Targeting transcription and translation of the antisense CCCCGG repeat expansion in C9ORF72 ALS/FTD

PROJECT SUMMARY
Amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) are devastating neurodegenerative
diseases with no cure to date. The most common genetic cause of ALS/FTD is a hexanucleotide GGGGCC
(G4C2) repeat expansion mutation in the C9ORF72 gene. The repeat is transcribed in both directions, producing
sense G4C2 and antisense CCCCGG (C4G2) transcripts that are translated into six dipeptide repeat (DPR)
proteins. The GGGGCC mutation is thought to cause ALS via three non-mutually exclusive mechanisms: a loss-
of-function mechanism due to reduced expression of C9ORF72 protein, and gain-of-function mechanisms due
to toxic repeat-containing RNA and/or DPRs. Sense and antisense RNA and DPRs accumulate in the central
nervous system of C9ORF72 ALS/FTD patients and have been shown to be toxic in multiple model systems. To
date, most studies have focused on the transcription and translation of the sense GGGGCC transcript. Hence,
we know very little about how the antisense CCCCGG RNA is transcribed and translated. Importantly, recent
clinical trials only targeting sense GGGGCC transcripts failed, suggesting that antisense CCCCGG RNAs and
their DPRs should be explored as therapeutic targets. In Aim 1 of this proposal, the yet-unknown transcription
start site (TSS) of the antisense transcript will be located using 5’ Rapid Amplification of cDNA Ends (RACE).
Upon locating putative TSSs, the site(s) will be functionally validated via CRISPR/Cas9 mutagenesis in
C9ORF72 patient-derived induced pluripotent stem cells (iPSCs). Then, I will target the TSS via CRISPR
interference (CRISPRi) - a catalytically inactive Cas9 enzyme will interfere with transcription of the antisense
strand, and the effects of blocking antisense strand transcription will be assessed in vitro using iPSC-derived
motor neurons. In Aim 2, antisense oligonucleotides (ASOs) will be used to selectively degrade antisense repeat
RNA. First, the candidate ASOs will be assessed for their ability to decrease DPR and transcript expression and
reduce cell death in vitro using iPSC-derived motor neurons. Next, candidate ASOs will be administered to mice
expressing 35 copies of the antisense CCCCGG repeat to determine whether they can rescue ALS/FTD
pathology and symptoms. Altogether, successful completion of this proposal will elucidate a new target
(antisense CCCCGG repeat RNA) of therapeutic interest for ALS/FTD. Such knowledge may be relevant for
multiple other neurodegenerative diseases which are caused by bidirectionally-transcribed repeat expansions,
including Huntington’s disease, spinocerebellar ataxias, and Friedreich ataxia.

Grant Number: 5F31NS141619-02
NIH Institute/Center: NIH
Principal Investigator: Mira Antonopoulos