title: Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA interference.
abstract: RNA interference (RNAi) is a mechanism that appears to control unwanted gene expression in a wide range of species. In Drosophila, RNAi is most effectively induced by double-stranded RNAs (dsRNAs) of over approximately 80 nucleotides (nt) and in mammalian cells an RNAi-like inhibition of gene expression has been shown to be mediated by dsRNAs of approximately 21-23 nt. To test if RNAi can be used to specifically down-regulate a human disease-related transcript we have used Drosophila and human tissue culture models of the dominant genetic disorder spinobulbar muscular atrophy (SBMA). A variety of different dsRNAs were assessed for the ability to inhibit expression of transcripts that included a truncated human androgen receptor (ar) gene containing different CAG repeat lengths (16-112 repeats). In Drosophila cells, dsRNAs corresponding to non-repetitive sequences mediated a high degree of sequence-specific inhibition, whereas RNA duplexes containing CAG repeat tracts only induced gene-specific inhibition when flanking ar sequences were included; dsRNAs containing various lengths of CAG repeats plus ar sequences were unable to induce allele-specific interference. In mammalian cells we tested sequence-specific small dsRNAs of 22 nt; these rescued the toxicity and caspase-3 activation induced by plasmids expressing a transcript encoding an expanded polyglutamine tract. This study demonstrates the feasibility of targeting a transcript associated with an important group of genetic diseases by RNAi.
authors.affiliation: Medical Genetics Branch, National Human Genome Research Institute and Neurogenetics Branch, National Institute of Neurological Diseases, National Institutes of Health, Bethesda, MD 20892, USA. ncaplen@nhgri.nih.gov
authors.forename: Natasha J
authors.initials: NJ
authors.lastname: Caplen
authors.forename: J Paul
authors.initials: JP
authors.lastname: Taylor
authors.forename: Victoria S
authors.initials: VS
authors.lastname: Statham
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Tanaka
authors.forename: Andrew
authors.initials: A
authors.lastname: Fire
authors.forename: Richard A
authors.initials: RA
authors.lastname: Morgan
mesh_terms: D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004195:Disease Models, Animal; D015536:Down-Regulation; D004330:Drosophila; D005240:Feasibility Studies; D020868:Gene Silencing; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D020966:Muscular Disorders, Atrophic / Q000235:genetics*; D010455:Peptides / Q000235:genetics* / Q000633:toxicity; D012313:RNA / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism*; D011944:Receptors, Androgen / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D018911:Trinucleotide Repeats
chemical_list: D008164:Luminescent Proteins; D010455:Peptides; D012330:RNA, Double-Stranded; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C097188:polyglutamine; D012313:RNA; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases