Summary A non-coding hexanucleotide repeat expansion in the C9ORF72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Patients harboring this expansion develop several unique histopathological hallmarks, including intranuclear foci composed of either sense or antisense RNA transcripts from the expanded repeats and dipeptide repeat proteins generated by non-canonical translation of the expanded RNA transcripts. To further investigate the pathological role of C9ORF72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9ORF72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features that are the hallmark of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptides repeat proteins. Finally, using a synthetic microRNA that targets human C9ORF72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9ORF72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy.

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GGGGCC (G 4 C 2 ) hexanucleotide repeat expansion (HRE) in the first intron of the C9ORF72 (C9) gene is the most common genetic cause of ALS and FTD, two devastating adult-onset neurodegenerative disorders 1,2 . Proposed disease mechanisms include a partial loss of the C9ORF72 protein function (C9ORF72 haploinsufficiency) and acquired toxicity of the repeat expansion 3 . Transcription of the C9ORF72 gene generates three transcript variants: V1, V2 and V3 (Fig. 1a). V1 is translated to produce a short protein isoform (222 amino acids), whereas V2 and V3 generate the most predominant C9ORF72 protein (481 amino acids), which functions in vesicular trafficking 4 . Located adjacent to the promoter region of the most abundant V2 transcript variant, the G 4 C 2 repeat expansion impairs its transcription, leading to C9ORF72 protein haploinsufficiency 5,6 , impaired function of myeloid cells 7,8 and diminished neuronal viability 9 . Both sense and antisense transcripts encompassing the HRE in V1 and V3 generate RNA foci and undergo translation into atypical, aggregation-prone dipeptide repeat (DPR) proteins in all open reading frames 10,11 . These unusual DPRs are toxic in several experimental model systems [12][13][14][15] . Despite important advances in elucidating the molecular pathology of the expanded hexanucleotide repeats, there are no meaningful therapies for C9ORF72-related ALS or FTD.ASOs can drive therapeutic effects by mechanisms that include splice-modulation or, if the ASO contains DNA, activation of endogenous RNase H 16 to degrade the target RNA. The broad bioavailability of ASOs in the central nervous system (CNS), including both neurons and glial cells 17 , has prompted development of ASOs as therapy for dominantly transmitted genetic disorders of the CNS (for example, ALS caused by mutations in the SOD1 gene).Here we report development of ASOs targeting C9ORF72 to treat ALS and FTD. Using different C9-related model systems, including patient-derived samples and two C9BAC transgenic mouse models 18,19 , we generated ASOs that specifically reduce levels of the transcripts harboring the HRE as well as their DPR products, with minimal effects on the most abundant V2 isoform, which does not contain the HRE. We show that modification of a subset of the phosphodiester internucleoside linkages significantly improves ASO tolerability without impairing potency. We demonstrate that, in a single patient harboring mutant C9ORF72 with the G 4 C 2 repeat expressions, repeated intrathecal dosing of the optimal ASO was well tolerated and led to significant and durable reduction in levels of cerebrospinal fluid (CSF) poly(GP). Results ASO suppresses C9ORF72 in fibroblasts and mouse neurons.Because haploinsufficiency of C9ORF72 is thought to be adverse, we developed ASOs that target only the 5′ end of transcripts V1 and V3 that bear the G 4 C 2 repeat expansion, sparing transcript V2. As it is not fully clear whether the repeat-containing intron is retained or spliced out, we focused our effort on ASO sequences targeting ...

ObjectiveTo define the natural history of the C9orf72 amyotrophic lateral sclerosis (C9ALS) patient population, develop disease biomarkers, and characterize patient pathologies.MethodsWe prospectively collected clinical and demographic data from 116 symptomatic C9ALS and 12 non–amyotrophic lateral sclerosis (ALS) full expansion carriers across 7 institutions in the United States and the Netherlands. In addition, we collected blood samples for DNA repeat size assessment, CSF samples for biomarker identification, and autopsy samples for dipeptide repeat protein (DPR) size determination. Finally, we collected retrospective clinical data via chart review from 208 individuals with C9ALS and 450 individuals with singleton ALS.ResultsThe mean age at onset in the symptomatic prospective cohort was 57.9 ± 8.3 years, and median duration of survival after onset was 36.9 months. The monthly change was −1.8 ± 1.7 for ALS Functional Rating Scale–Revised and −1.4% ± 3.24% of predicted for slow vital capacity. In blood DNA, we found that G4C2 repeat size correlates positively with age. In CSF, we observed that concentrations of poly(GP) negatively correlate with DNA expansion size but do not correlate with measures of disease progression. Finally, we found that size of poly(GP) dipeptides in the brain can reach large sizes similar to that of their DNA repeat derivatives.ConclusionsWe present a thorough investigation of C9ALS natural history, providing the basis for C9ALS clinical trial design. We found that clinical features of this genetic subset are less variant than in singleton ALS. In addition, we identified important correlations of C9ALS patient pathologies with clinical and demographic data.

