected in the lungs of mice using either influenza or control siRNAs with PEI. Administration of either of the influenza-specific siRNAs reduced viral titers in infected mouse lungs and a combination of anti-NP + anti-PA siRNAs was most effective. A reduction of viral titers ranging from 1 to 3 log 10 was seen and siRNA treatment was effective even when given 5-24 h after infection. Thomas and colleagues reported that deacylated PEI (PEI187) improved delivery of plasmid DNA in mice by iv administration 10 4 -fold and dramatically improved pulmonary targeting [131] . The deacylated PEI187 was used to deliver anti-NP siRNAs in mice by retroorbital iv injection using the same in vivo influenza model system described by Ge. A 94% reduction in viral titers was achieved with administration of a single 120-Ag dose of siRNA. However, these results are similar to those reported previously by Ge using similar siRNA doses with unaltered PEI.

Atelocollagen is a peptic digest of collagen with low immunogenicity. Takeshita and colleagues report the use of atelocollagen to assist in iv delivery of siRNAs in a bone tumor metastasis model system in mice [143] . Luciferase-expressing PC-3M-luc-C6 cells were injected into nude mice. After 4 weeks, unmodified siRNAs were administered at a dose of 50 Ag (2.5 mg/kg) by iv injection either with or without 0.05% atelocollagen, and luciferase expression was measured by in vivo bioluminescence 24 h later. The control siRNA or the naked anti-Luc siRNA had no effect. The anti-Luc siRNA administered with atelocollagen showed a 90% reduction in luciferase light emission. Tissue distribution of siRNAs was assessed using a RPA. Atelocollagen was seen to facilitate delivery into liver, lung, spleen, and kidney. Innate immune stimulation was tested (2 h postinjection) and siRNAs delivered by this approach did not result in a detectable increase in IL-12 or IFN-a levels; however, the single time point examined may have been inadequate to support this conclusion fully. Two genes thought to be involved in metastasis were targeted, enhancer of zeste homolog 2 (EZH2) and 3V-hydroxykinase p110-a-subunit (p110-a). Anti-EZH2 or anti-p110-a siRNAs were administered iv at a dose of 50 Ag on days 3, 6, and 9 after injection of tumor cells, and animals were imaged for the presence of bone implants. Use of control or naked siRNAs had no effect on tumor development; however, both anti-EZH2 and anti-p110-a siRNAs gave dramatic reductions in the number of bone lesions when administered with atelocollagen.

Although some researchers have reported success in the use of unmodified siRNAs with naked iv administration, a number of studies report that improved uptake and efficacy are seen when siRNAs are administered as a complex with some kind of polymer that facilitates delivery and/or tissue targeting.

Injection of bacterial LPS into mice triggers secretion of tumor necrosis factor (TNF-a) and other cytokines by macrophages, resulting in a septic shock-like syndrome and death from cardiovascular collapse. Sorenson and colleagues describe the use of an unmodified anti-TNF siRNA to rescue mice from what would otherwise be a fatal dose of LPS [198] . Mice were given the anti-TNF siRNA in cationic DOTAP liposomes by ip injection (dose was not stated) 18 h before administration of 350 Ag of bacterial LPS. Only 1/8 mice given a mismatch control siRNA bsurvivedQ this treatment, while 12/16 mice given the anti-TNF siRNA bsurvived.Q The precise meaning of bsurvivalQ in this experiment, however, is not entirely clear since the mice were euthanized based on a subjective evaluation of morbidity. Cytokine levels were measured in peritoneal lavage fluid and TNF-a was reduced by~75% in the anti-TNF-treated animals, while IL-1a levels were unchanged.

Yin and colleagues used ip delivery of siRNAs with cationic lipid to study the role that heat-shock proteins (HSPs) have on thermotolerance and cardiac ischemiareperfusion injury in mice [115] . Unmodified siRNAs targeting heat shock factor 1 (HSF1) were given at a dose of 25 Ag (1.2 mg/kg) by ip injection with cationic lipid; 72 h after siRNA injection, mice were subjected to wholebody hyperthermia (WBH), elevating rectal temperature to 428C for 15 min. Cardiac ischemia-reperfusion injury was performed 48 h after WBH treatment. HSF1 is usually induced to fourfold control levels by WBH. Treatment with the anti-HSF1 siRNA blocked elevation of HSF1 mRNA (HSF1/GAPDH ratio) relative to an amine-alone control (not a control siRNA). Further, treatment with the anti-HSF1 siRNA resulted in increased size of myocardial infarction following ischemic injury and increased mortality from WBH, as would be expected if the protective effects of HSPs were removed by siRNA treatment.

Urban-Klein and colleagues used linear low-molecularweight PEI as a carrier to deliver unmodified siRNAs by ip administration in nude mice with sc implants of SKOV-3 tumors [199] . The ability of PEI to protect unmodified siRNAs from degradation in serum was examined and naked siRNAs were degraded within 15 min of exposure to serum, while~2/3 of the siRNAs complexed with PEI were intact after 6 h. Intraperitoneal administration of siRNA/PEI led to highest accumulation in muscle and tumor and to a lesser extent in liver. SKOV-3 tumors were implanted into the flanks of nude mice and a siRNA specific for the HER2 oncogene was administered three times a week at a dose of 0.6 nmol (~8 Ag) starting when tumors reached a size of 10 mm 2 . Control or naked siRNAs did not influence tumor growth but the anti-HER2 siRNA administered with PEI led to a marked reduction in tumor size as well as an average of 50% reduction in HER2 mRNA in excised tumors.

