Advanced Drug Delivery Reviews 59 (2007) 87 100 www.elsevier.

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Gene manipulation through the use of small interfering RNA (siRNA): From in vitro to in vivo applications
Lekha Dinesh Kumar a , Alan R. Clarke b,
a

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India b School of Biosciences, Cardiff University CF10 3US, United Kingdom Received 15 September 2006; accepted 4 March 2007 Available online 20 March 2007

Abstract The conventional approach to investigate genotypephenotype relationships has been the generation of gene targeted murine strains. However, the emergence of RNAi technologies has opened the possibility of much more rapid (and indeed more cost effective) genetic manipulation in vivo at the level of the transcriptome. Successful application of RNAi in vivo depends on intracellular targeted delivery of siRNA/shRNA molecules for efficient knockdown of the desired gene. In this review, we discuss the rationale and different strategies of using siRNA/shRNA for accomplishing the silencing of targeted genes in a spatial and /or temporally regulated manner. We also summarise the steps involved in extending these approaches to in vivo applications, with a specific focus upon the development of silencing in the mouse. 2007 Elsevier B.V. All rights reserved.
Keywords: siRNA; shRNA; Transgenic; Transfection; Target validation; Lentiviral; Adenoviral; Therapeutic; siRNA design; Methods of delivery; Mammalian system

Contents Introduction . . . . . . . . . . . . . . . . . . . Discovery of RNAi . . . . . . . . . . . . . . . Mechanism of silencing by RNAi . . . . . . . Designing efficient siRNAs. . . . . . . . . . . Target validation in vitro and in vivo . . . . . . Vectors for siRNA manipulation in vivo . . . . Modification of siRNA and the use of aptamers Plasmid and viral delivery systems . . . . . . . 8.1. Lentiviral vectors . . . . . . . . . . . . 8.2. Retroviral vectors . . . . . . . . . . . . 8.3. Adenoviral vectors. . . . . . . . . . . . 9. Successful gene silencing in the mouse . . . . 10. SiRNA as novel therapeutic . . . . . . . . . . 11. Future prospects and conclusions . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. 4. 5. 6. 7. 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 88 88 89 90 91 92 93 93 93 93 94 94 96 96

This review is part of the Advanced Drug Delivery Reviews theme issue on Opportunities and Challenges for Therapeutic Gene Silencing using RNAi and microRNA Technologies. Corresponding author. Tel.: +44 2920 874609; fax: +44 2920 874116. E-mail address: clarkear@cf.ac.uk (A.R. Clarke).

0169-409X/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2007.03.009

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1. Introduction The transfer of genes through genetic engineering produces transgenic organisms. By physical manipulation, genetic modifications are introduced into an animal in one generation without compromising or limiting the overall pool of genetic information. This either results in the simple addition of genetic material, usually termed a transgene, or the modification of an existing allele, which may include gene deletion to create a knockout allele. The primary objective of transgenesis is to determine the effect of gene modification on the phenotype compared to its normal counterpart, and so to infer a genotype phenotype relationship. There are three principal mechanisms by which transgenic animals are produced: first, microinjection of cloned gene(s) into the pronucleus of a fertilized ovum; second, injection of genetically manipulated embryonic stem cells into blastocysts and third, by exposure to vehicles such as retroviruses that carry a desired transgene. Addition transgenesis, where the transgene is introduced into the genome is relatively rapid, and a plethora of addition transgenic strains have been generated over the last decades. However, this approach suffers from three main drawbacks: lack of control over transgene copy number and over the transgene integration site, and also it is limited to the addition of genetic material such that it is difficult to model genetic inactivation. These difficulties can lead to the development of inappropriate phenotypes and misleading experimental conclusions. The generation of knockout mice by gene targeting circumvents many of these difficulties, but remains a relatively slow process. This requires the engineering of a targeting vector and its subsequent use in embryonic stem (ES) cells to mediate gene targeting through homologous recombination. Successfully targeted ES cells are then injected into mouse blastocysts, with subsequent germ line transmission of the mutant allele. This approach produces constitutive mutations, with all cells within the organism carrying the mutant allele. Where tissue specific mutations are desired, the Crelox and FlpFrt systems [13] have been used to create conditional alleles. Using these approaches, tissue restricted expression of Cre or Flp drives the deletion of loxP/Frt flanked sequences, usually engineered to result in inactivation of the target allele. A further level of control has been added to this approach by using the Cre-lox/ Flp-Frt systems in conjunction with and the tetracyclineregulated transcriptional system. In this combinatorial approach, expression of Cre/Flp occurs only when the drug tetracycline is administered, allowing precise temporal and spatial control over Cre-or Frt mediated gene deletion [46]. Whilst genetically engineered mice have many strengths, their development is time intensive and requires significant effort and cost. This is particularly difficult for high throughput strategies, or where the required technology and expertise are not available. Hence, the development of more rapid and inexpensive approaches would be of great benefit to the scientific community. The designation of small RNAs as molecule of the year in 2002 has highlighted new possibilities for the use of this technology in creating knockouts and in selective gene silencing, with enormous potential to impact

