Gene silencing through RNA interference (RNAi) in vivo: Strategies based on the direct application of siRNAs

Journal of Biotechnology 124 (2006) 12–25

Review

Gene silencing through RNA interference (RNAi) in vivo: Strategies based on the direct application of siRNAs Achim Aigner ∗ Department of Pharmacology and Toxicology, Philipps-University Marburg, Karl-v.-Frisch-Strasse 1, D-35033 Marburg, Germany Received 8 July 2005; received in revised form 25 October 2005; accepted 1 December 2005

Abstract RNA interference (RNAi) offers great potential not only for in vitro target validation, but also as a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene, e.g. in tumor therapy. Since it relies on small interfering RNAs (siRNAs), which are the mediators of RNAi-induced specific mRNA degradation, a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on (viral) vector delivery may be of only limited clinical use. The more desirable approach is to directly apply catalytically active siRNAs. This review highlights the recent knowledge on the guidelines for the selection of siRNAs which show high activity in the absence of non-specific siRNA effects. It then focuses on approaches to directly use siRNA molecules in vivo and gives a comprehensive overview of in vivo studies based on the direct application of siRNAs to induce RNAi. One promising approach is the in vivo siRNA delivery through complexation of chemically unmodified siRNAs with polyethylenimine (PEI). The anti-tumoral effects of PEI/siRNA-based targeting of tumor-relevant genes in vivo are described. © 2005 Elsevier B.V. All rights reserved. Keywords: RNAi; siRNAs; In vivo siRNA delivery; Polyethylenimine; PEI; Gene-silencing

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of RNAi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-specific siRNA effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RNAi in vivo through direct application of siRNA molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: GFP, green fluorescent protein; HER-2, human epidermal growth factor receptor-2; PEI, polyethylenimine; PTN, pleiotrophin; RISC, RNA-induced silencing complex; RNAi, RNA interference; s.c., subcutaneous; siRNA, small interfering RNA ∗ Tel.: +49 6421 286 2262; fax: +49 6421 286 5600. E-mail address: [email protected]. 0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.12.003

A. Aigner / Journal of Biotechnology 124 (2006) 12–25

5. 6. 7.

In vivo siRNA delivery through PEI complexation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anti-tumoral effects of PEI-complexes siRNAs targeting the tumor-relevant genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The analysis and understanding of gene function has enormously benefited from functional genomics approaches including the use of antibodies, low molecular weight pharmacological drugs and gene-targeting techniques. Over the past two decades, the latter have included the use of antisense oligonucleotides and ribozyme-targeting as well as, most recently, RNA interference (RNAi). The specific inhibition of gene expression through antisense oligonucleotides (ODN) was first introduced in 1978 (Stephenson and Zamecnik, 1978; Zamecnik and Stephenson, 1978) and further explored since the mid-1980s when certain backbone modifications were established to increase the activity and stability of ODNs. Furthermore, techniques for packaging and delivery of the net negatively charged ODN compounds to cells in culture were developed including the use of cationic and anionic lipid formulations. While some recent randomized trials which turned out unsuccessful have dampened enthusiasm for antisense ODN therapeutics, several antisense ODN compounds are still in late-stage preclinical or clinical development (see Gleave and Monia, 2005 for review). The discovery of catalytically active RNAs (ribozymes) in the early 1980s (Cech et al., 1981; Kruger et al., 1982; Guerrier-Takada et al., 1983) showed for the first time that RNA molecules, beyond their role in the transfer of genetic information, can act as enzymes independent of the presence of proteins. Since these enzymatic functions of ribozymes include the breaking of covalent bonds in RNA molecules with high sequence specificity, the development of ‘transacting’ ribozymes like, e.g., the hammerhead ribozyme derived from the structure introduced by Haseloff and Gerlach (1988) with simplified catalytic motifs in the late 1980s and early 1990s represented a second step in nucleic acid-based gene-targeting. The therapeutic use of ribozymes, however, meets several problems including poor delivery into the target tissue/target cell as well

