Variant-selective stereopure oligonucleotides protect against pathologies associated with C9orf72-repeat expansion in preclinical models

Hélène Tran ,Shaunna S Berkovitch ,Ann Durbin ,M Braun ,Xiheng Hu ,Kenneth A Longo ,Juili Dilip Shelke ,Jean-Cosme Dodart,Yuanjing Liu,Yuan Yin,Robert H Brown,Chandra Vargeese,Pachamuthu Kandasamy,Fangjun Liu,Jörg Ruschel,Zhong Zhong,Naoki Iwamoto,Hailin Yang,Michael Byrne,Amy Donner,Hyun Gyung Jang

doi:10.1038/s41467-021-21112-8

Abstract

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.

Highlights

A large G4C2-repeat expansion in C9orf[72] is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)
We report the discovery of an oligonucleotide-targeting sequence called Splice Site-1b (SS1b) at the exon 1b–intron 1 junction of C9orf[72] that unexpectedly yields preferential RNase H-mediated knockdown of V3 and intron-1-containing transcripts with stereopure oligonucleotides
The potency and preferential activity we observed in cellular models under freeuptake conditions translated in vivo to a mouse model

Summary

Introduction

A large G4C2-repeat expansion in C9orf[72] is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. A large hexanucleotide-repeat (G4C2) expansion in the first intron or promoter region of C9orf[72] is the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)[1,2,3]. Accumulation of repeat-containing transcripts can produce motor neuron deficits and C9-ALS/FTD-like symptoms in animal models[9,10,11,14]. The repeat expansion leads to the generation and accumulation of neurotoxic dipeptide-repeat proteins (DPRs)[5,7,9,10,11,14,19,20]

Objectives

Methods

Results

Conclusion