upon the transgenic field. The inherent capacity of these small RNAs to shut down gene expression in a temporally and spatially controlled manner has opened up a plethora of opportunities for its use. In this review we focus on recent siRNA developments aimed at delivering efficient gene knockdown in vitro and upon the various advantages and disadvantages when extrapolated to the in vivo setting. 2. Discovery of RNAi RNA interference, most commonly referred to as RNAi, is a naturally occurring mechanism employed by the cell to mediate gene regulation. The short stretches of 23 to 25 nucleotide double stranded RNAs, which bring about the silencing of genes, are referred to as siRNAs (small interfering RNAs). These siRNAs are capable of degrading mRNAs that are complementary to one of the siRNA strands. The mechanism of gene silencing was first noted in plants in 1990 by Napoli et al. [7] and later confirmed by Van Blokland [8], where it was termed co-suppression as it resulted in the repression of both the transgene and endogenous gene. siRNA mediated suppression was confirmed as a mechanism by Fire et al. [9], who showed a similar transient phenomenon occurred when double stranded RNA was injected into in C.elegans. Subsequently, stable silencing has been achieved by the enforced expression of short hairpin RNAs (shRNAs) in different model systems such as C elegans, Drosophila and plants. [1012]. 3. Mechanism of silencing by RNAi Gene silencing using RNA interference (RNAi) utilises small double stranded RNAs of 23 to 25 nucleotide length to silence the desired complementary mRNA. The mechanisms underlying this functionality have become clearer with the recognition of the role-played by the enzyme dicer (an RNase III family member. Following cleavage of the double stranded RNAs (dsRNAs) by dicer,) into 2325 nucleotide lengths [13] they associate with a protein assembly called the RNA Induced Silencing Complex (RISC) and subsequently recognize specific mRNAs through base pairing. The 3 unpaired region of the small RNA, referred to as the seed sequence zipper up with the 5 region of the binding site on the target RNA so guiding the RISC to its appropriate target. This complex harbours specific catalytic activity (Slicer) that has been suggested to selectively degrade one of the strands [1416] leaving the other to be associated with the complex to target further fresh messenger RNAs (Fig. 1). Apart from the role of siRNA in post transcriptional gene silencing (PTGS) a potential role in Transcriptional Gene silencing (TGS) has been demonstrated whereby dsRNA corresponding to non-transcribed sequences can direct DNA methylation and transcriptional repression in a range of organisms [1722]. This potential involvement of small RNAs in cell division has also been studied using mutants lacking the RNAi machinery in dividing yeast cells [23]. This has shown that this machinery is required to trigger deletion and reshuffling of some DNA sequences during cell division. Many

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Fig. 1. Schematic diagram of the mechanism of RNA interference. Dicer binds to dsRNA and digests it into 21 to 23 nucleotide duplexes. These in turn are incorporated into the RISC, which has been suggested to eliminate one of the strands and so initiate a cyclical process as the siRNA associates with new target molecules.

other functions have been proposed for small RNAs including transposition, stem cell maintenance, defence against viruses, and the regulation of hox clusters, so implicating a diverse requirement for small RNAs in different cellular activities [24 31]. Given that the majority of disease processes are driven by changes in host gene expression, or by RNA from an infectious agent, the use of RNAi technology to down regulate mutant or mis-expressed genes represents an attractive therapeutic strategy. Such an approach carries the significant advantage that it is reliant upon the inbuilt accuracy of Watson-Crick pairing of siRNA, which should confer a very high degree of specificity, although as discussed below specificity does remain an issue with this technology. Such siRNA-based systems could prove superior to conventional drug technology since they mimic a natural process as well as potentially reducing the offtarget side effects involved in a conventional drug. 4. Designing efficient siRNAs Several different parameters govern the efficient design of siRNAs. A BLAST search of the designed siRNA is first performed to increase the likelihood that a single gene is targeted, with any potential polymorphisms and mutations taken into account. Since siRNA synthesis predominantly occurs in the cytoplasm, intronic sequences are usually avoided. The 5

and 3 untranslated regions are also usually avoided, with siRNAs designed to target 50 to 100 nucleotides downstream of the start codon. The GC content is usually maintained at approximately 50% and strings of three or more G or C residues avoided within the siRNA region. Earlier studies in drosophila [32] have indicated that the target recognition process is highly sequence specific but not all positions of a siRNA molecule contribute equally to target recognition. This has been shown to be particularly crucial at the centre of the siRNA; and any mismatch with the target RNA may prevent binding or target cleavage. However, a 19 bp long siRNA duplex with up to four mismatches (with respect to the target) at the terminal positions still mediated efficient gene silencing in HeLa cells [33] suggesting that base pairing with the target mRNA is not the sole factor in determining efficient activity. Standard siRNAs are designed to be 21 to 23 nucleotides in length with a preference for uridine residues in the 2-nucleotide 3 overhang [32]. Uridine can be replaced by 2deoxythymidine (without any loss of siRNA activity) to confer enhanced nuclease resistance of the duplex in mammalian transfections. In mammalian systems duplex length itself represents one critical factor, with 17 bp siRNA duplexes reported to be inactive, even in the presence of additional target-specific overhangs. However, the observation that a 19 nt long siRNAwith only a 15 nt target-specific stretch [33] was able to mediate significant knock-down may have general implications for target specificity and off target effects of siRNAs.

