siRNA delivery technologies for mammalian systems

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siRNA delivery technologies for mammalian systems David B. Rozema and David L. Lewis Inhibition of gene expression using the RNA interference (RNAi) pathway is rapidly becoming the method of choice for studying gene function in mammalian cells. However, successful knockdown of the target gene requires efficient delivery of short interfering RNAs (siRNAs). Several technologies have been developed that enable effective delivery of siRNAs to both cells in culture and whole animals.These technologies will allow the use of RNAi to study gene function in mammalian model systems in which classical methods are often limited and costly.

David B. Rozema David L. Lewis* Mirus Corporation 505 S. Rosa Road Madison WI 53719, USA *e-mail: [email protected]

▼ Knowledge of the function of a gene product is key to understanding basic biological processes and the role of the gene product in a particular disease. Recently, a powerful new technique has been described for determining gene function in mammalian cells. This method exploits the RNA interference (RNAi) pathway, which is present in a wide variety eukaryotic organisms [1,2]. RNAi is induced by the introduction of double stranded RNA (dsRNA) into the cell where it is cleaved by the action of Dicer enzyme into short dsRNA molecules, 21–25 bp in length, called short interfering RNAs (siRNAs). siRNAs interact with proteins in the cytoplasm to form a ribonucleoprotein complex known as dsRNAinduced silencing complex (RISC). Using the antisense strand of the siRNA as a guide, RISC associates with and cleaves the mRNA of identical sequence. The cleaved mRNA is then degraded by nonspecific RNases. It is likely that RISC acts as a true enzyme and that it cycles to cleave additional target mRNA molecules. Because introduction of long dsRNA also induces components of the interferonresponse pathway in non-embryonic mammalian cells, RNAi is usually induced by introducing siRNAs into cells [3,4]. Using siRNA, the level of knockdown of the target mRNA can be close to 20-fold and lasts typically for a few days in dividing cells before

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mRNA levels begin to recover. Data from microarray analyses show that mRNA knockdown using siRNA is target specific, although care must be taken to avoid using siRNAs that contain sequences that have significant similarity to off-target mRNAs [5–7]. Using RNAi, it is possible to determine the function of a gene using a robust, reverse-genetic approach. Several considerations should be taken into account when planning a knockdown experiment using siRNA. First, the siRNA must be properly designed against the target mRNA sequence. There are several web-based algorithms available to aid siRNA design (Box 1). However, even using state-of-the-art siRNAdesign software does not guarantee that a siRNA will have optimal activity. It is often necessary to design several siRNAs before finding one that gives satisfactory knockdown. Second, the levels of the target mRNA and the corresponding protein product should be monitored to confirm the siRNA is active. Northern analysis, RNase protection and reverse transcriptase–quantitative PCR can be used to quantify mRNA, and protein levels can be monitored by either immunochemical methods or activity. It is important to point out that the level of protein knockdown depends on the half-life of the protein; obviously, proteins with longer half-lives will take longer to become depleted than those with shorter half-lives. Finally, testing the activity of a given siRNA requires effective methods to introduce the siRNA into the cells and animal under study. Frequently, this step is given the least consideration when planning an RNAi experiment, but poor delivery of siRNA is often the reason for poor knockdown. In this review, we examine the reagents and techniques that have been developed to enable effective siRNA delivery to a variety of mammalian cell types in culture and in vivo.

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Box 1.Web-based siRNA design tools • The Whitehead Institute (http://jura.wi.mit.edu/pubint/ http://iona.wi.mit.edu/siRNAext) • Oligoengine (http://www.oligoengine.com) • Dharmacon (http://www.dharmacon.com) • Ambion (http://www.ambion.com/techlib/misc/ siRNA_finder.html)

Delivery of siRNAs to mammalian cells in culture Simple addition of naked, unmodified siRNAs to the culture media that overlies mammalian cells does not result in effective knockdown of the target gene [8]. There are three primary reasons for this: (i) mammalian cells appear to lack the effective dsRNA-uptake machinery that is found in other species such as Caenorhabditis elegans; (ii) siRNA is highly charged and cannot pass freely through the cytoplasmic membrane; and (iii) uptake of siRNA by fluid-phase endocytosis does not result in the release of siRNA into the cytoplasm. If siRNA is not delivered effectively to the cytoplasmic compartment, it will not interact with other RISC components and, thus, will not induce RNAi. In this section, we will focus on the use of chemical and viral transfection methods to deliver siRNA to cells in culture.

