Allele-selective inhibition of mutant atrophin-1 expression by duplex and single-stranded RNAs

Biochemistry. 2014 Jul 22;53(28):4510-8. doi: 10.1021/bi500610r. Epub 2014 Jul 11.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Ataxin-3
  • Cell Line
  • Gene Expression Regulation*
  • Humans
  • Huntingtin Protein
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Mutagenesis, Insertional*
  • Myoclonic Epilepsies, Progressive / drug therapy
  • Myoclonic Epilepsies, Progressive / genetics
  • Myoclonic Epilepsies, Progressive / metabolism
  • Myoclonic Epilepsies, Progressive / pathology
  • Nerve Tissue Proteins / biosynthesis*
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Nuclear Proteins / biosynthesis
  • Nuclear Proteins / genetics
  • RNA, Double-Stranded / metabolism*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Repressor Proteins / biosynthesis
  • Repressor Proteins / genetics
  • Trinucleotide Repeat Expansion*

Substances

  • HTT protein, human
  • Huntingtin Protein
  • MicroRNAs
  • Nerve Tissue Proteins
  • Nuclear Proteins
  • RNA, Double-Stranded
  • RNA, Messenger
  • Repressor Proteins
  • atrophin-1
  • ATXN3 protein, human
  • Ataxin-3