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Recently, Kim et al. [34] have shown that some siRNAs of length 2527 appear to have greater potency than synthetic 21mer siRNAs directed to the same target site in HeLa cell lines. They synthesised RNA duplexes of varying length containing 3 overhangs, 5overhangs or blunt ends were tested for their relative potency in several reporter systems. Using duplex RNA at several concentrations, they observed increased potency with duplex lengths up to 27 bp. Increased potency was observed even for siRNAs with 5 overhangs or blunt ends whereas reduced efficacy was observed for siRNAs with longer than 27 bp stems. Importantly, the 27mers do not induce interferon or activate PKR. Siolas et al. [35] have also reported synthetic shRNAs with 29-base-pair stems and 2-nucleotide 3 overhangs are more potent inducers of RNAi than shorter hairpins. The improved efficacies of the longer forms of siRNA, termed disRNAs (Dicer-substrate siRNAs), is postulated to result from their recognition and cleavage by Dicer, followed by their more efficient incorporation into the RISC complex. This interpretation is supported by observations that Drosophila Dicer is not only instrumental in handing over siRNA to the RISC, but is itself a component of the latter [36,37]. In an effort to determine the requirement for phosphorylation in RNAi, Elbashir et al. and Nykanen et al. have shown that synthetic siRNAs bearing a 5 hydroxyl can efficiently mediate RNAi both in vitro in Drosophila embryo lysates and in cultured human cells [3840]. In the Drosophila in vitro system an endogenous kinase rapidly converts the 5 hydroxyl group to a phosphate. Blocking siRNA phosphorylation by substituting the 5 hydroxyl with a methoxy moiety completely blocks the RNAi in Drosophila embryo lysates. Furthermore, 5 phosphorylated siRNAs more efficiently trigger RNAi in vivo in Drosophila embryos than comparable 5 hydroxyl containing siRNAs [41]. 5 hydroxyl containing synthetic siRNAs that trigger RNAi in culture mammalian cells [16,38,39], in mice [42,43] and perhaps even in plants [44] may likewise be phosphorylated by a cellular kinase prior to their entry in the siRNA pathway. Consistent with this idea, 5 hydroxyl containing siRNAs are rapidly 5 phosphorylated in HeLa cells. Further studies by Harborth et al. [45] showed that gene silencing was not perturbed when fluorescent chromophores were conjugated to the 5-end or 3-end of the sense siRNA strand and the 5-end of the antisense siRNA strand, but conjugation to the 3-end of the antisense siRNA abolished gene silencing. Blocking the 5-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3 terminus or blocking of the termini of the sense strand had no negative effect. Finally, the accessibility and secondary structures of the target RNA is also an important factor in determining the silencing efficiency in vivo [46,47]. Guidelines such as those outlined above are not absolute, nor do they guarantee that particular siRNAs will function in all systems alike. The overall message is that a particular designed siRNA need not work with a particular target, and therefore a number of different siRNAs will routinely need to be tested. Recently, various algorithms have been developed for siRNA design that should significantly enhance this process [4857].

5. Target validation in vitro and in vivo In vivo analysis of phenotype/genotype relationships has become a central tenet of modern biology. The predominance of this approach is becoming clear not only for fundamental research, but also for target validation in translational programmes. Phenotype/genotype relationships are routinely established by modulating gene expression at the DNA, RNA or protein levels. This is conventionally achieved by creating a knock out murine strain using homologous recombination. The advantage of this approach is that the effect of the particular gene mutation can be observed constitutively in either a normal setting or in a particular disease model. However this method is somewhat limited by the fact that it is laborious and because it sometimes manifests in milder phenotypes or is inconsistent with the in vitro predictions. These limitations may potentially be addressed at the transcript level by RNA interference [58] using antisense RNA [59], dsRNA [60,61] or siRNA [62] to induce homology dependent degradation of the target mRNA. The use of antisense RNA carries some inherent disadvantages, including poor stability, a dependence upon the expression levels of the antisense strand, and limited penetration to the appropriate target [63]. A major problem in using dsRNA for target validation is the non-specificity of gene silencing by dsRNA and the limitation of its size due to the generation of an interferon response by long dsRNAs, which can lead to the shutdown of gene expression or cell death [64]. Moreover transfection efficiency remains a difficulty with longer molecules. These problems were partially overcome by the development of small 2325 nt siRNA as effective mediators of gene silencing [65]. However, as a consequence of the instability of exogenously introduced naked siRNAs, target validation has remained difficult in vivo. Despite the advent of modification strategies to prevent in vivo degradation (see below, and [66]), there is still a necessity to test each siRNA independently, which is time consuming and costly. An alternative strategy has been to generate short siRNAs by digesting dsRNA by E. Coli RNAse III, which generates a pool of endonuclease specific siRNAs, termed esiRNAs. In comparison to chemically synthesised siRNAs, the use of a pool of esiRNAs increases the likelihood of efficient knockdown, and functional gene knock down by esiRNA has been shown to silence various endogenous genes in mammalian cells [67,68] and also in mammalian post-implantation embryos [69]. One particular advantage of using small-pooled siRNAs is the likelihood of achieving simultaneous knockdown of different isoforms of the same gene bringing about complete silencing of the gene. Although the use of esiRNAs is efficient, cost effective, and relatively quick [70] there remain some potential problems. Any residual unprocessed long dsRNA may activate RNA-dependent protein kinase (PKR), and so may result in non-specific translational inhibition [71]. This particular difficulty can be circumvented by gel purification of 21 23 mer siRNAs prior to transfection. A further concern is the potential for increased off-target effects, which have been reported [72] and also competition from less efficacious siRNAs

