R E V I E W
`
`RNAi therapeutics: a potential new class of
`pharmaceutical drugs
`
`David Bumcrot, Muthiah Manoharan, Victor Koteliansky & Dinah W Y Sah
`
`The rapid identification of highly specific and potent drug candidates continues to be a substantial challenge with traditional
`pharmaceutical approaches. Moreover, many targets have proven to be intractable to traditional small-molecule and protein
`approaches. Therapeutics based on RNA interference (RNAi) offer a powerful method for rapidly identifying specific and potent
`inhibitors of disease targets from all molecular classes. Numerous proof-of-concept studies in animal models of human disease
`demonstrate the broad potential application of RNAi therapeutics. The major challenge for successful drug development is
`identifying delivery strategies that can be translated to the clinic. With advances in this area and the commencement of multiple
`clinical trials with RNAi therapeutic candidates, a transformation in modern medicine may soon be realized.
`
`RNAi is a fundamental cellular mechanism for silencing gene expression
`that can be harnessed for the development of new drugs1,2. The reduc-
`tion in expression of pathological proteins through RNAi is applicable
`to all classes of molecular targets, including those that are difficult to
`modulate selectively with traditional pharmaceutical approaches involv-
`ing small molecules or proteins. Consequently, RNAi therapeutics as a
`drug class have the potential to exert a transformational effect on mod-
`ern medicine. In RNAi, the target mRNA is enzymatically cleaved, lead-
`ing to decreased abundance of the corresponding protein, and specificity
`is a key feature of the mechanism. Synthetic small interfering RNAs
`(siRNAs) leverage the naturally occurring RNAi process in a manner
`that is consistent and predictable with regard to extent and duration
`of action. In addition, viral delivery of short hairpin RNAs (shRNAs)
`represents an alternative strategy for harnessing RNAi. Both nonviral
`delivery of siRNAs and viral delivery of shRNAs are being advanced as
`potential RNAi-based therapeutic approaches.
`In this review, we provide an overview of the molecular mechanism
`of RNAi; the in silico design of siRNAs and shRNAs that are specific for
`a target of interest, in the context of current concepts relating chemical
`structure to specificity and potency; the use of chemical modifications
`that confer stability against exo- and endonucleases present in biologi-
`cal fluids and tissues; and strategies for facilitating cellular delivery in
`vivo through conjugation, complexation and lipid-based approaches
`to facilitate cellular uptake. We summarize the numerous publications
`to date demonstrating the robust efficacy of RNAi in animal models of
`human disease upon direct (local) as well as systemic administration.
`These proof-of-concept studies support RNAi as the basis for a new
`therapeutic approach that has the potential to change the treatment of
`human disease. Most importantly, as we will discuss, clinical trials have
`
`Alnylam Pharmaceuticals, Inc., 300 Third Street, Cambridge, Massachusetts
`02142, USA. Correspondence should be addressed to D.W.Y.S.
`(dsah@alnylam.com).
`
`Published online 15 November; doi:10.1038/nchembio839
`
`recently commenced, with RNAi therapeutic candidates under study for
`treatment of age-related macular degeneration (AMD) and respiratory
`syncytial virus (RSV) infection.
`
`Molecular mechanism of RNAi
`The RNase Dicer initiates RNAi by cleaving double-stranded RNA
`substrates into small fragments of about 21–25 nucleotides in length
`(Fig. 1). These siRNA duplexes are incorporated into a protein complex
`called the RNA-induced silencing complex (RISC; Fig. 1). Biochemical
`analysis identified Argonaute 2 (Ago2) as the protein in RISC responsible
`for mRNA cleavage3, and the crystal structure of RNA-bound Ago2 has
`been reported, revealing key interactions4.
`Before RISC activation, the sense (nonguide) strand of the siRNA
`duplex is cleaved by Ago2, in the same manner as it cleaves mRNA sub-
`strates5,6. Preventing sense-strand cleavage by chemical modification
`can reduce siRNA potency in vitro; however, experimental context is
`important, as siRNAs with highly stabilized (uncleavable) sense strands
`can be highly active.
