Identification of selective and non-selective C9ORF72 targeting in vivo active siRNAs

James W Gilbert; Zachary Kennedy; Bruno M D C Godinho; Ashley Summers; Alexandra Weiss; Dimas Echeverria; Brianna Bramato; Nicholas McHugh; David Cooper; Ken Yamada; Matthew Hassler; Hélène Tran; Fen Biao Gao; Robert H Brown Jr; Anastasia Khvorova

doi:10.1016/j.omtn.2024.102291

Identification of selective and non-selective C9ORF72 targeting in vivo active siRNAs

Mol Ther Nucleic Acids. 2024 Jul 31;35(3):102291. doi: 10.1016/j.omtn.2024.102291. eCollection 2024 Sep 10.

Authors

Affiliations

¹ RNA Therapeutic Institute, Worcester, MA 01655, USA.
² Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA 01655, USA.

Abstract

A hexanucleotide (G₄C₂) repeat expansion (HRE) within intron one of C9ORF72 is the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). C9ORF72 haploinsufficiency, formation of RNA foci, and production of dipeptide repeat (DPR) proteins have been proposed as mechanisms of disease. Here, we report the first example of disease-modifying siRNAs for C9ORF72 driven ALS/FTD. Using a combination of reporter assay and primary cortical neurons derived from a C9-ALS/FTD mouse model, we screened a panel of more than 150 fully chemically stabilized siRNAs targeting different C9ORF72 transcriptional variants. We demonstrate the lack of correlation between siRNA efficacy in reporter assay versus native environment; repeat-containing C9ORF72 mRNA variants are found to preferentially localize to the nucleus, and thus C9ORF72 mRNA accessibility and intracellular localization have a dominant impact on functional RNAi. Using a C9-ALS/FTD mouse model, we demonstrate that divalent siRNAs targeting C9ORF72 mRNA variants specifically or non-selectively reduce the expression of C9ORF72 mRNA and significantly reduce DPR proteins. Interestingly, siRNA silencing all C9ORF72 mRNA transcripts was more effective in removing intranuclear mRNA aggregates than targeting only HRE-containing C9ORF72 mRNA transcripts. Combined, these data support RNAi-based degradation of C9ORF72 as a potential therapeutic paradigm.

Keywords: C9ORF72 amyotrophic lateral sclerosis, ALS; MT: Oligonucleotides: Therapies and Applications; dipeptide-repeat proteins; frontotemporal dementia, FTD; mRNA localization; oligonucleotide therapeutics; siRNA.