key: cord-1025818-jfug80yu
authors: Aigner, Achim
title: Applications of RNA interference: current state and prospects for siRNA-based strategies in vivo
date: 2007-04-25
journal: Appl Microbiol Biotechnol
DOI: 10.1007/s00253-007-0984-y
sha: 8c5eb0eff7abf239b26aed8f379cc26c411ee876
doc_id: 1025818
cord_uid: jfug80yu

Within the recent years, RNA interference (RNAi) has become an almost-standard method for in vitro knockdown of any target gene of interest. Now, one major focus is to further explore its potential in vivo, including the development of novel therapeutic strategies. From the mechanism, it becomes clear that small interfering RNAs (siRNAs) play a pivotal role in triggering RNAi. Thus, the efficient delivery of target gene-specific siRNAs is one major challenge in the establishment of therapeutic RNAi. Numerous studies, based on different modes of administration and various siRNA formulations and/or modifications, have already accumulated promising results. This applies to various animal models covering viral infections, cancer and multiple other diseases. Continuing efforts will lead to the development of efficient and “double-specific” drugs, comprising of siRNAs with high target gene specificity and of nanoparticles enhancing siRNA delivery and target organ specificity.

After antisense technologies and ribozymes, in the late 1990s a novel mechanism for gene-targeting was discovered: RNA interference (RNAi). It soon became clear that RNAi represents a particularly efficient and-at least in vitro-easy-to-use method for the knockdown of the expression of a selected target gene. Consequently, RNAi is now a wellestablished method for high-throughput analyses as well as for functional studies in vitro, including mammalian cells.

Many pathological conditions rely on the aberrant expression of endogenous normal or mutant genes causing, e.g., alterations in signal transduction pathways, cellular proliferation, apoptosis, or resistance toward external factors. Additionally, the infection of an organism can lead to the introduction and expression of foreign genes. While the inhibition of the activity of (aberrant) gene products, e.g., through small molecule inhibitors or inhibitory antibodies is one major focus in therapy, much attention has now shifted to an earlier step, i.e., the initial knockdown of the specific gene of interest through RNAi. However, for the in vivo application of RNAi in mammals as a therapeutic approach for reversing a pathological condition as well as for the study of particular gene functions, sophisticated strategies for the induction of RNAi are needed.

RNAi is a naturally occurring, sequence-specific mechanism for gene silencing. Its discovery in the nematode C. elegans (Fire et al. 1998 ) was awarded the 2006 Nobel prize for physiology or medicine. However, soon it became obvious that RNAi, although somewhat more complicated, also exists in higher organisms including mammals. RNAi relies on an intracellular multistep process, which can roughly be divided into the initiation phase (see below) and the subsequent effector phase. In the effector phase ( Fig. 1,  left) , which represents the actual RNAi mechanism, small, 21-23 bp double stranded RNA molecules (small interfering RNAs, siRNAs), are incorporated into the RNA-induced silencing complex (RISC; Hammond et al. 2000) . Upon adenosine triphosphate (ATP)-dependent unwinding of the double-stranded siRNA molecule through an RNA helicase activity into a single-stranded, so-called guidance RNA (Nykanen et al. 2001) , the now activated RISC (RISC*) binds to its target mRNA molecule (Martinez et al. 2002; Nykanen et al. 2001 ). This process is mediated through the sequence-specific hybridization of the guidance RNA to the mRNA target site and brings RISC into close proximity to its target mRNA molecule, which is then cleaved by the RISC nuclease Argonaute 2 (Ago 2) and rapidly degraded due to its now unprotected ends (Liu et al. 2004; Rand et al. 2004; Rivas et al. 2005 ). Since RISC is recovered for subsequent rounds, this represents a catalytical process leading to the selective reduction in specific mRNA molecules and thus resulting in decreased expression of the targeted gene. The mechanism also demonstrates the pivotal role of siRNA molecules in initiating RNAi and established the delivery of siRNA molecules as sufficient for RNAi induction (Elbashir et al. 2001a, b) .

