Introduction

The salient feature of many neurodegenerative disorders is the selective vulnerability of precise classes of neurons. A complex combination of aetiological factors impairs the pursuit to understand such progressive and selective loss of neuronal populations. Nevertheless, the identification of the genetic basis of various neurodegenerative diseases has provided invaluable insights into the understanding of some pathogenic processes. Indeed, the immediate application of this genetic knowledge is the development of animal models aimed at recapitulating the main clinical aspects of human diseases. Ultimately, translation of pathogenic molecular networks that lead to neuronal degeneration into a therapeutic prospective relies on these animal models.¹

Experimental modelling of neurodegenerative disorders associated with loss of function can be soundly carried out by gene-targeted disruption through homologous recombination in embryonic stem cells. However, it usually precludes local and temporal genetic intervention in complex genetic backgrounds of rodents or non-human primates. Furthermore, for human genes that have no mouse orthologues, this species scale-up is crucially required.² An alternative mode of specific gene silencing would not only help in saving time but would also help in bridging the gap that exists between rodents and humans, for both experimental and therapeutic purposes.

Concerning dominantly inherited neurodegenerative diseases associated with gain-of-function mechanisms, the generation of animal models can be empirically achieved by overexpression of the suspected human mutated gene through a transgenesis approach or through local delivery of the related gene. This process has been successfully achieved in a wide variety of species ranging from worms to rats and has a real chance of success in non-human primates.^{1, 3} When disease-causing genes give rise to products with novel neurotoxic functions, the multifactoriality of neuropathologic characters of the related disease often impede the assignment of efficient therapy. In this context, an omni-directional treatment would have to prevent most of the cellular and molecular aberrations that lead to the neurodegenerative disease. The most straightforward therapeutic scheme would therefore be to knock down the mutated genetic product that governs all pathogenic processes.

RNA interference (RNAi) is an evolutionarily conserved innate process of post-translational gene silencing. RNAi promotes a highly specific degradation of an mRNA target through the use of homologous double-stranded RNA (dsRNA) of 19–23 nucleotides (nt) called small interfering RNA (siRNA). Antisense siRNA strand incorporated into the RNA-induced silencing complex (RISC) guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific manner. Importantly, siRNA can be generated from single-stranded RNA precursors, which form a hairpin structure. These short hairpin RNAs (shRNAs) can therefore be synthesized from a DNA template under the control of an RNA polymerase (Pol) II or III promoter. shRNAs are then cleaved by the multidomain ribonuclease, Dicer, to produce mature siRNA.^{4, 5, 6, 7}

Gene transfer technology based on recombinant adenoviral (AdV), adeno-associated viral (AAV) or lentiviral (LV) vectors offers the opportunity to manipulate gene expression in a wide range of mitotic and post-mitotic cells of different species origin. The persistence of virally delivered genetic instruction allows a genetic manipulation at the whole body level through transduction of the mouse germ line (and potentially to rats and non-human primates) or at precise sites of the central nervous system, which is in accordance with the chronic aspect of neurodegenerative disorders.^{3, 8, 9} The combination of RNAi and viral technology, therefore, provides a powerful means to mimic neurodegenerative disorders associated with loss-of-function mechanisms. Additionally, viral-mediated RNAi is a highly relevant therapeutic tool for neurodegenerative diseases associated with gain-of-function mechanisms such as polyglutamine disorders, Parkinson's disease or amyotrophic lateral sclerosis (ALS).

Viral platforms for shRNA delivery

Suitable activation of RNAi machinery in mammalian cells requires expression of short dsRNA species in the cytoplasm (<30 nt). shRNA made by both sense and antisense strands in the context of a hairpin (ranging from 6 to 12 nt) may overcome a nonspecific inhibitory response from the interferon pathway induced by endogenous dsRNA-dependent protein sensors.¹⁰ shRNAs derived from viral platforms are synthesized by host transcriptional machinery and transported (a step sensitive to the hairpin nature) to the cytoplasm by exportin-5, a nucleocytoplasmic transport factor involved in the nuclear export of both micro-RNA and shRNA.^{11, 12} Once in the cytoplasm, shRNAs are engaged by the endogenous RNAi machinery to drive sequence-specific cleavage of the mRNA target (Figure 1). Importantly, according to the small size of commonly used RNA Pol II or III promoters for driving expression of shRNA, cloning capacity for different viral vectors is no longer the choice-limiting factor. Cellular tropism and the integrative or non-integrative nature of the vectors and their clinical application potential, therefore, become the main criteria of choice.