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as their marked instability. In fact, the stability of allRNA ribozymes in human serum is less than 6 s due to their rapid degradation (Jarvis et al., 1996). Attempts to circumvent these problems include extensive chemical modifications of the ribozyme backbone or the delivery of plasmid or viral vectors with subsequent intracellular expression of ribozymes. While numerous applications of ribozymes for the specific downregulation of the expression of a target gene in cultivated cells have been described, in vivo and therapeutic applications are very limited. Most recently, RNA interference (RNAi) has been introduced as a potent naturally occurring biological strategy for gene silencing. Initially, Fire et al. (1998) identified double-stranded RNA molecules (dsRNA) as the mediator of gene silencing in C. elegans. These studies also explained earlier findings in the same organism on gene silencing by antisense as well as by sense oligonucleotides (Guo and Kemphues, 1995), and on gene-silencing in plants (Jorgensen, 1990). Subsequently, it turned out that RNAi is not restricted to any specific organism but rather, while initially described under different names (post-transcriptional gene silencing, PTGS, quelling, co-suppression), appears to be present in most eukaryotic organisms.

2. Mechanism of RNAi RNA interference (see Fig. 1) is mediated by small interfering RNAs (siRNAs) which are intracellularly generated from long endogenous or exogenous double-stranded RNA molecules (dsRNAs) through the cleavage activity of a ribonuclease III-type protein (Hamilton and Baulcombe, 1999; Zamore et al., 2000; Bernstein et al., 2001; Elbashir et al., 2001b). More specifically, the protein Dicer which typically contains an N-terminal RNA helicase domain, an RNA-binding so-called Piwi/Argonaute/Zwille (PAZ) domain, two RNase III domains and a double-stranded RNA bind-

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Fig. 1. Mechanism of RNA interference (RNAi) in mammalian systems.

ing domain (dsRBD) (see Collins and Cheng, 2005 for review), leads to the formation of short 21–23 nt duplexes with a symmetric 2 nt overhang at the 3 -end and a 5 -phosphate and 3 -hydroxy group referred to as siRNAs. This siRNA molecule is incorporated into a nuclease-containing multi-protein complex called RNA-induced silencing complex (RISC) (Hammond et al., 2000) which becomes activated (RISC* ) upon the loss of one strand of the siRNA duplex by an RNA helicase activity (Nykanen et al., 2001). By binding of the now single-stranded siRNA to a complementary target RNA molecule, it sequence-specifically guides the RISC complex to the target RNA and induces endonucleolytic cleavage of the mRNA strand within the target

site. Due to the generation of unprotected RNA ends, this cleavage leads to the rapid degradation of the entire mRNA molecule while the RISC complex is recovered for further cleavage cycles. From this mechanism it becomes clear that the intracellular presence of siRNA molecules complementary to the target mRNA is of critical importance for the induction of RNAi. Consequently, several studies have focussed on the optimization of siRNAs with regard to target specificity and targeting efficacy as well as on the delivery of siRNAs in vitro and in vivo. Early studies led to the development of a set of guidelines for the selection of siRNAs appropriate

A. Aigner / Journal of Biotechnology 124 (2006) 12–25

for a given sequence (Elbashir et al., 2001b, 2002) mainly based on structural attributes like the optimal length (19–25 bp), the requirement of a 3 dinucleotide overhang (Elbashir et al., 2001c) and a low G/C content ranging between 36% and 52% (Elbashir et al., 2002; Holen et al., 2002). More recent studies further refined these selection criteria showing that the thermodynamic flexibility of the duplex 3 -end (i.e. positions 15–19, sense strand), but not of the 5 -end, correlates with silencing efficacy and that the presence of at least one A/U bp in this region, which decreases the internal stability of the 3 -end, increased silencing efficacy (Reynolds et al., 2004). Furthermore, internal repeats or palindrome sequences which favor the formation of internal fold-back structures reduce the concentration of functional siRNAs and hence the silencing potential. Further criteria which enhance the silencing potency include an A in position 3 and a G at position 13 of the sense strand as well as the absence of a G or C at position 19; however, the data also suggest that additional discriminants for functional duplex selection are still to be discovered and defined (Reynolds et al., 2004). Among the sequence-specific criteria, however, the greatest individual impact on siRNA efficacy has a U in position 10 of the sense strand and hence at the site of the RISC-mediated cleavage of the target mRNA (nucleotides 10–11) indicating that RISC, like most endonucleases (DonisKeller, 1979) preferentially cleaves 3 of U rather than any other nucleotide (Reynolds et al., 2004). From several studies it is suggested that the siRNA efficacy is determined by siRNA-specific properties rather than by target mRNA properties. Other data, however, also show possible correlations between siRNA efficacy and target mRNA secondary structure and accessibility (Bohula et al., 2003; Holen et al., 2002; Lee et al., 2002; Ding and Lawrence, 2003; Vickers et al., 2003; Xu et al., 2003; Kretschmer-Kazemi Far and Sczakiel, 2003). Several criteria have been incorporated into computational siRNA design tools, some of which being accessible to the public and allowing the design of siRNAs for any given target sequence. Since the desired high potency of any designed siRNA, however, is still hard to fully predict from these criteria, and to enhance RNAi efficacy, many scientists tend to switch to pools of siRNAs, which are synthetically produced or made by recombinant Dicer, rather than using a single siRNA.

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Furthermore, most recently it was also shown that longer synthetic siRNA duplexes (25–30 nucleotides) can be up to 100-fold more potent as compared to 21 bp siRNAs since they represent a substrate of Dicer directly linking the production of siRNAs to their incorporation into the RISC complex (Kim et al., 2005a). In addition to RNAi, small dsRNAs were demonstrated to be also involved in RNA-directed DNA methylation (RdDM), which operates at the level of the nuclear genome and was the first described RNA-guided epigenetic modification of the genome (see Kawasaki and Taira, 2005; Matzke and Birchler, 2005; Mathieu and Bender, 2004 for review). Initially, RdDM was discovered and extensively studied in plants revealing that small RNAs with sequence homology to promoter regions can trigger promoter methylation and transcriptional gene silencing (see Matzke and Birchler, 2005 for review). However, some of the components of the RdDM machinery identified to be involved in plants are present also in mammals. While reports regarding RdDM in mammalian cells are somewhat conflicting so far, two recent studies in fact show that siRNAs sequence-specifically induce DNA methylation and transcriptional gene silencing in human cells (Kawasaki and Taira, 2004; Morris et al., 2004). Thus, the critical importance of siRNAs in both mechanisms has established a link between RNAi and RdDM.

3. Non-specific siRNA effects The sequence-specific binding of the siRNA to its target mRNA is required for the nucleolytic activity of the RISC complex and predicts a high sequence specificity of RNA interference. However, this specificity is questioned by studies which show off-target effects indicating non-specific RNAi-induced gene silencing upon the introduction of a gene-specific siRNA. Thus, siRNAs may cross-react with targets of limited sequence similarity when regions of partial sequence identity between the target mRNA and the siRNA exist. In fact, in some cases regions comprising of only 11–15 contiguous nucleotides of sequence identity were sufficient to induce gene-silencing (Jackson et al., 2003). However, the prediction of these off-target activities is difficult so far.

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Finally, an issue that needs to be considered especially for the in vivo application of RNAi is the activation of the interferon system leading to non-specific effects (Hornung et al., 2005; Sledz et al., 2003; Bridge et al., 2003; Elbashir et al., 2001a). The interferon system is induced when double-stranded RNA molecules, derived for example from viral replication or viral gene expression, activate a multi-component signalling complex. Hence, it represents the first line of defence against viral infection (Stark et al., 1998). The detection of long dsRNA may occur through the serinethreonine kinase PKR (Katze et al., 1991; Meurs et al., 1992; Williams, 2001) or the Toll-like receptor 3 (TLR3) (Alexopoulou et al., 2001) and lead to the production of type I interferon (IFN-alpha and IFN-beta) especially in plasmocytoid dendritic cells (PDC, also called interferon-producing cell, IPC). Subsequently, this results in the upregulation of many interferoninduced genes and/or inhibition of cellular protein synthesis and induction of apoptosis. While this effect is particularly true for long dsRNA molecules essentially preventing them from being used as inducers of RNA interference in mammalian systems, the development of synthetic siRNAs (Zamore et al., 2000; Caplen et al., 2001; Elbashir et al., 2001a,b) largely circumvents this problem since they seem to be too small. Nevertheless, some studies demonstrate the induction of components of the interferon system by showing significant non-specific changes in gene expression as a consequence of the delivery of siRNAs (Sledz et al., 2003; Bridge et al., 2003; Judge et al., 2005). However, only some synthetic siRNAs tested caused these effects indicating that the ability to induce the interferon system depends on the siRNA sequence (Sledz et al., 2003; Bridge et al., 2003) as well as, in the case of in vitro transcribed siRNAs, on the 5 initiating triphosphate (Kim et al., 2004). Furthermore, it was shown more recently that, through TLR7, certain siRNA sequences were potent inducers of IFN-alpha in PDC (Hornung et al., 2005), but not in other cell lines (Kim et al., 2004), indicating that synthetic siRNAs represent an excellent tool for sequence-specific downregulation of target genes in cell lines vitro, but that immunostimulation by siRNAs needs to be considered whenever PDC or other TLR7-expressing cells are present in vitro or in vivo. While this effect is independent of the GU content of the siRNAs, it was shown to rely on the administration of si-

RNAs in the presence of transfection reagents as nonviral delivery systems (Ma et al., 2005; Judge et al., 2005), and immunostimulatory RNA motifs (e.g., 5 UGUGU-3 ) were identified allowing the dissection of immunostimulatory and target silencing effects of a given siRNA molecule (Judge et al., 2005; Hornung et al., 2005). Hence, for the application of siRNAs in vivo in mammalian system, the (unwanted) interferon response needs to be kept in mind and may be avoided or at least reduced by (i) a rational design of siRNAs aiming at selecting non-stimulating siRNA sequences, (ii) using the lowest siRNA dose effective for targeting, (iii) optimizing siRNA delivery methods regarding decreased interferon induction, and (iv) defining acceptable levels of interferon system activation.

4. RNAi in vivo through direct application of siRNA molecules Despite these siRNA specificity and efficacy issues, the therapeutic potential of siRNAs for the treatment of various diseases is in principle very promising. This is particularly true for disorders that are based on the expression of mutant genes including dominantnegative proteins, of aberrant splicing isoforms or overexpressed genes leading to gain-of-function effects. However, limitations of transfer vectors may turn out to be the rate-limiting step in the development of RNAi-based therapeutic strategies. Approaches to solve these problems include the use of DNA vectors encoding double-stranded RNA. In this case, a partially palindromic hairpin loop mRNA with the desired sequence is expressed from a plasmid. The double-stranded RNA segments are then recognized and cleaved by Dicer. In principle, these DNA-based systems offer the advantage of constitutive expression of siRNAs with a potentially higher level and unlimited duration of gene silencing, and allow the incorporation of regulatory elements to the promoter region of the expression plasmid resulting in tissue-specific silencing. On the other hand, however, they are still based on the viral or non-viral delivery of the DNA and present problems similar to those of gene therapy including safety concerns and heightened regulatory scrutiny. Hence, the direct use of siRNA molecules may offer advantages and may be preferable over DNA-based

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strategies. However, the instability of siRNA molecules in vivo as well as their often poor uptake into target tissues/target organs remains an important issue and a major obstacle. Obviously, the stabilization/protection of siRNAs as well as the development of efficient methods for in vivo siRNA delivery is required. More recently, several in vivo studies have been published based on high-pressure, large-volume tail vein injections which allow the systemic delivery of siRNAs, local delivery, e.g., to the lungs, the use of cationic liposomes, polyplexes, modified siRNAs and/or electroporation. Table 1 gives an extensive overview summarizing siRNA-based in vivo proof-ofprinciple studies as well as papers which describe the targeting of various disease-relevant genes. Many systems, however, show major disadvantages such as -

relying on very high amounts of siRNAs, only allowing local rather than systemic treatment, being restricted to a site-specific injection, and/or requiring extensive chemical modifications of the siRNA molecules.

