AAV-based gene therapy mediated RNA editing as a therapy for C9ORF72-ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons (MNs). New therapeutics are urgently needed. However, developing effective treatments for ALS has been difficult be-cause pathogenesis begins long before patients are diagnosed, ALS disease course changes over time, and human genetics suggests that ALS is a collection of disease subtypes, suggesting that personalized medicine might be necessary.

The most common known genetic cause of ALS is a GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) within the first intron of the gene C9orf72. In addition, HRE in C9orf72 is the most common cause of frontotemporal dementia. Therefore, therapeutics designed specifically against HRE would benefit a large number of patients. Since HRE mutations are associated with both sense and antisense transcription, which are both toxic to neurons, we initially set about generating a gene therapeutic that used CasRx to specifically degrade both sense and antisense transcripts. As part of this effort, we modeled C9orf72-ALS using isogenic induced pluripotent stem cell (iPSC)-derived motor neurons (MNs), which we found showed many mutation-associated phenotypes. However, during the course of this project, Prof. Adri-an Isaacs published the exact same result, necessitating a change in direction.

Instead of targeting HRE-containing RNA for degradation, we proposed epigenetically silencing C9orf72 to prevent transcription, preventing the need for degradation of the RNA. This would have the advantage of simultaneously reducing sense and antisense transcription as well as reducing any effects coming from R-loops, including DNA damage, which has been shown to contribute to C9orf72-ALS. We identified multiple candidate epigenetic silencing vectors that will be further tested in future experiments.