Direct injection of siRNAs into a target tissue can allow for lower dosing relative to systemic administration, reduce the risk of systemic toxicity, and permit access to otherwise protected sites such as the CNS or the interior of the eye. As mentioned previously, the only FDA-approved antisense drug (Vitravene) is given by intraocular injection to treat CMV retinitis. Intraocular injection is therefore an obvious approach for in vivo use of siRNAs for both research applications and pharmaceutical development. Reich and colleagues described the use of unmodified siRNAs administered by intraocular injection to inhibit neovascularization following laser photocoagulation [200] . To demonstrate the feasibility of the method, recombinant adenovirus expressing enhanced green fluorescent protein (EGFP) were injected subretinally in the eyes of mice with or without 20 pmol (~0.27 Ag) of an anti-EGFP siRNA, and retina were examined at 48 h. The anti-EGFP siRNA almost totally eliminated EGFP fluorescence, even using this very low dose. Administration of a similar low dose of an anti-VEGF siRNA reduced the area of choroidal neovascularization after laser photocoagulation injury by~75%.

Shen and colleagues also reported the use of a siRNA that disrupts the VEGF pathway to reduced neovascularization following laser photocoagulation in mice [201] . The compound studied, Sirna-027, is a chemically modified siRNA that targets VEGFR1 and is under development as a therapeutic for treatment of age-related macular degeneration (see below). SiRNA was administered as a direct intravitreal injection at doses of 1.5 or 0.5 Ag in 1 Al PBS on days 1 and 7 following laser-induced rupture in Bruch's membrane. On day 14, choroidal neovascularization was visualized using fluoresceinlabeled dextran and fluorescence microscopy. Neovascularization was reduced by 56% using the 1.5-Ag dose compared with injection of a control siRNA (in the other eye) and by 66% using the 0.5-Ag dose compared with injection of PBS alone. A dose of 7.5 Ag given by periocular injection (a less traumatic method of administration) on days 1, 4, 7, 10, and 13 resulted in a 45% reduction in neovascularization following laser treatment. Treatment with Sirna-027 was also protective against oxygen-induced ischemic retinopathy. Both VEGFR1 mRNA and protein were demonstrated to be specifically reduced by injection of Sirna-027. Distribution of a BrdU-labeled siRNA after intravitreous injection was studied. After 6 h, labeled siRNA was detected in ganglion cells and by 12-24 h the siRNA was present in ganglion cells and the inner nuclear layer. By 48 h, staining was absent in ganglion cells but was increased in photoreceptor cell bodies. Injected siRNA was no longer detectable at 5 days postinjection.

Ocular infection with herpes simplex virus (HSV) can induce stromal keratitis and neovascularization. Kim and colleagues used unmodified siRNAs targeting three genes in the VEGF pathway (VEGFA, VEGFR1, and VEGFR2) to block angiogenesis from HSV infection [202] . A cocktail of all three siRNAs was administered either by subconjunctival injection (10 Ag) or by tail vein iv injection (40 Ag) complexed with a RPP polymer (see above). Using the anti-VEGF cocktail, both methods of treatment significantly improved the clinical angiogenic score relative to the control siRNA. Subconjunctival administration reduced VEGF mRNA in infected cornea approximately sixfold, while system administration achieved only approximately twofold reduction (using a fourfold higher dose of siRNA).

Antisense oligonucleotides have a long history of successful use in neuroscience research [203] . Although charged nucleic acids do not readily cross the bloodbrain barrier, oligonucleotides can be directly injected into the CNS. Compared with plasma and many tissues, the CNS is a relatively bnuclease-poorQ environment and antisense oligonucleotides with minimal modification have been employed with good results [204] . It is therefore not surprising that direct injection of unmodified siRNAs into the CNS has met with success. Dorn and colleagues reported the use of minimally modified siRNAs administered using an implanted intrathecal pump in rat CNS to modulate chronic neuropathic pain [205] . RNA duplexes had 2V-O-(2-methoxyethyl)-modified residues (MOE-modified) with phosphorothioate linkages comprising the 2-base 3V overhangs on each strand and targeted the pain-related cation-channel P2X 3 . SiRNAs were administered by continuous infusion at a dose of 400 Ag/day (20 mg/kg) for 6-7 days. Dorsal root ganglia showed~40% reduction in P2X 3 mRNA and protein. Rats exhibited an attenuated pain response in several models of neuropathic pain, including tactile allodynia and agonist-induced mechanical hyperalgesia.

Thakker and colleagues reported knockdown of two targets in mouse brain using direct CNS administration of 3V-overhang MOE-modified siRNAs. Alzet osmotic minipumps delivered 400 Ag/day of siRNA (20 mg/kg) into the dorsal third ventricle. In the first series of studies, anti-EGFP siRNAs were infused into EGFP-expressing mice. Following 2 weeks of infusion, the effect on EGFP fluorescence ranged from no suppression (in the locus coeruleus and the spinal trigeminal nucleus) to 50% suppression (in the dentate gyrus and hippocampus) [206] . SiRNAs targeting the dopamine transporter (DAT) gene were also infused and were found to reduce DAT mRNA levels and produce a temporal hyperlocomotor response similar to that seen using a small-molecule antagonist of DAT [206] . Infusion of siRNAs targeting the serotonin transporter (SERT) reduced SERT mRNA levels and produced an antidepressant-like response similar to that observed from administration of Citalopram, a small-molecule serotonin-reuptake inhibitor [207] .

