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Superior Gene Silencing Using Adenoviral Vectors

re 3A). Finally, these two adenoviral vectors were used to infect PC12 cells that had been created to express normal (Q28) or (Q166) mutant ataxin-3. Again, the viruses were able to specifically target the mutant allele (Figure 3B). These results indicate the utility of viruses for delivering siRNAs that target allele-specific disease genes.

Figure 3. Allele-specific Silencing with Adenoviral Vectors Expressing an siRNA (2). Cos-7 cells were co-transfected with the Atx-3-Q28-GFP (wt) and Atx-Q166 (mutant) constructs and then infected (MOI = 50) with adenovirus expressing siRNAs that had been designed such that a single nucleotide polymorphism (SNP) was in the middle of the target sequence. The siRNA produced by Ad-G10i was perfectly complementary to the wild type construct, whereas that of Ad-C10i was complementary to the mutant construct. (A) Green: GFP. Red: immunofluorescence of the polyQ construct detected with 1C2 antibody. Ad-G10i reduced wt ataxin-3 levels 95% but did not affect mutant ataxin-3 levels. Conversely, Ad-C10i reduced mutant ataxin-3 levels 91% but had no effect on WT ataxin-3 levels. (B) PC12 cells expressing WT (left) or mutant ataxin-3 (right) were infected (MOI = 100) with the Ad-G10i or Ad-C10i virus. Western analysis was performed with antibodies to Ataxin-3 and GAPDH (as a control), and the siRNAs produced by the viruses were able to specifically silence the allele to which they were designed.


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