Figure 1.

Viral-based delivery of RNAi instructions. LV-based delivery of RNAi information. Single-stranded positive sense RNA genome following reverse transcription gives rise to the proviral genome, which is integrated into the host genome. shRNAs synthesized by RNA Pol III promoter are then exported into the cytoplasm by exportin-5. Once in the cytoplasmic compartment, shRNA is cleaved by the dsRNA-specific endonuclease Dicer, which generates an siRNA. siRNA is then incorporated into the RISC assembly pathway and, following thermodynamic rules, submitted to an unwinding step that leads to exclusion of the sense strand. Active RISC, loaded with antisense strand, is guided to, through complementary recognition, the mRNA target, which then acts as an endonuclease to degrade it.

Full figure and legend (136K)

AdV-mediated expression of shRNA

Several AdV systems have been designed to produce shRNA in vitro and in vivo (Figure 2a, b, d and f). Most of them use the small nuclear RNA U6 (U6) and nuclear RNase P H1 RNA (H1) Pol III promoters. Both U6 and H1 have a compact and simple organization, lying upstream of the transcribed region, and are constitutively and ubiquitously active. In their simplest organization, RNAi-AdV contain either the U6 or H1 promoter to drive the synthesis of shRNA^{13, 14} (Figure 2a). A variation on this theme uses a U6-based tandem system to transcribe independently both sense and antisense strands, which, following annealing of both strands, compose the siRNA duplex¹⁵ (Figure 2b). Nevertheless, it has been shown that tandem-type vectors present less silencing activity than hairpin-type vectors.¹⁶ A higher yield of annealing and the presence of the loop in the hairpin system (which plays an important role in the transport of shRNA to the cytoplasm^{11, 12, 17}) might explain this enhanced silencing activity.¹⁶ Addition of a reporter gene expression cassette either upstream or downstream of the Pol lll-shRNA transcription unit allows the tracing of transduced cells in which silencing occurs^{18, 19, 20} (Figure 2d). The first in vivo demonstration of virally promoted RNAi in the nervous system made use of a bipartite AdV system containing a DsRed expression cassette and an shRNA directed against EGFP under the control of a modified version of the cytomegalovirus (CMV) Pol II promoter.¹⁹ This AdV vector was delivered in the striatum of transgenic mice expressing EGFP. At 5 days after injection, a robust decrease in the expression of EGFP was observed in the transduced striatal region.¹⁹

Figure 2.

Architecture of silence. Schematic view of different viral platforms designed to deliver RNAi instructions. (a–c) Minimalist design for shRNA delivery. shRNA synthesis can be under the control of the Pol III (usually U6 or H1) or Pol II promoter. (b) Tandem-type system for independent synthesis of sense and antisense strand that will anneal on their own to the future siRNA structure. (c) Siamese system incorporates two identical H1-shRNA cassettes in the proviral form of the LV. (d, e) Bipartite vector incorporates a Pol III or II promoter for synthesis of shRNA and a Pol II promoter for expression of a reporter or a drug resistance gene. Each expression cassette can be upstream or downstream of each other in a head-to-head or tail-to-head orientation (d). (e) The H1 promoter drives expression of shRNA and the Pol II promoter drives expression of either EGFP reporter gene or silencing-resistant form of the mRNA targeted by the shRNA. (f–i) Regulatable RNAi system. (f, g) Tet-off system, where, in the presence of Dox, the expression of shRNA is prevented. (h) Cre–Lox-dependent activation of shRNA synthesis. (i) Cre–Lox-dependent inactivation of shRNA synthesis. For further description of the different viral systems engineered to deliver RNAi instructions, refer to text. AdV, adenovirus; AAV, adeno-associated virus; LV, lentivirus.