Transfection of siRNA using chemical delivery agents Because siRNA is a nucleic acid, the basic principles that underlie the design of transfection reagents to deliver siRNA are similar to those used to design delivery agents for other nucleic acids, most notably plasmid DNA. Nucleic acid transfection reagents have two basic properties. First, they must interact in some manner with the nucleic acid cargo. Most often this involves electrostatic forces, which allow the formation of nucleic acid complexes. Formation of a complex ensures that the nucleic acid and transfection reagent are presented simultaneously to the cell membrane. Complexes can be divided into three classes, based on the nature of the delivery reagent: lipoplexes; polyplexes; and lipopolyplexes. Lipoplexes are formed by the interaction of anionic nucleic acids with cationic lipids, polyplexes by interaction with cationic polymers, and lipopolyplexes by interaction with both cationic lipids and cationic polymers [9]. The second basic property that transfection reagents must possess is the ability to fuse and/or disrupt biological membranes to deliver the nucleic acid to the cell cytoplasm. Because of the lipid composition of both the cell membrane and lipoplexes, it is reasonable to suggest that lipoplexes fuse directly with the cell membrane to deliver

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nucleic acids. However, there appears to be no correlation between fusion of lipoplexes with the cell membrane and transfection [10,11]. Indeed, fluorescence and electron microscopic studies indicate that lipoplexes enter the cell via endocytosis [12,13] and subsequent disruption of the endosomal/lysosomal membrane by the delivery reagent results in the release of nucleic acid to the cytoplasm. Endosome disruption can occur through either denaturation of the membrane bilayer structure or by increased osmotic pressure, which causes the membrane to swell and burst, or by a combination of both [14]. The cationic lipids used to prepare lipoplexes almost certainly disrupt endosomes through interactions between lipids of the lipoplex and lipids of the endosomal membrane. In addition to membrane disruption, it is proposed that displacement of the nucleic acid from the lipoplex occurs by exchange of the anionic lipids in the endosomal membrane with cationic lipids of the lipoplex [15]. Cationic-lipid-based reagents are the most common commercially available transfection reagent. Agents that cause endosomal disruption by increased osmotic pressure rely on acidification of the endosomes as they mature into lysosomes. During endosomal maturation, the pH of the lumen drops from ~7 to ~5. Acidification of the endosomal compartment is exploited by many viruses in order to facilitate release [16]. To mimic this viral activity, many cationic polymers used in polyplex-type transfection reagents have pH-dependent functional groups with pKa values of 5–7. The effect of these buffering groups is to increase the number of protons that are required to decrease endosomal pH. It has been postulated that the increased number of protons and, as a consequence, their counter-ions increases the osmotic pressure of the endosome and leads to membrane rupture [14]. The most widely studied and utilized cationic polymer that is proposed to utilize this mechanism is polyethylenimine (PEI) [17]. Lipopolyplex reagents combine the action of cationic lipids and polymers to deliver nucleic acids. Addition of histone, poly-l-lysine and protamine to some formulations of cationic lipids results in levels of delivery that are higher than either lipid or polymer alone. The combined formulations might also be less toxic [18,19]. At Mirus Corporation, we have developed a lipopolyplex transfection reagent called TransIT-TKO® to deliver siRNA, which is composed of a charge-dense polycation and a cationic lipid. We find that this charge-dense polymer forms complexes with siRNA that are resistant to disassembly in physiological solutions, including serum. This allows complexes to be added directly to cell culture media that contains serum. A list of transfection reagents

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RESEARCH FOCUS Table 1. Commercially available siRNA transfection reagents

a

Complex type

Name

Source

Lipoplex

siPORT™ Lipid Metafectene™ Lipofectamine™, Oligofectamine™ RNAiFect iFECT™

Ambion (http://www.ambion.com) Biontex (http://www.biontex.com) Invitrogen (http://www.invitrogen.com) Qiagen (http://www.qiagen.com) Molecula (http://www.molecula.com) Qbiogene (http://www.qbiogene.com)

