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Human Molecular Genetics, 2003, Vol. 12, Review Issue 2 R279-R284
DOI: 10.1093/hmg/ddg275
© 2003 Oxford University Press

Therapeutic gene silencing in the nervous system

Matthew J.A. Wood1,*, Barbara Trülzsch1, Amr Abdelgany2 and David Beeson2

1Department of Human Anatomy and Genetics, South Parks Road, Oxford University, Oxford OX1 3QX, UK and 2Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU, UK

Received July 28, 2003; Accepted August 4, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 TECHNOLOGIES FOR THERAPEUTIC...
 RNA INTERFERENCE
 GENE SILENCING FOR...
 UNIQUE CHALLENGES FOR GENE...
 THE FUTURE
 REFERENCES
 
Progress in the understanding of RNA biology has brought into focus the prospect of using RNA-based therapeutics as a novel approach to treat human disease. In particular, following the discovery of the RNA interference (RNAi) pathway, the emergence of technology based on small interfering RNA (siRNA) now offers a powerful and highly specific tool for therapeutic gene silencing. Many neurological diseases, including neurodegenerative disorders, tumours and retinal disease are likely candidates to benefit from such advances. The challenges ahead will be to identify appropriate disease gene targets and, crucially, to understand the biological parameters that determine safe, precise and effective delivery and function of RNA-based therapeutic molecules within the unique environment of the nervous system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
TECHNOLOGIES FOR THERAPEUTIC...
 RNA INTERFERENCE
 GENE SILENCING FOR...
 UNIQUE CHALLENGES FOR GENE...
 THE FUTURE
 REFERENCES
 
Recent advances in our understanding of RNA biology continue to reveal that the functions of RNA are more complex than was once thought. In addition to its fundamental function encoding genetic information as ribonucleic acid, other forms of RNA have been found to have catalytic properties—the RNA enzymes or ribozymes—the substrates for which are other RNA molecules. And more recently, an intrinsic cellular gene silencing mechanism centred on RNA—the RNA interference (RNAi) pathway—has been uncovered in organisms from worm to fly to mouse and man. Such discoveries have opened up the field of RNA therapeutics and offer the prospect of RNA-based gene silencing technologies being developed as therapies for human disease.

Neurological diseases are one class of disorders likely to benefit from advances in RNA therapeutics. Not only are such diseases major, and increasing, causes of mortality in developed nations such as the UK, but few effective treatments are available and there is therefore an urgent need to develop novel therapies that effectively target the molecular pathology underlying these disorders. There are further reasons why the development of such ‘gene therapies’ might be especially appropriate for the nervous system (1). First, more unique gene sequences are expressed in brain than in any other tissue, and as a consequence many genetic diseases display a neurological phenotype. Second, the insulation of the brain from the systemic vascular system by the blood–brain barrier (BBB) and blood–cerebrospinal fluid (CSF) barrier, and the complex structural organization of the brain itself, with many neurological diseases having a predilection for affecting specific neuronal sub-populations, argue for precise local therapeutic intervention. Thus, technologies that allow effective and precisely targeted gene silencing within the complex environment of the nervous system are likely to have significant therapeutic potential.


   TECHNOLOGIES FOR THERAPEUTIC GENE SILENCING
TOP
 ABSTRACT
 INTRODUCTION
 TECHNOLOGIES FOR THERAPEUTIC...
RNA INTERFERENCE
 GENE SILENCING FOR...
 UNIQUE CHALLENGES FOR GENE...
 THE FUTURE
 REFERENCES
 
A number of technologies offer the potential for specific therapeutic gene silencing. A common strategy in recent years has been the use of antisense oligonucleotides that down-regulate gene expression by translational blockade and induction of mRNA degradation, primarily through the action of RNase H ribonuclease (2). Although this approach remains useful in certain experimental contexts, it has suffered from poor efficacy and lack of specificity in mammalian systems, and two recent studies comparing it directly with RNAi attest to the superior potency and efficiency of the latter (3,4).