Full figure and legend (203K)

An important contribution in the development of RNAi viral vectors came with the demonstration of cell-specific gene silencing.²¹ The superfactant protein C (SP-C) promoter has been successfully used to exclusively produce shRNA in alveolar epithelial type II cells. The type II cell-specific inhibition of endogenous gene has been demonstrated both in vitro in lung organ culture and in vivo following intrabronchial administration of AdV.²¹

These experiments summarize the role of AdV in delivering shRNA. AdV has several advantages including its large insert capacity, its relative ease of manipulation and the fact that it is the most used and well-studied virus employed in gene therapy to date. This vector does, however, have its limitations. A strong immune response against AdV vectors has been observed in most vector types, with the exception of the 'gutted' helper-dependent form.²² This immunotoxicity may be of a lesser concern for delivery in the CNS, which has the possibility of being immune privileged. However, in other applicable tissues or whole body administration, specific targeting techniques must be employed, such as transductional or transcriptional modalities.²³ Also, upon administration of AdV, the resulting gene expression can be short-lived, which may preclude this virus from being utilized in many neurodegenerative models and therapies.

AAV-mediated delivery of shRNA

AAVs have also been used as an shRNA delivery platform with a similar configuration as AdV (Figure 2a and d). It ranges from a basic shRNA source using either the U6 or H1 promoter²⁴ to the commonly used bipartite vectors that, in addition to the shRNA transcription unit, incorporate a reporter or drug resistance expression cassette under the Pol II promoter.^{25, 26, 27, 28, 29, 30, 31} In the latter bipartite configuration, other promoter possibilities have been evaluated for synthesis of hairpin-type structure.^{25, 26} One such possibility is an optimized human tRNA^met-derived (MTD) Pol III-type promoter. Another candidate is a U6+1 version of the U6 promoter, which confers to the sense strand an additional 5' guanosine. Finally, a third possible promoter construct is the U6+27 system. The U6+27 construct contains the first 27 nt of U6 snRNA, which directs methylation of the 5'--phosphate in order to stabilize and increase its accumulation of the transcript. Using transient transfection, a higher level of expression was obtained with these new promoters compared to the H1 and U6 promoter. It is interesting to note that the MTD promoter gave the more effective gene silencing activity over different Pol III promoters that were tested and should therefore be evaluated in a neural context.^{17, 19, 26} Nevertheless, AAV carrying a U6 or H1 promoter have been demonstrated as potent and long-lasting promoters of shRNA synthesis both in vitro (up to 5 months)²⁹ and in vivo in the CNS (up to 4 months).^{27, 30, 31}

AAV represents an attractive vector choice owing to its low immunogenicity and small size (making it ideal for applications requiring diffusion into other target areas). Another advantage is its ability to exist as a stable episomal form, resulting in lasting gene expression.³² Some disadvantages, however, are its small insert capacity, which can be a concern for applications that necessitate expression of a large transgene simultaneously with shRNA,³³ and its relative difficulty in producing. Owing to recent advances in production and purification techniques,³⁴ this vector is nevertheless a fitting vector for neurodegenerative models and therapies.

LV vector-based delivery of shRNA

In its simplest form, RNAi-LV contains either the U6 or H1 promoter to deliver the desired shRNA in a long-term manner both in vitro (up to 4 months)^{35, 36, 37} and in vivo (up to 6 months in mouse striatum)³⁷ (Figure 2a). Another single Pol III-based system has been developed with a simian immunodeficiency virus- or human immunodeficiency virus (HIV-1)-derived vector. By introducing the H1-shRNA cassette into the 3' long terminal repeat (LTR) of the viral genome, following retrotranscription and genomic integration, the proviral form will therefore contain two synthesis sources of the shRNA^{38, 39} (Figure 2c, e and g). The same duplication of shRNA source has also been used in bipartite configuration.^{33, 40} Nevertheless, most RNAi-LV (HIV-1- or equine infectious anaemia virus (EIAV)-derived vector) platforms incorporate U6- or H1-based shRNA delivery between the two LTRs and comprise a Pol II reporter or drug resistance gene cassette.^{16, 18, 41, 42, 43, 44} Another U6-driven tandem type has also been designed and it was confirmed that hairpin-type shRNA showed an enhanced suppressive activity than the tandem type¹⁶ (Figure 2b).

The most attractive advantage of LV is their ability to integrate into the host cell genome and thereby lead to a persistent expression of transgene as well as shRNA. Indeed, RNAi-LV have been successfully applied in the development of transgenic 'knockdown' mice.^{39, 42} Additionally, the low immunogenicity and large transgene capacity of LV have broadened their potential as effective gene transfer vectors for clinical purposes.

