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 ...

Protein misfolding is a common theme in neurodegenerative disorders including Huntington's disease (HD). The HD-causing mutant huntingtin protein (mHTT) has an expanded polyglutamine (polyQ) stretch that may adopt multiple conformations, and the most toxic of these is the one recognized by antibody 3B5H10. Here we show that the 3B5H10-recognized mHTT species has a slower degradation rate due to its resistance to selective autophagy in human cells and brains, revealing mechanisms of its higher toxicity.

Expansions of a G4C2 repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Proposed disease mechanisms include a gain of toxic functions of the G4C2 repeats, implying that selective reduction in levels of the repeat-containing transcripts would represent a treatment strategy for this disorder. In the present study, using C9-ALS/FTD patient derived cells and C9ORF72 BAC transgenic mice, we have generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G4C2 repeat containing transcripts in both the sense and anti-sense strands of C9ORF72 and effectively suppress tissue levels of polyGP dipeptides. In a single patient harboring mutant C9ORF72 with the G4C2 repeat expressions, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of CSF polyGP.

Significance Classical drug discovery identifies inhibitors that block the activities of pathogenic proteins. This typically relies on a measurable biochemical readout and accessible binding sites whose occupancy influences the activity of the target protein. These requirements make many pathogenic proteins “undruggable.” Here, we report a strategy to target these undruggable proteins: screening for compounds that directly bind to the undruggable target and rescue disease-relevant phenotypes. These compounds may suppress the target’s pathogenic functions via direct binding to it. We applied this strategy to the mutant HTT protein, which is an undruggable protein that causes Huntington’s disease (HD). We revealed desonide, an FDAapproved drug, as a possible lead compound for HD drug discovery.

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GM3 synthase deficiency (GM3SD) is an infantile-onset epileptic encephalopathy syndrome caused by biallelic loss-of-function mutations in ST3GAL5. Loss of ST3GAL5 activity in humans results in systemic ganglioside deficiency and severe neurological impairment. No disease-modifying treatment is currently available.Certain recombinant adeno-associated viruses (rAAVs) are capable of crossing the blood-brain barrier to induce widespread, long-term gene expression in the central nervous system (CNS), and represent a promising therapeutic strategy. Here, we show that a first-generation rAAV-ST3GAL5 replacement vector employing a ubiquitous promoter restored tissue ST3GAL5 expression and normalized cerebral gangliosides in patient-derived iPSC neurons and brain tissue from St3gal5 knock-out mice, but caused fatal hepatotoxicity when administered systemically. In contrast, a second-generation vector optimized for CNS-restricted ST3GAL5 expression, administered by either intracerebroventricular or intravenous route at postnatal day 1, allowed for safe and effective rescue of lethality and behavior impairment in symptomatic GM3SD mice up to a year. These results support further clinical development of ST3GAL5 gene therapy.