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within the pool, which may reduce the overall efficiency. A final practical concern lies in the difficulties associated with identifying of the most effective siRNA molecule from the pool, so potentially limiting the usefulness of the pool. Despite the advances outlined above, perhaps the most significant technical difficulty in target validation by siRNA remains the potential for off-target activity, with a given siRNA knockdown potentially affecting several proteins in the same or different pathways especially if there are genes sharing different degrees of homology. Hence the design of specific siRNAs is paramount for accurate target validation, a difficulty that may be progressively overcome through the use of enhanced siRNA design strategies and algorithms. 6. Vectors for siRNA manipulation in vivo siRNA is emerging as a powerful tool not only in studying gene function, but also as a potential new basis for the treatment of human disease. However, accurate tissue specific delivery remains a significant in vivo challenge, as does achieving stable transfection in transgenic models. In the case of the liver, hydrodynamic tail vein injection appears to deliver good targeting [7375]. Beyond this organ, conjugation of siRNAs with molecules or biopolymers that specifically interact with cell surface receptors of the target tissues has been considered for tissue specific delivery of the silencing molecules. The main strategies adopted for targeting siRNA to specific tissues are described below. A further difficulty is that silencing by siRNA is transient. This difficulty could be overcome by the generation of siRNA through expression of a cloned shRNA known as short hairpin RNA. Here, complementary regions spaced by a non-complementary small loop cause the transcript to fold back on itself forming a short hairpin in a manner analogous to natural microRNA. Due to their well defined initiation and precise termination sites, the Pol III promoters have proven to be the promoters of choice for driving shRNAs, such as the human ribonuclease P RNA component H1, or the human U6 or mouse U6 small nuclear RNA promoter [7678]. Constructs containing the H1 promoter are significantly less effective in mice than those containing the U6 promoter, whereas both promoters functioned equally well in cultured cells [78]. In the U6-based expression platforms, the presence of a G at the transcription start position appears critical. This does not, however, represent a serious limitation, since a G in the first position of the sense strand of siRNA (the first transcribed nucleotide) is positively correlated with siRNA functionality [79]. Transcription by Pol III is initiated at a precise position outside of the promoter sequence and terminates upon encountering a stretch of 46 thymidines in the expression cassette. Thus, for expression of an shRNA, an expression cassette requires, in the following order: the top strand of the hairpin, the hairpin loop, the bottom strand of the hairpin, and the terminator, all placed immediately downstream of the promoter (Fig. 2). Recognition and processing by the RNAi machinery converts the shRNA into the corresponding siRNA that is subsequently processed by dicer to 21 to 23 nucleotide

siRNAs in vivo [80,81]. Several different strategies have been developed for the construction and expression of shRNA expression vectors [8289]. A common drawback of constructing shRNA vectors, irrespective of the method used, is the difficulty in confirming the sequence of the hairpin region using automated sequencing protocols. Thus, it has been widely reported that hairpin templates can lead to sequencing reactions that terminate prematurely, at sites adjacent to or just within the region that encodes the hairpin stem, most likely due to the inability of the polymerase to read-through the highly structured template [81,9092]. Although this phenomenon is commonly encountered, it does not affect all hairpins equally and is very likely dependent on the strength of secondary structures that are unique to each sequence. As an alternative, McIntyre [93] found that inclusion of a unique restriction enzyme (RE) site within the loop sequence allows the vector to be linearised and sequenced in two separate reactions; one for the sense and one for the antisense. An alternative strategy to address the sequencing problems derives from the use of vectors that contain dual Pol III promoters that drive the expression of two short complementary RNA strands from a single 19 bp DNA [94,95]. These vectors appear as effective as the shRNA encoding vectors, but are more robust for cloning because of the lack of secondary structure in the siRNA coding region, and incur lower level of DNA synthesis errors due to the shorter sequence. One interesting feature of the Pol III promoters is that they can be relatively easily adapted into inducible promoters [96], a feature of significant importance for functional studies and screening. The CMV promoter has also been used for siRNA production in vivo [97]. This is a much stronger promoter than the Pol III promoters, and allows more siRNA molecules to be transcribed from a given amount of DNA vector. However, since the CMV promoter is a RNA polymerase II (Pol II)

Fig. 2. Representation of a typical shRNA viral based vector system. Shown here are two expression cassettes, one with a U6 promoter for the expression of the shRNA and a GFP cassette to mediate reporter gene expression. puc ori-origin of replication ; F1 ori origin of replication; ampR ampicillin resistance gene for bacterial selection ; purR puromycin resistance for mammalian selection 3LTR left terminal repeats; 5LTR left terminal repeats.