Nevertheless, the consequence of stable delivery offered by retroviruses is the risk of insertional mutagenesis by activation of cellular proto-oncogenes.⁴⁵ To our concern, risks of oncogenic transformation are less of a concern for neurons, but more for glial cells, which retain a mitotic competence. As integration of retroviral DNA occurs in transcriptionally active regions and may therefore be independent of the cell type, analysis of viral integration in neural cells would reveal the potential for insertional oncogenesis of LV in this context. Conjointly, an important gain in viral safety could be achieved by directing integration to specific target sites through the use of viral integrase fused with a sequence-specific DNA-binding protein,⁴⁶ or through the use of integration-defective LV vectors.⁴⁷

Viral-based reversible and irreversible regulated delivery of shRNA

Regulatable RNAi activation represents a valuable means to better understand, in an experimental pathological context, which molecular mechanisms are irreversibly committed, when this commitment occurs during the course of the disease or if a neuron can recover from these pathogenic mechanisms once the disease has been declared. Additionally, controllable delivery of RNAi instruction may be solicited for clinical safety and efficacy. Indeed, a constant recruitment of RNAi machinery may lead to undesirable effects by competing with the endogenous mechanism of gene regulation dependent on RNAi. Several reversible (drug-dependent) and irreversible (Cre–LoxP-dependent) regulated viral systems have been described for ubiquitous or cell-specific gene silencing.

A reversible doxycycline (Dox)-based shRNA expression cassette has been designed in an AdV vector. A tetracycline operator sequence (tetO) was introduced downstream of the TATA box of the H1 promoter. Binding of the prokaryotic tetracycline repressor (TetR) protein to the tetO sequence can be prevented by the tetracycline analogue, Dox. In the presence of Dox, TetR (delivered in trans through an additional AdV) no longer binds to the tetO sequence, and transcription of the shRNA is therefore initiated⁴⁸ (Figure 2f). Using LV vectors, another Dox-dependent system of shRNA synthesis has been described.⁴⁰ In this situation, TetR was fused to the Kruppel-associated box (KRAB) repressor domain of human Kox1, a zinc-finger protein that has an in cis transcriptional repressive activity on both Pol II and III promoters over a distance of 3 kb from its binding site. A tetO sequence was placed upstream of the H1 promoter cloned into the 3'LTR of a self-inactivated LV. Following duplication of the 3'LTR and genomic integration, the two tetO-H1-shRNA cassettes flanked an EF-1(Pol II)-EGFP expression cassette. In the absence of Dox, both the reporter gene and Dox-regulatable H1 cassette are silenced in a TetR-KRAB-dependent manner, whereas in the presence of Dox, a rapid and highly Dox-sensitive production of shRNA is achieved⁴⁰ (Figure 2g).

A conditional activation of shRNA synthesis was demonstrated using the Cre–LoxP system by LV vectors.^{39, 49} The TATA box of the U6 promoter was replaced by a modified LoxP site (LoxP^TATA) mimicking the original TATA box. The second LoxP^TATA site was located immediately upstream of the shRNA. Both LoxP^TATA sites were separated by a sequence comprising a reporter gene under the control of the CMV promoter ensuring a transcriptional barrier. Following the action of Cre recombinase, which deletes the sequence flanked by two oriented LoxP sites, only one modified LoxP^TATA site remained and reconstituted the TATA box required for U6 activity (Figure 2h). Indeed, an efficient synthesis of shRNA was achieved by this Cre-mediated site-specific recombination both in vitro³⁹ and in vivo.⁴⁹

Alternatively, a conditional repression has been achieved in a Cre-dependent manner.⁴⁹ In this vector, the U6-shRNA cassette upstream of the CMV-EGFP expression cassette is flanked by two LoxP sequences. The first LoxP sequence is placed in an upstream region of the U6 promoter partially dispensable for its activity. The second is located downstream of the CMV-EGFP cassette. Cre-mediated recombination (Cre was delivered by an AdV vector) led to the excision of the U6-shRNA-CMV-EGFP transcription unit and terminated per se the constitutive synthesis of the shRNA as well as reporter gene⁴⁹ (Figure 2i).

In the light of this impressive development in viral platforms allowing for manipulation of RNAi in a time- and space-dependent manner, a plethora of neurodegenerative gene functions and therapeutic concepts can therefore be addressed. Concerning therapeutic application, drug-regulatable systems represent the most attractive way to control RNAi information in a sensitive and reversible manner. Although reversible control of transgene expression has been successfully achieved using AdV, AAV or LV in the rat brain,^{50, 51, 52, 53, 54, 55} feasibility of tetracycline-based reversible silencing through viral vectors needs to be demonstrated in vivo.