In a natural, experimental or therapeutical setting, siRNAs can be directly or as precursor molecules introduced into a target cell through different strategies (Fig. 1, upper part) . This includes viral or nonviral delivery of DNAs, which are transcribed into long, double-stranded RNA molecules. In the so-called initiation phase, these dsRNAs are cleaved into siRNAs by the multiprotein complex "Dicer", which con-tains an N-terminal helicase domain, an RNA-binding socalled Piwi/Argonaute/Zwille (PAZ) domain, two RNAse III domains and a double-stranded RNA binding domain (Bernstein et al. 2001; Collins and Cheng 2005) . Commercially available systems explore this mechanism by providing DNA vector constructs coding for short hairpin RNAs (shRNAs): The double-stranded region of the shRNA is formed through hairpin formation and intramolecular hybridization and is recognized by Dicer, leading to the formation of siRNAs homologous to the target gene of interest. Alternatively, shRNA molecules can be directly introduced into the cell. However, one major disadvantage of long double-stranded RNA molecules, either directly introduced or intracellularly transcribed, is the induction of a cellular immune response through activation of the interferon system. The direct delivery of siRNA molecules into the target cell strategy largely avoids this problem, although some interferon-stimulating sequences are known as well. Furthermore, it does not require the action of Dicer (Bridge et al. 2003; Hornung et al. 2005; Sledz et al. 2003) .

Systematic studies on targeting efficacies have shown that optimal siRNAs can be deduced according to certain selection rules. This includes an optimal length of 19-25 bp and a guanine-cytosine content between 30 and 52%, symmetric two nucleotide 3′ overhangs as well as specific nucleotides at certain positions (see, e.g., Dykxhoorn and Lieberman 2006 for review). Based on these already established criteria, several computer-based algorithms allow the identification of optimal siRNA sequences for any given gene of interest. One example is the siRNA Design Software (SDS) by the University of Hong Kong which combines algorithms from different companies and is accessible through the internet (http://i.cs.hku.hk/~sirna/ software/sirna.php). Nevertheless, any presumably optimal siRNA still requires extensive testing. This refers to a high targeting efficacy, which is, among others, also determined by variations in the accessibility of the target mRNA at different positions, as well as to the absence of any unwanted side effects. In fact, nonspecific silencing of genes due to only partial sequence homology has been described (Jackson et al. 2003) . Furthermore, in vivo some siRNA sequences, as well as longer dsRNA molecules, have been shown to activate the innate immune system leading to nonspecific effects due to the stimulation of inflammatory responses (Heil et al. 2004; Judge et al. 2005; Sioud 2005; Sledz et al. 2003) . This phenomenon seems to depend on the presence of GU-rich sequences as well as on the formulation and amount of siRNAs (Heidel et al. 2004; Ma et al. 2005; Sioud and Sorensen 2003) , and these aspects need to be considered for any therapeutic siRNA application in vivo.

Since the discovery of the pivotal role of siRNAs for inducing RNAi (Elbashir et al. 2001a, b) , the direct application of siRNA molecules has been explored in vitro and in vivo. In vitro, several transfection reagents allow the delivery of siRNAs in mammalian cells in the presence or absence of serum. The in vivo application of siRNAs, however, requires the development of more sophisticated formulations and/or the identification of optimal modes of administration (see below). Several proof-of-principle studies have shown the delivery of fluorophor-labeled siRNA molecules into various organs (see, e.g., Bradley et al. 2005a, b; Pirollo et al. 2006; Sioud and Sorensen 2003) . Beyond that, the specific in vivo knockdown of artificially introduced reporter genes like GFP or luciferase, or various endogenous target genes, has been described. The target organ was often the liver, but gene targeting in other organs, in other parts of the body, or in tumor xenografts has been reported as well (Table 1) . Taken together, these studies provide valuable insights into the delivery and efficacy of siRNAs for the induction of RNAi.