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promoter, the resulting transcripts are normally capped at the 5end and tailed at the 3-end with a long poly (A) sequence. It appears that such modifications are well tolerated as shown in the efficacy assays. In order to target the expression of siRNAs to specific tissues, a tissue specific promoter could conceivably replace one of the dual promoters; however, such an approach has yet to be reported in the literature. Due to their potentially higher ability to deliver different spatial and temporal expression patterns, these types of vector-based siRNA strategies may prove particularly potent for in vivo strategies and for siRNA-based gene-therapy. shRNAs have been successfully used to down regulate a set of gene products associated with self-renewal regulatory functions in mouse embryonic stem cells [26]. Furthermore, successful suppression of SOD2 (Mn superoxide dismutase) has been reported using shRNA under the control of a pol II promoter (ubiquitin C) [98]. Inducible siRNA strategies have also been developed by a number of groups [99107]. For example, a doxycycline inducible form of the RNA polymerase III H1 promoter has been developed to drive siRNA expression where the addition of doxycycline into the medium leads to expression of siRNA and in turn the down regulation of the target gene [101]. Watering et al. [96] have also developed a doxycycline-inducible vector that inducibly down regulates catenin/TCF activity in colorectal cancer cells. Similarly Czauderna [108] et al have suppressed the two catalytic subunits of PI 3-kinase in an inducible manner in an attempt to assess the role of this protein in conferring invasiveness both in vitro (human prostate cancer cells (PC-3)) and in vivo (in a prostate cancer mouse model). Finally, vector-based siRNA-(or shRNA) libraries and chemically synthesized oligonucleotide libraries are being developed for high-throughput genome-wide screening [95,109114]. Such genome-wide RNAi surveys of gene function have remained out of reach in mammalian systems, but recently two groups have reported tools to allow RNAi mass screening of mammalian genes. Berns et al. [111] used their library to search for genes that affect the function of p53, a tumour-suppressor gene that arrests cells with damaged DNA. The shRNA library described by Paddison et al. [110] incorporates an elegant system for shuttling the inserts into any destination vector simply using bacterial mating. They report the construction and application of an shRNA library targeting 9610 human and 5563 mouse genes. They subsequently used this library for a genetic screen designed to report defects in human proteasome function. Such libraries represent valuable new tools for gene analysis and discovery, and may be further extended by the development of true genome wide libraries, that encode substantially all permutations of 19-mer siRNA to target both poly (A) tailed and untailed transcripts. One further approach is to generate semi-random siRNA libraries from an mRNA source. In this case, a specific mRNA population is converted into double stranded cDNA, which is then cut into short DNA fragments and inserted into a vector for encoding siRNA. The advantage of this type of library is that it can be relatively simple (of the order of 106 species) but be able to cover a substantial part of the genome. The limiting aspect of

the approach is that a library made in this way will only be useful for the same organism, or even only for the same cell type, whereas other fully randomized libraries can be used in all cell types and all relevant organisms. There have been several reports [115117] of such semi-random siRNA libraries, for example in the construction of an siRNA library from mouse embryos. 7. Modification of siRNA and the use of aptamers A major challenge to the potential use of siRNA-based therapies is the requirement for safe and efficient delivery of naked siRNA drugs directly to diseased cells in vivo. It requires the ability of intact siRNAs to migrate through the body without being degraded by serum based endonucleases, to penetrate the specified diseased cell, enter the cytoplasm and finally to bring about silencing [64]. The problem of degradation is beginning to be addressed by chemical modification prior to in vivo administration. Different modifications have been used, including 2-O-methyl or fluoryl modification and phosphorothioate modifications [66,118]. Furthermore, conjugation of cholesterol to the 3 end of the sense strand of a siRNA molecule by means of a pyrrolidine linker (thereby generating chol-siRNA) has been demonstrated to protect the siRNA against degradation in cell culture. Such modified siRNAs and vector-based shRNAs, if delivered by hydrodynamic tail vein injections, have been shown to bring about the silencing of the target gene in vivo in mice [42,73,119]. The major disadvantage of this method is that 20% of the body weight is injected into the mouse-tail vein within 7 seconds, with frequent complications leading to heart failure in the recipient animal. Even though this method has limited application due to its severity, it is the first example of successful in vivo gene manipulation and target validation in mammalian systems. These modifications reduce the likelihood that the siRNA is degraded by serum endonucleases. Ellington and Szostak [120] coined the term aptamer for such nucleic acid based ligands and several of them have been generated against different molecular targets of therapeutic interest. They are typically from 15 to 40 nucleotides in length and can be composed of DNA, RNA or nucleotides with a chemically modified sugar backbone such as 2-O-methyl or phosphorothioate. This provides them with considerable stability against the degrading nucleases in the serum. The biggest advantage of aptamers is that they can function at low concentrations with great specificity and accuracy. Once prepared, they can be stored without degradation or can be cloned and used as expression cassettes. Protein aptamers can distinguish between closely related members of a given protein family or between different functional or conformational states. The simultaneous application of siRNA and aptamers against the transcription factor NF-kB has been reported to deliver 90% knockdown; while either method alone only achieved 60% [121]. Because of their small size and ease of synthesis with low cost, aptamers are rapidly becoming the preferred agents for both extracellular and intracellular validation of targets. Other possible solutions include the use of biopolymers around the siRNA which may be charged as well

