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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons, causing progressive muscle weakness and respiratory failure. The presence of an expanded hexanucleotide repeat in chromosome 9 open reading frame 72 (C9ORF72) is the most frequent mutation causing familial ALS and frontotemporal dementia (FTD). To determine if suppressing expression of C9ORF72gene products can reduce toxicity, we designed a set of artificial microRNAs (amiRNA) targeting the human C9ORF72gene. Here we report that an AAV9-mediated amiRNA significantly suppresses expression of the C9ORF72mRNA, protein, and toxic dipeptide repeat proteins generated by the expanded repeat in the brain and spinal cord of C9ORF72 transgenic mice.

Expansions of a G4C2repeat in the C9ORF72gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Using C9-ALS/FTD patient-derived cells and C9ORF72BAC transgenic mice, we generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G4C2repeat-containing transcripts and effectively suppress tissue levels of poly(GP) dipeptides. ASOs with reduced phosphorothioate content showed improved tolerability without sacrificing efficacy. In a single patient harboring mutant C9ORF72with the G4C2repeat expansion, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of cerebrospinal fluid poly(GP). This report provides insight into the effect of nucleic acid chemistry on toxicity and, to our knowledge, for the first time demonstrates the feasibility of clinical suppression of the C9ORF72gene. Additional clinical trials will be required to demonstrate safety and efficacy of this therapy in patients with C9ORF72gene mutations.