Polyplex

jetSI™

Lipopolyplex

TransIT-TKO TransMessenger

Mirus (http://www.genetransfer.com) Qiagen (http://www.qiagen.com)

Information unavailable

siPORT™ Amine GeneSilencer™ RiboJuice™ GeneEraser™ Targetfect-siRNA

Ambion (http://www.ambion.com) Gene Therapy Systems (http://www.genetherapysystems.com) Novagen (http://www.novagen.com) Stratagene (http://www.stratagene.com) Targeting Systems (http://www.targetingsystems.com)

®

a

Abbreviation: siRNA, short interfering RNA.

available for siRNA delivery and their source is shown in Table 1. The effectiveness of transfection reagents varies according to cell type and it is incumbent on the investigator to determine which reagent is optimal for their cell line of interest. In addition, factors such as cell density, passage number and media components can affect transfection efficiency to varying degrees. Finally, although chemicalbased transfection reagents allow the delivery of siRNA to most cell types, there are some commonly used cell types, such as cell lines derived from T cells and some primary cells, that remain recalcitrant to chemical transfection. For problematic cell types, electroporation is an alternative delivery method [20]. However, although electroporation gives high transfection efficiency once optimized, it can also kill a high proportion of cells.

Delivery of siRNA expression vectors An alternative to delivering synthetic siRNA to induce RNAi is to use a vector that expresses siRNA in the cell. This approach was described originally in a flurry of publications in which RNA polymerase III (Pol III) promoters were used to drive the expression of siRNA transcripts, either as separate sense and antisense strands that anneal in the cell or as a single short hairpin that contains both siRNA strands [21–29]. Although a plasmid vector has been described that utilizes the Pol II promoter to produce siRNA, Pol III has the advantage of initiating and terminating RNA transcripts at well-defined positions, which allows investigators to design constructs that produce the intended siRNA [30,31]. The Pol III promoters used most often for siRNA expression are the human and mouse U6 promoters and the

human H1 promoter, but constructs that utilize the tRNAVal promoter are also described [32]. Each promoter is <300 bp in length and can also be incorporated into fragments generated by PCR for rapid testing of siRNA designs [33]. Many of the plasmid vectors contain selectable markers that can potentially be used to generate stable knockdown cell lines. siRNA expression vectors are delivered to cells using the reagents developed for the delivery of plasmid DNA. A list of siRNA expression vectors and their properties is presented in Table 2.

Viral methods for delivering siRNA expression cassettes The delivery of siRNA expression vectors can be accomplished using viruses rather than chemical and mechanical methods. Similar to the siRNA expression plasmids described above, Pol III promoters are used most frequently to drive siRNA expression form viral genomes. RNAi induced by viral delivery was demonstrated first using retroviral vectors based on the mouse stem cell virus (MSCV) and the Moloney murine leukemia virus (MoMLV) [34,35]. Production of high-titer, pantropic viral particles that contain the vesicular stomatitis virus G glycoprotein allows retroviruses to infect a number of cell types, including primary cells [36,37]. One drawback of retroviral systems based on MoMLV and MSCV is that they require cell division for expression. By contrast, retroviral vectors derived from lentivirus can stably infect non-dividing cells. Lentiviral vectors that contain Pol III siRNA-expression cassettes have been used to transfect a number of cell lines in vitro, including HeLa, HEK293, mouse embryonic fibroblasts, primary T cells and hematopoietic stem cells [29,38–42]. Because retroviruses

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RESEARCH FOCUS Table 2. Commercially available siRNA expression vectors Product line

