Self-duplexing CUG repeats selectively inhibit mutant huntingtin expression

Abstract

Huntington’s disease (HD) is a neurodegenerative genetic disorder caused by the expansion of the CAG repeat in the translated sequence of the HTT gene. This expansion generates a mutant huntingtin protein that contains an abnormally elongated polyglutamine tract, which, together with mutant transcript, causes cellular dysfunction. Currently, there is no curative treatment available to patients suffering from HD; however, the selective inhibition of the mutant allele expression is a promising therapeutic option. In this study, we developed a new class of CAG repeat-targeting silencing reagents that consist of self-duplexing CUG repeats. Self-duplex formation was induced through one or several U-base substitutions. A number of self-duplexing guide-strand-only short interfering RNAs have been tested through transfection into cells derived from HD patients, showing distinct activity profiles. The best reagents were highly discriminatory between the normal and mutant HTT alleles (allele selectivity) and the HTT transcript and other transcripts containing shorter CAG repeats (gene selectivity). We also demonstrated that the self-duplexing CUG repeat short interfering RNAs use the RNA interference pathway to elicit silencing, and repeat-targeting reagents showed similar activity and selectivity when expressed from short hairpin RNA vectors to achieve more durable silencing effects.

Mutant CAG Repeats Effectively Targeted by RNA Interference in SCA7 Cells

“…A similar upregulation caused by other CAG repeat–targeting ONs was observed for normal huntingtin protein [18]. Some aspects of the mechanism, by which these so-called miRNA-like siRNAs function, was previously investigated [21,25] proving the AGO2-dependent activity and suggesting the cooperative action of multiple RNA-induced silencing complexes (RISCs) bound to the expanded CAG repeat tract.…”

“…Next, we compared the efficiency and selectivity in the downregulation of the ataxin-7 protein for ATXN7 sequence-specific siRNA (siATXN7) and selected a CAG repeat–targeting ON: self-duplexing (sd)-siRNA A2 [21]. We investigated the activity of siATXN7 and A2 (Figure 2A) at 24, 48 and 72 h post-transfection at 100 nM.…”

“…We tested sd-siRNA G2 [21], W1316 duplex [18] and A2F and A2M [26] (Figure 2A). A2F possesses a chemical modification pattern containing mainly 2′-fluoro (2′F) as well as two 2′- O -methylo (2′ O Me) nucleotides from the 3′ end, while A2M contains 2′F, 2′ O Me and phosphorothioate modifications.…”

“…CAG repeat–targeting RNAi reagents containing base substitutions were successfully tested for HD, SCA3 and DRPLA, and various types of reagents were developed for this strategy, including short duplexes, self-duplexing guide-only siRNAs, shRNA, and chemically modified single-stranded siRNAs [17,18,19,20,21,22,23,24]. These reagents formed mismatches with their target and triggered translational inhibition, rather than transcript degradation [25].…”

Reduction of Huntington’s Disease RNA Foci by CAG Repeat-Targeting Reagents

“…In order to achieve high preference in the silencing of mutant alleles, CAG repeat-targeting siRNAs have been modified to form base mismatches with the target sequence and induce a mechanism similar to that of miRNAs (Hu et al, 2010; Fiszer et al, 2011, 2013, 2016; Yu et al, 2012; Liu et al, 2013). In mutation-targeting approach also oligomers acting as translational blockers were developed as potential therapeutics for HD and spinocerebellar ataxia type 3 (SCA3) (Hu et al, 2009; Fiszer et al, 2012).…”