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RNA Knockdown As a Potential Therapeutic Strategy in Parkinson's

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RNA Knockdown As a Potential Therapeutic Strategy in Parkinson's Gene Therapy (2006) 13, 517–524 & 2006 Nature Publishing Group All rights reserved 0969-7128/06 $30.00 www.nature.com/gt RNA knockdown as a potential therapeutic strategy in Parkinson’s disease FP Manfredsson1,2, AS Lewin2,3 and RJ Mandel1,2 1Department of Neuroscience, McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL, USA; 2The Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA and 3Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA Parkinson’s disease is a prevalent progressive degenerative interference can theoretically be applied to Parkinson’s disease disorder of the elderly. There is a current need for novel since over-expression of various proteins is known to kill the therapeutic strategies because the standard levodopa phar- dopamine neurons of the substantia nigra in animal models macotherapy is only temporarily efficacious. Recently, there and in familial forms of Parkinson’s disease. Potential RNA have been some high-profile successful preclinical results interfering strategies and caveats are discussed in this review. obtained in animal models of neurological disorders using Gene Therapy (2006) 13, 517–524. doi:10.1038/sj.gt.3302669; small interfering RNAs delivered by viral vectors. RNA published online 3 November 2005 Keywords: adeno-associated virus; ribozyme; interfering RNA Introduction eukaryotic kingdoms. The major enzymes involved are highly conserved amongst species, indicating that the Parkinson’s disease (PD) is one of the most common response is an important cellular mechanism. Work into neurological disorders, second only to Alzheimer’s dis- the world of naturally occurring siRNA has shown that ease, affecting approximately 2% of adults. Although the this response is not only involved in protecting the cause of the idiopathic form of the disease is yet to be genome from threats such as viruses,1 but is also established, recent advances in the study of certain familial mechanistically related to microRNA (miRNA) path- forms of the disease have shed some light on the overall ways that provide crucial regulation of temporal control process of neurodegeneration in PD. Still, much remains to of development. The understanding of the mechanisms be understood about the disease. The recent development involved in RNA interference has progressed dramati- of new techniques such as the use of short RNA molecules cally in the past several years. Briefly, long double- like small interfering RNAs (siRNA) have gained increas- stranded RNA are processed by an RNase III enzyme ing popularity as tools to knockdown certain cellular named dicer to shorter (21–23 nt) siRNA complexes. The mRNAs, and these tools have been valuable in elucidating actual gene-silencing is processed by a group of proteins molecular pathways and players involved in PD. Still, the collectively termed the RISC (RNA-induced silencing ultimate goal is to treat the disease, and the current complex), which serves to bring the siRNA to the vicinity understanding of the potential etiology of PD makes it an of a complimentary mRNA and provides the catalytic attractive target for a siRNA therapy. One common feature enzymes involved in the cleavage of the mRNA.2–4 amongst the various forms of the disease is protein miRNA works along a similar pathway, however, naturally accumulation and aberrant protein clearance. Hence, a occurring miRNA precursors are short (70 bp) non-coding reduction in translation of specific proteins may suffice to hairpin sequences that are also processed by dicer into relieve the cellular stresses. In addition, a number of cell short stretches of single-stranded RNA. Unlike siRNAs, death signaling events likely to be involved in all forms of miRNAs contain short mismatches in the sequences PD are also potential siRNA targets. complementary to their target mRNAs, and miRNAs are thought to inhibit translation rather than lead to the degradation of mRNA. miRNAs are extremely important RNA interference in gene regulation, and certain neuronal disorders such as fragile X are linked to the miRNA pathway.5 RNAi is a response to double-stranded RNA that has The siRNA system has been commandeered to create been identified in numerous organisms in all three a valuable tool in order to study the effects of knocking down certain transcripts. Experimentally, one scans a Correspondence: Dr RJ Mandel, Department of Neuroscience, target gene for suitable target sequences 21–23 nt long McKnight Brain Institute, University of Florida College of Medicine, and designs double-stranded RNAs to target these sites. PO Box 100244, Gainesville, FL 32610-0024, USA. Scanning is typically assisted by one of several public- E-mail: [email protected]fl.edu Received 21 June 2005; revised 16 September 2005; accepted 19 access computer programs that incorporate experimental September 2005; published online 3 November 2005 ‘rules’ for designing effective siRNAs. These rules RNA