Human Molecular Genetics

Human Molecular Genetics 2004 13(Review Issue 2):R275-R288; doi:10.1093/hmg/ddh224

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Human Molecular Genetics, Vol. 13, Review Issue 2 © Oxford University Press 2004; all rights reserved

RNA interference: from model organisms towards therapy for neural and neuromuscular disorders

Steven D. Buckingham¹, Behrooz Esmaeili¹, Matthew Wood² and David B. Sattelle^1,*

¹MRC Functional Genetics Unit and ²Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK

Received June 15, 2004; Accepted July 19, 2004

	ABSTRACT

TOP ABSTRACT THE RNAi MECHANISM—A BRIEF... APPLYING RNAi TO MODEL... RNAi IN WORM FLY... GENOME-WIDE RNAi SCREENS MUSCULAR DYSTROPHY SPINAL MUSCULAR ATROPHY FRAGILE X SYNDROME ALZHEIMER'S DISEASE POLY(Q) DISEASES PARKINSON'S DISEASE DEVELOPMENTAL DISORDERS ADDICTION RNAi, MODEL ORGANISMS AND... CONCLUSIONS AND PERSPECTIVES NOTE ADDED IN PROOF REFERENCES

 
Experimental RNA interference (RNAi) leading to the selective knockdown of gene function is induced by introducing into cells either double stranded RNA (dsRNA), or short interfering RNA (siRNA) fragments into which dsRNA is cut. The siRNA triggers degradation of homologous messenger RNA (mRNA). Widely used as a research tool in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster and mouse to investigate the function of individual genes, RNAi has also been deployed in genome-wide, specific gene-knockdown screens. Recent rapid progress in the application of RNAi to mammalian cells, including neurons and muscle cells, offers new approaches to drug target identification and validation. Advances in targeted delivery of RNAi-inducing molecules has raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. Here, we review examples of the application of RNAi to worm, fly and mouse models of such diseases aimed at understanding their pathophysiology and we address problems to be solved in developing RNAi-based therapies.

RNA interference (RNAi) is a powerful new gene knockdown technique that permits tissue-specific, temporally controlled suppression of gene expression. It exploits a highly conserved, endogenous mechanism thought to play a role in protection against double stranded RNA (dsRNA) viruses (1) and genome-invading transposable elements (2,3), as well as helping preserve genome stability in the germ line. When dsRNA is introduced into the cytoplasm of cells it is cut into short interfering RNAs (siRNAs) which ‘label’ homologous mRNA for degradation (Fig. 1). Thus, the expression of a particular gene can be suppressed by introducing dsRNA whose antisense strand sequence matches the mRNA sequence. In some organisms, such as Caenorhabditis elegans, the effect spreads to tissues remote from the site of application, allowing knockdown of the selected gene throughout the organism, although this effect is limited in certain tissues, including the nervous system (4).

View larger version (24K): [in this window] [in a new window]   Figure 1. The mechanism of RNAi which results in post-transcriptional knockdown of a gene product. RNAi is initiated by the application (soaking or injection) of dsRNA or by transfecting target cells with either siRNA, or with vectors encoding shRNAs. DICER cuts dsRNA into siRNAs, which are then directed onto the RISC where they direct the breakdown of complementary mRNA.

 
Gene knockdown by RNAi is temporary, but can be rendered stable by transfecting target tissues or cell lines so that they express dsRNA autonomously. Such expression can be constitutive or under the control of an inducible promoter, allowing the experimenter to induce knockdown at a specified time. Some non-specific RNAi effects have been reported (5), but with appropriate controls and physiological follow-up experiments RNAi has quickly become one of the most potent tools of modern reverse genetics. The versatility of the technique has led to a number of exciting applications. For instance, RNAi can be used in drug/chemical target validation. RNAi can target specific spliced exons, enabling the investigation of the functional roles of alternatively spliced forms of a gene. Furthermore, one of the most exciting opportunities offered by RNAi is the ability to identify all candidate genes required for certain physiological processes using genome-wide RNAi screens (6–10).

RNAi is particularly powerful when applied to genetic model organisms such as the fruitfly, Drosophila melanogaster, the nematode, C. elegans and the mouse, whose genomes have been fully sequenced and for which a wealth of genetic, physiological and some interactome data has been accumulated. Several models of human neural and neuromuscular disorders are available in these three experimental models (11–15). However, although the application of RNAi as a research tool or as a potential new therapy has been reviewed extensively (16–29), surprisingly few reviews have specifically assessed the application of RNAi to model organisms in the context of understanding neural and neuromuscular disorders. Here, we illustrate how applying RNAi in model organisms is opening up new experimental avenues in the study of neural and neuromuscular diseases. We also discuss briefly the possible application of RNAi to the development of new therapies for neural and neuromuscular disorders.

	THE RNAi MECHANISM—A BRIEF DESCRIPTION

TOP ABSTRACT THE RNAi MECHANISM—A BRIEF... APPLYING RNAi TO MODEL... RNAi IN WORM FLY... GENOME-WIDE RNAi SCREENS MUSCULAR DYSTROPHY SPINAL MUSCULAR ATROPHY FRAGILE X SYNDROME ALZHEIMER'S DISEASE POLY(Q) DISEASES PARKINSON'S DISEASE DEVELOPMENTAL DISORDERS ADDICTION RNAi, MODEL ORGANISMS AND... CONCLUSIONS AND PERSPECTIVES NOTE ADDED IN PROOF REFERENCES

 
Since the initial ground-breaking investigations of Fire et al. on C. elegans (30), our understanding of the RNAi mechanism has greatly increased (31,32). In Figure 1 the key molecular components for RNAi are summarized. The dsRNA is taken up into cells [in the case of C. elegans possibly by SID-1, an RNA transporter (33)] where the dsRNA strands are cut by DICER (an RNAse III) into duplexes (siRNAs) of about 21 nt length with about 2 nt 3' overhangs. The two strands of each siRNA are unwound in an ATP-dependent process (34) and attach themselves to the RNA-induced silencing complex (RISC), where they bind to target mRNA of perfectly complementary sequence. DICER associates with R2D2 (homologous to RDE-4 in C. elegans), which does not affect DICER's catalytic function but serves to pass the siRNA on to the RISC (35). The RISC is a supramolecular structure composed of at least five molecules [AGO2, VIG-1, ARMI, AUB and FXR-related (36,37)] in Drosophila. In Drosophila, C. elegans and mammals the RISC also comprises TUDOR-SN, which is probably the catalytic component (37). When the RISC binds to the target mRNA, the latter is degraded, thereby preventing its translation.

dsRNA can be introduced to C. elegans by microinjection into the animal or even soaking the animals in a medium containing dsRNA. RNAi gets into all the cells but with varying efficiency (38). The finding that dsRNA can be introduced by feeding worms with bacteria transformed to express dsRNA targeted against a gene of interest paved the way for the development of genome-wide, systematic RNAi screens.