Use of a cationic lipid delivery agent may enable lower doses of siRNAs to be used for these kinds of studies and can be safe for use in the CNS. Luo and colleagues delivered an unmodified control siRNA or a siRNA specific for the delta opioid receptor (DOR) via an implanted intrathecal catheter in the lumbar spine of rats [117] . Dose ranged from 0.5 to 4.0 Ag/day given as a 2 Ag/10 Al solution in the cationic lipid i-Fect (very low dose of 2.5-20 Ag/kg). Fluorescence signal was readily visualized in the spinal cord and dorsal root ganglia using a dye-labeled siRNA with cationic lipid but not with unassisted infusion. DOR protein levels were markedly reduced when examined by immunohistochemistry 72 h after the last dose of siRNAs in anti-DOR-treated animals but not mismatch control-treated animals. Levels of the related mu opioid receptor were unchanged, demonstrating specificity. Functionally, treatment with the anti-DOR siRNA specifically blocked the antinociceptive effect of a DOR agonist. Although eight groups have published data showing successful use of siRNAs in CNS studies, two groups have reported negative results [208, 209] , both using siRNAs that had been validated in vitro.

A number of groups have reported success using direct injection into or adjacent to tumor masses using naked and/or complexed siRNAs. Aharinejad and colleagues reported direct injection of unmodified siRNAs into subcutaneous masses of the human mammary tumor cell line MCF-7 in nude mice [210] . Injections were started after tumors were established (beginning 24 days after implantation) at a dose of 10 Ag once every 3 days for 2 weeks. Two different siRNAs specific for murine colonystimulating factor (CSF)-1 and the CSF-1 receptor (c-fms proto-oncogene) were employed. CSF-1 signaling recruits macrophage infiltration of tumors and several macrophage-derived factors are believed to facilitate metastasis and tumor growth (matrix metalloproteases and VEGF). SiRNAs were tested in vitro and the most active sequences were employed for subsequent in vivo experiments. Both CSF-1 and CSF-1R mRNA levels were reduced in tumor tissue. Consistent with the original hypothesis, both metalloprotease levels and VEGF levels were also reduced in tumors by this treatment and tumor growth was reduced by~50%.

Leng and Mixon used cationic peptides to facilitate delivery of unmodified siRNAs by direct injection into human breast cancer MDA-MB-435 implants in nude mice [211] . The siRNA employed was specific for Raf-1, a member of the Raf kinase family involved in Ras/MEK signaling. A series of structurally different histidinelysine peptides were synthesized and compared with DOTAP liposomes for their ability to deliver siRNAs in mice. Injections contained 12 Ag peptide complexed with 4 Ag siRNA and were administered every 5 days for two or three doses. The H 3 K4b peptide/anti-Raf-1 siRNA complex was most effective and reduced tumor size by 60% relative to treatment with the control siRNA/peptide and was superior to the liposomal preparation.

Atelocollagen has also been used to assist with direct intratumor injections. Takei and colleagues described injection of an unmodified anti-VEGF siRNA in 50 Al of a 1.75% solution of atelocollagen in PBS into established human prostate cancer PC-3 cell implants in nude mice [212] . Dose-response curves were performed using 1, 5, and 10 AM concentrations of the siRNA (0.67-6.7 Ag); injections were done once every 10 days and tumor response was assessed at 40 days. Treatment with the anti-VEGF siRNA reduced tumor volume in a dosedependent fashion and, using the highest dose, tumors were~80% smaller than those in control siRNA-treated mice. An 80% reduction in VEGF protein was observed in day 17 tumors treated with 10 AM anti-VEGF siRNA. Properties of atelocollagen as a delivery agent were also described. Tumors injected with a fluorescent dye-labeled siRNA with atelocollagen had significantly more labeled cells (also bbrighterQ) than tumors injected with naked siRNAs. Further, intact 32 P-labeled siRNA could be detected in tumors 7 days after injection with atelocollagen, whereas naked siRNAs were mostly undetectable only 1 day postinjection.

Nucleic acids can be absorbed through epithelial or mucosal surfaces. Maeda and colleagues described the use of this delivery route to administer unmodified siRNAs in a murine deafness model system [118] . The GJB2 gene (gap junction protein, h-2) encodes a transmembrane protein that is involved in potassium recycling during auditory signaling. Allelic variants of this gene are implicated in some severe forms of hereditary deafness. A dominant-negative mutant of this gene (GJB2 R75W ) was fused to EGFP and was used to create a model of human deafness in mice. Anti-GJB2 R75W siRNAs were delivered to the cochleae of mice in a mixture of DOTAP/cholesterol liposomes (GeneSHUTTLE) using 0.5 Ag siRNA in 2 Al soaked in a piece of gel foam, which was surgically placed against the round window membrane. Administration of the anti-GJB2 R75W siRNA reduced expression of GJB2 R75W mRNA by 70% without affecting levels of normal murine Gjb2 and reversed the auditory impairment caused by expression of the mutant human gene in mouse cochleae.

