Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and translation inhibition of mutant transcripts.

Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington's disease (HD). CAG repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.