Initially, it appeared that RNAi might be restricted to invertebrates. Long (>30 nt) dsRNA applied to mammalian cells was generally found to be impractical because it evoked the interferon response or a non-specific inhibition of protein synthesis through dsRNA-dependent protein kinases. Although this problem is not generally observed in invertebrate models (39), some non-specific effects of dsRNA on Drosophila S2 cells have been reported (40). The initial lack of success in applying RNAi to mammalian models was eventually overcome by the discovery that introducing siRNAs, the products of DICER action on dsRNA, directly into the cytoplasm either by conventional transfection, or through vectors expressing siRNAs (41), produces effective and specific silencing, without evoking a global shutdown of protein synthesis. Therefore, although vertebrate genetic models such as mouse and rat do not have the advantages of short generation time and simplicity offered by invertebrates, they do have a role to play in modelling human disease using RNAi by virtue of their closer similarity to man—less than 150 mouse genes lack a human equivalent.

Gene knockdown by RNAi is post-transcriptional and so does not invoke any compensatory transcription, as is the case for gene deletion. RNAi is specific and, in most cases, sensitive to even a single nucleotide mismatch. The short-hairpin RNA (shRNA) encoding transgenes can be placed under the control of an inducible promoter, such as a tissue-specific promoter. RNAi is also dose dependent, so the degree of knockdown can be controlled, although this is limited in C. elegans where RNAi amplification through RNA-dependent RNA polymerase activity is evoked.

The advantages of RNAi over techniques such as gene knockout, reside in its ability to reduce, in a dose-dependent manner, the expression of an mRNA without the dangers of embryonic lethality and with reduced risk of compensatory gene regulation. In addition, RNAi is fast, less expensive and in many cases almost as effective as genomic gene deletion. For example, applying RNAi to Drosophila using dsRNA directed against lush, white and dGq produced phenotypes indistinguishable from gene knockout (42). Applying RNAi to C. elegans is particularly easy and can even be achieved either by ‘feeding’ with appropriate constructs engineered into the Escherichia coli strain on which the worms feed in the laboratory (43), or by simply adding dsRNA to the culture medium (44), both of which are simpler than injection procedures. Moreover, RNAi can be combined very effectively with established functional genetic approaches. For example, bidirectional control of a gene can be effected by transfecting cells with a gene and balancing its action with RNAi (45). The use of GeneGun technology enables single copy gene inserts, and hence more robust RNAi control.

	APPLYING RNAi TO MODEL ORGANISMS

TOP ABSTRACT THE RNAi MECHANISM—A BRIEF... APPLYING RNAi TO MODEL... RNAi IN WORM FLY... GENOME-WIDE RNAi SCREENS MUSCULAR DYSTROPHY SPINAL MUSCULAR ATROPHY FRAGILE X SYNDROME ALZHEIMER'S DISEASE POLY(Q) DISEASES PARKINSON'S DISEASE DEVELOPMENTAL DISORDERS ADDICTION RNAi, MODEL ORGANISMS AND... CONCLUSIONS AND PERSPECTIVES NOTE ADDED IN PROOF REFERENCES

 
The use of model organisms has been of fundamental importance to functional genetics. Often, with complete genomes sequenced and annotated, these organisms (including C. elegans, D. melanogaster, zebra-fish, puffer-fish and mouse) form the focus of networked research communities sharing a vast body of published and unpublished data. The most extensive studies to date have been performed on worm, fly and mouse, or cells derived therefrom, and these are the focus for this review (Fig. 2). A new, publicly available database stores the results of RNAi experiments (http://www.rnai.org) (50). Examples of the impact of RNAi are found in studies on C. elegans models of Alzheimer's disease (51), polyglutamine [poly(Q)] repeat diseases (52), spinal muscular atrophy (53), Drosophila models of poly(Q) repeat diseases (54), spinal muscular atrophy (55), amyotrophic lateral sclerosis (56), Parkinson's disease (57) and a vertebrate (cell line) model of spinal muscular atrophy (58). The high degree of conservation of genes controlling fundamental physiological and developmental processes is the basis of a homology-based strategy, in which genes underlying human inheritable diseases are identified and either invertebrate orthologues sought or transgenic lines raised, which express the human gene heterologously. Identifying the functions of the invertebrate orthologues and the effects of disease-related mutations can then provide insights into the functions of the human counterparts and the aetiology of the disease states.

View larger version (66K): [in this window] [in a new window]   Figure 2. Molecular components of the RNAi mechanism involved in digestion of dsRNA into siRNA and RISC formation. The RISC components appear to be conserved across species with a few exceptions. For example, FXR has been associated with the RISC complex in humans and Drosophila but not in C. elegans. As the human RISC has not yet been fully characterized, micro-RNA associated protein complex, eIF2C2, Gemin3 and Gemin4 are omitted from the table (47–49). C, C. elegans, D, Drosophila; H, human. Drosophila image: http://www.hagvanlaralemi.addr.com/guzellikler/guzellik1-80.php; mouse image: http://www.csms.edu/csri/korenberg/mousemolecular.html.

