Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration

Abstract

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 ...

Clinical Trial Designs in Amyotrophic Lateral Sclerosis: Does One Design Fit All?

“…There has been a recent trend toward subgroup-targeted therapy based not on phenotype but on genotype. RNA silencing (siRNA) and antisense technologies trials targeting specific genes have, thus far, enrolled patients with the specific mutations [6,7]. This type of subgrouping will likely continue, and while nongene-targeted therapies may not exclude participants by gene status, future trials may well stratify analysis by gene mutation.…”

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…However, targeted knockdown C9orf72 failed to recapitulate the same pattern of gene expression noted in fibroblasts from ALS patient cells carrying C9orf72 mutations, implying that a simple loss of C9orf72 function is unlikely to explain the observed pattern of RNA dysregulation in C9orf72 mutant cells [67]. In addition, C9orf72 knockdown was not associated with adverse effects or neuronal loss in mice receiving ASOs [67]. Knockdown of the mutant C9orf72 allele also restored expression for several affected genes in C9orf72-mutant iPSC-derived neurons [66,68], indicating that this therapeutic strategy may prove effective and safe in humans with disease.…”

“…The C9orf72 protein is ubiquitously expressed but highly enriched in neurons [66,68,70], with a proposed function in endosomal trafficking [71]. However, targeted knockdown C9orf72 failed to recapitulate the same pattern of gene expression noted in fibroblasts from ALS patient cells carrying C9orf72 mutations, implying that a simple loss of C9orf72 function is unlikely to explain the observed pattern of RNA dysregulation in C9orf72 mutant cells [67]. In addition, C9orf72 knockdown was not associated with adverse effects or neuronal loss in mice receiving ASOs [67].…”

“…Pathogenic hexanucleotide expansion mutations in C9orf72 are associated with distinct changes in gene expression that only partially overlap with those induced by TDP43 or FUS dysregulation [66][67][68][69]. Human iPSCs carrying disease-associated C9orf72 expansions exhibited prominent downregulation of several essential genes [68], including those involved in synaptic transmission, cell adhesion, and membrane excitability.…”

“…Human iPSCs carrying disease-associated C9orf72 expansions exhibited prominent downregulation of several essential genes [68], including those involved in synaptic transmission, cell adhesion, and membrane excitability. Importantly, this effect was strikingly muted in mutant C9orf72 fibroblasts [67], suggesting that many of the differentially expressed genes are unique to neurons. The C9orf72 protein is ubiquitously expressed but highly enriched in neurons [66,68,70], with a proposed function in endosomal trafficking [71].…”