Viral-mediated silencing of dominant pathogenic genes

Combination of the high specificity of RNAi-based gene silencing with the long-lasting transfer of genetic information achieved by viral vectors in vivo offers the most promising therapeutic option for neurodegenerative diseases. Therapeutic schemes mandating RNAi to target dominantly inherited genes have led to promising results for three neurodegenerative disorders. These include two polyglutamine (polyQ) disorders and a familial form of ALS.

Spinocerebellar ataxia and Huntington's disease

PolyQ repeat disorders are a heterogeneous group of severe neurodegenerative diseases including Huntington's disease (HD) and a number of spinocerebellar ataxias (SCAs). PolyQ disorders are caused by the expansion of the trinucleotide repeat (CAG)_n within the respective gene, which results in an abnormal polyglutamine tract in the related protein. The presence of a polyQ tract results in a gain of function of the mutant protein associated with the formation of intranuclear and cytoplasmic aggregates, which mediates cytotoxic effects in different neuronal cell types. The age of onset and the rate of progression are inversely correlated to the size of polyQ expansion. However, the precise mechanism(s) by which a mutant polyQ form mediates pathogenesis in different cell types remains unknown.

Autosomal dominant SCAs are a diverse group of clinically and genetically slow progressive neurodegenerative disorders that initiate mostly in the fourth decade of life. Clinical signs are variable and include uncoordination (ataxia) of gait and stance, spasticity, cognitive impairment, disarthria or opthtalmoplegia, tremor and epilepsy. SCAs are characterized by the degeneration of Purkinje cerebellar neurons, spinocerebellar tracts and some brain stem nuclei.⁵⁶ A subclass of SCA is caused by a gain-of-function mechanism linked to expansion of polyQ in up to seven different genes such as the ataxin-1 gene.⁵⁶ Interestingly, when SCA was modelled in mice, it led to ataxia and degeneration of the cerebellum.^{57, 58, 59, 60} Although these models have increased the broader understanding of the general pathogenic aspect of disease, the precise molecular pathogenic mechanism resulting from the polyQ-expanded Ataxin-1 protein remains elusive. Davidson and co-workers³⁰ have used an AAV-based inducement of RNAi to silence the mutated 82Q-expanded ataxin-1 in mice. An AAV serotype 1, carrying shRNA against ataxin-1 under the control of the H1 promoter and a GFP reporter gene under the control of the CMV promoter (AAV-shSCA1), was injected in cerebellar lobules of SCA1 mice (Figure 2d). AAV-shSCA1-mediated silencing of Ataxin-82Q led to a significant reduction of intraneuronal inclusion and improved cerebellar pathology, as observed by a reduced thinning of the molecular layer of mice cerebellum. Importantly, silencing of Ataxin-82Q in 5–10% of cerebellar Purkinje cells significantly improved motor performance of ataxic mice.³⁰

HD is a neurodegenerative disease characterized by a selective loss of striatal medium spiny GABAergic neurons, and later cortical neurons. HD leads to involuntary Choreic movements, emotional disturbance, dementia and death 10–20 years after diagnosis. This neurodegenerative disorder is caused by the expansion of polyQ tract in the N-terminus of Huntingtin (Htt) protein. The N171-82Q mouse model for HD overexpresses the first 171 amino acids of Htt, which encompasses an expansion of 82 glutamines.³¹ These mice share many features of human diseases, including progressive and selective loss of striatal neurons. HD mice develop ataxia, hindlimb weakness and clasping. They prematurely die at approximately 4–6 months of age.⁶¹ At the histopathological level, N171-82Q mice showed Htt-positive cytoplasmic and nuclear aggregates in the cortex and striatum.⁶¹ Intrastriatal injection of a bipartite AAV vector for Htt-shRNA (U6-based; Figure 2d) significantly reduced the expression of htt mRNA and protein, and led to the loss of htt-reactive intranuclear inclusions at late stage. Furthermore, AAV-mediated silencing of mutant Htt substantially improved motor performance of HD-N171-82Q.³¹