 

	RNAi IN WORM FLY AND MOUSE MODELS

TOP ABSTRACT THE RNAi MECHANISM—A BRIEF... APPLYING RNAi TO MODEL... RNAi IN WORM FLY... GENOME-WIDE RNAi SCREENS MUSCULAR DYSTROPHY SPINAL MUSCULAR ATROPHY FRAGILE X SYNDROME ALZHEIMER'S DISEASE POLY(Q) DISEASES PARKINSON'S DISEASE DEVELOPMENTAL DISORDERS ADDICTION RNAi, MODEL ORGANISMS AND... CONCLUSIONS AND PERSPECTIVES NOTE ADDED IN PROOF REFERENCES

 
C. elegans
The nematode C. elegans is the most thoroughly understood experimental animal for investigating human disease-related genes (Fig. 3). C. elegans provides a powerful genetic tool kit for studying development and neurobiology, as its complete cell lineage is known, its synaptic connections mapped and its genome fully sequenced. Moreover, it has been estimated that 50% of human disease genes have a C. elegans counterpart (12). Therefore, a great deal can be discovered about human biology and disease. The animal is particularly well suited for neurobiological studies. With only 302 neurons, the animal is able to perform a diverse repertoire of behaviours. Furthermore, many of the classical vertebrate neurotransmitters are used in the nervous system and at neuromuscular junctions.

View larger version (37K): [in this window] [in a new window]   Figure 3. RNAi in the worm. (A) Schematic diagram of C. elegans GABAergic neurons: the name of all neurons are indicated below the cell bodies (white circles); DC dorsal nerve cord, CO, commissure; VC, ventral nerve cord. (B) Wild-type expression morphology of GABAergic motorneurons visualized by unc-47::GFP a GABAergic marker. (C) Commissural defects associated with mig-15 RNAi knockdown. (D) Similar axon guidance defects seen in mig-15(rh148) mutant animals (48). Transgenes expressing gfp hairpins are triggered for RNAi. (E) The expression pattern of myo-3::GFP in body wall muscle cells. (F) myo-3::gfp hairpin efficiently reduces the expression pattern of myo-3::GFP in body wall muscle cells (47). Width of field (B,C,D) is 100 µm.

 
Although C. elegans has proved particularly useful in applying functional genomics, its many advantages have been somewhat offset by its limited usefulness for physiological studies owing to its small size and the inaccessibility of its cells. Electrophysiology is a case in point, but in recent years the development of methods to record in semi-intact preparations from body wall muscle and neurons (59) and even optical recordings of neural activity (60) have enabled important advances, although physiological approaches remain technically demanding. Recently, however, culturing of C. elegans embryonic muscle cells and neurons, which are readily accessible to RNAi, has been reported (61), so this aspect of physiology is set to develop rapidly. C. elegans has also been of particular use in unravelling many aspects of the mechanism of RNAi, which is highly conserved from worm to man.

D. melanogaster
Drosophila also has a powerful genetic toolkit, including a wealth of behavioural mutants and is more convenient for physiological studies than C. elegans. Again, many of the neurotransmitters found in vertebrates are also found in Drosophila, though unlike C. elegans the neurotransmitter at the neuromuscular junction is L-glutamate rather than acetylcholine. RNAi cannot be introduced by feeding dsRNA to Drosophila, but Drosophila has been stably transformed to express an extended hairpin loop dsRNA (62). The resulting specific suppression of genes was found to be heritable. For transient knockdown, dsRNA can be successfully introduced into adult Drosophila by injection of dsRNA into the abdomen, a technique which has been shown to produce effective gene knockdown of a lacZ transgene and GM06434 (the Drosophila homologue of the C. elegans gene nrf—nose resistant to fluoxetine) by RNAi in the nervous system (63).

A number of cell lines from Drosophila are available and offer particular advantages for detailed genome-wide, functional analyses (64). Cultured cells provide readily available material for physiological and molecular studies without the variability and additional preparation associated with animal tissues. Many techniques, such as calcium imaging and electrophysiology, benefit from using isolated cells, and employing stable cell lines avoids the variability associated with primary cultures. Experimenters are often able to obtain cell lines that do not express a human disease orthologue, so that the gene of interest can be expressed heterologously, with the important consequence that the subunit composition of the expressed protein is known. For example, using the Drosophila S2 cell line stably transfected with an insect muscarinic acetylcholine receptor allowed detailed analysis of the spatiotemporal aspects of calcium signalling (65), and RNAi knockdown to silence downstream calcium signalling genes revealed the requirement, for calcium signalling in these cells, of IP3 receptors and the SERCA pump, but not ryanodine receptors (66). The amenity of cell lines to rapid assays, such as cell death or dye-based assays, is of considerable benefit in developing genome-wide RNAi screens. RNAi knockdown has also been used to dissect the insulin signalling pathway (67). Studies combining RNAi and S2 cells have been deployed to examine the role of the S13 subunit, a component of the 19S complex, in the proteolytic activity of the 26S proteasome (68) and the role of ß-catenin in WNT signalling (69).

Although the nervous system of D. melanogaster is comparatively resistant to RNAi, genomic cDNA fusions encoding dsRNA were effective in Drosophila neurons (42). Unfortunately, no equivalents of the rrf-3 or the eri-1 mutations that are hypersensitive to RNAi in the nervous system of C. elegans have yet been described in Drosophila.

Vertebrate models: mouse
With the recent completion of the mouse genome, mouse models offer advantages that stem from their closer similarity to man, and hence greater ease of application of findings to human disease. Also, a number of projects to integrate expression data into brain maps have been initiated, including the Allen Brain Atlas project at the Allen Institute for Brain Science in Seattle, WA, USA, and the Mouse Atlas Project at the University of California at Los Angeles (http://www.loni.ucla.edu/MAP/About_MAP/index.html).

The power of the mouse model in RNAi studies is particularly evident in studies relating disease pathogenesis to the development of RNAi-based therapies. The demonstration that siRNAs of 21–23 nt transfected directly into mammalian cells could bypass the host interferon response provided one strategy for the application of RNAi to mammalian systems. The other approach, as for Drosophila, has been the development of shRNA expression systems (70,71). The generation of inducible mammalian shRNA systems and also a more rational approach to shRNA design, perhaps incorporating more design features of mouse and human microRNAs, will lead to greater efficiency and specificity in the application of this approach. Moreover, the development and optimization of a range of viral delivery vectors for shRNA expression will allow efficient transduction of mammalian cells and widespread application both in vitro and in vivo. Finally, shRNA expression can be used to create germline transgenic mice, complementing standard gene knock out approaches (72). Once fully exploited, this method will provide a powerful means for tissue-specific, inducible and reversible gene suppression in mice. It should also offer a route for transgenic RNAi in animals where homologous recombination is not possible owing to the lack of suitable ES cell lines.