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containing a specific receptor ligand to target delivery, as well as the use of viral based vector systems, discussed below. 8. Plasmid and viral delivery systems shRNAs have been constructed in plasmid vectors in order to drive their expression in vivo [80,86,87,91,92]. Although mammalian cell based transfection of these DNA based constructs has been achieved, primary neural and hemopoietic cells have proven difficult to transfect. Furthermore, the in vivo delivery of these plasmids and their target integration has still not been reported successfully for transgenic analysis or for plasmid shRNA based therapies. Viral based vectors have therefore been developed as an alternate strategy. Although plasmid vectors are easy to handle and transfect, virus vectors can deliver higher transfection efficiencies, and both lentiviruses and retroviruses have been used extensively to deliver stable knockdown by integration into the genome. A number of viruses have been developed, however interest has centered on four types; retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses and herpes simplex virus type 1. 8.1. Lentiviral vectors Lentiviruses possess a number of features that make them attractive delivery systems. Lentiviruses are a type of retrovirus that can infect both dividing and non dividing cells because their virus shell can pass through the intact membrane of the nucleus of the target cell [122124]. Given their ability to infect non-dividing cells, they can be used to target terminally differentiated cells such as neurons, macrophages, haematopoietic stem cells, muscle and liver cells, for which previous gene therapy methods could not be used. With lentiviral vectors there is no need for ex vivo treatment, and the target cells are simply recognised through their cell membrane receptor proteins. Furthermore immune responses to lentiviruses have not yet been reported [124]. In terms of cell type specificity, this is necessarily limited by the type of lentivirus used, but can deliver tight selectivity, for example through the use of HIV based vectors to infect helper T cells and other macrophages. Given this host of advantages, lentiviral vectors have been widely used to clone and express many shRNAs, bringing about stable down regulation of many genes [125132]. A major advantage of the Lentiviral gene delivery system is that transgenes expressed from lentiviruses are not silenced during development and can be used to generate transgenic animals through infection of embryonic stem cells (ES) cells or embryos. [123,133,134]. Using this approach, Rubinson et al. [123] used a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progenies in transgenic mice. A general method for knocking down GFP RNAs in pre-implantation mouse embryos were also reported [122,135]. Furthermore, Moffat et al. [127] have used lentiviral short hairpin RNA (shRNA) libraries to target both the human and murine genomes. These libraries contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified con-

structs. To test the utility of the library for arrayed screens, they developed a screen that identified genes required for mitotic progression in human cancer cells. This screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; so validating this strategy and providing a widely applicable resource for loss-offunction screens. 8.2. Retroviral vectors Retroviruses are a class of enveloped viruses, the genome of which is containing in a single stranded RNA molecule of 10 kb. Following infection, this genome is reverse transcribed into double stranded DNA and integrated into the host genome. Retroviral vectors are most frequently based upon the Moloney murine leukaemia virus (Mo-MLV), which is an amphotrophic virus, capable of infecting both mouse and human cells, enabling vector development in cells of both species. The viral proto-oncogene is routinely replaced by the gene of interest and transgene expression can either be driven by the promoter/ enhancer region in the 5 LTR, or by alternative viral or cellular promoters. Effective ShRNA gene knockdown has been achieved using retroviral vectors and many shRNA libraries have been made for functional genome analysis [136141], with three large-scale siRNA retroviral libraries now reported [90,111]. A requirement for retroviral integration and expression of viral genes is that the target cells should be dividing. This restricts use to proliferating cells in vivo or ex vivo where cells can be more efficiently transduced. Although a disadvantage in many scenarios, this apparent limitation can prove useful if the objective is cancer therapy in vivo, as the tumour cells will be preferentially targeted. Thus, Roth et al. [142] used a retroviral vector to successfully transfer wild-type p53 into human nonsmall cell lung, a strategy that could theoretically be extrapolated to deliver shRNA. Unfortunately, transgene expression has also been reported to be reduced by inflammatory interferons [143]. Furthermore, retroviruses are inactivated to an extent by two elements in human sera in vivo, the c1 complement protein and an anti-alpha galactosyl epitope antibody, the latter thought to provide a species barrier for the horizontal transmission of retrovirus [144,145]. One of these difficulties may be overcome by the development of complement-resistant retroviral vectors, which may pave the way for more efficient retroviral mediated delivery of shRNA. 8.3. Adenoviral vectors Adenoviruses (Ads) are among the most commonly used vectors for gene therapy, second only to retroviruses [146]. They are 35 kb DNA viruses, the lifecycle of which does not involve integration into the host genome, but replication as episomal elements within the nucleus of the host cell. For the development of vectors, up to 30 kb of viral genome may be replaced [147,148] to generate vectors which can deliver prolonged in vivo transgene expression [149]. Ad vectors show high transduction efficiencies in both dormant and dividing cell

