Allele-specific silencing of dominant disease genes

Abstract

Small interfering RNA (siRNA) holds therapeutic promise for silencing dominantly acting disease genes, particularly if mutant alleles can be targeted selectively. In mammalian cell models we demonstrate that allele-specific silencing of disease genes with siRNA can be achieved by targeting either a linked single-nucleotide polymorphism (SNP) or the disease mutation directly. For a polyglutamine neurodegenerative disorder in which we first determined that selective targeting of the disease-causing CAG repeat is not possible, we took advantage of an associated SNP to generate siRNA that exclusively silenced the mutant Machado-Joseph disease/spinocerebellar ataxia type 3 allele while sparing expression of the WT allele. Allele-specific suppression was accomplished with all three approaches currently used to deliver siRNA: in vitro-synthesized duplexes as well as plasmid and viral expression of short hairpin RNA. We further optimized siRNA to specifically target a missense Tau mutation, V337M, that causes frontotemporal dementia. These studies establish that siRNA can be engineered to silence disease genes differing by a single nucleotide and highlight a key role for SNPs in extending the utility of siRNA in dominantly inherited disorders.

Fig. 1.

RNAi-mediated suppression of expanded CAG repeat-containing genes. Expanded CAG repeats are not direct targets for preferential inactivation (a), but a linked SNP can be exploited to generate siRNA that selectively silences mutant ataxin-3 expression (b–f). (a) Schematic of cDNA encoding generalized polyQ-fluorescent protein fusions. The bars indicate regions targeted by siRNAs. HeLa cells cotransfected with Q80-GFP, Q19-RFP, and the indicated siRNA. Nuclei are visualized by 4′,6-diamidino-2-phenylindole staining (blue) in merged images. (b) Schematic of human ataxin-3 cDNA, with bars indicating regions targeted by siRNAs. The targeted SNP (G987C) is shown in color. In the displayed siRNAs, red or blue bars denote C or G, respectively. (c) Quantitation of fluorescence in Cos-7 cells transfected with WT or mutant ataxin-3 (Atx)-GFP expression plasmids and the indicated siRNA. Fluorescence from cells cotransfected with siMiss was set at 1. The bars depict mean total fluorescence from three independent experiments ± SEM. Rel. Fl., relative fluorescence. (d) Western blot analysis of cells cotransfected with the indicated ataxin-3 expression plasmids (Upper) and siRNAs (Lower). Appearance of aggregated, mutant ataxin-3 in the stacking gel (seen with siMiss and siG10) is prevented by siRNA inhibition of the mutant allele. (e) Allele specificity is retained in the simulated heterozygous state. Western blot analysis of Cos-7 cells cotransfected with WT (Atx-3-Q28-GFP) and mutant (Atx-Q166) expression plasmids along with the indicated siRNAs (mutant ataxin-3 detected with 1C2, an antibody specific for expanded polyQ, and WT ataxin-3 detected with anti-ataxin-3 antibody). (f) Western blot of Cos-7 cells transfected with ataxin-3-GFP expression plasmids and plasmids encoding the indicated shRNA. The negative control plasmid, phU6-LacZi, encodes siRNA specific for LacZ. Both normal and mutant proteins were detected with anti-ataxin-3 antibody. Tubulin immunostaining is shown as a loading control in d–f.

Fig. 2.

shRNA-expressing adenovirus mediates allele-specific silencing in transiently transfected Cos-7 cells simulating the heterozygous state. (a) Representative images of cells cotransfected to express WT and mutant ataxin (Atx)-3 and infected with the indicated adenovirus (Ad) at 50 multiplicities of infection. Ataxin-3-Q28-GFP (green) is directly visualized, and ataxin-3-Q166 (red) is detected by immunofluorescence with the 1C2 antibody. Nuclei visualized with 4′,6-diamidino-2-phenylindole staining in merged images. An average of 73.1% of cells coexpressed both ataxin-3 proteins with Ad-LacZi. (b) Quantitation of mean fluorescence from two independent experiments was performed as described for a. Rel. Fl., relative fluorescence. (c) Western blot analysis of viral-mediated silencing in Cos-7 cells expressing WT and mutant ataxin-3 as described for a. Shown are mutant ataxin-3 detected with 1C2 antibody and WT human and endogenous primate ataxin-3 detected with anti-ataxin-3 antibody. (d) shRNA-expressing adenovirus mediates allele-specific silencing in stably transfected neural cell lines. Differentiated PC12 neural cells expressing WT (Left) or mutant (Right) ataxin-3 were infected with adenovirus (100 multiplicities of infection) engineered to express the indicated hairpin siRNA. Shown are Western blots immunostained for ataxin-3 and GAPDH as loading control.

Fig. 3.

Allele-specific siRNA suppression of a missense Tau mutation. (a) Schematic of human Tau cDNA with bars indicating regions and mutations tested for siRNA suppression. Of these, the V337M region showed effective suppression and was studied further. Vertical bars represent microtubule-binding repeat elements in Tau. In the displayed siRNAs, blue and red bars denote A and C, respectively. (b) Western blot analysis of cells cotransfected with WT or V337M Tau-EGFP fusion proteins and the indicated siRNAs. Cells were lysed 24 h after transfection and probed with anti-Tau antibody. Tubulin immunostaining is shown as a loading control. (c) Quantitation of fluorescence in Cos-7 cells transfected with WT Tau-EGFP or mutant V337M Tau-EGFP expression plasmids and the indicated siRNAs. The bars depict mean fluorescence and SEM from three independent experiments. Fluorescence from cells cotransfected with siMiss was set at 1. Rel. Fl., relative fluorescence.

Fig. 4.

Allele-specific silencing of Tau in cells simulating the heterozygous state. (a) Representative fluorescent images of fixed HeLa cells cotransfected with flag-tagged WT-Tau (red), V337M-Tau-GFP (green), and the indicated siRNAs. An average of 73.7% of cells coexpressed both Tau proteins with siMiss. Although siA9 suppresses both alleles, siA9/C12 selectively decreased expression of mutant Tau only. Nuclei were visualized with 4′,6-diamidino-2-phenylindole stain in merged images. (b) Quantitation of mean fluorescence from two independent experiments was performed as described for a. Rel. Fl., relative fluorescence. (c) Western blot analysis of cells cotransfected with flag-tagged WT-Tau and V337M-Tau-EGFP fusion proteins and the indicated siRNAs. Cells were lysed 24 h after transfection and probed with an anti-Tau antibody. V337M-GFP Tau was differentiated on the basis of reduced electrophoretic mobility due to the addition of GFP. Tubulin immunostaining is shown as a loading control.