Palliser and colleagues used unmodified siRNAs in a cationic lipid carrier as a bmicrobicideQ applied intravaginally to block HSV2 infection in mice [119] . Mice were vaginally infected with 2 Â 10 4 pfu (~2Â LD 50 dose) of HSV2 and were given control or anti-HSV2 siRNAs at bÀ2 h and 4 hQ time points or at b+3 and +6 hQ time points. Duplexes targeting several viral genes were compared and one targeting the viral UL29 was most potent in suppressing virus production in vitro. SiRNAs were administered intravaginally as described at a dose of 0.5 nmol (~7 Ag) in 12-Al volume complexed with Oligofectamine. Mice receiving the anti-UL29 siRNA had~80% survival at 15 days, while mice receiving the control or no siRNA had 20-25% survival. Mice receiving a siRNA targeting a different viral gene, UL27, had~60% survival. Importantly, no evidence for activation of the innate immune system was detected following siRNA treatment in the absence of viral infection (IFN-h, STAT1, and OAS1 mRNA levels were tested by qRT-PCR). Thus, two independent anti-HSV2 siRNAs showed similar biological effects with no evidence of immune stimulation, so the antiviral effect could be attributed directly to RNAi-mediated suppression. Delivery of siR-NAs in liposomal or cationic lipid bpaintQ might similarly be useful for other epithelial surfaces, such as oral or rectal mucosa.

Antisense oligonucleotides have been administered by inhalation to treat asthma and other pulmonary diseases [213, 214] . Similarly, several groups have delivered siRNAs by intranasal or intratracheal infusion in mice. Massaro and colleagues describe intranasal administration of both siRNA and small-molecule compounds to mice in Infa-Surf (replacement pulmonary surfactant, more commonly used to treat neonatal severe respiratory distress syndrome) [215] . An unmodified siRNA targeting GAPDH was administered intranasally in lightly anesthetized mice at a dose of 10 Ag in 10-to 20-Al volume. At 24 h postinfusion, GAPDH protein levels were reduced by 50% or more in lung, heart, and kidney, but not in liver or brain. At 7 days, GAPDH was further suppressed to 67% control levels in lung but other tissues showed limited or no effect.

A number of groups are exploring the use of siRNAs to treat respiratory viruses using direct pulmonary delivery in mammals. Tompkins and colleagues used unmodified siRNAs specific to viral NP and PA proteins to protect mice from lethal infection with influenza virus [157] . SiRNAs in PBS were administered iv by HD tail vein injection 16-24 h prior to infection (single dose of 50 Ag, or~2.5 mg/kg). Influenza virus was then coadministered with siRNAs complexed with the cationic lipid Oligofectamine by direct intranasal infusion in mice at a dose of 1.5 nmol (20 Ag, or~1 mg/kg). Viral titers in lung tissue were reduced as much as 63-fold using anti-influenza siRNAs compared with the control siRNA and survival was significantly improved, especially in mice receiving a combination of anti-NP + anti-PA siRNAs. Bitko and colleagues described the use of unmodified siRNAs to treat pulmonary infection with respiratory syncytial virus (RSV) and parainfluenza virus in mice by targeting the viral P gene for both viruses [216] . SiRNAs in PBS were administered either naked or complexed with the cationic lipid TransIT-TKO by direct intranasal infusion in mice at a dose of 35 Ag in each nostril (70 Ag total dose, or 3.5 mg/kg). Treatment with naked siRNAs was~70% as effective as with cationic lipid. Mice receiving antiviral siRNAs showed decreased lung pathology and overall distress compared with mice receiving control siRNAs. Although the greatest improvement was seen when treatment was started at the time of infection, some benefit was seen when siRNA treatment was given 1, 2, and even 3 days postinfection.

The severe acute respiratory syndrome coronavirus (SCV) has been the subject of intense investigation in recent years and development of any kind of therapeutic method to treat this virus other than supportive care is a high priority. Li and colleagues describe the use of unmodified siRNAs delivered intranasally in D5W in rhesus macaques (Macaca mulatta) [217] . Delivery of siRNAs targeting a reporter construct were first tested using D5W or InfaSurf via intranasal delivery in mice and the D5W carrier gave superior results and was therefore employed in the primate studies. A mixture of two siRNAs specific for the Spike protein-coding and ORF1b regions was administered 4 h prior to infection (single dose), at the time of infection (and at 1, 3, and 5 days postinfection), or 4 h postinfection (and at 1 and 3 days postinfection). Each dose was 30 mg in 3 ml D5W and total cumulative dosing was 30 to 120 mg siRNA (10-40 mg/kg). The severity of symptoms was improved in all treatment groups. Animals were tested at 4 days postinfection for the presence of SCV RNA using a qRT-PCR assay (from oropharyngeal swab samples); all control animals were positive for SCV viral RNA, while 75% of anti-SCV siRNA-treated animals had no detectable viral RNA.

Electroporation is a useful tool to transfect siRNAs in vitro into cell lines that are refractory to cationic lipidmediated transfection. Interestingly, it is also being employed to assist with siRNA delivery in vivo. Golzio and colleagues described electroporation-assisted delivery of an EGFP expression plasmid with or without an unmodified anti-EGFP siRNA following direct injection into mouse muscles in PBS [171] . EGFP fluorescence was specifically reduced by the anti-EGFP but not the control siRNA; targeting of an endogenous target was not described. Schiffelers and colleagues used electroporation to assist with delivery of unmodified siRNAs targeting TNF-a in mice with collagen-induced arthritis [170] . Injection of an EGFP expression plasmid into the knee joint of mice showed local expression of EGFP only if electroporation was employed. Coadministration (injection + electroporation) of 2 Ag of a luciferase expression plasmid with 10 Ag of either the anti-EGFP (control) or the anti-Luc siRNAs showed 90% suppression of luciferase activity when the anti-Luc siRNA was used. Collageninduced arthritis was induced by injection of mice with type II collagen and inflammation was triggered with ip injection of LPS 3 days after the second dose of collagen. Anti-TNF-a siRNA treatment was initiated 3 days after LPS treatment at a dose of 10 Ag (direct injection with electroporation into the knee joint) and was administered weekly for three doses. Animals treated with the anti-TNF-a siRNA showed a marked improvement in inflammation and reduced arthritis score relative to control animals. Similarly, Inoue and colleagues used electroporation to assist with delivery of an unmodified siRNA targeting TNF-a in rats with collagen-induced arthritis [172] . In this case, 0.8 nmol (~11 Ag) of the anti-TNF-a siRNA or a control siRNA was injected into the knee joint in rats in cationic lipid (siPORT Amine) followed by electroporation. TNF-a mRNA levels were reduced in synovial tissue and inflammation was markedly reduced as assessed by both paw swelling and histological examination in anti-TNF-a-treated animals relative to controls.