`
`Role of chemical modifications
`Small-molecule pharmaceutical drugs, almost without exception, meet
`the ‘Lipinski Rules’, criteria including high lipophilicity and molecular
`weight of not more than 500. In sharp contrast, siRNAs naturally lack
`these drug-like properties owing to their large size (two turns of a nucleic
`acid double helix), nearly 40 anionic charges due to the phosphodiester
`backbone, and high molecular weight (over 13 kDa). In aqueous solu-
`tion, with their sugar-phosphate backbone exposed to water, siRNAs
`are extremely hydrophilic and heavily hydrated. Furthermore, siRNAs
`are unstable in serum as a result of degradation by serum nucleases,
`contributing to their short half-lives in vivo7. Although the molecular
`weight of siRNAs cannot be reduced, these molecules can be made more
`‘drug-like’ through judicious use of chemical modification to the sugars,
`backbone or bases of the oligoribonucleotides.
`Chemically modified siRNA duplexes have been evaluated in cell-
`based assays and in animal models. The modifications discussed are
`
`NATURE CHEMICAL BIOLOGY VOLUME 2 NUMBER 12 DECEMBER 2006
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`© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology
`
`Alnylam Exh. 1076
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`R E V I E W
`
`Synthetic
`siRNAs
`
`dsRNA
`
`Dicer
`
`Cleavage
`
`siRNA
`
`Strand separation
`
`Natural
`process of
`RNAi
`
`RISC
`
`Complementary pairing
`
`mRNA
`downmodulation
`
`mRNA
`
`mRNA
`degradation
`
`(A)n
`
`Cleavage
`
`(A)n
`
`Figure 1 Cellular mechanism of RNA interference. Long double-stranded RNA (dsRNA) is cleaved, by the enzyme Dicer, into small interfering RNA (siRNA).
`These siRNAs are incorporated into the RNA-induced silencing complex (RISC), where the strands are separated. The RISC containing the guide or antisense
`strand seeks out and binds to complementary mRNA sequences. These mRNA sequences are then cleaved by Argonaute, the enzyme within the RISC
`responsible for mRNA degradation, which leads to mRNA down-modulation. A, adenosine.
`
`shown in Figure 2. Stability against nuclease degradation has been
`achieved by introducing a phosphorothioate (P=S) backbone linkage at
`the 3′ end for exonuclease resistance and 2′ modifications (2′-OMe, 2′-F
`and related) for endonuclease resistance8–10. An siRNA motif, consisting
`entirely of 2′-O-methyl and 2′-fluoro nucleotides, has enhanced plasma
`stability and increased in vitro potency. At one site, this motif shows
`>500-fold improvement in potency over the unmodified siRNA11. Using
`phosphatase and tensin homolog (PTEN) as a target, the effect of 2′
`sugar modifications such as 2′-fluoro-2′-deoxynucleoside (2′-F), 2′-O-
`methyl (2′-O-Me) and 2′-O-(2-methoxyethyl) (2′-O-MOE) in the guide
`and nonguide strands was evaluated in HeLa cells. The activity depends
`on the position of the modification in the guide-strand sequence. The
`siRNAs with modified residues at the 5′ end of the guide strand seem
`to be less active than those modified at the 3′ end. The 2′-F sugar is
`generally well tolerated on the guide strand, whereas the 2′-O-MOE
`modification results in loss of activity regardless of placement position
`in the construct. The incorporation of 2′-O-Me and 2′-O-MOE in the
`nonguide strand of siRNA does not have a notable effect on activity12.
`Sugar modifications such as 2′-O-Me, 2′-F and locked nucleic acid (LNA,
`with a methylene bridge connecting 2′ and 4′ carbons) seem to be able to
`reduce the immunostimulatory effects of siRNAs (see below).
`Duplexes containing the 4′-thioribose modification have a stability
`600 times greater than that of natural RNA13. Crystal structure studies
`reveal that 4′-thioriboses adopt conformations very similar to the C3′-
`endo pucker observed for unmodified sugars in the native duplex14.