Thus, several approaches are unlikely to be useful in humans or will only allow the targeting of genes in a very restricted number of organs which may not be relevant for certain diseases. E.g., in previous studies it was shown that upon intravenous injection of siRNAs the liver is the primary site of siRNA uptake (Zhang et al., 1999; Lewis et al., 2002), certainly identifying liver diseases as an optimal system for studying therapeutic siRNA effects. For other diseases, however, the development of more generally applicable delivery platforms is required.

5. In vivo siRNA delivery through PEI complexation One recently described method for the efficient protection and delivery of siRNAs in vitro and in vivo relies on polyethylenimine complexation. Polyethylenimines (PEIs) are synthetic linear or branched polymers which are available in molecular weights ranging from <1 kDa to >1000 kDa. They possess a protonable amino group in every third position and a high cationic charge density (Boussif et al., 1995; Behr, 1997) which allows them to form non-covalent interpolyelectrolyte com-

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plexes with DNA (Boussif et al., 1995). These small colloidal particles are efficiently taken up by cells through endocytosis and, intracellularly, buffer the low endosomal pH. Due to the so-called ’proton-sponge effect’ leading to enhanced influx of protons and water, endosomes eventually burst and complexes are released into the cytoplasm. On the basis of this mechanism, certain PEIs have been introduced as transfection reagents in a variety of cell lines and in animals for DNA delivery (for review, see Kichler, 2004; Wagner et al., 2004 and references therein). In general, low molecular weight PEIs (<25 kDa) with a branched rather than linear structure are superior for gene transfer, and higher molecular weights lead to increased cytotoxicity (Fischer et al., 2003). Besides the degree of branching and their molecular weight, the DNA/PEI ratio (defined as nitrogen/phosphorus (N/P) ratio) is critical for the PEI’s transfection efficacy and lack of cytotoxicity. More recently, it was shown that in addition to their use as DNA transfection reagents, PEIs also allow the complexation of RNA molecules like ribozymes (Aigner et al., 2002) or siRNAs (Urban-Klein et al., 2005; Grzelinski et al., submitted for publication). In fact, PEI complexation of siRNA molecules, comparable to DNA, leads to the formation of stable and uniformly sized particles as analyzed by atomic force microscopy (AFM). These complexes completely cover the siRNAs and are sufficiently large for endocytotic uptake (Grzelinski et al., submitted for publication). More importantly, upon PEI complexation siRNAs are efficiently protected against nucleolytic degradation both in vitro in the presence of RNase and in vivo in the presence of serum nucleases. In tissue culture, PEI/siRNA complexes are internalized by tumor cells within a few hours leading to the intracellular release of siRNA molecules which display full bioactivity. In vivo, upon intraperitoneal or subcutaneous injection of PEI/siRNA complexes into athymic nude mice, but not upon similar treatment with noncomplexed siRNAs, intact radioactively labeled siRNA molecules are detected in several organs including subcutaneous tumors, muscle, liver, kidney and, to a smaller extent, lung and brain. Notably, no siRNAs are found in the blood indicating that despite the use of nonperfunded organs the detected signals are derived from siRNA molecules actually internalized by the cells of the respective target organ (Urban-Klein et al., 2005).

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Table 1 Overview of in vivo studies based on the direct application of siRNAs to induce RNAi Target tissue

Delivery route

siRNA formulation

Target gene(s)

Aim of study/disease

Reference

S.c. HeLa xenograft Liver

Intratumoral Intravenous (hydrodynamic transfection) Local into the liver

Cytofectin GSV Unmodified

GFP GFP

Downregulation of GFP Downregulation of GFP

Bertrand et al. (2002) Lewis et al. (2002)

Unmodified

Luciferase

McCaffrey et al. (2002)

Unmodified Unmodified

VEGF HBsAg

Unmodified

HBsAg

Inhibition of HBV replication

Klein et al. (2003)

Eye Various

Intraperitoneal Intravenous (hydrodynamic transfection) Intravenous (hydrodynamic transfection) Subretinal Intravenous/intraperitoneal

Downregulation of cotransfected luciferase Tumor growth inhibition Inhibition of HBV replication

Unmodified Liposomes

VEGF –

Reich et al. (2003) Sioud and Sorensen (2003)