	GENOME-WIDE RNAi SCREENS

TOP ABSTRACT THE RNAi MECHANISM—A BRIEF... APPLYING RNAi TO MODEL... RNAi IN WORM FLY... GENOME-WIDE RNAi SCREENS MUSCULAR DYSTROPHY SPINAL MUSCULAR ATROPHY FRAGILE X SYNDROME ALZHEIMER'S DISEASE POLY(Q) DISEASES PARKINSON'S DISEASE DEVELOPMENTAL DISORDERS ADDICTION RNAi, MODEL ORGANISMS AND... CONCLUSIONS AND PERSPECTIVES NOTE ADDED IN PROOF REFERENCES

 
Invertebrates have proved useful in high-throughput RNAi screens. The ease with which RNAi can be applied has prompted the development of a library of bacteria expressing dsRNAs representing 86% of the estimated 19 000 C. elegans genes (6,10), greatly facilitating genome-wide screens of this species using RNAi. A similar genome-wide screen using the RNAi-hypersensitive mutant rrf-3 (PK1426) has also been conducted (8).

Approaches that apply global RNAi screens to ageing-related disorders are likely to be of considerable benefit. A systematic genome-wide RNAi search for genes whose inactivation lengthened lifespan in C. elegans, confirmed a link between mitochondrial function and longevity, but showed for the first time that this is not due simply to reduced free radical production in long-lived worms, but that the link between mitochondrial function, insulin signalling and longevity is complex, involving several parallel pathways (73). Furthermore, genome-wide RNAi studies have the potential to contribute to research areas that suffer from a lack of viable mutants. G-protein coupled receptors (GPCRs), for instance, are expected to underlie many cellular and physiological mechanisms, yet there is a paucity of GPCR mutants. A genome-wide search of over 60 GPCRs, however, highlighted 13 GPCR genes (74) with behavioural RNAi phenotypes, particularly featuring locomotion and reproduction. Receptors to neuropeptide Y (NPY) featured strongly in this set, a finding consistent with the predominance of NPY in the body wall and reproductive tract of nematodes. Genome-wide screens of C. elegans have also lead to the discovery of TAC-1, a major regulator of microtubule length (75), and novel genes involved in transposon silencing, a phenomenon which prevents transposon activity in the germ line, as well as many genes involved in early embryognesis (76).

A global RNAi screen by Nollen et al. (77) has identified key molecular components that participate in poly(Q) aggregation in C. elegans. Such a global approach to the identification of proteins involved in poly(Q) disease pathogenesis could accelerate progress in Huntington's disease therapy by indentifying novel drug target candidates. Similarly, a large-scale RNAi screen (43% of the Drosophila genome) on wing imaginal disc-derived cl-8 cells was used to uncover genes essential in Hedgehog signalling (78).

The ability to extend genome-wide RNAi screens from worm and fly to mouse and ultimately to human model systems will be very powerful. Two groups have recently described the construction of first generation mouse and human RNAi libraries, and have provided some preliminary biological validation (79,80). The former provided evidence that this approach could be used to screen for new modulators of p53-dependent proliferation arrest, whereas the latter was successfully screened to identify gene products playing a critical role in proteasome function. The construction of second generation mammalian RNAi libraries is now underway incorporating improved vector technology and rational shRNAi design.

Interpretation of genome-wide screens assumes that gene silencing is specific. It has been shown (81) that targeting RNAi against three genes in human carcinoma cell lines produced similar changes in mRNA levels for those genes as determined by DNA microarrays, but with no measurable effect on non-targeted genes. However, another study showed that in cultured human cells siRNAs can affect off-target mRNAs with as few as 11 nt in common (82). This indicates that although the majority of RNAi studies report high specificity, results from RNAi experiments must be confirmed using complementary techniques, including the generation of mutants of the genes of interest. This is particularly important in the context of whole-genome approaches, where it is impractical to optimize each siRNA for specificity.

Genome-wide uses of RNAi have understandably provoked considerable interest, placing a premium on finding ways to generate RNAi libraries rapidly. A recent report describes an enzymatic method for generating a shRNAi library that was successful when used with a variety of mammalian cell cultures, and which can be combined with high-throughput screening to find optimal shRNAi clones (83). This approach begins with random cutting of source DNA followed by a ligation to a hairpin adaptor resulting in a shRNAi library that has a high proportion of inverse repeats. To select the optimum shRNA, the target gene is fused with a thymidine kinase gene and inserted into a host cell line. Cells expressing efficient shRNA libraries survive exposure to ganciclovir owing to the thymidine kinase activity, allowing the effective libraries to be recovered using PCR amplification. With the emergence of these resources, the time is clearly ripe for such applications to be made to these genetic model organisms.

	MUSCULAR DYSTROPHY

TOP ABSTRACT THE RNAi MECHANISM—A BRIEF... APPLYING RNAi TO MODEL... RNAi IN WORM FLY... GENOME-WIDE RNAi SCREENS MUSCULAR DYSTROPHY SPINAL MUSCULAR ATROPHY FRAGILE X SYNDROME ALZHEIMER'S DISEASE POLY(Q) DISEASES PARKINSON'S DISEASE DEVELOPMENTAL DISORDERS ADDICTION RNAi, MODEL ORGANISMS AND... CONCLUSIONS AND PERSPECTIVES NOTE ADDED IN PROOF REFERENCES

 
RNAi knockdown of genes in Drosophila and C. elegans to mimic loss of function mutations is helping to elucidate the mechanisms of a number of muscle wasting diseases. These are degenerative disorders marked by progressive paralysis of body muscle ending in premature death. Duchenne muscular dystrophy (DMD), the major muscular dystrophy in children, results in a mean death age of 25.3 years, which can be shortened further due to cardiomyopathy complications (84).