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types. They also deliver high levels of short-term expression that carries clear benefits for siRNA technology. In a pilot study, adenoviral vectors expressing p53 siRNA have been used to successfully target breast cancer MCF-7 and lung carcinoma A549 cells, suggesting these viral vectors may have some in vivo functionality in cancer therapy [146]. Tissue-specificity and vector targeting remain key issues to be resolved with this vector system. Despite this, improved Ad vector systems for transgene expression, liver-targeted gene therapy, and the delivery of small interfering RNA are being developed [141,150154]. For example, a library of 742 siRNA adenoviral based vectors was constructed that cover 7,914 human genes [141]. To generate this library, the siRNA encoding DNA fragments were cloned in a high-throughput fashion into pRetroSuper (pRS), a previously reported retroviral vector containing an shRNA expression cassette. As part of its validation, this library has been used to discover novel members of the NF-kB pathway [95]. A second adenoviral siRNA library, generated commercially by Galapagos Genomics, contains knockdown re-agents for over 4900 human transcripts. In this library, three adenoviral vector encoded siRNAs were designed for each gene. This three-fold redundancy in siRNA sequences gives a 90% probability that the mRNA for any given gene will be knocked-down by at least 75%. Even though preparation of an ideal vector is difficult, adenoviruses and retroviruses have been found to be well suited to the transduction of some tissues such as the gut epithelium with subsequent transgene expression [155]. An ideal vector system should contain minimal foreign sequences to minimise the risk of eliciting an immune response. Given their smaller size, viral promoters possess an advantage in this respect, although they do carry the risk of being silenced during transgene expression [156]. With respect to the expression of shRNAs, HI or U6 promoters remain the promoters of choice. However, the establishment of tight cell specificity remains an issue, and significant effort is still required to develop ideal vectors for transgene studies and possible human therapy. Where human therapy is the objective, there are considerable issues associated with the use of viral vectors, and molecular encapsulation is currently the preferred approach, primarily through the use of aptamers as discussed above. Micromanipulation using nano carriers is now also being considered as an alternative to deliver tissue specific silencing. This approach relies upon packaging the plasmid DNA encoding the short interfering siRNA condensed by poly-L-lysine (PLL) into a Multifunctional Envelope type Nano Device (MEND). When used to target the luciferase gene, a 96% inhibition of activity has been reported in a co-transfection study [157]. Hence MEND has been shown to be a promising gene delivery system for siRNA expression plasmids with less heterogeneity associated with conventional transfection. However, its use in vivo remains to be established. 9. Successful gene silencing in the mouse From the above discussion it is clear that RNAi is now widely being used to bring about knockout phenotypes in various

animal cultured cells, and that this technology is now being applied in vivo. In mammalian systems, an initial problem encountered was that sequences in excess of 30 bp elicit an interferon response by inactivating oligoadenylate polymerase and repressing IkB [64]. Such a difficulty has largely been overcome by the development and use of strategies based on shorter molecules [38]. However, it should be noted that, even with this potential impediment, dsRNA has been used to successfully transfect mammalian cells [158160]. Padisson et al. [60] have demonstrated that long dsRNAs can induce potent and specific gene silencing in mouse embryonic cell lines embryonal carcinoma cell lines, in normal mouse embryonic stem cells, and in some mouse undifferentiated somatic cells. Indeed, sequence specific silencing in mouse embryonal tetracarcinomal cells and undifferentiated mouse embryonal stem cells by dsRNA has also been demonstrated without eliciting an interferon response [159,160], Low in vivo transfection efficiencies and the reliance upon transient siRNA have also proven problematic [161,162], difficulties that have at least been partially addressed by the stable transfection of short hairpin RNA (shRNA) cloned into various viral and plasmid vectors [83,84]. Despite the advances discussed in this review, and the fact that RNAi has established itself as a practical in vitro approach, their application still remains more challenging in vivo. However, functional knockdown has been achieved by expressing shRNA from adenoviral and lentiviral based vectors and various other methods in mice in vivo [103,122,163170], and indeed the use of vector based systems to express siRNA is emerging as an alternative tool for cre-lox mediated knockdown of genes in transgenics [171]. Several constitutive uses of knockdown technology have now been reported; for example ES cellderived embryos expressing an shRNA against p120 RasGTPase-activating protein (Rasgap) have been shown to completely silence the endogenous gene and so resemble a conventional knockout [136]. Furthermore, Carmell et al [165] have demonstrated successful silencing following germline transmission of siRNA against the DNA repair gene Neil, a novel DNA N -glycosylase. Given the volume of new approaches being developed in vitro, and the first clear successes in vivo, it seems very likely that RNAi will rapidly establish itself as a truly useful in vivo tool within mammalian systems. The different strategies that may be used to bring about silencing in vivo are illustrated in Fig. 3. 10. SiRNA as novel therapeutic Beyond its use in functional genomic studies in transgenics, siRNA has the potential to be developed as a novel therapeutic drug. To be a successful drug, a molecule must overcome a set of hurdles. First, it must be able to be manufactured at reasonable cost and administered safely and conveniently. Second, it must be sufficiently stable and accumulate to an effective level in the target cell. Third, it must exert its effect on or within the target cell. Finally, it must do all these without causing significant toxic effects in either target or non-target tissues. Thus, the issues of delivery and targeting pose significant hurdles for the