`Stretches of 4′-thio-RNA were well tolerated in both the guide and non-
`guide strands. However, optimization of both the number and the place-
`ment of 4′-thioribonucleosides is necessary for maximal potency. These
`optimized siRNAs are generally equipotent with or superior to native
`siRNAs and show increased thermal and plasma stability. Furthermore,
`substantial improvements in siRNA activity and plasma stability have
`been achieved by judicious combination of 4′-thioribose with 2′-O-Me
`and 2′-O-MOE modifications15.
`
`As mentioned, phosphorothioate (P=S) modifications are gener-
`ally well tolerated on both strands and provide improved nuclease
`resistance. The 2′,5′-phosphodiester linkages seem to be tolerated in
`the nonguide but not the guide strand of the siRNA16. In the borano-
`phosphate linkage, a nonbridging phosphodiester oxygen is replaced
`by an isoelectronic borane (BH3-) moiety. Boranophosphate siRNAs
`have been synthesized by enzymatic routes using T7 RNA polymerase
`and a boranophosphate ribonucleoside triphosphate in the transcrip-
`tion reaction. Boranophosphate siRNAs are more active than native
`siRNAs if the center of the guide strand is not modified, and they
`may be at least ten times more nuclease resistant than unmodified
`siRNAs17,18.
`siRNA duplexes containing the 2,4-difluorotoluyl ribonucleoside
`(rF) were synthesized to evaluate the effect of noncanonical nucleoside
`mimetics on RNA interference. Thermal melting analysis showed that
`the base pair between rF and adenosine is destabilizing relative to a uri-
`dine-adenosine pair, although it is slightly less destabilizing than other
`mismatches. The crystal structure of a duplex containing rF-adenosine
`pairs shows local structural variations relative to a canonical RNA helix.
`As the fluorine atoms cannot act as hydrogen bond acceptors and are
`more hydrophobic than uridine, a well-ordered water structure is not
`seen around the rF residues in both grooves. Rapid amplification of
`5 complementary DNA ends (5′-RACE) analysis confirms cleavage of
`target mRNA opposite to the rF placement site19,20.
`Certain terminal conjugates have been reported to improve or direct
`cellular uptake. For example, siRNAs conjugated with cholesterol
`improve in vitro and in vivo cell permeation in liver cells6. As described
`below, cholesterol and an RNA aptamer conjugation show promise in
`animal models.
`
`Design considerations for potency and specificity
`Critical design concerns in the selection of siRNA duplexes for therapeu-
`tic use are potency and specificity. There are two major considerations
`
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`R E V I E W
`
`with regard to siRNA specificity: ‘off-targeting’ due to silencing of genes
`sharing partial homology with the siRNA, and ‘immune stimulation’
`due to the engagement of components of the innate immune system by
`the siRNA duplex. A combination of bioinformatics methods, chemi-
`cal modification strategies and empirical testing is required to address
`these issues.
`Concomitant with the first description of the structure of active
`siRNAs, a set of ‘rules’ was proposed for selecting potent siRNA
`duplex sequences21,22. Several groups have subsequently developed
`more sophisticated extensions of these largely empirical criteria, lead-
`ing to the development of algorithms for siRNA design23,24. Recent
`biochemical studies of the molecular mechanism of RNA interference
`have highlighted some key features of potent siRNA duplexes (Fig. 3).
`Most notably, it has been found that the efficiency with which the
`guide strand is incorporated into the RISC complex is perhaps the
`most important factor determining siRNA potency. Because siRNA
`duplexes are symmetric, the question arose of how the RISC machinery
`is able to determine which strand to use for target silencing. Insight into
`this enigma came from careful analyses of microRNAs (miRNAs), the
`endogenous counterparts of siRNAs. Examination of the sequences
`of a large number of vertebrate and invertebrate miRNA precursor
`sequences showed that the predicted thermodynamic stabilities of the
`two ends of the duplex are unequal25,26. Specifically, calculating the ∆G
`for the several base pairs at each end of the duplex revealed that the
`5′ end of the mature miRNA pairs less tightly with the carrier strand
`than does the 3′ end. In short, miRNA precursors show thermodynamic
`asymmetry. It was hypothesized that components of the RISC machin-
`ery select the guide strand based on this asymmetry.