Liver Various

Intravenous pulse injection Intraperitoneal

Unmodified Liposomes

Fas TNF-␣

Peritoneal cavity Liver

Intraperitoneal Intravenous (hydrodynamic transfection) Intrathecal

Liposomes Unmodified

␤-catenin Caspase-8

Unmodified

Intravenous

Unmodified

Cation channel P2X3 CEACAM6

Ocular neovascularization Detection of FITC-labeled siRNA Fulminant hepatitis Sepsis after lipopolysaccharide injection Tumor growth inhibition Fas-mediated apoptosis/acute liver failure Chronic neuropathic pain

Intraperitoneal Intravenous

Liposomes PEI complexation

Intravenous (renal vein hydrodynamic transfection) Intravenous high or low pressure Testes

Unmodified

IL-12p40 Influenza virus genes Fas

Unmodified

Liver Fibrosarcoma xenografts Liver Liver

Pancreatic adenocarcinoma xenograft Peritoneal cavity Lung Kidney Liver Orthotopic germ cell tumor xenograft S.c. neuroblastoma xenograft Liver and jejunum S.c. prostate carcinoma xenograft Lung S.c. prostate carcinoma xenograft Liver metastasis Lung

Song et al. (2003) Sorensen et al. (2003) Verma et al. (2003) Zender et al. (2003) Dorn et al. (2004)

Tumor growth inhibition

Duxbury et al. (2004)

Inflammation Influenza virus infections

Flynn et al. (2004) Ge et al. (2004) Hamar et al. (2004)

Fas

Renal ischemia-reperfusion injury Fas downregulation in liver

Atelocollagen

HST-1/FGF-4

Tumor growth inhibition

Minakuchi et al. (2004)

Intravenous Intravenous

RGD-PEG-PEI Chemically modified

VEGF-R2 ApoB

Schiffelers et al. (2004) Soutschek et al. (2004)

Intratumoral Intravenous (hydrodynamic transfection) Subcutaneous Intravenous Intranasal

Atelocollagen Unmodified

VEGF Nucleoprotein, acidic polymerase bcl-2 bcl-2 HO-1

Tumor growth inhibition Reduction of apoB and total cholesterol Tumor growth inhibition Influenza virus infections Tumor growth inhibition Metastasis growth inhibition Functional analysis in lung ischemia-reperfusion injury

Yano et al. (2004) Yano et al. (2004) Zhang et al. (2004)

Liposomes Liposomes Unmodified

Heidel et al. (2004)

Takei et al. (2004) Tompkins et al. (2004)

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Brain

Filleur et al. (2003) Giladi et al. (2003)

Intranasal

Pancreatic islet

In situ perfusion/intravenous

S.c. breast cancer xenografts

Intravenous

Muscle Kidney

Intramuscular Intravenous

Brain S.c. HeLa xenografts

Intra-cerebroventricular Intratumoral

Antigen-presenting cells Breast tumor xenograft

Intradermal Intratumoral

Metastatic breast cancer cells

Unmodified or TransIT-TKO Unmodified Cationic cardiolipin analogue Electropulsation Liposomes

RSV-P, PIV-P

Respiratory viral diseases

Bitko et al. (2005)

–

Detection of fluorescing siRNA Tumor growth inhibition

Bradley et al. (2005)

Downregulation of GFP Role of V2R in water/sodium homeostasis Downregulation of luciferase Enhancement of cisplatin anticancer effect Cancer vaccine potency Breast cancer

Golzio et al. (2005) Hassan et al. (2005)

Blockage of breast cancer metastasis Anti-apoptosis in retinal ganglion cells Acute lung injury Hearing loss Coxsackieviral cytopathogenicity Tumor growth inhibition Inhibition of tumor growth

Liang et al. (2005)

Downregulation of target genes Collagen-induced arthritis

Sato et al. (2005)

c-raf GFP V2R

Intravenous

JetSI + DOPE Inactivated HVJ suspension Unmodified Histidine-lysine complex Unmodified

CXCR4

Optic nerve stump

Local injection

Unmodified

c-Jun, Bax, Apaf-1

Lung Ear Coxsackievirus/various organs

Intratracheal instillation Local Intravenous (hydrodynamic transfection) Transurethral Systemic

