Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide

Abstract

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 ...

Biological basis and psychiatric symptoms in frontotemporal dementia

“…Antisense oligonucleotide (ASO) dependent repeat RNA degradation is promising and realistic therapeutic strategy in C9orf72 FTLD/ALS, 97 and a recent report described a C9orf72 repeat positive ALS patient intrathecally administered with an ASO called afinersen, a newly developed mixed backbone gapmer ASO which selectively targets GGGGCC repeat‐containing C9orf72 transcripts. CSF analysis of this ALS patient revealed dramatic reduction of poly‐GP DPR and partial clinical recovery, suggesting the feasibility of clinical suppression of repeat containing C9orf72 gene through ASO 98 . Meanwhile, BIIB078, another ASO for C9orf72 ALS, was in a phase I clinical trial, but was reported to be discontinued on March 28, 2022, after no efficacy was found in any of the secondary outcomes, although detailed data have not been published.…”

“…CSF analysis of this ALS patient revealed dramatic reduction of poly-GP DPR and partial clinical recovery, suggesting the feasibility of clinical suppression of repeat containing C9orf72 gene through ASO. 98 Meanwhile, BIIB078, another ASO for C9orf72 ALS, was in a phase I clinical trial, but was reported to be discontinued on March 28, 2022, after no efficacy was found in any of the secondary outcomes, although detailed data have not been published.…”

Gene Therapy in Amyotrophic Lateral Sclerosis

“…An important point to note is that only C9orf72 variants 1 and 3 (which carry the repeat expansion mutation) were targeted by ASOs without affecting variant 2 expression [69]; therefore, C9orf72 abundance post treatment remained fairly similar between transgenic and wild-type animals. Furthermore, another study showed proof of concept in a single human, where intrathecal Afinersen (ASO5-2) was effective at safely suppressing C9orf72 transcripts and had an 80% reduction in poly(GP) dipeptide levels with functional stability in this individual over an 18 month period [70]. Notably, a phase I clinical trial of ASOs targeting C9orf72 variants 1 and 3 (BIIB078) was recently completed by Ionis Pharmaceutical and Biogen Inc., in January 2022, and although it was well tolerated, it did not show any clinical benefit (ClinicalTrials.gov Identifier: NCT03626012) [71].…”

Development of a sensitive trial-ready poly(GP) CSF biomarker assay forC9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis

“…Levels of poly(GP) in CSF were not found to correlate with clinical disease markers or neurofilament CSF levels, a non-disease specific biomarker of neurodegeneration 17 24. Encouragingly, ASO treatment of mouse models has been shown to lead to durable, decreased poly(GP) levels both in brain tissues and mouse CSF, and a recent study showed reduction in CSF poly(GP) levels in in a person with C9orf72 ALS, showing that CSF poly(GP) levels could be used as a pharmacodynamic biomarker 16–18 25…”