Abstract
Spinocerebellar ataxia type 7 is a polyglutamine disorder caused by an expanded CAG repeat mutation that results in neurodegeneration. Since no treatment exists for this chronic disease, novel therapies such post-transcriptional RNA interference-based gene silencing are under investigation, in particular those that might enable constitutive and tissue-specific silencing, such as expressed hairpins. Given that this method of silencing can be abolished by the presence of nucleotide mismatches against the target RNA, we sought to identify expressed RNA hairpins selective for silencing the mutant ataxin-7 transcript using a linked SNP. By targeting both short and full-length tagged ataxin-7 sequences, we show that mutation-specific selectivity can be obtained with single nucleotide mismatches to the wild-type RNA target incorporated 3′ to the centre of the active strand of short hairpin RNAs. The activity of the most effective short hairpin RNA incorporating the nucleotide mismatch at position 16 was further studied in a heterozygous ataxin-7 disease model, demonstrating significantly reduced levels of toxic mutant ataxin-7 protein with decreased mutant protein aggregation and retention of normal wild-type protein in a non-aggregated diffuse cellular distribution. Allele-specific mutant ataxin7 silencing was also obtained with the use of primary microRNA mimics, the most highly effective construct also harbouring the single nucleotide mismatch at position 16, corroborating our earlier findings. Our data provide understanding of RNA interference guide strand anatomy optimised for the allele-specific silencing of a polyglutamine mutation linked SNP and give a basis for the use of allele-specific RNA interference as a viable therapeutic approach for spinocerebellar ataxia 7.
Highlights
Spinocerebellar ataxia 7 (SCA7) is a late onset neurodegenerative disease which presents with a classic autosomal dominant ataxia but which is uniquely associated with macular degeneration [1]
These short hairpin RNAs (shRNAs) were screened in an assay in which a short 60 bp target of either the mutant or the wild-type ataxin7 gene sequence was inserted in the 39UTR of Renilla luciferase and normalised to background Firefly luciferase (Figure 1B)
Nucleotide mismatches incorporated at shRNA positions 11-16 all showed effective discrimination between the wild-type and mutant targets (Figure 2A), shR-P15 demonstrating the greatest discrimination with the wild-type target minimally affected retaining 90% expression and the mutant knocked down to nearly 50% relative to a non-specific shRNA control (p,0.05)
Summary
Spinocerebellar ataxia 7 (SCA7) is a late onset neurodegenerative disease which presents with a classic autosomal dominant ataxia but which is uniquely associated with macular degeneration [1] It is part of a group of nine known polyglutamine (polyQ) disorders which share expanded (CAG) repeat mutations that translate into polyQ tracts [2]. Most RNA interference (RNAi)-based therapeutic approaches to suppress the expression of toxic polyQ proteins have been demonstrated in mouse models of polyQ disorders, including SCA1, SCA3 and HD [10,11,12] While important, these studies have targeted suppression of the human disease transgene in the mouse and have not investigated the effects of concomitant wild-type allele suppression or investigated the mechanistic basis for specific suppression of polyQ disease alleles
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