Beyond the detection of the downregulation of an endogenous target gene, the siRNA-mediated RNAi for therapeutical purposes has been explored. Target organs include liver, kidney, lung, eye, ear, heart, pancreas, tumors, blood, as well as the central nervous system, and the peritoneum (see Table 1 for an overview), using locally or systemically administered siRNAs in various formulations.

A large body of studies refers to the treatment of cancer with the primary goals being the inhibition of tumor growth. Target molecules usually represent genes that have been shown previously to be relevant or rate-limiting for tumor growth, including growth factors and receptors as well as antiapoptotic or downstream signal transduction proteins in tumor cells. Typically, these studies involve subcutaneous or orthotopic xenografts of different tumor entities in mice, and employ various strategies for local or systemic administration of a wide variety of siRNA formulations (see below). In some cases, the antiangiogenic effect after siRNA delivery to tumor endothelial cells, rather than an inhibitory effect on tumor cells, was explored (Santel et al. 2006a, b) , or simultaneous targeting of tumor growth and tumor angiogenesis both contributed to the antitumorigenic effects observed (Grzelinski et al. 2006 ). Additionally, in some studies the blockage of cancer metastasis, e.g., to the lung, liver or bone has been achieved. Taking into consideration that a large number of cancer patients die from metastases rather than the primary tumor, this represents another very relevant approach in cancer therapy.

Several studies focus on viral gene products, taking into consideration that options for protection or therapy through current antiviral drugs or vaccination strategies are rather limited. Thus, novel RNAi-based approaches to battle viral infections rely on the specific siRNA-mediated knockdown of virus-specific genes. Animal models infected with various viruses including hepatitis virus, influenza virus, respiratory syncytial virus (RSV), SARS corona viruses, or ebola virus have been employed, and primary goals were the reduction in virus titers and protective effects including, when lethal doses were applied, prolonged survival rates upon specific siRNA treatment.

Beyond studies related to cancer or viral infection, several target molecules with proven relevance in other pathologies have been selected. Examples include Fas in hepatitis, vascular endothelial growth factor (VEGF) in macular degeneration due to extensive ocular neovascularization or tumor necrosis factor alpha (TNF-α) in arthritis, and these studies aim at the establishment of novel, improved therapeutic avenues through siRNA-mediated gene silencing. As it can be seen from Table 1 , some target genes are relevant in more than one pathology (e.g., Fas in fulminant hepatitis, in renal ischemia-reperfusion injury or in hemorrhagic shock and sepsis in the lung; VEGF in age-related macular degeneration or in tumor growth/tumor angiogenesis; caspase-8 in liver failure or in sepsis), and thus, specific siRNAs may represent drugs applicable for the treatment of different diseases. Some studies also compare siRNA-based therapeutic strategies with already established drugs and discuss decreased side effects and/or higher specificity, or additive effects upon combination of both.

Other papers rather aim at the further elucidation of physiological processes through in vivo knockdown of a certain target gene. Examples include OATC3 in blood-brain barrier transport, V2R in water and sodium homeostasis, or HO-1 in lung ischemia-reperfusion injury (Table 1) .