A second approach has been the exploitation of catalytic nucleic acid biology, most notably that of ribozymes. Awareness of the catalytic properties of RNA emerged from the work of Cech and Altman in the early 1980s (5,6). This revealed a number of naturally occurring catalytic RNA species, in particular those of the hammerhead and hairpin motifs, with the ability to regulate gene expression through the cleavage of phosphodiester bonds (7). The principle advantages of ribozymes for gene silencing are their enzymatic properties, and consequent ability to cleave many target mRNA molecules, their high specificity, and their action prior to protein translation. However, given their vulnerability to ribonuclease degradation, significant efforts have been made to enhance their stability and function in vivo through chemical modification (8,9). Although naturally occurring DNA counterparts to ribozymes have not yet been found to exist, such DNA enzymes, or DNAzymes, have been developed synthetically, with similar structural and functional properties to the hammerhead class of ribozyme (10). Their deoxyribonucleic acid content confers greater stability than that typically found for ribozymes, however chemical synthesis is usually required for their generation, although a novel method for the ‘expression’ of DNAzymes has recently been described (11).

In addition to antisense and catalytic nucleic acid approaches, the range of methodologies available for targeted gene silencing has been significantly boosted by the recent, and well documented, discovery of RNAi.


   RNA INTERFERENCE
TOP
 ABSTRACT
 INTRODUCTION
 TECHNOLOGIES FOR THERAPEUTIC...
 RNA INTERFERENCE
GENE SILENCING FOR...
 UNIQUE CHALLENGES FOR GENE...
 THE FUTURE
 REFERENCES
 
RNAi is the natural process of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNA (dsRNA) homologous in sequence to the target gene (12,13). This was first clearly elucidated in 1998 by Fire and colleagues in studies in the nematode worm C. elegans, where gene silencing by dsRNA was unexpectedly discovered to be more effective than using either the sense or antisense strand alone (14). Given the extraordinary potency and efficacy of RNAi as a gene silencing tool, and the ease with which dsRNA can now be introduced into cells and tissues, it has significant potential for human therapeutic purposes.

The mechanism of gene silencing by RNAi is now known to proceed via a highly conserved two-step process (15). First, long dsRNAs are cleaved by the ribonuclease Dicer, generating small interfering RNAs (siRNAs), 21–23 nucleotides in length (16,17). Subsequently the single-stranded antisense siRNA associates with a nuclease complex—the RNA interference silencing ribonucleoprotein complex (RISC)—and guides target RNA cleavage (18,19).

In mammalian somatic cells the introduction of long dsRNA typically activates defence mechanisms that lead to a non-specific reduction in cellular mRNAs. However, Tuschl and colleagues (20) demonstrated that this defence response could be bypassed and effective gene silencing brought about by the direct introduction of siRNA duplexes. siRNA-mediated gene silencing carried out in this fashion generally results in highly efficient gene silencing in cell culture for periods of up to about 96 h, although variables such as siRNA efficacy, transfection efficiency, cell type and protein stability might all contribute to observed differences in the effectiveness of siRNA-mediated RNAi. Subsequently, several plasmid expression systems have been developed for gene silencing in mammalian systems, by expressing siRNAs from short hairpin RNA (shRNA) stem–loop precursors driven off RNA polymerase III promoters (21–23). This approach offers the potential for stable gene silencing and, taking it one step further, several viral vector systems have now demonstrated utility for shRNA-mediated gene silencing (24–26) (Fig. 1).



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  		Figure 1. Therapeutic gene silencing using the RNAi pathway. In organisms such as the nematode worm C. elegans, on entry into the cell, long double-stranded RNA (dsRNA) enters the RNAi pathway and is processed by the Dicer nuclease protein complex to yield short interfering RNA (siRNA) duplexes of 21–22 nucleotides in length. The antisense strand of the siRNA duplex is the agent of RNA interference (RNAi), and it subsequently associates with the RNA interference silencing ribonucleoprotein complex (RISC), and is utilized to guide homology-dependent target mRNA cleavage, resulting in target mRNA degradation and specific gene silencing. In mammalian cells, therapeutic gene silencing using the RNAi pathway can be brought about by the direct introduction of siRNA duplexes, or by short hairpin RNA (shRNA) generated from plasmid or viral expression systems using RNA polymerase III promters, such as U6.

