Allele-Selective Inhibition of Mutant Atrophin-1 Expression by Duplex and Single-Stranded RNAs

Abstract

Dentatorubral-pallidoluysian atrophy (DRPLA) is a progressive neurodegenerative disorder that currently has no curative treatments. DRPLA is caused by an expansion of a CAG trinucleotide repeat region within the protein-encoding sequence of the atrophin-1 (ATN-1) gene. Inhibition of mutant ATN-1 protein expression is one strategy for treating DRPLA, and allele-selective gene silencing agents that block mutant expression over wild-type expression would be lead compounds for therapeutic development. Here we develop an assay for distinguishing mutant from wild-type ATN-1 protein by gel electrophoresis. We use this assay to evaluate duplex RNAs and single-stranded silencing RNAs (ss-siRNAs) for allele-selective inhibition of ATN-1 protein expression. We observed potent and allele-selective inhibition by RNA duplexes that contain mismatched bases relative to the CAG target and have the potential to form miRNA-like complexes. ss-siRNAs that contained mismatches were as selective as mismatch-containing duplexes. We also report allele-selective inhibition by duplex RNAs containing unlocked nucleic acids or abasic substitutions, although selectivities are not as high. Five compounds that showed >8-fold allele selectivity for mutant ATN-1 were also selective for inhibiting the expression of two other trinucleotide repeat disease genes, ataxin-3 (ATXN-3) and huntingtin (HTT). These data demonstrate that the expanded trinucleotide repeat within ATN-1 mRNA is a potential target for compounds designed to achieve allele-selective inhibition of ATN-1 protein, and one agent may allow the targeting of multiple disease genes.

Phosphorothioates, Essential Components of Therapeutic Oligonucleotides

“…A single phosphorothioate is often placed at the 3¢ terminus at both strands to protect against degradation [83]. In order to bypass the requirement of a carrier for cellular uptake of the double stranded siRNA, a fully …”

Engineering Duplex RNAs for Challenging Targets: Recognition of GGGGCC/CCCCGG Repeats at the ALS/FTD C9orf72 Locus

“…We have previously tested duplex anti-CAG RNAs with central mismatches as inhibitors of huntingtin, ataxin-3, and atrophin-1 expression (Hu et al, 2014). We found that these duplexes do not promote cleavage of their targets but can be potent and allele-selective inhibitors of protein expression.…”

Mutant CAG Repeats Effectively Targeted by RNA Interference in SCA7 Cells

“…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].…”