It should be noted that, although the therapeutic potential of siRNAs has only been explored in the recent years, first siRNA-based drugs are already in clinical trials. This includes ALN-RSV01 (Alnylam Pharmaceuticals) for targeting the human RSV after viral infection, which is the first example of an antivirus siRNA-based therapeutic in a phase I clinical study. Other companies aiming at the development of RNAi therapeutics for viral diseases include Nastech/Galenea, which are expected to start clinical trials in 2007. Benitec, in collaboration with City of Hope in Duarte, California, has developed a multi-RNA therapeutic for treatment of AIDS lymphoma. Furthermore, age-related macular degeneration (AMD) was treated with Sirna-027 (Sirna Therapeutics) targeting the VEGF receptor VEGFR1 (Shen et al. 2006) , and resulted in stabilization or even improvement of visual acuity (Whelan 2005) . A 24month phase II study to evaluate multiple doses of Sirna-027 (also termed AGN211745) in the treatment of subfoveal choroidal neovascularization associated with AMD is currently recruiting patients. Targeting the ligand (VEGF) rather than its receptor, Cand5 (Acuity Pharmaceuticals) has been employed for treatment of the same disease, and in the so-called C.A.R.E™ trial showed no adverse effects related to the drug. Cand5, which is now named Bevasiranib, was the first siRNA to enter both phase I and II clinical trials. Recently, SR Pharma plc announced the start of a phase I clinical trial with RTP-801i, an siRNA therapeutic licensed from its subsidiary Atugen AG that targets a gene product involved in the progression of AMD. The same company has most recently announced that the FDA has approved an investigational new drug (IND) application for a second siRNA therapeutic, AKIi-5 being developed for the treatment of acute kidney injury. AKIi-5 is expected to reduce the frequency of postsurgery acute kidney injury in high-risk patients undergoing major cardiovascular surgery. Finally, after successful completion of experiments demonstrating its therapeutic efficacy in animal models of pancreatic cancer, Atu027 is scheduled to enter human clinical trials in 2007. In addition, several other pharmaceutical or biotechnology companies are pursuing collaborative or internal projects for the development of drugs based on RNAi (see, e.g., Behlke 2006 for review).

Advantages of the direct application of siRNAs, rather than DNA-based constructs coding for long dsRNA, include the relatively easy chemical synthesis of small RNA molecules, the lower probability of nonspecific side effects (see above) and the safety due to the fact that siRNA delivery is based on nonviral transfer strategies and siRNAs cannot integrate into the genome. On the other hand, successful siRNAbased gene targeting relies on several preconditions: protection of the rather instable siRNA molecules from nucleolytic degradation by serum nucleases, efficient cellular uptake and subsequent intracellular release into the cytoplasm, as well as the absence of intracellular immune responses, in vivo toxicity or rapid elimination in liver or kidney.

Strategies for the in vivo application of siRNA molecules include local as well as systemic modes of administration as detailed in Table 2 . However, many studies rely on the use of relatively high amounts of siRNAs. Bearing in mind that intracellular immune responses have been shown to be concentration-dependent, this may increase the risk of nonspecific effects in addition to other side effects and cost considerations. When siRNAs are administered locally, lower doses are sufficient since nonspecific delivery to other organs as well as renal or hepatic elimination are reduced. This approach, however, is invasive and limited to tissues that are sufficiently accessible. With regard to systemic application, several studies rely on the hydrodynamic transfection of siRNAs, i.e., the rapid (∼20 s) high-pressure injection of large volumes (up to 2 ml) of siRNA-containing solution. Hydrodynamic injection has led to the efficient induction of RNAi in liver as well as in kidney, lung, pancreas, and spleen and is probably due to the transient enhancement of membrane-permeability. However, in animals, side effects have been observed (Zhang et al. 2004a, b) , and in man, this method is not applicable at all.

Many groups have employed different approaches for the formulation of siRNAs in carrier systems (Table 3) , which deliver their siRNA "payload" into the target tissue and target cell, some of them already being known as DNA delivery techniques in gene therapy or antisense targeting.

Various liposomes/cationic lipids can be considered as examples of nonviral envelopes that protect siRNAs, thus increasing serum stability, reducing renal excretion and mediating siRNA uptake into the cells through endocytosis. The comparison of neutral versus cationic liposomes also reveals that the biodistribution as well as the uptake into macrophage seems to be dependent on their charge (Landen et al. 2005; Miller et al. 1998) , thus emphasizing the need for the further development and analysis of different liposomal particles. SNALPs (stable nucleic acid lipid particles) have been used for siRNA-mediated targeting of an Ebola-virus-specific gene (Geisbert et al. 2006) or ApoB. This is also the first study that describes the systemic efficacy of formulated siRNAs in a nonrodent species (Zimmermann et al. 2006) . Nanoparticles Another strategy allowing the protection and cellular delivery of siRNAs is the formation of nanoparticles with positively charged macromolecules. Based on electrostatic interactions, complexes are formed with atelocollagen (Banno et al. 2006; Minakuchi et al. 2004; Takei et al. 2004; Takeshita et al. 2005) , chitosan (Pille et al. 2006) , or polyethylenimine (PEI).