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Fig. 3. A schematic summary of several different methods of siRNA delivery in vivo leading to silencing of the target gene within the cell, These include: Hydrodynamic tail vein injection of mice by which modified siRNAs are delivered; Viral shRNA vectors, with stable integration into the genome and the production of siRNAs; Lipofection of plasmid shRNA vectors which are then maintained as episomal elements for the production of siRNAs. Each of these methods could independently bring about silencing.

development of siRNA as a successful drug. It must also be administered in a dose that is not immunogenic and it should not produce deleterious off target effects. Moreover, it has to fulfil the basic qualities of a drug with respect to its ease of manufacturing, cost, convenience and safety of administration. Lieberman and group [167] recently demonstrated the ability of RNAi to silence expression of the Fas gene in mice and so protect them from fulminant hepatitis. However the delivery method (high-pressure tail-vein injection) is associated with significant mortality, almost certainly ruling this out as a human therapeutic strategy. A further major obstacle is cellular uptake of the RNAi therapy. The difficulty here is that a charged oligonucleotide will not pass through a lipid layer, which it must do in order to enter a cell. To solve the problem of cell penetration, most researchers have either complexed the RNA with a lipid or modified the RNA's phosphate backbone to minimize its charge. Another potential solution to the many in vivo difficulties is the use of polycation-based nucleic acid self-assembly systems (polyplexes) for the delivery of bioactive agents [172]. Polymer modification ensures stability in the bloodstream and allows the incorporation of cell specific ligands that can be used for cellular and site specific targeting. These vehicles are currently being developed and evaluated to optimise the in vivo delivery of plasmid DNA and short interfering RNA (siRNA) as an approach for gene expression and silencing in the treatment of infectious diseases and cancer.

Even assuming that the delivery problems could be solved, other questions remain, including that of whether therapeutic levels of RNAi may be toxic. Another unanswered question is what effect an excess of RNA from outside the cell may have on the normal function of the RISC, the complex at the heart of the RNAi mechanism. The number of RISCs in the cell is unknown, and one concern is that the amount of RNA needed to have a therapeutic effect may occupy all the available complexes. Assuming that the endogenous level of RISC is there for a purpose, the introduction of siRNA to an unprecedented level may be predicted to cause side effects. Despite the questions and unsolved problems, the development of RNAi therapy is being actively pursued, and recently a number of successful pilot experiments have been reported. For example, Xia et al. [173] have shown that upon intracerebellar injection of recombinant adeno-associated virus (AAV) vectors expressing short hairpin RNAs against the SCA1 gene improved motor coordination, restored cerebellar morphology and resolved characteristic ataxin-1 inclusions in Purkinje cells of SCA1 mice. This silencing inhibited the production of a neurotoxic protein, suggesting that this technology may also be helpful against other degenerative neurological diseases caused by neurotoxic proteins, such as Alzheimer's disease. Furthermore, Howard et al. [174] have reported use of a chitosan-based siRNA nanoparticle delivery system to knockdown EGFP both in vivo and in vitro.

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11. Future prospects and conclusions RNAi, originally considered to have evolved as a cellular defence mechanism [175177], is now being exploited for genetic manipulation. This approach has already proven itself an indispensable tool for elucidating molecular pathways and phenotype/genotype relationships, and has many potential advantages over existing technologies. For example, in comparison to conventional knockouts, which require engineering of both alleles, RNAi based knockouts can be created more simply by expression of the vector mediating RNAi anywhere in the genome. RNAi is also a powerful tool for generating hypomorphic alleles to study gene dosage effects or mimicking disease pathologies. Off target effects of RNAi are a concern that has been raised recently [72], however, this issue may be overcome by refinement of the algorithms used for designing siRNA, as well as through chemical modification of the siRNA or even encapsulation in a nano device [178]. Since most human diseases are polygenic, the use of multiple siRNAs also offers great potential for model development of complex disease models. Currently, this technology is in a period of rapid development in relation to its in vivo application. Difficulties do remain, for example in delivering the exact titre of siRNA required to mediate efficient knock down of siRNA, since over administration could create problems in exhausting the pools of Dicer, a known limiting factor for normal gene regulation [179]. However, there is a clear requirement for the development of novel strategies, both in the mouse to speed the process of genotype/phenotype analysis, and also in those species where we remain limited in our ability to manipulate the genome. Assuming the technical issues discussed here are resolved, such as those surrounding limiting factors such as RISC or exportin-5 (the latter of which excludes siRNAs from non-nucleolar areas of the nucleus [179]), the future for RNAi technologies in both basic and applied science is clearly very promising. References
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