`
`Experimental evidence supporting the asymmetry hypothesis has
`been derived from studies using chemically synthesized siRNAs in trans-
`fection experiments. Through an elegant assay in which each strand
`of the siRNA targets a different reporter gene, Schwarz et al. were able
`to quantify the relative efficiency of RISC incorporation for each of
`the two strands25. They found that the RISC machinery preferentially
`incorporates the strand whose 5′ end binds less tightly with the other
`strand. In fact, strand selection could be switched by making a single
`nucleotide substitution at the end of the duplex to alter relative bind-
`ing of the ends. A similar conclusion was reached by another group
`based on in vitro screening of a large collection of siRNAs with varying
`potency26,27. Thus, designing siRNAs with relatively weaker base pairing
`at the 5′ end of the desired guide strand may increase the likelihood of
`obtaining a potent duplex.
`The issue of off-target silencing has been the subject of intensive
`study in a number of different laboratories over the past several years.
`Transcriptional profiling studies have confirmed that siRNA duplexes
`can potentially silence multiple genes in addition to the intended tar-
`get. As expected, genes in these so called off-target ‘signatures’ contain
`regions that are complementary to one of the two strands in the siRNA
`duplex28–30. More detailed bioinformatic analyses have revealed that
`the regions of complementarity are most often found in the 3′ UTRs
`of the off-target genes31. This immediately suggested a microRNA-like
`mechanism, because miRNAs generally interact with the 3′ UTR region
`of their targets. Evidence in support of this concept came from a closer
`look at the determinants of siRNA off-targeting. It was discovered that
`sequence complementarity between the 5′ end of the guide strand and
`the mRNA is the key to off-target silencing31,32. The critical nucleo-
`
`O
`
`B
`
`O
`
`O
`
`B
`
`O
`
`O
`
`B
`
`O
`
`O
`
`B
`
`O
`
`O
`
`B
`
`O
`
`O
`
`B
`
`S
`
`O
`
`OH
`
`Sugar: Ribo
`
`OO
`
`2´-O-Me
`
`OO
`
`O
`
`O
`
`F
`
`2´-O-MOE
`
`2´-Deoxy-2´-fluoro (2´-F)
`
`O
`
`O
`
`LNA
`
`O
`
`OH
`
`4´-Thio
`
`O
`
`B2
`
`O
`
`RO
`
`P
`
`O
`
`O
`H3B
`
`B1
`
`O
`
`O
`
`B2
`
`O
`
`RO
`
`O
`
`B1
`
`O
`
`P
`
`O S
`
`O
`
`B2
`
`O
`
`RO
`
`O
`
`B1
`
`O
`
`P
`
`O O
`
`Backbone (R = OH or 2´-modified): Phosphate (P=O)
`
`Phosphorothioate (P=S)
`
`Boranophosphate
`
`RO
`
`RO
`
`RO
`
`OH
`
`O X
`
`OP
`
`3´
`
`O
`OH
`
`O
`
`HN
`
`N
`
`O
`
`O
`Conjugate: siRNA-cholesterol (X = O or S)
`
`F
`
`O
`
`O
`
`F
`
`5´
`
`HO
`HO
`
`O
`
`OH
`
`Base: 2,4-Difluorotoluyl (DFT)
`
`Figure 2 Chemical modifications of siRNAs. Shown are structures of sugar, backbone and base modifications and of the cholesterol conjugate.
`
`NATURE CHEMICAL BIOLOGY VOLUME 2 NUMBER 12 DECEMBER 2006
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`

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`5´
`
`3´
`
`Double overhang
`
`Cleavage
`site
`
`Seed region
`
`3´
`Nonguide
`
`5´
`
`Guide
`
`Weak base pairing
`increases potency
`
`Important for cleavage
`specificity
`
`Chemical modification
`reduces off-targeting
`
`
`
`Figure 3 Critical nucleotide positions in siRNAs. Nucleotides that are important for potency, mRNA
`recognition, mRNA cleavage and cleavage specificity, including minimization of off-targeting, are shown.