PEIs are synthetic linear or branched polymers available in a wide range of molecular weights (Godbey et al. 1999; Tang and Szoka 1997) . Due to the presence of a protonable amino group in every third position, leading to a high cationic charge density at physiological pH, PEIs are able to form noncovalent complexes with DNA as well as small RNA molecules like siRNAs (Urban- Klein et al. 2005) or ribozymes (Aigner et al. 2002) . This siRNA complexation results in the complete protection against degradation in the presence of serum or RNase A and allows the efficient cellular uptake of the PEI/siRNA complexes through endocytosis. For any siRNA formulation, the release from endosomes is critical for siRNA delivery. In the case of PEI, the so-called "proton-sponge effect" postulates improved transgene delivery by cationic complexes, which contain H + -buffering polyamines, based on enhanced endosomal Cl − accumulation and subsequent osmotic swelling and lysis (Behr 1997; Boussif et al. 1995) . This effect may also apply for PEI-mediated siRNA delivery into the cytoplasm. Additionally, to further enhance the efficacy of PEI complexes through membrane-destabilization, the conjugation of melittin analogs to PEI has been described (Boeckle et al. 2005 (Boeckle et al. , 2006 Shir et al. 2006) . It should be noted, however, that by far not all PEIs are suitable for the transport of nucleic acids like siRNAs (Hassani et al. 2005; Werth et al. 2006) . Table 2 In vivo application of siRNAs for the induction of RNAi: modes of administration of naked or formulated siRNAs

Hydrodynamic transfection Bradley et al. 2005a, b; Duxbury et al. 2004; Giladi et al. 2003; Hamar et al. 2004; Heidel et al. 2004; Hino et al. 2006; Klein et al. 2003; Lewis et al. 2002; Liang et al. 2005; Matsui et al. 2005; Merl et al. 2005; Sato et al. 2005; Song et al. 2003; Tompkins et al. 2004; Zender et al. 2003 Intravenous (without high pressure) Bradley et al. 2005a, b; Chien et al. 2005; Ge et al. 2004; Hassan et al. 2005; Miyawaki-Shimizu et al. 2005; Morrissey et al. 2005a, b; Schiffelers et al. 2004; Soutschek et al. 2004; Yano et al. 2004; Takeshita et al. 2005 Intraperitoneal Filleur et al. 2003 Flynn et al. 2004; de Jonge et al. 2005; Ocker et al. 2005; Sorensen et al. 2003; Verma et al. 2003; Urban-Klein et al. 2005; Yin et al. 2005 Intramuscular Golzio et al. 2005 Intratracheal Lomas-Neira et al. 2005 Perl et al. 2005 Intranasal Bitko et al. 2005 Zhang et al. 2004a , b Subretinal Reich et al. 2003 Intraocular Herard et al. 2005 Intradermal Kim et al. 2005 Subcutaneous Yano et al. 2004 Intrathecal Dorn et al. 2004 Luo et al. 2005 Stereotactic injection to hypothalamus Makimura et al. 2002 Infusion into the ventricular system (brain) Hassani et al. 2005; Thakker et al. 2004 Thakker et al. , 2005 Intrathecal infusion using miniosmotic pump Dorn et al. 2004 In situ perfusion/intravenous (pancreatic islet) Bradley et al. 2005a , b Intracardiac Bollerot et al. 2006 Intratumoral Bertrand et al. 2002 Ito et al. 2005; Leng and Local injection and electroporation (mouse joint) Schiffelers et al. 2005 In vivo studies in xenografted mice have demonstrated that the i.p. injection of PEI-complexed siRNAs, but not of naked siRNAs, resulted in the delivery of intact siRNA molecules into the subcutaneous tumors, leading to antitumorigenic effects when targeting for example the receptor HER-2 in s.c. ovarian carcinoma xenografts (Urban- Klein et al. 2005) or the growth factors pleiotrophin (PTN, s.c. glioblastoma) (Grzelinski et al. 2006) or VEGF (s.c. prostate carcinoma; Hobel et al., unpublished data) . Likewise, intrathecal delivery of PEI-complexed specific siRNAs led to successful PTN targeting and tumor inhibition in an ortotopic glioblastoma mouse model (Grzelinski et al. 2006) , or to the knockdown of the Nmethyl-D-aspartate (NMDA) receptor subunit protein NR2B in a rat model (Tan et al. 2005) . PEI complexation of siRNAs has also been employed in antiviral therapy studies (Ge et al. 2004; Geisbert et al. 2006) . Furthermore, PEI/ siRNA complexes are a good example for the introduction of chemical modifications to enhance tissue specificity and in vivo biocompatibility, to reduce immunogenicity and toxicity and to increase siRNA delivery through improved endocytosis and intracellular siRNA release. This includes full deacetylation of PEI (Thomas et al. 2005) , the introduction of novel low molecular weight PEIs (Werth et al. 2006) , and the coupling of PEI to other macromolecules like polyethylene glycol (PEG), either alone (Mao et al. 2006) or in combination with a ligand for tissuespecific targeting (RGD peptide for the recognition of tumor vasculature; Schiffelers et al. 2004 ).