DMD is associated with mutations in the dystrophin gene, which encodes key components of a muscle complex comprising transmembrane and cytoplasmic proteins. A number of these proteins have orthologues in C. elegans (85). The discovery that a homologue of the dystrophin gene (dys-1) is present in C. elegans led Grisoni et al. (85) to search for nematode homologues of other members of the dystrophin complex. A number of homologues were found, and RNAi knockdown of the conserved orthologues resulted in phenotypes similar to that seen in dys-1. A homologue (dmDp186) of the human dystrophin gene occurs in Drosophila, and the function of dystrophin and related genes are being investigated using RNAi. Mouse models of DMD have not yet been investigated using RNAi.

RNAi can be applied in the context of a positive transgenic background to provide bidirectional control of gene expression. For instance, a protein-coding gene and a shRNA directed against that gene, each under the control of different inducible promoters, can be transfected into the same cell. This approach has been applied to C. elegans to enable bidirectional control of transmembrane calcium flux in an attempt to unravel the poorly understood involvement of elevated intracellular calcium in DMD (45). EGL-19 is a C. elegans calcium channel and Mariol and Segalat (45) used the gain-of-function mutation to enhance the signal and RNAi knockdown of egl-19 to diminish it. Enhancing the calcium signal resulted in enhanced muscle degeneration, and this was blocked by RNAi knockdown of egl-19. Clearly, there is considerable potential for the application of RNAi to worm and fly models of this disease.

In humans, the X-linked form of Emery-Dreifuss muscular dystrophy is caused by the loss of function of emerin, a nuclear membrane LEM-domain protein. Emerin is a member of the lamin class of proteins that form networks of filaments in the inner nuclear envelope and are essential for maintaining nuclear shape, DNA replication and transcription (86). Although the C. elegans equivalent to emerin, EMR-1, is homologous to its vertebrate counterpart, RNAi knockdown of emr-1 produced no detectable phenotype in C. elegans at any stage of development (87). This suggested that functional loss of EMR-1 was being compensated for by another pathway. To test the hypothesis that EMR-1 overlaps functionally with the structurally similar MAN-1, for which no function was known, it was established that RNAi knockdown of MAN-1 alone was lethal in 15% of embryos, but MAN-1 knockdown in the absence of EMR-1 was lethal to all embryos (88). Thus, applying RNAi approaches using the worm model suggests that EMR-1 and MAN-1 overlap functionally, raising hopes for a gene replacement strategy as a therapy for Emery-Dreifuss muscular dystrophy.

	SPINAL MUSCULAR ATROPHY

TOP ABSTRACT THE RNAi MECHANISM—A BRIEF... APPLYING RNAi TO MODEL... RNAi IN WORM FLY... GENOME-WIDE RNAi SCREENS MUSCULAR DYSTROPHY SPINAL MUSCULAR ATROPHY FRAGILE X SYNDROME ALZHEIMER'S DISEASE POLY(Q) DISEASES PARKINSON'S DISEASE DEVELOPMENTAL DISORDERS ADDICTION RNAi, MODEL ORGANISMS AND... CONCLUSIONS AND PERSPECTIVES NOTE ADDED IN PROOF REFERENCES

 
Spinal muscular atrophy (SMA) is associated with lower motor neuron loss in the spinal cord caused by the mutations in the survival motor neuron protein, SMN. However, it is not clear how the loss of SMN is linked to the death of neurons. Worm, fly and mouse models of SMA have been generated.

C. elegans also has an orthologue of SMN, which is ubiquitously expressed (53). Knockdown of the worm SMN protein using RNAi results in poor embryonic viability and severely uncoordinated locomotion (86). The results of yeast two-hybrid experiments suggested that some C. elegans SMN protein interactions are similar to their human equivalents (86). Drosophila has an orthologue of SMN (dSMN) (55) and has been used as a model of this disease. A Drosophila smn gene point mutation results in a phenotype that includes abnormal motor behaviour, functionally impaired neurons and altered synaptic transmission (55). RNAi suppression of SMN in Drosophila S2 cells resulted in a significant increase in apoptosis (89) through a pathway involving the caspases DRONC and DRICE. The effect was reversed by the caspase inhibitor, Z-VAD-fmk, suggesting a possible target for novel therapies. Mouse models of SMA are available (90,91), but gene deletions of SMN are lethal, although heterozygous SMN^+/– mice survive and have up to 46% reduction of SMN and display motor neuron degeneration similar to that seen in spinal muscular atrophy type 3. More recently, RNAi has been shown to be effective in knocking down expression of SMN in mouse P19 cells (Fig. 4) (58), suggesting that this approach may provide a convenient model for the investigation of SMA.

View larger version (84K): [in this window] [in a new window]   Figure 4. Silencing of mouse SMN expression by RNAi. Mouse SMN expression (shown in green) in embryonal P19 cells as determined by immunofluorescence cytochemistry, with propidium iodide nuclear staining shown in red: (A) 72 h after transfection of scrambled siRNA controls and (B), 72 h after transfection of specific siRNAs against the mouse SMN sequence. Field width (A and B) is 150 µm. The authors are indebted to Dr Barbara Trulzsch for this illustration.

 

	FRAGILE X SYNDROME

TOP ABSTRACT THE RNAi MECHANISM—A BRIEF... APPLYING RNAi TO MODEL... RNAi IN WORM FLY... GENOME-WIDE RNAi SCREENS MUSCULAR DYSTROPHY SPINAL MUSCULAR ATROPHY FRAGILE X SYNDROME ALZHEIMER'S DISEASE POLY(Q) DISEASES PARKINSON'S DISEASE DEVELOPMENTAL DISORDERS ADDICTION RNAi, MODEL ORGANISMS AND... CONCLUSIONS AND PERSPECTIVES NOTE ADDED IN PROOF REFERENCES

 
The native RNAi mechanism may itself be implicated in at least one disorder. Fragile X is an inheritable human mental retardation disorder that is associated with low or failed expression of FMR1 (92,93). Recent studies have shown that Fragile X may represent a disease resulting directly from a disorder in the operation of the native RNAi mechanism (92,93). There are mouse (94), worm (95) and fly (96) models of this disease, and the Drosophila homologue of FMR1 was recently shown (97) to include ARGONAUTE 2, a component of the RISC, and to be closely associated with DICER. As Drosophila and especially C. elegans are so amenable to RNAi, and have furnished the most detailed understanding of RNAi so far, the presence of a candidate FMR1 homologue merits further investigation.

