Allele-specific silencing of dominant disease genes

TLDR

siRNA offers therapeutic potential for silencing dominantly acting disease genes, especially when mutant alleles can be selectively targeted. The study demonstrates that allele‑specific silencing of disease genes can be achieved in mammalian cells by targeting either a linked SNP or the disease mutation itself. Using mammalian cell models, the authors designed siRNA to target a disease‑causing CAG repeat via an associated SNP in Machado–Joseph disease, optimized siRNA against the Tau V337M missense mutation, and delivered the constructs as in‑vitro duplexes, plasmid‑encoded, or viral short‑hairpin RNA. Allele‑specific suppression was successfully achieved with all three delivery approaches, and the work shows that siRNA can be engineered to silence genes differing by a single nucleotide, underscoring the importance of SNPs for treating dominantly inherited disorders.

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.