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RNAi in Neurodegenerative Diseases—What's the Therapeutic Potential?
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By Zuoshang Xu, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605
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Zuoshang Xu led this live discussion on 18 March 2003. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.
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 View Transcript of Live Discussion — Posted 28 August 2006
Background Text
By Zuoshang Xu
Diseases caused by dominant, gain-of-function mutations develop in people bearing one mutant and one wild-type copy of the gene. Some of the best-known examples of this class are neurodegenerative diseases, including Huntington's, some forms of amyotrophic lateral sclerosis (ALS) and rare, familial forms of the otherwise common Alzheimer's and Parkinson's diseases (Taylor et al., 2002). In all these diseases, the exact pathways whereby the mutant proteins cause cell degeneration are not clear, but the origin of the cellular toxicity is known to be the mutant protein. Thus, selectively lowering or eliminating the mutant protein is a key step in developing effective therapies. Until recently, it was not clear how specific down-regulation of a wide variety of mutant proteins could be achieved. But now, new advances in RNA interference (RNAi) raise the possibility that RNAi can be developed and eventually applied as a therapeutic means for these neurodegenerative diseases.
When exposed to double-stranded RNA (dsRNA), eukaryotic cells respond by destroying their own mRNA that shares sequence with the dsRNA. This phenomenon is called RNA interference (RNAi). A similar cellular process was first discovered in plants, where it is called co-suppression (Napoli et al., 1990; Smith et al., 1990; van der Krol et al., 1990), and in the fungus Neurospora crassa, where it is known as quelling (Romano and Macino, 1992; Cogoni et al., 1996). Since its first description in animal cells (Fire et al., 1998), a growing number of investigators have been using RNAi for reverse genetics to investigate gene functions in cells and animals (Kennerdell and Carthew, 1998; Ngo et al., 1998; Lohmann et al., 1999; Sanchez Alvarado and Newmark, 1999; Gonczy et al., 2000; Svoboda et al., 2000; Wianny and Zernicka-Goetz, 2000; Yang et al., 2001). Expression of virtually any gene can be disrupted by delivering dsRNA corresponding to that gene's sequence. Capping this broad-based application are several new reports where RNAi has been used for genome-wide scans of gene function (Dillin et al., 2002; Ashrafi et al., 2003; Kamath et al., 2003; Lee et al., 2003).
The exact mechanism of RNAi has not been worked out completely, but current biochemical evidence suggests a four-step process. First, Dicer, an enzyme in the RNase III family (Bernstein et al., 2001), initiates ATP-dependent fragmentation of long dsRNA into 21-25 nucleotide double-stranded fragments, termed small interfering RNAs (siRNAs) (Zamore et al., 2000; Bernstein et al., 2001; Nykanen et al., 2001). Second, ATP-dependent unwinding of the siRNA duplex remodels the complex to generate an active RNA-induced silencing complex (RISC) (Hammond et al., 2000; Nykanen et al., 2001). Third, the RISC recognizes and cleaves a target RNA complementary to the guide strand (the antisense strand) of the siRNA (Hammond et al., 2000; Nykanen et al., 2001). Finally, the RISC releases its products and goes on to catalyze a new cycle of target recognition and cleavage (Hutvágner and Zamore, 2002).
It was clear from the beginning that RNAi would be a powerful tool for probing gene function, however, its uses in differentiated mammalian cells had been limited because long dsRNA elicits an interferon reaction that leads to apoptosis (McManus and Sharp, 2002). But then in 2001, two groups showed that synthetic siRNAs can mediate efficient RNAi without eliciting the interferon response in mammalian cells (Caplen et al., 2001; Elbashir et al., 2001c ). This opened the floodgates for applying RNAi to test the functions of a great number of genes in mammalian cells, as attested by the wealth of recent publications on this topic.
In the less than two years since the original report on gene silencing using siRNA in differentiated mammalian cells, the technology for delivering siRNA into cells in culture and in vivo has improved rapidly. Most notable is the method of synthesizing small hairpin RNA (shRNA) from plasmid constructs directly in cells. A popular approach uses type III RNA polymerase III (Pol III) promoters (Paule et al., 2000), which offer several advantages. First, this class of RNA polymerases naturally produces small, non-coding transcripts such as U6 small nuclear RNA (snRNA) and H1 RNA. Second, their natural transcripts are neither capped at the 5' nor polyadenylated at the 3' ends, and therefore resemble siRNA. Third, all of their promoter elements are located 5' to the transcription initiation site, therefore allowing convenient design of transcript sequences. Fourth, transcription directed by these promoters initiates at defined nucleotides (e.g., a G for the U6 promoter or an A for the H1 promoter) and terminates when the transcription encounters four or more Ts in succession (Bogenhagen et al., 1980). Incidentally, the transcripts also carry 3' overhangs of one to four Us (the termination sequence), a structural feature similar to what has been defined in vitro for effective siRNAs (Elbashir et al., 2001a ).
Using this strategy, numerous groups have demonstrated that shRNAs transcribed in cells can trigger degradation of corresponding mRNAs, probably because shRNA is processed into siRNAs (Brummelkamp et al., 2002b; Jacque et al., 2002; Lee et al., 2002; McManus et al., 2002; Miyagishi and Taira, 2002;Paddison et al., 2002; Paul et al., 2002; Sui et al., 2002; Yu et al., 2002). Recent work suggests that tRNA promoters, which are also transcribed by Pol III, may facilitate export of shRNA to the cytoplasm, where Dicer resides (Kawasaki and Taira, 2003). An alternative to the PolIII promoter is a strategy developed by Cullen and colleagues, in which a PolII CMV promoter was used to direct synthesis of shRNA (Zeng et al., 2002). This is more complex because one must incorporate the shRNA coding sequence into the stem of a microRNA precursor (pre-miRNA), but provides for more exquisite developmental or tissue-specific regulation.
