Summary Hexanucleotide expansions in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9ORF72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9ORF72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but no motor dysfunction. Hexanucleotide expansions caused age-, repeat length- and expression level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9ORF72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain-of-toxicity as a central disease mechanism caused by repeat-expanded C9ORF72 and establish the feasibility of ASO-mediated therapy.

Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.E xpanded hexanucleotide repeats in the first intron of the chromosome 9 open reading frame 72 (C9orf72) gene were recently identified (1, 2) as the most common genetic cause of ALS, frontotemporal degeneration (FTD), or concomitant ALS/ FTD (3, 4). The mechanisms by which the expanded repeats cause neurodegeneration are unknown, but leading candidate mechanisms are RNA-mediated toxicity, loss of the C9orf72 gene function (from reduced C9orf72 produced by the allele with the expansion), or a combination of the two.RNA-mediated toxicity from nucleotide repeat expansion was initially described for CUG expansion in the RNA encoded by the DMPK gene in myotonic muscular dystrophy (5). A consensus view is that RNA toxicity plays a crucial role in a variety of repeat expansion disorders (6). A hallmark of these disorders is the accumulation of expanded transcripts into nuclear RNA foci (7). RNAs harboring a long stretch of repeats are thought to fold into stable structures and sequester RNA binding proteins, which, in turn, sets off a molecular cascade leading to neurodegeneration (7). In myotonic dystrophy, sequestration and functional disruption of the muscleblind-like family of RNA binding proteins are associated with specific splicing and expression changes in affected tissues (5,(8)(9)(10)(11)(12).Sequestration of one or more RNA binding proteins into pathological RNA foci has ...