In addition to chemical and electrical methods to promote delivery of siRNAs in vivo discussed above, Kim and colleagues described a mechanical method using a gold nanoparticle gene gun in the context of improving the immune response in mice to DNA vaccines [174] . Infection with human papillomavirus type 16 (HPV-16) is associated with development of cervical carcinoma. Attempts to immunize mice against HPV-16 through the use of a DNA vaccine (a plasmid expressing the E7 gene of HPV-16) resulted in an incomplete response, partially due to the transient nature of antigen presentation by resident antigen-presenting cells (APCs). The authors tested if coadministration of the HPV-16 E7 plasmid with siRNAs targeting the proapoptotic genes BAX and BAK would prolong survival of the APCs and improve vaccination. Nucleic acids were coated onto gold microcarriers and delivered subdermally using the Helios gene gun at a dose of 2 Ag plasmid coadministered with 0.2 Ag unmodified anti-BAK + anti-BAX siRNAs. Vaccine delivered with the BAK + BAX siRNAs increased the number of antigen-specific IFN-g + CD8 + T cell precursors. Further, mice receiving the combined vaccine (E7 with BAX + BAK siRNAs) showed a marked reduction in tumor burden when injected with TC-1 cells expressing the E7 antigen compared with mice that received the E7 vaccine with the control siRNA.

Kinoshita and Hynynen described the use of microbubbles from focused ultrasound (sonoporation) to deliver siRNAs to cells in vitro [218] . Tsunoda and colleagues used sonoporation to deliver an EGFP expression plasmid with or without unmodified anti-EGFP siRNA to cardiac tissue in mice [173] . The EGFP expression plasmid was injected into the left ventricle of anesthetized mice (500 Ag in 400 Al PBS with 100 Al BR14 microbubbles) and transthoracic ultrasound stimulation was applied. EGFP fluorescence was detected in the cardiac ventricular wall and in the coronary arteries. Co-injection of 40 Ag of the anti-EGFP siRNA suppressed EGFP fluorescence.

Over 30 pharmaceutical and biotechnology companies have declared an interest in or have an active drug development program already under way in RNAi-based therapeutics. A number of these companies and their drug development programs are outlined in Table 2 . In addition, many large pharmaceutical companies that are traditionally associated with small-molecule drug development also have active internal or collaborative RNAirelated drug development projects.

Sirna Therapeutics reported interim results of a Phase I study of Sirna-027 at the American Society of Gene Therapy conference in June 2005 [219] . Sirna-027 is a chemically modified siRNA drug that targets VEGFR1 to treat age-related macular degeneration (AMD). The study involved administration of a single escalating dose of the drug by direct intravitreal injection. Doses from 100 to 800 Ag were administered and no significant toxicity was observed. During the brief observation period, visual acuity stabilized and even improved in a dose-dependent manner. Preclinical studies of Sirna-027 in a mouse model system have also been reported (with Johns Hopkins University) [201] .

Acuity Pharmaceuticals reported results of a Phase I clinical trial of Cand5, a siRNA drug targeting VEGF to drug to treat AMD, at the October 2005 meeting of the American Academy of Ophthalmology. Fifteen patients were administered Cand5 at five escalating dose levels up to 3.0 mg per eye by direct intravitreal injection. No adverse effects that related to the actual siRNA drug were reported; however, the expected side effects such as subconjunctival hemorrhage, etc., were observed (normal for this route of drug administration). No systemic delivery was detected. Patients are currently being enrolled in a Phase II clinical trial.

Alnylam Pharmaceuticals has drug development programs ongoing for multiple indications and has specifically partnered with Novartis, Merck, and Medtronic for certain projects. The most advanced drug in their pipeline targets RSV and an IND was filed on November 1, 2005, with the FDA for their lead compound, ALN-RSV01. Treatments for pandemic influenza are next in the development pipeline and Alnylam plans to file an IND in this field later in 2006.