 
Despite rapid progress in the RNAi field and the undoubted therapeutic promise of this technology, there remain important questions to resolve before clinical application becomes a realistic prospect. The possibility of non-specific effects on gene expression is a major concern. However, two recent studies in kidney and lung cells, taking a genome-wide approach, have shown that RNAi is likely to be highly specific in mammalian systems and did not detect non-specific mRNA degradation by transitive RNAi, as occurs in plants and worms (27,28). This will require confirmation in other tissues and in vivo. Although siRNAs appear effectively to bypass the interferon defence response, a single report to date suggests that this may not be equally true of shRNAs (29). Finally, given that the RNAi pathway can be saturated (30), it is conceivable that the natural functions of this pathway such as gene regulation and the control of chromatin structure and function (31), could be usurped. To what extent such concerns are relevant in the human therapeutic context will require further study.


   GENE SILENCING FOR NEUROLOGICALDISEASE
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 ABSTRACT
 INTRODUCTION
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 RNA INTERFERENCE
 GENE SILENCING FOR...
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 THE FUTURE
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Recent studies have shown that the potential of RNAi extends to applications in the mammalian nervous system. Effective gene silencing using RNAi has been demonstrated in mouse neuroblastoma cells (32), in our own work in P19 cells (33) and neural stem cells (unpublished observations), and in primary cortical and hippocampal neurons, where silencing of the endogenous neuronal genes, microtubule associated protein 2 and RNA binding protein YB-1 genes was shown (34). Moreover, gene silencing by adenovirus vector shRNA expression has also been demonstrated in mouse brain in vivo (26). The challenge ahead, therefore, will be to identify neurological diseases and gene targets where gene silencing is likely to have therapeutic benefit, and optimize the technology and its delivery in order to achieve this, ultimately in the clinic.

Many of the major neurological diseases that are significant causes of mortality in developed nations are likely to be suitable targets for therapeutic gene silencing; these include central nervous system (CNS) tumours and neurodegenerative disorders. In addition, retinal disorders, on account of their accessibility, nervous system infections, given the intense focus of the RNAi field in this area (35), prion diseases (36) and pain (37), are other potential areas for therapeutic development.

Neurodegenerative disorders can be classified aetiologically into familial or sporadic in origin. At least nine of the former, including Huntington's disease and spinobulbar muscular atrophy (SBMA), are caused by unusual mutations involving trinucleotide repeat expansions. In a model of SBMA it has been shown that the cellular phenotype can be rescued by RNAi targeting the disease transcript, but only when dsRNAs corresponded to or included nonrepetitive sequences, i.e. it was gene- but not allele-specific (38). This is an important observation for cases where loss of function of the normal allele might have clinical impact. However, in dominant familial disease where the disorder is caused by a point mutation, the exquisite specificity of RNAi would be predicted to allow allele-specific silencing. Proof of principle for this approach has recently been elegantly demonstrated by Davidson and colleagues (39), where a missense Tau mutation (V337M) underlying the disorder frontotemporal dementia with parkinsonism was specifically silenced. This report also confirmed that allele-specific silencing of trinucleotide repeat mutations was not possible, but an alternative allele-specific strategy, targeting a single nucleotide polymorphism (SNP) in linkage disequilibrium with the disease-causing expansion, was successful using siRNA duplexes as well as plasmid and viral expression of shRNA. Successful allele-specific silencing by RNAi has been rapidly confirmed in other reports targeting the TorsinA gene underlying a form of dystonia (40), and in our own work targeting dominantly inherited mutations in the muscle acetylcholine receptor that cause the rare neuromuscular disorder, slow channel myasthenic syndrome (41). Such technology is likely to have application to dominantly inherited forms of the more common neurodegenerative disorders, as well as to disorders outside the nervous system (42). For sporadic neurodegenerative disease the optimal gene targets are less clear at present, however, the silencing of transcripts directly implicated in cellular pathways underlying disease pathogenesis might well have therapeutic benefit. These may include, critical components of the apoptotic pathway, for example caspase 3 (43), or enzymes central to the generation of toxic oxygen free radical species, for example neuronal nitric oxide synthase (nNOS) (44,45).

