Amyotrophic lateral sclerosis
(ALS) is a devastating neurodegenerative disease that causes motor neuron degeneration, paralysis, and death. Approximately 10% of ALS cases are familial and 90% are sporadic. Gene mutations are known to underlie familial ALS. The genes where mutations cause familial ALS include Cu, Zn superoxide dismutase (SOD1), Alsin, senataxin, dynactin, VAMP-associated protein B (VAPB), and TAR DNA binding protein 43
KD (TDP-43) (25
). Mutations in all these genes except Alsin are dominantly inherited. In contrast to the familial cases, no obvious cause is known for sporadic ALS. However, recent studies have discovered TDP-43 as a prominent component in the ubiquitin-positive intracellular inclusions in sporadic ALS (2
), thus suggesting that TDP-43 is involved in the pathogenesis of sporadic ALS.
Mutations in the SOD1 gene were the first discovered genetic cause for ALS and they cause ~20% of familial ALS cases (37
). In the past 16 years following this discovery, much progress has been made in our understanding of the mechanism whereby the mutant SOD1 causes this disease (5
). Among the most important findings is the proof that mutant SOD1 causes motor neuron degeneration by a gain of a toxic property rather than a loss of the enzymatic function of SOD1. First, there is no correlation between the retention of the enzyme activity and the disease-causing propensity in the SOD1 mutants. While some mutations retain normal levels of superoxide dismutation activity, others lose almost all the enzyme activity (8
). In addition, the presence of mutant enzyme does not affect the activity and stability of the normal enzyme despite the formation of mutant-wild type heterodimer (7
). Second, transgenic mice expressing the mutant SOD1 develop motor neuron degeneration and ALS without lowering the level of superoxide dismutase activity (19
). Third, neither overexpression of the wild-type SOD1 nor deletion of the SOD1 gene leads to ALS in mice (19
), indicating that alteration in normal SOD1 activity is not a direct cause of this disease. Fourth, overexpression of wild-type SOD1 does not alleviate, but instead, accelerates the disease; and knockout of the endogenous SOD1 does not significantly alter the course of the disease (10
), indicating that the level of the superoxide dismutase activity is not related to the pathogenesis of ALS.
Since a toxic property in the mutant SOD1 causes motor neuron degeneration, we can predict that the higher the mutant protein expression, the stronger the toxicity, and consequently, the more severe the disease. Indeed, in different transgenic lines that express mutant SOD1, the higher the expression levels, the more severe the disease, as manifested by earlier disease onset and more rapid disease progression (12
). With this knowledge, we can conclude that lowering the mutant SOD1 expression will be therapeutic and RNA interference (RNAi) may be harnessed for silencing the mutant SOD1 expression (16
RNAi is a widely conserved eukaryotic function (35
). Triggered in cells by double-stranded RNA (dsRNA), RNAi destroys the target RNA that shares sequence homology with the dsRNA. The main steps of the RNAi mechanism can be simplified as the following steps (): First, Dicer, an enzyme of the RNase III family, initiates ATP-dependent fragmentation of long dsRNA into 21–25 nucleotide double-stranded fragments, termed small interfering RNAs (siRNAs). Second, the siRNA duplexes bind with proteins Dicer and TRBP (or R2D2 for invertebrates), which facilitate the formation of a siRNA/multi-protein complex called RISC loading complex (RLC). Third, the siRNA duplex in RLC unwinds, which involves the protein Ago2 to cleave the passenger strand) to form an active RNA-induced silencing complex (RISC) that contains a single-stranded RNA (called the guide strand). Fourth, the RISC recognizes the target RNA by Watson–Crick base pairing with the guide strand and cleaves the target RNA. Finally, the RISC releases its cleaved product and goes on to catalyze a new cycle of target recognition and cleavage (39
FIG. 1. RNAi therapeutic strategies: (1) siRNA may be delivered directly to the CNS to silence disease genes; (2) Pol III constructs synthesizing shRNA; or (3) Pol II constructs synthesizing miRNA can be placed in viral vectors and delivered into the CNS cells (more ...)
In differentiated mammalian cells, long dsRNA activates RNA-dependent protein kinase PKR and type I interferon response, which leads to a nonspecific global translation depression and apoptosis (18
). However, this nonspecific reaction can be circumvented by introduction of synthetic siRNA (11
), which can go into the RNAi pathway similar to the siRNAs produced from long dsRNA (, #1). Alternatively, RNAi may be triggered by a short hairpin RNA (shRNA) synthesized by a Pol III promoter (, #2) or by a microRNA (miRNA) that can be synthesized by either a Pol II or Pol III promoter (, #3) (9
). The shRNA is a single-stranded RNA folded into a simple hairpin, composed of a perfectly base-paired stem of 21 to 23
nt and a loop. It is synthesized by a Pol III promoter (e.g.