Many companies have reported preclinical data validating their siRNA drug development projects in vivo in mammalian model systems. Nastech Pharmaceuticals (working with Mayo Clinic) presented data on the use of an anti-TNF-a siRNA delivered using their peptide carrier system to treat rheumatoid arthritis at the November 2005 American College of Rheumatology meeting. Alnylam Pharmaceuticals has published preclinical work relating to treatment of hypercholesterolemia using anti-ApoB siRNAs [65] and Sirna Therapeutics has published preclinical work relating to treatment of hepatitis B infection (with Protiva Biotherapeutics, who provided the SNALP delivery system) [52, 63] . Calando Pharmaceuticals published results of a collaborative study relating to siRNA treatment of Ewing sarcoma (with Children's Hospital of Los Angeles) [134] and Galenea has described the use of siRNAs to treat influenza in mice (work done at MIT) [197] . Intradigm has published a number of collaborative studies done relating to siRNA treatment of cancer, arthritis, ocular diseases, and viral diseases [130, 170, 194, 202, 217] . Abbott described the use of siRNAs in a mouse cancer model system [220] and Novartis has reported the use of siRNAs in CNS studies [205] [206] [207] . TransGenex Nanobiotech's chitosan delivery system was employed in a report using ddRNAi to treat RSV (University of South Florida) [221] and NeoPharm has published the use of their liposomes to deliver siRNAs in two mouse cancer model systems (one with Georgetown University Medical Center) [127, 128] . Mirus has published the use of their hydrodynamic delivery system in mice [149] and larger mammals (with the University of Wisconsin, Madison, WI, USA) [155] .

Considering the relative youth of the RNAi field, a remarkable number of in vivo studies have already been published that relate to preclinical development of siRNA drugs or the use of siRNA methods to validate targets for small-molecule drug development projects.

RNA interference has proven to be an extremely potent and versatile tool to specifically reduce expression of targeted genes. Use of this technology has rapidly moved from in vitro cell culture studies to in vivo administration in mammals, both to address questions of basic biology and in drug development programs. RNAi-based drugs are already in clinical trials and it is hopeful that a siRNA therapeutic will receive FDA approval in the not too distant future.

As understanding of the basic biochemistry and biology of RNAi advances, it is becoming clear that extra controls should be employed when using siRNAs in vivo to avoid the risk of misinterpreting results due to unintended stimulation of the innate immune system or off-target effects. Few published studies actually measured cytokine levels. Most simply employed bcontrolQ siRNAs to ensure that immune stimulation or other unintended events did not occur. Given the sequence-specific nature of innate immune responses, confidence in these kinds of controls may be misplaced, even if modified siRNAs that have a lower incidence of immune stimulation are employed. Measurement of serum IFN-a, TNF-a, and possibly IL-12 levels at~4-6 h posttreatment should be considered, especially when systemic administration methods are employed. If local administration is employed, assays should include extracts prepared from the injected site/ organ as well as from serum.

Off-target effects can be difficult to identify and are also sequence specific. Microarrays have been used to assess the extent of OTEs at the gene expression level on a genome-wide scale in studies done in cell culture; however, this kind of experiment has not been reported yet in vivo and these kinds of data might be difficult to interpret. One approach to control for OTEs would be to require the use of two (or more) different siRNAs against the same target with the expectation that both result in suppression of the targeted gene and produce the same biological response (phenotype). Few of the in vivo studies reported to date confirmed results using two independent siRNA sequences. Further, few of the studies discussed here performed any kind of dose-response optimization of the siRNA reagent in vivo and instead simply reported results using a single dosing regimen. Even though these studies are expensive to perform in mammals, it is important to characterize the system fully and this kind of information may uncover dose-dependent side effects. Actual OTE-related events will probably show variation between individuals, especially in outbred populations, making these controls even more important to include in this setting.

Although some labs have reported success using in vivo administration of naked siRNAs, a greater number of investigators reported that using some kind of delivery system improved results. Route of administration and choice of which delivery tool to use will be crucial to success. Other than hydrodynamic delivery, too few in vivo studies have been published using any other single method to accumulate the amount of information needed to assess functional performance adequately (with different target genes and different target tissues) or permit a general endorsement of that method, although several approaches do seem quite promising. Unfortunately, many of the most advanced delivery systems employed in the studies discussed above are not available to most researchers. Widespread adoption of RNAi using synthetic siRNAs as an in vivo research tool will accelerate as more effective mammalian delivery tools become commercially available. If the target tissue or system of study permits the use of a local delivery approach, this method may be easier to perform using boff the shelfQ tools and offer greater assurance of success than systemic iv administration.

Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide

Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide

Injected anti-sense RNAs specifically block messenger RNA translation in vivo

Inhibition of SV40 gene expression by microinjected small antisense RNA and DNA molecules

Molecular mechanisms of action of antisense drugs

Chemistry and biochemistry of 2V,5V-oligoadenylate-based antisense strategy

Oligonucleotide analogues: an overview

Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action

Toxicology of antisense therapeutics

A randomized controlled clinical trial of intravitreous fomivirsen for treatment of newly diagnosed peripheral cytomegalovirus retinitis in patients with AIDS

Progress in antisense technology

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans

RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals

RNA interference is mediated by 21-and 22-nucleotide RNAs

Role for a bidentate ribonuclease in the initiation step of RNA interference

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Passengerstrand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes

Argonaute2 cleaves the antiguide strand of siRNA during RISC activation

Argonaute2 is the catalytic engine of mammalian RNAi

Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity

Purified Argonaute2 and an siRNA form recombinant human RISC

TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing

TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing

Human RISC couples microRNA biogenesis and posttranscriptional gene silencing

Mechanisms of gene silencing by double-stranded RNA

Unlocking the potential of the human genome with RNA interference

Revealing the world of RNA interference

Assembly and function of RNA silencing complexes

Post-transcriptional gene silencing by siRNAs and miRNAs

Asymmetry in the assembly of the RNAi enzyme complex

Functional siRNAs and miRNAs exhibit strand bias

An algorithm for selection of functional siRNA sequences

Rational siRNA design for RNA interference

Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference

Design of a genome-wide siRNA library using an artificial neural network

Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy

The silent revolution: RNA interference as basic biology, research tool, and therapeutic

RNA interference: from gene silencing to gene-specific therapeutics

Small interfering RNA for experimental cancer therapy

Delivering RNA interference to the mammalian brain

State-of-the-art modified RNAi compounds for therapeutics

The therapeutic potential of RNA interference

In vivo application of RNA interference: from functional genomics to therapeutics

Short interfering RNA (siRNA): tool or therapeutic?