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. Approximately 20% of familial ALS cases are caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Rodents expressing mutant SOD1 transgenes develop progressive, fatal motor neuron disease and disease onset and progression is dependent on the level of SOD1. We investigated the possibility that a reduction in SOD1 protein may be of therapeutic benefit in ALS and screened 30,000 compounds for inhibition of SOD1 transcription. The most effective inhibitor identified was N-{4-[4-(4-methylbenzoyl)-1-piperazinyl]phenyl}-2-thiophenecarboxamide (Compound ID 7687685), which in PC12 cells showed an EC50 of 10.6 microM for inhibition of SOD1 expression and an LD50 >30 microM. This compound was subsequently shown to reduce endogenous SOD1 levels in HeLa cells and to exhibit a modest reduction of SOD1 protein levels in mouse spinal cord tissue. These data suggest that the efficacy of compound 7687685 as an inhibitor of SOD1 gene expression is not likely to be clinically useful, although the strategy reported could be applied broadly to screening for small molecule inhibitors of gene expression.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are detected in 20% of familial and 3% of sporadic amyotrophic lateral sclerosis (ALS) cases. Although mutant SOD1 is known to induce motor neuron death via multiple adverse acquired functions, its exact pathogenic mechanism is not well defined. SOD1 toxicity is dose dependent; levels of mutant SOD1 protein in transgenic mice determine disease susceptibility, onset and rate of progression. We therefore sought to identify small molecules that reduce SOD1 levels by inhibiting the SOD1 promoter. We tested pyrimethamine (previously reported to suppress SOD1 expression), several compounds currently in trials in human and murine ALS, and a set of 1,040 FDA-approved compounds. In a PC12 cell-based assay, no compounds reduced SOD1 promoter activity without concomitant cytotoxicity. Additionally, pyrimethamine failed to repress levels of SOD1 protein in HeLa cells or homogenates of liver, spinal cord and brain of wild-type mice. 34 compounds (including riluzole, ceftriaxone, minocyclin, PBA, lithium, acetylcysteine) in human and mouse ALS trials and an additional set of 1,040 FDA approved compounds also showed no effect on SOD1 promoter activity. This present study thus failed to identify small molecule inhibitors of SOD1 gene expression.

Background An intronic GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), referred to as C9ALS/FTD. No cure or effective treatment exist for C9ALS/FTD. Three major molecular mechanisms have emerged to explain C9ALS/FTD disease mechanisms: (1) C9ORF72 loss-of-function through haploinsufficiency, (2) dipeptide repeat (DPR) proteins mediated toxicity by the translation of the repeat RNAs, and more controversial, (3) RNA-mediated toxicity by bidirectional transcription of the repeats that form intranuclear RNA foci. Recent studies indicate a double-hit pathogenic mechanism in C9ALS/FTD, where reduced C9ORF72 protein levels lead to impaired clearance of toxic DPRs. Here we explored whether pharmacological compounds can revert these pathological hallmarks in vitro and cognitive impairment in a C9ALS/FTD mouse model (C9BAC). We specifically focused our study on small molecule inhibitors targeting chromatin-regulating proteins (epidrugs) with the goal of increasing C9ORF72 gene expression and reduce toxic DPRs. Results We generated luciferase reporter cell lines containing 10 (control) or ≥ 90 (mutant) G4C2 HRE located between exon 1a and 1b of the human C9ORF72 gene. In a screen of 14 different epidrugs targeting bromodomains, chromodomains and histone-modifying enzymes, we found that several bromodomain and extra-terminal domain (BET) inhibitors (BETi), including PFI-1 and JQ1, increased luciferase reporter activity. Using primary cortical cultures from C9BAC mice, we further found that PFI-1 treatment increased the expression of V1-V3 transcripts of the human mutant C9ORF72 gene, reduced poly(GP)-DPR inclusions but enhanced intranuclear RNA foci. We also tested whether JQ1, an BETi previously shown to reach the mouse brain by intraperitoneal (i.p.) injection, can revert behavioral abnormalities in C9BAC mice. Interestingly, it was found that JQ1 administration (daily i.p. administration for 7 days) rescued hippocampal-dependent cognitive deficits in C9BAC mice. Conclusions Our findings place BET bromodomain inhibitors as a potential therapy for C9ALS/FTD by ameliorating C9ORF72-associated pathological and behavioral abnormalities. Our finding that PFI-1 increases accumulation of intranuclear RNA foci is in agreement with recent data in flies suggesting that nuclear RNA foci can be neuroprotective by sequestering repeat transcripts that result in toxic DPRs.

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) accounts for most cases of familial ALS and frontotemporal dementia (FTD). To determine if suppressing expression of C9ORF72 gene products can reduce toxicity, we designed a set of artificial microRNAs (amiRNA) targeting the human C9ORF72 gene. Here we report that an AAV9-mediated amiRNA significantly suppresses expression of the C9ORF72 mRNA, protein, and toxic poly-dipeptides generated by the expanded repeat in the brain and spinal cord.