. RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5820-5. PubMed.


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  1. RNAi Therapy Works in Animal Models

    Scott Harper et al. recently demonstrated that RNA interference (RNAi) can treat Huntington disease in an animal model (Harper et al., 2005). This work, together with a previous published experiment from the same group on treatment of spinal cerebellar ataxia (Xia et al., 2004), and two other experiments on treatment of ALS (Ralph et al., 2005; Raoul et al., 2005), demonstrates the concept of RNAi therapy for neurodegenerative diseases.

    The common approach in these experiments was to deliver RNAi using viral vectors. All showed in vivo knockdown of the target gene and phenotypic improvement. These are very encouraging developments that bring RNAi one step closer to clinical application. Here I provide some background about these experiments and discuss some challenges that we still need to meet in order to realize the full therapeutic potential of RNAi.

    In general, genetic disorders can be caused by two types of genetic mutations. One causes the gene to lose its function and the other causes the gene to gain a function—the gain could be either a novel function or an enhancement of an existing function. The treatment strategy for the loss-of-function type of mutations is to deliver to the patient a normal copy of the mutated gene so that its function can be replaced. But for a long time the conceptual framework for treatment of gain-of-function mutations was less obvious. However, RNAi has provided a conceptual basis for this kind of treatment (see ARF Live Discussion).

    RNAi is a cellular function that is widely conserved in eukaryotes. Triggered by double-stranded RNA (dsRNA), RNAi destroys the target RNA that shares sequence homology with the dsRNA. The mechanism of RNAi is not understood completely, but a key executor is a protein complex called RNA-induced silencing complex, or RISC, which contains proteins and a single-stranded RNA (guide strand) derived from the dsRNA that the cells are originally exposed to. This guide strand is capable of recognizing the target RNA by Watson-Crick base pairing. After the pairing, RISC cleaves the target RNA. Thus, RNAi can destroy the target RNA specifically. When the target is an mRNA, RNAi can selectively knock down the expression of the target protein (Tomari and Zamore, 2005). This makes RNAi ideal for treatment of diseases caused by gain-of-function type of genetic mutations (see ARF Live Discussion).

    A serious challenge to the success of RNAi therapy is the delivery of RNAi in vivo. Authors of the above four papers met this challenge by demonstrating that viral vector-delivered RNAi can treat neurodegenerative diseases in animal models. These experiments used viral vectors that express short hairpin RNA (shRNA) that can be processed to short RNA duplexes called small interfering RNA (siRNA) (Shi, 2003). The siRNAs mediated RNAi against mutant ataxin-1, SOD1, and huntingtin mRNAs, which cause spinal cerebellar ataxia 1 (SCA-1), amyotrophic lateral sclerosis (ALS) and Huntington disease, respectively. These successes are not surprising since all of these diseases are caused by gain-of-function gene mutations. Although the nature of the gained function is not clear, it is clear that disease severity correlates with the levels of mutant protein. Therefore, lowering the mutant protein levels will protect neurons from the toxic effects of these proteins.

    What are the future challenges in developing RNAi therapy? Here I raise a few: First, how long is shRNA expressed after the viral delivery and to what extent and for how long is the target gene silenced? These questions have not been answered in these experiments. Therefore, it is not clear whether the observed therapeutic effects are a result of transient silencing or a sustained silencing of the target genes. A related question is, what is the best time to administer the RNAi therapy, before or after the disease onset? In all experiments the RNAi therapy was administered before the disease onset. It will be important to determine whether, by administration of RNAi therapy after the onset of the disease, the disease can be reversed.

    Next, as a general rule, is it necessary to administer mutant-allele-specific RNAi? In these four cases the siRNAs were not specifically designed to silence the mutant gene because the mutant genes are human transgenes that differ in sequences from the mouse endogenous genes. Silencing the transgenes does not affect expression of endogenous mouse proteins. In human patients, however, shRNAs would silence both the mutant and the wild-type gene expression. This may be problematic because the wild-type functions of these genes are necessary for normal cellular function, and animal experiments show that in some cases they are essential (Cattaneo et al., 2001; Ding et al., 2003). In humans, the phenotypes for lack of these genes are not known, but they could be serious, given the animal data. To overcome this problem, allele-specific silencing or RNAi with replacement gene therapy may be necessary (Ding et al., 2003; Miller et al., 2003; Xia et al., 2005).

    Third, are the viral vectors the best way for delivery of RNAi therapy? Delivery using viral vectors carries risks associated with gene therapy (Kimmelman, 2005). An alternative is to deliver siRNA directly to patients. A recent study has demonstrated that chemically modified siRNAs can be systemically administered and can knock down specific genes in multiple tissues (Soutschek et al., 2004; see ARF related news story). However, siRNAs with chemical modifications that allow the siRNA to penetrate the blood-brain barrier have not been reported. Nevertheless, siRNA or chemically modified siRNA may be directly delivered to the CNS to induce specific knockdown (Thakker et al., 2004). Direct delivery to the CNS does carry disadvantages, such as risks and costs associated with surgery and constant consumption of drugs. Long-term risks of toxicity with chronic administration of siRNA or chemically modified siRNA are yet to be determined.

    In conclusion, the new results are exciting, and they are the first steps toward developing RNAi therapy. There is little doubt that most, if not all, mammalian cells possess the RNAi mechanism. The key to therapy rests on the development of highly efficient and safe delivery methods. If this can be achieved, RNAi can not only be used to treat genetic disorders that are caused by dominant, gain-of-function mutations, but also sporadic diseases where the disease pathways are understood. For example, if the amyloid hypothesis for Alzheimer disease is correct (Hardy and Selkoe, 2002), RNAi can be used to downregulate BACE and/or other proteins that are involved in the production of Aβ, a substrate for β amyloid that is postulated to cause neuronal degeneration.


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    . Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis. Aging Cell. 2003 Aug;2(4):209-17. PubMed.

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    . RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5820-5. PubMed.

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    . Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat Med. 2005 Apr;11(4):429-33. PubMed.

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    . Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference. Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17270-5. PubMed.

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    . RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med. 2004 Aug;10(8):816-20. PubMed.

    . An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase. J Neurochem. 2005 Jan;92(2):362-7. PubMed.

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