	ALZHEIMER'S DISEASE

TOP ABSTRACT THE RNAi MECHANISM—A BRIEF... APPLYING RNAi TO MODEL... RNAi IN WORM FLY... GENOME-WIDE RNAi SCREENS MUSCULAR DYSTROPHY SPINAL MUSCULAR ATROPHY FRAGILE X SYNDROME ALZHEIMER'S DISEASE POLY(Q) DISEASES PARKINSON'S DISEASE DEVELOPMENTAL DISORDERS ADDICTION RNAi, MODEL ORGANISMS AND... CONCLUSIONS AND PERSPECTIVES NOTE ADDED IN PROOF REFERENCES

 
Alzheimer's disease is associated with the accumulation of insoluble plaques of amyloid protein (Aß) in the central nervous system, as well as intracellular microfibrillar tangles, along with loss of cholinergic neurons projecting from the basal forebrain to the hippocampus and amygdala. Plaque deposition is a result of overproduction of Aß through the activity of -secretase, of which the presenilins are thought to be a component. In the only C. elegans model of Alzheimer's disease currently available, worms are engineered to express the human Aß polypeptide (14,51) in response to, for example, heat shock. This model was used in microarray studies to identify the genes up-regulated and down-regulated as a result of the heterologous expression of Aß (98), and should also prove useful in global RNAi approaches designed to identify genes regulating the secretion of Aß. Although this has not yet been attempted on this model, a similar approach was recently applied to a C. elegans model of poly(Q) expansion diseases, leading to the discovery of 186 genes controlling poly(Q) expansions and age-dependent protein misfolding (77). In the worm Alzheimer's disease model, the Aß is expressed only in muscle. Clearly, there is a need for improved invertebrate models of Alzheimer's disease, particularly for a C. elegans model in which human Aß is expressed in neurons and is secreted.

A Drosophila model of Alzheimer's disease has been generated that expresses APP (99), but it is not known if the APP is processed into Aß. More recent models of Alzheimer's disease have been reported in which Aß is expressed in the CNS, resulting in neurodegeneration and shortened lifespan (100,101). These models offer exciting possibilities for RNAi-based studies.

However, RNAi applied to Drosophila cell lines is a potentially powerful alternative approach in determining the pathways involved in the development of Alzheimer's, as the Drosophila equivalent to human presenilins PS1 and PS2 (PSN) appears to be involved in similar pathways to its vertebrate counterparts (102). Early onset familial Alzheimer's, for example, is associated with mutations in presenilin genes PS1 and PS2 which lead to over secretion of Aß. RNAi knockdown of PSN results in a blockage of -secretase activity (102), providing confirmation of this gene's role in the -secretase pathway. Another study (103) used RNAi to reduce expression of two genes, aph-1 and pen-2, in Drosophila S2 cells. Knockdown of these genes resulted in reduced proteolytic cleavage of Aß precursor protein and Notch substrates and reduced production of processed presenilin, suggesting that they are required for the action of, and the accumulation of, the -secretase.

The potential for RNAi in studies using these models is considerable and has yet to be fully exploited in Drosophila S2 cells, but similar approaches using COS cells have helped elucidate the identity of the -secretase. The increased levels of Aß in Alzheimer's disease caused by the -secretase is thought to be counteracted by -secretases that cleave the precursor protein in the middle. The identity of the -secretase is uncertain, but a recent study (104) showed that RNAi knockdown of each of three ADAM (‘a disintegrin and metalloprotease’) proteins, ADAM9, ADAM10 and ADAM17, in human glioblastoma A172 cells, which have elevated endogenous -secretase, indicating that all three of these proteins are involved in the -secretase activity. An extension of this approach in mammalian systems will also enable potential gene targets for therapeutic intervention to be identified. For example, siRNAs targeting the ß-secretase, BACE1, were shown to reduce APP production in mouse cortical neurons, offering a potential therapeutic approach for AD (105).

	POLY(Q) DISEASES

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There are at least nine human neurodegenerative disorders that are caused by expansion of the CAG trinucleotide repeat. RNAi has recently been performed on two prevalent poly(Q) diseases, Huntington's disease (HD) and spinobulbar muscular atrophy (SBMA). HD is an autosomal dominant hereditary brain disorder that is progressive and fatal. Mutations in the Huntingtin gene result in involuntary movement (chorea), cognitive impairment and psychiatric problems such as depression and anxiety (106). HD is caused by expansion of a CAG trinucleotide repeat in exon-1 of the Huntingtin gene. This expansion elongates the N-terminal poly(Q) stretch of the protein, resulting in aggregation and the formation of neuronal intranuclear inclusions. A number of treatment strategies have been proposed that target Huntingtin proteolysis, aggregation and transcription (107).

SBMA is an X-linked motorneuron disease that occurs at adulthood (108). This progressive disease results in the loss of motorneurons in the lower spinal cord and the brain stem and is caused by CAG trinucleotide expansion in the first exon of androgen receptor (AR). Unaffected individuals have between 11 and 35 poly(Q) repeats as opposed to 38 and 62 repeats in SBMA individuals (109). This poly(Q) expansion results in intranuclear aggregates formation in tissues where the AR gene is normally expressed.

One laboratory (77) made transgenic strains that expressed poly(Q₃₅) expansion fused with yellow fluorescent protein and used it in a global RNAi screen. This screen identified 186 genes that resulted in premature appearance of protein aggregates. The genes identified are mainly involved in RNA metabolism, protein synthesis, protein folding, protein degradation and protein trafficking. It would be interesting to see whether the same genes control other protein aggregation diseases such as Alzheimer's.