Alternatively, the goals of increased systemic siRNA stability and serum half-life have also been achieved Bradley et al. 2005a, b; Duxbury et al. 2004; Giladi et al. 2003; Heidel et al. 2004; Hino et al. 2006; Klein et al. 2003; Lewis et al. 2002; Liang et al. 2005; Matsui et al. 2005; Merl et al. 2005; Sato et al. 2005; Song et al. 2003; Tompkins et al. 2004; Zender et al. 2003 Chemically modified, naked Braasch et al. 2004 Elmen et al. 2005; Soutschek et Schiffelers et al. 2004 through extensive chemical modifications of the siRNA strands including the introduction of phosphorothioate (Braasch et al. 2003 (Braasch et al. , 2004 , 4′ thioribose (Dande et al. 2006) or methylene linkages between positions 2′ and 4′ (locked nucleic acids, LNAs; Braasch et al. 2003; Elmen et al. 2005) , or multiple 2′ modifications (Amarzguioui et al. 2003; Harborth et al. 2003; Holen et al. 2002 Holen et al. , 2003 . Additionally, chemical conjugation of siRNAs, e.g., to cholesterol (Soutschek et al. 2004) or to a protamin-antibody fusion protein led to enhanced efficacy and specificity in tissue uptake. For other strategies, refer to Table 3 .

RNAi has already proven to be a very efficient and specific method for the knockdown of physiologically or pathologically relevant genes of interest. Notably, this also applies to so-called "nondruggable" genes, thus opening new therapeutic avenues. Still, the therapeutic applicability and success of siRNAs will largely depend on their efficient and safe in vivo delivery avoiding unwanted side effects.

Reflecting the high relevance of RNAi, many studies have been published or are ongoing, which will finally allow to identify optimal strategies based on already promising results. This also refers to the first clinical trials, which are completed or ongoing. Most likely, one major advantage of formulated, modified, or unmodified siRNAs for gene knockdown will be their "double specificity", i.e., the combination of a high target gene specificity through optimal siRNA sequences and an at least somewhat increased target organ specificity through sophisticated delivery vehicles like liganded nanocarriers.