These technical advances raise the possibility that specific constructs can be designed to express shRNA in vivo to silence target genes. For example, constructs may be inserted into a virus for transducing cells in vivo. Such a strategy may become a therapeutic intervention for diseases caused by dominant, gain-of-function gene mutations. In addition, these constructs may be used to inhibit expression of genes to investigate gene function by transfection in cultured cells or by transgenic approach in vivo. The initial experiments demonstrating the feasibility of these strategies have already been carried out. Both viral vector- and transgene-directed synthesis of shRNA have been shown to mediate inhibition of endogenous genes in cultured cells and in vivo (Brummelkamp et al., 2002a ; Hasuwa et al., 2002; Xia et al., 2002; Rubinson, 2003; Tiscornia et al., 2003). The therapeutic potential of RNAi has already been tested in several cellular models of diseases. Efficacy of RNAi has been demonstrated against viral infection (Gitlin et al., 2002; Jacque et al., 2002), cancer cell proliferation (Brummelkamp et al., 2002a ; Wilda et al., 2002; Cioca et al., 2003) and polyglutamine diseases (Caplen et al., 2002; Xia et al., 2002). Most recently, Judy Lieberman's group reports in the February 10 online Nature Medicine that intravenous injection of siRNA duplexes that target the gene encoding the Fas receptor inhibited apoptosis, protected against liver fibrosis, and prolonged survival in a mouse model of autoimmune hepatitis (Song, 2003). The list will surely increase rapidly in the near future.
To treat dominant genetic disorders of the gain-of-function type, ideally one should seek to silence expression of the mutant protein selectively, thereby allowing the wild-type allele to continue functioning. Given that the vast majority of gene mutations that cause dominant diseases are single nucleotide changes, one wonders whether RNAi mediated by siRNA can discriminate mutant from the wild-type mRNA with single nucleotide specificity. Current literature presents conflicting answers to this question. siRNAs that differ from the sequence of their target RNA at one or more nucleotides retain efficacy in some cases (Boutla et al., 2001; Holen et al., 2002) and lose activity in others (Boutla et al., 2001; Elbashir et al., 2001b ; Brummelkamp et al., 2002a ; Brummelkamp et al., 2002b; Yu et al., 2002). One recent report concludes that siRNAs cannot differentiate RNAs with single nucleotide difference (Zeng and Cullen, 2003). In collaboration with Phillip Zamore's and Yang Shi's groups, we have investigated the potential of siRNA to selectively silence the expression of mutant Cu, Zn superoxide dismutase (SOD1), which causes motor neuron degeneration and ALS by gaining a toxic property (Cleveland and Rothstein, 2001). Our data suggest that siRNA sequences that selectively silence the mutant can be found by screens using in vitro RNAi reactions and transfected cells (unpublished observation). The rules in designing the siRNA that can silence genes with single nucleotide specificity are currently unclear and remain to be further investigated.
The potential of using RNAi for therapy is not limited to directly silencing pathogenic genes or disease-causing mutant genes. As disease mechanisms become increasingly clear, its application can be expanded to silence genes involved in known pathogenic pathways. For example, an obvious target for treatment of Alzheimer's disease is the b-site APP-cleaving enzyme BACE, which is required for the production of Ab peptide (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001) and is present at elevated levels in the cortex of people with AD than controls (see related ARF news story). If such a strategy is successful, one can envision that RNAi could be applied not only to familial disease with identified dominant gene mutations, but also to sporadic disease.
While the therapeutic potential of RNAi is real and will undoubtedly grow, urgent problems must be resolved to prepare this technology for eventual human trials. First, how can siRNA be delivered in vivo? One way is to administer siRNA directly. This will require its modification or formulation with other agents to increase its stability and enable it to enter cells. While this method might work in treating diseases of peripheral organs, its uses for CNS diseases have not been well supported. A gene therapy approach using viral vectors offers an alternative. The concept has not yet been proven for CNS diseases in humans, but animal experiments indicate that long-term transgene expression in the CNS cells is achievable using AAV or lentiviral vectors (Kordower et al., 2000; Azzouz et al., 2002; Fu et al., 2002; Muramatsu et al., 2002; Wang et al., 2002). Human trials in peripheral disease have had limited success but serious problems remain (Verma, 2002). To overcome the threshold of clinical application, answers to other important questions are also eagerly awaited: Is inhibiting expression of mutant proteins, or proteins involved in pathogenesis, sufficient to prevent, slow, stop, or even reverse the disease? What is the maximal therapeutic effect achievable by RNAi? What's the right cell type for inhibition? Are there long-term adverse effects from expressing high levels of shRNA?
I suggest we address these questions during the discussion:
1. What are the key technical obstacles to overcome in moving RNAi therapy forward in ALS, Huntington's, Parkinson's and Alzheimer's?
2. What is the optimal delivery method for RNAi, direct administration of siRNA or gene therapy?
3. What questions about RNAi in the CNS do we need answers for before therapeutic trials?
4. What are the potential targets in neurodegenerative diseases for RNAi therapy?
5. Short of therapy, what are the most fruitful research questions to address with current RNAi technology?
Acknowledgement
I thank Gabrielle Strobel and Phillip Zamore for editing and suggestions.
For a news summary of two experimental therapeutic approaches, see ARF related news story
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