Familial ALS associated with mutation in SOD1

ALS is a late-onset incurable neurodegenerative disease characterized by the selective and progressive loss of motoneurons in spinal cord, brainstem and cerebral cortex. The selective loss of these motoneurons leads to a progressive weakness and skeletal muscle atrophy, and ultimately to death. Disease progression is rapid, with a mean survival of 3 years. One-fifth of familial ALS (FALS) cases are linked to dominantly inherited mutations in the ubiquitously expressed Cu–Zn superoxide dismutase (SOD1) gene. Remarkably, FALS pathology is faithfully mimicked in transgenic mice or rats that overexpress various SOD1 mutations. SOD1 mice develop severe motor impairment starting in the hindlimbs that progresses rostrally over weeks or months, resulting in death. A wealth of studies in FALS patients and transgenic mice has demonstrated that SOD1 mutations cause motoneuron death by a gain of novel unknown toxic properties rather than by an altered dismutase activity.^{62, 63} This explains the rationale to shut down the expression of this deleterious gene that controls the whole pathogenic process. Three independent studies have shown the effectiveness of RNAi to silence the pathogenic SOD1 mutant gene in ALS mice.^{33, 44, 64} In our study, a vesicular stomatitis virus G protein-pseudotyped LV vector was engineered to contain phosphoglycerate kinase-EGFP transcription unit and the H1-shSOD1 cassette (Figure 2e). We first demonstrated that direct delivery of LV-shSOD1 in lumbar spinal cords of ALS mice promoted the silencing of mutated gene in both motoneurons and non-neuronal cells in a long-term manner. LV-mediated silencing of SOD1 led to a significant neuroprotective effect and delayed both the apparition of the first signs of motor impairment and the decline of neuromuscular function.³³

Azzouz and colleagues made use of an EIAV pseudotyped with rabies-G envelope glycoprotein carrying H1-shRNA directed against SOD1 and the CMV promoter driving the LacZ reporter gene (Figure 2d). Rabies-G envelope glycoprotein has the property to enhance gene transfer to selected populations of motoneurons through the retrograde transport of viral particles delivered intramuscularly.⁶⁵ The functional impact of RNAi-based silencing of SOD1 mutant on the disease was demonstrated by an improved motoneuron survival, which correlated to a significant delay in disease onset and an extension of lifespan. The latter beneficial effect is likely attributable to a greater number of targeted motoneurons, such as those controlling vital functions (i.e. respiration and feeding).

Finally, Cleveland and co-workers⁶⁴ delivered into lower leg muscles of ALS mice a serotype 2 AAV with a bipartite configuration (Figure 2d), which allowed for delivery of an shRNA against SOD1 and expression of EGFP reporter gene. When injected intramuscularly, AAV-2 transduced mature post-mitotic muscle fibres with high efficiency,⁶⁶ but, to a lower extent, was also taken up at the level of nerve endings and transported back through axonal transport to reach the motoneuron soma.⁶⁷ Although no evidence was provided for a reduction of mutated SOD1 levels in muscles, the authors showed decreased levels of SOD1 mutant in transduced motoneurons. Interestingly, AAV-based silencing of SOD1 mutant led to an improvement of hindlimb strength in ALS mice.⁶⁴

Altogether, these studies demonstrate the therapeutic potential of RNAi and also highlight the potential of RNAi in the understanding of pathogenic mechanisms by targeting different cell types.^{33, 44, 64} As all these therapeutic schemes were applied before the apparition of the first signs of motor impairment, it is now important to examine if viral-mediated silencing of SOD1 mutant at disease onset will have an equivalent benefit on the disease progression. Indeed, we still do not know whether neurons can recover from previously initiated pathogenic events or whether the RNAi machinery in damaged neurons is still functionally able to drive an efficient knockdown of SOD1 mutant mRNA.

Additionally, translation into the clinic raises several questions about safety of both clinical protocols. Although intraspinal injection of LV offers the advantage to directly target specific motor neuron populations, such as those controlling breathing, its regional therapeutic feature would require multiple intraspinal interventions. Although such a surgical procedure for intraspinal manipulation has been achieved in non-human primates,⁶⁸ and although LV has been demonstrated as an efficient gene transfer vector for neurons in a non-human primate model of Parkinson's disease,⁶⁹ intraspinal delivery of LV has to be evaluated for both safety and gene transfer efficiency.