Promoter

Type

Drug marker

Virus production

Source

LineSilence™ SilenCircle™

U6 U6

PCR Plasmid

No No

No No

Silencer™ pSIREN

hU6, mU6, H1 hU6

PCR and plasmid Plasmid

Yes Yes

GeneSuppressor™

hU6

Plasmid

Yes

psiRNA™ siXpress™ pSUPER™

H1 hU6, mU6, H1 H1

Plasmid PCR and plasmid Plasmid

Yes No Yes

No Retrovirus, adenovirus Retrovirus, adenovirus Adenovirus No Retrovirus

Allele Biotechnology and Pharmaceuticals (http://www.allelebiotech.com) Ambion (http://www.ambion.com) BD Biosciences (http://www.clontech.com) Imgenex (http://www.imgenex.com)

pRNA

U6, H1

Plasmid

Yes

Retrovirus, adenovirus

Invivogen (http://www.invivogen.com) Mirus (http://www.genetransfer.com) Oligoengine (http://www.oligoengine.com) GenScript (http://www.genscript.com)

a

Abbreviation: siRNA, short interfering RNA.

such as MoMLV and lentivirus integrate into the host genome, they can be used to generate stable knockdown cell lines with relatively high efficiency. A third viral system described recently for the expression of siRNAs is based on adenovirus. Like lentivirus, adenovirus does not require cell division for expression and infects many cell types in culture. Adenovirus rarely integrates into the host genome and strains used for gene transfer are generally replication defective. Recombinant adenoviral vectors are usually produced by cotransforming Escherichia coli with a shuttle plasmid that contains the siRNA expression cassette together with a plasmid that contains a modified adenoviral genome. Recombinant adenoviral plasmid is used to transfect mammalian packaging cell lines to produce recombinant viral particles. Shen et al. have engineered an adenoviral vector that contains the Pol III H1 promoter [43], and several viral vectors that contain Pol III promoters for expression of siRNAs are available commercially (Table 2). Xia et al. have constructed an adenoviral construct that contains a modified cytomegalovirus promoter and a minimal simian virus 40 polyadenylation cassette [44]. This construct differs from most others designed to produce siRNAs because it requires transcription by Pol II. Xia et al. report that it is crucial to have the siRNA-expression cassette immediately downstream of the transcription start site and to use a minimal polyadenylation signal sequence [44].

In vivo delivery of siRNA and siRNA-expression constructs Although valuable insights into gene function can be gained using siRNA-mediated gene knockdown in cell-culture

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systems, complete understanding of gene function often requires that it is studied in the context of a whole organism. This is especially true when determining the role of a gene in a disease in which cell-culture models are either inadequate or nonexistent. Whereas the delivery of siRNA to cells in culture requires entry into the cell and release to the cytoplasm, in animals siRNA must also gain access to the target cells and tissues. This is a significant barrier that has hampered the development and clinical application of nucleic acid-based therapies. In the following sections, we describe delivery strategies used to tackle the problem of gene delivery in vivo. Many of these strategies are likely to be directly applicable to delivery of a siRNA-expression construct and, with minor modification, to delivery of siRNA itself. However, only a very limited number of studies that directly address the delivery of these molecules have been published. Several strategies have been employed to deliver gene constructs and other nucleic acids, such as antisense oligonucleotides and ribozymes, to tissues in vivo. Many of these strategies have grown out of the field of gene therapy and can be broken down into two broad categories, viral and non-viral. Viral constructs have the advantage of efficient delivery but often induce serious immune effects in the host [45]. The use of fully ‘gutted’ viral vectors, which are unable to produce viral proteins, minimizes the immune response [46]. Non-viral approaches include the delivery of naked nucleic acid by a physical method, such as electroporation, gene gun and hydrodynamic injection, and delivery of nucleic acids by synthetic carriers, some of which are similar to those described for transfection of cells in culture [47]. Non-viral approaches have several

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RESEARCH FOCUS potential advantages over viral approaches for delivering nucleic acids, including lowered or absent immunogenicity enabling repeat administrations, and lower production costs [48].