`
`immune regulator engaged by antisense oligo-
`nucleotides38. In the case of siRNAs, it seems
`to be TLR-7 that is the mediator of immune
`stimulation36.
`Several possible strategies exist for avoid-
`ing immune stimulation by siRNA duplexes,
`including avoidance of the offending sequences
`during siRNA design and chemical modifi-
`cation to inactivate the motifs. The former
`approach is not feasible at present because
`the full spectrum of stimulatory motifs has
`not been identified. Evidence supporting the
`latter approach comes from studies in which
`chemical modifications at the 2′ position of
`nucleotides within putative TLR-7–interact-
`ing sequences eliminate immune stimulation
`without compromising silencing activity36,39. Another possibility would
`be to use siRNA delivery strategies that avoid the cell types responsible
`for immune stimulation.
`Prediction of the nucleotide sequence and chemical modifications
`required to yield an ideal siRNA duplex remains a work in progress.
`Still, the recent advances described above have allowed the development
`of design algorithms that greatly increase the likelihood of success. It is
`nonetheless important to note that the relevance of in vitro measure-
`ments of potency and specificity to in vivo activity in a therapeutic set-
`ting has yet to be established. For example, the spectrum of off-target
`genes identified in tissue culture studies can differ depending on the
`method by which siRNAs are introduced into cells40. Also, the induc-
`tion of an innate immune response by certain siRNA sequences is cell
`type dependent41. At present, the most prudent and robust strategy is to
`synthesize and screen a substantial library of siRNA duplexes for each
`target of interest (perhaps even ‘tiling’ the entire messenger RNA) to
`identify the most promising candidates.
`
`tides were found to be positions 2–8, counting from the 5′ end of the
`guide strand (Fig. 3). This corresponds to the so-called ‘seed region’ of
`miRNAs, which has been shown to determine miRNA specificity33.
`Two strategies for avoiding seed region–mediated off-targeting can be
`envisioned. The first is simply to ensure that nucleotides complementary
`to positions 2–8 of the guide strand are unique to the intended target.
`Though theoretically possible, this approach may prove impractical, as
`the universe of possible seed-region heptamers is only 16,384 distinct
`sequences. Even if the homology is restricted to the 3′ UTR, it may prove
`difficult to identify siRNA duplexes satisfying the criteria of potency and
`specificity. As one alternative, recent published work has reported that
`off-targeting can be substantially reduced by chemical modification of
`nucleotides within the seed region34. Specifically, the introduction of a
`2′-O-Me modification into nucleotides within the seed region was shown
`to inactivate the off-target activity of the siRNA without compromising
`silencing of the intended mRNA. In fact, introduction of the modifica-
`tion at a single nucleotide position (position 2, Fig. 3) is sufficient to
`suppress the majority of off-targeting. The mechanism, anticipated by
`recently published crystal structure data, appears to involve perturbation
`of RISC interaction with the modified nucleotide.
`Interactions outside of the seed region can also substantially affect
`siRNA specificity. Although the seed region seems to be critical for
`mRNA recognition, notable mRNA cleavage requires more extensive
`base pairing between the siRNA and the target32. In a recent study,
`Schwarz et al. designed siRNAs capable of distinguishing between
`mRNA targets that differ by only one nucleotide35. They showed that
`target selectivity depends on the location of the mismatch between
`the siRNA and the mRNA. Whereas positioning the mismatch within
`the seed region imparts a certain degree of selectivity, positioning the
`mismatch further 3′ in the guide strand (especially at positions 10
`and 16, Fig. 3) produces highly discriminatory siRNAs. The authors
`hypothesized that mismatches at these positions are particularly dis-
`ruptive to the helical structure of the siRNA–mRNA complex required
`for target cleavage.