Drosophila S2 cells were engineered to express a portion of human ar gene with CAG tracts of 26, 43 or 106 repeats tagged by green fluorescent protein (GFP) (110). Cells carrying the CAG repeats of 43 and 106 developed GFP aggregates and aggresomes with 106 aggregates being formed much faster than 43 CAG repeats. Using RNAi directed against AR protein, Caplen et al. (110) showed loss of ARGFP aggregates by 80% in co-transfected S2 cells. Therefore, RNA interference could have considerable therapeutic potential in poly(Q) neurodegenerative disorders.

	PARKINSON'S DISEASE

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Where a disorder results from a reduction in gene expression, RNAi can be used to mimic gene function loss. Parkinson's disease, for example, is associated with loss of dopaminergic neurons, and as with SMA, RNAi will be an important approach for exploring the molecular mechanisms underlying Parkinson's disease. Recently, this approach has been used in combination with overexpression studies to explore the role of Parkin, an E3 ubiquitin ligase, in dopamine neuron degeneration in Drosophila. Overexpression of Parkin was shown to degrade its substrate (Pael-R) and suppress its toxicity, whereas interfering with endogenous Parkin promoted substrate accumulation and augmented its neurotoxicity (111).

Viral-mediated RNAi has been used (112) to block dopamine synthesis in mid-brain neurons of adult mice. This study used an adeno-associated virus vector in which a U6 promoter drove the expression of shRNA directed against tyrosine hydroxylase, an enzyme required for the production of dopamine. This was injected stereotactically into the substantia nigra of one side of the brain and a similar vector promoting the expression of a randomized shRNA injected into the other side. GFP expression was observed in both halves of the brain, but dopamine staining was reduced only in the side of the brain into which the anti-tyrosine hydroxylase shRNA had been injected. The resultant behavioural deficits included loss of motor performance: bilateral shRNA knockdown of dopamine synthesis resulted in reduced motor activity in response to amphetamine (a well-established dopamine-dependent behaviour) and poorer performance in the rotarod test. Although only 30–40% protein knockdown was reported, these results are similar to previously established toxin-induced models of dopaminergic neuron loss, suggesting that RNAi-induced knockdown of dopamine synthesis furnishes a reasonably faithful model of Parkinson's disease.

	DEVELOPMENTAL DISORDERS

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RNAi knockdown removes mRNA leaving the parent gene intact. This can be an advantage over gene knockout studies, where the absence of a functional gene may induce compensatory expression. This may explain some contradictory findings in research into double cortical syndrome. This neurological disorder is associated with the doublecortin (DCX) gene, but gene deletion approaches had failed to mimic the syndrome. Mice in which the DCX gene is deleted develop normal cortices. However, directing plasmid-mediated dsRNA against DCX caused disruption of radial migration of developing cortical neurons (113), suggesting that neurons lacking the DCX gene select an alternative migration mechanisms. The DCX knockdown model might therefore serve as a useful experimental model of double cortical syndrome. To date there are no known worm or fly models of DCX.

	ADDICTION

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According to the National Center for Disease Control and Prevention, there are over 20 000 alcohol-induced deaths each year in the US alone, not including motor vehicle fatalities (http://www.cdc.gov). The neural and genetic basis of ethanol addiction is poorly understood, but is a complex phenomenon involving several receptor types (114). RNAi offers a complementary approach to dissecting the roles of these receptors in ethanol addiction. GABA receptors have a possible role in addiction, and RNAi reduction in expression of the R1 subtype of the GABA receptor in D. melanogaster reduced the behaviour-impairing effects of alcohol (115).

Nicotine addiction, despite its well-known health hazards, is common around the world (116). RNAi is a promising approach for unravelling the gene networks known to be involved in invertebrate models of nicotine tolerance (117).

	RNAi, MODEL ORGANISMS AND THE DEVELOPMENT OF NEW THERAPIES

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RNAi-mediated gene knockdown offers many opportunities for the development of new therapies, especially when coupled with the advantages of using model organisms. Initial studies into the application of RNAi as a therapy in non-neural diseases have been encouraging. Preliminary trials using a mouse model of hepatitis have suggested that RNAi may be of use in the treatment of hepatitis (118–120). Successful application of RNAi to mouse models of various cancers (121,122) and even RNAi approaches aimed at combating malaria (123) have been pursued.

Invertebrate models also play a part in exploring possible routes to RNAi therapies. Progressive SBMA, or Kennedy syndrome, is a subtype of SMA that affects adult males. It is caused by an expansion of a CAG repeat in the first exon of the AR resulting in a receptor with a string of 38–62 glutamines instead of the normal 9–36 (108). To determine the potential usefulness of RNAi as a basis for therapy for SBMA, RNAi was tested for its ability to block transcription of a truncated AR containing CAG repeats in Drosophila cells (110). dsRNAs suppressed transcription only when encoding CAG repeats were flanked by receptor-coding regions: those encoding CAG repeats alone had no effect, pointing to the potential usefulness of RNAi as a means of selectively inhibiting a gene associated with a polyglutamine disorder (110).

Enhanced methods of driving RNAi interference
As our understanding of the basic mechanisms of RNAi increases, new methods for improving RNAi efficacy will emerge. In addition, the sequence requirements for optimum RNAi knockdown efficiency have been examined (124). Successful therapeutic RNAi depends upon efficient transfection. The use of lipofection as the means of introducing the siRNAs led to low transfection rates in some cells and transience in the silencing effect. These problems were overcome by the use of vector-based siRNA expression systems using RNA polymerase III promoters (71,125–131). It has been shown (132) that siRNA vectors transfected into embryonic mouse stem cells suppresses ubiquitously expressed GFP throughout the body, indicating that this technique works across the animal and also indicates that this approach can be used for species for which embryonic stem cell lines are not available.

Lentiviruses have been shown to improve the efficiency of delivery to a variety of different cell types, thus improving the potential usefulness of RNAi as therapy (133–136). Lentivirus-mediated transfection of shRNA was shown to be long term and mediate stable RNAi in a dose-dependent manner (134). The shRNAs are effective at bringing about RNAi and can be synthesised in vitro or transcribed from RNApol III in vivo, allowing the development of stably transfected, gene-suppressed cell lines (137).