Hydrodynamic intravascular injection We observed that delivery of naked siRNA by standard injection methods, either directly into tissue or indirectly via the blood stream or peritoneum, does not result in efficient siRNA uptake or appreciable siRNA biological activity. A non-viral method that delivers siRNA effectively in vivo is ‘high-pressure’ or ‘hydrodynamic’ tail-vein injection of mice [49–51]. The hydrodynamic method, which involves rapid injection of a large volume into the tail vein, was developed originally to deliver plasmid DNA [52,53]. Rapid injection causes a transient increase in venous pressure and extravasation of the nucleic acid. The nucleic acid is rapidly internalized when presented to parenchymal cells in this manner. Analysis of the distribution of plasmid DNA by reporter-gene expression indicates that the liver is targeted most effectively using this technique, with 5–40% of hepatocytes showing reporter-gene expression [52,53]. Interestingly, non-parenchymal cells in the liver, such as Kupffer cells, endothelial cells and the bile-duct epithelium, do not appear to be transfected. Other organs, including the kidney, pancreas, spleen, lung and heart are also transfected, but the efficiency of delivery of plasmid DNA is at least 10-fold lower than in liver [52]. Some transient toxicity is associated with this procedure. Liver enzyme levels in the serum are elevated 1 day after injection but return to near normal levels by day 2 [52,53]. There is also histological evidence of limited hepatocyte necrosis [53]. Delivery of siRNA to the liver using the hydrodynamic approach appears to be more effective than delivery of plasmid DNA. We have shown that delivery of siRNA inhibits the expression of green fluorescent protein (GFP) in the majority of hepatocytes in GFP-transgenic mice [50]. Song et al. used multiple hydrodynamic injections of siRNA to knockdown expression of the endogenous Fas receptor in hepatocytes by 90% [51]. It is not known why siRNA delivery is more efficient than plasmid DNA, but it might relate to its relatively small size and the fact that very low intracellular concentrations of siRNA are likely to be sufficient to elicit gene knockdown. In addition, siRNA acts in the cytoplasm and does not require nuclear import, which is a significant barrier to delivery of biologically active plasmid DNA [54]. However, codelivery using hydrodynamic tailvein injection of siRNA expression vectors and plasmid DNA vectors that contain the target genes results in knockdown of the target gene [49,55].

The hydrodynamic tail-vein injection procedure for delivering naked siRNA and siRNA-expression plasmids is a promising tool for gene-function and target-validation studies in mice. It is robust, relatively easy to perform and requires no specialized equipment. In larger mammals, such as rabbits and non-human primates, direct delivery of plasmid DNAs that contain gene-expression cassettes to the liver has been accomplished using a catheter-based approach [56]. Delivery of plasmid DNA using hydrodynamic injection to other organs has also been explored [57]. We believe these methods could easily be adopted for delivery of siRNA and siRNA expression vectors.

Synthetic delivery vehicles A second non-viral approach for delivering either siRNA or siRNA-expression vectors to cells in vivo uses synthetic vehicles. Although this approach is only now being investigated for delivery of these molecules, it has been investigated widely in several laboratories as a way to deliver geneexpression constructs for gene therapy [58]. Although complexes can be injected directly into the target tissue, this route of delivery is generally inefficient and only transfects cells near the site of injection. It is conceivable that intravenous administration of complexes might allow uptake by many more target cells. However, for this approach to be viable, the delivery vehicles used must not only overcome cellular barriers to delivery, but must also form stable complexes with the nucleic acid that remain intact as they traverse the vasculature en route to the target cells. Synthetic vehicles designed for the in vivo delivery of nucleic acids often contain cationic polymers and/or lipids. It is important to note that although the cationic nature of polymers and lipids is essential for complex formation, positively charged agents have a tendency to be toxic following systemic administration and have low transfection efficiencies when used to deliver gene-expression constructs [59,60]. Toxic manifestations of systemically administered cationic nucleic acid complexes can range from agglutination of red blood cells to potent inflammatory reactions and elevated concentrations of liver enzymes in the serum [60,61]. Cationic complexes also tend to aggregate in physiological conditions and might accumulate in capillary beds of the lung, which would be lethal [60,62]. Furthermore, after intravenous injection, cationic nucleic acid complexes also encounter cells such as macrophages, monocytes, neutrophils, platelets and erythrocytes, which are important potential mediators of immunity [63]. The most commonly used strategy to avoid nonspecific, undesirable physiological effects of the delivery vector is to attach uncharged, hydrophilic polymers. These polymers inhibit nonspecific hydrophobic and charge–charge