`A second mechanism whereby siRNA duplexes can induce unin-
`tended effects is through stimulation of the innate immune system in
`certain specialized immune cell types. It has been demonstrated that
`siRNA duplexes harboring distinct sequence motifs can engage Toll-like
`receptors (TLRs) in plasmacytoid dendritic cells, resulting in increased
`production of interferon36. Such immune stimulation could pose a sig-
`nificant problem in a therapeutic setting. This phenomenon is reminis-
`cent of the results of earlier studies with DNA antisense oligonucleotides
`in which distinct sequences (so-called CpG motifs) were shown to be
`immunostimulatory37. Subsequent studies established that TLR-9, the
`receptor for unmethylated CpG-containing pathogen DNA, is the innate
`
`Proof of concept for local RNAi in animal models
`During the past several years, numerous studies have been published
`demonstrating efficacious silencing of disease genes by local adminis-
`tration of siRNAs or shRNAs in animal models of human disease. Both
`exogenous and endogenous genes have been silenced, and promising
`in vivo results have been obtained across multiple organs and tissues.
`Efficacy has been demonstrated for viral infection (respiratory and
`vaginal), ocular disease, disorders of the nervous system, cancer and
`inflammatory bowel disease (Fig. 4). An important aspect of these
`proof-of-concept studies is that they have supported the expected high
`specificity of RNAi.
`Local RNAi can protect against both respiratory42,9and vaginal43 viral
`infections. Two reports illustrate efficacious direct delivery of siRNA to
`the lung in rodent and monkey models of RSV, influenza and severe
`acute respiratory syndrome (SARS) infection with and without lipid
`formulation. In mouse models of infection, pulmonary viral titers of
`RSV and parainfluenza were reduced by more than 99% with intra-
`nasal delivery of siRNAs formulated with TransIT-TKO, a cationic
`polymer–based transfection reagent, targeting RSV and parainfluenza
`virus, respectively42. In addition, siRNA targeting RSV reduced pulmo-
`nary pathology, as assessed by respiratory rate, leukotriene induction
`and inflammation. These positive proof-of-concept studies in mice have
`led to clinical trials of RNAi therapeutics targeting RSV.
`Another system for which there have been multiple examples of effica-
`cious local delivery of siRNA is the eye, where proof of concept has been
`successfully achieved in animal models of ocular neovascularization and
`scarring using saline and lipid formulations44–46. Intravitreal injection
`
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`tumor regression in a xenograft model of prostate cancer68. Viral and
`vector-based delivery of shRNAs directly to the tumor site69,70 has also
`been used effectively in mouse models of adenocarcinoma, Ewing sar-
`coma and prostate cancer. Of the multiple delivery strategies that have
`been effective in mouse tumor models, the aptamer approach has the
`potential of substantially simplifying delivery, if an aptamer is available
`for a tumor-specific receptor such as prostate-specific membrane antigen
`(PMSA) and the large-scale synthesis of such a construct is feasible.
`For inflammatory bowel disease, direct delivery of siRNA target-
`ing tumor necrosis factor-α (TNF-α) with a Lipofectamine formula-
`tion has recently been shown to reduce not only TNF-α abundance
`but also colonic inflammation after administration by enema71. This
`report, together with a study of siRNA targeting herpes simplex virus-2
`(ref. 43), suggests that mucosal surfaces are accessible with liposomal
`siRNA formulations.
`
`Proof of concept for systemic RNAi in animal models
`Over the past several years, a number of studies have been published
`demonstrating the silencing of disease genes by systemic administration
`of siRNAs (Fig. 4; reviewed in refs. 1,72). In some of these studies, silenc-
`ing of endogenously expressed genes has shown promising in vivo results
`in different disease contexts. For example, efficacy has been demonstrated
`in mouse models of hypercholesterolemia and rheumatoid arthritis. In
`other work, systemic RNAi targeting exogenous genes has shown promise
`in models of viral infection (hepatitis B virus (HBV), influenza virus,
`Ebola virus) and in tumor xenografts. Critical to the success of most
`of these studies has been the use of chemical modifications or delivery
`formulations that impart desirable pharmacokinetic properties to the
`siRNA duplex and that also promote cellular uptake in tissues.