Potential applications for RNAi to probe pathogenesis and develop new treatments for ocular disorders

Prospects of RNA interference therapy for cancer

Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells

Surface calreticulin mediates muramyl dipeptide-induced apoptosis in RK13 cells

Functional polarity is introduced by Dicer processing of short substrate RNAs

Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs

RNA interference using boranophosphate siRNAs: structure-activity relationships

Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

siRNA function in RNAi: a chemical modification analysis

RNA interference in mammalian cells by chemicallymodified RNA

Tolerance for mutations and chemical modifications in a siRNA

Phosphorothioate oligodeoxynucleotides: antisense or anti-protein?

Fully 2V-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA

Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2V-O-methyl RNA, phosphorothioates and small interfering RNA

Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality

In vivo activity of nuclease-resistant siRNAs

Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication

Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

Biomembrane-permeable and ribonuclease-resistant siRNA with enhanced activity

Peptide-oligonucleotide conjugates for the delivery of antisense and siRNA

Biodistribution of phosphodiester and phosphorothioate siRNA

From bugs to drugs: therapeutic immunomodulation with oligodeoxynucleotides containing CpG sequences from bacterial DNA

Immunotherapeutic utility of stimulatory and suppressive oligodeoxynucleotides

Modulation of Toll-like receptor 9 responses through synthetic immunostimulatory motifs of DNA

Recent advances in immunostimulatory CpG oligonucleotides

Toll-like receptors: a family of patternrecognition receptors in mammals

Innate immune responses to infection

Detection of foreign RNA: implications for RNAi

Sensing the danger in RNA

Immune responses to dsRNA: implications for gene silencing technologies

How cells respond to interferons

The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses

Activation of the interferon system by short-interfering RNAs

Structural requirements of double-stranded RNA for the activation of 2V,5V-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells

Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase

Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3

Exogenous siRNA mediates sequence-independent gene suppression by signaling through Toll-like receptor 3

Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3

Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA

Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization

Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8

Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7

Lack of interferon response in animals to naked siRNAs

Cationic liposome-mediated delivery of siRNAs in adult mice

Cationic lipids enhance siRNA-mediated interferon response in mice

Activation of the mammalian immune system by siRNAs

Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA

Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo

Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus

Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways

A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites

Expression profiling reveals off-target gene regulation by RNAi

Nonspecific, concentrationdependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs)

Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells

Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs

MicroRNAs: small RNAs with a big role in gene regulation

MicroRNA biogenesis: coordinated cropping and dicing

Small RNAs with imperfect match to endogenous mRNA repress translation: implications for off-target activity of small inhibitory RNA in mammalian cells

siRNAs can function as miRNAs

MicroRNA-directed cleavage of HOXB8 mRNA

Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation

Specificity of microRNA target selection in translational repression

siRNA-mediated off-target gene silencing triggered by a 7 nt complementation

High doses of siRNAs induce eri-1 and adar-1 gene expression and reduce the efficiency of RNA interference in the mouse

Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells

Efficient delivery of small interfering RNA for inhibition of IL-12p40 expression in vivo

Silencing heat shock factor 1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia-reperfusion injury in mice

Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels

An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons

In vitro and in vivo suppression of GJB2 expression by RNA interference

An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection

Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA

U.S. Food and Drug Administration approval of AmBisome (liposomal amphotericin B) for treatment of visceral leishmaniasis

Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol

Liposomes modified with cyclic RGD peptide for tumor targeting

Importance of lateral and steric stabilization of polyelectrolyte gene delivery vectors for extended systemic circulation

Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties

CD40 antisense oligonucleotide inhibition of trinitrobenzene sulphonic acid induced rat colitis

Systemic delivery of Raf siRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer

Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine

Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle

Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung

Target-specific gene silencing by siRNA plasmid DNA complexed with folate-modified poly(ethylenimine)

Cyclodextrin-modified polyethylenimine polymers for gene delivery

Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma

Transferrinpolycation conjugates as carriers for DNA uptake into cells

Cellspecific targeting of nanoparticles by multivalent attachment of small molecules

Protamine sulfate enhances lipidmediated gene transfer

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors

Peptide-based strategy for siRNA delivery into mammalian cells

Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells

Highly efficient small interfering RNA delivery to primary mammalian neurons induces microRNA-like effects before mRNA degradation

Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells

Efficient delivery of small interfering RNA to bonemetastatic tumors by using atelocollagen in vivo

Specific delivery of therapeutic RNAs to cancer cells via the dimerization mechanism of phi29 motor pRNA

Reconstituted influenza virus envelopes as an efficient carrier system for cellular delivery of smallinterfering RNAs

Rad51 siRNA delivered by HVJ envelope vector enhances the anti-cancer effect of cisplatin

Efficient delivery of RNA interference effectors via in vitro-packaged SV40 pseudovirions