In contrast to chronic neurodegenerative disorders, no reports as yet have investigated the potential of RNAi in models of acute neurodegenerative disease, such as stroke. However it is clear that susceptibility to acute neurotoxicity and stroke may arise from dominantly inherited mutations in genes such as Notch3 (46) or Presenilin 1 (47), and thus allele-specific gene silencing might have application here also. The more likely alternative will be strategies to develop RNAi as a neuroprotective tool, along the lines of recent work using antisense oligonucleotides against critical molecular targets causing neurotoxicity in stroke models, for example bcl-2 or inducible NOS transcripts (48,49).

Given the clinical significance of CNS tumours, in particular malignant glioma, which is largely refractory to current conventional therapies, a number of recent reports have investigated and demonstrated the efficacy of gene silencing approaches in tumour models. No published studies have yet investigated RNAi technology but these are surely on the way. A critical question here is the selection of suitable therapeutic targets. In two separate studies, ribozyme silencing of growth factor receptor transcripts has provided encouraging results. In the first, silencing of the hepatic growth factor receptor inhibited glioma tumour growth and promoted tumour cell apoptosis (50), whilst in the second, targeting the epidermal growth factor receptor effectively reversed a malignant glioma phenotype in vitro (51). Another approach has been to target components of signal transduction pathways, for example DNAzyme silencing of protein kinase C alpha expression was shown to induce tumour cell apoptosis and, importantly, prolong animal survival in combination with an angiogenesis inhibitor (52,53). An important problem in neuro-oncology is that of acquired resistance to chemotherapeutic agents, and therefore a further strategy has been to attempt to enhance tumour chemosensitivity by, for example, silencing the DNA methyl-transferase gene (54). In this case the authors concluded that incomplete down-regulation of the target gene using antisense or ribozyme approaches was not sufficient to overcome drug resistance, and the greater potency of RNAi may therefore have an important role here. Future progress in this field will certainly benefit from the enormous focus on the application of siRNA technology to cancer therapeutics in general (55).

Finally, one region of nervous system with considerable promise for RNA-based therapies is the retina, both on account of its accessibility, in contrast to the rest of the CNS, and the devastating nature of many retinal disorders. Proof of principle for a therapeutic gene silencing approach has come from a number of elegant studies utilising ribozyme technology in models of retinitis pigmentosa, a disorder involving the death of retinal photoreceptors. Given the genetic heterogeneity of this condition, mutation-independent approaches to therapy are being developed to bypass such diverse genetic aetiology (56); for example, targeting degenerate sites or untranslated regions in retinal transcripts (57), or targeting all known human rod opsin mutations (58). In other experiments, ribozymes targeting the P23H mutation in rhodopsin have been shown to slow photoreceptor degeneration in transgenic rats for prolonged periods (59,60). The recent development of allele-specific RNAi technology will almost certainly find application in diseases of the retina, and the work to date in this disease group suggests that it may be one of the first to enter clinical evaluation using therapeutic gene silencing.