knockdown as a potential therapeutic strategy in Parkinson’s disease FP Manfredsson et al 518 include avoiding runs of four or more guanosines or an alternative to siRNA lipid complexes, electroporation adenosines, keeping the base composition between 30 of ‘naked’ RNA has been shown to be effective in and 55% G+C, and identifying sites with asymmetric delivering siRNA to a limited number of target organs, thermal stability so that the antisense strand is preferen- including the visual cortex and the CA1 region of the tially incorporated into RISC.6–9 As an alternative for hippocampus.20 As with lipid complexes and RNA- rational design of siRNAs, several groups have devel- nanoparticles, however, electroporation leads to only a oped strategies for creating and screening siRNA transient reduction of gene expression, and the prospect libraries targeted to a gene of interest.10–13 This approach of sequential re-administration to the brain is unattrac- is valid because some of the most effective siRNAs that tive in a therapeutic setting. have been described in the recent literature would have Viral vectors provide the most suitable means of been avoided according to the current design algorithms. delivering and expressing siRNA for long-term therapy. Several recombinant viruses have been used for gene Delivery of siRNA therapy in the brain, but the short duration of transduc- Short interfering RNAs can be introduced to mammalian tion and the proinflammatory properties of vectors based cells and experimental animals either as RNA or as DNA on adenovirus and herpes simplex virus have restricted clones that code for the double-stranded RNA. When their use to antitumor applications. Lentivirus vectors DNA clones are used, they are often designed to produce and recombinant adeno-associated virus (rAAV) vectors short hairpin RNA (shRNA) that is processed to form have been used for long-term transduction of the brain siRNA in the cell. For cultured cells and for temporary and particularly for gene therapy in animal models of dosing to tissue, direct delivery of siRNA has distinct neurodegenerative disorders such as Huntington’s dis- advantages: short RNA molecules are commercially ease (HD),21,22 spinocerebellar ataxia23 and PD.24,25 Both available in pure form. Double-stranded RNA can be virus types can infect nondividing cells and lead to long- stabilized by chemical modifications in both strands of term gene expression. Lentiviral vectors give a more the duplex.14–16 Certain ribose modifications in the sense localized response and can contain a larger ‘payload’ in strand have the added advantage of increasing the terms of passenger genes, but delivery of siRNA does not incorporation of the antisense strand of the duplex into exceed the 4.7 kb carrying capacity of AAV. While rAAV RISC, therefore increasing cleavage of the intended does not provoke an inflammatory response, infection mRNA. Using RNA also permits accurate dosing of the does stimulate humoral immunity, and most people have cells, since a known quantity of RNA is added. The circulating antibodies to AAV.26,27 Indeed, pre-immuni- disadvantage of direct RNA treatment is that a large zation of rats with AAV2 prevents striatal transduc- amount of siRNA is required (typically 10–100 nM)and tion with rAAV2 vectors.28 Several rAAV serotypes the effect is temporary (3–4 cell generations). Using a high have been used to deliver marker genes or therapeutic dose of siRNA may saturate the nuclear-cytoplasmic genes to the midbrain, but to date, rAAV1 and rAAV5 transport of miRNAs and thus lead to non-specific effects. appear to provide the widest area of transduction, while Plasmids and viral vectors can be used to deliver gene expression mediated by rAAV2 infection is more shRNAs, which are usually produced under the control precisely located near the site of injection.29 siRNA of RNA polymerase III (pol III) promoters. Pol III therapeutics will almost certainly need to specifically promoters generate large amounts of transcript, but target the SN. rAAV2 has the serendipitous feature that it much of it is sequestered in the nucleus away from the transduces the nigral DA neurons with higher efficiency cytoplasmic RISC. Consequently, some investigators have than other neurons in the anatomical vicinity providing switched to pol II promoter systems for the expression of specific nigrostriatal transduction.29–31 shRNA17 or have disguised their siRNA hairpins as RNA interference holds a valuable potential as a miRNA precursors to facilitate transport to the cyto- future therapeutic, it could be used to simply knock- plasm.18 Delivery with viral vectors has the advantages down a specific disease gene. siRNA is selective for fully that hard-to-transfect primary cells can often be infected complementary mRNA targets, and short mismatches and that expression of the RNA hairpin may remain stable has been shown to significantly lower the efficacy of in nondividing cells or for many generations in dividing target mRNA degradation.32 Nevertheless, as few as 11 cells, if an integrating virus is employed.
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