`
`Systemic RNAi
`
`Lung
`Influenza
`Tumor
`Liver
`HBV
`Hypercholesterolemia
`Joint
`Rheumatoid arthritis
`
`of siRNA targeting vascular endothelial growth factor (VEGF) recep-
`tor-1, formulated in phosphate-buffered saline, was effective in reduc-
`ing the area of ocular neovascularization by one-third to two-thirds
`in two mouse models44. In addition, siRNAs targeting VEGF and the
`transforming growth factor-β receptor type II, formulated with TransIT-
`TKO, were injected directly into the mouse eye, resulting in inhibition
`of laser photocoagulation–induced choroidal neovascularization45 and
`latex bead–induced collagen deposition and inflammatory cell infiltra-
`tion46, respectively. As with the lung, multiple siRNA formulations were
`effective in the eye. These encouraging proof-of-concept studies in ani-
`mal models have led to clinical trials of siRNAs targeting the VEGF
`pathway in AMD.
`In the nervous system, RNAi has been particularly useful for vali-
`dating disease targets in vivo. Again, several formulations, including
`saline, polymer complexation and lipid or liposomal formulations, have
`been efficacious for delivering siRNAs locally to the nervous system
`in numerous disease models. The simplest mode of delivery is intra-
`cerebroventricular, intrathecal or intraparenchymal infusion of naked
`siRNA formulated in buffered isotonic saline, which results in silencing
`of specific neuronal molecular mRNA targets in multiple regions of
`the central and peripheral nervous systems47–50. With naked siRNA
`formulated in buffered isotonic saline, doses of 0.4 mg per day are typi-
`cally required for effective target gene silencing. Polymer complexation
`and lipid or liposomal formulations such as polyethylene imine (PEI),
`iFECT, DOTAP and JetSI/DOPE facilitate cellular uptake and reduce the
`doses of siRNA required for effective neuronal target silencing in vivo to
`approximately 5–40 µg51–54.
`Local viral delivery of shRNA to the nervous system has been
`reported in vivo with adenoviral, adeno-associated viral (AAV) and
`lentiviral delivery in normal mice55 as well as
`in animal models of spinocerebellar ataxia56,
`Huntington disease57,58, amyotrophic lateral
`sclerosis (ALS)59,60 and Alzheimer disease61,
`where abnormal, disease phenotypes includ-
`ing behavior and neuropathology were nor-
`malized. Notably, all of the in vivo studies to
`date have targeted genes expressed in neurons;
`it remains to be seen whether silencing in vivo
`can be achieved in other nervous-system cell
`types such as oligodendrocytes and astrocytes.
`Moreover, for endogenous neuronal tar-
`gets, expression of the target gene is typically
`reduced only partially, and in some cases by as
`little as 10–20%, yet this modest reduction in
`mRNA results in a marked effect on the specific
`behavior appropriate to the targeted gene.
`For application to oncology, direct delivery
`of siRNAs and viral delivery of shRNAs to
`tumors have been successful in inhibiting
`xenograft growth in several mouse models. A
`number of approaches—including lipid-based
`formulation (TransMessenger62) and complex-
`ation with PEI63, cholesterol-oligoarginine64, a
`protamine-Fab fusion protein65 and atelocolla-
`gen66,67—have been shown to facilitate delivery
`into tumor cells. Notably, these siRNA delivery
`approaches are effective with several or even a
`single intratumor injection of siRNA, at micro-
`gram doses. Very recently, aptamer-siRNA chi-
`meric RNAs have also been used successfully
`to facilitate siRNA delivery in vivo, resulting in
`
`Direct RNAi
`
`Lung
`RSV
`Flu
`SARS
`
`Eye
`
`AMD (wet)
`Nervous system
`Depression
`Alzheimer disease
`Huntington disease
`Spinocerebral ataxia
`ALS
`Neuropathic pain
`Encephalitis, West Nile virus
`Tumor
`Glioblastoma
`Human papillomavirus
`Prostate
`Adenocarcinoma
`Digestive system
`Irritable bowel disease
`Vagina
`HSV
`
`Figure 4 Organs for which RNAi proof of concept has been demonstrated. Direct RNAi represents
`local delivery of RNAi, and has been carried out successfully to specific tissues or organs, including
`lung, eye, the nervous system, tumors, the digestive system and vagina. Systemic RNAi represents
`intravenous delivery of RNAi and has been carried out successfully to lung, tumors, liver and joint.
