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

Abstract

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.

Antisense oligonucleotides for Alzheimer's disease therapy: from the mRNA to miRNA paradigm

“…The strength of this approach is selective elimination of only mutant huntingtin without loss of wild-type huntingtin preserving its physiological function. However, the PRECISION-HD1 and 2 trials were both terminated this year for lack of efficacy ( www.clinicaltrials.gov ; NCT03225833 and NCT03225846 respectively, accessed 2021-10-27), suggesting more work is needed to fully understand the mechanism [25] . Several other ASOs for treatment of neurodegenerative diseases are currently in clinical trials or at preclinical stages of development, as reviewed elsewhere [ 15 , 26 ].…”

Implications of Poly(A) Tail Processing in Repeat Expansion Diseases

“…Interestingly, some of the mutant mRNA-targeting oligonucleotides may cause deadenylation. Our recent study, focusing on the mechanisms leading to the allele-selective silencing of HTT , demonstrated that a specific self-duplexing siRNA (sd-siRNA) [ 153 ], which is described as miRNA-like siRNA, caused the rapid deadenylation of the mutant transcript and translational inhibition [ 154 ]. It remains to be investigated how deadenylation is activated in this therapeutic strategy using an endogene of HTT .…”

What, When and How to Measure—Peripheral Biomarkers in Therapy of Huntington’s Disease

“…They contain specific nucleotide substitutions that result in non-canonical base pairs in interaction with the CAG repeat sequence. Testing of these siRNAs in HD cell models led to the activation of the silencing mechanism preferentially for the mutant allele, due to the binding of multiple RISCs to an extended sequence of repeats [ 28 , 29 ]. Additionally, this strategy is also effective for other polyQ diseases [ 30 , 31 , 32 ].…”