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Abstract
In several human polyglutamine diseases caused by expansions of CAG repeats in the coding sequence of single genes, mutant transcripts are detained in nuclear RNA foci. In polyglutamine disorders, unlike other repeat-associated diseases, both RNA and proteins exert pathogenic effects; therefore, decreases of both RNA and protein toxicity need to be addressed in proposed treatments. A variety of oligonucleotide-based therapeutic approaches have been developed for polyglutamine diseases, but concomitant assays for RNA foci reduction are lacking. Here, we show that various types of oligonucleotide-based reagents affect RNA foci number in Huntington’s disease cells. We analyzed the effects of reagents targeting either CAG repeat tracts or specific HTT sequences in fibroblasts derived from patients. We tested reagents that either acted as translation blockers or triggered mRNA degradation via the RNA interference pathway or RNase H activation. We also analyzed the effect of chemical modifications of CAG repeat-targeting siRNAs on their efficiency in the foci decline. Our results suggest that the decrease of RNA foci number may be considered as a readout of treatment outcomes for oligonucleotide reagents.
Highlights
A group of human neurodegenerative diseases is caused by the expansion of CAG repeats located in the coding sequence of single functionally unrelated genes; these disorders are called polyQ diseases
We used a set of chemically synthesized ONs that differed with regard to their (I) targeted sequence, (II) anticipated mechanism of activity (RNAibased, RNase H-inducing, and a “blocker”), and (III) pattern of chemical modification (pure RNA ONs and RNAs containing 2 -fluoro (2 F), 2 -O-methylo (2 OMe), and phosphorothioate (PTO) modifications, DNA gapmers with 2 OMe and PTO, as well as locked nucleic acid (LNA) oligomer)
Selected small interfering RNA (siRNA) (CAG/CUG, A2, G2, A2F, and A2M), antisense oligonucleotide (ASO) CTG, and LNA CTG were delivered by lipid-based transfection
Summary
A group of human neurodegenerative diseases is caused by the expansion of CAG repeats located in the coding sequence of single functionally unrelated genes; these disorders are called polyQ diseases. The most thoroughly analyzed member of this group of disorders is HD, caused by expanded CAG repeats in the first exon of the HTT gene. Both RNA and protein products from the mutant allele are proposed to be involved in the pathogenic process; the most promising therapeutic approaches are designed to target the mutant transcripts. Increased retention of mutant RNA in the nucleus is associated with compromised functions of proteins that bind to mutant RNA (Jazurek et al, 2016); examples of such malfunctions are alternative splicing abnormalities (Mykowska et al, 2011; Sathasivam et al, 2013; Cabrera and Lucas, 2016) and other gene expression alterations (Sharma and Taliyan, 2015). RNA foci are increasingly considered undesired toxic structures, rather than a protective cellular self-defense
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