Safety of AAV-based gene transfer to skeletal muscles of humans has already been demonstrated.⁷⁰ Nevertheless, according to the intracellular feature of the RNAi-based therapeutic modality, several crucial issues have to be addressed in non-human primates. These include the efficient gene transfer to motoneurons through a retrograde transport mechanism, the feasibility and safety of delivery of viral gene transfer vectors to vital muscles such as the diaphragm or the scale-up of viral load required to gain access to a clinically relevant number of neuromuscular junctions. Clinical application of virally delivered RNAi instruction for ALS now depends on this translational research.

Perspectives

RNAi-based functional analysis of genes associated with human recessively inherited neurodegenerative diseases can be addressed through an impressive development of various viral platforms. AdV, AAV and LV offer opportunities to deliver RNAi instruction in either a ubiquitous or cell-specific manner, and also in a constitutive or regulated manner. Moreover, according to their broad tissue tropism, these RNA instructions can be delivered either at the whole body level or in a precise site of the CNS of any genetic background or origin species. The former can be achieved using LV vectors, owing to their integrative nature, by the transduction of fertilized oocytes or embryonic stem cells to generate an RNAi animal. AdV, AAV and LV have already been used to efficiently deliver genetic instructions in precise regions of rodents and non-human primates CNS.^{3, 8, 71} RNAi-based silencing of a specific gene in a precise neuronal population of newborn or adult animals offers the opportunity to bypass developmental compensatory mechanisms that can take place when a gene disruption is achieved. Interestingly, silencing can also be utilized to shunt some species or strain-dependent intrinsic features.^{72, 73, 74}

Despite the clear therapeutic potential of RNAi-based gene therapy,^{30, 31, 33, 44, 64} several issues are challenging translation towards the clinic. The first concerns the RNAi-mediated depletion of the wild-type allele. Indeed, the dominant trait of inheritance of neurodegenerative disorders often implies that only one allele harbours the causative mutation. Therefore, silencing of the wild-type gene could have a deleterious effect, owing to the loss of normal physiological functions, as illustrated in the genetic disruption of endogenous huntingtin.⁷⁵ To circumvent this limitation, allele-specific siRNAs discriminating between wild type and mutant allele have been designed.^{76, 77, 78, 79} Nevertheless, for neurodegenerative diseases associated with a large number of different point mutations in the same gene, such as ALS-linked mutations in SOD1, a selective silencing may not be clinically appropriate. Indeed, design of allele-specific siRNA is not so straightforward. Moreover, the silencing efficiency of the allele-specific siRNA may vary with the nature and the position of the point mutation. Finally, all the safety concerns of each newly designed siRNA will have to be addressed before its translation into the clinical situation. An interesting alternative consists of a gene replacement technology that potentially allows for the knockdown of the vast majority of mutated genes and the synthesis of a wild-type protein refractory to RNAi-based silencing.³³ By retaining normal gene function and switching off a potential broad range of mutations, this system opens a therapeutic avenue to a wide variety of dominantly inherited neurodegenerative disorders.

As with any therapeutic scheme, the combination of gene therapy and RNAi raises several safety concerns that will have to be addressed before translation into the clinic. One of these concerns the mechanism of action of RNAi, which relies on overexpression of shRNA and the use of the endogenous RNAi machinery. It cannot be excluded that such a utilization might disturb biological events regulated by RNAi, including chromatin remodelling, gene regulation or susceptibility to viral infection. These do not include idiosyncratic off-target effects that are directly linked to the molecular nature of siRNA and which can be evaluated by microarray analysis during initial screening of siRNA. Although therapeutic benefits of RNAi overwhelm these issues in preclinical models, they have to be anticipated for further clinical development. Feasibility of a clinical intervention using RNAi for ALS or polyQ disorders does not rely anymore on a homeric movement but more on a realistic progression in which other several neurodegenerative diseases will be imbricated. Of these, Parkinson's disease is the most favourable to gene therapy based on the localized site of intervention and the discrete number of neurons that have to be targeted to provide a significant altering of the disease progression. Finally, studies in humans and in experimental paradigms of neurodegeneration should help us identify common cell death pathways leading to neuronal loss. This understanding of neuronal death mechanisms should therefore allow the identification of potential targets for RNAi-based gene silencing and open therapeutic perspectives for sporadic cases of neurodegenerative disorders.

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Acknowledgements

We thank Chris Towne for critical comments on the manuscript. We are grateful to Nicolas Bouche for art colour figures. Our work was supported by the Swiss National Science Foundation and the ALS association (ALSA).