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RESEARCH FOCUS interactions, thereby increasing the ability of the vectors to circulate in vivo, and decreasing toxicity. The most widely used hydrophilic polymer is polyethyleneglycol (PEG). PEG has been used to modify both liposomes and polyplexes in gene-delivery studies [64–70]. In addition to PEG, several groups have used other hydrophilic polymers, such as polyhydroxypropylmethacrylic acid and cyclodextrins, to stabilize complexes and limit toxicity [71,72]. A second strategy to decrease the toxicity of positively charged nucleic acid complexes is to ‘recharge’ the cationic complexes by adding a polyanion. When absorbed on macrosurfaces in aqueous solution, polycations and polyanions form layered structures, and a similar phenomenon takes place on the surface of polycation-condensed plasmid DNA when further complexed with a polyanion [73,74]. Recharged particles can be prepared with either a net negative or net zero charge that do not aggregate under physiological solutions and have improved serum stability. Tail-vein injection of PEI complexes recharged with polyacrylic acid results in reporter-gene expression in mouse lungs and reduced toxicity [75]. Investigations are underway in several laboratories to determine if synthetic delivery vehicles developed for gene transfer can deliver siRNA in vivo, or if other formulations are required.

Viral vectors for in vivo delivery In principle, viral vectors developed for delivery of siRNAexpression cassettes to cells in vitro could be used for delivery in vivo. Retroviral vectors based on MoMLV that contain a Pol III siRNA-expression cassette have been used recently to induce RNAi in the limb bud of developing chicks [76]. Work from Beverly Davidson’s laboratory also shows that it is possible to inhibit expression of a target gene in mouse brain and liver using adenovirus constructs that contain minimal Pol II promoters to drive expression of a hairpin siRNA [44]. Recently, lentiviral vectors have been used to deliver Pol III-dependent siRNA-expression cassettes to mouse embryonic stem cells and fertilized eggs [42,77], and transferring these cells to pseudopregnant mice generates transgenic animals. The use of lentivirus to create transgenic mice is less challenging technically than standard pronuclear injections, and lentivirus integrants appear to be less sensitive to gene silencing during development than other retroviral integrants [78,79]. A study by Rubinson et al., detected target-gene knockdown in all tissues examined, including brain, liver, spleen and thymus. Tisconia et al. took this approach a step further to show germline transmission of knockdown of GFP expression in GFP transgenic mice. That knockdown is detected in F1 mice indicates that siRNA expression from the Pol III promoter has not

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been silenced. However, more extensive analyses of the progeny are needed to determine if this technique provides stable, heritable knockdown in all tissues.

Conclusion It is a fortunate coincidence that the phenomenon of RNAi was discovered when the sequences of the human and mouse genomes were fully revealed. By exploiting the power of RNAi and using it as a tool, it is theoretically possible to determine the function of any gene by designing and delivering siRNAs. In this review, we have focused on current non-viral and viral strategies to deliver siRNA and siRNA-expression vectors to mammalian cells in culture and whole animals. This field is moving rapidly and new strategies are continually coming to fruition, especially for in vivo delivery. Future research will be directed at improving the efficiency of in vivo delivery of siRNAs to specific organs as well as limiting toxic side-effects. Continued development of gutted viral vectors and biocompatible synthetic delivery molecules, perhaps in combination with organ-specific targeting ligands, will further expand the use of RNAi technology to study gene function.

Acknowledgements We thank Jennifer Grenier for critically reading the manuscript.

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So – how are we doing? TARGETS reviews advances in genomics and proteomics that will impact on drug and target discovery. Coverage includes new technologies, new applications of existing technologies, and current problem areas. We welcome constructive comments about the content of TARGETS and encourage you to email the editorial team with your suggestions and comments. Your comments and feedback are important in helping us shape this journal – we aim to provide information that you need. Please send your comments to: Dr Joanna Owens, Editor, TARGETS Drug Discovery Today Publications Elsevier 84 Theobalds Road, London UK WC1X 8RR e-mail:[email protected]

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