RNA interference in adult mice

Efficient delivery of siRNA for inhibition of gene expression in postnatal mice

RNA interference targeting Fas protects mice from fulminant hepatitis

Small interfering RNA inhibits hepatitis B virus replication in mice

Caspase 8 small interfering RNA prevents acute liver failure in mice

Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA

Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice

A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs

Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury

Protection against lethal influenza virus challenge by RNA interference in vivo

Use of RNA interference to prevent lethal murine West Nile virus infection

Targeting 2A protease by RNA interference attenuates coxsackieviral cytopathogenicity and promotes survival in highly susceptible mice

In vivo delivery of caspase-8 or Fas siRNA improves the survival of septic mice

Toward a gene therapy for dominant disease: validation of an RNA interference-based mutation-independent approach

Sequence-specific suppression of mdr1a/1b expression in mice via RNA interference

Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases

Gene silencing in rat-liver and limb grafts by rapid injection of small interference RNA

RNA silencing in vivo reveals role of p22phox in rat angiotensin slow pressor response

Delivery of siRNA and siRNA expression constructs to adult mammals by hydrodynamic intravascular injection

Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA

Exploring RNA interference as a therapeutic strategy for renal disease

RNAi-induced gene silencing by local electroporation in targeting brain region

Effects of treatment with small interfering RNA on joint inflammation in mice with collageninduced arthritis

Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery

Electro-transfer of small interfering RNA ameliorated arthritis in rats

Sonoporation using microbubble BR14 promotes pDNA/ siRNA transduction to murine heart

Modification of professional antigen-presenting cells with small interfering RNA in vivo to enhance cancer vaccine potency

Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA

Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice

Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures

Cytotoxicity issues pertinent to lipoplex-mediated gene therapy invivo

In vivo characteristics of cationic liposomes as delivery vectors for gene therapy

DOTAP (and other cationic lipids): chemistry, biophysics, and transfection

Side-effects of a systemic injection of linear polyethylenimine-DNA complexes

Effects of structure of betacyclodextrin-containing polymers on gene delivery

Self-assembling nucleic acid delivery vehicles via linear, water-soluble, cyclodextrin-containing polymers

Mutant V599EB-Raf regulates growth and vascular development of malignant melanoma tumors

Rapid intravascular injection into limb skeletal muscle: a damage assessment study

In vivo gene silencing in Plasmodium berghei-A mouse malaria model

SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth

RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine

Systemic siRNA-mediated gene silencing: a new approach to targeted therapy of cancer

Silencing of CXCR4 blocks breast cancer metastasis

An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis

Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery

Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line

Transporting silence: design of carriers for siRNA to angiogenic endothelium

Tumor-targeted gene therapy: strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/DNA complexes

Toll-like receptors on tumor cells facilitate evasion of immune surveillance

Inhibition of influenza virus production in virus-infected mice by RNA interference

Gene silencing by systemic delivery of synthetic siRNAs in adult mice

RNAimediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo

Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model

Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1

Inhibition of ocular angiogenesis by siRNA targeting vascular endothelial growth factor pathway genes: therapeutic strategy for herpetic stromal keratitis

Antisense oligonucleotides for target validation in the CNS

End-capped antisense oligodeoxynucleotides effectively inhibit gene expression in vivo and offer a low-toxicity alternative to fully modified phosphorothioate oligodeoxynucleotides

siRNA relieves chronic neuropathic pain

Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference

siRNA-mediated knockdown of the serotonin transporter in the adult mouse brain

Lack of efficacy of 'naked' small interfering RNA applied directly to rat brain

Central administration of small interfering RNAs in rats: a comparison with antisense oligonucleotides

Colony-stimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice

Small interfering RNA targeting Raf-1 inhibits tumor growth in vitro and in vivo

A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics

DNA antisense therapy for asthma in an animal model

Discovery and development of respirable antisense therapeutics for asthma

Noninvasive delivery of small inhibitory RNA and other reagents to pulmonary alveoli in mice

Inhibition of respiratory viruses by nasally administered siRNA

Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in rhesus macaque

A novel method for the intracellular delivery of siRNA using microbubble-enhanced focused ultrasound

First clinical data on RNAi

Evaluating hypoxia-inducible factor-1alpha as a cancer therapeutic target via inducible RNA interference in vivo

Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene

Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo

Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis

Hypoxia inducible factor-1 activation by prolyl 4-hydroxylase-2 gene silencing attenuates myocardial ischemia reperfusion injury

Blockage of the macrophage migration inhibitory factor expression by short interference RNA inhibited the rejection of an allogeneic tracheal graft

Silencing T-bet defines a critical role in the differentiation of autoreactive T lymphocytes

Silencing of fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis

In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury

Small interfering RNA-mediated functional silencing of vasopressin V2 receptors in the mouse kidney

siRNA-induced caveolin-1 knock-down in mice increases lung vascular permeability via the junctional pathway

Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer

Variants of bcl-2 specific siRNA for silencing antiapoptotic bcl-2 in pancreatic cancer

Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo

Characterization of the bystander effect of somatostatin receptor sst2 after in vivo gene transfer into human pancreatic cancer cells

Requirement for sphingosine 1-phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference

Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo

Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer

RNA interference targeting transforming growth factorbeta type II receptor suppresses ocular inflammation and fibrosis

Down-regulation of apoptosis mediators by RNAi inhibits axotomy-induced retinal ganglion cell death in vivo

siRNA targeted against amyloid precursor protein impairs synaptic activity in vivo

Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake

Attenuation of alpha2A-adrenergic receptor expression in neonatal rat brain by RNA interference or antisense oligonucleotide reduced anxiety in adulthood

The author thanks Drs. John Rossi, Bryan Williams, Edouard Cantin, and Scott Rose for helpful discussions and for reading the manuscript.