Transcriptional and phenotypic comparisons of Ppara knockout

Delivery of unmodified bioactive ribozymes by an RNAstabilizing polyethylenimine (LMW-PEI) efficiently down-regulates gene expression

Tolerance for mutations and chemical modifications in a siRNA

Ex vivo and in vivo delivery of anti-tissue factor short interfering RNA inhibits mouse pulmonary metastasis of B16 melanoma cells

Controlled release of small interfering RNA targeting midkine attenuates intimal hyperplasia in vein grafts

Progress towards in vivo use of siRNAs

The proton sponge: a trick to enter cells the viruses did not exploit

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

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

Inhibition of respiratory viruses by nasally administered siRNA

C-versus N-terminally linked melittin-polyethylenimine conjugates: the site of linkage strongly influences activity of DNA polyplexes

Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes

Widespread lipoplex-mediated gene transfer to vascular endothelial cells and hemangioblasts in the vertebrate embryo

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

RNA interference in mammalian cells by chemicallymodified RNA

Biodistribution of phosphodiester and phosphorothioate siRNA

Successful incorporation of short-interfering RNA into islet cells by in situ perfusion

Gene silencing in the endocrine pancreas mediated by shortinterfering RNA

Induction of an interferon response by RNAi vectors in mammalian cells

Novel cationic cardiolipin analoguebased liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo

Structural domains in RNAi

Improving RNA interference in mammalian cells by 4′-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2′-O-alkyl modifications

Reconstituted influenza virus envelopes as an efficient carrier system for cellular delivery of small-interfering RNAs

Rad51 siRNA delivered by HVJ envelope vector enhances the anticancer effect of cisplatin

Expression profiling reveals off-target gene regulation by RNAi

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

Efficient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor alpha in experimental arthritis

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

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

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

Intraperitoneal delivery of liposomal siRNA for therapy of advanced ovarian cancer

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

Silencing of CXCR4 blocks breast cancer metastasis

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

Argonaute2 is the catalytic engine of mammalian RNAi

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

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

Cationic lipids enhance siRNA-mediated interferon response in mice

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

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

vivo potentialities of EWS-Fli-1 targeted antisense oligonucleotidesnanospheres complexes

Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi

Sequencespecific suppression of mdr1a/1b expression in mice via RNA interference

RNA interference in adult mice

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

Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes

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

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

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

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

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

ATP requirements and small interfering RNA structure in the RNA interference pathway

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

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

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

Intravenous delivery of antiRhoA small interfering RNA loaded in nanoparticles of chitosan in mice: safety and efficacy in xenografted aggressive breast cancer

Tumor-targeting nanoimmunoliposome complex for short interfering RNA delivery

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

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

Purified Argonaute2 and an siRNA form recombinant human RISC

A novel siRNAlipoplex technology for RNA interference in the mouse vascular endothelium

RNA interference in the mouse vascular endothelium by systemic administration of siRNA-lipoplexes for cancer therapy

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

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

Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis

Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1

EGF receptor-targeted synthetic double-stranded RNA eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice

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

Cationic liposome-mediated delivery of siRNAs in adult mice

Activation of the interferon system by short-interfering RNAs

RNA interference targeting Fas protects mice from fulminant hepatitis

Antibody mediated in vivo delivery of small interfering RNAs via cellsurface receptors

Gene silencing by systemic delivery of synthetic siRNAs in adult mice

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

Exploring RNA interference as a therapeutic strategy for renal disease

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

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

Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo

Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat

The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes

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

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

Protection against lethal influenza virus challenge by RNA interference in vivo

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

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

A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes

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

First clinical data on RNAi

Single-walled carbon nanotubesmediated in vivo and in vitro delivery of siRNA into antigenpresenting cells

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

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

Caspase 8 small interfering RNA prevents acute liver failure in mice

Hydroporation as the mechanism of hydrodynamic delivery

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

Small interfering RNAs targeting mutant K-ras inhibit human pancreatic carcinoma cells growth in vitro and in vivo

RNAi-mediated gene silencing in nonhuman primates

Acknowledgments The author's work reported herein was supported by grants from the Deutsche Forschungsgemeinschaft (AI 24/5-1 and Forschergruppe Nanohale, AI 24/6-1) and by the Deutsche Krebshilfe.