, U6) as in the nucleus (44
). In contrast, the miRNA is synthesized as a long transcript, called primary miRNA (pri-miRNA) (27
). The pri-miRNA need to be processed by RNase III enzyme Drosha and its partner DGCR8 (or Pasha in invertebrates) to form precursor miRNA (pre-miRNA), which is ~70
nt long and folds into a hairpin structure composed of an imperfectly base-paired stem and a loop. The pre-miRNA then shares the same downstream processing pathway with the shRNA. Both are exported by exportin 5/RAN-GTP from the nucleus to the cytoplasm, where they are further processed to form a single-stranded miRNA or siRNA. This processing step is tightly coupled with loading the guide strand of miRNA or siRNA into the RISC, which is capable of either cleaving the target RNA if the target perfectly complements the miRNA in sequence, or mediating translational gene silencing if the miRNA mismatches the target RNA at multiple base-pairs ().
Because RISC recognizes its target by Watson–Crick base pairing with the guide strand of the siRNA, destruction of the target RNA can be specific (43
). This property of RNAi can be harnessed to target specific mRNA species for destruction, and therefore, to silence the expression of the toxic protein encoded by the mRNA for therapy. Thus far, RNAi therapy for CNS diseases has been delivered by direct administration of synthetic siRNA or genes that encode shRNA or miRNA using viral vectors. Both delivery methods have shown therapeutic efficacy in animal models for neurodegenerative diseases, and thus, are potential therapeutic strategies for humans (15
The viral delivery method (gene therapy) delivers transgenes that are composed of a promoter and a desired transgene cassette to the CNS cells. In recent years, viral vector technology for gene delivery into the brain has improved substantially (33
). A sustained expression extending beyond 12 months has been achieved using recombinant adenovirus (RAd)-, lentivirus-, adeno-associated virus (AAV)-, and herpes simplex-1 virus-derived vectors in animal studies (32
). Viral vector systems, including both adenovirus and AAV, have been developed to the point where clinical gene therapy for brain diseases is now possible for both acute and chronic central nervous system pathologies. Taking advantage of these viral vectors, several groups have tested viral delivery of RNAi therapy for neurological diseases. These tests have shown efficacy in models for polyglutamine diseases, Alzheimer disease, and ALS (13
In these studies, two transgenes were incorporated into AAV or lentiviral vectors. One transgene expressed a marker gene (e.g.
, EGFP) driven by a Pol II promoter (e.g.
, CMV) and the other expressed a shRNA driven by a Pol III promoter (e.g.
, U6). The marker gene provided an indicator as to efficiency with which the transgene is delivered. The shRNA silences the expression of the mutated disease gene or a gene that is in the disease pathway. In the cases of polyglutamine diseases and Alzheimer disease, the viral vectors were directly injected into the relevant disease areas in the brain. However, in treating ALS, the limited spread of virus presents a particular challenge for administering the virus to wide groups of motor neurons distributed along the spinal cord and in the motor cortex. Injection into a single or a few spots can only cover a small fraction of motor neurons that are degenerating (). Indeed, injection of a lentivirus delivering RNAi against human SOD1 into the spinal cord of an ALS mouse model led to a local functional improvement but no extension of survival (40
FIG. 2. A schematic illustration of different methods of administering viral vectors for delivering RNAi to spinal motor neurons: (A) direct spinal cord injection, (B) muscle injection, and (C) nerve injection. Different colors mark the different motor neuron (more ...)
Because of the limitation in injecting the viral vector directly into the CNS, other groups have injected the virus into muscles (). These experiments have demonstrated that some virus such as adenovirus, AAV2, and Rabies glycoprotein-pseudotyped lentivirus can be taken up by the nerve terminals and retrogradely transported to the spinal cord motor neurons (3
). However, because each muscle only receives a small number of axonal projections, injection of many muscles were required to cover multiple motor neuron pools. Because of the large tissue mass of the muscles, these injections also require relatively large doses of the virus. Perhaps to reduce the required dose, Ralph and colleagues injected a lentivirus delivering RNAi against mutant SOD1 into the muscles in 7-day-old mice, where muscle mass is small and immune response underdeveloped. They observed retrograde transport of the virus and the transgene expression. This treatment resulted in a large extension (~70%) in lifespan of the mutant SOD1 mouse model (38
). This is an encouraging result for RNAi therapy but the treatment may not be practical in humans. Administration of lentivirus to adult humans in muscles throughout the whole body will require even larger doses of virus, which will not only increase the cost, but also put the patient at risk due to untoward side effects, including immune responses, transduction of irrelevant cells (e.g.
, germline cells), and the potential to cause insertion mutagenesis and neoplasia formation (28
). In addition, it was unclear from this experiment whether silencing of mutant SOD1 at the disease onset will be effective, because the therapy was administrated long before the disease onset in the mouse model. In order to improve the delivery of viral vectors to motor neurons, we administered viral vectors by nerve injection.