   UNIQUE CHALLENGES FOR GENE SILENCING IN THE NERVOUS SYSTEM
TOP
 ABSTRACT
 INTRODUCTION
 TECHNOLOGIES FOR THERAPEUTIC...
 RNA INTERFERENCE
 GENE SILENCING FOR...
 UNIQUE CHALLENGES FOR GENE...
THE FUTURE
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The nervous system, in particular the CNS, has several characteristic properties that are likely to present important challenges to the development of RNA-based therapeutic gene silencing. The unique cell biology of mammalian neurons is still relatively poorly understood, in particular relating to the processing, transport and functions of RNA. Added to general concerns about the saturability of the RNAi pathway, neurons, in particular, are being found to contain a high number of small cellular microRNAs that probably require processing via the RNAi pathway, appear to form important components of neuronal ribonucleoprotein complexes (61), and may even be directly associated with neurological disease (62). A second feature is the extraordinary distances over and precision with which mRNA species are transported in neurons, to reach distant axon terminals or dendrites prior to local translation (63,64). Given such special neuronal properties, to what extent they pose additional complexity upon successful intervention with RNA-based therapies remains to be seen.

A second area concerns the unique immunological properties of the CNS—a so-called immunologically privileged site. Given the possibility that RNAi via the expression of shRNA constructs may elicit an interferon response (29); although this has yet to be confirmed independently or shown in vivo, such a response might manifest unusually within the CNS immune environment and will need to be looked for specifically and its potential consequences studied. In addition, despite its relative immunological privilege, it is well established that viral vector delivery systems elicit immunological responses in the brain (65,66), and therefore not only is this a concern where they might be used for RNAi delivery, but it is conceivable that immune responses might also be generated to the RNA-based therapeutic molecules themselves.

Finally, the structural and functional complexity of the nervous system presents considerable obstacles to the delivery of therapeutic molecules such as siRNAs. The CNS consists of parenchymal and ventricular compartments insulated from the systemic vascular system by the BBB and blood–CSF barrier, respectively. To what extent therapeutic siRNAs can traverse these barriers via systemic delivery, whether or not chemical modification can enhance delivery by this route, or whether in fact direct delivery via either the intraparenchymal or intraventricular routes is feasible, as appears the case for antisense oligonucleotides (67), remain to be determined. Moreover, given the organizational complexity of the CNS, and the predilection of many neurological diseases to affect specific neuronal sub-populations, the requirement for precise targeting of siRNAs to specific cell populations will probably be essential. It is conceivable that chemical modification of siRNAs could in some way facilitate this, but it would seem more likely that viral systems for siRNA delivery will be needed (24–26), a number of which already have well-documented utility for the nervous system.


   THE FUTURE
TOP
 ABSTRACT
 INTRODUCTION
 TECHNOLOGIES FOR THERAPEUTIC...
 RNA INTERFERENCE
 GENE SILENCING FOR...
 UNIQUE CHALLENGES FOR GENE...
 THE FUTURE
REFERENCES
 
Gene silencing, in particular that based on the potency and specificity of RNAi technology, has clear potential as a novel therapeutic strategy for disorders of the nervous system, many of which are currently untreatable. A range of neurological diseases from chronic neurodegenerative disorders to tumours and stroke are all candidates for the development of therapeutic gene silencing. Given proof of principle for allele-specific gene silencing using RNAi, the number of potential neurological disease targets is increased further to include many dominant genetic disorders and disease-linked SNPs. Whether the undoubted potential of RNA-based therapeutics can be achieved given the very particular biological constraints imposed by the nervous system remains to be seen. Central to this enterprise, therefore, will be the need to develop a greater understanding of those critical biological variables determining safe and precisely targeted RNA-based therapeutic intervention in brain.


   ACKNOWLEDGEMENTS
 
We would like to acknowledge the MRC (UK), BBSRC (UK), The Wellcome Trust, The Marie Curie Foundation and Somerville College Oxford, for their support of the authors.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +44 1865272419; Fax: +44 1865272420; Email: matthew.wood{at}anat.ox.ac.uk Back


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 RNA INTERFERENCE
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S. D. Buckingham, B. Esmaeili, M. Wood, and D. B. Sattelle
RNA interference: from model organisms towards therapy for neural and neuromuscular disorders
Hum. Mol. Genet., October 1, 2004; 13(suppl_2): R275 - R288.
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