Unmodified Liposomes Unmodified

KC, MIP-2 GJBR75W CVB 2A

Liposomes Cationic cardiolipin liposomes Unmodified

PLK-1 Raf-1

Unmodified

TNF␣

Protamin-antibody fusion protein Unmodified

c-myc, MDM2, VEGF TGF-␤1

Tumor growth inhibition

Song et al. (2005)

Glomerulonephritis

Takabatake et al. (2005)

PEI complexation

HER-2

Tumor growth inhibition

Urban-Klein et al. (2005)

Bladder cancer Prostate cancer xenografts Liver and limb grafts Mouse joint S.c. melanoma xenografts Kidney S.c. ovarian carcinoma xenografts

Intravenous (hydrodynamic transfection) Local injection and electroporation Intravenous or intratumoral Renal artery and electroporation Intraperitoneal

Luciferase Rad51

Chien et al. (2005)

Bak, Bax Raf-1

DsRed2, GFP

Hassani et al. (2005) Ito et al. (2005) Kim et al. (2005b) Leng and Mixson (2005)

Lingor et al. (2005) Lomas-Neira et al. (2005) Maeda et al. (2005) Merl et al. (2005) Nogawa et al. (2005) Pal et al. (2005)

Schiffelers et al. (2005)

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Nose after viral infection

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6. Anti-tumoral effects of PEI-complexes siRNAs targeting the tumor-relevant genes The biological efficacy of this PEI/siRNA complexation was demonstrated in vivo in different mouse tumor models by targeting different tumor-relevant proteins including the HER-2 (neu/c-erbB-2) receptor and the growth factor pleiotrophin (PTN). The HER-2 proto-oncogene belongs to the epidermal growth factor (EGF) receptor family with heterodimeric, HER-2 containing receptor combinations showing superior signal-transducing, anti-apoptotic and cell growth-stimulating capabilities (Tzahar et al., 1996). HER-2 overexpression has been observed in a wide variety of human cancers and cancer cell lines, and has been generally linked to an unfavorable prognosis and more aggressive malignant behavior of tumors (e.g., Slamon et al., 1989). The humanized monoclonal anti-HER-2 antibody trastuzumab (Herceptin (R)) has been approved for the adjuvant treatment of advanced breast and ovarian cancer (McKeage and Perry, 2002 for review, Bookman et al., 2003) and several low molecular weight inhibitors are being developed and/or in clinical studies emphasizing the clinical relevance of HER-2 targeting. In several studies, the relevance of HER-2 (over-)expression on tumor growth has been further established by gene-targeting approaches including ribozymes (Juhl et al., 1997; Thybusch-Bernhardt et al., 2001; Suzuki et al., 2000; Czubayko et al., 1997). In addition, more recently synthetic HER-2 siRNAs were shown in vitro to reduce HER-2 expression resulting in growth inhibition or apoptosis and upregulation of HLA class I expression in different tumor cell lines (Choudhury et al., 2004). In another work, HER-2 silencing upon retrovirus-mediated siRNA transfer led to slower proliferation, increased apoptosis and changes in cell cycle-associated and pro-/anti-angiogenic factors in breast and ovarian cancer cell lines in vitro. Upon subcutaneous (s.c.) injection of these in vitro pretreated cells, decreased tumor growth was observed (Yang et al., 2004). In summary, these data identify HER-2 as an attractive candidate target molecule in the development of anti-tumoral strategies based on RNAi-mediated gene-silencing. Pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) is a secreted growth factor which shows mitogenic, chemotactic, angiogenic and transforming activity (Fang et