`Specific disease models are indicated where efficacy was achieved.
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`R E V I E W
`
`In 2004, Soutschek et al. demonstrated effective silencing of the
`apolipoprotein apoB in mice by intravenous administration of choles-
`terol-conjugated siRNA duplexes6. Three daily injections of cholesterol-
`conjugated siRNA at a dose of 50 mg kg–1 resulted in silencing of the
`apoB mRNA by 57% and 73%, respectively, in the liver and jejunum, the
`two principal sites for apoB expression. The mechanism of action was
`proven, by 5′-RACE, to be RNAi mediated. This mRNA silencing pro-
`duced a 68% reduction in apoB protein abundance in plasma and a 37%
`reduction in total cholesterol. These therapeutically relevant findings
`were completely consistent with the known function of apoB in lipid
`metabolism. Cholesterol conjugation imparted critical pharmacokinetic
`and cellular uptake properties to the siRNA duplex.
`Further advances in systemic RNAi with optimized delivery have
`recently been reported. Recently, Zimmermann et al. made use of siRNA
`duplexes formulated in stable nucleic acid lipid particles (SNALPs39)
`to recapitulate the silencing of apoB in mice73. In rodent studies, the
`silencing produced by a single dose of SNALP-formulated siRNA at
`2.5 mg kg–1 was greater than that reported in the earlier study using
`cholesterol-conjugated siRNAs. More importantly, therapeutic silencing
`of apoB was also demonstrated in nonhuman primates. A single dose
`of 2.5 mg kg–1 siRNA encapsulated in the SNALP formulation reduced
`apoB mRNA in the livers of cynomolgus monkeys by more than 90%.
`As in the mouse experiments, apoB silencing was accompanied by sub-
`stantial reductions in serum cholesterol (>65%) and low-density lipo-
`proteins (>85%). Furthermore, silencing was shown to last for at least
`11 d after a single dose. In addition, the treatment seemed to be well
`tolerated, with transient increases in liver enzymes as the only reported
`evidence of toxicity. This primate study represented an important step
`forward in the development of systemic RNAi for therapeutic applica-
`tions. Moreover, the general applicability of SNALP formulations for
`hepatic delivery of siRNA has been demonstrated in animal models of
`HBV and Ebola virus infection74,75.
`In mouse tumor xenograft models, the efficacy of systemic RNAi has
`been demonstrated using a variety of delivery strategies (reviewed in refs.
`1,2,76). Systemically delivered cationic cardiolipin liposomes containing
`siRNA specific for Raf-1 inhibit tumor growth in a xenograft model of
`human prostate cancer77. Vascular endothelial growth factor receptor-2
`(VEGF-R2)-targeting siRNAs complexed with self-assembling nanopar-
`ticles consisting of polyethylene glycol-conjugated (PEGylated) PEI with
`an Arg-Gly-Asp peptide attached at the distal end of the PEG accumulate
`in tumors and cause inhibition of VEGF-R2 expression. Intravenous
`administration of these complexes into tumor-bearing mice inhibits
`both tumor angiogenesis and growth rate78. Simpler PEI formulations
`have also shown efficacy in xenograft tumor models79, as have complexes
`of siRNA duplexes with atelocollagen. Systemic administration of atelo-
`collagen–siRNA complexes has marked effects on subcutaneous tumor
`xenografts66 as well as bone metastases76. Another recently described
`delivery strategy made use of a recombinant antibody fusion protein to
`achieve cell type–specific delivery. As described above, Song et al. fused
`the nucleic acid binding protein protamine to the C terminus of a frag-
`ment antibody (Fab) targeting the HIV-1 envelope protein gp160. After
`systemic administration, the Fab-protamine fusi