Inducing RNAi through viral transfection has been applied successfully to a number of mammalian systems. Viral-mediated delivery was shown to reduce expression of target genes in the brain and liver (138), including a reduction in poly(Q) accumulation in cells in model of neurodegeneration, whereas viral-mediated RNAi directed against the tyrosine hydroxylase (the enzyme that produces dopamine) gene, Th, produced distinct changes in behaviours associated with the mid-brain (112). Viral transfection can be localized to allow tissue-specific RNAi.

However, despite these improved transfection methods, an inherent variation in the effectiveness of RNAi remains. This requires careful attention to siRNA design. The effectiveness of siRNAs in producing RNAi is dependent upon the siRNA sequence (139), but the effectiveness of a sequence is difficult to predict. However, the efficiency of any siRNA against a gene can be rapidly assessed by measuring their ability to suppress signals from reporter-target constructs linked to the gene of interest (enhanced green fluorescent protein, or red fluorescent protein or Renilla luciferase) (140). Alternatively, a PCR-based strategy (141) is available which allows rapid screening of candidate siRNAs without subcloning. A complementary approach to enhancing the effectiveness of RNAi is to exploit some known component of the RNAi pathway. For instance, uptake of dsRNA from soaking solution can be enhanced by transfecting cells with SID-1 (33).

RNAi induction is, however, also variable between tissues, with some tissues, notably the nervous system, being resistant to its effects (142), although knockdown in neurons can be effected by using very high concentrations of dsRNA. This has been overcome much more effectively by the discovery of a mutant strain (rrf-3) that is hypersensitive to RNAi (4,8). Loss of RRF-3, which encodes an RNA-directed RNA polymerase, increased sensitivity of the worms to RNAi especially in the nervous system (4). For example, feeding a genome-wide RNAi library to a standard (Bristol N2) strain revealed the phenotypes of 10% of genes (6), but this was elevated to 23% when using rrf-3 worms (8).

The resistance of the nervous system to RNAi has also been addressed by transforming worms with a transgene directing the expression of the dsRNA as a hairpin construct. This approach is, however, highly labour intensive. To circumvent this, the development of ‘pWormgate’, a high-throughput cloning system allowing the rapid generation of hairpin RNAi constructs for most C. elegans genes, has recently been reported (143). In addition, by using the eri-1 (146) mutant C. elegans siRNase can be removed from neurons, leading to enhanced RNAi. Transformation of the worms using this system is accomplished conveniently using a gene gun (144). Interestingly, resistance to RNAi in neurons is also a feature of vertebrates, although a recent study successfully transfected hippocampal neurons in culture to produce synthetic siRNA, which was found to promote effective RNAi (145).

Targeting RNAi to particular tissues
One of the advantages of RNAi over gene knockout is the ability to restrict gene knockdown to specific tissues or even cell types. This is important where a disease is a result of a mutation in an essential gene. SMA is associated with mutations in the SMN-1 gene (146), but null mutations of SMN-1 are embryonically lethal (91,147), making the generation of vertebrate models of this disease complex. The use of tissue-specific hairpins will facilitate the development of new cell targeting strategies (58).

Various approaches have been used to ensure that RNAi is tissue or time specific. One approach is to place a vector under the control of a tissue or developmentally specific promoter. In one set of studies (148,149) dsRNA was under the control of an oocyte-specific promoter in mouse oocytes, and this resulted in healthy adults with efficient RNAi occurring only in the context of oogenesis. Some studies have placed heterologous dsRNA expression under the control of an inducible promoter, such as the tetracycline-dependent promoter, so that dsRNA expression can be induced at will (150–152). Thus, by placing hairpin siRNAs under the control of a tissue-selective promoter and an inducible promoter (e.g. antibiotic or heat-shock) can allow tissue- and time-specific RNAi.

The success of RNAi-based therapies will also depend in part upon the ability to control the timing of RNAi induction. One approach to this is to place the expression of dsRNA-encoding transgenes under the control of an inducible promoter. This approach has proved successful in human prostate cancer cells, in which suppression of two phosphoinositol 3-kinase catalytic subunits was placed under the control of a tetracycline-induced promoter (153), in a murine carcinoma cell line (151) in which expression of presenilin-1 was controlled. Similar approaches to controlling the timing of RNAi applied to nervous tissue or muscle have yet to be reported.

	CONCLUSIONS AND PERSPECTIVES

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RNAi is now the most potent gene knockdown method used in animal systems. It has progressed from nowhere to being the leading method in just 5 years. The implications of the availability of a technique which allows, for the first time, the time- and tissue-specific knockdown of genes or their specific splice variants, expressed either transiently or stably, constitutively or under the control of an inducible promoter, and furthermore which is usually simple to apply, are hard to underestimate. In addition, it offers potential cost savings by avoiding, in some cases, the need to generate transgenic mice.

The application of RNAi to cell lines, primary cultures and embryonic stem cells offers previously inconceivable opportunities for basic research, especially using genome-wide RNAi screens. Cultured cells are convenient in high-throughput assays such as imaging. Rapid screening can be effected using transfected cell microarrays, in which cells are plated onto slides on which gelatin spots have been printed (154). RNAi can be induced in such arrays by including vectors expressing shRNAs in the gelatin spots.

Preliminary investigations of RNAi as a therapy using model organisms have been most encouraging, and the two main hurdles to this new form of therapy (target specificity and control) are rapidly being overcome. The simple intravenous application of dsRNA is unlikely to succeed, as it will be rapidly degraded by nucleases, unless chemically altered to prevent such breakdown. However, new vectors, such as pSUPER, under the control of PolII or III promoters offer particularly exciting avenues for the future. Delivery of such vectors can be made tissue-specific by adding genes encoding cell-recognition molecules. Furthermore, research is being undertaken to develop non-viral-mediated siRNA delivery (155,156). However, more research needs to be done before what has been achieved in animal models can be transferred safely to humans.

	NOTE ADDED IN PROOF

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Spinocerebellar ataxia type 1 (SCA1), a poly(Q) expansion disease, has been successfully suppressed by RNAi in a mouse model of this disease (161).

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: + 44 1865272145; Fax: + 44 1865282651; Email: david.sattelle{at}anat.ox.ac.uk

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