al., 1992; Bowden et al., 2002; Wellstein et al., 1992; Milner et al., 1989; Czubayko et al., 1994; Schulte et al., 1996; Choudhuri et al., 1997). While PTN expression is tightly regulated during embryogenesis and very limited in normal adult tissues, a marked PTN upregulation is seen in several human tumors including breast cancer (Zhang et al., 1997), testicular cancer (Aigner et al., 2003), lung cancer (Jager et al., 1997), melanoma (Czubayko et al., 1996), meningiomas (Mailleux et al., 1992), neuroblastomas (Nakagawara et al., 1995), prostate cancer (Fang et al., 1992), pancreatic cancer (Klomp et al., 2002) and choriocarcinoma (Schulte et al., 1996). In previous studies, gene-targeting of PTN or of the PTN receptors, ALK and RPTPzeta, indicated a contribution of PTN-activated signaling pathways in various tumors including melanomas (Czubayko et al., 1996; Aigner et al., 2002), pancreatic cancer (Weber et al., 2000) and glioblastomas (Grzelinski et al., 2005; Powers et al., 2002; Muller et al., 2003). The application of PEI-complexed siRNAs targeting the HER-2 receptor resulted in HER-2 downregulation in SKOV-3 ovarian carcinoma cells in vitro, while PEI-complexed, non-specific siRNAs or naked, HER-2-specific siRNAs had no effect. Moreover, systemic treatment of mice bearing subcutaneous ovarian carcinoma xenografts through intraperitoneal injection of HER-2-specific PEI/siRNA complexes led to siRNA-mediated HER-2 downregulation on mRNA and protein levels. This was paralleled by a marked reduction of tumor growth (Urban-Klein et al., 2005). Likewise, in vitro treatment of U87 glioblastoma cells with PEI-complexed PTN-specific siRNAs resulted in a robust PTN reduction leading to decreased proliferation and soft agar colony formation. Again, more importantly, in vivo treatment of nude mice through systemic application (subcutaneous or intraperitoneal) of PEI-complexed PTN siRNAs significantly inhibited the s.c. tumor growth of U87 glioblastoma cells. The same was true in a clinically more relevant orthotopic mouse glioblastoma model with U87 cells growing intracranially and treatment of mice through intrathecal injection of PEI/siRNA complexes (Grzelinski et al., submitted for publication). Hence, the complexation of chemically unmodified siRNAs with polyethylenimines proves to be a highly efficient and ease-to-use system for the in vivo application of siRNAs. For clinical use of siRNAs, this PEI-based non-viral delivery method may represent a

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potentially powerful tool to achieve maximal function of siRNAs in vivo. 7. Conclusion Several delivery methods for siRNAs are currently under investigation. However, their ultimate success will depend on many very relevant parameters including (i) siRNA protection, (ii) transfection efficacy, (iii) toxicity and absence of non-specific effects, (iv) efficacy also when using small amounts of siRNAs, (v) applicability in various treatment regimens and in various diseases as well as (vi) the ability of the transfer vectors to overcome numerous biological barriers after systemic or local administration to reach their target tissue/organ. Taking these aspects into consideration, polyethylenimine complexation may represent a promising avenue for therapeutic siRNA applications in vivo. The same may be true for other delivery strategies which are currently in development and which will eventually determine the therapeutic potential of siRNA-based RNA interference strategies. Acknowledgment The work of A. Aigner is supported by the Deutsche Forschungsgemeinschaft (AI 24/5-1). References Aigner, A., Brachmann, P., Beyer, J., Jager, R., Raulais, D., Vigny, M., Neubauer, A., Heidenreich, A., Weinknecht, S., Czubayko, F., Zugmaier, G., 2003. Marked increase of the growth factors pleiotrophin and fibroblast growth factor-2 in serum of testicular cancer patients. Ann. Oncol. 14, 1525–1529. Aigner, A., Fischer, D., Merdan, T., Brus, C., Kissel, T., Czubayko, F., 2002. Delivery of unmodified bioactive ribozymes by an RNA-stabilizing polyethylenimine (LMW-PEI) efficiently down-regulates gene expression. Gene Ther. 9, 1700–1707. Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001. Recognition of double-stranded RNA and activation of NFkappaB by Toll-like receptor 3. Nature 413, 732–738. Behr, J.P., 1997. The proton sponge: a trick to enter cells the viruses did not exploit. Chimia 51, 34–36. Bernstein, E., Caudy, A.A., Hammond, S.M., Hannon, G.J., 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366. Bertrand, J.R., Pottier, M., Vekris, A., Opolon, P., Maksimenko, A., Malvy, C., 2002. Comparison of antisense oligonucleotides and

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